OPTIMIZATION OF PLANT NUTRITION

Developments in Plant and Soil Sciences

VOLUME 53

The titles published in this series are listed at the end of this volume.

Optimization of Plant Nutrition

Refereed papers from the Eighth International Colloquium for the Optimization of Plant Nutrition, 31 August - 8 September 1992, Lisbon, Portugal

Edited by

M.A.C. FRAGOSO National Agronomy Research Station National Institute for Agrarian Research (IN/A) Oeiras, Portugal

and

M.L. VAN BEUSICHEM Department of Soil Science and Plant Nutrition Wageningen Agricultural University Wageningen, The Netherlands

Managing editor

A.HOUWERS Department of Microbiology Wageningen Agricultural University Wageningen, The Netherlands

Partly reprinted from Plant and Soil, Volume 154, No. 1 (1993)

SPRINGER-SCIENCE+BUSINESS MEDIA, B.V.

Library of Congress Cataloging - in - Publication Data

International Colloquium for the Optimization of Plant Nutrition (8th , 1992 , Lisbon, Portugal>

Optimization of plant nutrition refereed papers from the Eighth International Colloquium for the Optimization of Plant Nutrition, 31 August-S September 1992. Lisbon, Portugal I edited by M.A.C. Fragoso and M.L. van Beusichem ; managing editor. A. Houwers.

p. co. -- Weve 1 opments in p 1 ant and so II sciences ; v. 53> Includes Indexes.

1. Crops--Nutrition--Congresses. 2. Plants--Nutrition­-Congresses. 3. Crops--Effect of minerals on--Congreses. 4. Fertilizers--Congresses. 5. Crops--Effect of stress on--Congresses. I. Fragoso, M. A. C. II. Beus1chem, M. L. van (Marinus Leonard>, 1949- III. Houwers, A. IV. Title. V. Series. SB112.2.I57 1992 631.8'11--dc20 93-33132

ISBN 978-90-481-4331-3

Printed on acid-free paper

All Rights Reserved

No part of the material protected by this copyright notice may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, or by any information storage and retrieval system, without written permission from the copyright owner.

© 1993 Springer Science+Business Media DordrechtOriginally published by Kluwer Academic Publishers in 1993 Softcover reprint of the hardcover 1st edition 1993

ISBN 978-90-481-4331-3 ISBN 978-94-017-2496-8 (eBook) DOI 10.1007/978-94-017-2496-8

Contents*

Preface by the Editors

Eighth International Colloquium for the Optimization of Plant Nutrition Committee of Honour Organizing Committee I Scientific Committee

International Association for the Optimization of Plant Nutrition Board I Standing Committee

PART A: PLANT ANALYSIS METHODS AND REFERENCE VALUES FOR PLANT MATERIAL

1. A rapid wet digestion method for plant analysis,

XIII

XIV XV

XVI

by A. Pequerul, C. Perez, P. Madero, J. Val and E. Monge 3 2. Comparison of techniques for nitrogen analysis in potato crops,

by M.W. Young, H.V. Davies and D.K.L. MacKerron 7 3. Evaluation of inorganic elements in agricultural products from Italian farms by instrumental

neutron activation analysis, by A. Moauro, L. Triolo, P. Avino and L. Ferrandi 13

*4. Determination of lead in white lupin by anodic stripping voltammetry, by M.M.P.M. Neto and A. De Varennes 19

*5. Determination of copper in different chloroplast preparations, by J.B. Arellano, M. Baron, A. Chueca and M. Lachica 25

6. Four new CII reference materials for the chemical analysis of plants: Pine needles, oak leaves, barley-straw and apple-fruit, by R.Ch. Daniel, P. Lischer and G. Theiller 31

7. Determination of leaf standards for apple trees and grapevines in northern Italy, by 0. Failla, G. Stringari, D. Porro and A. Scienza 37

8. Interpretative indices for leaf analysis in vineyards of the Portuguese region of Bairrada, by C. Pachecho, F. Calouro and A. Andrade 43

9. Relationship between aboveground dry weight and N, P and K concentrations in grassland species: A guide for the diagnosis of plant nutrient status, by A. Peeters and V. Van Bol 49

PART B: NUTRITIONAL CHARACTERISTICS AND MANAGEMENT OF GROWTH MEDIA

* 1. Potassium supplying capacity of northeastern Portuguese soils, by E.A.C. Portela 57

*2. Nutritional disorders between potassium, magnesium, calcium, and phosphorus in soil, by S.T. Jakobsen 65

3. The influence of the soil phosphate capacity factor on soil and plant phosphorus critical levels of different vegetables, by R.F. Novais, J.C.L. Neves, N.F. Barros, V.W.D. Casali and A.S. Fabres 73

*Contributions indicated with an asterisk are reprinted from Plant and Soil, Volume 154, No. I (1993).

VI

4. The use as fertilizer of combined primary I secondary pulp-miii sludge, by F. Cabral and E. Vasconcelos 77

5. The use of industrial wastes as manures: A case study with effluent mud from an olive oil processing plant, _ by D. Ana<;, H. Hakerlerler and M.E. Irget 83

6. Influence of container size and substrate mineral composition on transplant growth and yield of broccoli cv. Green Duke by A.M. Simoes, F. Calouro, E. Abrantes and E. Sousa 87

7. Mulch and topdressed nitrogen effects on bell pepper, by P.D. Castellane, R.K. Fujimura, J.A.C. de Araujo and M.E. Ferreira 93

*8. Long-term effects of gypsiferous coal combustion ash applied at disposal levels on soil chemical properties, by R.F. Korcak and W.D. Kemper 97

9. Effects of com posted municipal waste and a paper mill waste com posted with bark on the growth of vegetable crops, by L.M. Brito and P. Hadley 101

10. Assessment of plant-available nitrogen in processed organic wastes, by W.M.F. Raijmakers and B.H. Janssen 107

11. Relations between nitrogen and phosphorus immobilization during decomposition of forest litter, by W.G. Braakhekke, H.A. Stuurman, H. van Reuler and B.H. Janssen 117

PART C: IMPROVING THE DIAGNOSIS OF THE NUTRITIONAL STATUS OF CROPS

1. Some factors affecting potassium nutrition of sour cherry trees, by E. J adczuk 127

2. The rootstock effect on some nutrient levels in leaves of apple tree cv. Granny Smith, by L. Duarte 133

3. Foliar diagnosis of sugarbeet: Mineral composition of leaves of different physiological age during the season, by M.A. Castelo Branco, M.G. Serrao, M.L. Fernandes, E.M. Sequeira, H. Domingues and F.P. Pires 137

4. Diagnosing nutritional status of sugarbeet by soil and petiole analysis, by M.D. Oliveira, C.F. Carranca, M.M. Oliveira and M.R. Gusmao 147

5. Simulation of maize yield response to combined effects of nitrogen fertilization versus irrigation and plant population, by J. Beltrao and J. Ben Asher 153

6. The use of diagnosis recommendation integrated system (DRIS) to evaluate the nutritional status of healthy and blight affected citrus trees, by E. Malavolta, S.A. Oliveira and G.C. Vitti 157

7. An expert system for diagnosing citrus nutritional status and planning fertilization, by D. Palazzo, G. Basile, R. D' Agostino, F. Intrigliolo, K. Chiriatti and C. Resina 161

*8. DRIS evaluation of persimmon (Diospyrus kaki L.), by I. Klein, L. Fanberstein and L. Viner 167

*9. A multivariate diagnosis approach applied to celery, by N. Tremblay, P. Auclair, L.-E. Parent and A. Gosselin 173

* 10. Design and analysis of mixture systems: Applications in hydroponic, plant nutrition research, by E. Schrevens and J. Cornell 179

PART D: NUTRIENT UPTAKE AND INTERACTIONS WITH PHYSIOLOGICAL AND BIOCHEMICAL PROCESSES

* 1. Genotype variability for physiological efficiency index of nitrogen in oats, by D. Isfan

*2. Mapping of genes for copper efficiency in rye and the relationship between copper and iron efficiency, by R. Schlegel, R. Kynast, T. Schwarzacher, V. Romheld and A. Walter

3. Effect of a transient anoxia on potassium uptake in cucumber, by G. Bertoni, J. Silvestre, J.M. Llorens, P. Morard and C. Maertens

*4. Differences in uptake kinetics of ammonium and nitrate in legumes and cereals, by T.P. Rao, 0. Ito and R. Matsunga

5. Influence of root temperature on potassium nutrition of tomato plant, by P. Cornillon and A. Fellahi

*6. Growth analysis of soil-grown spinach plants at different N-regimes, by E. Smolders, J. Buysse and R. Merckx

7. Nitrate assimilation by wheat species at low rates of nitrogen supply, by A.U. Jan and D.J. Pilbeam

8. Influence of nitrogen availability on growth and development of tomato plants until fruit­setting, by Y. Dumas, J. S. Quijada and M. Bonafous

*9. Effects of fertilization levels in two farming systems on senescence and nutrient contents in potato leaves, by A. Berchtold, J.-M. Besson and U. Feller

10. Relationship between biochemical indicators and physiological parameters of nitrogen and physiological plant age, by J.L. Valenzuela, M. Guzman, A. Sanchez, A. del Rfo and L. Romero

PARTE: MINERAL COMPOSITION IN RELATION TO CROP GROWTH, YIELD AND PRODUCT QUALITY

1. An overall approach to plant nutrition through the use of square diagrams,

VII

189

197

203

207

213

219

227

235

243

251

by P. Morard, A. Bernadac and G. Bertoni 261 2. Nutrient uptake by To!Jlouse violet (Viola odorata var. parmensis) during its developmental

cycle, by A. Shan Sei Fan and P. Morard 269

3. Effect of nitrogen on growth of broad beans, by M.G. Palha, C.F. Carranca, M.L. Fernandes and M.A. C. Fragoso 277

*4. Nitrogen uptake in relation to water availability in wheat, by J.P. De M.E. Abreu, I. Flores, F.M.G. De Abreu and M.V. Madeira 283

5. Effect of nitrogen-form on plant growth and nutrient composition of creeping bentgrass, by J.N. McCrimmon, K.J. Karnok and H.A. Mills 291

6. Sulphur status of maize leaves in relation to sulphur content and arylsulphatase activity in soil, by W.J. Melo, E.M. Quinones, M.O. Marques, M.A. Nogueira, R.A. Chelli and S.A.S. Leite 299

7. Soil properties and mineral content of leaves in fig orchards producing high-quality fruits, by U. Aksoy and D. Ana~ 305

8. Changes in K, Ca and Mg contents in different parts of fig fruit during development, by U. Aksoy and D. Akyuz 309

VIII

9. Effects of phosphorus and lime application on leaf mineral composition of olive trees grown in a schist soil, by P.V. Jordao, A. Mantas, M. Centeno and M.L. Duarte 313

10. Effects of paclobutrazol application and fruit load on microelement concentrations in peach leaves, by E. Monge, P. Madero, J. Val and A. Blanco 319

11. Effects of P-supply on growth and P-micronutrient interactions of potted peach seedlings, by M. Tagliavini, B. Marangoni and P. Grazioli 325

12. Effects of organic waste fertilization and saline irrigation on mineral composition of tomato leaves and fruits by I. Gomez, J. Navarro-Pedrefio and J. Mataix 333

PART F: RESPONSE TO FORM, RATE AND MANNER OF APPLICATION OF FERTILIZERS

1. Ammonia volatilization from compound nitrogen-sulfur fertilizers, by 0. Oenema and G.L. Velthof 341

2. Fertilizer recommendation scheme for phosphorus based on nutrient cycling in permanent pastures in the Basque Country, northern Spain, by A.G. Sinclair, M. Rodriguez, M. Oyanarte and G. Besga 351

3. Response of gamma medic (Medicago rugosa Desr.) grown on a calcaric cambisol to different rates of fertilizer phosphorus, by M.O. Torres, A.S.V. Costa and F. Calouro 355

4. Comparison of row and broadcast N application of N efficiency and yield of potatoes, by G. Hofman, P. Verstegen, P. Demyttenaere, M. Van Meirvenne, P. Delanote and G. Ampe 359

5. Phosphorus dynamics and mobility in the diffusion zone of band-applied phosphorus fertilizer by W. Werner and B. Strasser 367

6. Effects of phosphorus, magnesium and molybdenum application rates on resident and introduced meadow species in a dry land pasture in mountainous massif of Sic6, Portugal, by M.O. Torres, F. Ca1ouro, I. Magalhaes, J. Santos and J. Gama 375

7. Effects of nitrogen, phosphorus and potassium application rates on the botanical composition of an irrigated sward, by M.O. Torres, F. Calouro and A. Barata 381

8. Effects of nitrate, phosphate and sulfate combinations on growth and kinetics of phosphate and sulfate uptake by eucalypt seedlings, by N.F. Barros, F.A.S. Ferreira, R.F. Novais, J.C.L. Neves and V.H. Alvarez V. 391

9. Influence of animal manures on extractable micronutrients, greenhouse tomatoes and subsequent Swiss chard crops, by P.R. Warman 397

10. Do seaweed extracts improve vegetable production?, by P.R. Warman and R.R. Munro-Warman 403

PART G: INFLUENCE OF FERTILIZERS ON YIELD AND QUALITY OF V ARlO US CROPS

1. Effect of conventional and multiple N application by fertigation on maize grain yields and NO:J -N residues, by P.H. Girardin, R. Trendel, J.-L. Meyer, M. Birgaentzle and P. Freyss

2. Yield and quality of irrigated summer-annual forages in southern Portugal as affected by nitrogen fertilization, by M.E.V. Lourengo, M.A.P. DaSilva and L.M.B. Mendes

411

417

IX

3. Variation in yield and macronutrient uptake in vining peas regardless of N fertilization, by M.E. Ferreira, M.L. Fernandes and M.A. C. Fragoso 425

4. Nitrogen fertilization of Phaseolus vulgaris for freezing, by C.F. Carranca, A. Ferreira, L. Andrada, M.L. Fernandes, M.E. Ferreira and M.A. C. Fragoso 429

5. The effects of different nitrogen, phosphorus, potassium fertilizer application on tomato seed properties, by N. Eryuce and S. Aydin 435

6. Influence of nitrogen nutrition on nutritional status and yield of 'Navelina' orange, by F. Intrigliolo, G. Fisichella, M. Tropea, G. Sambuco and A. Giuffrida 439

7. Effect of NK fertilization on leaf nutrient content and fruit quality of 'Valencia late' orange trees, by C.F. Carranca, J. Baeta and M.A. C. Fragoso 445

8. Effects of lime and phospho gypsum on citrus, by G.C. Vitti, L.C. Donadio, E. Malavolta and J.R.M. Cabrita 449

9. Influence of soil and leaf applications of micronutrients on yield and fruit quality of Citrus sinensis Osbeck, variety Pera, by G.C. Vitti, L.C. Donado, R.D. Delarco, E. Malavolta and J.R.M. Cabrita 453

10. Effects of fertilization on apple tree development, yield and fruit quality, by D. Scudellari, B. Marangoni, D. Cobianchi, W. Faedi and M.L. Maltoni 457

11. Effects of Ca nutrition levels on growth and yield of wheat and two cvs. of triticales, by M.C. Matos, M.A. Nunes and E. Pinto 463

PART H: EFFECTS OF HEAVY METAL STRESS ON CROP BEHAVIOUR

1. Trace elements and isoenzyme activities in white lupin, by A. De Varennes and I. Carvalho 4 71

*2. Effect of iron chlorosis on mineral nutrition and lipid composition of thylakoid biomembrane in Prunus persica (L.) Bastch., by E. Monge, C. Perez, A. Pequerul, P. Madero and J. Val 477

3. Iron stress responses of chlorosis-susceptible and chlorosis-resistant cultivars of pepper (Capsicum annuum L.), by G.W. Welkie 483

4. Are manganese and iron deficiencies so similar? Fluorescence, a way of study, by J. Val, C. Perez, P. Madero, A. Pequerul and E. Monge 491

5. Specificity of iron in some aspects of soybean (Glycine max L.) physiology, by P. Madero, A. Pequerul, C. Perez, J. Val and E. Monge 497

6. Specificity of manganese in some aspects of soybean (Glycine max L.) physiology, by C. Perez, P. Madero, A. Pequerul, J. Val and E. Monge 503

7. The effect of liming and organic manuring on the reduction of copper toxicity in an acid soil of granitic origin of Dao wine region, Portugal, by R.M.S. Dias, J.C. Soveral-Dias and A.S.V. Costa 509

8. Some effects of different levels of lead on berseem, by C.M. DosSantos, M.M.P.M. Neto and A. De Varennes 517

*9. Evaluation of structural and physiological plant characteristics in relation to the distribution of cadmium in maize inbred lines, by P.J. Florijn, J.A. Nelemans and M.L. van Beusichem 523

10. Micronutrient content in graminaceous and leguminous plants contaminated with mercury, by J.J. Lucena, L.E. Hernandez, S. Olmos and R.O. Carpena-Rufz 531

X

PART I: MACRONUTRIENTS AND ENVIRONMENTAL STRESS

1. Effect of nutritional stress on photosynthesis rate of potato (Solanum tuberosum L.), by T. Mehouachi and R. Lemeur

2. Reactions of three soybean cultivars to interruptions in phosphorus supply, by H.E.P. Martinez, R.F. Novais, L.A. Rodrigues, L.V.S. Sacramento and R.A.R. Junior

*3. Effect of withdrawal of phosphorus on nitrate assimilation and PEP carboxylase activity in tomato, by D.J. Pilbearn, I. Cakmak, H. Marschner and E.A. Kirkby

4. Susceptibility of sweet pepper (Capsicum annuum L.) cultivars to the calcium deficiency disorder 'Blossum end rot', by P.S. Morley, M. Hardgrave, M. Bradley and D.J. Pilbeam

*5. Hydraulic properties of sphagnum peat moss and tuff (scoria) and their potential effects on water availability, by F.F. da Silva, R. Wallach andY. Chen

6. Influence of NaCl treatment on Ca, K and Na interrelations in maize shoots, by R. Izzo, A. Scagnozzi, A. Belligno and F. Navari-Izzo

*7. Effects of environment on the uptake and distribution of calcium in tomato and on the incidence of blossom-end rot, by P. Adams and L.C. Ho

8. Complete environmental effluent disposal and reuse by drip irrigation, by G. Oron and J. Beltriio

9. Effect of salinity on the nutritional level of the avocado, by E. Lahav, R. Steinhardt and D. Kalmar

* 10. Interaction of salinity and enhanced ammonium and potassium nutrition in wheat, by A. Shaviv and J. Hagin

11. Response of wild subclovers to soil calcium in xeric and acid Spanis soils, by J. Pastor, A. Martfn and S. Oliver

PART J: MAXIMIZATION OF NUTRIENT UTILIZATION IN RELATION TO ENVIRONMENTAL PROTECTION

1. Impact of intensive acgriculture on resources and environemnt,

541

547

555

563

569

577

583

589

593

597

603

by K. Mengel 613 2. Efficient management of nitrogen fertilization in modern cropping systems,

by A. Amberger 619 3. A review of internal cycling of nitrogen within trees in relation to soil fertility,

by P. Millard 623 4. Growth, nitrogen uptake and internal cycling in Eucalyptus globulus seedlings in relation to

nitrogen supply, by P.O. Carvalho, M.C. Caldeira, P. Millard and J.S. Pereira 629

*5. A method to optimize N-application in relation to soil supply ofN, and yield of potato, by D.K.L. MacKerron, M.W. Young and H.V. Davies 635

6. Nitrogen fertilization of potato and maize in relation to yield, quality of the production and risks to the environment, by J.P. Goffart and J. Guiot 641

7. Controlled supply of fertilizers for increasing use efficiency and reducing environmental damage, by A. Shaviv 651

8. Local resource management in computer aided farming: A new approach for sustainable agriculture, by E. Schnug, D.P. Murphy, S.H. Haneklaus and E.J. Evans

9. Suitability for agricultural use of sediments from the Maranhao reservoir, Portugal, by R. Fonseca, F.J.A.S. Barriga and W.S. Fyfe

Subject Index

Author Index

XI

657

665

673

679

Preface

The world-wide shortage of plant production menacing the survival of many people demands for more and better research, particularly on how to increase food and where it is most needed. Major problems of international concern for the scientific community are the availability in soil media of macro and micro nutrients and the efficiency of nutrient uptake by plant roots, the interactions between nutrients and other factors, the distribution of nutrients in different plant species, biochemical functions of nutrient elements, and their contribution to plant growth, yield and product quality. Feasibility and profit are also permanent concerns about plant nutrition in crop management, to which new require­ments are now imposed by the need to decrease pollution hazards, a problem of prime importance to preserve the environment of the future.

A deeper insight into basic knowledge is further required as well as into practical problems in the domains of agriculture, horticulture, and forestry. Such has been the concern of the International Association for the Optimization of Plant Nutrition (IAOPN) since 1964, promoting International Colloquia every four years as an opportunity for scientists concerned with plant nutrition to report new findings and to exchange ideas, experiences, and techniques. The Eighth International Colloquium for the Optimization of Plant Nutrition was hosted by Portugal and held in Lisbon from 31 August to 8 September 1992, with 280 delegates from 34 countries. Its success was attributable not only to the high scientific standard of the meeting and the opportunities given for discussions and exchange of ideas, but also to the memorable traditional Portuguese hospitality and to the pleasurable social programme for delegates and accompanying persons.

The scientific programme, in total 202 oral and poster presentations, covered a wide field of plant nutrition, varying from principles and methods in inorganic analysis of soils and crops, to soil fertility evaluation, the use of nutrients and their requirements by crops, as well as the biochemical functions and the changes caused by nutrient imbalances, deficiencies or excesses, or environmental stress conditions as resulting from alkalinity and salinity. Yield responses and the quality of crop products have proved frequent objects of study when dealing with fertilizers, their efficient and restricted use being encouraged to avoid environment degradation.

The present Proceedings includes 103 refereed full papers that clearly reflect the main themes of the Coltoquium. As editors, we are grateful to the authors for their efforts to follow the instructions for manuscript preparation and to meet the editorial standards. The very important, invisibly incorporated contributions of numerous scientists who kindly agreed to referee one or more papers, are gratefully acknowledged. Thanks are also due to those whose support and advice has never failed, in particular to the Colloquium Assistants and those regular willing horses who helped to make the event so success­ful.

Lisbon and W ageningen July 1993

M.A.C. Fragoso M.L. van Beusichem

Eighth International Colloquium for the Optimization of Plant Nutrition

COMMITTEE OF HONOUR presided by His Excellency the PRESIDENT OF THE REPUBLIC OF PORTUGAL

Ministers of: Planning and Territorial Administration Agriculture Environment and Natural Resources Foreign Affairs

Presidents of: Council of University Rectors Municipality of Lisbon Municipality of Oporto National Institute for Agrarian Research National Board for Scientific and Technological Research Commission of the European Communities Institute for Tropical Scientific Research Institute of Port Wine Institute of Madeira Wine Calouste Gulbenkian Foundation Luso-American Foundationfor the Development 'Casado Douro' 'Comissao de Viticultura da Regiao dos Vinhos Verdes'

Assistance and grants to the Colloquium by the above mentioned Institutions is gratefully ack­nowledged.

Thanks are also extended to Portuguese private entities for financial support.

Eighth International Colloquium for the Optimization of Plant Nutrition

ORGANIZING COMMIITEE

J .E. Mendes Ferrao, Chairman IICT J.C. Soveral Dias, Vice-Chairman INIA

M.A.C. Fragoso, Programme Coordinator INIA

L. Sefarim Bento, Secretary

M. Mayer Gon~alves, Treasurer

COLLOQUIUM ASSISTANTS

C. Moreira Ramos E. Leitao M. Rui Santos C. Alves Pacheco P. Vasconcelos Jordao

LQARS-INIA CEPTA-IICT CEPTA-IICT LQARS-INIA LQARS-INIA

SCIENTIFIC COMMIITEE

President

INIA

IICT

J. Quelhas dos Santos*, ISA, University of Lisbon, Portugal

Members M.L. Van Beusichem*, Wageningen Agricultural University, Netherlands J.F. Coutinho, UTAD, Vila Real, Portugal J.C. Soveral Dias, LQARS-INIA, Lisbon, Portugal F. Santos Henriques, UNL, Monte Caparica, Portugal E.A. Kirkby, University of Leeds, United Kingdom P. Martin-Pn!vel, IRFA-CIRAD, Montpellier, France P. Morard, ENSA, Toulouse, France T. Moreira, University of Evora, Portugal J. Santos Pereira, ISA, University of Lisbon, Portugal E. Menezes Sequeira*, EAN-INIA, Oeiras, Portugal A. de Varennes*, ISA, University of Lisbon, Portugal

* Thanks are due for assisting M.A.C. Fragoso in the Editorial Committee.

International Association for the Optimization of Plant Nutrition as at 1 July 1993

BOARD

P. Martin-Prevel, President M.L. Van Beusichem, Vice President M. Braud, Secretary-General, c/o CIRAD, P.O. Box 5035, 34032 Montpellier, France R.Ch. Daniel, Treasurer

STANDING COMMITTEE

D. Anak, Turkey G. Argyriadis, Greece M.L. Van Beusichem, The Netherlands J. Baier, Czech Republic M. Braud, France E. Barberis, Italy A. Cottenie, Belgium L. Castaing, France R.Ch. Daniel, Switzerland J. Ekorong, Cameroon M.M. El-Fouly, Egypt M.A.C. Fragoso, Portugal P. Fregoni, Italy G. Gonzalez Garcia, Spain H. Hansen, Denmark D. Isfan, Canada

E.A. Kirkby, United Kingdom P. Kozma, Hungary D. Lachia, Spain A. Lekchiri, Morocco L. Mansson, Sweden P. Martin-Prevel, France J.E. Mendes Ferrao, Portugal P. Morard, France J. Mpller Nielsen, Thailand B. Osseni, Ivory Coast N. Rossi, Italy L. Sanchez de la Fuente, Spain E. Skalska, Czech Republic G. Theiller, France A. Vaez Zadeh, Iran G.W. Welkie, United States of America

A

Plant analysis methods and reference values for plant material

M.A. C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 3-6, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-056

A rapid wet digestion method for plant analysis

A. PEQUERUL, C. PEREZ, P. MADERO, J. VAL and E. MONGE Department of Plant Nutrition, Estaci6n Experimental de Aula Dei, CSIC, Aptdo. 202, 50080 Zaragoza, Spain

Key words: AAS, AES, dry ashing, ICP, lucerne, macronutrients, Medicago sativa L., micronut­rients, plant analysis, wet digestion

Abstract

Analysis of nutrients in plant material requires previous digestion. Although a variety of digestion methods are used, they are usually time-consuming procedures to digest and prepare the samples. Calcination methods, using classical muffles furnaces, allow the treatment of a high number of samples but the process requires at least 24-48 hours. Sulfuric acid based wet digestion methods, have generally the inconvenient of low Fe and AI recoveries (Bowman, 1989), and the analysis is restricted to ICP. In this work, total P, Ca, Mg, K, Fe, Mn, Zn, and Cu, in lucerne leaves (Medicago sativa L.) were determined. The solutions for analysis were prepared by an improved wet digestion method (7-8 min) based on the addition of hydrogen peroxide to the sample previously introduced in concentrated HN0 3 , followed by moderate heating (100°C). Ca, Mg, Fe, Mn, Zn and Cu were determined by AAS and ICP, K, by AES, and P by colorimetry. Standard addition of iron in the leaf samples avoided the interference of HN0 3 in AAS determination of Fe. The results are compared with the obtained by classical dry calcination (muffle) and other wet methods.

Abbreviations: AAS =atomic absorption spectrometry; AES =atomic emission spectrometry; ICP = inductively coupled plasma.

Introduction

Plant material analysis provides estimations of levels of plant nutrients and is used as a tool to investigate nutritional imbalances, physiological disorders, etc. Nutrient analysis requires pre­liminary treatment of leaves and other plant material. One of the analytical bottlenecks, is the digestion of samples. The time required by traditional dry ashing (Jones, 1984; Jones et al., 1991; Pinta and DeWele, 1975) or wet digestion (Jones, 1984; Jones et al., 1991) is quite long or else a low number of samples can be processed in case of using microwave ovens.

To determine cations in solution, the use of ICP has obvious advantages over other methods,

because at the temperature reached (above 6000°C), complexes and/or organics that may exit, are converted to elemental forms (Bow­man, 1989; Halvin and Soltanpour, 1980). How­ever, ICP is an expensive technique which is not available in many laboratories.

A rapid and accurate wet digestion method is presented for the preparation of samples for nutrient analysis (P, Ca, Mg, K, Fe, Mn, Zn and Cu) by AAS (equipment available in most plant nutrition laboratories), as well as by ICP. The method here proposed, is based on the oxidation and solubilization of plant organic matter when H 2 0 2 (33%) :s directly added to the sample in concentrated HN0 3 and heated during a short time interval.

4 Pequerul et a!.

Material and methods

Leaves of lucerne (Medicago sativa L.) were sampled according to the general procedure used for foliar diagnosis. In brief, they were washed and dried for two days at 65 oc and ground to pass through a 60 mesh screen.

Ca, Mg, Fe, Mn, Zn and Cu were determined by AAS, K by AES (Pinta and C.I.I., 1973; Pinta and DeWele, 1975) and P was analyzed by molybdate-blue colorimetry (Jones, 1984).

The methods used to digest the samples were:

i) Dry ashing (Jones, 1984) 1 g of dry material was weighed in a high form crucible and placed in a muffle furnace for 24 hours at 500°C. Once cooled, it was wet with a few drops of distilled water, and 4 mL of diluted (1: 1) HN0 3 (density 1.3 g mL - 1 ) were carefully added. The solution was evaporated to dryness on a hot plate (100-l20°C), and set in the muffle furnace during two hours. Once cooled, 10 mL of diluted ( 1 : 1) HCl (density 1.18 g mL - 1) were added and heated on the plate. The solution was filtered, transferred to volume flask and diluted with H 2 0 up to 25 mL.

Wet acid digestion procedures:

ii) HN03 /HC!0 4 digestion (Jones, 1984) 5 mL of HN0 3 (70%) and 1.5 mL of HClO 4

( 60%) were added to 0.5 g of sample, and the solution heated until the disappearance of the brown fumes. It was then cooled, 5 mL of diluted (1: 1) HCl (density 1.18 g mL - 1 ) added, and finally diluted with H 20 up to 25 mL solu­tion.

iii) Modified HNO) HC!O 4 digestion The procedure was basically the same as previ­ous but modified in our laboratory by the addi­tion of 7 mL H 2 0 2 (33% ).

iv) HN0 3 1H2 0 2 wet digestion (Jones ct a!., 1991) 8 mL of HN0 3 were added to 0.5 g of sample and let stand overnight. The solution was then heated for one hour at l20°C on hot plate, and several additions of 4 mL 33% H 20 2 were made until the digest was colorless. The residue was

taken to dryness at low heat (80°C), cooled and diluted with (1:10) HCl (density 1.18g mL- 1).

v) Improved HN03 /H2 0 2 digestion 5 mL of HN0 3 were added to 0.5 g of sample in a 250 mL dry flask and stirred. Thus, all the material was wet. Then 4 mL of 33% H 20 2 were carefully added in a well ventilated hood and slightly stirred after the addition. It was heated on a hot plate and a strong effervescence was produced. When the brown fumes were less dense (7-8 minutes), the solution was allowed to cool. A slightly yellow dissolution and a small white solid quantity in suspension still remained. The solution was filtered, washed with 5 mL of (1: 1) HCl (density 1.18 g mL - 1) and diluted up to 25 mL with distilled H 20.

Results

Several procedures were tested: a conventional wet digestion (ii); a method (iii) based on a classical HN0 3 /HC10 4 wet procedure, modified in our laboratory by the addition of 7 mL H 2 0 2 ;

a method ( iv) reported by Jones et a!. ( 1991) and a method similar to previous (v) but using different proportions of HN0 3 (5 mL) and H 2 0 2

( 4 mL), also modiffiying the sequence of addition of reagents. The time of digestion, and the concentration in base to dry matter of macro and microelements are shown in Table 1. The results were compared with those obtained by tradition­al dry ashing (i) for organic matter destruction (Jones, 1984), which were taken as a reference, as this method is widely used in our laboratory and a large data bank of analysis in many higher plants, is available.

In digestions (iii) and (v), a small quantity of white solid remained in suspension, indepen­dently of the amount of H 2 0 2 added, and did not disappear either when the time of digestion was increased or with the increased addition of HN0 3 or HC104 .

The concentrations of P, Ca, Mg, K, Mn, Zn and Cu obtained by methods (ii), (iii), (iv) and (v) were not significatively different from those obtained by dry ashing (i). However, the values

A rapid wet digestion method for plant analysis 5

Table 1. Nutrient element concentrations following the different analytical methods used and the time required for digestion of samples

Method Time p Ca Mg K Fe Mn Zn Cu

(i) 30 h 0.31 ± 0.00 1.38 ± 0.02 0.26 ± 0.00 3.32 ± 0.02 188.81 ± 1.63 19.50 ± 0.21 47.11±0.62 6.30 ± 0.09 (ii) 3h 0.31 ± 0.01 1.15 ± 0.05 0.24 ± 0.01 2.63 ± 0.13 105.00 ± 4.16 18.00 ± 4.16 53.17 ± 6.61 5.42 ± 0.22 (iii) 30min 0.33 ± 0.01 1.26 ± 0.01 0.27 ± 0.00 2.85 ± 0.30 111.33 ± 1.17 16.83 ± 1.01 59.50 ± 1.04 5.58 ± 0.08 (iv) 24 h 0.32 ± 0.02 1.35 ± 0.10 0.26 ± 0.01 3.33 ± 0.02 173.67 ± 1.23 18.63 ± 0.10 61.35 ± 2.03 6.25 ± 0.04 (v) ?min 0.32 ± 0.00 1.31 ± 0.05 0.28 ± 0.01 3.34 ± 0.02 114.19 ± 3.45 19.68 ± 0.65 52.35 ± 4.41 6.15 ± 0.07

i, ii, and iv methods were reported by Jones (1984 and 1991 respectively). Methods iii and v were developed in our laboratory. Macroelements are given in % dry matter and microelements in mg kg - 1 dry matter. The results are the average of eight replications± standard error.

of iron analyzed by the methods (ii), (iii), (v) and (iv) were lower ( 60~ 70% and 91% respec­tively).

In order to investigate the low iron recovery in wet method (v), known amounts of iron (100, 200, 300 and 400 f.Lg) were added to each sample, corresponding to hypothetical iron concentra­tions in the plant of 200, 400, 600 and 800 mg kg -t respectively, then the digestion of samples (8 replications) was carried out (Table 2).

In all cases, when iron was added, an increase in the concentration of this element over that obtained by digestion without the addition was observed. The optimum quantity estimated was of 300 f.Lg of Fe per 0.5 g of sample, which gave a recovery of 96% compared with the obtained by the dry method.

The ICP analytical data were similar to those obtained by AAS (results not shown).

Table 2. Effect of different standard additions of Fe on the concentration of the element in the plant sample

Method

( i) Dry ashing ( iv) Original (v) Improved with iron addition

Iron added (mg kg - 1 )

200.00 400.00 600.00 800.00

Concentration

188.81 ± 1.63 173.67 ± 1.23

114.19 ± 1.34 127.00 ± 1.72 181.50 ± 1.52 182.50 ± 1.69 182.70 ± 1.43

The results for all methods are the average of eight replications± standard error.

Discussion

Preliminary studies on the amounts of reagents, specially hydrogen peroxide, and the adequate time of reaction for the total destruction of organic matter were necessary to select the most adequate method.

In the preliminary assays the addition of different amounts (7~13 mL) of hydrogen perox­ide to HC104 /HN0 3 mixture (iii) promoted the acceleration of the oxidation process. We ob­served that hydrogen peroxide did not influence the recovery of macro and microelements but the time of digestion was considerably lower (Table 1) than in the original method ( ii).

HN0 3 /H20 2 has been previously used (Hal­vin and Soltanpour, 1980; Jones et al., 1991), without any improvement over other mixtures used in wet methods. Although in the improved method (v) a HN0 3 /H20 2 mixture was used, important changes were observed. In the previ­ous method (iv), colourless solutions were ob­tained after an extended digestion (several hours), while in the improved method (v) slightly yellow dissolutions and a small white solid quantity remained in suspension. Except for the case of iron, interferences in the recovery of the elements were not detected (Table 1). It can be thought that the low recovery of iron could be assigned to precipitation of this element within the white solid, but a very sensitive direct assay of iron (color reaction with a,a '-dipyridyl) in the precipitate was negative.

In method (v), the addition of hydrogen perox­ide was accomplished in one step, in contrast to several successive additions reported elsewhere

6 A rapid wet digestion method for plant analysis

(Jones et al., 1991). The amount of H 2 0 2 added to accelerate the oxidation of organic matter, was minimized, so that it did not influence the recovery of the elements (Table 1 ), and also, the risk of losses through splashes and the use of a hazardous material decrease.

The low iron concentration measured (Table 1) was due rather to the interferences in AAS than to incomplete digestion, as both HN0 3

(methods iii and iv) and HCIO 4 (method iii) reduce the atomic absorption of iron at 248.33 nm. As usual iron concentrations in lucerne leaves vary between 30 and 250 mg kg - 1 (Jones et al., 1991) and the values obtained by the dry method were 180-190 mg kg - 1, the optimum quantity of iron added was estimated to be 300 !Lg in 0.5 g of sample (Table 2), which corresponded to a theoretical iron concentration of 600 mg kg - 1 (approximately 3 times the usual concentration). The interference of HN0 3 was thus minimized as the averages and standard errors obtained for this method (iv) (182.50 ± 1.69) compared with those obtained for dry ashing (I) (188.81 ± 1.63) were very close.

In the proposed method (v), a much more reduced time of digestion (7-8 minutes) and a subsequent filtering of the resulting solution is sufficient to recover the macro and microele­ments Ca, Mg, K, Mn, Zn and Cu as well as P (Table 2). Therefore, the improved method proves to be rapid and accurate, and does not show significant differences when compared to others methods widely used.

The method is currently assayed in different plant material, sugar beet and soybean (leaves, roots) cultured in hydroponic conditions, and

apple, pear and peach tree leaves m field con­ditions.

Acknowledgements

The authors thank Mrs M A Garcia, C Lope and Ms C Fustero for their excellent technical assis­tance. Work carried out under research project CONAI-DGA: PCA-4/91.

References

Bowman R A 1989 A rapid plant digestion method for analysis of P and certain cations by inductively coupled plasma emission spectrometry. Comun. Soil Sci. Plant Anal. 20, 539-553.

C.I.I. 1969 Comite Inter-Institutos para el estudio de tecnicas analiticas. Metodos de rcfcrencia para la determinacion de elementos minerales en vegetales. An. Edafol. Agrobiol. 38, 513-521.

Halvin J Land Soltanpour P N 1980 A nitric acid plant digest method for use with inductively coupled plasma. Commun. Soil Sci. Plant Anal. 11, 969-980.

Jones Jr J B 1984 Plants. In Official Methods of Analysis of the Association of Official Analytical Chemists. Ed. S Williams. pp 38-64. Association of Official Analytical Chemists, Arlington, Virginia 22209, USA.

Jones Jr J B, Wolf B and Mills H A 1991 Plant Analysis Handbook. Micro-Macro Publishing, Athens, Georgia 30607, USA. 213p.

Pinta M and C.l.I. 1973 Methodes de reference pour la determination des elements mineraux dans les vegetaux. Determination des elements Ca, Mg, Fe, Mn, Zn et Cu par absorption atomique. Oleagineaux 28, 87-93.

Pinta M and DeWele J 1975 Etalons vegetaux pour !'analyse foliaire. In Le Controle de 1' Alimentation des Plantes Cultivees. Ed. P Konza. pp 159-172. Akademiai Kidao, Budapest.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 7-11, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-017

Comparison of techniques for nitrogen analysis in potato crops

M.W. YOUNG, H.V. DAVIES and D.K.L. MacKERRON Scottish Crop Research Institute, Invergowrie, Dundee, DD2 5DA, UK

Key words: Dumas combustion, Kjeldahl digestion, near infra-red reflectance spectroscopy, nitrogen, NIR, potato

Abstract

Potato leaf, stem and tuber samples have been analysed for nitrogen concentration [N], by three methods; Kjeldahl digestion, Dumas combustion, and near infra-red reflectance (NIR) spectroscopy. Models to estimate [N] from NIR have been developed using either Kjeldahl or Dumas for calibration. Estimates of [N] from all three methods, are highly correlated. However, [N] measurements by Kjeldahl and Dumas are not in agreement. Kjeldahl has a recovery rate of ca. 98% organic-N and variable recovery of inorganic-N. Dumas measures both organic- and inorganic-N with a recovery rate of 100%. The suitability of Kjeldahl as an analytical technique must be questioned when NIR methods are available which will give accurate [N] values quickly, safely and cheaply.

Introduction

One of the most important plant nutrients, essential for potato production, is nitrogen. Fertilizer nitrogen is required to supplement soil nitrogen which may be present from various sources (Tinker, 1979) and correct application rates are dependent on many factors (Kunkel et a!., 1983). Analysis of total nitrogen in the soil does not indicate nitrogen availability (Green­wood et a!., 1986; Jollans, 1985). In the absence of a generally accepted method of assessing soil nitrogen status (Goh and Haynes, 1986; Tinker, 1979), the estimation of correct application rates of nitrogen fertilizer is difficult (Tinker, 1979).

Tissue analysis may be used to assess the nutrient status of a plant (Batey, 1977) and has the advantage that plant development reflects the growing environment of the plant, both above and below the ground (Batey, 1977; Gupta and Saxena, 1976).

Materials and methods

In 1990 and 1991, irrigated field experiments

were carried out using two vanetles of potato, Cara and Maris Piper, to investigate the relation between nitrogen uptake and total plant fresh weight and to investigate methods for nitrogen analysis of plant tissues. Standard applications of P and K were given and standard pesticide applications were made throughout the season. The nitrogen treatments were 0, 40, 80, 160, 80 + 80 (split application, 1990 only) and 240 kg N ha- 1 •

At each of five harvest dates throughout the first growing season 48 eight-plant samples (foliage and tubers) were lifted, resulting in 223 leaf and stem samples and 231 tuber samples. A sub-sample was taken, washed thoroughly and split into leaf, stem (including petioles and recovered roots) and tubers. These were oven­dried at 90 oc. All oven-dried samples were ground using a Retsch mill with 0.5 mm grid. Stem samples were coarsely ground with a hand mincer before being milled.

The nitrogen concentration of all oven dried material was determined by the Dumas combus­tion method using an automated CHN analyser (Carlo Erba Strumentazione Nitrogen Analyser 1500). A sub-set of 52 samples covering the

8 Young et al.

Table 1. Standard errors of calibration (SEC) of NIR [N] estimates of Kjeldahl (N] and Dumas [N] of the calibration sets of the three tissue types in 1990

Standard

Dumas Kjeldahl

Standard error of calibration

Leaf

0.298 0.184

Stem

0.272 0.164

Tuber

0.121 0.081

range of N concentration found was chosen to give similar representation of variety, harvest date and applied N. These samples were ana­lysed by Kjeldahl digestion using a Tecator Kjeltec Auto 1030 Analyser.

All samples were scanned, in duplicate, by near infra-red reflectance spectroscopy (NIR) using a Brann + Leubbe Infra-Alyser 260. The data from 40 of the samples analysed by Kjeldahl were analysed using principal component analy­sis and combined with the results from the Dumas and Kjeldahl analyses to produce models to simulate (N] as estimated by both Kjeldahl and Dumas for each type of material. The standard errors of calibration (the square root of the residual mean square) between NIR (N] estimates and Dumas and Kjeldahl [N] estimates are listed in Table 1. The residual 12 samples analysed by Kjeldahl were used as a check on the consistency of calibration. In the second growing season, 200 leaf and stem, and 198 tuber samples were produced and all were analysed by both NIR and Dumas combustion.

Results

Estimates of nitrogen concentration, (NJ, made by Dumas and Kjeldahl were closely correlated (Table 2). However, the Dumas method gave higher values for (N] in all three tissue types (leaf, stem and tuber) than did the Kjeldahl method (Fig. 1 ).

Strong linear relations were found between the NIR fitted values for (N] and those of both Kjeldahl (Table 3) and Dumas (Table 4). These relations are illustrated in Figure 2. The data presented in Figure 2 a-c are from all 52 of the samples analysed by Kjeldahl, including the 40 used in calibration. The data in Figure 2 d-f are

a 7.5

z ..._.

:c 5.5 Cil '0 Qi 2'3.5

1.5 1.5

b

~4.5

:c Cil '0 Qi 2.5 2'

0.5 0.5

c 3.5

~ :c 2.5 Cil '0 Qi 2'1.5

h

/

+

3.5 5.5 7.5

Dumas [N]

/ /

/ /

/

/ /

/ / +

/ / t

2.5 4.5 6.5 Dumas [N]

/ /

/ +

0.5-J=-----.------.----.-0.5 1.5 2.5 3.5

Dumas [N] Fig. 1. Relations between estimates of [N] by Kjeldahl and Dumas methods in 1990. a -leaf; b- stem; c- tuber; con­tinuous line- fitted regression; dashed line- 1: I relation. Note: scales on a) differ from those on b) and c).

a

7.0 t

......, i ~ z ~X ~5.0 0 <>

~"~ z /ix

3.0 , Cl

<> X

1.0 1.0 3.0 5.0 7.0

Kjeldahl [N]

b

6.0

~4.0 o" a: ~ z •

2.0 • I

,p~ 0.0

0.0 2.0 4.0 6.0 Kjeldahl [N]

c 4.0

3.0 ~

......, .. ·~ z <> olx ~2.0 ~

I z :P 1.0 ~

0.0 0.0 1.0 2.0 3.0

Kjeldahl [N]

4.0

Comparison of techniques for nitrogen analysis 9

d

7.0

z ~5.0

z 3.0

~X 1.0 -t----.----------.--------..---

1.0 3.0 5.0 7.0 Dumas [N]

e

6.0 foo X

~l-lfJ ~4.0

l« X

x xl olo'O< X X

a: X fx" )( z ~x"X

2.0

0.0 0.0 2.0 4.0 6.0

Dumas [N]

f 4.0 X xX O

3.0 X~<> ~M z ~)f /'I' ~2.0 z

1.0

0.0 0.0 1.0 2.0 3.0 4.0

Dumas [N]

Fig. 2. Relations between estimates of (N] by NIR and by Kjeldahl (a-c) and Dumas (d-f) methods on 1990. a,d- leaf;, b,e- stem; c,f- tuber. Hollow crosses indicate samples used in the calibration.

10 Young et al.

Table 2. Regression coefficients for the relation between estimates of [N] by the Kjeldahl (Y) and Dumas (X) methods for three tissue types in 1990

Leaf Stem Tuber

X coefficient 0.841 0.607 0.862 Standard error of X coefficient 0.016 0.009 0.011 Constant 0.268 0.276 0.074 Standard error of Y estimate 0.079 0.025 0.020 Regression coefficient (r) 0.992 0.995 0.996 Number of observations 52 52 52

Table 3. Regression coefficients for the relation between estimates of [N] by the NIR (Y) and Kjcldahl (X) methods for three tissue types in 1990

Leaf Stem Tuber

X coefficient 0.981 0.949 0.986 Standard error of X coefficient 0.019 0.024 0.017 Constant -0.001 0.000 0.000 Standard error of Y estimate 0.087 0.471 0.027 Regression coefficient (r) 0.991 0.984 0.993 Number of observations 52 52 52

Table 4. Regression coefficients for the relation between estimates of [N] by the NIR (Y) and Dumas (X) methods for three tissue types in 1990

Leaf Stem Tuber

X coefficient 0.991 1.001 0.993 Standard error of X coefficient 0.012 0.014 0.011 Constant -0.080 0.010 -0.083 Standard error of Y estimate 0.061 O.Q38 0.019 Regression coefficient (r) 0.984 0.980 0.985 Number of observations 223 223 231

from all 223 samples analysed by Dumas and NIR, again including the 40 used in calibration. Values of [N] by NIR were found to give good estimates of both the Dumas and Kjeldahl val­ues. The agreement between Kjeldahl and NIR estimates was slightly better than between Dumas and NIR. This may be due to NIR and Kjeldahl measuring predominantly organic nitro­gen, whilst Dumas measures all nitrogen within any sample both organic and inorganic (Carlo Erba Strumentazione, 1986). Kjeldahl measures reduced N (organic N and NH;) and NIR quantifies the amount of protein present via protein reflectance data, which is in turn calib­rated with N concentration (Korcak et a!., 1987).

The relations between Kjeldahl [N] and Dumas [N] in 1990 and between Dumas [N] and NIR [N] in each year were not significantly different between cultivars. Regression analysis of the data from 1990 and 1991 combined ( 423 data points for leaf and stem, 429 data points for tuber) showed that Dumas [N] accounted for 94.8%, 94.2% and 94.6% of the variance in NIR [N] (p > 0.001 ), for leaf, stem and tuber materi-

a 7.0

Q)

1ii -~5.0 w >- 3.0

//.

... "· 6 •• .... ... ...

--'. h •

/-.

; .o+--~~~~~,---~~,----1.0

b 6.0

Q)

~4.0 ~ w >- 2.0

3.0 5.0 7.0

Dumas [N]

0.0+--~--~-~~

0.0 2.0 4.0 6.0

Dumas [N] c 4.0

/.

Q) 3.0 /.

/.

1ii " ,.., E /.

:vi 2.0 ""' .!. w .%

>- 1.0 "/.

·/-.. /. . "/-/

0.0 0.0 ; .0 2.0 3.0 4.0

Dumas [N1 Fig. 3. Comparison of average fit of data to the PCA calibration between NIR spectra and Dumas [N] in successive years. a -leaf, b- stem; c- tuber; __ 1990; ...... 1991; ------ 1 : 1 (perfect fit is indicated by 1 : 1 line).

a! respectively. Addition of year as a factor was statistically significant (p = 0.005, p-0.056, p < 0.001 for leaf, stem and tuber respectively) but only accounted for a further 1.1%, 0.1%, 2.2% of the variance, for leaf stem and tuber respec­tively. Although these changes arc statistically significant, their effect on the regression equa­tion and hence on the estimated values of [N] are small (Fig. 3). The lines represent the average fit of the data to the calibration, with perfect fit being represented by the 1: 1 line. The lines for 1990 correspond to the regression equations in Table 4.

Discussion

Near infra-red spectroscopy is the cheapest, quickest and easiest of the three methods of N analysis studied. It is also safer than the Kjeldahl method with its hazardous mineral acid diges­tions. However, NIR spectroscopy is not an absolute method and requires calibration, for each tissue-type, against an accepted standard. NIR gave good correlations with both Dumas combustion and Kjeldahl digestion methods. The reference analytical technique used in calibration for NIR needs to be chosen with a view to the required purpose. If an estimate of reduced or organic [N] is required, then Kjeldahl analysis may be the most appropriate reference. How­ever it should be recognized that in Kjeldahl analysis, which is known to have a recovery rate of ca. 98-99% of the digestible N, an unpredict­able proportion of nitrate within a sample will be digested, unless precautions are taken to ensure all nitrate is reduced to ammonia during the digestion (Wetselaar and Farquhar, 1980), lead­ing to a variable overestimate of reduced N and an underestimate of total N. If, on the other hand, measurement of total N is required, then the Dumas combustion method should be used as the standard. The difference in regression coefficents between stem material (0.607) and leaf and tuber materials (0.841 and 0.862 respec­tively) probably reflects higher nitrate concen­tration in the stem. Where N-nutrition of a crop is being considered (MacKerron et a!., 1993), Dumas is the preferred standard, given that 100% of the sample is measured. Closely com-

Comparison of techniques for nitrogen analysis 11

parable relations for the three tissue-types be­tween NIR [N] and Dumas [N], and also be­tween NIR [N] and Kjeldahl [N] (Tables 3 and 4), should not be taken to indicate that the tissues share a common calibration. Separate calibrations were performed against spectral re­flectance for each tissue-type. The similarity of the resulting relations is due to the method of calibration being equally applicable to each tissue-type.

Acknowledgements

This work was supported by the Great Britain Potato Marketing Board and Scottish Office Agriculture and Fisheries Department. The au­thors would like to thank Ailsa Smith and Sigrun Holdhus for their technical assistance.

References

Batey T 1977 Prediction by leaf analysis of nitrogen fertilizer required for winter wheat. J. Sci. Food Agric. 28, 275-278.

Goh K M and Haynes R J 1986 Nitrogen and agronomic practice. In Mineral Nitrogen in the Plant-Soil System. Ed. Haynes pp 379-468. Academic Press, London.

Greenwood D J, Neeteson J J and Draycott A 1986 Quan­titative relationships for the dependence of growth rate of arable crops on their nitrogen content, dry weight and aerial environment. Plant and Soil 91, 281-301.

Gupta A and Saxena M C 1976 Total nitrogen concentration in leaves of potatoes (Solanum tuberosum L.) as an index of nutritional status. J. Agric. Sci. (Cambridge) 87, 293-296.

Jollans J L 1985 Using fertilizers on the farm. In Fertilizers in UK Farming. Centre for Agricultural Strategy Report 9. p 50-64.

Korcak R F, Norris K H, Walker V and Bhargava J N 1987 Measurement of apple leaf total nitrogen by near-infrared reflectance. HortScience 22, 308-309.

Kunkel R, Thornton R E and Holstad N M 1983 Petiole levels- what do they mean? Proceedings of the 1983 Washington Potato Conference and Trade Fair. pp. 103-111.

MacKerron D K L, Young M Wand Davies H V 1993 A method to optimize N-application in relation to soil supply of N and yield in potato. Plant and Soil 154, 139-144.

Tinker P B H 1978 Uptake and consumption of soil nitrogen in relation to agronomic practice. In Nitrogen Assimilation of Plants. Eds. EJ Hewitt and CV Cutting. pp 101-122. Academic Press, London.

Wctselaar R and Farquhar G D 1980 Nitrogen losses from tops of plants. Adv. Agron. 33, 263-302.

M.A.C. Fraf?oso and M.L. van Beusichem (eds.). Optimization of Pla111 Nutrition, 13-17, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-003

Evaluation of inorganic elements in agricultural products from Italian farms by instrumental neutron activation analysis

A. MOAURO, L. TRIOLO, P. AVINO and L. FERRAND! ENEA, INN, CRE Casaccia, P.O. Box 2400, via Anguillarese 301, Rome, Italy

Key words: ecotoxic elements, INAA, neutron activation analysis

Abstract

Instrumental neutron activation analysis (INAA) has been used to determine non destructively as many as 25 minor and trace elements in some crops collected from Italian farms, in different pollution conditions. For quantitative evaluations, some reference materials have been used, prepared by a group of 38 Analytical Institutes (CII) and analyzed in a intcrcomparison campaign. No large differences were found among the examined areas, but a comparison among our values and those recommended or considered provisionally safe by WHO, has shown that our data for Cr, Co and Cu exceeded these limits. The highest values were found for tobacco leaves, collected in a country area of central Italy.

Introduction

The problem of evaluating ecotoxic elements in common edible crop plants (as fruit and cereals) is becoming ever more important in view of the long term accumulation effects on human health. Some trace elements are considered essential but become hazardous to health when safety limits are exceeded (Alegria et a!., 1990).

In this paper the determination of microele­ments has been performed on some crops largely used in the Italian diet, either directly (fruits and tomatoes) or through by-products of wheat (bread and pasta). Due to the number of ele­ments to be determined, in so many samples and to tedious dissolution operations required by normal analytical techniques, Instrumental Neu­tron Activation Analysis (INAA) has been se­lected. After neutron irradiation, as many as 25 elements can be measured as their radioactive products, without any chemical treatment, also avoiding possible contaminations (Djingavova et a!., 1991 ). For Pb, Cd and Ni a different proce­dure is required (e.g. AESIICP). To perform quantitative evaluations, some plant reference materials have been used, prepared and analyzed

in an intercomparison campaign by a Committee of 38 European analytical Institutes (CII), to whom ENEA gives the NAA support (Moauro and Carconi, 1991). The farms who supplied the crops have been chosen in three Italian areas, with supposedly different pollution level, due to heavy traffic, industries and use of composts and fertilizers.

Experimental

Preparation

The sampling of the products was performed in September 1990, in three regions, Emilia, Cam­pania and Basilicata, in the north, centre, and south of Italy respectively. In Emilia tomatoes, apples, pears, bread and durum wheat were sampled, in Campania tomatoes and apples, in Basilicata tomatoes and durum wheat. An extra test was performed on tobacco leaves sampled in Campania. Many industrial activities (relevant to ceramics, cast iron, fertilizer products) and heavy motorized traffic are mostly present in the Emilia region (Bologna province) and to a lower extent

14 Moauro et al.

in the southern districts. About 5 kg of each product were sampled randomly, in the selected areas, washed with dionized water, pounded in a agate mortar, to avoid metallic contaminations, and dried at 70°C in an air current. A good homogeneity was reached for tomatoes, tobacco and wheat; some hygroscopicity occurred in pear and apple samples, because of the fructose content.

Irradiation and gamma spectrometry

Two types of irradiation were performed on five aliquots of the samples in the TRIGA reactor of ENEA Casaccia (Rome): a short one (60s), in the pneumatic device at 2 10 13 n em - 2 s - 1 , a long one (105 s), in the rotating device, at 210 12 n em -z s - 1 , both in polyethylene vials. The same procedure has been applied on aliquots of three reference materials (Pomme Fruit, Oeillet and Lucerne) prepared and analyzed by CII. After irradiation, the samples and standards were measured at convenient time intervals, by using a computerized multichannel analyzer, connected to a Ge (HP) detector.

Table 1. Elemental concentration in fruit samples (ftg g -I ±a)

Apples

Emilia

Yellow Spur

AI 28.3 ± 8.0 28.0 ± 7.6 As 0.030 ± 0.001 0.09 ± 0.04 Br 0.59 ± 0.06 0.24 ± 0.05 Ca 587 ± 44 402 ±59 Cl 84.0 ± 16.0 87.7 ± 9.9 Co 0.036 ± 0.002 0.048 ± 0.005 Cr 0.15 ± O.Dl 1.13 ± 0.21 Cu 3.70±0.10 5.18 ± 0.07 Fe 21.9 ± 2.0 29.4 ± 1.5 Hg n.d. n.d. La 0.10 ± O.Dl 0.04 ± 0.01 Mn 5.39 ± 0.57 735 ± 0.57 Mo 0.17 ± 0.02 0.57 ± 0.32 Rb 0.61 ± O.D7 n.d. Sb 0.06 ± 0.02 0.03 ± 0.01 Sc 0.004 ± 0.0003 0.002 ± 0.0001 v 9.50 ± 0.10 9.00 ± 0.50 Zn 2.54 ± 0.78 5.58 ± 0.20

n.d. =not detected.

Results and discussion

As can be seen from the Tables 1-3, the follow­ing conclusion can be drawn: a) there are no large differences among the sampling areas; only aluminium, arsenic and chromium values, seem to be higher in the northern area. b) in the southern area, bromine values are larger, proba­bly due to the large use of methylbromidc, as a nematocide; c) durum wheat generally shows higher values of AI, As, Br, Cu, Fe and Zn than bread wheat; d) a peculiar case is tobacco; leaves, sampled in a Campania's rural area have the highest elemental concentrations. This fact can be plausibly explained by the broad leaf surface, exposed to the atmospheric particles; e) the elements in wheat samples can arise only from the soil, as the grains are well shielded by their own teguments. The weekly intakes in the human diet, due to the examined crops, are reported in Table 4 and 5 respectively. The concentration range and the contribution to the diet for each element, based on the consumption rate foreseen by the Italian Nutritional Institute, for a mean body weight of 58.7 kg are shown in Table 5. It can be seen that for Co, Cr and Cu

Pears

Campania Emilia

Golden Conference

33.3 ± 1.5 86.5 ± 4.5 0.25 ± 0.09 n.d. 0.21 ± 0.03 n.d. 269 ± 75 n.d. 92.6 ± 4.5 121 ± 24 0.044 ± 0.003 0.26 ± 0.01 1.49 ± 0.58 1.57 ± 0.09 4.98 ± 0.84 15.3 ± 3.14 28.5 ± 6.8 14.0 ± 1.0 0.010 ± 0.001 0.006 + 0.002 0.08 ± 0.01 n.d. 2.7S ± 0.15 4.60 ± 0.14 0.24 ± O.D7 n.d. 14.1±1.2 16.1±1.7 O.o? ± 0.01 n.d. 0.002 ± 0.001 0.004 ± 0.001 8.64 ± 0.30 0.12 ± 0.01 6.89 ± 0.9S 15.0 ± 1.0

Evaluation of minor and trace elements by IN AA 15

Table 2. Elemental concentration in tomatoes and tobacco samples ( ~ g g - 1 ± u)

Tomatoes Tobacco

Emilia Campania Basilicata Campania2

Al 32.9 ± 0.9 18.81 ± 1.15 n.d. 3300 ± 100 As 0.10 ± 0.07 n.d. n.d. 0.79 ± 0.10 Br 7.48 ± 0.25 1.01 ± 0.02 40.3 ± 1.1 54.0 ± 0.38 Ca% 0.25 ± 0.03 n.d. 0.16 ± 0.03 2.87 ± 0.18 Ce n.d. n.d. 0.16 ± 0.02 4.53 +0.41 Cl% 0.55 ± 0.02 0.54 ± 0.09 0.67 ± 0.03 0.37 ± 0.01 Co 0.11 ± 0.01 0.042 ± 0.004 0.100 ± 0.004 0.41 ± 0.03 Cr 2.53 ± 0.13 1.72 ± 0.03 1.16 ± 0.08 3.89 ± 0.73 Cs n.d. 0.010 ± 0.003 0.04 ± 0.01 0.81 ± 0.07 Cu 10.8 ± 0.3 13.0 ± 1.6 (9.58) 15.4 ± 1.8 Eu n.d. n.d. 0.03 + 0.01 1.27 + 0.13 Fe 77.3 ± 0.63 72.9 ± 2.3 78.4 ± 5.4 936 ± 73 Hg n.d. n.d. 0.010 + 0.004 n.d. La 0.071 ± 0.003 n.d. 0.116 ± 0.021 2.86 ± 0.25 Mn 9.60 ± 0.33 15.9 ± 0.5 16.6 ± 0.9 89.2 ± 7.6 Mo 1.52 ± 0.18 O.Dl (0.08) n.d. Rb 14.8 ± 0.4 33.6 ± 7.0 18.5 ± 0.7 62.3 ± 11.8 Sb 0.031 ± 0.005 0.019 ± 0.007 0.021 ± 0.004 0.27 Sc 0.003 ± 0.001 0.001 ± 0.000 0.010 ± 0.001 0.17 ± 0.03 Se (0.08) 0.03 ± 0.001 0.02 ± 0.002 (0.06) Sr n.d. 5.00 ± 0.27 8.41 ± 0.43 80.5 ± 0.3 v n.d. n.d. n.d. 2.71 ±0.31 Zn 28.0 ± 1.1 25.5 ± 1.9 24.1 ± 0.4 34.2 ± 1.7

n.d. = not detected.

Table 3. Elemental concentration in wheat samples (~gg- 1 ± <7)

Durumwheat Bread wheat

Emilia Basilicata 1 Basilicata 2 Emilia

Al 51.5 ± 1.0 20.5 ± 4.8 31.3 ± 5.0 20.8±7.7. As (0.15) 0.012 ± 0.009 0.05 ± 0.02 0.33 ± 0.001 Br 1.88 ± 0.05 2.18 ± 0.10 2.05 ± 0.09 3.29 ± 0.27 Ca 800 ± 40 452 ± 16 760 ± 30 716 ± 44 Cl 774 ± 23 678 ± 70 713 ± 42 513 ± 37 Co 0.049 ± 0.005 0.026 ± 0.002 0.045 ± 0.002 0.019 ± 0.001 Cr 0.44 ± 0.02 0.50 ± 0.06 (0.42) 0.33 ± 0.02 Cu 8.42 ± 0.63 8.86 ± 0.65 7.32 ± 2.35 n.d. Fe 75.7 ± 18.9 47.5 ± 1.9 61.3 ± 8.9 38.3 ± 3.7 Hg n.d. n.d. n.d. n.d. La 0.012 ± 0.003 0.027 ± 0.004 0.024 ± 0.004 0.011 ± 0.001 Mn 58.3 ± 3.1 27.9 ± 4.3 60.5 ± 6.0 35.3 ± 2.8 Mo 0.78 ± 0.14 0.76 ± 0.13 0.82 ± 0.66 n.d. Rb 4.41 ± 0.28 n.d. 4.90 ± 0.27 2.37 ± 0.04 Sb 0.019 ± 0.005 0.002 ± 0.005 0.005 ± 0.003 (0.06) Sc 0.004 ± 0.001 0.002 ± 0.000 0.003 ± 0.001 0.001 ± 0.0001 Se 0.05 ± O.Dl n.d. 0.11 ±0.01 n.d. Sr 3.19 ± 0.36 4.98 ± 0.55 6.20 n.d. Zn 43.3 ± 0.5 25.3 ± 1.2 42.1 ± 1.9 24.5 ± 0.1

n.d. =not detected.

16 Moauro et al.

Table 4. Range of concentrations in crops (11-g g ' dry weight)

Apples Pears Tomatoes Wheat Tobacco

AI 28.0-33.3 86.5 18.0-33.0 20.5-51.5 0.33% As 0.03-0.25 n.d. 0.10 0.01-0.15 0.79 Br 0.20-0.60 n.d. 1.00-40.0 1.88-3.29 54.0 Cl 84.0-93.0 121.0 0.50-0.70% 513-774 0.37% Co 0.04-0.05 0.26 0.04-0.11 0.02-0.05 0.41 Cr 0.15-1.49 1.57 1.16-2.53 0.33-0.50 3.89 Cu 3.70-5.18 15.3 9.60-13.0 7.32-8.96 15.4 Fe 21.9-29.4 14.0 72.9-78.4 38.3-75.7 936 Hg 0.01 0.006 0.01 n.d. n.d. Mn 2.78-5.39 n.d. 9.60-16.6 27.9-60.5 89.2 Mo 0.17-0.57 n.d. 0.08-1.52 0.78-0.82 n.d. Sb O.!J3-0.o7 n.d. 0.02-0.03 0.01-0.06 0.27 Se n.d. n.d. 0.02-0.08 0.05-0.11 0.06 v 8.64 n.d. n.d. n.d. n.d. Zn 2.54-6.89 15.0 24.0-28.0 24.5-43.4 34.2

n.d. = not detected.

Table 5. Mean weekly intake for crops compared with WHO values (mg)

AI As Co

min max min max min

Bread wheat 27.1 0.039 0.026 Durum

wheat 10.2 25.6 0.005 O.o75 0.015 Tomatoes 14.6 25.6 0.078 O.G78 0.031 Apples 14.9 17.7 0.016 0.133 0.021 Pears 14.5 0.044

Total values 81.3 110.5 0.138 0.325 0.137 WHO values 410.9 0.822 8.4

*corresponding to 0.021 mg of vitamin B w

the contribution of crops exceeds the total elemental intake recommended or considered safe by the World Health Organization.

Conclusions

Our survey on some Italian crops has shown a content of Co, Cr and Cu higher than that reported by Alegria et al. (1990) in Spain or by Sun et al. (1991) in China, although the large values of Cu can be attributed to the use of CuS04 • A more complete sampli.1g has been envisaged to obtain a better statistical evalua­tion; in this frame INAA will play an important role for the large number of the samples and the elements involved; en reference materials, as similar as possible to the crops, will be used. The

Cr Cu Hg Se

max min max min max min max

0.43

0.025 0.21 0.25 3.64 4.45 0.025 0.055 0.086 0.90 1.97 7.44 10.1 0.008 0.016 0.062 0.027 0.08 0.79 1.97 2.76 0.005

0.26 2.57

0.208 1.88 3.67 15.6 19.9 0.013 0.040 0.117 w-'* 0.35 1.40 14 21 0.206 0.350 1.40

AES/ICP technique (with preliminary micro­wave digestion of the sample) will allow the determination of Pb and Cd.

Acknowledgement

The authors are grateful to Mr F Nanna for support in sampling and analytical operations.

References

Alegria A, Barbera R and Fasse R 1990 Influence of environmental contamination Cd, Co, Cr, Ni, Pb and Zn content of edible vegetables, safety and nutritional aspects. J. Micronutr. Anal. 8, 91-104.

Djingavova R. Arpadiou S and Kuleff I 1991 INAA and

Evaluation of minor and trace elements by INAA 17

flame AAS of various vegetable reference materials. Fre­scnius, Anal. Chern. 339, 181-186.

Djingavova R, Arpadiou S and Kuleff I 1991 INAA and flame AAS of various vegetable reference materials. Fre­senius, Anal. Chern. 339, 181-186.

Moauro A and Carconi P L 1991 A European intercom­parison of vegetal standard reference materials, based on INAA and some non nuclear spectrochemical techniques. J. Radioanal. Chern. 151, 149-157.

Sun L, Lu F, Su R, and Zhen H 1991 Determination and evaluation of some trace elements in Chinese foodstuffs. J. Radioanal. Chern. 151, 277-285.

Guzzi G. Colombo A, Girardi F, Pietra R, Rossi G and Toussaint N 1977 Comparison of various analytical tech­niques for homogeneity test of candidate standard refer­ence materials. J. Radional. Chern. 39, 263-276.

M.A. C. Fragoso and M.L. van Beusichem (eds.) Optimization of"plant nutrition 19-23, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-026

Determination of lead in white lupin by anodic stripping voltammetry

MARIA M.P.M. NET0 1 ' 2 and AMARILIS DE VARENNES 1

1 Agricultural Chemistry Department, Institute of Agronomy, Technical University of Lisbon, Tapada da Ajuda, 1399 Lisboa, Portugal; 2Research Centre of Electrochemistry and Kinetics, University of Lisbon, Calr;ada Bento da Rocha Cabral 14, 1200 Lisboa, Portugal

Key words: heavy metals, lead, Lupinus albus L., stripping voltammetry, white lupin

Abstract

An anodic stripping voltammetry method for the simultaneous determination of trace heavy metals in nutrient solutions, soils and plants has been developed at a hydrodynamic electrochemical sensor. Several parameters were optimized in order to enhance sensitivity. Calibration curves in different media are presented. The study of the uptake of lead by white lupin (Lupinus albus L.) was carried out. Toxicity symptoms were observed and compared with levels of lead measured in roots and leaves.

Introduction

Plant nutrition studies show that higher plant species require a certain amount of trace heavy metals. However, toxic heavy metals may also occur in the environment being available to plants from soil and aerosol sources.

Lead is a non-essential element known to be very poisonous to humans due to its cumulative nature (Mosbaek et a!., 1989). Investigations have been concerned with lead contamination of agricultural products and its introduction in the food chain. Contamination is mainly caused by industrial and car emissions.

Consequently, the quantitative determination of trace amounts of heavy metals such as lead in soils and plant organs is a very important ana­lytical problem. Spectrochemical methods, par­ticularly atomic absorption spectrometry, arc widely used. Alternatively, electrochemical methods have been employed more recently (Brett and Neto, 1986; Chizzola, 1989), and new hydrodynamic electroanalytical sensors have been developed (Brett and Brett, 1986). They allow simultaneous determination of four to six clements. Results are produced very quickly and frequently. Hydrodynamic characteristics as well

as regular calibration ensure good reproducibil­ity, accuracy and low detection limits.

In the present work, white lupin was grown in nutrient solutions, with different lead concen­trations. The determination of lead in roots and leaves was performed, after acid digestion, by linear sweep anodic stripping voltammctry. The sensor was a glassy carbon wall-jet disc electrode with a thin mercury film electrochemically depo­sited in situ. Reproducible results were obtained with satisfactory detection limits at submicromo­lar levels. The technique was also studied for the simultaneous determination of copper and lead.

Materials and methods

Voltammetric method

In trace analysis the achievement of a sufficient high signal-to-noise ratio is required. Stripping voltammetry is a very successful method in order to increase the measured signal (Vydra ct a!., 1976; Wang, 1985). Firstly, a preconcentration step is used to deposit the elcctroactivc species on the electrode, under potentiostatic control conditions; secondly, a stripping step is carried

20 Neto and De Varennes

out for the actual measurement. Here, the strip­ping step was performed by a linear potential scan in the anodic direction, for the oxidation of the metals previously deposited on the electrode. Peak currents are measured at the suitable stripping potentials.

Electrochemical detection

The hydrodynamic electrochemical sensor em­ployed was of the wall-jet type. It exhibits very high sensitivity, ease in use and ease of mainte­nance and well defined hydrodynamics (Brett and Neto, 1986).

A fine jet of analyte issuing from a circular nozzle impinges on the centre of a disc electrode perpendicular to the jet: the solution then spreads out radially. Under laminar flow any species which reaches the disc electrode and subsequently leaves the diffusion layer cannot react again. This is very useful from the ana­lytical point of view.

Apparatus and reagents

Oxford Electrodes electrochemical equipment (including triangular wave generator and poten­tiostat) was used. Voltammograms were regis­tered on a Philips PM8143 X-Y-t recorder. The wall-jet electrode cell was of the Fleet and Little design and has been described previously (Al­bery and Brett, 1983). The working electrode was a glassy carbon disc in a Kel-F sheat. A platinum tube counter electrode and an Ag/ AgCI reference electrode completed the cell. Solution was pulled through the wall-jet detector by means of a LKB P1 peristaltic pump placed downstream.

Solutions were prepared with distilled, deion­ized water and deoxygenated by bubbling oxy­gen-free nitrogen. All reagents were of analyti­cal-reagent grade. Stock solutions of 10- 4 M Pb(CH 3C00) 2 3H2 0 and 10-4 M CuS0 4 5H2 0 in supporting electrolyte were prepared to make standard additions; a stock solution of 10- 2 M Hg(II) was prepared from Hg(N0 3 ) 2 by dissolu­tion in HN0 3 at pH 2 and kept in the dark; this was added to the electrolyte solution such that the concentration of Hg(II) was 5 x 10- 5 M m the plating solution (Florence, 1984).

General procedure

The glassy carbon electrode was hand polished with 0.3 1-Lm alumina made into a slurry with triple distilled water and electrochemically pre­treated as follows (Edmonds et a!., 1980): de­aerated supporting electrolyte with 5 x 10- 5 M Hg(II) was sucked through the wall-jet cell and the electrode was polarized to -1.0 V vs. Ag/ AgCl for 2 min. The potential was then shifted to +0.5 V vs. Ag/AgCl at high sweep rate to strip off mercury. This treatment was repeated twice or three times, to get an active and reproducible electrode surface.

A thin mercury film was co-deposited with copper and lead on the glassy carbon substrate at -0.95 V vs. Ag/AgCl, for 1 min, in order to ensure limiting current deposition. Potential was then scanned anodically to a slightly positive potential to strip off metals and record the anodic stripping voltammogram. In linear scan stripping at the mercury film electrode, the peak height is proportional to the concentration of the species being striped. Calibration curves for copper and lead were obtained by the standard addition method. Flow rates were calibrated volumetrically every day.

Plant material

White lupin seeds (Lupinus albus L. var. Estoril) were germinated in moist cottonwool and grown hydroponically in a controlled environment. The nutrient solution contained 6 mM Ca(N03 ) 2

4H20, 6 mM KN0 3 , 2.5 mM MgS0 4 7H20, 1 mM KH 2P0 4 , 100 ~-LM H 3B03 , 100 ~-LM MnS04 4H20, 30 fLM ZnS0 4 7H20, 1 ~-LM Na2 Mo0 4 2H20, 0.1 fLM CuS04 5H20, 0.1 ~-LM CoC12 6H2 0 and 75 ~-LM Fe-EDTA. Six lead treatments were used: no Pb supply or 5, 20, 50, 100 and 250 mg L - 1 Pb supplied as Pb(CH3C00) 2

3H2 0. A light regime of 10 h light and 14 h dark was

used; the light intensity was 500 fLM foton m -z s - 1 supplied by day light fluorescent tubes. Temperature was kept at 18°C with constant 55% humidity.

Leaves and roots of 14 days old plants were used for analysis. Roots were washed with dis­tilled water and blotted dry with filter paper.

Determination of lead by anodic stripping voltammetry 21

Leaves and roots were weighed, oven dried at 105aC and weighed again. The plant material was then burned at 500°C and ashes were digested three times with 10 mL of 0.3% (v/v) HN03 and used for lead analysis, after dissolution in 10 mL of the background electrolyte.

Results

Linear sweep anodic stripping voltammograms of copper and lead in different media are shown in Figure 1. In both nutrient solution and nitrate medium the anodic stripping peak potential of copper is located close to the potential of the anodic dissolution of mercury; in hydrochloric acid media, separation between copper and mercury peak is adequate. The presence of citric acid is essential to eliminate interference of iron (III). Potential peaks for lead in nutrient solu-

tion, hydrochloric acid and nitric acid were -0.425 V, -0.680 V and -0.470 V, respectively.

The effects of preconcentration time and potential on peak height were investigated (Fig. 2). A preconcentration time of 1 min was adopted to prevent the formation of a too thick mercury film. At potentials more negative than -1.0 V the hydrogen evolution on the electrode surface disturbs the deposition of metals; conse­quently, electrode response becomes poorly re­producible.

Calibration curves for different concentration ranges (10~ 6 M, 10~ 7 M and 10~ 8 M) for copper and lead were obtained by the standard addition method. The plots are linear as shown in Figure 3.

The determination of lead in roots and leaves of white lupin was carried out in nitric acid/ citric acid. A typical linear sweep anodic stripping voltammogram is shown in Figure 4. Results are

Fig. 1. Linear sweep anodic stripping voltammograms for the redissolution of lead and copper at the wall-jet mercury thin film disc electrode, formed on glassy carbon substrate. Deposition time: 60s at -0.95 V vs. Ag/AgCl; fiow rate: 0.042 cm3 s -\ scan rate: 200 mY s- 1 • Solution :a) 4 x 10-6 M Pb(II), 5 x 10-5 M Hg(II) in nutrient solution; b) 7 x 10-7 M Pb(II), 7 x 10-7 M Cu(II), 5 x 10-5 M Hg(II) in 5 M HCl-0.1 M citric acid; c) 10-7 M Pb(ll), 10- 7 M Cu(II), 5 x 10-5 M Hg(II) in 0.7 M HN03 -0.1 M citric acid.

35.00 (>) 14 (b)

30.00 Pb 12

< 25.00 ,;:--;, 20.00

1

<( 10

t ]

~ 1S.OO . a.. 10.00

: 0.

5.00

so 100 150 200 250 ·1.1 ·1 -0.9 -o.a -0.7

Deposition time/s Pr.,c;once-ntr:ation potentiaiN vs. AgiAgCI

Fig. 2. Effect of (a) deposition time and (b) preconcentration potential on the peak height for 7 x 10-7 M solutions of copper and lead in 5 x 10-5 M Hg(II), 5M HCl-0.1 M citric acid. Flow rate: 0.042cm 3 s 1 ; scan rate: 200mV s- 1 .

22 Neto and De Varennes

1-c ~ ::>

" -"'

"' "' (l_

< ~ c ~

a '"' ~ ~

o._

250.00

200.00

150.00

100.00

50.00

0.00

0

. 20.00

.&. SM HC!-0.1 MCitri-::

acid

• Nutrient solution

2 4 6

Pb{ll) concentration/).lM

5M HCI·O.l M·

Citric acid

16.00 -4-- Nutrient solution

12.00 -)(- 0.73M HNC>j ·0.1M

Citric ~dd

8.00

4.00

0.00

0.2 0.4 0.6 0.8

Pb(ll) concentration/~1M

8 10

1.2

Fig. 3. Anodic stripping voltammetry of Pb(II) at the mer­cury-film wall-jet disc electrode in the media mentioned on the inset; experimental conditions as in Figure 1. Calibration plots constructed using standard addition method.

Pb

O.lV vs. Ag/AgCl

Cu

Fig. 4. Typical anodic stripping voltammogram at the wall­jet electrode for a plant extract.

presented in Table 1. As can be observed, the lead content was always higher in the roots than in the corresponding leaves. Furthermore, lead accumulated preferentially in the roots, as the level of lead in the nutrient solution was in­creased. A relatively high background was de­termined in the plants, probably due to high traffic in a bridge above the trial place.

Root growth was stimulated by low levels of lead in the nutrient solution (20 and 50 mg L -I Pb); toxicity was apparent in plants cultivated with 250 mg L -I Pb. These plants showed a slight leaf chlorosis and had a lower weight.

Discussion

The successful application of anodic stripping voltammetry at a thin film wall-jet electrode to the study of the uptake of lead by white lupin has been demonstrated. Low detection limits and high sensitivity were achieved and there was no noticeable interference effect of the other species in analysis for lead using plant extracts.

Oxidation peaks are well defined but the behaviour of copper is markedly influenced by the supporting electrolyte, as reported before by Edmonds et al. (1980). The mixture nitric acid/ citric acid was the preferred supporting elec­trolyte, as narrowest peaks and best wave defini­tion were found.

Lead was accumulated in lupin roots prevent­ing a high leaf level. In fact, it has been de­scribed that lead is bound in the "free space" and cells walls at the root level, and its transloca­tion is therefore limited (Malone et al., 197 4).

Low and medium levels of lead in the nutrient solution had a stimulating effect in root growth. Growth stimulation by low levels of lead have been described in other plants, as tomato and egg-plant (Khan and Khan, 1983). Wh;<~ lupin was tolerant to the presence of

k"d in lin:: nutrient solution up to the level of 1uU mg L -I and can therefore be considered to exhibit low sensitivity towards lead. In fact, Avena sativa, one of the least sensitive species in the study of Fiusello and Molinari (1973) showed impaired growth when grown in a nutrient solu­tion with lOM- 4 Pb.

The critical toxic level in this study proved to

Determination of lead by anodic stripping voltammetry 23

Table 1. Effect of lead on white lupin weight and lead content

Level of Pb in the Plant fresh weight Plant dry weight Level of Pb in the plant nutrient solution (g) (f.lg) (f.lg g _,dry weight) (mg L _,)

Roots Leaves Roots Leaves Roots Leaves

0 0.734" 1.042" 56,a.h 126"·"· 42" 4.6" 5 0.803' b 0.953" 63" 138" 249' 6.4'

20 1.084"·' 1.180" 85' 170" 1535 7.4" 50 l.J2Jb.c. 1.159" 76"' 165' 2114 12.2"

100 0.908" b' 0.952" 71 "·' 138' 4594 34.4 250 0.330 0.456 39b 86b 5416 104.8

Within a column values followed by the same letter are not significantly different as judged by the Scheffe F-test at 0.05 probability.

be 250 mg L _, Pb in the nutrient solution. However, for soil-grown white lupin a higher level is expected since soils can bind large quantities of lead and make it less available for plant uptake.

Acknowledgement

We would like to thank Mrs Gra~a Sanches for her technical assistance.

References

Albery W J and Brett C M A 1983 The wall-jet ring-disc electrode: Part II, collection efficiency, titration curves and anodic stripping voltarnmetry. J. Electroanal. Chern. 148, 211-200.

Brett C M A and Brett A M C F 0 1986 Hydrodynamic electrodes In Electrode Kinetics: Principles and Meth­odology, Comprehensive Chemical Kinetics, Vol. 26. Eds. C H Bamford and R G Compton. pp 355-441. Elsevier, Amsterdam.

Brett C M A and Neto M M P M 1986 Trace-metal in hydroponic solutions. J. Chern. Soc., Faraday Trans. 1, 82, 1071-1079.

Chizzola R 1989 Metallic trace elements in herbs and spices in Austria. Acta Hortic 249, 89-96.

Edmonds T E, Guogang P and West T S 1980 The differen­tial pulse anodic stripping voltammetry of copper and lead and their determination in EDTA extracts of soils with the mercury film glassy carbon electrode. Anal. Chirn. Acta 120, 41-53.

Fiussello N and Molinari M T 1973 Effects of lead on plant growth. Allionia 19, 89-96.

Florence T M 1984 Recent advances in stripping analysis. J. Electroanal. Chern. 168, 207-218.

Khan S and Khan N N 1983 Influence of lead and cadmium on the growth and nutrient concentration of tomato (Lycopersicum esculentum) and egg-plant (Solanum melongena). Plant and Soil 74, 387-394.

Malone C D, Koeppe D E and Miller R J 1974 Localization of lead accumulated by corn plants. Plant Physiol. 53, 388-394.

Mosbaek H, Tjell J C and Hovmand M F 1989 Atmospheric lead input to agricultural crops in Denmark. Chemosphere 19, 1787-1799.

Vydra F, Stulik K and Julakova E 1976 Electrochemical Stripping Analysis. Ellis Horwood Limited, Chichester, 283 p.

Wang J 1985 Stripping Analysis- Principles, Instrumentation and Applications, VCH Publishers, Inc. Deerfield Beach, Florida, 160 p.

Reprinted from Plant and Soi/154: 1-5, 1993.

M.A. C. Fragoso and M.L. van Beusichem ( eds.) Optimization of plant nutrition 25-29, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-104

Determination of copper in different chloroplast preparations

J.B. ARELLANO, M. BARON, A. CHUECA and M. LACHICA Estaci6n Experimental del Zaidin, CSIC, 18008 Granada, Spain

Key words: chloroplast, copper determination, electrothermal atomic absorption spectrometry

Abstract

The determination of total Cu is not often correlated with states of deficiency in plant material. This fact makes it necessary to look for biologically active Cu. Suspensions of thylakoid membranes and photosystem II particles, properly diluted with 13 mM nitric acid, were used for this purpose. The presence of a minute quantity of an antifoaming agent, such as 1-octanol, is essential when an aliquot of the slurry is injected into the graphite furnace of the atomic absorption spectrophotometer. Good agreement was obtained between our results and those obtained by a classical dry combustion method. Reproducibility was better than 5% when expressed as relative standard deviation.

Introduction

Copper is an essential microelement for the development of algae and higher plants (Baron and Sandmann, 1988), yet analytical results for total foliar copper are rarely correlated with Cu deficiency in higher plants. This points to the existence of biologically inactive Cu in leaves .It is therefore necessary to determine biologically active Cu to overco~e the problems of the analytical diagnosis of its deficiency (Lopez Gorge et a!., 1985).

The total concentration of leaf Cu ranges between 1 and 15 fLg g -l dry weight. The main pool of active Cu is located in the chloroplast, with 50% linked to plastocyanin, a blue Cu­containing protein which plays a role in photo­synthetic electron transport as the electron donor of photosystem I (PS I). However, total chloro­plast Cu content was found to be two to four times higher than that of plastocyanin (Droppa and Horvath, 1990). The physiological role of this 'extra Cu' is not known, and some authors consider it to be associated with photosystem II

(PS II) (Baron et a!., 1990, 1992; Sibbald and Green, 1987).

The goal of our study was to search for Cu­binding sites related to PS II in chloroplasts. The Cu content of thylakoid membranes and PS 11-enriched particles was determined to analyze the contribution of the different pools of active Cu in the chloroplast and to use this parameter for the diagnosis of Cu deficiency in higher plants. From the analytical point of view two main problems were encountered: The very small quantity of material available for analysis and the need to simplify sample preparation to avoid contamina­tion. Both problems could be solved by the use of flameless atomic absorption spectrometry, which allows the injection of a sample suspen­sion (slurry) into the graphite tube. The use of these slurries in electrothermal atomic absorp­tion spectrometry (ETAAS) has been widely reported since its description for the determina­tion of lead (Brady eta!., 1974). We have used it previously for the determination of Cu in plant materials (Lachica and Mingorance, 1984), since it has the advantage of direct samples injection

26 Arellano et al.

into the graphite tube by conventional proce­dures (Stephen eta!., 1985).

Materials and methods

A Perkin-Elmer model 503 atomic absorption spectrophotometer equipped with a HGA-400 graphite furnace was used.

All the reagents were of analytical-reagent grade. High-purity deionized water in quartz was used. Working standard solutions were obtained by diluting a standard stock solution of Titrisol from Merck (1.000 g Cu L -I in water). All the labware was washed in detergent solution, rinsed with water, soaked in 25% (v/v) HN0 3 and thoroughly rinsed with deionized water. Treat­ments in detergent and nitric acid solutions were carried out by sonication for 30 min.

Plants

Pea (Pisum sativum L., cv. Lincoln) plants were grown hydroponically in a Hewitt full nutrient solution in a growth chamber for 4 weeks (Baron and Sandmann, 1988).

Spinach (Spinacea oleraceae L.) was obtained from a local market. Thylakoid membranes and PS II particles were isolated according to Berth­old et a!. (1981) as modified by Ford and Evans (1983). Leaves were ground in 50 mM sodium phosphate pH 7 .4, 5 mM MgC1 2 and 300 mM sucrose at ooc for 15 s up top speed (Sorvall

Omnimixer), the homogenate was filtered, and after 4 washes followed by centrifugation, a thylakoid pellet was obtained. This pellet was resuspended in a stock buffer (50 mM Mes pH 6.5, 400 mM sucrose, 5 mM MgC1 2 and 15 mM NaCl) and used for the measurements of the Cu content of thylakoid membranes. For the isola­tion of PS II particles, Triton X-100 in a ratio 25:1 (detergent/chlorophyll, w:w) was added, and the suspension was stirred for 25 min and centrifuged at 40 000 g for 25 min. The resulting pellet was suspended in the stock buffer as described, and represented the PS II particles used.

Sample preparation

The slurry was shaken vigorously in a Vortex mixer for 1 min. Aliquots of 25 1-1L were then pipetted into a 2 mL test tube. A known volume of 13 mM HN0 3 , adapted to the sample Cu concentration, as well as 5 1-1L 1-octanol were added to the slurry and mixed in a Vortex mixer. Thylakoid membranes were previously treated with a tissue homogenizer.

A modification of the Co mite Inter-Ins titus d'Etude des Techniques Analytiques de Diag­nostic Foliaire procedure (C.I.I., 1969) was used as reference for the determination of the accura­cy of the proposed method: 50 1-1L slurry were poured into a platinum microcrucible, dried at 10SOC and ashed at 450°C for two hours, cooled and the residue solubilized in diluted HCI.

Table 1. Instrumental parameters for electrothermal atomic absorption spectrometry

Atomic absorption spectrophotometer Analytical wavelength Slit width Deuterium background correction Mode Graphite tube Gas

Sample injection volume

Graphite furnace program Step Temp. (0 C)

Dry 100 Dry 130 Char 900 Atomize 2250 Cleaning 2650

324.7 nm 0.7nm

yes peak height normal nitrogen 20f.LL

Ramp (s)

20 10 10 0

Hold (s)

10 10 10 3 3

300 300 300 stop 300

Instrumental analysis

The instrumental parameters used are given m Table 1. Manually inject 20-j.LL well agitated (with Vortex mixer) blank, standard or sample into the graphite furnace. Perform at least 2 measurements for each blank, standard and sample.

Results and discussion

Sample preparation

Previous homogenization of thylakoid membrane samples The difference between PS II particles and thylakoid membranes made a pretreatment of the latter necessary due to their non-uniform particle dispersion even after mechanical shaking and especially after having been frozen. After defrosting, the very coarse thylakoid membranes may give rise to volumetric errors from pipetting and to variations in the size and number of particles sampled. Occasionally, the micropipet was clogged. Some of the problems related to slurry heterogeneity may be overcome by treat­ing the whole sample with a tissue homogenizer for about one minute. The errors can thus be minimised by working with smaller particle sizes and narrower particle-size distributions, as pointed out by Holcome and Majidi (1989). The results presented in Table 2, for homogenized and non-homogenized samples, showed a signifi­cant difference between the 2 blocks and a higher relative standard deviation (RSD) for the non-homogenized samples.

Addition of 1-octanol When slurries of well-homogenized thylakoid membranes or PS II particles diluted with 13 mM

Determination of copper in chloroplasts 27

HN0 3 were used, reproducibility was very poor. For some duplicate injections, there was either a very low signal or no signal at all. However, there were no problems with the synthetic solu­tions used for calibration. This differential be­haviour between samples and standards indicated that the problem came from the composition of the samples. In fact, the simple observation of the graphite tube port could account for the situation when a very low signal was obtained. Due to the surface tension of the slurry the droplet remained suspended in the inner part of the port. On starting the furnace program, the gas flow ejected part of the sample and a very low signal or even no signal was recorded. The high surface tension of the slurry was related to the nature of the material and also to the presence of a wetting agent added during the preparation of the material for analysis. In addition, some foam was produced, introducing air bubbles and making very difficult a reproduc­ible sampling of the slurry (Fuller and Thomp­son, 1977). A similar difficulty had been de­scribed by Temminghoff (1990) when sample plus a matrix modifier, separated by a small amount of air, are injected together. To over­come these difficulties, a very small amount of 1-octanol was added to decrease the surface tension of the sample. The data presented in Table 3 show an almost doubled RSD for sub-

Table 3. Effect of the presence of 1-octanol on pea PS II particles

Without 1-octanol

With 1-octanol

Peak height (A)

0.116-0.120-0.121-0.110-0.127-0.130-0.117-0.117-0.105-0.123-0.114-0.102 Mean= 0.117 RSD=7.10%

0.119-0.122-0.126-0.125-0.133-0.124-0.121-0.125-0.124-0.122-0.130-0.118 Mean= 0.124 RSD = 3.44%

Table 2. Comparison between homogenized and non-homogenized spinach thylakoid membranes'

Thylakoid (ng mL _,)

Homogenized 1139- 1217- 1178- 1148- 1178- 1227- 1227- 1286- 1109- 1276- 1129 Mean= 1192 RSD =4.96%

Non-homogenized 1453- 1513- 1513- 1266- 1227- 1306- 1306- 1217- 1257- 1276- 1286- 1207 Mean= 1319 RSD=8.39%

' Each result is the average of 3 determinations.

28 Arellano et al.

samples without 1-octanol. This problem had previously pointed out by Hutchinson et a!. (1986). Two subsamples were prepared from a unique suspension of thylakoid membranes and each subsample was used for 12 different injec­tions. The concentration of 1-octanol was not critical, since it was shown that foam and air bubbles disappeared with its mere presence.

Addition of nitric acid The use of HN0 3 not only facilitated the ex­traction of Cu into the slurry medium (Bendicho and de Loos-Vollebregt, 1991), but it was also useful as a matrix modifier (Miller-Ihli, 1992). A highly dilute acid solution was used to protect the graphite tube against rapid deterioration.

Evaluation of the method The reliability of the method was assessed by determining Cu in thylakoid membranes with the proposed method and with that recommended by the C.I.I. (1969) for plant materials, without removing silica since it is not present in this material. The results (Table 4) showed that the agreement between the two methods was better than 90%.

The reproducibility of the method was tested by analyzing six replicates of a PS II particle sample. Statistical analysis of individual results (576; 554; 523; 584; 526; and 561 ng mL _,)gave 4.55% for the RSD.

The method proposed is thus quite reliable, in addition to its simplicity and rapidity. From the biochemical and physiological points of view, the localization of new Cu-binding sites in the chlo­roplasts could allow the correlation between analytical results (pools of 'active Cu' in photo­synthesis), biochemical parameters (activity of electron transport) and physiological aspects (diagnosis of a Cu-deficiency or toxicity status of the plant). We have demonstrated previously

Table 4. Comparison of methods using pea thylakoid mem­branes

Method

C. !.I. Proposed

"Mean of 4 results.

187 ± 5 170 ± 8

(Lopez Gorge et a!., 1985) a close relationship between the pattern of Cu. content of thylakoids and the PS !-dependent electron transport rate, as a function of Cu concentration in the medium. Similar experiments in relation to PS II are in progress.

References

Baron M, Lachica M, Chueca A and Sandmann G 1990 The role of Cu in the structural organization of PS II mem­branes. In Current Research in Photosynthesis. Ed. D Baltscheffsky. pp 303-306. Kluwer Academic Publishers, Dordrecht.

Baron M, Lopez Gorge J, Lachica M and Sandmann G 1992 Changes in carotenoids and fatty acids photosystem II of Co-deficient pea plants. Physiol. Plant. 84, 1-5.

Baron M and Sandmann G 1988 Activities of Co-containing proteins in Cu-depleted pea leaves. Physiol. Plant. 72. 801-806.

Bendicho C and De Loos-Vollebregt M T C 1991 Solid sampling in electrothermal atomic absorption spectrometry using commercial atomizers. J. Anal. At. Spectrom. 6, 353-374.

Berthold D A, Backock G T and Yocum C F 1981 A highly resolved oxygen-evolving photosystem II preparation from spinach thylakoid membranes. FEBS Lett. 134, 231-234.

Brady D V, Montalvo J G, Jung J and Curram R A 1974 Direct determination of lead in plant leaves via graphite furnace atomic absorption. At. Absorpt. News! 13, 118-119.

C.I.I. (Comite Inter-Instituts d'Etude des Techniques Analytiques de Diagnostic Foliaire) 1969 Methodes de reference pour Ia determination des elements mineraux dans les vegetaux. Oleagineux 24, 497-504.

Droppa M and Horvath G 1990 The role of Cu in photo­synthesis. Crit. Rev. Plant Sci. 9, 111-123.

Ford R C and Evans M C W 1983 Isolation of a PS II preparation from higher plants with highly enriched oxy­gen-evolution activity. FEBS Lett. 160, 159-164.

Fuller C V and Thompson I 1977 Novel sampling for the direct analysis of powders by atomic-absorption spec­trometry. Analyst 102, 141-143.

Holcombe J A and Majidi V 1989 Error analysis for sampling of slurries: Volumetric errors. J. Anal. At. Spectrom. 4, 423-425.

Hutchinson D J, Disinski F J and Nardelli C A 1986 Determination of copper in infant formula by graphite furnace atomic absorption spectroscopy with a L'vov platform. J. Assoc. Off. Anal. Chern. 69, 60-64.

Lachica M and Mingorance M D 1984 Direct determination of copper in plant material by electrothermal atomic absorption spectrometry. Proc. VI Coli. Int. pour !'Optimi­sation de Ia Nutrition des Plantes. Vol. I, pp 323-330.

Lopez Gorge J, Lastra 0, Chueca A and Lachica M 1985 Use of photosynthetic parameters for the diagnosis of

copper deficiency in Pinus radiata seedlings. Physiol. Plant. 65, 508-512.

Millcr-lhli N J 1992 A systematic approach to ultrasonic slurry GFAAS. At. Spectrosc. 13, 1-6.

Sibbald P R and Green B R 1987 Copper in PS II. Associa­tion with LHC II. Photosynth. Res. 14, 201-209.

Stephen S C, Littlejohn D and Ottaway J M 1985 Evaluation of a slurry technique for the determination of lead in

Determination of copper in chloroplasts 29

spinach by electrothermal atomic-absorption spectrometry. Analyst 110, 1147-1151.

Temminghoff E J M 1990 Signal stabilization in electrother­mal atomic absorption spectrometry by means of addition of butanol. J. Anal. At. Absorpt. News!. 13, 118-119.

ReprintedjromPlantandSoill54: 7-11, 1993.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Oprimizarion of Planr Nurrition, 31-35, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-031

Four new CII reference materials for the chemical analysis of plants: Pine needles, oak leaves, barley-straw and apple-fruit

R.Ch. DANIEL 1 , P. LISCHER1 and G. THEILLER2

1Swiss Federal Research Station for Agricultural Chemistry and Hygiene of Environment, CH-3097 Liebefeld-Bern, Switzerland; 2/NRA-Laboratoire d'analyses vegetates, F-33883 Villenave-d'Ornon, France

Key words: apple, barley chemical analysis, major elements, mmor elements, oak leaves, pine needles, plant analysis, reference materials

Abstract

The CII reports on four new reference materials in the fields of human nutntwn, agronomy and environment. Reference values are given for the major elements (N, P, K, Ca, Mg, Na), the minor elements (AI, Cu, Fe, Mn, Zn) and the ash content (often necessary for X-Ray fluorescence analysis). Accuracy and precision are discussed. These four new substances complete the 21 reference materials offered already by the en.

Introduction

Created in 1959 during the Colloquium on the mineral nutrition of plants in Abidjan (Ivory Coast), the 'Comite Intcr-Instituts d'etude des techniques analytiqucs (CII)' has for aim to place reference materials at the disposal of its members and the interested scientific communi­ty. This approach has evolved from pioneer work to become nowadays essential in the procedures concerning the implementation of Good Labora­tory Practices (GLP) and the accreditation of laboratories.

From its foundation to 1984, the CII has characterized nineteen reference plant materials: Leaves of: apple Cox'orange, apple Golden, artichoke, beetroot, codia discolour, coffee, cot­ton, eucalyptus, hevea, maize, olive, orange, palm, peach, tobacco, vine; as well as a hay, a lettuce and a rice-straw. (Daniel, 1984a; Daniel et a!., 1991a; Pinta et a!., 1975; Pinta et a!., 1976).

The availability of the four new reference

materials comes after the recent characterization of the following materials: alfalfa, cabbage, carnation-straw, ray-grass, peat, pine bark, (Daniel eta!., 1991b; Novozamsky eta!., 1993).

Experimental

Each participating laboratory had to determine as many as possible from the following parame­ters: crude ash, Kjeldahl nitrogen, P, K, Ca, Mg, Na, AI, Cu, Fe, Mn, and Zn. The methods used are briefly described in Table 1. A more detailed description is given in preceeding articles (Anon­ymous, 1969; Daniel eta!., 1991b; Pinta, 1973).

The elements were determined by inductively coupled plasma atomic emission, flame atomic absorption, molecular spectroscopy, and flame photometry. Nitrogen was determined by the method of Kjeldahl (digestion/distillation) and the crude ash (the residue obtained after the first calcination) by gravimetry.

32 Daniel et al.

Table 1. Distribution in groups of the analytical methods

Group number Methods

Total and non-destructive methods -solubilization of the ashes with HCl, followed by treatment of the residue with HF - alkaline fusion of the ashes -wet oxydation with the use of HF -neutron activation analysis -X-ray fluorescence spectrometry

II Solubilization of the ashes with HCl or HN03 ; no treatment with HF

lii Wet oxydation methods with no use of HF, including digestions under pressure (autoclaves) and micro-wave ovens

Ashing: Muffle oven with a temperature rise from cold to 450'C in 2 hours. A temperature at which the sample remains for another 2 hours.

Results and discussion

The comparison of the results from the three groups of methods of mineralization/ digestion for the elements: P, K, Ca, Mg, Na, AI, Cu, Fe, Mn, Zn, was based on an analysis of variance (p = 0.05) of the values obtained according to the CII classical method of calculation (De La Roche and Govindaradju, 1973). This method

involves two notions: the mean and the trimmed mean. The comparisons presented in Table 2 are based on the mean. The values obtained by inductively coupled plasma atomic emission or atomic absorption spectrometry are not differen­tiated. A comparison of these two methods has shown good agreement (Hoquellet eta!., 1988).

On the basis of the abov~ comparisons, the differences observed for the four materials seem

Table 2. Multiple comparisons between the three groups of analytical methods

Groups p K Ca Mg Na

Maritime Pine needles II II (16/9) II III (16/6) II/III (9/6)

II II (18/Y) II III (18/8) IIIIIJ (9/8)

I III ( 18/10) II III (18/8) II/III (10/8)

II II (22/11) II III (22/9) Ill III (11/9)

=I

=I

=I

=/L

=/L

= No significant difference between the two methods. =I Significant difference between the two methods.

=I

Oak leaves

=I =L

Barley-straw

Apple-fruit

=L No significant difference, but at the limit of the 0.05 probability level,

AI Cu

=I =I

=I

= /L Significant difference between the two methods, but at the limit of the 0.05 probability level.

Fe Mn

Blanks indicate that no reliable comparison could be made, usually due to an insufficient number of values (<4). ( ... I ... ) Number of laboratories in each group.

Zn

=I

to be fortuitous. Indeed, not a thing concerning the sampling, the different treatments of prepa­ration, or the storage allows the suspicion of soil or other contamination as in the case of ray-grass and peat (Daniel et al., 1991b). The only other plausible explanation is the number of determi­nations in the different groups of methods. There are between 11 and 24 results in group I, but only between 4 and 10 in groups II and III. As a measure of precaution, these differences should however be considered as possible un­known effects in the mineralization/ digestion of the materials. The analyst should therefore pay particular attention to this part of the analysis.

The CII-laboratories were instructed to use preferentially one of the methods in group I (Total and non-destructive methods). The calcu­lated reference values (Table 3) are based on these results.

The reference values are calculated using robust statistics (Daniel et al., 1991a, b; Hampel et al., 1986; Huber, 1981) which eliminate the problems allied to outliers. In regard to the precision, it can be considered excellent for the

Table 3. Reference values (calculated on dry matter)

Ash N p K

(gkg-1)

CII Reference materials for plant analysis 33

ash content and the major elements with an exception for sodium. Indeed, none of the co­efficients of variation exceed 7. 7% and more than half of them are smaller or equal to 5%. The precision for sodium is less but, if one takes into account the concentrations involved, it is under these circumstances at the very least acceptable. For the minor elements, coefficients of variation in the range of 10% are usually seen as satisfactory. The majority of them lie within this range. The dependence of the coefficient of variation for aluminum and copper on the con­centration is evident. It is emphasized that for copper, measured by flame atomic absorption spectrometry (the great majority of the results), the limit of determination is only slightly below the usual content of the element in plants. Such concentrations are however too high to be mea­sured easily by flameless atomic absorption spec­trometry in graphite furnace without high dilu­tions and thus in both cases high coefficients of variation can be expected. As shown in Table 4, there is good agreement between the values of group I (used for the calculation of the reference

Ca Mg Na AI Cu Fe Mn Zn

(mgkg- 1)

Maritime pine needles n 16 17 12 14 15 14 14 12 18 18 17 18

Reference values 19.2 8,14 0,81 3,10 2,16 1,13 0,85 212 3.1 83 28.2 22.4 Standard deviation 1.4 0.55 0.03 0.16 0.09 0.03 0.09 12 0.9 9 1.5 2.0

Coefficient of variation (%) 7.2 6.7 3.7 5.0 4.1 2.6 10.5 5.6 28.1 10.6 5.2 8.8

Oak leaves n 18 19 14 16 16 16 15 14 21 21 21 19

Reference values 51.8 22.8 1.53 9.23 7.80 1.88 0.06 158 9.3 135 1684 24.5 Standard deviation 2.1 0.7 0.06 0.39 0.24 0.08 0.01 13 0.9 10 102 1.5

Coefficient of varition (%) 4.2 3.2 3.9 4.2 3.2 4.4 25.5 8.5 9.5 7.7 6.1 6.0

Barley straw n 18 18 11 12 15 12 12 11 15 15 16 17

Reference values 75.6 5.95 0.79 23.2 3.49 0.84 2.20 79 3.5 78 15.7 8.3 Standard deviation 4.9 0.29 0.06 1.0 0.19 0.04 0.22 15 0.6 4 1.3 0.9

Coefficient of variation (%) 6.5 4.9 7.5 4.5 5.5 4.4 10.3 18.2 16.4 5.7 7.9 10.7

Apple-fruit n 18 24 16 18 19 18 15 12 22 22 23 24

Reference values 42.2 13.4 1.79 15.1 1.57 0.95 0.06 21.9 7.5 55 11.1 12.6 Standard deviation 3.3 0.7 0.08 0.6 0.09 0.05 0.02 4.7 0.9 6 1.3 1.0

Coefficient of variation (%) 7.7 5.0 4.6 3.9 5.7 5.4 26.7 21.5 11.8 10.7 12.2 8.2

n: number of results used to calculate the reference value.

34 Daniel et al.

Table 4. Accuracy assessment. Comparison between the reference values and those obtained from non-destructive neutron activation analysis and X-ray fluorescence spectrometry

K

(gkg-1)

Pine needles RV 3.10 NAA2 3.16

Oak leaves RV FX

Barley-straw RV 23.2 NAA1 NAA2 22.8 FX

Apple-fruit RV 15.1 NAA1 FX 15.7

All the results are calculated on dry matter; RV: Reference values (Table 3);

Ca

2.16 2.17

3.49 3.15 3.42 3.54

1.57

1.58

Mg Na

1.13 0.85 1.24 0.78

0.84 2.20

0.88 2.18

0.06 0.06

AI Cu Fe Mn Zn

(mg kg - 1)

212 83 28.2 22.4 213 77 27.8 19.9

9.3 135 1684 24.5 10.3 131 1732 23.6

79 3.5 78 15.7 8.3 7.4

72 80 15.2 7.7 4.3 77 15.9 7.8

7.5 55 11.1 12.6 59 11.4

7.0 52 11.1 11.9

NAA I. NAA2, FX: Three independent CII-Laboratories using non destructive neutron activation analysis and X-ray fluorescence spectrometry.

values) and those from activation analysis and X-ray fluorescence spectrometry of pressed pow­ders.

Conclusions

The aim of this article is firstly to present the reference values for the elements NKjeldahl, P, K, ea, Mg, Na, AI, eu, Fe, Mn, Zn, and the ash content of the four new reference materials: pine needles, oak leaves, barley-straw and apple-fruit. Secondly, it shows that the accuracy and preci­sion of the values are good.

Moreover, from the restricted methodological study carried out, it appears that all the miner­alization/ digestion methods used lead to equiva­lent results. Where small variations are observed

Table 5. Reference materials available

for certain elements, no plausible source of errors, which could come from the nature of the material or the analytical methodology, can be ascertained to explain the observed differences. One must assume them fortuitous, the analyst should however pay particular attention at this stage of the analytical procedure.

The en can nowadays offer 25 reference materials for chemical analysis in the fields of human nutrition, agriculture and animal nutri­tion, organic substrates and soil improvers, as well as for the environment and forestry; they are shown in Table 5. A preceding study (Daniel et a!., 1984b) has shown that the chemical composition of the en reference materials re­mained at least unaltered for a period of 10 years. Since no indication have up now modified his opinion, one can assume that the new materi­als will behave in the same manner.

Human nutrition directly indirectly"

apple-fruit, cabbage, lettuce,

Agricultural and animal nutrition Plant substrates and Soil improvers of organic nature Environment and forestry

Golden apple-, maize-, olive-, orange-, peach-, vine-leaves and rice-straw hay, alfalfa, rye-grass, codia discolor-, cotton-, tobacco-, sugarbeet-leaves barley-straw, pine bark, peat

carnation, pine needles, eucalyptus-, hevea-, oak-leaves

": through the foliar diagnostic (the initial trend of the CII).

Acknowledgements

The authors wish to thank particularly D Ruf for computation of part of the data, and J Paul for the english translation and fruitful discussions and comments. Our thanks go also to the mem­bers of the CII, without which this work could not have been carried out.

List of the CI/ Members

Centro de Recursos Naturales, Sevilla (E), In­stitut Europeen de I'Environnement, Bordeaux (F), lnstitut Nat. Agronomique, Paris (F), Esta­cion Experimental del Zaldin, Granada (E), Faculte d'Agronomie, Gent (B), CIRAD, Mont­pellier (F), Istituto di Chimica Agraria, Bologna (I), Station des Plantes Ornementales, Melle (B), Institut de Recherches Chimiques, Ter­vuren (B), ORSTOM, Bondy (F), SADEF, Aspach-le-Bas (F), Office d'equipement hy­draulique de Ia Corse, Bastia (F), Station Federate de Recherches en Chimie Agricole et sur )'Hygiene de I'Environnement, Liebefcld­Berne (CH), Universite Agronomique, Wagen­ingen (NL), INRA, La Grande Ferrade (F), ENSAT, Toulouse (F), Centre de Recherche de Gorsem, St. Truiden (B), Universita Catholica di Piacenza, Piacenza (I), Faculte des Sciences Agronomiques, Michamps (B), Escucla Superior de Agricultura, Lerida (E), Institut d'Hydraulique Agricole et des Herbages, Falenty (PL), Centrum voor Technologisch Onderzoek, Gent (B), ENEA Casaccia, Roma (I), INRA, Champenoux (F), Laboratorio Analisi Terreni Regione Emilia Romagna, Settefonti (I), CNRS, Vernaison (F), Institut Federal de Recherches forestieres, Birmensdorf ( CH), Istituto Agraria Provinciale, San Michelle all' Adige (I), Institut Paul Scherrer, Wiirenling­en (CH), E.E.H.E.I.C.S., Strasbourg (F), G.S.F., Neuherberg (D).

References

Anonymous 1969 Methodes de reference pour Ia determina­tion des elements mineraux dans lcs vegetaux. Oleagineux 24, 497-504.

Daniel R Ch et les Membres du en 1984a Les etalons

CI/ Reference materials for plant analysis 35

vegetaux du en. Resultats complementaires. In Actes VIe Colloque International pour !'Optimisation de Ia Nutrition des Plantes Montpellier 1984. Ed. P Martin-Prevel. Vol. 3. pp 837-846. AIONP/CIRAD Publishers, Montpellier, France.

Daniel R Ch, Bonvalet A et les Membrcs du CII 1984b Stabilite des etalons vegetaux du CII. Qu'en est-il apres 10 ans? Une comparaison d'anciens et de nouveaux resultats. In Actes VIe Colloque International pour l'Opimisation de Ia Nutrition des Plantes Montpellier 1984. Ed. P Martin­Prevel. Vol. 3. pp 831-835. AIONP/CIRAD Publishers, Montpellier, France.

Daniel R Ch, Lischer P, Bermond A, Ducauze C J et les Membres du CII 1991a Laquelle est Ia "Vraie valeur"? Une approche statistique pour determiner Ia teneur en nitrates des etalons vegetaux du CII. Recherche Agronom. Suisse 30, 11-21.

Daniel R Ch, Lischer P, Ruf D, Theiller G and the CII Members 1991b Statistical approches to the determination of reference values for new en standard substances: alfalfa, rye-grass, peat, and pine bark. Recherche Ag­ronom. Suisse 31, 117-126.

De Ia Roche H and Govindaraju K 1973 Etude cooperative sur un verre synthetique VS-N propose comme etalon analytique pour le dosage des e!Cments traces dans les silicates. Analusis 2, 59-70 mentioned in Pinta M 1977 Le Comite Inter-Instituts. Echantillons de reference pour !'analyse vegetale. 1. Azote, phosphore, potassium, cal­cium, magnesium, manganese, zinc. Geostandards News!. 1, 25-30.

Hampel F R, Ronchetti EM, Rousseeuw P J and Stahel W A 1986 Robust Statistics; the Approach based on Influence Fonctions. John Wiley & Sons, New York, 502 p.

Huber P 1981 Robust Statistics. John Wiley & Sons, New York, 308p.

Hocquellet P, Theiller G ct lcs Membrcs du CII 1988 Evaluation dans le cadre du Comite Inter-Instituts de Ia spectrometric par plasma it couplage inductif (ICP) pour !'analyse foliaire. In Proceedings 7th International Col­loquium for the Opimization of Plant Nutrition. Nyborg 1988. Ed. H. Hansen. pp 15511-8.

Novozamsky I, Houba V J G, Daniel R Ch and the Members of CII 1993 Certification of cabbage and carnation samples and their use in an international proficiency study. Fre­senius J. Anal. Chern. 345, 198-201.

Pinta M 1973 Methodes de reference pour Ia determination des elements mineraux dans les vegetaux. Determination des elements par absorption atomique. Oleagineux 28, 87-92.

Pinta M et CII 1975 Etalons vegetaux pour !'analyse foliaire. Analusis 3, 345-353.

Pinta M, Van Schouwenburg J Ch, Bonvalet A, Lachica M and Herman P 1976 Les etalons vegetaux pour !'analyse foliaire; resultats complementaires obtenus pour les elements sodium, chlore, soufre, bore, oligo-elements. In IVe Colloque International sur le Controle de I' Alimenta­tion des Plantes Cultivees. Comptes rendus. Gand 1976. Ed. A Cottenie. Vol. II. pp 41-52. Rijksuniversiteit Pub­lisher, Gent, Belgium.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 37-41, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-203

Determination of leaf standards for apple trees and grapevines in northern Italy

0. FAILLA\ G. STRINGARI2 , D. PORR0 2 and A. SCIENZA1' 2

1 Institute of Arboriculture, University of Milan, via Celoria 2, 20133 Milan, Italy; 2 Agrarian Institute of S. Michele Trento, Italy

Key words: apple tree, grapevines, leaf analysis, leaf standard

Abstract

The survey approach is the most economical and reliable method to establish provisional desirable ranges for leaf analysis interpretation. A three year local survey was undertaken in apple orchards and vineyards of Trentino (north-eastern Italy). Planting were chosen in collaboration with the local advisory service to represent the whole district (locations, soil types, cultivars, rootstocks, planting design and management). Leaf samples were collected in two sampling times according to the standard method. Macro and micro nutrients were determined following a standard laboratory procedures. Results were statistically processed in relation to location and field data. Provisional desirable nutritional ranges have been established for the three main apple cultivars (Golden Delicious, Red Delicious and Canada Reinette) and for grapevines. The importance of adjusting the ranges according to the seasonal weather course became evident in the course of the work.

Introduction

Leaf analysis is the main diagnostic test in evaluating the nutritional status of deciduous fruit orchards (Sadowski, 1990). Because of the strong interaction between mineral nutrition and environmental factors, diagnostic ranges need to be refined on a local scale (Lalatta, 1987). According to Webster (1990), "provisional desir­able ranges can be established by surveys with great economy of time and money, relative to ranges established entirely by field trials".

From this basis, a three year nutritional sur­vey, in apple orchards and in vineyards was undertaken in Trento district ( = Trentino; north­eastern Italy).

In Trentino, apple trees (12,000 ha; 300,000 t) and grapevines (8,700 ha; 125,000 t) are the most important intensive cultures, mainly devoted to high quality crops: about 65% of apples are kept for controlled atmosphere long storage and 50%

of grapes are used to produce controlled origin and sparkling wines.

Methods

Plantings were chosen by the local Provincial Advisory Service. These represented the whole district, in relation to different locations (val­leys), altitude, soil types, cultivars, rootstocks, planting design, training system and cultural practices (Tables 1, 2). Plantings were chosen only if they yielded in a satisfactory way, had an balanced vegetative status and produced high quality fruits.

The survey lasted three years (1987-89) to include possible differences in seasonal weather (Fig. 1).

At each planting, leaves were sampled two times per year. In apple trees, at the end of June and of July, mature leaves were collected from

38 Failla et a/.

Table 1. Cultivars, rootstocks and locations of apple orchards and vineyards chosen for the survey to represent the whole district

Cultivar

Apple trees ( 115) Golden delicious (85) Red delicious ( 16) Canada Reinette (8) Other (6)

Grapevines (52)

Rootstock

Seedlings MM106 (7) M7 (8) M26 (28) M9 (28) Other (7)

Location

Val di Non (50) Adige Valley ( 18) Vallagarina ( 11) Valsugana (14) Sarca Valley ( 11) Cembra Valley ( 4)

Cabcrnct s. (2) Chardonnay (17) Lambruscof.f. (1) Marzemino (3) Merlo! (2) Moscato b. (1) MiillerT. (4) Nosiola (1)

Kober 5BB (28) Teleki 5C (10) S.0.4 (3) 101/14 ( 4) Other (6)

Adige Valley (21) Vallagarina ( 12) Sarca Valley ( 12) Cembra Valley ( 6)

Pi not b. (I) Pinot g. (3) Pinot n. (3) Schiava (8) Teroldego (5)

Table 2. Soil characteristics of the different locations (valley) included in the survey

Location

Adige Valley and vallagarina

Cembra Valley Sarca Valley Valsugana

Val di Non

Soil characteristics

alluvial soil that in the valley bottom are sandy-loam, without skeleton, deep, with superficial water table, rich in organic matter (2.5~3.0% ), neutral or slightly alkaline (pH 7.5), fertile (available P2 0 5 * 60~70 J.Lg g-'; exchangeable K,O** 200~250 J.Lg g '). On the hills, soils are sandy-loam, rich in skeleton, calcareous ("active" carbonate 3% ), less fertile than the valley bottom ones. sandy-loam, originated from porphyric rock soils. alluvial, superficial, sandy-loam, calcareous, rich in organic matter (2.5~3%) but low in fertility soils. sandy-loam, rich in skeleton, superficial, acidic (pH 5.5~6.5), rich in organic matter (>3% ), low in fertility soils (particularly low in available P2 0 5 - <20 J.Lg g- 1).

sandy-clay, calcareous ("active" carbonate 3~4% ), rich in organic matter ( 4~6% ), fertile soils.

*Olsen method; **ammonium acetate method.

240 1987 1988

[: 1989

200

E E

.u _.J _.J 120 <! 0.: lL 2: z w <! 80 40

1-

0:::

40 20

0

A 5 N D

Fig. 1. Meteorological course recorded in the years of the survey. 0 =rainfall; 6 =temperature.

the lower third of medium vigour shoots (80 leaves per orchard from 10 trees). In grapevines, at fruitset and at veraison, mature leaves were collected from fruiting medium vigour shoots; the leaf opposite the proximal cluster at fruitset and the fourth leaf above the distal cluster at veraison ( 40 leaves per vineyard from 20 vines) were collected.

Leaves were washed (0.5% citric acid), dried (70°C), ground and screened (0.5 mm). N was determined by the Kjeldahl method or by near­infrared reflectance spectroscopy. The other nu­trients (P, K, Ca, Mg, Fe, Mn, B, Zn and Cu) were determined by plasma emission spec­trometry, after dry ashing ( 450°C) and treating the ash with HCl (Martin-Prcvcl et al., 1984).

The nutritional status of orchards and vine­yards was studied through the frequency dis­tribution of macro and micro nutrients. The importance and the statistic significance of the main environmental and cultural factors affecting plant nutritional status, was evaluated by analy­sis of variance or by Student's t-test.

Results and discussion

In this report, the method to establish provision­al desirable ranges is presented and discussed in a general way. For detailed information, readers are referred to Failla et al. (1991, 1992).

The first step was to study ranges and fre­quency distributions: macronutrient, boron and

Leaf standards for apple trees and grapevines 39

iron levels had a normal distribution, while manganese, zinc and copper frequency distribu­tion were not normal due to fungicide spray contaminations (Table 3).

The second step was to compare the lower borders of the nutritional ranges with the de­ficiency levels reviewed by Gagnard (1984) for apple trees and by Champagnol (1984) for grapevines:

- in apple orchards, nutrients levels were always above the published deficiency levels;

- in vineyards the main discordance related to iron and boron levels ( 43% of vineyards at fruitset and 19% at veraison had Fe levels below 100 f.Lg g -I; 15% of vineyards at veraison had B levels below 15 f.Lg g- 1).

The third step was to compare the nutritional ranges with the reference ranges established for apple trees by surveys made in other Italian regions (Lalatta, 1987), and, for grapevines, developed in France (Loue ct al., 1984), and often used also in Italy.

According those ranges 40 and 50% of apple orchards should have been in a sub-optimal nutritional status for potassium and magnesium respectively; and 50% of vineyards should have been in a sub-optimal status for nitrogen, phos­phorus and potassium.

These conclusions were unacceptable because all the surveyed plantings were of good prod­uctive and vegetative status. These results there­fore strongly suggested that provisional desirable ranges for Trentino's apple orchards and vine-

Table 3. Nutritional ranges recorded in sound apple orchards (115) and vineyards (52) of Trentino in the 87-89 three year survey

Nutrient Apple tree Grapevines ond.w.

end of June end of July fruitset

N(%) 1.70-3.20 1.40-3.00 1.85-3.10 p (%) 0.14-0.45 0.10-0.40 0.10-0.35 K(%) 0.90-2.60 0.90-2.50 0.75-1.85 Ca(%) 0.75-3.05 1.00-2.60 1.20-3.35 Mg(%) 0.16-0.56 0.14-0.60 0.16-0.43 Fe(!Lgg- 1)" 35-100 40-150 45-300 Mn (!Lgg- 1 ) 10-(*) 8-(*) 10-(*) B(!Lgg-') 14-60 11-50 15-40 Zn(!Lgg-') R-(*) 15-( *) 25-(*) Cu(!Lgg- 1 ) 2-(*) 1-(*) 4-(*)

"Values greater the upper limits were excluded because probably contaminated by soil. They did not exceed 5%. ( *) Due to fungicide residues, upper limits with a physiological meaning cannot be determined.

veraison

1.60-2.80 0.10-0.35 0.70-1.90 1.70-4.00 0.15-0.48

50-350 30-(*)

6-35 15-( *) 3-(*)

40 Failla et al.

yards should be proposed according to the actual nutritional status of sound Trcntino's apple or­chards and vineyards.

The fourth step was to compare the ranges with those recorded by our laboratory in other northern and central Italian regions (Piedmont, Lombardy, Emilia-Romagna and Tuscany) (Lalatta et al., 1992; Failla et al., 1993):

- Trentino apple orchards were lower in nitro­gen, magnesium, iron and manganese and higher in phosphorus calcium and zinc;

- Trentino vineyards were lower in calcium, magnesium and boron.

These results reinforced the idea that soil and climatic conditions of Trentino determined im­portant difference in the plant nutritional status relative to other nearby districts.

The fifth step was to decide if the ranges should be general or if they should be modified

according to different genetic and environmental factors, studying the importance and the statistic significance of the main sources of variation (Table 4):

-In apple tree, the major nutritional differ­ences were ascribed to the cultivar and to the rootstock, followed by the seasonal weather conditions and by the location:

-in grapevines, the seasonal weather condi­tions was the most important factor able to modify the plant nutritional status, while the cultivar determined only minor differences in nitrogen and calcium levels.

In conclusion we decided to adopt, for each sampling period, one provisional desirable range from each one of the three main apple cultivars (Table 5) and one general range for grapevines (Table 6). Yearly, according to the average nutritional status of a reference network of apple

Table 4. Main genetic, cultural and environmental factors affecting the plant nutritional status in Trcntino's apple orchards and grapevines (in order of their importance)

Nutrient Apple tree Grapevines

Nitrogen cultivar, year year, cultivar, location, nitrogen nitrogen fertilization fertilization

Phosphorus cultivar, rootstock, year location, nitrogen fertilization

Potassium cultivar, rootstock, location magnesium availability nitrogen fertilization

Calcium cultivar, rootstock, year, year, cultivar, location location

Magnesium cultivar, rootstock, year, year, potassium availability location

Boron rootstock, year, location, year, location nitrogen fertilization

Table 5. Provisional desirable nutritional ranges for the three main apple cultivars grown in Trentino

Nutrient End of June End of July ond.w.

Golden d. Red d. C. Reinette Golden d. Red d. C. Reinette

N(%) 2.10-2.70 2.30-2.90 1.90-2.50 2.00-2.60 2.10-2.70 1.80-2.40 P(%) 0.18-0.26 0.18-0.26 0.24-0.36 0.16-0.24 0.16-0.24 0.26-0.38 K(%) 1.50-2.10 1.30-1.90 !.10-1.70 1.30-1.90 1.10-1.70 1.00-1.60 Ca(%) 1.20-1.80 1.00-1.60 1.20-1.80 1.40-2.00 1.10-1.70 1.40-2.00 Mg(%) 0.24-0.36 0.24-0.36 0.26-0.32 0.24-0.36 0.27-0.39 0.24-0.36 Fe(J.tgg- 1 ) 35-100 35-100 35-100 40-150 40-150 40-150 Mn(J.tgg 1 ) >10 >10 >10 >8 >8 >8 B(J.tgg-') 20-40 20-40 20-40 20-40 20-40 20-40 Zn (J.tg g- 1) >8 >8 >8 >15 >15 >15 Cu (J.tgg- 1) >2 >2 >2 >1 >1 >I

Table 6. Provisional desirable nutritional ranges for grapevines in Trcntino

Nutrient Fruitset Veraison ond.w.

N(%) 2.20-2.70 1.75-2.25 P(%) 0.15-0.25 0.15-0.25 K(%) 1.10-1.50 1.00-1.50 Ca (%) 1.90-2.70 2.40-3.20 Mg(%) 0.20-0.34 0.20-0.40 Fe(fLgg- 1) >45 >50 Mn (!Lg g- 1) >25 >30 B(!Lgg-1) 18-32 15-30 Zn (!Lg g 1) >25 >15 Cu((Lgg- 1) >4 >3

orchards and of vineyards, the ranges should be adjusted to take into account seasonal weather effects on plant nutritional status.

References

Champagnol F 1984 Element de Physiologie de la Vigne et de Viticulture Generale. SARL, Montpellier, 351 p.

Failla 0, Stringari G and Agnolin C 1991 L'applicazione della diagnostica fogliare nei meleti del Trentino. Frutticol­tura 10, 23-28.

Leaf standards for apple trees and grapevines 41

Failla 0, Scienza A, Stringari G and Mescalchin E 1991 Risultati di un triennio di applicazione della diagnostica fogliare nei vigneti del Trentino. Vignevini 9, 65-70.

Failla 0, Stringari G, Porro D and Scienza A 1993 Vignevini 3, 77-82.

Gagnard J 1984 Pommier. In L"Analyse Vegetale dans le Controle de !'Alimentation des Plantes temperees & tropicales. Eds. P Martin-Prevel, J Gagnard and P Gautier. pp 234-257. Lavoisier, Paris, France.

Lalatta F 1987 Metodi ed interpretazione delle analisi fogliari in frutticoltura. Frutticoltura 617, 71-76.

Lalatta F, Failla 0 and Stringari G 1992 Monitoraggio dello stato nutrizionale di piante arboree in rclazione al rischio di inquinamento idrico. Riv. di Agron. 26, 4 Suppl., 706-713.

Loue A, Gagnard J and Morard P 1984 Vigne. In L'Analyse Vegetale dans le Controle de !'Alimentation des Plantes tempcrecs & tropicales. Eds. P Martin-Prevel, J Gagnard and P. Gautier. pp 197-233. Lavoisier, Paris, France.

Martin-Prevel P, Gagnard J and Gautier P 1984 Generalites. In L'Analyse Vegetale dans le Controle de !'Alimentation des Plantes temperees & tropicales. Eds. P Martin-Prevel, J Gagnard and P Gautier. pp 1-161. Lavoisier, Paris, France.

Sadowsi A 1990 International Symposium on Diagnosis of Nutritional Status of Deciduous Fruit Orchards. Acta Horticultune 274, 526 p.

Webster D H 1991 Role of field trials in development and refinement of soil and tissue diagnostic criteria. Acta Hortic. 274, 509-513.

M.A.C. Fragoso and M.L. van Beusichem (eds.). Optimization of Plant Nutrition, 43-47, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-077

Interpretative indices for leaf analysis in vineyards of the Portuguese region of Bairrada

CECILIA PACHECH0 1 , FATIMA CALOUR0 1 and ANABELA ANDRADE 2

1/NIA, Laborat6rio Quimico Agricola Rebelo da Silva, Tapada da Ajuda, 1300 Lisboa, Portugal; 2 DRABL, Estac;ao Vitivinicola da Beira Litoral, 3780 Anadia, Portugal

Key words: foliar analysis, leaf blades, leaf indices, petioles, vineyards

Abstract

Leaf analysis is an important tool for evaluating the nutritional status of vineyard. During four years, in the Portuguese region of Bairrada, 52 vineyards were observed and leaf samples were collected at full bloom. The study included the most representative regional grape-vine varieties (M. Gomes, Bical, Cerceal and Baga) and the main objective was the establishment of interpretative indices for foliar diagnostics. In each vineyard an experimental plot with 40 plants was installed in order to collect soil and leaf samples as well as to estimate the plant yield. Interpretative indices for N, P, K, Ca, Mg and B are presented, both for blades and petioles, taking into account the differences that were found between varieties. Significant simple correlations were found between N, P, K, Ca, Mg and B petiole and blade concentrations, suggesting the undifferentiated use of both foliar tissues for vineyard nutritional status diagnosis.

Introduction

Leaf analysis is an important tool to evaluate crops nutritional status. Historically it is associ­ated with vineyard since it was firstly used in this crop (Lagatu and Maumc, 1926).

Regional interpretative foliar reference values are requested because of the influence of differ­ent environmental conditions and variety I root­stock system on the plant nutritional status.

The Portuguese region of Bairrada is one of the main producing quality wines in the country; however, experimental work, supporting the establishment of the interdependence of yield (or well defined quality characteristics of the grapes) and the plant nutritional status is lacking. There­fore reference values for the leaf-mineral compo­sition are unknown for regional varieties and the results of leaf analysis have little application.

Because of its easy application, as a way to establish standard foliar values, Kenworthy methodology was used (Kenworthy, 1967), a!-

lowing for the establishment of criteria to com­pare the nutritional status of vineyards and to advise fertilizations.

According to the named author, a standard value may be considered as a mean for the range composition of plants without symptoms; but some restrictions must be imposed to this con­cept, because of yield, as well as some quality characteristics of the grapes are not always straightly related with leaf mineral concentra­tions. This is mainly due to external influencing factors, such as environmental and management practices, and internal ones, such as rootstock and variety. So, it is important to establish standard foliar values or normal range nutrition­al levels according to the local varieties and producing conditions.

This paper looks into the assessment of region­al interpretative indices for nutritional diagnostic through leaf analysis, concerning the main re­gional vine-grapes varieties, and leaf-tissue for chemical analysis.

44 Pacheco et al.

Materials and methods

In 1988, fifty two highly productive and appar­ently balanced vineyards, were selected in the Portuguese region of Bairrada. Four different varieties were chosen (M. Gomes, Bical, Cerceal and Baga) because of their regional economic importance. Most of the selected vineyards were established in soils with pH (H20) values higher than 6.5, low available P and high available K levels (Pacheco, Calouro and Andrade, 1990) and clay levels higher than 15%.

An experimental plot with forty plants was installed in each vineyard.

During four years (1988-1991), in each plot, the basal cluster opposite leaf of the grape-vines was collected at full bloom and N, P, K, Ca, Mg and B concentrations were determined, both in the blade and the petiole. Analytical methods are described by Duarte et a!. (1989). The grape yield per plot was also evaluated.

Plant nutritional status interpretative indices were established according to Kenworthy meth-

odology (1967). Coefficients of vanatiOn were estimated as well as the 95% confidence intervals about each nutrient mean.

Student's t-test was used in order to compare varieties concerning both petiole and blade min­eral concentrations (a= 0.05).

Simple correlations between petiole and blade mineral concentrations were also estimated.

Results and discussion

Mean mineral composition of the leaves and variability

Tables 1 and 2 show the means and the co­efficients of variation of the mineral composition of blades and petioles for each variety under survey.

According to the reference values proposed by Kenworthy (1967), the vineyards under survey show, on average, a balanced nutritional status.

Coefficients of variation are higher than those

Table 1. Mean mineral concentrations of petioles of grape varieties M. Gomes, Bical, Cerceal and Baga

Nutrients M. Gomes Bical Cerceal Baga

Mean CV(%) Mean CV(%) Mean CV(%) Mean CV(%)

N(%) 0.89 31.8 0.81 28.4 0.92 24.0 0.98 27.2 P(%) 0.24 47.7 0.18 53.3 0.26 43.4 0.27 59.7 K(%) 2.22 28.0 1.57 40.3 2.32 32.9 2.46 38.7 Ca(%) 2.41 21.5 2.60 15.9 2.22 25.4 1.47 31.5 Mg(%) 0.42 33.3 0.58 36.3 0.57 37.4 0.32 40.9 B (mgkg- 1 ) 33 21.5 36 23.4 37 19.6 32 18.3

CV: Coefficient of variation.

Table 2. Mean mineral concentrations of blades of grape varieties M. Gomes, Bical, Ccrccal and Baga

Nutrients M. Gomes Bical Cerceal Bag a

Mean CV(%) Mean CV(%) Mean CV(%) Mean CV(%)

N(%) 2.88 11.4 2.66 11.8 3.01 10.9 2.78 11.6 P(%) 0.21 18.3 0.19 18.2 0.20 16.8 0.21 18.7 K(%) 0.84 27.6 0.86 27.5 1.09 24.5 1.02 32.4 Ca (%) 2.07 29.0 2.07 22.2 1.94 25.0 1.46 34.9 Mg(%) 0.17 26.3 0.22 29.1 0.22 22.4 0.22 27.8 B (mgkg- 1 ) 42 26.6 44 27.5 43 29.9 44 22.6

CV: Coefficient of variation.

Interpretative indices for leaf analysis in vineyards 45

reported by Kenworthy (1967) for vineyards and by Pacheco (1987) for table-grape vines varieties in the Portuguese region of Alenquer, except for boron.

In the region of Bairrada the vineyards show great variation concerning fertilizer management practices and, within the same vineyard, differ­ent rootstoks can be found. This explains the obtained results.

Reference values for leaf mineral concentrations

Different management practices, namely fertili­zations, were used in the vineyards under survey, which caused several sources of variation con­cerning foliar mineral concentrations.

Significant differences (p < 0.05) were found between varieties regarding some of the mean foliar nutrient concentrations, both in blades and petioles:

Concerning the mean mineral concentrations of petioles, Bical shows significant lower levels of N, P, and K as compared with M. Gomes,

Cerceal and Baga varieties. On the contrary, significant differences were found between var­ieties, concerning petiole-Ca: Bical shows the highest and Baga the lowest concentration level. Regarding Mg and B, Bical and Cerceal show significant higher petiole concentrations as com­pared with M. Gomes and Baga.

Significant differences were also found be­tween varieties, concerning blade mineral con­centrations. This is the case of Bical, that shows the lowest levels of N and P, as compared with the other varieties; Baga shows the lowest blade­Ca concentration, and M. Gomes the lowest Mg; no significant differences were found concerning blade-B.

These facts show the need for the establish­ment of different reference values for leaf miner­al concentrations, at least for some nutrients, according to the variety, or group of varieties, which was done (Tables 3 and 4).

These results contradict the findings of Ken­worthy (1967) who supports the universal appli­cation of the same foliar reference values for

Table 3. Reference values for mineral concentrations of petioles of grape varieties M. Gomes, Bical, Cerceal and Baga

Nutrients M.Gomes Bieal Cerceal Baga

RF CV(%) RF CV(%) RF CV(%) RF CV(%)

N(%) 0.94 28.5 0.81 28.4 0.94 28.5 0.94 28.5 P(%) 0.26 53.9 0.18 53.3 0.26 53.9 0.26 53.9 K(%) 2.35 34.9 1.57 40.3 2.35 34.9 2.35 34.9 Ca (%) 2.41 21.2 2.60 15.9 2.22 25.4 1.47 31.5 Mg(%) 0.42 33.5 0.58 36.5 0.58 36.5 0.32 40.9 B(mgkg-') 33 19.6 36 21.6 36 21.6 33 19.6

RF: Reference value; CV: Coefficient of variation.

Table 4. Reference values for mineral concentrations of blades of grape varieties M. Gomes, Bical, Cerceal and Baga

Nutrients M. Gomes Bical Cerceal Baga

RF CV(%) RF CV(%) RF CV(%) RF CV(%)

N(%) 2.92 14.4 2.66 11.8 3.01 10.9 2.92 11.4 P(%) 0.21 18.2 0.19 18.2 0.21 18.2 0.21 18.2 K(%) 0.85 27.3 0.85 27.4 1.04 30.4 1.04 30.4 Ca (%) 2.04 26.3 2.04 26.3 2.04 26.3 1.46 34.9 Mg(%) 0.17 26.3 0.22 27.1 0.22 27.1 0.22 27.1 B (mg kg_,) 43 25.5 43 25.5 43 25.5 43 25.5

RF: Reference value; CV: Coefficient of variation.

46 Pacheco et al.

Table 5. Normal range values for mineral concentrations of petioles of grape varieties M. Gomes. Bical, Cerceal and Baga

Nutrients M. Gomes Bical Cerceal Baga

N(%) 0.89-0.99 0.72-0.90 0.89-0.99 0.89-0.99 p (%) 0.23-0.28 0.14-0.22 0.23-0.28 0.23-0.28 K(%) 2.20-2.51 1.32-1.81 2.20-2.51 2.20-2.51 Ca(%) 2.23-2.59 2.44-2.76 1.97-2.48 1.34-1.60 Mg(%) 0.38-0.47 0.51-0.64 0.51-0.64 0.29-0.36 B (mgkg- 1) 31-34 34-39 34-39 31-34

each species independently of the variety and environmental conditions.

The obtained yields reached 10 tha-I which is higher than the average of regional yields. Therefore, at the present stage of the survey, the obtained results were considered as the regional normal range nutritional levels (Tables 5 and 6).

Relationships between blade and petiole mineral concentrations

High significant (p ~ 0.001) simple correlations were found between blade and petiole mineral concentrations, except in the case of boron. These results suggest the undifferentiated use of both foliar tissues for chemical analysis in order to diagnose the nutritional status of vineyards. This fact has been already reported by several authors, namely Bertoni and Morard (1982).

Concerning B, the coefficient of simple corre­lation, although highly significant, is low (r =

0.314) which may be due to the lower variation of the petiole B concentration as compared with the blade's one (Tables 1 and 2). The fact is that B is accumulated in the blade rather than in the petiole, leading to more stable petiole-levels. Consequently blades are more sensitive to changes on B-concentration and therefore they should be used in leaf analysis especially in the cases of boron toxicity identification (Cook and Wheeler, 1978).

Conclusions

The obtained results suggest that within the same environmental conditions and similar yields, dif­ferent grape-vine varieties show different leaf mineral concentrations. This suggest the need for the establishment of different reference values for the interpretation of leaf analysis results.

The correlations found between blade and

Table 6. Normal range values for mineral concentrations of blades of grape varieties M. Gomes, Bical, Cerceal and Baga

Nutrients M. Gomes Bical

N(%) 2.75-2.89 2.54-2.78 P(%) 0.20-0.21 0.17-0.20 K(%) 0.79-0.91 0.79-0.91 Ca (%) 1. 92-2.16 1.92-2.16 Mg(%) 0.16-0.19 0.21-0.23 B(mgkg- 1 ) 42-45 42-45

Table 7. Simple correlations between petiole and blade mineral concentrations

Blades

N p

K Ca Mg B

Petioles

N

0.688***

n=52; ***-p~0.001

p K Ca

0.601 *** 0.723***

0.888***

Cerceal Baga

2.86-3.12 2.75-2.89 0.20-0.21 0.20-0.21 0.96-1.11 0.96-1.11 1.92-2.16 1.31-1.61 0.21-0.23 0.21-0.23

42-45 42-45

Mg B

0.714*** 0.314***

Interpretative indices for leaf analysis in vineyards 47

petiole mineral concentrations suggest the un­differentiated use of both tissues for the diag­nosis of the nutritional status of the vineyard. However, and concerning boron toxicity, blades should be used.

Acknowledgements

The authors are grateful to the staff of EVBL and LQARS who carried out the collecting and analytical work as well as text processing.

References

Bertoni G and Morard P 1982 Blades or petioles analysis as a guide for grape nutrition. Comm. Soil Sci. Plant Anal. 13, 593-605.

Cook J A and Wheeler D W 1978 Use of tissue analysis in viticulture. In Soil and Plant tissue Testing in California. pp 14-16, University of California, Berkeley, USA.

Kenworthy A L 1967 Plant analysis and interpretation of analysis for horticultural crops. Soil Test. Plant Anal. 2, 59-75.

Lagatu H and Maume L 1926 Diagnostic de !'alimentation d'un vegetal par !'evolution chimique d'une feuille convcn­ablement choisie. C. R. Acad. Sci. 182, 635-655.

Pacheco C A 1987 Estado nutritive de vinhas de uva de mesa da regiao de Alenqucr- Informa~ao preliminar. Vida Rural, ano 35°, 20/87 (1441), 26-29.

Pacheco C A, Calouro F and Andrade A 1990 Indices interpretativos de analise foliar de vinhas da regiao da Bairrada- Informa~a preliminar. Acta Hortic 7, 390-395.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 49-54, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-126

Relationship between aboveground dry weight and N, P and K concentrations in grassland species: A guide for the diagnosis of plant nutrient status

A. PEETERS and V. VAN BOL Laboratoire d'Ecologie des Prairies, Universite Catholique de Louvain, Place Croix du Sud 2, Bte 5, B-1348 Louvain-la-Neuve, Belgium

Key words: Anthriscus, dilution curve, grasses, macronutrient status

Abstract

In a field experiment, the dilution curves of 9 grass species and one dicot species, Anthriscus sylvestris, were compared. The P, K and Mg contents have been related with aboveground dry weight and N content. The N dilution in the dry matter could be described by only one equation for all species (N = 6 x Y- 0 .63 ). The differences between species for P were very small, but Anthriscus sylvestris was always richer than the grasses in K and Mg. The sorting out of the forage samples are thus necessary to determine the nutrient status of these two cations in permanent grasslands. Despite big differences in production, the equations of the nine grass species are very similar for all these nutrients (N, P, K and Mg). The parameters of these equations given here can be used as a guide to determine a satisfactory nutrition.

Introduction

The perfecting of expert systems for the diag­nosis of plant nutrient status requires quantifying several parameters. Without any growth-limiting factors, except elimatic factors, it is possible to define, for a given species, general curves of nutrient dilution in terms of harvested biomass (Eq. 1) or in terms of its nitrogen content (Eq. 2) (Greenwood et a!., 1990; Lemaire and De­noix, 1987; Lemaire and Salette, 1984; Salette and Huehe, 1991).

Nutrient(% DM) = a*Y-b

Y: yield (TDMha- 1) (1)

Nutrient (% DM) = c + dN

N: nitrogen content(% in DM) (2)

The following equations (Eqs. 3-4) ean also be considered for nitrogen.

Nitrogen(% DM) = e*10(-t•Yietd) (3)

Nitrogen (% DM) = Nmin + (Nmax- Nmin)

*exp(-0.25xY) (4)

Y: yield (T DM ha _,)

N min and N max: lower and higher values of

the experimental curve

These curves take into account the dilution effect of the nutrients into the dry matter during the growth. The parameters of the curves are very similar for cultivated grasses (Lemaire and Salette, 1984) but it is interesting to know if they are also similar for less productive and grasses adapted to more oligotrophic conditions, or even for dicots which are known to have higher mineral contents than grasses. These high miner­al contents of dicots could be only the con­sequence of a lower yield. It is well known indeed that the mineral content of the plants

50 Peeters and Van Bol

decreases with the age of these plants and thus also with their yield. Consequently, if a plant is less productive than another it is normal that, at a given time, its nutrient content is higher. Thanks to these equations, calculated for each species, is is possible to compare the nutrient contents of these species for equivalent yields. It is thus possible to verify if there is a "species effect" or not. If this species effect does not exist, the botanical assessments could not be necessary for nutrient diagnosis and the sampling method could be much simplified in grassland studies.

Methods

The spring growth of 9 grass species was com­pared in a field experiment: Agrostis stolonifera, Alopecurus pratensis, Arrhenatherum e/atius, Dactylis glomerata, Elymus repens, Lolium perenne, Phleum pratense, Poa pratensis, Poa trivia/is, and one dicot species: Anthriscus sylves­tris.

The experimental design was a split-plot (species-cutting date) with four replicates. It included 6 cuts on successive plots in order to describe the yield and mineral content evolution during the first cycle, until seed formation of most species (mid-July). The cutting height was 5 em. Each plot received a high nitrogen fertili­zation (100 kg N ha - 1) in March. The P and K fertilization was 35 and 100 kg ha - 1 respectively.

The forage samples were dried at 105°C and mineralized at 450°C. The minerals were solubil­ized in nitric acid and determined by spec­trometry of atomic absorption for the cations and by colorimetry for phosphorus. The nitrogen content was determined by the Kjeldahl method.

Results

The dilution curve of nitrogen of all species is shown in Figure 1. The three models (equations 1, 3 and 4) can describe this type of curve (Table 1). For equation 1, the exclusion of the yields below 1 ton ha - 1 increases the determination coefficient. For the other models (equations 3

%N

7

6

5

4

3

2

o4-~-r~~~--r-~~~-r~~~~r-~

0 2 4 6 8 10 12 14 16

Dry weight (t ha·1)

Fig. 1. Relationship between aboveground dry weight and %N for all species (10 species, n = 240).

and 4), the data lower than 1 t ha - 1 have not such influence. The percentage of variance ex­plained is not improved by removing the data of low yields.

In order to compare these results with those of Greenwood et al. (1990), Lemaire and Denoix (1987), Lemaire and Salette (1984) and Salette and Huche (1991), the attention has been fo­cused on equation 1. The parameters a and b seem to be similar for each species (Table 2). A statistical analysis of the regression has evaluated the gain in precision when parameters a and b varies (Tables 3, 4). The analysis includes mea­surements of the improvements in fit brought about by successive increases in the complexity of the models. The analysis measures the adequation of the general parameters with more particular parameters up to parameters suited to each species. The first two columns identify the models. The last two columns give the MS to the additional parameters of the more complex over the less complex model and the probability levels of this comparison. Nearly 92% of the entire variance was removed by equation 1 (Table 3). Models 2, 3 and 4 which utilise parameters a and/ or b adapted to each species, are each time different between them and with model 1 (gener­al equation). The comparison of models 5 to 14 with model 1 (Table 4) allows to compare the similarity of parameters a and b of each species with the general parameters. In the case of five species, there is no significative difference be­tween the tested model and model 1. It is Agrostis stolonifera, Alopecurus pratensis, Dac-

Plant nutrient status and grassland species 51

Table 1. Regression equations and determination coefficients of 3 models of N dilution into dry matter

Equation 1

Equation 3

N (% DM) = 4.96 x Yield- 049

N (% DM) = 6.12 x Yield -o. 63

N (% DM) = 5.27 X 10(-0o6xYkldJ

N (% DM) = 5_11 X lO<-o ooxYie!dl

R'

0.86 0.92 0.92 0.91

n y

240 all data 202 >1 Tha- 1

240 all data 202 >1 Tha- 1

Equation 4 N (% DM) = Nmin + (Nmex- Nmin) X exp(-0.25 X Y) N (% DM) = Nmin + (Nm"- Nm,J x exp(-0.25 X Y)

0.95 0.94

240 all data 202 >1 Tha 1

Yield in TDMha- 1 •

Nmin = 1 and Nm"' = 6.

Table 2. Parameters of the regression equations (equation 1) between nitrogen and dry matter yield above 1 ton ha _,

a b R' n

Agrostis stolonifera 6.10 0.62 0.97 21 Alopecurus pratensis 6.76 0.68 0.91 24 Anthriscus sylvestris 5.90 0.54 0.95 15 A rrhenatherum elatius 6.75 0.62 0.94 22 Dactylis glomerata 6.91 0.67 0.97 23 Elymus repens 5.63 0.52 0.91 15 Lolium perenne 5.70 0.66 0.95 22 Phleum pratense 6.21 0.63 0.96 23 Poa pratensis 6.17 0.67 0.96 17 Poa trivia/is 4.88 0.59 0.98 20

All species together 6.12 0.63 0.92 202 Minimum 4.88 0.52 Maximum 6.91 0.68

tylis glomerata, Phleum pratense and Poa praten­sis. On the other hands, for Anthriscus sylvestris, Arrhenatherum elatius, Elymus repens, Lolium perenne and Poa trivialis, the models differ significatively.

Phosphorus did not show big differences be­tween the grass species. However, A. sylvestris was richer in this element at high yield or low concentration of nitrogen (Tables 5 and 6). The regression equations of P (% DM) with nitrogen (% DM) gave similar slopes for all species (0.09 to 0.12) but the origin of the line was different for the grasses (0.13) and Anthriscus (0.28) (Table 6). So, Anthriscus was slightly richer in P at low N-concentration. Figures 2 and 3 show the curves of P in relation with aboveground dry weight and nitrogen content.

The case of potassium is completely different of the two preceding elements. A. sylvestris was always more concentrated in this element than the grasses by more or less 2 g 100 g DM- 1 • It is thus really an accumulator of potassium. It uses more potassium than the grasses to sustain a certain level of dry matter or for a given nitrogen concentration of this dry matter (Figs. 4 and 5; Tables 5 and 6).

For Mg, A. sylvestris was again systematically

Table 3. Regression analysis (equation 1) of ln %Non ln Y (yields above 1 tonha- 1)

No. Models

0 a fixed, b = 0 1 a, b fixed 2 a fixed, b different for each species 3 b fixed, a different for each species 4 a, b different for each species

Legend: p: number of explicative parameters. RSS: residual sum of square. df: degree of freedom. RMS: residual mean square. MS: mean square.

p RSS

1 44.28 2 3.69

II 2.49 11 2.21 20 2.00

df RMS %Variance MS due to additional accounted for parameters

Compared models

2

201 0.220 200 0.01844 91.6 40.592 191 0.01302 94.1 1.201** 191 0.01155 94.8 1.481 ** 182 0.01097 95.0 1.690** 0.489**

*, **: the probability levels for the improvements in fit were calculated using the residual MS: **: p < 0.01, *: p < 0.05.

3

0.209*

52 Peeters and Van Bol

Table 4. Regression analysis (equation 1) of In %Non In Y (yields above 1 tonha- 1 )

No. Models p RSS df RMS %Variance MS due to additional Probability accounted for parameters levels for the

comparison with model 1

0 a fixed, b = 0 1 44.28 201 1 a and b fixed 2 3.69 200 0.018 5 a, b fixed except for Agrostis stolonifera 4 3.68 198 0.019 -0.01 0.010 NS 6 a, b fixed except for Alopecurus pratensis 4 3.63 198 0.018 0.01 0.060 NS 7 a, b fixed except for Anthriscus sylvestris 4 3.56 198 O.D18 0.03 0.130 8 a, b fixed except for Arrhenatherum elatius 4 3.39 198 0.017 0.07 0.300 9 a, b fixed except for Dactylis glomera/a 4 3.59 198 0.018 0.02 0.100 NS

10 a, b fixed except for Elymus repens 4 3.57 198 0.018 0.02 0.120 11 a, b fixed except for Lolium perenne 4 3.29 198 0.017 0.10 0.400 12 a, b fixed except for Phleum pratense 4 3.68 198 0.019 -0.01 0.010 NS 13 a, b fixed except for Poa pratensis 4 3.63 198 0.018 0.01 0.060 NS 14 a, b fixed except for Poa trivia/is 4 2.97 198 0.015 0.19 0.720

Legend: see Table 3.

Table 5. Parameters of the regression equations between the DM contents of 3 mineral elements and dry matter yield

n K p Mg

a b R' a b R' a b R'

A. sylvestris 20 8.73 0.21 0.82 0.78 0.26 0.83 0.29 0.19 0.51 All grasses 160 5.64 0.31 0.66 0.79 0.45 0.87 0.22 0.34 0.76 L. perenne 20 6.50 0.38 0.96 0.82 0.47 0.95 0.19 0.27 0.91 D. glomera/a 20 7.91 0.40 0.94 0.88 0.48 0.97 0.21 0.27 0.79

Legend: the values of a and b were obtained by fitting Eq. 1.

Table 6. Parameters of the regression equations between the DM contents of 3 mineral elements and nitrogen

n K

c d R'

A. sylvestris 20 4.3 0.80 0.79 All grasses 160 1.9 0.66 0.64 L. perenne 20 1.5 0.92 0.98 D. glomerata 20 1.9 0.90 0.94

Legend: the values of c and d were obtained by fitting eq. 2.

%P

0.

0.

0.

0.

0.

0.

0.

0.1

0. 0 10 12 14 16

Dry weigh1 (1 ha·1)

Fig. 2. Relationship between aboveground dry weight and %P for Anthriscus sylvestris and the grasses (n = 20 and 160).

p Mg

c d R' c d R'

0.28 0.09 0.90 0.14 0.030 0.60 0.13 0.11 0.94 O.D7 0.027 0.78 0.13 0.12 0.98 0.08 0.023 0.93 0.14 0.11 0.98 0.09 0.020 0.82

%P

0.

0.

0.

0.

0.

0.

0.

0.1

0. 0 3 4 6

%N

Fig. 3. Regression between P and N contents for Anthriscus sylvestris and the grasses (n = 20 and 160, respectively).

0 4 10 12 14

Dry weight (t ha-1)

Fig. 4. Relationship between aboveground dry weight and %K for A. sylvestris, D. glomerata and L. perenne (n = 3 x 20).

%K

0 2 3 4 5 %N

Fig. 5. Regression between K and N contents for A. sylves­tris, D. glomerata and L. perenne (n = 3 x 20).

richer (more or less 0.1 g 100 g DM _,) than the grasses on the basis of N content or dry matter yield (Figs. 6 and 7; Tables 5 and 6).

%Mg

.. c c

c

10 12 14 Dry weight (t ha·1)

Fig. 6. Relationship between aboveground dry weight and %Mg for A. sylvestris, D. glomerata and L. perenne (n = 3 x 20)..

Plant nutrient status and grassland species 53

2 3 %N

Fig. 7. Regression between Mg and N contents for A. sylvestris, D. glomerata and L. perenne (n = 3 x 20).

Discussion

The parameters of the logarithmic equations of N dilution ( eq. 1) of each species are thus not strictly identical but the general equation is nevertheless quite satisfactory for practical pur­poses. From this point of view, there is no reason to distinguish Anthriscus sylvestris from the grasses. The general curve can be written as follows: %N = 6.1 x Y- 0.63 .

These results seem to be contradictory with those of Poorter et a!. (1990). These authors have shown that fast growing species have a higher total organic nitrogen concentration per unit plant weight and that they allocate more nitrogen to the leaves. However, their range of species include species which are much more oligotrophic and much more slow growing than the species of this experiment. Their experiment was also carried out in growth room excluding self shading of the plants. These artificial con­ditions are of course very different from the field conditions of this experiment.

The parameters of the logarithmic equation ( 6.1 x y-o 63 ) are similar with those of Lemaire and Salette (1984): 5.3 x Y-048 .The results are also consistent with Greenwood et a!. (1990) for a large range of C3 plants (5.7 x y-o 5 ). These values are thus reliable to determine the status of N nutrition.

The species are not very different for the evolution of P content. Nevertheless, the P content of Anthriscus sylvestris decreased a little bit more slowly than the grasses. At the oppo­site, there was a big difference between Anthris-

54 Plant nutrient status and grassland species

cus sylvestris and the grasses for the evolution of K and Mg contents. The sorting out of the dicots species in the forage samples could thus be very important to assess the nutrition status for the cations. The differences between the grasses are not negligible for K but they are very small for P and Mg. The general equations for all grass species are defined in Table 7, they are com­pared with those that Salette and Huche ( 1991) consider as satisfactory for a good K and P nutrition. The values of this experiment are higher, indicating a higher nutrition. These high

Table 7. General regression equations (Eqs. 1 and 2) be­tween P, K and Mg on one hand and aboveground dry weight and nitrogen content on the other hand, and comparison of their parameters with those that Salette and Huche (1991) recommend for a satisfactory nutrition.

Equation 1: Y =Above ground dry weight

K p

Mg

a

5.6 0.8 0.2

a'

4.40 to 5.00 0.45 to 0.62

b

0.31 0.45

Equation 2: Y =Nitrogen content

c' d

K 1.91 1.40 to 1.80 0.66 p 0.13 0.13to0.17 0.11 Mg 0.07

Legend: a, b: parameters of Eq. 1.

b'

0.30 0.30 to 0.40 0.34

d'

0.50 to 0.55 0.06 to 0.07 0.03

a', b': equivalent parameters of Salette and Huche (1991). c, d: parameters of Eq. 2. c', d': equivalent parameters of Salette and Huche (1991).

values are not necessarily desirable for K because there could be a luxury consumption for this element, but it is certainly not the case here for P.

Finally, the interactions between physiological state and individual species are small for N and P contents, but for K and Mg, Anthriscus sylvestris appears to be very different from the grasses and can accumulate these elements much more than them.

References

Greenwood D J, Lemaire G, Gosse G, Cruz P, Draycott A and Neeteson J J 1990 Decline in percentage N of C3 and C4 crops with increasing plant mass. Ann. Bot. 66, 425-436.

Lemaire G and Denoix A 1987 Croissance estivale en matiere seche de peuplements de fetuque e!evee et de dactyle dans !'Ouest de Ia France. Agonomie 7, 373-380.

Lemaire G and Salette J 1984 Relation entre dynamique de croissance et dynamique de prelevement d'azote pour un peuplement de graminees fourrageres. I. Etude de l'cffet du milieu. Agronomic 4, 423-430.

Lemaire G and Salette J 1984 Relation entre dynamique de croissance et dynamique de pn§Ievement d'azotc pour un peuplement de graminees fourrageres. II. Etude de Ia variabilite entre genotypes. Agronomic 4, 431-436.

Poorter H, Remkes C and Lambers H 1990 Carbon and nitrogen economy of 24 wild species differing in relative growth rate. Plant Physiol. 94, 621-627.

Salette J and Huche L 1991 Diagnostic de l'etat de nutrition d'une prairie par !'analyse du vegetal: Principes, mise en oeuvre, exemples. Fourrages 125, 3-18.

B

Nutritional characteristics and management of growth media

M.A. C. Fragoso and M.L. van Beusichem ( eds.) Optimization of plant nutrition 57-64, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-112

Potassium supplying capacity of northeastern Portuguese soils

ESTER A.C. PORTELA Department of Geosciences, University of Tras-os-Montes e Alto Douro, 5000 Vila Real, Portugal

Key words: illite, K release, K reserve, K uptake, mica, non-exchangeable K, potassium, ryegrass

Abstract

In Portugal, the response to K application is often inconsistent with the Egner-Riehm values for available K. This is partly related to high K reserves of some soils. Twenty surface soils representative of different parent materials from NE Portugal were studied to determine their K supplying capacity. Continuous cropping with perennial ryegrass permitted the assessment of the relative ability of soils to release non-exchangeable K. Soils were classified in the range of available K from medium to very high. However, their ability to supply K in the short and long term are very different. In some soils K status measured by plant growth does not fall appreciably, whilst others are rapidly exhausted, and 30% of them are very deficient in K. The supplying capacity varied both with the nature of the parent material and the degree of weathering. The soils deficient in K are those derived from basic rocks and those with more weathered clay minerals. This is the case of the soils with the largest content of organic matter where the dominant clay minerals were kaolinite and vermiculite. Soils that have the highest capacity for supplying K are highly micaceous, like those developed from mica schists, phyllites or river alluvium. In fact the amount of K released from non-exchangeable form is well correlated with the amount of illite in the clay fraction. Soil types and K buffer power coupled with available K must be taken into account when planning any application of K.

Introduction

Most of the methods used for determining avail­able K are based on the extraction of the exchangeable form. The method of Egner­Riehm, which is an approximate measure of exchangeable K, has been systematically used in Portugal. However it has been recognized that exchangeable K is an indifferent index of K availability for many soils (Alves, 1968; Alves et al., 1979; Cooke, 1982). One reason for this partial failure of the exchangeable K to account for uptake is the release of K from a non­exchangeable form during cropping.

The mineralogical composition of the soil has an enormous influence on K dynamics. Total soil K reserves are generally large, although the distribution of K forms differs from soil to soil as

a function of the dominant minerals present. Knowing soil K reserves and distribution of K forms coupled with mineralogical data should reveal more about the status of soil K in the major soil types of the region. Although the release of non-exchangeable K depends on the soil mineralogy there have been few successful attempts to relate it to mineralogical composition of soils.

The main objective of this study is the assess­ment of the K supplying power of soils of NE (Tnis-os-Montes region) Portugal. The relative ability of soils to release non-exchangeable K was evaluated by biological and chemical meth­ods. In addition, soils were examined by X ray diffraction to determine wh~ther differences could be established in their mineralogical composition.

58 Portela

Materials and methods

Soil analysis

Soil samples were taken from the 0-20-cm sur­face layer of 20 sites of NE Portugal. The physico-chemical characteristics of the soil sam­ples are given in Table 1. The soils represented the major soil types and were associated with a wide range of parent materials (Table 2) and climatic conditions. Most of the soils fall in the range of available K regarded as 'medium', 50-100 mg kg -l (Table 3). The soils received little or no K fertilizers up to the time of sampling. Only soil 9 had been limed, in the previous year.

Mineralogical analyses was done on the <2 J.Lm and 2-20 J.Lm soil fractions by X-ray diffraction. The methodology for preparing soil samples and estimation of mineral abundance is described by Silva (1983). Estimates of the amount of mineral specimens present should be regarded as semi-quantitative.

The 20 soil samples were each analyzed for several forms of soil K. The K concentration in soil solution was obtained by displacement procedure (Adams, 1974); exchangeable K was

Table 1. Chemical and physical properties of the soils

determined with 1N NH4 0Ac (pH 7); 1N boil­ing HN0 3 was used to determine the reserve of non-exchangeable K and HF digestion to extract total K (Knuden et a!., 1982); available K was extracted with NH4-lactate-acetic acid (pH 3.75). Analyses were made on air dried soils, K in the extracts was measured with an EEL flame photometer.

K buffer power was given by the slope of the line relating the change in exchangeable K (6 Kex) and the change of the Kin soil solution (6 Ks). Buffer curves of soils were obtained by adding, in the laboratory, graded doses of KCI to subsamples of 1 kg of soil. After treatment, the soils were maintained at a room temperature of 18°C ± 2oc during 21 days at 33 kPa moisture tension. At the end of this period soils were analyzed for exchangeable K and solution K. The resulting plots showed an approximately linear relationship, and buffer power was de­termined as 6 Kex/ 6 Ks.

Exhaustion experiment

Soils were air-dried and passed through a 2-mm sieve. One kg of each soil was placed in plastic

Soil Particle size (%) Organic pH 1:2.5 Exchangeable cations (cmol, kg- 1 )

matter >20 f.Lm 2-20 f.Lm <2f.Lm (%) H 20 KCl Ca Mg K Na Ac CECc

l 79.5 13.3 7.2 1.0 5.5 4.2 1.23 0.17 0.18 0.03 1.34 2.95 6 78.2 15.3 6.5 0.5 5.5 3.8 1.39 0.11 0.11 0.03 0.97 2.61

15 79.8 11.5 8.7 3.0 5.4 4.3 1.35 0.40 0.10 0.07 0.86 2.78 9 73.9 15.5 10.6 0.9 6.1 5.0 3.05 0.27 0.17 0.03 0.16 3.65

10 74.7 14.8 10.5 0.6 5.1 3.9 4.09 1.58 0.16 0.07 1.70 7.60 2 54.4 37.7 8.6 0.6 6.5 3.8 7.66 2.20 0.10 0.08 0.70 10.74 4 57.8 32.4 9.8 0.7 7.3 5.8 8.07 0.54 0.12 0.08 0.12 8.93 8 78.5 12.7 8.8 0.5 5.7 3.9 4.34 1.81 0.21 0.07 1.90 8.33

17 63.3 26.9 9.8 0.7 5.5 4.1 2.14 0.20 0.25 0.03 0.86 3.48 11 61.7 30.3 8.0 1.0 5.5 3.9 0.71 0.18 0.19 0.03 1.39 2.50 18 59.6 30.3 10.1 1.1 5.5 3.8 3.52 1.07 0.16 0.05 1.20 6.00 19 61.5 27.5 11.0 1.4 5.4 4.0 1.92 0.33 0.43 0.05 1.05 3.78 20 63.7 24.0 12.3 0.8 5.3 3.8 1.73 0.43 0.16 0.04 1.24 3.60 12 68.4 16.1 15.5 1.8 6.5 5.1 5.64 5.60 0.21 0.04 0.16 11.65 13 54.3 18.4 27.3 1.7 6.4 4.5 11.06 6.00 0.11 0.18 0.23 17.58 14 60.8 26.3 12.9 1.1 6.1 4.3 5.44 2.72 0.08 0.09 0.30 8.63 5 59.4 31.8 8.8 6.0 5.3 3.9 1.48 0.28 0.15 0.07 3.11 5.09

16 71.7 20.5 7.8 12.5 4.7 4.1 0.40 0.30 0.18 0.05 3.11 4.04 7 70.1 19.1 10.8 0.7 5.2 4.1 1.01 0.18 0.11 0.03 0.87 2.20 3 39.5 47.7 12.8 1.4 7.1 6.1 6.40 1.13 1.13 0.14 0.14 8.94

Ac- exchangeable acidity; CECe- effective cation exchange capacity.

K supplying capacity of NE Portuguese soils 59

Table 2. Soils, taxonomy, parent materials and mineralogy of clay and silt fractions

Soil Soil taxonomy Parent material Mineralogy

<2J.1m 2-20j.lm

K M v I-V Cl-V K Mi M v Cl 0

1 Carrazeda Humic cambisol Alkaline granites 4 4 2 6 Pereda Distric cambisol Alkaline granites 3 2 2 2 3 2 15 S. Andre Humic camhisol Alkaline granites 3 2 1 2 4 9 Malhadas Aplic aliso! Calcic granites 3 2 2 2 10 Miranda Distric cambisol Calcic granites 2 3 2 2 2 2 2 Carrascal Eutric cambisol Mica schists 3 2 1 2 3 2 4 Muxagata Aplic luvisol Mica schists 3 2 2 2 3 8Sendim Eutric leptosol Mica schists 3 3 2 2 2 17 V. Real Distric cambisol Mica schists 3 3 2 I 11 Vimioso Distric cambisol Phyllites 3 3 3 3 18 Alfandega Distric cambisol Phyllites 3 3 2 3 19 Curopos Distric cambisol Phyllites 2 2 2 2 2 2 20 Mirandela District cambisol Phyllites 2 3 2 2 2 12 Bragan<;a Chromic luvisol Peridotites 2 4 1 1 2 4 13 Izeda Chromic vertisol Amphibole schists 1 4 1 4 14 Frieira Chromic leptosol Amphibole schists 2 4 2 1 3 5 Reboredo Humic cambisol Schists and quartzites 3 3 2 2 2 16 Montalegre Humic cambisol Hornfelses 3 1 2" 2 1 2 2 7 Sanhoane Aplicalisol Coarse sediments 4 1 2 2 3 3 Vilari<;a Eutric fluvisol River alluvium 2 3 2 2 3 2

K- kaolinite; I- illite; M- montmorillonite; V- vermiculite; I-V- interlayered illite-vermiculite; Cl-V- interlayered chlorite-vermiculite; Cl- chlorite; 0- others 'Al-vermiculite 1 = <10%; 2 = 10-20%; 3 = 20-40%; 4 = 40-60%.

Table 3. Forms of potassium and buffer power of the soils

Soil Soil solution K Exchangeable K Buffer power K-reserve TotalK Available K20 (mgL ') (mg kg - 1) (mg kg - 1 ) (g kg -1) (mg kg - 1 )

1 22.0 70 2.3 336 47.2 123 6 7.0 43 2.1 1528 23.2 78

15 5.2 39 1.0 335 44.8 91 9 7.8 66 1.7 644 26.8 96

10 14.1 62 3.2 534 31.6 104 2 2.7 39 2.9 1455 29.0 65 4 4.2 47 2.3 632 27.2 80 8 8.2 82 2.0 2703 25.6 106

17 15.0 98 1.7 698 28.0 159 11 12.5 74 1.5 523 29.6 147 18 10.5 62 2.4 713 33.2 135 19 51.0 168 1.9 301 30.0 312 20 12.0 62 1.7 435 32.0 100 12 8.6 82 6.0 191 3.6 131 13 0.4 43 6.3 78 4.0 81 14 5.9 31 5.3 51 1.2 55 5 5.2 59 1.3 187 14.2 90

16 6.5 70 0.7 581 13.4 90 7 4.3 43 1.9 239 10.4 85 3 92.0 440 5.1 2067 34.6 676

60 Portela

pots and cropped in triplicate in the greenhouse. Those with pH(H20) ""'5.5 were limed with CaCO 3 . A basal nutrient solution (Portela, 1989) was thoroughly mixed with the soils before seeding. A 250 mg addition of K was made to half of the pots. Soils were seeded with Latium perenne, cv. 'Victorian', to obtain an approxi­mate plant density of 100 plants per pot. Dis­tilled water was added daily to bring the soils to 33 kPa moisture tension. Dry matter production was measured at intervals by cutting the grass 3 em above the soil. After each cutting, all pots received 100 mg N and 100 mg P, and those pots treated with K received 50 mg K. After a period of 5 months all pots were dressed with the same basal nutrient solution given at the beginning of the experiment. The number of cuttings varied according to the soil. The last harvest was taken when a response to K was first observed. Soils that never responded to K application were harvested on the 375th day of growth. Soils were separated from the roots and analyzed for ex­changeable K. Plant material was dried at 65°C for 48 hours, weighed, ground and digested in

nitric-percloric mixture. K was determined by flame photometry.

Results

Available K (Table 3) is well correlated with soil solution K(r = 0.978, p < 0.001) and with ex­changeable K(r=0.991, p<0.001). Thus the relative merits of any of these indexes arc the same. The buffer power of soils are generally low, particularly those with the highest content of organic matter.

The results of the exhaustion experiment are summarized in Table 4. The data listed refer to the pots which have not been fertilized with K. If K concentration in plants at the first harvest is taken as a measure of K supply, this is poorly correlated with available K (r = 0.629, p < 0.01 ), and this index only explains 40% of variation. The K concentration of the first cutting is, for some soils, lower than the level considered sufficient for optimum growth of ryegrass (16 g kg- 1 by Cook, 1982). Surprisingly, out of the 10

Table 4. Dry matter yield of Lolium perenne, K concentration of the first and the last cuttings, K uptake per kg of soil, fall in exchangeable K and release of non-exchangeable K

Soil No of Dry K concentration (g kg - 1 ) K uptake Fallin Release of non-exchangeable K" cuttings matter (mg kg - 1) exchangeable K

(g) 1st cut last cut (mgkg- 1 ) (mgkg- 1 ) (%)

1 7 27 18.6 4.6 282 55 227 80 6 7 27 11.6 9.1 424 16 408 96

15 3 16 8.4 4.7 97 16 81 84 9 6 23 13.0 5.7 228 35 193 85

10 15 49 21.4 4.9 728 39 689 95 2 15 52 16.2 17.1 941 16 925 98 4 15 50 16.8 8.6 982 16 966 98 8 15 52 37.7 12.5 1275 43 1232 97

17 8 31 24.5 7.2 499 70 429 86 11 8 32 24.4 10.2 618 39 579 94 18 15 55 32.8 10.2 1246 31 1215 98 19 8 34 31.2 12.4 752 105 647 86 20 15 54 17.0 6.7 884 35 849 96 12 6 26 18.2 4.9 278 39 239 86 13 2 10 5.1 3.6 43 4 39 91 14 2 10 7.4 3.7 51 4 47 92 5 2 11 5.6 3.4 50 8 42 84

16 2 12 4.3 2.5 41 16 25 61 7 2 11 6.3 5.0 61 8 53 87 3 15 54 40.2 16.3 1577 402 1175 75

"Non-exchangeable K was calculated from total K uptake by grass minus fall in exchangeable K.

soils classified as 'medium', 8 are K deficient, particularly soils 5, 7, 13, 14, 15 and 16, which showed a prompt response to K application. The remainder were maintained under cropping until 4 to 12 months of growth. In these soils the percentage of K released from non-exchangeable form is ?75%.

Figure 1 gives some typical curves of the cumulative uptake of K by ryegrass selected from soils that have the same initial exchange­able K. The rate of K uptake can be obtained from the slope of the curve when the cumulative

·c; "' 1200 .. D'l

1000 -"' D'l E 800

~ 600 L. D'l

"' >-. L. 400 >-. .0

"' 200 -"' .E a.

"' ~

K supplying capacity of NE Portuguese soils 61

K uptake is plotted against days of growth. The amount of K absorbed by the crop is approxi­mately linearly related with the growth period in all soils. So it is possible to fit regression equa­tions to the observed values (Table 5). If we consider the percentage of non-exchangeable K released from most of the soils (Table 4), it can be seen that the rate of K uptake is approximate­ly the same as the rate of K released. It is possible to distinguish two rates of K release in soils that have been maintained until the 15th cut (Fig. 1 ). One initial rate applies to 5 months of

Days of growth

Fig. 1. Cumulative K uptake by rycgrass in four soils, with similar initial exchangeable K.

Table 5. Regression equations and regression coefficients for the relation between cumulative K uptake and days of growth of the soils maintained under 3 to 12 months cropping

Soil No of Regression equation cuttings of initial observations"

1 7 y, = 1.54x, +45.8 6 6 y, = 2.85 x, -17.1 9 6 y, = 1.46 x, + 25.4

10 15 y 1 =5.06x, -62.2 2 15 y, = 4.43 x, + 30.3 4 15 y, = 3.91 x, + 17.6 8 15 y, =4.73x 1 +26.6

17 8 y, = 2.57x 1 + 56.0 11 8 y1 = 3.48x, + 12.7 18 15 y, = 6.43 x, -10.2 19 8 y, = 3.85 x, + 80.1 20 15 y, = 5.17 x, + 68.2 12 6 y, = 1.73 x, + 42.0 3 15 y, = 6.99 x, + 12.8

"This refers to the first 6 to 8 cuttings. "This refers to the last 8 cuttings.

2 r

0.963 0.979 0.973 0.978 0.982 0.988 0.982 0.977 0.983 0.963 0.978 0.988 0.943 0.956

Regression equations of last observationsb

Y2 = 0.75 x2 + 379 y, = 1.49 x, + 256 y, = 1.34 x, + 364 y, = 1.87 x, + 408

y2 = 1.67 x2 + 475

y2 = 1.25 X 2 + 419

y, = 2.34 x, + 491

r'

0.96 0.937 0.879 0.862

0.929

0.940

0.924

62 Portela

growth, and a second rate applies to the remain­ing period. In Table 5 these rates (mg kg - 1

day - 1) are represented by the angular coefficient of the two regression equations, y1 and y2

respectively. The first rate corresponds to a higher amount of K diffused from interlayer position, probably from K fixed close to the edges of clay mineral, and a second rate of release of K, which has to diffuse from the core of the micaceous minerals (Sinclair, 1979).

It is evident that some soils are more rapidly exhausted (soils 1, 6, 9, 12 and 17), but others (soils 2, 3, 4, 8, 18 and 20) continue to release K at an appreciable rate for a very long time. It seems that soils with an initial rate of release ~3.5 mg kg- 1 day- 1 could be considered as good K suppliers.

As shown in Table 2 the mineralogy of the clay and silt fractions depends on the nature of parent material from which the soil is derived. Similarly, the total K content of the soils is connected with the parent material and the mineralogy. Correlations were sought between the estimates of clay mineral constituents of the soils and the values for non-exchangeable K released by intensive cropping (Table 4). Since the mineralogical analyses of the soils are not quantitative estimates of mineral abundance, some simplification is necessary in order to relate the uptake of non-exchangeable K and miner-

1400 0 Ul

"';0'1 1200 ~

alogy. So, a mean value of the ranges given in the footnote of Table 2 was taken. The simplifi­cation allows some assessment of the variation between soils. The best correlation (r = 0.822, p < 0.001) was achieved between non-exchange­able K uptake and the percentage of illite in soil (Fig. 2). Adding a term for the percentage of mica from the silt fraction did not improve significantly the correlation. Although some studies demonstrated significant K release from the silt fraction during cropping (Doll et a!., 1965; Feigenbaum and Levy, 1977), the influence of this fraction is not evident from this study. Inspection of the deviations from the regression showed that a large portion of the variation was due to a few soils, namely soils 2, 4 and 10. When the results from these three soils were omitted the correlation was improved (r = 0.903, p < 0.001) and only 19% of the variation re­mained unaccounted for. As shown in Table 5, these three soils have an appreciable rate of K release, if only the percentage of illite is consid­ered. Table 2 shows that besides the presence of illites, soils 2, 4 and 10 also have certain amounts of montmorillonite. Rich (1968), suggested that the combined presence of illites and montmoril­lonite may promote the release of K to plants. Niederbudde and Fischer (1980) studied this phenomenon with pure specimens and reached the conclusion that the presence of smectites

a• • 3. 18

?1000 2• e4

::c 800 • ..S!! .10

20 .0

600 e19 _,,

Cl (:,! 0'1 .17 c 400 .6 Cl ;:::

12 1 u X 200 •• 9 (:,! c 13 15 ~14 0 z 0 .5 1.5 2 2.5 3 3.5 4

lllite,•t. Fig. 2. Relationship between the percentage of illite in soil and release of non-exchangeable K.

helped to maintain K in solution at a lower level. Thus the amount of K diffused from interlayer position was higher. The results from this study suggest that a good K supplier, illite, combined with a mineral that keeps low K in soil solution, montmorillonite, would enhance the K release from non-exchangeable form.

The K reserves (extraction with HN0 3 ) were plotted against the non-exchangeable K ab­sorbed by the crop. The correlation (r = 0.678, p < 0.001) between them revealed that the meth­od of boiling HN0 3 only explains 46% of var­iance among soils. In fact, there were some discrepancies. For example, in soils 6, 9, 15 and 16 the HN0 3 extracted a large amount of K when compared with K released by exhaustive cropping and underestimated non-exchangeable K in soils 4, 18 and 20.

Discussion

Soils from mountainous areas of NE Portugal, 1000 to 1450 mm of rainfall and classified as Humic cambisols (soils 1, 5, 15 and 16) show the highest degree of weathering. Gibsite, which is reported to be common, in upper and subsurface horizons of such soils (Silva, 1983), was not detected in the surface horizons of the soils studied. However, there is a high percentage of kaolinite and the presence of vermiculite and/ or At-vermiculite in the clay fraction, which indi­cates a high degree of weathering. These soils release small amounts of K, in spite of the high content of total K in the soils, particularly in soils 1 and 15 (Table 4). Obviously, in soils developed from alkaline granites the K in bear­ing minerals is very tightly held in feldspars and micas of coarser fractions. In addition, those with the highest percentage of organic matter (soils 5, 15 and 16) are very K deficient and gave a prompt response to K application. The low K buffer power of these soils might also be respon­sible for their limited K supply, due to their high susceptibility to K leaching. Fertilizer K recom­mendations in these soils should compensate for their low K buffer power and for the high precipitation occurring in these areas.

A second group of soils, developed on basic

K supplying capacity of NE Portuguese soils 63

rocks (soils 12, 13 and 14), have low K reserves due to low content of K bearing minerals. However, as they have higher K buffer power than any other group of soils, the K is less susceptible to leaching.

The largest and most representative group of soils in Tnis-os-Montes are Distric and Eutric cambisols. They are derived either from mica schists or phyllites, are less weathered, and have an appreciable percentage of micaceous minerals in both clay and silt fractions (soils 2, 4, 8, 11, 17, 18, 19 and 20). This group releases an extremely high percentage of K from non-ex­changeable form, with an initial rate of K release of 2.6 to 6.4 mg kg 1 day_,. The only Fluvisol studied (soil 3) behaved similarly to soil 18, however its rate of release was higher in the second stage. In soils derived from alluvial material, the K is less tightly held, and, there­fore, more readily available to plants (Binnie and Barber, 1964; Talibudeen and Dey, 1968).

It is difficult to use data obtained in pot trials for assessing the field situation since the volume of soil from which K is removed by plants is much smaller in pots. Besides, as shown by Seffens (1986), the test plant, ryegrass, has a great potential for exploiting soil K from the non-exchangeable pool. The high density of the root system is able to reduce K in the soil solution, promoting release of K from the inter­layers. Weber and Grimme (1986) suggested that one year of intensive cropping with ryegrass was approximately equivalent to 10-15 years of nor­mal cropping in the field. However the results of the pot experiment can be used as a guide in assessing the field situation.

Since available K does not fully describe K supplying power of the soils, soil types and K buffer power must also be taken into account when planning any application of K to the soil in the interest of fertilizer efficiency and economy.

Acknowledgements

PROCALFER-AID provided some financial support for this research. We wish to thank J M V Silva and J L Ahlrichs in the identification of clay minerals.

64 K supplying capacity of NE Portuguese soils

References

Adams F 1974 Soil solution. In The Plant Root and its Environment. Ed. E W Carson. pp 441-482. University Press of Virginia, Charlottesville.

Alves J A 1968 Fertiliza.;ao do trigo. Revista Agron6mica 31 (Tomas I e II): 1-10.

Alves J A, Nogueira M G B, Santos AD and Tavares M M S 1979 Fertiliza.;ao Mineral e Correc.;ao do Solo. I. Fer­tiliza.;ao Mineral. Direc.;ao Geral dos Servi<;os Agricolas­III Plano de Fomento (1968-1973), Lisboa.

Binnie R R and Barber S A 1964 Contrasting release characteristics of potassium in alluvial and associated upland soils of Indiana. Soil Sci. Soc. Am. Proc. 28, 387-390.

Cooke G W 1982 Fertilizing for Maximum Yield, 3rd ed. Macmillan, New York, 465 p.

Doll E C, Mortland M M, Lawton K and Ellis B G 1965 Release of potassium from soil fractions during cropping. Soil Sci. Soc. Am. Proc. 29, 699-702.

Feigenbaum S and Levy R 1977 Potassium release in some saline soils of Israel. Geoderma 19, 159-169.

Knuden D Peterson G A and Pratt P F 1982 Lithium, sodium ;nd potassium. In Methods of Soil Analysis, Part 2, 2nd ed. Eds. A L Page et al. pp 225-246. American Society of Agronomy, Madison.

Niederbudde E A and Fischer W R 1980 Clay mineral transformations in soils as influenced by potassium release from biotite. Soil Sci. 130, 225-231.

Portela E A C 1989 A valia<;ao da Disponibilidade de Potassio em Solos de Tras-os-Montes. Universidade de Tras-os­Montes e Alto-Douro, Vila Real. 220 p.

Rich C I 1968 Mineralogy of soil potassium. In Role of Potassium in Agriculture. Eds. V J Kilmer, S E Younts and N C Brady. pp 79-108. American Society of Agronomy, Madison.

Silva J M V 1983 Estudo mineral6gico da argila e do limo de solos derivados de granitos e rochas basicas da regiao de Tras-os-Montes. Garcia de Orta (Estudos Agron6micos) 10, 27-36.

Sinclair A H 1979 Availability of potassium to rye grass from Scottish soils. II. Uptake of initially nonexchangeble potas­sium. J. Soil Sci. 30, 775-783.

Steffens D 1986 Root system and potassium exploitation. In Nutrient Balances and the Need for Potassium. pp 97-108. International Potash Institute, Berne, Switzerland.

Talibudeen 0 and Dey S K 1968 Potassium reserves in British soil. II. Soils from different parent materials. J. Agric. Sci. Camb. 71, 405-411.

Weber M and Grimme H 1986 The K- supplying capacity of soils developed from loess before and after intensive cropping with ryegrass. pp 1006-1007. XIII Congress of International Soil Science, Hamburg.

Reprinted from Plant and Soi/154: 13-20, 1993.

M.A. C. Fragoso and M.L. van Beusichem ( eds.) Optimization of" plant nutrition 65-72, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-008

Nutritional disorders between potassium, magnesium, calcium, and phosphorus in soil

S.T. JAKOBSEN Department of Agricultural Sciences, Royal Veterinary and Agricultural University, Agrovej 10, DK 2630 Taastrup, Denmark

Key words: activity ratio, ammonium phosphate, chloride, maize, sandy loam, soil extracts, sulphate, superphosphate, unbalanced nutrition

Abstract

A new soil testing procedure has been used to demonstrate the effect of an overfertilization by potassium during the preceding years. The total concentration of cations was governed by the amount of soluble anions and the proportion between the different cations was dependent on exchange reactions and is described by activity ratio. High activity ratio between potassium and calcium induced Ca-defi.ciency, which resulted in a restricted root functioning shown by periodic decreases of nutrient uptake rates and plant growth rate. P-defi.ciency restricted root growth, but although ammonium phosphate was most effective to increase P-concentration in soil extracts and P-absorption by plants, ordinary superphosphate gave the highest yield and the best utilization of the absorbed phosphorus, magnesium, and calcium.

Introduction

Transport of cations in soils is accomplished by mass flow or diffusion of dissolved ions in the soil solution. The rate of transport and of nu­trient uptake by plants is dependent on the activity of the nutritive ions in the soil solution and on the relation between the dissolved ions (Hansen, 1972).

The total amounts of dissolved cations are governed by the amounts of anions, and the majority of these are made up by nitrate, chlo­ride, sulphate, and hydrogen carbonate of which chloride and sulphate are the most stable during the growing period (Jakobsen, 1992b).

The proportion between activity of cations is maintained by exchange reactions between cat­ions in the soil solution and exchangeable cations on the surfaces of soil colloids. Within a consid­erable range of activities the proportion between the activity of different cations has been de-

scribed by a straight line equation (Jakobsen, 1992a):

aK Kex --=A+C-­vaCa Caex

where aK and aCa are the activities and Kex and Caex are the exchangeable amounts of potassium and calcium, respectively. A and C are con­stants.

Formation of ion pairs and complexes, and precipitation of compounds reduce activities of phosphate and calcium in the soil solution (Adams, 1971). The relation between phosphate and calcium is complicated as these ions also influence each other in uptake and translocation to the growing points in roots (Jakobsen, 1979). If calcium ions are not constantly translocated to the growing point, its biological functioning will be destroyed (Marinos, 1962).

Potassium in soils is leached very little where

66 Jakobsen

application and removal by the crop is balanced (Hansen and Petersen, 1975). If year by year more potassium is applied than removed, it accumulates in the soil. Activity ratio aK/-vaCa increases and uptake of calcium and magnesium reduces which may result in deficiencies.

Potassium is often applied as muriate of pot­ash. The chloride ions increase the solubility of cations during the year of application, conse­quently, a potential deficiency of the divalent cations is not recognized. As chloride ions are easily leached, when percolation exceeds evapo­ration, malnutrition may develop in the follow­ing years resulting in reduced yield and quality of the crop.

The purpose of this paper is to show the effect of an overfertilization by potassium on uptake and utilization of magnesium, calcium, and phos­phorus.

Materials and methods

The soil and plant samples were taken from a second year field experiment. The soil was a phosphorus deficient sandy loam with a pH (H2 0) = 6.4 The crop was Zea mays L. in both years. The first year treatments included applica­tion of 0, 75, and 150 kg ha -J as muriate of potash. Each K-treatment was repeated 18 times yielding a total of 54 plots. About 75 kg K ha -I were removed by the crop. The activity ratio aK/-vaCa measured in soil extracts before sow­ing the second year crop increased from 2.4 in treatments without K-application to 4.5 and 6.2 (J.Lmol/kg soil) 112 for application of 75 and 150 kg ha -I, respectively.

In the second year these treatments were combined with 3 P-treatments: without phos­phate, and 45 kg P ha- 1 as either ammonium phosphate or ordinary superphosphate. Each plot was split up into 2 subplots. One of the subplots did not receive potassium. The second was fertilized by 75 kg K ha-l as muriate of potash. Totally the experiment consisted of 108 plots. During June and July the crop was in­spected frequently and occurrence of visual de­ficiency symptoms was noted.

Germinating seedlings with roots and adhering soil were sampled daily for eight days. Each day

10 plants from each of 36 plots = 12 from each P-treatment were selected. The adhering soil and the roots were extracted by deionized water (1 part of soil to 2 parts of water). Activities of potassium and calcium were determined by ion­selective electrodes, and concentration of phos­phate in the same water extract was determined colorimetrically by the molybdenum blue meth­od. A detailed description of the extraction and analytical method is given elsewhere (Jakobsen, 1992a).

The tops of plants were dried, weighed, and analyzed for content of potassium, magnesium, and calcium by flame photometry and phosphor­us by the vanadate molybdenum method after incineration at 500°C.

From July 6 to July 16, 5 plants from each of 36 plots were sampled daily. After drying, weighing, and chemical analyses the mean up­take of nutrients from 12 plots of each of the 3 P-treatments was calculated.

Results

Figure 1 shows results of the analyses of soil extracts. The mean decrease of phosphate until June 2 is supposed to be caused by fixation to the soil minerals. The very low concentration of phosphate on June 2 in the treatment without application of phosphate coincided with the appearance of P-deficiency symptoms in the plants on that date. The lesser fixation of phos­phate in treatments with ammonium phosphate was reflected by an increased uptake of phos­phorus (Fig. 2a).

The activity of calcium, Figure 1b, was highest in treatments fertilized with superphosphate, which contains 2 moles of calcium sulphate per 1 mole of calcium phosphate. An opposite effect on concentration of phosphate and activity of calcium is demonstrated.

On June 7, P-deficiency symptoms, character­ized by a purple to violet colouring of the leaves, were obvious and Ca-deficiency symptoms characterized by deformed, curled leaves were developing. The following days the Ca-deficiency appeared in the new leaves, while P-deficiency decreased. From June 10, Ca-deficiency disap-

o,4 a

'5 en 0,3 ,­

I

Ol ..l<:: 0 0,2

E E a.. 0,1

0

0 30/5

0,4 b ·c; en

or;- 0,3

~ ~ 0,2

E ~ 0,1

ro 0

30/5

31/5

31/5

Interactions between cations and phosphorus 67

1/6 2/6 3/6 4{6 5/6 6/6

1/6 2/6 3/6 4/6 5/6 6/6 Date

~ WithoutP B Ammonium phosphate Superphosphate

Fig. 1. Concentration of phosphorus a and activity of calcium bin extracts of rhizosphere soil, respectively, sampled daily from 3 P-treatments.

peared and P-deficiency symptoms increased again. On June 15, P-deficiency was evident and Ca-deficiency symptoms were developing again.

Notes on visual judgments were taken until mid-July. As a conclusion it was written: In many plots alternating phosphorus and calcium deficiency symptoms were observed. Plots with­out P-application showed a constant phosphorus deficiency with varying intensity. The symptoms were most evident in sub plots receiving potas­sium chloride.

Figure 3 shows curves for uptake of phosphor­us and potassium from July 6 to July 16 and for the same period. Figure 4 shows uptake of calcium and yield curves. Ammonium phosphate still increased the absorption of phosphorus more than superphosphate. On the other hand superphosphate increased the uptake of potas­sium more than ammonium phosphate. The difference between the effect of the two fertiliz-

ers on uptake of nitrogen, magnesium, and calcium was insignificant.

The maize crop was harvested in October. Relations between nutrient uptake and yield of dry matter arc shown as yield/utilization curves in Figures 5 and 6. The amount of absorbed nutrient is used as abscissa and the corre­sponding yield as ordinate. A high yield per unit absorbed nutrient, i.e. a good utilization of that nutrient, indicates that the nutrient is in mini­mum. In Figures 5 and 6 the three points at every curve represent increased application of potassium the preceding year from 0 to 75 to 150 kg K ha - 1 . The direction of the increases is indicated by arrows.

As long as both yield and nutrient uptake increase, the increased yield might be caused by the increased nutrient absorption. If the yield curve turns off, as it does for the highest amounts of potassium in Figure Sa, another

68 Jakobsen

co ..c

3

0 2 E "'0 Q) ..c 0 en -{61 ll..

0 30/5

16

'7 12 co ..c C) ~ ..... Q) :1= co E ~ Cl 4

31/5 1/6 2/6 3/6

b -Without P

•••• Ammonium phosphate

""" Superphosphate

4/6 5/6 6/6 7/6

0+---.---.---.---.---.---.---.-~ 30/5 31/5 1/6 2/6 3/6 4/6 5/6 6/6 7/6

Date Fig. 2. Absorbed amounts of phosphorus a and dry matter yield of maize seedlings b, respectively, sampled daily from 3 ?-treatments.

parameter is limiting. When replacing potassium at the abscissa by another plant nutrient, it is possible to show the effect on both crop uptake and utilization of that particular nutrient, thus demonstrating antagonism between plant nutri­ents.

Figure 5 shows the utiliziation of potassium Sa, magnesium 5b, and calcium 5c for treatments receiving 75 kg K ha - 1 in the experimental year. The potassium curves turn off from the minimum position where more than 75 kg K ha - 1 were applied the preceding year. The magnesium and calcium curves have the opposite course. The two nutrients became limiting where the soil was

200

-'m 160 ..c 0 E120

"'0 Q)

..c 0 (/) 80 ..c co ll..

co ..c

40

900

osoo E

a

/ ____ ... /

--------·----... ~':'-,'/!.~.'. '''

b

-WithoutP

···Ammonium phosphate

· · · · · · Superphosphate

/ /

/ /

/ I

I I

I I

I . /---- .· / .

0+--.--,-~r--.--.--.--.--.--,-~ 6/7 7/7 8/7 9/7 10/7 11/7 12/7 13/7 14/7 15/7 16/7

Date

Fig. 3. Absorbed amounts of phosphorus a and potassium b, respectively, measured in plants sampled daily from July 6 to July 16 from 3 ?-treatments.

overfertilized with potassium the year before i.e. where the activity ratio between potassium and calcium in soil extracts became too high. This indicates antagonism between the effect of resid­ual potassium and uptake and utilization of magnesium and calcium.

Figure 6 a and b show yield/utilization curves for phosphorus without and with application of 75 kg K ha-l in the experimental year, respec­tively. It is clearly demonstrated that overfertili­zation by potassium the preceding year also was antagonistic to uptake and utilization of phos­phorus, and this effect was intensified by a supplementary application of muriate of potash in the experimental year, evidently because of an

200

~ro 1so .c. 0 E -g1oo -e 0 (/) .0

: 50

0

Cll .c

15

~ 10 .c.

ro 'lil E ~ 5 0

b

-WithoutP

·--·Ammonium phosphate

...... Superphosphate

0~-r--~~--~~--~~--~-.r-• W W W ~ 1~1W1~1~1~1W1W

Date Fig. 4. Absorbed amounts of calcium a and dry matter yield b of plants sampled daily from July 6 to July 16 from 3 ?-treatments.

increased P-deficiency in treatments without ap­plication of phosphate and an increased Ca-dc­ficiency in treatments with application of phos­phate.

Application of superphosphate caused a higher yield and a better utilization of absorbed mag­nesium, calcium, and phosphorus as superphos­phate also contains calcium sulphate. Therefore, it is assumed that although ammonium phos­phate gave a better starting effect, see Figure 2, the gypsum in superphosphate had an effect at harvest time. It might then be reasonable to imagine that calcium deficiency restricted the root function and plant growth up to harvest time (Jakobsen, 1993b).

Interactions between cations and phosphorus 69

Discussion

Although most of the results show an interaction between Ca-deficiency and P-deficiency, the main reason for Ca-deficiency was caused by an overapplication of potassium in preceding years. This is demonstrated by the yield/utilization curves in Figure 5. The effect on Mg-deficiency may be important for the quality of the crop but magnesium retranslocates in plants and is not directly involved in functioning of roots.

Calcium is essential for the functioning of roots. From the work of Marschner (1974) and others follows that calcium is not retranslocated from top to roots in plants. Marinos (1962) showed that new plant cells in growing points were destroyed if they were not supplied with calcium. From these statements follows that calcium must be taken up from the soil surround­ing the root tips and translocated to the growing points in the roots without interruption (Jakob­sen, 1979).

An uninterrupted uptake and translocation of calcium to the growing points of roots is fur­nished by a simultaneous translocation of phos­phate, which may take place by formation of ion pairs or complexes between calcium and phos­phate before or after absorption. As proposed by Jakobsen (1979), a promotion of the transloca­tion of Ca-ions would be important if hydrogen carbonate developed by root respiration forms ion pairs or complexes with calcium. Transloca­tion of hydrogen carbonate from the centre to the surface of the root then attempts to keep the calcium ion away from the growing point.

Uptake and translocation of phosphate have the opposite direction, and owing to formation of complexes between calcium and phosphate, the translocation of calcium to the growing point is secured as long as phosphate is taken up and translocated. But a high concentration of calcium relative to that of phosphate outside the roots might counteract a temporary uptake of phos­phate (Jakobsen, 1979). Such an effect would explain the observed alternating visual deficiency symptoms of calcium and phosphorus in June and July.

A fall in amount of absorbed phosphorus and a stagnation of growth from June 6 to June 7, see Figure 2a, was also found for the nutrients

70 Jakobsen

I ctl

..c: en

..:.::

..c: .... a:> .... .... ctl

160

150

140

130

160

150

E 140 > ....

"C ..... 0

"C a:>

>

130

160

150

140

130

a

")-----.-----

~~~~--~--~~--~--

~

2200 2400 2600 2800 3000 3200

1500

c

K in crops, equivalents ha - 1

Without P

lt, 1 e •llr----11 P in NH4 H2 P04, 45 kg ha-

A.-- -A. P in Superphosphate, 45 kg ha- 1

1700 1900 2100

Mg in crops, equivalents ha - 1

/ .. -.. I\

/ /

/

/ilc-­/ \

•

~\-r---.--~-r-----.----,-1800 2000 2200 2400 2600 2800

Ca in crops, equivalents ha - 1

Fig. 5. Yield/utilization curves for potassium a, magnesium b, and calcium c. Absorption as function of increasing activity ratio between potassium and calcium (indicated by arrows) and after fertilization by 75 kg K ha _, as potassium chloride.

Interactions between cations and phosphorus 71

160 a

150 • -f--.

' 140 )\

I C'C • ...c: C'l 130

Without KCl, 1982 ..:.:::

~ -= .... Cl:l

1700 1800 1900 - 2000 2100 2200 2300 -C'C P in crops, equivalents ha - 1 E > 160 b

---------....

"'C / - / 0 / I

"'C / / Ci) 150 / / >- / //(

/

it/ ¥

140 --( .. •

130 75 kg ha- 1 K in KCl, 1982

~ 1700 1800 1900 2000 2100 2200 2300

p in crops, equivalents ha - 1

•------• Without P

A.-- -A. P in Superphosphate, 45 kg ha-

Fig. 6. Yield/utilization curves for phosphorus. Absorption as function of increasing activity ratio between potassium and calcium (indicated by arrows), in treatments without a, and in treatments with application of potassium chloride, b.

nitrogen, magnesium and calcium. During the same 24 hours the absorption of potassium and growth of the seedlings stagnated, see Figure 2b. The general decrease or stagnation of nutrient uptake indicates that the root function was affected which could be caused by a temporary lack of calcium absorption and translocation to the growing points (Jakobsen, 1979).

The growth rate and nutrient uptake rates were slow until July 10. In the journal of the experiment was noted: On July 6 many new

roots were developed from the basis of the stems. Symptoms of P-deficiency were untypical and Ca-dcficiency was observed in many plants. On July 11, Ca-deficiency symptoms were more distinct than the day before and the new roots developed on July 6 were brownish. These roots were longer in plots fertilized with phosphate than without phosphate. On July 12, P-deficiency symptoms in plots without phosphate application were typical again. These descriptions of plants agree very well with the shape of curves shown

72 Interactions between cations and phosphorus

in Figures 3 and 4. Especially in the treatments with ammonium phosphate the growth rate and nutrient uptake rates were reduced during the days of Ca-deficiency. Note: The visible de­ficiency symptoms are delayed a little in occur­rence relative to the effect shown by the plant analyses.

An overfertilization with potassium is more common than generally assumed. It is difficult to repair as potassium is not leached (Hansen and Petersen, 1975) until the activity ratio, aK/ vaCa, is too high (Jakobsen, 1993a). By applica­tion of chloride the total concentration of cations in the soil solution increases, whereby the uptake of calcium and magnesium sometimes increases. That was the case in the first year experiment.

In the second year experiment the application of potassium chloride had the opposite effect and that was caused by a restricted diffusion of phosphate in soil (Fig. 1) and uptake of phos­phorus by plants (Fig. 2). Phosphorus is essential for development of roots and an early develop­ment of roots is essential for the diffusion of phosphate to the plant roots. Precipitation of calcium phosphate and formation of ion pairs and complexes between calcium and phosphate restricted diffusion and absorption of phosphate by plants.

The total amounts of exchangeable cations at the soil colloids are restricted by the exchange­able capacity, which is nearly a constant. There­fore, it is difficult to decrease the nominator, aK, and to increase the denominator, vaca, substan­tially. A temporary increase of calcium in the soil solution· is carried out by application of soluble salts. Although application of potassium chloride increases the activity of calcium, because of exchange reactions, it also increases the activity ratio, aK/vaCa, and thereby the potential risk of Ca-deficiency. If the soil is low in available phosphate a P-deficiency will be­come more obvious.

By fertilizing with ordinary superphosphate both calcium phosphate and calcium sulphate are

applied. In spite of the fact that superphosphate had a lesser effect on the early uptake of phos­phorus than ammonium phosphate, superphos­phate is recommended when the antagonistic effect of potassium is pronounced.

Acknowledgements

The author would like to thank W H Eppen­dorfer for his professional criticism of the manu­script and together with Jette Youdcn for criti­cism of the English translation.

References

Adams F 1971 Ionic concentrations and activities in soil solutions. Soil Sci. Soc. Am. Proc. 35, 420-426.

Hansen E M 1972 Studies on the chemical composition of isolated soil solution and the cation absorption by plants. Plant and Soil 37, 589-607.

Hansen L and Petersen E F 1975 Losses of nutrients by leaching in agricultural production. Tidsskr. Planteavl. 79, 670-688.

Jakobsen S T 1979 Interaction between phosphate and calcium in nutrient uptake by plant roots. Commun. Soil Sci. Plant Anal. 10, 141-152.

Jakobsen S T 1992a Interaction between plant nutrients. I. Theory and Analytical Procedures. Acta Agric. Scand., Sect. B, Soil and Plant Sci. 42, 208-212.

Jakobsen S T 1992b Interaction between plant nutrients. II. Effect of Chloride on Activities of Cations in Soil Solution and on Nutrient Uptake by Plants. Act Agric. Scand., Sect. B, Soil and Plant Sci. 42, 213-217.

Jakobsen S T 1993a Interaction between plant nutrients. III. Antagonism between Potassium, Magnesium and Calcium. Acta Agric. Scand., Sect. B, Soil and Plant Sci. 43, l-5.

Jakobsen S T 1993b Interaction between plant nutrients. IV. Antagonism between Potassium, Magnesium and Calcium. Acta Agric. Scand., Sect. B, Soil and Plant Sci. 43, 6-10.

Marinos N G 1962 Studies on submicroscopic aspects of mineral deficiencies. I. Calcium deficiency in the shoot apex of barley. Am. J. Bot. 834-849.

Marschner H and Richter Ch 1974 Calcium translocation in roots of maize and bean seedlings. Plant and Soil 40, 193-210.

Reprinted from Plant and Soi/154: 21-28, 1993.

M.A. C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 73-76. 1993. © 1993 K!ttwer Academic Publishers. PLSO IAOPN-087

The influence of the soil phosphate capacity factor on soil and plant phosphorus critical levels of different vegetables

ROBERTO F. NOVAIS 1 , JULIO C.L. NEVES 1 , NAIRAM F. BARROS 1 ,

VINCENTE W.D. CASALI 2 and ANTONIO S. FABRES3

1 Departmento de Solos, Universidade Federal de Viqosa, 36570-000, Viqosa, MG, Brazil, 2 Departamento de Fitotecnia, Universidade Federal de Viqosa, 36570-000, Vi9osa, MG, Brazil and 3Cenibra Florestal, 36163-000, Bela Oriente, MG, Brazil

Key words: available phosphorus, phosphate buffering capacity, plant critical P level, soil critical P level, tropical soils

Abstract

A glasshouse experiment was carried out to study the effect of soil characteristics on soil and plant critical levels of P for the growth of tomato (Lycopersicum esculentum) and peruvian carrot (Arracacia xanthorrhiza), in a first cultivation, and cucumber (Cucumis sativus) or lettuce (Lactuca sativa) in a second cultivation. Samples of six soils received six rates of soluble P (0, 100, 200, 450, 700, and 1000 mg of P dm - 3 of soil). Soil available P was determined by Mehlich-1 and Bray-1 extractants. Plant tops were harvested, oven dried, and total-P was determined. For lettuce, leaf tissue inorganic P (PJ, organic P and total acid soluble P (P,J were determined. Soil and plant P critical levels varied for species and soils, and both varied inversely with the phosphate capacity factor (PCF). Critical concentrations of P,s and Pi varied inversely with soil characteristics associated with soil P adsorption and accounted for the correlation verified between plant critical level and PCF.

Introduction

The soil P available to plant growth is variable with the extractant solution and with soil charac­teristics related to the phosphate capacity factor (PCF), such as soil clay content, and soil P maximum adsorption (Novais and Kamprath, 1978). Hence, an adequate evaluation of soil P-status has to include both the extractable value and a measurement of the PCF.

It has been widely observed that the P critical level varies among soils and this variation is closely related to the PCF (Freire et a!., 1979). Further, Muniz et a!. (1985) observed that the P critical levels in soybean plants were also sig­nificantly dependent on soil characteristics re­lated to PCF. Later on, Fabres et a!. (1987) observed that the variations of the plant critical level in lettuce cultivated in different soils were

significantly correlated with PCF. They observed that this relationship was a function of the differential accumulation of inorganic P in the plant tissues depending on the characteristics of the soil used.

This work had as objective to evaluate the effect of the PCF on the soil and plant critical levels for the growth of four vegetables in different soils.

Material and methods

Superficial soil samples (0-20 em) with different PCF and very low available P (Table 1) were throughly mixed with increasing rates of soluble P (0, 100, 200, 450, 700 and 1000 mg of P dm - 3

of soil), applied as KH 2P04 and NH4 H 2P0 4 ,

and left incubating for seven months. They were

74 Novais et al.

Table 1. Chemical and physical characteristics of the soil samples used

Soil pH P' PMA2 I' PBC' AE' K" EP 7 FC8

(H,O)

LE-I 5.8 8 0.430 6.48 119 0.059 O.D75 45 28 LV-I 4.4 2 0.524 7.12 150 0.135 0.158 33 24 LV-2 4.5 7 0.621 6.67 109 0.165 0.165 18 23 LV-3 4.2 5 1.110 7.97 149 0.270 0.290 11 31 LE-2 5.0 1 1.497 7.54 400 1.040 0.620 5 30 LE-3 4.9 4 1.480 8.46 230 0.553 0.494 4 33

1Mehlich-1 extractant (JJ-g Pg 1 soil); 'p maximum adsorption (mgPg- 1 soil); 'Intensity factor as+ pCa + pH,PO,; 'Phosphate buffering capacity (moles of P.18- 8 g soil per unit of phosphate potential; 5P adsorption energy in mL JJ-g- 1 ; "Freundlich K constant; 'Equilibrium P (JJ-gL -I) after shaking 50 JJ-gPmL 1 of 0.01 M CaCI, solution for one hour, with the soil sample in a 1:10 soil: solution ratio; 'Water field capacity (%, in volume).

then limed to raise their pH(H20) to 6.0 and received a basic fertilization with all nutrients minus P.

In plastic pots containing 4.5 dm 3 of soil two plants of tomato (Lycopersicum esculentum) or peruvian carrot (Arracacia xanthorrhyza) were grown in a first cultivation, and cucumber (Cucumis sativus), or lettuce (Lactuca sativa in a second one, in the same substract used previous­ly. Lime and basic fertilization (minus P) were applied after each cultivation.

Nitrogen was applied as NH 4N0 3 solution to the soil surface during the plant growth period.

After 25 days of plant growth for tomato and cucumber, and 75 days for the other vegetables, the shoots were harvested, oven-dried, and ana­lysed for total P concentration. For lettuce, fresh mature leaf tissues were also sampled to de­termine inorganic P, organic P, and total P soluble in a 0.2 N HClO 4 solution, according to the procedure described by Hogue et a!. (1970); total P in oven-dried leaf tissues was also de­termined (Fabres eta!., 1987).

Regression equations between plant dry

weight and rate of P were fitted and the P critical rates corresponding to 90% of the estimated maximum growth, for each vegetable and soil, were determined. Soil P critical level, for each vegetable in every soil, was estimated by insert­ing the P critical rate values in the equations relating available soil P with P rate applied. Plant critical level for each vegetable in every soil was established by inserting P critical rate in the equations relating plant P with P rate.

Correlations between critical levels and soil PCF related characteristics were obtained.

Results

The soil P critical levels (S.CL) for the ex­tractants tested presented a small variation among species but a large one among soils (Table 2). Cucumber, with the greatest maxi­mum growth, presented the lowest S.CL, where­as lettuce, with the smallest one, had the highest S.CL.

The variation of the S.CL among soils can not

Table 2. Soil P critical levels (JJ-gPg- 1 soil) by Mehlich-1 (M) and Bray-1(B) extractants, for 90% of the estimated maximum growth (MG ), in g of dry weight per pot, of different vegetables in each of the soil samples used

Soil Tomato Peruvian carrot Cucumber Lettuce

M B MG M B MG M B MG M B MG

LE-I 254 246 12.0 276 268 9.8 219 213 12.5 290 282 5.7 LV-1 242 310 10.7 229 293 7.0 176 224 10.6 301 387 4.9 LV-2 313 339 12.2 237 254 6.3 209 224 13.8 293 317 5.2 LV-3 253 305 12.4 269 325 9.0 170 203 16.1 269 325 4.9 LE-2 81 49 14.4 72 47 6.8 75 49 14.4 132 86 3.4 LE-3 243 216 13.3 189 168 5.9 169 150 16.9 269 239 4.6 Mean 231 244 12.5 212 226 7.5 170 177 14.1 259 273 4.8

be ascribed to vanatwn of differential plant growth at this optimum condition in the different soils (Table 2), since this variation was minimum and not related to the estimated S.CL values. However, S.CL was highly correlated (negat­ively) with soil P characteristics closely related with PCF, as phosphate buffering capacity (Table 3). This result indicates that for a given P extractant solution the establishment of an unique S.CL for all kind of soils, with a broad PCF variation, can lead to great mistakes in the interpretation of soil P analysis. The error will be smaller if an unique S.CL is considered for any of the vegetables tested for a given soil, though some variation among species can be significant in some comparison as in lettuce versus cucumber (Table 2).

The non-significant correlation between soil clay content and S.CL was probably due to the great variation in clay quality of the soils.

Similarly to the S.CL results, the plant critical

Plant critical P levels 75

levels varied among species and more intensively among soils (Table 4). This variation was also significantly correlated with soil characteristics related to PCF (Table 5). For example, tomato plants presented a critical level of 0.34% in that soil with the highest phosphate buffering capaci­ty (PBC) - soil LE-2, and a critical level as high as 0.60% in the second lowest PBC soil- LE 1 (Table 1).

When P of lettuce plants was fractionated (Fabres et a!., 1987), the critical inorganic P levels were negatively and significantly corre­lated with different soil characteristics related to the soil PCF. This indicated that in those soils with lower PCF, there is much more inorganic P in the plant tissue than in those higher PCF soils. Higher levels of P in soil solution (intensity factor) under the former situation seemed to cause a higher P influx and some 'luxury' P accumulation as inorganic form in the plants (Bieleski and Ferguson, 1983).

Table 3. Linear correlation coefficients between soil samples characteristics and the soil P critical levels as extracted by Mehlich-1 (M) and Bray-1 (B) for the growth of different vegetables

Soil Tomato Peruvian carrot Cucumber Lettuce characteristics'

M B M B M B M B

Clay -0.48 -0.68 -0.63 -0.76 -0.52 -0.75 -0.57 -0.76* PMA -0.61 -0.67 -0.71 -0.70 -0.76* -0.81 * -0.70 -0.72 PBC -0.95** -0.95** -0.96** -0.92** -0.96** -0.99** -0.95** -0.90* A.E. -0.94** -0.93** -0.95** -0.90* -0.94** -0.96** -0.99** -0.91* K -0.77 -0.81 * -0.87* -0.85* -0.88* -0.92** -0.83* -0.82* E.P. 0.37 0.41 0.60 0.54 0.63 0.62 0.55 0.53

'For identification, see foot-notes in Table 1; *, **-significant at 5 and 1% levels, respectively.

Table 4. Plant total P critical levels for 90% of the estimated maximum growth of different vegetables in each of the soil samples used

Soil

LE-1 LV-1 LV-2 LV-3 LE-2 LE-3 Mean

'Fabres et al. (1987).

Tomato

(%)

0.60 0.57 0.53 0.47 0.34 0.51 0.50

Peruvian carrot

0.52 0.42 0.45 0.40 0.29 0.32 0.40

Cucumber Lettuce'

0.65 0.67 0.60 0.61 0.58 0.53 0.52 0.52 0.49 0.47 0.48 0.40 0.55 0.53

76 Plant critical P levels

Table 5. Linear correlation coefficient between soil sample characteristics and the plant total P critical levels for the growth of different vegetables

Soil Tomato Peruvian Cucumber Lettuce' characteristics I carrot

Clay -0.56 -0.75 -0.79* -0.83* PMA -0.80* -0.93** -0.97** -0.91 * PBC -0.88* -0.85* -0.69 -0.58 A.E. -0.91* -0.74 -0.58 -0.45 K -0.87* -0.96** -0.91 * -0.84* E.P. 0.77 0.87* 0.97** 0.96**

1 For identification, see foot-notes in Table I; 'Fabres et al. ( 1987); *, * * - significant at 5 and 1% levels, respectively.

Discussion

As the soil PCF increases, the exhaustion of the extracting power of the acid extractants causes an underestimation of the available P. Hence, the critical level is smaller under higher PCF condition than under the lower ones as vastly documented in the literature. In spite of that, an unique soil critical level for any soil type and plant species is frequently used everywhere.

For plant critical level the situation is similar. Jones et a!. (1991) consider tomato leaf P con­centration between 0.25 to 0.75 as sufficient. This broad range makes it impossible to assure, for example, if a plant with 0.5% of P has an adequate P status or not. The present work indicates that this inexactness in defining a point as sufficient or critical level instead of a range is also dependable on soil characteristics related to PCF. The higher accumulation of inorganic P in the plant tissue growing in soils with low PCF and vice-versa seems to be the reason for this observation (Fabres et a!., 1987).

References

Bieleski R L and Ferguson I B 1983 Physiology and metabo­lism of phosphate and its compounds. In Inorganic Plant

Nutrition- Encyclopedia of Plant Physiology. Eds. A Uiuchi and R L Bieleski. pp 422-449. Springer-Verlag, Berlin.

Fabres A S, Novais R F, Neves J C L, Barros N F and Corderio A T 1987 Niveis criticos de diferentes fra~oes de f6sforo em plantas de alface cultivadas em diferentes solos. Rev. Bras. Ci. Solo 11, 51-57.

Freire F M, Novais R F, Braga J M, Fran~a G E, Santos H L and Santos P R R S 1979 Aduba~ao fosfatada para a cultura da soja (Glycine max (L) Merrill) baseada no f6sforo disponivel e no 'fator capacidade'. Rev. Bras. Ci. Solo 3, 105-111.

Hogue E, Wilcox G E and Cantliffe D J 1970 Effect of soil phosphorus levels on phosphorus fractions in tomato leaves. J. Am. Soc. Hortic. Sci. 95, 174-176.

Jones Jr J B, Wolf B and Mills H A 1991 Plant Analysis Handbook. Micro-Macro Publishing. Inc., Athens, 213 p.

Muniz A S, Novais R F, Barros N F and Neves J C L 1985 Nivel critico de f6sforo na parte aerea da soja como variavel do fator capacidadc de f6sforo do solo. Rev. Bras. Ci. Solo 9, 237-243.

Novais R F and Kamprath E J 1978 Phosphorus supplying capacity of previosuly heavily fertilized soils. Soil Sci. Soc Am. J. 42, 931-935.

M.A. C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 77-81, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-021

The use as fertilizer of combined primary I secondary pulp-mill sludge

F. CABRAL and E. VASCONCELOS Department of Chemical Agriculture, Instituto Superior de Agronomia, Tapada da Ajuda, 1399 Lisboa, Portugal

Key words: Lactuca sativa L., organic fertilizer, pulp-mill sludge

Abstract

An experiment was carried out with lettuce plants using PVC pots filled with a Cambic Arenosol, in order to define critical application levels of combined primary/secondary pulp-mill sludge to that crop, as well as to study its effect on physical and chemical characteristics of the soil. From the results obtained no yield reduction was observed, in contrast with the results obtained from studies undertaken with the primary sludge, where amounts larger than 50 t ha-l led to a significant yield reduction. Concerning soil characteristics, significant differences on pH, organic matter and exchangeable sodium percentage values were detected as well as on Ca/Mg ratio.

Introduction

In Portugal, the paper processing industry has undergone a considerable growth recently. One third of the mainland area of Portugal is oc­cupied by forests.

The production of pulp-mill sludge, the main by-product of this industry, is around 35 000 t ha- 1 (dry matter) year- 1 Santos ct al., 1990).

Large amounts of this sludge arc being ac­cumulated in the neighbourhood of the paper­mills, causing serious handling and pollution problems, especially under an increasingly tight economic situation and strong environmental protection policies.

Since there are few alternatives for its practical use, its utilization in raw as an agricultural fertilizer might constitute an extremely effective contribution not only to minimize pollution problems but also to yield stabilization or in­crease, as well as, if properly managed, to the maintenance or improvement of soil fertility (Zibilskc, 1987).

There are several types of sludge from the paper processing industry, namely the primary, combined primary I secondary and secondary sludge, that are similar concerning its chemical composition, except for nitrogen content and release that are greater for secondary sludge than for primary or combined sludge ( Garau et al., 1986).

One of the main limitations attributed to land application of combined and secondary sludge, is the concern for groundwater contaminations caused by leaching of nitrates (Harkin, 1982). However, other authors (Bockheim et al., 1988) verified that an application level of combined sludge of 32 t ha-l in a forest ccossystem will not contaminate groundwater at depth of ?1.6 m.

According to this, it is clear that more in­formation is necessary to develop guidelines for the agricultural utilization of these materials, that are conducive to plant growth and the maintenance of soil productivity.

This study was designed to determine the effects of combined primary I secondary pulp-mill

78 Cabral and Vasconcelos

sludge on the yield of lettuce plants (Lactuca sativa L.) as well as on soil physical and chemical characteristics.

Material and methods

Experimental details

An experiment, with a completely randomized design, was carried out in a greenhouse of the Agricultural Chemistry Department, using PVC pots (0.038 m2 of available area and 0.25 m of depth), filled with 11.7 kg of the arable layer (fraction< 5 mm) of a Cambic Arenosol- FAO classification (Table 1).

Treatments were replicated three times and the amounts of combined primary I secondary pulp-mill sludge consisted of: 0; 30; 60; 90 tha-t (Table 2). The rate of addition of sludge to the soil in the pots, was calculated taking into consideration the available area of each pot (0.038 m2 ).

In every treatment, an addition of fertilizer at planting consisted of 0.5 g N/pot (ammonium nitrate solution), 0.44 g P/pot (superphosphate 18%) and 0.83 g K/pot (potassium sulphate p.a.). Two nitrogen top-dressing fertilizations consisting of 0.5 g N/pot (ammonium nitrate solution) were carried out.

Table 1. Soil physical and chemical characteristics

Coarse sand (%) 70.70 Exc. cations Fine sand (%) 17.00 (cmol( +) kg- 1 )

Silt(%) 9.70 Clay(%) 2.00 Ca 0.94 Organic matter ( %C x 1, 724) 0.51 Mg 0.17 pH(H 20) 5.70 Na 0.12 P (Egner-Riehm) (fLg g - 1) 7.60 K 0.14 K (Egner-Riehm) (fLg g - 1) 43.00

Table 2. The physical and chemical composition of combined primary I secondary sludge (dry matter)

Moisture (%) 76.68 Mn (fLg g- 1) 40.64 Or g. matter (%) 90.01 Cu(fLgg- 1 ) 3.49 p (%) 0.25 Ni (fLgg- 1 ) 26.00 N-Kjeldahl (%) 2.79 Pb(fLgg- 1 ) 98.00 Ca(%) 3.14 Zn (fLg g- 1) 7.56 K(%) 0.66 Hg (fLg g - 1) 3.00 Na(%) 0.26 Cd (fLg g- 1 ) <0.50 pHdil.l:5 7.13 C/N 18.70

One lettuce plant (Lactuca sativa L. cv. attrac­tion kingston) was transplanted per pot. At the beginning of the experiment, pots were watered to 70% of soil field capacity, with demineralized water. After the initial wetting the water content in soil was controlled by weighing pots twice in a week.

At the end of the experiment plants were harvested, weighed, and chemically analysed. Equally soil samples from each pot were col­lected and analysed.

Analytical methods

Organic matter in the soil was calculated by multiplication of the percentage of organic car­bon by the factor 1.724, based on the assumption that soil organic matter is 58% carbon. Organic carbon was measured by dry combustion at 1200°C, followed by measurement of the C0 2

evolved by Strohlein equipment. Organic matter in the sludge was determined

by weight loss in a furnace at 350-400°C after 7-Sh.

Exchangeable cations in the soil were deter­mined by atomic absorption spectrophotometry, after extraction by the Mehlich method, using a barium chloride - triethanolamine solution of pH 8.1 (Mehlich, 1953). Available phosphorus and potassium in the soil, were determined by colorimetric and flame emission photometric methods respectively, after extraction using an ammonium lactate-acetic acid solution of pH 3.75 (Egner eta!., 1960). Nitrogen in sludge and plant tissues, was determined by the Kjeldhal method (Jackson, 1958), using a Tecator equip­ment.

All the other mineral elements, were deter­mined by atomic absorption spectrophotometry (Pye-Unicam SP-9) after a hydrochloric miner­alization of the ash (Marti and Munoz, 1957), except for phosphorus which was determined by the vanadomolybdophosphoric yellow colour method (Koening and Johnson, 1942) in a Hitachi U-2000 spectrophotometer.

Statistical analysis

Results from the study were analysed by one­way ANOVA, followed by Scheffe F-test at p =

0.05.

Combined primary I secondary pulp-mill sludge as fertilizer 79

40 34.2 b

30

g/pot 2 o

10

0

01 301 601 901

Pulp mill sludge (t/ha)

Fig. 1. Dry matter of lettuce plants at the end of the experiment.

Results

Yield of lettuce plants is shown in Figure 1. From this figure, it is possible to conclude that, larger amounts of sludge led to a yield increase, though significant differences are observed only between treatments concerning smaller amounts ( 0 and 30 t ha -I) and the highest amount of sludge applied (90t ha- 1).

Results obtained from soil analysis at the end of the experiment are shown in Table 3. pH (H20), exchangeable sodium, exchangeable so­dium percentage (ESP) values, Ca/Mg ratio as well as organic matter content, significantly in­creased with the increase in the amounts of

sludge applied. Concerning other parameters analysed, no significant differences between treatments are observed.

Table 4 shows the results obtained from leaf analysis at the end of the experiment. Except for copper and potassium, all other mineral ele­ments are affected with the increase in the amounts of sludge applied. In fact, a pronounced increase of sodium content in the plants is observed when the amounts of sludge are larger. Concerning other nutrients, amounts of sludge larger than 60 t ha _, led to a significant reduc­tion in lettuce plants. However, though a de­crease on N, P, Ca, Mg, Fe, Mn and Zn content in the plants is observed when the amounts of sludge applied to soil are larger than 60 t ha -I, the uptake by lettuce plants of these elements is not significantly affected (Table 5). This evi­dence, suggests that differences obtained from leaf analysis might be due to a dilution effect. On the other hand, sodium uptake strongly increased when the amounts of sludge increased.

Discussion

Comparing with the results obtained from the primary sludge, where amounts larger than 50 t ha -I led to a pronounced yield reduction of

Table 3. Some physical and chemical characteristics of the soil at the end of experiment

Treat. pH(H20) Org. matter p K Exchangeable cations ESP Ca/Mg (tha-') (%) (%)

(flgg-1) Ca Mg Na K

(cmol( +)kg - 1)

0 5.00b* 0.83 b 25.91 a 30.15 a 1.62 a 0.32 a 0.22c 0.05 a 9.95 c 5.06 b 30 5.05 b 0.85 b 27.65 a 27.11 a 1.72a 0.30 a 0.23 be 0.06 a 9.96c 5.73 a 60 5.30 a 1.07 a 24.45 a 25.45 a 1.84 a 0.31a 0.30b 0.07 a 11.90 b 5.93 a 90 5.90 a 1.09 a 28.37 a 21.30 a 1.85 a 0.30 a 0.40 a 0.04a 15.44 a 6.16 a

* Different letters indicate significant differences between treatments.

Table 4. Leaf analysis of lettuce plants at the end of the experiment

Treat. N p K Ca Mg Na Fe Cu Mn Zn (t ha _,) (%)

(flgg-1)

0 4.05 a* 0.70 a 3.28 a 1.59 a 0.52 a 0.09 d 335.3 a 17.6 a 427.5 a 95.4 a 30 3.75 a 0.68 a 3.29 a 1.52 a 0.52 a 0.50c 295.8 ab 20.3 a 432.8 a 85.1 a 60 3.59 ab 0.59 b 3.19 a 1.02 b 0.44 ab 0.84 b 291.5 b 14.0 a 281.3 ab 65.3 b 90 3.15 b 0.54 b 3.10 a 0.86 b 0.40b 1.05 a 295.5 ab 17.7 a 257.0 b 54.9 b

* Different letters indicate significant differences between treatments.

80 Cabral and Vasconcelos

Table 5. Nutrient uptake by lettuce plants at the end of the experiment

Treat. N p K Ca Mg Na Fe Cu Mn Zn ( t ha _,)

(mg/pot)

0 1006.0 a* 183.5 a 852.7 a 413.7a 134.7 a 23.2 d 8.7 a 0.5 a 11.3 a 2.4 a 30 1086.5 a 178.9 a 865.9 a 397.3 a 136.1 a 131.9c 7.8 a 0.5 a 11.5 a 2.2 a 60 1098.9 a 179.3 a 973.9 a 312.7a 135.7 a 256.9 b 8.9 a 0.6 a 8.5 a 2.0 a 90 1044.6 a 180.1 a 1026.9 a 284.8 a 133.3 a 348.5 9.8 a 0.6 a 8.5 a 1.8a

*Different letters indicate significant differences between treatments.

different crops (Cabral et al., 1990; Santos et al., 1990; Vasconcelos and Cabral, 1992), the use as an organic fertilizer of combined primary I sec­ondary sludge did not cause any yield reduction of lettuce plants and particularly when the amount of sludge applied was 90 t ha - 1 a signifi­cant increase in the yield is observed. Disappear­ance of that effect might be due to the high CIN ratio (900-1000) of primary sludge that leads to a temporary imobilization of nitrogen in soil in contrast with combined primary I secondary sludge that shows a CIN ratio (15-25) conducive to promote nitrogen mineralization.

Yield differences obtained to lettuce plants, might result from the increase on pH values as well as from the increase on organic matter content in the soil. There is no reason to believe that the largest amount of nitrogen vehiculated trough the largest amount of sludge applied, might have a direct effect on the yield, since the uptake by the plants of this nutrient is not significantly different between treatments.

The increase on pH, organic matter, ex­changeable sodium, ESP values as well as on CaiMg ratio, when larger amounts of sludge are applied, was already expected because these results were equally obtained from some experi­ments we have undertaken with the primary sludge, which composition is not very different from that of the combined primary I secondary sludge (Vasconcelos et al., 1990).

Concerning crop development, the increase of exchangeable sodium and ESP values as well as CaiMg ratio, might lead to serious troubles if the right precautions are not taken into consid­eration. In fact, when a crop is particularly exigent to magnesium, larger amounts of com­bined sludge should not be applied in order to prevent the appearance of a deficiency in that nutrient in the plants.

Conclusions

From the results obtained from the use as organic fertilizer of combined primary I secondary sludge to lettuce plants it is possible to draw the following conclusions:

Combined primary I secondary sludge appears to be an organic fertilizer with the additional advantage, because of its liming capacity, to acid soils.

Amounts of 90 t ha _, caused, in the soil and crop tested, a significant increase in yield.

Concerning soil pollution, it is known that succeeding applications of large amounts of this sludge, in the soil tested, can lead to some problems, notably an increase of exchangeable sodium and ESP values, as well as CaiMg ratio. However, taking into consideration the rule 2781 86 from EEC, no heavy metal pollution is expected, so long as the applications of sludge do not exceed 70-80 t ha - 1 year - 1 •

Acknowledgement

The authors acknowledge Mrs Gra<;a Sanches for technical assistance.

References

Bockheim J G, Benzel T C and Rui-Lin Lu Thiel D A 1988 Groundwater and soil leachate inorganic nitrogen in a Wiscosin red pine plantation amended with paper industry sludge. J. Environ. Qual. 4, 729-734.

Cabral F, Vasconcelos E and Monjardino P 1990 Biodeg­rabilidade no solo das lamas celul6sicas. Proceedings da 2' Conferencia Nacional sobre a Qualidade do Ambiente, E1-E5.

EEC 1986 Official Journal of the European Communities, 4/07/86. Council Directive of 12 June 1986.

Combined primary I secondary pulp-mill sludge as fertilizer 81

Egner H, Riemh H and Domingo W R 1960 Un­teresuchungen iiber die chemische Bodenanalyse als Grund­lage fiir die Beurteilung des Niihrstoff-zustandes der Boden. II Chemische Extraktionsmethoden zur Phosphor­und Kaliumbestimmung. Kung!. Lantbr. Hiingsk. Ann. 26, 199.

Garau M A, Felipo M T and Ruiz de Villa M C 1986 Nitrogen mineralization of sewage sludge in soils. J. Environ. Qual. 15, 225-228.

Harkin J M 1982 Wise use of Wisconsin's papermill sludge. In Long Range Disposal Alternatives for Pulp and Paper Sludges. Eds. C A Rock and J A Alexander. pp 65-78. Univ. of Maine Press, Orono, M.E.

Jackson M L 1958 Soil Chemical Analysis. Ed. Prentice-Hall, Inc., Englewood Cliffs, NJ, USA.

Koening R A and Johnson C R 1942 Colorimetric determi­nation of phosphorus in biological materials. Ind. Eng. Chern. Anal. 14, 155.

Marti F B and Munoz J R 1957 Flame Photometric. Ed. Elsevier Publishing Company, Amsterdam.

Mehlich A 1953 Rapid determination of cation and anion exchange properties and pH of soils. J. AOAC 36, 445.

Santos J Q, Vasconcelos E and Cabral F 1990 Utiliza~ao das lamas celul6sicas como fertilizantc. Proceedings do IV Encontro Nacional do Saneamento Basico, pp 386-297.

Vasconcelos E, Cabral F and Monjardino P 1990 Impacto no solo da aplica~ao de lamas celul6sicas. Pedon 9, 95-101.

Vasconcelos E and Cabral F 1992 Estudo comparativo da utiliza~ao como fcrtilizante de lamas celul6sicas dos tratamentos primario e secundario. Proceedings da 3' Conferencia Nacional sabre a Qualidade do Ambiente, pp 727-735.

Zibilske L M 1987 Dynamics of nitrogen and carbon in soil during papermill sludge decomposition. Soil Science 1, 26-33.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 83-86, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-038

The use of industrial wastes as manures: A case study with effluent mud from an olive oil processing plant

D. ANA<;, H. HAKERLERLER and M.E. lRGET Department of Soil Science, Faculty of Agriculture, Ege University, TR-35100 Bornova, Izmir, Turkey

Key words: effluent mud/sludge, heavy metals, industrial wastes, plant nutrients, reuse techniques, soil amendments, toxic elements

Abstract

The effect of different effluent mud (EM) and nitrogen (N) combinations on biomass production, major and minor nutrient and toxic element concentrations of maize were studied. Nitrogen enriched EM applications improved the yield and lowered toxic element and sodium concentrations. Excluding the lowest rate of EM, all tested effluent doses resulted in poor growth.

Introduction

The growing industry in Turkey has brought public concern over the health hazard as a consequence of waste release into the environ­ment. Activities of humans generate enormous amount of refuse. Many disposal alternatives are present; however, the safest handling method is the recovery of the resource. Recent related studies by Anar; and Hakerlerler (1991 ), Haker­lerler and Hafner ( 1984) and Hafner et a!. ( 1978) report the importance of reuse techniques in Turkey of which composting was found worth mentioning.

Olive is one of the major crops in Turkey and oil processing plants are widely spread in the western part. The large volumes of effluent mud (EM) generated after the sewage treatments cause great disposal problems for the processors. In the mean time, the soils of this region are low in organic matter. In this respect, the EM was thought to have value for agriculture as an

alternative to animal manure which is scarce and expensive.

The objective of this study was to investigate the applicability and the consequential effects of EM on plants when enriched with nitrogen (N) fertilizers. The reported results are from a EM-N factorial case study carried out with the maize plant.

Methods

Layout and treatments

Maize seeds of cultivar Cargill 1967 were germi­nated and further grown in a soil + EM growing media as two seedlings per pot. In the prepara­tion of the media, 10 kg of loamy textured and slightly alkaline alluvial soil was amended by 4 different levels ( 0-200-400-800 g pot- 1 ) of EM and 3 different levels (0-300-600 mg kg_,) of N

84 Ana~ et al.

in the form of (NH 4 ) 2S04 . The 12 combinations of EM and N treatments were fully randomized with five replications (4 x 3 x 5 = 60). Physical and chemical composition of the EM is given below as total contents.

Primary elements

(%) N __l'_ ~ ~ Na Fe Al

0.3 2.0 14.5 0.99 0.48 0.32 1.4

Secondary elements~ Zn Mn Ni Pb Sb Sn - -

(mg kg- 1) 700 109 201 171 126 102 272

Trace elements Cu Mo Cd Co Cr Tl

(mg kg- 1) 18 2.9 1.87 4 20 17

. pJ-K:/N O.M. Moisture (70'C) Other properties 9.Ti:61 83 % 33 _5%

Ca(OH) 2 and Al 2(S0 4 ) 3 and praestol were the chemicals used to precipitate the mud during the sewage treatment in the plant. Adequate levels of P (80mgkg- 1 ) and K (100mgkg- 1 ) were added to the growing media as base dressings. Half of N was given as base and the other half later as supplementary dressing within the vege­tation period.

Sampling and analysis

After a 2 month (1 May-Hl July 1991) growing period, the aerial parts of the two maize plants in each pot were harvested, dried at 60aC and ground. Biomass yield was determined and sam­ples were analyzed for their N by a distillation method, P by colorimetry, K, Ca and Na by fiamephotometry and Mg, Fe, Zn, Mn, Cu, Mo, Cd, Cr, Co, Ni, Pb, Sb, Sn, AI and Tl by AA spectrometry.

Results

The biomass production and mean concentra­tions of the 20 elements examined with respect to N and EM applications at different levels are presented in Table 1. The studied elements were considered as 3 groups: major plant nutrients, minor nutrients and toxic elements. For simplici­ty of discussion, in each case, effects of N rates are discussed first, followed by EM.

The yield response to the increasing amount of N applied was very noticeable and improved the yield 6.38 times as compared to the control treatment. However, addition of EM exerted

adverse effect and progressively depressed the yield more than 2 times.

Nitrogen increments resulted in a considerable variability on the major nutrients as could be seen in the increases in N and Mg contents and the decreases in P, K and Na. However, different levels of applied EM resulted in unfavourable conditions i.e. low N concentrations and 10 fold increase in Na.

As for the minor nutrients, the increasing N rates had no consistent effect on Mn and Cu, considerably lowered Zn and Mo and increased Fe concentrations. In this regard, additions of EM lowered all of the minor nutrients excluding Mo which was originally high.

The relationships between the treatments and the toxic element concentrations are also shown in Table 1. It is worth mentioning here that higher N rates greatly reduced the concentra­tions. Whereas in the case of EM, increases were evident excluding Sb and Pb.

Discussion

When waste materials are used as soil amend­ments, attention should be focused on the Na content and the C/N ratio of the material. In similar trials, it is found that depressions on yield frequently accompany high leaf Na concentra­tions. This is, in general agreement with the previous results which refer to originally high Na content of the waste materials (Mengel and Kirkby, 1987).

In the presence of wider C/N ratios, microbial competition is what determines the availability of soil nutrients for higher plants. It appears from this work that addition of N narrowed the C/N ratio which further contributed to the vigorous growth of the experimental plant. Consequently, Na concentration were diluted in the aerial tissues resulting in Na reduction at higher rates of N applications (Arnon, 1975). However, the effects were reversed by the addition of EM due to its chemical composition.

The increase in Fe concentrations by high N applications may as in the case of Na be attribu­ted to the narrowed C/N ratio and enhanced decomposition of EM. Root vicinity pH might have been lowered by the probable release of

Tab

le 1

. B

iom

ass,

maj

or a

nd m

inor

pla

nt n

utri

ents

and

tox

ic e

lem

ents

in

mai

ze a

s af

fect

ed b

y ap

plic

atio

n of

nit

roge

n fe

rtili

zer

(N)

and

effl

uent

mud

(E

M)

Bio

mas

s N

p

K

Ca

Mg

Na

Fe

Zn

Mn

Cu

Mo

Cr

Co

Ni

Cd

Sn

Sb

Tl

Pb

AI

(g/p

ot)

dm%

d

m (

mg

kg

1 )

Nu

75.R

70

1.12

0.

370

2.48

1.

23

0.36

2 72

9 80

44

67

5.

55

4.11

4.

39

2.98

9.

71

1.50

8 40

.0

17.5

11

.06

6.01

20

4 N

l 38

3.35

7 1.

68

0.25

5 1.

42

1.07

0.

482

211

249

25

70

4.40

1.

93

1.15

0.

78

2.36

0.

516

32.3

12

.8

2.95

3.

63

227

N,

484.

558

2.15

0.

263

1.42

1.

20

0.45

6 14

6 31

5 29

65

5.

75

2.04

1.

20

0.85

2.

24

0.58

0 29

.7

16.1

3.

55

3.95

26

3 L

SD

' 36

.361

0.

13

0.02

4 0.

11

0.11

0.

052

153

39

4 ns

ns

0.

61

0.29

0.

27

1.01

0.

110

6.0

1.6

2.20

0.

81

35

EM

0 38

6.22

9 1.

81

0.21

2 1.

41

1.16

0.

391

80

261

34

66

5.46

1.

58

1.03

0.

82

2.31

0.

571

26.1

15

.3

3.96

3.

77

232

EM

I 38

7.54

0 1.

84

0.33

3 1.

90

1.26

0.

507

223

266

37

68

7.00

2.

27

2.59

1.

62

6.47

0.

981

37.6

15

.8

7.20

5.

02

295

EM

2 29

7.87

4 1.

58

0.31

0 2.

00

1.14

0.

430

366

162

34

57

4.46

2.

52

2.57

1.

77

4.87

0.

959

36.0

15

.6

4.42

4.

90

198

EM

1 18

6.73

6 1.

36

0.32

9 1.

79

l.l2

0.

404

781

169

26

77

4.00

4.

39

2.80

1.

95

5.42

0.

961

36.2

15

.1

7.83

4.

43

202

Lso·~

41.9

86

0.15

0.

028

0.13

ns

0.

060

177

45

5 12

1.

26

0.70

0.

34

(J.3

1 1.

16

0.12

7 7.

0 ns

2.

55

ns

40

'p "

'0.0

5.

._

;:

;:::,..

;::: "' " ~ ;t

~ "' R "' ~ "' ~

~

;:

;::: ;;; "' 00

U

1

86 Industrial wastes as manures

organic acids during the mineralization. Fe up­take was ensured and notified by . improved growth (Mengel and Kirkby, 1987). However, high Ca content of the EM with wide C/N ratio restricted Fe uptake and resulted in poor growth. A similar trend was not attained for other minor nutrients probably due to the known antagonistic relations among metals (Arnon, 1975).

Lower concentration of toxic elements accom­panying the increasing amounts of applied N may be ascribed to the dilution effect resulting from rather higher biomass yield (Arnon, 1975). None of these elements were found above the threshold values previously reported (Anony­mous, 1983; Austenfeld, 1979; Mengel and Kirkby, 1987; Scheffer and Schactschabel, 1984; Scholl and Metzger, 1981). Even AI concen­trations were not found beyond the critical level (Peterson and Girling, 1981). The effluent mud which has a quite high C/N ratio is advised to be used only after fermentation.

References

Ana~ D and Hakerlerler H 1991 Evaluation of plant nutrient and heavy metals in the composted solid wastes of Izmir. In Urban Ecology Proceedings. Eds. M Oztiirk, 0 Erdem

and G Gork. pp 99-109. Ege University Press, Izmir. Tiirkiye.

Anonymous 1983 Schwermetalle in der Nahrung: Akute Gefahrdung fiir Mensch und Tier. Heft 6. Darmstadt.

Arnon I 1975 Mineral Nutrition of Maize. International Potash Institute, Bern, Switzerland, pp 210-241.

Austenfeld F A 1979 Zur Phytotoxizitat von Nickel- und Kobaltsalzen in Hydrokultur bei Phaseolus vulgaris. Z. Pflanzenernaehr. Bodenkd. 142, 786-791.

Hakerlerler H und Hafner W 1984 Schwermetallbelastung von Olivcnanlagen durch Immissionen einer Diingemittel­fabrik. Z. Pflanzenernaehr. Bodenkd. 147, 523-526.

Hafner W. Kovanci I and Hakerlerler H 1978 EinfluB von Miillkompost und Eisenverbindungen auf die Eisen-, Zink­, und Manganaufnahme von Sonnenblumen und Mais im Gcfassvcrsuch. Z. Pllanzenernaehr. Bodenkd. 141, 547-555.

Mengel K and Kirkby E A 1987 Principles of Plant Nutrition. International Potash Institute, Bern, Switzerland. pp 589-601.

Peterson P J and Girling C A 1981 Other trace metals. In Effect of Heavy Metal Pollution on Plants. Ed. N W Lepp. Vol. 1. pp 213-263. Applied Science Publishers, London and New York.

Scheffer F und Schachtschabel P 1984 Lehrbuch der Boden­kunde. Ferdinand Enke Verlag, Stuttgart. 442 p.

Scholl G und Metzger F 1981 Erhcbungen iiber die Thallium Belastung von Nutzpflanzen auf kontaminierte Boden im Raurn Lengerich. Landwirtsch. Forsch. 38, 216-224.

M.A.C. Fraxoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 87-92, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-080

Influence of container size and substrate mineral composition on transplant growth and yield of broccoli cv. Green Duke

ANA MARIA SIMOES 1 , FATIMA CALOUR0 1 , EDUARDO ABRANTES 2

and EDMUNDO SOUSA2

1/NIA, Laborat6rio Qufmico Agricola Rebelo da Silva, Tapada da Ajuda, 1300 Lisboa, Portugal and 2/NIA, Departamento de Horticultura e Floricultura, Qta do Marques, 2360 Oeiras, Portugal

Key words: Brassica oleracea L. broccoli, container cell size, leaf area, nursery, plant hardness, plant height, plant dry weight, substrates

Abstract

Broccoli (Brassica oleracea L. cv. Green Duke) was sown and grown for 6 weeks in containers with different volumes and substrates and harvested after 8 and 9 weeks after transplantation. The aim of the study was to estimate the effect of container size and the type of substrate on plant growth, in the nursery, and their long-range effects on final yield in the field. In the nursery the variables analysed were the plant height, leaf dry weight and leaf area per plant. In the field, the final yield, the number of plants with head, and the marketable yield at two different harvest dates were assessed. The obtained results suggest that containers with 21-31 mm width and 71-75 mm depth, in combination with richer composition substrates, (N, 180-210mg L -I; P20 5 , 120-240mg L- 1 , and K20, 220-270mg L- 1) are more appropriate for transplant growth.

Introduction

Little information is available concerning con­tainer size and substrate mineral composition effects on transplant growth of cruciferous plants and the subsequent effect on their yield crop earliness and maturation uniformity, as well as on the yield itself.

The reported results on the container size effect on both crop earliness and yield are unstable: Chinese cabbage earliness increases with increasing container size (Kratky et a!., 1982) and the same result was obtained by Whitwell and Crofts (1972) in cauliflower; no significant effects were found by Dufault and Waters Jr. (1985) in broccoli, but both kinds of results are reported by Magnifico et a!. (1980) and Dufault and Waters Jr. (1985).

Concerning yield, increasing container size leads to higher yields in Chinese cabbage (Kratky et al., 1982) and cabbage (Miller et a!.

1969); no significant effect was found by Cox et a!. (1982), Csizinzky (1983) and Dufault and Waters Jr. (1985) in cauliflower; Magnifico et a!. (1980) report an increasing effect on broccoli yield with increasing container size, whereas Dufault and Waters Jr. (1985) found no relevant effect on the same variable.

So, the choice of nursery plant conditions is mainly a critical economic decision, because costs of raising and transplantation are usually very high. However, transplanting plants is pre­ferred to direct seeding, because it reduces seed requirement, it avoids thinning and provides optimal plant spacing.

The present paper deals with the obtained results of an experiment carried out both in the nursery and in the field, with broccoli, cv. Green Duke, aiming to establish the influence of container cell size and substrate mineral compo­sition on transplant growth and their long-range effects on yield earliness and yield, in the field.

88 Simoes et al.

Materials and methods

The experimental work was carried out during 1991, from 14 February to 27 March in the nursery, and from 27 March to 29 May in the field, with broccoli, cultivar Green Duke, which has a short vegetative cycle (about 85 days).

Nursery experiment

Split plot experimental design was used in the nursery experiment, arranged into complete ran­domized blocks, with four replications. Main plots were assigned to containers and subplots to substrates. Four different types of containers and substrates were used, with the characteristics described in Tables 1 and 2.

Plants were harvested at 12, 23 and 32 days after emergence and the following aspects were considered: plant height (from ground level to terminal meristematic tissue), fresh and dry weights, leaf area and the ratio leaves dry weight: leaf area, in order to estimate plant hardness.

Field experiment

A field experiment was carried out in a sandy soil, with pH(H20) 5.8 and low organic matter content (0.60% ). Available phosphorus and potassium contents were 95 mg kg- 1 P20 5 and

Table 1. Container characteristics

Container No of Cell Cell Cell cells width (mm) depth (mm) volume (cc)

A 128 37 65 35 B 216 31 75 37 c 294 21 71 27 D 288 22 55 12

Table 2. Chemical characteristics of the substrates

Substrate pH(H 20) Nitrogen Phosphorus Potassium

(mgL j)

sl 6.0-6.5 250 290 290 s, 6.0-6.5 210 240 240 s, 5.0-6.0 180 120 120 s4 6.0-6.5 180 210 210

66 mg kg_- 1 K 20, respectively. At plant trans­plantation, lOOkg ha- 1 of nitrogen, lOOkg ha- 1

of phosphorus (P2 0 5 ) and 150 kg ha-l of potas­sium (K2 0) were applied.

Plants were arranged in the field, as they were arranged in the nursery, leading to identical main and subplots, even with another randomi­zation, each one of them with 160 and 40 plants, respectively, spaced at 60 x 50 em. Top dress fertilization with 60 kg ha-l of nitrogen was applied, four weeks after plant transplantation and as the plant head thickened.

Two harvests were performed, and the follow­ing aspects were considered: plant height, head diameter and weight.

Results

Analysis of variance was performed, both with nursery and field data, in order to evaluate the effect of both container cell size and type of substrate on transplant growth, and to test if the long-range effects were retained in the field.

Transplant growth

Plant height The interaction between container size and sub­strate did not affect plant height significantly. On the contrary, container cell size and substrate show a significant mean effect on the height of plants, harvested at 12, 23 and 32 days after emergence.

Higher plants were obtained, on average, with container C and, with the exception of the first harvest, container D gave lower plant height (Table 3), suggesting that plant height is mainly

Table 3. Mean effect of container cell size on nursery plant height (em) (Duncan test, a = 0.05)

Container Harvest

1st 2"d 3'd

A 2.36 b 4.07 c 4.95 b B 2.48 ab 4.31b 5.11 a c 2.66 a 4.51 a 5.10 a D 2.54 ab 3.52 d 3.70c s,( ±) 0.0586 0.0404 0.0436

reduced by reducing container volume in associa­tion with depth, especially at the most advanced vegetative stages.

Concerning substrate mean effect, lower plants were obtained with substrate S2 , at the first harvest, and with substrate S4 at the second and third harvests (Table 4).

Plant dry weight As in plant height, the interaction container x substrate did not significantly affect nursery plant dry weight.

Container cell size shows a significant mean effect on plant dry weight, at all the performed harvests, while substrate mean effect was signifi­cant only at the second and third harvests.

In the smallest container, plant weights were highest at the first harvest, whereas at the second and third harvest container C (with the smallest width and medium depth) shows the highest value, as well as container B (the deepest one) at the third harvest (Table 5).

Concerning substrate effects, there are no significant differences at the first harvest; at the second and third, S3 leads to plant weights which

Table 4. Mean effect of substrate on nursery plant height (em) (Duncan test, a = 0.05)

Container Harvest

]st 2"d 3'd

sl 2.54 a 4.16 a 4.94 a s, 2.41b 4.13 a 4.61 be s, 2.58 a 4.21 a 4.83 ab s, 2.51 a 3.92 b 4.48c s,( ±) 0.0316 0.0497 0.0862

Table 5. Mean effect of container cell size on nursery plant dry weight (g) (Duncan test, a = 0.05)

Container Harvest

1st 2"d 3'd

A 0.153 c 0.672 c 1.52 be B 0.161 c 0.673 c 1.35 c c 0.254 b 0.922 b 1.66 b D 0.322 a 1.251 a 1.88 a s,( ±) 0.00507 0.0239 0.0640

Broccoli: container size and substrate influence 89

Table 6. Mean effect of substrate on nursery plant dry weight (g) (Duncan test, a = 0.05)

Container Harvest

1 ~I 2"d 3'd

sl 0.222 a 0.942 a 1.72a s, 0.218 a 0.884 b 1.65 a s, 0.226 a 0.866 b 1.61 a s 4 0.224 a 0.826 b 1.44 b s,( ±) 0.0054 0.0237 0.0508

are similar (p > 0.05) to those obtained with higher levels of N, P and K (Table 6).

Plant leaf area Container x substrate interaction shows a signifi­cant mean effect on nursery plant leaf area, at the first harvest, and a non-significant effect at the second and third.

At the first harvest, combinations of container D (the smallest one) and any of the substrates show higher plant mean leaf area (p """0.05); lower values were due to combinations with containers A and B, with no significant differ­ences (p > 0.05) between them (Table 7).

At second plant harvest, significant effects on plant leaf area were found, due to container cell size, while at the third no significant differences were found; however, the calculated F value was close to the significance limit (p = 0.06); signifi­cant mean effects on the variable were found at both plant harvests, due to the type of substrate.

At both harvests, container C shows on aver­age the highest mean plant leaf area, although with no significant differences from container D (Table 8); concerning substrate mean effects, S4

shows the lowest plant mean leaf area in both harvests (Table 9).

Table 7. Container cell size x Substrate interaction mean effect on nursery plant leaf area (cm2)- 1" harvest (Duncan test, a = 0.05)

Substrate Container

A B c D

sl 11.75 e 12.50 e 23.50 abc 27.75 a s, 11.00 e 10.75 e 19.25 cd· 25.25 ab s, 12.00e 12.75 e 20.00 cd 25.25 ab s, 11.25 e 15.75 de 22.25 be 20.75 b s,( ±) 1.415

90 Simoes et al.

Table 8. Mean effect of container cell size on nursery plant leaf area (em') (Duncan test, a = 0.05)

Container Harvest

2"' 3"'

A 106.56 b 214.27 ab B 111.50 b 196.99 b c 137.38a 238.21 a D 140.75a 212.59 ab s,( ±) 3.633 8.983

Table 9. Mean effect of substrate on nursery plant leaf area (em') (Duncan test, a= 0.05)

Substrate Harvest

2"" 3rd

s, 133.06 a 236.61 a s, 130.50 a 221.86 a s, 125.31 a 217.41 a s, 107.31 b 186.18 b s, ( ±) 4.731 6.989

Ratio dry weight: leaf area (plant hardness) Interaction container x substrate mean effect on the plant hardness was significant at the first harvest and did not affect the variable at the second or third.

Higher mean values for plant hardness were obtained with combinations S4D, S3D and S2B; the lowest plant hardness value was obtained with combination S 1C, which did not show a significant difference (p > 0.05) from combina­tion S3C (Table 10).

At the second and third plant harvests, container size shows significant mean effect on plant harness; so docs substrate type at the second one.

Table 10. Container x Substrate interaction mean effect on nursery plant hardness- 1" harvest (Duncan test, " = 0.05)

Substrate Container

A B c D

s, 3.67 be 3.80 be 3.09 d 3. 73 be s, 3.77 be 4.01 ab 3.72 be 3.84 abc s ' 3.70 be 3.59 be 3.53 cd 4.01 ab s, 3.77 be 3.55 c 3.73 be 4.25 a s,(±) 0.119

Table 11. Mean effect of container cell size on nursery plant hardness (Duncan test, a= 0.05)

Container Harvest

2"" 3'"

A 5.09 be 6.63 b B 4.89 c 6.20 be c 5.21 b 6.08 c D 6.34 a 7.33 a s,( ±) 0.0875 0.156

Container D (the smallest one) shows the highest plant hardness mean value, at both harvests, significantly (p ~ 0.05) higher than the other ones (Table 11).

Concerning substrate mean effects, S4 leads to the variable highest mean values at the first and second harvests, although it shows no significant differences (p > 0.05) from the other substrates in the third one (Table 12).

Broccoli yield

Yield crop Analysis of variance I covariance was performed in order to test long-range effects of both container cell size and the type of substrate on the yield crop. The number of plants per plot was used as the covariate.

Although no significant mean effects were found on yield crop, both due to nursery container cell size (F 13 ,81 = 0.586 p > 0.05) or substrate (F 13 ;351 = 2.524 p > 0.05) the obtained results show that the highest yield crop mean values were reached with plants coming from different combinations of B and C containers (the deeper ones) with S1 , S2 and S3 substrates (Table 13).

Table 12. Mean effect of substrate on nursery plants nursery plant hardness (Duncan test, "= 0.05)

Substrate Harvest

2"" 3"'

s, 5.35 b 6.57 a s, 5.14 b 6.52 a

s3 5.26 b 6.40a s, 5.77 a 6.74 a s,( ±) 0.0912 0.172

Table 13. Obtained mean yield corp values, according to different container and substrate combinations (g.10.25 plants_,)

Substrate Container

A B c D

s, 1392 1726 1855 1352 s, 1617 1854 1789 1431 s, 1406 1735 1678 1742 s, 1385 1586 1642 1456 s, ( ='-) 184.7

Regarding the number of plants with formed head, analysis of variance shows a significant mean effect of the container size on the named variable (F13 ,~ 1 = 3.757*): on average, experi­mental treatments with container D show a significantly (p ~ 0.05) lower number of plants with formed head (eight versus eleven plants).

Analysis of variance was also performed in order to test the effect of experimental treat­ments on height and head diameter. No signifi­cant results were obtained. On average, plants have shown 11.57 em of height and 10.36 em of head diameter, in field plots.

Yield crop quality Frequency distribution of plants showing market­able heads was performed according to harvest­ing time and experimental treatments (Fig. 1 ).

The obtained results show that the first har­vesting time was associated with the majority of formed heads, in relation to total crop yield. The

100

90

60

..........

..............

20 ·········

10 ...........

.......

l ....

........ .............

........

.......... ..............

......

. ...........

1 ............

L AS1 AS2 AS3 AS4 851 BS2 BS3 854 C51 CS2 CS3 CS4 051 DS2 053 054

Experimental treatments

] ~ 1st harvest - 2nd harvest I

Fig. /. Distribution of marketable yield according to ex­perimental treatments.

Broccoli: container size and substrate influence 91

AS1 AS2 AS3 AS4 851 BS2 863 BS4 CS1 CS2 CS3 CS4 051 052 DS3 054

Experimental treatments

j tz2(]1 st harvest - 2nd harv~

Fig. 2. Distribution of yield earliness according to ex­perimental treatments.

majority of the plants that were transplanted from experimental treatments associated with container D (the smallest one) were not ready for harvesting at the first date (Fig. 2). These results also show that marketable head forming was associated, especially, with the first harvest­ing date.

Conclusions

Increasing container volume, especially through the increasing of its depth, increases nursery plant height and weight; hardness is decreased. Although with no significant effects, those plants were associated with greater earliness and better quality yields.

Unstable results were found concerning sub­strate effects, showing that more experimental work is needed on the subject. The obtained results suggest that richer substrates should be used, especially for the earlier plant develop­ment stages in the nursery .

Acknowledgements

The authors are grateful to the 'Herdade Gil Vaz' staff for collaboration in the field experi­ment, as well as the LQARS staff, who carried out the analytical work and text processing.

92 Broccoli: container size and substrate influence

References

Cox E, Biddington N, Dearman A, Hawkins W and Romer G 1982 Transplant establishment: spacing of plant raising nodules. In National Vegetable Research Station, Welles­bourne, Warwick, England. 33rd Annual Report 98 p.

Csizinszky A A 1983 Effect of transplant container cell size and fertilizer levels on cauliflower and cabbage yields in full bed mulch culture. Hort Science 18, 566 (Abstr.).

Dufault R J and Wateres Jr L 1985 Container sizes influences broccoli and cauliflower transplant growth but not yield. Hart Science 20, 682-684.

Kratky B, Wang J K and Kubojiri K 1982 Effects of container

size, transplant age and plant spacing on chinese cabbage. J. Am. Soc. Hortic. Sci. 107, 345-347.

Magnifico V, Bianco V and Fortunato I M 1980 The effect of seeding size at transplanting on the production characteris­tics of broccoli. Ann. Fac. Agric. Univ. Bari 31, 717-731.

Miller C H, Splinter WE and Wright F S 1969 The effect of cultural practices on the suitability of cabbage for once­over harvest. J. Am. Soc. Hortic. Sci. 94, 67-69.

Whitwell J D and Crofts J 1972 Studies on the size of cauliflower transplants in relation to field performance with particular reference to date of maturity and length of cutting season. Expt. Hortic. 23, 34-42.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 93-96, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-030

Mulch and topdressed nitrogen effects on bell pepper

P.D. CASTELLANE, R.K. FUJIMURA, J.A.C. de ARAUJO and M.E. FERREIRA Department of Horticulture, UNESP University, Campus of Jaboti cabal, SP, Brazil

Key words: Capsicum annuum, fruit production, leaves mineral content, mulch, pepper, soil test values

Abstract

The objective of this research was to measure the effects of the black polyethylene mulch and the rates of nitrogen ( topdrcssed fertilization) on bell pepper (Capsicum annuum L. 'Ikeda') yield. Some soil characteristics, after crop development were also evaluated. The experimental design was a randomized block, with four replications using a 2 x 3 factorial (presence and absence of mulch and 0, 40, and 80 kg ha - 1 of N, applied five times at every 25 days after seedlings transplanting). Higher values for the soil temperature, lower rates of leaching of K, Ca, and Mg, higher plant growth, and a higher total fruit production were obtained in the mulched areas. With increasing N supply leaching of K, Ca, and Mg rose. There was no effect of N on total fruit production.

Introduction

In more developed countries, there is a constant concern about the dangers of water contamina­tion caused by excessive use of nitrogen fertilizer (BJorn-Zandstra, 1989; Newbould, 1989). Sever­al alternatives to minimize this problem have been suggested. Among them, the establishment of better practices for crop management have been discussed with the aims of decreasing N rates of fertilization.

One of the practices that can decrease nutrient loss by leaching and, consequently increase utili­zation by crops, is the use of mulch with a polyethylene film. Locascio et a!. (1985) ob­served that in these conditions, there were great­er absorption and efficiency of applied N for bell pepper, and that more fruits were produced than under unmulched conditions. Polyethylene mul­ches may also favour crop growth by increasing soil temperature, preserving soil moisture and decreasing the incidence of insects, viruses and, weeds (Asicgbu, 1991; Courter and Oebker, 1964; Decoteau et a!., 1990; Gerard and Cham­bers, 1967).

In Brazil, black polyethylene is used tradition-

ally for the strawberry crop, but its use has been extended for other crops such as tomato and bell pepper. In the latter case, however, there are limited data on N management use under such mulching condition. The objective of this experi­ment was to evaluated the influence of black polyethylene mulch and N topdressing rates on the growth and yield of 'Ikeda' bell pepper. Soil temperature, leaves composition and soil test values after crop growth also were evaluated.

Materials and methods

The experiment was conducted from March to July 1991, in a sandy clay soil (organic matter 3,1% and pH 5,6) of the Jaboticabal county (21 °15' South, 48°18' West Gr. and, altitude 550 m). Fertilizers were applied before planting and prior to mulching to provide 40 kg ha - 1 N, 60 kg ha - 1 P and, 66 kg ha - 1 K. The factorial 2 x 3 was arranged in a randomized block design with four replications. The treatments consisted in the presence and absence of black poly­ethylene mulch (0.03 mm thickness) associated with 0, 200 and, 400 kg ha - 1 total N rates, in a

94 Castellane et al.

urea form, split five times and applied every 25 days after planting. After each application, 0.5 em of water was applied by sprinkler irriga­tion. The seedlings were produced in a substrate containing carbonized rice straw, soil and, farmyard manure (1: 1: 1 by volume) and trans­planted at seven weeks old at a spacing rate of 1.2 x 0.5 m. Prior to transplanting the mulching was placed in beds of 0.35 m wide. The topdres­sed fertilizer was placed on opposite sides and 15 em away from the plants. In order to place the urea onto the soil, the polyethylene film was perforated with 1.5 em diameter holes. Every experimental plot was formed by 4 rows, total­ling 24 plants, and only on the central rows were measurements made.

During the experimental period there was a total rainfall of 41.3 em. In the absence or lack of rainfall, sprinkler irrigation were applied twice a week. Under these conditions 3.0 em of water per week was provided. The control of mites, insects and, fungal diseases was carried out weekly with dimethoate or methamidophos (mites and insects) and metalaxyl or chloro­thalonyl (fungal diseases).

The plants height was measured from the soil surface up to the apical buds, and the stem diameter 2 em far away from the soil surface. Youngest and fully expanded leaves samples were taken at flowering and fruit setting stage, and measured the dry weight matter and, also made the tissue analysis. The leaves were first washed with tap water and after oven dried and ground in a Wiley mill type and analyzed for N (micro-Kjeldahl), P (vanadium molybdate), K, Ca and Mg (atomic absorption spectrophotom­etry). The fruits were harvested weekly, between 6 May and 22 July (Autumn-Winter). After harvesting, soil samples (0-20 em) were taken from the different treatments and analyzed. Soil temperature measurements (°C) at 5 em depth were taken both in the morning and in the afternoon.

Results and discussion

The plants height and the stem diameter were affected by the mulching treatment from 37 days after transplanting. No effect of N was observed

in this treatment, however, the unmulched plants presented some N deficiency symptoms, mainly after heavy rainfall.

The amount of dry matter on the sampled leaves were higher in the mulching treatments, and also there was a positive N effect (Table 1). On average the N leaves content was 25.8% greater with mulch, and this could be considered as a consequence of the greater efficiency in the absorption of this nutrient by the mulched plants, similarly that was observed by Locascio et al. (1985). There was no mulch effect on the leaf P content, probably due to the high initial P soil level. The opposite occurred for the K, that similarly to N also is leached in the Brazilian soils, being it's amount on the leaves about 27.7% higher in mulched plants. For Ca and Mg also there were higher values with mulch. The amount of rainfall (40.7 em) were greater from transplanting time to leaves sampling time and, on account of it, probably there were greater N, K, Ca and Mg leaching in the unmulched soil. Topdressed N significantly increased the dry matter and nutrients leaves content. Leaf nu­trients concentrations were consistent with the values presented by Fontes and Monnerat (1984).

As the N supply increased a fall in soil pH also occurred along with decreased amounts of ex­changeable K, Ca and Mg (Table 2). This effect was more intense in the unmulched soil and, K was the most effected. No significant mulch x nitrogen interaction was observed.

Total fruits yields (Table 3) were about 55% higher with mulch. In agreement with the finding of Locascio et al. (1985). There was no influence of topdressed N on yield. Early yields (up to 20 May) were not stimulated by mulch, in accord­ance with the finding of Wien and Minotti (1987) who showed that there was no consistent effect of mulch in this respect. However, the N fertili­zation decreased early production.

Soil temperature at 5 em depth was higher in the mulched treatments, mainly in the afternoon and on days without rainfall. In just ten days was the soil temperature in the mulched areas greater than to 30°C, reaching at maximum 33.8°C. Gosselin and Trudel (1986) reported maximum fruit production of bell pepper when the root zone was nearly 30°C.

Mulch and topdressed nitrogen effects on bell peppper 95

Table I. Leaf dry weight, concentrations (% of dry matter) and contents (mg leaf- 1 ) of N. P, K. Ca. and Mg in leaf tissue of bell pepper as influenced by much and N rates

Treatments Dry weight N p K Ca Mg (gllcaf)

(%) (mg/lcaf) (%) (mg/lcaf) (%) (mglleaf) (%) (mg/leaf) (%) (mglleaf)

Mulch Yes 0.24 a" 3.57 a 8.57 a 0.20 a 0.46a 5.05 a 12.11 a 4.24 a 10.17a 0.43a 1.03 a No 0.19 b 3.59 a 6.81 b 0.23 a 0.43 a 4.99 a 9.48b 3.97 a 7.54 b 0.42a 0.79 b

Nitrogen (kg ha -I) 0 0.15 b 3.43 a 5.14 b 0.22 a 0.34b 4.86 a 7.28b 3.96 a 6.02 b 0.40 a 0.60 b

200 0.24 a 3.69 a 8.86 a 0.22 a 0.53 a 5.10 a 12.24 a 4.25 a 10.20 a 0.44a 1.06a 400 0.24 a 3.61 a 8.66a 0.20a 0.48a 5.10 a 12.24a 4.10 a 98.3 a 0.43a 1.03 a

"Means followed by the same letter do not differ (Tukey's test, p < 0.05).

Table 2. Final soil test values" (0-20 em depth) as influenced by black polyethylene mulch and N rates

Treatments pH Exchangeable (meg/ 100 mL air dry soil)

K Ca Mg

Mulch Yes 5.3 ab 0.43 a 7.30 a 0.91 a No 5.3 a 0.25 b 6.77 a 0.70 b

Nitrogen (kg ha -I) 0 5.6 a 0.45 a 8.65 a 0.90 a

200 5.3 ab 0.29 b 7.35 b 0.81 ab 400 5.0b 0.28 b 5.10 c 0.69 b

"pH (CaCI 2 0.01 M); K, Ca and Mg were extracted by the resin method. b Means followed by the same letter do not differ (Tukcy's test, p < 0.05).

Table 3. Early yield (from 5th up to May 20th) and total yield for 'Ikeda' bell pepper as influenced by mulch and N rates

Treatments Early yield

(tha- 1 ) (%oftotal)

Mulch Yes 5.06 20.22 a' No 3.36 21.28 a

Nitrogen (kg ha -I) 0 4.56 23.38 a

200 3.86 19.15 b 400 4.10 20.24 b

Total yield (tha-I)

24.54 a 15.79 b

19.50 a 20.16a 20.25 a

"Means followed by the same letter do not differ (Tukey's test, p < 0.05).

In this experiment the use of black poly­ethylene mulch increased the 'Ikeda' bell pepper yield. This resulted from the increased soil temperature, decreased nutrient leaching (0-20 em depth) and, the greater nutrient absorp­tion by the crop. Finally, it was concluded that the present results is possible to eliminate the topdressed N fertilization.

Acknowledgements

The authors thank to Rubens Sader and Sergio Antonio De Bartoli from the UNESP University (Campus of Jaboticabal, S. Paulo, State, Brazil), for the English revision.

References

Asiegbu J E 1991 Response of tomato and eggplant to mulching and nitrogen fertilization under tropical con­ditions. Sci. Hortic. 46, 33-41.

BJorn-Zandstra M 1989 Nitrate accumulation in vegetables and its relationship to quality. Ann. Appl. Bioi. 115, 553-561.

Counter J W and Oebker N F 1974 Comparisons of paper and polyethylene mulching on yields of certain vegetable crops. Am. Soc. Hortic. Sci. Proc. 85, 526-531.

Decouteau DR, Kasperbauer M J and Hunt P G 1990 Bell pepper plant development over mulches of diverse colors. HortScience 25, 460-462.

Fontes PC Rand Monnerat PH 1984 Mineral nutrition and fertilization of peppers crops. lnf. Agropec. 10, 25-31.

Gerard C J and Chambers G 1967 Effect of reflective

96 Mulch and topdressed nitrogen effects on bell peppper

coatings on soil temperature, soil moisture and the estab­lishment of fall bell peppers. Agron. J. 59, 293-296.

Gosselin A and Trudel M J 1986 Root-zone temperature effects on pepper. J. Am. Soc. Hortic. Sci. 111, 220-224.

Locascio S J, Fiskell J G A, Graetz D A and Hauck R D 1985 Nitrogen accumulation by pepper as influenced by mulch and time of fertilizer application. J. Am. Soc. Hortic. Sci. 110, 325-328.

Newbould P 1989 The use of nitrogen fertilizer in agriculture. Where do we go practically and ecologically? Plant and Soil 115, 297-311.

Wien H C and Minotti P L 1987 Growth, yield, and nutrient uptake of transplanted fresh-market tomatoes as affected by plastic mulch and initial nitrogen rate. J. Am. Soc. Hortic. Sci. 112, 759-763.

M.A. C. Fragoso and M.L. van Beusichem (eds.) Optimization of"plant nutrition 97-100, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-043

Long-term effects of gypsiferous coal combustion ash applied at disposal levels on soil chemical properties

R.F. KORCAK and W. DORAL KEMPER Fruit Laboratory and National Program Staff, USDA, Agricultural Research Service, BARC-W, Beltsville, MD 20705, USA

Key words: apple orchard, boron, calcium, magnesium, mineralogy, spent bed ash, sulfur

Abstract

Currently, there is renewed interest in the agricultural utilization of coal combustion byproducts. Field sites where high rates ( 112 Mg ha - 1) of high gypsum coal combustion spent bed ashes were surface applied in 1980 within fruit tree orchard rows were identified and sampled with depth. The objective of this study was to examine the effects on long-term exposure/leaching of these materials on soil profiJe chemical properties. When applied, the material had an aqueous pH of 12.5 and consisted of about 52% calcium sulfate, 33% calcium oxide and 15% coal ash residues. Eleven years after ash application, soil pH is significantly higher in the top 66 em of the treated sites compared to unamended sites. This has been accompanied by increases in extractable and total calcium and total boron and sulfur with a concomitant reduction in extractable magnesium. Remaining pieces of the applied spent bed material are composed primarily of calcite and quartz with some gypsum associated with large pieces.

Introduction

It is forecast that the burning of coal for power production will generate nearly 100 million tons of ash per year by the year 2000 in the United States alone. Atmospheric fluidized bed combus­tion (AFBC) technology represents an alterna­tive to conventional coal power production. Briefly, AFBC involves mixing fine grain coal and a sorbent (usually limestone) in a furnace with a 'fluid-bed' achieved by injecting air. The sorbent acts as an absorber of S from the coal during combustion. The resultant byproducts, the spent bed material and the captured fly ash, are dry, alkaline materials. Although variable, the spent bed ash is high in gypsum and un­reacted calcium oxide.

A number of studies have reported the poten­tial for utilizing these materials in agriculture (Korcak 1980, 1985; Stout et al., 1979; Terman, 1978). However, there arc still concerns regard­ing potential detrimental effects of high applica-

tion rates on crop growth caused by high salinity, high pH and induced phytotoxicities, particularly boron (Adriano et al., 1980).

In 1980 we applied spent bed material as a soil 'cap' about 5 em thick within the rows of an established, young apple orchard at rates up to 112 Mg ha - 1 • Data is needed on the long-term fate of coal combustion by-products in agricul­ture since most studies in the literature deal with greenhouse pot experiments or short-term, low application field studies. This report examines pertinent chemical factors within the soil profile eleven years after application from a relatively high initial application of spent bottom ash.

Materials and methods

The apple orchard site from the original experi­ment was a Rumford loamy sand soil on a 2 to 5% slope. These soils arc deep and well-drained containing some clay but little silt with an initial

98 Korcak and Kemper

surface aqueous pH of 5.8 and about 1.5% organic matter. The AFBC spent bed material was applied as a surface cap in 1980 and re­mained on the surface until the original experi­ment terminated in 1985. At this time the plots were plowed to a depth of 25 em and planted to a succession of grass crops. Triplicate treated (112 Mg ha - 1 , spent bed ash) and control (un­amended) plot areas from the original experi­ment (Korcak, 1988) were located and soil samples were taken at depths of 0-24, 25-66, 67-86, and 87-114 em in the fall of 1991. These samples were air-dried and sieved through a 2 mm sieve prior to analysis. Soil pH (1: 1 0.1 M CaCU, extractable Ca and Mg (1 N ammonium acetate), total Ca, Mg, and B (nitric/perchloric acid extractable), and total S (Leco total com­bustion) concentrations were determined.

The spent bed material, when applied, had an aqueous pH of 12.5 and contained 52% CaS04 ,

33% CaO, 0.6% CaS0 3 , 0.8% MgO, 0.3% NaCI, 0.2% P20 5 , 4.5% R 2 0 3 (primarily Fe and AI oxides), and 7% Si0 2 plus insoluble matter. Trace element concentrations were 1.92% AI, 1.02% Fe, 39mg kg- 1 Cu, 112mg kg- 1 B, 220 mg kg - 1 Mn, and 80 mg kg - 1 Zn. Additional details on the material have been reported by Korcak (1988). The by-product was applied to four apple (Malus domestica Borkh.) types: 'Spuree Rome' on M9, 'Redchief Delicious' on M7A, 'Sturdespur Delicious' on M9, and 'Red­chief Delicious' on M9 interstem and MM106 rootstock.

Statistical comparisons of the triplicate means were made by t-test, significance differences reported are at the 5% level.

Results

Soil profile pH was significantly elevated by the spent bed treatment down to 66 em (Fig. 1 ). The water table fluctuates between 66 and 86 em depths. Therefore results in and below this level may be affected by lateral movement of water. This probably accounts for the treated soil hav­ing lower pH than the check treatment at 86 em, but higher pH than the check at 114 em depth (Fig. 1 ). Correspondingly, extractable and total soil Ca were elevated by spent bed treatments

pH (0.01 M CaCI2)

Extractable Ca

114

Total Ca (%)

D Control

D Control

lillillJ Treated

D Control

[JJ Treated

Fig. 1. Soil profile pH, extractable Ca (mgkg- 1 ) and total Ca values from triplicate horizon samples from spent bed ash treated ( 112 Mg ha _,) and unamended controls. *Implies significance between treatments within horizons.

(Fig. 1). The higher ratio of Ca to Mg in the applied material is reflected in the significant reduction in extractable Mg with spent bed treatment, however, total Mg was significantly higher in the surface 25 em of soil compared to control soils (Fig. 2).

The high total soil Ca in the surface 25 em of soil and the higher total Mg versus the lower extractable Mg in the profile is partly due to the original cementitious nature of the applied spent bed ash. Immediately after initial application and wetting, the material set-up as a porous cement. Upon plowing pieces of this material ranging from <1 to about 5 em in diameter are common

a , ~

.! 25 c e "'" .!! ... Q. 86 w ~ ,,

~

~

~

10

J.

I*

I*

20 30 40

Extractable Mg

D Control

J • Treated

I*

50 60 70

D Control

114 t======~=~~~L_ _ ___,__C±Jg·)~U~rrere"'_at~etiJd 600 700

Total Mg

Tolal ll

Fig. 2. Soil profile extractable and total Mg and total B (mg kg -I) values from triplicate horizon samples from spent bed ash treated (112 Mg ha -I) and unamended controls. *Implies significance between treatments within horizons.

in the plow layer. X-ray diffraction analysis of the smaller pieces showed a predominance of calcite and quartz, some ettringite (a Ca-Al-S mineral) with very little if any gypsum present. Gypsum was found in the larger pieces. There­fore, a portion of the original Ca applied still remains in the slightly soluble calcite form and it is probable that a portion of the applied Mg remains in the calcite as a contaminant, in both cases relatively unextractable.

Total B has been significantly increased from the surface to below 66 em (Fig. 2). These higher B levels were not reflected in increased apple

Long-term effects gypsiferous coal ash 99

tissue concentrations of B throughout the first six years after application (Korcak, 1988).

Total Sin the plow layer (0-25 em) was 43 mg kg -t in the treated area and 16 mg kg - 1 in the control soils. In the subsoil ( 66 em) the S content of the treated area was 120 mg kg -t and 65 mg kg - 1 in the control area.

Discussion

Most literature on the agricultural utilization of spent bed ash centers around use at rates at or near the soil lime requirement. Horticultural utilization at relatively high rates may be a practical consideration since the material, in orchard settings, can be surface applied and remain in place for long periods. Results of the original apple study showed, in general, good growth and productivity of the trees and an enhanced Ca status (Korcak, 1988). This study has shown that the Ca, Mg, B, and S relation­ships are altered not only in the surface horizons but also throughout the profile.

The CaSO 4 in the spent bed material allows greater movement of Ca within the soil profile due to the greater solubility of CaSO 4 • After hydration of CaO to Ca(OH) 2 it converts slowly to the more insoluble CaC0 3 or calcite as indicated by the presence of calcite in eleven­year-old pieces of spent bed, however the rate of conversion is unknown. Current studies are aimed at more closely following the mineralogy of spent bed ash and other coal combustion residues.

A potential area of concern is the imbalance created in the Ca to Mg ratio when evaluated on an extractable nutrient basis. This situation can be corrected by application of MgSO 4 as a soil amendment or, for orchards, as a foliar spray. Alternatively, the use of dolomitic limestone as part of the absorbent during coal combustion would greatly increase the Mg content of the residue and lessen the potential for Ca: Mg imbalances.

Further research is in progress on the trace element relationships in these soils which in­cludes both chemical analysis and the growth of indicator crops on these soils which have been

100 Long-term effects gypsiferous coal ash

exposed to high rates of spent bed residues for long periods.

Acknowledgement

The authors express their appreciation to Ms Jody Solem, Chemistry Department, North Dakota State University for assistance with x-ray diffraction analyses.

References

Adriano D C, Page A L, Elseewi A A, Chang A C, and Straughan I 1980 Utilization and disposal of fly ash and other coal residues in terrestrial ecosystems: A review. J. Environ. Qual. 9, 33-344.

Korcak R F 1980 Fluidized bed material as a lime substitute and calcium source for apple seedlings. J. Environ. Qual. 9, 147-151.

Korcak R F 1985 Effect of coal combustion wastes used as a lime substitute in nutrition of apples in three soils. Plant and Soil 85, 437-441.

Korcak R F 1988 Fluidized bed material applied at disposal levels: Effects on an apple orchard. J. Environ. Qual. 17, 469-473.

Stout W L, Sidle R C, Hem J L, and Bennett 0 L 1979 Effects of fluidized bed combustion waste on the Ca, Mg, S, and Zn levels in red clover, tall fescue, oat, and buckwheat. Agron. J. 71, 662-665.

Terman, G L, KilmerV J, Hunt C M, and Buchanan W 1978 Fluidized bed boiler waste as a source of nutrients and lime. J. Environ. Qual. 7, 147-150.

Reprinted.from Plant and Soi/154: 29-32, 1993.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 101-105, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-149

Effects of composted municipal waste and a paper mill waste composted with bark on the growth of vegetable crops

LUIS MIGUEL BRITO and PAUL HADLEY Ponte de Lima Higher School of Agriculture, Viana do Castelo Polytechnic, Refoios, 4990 Ponte de Lima, Portugal and Department of Horticulture, School of Plant Sciences, University of Reading, Whiteknights, Reading, RG6 2AS, UK

Key words: ammonia toxicity, Brassica oleracea, compost, electrical conductivity, heavy-metals, Lactuca sativa L.

Abstract

There is increasing interest in the use of composted organic wastes for intensive vegetable production. Here, systematically designed fertilizer experiments with factorial combinations of organic wastes, a town waste compost and a paper mill waste composted with bark, and top-dress ammonium nitrate were carried out on summer cabbage (Brassica oleracea var. capitata cv. Lima) and randomised block designed experiments were conducted to evaluate seedlings emergence and plant growth rates on lettuce (Lactuca sativa L. cvs. Serda and Animo). Summer cabbage responded positively to base organic wastes in the absence of top-dressed ammonium nitrate whilst high base organic waste application rates caused cabbage growth reduction. There was clear evidence that lettuce growth rates increased with increasing concentration of composted paper mill waste whereas negative responses occurred with composted town waste. Inhibitory effects of town waste compost on lettuce seedling emergence and plant growth were likely to be the result of a combination of the high electrical conductivity, ammonia toxicity and a low degree of stabilisation of this compost. Field application rates of composts were limited by compost heavy-metal concentration to rates of 27 t ha-l year -l and 32 t ha-l year -r respectively for paper mill waste com posted with bark and town waste compost.

Introduction

With the recent interest in decreasing air and water pollution, organic wastes are being advo­cated for utilization on cropped land rather than by simple disposal as crop yields can be in­creased and long-term soil productivity improved by the use of organic wastes (Brady, 1984). Despite beneficial agronomic effects of organic wastes, the random application of these products in agriculture is restricted because of actual or potential environmental hazards due to the pres­ence of heavy metals, organic toxicants and disease producing organisms (Danneberg et a!., 1981) as well as excessive salt concentration and induced nitrogen deficiency (Juste et a!., 1987),

however, there is a need for clear quantitative data to determine both positive and negative effects of application of organic wastes to ag­ricultural land. Here we describe studies de­signed to quantify the response of vegetable crops to increasing levels of two contrasting composted organic wastes: (i) a separated and composted town waste and (ii) a paper mill waste composted with bark.

Methods

Experiments were carried out at Ponte de Lima Higher School of Agriculture, NW Portugal, with summer cabbage (Brassica oleracea var.

102 Brito and Hadley

capitata cv. Lima) as the test plant for field experiments and lettuce (Lactuca sativa L. cvs. Animo and Serda) for greenhouse experiments. Two systematically designed fertilizer experi­ments (Cleaver eta!., 1970) were arranged in the field with three randomized blocks experiment - 1

and two large plots ( 100 m 2 ) block- 1 • The large plots were arranged to give factorial combina­tions of 5 levels of each organic waste compost (town waste compost and paper mill waste com­posted with bark) calculated on the basis of the organic matter content and 5 levels of top dress ammonium nitrate. In the first experiment, the rates of application of compost waste organic matter were 0, 3, 6, 9 and 12 t O.M. ha - 1 and increased systematically from sub-plot to sub­plot along one axis of the main plot and the levels of top-dress ammonium nitrate were 0, 70, 140, 210 and 280 kg N ha - 1 and increased systematically along the second axis. In the second experiment, a similar approach was taken except that the 5 organic matter applications were split to give rates of application equivalent to 1/3 of that of the first experiment. Cabbage seedlings were planted with 3-4 leaves after selection for uniformity, on the 22nd April 1991 and plants were harvested on the 5th July 1991. Greenhouse experiments included (i) a pot ex­periment, laid out as a randomised block design with 4 replications, to study the effect of organic composts mixed with a sandy loam subsoil at 5 ratios by volume (0%, 25%, 50%, 75% and 100% compost) on lettuce (cv. Amino) growth rates, and (ii) an experiment, arranged as a randomised block design with factorial treatment structure and 3 replications, to study the effects on lettuce (cv. Serda) seedling emergence of composts mixed with a sandy loam topsoil at 6 ratios by volume (0%, 17%, 33%, 50%, 67% and 100% compost) and with ammonium nitrate applied at rates equivalent to 0 and 200 kg N h -l

a .

Results

Chemical characteristics and mineral composi­tion found for organic waste composts is listed in Table 1. Both organic materials typically contained significant heavy metal contamination.

Table I. Chemical characteristics and mineral composition of waste composts

Component Compost

Town waste Paper mill waste

pH 8.3 7.5 E.C.(mScm- 1 ) 3.46 2.12 Organic matter (%) 56 44 Total-N (%) 1.2 0.62 NH; -N (mg kg- 1 ) 1850 n.d. No; -N (mg kg- 1 ) 229 79 C/N 27.1 41.2 MG(%) 0.15 0.14 Ca (%) 2.6 12.2 Fe(%) 1.52 0.52 Ni (mg kg I) 56.0 90.4 Cr (mg kg 1 ) 63.4 61.4 Cd (mg kg -I) 4.5 5.5 Mn(mgkg- 1 ) 325.3 234.4 Cu (mg kg- 1) 363.4 19.4 Zn (mg kg -I) 936.9 45.7

Zinc and copper contamination was considerably higher in town waste than in the paper mill waste whereas the reverse was true for nickel and cadmium contamination. The response of sum­mer cabbage to base organic waste composts and top-dress ammonium nitrate is shown in Figure 1. Yield of summer cabbage increased dramati­cally with top dressed ammonium nitrate, how­ever, responses to organic waste amendments were smaller. Negative effects of organic applica­tions were apparent at the highest levels of inorganic nitrogen applications.

Lettuce seedlings transplanted into the com­post mixtures with town waste compost died or showed insignificant growth (Table 2) whereas mean plant dry weight accumulation day - 1 ,

increased for all paper mill compost treatments up to the second harvest (33 days after trans­planting) and, thereafter, declined for less than 50% compost treatments but continued to in­crease up to the third harvest ( 49 days after transplanting) for the other compost treatments. For the paper mill waste compost, raising com­post concentration did not significantly affect emergence of lettuce seedlings (Table 3). In contrast, raising town waste compost above 50% compost drastically reduced emergence of lettuce seedlings.

Organic wastes in vegetable production 103

(a)Town waste compost

(b)Paper mill s l udge compost

Fig. 1. Effect of increasing levels of base organic matter applications of (a) town waste compost and (b) paper mill sludge compost (tOM ha ') and of top-dress applied ammonium nitrate (hg N ha _,) on the yield response of summer cabbage ( t ha _, ).

Discussion

The growth response of summer cabbage to low rates of ammonium nitrate application and the smaller responses to organic waste amendments are likely to be related to the N availability characteristics of the materials. Ammonium ni­trate is soluble, and the N is immediately avail­able for uptake when the fertilizer is applied to the soil. Organic N is initially unavailable when

applied to the soil and it has to be released through microbial degradation. The negative growth response to large applications of both mineral N and organic composts, is probably a consequence of the high salt concentration (mea­sured as electrical conductivity - EC) of com­posts leading to high osmotic pressure in the soil solution. The main cause of lettuce death and of the inhibition of lettuce seedling emergence observed with town waste compost was also

104 Brito and Hadley

Table 2. Effect of town waste compost and paper mill waste compost in a range of volume combinations with subsoil on dry matter accumulation (g/plant) lettuce shoots

Compost treatment Dry weight (g/ plant) of lettuce shoots

Days after transplanting

Control (subsoil) Town waste 25% Town waste 50% Town waste 75% Town waste 100% Paper mill waste 25% Paper mill waste 50% Paper mill waste 75% Paper mill waste 100%

16

0.20 0.10 0.03 0.05 0.00 0.26 0.31 0.30 0.31

33

0.55 0.11 o.m 0.07 0.00 1.85 1.90 1.72 1.77

49 62

0.88 0.75 0.22 0.12 0.00 0.00 0.15 0.09 0.06 0.00 2.89 3.47 4.26 5.28 4.25 5.85 6.02 8.73

Table 3. Effect of town waste compost and paper mill waste compost in a range of volume combinations with topsoil and 0 and 200 kg N ha _, of supplied ammonium nitrate (kg N ha _,), on the emergence (%) of lettuce seedlings

Compost treatment Lettuce seedling emergence (%)

Supplied ammonium nitrate (kg N ha _,)

Control (topsoil) Town waste 17% Town waste 33% Town waste 50% Town waste 67% Town waste 100% Paper mill waste 17% Paper mill waste 33% Paper mill waste 50% Paper mill waste 67% Paper mill waste 100%

0

43.3 63.3 63.3 56.0 44.3 0.0

48.7 44.3 42.0 53.7 43.7

"- likely to be the result of the high EC and in addition the low degree of stabilisation of this compost. The conclusion that this compost was less stabilised than should be aimed for composts of this type is supported by its high ammonia concentration (typically greater than 1850 mg kg - 1), strong odour and the fact that it was attacked by a range of fungi during the period of the experiment. Ammonia gas liberation was reported by Katayama et al. (1985) to be the main inhibitory factor of immature sludge com­post on plant growth while Zucconi and Bertoldy (1987) suggests a maximum limit of 400 mg kg -I of NH; - N for town waste composts which is signficantly lower than for the town waste com­post examined here. Heavy metal contamination

200

46.7 75.0 59.7 63.7 18.3 0.0

24.7 21.3 18.7 19.0 26.7

of the organic materials was typically high. Land application rates of composts could be limited on the basis of their cadmium and zinc concen­tration to rates of 27 t ha - 1 year - 1 and 32 t ha- 1

respectively for paper mill waste composted with bark and town waste compost.

Acknowledgements

This research is sponsored by the Portuguese Ministry of Education (Instituto Nacional de Investigat;ao Cientffica). The authors thank Prof Dr J Quelhas dos Santos, Technical University of Lisbon.

References

Brady N C 1984 The Nature and Properties of Soils. Macmillan, New York, 677 p.

Cleaver T J, Greenhood D J and Wood J T 1970 Sys­tematically arranged fertilizer experiments. J. Hort. Sci. 45, 457-469.

Danneberg 0 H, Storchschnabel G and Ullah S M 1981 The analysis of nitrogen and humus in connection with a field test for the fertilization with straw and sewage sludge. In The Influences of Sewage Sludge Application on Physical and Biological Properties of Soils. 11-24. D. Reidel Publishing Company, Brussels.

Juste C, Solda P and Lineres M 1987 Factors influencing the

Organic wastes in vegetable production 105

agronomic value of city refuse composts. In Environmental Effects of Organic and Inorganic Contaminants in Sewage Sludge. pp 388-398. D. Reidel Publishing Company, Brussels and Luxemburg.

Katayama A, Hirai M, Shoda M, Kubota Hand MoriS 1985 Inhibitory factor of sewage sludge compost for growth of Komatsum Brassica campestris L. var. rapiferefroug. En­viron. Pollut. 38, 45-62.

Zucconi F and Bertoldi M 1987 Compost specifications for the production and characterization of composts from municipal solid waste. In Compost: Quality and Use. Eds. M Bertoldy, M P Ferranti, P L'Hcrmite and F Zucconi. pp 30-50. London.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 107-115, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-204

Assessment of plant-available nitrogen in processed organic wastes

W.M.F. RAIJMAKERS and B.H. JANSSEN Department of Soil Science and Plant Nutrition, Wageningen Agricultural University, P.O. Box 8005, 6700 EC Wageningen, The Netherlands

Key words: C-N ratio, double-pot technique, nitrogen mineralization, organic fertilizer, recover fraction, relative effectiveness

Abstract

In the near future large amounts of animal manure and organic waste products will be processed and used as organic fertilizers. When the rate at which processed organic materials (POMs) release nutrients can be predicted, chemical fertilizer application rates can be reduced accordingly. The assessment of nitrogen availability was studied and N availability was related to chemical properties for a number of POMs. A second purpose of the experiment was to evaluate the suitability of the so-called double-pot technique. Using that technique, the uptake of N by perennial ryegrass (Lolium perenne L., cv. Trani) was measured in a 62-days experiment. Availability of N was expressed by the relative effectiveness (RE), i.e. the ratio of the fractions of N recovered from a particular POM and from the reference chemical fertilizer. RE ranged from 0.06 to almost 1 for the various POMs. The calculated fraction mineralized organic N (FMON) ranged from 0.04 for well composted plant material to 0.6 for industrial sewage sludge. FMON was found to be determined by both the C-N ratio and the decomposability of the POMs. For POMs with a high content of inorganic N the assessment of FMON could be improved by leaching inorganic N before use. It was concluded that the double-pot technique enables assessment of the availability of a single nutrient, irrespective of the supply of other nutrients.

Introduction

Objectives

It is to be expected that in the near future large amounts of animal manure and other organic products, such as the organic fraction of munici­pal solid waste, will be processed and marketed as organic fertilizers and soil amendments. This is a result of governmental measures taken to relieve environmental problems. Farmers may be interested in such products because of the or­ganic matter they contain, but also for their nutrients. Use of such products implies that the application rate of chemical fertilizers can be reduced. It is then necessary to know the rate at which processed organic materials (POMs) re­lease nutrients. The rate of N release is rather

unpredictable and this is one of the reasons why farmers hesitate to use these products. This paper describes a pot experiment carried out to assess the release of N from a number of quite different POMs was related the uptake of N by the test plants to the chemical properties of the POMs, such as organic and inorganic N content, and C-N ratio and decomposability of the or­ganic compounds.

In preliminary experiments it proved difficult to assess the availability of organic N in POMs having a large fraction of inorganic N. The contribution of mineralized organic N to N uptake is then small compared to that of inor­ganic N initially present. Such an overruling effect of inorganic N also appears in a study of Van Erp and Van Dijk (1992). This difficulty may be lessened by leaching most of the inor-

108 Raijmakers and Janssen

ganic N from the POMs before use. This was verified by comparing leached and non-leached aliquots of the POMs.

This study was also intended to evaluate the suitability of the so-called double-pot technique (Janssen, 1990) for assessment of the availability of a single nutrient, irrespective of the supply of other nutrients by the POMs.

Theoretical considerations

Relative effectiveness Available N in organic products is usually mea­sured as the amount of N mineralized during a certain incubation period or as the amount of N taken up by plants. Commonly used indices for comparing fertilizers are relative effectiveness (RE) and substitution rate (Barrow, 1985; Bar­row and Bolland, 1990; Chien et al., 1990). The comparison can be based on either yield or nutrient uptake. The most accurate indication for nutrient release will be achieved by compar­ing nutrient uptake (Van Burg, 1963). For that reason we used RE as the ratio of the recovery fraction (RF) of the POM to that of the refer­ence chemical fertilizer. RF is the fraction of the applied N that has been taken up by the plant.

The value of RF usually is less than 1, because part of the nutrient may be lost by volatilization, denitrification, or leaching, or simply remain in the soil. Such may be the fate of initially applied inorganic N in POM or CF as well as of organic N once it has been mineralized, since they exist as NH; or NO~ or both. Consequently the values of RF will be the same for inorganic and mineralized organic N.

POMs contain N in inorganic and organic forms. Hence, the over-all RE of POMs may be considered to consist of at least two parts:

(1)

where F refers to fraction of N present in POM and the subscripts t, o and i refer to total, organic and inorganic respectively. Since RF of initially added inorganic N in POMs and refer­ence fertilizer are equal, is RE; = 1. As REo = FMON x RE;, where FMON is the fraction of mineralized organic N, it follows:

RE-F FMON=RE = t I

o Fo (2)

Mineralized organic N (MON) can be calculated as the product of FMON and the quantity of applied organic N (A 0 ). Because F; = Ai/A, and Fo = Ao/A" it follows:

(3)

where A, and A; are applied total and inorganic N, respectively.

Nitrogen mineralization The fraction of organic N that is mineralized from an organic substrate depends on charac­teristics of that substrate and of the micro-organ­isms involved, and on external conditions. The relevant characteristics of the micro-organisms are C-N ratio of their cell material and the efficiency of substrate utilization, in other words the dissimilation-assimilation ratio. Given a set of external conditions and a certain microbial population, the rate of N mineralization is direct­ly related to the rate of decomposition of the organic substrate and of the organic N content (usually indicated by the C-N ratio), as was shown by, among others, Gilmour et al. (1985), Melillo et al. (1982), Schulz (1988), and Smith and Hadley (1990). In general, it can be said that proteins are decomposed more readily than most other N-containing compounds. During de­composition easily decomposable compounds are broken down first and the remaining organic substrate will become increasingly resistant to decomposition. Hence composted materials and animal manures will be more resistant than "fresh" organic materials. A possible index for decomposability is lignin content, occasionally in combination with the content of one or more other chemical compounds (Tian et al., 1992a,b).

The relation between FMON and C/N of equally decomposable substrates is negative. If the substrates are decomposed by the same type of micro-organisms the relation is linear, as was derived from experiments by Jensen (1929). Organic materials with a high C-N ratio initially

have a negative mineralization (N immobiliza­tion). Because of the immobilization of N and the dissimilation of C, the C-N ratio of the remaining organic material gradually decreases during decomposition. After a shorter or longer period, sufficient organic N is present to switch from immobilization to mineralization.

For substrates with an equal C-N ratio, the rate of N mineralization during a certain time interval is determined by the rate of decomposi­tion of the organic material. In principle this also holds for the rate of N immobilization. It may happen, however, that the quantity of available inorganic N is less than the amount required by the micro-organisms. As a result, the decomposi­tion process slows down and the relationship between substrate decomposability and rate of (negative) N mineralization is blurred (Fog, 1988). The effect of substrate decomposability on the rate of N mineralization is thus expected to be most obvious for organic materials with low values for the initial C-N ratio.

The double-pot technique When fertilizers are compared, precautions must be taken to ensure that the supply of nutrients other than the one under study is non-limiting and equal for all treatments. This can easily be achieved in the case of straight fertilizers, but in the case of POMs this is impossible, because it is usually not known beforehand which fraction of nutrients present will become available to the plant during the uptake period. Such problems can be overcome using the so-called double-pot technique (Janssen, 1990), where plants are able to take up nutrients simultaneously from two compartments. An upper pot contains the sub­strate to be tested and a container underneath is filled with nutrient solution. When a nutrient is omitted from the nutrient solution, uptake of that nutrient is only possible from the upper pot. Possible differences between the different POMs in their ability to supply nutrients other than the one under investigation are unlikely to affect the growth of the test plants, because these nutrients are provided in ample amounts by the solution below.

Plant-available N in organic wastes 109

Materials and methods

Eight POMs (Table 1), leached and non­leached, were compared with NH4N0 3 as chemi­cal fertilizer (CF). POMs and CF were applied at 4 levels. Leaching of the POMs was by repeated (2-6 times) extraction with 0.01 M CaC1 2 fol­lowed by centrifuging (20 min., 1600 g). The leached and non-leached POMs were analyzed for dry matter content (10SOC), and, after drying (30°C) and grinding (0.5 mm), for organic matter (loss-on-ignition at 850°C), carbon (dichromate­oxidation), and total N. For total N determi­nation, POMs were digested in a mixture of sulphuric and salicylic acid and Se, to which H 2 0 2 was added. Total N was determined in the digests by the indophenol blue method. Concen­trations of NO~ -Nand NH; -N were determined in the supernatant of centrifuged suspensions of fresh POMs in 0.01 M CaCI2 (1: 10 mass ratio, shaking for 2 hrs). Analytical data of the POMs before and after leaching are listed in Table 2. Osmolarity was measured in the supernatants of centrifuged suspensions of fresh POMs in de­mineralised water (mass ratio 1 : 10, shaking for 2 hrs; Osmometer 030, Gonotec; results not shown).

The POMS described in Table 1 may be classified into three categories: (I) composted plant material (RC, AEC), (II) materials partial­ly or completely consisting of composted or digested animal manure (SMC, CPM, DPS), and (III) "fresh", com posted nor digested materials (ANR, SDP). This classification is supported by observations by Van Lune and Hassink (1991), who reported decomposition rates of cow ma­nure and a product equivalent to SMC (Group II) to be 2-3 times higher than of material comparable to AEC and EC. Based on the similar rate of decomposition of the latter two materials, EC fits in Group I. The resistance against microbial decomposition is thus expected to be relatively high in the composted materials of Groups I and II, and relatively low in the materials of Group III, although it should be noted that any discrete classification of decom­posability remains somewhat subjective.

Table 2 shows relatively high fractions of inorganic N for DPS, CPM, and SMC, and

110 Raijmakers and Janssen

Table 1. Codes, names and descriptions of POMs and chemical fertilizer

Code Name

RC Reference compost

AEC Aerobic compost

ANR Anaerobic residue

SMC Spent mushroom compost

CPM Composted pig manure

DPS Digested pig slurry

EC Earthworm compost

SDP Sludge dairy production CF Chemical fertilizer

Description

Equal to AEC, but dried at 70°C, to inhibit microbial processes during long term storage Organic fraction of municipal solid waste, separated at the source and eomposted outdoors. Organic fraction of municipal solid waste, separated at the source. Residue after the first phase (hydrolysis) of anaerobic digestion. Mixture of horse and chicken manure, straw, peat and gypsum, used as substrate for mushroom culture. Mixture of pig dung and wood chips. Pigs were housed for 2 years on a frequently mixed 70-cm thick bed. Solids and fluid were separated following anaerobic digestion. After aerobic treatment (nitrification), fluid was condensed, mixed with dried solids and pelletized. Excrement of earthworms fed on 50: 50 mixture of apple pulp and agroindustrial waste water treatment sludge. Waste water treatment sludge from a cheese factory. NH 4NO, (Merck)

Table 2. Mass fraction of dry matter (DM) in fresh POMs, and mass fractions of organic matter (OM), organic carbon (C), total N (N,), inorganic N (NJ, and organic N (NJ in dry matter, inorganic N as fraction of total N (F,) and C-N ratio of organic N (C/N). n.a. =not analyzed

DM OM c (g g-,)

(gkg-')

Non-leached POMs RC 0.98 331 149 AEC 0.56 319 145 ANR 0.14 915 581 SMC 0.35 637 293 CPM 0.44 762 367 DPS 0.88 679 281 EC 0.36 523 238 SDP 0.09 764 364

Leached POMs RC 0.49 n.a. 179 AEC 0.49 n.a. 133 ANR 0.13 n.a. 579 SMC 0.22 n.a. 291 CPM 0.22 n.a. 364 DPS 0.36 n.a. 334 EC 0.36 n.a. 221 SDP 0.10 n.a. 350

Chemical fertilizer CF 1.00

equally low fractions of inorganic N for the other POMs. Inorganic N fractions of POMs initially high in inorganic N were clearly reduced by leaching. C/N of most POMs is virtually unaf­fected by the leaching procedure. The only

N, N, No F, C/N

(g g -[)

13.9 0.25 13.6 0.02 11 12.1 0.22 11.8 0.02 12 28.0 0.23 27.8 0.01 21 17.6 2.19 15.4 0.12 19 20.1 4.12 16.0 0.20 23 42.4 36.49 5.9 0.86 48 10.5 0.29 10.2 O.D3 23 69.2 1.79 67.4 O.D3 5

13.6 0.08 13.5 0.01 13 11.7 0.37 11.3 O.D3 12 26.5 0.31 26.2 0.01 22

14.4 0.33 14.1 0.02 21 19.7 0.17 19.5 0.01 19 24.0 2.58 21.4 0.11 16 9.4 0.05 9.4 0.00 24

57.9 2.37 55.5 0.04 6

349.8 349.83 1.00

exception is DPS, of which the calculated initial C/N probably is inaccurate due to the low F 0 •

A pot experiment was carried out in a green­house in the spring of 1992. Available N in various POMs was measured by N uptake by

perennial rye grass (Lolium perenne L., cv. Trani). The experiment was continued for 62 days (24 hrs mean temperature was 21 °C, the photoperiod was maintained at 16 hrs, using artificial illumination). During that period the grass was cut once or twice, depending on its growth rate. The nutrient solution was renewed every 2 to 3 weeks. At the end, shoots and roots (in both compartments) were harvested. Roots in the upper pots were separated from the sand­POM mixture by sieving and washing with de­mineralised water. Residual inorganic N in the pot was measured by extraction of the sieved sand-POM mixture (1 : 10 mass ration, 0.01 M CaClJ. After drying (70°C) and weighing, plant material was analyzed for total N by the method described above. Nitrogen uptake was calculated as the sum of N in cuts and finally harvested material minus seed N (15 mg per pot).

The experimental set-up is shown in Figure 1. Roots of seedlings pass through the gauze bot­tom of the upper pot and reach the nutrient solution in the tank below. Perforated buckets are used to keep the roots separated. The upper pots (effective volume 2.1 L) were filled with a mixture of inert quartz sand and POM (not ground, sieve 10 mm) or CF. Perennial ryegrass was sown in the upper pots. The composition of the nutrient solution in the tank was (mM): MgS04 0.75, KH 2P0 4 3.0, K2S04 1.0, CaC12

3.0, and trace elements (mg L - 1): B 0.5, Mn 0.05, Zn 0.05, Cu 0.02, Mo O.Gl, Fe 1.0, added as Fe-citratc. Pots with leached and non-leached

Fig. 1. Schematic section of the double-pot equipment. Left: general view of tank and pots. Right: close-up of one experimental set.

Plant-available N in organic wastes 111

POMs were systematically distributed over 6 tanks. Additionally each tank held 4 pots with CF. For POMs the number of replicates varied from 1 to 3. On each tank (150 L) 28 upper pots were randomly placed and rotated every other day.

Maximum application of non-leached POMs was such that (i) the amount of available N (estimated as 90% of inorganic N plus 20% of organic N in average, as derived from prelimin­ary experiments) did not exceed 400 mg N I pot, (ii) that the osmotic potential of the soil solution was below 0.25 MPa, and (iii) that total moisture content did not exceed 55% of the maximum water-holding capacity of the mixture of POM and quartz sand. In all pots moisture content was kept at that level by daily weighing and watering.

For leached POMs maximum application rates of organic N could be much higher than for non-leached POMs, without causing salt dam­age. The amount of inorganic N per pot was adjusted to the same level for each type and application level of POM. Based on preliminary experience, it was estimated that 100 mg inor­ganic N per pot would be sufficient as initial quantity. By this criterion, maximum application rates also depended on residual inorganic N in the POMs after leaching. Maximum application rates for leached ANR and SDP were, however, lower due to limited amounts of leached POMs. Where necessary, the difference between inor­ganic N initially present in the applied leached POMs and the 100 mg N per pot was made up by addition of NH4N0 3-solution. Application rates of CF were set to cover the range of expected N uptake in the pots containing POMs.

Results

Figure 2 shows the relationships between N recovery and total applied N, where recovery refers to N taken up by the plants. Residual inorganic N at harvest was negligible and most plants showed signs of N deficiency, indicating efficient extraction of inorganic N from the upper pots. For leached POMs (Fig. 2B,D) applied N was corrected for additionally applied NH4N0 3 , and recovered N for N recovered from

112 Raijmakers and Janssen

Recovered N (mg/pot)

1000

A

500

0

1000

c

500

.. • !:>

··>~· o_ .. 0 ... o 0 -:· .. 0 .... ·

0 1000 2000

'RC . AEC . ANR

0 SMC

A CF

~

0

,--­CPM . DPS . EC 0

SDP A

'----

3000

B -

RC . AEC . ANR

0

s~c

-

a 0 -0 • •····· •... ·· •

... ri. ~' "-"'"·

D

• . .. .. · ... -~--·~~- ·o ---~. -G

0 1000

..•. • 2000

,--­CPM . DPS . EC 0

SDP ~

3000

Applied total POM-N or fertilizer-N (mg/pot)

Fig. 2. Relation between recovered N and applied total N in POM or reference fertilizer. A, C: non-leached POMs, B, D: leached POMs.

that additionally applied N, assuming a RF equal to RF of CF. The slopes of the lines in Figure 2 represent RFs (Table 3). Lines were calculated by linear regression, forcing zero as Y-axis inter­cept.

The RF for the reference chemical fertilizer was 0.86, and hence RE, of the POMs was calculated as RF/0.86. Among the non-leached POMs, high values of RE, (Table 3) were found for DPS, a product with a large fraction of inorganic N (Table 2), and for SDP, low in inorganic N, but apparently containing easily decomposable N compounds. Because the RF of non-leached DPS was 0.90, its RE, just exceeded 1, and consequently the calculated REo became unrealistically high. Low values of RE, were found for composted plant materials (RC, AEC, EC), all containing a low fraction of inorganic N. POMs high in inorganic N (SMC, CPM, DPS) had higher values of RE, than their leached equivalents, the difference obviously being caused by the removal of inorganic N. POMs low

in inorganic N had similar values of RE, as their leached equivalents, apparently because both leached and not-leached materials had almost equal contents of inorganic N. An exception was AEC, which had an unexpectedly high value of RF when not leached and a normal value when leached (as compared to RC). Among the leached POMs, SDP ranked first and ANR second in RE,, probably on the basis of easily mineralized organic N.

Mineralized organic N was calculated by Equation 3. The slopes of the lines in Figure 3 indicate the fraction of mineralized organic N (FMON). These fractions arc equal to REo (Table 3). Table 3 shows a wide range in REa for the various PO Ms. In Figure 4, FMON of the leached POMs is plotted against initial C/N of the organic material. Obviously no simple rela­tion exists. An FMON of 0.05 to 0.10 was found for POMs varying in C/N from 12 to 24. The highest FMON was found for the fresh materials SDP and ANR. Lowest values were found for

Mineralized organic N (mg/pot)

1000

A

500 0

0

0 •• - • -~~ ... • 0

1000

500

0 .•. 0

c

o .. o··· o

1000 2000

-AC • AEC .

ANA 0

SMC 6

0

3000 0

Plant-available N in organic wastes 113

B

o_.o· o

.-·:.1(~·~¥"-· ..•. ·---·

D

-"' t;;

~ D.'

• ·• • • :~ •• ~~- ~-<>

1000 2000

.-­AC .

AEC . ANA

0 SMC

6 '---

... ----· •

r--CPM • DPS . EC

0 SOP

6

3000

Applied organic N (mg/pot)

Fig. 3. Relation between mineralized organic N and applied organic N. A, C: non-leached POMs, B, D: leached POMs.

Table 3. Recovery fraction (RF) and relative effectiveness (REJ of total fertilizer N, and of organic N (REolb. NL and L: not leached and leached

RF RE, RE,

NL L NL L NL L

RC 0.06 0.04 O.o? 0.05 0.05 0.05 AEC 0.16 0.06 0.18 O.D7 0.17 0.04 ANR 0.16 0.16 0.18 0.19 0.18 0.18 SMC 0.27 O.D7 0.31 0.08 0.21 0.06 CPM 0.25 0.09 0.30 0.10 0.12 0.09 DPS 0.90 0.16 1.05" 0.18 1.34" 0.08 EC 0.05 0.04 0.06 0.05 0.04 0.04 SDP 0.46 0.53 0.54 0.61 0.53 0.60 CF 0.86

"For explanation see text; b RE" is equal to the fraction of mineralized organic N.

RC, AEC, and EC, all well composted plant materials, while SMC, a mixture of manure and straw and partly decomposed by fungi, had a slightly higher FMON. POMs derived directly from animal manure (CPM and DPS) had a FMON which was almost twice as high as that of composted plant material.

FMON (g/g)

0.6 ASDP Group I

• Group II

• Group Ill ...

0.4

0.2 .A.ANR

DPS CPM

AEC RC. • SMC

• EC •• • 0

0 10 20 30

Initial C/N substrate (g/g)

Fig. 4. Relation between the fraction of mineralized organic N and the initial C-N ratio of the organic fraction of the leached POMs.

114 Raijmakers and Janssen

Discussion

Although complete data on the uptake of other nutrients are not yet available, the double-pot technique seems well suited for this application. Clear signs of N deficiency and the absence of other deficiency symptoms justify this conclu­sion.

The POMs included in this study varied widely in origin. This variation was reflected in the various parameters (Table 2). Firstly, a distinc­tion should be made on the basis of initial inorganic N content between SMC, CPM and DPS having an Fi ~ 0.12 and the other POMs having an Fi ~ 0.03. The POMs with a high Fi, also have a high RF, due to the high availability of inorganic N. The value of RF for DPS appeared to be slightly higher than for the chemical fertilizer. Two reasons can be put forward to explain this. The first is that grass prefers nitrate to ammonium and DPS contains mainly nitrate, while CF supplies both nitrate and ammonium. Secondly the relation between N uptake and applied N appeared to be slightly curvilinear (Fig. 2). This implies that RF at high N rates was somewhat lower than at low N rates. Because N rates for CF were higher than for DPS, calculated RF was a little lower for CF than for DPS. A curvilinear description of the relation between N uptake and N application would give a more precise estimate of RF. Because the application rates of POM-N were generally rather low, a linear-response compari­son can be used, according to Chien et a!. (1990).

RE, as such is not very useful in practice, because its value depends on the experimental conditions. A distinction should be made be­tween inorganic N, immediately available (REJ, and organic N, which can be considered to behave as a slow release fertilizer (REo) (Eq. 2). For a balanced fertilizer application, both N sources should be taken into account. Ex­perimentally determined values for REo should be adjusted to account for variable environmen­tal conditions, especially temperature and mois­ture, and length of growing period. The use of temperature sum might be appropriate in this respect, e.g. the 62 days of our experiment at 21°C roughly corresponds to the months May,

June and July under average weather conditions in the Netherlands.

Comparison of the RE, of a non-leached POM with the REo of its leached equivalent gives an indication of the relative contribution of REo to RE, (Table 3). As reflected by the differences in REo between leached and non-leached POMs, assessment of RE., can be inaccurate because of large responses to the inorganic N contained in non-leached POMs. The same problem was observed in a study of Van Erp and Van Dijk (1992). The correction for initial inorganic N content is much larger for non-leached POMs than for the leached POMs. Hence, for the calculation of RE 0 , a possible inaccuracy in RF of CF is of minor importance in the case of leached POMs, and their values of RE 0 were thus considered more reliable. For POMs, low in inorganic N, leaching had practically no effect on Fi and RE,. Consequently the accuracy of the calculated REo can be expected to remain un­changed. By far the highest value for REo was obtained with SDP, waste water treatment sludge from a cheese factory. The low C/N (5-6) points to the presence of easily decomposable proteins. The relatively high REo value for ANR can be attributed to the fact that this organic fraction of municipal waste had only been submitted to hydrolysis.

The FMON was calculated assuming an equal RF for both initially present inorganic N and mineralized N (Eq. 2). This assumption results in a maximum estimate of RE 0 • However, it is possible that the RF of mineralized N was higher than that of initially present inorganic N, due to the fact that inorganic N initially present may be more readily lost during early growth, than N released by slow mineralization during the later part of the growth period. Had N resulting from mineralization entirely been recovered by the plants, values of REo would be 1/0.86 = 1.16 times lower than the RE 0 presented in Table 3.

The absence of a simple relation between C/N and FMON in Figure 4 is in line with the concept presented in the Introduction, that decom­posability of organic matter is a key characteris­tic for N mineralization. As we did not measure any index of the decomposability of organic material, such as lignin content, we have to confine ourselves to the semi-quantitative classi-

fication (in Materials and methods) based on the original composition, the type of processing of the POMs and data of Van Lunc and Hassink (1991). The POMs of Group III have, for a given C-N ratio, a higher value of FMON than those of Group I, while the values for Group II are in between. Although the number of data is too small to justify regression analysis per group, the results confirm the findings of e.g. Tian et al. (1992b), that both C-N ratio and decomposabili­ty determine the rate of N mineralization.

Acknowledgements

The authors are much indebted to Dr W G Keltjens, Dr M L Van Beusichem, and Dr M A J Van Montfort for their stimulating advice, and to Mr J A Nelemans and the students P G Groeneveld, F M M Jenniskens, and J H Keus for major contributions to the experimental work. This study was made possible due to grants by the Netherlands Agency for Energy and the Environment (NOVEM), NMI, and LEB-fund. We also thank the companies provid­ing the various POMs, especially YAM N.Y., which donated a large quantity of reference compost.

References

Barrow N J 1985 Comparing the effectiveness of fertilizers. Fert. Res. 8, 85-90.

Barrow N H and Bolland M D A 1990 A comparison of methods for measuring the effect of level of application of the relative effectiveness of two fertilizers. Fert. Res. 26, 1-10.

Chien S H, Sale P W G and Friessen D K 1990 A discussion

Plant-available N in organic wastes 115

of the methods for comparing the relative effectiveness of phosphate fertilizers varying in solubility. Fert. Res. 24, 149-157.

Fog K 1988 The effect of added nitrogen on the rate of decomposition of organic matter. Bioi. Rev. 63, 433-462.

Gilmour J T, Clark M D and Sigua G C 1985 Estimating net nitrogen mineralization from carbon dioxide evolution. Soil Sci. Soc. Am. J. 49, 1398-1402.

Janssen B H 1990 A double-pot technique as a tool in plant nutrition studies. In Plant Nutrition- Physiology and Ap­plications. Ed. M L Van Beusichem. pp 759-763. Kluwer Academic Publishers, Dordrecht, The Netherlands.

Jensen H L 1929 On the influence of the carbon: nitrogen ratios of organic material on the mineralisation of nitrogen. J. Agric. Sci. Camb. 19, 71-82.

Melillo J M, Aber J D and Muratore J F 1982 Nitrogen and lignin control of hardwood leaf litter decomposition dy­namics. Ecology 3, 621-626.

Schuiz E 1988 N-Transformationsprozesse beim Abbau von organischer Primiirsubstanz im Boden in Abhiingigkeit von ihrer Stabilitiit und dem C/N-Verhiiltnis. Arch. Ackcr­Pflanzenbau Bodenkd. 32, 577-582.

Smith S R and Hadley P 1990 Carbon and nitrogen miner­alization characteristics of organic nitrogen fertilizers in a soil-less incubation system. Fert. Res. 23, 97-103.

Tian G, Kang B T and Brussaard L 1992a Effects of chemical composition on N, Ca and Mg release during incubation of leaves from selected agroforestry and fallow plant species. Biogeochem. 16, 103-119.

Tian G, Kang B T and Brussard L 1992b Biological effects of plant residues with contrasting chemical compositions under humid tropical conditions- decomposition and nu­trient release. Soil Bioi. Biochem. 24, 1051-1060.

Van Burg P F J 1963 The agricultural evaluation of nitro­phosphates with particular reference to direct and cumula­tive phosphate effects, and to interaction between water­solubility and granule size. The Fertiliser Society Proc. 75, 7-54.

Van Erp P J and Van Dijk T A 1992 Fertilizer value of pig slurries processed by the Promest procedure. Fert. Res. 32, 61-70.

Van Lune P and Hassink J 1991 Hogere aardappelopbrengst en mindcr zicktcs met YAM GFT-compost. YAM Medel­ingen 2, 12-15.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 117-123, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-209

Relations between nitrogen and phosphorus immobilization during decomposition of forest litter

W.G. BRAAKHEKKE, H.A. STUURMAN, H. VAN REULER and B.H. JANSSEN Department of Soil Science and Plant Nutrition, Wageningen Agricultural University, P.O. Box 8005, 6700 EC Wageningen, The Netherlands

Key words: decomposition, CaC12-extraction, forest litter, incubation, nitrogen immobilization, phosphorus immobilization, shifting cultivation

Abstract

Field trials in the shifting cultivation system of the Tal region (south-west Ivory Coast) indicated that observed low crop yields were caused by immobilization of nitrogen and phosphorus during decomposi­tion of plant residues left after clearing the forest vegetation. To investigate this hypothesis, litter was collected and divided into three fractions: leaves, twigs and branches. Ground samples of these fractions were mixed with quartz sand and nutrient solution with various N and P concentrations, and incubated in plastic bags at 25°C. Samples were extracted with 0.01 M CaC1 2 and analyzed for NH4 ,

N0 3 , and PO 4 before and after the incubation. In all litter fractions the extractable amounts of N and P decreased at rates depending on the amounts applied. Maximum rate of N immobilization was 1 g kg - 1

litter in the twigs fraction. Maximum rate of P immobilization exceeded 0.3 g kg - 1 litter, which was the highest rate of P addition in the experiment. Addition of one nutrient increased the immobilization rate of the other. It is concluded that the observed low crop yields may indeed result from immobilization of N and P by forest remnants. The mutual stimulation of immobilization of N and P may lead to negative crop responses to fertilization.

Introduction

During decomposition of organic material added to soil, nitrogen and phosphorus can either be mineralized or immobilized, depending on the chemical composition of the organic materials that are decomposed and on the decomposer species involved. Net mineralization increases nutrient availability to plants. Consequently, it should increase crop yield and reduce the amounts of inorganic fertilizer that are needed for optimal crop production. Immobilization, on the other hand, decreases the availability of nutrients temporarily and reduces the recovery of inorganic fertilizer and the crop response to fertilization. Nutrient immobilization is an im­portant factor in low-input agriculture, where

insufficient fertilizers are available to compen­sate for immobilization.

Crop yields and yield responses to inorganic fertilizer may depend on the chemical composi­tion of organic materials present in or added to the soil, particularly on the C-N ratio and the C-P ratio of these materials. Depending on whether these ratios are below or above a critical value, N and P will be mineralized or immobil­ized during decomposition. Critical values of the C-N and C-P ratio are roughly 30 and 300, respectively. The rationale behind these critical ratios is that the respective C-N and C-P ratios of soil microbial biomass are about 10 and 100, and that the ratio of C-dissimilation to C-assimilation is about 2: 1. Consequently, the requirements of the microbes are matched when the ratio C: N: P

118 Braakhekke et al.

in the substrate is about 1:30:300. At C-N ratios >30 or C-P ratios >300, nitrogen or phosphorus become limiting to microbial growth and de­composition of the organic material. In these circumstances, addition of inorganic N or P may increase the decomposition rate (Munevar and Wollum, 1977). In practice there may be a considerable variation in critical C-N and C-P ratios, depending on the kind of organic material and the decomposer species involved (McGill et a!., 1981).

Much research has been carried out on miner­alization of N and P. Several authors have reported effects of P addition on N mineraliza­tion or vice versa (e.g. Blair, 1988; Cole and Heil, 1981; Dalal, 1977; Enwezor, 1976; Munevar and Wollum, 1977). Less attention has been paid to immobilization of N and P. Effects of availability of P on immobilization of N have not been reported to our knowledge. An in­crease of P immobilization by application of N has been reported by Dalal (1977). As both nutrients are essential to microbial activity, it is to be expected that shortage of one of these two nutrients may hamper mineralization as well as immobilization of the other nutrient.

The present paper reports on a study under controlled conditions of the N and P immobiliza­tion capacity of litter from the Tal forest in south-west Ivory Coast. The experiment has been carried out as a part of a soil fertility study in the Tal region (Van Reuler and Janssen, 1989). In this region the common agricultural system is shifting cultivation w1th upland rice as the main food crop. In preparing the fields the forest is cleared by slash and burn. The yields of rice were found to be lower on fields that were cleared in primary rain forest than on fields cleared in secondary forest that had been culti­vated previously (Van Reuler and Janssen, 1989). These results opposed to the general idea that yields would be higher after primary forest, having a higher nutrient store than secondary forest where the soil has been cultivated before.

A difference in nutrient immobilization might be one of the causes of the lower yields on fields cleared from primary forest. After clearing pri­mary forest, more wood, roots and litter are left behind in the field than after clearing secondary forest. In the Tal region these organic materials

are known to be extremely poor in phosphorus and nitrogen (Van Reuler and Janssen, 1989). Therefore, it can be expected that they immobi­lize available P and N initially present in the soil or supplied as inorganic fertilizer. The temporary nutrient losses due to immobilization can cause a reduction in crop yield, which will be larger in fields where more organic material remains after clearing, i.e. in fields cleared from primary forest.

Materials and methods

Materials

Litter was collected in a 20-year-old secondary forest 8 km south of the village Tal. The original forest is classified as tropical lowland evergreen seasonal forest. Data on the species composition are given by Vooren (1985). The litter was separated in three fractions: branches >3 em diameter, twigs <3 em diameter, and leaves. The branches were partly decomposed and contami­nated with soil due to biological activity. Appar­ently, they had been in the forest floor for a longer time than the leaves and twigs, probably due to their lower decomposition rate. This can also be inferred from the fact that the C-N ratio and total carbon content of the branches are lower than those of the twigs. The litter fractions were dried, ground and analyzed for total C, N and P and for CaC1 2 -extractable N and P (see Table 1).

Incubation

Ground litter samples of 3.00 gram were mixed with 27.00 gram of quartz sand and 10 mL of a nutrient solution, and incubated in sealed plastic bags (0.02 mm polyethylene, size 10 x 15 em), which were stored in the dark at 25°C. Separate bags were prepared for different incubation periods.

Sixteen nutrient solutions were used of a 42 NP factorial design. Nitrogen rates were 0, 0.49, 0.95, and 1.45 g N kg -I litter; phosphorus rates were 0, 0.10, 0.19, and 0.30g P kg- 1 litter. The choice of N and P rates was based on calcula­tions with the model of Janssen (1984), which

Nand P immobilization 119

Table I. Chemical analysis of three litter fractions: total contents of C, N and P; initial extractable N- and P contents (in g per kg litter) and ratios of organic C, N and P. Nmin ~ NO,-N + NH,-N; P-CaCI, ~ P extractable in 0.01 M CaCI,; N org ~ Ntotal­Nmin; P org ~ Ptotal- P-CaC1 2

Fraction CTotal NTotal P Total Nmin

Branches 193 3.59 0.255 0.056 Twigs 440 6.21 0.253 0.080 Leaves 309 10.01 0.476 0.123

gave a preliminary estimate of the maximum immobilization capacity. Nitrogen was added as nitrate with K, Ca and Mg as accompanying cations, in an equivalent ratio of 4: 1: 1. P was added as KH 2P04 .

The experiment was carried out in duplicate. Four of the N-P combinations were not applied to the branches, because of Jack of material (N1P2, N2P1, N2P3, N3P2). Incubation periods were 10, 17, 24 and 45 days for the leaf fraction; 24 and 45 days for the twigs and branches. Multiple regression was used to analyse the results.

Chemical analysis

To prevent separation of sand and litter, the sand-litter mixtures were moistened before sub­sampling for chemical analysis. Total organic carbon was measured by oxidation with K2 Cr2 0 7

and sulphuric acid, followed by spectrophoto­metric determination of Cr3 + (Walinga et a!., 1992). Total N and P were determined spectra­photometrically, using an automated continuous flow system, after digestion with sulphuric acid, selenium and salicylic acid (Novozamsky et a!., 1983). Available N (N0 3 and NH4 ) and P were measured spectrophotometrically, after extrac­tion of the moist samples with 0.01 M CaCl 2

(Houba et a!., 1986).

Results

Net immobilization of N and P was calculated as negative mineralization of N and P by subtract­ing the initial CaCI 2-extractablc N and P amounts and the added N and P amounts from the final CaCI 2 -extractable N and P amounts after incubation. In this way some chemical fixation that may have reduced the extractable

P- CaC1 2 C/N org C/Porg N org/P org

0.009 55 785 14 0.044 72 2105 29 0.057 31 737 24

amounts is included in immobilization. As there was no soil involved in the experiment, fixation of extractable N by clay minerals is assumed to be negligible.

Some of the decrease of extractable P was due to P fixation by impurities in the quartz sand. In control bags (sand without litter) that had re­ceived the highest P rate, about 9 mg P per kg sand was not recovered by extraction with CaC1 2

after 45 days incubation. As its size may depend on the P application rate and the amount of litter, it was not possible to correct the data for this Joss of P, which may have resulted in an overestimation of P immobilization with 30% at most.

Carbon dissimilation

After 45 days of incubation the total C content of the mixtures was measured. The dissimilation of carbon was calculated as the difference be­tween initial and final amount and expressed as percentage of the initial amount of C. As this is a rather insensitive measure of the decomposition rate over short periods, the coefficients of vari­ation are high (0.7 for branches, 0.4 for twigs, 0.15 for leaves). None the Jess, a significant difference in dissimilation rate was found be­tween the three litter fractions: the branches lost 13.5% of their initial C content, the twigs 7.2% and the leaves 21.2%.

Immobilization of N and P

After 10 days incubation an increase in inorganic N content was measured in the leaf fraction, but after 17 days this had turned into a decrease (not shown). Probably this short N flush at the start of the incubation was an artefact due to rapid mineralization of microbial biomass that had died during drying of the litter. Because net

120 Braakhekke et al.

immobilization hardly increased between 24 and 45 days, it is probable that in the leaves and the branches an initial N immobilization phase was followed by N mineralization at the end of the incubation period.

The decrease in extractable N after 45 days in branches, twigs, and leaves, depends on the rate of added N and P (Fig. 1). Even at zero N addition there was some N immobilization, which was equal to the initial inorganic N con­tent of the litter.

In general, N immobilization was equal to the available inorganic N until a maximum N im­mobilization capacity was reached, which dif­fered among litter fractions and depended on whether or not P was added. With addition of sufficient P, the maximum N immobilization after 45 days was about 0.6 g N kg -I for branches and 1. 0 g N kg -J for twigs. Due to variation between duplicates such a value could not be established for leaf litter. When no P was added, the maxi­mum N immobilization was only 0.3 g N kg -I for branches and 0.8g N kg- 1 for twigs. Evidently, the N immobilization capacity in the two woody litter fractions was reduced due to lack of avail­able P. In the leaf fraction a P effect is obscured by large variation between duplicates.

The decrease in extractable P after 45 days in branches, twigs and leaves also depends on the P and N addition (Fig. 2). At zero P addition there still was a small decrease in extractable P, which was equal to the initial extractable P content of the litter.

The observed decrease in extractable P may have been caused not by microbiological im­mobilization alone. Some P sorption by the litter may have occurred and part of the sorbed P may not have been extracted with CaC12 • This may result in an overestimation of the microbial immobilisation of P. The fact, however, that the decrease in extractable P was smaller at the NO level than at the higher N levels stresses the role of the microbiological processes. In judging the P immobilization capacity of the litter, the fixa­tion by the quartz sand should also be taken into account.

In general, P immobilization increased with the rate of P addition. The P-lines did not level off to a horizontal line. Apparently, the maxi­mum P immobilization capacity was not yet

Increase in extractable N vs. N addition

~ -0.2

:1:: ~ -0.4

~ .9 -0.6

c .E -0.8 z !!!

-1.0 Qj -a

"',,, ~······--·--·-~

·· .. -·-·--:-·-·-·-··

Branches ························... •

-1 ·2 L-o.J...._..__o . ._2_._o_..4_..-:o...L.6:-'-..,.o...,.8_._.,...1.7o_,_,_1,.....2:-'-:1...L.4:-'--:-1 "'::.6--'

0

~ -0.2

~ Ol -0.4

~ .9 -0.6 c .E z -0.8

!!! Qj -1.0 -a

Twigs -1.2

0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6

Or-r---------------------------,

~ -0.2 ;E

~ -0.4 z .9 -0.6 c .E

-0.8 z !!! Qi -1.0 -a

Leaves -1 ·2 '--oL-'-o-".'-2 _._o_...4__._o_..,.6_._..,-o.78_._1_..o.,...-~-....,1_.,.2:-'--:-1 ....,.4_._.,...1 ."'6-'

N addition (g N/kg litter)

• PO • P1 • P2 • P3

Fig. 1. Change in CaCl,-extractable N after 45 days incuba­tion at 25°C in three litter fractions form a tropical rain forest plotted against the amounts of N added, at different rates of added P. The dotted line indicates immobilization of all available N, i.e. added N plus the initial Nmin content.

Increase in extractable P vs. P addition

~ ,g C)

-"" a::- -0.1

~ a... Q)

:g -0.2

t5 ~

~ C1l -0.3

'il3 '0

Branches

0 0.04 0.08 0.12 0.16 0.2 0.24 0.28 0.32

0.-.-----------------------------, ~ ,g C)

-"" a::- -0.1

~ a... Q) :;:; -0.2 C1l t5 ~ x Q)

.l!l -0.3 Qj '0

Twigs 0 0.04 0.08 0.12 0.16 0.2 0.24 0.28 0.32

~ ,g 0.-.----------------------------,

C)

-" a::- -0.1

~ 0.. Q) :g -0.2

t5 C1l ~ Q)

.m -0.3 Qj '0

Leaves ·. '•,

0

• NO

0.04 0.08 0.12 0.16 0.2 0.24 0.28 0.32

P addition (g P /kg litter)

N1 • N2 • N3

Fig. 2. Change in CaC1 2-extractable P after 45 days incuba­tion at 25°C in three litter fractions from a tropical rain forest plotted against the amounts of P added, at different rates of added N. The dotted line indicates immobilization of all available P, i.e. added plus the initially available P.

N and P immobilization 121

reached at the highest P addition of 0.3 g P kg -I litter. Evidently, the model calculations on which the P rates were based gave an underestimation of the maximum P immobilization capacity. At the higher P rates not all of the added P was immobilized. In branches and leaves no effect of N addition on P immobilization could be dis­tinguished. In the twigs, however, there was a distinct effect of N shortage on the P immobiliza­tion. Although the maximum P immobilization capacity had not yet been reached, the fraction of added P which was immobilized was smaller at the NO and Nl addition rates than at the N2 and N3 rates. This indicated a reduction of the P immobilization rate due to lack of available N.

Discussion

Decomposition

In general, woody material is more resistant to decomposition than leaves, so the ranking of C dissimilation percentages is expected to be: leaves> twigs> branches. Indeed the leaf frac­tion appears to be the least resistant (21.2% dissimilation). The branches, however, are de­composing faster than the twigs (13.5 vs. 7.2% dissimilation). This is probably due to the fact that the branches were already partly decom­posed and contaminated with soil, which could have caused an enrichment of N and P. This is also indicated by the lower C-N and C-P ratios of the branches.

Due to the large coefficients of variation in the C dissimilation measurements the present experi­ment allowed no definite conclusions on the effect of nutrient additions on C dissimilation rate. Results of Enwezor (1967) indicate that C0 2 production may be independent of P addi­tion, while P immobilization is directly propor­tional to the P addition rate, as in the present experiment. This indicates that P deficiency may either cause an increase of the C-P ratio of the microbial biomass (less luxurious P consump­tion), or an increase of the dissimilation-assimila­tion ratio (more wasteful respiration). However, the changes in these ratios will be limited, so that it is only to he expected that C dissimilation will be affected by severe nutrient deficiency, as is

122 Braakhekke et al.

shown by many instances in literature (McGill et a!., 1981).

Immobilization of N and P in the field

In their study on the traditional shifting cultiva­tion system in the Tal region Van Reuler et a!. (in preparation) found that the amount of litter in the rain forest was 5700 kg ha-t. About 20% of this material remains in the field after cutting and burning. This means that, at a change in available N and P of 0.6 g N kg -t and 0.3 g P kg -t in 45 days, the remaining litter alone can immobilize 684 g N ha-t and 342 g P ha-t in 45 days, under conditions comparable to the incu­bation experiment. To appreciate these amounts, it should be realized that a production of about 600 g of rice is possible per g P uptake, so that a decrease of 342 g available P per ha is equivalent to the P uptake by a rice crop of about 200 kg ha- 1, which is 20-25% of the yield commonly attained by local farmers (De Rouw, 1991).

The potential for N and P immobilization will be much greater under field conditions, because the growing season is about twice as long as the incubation period in this experiment and because the litter is only a small fraction of the total plant biomass that remains after clearing the forest. Decomposition of roots especially is expected to cause a considerable immobilization. This means that N and P application rates should largely exceed the crop requirement in the first year after clearing. In subsequent years, part of the immobilized nutrients may become available, but this is not utilized by agricultural crops when the fields are abandoned after one or two seasons, which is the common practice.

Relation between immobilization of N and P

The present experiment shows that low availabil­ity of N and P not only affects the mineralization - as has frequently been reported in literature (Muncvar and Wollum, 1977; and references therein) but also the immobilization of these nutrients. This is understandable, because both mineralization and immobilization are results of microbial activity, which is the prime process affected by the nutrient deficiency. While it is self-evident that N immobilization

cannot occur when there is no inorganic N available, the experiment has also shown that N immobilization can be hampered by shortage of available P and vice versa. It appears to be strongest in the fraction with the highest C-N and C-P ratio, i.e. in the twigs.

A consequence of this mutual influence of N and P during immobilization is that, on soils that are low in both nutrients, immobilization of the one nutrient may be stimulated by application of the other. This may cause unexpected negative responses of crops to fertilization. For example, N application may increase immobilization of available P and, consequently, decrease recovery of fertilizer P by the crop. At low rates of fertilizer P application this might cause a reduc­tion in yield. At high P application rates, no effect on crop yield is to be expected, but the negative effect on P recovery will remain.

Usually the effect of N and P application on P and N recovery in the crop is positive: N applica­tion increasing P recovery and P application increasing N recovery. Fertilizer trials in the Tal region indicate, however, that a negative effect of N application on P recovery may also occur in practice (RJ Hijmans, unpublished results). The relation between N and P immobilization ob­served in the present incubation experiment may explain such negative effects. Conversely, a decrease in N recovery after P fertilization, due to stimulation of N immobilization, is also pos­sible.

References

Blair J M 1988 Nitrogen, sulphur and phosphorus dynamics in decomposing deciduous leaf litter in the southern Appalachians. Soil Bioi. Biochem. 20, 693-701.

Cole C V and R D Heil 1981 Phosphorus effects on terrestrial nitrogen cycling. In Terrestrial Nitrogen Cycles. Eds. F E Clark and T Rosswall. pp 363-374. Ecological Bulletins, Vol. 33. Stockholm.

Dalal R C 1977 Soil organic phosphorus. Adv. Agron. 29, 83-117.

De Rouw A 1991 Rice, wheat and shifting cultivation in a tropical rain forest. A study of vegetation dynamics. Doctoral thesis, Wageningen Agricultural University, The Netherlands. 263 p.

Enwezor W 0 1967 Significance of the organic C: organic P ratio in the mineralization of soil organic phosphorus. Soil Sci. 103, 62-66.

Houba V J G, Huijbregts A W M, Novozamsky I and Vander Lee J J 1986 Comparison of soil extractions by 0.01 M CaCl,, by EUF and by some conventional extraction procedures. Plant and Soil 96, 433-437.

Janssen B H 1984 A simple method for calculating decompo­sition and accumulation of 'young' organic matter. Plant and Soil 76, 297-304.

McGill W B, Hunt H W, Woodmansee R G and Reuss J 0 1981 Phoenix- A model of the dynamics of carbon and nitrogen in grassland soils. In Terrestrial Nitrogen Cycles. Eds. F E Clark and T Rosswall. pp 363-374. Ecological Bulletins, Vol. 33. Stockholm.

Muncvar F and Wollum A G 1977 Effects of the addition of phosphorus and inorganic nitrogen on the carbon and nitrogen mineralization in andcpts from Columbia. Soil Sci. Soc. Am. J. 41, 540-545.

N and P immobilization 123

Novozamsky I, Houba V J G, Van Eck Rand Van Vark W 1983 A novel digestion technique for multi-element plant analysis. Commun. Soil Sci. Plant Anal. 14, 239-249.

Van Rculcr H and Janssen B H 1989 Nutritional constraints in secondary vegetation and upland rice in south-west Ivory Coast. In Mineral Nutrients in Tropical Forest and Savanna Ecosystems. Ed. J Proctor. pp 371-382. Blackwell Scientific Publications, Oxford.

Vooren A P 1985 Patterns in tree and branch-fall in a West African rain forest. Report D 85-05. Department of Silviculture, Wageningen Agricultural University.

Walinga I, Kithomc M, Novozamsky I, Houba V J G and Van der Lee J J 1992 Spectrophotometric determination of organic carbon in soil. Commun. Soil Sci. Plant Anal. 23, 1935-1944.

c

Improving the diagnosis of the nutritional status of crops

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 127-132, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-191

Some factors affecting potassium nutrition of sour cherry trees

EWAJADCZUK Department of Pomology, Warsaw Agricultural University, Nowoursynowski 166, PL-02-766 Warszawa, Poland

Key words: fruit load, leaf potassium, potassium fertilization, Prunus avium, rootstock, sour cherry

Abstract

In autumn 1980 five potassium fertilizer treatments were started in a three-year-old sour cherry orchard planted on silty loam, alluvial soil low in potassium (K). Trees of cultivars (cvs) 'North Star' and 'Nefris' were grown on two rootstocks. During the nine-year period of study, rootstock was the primary factor affecting K nutrition; Prunus avium, compared with P. mahaleb favoured higher leaf K concentration. The rootstock effect depended on cultivar and season. Cultivar itself also modified leaf K concentration but only in trees grown on P. avium. Seasonal variations in leaf K concentration were considerable and they were apparently related to weather conditions and fruit load. Potassium fertilization applied in different doses resulted in a significant increase of leaf K concentration. After the first four-year period of study annual doses of 160 kg K ha - 1 resulted in the greatest increments of leaf K, later all the fertilizer treatments gave similar results. K fertilization was more effective in increasing leaf K concentration of trees grown on P. avium than of those grown on P. mahaleb stock. Effects of fertilization varied also depending on season.

Introduction

Nutrient absorption and translocation within the plant are strongly affected by many factors. However, relatively little information is available concerning factors affecting potassium nutrition of sour cherry trees.

Potassium plays an important role in mineral nutrition of sour cherry. According to Yang­Petersen (1984) sour cherry trees take up from 1 ha about 65 kg K.

Procbsting and Kenworthy (1954) reported that optimal mineral element contents of the cultivar 'Montmorency' can vary depending on geographical latitude, and accompanying light intensity, temperature and moisture conditions. Archibald and Cline (1962) reported that leaves of 'Montmorency' sour cherry in dry and warm years contained less N, P, K and Mg and more Ca than in wet years. Archibald and Cline (1962) found that cherry trees grown on soil maintained

under sod contained more K in their leaves than those from clean cultivated soil. Lenz and Biinemann (1974) did not find any significant differences in the leaf K content of 'Schatten­morelle' cherry due to soil management long the rows (herbicides vs weeding by hand). In the same paper, Lenz and Biinemann inform that fertilizer doses of 66.5 kg K ha -I or 133 kg K ha - 1 did not affect leaf K content. Holubowicz (1981 ), on the soil rich in potassium, noted that potassium fertilization had little influence on the content of this element in leaves. Yang-Petersen (1984) reported that 166kg K ha- 1 significantly increased leaf K concentration in comparison with the unfertilized control. Werner (1977) noted an increased leaf K concentration only when 249 kg K ha - 1 year had been applied. In the study of Sadowski et a!. (1982) long-term K fertilization at the rate of 83 kg K ha - 1 year induced an increase of leaf K content, with no influence on the yield.

128 Jadczuk

Genetic factors as cultivar and/ or rootstock may also alter the uptake of mineral elements. Kirkpatrick (1960) found that sour cherry trees on Pr. mahaleb were worse nourished with potassium than trees on Pr. avium stock. The influence of rootstock was also discussed by Prodanov and Vitanova (1977), Sadowski et a!. (1982), Hanson and Perry (1986) and by Ugolik and Holubowicz (1990). An effect of tree age upon leaf K content was noted by Archibald and Cline (1962) and by Smith et a!. (1962); young sour cherry trees usually contained more K in their leaves than older ones.

The main objective of this experiment was to study the effects of rootstock, cultivar, K fertili­zation and vegetation season upon K nutritional status of sour cherry trees planted on alluvial soil showing a low available K content.

Methods

The experiment was carried out in the years 1981-1989 at the Experimental Field of the Warsaw Agricultural University, situated in the post-glacial valley of the Vistula River at War­saw-Wilan6w. The soil was alluvial silty loam, low in K; available K content (Egner-Riehm method, extraction with Ca lactate -lactic acid solution at pH 3.6) amounted to 5.7, 4.2 and 3.1mg 100g- 1 in soil layers of0-15, 15-30 and 40-60 em, respectively. In autumn 1977 sour cherry trees, cvs 'North Star' and 'Nefris' on two rootstocks were planted at the distance of 5 x 3m. In 1980 leaf K content was within the deficient range, below 1% of dry matter (d.m), except for 'North Star' on Prunus avium root­stock. Nevertheless, no potassium deficiency symptoms were observed.

The experiment was set up in a split-plot factorial randomized block design with 8 replica­tions. Five fertilizer treatments, distributed as subblocks, were started in autumn 1980: (1) K0

check, no potassium; (2) K 166 annua1-166 kg K ha - 1 every year; (3) K 664 -664 kg K ha - 1 once in every four years; ( 4) K332-332 kg K ha -I once in every four years; (5) K 332 dcep·herb-in first four­year cycle (1981-1984) a single fertilizer dose of 332 kg K ha - 1 , as in treatment 4, but in the form of concentrated solution into holes 37 em deep

drilled at the distance of 75 em from the truck (two holes per tree), then in the second cycle (1985-1989) the fertilizer was spread within the herbicide strips only at the dose of 332 kg K ha- 1

in autumn 1984 and of 166 kg ha - 1 in following years. Potassium chloride was used as K fertilizer in all treatments. Nitrogen, in form of ammo­nium nitrate, was applied in all treatments in the equal dose of 100 kg N ha- 1 every year.

'Small plots' consisted of four cultivar/root­stock combinations: two cvs- 'Nefris' and 'North Star', each on two seedlings rootstocks- Pr. mahaleb and Pr. avium; there were two trees per plot.

Mid-shoot leaves were sampled shortly after harvest every year. Leaves were oven-dried at 60°C, ground and dry ashed in muffle furnace at 500°C. Potassium content was determined by flame photometry.

All the data were subjected to analysis of variance in the split-plot design. The significance of differences between treatment means was estimated using HSD based on the test of Tukey.

The average many years' rainfall amounted to 505 mm and mean yearly temperature was 7.7°C in this location. Considering deviations of rain­fall and of temperatures from the many years' means in a particular season, the years of this study were classified as wet, medium, dry or very dry. Minimum temperatures occurring in some years caused variations in fruit load. Spring frost in 1986 resulted in ca 50% crop reduction; in 1987 extremely low temperatures in January and March caused a complete killing of flower buds, resulting in nil crop year and as a consequence of that the 1988 crop was extremely abundant; cropping in the remaining years was considered as normal.

Results

Effects of vegetation season and of rootstock and their interaction

Leaf K concentration depended to a great extent on the season of vegetation (Fig. 1 ). In the first four-year cycle of study it was the lowest in the years 1982 and 1983 when prolonged periods of

Some factors affecting K nutrition of sour cherries 129

leaf K.,.

1.5

1.3

1.1 ___2_ ,Q2_

0.9 ~ ~

__JL ~ ,2-0.7

iil " . .!! .!! 0.5

-b >- E 0.3

-5 " t ~ >- '6

0 . -5 ~ • >

g E=5 '5

.E .lo .2.;:

~~ ,_-;;; " g >--5-...C ~ E.<: -5

0.1 81 82 83 84 85 86 87 88 89

Fig. 1. Leaf K concentration of sour cherry depending on vegetation season; mean values for two rootstocks and two cvs, unfertilized treatment. Bars having a common letter are not significantly different by the test of Tukey at p = 0.05.

drought occurred and the highest in the year 1981 when rainfall was abundant.

In the second cycle (1985-1989) leaf K content was the highest in the year 1987, in the absence of fruits, as all the flower buds were killed during the preceding severe winter and was relatively high in 1986 when about 50% of flowers were damaged by spring frost. A low leaf K content was noted in that cycle in 1988 when the fruit load was very heavy and in 1989 which was relatively dry, with a medium fruit load.

Over the whole nine-year period of study, sour cherry trees grown on Prunus avium seedling rootstock showed always a significantly higher leaf K concentration than trees budded on Prunus mahaleb (Fig. 2). In the check (unfertil­ized) treatment, trees on Pr. avium contained, on the average, 1.21% of K on dry matter basis while trees on Pr. mahaleb only 0.72%. How­ever, the rootstock effect was different in differ­ent seasons; it was the most pronounced in the year 1987, followed by 1985, 1988 and 1984.

Interaction of rootstock and cultivar

A significant interaction of rootstock and cultivar upon leaf K concentration was found (Fig. 3); it means that leaf K concentration was affected by the rootstock in different manner depending on the scion cultivar and that the differences due to cultivar depended on the rootstock used. Prunus

1.6

leaf K.,.

D Pr. mahaleb ffiiillJ Pr. avlum

81

LSD I 0.05

82 83 84 85 88 87 88 89

Fig. 2. Leaf K concentration of sour cherry depending on rootstock and season; mean values of two cultivars, unfertil­ized treatment. HSD value indicated is valid for comparisons of means for rootstocks on a particular season.

leaf K%

1.3 0North Btaf mm1Nefrl•

1.1 tso 0 .05 I

0.9

0.7

0.6

0.3

0.1 Pr. mahaleb Pr. avlum

Fig. 3. Leaf K concentration of sour cherry depending on rootstock and cultivar; mean values of nine years, unfertil­ized treatment. HSD value indicted is valid for comparisons of means for rootstocks within a particular cultivar or for means for cultivars on a particular rootstock.

avium rootstock, compared with Prunus mahaleb, significantly increased leaf K concen­tration in both cultivars under study, however, the effect of rootstock was more pronounced in the cv 'North Star' than in 'Nefris'. Cultivar 'North Star' contained more K in its leaves than 'Nefris', but the difference between cultivars in that respect was significant only for trees grown on Prunus avium.

As a result of the cumulative effect of a combination of both factors, leaf K concentra­tion of the cv 'North Star' on Pr. avium was, on the average for the nine-year period of study, by 87% higher than that of the cv 'Nefris' on Pr. mahaleb.

130 Jadczuk

Table 1. Effect of K fertilization of leaf K content (% d.m.) in different seasons; mean values for both cultivars and both rootstocks

Fertilizer Year Average of treatment nine years

1981 1982 1983 1984 1985 1986 1987 1988 1989

Ko-check 1.05a 0.79a 0.83a 0.91a 0.98a 1.15a 1.46a 0.93a 0.74a 0.97a

Kl66 annual 1.19ab !.lOb 1.15c 1.35c 1.37bc 1.36b 1.80b 1.26c 1.28c 1.32c

K664 1.41c 1.18b l.!Oc 1.18b 1.42c 1.48c 1.80b 1.32c 1.22bc 1.34c

K_B2 1.31bc 1.06b l.OObc l.OOa 1.28bc 1.38b 1.75b 1.09b 1.10b 1.22b 332 deep or herb 1.20ab 0.89a 0.94ab 0.94a 1.22b 1.36b 1.74b 1.20bc 1.13b 1.18b

Columns having a common letter are not significantly different by the test of Tukey at p = 0.05.

Interaction of K fertilization and season

Potassium applied to the soil in different doses resulted generally in a significant increase of leaf K concentration of sour cherries, except for the treatment with local profound fertilization (in the first four-year cycle of study) which was manifestly ineffective (Table 1). In the first two years (1981 and 1982) the single doses of 664 kg K ha -I or 332 kg K ha -I applied once (in autumn 1980) were more effective in increasing leaf K concentration than the dose of 166 kg K ha -I applied annually in successive years. Start­ing from the fourth year regular annual fertiliza­tion in the dose of 166 kg K ha -I had the greatest effect upon leaf K concentration.

In the second cycle of the experiment (1985-1989), all fertilizer treatments, including the modified treatment 5 (with application of the fertilizer to the herbicide strips only) gave similar effects resulting in significant increments of leaf K concentration every year.

leaf K,.

1.9 CJ KO -K 1135 annual 1.7

§ K66-4

1.5 lll!lll K 332

S K 332 deep-herb.

1.3

1.1

0.9

0.7

0.5 Pr. mahaleb Pr. avlum

Fig. 4. Effect of K fertilization upon leaf K concentration of sour cherry depending on rootstock; mean values of two cultivars and nine years. Bars having a common letter are not significantly different by the test of Tukey at p = 0.05.

Interaction of K fertilization and rootstock

All the fertilizer treatments were more effective in increasing leaf K concentration when cherry trees were grown on Prunus avium than when they were grown on Prunus mahaleb stock (Fig. 4).

Discussion

The results of this study have confirmed that root­stock is a primary factor affecting leaf K concen­tration of sour cherry. This is in accordance with the reports of Kirkpatrick (1960), Sadowski eta!. (1982), Hanson and Perry (1986) and of Ugolik and Holubowicz (1990). A higher leaf K content of cherries grown on Prunus avium, compared with those on Prunus mahaleb, is apparently due to genetically determined ability to K uptake by a rootstock itself. In nursery experiments (Sadow­ski et a!., 1987) and in sand culture experiment (Lesczynski and Sadowski, 1987) it was found that leaf K content of Pr. avium itself was always higher than that of Pr. mahaleb. In the present study additional information has been obtained concerning interactions of rootstock with the cul­tivar or season. However, the reasons why the rootstock effect upon leaf K concentration was higher in one cultivar than in another or why the rootstock effect was higher in some particular seasons are not quite clear.

Leaf K concentration fluctuated considerably depending on the vegetation season. Likewise in the reports of Proebsting and Kenworthy (1954) and of Archibald and Cline (1962) leaf K con­centration was relatively low in dry years. This may be explained by an apparently decreased

Some factors affecting K nutrition of sour cherries 131

root actiVIty (due to temporary drought) in the surface soil layer which, contains more K than deeper layers.

Fruit load was another factor provoking seasonal variations of leaf K. The absence of fruits which apparently compete with leaves for potassium favoured a higher leaf K concentra­tion. Similar conclusions were drawn by Maggs (1963) and Hansen (1973) who compared fruit­ing and non-fruiting (deblossomed) apple trees.

Van-Petersen (1984) mentions differences in leaf K level of different cultivars. Those were also found between the cvs 'North Star' and 'Nefris' in the present study. However it is worth noting that in our study the cultivar effect depended on the rootstock used; on Prunus mahaleb it was non-significant.

From not numerous reports on potassium fertilizer trials in sour cherry orchards it may be concluded that fertilization improved the K status of sour cherry trees when soil K content and leaf K concentration were relatively low (Sadowski et a!., 1982; Yang-Petersen, 1984; Werner 1977) while no effects were noted when potassium was abundant in soil and trees (Holubowicz, 1981; Lenz and Biinemann, 1974). The alluvial soil under study contained little available potassium, apparently due to the high fixing capacity of some clay minerals. Under those conditions regular and rather high doses of K were the most effective. Special attention should be called to the fact that fertilization was strikingly more efficient in increasing K concen­tration in leaves of trees grown on Pr. avium rootstock than leaf K concentration of trees on Pr. mahaleb, even without fertilization. Obvious­ly, roots of Pr. mahaleb hardly absorb K ions from the soil and it is probably difficult to overcome their resistance in that respect even with heavy K fertilizer doses. Therefore, Pr. mahaleb should be definitely avoided as root­stock for sour cherries planted on soils showing a low available K content.

Acknowledgements

The author is indebted to the Stefan Batory Foundation for financial support which enabled to cover expenses of travel to Lisbon for partici-

pation in the VIII International Colloquiuim for the optimization of Plant Nutrition. Thanks arc due to Prof A Sadowski for critical reading and correction of the manuscript.

References

Archibald J A and Cline R A 1962 Factors affecting leaf nutrient composition of Montmorency cherry. Ann. Rep. Exp. Sta. Prod. Lab. Vineland, Ontario, pp 39-41.

Hansen P 1973 The effect of cropping on the growth and uptake of nutrients by apple trees at different levels of nitrogen. potassium, magnesium and phosphorus. Acta Agric. Scand. 23, 87-92.

Hanson E J and Perry R L 1986 A comparison of the nutrient uptake efficiency of 'mazzard' and 'mahaleb' cherry root­stocks. HortScience 21, 669.

Hotubowicz T 1981 Wptyw zr6:i:nicowanego nawo:i:enia na plonowanie dw6ch odmian wisni Schattenmorelli i Koroser 2: Sad w 5-8 roku po posadzeniu (Effect of different fertilization upon yield of Schattenmorelle and Koroser sour cherries 2: Orchard in the 5th-8th year after planting). Roczn. Akad. Roln. Poznaniu 129, 133-140.

Kirkpatrick J D 1960 A study of Prunus cerasus var. Montmorency in Western New York: Interrelations of rootstocks, soil composition, root and soil populations of stylet bearing nematodes, leaf composition, fruit quality, yield and tree vigor. Diss. Abstr. 21, 282-289 (Hort. Abstr. 1961, 31, No. 3885).

Lenz F and Biinemann G 1974 EinfuB von Bodenpflegenaus­nahmen und unterschiedlicher N, K Versorgung auf das Wachstum und den Ertrag von Sauerkirschen. Erwerbsobst bau 16, 133-136.

Leszcyr\ski A and Sadowski A 1987 Reakcja r6znych podktadek jabtoni i wisni na r6:i:ny poziom odzywiania potasem (Response of different rootstocks used for apple and sour cherry trees do different level of potassium nutrition). Pr. Inst. Sadow Kw. (Skierniewice) ser. C, or 1-4/93-96, 103-105.

Maggs D H 1963 The reduction in growth of apple trees brought about by fruiting. J. Hortic. Sci. 38, 119-128.

Prodanov G and Vitanowa I 1977 Influence of the rootstock on the contents of nitrogen and some other elements in sour cherry leaves. Ovoscharstvo 56, 36-37.

Proebsting E L and Kenworthy A L 1954 Growth and leaf analysis of Montmorency cherry trees as influenced by solar radiation and intensity of nutrition. Proc. Am. Soc. Hortic. Sci. 63, 41-48.

Sadowski A, Dziuban R, Kowalik P, Kramarczyk W, Lun­iewska B and Wojtalewska E 1987 Potassium nutrition of apple and sour cherry nursery trees as affected by root­stock, cultivar and fertilization. J. Pl. Nutr. 10, 2025-000.

Sadowski A, Jadczuk E, Scibisz K and Dabrowska W 1982 Fertilization of sour cherries. Abstracts XXIst Inter. Hort. Congress, Vol. I, 1266.

Smith C C, Fleming H K and Kardos L T 1961 Leaf composition and performance of sour cherry trees as

132 Some factors affecting K nutrition of sour cherries

influenced by fertilizer and soil management. Bull. Pa Agric. Exp. Stat. 683, 12 p. (Hort. Abstr. 32(2), No. 2472).

U golik M and Holubowicz T 1990 The influence of rootstock and cultivar on the leaf content of nutrient elements, growth and yield of three sour cherry cultivars. Acta Hortic. 274, 491-499.

Yang-Petersen 0 1984 Fertilization with nitrogen and potas­sium in sour cherries (Prunus cerasus). Tidsskr. Planteavl 88, 81-90.

Werner H 1977 Einflul3 unterschiedlicher NPK-Gaben auf den Mineralstoffgehalt des Bodens, die vegetative Leistung und due Blattnahrstoffe bei 'Schattenmorelle' auf F 12/1. Erwerbsobstbau 19, 4-6.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 133-136, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-075

The rootstock effect on some nutrient levels in leaves of apple tree cv. Granny Smith

LUISA DUARTE INIA, Laborat6rio Qufmico Agricola Rebelo da Silva, Tapada da Ajuda, P-1300 Lisboa, Portugal

Key words: apple tree, calcium, copper, Granny Smith, leaf analysis, magnesium, manganese, nitrogen, phosphorus, potassium, rootstock, zinc

Abstract

The rootstock effect on nutrient concentrations in the leaves of apple trees, cv. Granny Smith, was evaluated using leaves from trees grafted on M 9 and MM 106, in Vila Real. The results point to higher N, Mg, Ca and Zn mean levels in apple trees and lower ones for P, K and Mn on M 9 as compared with MM 106. The Cu mean concentration was not affected by rootstock.

Introduction

Grafting leads to a stock/scion association that involves a disharmony which is exploited in order to decrease plant vigour and to increase fruit yield.

The physiology of the influence of the stock on scion development is not well known (Hulme and Rhodes, 1971). According to Jones et a!. (1984), mineral composition of the xylem sap, below and above the grafting zone is distinct, being the mineral concentration above the graft­ing zone lower as the stock has dwarfing effects. Cooper and Thompson (1972) comment on the occurrence of higher Ca concentration in the bark stock than in the bark scion. Saric et a!. (1977) note that K and Ca absorption is primari­ly determined by stock nature whereas the scion determines, uptake of N, P and Mg.

Vurmirovic (1978) observed some differences among foliar concentration of cv. Star king grafted on several stocks, showing that N, Ca, Mg and P leaf concentration reached higher levels in trees grafted on M 9 as compared with on MM 106, while the K-leaf was lower in trees on M 9. The cv. Granny Smith on M 9 as compared with MM 106 leads to a less vigorous system. According to Westwood (1978) scale,

with seven dwarfing degrees, the M 9 dwarfing rootstock is classified as the 6th scale degree while MM 106, scmidwarfing rootstock, is clas­sified as the 5th one, which leads to relevant pomological differences, concerning, namely, tree size and yield efficiency.

The present paper about the effect of the rootstock on the leaf mineral concentration of cv. Granny Smith is extracted from a dissertation which deals with the influence of the some management practices on foliar levels (Duarte, 1989).

The use of leaf analysis as a tool to diagnostic the nutritional status of apple tree, cv. Granny Smith, should take into account the rootstock type, besides the influence of the other manage­ment practices on foliar levels (Duarte, 1989).

Materials and methods

The experimental work was carried out in the Portuguese region of Tnis-os-Montes, Vila Real, in apple orchards, cv. Granny Smith. These orchards were established in 1980 (Castro and Pestana, 1980) in a schist soil of variable per­meability, low pH and low cation exchange

134 Duarte

capacity, high in available K with variable amounts of available P (Castro, 1984).

The trees (sixteen per treatment) were grafted on different stocks, M 9 and MM 106, obtained from the same nursery (Tapada da Ajuda, Lis­boa) and they were spaced (3 x 1m). During 1984 and 1985, leaves from the middle of the current season's terminal shoots (Chapman, 1966) were collected every 15 days, in the morning, 64 days after full bloom (DAFB) until 203 DAFB, in 1984, and from 76 DAFB till 211 DAFB, 1985. Eight leaves per tree were col­lected, according to Moon and Hymas (1964), in order to obtain eight composite samples of 16 leaves, corresponding to each one of the ex­perimental treatments (Duarte, 1989). In 1984, eleven collects were performed from the eight experimental treatments, corresponding to 88 samples in each orchard. In 1985, ten collects were performed and 80 samples per orchard were analysed.

Leaf samples preparation for analytical pur­poses and mineral analysis were performed ac­cording to Duarte et al. (1979). The yield of apples per tree was weighed.

The underlying distributions for foliar concen­tration throughout the leaf collecting period and between 90 and 120 days after full bloom (DAFB) were normal. Therefore Student's 't' test for large samples (Snedecor and Cochran, 1967; Steel and Torric, 1980) were applied in the

statistical analysis. At harvesting, the underlying distribution for foliar concentration was not normal and the Wilkoxon's non-parametric test was used (Snedecor and Cochran, 1967; Steel and Torrie, 1980).

Results and discussion

The rootstock effect on foliar concentration was evaluated through mean foliar levels comparison between cv. Granny Smith, on M 9 and on MM106.

The orchards were established on the same soil type under the same climate conditions and they were subjected to identical managements practices, so that the observed differences through leaf analysis results can be attributed to a rootstock effect. Within the same year, the yield, in both orchards was not significantly different between orchards (Duarte, 1989). In 1984, cv. Granny Smith on M 9 reached 77.4tha- 1 and on MM106 77.5tha- 1 . In 1985, the yield was 91.0 t ha - 1 and 93.6 t ha - 1 for the trees on M 9 and on MM 106, respectively.

Table 1 shows the observed average of the mean nutrient levels occurred over the collecting period, while Table 2 shows the leaf analysis results obtained of the usual collecting date for apple leaves (Chapman, 1966). The results are

Table 1. The rootstock effect on the leaf mineral concentration of the cv. Granny Smith. Comparison between the average of the mean nutrient levels observed throughout the leaf collecting period, in two years (1984 and 1985)

M.9 MM.106 M.9 MM.106 M.9 MM.106 M.9 MM.106 M.9 MM.106 M.9 MM.106 M.9 MM.l06 M.9 MM.106

N(%) p (%) K(%) Mg(%) Ca (%) Mn(mgkg- 1 ) Zn (mg kg- 1 ) Cu (mg kg_,)

1984 y 2.25 2.23 0.16 0.17 1.32 1.69 0.23 0.17 1.27 1.09 139 172 32 27 10.0 9.6 Sy 0.162 0.021 0.326 0.026 0.156 26.0 5.0 2.42 't' 1.05 NS 2.93** 7.63*** 16.50*** 7.62*** 8.46*** 5.75*** 1.01 NS [174]

1985 y 2.23 2.14 0.16 0.18 1.29 1.68 0.21 0.14 1.63 1.52 127 172 25 23 9.9 10.4 Sy 0.141 0.018 0.226 0.024 0.226 45.0 4.0 1.88 't' 4.43*** 5.00*** 10.83*** 19.63*** 3.07** 6.29*** 2.93** 1.50 NS [158]

NS- p > 0.05; *- p ~ 0.05; **- p ~ 0.01; ***- p ~ 0.001. Sy -standard error of the mean. 't'- Student's 't' statistics calculated value.

The rootstock effect on foliar mineral concentration 135

Table 2. The rootstock effect on the leaf mineral concentration of the cv. Granny Smith. Comparison between the average of the mean nutrient levels observed between 90 and 120 days after full bloom in two years (1984 and 1985)

M.9 MM.106 M.9 MM.l06 M.9 MM.l06 M.9 MM.106 M.9 MM.106 M.9 MM.106 M.9 MM.106 M.9 MM.106

N(%) p (%) K(%) Mg(%)

1984 y 2.42 2.34 0.17 0.18 1.57 1.91 0.24 0.18 Sy 0.146 0.013 0.158 0.023 '!' 1.82* 2.09** 7.34*** 8.87*** [46]

1985 y 2.27 2.17 0.18 0.19 1.42 1.79 0.22 0.15 Sy 0.112 0.010 0.136 0.023 't' 2.97** 4.63*** 9.14*** 10.83*** [46]

NS-p>0.05; *-p~0.05; **-p~0.01; ***-p~O.OOl. s, -standard error of the mean. 't'- Student's 't' statistics calculated value.

expressed in the dry matter at 100-105°C. Both obtained results lead to identical inferences.

At harvesting, in 1984, the P (Z = 2.363**, N1 = N2 = 8), the K (Z = 3.308***, N1 = N2 = 8) and the Mn (Z = 2.836***, N1 = N2 = 8) foliar concentration were higher on the trees on MM 106, while the Mg (Z = 3.361 ***, N1 = N2 = 8), the Ca (Z = 2.678**, N1 = N2 = 8) and Zn ones (Z = 3.098***, N1 = N2 = 8) were lower than in trees on M 9. In 1985, the P (Z = 2.941**, N1=N2=8), the K (Z=3.151***, N1 = N2 = 8), the Mn (Z = 3.361 ***, N1 = N2 = 8) and the Cu (Z = 3.361 ***, N1 = N2 = 8) foliar concentration were higher in the trees on MM 106, while the N (Z = 2.153**, N1 = N2 = 8) and Mg ones (Z = 3.361 ***, N1 = N2 = 8) were lower than in the trees on M 9.

The obtained results show significant differ­ences (p ~ 0.05) between cv. Granny Smith grafted on M 9 and MM 106 for the most of the determined foliar nutrient mean levels as re­ported by Vurmirovic (1978). Most pronounced differences were obtained between the cation species K +, Ca Z+, and Mg 2+. Leaves from the rootstock MM 106 showed significantly higher K+ concentrations and lower Mg2 + and Ca 2 + concentrations than leaves of the rootstock M 9. It is inferred that the roots of the MM 106 have a more vigorous K + uptake system and this depres­ses the uptake of Mg2 + and Ca2 + because of antagonism (Mengel and Kirkby, 1987). The

Ca(%) Mn(mgkg- 1 ) Zn (mgkg ') Cu(mgkg- 1 )

1.25 1.10 156 190 36 30 9.9 10.2 0.128 22.5 3.6 0.89 3.74*** 5.26*** 5.69*** 1.26 NS

1.52 1.34 95 136 24 21 9.2 9.4 0.140 35.6 3.9 1.97 4.43*** 3.97*** 2.29* 0.19 NS

effect on the N concentration was not so clearly cut. Mn leaf concentration were higher in the tree leaves on MM 106 than on M 9 and the reverse was the case with Zn. In the contrast to the other nutrients Cu mean foliar levels did not show significant differences between both root­stock/ cultivar association, in accordance with the findings of Simons and Swiader (1985). How­ever, in 1985, the Cu-leaf concerning the har­vesting time was higher in the trees on MM 106.

Only small but significant differences in P foliar concentration were observed between the two treatments by either means of sampling for both years. In findings of Ferreira (1978, person­al communication) working with cv. Golden Delicious are relevant to this observation, since this author notes that differences of only 0.01% in leaf-P lead to visual symptoms of deficiency when the value range were between the 0.12% and 0.13% on the dry matter.

Conclusions

The presented results suggest that the rootstock species lead to different leaf mineral composition of the cv. Granny Smith.

The dwarfing rootstock/ cultivar system ( cv. Granny Smith on M 9) shows higher foliar nu­trient mean levels except for P, K and Mn, Cu one that does not seem to be affected by root-

136 The rootstock effect on foliar mineral concentration

stock species, at least on usual collecting date and the mean value obtained during the period between the 9th and 30th week after full bloom.

Leaves from the rootstock MM 106 were high­er in P, K and Mn concentrations but lower in the concentration of Mg and Ca and also slightly lower in N concentration. It is assumed that the rootstock MM 106 has a more efficient uptake system for K+ than the rootstock M 9. High K+ uptake of the rootstock MM 106 may have sup­pressed the uptake of Mg2 + and Ca2 + due to antagonism.

References

Castro R A N 1984 CondU<;ao de mac1euas nas primeiras idades: Alguns factores influentes no seu comportamento com principal relevo para efeitos de variac;:ao na altura da enxertia e na altura da poda a transplantac;:ao. Dissertation, ISA/UTL, Lisboa. 205 p.

Chapman H D 1966 Diagnostic Criteria for Plant and Soils. Division of Agricultural Sciences, University of California. 793 p.

Cooper R E and Thompson A H 1972 Solution culture investigations of the influence of Mn, Ca, B and pH on internal bark necrosis of 'Delicious' apple trees. J. Am. Soc. Hortic. Sci. 97, 138-141.

Duarte L 1989 Teores foliares de alguns nutrientes na

madeira cv. Granny Smith: Efeito do cavalo, das tecnicas de enxertia e da proveniencia das arvores nos teores foliares e sua evoluc;:ao temporal em dois locais distintos. Dissertation, LQARS, INIA, Lisboa. 228 p.

Hulme A C and Rhodes M J C 1971 Pome Fruits. In The Biochemistry of Fruits and Their Products. Ed. A C Hulme. Vol. 2, pp 333-370. Food science and technology: A series of monographs. Academic Press, London.

Jones 0 P et a!. 1984 Rootstock/scion interactions: Apple. Report from East Mailing Research Station for 1983. 60 p.

Mengel K and Kirkby E A 1987 Principles of Plant Nutrition. International Potash Institute, Bern. 137 p.

Moon F E and Hymas G K 1964 Variations in the composi­tion of apple leaves and the errors associated with sam­pling. J. Sci. Food Agric. 15, 201-208.

Saric M R et a!. 1977 The influence of the rootstock and scion on ion uptake and distribution. Vitis 16, 174-183.

Simons R K and Swiader J M 1985 The effects of apple dwarfing rootstocks on leaf nutrient element composition in stoolbed production. J. Plant Nutr. 8, 933-943.

Snedecor G M and Cochran W G 1967 Statistical Methods. 6th ed. Iowa State University Press, Ames, IA. 534 p.

Steel R G D and Torrie J H 1980 Principles and procedures of statistics, a biometrical approach 2nd ed. McGraw-Hill Book Company, New York. 633 p.

Vurmirovic M 1978 Effect of different rootstocks on the content and dynamics of macro-metabolic elements in the leaf of Starking apple trees. Arhivza Poljoprivredne Nauke 31, 103-113 (abstract).

Westwood M N 1978 Temperate-Zone Pomology. Ed. W H Freeman and Company, San Francisco, CA. 428 p.

M.A.C. Fragoso and M.L. van Beusichem (eds.). Optimization of Plant Nutrition, 137-146, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-129

Foliar diagnosis of sugarbeet: Mineral composition of leaves of different physiological age during the season

M.A. CASTELO BRANCO, M.G. SERRAO, M.L. FERNANDES, E.M. SEQUEIRA, H. DOMINGUES and EP. PIRES National Agronomy Research Station, INIA, Quinta do Marques, 2780 Oeiras, Portugal

Key words: age-dependent concentration, Beta vulgaris L., leaf composition, season-dependent concentration, sugarbeet, tissue analysis

Abstract

Variations of the concentration of several elements (N, P, K, Ca, Mg, Na, Zn, Cu, Fe, Mn, B, Mo) in the blades of four types of sugarbeet leaves collected 94, 114, 134 and 157 days after sowing were evaluated, The types of leaves chosen were the following: a young centre leaf (B), the youngest fully expanded leaf near B (A2 ), the next older leaf below A 2 (A 1 ) and a leaf of the oldest leaves whorl (C). The blade concentrations of N, P, K, Zn and Cu decreased from young to the more mature leaves whereas Ca, Mg, Na, Mn and Mo concentrations increased, No well defined pattern was observed forB and Fe variation, especially for iron, Although significant differences were found for some element concentrations in leaves A 2 and A 1 collected at the same time, the range of concentrations in both types of leaves was adequate for those elements except for Ca, Fe, Mn and Na. Phosphorus, Cu, Zn and Mn concentrations in leaves A 2 and A 1 changed systematically from one sampling to the next which suggests that both leaves A 2 and A 1 show the greatest sensitivity to changes in the supply of these nutrients. The blades of leaves A 1 showed to be more suitable for assessing the nutritional status of Ca, Mg and Na in sugarbeet. The young centre leaf (leaf B) seems to reflect most the iron status. For Mo and B concentrations, it appears that both leaves A 2 and A 1 can be sampled for diagnostic purposes, If leaves A 2 and/or A 1 are chosen, the sampling periods need to be closer for Ca, Na, Fe and Mn foliar diagnosis.

Introduction

Among the several steps involved in foliar analy­sis technique, sampling procedures deserve care­ful consideration,

The plant part selected for sampling should give a sharp transition from a concentration reflecting a deficiency of the nutrient in question to one that indicates an adequate supply of the nutrient (Ulrich et a!., 1959). For the sugarbeet, one of the youngest fully matured leaves best meets this criterion (Ulrich et a!., 1959). This type of leaf is located about mid-way between the young centre leaves and the leaves of the oldest leaf whorl.

Seasonal variations in the nutrient concentra­tions of sugarbeet leaves have been observed for leaves changing in physiological age and for leaves of the same physiological age (Ulrich and Hills, 1973). This fact should be taken into account for interpreting leaf analysis data (Bould, 1983).

Previous work on the concentration of some nutrients in young matured leaves of autumnal sugarbeet, collected at rather close intervals during the growing season, has shown anomalous variations which were attributed to some error of the sampling procedure, namely in the choice of the adequate mature leaf.

The objective of the study was to determine

138 Castelo Branco et al.

the influence of leaf position and plant age on the foliar nutrient concentrations of autumnal sugarbeet plants. Recently mature leaves desig­nated A 2 and A 1 were analyzed in particular.

Methods

Sugarbeet cultivar 'Kaweinterpoly' leaves were taken from a field experiment conducted in 1982-1983, on an alluvial calcareous loam soil at Tagus Valley (Almeirim). The fertility analysis of a composite soil sample (0-40 em depth) from the experimental area showed that N, P, K, Ca and Cu were high (Table 1).

The sowing was done on the 2nd of December 1982. Just before the sowing, the field was fertilized with 56 kg ha -I of N, 112 kg ha -I of P, 112 kg ha -I of K and 25 kg ha -I of borax. On the 16th of February, the field received a nitro­gen surface dressing at the rate of 61.5 kg ha -I.

The field size was 2.5 ha, it was divided rough­ly into six equal areas, and the sampling was done at right angles to the crop rows according to Ulrich et al. (1959) and Ulrich and Hills

Table 1. Some properties of the alluvial calcareous loam soil from the experimental area (0-40 em depth)

Sand" Silt" Clay" pH (H,O)" Organic matter" Total N" Conductivity (saturation extract)" Water saturation percentage" Water soluble cations (saturation extract)" Ca Mg K Na Available Pb Available K" Available Cu' Available Fe' Available Mn' Available Zn'

"Silva et al. (1975). bEgner et al. (1960). 'Lindsay and Norvell ( 1978).

66.0% 25.4% 8.6% 7.70

13.57 g kg-[ 0.96 g kg-[ 1.80dSm- 1

48.2

12.61 meq L _, 2.91 meq L _, 0.33 mcq L _, 5.20meqL- 1

0.344 g kg l 0.184gkg-l 0.037 g kg-[ 0.013 g kg-[ 0.015 g kg-[ 0.001 g kg-[

(1973). In each area, leaves from twenty plants were collected, at about three weeks intervals, during the growing season (94, 114, 134 and 157 days after sowing). Four types of leaves (Fig. 1) were chosen in each plant: a young centre leaf (B), the youngest fully expanded leaf near B(Az), the next older leaf below A 2 (A 1), and a leaf of the oldest leaves whorl (C). The first picking was done when it was possible to dif­ferentiate these four types of leaves.

The samplings should have been done until harvest (27th July). However, a disease in plants occurred late in the season therefore the proper sampling of the different types of leaves at harvest was not possible.

The leaf blades were separated from the petioles, and the blades were rinsed first with tap water, after with distilled water and at last with distilled-deionised water. After drying at 70°C, during 48 h, the leaf blades samples were ground to pass through a 1 mm sieve.

Nitrogen was determined by the Kjeldahl method. The concentrations of P, K, Ca, Mg, Na, Zn, Cu, Fe, Mn, and Mo were determined after dry ashing ( 480°C) and dissolution of the ash in hydrochloric acid. The concentrations of K, Ca, Mg, Na, Zn, Cu, Fe and Mn were determined by atomic absorption spectropho-

c

Fig. 1. Types of sugarbeet leaves sampled (Adapted from Draycott, 1972).

tometry (Silva et al., 1975). Mo and P concen­trations were determined spectrophotometrically according to Purvis and Peterson (1956) and ADAS (1986), respectively. Boron concentration was determined after addition of calcium hy­droxide to the plant tissue and ignition to a grey ash which was taken up with sulphuric acid 1 M solution; the 1,1 '-dianthrimide colorimetric method was then applied to the clarified extract (Garfinkel and Pollard, 1952). The nutrient concentrations were expressed on a dry matter basis.

Analysis of variance of the nutrient concen­trations was made. The arcsine transformation ('angular transformation') of the nutrient data was made before the analysis of variance (Sokal and Rohlf, 1981).

Results

Leaf position

Nitrogen concentration was highest in young leaves and decreased as the leaf age increased (Fig. 2). The differences in the N concentration between leaves B, A 2 and A 1 decreased through­out the season.

Phosphorus concentration decreased in the order B > A 2 > A 1 > C (Fig. 3). At the 1st sampling, the four types of leaves were always significantly different. At the 2nd sampling there was a little difference in P concentration between leaf A 1 and C; the same was observed between leaf A 2 and A 1 at the last sampling.

Potassium showed a tendency to decrease from leaf B to leaf C (Fig. 4). However, these variations were not significant until the last sampling in which there was a significant differ­ence between leaf B and leaves A 2 and A 1 .

Also, there were significant differences between these last types of leaves and leaf C. No differ­ences between leaves A 2 and A 1 were found throughout the season.

Calcium increased rapidly from the young centre leaf to the oldest leaf, with the exception of leaves A 2 and A 1 at the last sampling, in which they had almost the same concentration (Fig. 5).

Magnesium concentration was highest in the

Foliar diagnosis of sugarbeet 139

oldest leaf (leaf C) then in decreasing order the highest levels were found in leaf A~> leaf A 2 and leaf B (Fig. 6). Leaves A 2 and A 1 were always different in Mg concentration, except at the last sampling.

The average concentration of Na differed among types of leaves (Fig. 7). It was highest in the oldest leaves (leaf C) and decreased from these leaves to the young centre leaves (leaves B). There was a small difference between leaves A 2 and A 1 at the last sampling, as observed for Ca and Mg.

At the 1st and the last sampling, Zn concen­tration decreased progressively from leaf B to leaf C (Fig. 8). At the 2nd sampling, Zn concen­tration tended towards to decrease, however, the concentration in leaf A 2 was lower than in leaf A 1 • At the 3rd sampling, the trend was the same, but the concentration in leaf A 1 and leaf A 2 was the same.

In general, Cu concentration decreased from leaf B to leaf C. These changes were always significant at the 2nd and 3rd samplings (Fig. 9). Leaves A 2 and A 1 were always different at all sampling times.

Iron variation in all types of leaves showed no consistent trend (Fig. 10).

Manganese concentration showed a tendency to accumulate in older leaves, except at the 1st sampling time in which small differences among the four leaf ages were found (Fig. 11 ). Leaves A 2 and A 1 were always different, except at the 1st sampling.

The trend variation of B concentration was similar at the 1st and the last sampling (Fig. 12). At the 1st sampling, the concentration of leaves A 2 and A 1 was bigger than in the others. However, this difference became more evident at the last sampling. At the 2nd and 3rd sam­plings, B concentration trend was a decrease from leaf B to leaf C, but small differences among the four leaves ages were found.

The highest Mo concentration was observed in the more mature leaves (Fig. 13).

Plant age

In general, the N concentration slightly de­creased with plant growth, in all types of leaves

140 Castelo Branco et a[.

100

80

60

40

20

0

30

25

20

15

10

5

0

75

60

45

30

15

0

FIg. 2. N

Flg.5. Ca

2 3 4

Flg.S. Zn

I?'Zll Leaf B

E:::3 Leaf Al

FIg. 3. P 10

8

6

4

2

0 2 3

Flg.6. Mg 25

20

1!!

10

5

0 2 3 4

Flg.9. Cu 50

40

30

20

10

0 4

Flg.12. 8 50r----------------.

40

30

20

10

Ol--~~~~~~LM~

2 3 4

Time of sampling

0 LeafA2

- LeafC

FIg. 4. K 50

40

30

20

10 I 0

2 3 4

Flg.7. Na 60

50

40

30

20

10

0 2 3 4

Flg.10. Fe 650

520

390

260

130

0 2 3 4

Fig. 13. Mo 1.0,------------,

o.e

0.6 t 0.4

0.2

O.Ol--...._._,...,__,.,"-.J''-""1,' 2 3 4

Time of sampling

Fig. 2-13. Positional variation in the N, P, K, Ca, Mg and Na concentration (g kg- 1) and Zn, Cu, Fe, Mg, B and Mo

concentration (mg kg- 1 ) of sugarbeet leaf blades.

(Fig. 14). However, in leaves A 2 , A 1 and C the N concentration tended to increase between the 3rd and the 4th samplings.

Analysis of variance indicated a significant interaction between phosphorus concentration in leaves and time of sampling. The phosphorus concentrations decreased from the 1st until the 3rd sampling and increased at the last sampling time (Fig. 15). In leaves A 2 and A 1 the differ­ences from one sampling to the next were always significant. As to potassium concentration, only in leaves C and at the last sampling time there was a signifi­cant decrease (Fig. 16). In leaves B, A 2 and A 1 ,

the concentration of the element was similar at all samplings.

The biggest changes of calcium concentration during the course of sampling occurred in leaves A, (Fig. 17). However, there was also a de­crease between the 2nd and the 3rd samplings in leaves B and an increase between the 1st and the 2nd sampling in leaves A 2 •

The magnesium levels in leaves B and A 2

showed very slight changes throughout the sam­pling period (Fig. 18). The magnesium concen­tration in leaves A 1 and C had a significant increase between the 1st and the 2nd samplings. Thereafter the Mg concentration in leaves A 1

decreased but not significantly until the last sampling while in leaves C, the Mg concentration increased until the 3rd sampling.

Sodium concentration in the blades of leaves A 2 and C showed a similar variation in succes­sive samples but in leaves C the decrease from the 3rd until the 4th sampling was significant while in leaves A 2 this is not observed (Fig. 19). In leaves A" there was a significant increase of the element from the 1st until the 2nd harvest and a decrease at the sequent samplings, espe­cially at the last one. Changes of this element in leaf B were insignificant.

Zinc concentration in leaves A 2 , A 1 and C showed a similar variation throughout the sam­pling period (Fig. 20). However, Zn levels in leaves A 1 and C were different from one sam­pling and another, while in leaves A 2 both 1st and 3rd samplings did not differ and the Zn concentrations were lower than at the other samplings.

The overall trend of the changes of Cu concen-

Foliar diagnosis of sugarbeet 141

tration in leaves B, A 2 and A 1 was similar (Fig. 21). By comparing the four samplings of these types of leaves, an increase of Cu concentration was quite evident at the 3rd harvest. The Cu concentration in leaves A 2 and A 1 was always different from one sampling to the next. In leaves B, only the leaves collected at the 3rd sampling had a distinct higher amount of this clement. The Cu concentration in leaves C at the 2nd sampling was lower than at all the other sampling times.

Fig. 22 shows that the Fe concentration in leaves B, A 2 and A 1 changed in a similar way throughout the sampling period, although the variations from one time of sampling to the next were especially evident in leaves B.

Manganese levels in leaves A 2 , A 1 and C were higher in successive samples with the exception of leaves A 2 and A, at the last sampling date, where Mn decreased (Fig. 23). On the other hand, Mn concentration in leaves B was general­ly invariable, with the exception of the 3rd sampling time.

In all types of leaves, boron concentration generally decreased until the 3rd sampling (Fig. 24). Subsequently there was a great increase of the nutrient in all blades, especially in leaves A 2

and A 1 .

In leaves B, the Mo concentration decreased until the 3rd sampling and thereafter a slight increase occurred while in the other types of leaves the reverse was observed (Fig. 25).

Discussion

Leaf position

Both nitrogen and phosphorus are very mobile in phloem. Sorensen (1962) and Coic et a!. (1962) reported that the young expanding laminae of sugarbeet contains more organic N than the older ones. The same results were found by Shahla ct a!. (1988). The very small differences between leaves B, A 2 and A 1 at the last sam­pling are perhaps due to the slowness of new growth late in the season while the rate of senescence increases, thereby lowering the rate of redistribution of N (Shahla et a!., 1988).

142 Castelo Branco et al.

Flg.14. N Flg.15. P Flg.16. K 100 10 ~0

eo 8 40

60 6 30

40 4 20

20 2 10 I 0 0 0

8 c 8 8 A2 A1 c

Flg.H. Ca Flg.18. Mg Flg.19. Na 30 2~ 60

25 20 50

20 40 15

15 30

10 10

20

5 5 10

0 0 0 c c

FIg. 20. Z n FIg. 21. C u FIg. 22. Fe 70 50 650 60 50

40 ~20

40 30 390

30

~ lm 20 260

20

0 10 130

0 0 8 A2 A1 c 0

FIg. 23. Mn Flg.24. B FIg. 25. Mo 120 50 1.0

100 40 0.8 80

30 0.6 60

40 20 0.4

20 10 0.2

0 0 0.0 8 A2 A1 c c

Type of leur Type of leur Type of leur

~ lst time of sampling [=:J 2nd time of sampling

E3 3rd time of sampling - 4th time of sampling

Fig. 14-25. Seasonal variation in the N, P, K, Ca, Mg and Na concentration (g kg - 1 ) and Zn, Cu, Fe, Mn, B and Mo

concentration (mg kg-]) of sugarbeet leaf blades.

Studies of the differences in N levels of blackcur­rent leaves due to leaf position (Bould, 1955) showed that the positional variation remains fairly constant at the last sampling.

To explain the highest P concentration in youngest leaves, Guha and Mitchell (1966) and Bouma (1967) reported that this element is possibly mobilized from the more mature leaves via the phloem to the younger tissues. Although leaves A 2 and A 1 were significantly different at the 1st and 2nd samplings, the P concentrations (2.3-5.7 g kg - 1) were within the intermediate range (1.0-8.0g kg- 1) established as adequate by Ulrich and Hills (1990).

Potassium is also an element of extreme mobility throughout the entire plant (Mengel and Kirkby, 1987). The small differences be­tween the four leaf ages until the 3rd sampling can be explained by extensive recycling of K in the plant (Shahla et a!., 1988) or by adequate amounts of K in the plant (Wilcox and Coffman, 1972). By the last sampling, K translocation from old to young leaves may explain the lower K concentrations in the older leaves (Fig. 4 ). Similar behavior was observed by Greenway and Pitman (1965). They verified that the K+ import into the oldest leaf was only slightly higher than the K + export. The K + export was redistributed from oldest to next younger leaf and successively to the younger one. It is obvious that the younger leaves were supplied with K + origina­ting from older tissues. The K concentrations (30.6-38.6g kg- 1 ) in leaves A 2 and A 1 were within the range of sufficiency (20.0-60.0 g kg - 1)

by Jones, Jr. et a!. (1991 ). The increase of Ca concentration in develop­

ing leaves tissues is often explained by the low mobility of calcium in phloem (Bittner and Buschamann, 1983; Mengel and Kirkby, 1987; Smith, 1986). Although Ca concentration in leaves A 2 and A 1 differed significantly until the 3rd sampling, only at the 2nd and 3rd samplings did the values vary from adequate levels (in leaf A 2 ) to high levels (in leaf A 1 ) (Jones Jr. et a!., 1991 ).

The trend variation of both Mg and Ca con­centrations was quite similar. The highest con­centrations occurred in the oldest leaves (C), then followed in decreasing order by leaves A 1

and A 2 , and youngest leaves. Brown and Irving

Foliar diagnosis of sugarbeet 143

(1942) and Shahla et a!. (1988) reported also similar results and established a close parallel between the Ca and Mg concentrations in sugar­beet. In spite of the concentration in leaves A 2

and A 1 differed significantly until the 3rd sam­pling, the concentration range was considered as adequate (Ulrich and Hills, 1990).

Sodium is an element considered to be rela­tively immobile in the phloem, therefore it accumulates in older leaves. This was observed in this study and by Shahla eta!. (1988). The Na concentration in leaves A 1 at the 2nd and 3rd samplings was higher than considered as adequate (0.2-37.0g kg- 1) by Ulrich and Hills (1990), whereas in leaves A 2 the levels were adequate at all samplings.

Zinc concentration was generally higher in younger leaves than in older ones. Zinc has been shown to be readily remobilized from old leaves to developing leaves if the plants are well sup­plied with Zn (Riceman and Jones, 1958). Zinc concentration in all types of leaves was higher than 8 mg kg - 1 found by Bowan and Viets (1956) in deficient leaves. However, at the 1st, 2nd and 4th samplings, Zn concentration was different in leaves A 2 and A 1 .

Copper shows an intermediate mobility within the plant, therefore Cu concentrations are usual­ly higher in younger leaves than in older leaves. In spite of leaves A 2 and A 1 being significantly different at all samplings, the Cu levels in both leaves A 2 and A 1 were higher than 7 mg kg-\ which is considered by Haddock and Stuart (1970) as adequate for sugarbeet.

As shown in Figure 10, iron did not move from leaf to leaf, although this element has an intermediate mobility in plant phloem (Kochian, 1991). Iron concentration in leaves A 2 and A 1

was much higher than considered as normal ( 60-150mg kg- 1) by Draycott (1972) at the 3rd and 4th samplings, although no differences were observed between leaves A 2 and A 1 at the same time of sampling.

Manganese is an element considered as im­mobile within the plant (Nable and Loneregan, 1984) which explains the higher concentrations in older leaves. At all sampling times, the Mn concentration in leaves A 2 and A 1 was signifi­cantly different. However, concentrations in both leaves A 2 and A, at the 1st sampling were

144 Castelo Branco eta!.

low and at the other samplings they were be­tween levels considered as adequate (26-360 mg kg- 1 ) by Vomel and Ulrich (1963).

Although there were only small differences in boron concentrations between leaves (especially until the 3rd sampling), boron tended to de­crease slightly from younger to older leaves. Boron is generally considered to be phloem immobile although its mobility may vary during plant development (Kochian, 1991 ). This fact may be related to the higher variations between leaves observed at last sampling. B concentration in leaf A 2 , at last sampling, was quite near the lower limit of the adequate range of concen­trations (35-200 mg kg -I) whereas the concen­tration in leaf A 1 was considered inadequate (Ulrich and Hills, 1990).

Old leaves contained more Mo than the younger ones. Normally, there is little if any movement of Mo out of leaves, when the levels are relatively low (Reuter, 1986). Although the concentrations in the leaf tissue were not high, the levels found in both leaves A 2 and A 1 were considered adequate for sugarbeet (Ulrich and Hills, 1969).

Plant age

From the results, it is clear that changes in the nutrient concentrations of sugarbeet leaves occurred with season.

Concentrations of N, P and B generally de­creased as the season progressed until the 3rd sampling, and thereafter increased. According to Haddock (1958), the increase of N late in the season could have resulted from root contact with deep soil N (probably nitrate) as the roots extended into subsoil. Shahla et a!. (1988) gave the same explanation for a similar increase observed in P concentration. The changes in P concentration throughout the season were much bigger than in N concentration as was expected because N concentrations were high even at the first sampling time. For boron concentration, the increase at last sampling could also be due to the root extension into subsoil where the applied boron may have been leached.

Potassium concentration remained almost in­variable throughout the season except at 3rd and

4th samplings of leaf C. The extensive recycling of Kin the plant may be an explanation (Shahla et a!., 1988).

The seasonal trends in the leaf concentration of Ca and Mg were similar as also reported by Brown and Irving (1942). However, for each one of these elements, the changes did not show the same trend in all types of leaves.

Conclusions

The blade concentrations of N, P, K, Zn and Cu decreased from young to the more mature leaves whereas Ca, Mg, Na, Mn and Mo concentrations increased. No well defined pattern was observed for B and Fe variation, especially for iron.

Although significant differences were found for some element concentrations in leaves A 2

and A 1 collected at the same time, the range of concentrations in both types of leaves was adequate for those elements except for Ca, Fe, Mn, and Na.

Phosphorus, copper, zinc and manganese con­centrations in leaves A 2 and A 1 changed sys­tematically from one sampling to the next which suggests that both leaves A 2 and A 1 show the greatest sensitivity to changes in the supply of these nutrients.

The blades of leaves A 1 showed to be more suitable for assessing the nutritional status of Ca, Mg and Na in sugarbeet.

The young centre leaf (leaf B) seems to reflect better the iron status in plant.

For Mo and B concentrations, it appears that both leaves A 2 and A 1 can be sampled for diagnostic purposes.

If leaves A 2 and/ or A 1 are chosen, the sam­pling periods need to be closer for Ca, Na, Fe and Mn foliar diagnosis.

Acknowledgements

The sugarbeet experiment was established on Administra<;ao Geral do A<;uar e do Aicool (AGA) land. The cooperation of Eng Duarte Amaral, Eng M Augusta Jeronimo, Eng Carmo

Silva, and Techn Eng Armando Silvestre (AGA) is particularly appreciated. Also, thanks go to Techn Engs Odete Monteiro and Armenio Oliveira, and to Technicians M Ema Faustino, M Celeste Campos, M Augusta Figueira, Teresa Vales, and M Helena Coelho (Esta<;ao Agron-6mica Nacional) for the soil analysis. Thanks are also due to Eng M Eugenia Balsa and M Regina Gusmao for kind assistance.

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Foliar diagnosis of sugarbeet 145

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Kochian LV 1991 Mechanisms of micronutrient uptake and translocation in plants. In Micronutrients in Agriculture. 2nd Edition. Eds. J J Mortvcdt, F R Cox, L M Shuman and R M Welch. pp 229-296. Soil Sci. Soc. Am. Inc., Madison, Wisconsin.

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Mengel K and Kirkby E A 1987 Principles of Plant Nutrition. 4th Edition. International Potash Institute, Bern, Switzer­land, 687 p.

Nable R 0 and Loneregan J F 1984 Translocation of manganese in subterranean clover (Trifolium subterraneum L. cv Seaton Park). I. The redistribution during vegetative growth. Aust. J. Plant Physiol. 11, 101-111.

Purvis E R and Peterson N K 1956 Methods of soil and plant analysis for molybdenum. Soil Sci. 81, 223-228.

Riceman D C and Jones D B 1958 Distribution of zinc and copper in subterranean clover (Trifolium subterraneum L.) grown in culture solutions supplied with graduated amounts of zinc. Aust. J. Agric. Res. 9, 73-122.

Shahla E M, Lee G S and Schmehl W R 1988 Nutrient concentration in sugarbeet senescing leaves during the season and six plant parts at harvest. J. Sugar Beet Res. 25, 11-27.

Silva A A, Alvim A J S and Santos M J 1975 Metodos de analise de solos, plantas e aguas. Pedologia, Oeiras 10, 153-544.

Smith F W 1986 Interpretation of plant analysis. Concepts and principles. In Plant Analysis. An Interpretation Manu­al. Eds. D J Reuter and S B Robinson. pp 1-12. Inkata Press, Melbourne, Sydney.

Sokal R Rand Rohlf F J 1981 Biometry. The Principles and Practice of Statistics in Biological Research. 2nd Edition. HW Freeman and Company, S. Francisco, USA, 859 p.

Sorensen C 1962 The influence of nutrition of the nitrogen­ous constituents of plants. III. Nitrate test and yield structure of fodder sugar beet leaves. Acta Agric. Scand. 12, 106-124.

Ulrich A and Hills F J 1969 Sugar beet nutrient deficiency symptoms. A colour atlas and chemical guide. Univ. California, Division of Agric. Sci., Berkeley, 36 p.

Ulrich A and Hills F J 1973 Plant analysis as an aid in fertilizing sugar crops: Part 1. Sugar beets. In Soil Testing and Plant Analysis. Eds. L M Walsh and J D Beaton. pp 271-288. Soil Sci. Soc. Am. Inc., Madison, Wisconsin, USA.

Ulrich A and Hills F J 1990 Plant analysis as an aid in fertilizer sugarbeet. In Soil Testing and Plant Analysis.

146 Foliar diagnosis of sugarbeet

Eds. R L Westerman, J V Baird, N W Christensen, P E Fixen and D A Whitney. pp 429-447. Soil Sci. Soc. Am. Inc., Madison, Wisconsin, USA.

Ulrich A, Hills F J, Ririe D, George A G and Morse MD 1959 I. Plant analysis- a guide for sugar beet fertilization. Calif. Agric. Exp. Stn. Bull. 766, 3-24.

Vomel A and Ulrich A 1963 Die Blattenalyse zur Ermittlung von Mangan Mangel bei Ruben. Z. Pflanzenernaehr Bodcnkd. 102, 28-45.

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M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 147-151, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-131

Diagnosing nutritional status of sugarbeet by soil and petiole analysis

M.D. OLIVEIRA, C.F. CARRANCA, M.M. OLIVEIRA and M.R. GUSMAO National Agronomy Research Station, IN/A, Quinta do Marques, 2780 Oeiras, Portugal

Key words: Beta vulgaris L., nitrate petiole analysis, NPK fertilization, soil testing, sugarbeet

Abstract

The main problem with nitrogen fertilization of sugarbeet is to decide when and how much N to apply for higher yielding quality crops. Soil tests for both N0 3-N and mineralizable-N are required as a basis for fertilizer recommendations. Besides soil N measurements, N0 3 levels in plant petioles along the growing cycle, and root sugar concentration at harvest, are assumed as sensitive indicators of the actual available-N and crop needs. A fertilizer experiment with sugarbcet 'Kawetanya' was conducted in an alluvial soil in Vila Franca de Xira. The plots were arranged on a randomized block design of the type 3 x 22 factorial for N, P and K, with 3 replicates. Nand K fertilizers affected root yield, as statistically denounced; sucrose concentration was also significantly affected by N and P applications; sugar yield was highly affected by applied N and significantly affected by N x K interaction; N0 3 concentrations in the petioles were not significantly affected by fertilization. Aerobic incubation and hydrogen peroxide methods to estimate mineralizable-N in soil were compared and a non-significant correlation was obtained.

Introduction

Practice confirms that special attention is re­quired for nitrogen fertilization of sugarbeet. Among the mineral elements, nitrogen has the greatest influence on physiology of the crop, affecting its various characteristics. Limited N application generally leads to high sucrose con­centration and high juice purity, but root weight and sucrose yield are restricted. Excessive avail­able nitrogen during the growing season can lower crop quality as vegetative growth is stimu­lated and therefore the sucrose concentration is reduced. Impurities in root composition are also increased which limit refined sucrose production (Dias and Oliveira, 1987).

The main problem with sugarbeet N-fertiliza­tion is then to decide how much and when to supply N. Analysis of soil nitrate is a provisional estimated availability of N to plants. Because some soil organic-N suffers mineralization to a plant available form, it needs to be considered as a nitrogen added source for the growing crop,

soil tests for both nitrate and mineralizable-N are advised as a basis for fertilizer recommendations (Roberts et al., 1972).

As sugarbeet accumulates much nitrate in leaves, particularly in the petioles, besides soil-N measurements, levels of nitrate in plant petioles during the growing season and concentration of sugar at harvest are regarded as sensitive in­dicators of the actual available-N for the crop (Dias and Oliveira, 1987; Roberts et al., 1972).

This report covers the results obtained from a one-year field fertilizer trial. It also evaluates some soil and plant tests that can be used to predict needs for sugarbeet optimum root weight and sucrose production.

Methods

A fertilizer experiment with irrigated sugarbeet (Beta vulgaris, L. cv. Kawetanya) was conducted in 1990 in a sandy clay loam soil in Vila Franca de Xira.

Tab

le

1.

Phy

sica

l an

d c

hem

ical

pro

pert

ies

of

the

sand

y cl

ay l

oam

soi

l

Dep

th

pH

O

rg.

C

Tot

al

Inm

gani

c-N

M

iner

aliz

a bie

-N

(em

) (H

,O)

(gk

g

')

N

(mg

kg

-1)

(mg

kg

-1

)

(g k

g-'

) N

H4

NO

, A

er.

H,o

, in

cub

... m

cth.

b

0-3

0

7.85

"0

.20

1

7.1

ct1

.57

0

0.8

2"

0.22

5 7

.70

" 2.

351

12

.77

" 3.

809

19

.34

" 9.

343

28

.81

" 15

.299

3

0-6

0

7.9

0"

0.15

2 5.

2 ±

0.8

99

0.62

± 0

.141

7.

39 ±

2.1

47

10.7

7 ±

2.4

09

9.59

± 8

.163

17

.94

± 9

.450

6

0-9

0

8.00

± 0

.124

3.

5 ±

1.

274

0.5

0±

0.2

29

6.98

± 2

.379

9.

36 ±

2.0

15

6.39

± 8

.940

1

0.5

4"

7.55

5

"Aer

. in

cub

=A

ero

hie

inc

ubat

ion:

bi-LO~

met

h.

=H

yd

rog

en p

erox

ide

met

ho

d.

Tabl

e 2.

C

orr

elat

ion

coe

ffic

ient

s fo

r m

iner

aliz

ablc

-N a

nd m

iner

ai-N

wit

h so

il p

H,

orga

nic

C a

nd t

otal

N i

n th

e pr

ofil

e

Dep

th

(em

)

0-3

0

30

-60

6

0-9

0

Min

eral

izab

le-N

Acr

. in

cub.

met

hJ

pH

O

rg.

C

n.s

. 0.

391

* n.

s.

n.s.

n.

s.

0.55

7***

To

ta1

N

n.s.

n.

s.

o.3

4r

Min

eral

-N

Hyd

rog.

pcr

ox.

met

h.tr

A

er.

incu

b. m

eth

.

pH

O

rg.C

T

ota

1N

p

H

Org

. C

n.s.

n.

s.

0.55

6***

n.

s.

0.5

02

" n.

s.

0.3

88

' n.

s.

n.s.

0.

351

* n.

s.

0.4

44

'*

0.4

75

'*

n.s.

0.

573*

**

CE

C

Ava

ilab

le

(cm

o1( +

)kg

1

) p

K

(mg

kg

1

)

1.3

76

" 0.

398

10

7.9

" 17

.491

12

7.0

"35

.76

7

1.01

6 ±

0.1

02

58.6

± 1

0.91

6 59

.8 ±

11.

071

1.04

2 ±

0.1

71

33

.6 ±

3.5

97

40.7

± 3

.376

Hyd

rog.

per

ox.

met

h.

To

taiN

p

H

Org

. C

T

ota

1N

n.s.

n.

s.

0.36

3*

0.5

34

"*

0.37

3*

n.s.

0

.46

9"

0.3

47

' 0

.52

5"

n.s.

0

.35

9'

0.5

25

***

aAer

. in

cub.

met

h.

=A

ero

bic

inc

ubat

ion

met

ho

d;

hHyd

rog.

per

ox.

met

h. =

Hy

dro

gen

per

oxid

e m

etho

d; n

.s.,

*,

**,

***

r-va

lucs

non

-sig

nifi

cant

an

d s

igni

fica

nt a

t 5

%.1

% a

nd

0

.1%

pro

babi

liti

es,

resp

ecti

vely

.

--1'>- c:>O a ::::-: " "" :::;· ., ~ ~

Plots were arranged as randomized block design with 3 replicates. The experiment was of the type 3 x 2 2 factorial for N, P and K involving 3 rates of urea (0, 75 and 150kgha- 1 of N), 2 rates of superphosphate 18% (0 and 100 kg ha -I of P) and 2 rates of potassium chloride (0 and 100 kg ha 1 of K). Both P and K fertilizers were broadcast before sowing while urea was incorpo­rated by disking into the soil when of seedbed preparation. Individual 6-row plots were 3m wide and 24m long. Irrigation was made by furrows applying 60 Lm -z water.

Previously to trial layout, soil samples were taken from each plot, at 0-30, 30-60 and 60-90 em depths to characterize the soil profile (Table 1 ).

Two methods were compared to determine mineralizable-N, aerobic incubation and hydro­gen peroxide oxidation. Mineral-N was esti­mated by adding the mineralizable-N to the inorganic-N. After harvesting, some soil samples were taken along the profile, in each plot, to evaluate residual amount of nitrogen in the soil mostly as resulting from the N-treatment. Ana­lytical methods used for soil samples are de­scribed in Carranca (1986).

Prior to harvest (July, August, September and October) some recently, fully mature expanded leaves, were sampled at random, according to Ulrich ct al. (1959). Petioles were separated, oven-dried at 70-80° C and analyzed for nitrate content (Dias and Oliveira, 1987).

Root yield was evaluated by harvesting plants in two central rows. Sucrose concentration was also estimated (Browne and Zcrban, 1941) to evaluate sucrose yield.

Analysis of variance was applied to residual-N in soil, root yield, sucrose concentration, sucrose yield and N0 3-N content in petioles. Correlation analysis was used to compare both methods for determining mineralizable-N in the soil and to relate mineralizable and mineral-N with soil pH, organic C and total N.

Results

In Table 1, main physical and chemical soil properties at 3 depths are shown. The moderate­ly alkaline soil was poor in organic matter and N

Nutritional status of sugarbeet 149

Table 3. Effect of N-treatment on residual inorganic-N and total N, at the end of growth cycle

Source of variation NO,-N TotalN

N 3.45* n.s. 5.09**

n.s., *, ** F-values non-significant and significant at 5% and 1% probabilities, respectively.

content. The mineralization capacity was moder­ate, varying from 23 to 12 mg kg 1 (by aerobic incubation) or 35 to 21 mg kg -I (by hydrogen peroxide), from the surface deeper into the soil profile, according to results in Carranca (1986).

Mineralizable-N, obtained by both aerobic incubation and hydrogen peroxide methods, and mineral-N were compared in each depth. Only for mineral-N values obtained from oxidation, at 30-60 em depth, a very significant r was found (r=0.48**).

Correlation coefficients for the relationships between mineralizable and mineral-N values with soil pH, organic C and total N are reported in Table 2.

Analysis of variance for residual amount of inorganic-N in soil at the end of growing cycle, showed a significant effect of N-treatment on ammonium content (F = 3.45*), with a slight increase for higher N levels, whereas residual nitrate was not significantly affected by applied N. Total N in the soil was statistically much affected by N rate (F = 5.09**). These results arc shown in Table 3.

Results of analysis of variance and the mean values of petiole nitrates, root yield, sucrose concentration and sucrose yield are presented in Table 4.

Discussion and conclusions

As organic matter content in soil was low, mineralizable-N values obtained by both ana­lytical methods were moderate. These values agree with those found by Carranca (1986) and Oliveira et al. (1989). That paper reports a slightly better correlation between the mineral-N values obtained by both methods, for similar soil conditions. From present results, mineralizable­N obtained by aerobic incubation was especially correlated with soil organic carbon; miner-

150 Oliveira et al.

Table 4. Mean and analysis of variance results of petiole nitrates, root yield, sucrose concentration and sucrose yield

Treatment Nitrate in the petioles (mg kg- 1 ) Root Sucrose Sucrose yield concentration yield

1st stage 2nd stage 3rd stage 4th stage (kg ha - 1 ) (gkg I) (kgha- 1)

NnPuKu 702.3 691.3 667.3 810.7 83,790 149 12,510 N0 P0 K, 757.3 781.0 948.3 527.3 85.150 152 12,950 N0P1K0 605.3 776.7 889.0 737.7 84,090 152 12,780 NIIP,K, 367.7 569.7 818.0 684.0 80,150 152 12,160 NIPOK(J 379.7 691.0 712.0 1150.7 103.630 144 14,920 N 1P0 K 1 1298.3 812.0 1945.0 1075.7 92,580 141 13,090 N 1P1K0 622.0 817.0 811.3 716.7 108,330 149 16,200 N,P,K 1 439.0 439.7 629.0 544.7 91,060 144 13,130 N2P0 K0 2109.7 844.0 1358.3 875.0 106,520 139 14,810 N 2P0K 1 363.7 848.3 1075.3 892.0 101,670 141 14,350 N 2P1K0 1364.7 596.7 865.0 706.3 99,090 147 14,520 N,P,K, 866.3 987.3 718.0 927.7 101,670 152 15,420

N n.s. n.s. n.s. n.s. 21.65*** 3.69* 9.50*** p n.s. n.s. n.s. n.s. n.s. 4.55* n.s. K n.s. n.s. n.s. n.s. 4.50* n.s. n.s. NXP n.s. n.s. n.s. n.s. n.s. n.s. n.s. NxK n.s. n.s. n.s. n.s. n.s. n.s. 3.58* PXK n.s. n.s. n.s. n.s. n.s. n.s. n.s. NxPxK n.s. n.s. n.s. n.s. n.s. n.s. n.s.

n.s., *, *** F-values non-significant, and significant at 5% and 0.1% probabilities.

alizable-N obtained by H 20 was also correlated with organic carbon but also with total N. Mineral-N by both methods showed similar rela­tionships either with organic carbon or total N. Soil pH was not significantly related to any method, probably because the low variation in soil pH down the profile.

Residual nitrate in soil shows that crop has removed all of it whatever N rate was applied. Ammonium accumulated at the end of growing cycle, probably resulting from a slow miner­alization rate of organic matter. This information is reinforced by accumulated total N.

Root yields ranged from 80, 150 to 108,330 kg ha -I, respectively at levels 0 and 75 kg ha -I of N. Lower yields were obtained by Oliveira et a!. (1989), varying from 24,400 to 68,300 kg ha -I for the same N levels, under similar soil type and crop management practices, but using another crop variety. Carter et a!. (1974) and Burnay (1983) also found root yields lower than those obtained in this experiment, even with higher N rates. Oliveira et a!. (1989) have also found a very significant effect of N fertilization on crop yield, but sucrose concen-

tration was not significantly affected by N-treat­ment. In the present experiment, sucrose con­centration in roots had a negative response to N applied, decreasing from around 150 to 145 g kg -I, respectively at 0 and 75 kg ha -I of N applied. No significant differences were found for sucrose concentration obtained with 75 or 150 kg ha -I of N application. Sucrose concen­trations obtained by Oliveira et a!. (1989) were higher as compared to this experiment ( 180-208 g kg -I) probably because crop variety was of Z type while the present one is of NZ type.

Nitrate content in petioles was not significantly affected by treatments, at any sampling time and varied from around 370 to 1,945 mg kg -t, re­spectively in first and third sampling stages. A significant correlation was found for nitrate in petioles sampled in September and sucrose con­centration. Levels of nitrate in petioles, at any time of sampling, were in general lower than the critical level ( 1,000 mg kg 1), indicating that plants were N deficient (Ulrich et a!., 1959) though with no N deficiency symptoms, which according to Gilbert et a!. (1981) appear for petiole nitrate levels around 70-200 mg kg- 1 (in

this experiment the lowest nitrate values were higher than 300mgkg- 1). The variability in N03-N content within the same N-level suggests that probably, in each sampling stage, leaves were harvested with different physiological life­time, or to analytical errors.

Under the present experimental conditions, the conclusions to take off this study are about the advisible N rate, and the most appropriate time to sample the leaves for N03-N measure­ments in the petioles as an indicator of the actual available-N for the crop. As N significantly affected sucrose concentration and root yield, 75 kg ha -I of N seems to be the recommended N rate for a higher sugarbeet production, with a high level of sucrose and probably a better root quality. Four to six weeks before the harvest seems to be the adequate sampling time to harvest the leaves for controlling root quality for sugar production.

Acknowledgements

Thanks are due to the laboratory staff, to J Baeta's statistical treatment and for paper revi­sion.

Nutritional status of sugarbeet 151

References

Browne C A and Zerban F W 1941 Physical and chemical methods of sugar analysis. Wiley, New York.

Burnay C H E C 1983 Beterraba sacarina (Beta vulgaris L.): Tecnicas culturais e ensaios de compara~ao de cultivares. Relat. Final do Curso de Eng. Agr6n. ISA, Lisboa.

Carranca C F 1986 Nitrogen availability and ammonium fixation in some maize cultivated soils of Portugal. Thesis for M.Sc. (Agric.) in Soil Science, Oeiras. 89 p.

Carter J N, Jensen ME and Bosma S M 1974 Determining nitrogen fertilizer needs for sugarbeets from residual soil nitrate and mineralizable nitrogen. Agron. J 66, 319-323.

Dias A and Oliveira M 1987 Nitrate reductase and petiole nitrate as indicators of the nitrogen nutrition status of field grown sugar beet. Agron. Lusit. 42 (3-4), 275-284.

Gilbert W A, Ludwick A E and Westfall D G 1981 Predicting in-season N requirements of sugarbeets based on soil and petiole nitrate. Agron. J. 73, 1018-1023.

Oliveira M D, Gusmao M R, Carranca C L and Gon~alves M G 1989 Some forms of nitrogen in a sugarbeet cultivated soil of Portugal. In Management Systems to reduce Impact of Nitrates. Ed. J C Germon. pp 197-209. Elsevier Applied Science. London.

Roberts S, Richards A W, Day M G and Weaver W H 1972 Predicting sugar content and petiole nitrate of sugarbeets from soil measurements of nitrate and mineralizable nitro­gen. J. Am. Soc. Sugar Beet Techno!. 17, 126-133.

Ulrich A, Ritie D, Hills F J, George A G and Morse MD 1959 1. Plant analysis. A guide for sugar beet fertilization. 2. Analytical methods for use in plant analysis. Calif. Agric. Exp. Stn. Bull. 766.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 153-155, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-188

Simulation of maize yield response to combined effects of nitrogen fertilization versus irrigation and plant population

J. BEL TRAO and J. BEN ASHER Unidade de Ciencias e Tecnologias Agrarias, Universidade do Algarve, 8000 Faro, Portugal and Jacob Blaustein Institute for Desert Research, Ben Gurian University of the Negev, Sde Boqer 84993, Israel

Key words: dynamic simulation model, irrigation, maize, nitrogen fertilization, plant population, Zea mays L.

Abstract

A dynamic simulation model is used to generalize the maize (Zea mays, L.) yield response to combined effects of nitrogen fertilization versus irrigation and plant population. Observed yields were generally raised according to the increase, in each of the three factors. However, the simulation model slightly underestimated the actual yield, with which the high applicability of the model is demonstrated for field conditions.

Introduction

Dynamic simulation is the most comprehensive way of defining actual maize yield as a function of yield factors at any moment and site (Jones and Kiniry, 1986). These aspects involve the evaluation of the influence of yield factors such as nitrogen, water, plant population and their interactions.

The objective of this work is to measure and simulate combined effects of nitrogen, irrigation and plant population density on the production of maize, in order to generalize maize product­ion functions.

Materials and methods

The experiments were carried out in Quinta do Morgado da Lameira, Algarve, South Portugal, from April until September 1985 (Beltrao, 1987). Table 1 gives the genetic parameters, according to the notations of Jones and Kiniry (1986): Pl- growing degree days from seedling emergence to the end of juvenile phase; P2-

photoperiod sensitivity coefficient; P5- growing degree days from silking to physiological maturi­ty; G2- potential kernel number; G5- potential growth rate, mg kernel! day. Sprinkle irrigation and the single line source were used for water application (Hanks et al., 1976) and its design and nitrogen amounts are shown in Figure 1. Average water application was calculated ac­cording to evaporation from a class A pan times a factor (k pan). Thus, the wettest zone (near the line source), had a value of k pan= 1.2, and the treatments near the two borders were the driest. The irrigation frequence was every 7 days. Sow­ing row space was 0. 75 m and its depth about 0.02-0.03 m. Plant population density PPD var­ied from 3 to 8 plants m -z. The harvest was about 150 days after sowing; mean plant water

Table 1. Genetic parameters

Corn variety

Pl Genetic P2 parameters P5

G2 G5

DEKALB XL-361

200 0.0

820 540

10.0

154 Beltrao and Ben Asher

Rep 3 . N3 N2 Y (H m

2' m

N4 . .Ni \ Rap q Rep 2

N2 N~/

J-'---J:!N.J-1 --'--+r-----'·--'N::c;)c_·ei· /4---_J

//l

----'-- 30 "'------¥

Scaie 1:500

Schematic representation of the single line source experiment. The numbers 1 to 9 denote the sprinklers. Water application is high along the sprinkler line. It is reduced linearly with the distance, and at the distance of 15 m from the sprinkler, line water application is zero. The plots marked by Nl to N4 denote nitrogen treatments with four replications. Application levels are, respectively, 40; 120; 200; and 280 kg 1 ha.

Fig. 1. Single line source experiment.

content during harvest was about 0.155 kg water kg- 1 grain. The dynamic simulation model, used to generalize the above effects, is based on CERES-Maize model (Jones and Kiniry, 1986), where CERES-Maize model is affected by addi­tional factors, such as 1) salinity (based on the model of Ben Asher, 1988); 2) groundwater level (based on a model that combines the model of Rijtema, 1965, and the model of van Genuch­ten, 1980); and 3) absorbed photosynthetically active radiation (based on the model af Gallo and Daughtry, 1986). The first step in the model calibration was to estimate the genetic parame­ters of this maize variety (Dekalb XL-361) and, then, to run the model iteratively at all observed conditions. The second step was to choose the most likely value of parameter (G2) and, then, to run the model, varying this parameter, at a constant value of the others, until the lower variation was obtained.

Results

The response of grain yield to combined effects of nitrogen and water, for a PPD = 6 plants m -z, is shown in Figure 2. The two lines (sim I1 and sim 12) represent the simulated yields of two irrigation levels during the growing season (Il-420 mm, 12 = 570 mm). The symbols (obs I1 and obs 12) in Figure 2 represent the results of the experiments of fresh grain yield obtained in Algarve.

The response of grain yield to combined effects of nitrogen and plant population density, for an irrigation level of I = 420 mm during the growing season is shown in Figure 3. The three curves drawn through Figure 3 represent the simulation values for three N levels ( 40; 120; and

sim 12

0

sim Il

LO

Nitrogen :t;ertilizati.on (Kg ha-l) o obs I 1 0 obs 12 - sim 1

Fig. 2. Effect of water and N application on simulated and observed grain yield (11 = 420; !2 = 570 mm).

situ N2"'!H

o sim Nl

obs Nl obs N2 obs N3 sim N

Fig. 3. Effect of N and PPD on simulated and observed grain yield (Nl = 40; N2 = 120; N3 = 200 kg ha- 1 ).

200 kg ha -J). The symbols ( obs N1, obs N2 and obs N3) in Figure 3 represent the results of the experiments. The abcissa axis gives the PPD values, ranged from 3.2 until 8.0 plants m -z.

Discussion

Figure 2 shows that observed yields always raised with the increase in the supply of N and water. On the other hand, the behaviour of simulated yields is different: 1) below 570 mm, water is the limiting factor and yield is always increased with the enhanced irrigation; 2) for both irrigation levels, the N effect is very pronounced below 120kg ha- 1 but not at higher levels; 3) if irrigation is greater than 420 mm of water, for higher N levels, it is suggested that plants (PPD = 6 plants m -z) have more N at their disposal than they really need, and, therefore, the yield remains constant.

Figure 3 shows that the effect of N is more pronounced than the effect of PPD. A large change in PPD is associated with small changes of absolute yield. Yield has always increased with the enhanced PPD; however, the increase was slight for the simulated yields and more pro­nounced for the observed fields. When N is low, observed yield increased linearly with PPD.

The relationships between simulated and ob­served yields were defined by the linear regres­sion equation Y =a+ bX (Beltrao, 1992), where Y is the simulated yield, a the constant, X the observed yield, and b the X coefficient. The results of the regression analysis between simu­lated and observed yields are given in Table 2 (for the combined effects of N fertilization and irrigation) and in Table 3 (for the combined effects of nitrogen fertilization and plant popula­tion density). Although coefficients of R2 = 0.77 and 0. 70, which are very acceptable in field conditions, on moderate slopes of 0.83 and 0.63, indicate that the model slightly underestimates yield.

Maize yield response to N and irrigation 155

Table 2. Regression analysis between observed and simu­lated yields (effects of N and water)

Constant Std. Err of Y Est R Squared No. of Observations Degrees of Freedom X Coefficient Std Err of Coef.

1452.4 646.4

0.771 11 9 0.83 0.15

Table 3. Regression analysis between observed and simu­lated yield (effects of N and PPD)

Constant 2349.7 Std. Err of Y Est 717.5 R Squared 0.702 No. of Observations 15 Degrees of Freedom 13 X Coefficient 0.63 Std Err of Coef. 0.11

References

Beltrao J 1987 Studies on pressurized irrigation systems in the Algarve. Report on activities for promotion to research assistant. University of Algarve, Faro, Portugal.

Beltriio J 1992 Generalization of combined effects of water and fertilizer on the yield function of irrigated crops. Ph.D. Thesis, Ben Gurian University of the Negev, Beer Sheva, Israel.

Ben Asher J 1988 Combined processes of ions and water uptake: a mathematical model and its implications. Israel Agresearch 2, 49-63.

Gallo K P and Daughtry C S T 1986 Techniques for measuring intercepted and absorbed photosynthetically active radiation in corn canopies. Agron. J. 78, 752-756.

van Genuchten M Th 1980 A closed form equation for predicting the hydraulic conductivity of unsaturated soils. Soil Sci. Soc. Am. J. 44, 892-898.

Hanks R J, Keller J, Ramusscn V P and Wilson G D 1976 Line source sprinkler for continuous variable irrigation crop studies. Soil Sci. Soc. Am. J. 40, 426-429.

Jones C A and Kiniry J R (Eds.) 1986 CERES-Maize: A Simulation Model of Maize Growth and Development. Texas A. & M. University Press, College Station, Texas, USA.

Rijtema P E 1965 An analysis of actual evapotranspiration. Pudoc, Wageningen, 109 p.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 157-159, l993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-157

The use of diagnosis recommendation integrated system (DRIS) to evaluate the nutritional status of healthy and blight affected citrus trees

E. MALAVOLTA1 , S.A. OLIVEIRA2 and G.C. VITTI3

1Center for Nuclear Energy in Agriculture, University of Sao Paulo, Piracicaba, SP, Brazil, 2 Department of Agricultural Sciences, University of Brasilia, Brasilia, DF, Brazil and 3 Escola Superior de Agricultura 'Luiz de Queiroz', University of Sao Paulo, Piracicaba, SP, Brazil

Key words: blight, citrus, DRIS, nutritional status, soil analysis

Abstract

Leaves from 16 healthy and 14 blight affected citrus trees (Pera and Natal on Rangpur lime) were analyzed for N, P, K, Ca, Mg, S, B, Cu, Mn and Zn. Soil samples were taken at 0-30 and 31-60 em depth in the dripline of the same trees, and pH, available P, exchangeable K, Ca and Mg and percentage of base saturation (V%) determined. No difference was found in the soil characteristics between sites with healthy or diseased trees. There were, however, marked differences in leaf analysis. Leaves from diseased plants had much lower concentrations of P and K and higher levels of Ca. Primary DRIS indices for all leaf nutrients were calculated with the aid of a computer program. The negative indices obey the following decreasing order of magnitude: blight affected- K, P, N, Mg, S, Zn, Mn, B, Cu; healthy- Mn, Cu, B, Ca P, Mg, Zn, N, K, S. The sum of absolute values of each individual index gave a value approximately three times greater in the case of blight affected trees. Therefore DRIS indices could be used as an additional tool for the diagnosis of blight affected trees.

Introduction

Citrus blight ('declinio') causes heavy loss in production in Brazil which eventually results in trees having to be replaced. Unless resistant rootstocks are used the abnormality shows up again, usually within a few years after replanting on the same site. The cause of the disease is unknown at present. For details see Prates et al. (1989). According to Wutscher and Hardesty (1979) blight trees have lower K concentrations in the leaves than healthy ones, and leaf- Mn was reported to be lower (Anderson and Calvert, 1970). The reduction in leaf K due to "declinio" was also reported by Hiroce (1984).

The subject of this paper is a comparison between leaf composition of healthy and blight affected trees by using Diagnosis and Recom­mendation Integrated System (DRIS) indices for

N, P, K, Ca, Mg, S, B, Cu and Zn (Beaufils, 1973).

Materials and methods

Samples were collected August 1984 from 14 diseased and 16 apparently healthy 12-15 years old trees from a grove of Pera and Natal on Rangpur lime located in the Northwest of the State of S. Paulo, Brazil. The third and fourth leaves from fruitless terminals were collected for analysis in the four quadrants of each tree. Each individual samples consisted of 30 leaves. Soil samples were taken in the dripline in the depths of 0-30 and 31-60 em. Leaf and soil samples were analysed by standard methods according, respectively, to Bataglia et al. (1983), and Raij and Quaggio (1983). DRIS indices were calcu-

158 Malavolta et al.

lated as described by Malavolta et al. (1989) using a computer program worked out by one of the authors (S.A.O.).

Results and discussion

Soil and leaf analysis

Results of soil analysis were similar at both depths near healthy and blighted trees.

From the leaf analysis data (Table 1) the following comments may be made concerning the average values:

Nitrogen. Although healthy plants have a higher

Table 1. Results of leaf analysis

Element Plants

Healthy Blight affected

N Percent range 2.5 - 2.9 2.0 -2.7 average - 2.7 -2.3

p

range 0.12- 0.18 0.09-0.11 average - 0.13 0.09

K range 1.0 - 2.0 0.58-1.4 average 1.4 0.84

Ca range 2.0 - 3.1 2.5 -4.6 average 2.8 3.7

Mg range 0.34- 0.63 0.35-0.76 average 0.47 0.49

s range 0.18- 0.30 0.20-0.26 average 0.24 0.22

B mgkg- 1

range 34-86 52-186 average 54 89

Cu range 7-15 8- 36 average 8 15

Mn range 22-55 29- 80 average 37 - 56

Zn range 13-21 13- 34 average -16 23

concentration, the level found in blight affected trees fall within the concentration range usually considered as satisfactory or adequate.

Phosphorus. Healthy plants have higher leaf- P, a result similar to that found by Wutscher and Hardesty (1979).

Potassium. Blight affected trees show a much lower K concentration.

Calcium. Probably as a consequence of lower K, leaves from diseased plants have higher Ca levels.

Magnesium and sulfur. No difference.

Boron, copper, manganese and zinc. The levels found in the leaves of healthy trees are lower in agreement with data reported by Wutscher and Hardesty (1979), except in the case of B. The average values were different, at the five per cent level, in the case of P, K, Ca, B, Cu, and Zn.

DRIS indices

The values found in the calculation of the pri­mary indices are shown in Table 2. It should be remembered that the imbalance for a given nutrient increases as the corresponding indices become more negative. The negative indices obey the following decreasing order of mag­nitude: blight affected trees- K, P, N, Mg, S, Zn, Mn, B, Cu; healthy plants- Mn, Cu, B, Ca, P, Mg, Zn, N, K, S.

It is clear therefore that K is the most limiting element in the case of blighted trees. On the other hand, high positive indices indicate either luxury consumption or excess. A zero value for a given index means balanced nutrition. By com­paring Tables 1 and 2 it seems that DRIS indices could be used as an additional diagnostic tool capable of separating healthy from blight affect­ed trees. By adding all indices irrespective of their signal a value which indicates the overall nutritional status is obtained: the greater the magnitude, the greater the imbalance. Healthy trees- 289, blight affected- 920. In the estima­tion of the DRIS indices it has been assumed

Table 2. Primary DRIS indices 1

Element Plants

Healthy Blight affected

N range -63 to+ 46 -202 to+ 11 average -18 to+ 21 -73 to+ 11

p

range -77 to+ 92 -26560+ 47 average -29 to+ 28 -139

K range -52 to+ 98 -502 to+ 31 average -24to+ 38 -227 to+ 31

Ca range -79to+ 51 + 8 to+ 163 average -38to+ 23 + 80

Mg range -75 to+ 81 -135 to+ 114 average -37to+ 40 -54 to+ 36

s range -44 to+ 23 -68 to+ 9 average -21 to+ 21 -34to+ 9

B range -81 to+ 66 -13 to+ 102 average -20to+ 31 -7 to+ 56

Cu range -94 to+119 -8 to+ 180 average -22 to+ 56 -8 to+105

Mn range -96 to+ 78 -20 to+ 186 average -42 to+ 41 -15 to+lOO

Zn range -71 to+ 66 -59 to+221 average -28 to+ 23 -38 to+ 122

1 Average of negative and positive values.

that yield per plant follows a normal distribution since actual data were not available.

Conclusions

No differences were found in soil characteristics between samples collected near healthy and blight affected trees.

DRIS in citrus 159

Leaves from diseased trees, however, differ markedly in their mineral composition, particu­larly with respect to P and K (lower level) and Ca (higher concentrations).

Primary DRIS indices provide a valuable means of discriminating between healthy and blight affected trees indicating their use as an additional tool of diagnosis.

References

Anderson C A and Calvert D V 1979 Mineral composition of leaves from citrus trees affected with decline of unknown etiology. Proc. Fla. State Hortic. Soc. 83, 41-45.

Bataglia 0 C, Furlani A M C, Teixeira J P F, Furlani P R and Gallo J R 1983 Metodos de analise quimica de plantas Instituto Agron6mico. Boletim Tecnico, 78.

Beaufils E R 1973 Diagnosis and recommendations inte­grated system (DRIS); a general scheme for experimenta­tion and calibration based on principles developed from research in plant nutrition. Bull. Soil Sci. 1.

Hiroce R 1985 Uso da analise foliar em citros para aduba~ao. An. I Simp6sio sobre Produtividade de Citros, Jaboticabal, S. Paulo, Brasil, 111-126.

Malavolta E, Vitti G C and Oliveira S A 1989 Avalia~o do estado nutricional das plantas- principios e aplica~oes. Associa~ao Brasileira para Pesquisa da Potassa e do Fosfato, Piracicaba, Sao Paulo.

Prates H S, Tubelis A, Salibe A A and Carvalho A M 1989 Evolu~ao do declinio de citros em pomares comerciais de laranjeira Pera conduzidos em latossolos do Estado de Sao Paulo. Laranja 10, 292-320.

Raij B van and Quaggio J A 1983 Metodos de amilise de solo para fins de fcrtilidade. Instituto Agron6mi co. Boletim Tecnico, 81.

Wutseher H W and Hardesty C 1979 Concentration of 14 elements in tissue of blight affected and healthy Valencia orange trees. J. Am. Soc. Hortic. Sci. 104, 9-11.

M.A.C. Fragoso and M.L. van Beusichem (eds.). Optimization of Plant Nutrition, 161-166, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-208

An expert system for diagnosing citrus nutritional status and planning fertilization

D. PALAZZ0 1 , G. BASILE 1 , R. D'AGOSTIN0 1 , F. INTRIGLIOL0 2 , K. CHIRIATTI 1 and C. RESINA1

1Metapontum Agrobios, SS. Jonica 106, km. 448.2, I-75010 Metaponto (MT), Italy and 2Citriculture Experimental Institute, Corso Savoia 190, I-95024 Acireale (CT), Italy

Key words: citrus, data base, expert system, fertilization, nutrient diagnosis

Abstract

The diagnosis of plant nutritional status is a fundamental step for correctly defining fertilizer recommendations to improve yields and fruit quality in Citriculture. With this purpose, the SEFEAG expert system prototype has been developed as a tool able to emulate cognitive processes and evaluate data for the assessment of Citrus nutritional status. SEFEAG is composed of subsystems for acquisition and evaluation of historical farm data, symptomatology and analytical data on soil, water and leaf nutrient contents. It is interfaced with a relational Data Base (DB) designed according the Entity Relationship (E/R) methodology and containing information about individual orchards, such as cultivar, productive performance, fertilization and other cropping operations, leaf, soil and water analyses. SEFEAG provides a diagnostic report as well as fertilization advice. The knowledge base of the expert system has been implemented using Nexpert Object shell, the DB with RDB (Relational Data Base) and Rally both in VMS (Virtual Memory System)- Digital environment. In the near future SEFEAG will run also on IBM and compatible PCs (Personal Computers).

Introduction

Several methods have been proposed for diag­nosing the nutritional state, and the fertilizer requirements, of Citrus orchards.

Leaf analyses provide a useful means of asses­sing nutrient status of perennial plants such as Citrus trees (Embleton et al., 1973). However, plant-testing poses some serious drawbacks. Ref­erence values may vary among varieties of the same species (Chapman, 1966; Intrigliolo and Starrantino, 1988) and with changes of pedo­climatic conditions (Legaz and Primo-Millio, 1989). Furthermore, leaf analyses point out if nutrients are deficient or excessive but provide no indication of causes of imbalance. Knowledge of soil properties, soil nutrient availability, climatic conditions, orchard performance and

cropping operations is, therefore, indispensable for refining leaf analysis data, and identifying causes of nutritional imbalance and suggesting corrective fertilization. Antagonistic and syner­gistic relationships between plant nutrients must also be taken into account.

For some nutrients, and when leaf analysis data are missing, other diagnostic tools can be used with a certain degree of success. For exam­ple, symptomatological analyses can easily indi­cate deficiencies of nitrogen, magnesium, iron, manganese and zinc, as well as excesses of chloride and boron (Chapman, 1968; Intrigliolo and Raciti, 1992). Analyses of available P and soil properties related to P immobilization (pH, calcium bicarbonate) can give reliable indications of plant P content; similarly, information on exchangeable K and other indexes of soil K

162 Palazzo et al.

availability and mobilization are good pre­dicators of K status of the plant (Mengel and Kirkby, 1987).

In any case, the reliability of every diagnostic tool is improved if, from time to time, data on cropping operations which can affect nutritional status are considered to account for the results.

It should be stressed that diagnostic methods such as plant analyses and soil tests do not indicate the amount of fertilizer needed to produce a given yield increase in conditions of deficiency (Beaufils, 1976). In order to make sound fertilizer recommendations, it is necessary to relate the diagnostic indexes to the amounts of nutrients required for optimum yields. There­fore, long and expensive field trials in the diverse cultivation areas are required.

Alternatively, the fertilization plan may be based on knowledge of the nutrient-budget of the plant-soil system. The main drawback of this method, when used for Citrus orchards, is poor understanding of some budget components. For an effective application of balance sheets, some terms should be evaluated in the light of the local factors. In many instances, rules-of-thumb as well as intuition and experience of the ag­ronomist can provide useful information.

For all these reasons, diagnosis of Citrus nutritional state and fertilizer recommendations are usually made by extension specialists or other advisory personnel who know the setting in which the advice is to be applied. However, it is beyond the capacity of the fertilization-expert to deal with heterogeneous situations, particularly with the proliferation of data relationships in the reasoning process, and with the high degree of uncertainty inherent in some pieces of informa­tion (Palazzo et al., 1992).

In the last decade, expert systems, a technolo­gy sub-area of artificial intelligence, have emerged as a field of research and development in agriculture, particularly suited for solving problems that are sufficiently difficult to require significant human expertise for their solution (Feigenbaum, 1981). Expert systems, whose construction has been accelerated by the intro­duction of specialized developmental tools (Richer, 1986), are able to fuse and to handle in an heuristic way different knowledge sources or even sources from different domains, as in the case of Citrus fertilization.

SEFEAG is an expert system based on differ­ent types of inputs, being implemented with human expertise, literature studies and field-trial results (Resina et al., 1992). It can diagnose Citrus nutritional status, and make fertilizer recommendations in order to improve Citrus productive performance and to reduce the en­vironmental impact of the fertilization.

Materials and methods

'SEFEAG expert system'

SEFEAG consists of a "knowledge base" for storing "facts" and "rules" of inference, an "inference engine" to perform the "reasoning" process, and a user interface.

The knowledge is organized as a collection of objects sharing common properties. The objects can inherit characteristics from a general object class and have their own specific attributes. Relationships among objects are defined using rules, where the left part specifies a condition or situation and the right part declares the action to perform.

Two strategies are available for evaluating the consequences of the initial condition given by the user: forward chaining and backward chaining. Under forward chaining, the reasoning proceeds from the fact to the conclusion. It is typical of diagnosis problems where there are many pos­sible hypotheses and limited initial data. Under backward chaining, evaluation proceeds in re­verse, from the hypothesis (conclusion to be proved) to the facts supporting the hypothesis.

As the information used by expert systems is often expressed in qualitative rather than quan­titative form, and the rules are not well-defined in a logical sense, a mechanism for modelling uncertainty is necessary. Moreover, reasoning with uncertainty takes on a greater importance because its application area concerns the fusion of information from many knowledge sources often leading to contradictory conclusions. In this case, the system should note the conflict and take appropriate action. For these reasons the system must provide a method of representing propositions expressed in natural language when their meaning is imprecise, and a non-categorical mechanism of inference must be employed. A

method of modelling uncertainty is based on the theory of fuzzy sets and fuzzy measures (Prade, 1985; Zadeh, 1979).

If two outcomes are possible for a given situation, they arc automatically generated to pursue reasoning about both, until an alternative which leads to an obvious contradiction is elimi­nated. The other alternative can be, then, select­ed with a degree of certainty.

Each proposition is assigned a Certainty Fac­tor ( CF) that indicates the reliability of the assertion. For instance, 'N leaf content is high' is a proposition that can be true or false, and its CF is a function defined in the domain [0, 14], assuming values in the range [2.2, 2.6], as showed in Figure 1.

The CF ranges from -1 to 1. Positive numbers indicate that the condition strengthens the belief

Fig. 1. CF function for the propositions 'N leaf content is low', 'N leaf content is optimum, N leaf content is high'.

1,n

1,1

I Analyses J

Expert system for Citrus fertilization 163

in the hypothesis, whereas negative numbers increase the belief in the negation of hypothesis. In the case of multiple CF, if a rule R1 supports the conclusion h with certainty CF1, and a second rule R2 supports the same conclusion with certainty CF2, the two certainty factors must be combined in a coherent manner (Short­liffe, 1976) to obtain a new certainty factor (CF') according to the following formula:

CF' = CF1 + CF2*(1 -ICF1i)

if CF1 * CF2 > = 0

CF' = (CF1 + CF2)/(1- min(ICF1I, ICF21))

if CF1 *CF2 < 0

'Data base'

The data base design has been developed with an E/R model (Batini et a!., 1986; Date, 1985). The main E/R classification structures are en­tities, relationships and domains. In the physical data base, entities and relationships are con­verted into tables according to normalization rules (Atzeni et a!., 1985). Each table has been identified through indexes or keys. The scheme resulting from the application of the E/R model is shown in Figure 2.

Fig. 2. Conceptual schema of the data base.

164 Palazzo et al.

Results and discussion

SEFEAG consists of four interagent compo­nents, a diagnostic subsystem, a decision making subsystem, a relational data base and a user interface (Fig. 3).

The first supplies a diagnostic report using different sources of information. It contains separate subsystems for managing the knowledge which derives from in-field observations, crop-

USER

Fertilization Diagnosis Plan

user interface

decision-making subsystem

diagnostic subsystem

Fig. 3. SEFEAG architecture.

Fig. 4. SEFEAG subsystems.

ping history, leaf analyses, soil and water analy­ses (Fig. 4).

The second proposes a fertilization plan en­abling the replenishment of nutrient removals and losses as well as the fulfilment of nutritional needs (elucidated during the diagnostic step) to improve fruit quality and eliminate nutritional imbalances (Fig. 4).

The third contains, in the form of tables, the information required for running SEFEAG, such as cultivar, productive performance, fertilization and other cultivation techniques, leaf, soil and water analyses (Basile et al., 1992). Its aim is to control the integrity and security of the informa­tion, ensuring the consistency of data and eliminating redundancy (Date, 1985). Moreover, it allows an easy access to the data in order to read and update.

The fourth allows an easy and friendly inter­action between SEFEAG and the end-user.

The output of each subdomain provides a judgement of the orchard nutritional condition (in terms of deficiency, normality or excess) for every macro and microelement. A certainty factor (CF) is associated with this diagnosis, indicating not only the reliability but also the magnitude of the asserted nutritional status.

As discussed above, the CF value for diagnosis depends on the number of the proving conditions that are satisfied and on the degree to which they are verified. By adopting certainty factors, out­puts of the different subdomains have the same unit of measure and their comparison is possible.

Several studies regarding the prediction of the crop nutritional state have shown that for a certain nutrient some diagnostic tools are more effective than others. Consequently, before in­dividual results are combined for the final diag­nosis, they are weighed taking into account the diagnostic power of the respective subdomains for each nutritional element (Table 1).

In the case of N, soil indexes (organic matter

Table 1. Diagnostic power of the different types of information, considered by SEFEAG, 0n the nutritional state of Citrus orchards

N p K Ca Mg Mn,Zn,Fe

Leaf analysis 0.8 0.8 0.65 0.6 0.8 0.65 Soil analysis 0.3 0.6 0.6 0.5 0.6 0.2 Visual analysis 0.5 0.3 0.6 0.2 0.55 0.8 Cropping history 0.2 0.6 0.4 0.1 0.2 0.3

organic N) are uncertain predictors of N availa­bility for the plant during the growing season (Mengel and Kirkby, 1987). Leaf analyses can give more reliable indications on Citrus orchard nutritional state, especially when fruit load and previous pruning operations are appropriately considered (Embleton et a!., 1973; Legaz and Primo-Millio, 1989). Visual analysis can give useful hints too.

For P, very little information is available from in-field observations, while leaf analysis, soil analysis and knowledge of previous P fertiliza­tion can all be useful for the diagnosis (Scuderi and Raciti, 1985).

As for K status, leaf analysis (which is normal­ly done when fruits are still on the plant) should be evaluated carefully, because the leaf K con­tent strongly depends on the fruit load. On the other hand, observations of fruit characteristics can be used with some reliability, as K has been found to affect fruit quality (Du Plessis and Kocn, 1988).

For Ca, the coefficients for the diagnostic power have been maintained low as few studies have considered the importance of assessing Ca status for Citrus orchards while, in the case of Mg, a high informative content has been as­signed to the leaf analysis.

For microelements, symptom evaluation has been considered as the most important diagnos­tic tool, leaf analysis being poorly correlated to the productive performance of Citrus species (Embleton et a!., 1973).

The combination of the CF values, after weighing with the diagnostic power of the differ­ent subdomains, produces a score which takes into account a great number of factors affecting plant uptake and assimilation. Also it has a probabilistic meaning, in the sense that high negative or positive values, obtained when there is agreement among the CFs, indicate deficiency or excess of a particular nutrient with high degree of certainty; on the other hand, values around 0 deriving from conflicting CFs suggest caution in diagnosing nutritional imbalances.

However, the diagnostic response shows if a nutrient should be added or withdrawn to re­cover the optimum nutritional status, but cannot be used to predict the amount of fertilizer required to compensate for crop removal and nutrient losses from the plant-soil system.

Expert system for Citrus fertilization 165

With the aim of formulating a fertilization plan, SEFEAG manages a balance sheet for the major nutrients (N, P, K).

Nutrient balance in Citrus orchards is subject­ed to a high degree of uncertainty, because there is a scarcity of knowledge about terms relative to N, P and K losses.

In this context, SEFEAG starts from reference evaluation obtained in similar agrosystems, and proceeds to a stepwise verification and correction of the balance terms using 'if ... then' rules. In this process, pedoclimatic and management conditions of the orchard, as well as hints de­rived from the reasoning flow inside other subdo­mains are taken into account (Palazzo et a!., 1992).

Moreover, SEFEAG utilizes the output of the diagnostic subsystem for correcting the balance of each single nutrients with a certain amount that could maintain or bring to an optimal level the plant reserves required for adequate vegeta­tive and productive performance. This amount is based on the magnitude of the diagnostic score, but in the first phases of SEFEAG application it can be only roughly estimated, as its calibration for plant response requires long field trials. However, running the expert system over several years and examining the variation of the CFs toward the optimum nutritional state will pro­vide useful feedback for judging the correctness of our estimates.

Acknowledgement.

The authors wish to thank Dr Allan Eaglesham for improving the English of the manuscript.

References

Atzeni P, Batini C and De Antonellis V 1985 La Teoria Rclazionalc dei Dati. Boringhicri, Torino. 200 p.

Basile G, Palazzo D, lntrigliolo F, Giuffrida A, Coniglione L and Resina C 1992 Data base on Citrus fertilization. Proc. Int. Soc. Citriculture (In press).

Batini C, Di Preta G, Lenzerini M and Santucci G 1986 La Progettazione Concettuale dei Dati. Franco Angeli, Milano. 314 p.

Beaufils E R 1976 Plant-soil-environment calibration (ORIS). 4th Intern. CoiL on the Control nf Plant Nutri­tion, Gent. VoL I, 21-36.

166 Palazzo et al.

Chapman H D 1966 Diagnostic criteria for plant and soils. Univ. Calif. Div. of Agr. Sci., Berkeley, CA. 54.

Chapman H D 1968 The mineral nutrition of Citrus. In The Citrus Industry. Eds. W Reuther, L D Batchelor and H J Webber. pp 127-289. Univ. Calif. Div. Agr. Sci., Ber­keley, CA.

Date C J 1985 An Introduction to Data J?ase Systems. Vol. II, 383 p. Addision-Wesley.

Doluschitz R and Schmisseur W E 1988 Expert systems: Application to agriculture and farm management. Comput. Electron. Agric. 2, 173-182.

Du Plessis S F and Koen T J 1988 The effect of N and K fertilization on yield and fruit size of Valencia. Proc. Sixth Int. Citrus Congress, Tel Aviv, Israel, 663-672.

Embleton T W, Jones W W, Labananskas C K and Reuther V 1973 Leaf analysis as a diagnostic tool and guide to fertilization. In The Citrus Industry. Vol. III. Ed. W Reuther. Univ. Calif. Div. of Agr. Sci., Berkeley, CA.

Feigenbaum E A 1981 Knowledge engineering in the 1980s. In Machine Intelligence. Ed. A Bond. lnfotech State of Art Report, Series 9, No. 3. Pergamon Infotech Limited, Oxford.

Intrigliolo F and Starrantino A 1988. Nutritional features of 16 clones of lemon. Proc. Sixth Int. Citrus Congress, Tel-Aviv, Israel, 673-679.

Intrigliolo F and Raciti G 1985. Nutrizionc c concimazionc degli agrumi. L'Informatore Agrario 18, 77-83.

Legaz Peredes F and Primo-Millio E 1989 Normas para Ia

fertilizacion de los agrios. Generalitat Valenciane, Fullate Divulgacio, 5 pages.

Mengel K and Kirkby E A 1987. Princiles of Plant Nutrition. 4th Edition. International Potash Institute, Bern, Switzer­land. 687 p.

Palazzo D, Resina C, Intrigliolo F, Chiriatti K, D'Agostino R, Linsalata F and Coniglione L 1992 An expert system for rationalizing the fertilization of Citrus orchards in order to avoid environmental pollution. Computational Mechanics. In Computer Techniques in Environmental Studies IV. Ed. P. Zannetti. pp 779-793. Computational Mechanics Publi­cations, Southampton, UK & Elsevier Applied Science, Barking, UK.

Prade H 1985 A computational approach to approximate an plausible reasoning with applications to expert systems. IEEE transactions on pattern analysis and machine in­telligence Pami-7, no. 3.

Resina C, Intrigliolo F, Chiriatti K, Palazzo D and Conig­lione L 1992 Sefeag: An expert system for Citrus fertiliza­tion. Proc. Int. Soc. Citriculture, In press.

Richer M H 1986 An evaluation of expert system develop­ment tools. Expert Syst. 3, 166-183.

Scuderi A and Raciti G 1985 In Trattato di Agrumicoltura. Ed. P Spina. pp 367-403. Edagricolc, Bologna.

Shortliffe E H 1976 Computer-Based Medical Consultations: Mycin. Elsevier-North Holland, New York. 146.

Zadeh L A 1979 A theory of approximate reasoning. Ma­chine Intelligence 9, 49-194.

M.A. C. Fragoso and M.L. van Beusichem (eds.) Optimization a_{ plant nutrition 167-172, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-214

DRIS evaluation of persimmon (Diospyrus kaki L.)

I. KLEIN\ L. FANBERSTEIN 1 and L. VINER2

1Institute of Horticulture, ARO, The Volcani Center, Bet Dagen 50250, Israel; 2Ministry of Agriculture, Extension Service, Raanana, Israel

Key words: Diospyrus kaki L., DRIS, leaf analysis, macronutrient profile, specific leaf weight

Abstract

The leaf macroelement profile of fruiting shoots of persimmon was characterized by a modified diagnostic and recommendation integrated system (DRIS), using SLW as a primary determinant of leaf mineral content. Leaf N, P, Ca, and Mg content was positively and linearly correlated with SLW when expressed on leaf area basis (1-tg mm - 2 ). Potassium had a negative and higher correlation to SLW when expressed on %DW basis. Mineral ratios relevant for the DRIS analysis were calculated using all four possible combinations of Area (A) and Weight (W) expressions (A/A, A/W, W/A and W/W) and correlated with leaf SLW. The particular expressions chosen for the DRIS analysis were based on their highest correlation to SLW and included N /K, P/K and Ca/Mg, based on the A/W expression of the respective nutrients and the reciprocal expression (W/A) for all other ratios. Derivation of DRIS norms were based on the mineral profile of highly exposed shoots (SLW of 15.0 ± 0.3 mg em - 2 ). Calculated indices of gradually less exposed shoots (SLW of 3.8-18.9 mg em - 2) revealed a strong exponential imbalance of N, K and P (increasingly positive) vs Ca and Mg (increasingly negative). The calculated Nutritional Imbalance Index (NII) value of leaves decreased exponentially as shoot leaf SLW decreased. The modified DRIS analysis detected successfully a distinct mineral profile of highly vigorous fruiting 'water shoots', as compared to regular fruiting shoots of comparable SLW.

Introduction

The DRIS analysis developed by Beaufils (1956; 1973) was considered to be either superior (Bev­erly et a!., 1984; Elwali and Gascho, 1984; Sumner, 1977; Walworth and Sumner, 1987) or supplementary to other methods of interpreta­tion of plant nutritional status (Alkoshab et a!., 1988; Kelling and Schulte, 1986). Originally, the method eliminated the leaf DW component in the analysis by using only element ratios in the calculation. Accordingly it was claimed that for the DRIS analysis the plant can be sampled at any time, rather than at standard physiological stages (Kelling and Schulte, 1986; Sumner, 1987; Walworth and Sumner, 1987). An additional advantage of the DRIS procedure is the assign­ment of relative nutrient limitations and exces­ses, in every analysis. In recent years, however,

an M-Dris modification was proposed to separate limiting from non-limiting nutrients (Halmark et a!., 1987). This modification re-introduced the dry weight component into the analysis.

DRIS norms for fruit tree crops have been derived only for a few species (Alkoshab et a!., 1988; Beaufils, 1956; Beverly et a!., 1984; Davee et a!., 1986; Parent and Granger, 1989; Righetti eta!., 1988; Sumner, 1986). The Data base used for fruit trees was considerably more restricted than that for annual crops. In some cases a DRIS analysis of the data for fruit trees was unsatisfac­tory and was considered only supplementary to the critical level or sufficiency range method. (Alkoshab et a!., 1988; Righeti et a!., 1988).

A modification of the DRIS procedure was proposed by Klein et a!., (1991c) for walnut spur leaves taking into account the effect of light on the leaf mineral profile, as a primary deter-

168 Klein et al.

minant. In this modification a mixed expression of mineral content ( %DW and fJ-g mm - 2 ), highly correlative to photosynthetic photon flux (PPF) and SLW were utilized in the calculation. The procedure for walnut was calibrated directly to the productivity of spurs as influenced by light exposure, and took into account in each mineral ratio (either in numerator or dcnumerator) the DW component (%DW) and the effect of light (JJ-g mm - 2 ). The macroelement mineral profile of walnut spur leaves was shown to be imbal­anced at decreasing light exposures (Klein et a!., 1991c), concomitantly with a decline in spur productivity (Klein et a!., 1991a).

The aim of the present work was to test if the DRIS analysis procedure developed for walnut spurs is applicable to other crops, i.e. persim­mon, and derive the norms to be used in future nutritional diagnosis of this crop.

Materials and methods

Three hundred leaf samples were collected in mid September 1990 from fruiting shoots of persimmon trees in a single orchard at the coastal plain of Israel. Samples were selected from various positions in the canopy to obtain variable SLW, representing variable light expo­sures. Leaves were sampled from three distinct shoot types available within the trees; a. Shaded short shoots from tree interior. b. Exposed vigorous shoots. c. Vigorous and long shoots arising from severe pruning cuts ('water shoots'). The respective shoot types carried 4-6, 8-12 and 16-20 leaves per shoot. Two to four middle shoot leaves and associated fruit (fruit analysis data is not presented), were sampled from each shoot and served as a single replicate.

Leaf area of each sample was measured on a Licor area meter, washed with soap and deion­ized water (x3), dried at 70°C and weighed. SLW (mg em - 2 ) was calculated from weight and area measurement and the data plotted for visual observation of SLW distribution, followed by selection for further analysis. One fourth of the samples were selected, representing a wide range of SLW, for grinding to 20 mesh and digestion for mineral analysis.

Leaf samples were digested with concentrated sulfuric acid and clarified with hydrogen perox­ide. Nitrogen was analyzed by the Nessler proce­dure (Chapman and Pratt, 1961) and P, K, Ca and Mg with plasma emission.

A balanced calculation of the DRIS procedure was used, as described in detail previously (Klein et a!., 1991c). The mineral content of leaves with a SLW range of 15.0 ± 0.3 mg em - 2 was used to derive the x/y (x and y representing various elements) and CV values for the norms of per­simmon. The mineral content of leaves with SLW range of 3.8-18.9 mg em - 2 were used for X/Y calculation and derivation of the appropri­ate indices.

Results

A range of specific leaf weights were found in each shoot type sampled (Fig. 1). Leaves on shaded interior shoots had a mean SLW of 8.0 ± 0.4, while leaves on exposed shoots from tree exterior and 'water shoots' had a SLW of 13.7 ± 0.3 and 13.5 ± 0.4, respectively.

Leaf N, P, Ca and Mg were linearly and positively correlated with SLW (Table 1) when mineral content was expressed on the basis of leaf area. In contrast, K had a higher and negative correlation to SLW, when expressed as %DW. The highest correlation of P (as JJ-g mm - 2 ) and K (as %DW), although statistically significant, were lower than the correlation of N, Ca and Mg (TabJ,c 1).

With the exception of N/K, P/K and Ca/Mg, the highest correlation of mineral ratios to SLW were found when the basis for calculation was W/A (weight/area, Table 1). The highest corre­lation of N /K, P/K and Ca/Mg occurred when the basis for calculation was A/W (area/weight). The later group of mineral ratios could be characterized by a linear and positive correlation to SLW, while the former group exhibited a second order negative correlation (Fig. 1 ).

The choice of the more important expression for the DRIS calculation was based on the highest correlation with SLW. Using the mineral ratios of highly exposed leaves with a SLW of 15.0 ± 0.3 as tentative norms for persimmon (Table 2), and a balanced equation of DRIS, the

r 2 ~0.379 3 P/K 10 (ANI) "

2 0

20

10

"' .s ~ 0

e:::

DRIS evaluation of persimmon 169

+ Exposed vigorous water shoots

o Exposed vigorous K Interior, shaded

20

10

0 15

10

0

r 2 ~0.777

Ca/Mg (ANI)

r 2 ~0.622 K/Mg (W/A)

"'@ 4 .....

.. r 2 ~ 0.691 r 2 ~ 0.687

K/Ca (W/A) <!) "'.-c:

~ 2

0 g

r 2 ~0.398 6 N/K (ANI)

4

2

0 8

6 . . )0( )C ~X

4

N/Ca (W/A) 2

0 10

6

4

2

0

r 2 ='l.607 3

N/lOP(W/A) 2

0

..

r 2 ~0.523 lOP/Mg (W/A)

r 2 ~0.635

lOP/Ca (W /A)

0 4 8 12 16 20 0 4 8 12 16 20

SLW (mg cm-2 ) Fig. 1. The choices of expressions of mineral ratios (W = %DW, A= J.Lg mm _,) of persimmon and their correlation with SLW for the ORIS analysis.

indices and the Nil value were zeroed (Fig. 2). The indices of less exposed leaves, having lower SLW showed an imbalance of their mineral profile. As SLW decreased, Ca and Mg became limiting (negative), balanced by an excess N > K > P. The DRIS indices and Nil values were polynomially (3d degree) related to SLW.

The DRIS indices and Nil value of leaves on highly vigorous 'water shoots', arising from se­vere pruning cuts, were different from that of the norm values, at identical specific leaf weights (Table 3). Water shoots had a positive IN> I P

indices and a negative t Ca = t Mg > t K val­ues. These differences, as well as the Nil value, exceeded the variation found within each cate­gory of leaves.

Discussion

Light is a primary determinant of fruitfulness (Klein et al., 1991a) and mineral content (Klein et al., 1991c; Weinbaum et al., 1989). Therefore a quantitative integration of its effect into miner-

170 Klein et al.

Table 1. Correlations and probabilities of mineral content and ratios with SLW in persimmon leaves

Expression

w (%DW)

W/W AlA

WIA

A/W

Parameter

N p

K Ca Mg

N p

K Ca Mg

N/10P N/K N/Ca N/Mg 10P/K 10P/Ca 10P/Mg K/Ca K/Mg Ca/Mg

N/lOP N/K N/Ca N/Mg 10P/K 10P/Ca JOP/Mg K/Ca K/Mg Ca/Mg

N/lOP N/K N/Ca N/Mg lOP/K 10P/Ca 10P/Mg K/Ca K/Mg Ca/Mg

Linear

r'

0.298 O.D38 0.226 0.209 0.009

0.515 0.267 0.170 0.770 0.627

0.006 0.042 0.395 0.207 0.068 0.249 0.063 0.395 0.176 0.231

0.526 0.348 0.613 0.600 0.207 0.522 0.450 0.588 0.548 0.610

0.302 0.398 0.016 0.164 0.379 0.070 0.170 0.000 0.056 0.777

al diagnosis is essential before limitations of other primary determinants (i.e. soil) can be diagnosed properly.

SLW was used m the present study as a substitute measure of PPF smce it has been shown to integrate leaf exposure to irradiance

p

0.0001 0.0780 0.0001 0.0001 0.3905

0.0001 0.0001 0.0001 0.0001 0.0001

0.4766 0.0652 0.0001 0.0001 0.0179 0.0001 0.0234 0.0001 0.0001 0.0001

0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001

0.0001 0.0001 0.2593 0.0002 0.0001 0.0159 0.0001 0.9789 0.0322 0.0001

Polynomial

r'

0.117 0.063 0.417 0.216 0.083 0.268 0.770 0.413 0.178 0.245

0.607 0.380 0.691 0.654 0.300 0.635 0.523 0.687 0.622 0.667

0.361 0.423 O.D38 0.180 0.383 0.109 0.211 0.012 0.072 0.777

p

0.0205 0.1661 0.0001 0.0003 0.0797 0.0001 0.0996 0.0001 0.0015 0.0001

0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001

0.0001 0.0001 0.3870 0.0014 0.0001 0.0281 0.0003 0.1189 0.1189 0.0001

(DeJong and Doyle, 1985; Klein et al., 1991b; Marini and Marini, 1983; Weinbaum et al., 1989). The relation to SLW is evident in persim­mon for N, P, Ca and Mg when the choice of mineral expression is based on leaf area, while that of K on %DW basis (Table 1). Similar

DR/S evaluation of persimmon 171

Table 2. Norm values" and relation of nutrient ratios to SLW" of persimmon leaves

Ratio Norm values

N/lOP 1.80 ± 0.270 N/K 2.45 ± 0.292 N/Ca 0.39 ± 0.026 N/Mg 2.34 ± 0.195 10P/K 1.03 ± 0.156 10P/Ca 0.18 ± 0.023 10P/Mg 1.06 ± 0.130 K/Ca 0.29 ± 0.042 K/Mg 1.79 ± 0.302 Ca/Mg 8.98 ± 0.588

'Derived from SLW of 15.0 ± 0.3 mg em 2

b For SLW range of 3.8-18.9 mg em_,.

.-.. ...... ...... 6 500

>< l1.l

400 '0 ::::: -l1.l (.) 300 ::::: ro ~

200 ..D

8 ...... E 100 l1.l

'E :::l 0 z

200

>< 100 l1.l

'0 ..s E l1.l 0 "5 :::l z

-100

-200 4 6 8

y~2179-510x+4lx2 -l.lx3

0 N y=386-75x+Sx 2-0.I3x3 0 P y=2847-77x+7x 2 0.19x 3

D K y=407-99x+8x2-0.23x3

]() 12 14 16

SLW (mg cm-2)

Fig. 2. The correlation of macroelement indices and Nil values (Meyer, 1975) of persimmon leaves with SLW.

results have been found in prunes (Weinbaum et a!., 1989) and Walnuts (Klein et a!., 1991c) and it may be universally true for all plants.

Correlation to SLW

Intercept X X 2

9.814 -0.989 0.0301 -0.500 0.229

5.640 -0.641 0.0196 22.640 -2.331 0.0671 -0.026 0.095

2.150 -0.255 0.0082 8.529 -0.908 0.0278 3.979 -0.477 0.0152

16.190 -1.801 0.0553 -1.714 0.721

Table 3. DRIS indices and Nil values of exposed leaves from 'water shoots', arising from severe pruning cuts in persimmon trees. ( ±SE)

Parameter Norms 'Water shoots'

SLW' 15.0 ± 0.3 15.0 ± 0.4 N 0 ± 3.1 11.2 ± 3.0 p 0 ± 1.6 6.2 ± 1.9 K 0 ± 0.05 -4.7 ± 0.4 Ca 0 ± 1.6 -7.0 ± 1.7 Mg 0 ± 3.1 -7.6 ± 2.8 Nil 0 ± 9.5 38.5 ± 9.8

'(mg em - 2 ).

Using the appropriate expression (%DW or f.tg mm - 2 ), the mineral content and ratios in leaves within the canopy could be described by a single curve, regardless of shoot type .

The modified DRIS analysis of persimmon from mid-shoot leaves of the current year growth was essentially similar to that of walnut spur leaves (Klein et a!., 1991c), with two minor exceptions; a. The correlation values of P to SLW were

considerably lower in persimmon as compared to walnut (0.267 vs 0.654 and 0.684 in two walnut cultivars, respectively. Table 1 and Klein et a!., 1991c). Phosphorus have been shown to accumulate in leaf blade as a soluble fraction when its supply is abundant (Skinner et a!., 1987) and under such conditions its correlation to light and SLW may be higher. Similarly, potassium (as %DW) had a low correlation value to SL W in persimmon (Table 1) and in one of two walnut cultivars tested

172 DRIS evaluation of persimmon

(Klein et a!., 1991c). Leaf potassium content is reduced considerably by heavy fruit load (For­shey 1969). Thus, a low K correlation to SLW may indicate cultivar specificity or low leaf K under heavy fruit load.

b. Unlike in walnut, in persimmon the Ca/Mg correlation to SLW was higher when expressed as A/W rather than as W/A and linearly related to SLW. Calcium and Mg move into exposed leaves via enhanced transpiration stream (Sprugel et a!., 1991 ). The linear relationship of Ca/Mg indicates a proportion­ally equal influx of both clements as transpira­tion of exposed leaves is increased. The modified DRIS analysis detected success­

fully a distinct mineral profile of highly vigorous fruiting 'water shoots', as compared to regular fruiting shoots of comparable SLW. The change in the mineral profile of 'water shoots' probably indicate a lower transpiration (decrease of Ca and Mg indices) and a better water use efficiency in these shoots, since their vigor was higher than that of comparably other exposed shoots.

References

Alkoshab 0, Righetti T Land Dixon A R 1988 Evaluation of DRIS, for judging the nutritional status of hazelnut, J. Am. Soc. Hortic. Sci. 113, 643-647.

Beaufils E R 1956 Mineral equilibrium in the foliage and latex of Hevea brasiliensis. Ann. Agron. 2, 205-218.

Beaufils E R 1973 Diagnosis and recommendation integrated system (DRIS). A general scheme for experimentation and calibration based on principles developed from research in plant nutrition. Soil Sci. Bull. No. 1, Univ. of Natal, S. Africa.

Beverly R B, Stark J C, Ojala J C and Embelton T W 1984 Nutrient diagnosis of 'Valencia' oranges by DRIS. J. Am. Soc. Hortic. Sci. 109, 649-654.

Chapman H D and Pratt P F 1961 Methods of Analysis for Soils, Plants, and Waters. University of California, Divi­sion of Agricultural Science, Berkeley, CA.

Davee D E, Righetti T L, Falahi E and Robbins S 1986 An evaluation of the DRIS approach for identifying mineral limitations on yield in 'Napoleon' sweet cherry. J. Am. Soc. Hortic. Sci. 111, 988-993.

DeJong T M and Doyle J F 1985 Seasonal relationship between leaf nitrogen content, photosynthetic capacity and leaf canopy light exposure in peach. Plant, Cell Environ. 8, 701-706.

Elwali A M 0 and Gascho G J 1984 Soil testing, foliar an.alysis, and DRIS as guides for sugarcane fertilization. Agron. J. 76, 466-470.

Forshey C G 1969 Potassium nutrition of deciduous fruits. HortScience 4, 39-41.

Hallmark, W B, Walworth J L, Sumner M E, deMooy C J, Pesek J and Shao K P 1987. Separating limiting from non-limiting nutrients. J. Plant Nutr. 10, 1381-1390.

Kelling K A and Schulte E E 1986 Review. DRIS as part of a routine plant analysis program. J. Fertil. Iss. 3, 107-112.

Klein I, Weinbaum S A, DeJong T M and Muraoka T T 1991a Relationship between fruiting, specific leaf weight and subsequent spur productivity in walnut. J. Am. Soc. Hortic. Sci. 116, 426-429.

Klein I, DeJong T M, Weinbaum S A and Muraoka T T 1991b Specific leaf weight and nitrogen allocation within walnut trees. HortScience 26, 183-185.

Klein I, Weinbaum S A, DeJong T M and Muraoka T T 199lc Spur light exposure as a primary external cause for derivation of ORIS norms in walnut trees. J. Plant Nutr. 14, 463-484.

Marini R P and Marini M C 1983 Seasonal changes in specific leaf weight, net photosynthetic, and chlorophyll content of peach leaves as affected by light penetration and canopy position. J. Am. Soc. Hortic. Sci. 108, 609-613.

Meyer J J 1975 Advances in the interpretation of foliar analysis of sugarcane in South Africa. Proc. S. Afr. Sugar Tech. Assn. 49, 1-9.

Parent L E and Granger R L 1989 Derivation of DRIS norms from a high-density apple orchard established in the Quebec Appalachian mountains. J. Am. Soc. Hortic. Sci. 114, 915-919.

Righetti T L, Alkoshab 0 and Wilder K 1988 Diagnostic biases in DRIS evaluations on sweet cherry and hazelnut. Comm. Soil Sci. Plant Anal. 19, 1429-1447.

Skinner P W, Matthews M A and Carlson R M 1987 Phosphorus requirements of wine grapes: Extractable phosphate of leaves indicates phosphorus status. J. Am. Soc. Hortic. Sci. 112, 449-454.

Sprugel D G, Hinckley T M and Schaap W 1991 The theory and practice of branch autonomy. Ann. Rev. Ecol. Syst. 22, 309-334.

Sumner ME 1977 Use of the DRIS system in foliar diagnosis of crops at high yield levels. Comm. Soil Sci. Plant Anal. 8, 251-268.

Sumner M E 1986 The diagnosis and recommendation integrated system (DRIS) as a guide to orchard fertiliza­tion. Food Fertil. Technology Center, Ext. Bull. 231, FFTC/ASPAC, Taipei, Taiwan, R.O.C.

Walworth J L and M E Sumner 1987 Diagnosis and Re­commendation Integrated System (DRIS). Advances in Soil Science. VI, pp 149-188. Springer-Verlag. New York.

Weinbaum SA, Southwick S M, Shackel K A, Muraoka T T, Krueger W and Yeager J K 1989 Photosynthetic photon flux influences macroclement weight and leaf dry weight per nnit leaf area in prune tree canopies. J. Am. Soc. Hortic. 114, 720-723.

Reprinted from Plant and Soi/154: 33-38, 1993.

M.A. C. Fragoso and M.L. van Beusichem (eds.) Optimization ofplant nutrition 173-177, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-023

A multivariate diagnosis approach applied to celery

N. TREMBLAY 1 , P. AUCLAIR2 , L.-E. PARENT2 and A. GOSSELIN3

1Research Station, Agriculture Canada, 430 Gouin Blvd, St-Jean-sur-Richelieu, Canada J3B 3£6; 2 Soil Science Department and 3 Horticultural Research Center, Faculty of Agriculture and Food, Laval University, Ste-Foy, Canada GlK 7P4

Key words: Apium graveolens L., celery, diagnosis system, DRIS, multivariate analysis, nutrient deficiency, principal component analysis, toxicity

Abstract

Celery (Apium graveolens L. var Dulce) is a high value crop affected at different growth stages by a variety of nutrient disorders. Each nutrient concentration can be corrected for its dependence on concentrations of other nutrients by recognizing plant composition as a closed system whose com­ponents add up to one. New variables zi are computed as logratioed values of individual nutrients, where each nutrient concentration is corrected for the geometric mean of all nutrient concentrations. The z, are used together with principal component analysis (PCA) to relate celery composition to yield, deficiency symptoms and quality parameters. A survey of commercial celery fields suggested that (1) celery growth is most often limited by P and N deficiencies associated with Fe toxicity; (2) K uptake is most likely to become limiting when the crop reaches 15 em in height; (3) blackheart incidence can be traced to low levels of K and Mg in external petioles, and ( 4) cracked stem incidence is related to low B when the crop is 30 em in height.

Introduction

Celery (Apium graveolens var. Dulce) is usually harvested at an early stage of maturity and the best-quality product depends upon rapid and continuous growth. The crop is considered as one requiring high input, relying on the use of transplants, heavy soil fertilization, irrigation and protection practices. Harvesting is done mechanically, and the hand-trimming performed at the packing plant can result in significant losses depending on the characteristics of exter­nal stems.

Since celery is very sensitive to nutritional disorders it is common that foliar applications of nutrients are used to prevent the appearance of symptoms. Still, growers frequently experience a wide variety of quality problems that can often be traced to nutrient deficiencies, either excesses or imbalances. High levels of N fertilization can cause a drastic reduction of the characteristic

celery flavour compounds (phthalide derivatives) (Van Wassenhove et a!., 1990). Nitrogen form and accompanying ion are involved in Fusarium suppression in celery (Schneider, 1985). Know­ing the inherent limitations of soil analyses, a reliable diagnosis system based on celery tissue nutrient concentration would be most helpful in determining the nutritional status of the crop and identifying appropriate corrective actions.

Most of the systems generally available for diagnostic purposes are based on the critical value approach (CVA) or sufficiency range ap­proach (SRA) and are used for only a few nutrients. Sanchez et a!. (1990) proposed CVA of 0.55% for P and 7.7% forK in celery petioles. Geraldson and Tyler (1990) reported SRA for N0 3-N, P0 4-P and K.

However, CVA and SRA validity have been seriously questioned (Holland, 1966). The Diag­nosis and Recommendation Integrated System (DRIS) often provides a better estimate for

174 Tremblay et al.

predicting nutrient shortages and responses than the CVA or SRA (Munson and Nelson, 1990). DRIS is basically a mathematical means of accounting for the interactive nature of relations among nutrients in the tissue (Walworth and Sumner, 1987). DRIS norms have been pro­posed ·for celery transplants (Tremblay et a!., 1990) and successfully tested by local celery growers.

Compositional data analysis (CDA) recognizes a structure of dependence among plant nutrient concentrations, the bounded sum constrains to one, and removes the curvature problem carried by crude components and by dual ratios or logratios when treated in isolation (Parent and Dafir, 1992). The procedure called row-centered logratioing consists of correcting any nutrient concentration X; for the geometric mean g(x) of all D nutrient concentrations of a compositional array to generate a new variable, z;:

(1)

and

Z; = log[x;fg(x)] (2)

The new variable z; can be used for several purposes. One is the drawing of a yield-z; curve with critical Z; levels and a sufficiency range for each z0 much like what is involved in setting CVA norms. Another is the computation of descriptive statistics from experimental or survey data and relating them to yield as is generally done in DRIS (Walworth and Sumner, 1987).

Moreover, CDA places on solid ground the use of multivariate analyses on compositional data (Parent and Dafir, 1992). More than a quarter century ago, Holland (1966) compared several approaches to the study of plant compo­sition and recommended principal component analysis (PCA) as the best available procedure to take into account the complex state of interrela­tionships among nutrients. He realized, though, that the variables are studied as a closed system, while actually they may well be related only in part to other variables, the remainder of the variation being associated with features outside the system. Ratkowsky and Martin (1974) used PCA followed by a varimax rotation on axes as it generally appears to provide increased clarity in

the interpretation of the relationships among mean fruit size, nutrient concentrations and bitter-pit in apples. Broschat (1979a; 1979b) used PCA to relate plant or soil chemical composition to plant ornamental characteristics.

PCA is a mathematical technique for summa­rizing a set of related measurements as a set of derived variates, frequently fewer in number, which are definable as independent linear func­tions of the original measurements (Holland, 1969). It offers the possibility of taking advan­tage of survey data obtained from commercial fields. Scouting networks applying integrated pest management (IPM) procedures offer the opportunity to efficiently obtain tissue samples which, once analyzed for their nutrient concen­trations, can be combined to yield, deficiency symptoms, disease susceptibility or other perti­nent variables, and processed through PCA to provide clues and generate grounded hypotheses for future research.

Therefore, the purpose of this study was to use a multivariate approach to diagnose the general status of celery crops grown in Quebec by relating tissue composition to yield and nutrition­al disorders.

Materials and methods

During the seasons 1989, 1990 and 1991, com­mercial celery fields visited by the Scouting and Research Organization of the South of Montreal Region and generally located on organic soils were selected for plot establishment. Two to five 30 x 30 m plots were laid out in 15 to 25 fields per year. In every plot, shoot samples of freshly planted celery plants (growth stage A) were randomly selected. For growth stages B (plant height 15 em), C (30 em) and D (50 em), the petiole of the tallest leaf was selected because large representative samples can be obtained with minimal effort, and because petiole tissue is more commonly used for diagnostic purposes in celery (Sanchez et a!., 1990).

All tissue samples collected were washed with tap and distilled water, dried at 70°C for 48 h and ground for elemental analysis. After wet ashing, N and P were determined colorimetrically, and

A multivariate diagnosis approach applied to celery 175

K, Ca, Mg, Fe, Mn, B and Zn by inductively coupled plasma spectrometry.

As close as possible to commercial harvest time, and after recording untrimmed weights, 24 randomly selected celery plants were trimmed and weighed again. Cracked stem (CS) and blackheart (BH) incidences were recorded as a proportion of affected plants. For the purpose of this study, fresh matter accumulation efficiency (FMAE) was measured from untrimmed weights as the weight of the aerial part at harvest divided by degree-days accumulated between planting and harvest (threshold temperature 7oC)

Computation of zi was done according to Parent and Dafir (1992). PCA was performed using SAS (Statistical Analysis System, Cary, NC, USA).

Results and discussion

The application of the procedures suggested by Parent and Dafir (1992) leads to an improvement of normal distributions of nutritional variables in almost 70% of cases studied.

Fresh matter accumulation efficiency (FMAE)

Depending on growth stage, between 261 and 270 observations were available for PCA on nutrient concentration data and fresh matter accumulation efficiency. At planting time (Stage A) the PCA's second Factor in importance ( y =

1. 77) associated a high FMAE to high Zn and low Mg concentration (data not shown). At growth stage B, the second Factor ( y = 1. 90) suggests a link among FMAE (0.78), zp (0.72) and zK (0.41). FMAE is at the same time inversely related to zFe(-0.71). This was a rather surprising development which merits fur­ther investigation. Only at this stage did K concentration appear to be of some importance for FMAE. According to Zink (1963) the crop accumulates up to 600 kg ha - 1 vs 40 kg P ha - 1 • K uptake (in kg ha - 1) follows an exponential growth-rate curve while N and P uptake remain relatively steady throughout the growing season ( Geraldson and Tyler, 1990). Our results suggest that much of this K uptake could be related to luxury consumption.

Table 1. First factors of the principal component analyses (PCA) among celery petiole nutrient concentration variables and fresh matter accumulation efficiency (FMAE) for growth stage C (30 em) and D (50 em). PCA was complemented by a Varimax rotation of axes

Variable Growth stage C Growth stage D

'N 0.84 0.41 'p 0.82 0.81 'K 0.20 0.22 'Ca -0.19 -0.D7 'Mg -0.17 -0.20 'B -0.06 -0.10 'Fe -0.30 -0.80 'Mn -0.30 0.31 'Zn -0.15 -0.05 FMAE 0.56 0.67

Eigenvalue ( y) 2.00 2.11

Table 1 shows the similarities in the first Factor of the PCA between growth stages C and D as illustrated by a relationship of the same trends among variables zN, zp and FMAE. The variable zFe is inversely related to FMAE par­ticularly in the stage D. Two conclusions can be reached on this basis. First, stages C and D seem to behave in the same way toward FMAE so they may be grouped for diagnostic purposes. Second, since the relationship is present in the first and most important Factor of the PCA, this constitutes a strong indication that FMAE at both stages was related to a high N and P concentration, and, to a lesser degree, to a low Fe concentration in celery petioles.

Blackheart incidence

PCA was performed on Stage D in an attempt to relate blackheart incidence to petiole nutrient concentrations. In the second Factor ( y = 1.41) only the following coefficients were higher than ±0.50: zK (-0.67), zMg (-0.51) and BH (0.80). Surprisingly, zca shows a mere 0.16. From these numbers it can be inferred that BH did not seem much related to Ca concentration but rather was induced by K and Mg deficiencies. Indeed, no BH was found in fields where K (Fig. 1) and Mg (not shown) petiole concentrations were high, while Ca concentration (Fig. 2) seemed to play no significant role.

Before reaching any conclusions: (1) it is

176 Tremblay et al.

0.35 s

0.30 s 1i)

"' ~ 0.25 "" s "' .s:; a iii s

"' 0.20 a

(.) c

"' '0 a '(3 0.15 .<:::

~ "' 0.10 s a .s:;

"" 1!10 a (.)

"' ro 0.05 a a " @J l!lt:lO lEI[;! a 0

0.00 f.,...,~.q<o-llllllll_ .... _ ........ ___ "ll"'IID-<,.,.....,..,~

1.50 1.60 1.70 1.80 1.90 2.00 2.10

ZK

Fig. 1. Relationship between blackheart incidence at harvest and petiole K concentration when the celery crop reached 50 em in height (Stage D). K concentrations were subjected to compositional data analysis (CDA) and transformed as indices 'K as suggested by Parent and Dafir (1992).

0.35 a

0.30 s 1i)

"' ~ 0.25 a "' .s:;

iii s

"' 0.20 (.) c

"' '0 a '(3 0.15 .<:::

~ "' 0.10 a a .s:;

"" s a a a a s (.)

"' ro 0.05 s "' Oa::J~

0.00 0.95 1.05 1.15 1.25 1.35 1.45

ZCa

Fig. 2. Relationship between blackheart incidence at harvest and petiole Ca concentration when the celery crop reached 50 em in height (Stage D). Ca concentrations were subjected to compositional data analysis (CDA) and transformed as indices 'Ca as suggested by Parent and Dafir (1992).

commercial practice to spray Ca solutions re­peatedly during the season to prevent BH occur­rence, and (2) the tissue sampled (external petiole) is not the one affected by BH (zone of active growth located at the heart of the plant). It is therefore possible that while BH is still strongly related to Ca deficiency, the Ca sprayed

accumulated on the sampled tissues masking the actual deficiency at the center of the plant. This possibility will have to be specifically addressed.

Cracked stem incidence

As BH is generally related to Ca deficiency, cracked stem (CS) is thought to be a mani­festation of B deficiency. A PCA conducted on Stage C confirms this view as Factor 4 ( y = 1.17) presents coefficients of -0.67 for CS and 0.75 for zB (no other coefficient exceeding ±0.27; data not shown). However, at Stage D, the CS-zB relationship is lost and CS (0.95) is more related to zK ( -0.49) than to zB(0.13) (Factor 4; y =

1.18). To prevent CS it is also a commercial practice to spray with B formula. It may be that, as for Ca and BH, the application of B during the season gradually builds a concentration of B in the petiole interfering with the expected relationship of deficiency.

In summary, with the help of the previously described multivariate diagnosis procedure, it can be hypothesized that celery growth in the field is most often limited by P and N deficiencies associated with Fe toxicity while K uptake is likely to become limiting when the crop reaches 15 em in height. Blackheart incidence can be traced to low levels of K and Mg in external petioles and cracked stem incidence is related to low B when the crop is 30 em in height. By virtue of the empirical nature of the approach, such inferences must however be speculative and will require further justification, e.g. by direct ex­perimentation.

Acknowledgements

The contribution of celery growers and scouts related to the Scouting and Research Organiza­tion of the South of Montreal Region is grateful­ly acknowledged. The authors wish to thank Yvon Perron for the mineral analyses.

References

Broschat T K 1979a Principal component analysis in horticul­tural research. HortScience 14, 114-117.

A multivariate diagnosis approach applied to celery 117

Broschat T K 1979b Relationships among soil and foliar nutrient levels and plant quality variables in field-grown Salvia determined by principal component analysis. J. Am. Soc. Hortic. Sci. 104, 748-751.

Geraldson C M and Tyler K B 1990 Plant analysis as an aid in fertilizing vegetable crops. In Soil Testing and Plant Analysis, 3rd Edition. Ed. R L Westerman. pp 549-562. Soil Sci. Soc. Am. Madison WI.

Holland D A 1966 The interpretation of leaf analyses. J. Hortic. Sci. 41, 311-329.

Holland D A 1969 Component analysis: an aid to the interpretation of data. Exp Agric. 5, 151-164.

Munson R D and Nelson W L 1990 Principles and practices in plant analysis. In Soil Testing and Plant Analysis, 3rd Edition. Ed. R L Westerman. pp 359-387. Soil Sci. Soc. Am., Madison WI.

Parent L E and Dafir M 1992 A theoretical concept of compositional nutrient diagnosis. J. Am. Soc. Hortic. Sci. 117, 239-242.

Ratkowsky D A and Martin D 1974 The use of multivariate analysis in identifying relationships among disorder and mineral element content in apples. Aust. J. Agric. Res. 25, 783-790.

Sanchez C A, Burdine H W and Guzman V L 1990 Soil testing and plant analysis as guides for the fertilization of celery on histosols. Soil Crop Sci. Soc. Fla Proc. 49, 69-72.

Schneider R W 1985 Suppression of Fusarium yellows of celery with potassium, chloride, and nitrate. Phytopatholo­gy 75, 40-48.

Tremblay N, Parent L E and Gosselin A 1990 Elaboration de normes DRIS provisoires pour des transplants de celeri. Phytoprotection 71, 129-136.

Van Wassenhove F A, Dirinek P J, Schamp N M and Vulsteke G A 1990 Effect of nitrogen fertilizers on celery volatiles. J. Agric. Food Chern. 38, 220-226.

Walworth J L and Sumner M E 1987 The diagnosis and recommendation integrated system (ORIS). Adv. Soil Sci. 6, 149-188.

Zink F W 1963 Rate of growth and nutrient absorption in celery. Am. Soc. Hortic. Sci. Proc. 82, 351-357.

Reprinted from Plant and Soi/154: 39-43, 1993.

M.A. C. Fragoso and M.L. van Beusichem (eds.) Optimization ofplant nutrition 179-186, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-010

Design and analysis of mixture systems: Applications in hydroponic, plant nutrition research

E. SCHREVENS 1 and J. CORNELL2

1 Faculty of Agricultural Sciences, Katholieke Universiteit Leuven, Wilem de Croylaan 42, B-3001 Heverlee, Belgium; 2/nstitute of Food and Agricultural Sciences, Department of Statistics, University of Florida, Gainesville, FL 32611, USA

Key words: nutrient solutions, mixtures, optimization

Abstract

This study demonstrates that nutrient solutions can be defined as 'mixture systems'. A general methodology for design and analysis of mixture optimization experiments is developed. The emphasis is centered on multivariate investigation of the zone of optimal solution properties as a function of the ion composition and the total ionic strength of the solution. The study of the effects of ion interaction on well-defined solution properties is also possible by this multivariate approach. This work is a valuable tool in mineral nutritional research, because for the first time the chemical feasibility conditions of such solution, combined with additional chemical, physiological or economical constraints, form the foundation of the statistical experimental design theory, which makes the optimization of complex mixtures of ions in relation to well-defined response variables possible.

Introduction

Nutrient solutions play an important role in many fields of scientific research: plant nutrition, plant and animal tissue cultures, fermentation technology and so on. Within the last decade, the use of hydroponic installations for horticultural production has grown exponentially. The need for 'optimal' nutrient solutions is widely felt. Although different nutrient solutions have been proposed by different authors for specific situa­tions, it seems that many investigators have chosen solutions based on trial and error ap­proaches or based on intuitive arguments, and that, where the results have been partially suc­cessful, invariably a specific solution is recom­mended as being optimal for the particular situation. The fact that a given nutrient solution has seemingly desirable properties, does not necessarily rule out the possible existence of

other solutions with equally desirable or better properties.

Another important argument for undertaking a systematic investigation of a nutrient system is that in many situations the user is not so much interested in finding a single point or mixture that is optimum for the system, but rather in finding a zone or group of optimal operating conditions. Finding a zone of optimal blends for a particular response or property of the solution, increases the likelihood that other properties might be optimized within this zone as well. In this context optimization means an as exact as possible description of the optimal zone as well as the boundaries of this zone.

In this study the emphasis is centered on the development of a method to optimize the nu­trient solution for hydroponic plant cropping as a research tool as well as a method for commercial plant production, making use of 'mixture

180 Schrevens and Cornell

theory'. Although the theory of mixture experi­ments dates from the late fifties and has been discussed extensively during the past two or three decades, this theory was never put to use in plant nutrition.

In this study for the first time the design and analysis of mixture experiments is applied to the problem of nutrient solutions (Schrevens, 1988).

Design and analysis of mixture experiments

The original mixture problem

The distinguishing feature of mixture experi­ments is that the independent variables represent proportionate amounts of the mixture rather than unrestrained amounts. These proportions must be nonnegative, and if expressed as frac­tions they must sum to unity. So in a q-com­ponent mixture if we let X; be the proportion of the i-th component in the mixture, then

0,: X;,: 1 fori= 1, 2, 3, ... q (1)

q

Lx;=1 (2) i=l

The q-components of this system are called 'mixture variables'. By virtue of the above re­striction, the totality of the unrestricted factor space of q dimensions is reduced to a ( q - 1) dimensional simplex, as was first noted by Claringbold (1955).

A most important consequence of the com­ponent dependencies in mixtures is the necessity for multivariate experimentation. This is because the effect of a single component can only be understood when studied in combination with the effects of one or more of the other com­ponents of interest. The accuracy of estimation of a single component effect increases not only with an increasing number of components but also with the design strategy used in looking at different combinations of the components.

If, in addition to the mixture variables certain other variables are present in the system where the latter variables are not bounded by the above restriction, they are called 'process variables' (Cornell, 1971; Scheffe, 1963).

In the original mixture problem developed by Scheffe (1958), the response is only a function of the proportions of the components present in the mixture and is not a function of the total amount of the mixture. In later generalizations (Piepel and Cornell, 1985; 1987), mixture models and designs where developed where the response depends on the total amount as well. In these cases the total amount could be viewed as a process variable when at the different amounts, a separate simplex or mixture design is set up and the experiments performed.

Experimentation over the whole simplex

In many mixture situations the region of interest is the whole simplex. In this case, experimental designs that have been proposed in the literature are the simplex lattice design (Scheffe, 1958), the simplex centroid design (Scheffe, 1963), the symmetric simplex design (Murty and Das, 1968), the simplex screening design (Snee and Marquardt, 1976) and axial designs (Cornell, 1975). While some of these designs are D-opti­mal and would be strong candidates for studying nutrient systems, most were developed in the chemical or other industries and were based on rather pragmatic and empirical grounds. For these designs specific mixture models were de­veloped and are based on the incorporation of the mixture constraints (Eq. 1 and 2) resulting in polynomial or other model forms: Scheffe canonical polynomials (Scheffe, 1958; 1963), Cox's mixture models (Cornell, 1975; Cox, 1971) mixture models with inverse terms (Draper and St John, 1977) and adaptations of some theoret­ical model to the mixture situation (Gorman and Cornell, 1985).

Mixtures with additional constraints on the component proportions

Frequently chemical, physical and/ or economical considerations impose additional restrictions on the component proportions. These restrictions are in the form of lower bounds ( 0 < L; '"""x;) and/or upper bounds (x;,: U; < 1). Due to these additional constraints on the component pro-

portions, the factor space of interest is reduced to a subregion of the ( q-1 )-dimensional simplex. So, in addition to constraints 1 and 2, it is possible that the following constraints are im­posed:

Single component constraints:

(3)

Multicomponents or multivariable constraints (Snee (1976):

where the L;, U; and A;j are constants. In situations were bounds of the form (3) are

specified, it may not be possible for every com­ponent to attain its lower bound or its upper bound. In such a situation the bounds are said to be inconsistent. Piepel (1983) presented a meth­od for checking what he called 'consistency' of the constraints in (3).

In some situations, constraints are imposed by the experimenter. For instance, if a good esti­mate of the optimum or sub-optimum is avail­able and a description of the immediate vicinity of this point is needed. The aim of such experi­ments is twofold. Firstly when in this limited region of interest no differences in response are found, then the experiment results in the de­scription of an optimal zone. Secondly, if signifi­cant differences are found, then by 'steepest ascent methods' the sub-optimum can be ameliorated (Box and Wilson, 1951).

In other situations the constraints (3) are inherent properties of the system, for instance dissociation, precipitation and complexation con­straints of nutrient solutions.

Experimentation in constrained mixture spaces

As a result of the nature of the additional constraints (Eq. 3 and/or 4), two possibilities can occur. 1. The resulting subspace is homomorphic with

the whole simplex. This occurs when lower bounds L; ~X; only are imposed or in some cases when the ranges L; - U; of the X; are equal in value. In such cases, the subregion is simplex shaped and the subregion is defined

Mixture systems 181

in terms of 'pseudocomponents' (Kurotori 1966; Crosier 1984, 1986) or 'L-pseudo­components' (Cornell, 1990). Since the sub­region is a simplex, designs used to explore a simplex region, such as the simplex-lattice or simplex-centroid designs, when expressed in the pseudocomponents, can be used.

2. The resulting subspace is a convex, irregular hyperpolyhedron.

In practice most mixture systems are subjected to constraints of the form (3) which results in constrained or irregularly-shaped factor spaces. In the case of an irregularly-shaped experimental region, the only way to achieve an optimal experimental design is the use of optimal design theory. For hypcrpolyheders of high dimen­sionality computer aided design of experiments becomes essential.

The methodology to construct discrete optimal designs in constrained mixture spaces, and dis­cussed in this work, consists of the following steps: initially one determines a list of candidate points by assuming some form of mathematical model. Then one chooses an optimal design criterion and an optimization algorithm that can be used to select a group of points from the candidate list. These steps are discussed further as: a. To generate a list of candidate points, the

'extreme vertices' algorithm 'of Me Lean and Anderson (1966) can be used. This algorithm generates only the extreme vertices of the irregularly-shaped hyperpolyhedron. Other algorithms that have been developed for the same purpose are XVERT (Snee and Mar­quardt, 1974), CONSIM (Snee, 1979), XVERTl (Nigam eta!., 1983). Once the list of extreme vertices has been generated, other boundary points such as the mid-points of the edges or the centroids of the two-dimensional faces, etc, of the hyperpolyhedron, can be defined. This list can eventually be extended with a number of interior checkpoints.

As an alternative to generating the list of extreme vertices from which the mid-points of the edges and I or the centroids of the faces are defined, one could generate a list of candidate points by imposing a grid of points over the experimental region. This second approach, that of imposing a grid of points,

182 Schrevens and Cornell

has not proven to be as successful or as popular as that of generating the extreme vertices.

b. Next comes the choice of an adequate mix­ture model to describe the response as a function of the mixture variables.

c. The next step is the choice of a criterion to be used to select an 'optimal' set of points from the candidate list. These points form an optimal design in the sense of allowing for the efficient estimation of the parameters in the model or for providing a value of the pre­dicted response, assuming the functional form, with the highest degree of accuracy. For design selection, with respect to optimal pa­rameter estimation, the following criteria have been proposed in literature: D-optimali­ty (Kiefer and Wolfowitz, 1959; Wald, 1943), A-optimality (Eifving, 1952) and E-optimality (Ehrenfeld, 1955). To select design points for optimal response estimation the following criteria are described: G-optimality (Kiefer and Wolfowitz, 1959; Smith, 1918) and V­optimality (Fedorov, 1972; Welch, 1984). In the optimization of nutrient solutions both D­and G-optimality are used.

d. The last step is the calculation of the optimal design using exchange algorithms. These iterating procedures start with a non-singular n-point design and then add and delete one or more candidate design points in order to minimize the selection criterion, resulting in the optimal set of points. A major advantage of exchange algorithms is that they can be used to expand a given n-point design to a m-point design (m > n) in an optimal way, which makes optimal sequential experimenta­tion possible. For D-optimality these algo­rithms were developed by Fedorov (1972) and Mitchell (1974a; 1974b). This was later extended to G- and V-optimality by Welch (1984) who made usc of branch-and-bound optimization algorithms.

Several optimal design criteria have been pro­posed and applied on constrained mixtures (Ken­nard and Stone, 1966; Mitchell, 1974; 1976; Nigam et al., 1983; Snee, 1975; Snee and Mar­quardt, 1974; 1976; Welch, 1984; Wynn 1970; Zemroch, 1986).

The problem of nutrient solutions

In the context of this study a nutrient solution is defined as an aqueous solution of a given number of chemical substances, whose effects on a certain process are of interest. The nutrient solutions for plant growth consist exclusively of inorganic ions (exception made for certain chelating agents). Some ions are essential, some are beneficial while still others may be toxic elements. The fact that plants need ions but the solution is made up of dissociated salts, imposes the major constraint upon nutrient solutions, namely the balance of charge: the sum of the cation equivalents must be equal to the sum of the anion equivalents. This constraint is the major reason for the impossibility of using classi­cal experimental designs (factorial-type designs) with nutrient solutions and the main argument for defining nutrient solutions as 'mixture' sys­tems, because it is easily understood that the ionic balance constraint equals the mixture con­straint (Eq. 1 and 2). Moreover dissociation, precipitation and complexation reactions further reduce the region of chemical feasibility. These additional constraints define the factor space as a 'constrained mixture' system (Eq. 3 and/ or 4). Furthermore, the total ionic strength can be considered as a process variable. Thus the prob­lem of experimentation with nutrient solutions in plant nutrition can be dealt with by using the theory of mixture designs and model forms.

Results

In what follows two simplified examples are presented to illustrate the application of mixture and optimal design theory in hydroponic, plant nutritional research.

Example 1. A whole simplex design to investigate the effects of cation composition of the nutrient solution on the head production in hydroponic chicory forcing

Nutrient solutions were made up consisting of each of the three cations K +, Ca + + and Mg + +

individually and combined. A six-point simplex

Table 1. The matrix of the design points (proportions), the mean and standard error of the mean for the head weight (g) for Example 1

Point K + ca ++ Mg H Mean Std err

1 0 0 128.1 6.2 2 0 1 0 Vertices 119.5 6.6 3 0 0 97.1 5.8

4 0 0.5 0.5 131.2 7.1 5 0.5 0 0.5 Edge 136.5 6.8 6 0.5 0.5 0 centroids 120.3 7.0

7 0.33 0.33 0.33 Overall 135.4 8.1 centroid

lattice design (points 1 to 6, Table 1) in the cation space (K +, Ca + +, Mg + +), combined with an average composition of anions (0.33 NO~,

0.33 H2PO~ and 0.33 SO~ - ), was set up to screen the effects of the different cations over the whole cation simplex . The six-point design was extended with the overall centroid (point 7, Table 1) as a check-point for testing goodness of fit of the proposed second-order model. The design consisting of points 1 to 6 is D-optimal with respect to a second order Scheffe canonical model. The design points are shown in Figure 1.

25 cut roots were assigned to each treatment solution and forced. At harvest, production and quality parameters were measured per root. The average head weights (g) and the standard deviation of the mean of the 25 roots per solution are listed in Table 1.

Potass ium

Moqnesiurn Calcium

Fig. 1. A six point simplex-lattice design in the cation factorspace , with the overall centroid as a checkpoint.

Mixture systems 183

The model was fitted to the data where the cations are expressed as proportions of half the total ionic strength, which was 50 mval L- 1•

The regression equation for the second order model of the yield (FW) as a function of the cation composition is :

FW= 128.0*K+ + 119.4*Ca ++ + 97 .0*Mg+ +

-12.2*K + *Ca ++ + 97.4*K+*Mg ++

+ 93 .3*Ca ++ *Mg ++

with R 2 = 0.995 and RA = 0.992. The estimated head fresh weight surface, generated from the model, is plotted in Figure 2.

Significance tests were performed on the co­efficient estimates of the nonlinear blending (crossproducts) terms in the fitted model to determine if the head fresh weight of the 50: 50 blends of the cations differed from the average of the head fresh weights corresponding to the single cation solutions. All three estimates ( -12.0, 97.6 and 93.2) were significantly differ­ent from zero (p < 0.01 ). Based on the signs of the coefficient estimates, head fresh weights of the two cation blends with Mg + + were signifi­cantly higher, meaning Mg + + blended synergisti­cally with K + and Ca + +. The average head fresh weights of the binary blend of K + and Ca + + was significantly lower than expected from additive blending of K + and Ca + +. These nonlinear blending characteristics of the two-cation blends are reflected in the nonplanar shape of the

Fig. 2. The FW of chicory heads in function of cation composition.

184 Schrevens and Cornell

estimated head fresh weight response surface that is plotted in Figure 2. Although generally not tested, the coefficient estimates (128.0, 119.4 and 97.0) of the linear blending terms in the model represent average head fresh weights of

. . 1 K+ C ++ M ++ solutions contammg on y , a or g , respectively.

The shape of the surface can be further ex­plored: -by calculating the expected response functions

along different axes of the cation space -by locating the stationary point by canonical

analysis of the response surface -by the evaluation of slope functions.

Component interaction effects can be evaluated:

-by comparing the estimated response value at points of interest with the estimated response at a reference mixture

-by computing total and partial effects. As a result of the analysis performed, this

response surface, along with the corresponding fitted model above, provides an accurate descrip­tion of the process under study, emphasizing the multivariate interactional (linear and nonlinear blending) nature of plant nutritional problems.

Example 2. A constrained mixture design to explore the optimal zone of cation composition of the nutrient solution for growth and development of tomato

To study the effects of potassium, calcium and magnesium and their interaction, an experimen­tal design is set up in the vicinity of the actual operating conditions for tomato, namely the cation composition of the standard solution of the Research Center for Soilless Cultures (point 13 of Table 2). The main question is 'How much deviation from the standard cation composition of the nutrient solution is allowed without sac­rificing the growth and development characteris­tics of tomato plants?'.

For each ion a lower bound of 50% less and an upper bound of 50% more was considered pos­sible, resulting in the following constrained ex­perimental region. Cations are expressed in proportions of half the total ionic strength of the nutrient solution (80 mval L - 1).

Table 2. The matrix of the candidate points for Example 2

Point K+ Ca 1-+ Mg++

1 0.66 0.22 0.12 2 0.22 0.66 0.12 3 0.60 0.22 0.18 Extreme 4 0.66 0.28 0.06 vertices 5 0.28 0.66 0.06 6 0.22 0.60 0.18

7 0.41 0.41 0.18 8 0.47 0.47 0.06 9 0.63 0.22 0.15 Edge

10 0.66 0.25 0.09 centroids 11 0.22 0.63 0.15 12 0.25 0.66 0.09

13 0.44 0.44 0.12 Overall centroid

0.22 ~ K+ ~ 0.66 (5)

0.22~Ca++ ~0.66 (6)

0.66 ~ Mg ++ ~ 0.18 (7)

Applying the XVERT algorithm to the con­straints above produced a region with six ex­treme vertices from which six edge centroids and an overall centroid point were generated. The matrix of the candidate points is shown in Table 2. The candidate list is plotted in Figure 3.

Out of these candidate points an 'optimal' design has to be selected. For this purpose, a model must be specified. Within this region it is assumed that the response can be approximated

Potassium Simplex Design

Magnesium Calcium

Fig. 3. The list of candidate points, consisting of the vertices, the edge centroids and the overall centroid.

reasonably well by a second-order Scheffe model which consisted of six terms. In the next step a D-optimal design is searched for with the use of Welch's branch-and-bound optimization algo­rithm. The number of design points necessary for supporting the fit of the quadratic model is six but it is recommended that at least two or three additional points be selected in order to cover the experimental region better than would be the case with the minimum number. The results are listed in Table 3.

The nine point design, consisting of the ver­tices, the centroids of the two longest edges of the polyhedron and the overall centroid was chosen. This nine-point design actually had a lower D-optimality value than all the other designs ranging in size from six to eleven points.

The experiment was carried out with these nine nutrient compositions. Per treatment de­structive growth analysis was carried out on ten plants. The effects on the total leaf biomass (FW in g) are reported here (Table 4 ). First of all a quadratic canonical polynomial was fitted to the data. The statistical tests of the model parame­ters showed that the nonlinear blending esti­mates were not significantly different from zero, so they were dropped from the model resulting in the following linear blending model:

FW= 102.8*K + + 109.2*Ca++ + 107.3*Mg++

with R 2 = 0.982 and RA = 0.980. The statistical evaluation of this model showed in an objective way that the response did not change with

Table 3. The 0-optimality criterion of designs with different numbers of treatments for Example 2'

Number 0-optimality Design consisting of criterion of points point numbers

6 132.5 1345613 7 126.5 12345613 8 125.5 123456713 8 125.5 1 2 3 4 56 813 9 125.4 1234567813

10 126.6 1 2 3(2) 4 56 7 8 13 10 126.6 1234(2)567813 11 126.6 1 2 3 4(2) 5 6(2) 7 8 13 11 126.6 1 2 3 4(2) 5(2) 6 7 8 13

"Numbers between parenthesis indicate replication of that particular point.

Mixture systems 185

Table 4. The matrix of the design points (proportions). the mean and the standard error of the mean for the total leaf weight (g) fo r Example 2

Point K' ca++ Mg++ Mean Std err

0.66 0.22 0.12 97.3 9.8 2 0.22 0.66 0.12 106.1 6.5 3 0.60 0.22 0.18 110.4 6.0 4 0.66 0.28 0.06 109.5 4.7 5 0.28 0.66 0.06 109.2 6.5 6 0.22 0.60 0.18 108.6 6.2

7 0.41 0.41 0.18 107.1 6.6 8 0.47 0.47 0.06 105.3 6.9

13 0.44 0.44 0.12 102.3 7.5

ff

IGI

11

l l

Fig. 4. Total leaf weight (g) of tomato in function of cation composition over an irregular shaped experimental region.

different cation composition within the region of interest. Thus Equations 5 to 7 give a first approximation of the zone of optimal response for the leaf weight. The response surface is shown in Figure 4. Of course, before claiming the zone as defined in equations 5, 6 and 7 as being optimal, other dependent variables or tomato plant characteristics , like production and quality , need to be investigated.

Conclusion

The application of mixture theory in terms of mixture designs and model forms is an indispens­able tool for investigating nutrient solutions m hydroponic plant nutritional research.

186 Mixture systems

In the optimization of nutrient solution composition, the emphasis is placed on multi­variate investigation of the zone of optimal response as a function of the ion composition and the total ionic strength of the solution. This multivariate approach makes the study of ion interaction effects on well defined response vari­ables possible. This work is a valuable tool in mineral nutritional research, because for the first time the chemical feasibility conditions of such solution, combined with additional chemical, physiological or economical constraints, form the foundation of the statistical experimental design theory, which makes the optimization of com­plex mixtures of ions in relation to response variables possible.

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Reprinted from Plant and Soi/154: 45-52, 1993.

D

Nutrient uptake and interactions with physiological and biochemical processes

M.A. C. Fragoso and M.L. van Beusichem ( eds.) Optimization of" plant nutrition 189-195, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-040

Genotypic variability for physiological efficiency index of nitrogen in oats

D. ISFAN Agriculture Canada, Research Station, 2560 Hochelaga Blvd., Sainte-Fay, Quebec, Canada GlV 213

Key words: Avena sativa L., breeding method, genotypes, nitrogen efficiency, nitrogen physiology, nitrogen uptake, nitrogen utilization, sustainable agriculture

Abstract

One of the methods for measuring the efficiency of N utilization by plants is the index of physiological efficiency of absorbed N (PEN) which for cereals is defined as the ratio of grain produced to the total N absorbed by the above-ground plant parts (grain and straw) at maturity. This index indicates how this absorbed N is used by the plant to produce grain. The objective of this work was to study the genotypic variability of PEN in oats (A vena sativa L.) and to what extent grain yield is related to PEN. Seven selected oat genotypes were studied under greenhouse conditions with 5 levels of added N including control (no additional N). At maturity the grain and straw were harvested separately and analyzed for total N. The results show that there was a highly significant variation among genotypes in both yield per pot and PEN. Grain yield was positively and significantly related to PEN (r = 0.95xx). The total N absorbed by plants was much less correlated with grain yield than PEN. The results suggest that PEN may be used in a plant breeding program to detect the potentially high yielding oat genotypes and to evaluate those capable of exploiting N input most efficiently.

Introduction

Nitrogen fertilizer is the key input in crop production and yield increase but at the same time it is one of the most energy-consuming nutrients and likely to contribute to surface and ground water pollution and to global warming when inefficiently used by crops. Therefore, improving nitrogen fertilizer efficiency is an important goal in crop production in a sustain­able agriculture.

One of the ways to increase nitrogen efficiency is to create cultivars with high nitrogen ef­ficiency. Hardy et a!. (1975) and Mertz (1976) pointed out that improving nitrogen efficiency of cultivars is an important objective in the breed­ing program. Many researchers found significant variations of nitrogen efficiency among cereals genotypes (Anderson et a!., 1984, 1991; Bock, 1984; Broadbent eta!., 1987; Buresh eta!., 1988; De Datta and Broadbent, 1988; Hamid, 1972;

Isfan, 1990; Isfan et a!., 1991; Ohm, 1976; Pearman eta!., 1977; Pino, 1979; Simonis, 1988; Tilman et a!., 1991; Welch and Yong, 1980; Wuest and Cassman, 1992). Bloom and Chapin (1981) and Bloom and Finazzo (1986) reported a genetic variation even in NH4 and N0 3 uptake in barley. In spring barley (Isfan, 1990) and in triticale (Isfan et a!., 1991) the physiological efficiency index of nitrogen varied significantly among genotypes and was positively and sig­nificantly related to grain yield.

In maize, the N efficiency is related to geno­type and consequently it may be improved dur­ing a breeding program (Beauchamp et a!., 1976; Chevalier and Schrader, 1977; Mollet a!., 1982). Broadbent et a!. (1987) also suggested that there is a considerable potential for exploiting genotypic differences in N utilization efficiency by crop.

As genetic selection is very often conducted at high levels of nitrogen application, the differ-

190 Isfan

ences between genotypes as to N uptake and N utilization can be masked (Kamprath et a!., 1982). Schmidt (1984) pointed out that cultivar development may need to be oriented on the production of genotypes that exploit inputs most efficiently, not on the development of genotypes that have superior yield only when high pro­duction inputs are used. In breeding for im­proved N use efficiency, it would seem desirable for both uptake efficiency and utilization ef­ficiency to be improved simultaneously (Moll et a!., 1982). Coffman and Smith (1991) pointed out that breeding for sustainability is mostly a process of fitting cultivars to an environment instead of altering the environment by adding inputs such as fertilizers, water, and pesticides. A higher efficiency would permit reducing N rate without reducing yield and profits and lead to a smaller proportion of N susceptible of being carried to surface and ground waters ( Capuro and Voss, 1981). Thus, the efficiency of N utilization by crops is one of the main objectives in agricultural production and management.

One of the methods for measuring the ef­ficiency of N utilization is the index of the physiological efficiency of absorbed (uptake) nitrogen (PEN) which for cereals is defined as the ratio of grain produced to the total nitrogen absorbed by the above-ground plant parts (grain plus straw) at maturity (Novoa and Loomis, 1981). The index indicates how absorbed nitro­gen is used by the plant to produce grain. It is related to many physiological processes such as absorption, nitrate reduction efficiency, nitrogen remobilization, translocation, assimilation and stockage (Novoa and Loomis, 1981). Anderson et a!. (1984) reported that genotypes differ in remobilization of N from leaves and stalk, which is important in the efficient utilization of maize plant nitrogen.

The capacity of using N more efficiently is seldom evaluated in cereal breeding programs although N is an important factor of the yielding capacity of cultivars, an expensive and poten­tially pollutant nutrient. The objective of this work was to study (1) the genotypic variability for physiological efficiency index of nitrogen and (2) to that extent that index is related to grain yield. If the grain yield is significantly related to

PEN, than this index can be used in the breeding program to detect the potentially high-yielding genotypes capable of exploiting nitrogen input most efficiently for a sustainable agriculture.

Materials and methods

The experiment was done in greenhouse con­ditions where the climatic and diseases influences on grain yield performance of cultivars are practically avoided and thus some genetic differ­ences between cultivars, like PEN, will appear more clearly. During the plant breeding program some genotypes can be thus eliminated by a first screening step in greenhouse before field experi­ments which are much more expensive. Coffman and Smith (1991) suggest that new techniques are needed to increase breeding efficiency and thus reduce the cost of seed. Evolving appropri­ate selection strategies and breeding methods constitutes a stage of the basic plant breeding research needed to develop germplasm for sus­tainable agriculture.

Seven oats (A vena sativa L.) cultivars were grown (Table 5) under greenhouse conditions, in pots with 3.5 kg air-dried sandy-loam soil and five nitrogen levels: 0, 80, 160, 240 and 320 mg of N as ammonium nitrate kg -I of dry soil. A rate of 200 mg P20 5 and 300 mg of K2 0 kg -I of dry soil was applied. The analyses of soil at the beginning of the experiment were as follows: 5.9% mg kg -I O.M., 13.9 mg kg -I N-NH 4 ,

15.7mg kg- 1 N-N0 3 , 77 mg kg- 1 P, and 80mg kg -I K. The experimental setup was a random­ized complete block design with a factorial combination of the seven cultivars (genotypes), five nitrogen fertility levels and four replications. Two weeks after sawing the plants were thinned to eight plants per pot. At maturity the grain of main shoot and tillers and the straw were har­vested separately, dried at 70°C in a forced-air oven, weighed, ground and then analyzed for total nitrogen (macro-Kjeldahl method). The main shoot and tillers were harvested and ana­lyzed separately because of their very different impact on grain yield and absorbed N, and their different response to cultivars and added nitro­gen. In this study the absorbed N is the total

amount of N absorbed (total N uptake) in the above-ground plant parts (grain plus straw) per pot at maturity, and the yield per pot (in mg) to the amount of absorbed N per pot (in mg). Results were analyzed statistically using SAS GLM procedure (1985).

Results and discussion

Grain and straw yield

Grain yield varied significantly among cultivars as well as among nitrogen treatments (Table 1). The yield varied from 8.9 to 26.8 g/pot in the low-yielding cultivar (LYC) Elgin and from 12.0 to 43.1 g/pot in the high-yielding cultivar (HYC) Cascade. The average yield over all treatments, was 21.2 and 32.2 g/pot for Elgin and Cascade, respectively. A positive interaction between cul­tivars and N treatments was found. Thus, the maximum yield increase due to added N was of 17.9 g/pot with LYC Elgin and as high as 21.1 g/ pot with HYC Cascade.

The yield differences among cultivars were due more to main shoot grain yield than to tillers grain. The latter one was influenced mostly by N fertilizer (Table 1 ). Thus, the total grain yield variation among cultivars was significantly re­lated to main shoot grain yield and not related to tillers' grain (Table 4). These results suggest that the main shoot grain yield is a very determinant factor in the differentiation of cultivars.

Physiological efficiency index of nitrogen 191

Nitrogen concentration

Nitrogen concentration(%) in grain as well as in straw varied significantly among cultivars and was higher in low- than in high-yielding cultivars (Table 1). As for the grain yield the differences between cultivars were due more to the main shoot grain concentration than to the tillers' grain. The added nitrogen had on the contrary a higher influence on tillers' than on main shoot N concentration.

A significant negative correlation (r = -0.91 **) was found between grain N concentration (in %) of cultivars (C) and grain yield (Y):

y = 50.8- 11.96 c

Thus, the average N concentration was of 1.71% in HYC Cascade and as high as 2.53% in the LYC Elgin. Kramer (1979) also found a negative correlation between grain N concentration and grain yield among winter wheat genotypes. Rizzi et al. (1991) found that N concentration in maize and particularly nitrate N concentration was genetically controlled and that maize plants differ in this respect.

The data also show that grain N concentration at a low N level (80 mg kg -J of soil) decreased in the high yielding cultivars compared to control treatment and increased in low yielding cultivars. This dilution effect may be due to N deficiency at this rate in the HYC since the yield increase was higher than in LYC. These results suggest that at low N level some genotypes tend to use this N to produce more grain than to store it in the straw

Table 1. Analysis of variance (mean squares) for grain, straw yield and nitrogen concentration (%) as related to cultivars and added nitrogen fertilizer

AN OVA OF Yield Nitrogen concentration (%) source

Main shoot Tillers' grain Total grain Straw Main shoot Tillers' grain Total grain Straw

grain grain

Cultivar (C) 6 405.7 63.3 264.5 66.1 1.672 1.201 1.475 0.048 Nitrogen (N) 4 229.4 1686.2 2811.5 1296.8 10.532 29.549 9.266 0.404 CxN 24 20.5 15.9 16.5 7.5 0.123 0.164 0.088 0.012 Error 105 2.4 3.1 1.5 1.1 0.011 0.011 0.008 0.002 F value" 46.3 70.6 257.1 143.8 138.7 347.2 175.4 28.2 CV% 10.1 15.2 4.5 5.5 5.2 6.4 4.5 11.2

"All F values are statistically significant at p = 0.05.

192 Isfan

or grain. This was true for HYC Cascade and not for LYC Elgin.

Conversely, at high N rate the N concentration in grain was continuously increasing in HYC compared to LYC. In latter genotype the trans­location of N in grain was less efficient and a higher N fraction of absorbed N was stored in straw in comparison with HYC.

Absorbed nitrogen

The results show that there was a significant genotypic variation with respect to the amount of absorbed N (N uptake) in grain as well as in straw (Table 2 and Fig. 3) but no correlation was found between this N and grain yield of oats (Table 4). The LYC Elgin had one of the highest amounts of absorbed N but the lowest grain yield (Table 3).

The data show that with the same amount of absorbed N a high-yielding cultivar (Cascade) can produce much more grain than a low-yield­ing cultivar (Elgin) (Fig. 1 ), or in other words, a HYC can produce the same grain yield but with much less N than a LYC. This may be one of the explanations why the grain yield of cultivars was so weakly related to absorbed N.

Figure 1 also shows that the differences be­tween cultivars arc much higher at higher ab­sorbed N and that at high N rates the HYC (Cascade) can absorb much more N than the LYC (Elgin).

The translocation efficiency was also related to genotype. Thus, the translocation of N from vegetative tissues to grain was higher in HYC Cascade than in L YC Elgin where an important amount of N remained in straw at high N rate.

Table 3. Absorbed nitrogen and grain yield as related to cultivars (overall nitrogen treatments)'

Cultivar Absorbed nitrogen (g pot -]) Grain yield (g poC 1)

Cascade 0.709 a' 33.2 a Elgin 0.678 b 21.2e Oxford 0.663 be 27.5 be Lamar 0.661 be 28.1 b Scott 0.665 c 27.9b Manic 0.644 c 24.8d Laurent 0.604 d 26.7 c

'Absorbed nitrogen in the above-ground plant part, grain and straw. ' Column values followed by same letter are not significantly different at 0.05 level of probability using Duncans's Multiple Range Test.

1. CASCADE

2. OXFORD

3. ELGIN

' 50 -0 Cl.

Ill 40

c:i 2 30 ..J

__ ... 3 w

> 20 ,..-"""'--z ;

,."'"' < 10 ; a: (!)

0 0.1 0.5 0.9 1.3

ABSORBED NITROGEN, g pot -l

Fig. 1. Grain yield as related to total absorbed nitrogen in the above-ground plant part (grains plus straw) in high (Cascade), low (Elgin), and medium (Oxford) grain yielding cultivars.

Table 2. Analysis of variance (mean squares) for absorbed nitrogen in the above-ground plant part and for the physiological efficiency index of nitrogen (PEN)

AN OVA DF Absorbed nitrogen PEN source

In grain In straw Grain + straw

Cultivar (C) 6 0.0180 0.0017 0.0207 770.6 Nitrogen (N) 4 2.8810 0.0611 3.7751 4862.3 CxN 24 0.0190 0.0006 0.0153 40.2 Error 105 0.0010 0.0001 0.0011 4.4 Fvalue" 295.9 65.8 414.5 164.7 CV% 6.1 12.3 5.0 4.5

'All F values are statistically significant at p ~ 0.05.

Our results suggest that the absorbed N is a rather weak criterion to detect the higher-yield­ing cultivars and that the way in which the absorbed N is used to produce grain is more important than the total amount.

Many other researchers studied the differences in absorbed N among cultivars. Spiertz (1978) found only small differences between wheat cultivars. A significant correlation between ab­sorbed N and grain yield was found in 7 spring barley genotypes (Isfan, 1990) and no correla­tion at all in 12 spring triticale (Isfan et a!., 1991). Some researchers found that wheat cul­tivars differ in absorbed N even late in the growing season (Halloran, 1981; Paccaud et a!., 1985).

In maize many workers found genotype differ­ences in absorption and in utilization of N (Beauchamp et a!., 1976; Chevalier and Schrader, 1977; Kamprath et a!., 1982; Moll et a!., 1982). Roth et a!. (1989) concluded that N uptake was the weakest predictor of N de­ficiency, had the largest spatial variability and was affected by factors that limited crop dry matter production in winter wheat. De Datta and Broadbent (1988) found statistically signifi­cant differences among rice genotype grain yield but not significant differences in total N uptake.

Physiological efficiency index of nitrogen

The physiological efficiency index of N which in this work is defined as the ratio of grain yield (in mg) to the amount of absorbed N in the above­ground plant dry matter production at maturity (in mg), varied significantly among cultivars at all N treatments (Table 5). Variation of the physiological efficiency of nitrogen among geno­types was also found in wheat (Hamid, 1972; Pearman et a!., 1977; Pino, 1979; Simonis, 1988); in barley (Isfan, 1990; Simonis, 1988) in triticale (Isfan et a!., 1991; Pino, 1979) and in rice (Buresh et a!., 1988).

In all cultivars the PEN was higher in control treatment and decreased with the increasing N rate (Table 5 and Fig. 2). Thus, the magnitude of PEN depends on the amount of available N but as the results indicate the genotype which had a high PEN at low N level had also a high PEN at high N level. Other studies on the subject have

Physiological efficiency index of nitrogen 193

BOr---------------------------,

2

• 3 ......

0

...... ... •,

1. CASCADE

2. OXFORD

'',,, ·-----·-----

160 320

ADDED NITROGEN mg kg-l of soil

Fig. 2. Physiological efficiency index of absorbed nitrogen in above-ground plant part in a high (Cascade), low (Elgin) and medium (Oxford) grain yielding cultivars.

also indicated that N efficiency was higher on low than on high N level (Buresh et a!., 1988; Moll et a!., 1982; Pearman et a!., 1977; Pino, 1979; Simonis, 1988).

Our results also show that grain yield of genotypes was significantly correlated to PEN at all level of N treatments (Table 4 and Fig. 3) and especially at medium N rate. The PEN was also higher in high-yielding cultivars. The data show that the way in which the absorbed N is used to produce grain is more important than the total amount of available N in soil or in plant.

The results of this study suggest that the differences in the physiological efficiency index of N among oat genotypes may be used in the breeding program to detect the potentially high­yielding genotypes capable of exploiting N input most efficiently. An ideal genotype could be one which absorbs relatively high amounts of N from soil and fertilizer, produces a high grain yield per unit of absorbed N and stores relatively little N in the straw.

Acknowledgement

Grateful thanks are due to Mr Antoine D'Avig­non for technical assistance.

194 Isfan

Table 4. Simple correlation coefficients (r) between grain yield and physiological efficiency index of nitrogen (PEN) and other independent variables

Variables correlated Added nitrogen (mg kg -I of soil)

Dependent Independent 0 80 160 240 320 variable variable

Grain yield PEN NS 0.93** 0.96** 0.93** 0.76* Grain yield Absorbed N NS NS NS NS 0.87* Grain yield Main shoot 0.83* 0.91** 0.93** 0.89**

Grain yield Grain yield Tillers' grain yield NS NS NS NS Yield increase' PEN 0.82* 0.91** 0.85* NS

" Yield increase due to added nitrogen fertilizer with respect to zero nitrogen treatment. NS: Not significant; *, **: significant at 5% and 1% levels, respectively. Each coefficient reprcscnts.seven data (cultivars) each one being the mean of four replicates.

Table 5. Physiological efficiency index of nitrogen as related to cultivars and added nitrogen fertilizer

Cultivar Added nitrogen (mg kg- 1 of soil) Mean

0 80 160 240 320

Cascade 71.4 a' 71.4a 56.4a 40.8 a 35.2a 55.0 a Laurent 64.1 b 64.1 b 50.5 b 37.7b 34.6a 50.2 b Lamar 63.3 b 62.3 be 47.8 c 35.3 c 34.1 a 48.6c Oxford 61.5 be 62.8 be 48.3 c 37.0 be 31.3c 48.2 c Scott 59.2c 59.3 c 48.6 be 37.8 b 32.7b 47.8c Manic 59.4 c 54.9d 4l.Od 33.0d 30.7 c 43.8 d Elgin 49.3 d 41.0e 30.2c 27.5 e 27.6 d 35.1 e Mean 61.2A 59.4B 46.1 c 35.6D 32.3E CV% 3.5 4.3 2.9 4.2 2.5

'Column values followed by same letter (a to e) are not significantly different at 0.05 level of probability using Duncan's Multiple Range Test. All mean values (A to E) are significantly different (using the same test).

.. 0 c. a

0 ..J !!:! >-a!: < a: (.!)

40

30

y=9.32 + 0.48x

r=0.96 ** CASCADE

I •

2 0 .__.___..___......__...J...._......a.... _ ___._ _ __. 30 40 50 60

NITROGEN PHYSIOLOGICAL

EFFICIENCY INDEX

g GRAIN g-10F ABSORBED N, (x)

Fig. 3. Grain yield of cultivars as related to the physiological efficiency index of absorbed nitrogen (Cascade: high yielding cultivar; Elgin: low yielding cultivar; Oxford and Manic: relatively medium yielding cultivars).

References

Anderson E L, Kamprath E J and Moll R H 1984 Nitrogen fertility effects on accumulation, remobilization, and parti­tioning of N and dry matter in corn genotypes differing in prolificacy. Agron. J. 76, 397-404.

Anderson W K, Scymur M and D'Antuono M F 1991 Evidence for differences between cultivars in responsive­ness of wheat to applied nitrogen. Aust. J. Agric. Res. 42, 363-377.

Beauchamp E G, Kannenberg L W and Hunter R B 1976 Nitrogen accumulation and translocation in corn genotypes following silking. Agron. J. 68, 418-422.

Bloom A J and Chapin F S 1981 Differences in steady-state net ammonium and nitrate influx by cold- and warm­adopted barley varieties. Plant Physiol. 68, 1064-1067.

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Broadbent F E, De Datta S K and Laureles E V 1987 Measurement of nitrogen utilization efficiency in rice genotypes. Agron. J. 79, 786-791.

Buresh R J, De Datta S K, Padilla J Land Samson M I 1988 Field evaluations of two urease inhibitors with transplanted lowland rice. Agron. J. 80, 763-768.

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Dalling M J 1985 The physiological basis of nitrogen redistri­bution during grain filling in cereals. In Exploitation of Physiological and Genetic Variability to Enhance Crop Productivity. Eds. J E Harper, L E Schrader and R W Howell. pp 55-71. American Society of Plant Physiolog­ists, Rockville, MD.

De Datta S K and Broadbent F E 1988 Methodology for evaluating nitrogen utilization efficiency by rice genotypes. Agron. J. 80, 793-798.

Fisher R A and Wall PC 1976 Wheat breeding in Mexico and yield increases. J. Aust. Ins!. Sci. 42, 139-148.

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Hardy R W F, Filner P and Hageman R H 1975 Nitrogen input. In Crop Productivity- Research Imperatives. pp 133-176. Michigan Agric. Exp. Station, East Lansing, MI.

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Isfan D, Cserni I and Tabi 1991 Genetic variation of the physiological efficiency index of nitrogen in triticale. J. Plant Nutr. 14, 1381-1390.

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Moll R H, Kamprath E J and Jackson W A 1982 Analysis and interpretation of factors which contribute to efficiency of nitrogen utilisation. Agron J. 74, 562-564.

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Pino I 1979 Economia del nitrogeno en cultivares de trigo (Triticum aestivun L.) y triticales (Triticosecale sp.). Magis­ter Sci. Thesis. Escuela Agronomia. Universidad Catolica de Chile, Santiago. 57 p.

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Roth G W, Fox R H and Marshall H G 1989 Plant tissue test for predicting nitrogen ·fertilizer requirements of winter wheat. Agron. J. 81, 502-507.

Schmidt J W 1984 Genetic contributions to yield grains in wheat. In Genetic Contributions to Yield Grains of Five Major Crop Plants. Ed. W R Fehr. pp 89-101. CSSA Special Publication Number 7. A S A, Madison, WI.

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Reprintedfrom Plant and Soi/154: 53-59, 1993.

M.A. C. Fragoso and M.L. van Beusichem (eds.) Optimization of plant nutrition 197-201, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-064

Mapping of genes for copper efficiency in rye and the relationship between copper and iron efficiency

R. SCHLEGEL, R. KYNAST, T. SCHWARZACHER1 , V. ROMHELD 2 and A. WALTER2

Institute of Plant Genetics and Crop Plant Research, Corrensstraf3e 3, D-06466 Gatersleben, Germany; 1John Innes Centre for Plant Science Research, Cotney, Norwich, NR4 7UJ, UK; 2/nstitute of Plant Nutrition, University of Hohenheim, 7000 Stuttgart, Germany

Key words: chromosome translocation, copper efficiency, DNA:DNA in situ DNA hybridization, physical mapping, phytosiderophores, rye, wheat

Abstract

A 4B/5R wheat-rye translocation line derived from the Danish wheat variety 'Viking' was revealed to be highly copper efficient. The chromosomal exchange includes a very small terminal segment of chromosome arm 5RL of rye which was physically mapped by genomic DNA: DNA in situ hybridiza­tion and chromosome analysis. The gene for Cu efficiency (Ce) is linked to a dominant hairy neck character from rye (Hal) and to two rye-specific leaf esterase loci (Est6, Est7), all of which are postulated to map to the distal part of 5RL. Genes coding for mugineic acid synthetase and 3-hydroxymugineic acid synthetase also on chromosome 5R are not included in the 4B/5R translocation and hence map outside the terminal 5R region. These genetic and molecular markers can be useful tools for large-scale screening in wheat breeding programmes.

Introduction

Because of the increasing costs and environmen­tal concerns the agriculture of industrial coun­tries faces the problem of reduction of fertilizer input in plant production, while developing countries are often confronted with an excess of toxic minerals as growth-limiting factors. There­fore, the high efficiency in acquisition of macro­and micronutrients as well as a high tolerance to toxicity stress has become an important task for plant breeding and breeding research. For both approaches- the nutritional efficiency and the stress tolerance - a genetic variability was found within and between species ( cf. Man yow a and Miller 1991; Schlegel et a!., 1991b). Although mineral nutrition and stress factors have been intensively studied during the past decade, only

few genetic results are available. Nevertheless, the data summarized from cereal research dem­onstrate oligogenic and polygenic control of efficiency and tolerance characters as well as a clustering of these genes in the homoeologous chromosome groups 4 and 5 (Schlegel et a!., 1991b). In rye, there are also loci on chromo­some 5R which could be utilized as alien sources for improvement of wheat and possibly other crops (Graham et a!., 1987, Schlegel et a!., 1991a).

On the other hand, the physiological causes of copper efficiency are still unknown. Among the several speculations and suggestions, which con­sider differences in root growth, Cu-mobilizing root exudates, different utilization and distribu­tion patterns within the plant etc., the relation­ship between phytosiderophore release and cop-

198 Schlegel et al.

per acquisition seems particularly meaningful for further research approaches (Takagi et al., 1984, Treeby et al., 1989).

Materials and methods

Plants

The hairy-necked line of hexaploid what 'Viking' and the wheat-rye addition lines 5R, 5RS and 5RL of the 'Holdfast'- 'King II' series were kindly provided by the Institute for Plant Science Re­search Cambridge (UK). Control seeds of 'Vik­ing', 'Holdfast' and 'King II' came from the Gatersleben Gene Bank collection. Copper ef­ficiency was investigated in greenhouse pot ex­periments ( cf. Schlegel et al., 1991a).

Genomic in situ DNA hybridization

Total genomic DNA of rye was labelled with digoxigenin-II-dUTP (Boehringer Mannheim) and hybridized in situ to squashed mitotic chro­mosomes of root tips from the wheat variety 'Viking' as described in detail by Heslop-Har­rison et al. (1991). Per slide 160 ng of labelled rye DNA together with 3.3 1-Lg of unlabelled wheat DNA as a competitor were applied in 40 f-LL 50% formamide in 2 X SSC (0.34M NaCl, 0.3 M Na-citrate). Slides were washed for a stringency greater than 85% and sites of probe hybridization were detected with fluorescein conjugated antibodies.

Phytosiderophores

Ten seeds from each line were germinated in Petri dishes on water-dampened filter paper for three days, and transferred to containers filled with distilled water. Root tips were taken to verify the karyologic status. After vemalisation (at temperature of +4°C with low light intensity for 8 weeks) the seedlings were transferred to aerated nutrient solution without iron supply (Tree by et al., 1989). The plantlets were then grown under controlled conditions (day I night 16/8 h, light intensity 230 1-LE m -z s- 1), tempera­ture 23/21 oc, rei. humidity 70-85%) and nutrient solution was changed every three days. After

two weeks, Fe deficiency chlorosis became vis­ible and root exudates were collected every three days for four hours starting two hours after the beginning of the daily light period. The root exudates of four successive collections were analyzed for the various phytosiderophores (deoxymugineic acid- DMA; mugineic acid­MA:; hydroxymugineic acid- HMA) by HPLC (Mori et al. 1987).

Results and discussion

In experiments using disomic rye-wheat chromo­some addition lines it was confirmed that chro­mosome 5R, and more specifically the chromo­some arm 5RL, is capable of giving wheat as high a Cu efficiency as rye under experimentally deficient soil conditions (Podlesak et al., 1990). The addition lines 5R and/or 5RL are marked by a monogenic hairy neck character (Hal) genetically mapped about 45 eM units from the centromere, 26 units to the Acol locus, 39 to Est2, 32 to Spl, 19 to wa2, 26 to XiaglO (cf. Schlegel and Melz, 1992) and 38.5% to ct2 (J Plaschke, pers. comm.). By this dominant mor­phological trait a spontaneous wheat-rye translo­cation derivative of the Danish wheat cultivar 'Viking'* was revealed which also showed copper efficiency (Schlegel et al., 1991a). The grain yield is slightly less than that of the 'Viking' control in the presence of sufficient copper (50 mg Cu/pot). Without copper supple­mentation, however, the genotypes react dif­ferentially. The grain yield is greatly reduced in the control line (97%)- a typical reaction of wheat grown on very Cu-deficient peaty soils -while the yield reduction in the translocation line ( 49%) is significantly lower than in sister

*This variety arose from a single plant selection of a French wheat x 'Jonquois'. It was bred by the Danish plant breeder Viggo Lund, head of the plant breeding station of the Danish Sugar Factories on the island of Lolland. 'Viking' was marketed in Denmark, but grown occasionally in England, whence the present seed sample is derived (I. Linde-Laursen, pers. comm.). It is likely that the variety was not very uniform and consisted of different genotypes and/or karyotypes. The segregation for morphological and isozyme traits within the variety supports this conclusion.

line: without the r tran ·location. The value · of traw yield correlate with tho.c of graio yield.

Pre ·umcd translo '<Ilion breakpoint arc lo­cal d in the di tal 4BL2.2. and SRL2.3 chromo­. me band rcgi n of wh at and rye, re pecti e­ly. Two funh r c tera. i oz me tructural gene (E t6 and £st7; Wehling et at., J 9 ) arc linked to the Ha 1 gcn and the copp r efficiency ( e) on the translocat d chrom orne egment f ry . The . h \ monogenic Mendelian inheritance.

Differential chromo me taining, b C- and -banding, and mei tic pairing tudie did n t

enable c mpletc characterization of the tran l -catcd rye ·cgment. Henc an advanced m leeu­lar detection procedure wa applied. As can be -een from igure 1 th re i a differentially labelled region on one pair of wheat chromo­orne aft r genomic in situ hybridization with

rye D A. The egmcnt i on the long arm of a wheat chr mosome (4B), thu confirming the f rmer re. ult. of chlegcl et al. ( 199la). H w­ever, the ize of th rye-derived egment is le

B 48

Mapping of genes 199

found.

l7 4BS.4BL-5RL SR

u acqui ·i-

break

f-Ig. I . ( ) Labcll~d rye chr malin on wheat chromo omc of a ' hem-rye Iran I at ion line of the varier · iking' after genomi D A : o, in •itu hybridi7.:Hion ( c~ arrow) and (B) a kar ogramatic drawing [the wh at and rye chr mo m I egments involved in th tran•location (arrows mark the po,;, iblc breakpoints) .

200 Sclllegel et al.

Tahf~ ( The approximate amounts (nmol/mL) of phytosiderophore~ in the root-wa~htng' after four samplings ( 11 /8: 11 / 11; II 1-1: 11 / 18 1991) and plant culture in Fe-deficient nutritional solution and IIPLC analysas

Plant matcraal DMA MA HMA

R)C (King II) 101 98 1102 Wheat-rye addataon SR 378( + )' 36(-) 30(-) Wheat·ryc addiuonSRS 665( +) 0( - ) 0(- ) Wheat-rye uddition SRL 204( +) 0(+) 0(+) Whe<&t ( llold!aot) 1079 () 0 Wheat (Viking) 679( +) 0(- ) 0(-) Whcat · ryc translocation (Viking) 1470( + ) 0(- ) 0(- )

"+ Po>itivc and - negative reaction in experiments of S Mori using the same samples of tester lines .

tion and chromosome SR of rye was identified to carry the gene for phytosiderophore synthesis­mugincic acid synthetase catalyzing synthesis of MA from 2' -deoxymugineic acid and the gene for 3-hydroximugineic acid synthetase catalyzing synthc ·i:. of HMA from AM (Mori ct at.. 1990) ­as well a the gene for raising Co-efficiency, it has been concluded that iron as weiJ as copper efficiency in rye are controlled by a common genetic system.

An initial study demonstrated that rye ecretcs mainly HMA under Fe-dcficicncy conditions. but also MA and DMA. This pattern is also found in the wheat-rye addition line SR. The results agree with the data of Mori et at. (1990). However, when the telocentric wheat-rye additions SRS and SRL arc tested neither the short arm nor the long arm alone affects the secretion of MA or HMA (Table I) .

With caution , either a complementary gene interaction can be assumed which produces an effect qualitatively similar to that of the whole chromosome SR ( intergenic complementation is also described for blue aleuron in barley, or for progressive necrosis and dwarfism in wheat, Borner and Mettin , 1989) or different genotypes can influence a different behaviour.

If preliminary data of S Mori are included (Table I), the long arm of chromosome SR also lcc.tds to the ~arne reaction as does the whole chromosome in our own studies. On the other hand, in Mori's investigation chromosome SR failed to produce AM and HMA. Further experi­ment are in progress to resolve these partially contradictory results.

In the SR /48 wheat-rye translocation line of "Viking', a comparable pattern to that of normal wheat is observed. It shows that the same trans-

located rye segment, which is critical for the copper efficiency, does not influence the iron efficiency via production of phytosiderophores. The genes postulated for AM and HMA synthe­tascs should be located outside the terminal region of SRL. An additional experiment carried out by S Mori (Tokyo) confirmed this assump­tion . Hence an independent inheritance of the two characters is suggested.

Acknowledgement

We thank Dr S Mori (Tokyo, Japan) for HPLC analysis of the tester lines, and Dr J S Heslop­Harrison (Norwich, UK) for help with genomic probing. Drs T Schwarzacher and R Kynast thank BP Venture Research for support.

References

Borner A, 0 Mcuin 1989 Genetische Grundlagen der Halm· verkurzung (Dwudismus) bcim Weizen und Moglich keiten dcr zuchtcrischen Nutzung. Kulturpftanze 37. 29-55.

Grahnm R D. Asher J S, Ellis P A and Shepherd K W 1987 Tran)fer to wheat of the copper efficiency factor carried on rye chromosome arm SRL. Plant and Soil 99. 107-114.

Heslop-Harrison. J S 1991 The molecular cytogenetics or plants. J Cell . Sci. 100. 15-21.

Heslop·Harrison J S. Schwarzacher T. Anamtbawat-Jonsson K. Lcatch A R. Shi M and Leitch I J 1991 Ln situ hybridization wnh automated chromosome denaturation. Techniquc·J . Mcth. Mot. Baot. 3. 109-116.

Manyo"a M and Miller T E t991 The genetics of tolerance to hagh maneral conccntr,uion~ an the tribe Trillceae- a review and update. Euphytaca 57. 175-185.

Mar.chner li. Trecby M and Romheld V 1989 Role of root-induced changes an the rhizosphere for iron acquisi-

tion m higher plants. Z Pftanzenernaehr. Bodenkd. 152, 197-204.

Mori S, Kishi-Nishizawa N and Fujigaki J 1990 Identification of rye chromosome 5R as a carrier of the genes for mugineic acid synthetase and 3-hydroxymugineic acid synthetase using wheat-rye addition lines. Jap. J. Genet. 65, 343-352.

Mori S, Nishizawa N, Kawai S, Sata Y and Takagi S 1987 Dynamic state of mugincic acid and analogous phyto­siderophores in Fe-deficient barley. J. Plant Nutr. 10, 1003-1011.

Podlcsak W, WernerT, Griin M, Schlegel Rand Hiilgenhof E 1990 Genetic differences in the copper efficiency of cereal. In Plant Nutrition- Physiology and Applications. Ed. M L Van Beusichem. pp 297-301. Kluwer Academic Publishers, Dordrecht.

Riimheld V and Marschner H 1986 Evidence for a specific uptake system for iron phytosiderophores in roots of grasses. Plant Physiol. 70, 175-180.

Schlegel R and Melz G 1992 Genetic linkage map of rye (Secale cereale L.). In Genetic Maps- Locus maps of complex genomes Ed. S J O'Brien. Cold Spring Harbor Press (In press).

Mapping of genes 201

Schlegel R, WernerT and Hiilgenhof E 199la Confirmation of a 4BL/5RL wheat-rye chromosome translocation line in the wheat cultivar 'Viking' showing high copper efficiency. Plant Breed. 107, 226-234.

Schlegel R, WernerT and Jakob F 1991b Mineral nutrition and genetical control in cereals. Vortr. Pftanzenziichtg. 20, 85-94.

Takagi S, Nomoto K and Takemoto T 1984 Physiological aspects of mugineic acid, a possible phytosiderophore of graminaceaous plants. J. Plant Nutr. 7, 469-477.

Treeby M, Marschner H and Riimheld V 1989 Mobilization of iron/ and other micronutrient cations from a calcareous soil by plant-borne, microbial and synthetic metal chelators. Plant and Soil 114, 217-226.

Wehling P, Schmidt-Stohn G and Wricke G 1985 Chromo­somal location of esterase, peroxidase and phospho­glucomutase isozyme structural genes in cultivated rye (Secale cereale L.). Theor. Appl. Genet. 70, 377-382.

Reprinted from Plant and Soi/154: 61-65, 1993.

M.A.C. Fraf?oso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 203-206, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-034

Effect of a transient anoxia on potassium uptake in cucumber

G. BERTONI, J. SILVESTRE, J.M. LLORENS, P. MORARD and C. MAERTENS Plant Physiology, ENSAT, 145 Avenue de Muret, F-31076 Toulouse, France

Key words: anoxia, cucumber, Cucumber sativus L., potassium efflux, potassium influx

Abstract

The effect of a transient anoxia ( 40 min.) on K uptake and release by roots of intact cucumber plants was studied using direct measurement of K and 0 2 concentrations in the dilute nutrient solution. Anoxia obtained by N 2 gas bubbling inhibited K uptake in 10-15 minutes and induced an increasing K release, which was first similar to, then greater than the calculated K efflux in the preceding aerobic phase. Aerobiosis restoration inhibited K release and was followed, after 10 minutes, by an apparently complete restoration of K uptake capacity. The results were discussed in relation to root cell membrane depolarization-repolarization assumptions.

Introduction

Oxygen deficiency in the root environment limits the nutrient absorption phenomena that are ruled by active mechanisms as shown for phos­phorus (Hopkins, 1956), nitrate (Rao and Rains, 1976) and potassium (Hiatt and Lowe, 1967). At low potassium concentrations (K < 1 mM), the absorption inhibition results in a decreased net K uptake or even in a net K release from the roots towards the nutritive medium (Hiatt and Lowe, 1967). The net K release appears rapidly, less than 10 min. after the onset of oxygen deficiency (Morard et al., 1990). However, the variation of the K release with time and the restoration of the root absorption capacity after anoxia dis­continuation have been little documented. The investigation of these two phenomena in nutrient solutions during an aeration-anoxia-aeration sequence is reported hereafter.

Methods

Cucumber (Cucumis sativus L., cv. Admirable) plants were grown in a glasshouse for 7 weeks on a nutrient solution gradually diluted until the following composition was reached: KN03 0.3,

KH 2P0 4 0.125, Ca(N0 3 ) 2 0.5 and MgS04

0.15 mM for macronutrients; Fe 6, B 0.5, Mn 0.5, Zn 0.08, Cu 0.02 and Mo 0.02 mg L _, for micronutrients. The plants were transferred into a growth chamber (relative humidity above 55%, 21-16°C day-night temperatures, 14 h photoperiod and 105 W m - 2 irradiance) one week before the beginning of the experiment. The anoxia experiment was carried out at the 13-15 leaf stage. Four plants (total root fresh weight: 530 g) were put into a sealed container filled with 4.3 L solution homogeneized using a magnetic stirrer. The diffuser immersed in the solution was connected to a three-way valve, fed either by an air pump or by a nitrogen gas source. The whole system was put on an au­tomatic balance so as to control water losses due to transpiration, which were assimilated to vol­ume variations due to water absorption and compensated for by injection of degassed dis­tilled water.

The experiment included three successive phases during which the oxygen concentration was controlled using a polarographic electrode connected to an oxymeter: (i) during the aerobiosis phase 1, the 0 2 concentration was maintained at 8 mg L -I through air bubbling: this phase was extended up to the moment when

204 Bertoni et al.

almost all the potassium in the nutrient solution was taken up by the plants; (ii) the 40 min. anoxia phase 2 was obtained through nitrogen bubbling: anoxia was maintained until the K release gave a K concentration (110 JLM) nearly equal to the initial K concentration (98 JLM) in phase 1, in order to provide similar initial con­ditions for K uptake in phase 3 and in phase 1; (iii) oxygen bubbling was restored in the final aerobiosis phase 3 and was maintained until the depletion of the solution K reached a level comparable to that observed in phase 1.

Potassium absorption (during aeration) and efflux (during anoxia) were calculated from the potassium concentrations CK measured at vari­ous times (CK(t)) in the solution using a ion electrode connected to a ionometer. Potassium net uptake Inet per unit fresh weight of root R in the solution volume V was obtained by derivation of the potassium depletion curve (dCK(t)/dt, equation 1) according to the parabolic fit method (Du Chateau eta!., 1972). The model developed by Claassen and Barber (1974), which can be applied solely to the depletion curves in phase 1 and phase 3, was used for the evaluation of the passive efflux E during aerobiosis (equation 2):

02 bubbling 1 N2 bubbling

Inet = (V/R) x (dCK(t)/dt) (1)

Inet = (Imax x CK(t))/(Km + CK(t))- E (2)

The parameters (maximum influx rate Imax, affinity constant Km and supposedly constant passive efflux E) were calculated using an itera­tive procedure (Marquardt, 1963). When efflux E was not negligible relatively to Imax, the values of Imax and Km obtained from equation 2 were not comparable to those issued from models without an efflux term. The comparison was made possible by calculating the apparent maximum influx Imax* and the apparent affinity constant Km*:

Imax* = Imax- E Km* = Km(Imax + E)/(Imax- E)

Results

During the first minutes of the experiment, the low potassium release observed was the result of the plant transfer into the solution used (dotted line, phase 1, Fig. 1 ). Then, the high aerobic

102 bubbling

120 1 Aerobic phase 1 Anoxia phase 2 . Aerobic phase 3 9 '

I I

0

i 8 100

f\ 7~ i" d en

a 80 'j K+ as c

I i • c 0 0 ~ 5 ~ ~ 60 I ~ .... .... c

I 4 c Gl Gl u u c 40 c 0 3 0 u u ~ 2 N

20 0

0 0 0 50 100 150 200

Time (min.) Fig. 1. Changes in 0 2 (e) and K (0) concentration in the nutrient solution during the three phases of the experiment. Parameter values (±S.E.) were: lmax1 1.90±0.13, E1 0.72±0.15J.tmolg- 1 h- 1 , Km1 6.6±1.3J.LM; E2a 0.69±0.01, E2b 1.52± 0.021-'molg- 1 h- 1 ; Imax3 2.28±0.08, E3 0.51 ±0.11 J.tmolg- 1 h-\ Km3 11.1 ±2.1 J.LM.

potassium absorption was reflected by the de­crease in solution concentrations (solid line, phase 1, Fig. 1). The modelization of this uptake period under the form of absorption kinetics (Fig. 2) showed that the net uptake rate de­creased with the ion concentration and zeroed for a K concentration close to 3 !LM. The calcu­lation of the parameters showed the occurrence of a high aerobic passive efflux (E1, phase 1, Fig. 1) as it represented approximately 38% of the maximum influx Imaxl.

During the first 15 minutes of N2 bubbling, as a result of the low K concentration range (3-10 !LM) and of the decreasing 0 2 concentration from 8 to 0.01 mg L - 1 (Fig. 1), the possible effect of the limitation of the 0 2 supply on the K uptake rate could not be characterized. Then, K net uptake was inhibited and a net K release was observed: (i) during the first 13 minutes of anoxia (phase 2, 0 2 < 0.01 mg L - 1), the net K release rate E2a measured from the increase in concentration in the solution (Fig. 1) was close to the passive aerobic efflux E1 calculated for phase 1; (ii) during the following 28 minutes, the release rate value was doubled (E2b, Fig. 1).

As aerobiosis was being restored (phase 2-phase 3 transition, Fig. 1 ), net K release was nearly immediately stopped and accompanied with a transient low reabsorption. However, normal potassium uptake was resumed only 10 minutes after the onset of air bubbling. As

.. -·-·

40

lmax* = 1.77

phase 3

lmax*= 1.18

._ . _. __ ...- .. phase 1

60 80 100

K concentration (JJM) Fig. 2. Kinetics of net potassium uptake rate during aeration prior to anoxia (phase 1) and after anoxia (phase 3). Km* and !max* are the apparent influx parameters.

Potassium fluxes during anoxia 205

inferred from K depletion modelization, K efflux during phase 3 was lowered to a level (E3 = 0.511Lmolg- 1 h- 1) close to that measured during the first aerobiosis phase (E1 = 0.72/Lmol g - 1 h - 1); the net potassium uptake was significantly higher than that observed prior to anoxia (Fig. 2). The comparison of uptake parameters between phases 1 and 3 (Fig. 1) suggests that this variation can be accounted for in terms of an increase in the maximum absorp­tion capacity and of a decrease in passive efflux.

Discussion

The passive efflux values calculated for the above runs (E=0.51-0.721Lmolg- 1 h- 1) are quite comparable to the values obtained using the same method on maize (0.55 /LIDO! g - 1 h - 1 ;

Claassen and Barber, 1974). The apparent Imax (1.18-1.77 /LIDO!g- 1 h- 1) and Km (15-18/LM; Fig. 2) values are lower than those (Imax = 2.17 /LIDO! g - 1 h - 1 , Km = 27 !LM) observed on cucumber by Cooil (1974). The difference seems to be related to the experimental conditions: in the experiment described above, the solution temperature (21 against 27°C) and the K concen­tration range (2-100 against 30-500 !LM) were lower than those used by Cooil (1974 ).

During anoxia, the potassium uptake inhibi­tion followed by potassium release are similar to the effects observed in other species (Buwalda et a!., 1988; Morard et a!., 1990). The similarity between the values of the calculated aerobic passive efflux (E1, E3) and those of the efflux measured during the first minutes of anoxia (E2a) suggests that the first effect of anoxia is the inhibition of the K influx. The later increase in efflux (E2b) might be accounted for by cell depolarization in relation to H+-ATP ase inhibi­tion (Buwalda et a!., 1988; Cheeseman and Hanson, 1979).

The method used allowed the change in potas­sium uptake during aerobiosis restoration to be followed. The data presented above showed that K absorption was restored within a time lapse (10 min) which was close to that observed for inhibition. These time intervals measured for the whole root system appear to be consistent with those measured for root cell depolarization

206 Potassium fluxes during anoxia

(6-7min.) and repolarization (3-4min., Buwalda et a!., 1988) on excised wheat roots.

In dilute solutions, potassium absorption from and efflux into the nutritive medium are thus good indicators of root respiratory activity, which can be used in experiments where the effect of rapid variations in the oxygen content of the medium is investigated. Besides, the absence of any negative effect of anoxia on later potassium uptake suggests that a short-duration anoxia is tolerated without damage by cucumber roots.

Acknowledgement

The authors thank Dr Daniel Sayag for the English text. This experiment was carried out at the Agronomy station, INRA Toulouse­Auzeville.

References

Buwalda F, Thomson C J, Steigner W, Barrett-Lennard E G,

Gibbs J and Greenway H 1988 Hypoxia induces membrane depolarization and potassium loss from wheat roots but does not increase their permeability to sorbitol. J. Exp. Bot. 39, 1169-1183.

Cheeseman J M and Hanson J B 1979 Energy linked potassium influx as related to cell potential in corn roots. Plant Physiol. 64, 842-845.

Claassen N and Barber S A 1974 A method for characterizing the relation between nutrient concentration and flux into roots of intact plants. Plant Physiol. 54, 564-568.

Cooil B J 1974 Accumulation and radial transport of ions from potassiums salts by cucumber roots. Plant Physiol. 53, 158-163.

Du Chateau P C, Nofziger D L, Ahuja L R and Swartzen­druber D 1972 Experimental curves and rates of change from piecewise parabolic fits. Agron. J. 64, 538-542.

Hiatt A J and Lowe R H 1967 Loss of organic acids, amino acids, K and Cl from barley roots treated anaerobically and with metabolic inhibitors. Plant Physiol. 31, 155-161.

Marquardt D W 1963 An algorithm for least-squares estima­tion of non linear parameters. J. Soc. lndust. Appl. Math. 11, 431-441.

Morard P, Maertens C, Bertoni G et Boisseau Y 1990 Influence de la respiration des racines sur l" absorption du potassium et des nitrates chez le ble. C.R. Acad. Sci. Fr. 311, III, 103-108.

Rao K P and Rains D W 1976 Nitrate absorption by barley. I. Kinetics and energetics. Plant Physiol. 57, 55-68.

M.A. C. Fragoso and M.L. van Beusichem (eds.) Optimization (){plant nutrition 207-212, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-228

Differences in uptake kinetics of ammonium and nitrate in legumes and cereals

THEERTHAM P. RAO, OSAMU ITO and RYOUICHI MATSUNGA1

International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru P.O., A.P. 502 324, India; 1 Present address: Tropical Agriculture Research Center, Ohwashi, Tsukuba, Ibaraki 305, Japan

Key words: ammonium uptake, cereals, kinetics, legumes, 15N, nitrate uptake, translocation

Abstract

Influx isotherms were obtained for nitrate and ammonium from three legumes, Cajanus cajan (L.) Millsp., Cicer arietinum L. and Arachis hypogaea L. and three cereals, Sorghum bicolor (L.) Moench., Pennisetum glaucum L. and Zea mays L. The transition in influx isotherms for both nitrogen sources was found to be within the concentration range (0.05-2.5 mM) tested. There were significant differences in Km and Vmax for ammonium between legumes and cereals. The difference in the kinetic properties for nitrate uptake between the two groups of plants only became apparent at the higher concentration tested. Legumes translocated absorbed nitrate and ammonium to shoots more rapidly than cereals. Results show that there are significant differences in uptake and translocation of ammonium and nitrate between legumes and cereals.

Introduction

To characterize nutrient uptake among different species of plants, the total amounts of the particular nutrient taken up by plants during the entire growth period have been often used. To compare any nutrient uptake process per se, however, kinetic parameters on the rate and affinity of membrane transport will be the most suitable indices as they are relatively indepen­dent of the dry matter production of the plants. Although nitrogen is a key nutrient for the growth and development of plants, little success has been made in inter- (Van de Dijk et a!., 1982) and intraspecific (Pace and McClure, 1986) comparison of uptake properties with the kinetic approach, mainly because of the ana-

ICRISAT Journal Article No. CP-818

lytical difficulties and poor availability of iso­topes ( 15N and 13N) which enable simple mea­surement of the element absorbed by plants during a short time period.

It is important to know the kinetic properties of each plant species not only for screening species or genotypes with high absorbing capaci­ty, but also for understanding and predicting the competition for nutrient uptake by plants grown in a mixture with other plant species in natural and agricultural ecosystems. The intercropping of a legume and cereal is widely practised by farmers in the semi-arid tropics (SAT), where water and nutrients are generally limiting. These limited nutrients in soil can be most efficiently exploited by intercropping a combination of crops which are complementary in nutrient up­take.

The present study was initiated to compare

208 Rao et al.

kinetic parameters of nitrogen uptake and trans­location among three legumes and three cereals which are commonly used as component crops in intercropping in the SAT.

Materials and methods

Plant material and culture conditions

Three legumes, Pigeonpea (Cajanus cajan (L.) Millsp. var. ICPL 87), Chickpea (Cicer arietinum L. cv. K850) and Groundnut (Arachis hypogaea L. cv. NCAC 17090) and three cereals, Sorghum (Sorghum biocolor (L.) Moench var. CSH5), Millet (Pennisetum glaucum L. var. WC 75) and Maize (Zea mays L. var. Ganga 5) were grown in a greenhouse for a month at an average temperature of 30°C. Twenty seeds of each plant species were sown in a wooden tray (150 mm x 450 mm and 120 mm depth) filled with sand which was thoroughly washed with tap water before the experiment. The plants were regularly supplied with Hoagland nutrient solution modi­fied by Johnson et a!. (1957) with 1/5 strength for legumes and 2/5 strength for cereals. Roots were carefully separated from sand with flowing tap water and subjected to uptake rate measure­ment.

Uptake rate measurement

Roots detached from a plant were placed in a 50 mL Erlenmeyer flask with 20 mL of 15N-la­belled N solution. The flasks were incubated at 30 ± 2°C for 2 hours by shaking them vigorously in the dark. The roots of intact plants were placed in a 100 mL glass tubes containing 75 mL 15N-labelled N solution. The tubes were incu­bated in the greenhouse with full sunlight at 30°C for 2 hours. Only 15N-labeled nitrogen salts as ammonium sulfate and potassium nitrate were added into the incubation solution at initial concentrations of 0.05, 0.1, 0.25, 0.5, 1.0 and 2.5 mM with three replications. The 15N abun­dances of nitrate and ammonium were 98 and 99.5 atom %, respectively. After incubation, the plant samples were dried in an oven at 70°C for 2 days and then digested with salicylic acid-sulfuric acid mixture and catalyst (K2SO/CuS04 5H20/ Se: 100/10/1). The nitrogen content was deter-

mined by distillation and titration with 0.005 M sulfuric acid. Nitrogen in the digest was concen­trated into a small volume of 0.5 N HCl solution by a modified Conway diffusion method (Yoneyama et a!., 1975). 15N abundance in the concentrated solution was analyzed by an emis­sion spectrometer (JASCO N-150).

Kinetic parameters, Km and Vmax, were calculated using weighed least squares regression analysis of the data according to Michaelis-Men­ten equations (Epstein, 1972). The results of experiments with detached and intact roots were pooled together as both gave almost identical kinetic parameters. Translocation rates of the absorbed nitrogen were estimated by the ratio of 15N in the shoots to that in whole plants during the incubation.

Results

Influx isotherms

The influx isotherms of nitrate and ammonium are shown in Figure 1. Since the influx rates at 2.5 mM are far off from the line expected with Michaelis-Menten equation in all cases, there seems to be at least two different uptake patterns within the concentration range tested here. The first component, uptake at low substrate concen­trations, is saturable and fits well with Michaelis­Menten kinetics. The appropriate modelling for the second component, uptake at higher concen­trations of the substrate, is difficult to be drawn due to the limited range of nitrogen concen­tration in the external medium.

The reciprocal plotting (Lineweaver-Burk plot) of both the external concentration of sub­strates and influx rates from five sets of data excluding the highest concentration gave straight lines with high correlations (0.82 < r2 < 0.99) (insets for legumes in Figure 1).

Kinetic parameters

The Km and Vmax calculated from the saturable first component (Fig. 1) are shown in Figure 2. The legumes had a higher Km for ammonium than the cereals. Millet had the lowest Km among the six plants (0.054 mol m - 3 ). The Km for nitrate was not very different in the legumes

Ammonium and nitrate uptake in legumes and cereals 209

0.008

0.006

2000~ . ~000 *

0o ·1t. 20 o

D

• * D

----------Millet

0.004 D Groundnut _______ * ___ ,

0.002

/I---·------Pig~O~Pe;--,

Chickpea

• Sorghum

~

.r:: .,-

Ol .:.:. 0.000 0 0

2 3 0 2 3 ·3 E Nitrate cone. ( mol m )

Cl.l 0.04 "@

Cl.l .:.:. <1l a_ 0.03 :J

300~ 200

100

1.\l HI: 20 0

*

e Pigeonpea

0.02 -------------- - - -Chickp~a- - - - •

------------Groundnut

0.01

0.000 2 3 0 2 3

-3 Ammonium cone. ( mol m )

Fig. 1. Influx isotherms of nitrate (top) and ammonium (bottom) for legumes (left) and cereals (right). Insets show Lineweaver­Burk plots for the influx of nitrate and ammonium for legumes. The lines were estimated with the influx rates at the lower five concentrations.

and cereals tested. Vmax was strikingly higher for ammonium than for nitrate. The Vmax for am­monium was higher for the legumes than for the cereals, whereas no clear trend was found among the species in the Vmax for nitrate. Assuming a linear relationship for the second component, a k was obtained from the influx rate at 2.5 mM and the simulated rate at 1.0 mM. The k was higher for ammonium than for nitrate. The legumes had a higher k for both nitrate and ammonium than did the cereals (Fig. 2).

Translocation

Since differences in the distribution percentage of N among the lower five concentrations were not significant, they were averaged. Nitrate was more readily translocated to shoots than am-

monium in all plant species (Table 1). For both nitrate and ammonium, legumes had higher distribution percentages to the shoot than cere­als, indicating a more rapid translocation in the legumes than in the cereals.

Discussion

The transition in influx isotherms was first dem­onstrated by Epstein (1972) and defined as a dual pattern of ion uptake. The saturable pattern according to the Michaelis-Menten equation was applied to both components. Since Km and Vmax are always lower in the first component than in the second, it was suggested that the first was controlled by a carrier-mediated process and the second by a passive diffusion process. The

210 Rao et al.

c

E 0 .s E ~

-c ::;;:

c .c 0>

.Y.

0 .s

0.200

0.150

0.100

0.050

0.000 n

Cc Ca Ah Sb Pg Zm

Plant species O.o15

I Nitrate

0.010 0 Ammonium

0.005

0.000 ..... L....Jc...-L..J __ L....L __ L....L __ L.....I. __ W-

Ah Sb Pg Zm

Plant species

0.030

_c 0.020

0> .Y.

0 .s ~ 0.010

E >

Ah Sb Pg Zm

Plant species

Fig. 2. Kinetic parameters for the uptake of nitrate and ammonium by legumes and cereals. The Km (mol M- 3 ) and Vmax (mol kg- 1 hr 1) were calculated from Lineweaver-Burk plots. The k (mol kg 1 hr 1 M 1 ) is the slope of the linear line between the influx rate at 2.5 mM and the simulated rate at 1.0 mM. Ce = Pigeonpea (Cajanus cajan L.), Ca =Chickpea (Cicer arietinum L.), Ah = Groundnut (Arachis hypogaea L.), Sb =Sorghum (Sorghum bicolor L.), Pg =Millet (Pennisetum glaucum L.) and Zm =Maize (Zea mays L.)

Table 1. Percentage distribution of nitrogen translocated to the shoot to nitrogen absorbed by three legumes and three cereals ( 15N in shoot/ 15 N in whole plants x 100)

Species N sources

Nitrate Ammonium

Pigeon pea 51 19 Chickpea 71 24 Ground nut 37 19 Sorghum 20 7.1 Millet 23 12 Maize 26 6.3

Mean 38 15 SE 3.4 1.3 CV% 20 19 LSD 10 3.7

Each value is an average of five treatments with different N concentrations. Data from the highest concentration (2.5 mM) were omitted as the influx and translocation of nitrogen may be driven by a different mechanism at this concentration (see text).

data obtained in the present study are also not compatible with a single saturable component (Fig. 1), again suggesting that the influx iso­therms for both nitrate and ammonium should consist of at least two components. The transi­tion point may be somewhere around 1.0 mM in both cases. Rao and Rains (1976) reported that in barley roots, the uptake rate of nitrate was accelerated as the external concentration ex­ceeded 0.5 mM. Approximately the same con­centration for the transition point has been reported for potassium with corn (Kochian and Lucas, 1982) and ryegrass (Glass and Dunlop, 1978), and for phosphate with corn (Nandi and Pant, 1984). It should also be noted that this transition in isotherms exists in all six plant species used in this study.

There has been a long debate on the charac­terization of the second component. A mul­tiphasic model has been proposed for potassium (Nissen, 1989) and phosphate (Singh and Pant, 1982), which can incorporate the present theory

Ammonium and nitrate uptake in legumes and cereals 211

on ion transport with multistate ion channels. In contrast, Kochian and Lucas (1982) and Vale et a!. (1987) indicated that the second phase of potassium uptake could be described with a linear term, suggesting the existence of two distinct uptake mechanisms. Although the pres­ent study does not provide confirmative evidence for the isotherm pattern of the second com­ponent of nitrate or ammonium uptake, the assumption of a linear component for concen­trations > 1 mM to calculate the k is supported by recent results with barley (Siddiqi et a!., 1990) and maize (Pace and McClure, 1986).

Significant differences were found between legumes and cereals in Km, Vmax and k for ammonium (Fig. 2). Since ammonium is not a predominant form of nitrogen under the upland conditions where the plants used in this study are commonly cultivated, those differences may not have a practical importance in the field. For nitrate, the only clear difference between legumes and cereals was observed in k (Fig. 2). Although k is a tentative index for uptake kinetics at concentrations > 1 mM, the results imply that at the high concentration range nitrate absorption in legumes is at a relative advantage compared to that in cereals. Following the appli­cation of ammonical fertilizer, nitrate concen­tration in the soil solution was maintained above 1 mM for a few weeks (Ito et a!., 1992).

The legumes were also found to be more efficient than cereals in translocating the ab­sorbed nitrogen from root to shoot (Table 1 ). Especially the translocation rate of nitrate in chickpea was strikingly higher than in other species. This may be partly associated with the highest transpiration rate (data not presented) in this species. Another factor that regulates nitrate translocation may be the distribution of nitrate reductase (NR) within the plant. Since the trans­location of ammonium is much slower than that of nitrate (Table 1 ), differences in translocation between legumes and cereals may be related to the NR activity in the roots. Root tips of Zea mays had a high level of in vitro NR activity (Oaks eta!., 1979) and, as a consequence, a high correlation was found between numbers of later­al roots (which should correlate with number of root tips) and nitrate reduction (Pan et a!., 1985). In Cicer arientinum, however, NR activity

was higher in shoots than in roots (Wasnik et a!., 1988). The rapid translocation in legumes may be due to the lower conversion of nitrate to ammonium and amino acids in legume roots than in cereal roots.

The present study confirms that influx iso­therms for both nitrate and ammonium have a transition at approximately 1 mM, if the first component operating at low concentrations is assumed to be saturable according to Michaelis­Menten kinetics. Significant differences in kinetic properties were found between legumes and cereals, implying that the nitrogen utilization may be improved by intercropping a proper combination of crops. Since the nutrient uptake may be influenced not only by kinetic properties but also by root morphological and metabolic traits (Robinson and Rorison, 1983), further comparative studies should be conducted to correlate these interspecific differences with root morphology and activities.

Acknowledgements

We extend our sincere thanks to Dr C Johansen and Dr J V D K Kumar Rao for their valuable and critical evaluation of this paper. Our thanks also go to Mrs Y Gayathri Devi for the help of graphic presentation.

References

Epstein E 1972 Active ion transport in cells and tissues. In Mineral Nutrition of Plants: Principles and Perspectives. pp 103-150. John Wiley and Sons, New York.

Glass A D M and Dunlop J 1978 The influence of potassium content on the kinetics of potassium influx into excised ryegrass and barley roots. Planta 141, 117-119.

Ito 0, Tobita S, Matsunaga R, Rao T P and Johansen C 1992 Inorganic nitrogen in soil water collected from soil under intercrop components sorghum and pigeonpea- A pot experiment. In Proceedings of the International Symposium on Nutrient Management for Sustained Prod­uctivity. Vol II. pp 16-18. Dept. of Soils, Punjab Agricul­tural University.

Johnson C M, Stout P R, Brayer T C and Carlton A B 1957 Comparative chlorine requirements of different species. Plant and Soil 8, 337-353.

Kochian LV and Lucas W J 1982 Potassium transport in corn

212 Ammonium and nitrate uptake in legumes and cereals

roots. 1. Resolution of kinetics into a saturable and linear component. Plant Physiol. 70, 1723-1731.

Nandi S K and Pant R C 1984 Temperature effect on kinetics of phosphate uptake by excised corn (Zea mays L. var Ganga 5) roots. Ind. J. Exp. Biol. 22, 564-567.

Nissen P 1989 Multiphasic uptake of potassium by corn roots. No linear component. Plant Physiol. 89, 231-237.

Oaks A, Aslam M and Boesel I L 1979 Intluence of amino acids as regulators of nitrate reductase in corn roots. Plant Physiol. 59, 391-394.

Pace G M and McClure P R 1986 Comparison of nitrate uptake parameters across maize inbred lines. J. Plant Nutr. 9, 1095-1111.

Pan W L, Jackson W A and Moll R H 1985 Nitrate uptake and partitioning by corn root systems. Differential effects of ammonium among genotypes and stages of root de­velopment. Plant Physiol. 77, 560-566.

Rao K P and Rains W 1976 Nitrate absorption by barley I. Kinetics and energetics. Plant Physiol. 57, 55-58.

Robinson D and Rorison I H 1983 Relationships between root morphology and nitrogen availability in a recent theoretical model describing nitrogen uptake from soil. Plant Cell Environ. 6, 641-647.

Siddiqi M Y, Glass A D M, Ruth T J and Rufty T W 1990 Studies of the uptake of nitrate in barley. I. Kinetics of 13NO; intlux. Plant Physiol. 93, 1426-1432.

Singh S P and Pant R C 1982 Kinetics of phosphate uptake in excised corn (Zea mays L. var Ganga 5) roots. Ind. J. Exp. Biol. 20, 324-326.

Vale F R, Jackson W A and Yolk R 1987 Potassium influx into maize root systems: Influence of root potassium concentration and ambient ammonium. Plant Physiol. 84, 1416-1420.

Van de Dijk S J, Lanting L, Lambers H, Posthumus F, Stulen I and Hofstra R 1982 Kinetics of nitrate uptake by different species from nutrient-rich and nutrient-poor habitats as affected by the nutrient supply. Physiol. Plant. 55, 103-110.

Wasnik K G, Varade P B and Bagga A K 1988 Nitrate reductase activity in chickpea (Cicer arietinum L.) leaves, roots and nodules in relation to moisture stress. Ind. J. Plant Physiol. 31, 324-327.

Yoneyama T, Arima Y and Kumazawa K 1975 Sample preparation from dilute ammonium solution for emission spectrographic analysis of heavy nitrogen. J. Sci. Soil Manure Jap. 46, 146-147.

Reprinted from Plant and Soi/154: 67-72, 1993.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 213-217. 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-137

Influence of root temperature on potassium nutrition of tomato plant

P. CORNILLON and A. FELLAH! lnstitut National de la Recherche Agronomique, Station d'Agronomie, Domaine St Paul, F-84143 Montfavet, France

Key words: Lycopersicon esculentum Mill., potassium, Q10, root surface, root temperature, tomato

Abstract

The interaction between root temperature and potassium concentration in nutrient solution has been studied in hydroponic culture on young tomato plant (Lycopersicon esculentum Mill.). These two factors affect plant growth but the potassium absorption only varies according to concentration in nutrient solution. The plants grown for 2 weeks in a nutrient solution maintained at 11 oc with 0.5 mM L - 1 of potassium synthesize 6.0 g of total dry matter with 0.58 g in roots corresponding to 952 cm 2 area; while at 3.0 mM L -t, the plant gives 6.1 g of total dry matter with 0.65 g in the roots corresponding to 1028 cm 2 . On the other hand, at 21 oc, we can see the following synthesis: at 0.5 mM of K ion, 6.43 total dry matter with 0.90 in the roots and a 1412 cm 2 area and respectively 7.03, 0.78 and 2096 cm 2 with 3.0 mM L - 1 of potassium. These results point out that the root surface are not limiting factors for potassium absorption by young tomato plants during a short period of treatment.

Introduction

Root temperature influences the uptake and metabolism of water and mineral elements. Among the mineral elements essential to plant growth, potassium is a major element, represent­ing 4 to 8% of the dry weight in plant tissue (Epstein, 1972).

Temperature control is currently a constant source of concern to farmers as they strive for energy savings on crops under shelter (La Malfa, 1990).

Our observations concern the role of substrate temperature and potassium concentration in the nutrient solution on tomato growth. Potassium nutrition is studied over time and cation content of the plant is indicated at the end of the trial.

Materials and methods

Choice of root temperatures and potassium concentrations

The experimental design is a split-plot device with the temperature as a mean treatment. The

temperatures selected, 10 oc and 20 oc, enable us to obtain slowed growth at 10 oc and normal growth at 20 oc. The temperature remains con­stant at root level due to the use of hydroponic culture and aeration using bubbles of compres­sed air.

Potassium nutrition was differentiated by the potassium content levels, 0.5 and a 3.0 mM potassium per liter, and by the renewal rate for the nutrient solution after 5, 4 and 3 days to maintain between 0.5 and 1.0 L of nutrient solution at the root level of each plant.

The 0.5 mM K ion nutrient solution contains 2.0 mM of Ca(N0 3 ) 2 , 0.5 mM of KH 2P04 ,

1 mM of NH4N0 3 and 0.5 mM of MgS0 4 • The 3.0 mM K + solution is made up of 1.5 mM of Ca(N0 3 ) 2 , 3.0 mM of KN0 3 , 0.5 mM of NH4H 2P0 4 and 0.5 mM of MgS0 4 • The neces­sary trace elements essential to plant growth are added to these solutions.

Crop conditions

Sowing occured on September 5, the plants were transplanted on September 25 at the two leaf

214 Cornillon and Fellahi

stage, one tomato by plastic pot containing 1000 mL of nutrient solution. The trial was conducted from October 9 to 22. Nutrient solu­tion samples were taken during this period and plant samples were taken at the end of the trial. The various organs of each plant were quickly separated: root, stem+ petiole and laminae. Each plant fraction was washed twice in de­mineralized water under the tap, dried and weighed.

Analyses

Cations were analyzed by atomic absorption spectrometry of the nutrient solutions and the plant matter previously put into solution, follow­ing the Comite Inter Institut method (CII, 1973).

Root surface was measured by image process­ing (Cuny, Roudot, 1991).

The variance analysis and the Newman-Keuls test were applied to the analytic data resulting from the 4 treatments and the 2 replications using the STAT-ITCF program.

Results

Growth

Table 1 presents the results of the influence of root temperature and potassium concentration on the accumulation of dry matter in the plant. The variance analysis shows an interaction effect between these 2 treatments on the ration shoot/ root.

Temperature acts on the root mass with a positive action of the high temperature which is significant at the 5% level; while potassium

Table 1. Influence of root temperature (10 and 20 oc) and potassium concentration (0.5 and 3.0 mM L 1) on dry matter synthesis by tomato plant (g/ plant)

10 oc 20 oc

0.5 3.0 0.5 3.0

Root 0.58 0.61 0.90 0.78 Stem + petiole 2.67 2.78 2.28 2.83 Laminae 2.75 2.71 3.24 3.43 Whole plant 6.00 6.10 6.43 7.03 Ratio shoot/ root 9.27 a 9.01 a 6.12b 8.01 a

solely influences the conductive system: stem and petiole.

The Q10 value varies not only as a function of potassium nutrition, but also- and especially­depending on the organ under study.

Q10 0.5mMKion 3.0mMKion

1.57 Root 1.84 Stem+ petiole 0.87

Laminae 1.24 Plant 1.13

1.08

1.45 1.29

The influence of temperature on the growth of conductive organs is negligible; maximum in­fluence appears to be on roots with a variation opposite to the other organs. On the whole plant, the QlO values are very similar.

The root surface measured by image analysis corresponds to strong influence by temperature and potassium.

11 oc 0.5 mM Kion 952cm 2

11 oc 3.0mM Kion 1028cm 2

21 oc 0.5mM Kion 1412 cm2

21 oc 3.0mMKion 2096 cm 2

But during the trial, even at low potassium concentrations, no symptoms of potassium de­ficiency were ever apparent.

Potassium uptake

Figure 1 shows the evolution of potassium con­sumption in the tomato plants as a function of

1<0- mg.pla"l/•-----_.o~-·----------~1~~~ 3,0mM

~-----0~~~~~--lQO. ~~~

" " " " " 20

" " " " "~•~. ====w:J.: _____ IlJ 20°C IO"C 0.5 mM

12 day

Fig. 1. Influence of root temperature and potassium concen­tration on potassium uptake by tomato plant (mg/plant/ period).

the treatment. It is seen that at 0.5 mM, the concentration is the principal limiting factor, whereas at 3.0 mM, temperature limits uptake during the first 9 days of the trial and then it becomes identical at the 2 temperatures. With our experimental design we have also limited the quantity of K + at the root level to obtain a better differenciation of potassium nutrition because you can give large quantity of an element with a low concentration.

The root surface has no influence since the plants absorb the same amount of potassium when the root system surface increases from 50 to 100 percent. Variations in the potassium con­tent of the roots do not seem to play a decisive role in its uptake (Table 2).

When the percentage of potassium is ex­pressed on the dry matter basis in the various organs analyzed, it is seen that it is always a function of the K ion concentration in the nutrient solution, whereas temperature solely affects concentration in the roots (Table 3). It

K nutrition of tomato 215

can be noted that there is no correlation between growth and the percentage of potassium in the analyzed organs; proper growth is obtained with low K ion content in the roots and the laminae.

The transport of potassium between the root and the shoot is not affected by the root zone temperature because the quantities present in the shoot are very similar: 256 mg plant -I at 10 ac and 254 mg plant at 20 oc.

The efficiency of potassium nutrition (mg of dry weight synthesized per mg of K ion ab­sorbed) is very high when the nutrient solution contains 0.5 mM of potassium, whatever the root temperature is (Table 4). Efficiency varies de­pending on the organ under consideration, how­ever for the stems and the petioles, its value is comparable, at both temperatures, for a given K + concentration.

Calcium and magnesium nutrition

Table 2 shows the percentages of calcium and

Table 2. Influence of root temperature (10 and 20 oc) and potassium concentration (0.5 and 3.0 mM L 1 ) on cation concentration of plant fraction of tomato (% of dry wt.)

Plant fraction Element 10°C 20°C

0.5 3.0 0.5 3.0

Root K 2.83 5.72 1.72 4.25 Ca 0.75 0.66 0.71 0.70 Mg 0.44 0.44 0.83 0.53

Stem+ K 3.66 8.88 3.82 9.05 petiole Ca 1.59 1.68 2.84 1.82

Mg 0.26 0.35 0.66 0.46

Laminae K 1.91 4.41 1.32 3.67 Ca 2.51 2.99 3.15 3.00 Mg 0.38 0.49 0.42 0.55

Table 3. Influence of root temperature and potassium concentration on cations concentration of tomato plant fractions (% of dry wt.) (Treatment rank based on the Newman Keuls test at 5% confidence interval. The results with the same letter are not different)

Plant fraction Element K+(mML- 1 ) Room temperature

0.5 3.0 10 'C 20°C

Root K 2.28 b 4.97 a 4.28 a 2.97 b Stem+ K 3.74 b 8.97 a

petiole Ca 2.21 a 1.75 b 1.63 b 2.33 a Laminae K 1.67 b 4.03 a

Mg 0.40 a 0.52 a

216 Cornillon and Fellahi

magnesium in the various plant organs as a function of the treatments and the Table 3 shows the significance at the 5% level.

Temperature causes an increase in the calcium and magnesium content in the laminae and in the conductive organs, while at the root level, only magnesium is involved. However, the potassium treatment has no clear influence on calcium and magnesium content in the various organs.

When considering the quantity of calcium and magnesium in the different parts of the plant, it is only the quantities present in the stem and the petiole which vary under the influence of the temperature and of the potassium. The quan­tities below are expressed in mg by organ.

Temperature lO"C 20 "C

K' concentration O.SmM 3.0mM O.SmM 3.0mM

42.4 46.6

6.92 9.71

64.8

15.0

51.5

13.1

At 10 oc, the quantity of Ca 2 + and Mg2 + increases which the concentration of K +, while at 20 oc, we observe the reverse phenomenon. However, the quantity in the conductive tissues is always higher at 20 oc than at 10 oc.

Discussion

Many authors have observed a reduction in potassium, calcium and magnesium content in plant tissue when growth occurs at a low tem­perature, compared to plants grown at normal temperatures (Kafkafi, 1990; Tindall et al., 1990). According to Bingham and Cumbus (1991), these observations depend on the refer­ence used: dry weight or fresh weight, especially for potassium.

In fact, our results, with the K+ content expressed on dry matter weight, show that nor­mal growth is obtained with lower potassium levels in the roots and the laminae, whereas the level is slightly higher in the stems and petioles. Plant's potassium nutrition is shown more con­clusively by checking potassium content in the conductive tissue: stem and petiole.

The absorption of potassium is independent of root temperature during the 12 days of observa­tion and this result supports the observations on

barley of Siddiqi et al. (1984) who has studied the same temperature during short periods. Engels et al. (1992) have noted a marqued influence of root zone temperature on nitrogen, potassium and calcium uptake by maize grown in water culture.

Unlike the results obtained by Bingham and Cumbus (1991), we have not observed any stable concentration of K + in the tissues at 10 ac and at 20 oc when potassium nutrition is near optimal. Indeed, the difference is felt when expressing the results as a function of the water content in the tissues since at low temperatures, there is greater dry weight due to the accummulation of soluble solids and starch in the cells and to the thick­ening of the pecto-cellulosic walls ( Cornillon, 1974). At 12 oc, there are accumulations of total dry matter in the roots, especially carbohydrates and also organic nitrogen.

Plants' response to variations in root tempera­ture depends on the species.

In our experiment, the transport of potassium between root and shoot is not affected by root zone temperature unlike Engels et al. (1992) findings. These authors have observed an in­creased translocation of K + , N and Ca + + from root to shoot after 3, 5 and 10 days treatment.

Efficient use of potassium occurs when the substrate is very poor in the element at the two temperatures, however such efficiency decreases when the supply of potassium is higher with a better plant growth (Table 4).

At low root temperature, the flow of sap within the plant undergoes changes in speed, with, in particular, a decrease at the collar level and in the roots. These variations can cause an increase in its needs for K ion to insure solution transfer and to meet its role of osmoticum in the

Table 4. Indices of efficiency of potassium (mg DM/mg K+ absorbed) for plants at 2 potassium concentration in solution (Treatment rank based on the Newman Keuls test at 5% confidence interval)

K+ (mM L - 1 ) K+ (mM L -I)

0.5 3.0

Root 47.13 a 20.58 b Stem + petiole 27.40 a 11.15 b Laminae 65.60 a 25.08 b Plant 40.88 a 16.13 b

cells. Our results highlight the correlation be­tween potassium nutrition and synthesis of con­ductive tissue.

In conclusion, it appears that tomato growth is not greatly affected even if there are great variations in the concentration of potassium in the substrate, when root temperature is favor­able. This normal growth corresponds to highly varied levels of potassium in the plant organs. Root surface is not a limiting factor in potassium nutrition since the same quantity of potassium is absorbed by plants having very different root systems. The lowest growth, noted at 10 oc, cannot be linked to potassium deficiency since tissue content corresponds to the range of stan­dard values.

References

Bingham I J and Cumbus I P 1991 Influence of root temperature on the potassium requirement of young tomato plants. Plant and Soil 133, 227-237.

K nutrition of tomato 217

C I I 1972 Methodes de reference pour Ia determination des elements mineraux dans les vegetaux. Oleagineux 28, 87-92.

Cornillon P 1977 Influence de Ia temperature du substrat sur Ia composition des racines et des limbes de tomate. Implications concernant !'absorption hydrique. Ann. Agron. 28, 277-289.

Cuny F and Roudot A C 1991 Germination et croissance pollinique in vitro du pollen de melon ( Cucumis me/a L.) apres irradiation gamma. En vir. Exp. Bot. 31, 277-283.

Engels C, Munkle L and Marschner H 1992 Effect of root zone temperature and shoot demand on uptake and xylem transport of macronutrients in maize (Zea mays L.) J. Exp. Bot. 43, 537-547.

Epstein E 1972 Mineral Nutrition of Plants: Principles and Perspectives. Wiley, New York, 412 p.

Kafkafi V 1990 Root temperature, concentration and the ratio NO; /NH; effect on plant development. J. Plant Nutr. 13, 1291-1306.

La Malfa G 1990 Melanzana. In Orticultura. Eds. V V Bianco and F Pimpini Coordinatore. pp 793-811. Patron Editore Bologna.

Siddiqi MY. Memon A Rand Glass ADM 1984 Regulation of K + influx in barley: effect of low temperature. Plant Physiol. 74, 730-734.

Tindall J A, Mills H A and Radcliffe D E 1990 The effect of root zone temperature on nutrient uptake of tomato. J. Plant Nutr. 13, 939-956.

M.A. C. Fragoso and M.L. van Beusichem (eds.) Optimization ofplant nutrition 219-226, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-042

Growth analysis of soil-grown spinach plants at different N-regimes

E. SMOLDERS, J. BUYSSE and R. MERCKX Laboratory of Soil Fertility and Soil Biology, Katholieke Universiteit Leuven, Kardinaal Mercierlaan 92, B-3001 Heverlee, Belgium

Key words: exponential nutrient addition, growth model, nitrogen productivity, shoot-root balance, soil-plant interaction, spinach

Abstract

Spinach plants were grown in pots under controlled conditions in three different soils (a loamy sand, a silt loam at low minerai-N level and a silt loam at the double mineral-N level). The nitrogen uptake pattern varied considerably between the three soil types and was used to validate an equation between the relative growth rate and nitrogen content. This equation is based on the growth response of spinach plants grown hydroponically at equal environmental conditions either at optimum nitrogen supply (complete nutrient solution) or with a relative nitrate addition rate of 0.30 day- 1 , 0.225 day- 1 or 0.15 day -I effecting an exponential increase in nitrogen uptake. Growth in potted soil was slightly overestimated. Part of this bias was explained by the lower shoot weight ratio observed for the soil grown plants. This was demonstrated by the improvement in growth predictions when using net assimilation rate rather than relative growth rate as the driving variable in the model.

Introduction

The soil to plant transfer of nutrients is the result from interactions between plant and soil pro­cesses. Nitrate uptake from soil is rather con­trolled by the plant processes owing to the high mobility of the ion in soils and the strong internal coupling between growth rate and ni­trate demand rate (Clarkson, 1986). Further­more, growth rate as well as biomass partitioning between above- and below-ground plant parts change with nitrogen supply.

In order to unravel the plant processes, the effects of nitrate concentration at the root sur­face on uptake rate and growth response have been major research items in plant nutrition (e.g. Bhat et a!., 1979). The role of nitrate concen­tration on uptake rate has however been chal­lenged recently (Clement et a!., 1978; De Wil­ligen and Van Noordwijk, 1987) and the difficul­ties pertinent to the use of nitrate concentrations to grow plants in steady state with their nutrition

resulted in a new growth technique (Ingestad and Lund, 1979) and the concept of nitrogen addition rate as the variable to which the plant responds (Ingestad, 1982).

Recently we adopted the technique of ex­ponential nutrient addition to investigate the influence of nitrogen supply on the growth and shoot: root partitioning of spinach plants. The general (linear) dependency of the relative growth rate (RGR) on the nitrogen level in the plant (e.g. Ingestad, 1981) was however not confirmed. This was due to the non-exponential growth pattern of spinach plants necessitating the introduction of a new growth equation ac­counting for this ontogenetic effect on the RGR/ nitrogen content relationship (Smolders and Merckx, 1992).

In this paper we examine to what extent such an equation can be used to predict plant growth in potted soils. Plant growth was followed in a growth chamber under other environmental con­ditions than at which the previous growth equa-

220 Smolders et al.

tion was derived. Therefore, a hydroponic ex­periment was repeated at these conditions to calibrate the growth equation. This equation was used to predict growth of spinach plants grown at equal environmental conditions in three different potted soils (a loamy sand, a silt loam at low mineral-N level and a silt loam at the double mineral-N level).

Materials and methods

Plant growth

General Spinach plants (Spinacia oleracea L., cv. Subito) were used as test plants in both the nutrient solution experiment and the potted soil experi­ment. The experiments were carried out in a growth cabinet (Weiss, 18'SP/ + 5Ju-Pa) with a 12h/12h day/night cycle. Light was provided with 24 SON IT lamps (Philips, 250W) and 24 HQI/T lamps (Philips, 250W). Photosynthetic photon flux density at canopy height was 575 ± 65 f.Lmol m- 2 s-t. During the day cycle, the air temperature was 18.5 ± O.S"C and the relative humidity was 75 ± 5%; at night these values were 14.5 ± O.SOC and 75 ± 5% respectively.

Nutrient solution experiment The lay out of this experiment was as described elsewhere (Smolders and Merckx, 1992) aside from following modifications: after a pregrowth period which lasted until 13 days after sowing (DAS) 60 plants were harvested and 180 plants were divided into 4 groups: 36 plants were grown at optimum N-supply and 3 groups of 48 plants at a relative nitrogen addition rate (RAR) of 0.30 day-\ 0.225 day- 1 or 0.15 day- 1 . Nitrogen addition started after 1 ( RAR = 0.30 day -t), 2 (RAR=0.225day- 1) or 3 (RAR=0.15 day- 1)

days of nitrogen starvation. The estimated initial nitrogen content was 67.5 f.Lmol per seedling which corresponded with the result of chemical analysis (70.3 f.Lmol per seedling). Twelve plants were harvested at 18, 22 and 26 DAS in the optimum treatment, at 19, 23, 27 and 31 DAS in the RAR = 0.30 day -t treatment, at 22, 27, 32 and 38 DAS in the RAR = 0.225 day- 1 treat-

ment and at 26, 34, 42 and 50 DAS in the RAR=0.15 day- 1 treatment. The relative N­uptake (in % of N-addcd) was almost 100% except for the last harvest of the RAR = 0.30 day -t treatment at which 20.6% of the cumula­tive N-dose was left in the nutrient solution as NO;.

Potted soil experiment The top layer (0-30 em) of two farmer's fields of contrasting texture were sampled and homogen­ised. The first was a loamy sand which was collected in March 1992 at the 'Proefstation voor de Groententeelt' in Sint-Katelijne-Waver (Bel­gium). The second was a silt loam collected at the same date in Bertem (Belgium).

Both soils were air dried for 5 days and sieved (2 mm). The soils were moistened to a final moisture content (g(g dry soil)- 1) of 20.0% (silt loam) or 16.5% (loamy sand) corresponding to a pF of 2.6 for both soils. Mineral-N content was highest in the loamy sand (1.83 meq NO; per 1.1 kg moist soil, the quantity used in one pot; NH; -N, extracted with 1 N KC1, was only about 3% of the mineral-N pool in the soil). NO; was added to the silt loam to obtain an equal final amount of mineral-N per pot (1.83 meq per 1.1 kg moist soil, N 1 treatment) or the double amount (3.66 meq per 1.1 kg moist soil, N 2 treatment). NO; (3.31 meq) was added to 1.1 kg moist soil for silt loam N 2. Other nutrients (K, Ca, Mg, P, S, Cl, Fe and trace elements) were added in the same molar proportion to the nitrate gift as in the complete nutrient solution used in the nutrient solution experiment (see e.g. Smolders and Merckx, 1992 for composition details). NO; (1.48meq) was added to 1.1 kg moist soil for silt loam N 1 and an equal amount of the other nutrients were added as in the N 2 treatment; the lower NO; input was compen­sated by the corresponding chloride salts. Other nutrients were added to the loamy sand in the same molar proportion to the nitrate amount present in the soil as in the complete nutrient solution in which all nitrate salts were replaced by chloride salts. All mineral salts were added as concentrated stock solutions. Fertilisation and moistening was carried out per 20 kg soil quan­tities which were mixed for about 10 min with a hand mixer.

Sixty 990 cm3 pots were filled per treatment. The pots were covered and incubated for 3 days at 18°C. Soil pH(CaC12 , 0.01M) after moistening and nutrient amendment was 5.2 (loamy sand) and 6.7 (silt loam). Seeds were germinated for 4 days on moistened paper and transferred to the pots (1/pot). An one em cover of polythene beads was layered on top of the soil surface to reduce evaporation. Soil moisture content was adjusted daily, alternating deionized water or nitrate-free nutrient solution additions. Plants were harvested at 10, 14, 19 and 24 days after transplanting (DAT). The shoot fresh weight was recorded and roots were recovered from the soil by washing. Tissue dry weight was recorded following three days drying at 70°C. Plant sam­ples were ground pending analyses.

Analyses

Tissue nitrate and total nitrogen content were analysed as described elsewhere (Smolders and Merckx, 1992). Soil solution was collected from a root-free soil subsample with an immiscible displacement technique using chloroform (Mubarak and Olsen, 1976). Nutrient solutions and soil solutions were analysed with ion chro­matography (Dionex, QIC analyser with AS-4A column).

Results and discussion

Nutrient solution experiment

The RGR of the plants grown in complete nutrient solution decreased during development while the nitrogen level in the plant was almost stable. At suboptimum exponential nitrogen addition, the RGR initially equalled the RAR but declined at higher growth stages while the total nitrogen concentration increased (data not shown). Similar results were obtained earlier (Smolders and Merckx, 1992) where we demon­strated that the equation

(1)

described growth adequately. [NLr represents

Growth analysis of soil grown spinach plants 221

the organic nitrogen level in the plant, W repre­sents the plant dry weight and k 1 , k 2 and k 3 are positive parameters. Since growth conditions were different here the parameters of Equation 1 could be different from what was obtained ear­lier (Smolders and Merckx, 1992). In order to relate the growth rate to the nitrogen taken up, Equation 1 was furthermore reformulated to describe the dependency of the RGR on the total nitrogen level (organic-N + nitrate-N) in the plant. At ample N-provision, spinach plants accumulate nitrate substantially. Nitrate is not 'growth active' as such and the RGR therefore levels off at the higher nitrogen concentrations in the plant. This can be accounted for as

1 dW RGR= Wdt

k 1([N]-k 2 ) (2)

in which [N] represents the total nitrogen level in the plant and k 1 , k 2 , k 3 and k 3 are all positive parameters. Equation 2 was fitted in its inte­grated form to the ln(W) data using the observed nitrate uptake curves of the four different nitro­gen treatments. The least squares were found as described earlier (Smolders and Merckx, 1992) and the parameters yielding the best fit (R2 = 0.985) were k 1 = 2.259 g day -J, k 2 = 1.12 mmol g- 1 , k3 =3.836g and k 4 =1.25mmol g- 1 • The resulting growth curves and the growth data are given in Figure 1. The growth equation is sum­marized graphically in Figure 2. At constant N-level in the plant, RGR is halved at a plant dry weight of 3.836g (=k3 ). At constant plant dry weight, RGR initially increases linearly with the nitrogen level in the plant tissue but saturates at higher levels. The lowest levels observed in the nutrient solution experiment were 1.6 mmol g -l. Hence, RGR lines below that level are extrapolated from the model.

Potted soil experiment

The nitrogen uptake pattern was different in the three different soils (Fig. 3). The soil nitrate content was exhausted by the last harvest in all cases. Nitrogen uptake continued up to the last harvest in the loamy sand, whereas it had ceased

D.15 day-1

30 50 Time (DAS)

Fig. 1. Growth curves (natural log scale) of the spinach plants grown at optimum N-nutrition, or at a suboptimum nitrogen

relative addition rate of 0.30 day- 1, 0.225 day- 1 or 0.15 day-'. Symbols are observed values and full lines are predicted growth

curves calculated using equation 2 integrated with respect to time.

RGR=relative growth rate, 1/day TNCP= total nitrogen concentration 1n plant, mmol/ g DWP=dry weight of the plant, g

Fig. 2. Plant dry weight RGR as a function of plant nitrogen concentration and plant dry weight calculated using Equation 2 which was fitted to the observed growth data of the nutrient solution experiment.

Growth analysis of soil grown spinach plants 223

Loamy sand Silt loam Silt loam N 1 N 2

3.5 3.5

0 3 3 0..

""-... c;2.5 2.5 E

' E 2 't 2 \.._

0 \ '

~ 1.5 ' 1.5 0

\ \ 0.. ""-... \

\

0 ' ' E \,k, E0.5 0.5

0 ------l 0 10 14 18 22 10 14 18 22 10 1 4 18 22

Time (OAT) Time (OAT) Time (OAT)

Nitrogen in plant (mmol/plont)

Nitrate in soil solution (mmol/pot)

Fig. 3. Nitrogen uptake and nitrate in the soil solution (note: one plant/pot) during growth (in days after seedling transfer into the soil, DAT) in three different potted soils. Points represent the observations, vertical bars their standard deviation and full lines are experimental curves fitted on the observations.

between the second and the third harvest in the silt loam N 1. Nitrogen uptake rate was highest in the silt loam N 2 but its value decreased sharply between the third and the last harvest. Net mineralisation was obvious in the loamy sand but insignificant in the silt loam soils (at any time, the contribution of NH; to the nitrogen uptake can be neglected).

The data of nitrogen uptake are depicted in Figure 3. Curves (Richards function) were fitted to these data and were used to predict growth combined with the equation relating nitrogen content with growth rate (the integration of Equation 2). Initial values of plant weight and nitrogen content for the three growth curves were those observed at the first harvest. Plant weight was most often underestimated (figures not shown). Several factors can be put forward to explain differences in productivity between soil and hydroponically raised plants. First, the availability of water and nutrients is always lower to soil-grown plants irrespective the precautions as taken in this experiment. Secondly, soil grown

plants have a higher carbon cost per unit root weight as hydroponically grown plants. This is indicated by the visual observation that root decay was already initiated in the potted soil experiment. Furthermore, we observed a higher root weight ratio (g root dry weight (g plant dry weight)····\) for the soil grown plants which again leads to a lower overall productivity. This latter observation perhaps is the main source of growth rate differences between our two experiments. To account for this, we preferred to analyse the growth in both experiments by a net assimilation rate (NAR) model (cf. Hirose et al., 1988). In a NAR model, growth rate is explained by shoot weight (Ws) and shoot nitrogen concentration contrasting Equation 2 in which growth rate is explained by plant weight and plant nitrogen concentration. It is furthermore essential for a complete growth analysis with a NAR model to quantify the distribution of biomass among shoot and root, the distribution of nitrogen within the plant and the way how these distributions are affected by the nitrogen supply. The rationale

224 Smolders eta!.

behind our preference for a NAR model is that small differences in nutrient and/ or water availa­bility to both soil- and hydroponically grown plants probably induce pronounced differences in biomass allocation without significantly affect-· ing theNAR.

We described biomass partitioning by a parti­tioning function dWs/dt/(dWr/dt) (i.e. the ratio between shoot and root growth rate). Shoot and root growth rate was calculated after fitting empirical growth curves to shoot and root weight data (polynomials for the first experiment, Rich­ards curves for the soil experiment). The parti­tioning function was related to the total nitrogen concentration in the plant (Fig. 4) and, in the absence of a partitioning model validated for spinach plants, a linear regression was made. Significantly more biomass was allocated to the shoots of plants grown in nutrient solution at high nitrogen supply compared to the soil grown plants whereas nitrogen stressed plants exhibited equal partitioning functions.

A NAR equation can be postulated along the same lines as follows for the selection of Equa­tion 2.

1 dW k; ([N]- k~)

NAR = W, dt = (([N]- k~) + k~)(k~ + ~) (3)

where k;, k~, k~ and k~ are positive parameters and [N] represents the total nitrogen concen­tration in the plant. In this model, the net assimilation rate of a shoot is explained by the nitrogen level in the plant and by the shoot dry weight. This contrasts the NAR model of Hirose eta!. (1988) in which the net assimilation rate is related to the nitrogen level only. The shoot dry weight is included as a regression variable in our NAR model to account for the fact that the NAR decreases at increasing growth stages.

Shoot nitrogen concentration almost equalled plant nitrogen concentration at any growth stage or nitrogen level in our experiments. Hence, we

~ 4.5 ,-----------------------------------------------, "--.. Hydroponics 0' 4 '-

~ 3.5 "--.. -3 3 "0 '--/ 2.5 c

15 2 u c 2 1.5 0' c c 0 0.5

Loamy sand

Silt loam

..

1.4 1.8 2.2 2.6 TNCP (mmol/g)

Hydroponics

<>Optimal N-·supply

o RAR 0.30 day-1

+ RAR = 0.225 day-1

o RAR = 0.1 5 day-1

0

0

3 3.4 3.8

Soil

• Loamy sand

.&. Silt loam (both N 1 and N 2)

Fig. 4. The biomass partitioning function P = dWsldtl(dW/dt) as a function of the total nitrogen concentration in the plant. W, and W, represent shoot and root dry weight respectively. Points represent the calculated values at the time at which plants were harvested, lines are linear regressions. p = -0.997 + 1.3397*TNCP, R2 = 0.926 for hydroponics, p = 0.195 + 0.6057*TNCP, R2 = 0.997, for loamy sand; p = 0.941 + 0.3036*TNCP, R2 = 0.805 for silt loam.

can use average plant nitrogen concentration in Equation 3 instead of shoot nitrogen concen­tration. Using the experimental line relating the partitioning function with the nitrogen level in the plant (Fig. 4), the integration of Equation 3 was fitted to the ln(W) data of the nutrient solution experiment. The least squares were found as described above and the parameters yielding the best fit (R2 = 0.984) were k; =

1.691 g day-\ k~ = 2.531 g, k~ = 1.07 mmol g - 1

and k~ = 0.69 mmol g - 1 . This equation was com­bined with the experimental nitrogen uptake curves and the experimental lines relating biomass partitioning with the N-level in the plant (one regression per soil type) to predict plant growth in the soil (Fig. 5). These predictions of plant growth were closer to the observations (R2 = 0.946 for loamy sand, R2 = 0.955 for silt loam N 1 and R2 = 0.971 for silt loam N 2) than the predictions of the overall growth model (Equation 2). Plant weight was still overesti­mated in loamy sand whereas a similar trend was absent in silt loam. Plant weight was under­estimated at 24 DAT in silt loam N 1 which might be due to the fact that the nitrogen level in the plant (1.08 mmol g - 1) is beyond the lowest level (±1.6mmol g- 1) used to construct Equa­tion 3.

The more accurate growth predictions through

~

o_ -2 :s: 0 '-.../

g' -3 _j

loamy sand

! I. I, 'I

Growth analysis of soil grown spinach plants 225

NAR analysis rather than with RGR analysis suggests that allocation related factors may ex­plain difference in productivity between soil- and hydroponically grown plants to some extent. This is consistent with the observation that the effect of a varying nutrient supply on plant productivity is associated with higher values of leaf area (or shoot weight) but only slight varia­tions in theNAR (Watson, 1947). However, the slower plant growth in the loamy sand compared to the silt loam and similar biomass partitioning in both soil types purport different net assimila­tion rates in both soils. It is difficult to pin-point the factor (water or nutrient availability, carbon cost per unit root weight) accounting for that difference.

The question remains how the growth equa­tions such as Equation (2) or (3) must be treated to analyse plant productivity in a wider range of soil types or, eventually, under other growth conditions. Parameters k 2 (k~) and k 4 (k~) de­scribe what part of the nitrogen pool in a plant is the active nitrogen pool. The RGR (NAR) is linearly related to this part. Most probably, this part is species specific and does not change considerably with the growing conditions. The k 3

(k~) parameter describes how the productivity decreases at higher growth stages (higher contri­bution of non growing tissue, effect of shading).

silt loam N 1

silt loam N 2

II

.. . I·

1 0 14 18 22 Time (DAT)

10 14 1(8 22 Time DAT)

10 14 1~ 2_2 Time (DAT)

26

Fig. 5. Dry weight (natural log scale) versus time (in days after seedling transfer into the soil, OAT) of spinach plants grown in three different potted soils. Points represent observed plant dry weights, full lines arc growth predictions based on the NAR model (Equation 3), the biomass partitioning function (Fig. 4) and the nitrogen uptake rate (Fig. 3).

226 Growth analysis of soil grown spinach plants

Neither this parameter is believed to change drastically for a range of growing conditions. The k 1 (k;) parameter is equivalent to the definition of nitrogen productivity (Agren, 1988) which changes with environmental conditions as e.g. the light intensity (Agren, 1985). If the different growing conditions have the same effect on the nitrogen productivity at any nitrogen level (as e.g. proposed in Equation 7 by Agren (1988) for the effect of other nutrients on nitrogen prod­uctivity), the growth pattern can be summarized in one equation with one adjustable parameter (k 1 or k;) for the soil type or environmental condition. To what extent these assumptions hold is scope for a further general approach. Furthermore, which of both growth analyses (RGR or NAR) is to be preferred is hard to say at this stage but the use of the NAR model requires an extra (important) equation relating biomass partitioning with nitrogen supply for which, to our knowledge, no simple model exists covering a range of environmental conditions.

Conclusions

The influence of nitrogen supply on the growth of spinach plants is at variance with the pattern observed for exponentially growing plants (Agren, 1985). From the growth response of these plants to exponential nitrogen addition in hydroponics, a growth model was introduced which accounted for the effect of growth stage on the nitrogen productivity.

Plant growth in three different potted soils was analysed with that model using the observed nitrogen uptake pattern. Plant growth was slight­ly overestimated. It was shown that part of the lower productivity could be accounted for by the lower shoot weight ratio of the soil-grown plants compared to the hydroponically grown plants.

Acknowledgements

Erik Smolders IS indebted to the Nationaal Fonds voor Wetenschapelijk Onderzoek (N.F.W.O.) for a research position as research

assistant. Jan Buysse acknowledges a grant of the onderzoeksfonds K. U. Leuven (project OT I 91/22).

References

Agren G I 1985 Theory for growth of plants derived from the nitrogen productivity concept. Physiol. Plant. 64, 17-28.

Agren G I 1988 Ideal nutrient productivities and nutrient proportions in plant growth. Plant Cell Environ. 11, 613-620.

Bhat K K S, Nye P H and Brereton A J 1979 The possibility of predicting solute uptake and plant growth response from independently measured soil and plant characteristics VI. The growth and uptake of rape in solutions of constant nitrate concentration. Plant and Soil 53, 137-167.

Clarkson D T 1986 Regulation of the absorption and release of nitrate by plant cells: a review of current ideas and methodology. In Fundamental, Ecological and Agricultur­al Aspects of Nitrogen Metabolism in Higher Plants. Eds. H Lambers, J J Neeteson and I Stulen. pp 3-27. Martinus Nijhoff Publishers, Dordrecht/Boston/Lancaster.

Clement C R, Hopper M J and Jones L H P 1978 The uptake of nitrate by Latium perenne from flowing nutrient solu­tion. J. Exp. Bot. 29, 453-464.

De Willigen P and Van Noordwijk M 1987 Roots, plant production and nutrient use efficiency. Ph.D. Thesis, Wageningen Agricultural University, Wageningen, 282 p.

Hirose T, Freijsen A H J and Lambers H 1988 Modelling of the responses to nitrogen availability of two Plantago species grown at a range of exponential nutrient addition rates. Plant Cell Environ. 11, 827-834.

Ingestad T 1981 Nutrition and growth of birch and grey alder seedlings in low conductivity solutions and at varied relative rates of nutrient addition. Physiol. Plant. 52, 454-466.

Ingestad T 1982 Relative addition rate and external concen­tration; driving variables used in plant nutrition research. Plant Cell Environ. 443-453.

Ingestad T and Lund A B 1979 Nitrogen stress in birch seedlings. I. Growth technique and growth. Physiol. Plant. 45, 137-148.

Mubarak A and Olsen R A 1976 Immiscible displacement of the soil solution by centrifugation. Soil Sci. Soc. Am. J. 40, 329-331.

Smolders E and Merckx R 1992 Growth and shoot :root partitioning of spinach plant as affected by nitrogen supply. Plant Cell Environ. 15, 795-807.

Watson D J 1947 Comparative physiological studies on the growth of field crops. II. The effects of varying nutrient supply on net assimilation rate and leaf area. Ann. Bot. 11, 375-407.

Reprinted from Plant and Soi/154: 73-80, 1993.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 227-233, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-051

Nitrate assimilation by wheat species at low rates of nitrogen supply

AMAN ULLAH JAN and D.J. PILBEAM Department of Pure and Applied Biology, The University of Leeds, Leeds LS2 9JT, UK

Key words: nitrate reductase activity, nitrogen, relative addition technique, relative growth rate, Triticum

Abstract

With evolution of Triticum from diploid to tetraploid and to hexaploid species grain yield has been increased, particularly with high rates of application of N fertilisers. The higher yield of 6n species is known to be due to higher harvest index but faster rates of uptake and assimilation of nitrate may also be important. Ten Triticum species were grown in 2.0 mol m - 3 nitrate and the activity of the enzyme nitrate reductase in the leaves was measured by in vivo assay after 15 days' growth. The rate of reduction of endogenous nitrate in the assay was highest in the hexaploid species, as was RGR. The rates of uptake of nitrate from 2.0 mol m - 3 solution were determined for each species, and then a hexaploid, a tetraploid and a diploid species were grown with three Sub-optimum rates of supply with the relative addition technique. RGR was positively correlated with nitrate reductase activity (NRA) and with the natural logarithm of total NRA in the leaf system, for all three species. However the plants were not in the stable state that the relative addition theory demands, and root: shoot ratios varied between rates of addition of nitrate. The lower values of NRA per gram of leaf material of Triticum monococcum appeared to be correlated with its higher root: shoot ratio than the ratios of the other two species.

Introduction

Yields of wheat crops per unit area have in­creased in the past century with the introduction of new cultivars, better management techniques and application of fertilisers. However, with the higher rates of fertiliser application now used in many countries there is great concern about their effects on the environment and on the rising cost of crop production. In the last few decades the idea of breeding low input cultivars has been put forward with the aim of producing more yield with less fertiliser application.

One of the main nutrients is nitrogen and the breeding of cultivars of wheat or other crops that can utilize nitrogen more efficiently could produce considerable environmental benefits.

Modern bread wheat (Triticum aestivum L.) contains many characters that can be selected for

in breeding programmes as it is a hexaploid species that arose from a tetraploid Triticum species. The tetraploid ancestor of bread wheat itself arose from a diploid Triticum species, and at both stages where chromosome number was increased the extra chromosomes came from a different genus, from Aegilops species.

The modern cultivars of Triticum aestivum give heavier yields than older cultivars with high nitrate supply, partly because they have a higher harvest index (Mengel, 1983). A key enzyme in the assimilation of nitrate is nitrate reductase [EC 1.6.6.1], and its characteristics are known to be heritable (Baligar and Barber, 1979; Clark, 1972; Dalling et al., 1985). Activity of this enzyme has been shown to be positively corre­lated with grain yield and grain protein content of cereals in some experiments, (Eilrich and Hageman, 1973; Reilly, 1976) although in other

228 Jan and Pilbeam

studies there has been no clear relationship between nitrate reductase activity (NRA) and organic nitrogen accumulation (Hageman, 1990). Despite these conflicting ideas the fact that nitrate reductase is the first enzyme of nitrate assimilation in plants means that study of this enzyme can give valuable information about the efficiency of nitrate assimilation by different species.

In this study NRA was measured in diploid, tetraploid and hexaploid Triticum species at different rates of nitrate supply to determine the efficiency of nitrate assimilation in the genus, particularly when the supply of nitrate is low.

Methods

Experiment 1

Seeds of ten Triticum species were germinated on moist filter paper with solid CaSO 4 supplied for 7 days. Similar seedlings were selected and roots were threaded through polythene mesh placed over 10 dm3 plastic containers, containing vigorously aerated nutrient solution (10 plants per container). Mineral nutrients were supplied in the concentrations (mol m- 3); MgS0 4 (1.5), KH 2P0 4 (1.5), K2S0 4 (1.25), Fe-EDTA (0.05), MnCI (0.01), H 3B03 (0.0306), Na2Mo0 4 (4.8 x 10 4 ), CuCI2 (0.001), CoS0 4 (2.0 x 10- 4),

ZnCI2 (0.001). Nitrate was added as 3:1 Ca(N03 ) 21 KN0 3 at a total rate of 2.0 mol m- 3 .

pH was adjusted to pH 6.0 by daily addition of saturated Ca(OH) 2 or 1.0 mol m- 3 H 2S04 • For the first two days nutrients were supplied at one fifth strength to prevent osmotic stress of the seedlings.

Experiment 2

In the second experiment plants were grown as above, except that nitrate was supplied according to the Relative Addition Technique (Ingestad and Lund, 1979). This theory is based on the assumption that if plant fresh weight increases exponentially with time, the uptake of nitrogen (in this case as nitrate) also increases exponen­tially according to the formula Nt = N0 *eRNt where N0 is the nitrogen content of the plant at

day 0, N 1 is the nitrogen content after t days and RN is the rate of increase in plant nitrogen (equivalent to the rate of supply of nitrate, RA). If this cumulative nitrate uptake is converted to natural logarithms there is a straight line rela­tionship between uptake and time. The rate of uptake of nitrate by each Triticum species was determined in the first experiment by measuring daily depletion of nitrate solutions, and in ex­periments where the relative addition technique was used daily additions of nitrate were made to the nutrient solution at a rate to maintain this straight line relationship. In the original use of this technique (Ingestad and Lund, 1979) there was a lag phase of 22 days before nitrogen was supplied in order to minimise the effects of internal nitrogen already present in the plants. As this lag phase was measured in a tree species a shorter lag phase was allowed for the Triticum in this study. After the initial 7 days with nitrogen only available from seed reserves ni­trate was supplied at a half the rate required for the first day of the growth in hydroponic culture, and subsequent daily additions were made at the appropriate rate from this starting value.

Plants were grown in controlled environments at 23°/18°C (day/night). Light was supplied at a photon flux density of approximately 290 f.Lmol photosynthetically active radiation m -z s -l with a photoperiod of 16 h. Relative humidity was 65%. Plants were harvested on day 15, and RGR was determined from the increase in weight of every individual plant over the 15 days. RGR of shoots and roots was determined from the differ­ence in weights of these parts in the harvested plants and representative plants sacrificed at the start of the experiment.

The in vivo nitrate reductase assay was based on the procedure of Klepper et al. (1971). Leaves were chopped into 1 em segments with a coleoptile cutter. These segments were placed in potassium phosphate buffer (100 mol m- 3 ) (pH 7.5) with 1% 1-propanol and were vacuum infiltrated for one minute and then incubated in an atmosphere of N 2 in a water bath at 30°C. Aliquots of buffer (0.5 cm 3 ) were collected at time intervals of 30, 60, 90, 120 and 150 minutes for determination of nitrite concentration by colorimetric analysis. NRA was calculated from a graph of nitrite produced against time. In all

experiments the assay was performed two hours before the end of the light period, and in experiment 2 this was 200 minutes after the final addition of nitrate.

Nitrate uptake was measured (as daily deple­tion of nitrate from the nutrient medium) by absorbance at 203 nm (Cawse, 1967).

All the species used are kept in the herbarium at The University of Leeds, except Triticum aestivum cv Slejpner bought from commercial suppliers.

Statistical analyses were carried out with the SAS system.

Results

When Triticum species were grown in an initial concentration of 2.0 mol m - 3 nitrate, NRA at day 15 ranged from 0.79 to 1.73 f.Lmol nitrite produced per gram fresh weight of leaf per hour and was positively correlated with RGR (Table 1 ). The activity was significantly greater in the hexaploid species Triticum aestivum and Triticum compactum than in the other species, but there were no overall differences between the tetra-

Table 1. Nitrate reductase activity (NRA) in the leaves, relative growth rate (RGR) of whole plants and root: shoot (R: S) ratio of different wheat species, grown in 2.0 mol m - 3

nitrate for 15 days

Species

Diploid T. boeoticum T. monococcum

Tetraploid T. dicoccoides T. dicoccum T. turgidum T. durum T. timopheevii T. polonicum

Hexaploid T. compactum T.aestivum

( cv Slejpner)

NRA*

1.05 de 0.93 dee

1.22b 1.15 be 0.99 cde 0.98 cde 0.79f 0.84 ef

1.73 a 1.66 a

R:S ratio

0.15 ed 0.14 ef I: 0.79

0.18 b 0.16 de 0.16 de 0.18 b 1:0.99 0.15 ed 0.14 ef

0.20 a 0.20 a 1: 1.15

NRA * = (f.Lmol NO, produced g _, fw h _,). Means of three replicates. Same letters within the columns are not signifi­cantly different (p = 0.05). The regression coefficient of determination for leaf NRA against whole plant RGR is R' =0.66.

Nitrate assimilation by wheat 229

ploid species as a group and the diploid species. The two hexaploid species had significantly high­er values of RGR than the other species.

In the second experiment diploid, tetraploid and hexaploid species (Triticum monococcum, Triticum durum and Triticum aestivum respec­tively) were grown with nitrate supplied by the relative addition technique at three rates for each species. The highest rate of supply (Sub­optimum I) was the rate of uptake measured for each species in the first experiment (data not shown), and the other two rates of supply were one half of the Sub-optimum I rate (Sub-op­timum II) and one third of the rate (Sub-op­timum III).

The rates of NRA after 15 days' growth at the three rates of supply, and the relative growth rates over the 15 days are shown in Table 2. For each species NRA was higher at the higher relative addition rates. NRA was considerably higher at the Sub-optimum I rate compared with the Sub-optimum II rate, although the difference in NRA between Sub-optimum III and Sub­optimum II was less noticeable. There was not a large difference between rates of NRA in these

Table 2. Nitrate reductase activity (NRA) in the leaves, relative growth rate (RGR) of whole plants and root: shoot (R: S) ratio of three species grown with three relative rates of addition (RA) of nitrate for 15 days

Species NRA* RGR(day- 1) R:SRatio

RAI T. monococcum 0.77 ± 0.08 0.13±0.004 1:0.78 T. durum 0.68 ± 0.05 0.13 ± 0.008 1:0.58 T. aestivum 0.62 ± 0.06 0.13 ± 0.004 1:0.58

RA II T. monococcum 0.38 ± 0.04 0.12 ± 0.010 I :0.77 T. durum 0.31 ± 0.02 0.07 ± 0.004 1:0.65 T. aestivum 0.33 ± 0.02 0.06 ± 0.009 1:0.50

RA III T. monococcum 0.24 ± 0.02 0.10 ± 0.011 1:0.60 T. durum 0.23 ± 0.02 0.07 ± 0.006 1:0.55 T. aestivum 0.31 ± 0.05 0.05 ± 0.003 1:0.50

NRA* = (f.Lmol NO, produced g- 1 fw h- 1). Means of three replicates ±SO. T. monococcum RAJ= 0.174, RA II= 0.087, RA III= 0.058 day- 1 ;

T. durum RAI=0.264, RA II =0.132, RA III=0.088day- 1 ;

T. aestivum RAJ= 0.234, RA II= 0.117, RA III= 0.078 day- 1 ;

RA I, RA II, RA III= suboptimum I, II, III resp.

230 Jan and Pilbeam

Q.22

0-20

0-18

0·16

Q-14

' • • .. 006

... , ... 0<>4

o-a>

• • • •• • • . ,. ....

• T. monococcum

+ T-durum

e T-aestivum

0..0 0-2 0-4 0.6 0-8 1·0 1·2 1·4 1·6 1·8 2·0

I'J1A(umol ~2 produced g-1 fw h-1)

Fig. 1. Relationship between nitrate reductase activity (NRA) per gram of leaf weight and relative growth rate (RGR) of T. monococcum, T. durum and T. aestivum plants grown at Sub-optimum I, Sub-optimum II and Sub-optimum III rates of nitrate supply, and in 2.0 mol m - 3 nitrate.

different species at any rate of relative addition. The RGR differed considerably between species, and also differed between values of RA for each species. However, contrary to the theory of the relative addition technique the values did not equal the values of RA.

The rates of NRA per gram of leaf material for each species grown in the second experiment are shown plotted against the RGR of the whole plant in Figure 1, and the values for the first experiments are included. In all three species

0·22

0-20

018

0·16

-0·14

~0-12 '0

;:;:o-10

~008 0

0·

0·

•

• ........... • •

• • .. ,. ... ... -3 -2 -1

... • • •

• T .monococcum

+ T -durum

e T. aest ivum

0

In NRA< umo1N02 Leaf System-1 h-1 l

2

Fig. 2. Relationship between In nitrate reductase activity (In NRA) of leaf system (natural logarithm of NRA in f.Lmol nitrite produced per hour) and relative growth rate (RGR) of T. monococcum, T. durum and T. aestivum plants grown at Sub-optimum I, Sub-optimum II and Sub-optimum III rates of nitrate supply, and in 2.0 mol m _, nitrate.

0.22

020

0.18

0.16

-'7 Oj4

;; 0.12

" -0.10 • g 0.08

Vi 0.06 a: ~ 0.04

0.02

0.00 -4

~ .~ • • •J I .J' • • • • •

-3 -2 -1 In total NRA in

• •

• •• • • •

•T. monococcum +T.durum eT.aestivum

0 leaf system

2

Fig. 3. Relationship between In nitrate reductase activity (NRA) of leaf system (natural logarithm of NRA in f.Lmol nitrite produced per hour) and relative growth rate (RGR) of the shoots of T. monococcum, T. durum and T. aestivum plants grown at Sub-optimum I, Sub-optimum II and Sub­optimum III rates of nitrate supply, and in 2.0 mol m - 3

nitrate.

RGR and NRA were positively correlated, al­though for Triticum monococcum, at least, the relationship could have been hyperbolic.

If these values of NRA are multiplied by the total weight of leaf material after 15 days, and the resulting values are plotted as their natural logarithms (In) against RGR of the whole plant, it can be seen that in each species there was a positive correlation (Fig. 2). In Figure 3 the natural logarithm of NRA of the leaf system is plotted against RGR of the shoot, and it can be seen that Triticum monococcum had a lower shoot RGR for any given value of In NRA than the other two species.

Discussion

It can be seen from Table 1 that when seedlings of different Triticum species were grown in the high concentration of 2.0 mol m - 3 nitrate there was a wide range of rates of NRA in the leaves after 15 days of growth. Big differences in NRA and rates of uptake of nitrate have been reported between different cultivars of Triticum (Gal­lagher et al., 1983; Rodgers and Barneix, 1988) and so the differences observed here are not

surprising. However, these differences are not obviously linked with the ploidy level of each of the species, except that the hexaploid species Triticum aestivum and Triticum compactum had significantly higher rates of NRA than the other species.

Furthermore, these species also had a sig­nificantly higher RGR over the 15 days, and there was a positive correlation between NRA and RGR in the ten species studied (Table 1). This correlation between rate of assimilation of nitrate and the rate of vegetative growth is expected as values of NRA have frequently been reported to be positively correlated with both vegetative and seed yield of plants (Eilrich and Hageman, 1973; Gonzalez Ponce et al., 1990).

The relative addition technique was used to grow a diploid, a tetraploid and a hexaploid species (Triticum monococcum, Triticum durum and Triticum aestivum respectively) at different rates of nitrate supply in order to determine the relationship between RGR and NRA in species of each ploidy level. It can be seen from the formula (Methods) that RGR equals the rate of relative addition (RA), and so plants were grown with different values of RGR by adjusting RA. However the technique did not work as expected as RGR did not equal RA (Table 2).

Although the rates of RA required to produce optimum growth were determined from the rate of uptake of nitrate by each species in the first experiment, even at the highest values of RA each species showed a considerably lower RGR in the second experiment. In all but two in­stances (Triticum monococcum at RA Sub-op­timum II and Sub-optimum III) RGR was less than RA, and so in the second experiment plants did not utilise the nitrate at the rate at which it was supplied. Similar discrepancies between RA and RGR at the higher rates of RA have been seen in barley (Hordeum vulgare L.) (Mattsson et al., 1991), although in that study there was close agreement between the two set of values at low RA.

However, despite this poor control of rate of growth by rate of nitrate addition, for all three species plants were produced with a range of values of RGR. Assay of NRA was carried out at the same time in the photoperiod, 200 minutes after the last addition of nitrate, so that activity

Nitrate assimilation by wheat 231

of the enzyme should have been constant in relation to induction by that day's supply of substrate and in relation to the light/ dark cycle. When RGR was plotted against natural logarithm of total NRA in the leaf system there was a progressively increasing relationship up to a saturation point, where values reached a plateau (Fig. 2). This relationship must be posi­tive as leaf weight is a major component of RGR of the whole plant, but if root: shoot ratio is constant the relationship between RGR and In NRA in the leaf system would be linear if the relationship between RGR and NRA per gram of leaf material is linear. It can be seen from Figure 2 that for Triticum monococcum this relationship between RGR and ln NRA followed the same pattern as the relationship between RGR and NRA per gram of leaf material. As root: shoot ratio was identical for the plants in experiment 1 and for plants grown at the Sub­optimum I and II rates of nitrate supply in experiment 2 this is an inevitable consequence of shoot weight being such a large component of the values plotted on both axes of the graph.

However, for Triticum durum and Triticum aestivum the values of root: shoot ratio were not so similar between treatments and experiments, especially when the ratios recorded in experi­ment 1 are compared with those for plants supplied at Sub-optimum rate I in experiment 2. For these two species, the relationship between RGR and In NRA in the leaf system followed a similar pattern to the relationship between RGR and NRA per gram of leaf material, but ap­peared to be more of a straight line and less hyperbolic (Fig. 2). This indicates that in experi­ment 1, where the RGR of Triticum durum and Triticum aestivum was much higher, the high values of NRA per gram of leaf material that did not give rise to increased whole plant RGR in direct proportion to lower values of NRA (Fig. 1) were providing enhanced growth of leaves relative to roots. Across a range of values of RGR of whole plants there was apparently a constant relationship with the capacity to assimi­late nitrate in the leaf system.

This interpretation seems to be confirmed in Figure 3 where for all three species there was a positive, linear relationship between the RGR of the shoots and In total NRA in the shoots. As

232 Jan and Pilbeam

the logarithm of the shoot weight is the major component of the values plotted on both axes there has to be a positive, linear relationship between the two sets of values unless the com­ponent due to NRA per gram causes a deviation from linearity. Although data for RGR of plant parts cannot be as accurate as data for RGR of whole plants (because the initial determinations of weight are made on different plants from the final determinations), Figure 3 shows that re­gardless of the root: shoot ratios of the plants there was a strong correlation between the total assimilation of nitrate in the shoots and the growth of the shoots. For both Triticum durum and Triticum aestivum, where in experiment 1 root: shoot ratios were much lower and RGR of the whole plants was much higher than in experi­ment 2, this relationship still held. However, it is difficult to determine if the correlation is strictly linear, particularly bearing in mind the difficul­ties with determining shoot, RGR and further experimentation is required to investigate this point.

The data in Figure 3 show that even at very low values of total NRA in the leaves there is some growth of the shoot. At very low rates of supply of nitrate the assimilation presumably occurs almost exclusively in the roots, and re­duced N exported to the leaves enables leaf growth and metabolism to continue so that there is still a supply of photosynthates going to the roots. It appears from Figure 3 that in Triticum monococcum a larger proportion of nitrate as­similated in the leaves goes to provide reduced N for root growth as for any value of In NRA in the leaf system shoot RGR is lower than in Triticum durum and Triticum aestivum. However, it should be remembered that for the Sub-optimum I and II rates of nitrate supply the root: shoot ratios were close to the ratio seen in plants grown in 2.0 mol m - 3 nitrate, whereas for all three RA rates Triticum durum and Triticum aestivum had a much higher root: shoot ratio than the plants grown in experiment 1. In order to determine the relationship between assimila­tion of nitrate in one plant part and the growth of that part the RA experiments need to be repeated with RA equalling RGR, as the relative addition theory demands, so that root: shoot ratios do not vary.

It is apparent from experiment 1 that the root: shoot ratio of Triticum monococcum is higher than the root: shoot ratios of Triticum durum and Triticum aestivum, and this is one of the reasons why Triticum aestivum has a higher harvest index and is higher yielding under culti­vation than the other species (Austin, 1982; Bhatt, 1976; Donald, 1970; Evans and Dun­stone, 1970). Although the comparatively small­er leaf system in Triticum monococcum poorly equips this species for the carbohydrate pro­duction needed for high grain yields the com­paratively large root system may equip it well for growth in nitrate-poor soils. However, it also seems likely that low rates of NRA per gram of leaf material poorly equip the species for rapid vegetative growth when the rate of nitrate supply is high, whereas with abundant nitrate Triticum aestivum has much higher values of NRA per gram of leaf material and much higher RGR of the plants.

Acknowledgements

The financial support of the Government of Pakistan as a studentship for one of us (AUJ) is gratefully acknowledged. Thanks are due to Dr C-M Larsson for valuable discussion on the use of the Relative Addition Technique and to Mr K Redshaw for maintaining viable stock of the Triticum species used.

References

Austin R B 1982 Crop characteristics and the potential yield of wheat. J. Agric. Sci. Camb. 98, 447-453.

Baligar V C and Barber S A 1979 Genotypic differences of corn for ion uptake. Agron. J. 71, 870-872.

Bhatt G M 1976 Variation of harvest index in several wheat crosses. Euphytica 25, 41-50.

Cawse P A 1967 The determination of nitrate in soil solution by ultraviolet spectrophotometry. Analyst 92, 311-315.

Clark R B 1972 Plant genotypic differences in the uptake, translocation, accumulation and use of mineral elements required for growth. Plant and Soil 72, 175-195.

Dalling M J, Halloran G M and Wilson J H 1975 The relationship between NRA and grain N productivity in wheat (Triticum aestivum L.). Aust. J. Agric. Res. 26, 1-10.

Donald C M 1968 The breeding of crop ideotypes. Euphytica 17, 385-403.

Eilrich G L and Hageman R H 1973 NRA and its relation­ship to accumulation of vegetative and grain nitrogen in wheat (Triticum aestivum) Crop Science 13, 59-66.

Evans L T and Dunstone R L 1970 Some physiological aspects of evolution in wheat. Aust. J. Biol. Sci. 23, 724-741.

Gallagher L W, Soliman K M, Rains D W, Qualset C 0 and Huffaker R C 1983 Nitrogen assimilation in common wheat differing in potential nitrate reductase activity and tissue nitrate concentration. Crop Sci. 23, 913-919.

Gonzalez Ponce R, Salas M L and Lamela A 1990 Nitrate reductase activity, grain yield and grain protein in wheat (Triticum aestivum) as affected by nitrogen fertilization under semi-arid conditions. In Plant Nutrition Physiology and Applications. Ed. M L Van Beusichem. pp 561-563. Kluwer Academic Publishers, Dordrecht.

Hageman R H 1990 Historical perspectives of the enzymes of nitrate assimilation by crop plants and potential for biotechnological application. In Inorganic Nitrogen Metab­olism in Plants and Microorganism, Uptake and Assimila­tion. Eds. W R Ullrich, C Rigano, A Fuggi and P J Aparicio. pp 3-11 Springer-Verlag, Berlin.

Ingestad T and Lund A-B 1979 Nitrogen stress in birch seedlings. I: Technique and growth. Physiol. Plant. 45, 137-148.

Nitrate assimilation by wheat 233

Klepper L, Flesher D and Hageman R H 1971 Generation of reduced nicotinamide adenine dinucleotide for nitrate reduction in green leaves. Plant Physiol. 48, 580-590.

Larsson C M, Mattsson M, Duarte P, Samuelson M, Ohlen E, Oscarson P, Ingermarsson B, Larsson M and Lundborg T 1992 Uptake and assimilation of nitrate under nitrogen limitation. In Nitrogen Metabolism of Plants. Eds. K Mengel and D J Pilbeam. pp 71-89. Oxford University Press, Oxford, UK.

Mattsson E, Johnsson E, Lundborg T, Larsson M and Larsson C-M 1991 Nitrogen utilization in N-limited barley during vegetative and generative growth. I Growth and nitrate uptake kinetics in vegetative cultures grown at different relative addition rates of nitrate-N. J. Expt. Bot. 42, 197-205.

Mengel K 1983 Responses of various crop species and cultivars to fertilizer application. Plant and Soil 72, 305-319.

Reilly M L 1976 The nitrate assimilation capacity of some Irish-grown wheat (Triticum vulgare) varieties. III. An in vivo assessment of nitrate reductase activity and its relation to productivity. Proc. Royal Irish Acad. 76, 567-576.

Rodgers C 0 and Barneix A J 1988 Cultivar differences in the rate of nitrate uptake by intact wheat plants as related to growth rate. Physiol. Plant. 72, 1221-126.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 235-241, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-063

Influence of nitrogen availability on growth and development of tomato plants until fruit-setting

Y. DUMAS, J. SUNIAGA QUIJADA and M. BONAFOUS National Institute for Agronomic Research, INRA, BP 91, F-84143 Montfavet, France

Key words: Lycopersicon esculentum, nitrogen supply, nutrient solution, tomato for processing

Abstract

The influence of N availability in the root medium on growth and development of tomato for the processing industry was studied during the period from sowing time until the beginning of fruit-setting. In a growth chamber or in a glasshouse sand pots were regularly rinsed with complete solutions with or without N before emergence and with 0, 2, 6 and 18 meq N L - 1 as ammonium-nitrate after emergence. The presence of N in the nutrient solution did not affect the germination process and the emergence rate. The absorption of N began immediately after emergence. When no N was supplied growth was reduced and ceased totally after 2 weeks. At a higher N supply shoot and root growth was stimulated and the truss appearance rate and the percentage of the fruits in total dry weight was increased. 6 meq N L - 1 in the nutrient solution seemed to be the optimal level for young tomato seedlings from emergence to early blooming.

Introduction

Nitrogen absorption by tomato plants has been widely studied. However, in these studies trans­plants have generally been used during their reproductive phase (Cornillon and Auge, 1980). Such results may be affected by plant traumatism during transplanting. Haag et al. (1978) and Halbrooks and Wilcox (1980) studied the miner­al nutrition of tomato seedlings (cultivars used for canning industry) but their research started 21 days after sowing. On the contrary Widders (1989) studied the effect of nitrogen and phos­phorus supply on dry matter accumulation in tomato transplants in the nursery from sowing to transplanting only.

Tomatoes for processing are currently sown directly in the field in order to reduce production costs. Once-over mechanical harvesting requires early concentrated ripening and foliage which is not developed. For proper management in the commercial production of tomato crops for the processing industry, a thorough understanding of

the nitrogen needs of tomato plants during their total development and especially during crop establishment is essential.

Therefore the influence of N supply on tomato growth and development has been studied during the whole period between sowing time and the beginning of fruit-setting.

Methods

Pot experiments were performed in a growth chamber and in a glasshouse. In each case, uniform-sized seeds of a common cultivar for processing industry (UC82 from Petoseed, Cali­fornia) were placed 1 em deep in fine inert sand (particle size between 0.8 and 1 mm). More than 98% of the seeds germinated.

Growth chamber experiment

Plantlets grew in 1.33 L pots filled with inert sand for up to 18 days. 9 to 560 seeds (of -3 mg

236 Dumas et al.

each) were sown per pot depending of the intended harvesting date. For the sake of accura­cy and in order to provide enough material for chemical analysis, sampling during germination required a greater number of seeds. Plants received light 16 hours per day; the temperature was 18oC during the night and 24oC during the day and the relative humidity was 70%. The effects of three nutrient solutions were com­pared, i.e. a complete solution ('N'), a solution without nitrogen ('ON') and deionized water with added traces of CaSO 4 ('00'). The solutions were supplied twice a day starting at the date of sowing. Pots could drain freely. There were four replications in a randomized design. Plants were sampled every day until day 10, and every 2nd day after that. Four pots per treatment were sampled at each sampling date.

Glasshouse experiment

Plantlets grew in 10 L pots filled with inert sand up to the beginning of fruit-setting. The weekly mean values of minimum air temperature were 15-16°C and the weekly mean values of maxi­mum air temperature were 30-32°C. The effects of four nutrient solutions were compared, i.e. a complete solution ('N') containing all the nu­trients and 6 meq N L -I, a complete solution overdosed in nitrogen ('3N') with 18 meq N L -I, a complete solution underdosed ('1 /3N') with 2 meq N L -I and a solution containing all the nutrients except N ('ON'). From the time of sowing, the solutions were supplied at least twice a day and pots could drain freely. The design was at random with four replications, i.e. four pots for each treatment and each sampling date (14, 22, 33, 47 and 60 days after sowing). After emergence, four plants were kept per pot. A treatment with soil was added (labelled 'ONs') to estimate its N supply in comparison to the nutrient solutions. It received the solution 'ON'. It was a clayey calcareous soil, poor in phosphor­us but well provided with potassium and mag­nesium and with a total nitrogen content of 1.2 g per kg of dry soil.

Nutrient solutions

The basic solutions were derived from Hoagland and Arnon (1950). They were considered to be

suitable for tomato (Cornillon and Maisonneuve, 1985). For the growth chamber experiments the composition of the complete solution was as follows: 6.5, 1.5 and 1.0 meq L -I for NO;, PO!-, and SO~-, respectively, and 3.5, 3.0, 1.0, 0.5, and 1.0 meq L -I forK+, Ca2 +, Mg2 +, NH:, and H+, respectively. In the glasshouse experi­ment the composition of the complete solution (N) was as follows: 3.0, 3.0, 3.0, and 2.0 meq L -I for NO~, PO!-, SO~-, and Cl-, respective-

. I ly, and 3.0, 2.0, 1.0, 3.0, and 2.0 meq L- for K+, Ca 2+, Mg2 +, NH:, and H+, respectively. In each experiment micronutrients (B, Cl, Cu, Fe, Mn, Mo, Zn) were added to the solutions and the pH was about 5.3.

Observations and measurements

In growth chamber experiments, germination, emergence, decrease in seed reserves, shoot height (using plant photocopies) and dry matter distribution in the plantlet were accurately ob­served and measured.

In glasshouse experiments, dry matter ac­cumulation in the different plant organs was measured on harvested plants. Between destruc­tive plant samplings and in order to better understand its changes with time, estimations of shoot dry matter per plant were performed using regressions with linear measurements of leaf length or truss length (Dumas, 1990) 18, 26 and 40 days after sowing.

Total N analyses were carried out on the harvested plants using the Kjeldahl method. Ethanol extracts of leaf-blades were analysed by spectrophotometry. The cell ultrastructure of the upper side leaf palisade tissue was studied by transmission electron microscopy.

The results were processed by analysis of variance and means were compared by the Newman-Keuls method.

Results

Germination, emergence and initial growth (experiment in the growth chamber)

Four days after sowing, germination was finished (98% radiclcs appearance) in all the treatments.

On day 6, 80% and 60% of the 'N' and 'ON' plants had emerged respectively, whereas plants from the '00' treatment were one day later. From sowing to day 6 the total seed + plantlet dry matter weight (DMW) continuously decreased for all the treatments alike. From day 6 on DMW began to increase for 'N' and 'ON' at the same rate until day 8, while it continued to decrease for '00' (Fig. 1). However, from day 6, shoot height in 'N' was greater than in 'ON' (data not shown). In fact, Figure 2 shows that, on day 6 and later, shoot DMW per plant was sig­nificantly increased at the higher N supply while root DMW was significantly lower for the 'N' treatment compared to the 'ON' and the '00' treatments. Thus, immediately after emergence root growth partly compensated for the inhibited shoot growth when nitrogen was limiting.

On day 9, total dry matter weight per plant was significantly different for the treatments 'N' and 'ON', and this characterized the beginning of the global effect of nitrogen on plant DMW (Fig. 1). This difference increased after day 10. As for treatment '00', total DMW per plant decreased until day 10 and then increased slowly. On day 14, plants of the treatment 'N' were at the 2 true leaves stage, those of the treatment 'ON' at the beginning of 1 true leaf stage and

16

14

12

4

El l=~

2!-x-x-x 0- I I I

2 4

I

10 12 14 16 18

Days after sowing

Fig. 1. Dry matter accumulation during the initial growth of tomato seedlings. For a same abscisse value, two points situated on two distinct curves mean that their ordinate 'Vialues are significantly different (p = 0.05).

2.25

2.00

1.75

"60 1.50 .§,

li 1.25 'a I; "'1.00

~ 0.75

0.50

0.25

0.00

N and growth of tomato seedlings 237

4

n ~

Shoots /--x--x Roots ~

10

Days after sowing

Fig. 2. Changes in shoot and root DMW during initial growth. For a same abscisse value, two points situated on two distinct curves mean that their ordinate values are signifi­cantly different (p = 0.05).

those of the treatment '00' at the 2 cotyledon stage.

Plant growth from emergence to fruit-setting (experiment in glasshouse)

In the treatment 'ON', plant growth was very slow and stopped 22 days after sowing but plants were still alive on day 60 at a stage of 3-4 small true leaves. Figure 3 shows an increase in the shoot dry matter accumulation rate for the 4 other treatments from day 14 to day 60 after sowing. During the period of strictly vegetative growth (until day 26, appearance of the first truss) treatments 'N' and '3N' had rather similar growth rates which increased with time. For the treatment '1/3N', the growth rate was always 3 to 4 times lower. The growth rate of the treat­ment 'ONs' was roughly equivalent to the growth rate of '3N'. This classification of the four treatments globally remained between the 1st truss appearance stage and the 1st truss bloom­ing stage. Changes appeared at day 40 (begin­ning of blooming) when '3N' plants started to grow faster and 'ONs' slower than those of the 'N' treatment.

From a general point of view, root growth was quite similar to shoot growth, except for some details. On day 14, 'l/3N' root DMW was significantly higher than '3N' DMW and was

238 Dumas et al.

250

~ 200

§ -a 150 l:l 0..

~ 100

] en 50

14

----.....-- l/3N

16 18 20 22 24 26

Days after sowing

14

12

§ 10

0.

---3N

-- ONs

25 30 35 40 45 50 55 60

Days after sowing

Fig. 4. Changes in cumulative total number of trusses per

20000 plant.

18000 ----.....-- 1/3N

i 16000

'i 14000

-a 12000 l:l 0.. 10000

~ 8000

0 6000

~ 4000

--N

------- 3N

- --o - ONs

2000

o~~::.!:::t==~-+--f---+-----l

25 30 35 40 45 50 55 60

Days after sowing

Fig. 3. Shot dry matter accumulation per plant after seedling emergence until fruit-setting. For a same abscisse value. two points situated on two distinct curves mean that their ordi­nate values are significantly different (p = 0.05).

equivalent to that of 'N'. Prior to the blooming stage, 'N' root DMW was significantly higher than that of '3N' but after day 40 this was reversed (data not presented).

Plant development

The first truss appeared on some plants for the treatments 'ONs', 'N' and '3N' on day 26 but for

'1/3N' on day 29. Figure 4 shows the progression of the number of trusses per plant. '1 I 3N' was delayed and characterized by a lower truss ap­pearance rate. 'N' was surpassed by '3N' at the beginning of the first fruits swelling, 54 days after sowing. 'ONs' was surpassed by '1/3N' as soon as blooming occured and during fruit swelling there even was a decrease in the number of trusses (i.e. trusses were aborted). On day 60, mean truss length was 14.8 mm for '1/3N' and 'ONs', 13.5 mm for 'N' and 11.0 mm for '3N' where many trusses were aborted. Fruit DMW charac­teristics on day 60 are presented in Table 1. Treatment 'N' was most efficient in early fruit production.

Aspects of plant metabolism

Nitrogen accumulation in the plant shoots was approximately related to DM accumulation, ex­cept that the treatment '3N' led to a much higher N accumulation than the treatment 'N' early on. This was due to a higher N-concentration in the plant (Table 2). When N was highly available (treatment '3N' compared to 'N'), there was overconsumption and lower efficiency. Nitrogen accumulation in the root system was about one third of that in the shoot in each treatment.

Table 1. Fruit DMW characteristics in the glasshouse experi­ment 60 days after sowing

Treat- Fruit DMW per plant Fruit DMW/total DMW ment (mg) (%)

1/3N 563 b* 8.1 b N 1857 a 13.0 a 3N 1012 b 5.4 b ONs 597 b 8.8 b

* the results followed by the same letter are not statistically different (Newman-Keuls method, p = 0.05).

Table 2. Mean nitrogen concentration (% of DM) in the plant for two treatments

Plant Treatment Number of days after sowing part

14 33 47 60

Shoot N 6.14 3.64 3N 8.13 7.06

Leaf- N 2.78 2.75 blade 3N 5.45 5.25

Figure 5 represents the pigment absorption spectrum of leaf ethanol extracts. The influence of N availability on leaf chlorophyll content was demonstrated by the two typical chlorophyll peaks for the wave lengths 434 and 664 nm. Absorbency between 300 and 350 nm is typical of the presence of polyphenols. Polyphenol content was considerably increased by low N availability.

Electron microscopy showed that chloroplasts were functional at emergence and had well

Absorbency

0.5

0.0

434 !'. 'I

334 i i. ; i

... , i ' / ; i ·,

./ \ i \ \ / \ ...

\ _, ~.

\ /~ , .. , \ ,.,. ..... _, ~ ' i

~·.... ', ,.. \ \ .... , \ \

.. _., ~

\ l I I

\\ II

\

300 400 500

Wave length (nm)

JN

N

1/JN

600

664

700

Fig. 5. Absorption spectrum of leaf-blade tomato pigments at the beginning of the blooming stage.

N and growth of tomato seedlings 239

structured thylakoids. A few days after emer­gence, the absence of nitrogen in the nutrient solution resulted in enormous starch accumula­tion in the chloroplasts of plants sampled at the beginning of the light period. Later, at the reproductive stage, the more available nitrogen was, the less starch there was in the chloroplasts. N deficiency led to increasing storage of starch which resulted in a deformation of the chloro­plasts and a dislocation of the thylakoids.

Discussion

Until emergence, no nitrogen was taken up by the seedlings. During this phase, the plantlets were completely dependent on seed reserves and therefore not affected by the external N supply. Catabolism exceeded anabolism, resulting in a DMW decrease. This phenomenon was already described for wheat (Pinto Contreras, 1981), for millet by Siband (1981) and for corn by Bourdu and Gregory (1983). In the same growth chamber experiment as the one described here, Suniaga Quijada (1991) showed that only potas­sium was absorbed between day 4 and day 6 and that the utilization of seed reserves between day 5 and day 10 was accelerated in the treatments 'N' and 'ON', resulting in quicker emergence and growth. On day 6, N absorption and assimilation began simultaneously with the onset of photo­synthesis. An influence of nitrogen on total DMW could be measured 3 days later i.e. on day 9 when the seed reserves were used up (Suniaga Quijada, 1991). At that time, the higher root DM production in the N-deficient treatments could be explained by a temporary carbohydrate accumulation in the root system as had been suggested by Champigny et a!. (1985) for wheat and by Just et a!. (1989) for sunflowers. Accord­ing to these authors, this accumulation should be possible by saving energy as there is little or no nitrate to be reduced and/ or by reorienting carbon flux when plants have to face limiting nitrogen conditions.

The glasshouse experiment has proved that N availability was a major growth factor. At the 2 true leaves stage (day 14 after sowing), the concentration '1 /3N' was not sufficient to allow optimal growth whereas '3N' did not permit a

240 Dumas et al.

growth rate much higher than 'N' although its total plant N accumulation was more than two times greater at the swelling fruit stage. These results are consistent with those reported by Widders (1989) for nursery plants. At the begin­ning of the reproductive phase, '1/3N' and '3N' were inferior in comparison with 'N' and so, for a diagnosis, a great accumulation of total dry matter ('3N') is not necessarily a good indicator of the expected production. Considering truss number and length and fruit DMW, it appeared that N deficiency ('1/3N' and 'ONs') resulted in a slower rate of appearance of new trusses. This is in line with the distribution priority for assimi­lates towards the reproductive organs, particu­larly swelling fruits (Bonnemain, 1975). On the contrary, N excess ('3N') resulted in high shoot growth and new truss differentiation, but in a delayed fruit growth and in high N-concentra­tions in the plants. In the present range of experimental concentrations, 'N' seemed to be an advisable level of nitrogen content in the nutrient solution for young tomato seedlings from emergence till early blooming.

Ethanol extract analysis gave an opportunity to characterize metabolism modifications related to N availability. High N availability resulted in chlorophyll synthesis (and green colour) whereas N shortage resulted in polyphenol synthesis (and yellowish colour). Indeed absorbencies between 300 and 400 nm are commonly associated with polyphenolic pigments and more precisely be­tween 330 and 340 nm with compounds derived from cinnamic acid (Monties, 1975). In fact polyphenols are always present in the tomato leaf, particularly chlorogenic acid (Monties, 1975) with an absorption maximum at 334 nm (Macheix, 1979). They are known to be able to inhibit plant growth regulators (Domenjou and Marigo, 1978) and are thus associated with slower growth (Marigo and Boudet, 1975). They are accumulated in ageing organs. But here their content increased with N shortage as a con­sequence of a disturbed protein metabolism. Ethanol extract analysis associated with leaf pigment measurements could be a potentially low-destructive tool for directly diagnosing N deficiency in young tomato plants (Suniaga Qui­jada, 1991).

Practically speaking, the management of the

nitrogen availability in the field deserves atten­tion. In the present work, plant behaviour in the treatment 'ONs' was interesting. Under condi­. tions of good water supply, no addition of N was necessary until the beginning of blooming. After­wards it became indispensable for avoiding any risk of reducing the production potential. The management of nitrogen fertilization of field tomato crops will be improved when the availa­bility of mineral N in the soil is better known throughout the crop cycle. In order to reduce the input and the leaching of N in sown tomato crops, no additional N will be necessary before the appearance of the first trusses.

References

Bonnemain J L 1975 Transport et distribution des produits de Ia photosynthese. In Photosynthese et Production Veget­ale. Ed. C Castes. pp 147-170. Gauthiers-Villars Pub­lishers, Paris, France.

Bourdu R and Gregory N 1983 Etude comparee du debut de Ia croissance chez divers genotypes de mais. Agron. 3, 761-770.

Champigny M L, Guiraud G, Soualmi-Boujemaa K, Talouizte A and Moyse A 1985 Le role des racines et des feuilles dans !'assimilation du nitrate; le cas de Ia jeune plante de ble. C. R. Acad. Agric. Fr. 71, 283-291.

Cornillon P and Auge M 1980 Cinetique d'absorption des elements mineraux par Ia tomate cultivee sous serre, consequences agronomiques. C. R. Acad. Agric. Fr. 46, 1242-1255.

Cornillon P and Maisonneuve B 1985 Effets de basses temperatures appliquees aux parties aerienne ou racinaire de Ia !ornate sur !'absorption d'elements mineraux et Ia fertilite pollinique. Agron. 5, 33-38.

Doumenjou N and Marigo G 1978 Relations polyphenols­croissance: role de l'acide chlorogenique dans le catabolisme auxinique chez Lycopersicon esculentum. Phy­siol. Veg. 16, 319-331.

Dumas Y 1990 Interrelation of linear measurements and total leaf area or dry matter production in young tomato plants. Adv. Hortic. Sci. 4, 172-176.

Haag H P, Oliveira G D de, Barbosa V and Silva Neto J M 1978 Nutricao mineral de hortalicas XXXII Marcha de absorcao des nutrientes pelo tomateiro (Lycopersicon esculentum Mill) destinado ao processamiento industrial. An. Esc. Sup. Agric. Luiz de Queiroz 35, 243-269.

Halbrooks M C and Wilcox G E 1980 Tomato Plant De­velopment and Elemental Accumulation. J. Am. Soc. Hortic. Sci. 105, 826-828.

Hoagland D R and Arnon D I 1950 The Water Culture Method for Growing Plants without Soil. Calif. Agric. Stn. Circ. 347 p.

Just D, Saux C, Richard C and Andre M 1989 Effect of

nitrogen stress on sunflower gas exchange. I. Photorespira­tion and carbon partitioning. Plant Physiol. Biochem. 27, 669-677.

Macheix J J 1979 Les esters hydroxycinnamiques de la pomme: identification, variations au cours de la croissance du fruit et metabolisme. These, Univ. Paris VI. 167 p.

Marigo G and Boudet A M 1975 Role des polyphenols dans la croissance. Definition d'un modele experimental chez Lycopersicon esculentum. Physiol. Plant. 34, 51-55.

Monties B 1975 Rayonnement ultraviolet et photosynthese. In Photosynthcsc ct Production Vegetate. Ed. C Castes. pp 193-215. Gauthier-Villars Publishers, Paris, France.

Pinto Contreras M 1981 Etude des coiits energetiques de la croissance de plantules de ble (Triticum aestivum) lors du

Nand growth of tomato seedlings 241

passage de !'heterotrophic a !'autotrophic. These INA-PG, Paris. 77 p.

Sihand P 1981 Croissance, nutrition et production du mil (Penisetum typhoides). Essai d'analyse du fonctionnement du mil en zone sahelienne. These d'Etat, Univ. de Mont­pellier. 302 p.

Suniaga Quijada J 1991 Nutrition azotee de la !ornate de type determine, issue de semis. Analyse de la croissance et du developpement au stadc jcunc. These de doctoral, Uni­versite de Rennes I. 180 p.

Widders I E 1989 Pretransplant treatments of N and P influence growth and elemental accumulation in tomato seedlings. J. Am. Soc. Hortic. Sci. 114, 416-420.

M.A. C. Fragoso and M.L. van Beusichem ( eds.) Optimization of plant nutrition 243-250, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-092

Effects of fertilization levels in two farming systems on senescence and nutrient contents in potato leaves

A. BERCHTOLD 1 , J.-M. BESSON2 and U. FELLER1

1 Institute of Plant Physiology, University of Bern, Altenbergrain 21, CH-3013 Bern, Switzerland; 2Swiss Federal Research Institute for Agricultural Chemistry and Environmental Hygiene, Schwarzen­burgstrasse 155, CH-3097 Liebefeld, Switzerland

Key words: farming system, fertilization, magnesium, nitrogen, potassium, potato, proteolytic enzymes, senescence, Solanum tuberosum L.

Abstract

The influence of fertilization on senescence and nutrient remobilization in potato leaves was investi­gated in two farming systems on a soil with a poor potassium availability. The 'Conventional' farming system followed good local practices including industrial products, while in the 'Bio-Dynamic' farming system industrial fertilizers and synthetic pesticides were avoided. Potassium concentrations in the dry matter of mature leaves varied over a wide range. Nitrogen compounds (protein, chlorophyll) were less affected, and phosphorus concentrations in the dry matter were similar. Magnesium and potassium concentrations in the leaves were negatively correlated. In both farming systems senescence was advanced in plants with a low nutrient supply. Alkaline pyrophosphatase and aminopeptidase activities (in general highest in expanding and mature leaves) were lower and endopeptidase activities peaked earlier on plots with low fertilizer levels. A high percentage of potassium was remobilized from senescing leaves on unfertilized plots, but the phosphorus concentration remained high at the end of the season. The results suggest that the differential net remobilization of nitrogen, phosphorus and potassium depended on actual source/sink relations in the plants.

Introduction

Leaf senescence leads to the death of the organ and is characterized by a net degradation of pro­teins and of chlorophyll as well as by changes in the enzyme pattern (Nooden and Leopold, 1988). Mobile nutrients can be remobilized from sen­escing leaves and translocated via the phloem to other plant parts (Hanway and Weber, 1971; Marschner, 1986; Schenk and Feller, 1990). Expanding leaves and tubers may act as major sinks in potato plants. In mature leaves nitrogen is mainly present in chloroplast constituents (e.g. proteins), which must be degraded prior to the export of the nitrogen during senescence (Feller, 1990). Proteins can be hydrolyzed by a series of exo- and endopeptidases to free amino acids

(Dalling, 1986; Ryan and Walker-Simmons, 1981). The activities of various peptide hydro­lases change differently during the senescence of corn (Feller et al., 1977) and wheat (Feller, 1983; Frohlich and Feller, 1992) leaves. Potato tubers and leaves are rich in proteinase inhibitors (Ryan 1973; Santarius and Belitz, 1978). How­ever, these inhibitors do not affect major endog­enous proteinases and it is therefore unlikely that they are involved in the control of intracel­lular proteolysis (Santarius and Belitz, 1978). The enzyme pattern changes during senescence in favour of catabolic enzymes (Feller, 1990; Frohlich and Feller 1991; 1992). Although sever­al aspects of senescence have been investigated in detail, only very restricted information con­cerning the effects of the nutrient status on leaf

244 Berchtold et al.

senescence, on the change in the enzyme pattern and on the nutrient redistribution in the field is available (Nooden and Leopold, 1988). Recently it was reported that the phosphorus nutrition does not play a major role in the control of leaf senescence in soybean (Crafts-Brandner, 1992).

A large field study (DOC) with three farming systems (Bio-Dynamic, Organic and Conven­tional) was started in 1978 on a loamy loess soil in Therwil near Basel (Besson and Niggli, 1991 ). Potato cultivation followed after a two year period with a grass/ clover mixture. Details con­cerning fertilization, plant protection, yield and some quality parameters for potato tubers were published previously (Besson et a!., 1991 ). This field study offered the unique chance to investi­gate the influence of the nutrient supply on senescence and nutrient remobilization. The aim of the work presented here was to identify in two farming systems the effects of fertilization on the nutrient dynamics in potato plants by analyzing macronutrient contents and selected enzyme activities in a particular leaf throughout the growing season.

Materials and methods

The experimental design of the 'DOC' field study on Birsmattenhof in Therwil near Basel was reported previously (Besson and Niggli, 1991). Plots with the different treatments consid­ered for the work presented here were available in 4 replicates on a loamy loess soil. Plants grown on fertilizer level 0 (unfertilized), on fertilizer level 1 and on fertilizer level 2 (2-fold

quantity applied on level 1) were analyzed for the farming systems "Bio-Dynamic" (D) and "Conventional" (C). The nutrient supply by fertilization in these two farming systems is listed in Table 1 for the growth seasons investigated. Potatoes (Solanum tuberosum L., cv. Ostara) were grown in 1981 and 1982 on different plots following a two year period with a grass/ clover mixture (Besson and Niggli, 1991; Besson et a!., 1991 ). The tuber yield as well as the availability of K and P in the soil before planting and after harvest are summarized in Table 2. The contents of mineral nitrogen in the soil were far less affected by fertilization than the contents of K and P (Alfoldi eta!., 1992). Leaf samples (each containing 6 well developed peripheral leaflets) were collected repeatedly during the growing season and transported on ice to the laboratory. At the first sampling date these leaflets were fully expanded at the top of the plant. To detect physiological changes in a particular leaf (time courses), leaflets from the same position were collected throughout the season. Samples for the analysis of enzyme activities and of nitrogen compounds were stored frozen (-20°C) prior to the extraction. Samples for the quantification of K, Mg, Ca and P were dried at lOOoC for 24 h. The dry matter was weighed and all results were computed on a dry matter basis.

Samples containing 6 frozen leaflets were extracted in 24 mL extraction buffer (50 mM Na­acetate pH 5 .4, 0.1% mercaptoethanol, and 1% polyvinylpolypyrrolidone) as described previous­ly (Feller, 1983). The homogenate was filtered through Miracloth (Calbiochem, San Diego) and the filtrate was used to analyze chlorophyll

Table 1. Nutrient supply by fertilization in the farming systems 'Bio-Dynamic' (DO, Dl, D2) and 'Conventional' (CO, Cl, C2). The values forDland Cl were calculated from Besson et al. (1991). Other plots in the same farming systems were fertilized with 200% of the nutrient quantities listed (D2, C2) or remained unfertilized (DO, CO)

Treatment Fertilization (kg ha-l)

N p K Ca Mg

1981 Dl 51 16 17 129 13 Cl 127 39 184 59 23

1982 01 84 26 46 134 16 Cl 123 33 142 38 14

Fertilization and senescence in potato leaves 245

Table 2. Yield (fresh weight of tubers) and nutrient availability in the soil (calculated from Besson et al., 1991, 1992). Nutrients in the soil were analyzed before fertilization and planting as well as after harvest in the farming systems 'Bio-Dynamic' (DO, D1, D2) and 'Conventional' (CO, C1, C2). Unfertilized plots (DO, CO) were compared with those on low (D1, C1) and higher (D2, C2) fertilizer levels

Measurement Farming system and fertilization level

DO D1

1981 Yield (t ha · 1) 15.4 20.6 K before planting (mg kg 1) 6.6 5.8 K after harvest (mg kg - 1 ) 5.0 5.2 P before planting (mg kg -1) 1.2 1.3 P after harvest (mg kg - 1) 1.1 1.3

1982 Yield (t ha -- 1 ) 20.5 33.6 K before planting (mg kg - 1) 5.0 5.0 K after harvest (mg kg - 1) 4.2 3.7 P before planting (mg kg - 1) 0.8 0.7 P after harvest (mg kg - 1) 0.7 0.9

(Strain et al., 1971 ), total proteins (Bradford, 1976) and free amino groups with a ninhydrin reagent (Cramer, 1958). A part of the extract was centrifuged for 10 min at 4000 g and the supernatant was desalted by centrifugation through Sephadex G-25 as described by Feller et al. (1977). The desalted extract was used to measure the activities of : aminopeptidase, with L-leucine-p-nitroanilide as substrate; carboxy­peptidase, with N-carbobenzoxy-L-phen­ylalanine-L-alanine as substrate; endopeptidase at pH 5.4 with azocasein as substrate (Feller, 1983); and pyrophosphatase (Salgo and Feller, 1986).

Samples of 100 mg dry matter were kept in glass tubes for 4.5 h at 540°C. The ash was dissolved in 0.5 mL 10 N HCl and 49.5 mL H 20 were added. This solution was used to determine P colorimetrically with a vanadate-molybdate reagent. The elements K, Mg and Ca were detected by atomic absorption spectrometry after appropriate dilution with 1.267 gL -I CsCl sup­rapur in 0.1 N HCl (for K) or with 13.37 gL -I LaC1 3 7 H 20 in 0.1 N HCl (for Mg and Ca).

Results

From the study in 1981 (fourth year after the change to different farming and fertilization procedures) it became evident that senescence

D2 co Cl C2

29.1 22.2 49.1 57.9 5.0 5.0 5.8 8.3 6.1 4.2 6.1 15.4 1.4 1.7 2.0 2.4 1.6 1.0 2.0 2.8

43.1 21.1 54.8 62.4 5.0 4.2 5.0 8.3 5.0 3.7 6.5 12.3 0.8 0.8 0.7 1.7 1.3 0.7 1.3 2.0

and nutrient remobilization in potato leaves strongly depended on the nutrient supply. Senescence, as judged by the net chlorophyll degradation, was advanced on unfertilized plots compared to the other treatments (Table 3). Potassium concentrations in the dry matter of mature leaves varied over a very wide range and were highest on C2 plots. Magnesium and calcium concentrations were far less influenced. Pyrophosphatase activity (an enzyme involved in biosynthetic processes) was lower on un­fertilized plots. Azocaseinase (a catabolic en­zyme) reached highest activities on well fertil­ized plots later than on plots with a low nutrient supply.

The investigation was repeated in 1982 (fifth season in the field study) on other plots subject­ed to the same farming and fertilization tech­niques. The main results of the two seasons were consistent (Table 3; Figs. 1, 2 and 3). The concentrations of some macronutrients are shown in Figure 1 and indicate that potassium was most severely affected by the fertilization level. In general, magnesium concentrations in the leaves were highest on unfertilized plots. On the other hand calcium concentrations in the dry matter were far less influenced and increased only slightly throughout the growing season. Phosphorus levels in the leaves were very con­stant. The highest percentage of the potassium present in mature leaves was remobilized on

246 Berchtold et al.

Table 3. Senescence and macronutrients in leaves of potato plants grown during summer 1981 in the farming systems 'Bio-Dynamic' (DO, D1, D2) and 'Conventional' (CO, C1, C2). Unfertilized plots (DO, CO) were compared with those on low (Dl, C1) and higher (D2, C2) fertilizer levels. Means of 4 replicates are listed. Values in the same row followed by the same letter are not significantly different at the p 0.05 level (Studcnt-Newman-Keuls test)

Date Farming system and fertilization level

DO D1 D2 co C1 C2

Potassium (mg K g- 1 dry matter) June 2 10.7 c 13.1 c 20.0 be 17.1 c 27.9 ab 31.8a June 30 13.0 d 14.2 d 22.0 c 13.6d 34.7 b 45.5 a July 28 5.7 c 8.6 c 13.2 c 8.9 c 26.8 b 46.9 a

Magnesium (mg Mg g- 1 dry matter) June 2 3.1 a 3.5 a 3.5 a 4.4a 2.8 ab 2.0b June 30 10.6 ab 10.0 ab 8.8 a 10.6 ab 5.9 b 3.5 c July 28 12.4 ab 13.5 ab 12.3 a 14.5 a 6.6 b 3.5 c

Calcium (mg Ca g - 1 dry matter) June 2 14.1 a 17.3 a 16.7 a 15.4 a 14.5 a 14.2 a June 30 36.5 ab 38.4 ab 39.4 a 32.3 ab 31.1 b 23.4c July28 36.9 ab 38.3 ab 40.5 a 37.5 ab 32.6 ab 28.3 b

Chlorophyll (mg chlorophyll g - 1 dry matter) June 2 3.4 b 3.5 b 3.7b 3.7 b 4.4 a 4.6 a June 30 3.1 b 3.1 b 3.3 b 3.3 b 4.7 a 5.4 a July 28 0.7 c l.Oc 1.5 be 1.2 be 2.0 ab 2.6 a

Pyrophosphatase activity (mmol h - 1 g 1 dry matter) June 2 5.4d 5.5 d 5.8 d 6.6 c 7.9b 8.6 a June 30 3.0a 3.0a 3.0 a 3.0 a 3.8a 4.4 a July 28 0.1 a 0.3 a 0.6 a 0.4 a 0.7 a 0.9 a

Azocaseinase activity (mg h - 1 g - 1 dry matter) June 2 5.2a 5.6a 5.6 a 5.3 a 4.6a 3.5 a June 30 21.8a 20.3 a 21.5a 18.7 a 20.9 a 19.1 a July 28 10.2c 13.6c 15.2 be 15.2 be 20.2 ab 21.7 a

unfertilized plots. In contrast, major quantities of phosphorus were exported only from the leaves on well fertilized plots.

The dry matter per leaflet was only slightly increased on plots with a high nutrient supply (Fig. 2). Protein and chlorophyll levels in mature leaves increased by 20 to 50% from unfertilized (DO, CO) to well fertilized (C2) plots. The net decrease in the chlorophyll and protein quan­tities was more pronounced in plants with a low nutrient supply. No accumulation of free amino acids was observed during the net degradation of proteins.

Pyrophosphatase activity was initially (June 8) very similar for all treatments, but it decreased more rapidly on unfertilized plots (Fig. 3). Simi­lar tendencies were observed for leucine amino-

peptidase. No clear trend was detected for car­boxypeptidase. The activity of this vacuolar enzyme remained high during the period of net protein degradation. Azocaseinase activity (endopeptidase) showed interesting time courses for the various treatments. At the end of the season this enzyme was more active in plants on well fertilized plots.

Discussion

The fertilization effects were more pronounced on the 'Conventional' than on the 'Bio-Dynamic' plots. However, it must be borne in mind that at the same fertilization level more macronutrients were applied to 'Conventional' plots (Besson et

Fertilization and senescence in potato leaves 247

Bio-Dynamic Conventional

•oo co1 EJD2 • CO C Cl El C2

Potassium

>. ... "C

Cll .s::. ...., c

Phosphorus

3

2

8 24 June

7 20 July

8 24 7 20 June July

Fig. 1. Concentrations of potassium, magnesium, calcium and phosphorus in the dry matter of potato leaves collected during summer 1982. Unfertilized plots (DO, CO) were compared with those on low (Dl, Cl) and higher (D2, C2) fertilizer levels in the farming systems 'Bio-Dynamic' and 'Conventional'. Means and standard deviations of 4 replicates are shown.

a!., 1991). Taking this fact into consideration, the influence of the fertilizer quantity on the parameters investigated in this study were similar for the two farming systems. Although phos­phorus, potassium and magnesium are mobile within the plant (Marschner, 1986; Schenk and Feller, 1990), a marked loss from senescing leaves was observed only for potassium. Our results lead to the conclusion that potassium was limiting on unfertilized plots. The concentration of this element in the dry matter of leaves varied over a very wide range. Considerable potassium quantities were lost from senescing leaves on

plots with a low potassium supply. Nitrogen compounds (chlorophylls, proteins) decreased later than potassium. No major effects of the growth conditions on the level of free amino acids was observed. This result indicates that the metabolism and the translocation of amino acids via the phloem were rapid enough to avoid an accumulation. The increased magnesium concen­trations in leaves with a low potassium level were most likely caused by a compensation during the nutrient uptake into the roots. A more rapid leaf senescence was detected in unfertilized plants. The activities of the investigated enzymes were

248 Berchtold et al.

0)

E

0)

0)

E

6

4

2

0

50 40 30 20 10-0

Bio-Dynamic Conventional • DO C D1 § D2 .co CC1 §C2

Chlo

I I ~~ J~ Proteins

T

T

I j~ 140· Free amino acids

0 E 2:

120· 100 80 60 40 20 0

200·

150

100

50·

n 8 24 June

I I I Dry weight

II II 7 20 8 24 7 20

July June July

Fig. 2. Nitrogen remobilization in potato leaves during summer 1982. Unfertilized plots (DO, CO) were compared with those on low (Dl, Cl) and higher (D2, C2) fertilizer levels in the farming systems 'Bio-Dynamic' and 'Conventional'. Means and standard deviations of 4 replicates are shown.

similar on a dry matter basis for all treatments in young leaves, but the changes in the enzyme pattern from anabolic (pyrophosphatase) to catabolic (endopeptidase) activities occurred ear­lier in plants with a low nutrient supply. Amino­peptidase was most active in young and mature leaves compared to senescing leaves. The changes in the enzyme pattern from anabolic to catabolic activities were clearly influenced by the availability of nutrients in the soil. The prop­erties of young leaves were less affected by

fertilization than the nutrient losses from senesc­ing leaves.

Acknowledgements

We thank Dr A Fleming for improving the English of the manuscript. Financial support for the DOC field study was provided by the Swiss Federal Office for Agriculture. The senescence

Fertilization and senescence in potato leaves 249

Bio-Dynamic Conventional

•oo cot §D2 • CO C C1 § C2

Pyrophosp

8 24 7 20 8 24 7 20 June July June July

Fig. 3. Activities of pyrophosphatase and of peptide hydro lases in potato leaves collected during summer 1982. Unfertilized plots (DO. CO) were compared with those on low (D1, C1) and higher (D2, C2) fertilizer levels in the farming systems 'Bio-Dynamic' and 'Conventional'. Means and standard deviations of 4 replicates are shown.

investigations were partially supported by Swiss National Science Foundation (Projects 3.067-0.81 and 31-30805.91).

References

Alfiildi Th, Mader P, Schachenmann 0, Niggli U and Besson J-M 1992 DOK-Versuch: vergleichende Langzeitunter­suchungen in den drei Anbausystemen biologisch­Dynamisch, Organisch-biologisch und Konventionell. III.

Boden: Nmin-Untersuchungen, 1. und 2. Frucht­folgeperiode. Schweiz. Landw. 32, 59-82.

Besson J-M and Niggli U 1991 DOK-Versuch: vergleichende Langzeit-Untersuchungen in den drei Anbausystemen biologisch-Dynamisch, Organisch-biologisch und Konven­tionell. I. Konzeption des DOK-Versuchs: 1. und 2. Fruch­tfolgeperiode. Schwei. Landw. 31, 79-109.

Besson J-M, Meyre S and Niggli U 1991 DOK-Versuch: vergleichende Langzeit-Untersuchungen in den drei An­bausystemen biologisch-Dynamisch, Organisch-biologisch und Konventionell. II. Ertrag der Kulturen: Kartoffeln, 1. und 2. Fruchtfolgeperiode. Schweiz. Landw. 31, 127-155.

Besson J-M, Michel V and Niggli U 1992 DOK-Versuch:

250 Fertilization and senescence in potato leaves

vergleichende Langzeituntersuchungen in den drei An­bausystemen biologisch-Dynamisch, Organisch-biologisch und Konventionell. II. Ertrag der Kulturen: Kunstwiesen, 1. und 2. Fruchtfolgeperiode. Schweiz. Landw. 32, 85-107.

Bradford M M 1976 A rapid and sensitive method for the quantification of microgram quantities of protein using the principle of protein dye binding. Anal. Biochem. 72, 248-254.

Crafts-Braodner S J 1992 Phosphorus nutrition influence on leaf senescence in soybean. Plant Physiol. 98, 1128-1132.

Cramer F 1958 Papierchromatographie, Verlag Chemie, Wcinheim, 102 p.

Dalling M J (Ed.) 1986 Plant Proteolytic Enzymes, Volume 1. CRC-Press, Boca Raton, 157 p.

Feller U K, Soong T-S T and Hageman R H 1977 Leaf proteolytic activities and senescence during grain develop­ment of field-grown corn (Zea mays L.). Plant Physiol. 59, 290-294.

Feller U 1983 Senescence and proteolytic activities in de­tached leaves and detached shoots of wheat. Physiol. Veg. 21, 93-102.

Feller U 1990 Nitrogen remobilization and protein degra­dation during senescence. In Nitrogen in Higher Plants. Ed. Y P Abrol. pp 195-222. Research Studies Ltd., Somerset, England.

Frohlich V and Feller U 1991 Effect of phloem interruption on senscence and protein remobilization in the flag leaf of field-grown wheat. Biochem. Physiol. Pflanzen 187, 139-147.

Frohlich V and Feller U 1992 Effect of phloem interruption on endopeptidase and aminopeptidase activities in flag leaves of field-grown wheat. Biochem. Physiol. Pflanzen 188, 13-21.

Hanway J J and Weber C R 1971 N, P and K percentages in soybean (Glycine max [L.] Merill) plant parts. Agron. J. 63, 286-290.

Marschner H 1986 Mineral Nutrition of Higher Plants. Academic Press, London, 674 p.

Nooden L and Leopold A C (Eds.) 1988 Senescence and Aging in Plants. Academic Press, San Diego, 526 p.

Ryan C A 1973 Proteolytic enzymes and their inhibitors. Annu. Rev. Plant Physiol. 24, 173-196.

Ryan C A and Walker-Simmons M 1981 Plant proteinases. In The Biochemistry of Plants, Volume 6, Proteins and Nucleic Acids. Ed. A Marcus. pp 321-350. Academic Press, New York.

Salgo A and Feller U 1986 Effects of the germination time on enzyme stabilities in extracts from wheat endosperms. Bot. Helv. 96, 273-282.

Santarius K and Belitz H-D 1978 Proteinase activity in potato plants. Planta 141, 145-153.

Schenk D and Feller U 1990 Effect of phloem interruption on leaf senescence and nutrient redistribution in wheat (Tri­ticum aestivum). In Plant Nutrition- Physiology and Ap­plications. Ed. M L van Beusichem. pp 121-125. Kluwer Academic Publishers, Dordrecht.

Strain H H, Cope B T and Svec W A 1971 Analytical procedures for isolation, identification, estimation and investigation of the chloropylls. In Methods in Enzymolo­gy, Vol. 23. Eds. S P Colowick and N 0 Kaplan. pp 452-476. Academic Press, New York.

Reprinted from Plant and Soi/154: 81-88, 1993.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 251-257, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-198

Relationship between biochemical indicators and physiological parameters of nitrogen and physiological plant age

J.L. VALENZUELA1 , M. GUZMAN 1, A. SANCHEZ1 , A. DEL RI0 2 and L. ROMER0 2

1 Departamento Biologia Vegetal, Campus Universitario de Almeria, E-04071 Almeria, Spain and 2Departamento Biologia Vegetal, Facultad de Ciencias, Universidad de Granada, E-18071 Granada, Spain

Key words: biochemical indicator, cucumber, nitrate reductase, tomato

Abstract

The families, Solanaceae and Cucurbitaceae, differ mainly in the size of the sink and the length of their biological cycle, although their phenological stages overlap in time to some extent. Leaves were sampled during the period of greatest metabolic activity, which coincided with a plural of morphological changes, in order to analyze nitrogen indicators and parameters. Nitrogen concentration decreased over time in the leaf, as did N0 3-ions, dry weight, amino acids, total and soluble protein. NH4-ions, nitrate reductase and productivity peaked during the period of greatest metabolic activity, coinciding with flowering and the initial and final phases of fruit ripening, and were lowest during foliar senescence. Total and soluble vegetative index (relation between mobile N, P, K and immobile Ca, Mg) declined, and transient alterations coincided with certain morphological and metabolic changes. All physiological alterations during the different physiological stages were seen in both species regardless of the size of the sink, however the magnitude of the alterations was conditioned by the sink and by exogenous and endogenous N levels. Organ senescence can be defined as a series of metabolic changes in the mature plant which include a decrease or increase in enzymatic activity, proteolyis, pigment degradation and death.

Introduction

The metabolic changes that occur during differ­ent phenological stages have generally not been taken in account in the interpretation of foliar analysis data, hence foliar analysis has fallen short of fully explaining the mechanisms respon­sible for the plant's life processes. The transition from one stage to the next is characterized by changes in biochemical activity and a restructur­ing of primary metabolism. These fluctuation influence the entire plant, and consequently the analytical data obtained in each phenological stage.

Biochemical and physiological indicators be­have in specific ways in each organ, and are also influenced by the characteristics of sink. Thus

changes in these indicators are the result of a steady exportation of nutrients from one organ to another.

The vegetative index (VI) provides informa­tion on the plant's metabolic activity throughout the growth cycles, when the different stages (flowering, pollination, fruit development and ripening) can occur in rapid succession. Rises and falls in nutrient levels in leaf have been interpreted (Smith, 1962; Ulrich, 1952) as lead­ing to changes in dry weight, an increase imply­ing that nutrients are more diluted unless the mineral supply compensates for this effect (Loneragan, 1968).

The vegetative index accurately reflects the plant aging process and consequent decrease in levels of three fundamental nutrients, as cell

252 Valenzuela et al.

membranes harden and become nonfunctional as a result of Ca accumulation. The vegetative index was proposed by Pijoan-Pascual (1976) and Guzman and colleagues (1984) as an attempt to correlate the concentrations to macronut­rients. According to these authors, N-P-K levels show a clear tendency to decline during the vegetative cycle, while Ca and Mg tend to increase. The vegetative index can therefore be summarized in the following formula:

N + 10P + K V.I. = Ca + Mg x 0.365

in which nutrients are expressed as percentages. This study examines the behaviour of bio­

chemical and physiological indicators in two horticultural species which differ in the length of their vegetative cycle (long in tomato and short in cucumber) and in the characteristics of their sink. The changes in the vegetative index in response to the amount of N applied to the growth medium were also studied.

Materials and methods

Growth conditions

The plants were grown in "Parra!" type ( tentshaped) greenhouses constructed of wood and stainless steel wire (frame) covered with clear 800 gauge polyethelene. Each greenhouse measured 20 m wide and had a useable ground surface area of 2000 m 2 • Localized fertilization­irrigation was achieved with a long-distance interlineal drip system at a flow rate of 4 L h - 1 •

The crops were high-yielding (under green­house conditions) annuals: Dutch cucumber (Cucumis sativus L. cv. Pepinex) and tomato (Lycopersicon esculentum Mill. cv. Dombo). A total of 16 plots was used to study each species with various treatments and replications. Flood­ing and nutrient leaching did not occur. Each plot measured 7m 2 and contained 14 plants.

Throughout the biological cycle of the crops, treatments of different N supply were applied: N 1 (7.5g m- 2), N2 (10.0g m- 2 ), N3 (20.0g m - 2 ) and N4 (30.0 g m - 2). Four replications per treatment were studied in each species. Fertiliza-

tion was supplemented with P (10.0 g m - 2), K (30.0g m- 2 ), Mg (0.15g m- 2 ), Mn (2.5mg m - 2), Fe (3.0 mg m - 2), Cu (2.0 mg m - 2 ) and Zn (1 mg m - 2 ). The nutrients were applied as am­monium nitrate, potassium nitrate or sulfate, except for P, which was applied as phosphoric acid.

The soil properties (20-40 em deep) were as follows: Texture: loamy-sand; sand ( 48.2% ), silt (36.1%), clay (15.7% ), organic matter (205 mg lOOg- 1); CaC0 3 equivalent (17%); active CaC0 3 (6.38%); P20 5 (22.75 f.Lg g- 1); K 20 (916f.Lg g- 1); pH (1:1) (8.20) and electrical conductivity (1: 2) (0.1058 S m - 1 ).

The properties of the irrigation water were: pH, 8.25; E.C., 0.2113 S m- 1; N, 0.82 f.Lg mL - 1;

P, 0.16JLg mL- 1 ; K, 14.76/-Lg mL- 1 ; Ca, 50.01 f.Lg mL - 1 ; Mg,55.80 f.Lg mL - 1 ; Na, 286.29 f.LL - 1 ; Cl 284.23 f.Lg mL - 1 ; Fe, 0.45 f.Lg mL - 1 ; Mn, 0.13 f.L g mL - 1 ; Zn, 0.05 JLg mL - 1;

Cu, 0.14 f.Lg mL - 1 ; Sodium adsorption ratio 48.45 and potassium adsorption ratio 2.46.

Analyses

Leaves were sampled every two weeks from the middle third of each plant. After washing in nonionic soap and des tilled water, part of this material was dried and another part was refriger­ated until fresh material analyses were carried out.

Dry material Dried, ground leaves were digested in sulfuric acid and macronutrients were determined with Wolf's method (1982). Extraction with 1N HCl was followed by a second sulfuric digestion to determine soluble macronutrients and N as NH; (Guzman et a!., 1986). N03-ions were deter­mined by aqueous extraction followed by reduc­tion in a Zn-Cd column (Aguilar et a!., 1982).

Total protein Dry plant matter was digested in sulfuric acid with an oxidizing agent. The values for N as NH; were multiplied by a correction factor to obtain protein concentration (A.O.A.C., 1970).

Fresh material

Nitrate reductase activity (NRA) was determined with method of Harper and Hageman (1972). Disks of plant tissue were placed in a test tube with 20 mL infiltration and incubation medium consisting of 50 mM KN0 3 , 100 mM phosphate buffer (pH 7.5) and 1% propanol. The samples were then vacuum-pumped and incubated for 60 min at 27°C (Bar-Akiva and Sternbaum, 1966). The nitrate (N02-ion) obtained was mea­sured colorimetrically as described by Snell and Snell (1949). The NRA was expressed in fA-IDOl N0 2 g - 1 fresh wt h - 1 .

Soluble protein One gram of plant material was homogenized with 10 mL tris-HCl buffer, and the homogenate was centrifuged. The superna­tant was added with 5 mL cold trichloracetic acid and centrifuged again. After discarding the supernatant, the precipitate was washed and oven-dried, then dissolved in 1 M sodium hy­droxide and heated for 10 min at 70°C. Soluble proteins were determined in this solution with the method of Bradford (1976). Soluble proteins was expressed in mg g - 1 fresh wt.

Soluble amino acids were determined with the spectrophotometric method proposed by Moores and Stein (1954). An 0.5 mL aliquot of the

Nitrogen biochemical indicators and plant age 253

homogenate was added with 1.5 mL ninhydrin reagent. After adding 8 ml 50% propanol, the solution was placed in a water bath at 100°C for 20 min and then read at 570 nm against a stan­dard glycocol-ninhydrin curve. The amino acids were expressed in f.tg glycocol g - 1 fresh wt.

Productivity Tomato and cucumber fruits suit­able for export should remain firm and uniform in color after picking and storage, and show no evidence of physical damage (scars, blemishes, cracks, etc.). After storage the produce should be uniform in shape and maintain good taste characteristics. The fruit production was ex­pressed in kg/pl./0.5 m -z.

Results and discussion

Nitrogen, a mobile macronutrient according to Loneragan's classification (1968), is exported from the leaf to other organs, a process which is independent of the endogenous or exogenous N concentration in both tomato and cucumber. Endogenous accumulation of N can occur as greater amounts of nutrients are applied, a situation that can curtail production. This was seen in both tomato and cucumber (Table 1) at a time which coincided with foliar senescence. Maximal N levels however did not coincide with

Table 1. Time course of N (mg g 1 d.wt.), productivity (kg/pl./0.5 m2 ) and dry weight (mg!leaf or leaflet) at different phenological stages

Plant age Phenological Treatments of nitrogen (days) stage' ----------------------------------

30 gm-2 ofN

N Prod. dr.w. N Prod. dr.w. N Prod. dr.w. N Prod. dr.w.

Tomato 115 1-2 45.0 a* 0.14 b 588 a 48.0 a 0.33 b 513 a 48.5 a 0.08c 576a 50.3 a 0.09 b 622 a 130 1-2-3 4l.Ob 0.14 b 564 a 42.0 b 0.60 a 551 a 45.0b 0.09b 512 b 44.8 b 0.10 a 435 b 145 1-2-3-4 41.0 b 0.70 a 395 b 43.0 b 0.17 c 406 b 43.5 c 0.15 a 399c 40.7 c 0.10 a 384 be 160 2-3-4 36.5 c 0.09c 400 b 38.8 c 0.12e 363 c 38.5 d 0.09 b 417 c 4l.Oc 0.08c 395 be 175 2-3-4-5 37.3 c 0.08d 400 b 37.5 d 0.09c 366 c 38.0 d 0.07 d 366d 39.0 d 0.06d 378c

Cucumber 110 1-2 27.0a 1.02 b 3356 a 29.8 a 1.11b 3512 a 32.8 a l.OOb 3391 a 34.8 a 1.01 b 3129 a 125 2-3-4 26.0 a 1.37 a 3036 b 25.0b 1.59 a 3509 b 25.8 b 1.51 a 3192 b 28.0b 1.53 a 2864c 140 3-4-5 20.8 c 0.67 c 2766c 21.5c 0.72c 3197 c 20.0c 0.79c 2734c 22.0c 0.78c 2600c

* Mean separation within columns by Duncan's multiple range test, 1% level. " 1: Flowering; 2: Fruit development; 3: Early fruit ripening; 4: Fruit ripening; 5: Foliar senescence.

254 Valenzuela et al.

peak productivity (at the end of the flowering period and during fruit development). Nitrogen reached lowest levels at the time of foliar senesc­ence. Thus changes in productivity are not di­rectly correlated with the amount of N applied or foliar N levels (Table 1).

The time course of NH; -ion concentration differs in the two species, with significant differ­ences between treatments and phenological stages in all cases. Highest concentrations in both tomato and cucumber were obtained in the treatment N4 • As the concentration of exogen­ous N rose, N0 3-ion levels also increased in the aerial parts of both species (Table 2). The differences in N0 3-ion concentration in the two species may result from the greater sensitivity of Solanaceae to Cl-ion, present in the high concen­trations in irrigation water (See Methods). Solanaceae have a mechanism to detect Cl- and N0 3-ions, based on the fact that high intracellu­lar concentrations of nitrates reduce considerably the influence of chlorides (Cram, 1973; van Diest, 1990). During the early phase of fruit ripening, NH; -ion concentrations were highest, possibly as a result of 1) proteolysis which releases ammonium ions for translocation to the fruit, and 2) increased the nitritoreductase activi­ty (Beevers and Hageman, 1983): foliar levels of N0 3-ions began to fall during this period, reach-

ing minimum values at foliar senescence in both species (Table 2).

Increased N content is characteristically associ­ated with greater dry weight. As shown in Table 1, dry weight in tomato rose in a linear fashion with treatments Np N2 and N3 , with no increase after treatment N 4 • In cucumber the first two treatments raised dry weight, whereas the other two lowered it as a result of excess N and its influence on the other nutrients. Increased N supply not only stimulates growth but also changes the plant's morphology. High N supplies stimulate shoot lengthening and modify leaf morphology. In tomato, N led to similar changes regardless of the amount applied or foliar level, with no statistically significant differences be­tween the different treatments.

The ability of N to increase dry weight of leaves declined gradually in both species as foliar senescence approached (Table 1). These de­creases are related to the gradual loss of meta­bolic activity in the leaf. Dry weight declines when the export of ions and organic compounds along with enzymatic disorganization/breakdown and inactivation, predominate over the importa­tion of nutrients. These phenomena are related to foliar and fruit senescence.

These annual plants accumulate considerable amounts of nitrates in a manner which is not

Table 2. Time course of nitrate reductase activity (JLmols N0 2 g- 1 fresh wt h- 1), ammonium (mgg- 1 dry wt.) and nitrate (meq g -I dry wt.) concentration at different phenological stages

Plant age Phenological Treatments of nitrogen (days) stage'

7.5 g m - 2 ofN 10gm 'ofN 20gm 2 ofN 30gm -2 ofN

NRA NH; No; NRA NH: No; NRA NH: NO; NRA NH: NO~

Tomato 115 1-2 4.58 b* 8.0c 198 a 5.04 b 9.0 be 210 a 5.06 b 9.3 d 224a 5.28 b 5.0 c 226 a 130 1-2-3 3.39c 7.0d 182 b 3.84c 9.5 b 190b 4.47 c 10.0c 198 b 3.74 c 1l.Oa 200b 145 1-2-3-4 5.49 a 10.5 a 170c 5.94 a 10.0 a 187 c 6.56 a 10.8 b 196 b 5.71 a 11.0 a 209 b 160 2-3-4 3.08 c 10.5 a 157 d 2.61 d 10.0 a 165 a 2.39 d 11.3a 173 c 3.15 d 10.5 b 188 c 175 2-3-4-5 1.43 d 9.0 b 155 d 1.82 e 8.8 c 169 d 1.68 e 8.3 e 174c 1.68 e 9.8 b 180 c

Cucumber 110 1-2 5.87 a 4.8 b 124 a 6.67 a 5.8 b 130a 5.86 a 7.8 b 149 a 5.92 a 8.0 b 150 a 125 2-3-4 4.67 b 6.8 a 113 b 5.62 b 6.8 a 113 b 5.24 b 8.0 a 111 b 4.82 b 9.5 a 124 b 140 3-4-5 3.70 c 4.5 c 92c 3.63 c 4.8 c 95 c 1.65 c 5.0c 90c 3.29 c 5.0 c 95 c

* Mean separation within columns by Duncan's multiple range test, 1% level. '1: Flowering; 2: Fruit development; 3: Early fruit ripening; 4: Fruit ripening; 5: Foliar senescence.

Nitrogen biochemical indicators and plant age 255

linearly correlated with the amount of N applied. As seen in Table 2, nitrate reductase activity (NRA) was not regulated by applied N, but was correlated with endogenous N0 3 levels. In tomato, enzyme activity was inhibited in associa­tion with a greater accumulation of nitrates. Inhibitions was less marked when nitrate levels fall (Treatments N 1 and N 4 ) (Table 2). The influence of endogenous nitrate concentrations on NRA was similar in cucumber leaves; highest levels of enzyme activity were recorded in plants treated with N2 , which had the lowest nitrate content of all fertilizers tested. The high levels of nitrates in treatments N 3 and N 4 inhibited NRA, whereas N 4 paradoxically behaved like N 1 ,

which reduced N0 3-ion in leaves (Table 2). Soluble amino acid concentration was not

directly related to nitrate reductase activity in tomato. Highest levels were obtained with treat­ment N2 in comparison with all other treatments, which led to similar amino acid levels. In cucumber, however, these two parameters were well correlated (Tables 2 and 3).

According to Streit and Feller (1982) and Santoro and Magalhaes (1983), NRA changes during leaf ontogenesis, and reaches highest values when leaf surface area is greatest. En­zyme activity peaked at the end of the flowering period and beginning of fruit ripening in tomato,

but at the beginning of fruit development in cucumber. In both species this parameter fell sharply after reaching maximum values, the activity of this enzyme involved in the assimila­tion of inorganic N decrease during senescence (Lauriere, 1983). The behavior of N0 3-ions was similar (Table 2), in contrast to the findings of Santoro and Magalhaes (1983), but in agreement with the observations of BJorn-Zandstra and Lampe (1983). We stopped supplying nitrates to tomato and cucumber plants at the end of the biological cycle in order to mobilize nitrate ions from their site of storage, i.e. vacuoles (Mar­tinoia et a!., 1983) to their site of reduction, i.e. the cytoplasm (Rufty et a!., 1982). The sudden drop in enzyme activity may be responsible for the rapid fall in productivity (Table 1) due to the limited rate of nitrate reduction.

Soluble amino acids show the same trends as nitrate reductase activity and N0 3-ions (Tables 2 and 3) in cucumber but not in tomato. During the beginning of fruit development, NRA de­clines along with amino acid concentration, al­though both parameters rise thereafter because of the increased demands of the main sink (fruit), then fall once again. This pattern was seen in all treatments except N4 ; amino acid concentrations rose steadily in this group as a result of proteolysis, a sign of approaching

Table 3. Time course of soluble aminoacid concentration (!Lg glycocol g -I fresh wt.), total (mg g -I dry wt.) and soluble protein concentration (mg g -I fresh wt.) at different phenological stages

Plant age Phenological Treatments of nitrogen (days) stage

7.5gm- 2 ofN lOgm-'ofN 20 g m- 2 ofN 30 g m- 2 ofN

Amin. P. tot. P. sol. Amin. P. tot. P. sol. Am in. P. tot. P. sol. Amin. P. tot. P. sol.

Tomato 115 1-2 1.21 b 281 a 116 a 1.21 b 300 a 177a 1.15 b 319 a 192 a 1.07c 314 a 206 a 130 1-2-3 0.90d 256 b 63 b 1.30 a 262 b 74 b 1.14 b 284 b 58 b 1.17b 280 b 56 b 145 1-2-3-4 1.39 a 250 b 57 c 1.32 a 263 b 56 c 1.26 a 278c 45 c 1.23 a 288 b 33 c 160 2-3-4 1.24 b 222 c 23 d l.llc 241 c 31 d 1.28 a 244d 23 d 1.24 a 262c 31 c 175 2-3-4-5 1.15 c 225 c 16 e 1.13c 230 d 18 e l.lOc 244d 21 d 1.25 a 256c 21 d

Cucumber 110 1-2 !.30 a 169 a 108 a 1.47 a 196 a 88 b 1.30 a 205 a 88 b 1.39 a 215 a lOOa 125 2-3-4 1.28 b 162 b 71 b 1.47a 156 b 89 a 1.24 b 158 b 93 a 1.29 b 175 b 85 b 140 3-4-5 1.28 b 130c 63 c 1.25 b 134c 44c 1.21 c 125 c 71c 1.26 c 137 c 57 c

*Mean separation within columns by Duncan's multiple range test, 1% level. "I: Flowering; 2: Fruit development; 3: Early fruit ripening; 4: Fruit ripening; 5: Foliar senescence.

256 Valenzuela et al.

senescence, which was delayed by high N levels (Table 1). In both tomato and cucumber, amino acids were accumulated in a manner unrelated to the supply of exogenous N (Table 3). The time course of this parameter followed the expected model for foliar senescence, declining most sharply during this period and reaching highest values during the flowering period and fruit development and ripening.

In general terms, totals and soluble protein concentrations were closely related to NH; and N0 3-ion levels (Tables 2 and 3). Morphological changes had effects similar to those of N concen­tration, foliar leaves decreasing gradually as foliar senescence progressed. High protein con­centrations appeared when large doses of N were applied in tomato, although this association was less evident in cucumber. Changes in protein concentration were easily detectable in leaf, where maximum levels occurred during flowering and fruit development, and minimum levels were recorded in the final stages of the biological cycle in both species (Table 3).

During the early stages of growth (flowering and fruit development), Vegetative Index (VI) was usually greater than one (Table 4). The VI declines gradually in tomato but sharply in cucumber because of the different length (long in tomato, short in cucumber) of the periods of

growth and fruit production. The decline in VI coincided with the transition between certain phenological stages and consequent increase in metabolic activity. Treatment N2 led to the highest values of total VI; maximum productivity was also obtained with this treatment in tomato (Table 1).

The soluble Vegetative Index is based on the same principle as total VI, but is based on easily extractable nutrients in the process of being incorporated into plant tissues. Values below unity are the most favourable. The soluble VI gives as accurate picture of the phenological changes occurring in the plant (Table 4) as well as the ionic changes and nutritional demand, detecting latent or transitory ionic deficiencies. In tomato, a transitory deficit occurred at the onset of the major developmental stages (flower­ing and fruit development). An early analysis could have detected and prevented the imbal­ance, thus preventing the drop in production. The suboptimal value of soluble VI resulting from treatment N 4 would call attention to the need to correct the nutritional imbalance.

Hence two clearly different stages can be identified: a relatively long period when en­zymatic activity, dry weight, vegetative index decline steadily, followed by a relatively short phase when amino acid and protein levels fall

Table 4. Time course of total and soluble Vegetative Index in different phenological stages

Plant age Phenological Treatments of nitrogen (days) stage"

7.5 g m _, ofN 10gm -2 ofN 20gm- 2 ofN 30 gm- 2 ofN

tot. sol. tot. sol. tot. sol. tot. sol.

Tomato 115 1-2 1.04* 0.36d 1.34 a 0.57 b l.lla 0.65 b 0.99 a 0.40d 130 1-2-3 0.84 b 0.52 b 1.09b 0.77 a 0.87 a 0.78 a 0.83 b 0.67 b 145 1-2-3-4 0.83 b 0.55 b 0.67 c 0.55 b 0.82c 0.75 a 0.64c 0.74 a 160 2-3-4 0.60c 0.67 a 0.54 d 0.59b 0.72d 0.54c 0.56 d 0.48c 175 2-3-4-5 0.48 d 0.47 c 0.55 d 0.48 c 0.50c 0.39 d 0.52e 0.40d

Cucumber 110 1-2 0.44 a 0.21 a 0.39a 0.26 a 0.43a 0.17 a 0.36a 0.29 a 125 2-3-4 0.35 b 0.20 b 0.35 b 0.18 b 0.30b 0.21 b 0.27 b 0.25 b 140 3-4-5 0.32 c 0.19 c 0.29c 0.17 c 0.28c 0.19 c 0.23 c 0.16 c

* Mean separation within columns by Duncan's multiple range test, 1% level. '1: Flowering; 2: Fruit development; 3: Early fruit ripening; 4: Fruit ripening; 5: Foliar senescence.

sharply. This sequence ends with senescence. Certain enzyme activities may therefore be use­ful as indicators of approaching senescence.

Acknowledgements

This study was carried out in greenhouses of the Centro de Investigaci6n y Desarrollo Horticola in El Ejido, Almeria (Spain), under the auspices of the Junta de Andalucfa (Andalusian Regional Government). We thank Mr F Alex, Mr F Capdevila and Mr J Rojo for their collaboration throughout the study, and Ms Karen Jane Shashok for translating the original manuscript into English.

References

Aguilar A, Romero L and Lachica M 1982 Determinacion de nitratos en tejidos vegetates. Proc. 5th Coli on the Control of Plant Nutr. Vol. 1. pp 11-15. Edizioni Comunita Nuova IPSA, Castelfranco Veneto, Treviso, Italy.

Association of Official Agricultural Chemist 1970 Methods of Analysis, AOAC, Washington DC, USA. pp 175-176.

Bar-Akiva A and Sternbaum J 1966 No enzymic reduction of the nitrate by means of ascorbic acid in citrus and other higher plants tissues. Physiologia Plant 19, 422-428.

Beevers L and Hageman R H 1983 Uptake and reduction of nitrate: Bacteria and higher plants. In Encyclopedia of Plant Physiology, New Series. Eds. A Liiuchli and R L Bieleski. Vol15A, pp 351-373. Springer-Verlag, Berlin and New York.

Blom-Zandstra A and Lampe J E M 1983 The effect of chloride and sulphate salts on the nitrate content in lettuce plants (Lactuca sativa L.). J. Plant Nutr. 6, 611-628.

Bradford M M 1976 A rapid and sensitive method for the quantification of microgram quantities of protein dye binding. Anal. Biochem. 72, 248-254.

Cram W J 1973 Internal factors regulating nitrate and chloride influx in plant cells. J. Exp. Bot. 24, 328-341.

Guzman M. Urrestarazu M and Romero L 1984 Nutritional variations in Junglans regia L. during its annual cycle.

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Proc. 5th Int. Coli. for the Optimization of Plant Nutr. Vol. 1, pp 251-260. Pierre Martin-Prevel, Montpellier, France.

Guzman M, Urrestarazu M and Romero L 1986 Active and total Fe in Castanea sativa and their relation to other nutrients. J. Plant Nutr. 9, 909-921.

Harper J E and Hageman R H 1972 Canopy and seasonal profiles of nitrate reductase in soy beans (Glycine max L. Merr.). Plant Physiol. 49, 146-154.

Lauriere C 1983 Enzymes and leaf senescence. Physiol. Veg. 21, 1159-1177.

Loneragan J F 1968 Nutrient requirements of plants. Nature (London) 220, 1307-1308.

Martinoia E, Heck U and Wienecken A 1981 Vacuoles as storage compartments for nitrate in barley leaves. Nature (London) 289, 292-294.

Moores Y and Stein W H 1954 A modified ninhydrin reagent for the photometric determination of amino acids com­pounds. J. Bioi. Chern. 211, 907-913.

Pijoan-Pascual J 1976 L'index vegetatif (LV.) dans le domainse de Ia fruticulture et Ia dynamique des valeurs pendant le cycle vegetatif. Proc. 4th Int. Coli. on the Control of Plant Nutr. Vol. 2, pp 187-198. Ghent, Brus­sels.

Rufty T W Jr, Yolk R J, McClure R R, Israel D Wand Raper C D Jr 1982 Relative content of NO; and reduced N in xylem exudate as an indicator of root reduction of concur­rently absorbed "NO;. Plant Physiol. 69, 166-170.

Santoro L G and Magalhaes A C N 1983 Changes in nitrate reductase activity during development of soybean leaf. Z. Pflanzenphysiol. 112, 113-121.

Smith P F 1962 Mineral analysis of plant tissues. Am. Rev. Plant Physiol. 13, 81-108.

Snell P F and Snell C T 1949 Colorimetric Methods Analysis. pp 904-805. D. Van Nostrand Company, Inc., New York.

Streit C and Feller U 1982 Changing activities of nitrogen­assimilating enzymes during growth and senescence of dwarf bean (Phaseolus vulgaris L.) Z. Pflanzenphysiol. 108, 273-281.

Ulrich A 1952 Physiological basis for assesing the nutritional requirement of plants. Am. Rev. Plant Physiol. 3, 207-228.

Van Diest A 1990 Accumulation of nitrate in higher plants. Its causes and prevention. In Nitrogen in Higher Plants. Ed. Y P Abrol. pp 441-460. John Wiley & Sons Inc., New York.

Wolf B 1982 A comprehensive system of leaf analysis and its use for diagnosing crop nutrient status. Commun. Soil Sci. Plant Anal. 13 1035-1059.

E

Mineral composition in relation to crop growth, yield and product quality

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 261-267, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-035

An overall approach to plant nutrition through the use of square diagrams

P. MORARD, A. BERNADAC and G. BERTONI Department of Plant Physiology, ENSAT, 145 avenue de Muret, F-31076 Toulouse, France

Key words: cucumber, graphical analysis, sorghum

Abstract

A methodological approach is put forward that is based on relating nutrient uptake to the composition of the nutrient medium, under systematic variation of the latter. The relation is plotted for each element in the form of a square diagram. A systematic analysis of long-term nutritional phenomena can thus be achieved. The potentialities of this method are illustrated by selected examples obtained with sorghum and cucumber, at the level of the organ and of the whole plant: (i) leaf senescence is characterized by the accumulation of calcium (a non mobile element) in parallel to potassium (a mobile element) depletion; (ii) the square diagrams show the maintenance of the contents of the essential macronutrients and the exclusion of non-essential elements when large variations in the nutrient medium composition occur; (iii) the comparison of the nutritional behaviour of the two species shows a better regulation of the balance of the internal mineral composition in sorghum than in cucumber. Through the above examples, this method appears to be valuable for the visualization of the complex nutritional mechanism involved during plant growth.

Introduction

The mineral nutrition physiology of whole plants during their development (from the seedling to the harvest stage) involves complex processes, in relation to organ differentiation and soil interac­tions. Yet, the understanding of the nutritional mechanisms in the whole plant is essential in agronomy. The various approaches used in plant nutrition can be listed in three categories: -The kinetic studies include all the investiga-

tions relative to root uptake rates (and occas­ionally translocation rates). The measure­ments are usually carried out over a relatively short time using seedlings or excised roots. Data interpretation is based on mechanisms operating at the cell level (Epstein and Hagen, 1952; Grignon et a!., 1972): the con­cept is thus physiological.

-The nutrient consumption balances, which are established from plant analyses at a given developmental stage and most frequently at the end of the cycle, allow the determination of the amounts of nutrients accumulated by the plants over longer periods (one or several months): the nutrient exportations by a crop can thus be inferred. These balances, ob­tained from field experiments or pot cultures, are used for the calculation of fertilizer appli­cation rates (Bertoni et a!., 1988; Lubet and Juste, 1985).

-The investigation of the plant response to controlled variations of the composition of the nutritive medium integrates the adaptation of the uptake and translocation mechanisms to the variations of the nutritive medium. This methodology requires accurate balances ob­tained in hydroponics from the well defined

262 Morard et al.

nutrient solutions used (Al-Ani and Ouda, 1972): the concept can be termed agro­physiological or ecophysiological. The latter method of approach, which is de­

veloped below, relies upon: -well-defined variations of the mineral compo­

sition of the nutritive medium through the gradual substitution of an essential macronut­rient in the nutrient solution: the method of the "systematic variations" is derived from the investigations of Homes ( 1961), De Wit et a!. (1963) and Van Schoor (1966).

-the graphical analysis of the consequences of these variations on the mineral composition of the plant so as to visualize the data: the method of the "square diagrams" is derived from clay geochemistry investigations (Sposito, 1981 ).

In similar approaches, the plant response to controlled variations of the composition of the nutrient solution was illustrated graphically with­out any transformation of the data (Demarty et a!., 1978); nutrient ratios have also been used in double logarithmic plots (Braakhekke, 1980).

Methods

Experimental procedure The plants were grown hydroponically in a greenhouse, using a non-circulating nutrient so­lution. As a result of the plant-solution ratio selected and of the harvest at an early stage, the composition of the medium did not vary sig­nificantly during the experiments.

The plant analyses were carried out as previ­ously described (Morard et a!., 1990). The total amounts of nutrients in the plant provided a good estimation of the uptake from the nutrient solution, since 90 to 99% originated from the nutritive medium whereas the remainder was derived from the seed.

Systematic variations The concentration of a macronutrient in the nutritive medium was varied stepwise from a maximum value, which was close to that of Hoagland's standard nutrient solution (K-ions 7,

Ca-ions 10, Mg-ions 3, H 2P0 4-ions 2, S04-ions 3 and N03 -ions 15 meq L - 1 ) to a minimum value for which no deficiency symptoms could be observed at the harvest stage. The minimum concentrations were pre-established for the cul­ture technique described above and varied de­pending on the plant investigated and on the harvest stage selected.

So as to keep the ionic sums constant, the macronutricnts were replaced with equivalent amounts of non-essential and non-toxic ele­ments: as usually practiced in such experiments, sodium was the substitute ion for the cations (K-ions, Ca-ins and Mg-ions) and chloride for the anions (N0 3-ions, H 2P04 -ions and S04 -

ions). Each series of culture concerned only one element. The same experimental procedure was applied to each of the six essential macronu­trients. Thus, for example, the following treat­ments were applied in the case of potassium (maximum concentration 7 meq L - 1 minimum concentration 0.1 meq L - 1 , number of decreas­ing steps 7):

K-ions 7 6 5 4 3 2 1 0.1

Na-ions 0 1 2 3 4 5 6 6.9

Square diagrams After a growth period of approximately one month on these different media, the plants were harvested and analysed (whether separated into organs or not). The data were expressed in ion equivalents, referring to the form in which the elements are taken up by the plant and not to the metabolized form present in the plant at harvest (e.g. the plant nitrogen content was expressed in meq N0 3-ions absorbed). The ionic composition of the medium and that of the plant could then be compared.

So as to facilitate the analysis and the interpre­tation of the data, a particular mode of plotting termed "square diagrams" was called upon. Each macronutrient, in the nutrient solution as well as in the plant, was expressed in percent of the sum of the elements of the same charge. Thus, for example, for potassium:

Approach to plant utrition through square diagrams 263

in the nutrient solution

K+(meqL- 1)xlOO Ks%=--~--~~--~~~~------~

K+ + Ca2 + + Mg 2+ + Na+(meq L - 1)

in the plant

Kp%

K+(meq 100 g d.m.- 1) x 100

K + + Ca2 + + Mg2 + + Na +(meq 100 g d.m. 1 )

Within the plant, the percentage is a ratio of contents, which is equivalent to a ratio of amounts taken up. This percentage, which corre­sponds to an equivalent fraction, represents the proportion of the element relatively to the total of the cations or anions absorbed. As it is a dimensionless value, the comparison between the nutrient medium and the plant is thus con­sistent.

Results and discussion

The method was first applied to the investigation of the macronutrient nutrition physiology of two plants: a monocot, sorghum, harvested at the 7-leaf stage (Bernadac, 1989) and a dicot, cucumber, harvested at the 4-leaf stage (Benavides, 1991). The same experimental procedure was used in both cases.

Plant selectivity

Plant selectivity corresponds to the preferential uptake of some elements (Epstein, 1972). Con­sequently, the elements are not present in the same proportions in the plant and in the nutritive medium (Collander, 1941). In the long term, the selectivity results from the involvement of vari­ous phenomena during plant growth: root ab­sorption, exsorption, translocation and utiliza­tion. These processes gradually induce differ­ences in composition between the plant and the supporting medium and between the various organs.

The selectivity is reflected in a square diagram by the position of the data points relatively to the diagonal (Fig. 1) which corresponds to equal proportions of a nutrient in the nutrient solution and in the plant tissues (there is then no selec-

100

.. c: ca 50 a:

50 100

Nutrient Solution Fig. I. Plant-nutrient solution square diagram illustrating different cases of plant selectivity. 1: selective accumulation; 2: selective exclusion; 3: uptake regulation.

tivity). When the data points are lying above the diagonal (curve 1, Fig. 1), there is a selective nutrient accumulation; on the contrary, when the points are below the diagonal (curve 2, Fig. 1), there is a selective nutrient exclusion. The de­gree of selectivity can then be estimated from the distance between the data points and the diag­onal. As shown by curve 3 (Fig. 1), the nature of the plant selectivity is dependent of the range of proportions in the nutrient solution: for the same element, selective accumulation may occur for a given proportion in the nutrient solution whereas selective exclusion will be observed for another proportion. As the plant tends to regulate its relative uptake, the selective uptake can be characterized by another parameter which is the slope (a) of the line in the high concentration range. An increasingly wider difference in slope relatively to the diagonal then reflects a greater extent of uptake regulation by the plant.

The nitrogen series of the experiments on sorghum, where chloride was substituted step­wise for nitrate in the nutrient solution, provides a valuable example (Fig. 2a). The square dia­gram clearly illustrates the selective accumula­tion of nitrate by the root system of sorghum with a maximum selectivity for low nitrate pro­portions in the nutrient solution. For a large

264 Morard et al.

100 100

0 0 0 0 0 0 0

K+ t: t: •• ., ., c: c: ••• Ql 50 Ql 50 • 0 0 s:. s:. s: s:

/',

Cl- Na+

0 /', /', /',

0 ........... ~

0 50 100 0 50 100

(a) Nutrient Solution (b) Nutrient Solution

Fig. 2. Relative proportions of N0 3-ions and Cl-ions (a), and K-ions and Na-ions (b) in nutrient solution versus whole plant.

variation of the nitrate proportion in the nutrient solution (from 5% to 75% of the sum of the anions), the nitrate uptake was regulated by the plant and the relative proportion of nitrogen (around 82%) was maintained constant in the biomass produced. When the relative nitrate concentration was lower than 5% of the anions present in the nutrient solution, a sufficient nitrogen content could no longer be maintained.

In contrast, the plant exerted a selective exclu­sion towards chloride ions during biomass pro­duction. For Cl-ions proportions ranging from 2% to 70% in the solution, the Cl plant content was below 5% of the sum of the anions ab­sorbed. The slight relative enrichment (23%) observed only for the highest chloride proportion (75%) in the nutrient solution might be inter­preted in terms of a mechanism of compensation of decreased nitrate uptake by enhanced chloride uptake.

The same pattern, which is characteristic of the relative uptake of an essential element com­pared to that of a non-essential element, was observed when potassium was replaced with sodium: Figure 2b shows high selective K-ion accumulation and high selective Na-ion exclu­sion.

Translocation between different organs

Over a given period, some mineral elements gradually accumulate within an organ (sink)

whereas others (mobile elements) can migrate and are redistributed between the various or­gans. The composition of these various organs results from the involvement of both phenom­ena.

The method presented here can be used for comparisons between the relative proportions of mineral elements in two organs in direct relation (e.g. leaf-stem) and the variation in mineral composition, which is induced by changes in the nutritive medium, can be investigated.

Different kinds of information can be obtained through the use of this methodology. Sorghum provides a demonstrative example (Bernadac 1989). The change in the relative proportions of K-ions and Ca-ions in the various leaves was analysed relatively to the composition of the sheath (the organ to which they were attached). The plants were harvested at the 7-leaf stage and the leaves were grouped in three lots, depending on their age (Morard, 1973): the younger leaves (YL), i.e. the leaves that appeared last and were completing their growth (leaves 6 and 7); the adult leaves (AL), i.e. leaves 3, 4 and 5; the older leaves (OL), i.e. the two leaves that appeared first (and the cotyledon).

On the square diagrams (Fig. 3), the relative proportions of K and Ca in these leaf classes were plotted against the relative contents of these elements in the sheaths. The distribution of the data points is markedly different from that observed in the preceding diagrams. Irrespective of the nutrient supply, the relative fractions of

Approach to plant utrition through square diagrams 265

100

.. ~ 50 ~

0 0

K+

50

Sheath

100

100

.. ~ 50 ~

Ca++

50

Sheath

100

Fig. 3. Relative proportions of K-ions and Ca-ions in sheath versus leaves. (D) younger leaves, (II) adult leaves, (•) older leaves.

the various elements varied little in the sheaths (abscissa). On the contrary, a large variation was observed for the relative fractions in the leaves (ordinate) in relation to their age: -the younger leaves had a relative proportion

of potassium and calcium that was similar to that in the sheath,

-the relative proportion of potassium de­creased and that of calcium increased with the age of the leaves.

Thus, data presentation in the form of square diagrams clearly illustrates the effect of tissue senescence on the mineral composition. Potas­sium becomes proportionally less represented as the leaf is ageing: this mobile element is redistri­buted from the older tissues towards the younger ones. On the opposite, calcium steadily accumu­lates in the tissues during their senescence; this phenomenon is classically assigned to the absence of redistribution of this element in the phloem. The opposite trends observed for the two phenomena illustrate the occurrence of a potassium-calcium antagonism in sor­ghum.

However, the square diagram does not reveal whether the decrease in potassium with the age of the leaves is rather relative (i.e. caused by calcium accumulation), or absolute (i.e. caused by retranslocation of potassium). Such plots, like other kinds of proportional representations, then

do not show the constancy of the sum of the cations contained in a given organ.

Comparison of the selectivity capacities of various species

Species or even cultivars or rootstocks are known to react differently to a variation in the mineral composition of the medium. The be­haviour of two different species, cucumber (Morard and Benavides, 1990) and sorghum (Morard et a!., 1990) was thus analysed and compared.

Among the macronutrients investigated, the behaviour of the cations is noteworthy. For low proportions of calcium in the nutrient solution, the selective accumulation of this element (Fig. 4a) was slight in sorghum but higher in cucumber. When the calcium concentrations in the nutrient solution were increased, the selec­tivity decreased in cucumber whereas selective exclusion was observed in sorghum. The degree of uptake regulation (reflected by the slopes of the lines connecting the data points), which seemed to be the same in the two plants, resulted in higher calcium contents in cucumber (around 50%) than in sorghum (around 10% ). This difference seems to be consistent with the higher affinity of dicot cell walls for calcium (Loneragan and Snowball, 1969; Sentenac and

266 Morard et al.

.. c 01 0: .!! 30 0

~

0

(a) Ca++

0

(b) K+

20

c 01 0: CD 10 0 ~

(c) Mg++

30

Nutrient Solution

40

Nutrient Solution

10

Nutrient Solution

60

80

20

Fig. 4. Comparison of the relative proportions of (a) Ca­ions, (b) K-ions and (c) Mg-ions in cucumber (•) and in sorghum (e): nutrient solution versus whole plant.

Grignon, 1981). Within the range of the potas­sium concentrations investigated, the plant selec­tivity always resulted in an accumulation which was higher in sorghum than in cucumber (Fig . 4b). As a result of plant uptake regulation, the selectivity of both plants decreased when the proportions of potassium in the nutrient solution increased. The increase in the relative mag­nesium contents (Fig. 4c) in cucumber was nearly proportional to the increase in relative content in the nutrient solution, but this was not the case in sorghum where the response was characterized by a levelling off at high concen­trations. The smaller slope of the line connecting the data points suggests the involvement of a stronger regulation of the relative magnesium contents in sorghum as compared to cucumber.

Conclusion

The expression of the whole plant content of an element as the ion equivalent absorbed in % of the content of the other elements of the same sign (anions or cations) provided an evaluation of the relative absorption of the ion considered, which could easily be related to relative varia­tions of the nutritive medium composition. This method could then be extended to the composi­tion of various consecutive plant fractions (e.g. conductive tissues -leaves). The enrichment in Ca and the impoverishment in K of the leaves with increasing plant age could thus be charac­terized.

The comparison of the relative absorption of N0 3-ions and Cl-ions or K-ions and Na-ions clearly showed the high affinity for essential elements (N03-ions and K-ions) and the exclu­sion of non-essential elements (Na-ions and Cl­ions). This selectivity results in the maintenance of the relative absorption of each of the elements by the plant at very low proportions for non­essential elements and at high proportions for N0 3-ions and K-ions.

The application of the same experimental procedure to sorghum and cucumber showed the effect of the species. In these two plants, the relative contents of the mineral elements, which are higher for potassium and lower for calcium in sorghum than in cucumber, are maintained at

Approach to plant utrition through square diagrams 267

levels that are likely to correspond to physiologi­cal requirements. However, in sorghum, the relative absorption of Mg is more strictly main­tained in the presence of large variations of the medium composition for this element.

The association of this mode of expression of the amounts absorbed with the use of systematic variations for plotting square diagrams provides an overall view of the long-term behaviour of plant absorption relatively to variations of the nutritive medium composition.

Acknowledgement

The authors thank Dr D R Sayag for correction of the English text.

References

Al-Ani T A and Ouda N A 1972 Distribution of cations in bean plants grown at varying K and Na levels. Plant and Soil 37, 641-648.

Benavides B 1991 La nutrition minerale du concombre: Selectivite de !'absorption racinaire. Ph.D. Thesis, INP Toulouse 177p.

Bernadac A 1989 Contribution a !'etude de Ia selectivite de !'absorption des macroelements par lc sorgho (Sorghum dochna f.). Ph.D. Thesis, INP Toulouse. 156p.

Bertoni G, Morard P and Espagnacq L 1988 Dynamique de !'absorption des elements mineraux chez !'ail (Allium sativum L.). Agrochimica 32, 518-530.

Braakhekke W G 1980 On Coexistence. Agricultural re­search reports 902, Pudoc, Wageningen. 164p.

Callander P 1941 Selective absorption of cations by higher plants. Plant Physiol. 16, 691-720.

Demarty M, Morvan C and Thellier M 1978 Exchange properties of isolated cell walls of Lemma minor L. Plant Physiol. 62, 477-481.

De Wit C T, Dijkshoorn W and Noggle J C 1963 Ionic balance and growth of plants. Verslagen Landbouwkundige Onderzoekingcn 69.15. Pudoc, Wageningen. 68p.

Epstein E 1972 Mineral Nutrition of Plants: Principles and Perspectives. Wiley, New York. 412p.

Epstein E and Hagen C E 1952 Kinetic study of the aborption of alkali cations by barley roots. Plant Physiol. 27, 457-474.

Grignon C, Rona J P and Heller R 1972 Analyse cinetique des flux de potassium dans les suspensions celluaires d' Acer pseudoplatanus L. C. R. A cad. Sci. Paris 275, ser. D, 2485-2488.

Homes M V 1961 "Systematic" methods in the determination of nutrient requirements of plants. Ann. Phys. Veg. Univ. Bruxelles 6, 99-136.

Loneragan J F and Snowball K 1969 Calcium requirements of plants. Aust. J. Agric. Res. 20, 465-478.

Lubet E and Juste C 1985 Cinetique de Ia production de matiere seehe et de prelevement d'clcments nutritifs par une culture irriguee de mais a haute potentialite de rendement. Agronomic 5, 239-250.

Morard P 1973 Contribution a !'etude de Ia nutrition potas­sique du sorgho. These Doctoral d'Etat, Toulouse, 199p.

Morard P and Benavides B 1990 Relative accumulation of macronutrient ions in different parts of cucumber (Cucumis sativus). Scient. Hortic. 44, 17-30.

Morard P, Bernadac A and Valles V 1990 Selectivity of the root absorption of nutrient ions in grain sorghum. J. Plant Nutr. 13, 249-268.

Sentenac H and Grignon C 1981 Model for predicting ionic equilibrium concentrations in cell walls. Plant Physiol 68, 415-419.

Sposito G 1981 The Thermodynamics of Soil Solutions. Clarendon Press, Oxford, UK. 223p.

Van Schoor G H 1966 La composition minerale de Zebrina pendula en fonction de proportions variables de potassium et de sodium du milieu nutritif. Bull. Soc. Roy. Bot. Belg. 99, 113-125.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 269-275, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-033

Nutrient uptake by Toulouse violet (yiola odorata var. parmensis) during its developmental cycle

A. SHAN SEI FAN and P. MORARD Department of Plant Physiology, ENSAT, 145 avenue de Muret, F-31076 Toulouse, France

Key words: calcium, macronutricnts, magnesium, nitrogen, phosphorus, potassium, sulfur, Viola, violet

Abstract

The aim of the investigations was to determine the macronutrient uptake of the Toulouse violet at various developmental stages so as to rationalize the fertilization of this ornamental crop. The total dry matter (TDM) produced by the soil grown crop amounted to 15.5 g per plant, i.e. 82 kg per are after 8 months. Biomass production, which was low at the beginning of growth, rose dramatically at the time of flowering to become intensified during the last months. The floral production represented only 3% of TDM. The macroelement concentrations of each plant organ were determined: the vegetative apparatus was characterized by high nitrogen and potassium concentrations whereas the roots accumulated calcium and the flowers phosphorus and sulphur. The macronutrient uptake kinetics paralleled that of dry matter production. As a result, the concentrations varied only slightly during the developmental cycle. However, potassium, magnesium and sulphur uptake was higher during the last months, at the end of flowering. The amounts accumulated by the reproductive apparatus represented only 1 to 6% of the total consumption, depending on the element considered. At the end of the cycle, the amounts taken up by the violet crop were N 358, P 33, K 451, Ca 184, Mg 75, and S 52 mg/plant. Under current crop management, for an average density of 5300 plants plants/ are, the amounts of macronutrients accumulated are thus N, 1.9; P20 5 , 0.4; K20, 2.9; CaO, 1.3; MgO, 0.7 and S03 , 0.8 fertilizer units per are.

Introduction

Widely grown in the market garden belt located in the northern fringe of the city, at the turn of the century, Toulouse violet cropping provided additional income to family farms: the acreage devoted to this crop by 600 growers then amounted to 25 ha. In 1985, there were only 7 families of violet growers on a surface of less than 0.5 ha. Now, fewer than 3000 flower bun­ches are commercialized every year, on a mostly regional market. The decline observed is to be assigned to the unfavourable economic environ­ment as well as to plant degeneration induced by exclusively vegetative multiplication.

A few years ago, a program of investigations and crop redevelopment was started: the aim was to promote the reintroduction of the Toulouse violet into its cropping area using soilless culture, after regenerating mother plants through in vitro culture (Morard and Roucolle, 1991 ).

As observed for other crops of minor economic importance, few investigations have been carried out relative to the violet. Deportes et a!. (1979) investigated the violet pathogens and reported data on nutrient uptake by leaves and flowers.

The aim of the investigations reported here was to determine the macronutrient accumula-

270 Shan Sei Fan and Morard

tion in the various plant organs so as to define the overall macronutrient requirements of the Toulouse violet.

Material and methods

The violet plants were grown on a 110m2 plot, in rotation with other market garden crops, on a family farm at Aucamville, 10 km north of Toulouse. The main characteristics of the alluvial soil were: pH H 2 0 7.6, pH KC16.5, CaC0 3

1.1 %, coarse sand 34%, fine sand 25%, silt 26%, clay 10%, organic matter 4.3%.

The plot was managed according to the tradi­tional regional practices (Morard et al., 1992). The average density was 53 plants m - 2 • Before starting the crop, 500 kg of farmyard manure per are and 6.1 nitrogen fertilizer units per are were incorporated into the soil. At the end of July, a 17-17-17 fertilizer was applied at the rate of 10 kg are - 1 . Excess stolons were removed as their development could affect flower production. The plot was watered manually.

Four samplings were carried out from the vegetative maturity stage (September). The dates of sampling and the main developmental stages are mentioned in Table 1. At the time of each sampling, 5 plants were harvested randomly on the plot. A fork was used to unearth the root system. After rinsing with demineralized water, the plants were separated into laminae, petioles, stolons, and roots. The samples were oven dried at 105°C, weighed and ground. During the flow­ering period (October to April), flowers were harvested weekly on 2 rows of 10 violet plants. After separation into corollae and stalks, the samples were regrouped by month, dried, weighed and ground.

The dry powders were used for the analytical determinations, after mineralization with sui-

phuric acid for total nitrogen and with nitric acid for the other elements. Nand P were determined by automatic visible absorption spectrophotom­etry. S was determined by turbidimetry. Atomic emission spectrophotometry was used for K and atomic absorption spectrophotometry for Ca and Mg (Shan Sei Fan, 1993). The statistical analysis of the data was carried out using the STAT­ITCF program. After variance analysis, the data were classed into groups, using the Newman and Keuls test at the 5% threshold.

Results and discussion

Biomass production

The Toulouse violet is a perennial, with a roset­te-shaped vegetative apparatus. The determina­tion of the biomass produced provides a descrip­tion of the growth and development rhythm. The total dry matter (TDM), the aerial dry matter (ADM), with laminae (L), petioles (P) and stolons (S), the root dry matter (RDM) and the floral dry matter (FDM) were distinguished (Table 2).

The total dry matter production was low initially, then increased rapidly during the flow­ering season to further intensify during the last months of the crop. At the end of the crop, the biomass produced was 15.5 g per plant, i.e. 11 times higher than the initial value in September.

The aerial dry matter constituted the major part of the biomass produced, with an average contribution of 75.6%. At the end of the crop, the vegetative development intensified with the return to favourable climatic conditions (photo­period lengthening, rise in temperature). Throughout the developmental cycle, the laminae amounted to 50% of the aerial dry matter. At the end of the crop, their contribu-

Table I. Dates of sampling in relation to the phenological cycle of the Toulouse violet

Planting Leaf cutting Vegetative Flowering End maturity of crop

I st flowering 2nd flowering peak peak

May Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Samplings 1 2 3 4

Table 2. Biomass production rhythm

Month TOM L

(g) (g/month) (%TOM)

September 1.33 54.1 November 3.02 0.84 37.8 February 6.98 1.32 32.3 May 15.54 2.85 30.4

p

10.5 18.5 16.4 23.0

Nutrient uptake by Toulouse violet 271

s ADM

18.1 82.7 15.9 72.2 17.1 65.8 28.5 81.9

ROM

17.3 21.2 29.5 14.9

FDM

6.6 4.7 3.2

TDM: total dry matter; L: laminae; P: petioles; S: Stolons; ADM: aerial dry matter; ROM: root dry matter; FDM: floral dry matter.

tion decreased as a result of the increasing development of stolons. The contribution of the petioles, which was significantly lower than that of the laminae, was nearly constant throughout the developmental cycle.

The root dry matter averaged 20% of the total biomass produced. Its contribution was equal to that of the petioles. After a sharp increase until February, the root mass was stabilized at the end of the crop, at the time when aerial biomass production was boosted.

The floral production had a limited incidence on the total biomass production by Toulouse

500,-------------------------~~~

;:: "' 0.. 400

"" "' ..@; 300 Cumulative production

_§

.:c 200 00 ·o: ;!: 100 >.

o I o•---~--~--¥===~--~--------~4

~ ~ - -~ - ~ . ~ Fig. 1. Floral production.

violet: the contribution of the floral dry matter was 3-7% of the TDM produced. The floral production rhythm is shown by the monthly change in the dry weight of flowers harvested per plant (Fig. 1 ). The patterns of change were similar for these two parameters: the two prod­uctivity peaks observed in October and March were separated by a very low yield period in December and January.

The annual production was 4.1 flowers per plant or 495 mg FDM. These values are in agreement with those reported by Carlier and Pointereau (1987), although high yield dis­crepancies can be observed from one year to the next (climatic conditions, pests) and from one farm to the next (crop management).

At the plot level, the yield was 68 kg ADM are - 1 for a plant density of 5300 plants are -l . The literature provides data obtained from leaf cutting: 100 kg ADM are -l for V. odorata var. semperflorens (Dcportes et al., 1979) and 70 kg ADM are - 1 for V. odorata var. parmensis (Carlier and Pointereau, 1987). The yield dis­crepancies can be assigned to various factors, such as the cultivar, the climatic conditions, the crop management or the leaf cutting technique.

Table 3. Average macronutrient concentrations (%DM) of the various plant organs

Organ N p K Ca Mg s Vegetative apparatus and root system

Laminae 2.52 a* 0.20 b 2.58 b 1.15 be 0.49b 0.34 a Petioles 1.56c 0.17 c 4.83 a 1.03 c 0.24 a 0.26 b Stolons 2.47 a 0.24 a 2.66 b 1.20 b 0.32c 0.34 a Roots 1.97 b 0.24 a 1.31 c 1.45 a 0.87 a 0.25 b

Reproductive apparatus Corollae 3.10 a 0.39 a 3.22 ns 0.50 ns 0.37 a 0.56 a Flower stalks 1.95 b 0.22 b 3.09 ns 0.52 ns 0.24 b 0.27b

*Mean separation in columns by Newman and Keuls test at 5% threshold.

272 Shan Sei Fan and Morard

Macronutrient concentrations

The changes in the macronutrient composition of the various organs of Toulouse violet in relation to the developmental stage are presented in Table 3.

The laminae were rich in nitrogen and sul­phur. The nitrogen, sulphur and magnesium concentrations increased with time. The calcium concentration varied markedly from one sam­pling to the next and decreased during the flowering season. The phosphorus and potassium concentrations remained nearly constant throughout the developmental cycle.

The petioles displayed high potassium concen­trations whereas the concentrations of the other elements were minimum. The N, P, K, Mg, and S concentrations were nearly constant during the developmental cycle of the Toulouse violet, but the calcium concentration decreased significantly from the onset of flowering.

The stolons were rich in nitrogen, potassium, calcium and sulphur. The nitrogen concentration increased with time. The calcium concentration decreased significantly as soon as flowering began; such a behaviour seems to be characteris­tic of the conductive tissues (petioles and stolons). The concentrations of the other ele­ments remained nearly constant in the course of time.

High concentrations of phosphorus, calcium and magnesium were found in the roots. The macronutrients did not vary significantly, with the exception of magnesium which decreased markedly with time.

The corollae were the richest in N, P, and S. The calcium concentration was relatively low with respect to the levels observed in the vegeta­tive apparatus and in the roots. The nitrogen concentration decreased steadily during the flow­ering season. The other clements did not vary significantly.

The flower stalks displayed macronutrient con­centrations which were significantly lower than those of the corollae, with the exception of potassium and calcium, for which the percen­tages were equivalent in the two organs. The K, Mg, and S rates were close to those of the petioles: the flower stalks had higher N and P concentrations but contained less K and Ca. The

nitrogen and potassium concentrations eventual­ly increased whereas the concentrations of the other elements remained nearly stable.

These data are close to those reported by Deportes et al. (1979). In Viola odorata var semperflorens, the variation ranges for the foliar concentrations (%DM) were: N 3.3-5.4/ P 0.23-0.41/K 4.2-5.7/Ca 0.6-1.4/Mg 0.2-0.3. The average concentrations in the flowers were: N 4.3/P 0.5/K 4.0/Ca 0.9/Mg 0.4.

Dry matter 14 r======-------cSOO Macronutricnt (g) per plant12 ~~ accumulation

(rug) per plant 400

Fig. 2. Biomass production and macronutrient uptake rhythms.

Table 4. Macronutrient accumulation at the end of the crop (mg/plant)

N P K Ca Mg S

Vegetative apparatus and root system 345 31 435 182 73 50

Reproductive apparatus 13 3 16 2 2 2

Whole plant 358 33 451 184 75 52

K

1.8% 1.3%

40.3%

N

Nutrient uptake by Toulouse violet 273

t:J laminae ~petioles

D stolons [ill roots till corollae II flower stalks

Ca

0.8% 0.6%

Mg

12.2%

s 9.0~79'·1.0%

37.9% 30.8% .

18.7%

45.4%

p

3.4%1.5% 14.9%~

27.1%~32.6% 20.4%

Fig. 3. Macronutrient accumulation at the end of the crop: distribution per organ (% ).

274 Shan Sei Fan and Morard

Macronutrient uptake

Figure 2 shows the amounts of macronutrients taken up by the violet plants during the growing season. The changes in the kinetics of macronut­rient uptake paralleled those of dry matter production, which reflects a relative constancy of the macronutrient concentrations during the whole developmental cycle of the plants.

The total amounts of macronutrients accumu­lated during the developmental cycle are listed in Table 4. The distribution of the macronutrients per organ at the end of the crop (May) is shown in Figure 3, where the surfaces of the circles are proportional to the total amounts of nutrients taken up.

The various elements are taken up in differing proportions, depending on the organs. For the vegetative apparatus and the root system, the following ratios were obtained: K 1/N 0.79/ Ca0.42/ Mg0.17/S0.12/P0.07. For the re­productive apparatus, the ratios were K 1/ N 0.81/P = Ca = Mg = S 0.13.

The laminae are the organs that accumulate the highest amounts of macronutrients, with the exception of potassium for which the largest part is found in the petioles. The stolons import mostly phosphorus, calcium and sulphur. Mag­nesium and calcium are the elements that are the most highly taken up by the root system which accumulates very little potassium.

As a result of the small amount of floral dry matter produced, the reproductive apparatus macronutrient uptake is low: the amounts repre­sent 1-6% of the total importations by the violet plants, depending on the elements.

Our results are in agreement with the data reported by Deportes et al. (1979) for Viola odorata var. semperflorens. The data can be compared to those reported for strawberry, which is a phenologically similar plant. N, P, K and Mg uptake is of the same order of mag­nitude, but calcium accumulation is higher in field-grown strawberries (Albregt and Howard, 1980). The soilless culture of strawberries on vertical columns (Morard and Lacroix-Raynal, 1989) is characterized by a higher consumption of N, P and K, in contrast to calcium and sulphur uptake.

Conclusion

The above investigations allowed the macronut­rient requirements of violet plants at various developmental stages to be defined. In relation to the general behaviour of perennials, the rhythm of biomass production by the Toulouse violet was relatively slow. The total dry matter production was 82 kg are -l and the floral pro­duction reached 2.6 kg are -I.

The vegetative apparatus displayed high nitro­gen and potassium concentrations. The roots concentrated calcium. The flowers had high phosphorus and sulphur concentrations but were poor in calcium.

At the end of a vegetative cycle, the consump­tion balance expressed in fertilizer units per are, was: N 1.9/P20 50.4/K20 2.9/CaO 1.3/MgO 0.7 I S03 0.8. The macronutrient uptake was low, as a result of the limited vegetative development of the plants. Under current conditions of crop management, macronutrient restitutions to the soil are quite high: at the end of the crop, the leaves and the roots are ground on the plot. Thus, the macronutrient exportations are exclu­sively due to the flowers.

The above investigations were part of a Ph.D. study relative to Toulouse violet culture, which also included micronutrient consumption bal­ances (Shan Sei Fan, 1993).

Acknowledgements

The authors thank Dr D R Sayag for improve­ment of the English text.

References

Albregts E E and Howard C M 1980 Accumulation of nutrients by strawberry plants and fruit grown in annual hill culture. J. Am. Soc. Hortic. Sci. 105 386-388

Carlier B and Pointereau P 1987 La Violette de Toulouse. Bilan d'enquetes et de travaux. Institut Agricole La Cadene. 11 p.

Deportes L, Gilly G. Cuany A, Mercier S, Poupet A and Marais A 1979 Contribution a !'etude de quelques prob­lemes techniques poses par Ia culture de Ia Violette. P. H.M. Revue Horticole 200 13, 21.

Morard P and Lacroix-Raynal C 1989 Uptake of macro­nutrients by strawberry plants in soilless culture. Soilless Culture 5, 31-45.

Morard P and Roucolle A 1991 La Violette de Toulouse. 1. La regeneration de la culture: une approche originate de recherche-devcloppcment. P.H.M. Revue Horticole 318, 49-50.

Nutrient uptake by Toulouse violet 275

Morard P, Cas bas N, Barandou P and Vidalie H 1992 La Violette de Toulouse. 2. Donnees historiques, ag­ronomiques et morphologiques. P.H.M. Revue Horticole 333, 33-35.

Shan Sei Fan A 1993 Contribution a !'etude de la physiologic de la Violette de Toulouse. Ph.D. Thesis. INP Toulouse.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 277-281, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-132

Effect of nitrogen on growth of broad beans

M.G. PALHA1 , C.F. CARRANCA2 , M.L. FERNANDES 2 and M.A.C. FRAGOS02

1Department of Vegetable and Ornamental Crops and 2National Agronomy Research Station, IN/A, Quinta do Marques, 2780 Oeiras, Portugal

Key words: broad beans, N fertilization, N2 fixation, Vicia faba L. minor

Abstract

The present study was undertaken to assess the contribution of the soil N, fertilizer N, and fixed N 2 by native Rhizobium on the growth of fababean. A one-year experiment using two spring uninoculated cvs. Vicia faba L. minor, 'Bianka' and Threefold White' for processing, was laid out on each of three sites. Each trial was of a split-plot block design with three N levels and three replicates. 'Sites' had a highly significant effect on plant growth, nodule weight and some soil characteristics. At flowering stage, applied N only significantly affected nodule production; at harvest, no significant effect of N on growth measurements and dry-matter yield was found. At flowering there were very big differences between cultivars in plant height, shoot and nodule dry-matter yield, but not at harvest. Also at flowering, both cultivars showed nitrogenase activity, at all sites. N treatment did not significantly affect soil pH, total N and inorganic-N during the growing cycle.

Introduction

Vicia faba L. minor suitable for the freezing industry is a leguminous crop that has been recently introduced to Portugal. Potential expan­sion of the spring varieties is somewhat limited where a processing factory has to deal with both vining peas and dwarf green beans, as broad bean harvest falls between the other two crops. Its potential high yields and protein content suggests broad bean could be among the most useful grain and forage legumes. Nitrogen nutri­tion depends on an unstable equilibrium between the fixation of atmospheric N 2 and the assimila­tion of available nitrogen in the soil. Studies on biologically fixed N 2 for maximum yield, espe­cially in soils of low N status, are thus required. There have been only a few reported quantita­tive estimates of N 2 fixation in field-grown faba­bean. These estimates have indicated that this crop is capable of satisfying its N needs largely from the atmosphere. More studies are essential on different sites in Portugal to ascertain if this

phenomenon is general in Portugal (Zapata et a!., 1987).

The present study was undertaken to assess the contribution of soil N, fertilizer N, and fixed N2 by native Rhizobium on growth of broad beans.

Methods

At three different sites: Gafanha da Vagueira (Aveiro), Seixal (Lourinha) and Irrigation Dept. in Coruche, a one-year experiment using two spring uninoculated cultivars of Vicia faba L. minor, 'Bianka' and 'Threefold White' was car­ried out using a split-plot block design. Three replicates and three N levels (0, 30 and 60 kg ha -I) were applied as ammonium nitrate before sowing. The plant population was 15 plants m -z.

Soil samples were taken at 0-20 em depth from each plot, either at sowing and before fertilizer application, or at flowering and or at

278 Palha et al.

Table 1. Soil physical and chemical characteristics

Site Texture pH Org. Total (H,O) mat. N

(gkg-')

Gafanha da Sandy 7.19 ± 7.8 ± 0.55 ± Vagucira O.D7 2.59 0.06

Lourinha Loamy 7.99 ± 7.3 ± 0.68 ± sand O.D7 2.34 0.06

Coruche sandy 5.85 ± 8.9 ± 0.55 ± 0.22 2.07 O.Q7

harvest. They were analyzed for pH, total N and inorganic-N by methods reported in Carranca ( 1986). Some relevant physical and chemical properties of the three soil types are summarized in Table 1.

At flowering stage, the following plant charac­teristics were measured: number of stems, nodes and nodules per plant; plant height; shoot, root and nodule dry-matter yield (after oven-drying at =70°C). Total N in the shoots, at two sites, was determined by the micro-Kjeldahl procedure. Nitrogenase activity was measured by acetylene reduction (Hardy et a!., 1968) on three single­plant samples per plot of each cultivar at each location. Nodulated roots were separated from shoots and incubated for one hour, at 27°C in a 10% air-acetylene mixture. Ethylene production was measured on a Perkin-Elmer Sigma 4 gas chromatograph equipped with a hydrogen flame detector and a Poropak N column. Acetylene

NH,-N N0 3-N CEC Available cmol( + )kg- 1) p K

(mg kg_,) (mg kg- 1 )

7.34± 9.46± 2.66 ± 0.27 320 ± 33 ± 1.49 2.80 47.20 6.94

6.13 ± 11.92 ± 8.38 ± 0.82 448± 142± 1.31 1.77 50.74 19.85

7.74± 15.38 ± 2.68 ± 0.23 103 ± 84 ± 2.22 3.43 14.63 31.63

reduction rates were expressed in nmoles ethyl­ene plant- 1 min- 1

Except for root, nodule weight and nitrogen­ase activity the plant characteristics previously described, together with pod and grain weight, were determined at harvest, for each cultivar at each site.

Results

As inferred from Table 1, the three light tex­tured soils had pHs varying from around 6.0 to 8.0 and low contents of nitrogen and organic matter.

During the growing cycle there was a non­significant effect of N treatment and cultivars on soil pH, total Nand inorganic-N. Only the factor "sites" had highly significantly effect on most soil characteristics. In Table 2 mean values of these

Table 2. Mean values of soil pH, total N and inorganic-N at three stages during the growth cycle of both cultivars, in the three sites

Sampling time Site pH TotalN NH 4-N N0 3-N (g kg -I)

(mg kg_,)

Pre-sowing Gaf. Vagueira 7.19 b* 0.55 b 7.34 a 9.46b Lourinha 7.99 a 0.68 a 6.13 a 11.92 b Coruche 5.85 c 0.55 b 7.74 a 15.38 a

Flowering Gaf. Vagueira 6.60 b 0.66 a 6.40 ab 17.26 a Lourinha 7.85 a 0.69 a 4.96 b 10.65 b Co ruche 5.74c 0.56 b 9.19 a 11.96ab

Harvest Gaf. Vagueira 6.65 b 0.57 a 7.00 a 15.33 b Lourinha 7.93 a 0.60 a 7.92 a 13.07 b Co ruche 5.36c 0.58 a 4.09 b 25.67 a

*Means separation within columns by LSD range test, p-0.01.

Tab

le 3

. A

naly

sis

of v

aria

nce

for

mea

sure

d pl

ants

cha

ract

eris

tics

and

nit

roge

nase

act

ivit

y, a

t fl

ower

ing

and

harv

est

stag

es

Sour

ce o

f S

tem

N

ode

Pla

nt h

eigh

t G

rain

yie

ld

Dry

-mat

ter

yiel

d A

cety

lene

va

riat

ion

pla

nt-

1 p

lan

t-1

(em

) (g

) re

duct

ion

rate

s S

hoot

P

od

Gra

in

Roo

t N

odul

e

Flo

wer

ing

stag

e S

ite

11

.6'"

70

.7**

* 43

.0**

* 13

1.5*

**

30.2

***

144.

4***

n.

s.

N

n.s.

n.

s.

n.s.

n.

s.

n.s.

3

.8'

Cul

tiva

r n.

s.

43.1

***

72

.8**

* 10

.9**

* n.

s.

12

.3'

Har

vest

sta

ge

Sit

e n.

s.

73.4

***

211.

9***

3

2.9

1"'

23

.1**

* 71

.0**

* 56

.1**

* N

n.

s.

n.s.

n.

s.

n.s.

n.

s.

n.s.

n.

s.

Cul

tiva

r n.

s.

n.s.

n.

s.

n.s.

n.

s.

n.s.

n.

s.

n.s

., *

, * *

*-F

-val

ues

non-

sign

ific

ant

and

sign

ific

ant

at 5

% a

nd 0

.1%

pro

babi

litie

s, r

espe

ctiv

ely.

Tab

le 4

. M

ean

valu

es o

f gr

owth

mea

sure

men

ts,

dry-

mat

ter

yiel

d, a

cety

lene

red

ucti

on r

ates

and

N u

ptak

e du

ring

the

gro

wth

cyc

le

Ste

m

Nod

e G

ran

yiel

d P

lant

hei

ght

Dry

-mat

ter y

ield

(g

plan

t -I)

p

lan

t-1

plan

t (k

gh

a-1 )

(e

m)

Sho

ot

Pod

G

rain

R

oot

Nod

ule

Flo

wer

ing

stag

e G

af.

Vag

ueir

a 2

.9b

14

.7 a

51

.81

a 18

.74

b 2.

61 b

0

.66

b

Lou

rinh

3 3.

5 a

14.6

a

56.3

6 a

27.0

8 a

3.3

4a

0.82

a

Co r

uche

3.

4 a

10

.9b

35

.09

b 11

.36

c 2.

28 b

0.

33 c

'B

iank

a'

3.2

a 14

.5 a

52

.76

a 20

.69

a 2.

80 a

0.

55 b

'T

hree

fold

Whi

te'

3.3

a 12

.4 a

42

.74

b 17

.43

b 2.

70 a

0.

65 a

N

I 3.

3 a

13.1

a

47.3

7 a

19.3

7 a

2.68

a

0.63

a

N,

3.3

a 13

.5 a

48

.78

a 19

.24a

2.

78 a

0.

62 a

N

l 3.

1 a

13.7

a

47.1

1a

18.5

8 a

2.77

a

0.56

a

Har

vest

sta

ge

Gaf

. V

aguc

ira

3.2

a 28

.0 a

22

,858

a

141.

62 a

68

.41

a 64

.21

a 34

.75

a L

ouri

nha

3.5

a 21

.4 b

14

,078

b

100.

50 b

42

.13

b 36

.27

b 22

.90

b C

oruc

he

3.5

a 2

1.5

b

9,87

5 b

76.1

6 c

38.2

5 e

26.9

0 b

11.6

9c

'Bia

nka'

3.

3 a

23.9

a

16,1

24 a

10

8.74

a

50.1

4 a

43.1

7a

24.4

7 a

'Thr

eefo

ld W

hite

' 3.

5 a

23.4

a

15,0

84 a

10

3.40

a

49.0

5 a

41.7

5 a

21.7

5 a

NI

3.5

a 23

.6 a

15

,884

a

107.

54 a

52

.88

a 44

.69

a 23

.52

a N

, 3.

4 a

23.3

a

15,3

69 a

10

4.41

a

45.9

5 a

442.

68 a

23

.99

a 3.

3 a

24.3

a

15.5

60 a

10

6.33

a

50.0

0 a

40.0

1 a

21.8

3 a

Mea

ns w

ithi

n a

colu

mn

follo

wed

by

the

sam

e le

tter

are

not

sig

nifi

cant

ly d

iffe

rent

at

1% p

roba

bilit

y.

n.s.

n.

s.

Ace

tyle

ne

redu

ctio

n ra

tes

nmol

C2H

4 pl

ant

1 m

in.

1

56.0

2 a

46.4

5 a

56.0

3 a

54.6

8 a

50.7

2 a

58.0

6 a

47.6

1 a

52.8

0 a

N u

ptak

e (k

gh

a-1

)

30.7

4***

n.

s.

10

.49

"

N u

ptak

e (k

g ha

-I)

104.

00 b

14

3.04

a

135.

52 a

11

1.52

b 12

7.18

a 12

1.96

a

121.

43 a

~

~

Q

~

~· .., ~

~

;::

0 ;::

()q

.., 0 :;; St- ~

<:J- .., 0 ;,

;,..

<:J-~

;,

;:: "' N

---.1

'D

280 Palha et al.

soil characteristics studied during the growing cycle are shown and significant differences iden­tified.

Table 3 summarizes the effects of sites and fertilizer on plant characteristics, at flowering and at harvest. Mean values of stem and node numbers per plant, plant height, dry-matter yield, nitrogenase activity and N uptake for each site, level of N-fertilizer and culture are pre­sented in Table 4.

At both flowering and harvest stages, N-fertil­izer had no significant effect on any of the plant characteristics, only at the former stage nodule dry-matter was significantly affected. Neither variety nor N-fertilizer and sites significantly affected acetylene reduction capacity, but there was considerable variability and the analysis of variance was performed using the logarithms of the data.

At flowering stage, there were highly signifi­cant differences between cultivars in node number, plant height and shoot dry-matter yield; nodule weight and N uptake appeared signifi­cantly different. 'Bianka' was the tallest cultivar and had the largest shoot dry-matter yield and N uptake (135.52 kg ha - 1). 'Threefold White' had the highest dry weight of nodules. At harvest, no significant difference between cultivars was de­tected.

A highly significant effect of sites on most plant characteristics was observed, at both sam­pling stages. The highest grain yield was found in Gafanha da Vagueira (22,858 kg ha- 1 ) and the lowest in Coruche (9 ,875 kg ha -I). These differ­ences in yield are probably due to the quite different climatic conditions in these two regions. Gafanha da Vagueira is a coastal site with higher humidity and a narrower daily temperature inter­val, while Coruche is a warmer and dried region. At flowering, N uptake was higher in Lourinha ( 143.04 kg ha -I) and lower in Gafanha da Vagueira (104.00kg ha- 1). N uptake in each cultivar varied from an average of 0.851 to 0.931 g per plant, respectively for 'Threefold White' and 'Bianka'.

Discussion and conclusions

Residual inorganic-N at 0-20 em depth, at flow-

ering and harvest, was not significantly affected by N treatment and cultivars, which indicates that fertilizer was probably recovered by the crop at the beginning of the growing cycle, immobilized or even lost. Values of pH and total N also did not significantly differ from the initial ones.

Both cvs. Bianka and Threefold White had well-nodulated roots respective of treatments or site; the highest nodule production was at Lourinha and the lowest at Coruche. Nitrogen­ase activity was not significantly affected by locations or cultivars or N-fertilizer.

N uptake was not statistically affected by nitrogen applied. The N uptake at flowering was higher at Lourinha than at Gafanha da Vagueira and was higher than 0.708 g per plant, obtained in a growth chamber experiment with no applica­tion of N fertilizer (Richards and Soper, 1979). Even the lowest N uptake obtained in this experiment was higher than those reported by Simoes eta!., (1990) for the same cultivar, under similar experimental conditions. With the best inoculation of V. faba (var. minor) grown in pots, Sherbecny et a!. (1977) obtained 0.191 g of N uptake per plant, at flowering stage.

The absence of a broad beans response to N treatment is in agreement with the results of another similar experiment, where no significant effect was obtained to the application of four N rates (0, 30, 60 and 90kg ha- 1) to 'Bianka' broad bean (Simoes et a!., 1990). Day et a!. (1979) verified that spring faba yield was unaf­fected by the application of 200 kg ha -J of N.

In spite of scarce references for this crop in Portugal, grain yield obtained in the present trial with minor varieties was quite significant, rela­tively to the productions got by Ferreira (1988) and Simoes et a!. (1990).

Because N treatment did not significantly affect nitrogenase activity and grain yield, it seems reasonable to conclude that up to 60 kg ha -J of fertilizer N was not inhibitory to N 2 -

fixation. However, there is no need to apply N fertilizer to these crop varieties, under the pres­ent field conditions, even as N-starter, because no significant crop response was observed at flowering stage, though residual soil N at 0-20 em depth was not statistically affected by N applied. Richards and Soper (1979) also found

no need to apply nitrogen as starter. According to Day et al. (1979), failure of response to nitrogen fertilizer indicates that though soils may be poor in nitrogen and organic matter the nodules may supply plant requirements in the presence of native Rhizobium specific to both spring broad beans cultivars.

Acknowledgements

The authors acknowledge J F Marques for help­ing with the nitrogenase activity assay and results interpretation, the Pedology Dept. staff for the analytical work, and those contributing for paper revision, in particular for criticism by Dr D J Greenwood.

References

Carranca C F 1986 Nitrogen availability and ammonium fixation in some maize cultivated soils of Portugal. Thesis for M. Sc. in Soil Science, Agric. Univ. Norway, Oeiras, 89 p.

Effect of nitrogen on growth of broad beans 281

Day J M, Roughley R J and Witty J F 1979 The effect of planting density, inorganic nitrogen fertilizer and supple­mentary carbon dioxide on yield of Vicia faba L. J. Agric. Sci., Camb. 93, 629-633.

Ferreira M E S 1988 Fava miuda para a industria de congelac;ao. Folhas de Divulgac;ao 2. INIA, DHF. 15 p.

Hardy R W F, Holsten R D, Jackson E K and Burns R C 1968 The acetylene ethylene reduction assay for N fixation: laboratory and field evaluation. Plant Physiol. 43, 1185-1207.

Richards J E and Soper R J 1979 Effect of N fertilizer on yield, protein content, and symbiotic N fixation in faba­bcans. Agron. J. 71, 807-811.

Simoes A M, Calouro F, Ferreira M E and Saltao B 1990 Resposta da cultura de fava miuda para congelac;ao a aplicac;ao de quatro niveis de adubac;ao azotada e pot­assica. Aetas de Horticultura 5, 217-222.

Sherbeeny M H El-, Mytton L R and Lawes D A 1977 Symbiotic variability in Vicia faba. 1. Genetic variation in the Rhizobium leguminosarum population. Euphytica 26, 149-156.

Yuch L-Y and Hensley D L 1993 Pesticide effect on acethylene reduction and nodulation by soybean and lima bean. J. Am. Soc. Hortic. Sci. 118, 73-76.

Zapata F, Danso S K, Hardarson G and Fried M 1987 Nitrogen fixation and translocation in field-grown faba­bean. Agron. J. 79, 505-509.

M.A. C. Fragoso and M.L. van Beusichem (eds.) Optimization of plant nutrition 283-290, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-168

Nitrogen uptake in relation to water availability in wheat

J.P. De M.E. ABREU, I. FLORES, F.M.G. De ABREU and M.V. MADEIRA Department of Environmental Sciences, Instituto Superior de Agronomia, Tapada da Ajuda, 1300 Lisboa, Portugal

Key words: nitrogen concentration, nitrogen content, nitrogen uptake, Trilicum aestivum L., wheat, water stress

Abstract

Nitrogen uptake and distribution in wheat (Triticum aestivum L.) arc dependent on environmental conditions and in particular on the water regime. Under Mediterranean conditions, where high water stress at the end of the crop cycle is frequent, nitrogen uptake can be reduced, affecting yield and quality of the grain. To disclose these relations a field experiment was carried out in Central Portugal. Wheat was grown on a clay soil (Vertisol) at three water treatments: rainfed (WO), with 80 mm of irrigation (W1) and with 50mm and 70mm irrigations (W2). All treatments received 50 kg ha- 1 of N prior to sowing and were top-dressed with 140 kg ha -I of N, splitted in two applications, Kjeldahl N was determined in green leaves (GL), yellow leaves (YL), stems (ST), chaff (CH) and grain GR). N uptake after an thesis was 40% of the total in W2, but was not noticeable in the other two treatments. N concentrations in the total above-ground plant dry matter, and in both YL and ST were not very different according to treatment, but water availability increased grain-N concentration. It seems, therefore, that grain protein concentration and N uptake can be substantially increased by late irrigations.

Introduction

Mediterranean conditions are characterized by a quickly developing water deficit after spring, due to both a rapidly increasing evaporative demand and reduced rainfall. Water regime influences nitrogen uptake, concentration and distribution within the plant, even when the absolute amount of nitrogen in the soil volume occupied by the roots is not limiting (Bauer et al., 1987; Camp­bell et al., 1983; Clarke et al., 1990).

Modelling nitrogen cycle demands a precise knowledge of these interactions and related plant responses which are still lacking for Mediterra­nean conditions. Detailed growth models simu­late N uptake using empirical relations derived from available data on N concentration in the plant parts in relation to development stage. It is

also important to know the current N concen­tration of leaves to be able to accurately simulate photosynthesis (Van Keulen and Seligman, 1987). Present wheat growth models do not account for the effect of water stress on maxi­mum concentration in the plant parts (Aslyng and Hansen, 1985; Groot, 1987; Van Keulen and Seligman, 1987; Stockle and Campbell, 1989). Good data on the effects of low water availabili­ty on late N uptake in wheat under field con­ditions are scarce. Most of these studies are conducted under conditions inflicting limited water stress either because storage capacity of the rooted soil is high or because of insufficiently dry summers (e.g. Bauer et al., 1987; Boatwright and Haas, 1961; Groot, 1987).

A field experiment with wheat was carried out in Central Portugal. The changes of the nitrogen

284 Abreu et al.

concentration and content in the above-ground plant parts was followed, under three water regimes.

Materials and methods

Spring wheat (Triticum aestivum L., cv. Pesudo) was grown in the fields of Tapada da Ajuda, Lisbon, Portugal (latitude: 38°42' N; longitude: 9° 11' W) in 1991.

The soils of the experimental area are Vertisols with a clay content of ca. 50%, a pH(KCl) of 7, an organic matter content of 4%, and very high measured extractable P and K. Plots differed in soil depth. Measured maximum rooting depths, observed by direct examination of soil cores, are shown in Table 2.

Three water treatments were imposed on the three plots; WO was rainfed, W1 was given an irrigation of 80 mm 20 days after anthesis, and W2 was irrigated with 50 mm and 70 mm, 4 and 15 days after anthesis, respectively. Soil water was monitored with a neutron moisture meter, in 0.10 m increments to 1.20 m, in three access tubes per plot.

Standard meteorological data were collected at a weather station not further than 100m, as well as above the crop. Ten-day average values of air

600

500

e E 400

n::'" 1- ~ we;; 0 ~ 300 w > a:::::: a..!l!

" E 200

" ~ 100

0 llill-,~.~

c::===J ETR

______._ Tav

r v _._ /

h. n. l. n ~. II Ill II Ill

Fev II Ill

Jan Mar

temperature, cumulative rainfall, and cumulative reference evaporation (Penman, 1948) are shown in Figure 1.

The soil was ploughed in autumn and disked twice before sowing, when 50 kg ha - 1 of N in ammoniacal form and 100 kg ha - 1 of P were incorporated. As split dressing, 70 kg ha - 1 (at tillering) and 70 kg ha - 1 (at stem elongation) of a nitro-ammoniacal fertilizer was applied.

Planting was on 24 January 1991. Planting rate was 180 kg ha - 1 of seed with a laboratorial germination percentage greater than 95%. After emergence (14 February) an average of 205 plants m - 2 were established. Zadoks decimal codes for the main stem and first side tiller were assessed. Tillering (14, 23), stem elongation (16, 31) and anthesis (65) occurred 41, 53 and 81 days after emergence respectively. Dead ripe maturity, when grain humidity reached 0.150 kg of water per kg of grain, was attained 115, 116 and 122 days after emergence, respectively for WO, W1 and W2.

In each treatment the plants of four sampling plots (0.5 m2) were harvested weekly. From each sampling plot 20 plants were selected and partitioned in four five plant subsamples: green (GL) and yellow (YL) leaves, stems (ST) (sheaths were included), chaff (CH) and grain (GR). Plant parts were oven dried (70oC) and

~ 1/

II Ill Abr

Ill Mai

v/

II Jun

25

20

15

10

5

0

Fig. 1. Time-course of cumulative precipitation (PRE C), Penman reference evaporation (ETR) and average temperature in the experimental site.

weighed. For N analysis the plant subsamples were pooled to give one composite sample per treatment and sampling period for each plant part. The composite sample was ground in a laboratory mill with a 1 mm screen and its N content was determined with a Kjeltec equip­ment.

Data were plotted against a normalized de­velopmental stage scale (Penning de Vries and Van Laar, 1982). The accumulated temperature at a given moment is divided by the thermal time required to reach an event. Emergence is event 0

5

-cf2. 4 16~ ..___.. r::: 0

N uptake in relation to water availability 285

and anthesis is 1; maturity (15% water content in the grains) corresponds to 2. Base temperatures of 3°C before anthesis and soc thereafter were used, realistic numbers for Mediterranean wheat cultivars (Pozo et al., 1987; Weir et al., 1984).

Results and discussion

The changes of N concentration of the aerial parts of the plants (Figs. 2 to 5 and Table l) is similar to that reported in the literature by other

-fr-- GLO

D· GL1

:p 3 -- ·*·--* ··*· GL2 co .. '--c

* YLO Q) 2 ....

(.)

-~, r::: --.- YL1 0 • (.)

z ---0----- YL2

0

0.00 0.50 1.00 1.50 2.00

Developmental stage Fig. 2. Green-leaf and yellow-leaf N concentration against a normalized developmental stage scale (base temperatures before and after an thesis are 3"C and 8"C, respectively). GLO and YLO, GLl and YLl, GL2 and YL2 correspond to treatments WO, W1 and W2. respectively.

Table 1. Changes in concentration of N (%) in plant parts in two extreme treatments

Days GLO YLO STO GRO SHO GL2 YL2 ST2 GR2 SH2 after em erg.

36 4.108 2.205 3.215 3.586 2.454 2.724 43 3.915 2.227 2.574 4.182 2.103 3.235 so 4.126 2.826 1.988 3.962 2.351 2.059 57 2.783 1.588 1.186 3.422 1.800 1.424 64 2.980 1.548 1.019 3.670 1.482 1.347 71 3.429 1.468 1.526 3.765 1.968 1.628 78 3.331 1.753 1.753 3.363 1.745 1.662 85 3.009 1.811 1.099 2.892 1.555 1.084 1.684 99 2.418 1.044 0.748 2.195 1.070 2.962 1.179 0.880 2.034 0.825

106 2.165 1.325 0.419 2.962 1.183 0.642 113 1.351 0.906 0.357 1.826 1.110 1.888 0.927 2.297 0.705 120 1.570 0.891 0.416 2.392 0.749

286 Abreu et al.

workers under similar conditions (e.g. Bauer et al., 1987; Boatwright and Haas, 1961; Karlen and Whitney, 1980).

Green-leaf N concentration depicts a quasi­linear decrease (Fig. 2), from about 3% at an­thesis to 1.5% at maturity in all treatments. However, a major difference is that W2 was able to maintain N concentration in the leaves around 3% throughout grain filling. This maintenance of N concentration is correlated with the mainte­nance of the green lea area index (Fig.8). In all treatments, the steep decrease in N concentra­tion after stage 0.6 was related to an unusual dry period (Fig. 1 ).

Yellow-leaf N concentration had an evolution parallel to GL N concentration (Fig. 2). Im­portant differences among treatments were not found. Nitrogen concentration at anthesis was around 1.7%, declining to about 1% at maturity.

'Stem' N concentration exhibited a non-linear evolution (Fig. 3). All treatments show a steep decline in N concentration during the first stages of growth. N concentrations then declined slowly from about 1% at anthesis to 0.5% at maturity.

Grain and chaff N concentrations depicted differences according to water treatment (Fig. 4). Greater concentrations in the grain were achieved for the wetter treatments, with an inverse effect in chaff N concentration. This might be related to the longer grain growth

5 -eft. 4 -r:::

0 :;::::; 3 co ,_ ..... r::: Cl) 2 t.) r::: 0 t.)

z 0

period of the wetter treatments, associated with a more intense translocation of GL and CH nitrogen to the grain. Moreover, W2 had a substantial uptake of N after anthesis, as dis­cussed later.

Above-ground N concentrations were derived from all above-ground parts using a weighted average. It was very similar in all treatments (Fig. 5), since the partial values did not exhibit big differences among them. Above-ground N content was very different (Fig. 6). This is a consequence of the increase of dry matter yield attained in the wetter treatments (Fig. 7).

The experimental plots differed in soil depth, limiting the maximum depth attained by the roots (Table 2). Since soil depths were smaller on the drier treatments there was an enhance­ment of the effects of irrigation. Water availabili­ty almost doubled N uptake after anthesis in W2 (when the drier treatments showed noN uptake) mainly through an equivalent increase in dry matter accumulation (Table 2). The magnitude of these late increases is beyond what we would expect based on our experience and relevant literature (Clarke et al., 1990; Doorenbos and Kassan, 1979; Entz and Fowler, 1989). This finds its explanation in a green leaf area survival increase (Fig. 8), extended grain growth period and enhanced root activity. This increase in root activity and the effect of water on soil strength

--D- STO

--*- ST2

0.00 0.50 1.00

Developmental stage

1.50 2.00

Fig. 3. Stem N concentration against a normalized developmental stage scale (base temperatures before and after anthesis are 3'C and S'C, respectively). STO, STl, and ST2 correspond to treatments WO, Wl and W2, respectively.

-'#. -c: 0

:;:::; Cl'l .... -c: Q) () c: 0 ()

z

5

4

3

2

0

--tr-- GRO

0 ····· GR1

--*-GR2

* ··· CHO

--11-- CH1

---0-- CH2

N uptake in relation to water availability 287

0.00 0.50 1.00

Developmental stage

1.50 2.00

Fig. 4. Grain and chaff N concentration against a normalized developmental stage scale (base temperatures before and after anthesis are 3"C and S"C, respectively). GRO and CHO, GRl and CHl, GR2 and CH2, correspond to treatments WO, Wl and W2, respectively.

5 -'#. 4 - ---D- PLO

c: 0

t:. PL 1 :;:::; 3 Cl'l .... *- PL2 -c: Q) 2 () c: 0 ()

z 0 0.00 0.50 1.00

Developmental stage

1.50 2.00

Fig. 5. Above-ground plant N concentration against a normalized developmental stage scale (base temperatures before and after anthesis arc 3"C and S"C, respectively). PLO, PLl, and PL2 correspond to treatments WO, Wl and W2, respectively.

might explain the differences encountered in rooting depth.

All treatments showed a halt in N uptake between heading and flowering, that might be related to an yet unexplained phenomenon, also observed by Campbell et al. (1983), that can even lead to loss of nitrogen from the plant. N uptake halted just after flowering in the case of the treatments WO and Wl, but continued almost

linearly until the end of grain filling in W2. The amount of above-ground N uptake after anthesis was at this treatment about 40% of the total. Such high values were also found by Campbell et al. (1983); Clarke et al. (1990); Spiertz and Ellen (1978). This means that as much as 47% of the grain N content can come from N uptake after anthcsis, if translocation from the roots is not taken into account.

288 Abreu et al.

-..-I

< co ..c: C)

~ ..... c:: Q) ..... c:: 0 0

z

120

100

80

60

40

20

0 0

---o- CNO

6: CN1

0.5 1.5 2

Developmental stage

Fig. 6. Above-ground N content against a normalized developmental stage scale (base temperatures before and after anthesis are 3oC and 8°C, respectively. CNO, CN1, and CN2 correspond to treatments WO, W1 and W2, respectively.

10 -..- 9 I < 8 co ..c: 7 t/) c:: 6 0 ..... 5 ........ .... 4 Q) ~ 3 co E 2 2:- 1 0

0 0

-D- DMO

6: DM1

--*- DM2

0.5

:¥··············

'~~/·

1.5 2

Developmental stage

Fig. 7. Above-ground dry-mater accumulation against a normalized developmental stage scale (base temperatures before and after anthesis are 3°C and soc, respectively). DMO, DM1, and DM2 correspond to treatments WO, WI and W2, respectively.

Table 2. Maximum root depth, dry matter (DM) and nitrogen in the aerial part of the plants

Treatment Maximum DMat DMat Harvest N uptake N uptake N harvest root depth anthesis maturity index until until index (±SE) (kg ha - 1) (kg ha _,) (%) an thesis maturity (%) (em) (kgha- 1) (kgha- 1)

wo 35.6( ± 4.6) 2812 4131 32.6 42.91 36.52' 74 WI 42.2( ± 6.2) 4544 6100 32.6 61.67 66.77 68 W2 52.8( ± 10.3) 4522 9067 40.9 71.26 118.55 86

'This value is smaller than the one until anthesis, what must be due to sampling error of D M.

N uptake in relation to water availability 289

2.5

---D-- wo

>< 2 Cll

········-/:,; ····· W1 't:l r::: ·- ...... C'CIN 1.5 CIJ I .._< C'CI E 'lU~ .!!!E 1 r:::~ Cll e (!) 0.5

\ .. A · ..

\1::. 0

0 20 40 60 80 100 120

Days after emergence Fig. 8. Time-course of green leaf area index in treatments WO, WI, W2.

The harvest indices for N were 0.74, 0.68 and 0.86, at WO, Wl, W2, respectively.

Conclusions

Some qualitative rules of thumb relating high grain-protein content to environmental condi­tions can be misleading. Postanthesis water stress and increased N availability in the soil do not always determine a higher grain protein, since retranslocation, postanthesis N uptake and growth respond differently to those, and other, conditions. This stresses the need for quantita­tive tools: simulation models.

The changes of N concentration in the differ­ent aerial parts of the plant under conditions of high availability of N was not very different from what is found in the literature and was relatively unaffected by water stress (except for GL and GR). This allows the curves fitted to the data to be used in assessing N deficiencies and in N modelling.

Acknowledgement

We thank Junta Nacional de Investiga<;ao Cien­tifica e Tecnol6gica (JNICT) for the financial support.

References

Aslyng H C and Hansen S 1985 Radiation, water and nitrogen balance in crop production. Field experiments and simulation models WATCROS and NITCROS. Hydrotech­nical Laboratory. The Royal Veterinary and Agricultural University, Copenhagen.

Bauer A, Frank A B and Black A L 1987 Aerial parts of hard red spring wheat. II. Nitrogen and phosphorus concentration and content by plant development stage. Agron. J. 79, 852-858.

Boatwright G 0 and Haas H J 1961 Development and composition of spring wheat as influenced by nitrogen and phosphorus fertilization. Agron. J. 53, 33-36.

Campbell C A, Davidson H R and McCaig T N 1983 Deposition of nitrogen and soluble sugars in Manitou spring wheat as influenced by N fertilizer, temperature and duration and stage of moisture stress. Can. J. Plant Sci. 63, 73-79.

Clarke J M, Campbell C A, Cutforth H W, DePauw R M and Winkleman G E 1990 Nitrogen and phosphorus uptake, translocation, and utilization efficiency of wheat in relation to environment and cultivar yield and protein levels. Can. J. Plant Sci. 70, 965-977.

Doorenbos J and Kassan A H 1979 Yield response to water. Irrigation and Drainage Paper No.33. FAO, Rome.193 p.

Entz M H and Fowler D B 1989 Response of winter wheat to N and water: growth, water use, yield and grain protein. Can. J. Pant Sci. 69, 1135-1147.

Groot J J R 1987 Simulation of nitrogen balance in a system of winter wheat and soil. Simulation Report No 13. Centre for Agrobiological Research. 69 p.

Karlen D L and Whitney D A 1980 Dry matter accumula­tion, mineral concentrations, and nutrient distribution in winter wheat. Agron. J. 72, 281-288.

Penman H L 1948 Natural evaporation from open water,

290 N uptake in relation to water availability

bare soil and grass. Proc. R. Soc. London. A, 194, 120-145.

Penning de Vries F W T and van Laar H H 1982 Simulation of plant growth and crop production. Simulation Mono­graphs. PUDOC, Wageningen. 309 p.

Pozo A H, Garcia-Huidobro R N and Villaseca S 1987 Relationship of base temperature to development of spring wheat. Expl. Agric. 23, 21-30.

Spiertz J H J and Ellen J 1978 Effects of nitrogen on crop development and grain growth of winter wheat in relation to assimilation and utilization of assimilates and nutrients. Neth. J. Agric. Sci. 26, 210-231.

Stockle C 0 and Campbell G S 1989 Simulation of crop response to water and nitrogen: an example using spring wheat. Transactions of the ASAE 32, 66-74.

Van Keulen H and Seligman N G 1987 Simulation of water use, nitrogen nutrition and growth of a spring wheat crop. Simulation Monographs. PUDOC, Wageningen. 310 p.

Weir A H, Bragg P L, Porter J R and Rayner J H 1984 A winter wheat crop simulation model without water or nutrient limitation. J. Agric. Sci., Cambridge 102, 371-382.

Reprintedfrom Plant and Soi/154: 89-96, 1993.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 291-297, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-235

Effect of nitrogen-form on plant growth and nutrient composition of creeping bentgrass

JAMES N. McCRIMMON 1 , KEITH J. KARNOK 2 and HARRY A. MILLS 3

1Department of Agronomy and Horticulture, Box 30003, New Mexico State University, Las Cruces, NM 88003, 2 Department of Agronomy, 3114 Miller Plant Sciences Building, University of Georgia, Athens, GA 30602 and 3Department of Horticulture 1114 Miller Plant Sciences Building, University of Georgia, Athens, GA 30602, USA

Key words: Agrostis palustris (Huds.), creeping bentgrass, nitrogen, nutrient solution

Abstract

The effect of nitrogen-form on the growth and nutrient composition of 'Penncross' creeping bentgrass [Agrostis palustris (Huds.)] was determined on turfgrass grown in nutrient solution culture under greenhouse conditions. The nutrient solutions consisted of a modified Hoagland's solution with the form of nitrogen as either nitrate, ammonium, urea, or a 50:50 ratio of nitrate:ammonium. Non­nitrogen and water (control) treatments were applied. TheN concentration was 150 mg N kg -I with the N sources being (NH4 ) 2SO 4 , Ca(N03 ) 2 • 4H20, and CO(NH2 ) 2 for the ammonium, nitrate, and urea treatments, respectively. The turfgrass was maintained at a height of 1 em and clipped twice weekly. Dry weight and macronutrient and micronutrient content were determined for shoot, root, verdure, and total plant at the end of the 10 week study. The nitrate treatment yielded the highest shoot dry weight while the urea treatment yielded the highest root, verdure, and total plant dry weight. Both the nitrate­and urea-treated plants yielded more root dry matter than did the ammonium-treated plants. Nitrogen form influenced macronutrient content in the root and verdure. The ammonium-treated plants had the highest N concentration in the roots compared to plants of the nitrate and urea treatments. The nitrate­and urea-treated plants resulted in higher P, Ca, and Mg concentrations in the root compared to those of the ammonium treatment. The urea-treated plants had the highest P, Ca, and Mg concentrations in the verdure. Nitrogen-form influenced micronutrient content in the shoot, root, and verdure. In the shoot tissue, urea-treated plants had the highest B concentration, ammonium-treated plants the highest Fe concentration, and nitrate-treated plants the highest Mn. Generally, the nitrate- and urea-treated plants had higher concentrations of micronutrients in the root tissue compared to the ammonium­treated plants and, in the verdure, nitrate-treated plants had the highest concentrations of mi­cronutrients.

Introduction

Creeping bentgrass is the best turfgrass for top quality golf greens (Beard, 1973). The creeping stolons at the surface of the ground allow roots and shoots to grow from the nodes thus enabling creeping bentgrass to form a fine textured turf of great density, uniformity, and quality (Beard, 1973). When properly maintained, it forms an

ideal turf that is preferred on golf greens (Juska and Hanson, 1959; Lapp, 1943).

The level and balance of nutrients in creeping bentgrass depend upon fertilizer applications, soil nutrients, and plant response. Although the plant must be provided all essential nutrients to obtain the best growth, nitrogen is generally the most limiting nutrient influencing plant growth in a given cropping system (Barker and Mills, 1980;

292 McCrimmon eta!.

Hageman, 1980). In turfgrass, nitrogen is re­quired in the largest amount and is applied in the largest quantity of any of the essential nutrients (Beard, 1973; Davis, 1962). The form, source, and rate of nitrogen applied affects the establish­ment and overall growth of the turfgrass plant. The main nitrogen forms for plants are am­monium and nitrate. They produce different physiological responses within plants (Cox and Reisenauer, 1973; Haynes and Goh, 1978), and plants vary in their ability to absorb and utilize ammonium and nitrate (Kirkby and Hughes, 1970; Lycklama, 1963).

The studies involving the effect of different nitrogen forms or nitrogen fertilizer sources on turfgrass have primarily investigated the effects of nitrogen upon various growth parameters and have not investigated what effect the nitrogen form has on other essential nutrients (Darrow, 1939; Sprague, 1934). The importance of the essential nutrients for proper plant nutrition is well established (Mengel and Kirkby, 1987). Although there has been work regarding the effect of nitrogen forms on other macronutrients such as phosphorus and potassium, there is a limited amount of work concerning the effect of nitrogen forms on the other macronutrients and the micronutrients in turfgrass. The addition of micronutrients to turf have been reported to affect growth (Killinger et al., 1943); improve turfgrass color and sod density (Deal and Engel, 1965); and affect micronutrient tissue concen­trations (Markland and Roberts, 1969; Wadding­ton et al., 1972).

Although the importance of nutrients other than nitrogen for proper turfgrass growth is well established, the actual concentrations of these nutrients in plant tissue when these nutrients arc adequately applied under different nitrogen forms is not well documented. Field studies utilizing different sources of nitrogen have not dealt solely with the effect of nitrogen-form on turfgrasses. In addition, the loss of nitrogen caused by leaching, denitrification, and/ or vol­atilization occurring after fertilizer application in the field presents problems in assessing the true effect of a given form of nitrogen on the turf.

Creeping bentgrass is utilized on golf course putting greens that are composed primarily of sand. These sand-based greens react similar to a

hydroponic system, thus, solution culture meth­odology was utilized to assess the effect of nitrogen-form on creeping bentgrass under con­trolled conditions in which nitrogen loss could be minimized. Therefore, the objectives of this study were to determine the effect of nitrogen form on: 1) plant growth and 2) elemental composition of creeping bentgrass.

Materials and methods

Plugs of creeping bentgrass ( 10.2 em diameter x 8.4 em deep) were taken from an established 'Penncross' creeping bentgrass putting green. Soil was thoroughly washed from the plugs, the roots were cut 2 em below the thatch layer, and plugs were grown in solution culture in the greenhouse. Each plug was placed in a separate 18 L solution culture vessel that contained 15 L of water. Water was changed weekly for 3 weeks, at which time nutrient solution treatments con­taining 15 L of nutrient solution were applied to the plants for the next 7 weeks. Nutrient solu­tions were applied as a modified Hoagland's solution at an initial pH of 6.4 to 6. 7 and were changed each week. The pH was monitored weekly and was not adjusted. The treatments were 100%NH:, 100%NO~, 50:50 NH: to NO~, urea, non-nitrogen, and water. The nitro­gen (N) concentration was 150 mg N kg - 1 with the N sources being (NH4 ) 2S04 ,

Ca(N0 3 L4H20, and CO(NH2 ) 2 for the NH:, NO~, and urea treatments, respectively. The 50:50 treatment received 75 mg N kg - 1 from each of (NH 4 ) 2S0 4 and Ca(N0 3 ) 2 ·4H2 0, while the non-nitrogen and water treatments received no nitrogen during the entire study. The water treatment did not receive any macro- or mi­cronutrients. All other treatments received phos­phorus (P) and potassium (K) as 40 mg P kg 1

and 50 mg K kg - 1 , respectively, from KH 2PO 4 ,

and 100 mg K kg - 1 from K2S0 4 • The NO~ and 50:50 treatments received 214 and 107 mg cal­cium (Ca) kg- 1, respectively, from Ca(N0 3 ) •

4H2 0. The 50:50, NH;, urea, and non-nitrogen treatments received 85 mg Ca kg - 1 from CaC1 2 ,

with the three former treatments receiving an additional 129 mg Ca kg - 1 from CaSO 4 • 2H 2 0 (50:50 obtained 22 mg) in order to provide a

total of 214 mg Ca kg -I. The NO~ treatment received 51 mg magnesium (Mg) kg -t from MgC12 • 6H 2 0 while the other treatments re­ceived 51 mg Mg kg -I from MgS0 4 • 7H 20. All treatments, except the water treatment, received the following micronutrient concentrations: 0.25 mg boron (B) kg- 1 as H 3B03 , 0.25 man­ganese (Mn) as MnC1 2 · 4H20, 0.02 mg copper (Cu)kg- 1 as CuS0 4 ·5H 20, 0.01mg molyb­denum (Mo)kg- 1 as Mo0 3 , 0.5mg zinc (Zn)kg- 1 as ZnS0 4 ·7H 20, and 5mg iron (Fe) kg- 1 as Fe-EDTA. Chloride (Cl) was pres­ent at a concentration of 150 mg Cl kg -I pro­vided by CaC1 2 and MgC1 2 • 6H20. Sulfate was the only nutrient to vary among the treatments.

Transpirational losses from the solutions were replaced weekly with deionized water. Nutrient solution samples were taken weekly for the old and new nutrient solutions for each solution and analyzed for differences in initial and ending values of each clement. Ammonium and nitrate values were determined using an AutoAnalyzer while other elements were analyzed using an inductively coupled argon plasma (ICAP) emis­sion spectrometer. Turf was clipped twice weekly and maintained at a height of 1 em. Roots, verdure, and final shoot clippings were harvested at the end of 10 weeks and dry weights were determined. Kjeldahl N was determined for the shoot, root, and verdure of each plant. Other essential elements were analyzed by ICAP fol­lowing dry-ashing.

The experiment was conducted as a complete­ly randomized block design with 4 replications. The data was subjected to analysis of variance utilizing the GLM procedure of the Statistical Analysis System (SAS), and LSDs were per-

Effect of nitrogen-form on plant growth 293

formed to determine mean separation (SAS, Version 6, SAS Institute, Cary, NC).

Results and discussion

Growth response

The NO~ treatment yielded greater shoot dry weight than the NH; and 50:50 treatments (Table 1). The NH; and 50:50 treatments re­sponded similarly, 0. 57 and 0. 54 g shoot -I, re­spectively. Urea yielded the greatest root and verdure dry matter and was different from all nitrogen treatments. The NO~ treatment yielded a greater amount of root dry matter compared to the NH; treatment. This is similar to what others have reported that various turfgrasses have yielded greater root growth when grown under high NO~ levels compared to those plants grown under 100% NH; (Darrow, 1939; Har­rison, 1934). The root dry weight for the NH;­treated plants was lower than both the non­nitrogen and water treatments. Others have reported less root growth for plants grown in 100% NH; (Bennett et a!., 1964; Glinski et a!., 1990; Maynard and Barker, 1969). This may be attributed to greater acidity in the rhizosphcre since NH; may lower the pH of the rhizosphere by several units (Barber, 1984; Smiley, 1974; Warnke and Barber, 1973). In the present study, the pH of the new NH; solutions each week were initially 6.4 to 6.7 but the pH decreased to 4.5 or lower within 24 to 36 hours. Therefore, the low root dry matter accumulation for the NH; -treated plants may have been caused by the low pH in the rhizosphere of these plants.

Table I. Effect of N-form on shoot, root, verdure. and total plant dry weight of creeping bentgrass

Total dry weight (g/ plant)

Shoot Root Verdure Total plant

No; 0.91 a 1.31 be 6.69 b 8.91 b Nil; 0.57 b 0.54 e 6.55 b 7.66 be Urea 0.63 ab 2.29 a 9.41 a 12.33 a 50:50 0.54 b 0.68 de 5.11 b 6.33 c Non-nitrogen 0.16 c 1.76 ab 6.48b 8.40 be Water 0.12 c 1.16 cd 5.6R b 6.96 be LSD 0 05 0.33 0.53 2.04 2.31

Means within a column followed by a common letter arc not significantly different based on the LSD (0.05).

294 McCrimmon et al.

Urea yielded the greatest amount of verdure and total plant dry matter (Table 1 ). There were no differences between the NO~ and NH; treatments for total plant dry weight. This is in-contrast to what previous studies have re­ported in which a variety of grasses receiving a portion or all nitrogen as NO~ produced greater dry matter than those receiving only NH; (Arnon, 1937, 1939; Mazur and Hughes, 1976; Sprague, 1934). Although the NO~ -treated plants yielded greater shoot and root dry matter compared to the NH; -treated plants, total plant dry matter was similar. The verdure dry matter was similar for both treatments. The verdure may act as a buffer zone for dry matter accumu­lation, thus total plant dry matter was not different since the verdure dry weight made up over 75% of the total plant dry matter of each of these treatments.

Macronutrient composition

The form of nitrogen had less effect on mac­ronutrient concentration in the shoot tissue than it did on concentrations in either the root or verdure tissue (Table 2). The urea-treated plants had the highest N concentration in the shoot tissue while there were no differences between the NO~ and NH; treatments. There were no differences among the treatments receiving N for concentrations of P, K, Ca and Mg. This is in contrast to other studies in which NO~ -treated plants resulted in higher concentrations of K, Ca, and Mg (Arnon, 1939; Cox and Reisenauer, 1973). Potential differences among treatments for these macronutrients in the shoot tissue did not result, possibly as a result of the duration of the study and due to some accumulation of these nutrients in the verdure tissue.

Table 2. Effect of N-form on the concentration of macronutrients in creeping bentgrass shoot, root, and verdure tissue

N-form ratio Macronutrient

N p K Ca Mg

Concentration (mg g -I)

Shoot No; 53.4 b 8.4 b 38.9 a 14.6 a 6.0 a NH: 54.8 b 8.7 b 40.1 a 16.0 a 7.3 a Urea 61.8a 8.8 b 38.0 ab 10.5 a 6.5 a 50:50 58.8 ab 8.2 b 36.6 ab 10.8 a 5.0 a Non-nitrogen 37.4 c 11.7a 30.5 b 17.8 a 9.0 a Water 29.7 d 2.6c 11.3c 15.3 a 5.7 a LSDO.O:'i 6.8 1.6 7.7 NS NS

Root No; 16.7 b 48.7b 9.9 b 48.6 b 2.3 a NH; 19.4 a 19.7cd 6.2 de 8.1 c l.Oc Urea 17.0 b 155.9a 5.5 e 203.3 a 1.8 ab 50:50 17.9 ab 22.8 c 7.7 cd 15.5 c 1.4 be Non-nitrogen 12.9 c 34.3 be 12.4 a 18.7 be 1.8 ab Water 14.3 c 1.9d 9.5 be 3.0c 1.8 ab

LSD"'" 2.3 20.6 2.2 32.7 0.5

Verdure No; 17.5 a 21.3 b 12.5 a 36.4 b 3.8 a NH: 17.3 a 5.2 c 10.7 a 7.7 c 2.3 b Urea 16.7 a 43.6 a 11.6a 71.5 a 3.7 a 50:50 22.1 a 11.2 be 11.6 a 16.3 c 2.8 b Non-nitrogen 15.5 a 8.1 c 9.6 a 13.1 c 2.8b Water 15.7 a l.Sc 3.2 b 6.0c 1.2c LSDuus NS 10.7 3.3 17.1 0.8

Means within a column followed by a common letter are not significantly different based on the LSD (0.05); NS =not significant at (p < 0.05).

The NH; -treated plants had the highest N concentration, while urea-treated plants had the highest concentrations for both P and Ca in the root tissue (Table 2). Urea enhanced the uptake of P and Ca as evidenced by the large concen­tration of these two nutrients in the roots of the urea-treated plants. This may be a result of a combination of enhanced uptake of P and Ca by these plants, in addition to adsorption of a Ca-P precipitate on the root surface thus resulting in high levels of these nutrients in the root tissue. The NO~ -treated plants had higher concentra­tions of P, K, Ca, and Mg in the roots compared to the NH; -treated plants. This is similar to what others have reported for K, Ca, and Mg concentrations in the root tissue of NO~ -treated and NH; -treated plants (Blair et a!., 1970; Cox and Reisenauer, 1973). The higher P concen­tration in the NO~ -treated plants was in contrast to what others have reported (Bennett et a!., 1964; Blair et a!., 1970).

Urea had the highest concentrations of P and Ca in the verdure tissue compared to the other treatments (Table 2). These high P and Ca concentrations, along with high concentrations of P and Ca in the roots of the urea-treated plants, indicated that urea-treated plants enhanced up­take of these two nutrients but the shoot tissue did not reflect the high level of uptake of these two nutrients. The NO~ and urea treatments had the highest Mg concentrations. Limited informa­tion is available as to the potential of the verdure as a site for accumulation of nutrients in turfgrass (Madison, 1962).

The total amount of each macronutrient on a total plant basis was highest for the urea treat­ment (data not shown). The amount of a given nutrient is a function of the dry weight and concentration. Urea yielded the greatest amount of root, verdure, and total plant dry matter. The concentrations of nutrients for the urea treat­ment were not always the highest, but the amount of dry weight provided by the urea treatment resulted in the highest values for total macronutrient amounts in the plant.

Micronutrient composition

There were significant differences between the NO~- and the NH; -treated plants for the con-

Effect of nitrogen-form on plant growth 295

centrations of each micronutrient in the shoot tissue, except Cu and Zn (Table 3). Compared to the other treatments, the NO~ -treated plants resulted in the highest concentration of Mn in the shoot tissue. Arnon (1939) reported similar results of higher levels for Mn in NO~ -treated plants compared to NH; -treated plants. The Mn concentration in the roots of the NO~ -treated plants was more than 5 times that of plants of either the NH; or 50:50 treatments. In addition, the NO~ treatment had the highest concentra­tion of Cu in the root tissue. Arnon (1939) found similar differences in root Mn and Cu concen­trations in plants treated with 100% NO~ versus those treated with 100% NH;. He suggested that if NH; was the sole source of N that the micronutrient supply may be the limiting factor for plant growth rather than N. The NO~ treat­ment resulted in the highest B and Cu concen­trations in the verdure tissue, while the 50:50 treatment had the highest Fe and Mo concen­trations. The NO~ treatment was significantly different from the -NH; and urea treatments for all micronutrients except Mo and Zn.

The high concentrations of certain micronut­rients such as Fe in the root tissue indicates the possibility of contamination from the soil on the original turfgrass plug. Although all plugs were thoroughly washed of soil initially, there is the possibility of contamination from soil, thus add­ing to the concentration of certain micronutrients such as Fe in the root tissue. This possible contamination cannot be discounted, but it is the opinion of the authors that any contamination and subsequent addition to the concentration of any micronutrient in the root tissue is minimal.

Urea had significantly higher total amounts of B, Mo, and Zn on a total plants basis compared to the other treatments (data not shown). The Cu amounts for the urea and NO~ treatments were not significantly different, while the total amounts for Fe and Mn were not different between these two treatments and the non-nitro­gen treatment. For the amounts of all micronut­rients, except Mo, the NO~ and NH; treatments were different with the higher total amounts associated with the NO~ -treated plants. Thus, NO~ -treated plants resulted in greater micronut­rient accumulation in the plant tissue.

In summary, N-form affected the growth and

296 McCrimmon et al.

Table 3. Effect of N-form on the concentration of micronutrients in creeping bentgrass shoot, root, and verdure tissue

N-form ratio Micronutrient

B Cu Fe Mn Mo Zn

Concentration (IJ.g g- 1 )

Shoot No; 24 ab 17 a 414 b 408 a 0.9 a 161 b NH~ 14cd 17a 1005 a 137 c 0.5 b 197 ab Urea 26 a 16 a 504 b 163 c 0.5 b 150 b 50:50 17 be 15 a 742 ab 164 c 0.4 b 174 ab Non-nitrogen 23 ab 15 a 773 ab 340 b 0.9 a 166 ab Water 9d 7b 379 b 167 c 0.4 b 221 a LSD 00, 7 3 478 56 0.3 55

Root NO-

3 31 a 67 a 79,121 a 1505 a 1.7b 1661 ab NH; 20 b 26 c 81,238 a 261 b 2.1 ab 805 c Urea 31a 52 b 51.963 b 1213 a 2.5 a 2217 a 50:50 21 b 49 b 75,004 a 251 b 1.7b 1165 be Non-nitrogen 32 a 48 b 81,397 a 1292 a 1.9 ab 1154 be Water 2c !Od 2088 c 136 b 0.2c 600 c LSDO 115 4 15 12,637 400 b 0.7 578

Verdure NO~ 17 a 30 a 11,679 b 941 a 0.7 be 317 ab NH; 6d 16cd 7,934 c 167 d 0.78 139 c Urea 14 b 21 be 6,918 c 570 b 0.7 be 390 a 50:50 llc 24 ab 14.952 a 253 cd l.Oa 288 b Non-nitrogen llc 24 ab 7,492 c 973 a 0.6 c 159 c Water 6d 12 d 1,473 d 356c 0.2 d 153 c LSD 0 _05 2 6 3,233 173 0.1 77

Means within a column followed by a common letter are not significantly different based on the LSD (0.05).

nutrient composition of creeping bentgrass. Urea resulted in the greatest root, verdure, and total plant dry matter. Nitrate-treated plants yielded more shoot dry matter than NH; -treated plants. Although there were differences in the tissue concentrations of various nutrients, all N-form treatments resulted in sufficient levels of each element in the shoot tissue. Nitrogen-form af­fected root and verdure macronutrient concen­tration, but had less effect on the shoot concen­tration. The NO~ and urea treatments resulted in similar micronutrient concentrations while the NH; treatment had lower concentrations. Under the conditions of the study, the NO~ and urea treatments provided the best combination of plant growth and nutrient composition for creep­ing bentgrass. Since creeping bentgrass is grown on putting greens that are primarily sand-based and that react similar to a hydroponic system, the results from this study suggest strongly that

the form of nitrogen applied to creeping bentgrass growing on these types of greens can affect both growth and nutrient content of the turf grass plant.

References

Arnon D I 1937 Ammonium and nitrate nitrogen nutrition of barley at different seasons in relation to hydrogen-ion concentration, manganese, copper, and oxygen supply. Soil Sci. 44, 91-113.

Arnon D I 1939 Effect of ammonium and nitrate nitrogen on the mineral composition and sap characteristics of barley. Soil Sci. 48, 295-307.

Barker A V and Mills H A 1980 Ammonium and nitrate nutrition of horticultural crops. In Hortic. Rev. Vol. 2. Eds. F G Dennis, D N Maynard and M N Rogers. pp 395-423. Avi Pub!., Westport, C N.

Barber S A 1984 Nutrient balance and nitrogen use. In Nitrogen in Crop Production. Ed. R D Hauck. pp 87-95. ASA, CSSA and SSSA, Madison, WI.

Beard J B 1973 Turfgrass: Science and Culture. Prentice­Hall, Englewood Cliffs, NJ. p 658.

Bennett W F, Pesek J and Hanway J J 1964 Effect of nitrate and ammonium on growth of corn in nutrient solution sand culture. Agron. J. 56, 342-345.

Blair G J, Miller M H and Mitchell W A 1970 Nitrate and ammonium as sources of nitrogen for corn and their influence on the uptake of other ions. Agron. J. 62, 530-532.

Cox W J and Reisenauer H M 1973 Growth and ion uptake by wheat supplied nitrogen as nitrate, or ammonium, or both. Plant and Soil 38, 363-380.

Darrow R A 1939 Effects of soil temperature, pH, and nitrogen nutrition on the development of Poa pratensis. Bot. Gaz. 101, 109-127.

Davis R R 1962 Nitrogen fertilization of turfgrasses. In Agronomy Abstracts p. 10. ASA, Madison, WI.

DealE E and Engel R E 1965 Iron, manganese, boron, and zinc: Effects on growth of Merion Kentucky bluegrass. Agron J. 57, 553-555.

Glinski D S, Mills H A, Karnak K J and Carrow R N 1990 Nitrogen form influence root growth of sodded creeping bentgrass. HortScience 25, 932-933.

Hageman R H 1980 Effect of form of nitrogen on plant growth. In Nitrogen Inhibitors-Potentials and Limitations, ASA Spec. Pub!. 38. Ed. D M Kral. pp 47-62. ASA, CSSA, and SSSA, Madison, WI.

Harrison C M 1934 Responses of Kentucky bluegrass to variation in temperature, light, cutting, and fertilizing. Plant Physiol. 9, 83-106.

Haynes R J and Goh K M 1978 Ammonium and nitrate nutrition of plants. Bioi. Rev. 553, 465-510.

Juska F V and Hanson A A 1959 Evaluation of cool season turfgrasses alone and in mixture. Agron. J. 51, 597-600.

Killinger G B, Blaser R E, Hodges E M and Stokes W E 1943 Minor elements stimulate pasture plants. Fla. Agric. Exp. Stn. Bull. 384.

Kirkby E A and Hughes A D 1970 Some aspects of ammonium and nitrate nutrition in plant metabolism. In Nitrogen Nutrition of the Plant. Ed. E A Kirkby. pp 69-77. University of Leeds, Leeds, UK.

Effect of nitrogen-form on plant growth 297

Lapp W S 1 943 A study of factors affecting the growth of lawn grasses. Penn. Acad. Sci. 17, 117-148.

Lycklama J C 1963 The absorption of ammonium and nitrate by perennial ryegrass. Acta Bot. Neerl. 12, 361-423.

Madison J H 1962 Turfgrass ecology. Effects of mowing, irrigation, and nitrogen treatments of Agrostis palustris Huds., 'Seaside' and Agrostis tenuis Sibth., 'Highland' on population, yield, rooting, and cover. Agron. J. 54, 407-412.

Markland F E and Roberts E C 1969 Influence of nitrogen fertilizers on Washington creeping bentgrass, Agrostis palustris Huds. I. Growth and mineral composition. Agron. J. 61, 698-700.

Maynard D N and Barker A V 1969 Studies on the tolerance of plants to ammonium nutrition. J. Am. Soc. Hortic. Sci. 94, 235-239.

Mazur A Rand Hughes T D 1976 Chemical composition and quality of Penncross creeping bentgrass as affected by ammonium, nitrate, and several fungicides. Agron. J. 68, 721-723.

Mengel K and Kirkby E A 1987 Principles of Plant Nutrition. Internation Potash Institute, Bern, Switzerland. 684 p.

SAS/STAT Guide for Personal Computers Version 6 1987 SAS Institute, Inc., Cary, NC.

Smiley R W 1974 Rhizosphere pH as influenced by plant, soil, and nitrogen fertilizers. Soil Sci. Soc. Am. Proc. 38, 795-799.

Sprague H B 1934 Utilization of nutrients by colonial bentgrass (Agrostis tenuis) and Kentucky bluegrass (Poa pratensis). NJ Agric. Exp. Stn. Bull. 570, 1-16.

Waddington D V, Moberg ELand Duich J M 1972 Effect of N source, K source, and K rate on soil nutrient levels and the growth and elemental composition of Penncross creep­ing, bentgrass. Agrostis palustris Huds. Agron. J. 64, 562-566.

Warncke D D and Barber S A 1973 Ammonium and nitrate uptake by corn (Zea mays L.) as influenced by nitrogen concentration and NH; /NO~ ratio. Agron. J. 65, 950-953.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 299-304, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-201

Sulphur status of maize leaves in relation to sulphur content and arylsulphatase activity in soil

W.J. MELO, E.M. QUINONES, M.O. MARQUES, M.A. NOGUEIRA, R.A. CHELLI and S.A.S. LEITE Departamento de Tecnologia, Faculdade de Ciencias Agrarias e Veterinarias, Rodovia Carlos Tonanni, Km 5. 14870-000 Jaboticabal, SP, Brasil

Key words: arylsulphatase, gypsum, maize, sulphur

Abstract

Maize was cropped in a soil treated with 0, 20 and 40 kg S ha-t as gypsum or gypsum+ ammonium sulphate. At 0, 21, 42, 63 and 84 days after sowing, soil samples were taken at 0-20 em depth and analysed for sulphate-S, nitric-perchloric digest-S and arylsulphatase activity. At 63 days after sowing, leaves were sampled and analysed for total S. Sulphur application significantly affected soil sulphate-S, soil NPDS and the soil arylsulphatase activity. The leaf-S content was also affected by sulphur application. The soil sulphate-S level was highly correlated with the leaf-S when soil samples were taken later in the maize cycle (r = 0.77***, for soil samples obtained at 84 days after sowing), whereas the correlations between soil arylsulphatase activity with leaf-S were highly significant when the samples were taken early in the plant cycle (r = 0.69*** and r = 0.52** for samples obtained at 42 and 0 days after sowing, respectively). The nitric-perchloric digest-S presented significant correlations in most the sampling times, but in a lower extent. The correlations between two of the soil-S fractions were significant or not, depending upon the sampling time.

Introduction

Sulphur is an essential nutrient for plant growth, occurring in soils mainly in organic forms, which can be transformed to inorganic forms, among them the sulphate ion, the most important form of sulphur taken up by the plants.

Some of the soil sulphur organic forms are the sulphur esters, that can be hydrolyzed to sul­phate, a reaction catalysed by the arylsulphatase enzyme.

The hydrolysis of the organic sulphate esters in soils depends upon a number of factors such as the concentration of the substrate, the extent by which the organic sulphate esters are protected against enzymatic hydrolysis, the activity and persistence of the extracellular sulphatases, and factors that regulate enzyme activity as pH, temperature, ionic content and composition,

including the inorganic sulphate-S, once sulphat­ase activity may be under feedback control.

It has been demonstrated that the arylsulphat­ase produced by microorganisms is positively correlated with the soil sulphate content, while the arylsulphatase from plant origin is negatively correlated (Ganeshamurthy and Nielsen, 1990). Hagerdon and Stott (1980) observed a positive correlation between the arylsulphatase activity and the content of soil organic matter. But there is not enough information about the relation­ships between soil sulphate-S, soil NPDS or soil arylsulphatase and the nutritional status of the plant with regard to sulphur. These soil parame­ters can be useful for predicting the soil capacity to supply sulphur to plants. So, the aim of this work was to vary the content of the soil sulphur forms by the application of ammonium sulphate and gypsum, alone or together, and to correlate

300 Melo et al.

soil sulphate-S, soil nitric-perchloric digest-S and soil arylsulphatase activity with the sulphur con­tent of maize plants.

Material and methods

The trial was conducted in a Typic Haplorthox (latossolo vermelho- escuro textura media) in the Jaboticabal County, Sao Paulo State, Brasil, a sandy soil (sand= 64.81%, silt= 8.45% and clay= 25.58%) contammg organic matter= 2.00%; N(total) = 0.08%; pH (0.01 M CaC12 ) = 4.5; P(extracted with resin)= 11.21 mg kg- 1 ;

K = 2.36, Ca = 17.28, Mg = 6.29, H +AI= 27.35 (g kg - 1), CEC = 4.37 eg kg-], V (base saturation)= 33%.

The experimental design was a randomized blocks with nine treatments (NOSO, NOS1, NOS2, USO, US1, US2, ASO, AS1 and AS2, where NO= without N at sowing; SO, S 1 , S2

mean 0, 20 and 40 t ha -J of S as ammonium sulphate, gypsum or ammonium sulphate+ gypsum; U = N as urea at sowing and A= N as ammonium sulphate at sowing), with three repli­cations. In the treatments that received ammo­nium sulphate, the doses of sulphur were com­pleted with gypsum. Gypsum and fertilizers were applied in the three growing seasons.

Table 1. Arylsulphatase activity in a Typic Haplorthox submitted to three doses of sulphur and cropped with maize

Treatment Arylsulphatase activity (mg kg_, PNPh _,)

o' 21 42 63 84

Growing season 1989/90 NOSO 20.4a 2 14.4a 10.8d 28.4e 13.8e NOS1 21.5a 18.8a 17.5d 45.7cd 51.8c NOS2 23.7a 13.3a 18.9cd 54.8bc 51.6c uso 16.4a 15.0a 13.6d 28.6e 37.4d US! 21.2a 17.6a 18.9cd 55.7b 65.5b US2 19.4a 14.4a 31.5ab 59.5b 96.0a ASO 23.4a 15.0a 18.2cd 38.8d 19.9e AS! 23.4a 15.0a 26.7bc 61.5b 34.ld AS2 22.2a 14.7a 38.2a 80.0a 51.6c

Growing season 1990/91 NOSO 50.0c 39.5e 41.4f 33.5c 16.5c NOS! 24.9d 53.3e 57.0e 34.8c 28.7d NOS2 78.5b 78.2b 75.7d 52.7ab 35.0cd uso 18.5d 38.5e 37.6f 23.3d 42.6c US! 54.9c 65.4c 88.3bc 46.9b 37.3cd US2 53.7c 79.5b 96.7b 57.7a 67.8a ASO 57.4c 55.5d 75.5d 44.7b 57.8b AS! 88.8a 86.5b 86.6c 59.2a 71.5a AS2 88.6a 103.4a 108.6a 61.4a 74.5a

Growing season 1991/92 NOSO 15.8f 22.3d 23.8g 15.7c 13.7cd NOSl 37.0cd 28.9cd 34.5f 21.2bc 15.8bcd NOS2 27.8de 33.lc 43.4ef 32.0a 21.2abcd uso 23.5cf 23.5d 39.4ef 21.5bc 12.9d USl 75.6a 34.1bc 65.Jc 25.2ab 24.6ab US2 32.4de 42.9ab 55.2d 31.6a 26.8a ASO 42.2c 28.5cd 44.8e 18.5bc 15.7bcd AS! 65.9b 35.9bc 76.2b 24.5abc 20.9abcd AS2 63.7b 48.0a 97.1a 25.7ab 22.9abc

'Days after sowing; 2In the same column and same growing season, means followed by the same letter are not different (Tukey, p < 0.05). NO= without Nat sowing; U = N as urea; A= N as ammonium sulphate at sowing and urea by the side-dress; SO, S,, S 2 =doses of S.

Before the experiment installation, the soil was subjected to liming with 2.8 tha-I of a dolomite limestone (CaO = 24.92%, MgO = 15.72%).

The N-P-K fertilization was made on basis of the soil chemical analysis, using ammonium sulphate (N = 20%, S = 24% ), urea (N = 45% ), triple superphosphate (P20 5 = 41%) and KCl (K2 0 = 60% ). Nitrogen (75 kg ha-l in the grow­ing seasons 1989/90, 1991/92 and 90 kg ha-l in the growing season 1990/91) was split in three times (1/5 at sowing, 2/5 at 30 days and 2/5 at 45 days after sowing); urea (N = 45%) was theN fertilizer used after sowing. Phosphorus (33, 50 and 45 kg ha-l P20 5 in the first, second and third

Sulphur in maize leaves 301

growing season respectively) was applied at sowing. Potassium (36, 60 and 90 kg ha- K20 in the first, second and third growing seasons re­spectively) was split in three equal doses ( 1/3 at sowing, 30 and 45 days after sowing). At sowing, it was also applied a mixture of trace nutrients (40kg ha- 1 in the growing seasons 1989/90, 1990/91 and 27 kg ha -I in the growing season 1991/92) containing Zn = 9%, B = 1.8%, Cu = 0.8%, Fe=3%, Mn=2% and Mo=0.1%. Gypsum (S = 16%) was split in two times (125kg ha- 1 at sowing in the treatments NOS~' NOS2 , US 1 and US2 and the difference to complete the S doses at 30 days after sowing). Gypsum and fertilizers were applied into the

Table 2. Sulphate-S in a Typic Haplorthox submitted to three doses of sulphur and cropped with maize

Treatment Sulphate-S (mg kg -I air dried soil)

01 21 42 63 84

Growing season 1989/90 NOSO 7.47a' 4.95g 1.83e 0.24c 0.61d NOS1 7.52a 43.70a 6.73cd l.OObc 4.85ab NOS2 7.29a 12.00f 8.60bc 0.48c 5.38a uso 7.66a 5.01g 1.23e 0.26c 3.11bc US1 7.51a 20.83c 6.48d 1.30bc 5.38a US2 7.62a 20.60c 10.27b 1.90abc 4.70ab ASO 7.70a 15.97e 3.07e 1.80abc 1.59cd AS1 7.66a 18.17d 8.70bc 2.87ab 3.79ab AS2 7.68a 22.93b 15.70a 3.80a 5.38a

Growing season 1990/91 NOSO 0.53c 3.58ef 2.95e 4.67f 0.50e NOS1 2.05bc 8.75d 18.73c 13.42e 4.77bc NOS2 3.64b 18.25c 19.70c 17.25c 6.53ab uso 0.61c 3.25f 4.85e 4.08f 2.10de US2 2.73b 36.58a 24.16b 20.50b 7.27a US1 2.88b 20.67b 20.61c 15.33cde 4.77bc ASO 3.94b 5.58e 16.44d 15.67cd 3.30cd AS1 3.94b 8.75d 22.80b 14.67de 3.93cd AS2 6.52a 22.25b 28.78a 22.92a 7.70a

Growing season 1991/92 NOSO 6.99bc 7.17e 6.94e 5.61d 5.70de NOSJ 7.13bc 11.42d 10.80d 7.24d 6.16cde NOS2 8.48ab 14.42c 13.85bc 9.86c 6.61cde uso 5.83c 8.04e 6.98e 6.35d 4.64e USl 7.28abc 12.73cd 13.51c 11.76c 7.44bcd US2 7.65abc 18.43b 12.38cd 10.23c 7.80bc ASO 7.13bc 12.15d 14.31bc 11.25c 9.21b ASl 8.24ab 18.42b 15.68b 14.20b 12.42a AS2 9.19a 22.35a 26.43a 18.14a 12.49a

1Days after sowing; 2 ln the same column and same growing season, means followed by the same letter are not different (Tukey, p < 0.05). NO= without Nat sowing; U = N as urea; A= N as ammonium sulphate at sowing and urea by the side-dress; SO, S1, S2 =doses of S.

302 Melo et al.

furrow at sowing and by the plant side-dress at 30 or 45 days after sowing.

Maize hybrid AG-403-B was cropped for three growing seasons in 6 x 10m plots with a density of 50,000 plants per ha. Soil samples were taken at 0-20 em depth each 21 days during the maize cycle, the first one before soil fertilization. The composite sample of each plot consisted of 20 single samples (10 in-rows and 10 inter-rows). The samples were air dried, sieved ( < 2 mm) and then analysed for NPDS (sulphur in the nitric-perchloric digest), inorganic sulphate (Vitti, 1989) and arylsulphatase activity (Tabatabai and Bremner, 1970a). The data were expressed on air dried basis.

Leaf sampling was made at 63 days after sowing by collecting the fourth leaf from the top of 20 plants per plot (Trani et a!., 1983). The middle one third of each leaf, after removing the central nervure, was washed (water, distilled water and deionized water), dried at 60-70°C, ground ( < 40 mesh) and then analysed for total­S (Vitti et a!., 1989).

Results and discussion

The soil pH (0.01 M CaClz) increased to 5.26 (mean of all the plots) soon after liming (63 days after the first sowing). In the two next growing

Table 3. Nitric-perchloric digest-S (NPDS) in a Typic Haplorthox submitted to three doses of sulphur and cropped with maize

NPDS (g kg -I air dried soil)

01 21 42 63 84

Growing season 1989/91 NOSO 0.36a2 0.35e 0.36e 0.35e 0.35e NOS! 0.36a 0.46d 0.46d 0.40de 0.37cde NOS2 0.37a 0.50cd 0.51cd 0.45cd 0.4lbcd usa 0.37a 0.39e 0.39e 0.36e 0.36de US! 0.37a 0.46cd 0.45d 0.40de 0.39bcde US2 0.37a 0.52bc 0.55bc 0.49bc 0.43abc ASO 0.38a 0.46cd 0.47d 0.43d 0.41bcd AS! 0.36a 0.58ab 0.61ab 0.54ab 0.45ab AS2 0.36a 0.61a 0.62a 0.57a 0.49a

Growing season 1990/91 NOSO 0.24a 0.25d 0.26e 0.22b 0.20c NOS! 0.27a 0.38bc 0.41cd 0.35a 0.25abc NOS2 0.26a 0.40abc 0.44bcd 0.37a 0.28a usa 0.25a 0.22d 0.27e 0.19b 0.20bc US! 0.26a 0.36c 0.41bcd 0.36a 0.27a US2 0.29a 0.38abc 0.45bc 0.40a 0.29a ASO 0.30a 0.42abc 0.39d 0.37a 0.25abc AS! 0.26a 0.44a 0.47ab 0.36a 0.26ab AS2 0.29a 0.44ab O.Sla 0.41a 0.28a

Growing season 1991/92 NOSO 0.44e 0.49de 0.34e 0.59e 0.65cde NOS! 0.47de 0.52cd 0.42d 0.56ef 0.74ab NOS2 0.5lcd 0.53cd 0.53c 0.67bc 0.64e uso 0.41e 0.37f 0.43d 0.52f 0.60e US! 0.51d 0.45e 0.56bc 0.72ab 0.70bcd US2 0.52cd 0.58bc 0.61ab 0.60de 0.71bc ASO 0.57bc 0.62ab 0.46d 0.59de 0.6le AS! 0.59b 0.52d O.SSbc 0.65cd 0.68bcd AS2 0.67a 0.66a 0.65a 0.78a 0.78a

1Days after sowing. 'In the same column and same growing season, means followed by the same letter are not different (Tukey, p < 0.05). NO= without Nat sowing; U = N as urea; A= N as ammonium sulphate at sowing and urea by the side-dress; SO, S1, S2 =doses of S.

Table 4. Leaf-S maize content when cropped in a Typic Haplorthox submitted to three doses of sulphur

Treatment Sin the Leaf(%)

89/901 90/91 91192

NOSO 0.30cd 2 0.33d 0.42e NOS! 0.32cd 0.41c 0.51d NOS2 0.36b 0.51b 0.57c uso 0.29d 0.51b 0.50d USl 0.32cd 0.49b 0.56c US2 0.37b 0.59a 0.70b ASO 0.33c 0.41c 0.59c AS! 0.38b 0.53b 0.59c AS2 0.43a 0.52b 0.75a

1Growing season. 2In the same column and same growing season, means followed by the same letter are not different (Tukey, p < 0.05). NO= without N at sowing; U = N as urea; A= N as ammonium sulphate at sowing and urea by the side-dress; SO, S1, S2 =doses of S.

seasons, the soil pH was 4.94 and 5.55 respec­tively (samples taken at 63 days after sowing).

The sulphur application significantly affected (F-test, p < 0.01) the arylsulphatase activity (Table 1), the soil sulphate-S (Table 2) and the soil NPDS contents (Table 3).

The leaf-S percentage was also significantly affected by the treatments (F-test, p < 0.01) in the three growing seasons (Table 4) and ranged from 0.29% to 0.75%, both these values higher than critical level of 0.20% for maize plants in Sao Paulo State (Trani et al., 1983).

Some authors have found that the sulphatase activity is correlated with soil organic matter, highly correlated with soil organic-S, has shown low significant negative correlations with sui-

Sulphur in maize leaves 303

Table 5. Significant correlations between maize leaf-S con­tent and soil inorganic sulphate-S, soil nitric-perchloric di­gest-S or soil arylsulphatase activity

Sampling Regression

Sulphate- S 421 0.66*** 2 y = 0.011x + 0.333 3

63 0.71 *** y = 0.013x + 0.356 84 0.77*** y = 0.032x + 0.291

NPDS 0 0.54 ** y = 0.574x + 0.245

42 0.44* y = 0.544x + 0.213 63 0.57** y = 0.481x + 0.240 84 0.53** y = 0.349x + 0.311

Arylsulphatase 0 0.52** y = 0.0027x + 0.358

21 0.47* y = 0.0023x + 0.378 42 0.69*** y = 0.0029x + 0.322

1Days after sowing. 'Significant (t-test) at p < 0.05 (*), p < 0.01 (**) or p < 0.001 (***). 3y =% of sulphur in the leaf.

phate-S, but did not appear to correlate sig­nificantly with total-S (Tabatabai and Bremner, 1970a; Tabatabai and Bremner, 1970b). Tabatabai and Bremner (1972) found no signifi­cant relationship between sulphatase activity and the amount of sulphur mineralized, whereas Speir (1977) suggested that sulphatase activity may be a useful indicator of the mineralizable-S that can be taken up by the plants.

Table 5 shows that the correlation of soil sulphate-S with leaf-S was greater when the soil samples were taken later in the maize cycle (r=0.71*** and r=0.74*** at 63 and 84 days after sowing). For the correlations of soil arylsul-

Table 6. Correlations between soil inorganic sulphate-S with soil arylsulphatase activity and soil NPDS

Arylsulphatase 01

21 42 63 84

NPDS 0

21 42 63 84

01 21 42 63

-0.21 0.12 -0.50** 0.14 -0.30 0.23 -0.04 0.48* -0.16 0.40*

0.74*** 0.09 0.78*** 0.34 0.66*** 0.49** 0.74*** 0.20 0.72*** 0.06

0. 75*** 0.81 *** 0.92*** 0.32 0.33

0.04 0.27 0.42* 0.25 0.01

0.82*** 0.89*** 0.96*** 0.05 0.12

0.52** 0.05 0.18 0.17

-0.01

1Days after sowing. 'Significant (t-test) at p < 0.05 (*), p < 0.01 (**) or p < 0.001 (***).

84

0.42* 2

0.24 0.53**

-0.14 -0.06

0.72*** 0.64*** 0.62*** 0.74*** 0.64***

304 Sulphur in maize leaves

phatase activity with leaf-S, the highest values were observed when the sampling was made earlier (r = 0.52*** and r = 0.69*** at sowing and 42 days after sowing respectively). The NPDS presented significant positive correlations with leaf-S in most of the samplings but in a lower extent than the sulphate-S. These data suggest that the sulphate-S can be a good param­eter to estimate the available-S to maize when samples were obtained later in the plant cycle, whereas arylsulphatase can be a good one when the samples were taken in the first stages of maize growing.

The correlations between soil sulphate-S with soil arylsulphatase activity (Table 6) greatly varied with the sampling time and only in a few cases they were significant. If soil sulphate-S and arylsulphatase activity were evaluated in the same sample, only when sampling was carried out at 42 days after sowing the r value was significant (r = 0.92***). This shows a marked influence of the climate conditions that precedes the sampling on the relation between those soil parameters and may be an explanation for corre­lation values found by different authors.

The other tested correlations (soil arylsulphat­ase activity x NPDS and soil sulphate-S x NPDS) were also affected by the time the sampling was made (Table 6).

Acknowledgement

The authors acknowledge the Funda<;ao de Am­para a Pesquisa do Estado de Sao Paulo (FAP­ESP) for the financial support.

References

Ganeshamurthy A N and Nielsen M E 1990 Arylsulphatase and the biochemical mineralization of soil organic sulphur. Soil Bioi. Biochem. 22, 1163-1165.

Hagerdon C and Stott D E 1980 Interrelations between selected soil characteristics and arylsulfatase and urease activities. Commun. Sol Sci. Plant Anal. 10, 935-955.

Speir T W 1977 Studies on a climosequence of soils in tussock grasslands. 2. Urease, phosphatase, and sulphatase ac­tivities of topsoils and their relationships with other prop­erties including plant available sulphur. N. Z. J. Sci. 20, 159-166.

Tabatabai M A and Bremner J M 1970a Arylsulfatase activity of soils. Soil Sci. Soc. Am. Proc. 34, 225-229.

Tabatabai M A and Bremner J M 1970b Factors affecting soil arylsulfatase activity. Soil Sci. Soc. Am. Proc. 34, 365-370.

Tabatabai M A and Bremner J M 1972 Distribution of total and available sulfur in selected soils and soil profiles. Agron. J. 64, 40-44.

Trani P, Hiroce R and Bataglia C 0 1983 Analise foliar: amostragem e interpreta9ao. Campinas, Funda9ao Cargill. 18p.

Vitti G C 1989 Avalia9ao e interpreta9ao do enxofre no solo e na planta. Jaboticabal, FUNEP. 37p.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 305-308, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-036

Soil properties and mineral content of leaves in fig orchards producing high-quality fruits

UYGUN AKSOY and DILEK ANA<:: Department of Horticulture, Faculty of Agriculture, Ege University, 35100 Bornova, Izmir, Turkey

Key words: Ficus carica, fig, fruit quality, plant nutrients, soil properties, weighted-rankit method

Abstract

Soil, leaf and fruit samples from Big and Small Meander Valleys, the main dried-fig producing region in Turkey, were analysed and evaluated. Quality parameters were determined as size, colour, acidity, soluble solids and ratio of defects, and were classified according to their relevance in fresh- and dried-fig production. The five top-ranking orchards were selected according to their overall quality. Soil properties and leaf nutrient status were also determined. Evaluations brought out the significance of nitrogen and potassium fertilization in commercial dried-fig production.

Introduction

Fig (Ficus carica L.) is a dominating crop in the western part of Turkey. The production is con­centrated in two valleys, the Big and Small Meander. The fig crop produced in this region, the Aegean, is almost completely dried and exported. The orchards are established with the vegetatively propagated Sarilop ( =Calimyrna) variety, which is known for high dried-fruit quality. The quality obtained in the Small Mean­der (SM) is somewhat inferior to that of the Big Meander (BM) due to the prevailing climatic conditions. The trees in the SM basin are grown as single trunks and, in some locations, irrigated in late winter, whereas in the BM they are left to grow in bush or multitrunk form. All these factors, together with the physical and chemical properties of the soil, exert significant effects on fruit quality. In a survey, fruit size, colour, cracking and sunscaled were noted as the major attributes in commercial dried-fig production (Aksoy, 1987). Further work comprised the soil and plant nutrient status of the selected fig orchards and their relation to fruit quality criteria. Results proved significant relationships between nutrient status and, particularly, dried-

fig quality (Aksoy et a!., 1987a; Ana<; et a!., 1992). Even though Turkey is the prime producer of dried figs, there is a lack of informa­tion with respect to critical values for the fertili­zation of fig trees. Since figs can be grown only in regions where a mediterranean climate pre­vails, world-wide data are also very limited. This study was set up to determine the soil properties and leaf nutrient status of the orchards produc­ing the highest fruit quality, to compare these results with the regional averages that are used as reference values, and to highlight the present fertilizer program.

Methods

In BM valley 24 and in SM valley 20 orchards were selected and analysed for their soil, leaf and fresh- and dried-fruit characteristics.

Fruit quality analyses

For every orchard, an overall evaluation of quality in terms of both fresh-and dried-fig crop was made by a 'weighted-rankit' (Michelson et a!., 1958) method in which the product is nu-

306 Aksoy and Anaq

merically evaluated by gtvmg weight to each attribute making up the general quality. The criteria considered important for fig quality were chosen according to Aksoy (1987) and Aksoy et a!. (1992) and each factor was assigned a rate value. These factors were then subdivided into classes, and each class derived a rank value.

Fresh-fruit samples were analysed for the following parameters: fruit diameter (15), neck length (8), ostiole width (10), receptacle thick­ness (10), average fruit weight (15), titratable acidity (8), total soluble solids (8), dry matter (10), ratios of cracked or split (8) and sunscalded (8) fruits. Dried-fruit attributes were average fruit weight (20), total soluble solids (TSS;lO)

titratable acidity (10), colour (15), firmness (15), ratios of cracked or split (15) and sunscalded (15) fruits. Values in parentheses represent the rate value of each selected criterion. In the final evaluation (Table 1), a general quality score for each orchard is calculated as the algebraic sum of fresh- and dried-fruit quality scores, and the top five ranking orchards are selected.

Soil and leaf analyses

Soil samples were taken from two depths (0-30 em and 30-60 em) and were evaluated in terms of texture, CaC0 3 , pH, total N, and available P and K contents (Table 2). The third

Table 1. Some of the fresh- and dried-fruit quality values of the selected orchards in BM (1-5) and SM (6-10)

Orchard Fresh fruit Dried fruit No

Diameter Ostiolc Receptacle Fruit Titratable Soluble Fruit Colour Firmness Severe Total (mm) Width Thickness Weight Acidity solids Weight Class Class Crack Sunscaled Quality

(mm) (mm) (g) (%) (%) (g) (%) (%) Score

48.3 6.1 2.1 60.6 0.149 21.8 17.2 3.4 2.6 9.0 30.0 616 2 40.9 5.4 2.3 56.5 0.152 20.6 17.3 3.7 2.7 2.0 19.0 616 3 48.9 5.4 2.8 64.6 0.198 19.4 19.3 3.4 2.3 3.0 21.0 597 4 44.1 4.6 2.5 39.5 0.318 24.7 15.7 3.3 2.0 3.0 17.0 586 5 38.3 5.8 2.8 53.9 0.153 23.9 18.8 3.6 2.6 4.0 36.0 586

6 50.6 8.6 4.2 65.1 0.155 19.0 19.8 3.4 1.8 0.8 10.9 645 7 49.3 6.9 3.6 65.4 0.129 20.9 28.0 4.4 1.8 5.0 6.0 644 8 51.6 8.6 4.7 66.4 0.140 20.4 19.3 3.2 2.6 11.7 17.8 638 9 50.3 8.5 3.3 64.3 0.115 21.0 18.3 3.5 1.9 1.7 15.9 622

10 51.0 8.9 3.8 66.2 0.137 19.2 19.5 3.9 1.9 10.0 9.2 604

Table 2. Soil properties and mineral content of the fig leaves from the selected orchards located in BM (1-5) and SM (6-10)

Orchard Soil Leaf (blade+ petiole) No

Depth Texture pH CaC0 3 N p K N p K Ca Mg (em) (%) (%) (mg/kg) (mg/kg) (%) (%) (%) (%) (%)

0-30 sandy loam 7.60 6.92 0.06 1.6 145 1.62 0.11 1.40 3.40 0.55 2 0-30 loamy 8.04 3.87 0.12 1.1 170 1. 75 0.08 1.41 3.30 0.57 3 0-30 loamy 6.66 trace 0.10 0.9 270 1.46 0.15 1.28 3.61 0.55 4 0-30 sandy loam 7.65 10.06 0.07 0.5 170 1.94 O.D7 1.11 3.79 0.72 5 0-30 sandy loam 6.99 1.74 0.04 0.3 120 1.49 0.10 1.37 2.78 0.74

6 0-30 sandy loam 6.20 1.09 0.07 3.4 95 1.63 0.08 1.05 4.73 0.70 7 0-30 sandy loam 6.45 0.91 0.05 2.9 85 1.50 0.08 1.53 4.15 0.50 8 0-30 sandy loam 6.81 1.28 0.05 8.4 125 1.89 0.10 1.48 4.70 0.59 9 0-30 sandy 6.78 1.09 0.04 2.7 75 1.50 0.08 1.53 4.15 0.50

10 0-30 sandy loam 6.51 1.28 0.04 2.1 75 1.61 0.09 1.47 4.05 1.07

MEAN 6.97 2.82 0.06 2.4 133 1.64 0.09 1.36 3.87 0.65

leaf from the base of each shoot was sampled at the onset of the fruit ripening period and ana­lysed for leaf N content by distillation method, for P by colorimetry, for K and Ca by flamephotometry, and for Mg by AA spec­trometry.

Results

The evaluation of quality scores revealed that the average of BM was slightly higher than SM even though higher individual total scores were obtained for the latter region among the top­ranking group (Table 1). The orchards selected for their total scores also made the top positions with respect to their dried-fruit quality.

The soil properties of the orchards which produced the highest-quality fruits can be de­scribed as sandy loam with two exceptions which have loamy texture. The pH of the analysed samples ranged from 6.12 to 6.94 in SM, and from 6.56 to 8.10 in BM, implying a slightly acid to alkaline property. Parallel to the pH values, CaC0 3 content varied within narrower limits (0.81-1.28%) in SM. In the other region, the range was from 1.00 to 10.84% (Table 2).

The average values for soil N were not con­stant in BM, varying between 0.04 and 0.20%. In SM, the variation was slight (0.04-0.07% ). In the case of P, the SM basin showed sufficiency in contrast to that of BM. Excluding the two orchards on loamy texture, all of the other soils were found inadequate in terms of K.

As for the leaf nutrient status, N content in BM ranged from 1.46 to 1.94% and in SM from 1.50 to 1.89% with an average value of 1.65% indicating sufficiency. The range of P in BM orchards was wider (0.071-0.147%) than in SM (0.080-0.100%). Moreover, the average P value (0.094%) was also at sufficiency level according to the reported results (Beutel et a!., 1983). The average K concentration was calculated as 1.36%. In the case of Ca, with an average value of 4.18%, SM seemed to be richer. In BM, it ranged within narrower limits (2.78-3.79%) par­allel with a lower average value. The average leaf Mg content was found to be 0.664%. Ex­cluding an orchard in SM having a Mg content of

Nutrient status of fig orchards 307

1.07%, all the others possessed values varying between 0.50 and 0.74%.

Discussion

In Turkey the fertilizer programs for fig orchards are based on general data guided by the regional average values obtained in a nutrient survey conducted in 115 fig orchards (Aksoy et a!., 1987b). Thus the regional average leaf nutrient values developed for the Sarilop variety are: N 1.65%, P 0.09%, K 1.19%, Ca 3.82% and Mg 0.71%. Beutel and coworkers (1983) give the critical adequate nutrient level in the leaves of fig trees as N 2.0-2.5%, K 1.0% and Ca 3.0%, and note that deficiency starts below 1.7% N and 0.7% K under California conditions. If these two critical nutrient concentrations associated with fig trees are compared, they are in agreement with the exception of N. The values obtained in this study for fig orchards producing high-quality fruits (Table 2) are in accordance with the regional mean values (Aksoy et a!., 1987b) for N, P, Ca and Mg with slightly higher K levels.

In the Sarilop variety high N is found to exert an adverse effect on dried-fig quality. Significant negative correlations were found between fruit size, colour and ratio of sunscalded fruits and leaf-N (Aksoy et a!., 1987a). Under Aegean conditions fig tress are rain-fed; however, in California all fig trees are irrigated. The differ­ence in leaf-N content might be due to higher N fertilizations in the irrigated California orchards. Leaf-K concentration averaging to 1.36% (Table 2) was higher than both the regional average value (1.19%) and the critical level (1.0%) given by Beutel and coworkers (1983). This result implies that a higher quality in dried-fig pro­duction is accompanied by a higher leaf-K level. This result is confirmed by previous research in which both leaf and soil K were found to be significantly correlated with light colour and soft texture, which are among the main features of Turkish dried-fig fruits. K also positively affected the number of healthy (no sunscald) fruits, TSS and titratable acidity contents of the fig fruits (Aksoy eta!., 1987a; Ana<; eta!., 1992).

The aim of finding out the soil properties and nutrient status of orchards producing high-qual-

308 Nutrient status of fig orchards

ity fruits in order to compare them with previ­ously determined critical concentrations for the evaluation of the fertilizer programs resulted m the recognition of the significance of N and K fertilization especially in dried-fig orchards.

References

Aksoy U 1987 Evaluation of the dried fig crop with respect to standard quality properties. J. Ege Univ. Fac. Agric. 23, 23-30.

Aksoy U, Ana~ D, Hakcrlcrler Hand Diizbastilar M 1987a Nutrient status of Calimyrna fig orchards in Big Meander Valley and their relationships with yield and quality. Tari§ Research and Development Center. Project No 006.

Aksoy U, Ana~ D, Eryiice Nand Yalta§ T 1987b Evaluation of the nutrient status of Calimyrna fig orchards in the Aegean Region. J. Ege Univ. Fac. Agric. 24, 21-35.

Aksoy U, Ana~ D and Giil N 1992 Interrelationships among fresh and dried fruit quality characteristics in Calimyrna figs. Proc. 1st Turkish National Horticultural Congress, pp. 427-430.

Ana~ D, Aksoy U, Hakerlerler H and Diizbastilar M 1992 Nutrient status of Calimyrna fig orchards in Small Meander Valley and their relationships with yield and quality. Tari§ Research and Development Center. Project NO 004.

Beutel J, Uriu K and Lillehand 0 1983 Leaf Analysis for California Deciduous Fruits. In Soil and Plant Tissue Testing in California. Ed. HM Reisenauer, UC Bulletin 1879, pp 15-18.

Michelson L F, Lachmann W Hand Allen D D 1958 The use of weighted-rankit method in variety trials. Soc. Hortic. Sci. Proc. 71, 334-338.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 309-312, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-037

Changes in K, Ca and Mg contents in different parts of the fig fruit during development

UYGUN AKSOY and DILEK AKYUZ Department of Horticulture, Faculty of Agriculture, Ege University, 35100 Bornova, Izmir, Turkey

Key words: calcium, Ficus carica L., fig, fruit development, fruit quality, magnesium, potassium

Abstract

Changes in the K, Ca and Mg contents and K/Ca ratios within the fig fruit were determined during the fruit development period. The trial was performed with the Bursa Black, Goklop and Sarilop (syn = Calimyrna) varieties in Erbeyli-Aydin, the main fig-producing province of Turkey. The K, Ca and Mg analyses were carried out on whole intact fruits and on the skin, meat and pedicel of the fruit. It was found that in the Goklop and Sarilop varieties, there was a rapid decline in the Ca content of the fruit prior to ripening, as opposed to an increase in Bursa Black. Significant differences were determined among varieties with respect to fruit K, Ca and Mg content. The results are discussed in terms of fruit cracking.

Introduction

In fig production, fruit cracking at the ostiolar­end and sunscald are accepted as the two most important physiological disorders. The average ratio of cracked fruits in an orchard may range from 8.1 to 28.6% within the same year (Aksoy, 1987). Many factors such as tree vigour, crop load, fruit size, pruning, irrigation, as well as heat stress and other abnormal weather patterns are known to be involved in most of the physio­logical disorders (Raese, 1989). A correct min­eral balance in the fruit is also necessary to avoid various disorders (Perring, 1984 ). The results so far of still continuing research proved that fruit cracking is triggered by the K and Ca levels and even more by the ratio of K/Ca in the fig fruit (Aksoy et a!., 1987).

According to the UN/ECE Standards (1988) for fresh figs (FFV-17), in extra class fruit crack­ing is not tolerated at all. For quality class I, cracks are not allowed to exceed 3 em, whereas a severely cracked fruit will not be accepted for

human consumption. Thus, high ratios of cracked fruits mean big economic losses.

The objective of this study was to determine the changes in K, Ca and Mg content in different parts of the fig fruit during development and to evaluate these changes in terms of fruit cracking.

Methods

Fruit samples were taken as 15 to 40 fruits from each of three trees of the Bursa Black, Goklop and Sarilop fig varieties located in the Fig Insti­tute Collection Garden in Aydin with two-week intervals from May 31st to August 8th, the onset of fruit ripening in 1990. The fruits were divided into three concentric zones; skin (the outermost coloured part of the fruit), meat (the white fleshy part) and pedicel (the amber-to-red coloured part consisting of flowers and fruitlets). The whole fruits and the described parts were cut into pieces, oven-dried at 65°C and ground. After wet digestion, K and Ca were determined

310 Aksoy and Akyuz

through fiamephotometry, Mg by AA spectro­photometry.

Results

The fruit development of the fig shows a double sigmoid curve: two rapid-growth phases and a slow phase in between. In all three varieties, the fruit weight changed according to the same pattern (Fig. 1). According to the weight incre­ment, the slow phase was found to occur be­tween June 13th and July 24th.

The two fig varieties included in the trial, Goklop and Bursa Black, are fresh varieties. Sarilop, which comprises most of the plantations in the west, is the standard variety for drying but can be seen in the fresh market as well. In the Bursa Black, Goklop and Sarilop varieties, the ratio of severely cracked fruits was 0.1, 13.2 and 5.2% , respectively. The ratio of mild cracks was 0.0, 46.0 and 23.0% and of healthy fruits 99.9, 40.8 and 71.8%. These figures reveal that Bursa Black can be described as resistant to cracking and Goklop as susceptible. Sarilop is ranked in between and can be termed intermediate.

At the onset of fruit development, K content was similar for all three varieties , ranging be­tween 1.43 and 1.52%. During the first rapid­growth period, there was a slight decrease in K content. It was followed by an increase during the slow phase; however, K content declined towards ripening. The variation trend of K in the skin, meat and pedicel resembled that in the whole fruit. Concerning the analysed fruit parts, higher concentrations were detected in the inner

, ___ Fig. 1. Change of fruit weight (g) during development.

parts, meat and pedicel during fruit develop­ment. In ripe fruits higher K levels were found in the skin compared to the pedicel. Maximum values were still obtained for the meat (Table 1).

The Ca content of all fruit parts was highest early in the season and decreased later on in Sarilop and Goklop. High Ca content in Goklop fruits was found to decrease in samples taken on June 13th. In Bursa Black higher Ca levels were attained at the second sampling date. In whole fruits and in the analysed parts of Bursa Black fruits, theCa content was lowest on July 24th. In the Goklop and Sarilop varieties, the lowest values appeared at ripening.

In Sarilop and Goklop fruits, Mg concentra­tions were higher at the onset of development. Although it showed fluctuations through the development period, a gradual decline appeared towards ripening. In Bursa Black fruits lower Mg levels were followed by higher concentrations at ripening in all parts and in the fruit as a whole.

Discussion

In fruit growth and development, Ca has re­ceived much attention because deficiency symp­toms are usually associated with the specific problem of Ca transport. Low Ca content or an imbalance as a result of high concentrations of other cations such as Mg, K or H may result in deficiency features (Ferguson and Drobak, 1988). Aksoy and coworkers (1987) report that as blade and petiole-K increased, the ratio of cracking was encouraged. High Ca content, on the other hand, favoured healthy fruits. Similar correlations were identified with leaf-Mg; how­ever, more significant ones were obtained with the ratio of K / Ca (Aksoy et a!., 1987).

The three varieties were found to have differ­ent tendencies for cracking and to vary signifi­cantly in fruit K, Ca and Mg contents. The changes in concentration of K and Ca during the fruit development period were in accordance with the results reported for grape berries (Possner and Kliewer, 1985). The K / Ca ratio for grape was characterized by two distinct plateaus. For figs such plateaus could not be observed (Fig. 2) . The change of K / Ca ratio was similar in the whole fruits and meat and pedicel of Goklop

K, Ca and Mg content of the fig fruit 311

Table 1. The mean K, Ca and Mg content(% dry weight) and K/Ca ratio of the fruit samples taken from Bursa Black, Goklop and Sarilop fig varieties

Sampling date

Bursa black* Giiklop* Sartlop*

Potassium (%) May31 June 13 July 11 July 24 August 8

Calcium(%) May31 June 13 July 11 July24 August 8

Magnesium(%) May31 June 13 July 11 July 24 August 8

Potassium I Calcium May31 June 13 July 11 July 24 August 8

1.50 1.41 1.49 1.36 1.46

0.54 0.95 0.47 0.24 0.84

0.24 0.39 0.26 0.16 0.52

2.78 1.48 3.17 5.67 1.74

2

1.05 1.22 1.44 1.10 1.62

0.75 0.98 0.83 0.29 0.45

0.26 0.29 0.39 0.16 0.43

1.40 1.24 1.73 3.79 3.60

3

1.53 1.80 1.85 1.01 1.74

0.48 0.60 0.59 0.16 0.68

0.22 0.30 0.32 0.11 0.34

3.19 3.00 3.13 6.30 2.55

4

1.72 1.53 1.56 0.95 1.47

0.46 0.51 0.38 0.14 0.66

0.18 0.30 0.25 0.10 0.48

3.73 3.00 4.10 6.78 2.23

1.43 1.56 1.34 1.30 1.34

1.35 0.48 0.79 0.72 0.17

0.41 0.27 0.30 0.35 0.14

1.06 3.25 1.70 1.80 7.88

2

1.10 1.05 1.01 0.95 1.09

1.24 0.60 0.89 0.88 0.30

0.40 0.27 0.44 0.45 0.18

0.89 1.75 1.13 1.08 3.63

3

1.51 1.47 1.42 1.54 1.11

0.88 0.39 0.72 0.61

0.18

0.35 0.24 0.34 0.39 0.17

1.72 3.77 1.97 2.52 6.17

4

1.82 1.84 1.65 1.48 1.11

1.04 0.45 0.67 0.48 0.16

0.29 0.22 0.24 0.27 0.14

1.75 4.09 2.46 3.08 6.94

1.52 2.10 1.60 1.52 1.02

1.15 1.05 0.97 0.66 0.20

0.58 0.31 0.40 0.35 0.12

1.32 2.00 1.65 2.30 5.10

2

1.34 1.20 1.30 1.36 1.31

1.06 1.08 1.32 1.13 0.44

0.68 0.42 0.60 0.68 0.26

1.26 1.11 0.98 1.20 2.98

3

1.53 1.74 1.83 1.49 1.45

0.72 0.81 0.82 0.52 0.43

0.55 0.38 0.41 0.28 0.22

2.12 2.15 2.23 2.86 3.37

4

1.78 1.65 1.64 1.29 1.12

0.81 0.78 1.02 0.42 0.43

0.42 0.30 0.31 0.20 0.28

2.20 2.12 1.61 3.07 2.60

LSD (%5) K variety 0.045 Ca variety 0.048 Mg variety 0.025 *FRUIT PARTS: part 0.069 part 0.073 part 0.039 1. Whole fruit 3. Meat date 0.059 date 0.062 date 0.023 2. Skin 4. Pedicel

~KJ~Ca=-------------------~ 10,

--- ---~--

0+--------r------~r-------,-------~ 31 May 13June 11 July 24July 8 Aug

1--~ -- ....... -·---1 Fig. 2. Change of whole fruit K/Ca ratio during develop­ment.

and Sarilop. Bursa Black fruits presented a different variation in terms of K/Ca ratio in the meat and pedicel, as well as in the whole fruit.

The K/Ca peak values in meat and whole fruit of Goklop and Sarilop were reached on August 8th, and in Bursa Black on July 24th. In Bursa Black, there was a drastic decline in contrast to the noticeable increases in the other two. Such a rapid decline at the ripe stage can be an explana­tion for the crack resistance of Bursa Black fruits combined with an extraordinary accumulation of Mg, which may act on the imbalance.

Perring (1984) notes that apples, predisposed to cracking because of low Ca concentrations, become more susceptible as K and Mg concen­trations increase. At ripe stage, supporting this result, fruits of Goklop possessing low Ca had higher levels of K and Mg parallel with a higher ratio of cracking.

The change of K/Ca ratio in the skin was similar in all varieties and varied from the other

312 K, Ca and Mg content of the fig fruit

parts in this respect. Contrary to the smaller variation in the skin, significant differences in meat and pedicel suggest their major role in ostiole-end cracks of fig fruits.

Trials on Ca applications prior to rapid K accumulation must follow these results to over­come cracking, especially in susceptible varieties.

References

Aksoy U 1987 Evaluation of the dried fig crop with respect to standard quality characteristics. J. Ege Univ. Fac. Agric. 23, 23-30.

Aksoy U, Ana~ D, Hakerlerler H and Diizbastilar M 1987 Nutrient status of Calimyrna fig orchards in Big Meander Valley and their relationships with yield and quality. Tari§ Res. and Dev. Center. Project no 006.

Ferguson I B and Drobak B K 1988 Calcium and the regulation of plant growth and senescence. HortScience 23, 262-266.

Perring M A 1984 Indirect influences on apple fruit mineral composition and storage quality. Proc. Vlth Int. Coli. for Optimization of Plant Nutrition, 1199-1206.

Possner D R E and Kliewer W M 1985 The localisation of acids, sugars, potassium and calcium in developing grape berries. Vitis 24, 229-240.

Raese T 1989 Important considerations about calcium on apples and pears. Good Fruit Grower, 31-35.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 313-318, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-110

Effects of phosphorus and lime application on leaf mineral composition of olive trees grown in a schist soil

P.V. JORDA0 1 , A. MANTAS 2 , M. CENTEN0 2 and M.L. DUARTE 1

1/N/A, Laborat6rio Quimico Agricola Rebelo da Silva, Tapada da Ajuda, 1300 Lisboa, Portugal; 2DRATM, Qta. do Valongo, 5370 Mirandela, Portugal

Key words: leaf analysis, liming, nitrogen, olive trees, phosphorus

Abstract

A field experiment was established in a schist soil, in the portuguese region of Tnis-os-Montes, aiming to study the effect of two nitrogen, phosphorus and lime application rates on leaf mineral composition and yields of olive trees (cv. Verdeal Transmontana). The results are inconsistent concerning nutrient behaviour according to the experimental year. However, main results point to a leaf-Zn decreasing during the third year of the trial as a result of nitrogen application. During the fourth and fifth years of the trial, the leaf-P increased due to both lime and phosphorus applications. In the fifth year, nitrogen application showed a positive effect on foliar Mn concentrations, while liming induced lower levels; in the same year the phosphorus fertilization increased the leaf-S.

Multiple linear regressions were established in order to obtain the relationship between leaf mineral composition and the expected yield.

Introduction

The relevance of leaf analysis as a tool to diagnose the crops nutritional status depends on the available knowledge on the factors affecting leaf mineral composition. In olive grove, as in the others fruit trees, management practices such as fertilization, pruning, pest and disease control have great influence on leaf mineral concen­tration. The level of fertilization is an influencing factor as the mineral concentration of the leaves reflects the variation of the nutrients supply, particularly in some of the development stages, which show higher correlations between leaf mineral composition and yields (Bould, 1972). Therefore it is important to develop knowledge concerning both fertilization effects on leaves mineral concentration and the delay on the response.

The present paper looks into the experimental results concerning the effect of two application rates of nitrogen, phosphorus, and lime on the

leaf mineral composition of olive trees (cv. Verdeal Transmontana) and its relationship with the yield.

Material and methods

In 1986, a factorial field experiment, arranged into complete randomized blocks with three replications, was set up in an olive grove, cv. Verdeal Transmontana, aged 25 years, with 156 trees ha -I, established in a schist soil at Miran­dela, in the portuguese region of Tnis-os­Montes.

Table 1 shows the average level of soil fertility prior to the first year of the trial (1986), before experimental treatments were established.

The yield was very low (0.74 kg tree -I in 1986) probably due to water stress, fertilization and pruning absence.

The effect of nitrogen, phosphorus and lime application on yield was studied during the first 3

314 Jordiio et al.

Table 1. Average of some characteristics of the soil before the establishment of experimental treatments (1986)

1986

0-20cm 20-50 em

Organic matter (%) 0.74 0.69 Available P2 0 5 (mg kg- 1) 15 14 Available K2 0 (mg kg - 1) 142 134 pH(H,O) 5.04 5.05

Exchangeable Bases Ca meg (100 g)- 1 0.72 0.74 Mg meg (100 g)- 1 0.43 0.45 K meg (100 g)- 1 0.20 0.20 Nameg (10ogr 1 0.17 0.17

Titratable H 3.90 4.02 Extracted Fe (mg kg -I) 19 18 Extracted Mn (mg kg -I) 83 77 Extracted Zn (mg kg - 1) 0.3 0.3 Extracted Cu ( mg kg- 1) 0.2 0.2 Extracted B (mg kg - 1) 0.36 0.35

years of the trial and the experimental results have been recently published (Centeno and Jordao, 1990).

Both nitrogen (N 1 = 0.60 and N 2 = 1.20 kg tree- 1 ) and phosphorus (P1 = 0.09 kg tree - 1 )

were applied annually, whereas lime (L 1 =

10 t ha - 1) was only applied in the first year of the trial (1986); nitrogen and phosphorus were pro­vide through ammonium nitrate (20.5% N) and calcium superphosphate (18% P20 5 ), respective­ly.

In all plots 0.67 kg tree - 1 • year - 1 of potassium chloride (60% K20) and 0.35 kg tree - 1 of borax (10.5% B) were equally applied, in the establish­ment year. Potassium chloride was also applied in the following years. In autumn, 1987, 3.5 kg tree- 1 of magnesium sulphate (16% MgO) were equally applied, in all plots, because leaf analysis control showed a low content of magnesium (0.5% ).

Nitrogen was applied in spring (March/April) whereas the other nutrient supply was performed in autumn.

In early spring of 1987, 1988 and 1991, the trees were pruned. Pruning was very severe in the first and the last years and lighter in the second one.

Pest and disease treatments were not carried out.

Soil and leaf samples were collected annually,

the first ones in early autumn, at 0-20 em and 20-50 em layer depth and the second ones in midwinter (January/February). Leaves were col­lected from the middle of the current season's terminal shoots. Preparation of soil and leaf samples for analytical purposes were performed according to Dias et a!. (1980) and Duarte eta!. (1989). The yield of each tree was weighted, separately.

Because of the olive tree natural tendency for alternated bearing, analysis of variance was performed annually, in order to get the ex­perimental treatments effect on the leaf mineral composition.

Duncan-test (a= 0.05) was used in order to establish the differences among means.

Step by step procedure was used and each one of the independent variables were introduced in the model at 0.05 confidence level.

For each level of experimental variables (N, P and lime) a regression model was fitted, as well as for the overall data.

Multiple linear regressions were established in order to obtain the relationship between leaf mineral composition and the expected yield.

Results and discussion

Effect of the experimental treatments on the leaf mineral composition

Both in 1987/88 and 1988/89, no significant effects on foliar mean mineral levels were found, whereas in the third year of the trial (1989/90) a significant nitrogen mean effect on leaf-Zn was found (Table 2): the increase of nitrogen appli­cation level results into a significant (p,:; 0.05) decreasing on leaf-Zn (Table 4), which is in accordance with Olsen (1972), who supports that nitrogen application induces a zinc foliar de­ficiency in citrus; in addition, the level of soil extractable zinc is considered low which may have strengthened the effect of the highest rate of nitrogen application.

In the fourth and fifth years of the trial (Table 3), both lime and phosphorus applications re­sulted into a significant (p,:; 0.05) increasing leaf-P, which may be related to both lime and

N, P and lime on leaf mineral composition of olive trees 315

Table 2. Analysis of variance. Calculated F17 ;141 values for different years and nutrients

Nutrient (%) 1987/88 1988/89 1989/90 1990/91 1991192

N 0.99 NS 0.82NS 0.73 NS 1.86 NS 1.41 NS p 2.21 NS 0.99NS 1.10 NS 7.29 *** 4.34 *** K 0.91 NS 2.00NS 0.62 NS 0.43 NS 2.18 NS Ca 0.32 NS 2.71 NS 1.98 NS 2.68NS 1.76 NS Mg 0.92 NS 1.39 NS 0.45 NS 0.54 NS 0.59NS s 3.78 * Fe 2.01 NS 1.02 NS 0.67 NS 1.40 NS 0.90NS Mn 1.75 NS 1.96 NS 1.59 NS 18.70 *** 8.46 *** Zn 0.87NS 1.59 NS 3.71 * 0.77NS 1.04 NS Cu 0.95NS 0.54NS 1.12 NS 1.23 NS 1.42 NS B 0.28NS 0.88NS 1.07 NS 0.43 NS 1.10 NS

NS- p > 0.05; *- ~ 0.05; **- p ~ 0.01; ***- p ~ 0.001.

Table 3. Nitrogen, phosphorus and lime mean effects on the foliar macronutrient concentrations (Duncan test, a = 0.05)

Nutrient (%) Year Sm(±) Nl N, Po PI Lo Ll

N 1987/88 0.302 2.08 a 2.13 a 2.10 a 2.10 a 2.10a 2.11 a 1988/89 0.012 1.85 a 1.91 a 1.88 a 1.89 a 1.87 a 1.90 a 1989/90 0.007 1.84 a 1.87 a 1.85 a 1.86 a 1.85 a 1.86 a 1990/91 0.015 2.07 a 2.00a 2.00 a 2.07 a 1.99 a 2.08 a 1991/92 0.022 1.96 a 1.97 a 1.90 a 2.03 a 1.96 a 1.97 a

p 1987/88 0.0013 0.128 a 0.120 a 0.124 a 0.124 a 0.124 a 0.125 a 1988/89 0.0017 0.109 a 0.108 a 0.109 a 0.108 a 0.104 a 0.113 a 1989/90 0.0022 0.131 a 0.129 a 0.126 a 0.134 a 0.124 a 0.136 a 1990/91 0.0022 0.141 a 0.129 a 0.121 b 0.149 a 0.118 b 0.152 a 1991/92 0.0013 0.135 a 0.134 a 0.126 b 0.143 a 0.129 b 0.140 a

K 1987/88 0.010 0.97 a 1.00 a 0.96 a 0.98 a 0.98a 0.97 a 1988/89 O.D15 1.14 a 1.11a 1.14 a 1.10 a 1.17a 1.07 a 1989/90 0.020 0.89 a 0.92 a 0.94 a 0.87 a 0.89 a 0.92 a 1990/91 0.013 0.95 a 0.95 a 0.95 a 0.95 a 0.97 a 0.93 a 1991192 0.009 0.67 a 0.65 a 0.64 a 0.66 a 0.68 a 0.61 a

Ca 1987/88 0.019 0.80a 0.82 a 0.82 a 0.80 a 0.80 a 0.81 a 1988/89 0.024 0.89 a 0.98 a 0.99 a 0.87 a 0.81 a 1.05 a 1989/90 0.017 0.81 a 0.83 a 0.79 a 0.85 a 0.76a I 0.88 a 1

1990/91 0.016 0.70 a 0.68 a 0.70 a 0.68 a 0.60 a 1 0.79 a I

1991/92 0.031 1.48 a 1.50 a 1.51 a 1.48 a 1.37 a 1.61 a

Mg 1987/88 0.003 0.071 a 0.072 a 0.072 a 0.070 a 0.069 a 0.073 a 1988/89 0.003 0.099a 0.101a 0.103a 0.098a 0.096a 0.107a 1989/90 0.002 0.108 a 0.110 a 0.108 a 0.109 a 0.105 a 0.113 a 1990/91 0.002 0.110 a 0.110 a 0.111 a 0.111 a 0.111 a 0.111 a 1991/92 0.004 0.157 a 0.159 a 0.163 a 0.153 a 0.149 a 0.166 a

s 1987/88 1988/89 1989/90 1990/91 1991192 0.001 0.160 a 0.164 a 0.156 b 0.168 a 0.159 a 0.165 a

I p =0.06.

316 Jordao et al.

phosphorus application effect on available phos­phorus of soil.

In 1991/92 leaf-S was determined and a signifi­cant effect on this nutrient level was found due to phosphorus application (Table 3), which may have been caused by the sulfur content of cal­cium superphosphate (12% S).

The mean effect of the interaction among nitrogen, phosphorus and lime on the leaf-Mn concentrations was significant in 1990/91 (Table 5): the highest Mn mean levels were found, for both rates of nitrogen application with phos­phorus, but no liming, whereas the lowest ones were obtained with lime application. The ob­tained results suggest that the simultaneous ef­fect of both nitrogen and phosphorus on Mn foliar concentrations depends on liming, which may be connected to a lower Mn unavailability due to soil-pH increase, as a result of liming.

In the following year nitrogen shows a mean effect on the nutrient levels: mean leaf-Mn level increased with the increase of nitrogen soil application, which has been already reported by

Jordao (1990), whereas liming resulted in a decrease of the leaf-Mn concentration (Table 4).

No significant effect on leaf-Ca concentrations was found due to lime application. Nevertheless, in 1989/90 and 1990/91, liming showed an al­most significant (p = 0.06) and positive mean effect on the leaf-Ca concentration (Table 3).

The Ca and Mg foliar level increase from 1989/90 to 1991/92 is simultaneously accom­panied by a leaf-K decrease, because of the average of yields in 1989 is significantly lower than in 1991/92, 2.8 kg tree - 1 and 7.1 kg tree - 1

respectively. This is probably due to a greater K removal.

The leaf-Mn increase from 1989/90 to 1991/92

Table 5. Mean effect of the interaction nitrogen X

phosphorus X lime on leaf-Mn concentration in 1990/91 (mg kg - 1 ) (Duncan test, a= 0.05)

82 c 53e

117 a 62d

103 b 67 d

120 a 69 d

Table 4. Nitrogen, phosphorus and lime mean effects on the foliar micronutrients concentrations (dm) (Duncan test, a= 0.05)

Fe (mg kg - 1 ) 1987/88 1.220 . 69 a 69 a 66 a 72a 65 a 73 a

1988/89 2.380 80 a 75 a 78 a 78 a 72a 83 a 1989/90 1.610 65 a 58 a 61 a 62 a 62 a 60 a 1990/91 1.245 75 a 68 a 71a 72a 69 a 74 a 1991/92 1.200 69 a 66 a 69 a 67 a 67 a 69 a

Mn(mgkg- 1) 1987/88 1.090 54 a 56 a 54 a 56 a 54 a 57 a 1988/89 1.980 79 a 87 a 75 a 84 a 89 a 78 a 1989/90 1.575 59 a 57 a 56 a 60 a 63 a 52 a 1990/91 1991/92 4.645 138 b 175 a 146 a 167 a 200 a 112 b

Zn (mg kg - 1 ) 1987/88 0.280 13.4a 12.0 a 12.8 a 12.7 a 12.9 a 15.8 a 1988/89 0.176 13.4 a 12.5 a 13.3 a 12.6 a 13.4a 12.5 a 1989/90 0.204 13.2 a 10.8 b 12.4 a 11.7a 11.7a 12.3 a 1990/91 0.390 9.5 a 8.7 a 8.7 a 9.5 a 9.6 a 8.6 a 1991/92 0.224 12.8 a 12.2 a 12.4 a 12.6 a 13.2 a 11.7 a

Cu(mgkg- 1 ) 1987/88 0.088 5.6 a 6.0 a 5.8 a 5.9 a 5.8 a 5.8 a 1988/89 0.113 5.4 a 5.6 a 5.5 a 5.5 a 5.3 a 5.7 a 1989/90 0.091 5.2a 5.2a 5.3 a 5.3 a 5.2 a 5.2 a 1990/9, 0.190 5.4 a 5.5 a 5.9 a 4.9 a 5.0 a 5.6 a 1991/92 0.170 7.1 a 7.3 a 7.0 a 7.4 a 7.6 a 6.7 a

B (mgkg-') 1987/88 0.302 16.3 a 16.3 a 15.8 a 16.8 a 16.2 a 16.4 a 1988/89 0.322 20.1 a 19.1 a 19.6 a 19.6 a 19.0 a 20.2 a 1989/90 0.437 18.4 a 18.4 a 17.4a 19.3 a 17.1 a 19.6 a 1990/91 0.287 17.4a 16.7 a 17.2 a 16.9 a 17.0 a 17.1 a 1991/92 0.229 15.8 a 15.0 a 15.0 a 14.8 a 15.5 a 15.3 a

N, P and lime on leaf mineral composition of olive trees 317

is due mainly to the N and potassium chloride fertilization effect and also to the liming decrease effect throughout the five years of the experi­ment.

Relationships between yield and leaf mineral concentrations

Simple correlations between annual yields and leaf mineral concentrations were established in order to measure the degree of variation of yield together with the mineral composition of the leaves of the previous year.

The results show significant (p ~ 0.05) and negative associations between yield and the concentrations of Ca and B on the leaves of the previous year suggesting that functional relations of dependence between yield and leaf mineral concentrations could be found.

A great variation in the yield was found during the experimental period, (Centeno and Jordao, 1990). Regression models were established with data corresponding to four years of the trial. On average, the yield varied between 0.030 and 28.15kg tree- 1 •

Table 6 shows the multiple linear regression models fitted to both the annual yields and the leaf mineral concentrations corresponding to the previous year.

Equation (1) shows that 22.76% of the ex­pected yield variation is due to leaf-K, Ca, Mg, Fe and B concentration at the end of the previ­ous season, suggesting that nitrogen, phosphorus

and lime applications, because of the olive grove characteristics (aged 25 and with little fertiliza­tion) resulted into unbalanced leaf mineral composition which is yield constraint, within the experimental nutrient range values.

Concerning the relationship between yield and leaf mineral concentrations, for a supply of 0.60 kg tree_, year -t of nitrogen, (N 1), equation (2) shows that leaf-N and B concentration are inversely related to yield, whereas Ieaf-P and Cu show a direct relation with it. This suggests that the additional available nitrogen, due to fertilizer application, was mainly used for plant vegetative vigour improvement, resulting into a probable increase of leaf-area. Consequently, leaf-P and Cu levels were diluted, reaching limitative levels. Therefore, low yields were obtained (8 kg. tree -I, on average), which may be the cause of boron accumulation on leaves.

The increase of nitrogen application ( +0.60 kg tree -I year -I of N) leads to higher yields ( Cen­teno and Jordao, 1990). Equation (3) shows that =43% of the yield variation, within experimental treatments with 1.20 kg. tree_, year -I of nitrogen (N2 ) is due to leaf-N, K, Fe and Mn concen­trations, which are directly related to yield. Higher nitrogen availability, resulting into higher yields suggest a preferential nutrient request for fructification organs. Thus, leaf nutrient levels become yield limitative.

Phosphorus supply lead, on average, to signifi­cant lower yields (Centeno and Jordao, 1990). About 50% of yield variation, in experimental

Table 6. Relations of dependence between leaf mineral composition and expected yield

Nutrients

Nitrogen

Phosphorus

Lime

Application rates

Y = 5.27 + 11.27 K -11.30 Ca + 103.56 Mg + 0.089 Fe- 0.898 B

0.60 kg.tree 1.year 1 Y = -38.21- 18.20 N + 107.07 P + 1.54 Cu- 0.935 B 1.20 kg.tree - 1.year -J Y = -54.50 + 14.41 N + 21.61 K + 0.128 Fe+ 0.096 Mn

0.00 kg. tree -l year-] 0.09 kg. tree - 1 .year- 1

Y = 1.33 + 1.83 Cu Y = -10.32 + 80.08 P + 49.67 Mg + 0.089 Fe+ 0.104 Mn- 0.683 B

R' %

22.76*** 1

40.22*** 42.87***

8.80* 50.49***

Okg.ha- 1 Y=23.42-20.68Ca 19.55*** 104 kg.ha -] Y = 16.90-9.35 N + 108.25 P + 0.083 Fe+ 1.47 Cu- 1.07 B 48.70***

n=48; Y-Yield (kg.tree- 1); N, P, K, Ca, Mg-dry matter%; Fe, Mn, Cu, B-dry matter mg kg-\ R2 -Coefficient of determination;*- p ~0.05; ***- p ~0.001; 11 regression fitted to overall obtained data (n = 96).

318 N, P and lime on leaf mineral composition of olive trees

treatments with phosphorus supply (P1), is due to leaf-P, Mg, Fe and B concentrations (equation 5). With exception for boron, these nutrient levels are directly related to yield. Leaf-P, al­though with significant (p ""0.05) increase as a result of the supply, is yield limitative, suggesting a preferential demand for both vegetative de­velopment on root growth (Hansen, 1979).

Liming shows a depressive mean effect on the yield (Centeno and Jordao, 1990) and a positive and significant one (p = 0.05) on leaf-P and Ca.

Equation 6, concerning experimental treat­ments without liming, suggests that leaf calcium accumulation at the end of the production season, may express a nutritional unbalanced status caused by the other nutrients supply and the obtained yields. Although without significant mean influence other than on leaf-P and Ca concentrations, experimental treatments corre­sponding to lime application lead to an unbal­anced leaf mineral composition which is partly responsible for lower yields. Equation 7 shows that =49% of yield variation is due to leaf-P, Fe and Cu as well as leaf-N and B, which are inversely related with yield.

Conclusions

In some years of the trial, nitrogen, phosphorus and lime applications show significant, (p"" 0.05) mean effects on leaf mineral concentra­tions, namely on P, Mn, Zn, and S as well as on Ca (p = 0.06).

The applied experimental fertilizations re­sulted into unbalance nutritional status of olive grove. This was partly responsible for the ob­tained yields, which were lower in the ex­perimental treatments with phosphorus and lime applications.

Multiple linear regression models, fitted to obtained data within the yield range values

(0.030 to 28.15kg tree- 1), show that the use of reference values for nutritional status diagnosis of olive trees should take into account, not only the adopted methodology (namely the leaf col­lecting date and its physiological age, and the used analytical procedure), but also the yield levels which are often omitted. These facts can condition the validity of the use of other author's leaf standards as a reference, whenever plant varieties, climate and soil conditions are distinct. The regression models, which were applied are incipient and must be improved further.

Acknowledgements

The authors wish to express their appreciation to Fatima Calouro for her advice, concerning the elaboration of this paper, as well as to Mr Henrique and Mr Martins, from DRATM, for their collaboration in field work and to LQARS staff for chemical analysis and processing text.

References

Bould C 1972 Mineral Nutrition of Fruit Plants. Proc. 18th ISHS. Congress, Vol IV, pp 151-154.

Centeno M and Jordao P 1990 Resposta do olival a aplica~ao de azoto, f6sforo e calcario num solo mediterraneo pardo de Tnis-os-Montes. Aetas de Horticultura III, 26, 307-312.

Dias J C Setal. 1980 Guia Pn\tico de Fertiliza~ao. Ministerio da Agricultura e Pescas, DGER, Laborat6rio Ouimico Agricola Rebelo da Silva, Lisboa. 72 p.

Hansen P 1979 Crop load and nutrient translocation. In Mineral Nutrition of Fruit Trees. Eds. D Atkinson, J E Jiikson, R 0 Sharples and W M Waller. pp 201-212. Butterworths, London.

Jordao P 1990 Efeito da aplica~ao de fertilizantes na com­posi<;ao mineral de folhas de oliveira. Tese de Mestrado, UTL, Instituto Superior de Agronomia, Lisboa 99 p.

Olsen S R 1972 Micronutrient interactions. In Micronutrients in Agriculture. Eds. J J Mortvedt, PM Giordano and W L Lindsay. pp 243-264. Soil Science Society of America, Inc., Madison, WI.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 319-323, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-053

Effects of paclobutrazol application and fruit load on microelement concentrations in peach leaves

E. MONGE, P. MADERO, J. VAL and A. BLANCO Estaci6n Experimental de Aula Dei, CSIC, Aptdo 202, 50080-Zaragoza, Spain

Key words: crop load, copper, iron, manganese, microelements, paclobutrazol, peach, Prunus persica (L.) Bastch., zinc

Abstract

'Catherina' peach trees (Prunus persica (L.) Bastch.) were soil-treated at full-bloom with paclobutrazol (Pbz) or left untreated and one month later fruit-thinned to leave four different cropping levels from full crop to none. Leaf samples were harvested on different dates from July 9 to 31 October and analysis of mineral elements made by AAS. The concentrations of Fe and Mn were always greater in leaves from Pbz treated trees than in the controls, while Zn and Cu concentrations remained unaffected. On all sampling dates, the concentration of Mn decreased linearly with increasing levels of cropping. No effects of the cropping level were observed in the concentrations of the other elements analysed.

Abbreviations: AAS-atomic absorption spectrometry, Pbz-paclobutrazol

Introduction

Peach growers are becoming increasingly inter­ested in the control of tree size as production costs rise. The use of plant growth regulators becomes an alternative for the grower to control the growth of trees. Paclobutrazol is a very effective growth inhibitor in peach (Blanco, 1987; 1988; Martin et a!., 1987). Besides its effects on shoot growth, this compound also inhibits leaf expansion and root growth (Aguirre and Blanco, 1990; Rieger and Scalabrelli, 1990), and promotes fruit growth. These effects have been explained in terms of changes in the sink­source relationships in the plant. In fact, effects on the concentration of different mineral ele­ments have been found (Martin et a!., 1987; Rieger and Scalabrelli, 1990).

Fruit development has been shown to inhibit root growth, which also reduces mineral nutrient uptake (Wright, 1989). Consequently, interac­tions between crop-load and paclobutrazol treat-

ments may exist on the absorption of different mineral elements. It is generally accepted that mineral element concentration in leaves of fruit trees is a good indicator of the nutritional status of the plant. To study the effects of paclobut­razol and of cropping on the concentration of different microelements in peach leaves, an experiment was designed in which trees were treated with paclobutrazol and left with different fruiting levels, and leaf analysis carried-out in different dates along the summer and autumn.

Material and methods

The experiment has been carried-out in on 24 adult peaches cv. Catherina on Nemaguard root­stock, of the A.L.M. Group in Fuentes de Ebro (Zaragoza, Spain), subjected to the usual cultur­al practices.

The experiment was designed as a 2 x 4 fac­torial in which Pbz and crop load were the

320 Monge et al.

factors. At full-bloom, four trees were treated with 2 g (a.i.) paclobutrazol dissolved in 1 L of water and poured on the soil around the tree trunk, and four other trees were left untreated. At the end of April, the trees, either treated or untreated with paclobutrazol, were fruit thinned, leaving 4 different levels of crop load, from none to full crop, so that at harvest the average number of fruits collected per tree were 4 ± 3, 171 ± 41, 292 ± 87, and 544 ± 135, corresponding to levels 1 to 4. In all, eight treatments were applied, arranged in randomized blocks with 3 replications, and using the tree as the experimen­tal unit.

On six different dates during the summer and autumn: 9 July (during phase III of fruit growth), 31 July (at fruit harvest), 17 September, 2,16 and 31 October, (at leaf fall), 20-25 fully de­veloped leaves from the middle part of the shoot were harvested from randomly selected shoots around the canopy from each tree. Leaf area of samples was determined with a ~T image-ana­lyser. To determine the mineral elements, the leaves, with their petioles removed, were care­fully washed with a soft brush and liquid soap (1%) and rinsed with tap and deionized water to eliminate surface contamination. Dry ashing was carried out following the methods of C.l.l. (1969) and Pinta and DeWele (1975). Fe, Mn, Cu and Zn were determined by atomic absorp­tion spectrometry (Pye Unicam SP9).

Analysis of variance and regression of data against the number of fruits per unit tree size were carried-out. When ANOVA resulted in significant differences, means were separated by a LSD test or by Duncan's multiple range test.

Results

The area of leaves was significantly affected by both factors (p < 0.001) at all dates of measure­ment: in Pbz treated trees, leaves were on average 49.5% smaller, while in trees of crop­ping levels 2, 3 and 4 they were 80.0, 72.7 and 61.5% smaller than those from trees with no crop.

The concentrations of Fe and Mn were greater in leaves of paclobutrazol-treated trees than in leaves of untreated trees on all sampling dates (Table 1). The effects were statistically signifi­cant except on 16 October for the Mn concen­tration. In general terms, the concentrations of Cu and Zn have not been affected by the application of the growth inhibitor. Only on 16 October, the concentration of Cu was signifi­cantly greater in leaves from paclobutrazol treated trees than in the controls.

Crop-load level had no effects on Fe, Zn, and Cu concentration except for certain sampling dates (Table 2). On the contrary, Mn has shown the same pattern of behavior throughout the

Table 1. Concentration (mg kg_,) of different microelements in leaves of 'Catherina' peach trees treated or not with Pbz and sampled in different dates along the summer

Element Pbz 9July 31 July 17 Sept. 20ct. 16 Oct. 31 Oct.

Fe Og 129.50 145.10 139.40 147.20 157.90 111.90 2g 156.00 180.70 153.60 168.90 182.40 132.30 Signif.

Mn Og 32.98 37.90 55.90 49.20 53.44 49.29 2g 41.67 47.98 69.83 53.30 63.71 62.56 Signif. NS

Zn Og 35.35 23.54 20.79 23.46 27.10 24.56 2g 35.38 25.75 19.71 23.29 24.00 23.21 Signif. NS NS NS NS NS NS

Cu Og 22.72 21.96 17.69 9.10 10.75 10.17 2g 24.02 25.73 19.17 9.52 13.48 11.04 Signif. NS NS NS NS NS

NS, *. ** and *** mean non-significant, or significant at the p < 0.05, p < 0.01 and p < 0.001 level respectively; Values averaged over crop-load levels.

Paclobutrazol and fruit crop effects 321

Table 2. Crop load effects on the concentration (mg kg- 1 ) of mineral elements in leaves of peach trees (Catherina/Nemaguard) harvested in different dates along the summer

Element Crop-load 9 July 31 July 17 Sept. 20ct. 160ct. 31 Oct.

Fe 1 143.00 161.10 144.10 141.80 157.70 121. 70' 2 149.10 149.10 148.50 164.50 163.70 114.30' 3 131.70 174.80 149.90 162.70 184.80 116.50' 4 147.10 166.80 143.50 163.10 174.50 135.90b Signif. NS NS NS NS NS

Zn 1 32.08' 25.21 19.75 21.46' 25.46 21.71 2 34.00"b 25.67 20.42 22.71 >b 24.58 27.33 3 39.17c 26.42 20.63 26.04c 27.04 22.92 4 36.21 b 21.33 20.21 23.29b 25.13 23.58 Signif. NS NS NS NS

Cu 1 25.40 23.71 18.79 10.08 12.33' 14.25 2 24.42 18.79 17.75 9.54 12.88'b 9.00 3 21.63 27.08 18.04 8.75 14.13b 9.46 4 22.04 25.79 19.13 8.88 9.13' 9.71 Signif. NS NS NS NS NS

NS, *, mean non-significant, or significant at the p < 0.05. Within columns, values followed by different superscript are significantly different at the p < 0.05 level following Duncan's Multiple Range test; Values averaged over paclobutrazol levels.

summer and autumn: the concentration de­creased linearly with increasing levels of crop­load. Figure 1 shows the pattern recorded on 31 July, as an example.

Analysis of Variance of data has shown that only for Mn the interaction between paclobut­razol and cropping level has resulted in signifi­cant differences. This agrees with the significant differences detected for the intercepts and the

80~-----------------------------,

+Pbz 01 ..-: 0, 40 <" l:i •t.

"' ' '~ "' 20 .... -Pbt

00 0,02 0,04 0,06 No. fruits I UTS

Fig. 1. Mn concentration in leaves from trees carrying different crop-loads and, either treated with paclobutrazol (6) or not (•). Paclobutrazol treated trees: y = 68.8-1050.8 x, (p,;;; 0.001; r = -0.888). Control trees: y = 54.0-753.8x, (p ,;;;0.001; r= -0.918).

slopes of the two lines obtained from the regres­sion analysis (Fig. 1).

Discussion

Pbz is a giberellin biosynthesis inhibitor (Hedden and Graebe, 1985) which effectively reduces growth in peach (Blanco, 1987; Martin et al., 1988). Although the use of this growth inhibitor is becoming increasingly important in commer­cial practice, its effects on micronutrient uptake have been little studied. The effects of Pbz on root growth and development (Rieger and Scalabrelli, 1990; Williamson et al., 1986) may influence water and nutrient uptake.

Throughout the summer, the levels of most microelements analysed were, in general terms, close to those considered optimum for the diag­nosis of peach tree nutrient status (Leece, 1976). Only Cu has shown higher concentrations than normal.

Swietlik and Miller (1985) found that apple trees treated with Pbz had a smaller concen­tration of leaf Mn than untreated trees, while Zn and Cu concentrations were not affected, and Rieger and Scalabrelli (1990) reported a reduc­tion of Fe and Mo and an increase in Mn when

322 Monge et al.

Pbz was supplied in the nutrient solution to peach rootstocks, which indicates difference in behavior within species.

In our experiment, the concentrations of Fe and Mn in leaves of peach trees cv. Catherina treated with the growth inhibitor were consis­tently greater than in the control leaves (Table 1). However, the reduction in leaf area pro­moted by Pbz (on average 49.5%) was relatively greater than the increase in Fe and Mn (on average 17% and 22% respectively), which may well indicate a reduction in mineral uptake by the root system and this could be confirmed by the lack of variation in the concentration of the other two microelements analysed.

The study of the nutrients relationship are useful for the interpretation of the nutritive status of plants (Abadia et a!., 1985). Several researchers reported an antagonism between Fe and Mn, which could lead to induce Fe chlorosis (Casero and Carpena, 1987; Roger eta!., 1974) probably due to a substitution of Fe by Mn in the biosynthesis of chlorophyll (Clairmont et a!., 1986). In our case, in spite of the antagonism reported, proportional increases in both ele­ments on Pbz treated leaves were found, and therefore, this ratio did not change in relation to untreated trees in any of the dates of sampling (data not shown).

Marschner (1986) reported that mineral nutri­tion can affect the number of fruits and I or seeds per plant. In our study, we have induced the opposite situation: the number of fruits per tree was controlled to create four cropping levels. The results (Table 2 and Fig. 1) show that the Fe, Cu and Zn concentrations were not affected by the fruiting level, while that of Mn decreased linearly with increasing levels of fruiting. This effect in concentration was greater in leaves of Pbz treated trees than in untreated trees as indicated by the slope of the line, so that when the trees carried a full crop the concentrations of Mn in treated and untreated trees was similar. A reduction of leaf size with increasing levels of cropping has also been recorded, which could be explained by the role of Mn in cell division and extension (Marschner, 1986).

The results of the experiment show significant effects of Pbz on Fe and Mn. This could well mean that leaves have a greater need for Fe and

Mn in Pbz treated peach trees. Besides, Pbz and crop-load interaction only occurs for Mn concen­tration in 'Catherina' peach leaves. Knowing the important roles of Fe and Mn in photosynthesis (Monge et a!., 1991; Monge and Val, 1990), work in progress has been designed to elucidate whether the changes in micronutrient levels affect photosynthesis.

Acknowledgements

The authors acknowledge A L M Group for the facilities provided to conduct the experiment, and particularly Mr J M Soriano, Dr J Negueroles, and Mr C Flamarique, ICI-Zeltia for gifts of Pbz and Mrs M A Gracia for her excellent technical assistance. Work carried-out under research projects CICYT AGR090-0792 and CONAI-DGA: PCA-4/91.

References

Abadia J, Nishio J N, Monge E, Montaiies L and Heras L 1985 Mineral composition of peach affected by iron chloro­sis. J. Plant Nutr. 8, 697-707.

Aguirre R and Blanco A 1990 Efecto del Paclobutrazol sobre el crecimiento de brotes y raices en patrones de melocoto­nero. Aetas de Hortic. 1, 90-95.

Blanco A 1987 Fruit thinning of peach trees (Prunus persica (L.) Batsch.): the effect of paclobutrazol on fruit drop and shoot growth. J. Hortic Sci. 62, 147-155.

Blanco A 1988 Control of shoot growth of peach and nectarine trees with paclobutrazol. J. Hortic. Sci. 63, 201-207.

Casero T and Carpena 0 1987 Relaciones nutritivas en melocotonero 'Sudanell'. Inv. Agrar.: Prod. Prot. Veg. 2, 19-30.

Clairmont K, Hagar W and Davies E 1986 Manganese toxicity to chlorophyll synthesis in tobacco callus. Plant Physiol. 80, 291-293.

C. !.I. 1969 Comite Inter-Institutos para el estudio de tecnicas analiticas. Metodos de referencia para la determinacion de elementos minerales en vegetates. An. Edaf. Agrobiol. 38, 403-417.

Hedden P and Graebe J E 1985 Inhibition of gibberellin biosynthesis by paclobutrazol in cell-free homogenates of Cucurbita maxima and Malus pumila embryos. J. Plant Growth Reg. 4, 111-122.

Leece D R 1976 Diagnosis of nutritional disorders by leaf and soil analyses and biochemical indices. J. Aust. Ins!. Agric. Sci. 42, 7-19.

Marschner H 1986 Mineral Nutrition of higher Plants. Academic Press, London. 674 p.

Martin C G, Yoshikawa F and La Rue J H 1987 Effect of soil applications of paclobutrazol on vegetative growth, prun­ing time, flowering, yield, and quality of 'Flavorcrest' peach. J. Am. Soc. Hortic. Sci. 112, 915-921.

Monge E, Montaiies L, Val J and Heras L 1991 El hierro modulator de la estructura y funci6n del cloroplasto. In Fijaci6n y Movilizaci6n Biol6gica de Nutrientes. Ed. J L6pez-Gorje. Vol. I. pp 85-108. CSIC, Madrid, Spain.

Monge E and Val J 1990 The role of oligoelements in higher plants I. Manganese. An. Aula Dei 20, 65-90.

Pinta M and DeWele G 1975 Etalons vegetaux pour !'analyse foliare. In Lc C6ntrole de !'Alimentation des Plantes Cultivees. Ed. P Kozma. pp 159-172 Akademiai Kiado. Budapest.

Rieger M and Scalabrelli G 1990 Paclobutrazol, root growth,

Paclobutrazol and fruit crop effects 323

hydraulic conductivity, and nutrient uptake of 'Nemaguard' peach. HortScience 25, 95-98.

Rogers E, Johnson G and Johnson D 1974 Iron induced manganese deficiency in "Sungold" peach and its effects on fruit composition and quality. J. Am. Soc. Hortic. Sci. 99, 242-244.

Swietlik D and Miller S 1985 The effect of paclobutrazol on mineral nutrition of apple seedlings. J. Plant Nutr. 8, 369-382.

Williamson J G, Coston D C and Grimes L W 1986 Growth responses of peach roots and shoots to soil and foliar­applied paclobutrazol. HortScience 21, 169-175.

Wright C J 1989 Interactions between Vegetative and Re­productive Growth. In Manipulation of fruiting. Ed C J Wright. pp 15-27. Butterworths & Co. Ltd. London.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 325-331, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-011

Effects of P-supply on growth and P-micronutrient interactions of potted peach seedlings

M. TAGLIAVINI, B. MARANGONI and P. GRAZIOLI Department of Horticulture and Forestry, University of Bologna, Via F. Re 6, 40126 Bologna, Italy

Key words: iron, manganese, peach nutrition, phosphorus, Prunus persica, P-micronutrient interac­tions, replant soil, zinc

Abstract

A short-term study was conducted to gain preliminary data on the use of phosphate as a potential means of increasing the post-transplant growth of peach in virgin and replant soils. Since phosphorus (P) is known to adversely affect micronutrient nutrition, P-micronutrient interactions are also reported and discussed. Peach seedlings (Prunus persica, L. Batsch) were grown under greenhouse conditions in pots filled with soils collected either from an aged peach orchard (replant soil) or from an adjacent virgin plot (virgin soil). The soils were enriched with 0 (control), 100 and 500 mg P (as monosodium phosphate) per kg of soil. Plants were arranged in a randomized block design with five replicates. P addition to soils resulted in a slight (less than 30% of added P) increase of bicarbonate-extractable P. Increasing P supply stimulated progressive growth in replant soil, while 100 mg kg -I was optimal in virgin soil. Concentrations of P and Mn in leaves were low in control soils but significantly increased with P supplements. Leaf Fe and Zn concentrations were not affected by P supply; leaf Fe concentrations were within the sufficiency range while Zn concentrations were generally low. Since the results refer to a preliminary approach with young peach seedlings, caution should be taken in extrapolating them to a field situation. Nevertheless, data suggest that the technique of mixing P in the planting hole deserves attention even in soils apparently well endowed with P.

Introduction

Deciduous fruit trees growing in soils with high phosphate buffer capacity seldom respond to surface-applied P. This because the poor down­ward movement of P in such soils (Neilsen et a!., 1990) makes it difficult to introduce phosphate into a significant part of the root zone. Orchards in the fruit growing area of northern Italy's Po Valley are frequently planted in soils that are considered naturally rich in P (Regione Emilia­Romagna, 1988) and, hence, scant attention has been given to pre-planting P fertilization. How­ever, it has been shown (Taylor, 1975; Taylor and Goubran, 1975) that the addition of super­phosphate to the planting hole may stimulate the growth of apple and peach trees despite the

presence of medium or high soil phosphate levels. The use of planting-hole P fertilization seems to be a particularly desirable strategy in orchard replants (Neilsen and Yortson, 1991; Slykhuis and Li, 1985) since high P supply is known to improve the establishing of same woody perennials (Neilsen et a!., 1990).

If large amounts of P are supplied, however, luxury uptake of P may occur (Tagliavini et a!., 1991), an effect that raises the ratios of P to Fe (De Kock and Wallace, 1965) and Zn in plant tissues (Loneragan et a!., 1979, 1982) and has often been associated with deficiency symptoms of the two micronutrients (Murphy et a!., 1981 ). Yet it is not clear whether the major interactions take place in the plant or in the soil. Indeed, large P-inputs decrease soil Zn diffusion rates

326 Tagliavini et al.

(Schropp and Marschner, 1977) and enhance Fe immobilization. Thus the present short-term study was designed to gain preliminary data on the use of phosphate as a potential means of increasing post-transplant growth of peach in soils that are considered naturally rich in P. The interaction of P-micronutrients is also examined and discussed.

Material and methods

Seeds of PSB2 peach rootstocks (Prunus persica, L. Batsch) were germinated in sand, grown for 20 days and then transplanted to 1.2 L pots. The latter contained sandy clay soil taken either from the site of an aged orchard grafted on peach seedling rootstock (replant soil) or from an adjacent virgin plot (virgin soil). The sites were located at the Cadriano Experimental Station of the University of Bologna in a Eutric Cambisol soil series. Selected soil physical and chemical characteristics are shown in Table 1. Soils were enriched with 0 (control), 100 and 500 mg P kg - 1

as NaH2P0 4 .H20: the fertilizer was mixed into soil just before transplanting. Monosodium­phosphate was chosen to minimize the effect of the accompanying ion (Na) on plant response. The control soils were analysed prior to the trial for P-fixation capacity: increasing amounts of KH 2PO 4 were added to the soils, and the bicar­bonate-extracted P fraction was determined after 24 hours of incubation at 40°C (Facco and Faleschini, 1987).

The potted peach seedlings were subsequently grown for 62 days under greenhouse conditions. Shoot length was measured at 11-13 day inter­vals. At the end of the trial, plants were pulled out and separated into stem, leaves and roots.

Table 1. Selected chemical characteristics of soils'

Soil pH Organic Total P" matter N (%)

Virgin 7.1 1.6 980 21 Replant 7.5 1.8 1110 23

Total leaf area was determined by a 'LI-COR LI 3000' portable area meter, and the weight of all tissues was determined after oven-drying at 6SOC for 48 hours. Phosphorus concentration in leaves was determined colorimetrically using phospho­molybdenum blue complex. Iron, Mn and Zn leaf concentrations were determined by atomic absorption spectrophotometry. Soil samples were collected from the pots at the end of the trial to determine total and bicarbonate-ex­tracted P (Olsen et al., 1955).

The experimental layout was a randomized block design with 5 replicates. Growth and mineral concentration data were subjected to the analysis of variance (2 soil types x 3 P levels). The standard error of the mean (SEM) was used as a precision measure of the treatment means (Nelson, 1989). Simple linear correlation of P to Fe and Zn leaf concentrations and simple linear regression of leaf P to soil P were performed. Leaf P and Mn concentrations were related by non-linear regression analysis.

Results

While the phosphorus bicarbonate-extracted fraction linearly increased at increasing P rate regardless of soil type (Fig. 1, less than 30% of added P was recovered by the method used. Seedling shoot length was not affected by soil type but plants positively responded to P fertili­zation (Fig. 2) just 25 days after treatment. From day 46 on, plants in soil with 500 mg P kg - 1

showed the best growth response (Fig. 2). Sig­nificant interaction of soil type and P supplement characterized total leaf dry weight (d.w.), leaf area and root d.w. (Table 2). This fact indicates that maximum leaf and root development in

K' Mg' Ca' Fed Mnd Znd

122 298 3300 36 24 0.9 124 186 3600 20 17 1.1

"Soil analyses according to Unichim (1986). Mineral elements as 11-g g -I

hBicarbonate-extracted. 'Extracted by BaCI2 +TEA. dExtracted by DTPA.

28

26

24

22

20

Bicarbonate-extracted P (}Jg g·1)

•

0

Y = 21.3 + 0.29 X

R2 = 0.89

•

• •

2 4 6 B 10 12 14 16 18

Soil-added P (}Jg g"1)

Fig. 1. Linear regression of bicarbonate-extracted P versus soil-added P. Circles and squares refer to replant and virgin soils, respectively

35

30

25

20

15

10

Shoot length (em)

o mg Kg-1

100 mg Kg"1

soo mg Kg" 1

5 10 15 20 25 30 35 40 45 50 55

days after transplant

Fig. 2. Shoot development as affected by P rates. Data are averages of plants grown in virgin and replant soils (n = 10).

virgin soil was reached by plants receiving 100 mg P kg - 1 , while in replant soil a significant further increase of leaf and root growth follow­ing the highest P rate was found (Table 2). The specific leaf weight and the shoot-to-root d. w. ratio were not affected by P (data not reported), although the shoot-to-root ratio was higher in plants grown in replant (2.2) than in virgin soil (1.7). Regardless of soil type, P application stimulated stem d.w. and increased the number of leaves per plant (Table 2), although no differences between the two P rates were found.

Increasing the rate of phosphate resulted in an increase in leaf P concentration (Table 3, al­though the response varied depending on soil

P-supply in peach 327

(Table 3) and the maximum was recorded in the replant soil at the highest P rate. Leaf iron and Zn concentrations were neither affected by P treatment and soil type (Table 3), nor were they linearly correlated toP leaf concentrations (data not reported). Phosphate application resulted in an increase of leaf Mn concentration (Table 3), the latter being related to leaf P concentration of plants in both soils (Fig. 3). At the end of the trial, interveinal chlorosis in older leaves, resem­bling symptoms of Mg deficiency, was found in all plants treated at the highest P rate, the symptoms being more severe in those grown in replant soil.

Bicarbonate-extractable soil P was a more suitable predictor of leaf P concentration than total soil P and soil-applied P (Table 4). Phos­phate enrichment still resulted in higher bicar­bonate-extractable P in soils at the end of the trial (Table 5). The extractable fraction in con­trol soils was about 4% of total P, while this percentage more than doubled at 100 mg kg - 1 of P and was four- to five-fold higher at 500 mg kg - 1 of P (Table 5).

Discussion

Positive peach growth response to P fertilization was found in this trial despite the presence of high levels of phosphate in control soils and their high P-fixation capacity. This evidence suggests that newly planted and replanted peach may benefit from pre-planting and localized applica­tion of phosphate even in soils apparently well endowed with P. These findings are in good accord with those reported by Taylor (1975) for peach and apple in nursery and field experi­ments. However, it should be borne in mind that our results were recorded in young ungrafted peach plants growing in pots, where overlap of P depletion zones may have occurred. Caution should therefore be taken in extrapolating these findings to a field situation, where root growth limitation is less likely, especially when vigorous rootstocks and low- to medium tree densities are employed. Trees may thus use such strategies as increasing the volume of soil explored by roots to improve their P uptake (Taylor and Goubran, 1976).

328 Tagliavini et al.

Table 2. Effects of P application (mg kg- 1 ) and soil type (V= virgin; R =replant) on leaf area (cm 2 plant- 1), leaf d.w. (mg plant- 1), leaf number, and stem and root d.w. (mg plant- 1)

Phosphorus 0

100 500 SEM" Significance

Soil Virgin Replant SEM Significance

Interaction Significance Interaction SEM

Leaf area

v

84 132 148

* * 11

"SEM =Standard error of the mean.

R

75 139 227

Leaf d.w.

v

432 643 748

* * 47

Leaf number

R

365 17 728 22 980 23

1 * * * b

21 20

NS

Stemd.w.

395 613 670

41

* * *

544 579

33 NS

Rootd.w.

v

462 823 748

* * 53

R

381 503 903

b * * * * * and NS =Significance at 0.1%, 1% level of probability and nonsignificance, respectively.

Table 3. Effects of P application (mg kg- 1 ) and soil type (V= virgin; R =replant) on P, Fe, Zn and Mn leaf concentrations

P(%d.w.) Fe(!Lgg- 1 ) Zn(!Lgg- 1 ) Mn(!Lgg- 1)

v R

Phosphorus 0 0.11 0.12

100 0.16 0.19 500 0.32 0.74 SEM" Significance

Soil Virgin Replant SEM Significance

Interaction Significance * * * Interaction SEM O.D3

"SEM = Standard error of the mean.

207 18 175 16 151 14 26 2

NSb NS

152 15 200 17

25 2 NS NS

v

24 28 40

* * 4

R

24 38 74

b * * *, * * and NS =Significance at 0.1 %, 1% level of probability and nonsignificance, respectively.

Untreated virgin and replant soils induced similar seedling growth, whereas maximum growth response was registered by the lower P application in virgin soil and the higher rate in replant soil. The lack of difference in growth of plants in virgin and replant soil likely indicates a low level of replant problems associated with the soil used. On the basis of our data, it would be incorrect to conclude that P application alone

overcomes the growth depression often found during the replanting of peach. A recent study (Tagliavini and Marangoni, 1992), has shown that P addition may stimulate the growth of peach seedlings in virgin soil but may not remedy the growth depression caused by the presence of decomposing peach root residues in soil.

Phosphorus deficiency symptoms as shown by purplish leaf were not visible, but control leaves

Leaf Mn concentration (ug g -1 d.w.) 100,-------------------------------------,

80

60

40

20

2 R ;Q,96

Y;85.5*(1-e -2.5*X)

. I o ...

. ·-

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

Leaf P concentration (% d.w.)

Fig. 3. Nonlinear regression of leaf Mn concentration versus leaf P concentration.

were dark green and reduced in area as com­pared to P-treated plants. Meristematic and expanding tissues of a plant are important sinks for P (Bieleski, 1973), so it is not surprising that the most pronounced effect of P treatments was to elicit an increase of leaf number and leaf area.

The increased P-leaf concentration and leaf

?-supply in peach 329

d. w. indicate an increase of P uptake in P­treated plants. This fact is a likely consequence of increased phosphate supply (Table 4), though it may also have resulted from a more rapid, P-induced development of the root system and hence a greater root surface for mineral uptake.

Values of P-leaf concentration for plants in control soils were close to the deficiency threshold for peach leaves (Shear and Faust, 1980). Leaf analysis also showed that the lower rate of P application ( 100 mg kg -I) to soils led to only a small increase in the level of P in leaves, suggesting that phosphate absorption and growth are closely coupled. Conversely, the high P rate (500mg kg- 1) resulted in higher leaf P concen­tration rather than a growth increase, implying that these plants had to some extent a luxury uptake of P.

Hydroponically grown peach with root-zone temperature of 16 and 24oC showed shoot P concentration of 0.37 and 0.52%, respectively, when P was raised in the growing medium to 5 mM (Tagliavini et a!., 1991 ). At these concen-

Table 4. Regressions and linear relations between soil-P characteristics and leaf P concentration

X

Total soil P' (mg g -I)

Bicarbonate­extracted P (mgg-I)

Soil-applied P (mg kg- 1)

y

LeafP (% d.w.) LeafP (% d.w.)

LeafP (% d.w.)

'Naturally-endowed soil P plus soil-applied P. bSignificance at 0.1% level of probability.

Regression equation Coefficient of determination

Y = -0.39 + 0.89 X 0.52•••b

Y = +0.02 + 2.82 X 0.74•••

Y = +0.01 + 0.0009 X 0.65•••

Table 5. Effects of P application (mg kg -I) on concentration of total P, bicarbonate-extracted P and ratio between bicarbonate­extracted and total P at the end of the trial

Soil pr<Jtt:~ Total P Bicarbonate- Ratio bicarbonate-(mgg-I) extracted P extracted P to total P (%)

(!Lgg-1)

Virgin 0 0.57 21 3.7

100 0.65 58 8.9 500 1.09 180 16.5

Replant 0 0.57 23 4.0

100 0.65 64 9.8 500 1.00 210 21.0

330 Tagliavini et al.

trations plants showed growth depression, a likely result of negative P-Fe and P-Zn interac­tions. The present study found neither symptoms of leaf Fe and Zn deficiency nor an effect of P on Fe and Zn concentration in leaves. In so far as Fe is concerned, this discrepancy may partially be explained by considering that antagonistic interactions are likely when high rates of P are superimposed on a Fe-deficient soil (Murphy et a!., 1981), a situation rather different from that of soils used in this trial (Table 1). Our soils had low values of DTPA-extractable Zn (Table 1), although not below the critical level determined for annual crops between 0.5 and 0.8mg kg- 1

(Brown eta!., 1971; Lindsay and Norvell, 1978). A low Zn supply in all the treatments is also indicated by the low Zn concentrations in leaves (Table 3) (Jones eta!., 1991). While Fe and Zn concentrations in leaves remained unaffected by P enrichment, the total leaf content of these micronutrients increased with P supplements as a result of the growth-stimulation caused by P. This fact helps to support the assumption that in our trial P did not interfere with Fe and Zn uptake.

Leaf Mn concentrations of plants in control soils were in the low range (Jones et a!., 1991), although they increased significantly to a normal range with P enrichment. This phenomenon, often found in the literature (Smilde, 1973), is attributed to the soil-acidifying effect of P, which increases the Mn uptake (Jackson and Carter, 1976; Lindsay and Stephenson, 1959).

Although the reported evidence comes from a preliminary approach using young plants and needs to be confirmed in a field trial, the tech­nique of mixing P in the planting hole deserves more attention, even in soils apparently well endowed with P.

References

Bieleski R L 1973 Phosphate pools, phosphate transport and phosphate availability. Annu. Rev. Plant Physiol. 24, 225-252.

Brown A L, Quick J and Eddings J L 1971 A comparison of analytical methods for soil zinc. Soil Sci. Soc. Am. Proc. 35, 105-107.

De Kock P C and Wallace A 1965 Excess phosphorus and iron chlorosis. Calif. Agric. 19 (12), 3-4.

Facco S and Faleschini F 1987 Retrogradazione del fosforo nel suolo. In Atti Convegno Fertilita del suolo e nutrizione delle piante. Sorrento (Italy), pp 385-387.

Jackson T L and Carter G E 1976 Nutrient uptake by burbank potatoes as influenced by fertilization. Agron. J. 68, 9-12.

Jones J B, Wolf B and Mills H A 1991 Plant Analysis Handbook. Micro-Macro Publishing Inc. Athens (USA), 213 p.

Lindsay W L and Norvell W A 1978 Development of a DTPA soil test for zinc, iron, manganese, and copper. Soil Sci. Soc. Am. J. 42, 421-428.

Lindsay W L and Stephenson H F 1959 Nature of reactions of monocalcium phosphate in soils: II. Dissolution and pre­cipitation reactions involving iron, aluminium, manganese, and calcium. Soil Sci. Soc. Am. Proc. 32, 18-22.

Loneragan J F, Grove T S, Robson A D and Snowball K 1979 Phosphorus toxicity as a factor in zinc-phosphorus interactions in plants. Soil Sci. Soc. Am. J. 43, 966-972.

Loneragan J F, Grunes D L, Welch R M, Aduayi E A, Tengah A, Lazar V A and Cary E E 1982 Phosphorus accumulation and toxicity in leaves in relation to zinc supply. Soil Sci. Soc. Am. J. 46, 345-352.

Murphy L S, Ellis R and Adriano D C 1981 Phosphorus­micronutrient interaction effects on crop production. J. Plant Nutr. 3, 597-613.

Neilsen G H, Hogue E J and Yorston J 1990 Response of fruit trees to phosphorus fertilization. Acta Hortic. 274, 347-359.

Neilsen G H, Neilsen D and Atkinson D 1990 Top and root growth and nutrient absorption of Prunus avium L. at two soil pH and P levels. Plant and Soil 121, 137-144.

Neilsen G H and Yorston J 1991 Soil disinfection and monoammonium phosphate fertilization increase precocity of apple on replant problem soils. J. Am. Soc. Hort. Sci. 116, 651-654.

Nelson L A 1989 A statistical editor's viewpoint of statistical usage in Horticultural Science publications. HortScience 24, 53-57.

Olsen S R, Cole C V, Watanabe F S and Dean L A 1955 Estimation of available phosphorus in soils by extraction with sodium bicarbonate. USDA Circular 939, 19 p.

Regione Emilia-Romagna Assessorato Agricoltura Servizio di Sviluppo Agricola 1988 Guida All'Interpretazione dei Risultati Dell'Analisi dei Terreni e alia Formulazione dei Consigli di Concimazione. Regione Emilia-Romagna, Bologna (Italy), 86 p.

Schropp A and Marschner H 1977 Wirkung haler phosphat­dungung auf die wachstumsrate den Zn-gehalt und das P/Zn-verhaltnis in weinreben (Vitis vinifera). Z. Pflan­zenernaehr. Bodenkd. 140, 525-529.

Shear C B and Faust M 1980 Nutritional ranges in deciduous tree fruits and nuts. Hortic. Rev. 2, 143-163.

Slykhuis J T and Li T S C 1985 Response of apple seedlings to biocides and phosphate fertilizers in orchard soils in British Columbia. Can. J. Plant Path. 7, 294-301.

Smilde K W 1973 Phosphorus and micronutrient metal uptake by some tree species as affected by phosphate and lime applied to an acid sandy soil. Plant and Soil 39, 131-149.

Tagliavini M, Hogue E J and Neilsen G H 1991 Influence of phosphorus nutrition and root zone temperature on growth and mineral uptake of peach seedlings. J. Plant Nutr. 14, 1267-1275.

Tagliavini M and Marangoni B 1992 Growth of peach as affected by decomposition of own root residues in soil. Plant and Soil 145, 253-260.

Taylor B K 1975 Response of newly planted peach and apple to superphosphate. Aust. J. Agric. Res. 26, 521-528.

P-supply in peach 331

Taylor B K and Goubran F H 1975 The phosphorus nutrition of the apple tree. I. Influence of rate of application of superphosphate on the performance of young trees. Aust. J. Agric. Res. 26, 843-853.

Taylor B K and Goubran F H 1976 Effects of phosphate and pH stress on the growth and function of apple roots. Plant and Soil 44, 149-162.

Unichim 1986 Analisi dei terreni agrari. Parte I: Metodi manuali. Unichim, Milano (Italy), 120 p.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 333-337, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-215

Effects of organic waste fertilization and saline irrigation on mineral composition of tomato leaves and fruits

I. GOMEZ, J. NAVARRO-PEDRENO and J. MATAIX Division Agroqufmica, Facultad de Ciencias, University of Alicante, P.O. Box 99, 03080-Alicante, Spain

Key words: organic residues, salinity, tomato

Abstract

The effects of saline irrigation and organic waste fertilization on mineral composition of leaves and fruits of tomato plants grown in a calcareous soil were studied during two culture seasons (spring­summer, autumn-winter). Sewage sludge and almond by-product were added along with three levels of salinity provided by NaCI. The results showed that sewage sludge affected N and P content. The major effect of saline irrigation was to increase Na concentration. In general, greater concentrations in leaves of N, P, K, Ca, Mg, Fe, Mn, Cu and Zn were found in the second season. Yield was greater in the first season. Treatments containing sewage sludge produced more number of fruits.

Introduction

Arid and semi-arid areas are deficient in fresh water of suitable quality for agricultural irriga­tion. In these regions substantial quantities of saline water are often available from agricultural drainage, food processing waste water and also ground water (Dinar et a!., 1986). It may be possible to use these resources in agricultural fields. However, saline soil and water contain excessive amounts of soluble salts for practical and normal production of most agricultural crops (Rhoades and Loveday, 1990). Excess salts in the root zone change the nutritional require­ments of plants in both the type and rate of fertilizers used (Cerda et a!., 1988). The in­fluence of plant nutrition is particularly relevant respect fruit (development and its mineral con­tent), because of the possible effects on its alimentary quality produced by deficiencies and toxicities of the different clements (L6pez-An­dreu et a!., 1987).

Waste water treatment originates an important amount of sewage sludge which is spread over fields used for agriculture because of its enorm-

ous input of organic matter (Diez, 1986). In the mediterranean coast, epicarp-mescarp of al­monds is sometimes used as fertilizer for agricul­tural purposes. Its N and P content is similar to organic fertilizers, but there is much more K (Gomez et a!., 1989).

Interactions between salinity and mineral nutrition are well studied (Bernstein et a!., 1974) but these have been less studied in soils amended with organic wastes. It has been known for several years that saline treatment of tomato plants improves fruit quality (Mizrahi and Pas­ternak, 1985), but this is accompanied by re­duced yields (Adams, 1986).

The purpose of the present work is to study the effect of using sewage sludge and almond by-product as fertilizers under saline conditions on leaves and fruits of tomato.

Methods

Nine treatments combining saline irrigation levels and organic waste fertilization were estab­lished. For each one, 26 tomato plants (cv.

334 Gomez et al.

Muchamiel) were cultivated in spring-summer: april to august (first season) and other 26 in autumn-winter: september to january (second season) , in the same 13 pots containing 15 kg of calcareous soil supplemented with 25 g of MgS0 4

(7% weight of Mg) and 100 g of superphosphate (18% weight of P). Ten tomato plants were selected to study their production in each treat­ment using an aleatory selection.

Saline levels were S0=0.5-l.OdS m- 1 (cur­rent water), Sl = 2.5 dS m - 1 and S2 = 4.5 dS m - 1 • NaCl was added to maintain the required electrical conductivity at 20oc. Plants were sup­plied with 0.5 liter of water per pot, six times a week with these saline solutions. Treatments were called BSO, BSl and BS2 (B: no organic fertilizer added); ASO, ASl and AS2 (A: soil amended with 0.5 kg pot- 1 of dry epicarp­mesoearp of almonds); JrSO, LSl and LS2 (L: soil amended with 0.5 kg pot -t of dry sewage sludge). Soil characteristics and chemical compo-

sition of the organic residues are shown in Table 1.

In each season, leaves samples were taken in the beginning of the fructification period (I corresponding with spring-summer season and II related to autumn-winter). Four plants from every treatment were analyzed in each sample. Leaves were washed and dried before analysis. Tomato fruits were harvested over the last two months in both seasons. Fruits were divided in groups, Fl for the first season, and F2 for the second. Number and weight of fruits were de­termined. Four tomato fruits (random sampling) from each treatment (in Fl and F2) were washed and cut into pieces. They were dried at 60°C for over a week until they were completely dry.

N was determined by a macro-Kjeldahl meth­od and elemental concentrations of P, K, Na, Ca, Mg, Fe, Mn, Cu, Zn and B were determined by ashing 0.5 g of dry matter for 6 hours at 500°C followed by digestion with hydrochloric acid. P

Table 1. Soil characteristics and elemental composition of organic residues

Characteristics

Clay Silt Sand Total carbonate Active lime Electrical cond. (1:5H 20) pH (1: H 20) Organic matter N (Kjeldahl) p

K Na Ca Mg Fe Mn Cu Zn B Cd Cr Hg Ni Pb

soil

(%) 15 (%) 58 (%) 27 (%) 55 (%) 2.1

0.39 (mS/cm-1)

7.6 (gkg-1) 13.6 (gkg-1) 1.31 (g kg I) 0.022 (gkg-1) 0.42 (g kg -I) 0.19 (gkg-1) 4.40 (gkg-1) 0.54 (mg kg- 1) 1.9 (mg kg- 1) 1.2 (mg kg- 1) 0.7 (mg kg- 1) 1.6 (mgkg- 1) (mg kg- 1) (mg kg- 1) (mg kg- 1) (mg kg- 1) (mgkg- 1)

Almond by-product

7.13

8.9 83.0 10.3 2.3 42.5 3.7

42.6 4.4

995 86

390 72 85

Soil: P (Burriei-Hernandop procedure); K, Na, Ca and Mg (ammonium acetate extraction); Fe, extraction). Organic residues: elements determined in acid digestion. -: not determined.

Sewage sludge

6.35

5.5 56.6 29.9 17.9 2.6 0.66

49.4 5.6

9700 115 272 905

79 4

12 1

18 2

Mn, Cu and Zn (DTPA

Tabl

e 2.

E

lem

enta

l co

mpo

siti

on o

f le

aves

(dr

y w

eigh

t).

Sta

tist

ical

AN

OV

A F

tes

t

Sam

ple

Tre

atm

ent

II

II

II

N(g

kg

-1 )

P

(gkg

-1 )

K

(gk

g-

1 )

BSO

BS

I

BS

2

ASU

A

S!

AS

2

LSO

LS

I

LS

2

S.(

A)

A.F

. S.

XA

.F.

S.(

SS

)

SS

.F.

S. >

<SS.

F.

2fl.

l

15.9

15.8

17

.3

2f1.

9 21

.6

26.8

24.3

25.4

22.6

3.

9 22

.2

3.7

25.9

3.

9

19.4

7.

7

22.9

4.

1 30

.2

2.3

27.8

2.

8

38.6

2.

2 35

.0

1.9

u =

u

" D

U

u =

3.

8 11

.6

u u

u ~

3.1

13.1

u ~

S.(

A):

Sal

init

y co

mpa

ring

A·t

reat

men

ts a

nd B

·tre

atm

ents

.

S.(

SS

): S

alin

ity

com

pari

ng L

-tre

atm

ents

and

8-t

reat

men

ts.

A.F

.: A

lmon

d by

-pro

duct

fer

tili

zati

on.

SS

.F.:

Sew

age

slud

ge f

erti

liza

tion

.

S. x

A.F

.: S

alin

ity

com

bine

d w

ith a

lmon

d by

-pro

duct

fer

tili

zati

on.

S. x

SS

.F.:

Sal

init

y co

mbi

ned

wit

h se

wag

e sl

udge

fer

tili

zati

on.

24.6

19

.0

21.2

23

.2

18.6

22

.7

22.6

22.9

24

.0

II

Na(g

kg

-1 )

0.8

4.2

12.6

0.

8 8.

0 13

.3

1.0

10.5

22.9

0.9

6.7

17.7

1.1

8.8

2f!.

9

0.6

7.3

12.1

II

Ca

(gkg

-1)

20.9

28

.4

32.4

22

.3

37.6

29

.1

30.3

33

.6

28.4

42.6

38

.7

50.1

44.9

47.4

53.0

42

.7

46.4

54

.3

II

Mg

(gk

g-1

)

6.3

6.4

8.6

6.3

8.5

7.6

7.6

6.0

5.8

9.1

7.3

7.0

8.2

8.4

8.9

6.6

8.3

9.5

Tabl

e 3.

E

lem

enta

l co

mpo

siti

on o

f fr

uits

(dr

y w

eigh

t).

Sta

tist

ical

AN

OV

A F

tes

t

Sam

ple

Tre

atm

ent

BSO

B

SI

BS

2

ASO

A

S!

AS

2

LS

O

LS

I

LS

2

S.(

A)

A.F

. S.

XA

.F.

S.(

SS

)

SS

.F.

S. x

SS

.F.

Fl

F2

N (

gkg

-1 )

17.2

12

.0

15.1

17.4

17.5

17.2

18.4

18.6

19

.5

16.1

14.1

12

.4

16.5

16.7

15

.9

Fl

F2

P(g

kg

-1 )

3.1

3.9

3.6

5.6

5.0

4.5

4.2

3.9

3.6

4.4

4.5

5.9

4.5

4.2

3.8

Fl

K(g

kg

-l

29.6

32.1

31

.7

37.3

36.1

35

.6

31.2

34

.7

32.1

S.(

A):

Sal

init

y co

mpa

ring

A-t

reat

men

ts a

nd

8-t

reat

men

ts.

S.(

SS

): S

alin

ity

com

pari

ng L

-tre

atm

cnts

and

8-t

reat

men

ts.

A.F

.: A

lmon

d by

-pro

duct

fer

tili

zati

on.

SS

.F.:

Sew

age

slud

ge f

erti

liza

tion

.

S.

x A

.F.:

Sal

init

y co

mbi

ned

wit

h al

mon

d by

-pro

duct

fer

tili

zati

on.

S.

x S

S.F

.: S

alin

ity

com

bine

d w

ith s

ewag

e sl

udge

fer

tili

zati

on.

F2

24.7

25.8

27

.8

25.3

20.8

17

.7

Fl

F2

Na(g

kg

-1

)

0.3

1.3

3.2

0.5

1.7

4.3

0.5

1.2

2.1

2.3

2.3

3.5

0.3

2.0

3.3

Fl

F2

Ca(g

kg

-1 )

3.4

2.8

2.9

2.5

2.6

2.6

3.1

2.7

2.1

2.7

1.7

1.4

1.9

1.1

1.1

Fl

F2

Mg

(gk

g-

1 )

1.0

1.1

1.2

1.4

1.3

1.4

1.2

1.2

1.3

1.3

1.2

1.5

1.5

1.3

1.3

Not

e: i

n F

2. s

alin

ity

(S.)

x o

rgan

ic f

erti

liza

tion

(A

. or

SS

.) a

re n

ot r

epre

sent

ed b

ecau

se t

here

wer

e no

t fr

uits

in

all

the

trea

tmen

ts.

II

Fe(

mg

kg

-1 )

!36

126

!54

113

119

168

181

130

109

Fl

182

196

217

258

269

236

230

2f!3

20

3

F2

Fe(m

gk

g-1

)

55

37

50

33

45

44

44

~

~

~

~

a M

• •

II

Mn

(mg

ks-1

)

37

58

52

45

56

59

28

26

42

Fl

76

81

112 67

86

110 22

32

60

F2

Mn

(mg

kg

-1)

10 6

II

Cu

(mg

kg

-1 )

12

14

II

18

16

12

12

II

10

Fl

18

15

2f1

25

21

27

2f1 21

22

F2

Cu

(mg

kg

-1

)

II

Zn

(mg

kg

-1 )

29

25

23

35

33

24

36

27

24

Fl

43

30

41

52

41

39

50

43

47

F2

Zn

(mg

kg

-1 )

n a w

26

TI ~

w

24

24

m

u 28

w

26

~

II

B(m

gk

g-

1 )

51

64

55

47

58

41

51

40

33

Fl

32

21

27

37

28

29

34

27

39

F2

B(m

gk

g-

1)

24

15

2f1

22

25 w

w

18

21

G

W

22

~

13

17

~ ~ 0 ~

;;::

~- ;;::

;:s !}

.... "' ., ~

~- i !:; ~

~

::t ~ ., 6· ;:s

<.;

l <

.;l

Ul

336 Gomez et al.

was analyzed by the method of Kitson and Mellon (1944), B with Azometine (Lachica, 1976) and the rest of the elements by atomic absorption spectroscopy.

Results

Elemental composition of leaves (dry weight basis) is showed for both periods of cultivation Table 2, as well as elemental composition and yield of fruits are in Tables 3 and 4. Statistical ANOVA F test was used to evaluate the effects of the treatments. Symbols in tables are ns- no significant difference, and the stars * * * , * * and * -significant differences between means at p = 0.001, 0.01 and 0.05 respectively.

The organic fertilization applied affected the mineral composition of leaves and fruits; N was specially affected by sewage sludge, increasing its concentration. P level in leaves were lower in L-treatment. The content of K was greater in A-treatments. In general, all the elements were affected by these wastes added to the soil, specially affected by sewage sludge addition.

The effects of saline irrigation levels mainly affected Na, increasing its concentration. How­ever, this was not the only effect because in leaves and fruits; P and B seemed to decrease with the increment of salinity.

The main differences noticed between the first and the second season of cultivation in leaves were the increment of the concentrations of some elements in the autumn-winter period. Macronutrients like N, P, K, Ca and Mg were greater as well as the micronutrients analyzed except B. For the fruits, the main difference

Table 4. Number and mean fresh weight of fruits

Sample Number Fresh weight (g) Treatment

F1 F2 F1 F2

BSO 16 0 38.2 0 BS1 27 6 62.3 21.4 BS2 34 0 63.4 0 ASO 12 0 95.9 0 AS1 22 10 87.2 23.1 AS2 23 2 64.3 6.8 LSO 43 2 42.1 18.5 LS1 65 10 54.3 45.2 LS2 66 2 43.4 40.7

between the two periods of cultivation was produced in the reduction of N, K, Ca and Cu concentration.

Number and fresh weight of fruits were notori­ous lower in autumn-winter than in spring-sum­mer. Treatments like BSO, BS2 and ASO were not able to produce fruits. The major number of fruits was obtained in treatments with sewage sludge added to the soil. Where the fertilizer applied was almond by-product, the greatest fresh weight was found.

Discussion

The salinity caused by NaCl added to irrigation water mainly produced an increment of Na in leaves and fruits.

The high mineralization rate of sewage sludge (Verdu et a!., 1992) as well as the important amount of N in this waste (Keefer et a!., 1986) may be responsible for the N concentration in L-treatments.

Almond by-product is able to increase the potassium of the soil (Gomez et a!., 1989) and this fact could promote a certain increment of its concentration found in fruits of A-treatments.

The low plant growth rate observed during the second season compared with the plants of the first could produce the differences in the elemen­tal composition observed in these two periods of cultivation (Fontes and Wilcox, 1984). It is important to notice that the increase of some elements in leaves has no incidence on increasing mineral content in fruits.

Number and fresh weight of fruits are ac­corded with the results obtained by other authors (Shalhevet and Yaron, 1973) because of the moderately tolerant salinity of tomato plant; this could be the responsible for not reducing the yield in S1 and S2 treatments. The highest fresh weight produced in A-treatments may has rela­tion with the K concentration. These organic wastes bring forward some important nutrients likeN.

References

Adams P 1986 Effect of salinity and watering level on the calcium content of tomato fruit. Acta Hortic. 190 253-259. ,

Bernstein L, Francois L E and Clark R A 1974 Interactive effects of salinity and fertility on yields of grains and vegetables. Agron. J. 66, 412-421.

Cerda A and Martinez V 1988 Nitrogen fertilization under saline conditions in tomato and cucumber plants. J. Hortic. Sci. 63, 451-458.

Diez T 1986 Agricultural use of sewage sludge in densely populated areas demonstrated at the example of the city of Munich. Mittcilgn. Dtsch. Bodcnkundl. Gesellsh. 49, 234-238.

Dinar A, Letey J and Vaux H J 1986 Optimal ratios of saline and nonsaline irrigation waters for crop production. Soil Sci. Soc. Am. J. 50, 440-443.

Fontes PC Rand Wilcox G E 1984 Growth and phosphorus accumulation in tomato cultivars. J. Plant Nutr. 7, 1651-1669.

Gomez I, Gomez B and Mataix J 1989 Evaluacion mediante un sistema EUF de la fertilidad pot:isica de un suelo compostado con pie! de almendra. Agrochimica 32, 458-467.

Keefer R F, Singh R N and Horvath D J 1986 Chemical composition of vegetables grown on an agricultural soil amended with sewage sludges. J. Environ. Qual. 15, 146-152.

Tomato fruits under salinity-waste fertilization 337

Kitson R E and Mellon M G 1944 Colorimetric determina­tion of P as molybdovanado phosphoric acid. Ind. Eng. Chern. Anal. Ed. 16, 379-383.

Lachica M 1976 Estudio sobre Ia determinacion de Boro en plantas con Azometina-H. In Proc. 4'" International Col­loquium on the control of Plant Nutrition. pp 53-61. Ed. Rijksuniversiteit, Gent, Belgium.

Lopez-Andreu F J, Esteban R M, Lopez G J and Collado J G 1987 Incidence of nutrition in acidity and mineral content of tomato fruits. Agrochimica 31, 27-32.

Mizrahi Y and Pasternak D 1985 Effect of salinity on quality of various agricultural crops. Plant and Soil 89, 301-307.

Rhoades J D and Loveday J 1990 Salinity in irrigated agriculture. In Irrigation of Agricultural Crops-Agronomy Monograph no 30. pp 1089-1142. Ed. ASA-CSSA-SSSA, Madison, USA.

Shalhevet J and Yaron B 1973 Effect of soil and water salinity on tomato growth. Plant and Soil 39, 285-292.

Verdu I, Gomez I, Burlo F and Mataix J 1992 lncidencia del fosforo en dos suelos calizos. Extraccion mediante EUF. Suelo y Planta 2, 151-161.

F

Response to form, rate and manner of application of fertilizers

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 341-349, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-114

Ammonia volatilization from compound nitrogen-sulfur fertilizers

0. OENEMA and G.L. VELTHOF NMI, Department of Soil Science and Plant Nutrition, Wageningen Agricultural University, P.O. Box 8005, 6700 EC Wageningen, The Netherlands

Key words: ammonia volatilization, ammonium sulfate, N-S fertilizer, soil pH, urea, urea hydrolysis

Abstract

Co-granulated urea and ammonium sulfate (AS) offer the potential for supplying both nitrogen (N) and sulfur (S) in a form readily available for plant uptake. Possible effects of a partial substitution of urea by AS on urea hydrolysis and ammonia (NH3 ) volatilization were examined in laboratory experiments. Losses of NH3 were measured from surface-applied urea, Ureas-5S (80% urea+ 20% AS), Ureas-lOS (60% urea+ 40% AS), and AS at rates equivalent to SO, 100 and 200 kg N ha -I to two dissimilar soil types, using vented closed flux chambers. Soil moisture potential ranged from -6.3 to -630 kPa and simulated rainfall from 0 to 10 mm. In the slightly acidic sandy soil, the percentage hydrolyzed urea was lower from Ureas-5S than from urea during the first 88 hrs after application, whereas no differences were observed in the lime-rich loamy sand, that had a very low urease activity. In the sandy soil, NH3

losses increased in the order AS< Ureas-lOS < Ureas-5S <urea, whereas this order was reversed in the lime-rich loamy sand. Increasing the N application rate increased the proportion of the fertilizer lost, but not the order of the fertilizers. Rainfall and increasing the soil moisture content significantly decreased NH3 losses, but did not alter the differences between fertilizers. In the sandy soil, losses were on average 35% less from Ureas-5S than from urea per unit N applied. This was attributed to the reduced rate of urea hydrolysis and the smaller rise in soil pH in the Ureas-5S relative to the urea treatment. The lower NH3 losses may lead to an increased efficiency of N from Ureas-5S relative to urea.

Introduction

Ammonia (NH 3 ) volatilization is an important loss mechanism of nitrogen (N) from surface­applied urea fertilizer. The NH3 loss generally increases as the rate of urea hydrolysis increases. Many studies have evaluated the factors that affect urea hydrolysis in soil and much effort has been put into developing and testing urease inhibitors (Bock and Kissel, 1988). Sulfate (SO~-) containing salts, particularly thiosulfates, have been shown to inhibit urease activity (Goos, 1985). This is not without relevance, the more so because sulfur (S) is an essential ele­ment for plant growth. There is also a growing need for S fertilization in large areas to sustain

optimum plant growth (Kanwar and Mudahar, 1986), notwithstanding the increases in S emis­sion into the atmosphere and atmospheric S deposition on land during the last 100 years (Brimblecombe et a!., 1989). A number of different S containing compounds exists in the natural environment (Kelly, 1988) and in fertiliz­ers (Kanwar and Mudahar, 1986), but many of those compounds have to be degraded to sulfate (SO~-) by microbiological processes before S becomes available for plant uptake. The S from so~- containing fertilizers has been proven to be immediately available for plant uptake (Korenta­jer eta!., 1984). The modest totalS requirement of the crop, in conjunction with the similarities between S and N with regard to their functioning

342 Oenema and Velthof

Table 1. Composition of fertilizers

Fertilizer TotalS

(%)

Ammonium sulfate (AS) 24.1 Ureas-5S (20% AS+ 80% urea) 5.2 Ureas-lOS (40% AS+ 60% urea) 10.2 Urea 0.0 Urea+ (NH 4 ) 2S0 4 5.2 Urea+ K,S04 4.9 Urea+ CaS0 4 5.2

in the plant and behaviour in soil-plant systems, has led to the manufacturing and use of com­pound N-S fertilizers. We have tested the ef­ficiency of S from various compound N-S fertiliz­ers (Dijksterhuis and Ocnema, 1990). The pres­ent paper focuses on the effects of a partial substitution of urea by ammonium sulfate (AS) on urease activity and NH3 volatilization. We expected that partial substitution of urea by AS through co-granulation of urea and AS could reduce the potential NH3 volatilization from this compound N-S fertilizer. This was verified in laboratory experiments. Urea hydrolysis and changes in soil pH around dissolved fertilizer granules, as main controlling factors on NH3

volatilization, were measured. The potential NH3 volatilization from AS, Ureas-5S and Ureas-10S (Table 1) was compared with that from urea.

Materials and methods

Two dissimilar soil types were used: a sandy topsoil (0-20 em) from a Typic Hapludoll, with 2.4% organic carbon, a pH of 5.5 and a high urease activity, and a lime-rich loamy sand taken from a Fluvaquent at a depth of 20-40 em, with 0.7% organic carbon, a pH of 7.7 and a low urease activity.

The rate of urea hydrolysis was estimated in duplicate in 500-g soil samples, supplied with no or with 92 mg N from urea containing fertilizers, following a two week incubation in glass jars at a temperature of 1SOC and a soil moisture tension of -63 kPa. Possible inhibitory effects of SO~­containing salts on urea hydrolysis were ex­amined by mixing powdered urea with powdered (NH 4 ) 2S04 , K2S04 and CaS0 4 in an urea N: S

TotalN N/S Ratio

21.1 0.88 40.9 7.9 35.6 3.5 46.2 40.9 7.9 34.0 7.0 36.1 7.0

ratio similar to that in Ureas-5S (Table 1). The application rate was equivalent to 184 mg urea N kg - 1 of sandy soil. The increase in NH; concen­tration relative to the control treatment was taken as measure of the apparent urea hydrol­ysis. Concentrations of NH; and NO; were measured by standard auto-analyzer techniques (US methods No 795-86T and 824-87T, respec­tively) after extraction of 100 g soil with 250 mL 1M KCl containing 5 mg L - 1 phenyl mercury acetate to terminate urease activity.

Changes in soil pH around dissolved fertilizer granules were monitored daily for one week after fertilizer application. The sandy soil, with a mean base neutralizing capacity of 37 mmolc kg - 1 per unit of pH, was packed to a uniform bulk density of 1.43 g em - 3 • The initial moisture content was 0.29 cm3 em - 3 of soil. Median sized granules of urea and Ureas-5S were applied at equal total N rates. The median granule size of urea and Ureas-5S was 3.1 mm whereas Ureas­lOS had a median granule size of 2.8 mm. It was assumed that contents of total N and urea were independent of granule size. Soil cores were taken with decapitated syringes (ID 0.85 em, height 5 em) at various distances from the loca­tion of the dissolved granule. Soil compaction in the soil cores was minimal ( <5% ), possibly due to the relatively high bulk density of the sandy soil. Cores were sectioned into 0.3-cm or 0.5-cm slices which were suspended in 3 and 5 ml demineralized water, respectively. The pH of the suspension was measured immediately with a micro glass electrode. All samplings and mea­surements were carried out in duplicate.

Potential NH3 volatilization was measured in vented closed flux chambers with a height of 15 em and a diameter of 24.2 em. The stationary chambers overlaid 8 kg of bare soil (thickness of

soil layers 8 em) supplied with fertilizer. An NH 3

trap, consisting of a 9-cm diameter Petri dish with 10 mL 0.5 M HCl, was placed at a height of 10 em inside the chamber. The driving force for NH3 volatilization was the NH3 partial pressure at the soil surface divided by a diffusive path length of about 10 em. The HCl in the NH3 traps was replenished each day during the first week and thereafter twice a week. The NH; concen­tration was analyzed by standard auto-analyzer technique. The performance of the vented closed flux chamber was compared with a flow-through flux chamber. In the latter chamber, de-am­moniated air was blown through the chamber at a constant flow rate of 2 L per min. All air entering and leaving the chamber passed a NH3

trap containing 20 mL 0.02 M H 3P0 4 •

Effects of soil type, N application rate, soil moisture potential, simulated rainfall, and mix­tures of so~- containing salts with urea fertilizer on NH 3 volatilization were measured in four experiments using the vented closed chamber system. All experiments had a completely ran­domized block design with two replicates, and were carried out under laboratory conditions at a mean temperature of 21 ac. In the first experi­ment, the NH3 volatilization from urea, AS, Ureas-5S and Ureas-lOS on both soil types was compared at three application rates, equivalent to 50, 100 and 200kg N ha- 1 • Fertilizers were surface-applied. Soil moisture potential was -63 kPa. In a second experiment, the effect of varying moisture potential, viz. -6.3, -63 and -630 kPa (equivalent to pF 1.8, 2.8 and 3.8, respectively) of the sandy soil was investigated at an application rate of 100 kg N ha -I via urea and Ureas-5S. In the third experiment, surface appli­cations of urea and Ureas-5S were compared at a rate of 100 kg N ha -I on the sandy soil, with and without rainfall. Rainfall was simulated via sprinkling. There were 7 rainfall treatments, viz. 1, 3, 5 and 10 mm of water applied 1 h after fertilization, 1 and 3 mm applied 2.5 days after fertilization and 3 mm applied 1 h after fertiliza­tion combined with 2 mm after 2.5 days. Volatili­zation of NH3 from mixtures of SO~- containing salts and urea was measured in the fourth experi­ment. Powdered urea was mixed with powdered (NH 4 ) 2S04 , K2S0 4 and CaS04 in a N: S ratio similar to that in Ureas-5S. The reference fer-

Ammonia volatilization from N-S fertilizers 343

tilizers urea and Ureas-5S were also powdered. All fertilizers were surface-applied at a rate equivalent to 100 kg N ha -I to the sandy soil, which had a moisture potential of -63 kPa.

Statistical analysis

Nonlinear regression models ( Genstat 5, 1987) were used to describe the cumulative NH3 vol­atilization. The best fit generally gave the Gom­pertz curve: Y=c exp(-exp(-b(t-m))), where Y is cumulative amount of volatilized NH 3 in mg N m- 2 , t is time in h and c, b, and m are constants. Differences in the coefficients c (upper asymptote) as a measure for total NH3 losses, and b (slope parameter) as a measure for the rate of NH 3 volatilization were analyzed statistically using the calculated standard errors and Stu­dent's t-distribution.

Results and discussion

Apparent urea hydrolysis

The apparent rate of urea hydrolysis in the sandy soil with a high urease activity was initially slightly lower in the treatments with Ureas-5S and Ureas-10S than in those with urea (Table 2). With time, differences between fertilizers became smaller. Concentration of NO; -N in­creased slightly from 17.5 ± 0.3 at the start to 21.1 ± 0.4 mg kg -I after 233 h of incubation, suggesting that the rate of nitrification was low. Differences between control and fertilized treat­ments were ,-;Q.9 mg NO; -N kg - 1 • Effects of (NH4 ) 2S04 , K2S04 and CaS04 additions to urea on the apparent urea hydrolysis in the sandy soil were rather small (data not shown). Additions of (NH4 ) 2SO 4 significantly (p < 0.05) reduced urea hydrolysis during the first 30 h of incubation, whereas the effect of K2S04 and CaSO 4 was most pronounced on days 2 and 3. Other S containing compounds (e.g. thiourea and thiosulfate) have been shown to inhibit urease activity much stronger, but many of those compounds have other disadvantages, like in­compatibility and toxicity (Bock and Kissel, 1988; Goos, 1985; Malhi and Nyborg, 1979).

The rate of urea hydrolysis was much lower in

344 Oenema and Velthof

Table 2. Apparent hydrolysis of urea from straight urea fertilizer, Ureas-5S and Ureas-10S applied at a rate of 184 mg urea N kg_, to a sandy soil and loamy sand in an incubation experiment at 15°C

Soil type Treatment Urea hydrolyzed as a percentage of applied*

19 h 45 h 64 h 88 h 160h 233 h

Sandy soil Urea 2" llb 25b 44b 80" n.d. ** Ureas-5S 1" 6" 14"' 34" 81" n.d. Ureas-10S 1" n.d. 17" 28" 79" n.d.

Loamy sand Urea 1" 2" 3" 4' 11" 20' Ureas-5S <1' 1" 2" 4' 13" 23" Ureas-10S <1" 1' 2' 5' 13" 23a

* For each column values are significantly different at p < 0.05 when followed by a different letter. * * Not determined.

the lime-rich loamy sand than in the sandy soil (Table 2). After 233 h only 20, 23 and 23% of the urea from urea, Ureas-5S and Ureas-lOS, respectively, was hydrolyzed in the lime-rich loamy sand, which was taken from below the plough layer. Generally, subsoils have a low urease activity because of their low organic carbon content and the absence of crop residues (Dick, 1984). Differences between fertilizers were small and not statistically significant in the lime-rich loamy sand (Table 2).

Changes in soil pH around fertilizer granules

Soil pH was increased around dissolved granules following urea and Ureas-5S application (Fig. 1 ). In the surface layer (0-0.3 em), soil pH in-

(a)

9.5

8.6

7.6

8.6

5.5

18

12 ~

'-"' ""' eq

<) 2 3

Distance (em)

r c.

creased from a mean of 5.5 to a maximum mean of 8.8 in treatments with urea on day 2 and to 8.7 in treatments with Ureas-5S on day 3 (data not shown). Similar rapid increases in soil pH underneath dissolved urea granules were ob­served by Black et al. (1987). The maximum soil pH values were confined to a hemisphere of soil with a radius of 1.5 em or less. Notable increases in soil pH were measured up to a distance of 5 em from the centre of the dissolved granule. The standard deviation (SD) of the pH measure­ments was ,:;0.1 units for the high-pH hemi­sphere under the dissolved granule, and for soil at >3.0 em from the dissolved granule. In other situations, the SD frequently exceeded 0.3 units, probably as a consequence of a slight difference in the distance of urea diffusion between the

(b)

0.75 ~ .= ~

0.50 g ~ ~

o.2s E

18

12 ~ \.~

>$' eq

0 <) 2 3 5

Distance (em)

Fig. 1. (a) Soil pH at various distances and depths from a dissolved Ureas-5S granule, 4 days after application to the sandy soil. (b) Soil pH of the urea microsite minus soil pH in the Ureas-5S microsite, 4 days after application to the sandy soil.

replicates. The pH-affected area was enlarged when evaporation was restricted by covering the soil. It seems likely that a dry soil surface layer limits the diffusion of dissolved urea into the soil when evaporation is high. From day 3 onwards, the increase in pH was higher for the subsurface layer than for the surface layer (Fig. 1). The rapid drop in pH of the surface layer was probably caused by NH3 volatilization (Ferguson eta!., 1984).

On days 2 to 4, the average pH rise was 0.15 units higher (p < 0.05) in the high-pH hemi­sphere underneath urea granules than under­neath Ureas-5S granules (Fig. 1). This may be attributed to the difference between urea and Ureas-5S in the amount of urea per granule and in urea hydrolysis. To account for the difference in total N content, the ratio between Ureas-5S granule weight and urea granule weight was 1.13, whereas the ratio of the total amount of urea in Ureas-5S granules and urea granules was 0.88 (Table 1 ). Hence, more alkalinity was produced around urea than around Ureas-5S granules. Moreover, Ureas-5S itself is slightly

(a)

Urea

'0 .!!! a. c.

"' z 0 ~ Ureas·SS

"' :c z Ureas-10S

AS

96 192 288 384 480

Time after fertilizer application (h)

Ammonia volatilization from N-S fertilizers 345

acidic. In a 1: 5 (wt/wt) ratio of fertilizer and water, the pH was 8.05 for urea, 5.02 for Ureas-5S, 5.10 for Ureas-lOS and 5.18 for AS.

Ammonia volatilization

Due to the larger driving force, rates of NH3

volatilization were slightly higher in the flow­through chambers than in the vented closed chambers, but the general NH3 volatilization pattern was similar (data not shown). However, the SD was more than twice as large for the flow-through chamber system than for the closed chamber system, possibly because of absorption of NH3 in condensation water in the tubes, and small changes in flow rate in the course of time. As our main interest was to detect possible differences between fertilizers, we preferred the closed chamber system because it was easy to use and sensitive to small differences between fer­tilizers.

Soil type had a strong effect on NH3 volatiliza­tion from broadcast N-S fertilizers (Fig. 2). In the sandy soil, cumulative NH3 volatilization

10 (b)

'0 .!!! a. c. 6

"' Ureas-1DS z 0 ~ C/) C/)

.Q 4

"' :c z

Time after fertilizer application (h)

Fig. 2. Cumulative NH3 volatilization from AS and urea-based fertilizers, applied at a rate equivalent to 100 kg N per ha to (a) a slightly acidic sandy topsoil with a high urease activity and (b) to a lime-rich loamy sand with a low urease activity. Standard deviation shown by bar at 499 h after fertilizer application.

346 Oenema and Velthof

increased in the order AS< Ureas-lOS < Ureas­SS <urea (p <0.01). The onset of NH3 volatili­zation was earlier for urea than for Ureas, probably because of the earlier mentioned differ­ence in the rate of urea hydrolysis. Increasing the N application rate increased the proportion of fertilizer N lost via volatilization, but did not alter the order of the fertilizers. At application rates equivalent to 50, 100 and 200 kg N ha -I a total of 2. 9, 7.5 and 10.4%, respectively, was lost from urea, 2.3, 4.4 and 8.1% from Ureas-SS and 1.7, 2.8 and 5.3% from Ureas-lOS. Comparison of these losses with the results of measurements using a variety of methods (Bouwmeester et a!., 1985; McGarry et a!., 1987 Reynolds and Wolf, 1987; Whitehead and Raistrick, 1990) suggests that volatilization in the present system was rather low. It is well established that static enclosures with limited air movement do not adequately simulate field conditions and, there­fore, will not adequately estimate actual losses in the field. On the other hand, static enclosures may adequately identify differences between fertilizers for a wide range of simulated con­ditions.

In the lime-rich loamy sand, cumulative NH3

volatilization increased in the order urea < Ureas-SS < Ureas-10s <AS (p < 0.01). Losses were highest for AS and this is the apparent reason why NH 3 volatilization was higher for Ureas than for urea. The low losses from urea must be attributed to the low urease activity and the high losses from AS to the high native soil pH. It should be noted that cultivated topsoils with such a low urease activity are not common and that a slow NH3 volatilization from urea

(Fig. 2b) is not typical. A marked difference between AS and urea in their response to differ­ences in soil type was also observed by Whitehead and Raistrick (1990). They found higher losses from AS than from urea in lime­rich soils.

Soil moisture potential had a marked effect on total NH3 loss (Table 3). Lowering the moisture potential of the sandy soil from -6.3 to -63 and -630 kPa increased the proportion lost from urea from 2.9 to 3.9 and 6.1 %, respectively, and from Ureas-SS from 1.4 to 2.7 and 3.9%, respec­tively. The lower losses at high soil moisture content suggest that the NH; concentration and pH were lower in the surface layer of the wet soil than in the surface layer of the dry soil, possibly because of the faster diffusive transport in wet relative to dry soil. Similar results have been obtained by Prasad (1976) and McGarry et a!. (1987), but others found higher losses at higher initial soil moisture content (Bouwmeester et a!., 1985; Reynolds and Wolf, 1987). Differences in temperature, soil drying due to differences in air humidity, and methods employed may have contributed to these contradictory results (Bouw­meester eta!., 1985; Reynolds and Wolf, 1987). The higher losses from the dryer soil in our study also suggest that urea hydrolysis was not reduced at a soil moisture potential of -630 kPa. Reynolds and Wolf (1987) observed that urea hydrolysis could occur at a soil moisture po­tential was as low as -1500 kPa, provided that relative humidity was high. The effect of soil moisture content was similar for both fertilizers; NH3 losses were much lower from Ureas than from urea in both wet and dry soils (Table 3).

Table 3. Effects of soil moisture potential on NH3 volatilization from broadcast urea and Ureas-5S. Cumulative NH 3 loss and the rate of NH3 volatilization were estimated by the upper asymptote (c) and the rate parameter (b), respectively, of the Gompertz curve. The application rate was equivalent to 100 kg N per ha of sandy soil

Fertilizer Moisture NH3 loss* Rate* potential (kPa) (%) (h -1)

Urea -6.3 2.78' 0.043b -63 3.67b 0.049'

-630 5.27' 0.046'b

Ureas-SS -6.3 1.38' 0.028' -63 2.56' 0.032'

-630 3.57b 0.020"

*Treatment means within a column followed by different letters are significantly different at p < 0.01. * * Correlation coefficient.

r2**

0.99 0.98 0.95

0.96 0.96 0.98

The rate of NH3 volatilization (parameter b) was significantly lower in Ureas-5S treatments than urea treatments, possibly as a result of the slower urea hydrolysis in Ureas-5S treatments.

It is well established that rainfall reduces NH3

volatilization of surface-applied urea, although light rain may also stimulate volatilization (Bouwmcester et al., 1985). Results of experi­ment 3 indicate that even a light rainfall of 1 mm, immediately after fertilizer application or after 2.5 days, significantly reduced NH3 losses from both urea and ureas-5S (Table 4). A heavy initial rainfall of 10 mm reduced NH3 losses by about a factor 10. Delaying the simulated rainfall from 1 h to 2.3 days after fertilizer application reduced losses from Ureas-5S much stronger than from urea. This was so because a significant fraction (> 1.5%) of the N from urea had already volatilized after 2.5 days, whereas Jess than 0.1% of the N from Ureas-5S was lost at that time. The lower initial losses from Ureas-5S than from urea also follow from the significantly lower value for the slope parameter b (Table 4), and must be attributed to the slow hydrolysis of urea from Ureas-5S. Total losses in experiment 3 were on average 32% lower in Ureas-5S treat­ments than in urea treatments, when losses from

Ammonia volatilization from N-S fertilizers 347

all treatments were expressed as a percentage of the urea treatment without simulated rainfall.

Results of experiment 4 showed a rapid re­sponse of NH3 volatilization to application of powdered fertilizers (Table 5). Losses were lower from mixtures of urea and (NH4 ) 2S04 ,

K2S0 4 and CaS0 4 than from straight urea, per unit N applied. The lower losses were probably the combined result of a slight reduction in urea hydrolysis and a smaller rise in soil pH. The rise in soil pH following application of mixtures of urea and so;- salts was lower because of CaC0 3 precipitation, induced by the addition of K2S0 4 and CaS0 4 (Fenn and Hossner, 1985). The lower alkalinity production due to the par­tial substitution of urea by AS will have also contributed to the smaller rise in soil pH in the treatments with Ureas-5S and urea+ (NH4 ) 2 SO 4 • We have no satisfactory explanation for the significantly lower NH3 Joss from urea + (NH4 ) 2S04 than from Ureas-5S (Table 5).

Conclusions

Partial substitution of urea by AS more than proportionally reduced the potential for NH3

Table 4. Effects of simulated rainfall on NH 3 volatilization from broadcast urea and Ureas-5S. Cumulative NH 3 loss and the rate of NH3 volatilization were estimated by the upper asymptote (c) and the rate parameter (b), respectively, of the Gompertz curve. The application rate was equivalent to 100 kg N per ha of sandy soil

Rainfall treatment (mm) Fertilizer NH3 loss* Rate* (%) W')

1h 2.5 days

0 0 Urea 3.67' 0.049' 0 0 Ureas-5S 2.56d 0.032'

0 Urea 3.11' 0.047' 0 Ureas-5S 2.06' 0.028'

3 () Urea 1.56' 0.042b 3 0 Ureas-5S 1.85' 0.034'" 5 0 Urea 0.84j 0.040b 5 0 Ureas-5S 0.841 0.028d

10 0 Urea 0.22 1 0.033'" 10 0 Ureas-5S 0.52' 0.027' 0 Urea 3.34" 0.045' 0 Ureas-5S 1.91 f 0.026" 0 3 Urea 3.06' 0.046b 0 3 Ureas-5S 1.42" 0.026" 2 3 Urea 2.01' 0.037' 2 3 Ureas-5S l.l1' 0.028'

*Treatment means within a column followed by different letters are significantly different at p < 0.01. '* Correlation coefficient.

rz**

0.98 0.96 0.99 0.93 0.99 0.96 0.77 0.98 0.67 0.77 0.96 0.95 0.91 0.98 0.96 0.98

348 Oenema and Velthof

Table 5. Effects of so:· salt addition to urea on NH3 volatilization. Cumulative NH 3 loss and the rate of NH3 volatilization were estimated by the upper asymptote (c) and the rate parameter (b), respectively, of the Gompcrtz curve. The application rate was equivalent to 100 kg N per ha of sandy soil. All fertilizer mixtures were applied as powders

Fertilizer NH 3 loss* Rate* r2**

(%) (h-I)

Urea 5.91" 1.12' 0.98 Ureas-5S 5.15b 0.79b 0.97 Urea+ (NH 4 ) 2S0 4 4.65" 0.85b 0.83 Urea+K 2S0 4 4.22d 0.79b 0.91 Urea+ CaS0 4 4.53' 0.78b 0.87

*Treatment means within a column followed by different letters are significantly different at p < 0.01. * * Correlation coefficient.

volatilization in the slightly acidic sandy soil. Increasing the AS : urea ratio decreased NH3

losses per unit N; losses were significantly lower from Ureas-lOS than from Ureas-5S. Total losses were on average 35% lower in Ureas-5S treat­ments than in urea treatments, when results of experiments 1-4 were combined, whereas only 20% urea was substituted by AS, to give a ration of urea N to total N of 0.88. A lower rate of urea hydrolysis and a smaller increase in soil pH around dissolved Ureas-5S granules relative to that around urea granules will have been respon­sible for the reduced NH3 loss. The largest difference between urea and Ureas-5S was re­corded in treatments with a simulated rainfall 2.5 days after fertilizer application. By contrast, losses were higher from AS and Ureas than from urea in the lime-rich soil with a high soil pH.

The lower NH3 losses from Ureas-5S will increase the amount of N available for plant uptake in acidic and slightly acidic soils and thereby possibly affect the efficiency of N from Ureas-5S relative to urea. Such effects should be taken into account when examining the response of crop yield to S application. For example, it is possible that the positive effect of Ureas-5S application to grassland in Ireland (Murphy, 1988) and to maize in Brasil (Boas, 1990) is due to a combination of S application and the in­creased efficiency of the urea fertilizer.

Acknowledgements

This research was supported by Hydro Agri. Mr D Koole kindly supplied the fertilizers. We

would like to acknowledge the valuable com­ments of two anonymous reviewers.

References

Black A S, Sherlock R R and Smith N P 1987 Effect of urea granule size on ammonia volatilization from surface-ap­plied urea. Fert. Res. 11, 87-96.

Boas R LV 1990 Alternatives para aumento da recuperacao do nitrogcnio da ureia pelo milho. MSc thesis, University of Sao Paulo, Brasil. 87 p.

Bock B R and Kissel D E 1988 Ammonia Volatilization from Urea Fertilizers. Bulletin Y-206. NFERC, TVA, Muscle Shoals, Alabama, USA. 189 p.

Bouwmeester R J B, Vlek P L G and Stumpe J M 1985 Effect of environmental factors on ammonia volatilization from a urea-fertilized soil. Soil Sci. Soc. Am. J. 49, 376-381.

Brimblecombe P, Hammer C, Rohde H, Ryaboshapko A and Boutron C F 1989 Human influence on the sulphur cycle. In Evolution of the Global Biogeochemical Sulphur Cycle. Scope 39. Eds. P Brimblecombe and A Lein. pp 77-124. John Wiley & Sons, New York.

Dick W A 1984 Influence of long-term tillage and crop rotation combinations on soil enzyme activities. Soil Sci. Soc. Am. J. 48, 569-574.

Dijksterhuis G H and Oenema 0 1990 Studies on the effectiveness of various sulfur fertilizers under controlled conditions. Fert. Res. 22, 147-159.

Fenn L B and Hossner L R 1985 Ammonia volatilization from ammonium or ammonium forming nitrogen fertiliz­ers. Adv. Soil Sci. 1, 124-169.

Ferguson R B, Kissel D E, Koelliker J K and Basel W 1984 Ammonia volatilization from surface applied urea: Effect of hydrogen ion buffering capacity. Soil Sci. Soc. Am. J. 48, 578-582.

Genstat 5 Committee 1987 Genstat 5 Reference Manual. Glarendon Press, Oxford. 749 p.

Goos R J 1985 Identification of ammonium thiosulfates as a nitrification and urease inhibitor. Soil Sci. Soc. Am. J. 49, 232-235.

Kanwar J S and Mudahar M S 1986 Fertilizer Sulfur and

Food Production. Martinus Nijhoff/Junk Publishers, Dor­drecht, The Netherlands. 247 p.

Kelly D P 1988 Oxidation of sulphur compounds. In The Nitrogen and Sulphur Cycles. Eds. J A Cole and S J Ferguson. pp 65-98. Cambridge University Press, Cam­bridge, UK.

Korentajer L, Byrnes B H and Hellums D T 1984 Leaching losses and plant recovery from various sulfur fertilizers. Soil Sci. Soc. Am. J. 52, 672-677.

Malhi S S and Nyborg M 1979 Rate of hydrolysis of urea as influenced by thiourea and pellet size. Plant and Soil 51, 177-186.

McGarry S J. O'Toole P 0 and Morgan M A 1987 Effects of soil temperature and moisture content on ammonia vol-

Ammonia volatilization from N-S fertilizers 349

atilization from urea-treated pasture and tillage soils. lr. J. Agric. Res. 26, 173-182.

Murphy M D 1988 An evaluation of Urea-S as a fertilizer for grassland. In Proc 12th General Meeting European Grass­land Federation. pp 343-347. Dublin, Ireland.

Prasad M 1976 Gaseous loss of ammonia from sulfur-coated urea, ammonium sulfate, and urea applied to calcareous soil (pH 7.3). Soil Sci. Soc. Am. J. 40, 130-134.

Reynolds C M and Wolf DC 1987 Effect of soil moisture and air relative humidity on ammonia volatilization from sur­face-applied urea. Soil Sci. 143, 144-152.

Whitehead DC and Raistrick N 1990 Ammonia volatilization from five nitrogen compounds used as fertilizers following surface application to soils. J. Soil Sci. 41, 387-394.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 351-354, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-058

Fertilizer recommendation scheme for phosphorus based on nutrient cycling in permanent pastures in the Basque Country, Northern Spain

A. G. SINCLAIR1 , M. RODRIGUEZ, M. OYANARTE and G. BESGA SIMA, Depto. de Agricultura, Gobierno Vasco, 48016 De rio, Vizcaya, Spain; 1 Present address: AgResearch, Invermay Agricultural Centre, Private Bag 50034, Mosgiel, New Zealand

Key words: dairy farms, fertilizer recommendations, maintenance, nutrient cycle, pasture, phosphorus

Abstract

A method for deriving fertilizer P requirements for maintaining ncar-maximum production in permanent rye grass/ clover pastures is proposed. The method is based on estimation of P transfers involved in P cycling in pastoral systems. Feasibility of the method was assessed by application to nine dairy farms in the Basque Country, Northern Spain. Results indicated that the proposed procedure could provide satisfactory estimates of amounts of P required to maintain high levels of pasture production. It was concluded that nutrient cycles are likely to become the best basis for the practical management of soil fertility in permanent pastures.

Introduction

Phosphate (P) fertilizer is generally considered to be necessary for achieving high levels of production in improved pastures in the Basque Country. In newly established rye grass I white clover pastures, large responses to P fertilizer have been observed, with the size of these responses being closely related to soil tests (Rodriguez, 1990). Subsequent to establishment, P fertilizer application is continued in order to maintain production at a high level (Rodriguez and Domingo, 1987) but amounts required are not well defined. Soil tests have only limited value for calculating the amount of P fertilizer which is required in repeated applications (e.g. annually) for the long-term maintenance of de­sired levels of pasture production. These mainte­nance P fertilizer requirements are more appro­priately calculated from a knowledge of the losses and gains of P in the pastoral system (Cornforth and Sinclair, 1982). This approach has been successfully employed to provide a P fertilizer recommendation scheme for pastoral

agriculture in New Zealand (Cornforth and Sin­clair, 1982, 1984). Pastoral systems in the Basque Country differ markedly from those in New Zealand in that (a) a large proportion of the pasture grown is cut and removed for indoor feeding rather than being directly grazed, (b) large quantities of supplementary animal feed (and consequently plant nutrients) are imported into the system, and (c) large amounts of excreta from housed animals are available for recycling to the pasture. The size of the transfers of P involved in this dynamic cycling system need to be known in order to deduce maintenance P fertilizer requirements. In this paper we propose and evaluate a P fertilizer recommendation scheme for Basque Country pastures based on estimation of components of the P cycle.

The P cycle

The P cycle proposed for calculating mainte­nance P fertilizer requirements is illustrated in Figure 1. The cycle is for a pastoral system

352 Sinclair et al.

E

Fig. 1. Phosphorus cycle in dairy pastures. A= P uptake by plants. B = P returned to soil in uneaten herbage. C = P added in supplements. D = P ingested by cows. E = P re­moved in milk. F = P excreted by cows. I = P lost in non­recycled excreta. K = P recycled in excreta. 0 = P added in fertilizer. N =Total P applied to the soil. M = P lost in the soil.

maintained at equilibrium and all the P transfers, including P fertilizer input, are on a continuous (e.g. per annum) basis.

P is taken up from the soil by the pasture (A) and a small portion returns directly to the soil in unused herbage when it decays in the field (B). The remainder is eaten by stock either while grazing or indoors. P ingested by stock (D) also includes that in supplements brought into the farm (C). Significant amounts of P are removed from the farm in milk and othc,r animal products (E). The remainder of the P ingested is excreted (F). Some of the P excreted while grazing or while housed is effectively lost (I) through being deposited in stock-camps, under shelter-trees, and in other non-productive areas or through inefficient recycling of excreta from housed ani­mals. The remainder (K) is returned to the productive area of pasture to be used once more for pasture growth. In the soil, P may be lost (M) through chemical changes which make it unavailable to plants and through leaching and erosion. To maintain the system in equilibrium, an input of P fertilizer (Q) is required such that:

Q=E+I+M-C

A scheme for quantifying the phosphorus cycle

needs to be assembled with specific information which has to come from the farms under consid­eration to give a sound P fertilizer recommenda­tion.

Estimating P cycle components

To assess the feasibility of calculating mainte­nance P fertilizer requirements from the P cycle, nine dairy farms in the Basque Country were selected and data collected from them to calcu­late P cycle components for individual farms. These farms were considered to be in an equilib­rium condition with pasture production near to the potential maximum. Calculations were made on a whole-farm basis but excluding any areas not used in the dairy operation. Subsequently, results were converted back to a per hectare basis. Methods used to calculate these P cycle components were as follows:

(i) P uptake in pasture (A). A is the product of annual pasture dry matter (DM) pro­duction and P concentration in the DM. Annual DM production was estimated from relevant local data. The average herbage P concentration was taken to be 0.46%, this value being derived from cut by cut analysis of high-producing pastures at 10 sites in the Basque Country (Rod­riguez and Ascacibar, 1988). Use of this value is supported by relationships derived for herbage P concentration in ryegrass/ clover pastures in New Zealand which predict 0.46% in productive pastures main­tained at 95% of their yield potential (Cornforth and Sinclair, 1982).

(ii) P recycled in uneaten herbage (B) was estimated as 10% of A, i.e. 90% pasture utilisation by grazing or harvesting was assumed.

(iii) P in supplements (C) was calculated from the total weights of individual supplements brought into the farm multiplied by con­centration factors which represented the mean analysis of several samples of each type of supplement.

(iv) P ingested by cows (D) was calculated on the assumption of 100% utilisation of

Fertilizer recommendation scheme for phosphorus 353

supplements and 90% utilisation of pas-ture.

(v) P removed in animal products (E) was calculated from total milk production and an average value of 1 g P per litre of milk (Agricultural Research Council, 1980). Other animal products in a diary farm were estimated to have negligible P con­tent.

(vi) Excreted P recycled to pasture (K). Inef­ficiencies of recycling occur from both grazing and housed animals (I). No mea­surements have been made in the Basque Country. We assumed 80% recycling ef­ficiency for animals while grazing, based on measurements in New Zealand (Corn­forth and Sinclair, 1982) and 90% from housed animals where farmers claimed all slurry was returned to pastures. A much lower efficiency ( 40%) was attributed to one farm (No 2, Table 1) where recycling was acknowledged to be very inefficient.

(vii) Maintenance fertilizer P input (Q). This is the quantity of fertilizer P necessary as an average annual application to maintain a long-term steady level of production. Only four farmers were able to provide suffi­cient information on fertilizer history to make reliable estimates of Q.

(viii) Loss of P in the soil. From studies of grassland systems in Europe and New Zealand, Karlovsky (1983) concluded that the efficiency of P utilisation, defined as the percentage of total P applied annually (N) which is taken up by pasture herbage annually in systems at equilibrium (i.e. 100 x A/N), ranged from 70 to 90%. These values produced accurate predic-

tions of maintenance P fertilizer require­ments for New Zealand pastures when allocated according to the P adsorbing properties of the soil: 90% for soils with low P adsorption, 80% for medium and 70% for highly adsorbing soils (Cornforth and Sinclair, 1982). We conducted a survey of P adsorbing properties of Basque soils and found all to be within the low to medium range (Sinclair, et a!., 1991). Therefore we have used a P efficiency value of 80% to estimate M (i.e. M =

0.25 A).

Results and discussion

Estimates of the annual P transfers (kg P ha -I) in the P cycle for each farm are given in Table 1. From these estimates the annual maintenance P fertilizer requirements (QEst) have been calcu­lated.

It is clear from data in Table 1 that P returned to the soil in excreta ano uneaten pasture is used with a relatively high efficiency. Phosphorus uptakes by pasture (A) are always much higher than P inputs in fertilizer (Q), which is only possible if the P inputs in excreta and plant residues are used efficiently. The data suggest that the different forms of P input are more or less equally effective and equal effectiveness will be assumed in the rest of this discussion.

Supplements (C) provide substantial amounts of P, thus reducing the amounts which would otherwise have to be applied as fertilizer. On average the amount of P added in supplements was only slightly less than the P lost in milk (E) and non-recycled excreta (I). The large amounts

Table 1. Summary of P cycle transfers (kg P ha- 1 year- 1 ) in nine Basque Country farms

Farm No 2 3 4 5 6 7 8 9

A 46.0 34.5 46.0 34.5 48.3 46.0 36.8 39.1 50.1 B 4.6 3.5 4.6 3.5 4.8 4.6 3.7 3.9 5.0 c 13.2 12.2 17.1 13.7 10.1 13.6 10.6 12.9 41.8 E 9.2 6.7 10.5 6.7 7.2 14.9 5.9 9.3 15.0

4.6 21.9 8.0 6.5 7.3 5.6 5.3 6.6 12.3 K 40.8 14.6 40.0 31.6 39.1 34.5 32.5 32.2 59.8 M ll.5 8.6 11.5 8.6 12.1 11.5 9.2 9.8 12.5 Q 17.9 25.1 24.0 0.0

OE-,t 12.1 25.0 12.9 8.1 16.5 18.4 9.8 12.8 0.0

354 Fertilizer recommendation scheme for phosphorus

of supplements used on farm 9 gave a very large P input which explains why good pasture pro­duction was being maintained without fertilizer P. Recycled excreta (K) was the largest P input on all farms except one, emphasising the value of efficient recycling.

Only four farms (1, 2, 6 and 9) were able to provide maintenance P fertilizer rates. Even these values may not be accurate maintenance rates because fertilizer policies have varied and were not well documented, and it is possible that soil P status had not yet reached a steady state but was still rising due to the relatively short history of fertilizer use. Comparison of the reported maintenance P fertilizer rates (Q) on these farms with rates estimated from the other P cycle components (QEsr) provides a partial test of the accuracy of the estimation procedure. QEsr was identical to Q on farms 2 and 9 and was about 6 kg P ha -I lower than Q on farms 1 and 6. On the other farms, where Q was not re­corded, QEsr was generally slightly lower than the P fertilizer rates which are commonly used (ASGAFAL, 1992; Dpto. Agricultura y Pesca, 1990). These differences are small in relation to the total amount of P being cycled through the system. In total, the results suggest that the proposed procedure could provide satisfactory estimates of amounts of fertilizer P necessary to maintain high levels of pasture production.

Improvements in the estimation of mainte­nance P fertilizer requirements will need better information on the individual P transfers in the P cycle. The greatest need is for more data on P losses in the soil. Also the efficiency of P recycling from both grazing and housed animals needs more detailed studies, and the assumption that P in excreta and plant residues is used with the same efficiency as P in fertilizer needs to be tested in view of the large amounts of P recycled in these forms.

In spite of some present limitations imposed by the shortage of existing data on some of the P transfers, we consider that nutrient cycles are likely to become the best basis for the practical management of soil fertility with permanent

pastures. Nutrient cycles do not only provide a logical, mechanistic basis for calculating mainte­nance fertilizer P requirements. They also draw attention to sources of major nutrient loss and so help to identify opportunities for improving nutrient efficiency, they provide a framework into which new information on nutrient pro­cesses can be incorporated, they identify im­portant areas for research and they contribute towards a better understanding of pasture nutri­tion.

References

Agricultural Research Council 1980 The nutrient require­ments of ruminant livestock. Commonwealth Agricultural Bureaux, Farnham Royal, Slough, England.

ASGAFAL 1992 Resultados de gestion tecnico econ6mica. Memorias de ASGAFAL. Ed. ASGAFAL, Vitoria-Gas­teis, Espana.

Cornforth I S and Sinclair A G 1982 Model for calculating maintenance phosphate requirements for grazed pastures. N.Z.J. Exp. Agric. 10, 53-61.

Cornforth I S and Sinclair A G 1984 Fertiliser and lime recommendations for pastures and crops in New Zealand. Ministry of Agriculture and Fisheries, Wellington, New Zealand.

Dpto. Agricultura y Pesca 1990 Anuario estadistico del sector agroalimentario. C.A.P.Y. 1989. Ed. Servicio Cen­tral de Publicaciones, Gobierno Vasco.

Karlovsky J 1983 Phosphorus utilization in grassland eco­systems. Proceedings of the XIV International Grassland Congress, Lexington, USA. pp 279-282.

Rodriguez M 1990 Desarrollo y evaluaci6n del sistema integrado de diagn6stico y recommendaci6n (ORIS) para Ia fertilizaci6n de las praderas permanentes. Tesis Doc­toralcs No 9. Universidad del Pais Vasco. Dpto. de Agricultura y Pesca. Ed. Servicio Central de Publicaciones, Gobierno Vasco.

Rodriguez M and Ascacibar M 1988 Poteneialidad productiva de las praderas naturales en el Pais Vasco. XXVIII Reunion Cientifica de Ia SEEP, Jaca, Huesca. pp 265-273.

Rodriguez M and Domingo M 1987 Fertilizacion nitro-fosfo­potasica en praderas naturales del Pais Vasco. Pastas XVII (1, 2) 203-218.

Sinclair A G, Rodriguez M and Oyanarte M 1991 Modelo de recomendacion de abono en base a los ciclos de nutrientes para las praderas de Ia Comunidad Autonoma del Pais Vasco. Informe Tecnico. Dpto. de Agricultura y Pesca. Ed. Servicio Central de Publicaciones, Gobierno Vasco.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 355-358, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-169

Response of gama medic (M edicago rugosa Desr.) grown on a calcaric cambisol to different rates of fertilizer phosphorus

M. ODETE TORRES, A. SERGIO V. COSTA and FATIMA CALOURO Laborat6rio Quimico Agricola Rebelo da Silva, Tapada de Ajuda, 1300 Lisboa, Portugal

Key words: calcaric cambisol, fertilizer phosphorus, gama medic, Medicago rugosa, mineral content of plants

Abstract

A pot experiment was conducted in order to study the response to different rates of fertilizer phosphorus (0.00, 0.75, 1.50 and 2.25 g P2 0 5 per pot) in gama medic (Medicago rugosa Desr.). A composite soil sample, taken at the first 20 em depth, in a calcaric cambisol from mountainous massif of Sic6, in the Portuguese region of Beira Litoral, was used. Significant differences among treatments (p ~ 0.05) were found. On average, the best results (80.3 g DM per pot) were obtained with the highest application rate. A linear response of dry matter production was obtained, in both cuts, suggesting that higher quantities of dry matter could be reached with higher application levels of phosphorus, which is related to the specific behaviour of this nutrient in this type of soils.

Significant simple correlations were found between dry matter production and plant magnesium and molybdenum concentrations, in both cuts, and phosphorus was significantly associated with dry matter only in the first cut.

Introduction

Although with great vanatwn, calcareous soils show generally medium to high fertility levels. Nevertheless, a great number of botanical species are negatively affected by their high calcium carbonate contents, especially because of the resulting phosphorus, and several mi­cronutrients, availability problems.

In highly P-fixing soils, as it is the one under study, Torres et al. (1988) reported that phos­phorus is the main limiting factor to crop yields.

In calcareous soils, phosphorus coming either from native soil sources, especially hydroxy­apatites, fluorapatites and their mixing forms (Olsen, 1953), or from applied phosphorus fer­tilizers, reacts with soil constituents resulting in fixation or retention processes (Sample et al., 1980).

According to Sample et al. (1980), soil phos­phorus retention is mainly due to ortophosphate

ion adsorption on calcium carbonate particles surface and to precipitation reactions, resulting in less soluble calcium phosphate compounds formation. The consequence is that phosphorus concentration in soil solution, and hence the available phosphorus, is decreased.

The present study was performed with the aim of getting some knowledge about the most adequate phosphorus levels to apply in this type of soils, in order to guarantee a proper plant mineral nutrition.

Materials and methods

A pot experiment was carried out at 'Horto de Qufmica Agricola Boaventura de Azevedo', in Lisboa, from 18 November, 1987 to 15 April, 1988.

A composite soil sample, taken at the first 20 em depth in a calcaric cambisol, was used. Its

356 Torres et al.

analytical characteristics, shown in Table 1, were obtained through the analytical methodology used at 'Laborat6rio Qufmico Agricola Rebelo de Silva' (Cottenie et al., 1982; Dias et al., 1980; Mehlich, 1953; Silva ct al., 1975).

Gama medic (Medicago rugosa Desr.) cv. Paragosa was used as experimental plant.

Pots with double wall, of white polyethilene, chemically inert (Kickrauckmann type) were used, each one with 9.5 kg of soil, air dried and screened through a plastic frame screen with 1 em mesh.

The pot experiment has been designed in randomized complete blocks with four replica­tions and four experimental rates of applied phosphorus (P0 - 0.00, P1-0.75, P2-1.50 and P3-

2.25 g of P20 5 per pot, corresponding to 0.00, 0.33, 0.66 and 0.98 g P) and a control. Pots of each replicate were grouped together and treat­ments were randomized within each replicate.

Potassium, magnesium and molybdenum were uniformly added to each pot (0.8 g of K, as KCl; 0.30 g of Mg, as MgS04 7H 20 and 0.17 mg of Mo, as Na2Mo4 .2H20) except to the control.

Magnesium and molybdenum were applied because of the reported results by Torres et al.

Table 1. Calcaric cambisol mean characteristics

Characteristics

Sand (%) Silt (%) Clay (%) Total carbonate (%) Active carbonate (%) Organic carbon - C (%) Available P-P20 5 (mg kg-!) Available K-K,O (mg kg - 1)

pH (KCl) pH (H,O) Exchangeable Ca (cmol( +)kg-') Exchangeable Mg (cmol( + )kg- 1 )

Exchangeable K (cmol( +)kg - 1 )

Exchangeable Na (cmol( + )kg- 1 )

Exchangeable Bases (cmol( +)kg 1)

Exchangeable H (cmol( + )kg- 1 )

Cation exchange capacity (cmol( + )kg- 1 )

Base saturation (%) Extractable Fe (mgkg- 1)

Extractable Mn (mg kg- 1)

Extractable Zn (mg kg-') Extractable Cu (mgkg-') Extractable B (mg kg- 1)

Extractable Mo (mg kg-')

(1988) suggesting the soil incapacity for provid­ing a proper plant nutrition with these elements.

Plant seeding was performed with 44 pre­germinated seeds, previously inoculated with the appropriate rhizobium strains (Rhizobium meliloti CC 169 and CC 2151). After seeding the stand of each pot was thinned to 20 plants.

During the trial, deionized water was added to soil surface in order to maintain soil moisture at 80% of field capacity.

After cutting, the plants were washed with running and deionized water, oven-dried for 24 hours at 70 oc and weighted.

Plant analysis was performed according to Ribas et al. (1988).

Results and discussion

Dry matter production

The lack of phosphorus application results, in the first cut, in plants with height less than established for cutting. So, dry matter product­ion was considered as zero.

No significant differences were found (p >

Chemical methods

16.0 25.2 58.8 60.5 Scheibler 10.3 Drouincau-Galet

1.2 Tinsley <11 Egner-Riehm 324 Egner-Riehm

7.41 Potentiometer 7.99 Potentiometer

15.38 Mehlich 1.38 Mehlich 0.31 Mehlich 0.09 Mehlich

17.16 Mehlich 0.00 Mehlich

17.36 Mehlich 100.00 Mehlich 568 Lakanen and Ervio 132 Lakanen and Ervio

0.90 Lakanen and Ervio 0.09 Lakanen and Ervio 0.57 Boiling water during 5 min. 0.03 Tiocyanate

Response of gama medic to fertilizer phosphorus 357

Table 2. Dry matter production (Duncan test, a = 0.05)

Experimental First cut Second cut Total treatments

(g/pot)

Control 0.00 d 1.40 d 1.40 d Po O.OOd 1.58 d 1.58 d pl 5.08 c 35.00 c 40.08 c P, 11.40 b 49.25 b 60.65 d p3 20.08 a 60.75 a 80.83 a sm (±) 0.662 2.225 2.035

0.05) between experimental treatment P0 and the control (Table 2), which is in accord with Torres eta!. (1988), who used a calcareous soil from the same Portuguese region.

Linear increasing dry matter production was obtained with increasing phosphorus application rates, in both cuts (Table 3, Fig. 1), suggesting that higher quantities of dry matter would be reached, with higher application levels of phos­phorus, which is related to the specific behaviour of this nutrient in this type of soils (Sample et a!., 1980). These results confirm, also, that phosphorus is the main limiting factor to dry matter production, which has been reported by Torres et a!. (1988).

Plant mineral composition

Tissue concentrations of phosphorus, potassium, magnesium and molybdenum, as well as the corresponding mean uptake values, are shown in Table 4.

The results show low phosphorus concentra­tions, especially in the second cut, even in the experimental treatment corresponding with the highest phosphorus application rate (P3 ), which is in accordance with the dry matter composition (mainly stems, with lower phosphorus contents, and a few leaves).

Concerning the molybdenum concentrations,

Table 3. Dry matter production response to increasing phos­phorus application rates

Cut

1 2 Total

Y=0.154+8.30X Y = 11.872 + 24.26 X Y = 7.082 + 34.34 X

R'

0.975 ** 0.867 * 0.971 **

Y- Dry matter production (g/pot); X- P,O, g/pot; R2 -

Coefficient of determination; *- p,:; 0.05; **- p,:; 0.01.

90,---------------------------------,

~ .e: 9 :2 0

30

20

10

0 0 0.5 1 1.5 2.5

P205 (g I pot)

! * ~st cut + 2nd cut x Total

Fig. 1. Dry matter response to increasing phosphorus appli­cation rates.

the obtained results show a remarkable differ­ence between the control and the P0 treatment which is related to the low extractable soil nutrient content (Table 1) in the control. The high nutrient level found in plants from the P0

treatment is probably a concentration effect due to low dry matter production (as a result of the lack of phosphorus application), and is related with higher available molybdenum in the soil, resulting from the nutrient application. This is confirmed by the nutrient concentration in the other experimental treatments with higher dry matter production as a result of dilution effect in plants mineral concentrations.

Correlation between dry matter production and mineral composition of plants

Simple correlations were established between dry matter production and plant concentrations on phosphorus, potassium, magnesium and molybdenum (Table 5), in order to measure the association of dry matter production and plant mineral composition.

In the first cut significant (p ~ 0.05) positive correlations were found between dry matter production and plant phosphorus and mag­nesium concentrations and negative correlations with molybdenum concentrations.

In the second cut no significant (p > 0.05) correlation was found between plant phosphorus concentration and dry matter production. Nega­tive correlations were found with magnesium

358 Response of gama medic to fertilizer phosphorus

Table 4. Mean concentrations anrl uptake of nutrients by gama medic

Cut Experimental Concentration Uptake treatment

p K Mg Mo p K Mg Mo (mgkg- 1)

(%) (mg/pot)

Control Po PI 0.17 2.81 0.23 0.309 9.9 142.7 11.7 1.57 E _,

P, 0.17 2.74 0.26 0.148 19.4 312.4 29.6 1.69 E _,

P, 0.18 2.76 0.26 0.156 36.1 554.2 52.2 3.13E- 3

2 Control 1 0.11 1.76 0.28 0.219 1.5 24.6 3.9 3.10 E _,

Po 0.10 1.78 0.31 4.040 1.6 28.1 4.9 6.38 E- 3

PI 0.10 1.84 0.16 1.057 35.0 644.0 56.0 3.70 E- 2

P, 0.11 1.76 0.17 0.860 54.2 867.0 83.7 4.24 E _,

P, 0.12 1.90 0.18 0.512 72.3 1145.0 108.4 3.08 E _,

1 Chemical analysis was performed using a composite sample resulting from the four replications of dry matter production.

Table 5. Simple correlations between dry matter production and phosphorus, potassium, magnesium and molybdenum plant concentrations

Cut

2

n'

12 20

Phosphorus

0.793*** 0.357NS

NS- p > 0.05; *- p,; 0.05; *** p,; 0.001.

(p ,-; 0.001) and molybdenum (p ,-; 0.05), which may be related to both high nutrient levels and low dry matter production in the treatments P0 ,

P1 and the control, showing a higher influence on the association of the variables. No significant correlations were found (p > 0.05) in both cuts between dry matter production and plant potas­sium concentration.

Conclusions

The obtained results confirm that phosphorus is the main limiting factor to dry matter production and suggest the application of high rates of phosphorus as a way to solve plant nutritional problems in this type of soil. Nevertheless, those results must be tested in field trials.

Acknowledgement

The authors are grateful to 'LQARS' staff who carried out analytical work and text processing.

Potassium

-0.249NS 0.374 NS

References

Magnesium

0.596 * -0.896 ***

Molybdenum

-0.604 * -0.480 *

Cottenie A 1982 Chemical Analysis of Plants and Soils. Laboratory of Analytical and Agrochemistry State Uni­versity, Ghani.

Dias J C S 1980 Guia Pnitico de Fertiliza~ao. Servi~o de Analise de Terra e de Analise Foliar DGER, LQARS, Lisboa. 100 p.

Mehlich A 1953 Rapid determination of cation and anion exchange properties and pH of soils. ADAC 36, 445-457.

Olsen S R 1953 Inorganic phosphorus in alkaline and calcareous soils. In Soil and Fertilizer Phosphorus in Crop Nutrition. Eds. W H Pierre and A G Norman. pp 89-122. Academic Press Inc., New York, USA.

Ribas M C 1988 Metodos de analise de material vegetal e terras. Scc~ao de Nutri~ao das culturas. MAPA, INIA, LQARS. Lisboa. 58 p.

Sample E C 1980 Reactions of phosphate fertilizers in soils. In The role of Phosphorus in Agriculture. Eds. F E Khasawneh, E C Sample and E J Kamprath. pp 263-304. Soil Sci. Soc. Am. Madison, Wisconsin, USA.

Silva A A 1975 Metodos de analise de solos, plantas e aguas. Pedologia, 10, 100 p.

Torres M 0, Costa A S V and Calouro F 1988 Prospec~ao dos factores de natureza nutricionallimitantes do desenvol­vimento de algumas especies forrageiras em solos de­rivados de calc:irio da Serra do Sic6. Pastagens e For­ragens, SPPF 9, 143-153.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 359-365, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-189

Comparison of row and broadcast N application on N efficiency and yield of potatoes

G. HOFMAN\ P. VERSTEGEN 1 , P. DEMYTTENAERE 1 , M. VAN MEIRVENNE 1 ,

P. DELANOTE 2 and G. AMPE 2

1Department of Soil Management and Soil Care, Faculty of Agriculture and Applied Biological Sciences, University of Gent, Coupure 653, B-9000 Gent, Belgium; 2School for Agriculture and Horticulture, Boeschepestraat 44, B-8970 Poperinge, Belgium; 3Centre for Agricultural and Horticultural Research, leperseweg 87, B-8800 Roeselare, Belgium

Key words: application techniques, N fertilization, N efficiency, potatoes, soil mineral N

Abstract

A small rooting depth, a poor root distribution and an inefficient fertilizer application by broadcasting N fertilizers on potatoes result in a restricted N efficiency. Therefore, an application of N fertilizers in the row has potential possibilities to enhance this N efficiency.

In 1990 and 1991, field experiments were set up with various N levels allowing to compare the effects of a row versus a broadcast N application on the nitrification rate, the mineral nitrogen evolution in the soil profile and crop growth and yield.

The nitrification rate was slower for the row application which minimizes the possible leaching losses in early growing period. The mineral N exploitation as well as crop yield varied between the two years indicating that the effect of row application depends on several external factors.

Introduction

Economical motives can be a mainspring for a more efficient use of fertilizers. However, the cost price of fertilizers compared to other pro­duction factors is rather low in developed coun­tries. Therefore, it is not surprising that in the eighties farmers in W-Europe didn't pay much attention to the efficient use and application of fertilizers. However, the increasing economical pressure on agriculture and the over-production for several crops the last years moved farmers interest from maximizing the production towards optimization with lower and more efficient use of production factors such as fertilizers (Nychas, 1989). A second factor, i.e. the protection of the environment, enhances this trend. N losses can be minimized by a better adaptation of the N fertilization to the N needs of the crop during the vegetation period by a split application (De-

myttenaere et al., 1991; Lorenz et al., 1985; Slangcn et al., 1989). A better placement of the fertilizers, e.g. by row application of fertilizers, seems to be another possibility to diminish N losses, creating higher efficiencies (De Wit, 1953). Possible beneficial effects are: -a better availability of mineral N, resulting in

a faster and/ or higher N uptake and yield; -a reduction of N losses by NH3 volatilization

and/ or leaching. Although this technique was already utilized

several years ago (Prummel, 1957), a revival of this application method is noted since more attention is paid to the protection of the environ­ment. Especially the possibilities of N fertiliza­tion in the row of sugar beets was studied in Western-Europe (Jensen et al., 1983; Van­dergeten and Vanstallen, 1991; Van Meirvenne et al., 1991). Since a few years, this technique was tested on other crops. Referring to the possible

360 Hofman et al.

beneficial effects, positive results of a row appli­cation can be expected for crops with large distances between the rows and/ or with a re­stricted root distribution, and for crops which are earthed up soon after a N fertilization. ·The potato crop meets to a large extend these con­ditions. Other potential benefits are a delay of nitrification of NH 4 -N keeping fertilizers, reduc­ing possible N0 3-N leaching losses, and a reduc­tion of NH3 losses by incorporation of the N fertilizers (Clay et a!., 1990; Fenne and Kissel, 1976; Rachpal-Singh and Nye, 1988).

The aim of this paper is to discuss the mineral N evolution in the soil profile under potatoes after a row and broadcast application of various levels of N and to evaluate the effect on growth and yield.

Materials and methods

During two consecutive years (1990 and 1991), a field trial was carried out on a silt loam soil in Poperinge (N-W Belgium) in order to investigate the possibilities of row application of fertilizer N in potato growing. Besides the two application methods, row versus broadcast, the experimental field design consisted of different N doses (as NH4N0 3 with 27% N) in 3 replicates. The various N doses were chosen in function of the calculated optimum N fertilization which amounted to 225 kg ha - 1 in 1990 and 210 kg ha - 1

in 1991. These N advices were based on the mineral N balance as described by Hofman et a!. (1983). The broadcast application was spread on

Table 1. Some informative soil and fertilization characteristics

Soil texture

pHKCI Cultivar Planting date Harvest date Soil N0 3-N content (0-60 em) before planting (kg N ha 1)

Fertilization (3 replicates) -type -doses (kg N ha -I)

control broadcast row

the soil surface just before planting. The row application was given through the 'Agroband' (Horstine Farmery), allowing planting and fer­tilization at the same time. The row fertilization was incorporated at both sides of the row at about 5 em below the tubers and at 7-10 em from the planting line. General information about the experimental set up is given in Table 1.

During the growing season, soil and plant samples were collected regularly in order to get information on N dynamics in the soil profile and plant growth. For the evaluation of the mineral N evolution in the soil profile, soil samples were taken at different distances in and between the rows (Fig. 1). At each of the 8 locations, soil samples were taken in layers of 30 em down to 0.6 m. As the average distance between two plants was 45 em in the row and 75 em between two rows, the sampling design covered the aver­age space available for one potato plant. At each date, this procedure was repeated three times and the final sample was a pooled mixture.

The N0 3-N contents in the two layers and the NH4-N contents in the top 30 em were deter­mined with a continuous flow auto-analyzer system after extraction with a 1 M KCl solution.

Results and discussion

N dynamics

Ridging resulted in a concentration of the broad­castN fertilizer in the row. By contrast, row applica-

1990 1991

silt loam silt loam 5.2 5.4 Bintje Bintje 29 April 21 May 12 September 2 October

57 70

NH 4N03 27% NH4 N0 3 27%

0 0 100-225-325 110-210-310 100-175-225 110-175-210 325 310

\

...... -.1. ...

Effects of N fertilizer application techniques on potato growing 361

plant row

~~~

.. :J ... : .. :

,j, ,I,*** o: 10 25 37.5 ! 75 distance (em)

o sampling point

• plant position and sampling point

Fig. 1. Soil sampling locations.

5

tion of fertilizers gave the highest concentrations at about 10 em from the planting row (Fig. 2) corresponding with the location of the fertilizer.

In both years, significant higher NH4-N con­tents were found on the treatments with row application compared to broadcasting during the first months of the growing season, confirming the results obtained by Van Meirvenne et al. (1991) during a three year study on sugarbeets. Figure 3 illustrates these differences between both fertilization techniques 1! month after application. These clear differences in NH4-N concentrations can only be attributed to a net delay of the nitrification rate. NH3 losses from NH4N0 3 on soils with a pHKci < 5.5 are negli­gible (Demeyer, 1993). As the residual N03-N showed the same trend for both application techniques during the growing season, the signifi­cant differences in NH4-N contents in the plow layer can not be explained by variations in NH4-N uptake. An interpolation program 'SURFER' (Golden Software Inc.) was utilized to draw an area passing the data points. These data points were used for the interpolation of the rest of the area, covering the available space for one potato plant.

6

2 6

Fig. 2. Nitrate nitrogen contents (layer 0-30 em) in the available area of one plant after broadcasting (left) and row application (right) of 210kgNha-'-date: 17 June 1991. Sampling points are indicated.

362 Hofman et al.

6

"' "' ~ < ~

5 '-- 6

~ 7 8

Fig. 3. Ammonium nitrogen contents (layer 0-30 em) in the available area of a plant after broadcasting (left) and row application (right) of 210 kg N ha -I- date: 8 July 1991. Sampling points are indicated.

In all cases however, NH 4-N concentrations at harvest were low, indicating that nearly all ammonium was nitrified during the growing season or taken up by the crop. Anyway, the slower nitrification rate, corresponding with higher NH 4-N concentrations, has the advantage that possible leaching losses under wet condi­tions in the early growing period are diminished. This can be a real advantage for crops with a restricted rooting depth such as potatoes, grow­ing on soils with a low water holding capacity.

Yields

For both years, total tuber yields at harvest showed significant second degree polynomials in function of the N-application rate (Fig. 4 ).

In 1990, yields were rather small due to the extreme dry weather conditions (a precipitation of only 90 mm instead of the normal 232 mm from May till September). The fresh tuber yield (>35 mm) had a mean of about 10% higher for the row treatment compared to the treatment with broadcast application. For the same N dose, differences between the row and broadcast treat­ments rised till more than 30% for tubers >50 mm. However, these differences were not statistically significant as large vanat10ns occurred between replicates (Table 2).

In 1991, the yields were much larger than in 1990. The control showed significant smaller yields compared to the other treatments. For tubers >50 mm, the differences in yield between the 0 N and the optimum N fertilization were still more pronounced. There were no significant differences between comparable N fertilizations for the two application methods. In contrast with the results obtained in 1990, the effect of appli­cation method was limited in 1991 and didn't show any trend (Table 2). However, for tubers >50 mm, the optimum fertilization amount was reached at a lower N level when applied in the row as illustrated in Figure 4 (bottom). These results are in agreement with data mentioned by Van Erp and Dijksterhuis (1991).

The different behaviour is possibly due to various climatological conditions. Fertilizer placement may increase nutrient uptake and crop yield when crops are growing under con­ditions of limited nutrient supply. This was the case in the dry conditions of 1990. In these conditions, mass flow is limited. Diffusion on the other hand is in favour of placement because the nutrient concentration is the soil solution by placement is higher (Steenbjerg, 1957). At last, in dry conditions quite high amounts of N03 -N remain in the top of the ridges after a broadcast application and doesn't become available for the

Effects of N fertilizer application techniques on potato growing 363

35000,----------------,

... " .0 ;j

'"'

1990, >35

_.---a----~

QQ.Q..Q...O Broadcasting ~·.!.•~Row application

0 100 200 300 400

N-fertiiization (kg N ha-1 )

.--... 45000,----------------,

" .c:

"0 _, 30000

1991, > 35 mm

oooooBroadcasl ing '!...*.!.*-"Row appl ical ion

0 100 200 300 400

N-f ert i I izat ion (kg N ha-')

" ..c 20000

"' -"'

" ..c 20000

"" -"'

v ....... I 0000

"

1990, >50 mm

0 • ------

Q.QQ.Q.9 Broadcasting ~·.!.•_-Row application

0 100 200 300 400

N-fertiiizalion (kg N ha-1 )

1991, >50 mm

ooooo Broadcasting ~*.!.*-*Row application

0 100 200 300 400

N-fert i I izat ion (kg N ha-')

Fig. 4. Fresh tuber yield >35 mm (left) and >50 mm (right) at harvest of 1990 (top) and 1991 (bottom) as a function of nitrogen fertilization.

crop. This theory doesn't apply for the wetter vegetation period in 1991.

Soil N03 -N residues at harvest

Mineral N residues in the soil to a depth of 60 em at harvest are given in Table 3 for the various treatments.

In 1990, moisture shortage induced stress and stopped growth very early. This resulted in large N-residues on all treatments. In comparison with a broadcast treatment, the row application showed generally lower N-residues, corre­sponding with higher yields.

In 1991, theN residues were much lower than the preceding year, corresponding with higher yields. Contradictory to the results obtained in 1990, the highest mineral N residues were found on the treatments with row application, which is in agreement with somewhat lower yields com­pared to the broadcast treatments. Comparable

variations in residual mineral nitrogen are men­tioned by Van Erp and Dijkstra (1991) and Van Erp and Titulaer (1992).

Conclusions

The effect of fertilizer placement is not un­equivocal positive. Nevertheless, a correct row application of N fertilizers, avoiding salt damage, will at least result in the same yields as a broadcast application. Special conditions, like a limited nutrient supply by moisture stress, show positive effects of a row application. Although not explicitely studied, applications in the row have potential benefits on soils with high pH and free CaC0 3 , inducing NH3 losses, and on soils which are susceptible to N0 3-N losses. As Van Erp and Titulaer (1992) mentioned already, fertilizer placement can be considered as an insurance premium for a good crop production.

364 Hofman et al.

Table 2. Absolute and relative yields of potatoes (figures with the same superscript letter indicate no significant differences at p = 0,05)

Treatment Tubers >35 mm Tubers >50 mm

(kg ha - 1 ) (%yield*) (kgha- 1 ) (%yield*)

1990 Control 22605 ± 2237 84.6" 10175 ± 1710 85.5" Broadcast 100kg N ha- 1 25175 ± 2009 94.2"b 10475 ± 1064 88.1' 225 kgN ha- 1 26720 ± 2023 100.0"b 11895 ± 3153 100.0' 325 kg N ha-l 26270 ± 3170 98.3"b 10740 ± 2748 90.3' Row 100 kg N ha-l 27210 ± 758 101.8'b 14796 ± 3300 124.4' 175 kg N ha -I 27885 ± 1026 104.4b 12920 ± 2583 108.6' 225 kg N ha-l 29180 ± 3346 109.2b 14435 ± 1947 121.4' 325 kg N ha 1 29005 ± 3928 108.6b 15085 ± 3051 126.8'

1991

Control 20220 ± 2336 54.2" 6020 ± 1010 35.4' Broadcast 110 kg N ha-l 34765 ± 972 93.1 be 14645 ± 3058 86.0b' 210kgNha- 1 37330 ± 1757 100.0b' 17020 ± 617 100.0b'ct 310kgNha- 1 38695 ± 264 103.7' 18645 ± 1144 109.6'd Row 110kg N ha- 1 33585 ± 3791 90.0b 13795 ± 1791 81.0b' 175kgNha 1 36210 ± 2475 97.0hc 18995 ± 2405 111.6" 210 kg N ha-l 37760 ± 4375 101.2b' 18125 ± 3033 106.5'd 310kgNha- 1 35260 ± 2248 94.5b' 18155 ± 2721 106.7'd

*The advised optimum fertilization (broadcast), 225kg N ha 1 in 1990 and 210kgNha- 1 in 1991 respectively, is given as 100%.

Table 3. Soil N03 -N residues (kg ha- 1 ) at harvest

1990

Dose Broadcast Row

ON 75 lOON 152 194 175N 236 225 N 346 269 325N 386 344

Acknowledgements

Financial support by I.W.O.N.L. (Institute for Encouraging Scientific Research in Industry and Agriculture, Brussels) is gratefully acknowl­edged. We thank the firms A.V.R.-Machinery and Horstine-Farmery for placing at our disposal the Agro-band.

1991

Dose Broadcast Row

ON 45 110N 58 91 175N 117 210N 100 120 310N 144 203

References

Clay D E, Malzer G L and Anderson J L 1990 Ammonia volatization from urea as influenced by soil temperature, soil water content, and nitrification and hydrolysis in­hibitors. Soil Sci. Soc. Am. J. 54, 263-266.

Demyttenaere P, Hofman G, Vulsteke G, Van Meirvenne M and Van Ruymbeke M 1991 Minimizing N0 3-N leaching losses under field-grown vegetables. Pedologic 41, 105-117.

Effects of N fertilizer application techniques on potato growing 365

Demeyer P 1993 Ammoniakvervluchtiging uit de bodem na toediening van ureum en ammoniumhoudende meststof­fen. PhD-thesis, Faculteit van de Landbouwkundige en Toegepaste Biologische Wetenschappen, RUG, 235 p.

De Wit C T 1953 A physical theory on placement of fertilizers. Doctoral thesis, Wageningen Agricultural Uni­versity, 71 p.

Fenn L B and Kissel D E 1976 The influence of cation exchange capacity and depth of incorporation on ammonia volatilization from ammonium compounds applied to cal­careous soils. Soil Sci. Soc. Am. J. 40, 394-398.

Hofman G, Van Ruymbeke M, Ossemerct CandIde G 1981 Nouvelles tcndanccs dans la formulation des avis de fumure bases sur l'examen du profil. Revue de !'Agricul­ture 34, 917-937.

Jensen V, Marcussen C and Smed E 1983 Nitrogen for sugar beet in Denmark, research and its utilisation. In Symposium Nitrogen and Sugar Beets. pp 293-303. Institut International de Recherches Betteravieres, Brussels, Bel­gium.

Lorenz H, Schlaghecken H and Eng! 1985 Gezielte Stickstoffversorgung- das kulturbegleitende N mio -Soll­werte-System (KNS-system). Dtsch. Gartenbau 13, 646-648.

Nychas A E 1989 Environmental policy with regard to fertilizers in arable farming/field production of vegetables in the E.C. In Congres Meststoffen, Milieu en Akker­bouw. pp 7-20. Missel BV, Doetinchem, The Netherlands.

Prummel J 1957 Fertilizer placement experiments. Plant and Soil 8, 231-253.

Rachpal-Singh and Nye P H 1988 A model of ammonia volatilization from applied urea. IV. Effect of method of urea application. J. Soil Sci. 39, 9-14.

Slangen J, Titulaer H and Rijkens C 1989 Nitrogen fertilizer recommendation with the KNS-system for iceberg lettuce (Lactuca sativa var. capitata) in field cropping. VDLUFA­Schriftreihe 28, 251-261.

Steen bjerg FA 1957 A theory on the placement of fertilizers. K. Vet. Landbohoejsk. Arsskr., 1-30.

Vandergeten J-P and Vanstallen M 1991 Influence de !'appli­cation localisee de doses raisonnees d'azote sur le rende­ment et la qualite industrielle de la betterave sucriere. In 54th Winter Congress. pp 297-318. Institut International de Recherches Betteravieres, Brussels, Belgium.

Van Erp P J and Titulaer H H H 1992 Rijenbemesting in de akkerbouw met vollegrondsgroenteteelt. Meststoffen, pp 10-15.

Van Meirvenne M, Vanstallen M, Hofman G, Vandergeten J-P and Demyttenaere P 1991 Influence of row and broadcasted N application on the evolution of the mineral nitrogen under sugar beet. In 54th Winter Congress. pp 445-453. Institut International de Recherches Bettera­vieres, Brussels, Belgium.

M.A. C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition. 367-373, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-161

Phosphorus dynamics and mobility in the diffusion zone of band-applied phosphorus fertilizers

W. WERNER and B. STRASSER Institute for Agricultural Chemistry, University of Bonn, Meckenheimer Allee 176, D-53115 Bonn, Germany

Key words: adsorbed Fe/Al-P, diammoniumphosphate, P-adsorption-intensity, P-diffusion, subsur­face-band application, triplesuperphosphate

Abstract

Diammoniumphosphate (DAP) increases, Triplesuperphosphate (TSP) decreases the pH in the diffusion zone. Because of this, P from TSP is adsorbed more strongly. SoP migrates out of the DAP band at a substantially higher rate. In comparison to DAP the P delivery from TSP to the plant root by diffusion and by mass flow is impeded. As the final result, DAP causes an essentially higher P uptake.

Introduction

Subsurface band-applied phosphorus often re­sults in higher P uptake by the plant when applied as diammonium phosphate (DAP) than as triple superphosphate (TSP). In order to give causal explanation of this observation, we in­vestigated the dynamics and mobility of phos­phorus in the diffusion zones of DAP- resp. TSP-fertilizer bands and in the rhizosphere of plant roots growing in the direct vicinity of different fertilizer bands.

Material and methods

Lay-out of the experiments

All experiments were carried out with the same soil. Main characterisation of the soil: subsoil of a Parahraunerde derived from Loess, Mec­kenheim, silty loam, pH: 5.8, P content (mg kg - 1): 161.0 Ca-P and 177.0 Fe/AI-P (frac­tionation according to Kurmies, 1972) and 4.0 watersolublc P (1: 10, 2h).

Diffusion experiments

For the diffusion experiments the soil was sieved, passing a 0.2-mm-scrccn.

General lay-out (see Fig. 1) The P diffusion out of a DAP, rcsp. TSP band (experiment 1) and also the delivery from the band to the surface of a root growing in the diffusion area (experiment 2) was tested with a modified experimental design according to Kuchenbuch (1983). Onto a soil block ( 42 mm diameter and 18 mm high) containing the fertil­izer band as a thin layer of ground fertilizer granules (equivalent to 23 kg ha 1 P) on its upper side (soil block 1) a second soil block (diameter 42 mm and 18 mm high in the first experiment and 12 mm high in the second) was placed (soil block 2). Both were separated by a net ( 425 mesh) so that only dissolved P from soil block 1 was able to migrate into soil block 2. The upper side of block 2 was also covered by such a net. The whole was standing on a porous plate and a water tension 0.01 MPa was installed. In order to investigate the P dynamics in different distances from the band (diffusion experiment 1)

368 Werner and Strasser

in expperiment 1

this surface was sealed

in experiment 2 rape seedlings were

planted on this surface

surface of soil block 1

mesh

SOIL BLOCK 1

surface of soil block 2

425 mesh

SOIL BLOCK 2

-----------+-----porous plate

SCHEMATIC DESCRIPTION OF THE GENERAL LAY-OUT OF THE EXPERIMENTS

+---lO mm ->

ot •U bl.ook: 1

1-IIUD- la7en bf IOU bloolr: 1 aptalOm.m ~ (!rtlUur bt.Dd

bot.t.om ot .oU Wook 1

42$ ...

(oop .... Uoc ta.• aoU bloa.ka)

.......

THIN -SLICING OF SOIL BLOCK 1 IN EXPERIMENT 1 {see text)

-- le7n of .oU block 1: I \.o U 111.m from t.b11 ro.ot R.J"tau

(0 t..o 3 mm from the fa'1.i..11ur bu:t.d)

boU.Om of .aU tlloc.lr. 1

D•t. C.Z:$ lllwb

(Mp.,.._UA& t.l:t.e 10U bloclu)

... ..

THIN-SLICING SOIL BLOCK 1

IN EXPERIMENT 2 (see text)

Fig. 1. Schematic description of the general lay-out of the model diffusion experiments and the way of getting soil samples by thin-slicing the treated soil.

P dynamics in the diffusion area of P fertilizer bands 369

and in different distances from the root surface (diffusion experiment 2), the upper soil blocks were frozen in liquid nitrogen and then thin­sliced (1-mm-slices) with a freeze microtome.

Diffusion experiment 1 Because the P diffusion out of the fertilizer band and the very initial reactions between soil and dissolved fertilizer were of the main interest the surface of soil block 1 was sealed to prevent water evaporation. After 12 hours of reaction the block was cut from its bottom (surface touching soil block 2) to its upper side, parallel to the net covered surface, into 1-mm-layers (see Fig. 1, below left) and these slices were analysed separately. Because of the cutting technique 3 mm of the soil block (top of the block) could not be cut and got lost.

Diffusion experiment 2 Because in this experiment the P dynamics in the rhizosphere of roots growing in the diffusion zone were of the main interest, rape seedlings were grown for one week on top of soil block 1 in order to get the rhizosphere of a two-di­mensioned root surface (rhizoplane). The time of reaction between soil and fertilizer was 1 to 7 days. Based on the results of the first experiment the height of the soil block 1 was reduced to 12 mm. So the distance between root surface and fertilizer band was 12 mm. To distinguish be­tween pure diffusion and root-induced mass flow, one half of the experiment was carried out without plants. In experiment 2 the soil blocks were cut, beginning with the upper side towards the bottom (see Fig. 1, below right) and also here 3 mm (bottom of the block) got lost.

Pot experiments Experiments in Kick-Brauckmann-pots with Helianthus annus, Sorghum bicolor and Zea mays were carried out with the same soil, passed through a 20-mm-screen. The P fertilisation was made with DAP resp. TSP as a subsurface band each 5 em below and 5 em aside the seed, 0.34 g P per pot equivalent to 23 kg P ha -I. Further fertilisation was 1 g per pot N as (NH4 ) 2S04 as solution broadcasted and mixed with the soil in the TSP-treatments and 0.7 g N per pot as (NH4 ) 2SO 4 as solution broadcasted and mixed

with the soil and 0.3 g in the DAP-treatment, where 0.3 g N are already applied with the DAP in the band. Also 1 g K as K2S04 and 0.3 g Mg as MgS0 4 were applied.

The plants were grown for 40 days under outdoor-conditions resp. greenhouse-conditions in the case of rainfall; beginning of the experi­ment: 30.05.1991.

Chemical methods

Watersoluble P: ratio of soil: deionised water 1:10 (Pw1: 10), shaken for two hours, after centrifugation P determination in the clear supernantant (to estimate the P intensity).

P fractionation according to Kurmies (1972): extraction in a ratio of 1: 15 (soil: solution), eliminating Ca-ions with alcoholical KCl-solu­tion, extracting Fe/Al-P (adsorbed P) with 1M NaOH and Ca-P with 1M H 2S04 , after centrifu­gation P determination followed in the clear supernantant (to estimate the P quantity. P determination according to Murphy and Riley (1962).

Results and discussion

Release from the band and trespassing to the root surface

It is well known, that the reaction products of watersoluble P-fertilizers and the (noncalcare­ous) soil are only found in the fraction of adsorbed P (e.g. Werner, 1970). The intensity of this P adsorption is mainly dependent on the pH of the soil (e.g. Barrow, 1987), but also the P saturation of the adsorbing surfaces determines the intensity of adsorption (e.g. Bache and Wiliams, 1971).

We hypothesised that, in the diffusion zone of a P fertilizer band, P from TSP as an acid fertilizer is adsorbed more strongly than P from DAP as an alkaline one. Owing to this, we expected more P coming out of DAP, resulting in a higher P concentration in the diffusion area and a larger diffusion distance. As a result of the first diffusion experiment, it could have been shown very clearly that TSP lowers the pH in the diffusion zone whereas DAP raises it. With DAP

370 Werner and Strasser

mg 1:10 watersoluble P (PW1:10)/100g pH-Value 160,-----------------------~--------------~----,6,5

140 0 TSP

X DAP 120 6

100

80 5,5

60

40 5

20

1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 distance from the fertilizer band (mm)

Fig. 2. Waterso!uble P and pH in the diffusion area of band applied DAP resp. TSP after 12 hours.

the P concentration in the soil solution (P wl: 10) is enormously higher and the diffusion distance is greater compared to TSP (Fig. 2). We assumed that the initial pH determinates the intensity of the P adsorption.

Within one day in the experiment without plants (second diffusion experiment), P from

DAP advances via diffusion closer to the net covering the surface of the upper block than P from TSP. Moreover with plants, an enormously greater amount of P is delivered to the root by massflow, caused by the root induced flow of water. On the contrary with TSP the root activity docs not influence the P transportation (Fig. 3).

mg adsorbed P (Fe/ Al-P)/IOOg

80~==========~------------------~~

60

40

20

0 DAP no plants

-8- DAP with plants

X TSP no plants

--*- TSP with plants

~ LSD 5%

0 0

0-1 1-2 2-3 3-4 4-5 5-6 mm distance from the root surface

6-7 7-8

Fig. 3. Influence of the plant root on the P delivery from a DAP resp. TSP fertilizer band within one day.

P dynamics in the diffusion area of P fertilizer bands 371

mg 1:10 watersoluble P (Pw1:10)/100g 20~~------------------------------------------,20

0 DAP no plants -8- DAP with plants ·.X TSP no plants

--*- TSP with plants ~ LSD 5%

15 15

0,5 1 1,5 2 2,5 3 3,5 4 4,5 5 5-6 6-7 7-8

mm from the root surface

Fig. 4. Influence of the plant root on the P concentration in the diffusion area of DAP resp. TSP fertilizer bands after 7 days.

Depletion and replenishment of the rhizosphere

After one week the P concentration within the soil block without plants was nearly equilibrated by diffusion for both fertilizers at the same level. But in the DAP diffusion area with plants, the P concentration (P w1: 10) in the rhizosphere is lower and towards the fertilizer band higher (Fig. 4). This means that with DAP the maxi-

mum depletion and at the same time the replen­ishing of the depleted area are considerably higher.

Intensity of the P adsorption

The results obtained in this compact and re­stricted system as described above show that the two fertilizers do not differ much concerning the

mg 1:10 waterso1ub1e P/IOOg 20~r=============~--------------------~

15

10

5

0 DAP

+ TSP

linear regression

0

+++

0

0

• 0.89071

+

Pw1:10 • -6,67664 + 0,24809 mg Fe/ A1~P oo

oL_~ __ _l __ _L __ ~ __ ~ __ L_ __ L_~--~---l---L--~--~~

30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 mg adsorbed P (Fe/ A1-P)/100g

Fig. 5. Relation between adsorbed P and Pwl: 10 in the diffusion area of a DAP resp. TSP fertilizer band after 7 days.

372 Werner and Strasser

diffusion distance (and by this the soil volume provided with P) as one could suppose on the basis of the results shown in Figures 2 and 3. So, in previous research we found an identical diffu­sion distance for both fertilizers. However, with the same concentration of adsorbed P (quantity of P) higher concentrations of dissolved P (in­tensity of P) occured in the DAP diffusion area (Strasser and Werner, 1991). In fact the striking point is the adsorption power of P by the soil in the diffusion zone. The relations of adsorbed to dissolved P (Q/1-relations) proof the stronger adsorption of P from TSP in comparison with DAP. A same amount of adsorbed P always results in a higher P concentration of the soil solution for DAP compared to TSP (Fig. 5). Their common regression line divides the cloud of points exactly into the TSP-group below the line (the equilibrium of the reaction is shifted towards the adsorbed P) and the DAP-group above the regression line.

Also in the pot experiments (results with Helianthus annuus) the enormous difference concerning the quality of the adsorbed phos­phates remains and can be shown very clearly (Fig. 6). While the regression line for DAP

mg 1:10 watersoluble P (Pw1:10)/100g

indicates an overproportional increase of the dissolved P as described by Fritsch (1986) for a comparable soil, for TSP it is practically a straight line and it is shifted much more towards the adsorbed P.

P uptake by the plant

All the plants tested respond to subsurface band application of DAP resp. TSP in the same manner, only differing in the degree of their reaction, that is mainly dependent on their common root development. As a complex im­pact in combination with root accumulation and a positive physiological effect of ammonia (Stras­ser and Werner, 1992) the better mobility of Pin the diffusion area of the DAP band causes a great additional P uptake and by this a substan­tially more efficient fertilizer use (Fig. 7).

Acknowledgement

This research was supported by Deutsche For­schungsgemeinschaft, DFG.

12,---------------------------------------~2--------, § PW•2.403+(-0.207'FeAI-P)+(0,0045'FeAI-P)

DAP: r • 0,980"7" 10

8 D

+ + +

6 D

D

4

D 2L_ ____ _L ______ L_ ____ ~ ______ J_ ____ _J ______ _L ____ ~

45 50 55 60 65 70 75 80 mg adsorbed P (Fe/ AI-P)/100g

Fig. 6. Relation between adsorbed P and P w I : 10 in the diffusion area of a DAP resp. TSP fertilizer band under sunflowers after 40 days.

P dynamics m the diffusion area of P fertilizer bands 373

Zea mays

' ' ! '

l

Hellanthus annuus

Sorghum blco1or

' 100 80 60 40 20 0 20 40 60 80 100 P uptake per pot (DAP•100%)

DDAP DTSP

:~gbr:a!:~~~~ea~:t~:em~~ some plant species with DAP resp. TSP subsurface band application after 40 days (N compensation

References

Bache B W and Wiliams E G 1971 A phosphate sorption index for soils. J. Sci. Soc. 22, 289-301.

Barrow J 1987 Reactions with variable-charge soils. Martinus Nijhof Publishers, Dordrecht.

Fritsch F 1986 Charakterisierung der Mobilitiit von durch langjiih-rige Diingung mit verschiedenen Phosphatformen angereicherten Bodenphosphaten. Diss., Bonn.

Kuchenbuch R 1983 Die Bedeutung von Ionenaustausch­prozessen im wurzelnahen Boden fiir die Pftanzen­verfiigbarkeit von Kalium. Diss., Giittingen.

Kurmies B 1972 Zur Fraktionierung der Bodenphosphate. Die Phosphorsiiure 29, 118-151.

Murphy J and Riley J P 1962 A modified single solution

method for the determining of phosphate in natural waters. Anal. Chim. Acta 27, 31-36.

Strasser B und Werner W 1991 Einftul3 der Phosphatunter­ful3diingung auf die P-Dynamik in dcr Diffusionszone und Phosphataufnahme der Pftanze. Kongre13band, VDLUFA­Schriftenreihe 33, 152-157.

Strasser B and Werner W 1992 Inftuence of subsurface banded P on the P dynamics in the diffusion area, the root development and the P uptake by the plant. Fourth International IMPHOS conference, file of abstracts of posters. p 10.

Werner W 1970 Untersuchungen zur Pfianzcnverfiigbarkeit des durch langj iihrigc Phosphatdiingung angereicherten Bodcnphosphats. Z. Pfianzenern. Diing. Bodenk. 126, 135-150.

M.A. C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 375-380, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-078

Effects of phosphorus, magnesium and molybdenum application rates on resident and introduced meadow species in a dry land pasture in mountainous massif of Sic6, Portugal

M.O. TORRES 1 , F. CALOURO\ I. MAGALHAES 2 , J. SANTOS2 and J. GAMA2

1 Laborat6rio Quimico Agricola Rebelo da Silva, Tapada da Ajuda, 1300 Lisboa, Portugal; 2 DRABL, Av. Fernao de Magalhaes 465, 3000 Coimbra, Portugal

Key words: Calcaric Cambisol, cocksfoot, dry land pasture, gama medic, perennial ryegrass, phosphorus, strawberry clover, subterranean clover

Abstract

A dry land pasture was established in a representative Calcaric Cambisol of mountainous massif of Sic6, in the portuguese region of Beira Litoral. The aime of the study was to evaluate the effect of a range of phosphorus, magnesium and molybdenum application rates on the establishment of some meadow species. The meadow species used were perennial ryegrass (Latium perenne L.) cv. Victorian, cocksfoot (Dactylis glomerata L.) cv. Currie, strawberry clover (Trifolium fragiferum L.) cv. Palestine, subterra­nean clover (Trifolium brachycalycinum Katzn. & Morley) cv. Clare and gama medic (Medicago rugosa Desr.) cv. Paragosa. There was no response on magnesium or molybdenum fertilization. Phosphorus application was not successful concerning grasses percentage of sward, which may be due to the lack of nitrogen fertilization. On the contrary, resident species percentage of sward was significantly (p ~ 0.05) reduced by the better establishment conditions of legumes, allowed by increasing phosphorus application rates. Bare ground percentage was significantly (p ~ 0.05) decreased by phosphorus fertilization.

Introduction

Mountainous massif of Sic6, an extensive area in the portuguese region of Beira Litoral, is mainly composed by calcareous Jurassic formations. The soils show great limitations to agricultural and forestation activities, especially in hill-side soils, comprehending an extensive area of 50000 ha. Due to slope characteristics of these soils, and to the high annual rainfall of 1200-1300 mm (Car­valho and Sa, 1991), concentrated in a short period of time, important erosion processes occur, resulting in frequent unproductive cal­careous outcrops. Thus, the remaining soils are thin, and pastures should become an important way of soil protection in great part of the region (Salgueiro and Moura, 1985).

However, although of great regional impor-

tance due to home production of the traditional cheese, known as "Queijo do Rabac;al" (Car­valho and Sa, 1991), the use of these soils through pastures establishment, for sheep utiliza­tion, has been difficult.

Previous experimental field results suggest a good adaptation of some meadow species to hill-side management of dry land conditions. Yet, the reduced and heterogenous vegetation growth shows the existence of some limiting factors to their proper development in this type of soils (Salgueiro and Moura, 1985).

Pot experiments were conducted with the aim of determining the nutritional factors involved (Torres et a!., 1988) and the obtained results point to phosphorus as the main restricting factor in plants growth. A good response to molybdenum application was also found, as well

376 Torres et al.

as plants low magnesium contents, suggesting a soil undesirable Ca/Mg ratio to plants nutrition.

The aim of the present work was to study the effect of the application of these nutrients on the establishment of some meadow species that previous experimental work has shown being well adapted to local environmental conditions.

Materials and methods

The trial lasted for three years (since October, 1988 till October, 1991) and was installed in a representative Calcaric Cambisol from moun­tainous massif of Sic6, in a field that was not cultivated or fertilized for a long time.

As a part of a larger measurement pro­gramme, pasture sward botanical composition was monitored over this period of time.

Before trial establishment, a composite soil sample was taken, at the first 15 em depth. Soil testing results show a pH(H20) value of 7.99, medium level of organic matter (2.1%), low level of available phosphorus (11 mg kg - 1 P2 0 5 ,

Egner Riehm method) and high level of avail­able potassium (324 mg kg - 1 K 20, Egner Riehm method), as well as an extractable molybdenum value of 0.03 mg kg - 1 Mo (tiocyanate method) and a Ca/Mg ratio of 15.

The trial was a fractional ( 5 x 2 x 2) factorial arranged into randomized complete blocks with­out confounding. Fifteen experimental treat-

Table 1. Experimental treatments

Experimental treatments

1-P0 Mg 1 Mo 1

2-P0 Mg0 Mo 1

3-P0 Mg 1 Mo0

4-P, Mg 1 Mo 1

5-P1 Mg0 Mo, 6-P1 Mg 1 Mo0

7-P2 Mg 1 Mo 1

8-P2 Mg0 Mo, 9-P, Mg 1 Mo0

10-P3 Mg, Mo, 11-P, Mg0 Mo 1

12-P, Mg 1 Mo0

13-P, Mg 1 Mo 1

14-P, Mg0 Mo, 15-P, Mg 1 Mo0

Prates (kg ha-l P20 5 )

0 0 0

150 150 150 300 300 300 450 450 450 600 600 600

ments were assigned to plots with 12m2 area each with four replications. Table 1 shows ex­perimental combinations of the factors.

Phosphorus, magnesium and molybdenum were applied as triple superphosphate ( 42% P20 5 ), magnesium sulphate (16% MgO) and ammonium molybdate (54% Mo).

Pasture seeding was performed with a seed mixture composed by 5. 0 kg ha- 1 of perennial ryegrass (Lolium perenne L.), cv. Victorian, 5.0 kg ha - 1 of cocksfoot (Dactylis glomerata L.), cv. Currie, 3.0 kg ha-l of strawberry clover (Tri­folium fragiferum L.), cv. Palestine, 4.0 kg ha - 1

of subterranean clover (Trifolium brach­ycalycinum Katzn. & Morley), cv. Clare, and 4.0 kg ha - 1 of gama medic (Medicago rugosa Desr.), cv. Paragosa. Seeds were previously inoculated with adequate rizobium strains: CC 169 and CC 2151 of Rhizobium meliloti for gama medic and Na 14-8 of Rhizobium trifoli for clovers.

Regression analysis was performed within each botanical group and bare ground in order to estimate each group response function to phos­phorus application. For each botanical group and bare ground three regression models were fitted, one for each group of phosphorus experimental levels in the presence or absence of magnesium and I or molybdenum. The homogeneity of regression coefficients was tested using F-test (Steel and Torrie, 1980).

Mg rates Mo rates (kgha- 1 Mg0) (kg ha -I Mo)

32 0.270 0 0.270

32 0.000 32 0.270 0 0.270

32 0.000 32 0.270 0 0.270

32 0.000 32 0.270 0 0.270

32 0.000 32 0.270 0 0.270

32 0.000

Pasture sward botanical composition evaluation

The pasture sward botanical composition was evaluated through Levy point quadrat method (Levy and Madden, 1933). The used apparatus was a frame, mounted on legs pointed, to facili­tate pushing into the soil, and carrying a row of ten steel pins 5 em apart.

In each one of the experimental plots ten vegetation sampling unities spaced 0.7 m each were examined. The point quadrat frame was systematicly and alternately placed perpendicu­larly to each side of an axis previously defined along plot length.

When the pin was projected vertically down through the vegetation all hits were recorded. Thus, for every 100 points examined, more than 100 hits may be recorded according to the number of storys and species of vegetation present.

The hits corresponding to bare ground, intro­duced meadow species and the resident ones were recorded.

The contribution of each species to the sward was obtained by the number of hits of each species in relation to the total number of hits. The relative contribution is called 'percentage of sward' and obtained by the formula:

Number of times a species is hit x 100 Total times vegetation is hit

Results and discussion

The effect of increasing phosphorus application rates, in the presence or absence of magnesium and/ or molybdenum, on the establishment of each one of the introduced meadow species, was studied through the evaluation of their percen-

Effect of P application on meadow species 377

tagc of sward variation. Percentage of sward, refercd to resident species variation as well as percentage of bare ground, were also evaluated.

Establishment of the introduced botanical species

The establishment of each one of the introduced meadow species is related, not only with nutrient application effects, but also with species interac­tions within the sward, which we were not able to evaluate. The balance of species within plant community is determined by individual plants fitness to the limited resources of area (Harper, 1978) and depends on each species competitive­ness.

Ryegrass and cocksfoot Ryegrass and cocksfoot percentage of sward were not significantly affected by the applied nutrient levels. On average, both species repre­sent a small part of the sward: 1.1% and 7.9% for rye grass and cocksfoot, respectively, which is certainly due to the lack of nitrogen application.

Strawberry clover Strawberry clover percentage of sward variation, due to the effect of nutrient application, is described by regression models (1), (2) and (3) on Table 2.

No significant differences were introduced on the response to phosphorus fertilization due to magnesium or molybdenum application (F[7;4J =

0.461 ). Strawberry clover percentage of sward response to phosphorus application is described by regression (4). The increase of the relative contribution of the species was higher from 0 to 150 kg ha - 1 of applied phosphorus.

Maximum species percentage of sward (22%) was obtained with 600 kg ha - 1 of applied phos-

Table 2. Effect of increasing phosphorus application rates, in the presence or absence of magnesium and/or molybdenum, on the strawberry clover percentage of sward

Magnesium (kgha-' MgO)

32 0

32

Molybdenum (kg ha- 1 Mo)

0.00 0.27 0.27

Y = 0.339 + 0.190X- 6.28 E - 4X2 + 6.20 E- 7X3

Y = 1.398 + 0.088X- 8.43 E -sX 2

Y = 0.664 + 0.182X + 5.54 E-'x' + 5.25 E-'x' Y = 0.856 + 0.152X- 4.17 E- 4X2 + 3.75 E- 7X3

Y=% of sward; X=kgha-' P2 0 5 ; *-p~0.05; **-p~O.Ol.

R'

0.991 * (1) 0.968** (2) 0.999** (3) 0.906** (4)

378 Torres et al.

phorus (P2 0 5 ), suggesting that higher nutrient levels would still increase strawberry clover stand representativity.

Subterranean clover Subterranean clover shows an identical behav­iour to strawberry clover: no significant differ­ences (p > 0.05) on its percentage of sward variation were introduced by the absence of magnesium or molybdenum fertilization (F17 ;4J =

0.761). Table 3 shows the regression models fitted to the species percentage of sward re­sponse to applied phosphorus; regression model ( 4) represents the species response fitted to overall obtained data.

Subterranean clover percentage of sward shows a high positive response to phosphorus application reaching its maximum value with 150 kg ha -I (=19.9% ). For higher phosphourus application levels it shows a slowly decreasing tendency.

Gama medic Magnesium or molybdenum fertilization absence did not affect significantly gama medic percen­tage of sward response to increasing phosphorus application rates (F14 ;9J = 2.177). The fitted re­gression models are linear (Table 4), suggesting

that higher phosphorus application rates would increase gama medic stand representativity.

Resident botanical species The increase of phosphorus soil application rates allows a better establishment of legumes re­sulting into a resident species percentage of sward decrease, which is linear for the present study conditions. No significant differences (p > 0.05) were found among these species response to phosphorus application, due to magnesium or molybdenum absence (F14 ;9J = 1.689), allowing for the fitting of regression ( 4), on Table 5, to the overall obtained data.

Percentage of bare ground

Percentage of bare ground variation, due to the experimental treatment effects, is described by regression models (1), (2) and (3), presented in Table 6.

No significant differences (p ""'0.05) were found among the percentage of bare ground variation with phosphorus application, due to magnesium or molybdenum absence (F17 ;4J =

0.486). Regression ( 4) was fitted to overall obtained data.

Table 3. Effect of increasing phosphorus application rates, in the presence or absence of magnesium and/or molybdenum, on the subterranean clover percentage of sward

Magnesium (kgha- 1 MgO)

32 0

32

Molybdenum (kg ha -I Mo)

0.00 0.27 0.27

Y = 8.82 + 0.024X Y = 3.28 + O.lOOX- 1.47 E - 4X 2

Y = 5.09 + 0.204X -7.18 E- 4X 2 + 7.04 E- 7X 3

Y = 4.28 + 0.180X- 6.19 E - 4X 2 + 5.99 E - 7X 3

Y=% of sward; X=kgha- 1 P2 0 5 ; Ns-p>0.05; **-p.,;:O.Ol.

0.346 NS (1) 0.713 NS (2) 0. 754 NS (3) 0.537** (4)

Table 4. Effect of increasing phosphorus application rates, in the presence or absence of magnesium and/or molybdenum, on the gama medic percentage of sward

Magnesium (kg ha -I MgO)

32 0

32

Molybdenum (kg ha -I Mo)

0.00 0.27 0.27

Y=% of sward; X= kgha 1 P,O,; ** -po<;:0.01; *** -p o<;:O.OOl.

Y = 3.22 + 0.027X Y = 1.41 + 0.033X Y = 1.55 + 0.027X Y=2.16+0.029X

R'

0.874** (1) 0.888** (2) 0.919** (3) 0.869*** (4)

Effect of P application on meadow species 379

Table 5. Effect of increasing phosphorus application rates, in the presence or absence of magnesium and/or molybdenum, on resident botanical species percentage of sward

Magnesium Molybdenum (kgha- 1 MgO) (kg ha _, Mo) R'

32 0.00 Y=24.11-0.017X 0.440 NS (1) 0 0.27 Y = 25.38- 0.018X 0.855* (2)

32 0.27 Y = 22.38- 0.013X 0.604 NS (3) Y = 23.96- 0.016X 0.566*** (4)

Y =% of sward; X= kg ha _, P20 5 ; NS- p > 0.05; *- p.;; 0.05; ***- p.;; 0.001.

Table 6. Effect of increasing phosphorus application rates, in the presence or absence of magnesium and/or molybdenum, on the percentage of bare ground

Magnesium (kg ha _, MgO)

Molybdenum (kgha- 1 Mo) R'

32 0

32

0.00 0.27 0.27

Y = 57.80- 0.353X + 0.0011X 2 - 1.12E- 6X3

Y = 57.74- 0.172X + 1.79E- 4X 2

0.849 NS (1) 0.903* (2) 0.949 NS (3) 0.949*** (4)

Y = 60.80- 0.381X + 0.0012X2 - l.llE - 6X3

Y = 59.5- 0.333X + 9.75E- 4X2 - 9.033E- 7X3

Y =% of bare ground; X- kg ha- 1 P20 5 ; NS- p > 0.05; *- p < =0.05; ***- p < =0.001.

Percentage of bare ground shows high decreas­ing rates with increase phosphorus application levels, especially from 0 to 150 kg ha-t P2 0 5 . On average, the application of 150 kg ha - 1P20 5 re­duces the percentage of bare ground from 59% to 30%. Higher phosphorus application levels give lower values for the variable, although with lower variation rates, reaching 17%, on average, with 600kgha- 1 of applied phosphorus (P2 0 5 ).

The percentage of bare ground variation, due to applied fertilization, shows indirectly the effect of nutrient application on the pasture sward, considered as a all. Because of the natural erosion occurrence this is of great impor­tance in hill-side soils whenever soil covering with natural vegetation is poor. In these soils, because of calcareous outcrops due to erosion processes, vegetation is scarce, offering a poor soil covering. Thus the importance of reducing bare ground through the establishment of botani­cal species with morphologic characteristics that offer a better soil covering and good quality pastures.

Mean pasture botanical composition

The regression models fitted allow for the predic­tion of different pasture botanical compositions

according to each one of the phosphorus applica­tion rates (Fig. 1).

High phosphorus application rates benefits in the calcaric soil under study, are shown through the percentage of bare ground decreasing, (=60% and 16% respectively for 0 and 600 kg ha-t of applied phosphorus, P20 5 ).

Although with slowly variation rate, resident botanical species percentage of sward decrease with increasing phosphorus application rates, improving pasture nutritive value because of legumes percentage of sward increase. Neverthe-

~Grasses

~ Gamamedic

, 50 300 450 600

Phosphorus (kg I ha P205)

- Strawberry clover

D Resident species

~ Subterranean clover

D Bare ground

Fig. 1. Mean effect of increasing phosphorus application rates on pasture sward botanical composition.

380 Effect of P application on meadow species

less, an unbalanced sward was obtained, with all rates of applied phosphorus, because of low grasses percentage of sward (9% ).

Conclusions

Legumes establishment was positively influenced by increasing phosphorus soil application rates. Gama medic results suggest that higher nutrient application levels would still increase its percen­tage of sward, at least in the establishment year.

Regarding the establishment of the introduced grasses the experimental results suggest the need for nitrogen applications.

Phosphorus soil application shows a positive effect on introduced botanical species dominance upon the resident ones, suggesting a better nutritive quality pastures.

A remarkable effect was found on the decline of the percentage of bare ground which, in addition with morphologic characteristics of the established legumes, will contribute to the de­crease of hill-side soils erosion processes.

Acknowledgements

The authors are grateful to Mr Antonio Mar­ques, Mr A S Videira da Costa, Mr Idilio Neto

and Mr Carlos Alarcao for their collaboration in the field work, as well as to the "LQARS" staff who carried out text processing.

References

Carvalho A G S and Sa H F C 1991 Caracteriza~ao Edafo­climatica e Demarca<;ao da Area de Produ<;ao do Queijo do Raba<;al. Aplica<;ao dos Estudos em Cartografia. Ed. Associa<;ao de Municipios para o Desenvolvimento da Serra de Sic6, Coimbra, Portugal, 32 p.

Levy E B and Madden E A 1933 The point method of pasture analysis. N.Z.J. Agric. 46, 267-279.

Salgueiro T and Moura F R 1985 Considerac;iies sobre a composi<;ao das pastagens de sequeiro para a regiao da Serra do Sic6. Bit. Soc. Port. Pastagens e Forragens 2, 10-13.

Steel R G D and Torrie J H 1980 Analysis of covariance. In Principles and Procedures of Statistics, a Biometrical Approach. Ed. C Napier and J W Maisel. pp 401-437. MacGraw-Hill Book Company, New York, USA.

Torres M 0, Costa A S V and Calouro F 1988 Prospec<;ao dos factores de natureza nutricional limitante do desenvol­vimento de algumas especies forrageiras em solos de­rivados de calcario da Serra do Sic6. Pastagens e Forragens 9, 143-153.

Harper J L 1978 Plant relations in pastures. In Plant Relations in Pastures. Ed. J R Wilson. pp 3-34. Common­wealth Scientific and Industrial Research Organization, Australia.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 381-390, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-081

Effects of nitrogen, phosphorus and potassium application rates on the botanical composition of an irrigated sward

M. 0. TORRES 1 , F. CALOUR0 1 and A. BARATA2

1 Laborat6rio Quimico Agricola Rebelo da Silva, Tapada da Ajuda, 1300 Lisboa, Portugal; 2DRARO Zona Agraria de Setubal, Rua do Antigo Matadouro, 2900 Setubal, Portugal

Key words: botanical composition, irrigated sward, nitrogen, phosphorus, potassium

Abstract

A fertilizer trial was conducted during two years on an irrigated sward mainly consisting of tall fescue (Festuca arundinacea Schreb.), cocksfoot (Dactylis glomerata L.), and white clover (Trifolium repens L.). The sward was established in 1966 on a haplic podzol soil and it was degraded due to inadequate fertilizer management practices. The aim of the experiment was to evaluate the effect of a range of nitrogen, phosphorus and potassium application rates on the botanical composition. Four different rates of nitrogen and potassium were considered as well as three different rates of phosphorus. The treatment means are compared and the sward botanical composition responses to N and K applications were established. The obtained results suggest that sward improvement is possible, through rational fertilizer applications, namely small amounts of nitrogen, in early and middle summer, and the split of the total amount of applied potassium into several applications.

Introduction

Sward botanical composition may influence ani­mal production by affecting feeding value of herbage (Ulyatt, 1978) and seasonality of her­bage accumulation (Grant et al., 1981).

Many factors, such as soil type, fertilizer use, grazing and cutting management and their combinations, can influence the botanical composition of the sward.

The present paper deals with the effect of fertilizers on the botanical composition of an old degraded sward due to inadequate fertilizer management practices. Several researchers have reported that high nitrogen application levels lead to an increase in the proportion of grasses (Fairey and Lefkovitch, 1990; Klapp, 1971; Lam­bert et al., 1986; Mass et al., 1962), whereas phosphorus and potassium fertilization favours the legumes (Schmitt and Brauer, 1979 cited by Mengel and Kirkby, 1987). If high inorganic nitrogen fertilizer is applied to legumes, fixation

of atmospheric nitrogen may be reduced mark­edly or even precluded (Marshner, 1986).

Fertilizer use can also reduce the participation of weed species in botanical composition of the sward (Conway, 1992, personal communication).

The main objective of the study was the improvement of the proportion of legumes in the sward through an adequate fertilizer manage­ment practice. Based on the study of Klapp ( 1971) an aim for a target value of 30% legumes was set.

Materials and methods

The study was conducted on a haplic podzol soil, on an irrigated sward at 'Posta Experimental de Pcgoes', in the west Portuguese region.

The pasture was established in 1966, with a grass-legume mixture based on tall fescue (Fes­tuca arundinacea Schreb.), cocksfoot (Dactylis glomerata L.), smooth bromegrass (Bromus iner-

382 Torres et al.

mis Leyss.), erect bromcgrass (Bromus erectus Hudson), Arrhenatherum elatius (L.) Beauvais ex. J. Pres! & K. Pres!., alfalfa (Medicago sativa L.) and white clover (Trifolium repens L.).

At present the sward is degraded and some of those species are vanished and were replaced by some weed ones. Tall fescue and cocksfoot are the prevailling species whereas white clover is the only one remainder introduced legume, although with a reduced contribution to sward composition (Sousa, 1992, personal communica­tion).

Usual mineral fertilization is composed of 50 kg ha - 1 of nitrogen, 150 kg ha - 1 of phosphor­us (P2 0 5 ) and 150kg ha- 1 of potassium (K20) applied in winter (November/December), and 150 kg ha-t of nitrogen, equally splited into 5 applications, performed monthly from April to August, immediately after cuttings.

The sward is irrigated and rotational grazing is allowed by milk cows during 3 days in the same field, and a rotation period of 21-24 days.

In 1989 a fertilizer experiment with nitrogen, phosphorus and potassium was installed on the sward and the influence of different fertilization rates on its botanical composition was evaluated over the last two years.

Before the establishment of the experiment, a composite soil sample was collected at 0-10 em depth; soil testing results show a neutral reaction (pH H 20 values of 6.5), low levels of organic matter (1.5%) and available potassium ( 49 mg kg - 1 K20, Egner-Riehm method) and high level of available phosphorus (137 mg kg- 1 P2 0 5 ,

Egner-Riehm method).

Experimental design and statistical analyses

The experiment was arranged into randomized complete blocks with four replications. The following ten combinations of a NPK ( 4 x 3 x 4) factorial experiment plus the usual fertilization were used as experimental treatments: 1 -N 0 P0 K0 ; 2- N0 P1K2 ; 3- N 1P1K2 ; 4- N2P1K2 ;

5-N 3 P1K 2 ; 6-N2P0 K2 ; 7-N 2 P1K0 ; 8-N2P1K1; 9-N2P1K3 ; 10-N3P2 K3 ; 11-usual fertilization.

All the treatments were assigned to plots with 40m 2 area each.

The following nutrient levels were used: 0,

120, 240 and 360 kg ha - 1 of nitrogen; 0, 80 and 120kg ha- 1 of phosphorus (P20 5 ) and 0, 120, 240 and 360 kg ha - 1 of potassium (K20).

Phosphorus was applied as single superphos­phate (18% P2 0 5 ) in January. Potassium was splitcd into two applications: in January together with phosphorus application and in June; potas­sium chloride (60% K20) was used. Nitrogen was splited into six applications, the first one in February and the following performed immedi­ately after cuttings, from April to August; am­monium nitrate (20.5% N) was used.

According to the main objectives of the ex­perimental work, analysis of variance was per­formed annually, for each one of the considered cuttings, in order to establish the effect of the experimental treatments on each one of the sward components.

Sward improvement was evaluated through the comparison of treatment mean results, corre­sponding to identical harvests in both years. Duncan-test (Duncan, 1955) was used in order to establish the differences among means.

Regression analysis was performed annually, in order to establish response functions to in­creasing nitrogen and potassium application rates for each one of the sward components. In order to prevent P and K confounding, nitrogen and potassium responses were established from ex­perimental treatments including 80 kg ha - 1 P20 5

and 240 kg ha - 1 K2 0 (nitrogen) and 240 kg ha - 1

N and 80 kg ha - 1 P20 5 (potassium).

Botanical composition evaluation

Botanical composition was evaluated in the first, third, fifth and seventh harvests, in sward sam­ples collected by random in the experimental plots. Six randomized sampling unities with 0.25 m2 area (0.5 m x 0.5 m) each were estab­lished, in each one of the experimental plots. These sampling unities were randomized at each harvest; the same sampling unities were used in both experimental years.

After sample homogeneization, a sub-sample of 250 g weight was taken and splited into 'grasses', 'legumes', 'other botanical species' and 'dead material'. Each one of these sub-sample components was oven-dried for 24 hours at 6SOC

Effect of N, P and K application rates on sward botanical composition 383

and then weighted. Botanical composition was evaluated on a dried matter basis.

Results and discussion

In the first year of the trial, analysis of variance shows that the experimental treatments effect on the sward botanical composition was significant for all botanical groups, in all performed har­vests, except for "the other botanical" species in the third and seventh cuts.

Dead material percentage, composed by dead or yellowish leaves, stems and clover infloresc­ences, was significantly affected by experimental treatments in the first, fifth and seventh cuts whereas no significant effects were obtained in the third harvest.

Differences among means show a similar treat­ments behaviour within all harvests and ex­perimental years; the obtained mean results, in each one of the performed harvests, show also the complementary nature of the growth curves of legumes (especially white clover) and temper­ate grass species. The result is a summer clover dominance followed by grass dominance in the late autumn, winter and spring, which was found in the 2"d year of the trial in the experimental treatment N0P1K2 (Table 7), which is according to Brougham et al. (1978).

First harvest was performed in early spring,

and experimental results (Table 1) show that the absence of nitrogen application (control and N0P1K2 treatment) decreases the percentage of grasses, confirming that high nitrogen application levels lead to an increase in the proportion of grasses (Fairey and Lcfkovitch, 1990; Klapp, 1971; Lambert et al., 1986; Mass ct al., 1962). Simultaneously, the percentage of legumes is significantly increased (p ,-: 0.05). The percen­tage of the other botanical species was also increased (p ,-: 0.05) in the control which is related to their lower nutritive needs, decreasing their sward proportion with fertilization increase (Lambert et al., 1986); in addition, the complete absence of any kind of fertilization leads to a lower canopy, and a light animal pasturing, favouring those species increase. Concerning the percentage of dead material, no significant dif­ferences were found among experimental treat­ments (p > 0.05).

The results presented in Table 1 suggest that the experimental treatment that includes the application of SO kg ha -I of P2 0 5 and 240 kg ha- 1 of K20, but no nitrogen, leads to the most balanced sward botanical composition, in early spring (=60% of grasses; =18% of legumes; =13% of other botanical species and =9% of dead material). The percentage of grasses was the lowest one, within experimental treatments (p ,-: 0.05), except for the control; on the con­trary, the percentage of legumes was the highest

Table 1. Mean sward botanical composition as affected by experimental treatments. 1st harvest, early Spring, 1990. Data represent treatment means. Values followed by the same letter are not significantly different (Duncan's multiple range test; "'= 0.05)

Treatment Grasses(%) Legumes(%) Other Dead botanical material (%) species(%)

NoPoKo 55.72 c 8.95 b 19.35 a 15.98 ab NOP!K, 59.69 c 18.23 a 13.35 be 8.73 ab N 1P1K 2 74.58 ab 7.97 b 9.13 c 8.32 ab N 2 P,K 2 72.61 b 2.29 c 9.45c 15.65 ab N 3 P1K, 83.71 ab 1.07 c 9.15 c 6.07 b N 2 P0 K 2 73.00 b 2.83 c 16.42 ab 7.75 ab N,P1K 0 77.67 ab 0.28 c 11.40bc 10.65 ab N,P,K 1 76.56 ab 1.65 c 14.64 ab 7.15 ab N,P1 K 3 81.35 ab 1.17 c 11.51 be 5.97 b N,P2 K 3 84.59 a 2.10 c 8.10c 5.21 b Usual

fcrtil. 78.58 ab 2.49 c 12.22 be 6.71 ab sm(±-) 6.79 2.46 3.10 2.75

384 Torres et al.

one (p ~ 0.05). This is associated both with the lack of nitrogen application, allowing legumes improvement without relinquishing their N2 fix­ing contribution, (Marshner, 1986), and the decrease of grasses percentage leading to a less shading effects on legumes.

Simultaneously, the application of phosphorus and potassium has stimulation effects on legumes development (Klapp, 1971; Schmitt and Brauer, 1979, cited by Mengel and Kirkby, 1987). Leg­umes are particularly responsive to potassium application, in soils with low levels of this ele­ment and with low fixation capacity, which is the present case. Experimental treatment N2P1 K0

shows the lack of potassium effect on the percen­tage of legumes, which was insignificant (almost zero) in all performed harvests, in both ex­perimental years.

Concerning phosphorus effects, no significant differences were found between treatments with and without the element, which is certainly related with the high levels of available phos­phorus in the soil.

Regarding the other botanical species mean percentage, no significant differences were found between experimental treatment N0P1K2 and the other ones, except the control.

The following harvest, performed in early summer, shows an identical sward response to experimental treatments (Table 2). The difficulty is, once again, the separation of the fertilization effects on sward botanical composition, from

those due to species seasonality. However, re­markable mean values were obtained, con­cerning percentage of dead material ( =23%) with no significant variation (p > 0.05). Appar­ently this is not related to the experimental treatments, but in addition with a small increase of the other botanical species, leads to a relative percentage of grasses decrease.

Next data were obtained in middle summer. The highest legumes percentage mean levels were obtained with treatments with no nitrogen application or with the application of 120 kg ha -I of the nutrient. Treatment N 0P1 K 2 leads to a sward composed by 86% of grasses and legumes, almost in equal proportions (Table 3).

Remarkable decrease on the percentage of the other botanical species was found and a corre­sponding percentage of legumes increase.

Last harvest, performed in early autumn, shows a relative percentage of dead material increase, legumes decrease, and the corre­sponding grasses increase. Experimental treat­ments without nitrogen application show the most favourable ratio grasses/legumes (Table 4).

In the second year of trial, the effect of the experimental treatments on sward botanical composition was similar to the observed in the first year: significant differences (p ~ 0.05) were found among treatments, in all harvests and variables except for the other botanical species group, in the seventh harvest.

Percentage of dead material was significantly

Table 2. Mean sward botanical composition as affected by experimental treatments, 3rd harvest, early Summer, 1990. Explanation see Table 1

Treatment Grasses(%) Legumes(%) Other Dead botanical material (%) species(%)

NoPoKo 40.59 ed 8.81 be 28.74 a 26.86 a N 0 P1K 2 35.17 d 25.26 a 14.91ab 24.66 a N,P1K 2 45.55 bed 10.38 b 14.97 ab 29.10 a N,P,K2 56.06 abc 2.44 be 16.30 ab 25.20 a N 3 P,K2 65.47 a 1.34 c 17.50 ab 15.69 a N 2P0 K 2 55.56 abc 1.93 c 20.59 ab 21.92 a N 2P1 K 0 69.76 a 0.05 e 12.92 b 17.27 a N 2 P1K 1 64.37 a 2.26 be 13.84 b 19.53 a N 2 P1 K 3 57.46 ab 4.59 be 15.92 ab 22.03 a N 3 P2K 1 62.95 a 1.47 c 16.20 ab 19.38 a Usual

fertile. 57.56 ab 3.53 be 14.60 ab 24.31 a sm(±) 4.80 2.46 2.32 6.07

Effect of N, P and K application rates on sward botanical composition 385

Table 3. Mean sward botanical composition, as affected by experimental treatments, 5th harvest, mid. Summer, 1990. Explanation see Table 1

Treatment Grasses(%) Legumes(%) Other Dead botanical material (%) species(%)

NoPoKo 44.30 c 31.52 b 15.48 a 8.70 a N 0 P,K2 44.59 c 41.13a 8.47b 5.81 ab NIPIK2 64.53 b 18.19c 8.81 b 8.47 a N 2P,K2 79.75 ab 4.99 d 8.54 b 6.72 ab N 3 P 1K 2 88.24 a 2.06d 5.68 cd 4.02 b N,POK2 80.90 ab 6.24 d 9.08 b 3.78 b N,PIKO 83.31 ab 0.49 d 9.79 b 6.41 ab N,PIKI 82.40 ab 4.68 d 7.87 be 5.05 ab N,PIK3 87.22 a 4.45d 4.61 d 3.72b N 3 P2 K 3 89.19 a 2.56 d 3.77 d 4.48b Usual

fertil. 79.22 ab 7.09 d 4.94d 8.75 a sm( ±) 5.74 2.63 0.75 1.10

Table 4. Mean sward botanical composition, as affected by experimental treatments, 7th harvest, early Autumn, 1990. Explanation see Table 1

Treatment Grasses(%) Legumes(%) Other Dead

NoPoKo 59.70 e 17.53 a N 11 P1K 2 60.17 de 20.97 a NIPIK2 72.99 be 7.03 b N 2P1K 2 81.61 abc 1.72 be N 3P,K2 85.05 a 0.68c N 2 P0 K 2 81.29 abc 1.48 be N 2 P1K 11 71.01 cd 0.04 c N,P,K 1 79.14 abc 1.40 he N,PIK3 82.56 ab 1.66 be N 3 P2 K 3 87.81 a 0.98 be Usual

fcrtil. 80.68 abc 3.27 be sm(±) 3.33 1.84

affected in the first, third and fifth harvests, but no significant differences among treatments were found in the last.

In early spring, besides a sward botanical composition behaviour similar to the one ob­served in the former year, a relevant increase of legumes proportion was found in experimental treatment without nitrogen and with 80 kg ha-l of P20 5 and 240kg ha- 1 of K20 (18.23% in the first year of the trial as compared with 26.58% in the second one), leading to a sward botanical composition composed by =77% of legumes and grasses (Table 5).

In early summer the same treatment leads to a

botanical material (%) species(%)

9.18 a 13.59 ab 9.84 a 9.02 bed 7.85 ab 12.13 abc 7.66 ab 9.01 bed 7.67 ah 6.60 d 8.34 ab 9.82 bed 8.07 ab 15.84 a 6.83 abc 12.63 abc 5.54 abc 10.24 cd 2.94 c 8.27 cd

4.75 be 11.30 abc 1.30 1.54

significant (p ~ 0.05) increase on the percentage of legumes (=38% of sward botanical composi­tion, the highest one), as well as to a relevant decrease in the grasses proportion (representing =33% of the sward botanical composition) and the related increase of the other botanical species (=22%). The sward shows an unbal­anced botanical composition, and the other treatments results (Table 6) suggest the need of the application of nitrogen to the soil (between 0 and 120 kg ha- 1 and same amounts of P and K) followed by an adequate animal grazing.

Nitrogen application, in early summer, still helps grasses development, although the begin-

386 Torres et al.

Table 5. Mean sward botanical composition, as affected by experimental treatments, 1st harvest, early Spring, 1991. Explanation see Table 1

Treatment Grasses(%) Legumes(%) Other Dead botanical material (%) species(%)

NoPoKo 51.21 e 11.30 b 27.09 a 10.40 b NOPIK2 50.24 e 26.58 a 16.58 b 6.50 de NIPIK2 68.88 d 10.89 b 13.61 c 6.62 de N,PIK2 77.61 be 3.33 d 12.60 cd 6.46 de N,PIK2 87.02 a 1.53 de 5.48f 5.97 ef N 2 P0 K 2 76.03 c 3.18d 15.36 b 5.43f N 2P,K 11 75.29 c 0.13 e 11.20 de 13.38 a N,PIKI 79.63 b 2.19 d 9.39 e 8.79 c N,P,K, 75.55 c 6.93 c 11.58 cd 5.94 ef N,P,K, 84.85 a 3.15 d 4.87f 7.13 d Usual

fcrtil. 71.82 d 9.53 b 12.77 cd 5.88 cf s,(±) 3.31 2.05 2.12 0.91

Table 6. Mean sward botanical composition, as affected by experimental treatments, 3rd harvest, early Summer, 1991. Explanation see Table 1

Treatment Grasses(%) Legumes(%) Other Dead

NoPoKo 42.51 d 11.52bc N 0P,K2 33.05 d 37.62 a N,P,K, 57.00 c 13.75 b N,PIK2 71.58 ab 3.80 cde N,PIK2 77.08 a 2.72 cde N 2 P0 K 2 64.55 abc 3.35 cde N,PIKO 75.77 a 0.04 c N 2P,K 1 73.70 ab 2.44 cde N,P,K3 61.46 be 10.62 be N 3P2 K 3 78.16 a 1.40 de Usual

fertil. 70.18 abc 5.23 bcde s,( ±) 4.18 2.72

ning of their natural decline, which is confirmed by the other experimental treatments results, and will prevent the great development of the other botanical species.

The obtained results in middle summer (Table 7) shows the same tendency. Experimental treat­ment N0P1K2 shows an almost equal proportion of legumes and grasses (=40% and =39%, respectively), showing the need of small amounts of nitrogen application.

The following harvest, performed in early autumn, shows a reduced proportion of the other

botanical material (%) species(%)

34.05 a 11.92 ab 21.90 b 7.43c 16.04 bed 13.21 a 14.34 bed 10.28 abc 11.32 cd 8.88 be 21.49 b 10.61 abc 12.05 cd 12.14 ab 13.71 bed 10.15 abc 19.29 be 8.63 be 10.37 d 10.07 abc

14.05 bed 10.54 abc 2.45 1.21

botanical species (less than 6% on all treat­ments) suggesting, in average, a sward botanical improvement due to fertilizing practices (Table 8).

Sward botanical composition responses to increasing N and K application rates

Response to increasing nitrogen application rates Sward percentage of grasses shows a linear and positive response to increasing nitrogen applica­tion rates, which was observed in both years of

Effect of N, P and K application rates on sward botanical composition 387

Table 7. Mean sward botanical composition, as affected by experimental treatments, 5th harvest, mid. Summer, 1991. Explanation see Table 1

Treatment Grasses(%) Legumes(%) Other Dead botanical material (%) species(%)

NoPoKo 37.32 e 23.62 b 27.51 a 11.55 a N 0 P,K 2 38.95 e 39.83 ab 10.75 be 10.47 a N 1P1K2 64.72 d 15.88c 9.38 be 10.02 ab N 2 P,K 2 80.14 abc 2.62 de 10.79 be 6.45 be N 3P1K2 88.92 ab 1.26 de 6.44 be 3.38 c N 2 P0 K 2 77.75 be 3.58 de 12.75 be 5.92 c N,P1K 0 86.14 ab 0.04e 9.30 be 4.52c N,P,K, 86.26 ab 1.46 de 7.20 be 5.08 c N,P,K 3 82.50 abc 4.72 de 8.06 be 4.72 c N 3 P2 K3 90.85 a 1.08 de 4.23 c 3.84 c Usual

fertil. 71.98 cd 7.84 d 13.40 b 6.78 b sm( ±) 3.69 2.04 2.06 1.06

Table 8. Mean sward botanical composition, as affected by experimental treatments, 7th harvest, early Autumn, 1991. Explanation see Table 1

Treatment Grasses(%) Legumes(%) Other Dead

NoPoKo 73.69 be 9.85 b N 0 P,K 2 70.74 c 15.86 a N,P,K, 79.34 abc 4.19 c N,P,K, 82.89 ab 0.88e N 3 P1K, 86.86 a 0.51 e N 2 P0 K 2 81.69 abc 1.03 e N 2 P,K 0 83.51 ab 0.00 e N,P,K, 84.35 ab 0.60 e N 2 P1K 3 84.94 abc 1.72c N 3 P2 K 3 86.17 a 0.56e Usual

fertil. 79.95 abc 3.73 cd sm(±) 3.54 0.75

the trial, in all performed harvests (Tables 9 and 10), which is according to Klapp (1971).

The regression models fitted to the percentage of legumes variation with nitrogen increase (Tables 9 and 10) show a remarkable decrease from 0 to 120 kg ha-l of applied N, followed by a flattening until the end of the range of N applications.

Regarding the other considered groups, the obtained results show that, in case of the other botanical species, a significant decrease with increased nitrogen application rates occurs, with

botanical material (%) species(%)

5.63 a 10.83 a 5.02 ab 8.38 a 4.01 ab 12.46 a 3.77 ab 12.46 a 2.08 b 10.55 a 5.22 ab 12.06 a 3.94 ab 12.65 a 2.38 b 12.28 a 3.66 ab 13.68 a 3.05 ab 10.22 a

3.60 ab 12.72 a 0.93 1.70

some exceptions (Tables 9 and 10). Dead materi­al was not significantly aff~cted by nitrogen application, except for the obtained results in the middle summer of the second experimental year, showing a significant decrease with increasing nitrogen application rates (Tables 9 and 10).

Response to increasing potassium application rates Almost in all performed observations, no signifi­cant response of the sward botanical composition

388 Torres et al.

Table 9. Response of sward botanical composition to nitrogen application- 1st experimental year

Sward groups(%)

Grasses

Legumes

Other botanical species

Dead material

early spring early summer middle summer early autumn

early spring early summer middle summer early autumn

early spring early summer middle summer early autumn

early spring early summer middle summer early autumn

R'

Y~62.12+0.058X 0.835 NS Y ~ 35.35 + 0.084 X 0.999 *** Y ~ 47.35 + 0.122 X 0.970 ** Y ~ 62.47 + 0.069 X 0.940 *

Y~ 18.65-2.79ln(X+ 1) 0.946 * Y ~ 25.79- 3.94ln(X + 1) 0.953 * Y ~ 42.14- 6.32ln(X + 1) 0.941 * Y ~ 21.27- 3.38ln(X + 1) 0.979 **

Y ~ 13.26- 0.739ln(X + 1) 0.963 * Y ~ 14.56 + 0.008 X 0.907 * y ~ 7.88 ± 1.471 Y ~ 9.82- 0.386ln(X + 1) 0.991 **

Y~9.69±4.140

y ~ 23.66 ± 5.670 y ~ 6.26 ± 1.854 y ~ 9.19 ± 2.267

X~ N kg.ha- 1 ; NS- p >0.05; *- p ~0.05; **- p ~0.01; ***- p ~0.001.

Table 10. Response of sward botanical composition to nitrogen application- 2nd experimental year

Sward groups(%)

Grasses

Legumes

Other botanical species

Dead material

early spring early summer middle summer early autumn

early spring early summer middle summer early autumn

early spring early summer middle summer early autumn

early spring early summer middle summer early autumn

x~Nkg.ha 1 ; NS-p>0.05; *-p~0.05; **-p~O.Ol.

to increasing potassium application rates was found (Table 11).

Nevertheless, legumes development was re­markably increased from 0 to 120 kg ha-t of

Y ~ 53.08 + 0.099 X Y~ 37.68 + 0.122 X Y~43.38+0.138X

Y~ 72.17 + 0.043 X

Y ~ 27.12- 4.09ln(X + 1) Y ~ 38.15- 5.86ln(X + 1) Y ~ 40.69- 6.38ln(X + 1) Y ~ 15.96- 2.62ln(X + 1)

Y~ 17.21-0.029X Y ~ 20.92- 0.028 X Y~9.34±2.041

Y ~ 5.08-0.008 X

Y ~ 6.72-0.002 X y ~ 9.95 ± 2.465 Y ~ 11.31-0.021 X y ~ 10.96 ± !.943

R'

0.963 * 0.927 * 0.949 * 0.952 *

0.959 * 0.975 ** 0.951 * 0. 993 **

0.886 * 0.941 *

0.919 *

0.761 NS

0.929 *

applied potassium in middle summer and early autumn, in the first year of the trial, followed by a flattening until the end of the range of K applications.

Effect of N, P and K application rates on sward botanical composition 389

Table 11. Response of sward botanical composition to potassium application- 1st experimental year

Sward groups(%)

Grasses

Legumes

Other botanical species

Dead material

early spring early summer middle summer early autumn

early spring early summer middle summer early autumn

early spring early summer middle summer early autumn

early spring early summer middle summer early autumn

X=K,O kgha '; NS-p>0.05; *-p.;0.05; **-p.;O.Ol.

Conclusions

The obtained results, although corresponding to a few years of trial, suggest that sward improve­ment is possible through an adequate fertiliza­tion.

The improvement of the under lied sward, with great predominance of grasses, should begin with no nitrogen applications, especially in early spring and early autumn, aiming legumes re­establishment allowance. In late spring and dur­ing the summer, small amounts of nitrogen should be applied in order to help grasses de­velopment, although the beginning of their natural decline. The aim is higher dry matter productions and a balanced sward botanical composition. Studies on the effect of applied nitrogen amounts between 0 and 120 kg ha-t on sward botanical composition are suggested by the obtained results. Potassium application should be performed in order to accelerate legumes re-establishment and, because of the soil characteristics, should be splited into several applications, each one of them composed by small amounts of the nutrient. Studies on the effect of applied potassium amounts between 0 and 240 kg ha -r are also suggested by the ob­tained results.

y = 79.82 ± 3.303 Y = 68.69- O.Q38 X Y=83.17±3.094 Y = 73.01 + 0.031 X

Y=0.35 +0.170ln(X+ 1) Y = -0.101 + 0.602ln(X + 1) Y = 0.592 + 0.757ln(X + 1) Y = 0.043 + 0.288ln(X + 1)

y = 11.68 ± 2.260 y = 14.74 ± 1.628 Y=7.70±2.210 y = 7.02 ± 1.116

Y=7.46±2.190 y = 21.01 ± 3.405 y = 5.48 ± 1.377 y = 11.93 ± 3.009

Acknowledgments

R'

0.840 NS

0.834 NS

0.672NS 0.788NS 0.952 * 0.991 **

The authors are grateful to the staff of 'Posto Experimental de Pegoes', Mr Carlos Ribeiro, Mrs Raquel Dias and Cristina Sempiterno, for their collaboration in the field work, as well as to 'LQRAS' staff who carried out chemical analysis and text processing.

References

Brougham R W, Ball P R and Williams W M 1978 The ecology and management of white clover-based pastures. In Plant Relations in Pastures. Ed. J R Wilson. pp 309-324. Commonwealth Scientific and Industrial Research Organization, Australia.

Duncan D B 1955 Multiple range and multiple F tests. Biometrics 11, 1-42.

Fairey N A and Lefkovitch L P 1990 Herbage production: Conventional mixtures vs. alternating strips of grass and legume. Agron. J. 82, 737-744.

Grant D A, Luscombe PC and Thomas V J 1981 Responses of ryegrass, brown-top and an unimproved resident pasture in hill country, to nitrogen, phosphorus, and potassium fertilisers 1. Pasture production. N. Z. J. Exp. Agric. 9, 227-236.

Klapp E 1971 Prados e Pastagens. Funda~ao Calouste Gul­bcnkian, Lisboa, Portugal. 872 p.

Lambert M G, 1986 Influence of fertiliser and grazing

390 Effect of N, P and K application rates on sward botanical composition

management on North Island moist hill country. 2. Pasture botanical composition. N. z. J. Agric. Res. 29, 1-10.

Marschner H 1986 Mineral Nutrition of higher Plants. Academic Press, London, 674 p.

Mass E F 1962 Yield response, residual nitrogen and clover content of an irrigated grass-clover pasture as affected by various rates and frequencies of nitrogen application. Agron. J. 54, 212-214.

Mengel K and Kirkby E A 1987 Principles of Plant Nutrition. 4' ed., International Potash Institute, Bern, Switzerland. 687 p.

Ulyatt M J 1978 Aspects of the feeding value of pasture. Proc. Agron. Soc. New Zealand 8, 119-122.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 391-395, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-086

Effects of nitrate, phosphate and sulfate combinations on growth and kinetics of phosphate and sulfate uptake by eucalypt seedlings

NAIRAM F. BARROS, FRANCISCO A.S. FERREIRA', ROBERTO F. NOVAIS, JULIO C.L. NEVES and VICTOR H. ALVAREZ V. Federal University of Vi9osa, Soil Science Department, 36570 Vi9osa, MG, Brazil; 1Riocell S.A., 92500 Guaiba, RS, Brazil

Key words: Eucalyptus, mineral nutrition, nitrogen, phosphorus, sulfur

Abstract

This trial was carried out to study the effect of nitrate, phosphate and sulfate on growth and kinetics of P and S uptake by Eucalyptus grandis seedlings. The seedlings had their taproot pruned and were placed in sets of geminated plastic pots, with the root system split and growing in solutions containing the three nutrients, or with one of them isolated in one of the geminated pots. Seedling shoot dry weight when the solution in both pots contained the three nutrients was higher than that in the other treatments, which did not differ. Absence of NO~ in the nutrient solution significantly reduced root dry weight. When both pots contained the three nutrients, the Km -SO~- was two to three times lower than that of the other four treatments. Influx of SO~- and H2PO~ increased when NO~ was supplied separately from so;- or H2PO~.

Introduction

Increased growth of plant roots in sites enriched with phosphorus (P) as compared to sites with low values for, or absence of, this nutrient has been observed by several researchers (Anghinoni and Barber, 1980; Drew and Saker, 1978; Mach­ado eta!., 1983; McClure, 1972). The need for external P for root growth is probably due to the fact the internally translocated P would not satisfactorily supply P to the growing points. Also, the distribution of photoassimilates and the capacity of meristematic regions to utilize them is thought to be dependent on the presence of external P. A similar phenomenon has been described for ions such as ammonium (An­ghinoni and Barber, 1988) and nitrate. As for nitrate, this effect is a consequence of the prefer­ential allocation of photoassimilates to roots growing in a nitrate-enriched medium (Hackett, 1972), as a result of the link between nitrate reduction in the shoot and its uptake by the roots

and the synthesis and transport of malate from the shoot to the roots, as proposed by Ben Zioni et a!. (1971). Drew et a!. (1973), however, suggested that the increased nitrate uptake in the nitrate-enriched root zone could have the follow­ing effects: a) increase in protein synthesis and other metabolites that stimulate root initiation and development which, in turn, are sinks for photoassimilates and growth regulators produced in the shoot; b) increased synthesis in situ of growth regulators which would drive photoas­similate transport to the considered root zone, with the subsequent stimulus to the initiation and growth of lateral roots.

Regardless of the way in which this phenom­enon works internally in the plant, it was demon­strated in barley that root growth depends on the presence of N and P in the external solution (Drew, 1975).

It can be suggested that fertilizer composition and placement play an important role in growth and nutrient uptake by eucalypt plants. This

392 Barros et al.

study was carried out to test the effect of nitrate, phosphate and sulfate, isolated or in combina­tion with each other in relation to the root system, on growth and kinetics of P and S uptake by eucalypt seedlings.

Methods

Seedlings of Eucalyptus grandis W. Hill ex Maiden were grown in a complete nutrient solution (Clark, 1975) for 40 days under green­house conditions. Fifteen days after transfer to the solution, their taproots were pruned in order to stimulate growth of the lateral roots. After 40 days, the seedlings were transferred to plastic pots, with the root system split and growing in 2. 7 L of nutrient solutions with one of the following combinations: c1: [ HNO~' Hz PO~' SO~-)] (HNO~, H2PO~, SO~-); C2 : (NO~, H2PO~, SO~-) (absence of NO~, H2PO~, SO!-); C 3 : (NO~)(H2PO~, SO!-); C4 :

(H2PO~) (NO~, so;-); C5 : (so;-) (NO~, H2PO~). The concentrations of NO~, H2PO~, and so;- in the solutions were 6.43 mM, 0.17 mM, and 0.56 mM, respectively. All other nutrients were equally supplied to each pot in the following concentrations: Ca, 102.4 mg L - 1; Mg, -14.4mg L- 1; K, -70.2mg L-\ and micronutrients as recommended by Clark (1975); reagents containing ammonium or sulfate in their composition were replaced with those containing sodium or chloride.

Twenty-nine days after treatment application, five replicates of each nutrient combination were transferred to a growth chamber, with a tem­perature of 25 ± 2oc and 8.000 lux for the kinetic study, while the other four replicates remained in the greenhouse for 47 days, after which the seedlings were harvested and separated into leaves, branches, stem and roots, according to nutrient combination, and the dry weight was determined. Leaf chemical analysis was per­formed to determine the concentration of differ­ent sulfur fractions.

In the growth chamber the nutrient solution was the same as that employed in the greenhouse experiment, except for the concentrations of nitrate, phosphate and sulfate, which were re-

duced to 200 fLM, 7.4f.LM, and 25.5 fLM, respec­tively. The solutions containing P and S were labeled with 2.22 x 104 bq L - 1 of 32P and 6.66 x 104 bq L - 1 1 of 35S, respectively.

All solutions were sampled before transferring the seedlings to the pots, 1.65 L capacity. Each set of geminated pots + seedling was weighted at the beginning of the study. Samples of 3.0 ml of the nutrient solution were taken from each pot at 1.40, 2.13, 2.70, 4.23, 4.81, 5.81, and 11.10 hours after the beginning of the study and stored at -20°C. Immediately after the last sampling time, each set of pots + seedling was weighted again; seedlings were separated into shoot and roots and the fresh weight was determined.

The activities of 32P and 35S were determined in a Beckmann liquid scintillation counter model LS 233.

The kinetic parameters, Km and Imax' of sul­fate uptake were estimated by fitting the data to the linear form of the Michaelis-Menten equa­tion, according to the modification of Lineweaver and Burk (1934). Due to the fast phosphate uptake, a mean influx value was estimated for a period of 2.7 hour of P absorp­tion.

Results and discussion

Biomass production

Leaf and shoot biomass production was higher in the nutrient combination c1' in which nitrate, phosphate and sulfate were supplied to both halves of the root system (Fig. 1 ). In all the other combinations there was a considerable reduction in shoot dry matter production. This reduction in growth is attributed to the in­capability of the root system to meet plant nutrient demand, because only a small portion of it is supplied with nutrients. Similar results were reported by Drew and Saker (1975) for barley plants. According to these researchers, the rela­tive growth rate of barley was recovered if nitrate or phosphate were supplied to the whole root system 14 days after being provided to one half of the root system, although the total plant dry matter production had been reduced. There

Phosphate and sulphate uptake by eucalypt seedlings 393

~ Ltovu

30 miD Stom

0 Roo!~ 24

18

12

~

c

0

I /2 2

12 NPS NPS NPS 0 N PS P NS

Fig. 1. Dry matter of different components of E. grandis seedlings as affected by combinations of nitrate (N), phos­phate (P) and sulfate (S) in relation to the root system.

was a trend of lower shoot dry matter yield when nitrate and sulfate were supplied in different pots (Fig. 1). The interaction of N and S has been reported for other woody species such as pecan (Hu and Sparks, 1992). According to Cram (1990), the mechanism of this interaction is not completely clear, and raises the possibility of a linkage between nitrate and sulfate uptake and reduction.

The supply of nitrate to one half of the root system and sulfate to the other half appeared to impair the assimilation of the two nutrients; strong leaf chlorosis was observed associated with low sulfate content in plants grown in treatment C5 , or low sulfate assimilation in plants in C 3 (Table 1). Similar results have been

reported for coffee plants grown in nutrient solution (Alves, 1991). A distinct feature of the plants in these two treatments was the relatively high level of arginine in the leaves, i.e., 46.9% of the aminoacid-N in C 5 and 29.0% in C3 as compared with 12.9% in c4 and 0.0 in cl and C2 • The opposite was observed with the levels of glutamine, which were lowest in C5 (23.6%) and c3 (37.5%) and highest in c, (63.2%) and c2 (60.8% ).

The decrease in shoot dry matter yield in the combinations where phosphate was supplied separately from nitrate and sulfate might be a consequence of low energy (ATP) in the roots for nitrate and sulfate uptake, assimilation, and transport to the shoot.

Higher root dry matter production was associ­ated with the presence of nitrate in the growing solution (Fig. 1 ). Roots maintained in the solu­tion without nitrate presented reduced growth despite the fact that their nitrate concentration is comparable to that of roots grown in a solution with this ion in both halves, i.e., about 0.24%. Therefore, this internal nitrate seems to consti­tute a metabolic inactive pool (Martin ct a!., 1981 ).

Sulfate and phosphate uptake

The supply of nitrate, phosphate and sulfate to both halves of the root system (treatment C 1 )

resulted in low Km -so;- as compared to the other nutrient combinations (Fig. 2). The reduc­tion in the affinity of the carrier for the ion may be caused by the increase of sulfate concen­tration (25.5 11-M) in the solutions of treatment C2 through treatment C5 , as compared to that of C 1 (12.75 11-M, in each half).

Table 1. Contents of total, proteinic and non-proteinic sulfur fractions in leaves of Eucalyptus grandis grown in solutions with different combinations of nitrate, phosphate and sulfate in relation to the root system

Combinations S-Fractions' (mg S/pot) Proteinic-S %of

Pot1 Pot2 Total Proteinic Non-proteinic total

c, {NPS ~NPS 74.39 nd nd nd c, NPS 0 50.85 44.20 6.65 86.92 c, N PS 54.14 36.56 17.58 67.17 c, p NS 48.04 42.28 5.76 88.01 c, s NP 33.64 28.60 5.04 85.02

'nd- not determined.

394 Barros et al.

i

= I .... 0

"' I e

"'

Fig. 2. Kinetic parameters, Km and Im,, of sulfate uptake by E. grandis seedlings as affected by combinations of ni· trate(N), phosphate (P) and sulfate (S) in relation to the root system.

According to Jensen and Konig (1982) the uptake of sulfate is regulated by the internal level of this ion in the roots by a feedback process, which activates the carrier when the intracellular sulfate concentration is low and depresses the carrier synthesis under high inter­nal sulfate concentrations.

The Imax for sulfate was very high when sulfate and phosphate were supplied to the root system in the same pot (Fig. 2). This suggests that sulfate uptake depends on phosphate for increas­ing the level of energy (ATP) in the roots.

Phosphorus uptake by plants growing in C3

and C4 was much higher than in other combina­tions (Fig. 3). Assuming that the influx of phos­phate follows the same pattern of sulfate influx, i.e., the mean influx in the period of 2.7 hour shows the same trend as the Imax for sulfate (compare Fig. 2 and Fig. 3), it could be specu­lated that the Imax for phosphate would increase when this ion is supplied with sulfate in the same pot and nitrate in the other (C3 ) and when phosphate was supplied alone (C4 ). Hence, a feedback effect would come from the shoot increasing the influx of phosphate into the roots.

200

150

100

50

q L--LI~/d2-I/_2LUN~PS~--O_LIGN~-LPS_L~Pd-N-S-L~S~-~N-P~~ NPS NPS

Fig. 3. Amount of phosphate and sulfate uptake in pots where these ions were present, during a period of 2.7 hours, by roots of E. grandis seedlings as affected by combinations of nitrate (N), phosphate (P) and sulfate (S) in relation to the root system.

References

Alves J D 1991 Respostas de mudas de cafe em solm;ao nutritiva a localiza~ao de nitrogenio f6sforo e enxofre no sistema radicular. D.Sc. Diss. Universidade Federal de Vi~osa, Vi~osa, MG, Brasil.

Anghinoni I and Barber S A 1980 Phosphorus influx and growth characteristics of corn roots as influenced by phosphorus supply. Agron. J. 72, 685-688.

Anghinoni I and Barber S A 1988 Corn root growth and nitrogen uptake as affected by ammonium placement. Agron. J. 80, 779-802.

Ben Zioni A, Vaadia Y and Lips H 1971 Nitrate uptake by roots as regulated by nitrate reduction products of the shoot. Physiol. Plant. 24, 288-290.

Clark R B 1975 Characterization of phosphates of intact maize roots. J. Agric. Food Chern. 23, 458-460.

Cram W J 1990 Uptake and transport of sulfate. In Sulfur Nutrition and Sulfur Assimilation in Higher Plants. Eds. H Rennenberg, C Brunold, L J De Kok and I Stulen. pp 3-11. SPB Academic Publishing, The Hague, The Nether­lands.

Drew M C 1975 Comparison of the effects of a localized supply of phosphate, nitrate, ammonium and potassium on the growth of the seminal root system, and the shoot in barley. New Phytol. 75, 479-490.

Drew M C and Saker L R 1975 Nutrient supply and the growth of the seminal root system in barley. II. Localized,

Phosphate and sulphate uptake by eucalypt seedlings 395

compensatory increases in lateral root growth and rates of nitrate uptake when nitrate supply is restricted to only part of the root system. J. Exp. Bot. 26, 79-90.

Drew M C and Saker L R 1978 Nutrient supply and the growth of seminal root system in barley. Ill. Compensat­ory increases in growth of lateral roots and in rates of phosphate uptake, in response to a localized supply of phosphate. J. Exp. Bot. 29, 435-451.

Drew M C, Saker L R and Ashley T W 1973 Nutrient supply and the growth of the seminal root system in barley. I. The effect of nitrate concentration on the growth of axies and laterals. J. Exp. Bot. 24, 1189-1202.

Hackett C 1972 A method of applying nutrients locally to roots under controlled conditions and some morphological effects of locally applied nitrate on the branching of wheat roots. Aust. J. Bioi. Sci. 25, 1169-80.

Hu H and Sparks D 1992 Nitrogen and sulfur interaction influences net photosynthesis and vegetative growth of pecan. J. Am. Soc. Hortic. Sci. 117, 59-64.

Jensen and Konig T 1982 Development of a regulation mechanism for so;- influx in spring wheat roots. Physiol. Plant. 55, 459-464.

Lineweaver H and Burk D 1934 The determination of dissociation constants. J. Am. Chern. Soc. 56, 658-666.

Machado R P, Novais R F, Sediyama C S and Borges A C 1983 Efeito da localiza9ao de doses de f6sforo em rela9ao ao sistema radicular sabre o comportamento da soja, com a utiliza9ao da tecnica de raizes subdivididas. Rev. Ceres 30, 295-307.

Martin F, Chemardin M and Ada! P 1981 Nitrate assimilation and nitrogen circulation in Austrian pine. Physiol. Plant. 53, 105-110.

McClure G W 1972 Nutrient distribution in root zones. Ill. Further studies on the effects of phosphorus distribution on corn and wheat. Can. J. Bot. 50, 2275-2282.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 397-401, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-103

Influence of animal manures on extractable micronutrients, greenhouse tomatoes and subsequent Swiss chard crops

P.R. WARMAN Department of Chemistry and Soil Science, Nova Scotia Agricultural College, P.O. Box 550, Truro, NS, Canada B2N 5£3

Key words: animal manures, DTPA-extractable micronutrients, Swiss chard, tissue composition, tomatoes

Abstract

Animal manures have been traditionally used as organic fertilizers; however, there is little documenta­tion on the use of manures for greenhouse crops, partly due to odours and the assumption that manure salts in greenhouse soils will be toxic to plants. For this study, three crops of greenhouse tomatoes were grown in large containers of Pugwash sandy loam and fertilized regularly with three rates (by volume) of three types of liquid manures or commercial fertilizer. Following each tomato crop, Swiss chard was grown in the treated soils without additional fertilization in order to determine residual nutrient response. Tomatoes and Swiss chard were harvested at maturity and crop yields evaluated. One specific tomato leaf from each plant and all chard tissue was dried, ground, and analysed for macronutrients and Cu, Mn and Zn. Soil samples were analysed for DPTA-extractable Cu, Mn, and Zn and then correlated with plant tissue concentration or nutrient uptake. Crop yields were positively influenced by the highest application rate of each manure amendment. Of all the treatments, only the complete fertilizer amendment produced phytotoxic levels of salts. The liquid pig manure produced the most fruit and leaf biomass for an equivalent application rate and the highest nutrient uptake by the subsequent Swiss chard crops. DTPA-extractable Mn and Zn was significantly correlated to plant Mn and Zn uptake.

Introduction

Animal manures have been used for thousands of years as fertilizers for field crops. Aside from a few short-term trials, manures have not been popular for greenhouse vegetable production, partly because of the concern for odour and soluble salts released from the manures. In fact, Adams (1986) in an extensive review of the mineral nutrition of the tomato crop, does not even mention the use of organic fertilizers. However, Ryan et al. (1985), Sims and Pill (1987), and Warman (1990b) have evaluated the use of manure amendments for tomato (Lycoper-

sicum esculentum) production; while Swiss chard (Beta vulgaris) has become a popular leafy vege­table for evaluating heavy metal availability, especially from sewage sludge-amended soils (Keeney and Walsh, 1975; Mahler et al., 1982; Sikora et al., 1980).

This study had a number of objectives; a) to determine the effect of three rates each of three different liquid manures on successive tomato crop yields; b) to test the residual effect of the amendments on unfertilized Swiss chard; c) to analyze plant tissue and DTPA-extractable Cu, Mn, Zn in order to compare soil content and plant micronutrient uptake.

398 Warman

Materials and methods

A greenhouse experiment was begun in August 1982 and continued until August 1984. Woodville sandy loam (Humo-Ferric Podzol) was placed in large fiber pots (18.51). The initial soil charac­teristics are described in Warman (1990a). One fertilized tomato crop (cv. Vendor and Toy Boy) was grown in each of the three years and Swiss chard (cv. Fordhook Giant) was seeded into the pots following each tomato harvest. Three rates (50, 100, 200 mL) of liquid chicken, dairy and pig manure were applied weekly (max. of 15 wks.) to the surface of the tomato pots, only; the Swiss chard was not fertilized. An additional 100 or 150 mL of water was added to the lower rates of manure so that all pots received 200 mL of fluid. The average dry weight analysis of the manures applied to the three tomato crops is given in Table 1. Chicken and dairy manure (without bedding) were collected from the NSAC barns and diluted 1: 10 and 1 :5, respec­tively, to provide an average solids content of 2-3%. The pig manure was not diluted; how­ever, it was the most variable in percent dry matter and, therefore, nutrient analysis. In addi­tion to the manures, there was a Control rate of zero fertilizer, a N-P-K treatment (1.5 g/wk 20-20-20 fertilizer), and a N-P-K-Ca-Mg treatment (1.5 g/wk 20-20-20 + 0.5 g CaC12 2H2 0 and 1.5 g MgS0 4 7H20/2 wk). Therefore, initially 12 treatments in a randomized complete block de­sign with three replicates.

Marketable tomatoes were harvested and weighed while the entire Swiss chard plant was removed at maturity. Tomato tissue samples from each plant consisted of the most recently mature leaf (blade and petiole) above the second flower cluster. The analysis of the manures, plant tissue and soils is as described in Warman (1990a). Data were analyzed using ANOVA;

treatment means were evaluated using the LSD multiple range test.

Results and discussion

The effect of the treatments on tomato yields is shown in Table 2. For the first crop, yield differences were not significant; however, treat­ment differences were significant for the second and third crops. A composted manure treatment replaced the Control for the third tomato crop since the second Swiss chard crop failed to grow in the Control treatment. Results from the 1984 tomato crop indicated that PM 100 and PM 200 were superior to the other treatments; this result was expected considering the PM treatments contained more total nutrients than the other manures. For each manure source, the 200 mL/ wk rate was superior to the 50 and 100 mL/wk additions. Even after three tomato crops had been amended in the same soil, soluble manure salts were not a problem, unlike the Ca + Mg­amended fertilizer treatment. There are interest­ing comparisons between crop yield and elemen­tal leaf tissue content. Highest yields were from the CM 200, DM 100 and DM 200 treatments in 1983; however, the leaf analysis (Table 3) does not indicate that DM 100 and DM200 would be that productive. Considering the elements ana­lyzed, the most nutrient-rich Vendor leaves were associated with the PM 100 and PM 200 treat­ments. One could conclude, therefore, that the tomato leaf sampled in this experiment was not a good indicator of eventual crop yield; another tomato leaf might have been more suitable.

The data for the first Swiss chard crop are found in Table 4. Treatment yield differences were highly significant, causing plant uptake of all elements to follow a similar pattern to yield. This occurred even though tissue P and Cu did

Table 1. Average dry weight analysis of the animal manures applied to the tomato plants'

Type D.M. N p K Ca Mg Mn Zn Cu (source)

(g kg -1) (mgkg- 1)

Pig 63.5 87 24 33 18 6 330 840 110 Dairy 135 34 10 14 9 3 220 160 100 Chicken 216 89 18 28 145 12 530 450 85

'A minimum of six samples of each type.

Effect of manures on tomatoes and Swiss chard 399

Table 2. Effects of treatments on greenhouse tomato yields (g plant-')"

Treatmentb Vendor Toy Boy 1984

1982 1983

CM 50 428 401cd 352bc CM 100 448 521de 489c CM200 630 729fg 1001d DM 50 413 653ef 1R3ab DMlOO 434 708fg 275abc DM200 524 847g 369bc PM 50 494 310bc 1080d PM 100 484 471cd 1446e PM200 525 562def 1469e NPK 503 361bcd 1159d NPKCaMg 627 68a 63a Controlc 456 218ab 446c

LSD 176 259 Mean± s.d. 497 ± 71

"Means followed by the same letter are not significantly different at p = 0.05 using LSD. b CM =chicken manure; DM =dairy manure; PM= pig manure; 50= 50 mL/wk liquid manure; 100 = 100 mL/wk liquid manure. 200 = 200 mL/wk liquid manure; NPK = 22.5 g 20-20-20 fer!.; NPK + CaMg = 22.5 g 20-20-20 fer!. + 4 g CaC1 2 2H,O + 10.5 g MgSO 4 • 7H2 0; Control = 0 fertilizer. c For 1984, this treatment is compost manure (450 g 1-1-1 analysis).

Table 3. Elemental leaf tissue content of 1983 Vendor tomatoes (g kg- 1)'

Treatmento N p K Ca Mg

CM 50 55.3e 3.0a 34.0ab 24.7cde 6.2dc CM100 50.0de 3.2a 32.0ab 27.7e 4.7bc CM200 41.3bcd 2.6a 33.0ab 25.8de 2.8a DM 50 33.0ab 3.5ab 34.4ab 21.8bcd 5.0cd DM100 29.0a 4.1ab 32.6ab 22.5be 4.5bc DM200 29.7a 3.6ab 29.4a 18.3ab 3.8abc PM 50 49.7cd 3.1a 37.4abc 26.3de 7.0e PM 100 45.3cd 5.4bc 53. 7cd 25.4de 5.3cd PM200 50.0dc 6.lc 43.7abc 19.8abc 5.1cd NPK 55.0e 4.3abc 61.5d 16.0a 3.4ab Control 40.0bc 3.2a 48.0bcd 23.0bc 4.6bc

LSD 9.0 1.9 16.8 5.4 1.5

'Means followed by the same letter are not significantly different at p = 0.05 using LSD. ' See Table 2 for designation of treatments applied to the tomatoes.

not vary between treatments. The residual effect of PM 200 was most apparent to DMY and tissue N, K, Mn and Zn. There are no data available for the second Swiss chard crop, but the results of the last experimental crop are shown in Table 5. The PM 100 and 200 treatments still produced highest DMY, tissue N, P, Mg, Mn and Zn, and were not significantly different than the CM 100 in tissue Cu. The Ca composition in chicken manure (Table 1) caused tissue Ca in the

CM 100 and CM 200 chard to exceed all other treatments.

For the soil sampled after the first tomato harvest, DTPA-extractable Cu, Mn and Zn were highly correlated (p = 0.01) with plant uptake; however, only tissue Mn and Zn were correlated (p = 0.05) with DPTA Mn and Zn. Following the first chard harvest, only DPT A Mn and Zn correlated with tissue (p = 0.05) and plant up­take (p = 0.01). The last soil samples to be

400 Warman

Table 4. 1983 Swiss chard dry matter yield (DMY) and tissue analysis'

Treatmenth DMY N p K Cu Mn Zn (g/pot)

(gkg-1) (mgkg- 1)

CM 50 5.73bc 14.0a 5.2 14.6abc 18.3 252a 53.9a CM 100 7.90cd 14.6a 5.3 19.4be 16.8 261a 58.0a CM200 12.00e 15.5a 4.5 22.0de 14.3 269a 57.2a DM 50 4.62ab 19.9abc 3.8 12.3ab 14.1 259a 66.4a DM100 6.65bcd 17.2ab 4.1 11.4a 15.2 372a SO.lab DM200 9.60de 17.3ab 6.5 16.4ad 15.9 358a 91.8ab PM 50 9.46de 16.3ab 3.7 17.1ae 13.6 440a 109.8b PM 100 12.37e 16.2ab 3.8 21.6ce 13.6 810b 161.3c PM200 19.02f 26.9d 4.4 36.7f 13.4 943b 188.2c NPK 9.50de 25.9cd 3.4 24.2e 12.0 819b 68.3a Control 1.88a 22.3bcd 2.7 16.8ad 15.7 410a 63.3a

LSD 3.11 6.4 7.3 226 38.2 Mean± s.d. 4.3 ± 1.0 14.7 ± 2.0

'Means followed by the same letter are not significantly different at p = 0.05 using LSD. b See Table 2 for designation of treatments applied to the tomatoes.

Table 5. 1984 Swiss chard dry matter yield (DMY) and tissue analysis'

Treatmenth DMY N p K Ca Mg Cu Mn Zn (g/pot)

(gkg-1) (mgkg 1)

CM 50 10.79ab 18.0ab 4.3ab 24.5bc 6.5a 8.9ab 6.3a 100a 19.9a CM100 14.70bc 17.8ab 5.3ab 26.3bc 9.4bc IO.Obc lO.Oc 123a 35.3a CM200 15.77bc 21.9b 5.9ab 24.1ac 11.5c 9.5ac 9.9c 88a 33.9a DM 50 9.14a 19.8ab 4.0a 26.0bc 5.4a 8.9ab 7.0ab lila 33.8a DM 100 10.44ab 18.9ab 4.5ab 22.2ab 6.3a 9.1ab 6.3a 107a 38.5a DM200 14.27ac 21.2ab 6.1b 34.8bc 6.4a 11.2c 7.8ac 129a 50.0a PM 50 14.35bc 27.5c 4.2ab 15.8ab 7.5ab 13.3d 8.9bc 174ab 198.1b PM 100 19.21c 31.7cd 5.6ab 10.5a 6.9a 15.6e 8.5ac 277c 269.9c PM200 26.28d 30.9cd 10.1c 29.8bc 5.9a 14.6de 9.1bc 251bc 340.5d NPK 11.06ab 35.0d 4.4ab 32.9bc 6.1a 7.7a 7.2ab 288c 49.8a Compost ll.lOab 17.1a 5.7ab 61.8d 8.6b 9.4ac 7.4ab 103a 37.7a

LSD 5.19 4.7 2.0 13.7 2.5 1.9 2.3 94 44.0

'Means followed by the same letter are not significantly different at p = 0.05 using LSD. b See Table 2 for designation of treatments applied to the tomatoes.

evaluated were taken after the second tomato harvest (Aug. 1983). Treatments caused highly significant differences in DPT A-extractable Cu, Mn and Zn. The two commercial fertilizer treat­ments produced the highest levels of DTPA Cu and Mn and the lowest pH, while the PM 100 and PM 200 treatments produced the highest levels of DTPA Zn (due to space limitations, none of the soils' data is shown).

In conclusion, manure salts were not a prob-

!em to tomatoes or Swiss chard. Manures con­tributed substantial amounts of all nutrients. The pig manure treatments, in general, produced the highest dry matter yield and most nutrient-rich Swiss chard crops. DTPA-extractable Mn and Zn were highly correlated to Mn and Zn uptake. Lastly, manure application rates must continue to be based on N and/ or P content since equiva­lent volumes of manures differ significantly in nutrients.

Acknowledgements

Thanks are extended to C Bishop for technical assistance in the field and laboratory and to the Nova Scotia Department of Agriculture and Marketing for financial support.

References

Adams P 1986 Mineral nutrition. In The Tomato Crop. Eds. J G Atherton and J Rudich. pp 281-334. Chapman and Hall, N.Y.

Keeney D Rand Walsh L M 1975 Heavy metal availability in sewage-sludge-amended soils. In Inter. Conf. on Heavy Metals in the Environ., Toronto, Ont., Oct. 27-31, 1975. pp 379-402.

Mahler R J, Bingham F T, Page A Land Ryan J A 1982

Effect of manures on tomatoes and Swiss chard 401

Cadmium-enriched sewage sludge application to acid and calcarious soils: effect on soil and nutrition of lettuce, corn, tomato, and swiss chard. J. Environ. Qual. 11, 694-700.

Ryan J, Harik S N and Shwayri R 1985 A short-term greenhouse evaluation of non-conventional organic wastes. Agric. Wastes 12, 241-249.

Sikora L J, Chaney R L, Frankos N Hand Murray C M 1980 Metal uptake by crops grown over entrenched sewage sludge. J. Agric. Food Chern. 28, 1281-1285.

Sims J T and Pill W G 1987 Composted sewage sludge and poultry manure as growth media amendments for tomato transplant production. Applied Agric. Res. 2, 158-163.

Warman P R 1990a Effects of animal manures and clover intercrops on barley and corn yields, and on tissue and soil copper, manganese and zinc. Bioi. Agric. Hortic. 6, 313-424.

Warman P R 1990b Fertilization with manures and legume intercrops and their influence on brassica and tomato growth, tissue and soil Cu, Mn and Zn. Bioi. Agric. Hortic. 6, 325-335.

M.A.C. Fragoso and M.L. van Beusichem (eds.). Optimization of Plant Nutrition, 403-407, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-102

Do seaweed extracts improve vegetable production?

P.R. WARMAN and T.R. MUNRO-WARMAN Nova Scotia Agricultural College and Coastal BioAgresearch Ltd., P.O. Box 550, Truro, NS Canada B2N 5E3

Key words: crop yield, seaweed extract, seed germination, tissue composition, vegetables

Abstract

Seaweed and seaweed extracts have been reported to improve seed germination, crop yield, disease resistance, frost hardiness, etc. This study evaluated the germination of pea and sweet corn seeds and the growth of five vegetables treated with seaweed extracts in experimental field plots. Commercial seaweed extracts (Maxicrop and Micro-Mist 300) and developmental kelp extracts (produced by ASL.) were tested for their effect on emergence and growth of various vegetable crops. At temperatures 18°C or below, both the Micro-Mist 300 and kelp extracts accelerated germination; however, when soil temperatures exceeded l8°C, none of the extracts positively influenced germination. Field experiments with beans, potatoes, cabbage, sweet corn and cucumbers were conducted in loamy sand and sandy loam soils near Truro, N.S. Treatments consisted of control, Micro-Mist 300, gypsum and as many as three rates of the developmental kelp extract. Micro-Mist 300 and kelp extracts were sprayed on the crop foliage at the rate and time recommended for the products. All treatments received N-P-K fertilizer according to the soil test recommendations for the particular vegetable. None of the seaweed amendments improved crop yield over the control; in fact, there were slight reductions in yields using the higher rates of kelp extract. Additionally, there were no significant differences in the elemental tissue composition of sweet corn and cabbage leaves, the two crops for which tissue analyses were conducted. We concluded that none of the seaweed or kelp extracts improved the growth of any of the vegetables tested.

Introduction

Seaweed has been used as a fertilizer for cen­turies, especially in regions with extensive coast­lines. Commercial seaweed extracts became available in the 1950's and have been reported to improve seed germination, root growth, flower production, fruit set, crop yield, disease resist­ance, frost hardiness, etc. (Booth, 1966; Senn, 1987; Stephenson, 1968). Seaweed extracts have been cited as a source of trace elements and growth regulators; however, researchers have found the response of the extracts to crops ranged from positive to negative.

Various fruits and vegetables have received foliar applications of seaweed extracts. For potatoes, Blunden and Wildgoose (1977) found a

significant increase in yield following application of a seaweed extract, while Kuisma (1989) and Morton (1991) found no yield increases. Lang and Langille (1984) and Dwelle and Hurley ( 1984), however, reported distinct varietal re­sponses to the use of seaweed sprays to different potato cultivars. Nelson and Van Staden (1984) found that nutrient-stressed greenhouse cucum­bers responded to the use of a seaweed concen­trate. In general, there are many other positive citations in Senn's 1987 book. Yet, because of the differences in soils, plants and seaweed extracts, world-wide evaluation of seaweed dips and sprays is still required.

This study evaluated the germination of pea and sweet corn seeds and the growth of five vegetables treated with seaweed extracts in ex-

404 Warman and Munro- Warman

perimental field plots. Our objective was to determine whether developmental extracts pro­duced by ASL from Ascophyllum nodosum were as good as commercial seaweed powders (Max­icrop and Micro-Mist 300) and if higher than recommended rates of ASL extracts would in­fluence plant growth.

Materials and methods

A. Seed soak and germination

Maxicrop (Zook & Ranck, Inc) and Micro-Mist 300 (Bio-Crop International Inc) were diluted according to the stated recommendations, i.e., 1:60 (w /v) and 1:2250 (w /v), respectively. Seeds of ten plant species were soaked overnight in either Maxicrop, Micro-Mist 300 or water. The soaked seeds were planted into Nova Mix 200 in fiber pots using 2 seeds I pot. At least five pots I plant species (blocks) were used. Pots were placed in a growth room at 20 ± 2°C. Pots were evaluated daily for emergence and growth.

Developmental kelp extracts were provided by ASL and tested against distilled water and Mi­cro-Mist 300 for their effect on germination at a concentration of 1:100 (w/v). Corn and pea seeds were soaked about 12 hours and then planted into fiber pots of Pro Mix. These experi­ments were conducted up to 12 days at tempera­tures below and above 18°C.

The elemental analysis of the seaweed extracts used in the majority of the seed soak and field experiments is shown in Table 1.

B. Field test plot evaluation

Potatoes, cabbage, sweet corn and cucumbers were grown in 1988 at Lower Onslow_, N.S., in a

Table 1. Dry weight analysis of seaweed extracts

Extract Na p K Ca

(g kg-])

Maxi crop 35 0.3 80 1.3 Micro-Mist 43 0.2 80 4.2

300 ASL#6 38 0.8 90 2.0

Pugwash sandy loam (water pH= 5.6). Beans were grown at Truro, N.S., in a loamy sand. For each crop a minimum of four treatments were used, set up in a randomized complete block design with at least three blocks. Treatments included a Control (water spray), Micro-Mist 300 (0.5% [w/v]) at the recommended time and rate of foliar application, ASL extract #6 (0.5% w/v) at the same time and rate of application as Micro-Mist 300, and ASL #6 at two and/or three times the rate of application and/ or fre­quency. Except for the bean experiment, a gypsum + fertilizer treatment was also used for comparison, since the Pugwash sandy loam test­ed low in available sulfate. Based on the Nova Scotia soil test recommendations for each crop, commercial N-P-K fertilizer and lime were ap­plied to all plots. All crops were hand-weeded; rotenone powder was applied as necessary for insect control. Only the marketable portion of each crop was included in the yield analysis.

Corn leaf and cabbage tissue samples were washed in distilled water, dried at 65 ± 2°C, ground through a 40 mesh stainless steel screen, digested using HN0 3 and analysed with a Jarrel­l-Ash 9000 !CAP spectrophotometer. Statistical analysis was performed using the ANOVA pro­gram on a T159 programmable calculator; where appropriate, LSD was calculated to indicate treatment mean differences.

Potatoes (cv. Superior) were planted on May 20 into two furrows per plot providing eight plants in total. Four blocks of six treatments were used. Treatments were Control, Gypsum (594 kg ha- 1), Micro-Mist 300 (1.48kg ha- 1 applied at the equivalent of 0.74 kg ha-l prior to blossom, 0.37 kg ha-l at early blossom, 0.37 kg ha-l 7-10 days after blossom), 1.48 ASL #6 (1.48kg ha- 1

applied identically to Micro-Mist 300), 2.96 ASL

Mg s Fe Mn Zn

(mgkg- 1 )

1.6 25 210 2.0 58 3.6 26 64 5.0 5.0

4.2 14 110 10 18

#6 (2.96 kg ha -I applied four times at the equivalent of 0.74 kg ha -I each), 4.44 ASL #6 ( 4.44 kg ha -I applied six times at the equivalent of 0.74 kg ha 1 ). Marketable yields were de­termined on September 6.

Cabbage (cv. Viking Golden Acre) was trans­planted (five plants/plot) June 7. The treatments and spray rates were the same as with the potatoes, except spraying was equally divided throughout the growing season. Plants were harvested beginning mid-August. Pie-shaped sec­tions were taken for tissue analysis from the largest three heads/plot.

Cucumber (Straight 8 and Double Yield Pick­ling) were transplanted into the field. Replicate 1 consisted of six or seven plants/plot of a pickling variety, while replicates 2 and 3 averaged four plants/plot of a slicing variety. Treatments con­sisted of Control, Micro-Mist 300 (1.48 kg ha -I applied at the equivalent of 0.74 kg ha -I 10 days after transplanting, 0.37 kg ha -I at pre-blossom, and 0.37 kg ha -I two weeks later), 1.48 ASL #6 (1.48 kg ha -I applied identically to Micro-Mist 300), 4.44 ASL #6 (4.44kg ha- 1 applied six times at 0.74kg ha- 1 about every 7-10 days), Gypsum ( 594 kg ha -I). Cucumbers which were at least 9 em long for picklers and 18 em long for slicers were harvested August 8 to September 11.

Sweet corn (cv. Butter Vee) was grown in three rows of 12 groupings, containing four seedlings/ group. Treatment groups were assigned in a completely randomized design to provide five replicates of six treatments: Control, Gypsum (30g/group), Micro-Mist 300 (2.6kg ha-\ two 15 mL sprays (0.5% w/v) in July), 2.6 ASL #6 (2.6 kg ha -\ two 15 mL sprays in July), 15.6 ASL #6 (15.6 kg ha -\ six 15 mL sprays from July 7-Aug. 1), Manure (approx. 1 litre beef manure I group). All treatments, excluding Manure, were sprayed periodically with 20-20-20 fertilizer solution. Leaf tissue samples from each plant were taken August 6 to 15 using the leaf immediately below the second maturing ear at silking. Corn ears were harvested August 15 to September 10 to provide an average of four ears/plot for yield analysis.

Seaweed extracts for vegetable production 405

Bush green and wax beans were grown in three replicates of four treatments containing about 25 plants/treatment. The treatments were Control, Micro-Mist 300 (1.48kg ha- 1 ; 0.74kg ha 1 full leaf stage, 0.37 kg ha · 1 at early bloom, 0.37 kg ha- 1 two weeks later), 1,48ASL #6 (1.48kg ha- 1 applied identically to Micro Mist 300), 4.44 ASL #6 ( 4.44 kg ha -I applied six times at 0.74kg ha- 1 about every five days). Yield data were collected six times from July 30 to August 15 even though the beans were severely affected by fungal diseases.

Results and discussion

A. Seed soak and germination

Of the 10 plant species evaluated in the seed soak trials at 20 ± 2°, corn, lettuce and peas germinated better with the Maxicrop and Micro­Mist 300 treatments than with the water Control. The germination of the other seven species was either unaffected by the commercial extracts or negatively affected compared to the Control.

At temperatures of 16-18°C, experimental kelp extracts and Micro-Mist 300 increased germination of corn and peas relative to the water Control. In contrast, when the germina­tion temperature was kept at 18-20°C for up to 12 days, kelp extracts and Micro-Mist 300 did not improve germination and growth of seed­lings.

B. Field test plot evaluation

The yield analysis showed that neither Micro­Mist 300 nor ASL #6 (at the same rate as Micro-Mist 300) improved crop yield compared with the Control (Table 2; data not presented for sweet corn). In addition, for all crops, the highest rate of ASL #6 (3 or 6 times the recommended equivalent) reduced crop yield compared with the Control, or was at least one std. dev. below the means of all treatments. Since the Micro-Mist 300 timing and concen­tration recommendations were followed, we can only conclude the seaweed extracts had no positive impact on crop yield. We would reserve judgment on the use of seaweed sprays on beans,

406 Warman and Munro-Warman

Table 2. Mean Potato (kg/pot), Cabbage (kg/head), Cucumber (kg/plant) and Bean (kg/plot) yields'

Treatmentb Potato Cabbage Cucumber Bean

Control 18.15ab 1.45 4.06 1.26a Gypsum 21.17b 1.76 4.45 Micro-Mist 17.71ab 1.55 4.05 1.18ab 1.48 ASL 16.47ab 1.46 4.30 1.09ab 2.96ASL 14.32a 1.21 4.44ASL 15.88a 1.23 3.87 0.95b

Mean± Std. Dev. 17.28 ± 2.34 1.44 ± 0.21 4.15 ± 0.23 1.12 ± 0.13

'Means followed by the same letter are not statistically different at p = 0.05 using LSD. b Control= Fertilizer (no spray); Gypsum= Fertilizer+ Gypsum; Micro-Mist= 1.48 kg ha- 1 Micro-Mist 300 +Fertilizer; 1.48 ASL=l.48kg ha- 1 ASL #6+Fertilizer; 2.96 ASL=2.96 kg ha- 1 ASL #6+Fertilizer; 4.44ASL=4.44kg ha-- 1 ASL #6+ Fertilizer.

Table 3. Cabbage elemental tissue composition

Treatmene p K Ca Mg s Mn Zn Cu B

(g kg -I) (mg kg -I)

Control 3.6 30.1 4.4 1.7 6.4 22 18 4.0 16 Gypsum 4.1 31.0 4.7 1.8 6.7 24 19 4.9 16 Micro-Mist 3.6 28.8 3.8 1.6 6.1 21 17 4.5 14 1.48 ASL 3.6 28.6 4.5 1.7 6.1 22 17 4.0 16 2.96ASL 3.9 28.4 3.7 1.7 6.2 22 18 4.1 13 4.44ASL 3.6 28.8 3.8 1.7 6.3 23 17 4.1 14

Mean 3.7 29.3 4.2 1.7 6.3 22 18 4.3 15 ±s.d. 0.2 1.0 0.4 0.1 0.2 0.4

'Control= Fertilizer (no spray); Gypsum= Fertilizer+ Gypsum; Micro-Mist= 1.48 kg ha -I Micro Mist 300 +Fertilizer; 1.48 ASL = 1.48 kg ha -I ASL #6 +Fertilizer; 2.96 ASL = 2.96 kg ha -I ASL #6 +Fertilizer; 4.44 ASL = 4.44 kg ha - 1 ASL #6 + Fertilizer.

Table 4. Corn ear leaf elemental tissue composition

Treatment' p K Ca Mg s Mn Zn Cu B

(gkg-1) (mg kg- 1)

Control 3.5 34.9 6.4 2.4 2.2 89 33 13.5 4.6 Gypsum 3.7 35.5 6.8 2.6 2.4 85 34 12.5 4.8 Manure 4.3 35.0 6.6 3.0 2.4 89 43 14.0 5.4 Micro-Mist 4.3 37.7 7.7 3.1 2.5 103 35 14.6 6.2 2.6 ASL 3.8 35.6 6.9 2.7 2.4 94 36 13.2 6.6 15.6ASL 3.5 36.1 6.5 2.5 2.6 89 39 13.1 4.4

Means 3.9 35.8 6.8 2.7 2.4 92 37 13.5 5.3 ±s.d. 0.4 1.0 0.5 0.3 0.1 6 4 0.7 0.9

"Control= Fertilizer (no spray); Gypsum= Fertilizer+ Gypsum; Manure= approx. I litre beef manure/ group; Micro-Mist= 2.6 kg ha -I Micro-Mist 300 =Fertilizer; 2.6 ASL = 2.6 kg ha -I #6 +Fertilizer; 15.6 ASL = 15.6 kg ha -I ASL #6 +Fertilizer.

however, because the ASL extract was not available to use early enough in their growth cycle and disease problems affected overall pro­duction. Elemental tissue analysis of cabbage and corn ear leaf showed no affect of any treatment on tissue composition (Tables 3 and 4).

To more effectively evaluate seaweed extracts, further field evaluation is needed to determine more appropriate application rates and timing.

Acknowledgement

The research was financed by a contract with Acadian Seaplants Ltd.

References

Blunden G and Wildgoose P B 1977 The effects of aqueous

Seaweed extracts for vegetable production 407

seaweed extract and kinetin on potato yields. J. Sci. Food Agric. 28, 121-125.

Booth E 1966 Some properties of seaweed manures. In Proc. Fifth Intern. Seaweed Symp., Halifax, N.S., pp 349-357. Pergamon Press, London.

Dwelle R B and Hurley P J 1984 The effects of foliar application of cytokinins on potato yields in south-eastern Idaho. Am. Potato J. 64, 293-299.

Kuisma P 1989 The effect of foliar application of seaweed extract on potato. J. Agric. Sci. 61, 371-377.

Lang D J and Langille A R 1984 Influence of plant growth stage and concentration of cytex and kinetin application on tuber yields of two potato cultivars. HortScience 19, 582-583.

Morton N 1991 The effects of seaweed extract on the yield of potatoes. Senior project, NSAC, partial fulfillment of B.Sc. degree, 34 p.

Nelson W R and Van Staden J 1984 The effect of seaweed concentrate on growth of nutrient-stressed greenhouse cucumbers. HortScience 19, 81-82.

Senn T L 1987 Seaweed and plant growth. T. L. Senn, Clemson, SC, 166 p.

Stephenson W A 1968 Seaweed in agriculture and horticul­ture. Faber and Faber, London.

G

Influence of fertilizers on yield and quality of various crops

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 411-415, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-155

Effects of conventional and multiple N application by fertigation on maize grain yields and NO; -N residues

P.H. GIRARDIN 1 , R. TRENDEe, J-L. MEYER1 , M. BIRGAENTZLE2 and P. FREYSS 3

1Laboratory of Agronomy, INRA, BP 507, F-68021 Colmar, France; 2Experimental Farm, F-68250 Rouffach, France; 3Lycee Agricole, F-67210 Obernai, France

Key words: fertigation, maize, multiple N application, N fertilization, nitrate, residual N

Abstract

When nitrogen is added to irrigation water (fertigation), N applications can be done in several dressings during the growing season. With this technique, it is possible to match the N requirements of maize crop. In such case, N efficiency should be enhanced, and, lower rates of nitrogen could be applied to avoid nitrate loss through leaching. In a two year field experiment the effect of N fertigation was compared with conventional N fertilization at two N rates (170 and 220 kg N ha -I). Grain yield, total biomass, N exportation and residual soil nitrogen were recorded. There was no influence of method of application on maize yields and yield components. But, the residual soil nitrogen was higher after multiple N applications, even at the low N rate. As no leaching was recorded, and, since theN recovery by the maize crop was the same for all the treatments, the difference in residual soil nitrogen was probably due to N immobilization by the soil biomass. The nitrogen applied late in the season (at tasselling) was not immobilized as fast as side-dressed nitrogen. N fertigation at the conventional N rate increases the risk of N leaching after maize harvesting, and to apply N at lower rates may decrease grain yield. So, if nitrate leaching is a potential risk, as it is generally the case in numerous irrigated areas, because of sandy or shallow loamy soils, N fertigation is not recommended so long as the N fertilization rates cannot be calculated more accurately.

Introduction

Recent developments of center-pivot irrigation systems in Europe allow to add N fertilizer solutions to the irrigation water in numerous irrigated areas. Generally, irrigation is applied in shallow soils which are loamy sands or sandy loams. The risk of leaching is very high in such soils, even if the amounts of water applied by the farmers equipped with a center-pivot system are low (5-10 mm). Multiple applications of N fertil­izer in irrigation water (fertigation), during the growing season of maize crop can be an effective method of reducing nitrate loss through leaching. Moreover, it is possible to match the N uptake of maize by using N fertigation, since it makes it possible to adapt the dressings to the crop's

requirements. A higher efficiency could be ex­pected with fertigation because N application is delayed until a time closer to that of actual plant uptake. However, the results from literature are contradictory. Gascho et al. ( 1984) observed that N which was applied in multiple applications through the irrigation water, produced yields equal to or lower than those obtained by conven­tional side-dress applications, even at lower rates of nitrogen fertilization. This conclusion is sup­ported by Maddox and Barnes (1985) with data from a four-year experiment. In contrast Rhoads et al. (1978) found a clear increase of grain yield with N application in seven dressings. Rehm and Wiese (1975) explained the inconsistancy of their results between years by the soil texture. Bundy (1985) in a review on timing of N applications on

412 Girardin et al.

maize concludes that a well-timed side-dress application can be as effective as multiple appli­cations in irrigated corn production. But if the residual soil nitrogen is taken into account, it seems that fertigation induces a higher post­harvest soil N content that the conventional side­dress application (Rehm and Wiese, 1975). Un­fortunately, very few data are available for the estimation of the environmental impact of ferti­gation. The farmers using this technique have no information on the potential N leaching. The objective of this study is to evaluate the effects of the N fertigation on grain yield and residual soil nitrogen.

Materials and methods

A two-year experiment was carried out on a site of the Rouffach Agricultural Station (Alsace, France). The soil (32% clay, 40% silt, 28% sand; 2.4% organic matter; pH: 6, 6, P20 5 , 16% and K20, 13%) is 0.8 m deep. The cultivar DEA was planted in late April (30-4-1990 and 17-4-1991, respectively) at 110,000 plants ha - 1 . 35 kg P and 100 kg K were applied before sowing. The rates and methods of N application are listed in Table 1. Individual plots were 1.50 m by 6.0 m and the treatments Du D 2 , D 3 and D 4 were replicated four times. Only two plots of the treatment T 0

(no nitrogen) were placed at each extremity of the experimental design as a reference, but this treatment was not included in the experimental design. The treatments were for 2 years running on the same plot. All N treatments were applied by hand in the form of urea, and, followed by 7 or 14 mm of irrigation water exclusive of pre­plant N, to simulate fertigation (Table 1 ). A

Table 1. Rates and methods of nitrogen application

moving ramp was used to apply 7 mm on one way and 7 mm on the way back. No run-off was observed on the experimental plots. During the growing season (last week of september ex­cluded) the maize crop received 196 and 210 mm of irrigation water, and, 228 and 187 mm of rainfall in 1990 and 1991 respectively. The irriga­tion was managed to keep 70% of the field capacity (100 mm).

Three soil samples were collected once a month at two depths (0-30 and 30-80 em) on each plot and mixed together to get one average sample per plot and per layer. NO~ -N was analyzed by a colorimetric method. Total N was measured by Kjeldahl procedure on grain sub­samples for each treatment. The treatment To (control without N) was used to estimate the soil nitrogen available from mineralization. The yield was measured from the production of each whole plot, harvested by machine.

Results and discussion

The quantity of N0 3-N in the soil profile was the same in each treatment plot before sowing in 1990 and 1991 (Fig. 1), so we may consider there was no after-effect of the 1990 N-treatments concerning the soil mineral nitrogen.

A multiple N application results in maize grain yields and yield components equal to those obtained by a conventional N side-dressing, for both nitrogen rates and both years (Table 2). In 1990 and 1991, 170 kg N ha - 1 should be enough to get the maximum grain yield. The extra nitrogen (220 -170 =50 N) could contribute either to enhance the nitrogen content of grains or dry matter, or, to increase the nitrate in the

Date N-treatment (kg N ha - 1 ) Irrigation (mm)

1900 1991 TO 01 D2 D3 D4 1990 1991

Preplan! 30-4 11-4 0 0 60 30 30 0 0 8 leaf stage 30-5 10-6 170 160 40 50 7 7 10 leaf stage 10-6 20-6 40 50 7 7 13 leaf stage 30-6 08-7 40 50 7 14 Tasseling 09-7 13-7 20 40 7 14 Total N 0 170 220 170 220 Treatment 170 220 170 220

conv conv 5X 5X

250

200

150

350

300

250

200

150

700

600

500

400

300

200

450

400

350

300

250

200

150

100

50

0 0 0

~ ~ m :!; m

"' ~ :l; "' :;; "' iii iii "' "'

01

(170N-CONV)

02

{220N-CONV)

0

~ ;?;

03

(170N-5X)

04

(220N-5X)

"' :s :;; :;;

m

Maize N fertigation and soil N residues 413

~ ~ ~ m

~ ~ m "'

" "' ~ ~ ~

Fig. 1. N0 3-N content in the soil profile during the period of the experiment (from March 1990 to October 1991); (o o): standard error.

414 Girardin et al.

Table 2. Yield, yield components and total N uptake as affected by rate and method of N application

Year N treatments

To 01 02 03 04 (0 N) (170 N-conv) (220 N-conv) (170 N-5X) (220N-5X)

Humidity (%) 1990 28.5* 28.8 a** 29.0 a 28.2 a 29.0a 1991 29.5 26.8 a 27.4a 26.0a 26.6 a

Number of ears- 1 (x 1000) 1990 106.8 107.2 a 108.2 a 107.2 a 107.3 a 1991 105.5 108.0 a 105.8 a 108.4 a 106.3 a

Number of grains ear -I

1990 244.2 323.2 a 329.0 a 330.9 a 346.1 a 1991 226.2 369.9 a 358.7 a 359.5 a 366.7 a

Thousand grain weight (g) (15% H,O) 1990 270.0 340.6 a 338.6 a 351.6 a 338.2 a 1991 255.6 335.5 a 345.4 a 333.6 a 334.4 a

Grain yield (Mg ha _,) (15% H,O) 1990 7.31 11.82 a 12.06 a 12.48 a 12.56 a 1991 5.79 12.98 a 12.80 a 12.68 a 12.73 a

Total N uptake (kgha--')*** 1990 120.0 264.8 c 270.2 c 262.2 c 287.9 c 1991 90.6 264.0 c 268.0 c 261.8 c 272.2 c

*Statistical treatments don't take into account T 0 because it was not included in the experimental design; **Data followed by the same letter are not significantly different at the 0.05 probability level; ***Root biomass was estimated as 10% of above ground plant part biomass, and the estimation of harvest index was 0.5.

soil profile. The quantity of nitrogen accumu­lated in the plant parts was not different between N treatments. But, at the time of maturity, residual soil nitrogen was higher for multiple N applications than for conventional ones (Table 3). The same phenomenon was observed in sandy soils by Plenet et al. (1991). At the rate of nitrogen (220 kg N ha - 1) calculated according to the recommendations, the nitrate level of the conventional treatment (D 2 ) which consisted only in two N applications was 45 and 49% (respectively in 1990 and 1991) of the five-appli­cation split treatment (D 4). The tendency was the same in 1991; at a low rate of nitrogen

(170 kg N ha - 1), there was a 81% increase of NO~ -N in the soil profile when N fertilizer was applied up to tasselling. Because of this excess of nitrogen in 1991 we can conclude that the quanti­ty applied during the growing season (170 kg N ha -- 1) was too high; the optimum rate was probably lower. In contrast, in 1990, 170 kg N ha - 1 seems to have been very close to the optimum rate, since the residual soil nitrogen was low (24 kg NO~ -N in the 0-80 em zone), and since the yield was not increased by an increment of 50 kg nitrogen.

However, the N mineralization estimated through the N uptake of T 0 was higher in 1990

Table 3. Residual N0 3 -N in the soil as the harvest time (27.09.1990 and 30.09.1991, respectively) (kgNha- 1)

Year

1990 1991

N treatments

To (ON)

13 8

D1 (170 N-conv)

21 ± 2 26± 8

02 (220 N-conv)

20 ± 7 35 ± 9

03 (170 N-5X)

24± 7 47 ± 8

04 (220 N-5X)

44± 9 70 ± 41

than in 1991 (148 and 106 kg N respectively). More nitrogen was available for plants in 1990, so the optimum rate of N fertilization should have been lower. Our previous assumptions don't agree with this conclusion, probably because of the imprecise estimation of N miner­alization. The treatment T 0 was for 2 years running on the same plot. This may explain why the grain yield obtained in 1991 was 20% lower than in 1990.

If the estimation of the optimum N fertilization rate was possible, the NO~ -N leaching should be avoided and multiple N applications could be used by the farmers. The fractioning of N ap­plied on maize, at the optimum rate, suppresses the risk of nitrate leaching at early growth stages. With the conventional technique, most of the nitrogen is applied between the 6 and the 8 leaf stages. If an excess of rainfall occurs a few days later, an important quantity of NO~ -N can be lost by run-off, and, in the shallow or sandy soils by leaching. The N fertigation removes this kind of risk.

Because the recommended N rates were too high in 1990 and 1991, it was impossible to test a sub-optimal level of nitrogen with the multiple application technique. Nevertheless, these ex­periments point out the difficulty to estimate, at the field level, the optimum rate of N fertiliza­tion. The corn grower who has irrigation equip­ment, has the two following options: either, to use the conventional method (preplant 50 N, and, the rest at 6-8 leaf stage) with the risk of leaching just after the second application, or, to use fertigation ( 4 to 8 N applications) with the risk of high residual N if the N rate is over­estimated.

Maize N fertigation and soil N residues 415

Even if the fertigation is cheap and easy to use, from an agronomic and environmental point of view, it is recommended to postpone the use of this method so long as the N fertilization rate cannot be estimated more accurately, or eventu­ally to grow a catch-crop after the maize (Lubet and Juste, 1979) to avoid N0 3 leaching due to an excess of residual N0 3-N in the soil profile.

Acknowledgement

This work was financially supported by RNED, France.

References

Bundy L G 1985 Review, Timing nitrogen applications to maximize fertilizer efficiency and crop response in conven­tional corn production. J. Fert. Issues 3, 99-106.

Gascho G J, Hook J E and Mitchell G A 1984 Sprinkler­applied and side-dressed nitrogen for irrigated corn grown on sand. Agron. J. 76, 77-81.

Lubet E and Juste C 1979 Effet de !'introduction d'une prairie temporaire, d'un cngrais vert et de !'exportation des residus de recolte sur les monocultures de mais implantces dans les sols sablo-limoneux du sud des Landes. C.R. Acad. Agric. Fr. 4, 295-309.

Maddox L D and Barnes P L 1985 Effects of time and rate of applied nitrogen and nitrapyrin on irrigated corn. J. Fert. Issues 2, 124-129.

Plenet 0, Lubet E, Desvignes P and Sombrun F 1991 Fertilisation azotee et composantes du rendement du mais: effets des niveaux et des modalites d'apport. In Physiologic et Production du mais. pp 366-382. AGPM, INRA, Paris, France.

Rehm G W and Wiese R A 1975 Effect of method of nitrogen application on corn grown on irrigated sandy soils. Soil Sci. Soc. Am. Proc. 39, 1217-1220.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 417-423, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-101

Yield and quality of irrigated summer-annual forages in southern Portugal as affected by nitrogen fertilization

MARIA ERMELINDA V. LOURENC,:O, MARIA DE LOURDES A. PIMENTA DaSILVA and LUIS MIGUEL B. MENDES Departmento de Fitotecnia, Universidade de Evora, Aptdo 94, 7001 Evora, Portugal

Key words: forage, nitrogen fertilization, nutritive value, pearl millet, Pennisetum glaucum, sorghum, Sorghum sudanense

Abstract

A study was conducted for two years (1990 and 1991) at nitrogen (N) levels of 0, 50, 100 and 150 kg N ha -I applied, with two summer-annual species: forage sorghum [Sorghum sudanense (Piper) Stapf] and pearl millet [Pennisetum glaucum (L.) R. Br]. Trudan 8 and Tifleaf 2 were the genotypes used for both crops respectively. The aims of this study were to evaluate the effect of N fertilization on forage yield and quality (crude protein content, dry matter digestibility and nitrate concentration). The amounts of irrigation water applied were 300 and 428 mm in 1990 and 1991 respectively. Forage was harvested in the vegetative stage three times a year. Nitrogen fertilization up to 100 kg N ha -I increased dry matter yield, shoot number and plant height. Crude protein and nitrate concentration showed the highest value at 150 kg N ha -I (159.3 and 10.2 g kg -I respectively). Dry matter digestibility was only significantly improved with the application of 50 kg N ha -I. Pear millet had a lower yield in the first two harvests, greater tillering capacity, shorter plants, higher leaf-to-stem ratio, higher protein content and higher dry matter digestibility than sorghum. The main conclusion of the study is that nitrogen fertilization increased dry matter yield and improved forage quality especially regarding protein content.

Introduction

In the southern region of Portugal (Alentejo), forage production in the summer is nihil under rainfed conditions and depressed under irrigation for most temperature species due to very high temperatures prevailing at this time of the year. The farmers usually face this situation by feeding the ruminants residues of small grain crops and poor quality hay which leads to low animal output or increases the production costs if the nutritional needs of the animals are met by the use of concentrates.

The utilization of summer annuals such as sorghum or pearl millet, which are drought resistant, in the irrigated areas with limited water availability might be an alternative for this prob­lem.

The effect of nitrogen (N) fertilization on such forages has been studied by several research workers elsewhere (Broyles and Fribourg, 1959; Elder and Denman, 1966; Muldoon, 1985) but in Portugal there is lack of research concerning the behaviour of those species under this kind of fertilizer treatment.

Methods

The experiment was conducted in 1990 and 1991 at the Mitra Experimental Station of the Uni­versity of Evora, located at 12 km away from Evora. The soil type was a gleysol, with pH(H20) 6.5, 0.452mg kg- 1 N, 145mg kg- 1 P, 100mg kg- 1 K in 1990, and pH(H2 0) 7.3,

418 Lourenfo et al.

0.226mgkg- 1 N, 172mgkg 1 Pand42mgkg- 1

K in 1991. The values of P and K were de­termined by the Egner-Riehm method (Riehm, 1958).

The treatments consisted of four N levels (0, 50, 100 and 150kg N ha- 1) and two species: forage sorghum [Sorghum sudanense (Piper) Stapf] and pearl millet [ Pennisetum glaucum (L.) R. Br]. Trudan 8 and Tifteaf 2 were the geno­types used for both species respectively.

The experimental layout was a split-plot with four replications, nitrogen levels as whole units, and species as subunits.

The subplots were 10m long, and comprised four rows at distance of 25 em. These were fertilized with 90 kg ha -I of potassium chloride in the second year. Seeding rates were 27 kg ha -I for sorghum and 10 kg ha -I for pearl millet, on 6 June 1990 and 3 June 1991. The plants were thinned to a plant population of 400000 plants ha -I. Weeds were controlled by hand in 1990 and by application of atrazine one month after seeding in 1991.

The experimental field was irrigated with a traditional sprinkler irrigation at seeding and when 50% of the available soil moisture was depleted, at 20 em depth, as indicated by ten­siometers. In this way, 300 (mm) and 428 (mm) were applied in 1990 and 1991 respectively. The most frequent and shortest irrigation interval was one week.

Forage was harvested in the vegetative stage at the time sorghum reached a height of about 30 em at the lowest level of N fertilization. The harvests were made on July 26th, August 23rd and October 4th in 1990, and July 25th, August 28th, and October 15th in 1991. The subplots were sampled by randomly harvesting three times 1 m in each of the two central rows. Plants were cut leaving a 5-10 em stubble height. After weighing the six random samples, from one of them after counting the number of tillers, five plants were used to measure plant height and percentage of leaves and stems.

Dry matter yield was determined after oven­drying at 6SOC for 48-72 hours. Crude protein was evaluated by Kjeldahl standard procedure (AOAC, 1975) and dry matter digestibility by the Tilley and Terry technique (Tilley and Terry, 1963).

Nitrate and cyanide were determined by the potentiometric method of ion-specific electrode (Cantliffe, 1970; Melo e Martinho, 1988).

The results were analysed as a split-split-split plot with years as whole units, nitrogen levels as subunits, species as sub-subunits and harvests as sub-sub-subunits.

Results

Dry matter yield

The values for this variable are presented in Table 1. The effect of nitrogen application was significant up to 100 kg ha -I level both years. The yields were greater in the second year of the study.

Mean dry matter yield for pearl millet, over harvests, was lower than for sorghum. Although the same trend was not shown for the third harvest where both species yielded similarly. For pearl millet dry matter yield was increasingly higher from the first to the third harvest but for sorghum the same did not happen in the third harvest.

Shoot number, plant height and leaf-to-stem ratio

Nitrogen application increased shoot number significantly up to 100 kg N ha -I especially for Tifteaf 2 (Fig. 1). Pearl millet showed the best tillering capacity. The number of shoots in­creased significantly from the first to the third harvest for Tifteaf 2. For Trudan 8 there was no significant difference between the last two har­vests.

In the second year of the study, plants were taller. That difference was particularly noticeable for sorghum (Fig. 2) which showed the highest values for plant height ( 45 em on the average). Plant height increased significantly with N appli­cation but only for sorghum up to 100 kg N ha -I. Plants were taller in the third harvest.

Leaf-to-stem ratio showed the highest values for pearl millet (Table 2) and decreased sig­nificantly from harvest to harvest for this species.

N fertilization of forages 419

Table 1. Dry matter yield (kg ha- 1) by year, nitrogen level. and harvest

Harvests Mean TOTAL

First Second Third

1990 Pearl millet

0 507 1170 1663 1113 3340 50 1438 2849 3586 2624 7873

100 1751 4470 5079 3767 11300 150 2071 4929 4595 3865 11595 Mean 1442 3355 3731 2842

Sorghum 0 1508 1622 2296 1809 5426

50 2929 3446 3776 3384 10151 100 3408 4297 4452 4052 12157 150 3447 5182 4736 4455 13365 Mean 2823 3637 3815 3425

1991 Pearl millet

0 1019 2340 3113 2157 6472 50 2468 4622 4281 3790 11371

100 3187 5081 5253 4507 13521 150 3738 4188 5043 4323 12969 Mean 2603 4058 4423 3694

Sorghum 0 2774 2637 1934 2448 7345

50 4245 4805 5185 4745 14235 100 5178 5690 5152 5340 16020 150 5727 5091 4592 5137 15410 Mean 4481 4556 4216 4418

LSD (0.05) for the means: years x species x nitrogen= 427; years x species x harvests= 358.

Forage composition

The values of crude protein content, dry matter digestibility and nitrate concentration are pre­sented in Table 3.

Crude protein content was higher in 1991, increased significantly with N fertilization up to the highest application rate, and showed higher values for pearl millet. The first harvest supplied a better quality forage in terms of crude protein content.

Dry matter digestibility was higher in 1991, increased significantly only from the control to the 50 kg ha - 1 nitrogen rate, was greatest for the pearl millet, and decreased from the first to the third harvest.

Nitrate concentration increased significantly

with nitrogen application from the 50 kg ha- 1 N to the 150 kg ha - 1 level. Pearl millet showed higher values than sorghum and from harvest to harvest the values decreased significantly.

With respect to the hydrocyanic acid (HCN) the results obtained for all the samples were below 0.06 mg kg -l hydrocyanic acid in both years.

Discussion

Total dry matter yield increased significantly up to an application rate of 100 kg N ha- 1 rate in both years (Table 1 ). The yields were greater in 1991 probably due to higher amounts of water applied, as required by tensiometers readings.

420 Louren~o et al.

1QQO Flret harveot

~L_----~~~----~m~--~~

Nitr-oQen Level& (kg ha-1)

Second harveot

Shoot number m-2 100 ... -.. 0

~ / ~

... 7 -----

0/ .. toO ... ...

0

0 " m ~

Nitrogen LeV&Io (kg ha-1)

Third harvest

•o e-----------------------1 ,LI ----~----~----~ o eo 100 ~

NltrO\Iffi Level& (kg ha-1)

~0 --------~----~-----

....

KO ~ 000 ·---

••o ... -----------&0 --

0 0 .. m ...

Nitrogen Levelo (kg ha-1)

Second harvest

Shoot runber m-2 ... I ... e-.-----·-------------

-"-----------------~ NO-~-----------·--------·--

... /

-~ .../ ... • .. e.---------------------..e.-----------------~ OL_----~----~~-----0 1(1 'tOO 160

Nllrollffi Levela (kg ha-1)

Third harveat

0 ... .. --

/ 0

/

... /

0~ -·--.. .. ,v --... ..

0 .. ""' "" Nitrogen Levels (kg ha-1)

Fig. 1. Shoot number per square meter for pearl millet(-) and sorghum(+) by nitrogen level, year, and harvest.

In both years, pearl millet yielded less dry matter than sorghum in the first two harvests, but not in the third harvest. This can be attribu­ted to the fact that several pearl millet tillers had elongated stems, being taller, in the third harvest

(Fig. 2), while in the previous harvests all the plants remained vegetative. According to Ong and Everard (1979) and Carberry and Campbell (1985) pearl millet, as a short day plant, when subjected to conditions of long photoperiods,

1990 Firat ha,.....t

7 Plant hel11ht (em) 0

..

.. c:======= -

-~~ .. --~

0

:..-0

60 ""' ...

NitrOgen Levela (kg ha -11

Second harvest

,.. Plant height (em)

..

.. /

/ /

.. '

0~

0 .. ... ,.. Nitrogen Levela (kg ha-11

Third harveat

•ol----~-------1

oL---~---~--~ 0 &o 100 '1&0

Nitrogen Levels (li!J ha-1)

N fertilization of forages 421

1991 Firat twveat

7 Plant height (em) 0

.. --

/ -v ..

0

f-0

0 10 1()() t60

NitrOgen Levelo (kg he-1)

Second harvest

,.. Plant height (em)

-----.. /

/ 7

v

..

..

.. 0

0 .. ... ,.. NitrOgen Levela (kg he-1)

Third harveet

·.L---~ .. ~--~ ... ~--~ Nitrogen Levela (kg he -1)

Fig. 2. Plant height for pearl millet (-) and sorghum ( +) by nitrogen level, year, and harvest.

higher than 13.5 hours, will flower later and the number of flowering tillers will decrease. Values such as that are registered during the summer in the Alentejo region of Portugal. Sorghum showed the lowest yield in the first harvest but

from the second to the third there was no significant difference.

Nitrogen fertilization increased the number of tillers per m 2 up to 100 kg N ha-l especially for pearl millet from the first to the third harvest

422 Lourenr;o et al.

Table 2. Leaf-to-stem ratio

Species Mean

Pearl millet Sorghum

Year 1990 6.6 1.2 3.9 1991 4.4 0.9 2.6 Mean 5.5 1.0

Harvests First 10.4 1.1 5.8 Second 4.3 1.2 2.7 Third 1.9 0.8 1.3

LSD (!l.05) for the means: genotypes= 0.9; years= n.s.; harvests = 1. 3; genotypes x years= 1.2; genotypes x harvests = 1. 8.

Table 3. Crude protein, dry matter digestibility and nitrates (gkg-1)

Crude Dry matter Nitrates protein digestibility

Year 1990 114.6 705.7 3.6 1991 122.7 693.4 4.2 LSD (0.05) 7.7 5.5 0.6

Nitrogen levels (kg ha - 1) 0 84.1 675.0 1.0

50 106.0 705.3 1.2 100 125.3 713.5 3.3 150 159.3 704.4 10.2 LSD (0.05) 4.5 21.0 0.5

Species Pearl millet 133.9 716.5 4.7 Sorghum 103.4 682.6 3.1 LSD (0.05) 4.1 4.7 0.5

Harvests First 137.5 744.1 5.6 Second 103.9 712.6 3.4 Third 114.6 641.9 2.7 LSD (0.05) 4.9 12.0 0.7

(Fig. 1 ). For sorghum this just happened from the first to the second harvest. This species responded to nitrogen mainly by growing taller than by tillering since the values for plant height were higher for this genotype and increased significantly with N application up to 100 kg N ha -I. The same did not happen with pearl millet. Plants were taller in 1991 and had higher yields than in 1990. This means that the difference in

dry matter yield was due mainly to stem elonga­tion.

As it would be expected leaf-to-stem ratio (Table 2) showed the highest values for pearl millet in the first year and decreased significantly from harvest to harvest for this genotype. Great­er leaf-to-stem ratios usually mean better quality at least in terms of protein content. In fact pearl millet showed the best value (Table 3) which approaches the values reported by Hanna et a!. (1989). The lowest values occurred in the second harvest were probably due to high temperatures that promote the transformation of cellular con­tent to cell wall components, and higher lignifica­tion of plant cell wall (Van Soest, 1984).

Pearl millet also showed the best values for dry matter digestibility which was influenced by nitrogen fertilization but only up to the 50 kg ha - 1 nitrogen rate. The first harvest supplied forage with the highest values. Thus, as com­pared with the traditional roughages which val­ues usually range between 400 and 600 g kg - 1

(Bento, 1990) the species studied supplied a much better quality forage, mainly with respect to pearl millet.

Nitrate concentration, even though significant­ly influenced by nitrogen fertilization up to 150 kg N ha -I and being greater for pearl millet, did not reach the critical value of 15 g kg - 1

considered unsafe by Reid and Jung (1973). The same also happened with respect to hydrocyanic acid concentrations which showed values of less than 0.06 mg kg 1 which are lower than the value of 200 mg kg - 1 considered dangerous by Kingsbury (1964).

Conclusions

Dry matter yield increased significantly up to 100 kg N ha - 1 in both years. Sorghum showed better yields than pearl millet for the first two harvests but in the third the results were similar. Forage sorghum responded to nitrogen fertiliza­tion mainly by stem elongations while pearl millet showed the greatest tillering capacity. The first harvest showed the lowest yield for both species.

Crude protein content increased significantly with N fertilization while with dry matter di-

gestibility this only happened from the control to the 50 kg ha- 1 rate. Pearl millet and the first harvest showed the best values for both vari­ables.

The main conclusion of the study is that nitrogen fertilization increased dry matter yield and improved forage quality especially regarding protein content.

References

Association of Official Analytical Chemists (AOAC) 1975 Official Methods of Analyses, 12th ed. AOAC, Washing­ton DC.

Bento 0 P 1990 Estudo comparativo do valor alimentar da aveia x ervilhaca conservada como feno e silagem. Tese de Doutoramento. Universidade de Evora, Evora, Portugal.

Broyles K R and Fribourg H A 1959 Nitrogen fertilization and cutting management of sudangrass and millets. Agron. J. 51, 277-279.

Cantliffe D Jet a!. 1970 The potenciometric determination of N0 3 and Cl in plant tissue. N.Y. Food Life Sci Bull. 3.

Carberry P S and Campbell L C 1985 The growth and development of pearl millet as affected by photoperiod. Field Crops Res. 11, 207-217.

Elder W C and Dennan C E 1966 Sudangrass and sudangrass hybrid research. Okla Agr. Exp. Stn. Processed Ser. 543.

N fertilization of forages 423

Hanna W W, Dujardin M, Monson W G 1989 Using diverse species to improve quality and yield in the Pennisetum genus. In Proceedings of the XVI International Grassland Congress. pp 403-404. Nice, France.

Kingsbury J M 1964 Poisonous plants of the United States and Canada. Prentice-Hall, Inc., Englewood Clifs, N.J.

Muldoon D K 1985 The effect of nitrogen fertilizer on growth, mineral composition and digestibility of a sorghum x sudangrass hybrid and Japanese barnyard mil­let. Aust. J. Exp. Agric. 25, 411-416.

Neto M P and Martinho 1988 Determina~ao potenciometrica de cianetos (glicosidos cianogenicos) com electrode espec­ifico, em alimentos para animais. Esta<;ao Zootecnica N acional. Vale de Santarem, Portugal.

Ong C K and Everard A 1979 Short day induction of flowering in pearl millet (Pennesetum typhoydes) and its effect of plant morphology. Exp. Agric. 15, 401-410.

Reid R Land Jung G A Forage animal stresses. In Forages. Eds. M E Heath et a!. pp 639-653. Iowa State University Press, Ames.

Reihm E 1958 Die ammoniumlaktatessigsounc-methode zur Beslimmung der leichtloslichen phosphonosoure in kar­bonotholligen Boden. Agrochimica 3.

Tilley and Terry 1963 A two-stage technique for the in vitro digestion of forage crops. J. British Grass. Soc. 18, 104-111.

Van Soest 1982 Environment and forage. In Nutritional Ecology of the Ruminant. 0 and B Books In, Corvalis, Oregon, USA.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 425-428, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-170

Variations in yield and macronutrient uptake in vining peas regardless of N fertilization

M.E. FERREIRA1 , M.L. FERNANDES2 and M.A. C. FRAGOS0 2

1Department of Vegetable and Ornamental Crops and 2 National Agronomy Research Station, IN/A, Quinta do Marques, 2780 Oeiras-Portugal

Key words: cultivar behaviour, macronutrients, N fertilization, nutrient uptake, nutrient removal, Pisum sativum L., vining peas

Abstract

Since the late seventies, pea (Pisum sativum L.) has been the main frozen vegetable in Portugal. Because farmers, as yet hardly familiar with the pea crop, usually apply excessive quantities of nitrogen fertilizers, four fertilizing trials were set up with cv. Jof on a sandy soil to study the effect of four nitrogen levels (0, 30, 60, and 90 kg ha-l) on grain yield and nutrient uptake (N, P, K, Ca, and Mg) at various development stages of the pea crop. Nitrogen treatments did not affect grain yield, the average value which was rather high: around 9 t ha-l ( tenderometer readings 100-110), disregarding harvest and processing losses. This suggests that the amount of nitrogen supplied by soil organic matter and biological N2 fixation was sufficient. Nutrient contents (percentage in dry matter) are reported for six stages of development. There was no influence of N level. Macronutrient removal was significantly different from year to year. It is concluded that N fertilization of peas is not required under the edaphic and climatic conditions studied.

Introduction

Frozen vegetables gradually became part of the Portuguese diet after 1973. Peas (Pisum sativum L.) represent 45% of these. The growing of vegetables for processing has greatly increased in the centre of Portugal where peas were intro­duced as a non-irrigated crop sown in late winter. Industry demand for peas stimulated farmers to occupy the sandy plains in Ribatejo. Plant population, formerly under 50 plants per square metre, was increased, as well as the amounts of nitrogen fertilizers used, despite an apparently good nodulation. This practice de­creases grain quality and increases production costs. Furthermore, it aggravates environmental pollution.

A study for a more judicious application of nitrogen was thus initiated, which aimed at determining the nutrient uptake patterns for

vining peas at certain stages of development, and at evaluating nutrient need. This should lead to a better balance of the mineral nutrition and better fertilizer recommendations in order to lower fertilization costs and to reduce pollution hazards in surface and underground waters.

Materials and methods

Four nitrogen levels, 0, 30, 60, and 90 kg ha -I, were studied during the period 1984/88 in a randomized block design with four replications. Similar sites were chosen on a representative Pliocene sandy soil in Ribatejo where the trials were laid out. Soil analysis values at sowing were: under 10 g kg -I for organic matter; 0.5-0.8 g kg -l for total N; 10-14 mg kg -l for N min, and 5.7-6.5 for pH.

Shortly before sowing, a dressing of a PK

426 Ferreira et al.

fertilizer (triple phosphate and potassium chlo­ride), at the rate of 46 kg ha- 1 of P and 87 kg ha _, of K was broadcast to the whole field, while the tree levels of nitrogen were applied as ammonium nitrate. Jof, a mid-late pea cultivar widely grown in Portugal, was sown in February at 70 plants per square metre, and grown as a non-irrigated crop to be harvested in May.

In the four years temperature and rainfall values were registered and averages varied, respectively, from 12.6 to 14.4°C and from 135 to 200mm.

Every year, samples of ten plants (roots ex­cluded) were collected at regular intervals, on each plot. Sampling occurred at the stages of development SD1, SD2, SD3, SD4, and SD5, respectively as defined by Knott (1987): first open flower, full flower, flat pod, pod swell, pod fill, and green wrinkled pod. This last stage was that of harvesting for freezing when pea tender­ness per plot gave 100-110 Tenderometer Read­ings (T.R.). At harvest time, plant weights and grain yields were evaluated irr a certain area (20m2 ) in each plot, but only for three years since in 1986 plants were damaged by heat.

Plant samples were dried at 70-80°C in a forced-air oven for dry matter (DM) evaluation, and ground in a Wiley mill ( 40-mesh) for mac­ronutrient assays. Analytical procedures were the micro-Kjeldahl technique for N determina­tion and nitroperchloric acid digestion (0.2 g/ 50 mL) for elemental determinations. Aliquots of the digest solutions were used for K determi­nation, by emission spectrophotometry (EEL); for Ca and Mg, by atomic absorption spectro­photometry (Perkin-Elmer 403), with Sr as the releasing agent; for P, by the yellow phos­phovanadomolybdate method, using a Unicam Pye spectrophotometer. Analytical results are expressed as percentage on DM basis.

As regards nutrient removal, data are reported as kg ha -I of each macronutrient, evaluated from plant weights and nutrient concentrations at harvest time.

Data were examined by analysis of variance and regression equations with growth stage were also determined.

Results and discussion

Since the analysis of variance applied to dry matter and macronutrient concentration data showed no significant differences among N levels, average values per year were used. Curves for fitted equations were drawn repre­senting four-year average values for plant dry matter production and macronutrient concentra­tion indices at the five critical stages of develop­ment as represented in Figure 1. The six selected developmental stages are equally spaced along the x-axis to enable a better pattern representa­tion of plant behaviour as regards nutrient con­centration, also for comparison between cul­tivars. Nutrient content per plant can be evaluated through Figure 1a.

Tables 1 and 2 deal with yield and nutrient removal values from only three-year experi­ments.

As shown in Figure 1a, during the Jof growing period, average values for dry matter growth rate of ten plants between stages 1 and 6 was 3.01 g per day. The greatest rate was between stages 2 and 3 (3.69 g) and the lowest between stages 1 and 2 ( 1. 98 g).

According to Figure 1b, from stage 1 to stage 6 mean N concentration decreased from 3.8 to 2.7%; again referring to the above cultivars, DSP and V, Jof was reported to have lower N concentration values by Ferreira and Fragoso (1990).

Mean P concentrations ranged from 0.505% to 0.395% (Figure lc). Identical values were found by McMahon and Price (1982) with a different cultivar, but lower than those of V and DSP (Ferreira and Fragoso, 1990).

Mean K concentrations in Jof as given in Figure 1d range from 2.88%, at stage 1, to 1.59%, at stage 6, which is in agreement with other authors (Ferreira, 1988; Geraldson et a!., 1973).

Fitted values found for Ca in Figure 1e show that cv. Jof follows the usual trend for pea cultivars, a fact not shown by cultivars V and DSP (Ferreira and Fragoso, 1990).

In Figure lf, mean Mg values range from 0.36 to 0.31% quite in agreement with values re­ported by Geraldson et a!. (1973) and Ferreira

Pea nutrient uptake and yield with N fertilization 427

;,o plants DM(J;) ~j(%lJM) , .. N \\) :: 4. OS - 0. 2SSC + S.Sse·l"1 f11 :: 0. 72

DH (g)" 33.98- 2.49SD t J.•lSSD1 n> D.7l

~ . ' . . ' ! ; :

0 ;troDM) kl '·" ·K(%DM) ,,,

p (\):: O.S6- S.?Oe-H' + S.19e- 1 " 1 R1 :: 0.42

~· 0

·~

eac%oM) Ca (\) = 1.29 - 4.40e·~B - l.59e·lH2

,,, •' = o.ss

' . ' Stages of development (SD)

" Days from sow uog

K (t) = 3.20- O.HSD + 7.17e·l502

:~ . . 0 •

+ • 0 0 . . .

Mg(%6M) OJI 0 Mg (\) "0.37 - l..17e·I!O + "J.60e·'"1

. ' . Stages of development (SD)

" Days from sowing

Fig. 1. Fitted equations of four-year average values at stages of development: !-first open flower; 2-full flower; 3-flat pod; 4-pod swell; 5-pod fill; 6-green wrinkled pod. (a)- DM of ten plants (b) to (f)-% concentration of N, P, K, Ca, and Mg, on DM basis. Reference per year: + 1984; e 1985; 0 1985; * 1988.

and Fragoso (1990), but lower than those sug­gested by McMahon and Price (1982).

Results are reported in Table 1 regarding macronutrient removal. No differences could be connected with N treatments. Except for K,

Table 1. Effect of year and N treatment on mean values of macronutrient removal by whole crop (kg ha - 1 )

N p K Ca Mg

Year 1984 97 b 31 a 116 a 81 a 25 a 1985 159 a 21 b 122 a 47 b 14 c 1988 158 a 21 b 114 a 75 a 20b

N treatment 0 kg ha _, 131 a 23 a 108 a 62 a 18 a

30 kg ha _, 142 a 24 a 119 a 64 a l9 a 60 kg ha _, 137 a 25 a 123 a 72a 21 a 90 kg ha 1 142 a 25 a 119 a 73 a 21 a

Means within a column followed by the same letter are not significantly different (p > 1% ).

differences were found between years for all the nutrients, certainly due to weather variations, in particular the rainfall values and distribution during the growth period in 1984 and 1988, consequently affecting dry matter production.

Table 2. Effect of year and N treatment on mean values of plant fresh weight and grain yield ( t ha - 1 )

Plant Grain

Year 1984 39 a lOa 1985 27 b 8b 1988 32 b 10 a

N treatment 0 kg ha _, 31 a 9a

30 kg ha- 1 32 a 9a 60kgha-' 34 a 9a 90 kg ha - 1 33 a 9a

Means within a column followed by the same letter are not significantly different (p > 1% ).

428 Pea nutrient uptake and yield with N fertilization

Both plant weights and grain yields showed significant differences between years (Table 2). Grain benefited from rainfall at stages 1 and 5 in both 1984 and 1988. Although N levels had no influence on yields, it is suggested that the increase of plant population in the presently reported trials may have contributed to such high yields, much higher than the national averages so far reported. This fact together with the lack of response to N fertilization and a remarkable nodulation (eventually efficient as judging from the pink coloured nodules), suggest that bio­logical N 2 fixation was the main N source.

Conclusions

Nitrogen fertilization of peas has not proved necessary. No influence of N dressings on both yield and macronutrient removal was detected. Suitable conditions for efficient biological N 2

fixation should be encouraged. Pea cultivar Jof is less demanding in nutrients

than former home grown cultivars. Nutrient uptake values fall in the common ranges of most known cultivars and follow similar nutrient pat­terns. This suggests it may become the standard vining pea in the main pea producing areas of Portugal.

Acknowledgements

Thanks are due to Dr Alan Scaife for text reading and criticism, also to Mrs M F Vargues, for assistance with assays, and to the Depart­ment of Water and Irrigation (INIA) for trial facilities.

References

Ferreira M E 1988 Variation in macronutrient content in peas for processing. Acta Hortic. 220, 267-274.

Ferreira M E and Fragoso M A C 1990 Criteria for fertiliza­tion of vining peas in Portugal. In Plant Nutrition- Physi­ology and Applications. Ed. M L van Beusichem. pp 723-727. Kluwer Academic Publishers, Dordrecht.

Geraldson C M, Klacan G R and Lorenz 0 A 1973 Plant analysis as an aid in fertilizing vegetable crops. In Soil Testing and Plant Analysis. Eds. L M Walsh and J D Beaton. pp 365-379. Soil Science Society of America, Madison, WI.

Knott C M 1987 A key for stages of development of the pea (Pisum sativum). Ann. Appl. Bioi. 111, 233-244.

McMahon C R and Price G H 1982 Nutrient Uptake Studies in Some Processing Vegetable Crops. Consolidated Fertiliz­ers Limited, Australia, 39 p.

Rankov V and Uzunova E 1988 On biological removal of nutrients with green pea yield. Acta Hortic. 220, 275-280.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 429-433, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-128

Nitrogen fertilization of Phaseolus vulgaris for freezing

C.F. CARRANCA\ A. FERREIRA2 , L. ANDRADA2 , M.L. FERNANDES 1 ,

M.E. FERREIRA3 and M.A.C. FRAGOS0 1

1 National Agronomy Research Station, 2 National Station of Technology for Agricultural Products and 3Department of Vegetable and Ornamental Crops, IN/A, Quinta do Marques, 2780 Oeiras, Portugal

Key words: beans, freezing industry, N balance, N requirement, Phaseolus vulgaris

Abstract

This paper reports some results of a N fertilizer experiment (20, 80, 140 and 200 kg ha -I) with irrigated Phaseolus vulgaris cv. Martingal, laid out for 3 years in a heavy textured soil. The treatment did not significantly affect pod yield, N removal or quality. At the end of the growing cycle, for the highest N levels, there was nitrate accumulation in the soil. Such facts seem to indicate that the lowest N level (20 kg ha - 1) was enough for yields higher than 10,000 kg ha - 1 , without decreasing the pod quality for deep freezing. Seasonal variations affected yield, N removal in pods and most quality characteristics with high significance, except for N removal in tops and alcohol insoluble solids which were not significantly affected.

Introduction

According to Piha and Munns (1987), nitrogen fixation by Phaseolus vulgaris L. is mostly unreli­able and N fertilization of field grown plants is recommended. Poor N2-fixation has been attrib­uted to difficulty of establishing effective symbioses in field, and to genetic variability in capacity to fix N 2 • Little is known regarding N requirements for this crop. According to FAO (1979) fertilizer requirements for high product­ion are 20-40 kg ha - 1 of N.

Climatic conditions also seem to be quite important, namely water availability and tem­perature. Excessive water and hot weather cause flower and pod drops, and increase incidence of diseases. Daily optimal mean temperatures range from 15 to 20°C. High temperatures also increase pod fibre content (FAO, 1979).

'Martingal' is a green bean cultivar of the fine type with interest for export to the Northern European market. Since mechanical harvesting is being introduced and new irrigated areas are potentially promising to this crop in Portugal, as

an alternative to maize and tomato in Portugal, it would provide raw material to fill up the gap between pea and pepper processing in freezing industry.

Results are summarized for a 3-year fertilizer experiment with irrigated Phaseolus vulgaris as affecting pod yield, N removal and quality characteristics for freezing industry.

Methods

Field experiments with Phaseolus vulgaris cv. Martingal were conducted in 3 years, in an alluvial soil of Quinta do Marques (Oeiras). Main soil characteristics are shown in Table 1.

The experimental design was on randomized blocks, with 4 N rates (20, 80, 140 and 200kgha- 1) as ammonium nitrate 20.5%, and 5 replicates. A basal dressing was applied (20kgha- 1) in all plots at sowing time; 2 dif­ferentiated top-dressings were also applied at stage of 1112-2 112 leaves and at flowering. Sprinkling irrigation was controlled to avoid N

430 Carranca et al.

Table I. Physical and chemical characteristics of the sandy clay loam soil

Year pH Or g. Total N Inorganic-N CEC Available (H,O) matter (gkg-1) (cmol(+)kg- 1)

(gkg-1) N-NH 4 N-N0 3 p K

(mgkg- 1 ) (mgkg l)

1987 7.80 15.8 1.31 6.97 1988 7.80 14.9 1.14 10.55 1989 7.80 14.7 1.03 16.84

leaching and the crop needs. The plant popula­tion was 36 plants m -z.

During the growing cycle (sowing, 1112-2 112

leaves, 3 112 leaves, flowering and harvest), soil samples were taken up in all plots, at 0-20 em depth, and were analysed for inorganic-N to evaluate the distribution of N in soil. Methods used for soil sample analyses follow those de­scribed in Carranca (1986).

In each plot, plant samples were collected at harvest time, evaluated as regards seed to pod weight ratio. Yield, N removal and pod physical and chemical characteristics- sieve size, weight per pod, pod bending, dry-matter (D.M.) and alcohol insoluble solids (A.I.S.)- were evaluated. Total N in the plant samples was determined by micro-Kjheldahl method, and N removal was evaluated as depending on top and pod yield. Pod bending was estimated according to Rodrigo eta!. (1977), and D.M. and A.I.S. as referred in Association of Official Analytical Chemists ( 1984). Pod samples were frozen in an air tunnel, after blanching in boiling water dur­ing 3 minutes (negative peroxidase activity) and cooled by water immersion. After freezing and storage at -20oc during 6 months, organoleptic characters were evaluated by a group of 8 trained panelists. For different aspects of colour, flavour and texture, a score from 1 (the least presence of a character) to 5 (the greatest presence of a character) was attributed (Adams et a!., 1981 ).

Through ANOVA, treatment effects were esti­mated as regards yield, N removal, pod charac­teristics, organoleptic assessment and residual inorganic-N in soil. The effects of seasonal variations and sampling time were also evaluated.

19.29 20.91 211 299 19.67 19.21 173 276 25.39 19.78 229 208

Table 2. ANOVA results for treatments effect on soil inor­ganic-N

Source of variation N-NH; N-No;

Year 70.77*** 12.75*** N u.s. 13.71 *** Year x N u.s. u.s. Time of sampling 24.12*** 23.72*** Year x time of sampling 54.98*** n.s.

u.s.,*,***= F-values non-significant and significant at 0.1% probability.

Results

Table 2 reveals the highly significant effects of climate and time of sampling on nitrate and ammonium contents in soil. N-trcatment only affected nitrate with a high significance.

Yearly, N did not significantly affect ammo­nium content, whereas nitrate was much sig­nificantly affected (Table 3). The different sam­pling time showed significantly high differences in the amount of both N-forms in soil.

In Table 4, residual inorganic-N values are shown as affected by N treatment and growing cycle stages. In soil, ammonium significantly decreased along the cycle while nitrate increased at harvest, especially in the years 1988 and 1989.

Tables 5 and 6 show the results of analysis of variance and means comparison of plant charac­teristics, for the 3-year experiment.

Results of the analysis of variance showed a non-significant effect of N treatment on yield, N removal, pod characteristics and organoleptic assessment. However, a high significant effect of climate (temperature, daylight or rainfall) on these plant parameters was observed, except for

Nitrogen fertilization of Phaseolus vulgaris 431

Table 3. ANOVA for soil inorganic-N in soil as affected by N and sampling time

Source of 1987 1988 1989 variation

N-NH; N-NO~ N-NH: N-NO; N-NH; N-NO-3

N n.s. 8.48*** n.s. 5.95*** n.s. 4.41 * * Sampling 13.91 *** 14.22*** 102.52*** 7.14*** 32.70*** 9.20***

n.s., **, *** = F-values non-significant and significant at 1% and 0.1% probabilities.

Table 4. Residual inorganic-N as affected by N treatment and growing cycle stages

Source of N-NH: (mg kg- 1) N-No; (mgkg- 1 )

variation 1987 1988 1989 1987 1988 1989

Nl 7.45a 13.34 a 10.02 a 14.92 b 17.76 b 20.51 b N, 6.60 a 13.17 a 10.24 a 14.99 b 22.09 b 22.75 b N3 6.79 a 12.50 a 10.24 a 20.83 ab 24.11 ab 25.81 ab N4 6.91 a 12.42 a 9.92a 24.83 a 32.40 a 35.72 a

Sowing 7.11 ab 10.63 c 16.84 a 19.29 a 19.78 b 25.39 b 1112-2 112 leaves 8.59 a 13.94 b 8.97b 7.36 b 19.60 b 20.47 b 3112 leaves 6.51 b 20.13 a 6.60b 18.84 a 23.10 b 18.81 b Flowering 4.62c 24.75 a Harvest 7.86 ab 6.74 d 8.01 b 24.23 a 33.89 a 40.11 a

Means within a column followed by the same letter are not significantly different (p > 1% ).

Table 5. Results of analysis of variance and mean values of pod yield, N removal and other pod characteristics

Years Pod N removal in N removal in Seed/pod Sieve size grading Pod yield top pod (gkg-1) (g kg I) weight (g)

(kgha- 1 ) <P ~6.5mm 6.5<¢~8.0mm 8.0<</J ~9.5mm

1987 13,590 a 84.23 a 84.84 a 89.6 a 49 b 473 b 475 a 3.7 a 1988 10,870 b 70.72 a 51.77 b 71.9 b 128 a 680 a 192c 3.2 b 1989 10,570 b 80.46 a 66.28 b 101.3 a 142 a 469 b 389 b 3.0c

Year n.s. *** *** N n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. Year x N n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s.

Means within a column followed by the same letter arc not significantly different (p > 1% ); n.s. and *** = F-valucs non­significant and significant at 0.1% probability.

Table 6. Results of analysis of variance and mean values of six organoleptic characteristics

Years Colour Texture Flavour'

Green' Uniformityb Soft' Firmd Fibrous'

1987 3.1 a 3.3 b 3.0 b 3.4 b 2.0 a 3.0 b 1988 3.1 a 4.0 a 3.7 a 4.2a l.Ob 4.0 a 1989 3.2 a 3.3 b 2.9 b 4.0 a 2.2 a 3.3 b

Year N n.s. n.s. n.s. n.s. n.s. n.s. Year x N n.s. n.s. n.s. n.s. n.s. n.s.

Means within a column followed by the same letter are not significantly different (p > 1% ); 'green: 1- very pale/5- very dark; buniformity: 1- non-uniform/5- very uniform; 'soft: 1- not soft/5- very soft; dfirm: 1- not firm/5- very firm; 'fibrous: 1- not fibrous/5- fibrous; 'flavour: 1- weak/5- strong; n.s. and *** = F-values non-significant and significant at 0.1% probability.

432 Carranca et al.

N removal in tops and A.I.S. values which were not significantly affected.

Discussion

Soil nitrate was accumulated with increasing N rates (Table 4). At N 3 and N 4 levels, the increase did not statistically differ, but was significantly higher than in the case of lower N-levels. A significant accumulation occurred at harvest, namely in 1988 and 1989. Ammonium was not accumulated with the increase in N rates, even at last sampling time, probably due to favourable nitrification conditions. Controlled irrigation to avoid nitrogen losses into the soil seems not to have caused deeper nitrate leach­ings, as observed by N-N0 3 accumulation in the above layer.

These facts indicate that the higher N applica­tions were not completely uptaken by the crop, namely N applied as 2nd top-dressing at flower­ing time, and it seems that as much as 80 kg N ha - 1 split only twice (one as top-dres­sing) would be efficiently used by the crop.

In Table 5 we may observe that pod yield significantly decreased from 13,590 kg ha - 1 in 1987 to 10,570 kg ha - 1 in 1989. Even the lowest yield exceeded the average yield in Portugal (8,000-9,000 kg ha - 1 ), according to the informa­tion given by Direc<;ao-Geral dos Mercados Agrfcolas e Industrias Agro-Alimentares (1991 ). Mack (1983) obtained higher yield (around 19,000 kg ha - 1), for the same plant population (36 plants m - 2 ).

N removal in the tops was not significantly different each year, varying from around 84 kg ha 1 in 1987 to 80 kg ha - 1 in 1989, while N removal in pods significantly decreased from 85kgha- 1 in 1987 to 66kgha- 1 in 1989 as a consequence of a significant decrease in total N.

Estimating N balance, an accumulation of nitrogen in soil was verified at 0-20 em depth, especially as nitrate, even at lowest N level applied.

Pod sieve size also significantly differed each year, with the highest number of 6.5-8 mm diameter pods in 1988, and the highest number of bent pods in 1987. Quality characteristics

given by organoleptic assessment, particularly colour, softness and fibrousness, depend on seed/pod ratio (w /w): a high ratio (> 100 g kg - 1 )

origins a non-uniform colour and tends to be fibrous (Campden Food Preservation Research Association, 1985). In the reported experiment, green beans showed a medium green colour (Table 6). A uniform colour, soft, firm and non-fibrous texture, and a natural green bean flavour of the pods were the characteristics observed, in 1988 at the spring cycle with 59 days, against 63 days in 1987 and 61 days in 1989. Mean daily temperature for the 3-year experiment was above 20°C the optimum rec­ommended by FAO (1979).

Under the studied field conditions, the lowest N rate (20 kg ha - 1) was enough for green bean yields higher than 10,000 kg ha - 1 , with no de­crease on pod quality for deep freezing, and with a higher N fertilizer efficiency. Such result agrees with the N-rate recommended by FAO (1979). Seasonal variations (temperature, daylight or rainfall) were quite important for pod quality.

Acknowledgement

The authors acknowledge those helping with analytical work and paper revision.

References

Adams M J, Bedford LV and Geering J 1981 A method for the sensory appraisal of quality of processed vegetables varieties. Technical Memorandum 278. Campden Food Preservation Research Association. Campden, 319 p.

Association of Official Analytical Chemists 1984 Official Methods of Analysis. 14th Edition. Washington, DC. pp 613-616.

Campden Food Preservation Research Association 1985 Campden quick frozen vegetable specification: Cut green beans. Glos. GL55 6 LD.

Carranca C F 1986 Nitrogen availability and ammonium fixation in some maize cultivated soils of Portugal. Thesis for M.Sc. in Soil Science. Oeiras. 89 p.

Direc9ao-Geral dos Mercados Agricolas c lndustrias Agro­Alimentares 1991 Plano sectorial. Frutos e Horticolas Transformados. Reg. (CEE), N. 866/90. Ministerio da Agricultura, Pescas e Alimentac;ao, Lisboa. 261 p.

FAO 1979 Yield response to water. In Fao Irrigation and Drainage Paper (33). Ed. Food and Agriculture Organiza­tion of the United Nations. pp 77-79. Rome.

Mack H J 1983 Fertilizer and plant density effects on yield performance and leaf nutrient concentration of bush snap beans. J. Am. Soc. Hortic. Sci. 108, 574-578.

Piha M I and Munns D N 1987 Nitrogen fixation capacity of field-grown bean compared to other grain legumes. Agron. J. 79, 690-696.

Nitrogen fertilization of Phaseolus vulgaris 433

Rodrigo M, Navarro A, Duran V J L and Saf6n J 1977 Selecci6n de indices de madurez de judfas verdes para conserva. Agroquimica y Tecnologia de Alimcntos 17, 95-110.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 435-438, 1993, © 1993 Kluwer Academic Publishers. PLSO IAOPN-041

The effects of different nitrogen, phosphorus, potassium fertilizer application on tomato seed properties

NEVIN ERYUCE and SENAY AYDIN Department Soil Science, Faculty of Agriculture, Ege University, TR-35100 Bornova, Izmir, Turkey

Key words: germination rate, germination vigor, Lycopersicon esculentum Mill, tomato

Abstract

Nitrogen, phosphorus and potassium were applied to L. esculentum Mill cv Rio Grande processing tomato in an experiment with five replications. The two nutrients, except the varying one were kept constant. The experiment was carried out under field conditions and thousand seed wt, germination rates and germination vigor of seeds were determined at the maturity stage. The highest N, P and K rates have resulted in the lowest values for all the properties, except thousand seed wt for K and N. Based on the LSD test, only the highest dose of each three nutrient was significantly different from the others for germination vigor. The means of doses vary significantly for the germination rate.

Introduction

Fertilizers not only increase the yield of process­ing tomatoes but aiso have some effect on seed quality characteristics. As reported by Toon et a!. (1991) seed related maternal effects can arise through the influences of both maternal geno­type and maternal environment on seed develop­ment. Numerous publications are available perti­nent to the influence of fertilizer applications on the yield of tomatoes. So far seed quality charac­teristics are influenced by nutrition of the mother plant has not been widely demonstrated. Alek­seev (1980) reported that K fertilisers and N top dressing during fruiting led to the best germina­tion and emergence of tomato seedling. Vadivelu (1983) achieved the best results with regard to seed yield and quality at N = 100 kg ha 1 , P = 44 kg ha -I and K = 82 kg ha 1 rates for tomato cv co 21 Seno et a!. (1987) observed that seed germination was higher in the absence of P and the effect of K on seed quality was variable, influence of P and K fertiliser levels on 1000 seed wt was not observed. George et a!. (1980) and Varis et a!. (1985) studied the effect of mineral

nutrient on tomato seed quality cv Moneymaker glasshouse adapted grown variety. Both re­searchers observed that higher mineral nutrition gave rise to a significant improvement in seed quality and the highest seed weight was at the levels of N = 100 g m -z, P = 43 g m -z, K = 166 g

-2 m . The production capacity of processing

tomatoes in Turkey is about 1.8 million ton per year including 3000,000 tons tomato paste (Vural eta!., 1992). Of this amount, 127.794 tons were exported during the 1991-92 trading season (Tomato News, 1993).

In Turkey, proscssors generally collect seeds from mother plants; produce the seedling and then distribute these to the growers. In seed selection, maturity and morphological properties of maternal tomato fruits arc taken into consid­eration. However no attention is paid to the nutrition of the mother plants.

The purpose of this study was to investigate the effect of N, P, K fertilizers on certain important seed quality characteristics such as thousand seed wt, germination rate and germina­tion vigor of processing tomatoes.

436 Eryuce and Aydin

Methods

Lycopersicon esculentum Mill cv Rio Grande processing tomato has been grown under field conditions in the Aegean Region located in the western part of Turkey where processing tomatoes are widely cultivated. Growing season begins by early sowing in March under protected conditions in the seed bed and the seedlings are transplanted at the beginning of May, when the temperature is suitable. The experiment was conducted on clay-loam soil, having pH 7.84, total soluble salt 0.08%, total N 0.118%, avail­able P 0.8mg kg- 1 , K 190mg kg- 1• The plots were arranged in a randomized block design with 5 replicates each having 5 levels for N, P and K. A total of 75 plots were prepared. The applica­tion rates were as follows N· O· 60· 120· 180· 240 kg ha -I, P: 0; 17; 35; 52; 7o kg h~ -I a~d K~ 0; 50; 100; 150; 200 kg ha -I, 0 kg ha -I refers to the control. The two nutrients excluding the varying one were kept constant (N = 120 kg ha - 1, P = 35 kg ha- 1 , K = 100 kg ha -I). Half of N and all of the other two fertilizers were applied as basal dressing in (NH4 ) 2S0 4 , triple super phosphate and K2SO 4 forms. The second part of N was applied as top dressing in (NH4 )N0 3 form.

Fruit samples were taken at harvest time from all plots randomly, seeds were extracted, washed and dried at room temperature.

Table 1. Results of ANOVA for N, P, K doses

Thousand seed wt Source of Variation Replicates

Treatments

Germination rate Source of Variation Replicates Treatments

Germination vigor Source of Variation Replicates Treatments

NDose

0.284 ns'

28.109**

0.553 ns 27.583**

0.682 ns 10.165**

For laboratory germination tests, seeds were placed on 2 layers of paper towel in petri dishes and then were moistened with distilled water. Each plate contained 100 seeds and was kept in the germinator at 25°C. When radicles were 1 mm long the seeds were considered to be germinated. Seed germinations were recorded daily from the second day to the eighth day. Percent of first counting was evaluated as germi­nation rate (speed of germination) and percent of total germination was evaluated as germination vigor (percent germination). Thousand seed wt was calculated after weighing 100 seeds (Scpetoglu, 1987).

The effects of the applied fertilisers on germi­nation rate, germination vigor and thousand seed wt were evaluated statistically by analysis of variance (ANOVA). Least significant differences (LSD) were used to compare treatment means when the F values were significant (Little and Hills, 1978).

Results

The thousand seed wt was significantly (p ~ 0.01) affected by each of the application of N, P and K doses (Table 1). Results were classified in different groups for three nutrients, except 120 and 180 kg ha -I N treatments which occured in homogeneous groups. In comparison with the

P dose Kdose

0.995 ns 0.684 ns

17.251 ** 49.011 **

0.348 ns 1.777 ns 31.617** 100.562**

1.088 ns 0.605 ns 10.195** 271.885**

": Means of (5) replicates arc not significantly different (p oS 0.01, F-test) for three seed properties.

Effects of fertilization on tomato seed properties 437

Table 2. Effect of N, P, K fertilizer on thousand seed wt, germination rate and germination vigor

Doses (kg ha _,) Thousand seed wt (g) Germination rate (%) Germination vigor (%)

N {) 3.32 c 73.2BC 98.4A 60 3.59 B 90.8A 99.2A

120 4.07 A 86.0 AB 99.2A 180 4.08A 64.4C 92.4A 240 3.74B 31.6 D 64.8B

p () 4.32A 74.2B 93.2A 17 3.48CD 97.2A 99.6A 35 3.64BC 96.8A 100.0 A 52 3.83 B 69.2B 92.8A 70 3.24D 52.4 c 80.8B

K 0 3.48 c 75.6B 99.6A 50 3.71 B 91.6 A 99.2A

100 3.34 D 81.6 B 99.6A 150 3.66 B 77.6B 96.0A 200 3.97 A 12.0C 41.2B

Means followed by the same letter in the same column are not significantly different by LSD test, p,; 0.01.

control, all N applications revealed higher seed weights but the highest dose showed a slight decrease (Table 2). Increasing P levels caused negative effect while the K levels had positive effect on the thousand seed wt.

Germination rate and germination vigor were affected by different doses of N, P, and K individually, as shown in the table of analysis of variance (Table 1). The maximum value of germination rate was achieved at 60 kg ha -I N application. The increasing doses resulted in a decrease and above 120 kg ha -I N applications, values were found below the control. According to the LSD test, the dose means were placed in different groups for each of the N applications for germination rate (Table 2). With P applica­tions, an increase was observed beyond the control dose; the maximum value reached was at 17 kg ha -I P which occurred in the same group as 35 kg ha -I P dose. The germination rate, reached a maximum at 50 kg ha -I K application; 100 and 150 kg ha -I K applications gave similar results although other doses were placed in separate groups (according to LSD test).

For germination vigor, the highest doses for each of the three nutrient applications were placed in the separate groups with significant decreases, the remaining doses placed in the same groups (Table 2).

Discussion

Table 1 revealed that application of N increased thousand seed wt up to 120 kg ha- 1 , germination rate up to 60 kg ha-t while germination vigor according to the LSD test was in the same group from control to 180 kg ha -IN application. At the highest dose a decrease was recorded, which caused formation of a separate group. The results implied that there could be no relation­ship between thousand seed wt and the other two traits.

According to the LSD test, the most economic N fertilizer doses were 120 kg ha -I for thousand seed wt, 60 kg ha -I for germination rate and control dose for germination vigor. Vadivelu (1983) reported that the best seed yield and quality was obtained in a 100 kg ha -I N applica­tion.

It seems certain that increasing N applications up to 180 kg ha -I in order to obtain a high tomato fruit yield, with equal importants of seed quality properties from economic point of view, will not cause any negative effect on germination vigor (Table 2).

P applications caused fluctuations in thousand seed wt, however, it gave a continuous decrease trend from controls. Germination rate increased from control to 17 kg ha -I P application and

438 Effects of fertilization on tomato seed properties

according to the LSD test, 35 kg ha ··I P applica­tion which gave similar results. Afterwards a decrease was recorded. Germination vigor in­creased up to 35 kg ha -I then a decrease was observed but this trend was not significantly different according to the LSD test (Table 2) except at the highest (70 kg ha -I) dose which showed a decrease. Results implied that germi­nation rate and germination vigor are not related to the thousand seed wt. When economic aspects of the situation are considered 17 kg ha -I P application seemed ideal for germination rate and control dose for the other two parameters. Seno et a!. (1987) after conducting similar ex­periments on a large scale applying 0-87-174 and 260 kg ha -I P applications to Rio Grande tomato and stated that germination rate and germination vigor values found at control appli­cations were the best ones. These results have been supported by the significant decrease in two parameters at 70 kg ha -I P application which was the highest dose given in our experiment.

In fertilizer usage for tomato fruit yield, in­creasing applications of P up to 52 kg ha -I dose did not have a negative effect on germination vigor.

Thousand seed wt showed a continuous ten­dency to increase by the increasing amount of K applications. Germination rate increased up to 50 kg ha -I by K application but it decreased at higher doses. Although means for germination vigor were in the same group from control to 150 kg ha ·I at K applications it showed a de­crease at the highest dose. When thousand seed wt reached the maximum by the application of 200 kg ha -I K dose the other two parameters showed a contrasting trend and decreased con­siderably, as it is shown in the LSD test (Table 2). From the economic aspect, the optimum dose for germination rate is 50 kg ha -I K, it is the control dose for germination vigor, however, it is 200 kg ha -I for thousand seed wt. Vadivelu (1983) suggested that the profitable best quality seed could most profitably be achieved by 82 kg

ha -I K application. Furthermore if K doses are increased up to 150 kg ha -I for yield of tomato fruit it will have no negative effect on germina­tion vigor.

Results showed that the economic doses for thousand seed wt were 120 kg ha- 1 for N, control dose for P and 200 kg ha -I for K. For germination vigor, the control doses of the three elements were the best. For the germination rate, 60kg ha- 1N, 17kg ha- 1 P and 50 kg ha- 1

K were efficient. We have found that seed weight has little, if

any, effect on germination rate and germination vigor.

References

Alekseev R V 1980 Effect of mineral fertilizers and her­bicides on the sowing quality of stored tomato seeds. Khimya v sle'skom Khozyaistve 18, 22-23.

George R A T, Stephens R J and Varis S 1980 The effect of mineral nutrient and the quality of seeds in tomato. In Seed Production. Ed. P D Hebblethwaite. pp 561-567. Butterworths, London.

Little T M and Hills F J 1978 Agricultural Experimentation. Wiley, New York. 350 p.

Seno S, Makawa J, Zanin A S Wand Mischan M M 1987 Effects of phosphorus and potassium levels on fruit charac· teristics and quality of tomato seeds. Hort. Basilcira 5, 25-28.

Scpctoglu H 1987 Sicak iklim tahillari uygulama teksiri E. 0. Ziraat Fak. 33 p.

Toon P G, Hines R J and Dieters M J 1991 Relationship between seed weight, germination time and seedling height growth in Pinus caribea Morelrt var. hondurensis Barrett and Golfari. Seed Sci. Techno!. 19, 397-402.

Tomato News 1993 (January) World Information Centre for the Processing Tomato Industry. Avignon-Cedex, France.

Vural H and Eser B 1992 Tarimsal Sanayi-Oniversite i§bir­ligine Ornek Yakla§im E. 0. Yayim Biilt:ll Tarimsal Uygulama ve Ara§tima Merkezi (Turkish with English summary).

Vadivclu K K 1983 Effect of spacing and manuring on seed yield and quality. Tamil nadu Agric. Oni. 108-109.

Varis S and George R A T 1985 The influence of mineral nutrition on fruit yield and quality in tomato. J. Hortic. Scienc. 60, 373-376.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 439-444, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-146

Influence of nitrogen nutrition on nutritional status and yield of 'Navelina' orange

F. INTRIGLIOL0 1 , G. FISICHELLA2 , M. TROPEA3 , G. SAMBUC0 2 and A. GIUFFRIDA1

1Istituto Sperimentale per l'Agrumicoltura, Acireale, Italy; 2Istituto di Chimica agraria, Universita di Catania, Italy; 3Istituto di Chimica agraria e forestale, Universita di Reggio Calabria, Italy

Key words: fruit quality, macronutrients, N fertilization, nutritional status, oranges

Abstract

The trial was carried out to extend our knowledge of the nutritional, yield and vegetative status in 'Navelina' orange. Different fertilization formulae were tested to determine which best satisfied nutritional requirements and improved yields. A simple randomized design was used to compare increasing doses of N (0-200-400-600-800-1000 g/tree). The different N fertilization treatments resulted in, to varying degrees, marked increase in N and Mg, and decrease in K and Ca. Allowing for annual variations, a particularly significant increasing trend in yield was observed in the treatments with up to 600 g N/tree. Higher doses resulted in lower yields and impaired fruit quality.

Introduction

Nutrition plays a fundamental role in rational­ized growing techniques and must be calibrated to the real requirements of the trees in order to optimize fruit yield, quality and quantity, reduce costs and safeguard the environment (Bar-Akiva and Gotfried, 1972; Du Plessis and Koen, 1988; Intrigliolo and Raciti, 1992; Legaz Paredes and Primo Millo, 1992).

In Italy studies on Tarocco' orange (Intrig­liolo et a!., 1990; Raciti et a!., 1984-85) have shown that administration of more than 400-600 g N I tree does not increase yield and results in lowered fruit quality. They also revealed that leaf levels of some elements, especially N, ob­served in blood oranges grown in Italy differ from standard values (Intrigliolo eta!., 1986-87; Intrigliolo et a!., 1990; Raciti et a!., 1984-85; Tropea ct a!., 1983). In fact, the nitrogen values 2.40-2.60% d.m. which Embleton et a!. (1973) considered as optimal are elevated for old line Tarocco' and 'Moro' oranges which manage to satisfy all vcgeto-productive requirements at values between 2.30-2.40% d.m. The other

values of the elements are similar to standard ones. More marked differences are observed in the N nutrition values of 'Tarocco' nucellar line orange which reaches optimal condition at 2.00-2.20% d.m. In this cultivar leaf levels of P and Mg were slightly lower than standard.

The 'Navelina' orange was introduced into Italy from Spain in 1968 because of its very early ripening and its excellent fruit quality, and is now widely grown (Terranova et a!., 1978-79).

The aim of this study was to investigate the nutritional, yield and vegetative status of 'Navelina' and to test some fertilization formulas in order to detect the most suitable nutritional conditions which satisfy nutritional requirements and improve yield.

Materials and methods

The study was performed in three orange grow­ing areas, in eastern Sicily (Ramacca, field A and Paterno, field C) and in Basilicata (Metaponto, field B) on 'Navelina' orange trees (Citrus sinen­sis (L.) Osbeck) grafted on sour orange (Citrus

440 Intrigliolo et al.

aurantium L.) for the first year (1985). Its aim was to detect the nutritional and vegeto-prod­uctive status determined by each orchard routine growing practice using different nutrients (Table 1). The study was then continued in fields A and B for a further four year period. The fields were split, and a simple randomized scheme initiated, and the nutritional and productive responses of the trees treated with increasing doses of N (0-200-400-600-800-1000 g/tree) obtained from ammonium sulfate were studied. Mineral superphosphate ( 450 g P20 5 /tree) and potassium sulfate (150 g K2 0/tree) were administered to all the trees.

A plot of 54 trees at a spacing of 400 trees ha -I was chosen in each orchard. The trees in field A and B were 7 years old, whereas those in field C were 12 years old.

Yield and number of fruit per tree were recorded. Twenty fruits were picked simulta­neously from each tree in all fields to evaluate the fruit quality characteristics, carpometric and

Table 1. Supply of nutrients in the three trial fields

Nutrient Field (g/ tree)

A B c N' 300 225 300 PO"

2 ' 600 450 220

K20 357 772

"N as ammonium sulfate in fields B and C, as ammonium nitrate in field A. "P2 0 5 as superphosphate and K,O as potassium sulfate in all fields.

Table 2. Chemical and physical characteristics of the fields'

Field Sand Silt Clay pH Total N lime total

(%) (%) (%o)

A 41.64 21.49 36.87 7.43 9.20 1.19 B 66.68 12.76 20.56 7.71 1.00 1.15 c 44.21 19.62 36.17 7.30 12.27 1.11

'Mean of the analysis of 6 composite sites. "Dichromate K oxidation. 'Olsen method. "Extracted with ammonium acetate, pH 7.

juice analyses. In addition soil characteristics were analysed in the 0-40 em soil layer (Table 2). Methods of the analyses were as described by SISS (1985). Soil in all three fields was very homogeneous and tree roots spread throughout the whole soil layer (0-40 em), especially in field C.

The results of the analyses of fruit, leaves and yield were subjected to variance analysis and the means compared with Duncan's test.

Results and discussion

Marked interfield differences in fruit yield and quality (Fig. 1) were revealed in the first study year when routine growing practices and diverse doses of nutritional fertilization were underway. In fact, in field C fruit yield ( 66 kg/ tree) was about twice that obtained in the other two fields, but the latter two presented superior quality fruit. The climatic differences between Sicily and Basilicata may have modified the fruit quality, while the yield may have been influenced by the greater age and development of the trees in plot c.

Comparison with the nitrogen standards of Embleton eta!. (1973) revealed that N tended to be optimum in all three fields in the first year (Fig. 2). However, a slight N deficiency was observed, regardless of the diverse soil and climatic conditions and nutrient supply. Fe and Ca leaf values were in the optimal-elevated range.

In the following four year study the fertiliza-

C/N Organic P2 0 5 K 20 Salt matter b avail.c avail.d cone. (%) (kgha- 1) (kgha- 1) (%)

7.10 1.45 1447 1058 0.09 6.60 1.30 1112 1103 0.82 6.80 1.29 1277 993 0.14

~ .................. 0 10 20 30 40 50 60 70 50 100 150 200 250

Yield (kg/tree) Fruit weight (g)

~~~ 0 2 4 6 8 10 12 14 0 0.2 0.4 06 08 1 1.2 \.4

TSS ("'o) Acidity (%)

Rind thickness (mm)

I~ FIELD

E:Z:ll B

Fruit consistency (kg)

Fig. 1. Mean yield values and main quality characteristics.

Urut3111 I Element (Dr~ Deficient Low Optimum High

mater

N % ® 0 2.2 2.4 26 29 @

p % I 0.09 0.12 ®0 0.16 0.29 @

K %

I 0.4 0.7 ® ~ 1.09 2.0 i

Ca % 1.6 3.0 ;ss ~~ 6.9

' ~---

Mg %+---

0.16 0.26 ~ ! 0.6 1.1

- -- ··-- lw'f-f---Fe /-i9 g·' 36 60 120 200

Mn 0 500 J19 g' 16 25 (C)(B) 200

----~----- --

Cu J19 g, 3.6 ; 0

s.o 1 (8) 16 i (c) 22

Zn g g·• '0)

100 ~C) 21 31 (8) 260

QFIHD

Fig. 2. Leaf values of nutritional elements grouped in Em­bleton's nutritional classes.

tion treatments caused marked leaf content var­iations of the elements studied. Treatment with N fertilization (Fig. 3) provoked a marked in­crease not only in N leaf content compared with the control trees, but also a varying increment of

Mineral nutrition of 'Navelina' orange 441

N leaf values in the three fields in accordance with increasing doses of N. In field B this increment rose progressively as the total N doses increased. Moreover, 400 g N I tree was sufficient to obtain N leaf values of 2.40% d.m. Diversely, field A was characterized by greater inertia and differed only after N doses of 600/800 g/tree (values between 2.37 and 2.43% d.m.). These differences may have been caused by the diverse soil and climatic condition of the trial fields. Leaf P and K levels (Fig. 3) were in the optimal nutritional standard range, partly due to the good supplies in the soil.

As previously reported in the 'Tarocco' orange in Sicily by Intrigliolo et a!. (1990) and Raciti et a!. (1984-85), the different doses of nitrogen fertilizer provoked contrasting, negligible effects of P leaf content in the 'Navelina' orange.

The increasing N determined a very statistical­ly significant reduction in the K levels (Bar­Akiva and Gotfried, 1972; DuPlessis and Koen, 1984; Intrigliolo eta!., 1990).

Ca and Mg (Fig. 3) nutritional leaf levels were optimal, regardless of the treatment. In field A both elements presented greater differences with a higher statistically significant regression be­tween N leaf levels and the N administrations, i.e. the higher the nitrogen doses, the lower the Ca and the greater the Mg leaf levels.

Comparison of fruit yield (Fig. 4) clearly revealed an increasing trend in both fields up to 600 g/tree. Yields then decreased at higher doses. Leaf N regression/yield in field A showed a marked reduction of yield correlated with the increasing nitrogen leaf content, whereas this was reduced in field B.

Fruit weight (Fig. 4) demonstrated that the progressive rise in nitrogen supply determined increased fruit size and leaf N values, underlin­ing the close relationship between this parameter and yield.

The increased N doses induced a generic deterioration in all the indices, which was much less evident than seen in the 'Tarocco' (Intrig­liolo et a!., 1990), especially in the nucellar clone. More apparent repercussions were seen in the rind thickness and fruit consistency (Fig. 5) which were both negatively influenced by high N doses.

442 lntrigliolo et a!.

N (%) FIELD A ,N (%) FIELD 8

i. c c c c

··~ B.

p (%) o.>P r-'-(%-')'---------------,

r = 0.059 n.s. r ;; 0.045 n.s.

K (%) '"'Kr-'-(%_;) ___________ ---,

r = 0.147 n.s.

~ A B·BCBCBGc~

r=0.313*"'

~

.ca (%) Ca (%)

C. C. • A r = 0.017 n.s.

i .-.s I . '

Mg (%)

r = 0.392 ** ~~ B B AB A A~ A r=0.194*

Nitrogen doses (g/tree)· Nitrogen doses (g/tree)

Different capital letters mean 0.01; significant differences:

*0.05, **0.01, ***0.001 significant levels for correlation coefficients.

Fig. 3. Four year mean and statistical comparison of leaf N, P, K, Ca and Mg in the various treatments. Regression between the doses of N administered and leaf N, P, K, Ca and Mg.

Mineral nutrition of 'Navelina' orange 443

FIELD A FIELD B 100Vield {kg/tree) •oo Yield (kg/tree)

r = 0.328 .. r = 0.273 **

~ ~·

Nitrogen doses (g/tree) Nitrogen doses (g/tree)

Yield (kg/tree) Yield (kg/tree)

r = 0.280 ** r=0.218*

N (%) N (%)

Fruit weight (g) Fruit weight (g)

·r~ . A .A.

260~ .B B B

Nitrogen doses (g/tree) Nitrogen doses (g/tree)

Fruit weight (g) Fruit weight (g)

r = 0.281 ** r = 0.282 ** ~·

N (%) N (%)

Different capital letters mean 0.01; significant differences:

*0.05, **0,01, ***0,001 significant levels for correlation coefficients.

Fig. 4. Four year mean and statistical comparison of yield and fruit weight in the various treatments. Regression between the doses of N administered and yield and fruit weight. Regression between leaf N values and yield and fruit weight.

Conclusions

As reported in Taro ceo trees (Intrigliolo et a!., 1990; Raciti et a!., 1984-85), N fertilization with doses up to 600 g/trcc increased fruit yield in both fields, whereas higher doses impaired some fruit quality characteristics to a lesser extent than

observed in Tarocco orange (Intrigliolo et a!., 1990; Raciti et a!., 1984-85).

Our study showed that the leaf levels of P, K, Ca and Mg fall within the standard optimum range (Embleton et a!., 1973), especially when N fertilization is balanced.

Diversely, optimum N leaf levels are slightly

444 Mineral nutrition of 'Navelina' orange

FIELD A FIELD B 12

Rind thickness (mm) Rind thickness (mm)

r=0.196* r=0.174*

Fruit consistency (kg) Fruit consistency (kg)

1;1 A A AB All" a r = 0.431 ** " __ " ___ _

Nitrogen doses (g/tree) Nitrogen doses {g/tree)

Different capital lettors mean 0.01; significant differences:

•o.os, **0.01, ***0.001 significant levels for correlation coefficients.

Fig. 5. Four year mean and statistical comparison of rind thickness and fruit consistency in the various treatments. Regression between the doses of N administered and rind thickness and fruit consistency.

lower (2.3% d.m.) than these reference values and coincide with the most elevated yield levels.

This confirms the validity in Italy of the standard values proposed by Embleton et a!. (1973), slight modifications being required only for nitrogen, and shows that they are useful in defining the nutritional requirements of the 'Navclina' orange.

References

Bar-Akiva A and Gotfried A 1972 Effect of nitrogen and potassium nutrition on fruit yield and quality and leaf mineral composition of 'Valencia' orange trees. Agroch­imica 16, 127-135.

Du Plessis S F and Koen T J 1988 The effect of N and K fertilization on yield and fruit size of Valencia. Proc. Sixth Int. Citrus Congress, Israel-Tel Aviv II, 663-672.

Embleton T W, Jones W W, Labanauskas C K and Reuthe W 1973 Leaf analysis as a diagnostic tool and guide to fertilization. Citrus Industry III, 183-210.

Intrigliolo F and Raciti G 1992 Nutrizione e concimazione deg1i agrumi. L'Informatore Agrario XLVIII (18), 77-83.

Intrigliolo F, Scuderi A, Raciti G and Giuffrida A 1986-87

Risposte vegeto-produttive dell'arancio agli interventi di concimazione organica. Annali Ist. Sperim. Agrumicoltura XIX-XX, 227-265.

Intrigliolo F, Tropea M, Raciti G, Sambuco G and Giuffrida A 1990 Nutrizione minerale dell'arancio. 4o Contributo: L'influenza di diverse dosi di azoto sullo stato nutrizionalc e sulla produzione del Tarocco nucellare e vecchia linea. Annali Ist. Sperim. Agrumicoltura XXIII, 45-69.

Lcgaz Paredes F and Primo Millo E 1992 Influencia de Ia fertilizacion nitrogenada en Ia contaminacion por nitratos de las aguas subterraneas. Levante Agricola XXXI, 317-318, 4-15.

Raciti G, Scuderi A, Intrigliolo F and Giuffrida A 1984-85 La produttivitit del Tarocco nucellare in rapporto allo stato nutrizionalc ed agli interventi di concimazione azotata. Annali !st. Sperim. Agrumicoltura XVII-XVIII, 147-170.

SISS 1985 Metodi normalizzati di analisi del suolo. Edag­ricole Bologna.

Terranova G, Starrantino A and Russo F 1978-79 L'arancio Navelina. Annali Ist. Sperim. Agrumicoltura XI-XII, 3-22.

Tropea M, Intrigliolo F, Sambuco G, Scuderi A, Radaelli L and Raciti G 1983 Nutrizione mineralc dcll'arancio. I contributo: Indagine sullo stato nutrizionale e sulla produzione del Tarocco nucellare e vecchia linea. Tecnica Agricola XXX, 201-226.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 445-448, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-127

Effect of NK fertilization on leaf nutrient content and fruit quality of 'Valencia late' orange trees

C.F. CARRANCA, J. BAETA and M.A.C. FRAGOSO National Agronomy Research Station IN/A, Quinta do Marques, 2780 Oeiras, Portugal

Key words: fruit quality, K fertilization, leaf composition, N fertilization, oranges, 'Valencia late' orange trees

Abstract

A study was made on the effects of N and K fertilizer treatments on the leaf composition of Citrus sinensis cv. Valencia late trees, as an index of their nutritional status, and on other quality factors. The experiment was laid out for 7 years in an orchard in a clayey calcareous alluvial soil. The plots were arranged on a randomized block design of the type 52 factorial, in partial confounding. Comparison of leaf composition, collected from the fruiting and non-fruiting shoots, 6-7 and 4-7 months old, respectively, has shown that either leaf type enables the diagnosis of the nutritional status of these trees. In both leaf types, N was positively related to fruit peel thickness and had a negative relation with juice percentage and soluble solids; in fruiting shoots, Mg was positively related to the fruit weight and inversely related to juice acidity; P was related to fruit shape. Juice acidity decreased with fruit weight increase, as well as measured fruit height. For N, P, K, Ca, and Mg estimated optimal intervals in leaves from non-fruiting shoots, expressed as gkg- 1 d. m. at 70°C, were respectively: (24-26), (1.0-1.5), (4.0-5.3), (33-53) and (3.1-3.7); regarding fruiting shoots, intervals were (19-21), (1.1-1.5), (2.8-3.3), (55-60) and (3.5-5.5). These values correspond to N fertilizing rates 123 ± 31 kg ha - 1 • Climate had a high significant effect on both nutrient levels in the leaves and fruit characteristics.

Introduction

Soil, plant and water analyses are important guides to improve fertilizer efficiency, to increase yields and quality, to prevent potential accumu­lation of nutrients in soils and groundwaters, and to preserve the soil, water and mineral re­sources. The response to fertilizers may be evaluated by comparing plant analysis with criti­cal nutrient levels and yields. According to Embleton et a!. (1978) terminal leaves from the non-fruiting shoots of the spring cycle, 5 to 7 months old, should be sampled; for Chapman (1960), 4 to 10 months old leaves in fruiting shoots should be sampled for analysis.

The effect of NK fertilization on leaf composi­tion, is reported as a guide for the nutritional

status, and for some fruit quality factors, m 'Valencia late' orange trees.

Methods

The details of the experiment were previously described (Carranca eta!., 1990). A 7-year trial was laid out in a clayey calcareous alluvial soil of an orchard at Estac;ao Nacional de Citricultura "Vieira Natividade" (Setubal, Portugal). N and K fertilizers were applied to 'Valencia late' trees. The plots were arranged as randomized block design of the type 52 factorial, in partial con­founding, with five N rates (60, 120,260,400 and 460 kg ha - 1 ) and five K2 0 rates (0, 44, 150, 256

446 Carranca et al.

Table 1. Some physical and chemical characteristics of the orchard soil

Depth Sand Silt Clay pH (em) (g kg ~j) (H20)

0-25 609 121 270 7.9 25-50 598 125 278 8.0 50-75 562 135 303 8.2

and 300kgha- 1), with uniform application of 45kgha- 1 of P20 5 .

Main soil characteristics arc reported in Table 1. Spring cycle leaves were collected yearly from

non-fruiting (nf) and from fruiting (f) shoots, respectively 4-7 months old and 6-7 months old. Leaf sampling, preparation and analytical proce­dures were according to Fragoso et al. (1974); fruits were sampled each year, as previously described ( Carranca et al., 1990). The methods used for fruit quality were reported in Fragoso et al. (1974).

Statistical treatment by ANOVA of two-way was applied regarding the effect of treatments and leaf chemical composition. The t-test was used to compare leaf mineral composition be­tween shoot types. Principal-component analysis was done either for leaf nutrient data and for fruit quality. Due to the important effect of the uncertain factor "Year" on leaf composition and fruit quality, analysis of covariance to eliminate this effect was done. Through a multiple regres­sion analysis an optimization model was found

O.M. Total N Available P Exchangeable K (g kg ~j) (g kg ~j) (mg kg~ 1 ) (cmol( +) kg- 1 )

10.8 7.4 7.2

0.88 45 0.74 0.65 26 0.36 0.55 14 0.36

for recommendations of N fertilization on this 'Valencia' orchard, and nutrient levels were estimated for the optimum nutritional status of the trees.

Results

In Table 2, some leaf macronutrients, sampled either from non-fruiting or fruiting shoots of the spring cycle, are shown.

The main effects of the N and K treatments on the mineral content in both leaf types are pre­sented in Table 3. It is evident that the climatic conditions strongly interfere with the nutrient levels in both type of leaves. The N treatment significantly affected either N, P, K and Ca con­tents in the leaves of the non-fruiting shoots and N and K contents in the fruiting shoots. In both leaf types, K decreases with the increase in applied N. The K treatment and the interactions Year x K and N x K have not significantly affect­ed both leaf contents.

Table 2. Leaf content of (N, P, K, Ca and Mg) for spring cycle leaves, non-fruiting and fruiting shoots (as expressed in gkg~ 1 d. m. at 70°C)

Non-fruiting leaf samples

N p K Ca Mg

26.02± 1.68± 6.23± 30.46± 2.86± 0.329 0.041 0.087 1.302 0.037

Table 3. ANOVA of two-way on leaf nutrient content

Source of Non-fruiting leaf samples variation

N p K Ca

Year N n.s. Year x N n.s. n.s. n.s. K n.s. n.s. n.s. n.s. Year x K n.s. n.s. n.s. n.s.

Fruiting leaf samples

N p K

20.56± 1.19± 3.54± 0.366 0.023 0.057

Fruiting leaf samples

Mg

n.s. n.s. n.s. n.s.

N

n.s. n.s.

p

n.s. n.s. n.s. n.s.

n.s., *, **, *** = F-valucs non-significant and significant at p ~ 5%, 1% and 0.1 %.

K

n.s. n.s.

Ca

36.76± 1.588

Ca

n.s. n.s.

n.s. n.s.

Mg

3.26± 0.107

Mg

n.s. n.s. n.s. n.s.

0,8

0,6

0 ' 4 %jwce ;f' 0,2

+ ss

-0,2

-0,4

-0,6

-0,8

rMg

weight

* "' IP

2'l[1,

,.,_height

* nftv1g

.>.:'~>Nfert

nik

IV niN

t +i~~ 3 9% peet thtckn<::ss

-1 -0,8 -0,6 -0,4 -0.2 0,2 0,4 0,6 . 0,8

Factor 1

Fig. 1. Principal-component diagram for leaf mineral con­tents and fruit characteristics.

In Figure 1, the principal-component analysis reveals how the mineral contents in the leaves and the fruit quality are distributed. As to leaf composition, Mg in the leaves from the fruiting shoots and N in the non-fruiting shoots, are the most important variables in the two main com­ponents. As to the fruit characteristics, the first component shows the opposite relationship be­tween the percent juice and soluble solids (SS) with peel thickness. Otherwise, Mg in the fruit­ing shoots is closely related to the fruit weight (W) and inversely related to the juice acidity (A). In both leaf types, N is closely related to peel thickness, and has a negative relation with percent juice and SS. Besides this, K in the leaves also has an opposite relationship with SS and percent juice, while P is related with fruit shape (height).

Due to the highly significant effect of climate on the nutrient contents in both types of leaves and on the fruit characteristics ( Carranca et a!., 1990), covariance analysis was used to eliminate this variable.

As reported by Carranca eta!. (1990), optimal fruit quality values are: 170 g for W, 95 g kg -t for SS and 8 g L -l as citric acid for A. A multiple regression analysis was applied to optimize the nitrogen requirement (Y) for 'Valencia late' trees, in function of these optimal fruit charac­teristics, significantly affected by applied N:

Y(kg ha- 1 ) = 1751 - 13.430 W-315.208 SS +

+ 452.320 A, F = 1079.677* (p < 5%)

For the optimal fruit yield and quality, the

Effect of NK fertilization on orange trees 447

Table 4. Estimated optimal nutrient levels in both non­fruiting and fruiting leaves for optimal yield and fruit quality, and comparison with the literature

Nutrients

N(gkg- 1 )

p (g kg -I) K (g kg 1)

Ca(gkg- 1)

Mg (g kg- 1 )

Non-fruiting leaves

24-26(0)" 1.0-1.5 (0) 4.0-5.3 (L)b 33-53 (0) 3.1-3.7 (0)

Fruiting leaves

19-21 (L) 1.1-1.5 (S)' 2.8-3.3 (L) 55-60 (S) 3.5-5.5 (S)

"(0) =optimum range (Embleton et al., 1978); b(L) =low range (Embleton et al., 1978; Chapman, 1960); '(S) = sufficient range (Chapman, 1960).

equation gives us the rate of 120 kg ha-t and a tolerance of ± 28 kg ha-t as the optimum N level to be applied to a 'Valencia late' orchard, as under the present experimental conditions. Based on this recommended N rate application, the leaf nutrient levels that correspond to the optimum nutritional status of the trees were estimated (Table 4 ).

Discussion and conclusions

Nutrient contents found in both leaf types (Table 2) are similar to those found by Fragoso ct a!. (1990) for the same orange tree, under similar climate. Therefore, K levels in both leaf types are lower than the levels therein referred, and the levels of Ca in the fruiting shoots are slightly higher.

Comparing leaf mineral composition in both shoot types, as guiding values for the nutritional status of these trees, a non-significant t-value was found, meaning that either type of leaf may be used for such purpose. For both types of shoots, N and P proved to be important leaf nutrients while for fruiting shoots only Mg proved so. The N and P, as indicators for fruit peel thickness and shape, and Mg as indicator for fruit weight and juice acidity.

The calculated model to optimize the applica­tion of N fertilizer to the examined orchard indicate that for N rates around 92 to 148 kg ha- 1 , with no K fertilization, the trees are at their best nutritional status. This fact agrees with Hernandez (1979) who also verified that the 'Valencia late' did not respond to N fertilization levels higher than 160 kg ha-t, either for yield or

448 Effect of NK fertilization on orange trees

fruit quality. The same author also had no response to K application probably due to soil reserves.

In Table 4, the estimated values in the leaves of non-fruiting shoots, corresponding to the optimum nutritional status of these trees, may be included in the optimal range referred by Em­bleton eta!. (1978), except forK included in the low range. In the fruiting shoots, N and K values are included in the low range referred by Chap­man (1960). However, according to this refer­ence N leaf values around 21 g kg -I may be considered satisfactory when no deficiency symp­toms are observed in the leaves; as to P, Ca and Mg resulting values may be included in the satisfactory range.

Acknowledgements

Acknowledgements are due to Esta~ao Nacional de Fruticultura "Vieira Natividade" for col­laborating in field experiments and to the labora­tory staff of the Pedology Dept. for sample preparation and analyses.

References

Carranca C F, Baeta J and Fragoso MAC 1990 A aduba<;ao NK e a qualidade de laranjas 'Valencia late' num aluvios-

solo de textura pesada (Setubal). In Aetas de Horticultura do I Congresso I be rico de Ciencias Horticolas. III ( 6): Fruiticultura. Eds. Associa<;ao Portuguesa de Horticultura e Fruticultura e Sociedad Espanola de Ciencias Horticolas. pp 95-100. Lisboa.

Chapman H D (1960) Criteria for the diagnosis of nutrient status and guidance of fertilization and soil management practices: Leaf and soil analysis in citrus orchards. pp 1-53. Manual Agric. Exp. 25. Stat. Ext. Service.

Embleton T W, Jones W Wand Platt R G 1978 Leaf analysis as a guide to citrus fertilization. In Soil and Plant-Tissue Testing in California. Bulletin 1879. Ed. H M Reisenauer. pp 4-9. Division of Agricultural Sciences, University of California, Berkeley.

Fragoso M A C, Ferreira L A B and Vicente M A F S 1974 Leaf analysis and fruit composition in orange trees. I. Preliminary studies. In I Congreso Mundial de Citricul­tura. Ed. 0 Carpena. pp 139-145. Int. Society Citricul­ture, Murcia-Valencia.

Fragoso MAC, Vicente M A F, Jordao P V and Calouro F 1990 Amilisc foliar e composi<;ao dos frutos em laranjeiras. II. indices foliares e a qualidade dos frutos. In Aetas de Horticultura do I Congresso Iberico de Ciencias Horticolas III (6): Fruticultura. Eds. Associa<;ao Portuguesa de Hor­ticultura e Fruticultura e Sociedad Espanola de Ciencias Horticolas. pp 166-171, Lisboa.

Hernandez J 1979 Efecto del fosforo, potasio y dosis de nitrogenio sobre el rendimiento, calidad del fruto y con­tenido foliar del naranjo Valencia. Cultivos Tropicales 1, 23-36.

M.A.C. Fragoso and M.L van Beusichem (eds.), Optimization of Plant Nutrition, 449-452, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-156

Effects of lime and phosphogypsum on citrus

G.C. VITTI 1 , L.C. DONADI0 2 , E. MALAVOLTA3 and J.R.M. CABRITA4

1 Escola Superior de Agricultura 'Luiz de Queiroz', University of Sao Paulo, Piracicaba, SP, Brazil; 2 Faculdade de Cifmcias Agrarias e Veterinarias, State University Julio de Mesquita Filho, Jaboticabal, SP, Brazil; 3Center of Nuclear Energy in Agriculture, University of Sao Paulo, Piracicaba, SP, Brazil; 4Citriculture Experimental Station, Bebedouro, SP, Brazil

Key words: citrus, limestone, phosphogypsum

Abstract

An experiment designed to study the effects of calcined dolomitic limestone (DL) and phosphogypsum (PG), either separately or in combination, on citrus production (Pera on Rangpur lime) and soil characteristics (Dark Red Latosol, sandy phase, acidic) was carried out in the Citriculture Experimental Station, Bebedouro, Sao Paulo, Brazil. A randomized block design with seven treatments and six replicates, ten trees per replicate, was used. The main conclusions were as follows: lower fruit yields were obtained both in the control and in the PG alone treatments; the largest leaching of soil K was due to the single PG application; the utilization of either DL alone or its combinations with PG (70: 30 and 50: 50) gave the highest yields due to the correction of soil acidity and improvement of the calcium supply.

Introduction

The effect of lime is usually confined, to a large extent to the upper soil layers. The acidity in the subsurface restricts root growth and uptake of nutrients, both from the soil itself and from fertilizers, and makes the plant more susceptible to drought. Phosphogypsum (PG), on the other hand, leaches down easily, increasing Ca satura­tion and reducing AI participation in the ex­change complex, thereby causing an ameliora­tion in the subsoil conditions. (Pavan et a!., 1984; Reeve and Summer, 1972). The objective of this study was to evaluate the effects of calcined dolomitic limestone (DL) and PG, ap­plied separately or in combination on the soil characteristics, yield and nutritional status of orange trees grown in the region of Bebedouro, S. Paulo, which is one of the most important citrus producing areas of Brazil.

Materials and methods

The experiment was carried out in a Dark Red Latosol, sandy texture, in Bebedouro, S. Paulo, Brazil. The general soil characteristics, respec­tively for the 0-20 and 20-40 em layers are the following: pH (CaC12)-4.2 and 4.1; organic matter-9.1 and 7.6g kg- 1 ; P(resin)-6 and 3mg L- 1 ; K+ -0.13 and 0.09cmol (+) L- 1 ;

Ca2 + -0.29 and 0.14cmol (+)L - 1 ; Mg2 + -0.19 and 0.09cmol (+) L- 1 ; H++AlH-4.2 and 4. 7 cmol ( +) L -l. The grove of the variety Pera (Citrus sinensis Osbeck) on Rangpur lime was planted in November 1984 at the spacing of 6.5 x 7.0 m. The treatments were the following: (1) control- NPK + micronutrients; (2) DL to raise base saturation (V%) to 70%; (3) PG at half the rate used in treatment ( 6); ( 4) D L + PG, 10 and 30%, respectively; (5) DL at half the rate

450 Vitti et al.

used in treatment (2); ( 6) PG to raise Ca level at 20-40 em to 2.0 cmol ( + )L - 1 , assuming that 2.5 t PG ha -I corresponds to 1 cmol ( +) Ca kg- 1; (7) DL+PG, 50% each, at a dosage estimated to raise V% to 70. All treatments received the same NPK and micronutrient fertili­zation as the control ( 1). The PG was a by product of the production of phosphoric acid using apatite from Patos, MG, Brazil, containing 180g kg- 1 Ca, 150g kg- 1 S, 12g kg- 1 Si0 2 ,

6.3 g kg - 1 fluorides and 3-7 g kg - 1 sexquioxides. The DL had 320g kg- 1 Ca and 147g kg- 1 Mg and its particles passed the sieve of 0.3 mm mesh. Treatments involving broadcast applica­tions of DL and PG were made initially in 1984 and repeated 1987 and 1989. Fertilizers accord­ing to the official recommendation were used in the planting holes and every year afterwards. Six replicates with 10 useful plants each were used. Weeds, pest and diseases control were made according to the usual practices of the region. In the remainder of this paper the treatments will be referred to as respectively: control, DL2, PGl, 70 + 30, DLl, PG2 and 50+ 50. Soil sam­ples were collected in April 1991 at three depths in the dripline. The general chemical characteris­tics were analyzed by the methods described by Raij and Quaggio (1983); so~- - S was assessed according to Vitti (1989). As is usual in these procedures, a volume of the sample was taken for analysis rather than a given weight. In the same month leaf samples were collected for analysis which was conducted by using the proce­dures described by Malavolta et al. (1989). Several fruit characteristics were measured in the main crop of the same year.

Results and discussion

Soil analysis

Table 1 shows the results of the chemical analy­sis. pH values varied from 4.08 found in the control to 5.27 which occurred in the treatment DL2, which received the highest limestone rate. In the treatments with PG alone the pH was equal to that of the control indicating therefore that phosphogypsum did not modify the soil pH under the experimental conditions. The levels of

Ca were significantly raised by the treatments which carried the amendments. Although PG moves down the profile much more easily than DL, no difference was observed in the calcium levels in the deeper layers. In the control and in the treatments wherein only PG was used the Mg levels were lower and the plants showed symp­toms of deficiency in agreement with Pavan (1984 ).

Exchangeable K was present at higher concen­trations in the limestone treatments which likely prevented its leaching due to the negative charges generated. The opposite, however, is shown in the treatments with PG alone thereby suggesting that K leaching was promoted by these treatments. In the plots receiving PG + DL available K was raised in the upper layer when compared with plants receiving PG alone. Alu­minium concentration was higher in the control and when only PG was applied. On the other hand, base saturation, V% (cation exchange capacity divided by the sum of bases times 100) was increased in the treatments containing DL, particularly in the upper layers. Finally the concentrations of SO~- - S were higher in all layers when PG was supplied.

Leaf analyses

Concentrations of nutrients are presented in Table 2. There was no significant change in leaf K, whereas the concentration of Ca was in­creased by the treatments carrying either DL or PG. The effect, however, was significant only in the treatment DLl. Both in the control and in treatments wherein PG was used in the absence of DL lower MG levels were found. A marked increase in leaf MG occurred in the 50+ 50 treatment showing therefore the advantage of this blend. PG increased S concentrations. In the treatments in which the higher limestone rate was used a reduction on leaf sulfur was ob­served, probably due to increased leaching of SO~ caused by high pH (Vitti, 1989). Manganese and Zn concentrations were not affected m a consistent manner by the treatments.

Yield and fruit quality

As shown in Table 3, statistical analyses dis-

Tab

le 1

. A

vera

ges

of c

hem

ical

cha

ract

eris

tics

Cha

ract

eris

tic

Dep

th

Tre

atm

ent

L.S

.D.

(em

) C

ontr

ol

DL

2 P

GI

70:3

0 D

Ll

PG

2 5

0:5

0

Tre

atm

t.

Dep

th

pH

00-2

0 4.

08dB

5.

27aA

4.

27dA

5.

00bA

4.

72cA

4.

25dA

5.

13ab

A

(CaC

I 2)

21-4

0 4.

30bc

A

4.53

aB

4.07

dB

4.20

cdB

4.

20cd

B

4.10

cdb

4.48

abB

0.

23

0.15

41

-60

4.08

bB

4.20

abC

4.

08bB

4.

17ab

B

4.!3

abB

4.

!3ab

AB

4.

32aC

Mg

00-2

0 0.

12dA

1.

07aA

0.

10dA

0.

57ca

0.

60cA

O

.!OdA

0.

77bA

(c

mol

( + )L

-I)

21

-40

O.!O

cA

0.40

aB

O.!O

cA

0.27

abB

0.

22bc

B

O.!O

cA

0.38

aB

0.17

0.

13

41-6

0 O

.!ObA

0.

20ab

C

O.!O

bA

0.17

abB

0.

13ab

B

O.!O

ba

0.27

aB

AI

00-2

0 1.

03aA

0.

03dC

0.

73bB

0.

13cd

B

0.32

cC

O.S

Oab

A

0.03

dB

(cm

ol( +

)L -I

) 21

-40

0.82

abB

0.

42cd

B

0.90

aA

0.70

abA

0.

58bc

B

0.77

abA

0.

30dA

0.

26

0.15

41

-60

0.88

aAB

0.

63ab

A

0.68

aB

0.73

aA

0.78

aA

0.83

aA

0.42

bA

Ca

00-2

0 0.

58cA

2.

63aA

0.

92cA

2.

28aA

1.

63bA

0.

97cA

2.

78aA

~

(cm

ol( +

)L -I)

21

-40

0.32

cA

0.92

abB

0.

55bc

A

0.93

abB

0.

60bc

B

0.63

bcA

1.

27aB

0.

51

0.39

~

41-6

0 0.

30aA

0.

55aB

0.

53aB

0.

58aB

0.

48aB

0.

68aA

0.

78aC

'"' 1:;"

K

00-2

0 O

.!S

bcA

0

.2la

bA

0.

15cA

0.

18bc

A

0.25

aA

0.16

cA

0.24

aA

.Q.,

(cm

ol( +

)L -I)

21

-40

0.12

bcB

0.

16aB

0.

14ab

cB

0.!3

abcB

0.

16ab

B

O.l

lcB

0.

15ab

cB

0.05

0.

03

~ 41

-60

0.10

abB

0.

10ab

C

O.!O

abB

0.

09bC

0.

10ab

C

0.14

aA

0.!

3ab

B

"' 00

-20

19da

65

aA

24dA

53

bcA

45

cA

25dA

61

abA

;::,

;:::

V

%

21-4

0 12

cB

32aB

17

bcB

20

bcB

23

bB

19bc

B

36aB

9.

11

6.12

~

41-6

0 !3

baB

22

ahC

19

abA

B

25aB

21

abB

23

aAB

26

aC

'1::: ;:::- a

s-so

!-00

-20

29bc

A

28bc

A

40cC

35

bcB

24

cA

64aB

50

abA

i:l

(m

gL

-1)

21-4

0 31

cA

29cA

70

bB

69bA

33

cB

126a

B

84bB

22

.57

15.4

2 ;::

:- a 41

-60

36eA

41

eA

93bc

A

73cd

A

65dB

12

3aA

1!

3abC

~

Ave

rage

val

ues

foll

owed

by

the

sam

e ca

pita

l le

tter

in

the

colu

mn

do n

ot d

iffe

r at

the

5%

lev

el (

Tuk

ey);

'1::

: "' A

vera

ge v

alue

s fo

llow

ed b

y th

e sa

me

low

cas

e le

tter

in

the

line

do

not

diff

er a

t th

e 5%

lev

el (

Tuk

ey).

.:

~ a ;:::

'"' ~· .: "' ~

Ul

......

452 Effects of lime and phosphogypsum on citrus

Table 2. Concentrations of nutrients in leaves (1)

Treatment Mn Zn N p K Ca Mg s

(mg kg- 1 ) (g kg-')

Control 400AB 220A 26A 1.6A 13.2A 28.6B 1.4DE 3.4A DL2 280B 160A 25A 1.5A 11.5A 33.8AB 2.5C 3.2A PG1 360B 170AB 25.7A 1.5A 12.0A 33.2AB 0.9E 3.6A 70:30 290B 160B 25.8A 1.6A 11.3A 32.8AB 2.2CD 3.1A DLl 880B 170AB 25A 1.5A 11.4A 38.2A 3.9B 3.2A PG2 750AB l40B 25.8A 1.5A 11.5A 33.1AB 3.7B 3.0A 50:50 430AB 160B 26A 1.6A 12.7A 30.7B 5.1A 3.1A L.S.D. 489.5 48.4 1.8 0.2 2.6 6.0 0.9 0.6

Values followed by same letter do not differ at the 5% level (Tukey).

Table 3. Fruit production in kg by 10 plants in two harvests ( 1)

Offseason Main

Control (1) 24.38AB 738.98B DL2 (2) 20.53B 835.38AB PG1 (3) 39.92A 740.76B 70:30 ( 4) 22.59B 887.60BA DLl (5) 27.15AB 883.53A PG2 (6) 27.64AB 740.00B 50:50 (7) 20.04B 828.76AB L.S.D. 15.87 128.71

Average values followed by same letter do not differ at the 5% level (Tukey).

closed significant differences both in the off season (minor) and main (major) harvests. In the latter yields were lower in the control as well as in the treatments receiving only PG. The fact that practically the same yields were obtained when either only DL or DL on association with PG was applied can be explained by taking into account two facts: in the planting hole, according to the recommendations, 1.0 ton of limestone per hectare was supplied to all plants; the sandy texture of the soil and the fineness of the particles of the DL used allowed a part of the amendment to move downwards as can be seen in Table 1. The DL containing treatments gave an increase in yield of roughly 12 kg of fruit per tree. No significant effects were found on fruit quality (size, total solids, acidity).

Conclusions

The following conclusions can be drawn. Phos­phogypsum applied in the absence of calcined

dolomitic limestone did not increase yields above the control levels and promoted leaching of K. The use of calcined dolomitic limestone blended with phosphogypsum gave the same yields as the treatments receiving only limestone. The appli­cation of calcined dolomitic limestone either in the presence or in the absence of phos­phogypsum improved soil chemical conditions both in the upper and lower layers. Higher levels of Ca, Mg and S in the leaves were found when the limestone phosphogypsum blends were ap­plied.

References

Malavolta E, Vitti G C and Oliveira S A 1989 Avali<;ao do, estado nutricional das plantas- principios e aplica<;iies. Associa<;ao Brasileira para Pesquisa da Potassa e do Fosfato, Piracicaba, Sao Paulo.

Pavan M A 1984 0 calcio como nutrientes para as cultwra5 .. Anais Seminarios f6sforo, calcio, magnesia, enxofre e micronutrientes - situa<;oes atual e perspectivas no agricul­tura. Manah, S. Paulo, pp 82-97.

Pavan M A, Bingham F T and Pratt P F 1984 Redistribution of exchangeble calcium, magnesium and aluminium follow­ing lime or gypsum application to a Brazilian Oxisol. Soil Sci. Soc. Am. J. 48. 33-38.

Raij B Van and Quaggio J A 1983 Meodos de Analise de Solo Para Fins de Fertilidade. Instituto Agronomico. Boletim Tecnico, 81p.

Reeve N G and Sumner M E 1972 Amelioration of subsoil acidity in Natal Oxisols by leaching of surface-applied amendments. Agrochemophysica 4, 1-6.

Vitti G C 1989 Enxofre no solo. In Interpreta<;ao de Analise Quimica de Solo e Blanta Para Fins de Fertilidade. Eds. L T Bull and C A Rosolem. pp 129-173. Funda<;ao de Estudos e Pesquisas Agricolas e Florestais, Botucatu, S.P., Brazil.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 453-456, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-158

Influence of soil and leaf applications of micronutrients on yield and fruit quality of Citrus sinensis Osbeck, variety Pera

G.C. VITTI 1 , L.C. DONADI0 2 , R.D. DELARC02 , E. MALAVOLTA3 and J.R.M. CABRITA4

1'Luiz de Queiroz' College of Agriculture, University of Sao Paulo, Piracicaba, SP, Brazil. 2College of Agriculture and Veterinary Medicine, Julio de Mesquita Filho State University, Jaboticabal, SP, Brazil. 3Center for Nuclear Energy in Agriculture, University of Sao Paulo, Piracicaba, SP, Brazil. 4 Citriculture Experimental Station, Bebedouro, SP, Brazil

Key words: citrus, micronutrients, foliar application

Abstract

The experiment was carried out between 19S9 and 1991 at the Citriculture Experimental Station, Bebedouro, SP, Brazil, in a grove of the variety 'Pera' on Rangpur lime. The effect of three levels (0, SO and 160 g per tree) of soil-applied 'Nutricitro 24S', a fritted source containing 20 g kg -I B, SO g kg -l Mn and 240 g kg - 1 Zn was evaluated either in the presence or in the absence of foliar sprays of 'Nutrimins citrus N', a commercial product which has 5 g kg -I B, 20 g kg -I Mn, 30 g kg -l Zn and 100 g kg- 1 N. The main conclusions were the following: (1) soil applications raised leaf B to adequate levels, whereas leaf sprays did not; (2) manganese concentration in the leaves was increased by soil applications; (3) the combination of soil and foliar treatments was capable of increasing both leaf Mn and Zn; (4) tree growth, yield and fruit quality were not significantly influenced by the treatments.

Introduction

Brazil is world leader both in citrus production and export of concentrated orange juice. Aver­age yields, however, are below the potential maximum, and constitute about half of those of the major competitors. The possibility exists that deficiencies of micronutrients, particularly of B, Mn and Zn are one of the factors contributing to low productivity (Malavolta, 1975).

This paper deals with an experiment designed to compare the effects of leaf sprays with soil applications of micronutrients, and to study their interrelation.

Methods

The experiment was carried out at the Citricul­ture Experimental Station of Bebedouro, SP,

Brazil. The soil is a Dark Red Latosol, medium texture. The grove of Citrus sinensis Osbeck variety Pera on Cleopatra rootstock was planted in November 19S5, with a spacing of 7 x 5 m.

Six treatments with 7 replicates, each one with 5 useful plants, in a randomized block design were used, namely: S0 F 0 or control; S0 F 1 -leaf application; S1F 0 -soil application; S1F 1 -soil and leaf application; S2F 0 - soil application at a rate twice of that used in treatment S1F0 ; S2F 1 -

soil application as in the preceding treatment and leaf spray.

The product used in the foliar treatments, trade name 'Nutrimins', contains Mn and Zn as chelates in the proportion of 20 and 30 g L -l, respectively; it also has 5 g L - 1 B and 100 g L - 1

N. It was diluted in the proportion of 1.5 g L 1

before spraying. Leaf applications with 10 liters of solution per tree were made in February and October 19S9 and 1990. Soil applications were

454 Vitti et al.

made with fritted B, Mn and Zn, a finely ground silicate called 'Nutricitro 248' containing the three micronutrients at concentrations of 20 g kg -l, 80 g kg -I and 240 g kg-\ respectively. The soil treatments were made in the planting holes, in 1985 at rates of 0, 20 and 40 g per plant, and in October 1989 and 1990 at rates of 0, 40 and 80 g. Actual treatments therefore were leaf sprays added to the 0, 1 and 2 levels of 'Nutricitro' and a continuation of soil applica­tions.

Soil samples were collected for analysis in 1985, the following results being obtained, re­spectively, for the 0-20 and 20-40 em layers: pH (CaCl2 )-4.2 and 4.1; organic matter-9.1 and 7.6 g kg -I; P (resin)- 6 and 3 mg L -\ K+- 0.13 and 0.01cmol(+) kg- 1 ; Ca2 + -0.29 and 0.14 cmol(+) kg-\ Mg2+ -0.19 and 0.09cmol(+) kg-\ H+ + Al3+- 4.2 and 4.7 cmol( +) kg- 1 • All plants received NPK fertilizers at uniform rates split into three applications each year during the rainy season (September to March or April). The second or third leaf after the fruit was taken for analysis in March 1990 and April 1991. Boron, manganese and zinc were determined according to Malavolta et al. (1989). On the same occasions soil samples were collected in the dripline, which coincides with the middle of the fertilization band, at a depth of 0-20 em. Boron, Mn and Zn were extracted from the soil samples using the Mehlich 1 procedure.

The height of the canopy and the diameter of the trunk were measured. Yield data were re­corded and fruit samples analysed (weight, diam­eter, peel thickness, soluble solids and acidity).

Table 1. Micronutrient levels (mg kg_,) in the leaves

Treatments Zn

89/90 90/91

SoFo 10.0D 1 9.0C S0 F 1 13.0BC 14.0B S1F0 12.0C 7.0C S,F, 15.0AB 16.0B S,F0 13.0BC 8.0C S2 F 1 15.0AB 2l.OA

c.v. (%) 8.77 18.64 L.S.D. 1.88 3.75

Mn

All results represent the average of 7 repli­cates.

Results and discussion

Leaf analysis

Table 1 presents the results of the leaf analysis conducted in the crop years of 1989/90 and 1990/91. Leaf B was raised only when the element was soil-applied; sprays to the leaves were not efficient probably due to the lack of mobility of B in the phloem. It is known in the case of another perennial, the coffee plant, that soil-applied boron is much more efficient than leaf sprays (Malavolta, 1993). When 80 g Nut­ricitro were given, leaf B reached an optimum level according to Grupo Paulista (1990) and Jones and Embleton (1969).

All treatments increased the Mn concentra­tion, although the significant difference in rela­tion to the control was due only to the S1F1

treatment, a combination of soil and leaf applica­tions. De Geus (1973) has pointed out that both soil and foliar applications are capable of cor­recting Mn deficiency in citrus.

All treatments caused an increase in leaf Zn in the year 1989/90. In the year 1990/91, however, only foliar applications gave significant results, although the Zn levels remained rather low.

Soil analysis

The data are presented in Table 2. In the

B

89/90 90/91 89/90 90/91

26.0B 19.0C 34.0C 36.0C 30.0A 25.0ABC 24.0C 39.0C 3l.OA 26.0AB 6l.OB 75.0B 33.0A 30.0A 64.0B 77.0B 3l.OA 25.0ABC 79.0A 104.0A 3l.OA 25.0ABC 78.0A 98.A

8.42 16.27 6.56 9.56 7.50 6.22 6.22 11.08

1 Figures followed by the same letter do not differ at the 5% level (Tukey). Main treatments refer to soil application, while secondary treatments correspond to leaf sprays.

Micronutrients and citrus yield and quality 455

Table 2. Micronutrient levels in soil (mg kg_,)

Treatments Zn Mn B

89/90 90/91 89/90 90/91 89/90 90/91

SoFo 9.29D 1 2.71B 27.5C 42.14B 2.86C 4.00B

S\IFI 20.81B 2.20DE 35.71AB 38.71B 4.19A 3.88B

S,F" 12.24D 7.70CD 27.57BC 38.43B 2.80C 5.31A S,F, 34.39A 10.44C 42.42A 42.0GB 3.45BC 5.62A S2F 0 19.05BC 22.26B 25.71C 39.71B 2.96C 4.47AB S2F 1 34.44A 29.49A 40.42A 50.57 A 3.95AB 4.72AB

c.v. (%) 21.26 27.44 15.08 10.46 18.72 16.56 L.S.D. 7.50 5.52 8.14 7.!3 1.02 1.26

'Figures followed by the same letter do not differ at the 5% level (Tukey). Main treatments refer to soil application, while secondary treatments correspond to leaf sprays.

sampling done in the 1989/90 season the concen­tration of Zn in the soil was increased by all treatments including leaf sprays. In the following year, however, the application to the leaves only had no effect. Soil Mn in 1989/90 was increased by leaf application either separately or in as­sociation with soil treatment. In the 1990/91 season the only significant difference was found when the highest rate of soil application was combined with leaf sprays. The level of B in 1989/90 was increased only in the treatments in which leaf sprays were present, a pattern not observed in 1990/91. The surprising result repre­sented by leaf sprays being capable of raising the level of micronutrients in the soil could be due to excess solution dripping into the ground. No other suggestion can be made to explain such a finding. The combination of soil and leaf applica­tions (treatments S 1 F 1 and S2F 1 ) revealed higher levels when compared with the control. This could be due to the fact that, in this case, part of the plant needs were met by the foliar sprays,

Table 3. Height (em) of the canopy and trunk diameter (em)

Treatments 1989/90

therefore imposing a lower demand on soil nutrients whose concentrations would build up as a consequence.

Height of the canopy and trunk diameter

The treatments had no effect on tree growth as shown in Table 3.

Fruit analysis

Results of fruit analysis are given in Table 4. The treatments had no effect on fruit characteristics in the year 1989/90. In the year 1990/91, how­ever, the treatment S2F0 (160 g Nutricitro 248) increased soluble solids, whereas S2F 1 (same plus foliar application) caused an increase in the percentage of juice.

Yield

The results are shown in Table 5. The statistical

1990/91

Height Diameter Height Diameter

SoFo 265A' 11.1A 292A 12.8 S0 F 1 264A ll.IA 292A 12.4 S1F 0 261A 10.9A 291A 12.5 S,F, 258A ll.OA 292A 12.5 S2FO 260A 10.9A 292A 12.0 S,F, 257A 10.7A 294A 12.0

C.V.(%) 3.6 3.3 2.3 3.0 L.S.D. 15 0.6 11 0.6

'Values followed by the same letter in a given year do not differ at the 5% level (Tukey).

456 Micronutrients and citrus yield and quality

Table 4. Effect of treatments on citrus fruit characteristics

Characteristic Year Treatments

SoFa SUFI SIFO SIFI S2F0 S,FI

Acidity 89/90 0.93A1 0.96A 0.87A 0.81A 0.85A 0.91A 90/91 0.80A 0.81A 0.79A 0.81A 0.84A 0.91A

Soluble 89/90 9.3A 9.1A 9.9A 9.5A 9.8A 10.0A solids 90/91 9.0A 9.5A 9.4A 9.6A 10.1B 9.6A

Percentage 89/90 56 A 59 A 56 A 54 A 56 A 59 A of juice 90/91 56B 53C 53C 53C 56B 62A

Height of 89/90 7.1A 7.4A 7.2A 7.2A 7.2A 7.2A fruit (em) 90/91 6.9AB 6.9AB 7.1A 6.9AB 6.7B 6.9AB

Diameter of 89/90 6.8A 7.0A 6.8A 6.8A 6.8A 6.8A fruit (em) 90/91 6.3AB 6.3AB 6.6A 6.4AB 6.2B 6.3AB

Peel 89/90 3.5A 3.7A 3.4A 3.5A 3.4A 3.5A thickness (mm) 90/91 3.4A 3.5A 3.5A 3.5A 3.5A 3.3A

Weight 89/90 187A 203A 195A 190A 197A 208A of fruit (g) 90/91 161A 161A 171A 170A 168A 172A

1Values followed by the same letter for a given year do not differ at the 5% level (Tukey).

analysis did not show any effect, although all the treatments caused small increases in yield.

Conclusions

The following conclusions can be drawn: (1) Soil applications raised leaf B to adequate levels, whereas foliar sprays did not. (2) The combina­tion of soil and leaf applications was capable of increasing the levels of Mn and Zn in the leaves. (3) Leaf Mn was increased when the element

Table 5. Effect of treatments on citrus yields (kg fruit/5 plants)

Treatment Year

1980/90 1990/91

S0 F0 267A1 361 SUFI 301A 379 SIFO 301A 395 S,FI 303A 390 S2 F0 279A 370 S,FI 289A 365

c.v. (%) 11.2 9.8 L.S.D. 53 59

1Values followed by the same letter do not differ at the 5% level (Tukey ).

was applied either in the soil or in the leaves. ( 4) Available B, Mn and Zn were raised by the corresponding leaf application. (5) There were no significant effects on tree growth, yield and fruit quality.

References

De Geus J G 1973 Fruit crops-citrus. In Fertilizer Guide for the Tropics and Subtropics. pp 510-516. Centre d'Etude d'Azote. Zurich, Switzerland.

Grupo Paulista 1990 Recomenda<;oes de aduba<;ao e calagem para citros. Laranja 11, 1-14.

Jones W W and Embleton T W 1969 Development and current status of citrus leaf analysis as a guide to fertiliza­tion in California. In First International Citrus Symposium, v. 3, pp 1669-1671. Riverside, California, USA.

Malavolta E, Vitti G C and Oliveira S A 1989 Avalia.;ao do Estado Nutricional das Plantas- Principios e Aplica<;oes. Associa<;ao Brasileira para pesquisa da potassa e do fosfato, Piracicaba, SP, Brazil. 201 p.

Malavolta E 1975 Nutri<;ao e aduba<;ao dos citros. In I Simp6sio Sobre Produtividadc de Citros. Ed. L C Donadio. pp 165-190. Fac. Ci. Agr. e Veterinarias, Jaboticabal, SP, Brasil.

Malavolta E 1993 Nutri<;ao Mineral e Aduba<;ao do Cafcciro. Editora Agronomica Ceres Ltda, Sso Paulo, SP, Brazil. 210 p.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 457-462, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-136

Effects of fertilization on apple tree development, yield and fruit quality

D. SCUDELLARI 1 , B. MARANGONI\ D. COBIANCHI 2 , W. FAEDI 2 and M.L. MALTONI 2

1 Department of Horticulture and Forestry, University of Bologna, Via F. Re 6, 40126 Bologna, Italy. 2Experimental Institute for Horticulture, Piazzale della Vittoria 15, 47100 Forli, Italy

Key words: fruit quality, fruit storage, Malus domestica Borkh., nitrogen, phosphorus, potassium, tree performance

Abstract

A comparative fertilization trial was conducted in Ferrara Province of country's northeastern Po Valley to assess the effects of varying combinations of N, P and K inputs in cvs. 'Hi Early' and 'Heavy Stripe' budded to rootstocks M 26 and MM 106. Four fertilization regimes were applied yearly in March and tested against untreated control: N-P-K (110, 26, 125kgha- 1); N (110kgha- 1); N (110kgha- 1 in split application), and N-K (110, 125 kg ha -I). The data collected over the nine trial years show that (i) no differences in trunk and canopy size or in pruning wood weight were induced by the regimes in either cultivar; (ii) N-P-K, N-K and N increased yield in 'Heavy Stripe' alone; (iii) N-K and N-P-K induced heavier average fruit in both cultivars; and (iv) tree vigour and yield on MM 106 were greater than on M 26. The effects of fertilization on fruit quality were moderate; the split-N application increased soluble solids in fruit. These data suggest that the N and P amounts employed in this study can be reduced in apple orchards.

Introduction

A well-calibrated fertilization regime in apple orchards is indispensable in controlling tree growth habit, in ensuring steady production over orchard life and in producing good-quality fruit (Giulivo, 1986). Moreover, nitrogen inputs in excess of the tree's capacity to use it for op­timum productivity (over-fertilization) accumu­lates in the soil and becomes increasingly vulner­able to a variety of loss mechanisms, including leaching and denitrification (Weinbaum et a!., 1992).

Up to the 1980s the application of nitrogen (N), phosphorous (P) and potassium (K), the main inputs in Italy's apple orchards, had gener­ally been administered together in conjunction with tree regrowth. In those areas where N and K inputs exceeded their rates of uptake by trees

(Batjer and Rogers, 1952), such problems as excess tree vigour and poor fruit quality were common (Gorini, 1985). Then, too, no account was taken of planting density, which for apple varies widely in relation to the extent of vigour determined by the rootstock-cultivar combina­tion, in determining input rates. For example, dwarfing rootstocks like M 9 and M 26 are more susceptible to competition for nutrients and water than vigorous rootstocks (Delver, 1982) because of the less extensive root system de­veloped by trees on the former (Rosati and Faedi, 1977).

Given the lack of experimental data on miner­al nutrition in apple, a comparative fertilization trial was conducted in Ferrara Province of Italy's northeastern Po Valley to assess the effects of varying combinations of N, P and K inputs in cvs. 'Hi Early' and 'Heavy Stripe' budded to

458 Scudellari et al.

rootstocks M 26 and MM 106. The results place particular emphasis on tree growth habit/yield and fruit quality traits.

Material and methods

The apple orchard was established in a loam soil with the following initial chemical traits: pH 8.2, total N 0.105% (analysed after Kjeldahl), ex­tractable phosphorous 6.0 mg kg - 1 (analysed after Olsen et a!., 1954), extractable potassium 90 mg kg - 1 (analysed after Merwin and Peech, 1950) and organic matter 1.3% (analysed after Lotti, 1956). One-year old trees of the Red Delicious, cv. 'Hi Early' (standard) and cv. 'Heavy Stripe' (Cooper 7SB2 spur) grafted to rootstocks M 26 and MM 106, were planted in February 1981. Each cultivar was assigned to an experimental plot of 1500 m2 , replicated three times per rootstock, which in turn was divided into five sub-plots of 300 m 2 each corresponding to the compared fertilization regimes. Each sub­plot included two rows with 8-16 trees depend­ing on planting density ('Hi Early' /MM 106: 4 x 4 m; 'Hi Early' /M 26: 4 x 3m; 'H. Stripe' I MM 106: 4 x 2.5 m; 'H. Stripe' /M 26: 4 x 2m). The subplots were contiguous, and the data were collected from the four central trees so as to avoid border effects between input regimes. The distance from these trees to the boundary of the sub-plot was 7 meters. Four fertilization regimes were applied yearly in March: N-P-K (110, 26, 125kgha- 1 , respectively); N (110kgha- 1); N (110 kg ha - 1 in split-application: 70% in March and 30% at postharvest); N-K (110, 125 kg ha -I, respectively) and tested against un­treated control. Supplies of nitrogen, phosphorus and potassium were applied as urea, mineral perphosphate and potassium sulphate while post­harvest distribution (mid-September) was applied as ammonium nitrate. During the training of the young trees (until 1986) N and P rates were 90 and 17.5 kg ha -I, respectively. Fertilizers were uniformly distributed over the surface of the whole sub-plot.

From 1982 onwards the interrow alleys were managed as green swards and the intrarows as grass-free swathes (herbicide) about two metres wide. The pruning wood, after chopping and the

mown grass were left as ground mulching. The orchard was trained to free palmette and wa­tered by trickle irrigation ( 8 L h - 1). The plant parameters of trunk diameter (measured 15 em above graft union), canopy size and pruning­wood weight and the production parameters of fruit yield, weight and size (the latter indicated by percentage of fruits with diameter over 70 mm) were recorded yearly. Each year, 30 fruits per fertilization regime were analysed at harvest for flesh firmness (using an 11-mm point diameter penetrometer), epicarp colour ex­pressed as Colour Space coordinates (Hunter, 1975) with a Chroma Meter CR-200 colorimeter (Minolta Corp.), soluble solids (with Atago PR-1 refractometer) and acidity (expressed as malic acid content). In addition, a fruit sample of about 20 kg per input regime was kept in post­harvest cold storage at 2 oc and 80-85% relative humidity (rh) for 150 days, removed and sight­examined for external signs of bitter pit and rot. All data were subjected to analysis of variance; separation of means for main factors was per­formed by the Student-Newman-Keuls test (p =

0.05).

Results

Vegetative-production parameters The overall data at trial end in 1989 showed that tree growth as determined by trunk cross-sec­tional area, canopy size and pruning-wood weight (Table 1) of the control was comparable to that of the trees under the various fertilizer regimes. Evident too is the greater vigour of rootstock MM 106 as compared to M 26 (Table 1).

The 1983-89 cumulative production data show no significant differences for the 'Hi Early' trees in the five regimes (Table 2). By contrast, the 'Heavy Stripe' control plants had significantly lower yield than the fertilized trees, except for those in the split-N treatment (Table 2). Of the two rootstocks, MM 106 induced greater yields in both cultivars than M 26 (Table 2). Average fruit weight of 'Hi Early' was found to be higher (over 180 g) as a result of the inputs N-K, N-P­K and split-N; the lowest values were recorded in control and, especially, in the N-only applica-

Fertilization of apple tree 459

Table 1. Vegetative parameters of the two apple cultivars

Factor 'Hi Early' 'Heavy Stripe'

TCSA" Canopyb Pruning TCSA Canopy Pruning (cm 2 ) size wood (em2 ) size wood

(m') (kg) (m') (kg)

Input regime No input 104 7.6 51 50 2.6 9 N-P-K 113 8.2 56 56 2.9 11 N 106 7.2 50 56 2.8 11 Split-N 107 7.7 50 50 2.5 9 N-K 109 7.5 52 53 2.6 10

Significance NS NS NS NS NS NS

Rootstock MM106 127 a" 8.8 a 64 a 60 a 2.9 a 12 a M26 88 b 6.5 b 45 b 46 b 2.4 b 8b

Significance

Interaction NS NS NS NS NS NS

"TCSA trunk cross-sectional area (1989); b 1989; 'Cumulative 1984-1989; "Mean separation within column by SNK test; NS, *, **Non-significant or significant at p = 0.05, 0.01, respectively.

Table 2. Yield parameters of the two apple eultivars

Factor

Input regime No input N-P-K N Split-N N-K

Significance

Rootstock MM106 M26

Significance

Interaction

"Cumulative 1983-1989. b Average 1984-1989.

'Hi Early'

Yield" Fruitb (kg) weight

(g)

297 177 be' 310 184 ab 303 172e 301 182 ab 311 186 a NS

342 a 179 266 b 181

NS

NS NS

' Mean separation within column by SNK test.

'Heavy Stripe'

Fruitb Yield Fruit Fruit size (kg) weight size (%) (g) (%)

85 ab 141 b 172 b 72b 88 a 167 a 183 a 82 a 83 b 162 a 180ab 75 ab 85 ab 156 ab 175 ab 75 ab 87 a 167 a 184a 80 a

87 172a 175 73 85 146 b 183 81 NS NS NS

NS NS NS NS

NS, *,**Non-significant or significant at p = 0.05, 0.01, respectively.

tion (Table 2). Similar results were registered for the percentage of fruit having a transversal diameter more than 70 mm (marketable). With 'Heavy Stripe' the yearly inputs of N-K and N-P-K resulted in a weight increase in com-

parison to the control; similar findings were registered for the percentage of marketable fruit (Table 2). The rootstocks exhibited no signifi­cant differences as to fruit weight and size (Table 2).

460 Scudellari et al.

Fruit quality The response of flesh firmness varied depending on input regime. 'Hi Early' showed less firmness under N-P-K than under the control and N-only regimes (Table 3), whereas 'Heavy Stripe' ex­hibited significant differences only between con­trol and N-K (Table 4). The soluble solids of the 'Hi Early' fruits increased only in the split-N regime as compared to control (Table 3), while the same treatment with cv. 'Heavy Stripe' significantly increased sugar concentration as compared to the control, N and N-K regimes (Table 4). The apple acidity values at harvest were lowest in the N and split-N treatments for 'Hi Early' (Table 3 ), wheres only in the split-N treatment were they significantly lower than in the other treatments for the spur 'Heavy Stripe' (Table 4). No significant differences were found between rootstocks as to fruit quality (Tables 3 and 4). Colour brightness (L* coordinate) ex­hibited the highest value only in 'Heavy Stripe' under the N and split-N regimes while no signifi­cant differences were found in 'Hi Early' (data non reported). The input regimes induced in 'Heavy Stripe' fruit a lighter shade of red (a* coordinate) than in control while in 'Hi Early' only those fruits under the N-P-K and N-K regimes had a lighter colour than control (data non reported).

Table 3. 'Hi Early' apple fruit quality (average 1984-1989)

Factor Flesh Soluble Acidity firmness solids (meg) (kg em_,) (%)

Input regime No input 7.5 a 12.0b 36.9 a N-P-K 7.2 h 12.2 ab 38.2 a N 7.5 a 12.3 ab 34.6 b Split-N 7.5 a 12.4 a 34.9 b N-K 7.3 ab 12.3 ab 38.1 a

Significance

Rootstock MM106 7.4 12.4 36.3 M26 7.4 12.1 36.8

Significance NS NS NS

Interaction NS NS NS

' Mean separation within column by SNK test. NS, *. **, ***Non-significant or significant at p = 0.05, 0.01 and 0.001, respectively.

Table 4. 'H Stripe' apple fruit quality (average 1984-1989)

Factor Flesh Soluble Acidity firmness solids (meq) (kg em_,) (%)

Input regime No input 7.5 a' 12.4 b 32.7 a N-P-K 7.3 ab 12.6 ab 32.9 a N 7.3 ab 12.2 b 33.2 a Split-N 7.3 ab 13.0 a 30.7 b N-K 7.2 b 12.4 h 33.8 a

Significance

Rootstock MM106 7.3 12.6 32.7 M26 7.3 12.5 32.6

Significance NS NS NS

Interaction NS NS NS

' Mean separation within column by SNK test. NS, *, **Non-significant or significant at p = 0.05, 0.01, respectively.

Fruit storage Cold-stored fruits of 'Hi Early' showed a higher incidence of bitter pit when grafted on M 26 as compared to MM 106 (Table 5). An increase of bitter pit of 'Hi Early' fruits on M 26 only, was found as a result of N-P-K and N-K regimes (Table 5); split-N and N-K regimes induced a higher incidence of rot-affected fruits (Table 5). In 'Heavy Stripe', by contrast, no differences in regime were noted in comparison to the disorder monitored (data non reported).

Discussion

Plant growth habit evaluated per the main pa­rameters monitored at the end of the nine test years showed no influence of any fertilizer input, not even nitrogen. This finding is further con­firmation that for apple trees N-enriched fertiliz­ers do not always lead to developmental incre­ments of their shoot-canopy system (Miller, 1983; Scudellari eta!., 1986; Wesley eta!., 1991).

Yield response to the fertilizer regimes on the other hand differed depending on cultivar. The cv. 'Heavy Stripe,' which is marked by the limited growth habit and high potential cropping typical of apple spurs, increased both yield, unlike the standard cv. 'Hi Early,' and fruit size

Table 5. Percentage of 'Hi Early' apple fruits with signs of post-storage bitter pit and rot (average 1984-1989)

Factor Bitter pit Rot (%) (%)

Input regime MM106 M26

No input 9 18 N-P-K 13 28 N 12 14 Split-N 11 18 N-K 10 25

Significance

Rootstock MM106 M26

Significance

Interaction SEM• 2.3

' Mean separation within column by SNK test. • Standard error for the interaction mean. NS, *Non-significant or significant at p = 0.05.

6b' 9 ab 7ab

12a lla

7 10 NS

NS

under inputs. These response differences can be ascribed to the fact that yield, as well as flower­bud induction and fruit set, depend on an adequate supply of both mineral elements in the soil and reserve substances in the plant's woody organs (Blasing et a!., 1990). It is also to be noted that, given the higher planting density of 'Heavy Stripe' as compared to 'Hi Early' and the trial's equal input rates for both cultivars, the nutrient competition was in all likelihood greater among the 'Heavy Stripe' than among the 'Hi Early' trees. For higher planting densities result in a smaller rooted volume per tree (Atkinson et a!., 1976) and a higher nutrient uptake per volume of soil (Atkinson, 1978). It may thus be posited that the lack of nutrient inputs restricted availability of reserve substances and the higher density planting led in cv. 'Heavy Stripe' control to a marked decline of yield and fruit size. The response per cultivar of the rootstocks MM 106 and M 26 to nutrients inputs was identical in that no significant 'input regime x rootstock' inter­action was found, excepting 'Hi Early' fruit storage. The nitrogen-potassium regime appears to have induced a greater fruit size in both cultivars, whereas phosphorous inputs showed no effects at all, a fact that can be attributed to apple's limited intake of this element (Haynes and Goh, 1980).

Fertilization of apple tree 461

The effects of the input regimes on the main fruit quality traits often elicited varying re­sponses depending on cultivar. Noteworthy is the fact that the split-N schedule resulted in a higher soluble solids content in fruits. On the whole, the recorded data seem to indicate that in soils of similar fertility to those in the present study, mineral fertilization plays a limited role in pro­moting tree growth habit and yield performance. Of undoubtedly greater importance are plant material (i.e. cultivar, rootstock, their vigour and nutritional needs), planting density and the crop management practices employed in the orchard. It should be considered that the miner­alization of pruning wood, dropped leaves and mown grass may ensure the restitution of most mineral elements taken up by the orchard (Greenham, 1980). These findings confirm that a reduction in the amounts of nutrients supplied by fertilization to apple trees is advisible ( Catzeflis, 1985; Haynes, 1990; Mantinger, 1986; Marks and Andrews, 1990) and can result in enhanced apple fruit quality traits and shelf-life.

Acknowledgements

The authors wish to thank the Ente Regionale di Sviluppo Agricolo della Emilia-Romagna (ERSA), which hosted the trial at its 'Azienda Diamantina', and the farm's personnel for their technical assistance. The research was funded by the Regione Emilia-Romagna as part of a pro­gramme managed by ERSO of Cesena (Fo).

References

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Atkinson D 1978 The use of soil resources in high density planting systems. Acta Hortic. 65, 79-89.

Batjer L and Rogers B 1952 Fertilizer applications as related to nitrogen, phosphorus, potassium, calcium and mag­nesium utilization by apple trees. Proc. Am. Soc. Hortic. Sci. 60, 1-6.

Blasing D, Atkinson D and Clayton K 1990 The contribution of roots and reserves to tree nutrient demands: implication for the interpretation of analytical data. Acta Hortic. 274, 51-69.

Catzeflis J 1985 Les essais de fumure minerale du pommier,

462 Fertilization of apple tree

en Suisse romande. Rev. Suisse Vitic. Arboric. Hortic. 17, 189-191.

Delver P 1982 Changes in nitrogen fertilization in Dutch orchards. Compact Fruit Tree 15, 57-72.

Giu!ivo C 1986 Tecnica colturale del terreno, fertilizzazione e irrigazione del melo. In Atti Convegno SOl 'La coltura del Melo verso gli anni '90'. Eds. J Youssef. pp 509-526. Cordenons (Pn). SOl Publisher.

Gorini F 1986 Relazione tra fertilizzazione, qualita e conser­vabilitit. In Atti Convegno SOl 'La fertilizzazione delle piante da frutto'. Eds. F Lalatta, G Bargioni and A Febi. pp 119-150. Verona. CCIAA Publisher.

Greenham D W P 1980 Nutrient cycling: the estimation of orchard nutrient uptake. Acta Hortic. 92, 345-352.

Haynes R J and Goh K M 1980 Distribution and budget of nutrients in a commercial apple orchard. Plant and Soil 56, 445-457.

Haynes R J 1990 Nutrient status of apple orchards in Canterbury, New Zealand. 1. Levels in soil, leaves and fruit and the prevalance of storage disorders. Commun. Soil Sci. Plant. Anal. 21, 903-920.

Hunter R S 1975 The measurement of appearance. Wi!cy­Interscience. New York.

Mantinger H 1986 Influenza della concimazione minerale su alcunc varietit di melo. Riv. Fruttic. 48, 37-42.

Marks M J and Andrews L 1990 The response of Bramley's seedling apple trees grown on different rootstocks to spring and autumn applied nitrogen. Acta Hortic. 274, 321-329.

Merwin H D and Peech M 1950 Exchangeability of soils potassium in the sand, silt, and clay fractions as influenced by the nature of the complementary exchangeable cation. Soil Sci. Soc. Am. Proc. 15, 125-128.

Miller S S 1983 Response of young 'Topred Delicious' apple trees to orchard floor management and fertilization. J. Am. Soc. Hort. Sci. 108, 638-642.

Lotti G 1956 La determinazione della sostanza organica nel tcrrcno agrario. Ann. Fac. Agr. Univ. di Pisa 17, 113-125.

Olsen S R, Cole C B, Watanabe F S and Dean C A 1954 Estimation of available phosphorus in soils by extraction with sodium bicarbonate. U.S.D.A. Cir. 939, 19.

Scudellari D, Faedi W, Marangoni B, Cobianchi D and Casalicchio G 1986 Risposta vegeto-produttiva del melo a differenti livelli di fertilita del terrcno. In Atti Convegno SOl "La Fertilizzazione delle Piante da Frutto". Eds. F Lalatta, G Bargioni and A Fcbi. pp. 217-228. Verona.

Rosati P and Faedi W 1977 Osservazioni su apparati radicali di portainnesti del melo in Romagna. In Atti Convegno SOl "Rinnovamento della Coltura del Melo". Eds. pp 15-120. Bologna. SOl Publisher.

Weinbaum SA, Johnson R S and DeJong T 1992 Causes and consequences of overfertilization in orchards. HortTechnol 2, 112-121.

Wesley R A, GreeneD W, Cooley D Rand Schupp J R 1991 Improving the growth of newly planted apple trees. HortScience 26, 840-843.

M.A. C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 463-467, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-163

Effect of Ca nutrition levels on growth and yield of wheat and two cvs. of triticales

M. CEU MATOS 1 , M. ANTONIETA NUNES 2 and EUGENIO PINT0 1

1 Estaqao Agron6mica Nacional, Quinta do Marques, 2780 Oeiras, Portugal. 2 lnstituto de Investigar;;ao Cientifica Tropical, CEPTA, Tapada da Ajuda Ap. 3014, 1301 Lisboa, Portugal

Key words: calcium deficiency, yield components, wheat, triticales

Abstract

Calcium deficiency is a frequent nutritional disorder in large areas of acid soils from the north and central-cast regions of Portugal. Triticalcs, in general, have been reported as more tolerant than other cereal crops to soil acidity. Using wheat as a reference cereal we evaluated some growth characteristics and productivity of cvs. Arabian and Borba (produced by Estac;ao de Melhoramento de Plantas, Elvas, Portugal) grown with 10, 100, 1000 and 2500 J.LM of Ca given as CaC12 in a modified Hoagland's nutrient solution. Dry matter production of vegetative plant parts (roots, culms and leaves) was higher in triticales than in wheat grown in the solutions having higher Ca concentrations. Borba showed the highest dry matter and grain weight production when grown at any Ca concentration. Optimal Ca nutrition level for grain production in wheat was 100 J.LM. The lowest Ca concentration level (10 J.LM) did not affect grain production in triticales, whereas in wheat the yield was reduced due to a great reduction in the number of grains per spike.

Introduction

Triticales which are hybrids between wheat and rye, have been referred to as the cereals of the future, because they can combine the rustic qualities of rye and the productivity of wheat. One characteristic of triticales is their ability to adapt to different ranges of altitude and adverse climatic conditions including semi aridity and poor soils. These hybrids have attracted great interest in many areas of the world so that in 1986 the total sown exceeded 1 million ha (Carnide, 1990). In Portugal, studies of this cereal have been in progress since 1952 (Villax et a!., 1954) and the total area under cultivation has increased from 7000 ha in 1986 to 50000 ha in 1990 (Bagulho, 1990). In Portugal, breeding programmes and some selection has been done to achieve varieties better adapted to the soils and climatic conditions of the northeast

(Guedes-Pinto, 1977) or to the centre-south (Bagulho and Barradas, 1977). In both these regions the acid soils predominate.

The poor structure of the acid soils associated with the low levels of Ca and Mg and the high amounts of soluble AI and Mn are often in­adequate for the growth of crops. Liming of the soil by application of CaC0 3 , Ca(OH) 2 or CaO is frequently used to overcome the problem of acidity. These compounds supply Ca2 + and in­duce an increase in soil pH by neutralizing H+. According to Mengel and Kirkby (1978) one of the effects of liming the soils is also to reduce the soluble AI by precipitation, reducing its toxicity. However in many regions is neither practical or economic to lime and therefore breed and select cultivars for tolerance to soil acidity is of paramount importance. In the present work, the performance of two high yielding portuguese varieties of triticales and one variety of wheat

464 Matos et al.

are compared in conditions of Ca deficiency, in order to evaluate its potential adaptability to acid soils where Ca deficiency is a problem.

Methods

Seeds of the following vanettes were obtained from the 'Esta<;ao de Melhoramento de Plantas' at Elvas, Portugal: Triticum aestivum L., var. Almansor (wheat, WA), Triticosecale Wittm, var. Arabian (Triticale, TA) and var. Borba (Tri­ticale, TB). These varieties have been locally produced using Mexican germplasm (Interna­tional Maize and Wheat Improvement Center).

Plants were supplied with nutrients using a half strength Hoagland's no 1 solution in which NH4N0 3 was replaced by Ca(N0 3 ) 2 • Calcium was supplied at four concentrations: 10, 100, 1000 and 2500 f-LM as CaC1 2 • Concentrations (meq.L _,) of the major elements other than Ca and Cl in the solution were as follows: N, 7.5; P, 1.5; K, 3; S, 2; Mg, 1. Each treatment was replicated four times. Pots were filled with about 2 kg of sand that had been previously washed with an acid solution (HCL, 20% ), followed by deionised water until the draining solution reached the pH of the water. The bottom of each pot was covered with a 1 em thick layer of granulate polyethylene to allow good drainage. Seeds were buried in the sand 1 em deep, and wetted with deionised water during germination which occurred in the middle of April. Ten days after germination the plants were thinned to 3 plants per pot and the four nutrient solutions differing in Ca concentrations were applied three times a week in a volume required to meet the losses of evapotranspiration i.e.: 100 mL at the beginning to 400 mL at the end of the experi­ment.

The three plants were harvested at different times which occurred 45, 75 and 105 days after sowing. At the last harvest, in the end of July, plants of all the varieties had reached the end of the growing cycle and mature grains had formed. Plant roots, leaves, culms, spikes and grain were oven dried separately at 85°C for two days and the various plant parts weighed. Only the results obtained at the last harvest are presented.

The plants were grown in a glasshouse

throughout the experiment, in natural daylight and photoperiod. During the experiment, air humidity varied between 60 to 70% and the average day temperature ranged from 10°C in April to 2SOC in July. The pots were ranged in blocks with plant varieties and Ca treatments randomized within the block. The t student's test was used to evaluate the statistical significance (at p > 95% level) of the difference between two averages.

Results

Triticales grown in the more concentrated solu­tions (1000 and 2500 f-LM Ca) produced signifi­cantly more vegetative dry matter than wheat in the same conditions (Fig. 1); in the other two treatments with low levels of Ca the difference between wheat and any of the two varieties of triticales were not so evident. The triticale var­ieties presented a similar vegetative dry weight production in all treatments, with Borba showing slightly higher values than Arabian. Differences between these two varieties are significant at 100 and 2500 f-LM Ca concentrations.

Concerning the weight of the grain per plant (Fig. 2) both triticales produced higher grain weights than wheat and were not much affected by Ca concentrations even the lowest (10 f-LM) which halved the highest wheat grain production.

14 0 WA llJ TB 1!!1 TA

12

;; 10

.c

"' ·q:; ~

"' u

0 6 E' +

~ 4

iii

10 100 1000 2500

Ca concentration in the nutrient solution { J-1-M)

Fig. 1. Dry matter accumulation (g plant- 1 ) in roots and shoots (except grain) of wheat (WA) and two varieties of triticales, Borba (TB) and Arabian (T A) grown in nutrient solutions at different calcium concentrations.

8 0 WA ll'J TB Ill TA

6

~ .c 4 ·~ 3

" ·~ (!)

2

0 10 100 1000 2500

Ca concentration in the nutrient solution ( !J.M)

Fig. 2. Grain weight per plant of wheat (WA) and of two varieties of triticales Borba (TB) and Arabian (TA) grown in nutrient solutions at different calcium concentrations.

These maximum seed weight occurred in plants provided with 100 p.,M Ca concentration. At this Ca concentration the difference between the grain weight of wheat and the triticales is not significant. Relative to wheat the Borba variety had about 3 times higher production than wheat at the lowest and highest Ca concentrations, which suggests that the Borba performs better than wheat or the Arabian when the environ­mental conditions are not good. Triticales seem not to be affected by low Ca concentrations as can be seen in the 10 p.,M treatment.

Figure 3 compares the number of spikes per plant, the weight of 100 grains and the number of grains per spike between varieties and indi­cates that the latter is responsible for the lower grain yield of wheat plants grown in the extreme Ca concentrations (10 and 2500 p.,M). The weight of hundred grains (Fig. 4) was slightly higher in wheat except for plants with higher Ca supply. Borba variety had again the best per­formance. The results on dry matter partition (Fig. 5) show that: 1) dry matter was pref­erentially allocated to leaves and culms; 2) the weight of reproductive matter (spikes and grain) represented 15 to 31% in wheat, 23 to 45% in Borba and 21 to 32% in Arabian; 3) the lowest and highest Ca concentrations reduce the alloca­tion to grain in wheat, but not in triticales.

Regarding the Ca content in the dry matter (Fig. 6) it was very clear that: 1) the grain of

Ca nutrition of wheat and triticales 465

40 II n. spikes per plant r:ill 100 grain wt.(g)

30 EJ n. grains per spike

20

10

WA TB TA

Fig. 3. Number of spikes per plant, weight of 100 grains and number of grains per spike in wheat (WA) and two varieties of triticales Borba (TB) and Arabian (TA) grown in nutrient solutions with 10 JLM (A) and 2500 JLM (B) calcium.

:g 3 -~

0> 0 0

0 .c 0>

~

10 100 1000 2500

Ca concentration in the nutrient solution ( !J.M )

Fig. 4. Weight of 100 grains (g) for wheat (WA) and two varieties of triticales Borba (TB) and Arabian (TA) grown in nutrient solutions at different calcium concentrations.

wheat and triticales grown in optimum condi­tions (100 p.,M) had, respectively, about 20 and 7 times less Ca than the leaves and 2) in the case of deficient Ca supply (10 p.,M) this element was immobilized in the leaves i.e. did not translocate

466 Matos et al.

0>

0>

0>

0>

10J.tM Ca

WA TB TA

1000 J.tM Ca

WA TB TA

100 J.tM Ca

WA TB TA

2500 11M Ca

WA TB TA

Fig. 5. Dry matter partitioning expressed by the weight of roots !J, shoots Ill, spikes [!i:J and grains D divided by the respective total dry weight, for wheat (WA) and two varieties of triticales, Borba (TB) and Arabian (TA) grown in nutrient solutions at different calcium concentrations.

50

G G

40

30 .c 0> 'ill ;: ~ 20

0

"a'-

10

10 100

Calcium concentration in the nutrient solution ()lM)

Fig. 6. Calcium concentration (percent of dry weight) in leaves (L) and grain (G) for wheat (WA) and two varieties of triticales, Borba (TB) and Arabian (TA) grown in nutrient solutions at different calcium concentrations.

to the meristematic tops. As a result, to maintain the required Ca content in the grain, the normal number of seeds could not be formed. This reduction affected very significantly the wheat variety.

Discussion

It is usually considered that cereals have a lower Ca requirement for growth (Hewitt, 1963), a conclusion essentially based on the observation that cereals can develop without symptoms until later stages of growth than legumes or herbs in similar diluted nutrient solutions low in Ca (Marsh, 1942; De Turk, 1941). More recently, Loneragan et al. (1968) have confirmed that, if the concentration of the nutrient solution is maintained in the rhizosphere through the use of adequate hydroponic techniques, plants can grow well and without visual symptoms of de­ficiency in very low Ca concentrations. The results of these authors showed that the cereals growth (vegetative dry matter) was nearly 100% of the maximal in a 10 p..M Ca solution (i.e. 2 x 10- 3 of the normal Hoagland's solution con­centration), whereas legumes and herbs showed up to a 50% reduction in similar conditions. Among cereals Secale cereale grew to the maxi­mum and Triticum aestivum showed a 20% reduction. In our present work using additions of fresh solution to compensate for evapotranspira­tion, the vegetative growth of Triticales varieties did not differ among Ca concentrations from 10 p..M to 2500 p..M whereas wheat seems to have an optimum at 100 p.M. This agreement of our results reinforces the idea defended by Ingestat (1982) for nitrogen, but also other major ele­ments, that the addition rate of the solution rather than its concentration has to be discussed for effects on growth.

The effects of Ca concentration levels were more pronounced in the grain yield, Borba being about 3 times more productive than wheat at the lowest and highest Ca concentrations. This was the first triticale variety included in the National Catalogue, classified by Bagulho and Carvalho (1988) as a complete triticale with high yielding ability and good morphological stability in the official tests, a potentiality that agrees with our

results. In gramineous there is a clear distinction between vegetative and reproductive phases because the growth of the shoot stops when the terminal spike appears, and our results suggest that corresponding Ca requirements are differ­ent. The reproductive buds seem to have re­quirements that were not fulfilled because of deficiencies in the transport of Ca in the plant and inadequate supply from the nutrient solu­tion. Processes leading to the formation of the optimal number of seeds were thus affected. The production of floral primordia or the polen, fertilization and the initial steps of the grain filling can be responsible for the reduction in the seed number. The rapid occurrence of these developmental stages includes numerous cell divisions, cell elongation and organization of cellular compartmentation and basically requires very active metabolism. It is well documented in the literature that Ca does not translocate easily to the plant tops and has however an important role in cell wall and membrane stabilization in the young active tissues (Demarty et a!., 1984; Kirkby and Pilbeam, 1984) which can be imme­diately related with the above mentioned physio­logical processes. Furthermore the participation of Ca (usually linked to calmodulin) in the regulation of numerous key enzymes in the cell metabolism (Frieden, 1984; Marme, 1985; Sane et a!., 1987) can justify the disturbancy in the floral development of wheat as reported here. Following work will attempt to precise the phenological steps affected and the Ca dose necessary to produce similar effect in triticales.

References

Bagulho F 1990 Perspectivas da cultura dos cereais de sequeiro em Portugal. Workshop Luso-Israelita. pp 251-260. Lisboa.

Ca nutrition of wheat and triticales 467

Bagulho F and Barradas M T 1977 0 mclhoramcnto de Triticales na ENMP. I" Reuniao Portuguesa sobre tri­ticales. Abs. 4. Vila Real.

Bagulho F and Carvalho P M 1988 Results and perspectives in triticales breeding. Ber. Akad. Landwirtsch. 266, 407-413.

Carnide V P 1990 Estudos citogeneticos e de aptidao for­rageira de dois hibridos interespecificos (Festulolium e Triticale). Thesis, Univ. Tnis-os-Montes e Alto Douro, Vila Real.

Demarty M, Morvan C and Thellier M 1984 Calcium and the cell wall. Plant Cell Environ. 7. 441-448.

De Turk E E 1941 Plant nutrient deficiency symptoms: physiological basis. Ind. Eng. Chern. Ind. Edn. 33, 648-653.

Frieden C 1989 The regulation of protein polymerization. T.I.B.S. 14, 283-286.

Guedes-Pinto H 1977 Projecto de Melhoramento de triticale (2" aproxima9ao). IPVR. Divisao de Genetica e Melhoramento de plantas, Vila Real. 18 p (Mimco).

Ingestad T 1982 Relative addition rate and external concen­tration; driving variables used in plant nutrition research. Plant Cell Environ. 5, 443-453.

Kirkby E A and Pilbeam D J 1984 Calcium as a plant nutrient. Plant Cell Environ. 7, 397-405.

Loneragan J F, Snowball K and Simmons W J 1968 Response of plants to calcium concentration in solution culture. Aust. J. Agric. Res. 19, 845-857.

Marme D 1985 The role of calcium in the cellular regulation of plant metabolism. Physiol. Veg. 23, 945-953.

Marsh R P 1942 Comparative study of calcium-boron metab­olism of representative dicots and monocots. Soil Sci. 53, 75-78.

Mengel K and Kirkby E A 1987 Principles of Plant Nutrition. International Potash Institute, Bern, Switzerland. 685 p.

Sane P V, Kumar N N, Baijal M, Singh K K and Kochhar V K 1987 Activation of nitrate reductase by calcium and calmodulin. Phytochem. 26, 1289-1291.

Villax E J, Mota M, Ponce-Dentinho A 1954 Dois novos triticales. Melhoramento VII, 29-64.

H

Effects of heavy metal stress on crop behaviour

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 471-476, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-027

Trace elements and isoenzyme activities in white lupin

AMARILIS DE VARENNES and ISABEL CARVALHO Department of Chemistry, University of Agronomy, Tapada da Ajuda, 1399 Lisboa, Portugal

Key words: isoenzymes, Lupinus albus L., nutrient stress, solution culture, trace elements, white lupin

Abstract

Some enzymatic activities are known to be affected by plant stress. In the present work we studied the effect of different levels of trace elements on some isoenzyme activities of white lupin (Lupinus albus L.). Plants were grown in nutrient solutions with variable concentrations of manganese, boron, zinc, copper, cobalt, molybdenum or iron, and were analysed for acid phosphatases, carboxylesterases and aspartate aminotransferases. The activities of some of the isoenzymes were related to the level of trace elements in the plant. Manganese deficiency resulted in the inhibition of one carboxylesterase isoenzyme. Toxic levels of the trace elements studied lead to the appearance of new zymographic forms or to the inhibition of isoenzymes present in non-stressed plants.

Introduction

Some plant enzymatic activities are known to be affected by nutrient deficiency. For instance, iron deficiency inhibited the activity of a per­oxidase isoenzyme in bean roots (Sijmons et a!., 1985), phosphorus deficiency induced the ap­pearance of a new phosphatase isoenzyme in wheat (Guthrie et a!., 1991; McLachlan et a!., 1987) and nitrogen deficiency induced the ap­pearance of three new esterase isoenzymes in broadbeans (Gates and Boulter, 1979). On the other hand, very little is known on how toxic levels of some nutrients can affect plant en­zymes, though metabolic unbalances are always associated with toxicity.

In the present work, we investigated if the isoenzyme patterns of aspartate aminotransfer­ase (EC. 2.6.1.1.), acid phosphatase (EC. 3.1.3.2.) and carboxylesterase (EC. 3.1.1.1.) were modified when lupin plants were grown in the presence of different levels of several trace elements. Isoenzyme phenotypes can be characterized for white lupin because

it is mainly autogamous and multiplied by seed.

Aspartate aminotransferase (AA T) plays a key role in hydrogen and carbon shuttles (Hatch, 1972; Heber and Hedlt, 1981) and in nitrogen distribution in plants. It catalyses the transfer of an amino group from aspartate to a-ketoglu­tarate:

aspartate+ a-ketoglutarate- oxaloacetate +

glutamate

Aspartate is needed in higher plants for the biosynthesis of several aminoacids and nu­cleotides (Goodwin and Mercer, 1983).

Carboxylesterase (CE) catalyses the hydrolysis of carboxylic acid esters to the free acid anions and alcohol. They are abundant in plants, where they are involved in lipid metabolism and pres­ent several zymographic forms (Schwartz et a!., 1964).

Acid phosphatase (AP) hydrolyses a wide range of orthophosphate monoesters and also catalyses phosphate transfer. Its activity is re-

472 Varennes and Carvalho

latcd to plant phosphorus status (McLachlan et al., 1987; Reid and Bieleski, 1970).

These enzymes are not known to be depen­dent on any of the trace elements studied. Therefore, changes in their activity were not expected a priori. They are also easy to detect with simple colorimetric methods.

The results obtained showed that particular isoenzyme species were sensitive to the level of some of the trace elements studied.

Material and methods

Plant material

White lupin seeds (Lupinus albus L. cv Estoril) were germinated in moist cottonwool at room temperature, in the dark. Seedlings were trans­planted to nutrient solutions which contained macronutrients and different levels of mangan­ese, boron, zinc, copper, cobalt, molybdenum and iron supplied as MnS04 , H 3B03 , ZnS04 ,

CuS04 , CoC12 , Na2Mo0 4 and Fe-EDTA, re­spectively.

Control-plants were grown in solutions con­taining 6mM Ca(N0 3 ) 2 , 6mM KN0 3 , 2.5mM MgS04 , 1 mM KH 2P04 , 100 JLM Mn, 100 JLM B, 30 JLM Zn, 0.1 JLM Cu, 0.1 JLM Co, 1 JLM Mo and 75 JLM Fe.

In the experiments carried out, plants were grown in solutions with the same level of nu­trients as in control-plants, apart from the level of one trace clement. Concentrations tested (apart from control) were 0, 1000 and 10000 JLM Mn; 1000, 2000, 4000 and 5000 JLM B; 0, 300, 1500 and 3000 JLM Zn; 1, 10, 25 and 50 JLM Cu; 1, 10, 50 and 100 JLM Co; 10, 100, 250, 500, 1000 and 2000 JLM Mo; 0, 750 and 7500 JLM Fe. In one experiment, plants were grown for one week with 75 JLM Fe and a second week with 7500 JLM Fe.

Plants were kept in a growth cabinet at 18°C and 55% humidity, with 14 h dark and 10 h light, at a light intensity of 500 JLmol foton m -z s -I supplied by day light fluorescent tubes.

Samples from leaves and roots were collected one, two and three weeks after transplant and were analysed for enzyme activity.

Enzyme separation

Leaves or roots were homogenised at ooc in 0.1 M Tris-HCl buffer, pH7.5, containing 0.25% Na thioglycollate and 0.25% K disulphide. The homogenates were centrifuged at low speed and the supernatants were immediately used or stored frozen at - 20°C until the following day. The protein samples were resolved by non-de­naturing polyacrylamide gel electrophoresis using 8% acrylamide gels. Electrophoresis was performed at 500 V and ooc until the tracking dye reached the bottom of the gel.

Enzymatic staining

Enzymatic activities were detected in the gels using the staining procedures described by Schwartz et al. (1963), Scandalios (1969) and Market and Hunter (1959) for aspartate amino­transferase, acid phosphatase and carboxylester­ase, respectively.

Trace element analysis

The content of trace elements was determined in control-plants and in plants where modifications of zymographic stains were obtained. Leaves or roots of two week old plants were dried at 105°C, weighed and then burned at 500°C.

To determine boron concentrations, the ash was dissolved in 0.1 M HCl and reacted at 55°C with 5% oxalate and 0.04% curcumin in ethanol. It was then dissolved in ethanol and the solution centrifuged. The absorbance of the supernatant was measured at 540 nm and the results obtained were compared with a standard curve.

To determine the concentration of the other trace elements, the ash was digested in 3 M HCl and their concentration was estimated by atomic absorption spectrophotometry.

The concentration of molybdenum in the plants was not determined.

Results

The results obtained for the three enzymes studied are shown in Figure 1. As can be

Trace elements and isoenzyme activities in lupin 473

Aspartate aminotransferase (RRT)

Leaues Roots

Rf c -Mn Mn 8 -zn zn Cu Co Mo -Fe Fe Rf C - Mn Mn 8 - Zn Zn Cu Co Mo -Fe Fe

0.10 0.10 -- -0.13 - - - - - - - - - - - '·'' - - - -------0.11 - - - - - - - - - - - 0.18 --- -------0.25 ----------- 0.25 -------0.50 o.s ..

Held phosphatase (AP)

Leaues Roots

Rf C Mn Mn 8 Zn Zn Cu Co Mo -Fe Fe Rf C - Mn Mn B - Zn Zn Cu Co Mo -Fe Fe

0.20

0.42 0.45 0.50 0.55

-----------iii --

------- -- - - --- iii ---0.20

0.42 0.45 0.50 0.55

-= --= ----- - ----

------= - - -- = ------

CarboHylesterase (CE)

leaues Roots

Rf c -Mn Mn 8 -Zn Zn cu co Mo-re Fe Rf c -Mn Mn 8 -zn zn cu co Mo-re Fe

0.09 ----------- 0.09 -----------

0.22 -------1·--- 0.22 -----------0.38-----------0.42- ---------0.55-----------0.58-----------

0.58-----------0.42- ---------

::~: ==========-Fig. 1. Zymograms for aspartate aminotransferase, acid phosphatase and carboxylesterase, obtained when white lupin was grown with different levels of several trace elements. C- control plants; -Mn, -Zn and -Fe- plants grown with no supply of that element; Mn, B, Zn, Cu, Co, Mo and Fe- plants grown with a high level of that element.

observed, all trace elements studied induced at least a modification in one enzyme.

Normal zymograms

Lupin leaves and roots from control-plants showed three bands with AAT activity with Rf values of 0.13, 0.18 and 0.25, four main bands with AP activity with Rf values of 0.2, 0.42, 0.45 and 0.55 and six main bands with CE activity with Rf values of 0.09, 0.22, 0.38, 0.42, 0.55 and 0.58.

Manganese-related isoenzyme patterns

Leaves or roots from two or three week old plants grown with 10000 !-LM Mn presented an additional AAT species with a Rf value of 0.1.

Three week old plants grown with 10000 j.LM Mn showed a new leaf AP isoenzyme with a Rf value of 0.50. Levels of manganese in the nutrient solution lower than 10000 !-LM lead to zymog­rams that were indistinguishable from those obtained with control-plants.

Plants with two or three weeks, but grown with no manganese supply, showed a marked decrease in the activity of one CE isoenzyme with a Rf value of 0.42.

Boron-related isoenzyme patterns

Root AAT isoenzymes were severely inhibited in plants with one week or more, when grown with 2000 !-LM B or higher. The other isoenzymes were not affected by the different levels of boron tested.

474 Varennes and Carvalho

Iron-related isoenzyme patterns

Roots from plants grown for one week or longer in 7500 p.,M Fe showed an inhibition of the CE isoenzyme with a Rf value of 0.55. Leaves of two week old plants grown with 7500 p.,M Fe or grown for one week with 75 p.,M Fe and a further week with 7500 p.,M Fe had a new AAT iso­enzyme with a Rf value of 0.1. No other altera­tions related to iron were detected.

Copper, molybdenum and zinc-related isoenzyme patterns

The roots of plants grown with high levels of these trace elements presented a new AAT isoenzyme with a Rf value of 0.1. Critical levels and plant age were: 2000 p.,M Mo for one week or older plants; 25 p.,M Cu or higher for one week or older plants and 1500 p.,M Zn for three weeks old plants or 3000 p.,M Zn for one week or older plants. The other isoenzymes were not affected by these trace elements.

Cobalt-related isoenzyme patterns

Leaves from plants grown for two weeks or longer in 50 p.,M Co or higher presented a new AP isoenzyme with a Rf value of 0.50. No other modifications related to cobalt were detected.

Level of trace elements

The concentration of trace elements in the nu­trient solution had a direct effect in the corre­sponding plant level. The results obtained are shown in Table 1.

Discussion and conclusions

The results presented show that different levels of some trace elements induce isoenzyme changes in the white lupin cultivar used in these assays.

The root AAT zymogram was altered by high levels of manganese, zinc, copper or molyb­denum. This non-specific reaction involved the appearance of a new band with low electro­phoretic mobility (Rf 0.10). Zinc and copper levels (molybdenum was not quantified) were higher in the roots than leaves, which can ex­plain why the leaves had the same zymograms as control-plants. In the leaves the same new band was induced by high levels of manganese or iron. The root zymogram was not altered by iron, though its level was higher than in the leaves.

Root AAT isoenzymes were sensitive to boron, whilst the corresponding leaves exhibited unchanged zymographic bands. It should be pointed out that boron concentrations were higher in the leaves than in the corresponding

Table I. Levels of the trace clements in white lupin leaves and roots

Level of the trace Level of the trace elements in the plant dry weight element in the nutrient solution a Mn B Zn Cu Co Fe

(mgg-1) (p,g g I) (mg g I) (p,gg I) (p,gg-1) (mgg-1)

Control (roots) 0.18 ± 0.02 84.61 ± 20.85 0.52 ± 0.03 23.70 ± 3.66 12.20 ± 1.85 1.54 ± 0.06

High level of the 10.39 ± 0.76 294.37 ± 36.99 13.76 ± 2.23 128.27 ± 4.58 980.16 ± 115.88 13.96 ± 2.60 element (roots)

Control (leaves) 1.49 ± 0.08 338.97 ± 53.29 0.18 ± 0.01 19.12 ± 1.64 9.98 ± 3.33 0.55 ± 0.10

High level of the 34.27 ± 1.28 1656.00 ± 146.69 1.56 ± 0.07 16.23 ± 2.50 315.14 ± 27.22 1.77 ± 0.08 element (leaves)

"A high level of each trace element in the nutrient solution corresponds to: 10000 p,M Mn; 5000 p,M B; 1500 p,M Zn; 25 p,M Cu; 50 p,M Co and 7500 p,M Fe.

roots; therefore, either a different boron dis­tribution or a different enzyme sensitivity has to be assumed.

Three to four AA T isoenzymes have been reported in higher plants. They have different subcellular locations and separate cytosolic, plas­tid, mitochondrial and microbody AAT forms have been described (for a review see Newton, 1983). Therefore, the three AA T bands detected in non-stressed plants probably correspond to true isoenzymes. These are tolerant to high levels of all the trace elements studied except boron. The fourth AA T band detected in stres­sed-plants was not formed at the expense of the others species. It is therefore not probable that the new form resulted from a different post­translational modification of the proteins. It could have arisen from a silent locus that became active as a consequence of the imposed stress. Further studies are needed to determine its cellular location and quaternary structure.

The zymographic stains of root AP were stable, showing no modifications with the con­centrations of trace elements tested. A new leaf AP with a Rf value of 0.50 was induced by a high level of manganese or cobalt. It is curious to note that though the manganese concentration was higher in the leaves than in the corre­sponding roots, the opposite was true for cobalt. Therefore, it can be concluded that leaves are more sensitive than roots towards cobalt, proba­bly due to a different cellular distribution of this element.

The CE zymogram was only modified by manganese deficiency (leaves and roots) or iron toxicity (roots). The isoenzyme with a Rf value of 0.42 seems to be manganese-dependent, whilst the isoenzyme with a Rf value of 0.55 is inhibited by a high level of iron. It should be noted that iron accumulates preferentially in lupin roots, so that its root level is four to eight times higher than the corresponding leaf levels.

Trace clements are toxic to plants; as a result, growth is impaired, toxicity symptoms develop and, if the stress is intense, plants will eventually die. In spite of this, few biochemical aspects of trace clements' toxicity are known, so far. Meta­bolic unbalances due to altered enzymatic ac­tivities are to be expected, since some trace elements will be able to inhibit enzymes by

Trace elements and isoenzyme activities in lupin 475

incorporation in the protein, through metal che­lation, noncovalcnt binding or esterification.

Different isoenzymes will vary in their capacity to form complexes with trace elements. This was shown to happen in the present work for carbox­ylesterases and iron. Only one isoenzyme was inhibited by high iron levels.

As far as we know, this is the first report of differences in isoenzyme tolerance to high levels of trace elements in plants. However, heavy metal pollution is known to select against certain allozyme genotypes in marine organisms and this has been interpreted as a result of different isoenzyme tolerance to these metals (Lavie and Nevo, 1987).

Our work also showed that a further con­sequence of trace elements' toxicity is the induc­tion of new enzyme species. Since no genetic analysis of white lupin was carried out, further work is needed to prove if these are true iso­enzymes or the result of altered post-translation­al modifications. However, since the new species were apparently not synthesized at the expense of forms present in non-stressed plants, the former is more likely to have happened.

Since AA T and AP arc involved in nitrogen and phosphorus usage, the formation of new isoenzymes species are likely to result in further metabolic impairment.

Acknowledgements

We would like to thank Dr Diamantino Rebelo for his generous gift of the lupin seeds. We also wish to thank Mrs Gra<;a Pereira Roque for her technical assistance.

References

Gates P and Boulter D 1979 Nitrogen regime and isoenzyme changes in Vicia faba. Phytochemistry 18, 1789-1791.

Goodwin T W and Mercer E I 1983 Introduction to Plant Biochemistry. Pergamon Press, Oxford, 677 p.

Guthrie R E, McLachlan K D and De Marco D G 1991 Acid phosphatases associated with phosphorus deficiency in wheat: partial purification and properties. Aust. J. Plant Physiol. 18, 615-626.

Hatch M D 1972 Separation and properties of leaf aspartate aminotransferase and alanine aminotransferase isoenzymes

476 Trace elements and isoenzyme activities in lupin

operative in the C4 pathway of photosynthesis. Arch. Biochem. Biophys. 156, 207-214.

Heber U and Hedlt H W 1981 The chloroplast envelope: structure, function and role in leaf metabolism. Annu. Rev. Plant Physiol. 32, 139-168.

Lavie B and Nevo E 1987 Differential fitness of allec isozymes in the marine gastropods Littorina punctata and Littorina neritoides, exposed to the environmental stress of the combined effects of cadmium and mercury pollution. Environ. Manage. 11, 345-349.

Market C and Hunter R 1959 The distribution of esterases in the mouse tissues. J. Histochem. Cytochem. 7, 42-49.

McLachlan K D, Elliott DE, De Marco D G and Garran J H 1987 Leaf acid phosphatase isozymes in the diagnosis of phosphorus status in field-grown wheat. Austr. J. Agric. Res. 38, 1-13.

Newton K J 1983 Genetics of mitochondrial isozymes. In Isozymes in Plant Genetics and Breeding. Eds. S D Tanksley and T J Orton. pp 157-174. Elsevier, Amster­dam.

Reid M S and Bieleski R L 1970 Changes in phosphatase activity in phosphorus deficient Spirodela. Planta 94, 273-281.

Scandalios J C 1969 Genetic control of multiple molecular forms of enzymes in plants: a review. Biochem. Genetics 3, 37-79.

Schwartz H M, Biedron S I, Von Holdt M M and Rehm S 1964 A study of some plaut esterases. Phytochemistry 3, 189-200.

Schwartz M K, Nisselbaum J S and Bodansky 0 1963 Procedure for staining zones of activity of glutamic ox­oloacetic transaminase following electrophoresis with starch gels. Am. J. Clin. Pathol. 40, 103-106.

Sijmons P C, Kolattukudy P E and Bienfait H F 1985 Iron deficiency decreases suberization in bean roots through a decrease in suberin-specific peroxidase activity. Plant Phy­siol. 78, 115-120.

M.A. C. Fragoso and M.L. van Beusichem ( eds.) Optimization of plant nutrition 477-482, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-052

Effect of iron chlorosis on mineral nutrition and lipid composition of thylakoid biomembrane in Prunus persica (L.) Bastch.

E. MONGE, C. PEREZ, A. PEQUERUL, P. MADERO and J. VAL Plant Nutrition Department, Estaci6n Experimental de Aula Dei, Aptdo 202, 50080 Zaragoza, Spain

Key words: fatty acids, iron chlorosis, lipids, nutrients, Prunus persica, peach tree, thylakoid lipids

Abstract

The effect of iron chlorosis on mineral, thylakoid lipids and fatty acids composition of field grown peach tree leaves was studied. Significant differences were found in iron extracted by using a,a '-dipyridyl (active iron), total iron, P, K, Cu and the P/Fe and Fe/Mn ratios. The levels of total chlorophyll, total glycolipids and phospholipids were reduced under iron chlorosis. A slight iron deficiency does not modify the fatty acid composition of thylakoid membranes, while a strong deficiency changes the proportion of some fatty acids.

Abbreviations: Chi- chlorophyll, DGDG- digalactosyldiglycerol, MGDG- mono-galactosyldiglycerol, PC- phosphatidycholine, PE- phophatidylethanolamine, PG- phophatidylglycerol, TLC- thin layer chromatography, 16: 0- palmitic acid, 16: 1 - palmitoleic acid, 16: It- trans-hexadecenoic, 18: 0- steric acid, 18: 1- oleic acid, 18:2 -linoleic acid, 18:3- linolenic acid

Introduction

Iron-chlorosis is a world-wide problem, particu­larly in arid and semi-arid regions. The soils of these naturally dry areas are of lime-induced chlorosis and frequently contain high concen­trations (more than 20%) of calcium carbonate. Although, iron is abundant in these soils, it is not readily available for uptake to many types of plants including peach trees.

Iron stressed plants shown visible symptoms in the youngest leaves, which become yellow (chlo­rotic) due to a decline of the Chi content and therefore have lower net photosynthesis (Terry, 1980). Leaves suffering from lime-induced chlo­rosis often have a content of total iron similar to, or ever higher than that of green leaves, indicat­ing a physiological inactivation of iron. Studies on Fe-chlorosis are often controversial on the significance of the some nutrient ratios, mainly P/Fe, K/Ca and Fe/Mn on the development of lime-induced chlorosis.

Iron deficiency preferentially inhibits the de­velopment of the photosynthetic membranes, and its effects are particularly confined to the chloroplasts, while other cell organelles seem to be unaffected (Platt-Aloia et a!., 1983). The membrane lipid composition of chloroplasts de­pends on plant nutritional status and in general, on all factors affecting the membrane staking degree (Horvath et a!., 1987; Murata et a!., 1990; Murphy and Woodrow, 1983).

Lipids are recognized as major chemical com­ponents of biomembranes. Thylakoid mem­branes contain lipids arranged in a double­layered structure. Most of the chloroplast acyl lipids are located in the lamellae (70%) and osmiophilic globuli. 70% of these lipids corre­spond to unsaturated chargeless galactolipids (MGDG and DGDG) and all the others (PG, PC and SQDG) are anionic lipids (Leech and Murphy, 1976).

Most of the knowledge about iron-deficient plants has been obtained with annual plants

478 Monge et al.

(bean, sugar beet, barley and sunflower) grown in nutritive media (with and without iron) and then subjected to iron stress. These plants are usually grown in greenhouse or culture chamber, under controlled conditions of light, photo­period, temperature and humidity (Newman, 1964; Nishio and Terry, 1983). However, cul­tures developed in field conditions affected by environmental stresses whose physiological ef­fects over the thylakoid lipid membranes have received little attention. The purpose of the present study is to show the influence of induced iron deficiency on nutrient elements, their ratios and chloroplast membrane lipid composition of healthy, moderate and strong chlorotic peach trees grown in field conditions.

Materials and methods

About 40-60 leaves (5th-8th fully developed in the year growth) of each tree were harvested on adult peach trees in July (Prunus persica L. cv. Miraflores) grown on calcareous soils (pH= 7.9) in Zaragoza (Spain).

Extractable iron

The determinations were carried out by using a,a '-dipyridyl in order to extract the active iron in leaves, according to Abadia et a!. (1984 ). The extracts were purified by a Sep-Pak, C 18 car­tridge (Millipore) and its absorbance measured at 522 nm.

Mineral elements determination

Leaves were carefully washed with a soft brush and liquid soap (1%) and rinsed with tap water and deionized water to eliminate surface con­tamination. Dry ashing was carried out following the methods of 'Comite Inter-Institutos' (C.I.I., 1969) and Pinta and DcWele (1975). Ca, Mg, Fe, Mn, Cu and Zn were determined by atomic absorption spectroscopy, K and N a by flame emission and P by the vanodomolibdophosphoric method.

Pigment determination

Pigments were extracted from leaf disks cut with a cork borer (0.358 cm2 ) which were ground with 5 mL of 100% acetone and a few mg of sodium ascorbate to prevent the formation of phaeophytins. The quantitative determination was made by reverse phase HPLC according to the method of Val and Monge (1990).

Polar lipids determination

Thylakoid polar lipids were extracted according to the method described by Bligh and Dyer (1959). Later they were resolved by TLC follow­ing the method of Tremolieres and Lepage (1971 ). Methyl-esters of each lipid were obtained by trans-esterification carried out by the metha­nol-sulphuric method (Calvo et a!., 1988). Quantification was performed by using gas chro­matography (HP 5840A GC), equipped with a flame ionised detector and a 2m column (15% DEGS in 80/100 Chromosorb). Methyl pentade­canoate (15: 0) was used as internal standard. The identification of each fatty acid was made by comparing their retention times with commer­cially available standards.

Results

The levels of total Chl were significantly de­creased under iron chlorosis (Tables 1a and b). We classified into three groups according to their contents in chlorophyll per leaf area. Leaves under severe iron deficiency (S) consisted of samples with a chlorophyll concentration below 10 nmol.cm - 2 (average 8.04 nmol.cm - 2 ); (L), between 15-35 nmol.cm - 2 (mild iron deficiency) with an average level of 20.75 nmol.cm - 2 and (C) above 35 nmol.cm- 2 control.

The levels of several macro (N, Ca, Mg and Na) and micronutrients (Mn and Zn) were not significantly affected by iron chlorosis, however P, K, Fe and Cu concentrations were significantly different under iron chlorosis (Tables 1a and 1b). The iron extracted with a,a '-dipyridyl decreased significantly in (S) and (L) cases and was also positively correlated with chlorophyll (Table 2). Phosphorus and P/Fe ratio were significantly

Effect of iron chlorosis on peach 479

Table la. Averages and standard deviations (St) of chlorophyll concentrations (nmol/cm 2 ) and macronutrient levels (mg/ 100 mg dry matter) of peach trees (cv. Miraftores) under several iron deficiency degrees (n = 10-12)

Sample Chl-t N p Ca Mg K Na P/Fe K/Ca

s Average 8.04 3.73 0.35 1.39 0.42 2.73 0.07 47.43 2.05 s St 1.75 0.47 0.04 0.31 0.03 0.20 O.Dl 6.16 0.44

L Average 20.75 3.69 0.30 1.31 0.46 2.37 0.08 33.51 2.44 L St 2.38 0.50 0.03 0.42 0.05 0.27 0.01 5.56 2.24

c Average 35.21 4.01 0.25 1.58 0.45 2.27 0.07 23.63 1.45 c St 3.39 0.31 0.02 0.21 0.05 0.32 0.01 2.07 0.24

Chi-t, total chlorophyll. Sample: C, control leaves; L, leaves under mild iron deficiency; S, severe deficiency.

Table lb. Averages and standard deviations (St) of chlorophyll concentrations (nmol/cm2), micronutrients levels (mgkg- 1) and active iron (f.'g/cm2 ) of peach trees (cv. Miraftores) under several iron deficiency degrees (n = 10-12)

Sample Chl-t Fe-a Fe Mn Cu Zn Fe/Mn

s Average 8.04 1.72 74.41 40.00 9.98 28.42 1.86 s St 1.75 0.31 5.78 0.88 0.67 1.80 0.18

L Average 20.75 2.54 88.93 41.05 10.83 30.19 2.17 L St 2.38 0.37 6.58 2.16 0.52 1.82 0.20

c Average 35.21 3.67 105.37 39.66 11.30 29.60 2.66 c St 3.39 0.41 4.40 1.67 0.54 1.17 0.19

Chl-t, total chlorophyll; Fe-a, active iron. Sample: C, control leaves; L, leaves under mild iron deficiency; S, severe deficiency.

Table 2. Regression equations and variance analysis among total chlorophyll and the significant nutrients

Elements Regression Coefficient Significant

Fe-a Y= 0.12 + 5.35X 0.94 p Y= 0.38- 3.77X 0.85 K Y= 2.82- 0.02X -0.59 Fe Y = 65.87 + 1.11X 0.91 Cu Y= 9.78 + 0.04X 0.64 P/Fe Y= 5.34- 8.69X -0.90 Fe/Mn Y= 1.60- 0.03X 0.89

** and *** mean significant at the p < 0.01 and p < 0.001 levels respectively. Y =elements; X= total chlorophyll.

higher in (S) and (L) cases under iron chlorosis, nevertheless, the Fe/Mn ratio was significantly different under the three treatments, but Ill­

creased as the total chlorophyll decreased. The analysis of lipid extracts performed by

TLC revealed the existence of MGDG, DGDG, PC and PG, but no SQDG was detected. Table 3 shows the average values (percent) as well as the standard deviations of different lipids and the total glycolipid (DGDt) and phospholipid (PLt)

concentrations. These lipids were differentiated in their major lipids: MGDG, DGDG and PG, PC, respectively.

Differences in total galactolipids (DGDt) amongst the treatments (C, LandS) were due to a diminution of MGDG in L samples, while under severe deficiency (S) the decrease in MGDG was also accompanied by a reduction in the DGDG concentration. Iron deficiency acts by diminishing galactolipids.

480 Monge et al.

Table 3. Percent composition of galactolipids and phospholipids from peach tree thylakoid membranes (cv. Miraflores), under several degrees of iron chlorosis

Sample GDG(t) MGDG DGDG PL(t) PG PC

c L s

60.42 56.23 51.68

22.44 ± 1.49 19.69 ± 1.72 20.26 ± 2.31

37.98 ± 2.32 36.54 ± 2.16 31.43 ± 2.12

39.58 43.76 48.32

28.91 ± 2.70 28.71 ± 1.63 22.26 ± 2.15

10.67 ± 1.06 15.05 ± 2.09 26.06 ± 2.74

The results are the average of six determinations ± standard error. Sample: C, control leaves; L, leaves under mild iron deficiency; S, severe deficiency.

Iron deficiency also alters the phospholipid levels. The percent of total phospholipids (PLt) increased with the degree of iron deficiency. Of the phospholipids, the iron deficiency affected mainly PC. In L cases, PG suffered no modi­fication, while PC was affected. However, S thylakoids have shown a slight diminution of PG corresponding with a strong relative increment of PC.

The fatty acid analysis reveal that the light iron deficiency (L) does not disturb the fatty acid composition of lipid thylakoid membranes, while a strong deficiency (S) changes the proportion of some fatty acids (Table 4).

The analysis of these results demonstrates that the 18: 3 proportion of PG and PC fractions, from S samples, decreases practically to half, while the 18: 0 proportion is increased conse-

quently. The same variation seems to occur with the 16: lt and 16: 0 fatty acids, while 16: lt is reduced from 40% to 23%, the 16:0 is increased in the same proportion. The acid trans-hexade­cenoic (16:lt) only could be detected in the PG, while cis is the usual form, amongst the other lipids.

Discussion

When yellowing of the leaves from plants grown in calcareous soils appears, it could be due to several causes. In most cases, it is caused by iron-deficiency which is determined by analysis of physiological active iron in leaves (Abadfa, et al., 1984 ). The high correlation between extract­able iron and Chl (Table 2) demonstrated that

Table 4. Percent composition of fatty acids from galactolipids and phospholipids from peach tree thylakoid membranes (cv. Miraflores), under strong iron deficiency

Sample Fatty cid MGDG DGDG PG

c 16:0 6.88 ± 0.70 20.80 ± 1.60 14.84 ± 1.80 s 8.10 ± 0.60 19.60 ± 1.50 39.93 ± 2.20

c 16: 1 4.21 ± 0.60 3.61 ± 0.60 40.70 ± 3.30" s 1.23±0.15 0.63 ± 0.20 23.48 ± 1.60"

c 18:0 1.06 ± 0.18 3.11 ± 0.24 0.70 ± 0.20 s 2.76 ± 1.80 12.65 ± 1.00 4.56 ± 0.50

c 18: 1 2.00 ± 0.24 1.82 ± 0.20 2.96 ± 0.20 s 2.68 ± 0.20 1.92 ± 0.22 6.38 ± 0.82

c 18:2 4.34 ± 0.60 1.91 ± 0.24 6.80 ± 0.80 s 5.58 ± 0.70 1.80 ± 0.24 8.60 ± 0.80

c 18:3 81.51 ± 6.20 68.75 ± 4.60 34.00 ± 2.60 s 79.64 ± 6.60 63.40 ± 5.80 17.06 ± 1.80

"Trans-hexadecenoic acid. The results are the average of six determinations± standard error. Sample: C, control leaves; S, leaves under severe deficiency.

PC

38.20 ± 0.26 44.40 ± 3.60

2.66 ± 0.30 traces

8.04 ± 0.80 19.90 ± 1.40

7.40 ± 0.60 6.75 ± 0.64

24.77 ± 2.10 18.30 ± 1.40

19.00 ± 1.40 10.64 ± 1.20

the lack of Chi in the peach tree leaves studied was induced by iron-deficiency.

In our study, phosphorus and total iron con­tents were affected by iron chlorosis. The P is probably involved in the interaction with iron nutrition (Bindra, 1980; DeKock, 1981). In chlorotic leaves, the P levels are higher than in the healthy ones, while the iron contents were lower in chlorotic than in control leaves. The P/Fe ratio was significantly affected in both chlorotic samples (Sand L). Although Abadfa et a!. (1985) and Heras et a!. (1976) pointed out the significance of the K/Ca ratio in chlorotic samples, in our experiment, the K/Ca ratio was significantly affected, nevertheless the Fe/Mn ratio was affected. This is in agreement with Bindra (1980), which has shown that there is a close relationship between Fe/Mn proportions and iron chlorosis.

Total polar lipid concentrations in thylakoid membranes from peach tree leaves diminished in relation to iron deficiency, but not all in the same proportion. A peculiarity of iron deficiency is the lack of thylakoid membrane staking or­ganization (grana) in the chloroplasts of leaf cells (Platt-Aloia eta!., 1983). In our study, this iron deficiency probably induced the lack of staking organization, diminutions of galactolipids and concomitantly an increase in phospholoids, being the PC the more increased. These results arc in agreement with those found by Abadfa ct a!. (1988), which could indicate that lipid levels depend on the species and growth conditions. It is necessary to bear in mind that peach tree chloroplasts, in our particular field conditions, were exposed to very high light intensities.

The lack of iron also modified the fatty acid composition. A lower unsaturated degree of galacto and phospholipids were due to an in­crease in saturated fatty acids. Similar results were found by Abadfa et a!. (1988) and Newman (1964).

Acknowledgements

The authors wish to thank M A Gracia for her excellent technical assistance. Work was carried­out under the following research projects CICYT AGR90-0792 and CONAI-DGA: PCA-4/91.

Effect of iron chlorosis on peach 481

References

Abadia A, Ambard-Bretteville F, Remy Rand Tremolieres A 1988 Iron-deficiency in pea leaves: Effect on lipid composition and synthesis. Physiol. Plant. 72, 713-717.

Abadia J, Monge E, Montanes L and Heras L 1984 Ex­traction of iron from plant leaves by Fe(II) chelators. J. Plant Nutr. 7, 777-784.

Abadia J, Nishio J N, Monge E, Montanes Land Heras L 1985 Mineral composition of peach leaves affected by iron chlorosis. J. Plant Nutr. 8, 965-975.

Bindra A S 1980 Iron chlorosis in horticultural and field crops. In Annual Review of Plant Sciences. Ed. C P Malik. Vol. II. pp 221-312. Kalyani, New Delhi, India.

Bligh E and Dyer W J 1959 A rapid method of total lipid extraction and purification. Can. J. Biochem. 37, 911-917.

C.I.I. 1969 Comite Inter-Institutos para el estudio de tecnicas analiticas Metodos de referencia para Ia determinacion de elementos minerales en vegetales. An. Edaf. Agrobiol. 38, 403-417.

Calvo M, Naval J, Lampreave F, Uriel J and Pineiro A 1988 Fatty acids bound to alfa-fetoprotein and albumin during rat development. Biochim. Biophys. Acta 959, 238-246.

DeKock P C 1981 Iron nutrition under conditions of stress. J. Plant Nutr. 3, 513-521.

Heras L, Sanz M and Montanes L 1976 Correction of iron chlorosis in peach trees and its effect on mineral content, nutrient ratios and yield. An. Aula Dei. 13, 261-289.

Horvath G, Melis A, Hideg E, Droppa M and Vigh L 1987 Role of lipids in the organization and function of Photo­system II studied by homogeneous catalytic hydrogenation of thylakoid membranes in situ. Biochim. Biophys. Acta 891, 68-74.

Leech M L and Murphy D J 1976 The coopertive function of chloroplasts in the biosynthesis of small molecules. In The intact chloroplast. Eds. J. Barber, pp 365-401. Topics in photosynthesis. Vol. 1. Elsevier, Amsterdam.

Murata N, Higashi S I and Fujimura Y 1990 Glycerolipids in various preparations of photosystem II from spinach chlo­roplasts. Biochim. Biophys. Acta 1019, 261-268.

Murphy D J and Woodrow I E 1983 Lateral heterogeneity in the distribution of thylakoid membrane lipid and protein components and its implications for the molecular organi­zation of photosynthetic membranes. Biochim. Biophys. Acta 725, 104-112.

Newman D W 1964 Effects of iron deficiency on chloroplast lipids. J. Exp. Bot. 15, 525-529.

Nishio J and Terry N 1983 Iron nutrition mediated chloro­plast development. Plant Physiol. 71, 688-691.

Pinta M and DeWele G 1975 Etalons vegetaux pour I' analyse foliaire. In Le controle de ]'alimentation des plantes cultivees. Ed. K P Vol. pp 159-172. Akademiai Kiado, Budapest.

Platt-Aloia K A, Thompson W Wand Terry N 1983 Changes in plastid ultrastructure during iron nutrition mediated chloroplast development. Protoplasma 114, 85-92.

Terry N 1980 Limiting factors in photosynthesis. I. Usc of iron stress to control photochemical capacity in vivo. Plant Physiol. 65, 114-120.

482 Effect of iron chlorosis on peach

Tremolieres A and Lepage M 1971 Changes in lipid composi­tion during greening of etiolated pea seedlings. Plant Physiol. 37, 911-917.

Val J and Monge E 1990 Violaxanthin cycle and fluorescence

in iron deficient maize leaves. Curr. Res. Photosyn. 4, 765-768.

Reprintedfrom Plant and Soi/154: 97-102, 1993.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 483-489, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-138

Iron stress responses of a chlorosis-susceptible and chlorosis-resistant cultivars of pepper (Capsicum annuum L.)

GEORGE W. WELKIE Biology Department, Utah State University, Logan, UT 84322-5305, USA

Key words: Capsicum annuum L., chlorosis, chlorophyll, iron stress, pepper, riboflavin excretion, root iron reduction

Abstract

A cultivar of pepper (Zehavi) known to produce Fe chlorosis in calcareous soils in Israel was compared to two 'normal' pepper cultivars in Hoagland's I nutrient solutions with no Fe and several concen­trations of FeEDDHA. A number of parameters were monitored to establish to what degree they might be linked in Strategy I plant responses to Fe stress. These included apical leaf chlorophyll, nutrient solution pH, riboflavin excretion, root reductive capacity and tissue mass. The Zehavi cultivar required more FeEDDHA to prevent chlorosis of apical leaves, but the chlorophyll content of its apical leaves of plants supplied with 2 mg L -J was almost equal to that of the other cultivars: Iron stressed Zehavi plants were unable to lower the nutrient solution pH as did plants of the other cultivars. Riboflavin excretion by Fe-stressed roots of Zehavi plants occurred, but concentrations were much lower than those of the other cultivars. Riboflavin concentrations were independent of nutrient solution pH decreases. Root Fe reductive activity for Fe-stressed Zehavi plants harvested over 14 days were mostly less than that of the other cultivars, but activity of all plants fluctuated with time. Use of some NH; in solutions to maintain low pH prevented chlorosis of Zehavi plants supplied with an intermediate Fe concentration.

Introduction

There are numerous reports of physiological response differences among cultivars of both monocotyledenous and dicotyledenous species to Fe-stress (Clark and Gross; 1986). The Zehavi cultivar of pepper (Capsicum annum L.), derived from a cross with cultivar Maor, develops chloro­sis when grown on calcareous soils, contains low amounts of Fe in leaves and regreened when Fe EDTA was added to soil (Shifriss and Eidel­mann, 1983). The pepper cultivar Yaglik was cultured in nutrient solution and when under Fe stress a large increase in Fe reducing activity was noted in a root zone 8-20 mm from the apex. The time of the change was correlated with a pH increase of the nutrient solution, an increase in the citric and malic acid content of the 8-20 mm

root zone, and an autofluorescence of the root zone (Landsberg, 1986). The pepper cultivar Sweet Italian when grown in liquid culture at different Fe concentrations yielded decreases in nutrient solution pH, reduced chlorophyll con­tent and riboflavin excretion into the nutrient solution by roots (Welkie et a!., 1990).

The following studies were undertaken to compare several physiological responses of the 'Fe-inefficient' pepper cultivar Zehavi with the 'Fe-efficient' parent cultivar Maor and the 'Fe­efficient' cultivar Sweet Italian. Of particular concern was the relationships between the pH changes of the nutrient solutions, riboflavin excretion, the Fe reductive capacity of the roots, and the development of chlorosis and any re­covery. A number of dicotyledenous species considered to be very Fe efficient have been

484 Welkie

reported to excrete riboflavin into nutrient solu­tions when under Fe stress (Miller et a!., 1984; Welkie and Miller, 1960; 1988; 1992; Welkie et a!., 1990). The availability of seed of the Zehavi cultivar from Israel possessing the recessive character for Fe chlorosis susceptibility made it possible to test for the relative importance of riboflavin excretion with other Fe-stress re­sponses in this and 'normal Fe-efficient cul­tivars'.

Materials and methods

Seeds of the three pepper cultivars were germi­nated in trays of vermiculite moistened with Hoagland's I nutrient solution (Hoagland and Arnon, 1935) with added 1 mM NH4N0 3 to maintain a lower pH and Fe at 0.5 mg L -I as Giegy Fe Sequestrene 138. The same solution was used when seedlings were transferred to trays of aerated solution culture when the 1st true leaf was emerging. After 10 days, selected seedlings were transferred to trays of deionized water for 1 h to rinse the roots of Fe. Single plants were transferred to aerated, 175 mL wrap­ped test tubes containing standard all N0 3

Hoagland's I nutrient solution and Fe at 0, 0.02, 0.2 and 2 mg L -I. Plants were maintained in a chamber with fluorescent and tungsten light at 645 iJ.-Em -z at plant height and a regime of 18 h: 6 h day: night cycle and a 28: 20°C air temperature cycle. The volume of solution in the tubes was maintained by the addition of diluted Hoagland I nutrient solution lacking Fe. The concentration added was half strength for 3 days, quarter strength for 4 days and eighth strength for 7 days. The eighth strength solution contained an added millimolar solution of KCl to avoid any K deficiency. Tubes were brought up to volume 3 times a day with daily additions ranging from 25 to 100 mL.

Nutrient solutions were sampled daily, and after pH and riboflavin were determined the samples were returned to the tubes. Riboflavin fluorescence was measured on an Aminco Bow­man Spectrophotofluoremeter at 460 nm and 530 nm activation and emission wavelength re­spectively. At several intervals, intact plants with

rinsed roots were used to measure root Fe reduction by the BPDS method (Romheld and Marschner, 1983). Discs were removed from the expanding apical leaves and used for chlorophyll determinations by the DMF method (Moran, 1982).

Several experiments were conducted, but data presented are from a single experiment with the maximum number of plants that could be main­tained in a growth chamber.

Results

There were no early differences of nutrient solution pH with growth of the pepper cultivars at different Fe concentrations. The pH of all solutions increased rapidly from the initial value of 5.0 to 8.0-8.2 by 6 days (Fig. 1). The Italian cv with no Fe caused a nutrient solution pH decrease on day 7 and progressive pH decreases to day 14. The Maor cv with no Fe caused a similar nutrient solution pH decrease on day 9 to day 14. In contrast, the Zehavi cv without Fe caused no nutrient solution pH decrease. The Italian cv supplied with 0.2 mg Fe L · 1 caused a slow progressive pH decrease in the nutrient solution from day 10 to 14. The Maar cv sup­plied with 0.2 mg Fe L -I caused a small pH decrease in the nutrient solution from day 11 to 14. In contrast the Zehavi cv supplied with 0.2 mg Fe L -I caused no pH decrease in the nutrient solution. All cultivars supplied with 2.0 mg Fe L -I caused no pH decreases of their nutrient solutions.

Trace amounts of riboflavin were detected in nutrient solutions of all three cultivars at each of the Fe concentrations by day 1 and the amounts increased each day for a week or more, but at variable rates (Fig. 2). The Zehavi cv without Fe caused the smallest increases of riboflavin in nutrient solutions during the first week of growth. This cultivar caused small increases and minor decreases of riboflavin in the nutrient solution during the second week. In contrast, the Maor and Italian cv without Fe caused large rapid increases in riboflavin up to day 13. The Zchavi cv supplied with 0.2 mg Fe L -I caused about 10-20 fold more riboflavin to accumulate in nutrient solution than when no Fe was sup-

8

7

6

5

8

7

6

5

8

7

6

5

0.0 mg Fe l:1

z---o­M---•---(······-h.······

~ ', A····~:>···· \ .. _\

2 3 4 5 6 7 8 9 10 11 12 13 14 Days

0.2 mg Fe L · 1

z---o­M---•--· I ······-1>.·· .. ··

2 3 4 5 6 7 8 9 10 11 12 13 14

Days

,-:.> •.·

2.0 mg Fe t: 1

z---{)--M ···•··· I ······-h.······

2 3 4 5 6 7 8 9 10 11 12 13 14

Days

Fig. 1. Influence of Fe concentration of nutrient solutions on their changes in pH with culture of an Fe-inefficient pepper cultivar Zehavi (Z) and two Fe-efficient pepper cultivars, Maor ( M) and Italian (I).

Iron stress responses in pepper cultivars 485

17.5-

15.0-

12.5 10.0

10.0 1.0

7.5 0.1

5.0 0.01

2.5 0.001

0

15.0-

12.5 10.0

8 ~10.0

" g 7.5 u: c ~ 5.0 0

.'0 C2 2.5

0

15.0_

12.5

10.0

7.5

5.0

2.5

0

1.0

0.1

0.01

0.001

10.0

1.0

0.1

0.01

0.001

2

2

2

I

2

2

3 4 5 6

0.0 mg Fe L-1

z --o---­M---·-­I ...... ,.,. ......

.li -·

7 "~J -;·~·

!i :

,._ •

-.1:,.

3 4 5 6 7 8 9 10 11 12 13 14 Days

0.2 mg Fe L-1

z---o---­M---•--

/'> I ...... ,.,. ..... .

/~ _.-

3

I I

3 4 5 6 7 8 9 10 11 13 14 Days

2.0 mg Fe L.: 1

z---{)--M---•--I ······A······ _.6. .... 6. .. --A-- /)_ ____ !:J.

/Y

I I

3 4 5 6 7 8 1011121314

2 3 4 5 6 7 8 9 10 11 12 13 14 Days

Fig. 2. Influence of Fe concentration of nutrient solutions on their content of excreted riboflavin with culture of an Fc­inefficient pepper cultivar Zehavi (Z) and two Fe-efficient cultivars, Maor (M) and Italian (I). A fluorescence value of 0.13 corresponds to I mg L -I riboflavin.

486 Welkie

plied. The Italian cv supplied with 0.2 mg Fe L -I caused slightly less riboflavin accumulation in the nutrient solution than was obtained with no Fe. The amounts on most days exceeded those for the Zehavi cv by 10 fold. The Maor cv supplied with 0.2 mg Fe L -I caused riboflavin accumula­tions that were about a third those of the Italian cv. The amount of riboflavin in cultures with 2 mg Fe L -I were all low, but that for Maor cv was only about half that for the Zehavi cv and only about an eighth that for the Italian cv.

The reductive capacity of the pepper cultivars over time were irregular (Fig. 3). The Zehavi cv with no Fe had low reductive capacity from day 5

0.0 mg Fe L1

M

5 7 9 11 12 13 14 Days

M

0.2 mg Fe L-1

2.0 mg Fe L-1

M

5 7 9 11 12 13 14 Days

Fig. 3. Influence of Fe concentrations of nutrient solutions on the Fc-reduction capacity of intact plants of the Fe­inefficient pepper cultivar Zehavi (Z) and two Fe-efficient cultivars Maor (M) and Italian (! ) at selected harvest days.

to day 14. The Maor cv with no Fe had medium to low Fe reductive capacity followed by medium to a very low Fe reductive capacity. The Italian cv with no Fe had high initial Fe reductive capacity followed by low to very low values and more low values. The Zehavi cv with 0.2 mg Fe L - I had low and very low Fe reductive values with more low values. The Maor cv with 0.2 mg Fe had the highest Fe reductive capacity of all at day 5 with one of the lowest by day 9, followed by some low and medium values . The Italian cv at 0.2 mg Fe L -I had medium Fe reductive values with the highest value at day 13. The Zehavi cv with 2.0 mg Fe L -l had medium Fe reductive values at day 5 and 7 with a very low value at day 9 and 11 , followed by high and medium values. The Maor cv had an initial medium and low Fe reduction value followed by very low values, medium values and a very low value. The Italian cv with 2.0 mg Fe L -I had an initial medium value followed by very low val­ues.

Chlorosis developed more rapidly in the Zehavi cv than in the other two pepper cultivars (Fig. 4) . By the fifth day, tip leaves of the Zehavi cv contained one third to one fourth the chloro­phyll present in the other cultivars cultured with no Fe. At this time chlorosis was less pro­nounced in plants cultured with 0.2 mg Fe L - I

and the tip leaves of the Zehavi cv contained 3 times as much chlorophyll as those with no Fe. The other cultivars contained one and a half and two times as much chlorophyll in their tip leaves as did the Zehavi cv. Plants of the Zehavi cv. cultured with 2.0 mg Fe L - I at d 5 contained about double the chlorophyll in tip leaves com­pared to those at 0.2 mg Fe L - I and differed little from the chlorophyll content of tip leaves from the other two pepper cultivars . Zehavi plants cultured with no added Fe had a minimum value for chlorophyll at 11 d and this was fol­lowed by an increase at 12 d and a subsequent decrease. Chlorosis in the other cultivars also increased over time , but their chlorophyll con­tent in tip leaves only reached low values com­parable to the value for the Zehavi cv. at 5 days.

The chlorophyll content in the tip leaves of Zehavi plants cultured at 0.2 mg Fe L - I de­creased slightly over time but also fluctuated.

30

25

20

15

10

5

0

30

25

20

15

10

5

0.0 mg Fe L-1

M M

M I

5 7 9 11 12 13 14 Days

5 7 9 11 12 13 14 Days

2.0 mg Fe 1.:1

5 7 9 11 12 13 14 Days

Fig. 4. Influence of Fe concentrations of nutrient solutions on the chlorophyll content from tip leaves of the Fe·ineffici­ent pepper cultivar Zehavi (Z) and the Fe-efficient cultivars Maor (M) and Italian (I) at selected harvest days.

The chlorophyll content of tip leaves of the Maor cv decreased a small amount at 7 d, increased above the 5 d value on d 9 and 11, but decreased small amounts on d 12 and 14. At this Fe concentration chlorophyll in tip leaves of the Italian cv were mostly less than that in those of the Maor cv. especially at d 12 and 13. None of the chlorophyll values were as low as those for Zehavi plants. No chlorosis was evident in those plants supplied with 2.0 mg Fe L - I. However, the chlorophyll content in tip leaves of Zehavi were moderately less than those of the other cultivars at 9 and 12 d , but closer to or equal to that of the other cultivars on other days.

Iron stress responses in pepper cultivars 487

Discussion

The most pronounced differences between the pepper cultivars in response to Fe stress was the amount of riboflavin excreted into the nutrient solutions. On day 5 when the first reduction was measured the average riboflavin value for solu­tions of the Maor and Italian cv. containing no Fe were 5 and ca. 10 times that of the Zehavi with no Fe. Although the solutions of cultivars with 0.2 mg Fe L -t had more riboflavin at 5 days, the riboflavin in solutions of Maor and Italian pepper plants were only 1.5 and 3 times that for the Zehavi plants. The solutions of Zehavi plants at 2.0 mg Fe L - 1 had ca. 1.4 times more riboflavin than those of the other two cultivars at this Fe concentration. The differ­ences in the pH values at day 5 were negligible. The root Fe reductive capacity on day 5 did not correspond well with the riboflavin values of the nutrient solutions from these plants. Increases of riboflavin over time did not appear to corre­spond to the changes in reductive activity. The riboflavin increases that occured with time in the Fe-stressed plants did not closely correspond to the degree of chlorosis reflected by chlorophyll in the tip leaves. Both the Maor and Italian pepper plants with less chlorosis excreted much more riboflavin than the Zehavi plants when under Fe stress, but the Italian peppers with moderately more chlorosis than the Maor plants did excrete larger amounts of riboflavin, but not in amounts proportional to the chlorosis . In the absence of chlorosis when plants were cultured in 2.0 mg Fe L - 1 , only low amounts of riboflavin were excreted, and the larger amounts excreted at the end of the culture period by the Italian cv. were not related to any difference in chlorophyll content.

In a number of studies of iron stress responses of dicotyledenous species there is good correla­tion between the solution pH decrease, initial chlorosis and increased root reductive capacity , that may or may not be followed by a pH increase recovery from chlorosis lowered root reductive capacity, and sometimes a second cycle like the first (Brown and Jolley, 1988). In this experiment the lack of an early pH decrease of the nutrient solutions of Zehavi and Italian

488 Welkie

pepper plants when chlorosis was evident and root reduction was high is uncommon when small volumes of nutrient solution are used. The pH decrease may have been masked by the rapid uptake of nitrate that was replenished with one half concentration and one quarter strength Hoagland's during the first week of growth. In some earlier studies Italian pepper cultured in small volumes caused small pH decreases of nutrient solutions within 4 or 5 d in Fe-stressed cultures if solutions were replenished with deion­ized water. These had a subsequent reduction of chlorosis and a second more pronounced pH decrease in the second week of growth. The depletion of the nitrate is likely a major factor since the pH of solutions of plants cultured with adequate Fe did not reach pH values of 8.0 and there was some pH decline. Some nitrogen deficiency of the oldest leaves was also evident in the earlier study in the second week, but not in experiments when diluted nutrient solution was used to replenish the volume of solution utilized. Landsberg's (Landsberg, 1986) report of a pH decrease by Fe-stressed Yaglik cv peppers after 4 d from 6.8 to 3.8 was dependent upon a low volume ( 600 mL) of solution containing only about a quarter of the nitrate present in Hoag­land's solution and no nutrients added during growth.

The root Fe reduction values are somewhat unexpected in this experiment with respect to the number of very high values observed when the solution pH was increasing, and the number of more moderately high values when the pH decreased most in Fe-stressed plants. Perhaps the Fe reduction activities measured in pH 5.3 buffer are not a true measure of what the Fe reductive activity is in the nutrient solutions, since the optimum pH in buffered solution for Fe-EDTA reduction is known to be about pH 5.3-5.5. (Cakmak et al., 1987). Also, the pH of the nutrient solution may not reflect the local pH changes in the modified root zones of high reductive activity if utilization and availability of nitrate is the dominating factor controlling nu­trient solution pH and masking zonal pH de­creases.

The significance of the excretion of riboflavin in large amounts from Fe-stressed plants is still unclear. If riboflavin exists in the cytoplasm in

the reduced state and is functioning as a reduc­tant as it is released into the cell wall, one would expect much higher Fe reduction activity as the amounts of riboflavin excreted increased rapidly. This was not evident. Root reductive capacity of both Fe-stressed Zehavi and Maor plants were at times very low when very large amounts of riboflavin were accumulating in their nutrient solution. There is a possibility that a specific form of riboflavin such as FMN is functioning in the Fe reduction process and that riboflavin is produced in excess as a precursor to meet the need. FMN but not FAD is known to be in­creased in tobacco roots when plants are Fe­stressed (Welkie and Miller, 1962). There is negative evidence for Fe-stressed tobacco forma­tion of the FMN-protein, flavodoxin, that is increased in some algae under Fe-stress (Huang et al. , 1992).

The limited capacity of the Zehavi cv to excrete riboflavin when under Fe stress relative to the other two cultivars, coincides with its apparent inability to lower nutrient solution pH and to utilize limited Fe adequately to maintain a high chlorophyll content. However, those plants that caused a lowering of the pH of the nutrient solutions did so only after prolonged growth, long after early high reductive activity and pro­gressive riboflavin excretion.

The chlorosis susceptibility of the Zehavi cv is inherited as a recessive gene (Shifriss and Eidel­mann, 1983). The inability of Zehavi cv to utilize Fe as effectively as the parent Maor cv or Italian cv is likely a quantitative rather than qualitative deficiency in some unknown factor in the Fe reduction mechanism of the root. The ability of Zehavi to increase greatly its riboflavin excretion with an intermediate Fe concentration provides evidence that control for this Fe stress response is not eliminated completely. Although there is no direct evidence of it, the ability of Zehavi to decrease the rhizosphere pH may be a quantita­tive rather than a qualitative response. This will require additional studies with less nitrate and intermediate Fe concentrations, between 0.2 and 2.0 mg L - 1 • Also, root growth on agar with pH color indicators may distinguish small local root pH changes from the bulk root pH changes as described by Marschner et al. (1982). To obtain a better understanding of Fe-stress induced

changes in the Fe reductive actiVIty of pepper rots of the cultivars, larger experiments with more frequent assays may be required.

Acknowledgements

This research was supported by the Agricultural Experiment Station, Utah State University, Logan, Utah 84322-4845. Seed of Maor and Zehavi peppers were supplied by Dr Chen Shif­riss, P.O. B6 Bet Degan 50250, Israel and Hazera Ltd. P.O.B. 1565 Haifa, Israel.

References

Brown J C and Jolley V D 1988 Strategy I and Strategy II mechanisms affecting iron availability to plants may be established too narrow or limited. J. Plant Nutr. 11, 1077-1098.

Cakmak I, van de Wetering D A M, Marschner H and Bienfait H F 1987 Involvement of superoxide radical in extracellular ferric reduction by iron-deficient bean roots. Plant Physiol. 85, 310-314.

Clark R B and Gross R D 1986 Plant genotype differences to iron. J. Plant Nutr. 9, 471-491.

Hoagland D R and Arnon D I 1938 (Revision 1 D I Arnon, 1950) The water-culture method for growing plants without soil. Calif. Exp. Sta. Circ. 347.

Iron stress responses in pepper cultivars 489

Huang 1-J, Welkie G Wand Miller G W 1992 Ferredoxin and flavodoxin in tobacco in response to iron stress. J. Plant Nutr. 15, 1765-1782.

Landsberg E-C 1986 Function of rhizodermal transfer cells in the Fe-stress response mechanism of Capsicum annuum L. Plant Physiol. 82, 511-517.

Miller G W, Pushnik J C and Welkie G W 1984 Iron chlorosis, a world-wide problem: The relation of chloro­phyll biosynthesis to iron. J. Plant Nutr. 7, 1-22.

Moran R 1982 Formulae for determination of chlorophyllous pigments extracted with N; N-dimethylformamide. Plant Physiol. 69, 1376-1381.

Romheld V and Marschner H 1983 Mechanism of iron uptake by peanut plants. Plant Physiol. 71, 949-954.

Shifriss C and Eidelman 1983 Iron deficiency chlorosis in peppers. J. Plant Nutr. 6, 699-704.

Welkie G W, Hekmat-Shoar H and Miller G W 1990 Responses of pepper (Capsicum annuum) plants to iron deficiency: Solution pH and riboflavin. In Plant Nutrition­Physiology and Applications, pp 207-211. Ed. M L van Beusichem. Kluwer Academic Publishers, Dordrecht.

Welkie G Wand Miller G W 1988 Riboflavin excretion from roots of iron-stressed and reciprocally grafted tobacco and tomato plants. J. Plant Nutr. 11, 691-700.

Welkie G Wand Miller G W 1960 Iron nutrition of Nicotiana tabacum L. in relation to riboflavin, riboflavin-5-phosphate and flavin adenine dinucleotide content. Plant Physiol. 315, 516-520.

Welkie G W and Miller G W 1992 Iron stress and salt stress responses of lettuce (Lactuca sativa L.) J. Plant Nutr. 15, 1757-1764.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 491-496, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-055

Are manganese and iron deficiencies so similar? Fluorescence, a way of study

J. VAL, C. PEREZ, P. MADERO, A. PEQUERUL and E. MONGE Plant Nutrition Department, Estaci6n Experimental de Aula Dei, CSIC, Aptdo 202, 50080 Zaragoza, Spain

Key words: chlorophyll fluorescence, electron transport, Fe deficiency, fluorescence quenching, Mn deficiency, photosystem II, sugar beet

Abstract

The symptoms of iron and manganese deficiencies consist of interveinal yellowing. There is an evident lack of color in plants affected either by manganese or iron starvation. In order to avoid influences from other Mn-containing compounds, we measured thylakoid membranes from Mn and Fe deficient soybean beet cellular suspensions and they were treated with DCMU to interrupt the flow of electrons to the plastoquinone pool. In these samples we carried out measurements of the kinetics of fluorescence and the results confirmed those obtained in leaves by modulated fluorescence techniques (Madero and Perez, personal communications). Cellular crude fluorescence measurements showed a decrease in the Fv /Fm ratio in both cases. Nevertheless, the reasons for these changes are different. Fv /Fm diminution induced by manganese deficiency results from a relatively lower concentration of active PS2 reaction centers compared with light harvesting structures. That is to say, that the energy collected by the antenna cannot be driven to open PS2 traps. Therefore, one part of this extra energy has to be dissipated in the form of LHC fluorescence and thus the antenna contribution to the total yield of fluorescence increases. More complicated is the case of the fluorescence changes observed in iron deficient leaves. Apart from cytochromes, the site of iron in PS2 is considered to be between the two quinones 0 8 and QA. Therefore, other causes than those of Mn-deficiency must be operative.

Abbreviations: F- Fluorescence (subscripts o, m and v define minimal, maximal and variable levels), -Fe- iron-deficient, - Mn- manganese-deficient, PS - Photosystem, qQ- photochemical quenching

Introduction

Chlorophyll fluorescence is a powerful tool in the study of plant nutritional deficiencies. For each quantum of energy captured in the red, chloro­phyll induces the promotion of an electron, from its fundamental state to an excited state. Absorp­tion in the blue zone of the spectrum causes an even greater excitation due to the fact that a quantum, in this region, is more energetic. Of all energy captured by the chlorophyll, one part is dissipated in non-radiative form, the other is used in the photosynthetic reactions (ATP

synthesis, reduction of NADP, .. ) and the other portion is lost in the form of fluorescence. Two main mechan­isms that reduce the fluorescence signal can thus be defined: photochemical quenching (qQ) and non-photochemical (qE).

The structure of the chloroplast from Mn­deficient plants depends on the severity of the deficiency. While a lack of light docs not modify the ultrastructure of the chloroplast ( Cheniae and Martin, 1968), severe deficiency produces the disappearance of the thylakoid grana (Mercer et a!., 1962). Other cellular organelles

492 Val et al.

such as mitochondria seem unaffected (Passing­ham et al., 1964).

Chlorophyll fluorescence, determined in leaf, has been used to investigate the state of the Mn in the plant, the ratio Fv (Fv = Fp- Fo) and Fo providing a good indication of the physiological state of the plant under stress of Mn (Kriedemann et a!., 1988). In a fluorescence study with intact leaves of Mn deficient wheat Kriedemann et al. (1985) found that in com­parison with controls, the deficiency results in a strong increase in Fo, Fv decrease, together with a clear loss of capacity to evolve 0 2 , and a reduction of the chlorophyll a/b ratio. These results confirmed those obtained by Simpson and Robinson (Simpson and Robinson, 1984) in spinach. These authors attribute the changes observed in the fluorescence parameters, as well as in the photosynthetic electron transport, to a substantial loss of PS2 reaction centers. This was confirmed through electron microscopy (Simpson and Robinson, 1984). Photosynthetic electron transport is affected by Mn deficiency, since it has been demonstrated that this element is indispensable in the photolysis of water and therefore, the first step in the sequence of photosynthetic electron transport chain is detrimentally affected (Edwards and Walker, 1983).

In natural conditions, iron deficiency presents similar symptoms to those of manganese; with interveinal chlorosis produced mainly in the younger leaves. A number of studies gf fluores­cence emission in Fe-deficient leaves have been carried out (Morales et al., 1990, 1991, 1992), which have shown that severe iron deficiency produces an irreversible decrease in the PS2 photochemistry.

To date, studies of the PS2 photochemistry isolated from the rest of the electron transport chain have not been undertaken, because of the difficulty of obtaining a thylakoid sample repre­sentative of the state of the leaf. The purpose of this work was to introduce a new experimental way to study isolated PS2 that reflects the gener­al state of the leaf. In particular, we investigate the changes in the kinetics of induction of fluorescence in cellular crude suspensions from leaves with heterogeneous pigmentation affected by manganese or iron deficiency.

Material and methods

Plant material

Chloroplasts were extracted from soybean plants (Glycine max, L. cv. Williams) hydroponically cultured in Hoagland's solution. The Fe-deficient and Mn-deficient plants were obtained by with­holding the corresponding nutrient element from the culture solution. All the plants were de­veloped in the same environmental conditions: temperature: 24°C; humidity: 80%; PPDF: 350 ,umol m - 2 s- \ 16 h photoperiod.

Cellular crude suspension

Chlorophyll fluorescence is today a well estab­lished test of photochemical activity of the PS2. It has also been demonstrated that it is especially useful in photoinhibition studies for damage in photosystem 2 (Haworth et al., 1986). Fluores­cence studies in leaves from plants which are iron or manganese stressed have been carried out (Kriedemann et a!., 1985; Morales et a!., 1991), but as yet experiments on the kinetics of fluorescence in thylakoids have not been under­taken. This is because of the problem of the heterogeneous chloroplasts population in leaves with different pigmentation zones, it is imposs­ible, by conventional methods, to extract a sample of chloroplasts representative of the leaf. Briefly, the difficulty is that the chloroplasts from iron or manganese deficient leaves are structural­ly altered, as demonstrated by electron micro­scopy which has been carried out in both sorts of leaves (Platt-Aloia et al., 1983 in iron, and Simpson and Robinson, 1984 in manganese). Green zones of the leaf exist that contain very similar chloroplasts to those of control leaves. In the processes that imply several centrifugations, it is very possible that the final pellet contains a majority of chloroplasts similar to those of con­trol and therefore will be unrepresentative of the deficient plants. In fact, when one attempts to accomplish fluorescence measures on chloro­plasts extracted by conventional methods, small differences are obtained when compared with those accomplished on chloroplasts from control leaves.

In our laboratory, we have solved the problem

Fluorescence in relation to Mn and Fe deficiency 493

of chloroplasts heterogeneity in leaves which are iron and manganese deficient, by avoiding the centrifugation. 20 g of leaves are homogenized by grinding with buffer (50 mM Tricine; 5 mM MgC1 2 ; 10 mM NaCJ; 330 mM Sorbitol, pH 7.8) and filtering through 4 layers of Miracloth. All the operations are carried out at 4oC and in a dimmed room light.

This method, of course, is not appropriate to accomplish protein, enzymes or metabolites studies exclusively from chloroplasts, but it has been demonstrated that it is extremely useful to carry out fluorescence experiments with chloro­plasts or thylakoids, since on this sort of experi­ments only the chlorophyll fluorescence is mea­sured, and this is only contained in the thyla­koids of the chloroplast.

Fluorescence of thylakoids

The kinetics of induction of chlorophyll fluores­cence in thylakoids was measured basically as described previously (Val and Baker, 1989). Briefly, fluorescence was measured in cellular suspensions with a final chlorophyll concentra­tion in the cuvette of 5 J.L g mL -J, to avoid reabsorption. The reaction medium (3 mL) contained 50 mM Tricine, 10 mM KCl, 5 mM

0.1

0.08

a.> 0.06 <:..l ~ a.> <:..l ~ 0.04 '-0 ;:::l

G:: 0.02

0

CoiJtro/

MgC1 2 , 5 mM NaF, 1 J.LM nigericine, 15 J.LM DCMU.

The home-made cuvette consists of two open­ings in the vertical faces, the first harbors the extreme of the fibreoptics that drives light (PPFD of 150 J.Lmol m - 2 s -J) from an halogen source stabilized and a cut off filter of 620 nm (>620 nm, 35-5404, Eating, Watford, UK); at 90 degrees the detector is located (FDP/2-92106, Hansatech, Norkfold, UK) that solely collects the fluorescence emission at 680 nm through an interference filter (35-4068, Baling, Watford, UK)

The analogical signal is withdraw in a compu­ter, during 1 second at a frequency of 4kHz, by a 12 bit resolution AID card (PCL-711S, Advan­tech) plus a software Unkelscope Jr. (Unkel Software, Massachusetts, USA). The ASCII file that contains the curve ( 4000 points) is processed and monitored through Excel 3.0 (Microsoft).

Results

Using the cellular crude DCMU-poisoned, the curves that are shown in Figure 1 have been obtained. The effects of the manganese de­ficiency in the PS2 photochemistry are translated

..... 401 801 1201 1601 2001 2401 2801 3201 3801

-0.02

Time (1 s)

Fig. 1. Kinetics of chlorophyll a fluorescence on DCMU poisoned cellular crude suspensions from Control, manganese-deftcicnt (-Mn) and iron-deficient (-Fe) soybean leaves.

494 Val et al.

in an increase in the contribution of the LHC to the fluorescence emission (Fo) at the same time as the yield of the PS2 (Fv) is reduced, both facts result in a decrease of the Fv /Fm ratio (Table 1 or, in other words, a decrease in the photo­synthetic efficiency of the PS2.

In the comparative case of iron deficiency (Fig. 1) there is no increase in the contribution of the antenna to the fluorescence emission (Fo), but on the other hand, Fv decreases dramatical­ly.

The study of the curves of the induction of variable fluorescence during the first second with Fo normalized is shown in Figure 2. In the three

Table 1. Leaf chlorophyll and fluorescence parameters of cellular suspensions from control, Mn-deticient and Fe-de­ticient soybean leaves

Parameters Source of cellular crude suspension

Control -Mn -Fe

Total leaf Chi 35.52 21.77 9.87 (11-g em_,)

Chla/b 3.62 3.58 3.88 Fo O.G3 0.04 0.03 Fm 0.19 0.09 0.06 Fv 0.07 0.05 0.04 Fv/Fm 0.74 0.58 0.56

cases (Control, -Fe and -Mn) the influence of the addition of DCMU can be observed. In the case of iron-deficiency (-Fe) a dramatic decrease of the value Fv coincides with the greater influence of the DCMU in the development of the PS2 photochemistry. In fact, they are the PS2 iron­deficient, those which are more affected when Q 8 is blocked. The PS2 from control, with blocked Q 8 , increase slightly the fluorescence emission during the first second of lighting and in the Mn-deficient the fluorescence emission by the DCMU addition does not vary.

These results can be more clearly analyzed in Figure 3, in which, the study of the quenching of fluorescence qQ is detailed. The values of qQ have been calculated through the equation:

Fv qQ = 1- (Fv)M

Where: Fv is the variable fluorescence without DCMU (Fv)M the variable fluorescence with DCMU

In Figure 3 it can be observed that during the first second of illumination the thylakoid from Mn-deficient, showed qQ values equal to zero, however, in thylakoids from Fe-deficient plants, after an initial increase, they descend pro­gressively to 0.2.

CoiJJrol

t; +DCMU -DCMUJ -Ft

-Mr

0 500 1000 1500 2000 2500 3000

Time (0. 75 s)

Fig. 2. Kinetics of variable chlorophyll a fluorescence on cellular crude suspensions from Control, manganese-deficient (-Mn) and iron-deficient (-Fe) soybean leaves. Fo was normalized in all the cases. The origin of scale in control and (-Fe) was displaced for a more clear study. Black solid lines indicate Fv measured on DCMU poisoned suspensions and dotted gray lines Fv on suspensions without DCMU.

Fluorescence in relation to Mn and Fe deficiency 495

0.8

0.6

0'" 0.4 cr -Fe

0.2

0

-0.2

Time (0. 75 s)

Fig. 3. Analysis of fluorescence photochemical quenching (qQ) calculated from the values of Figure 2. The equation applied is given in the text.

These results indicate that in the first instants of illumination, the light intensity applied to the cellular crude without DCMU is sufficient, in the case of manganese deficiency, to thoroughly reduce QA. This is not in the case for iron deficiency, in which it is necessary to add DCMU to reach the maximum fluorescence.

Discussion

The data described above confirm that obtained in our laboratory studying fluorescence emission in the leaf (Madero and Perez, personal com­munication). In the case of -Mn, an increase in Fo and a decrease of the Fv /Fm ratio is ob­served. Since it has been possible to isolate the contribution of the PS2 through the blockage of Q 8 with DCMU and to eliminate the effect of the proton gradient by the addition of nigericine, it can be deduced that the presence, in principle undisturbed, of both light captators and of a thoroughly functional PSI, cause the probability of transmission of excitation energy received by the antenna of the PS2 toward open photochemi­cal traps to be very low due to the small number of PS2 reaction centers (Simpson and Robinson, 1984); in this way, there-emission of light would increase in the form of fluorescence and there-

fore would increase the Fo contribution due to its own antenna (Kriedemann et al., 1985). On the other hand, the decrease in the photo­synthetic capacity of the PS2 affected by Mn deficiency (Kriedemann et al., 1985) would make Fv reduce. The data from Abadla et al. (1986) also could be interpreted according to this hypothesis, since when comparing electropho­resis of manganese-deficient sugar beet plants with those of control plants, it is observed that the first have a smaller content in PS2 reaction centers.

These facts could indicate that in the con­ditions of the culture chamber, the soybean control has a thoroughly functional photo­synthetic apparatus (Fv/Fm = 0.74) with the LHC system connected to the reaction center of the PS2. The Mn-deficient soybean has dimin­ished its photosynthetic capacity (Fv /Fm = 0.58) due to the lack of PS2 reaction centers. Further­more, the value of qQ = 0 indicates that all the energy collected by the antenna is driven to the small number of remaining PS2 reaction centers. In other words, all the PS2 traps are open, and without the addition of DCMU, QA is fully reduced.

A different situation is observed in the case of iron deficiency, the photosynthetic efficiency is also very diminished, as would be expected at

496 Fluorescence in relation to Mn and Fe deficiency

the sight of the low value of total chlorophyll in leaf (Table 1) and the low Fv/Fm value (0.56), but in this case the qQ ascends above zero, which indicates that QA can not be completely reduced without the blockage of QB by DCMU. This indicates that, upon illuminating a cellular suspension from Fe-deficient leaves, a significa­tive percentage of the energy is used to carry out photochemical reactions which quench the yield of fluorescence. This maybe the reason for which the leaves under severe iron deficiency, with very low chlorophyll concentration, can survive and still can perform photosynthesis-related re­actions.

The results from Table 1 confirm the drawn hypothesis. The Mn is a part of the PS2, specifi­cally of the photosynthetic oxygen evolving cen­ter. Thus it can be expected that manganese deficiency induced a diminution of PS2 reaction centers. On the other hand, and in contrast with the results obtained by Kriedemann et a!. (1985), in our conditions and with the plant material used in this work, meaningful changes in the chlorophyll a/b ratio have not been detected, which is logical if taking into account that only two molecules of chlorophyll a in the whole PS2 core complex are contained, in front of the several hundreds of chlorophylls of the LHC system. This can mean that under Mn deficiency the stoichiometry of the PS2 antenna remains unaltered.

Acknowledgements

Our thanks to Ms MA Gracia for her expert technical assistance. The work was carried-out under research projects CONAI-DGA: PCA-4/ 91 and CICYT AGR90-0792.

References

Abadia J. Nishio J N and Terry N 1986 Chlorphyll-protein and polypeptide composition of Mn-deficient sugar beet thylakoids. Photosyn. Res. 7. 379-381.

Cheniae G and Martin I F 1968 Sites of manganese function in photosynthesis. Biochim. Biophys. Acta 153, 819-837.

Edwards G and Walker D 1983 C3 , C4 : Mechanism, and cellular and environmental regulation of photosynthesis. Blackwell. Oxford.

Haworth P, Baker N, Percival M P and Beckwith P B 1986 Modification of the photosystem II light-harvesting chloro­phyll a/b protein complex in maize during chill-induced photoinhibition. Biochim. Biophys. Acta 851, 86-92.

Kricdcmann P E, Graham R D and Wiskich J T 1985 Photosynthetic dysfunction and in vivo changes in chloro­phyll a fluorescence from manganese-deficient wheat leaves. Aust. J. Agric. Res. 36, 157-169.

Kriedemann P E and Anderson J E 1988 Growth and photosynthetic responses to manganese and copper de­ficiencies in wheat (Triticum aestivum) and barley grass (Hordeum glaucum). Aust. J. Plant Physiol. 15, 429-446.

Mercer F V, Nittin N and Possingham J V 1962 The effect of manganese deficiency on the structure of spinach chloro­plasts. J. Cell Bioi. 15, 379-381.

Morales F, Abadia A and Abadia J 1990 Characterization of the xanthophyll cycle and other photosynthetic pigment changes induced by iron deficiency in sugar beet (Beta vulgaris L.). Plant Physiol. 94, 607-613.

Morales F, Abadia A and Abadia J 1991 Chlorophyll fluorescence and photon yield of oxygen evolution in iron­deficient (Beta vulgaris L.) leaves. Plant Physiol. 97, 886-893.

Morales F, Susin S, Ahadia A and Abadia J 1992 Photo­synthetic characteristics or iron chlorotic pear (Pyrus communis L.). J. Plant Nutr.

Platt-Aloia K A, Thompson W Wand Terry N 1983 Changes in plastid ultrastructure during iron nutrition mediated chloroplast development. Protoplasma 114, 85-92.

Possingham J V, Vesk M and Mercer F V 1964 The fine structure of the leaf cells of manganese deficient spinach. J. Ultrastruct. Res. 11, 68-83.

Simpson D J and Robinson S P 1984 Freeze-fracture ultra­structure of the thylakoid membranes in chloroplasts from manganese deficient plants. Plant Physiol. 74, 735-741.

Val J and Baker N 1989 Light-dependent, chilling effects on phosphorylation of thylakoid proteins and consequences for associated photochemical activities in maize. Physiol. Plant. 77, 420-426.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 497-502, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-057

Specificity of iron in some aspects of soybean (Glycine max L.) physiology

P. MADERO, A. PEQUERUL, C. PEREZ, J. VAL and E. MONGE Plant Nutrition Department, Estaci6n Experimental de Aula Dei, CSIC, Aptdo 202, 50080 Zaragoza, Spain

Key words: Glycine max L., iron deficiency, macro- and micronutrient composition, photosynthetic pigments, soybean

Abstract

In this study, we have analysed the influence of iron deficiency on mineral composition of iron-chlorotic leaves in different developmental stages from plants hydroponically cultured. Significant differences in Ca, total Fe and active Fe between control and chlorotic samples were found. Ca contents increased in deficient leaves. Changes in photosynthetic parameters (pigment composition and fluorescence) were also investigated. Iron deficiency affected photosynthetic pigments to different extents: the leaf concentration of total chlorophyll and B-carotene showed decreases higher than xanthophylls. The photosynthetic capacity measured by chlorophyll fluorescence, was lower in chlorotic leaves. Altera­tions in the morphology of the plant were observed in iron-deficient plants since leaf size and root length were significantly lower.

Abbreviations: AAS- Atomic absorption spectroscopy, F- Modulated fluorescence (subscripts v and p define variable and maximal levels), PS- Photosystem

Introduction

Iron deficiency is one of the most studied nutri­tional disorders due to its agronomical impor­tance. Wide differences in the susceptibility to chlorosis has been reported in several plant species and different varieties of the same specie. In susceptible species, iron deficiency can be induced by several causes, like the interaction with other ions (Bindra, 1980). Studies of Fe­chlorosis are often controversial about the mean­ing of some ratios between nutrients, mainly P/Fe, K/Ca, and Fe/Mn, on the development of lime-induced chlorosis. In field grown conditions the chlorosis in soybean (Glycine max L.) can cause yield reduction if grown on calcinomorphic soils. The intensity of this chlorosis is related with physiological and nutritional changes and is

affected by the state of iron (Uvalle-Bueno, 1985).

Iron is an element with little mobility in plants, for this reason the growing zones of plants are the most affected and the young leaves are chlorotic. In green leaves, approximately 80% of the iron is located in the chloroplasts (Terry, 1980), thus, iron deficiency affects pro­cesses located in the chloroplasts. In higher plants, one of the most obvious effects of iron deficiency is the development of chlorotic leaves. This chlorosis is associated with a loss of not only chlorophyll but also all thylakoid constituents.

The reduction in thylakoid membranes during iron deficiency is accompanied by decreases in all photosynthetic pigments. (Monge et al., 1987; Monge et al., 1991; Terry and Abadia, 1986).

The efficiency of PS2 photochemistry has been

498 Madero et al.

found to be decreased in plants affected by Fe deficiency. Several researchers have detected decreases in the ratio of variable to maximum fluorescence (Fv I Fp) arising from PS2 both in maize (Val and Monge, 1990) and sugar beet grown in controlled environments (Morales et al., 1990).

Material and methods

Plant material

Soybean (Glycine max L.) plants, were hydro­ponically cultured for 45 days in Hoagland's nutrient solution. Plants were developed in a culture chamber (PFD 350 J.Lmol m -z s -I, 24°C, 80% humidity, 16h of hight period). Iron de­ficiency was obtained by no addition of the element to the culture solution. The culture solutions were tested by AAS to check the absence of iron.

The leaves usc for the experiments were taken according to their physiological developmental stage, old (0) and young (Y) leaves from control (C) and iron deficient (-Fe) plants.

Pigment analysis

Photosynthetic pigments were extracted from 10 control and iron deficient leaf discs of a known area (0,358 cm2 each) by grinding in a mortar with 10 mL of cold 100% acetone, in presence of a few mg of sodium ascorbate to avoid the formation of phaeophytins. All extraction proce­dures were carried out in dimmed room light. The extracts were stored at - 30°C until used.

Pigment analysis was carried out by rp-C 18

HPLC (Val and Monge, 1990). The flow rate was 2 mL min_,, and the two isocratic steps were: 1.75% methanol, 1.75% dichloromethane and 96.5% acetonitrile for the first phase, and the second mobile phase 50% acetonitrile and 50% ethyl acetate. The absorbance readings were monitored at 440 nm. All solvent were HPLC grade.

Mineral composition

To determine the mineral elements, the leaves were carefully washed with a soft brush and

liquid soap (1%) and rinsed with deionized water to eliminate surface contamination. Dry ashing was carried out following the methods of C.I.I. and Pinta (Pinta and DeWeler, 1975) Ca, Mg, Fe, Mn, Cu and Zn were determined by atomic absorption spectroscopy, K by flame emission, P by the vanadomolidophosphoric method (Jones-Jr, 1984) and N by Kjeldhal. Extractable active iron per weight unit were analysed by using 1 g of fresh leaf tissue. Little fragments were incubated in 10 mL of a,a '­dipyridyl (83 mM, pH 3.0) during 48 h. The extracts were purified by using C 18 Sep-Pack (Waters) cartridges (Abadfa et al., 1984), and the absorbance measured at 522 nm.

Plant measurement

Leaf areas Leaf areas were determined with an image analyser ~T (Leaf Area Meter).

Humidity In ten discs, of 1.747 cm2 , the difference between fresh and dry weight was determined, considerat­ing the % humidity this difference/fresh weight x 100.

Statistical analysis of data was carried-out by AN OVA.

Chlorophyll fluorescence

Modulated chlorophyll-a fluorescence was mea­sured at room temperature using a pulse am­plitude modulation fluorometer (Hansatech). The signals were recorded for 20 seconds at a frequency of 10Hz by a 12 bit resolution AID card (PCL-711S, Advantech) and a Labtech Acquire software (Lab. Tech. Corp) installed in a 386-Personal Computer. The ASCII file that contains the curve (200 points) was processed and monitored through Excel 3.0 (Microsoft).

Fluorescence determinations were preceded by a 30-min. period of dark adaptation and were carried out on the upper leaf surface. Fluores­cence was excited with a measuring beam of weak light from a pulsed light-emitting diode and

a cut off filter 620 nm to obtain Fo (all reaction centres open). Detection was measured at 700 nm. Maximum yield of fluorescence, Fp, was determined by application of white light (800 11-mol m- 2 s- 1). The variable fluorescence, Fv, is given by the difference between Fp and Fo.

Results

Mineral composition

The results obtained from the analysis of mineral composition (Table 1) of C and -Fe soybean plants indicated that the levels of several mac­ronutrients (Mg, K, P and N) and Cu were not significantly affected either by the iron supply or by the development stage of the leaf. By con­trast, Mn in 0 and Zn in Y increased. Significant differences by treatment, as well as by the age of leaf in Ca, Fe and active Fe (extracted with a,a '-dipyridyl) have been found. The concen­tration of Ca was higher in -Fe than in C leaves,

Iron in soybean physiology 499

and obviously, the total and active iron contents were significantly higher in C than in -Fe.

Photosynthetic parameters

Photosynthetic pigments have been studied in both C and -Fe samples, and the results are given in Table 2. The analysis revealed significant differences for all the pigments studied. Iron deficiency reduced leaf concentration of nco­xanthin, violaxanthin and B-carotene. However, antheraxanthin and zeaxanthin increased in re­sponse to iron deficiency. Although we found that lutein decreased significantly in -Fe leaves; this change is only observed in Y leaves while in 0 leaves remained unaltered.

Chlorophylls a and b were considerably re­duced by the effect of the deficiency (25% and 60% in 0 and Y leaves respectively). However, the chlorophyll a/b ratio remained unchanged.

Chlorophyll fluorescence parameters showed that the photosynthetic efficiency was affected by iron deficiency. The Fv/Fp ratio was higher inC leaves and did not change when working with

Table 1. Leaf macro and micronutrients concentration in control and iron deficient soybean. The analyses were made in two developmental stages: old and young leaves (average± standard error of six determinations). Statistical analysis of variance was made considering two main effects: iron deficiency (treat) and leaf age (age)

Control leaves Deficient leaves

Old Young Old Young

Macronutrients Ca 2.07 ± 0.4 1.24 ± 0.50 2.67 ± 0.40 1.62 ± 0.08 Mg 0.42 ± 0.06 0.41 ± 0.04 0.63 ± 0.07 0.58 ± 0.03 K 3.06 ± 0.25 2.74 ± 0.23 2.72 ± 0.20 2.96 ± 0.25 p 0.90 ± 0.06 0.91 ± 0.08 1.00 ± 0.03 0.93 ± 0.08 N 4.57 ± 0.29 4.93 ± 0.29 4.10 ± 0.10 5.34 ± 0.38

Micronutrients Mn 60.17 ± 7.20 45.69 ± 7.56 68.88 ± 6.63 37.00 ± 5.81 Cu 8.15 ± 0.84 6.30 ± 0.50 7.06 ± 0.43 7.36 ± 0.70 Zn 22.50 ± 3.62 30.56 ± 2.52 19.15 ± 4.70 33.50 ± 1.60 Fe 197.00 ± 20.50 178.62 ± 21.50 60.50 ± 24.50 39.20 ± 19.80 Active-Fe 22.17 ± 2.35 20.52 ± 1.90 10.64 ± 0.95 8.34 ± 0.50

Ratios K/Ca 1.48 ± 0.30 2.21 ± 0.40 1.02 ± 0.09 1.83 ± 0.10 P/Fe 45.68 ± 5.40 50.95 ± 6.25 165.29 ± 18.25 237.24 ± 22.34 Fe/Mn 3.27 ± 1.25 3.91 ± 1.30 0.88 ± 0.30 1.06 ± 0.50

Macroelements are given in mg 100 mg - 1 dry weight and microelements are given in mg kg 1 dry weight. NS, **, and *** mean non-significant, or significant at the p < 0.01, and p < O.O(Jl level respectively.

Sig. level

Treatm. Age

NS NS NS NS NS NS NS NS

NS NS NS NS

500 Madero et al.

Table 2. Pigment leaf concentration in control and iron deficient soybean. The analyses were made in two development stages: old and young leaves. (Average± standard error of six determinations). Statistical analysis of variance was made considerating two main effects: iron deficiency (treat) and leaf age (age)

Photosynthetic Control leaves Deficient leaves Sig. level parameters

Old Young Old Young Treatm. Age

Pigments Neoxanthin 2.53 ± 0.23 1.40 ± 0.32 2.15±0.19 1.20 ± 0.14 Violaxanthin 3.13 ± 0.21 1.67 ± 0.14 1.97 ± 0.20 0.73 ± 0.09 Antheraxanthin 0.12 ± 0.02 0.13 ± 0.02 0.48 ± 0.03 0.36 ± 0.02 Lutein 7.56 ± 0.42 5.52 ± 0.35 7.58 ± 0.43 3.20 ± 0.26 Zeaxanthin 0.02 ± 0.01 0.06 ± 0.02 0.45 ± 0.03 0.67 ± 0.04 Chlorophyll b 11.70 ± 0.44 6.08 ± 1.10 9.10 ± 0.72 2.33 ± 0.79 Chlorophyll a 39.45 ± 0.90 21.62 ± 2.10 29.40 ± 0.90 9.04 ± 1.10 13-carotene 4.87 ± 0.46 2.50 ± 0.34 3.34 ± 0.40 0.91 ± 0.36

Fluorescence Fv/Fp 0.08 ± O.o? 0.8 ± 0.06 0.755 ± 0.05 0.57 ± 0.06

Pigment concentration are given in }kg em_,_ NS, **, and *** mean non-significant, or significant at the p < 0.01, and p < 0.001 level respectively.

Table 3. Leaf area and humidity, and root length and humidity of control and iron deficient plants (average± standard error of fifty determinations)

Length (em) Humidity (%)

Area (cm 2 )

Humidity (%)

Control plants

Roots 64.50 ± 1.86 93.24 ± 0.17

Leaves Old

65.38 ± 4.50 81.16 ± 2.50

Young

25.71 ± 3.85 82.41 ± 3.05

Deficient plants

54.93 ± 1.42 92.80±0.19

Old

23.72 ± 2.25 82.73 ± 2.15

Young

11.92 ± 3.28 82.96 ± 2.25

Sig. level treatm.

NS

NS

NS, ** and *** mean non-significant, or significant at the p < 0.01, and p < 0.001 level respectively.

different-aged leaves. In -Fe leaves this ratio decreased from 0.75 (0) to 0.57 (Y).

Leaf and root measurements

The data of Table 3 demonstrated that soybean plants under iron deficiency experimented a highly significant reduction of root elongation and in both 0 and Y leaf area (Table 3). However, the percentage of humidity was the same in all cases.

Discussion

For many years, a great number of authors have approached the study of iron deficiency in photo-

synthetic organisms. Numerous data from plants grown in natural conditions and also in con­trolled environmental conditions have been re­ported (Monge et al., 1991; Terry and Abadfa, 1986). The extrapolation of the results obtained in plants grown in hydroponic solution without iron to field grown iron-deficient plants, has always been the greatest limitation in the in­vestigation of the causes of iron deficiency. In the field, there are numerous mechanisms that produce Fe-chlorosis: high pH, presence of lime in the soil, improper capacity of iron uptake by the plant, etc. (Bindra, 1980). As can be seen, a great number of factors can give, as a result, the characteristic symptoms of iron deficiency exist, and therefore the number of variables is large. Thus, even taking into account that the study of

iron deficiency in plants grown over inert sup­ports and controlled conditions, is a laboratory approximation to the real problem of iron de­ficiency, it is, to date, the way of isolating iron deficiency in plant tissues as an only variable.

In this work, the effect of iron deficiency in mineral composition of soybean cultivated in controlled conditions has been studied, in order to analyse the incidence that this deficiency causes in some macro (Ca, Mg, K, P, and N) and micronutrient (Mn, Cu, Zn, Fe) concentrations. Moreover, the photosynthetic efficiency and ef­fects in leaves and roots were investigated. Concerning macronutrients, we only found sig­nificant increases in Ca levels in -Fe respect to C leaves. The level of Ca was lower in Y than in 0 leaves, in both cases C and -Fe which is logical if we consider that Ca mobility, from cell to cell and through phloem, is very low. Variations of Ca content in peach iron deficient leaves grown under field conditions have been described (Abadia et a!., 1985).

The absence of iron in nutritive solution in­duced decreases in 0 and Y total iron and active iron concentrations. Considering the active to total iron rate in both, 0 andY leaves (Table 1), we found that extracted iron is lower in C (11% in both leaves) than in -Fe leaves (17 and 23% respectively). These observations confirm that, in both, hydroponical cultures and natural con­ditions (Abadia et a!., 1984), physiologically active iron was relatively more available in deficient leaves than in controls.

In field conditions, an interaction among iron and other elements absorption has been reported (Bindra, 1980). High levels of several minerals (Ca, P, N, Mn, Cu) in soil contributing to iron chlorosis presumably because they are involved in interaction with iron nutrition, although iron deficiency per se may inhibit absorption of some elements. Therefore, in iron chlorosis, the ratios are good indicators of nutritional status of plants (Dekock, 1981; Procopiou and Wallace, 1982) and appear to be useful for diagnosis, but only, in field conditions. In our study, the K/Ca, P/Fe and Fe/Mn ratios were significantly affected by induced iron deficiency which is logical if we bear in mind that Ca and Fe were the only elements which suffer significant variations. From these observations it could be concluded

Iron in soybean physiology 501

that the ratios are less relevant in hydroponic cultures than in the field as, in this conditions, the plants are not subject to environmental variations because, all the elements in nutritive solutions, are readily available by the root sys­tem.

Leaf concentration of photosynthetic pigments inC and -Fe leaves was investigated (Table 2). It was previously found that iron deficiency de­creased more the amount of Chis and B­carotenes than xantophyles (Monge eta!., 1987; Morales eta!., 1990). Our data showed that iron deficiency decrease photosynthetic pigments to different extents. Iron deficiency decreased nco­xanthin, B-carotene and chlorophylls.

Conversely, the lutein, antheraxanthin and zeaxanthin were less affected. In 0 iron deficient leaves the lutein concentration remain un­changed with regard to C, but decreased in young leaves. Iron deficiency induced increased in the ratio carotenoids versus chlorophylls, which is due to a relative enrichment in lutein (Val et a!., 1987). Our data corroborates this: we observed that in old leaves, the carotenoids/ chlorophyll ratio was 0.24 and 0.28 in C and -Fe respectively while it increased from 0.29 to 0.37 in young leaves.

In this work, modulated chlorophyll fluores­cence determined in leaves, has been used to investigate the photosynthetic efficiency of soy­bean leaves under iron deficiency. The observed changes in Fv I Fp could indicate a substantial lack of PS2 reaction centres. The efficiency of PS2 from leaves affected by iron deficiency has been found to be decreased in sugar beet (Morales eta!., 1990, 1991). We found significant decreases in the photochemical efficiency of PS2 in -Fe leaves of soybean exposed to controlled light after short dark-adaptation times. (Table 2). Young -Fe leaves provides lower Fv/Fp values than the older ones which is in agreement with the reduction in photosynthetic pigments shown in the same table.

Iron-deficient plants showed morphological changes in leaf and root size (Table 3). This could be interpreted as a general response of the plant to avoid the collapse that could cause the indiscriminated uptake of nutritive element with, probably, can not be processed due to the disability of the iron-deficient leaves to carry out

502 Iron in soybean physiology

photosynthesis. This data contrasts with that reported by Terry in sugar beet (Terry, 1979). This author observed that iron stress had re­markably little effects on many attributes of leaf grown including: leaf thickness, fresh weight per unit area or on leaf tissue volume.

From all the above mentioned data, it can be concluded that the lack of iron in the culture solution produces especially in the younger soy­bean leaves, a decrease of the leaf-iron concen­tration. This causes a diminution of chlorophylls and, to a lesser degree, of the rest of photo­synthetic pigments which gives cause for the yellowing of the leaves. The lack of chlorophyll induces a decrease of the photosynthetic yield that produces decreases in the leaf and root sizes.

Acknowledgements

Authors wish to thank Mrs Ma Angeles Gracia for her invaluable technical support and Dr Alvaro Blanco for his help to make the statistical analysis. Work carried-out under research pro­ject CICYT AGR90-0792 and CONAI-DGA: PCA-4/91.

References

Abadia J, Monge E, Montanes L and Heras L 1984 Ex­traction of iron from plant leaves by Fe (II) chelators. J. Plant Nutr. 7, 777-784.

Abadia J, Nishio J N, Monge E, Montanes L and Hcras L 1985 Mineral composition of peach leaves affected by iron chlorosis. J. Plant Nutr. 8, 697-707.

Bindra A S 1980 Iron chlorosis in horticultural and field crops. Annu. Rev. Plant Science II, 221-321.

C.I.I. 1969 Comite Inter-Institutos para el estudio de tecnicas analiticas: metodos de referencia para Ia determinacion de elementos minerales en vegetales. An. Edafol. Agrobiol. 38, 13-521.

De Kock P C 1981 Iron nutrition under conditions of stress. J. Plant Nutr. 3, 167-175.

Jones Jr J B 1984 Plants. In Official Methods of Analysis of the Association of Official Analytical Chemists. Ed. S Williams. pp 38-64. Association of Official Analytical Chemists, Arlington, VA.

Monge E, Val E, Heras L and Abadia J 1987 Photosynthetic pigment composition of higher plants grown under iron stress. Prog. Photosyn. Res. 4, 201-204.

Monge E, Montanes L, Val J and Heras L 1991 El hierro modulador de Ia estructura y funcion del cloroplasto. In Fijacion y Movilizacion Biologica de Nutrientes. Vol I. Aspectos Fisiologicos y de Estres. Ed. J. Lopez Gorge. pp 85-108. C.S.I.C., Madrid.

Morales F, Abadia A and Abadia J 1990 Characterization of the xanthophyll cycle and other photosynthetic pigment changes induced by iron deficiency in sugar beat (Beta vulgaris L.). Plant Physiol. vol. 607-613.

Morales F, Abadfa A and Abadfa J 1991 Chlorophyll fluorescence and photon yield of oxygen evolution in iron­deficient sugar beet (Beta vulgaris L.) leaves. Plant Phy­siol. 97, 886-893.

Pinta M and De Weier G 1975 Etalons vegetaux pour !'analyse foliare. In Le C6ntrole de !'alimentation des Plantes Cultivees. Ed. P Kozman. pp 159-172. Akadcmiai Kiado, Budapest.

Procopiou J and Wallace A 1982 Mineral composition of two populations of leaves (green and iron chlorotic) of the same age all from the same tree. J. Plant Nutr. 5, 811-820.

Terry N 1979 The use of mineral nutrient stress in the study of limiting factors in photosynthesis. In Photosynthesis and Plant Development. Eds. R Marcelle, H Clijsters and M V Poucke. pp 151-160. W. Junk, London.

Terry N 1980 Limiting factors in photosynthesis. I. Use of iron stress to control photochemical capacity in vivo. Plant Physiol. 65, 114-120.

Terry N and Abadfa J 1986 Function of iron in chloroplasts. J. Plant Nutr. 9, 609-646.

Uvalle-Bueno J X 1985 Fertilizacion foliar en soya Glycine max (L.) Merill, para el control de Ia chlorosis. Agric. Tee. Mex. 11, 3-17.

Val J, Monge E, Heras L and Abadfa J 1987 Changes in photosynthesis pigment composition in higher plants as affected by iron nutrition status. J. Plant Nutr. 10, 995-1001.

Val J and Monge E 1990 Violaxanthin cycle and fluorescence in iron-deficient maize levels. Curr. Rese. Photosyn. 4, 765-768.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 503-507, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-054

Specificity of manganese in some aspects of soybean (Glycine max L.) physiology

C. PEREZ, P. MADERO, A. PEQUERUL, J. VAL and E. MONGE Department of Plant Nutrition, Estaci6n Experimental de Aula Dei, CSIC, Aptdo. 202, 50080 Zaragoza, Spain

Key words: chlorophyll fluorescence, manganese deficiency, photosynthetic pigments, soybean (Glycine max L.)

Abstract

Mineral composition, photosynthetic pigments and chlorophyll fluorescence parameters of hydro­ponically grown soybean plants were differently affected by low Mn concentration and by the age of the leaf. Whereas macronutrients were mostly affected by the leaf age, photosynthetic pigments were also affected by Mn leaf concentration. Nutrient imbalances in hydroponically cultured Mn-deficient plants were not detected, and therefore, all the studied changes in photosynthetic pigments, fluorescence, root length and leaf area, could only be attributed to the Mn status.

Abbreviations: AAS-Atomic Absorption Spectrometry, Chi-chlorophyll; Fv-Variable fluorescence, Fp-Maximum yield of fluorescence at non saturating actinic light.

Introduction

Studies on the interaction between Mn uptake and other divalent cations have been reported (Bowen, 1969). Under field conditions mangan­ese deficiency is usually confined to plants grow­ing in highly leached tropical soils or high-pH soils with a large organic matter content. (Marschner, 1986). Interactions between the absorption of Mn2 +, and the micronutrient dival­ent cations have been studied (Bowen, 1981; Chinnery and Harding, 1980). In this way, Ca seems to increase the uptake of Mn and other divalent cations (Maas et al., 1969).

Manganese plays an important role in the metabolism of plants, particularly in processes of activation of different enzymes, chlorophyll synthesis and photosynthesis (Campbell and Nable, 1988). In leaf tissues, manganese is asso-

ciated with proteins of the oxygen evolving system and is indispensable for the generation of the photosynthetic energy flow (del Rio et al., 1983). The photosynthetic electron transport is affected when Mn deficiency occurs, since the first step of the electron transport chain is impaired (Edwards and Walker, 1983).

The chloroplast structure in Mn-deficient plants depends on the severity of the deficiency. A severe deficiency produces the disappearance of the grana! thylakoids while a light deficiency does not modify the ultrastructure of the chloro­plast (Cheniae and Martin, 1968). It also de­creases the reduction of C02 , nitrites and sul­phates, and the content of chlorophyll is dramatically reduced, while the rates of respira­tion and transpiration remain unchanged (Ohki et al., 1981).

In this work, the changes produced in mineral

504 Perez et al.

nutnt1on, photosynthetic pigments, photo­synthetic efficiency and morphology of Mn-de­ficient soybean plants have been studied.

Material and methods

Plant material

Soybean (Glycine max L.) plants were hydro­ponically cultured for 45 days in Hoagland's nutrient solution either containing Mn (control) or not (deficient). Plants were grown in a culture chamber (PFD 350fLmolm- 2 s-\ 24°C, 80% humidity, 16 h of light period). Manganese de­ficiency was obtained by no addition of the element to the culture solution. All the solutions were tested by AAS to certify the absence of Mn in the final culture solution.

The leaves used for the experiments were taken from three different parts of the plant: the upper (young leaves), middle intermediate leaves) and lower (old leaves).

Pigment analysis

Photosynthetic pigments were extracted from 10 leaf discs of known area (0, 358 cm2 each) by grinding in a mortar with 10 mL of cold 100% acetone, in the presence of a few mg of sodium ascorbate to avoid the formation of phaeophytins. All extraction procedures were carried out in dimmed room light. The extracts were stored at - 30°C until used.

Pigment analysis were carried out by rp-C 18

HPLC (Val and Monge, 1990). The flow rate was 2 mL min- 1 , and the two isocratic steps were: 1.75% methanol, 1.75% dichloromethane and 96.5% acetonitrile for the first phase, and the second mobile phase 50% acetonitrile and 50% ethyl acetate. The absorbance readings were monitored at 440 nm. All solvents were HPLC grade.

Mineral composition

The leaves were carefully washed with a soft brush and liquid soap (1%) and rinsed with tap water and deionized water to eliminate surface contamination. We have washed leaves carefully

to avoid Mn leaching (when analysis was per­formed without the washing procedure the re­sults were the same). Dry ashing was carried out following the methods of C.I.I. and Pinta (Pinta and DeWeler, 1975). Ca, Mg, Fe, Mn, Cu and Zn were determined by AAS, K by flame emis­sion, P by the vanodomolibdo phosphoric meth­od (Jones, 1984) and N by Kjeldhal.

Plant measurement

Leaf areas Leaf areas were determined with an image analyser ~T (Leaf Area Meter).

Humidity In ten discs of known area (1.747 cm2) the difference between fresh and dry weight was determined, considering the % humidity as the quotient between this difference and fresh weight x 100.

Statistical analysis of data was carried out by ANOVA.

Chlorophyll fluorescence Modulated chlorophyll-a fluorescence was mea­sured in the middle of the leaf at room tempera­ture using a pulse amplitude modulation fluoro­meter (Hansatech). The signals were recorded during 20 seconds at a frequency of 10Hz by a 12 bit resolution AID card (PCL-711S, Advan­tech) and Labtech Acquire software (Lab. Tech. Corp.) installed in a 386-Personal Computer. The ASCII file that contains the curve (200 points) was processed and monitored through Excel 3.0 (Microsoft).

Fluorescence determinations were preceded by a 30-min. period of dark adaptation and were carried out on the upper leaf surface.

Fluorescence was excited with a measuring beam of weak light from a pulsed light-emitting diode and a 620 nm cut off filter to obtain Fo (all reaction centres open). Detection was measured at 700 nm. Maximum fluorescence yield, Fp, was determined by application of white light (800 fLmOl m -z s -I) to close the reaction centres. The variable fluorescence, Fv, was calculated by the difference between Fp and Fo.

Results

Mineral composition

Leaf concentration of macro- and micronutrient was not significantly affected by Mn deficiency (Table 1). According to the age of leaf, the distribution of Macroelements followed the same tendency in control and Mn-deficient leaves. Respect to micronutrients, with the exception of Mn whose highest values were recorded in the older leaves and the lowest in the intermediates, the rest were not significantly affected either by the age of the leaf or by Mn deficiency.

Manganese in soybean physiology 505

Photosynthetic pigments

It is remarkable that the extent of the decrease of all the photosynthetic pigments was uniform when Mn-deficient leaves were compared with controls of the same age (Table 2). That is to say, the stoichiometry between pigments re­mained unaltered. The ag~;: of leaf also affected the distribution of photosythetic pigments, since the higher concentrations occurred in old leaves, and the lower in the young ones. The chlorophyll a/b ratio was not affected either by Mn-de­ficiency or the leaf age.

The chlorophyll fluorescence parameters re-

Table 1. Leaf macro and micronutrient concentration in control and Mn-deficient soybean. The analysis were made in three developmental stages: old, intermediate and young leaves (average± standard error of six determinations)

Control leaves Deficient leaves

Old lnterm. Young Old lnterm. Young

Macro Ca 2.75 ± 0.04 1.33 ± 0.15 1.31 ±0.14 2.15 ± 0.30 1.61 ± 0.08 0.81 ± 0.10 Mg 0.54 ± 0.07 0.42 ± O.D3 0.44 ± 0.04 0.49 ± 0.02 0.40 ± 0,01 0.42 ± O.D3 K 3.01 ± 0.24 3.32 ± 0.25 2.94 ± 0.25 2.85 ± 0.18 3.71 ± 0.20 3.45±0.17 p 1.07 ± 0.02 0.94 ± 0.08 0.91 ± 0.06 1.08 ± O.D3 1.05 ± 0.05 0.94 ± 0.08 N 4.37 ± 0.29 5.16 ± 0.29 5.35 ± 0.26 4.49 ± 0.10 5.03 ± 0.09 5.34 ± 0.16

Micro Mn 73.19 ± 8.00 24.43 ± 2.60 48.69 ± 5.00 19.88 ± 2.60 9.75 ± 1.30 13.38 ± 1.80 Cu 6.81 ± 0.35 5.37 ± 0.63 6.00 ± 0.45 5.06 ± 0.33 4.81 ± 0.62 5.56 ± 0.46 Zn 24.75 ± 2.60 32.31 ± 3.96 34.56 ± 2.50 25.06 ± 2.70 30.81 ± 3.00 34.50 ± 1.60 Fe 207.00 ± 22.00 238.00 ± 24.00 195.00 ± 18.00 233.00 ± 25.00 250.00 ± 28.00 160.00 ± 19.00

Macroelements are given in mg 100mg- 1 dry weight and microelements in mgkg- 1 dry weight.

Table 2. Pigment leaf concentration (f.Lg em_,) and fluorescence parameters in control and manganese deficient soybean. Analysis were made in three development stages: old, intermediate and young leaves (average± standard error of six determinations)

Control leaves Deficient leaves

Old In term. Young Old In term. Young

Pigments Neoxanthin 2.05 ± 0.11 1.63 ± 0.18 1.48 ± 0.22 1.65 ± 0.15 1.39 ± 0.14 1.20 ± 0.16 Violaxanthin 2.50 ± 0.17 2.46 ± 0.26 2.36 ± 0.32 1.87 ± 0.14 1.09 ± 0.19 1.82 ± 0.25 Antheraxanthin 0.12 ± 0.02 0.10 ± 0.03 0.15 ± 0.02 0.12 ± 0.04 0.16 ± 0.04 0.10 ± 0.02 Lutein 6.67 ± 0.34 5.78 ± 0.27 5.54 ± 0.55 5.42 ± 0.37 4.95 ± 0.52 3.85 ± 0.44 Zeaxanthin O.D7 ± 0.02 0.06 ± 0.09 0.09 ± 0.02 0.13 ± 0.09 0.09 ± O.D3 0.05 ± 0.02 Chlorophyll b 10.70 ± 0.44 8.22 ± 0.09 7.28 ± 0.79 7.90 ± 0.63 6.90 ± 0.74 4.77 ± 0.49 Chlorophyll a 35.17 ± 1.33 26.14 ± 2.63 25.23 ± 4.11 27.27 ± 2.14 24.06 ± 3.14 16.99 ± 1.79 13-carotene 3.87 ± 0.42 3.34 ± 0.34 2.93 ± 0.34 3.05 ± 0.40 2.97 ± 0.30 2.05 ± 0.26 Chi a/Chi b 3.29 ± 0.32 3.18 ± 0.48 3.46 ± 0.25 3.45 ± 0.87 3.44 ± 0.42 3.56 ± 0.39

Fluorescence Fv/Fp 0.81 ± 0.07 0.83 ± 0.09 0.80 ± 0.06 0.74 ± 0.6 0.62 ± 0.05 0.62 ± 0.05

506 ?erez et al.

Table 3. Leaf area and humidity, and root length and humidity of control and manganese deficient plants (average± standard error of fifty determinations)

Length (em) Humidity (%)

Control plants

Roots

62.20 ± 2.18 94.13 ± 0.19

Leaves

Old In term. Young

Deficient plants

Roots

56.23 ± 1.23 92.77 ± 0.24

Leaves

Old Interm. Young

Area (em') Humidity(%)

60.97 ± 4.50 45.09 ± 4.28 20.82 ± 3.85 52.68 ± 4.25 45.09 ± 4.17 20.15 ± 3.28 85.31 ± 2.25 84.46 ± 2.50 84.92 ± 2.70 84.74 ± 3.05 84.75 ± 2.15 86.19 ± 2.98

corded allows to deduce that the PS2 efficiency decreases in Mn-deficient leaves. The Fv /Fp ratio was almost constant in all three kinds of control leaves. By contrast, the highest value of this ratio was obtained for old leaves, while the intermediate and young leaves, with equal ratios, showed the lowest values.

Plant measurements

Leaf area, root length and humidity (difference between dry and fresh weight) were measured (Table 3). Old Mn-deficient leaves showed a slightly smaller size than controls. In inter­mediate and young leaves, we could not find any difference.

Root length of Mn-deficient plants was smaller than in control plants. Humidity of leaves and roots was not altered either by Mn deficiency or leaf age.

Discussion

Plant uptake of nutrients is affected by numerous soil and plant factors, being the two most im­portant the concentration of the available forms in the soil and plant genotype (Reisenauer, 1986). Solution culture techniques are employed when it is necessary to exert a greater control over the root environment than in soil-grown plants.

In dicotyledonous plants an intercostal chloro­sis of the younger leaves is the most distinct symptom of manganese deficiency, while in other

species the etiology of the deficiency is mani­fested in other ways (Windsor and Adams, 1987). In our conditions, the lower chlorophyll contents of Mn-deficient young leaves arc indica­tive of the typical chlorosis in this sort of plant (Table 2). The critical deficiency levels of Mn are between 10-20 mg kg - 1 weight dry in mature leaves. Net photosynthesis and chlorophyll de­cline rapidly below this level (Ohki et al., 1981). In our case, we found that all control leaves have Mn contents higher than 20 mg kg - 1 , and only intermediate Mn-deficient leaves have a Mn concentration below 10 mg kg - 1 • Surprisingly, although the lower photosynthetic pigment con­tents were found in young leaves, their Mn concentration was almost 40% higher than in intermediates (Table 1). This decline of Mn concentration in Mn-deficient leaves was also observed in intermediate control leaves. It could mean that total Mn is different from physiologi­cally active Mn. This fact, which deserves further investigation, was not contradictory to the modu­lated fluorescence measurements (Table 2). In spite of the higher Mn content of intermediate leaves, the photosynthetic efficiency expressed as Fv/Fp, had the same low value as in young leaves. These observations could be explained if we bear in mind that under Mn-deficiency the thylakoid membrane contains less PS2 reaction centre particles (Simpson and Robinson, 1984), and therefore its capacity to carry out photo­synthesis must be lower. However, when the ratio Chi a/b is calculated (Table 2), it reveals a constant value and thus we can assume that this lack of PS2 particles does not modify the stoi-

chiomctry of the light harvesting complex, which could be logical if taking into account that PS2 reaction centre only contains 4-6 chlorophyll a, and 1-2{3-carotene molecules (Montoya et al., 1991), compared with the several hundreds contained in the light harvesting structures.

From all the above mentioned data it can be concluded that the lack of manganese in the culture solution produces, especially in the inter­mediate soybean leaves, a decrease of the Mn concentration. Although the photosynthetic ef­ficiency was the same in Mn-deficient inter­mediate and young leaves. Furthermore, it has been found that manganese deficiency modifies slightly root length and does not modify leaf area by contrast with Fe-chlorosis which diminishes both parameters (Madero, personal communica­tion). Although no significant effects of Mn deficiency on the other elements examined were found, a certain increase in iron content was recorded in intermediate and old leaves from Mn deficient plants. In this sense, interactions be­tween iron and Mn was reported (Chinnery and Harding, 1980). They found that the concen­tration and amount of manganese in the shoots decreased with increased iron concentration in the solution, and probably an oxidation of iron by manganese.

Acknowledgements

Authors wish to thank Mrs M A Gracia for invaluable technical support, and Dr Alvaro Blanco for his help to make the statistical analy­sis. M C Perez gratefully acknowledges the support of a Fellowship from IberCaja during the course of these studies. Work carried out under research projects PGC PB89-0060.

References

Bowen J E 1981 Kinetics of active uptake of boron, zinc, copper and manganese in barley and sugarcane. J. Plant Nutr. 3, 215-223.

Manganese in soybean physiology 507

Bowen J E 1969 Absorption of copper, zinc and manganese by sugarcane leaf tissue. Plant Physiol. 44, 255-261.

Campbell L C and Nable R 0 1988. Physiological functions of manganese in plants. In Manganese in Plants and Soil. Eds. R D Graham, R J Hannam and N C Urcn. pp 139-154. Kluwer Academic Publishers, Dordrecht.

Cheniae G and Martin I F 1968 Sites of manganese function in photosynthesis. Biochem. Biophys. Acta 153, 819-837.

Chinnery L E and Harding C P 1980 The effect of ferrous iron on the uptake of mangese by ]uncus effusus. Ann. Bot. 46. 409-412.

Edwards G and Walker D 1983 C,, C4 : Mechanisms and cellular and environmental regulation of photosynthesis. Oxford.

Jones Jr J B 1984 Plants. In Official Methods of Analysis of the Association of Official Analytical Chemists. Eds. S Williams. pp 38-64. Association of official Analytical Chemists, Arlington, Virginia 22209 USA.

Maas E V, Moore D P and Mason B J 1969 Influence of calcium and magnesium on manganese absorption. Plant Physiol. 44, 796-800.

Marschner H 1986 Mineral Nutrition of Higher Plants. First, Academic Press Inc, San Diego, CA, 674 p.

Montoya G, Yruela I and Picorel R 1991 Pigment stoi­chiometry of a newly isolated D1-D2-Cyt b559 complex from the higher plant Beta vulgaris L. FEBS Lett. 283, 255-258.

Ohki K, Wilson D 0 and Anderson 0 E 1981 Manganese deficiency and toxicity sensitivities of soybean cultivar. Agron. J. 72, 713-716.

Pinta M and DeWeler G 1975 Etalons Vegetaux pour !'analyse foliare. In Le C6ntrole de !'alimentation des Plantes Cultivees. pp 159-172. Akademiai Kidao, Budap­est.

Reisenauer H M 1986 Determination of plant-available soil manganese. In Manganese in Soil and Plants. Eds. R D Graham, R J Hannam and N C Uren. pp 87-98. Kluwer Academic Publishers. Dordrecht.

del Rio LA, Lyon D S, Olah I, Glick B and Salim M L 1983 Immunotechnocytochemical evidence for a peroximal localization of manganese superoxide dismutase in leaf protoplasts from a higher plant. Planta 15, 216-224.

Simpson D J and Robinson S P 1984 Freeze-fracture ultra­structure of the thylakoid membranes in chloroplast from manganese deficient plants. Plant Physiol. 74, 735-741.

Val J and Monge E 1990 Violaxanthin cycle and fluorescence in iron-deficient maize levels. Cur. Res. Photosyn. 4, 765-768.

Winsor G and Adams P 1987 Diagnosis of mineral disorders in plants. Her Majesty's Stationary Office, Glasshouse Crops, London.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 509-516, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-205

The effect of liming and organic manuring on the reduction of copper toxicity in an acid soil of granitic origin of Dao wine region, Portugal

R.M.S. DIAS, J.C. SOVERAL-DIAS and A.S.V. COSTA Laborat6rio Quimico Agricola Rebelo da Silva, IN/A, Tapada da Ajuda, 1300 Lisboa, Portugal

Key words: acid soil, copper phytotoxicity, liming, organic manuring, ryegrass, soil pollution, vineyard

Abstract

Due to repeated application of Cu fungicides to vineyards there was an excessive accumulation of Cu in soil surface layer, reaching, sometimes, toxicity levels for some annual crops. Using an acid soil of granitic origin from the Dao wine region, containing 150 mg kg -t of total Cu, a pot experiment was carried out. This consisted of 12 treatments (with 4 replicates) at different levels of lime and dairy manure and, in some treatments, an additional application of 250 mg kg -I of Cu as sulphate with annual rycgrass as the test crop. The results clearly show the root damage induced by excess Cu and its negative effect on the ryegrass P uptake. They also show the positive effect of liming on reducing phytotoxicity and decreasing Cu solubility in the soil and its absorption by the plant. High organic manuring levels had a significant positive effect on yield, but did not, however, decrease the extractable Cu level in soil.

Introduction

The use of fungicides containing Cu began in the 19th century. The earliest report of Cu toxicity in vineyard soils dates from 1919 (Maquene and Demoussy), but it was only in the fifties, and especially in France, that attention was called to this problem (Anne and Dupuis, 1953; Drouineau and Mazoyer, 1953; Delas, 1963).

Copper in excess interferes with the plant's capacity to absorb and/ or translocate other nu­trients (Loue, 1988; Struckmeyer et al., 1969) inhibiting root elongation and adversely affecting the permeability of the root cell membrane (Sowell et al., 1957; Woolhouse and Walker, 1981 ).

Copper in excess also has a destructive effect on the integrity of the chloroplast membrane, leading to a decrease in photosynthetic activity (Eleftheriou and Karataglis, 1989; Struckmeyer et al., 1969). Yet, little is known on how its excess interferes with these mechanisms.

Several factors influence the level of available

copper in soil: total Cu content; organic matter (Delas, 1963; Hodgson et al., 1966; McLaren and Crawford, 1973a,b; Stevenson and Fitch, 1981); pH (Delas, 1963; McLaren and Crawford, 1973a,b); clay type and content (Delas, 1963; Pickering, 1979; Si\lanpaa, 1972, 1982); oxide type and content (McBride, 1981; Pickering, 1979); redox potential (Iu et al., 1981; Jarvis, 1981; Mitchell, 1972; Sillanpaa, 1972;); inter­action with other elements; nature of other elements associated with copper (Kiekens et al., 1984; Mitchell, 1972); microorganisms (McBride, 1981).

For the same total copper content of the soil, the level of available copper is higher in an acid, sandy soil, with low organic matter and low CEC, characteristics observed in many vineyard soils in Portugal (Pacheco, 1989).

The proportion of soluble/insoluble copper organic complexes depends mainly on the nature of the organic colloid, on the degree of satura­tion with copper and on the pH (Stevenson and Fitch, 1981 ). The higher the degree of saturation

510 Dias et al.

and the complex molecular weight, the more insoluble is the complex (Stevenson and Fitch, 1981). Also, copper complexes involving phenolic, hydroxyl and carboxylic groups are relatively weak, whereas copper porphyrin com­plexes are more stable (Goodman and Ches­chire, 1976). The copper-humus-clay complexa­tion also decreases copper availability to the plants (Stevenson and Fitch, 1981).

The increase of soil pH above 6.0 (Cavallaro and McBride, 1980) inducing the hydrolysis of hydrated copper ion leads to a stronger adsorp­tion of this element to the clay minerals (Delas, 1963; Krauskopf, 1972; McBride, 1981; Picker­ing, 1979) and to the organic matter (Delas, 1963; McLaren and Crawford, 1973a,b). As pH increases the size of organic colloids of high molecular weight diminishes thus increasing con­siderably the surface were Cu can be adsorbed. In calcareous soils copper precipitates with hy­droxides and carbonates (Delas, 1963).

This study was carried out in Laborat6rio Quimico Agricola Rebelo da Silva - Lis boa -aiming to assess the effect of pH increase and animal manuring in reducing copper toxicity in an acid soil of granitic origin receiving Cu inputs as copper fungicides during many years. In some treatments there was an additional application of 250 mg kg - 1 of copper as sulphate.

Materials and methods

Experimental design The pot experiment was set up in complete randomised blocks, with 12 treatments and 4 replicates.

Plant and soil The test crop was annual ryegrass (Lolium mul­tiflorum, Lam., cv. Tama). The soil used was the plow layer (0-20 em) of an acid soil of granitic origin from a vineyard in the Dao wine region­Portugal, high in copper due to repeated applica­tions of copper fungicides. Some analytical de­tails are shown in Table 1.

Soil treatments The treatments involved different rates of limes­tone (0; 9.75; 19.50; 29.25 and 39.00 g/pot) and

Table 1. Some characteristics of the soil used in the pot experiment

Clay content (%) 6.6 Organic matter (%) 0.74 P available (mg kg- 1 ) 32 K available (mgkg- 1 ) 209 pH(H,O) 5.6 pH(CaCI,) 4.9 Lime requirement (tha~ 1 ) 6 Exch. AI (cmol( +) kg~ 1 ) 0.35 Total Cu (mg kg~ 1 ) 148 Extractable Cu (mg kg~ 1 ) 39.5

dairy manure (0; 23.21; 46.43 and 92.86g/pot) and in some treatments application of 250 mg kg- 1 of copper as sulphate, as shown in Table 2.

Pot experiment procedure Air dried soil was passed through a plastic screen of 10 mm openings and stored until used.

In the glasshouse, polyethylene pots were filled with 9.75 kg of dry soil.

Basal fertilizers were applied on 4 occasions: the first, 5 days prior to sowing, included 500 mg N/pot, 655 mg P/pot and 1245 mg K/pot; the other 3 applications included 500 mg N /pot, supplied as a NH4N0 3 solution, after the 1", 2"ct and 3rct cut. The salts used were NH4N03 ,

Ca(H2P04 ) 2 H 20, K2S04 and MgS0 4 7H20, and were all pro-analysis.

The limestone and the dairy manure were also thoroughly mixed into the soil 5 days prior to sowing. Pots were watered with deionised water and held at 50% field capacity. Some analytical

Table 2. Application rates of copper (as sulphate), limestone and animal manure applied in the different treatments

Treatment

OCu+OL+OM -

0Cu+2L+OM -

1Cu+OL+OM 250 1Cu+1L+OM 250 1Cu+2L+OM 250 1Cu+3L+OM 250 1Cu+4L+OM 250 1Cu+OL+1M 250 1Cu+OL+2M 250 1Cu+OL+4M 250 1Cu+2L+1M 250 1Cu+2L+2M 250

Limestone (gpot~ 1 )

19.50

9.75 19.50 29.25 39.00

19.50 19.50

Dairy manure (gpoC 1 )

23.21 46.43 92:86 23.21 46.43

Liming and animal manuring effect on a Cu contaminated soil 511

characteristics of these amendments are shown in Tables 3 and 4.

On 24 December, 2.2 g ryegrass seeds were sown per pot (with 380 cm2 surface) 1.5 em deep, in 5 rows separated 3.5 em apart. The pots were watered just to keep the soil surface moist, until germination, after which the soil was maintained at 70% of field capacity.

Four cuttings- tops cut 3 em above soil level­were made (13 Feb., 18 March, 9 April and 3 May). The harvested plants were dried at 65oC for 45h and their yield expressed as g dry wt/pot. They were then ground in a stainless steel mill prior to analyses.

One week after the last cut a representative soil sample was taken from each pot.

Chemical analyses The soil samples were sieved through a 2 mm stainless steel mesh. The soil parameters ana­lysed were: pH(H20) in a 1:2.5 soil/water suspension after lh of contact; pH(CaC1 2 ) in a 1:2.5 soiljCaC12 suspension after 1h of contact; total organic C (Tinsley, 1950); extractable Cu and Mn (Lakanen and Ervio, 1971); total Cu (digested in a aqua regia solution and deter­mined by atomic absorption spectrophotometry); exchangeable bases and H (Mehlich, 1953 ); exchangeable AI (LQARS, 1977); available P and K (Egner-Riehm method; Riehm, 1958); clay content (Silva, 1975); lime requirement (Ojea and Taboadela, 1957).

The plant material of the four cuttings of each pot was analysed together as, in some treat­ments, yield was very low. The plant material

Table 3. Analytical characteristics of the limestone used in the pot experiment

Fineness

Ca(%) Mg(%)

>2mm 2-0.7lmm

<0.71 mm

Neutralizing value expressed as CaCO 3

0% 34.5% 65.4%

38.45% 0.43%

95%

Table 4. Analytical characteristics of the dairy manure used in the pot experiment

Dry matter Or g. matter Ash N p K Cu (%) (%) (%) (%) (%) (%) (mgkg- 1)

84 45.1 38.9 !.93 0.60 0.54 55.7

was prepared and analysed according to Duarte et al. (1977).

Results and discussion

As a result of the addition of copper sulphate, ryegrass presented leaves thinner than usual in this cultivar, this aspect being more pronounced in those treatments with little or no lime or manure (lCu+OL+OM, 1Cu+1L+OM, 1Cu+OL+1M, and 1Cu+OL+2M). These treatments also had a lower germination rate and the seedlings tended to bend at soil level. In some replicates of 1 Cu + 0 L + 0 M the plants even failed to survive after the 1st cut. No chlorosis was observed.

At the end of the pot experiment, when a soil sample was taken from each pot, it was observed that Cu sulphate application severely depressed elongation of the roots, which were short and branched. Lime and dairy manure applications favored root development in a similar way as they favored shoot vigour, but roots never reached the same size as those receiving no copper sulphate.

Figure 1 presents the accumulated yield of ryegrass.

Those treatments rece1vmg copper sulphate had a ryegrass Cu content (Table 5) already in the range (greater than 20 mg kg- 1) considered toxic to plants and reported to be toxic to sheep (20-30 mg kg -I in feeds and forages) but not to other domestic animals (Gupta, 1979).

100.0 /

80.0

8. 60.0

~ (;> ! 40.0

20.0

0.0 2

Treatmept.l: 1- OC\I+OL+OM 7-1Cu+4L+DM 2-0Cu+2L+0M 8-lCu+OL+lM

· · · · 3-lCu+OL+OM 9-1Cu+DL+2M · 4- 1Cu+1L+0M 10- 1Cu+OL+4M 5-1Cu+2L+OM 11· 1Cu+2L+1M 6-1Cu+3L+OM U- 1Cu+2L+2M

~ ....

J: ~ 4 5 6 7 8 9 10

'lleatments

Fig. 1. Accumulated ryegrass yield.

11 12

512 Dias et al.

Table 5. Mean effect of treatments on accumulated yield and ryegrass content of some nutrients

Treatment Accum. yield (g dry wt pot - 1)

OCu + OL+ OM 72.1 0Cu+2L+OM 78.7 1Cu+OL+OM* 0.4 1Cu+1L+OM 24.3 1Cu+2L+OM 37.0 1Cu+3L+OM 44.5 1Cu+4L+OM 52.2 1 Cu + OL+ 1M 1.5 1Cu+OL+2M 19.7 1Cu+OL+4M 43.7 1Cu+2L+1M 42.7 1Cu+2L+2M 46.5

sm( ±) 1.321 LSD (p < 0.05) 3.8 c.v. (%) 6.8

*insufficient vegetal material for analyses; sm =standard deviation; LSD= Least Significant Difference; c.v. =coefficient of variation.

N (%)

2.28 2.16

3.71 3.41 3.14 2.84 3.90 3.68 3.15 3.10 3.02

0.073 0.24 4.7

Copper in excess affected the plant content of certain nutrients, especially P. This aspect was also observed by Spencer (1966) and seems to be the consequence of two factors: drastic reduction of the root absorbing surface (Delas, 1963; Drouineau and Mazoyer, 1953; Foy eta!., 1978; Reuther and Labanauskas, 1966); membrane damage leading to increased rate of phosphate leakage from the roots (Woolhouse and Walker, 1981 ). The relation between P plant content and accumulated yield is presented in Figure 2. The following linear regression was obtained between the dry matter (DM in g dry wt/pot) and P content (% ):

DM = -1.159 + 181.226P with r2 = 0.89***

(p < 0.001).

As expected, ammonium nitrate fertilization and copper sulphate application acidified the soil (Table 6).

In those treatments receiving Cu sulphate, plants had a much higher Mn content, without reaching a toxicity level ( 500 mg kg -I, according to Cottcnic ct a!., 1976). This may in part result from soil acidification due to copper sulphate, but here we would also expect higher ext. Mn

p K (%) (%)

0.37 3.94 0.43 4.82

0.11 3.41 0.17 3.32 0.27 3.74 0.31 3.94 0.09 3.51 0.10 3.44 0.26 3.55 0.25 3.46 0.29 3.66

0.008 0.097 O.D3 0.27 6.7 5.2

Yield (g dry wt/pot)

Mn

(mgkg

108 99

302 309 189 96

314 342 400 261 275

14.341 48 11.7

1)

Cu

12 8

23 25 23 19 25 23 24 21 23

0.894 3 8.7

100.0.---------------

60.0

40.0

20.0

0.0 +----'-1------,----,-----,---j

0.00 0.10 0.20 0.30 0.40 0.50

Ryegrass P content (%)

Fig. 2. Relation between plant P content and ryegrass yield.

Liming and animal manuring effect on a Cu contaminated soil 513

Table 6. Mean effect of treatments on some analytical characteristics of the soil

Treatment pH (H,O)

OCu+OL+OM 4.95 0Cu+2L+OM 5.80 1Cu+OL+OM 4.60 1Cu+1L+OM 5.20 1Cu+2L+OM 5.60 1 Cu + 3 L +OM 6.00 1 Cu+4L+ OM 6.20 1Cu+OL+1M 4.60 1 Cu + OL+2M 4.90 1Cu+OL+4M 5.15 1Cu+2L+1M 5.50 1 Cu+ 2 L +2M 5.75

sm( ±) 0.067 LSD (p < 0.05) 0.19 c.v. (%) 2.5

levels in the soil, which was not observed. It may also be related to root cell membrane damage due to excess copper.

Liming effect on a soil contaminated with copper due to repeated applications of Cu containing fungicides

Although in this study a strictly comparable control was not available, at this level of ex­tractable copper in the soil ( 40-50 mg kg -I) no visual symptoms of copper toxicity in ryegrass were observed.

The rate of limestone applied (19.50 g/pot) was equivalent to a 1.0 lime requirement. This rate led to a significant (p < 0.05) soil pH increase from 4.95 to 5.80, which was favourable to ryegrass, significantly (p < 0.05) increasing yield and reducing the plant Cu content from 12 to 8 mg kg -I, both in the sufficiency range.

P availability increased, as expected, leading to a higher concentration in the plant.

The extractable Cu level in soil was decreased, but not significantly, still remaining very high. It is interesting to confirm that, comparing 0 Cu + 0 L + 0 M and 0 Cu + 2 L + 0 M with 1 Cu + 0 L + 0 M and 1 Cu + 2 L + 0 M (with equal rates of liming), pH increase had a greater effect on the soil treated with copper than on the same soil contaminated through the years with Cu fun­gicides.

P avail. Cu ext. Mn ext. (mg kg- 1) (mg kg- 1) (mg kg- 1)

69 47.8 18.0 82 43.0 17.7 73 254.1 16.9 74 235.5 14.4 70 228.0 14.7 74 205.8 14.9 70 175.8 15.6 80 247.5 17.6 82 248.3 16.1

101 253.1 19.7 80 249.6 15.2 89 255.5 17.6

2.767 3.989 0.540 8 7.0

11.4 1.6 3.9 6.5

Effect of liming a soil contaminated with Cu applied as sulphate

Liming induced a soil pH increase ranging from pH 4.8 (1Cu+OL+OM) to pH 6.2 (lCu+ 4L+OM).

Yield increase was positively related to soil pH increase, as shown in Figure 3. The following regression equation was obtained between DM (g dry wt/pot) and pH(H20):

DM = -353.96 + 110.98 pH -7.37 pH2 with

r2 = 0.99** (p < 0.01).

It was observed that the root development increased with rising soil pH.

Increases in soil pH were associated with significant decreases of Cu extractable level in soil, from 254.1mgkg- 1 (1Cu+OL+OM) to 175.8mgkg- 1 (1Cu+4L+OM) (Fig. 4). The plant copper content was only significantly de­creased in 1 Cu + 4 L + 0 M, corresponding to a soil pH of 6.2. Despite the evidence that in­creased pH decreases Cu availability, this effect is not always shown in plant uptake (Jarvis, 1981). In some cases Cu concentration decreased (Merry et a!., 1981); in others it was not sig­nificantly affected (Gupta, 1972; 1979). In fact, plants seem to be more efficient in Cu absorption than extracting reagents at higher pH levels (Loue, 1988; Sillanpaa, 1972). Also, pH seems to have an enhanced effect when Cu is added to

514 Dias et al.

Yield (g dry wt/pot) 60.0

.. 50.0

... 40.0

~·

30.0

.. 20.0

10.0

0.0 _.&___t _ __L__L___L _ __]______L _ __L__L__)

4.6 5.2 5.6 6.0 6.2 6.5 Soil pH(HP) after the trial

Fig. 3. Effect of soil pH increase on accumulated yield.

Plant Cu content Soil Cu ext. 30.---------------,300

25 ~~~ ~~~ ...•... 250

~~~

- "!7"·'"'·.;:.. -- -· ... ... 20

-.. ·. 200 ' ' ' '

15 150

10 100

5 50

0 I •- Plant Cu content _,._Soil Cu ext. I 0

4.6 5.2 5.6 6.0 6.2

Soil pH(HP) after the trial

Fig. 4. Soil pH effect on ryegrass Cu content (mg kg- 1 ) and soil Cu ext. level (mg kg -•).

the soil, compared to that already present (Loue, 1988).

The results obtained make us suppose that, for this level of Cu contamination, a soil pH above 6.2 would be more favourable to ryegrass, as it would lead to a greater decrease of the available Cu level in soil.

Liming also increased P absorption of rye­grass. This could be the result of a better root development (thus exploring a larger volume of soil) and to a positive effect on the root cell membrane, damaged by Cu phytotoxicity .

Effect of animal manuring on a soil contaminated with Cu applied as sulphate

High levels of animal manure significantly (p < 0.05) enhanced ryegrass yield (Fig. 5) and also root development.

Although shoot vigour was increased with manure application, neither the high plant Cu content nor the extractable Cu level in the soil decreased. Manure application may have led to the complexation of copper in soluble organic complexes, and the duration of the pot experi­ment (19 weeks) was possibly too short for the

Yield (g dry wt/pot)

0 23.25 46.43 69.75 92.86

Manure application (g/pot)

Fig. 5. Effect of dairy manure application on accumulated yield.

Liming and animal manuring effect on a Cu contaminated soil 515

stabilisation of Cu organic complexes. As Tiller and Merry (1981) state 'Soil organic matter has a recognised key role on the control of Cu solubili­ty in contaminated soils, but the question of 'reversion' of pollutant Cu in relation to its reaction with organic matter and its time scale has still to be resolved'. Another possible reason is that perhaps the Lakanen method extracts all types of Cu organic complexes, not being selec­tive to the soluble ones, which are more avail­able to the plant.

Manure application induced a significant (p < 0.05) increase of ryegrass P content. This could lead to a better development of the root system, and to a higher P availability in the soil (applied through basal fertilization and animal manure). Organic matter also favored P availability, name­ly through P complexation with humic acids easily available to the plant, anionic exchange of phosphate with humate, and the coating of oxides and clays with humus, reducing their capacity to immobilise phosphate.

The extractable Mn level in soil increased with heavy manure applications (1 Cu + 0 L + 4 M) and so did plant Mn content, as already observed by Sillanpaa (1972).

Effect of animal manuring on a soil contaminated with Cu applied as sulphate, and under moderate liming

A moderate rate of liming (19.50g/pot) and manure application at rates 23.21 and 46.43 g/ pot induced significant increases (p < 0.05) in ryegrass yield.

Neither plant Cu content nor extractable Cu level in soil decreased with manure application.

Available P level in soil was significantly (p < 0.05) increased due to the contribution of ma­nure and to the positive effect of organic matter on P availability through the already mentioned mechanisms. There was also a significant in­crease of plant P uptake probably due to a higher P availability and also to a positive effect of organic matter on P absorption.

Conclusions

Ryegrass grown in an acid soil of granitic origin,

with 150 mg kg - 1 of total Cu and 40 mg kg - 1 of extractable Cu showed no visual symptoms of Cu toxicity. Yet, at this level of ext. Cu in the soil, copper may already be near the toxicity threshold for this crop, beyond which biomass production decreases. Liming leads to a signifi­cant (p < 0.05) decrease in plant Cu content, which may have contributed to the significant yield increase.

Cu toxicity showed a strong negative effect on root and shoot development of ryegrass, and P nutrition was negatively affected.

Liming a soil heavily contaminated with Cu induced a significant decrease in the soil ex­tractable Cu level. The ryegrass yield was sig­nificantly (p < 0.05) increased, but the plant Cu content was significantly lower only at the high­est lime rate. These results may allow us to predict that, for this level of Cu contamination, a soil pH(H20) above 6.2 would be even more favourable to ryegrass, as it would lead to a greater decrease of the available Cu level in the soil.

Heavy rates of dairy manure applied to a soil highly contaminated with Cu increased signifi­cantly (p < 0.05) the ryegrass yield, without reducing plant Cu content nor extractable (Lakanen method) Cu level in the soil. Organic matter added to the soil had a positive effect on P nutrition of ryegrass.

References

Ann P and Dupuis M 1953 Toxicite du cuivre a l'egard de quelques plantes cultivees. C.R. Acad. Agric. de France 39, 58-61.

Cavallaro N and McBride M B 1980 Activities of Cu'• and Cd 2 + in soil solutions as affected by pH. Soil Sci. Soc. Am. J. 44, 729-732.

Cottenie A, Dhaese A and Camerlynck R 1976 Plant quality response to uptake of polluting elements. Qual. Plant. 26, 293-319.

Delas J 1963 La toxicite du cuivre accumule dans les sols. Agrochimica 7, 258-288.

Dias R M S 1991 Toxicidade de cobre num solo lit6lico nao humico derivado de granito. Contribui<;ao para o seu estudo. Relat6rio de Estagio do Curso de Engenheiro Agr6nomo, !SA, Lisboa, 120 p.

Drouineau G and Mazoyer R 1953 Toxicite du cuivre et evolution des sols sous !'influence des antiparasitaires. C.R. Acad. Agric. 39, 390-392.

516 Liming and animal manuring effect on a Cu contaminated soil

Eleftheriou E P and Karataglis S 1989 Ultrastructural and morphological characteristics of cultivated wheat growing on copper polluted fields. Bot. Acta 102, 134-140.

Foy C D, Chaney R Land White M C 1978 The physiology of metal toxicity in plants. Annu. Rev. Plant Physiol. 29, 511-566.

Goodman B A and Cheshire M V 1976 The occurrence of copper-porphyrin complexes in soil humic acids. J. Soil Sci. 27, 337-347.

Gupta U C 1979 Copper in agricultural crops. In Copper in the Environment. Part I. Ecological Cycling. Ed. J 0 Nriagu. pp 255-288. John Wiley & Sons, Inc., NY, USA.

Gupta U C 1972 Effects of manganese and lime on yield and on concentration of manganese, molybdenum, boron, copper and iron in the boot stage tissue of barley. Soil Sci. 114, 131-136.

Hodgson J E, Lindsay W L, and Trierweiler J F 1966 Micronutricnts cation complexing in soil solution. II: complexing of Zn and Cu in displaced solution from calcareous soil. Soil Sci. Soc. Am. Proc. 30, 723-726.

lu K L, Pulford I D and Duncan H J 1981 Influence of waterlogging and lime or organic matter additions on the distribution of trace metals in an acid soil. II: zinc and copper. Plant and Soil 59, 327-333.

Jarvis S C 1981 Copper concentrations in plants and their relationship to soil properties. In Copper in Soils and Plants. Eds. J F Loneragan, A D Robson and R D Graham. pp 265-285. Academic Press, Sydney, Australia.

Kiekens L and Camerlynck R 1982 Transfer characteristics for uptake of heavy metals by plants. Landwirtsch. Forsch. 39, 255-261.

Krauskopf K B 1972 Geochemistry of micronutrients. In Micronutrients in Agriculture. Eds J J Mortvedt, P M Giordano and W L Lindsay. pp 7-36. Soil Sci. Soc. Am., Inc., Madinson, Wisconsin, USA.

Lakanen E and Ervio R 1971 A comparison of eight extractants for the determination of plant available mi­cronutrients in soils. Acta Agr. Fenn. 123, 223-232.

Loue A 1988 Los Microelementos en Agricultura. Ed. Mundi-Prensa, Madrid, 354p.

LQARS 1977 Sector Fertilidade do Solo. Serie Divulga~ao. Documenta<;ao 2. DGSA, Lisboa, 39p.

Maquenne L and Dcmoussy E 1919 Sur !a richesse en cuivre des terres cultivees. C.R. Acad. Sci. 169, 937.

McBride M B 1981 Forms and distribution of copper in solid and solution phases of soils. In Copper in Soils and Plants. Eds. J F Loneragan, A D Robson and R D Graham. pp 25-45. Academic press, Sydney, Australia.

McLaren R G and Crawford D V 1973a Studies on soil copper. I: the fractioning of copper in soils. J. Soil Sci. 24, 172-181.

McLaren R G and Crawford D V 1973b Studies on soil copper. II: the specific adsorption of copper by soils. J. Soil Sci. 24, 443-452.

Mehlich A 1953 Rapid determination of cation and anion exchange properties and pH of soils. J. AOAC 36, 445-457.

Merry R H, Tiller KG and Alston AM 1986 The effects of contamination of soil with Cu, Pb, and As on the growth and composition of plants. I. Effects of season, genotype, soil, temperature and fertilizers. Plant and Soil 91, 115-128.

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Congresso da Vinha c do Vinho. pp 69-78. Confedera~ao dos Agricultores de Portugal, Luso, Portugal.

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M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nwrition, 517-521, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-117

Some effects of different levels of lead on berseem

CLAUDIA MARQUES DOS SANTOS, MARIA M.P.M. NETO and AMARILIS DE VARENNES Department of Chemistry, University of Agronomy Tapada da Ajuda, 1399 Lisboa Portugal

Key words: bcrseem, cambic arcnosol, eutric vertisol, heavy metal, lead, Trifolium alexandrinum L.

Abstract

Berseem plants (Trifolium alexandrinum L.) were cultivated in nutrient solutions containing different concentrations of lead and grown in a controlled environment with respect to light, temperature and humidity. Plants were analysed for dry weight, root length and chlorophyll and lead content. High levels of lead induced transient chlorosis in some leaves. Later on, some of the chlorotic leaves turned red. Chlorotic and red leaves had lower chlorophyll content. Weight and root length were affected by high concentrations of lead. Berseem plants were also cultivated in two different soils. The soils were dressed with macronutrients and seven levels of lead. After one month, shoots were weighed and analysed for lead and several nutrients. Plant weight was influenced by the concentration of lead present in the soils. In one soil the concentration of lead also influenced the plant content of some trace elements.

Introduction

There is a particular concern that toxic elements like lead might cause a problem to man and animals due to its accumulation in crops. The presence of lead in soils can affect the nutrient concentration and growth of plants (Brayer et al., 1972) though, due to an exclusion phenom­enon and low mobility of lead inside the plants, the shoots tend to show lower lead contents than the roots (Baker, 1981; Motto et al., 1970). Metal uptake varies with soil characteristics. Soils with high cation-exchange capacity tend to bind lead, making it less available for plant uptake (Carlson and Rolfe, 1979). Therefore, the risk of lead accumulation in edible plants is higher when these arc grown in light soils, with low organic matter content.

The aim of the experiments carried out was to study the uptake and effect of lead on the growth of berscem (Trifolium alexandrinum L.). In some experiments, berseem plants were culti­vated in nutrient solutions containing different levels of lead, to evaluate the effect of lead on

seed germination and on plant growth. The plants were also analysed for lead content. In another experiment, bersccm plants were grown in two different soils, in respect to organic matter and texture. Plant growth and the con­centration of lead and several other elements were evaluated.

Material and methods

Experiments in nutrient solutions

Berseem seeds (Trifolium alexandrinum L.) were germinated in the dark for one week in paper filters wetted with 10 mL of a solution with the following composition: 6mM Ca(N0 3 ) 2 , 6mM KN0 3 , 2.5 mM MgS0 4 , 1 mM KH 2P0 4 ,

100 J.LM H 3B0 3 , 100 J.LM MnS0 4 , 30 J.LM ZnS0 4 , 1 J.LM NaMo0 4 , 0.1 J.LM CuS0 4 ,

0.1 J.LM CoC1 2 and 75 J.LM Fe-EDTA. The seed­lings were transplanted to plastic vessels filled with 1.2 dm 3 of the solution described above, supplemented with six different lead concentra-

518 dos Santos et al.

tions supplied as Pb acet2te (0, 20, 200, 500, 1000 and 2000 mg L -I Pb). Plants were kept in a growth chamber at 18°C and 55% humidity, with 14/10 h dark/light periods at a light intensity of 500 J.Lmol foton m- 2 s- 1 supplied by day light fluorescent tubes. Two weeks after transplant, plants WGre collected and rinsed with distilled water. Roots and shoots were separated, dried at 105°C and weighed. This experiment was re­peated four times and, in each experiment, ten plants were weighed for each level of lead in the solution. These lots of ten plants were then pooled and processed together for lead analysis.

Inhibition of root growth by lead

Berseem seeds were sown in Petri dishes lined with filter paper wetted with 10 mL of the nutrient solution already described, sup­plemented with six levels of lead (0, 20, 200, 500, 1000, 2000 mg L -J Pb). Plants were grown in the dark in the same growth chamber for ten days. One hundred roots were measured (length) for each level of lead in the solution.

Chlorophyll determination

Chlorophyll was extracted in 80% acetone and its concentration was estimated by spectropho­tometry as described by Arnon (1939). Four replicates (leaves) were done for leaves with different pigmentations.

Pot experiments

A pot experiment was carried out in the same growth chamber, with two different soils. The soils used, a clay ( eutric vertisol) and a sandy soil (cambic arenosol), were arable top soils (0-25 em) from Queluz and Pegoes. The characteris­tics of the soils are given in Table 1. In each pot 2 kg (sandy soil) or 1.2 kg (clay soil) of soil received a basal dressing of NPK (7: 21: 21) at 0.05 g N, 0.15 g P and 0.15 g K per kg soil. The sandy soil was also dressed with 0.05 g Mg per kg of soil, supplied as Mg sulphate. Seven levels of Pb (0, 20, 100, 200, 350, 500, 2000mg kg- 1)

applied as Pb acetate, with four pots for each level, were used. Twenty five seeds were sown in each pot. Plants were watered with distilled

Table 1. Characteristics of the top soils used

Characteristics Origin of soil

Queluz Pegiies

Organic matter % 3.11 0.94 pHH,o 6.7 6.3 pHK~I 5.5 5.0 K 2 0(mgkg- 1) 111 44 P20 5 (mgkg-') 81 26 Zn (mg kg 1) 2.5 0.8 Fe (mg kg- 1) 135 38 Cu (mg kg-') 6.3 0.8 Mn (mg kg_,) 263 9

Organic matter was estimated by Sthrolein apparatus. The pH was determined by potenciometry. Nutrients were ex­tracted by the Egner Rhiem solution. P was determined by colorimetry, K by flame photometry and the others by atomic absorption spectrophotometry.

water daily to maintain 50% waterholding capacity. Shoots were collected after one month, oven dried at 105°C and weighed. All the plants from each pot were weighed individually. The plants from each pot were then pooled and processed together for lead and nutrient analysis.

Lead and nutrient analysis

Dried leaves and roots were burned overnight at 500°C. The ashes were then digested twice with 10 mL of 3.3% HN0 3 at 100°C. The clear residue was made up to 100 mL with 3.3% HN0 3 . The elements Cu, Fe, Mn, Zn, K, Na, Ca, and Mg were measured by atomic absorption spectrophotometry (Pye Unicam SP9). Lead was determined by anodic stripping voltammetry with a hydrodynamic electrode (Brett and Neto, 1989).

Statistics

The significance of differences between treat­ments were determined by one factor ANOVA using the Scheffe F-test at 0.05 probability.

Results

Symptoms and chlorophyll content

Plants cultivated in nutrient solutions with lead (20 mg L -J Pb or higher), presented transient

chlorosis when lead was present since germina­tion. Some of the yellow leaves turned red after a few days. Yellow and red leaves had a lower level of chlorophyll when compared with normal green leaves. Chlorophyll content was 3.330 ± 0.646 (green leaves), 0.776 ± 0.410 (yellow leaves) and 0.805 ± 0.740mg g- 1 fresh weight (red leaves). Mean values obtained with yellow and red leaves were not significantly different as judged by the Scheffe F-test at 0.05 probability.

Levels of lead of 500 mg L -I Pb or higher, reduced root and shoot dry matter accumulation as shown in Table 2.

Accumulation of lead

The results obtained in the experiment carried out in nutrient solutions are presented in Table 2. Shoots presented lower lead concentrations than the corresponding roots, for all the levels of the metal tested. These concentrations were not significantly different, for the lower levels of lead in the solution (0, 20 and 200 mg L - 1). They became higher for lead levels of 500 mg L -I or more in the solution. Roots presented high levels of lead; 200 mg L -I Pb in the solution was sufficient to cause a significant increase of the metal in the roots.

Root growth

All the seeds used in this assay germinated, independently of the level of lead present. Root growth inhibition was observed for high levels of lead (1000 mg L -l Pb or higher). Root length values are presented in Table 3.

Effects of lead on berseem 519

Table 3. Mean values for the root length of plants grown with different Pb levels

Pb in the nutrient solution (mg L - 1 )

0 20

200 500

1000 2000

Root length (em) per root

6.3a,b 7.2a 6.0a,b 6.3a,b 5.3b 3.0

Values followed by the same letter are not significantly different as judged by Scheffc F-test at 0.05 probability.

Pot experiment

Plants cultivated on the clay soil had a higher dry weight for all the levels of lead used, as com­pared to plants grown in the sandy soil (Table 4). The presence of lead in both soils even for the lowest level assayed, inhibited plant dry matter accumulation. The levels of lead in the plants cultivated in both soils are shown in Table 4. Plants grown in the clay soil presented similar lead content in their shoots for all the lead levels used except for the highest (2000mg kg- 1 Pb). Plants grown in the sandy soil with 350 mg kg -I Pb or more, had higher lead content in their shoots as compared with control or with plants grown with lower lead levels.

Of all the elements analysed, only iron and zinc were influenced by lead concentration in the sandy soil. In fact, plants grown in the sandy soil with 2000 mg kg -I Pb had higher iron and zinc levels as compared with the other plants. Con­trol-plants had 0.388 ± 0.093 mg g -I Fe and 0.093 ± 0.011 mg g -I Zn and plants cultivated with 2000 mg kg -I Pb had 5.445 ± 0.298 mg g -I Fe and 0.663 ± 0.069 mg g -I Zn. No influence on

Table 2. Mean dry weight and Pb content of plants cultivated in nutrient solutions with different levels of Pb

Pb level in the Dry weight per plant (mg) Level of Pb (P-g g - 1)

solution (mgL - 1)

Roots Shoots Roots Shoots

0 36" 86" 88" 34" 20 30"·b 67"·' 3000" 50"

200 24"·b 41 o.b 24445 90" 500 16b 22b.c 70375 306

1000 6b 8b 81000 984

Values within a column followed by the same letter are not significantly different as judged by Sheffe F-test at 0.05 probability.

520 dos Santos et al.

Table 4. Mean dry weight and lead content of shoots from plants cultivated with different levels of Pb

Pb level in Plant dry weight (mg) Pb content (mg kg -l plant soil (mg kg- 1 ) dry weight)

Pegoes Queluz Pegoes Queluz

0 92 252 19" 21' 20 61 "·b 199' 26' 24'

100 73' 185' 46'·b 27' 200 soh 184' 62'.b 33' 350 31' 115h 78b 32' 500 25' 117b 205 24'

2000 24 182' 514 61

Values within a column by the same letter are not significantly different as judged by Scheffe F-test at 0.05 probability.

nutrient content related to lead was observed in plants grown in the clay soil.

Discussion and conclusions

Berseem germination was not affected by lead. The same result was reported for tomato and egg-plant by Khan and Khan (1983). However, soon after germination lead was already toxic to seedlings, as inhibition of root growth was ob­served for 1000 mg L - 1 Pb and higher.

Toxicity towards roots and shoots was appar­ent when plants were cultivated in nutrient solutions containing lead. Not only the dry weights were smaller but the leaves also pre­sented chlorosis. Increases in lead content in plants were accompanied by decreases in growth. For the same level of lead in the solution, roots showed a higher lead content than shoots. Lead accumulation in roots has previously been de­scribed for several plants (Chizzola, 1989). How­ever, the level of lead in the shoots also in­creased when the amount of lead in the solution became higher, although it never reached the levels present in the roots. High background levels of lead are always observed in our experi­ments. This is due to intensive traffic on a bridge above the trial place.

The simple presence of lead reduced the dry matter accumulation of plants cultivated in the two soils used. Dry weights of the plants culti­vated in the clay soil were higher than those cultivated in the sandy soil. This was probably due to a greater fertility of the clay soil and also to its higher cation-exchange capacity that con­tributed to retain more lead. In fact, lead is

easily fixed in soils and only readily soluble lead compounds are absorbed by plants ( Chizzola, 1989).

Under certain conditions, including low soil cation-exchange capacity, large amounts of lead can be taken up by higher plants (Chizzola, 1989). This was the case for plants grown on the sandy soil.

Lead taken up by roots generally has no toxic effect on plants except at extremely high concen­trations (Miller and Koeppe, 1971; Rolfe, 1973). However, our results showed that for berseem grown in soils, even a low level of lead (20 mg kg -l Pb in the soil) was enough to inhibit plant dry matter accumulation, although visual symp­toms were absent in those plants. In the experi­ments carried out in nutrient solutions, inhibition of plant dry matter accumulation was only ob­served when the level of lead reached 500 mg L 1 Pb. This result is in apparent contradiction with the former, but not only were the plants of different age (2 weeks as opposed to 4 weeks), also the fact that a larger plant variation was observed in the experiments in nutrient solu­tions, made it more difficult to obtain significant differences. Furthermore, soil-grown berseem was dependent on N 2 fixation, as the supply of combined nitrogen was kept lO a minimum. It is possible that rhizobia were m Jre sensitive to lead than plant itself. In fact, soil microbial activity is affected at low soil heav· m:Ltls' concentrations (Brookes and McGrath 1%4).

Although the preser · of lead is to be ex­pected in the shoots o. plants grown in lead contaminated soils, our results showed that the toxic effect of lead is less pronounced when the soil has the capacity to retain lead ions. In fact,

in plants grown in the clay soil, the lead content of shoots was relatively low. The only exception was the case in which the level of lead in the soil was extremely high, a situation rarely encoun­tered in cultivated soils.

Acknowledgement

We thank Mrs Cra<;a Roque for her technical assistance.

References

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Baker A J M 1981 Accumulators and excluders strategies in the response of plants to heavy metals. J. Plant Nutr. 3, 643-654.

Brett C M A and Neto M M P M 1989 Voltamemetric studies and stripping voltammetry of Mn (ll) at the wall-jet ring­disc electrode. J. Electroanal. Chern. 258, 345-355.

Effects of lead on berseem 521

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Broyer T C, Johnson C M and Paull R E 1972 Some aspects of lead in plant nutrition. Plant and Soil 36, 301-313.

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Chizzola R 1989 Metallic trace elements in herbs and spices grown in Austria. Acta Hortic. 249, 89-96.

KhanS and Khan N N 1983 Influence of lead and cadmium on the growth and nutrient concentration of tomato (Lycopersicum esculentum) and egg-plant (Solanum me/ogena). Plant and Soil 74, 387-394.

Miller R J and Koeppe DE 1971 Accumulation and physio­logical effects of lead in corn. Proc. 4th Annual Conf. on Trace Substances in Environ. Health. pp 186-193. Uni­versity of Missouri, Columbia

Motto H L, Daines R H, Chilko D M and Motto C K 1970 Lead in soils and plants: its relationship to traffic volume and proximity to highways. Environ. Sci. Techno!. 4, 231-237.

Rolfe G L 1973 Lead uptake by selected tree seedlings. J. Environ. Sci. Qual. 2, 153-157.

Walsh G E, Ainsworth K A and Rigby R 1979 Resistance of red mangrove (Rhyzophora mangle L.) seedlings to lead, cadmium and mercury. Biotropica 11, 22-27.

MAC. Fragoso and M.L. van Beusichem (eds.) Optimization of plant nutrition 523-529, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-135

Evaluation of structural and physiological plant characteristics in relation to the distribution of cadmium in maize inbred lines

P.J. FLORIJN, J.A. NELEMANS and M.L. VAN BEUSICHEM Department of Soil Science and Plant Nutrition, Wageningen Agricultural University, P.O. Box 8005, 6700 EC Wageningen, The Netherlands

Key words: cadmium, desorption, genotypic variation, maize inbred lines, morphological parameters, nutrient solution culture, organic acid, root, xylem, Zea mays L.

Abstract

To establish the structural and physiological characteristics related to the genotypic variation in Cd distribution between maize inbred lines ('shoot Cd excluders' and 'non-shoot Cd excluders'), shoot and root morphological parameters were studied on plants grown in nutrient solution. Furthermore, the xylem sap composition and the desorbability of Cd from roots of these inbreds have been compared. No relationship between the morphological characteristics of either shoot (specific leaf area and leaf area ratio) or root (specific root length, specific surface area, and average diameter) and Cd distribution could be assessed. Cadmium concentrations in the xylem exudates from 'non-shoot Cd excluders' were higher than those from 'shoot Cd excluders', but not related to citrate and malate concentrations. The absolute and relative amounts of Cd desorbed from roots of 'shoot Cd excluders' were about twice as high compared to those of the 'non-shoot Cd excluders', especially at the lowest Cd concentration in solution. The absence of a relationship between shoot or root morphological parameters and Cd partitioning and the differences between both groups in the amounts of Cd desorbed, even at similar root Cd concentrations, indicate that the differential Cd distribution between 'shoot Cd excluders' and 'non-shoot Cd excluders' may be related to the observed differences in root Cd concentration, desorption characteristics and binding capacity of Cd inside and/ or outside the root and its distribution within the roots.

Introduction

Metal uptake from contaminated soil is different between plants. Two basic strategies of plant response to heavy metal toxicity may occur, accumulation and exclusion (Baker, 1981 ). Ac­cumulators can concentrate the metals in plant parts from low or high background levels, where­as excluders have a more or less constant low shoot level over a wide range of external concen­trations.

Both strategies seem to occur in maize inbred lines grown on Cd-contaminated soil (Hinesly et a!., 1978), which may result from a differential

whole-plant uptake and/ or distribution between shoots and roots. In an earlier article, we showed that total uptake of Cd by maize inbred lines was similar, whereas the partitioning of Cd between roots and shoots was very different (Florijn and Van Beusichem, 1993a). We distinguished two main groups of inbreds: a group with low shoot but high root Cd concentrations ('shoot Cd excluders') and a group with similar shoot and root Cd concentrations ('non-shoot Cd exclud­ers'). No relationship between the Cd concen­trations in shoots or roots and the corres­ponding dry matter yields of both groups of inbreds was observed, while environmental con-

524 Florijn et al.

ditions, such as pH and level of Cd supply, were not related to the variation in Cd distribution (Florijn and Van Beusichem, 1993b).

Genotypic variation in Cd distribution may be related to structural or physiological differences located in either shoots or roots. Root length, root surface area or root CEC (Boot, 1989; Nishizono et al., 1987) may be important in this respect. The Cd speciation in the roots and the binding capacity of root tissue components, the formation of granules (Khan et al., 1984; Rauser and Ackerley, 1987) or complexation of Cd to metal-binding peptides inside the cell (Rauser, 1990; Steffens, 1990) may also play a role in a reduced Cd transfer from roots to shoots. Furthermore, physiological processes like xylem loading (Bowling, 1981; Kochian 1991) may determine the Cd transport to the shoots. Final­ly, morphological leaf parameters determining the allocation of the xylem flux may be involved in the differential Cd transfer. Another potential explanation for a differential Cd transport may be the possible redistribution of Cd within the phloem system.

To establish the structural and physiological characteristics leading to the observed differ­ences in Cd distribution within maize inbred lines, shoot and root morphological parameters were studied on plants grown in nutrient solu­tion. Furthermore, the xylem sap composition and Cd adsorption to the roots of these inbreds have been compared. Equilibrium concentra­tions of Cd complexes occurring in the xylem sap were calculated to study the significance of complexation in Cd transport.

Materials and methods

Six maize inbred lines, representatives of the two main groups of inbreds ('shoot Cd excluders': B73, H99, and H96; 'non-shoot Cd excluders': B37, H98, and N28) and different in Cd dis­tribution (Florijn and Van Beusichem, 1993a), were used in the experiments. Four seedlings of each of the six inbreds were fixed in a plastic disc and transferred to either a 50-L or a 150-L container, in a growth chamber at 20/20aC day (16h)/night (8h), dew point 17.7/17.7°C, and light intensity 140 W m 2 (HPL comfort and

Son-T). Plants were grown in aerated flowing nutrient solutions (Florijn and Van Beusichem, 1993a). Instead of Fe-EDTA, FeS04 (10 fLM) was added. The iron supply was repeated daily during the following 9 days, then increased to 20 fLM for seven days and to 30 fLM for the rest of the experiment. The nutrient solutions were renewed eight days after the start of the experi­ment and then every four days until harvest. Cadmium (as CdC12 ) was added to the nutrient solutions 18 days after transfer of the plants. Three treatments (0, 10, and 30 fLg Cd L - 1) were introduced and the Cd concentrations were de­termined daily and adjusted to initial values. These external Cd concentrations did not affect dry matter yields of both 'shoot Cd excluders' and 'non-shoot Cd excluders' (Florijn and Van Beusichem, 1993a; 1993b). Solution pH was maintained at 5.50 ± 0.10.

Plants were harvested 32 days after transfer to the nutrient solutions and separated into shoots and roots for fresh and dry weight determi­nations and for chemical analysis. Roots were washed in two aliquots of demineralized water for one min, dried between tissues and weighed. Plant material was dried at 70°C for at least 24 h and ground in a stainless steel grinder. Cadmium concentrations were determined by atomic ab­sorption spectrometry (AAS) after digestion in a H 2S04 -HC10 4-HN0 3 mixture, as described pre­viously (Florijn et al., 1991; Florijn and Van Beusichem, 1993a). The concentrations of Cd in nutrient solutions were determined directly by AAS after acidification with 0.8 M H 2SO 4 ( 4.5 I 0.5 v /v).

Shoot and root morphological parameters, xylem sap composition (Cd, Mn, Fe, Cu, Zn, Ca, Mg, P, citrate, and malate) and the de­sorbability of root Cd were studied in three successive experiments. In the first experiment, plants were grown on eight 50-L containers, giving a triplicate for each Cd treatment (10 and 30 1-Lg Cd L - 1) and a duplicate for the control. The leaf area was determined with a leaf area meter (LICOR 3100). The specific leaf area (SLA, cm2 g- 1 dry wt. 1ear) and the leaf area ratio (LAR, cm2 g - 1 dry wt.plant) were derived from the primary data. Root length determinations were carried out on subsamples of 5 g fresh root material, according to the line-intercept method

Plant characteristics in relation to Cd distribution in maize 525

(Bland and Mesarch, 1990). The specific root length (SRL, m g- 1 dry wt.,001 ) was calculated. Because the specific gravity of the roots was close to unity and their dry matter contents were not different (preliminary investigations), the specific surface area ( SSA, m 2 kg - 1 dry wt. root)

and average diameter (mm) could be assessed. In another experiment, plants were grown on

three 150-L containers with either 0, 10, or 30 f.(-g CD L - 1 in solution. Four discs with 4 plants of each inbred were placed on each container. The collection of xylem exudates started 5 h after the light was turned on, and 3 h after adjustment of the solution Cd concentrations to preset values. Plants were decapitated 2 em above the upper­most roots. The exudate was discarded for 10 min to avoid phloem and cell tissue contami­nations (Andersen and Brodbeck, 1989; Arm­strong and Kirkby, 1979) and collected continu­ously in preweighed vials over an exactly 60-min period, using Pasteur pipets. Xylem exudate of all plants per disc were treated as one sample. The samples were weighed immediately after collecting and deep frozen until use. Exudate pH was determined with a combined glass/reference electrode and the osmolarity of the xylem sap was determined with an Osmomat 030 (Gonotec). Citrate and malate were determined by enzymatic procedures (Boehringer Mannheim GmbH; Anonymous, 1989), based on the de­crease or increase of the absorption of NADH (340 nm) as a result of specific enzymic oxidation of NADH or reduction of NAD+. Preliminary investigations in our laboratory showed that Cd had no effect on measured organic acid concen­trations. The concentrations of Cd and Zn were determined by AAS and of other elements (Mn, Fe, Cu, Ca, Mg, and P) by Inductively Coupled Plasma-Atomic Emission Spectrometry (ICP­AES), after acidification with H 2S04 . Equilib­rium concentrations of Cd complexes with citrate

and malate were calculated with the use of a computer program for the calculation of ionic speciation in soil-water systems (ECOSA T) (Keizer et a!., 1991). Osmolarity, pH and the concentrations of the two organic acids and of all measured elements were used as input data for the calculation of Cd speciation in the xylem sap.

ln the third experiment, roots of plants grown at two Cd treatments (10 or 30 f.(-g Cd L -I) were allowed to desorb Cd for 2 h according to the procedure of Cd desorption proposed by Rauser (1987), slightly modified according to prelimin­ary investigations. Ten grams of fresh roots were washed for one minute in demineralized water and subsequently transferred into 200 mL ice­cold 50 mM CaC1 2 • The solutions were continu­ously stirred during root desorption. Samples were taken from the desorption medium at regular intervals over a 120-min period.

Results

The mean values for the root parameters specific root length (SRL), specific surface area (SSA), and average diameter of both groups of maize inbreds are given in Table 1. No significant differences between both groups were observed, neither in the control nor in the Cd treatment. No effect of Cd application on these parameters was observed.

In Table 2, the specific leaf area (SLA), the leaf area ratio (LAR), and the shoot/root ratio of the inbreds are presented. Both the SLA and the LAR were not significantly different between both groups of inbreds. The shoot/root ratio of the 'shoot Cd excluders' tended to be lower compared to the 'non-shoot Cd excluders', al­though not significantly. Cadmium application did not affect these three parameters, except for

Table 1. Specific root length (SRL, m g- 1 dry wt., 001 ), specific surface area (SSA, m2 kg- 1 dry wt.coot), and average diameter (mm) of roots of 'shoot Cd excluders' (B73, H99, and H96) and 'non-shoot Cd excluders' (B37, H98, and N28), grown for 32 days in nutrient solution. Cadmium (30 JLg Cd L 1) was added 14 days before harvest. Values given ± standard deviation

Shoot Cd excluders Non-shoot Cd excluders

SRL

-Cd

106 ± 23 120 ± 23

+Cd

113 ± 30 128 ± 18

SSA

-Cd

146 ± 19 147 ± 16

+Cd

151 ± 24 153 ± 18

Average diameter

-Cd

0.45 ± 0.04 0.40 ± 0.05

+Cd

0.44 ± 0.07 0.38 ± 0.02

526 Florijn et al.

Table 2. Specific leaf area (SLA, cm 2 g- 1 dry wt.'"0,), leaf area ratio (LAR, em' g 1 dry wt.P1,,), and shoot/root ratio of dry matter yields of 'shoot Cd excluders' (B73, H99, and H96) and 'non-shoot Cd excluders' (B37, H98, and N28), grown for 32 days in nutrient solution. Cadmium (30 flg Cd L 1) was added 14 days before harvest. Values given ± standard deviation

Shoot Cd excluders Non-shoot Cd excluders

SLA

-Cd

273 ± 21 274 ± 42

+Cd

280 ± 41 294 ± 43

the LAR of 'non-shoot Cd excluders', which was significantly higher after Cd application.

Xylem sap concentrations of Cd, citrate, and malate of five inbred lines are given in Table 3. The Cd concentrations in the xylem exudate of both groups of inbreds increased at the higher level of Cd application and were considerably higher for 'non-shoot Cd excluders' compared to 'shoot Cd excluders'. The concentrations of both citrate and malate in the xylem sap of nearly all inbreds were either similar or slightly enhanced after Cd application.

The clement concentrations (Mn, Fe, Cu, Zn, Ca, Mg, and P), pH and osmolarity of the xylem exudate of two inbreds (the 'shoot Cd excluder' B73, and the 'non-shoot Cd excluder' N28) are presented in Table 4. The Zn and Ca concen­trations in the xylem exudate from B73 were higher compared to N28. No other differences between the inbreds were observed, neither in the concentrations of other elements nor in osmolarity. The pH was about 5.5 for both maize inbred lines (Table 4).

The amounts of Cd desorbed from roots of five maize inbred lines during 2 h are presented in Figure 1. The 'shoot Cd excluders' desorbed much higher amounts of Cd from the roots compared to the 'non-shoot Cd excluders'. The

LAR

-Cd

124 ± 5 121 ± 9

+Cd

131 ± 19 146 ± 16

Shoot I Root ratio

-Cd

4.0 ± 1.3 5.2 ± 1.2

+Cd

4.5 ± 1.0 5.5 ± 0.8

Table '4. Concentrations of Mn, Fe, Cu, Zn, Ca, Mg, and P (f!.M), the pH and the osmolarity (MPa) in xylem exudates from two maize inbred lines, grown for 32 days in nutrient solution. Cadmium (30 flg L -I) was added 14 days before harvest. Values given ± standard deviation. (n = 4)

Maize inbred line

B73 N28 Shoot Cd excluder Non-shoot Cd excluder

Mn 15.0 ± 12.6 15.2 ± 11.8 Fe 5.7 ± 5.8 5.2 ± 9.0 Cu 0.4 ± 0.5 0.9 ± 1.5 Zn 7.8 ± 0.3 2.7 ± 0.9 Ca 1430 ± 160 1160 ± 70 Mg 1260 ± 310 1610 ± 130 p 2960 ±410 2180 ±290 pH 5.6 ± 0.3 5.5 ± 0.1 Osmolarity 0.146 ± 0.020 0.136 ± 0.005

amounts of Cd desorbed from the roots did not increase between 10 min and 2 h.

Table 5 shows the shoot Cd concentrations, total Cd concentrations of the roots, and the root-desorbable Cd fraction of each inbred line. Shoot Cd concentrations of 'non-shoot Cd ex­cluders' were much higher for both Cd treat­ments compared to 'shoot Cd excluders'. The absolute and relative amounts of Cd desorbed from roots of the two 'shoot Cd excluders' B73

Table 3. Xylem concentrations (f!.M) of Cd, citrate, and malate of five maize inbred lines, grown for 32 days on nutrient solution. Cadmium (10 or 30 flg Cd L -I) was added 14 days before harvest. Values given ± standard deviation. (n = 4)

Cd Citrate Malate

+Cd (10) +Cd (30) -Cd +Cd (30) -Cd +Cd (30)

Shoot Cd excluders B73 0.5 ± 0.3 0.9 ± 0.4 140 ± 20 170 ± 20 700 ± 160 620 ± 170 H99 <0.1 0.5 150 100 400 500

Non-shoot Cd excluders B37 2.2 ± 0.8 4.2 ± 0.4 170 ± 10 300 ± 30 370± 40 730 ± 280 H98 5.0 ± 0.5 7.8±0.9 100 ± 20 120 ± 10 260± 70 260 ± 110 N28 2.0 ± 0.4 4.5 ± 0.9 80 ± 10 100 ± 10 210± 30 200± 40

Plant characteristics in relation to Cd distribution in maize 527

80,-,---------------------------------,

70

30

20

10

Shoot Cd excluders a 873

X H99

Non-shoot Cd excluders + 937

o H98

o N28

(min.)

Fig. 1. The amounts of Cd desorbed from the roots of 5 maize inbred lines, grown for 32 days in nutrient solution. Cadmium (30 fLg L - 1 ) was added 14 days before harvest.

and H99 were higher compared to the three 'non-shoot Cd excluders', especially at the lowest Cd concentration. The root-desorbable Cd frac­tion of the group of 'non-shoot Cd excluders' increased significantly at a higher external Cd supply (p < 0.05), whereas that of 'shoot Cd excluders' remained constant.

Discussion

The maize inbred lines grown in Cd-containing nutrient solutions showed remarkable differences in shoot/root distribution of Cd (Table 5) and may be characterized according to earlier in­vestigations (Fiorijn and Van Beusichcm, 1993a)

by 'shoot Cd excluders' and 'non-shoot Cd excluders'.

Shoot morphological parameters, like specific leaf area (SLA) and leaf area ratio (LAR) were similar for both groups of maize inbred lines, independent of Cd application (Table 2). In addition, no significant differences in the shoot/ root ratio were obtained between both groups of inbreds (Table 2), and Cd application had no effect on dry matter yields (Fiorijn and Van Beusichem, 1993b). Therefore, shoot mor­phological parameters may not be involved in the variation in shoot Cd concentrations among the inbreds. Also the root parameters were not significantly different for 'shoot Cd excluders' and 'non-shoot Cd excluders' (Table 1 ). These observations in combination with the similarity in shoot/root ratio of both groups of inbreds (Table 2), provide circumstantial evidence for the absence of a definitive role of root mor­phological parameters and root growth in the explanation of the differential Cd distribution. The considerable variation in the distribution of Cd or more precise the ability to retain Cd in the roots, as observed by 'shoot Cd excluders', may therefore be attributed to differences in xylem loading and I or a dissimilar binding capacity of Cd outside or inside the root cell.

The Cd concentrations in the xylem sap of 'shoot Cd excluders' and 'non-shoot Cd exclud­ers' differed significantly for each Cd treatment (Table 3). The roughly two-fold increase of the Cd concentrations in the xylem sap was also observed for the Cd concentrations in the shoots

Table 5. Cadmium concentrations (fLg g- 1 dry wt.; n = 2 or 3) in shoots and roots and the root-desorbable Cd fraction (% between parentheses) of 'shoot Cd excluders' and 'non-shoot Cd excluders', grown for 32 days in nutrient solution. Cadmium (10 or 30 fLg Cd L - 1 ) was added 14 days before harvest. Values given ± standard deviation

Shoot Cd concentration

+Cd (10)

Shoot Cd excluders B73 1.3 ± 0.4 H99 H96

0.8 ± 0.2 2.0 ± 0.4

Non-shoot Cd excluders B37 23.6 ± 3.7 H98 13.9 ± 2.7 N28 15.2 ± 1.4

"n.d.: not determined.

+Cd (30)

9.0 2.5 ± 1.1 5.8 ± 1.4

54.6 ± 3.0 37.8 ± 6.7 39.0 ± 9.4

Root Cd concentration

+Cd (10)

114.8 ± 4.3 (18.0 ± 7.0) 32.0 ± 3.2 (16.3 ± 8.1) 61.2 ± 7.5 (n.d.)'

24.7 ± 4.0 (3.7 ± 3.5) 25.4 ± 3.5 (3.0 ± 2.7) 30.4 ± 2.9 (5.7 ± 5.1)

+Cd (30)

343.5 (13) 112.3 ± 26.6 (15.7 ± 2.3) 128.4 ± 21.3 (n.d.)

49.9± 9.6 (8.7±4.7) 71.6 ± 9.9 (9.3 ± 1.2) 70.5 ± 20.9 (10.3 ± 3.2)

528 Florijn et al.

in this particular experiment. This suggests that Cd is hardly retranslocated in the maize plants. Senden and Wolterbeek (1990) suggested that complexation of Cd in the xylem sap may result in a higher efficiency of Cd transport, leading to elevated Cd levels in the leaves. Although a considerable variation of organic acid concen­trations in the xylem exudates occurred between inbreds, no relationship between these concen­trations and Cd concentrations in the xylem sap was observed (Table 3). Slightly higher organic acid concentrations were obtained after Cd ap­plication (Table 3). The concentrations of other elements in the xylem exudates of B73 and N28 were either similar or slightly different (Table 4). Furthermore, calculations of the equilibrium concentrations of Cd complexes with citrate and malate, revealed the negligible role of these complexes in Cd transfer. Only 0.01 to 0.1% of the total Cd concentration was transported in a complexed form, because of the high Ca and Mg concentrations in the xylem sap (Table 4). From these calculations it may be concluded that Cd is transported in the xylem sap mainly as a free ion. This emphazises the minor role of Cd complexation in the explanation of the differ­ences in xylem sap (or shoot) Cd concentrations of the inbreds. The xylem loading process is therefore of particular importance to explain the variation in Cd concentrations in the xylem sap.

Cadmium adsorbed to the roots may be re­moved by a CaC12 solution (Rauser, 1987). The maximum amounts of Cd were removed within 10 min, when roots of Cd-treated plants were exposed to a solution of 50 mM CaC12 (Fig. 1). A similar period of time was reported by Rauser (1987) for roots of Agrostis and maize seedlings. Differences in Cd desorption between both groups of inbreds may partly be explained by the lower root Cd concentrations of 'non-shoot Cd excluders' compared to 'shoot Cd excluders' (Table 5). Only H99 did not support this expla­nation. At an external Cd concentration of 10 f.Lg Cd L -I, root Cd concentrations of the 'non­shoot Cd excluders' were comparable to those of the 'shoot Cd excluder' H99. However, the amounts of Cd desorbed from roots of the 'non­shoot excluders' were about one third to one fifth of H99 (Table 5). Thus, at similar total root Cd concentrations, 'shoot Cd excluders' dis-

played a different exchange ability compared to 'non-shoot Cd excluders'. Another possible ex­planation for the differential Cd desorption may be found in the relative amounts of Cd adsorbed to the roots. Table 5 shows that 'shoot Cd excluders' desorb about 15% of the total amount of Cd of the roots and that this percentage was independent of the level of external Cd supply. The amounts of Cd desorbed from roots of the 'non-shoot Cd excluders' were much lower and increased from about 4% to 9% when the external Cd concentration was increased. Cad­mium saturation of the media can not be the limiting factor to explain these differences bet­ween the inbreds (Fig. 1). This consistent differ­ence clearly indicates that even at a similar Cd content of the roots the amounts of Cd adsorbed to structural tissues and probably the distribution within the roots is different between 'shoot Cd excluders' and 'non-shoot Cd excluders'. This part is currently being investigated in more detail.

Acknowledgements

The help of 0 Gaborieau (ESA, Angers, France) and A Bobbink in the experiments is gratefully acknowledged. The seeds of the maize inbred lines were generously provided by D J Van Der Have Seeds BY (Kapelle, The Nether­lands).

References

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Reprinted from Plant and Soil154: 103-109, 1993.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 531-537, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-159

Micronutrient content in graminaceous and leguminous plants contaminated with mercury

J.J. LUCENA, L.E. HERNANDEZ, S. OLMOS and R.O. CARPENA-RUIZ Department of Agricultural Chemistry, Aut6noma University, 28049 Madrid, Spain

Key words: Graminaceae, iron, Leguminosae, manganese mercury, soil extractants

Abstract

Toxic residues produce pollution in soils and plants due to heavy metals. Mercury toxicity can cause disorders in the micronutrients status of plants. In this paper the uptake/ availability ratio for several micronutrients is evaluated in relation to the Hg contamination of plants. Samples of soils and plants were taken from a zone polluted by mercury. AAc-EDTA (Lakanen and Ervio) and AB-DTPA (Soltanpour and Schwab) methods were used to evaluate Hg and micronutrient availability in soils. Plants sampled in the field were classified as graminaceous and leguminous. Appropriate correlations have been found between Hg extracted by the Lakanen and Ervio method, and with the Soltanpour and Schwab method and Hg content by plant shoots, the relationship being more significant using the latter technique. Increased iron uptake was observed with high mercury contamination in the soil, although the availability of iron in the soils remained unaltered. The implications of these results are discussed in the context of a mechanism used by the plant to alleviate Hg toxicity.

Introduction

Mercury is highly toxic in the environment even when it is present at low levels, usually the form of the gaseous alkyl compounds (methyl and ethyl-mercury). Common Hg concentration in non polluted soils vary from 10 to 500 p.,g kg -l,

depending on the parent material. In soils, Hg occurs as oxides, phosphates and carbonates, and in reducing environments as sulphides (Langerwerff, 1972). Since these minerals arc rather insoluble, inorganic Hg is considered to have low mobility in soils; this mobility being reduced at high pH (Lindsay, 1979). Hg can also be adsorbed on to the surfaces of soil particles such as clay minerals, metal oxides and organic matter. Molecular sorption is also possible (Friburg, 1972). Substances containing sulphur (cysteine, thiourea) are the best complexing agents for mercury.

The total concentration of an element in the soil does not provide information about its

availability to the plant, since different soil factors can affect the movement of the element from the solid phase into the plant roots (Tiller, 1989). Several chemical extractants has been used for assessing the fraction of an element that is available for plants such as AAc-EDTA (am­monium acetate and ethylene-diamine tetraacetic acid) (Lakanen and Ervio, 1971) and AB-DTPA (ammonium bicarbonate and diethylene-tri­aminc-pentaacetic acid) (Soltanpour and Schwab, 1977). The former was accepted by FAO as an indicator of the availability of toxic metals for plants (FAO, 1972). Garate and Lucena, 1983, observed that lead extracted using the AAc-EDTA method correlated well with lead content in graminaceous plants. The AB­DTPA method is a modification of the Lindsay and Norvell test (1978), but includes the ex­traction of macronutrients. Thus, this method is considered as universal (Jones, 1991). Bingham et al., 1975, successfully used DTPA as an extractant to determine available Cd. Lucena et

532 Lucena et al.

a!. (1992) found a relationship between Hg content in plant shoots and Hg extracted by both methods that was independent of the total mer­cury in the soil.

Normal values of mercury found in non pol­luted plants ranges from 10 to 200 JLg kg -I DW, but those living near mercury mines contain 500 to 3500 JLg kg- 1 DW (Goldwater, 1976), or even up to 14000 JLg kg 1 DW (Siegel et a!., 1987). Mercury uptake by roots is usually easier from organic sources, such as pesticides, than from inorganic ones (Tiller, 1989). Also foliar absorp­tion is possible (Mosbaek et a!., 1988). A mecha­nism has been described for Hg toxicity action in plants which involves the binding of mercury by thiol groups of proteins, for example in o-amino­levulinic acid dehydratase, that regulates the synthesis of chlorophyll and other specialized linear tetrapyrroles found in plants (Prasad and Prasad, 1987).

Despite the high tolerance that some plant species have to Hg toxicity (Lucena et a!., 1992; Siegel et a!., 1987), mercury can interact with other metals, so the metal composition, and activity of metals in plant can be seriously modified. Very little has been reported about mercury-heavy metal interactions and even less about the relationship between mercury and the essential metals. In this paper the uptake/ availa­bility ratio for several micronutricnts is evaluated as a function of the Hg concentration in legumin­ous and graminaceous plants grown close to a highly polluted area.

Site 1

4Km

[SitE' 2

250m SitE' 3

ZonE' I

Cinebar mine

Toxic rE'siduE's 0 -dE'pasil

D- rE'd ox idE's t rE'aimE'nt plant

river

Materials and methods

Sampling

Soil and plant samples were taken in springtime from six different sites (plots) (Fig. 1 ). Sites 1 to 3 were chosen in the same riverside (Zone I), and sites 4 to 6 were on the banks of a different river (Zone II). Sites 3 and 5, in zones I and II, respectively were near the location where highly polluted residues has been accumulated until three months before sampling. Percolates from these deposits watered these sites for ten years. The gallery mine near site 4, and the red oxides treatment plant near site 3 were closed at least 10 months before the sampling time, so air pollution was absent in that time.

The surface of the plots varied among 50 to 250m 2 • For soil analysis, 7 to 15 subsamples from each site were taken from the first 15 em of soil. Concentration of elements were determined by sampling whole shoots of leguminous and graminaceous plants grown on the selected soils.

Soils

A basic characterization of the soils is shown in table 1. Standard analytical methods were used (M.A.P.A., 1983), except for mercury. Total mercury was determined using a modification of the method proposed by Delft and Vos (1988): 0.400 g of an air dried and sieved (c:fJ < 2 mm) sample was digested with a mixture of acids

ZonE' II ~ -sit"'4] ~ 300m

Toxic rE'siduE's 0 -SitE' 5 dE'posit

1.5 Km

SitE' 6

Fig. 1. Scheme of the localization of the six sampling sites

Micronutrients in Hg contaminated plants 533

Table 1. Chemical and physical characterization of the studied soils

Soil pH Organic CaC0 3, 4 matter

H 20 KCI (%)

Zone I 1 5.52 5.24 0 5.7 2 5.50 5.13 0 3.0 3 6.81 6.14 0 3.8

Zone II 4 7.11 6.50 0 7.8 5 7.68 6.99 3.1 5.7 6 6.30 5.85 () 3.3

(HCl: HN0 3 : HF, 1:3: 5) for 24 hours at 100°C in hermetic teflon digesters. Then samples were allowed to react with H 2 0 2 for 5 hat 100°C, and after cooling were filtered and made up 100 mL.

Available Fe, Mn, Cu, Zn, Cd and Hg were determined using the methods of Lakancn and Ervio (1971) and Soltanpour and Schawb, (1977). In the former, 10 g of soil was extracted for 30 min with 100 mL of an extractant solution containing 0.02 M EDTA (Titriplex III, Merck) in 0.5 M Acetic/Ammonium Acetate buffer (pH 4.65). The Soltanpour and Schwab extractant medium consisted of 0.01 M DTPA (Titriplex V, Merck) in 1 M ammonium bicarbonate buffer at pH 7.6.

Plants

Shoots of plant samples were carefully washed to elliminate the possibility of particle deposits, then oven dried at 60°C for 24 h and ground. Amounts of 1.000 g were treated with 20 mL 30% HN0 3 (from HN03 65% Merck, Hg con­tent less than 5 · 10 -?%) in tightly closed teflon reactors for 4 hours at 100°C. Samples were filtered and made to 50 mL with water.

Analytical determinations

Hg was determined in each soil and plant extract using a Perkin-Elmer 4000 Atomic Absorption spectrophotometer provided with Hydride generator MSH-20. Standards were prepared in the same matrix as samples to avoid interfer­ences and an excess KMnO 4 solution was added when DTPA or EDTA extracts were measured.

Texture Hg-total Clay Silt Sand (mg kg- 1 )

65 26 9 silt-loam <1 53 15 32 silt-loam 25 45 23 32 loam 781

37 33 30 clay-loam 288 31 25 55 loam 1462 31 29 40 loam 188

Fe, Mn, Cu and Zn were determined by A.A. using an air-acetylene flame and the appropriate hollow cathode lamps.

Statistics All digestions and extractions were prepared in triplicate. Correlation analysis was used to de­termine the relationships among the different parameters. (Steel and Tonie, 1980).

Results and discussion

Availability of elements in soils

Table 2 shows the results of available Hg, Fe. Mn, Cu, Zn and Cd in the soils of the 6 sites, all determined using both the AAc-EDTA and AB­DTPA methods. Available Hg content showed a high variability, ranging over 104 units, which did not correlate significantly with total mercury (Table 1 ). The greatest amount of available mercury was found in soil 3, whereas the most total mercury was present in soil 5.

Correlations were found between both extrac­tion methods (Table 3). In spite of the low number of data (six pairs), we observed that both methods show statistically significant correlation for Hg, Mn, Cu and Zn, but not for Fe (although there is a similar trend) and Cd. This could be expected, since both extractants contained chelating agents, and their behaviour in respect of divalent cations is similar at the same pH (Lindsay, 1979). As Ca and Mg are the most abundant divalent ions, they behave similarly to both divalent-metal EDTA or DTPA chclates

534 Lucena et al.

Table 2. Extractable Hg, Fe, Mn, Cu and Zn in the six soils studied (mg kg- 1 )

Hg Fe Mn Cu Zn Cd

AAc-EDTA Zone I

1 <0.01 524 383 4.8 5.0 0.06 2 <0.01 188 184 2.3 2.3 0.06 3 270 168 175 2.9 3.9 0.13

Zone II 4 0.024 760 104 6.6 11.1 0.13 5 0.173 595 240 23.0 110 0.50 6 0.021 221 164 3.6 4.1 0.10

AB-DTPA Zone I

1 0.039 174 78 3.1 3.8 0.12 2 0.108 64 23 1.2 1.8 0.12 3 203 53 35 1.4 2.0 0.06

Zone II 4 0.050 128 12 3.7 5.6 0.01 5 0.232 89 17 12.3 24.1 0.08 6 0.064 69 22 2.0 2.3 0.03

Table 3. Correlation parameters for elements extracted by the Lakanen and Erviii (x value) versus AB-DTPA methods (y). Numbers in parenthesis indicate correlation analysis calculated from the logarithm of data, when data ranged more than two orders of magnitude and significance was found for non-transformed data

Element Intercept x coefficient

Hg 0.062 0.754 1.000** (0.232) (0.816) (0.985**)

Fe 41.7 0.132 0.717 NS Mn -15.1 0.222 0.876* Cu 0.103 0.533 0.998** Zn 2.021 0.202 0.995**

( -0.007) (0.688) (0.986**) Cd 0.073 -0.015 0.057 NS

*, ** significant at 5 and 1% level respectively. NS =not significant.

over a wide pH range. However, Fe-EDTA and Fe-DTPA complexes were found to be pH de­pendent, since they are going to be affected in different way by the abundant divalent ions.

There was no correlation between any single metal concentration and that of mercury (Re­gression analysis not shown), so variations of Hg level in the soil do not appear to depend statisti­cally on the concentrations of the other metals.

Mineral content of plants

Table 4 presents the microelement concentration in shoots of graminaceous and legume plants. Cd concentrations were below the detection limit (<0.01 mg kg- 1 ) for all samples. The Hg results

were in agreement with the high Hg pollution expected in sites 3 and 5. In site 3 we observed that some plants, from the ditches that had been flooded with the effluents from the toxic de­posits, were necrotic and dried, but those taken for analysis did not show any toxicity symptoms. Thus, both plant types, graminaceous and leguminous took up high amounts of Hg (100 and 50 mg ·kg -I, respectively) without display­ing visual toxicity symptoms.

The regression analyses for the correlations between each micronutrient and the mercury content in plant shoots shows that Fe concen­tration in graminaceous and leguminous corre­lated significantly at the 1% level to the amount of Hg in shoots (r = 0.998 and r = 0.992 respec-

Micronutrients in Hg contaminated plants 535

Table 4. Concentrations of Hg, Fe, Mn, Cu and Zn in gramineous and leguminous plants from the six plots studied. Data in mg·kg- 1 DW

Hg Fe

Graminaceae Zone I

1 2.5 127 2 6.7 119 3 102 1226

Zone II 4 2.6 79 5 8.0 116 6 1.2 57

Leguminosae Zone I

1 1.8 95 2 3.0 92 3 56 486

Zone II 4 2.3 136 5 9.2 148 6 1.7 75

tively): The larger the concentration of Hg, the larger the accumulation of Fe. A correlation also exists between Mn and Hg concentrations in graminaceous (r = 0.925, significant to the 1% level), but no in legume. Our findings are in agreement with those of Barghigiani et al., 1988, that studied the accumulation of Hg and other metals in plants of Mt. Etna (Sicily). They observed an increasing relationship (almost linear) between bioconcentration (plant/ soil ratio of the metals) of Fe and of Hg, but not in

Mn Cu Zn

57 9 26 74 8 24

151 8 21

36 5 20 32 8 32 22 6 16

83 12 34 58 11 27 62 12 30

38 8 39 33 10 38 38 12 27

the case of Mn and of Hg. Those results showed that exists an accumulation of iron in shoots when Hg bioconcentration increased.

The potential toxicity of a soil is often ex­pressed in terms of the total amount of the toxic element in the soil. However, in agreement with other workers (Shaw and Panigrahi, 1986) our results indicate that this parameter does not give sufficient information about the degree of pollu­tion. Our data suggest there is a better correla­tion between Hg concentration in plant and in

Table 5. Correlation parameters for Hg and Fe concentrations in plant shoots (y) soil extracts (x). Numbers in parenthesis indicate correlation analysis calculated from the logarithm of data, when data ranged more than two orders of magnitude and signification was found for non-transformed data

Extractant Element Intercept x coefficient

Graminaceae AAc-EDTA Hg -3.69 0.37 0.972 * *

(0.708) (0.13) (0.370)NS Fe 639 -0.85 0.466NS

AB-DTPA Hg 4.15 0.48 0.997 * * (1.011) (0.45) (0.943) * *

Fe 705 -4.34 0.437 NS

Leguminosae AAc-EDTA Hg -0.91 0.204 0.988 * *

(0.59) (0.172) (0.576) NS Fe 258 -0.205 0.335 NS

AB-DTPA Hg 3.57 0.258 0.991 * * (0.88) (0.398) (0.955) * *

Fe 309 -1.423 0.422 NS

* * *significant at 5 and 1% level respectively; NS = not significant.

536 Lucena et al.

the extracts (Table 5). The best correlation was found with data derived from using the AB­DTPA method. Interestingly, correlations for other elements are not as significant, despite both methods having been proved useful for micronutrients over a wide range of soils. In our work, the amount of Fe in the plant was affected by Hg content. Thus, our data suggest that Hg is a more important factor in the behaviour of Fe in the plant than its availability in the soil. Similar results were found for Mn in graminace­ous, but not in legume.

In recent work, a redistribution of Mn was found to occur in some Lactuca species in relation to the Cd toxicity resistance (Garate et a!., 1992). The results indicated that a greater flux of Mn to the shoots might prevent the action of the Cd on the mangan-protein responsible for the photolysis of water during photosynthesis. With the present work, it is possible that we have identified another mechanism of resistance to a heavy metal toxicity. Since Hg forms com­plexes with thiol groups, it may compete for Fe sites in some enzymatic systems. A better redis­tribution of Fe from roots to shoots may alleviate this competition, so that a large accumulation of mercury in these plants would not result in the expression of toxicity symptoms. However this hypothesis needs to be proved through further studies.

Conclusions

As air pollution of Hg can not explain the differences of Hg accumulation among sites, we conclude that sites 3 and 5 present Hg contami­nation in the soil due to the presence in the past of toxic residues nearby. Plants grown in these sites obtain Hg mainly from the soil.

The results indicated that there was a correla­tion between mercury concentration in shoots and Hg extracted by the AB-DTPA method and, with less significance, using the AAc-EDTA extractant, but not with the total amount of Hg in the soil.

Also we have observed that Fe accumulates in the shoots of plants polluted with mercury. This may represent a resistance process in the plant to

avoid Hg toxicity. Further research, comparing these results with greenhouse experiments, is needed to prove this idea.

Acknowledgements

This work has been partly supported by C.I.C.Y.T. project AGR90-0286.

We thank Dr David Cook for his helpful review of the manuscript and correcting the english language. We also acknowlege the contri­bution of M F Camara for her technical assist­ance.

References

Barghigiani C, Bargagli R, Siegel B and Siegel S M 1988 Source and selectivity in the accumulation of mercury and other metals by the plants of Mt. Etna. Water, Air, Soil Pollut. 39, 95-408.

Bingham F T, Page A L, Hahler R J and Ganje T J 1975 Growth and cadmium accumulation of plants grown on a soil treated with cadmium-enriched sewage sludge. J. Environ. Qual. 4, 207-211.

Delft W and Vos G 1988 Comparison of digestion procedures for the determination of mercury in soils by cold-vapour atomic absorption spectrometry. Anal. Chim. Acta 209, 147-156.

FAO 1972 Micronutrients in Soils and Agriculture. F.A.O. Soil Bulletin No. 17. Rome. 74 p.

Friberg L 1972 Mercury in the Environment. CRC Press, Cleveland. 186 p.

Garate A, Ramos I and Lucena J J 1992 Efecto del Cadmio sobre la absorci6n y distribuci6n de Mn en distintas variedades de Lactuca. Suelo y Planta 2, 581-591

Garate A and Lucena J J 1983 Estudio del plomo en el sistema suelo-planta. Relaci6n con factures edaficos pH, textura y materia organica. Anal. Edafol Agrobiol. 42, 1111-1119.

Goldwater L J 1976 El mercurio en el medio ambiente. In Qufmica y Ecosfera. pp 383-390. Hemann Blume Eds, Madrid.

Jones J B Jr 1991 Universal soil extractants: their composi­tion and use. Commun. Soil Sci. Plant Anal. 21, 1091-1101.

Lakanen E and Ervio R 1971 A comparison of eight extractants for the determination of plant available mi­cronutrients in soils. Acta Agric. Scand. 17, 131-139.

Langerwerff J V 1972 Lead, mercury and cadmium as environmental contaminants. In Micronutrients in Agricul­ture. Eds. J J Mortvedt, PM Giordano and W L Lindsay. Soil Sci. Soc. Am. Madison. Wis., pp 593-630.

Lindsay W L 1979 Chemical Equilibria in Soils. John Wiley & Sons, New York 449 p.

Lindsay W Land Norvell W A 1978 Development of a DTPA soil test for zinc, iron, manganese and copper. Soil Sci. Soc. Am. J. 42, 421-428.

Lucena J J, Carpena-Ruiz R 0, Hernandez L E and Olmos S 1992 Disponibilidad de mercurio en suelos contaminados. IV Congreso Nacional sobre Nutrition de las Plantas. Alicante. pp 81-88.

Ministerio de Agricultura Pesca y Alimentaci6n 1983 In Metodos Oficiales de Am\lisis. Vol III. M.A.P.A. Spain. 532 p.

Mosbaek H, Tjell J C and Seve) T 1988 Plant uptake of airborne mercury in background areas. Chemosfere 17, 1227-1236.

Prasad D D K and Prasad A R K 1987 Effect of lead and mercury on chlorophyll synthesis in mung bean seedlings. Phytochem. 26, 881-883.

Micronutrients in Hg contaminated plants 537

Shaw B P and Panigrahi A K 1986 Uptake and tissue distribution of mercury in some plant species collected from a contaminated area in India: Its ecological implica­tions. Arch. Environ. Contam. Toxicol. 15, 439-446.

Siegel S M, Siegel B Z, Barghigiani C, Aratani K, Penny P and Penny D 1987 A contribution to the environmental biology of mercury accumulation in plants. Water, Air, Soil Pollut. 33, 65-72.

Soltanpour P N and Schwab A P 1977 A new soil test for simultaneous extract of macro and micro nutrients in alkaline soils. Commun. Soil Sci. Plant Anal. 8, 195-207.

Steel R G D and Torrie J M 1980 Principles and Procedures of Statistics. 2nd Ed. McGraw Hill, New York. 633 p.

Tiller K G 1989 Heavy metals in soils and their environmen­tal significance. Adv. Soil Sci. 9, 113-142.

I

Macronutrients and environmental stress

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 541-546, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-212

Effect of nutritional stress on photosynthesis rate of potato (Solanum tuberosum L.)

T. MEHOUACHI and R. LEMEUR Ecole Superieure d' Agriculture de Mograne, 1121 Zaghouan, Tunisie and Laboratory of Plant Ecology, University of Gent, Coupure links 653, 9000 Gent, Belgium

Key words: leaf nutrient concentration, net photosynthesis rate, nutrient stress, potato, Solanum tuberosum L.

Abstract

The objective of this research is to study the effect of nutritional stress of nitrogen (N), phosphorus (P) and potassium (K) on the photosynthetic assimilation of two potato varieties ('Spunta' and 'Desiree'). Six different levels of NPK concentration were chosen from the standard Steiner nutrient solution. The net photosynthesis rate was determined by measurement of C02-exchange in leaf cuvettes. Results showed that nutritional stress affects negatively the net photosynthesis and below a threshold nutritional level (dependent on the variety) the net photosynthesis reduced considerably. A correlation study between the N, P and K concentration in the leaves and the net photosynthesis was also carried out. The study showed that nitrogen had the greatest effect on net photosynthesis, whereas, potassium and phosphorus had less effect than nitrogen.

Introduction

The relationship between the photosynthesis and mineral nutrition has been studied in several species (Natr, 1972). In fact, net photosynthesis (Pn) has been proposed as a suitable criterion for optimal mineral nutrition because photosynthesis represents the basic process of new organic matter formation (Nghia et al., 1981 ). Reports of the effects of mineral nutrients on photo­synthesis are varied. Several studies have illus­trated a positive relationship between leaf nitro­gen concentration and photosynthesis in various species (Keuken et al., 1989; Khamis et al., 1990; Marshall and Vos, 1991). Information on the effect of phosphorus on rates of photo­synthesis is scarce. Sheriff et al. (1986) using Eucalyptus seedlings and Terry and Ulrich (1973) using Beta vulgaris found that decreasing phosphorus supply caused a decrease in Pn. Whereas, Osman et al. (1977) did not find this in leaves of Triticum aestivum. Net photosynthesis

was related to potassium status by Terry and Ulrich (1973) in Beta vulgaris. But Cao and Tibbitts (1991) and Osman et al. (1977) did not find this link respectively for Solanum tuberosum and Triticum aestivum.

For potato there is relatively little information on the relationship between the mineral ele­ments and the photosynthesis (Firman and Allen, 1988). Thus, the effects of mineral nu­trients on photosynthetic characteristics of potato leaves need to be examined for a fuller appreciation of the role of nutrients on the productivity of potato plants. In this study, we investigate, under controlled environmental con­ditions, the effects of nutritional stress on the net photosynthesis and the parameters defining the mathematical relationship between photo­synthesis rate and photon flux density (i.e. func­tional parameters of photosynthetic light re­sponse curve). The relationship between photo­synthesis and nutrients concentration of the leaves is also studied.

542 Mehouachi and Lemeur

Material and methods

Plant culture and treatment design

The experiment was conducted in three growth rooms using the nutrient film technique (NFT). The environmental conditions were controlled: a light period of 12 hours, a photosynthetic photon flux density of 300 11-mol.m- 2 .s-\ 21°C during the light and 1SOC during the dark, 65% of relative humidity.

Six treatments with N (nitrogen), P (phos­phorus) and K (potassium) were established. The mineral composition of nutrient solutions was based on the formulae by Steiner (1969) which represents, in this study, the control (T2). Composition changes were made in comparison with the control solution: solution T1 is consid­ered to represent excess of nutrients; while, T3 to T6 are considered to be a reduction series of NPK (the reduction being a factor of 2). Thus, the concentrations of these three elements, for the six treatments (T1 to T6), are presented in Table 1. Corresponding electric conductivities (measured at 25°C) are also given in Table 1.

Two potato varieties 'Spunta' and 'Desiree' were used in this experiment. Plants were grown in plastic pots (PVC) containing vermiculite. The nutrient solution was circulated continously through a pump at 60 L h-I. Every day, the electric conductivity and the pH (6.0) were checked.

Measurement of photosynthesis rates

Leaf gas exchange measurements were taken 60 days after planting on the fourth leaves from the tip of the main stem of each plant. These

measurements were carried out in the phytotron unit of the Laboratory of Plant Ecology. A detailed description of the unit has already been given by Lemeur (1991). Net photosynthesis rates were measured using a C02 gas analysis circuit (infra-red gas analyser, type ADC 225 MK 3 Differential modus) connected with three leaf chambers with environmental control (air temperature: 21 oc, relative humidity: 65% ). Photosynthesis of samples leaves is determined operating in the open system mode at different light intensity steps (obtained from a grid of high pressure mercury vapour lamps). Corresponding photosynthesis rates are derived, after the cali­bration of the corresponding instruments, from the C0 2 concentration difference between inlet and outlet of the leaf chamber. Finally, the photosynthesis rate was calculated following the Equation:

D(C0 2 ) x F 1 273.15 Pn = A x 22.4 x 273.15 + TL

Pr 100 X 1013 X 3600

Pn: the net photosynthesis rate (11-mol C0 2 m - 2

s -I), D(C0 2): the C02 concentration gradient be­tween the in- and outlet of the leaf chamber (L - 1

C0 2 L - 1 air), F: the flow rate (L h - 1 ),

A: the leaf area contained in the chamber (dm2),

TL: the leaf chamber temperature (°C), Pr: the atmospheric pressure (HPa), 100/3600: a correction factor to convert dm 2 of leaf area to m2 and L h- 1 to L s- 1 •

The values of the photosynthesis rate combined with the corresponding quantum flux density

Table 1. Electrical conductivity and nutrient concentration of the six treatments used in the experiment (T1: excess, T2: control and T3, T4, T5, T6: deficit)

Elect. conduct. Concentration (mmol L- 1)

(mss-') K+ NO~ H,PO~

Tl 2.70 28.8 2.0 14.0 T2 1.42 14.4 1.0 7.0 T3 0.90 7.2 0.5 3.5 T4 0.72 3.6 0.25 1.75 T5 0.54 1.8 0.12 0.87 T6 0.45 0.9 0.06 0.43

Effect of nutritonal stress on photosynthesis rate 543

gave the so called 'photosynthesis light response curve' which can be expressed following one of the equations described by Thornley (1976):

o:c (I- Ic) Pn(max) Pn=--~~~~~~~

o:c (I- Ic) + Pn(max)

Where: Pn: the net photosynthesis rate (11-mol C0 2 m - 2

s -1 ),

I: the photon flux density (11-mol m- 2 s- 1).

The functional photosynthetic parameters, de­rived from the statistical fit to the measured points, are: Pn(max): the maximum net photosynthesis rate (11-mol C02 m- 2 s- 1),

Lc: the light compensation point (11-mol m -z s -1), o:c: the quantum efficiency at the level of the light compensation point (11-mol C0 2m -zs - 1 /

11-mol m- 2 s- 1 )

Rd: the dark respiration rate (11-mol C0 2 m -z s -1).

Mineral analysis

The leaves near the fourth leaf were used for mineral analysis. After drying these leaves in the oven at 70aC during 3 days and after mixing, samples of dry matter were analyzed in the Laboratory of Analytical and Agrochemistry (University of Gent) using the methods de­scribed by Cottenie et a!. (1982). Total nitrogen (N) was measured by Kjeldahl method using salicylic acid-thiosulphate reduction of nitrate-N. Phosphorus (P) was determined by absorbance at 430 nm with a spectrophotometer (type CE 373 Linear Redout Grating). The potassium (K) was measured by emission in an air-propane flame at 768 nm with a flame-photometer (type appendorf ELEX 6361).

Results

The results, as illustrated in Table 2 and by Figure 1, show that net photosynthesis rates decreased rapidly with nutritional deficit. This is true for the low (100 11-mol.m -z .s- 1) and high (1000 fLmol.m- 2 .s- 1) light intensity. At severe

Table 2. Standard deviations of net photosynthesis rates at selected photon flux densities and for different nutrient levels (Tl: excess, T2: control and T3, T4, T5, T6: deficit)

Photon flux densities {!.tmol m- 2 s- 1 )

100 300 500 1000

'Spunta' Tl 0.14 0.67 0.75 0.79 T2 0.06 0.43 0.83 1.51 T3 0.09 0.59 1.03 1.72 T4 0.12 0.40 0.58 0.82 T5 0.40 0.83 1.05 1.30 T6 0.08 0.20 0.27 0.34

'Desiree' Tl 0.53 0.94 1.11 1.32 T2 0.33 0.27 0.84 1.78 T3 0.07 0.11 0.19 0.33 T4 0.13 0.48 0.72 1.01 T5 0.33 0.70 0.89 1.13 T6 0.09 0.12 0.12 0.13

deficiency the rates of photosynthesis were de­pressed considerably. For example, at T6 ( 0.45 mS. s - 1 ) the net photosynthesis rate is less than 10% compared to the rates found for control plants. On the other hand, the excess solution (Tl: 2.70mS.s- 1) increased Pn for 'Spunta' and 'Desiree' by respectively 8 and 14% in comparison with the control plants.

The comparison between varieties show that 'Spunta' gives higher values of Pn than 'Desiree', both at low and high levels of light intensity and the solutions T1 to T4. However, at severe deficiencies, in particular for the solution T5, 'Desiree' gives higher values of Pn. In com­parison with the control, the reduction of Pn with nutritional deficient, as illustrated by rela­tive deviations in Figure 2, is relatively more pronounced for 'Spunta' than 'Desiree'. For example, the reduction of Pn is 19 and 49% at respectively T3 and T5 for 'Desiree'. Corre­sponding values of reduction for 'Spunta' are respectively 27 and 75%. It can be observed also from Table 2 and Figure 1 that there was a great difference between Pn at control level (T2) of the two varieties studied. 'Spunta' gives 20% more than 'Desiree'.

The results of the nutritional stress on photo­synthetic parameters are presented in Table 3. These results show an increase of the maximum of photosynthesis (Pmax) and a decrease at nutritional deficit in comparison with the control.

544 Mehouachi and L emeur

25 rPn~(~u~m~ol~c~o~2/~m~2/~·~l---------------------------, '$pun ta'

20

15 ~~;

·: ~~:: ~---------o~~~~~==~======================_]T~6--~

-5 ~----~----~----~------~----~-----" 0 2 00 .. oo eoo soo 100 0 1200

Photon llux density ( "'mo1/m2/s )

Pn ( umol co2/m2/ s ) 20~~~~~~---------------------------,

-5L-----~----~----~~----~----~----~ 0 200 400 600 600 1000 1200

Pho,on flux density ( umol/m2/a )

Fig. 1. Light response curves of net photosynthesis rate of two potato varieties ('Spunta' and 'Desiree' ) under different levels of nutritional stress (Tl : excess, T2: control, and T3, T4, TS, T6) . Each light response curve represents the average of three replicates measurements. The standard deviations of the measurements are represented in Table 2.

( " )

- 'Spunla' fii\\ID 'Ohir~e· Fig. 2. Comparison between varieties based on the relative deviation ( % ) of the net photosynthesis rate of stressed plants compared to control plants .

The quantum efficiency (ac) decreased with nutritional deficit. At severe deficiency (T5 and T6) , the reduction of ac was higher with 'Spunta' than 'Desiree' . The values of the dark respiration were decreased as a result of decreas­ing electric conductivity (deficit) . This decrease was considerable at severe deficiency particularly with 'Spunta' . For light point compensation (Lc), there was a tendency that the nutritional deficit increases the values of this photosynthetic pa­rameter. But in our study we can not generalize this finding.

For further investigations of the link between photosynthesis and mineral nutrition, we also studied the relationship between leaf nutrient concentration and net photosynthesis rate using single linear regression. The values of Pn at 1000 p,mol m- 2 s- 1 were used because this light intensity represents, according to our opinion, a realistic level for natural conditions. Results, presented in Table 4, showed a strong relation between Pn and leaf N concentration of the two varieties (r2 = 0.97 et 0.91 ) . For phosphorus, the relation varied with variety. It is significant for 'Spunta ' (r2 = 0.85) but not significant fo r 'De­siree' (r2 = 0.41) . On the other hand, the rela­tionship between leaves K concentration and Pn was not significant both for the two varieties (r2 = 0.37 et 0.59).

Discussion

From the results mentioned above it follows that the net photosynthesis rate is strongly affected by nutritional stress in particularly with nutri­tional deficit. The decline of quantum efficiency and dark respiration might explain the reduction of Pn with nutritional deficit . The quantum efficiency is a measure for conversion of photon energy (mole photons) into biochemical energy (fixation of mole C0 2) . Hence, lowered values of quantum efficiency illustrate that the electron transport in chloroplasts of stressed plants is reduced , in light reactions, in comparison with control plants. Khamis et al. (1990) observed a small decline of the quantum yield for N-stressed plants of Z ea mays L. cv Contessa. The decline of dark respiration seems to be related especially

Effect of nutritonal stress on photosynthesis rate 545

Table 3. Values of 'Functional photosynthetic parameters' obtained from the light response curve of net photosynthesis rate under different levels of nutritional stress (excess: T1 and deficit: T3, T4, T5, T6; T2: control) of two potato varieties ('Spunta' and 'Desiree'). pmax; maximum of net photosynthesis rate; a,: quantum efficiency; L,: light point compensation; Rd: dark respiration

pm" a, (10- 2 ) L, Rct(l0- 1 )

(!Lmolco2 m- 2 s- 1 ) 2 (!Lmolco 2 m- 2 s- 1 ) 2 (1-'molm-'s- 1 ) (!-'mol co2 m 2 s -1)2

'Spunta' T1 28.1a 1 0.78 6.3a 0.60 7.5d 1.53 -7.1a 0.6 T2 26.1a 1.59 5.9a 0.15 8.4d 0.79 -6.3a 0.9 T3 17.7b 1.10 5.1b 0.17 10.4dc 1.58 -3.9b 0.7 T4 10.8c 1.93 3.6cd 0.92 10.6cb 0.33 -3.7b 0.6 TS 5.6d 1.71 2.3d 0.70 13.9a 2.78 -3.2b 0.2 T6 1.3e 0.47 0.6e 0.10 12.8b 1.93 -0.8c 0.1

'Desiree' T1 20.3a 1.80 6.9a 0.97 9.2d 0.56 -6.4a 0.9 T2 19.4a 1.69 5.6a 0.90 9.9d 0.15 -6.7a 0.9 T3 14.3b 0.64 5.3b 0.95 9.6dc 0.20 -6.0ab 1.2 T4 10.1cd 1.09 3.9cd 0.14 13.0cb 1.20 -5.1bc 0.6 T5 lO.Od 1.76 3.2d 0.63 15.4a 2.87 -4.6c 0.2 T6 1.2e 0.14 l.Oe 0.20 12.1b 2.92 -l.Od 0.1

'Significant differences among means by Duncan's Multiple Range Test, 5% level.

Table 4. Equations of single regression between the net photosynthesis rate and leaf nutrient concentration (N, P, K) of the potato varieties ('Spunta' and 'Desiree'). y: P" in !-'mol C02 m- 2 s- 1 ; x: nutrient concentration in % of leaf dry matter

Equation of regression

'Spunta' N y= -11.1164+4.2982x p y = -44.3694 + 87.1697 X

K y = -41.8589 + 7.4321 X

'Desiree' N y = -9.9251 + 3.5261 X p y = -22.3251 + 47.6242 X

K y = -24.2784 + 4.8765 X

**: significant at 0.01; ns: not significant.

2 r

0.97** 0.85** 0.37 ns

0.91** 0.41 ns 0.59 ns

to deficiency of N and Pin the leaves (Bottrill et al., 1970). The observed reduction of the maxi­mum photosynthesis, resulting from nutritional deficit, means that the biochemical reactions of the photosynthetic assimilation are inhibited.

Results of this research showed in particular a strong relation between Pn and the N concen­tration in the leaf. This is in agreement with many authors (Keulen et al., 1989; Khamis et al., 1990; Marshall and Vas, 1991). In a pot experiment with potato, Vas and Oyarzun (1987) observed a direct relation between photo­synthetic capacity and the concentration of N in

leaf dry matter. Decreased rates of photo­synthesis with nitrogen deficit could reflect de­creases in chlorophyll content (Chapin et al., 1986). Also, the dark reactions (biochemical reactions) could be disturbed by N deficiency. In fact, it was found that the protein content, especially the ribulose 1,5-bisphosphate carbox­ylase/oxygenase (RUBISCO) which constitutes the most important protein in leaves of c3 plants, decreased with N deficiency (Millard and Catt, 1988). In this study, the effects of phos­phorus on Pn depends on variety. This is in agreement with many reports cited in the litera­ture (Natr, 1972). It seems that diminished rates of Pn with a decrease of P concentration in the leaves can be associated with decreased rates of production of ATP and NADPH by the light reactions of photosynthesis. Evidence that P deficiency affects ATP and NADPH was ob­tained by other authors (Robinson and Giersch, 1987). For potassium, the relationship between leaf K concentration and Pn was not significant in this study. Cao and Tibbitts (1991) found that gas exchange measurements in situ on leaflets of Solanum tuberosum L. demonstrated no signifi­cant differences among different K treatments in C0 2 assimilation rate. In fact, it has been shown that K deficiency has an indirect effect on the photosynthesis. Several authors (Fisher, 1971;

546 Effect of nutritonal stress on photosynthesis rate

Humble and Hsiao, 1970) suggested that move­ments of K ions into the guard cells could play a decisive role in increasing their turgor and conse­quently the stomata aperature. Terry and Ulrich (1973) found a decrease in Pn of K deficient Beta vulgaris leaves as due to the decrease in both stoma aperture and rate of internal C0 2 fixation.

This study show also that the variety 'Spunta' gives higher values of Pn than the variety 'Desiree'. But, at severe deficiency (O.SmS.s- 1),

'Spunta' appeared to be more strongly damaged resulting in lower values of Pn (1983).

Acknowledgement

The authors would like to express their thanks at Prof Dr ir M Verloo for laboratory facilities m mineral analysis.

References

Bottrill D E, Possingham J V and Kriedemann P E 1970 The effect of nutrient deficiencies on photosynthesis and respi­ration in spinach. Plant and Soil 32, 424-438.

Cao W and Tibbitts T W 1991 Potassium concentration effect on growth, gaz exchange and mineral accumulation in potatoes. J. Plant Nutr. 14, 525-537.

Chapin Ill F S, Shaver G R and Kedrowski 1986 En­vironmental controls over carbon, nitrogen and phosphor­us fractions in Eriophorum vaginatum in Alaskan tussock tundra. J. Ecol. 74, 167-195.

Cottenie A, Verloo M, Kiekens L, Velghe G and Camerlynck R 1982 Chemical analysis of plants and soils. Laboratory of Analytical and Agrochemistry, University of Gent, Bel­gium.

Firman D M and Allen E J 1988 Field measurements of the photosynthetic rate of potatoes grown with different amounts of nitrogen fertilizer. J. Agric. Sci., Camb 111, 85-90.

Fischer R A 1971 Role of potassium in stomatal opening in the leaf of Vicia faba. Plant Physiol. 47, 555-558.

Humble G D and Hsiao T C 1970 Light- dependent influx and efflux of potassium of guard cells during stomatal opening and closing. Plant Physiol. 46, 483-487.

Keulen H Van, Goudrian J and Seligman N G 1989 Model-

ling the effect of nitrogen on canopy and crop growth. In Plant canopies: Their growth, Form and Education. Eds. G Russel, B Marshall and P G Jarvis. pp 83-104. SEB Seminar Series 31, Cambridge University Press, Cam­bridge.

Khamis S, Lamaze T. Lemoine Y and Foyer C 1990 Adapta­tion of the photosynthetic apparatus in maize leaves as a result of nitrogen limitation. Relationship between electron transport and carbon assimilation. Plant Physiol. 94, 1436-1443.

Lemeur R 1991 Measurement of photosynthesis: Method­ology and interpretation. Note 1. Laboratory of Plant Ecology, University of Gent, Belgium.

Marshall B and Vos J 1991 The relation between the nitrogen concentration and photosynthetic capacity of potato (Solanum tuberosum L.) leaves. Ann. Bot. 68, 33-39.

Millard P and Catt J W 1988 The influence of nitrogen supply on the use of nitrate and ribulose 1 ,5-bisphosphate carbox­ylase/oxygenase as leaf nitrogen stores for growth of potato (Solanum tuberosum L.). J. Exp. Bot. 39, 1-11.

Natr L 1972 Influence of mineral nutrition on photosynthesis of higher plants. Photosynthetica 6, 80-99.

Nghia P T N, Natr L and Fialova S 1981 Changes in photosynthetic rate of spring barley induced by removal of nitrogen or phosphorus deficiency. Photosynthetic 15a, 216-220.

Osman AM, Goodman P J and Cooper J P 1977 The effects of nitrogen, phosphorus and potassium on rates of growth and photosynthesis of wheat. Photosynthetica 11, 66-75.

Robinson S P and Giersch C 1987 Inorganic phosphate concentration in the stroma of isolated chloroplasts and its influence on photosynthesis. Aust. J. Plant Physiol. 14, 451-462.

Sheriff D W, Nambiar E K S and Fife D N 1986 Relation­ships between nutrient status, carbon assimilation and water use efficiency in Pinus radiata (D.Don) needles. Tree Physiol. 2, 73-88.

Steiner A A 1969 Enkele problemen van het wortelmilieu van de plant. Vakblad voor Biologen, 61-66.

Terry Nand Ulrich A 1973 Effects of potassium deficiency on the photosynthesis and respiration of leaves of sugar beet. Plant Physiol. 51, 783-786.

Terry N and Ulrich A 1973 Effects of phosphorus deficiency on the photosynthesis and respiration of leaves of sugar beet. Plant Physiol. 51, 43-47.

Thornley J H M 1976 Mathematical models in plant physi­ology. Acad. Press. London, 318 p.

Vos J and Oyarzun P J 1987 Conductance of potato leaves: effects of age, irradiance and leaf water potential. Photo­synth. Res. 11, 253-264.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 547-554, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-029

Reactions of three soybean cultivars to interruptions in phosphorus supply

HERMINIA E.P. MARTINEZ\ ROBERTO F. NOVAIS 2 , LUCIANA A. RODRIGUES 3 ,

LUIS V.S. SACRAMENT0 4 and ROBERTO A.R. JUNIOR5

1Crop Science and 2Soil Science Department, Federal University of Vifosa (UFV), 36570 Vifosa, MG, Brazil, 3Forestry Engineering, 4Soil and Plant Nutrition, UNESP, 18600 Botucatu, SP, Brazil and 5 UFV, Vifosa, MG, Brazil

Key words: phosphorus absorption, phosphorus partition, phosphorus stress, soybean cultivars

Abstract

Three soybean cultivars (Santa Rosa, Doko and UFVl) were submitted to 0, 4, 8 or 12 days of P omission periods in nutrient solution. Following the omission treatments, the plants were returned to a complete solution until they were 36 days old. Dry matter (DM) yield, phosphate content (PC), plant P-concentration (P% ), inorganic-P (Pi) and organic-P (Po) concentrations in fresh material were determined. Short P-omission periods affected DM and PC only slightly, and longer ones caused a reduction on these. Roots supported longer P-omission periods without losses in DM than shoots. With longer P-omission periods, DM yield decreases were followed by Pi and P% increases at the basal and intermediate leaves. The variations of root parameters were smaller than those of the shoots, showing that homeostasis is maintained for longer periods of time in roots when compared with shoots. Small differences were found between the genotypes.

Introduction

Phosphorus diffusion plays an important role in P-dynamics at the soil/root interface and in plant P-absorption. Soil water content is one of the most important factors regulating soil P-diffu­sion.

Phosphorus-diffusion in pure water is more than 200 times greater than that observed in soil with a high P-concentration in the soil solution, and even 2.105 times higher when a soil poor in P is considered (Bieleski, 1973). Small fluctua­tions in soil water content can be accompanied by pronounced variations in P uptake.

It is well known that P-stressed plants increase their P-absorption capacity (Clarkson, 1985; Faquim et a!., 1990; Lefebvre et a!., 1990; McPharlin and Bieleski, 1989) by modifying their kinetic parameters (Km, Vmax and Cmin) or by morphological adjustments, such as shoot/root ratio (Romer et al., 1989). The response is under

genetic control (Schj0rring and Nielsen 1987). In addition, the P-pool size in, and remobilization rates from the vacuoles into the cytoplasmic organic-P may differ in various genotypes.

Assuming that the type and the degree of these adjustments are under genetic control it should be possible to select genotypes which compensate short drought-induced P-stress periods more efficiently than others. Conse­quently a smaller reduction in their final yield would result.

This study investigated the effect of exposing three soybean (Glycine max L. Merrill) cultivars to various P omission periods followed by re­supply, on the yield, P content and concentration and P-fractions in the tissue.

Material and methods

Three soybean cultivars (Santa Rosa, Doko and

548 Martinez et al.

UFVl) were grown for 12 days in pots con­taining eight and a half liter of a modified Clark nutrient solution (Clark, 1975), containing 69 f.tmol L -l P. Subsequently, they were sub­mitted to P omission periods of 0, 4, 8 or 12 days. After the P-starvation the plants were transfered back to the initial complete solution until they were 36 day old. Two plants were maintained in each pot. The nutrient solution was renewed in 4 day intervals. The pH was adjusted daily to 5.0 ± 0.5. The upper, inter­mediate and basal leaves, stems and roots of one of the plants were harvested separately. The plant parts were rinsed in water, oven dried, weighed, grounded and analysed for P.

Tissue P-fractions were determined in all parts of the second plant from each pot. One gram of tissue was soaked in 0.2 N HC10 4 and cen­trifuged at 5000 g, according to the acid ex­traction procedure described by Smillie and Krotkov (1960), as modified by Hogue et a!. (1970). Inorganic fraction (Pi) was determined in the HClO 4 extract.

The acid soluble P-fraction (Pts) was deter-

40

150

30

100

10 .... )(,

.__ -- -· ~· :.·~·~ ·..: ·--·<

mined after destruction of the HClO 4 extract with nitroperchloric acid. The organic P-fraction (Po) was calculated as the difference between Pts and Pi. All P-determinations were made colorimetrically by reduction of phos­phomolybdate with ascorbic acid. Phosphate content (PC) was calculated from plant P con­centration (P%) and dry matter (DM) yield data. All data were submitted to analyses of variance and regression. Twelve regression models were tested. The model with level of significance greater or equal to 10% of probab­ility, and with greater R2 was selected.

Results

For the Santa Rosa cultivar, total plant, shoot, leaf and stem DM yield increased with short P-omission periods, decreasing with longer ones. The greatest DM yield occurred after about two days of starvation. For Doko cultivar, total, leaf and shoot DM yield decreased linearly; stems presented the same trend observed for Santa

1.0

00 If-·--· -{;1· --· --{lf-. --. 00 1-------'----'----'--' 0.0 L---L---"--....:::..J

12

1000

~ ~

~ 500<~-----e-

OOL---~----~--~0~12 P-Omission (days)

1000

" .! sao 0 ~

12

12

P-Omission (days)

0

P-Omission (days)

0 _ Upper leaves

0 -- Intermediate leaves

~---·- Basal 1~::i:lves 'V --·-· Shoots

I2J --·- Roots

Y -···- Total plant X · Leaves

e--- Stems

12

Fig. 1. Dry matter yield (OM yield-A), phosphorus content (PC-B), dry matter P concentration (P%-C), inorganic-? (P,-D) and organic-? (P0 -E) in parts of Santa Rosa cultivar soybean plants as function of the duration of the ?-omission period.

40

150

30

~ 100

?::~:~~-:-:.~ 1ol .,~.~.~.~•.o.~.o ~ lr---~·~·

,.... .. 20 ";0

~

I<: 50

00 P--·--. 1!-· -fl!·--·-,~ 00

l2

1000 1000

i i "" M "" M 500 " " (!.

500

~

00

12

P-Omlssion (days)

Reactions to interruptions in phosphorus supply 549

··~-... ~ -.. -. 'V .. _ ... ,

.Y.. 'V ~

·'"·---~·"c·~·: ~~ ·- -,-

-·--.$-. 7!1-· --·-

B

12

l

12

P-Omission (days)

l.O ,·--·--J?J

00

o-­o ---6--­'V- -· ¢--­~- -X . ----

'•. ',,

~·,

P·Ornission (days)

Upper leaves

Intermediate leaves

Basal leaves

Shoots

Roots

Total plant

Leaves

Stems

12

Fig. 2. Dry matter yield (DM yield-A), phosphorus content (PC-B), dry matter P concentration (P%-C), inorganic-? (P,-D) and organic-P (P0 ·E) in parts of Doko cultivar soybean plants as function of the duration of the ?-omission period.

40

30

: , ~ 2d

, .2. .. - ~.- .,

- ~ .. - '"'' • 10 .. ~~·"''"'"·~~·~·-· ,..,._."~·".

~-. --·0'--. --. r;Y--- ---00 A

12

1000

i " 3

500

~

00

12

P-Omission (days)

i "' "' "

(!.

150

100 ~

50 X .......

X

if:::.:.·.: ~l(i..:.:: :_--:~-- -. -_-_

00 L---~--~-~~ 12

1000

500

00

0 12

.. P-Omission (days)

l.O

!': 0.5 v ·-·-~ ~--=--~~~·~--~ rr - - ----~-----

00

12 P-Omission (days)

o-- Upper leaves

0 -- Intermediate leaves

6-- Basal leaves

V'- - Shoots

¢ --·- Roots

~ _.,,_ Total plant

X Leaves . ---- Stems

Fig. 3. Dry matter yield (DM yield-A), phosphorus content (PC-B), dry matter P concentration (P%-C), inorganic-? (P,-D), organic-P (P"·E) in parts of UFVl cultivar soybean plants as function of the duration of the ?-omission period.

Tabl

e I.

Dry

mat

ter

yiel

d (O

M-g

), P

-con

cent

rati

on (

P%

) an

d P

-con

tent

(P

C)

regr

essi

on e

quat

ions

and

R2

for

uppe

r le

aves

(U

L)

inte

rmed

iate

lea

ves

(IL

), b

asal

lea

ves

(BL

),

tota

l le

aves

(L

), s

tem

s (S

), s

hoot

s (S

H),

roo

ts (

R),

and

tot

al p

lant

(T

P)

of s

oybe

an p

lant

s as

fun

ctio

n of

the

dur

atio

n of

the

P-o

mis

sion

per

iod

Sant

a R

osa

R'

OM

Yie

ld

L

Y ~ 1

0.2

+ 3

.355

fX-

1.1

88

"X

81.9

5 s

Y ~ 8

.5 +

2.5

33 fx

-o

.93

7"X

52

.69

R

Y ~ 2

.5 +

0.3

37

X-

0.0

27

''X2

99.8

9 S

H

Y ~ 1

8.7

+ 5

.88

8fX

-2

.12

5X

" 69

.70

TP

Y

~ 2

1.1

+ 7

.02

0fX

-2.

463X

**

81.2

9

P%

U

L

Y~Y~0.73

IL

Y ~ 0

.46

+ O

.Oll

X*

" 74

.96

BL

Y

~ 0

.41

-0.

016C

+ 0

.003

X2'"

98.0

2 s

Y~Y~0.47

R

Y ~ 0

.72

+ 0

.04

7X

-0.

004X

2*

95.7

PC

L

Y

~ Y~ 5

5.5

s Y

~ 3

9.5

+ 1

4.9

05

fX-

5.4

30

X"'

95

.65

SH

Y

~ 9

1.6

+ 2

9.92

3 fx

-w

.o54

X"

73.2

3 R

Y

~ 1

7.5

+ 3

.43

9X

-0

.30

-2X

2"

92.4

5 T

P

Y ~ 1

09.3

+ 4

2.1

08

fX-

96.5

6 13

.662

X**

*

', "

, "'

Sig

nif

ican

t at

p <

0.1

0; p

< 0

.05

and

p <

0.0

1.

DO

KO

Y ~ 1

2.3

-0.

298*

*X

Y ~ 1

0.5

+ 2

.39

0fX

-0

.92

2X

" "Y~Y~2.7

Y ~ 2

3.7

-0

.55

0X

" Y

~ 2

6.4

-0

.55

5X

"

"Y~"Y~0.70

"Y~Y~0.47

Y ~ 0

.40

-0

.06

8fX

-0

.03

4X

" "Y~Y~0.42

Y ~ 0

.94

+ 0

.15

lfX-

0.0

77

X"

Y~Y~45.7

Y~48.6+ 1

1.3

9fX

-4.8

9X

'**

"Y~"Y~98.6

Y ~ 3

0.0

+ 2

.931

X +

0.1

55X

2*

"

Y~ 1

40

.7-3

.49

8X

"'

R'

UF

V1

71.4

5 Y

-Y9

.9

50.8

2

51.8

7

~ "Y~ 9

.4

~ 3

. 7 +

0. 7

09 fX

-0

.29

1X

' ~"¥~19.3

59.5

4 "Y

~ "Y

~ 22

.7

98.7

4

77.0

3

"Y~"Y~0.64

y ~ y

~ 0

.46

Y ~ 0

.38

+ 0

.006

X +

0.0

09X

2**

Y

~ 0

.46

-O

.OlO

X"

Y ~ 0

. 78

-0.

015X

*

Y~Y~SO.l

64.4

8 "Y

~ "Y

~ 38

.1

"Y~"Y~ss.2

91.3

6 71

.16

Y ~ 2

7.7

+ 6

.43

5fX

-2

.91

9X

"'

"Y~"Y~ll1.8

R'

79.4

6

98.4

5 87

.09

91.4

8

90.9

9

Ul

Ul

0 ~ ~- ;:: ~ ~ ¥2-

Reactions to interruptions in phosphorus supply 551

Rosa, and roots did not present significant vari­ation. For UFV1 cultivar, there was not signifi­cant variation in DM yield of all aerial plant parts, although the scattered points had the trend described for Santa Rosa (Figs. 1a, 2a, 3a, Table 1). Curves for PC content had similar patterns to those for DM yield. Only leaves of Santa Rosa, and leaves, shoots and roots of Doko presented differences in the curve fit (Figures 1b, 2b, 3b, Table 1). Roots of Santa Rosa and Doko presented the smallest variation in DM yield and in PC (Figs. 1a, 1b, 2a, 2b, 3a, 3b, Table 1).

Higher PC and DM yield of Santa Rosa shoots were not correlated with tissue P%, whereas for roots this did happen. Root P% curve was similar to root DM yield, with maximum values between 5,5 to 6,0 days (Figs. 1a, 1c, Table 1). For all cultivars, a decrease in root P% was followed by an increase in basal leaf concen­tration, whereas higher DM yield was related to lower basal leaf P% (Figs. 1a, 1c, 2a, 2c, 3a, 3c, Table 1). For Santa Rosa cultivar, the decrease in root P% followed by increase in basal leaf P% occurred after a longer period of P-omission (about five days) than for Doko (about one day). For UFV1 this pattern occurred with all range of P-omission periods. There was no significant variation in P% of intermediate leaves for Doko and UFV1, and for Santa Rosa increases were linear. P% in stems did not vary significantly for Santa Rosa and Doko, but decreased linearly for UFV1 (Figs. 1c, 2c, 3c, Table 1).

For all cultivars, there was great variation in Pi-fraction, especially in basal leaves, with de­creases with short P-omission periods followed by sharp increases with longer ones. It is im­portant to emphasize that the lower Pi concen­trations coincided with the higher DM yield, while the higher Pi concentrations correlated with the smaller DM yield for longer P-omission periods. This same trend was observed for inter­mediate leaves, but variation was less pro­nounced. No significant variation in Po was verified for any cultivar and plant part, except for upper leaves and stems of Santa Rosa. In general, variation in P-fractions due to treat­ments was small in upper leaves and roots and was greatest for the basal and intermediate leaves (Figs. ld, le, 2d, 2e, 3d, 3e, Table 2).

~8~~ ~§~~

(€~~~ re~g~

00-.:tt"''O\

~~~~~ ....... -....... --II II II II II 1~1~1~1~1~

II II II II II <~ <>- <~ <>- <:>c

0\~V-

Q~r--:rf'i~ OI.OVNN N---0\ II II II II II

1).; 1;>c 1)-o 1;>c 1;>c II II II II II

<:>c <:>c <:>c <:>c <:>c

-0 0 v

"'

552 Martinez et al.

Discussion

Differences in DM yield and PC were not significant for the different cultivars, probably due to their narrow genetic basis. For all cul­tivars, total DM yield and PC were correlated. Higher shoot and root DM yield was followed by higher PC. Differential response patterns were verified for shoot and root (Figs. 1a, 1b, 2a, 2b, 3a, 3b, Table 1) probably due to their different internal homeostasis mechanisms along different P-omission periods.

For Santa Rosa cultivar, shoot DM yield and PC were increased with two day P-omission periods. For roots, this period was of six days. In short P-omission periods (around two days), variation in root DM yield was small, but total plant PC increased considerably, indicating greater P-absorption per root weight unit (Figs. 1a, 1b, Table 1). If the greater absorption resulted from kinetic absorption parameter modulation or from plant shoot/root adjustment it is not clear, because root length or surface area were not determined. Results obtained by the author (Martinez et al., 1991), for the same soybean cultivars, showed that P-starved plants had greater P-absorption Vmax. This higher P absorption can result in plant DM yield recover or not depending on the degree of P-starvation.

Doko cultivar presented, in general, linear decrease in total plant, shoot and leaf DM yield, and PC with P-starvation. However, scattered points showed the same trend observed for Santa Rosa cultivar (Figs. 2a, 2b, Table 1).

Despite few significant response curves relat­ing UFV1 plant performance and P-omission periods, for this cultivar, the results presented the same variation pattern of the other cultivars (Figs. 3a, 3b, Table 1).

The greatest PC and total DM yield was not related to greater tissue P-concentrations (P%, Pi and Po). For Santa Rosa, the greatest P­absorption correlated with the lowest basal leaf Pi and P%, which occurred at about 2.5 to 3.0 day omission period (Figs. 1a, 1b, 1c, 1d, 1e, Tables 1, 2). Thus, vacuolar P-fraction (Pi) may not be a good plant indication about P-status, at least in basal and intermediate leaves (Figure ld, Table 2). Hart and Collier (1991) obtained high correlation between DM yield and Pi in upper

leaves for white clover and lotus, but not for ryegrass. In the present work DM yield of all cultivars correlated with Pi concentration of the upper leaves (Figs. 1a, 1d, 2a, 2d, 3a, 3d, Tables 1, 2).

For Santa Rosa, DM yield increased with two day P-omission. At the same time, total PC also increased, suggesting that short stress periods causes an enhancement in P-absorption. On the other hand, Po (cytoplasmic-fraction) of the upper leaves and Pi (vacuolar-fraction) of the basal and intermediate leaves decreased. Sup­posing that Po in upper leaves was in an exces­sive amount without any P-omission, this de­crease in connection to basal leaves Pi mobiliza­tion could result in enhancement of P-utilization efficiency (Figs. 1d, 1e, Table 2). According to several authors reported by Pereira (1991), high cytoplasmic-P concentrations can cause a de­crease in photosynthesis and modify the partition of carbohydrate in photosynthetic and glucogenic tissue. However, a casual variation need to be considered because P% was the same in both cases, no omission or two days P-omission.

For Santa Rosa, increased greater P-omission periods up to about six days resulted in root DM yield increases, while shoot DM yield decreased. This indicates an adaptation of roots to explore a greater surrounding volume, and occurs together with greater absorption rates per root weight unit. However, final total DM yield and PC were not recovered with longer P-omission periods. P-omission periods greater than six days resulted in DM yield and PC decreases in shoots and roots of Santa Rosa cultivar, showing that the adjustment mechanisms failed in compensating previous losses (Figs. 1a, 1b, Table 1). Burns (1987) omitted N, P and K supply to lettuce plants and observed relative growth rates (RGR) decreases when these nutrients in the tissue fell below their critical levels. Nutrient resupply aided in RGR recover at the level of the un­stressed control plants, but DM losses were not totally recovered at the end of the plant cycle.

Roots presented several differences in their responses to P-omission/reposition when com­pared with shoots. For Santa Rosa, root DM yield decrease was smaller than that of shoot, and with 12 days P-omission period was 7.6% higher than the value obtained without P-omis-

sion. Maximum root DM yield, PC and P% concentration occurred after, approximately, same omission periods, and the shapes of the curves relating these parameters and time of omission were also similar. Variation of all mea­sured parameters for roots was lower than for shoots, indicating that their growth is not so variable with omission treatments (Figs. 1a, 1b, 1c, Table 1). Wieneke (1990) observed in sor­ghum plants submitted to different P rates that PC in roots was less affected than that of shoots. The author considered that the maintenance or enhancement in root PC during P-starvation is due to photosynthate partition alterations in favor of roots. According to that work, Pi could be supplied to the root through the phloem, together with photosynthates.

Variation in shoot DM yield for Doko was also lower than in root DM yield, although the relationship between DM yield, PC and P% of root was not clear in this case. For UFV1, root DM yield, PC and P% were correlated, but the root DM yield was not clearly favoured in relation to shoot growth.

Partition of the absorbed P was modified by the degree of plant P-stress. For Santa Rosa, without P-omission, 16.3% of the total P was allocated in the roots, and the mean value increased to 23.1% with 8 day P-omission, fol­lowed by a subsequent decrease to 16.1% with 12 day P-omission. For Doko, relative root P­allocation decreased from 21.5% without P­omission to 16.1 with four day omission period. For greater omission periods, P-allocation re­mained nearly constant. Up to eight day omis­sion period, the amount of P allocated in roots of UFV1 remained around 22.3%, decreasing to 16.7% with 12 day P-omission. Apparently homeostatsis in the roots was maintained over that of shoots. However, after a certain degree of stress (omission periods longer than eight days for Santa Rosa and UFV1, and four days for Doko), plant tops became preferential sinks for the absorbed P. This agrees with the fact that root P% decrease corresponds to basal and in low degree to intermediate leaf P% increase. However, the mechanism through which root and shoot P-concentration act on the partition of the absorbed Pis an open question. Nerson eta!. (1987) studied P-stress recovery in muskmelon

Reactions to interruptions in phosphorus supply 553

plants grown under N or P defficient nutrient solutions for a period of four weeks. It was verified that after placing the P-stressed plants to the solution with normal P content, the plants showed old leaf expansion before producing new ones. The same did not occur with theN-stressed plants. Therefore, P-deficiency may cause basal leaves to be preferential sinks for photosynthates and P.

With respect to phosphate fractions, it is evident that the increase in P% of basal leaves is due to a sharp increase in Pi, and, to a lesser degree, in Po. Intermediate leaves followed the same trend, however with lower increases than basal leaves (Figs. lc, ld, le, 2c, 2d, 2e, Table 1, 2).

There is evidence in the literature that basal leaves are the major sources of Pi during P-stress periods. For soybean plants it seems to be true. Martinez et a!. (1990) determined P-fraction kinetics in four soybean cultivars, including Santa Rosa and UFVl. Twenty nine day old plants were submitted to 0, 1, 2, 4 and 8 day P-omission periods, and the P-fractions Pts, Pi and Po were determined in all plant parts at the end of each omission period. For all plant parts of all cultivars there was a sharp decrease in Pi levels. This decrease was most evident for roots and basal leaves (around 40% after two day P-omission). Nevertheless, after certain period of omission, basal leaves seem to act as major sinks of Pi, as demonstrated in the present study. Similar results were observed by Cogliatti and Clarkson (1983) in P stressed potato plants. The data now presented allows the inference that the cytoplasmic P-pool size in the metabolically active plant parts (upper leaf Po) somehow influences the amount of P transported to the shoots, and the sink/ source relationships of the basal leaves. On the other hand, it is clear that in shoots, the events related to the P-internal concentration recovery precede those of the growth recovery.

Katz et a!. (1986) related high P-absorption rates per unit of root DM of tomato plants submitted to P-omission periods of less than one day. According to these authors, plants sub­mitted to longer P-omission periods attained higher P-tissue level, and despite of that, their P-absorption rates were higher than those ob-

554 Reactions to interruptions in phosphorus supply

served in plants submitted to short omission periods. Those results show that under greater P-stress, there is a fail in the repression of the absorption mechanisms, even when high tissue concentration is attained. Thus, metabolically driven processes seem to be involved in the regulation of absorption rate.

Acknowledgements

The authors thank the 'Conselho Nacional de Desenvolvimento Cicntifico e Tecnol6gico' -CNPq for the financial aid, and the Soil Science Department of the Federal University of Vi<;osa for use of their facilities.

References

Bieleski R L 1973 Phosphate pools, phosphate transport, and phosphate availability. Annu. Rev. Plant Physiol. 24, 225-252.

Burns I G 1987 Effects of interruptions in N, P or K supply on the growth and development of lettuce. J. Plant Nutr. 10, 1571-1578.

Clark R B 1975 Characterization of phosphatase of intact maize roots. J. Agric. Food Chern. 23, 458-460.

Clarkson D T 1985 Factors affecting mineral nutrients acquisition by plants. Annu. Rev. Plant Physiol 36, 77-115.

Cogliatti D H and Clarkson D T 1983 Physiological changes in. and phosphate uptake by potato plants during develop­ment of and recovery from phosphate deficiency. Physiol. Plant. 58, 287-294.

Faquin Vet al. 1990 Cinetica da absor9ao de fosfato em soja sob inftuencia de micorriza vesiculo-arbuscular. Rev. Bras. Ci. Solo 14, 41-48.

Hart A L and Collier W A 1991 Inorganic phosphorus and DNA concentration in the leaf tissue of some forage species. Grass Forage Sci. 46, 167-171.

Hogue E et al. 1970 Effect of soil phosphorus levels on phosphate fractions in tomato leaves. J. Am. Soc. Hortic. Sci. 95 174-176.

Katz K B et al. 1986 Effect of phosphate stress on the rate of phosphate uptake during resuply to deficient tomato plants. Physiol. Plant. 67, 23-28.

Lefebvre D D et al. 1990 Response to phosphate deprivation in Brassica nigra suspension cells. Plant Physiol. 93, 504-511.

Martinez H E P et al. 1990 Cinetica das fra9oes fosfatadas em quatro variedades de soja (Glycine max (L.) Merrill). Anais da XIX Reuniao Brasileira de Fertilidade do Solo e Nutri9ao de Plantas. p 215. Abstract.

Martinez H E P et al. 1990 Cinetica da absor9ao de f6sforo por !res variedades de soja sob diferentes estados nut­ricionais. Anais do III Congresso Brasileiro de Fisiologia Vegetal. p 41. Abstract.

McPharlin I R and Bieleski R L 1989 Chemical nature of P efflux from P-adequate Spirodel/a and Lemna plants. Physiol. Plant. 76, 95-99.

Nerson H et al. 1987 Nitrogen and phosphorus stress repair in muskmelon (Cucumis melo) seedlings. J. Plant Nutr. 10, 1835-1841.

Pereira P R G 1991 Eficiencia da utiliza9ao de fra9oes de f6sforo na soja e regula9ao da coloniza9ao micorrizica. Ph. D Thesis. Universidade Federal de Yi9osa, Yi9osa 188 p.

Romer W et al. 1989 The relationship between phosphate absorption and root length in nine wheat cultivars. In Structural and Functional Aspects of Transport in Roots. Eds. B C Loughman, et al. pp 123-125. Kluwer Academic Publishers, Dordrecht.

Schj0rring J K and Nielsen N E 1987 Root length and phosphorus uptake by four barley cultivars grown under moderate deficiency of phosphorus in field experiments. J. Plant Nutr. 10, 1289-1295.

Smillie R M and Krotkov G 1960 The estimation of nucleic acids in some algae and higJ:!er plants. Can. J. Bot. 38, 31-49.

Wieneke J 1990 Phosphorus efficiency and phosphorus re­mobilization in two sorghum (Sorghum bicolor (L.) Moench) cultivars. Plant and Soil 123, 139-145.

M.A. C. Fragoso and M.L. van Beusichem (eds.) Optimization of plant nutrition 555-561, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-050

Effect of withdrawal of phosphorus on nitrate assimilation and PEP carboxylase activity in tomato

D.J. PILBEAM\ I. CAKMAK2 '3 , H. MARSCHNER2 and E.A. KIRKBY 1

1Department of Pure and Applied Biology, University of Leeds, LS2 9JT, UK; 2/nstitute of Plant Nutrition, University of Hohenheim, Postfach 70 05 62, D-7000, Stuttgart 70, Germany; 3Present address: Department of Soil Science, Faculty of Agriculture, Cukurova University, Adana, Turkey

Key words: Lycopersicon esculentum, nitrate assimilation, PEP carboxylase, phosphorus deficiency, tomato

Abstract

Tomato plants (Lycopersicon esculentum) grown in a complete nutrient solution for 8 days were transferred to a P-free solution of pH 6.0. Within 2 days of transfer the rate of alkalinization of the nutrient solution declined and by 4 days the solution had become acid. Nitrate transferred from roots to leaves was depressed over this period, and the rate of nitrate reductase activity in the leaves (the main site of assimilation of nitrate in tomato) had declined by 60% within 5 days of transfer. The activity of PEP carboxylase in the leaves of the P-deficient plants increased after 3 days, eventually becoming 3 times greater than in the leaves of plants adequately supplied with P. The PEP carboxylase activity in the roots of the P-deficient plants increased within 2 days, becoming 4 times greater after 8 days' growth. These results are discussed in relation to mechanisms for enhancement of P acquisition and maintenance of cation and anion uptake during P-deficiency.

Introduction

The acquisition of nutrients of low mobility in soils, such as phosphorus, is strongly influenced by morphological and physiological properties of roots. One of the responses of plants to P deficiency is to favour root growth relative to that of the shoot (Anghigoni and Barber, 1980; Heuwinkel et a!., 1992) probably because of a stronger sink competition by roots for photo­synthates and possibly by changes in the phytohormone balance between shoots and roots. The roots of P-deficient plants are typical­ly long and slender (Anghigoni and Barber, 1980) and often with abundant root hairs (Fohse and Jungk, 1983) so that a very large root surface is exposed to the soil. Additionally there are a number of root-induced changes in the rhizosphere that result from P deficiency and which also enhance the availability of soil P to plants.

Acidification of the substrate is a typical re­sponse of plants to P deficiency. For rape (Bras­sica napus L.) Moorby eta!. (1988) have demon­strated that this acidification results from net H+ excretion from the roots associated with an increase in the ratio of cations to anions taken up, although uptake of anions still slightly ex­ceeded uptake of cations. This acidification oc­curs in P-deficient plants even when supplied with N0 3-N, a form of N-nutrition that normally gives rise to excess anion over cation uptake and an increase in rhizosphere pH (Kirkby 1968). In tomato (Lycopersicon esculentum Mill.) grown with nitrate as the N-source P deficiency has a marked effect in decreasing rhizosphere pH due to a considerable decrease in the uptake of nitrate, which normally represents approximately 80% of the total anion uptake (Heuwinkel et a!., 1992). Anion uptake thus became greatly de­pressed, and even though there was a decrease in the uptake of potassium and calcium was ex-

556 Pilbeam et al.

ceeded by cation uptake, with a concomitant accumulation of organic anions in the roots to maintain charge balance.

Accumulation of organic anions in plants has been shown to be matched by an increase in activity of the enzyme phosphoenolpyruvate car­boxylase (PEP carboxylase, EC 4.1.1.31). Ac­cording to Hoffiand et al. (1990) organic acids exuded from the roots of P-deficient rape plants resulted mainly from an increase in PEP carbox­ylase activity in the shoots. Changes in the activity of this enzyme are an important com­ponent of the 'pH stat' mechanism that main­tains cellular pH within a narrow range (Davies, 1986), a mechanism that is particularly important in neutralising the OH- equivalents produced during the assimilation of nitrate.

The increase in PEP carboxylase activity in response to P-deficiency raises the question as to how this affects the functioning of the pH stat, and the assimilation of nitrate. In this paper we report the findings of experiments carried out on young tomato plants supplied with nitrate-N to test whether changes in PEP carboxylase activity following withdrawal of P occurred prior to or after changes in the rate of nitrate assimilation.

Materials and methods

Growth of plants

Seeds of tomato (Lycopersicon esculentum Mill. cv Ailsa Craig) were germinated in peat and after 2 weeks similar sized seedlings were trans­ferred to aerated nutrient solutions in 10 dm 3

containers ( 4 plants per container). After 4 days in one-fifth strength nutrient solutions the plants were then supplied with nutrients as follows (molm- 3 ): Ca(N0 3 ) 2 2.0; K2S04 0.7; KH 2P04

0.25; KCl 0.1; MgS04 0.5; Fe EDTA 0.08, H 3B0 3 1 x 10-2 ; CuS0 4 0.2 x 10-3 ; MnS04

0.5 X 10- 3 ; Na2Mo0 4 0.1 X 10- 3 ; ZnS0 4 0.5 X

10- 3 . Plants were grown in a constant environ­ment room with a photoperiod of 16/8h light/ dark (light intensity 38 Wm - 2); temperature was 23° I 18°; RH65- 75%; pH was adjusted daily to pH 6.0 by titration with 5 mol m - 3 H 2SO 4 or 200 mol m - 3 NaOH.

After 4 days plants were transferred to new

solutions, and for half the plants KH 2PO 4 was replaced by K2SO 4 at an equivalent K + ion concentration. Plants were harvested 5 hours after the start of the light period 1, 2, 3, 5 and 8 days after withdrawal of P. After decapitation and collection of xylem sap for 15 minutes shoots were weighed, and nitrate reductase and PEP carboxylase activities assayed in leaves 2 and 3. Roots were weighed, and the activities of both enzymes determined in the material taken ran­domly from all parts of the root system. The remaining material was dried at 95° for 2 days, and P concentration in the dry material was determined. In a second experiment plants were allowed to stand in 350 cm 3 CaSO 4 solution ( 0. 5 mol m- 3 ) for one hour, and the efflux of nitrate from the roots was determined. The plants were then returned to nutrient solutions for one hour before harvest.

In vivo nitrate reductase assay

The method was based on Klepper et al. (1971). Discs of leaf material (11 mm diameter) were cut from areas of leaf away from main veins with a cork borer. Approximately 0.3 g of weighed discs were placed in 10 cm3 potassium phosphate buf­fer (100mol m- 3 ), pH 7.5, containing 1% (v/v) 1-propanol, in a darkened flash. The flask was vacuum infiltrated for 1 minute, was flushed with N 2 gas for 30 seconds and was then sealed and incubated at 30°. Samples of buffer (0.5 cm3 )

were removed at 30 minute intervals for determi­nation of nitrite concentration by colorimetric analysis. Nitrate reductase activity (NRA) was calculated from the slope of a graph of nitrite concentration against time. In the second experi­ment roots were cut into 1 em segments, and approximately 0.6 g of weighed material taken at random from all the parts of the root system was assayed as above.

PEP carboxylase assay

The extraction and assay of this enzyme was based on Marques et al. (1983), as used by Schweizer and Erismann ( 1985). Desalted ex­tracts of plant material were incubated with PEP, malate dehydrogenase [EC1.1.1.37] and NADH, and the disappearance of NADH during the

Effect of P deficiency on nitrate assimilation and PEP carboxylase 557

conversion to malate of oxalacetate formed from the PEP was followed in a spectrophotometer at 340nm.

Other measurements

Protein concentrations were determined by the method of Bradford (1976), nitrate concentra­tions in xylem saps by the colorimetric method of Cataldo et a!. (1975), nitrate concentrations in the nutrient solutions by absorbance at 203 nm (Cawse, 1967) and P concentrations by the vanadate-molybdate colorimetric method.

Results

The amounts of H+ /OH- equivalents released into the growth medium per plant are shown in Figure 1. Plants adequately supplied with P continued to release OH- equivalents from their roots into the nutrient solution in increasing amounts throughout the growth period. For the plants deprived of P, however, the rate of OH­release slowed down as early as two days after the onset of the treatment, and by 4 days a switch had occurred from net release of OH- to net release of H +.

The rate of nitrate reductase activity (NRA) in leaf discs and root segments is shown in Table 1. NRA was higher in leaves than roots, and in both tissues the rate was depressed in the plants not supplied with P. However, this depressed rate was apparent only after 3-5 days of with­drawal of P, although in one of the two experi­ments it may have been occurring earlier.

By this time the plants without P supply were already taking up less nitrate than the plants with normal P supply (Fig. 2). This decrease in net uptake was partly accounted for by an increased rate of efflux of nitrate from roots of the P­deficient plants (Table 2), but this increased efflux accounted only for a small proportion of the shortfall. In the P-deficient plants a decrease in the transport of nitrate from roots to shoots in the xylem occurred within a short time of P withdrawal (Table 2).

Along with these changes in the rates of uptake, assimilation and transolocation of

"' -

0.5

0.4

~ 0.3 ~ ..

"1 .. :::

> "' "' 0" "0 CIJ 0.2

l .., J:

-;:o "' a. 0.1

+ p 1/.

- p [',.

0 E E 0 ----------------

)( "' .2~ - CIJ w iii 0.1

>

"' 0" CIJ

+ 0.2 J:

-2 0 2 4 6

Time from withdrawal of P (days)

8

Fig. 1. Rate of efflux of OH- /H+ equivalents from roots of tomato plants grown with or without phosphate, as de­termined by daily titration of acid or base to nutrient medium to restore pH to 6.0. The break in the line represents change of nutrient solutions. (Values are means calculated per plant for 6 containers per treatment. Error bars represent standard deviations about means, and where not shown are smaller than symbols).

nitrate there were large increases in the activity of PEP carboxylase in both leaves and roots of the P-deficient plants (Fig. 3). Compared with the plants supplied P, in the plants where P was withdrawn the activity of this enzyme increased within three days in the leaves and within two days in the roots.

During the first five days after withdrawal of P there was no significant change in dry weight of the plants, but by eight days after withdrawal the dry weight of the P-deficient plants was only one third of the weight of the P-sufficient plants

558 Pilbeam et al.

Table 1. Rate of reduction of endogenous nitrate in roots and leaves (2 and 3) of tomato plants grown with or without phosphate. (Values are means of 4 replicates ± standard deviations, results from 2 separate experiments)

Nitrate reductase activity Time after withdrawal of phosphate (days) (fl. moles NO; produced g- 1 fwt h - 1)

2 3 2

3 5 8

+P

Roots

0.96 ± 0.43 1.28 ± 0.19 1.29 ± 0.22

(Fig. 4). The concentration of P in the dry matter decreased quickly after the withdrawal of P and was noticeably lower in the samples taken even as soon as one day after withdrawal.

2.8

2.6

2.4 ~ + p ... I 2.2 >- -P ,-,.

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Time from withdrawal of p (days)

Fig. 2. Rate of uptake of nitrate by tomato plants grown with or without phosphate. The break in the line represents change of nutrient solutions (Values are means calculated per plant from daily measurement of nitrate concentration in 6 containers per treatment. Error bars represent standard deviations about means).

Leaves

3.28 ± 0.33 3.83 ± 0.31 3.22 ± 0.34 2.50 ± 0.33 3.12 ± 0.88 2.61 ± 0.67 1.90 ± 0.63

'T c

E c 100

"' -0 ~

c.

'I., E "0

50

-P

Roots

1.01 ± 0.43 1.19 ± 0.42 1.11±0.13

LEAVES

Leaves

3.39 ± 0.06 3.70 ± 0.47 2.79 ± 0.55 1.81 ± 0.16 1.91 ± 0.46 1.11 ± 0.17 0.17±0.05

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~ 15

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~ 50

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ROOTS

0+-----~----~----~------~ 0 2 4 6

Time from withdrawal of P (days)

8

Fig. 3. Activity of PEP carboxylase in Leaves 2 and 3 and roots of tomato plants grown with or without phosphate. (Values are means of 4 replicates. Error bars represent standard deviations about means, and where not shown arc smaller than symbols. Values are from 2 experiments).

Effect of P deficiency on nitrate assimilation and PEP carboxylase 559

Table 2. Flux of nitrate in xylem and efflux of nitrate from roots of tomato plants grown with or without phosphate. (Values are means of 4 replicates ± standard deviations, results from 2 separate experiments)

Time after Flux of nitrate in xylem Efflux of nitrate into 0.5 mol m _, withdrawal of (fLmoles NO; plant - 1 h - 1 ) CaS0 4 (!Lmoles N0 3 plant_, h - 1)

phosJ?hate (days)

1 2 3 2

3 5 6

--3: "0

I 01

01 E

c: ~

!': c ., " c: 0

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01

:;: 01 ., 3:

> 0

10

8

6

4

2

0

2.5

2.0

1.5

1.0

0.5

0.0

+P

3.41 ± 0.25 5.51 ± 0.59 5. 74 ± 1.16 3.52 ± 0.34 3.81 ± 0.66 6.20 ± 0.66

13.07 ± 2.60

\

0 2 4 6

+P"' -p~

0 2 4 6 Time from withdrawal of

(days)

-P

2.44 ± 0.49 4.27 ± 1.30 4.17 ± 1.34 2.05 ± 0.39 2.17 ± 0.66

L

1.45 ± 0.54 1.83 ± 0.64

8

8 p

+P -P

0.081 ± 0.048 0.124 ± 0.039 0.105 ± 0.045 0.265 ± 0.085 0.113 ± O.D38 0.265 ± 0.110

Discussion

As has previously been observed, tomato plants supplied N0 3-N acidify the nutrient solution when the supply of P is limiting (Heuwinkel et a!., 1992). Acidification was apparent in this experiment after 4 days, but a decline in alkalini­zation of the nutrient solution occurred two days after withdrawal of P.

In nutrient-sufficient plants supplied with ni­trogen as nitrate, total anion uptake exceeds total cation uptake and electroneutrality is main­tained either by net uptake of H + equivalents from the growth medium or net efflux of OH­equivalents. In the plants nitrate is reduced to ammonium, and the OH- equivalents produced are neutralized by the dissociation of carboxylic acids in order to stabilize cellular pH (Raven and Smith, 1978).

In this regulation of cellular pH PEP carbox­ylase plays a key role. It is stimulated by increase in pH, leading to enhanced carboxylation of PEP (C3) to oxalacetate (C4), and an additional carboxyl group is made available for dissociation (Davies, 1986).

In the tomato plants subjected to P deficiency there was a decrease in the rate of uptake and assimilation of nitrate, yet PEP carboxylase activity showed a marked increase. The rate of nitrate uptake dropped significantly within 3 days (Fig. 2), and as the increased efflux of nitrate

Fig. 4. Concentration of phosphorus (upper graph) in leaves 2 and 3 (L) and roots (R) and dry weight per plant (lower graph) of tomato plants grown with or without phosphate. (Values are means of 4 replicates. Error bars represent standard deviations about means, and where not shown arc smaller than symbols)

560 Pilbeam et al.

from the roots was not large enough to account for the decrease in net uptake (Table 2) there must have been a decrease in influx of nitrate. The transport of nitrate moving from the roots to the shoots decreased rapidly (Table 2), and there was a rapid decrease in the rate of reduction of nitrate in both roots and leaves (Table 1 ).

Despite the decrease in nitrate uptake and reduction in the P-deficient plants the activity of PEP carboxylase increased markedly, particular­ly in the roots. Increases in the activity of this enzyme are normally associated with increases in cellular pH, or with a drain on carbon skeletons from the tricarboxylic acid cycle during amino acid biosynthesis. It is difficult to envisage how either process would be enhanced by P de­ficiency, unless these plants release large amounts of organic acids from their roots, or an enhanced net excretion of H+ raises the cellular pH. It certainly appears that as PEP carboxylase activity in the leaves increased at the same time as the rate of nitrate reduction decreased there is not the direct link between assimilation of nitrate and synthesis of organic acids that the pH stat mechanism implies.

It has been suggested that PEP carboxylase activity increases in the green alga Selenastrum minutum during P limitation as a means of releasing P from PEP (Theodorou et a!., 1991), but it remains unclear whether this mechanism also exists in P-limited higher plants.

The decrease in NRA in the leaves appeared to precede the decrease in rate of uptake of nitrate in one of the two experiments, although in the other it seemed to occur after the rate of uptake had slowed. This roughly matched the time at which decrease in the rate of alkaliniza­tion of the growth medium occurred although decrease in both uptake and reduction of nitrate appears to have occurred slightly later. This compares with a study on rape, in which the onset of acidification occurred two days before a decrease in NRA (Moorby et a!., 1988). How­ever, in that study on rape NRA was measured in vitro, and so it was the capacity of nitrate reductase to reduce exogenous nitrate that was determined. In our study it was the reduction of endogenous nitrate in the leaves that decreased, and this followed the decrease in transport of nitrate in the xylem after withdrawal of P. It has

been suggested that OH- efflux by plants sup­plied with N0 3-N is linked to the reduction of nitrate rather than nitrate uptake (Deane-Drum­mond, 1982; Deane-Drummond, 1984; Eddy and Hopkins, 1985; Eisele and Ullrich, 1975) and in this study decrease in the rate of change of pH certainly seems to have occurred before a signifi­cant decrease in the rate of uptake of nitrate.

However, decrease in net nitrate uptake after more than three days of P withdrawal of (Fig. 2) and a corresponding shift in cation: anion uptake ratio may have contributed to the lowering of external pH, as has been observed in P-deficient rape (Hedley eta!., 1982; Hoffland eta!., 1989a; Moorby et a!., 1988 ;Schjorring, 1986), chickpea (Cicer arietinum L.) (Le Bot eta!., 1990), white lupin (Lupin us alb us L.) (Dinkelaker et a!., 1989) and tomato (Heuwinkel et a!., 1992).

In another experiment with tomato plants (Heuwinkel eta!., 1992) the efflux of H+ equiva­lents exactly matched the excess uptake of cat­ions over anions, and so the acidification of the growth medium by P-deficient tomato plants could be attributed to the increase in net excre­tion of H+ only. However, in rape (Hoffland et a!., 1989b) and white lupin (Dinkelaker et a!., 1989) organic acids are excreted into the rhizo­sphere by P-deficient plant. In P-deficient chick­pea plants organic anions accumulate in the roots (Le Bot et a!., 1990) and this may also be the case in tomato.

The enhanced PEP carboxylase activity both in leaves and roots of tomato plants after with­drawal of P strongly suggests that there is en­hanced synthesis of organic acids. Increased PEP carboxylase activity in P-deficient plants has previously been seen in rape (Hofftand et a!., 1990), but in that species the increase appears to be confined to the leaves. The carbon source for PEP carboxylation is Hco; (O'Leary, 1982), and so enhanced PEP carboxylase activity in roots of plants in hydroponic culture could lead to enhanced uptake of Hco; and acidification of the growth medium by H+ released in the H 2C0 3 :;:::::: H+ + Hco; dissociation. Increased PEP carboxylase activity in roots could provide a simple means of maintaining cation uptake by increasing the uptake of HCO; as accompanying anions.

The rapid stimulation of PEP carboxylase

Effect of P deficiency on nitrate assimilation and PEP carboxylase 561

activity in P-deficient roots and leaves may therefore be a response to a shift in cation: anion uptake ratio, but may also be a mechanism for the enhancement of P acquisition by increasing the rates of either H+ or organic acid excretion.

Acknowledgements

The authors are indebted to Christine Hengeler for help with PEP carboxylase assays and to the British Council and the Deutscher Akademischer Austauschdienst (DAAD) for financial support.

References

Anghigoni I and Barber S A 1980 Phosphorus influx and growth characteristics of corn as influenced by phosphorus supply. Agron. J. 72, 685-688.

Bradford M M 1976 A rapid and sensitive method for the quantification of microgram quantities of protein using the principle of protein dye binding. Anal. Biochem. 72, 248-254.

Cakmak I and Marschner H 1990 Decrease in nitrate uptake and increase in proton release in zinc deficient cotton, sunflower and buckwheat plants. Plant and Soil 129, 261-268.

Cataldo D A, Haroon M, Schrader L E and Youngs V L 1975 Rapid colorimetric determination of nitrate in plant tissue by nitrate of salicylic acid. Commun. Soil Sci. Plant Anal. 6, 71-80.

Cawse P A 1967 The determination of nitrate in soil solutions by ultraviolet spectrophotometry. Analyst 92, 311-315.

Davies D D 1986 The fine control of cytosolic pH. Physiol Plant. 67, 702-776.

Deane-Drummond C E 1982 Mechanisms for nitrate uptake into barley (Hordeum vulgare L. cv Fergus) seedlings grown at controlled nitrate concentration in the nutrient medium. Plant Sci. Lett. 24, 79-89.

Deane-Drummond C E 1984 Mechanism of nitrate uptake into Chara corallina cells: lack of evidence for obligatory coupling to protein pump and a new NO; /NO~ exchange model. Plant Cell Environ. 7, 317-323.

Dinkelaker B, Romheld V and Marschner H 1989 Citric acid excretion and precipitation of calcium citrate in the rhizo­sphere of white lupin (Lupinus a/bus L.) Plant Cell Environ. 12, 285-292.

Eddy A and Hopkins P G 1985 The putative electrogenic nitrate-proton symport of the yeast Candida uti/is. Com­parison with the systems absorbing glucose or lactate. Biochem. J. 231, 291-297.

Eisele R and Ullrich W R 1975 Stoichiometry between photosynthetic nitrate reduction and alkalinization by Ankistrodesmus braunii. Planta 127, 117-124.

Fohse D and Jungk A 1983 Influence of phosphate and

nitrate supply on root hair formation of rape, spinach and tomato plants. Plant and Soil 74, 359-368.

Hedley M J, Nye P H and White R E 1982 Plant induced changes in the rhizosphere of rape (Brassica napus var. Emerald) seedlings. II. Origin of the pH change. New Phytol. 91, 31-44.

Hcuwinkel H, Kirkby E A, Le Bot J and Marschner H 1992. Phosphorus deficiency enhances molybdenum uptake by tomato plants. J. Plant Nutr. 15, 549-568.

Hoffland E, Findenegg G R and Nelemans J A 1989a Solubilization of rock phosphate by rape. I. Evaluation of the role of the nutrient uptake pattern. Plant and Soil 113, 155-160.

Hoffland E, Findenegg G R and Nelemans J A 1989b Solubilization of rock phosphate by rape. II. Local root exudation of organic acids as a response to P-starvation. Plant and Soil 113, 161-165.

Hoffland E, Nelemans J A and Findcncgg G R 1990 Origin of organic acids exuded by roots of phosphorus stressed rape (Brassica napus) plants. In Plant Nutrition- Physi­ology and Applications. Ed. M L van Beusichem. pp 179-183. Kluwer Academic Publishers, Dordrecht.

Kirkby E A 1968 Influence of ammonium and nitrate nutrition on the cation-anion balance and nitrogen and carbohydrate metabolism of white mustard plants grown in dilute nutrient-solutions. Soil Sci. 105, 133-141.

Klepper L, Flesher D, Hageman R H 1971 Generation of reduced nicotinamide adenine dinucleotide for nitrate reduction in green leaves. Plant Physiol. 48, 580-590.

Le Bot J, Alloush G A, Kirkby E A and Sanders FE 1990 Mineral nutrition of chickpea plants supplied with NO; or NH,-N. II. Ionic balance in relation to phosphorus stress. J. Plant Nutr. 13, 1591-1601.

Marques I A, Oberholzer M J and Erismann K H 1983 Effects of different inorganic nitrogen sources on photo­synthetic carbon metabolism in primary leaves of nonnodu­lated Phaseolus vulgaris L. Plant Physiol. 71, 555-561.

Moorby H, White R E and Nye P H 1988 The influence of phosphate nutrition on H ion efflux from the roots of young rape plants. Plant and Soil 105, 247-250.

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Reprintedfrom Plant and Soi/154: 111-117, 1993.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 563-567, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-049

Susceptibility of sweet pepper (Capsicum annuum L.) cultivars to the calcium deficiency disorder 'Blossom end rot'

PHILIPS. MORLEY1 , M. HARDGRAVE2 , M. BRADLEY2 and D.J. PILBEAM1

1Department of Pure and Applied Biology, University of Leeds, Leeds LS2 9fT, UK and 2 Horticulture Research International, Stockbridge House, Cawood, Selby, YOB OTZ, UK

Key words: 'blossom end rot', calcium, Capsicum annuum, pepper, physiological sink, sun scorch

Abstract

Fifteen cultivars of sweet pepper (Capsicum annuum L.) were grown hydroponically on rockwool under standard glasshouse conditions from January to November. Significant differences in varietal suscep­tibility to Blossom end rot (BER) were found. This was related to fruit load over the growing season. Analysis of tissue showed no significant differences in fruit calcium concentration between cultivars. Calcium concentration was seen to vary in different areas of the fruit. Data are discussed in relation to differences in susceptiblity of sweet pepper cultivars to BER, particularly with respect to distribution of calcium.

Introduction

Calcium deficiency disorders pose a significant problem in the cultivation of many horticultural crops. An increase in such disorders has been seen over the past few years, particularly in the glasshouse crop industry, as soilless growing techniques become more widespread. There is also the commercial desire to produce the largest quantity of saleable fruit per plant, which in itself may cause certain nutritional stresses with­in the plant.

The calcium deficiency disorder of fruit 'Blos­som end rot' (BER), is one of the most im­portant nutritional disorders of the Sweet pepper plant. The first symptoms of BER is often the appearance of a small necrotic grey /brown area of tissue towards the distal end of the fruit. The symptom is a result of cellular degradation, the colouration due to leakage of phenolic precur­sors from the vacuoles of cells and their sub­sequent oxidation to polyphenols (Decock et al., 1975). Sunken, discoloured areas of dead tissue may enlarge to include a large part of the distal end of the fruit. BER often leads to secondary

infection by fungal pathogens (Marschner, 1986), combining to make the fruit commercially worthless. Bangerth (1979) suggests reduced storage and shelf life might be a consequence of less extreme calcium deficiency in fruit. This might also increase the susceptibility of fruit to other diseases (Guttridge and Bradfield, 1983). Other workers (Mason, 1979) suggest that tissue which is susceptible to calcium shortage may be sprayed with a calcium salt to correct any de­ficiencies thus overcoming the internal distribu­tion calcium shortfall, although in peppers this can sometimes cause skin pitting (J anse and de Kreis, 1988). This study looks at the different susceptibilities of several cultivars of Sweet pep­per to the calcium deficiency disorder BER. The physiological basis of observed differences is investigated. It has also been suggested that the phenomenon of 'sun scorch' of pepper fruit, where localised patches of dead and dried fruit tissue up to 5 em in diameter occur, may also, in part, be a consequence of local calcium de­ficiency (N Dungey, 1991 pers. comm.). The role of calcium in relation to 'sun scorch' is also investigated.

564 Morley et al.

Methods

Twenty plants each of fifteen cultivars of Cap­sicum annuum L. were grown on a rockwool system in a glasshouse at HRI Stockbridge House (Cawood UK) during the 1991 season. Plants were spaced 45 em apart, edge effects were minimised by the presence of a pair of 'guard' plants at the end of each row. Plants were fed (Table 1) and watered automatically using a dripfeed system. The glasshouse environ­ment was monitored and controlled automatical­ly to give optimal growing conditions throughout the season (Mean relative humidity= 50-85%; min/max temp= 19°C/21°C). Ripe fruit were harvested weekly, when they were individually weighed and graded (Healthy, BER and Sun scorch). Results were statistically analysed using 'Duncans multiple range test' (SAS software package). Three cultivars showing a range of susceptibility to BER (Evident, Sisi and Ben­digo) were further studied. Here analysis of the calcium content of different areas of the fruit was carried out. Fruits were equally sectioned into proximal, middle and distal areas. Tissue was then weighed and oven dried for 36 hours at 60°C. Preparation for calcium determination using atomic absorption spectrophotometry was then carried out following the method described by MAFF/ADAS (1981).

Table 1. Nutrient concentrations in the rockwool blocks during the 1991 season

Nutrient Concentration

N03 -N 150-200 NH 4-N 10-15 K 300-500 Ca 180-250 Mg 30-50 p 30-50 Fe 0.6-3.0 Zn 0.2-2.0 Mn 0.3-1.0 Cu 0.02-1.0 B 0.2-2.0 Na 0-100 Cl 0 s 0

pH 5.8-6.8 Ec 2.0-3.0

Results

Figure 1 shows the yield of BER affected fruit, the yield of healthy fruit and the proportion of the total fruit (both by weight and number) that were affected by BER in experiments carried out during the 1991 season. The highest yield of fruit over the entire season was found in the highly BER susceptible cultivar 'Evident' whereas lower yielding cultivars such as 'Bendigo' and 'Sisi' had a lesser incidence of BER affected fruit. These three cultivars, highlighted in the Figures, show a range of BER susceptibilities and were chosen for further investigation. The cultivar 'Evident' had a higher total yield than any other cultivar, both by weight and number (data not shown). It also produced the greatest yield of BER fruit. Mean weights of individual affected fruit from all cultivars are shown in Figure 2 and in all cases they were lower than the weights of healthy fruits. The mean fruit weight of the least BER susceptible cultivar, 'Bendigo', was significantly lower than that of the highly susceptible 'Evident'. However, this correlation did not hold for all cultivars. One example is 'Cubico' where mean fruit size was large (190.4 g) and incidence of BER was low. Figure 3 shows weekly numbers of healthy and BER affected fruit of all cultivars. It shows that the incidence of BER matched times of high overall yield. The concentrations of calcium in proximal, middle and distal areas of healthy fruit of the cultivars Sisi, Bendigo and Evident are shown in Figure 4. There was a uniform decline in calcium concentration with increased distance from the pedicel. This ranged from 1.5-1.8 mg g - 1 dry weight in proximal tissue (9.86 x 10-2-11.9xl0-2 mgg- 1 fresh weight, data not shown) to l.0-1.2mgg- 1 dry weight (6.57 x 10-2-7.89X10- 2 mgg- 1 fresh weight, data not shown), in distal tissue. The concentrations of calcium in the distal end of BER affected fruit of all three cultivars are also shown. It can be seen that the calcium concentrations were significantly lower than in other areas. The concentrations were approximately 0.6 mg g - 1 dry weight (3.96 x 10-2 mg g - 1 fresh weight, data not shown), with no significant differences between cultivars.

Up to 3% of the fruit of some of the cultivars

IS

'" l! .c ., ~ "'- '0 .. ., u

'0 .c 0 - .r:.2 UCI.C.

s;~, -CI .&. .,_ " .. !!

:;:

ffi s~

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20

...

Lo.IL.o.JOOZt--<-<-<a::-<<--<­-J z o o - :::: - ·o ::.:: w :::::£ ~ rn t- li1 W<--::X::W..J-<a:rna:::z-t-CI o-o.ma...o.=:Jm::Jt--<<CI'ILLI--<o::z:::~.-~-a:::!NZC...::l ...J

-<wcw>-<<-<< < < m O.II.JL&--':::Ia.. en >

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Blossom end rot in sweet pepper fruit 565

"'

:X: 0 LJI.~IL..IL..

... -8~.8'0

... uu.8., .8

u.C .c"'

;fl., .,

LUI.&.IOOZt-<<-<a:<-<-<­...JZOO-:Z:-C!I:::.LLI~I-CI'It-10 w.<-- :X:: w ...J-< c::: en a:; z- ..... "' o-amo..o::~m:Jt-<<tnUJ-<a::z:J-'-a:::lNZC..:l ...J

<t.tJ<.JI.a.l><<<< < < I:D. OLI.ILL....J::::IQ. ~ >

Fig. 1. Total fruit yield, yield of BER affected fruit, yield of healthy fruit and percentage yield (by weight and by number of fruit) of the 15 cultivars of Capsicum annuum L. grown during the 1991 season (Bars with same letter= no significant difference). Total n = 12539.

were affected by 'sun-scorch' during the growing season. Incidence of scorch did not increase at times of heavy fruit load (data not shown) and when 'sun scorched' fruits were sampled the calcium concentrations in the affected and heal­thy tissue showed no significant differences (Healthy tissue calcium concentration = 0.96mgg- 1 dry weight+/- 0.11 n=14; sun­scorched tissue, calcium concentration = 0.98 mg g -I dry weight +I- 0.12 n = 14).

Discussion

The results indicate that there is a link between the total yield of fruit and the incidence of BER. This is true of the cultivar 'Evident' where a high

total yield was correlated with a high yield of BER affected fruit and lower yielding cultivars such as 'Bendigo' had lower levels of incidence of BER. This suggests that a larger yield of fruit creates a greater sink for available calcium within the plant and follows the proposal of Chiu and Bould (1976) that it is the distribution of calcium within plants which is of primary impor­tance in causing BER and moreover that fruit load affects this. Results of earlier experiments (data not shown) indicate that there is no signifi­cant difference in the rate of uptake of calcium by the different cultivars.

Another possible explanation for the differ­ences between cultivars in susceptibility to BER could be that the critical calcium concentrations in fruit, which lead to occurrence of BER, may

566 Morley et al.

II.IIUOO::;:t-<<<a::<<-<~ -IZOO-:Z:-O!.::II.l::l:t-<nt-10 LU<--:X::II.I..J<a::<nii::Z:-1-CII 0-0alC..O::::lal::::ll-<<<nw~ <II:Z:J..J-II:::I:NZC..:::!; -1

oo<LUOW><<<< -< -< Cl 0"-l"--I:::!;C.. <n >

Fig. 2. Mean weights of BER affected and healthy fruits of the 15 cultivars of Capsicum annuum L. grown during the 1991 season (Bars with same letter= no significant differ­ence).

WEEK

Fig. 3. Weekly numbers of healthy and BER affected fruit of all the cultivars grown during the 1991 season.

vary. However, although there were differences in calcium concentrations between the areas of individual fruits, cultivar type had no effect on concentration. In all fruit, calcium concentration was found to decrease with increasing distance from the pedicel and tissue affected by BER had significantly lower calcium concentrations than healthy tissue. The apparent critical calcium

IZ!PROxiMAL

.MIOOLE

Fig. 4. Concentration of calcium in tissues of the proximal, middle and distal areas of healthy and BER affected fruit of three cultivars of Capsicum annuum L. (A= Sisi; B = Bendigo; C =Evident; n = 14; error bars= 95% confidence interval).

concentration was between 0.6 and 1.0 mg g -I dry weight in the three cultivars studied.

If BER is partly caused by competition be­tween sinks for calcium it would be expected that increases in the rate of fruit set and fruit expan­sion during the season may increase the risk of BER occurring (Ho and Adams, 1989). In this study, the highest incidence of BER occurred at weeks 8 and 19. At both these times the yield of healthy fruit was considerably higher than in the immediately preceding weeks. Ho (pers. comm) suggests that the incidence of BER in tomato fruit is negatively correlated with xylem density in tissue. If this is also the case in sweet pepper, cultivars with lower density of xylem in suscep-

tible areas of fruit tissue would be particularly at risk during flushes of fruit growth. Possible differences in densities of xylem vessels in fruit of different pepper cultivars would be a more plausible cause of different susceptibility to BER than differences in sink competition alone as BER affected fruits were shown here to be significantly smaller than healthy fruits. This should make the calcium requirement of affected fruits lower. Therefore they should have been less at risk of developing BER. The decreased size could also be further evidence of restricted xylem transport in BER affected fruit. This possibility is currently under investigation.

The phenomenon of 'sun-scorch' (superficially like the symptoms of BER) seems to occur randomly throughout the season. It does not appear to be a consequence of poor calcium nutrition as has been thought. Indeed symptoms similar to 'sun-scorch' have been produced in the laboratory by exposing fruit tissue to the heat and light of a 60 W incandescent light bulb. This suggests to us that occurrence of 'sun-scorch' is a consequence of specific sunshine events. Small differences between cultivars might be simply due to variation in fruit shading by leaves. Indeed initial observations support this. The least 'sun-scorch' susceptible cultivars do seem to have denser foliage.

Acknowledgement

Thanks are due to Quentin Cleal for his valuable discussion and ideas and to the Science and

Blossom end rot in sweet pepper fruit 567

Engineering Research Council for a studentship for one of us (P.S.M.).

References

Bangerth F 1979 Calcium related physiological disorders of plants. Annu. Rev. Phytopathol. 17, 97-122.

Chiu T F and Bould C 1976 Effect of shortage of calcium and other cations on 45Ca mobility, growth and nutritional disorders of tomato plants (Lycopersicon esculentum). J. Sci. Food Agric. 27, 969-977.

Decock P C, Dyson P W, Hall A and Grabowska F 1975 Metabolic changes associated with calcium deficiency in potato sprouts. Potato Res. 18, 573-581.

Guttridgc C G and Bradfield E G 1983 Root pressure stops blossom-end rot. Grower 100, 25-26.

Ho L C and Adams P 1989 Calcium deficiency- a matter of inadequate transport to rapidly growing organs. Plants Today 2, 202-207.

Janse J and deKreis C 1988 Capsicums: Pitting linked especially with high calcium in fruit. Groenten Fruit 44, 40-41.

MAFF/ADAS 1981 The analysis of agricultural materials. Second Edition. H.M.S.O., London, pp 30-31.

Mason J L 1979 Increasing calcium content of calcium­sensitive tissues. Commun. Soil. Sci. Plant Anal 10, 349-371.

Marschner H 1986 Mineral Nutrition of Higher Plants. Academic Press, London. 674 p.

M.A. C. Fragoso and M.L. van Beusichem (eds.) Optimization qf"plant nutrition 569-576, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-134

Hydraulic properties of sphagnum peat moss and tuff (scoria) and their potential effects on water availability

F.F. da SILVA, R. WALLACH and Y. CHEN Department of Soil and Water Science, Faculty of Agriculture, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel

Key words: predictive model, unsaturated hydraulic conductivity, water characteristic curve, water availability

Abstract

The potential rate of water and nutrient supply to plant roots depends on the hydraulic properties of the container medium (growth medium, substrate), primarily on its unsaturated hydraulic conductivity, which is a measure of the medium's resistance to water flow. Water availability to plants grown in containers is usually being evaluated using criteria based exclusively on water characteristic curves of the medium in which the plant is grown. This approach is challenged in the present paper. We hypothise that the coarse structure of peat moss as well as of other container media may result in a sharp decrease in hydraulic conductivity, as the water content of peat is reduced. Transient changes in unsaturated hydraulic conductivity may result in reduced water uptake by plant roots. The objectives of this research were to determine the hydraulic properties of sphagnum peat moss and to evaluate their potential effects on water availability. Tuff (granulated volcanic ash) and its mix with peat were also tested for comparison. Water characteristic curves (drying and wetting cycles) and saturated hydraulic conductivity were measured. A predictive mathematical model was used to calculate the unsaturated hydraulic conductivity of the media. Measured water retention and saturated hydraulic conductivity data were used to estimate model parameters by a nonlinear least-squares curve-fitting technique. Model predictions of unsaturated hydraulic conductivity were validated by direct measurements. Results showed that,sharp variations in hydraulic conductivity occur in a very narrow suction range (0-2.5 kPa). In this range a decrease of more than three orders of magnitude in the unsaturated hydraulic conductivity was observed for peat. A similar trend was observed for the other media tested. This suggests that the approach that has been commonly used for determinations of water availability and for irrigation scheduling in container media may provide inaccurate predictions as to potential plant response.

Introduction

Greenhouse agriculture is a rapidly growing crop production system aimed to grow ornamentals or out of season crops, and to achieve high yields at the best attainable quality. The use of container media in greenhouse agriculture is enhanced in areas where local soils are not suitable for the growth of intensive crops. Peat is among the

most widely used container media in Northwest Europe (Bunt, 1988) as well as in Israel.

Peat is a general name for many types of partially decomposed plant residues. The actual nature of peat is determined by its botanical origin and the climatic conditions prevailing during its formation (Puustjarvi, 1977). Peat bogs are formed under climatic conditions of high precipitation, low evaporation, low annual

570 da Silva et al.

solar radiation and low summer temperatures. The most important peat forming plants are Sphagnum spp. (Raviv et a!., 1986).

To support optimal growth a high content of available water and an adequate air supply are considered the most important physical charac­teristics required in a container medium (Beard­sell et a!., 1979a,b; Bilderback, 1985; Chen et a!., 1980; De Boodt and Verdonck, 1972; Hanan eta!., 1981; Riviere eta!., 1990; Spomer, 1974). Knowledge of the water characteristic curve and of the transpirational demand, and selection of a container medium which contains sufficient air at low suctions has been considered a good basis for irrigation management. However, the poten­tial rate of water and nutrient supply to plant roots depends strongly on the hydraulic conduc­tivity and on the hydraulic gradients prevailing in the medium.

Greenhouse studies have showed that many growers do not irrigate frequently enough and when they do, they apply to much water. Kar­lovich and Fonteno (1986), concluded in their work that under the watering ranges used, the total volume of water in the container at any time may be of greater importance than the suction at which the water is held. However, reduced plant growth has been reported for increasing water suction in container media (De Boodt and Verdonck, 1972; De Boodt et a!., 1974; Johnson et a!., 1981 ), thus indicating that the availability of water and nutrients may be reduced. Experimental work on water loss in container-grown plants shows a progressive de­cline in the rate of transpiration as the water content in the medium is reduced (Bunt, 1988).

The concept of water availability has long served as a criterion for irrigation management. De Boodt and Verdonck (1972) introduced the concept of 'easily available water', EAW, defined as the difference between the water content at 1 kPa and at 5 kPa. These authors also defined 'water buffering capacity', WBC, as the differ­ence between the water content of the medium at 5 and at 10 kPa. The definitions suggested by De Boodt and Verdonck (1972) are widely used in container media research and they differ from earlier approaches. For example, White and Mastalerz (1966) introduced the concept of container capacity, CC, defined as the amount of

water retained in a containerized medium after drainage from saturation has ceased, but before evaporation has started. A combination of mathematical functions for the water characteris­tic curve and container geometry was later shown to provide a more consistent description of container capacity (Bilderback and Fonteno, 1987; Fonteno, 1989). According to this ap­proach, CC is the total volume of water in the container, as given by its water characteristic curve, divided by the container volume. This parameter describes the maximum water reten­tion capacity of the medium. Bilderback and Fonteno (1987), defined available water, AW, as the difference between CC and the water held at permanent wilting point (PWP = 1500 kPa). However, although such a value may represent an endpoint for plant survival, the proposed range does not represent an optimum plant growth in containers, and water is not equally available over the range from CC to PWP. The approach proposed earlier by De Boodt and Verdonck (1972) recognizes different degrees of availability. In the range of 1 to 5 kPa water is assumed to be easily available (EAW), whereas in the range of 5 to 10 kPa water is assumed to be available to a lesser degree, as the term 'buffering capacity' suggests.

Water and nutrient availability to plants de­pend not only on the water content, 0, but also on the actual water fluxes which are strongly affected by the momentary value of the hydraulic conductivity, K. This is especially important in container media where significant changes in () and K occur in the above mentioned suction ranges (Wallach et a!., 1992a, b), which practi­cally occurs between irrigations. Transient changes in K may result in reduced water uptake by the plant. A stress situation may evolve regardless of the total amount of water in the container, which may appear to be sufficient for plant transpiration. The question of whether water is or is not available for plant growth cannot be answered without determination of the medium's unsaturated hydraulic conductivity. In contrast to the relatively easy measurement of water characteristic curves, many technical dif­ficulties are involved in measurements of hy­draulic conductivity under unsaturated condi­tions. In addition, these measurements are tedi-

ous and common measuring devices cannot effi­ciently cover wide range of variation (Klute and Dirksen, 1986). In recent years, efforts have been made to apply various mathematical func­tions to describe and predict the hydraulic prop­erties of container media. Fonteno et al. (1981), Fonteno and Bilderback (1983) and Karlovich and Fonteno (1986) applied regression analysis to describe water characteristic curves of a wide variety of container media used in horticulture. Milks et al. (1989) and Wallach et al. (1992a, b) applied the nonlinear equation proposed by van Genuchten (1980) to fit retention data collected for several container media. This equation can be written as follows:

(1)

where h is the water suction (kPa) and a (kPa _,), n and m are parameters that can be determined by curve-fitting techniques. In Equa­tion 1, E> is the dimensionless water content, sometimes called effective saturation:

()-() E>=---r

(),- ()r (2)

The subscripts s and r indicate saturated and residual values of water content, () (cm3 em - 3 ),

respectively. The main advantage of Equation 1 is the possibility of its combination with a predic­tive model for the unsaturated hydraulic conduc­tivity, thus forming the basis for a combined hydraulic model. According to the model de­veloped by Mualem (1976), the relative hy­draulic conductivity, Krei can be calculated by the following equation:

(3)

where x is a dummy integration variable. Solving Equation 1 for h = h(E>), substituting the re­sulting expression into Equation 3 and assuming m = 1-1/n leads to:

(4)

(van Genuchten, 1980; Equation 8) where 0 < m < 1. In terms of h, Equation 4 becomes:

Hydraulic properties of peat moss and tuff 571

K (h)= [1- (aht- 1[1 + (aht]-m] 2

rei [1 + (ahtr'2 (5)

(van Genuchten, 1980; Equation 9). Multiplying Equation 4 and Equation 5 by the value of Ks will provide the K(E>) and K(h) functions, re­spectively.

Wallach et al. (1992a,b) applied this closed­form analytical solution of Mualem's (1976) model, and determined K(h) curves of several container media extensively used in Israel. In the present study we followed this approach to apply the above combined hydraulic model to describe the hydraulic properties of sphagnum peat moss.· Tuff (scoria, granulated volcanic ash) and a mix of tuff and peat were also tested for comparison. Besides an accurate description and curve fitting of the water characteristic curves of the media (drying and wetting cycles), the calculation of a reliable unsaturated hydraulic conductivity func­tion is of vital practical importance to the de­velopment of an efficient management regime of irrigation and fertilization for peat and other container media.

Materials and methods

The media tested were (Table 1): (i) PEAT-a medium textural grade of sphagnum peat moss from Germany (Klasman); (ii) TUFF- 'red' tuff originating from granulated volcanic ash (scoria) and consisting of particles ranging in size from 0 to 8 mm (Wallach et al., 1992a). Tuff was ob­tained from a tuff strip mine by crushing large particles (diameter of 30 to 50 mm), then sieving the crushed material through an 8-mm sieve; and (iii) MIX=PEAT+TUFF (40%: 60% by vol­ume). This mix was chosen because of its exten­sive use in Israel.

Three replicates of the media were packed in sintered glass funnels. Air dried peat was previ­ously soaked in deaerated water for 48 h, then packed in the funnel. The medium was allowed to drain until the water reached a level slightly higher than the porous plate of the funnel. Peat material was added to the funnel and its volume was corrected to 500 mL, which corresponded to a height of 6.6 em in the funnel. The tuff materi-

572 da Silva et al.

Table 1. Description of the tested container media

Medium

Symbol

PEAT TUFF

MIX

Description

Sphagnum pet moss 'Red' tuff (scoria, granulated volcanic ash) PEAT+TUFF (40%: 60% vol/vol)

a] was carefully wetted to a water content of about 5% by volume prior to sampling to reduce segregation of fine particles to a minimum while allowing uniform packing. The mix of peat and tuff was prepared using prewetted tuff and presoaked peat that was allowed to drain excess water. After packing, all the media were slowly wetted to saturation during a 24 h period and left saturated for an additional 24 h. Water charac­teristic curves were then measured by the han­ging-water-column method (Klute, 1986). Mois­ture retained was determined at suctions be­tween 0 and 12 kPa, at small increments (main drying curve), and back to 0 (primary wetting scanning curve). Suction was applied using a hanging water column of different lengths (Wal­lach et al., 1992a). Saturated water content, es, was taken as the water content achieved at the end of the saturation procedure. Saturated hy­draulic conductivity, Ks, was determined using a modified (Wallach et al., 1992a) constant-head method (Klute and Dirksen, 1986). Previous to the determination of Ks, the range of validity of Darcy's law was identified by applying a series of hydraulic gradients and measuring the resulting flux, q. Parameters a, n, m = 1-1/n, and e, in Equation 1 were estimated for the arithmetic average of each set of three replicates. The measured value of e, was used for the curve­fitting of the drying cycle of the O(h) curve, whereas for the wetting O(h) curve a maximum (saturated) value was used for e, being the highest value measured at the end of the experi­ment (Ow). The value of e, was assumed to be the same for the drying and wetting data and was obtained from curve-fitting of the drying reten­tion data, as suggested by van Genuchten (1980). A detailed description of the nonlinear least squares curve-fitting technique used for this purpose is given by van Genuchten (1978). The

Bulk density (gcm- 3 )

0.068

1.091 0.585

Porosity (cm 3 em - 3 )

0.956

0.587 0.730

Maximum particle diameter (em)

0.4

0.8 0.8

drying cycle of K(h) was calculated by multiply­ing the drying K,e1 (h) relationship obtained from the model by Ks. The wetting cycle of K(h) was obtained by multiplying the corresponding K,e1 (h) relationship by Kw, which corresponds to the hydraulic conductivity of the medium at the water content ew. This assumption was made due to the fact that no matching value of K was measured, except for K 5 • The calculated K(h) curve (drying cycle) was directly validated using the long column version (50 em) of the steady­state flux control method (Klute and Dirksen, 1986). According to this method, a steady-state flow of water is established in a vertical, uni­formly packed and previously saturated column, with its lower end kept at equilibrium with a free water surface. The discharge, Q (em 3 min -J) is regulated so that the flux, q (em min -l) is less than K5 • Upon reaching a steady-state condition, the moisture distribution along the upper region of the column is expected to be relatively con­stant. A constant water-suction distribution, i.e., a unit hydraulic gradient, should, therefore, be established in that part of the column. Under these conditions, K is numerically equal to q. This process was then repeated at a series of decreasing flow rates, and each time q and h were recorded. Two replications of the tested media were packed in 50 em long, 10 em diam­eter (i.d.) columns. Water flux in the columns was controlled using a peristaltic pump. Water suction values were measured at various loca­tions using tensiometers (Wallach et al., 1992a).

Results and discussion

The media tested differ significantly in their water retention properties. The highest 8, was exhibited by peat, almost twice as high as that of

Hydraulic properties of peat moss and tuff 573

Table 2. Hydraulic model parameters: measured values of 0, and 0.,., and fitted values of Or, a and n

Medium

PEAT

TUFF

MIX

Curve

d w

d w d w

Model parameters

fis (cm 3 em_,)

0.901

0.548

0.708

d- drying curve; w- wetting curve. "IJr < 0.001; changed to fit with fir= 0.0.

Ow (em' em_,)

0.810

0.450

0.631

the tuff material (Table 2). Approximately 50% of the water of saturated peat was lost when suction was increased to 2.5 kPa. As suction was further increased water loss was less drastic, and at the highest suction applied (h = 12 kPa) the medium still held more than 20% water by volume. For tuff, water loss in the 0 to 2.5 kPa range was very sharp (about 75% ), and the volume of water held at h = 12 kPa was only slightly higher than 10% of volume. Peat ex­hibited a higher water holding capacity. This property is of particular importance when im­proved water retention of container media is required. For example, the amendment of tuff with the peat resulted in an increase in water content at any given suction. Peat also exhibited large differences between drying and wetting curve (hysteresis), probably because as suction increased and the material dried out it became more hydrophobic (Beardsell and Nichols, 1982). Consequently it would become increasing­ly more difficult to rewet the medium. After one cycle of drying and wetting, the water content at zero suction, fJw, was considerably lower than (Js

(Table 2), probably due to entrapped air. Comparison of fitted curves with observed

data gives some insight into the accuracy of the model. In Figure 1, the nonlinear least-squares fit of Equation 1 is presented along with the observed data. It should be noted that of the five parameters of the model, (Js and (Jw were mea­sured whereas a, n, m = 1- 1/n, and fJ, were calculated to fit the measured data (Table 2). A good agreement between measured and fitted retention data was obtained, throughout the tested suction range. Only slight deviations from observed data were observed, with the exception of the wetting curve of peat. However, the high

/"""'.. n I

E 0

n

E 0

~

+-' c Q)

+-' c 0 u

L Q)

+-' 0 5:

0.0" 0.0 0.079 0.079 0.0" 0.0

k Pa -I

0.264 0.582 0.324 0.387 0.311 0.912

n

1.390 0.345 2.186 2.534 1.387 0.268

r'

0.998 0.989 1.000 0.999 0.997 0.985

1.0 .,.------------------------,

0.8

0.6

0.4

0.2

PEAT • drying o wetting

1.0 ,--------------------,

0.8

0.6

TUFF • drying o wetting

1.0 .,.-------------------,

0.8

0.6

0.4

0.2

0

MIX

2 4 6 8

• drying o wetting

10

Water Suction, h (kPo)

12

Fig. 1. Measured (symbols) and fitted (lines) water retention curves of the tested media.

574 da Silva et al.

coefficients of determination of the nonlinear regression (r 2 > 0.99) indicate that the estimation of both drying and wetting curves was accurate for the media tested (Table 2).

Predicted K(h) curves were validated by direct measurement in the range 0 to 2.5 kPa. Mea­sured values of K were in good agreement with predicted K(h) curves (Fig. 2). All three media exhibited high values of Ks (Table 3). However, as suction was increased from 0 to 2.5 kPa, K(h)

1 o' . measured

10° -predicted

10-1

10-2

10-J ..., 10-4

i PEAT c 10-5

E E 101 0 . measured

'-"

~ 10° -predicted

>. 10-1 +-' ·:;:

10-2 :;:; 0 ::J 1 0-J v c 0 10-4

0 TUFF 0 10-5

::J 0 101 L

v . measured >-,

10° -predicted I

10-1

1 o-2

10-J

10-4

10-5 MIX

0.0 0.5 1.0 1.5 2.0 2.5

Water Suction, h (KPa) Fig. 2. Measured (symbols) and predicted (lines) hydraulic conductivity of the tested media.

Table 3. Saturated hydraulic conductivity, K,, of the tested container media, and water flux density, q, at which K, measurements were performed

Medium K, q (cmmin- 1) (cmmin- 1 )

PEAT 4.91 1.47 TUFF 7.32 3.67 MIX 5.42 1.35

decreased by approximately three orders of magnitude for peat and by approximately four orders of magnitude for tuff. In Table 4, the values of (} and K are given for suctions of 0 (saturation), 1, 5 and 10 kPa. These values were considered as the most important to plant growth by De Boodt and Verdonck (1972). The decrease in the predicted value of K as suction increases from 1 to 5 kPa, i.e., over the range of 'easily available water' of peat is of approximately two orders of magnitude. This means that to main­tain the water flux from the medium to the roots prevailing in a container at an average suction of 1 kPa the hydraulic gradient in the root-medium interface would have to increase by the two orders of magnitude when the corresponding suction is increased to 5 kPa. Addition of peat to tuff yielded a medium exhibiting a lesser degree of reduction in hydraulic conductivity with suc­tion when compared to pure tuff.

The results suggest that in addition to O(h), knowledge of K(h) may provide a very useful tool in irrigation scheduling. First, K(h) allows the grower to predict the potential reduction of water fluxes in the medium. For example, if the limiting suction is reduced from 5 kPa to 3 kPa, the corresponding limiting hydraulic conductivity will be increased by approximately a factor of 10. In addition, when potential evapotranspiration reaches its higher levels the plant may exhibit a water stress. Even partial dehydration of the plant may induce stomatal closure, thus limiting photosynthesis when light and temperature would otherwise permit high photosynthetic rates (Huck, 1984). Small irrigation pulses dur­ing periods of high demand could contribute to shift potential uptake rates to considerably high­er values, probably sufficient to reduce the effects of stomatal closure.

Hydraulic properties of peat moss and tuff 575

Table 4. Water content and hydraulic conductivity of the tested media at saturation (h = 0 kPa) and at values of water suction within the range of 'easily available water' and "water buffering capacity" (De Boodt and Verdonck, 1972)

Medium

PEAT TUFF

(J K II h(KPa) (cm 3 em_,) (em min-') (em' em_,)

0 0.901 4.91 0.548 0.587 1.5 X 10- 2 0.191

5 0.327 1.7 X 10-4 0.097 10 0.251 2.2 X 10- 5 0.087

Conclusions

Optimization of irrigation and fertilization in container-grown crops aimed to attain high yields, requires a comprehensive understanding and quantification of the mechanisms that con­trol water transport under saturated and unsatu­rated conditions. The use of a mathematical model to predict the unsaturated hydraulic con­ductivity is a useful tool for the complete charac­terization of the hydraulic properties of container media of horticultural importance. The results of this study showed that sharp variations in hydraulic conductivity occurred in a very narrow suction range (0-2.5 kPa). The sharp decrease in K(h) with increasing suction indi­cates that water in this range may be unequally available to the plant. The common approach to determine water availability in container media (Bilderback and Fontcno, 1987; De Boodt and Verdonck, 1972) may, therefore, provide inaccu­rate predictions as to potential plant response. Improvement of water and nutrient use ef­ficiency largely depends on improving synchroni­zation of supply and demand. Van Noordwijk (1990) concluded that the ratio of 'uptake con­centration' (current nutrient uptake rate divided by current water uptake rate) is a key parameter in the dynamics of nutrient use efficiency. In addition to O(h) and K,, K(h) should be utilized as an important parameter when considering a more efficient irrigation management in green­houses. The observed variations in unsaturated hydraulic conductivity may become particularly important when daily potential evapotranspira­tion reaches its maximum levels. Knowledge of

MIX

K (J K (em min_,) (cm 3 cm- 3 ) (cmmin- 1 )

7.32 0.708 5.42 5.5 X 10-3 0.438 1.1 X 10- 2

2.1 X 10-6 0.250 1.2 x w-' 6.8 X 10-8 0.193 1.6 X 10- 5

K(h) can contribute to avoid or reduce water stress conditions. The addition of peat to tuff modified the hydraulic properties of the mix, when compared to pure tuff. The mix is subject­ed to decreases in water content and in hydraulic conductivity to a lesser degree than pure tuff.

Acknowledgement

The authors wish to thank the Secretary of State for Science and Technology of Portugal for the grant awarded in the framework of the EEC.

References

Beardsell D V and Nichols D G 1982 Wetting properties of dried-out nursery container media. Sci entia Hortic. 17, 49-59.

Beardsell D V, Nichols D G and Jones D L 1979a Physical properties of nursery potting mixtures. Scientia Hortic. 11, 1-8.

Beardsell D V, Nichols D G and Jones D L 1979b Water relations of nursery potting media. Scientia Hortic. 11, 9-17.

Bilderback T E 1985 Physical properties of pine bark and hardwood bark media and their effects with four fertilizers on growth of /lex x 'Nelie R. Stevens Holly'. J. Environ. Qual. 3, 181-185.

Bilderback T E and Fonteno W C 1987 Effects of container geometry and media physical properties on air and water volumes in containers. J. Environ. Hortic. 5, 180-182.

Bunt A C 1988 Media and Mixes for Container-Grown Plants. Unwin Hyman, London. 309 p.

Chen Y, Banin A and Ataman U 1980 Characterization of particles, pores, hydraulic properties and water-air ratios of artificial growth media and soil. Proc. 5th. Int. Congr. Soilless Culture. pp 63-82. Wageningen.

576 Hydraulic properties of peat moss and tuff

De Boodt M and Verdonck 0 1972 The physical properties of the substrates in horticulture. Acta Hortic. 26, 37-44.

De Boodt M, Verdonck 0 and Cappaert I 1974 Determi­nation and study of the water availability of substrates for ornamental plant growing. Acta Hortic. 35, 105-111.

Fonteno W C 1989 An approach to modeling air and water status of horticultural substrates Acta Hortic. 238, 67-74.

Fonteno W C and Bilderback T E 1983 Physical property changes in three container media induced by a moisture extender. Hort Science 18, 570.

Fonteno W C, Cassel D K and Larson R A 1981 Physical properties of three container media and their effect on poinsettia growth. J. Am. Soc. Hortic. Sci. 106, 736-741.

Hanan J J, Olympios C and Pittas C 1981 Bulk density, porosity, percolation and salinity control in shallow, freely draining, potting soils. J. Am. Soc. Hortic. Sci. 106, 742-746.

Huck M G 1984 Water flux in the soil-root continuum. In Roots, Nutrients and Water Influx, and Plant Growth. ASA special publication, No. 49. pp 47-63.

Johnson C R, Ingram D L and Barret J E 1981 Effects of irrigation frequency on growth, transpiration and acclimati­zation of Ficus benjamina L. Hort Science 16, 80-81.

Karlovich P T and Fonteno W C 1986 Effect of soil moisture tension and volume moisture on the growth of Chrysan­themum in three container media. J. Am. Hortic. Sci. 111, 191-195.

Klute A 1986 Water retention, laboratory methods. In Methods of Soil Analysis, part 1. Physical and Miner­alogical Methods. Agronomy Monograph, No.9 (2nd ed.). pp 635-660. Madison, Wisconsin USA.

Klute A and Dirksen C 1986 Hydraulic conductivity and diffusivity, laboratory methods. In Methods of Soil Analy­sis, part 1. Physical and Mineralogical Methods. Agronomy Monograph, No.9 (2nd ed.). pp 687-732. Madison, WI.

Milks R R, Fonteno W C and Larson R 1989a Hydrology of horticultural substrates, I. Mathematical models for mois­ture characteristics of horticultural container media. J. Am. Soc. Hortic. Sci. 144, 48-52.

Mualem Y 1976 A new model for predicting the hydraulic conductivity of unsaturated porous media. Water Resour. Res. 12, 513-522.

Puustjarvi V 1977 Peat and its uses in horticulture. Tur­veteollisuusliitto Ry. Pub. 3, Helsinky. 162 p.

Raviv M, Chen Y and Inbar Y 1986 Peat and peat substitutes as growth media for container-grown plants. In The Role of Organic Matter in Modern Agriculture. Eds. Y Chen and Y Avnimelech. pp 257-287. Martinus Nijhoft, The Hague.

Riviere L M, Foucard J C and Lemaire F 1990 Irrigation of crops according to the substrate. Sci. Hortic. 43, 339-349.

Spomer L A 1974 Optimizing container soil amendment: the 'threshold proportion' and prediction of porosity. Hort Science 9. 532-533.

Van Genuchten M Th 1978 Calculating the unsaturated hydraulic conductivity with a new closed-form analytical model. Research report 78-WR-08. Dept. of Civil Eng., Princeton Univ., New Jersey.

Van Genuchten M Th 1980 A closed-form equation for predicting the hydraulic conductivity of unsaturated soils. Soil Sci. Soc. Am. J. 49, 12-19.

Van Noordwijk M 1990 Synchronisation of supply and demand is necessary to increase efficiency of nutrient use in soilless culture. In Plant Nutrition- Physiology and Applications. Ed. M L van Beusichen. pp 525-531. Kluwer Academic Publishers, Dordrecht.

Wallach R, da Silva F F and Chen Y 1992a Hydraulic characteristics of tuff (Scoria) used as a container medium. J. Am Soc. Hortic. Sci. 117, 415-422.

Wallach R, da Silva F F and Chen Y 1992b Unsaturated hydraulic characteristics of composted agricultural wastes, tuff and their mixtures. Soil Sci. 434-441.

White J Wand Mastalerz J W 1966 Soil moisture as related to container capacity. Proc. Am. Soc. Hortic. Sci. 89, 757-765.

Reprinted from Plant and Soi/154: 119-126, 1993.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 577~582, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-061

Influence of NaCI treatment on Ca, K and Na interrelations in maize shoots

R. IZZO\ A. SCAGNOZZI2 , A. BELLIGN0 1 and F. NAVARI-IZZ0 2

1/stituto di Chimica agraria, Universita di Catania, Catania, /95123, Italy; 2/stituto di Chimica agraria, Universita di Pisa, /-56124 Pisa, Italy

Key words: chlorophyll, NaCI stress, maize, nutrients, water status, Zea mays L.

Abstract

The growth, water status and the uptake of the major ions (K +, Ca Z+, N a+ and Cl ~) were investigated during a 9-day-stress period in seedlings of maize (Zea mays L. cv. Summer II) grown in a half strength Hoagland's 2 solution containing 0.24 M NaCI. NaCl induced a reduction in maize growth and significantly reduced the chlorophyll a, chlorophyll b, and total chlorophyll, the leaf water potential ('l'w) and leaf solute potential ('l'7T), but leaf turgor potential ('l'p) remained constant. In treated seedlings relative leakage ratio (RLR) was greater than in the control. At each sampling date the ion contents in the stressed seedlings were substantially higher than in the control, with the exception of Ca2 + after nine days of treatment. At the end of the experiment K + reached the maximum uptake, accompanied by the lowest level of Na +. At the same date in the stressed seedlings the Ca2 + /K + ratio was more than twice higher than in the control, indicating that the increase of K + might oppose to the uptake of Na +.

Introduction

Many studies have been carried out on the mechanism and nature of salt stress, using differ­ent salts, concentrations and stress periods, as well as different crop species.

The alteration of cell membranes is generally considered to be the first effect of the stress injury, leading, in turn, to the inability of the cell to retain solutes, therefore the uptake and trans­location of micro- and macro-nutrients are af­fected (Izzo eta!., 1991). Nevertheless, no clear conclusive results have been reached. It is ex­tremely significant, therefore, to interprete the relation between salt treatments and the balance of nutrients because of the importance of the nutrients for the osmotic adjustment mechanism (Weimberg, 1987) which can play a role in the crop capacity of overcoming and/ or in adapta­tion to stress conditions. Yeo and Flowers ( 1986) have demonstrated, however, that the alterations

caused by NaCI depend more on the toxicity of Na + and Cl ~ than on osmotic effects.

A visible effect of the salt treatment is a reduction in growth and yield of crops (Izzo et a!., 1991; Navari-Izzo et a!., 1988a,b). Quite different results, however, were reached by El­Kady et a!. (1981) who observed an increase in dry matter with salinity up to 4,000 mg L ~J and the opposite at higher levels of salinity. More recently, we noticed that the dry matter pro­duced by maize shoots decreased progressively with increasing salt concentration, whereas it decreased in roots at 0.16 M NaCI, but not at 0.08 M NaCI (Izzo et a!., 1991). In the same experiment, the uptake and accumulation of nutrients were shown to be affected by salt treatments.

Calcium is necessary since it is the most effective in maintaining membrane integrity, and regulates ion transport (Hanson, 1984) in order to reduce the dangerous effects of salinity on

578 Izzo et al.

plant growth (Cramer et a!., 1986; Kent and Uiuchli, 1985). Calcium has been shown to reduce the negative effects of NaCl by inhibiting Na uptake (Cramer et a!., 1985) and reducing the leakiness of membranes (Leopold and Wil­ling, 1984).

Also potassium, when in adequate amounts, is necessary not only as cofactor for many en­zymes, but particularly for cell expansion (Leigh and Wyn Jones, 1984).

The present experiment, based on results obtained previously, aimed to investigate the response of maize seedlings using a higher con­centration of NaCI. Emphasis was given to the possible Ca2 +, K + and Na + interrelations which might overcome the deteriorative effect of NaCI on maize growth.

Materials and methods

Maize seeds (Zea mays L. Summer II, specific purity 98% and germinability >93%) from Funk's Ciba Geigy were soaked in a Dubnhoff bath for 24 h at 21 ac under continuous shaking and aeration. After imbibition, seeds were germinated in the dark at 24° ± 1 oc for 24 h on moist filter paper in Petri dishes. The dishes were then transferred into a growth chamber with a 21°-15°C day/night temperature, a 16h photoperiod, an 85% relative moisture and a light intensity of 280 JLE/m, obtained with fluorescent (F27T-CW-VHO, Sylvania) and in­candescent (75W, Philips) lights. After 48 h, seedlings were transferred in a half-strength aerated Hoagland's 2 solution ('1'1r = -0.03 MPa), renewed every two days.

Fourteen days after sowing, three replicates of 50 seedlings each were collected. Scutellum and endosperm were removed and roots and shoots were separated. Shoot bases (2 em) were rinsed quickly with distilled water to wash out possible salt surface contamination. Length, and fresh and dry weight of shoots were then determined. Leaf water potential ('l'w) and relative water content (RWC) were measured in 10 seedlings in triplicates, according to a method described previously (Navari-Izzo et a!., 1990). The rela­tive leakage ratio (RLR) was determined accord­ing to Navari-Izzo et a!. (1989) and chlorophyll

extraction followed Moran's method (1982). The remaining seedlings were divided into two sets, one of which was subjected to salt stress by adding NaCl to the growth solution in order to achieve a final NaCl concentration of 0.24 M. The final solution had an osmotic potential of -1.32 MPa, determined by a freezing-point osmometer (Roebling microosmometer, Vogel). The other set of seedlings was maintained in the Hoagland's 2 solution as a control.

Three, 6 and 9 days after stress imposition three replicates of 150 seedlings each were col­lected and analysed for the same parameters determined before the stress imposition.

Three replicates of ten seedlings were sampled on each date to determine pressure-volume curves by using a pressure chamber (Navari-Izzo et a!., 1990).

On each sampling date shoot samples were immediately lyophilized and stored at -20 oc till analyzed. Aliquots were used for residual humidity determination and quantification of K, Na and Ca by a Perkin-Elmer model 373 atomic absorption spectrophotometer, after sample wet digestion with cone. HN0 3 • Chloride ions were extracted and determined according to Binzel et a!. (1987).

The two-way analyses of variance was applied to the data. LSDs at p = 0.01 were also de­termined.

Results

During the 9-day-stress-period, the 0.24 M NaCI ('1'1r = -1.32 MPa) inhibited Zea mays shoot elongation (Fig. 1) and fresh and dry matter production (Fig. 2). Stress conditions induced a reduction in 'l'w, but the decrease in '1'1r de­tected on all sampling dates was apparently sufficient to compensate completely for the de­crease in 'l'w, so that 'l'p remained virtually constant (Table 1). Symptoms of salt injury, such as leaf tip burn and wilting, did not appear.

In the shoots from the treated seedlings cel­lular leakage was higher than in the control, but it did not increase with time (Table 1).

Table 2 shows the values of the chlorophyll content: chlorophyll a was always the most abundant, independent of the treatments. Chlo-

Sodium chloride effect on Ca, K and Na in maize 579

39

~-I LSD= O.Ql

37

35 / ~ 33 c. e 31 ./ ---~ Control -!:0

"' 29

L ---R~r---- Stress "" =

~ 27

~ 25 "'

23

21 LSD= 0.01

19 ___ ..........,

14 17 20 23 26

Days

Fig. 1. Length of Zea mays L. shoots subjected to NaCI treatment during the growth period.

3,5

3 ~ = ~2,5

~ 2 ~ ~ 1,5

8 1 .::. "' 0,5

0 14

..a--------- I LSD = 0.01

~_......-------+--------+1 LSD= 0.01

17 20

Days

23

LSD= 0.01 LSD= 0.01

26

-- Fresh Weight _._...... Fresh Weight _____..._ Dry Weight --~;r-- Dry Weight Control Stress Control Stress

Fig. 2. Fresh and dry weight evolution of Zea mays L. shoot subjected to NaCl treatment during the growth period.

Table 1. Water status and RLR of Zea mays L. seedlings subjected to NaCl treatment during the growth period

Days 'l'w(MPa) 'l'1r(MPa) 'l'p(MPa) RWC(%) RLR

Control Stress Control Stress Control Stress Control Stress Control Stress

14 -0.1a -1.3b l.Zb 98.2c 0.3b 14 + 3 -0.2b -0.3c -1.2a -1.3b l.Oa l.Oa 96.lc 89.3a 0.4c O.Sd 14 + 6 -0.3c -0.4d -1.3b -1.4c l.Oa l.Oa 96.5c 93.0b 0.1a 0.7d 14 + 9 -0.3c -0.4d -1.3b -1.4c l.Oa l.Oa 95.0b 90.6a 0.2b 0.7d

For comparisons among means the analysis of variance test was used. For each component means with different letters arc significantly different at the p = 0.01.

rophylls progressively increased during growth in control seedlings. Three days after the salt treat­ment, stressed and control seedlings showed similar values, but after six, and again after nine

days of treatment, chlorophylls decreased sig­nificantly in the stressed seedlings showing the highest decrease rate after six days.

Chi-a and Chl-b ratios were stable in the first

580 Izzo et al.

Table 2. Evolution of chlorophyll content in shoots of Zea mays L. seedlings subjected to NaCl treatment during the growth period (mg/seedling)

Days Total Chl Chl-a Chl-b

Control Stress Control Stress Control Stress

14 1.3a l.la 0.2a 14 + 3 4.9c 4.6c 3.7c 3.6c l.lb l.Ob 14 + 6 5.3d 1.5ab 4.0d 1.2ab 1.4b 0.3a 14 + 9 5.9d l.la 4.4d 0.8a 1.6c 0.4a

For comparisons among means the analysis of variance test was used. The significance of the letters is the same as in Table 1.

Table 3. Concentrations of Cl , Ca'', Na + and K' (f.Leq/plant) and cationic ratios in shoots of Zea mays L seedlings subjected to N aCl treatment during the growth period

Days Cl- Ca 21 Na+ K+ Ca2 + /K+ Ca 2+ /Na+ K+ /Na +

Control Stress Control Stress Control Stress Control Stress Control Stress Control Stress Control Stress

14 6a 30a 4a 122a 0.24 7.50 30.50 14 + 3 6a 1402d 56b SSe 9b 1019f 234b 379e 0.24 0.22 6.22 o.os 26.00 0.37 14 + 6 Sa llOOc 67b 94d 26c 729e 254b 290c 0.26 0.32 2.5S 0.13 9.77 0.40 14 + 9 Sa 990b 84c 55b 27c 568d 332d 507f 0.25 0.11 3.11 0.10 12.30 0.89

For comparisons among means for each ion the analysis of variance test was used. The significance of the letters is the same as in Table 1.

6-day period, but at the end of the experiment the ratio decreased because of the increase in Chl-b content.

Table 3 shows the contents of Cl-, Ca2 +, Na +, and K + (!Leg/plant) and the cationic ratios in maize shoots during the stress experiment. In the control shoots, K' was always the most abun­dant ion with a concentration always four times as high as that of Ca21 . The concentration of Na + was very low and gradually increased up to 20 d, remaining constant thereafter, although its increase rate was higher than that of Ca2 +. Three days after stress imposition, the plants had absorbed one hundred times as much N a+ as control plants, but afterwards, although Na + uptake remained higher than in the control, became 28 and 21 times as low respectively.

In stressed plants Cl- showed a higher concen­tration than in the control, paralleling the trend of Na'.

Discussion

In the conditions outlined here, NaCI caused slow growth of the maize seedlings resulting in a reduction in shoot length (Fig. 1) and fresh and dry matter production (Fig. 2).

Moreover, shoots were affected by salinity more than roots as shown by their ratio decrease (Izzo et al., personal communication). Cramer et al. (1989) found the opposite in barley, whereas Bogemans et al. (1990) observed the greatest reduction in shoot growth at the latest stage. Evident differences in fresh matter between control and stressed seedlings were observed, indicating a possible direct involvement of tissue water content in the plant response to the treatment. During the experiment, different val­ues of fresh to dry matter ratio were observed in the control and treated shoots (Fig. 2). After an increase due to the intense growth, this ratio had a progressive decrease in the control seedlings.

On the contrary, in treated shoots this ratio did not change in the first three days of the stress imposition, whereas it showed a strong decrease after six days. Since fresh matter remained constant, the decrease in the FM to DM ratio could be ascribed to a decrease in water content. This might lead to an increase in the concen­tration of compounds which might, thus, induce an osmotic adjustment. From the data in Table 1 it is evident that an osmotic adjustment occurred as 'l'w lowered. The water status attained .in the present experiment indicated that the decrease in '¥77 resulted in a virtually complete turgor

Sodium chloride effect on Ca, K and Na in maize 581

maintenance as the plants grew. Thus, the reduc­tion in growth could not have been caused by a sustained turgor loss. This is consistent with the observations of Termaat et a!. (1985) for salt­stressed wheat and barley. Furthermore, since turgor was maintained, the decrease in 'P1r should not have been caused by dehydration, and true accumulation of solute by shoot cells should have occurred. The physiological mainte­nance of adequate turgor could act as an adapta­tive mechanism to avoid leaf water deficits asso­ciated with an excess of salinity.

Salt stress induces such alterations in cell membranes, that their ability to retain solutes is impaired (Sanchez-Diaz et a!., 1982). As a consequence, the electrolyte efflux from the tissue is favoured, thus compromising the shoot vitality. The higher RLR values observed in our stressed seedlings, at any growth stage, indicated increased disorganization and permeability of the membranes (Table 1).

Three days after stress imposition the total chlorophyll contents of the treated plants had the same value as in the control, where chlorophyll increased progressively. In the treated shoots chlorophyll was more than 65% and 25% lower after six and nine days respectively. Although both chlorophyll-a and chlorophyll-b decreased over the whole experimental period, chlorophyll­a appeared to be the more affected (Table 2). In agreement with our results Garcia et a!. (1987) found that chlorophyll content was more reduced in maize seedlings treated with NaCl and this may be due to the complementary action of Na + and Cl- ions on the structure and function of chloroplasts. The decrease in chlorophylls in Vigna mungo following NaCI treatment was ascribed to the suppression of a specific enzyme involved in the synthesis of green pigments (Ashraf, 1989).

The presence of NaCl in the medium resulted in the accumulation of Cl-, which could lead to growth reduction through photosynthesis inhibi­tion (Munns and Termaat, 1986) related to the lower production of chlorophyll (Table 2).

The fourteen-day-old maize shoots presented an ion concentration comparable with that ob­served in previous reports (Izzo et al., 1989; Izzo et al., 1991). Ca 2 +, Na+ and K+ increased progressively during seedling growth, although with a decreasing rate (Table 3). In the treated

shoots, the concentration of all three ions had a dramatic increase always higher than in the control, except for Ca2 +, which at the latest stage showed a significant reduction.

Calcium enhances the selective uptake of K + in the presence of Na + by reducing Na + accumu­lation (Bogemans et al., 1989). A low Ca2 + concentration (Lehaye and Epstein, 1971) or a high Na+ /Ca 2 + ratio (Cramer eta!., 1989) alters the membrane function, increasing its per­meability. The consequence is an increase in Na + and Cl passive transport (Cramer eta!., 1985).

The 0.24 M NaCI used in the present experi­ment was probably high enough to justify not only the initial dramatic concentration of Na +, but also the high values in shoots during growth.

Analyses of maize roots and shoots (Izzo eta!, personal communication) showed that the root concentration of the three cations was much lower than in the shoots, which indicates that they do not accumulate in roots, but are translo­cated to shoots. Sodium is known to be a possible substitute for K in its non-specific func­tions, i.e. the replacement of vacuolar K (Bes­ford, 1978a,b). Thus, a high Na substitution is likely to occur in plants which allow more Na to be translocated to and accumulated in the shoots (Flowers and Lauchli, 1983). Calcium deficiency was reported by Maas and Grieve (1987) in salt-stressed maize shoots. These authors also found that a high Na + /Ca2 + ratio could produce nutritional imbalance and Ca 2 + deficiencies. In­hibition of Ca2+ transport and decrease of Ca2 + concentration in tissues caused by salinity was also observed by Cramer et a!. (1989), which furthermore reported that Ca2 + was displaced by Na + from membranes, thus resulting in a K + efflux. The increase of Ca2 + concentration in treated shoots, till the sixth day of treatment, was probably sufficient to prevent the enhance­ment of Na + uptake which, despite its high levels, continued to decrease during the whole experimental period. Thereafter, also the in­creased K + concentration might be considered as an attempt by the plant to restrict Na + uptake. This should occur especially during the last experimental period, when high levels of K + accompanied the decrease in Ca 2 + and reached the level of Na +.

Also the increase in chlorophyll b, which partially contributes to maintain the Chi-a to

582 Sodium chloride effect on Ca, K and Na in maize

Chl-b ratio values similar to those of the control (Table 2), appears to be affected by the increase in K +. The interrelation among the three cat-

+ + C 2+/K+ d ions, expressed by the K /Na , a an Ca2 + /Na + ratio (Table 3), suggests that ion ratios could be even more important than abso­lute ion concentration in the regulation of up­take, at least at certain ion levels.

References

Ashraf M 1989 The effect of NaCl on water relations, chlorophyll, and protein and proline contents of two cultivars of blackgram (Vigna mungo L.). Plant and Soil 119, 205-210.

Bcsford R T 1978a Effect of replacing nutrient potassium by sodium on uptake and distribution of sodium in tomato plants. Plant and Soil 50, 399-409.

Besford R T 1978b Effect of sodium in the nutrient medium on the incidence of potassium-deficiency symptoms in tomato plants. Plant and Soil 50, 427-432.

Binzel M L. Hasegawa P M, Rhodes D, Handa S, Handa A K and Bressan R A 1987 Solute accumulation in tobacco cells adapted to NaCI. Plant Physiol. 84, 1408-1415.

Bogemans J, Stassart J M and Neirinckx L 1990 Effect of NaCl stress on ion retranslocation in barley. J. Plant Physiol. 135, 753-758.

Cramer G R, Liiuchli A and Epstein E 1986 Effects of NaCl and CaC1 2 on ion activities in complex nutrient solutions and root growth of cotton. Plant Physiol. 81, 792-797.

Cramer G R, Epstein E and Liiuchli A 1989 Na-Ca interac­tions in barley seedlings: Relationship to ion transport and growth. Plant Cell Environ. 12, 551-558.

Cramer G R, Liiuchli A and Polito U S 1985 The displace­ment of Ca2 + by Na + from the plasmalemma of root cells: A primary response to salt stress? Plant Physiol. 79, 207-211.

El-Kady M M, Mansour M A, Abou-El-Seound I and El Shewiith A E 1981 A comparative study on two Mexican wheat varieties and the local variety Giza 155 grown under different levels of salinity. Monoufia J. Agric. Res. 4, 1-21.

Flowers T J and Liiuchli A 1983 Sodium versus potassium: Substitution and compartmentation. In Encyclopedia of Plant Physiology: Inorganic Plant Nutrition. New Series. Vol. 15B. Eds. A Liiuchli and R L Bieleski. pp 651-681. Springer-Verlag, Berlin.

Garda A L, Torrecillas A, Leon A and Ruiz-Sanchcz MC 1987 Biochemical indicators of the water stress in maize seedlings. Bioi. Plant. 29, 45-48.

Hanson J B 1984 The function of calcium in plant nutrition. In Advances in Plant Nutrition. Vol. 1. Eds. P B Tinker and A Liiuchli. pp 149-208. Praeger, New York.

Izzo R Navari-Izzo F and Quartacci M F 1989 Growth and mine,ral content of roots and shoots of maize seedlings in response to increasing water deficits induced by PEG solutions. J. Plant Nutr. 12, 1175-1193.

Jzzo R Navari-Izzo F and Quartacci M F 1991 Growth and mine,ral absorption in maize seedlings as affected by increasing NaCl concentrations. J. Plant Nutr. 14, 687-699.

Kent L M and Liiuchli A 1985 Germination and seedling growth of cotton: Salinity-calcium interactions. Plant Cell Environ. 8. 155-159.

Lehaye P A and Epstein E 1971 Calcium and salt toleration by bean plants. Physiol. Plant 25, 213-218.

Leigh R A and Wyn Jones R G 1984 A hypothesis relating critical potassium concentrations for growth to the dis­tribution and functions of this ion in the plant cell. New Phytol. 97, 1-14.

Leopold A C and Willing P 1984 Evidence for toxicity effects of salt membranes. In Salinity Tolerance in Plants: Strate­gies for Crop Improvement. Eds. R C Staples and G H Toenniessen. pp 67-76. Wiley, New York.

Lynch J and Liiuchli A 1984 Potassium transport in salt­stressed barley roots. Planta 161, 295-301.

Maas E V and Grieve C M 1987 Sodium-induced calcium deficiency in salt-stressed corn. Plant Cell Environ. 10, 559-564.

Moran R 1982 Formulae for determination of chlorophyllous pigments extracted with N,N-dimethylformamide. Plant Physiol. 69, 1376-1381.

Munns R and Termaat A 1986 Whole plant responses to salinity. Aust. J. Plant Physiol. 13, 143-160.

Navari-Izzo F, Izzo R, Bottazzi R and Ranieri A M 1988a Effect of water stress and salinity on sterols in Zea mays shoots. Phytochem. 27, 3109-3115.

Navari-Izzo F, Izzo Rand Quartacci M F 1988b Phospholipid and sterol alterations associated with salinity and water stress in maize roots. Plant Physiol. (Life Sci. Adv.) 7, 137-142.

Navari-Izzo F, Izzo R, Quartacci M F and Lorenzini G 1989 Growth and solute leakage in Hordeum vulgare exposed to long-term fumigation with low concentrations of SO,. Physiol. Plant 76, 445-450.

Navari-Izzo F, Quartacci M F and Izzo R 1990 Water-stress induced changes in protein and free aminoacids in field­grown maize and sunflower. Plant Physiol. Biochem. 28, 531-537.

Sanchez-Diaz M F, Aparicio-Tejo P, Gonzales-Murua C and Pena J I 1982 The effect of NaCl salinity and water stress with polyethylene glycol on nitrogen fixation, stomatal response and transpiration of Medicago sativa, Trifolium repens and Trifolium brachycalcinum (subclover). Physiol. Plant. 54, 361-366.

Termaat A, Passioura J B and Munns R 1985 Shoot turgor does not limit shoot growth of NaCl-affected wheat and barley, Plant Physiol. 77, 869-872.

Weimberg R 1987 Solute adjustment in leaves of two species of wheat at two different stages of growth in response to salinity. Physiol. Plant. 70, 381-388.

Yeo A R and Flowers T T 1986 Salinity resistance in rice (Oryza sativa L.) and a pyramiding approach to breeding varieties for saline soils. Aust. J. Plant Physiol. 13, 161-173.

M.A. C. Fragoso and M.L. van Beusichem (eds.) Optimization of plant nutrition 583-588, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-223

Effects of environment on the uptake and distribution of calcium in tomato and on the incidence of blossom-end rot

P. ADAMS and L.C. HO Horticulture Research International, Littlehampton, West Sussex, BN17 6LP, UK

Key words: blossom-end rot, calcium, greenhouse environment, humidity, root-temperature, salinity, tomato, transpiration

Abstract

Studies of Ca uptake and distribution in relation to environmental variables were used to relate Ca status of tomato fruit to blossom-end rot (BER) incidence. Ca uptake was highly correlated with solar radiation and root temperature. The rate of Ca uptake decreased linearly with increasing salinity. High humidity reduced Ca import by the leaves but increased that by the fruit. While total plant dry weight was reduced more than fruit dry weight by salinity, total Ca uptake and the Ca content of the fruit were decreased similarly. Thus, the concentration of calcium in the fruit was substantially reduced by salinity. The distal half of the fruit contained Jess Ca than the proximal half. The lowest %Ca was found in the distal placenta and locular tissues, where BER first develops. The incidence of BER was often stimulated more by high salinity achieved with the addition of major nutrients than with NaCI. The cause of BER is usually an interaction between the effects of irradiance and ambient temperature on fruit growth and the effects of environmental stress on calcium uptake and distribution within the whole plant.

Introduction

Blossom-end rot is a local deficiency of Ca in tomato fruit. Although this may be caused by dryness or an inadequate supply of Ca in the root zone, it frequently occurs when the mois­ture and Ca contents of the substrate are fully adequate. In these circumstances, the most likely causes of the disorder are poor Ca uptake by the roots and/ or inadequate distribution of Ca to the fruit at a period of high Ca demand.

The uptake of Ca is reduced by osmotic stress (Ehret and Ho, 1986) or by cation competition (Raleigh and Chucka, 1944) in the root zone. As Ca movement in tomato is virtually confined to the xylem, transport of the absorbed Ca to the shoot is either inhibited by high humidity (Adams and Holder, 1992) or salinity (Ho, 1989). While the concentration of Cain the fruit is intrinsically low (about one tenth of that in the

leaves), the transport of Ca within the fruit to the distal half is very poor and is restricted further by high salinity (Ehret and Ho, 1986).

The data reported here show how changes in environmental factors such as light, temperature, humidity and salinity affect both the uptake and distribution of Ca. The tissues most vulnerable to BER are identified from the concentrations of Ca within the fruit.

Materials and methods

The experimental plants were propagated in rockwool cubes, and grown in NFf systems, except where noted otherwise (humidity experi­ment). A fogging system was used to increase humidity and ventilation to reduce it; all humidi­ty treatments were maintained at the same air temperature (Adams and Holder, 1992). The

584 Adams and Ho

actual mean day I night vapour pressure deficits (kPa) achieved were: 0.1/0.1, 0.20/0.15; 0.1/ 0.8, 0.21/0.45; 0.8/0.1, 0.47/0.16; 0.8/0.8, 0.55/ 0.50. Increased salinity was achieved by adding NaCl to the basic nutrient solution, except when macronutricnts were used for comparison with NaCl (Table 3). Then a mixture of Ca(N0 3 ) 2

and KN0 3 was used that supplied N, K and Ca in the same ratios as in the basic solution. All crops were grown under semi-commercial con­ditions using the layering system and received solutions containing nutrients within the follow­ing concentration ranges (mg L - 1): 175-200 N, 30-40 P, 350-400 P, 175-200 Ca, 70-80 Mg, 10-12 Fe, 0.7-1.0 Mn, 0.4-0.5 B, 0.4-1.0 Zn, 0.2-0.3 Cu and 0.05-0.1 Mo. The glasshouse atmospheres were enriched with C0 2 to 1000 f1L L -I until late April, when frequent venting rendered it impracticable.

Plant material, including fruit, was dried for 48 h at 80°C and ground to pass a 2-mm sieve. The Ca content of the ashed (560°C) material was determined by atomic absorption spectro­photometry. K was estimated by flame photo­metry.

Results

Responses to light and temperature and humidity

The relationship between water absorption and Ca uptake was investigated using tomato plants grown in NFT. Water uptake during short periods (hours or a day) was closely related to solar radiation over the range 4 to 13 MJ m -z d- 1 (r = 0.95), i.e., water uptake was stimulated as transpiration increased with irradiance. The uptake of Ca was linearly related to that of water (r = -0.97; Fig. 1). However, the apparent con­centration at which Ca was absorbed decreased from 120 mg L -I to 91 mg L - 1 as the rate of water uptake increased. Ca uptake also in­creased with water uptake as the root tempera­ture was increased from 14 to 26oC (Fig. 2). The apparent concentration of Ca absorption was slightly lower at 14 and 26oC (96 and 98 mg L -I respectively) than at 18 and 22oc (both 104 mg L - 1). Therefore, Ca uptake can be stimulated by

100 0

>- 0 "' "0 90 ..... c 0

"' Ci 80 .....

Ol

E ai -"' 70 "' 0.. ::J

"' 60 u 0

50 0.4 0.6 0.8 1.0 1.2

Water uptake, 1/plant/day Fig. 1. Relation between the uptakes of Ca and water by fruiting tomato plants grown in NIT. The data represent the relationship for daily irradiances inside the glasshouse over the range 4-13 MJ m -z during September.

100 >. (\) /o 26°C "U 90 .........

....... c o 22°C (\)

o/ 18°C 0. 80 ......... Ol

~'C E Q) 70

..::.:: (\)

....... 0. ::J 60 (\)

0 50

0.6 0.8 1.0 Water uptake, !/plant/day

Fig. 2. Effect of root temperature on Ca and water uptake by tomato plants grown in NIT over a period of 10 weeks ending in April.

increasing either the root temperature or transpi­ration rate, but the effects on the ratio of absorbed Ca to water are different.

Environment, calcium uptake and blossom-end rot 585

The distribution of Ca to the leaves and fruit was studied in tomato plants grown in rockwool. Although high humidity restricts Ca distribution to the leaves, the concentration of Ca (%)in the expanding leaves was unaffected by vapour defi­cits (held constant day and night) in the range 0.15-0.65 kPa. However, as the dry weight of the leaves decreased at high humidity, so did the total amount of Ca accumulated per leaf (p < 0.01; Fig. 3). The reduction in leaf size may be related to an inadequate supply of Ca and K, since movement of both nutrients is reduced at high humidity (Adams, 1991). In contrast to the leaves, the %Ca and the total amount of Ca accumulated in the fruit increased with high humidity during the day but was not significantly affected by humidity at night (Table 1 ). The

13 1.0

12 -"' 0.8 ~ 11>

<:::: C1l E 11

0

"' :;·

c 0.6 ~ 11> c 10 0 u

"' u 9 0

0.4

8 0.2

0.1 0.3 0.5 0.7

Vpd, kPa

Fig. 3. Relation between the Ca content of tomato leaves in mid-February (fifth below the top) and the ambient vapour pressure deficit, which was held constant throughout the day and night (cv. Counter grown in rockwool). e, %Ca; o, mg Ca.

'<

leaves and fruit therefore respond differently to changes in humidity.

Responses to salinity

The effect of salinity on the Ca status of the fruit was studied in conjunction with dry matter partitioning. Increasing salinity reduced the total dry weight per plant and that of the fruit, but increased the proportion of the total dry matter in the fruit (Table 2). The same difference in salinity caused a much greater decrease in Ca uptake and in the %Ca and total Ca content of the fruit, but had little effect on the proportion of the total Ca in the fruit (Table 2). Observa­tions from another experiment showed that the rate of Ca uptake was reduced linearly from 143mg d- 1 at 3mS cm- 1 to 88mg d- 1 at 15mS cm- 1 (r= -0.95).

Apart from the common osmotic effect, the source of salinity had a considerable influence on the quality and composition of the fruit. A high proportion of the early fruit grown at high salinity (17mS cm- 1 ) with extra major nutrients developed BER despite the high concentration of Ca in the solution. In contrast, the incidence of BER was negligible when NaCl was used to increase the salinity, even though the concen­tration of Na in solution was excessive (Table 3). Both sources of salinity increased the acidity of the fruit juices to a high level, but additional K from the added major nutrients stimulated extra acid production; this was evident when the amount of acid per fruit was calculated. Surpris­ingly, fruit grown with extra major nutrients did not accumulate more Ca than those grown with NaCl, but they had a higher K content. The data suggest a marked antagonism between K and Ca ions affecting Ca uptake whereas Na ions appear to have little effect.

Table 1. Effect of day and night (din) humidity on the concentration of Ca (%in dry matter) and on the total Ca content (mg) in young tomato fruit in February (20 d after an thesis; cv. Counter grown in rockwool; sown in October)

Ca(%) Caper fruit (mg)

Humidity treatment (din), vpd (kPa)

0.8i0.8

0.066 0.89

0.8i0.1

0.066 0.91

0.1i0.8

0.082 1.13

0.1i0.1

0.079 1.04

Significance, p

Day

0.001 0.001

Night

n.s. (0.07)

586 Adams and Ho

Table 2. Effect of salinity on dry matter and Ca accumulation, and on their distribution to the fruit (cv. Counter; sown in January and sampled in April)

Salinity level (mS em_,) Significance, p

5 12

Total dry matter per plant (g) 165 111 <0.001 Dry matter in fruit (g) 46.7 40.5 n.s.

Total dry matter in fruit (%) 28.3 36.5 <0.001 Ca concentration in whole 1.94 1.52 <0.001

plant dry matter (%) Ca concentration in fruit 0.91 0.64 <0.001

dry matter (%) Total Caper plant (mg) 3196 1688 <0.001 Cain fruit (mg) 42.4 25.8 0.003 Total Cain fruit (%) 1.33 1.53 n.s.

Table 3. Effect of the composition of the nutrient solution on the proportion (%) of harvested fruit with blossom-end rot (BER) and on the chemical composition of the fruit sampled in June (cv. Counter; sown in December). All plants were grown at a constant salinity of 17 mS em - 1 , of which the basic nutrient solution contributed 3 mS em - 1

Average nutrient content of solution (mgL- 1 )

Ca

K Na

Salinity source

Macronutrients NaCl

1570 230

5486 420 120 4500

Harvest period Fruit with BER (%) April 100 0

May 94 0.5 June 9 0

Acidity of fruit Meq/100 mL juice 12.6 11.4

Mineral content of fruit (% in dry matter)

The distribution of Ca in tomato fruit

Meq/fruit

Ca K

The distribution of Ca between the different tissues of mature green fruit ( 45-day old) was assessed by dividing them into equal proximal

4.2 2.9

0.024 0.027 3.27 1.64

and distal halves. The distal half was then subdivided into two parts, pericarp and placenta. The proximal half had the highest concentration of Ca and 64% of the total Ca content (Table 4). The concentration of Ca was lower in the distal

Table 4. Distribution of Cain tomato fruit (cv. Counter; sown in December and sampled in May)

Dry weight Total Ca Ca Total per fruit dry weight (%) per fruit Ca (g) (%) (mg) (%)

Proximal half (complete) 2.11 50.7 0.208 4.38 63.7 Distal pericarp 1.14 27.4 0.131\ 1.65 24.0 Distal placenta and 0.91 21.9 0.094 0.85 12.4

associated locular contents contents

Environment, calcium uptake and blossom-end rot 587

half than in the proximal half, and was lowest in the distal placenta.

Discussion

The uptake of Ca is determined by root function and transpiration rate. Increasing the root tem­perature stimulated Ca uptake in proportion to water uptake (Fig. 2) whereas higher transpira­tion rates increased the rate of water uptake more than that of Ca (Fig. 1). Nevertheless, factors that stimulate water uptake increase Ca uptake.

However, the uptake of Ca does not necessari­ly determine the Ca status of the fruit, as the accumulation of Ca by fruit is inversely related to the transport of Ca to the leaves. Thus, when leaf size and transpiration were reduced by high humidity, the accumulation of Ca by the leaves was decreased (Fig. 3) while the Ca content of the fruit increased (Table 1). Therefore, in order to meet the requirement of Ca for rapid fruit growth, high rates of transpiration should be avoided. Furthermore, as the uptake of water is much higher during the day than at night, high humidity in the day would increase the Ca status of the fruit more than the same humidity at night. Thus, although the proportion of newly absorbed Ca moving into the fruit at night is greater than that during the day, and can be enhanced by high humidity (Ho, 1989), more Ca is absorbed during the day than at night and the increase in Ca in the fruit due to high humidity at night is relatively small.

Salinity reduces Ca uptake mainly by restrict­ing water uptake. However, when the increased salinity is achieved by addition of major nu­trients, Ca uptake may be restricted further by competition from K and possibly from Mg (Raleigh and Chucka, 1944). The very high incidence of BER induced when the salinity was increased with major nutrients may have been due to both a low Ca level and a high concen­tration of free acids in the affected tissues (Table 3). A high concentration of organic acids may reduce the availability of Ca in the tissues and so render the fruit more susceptible to BER. Both the uptake and transport of Ca within the plant (Ehret and Ho, 1986), as well as xylem develop-

ment inside the fruit (Beida and Ho, 1993) are restricted by high salinity. Hence, salinity has a profound effect on the induction of BER.

The lowest concentration of Ca was found in the distal locular tissue (Table 4) rather than in the distal pericarp, where the external symptom of BER occurs. In fact, the locular tissue is where the earliest symptom appears, before it extends into the placenta (internal BER), or to the blossom-end pericarp (external BER; Adams and Ho, 1992). Therefore, the concentration of Ca in this tissue during the early stage of de­velopment is the best index of the Ca status of the fruit in relation to susceptibility to BER.

In this study, we identified the environmental factors causing BER, and these are summarised in Figure 4. The basic cause of BER is a lack of co-ordination between the transport of assimi­lates by the phloem and of Ca by the xylem during rapid cell enlargement in the distal

~ Lir ~ _ -~ T'm'''"'""

Photosynthesis l LEAF ~ Humidity

Transpiration

ROOT

Sucrose transport (Phloem)

Calcium transport (Xylem)

FRUIT Cell

enlargement

~------!Salinity Water

Uptake of water and calcium

Nutrients

Fig. 4. A summary of factors affecting the uptake and distribution of Ca by tomato plants, and the rate of fruit growth.

588 Environment, calcium uptake and blossom-end rot

placenta tissue, i.e., an interaction between the rates of fruit growth and of Ca acquisition at the distal end of the fruit. Whilst changes in the environment have a marked influence on the incidence of BER, genetic susceptibility is also a major cause of the disorder (Adams and Ho, 1992).

References

Adams P 1991 Effect of diurnal fluctuations in humidity on the accumulation of nutrients in the leaves of tomato (Lycopersicon esculentum). J. Hortic. Sci. 66, 545-550.

Adams P and Ho L C 1992 The susceptibility of modern tomato cultivars to blossom-end rot in relation to salinity. J. Hortic. Sci. 67, 827-839.

Adams P and Holder R 1992 Effects of humidity, Ca and salinity on the accumulation of dry matter and Ca by the leaves and fruit of tomato (Lycopersicon esculentum). J. Hortic. Sci. 67, 137-142.

Beida R M and Ho L C 1993 Salinity effect on the network of vascular bundles during tomato fruit development. J. Hortic. Sci. 68, 557-564.

Ehret D L and Ho L C 1986 Translocation of calcium in relation to tomato fruit growth. Ann. Bot. 58, 679-688.

Ho L C 1989 Environmental effects on the diurnal accumula­tion of 45 Ca by young fruit and leaves of tomato plants. Ann. Bot. 63, 281-288.

Raleigh S M and Chucka J A 1944 Effect of nutrient ratio and concentration on growth and composition of tomato plants and on the occurrence of blossom-end rot of the fruit. Plant Physiol 19, 671-678.

Reprinted from Plant and Soill54: 127-132, 1993.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 589-592, 1993. © 1993 Kluwer Academic Publishers. Printed in the Netherlands. PLSO IAOPN-002

Complete environmental effluent disposal and reuse by drip irrigation

GIDEON ORON and JOSE BELTRAO Ben-Gurion University of the Negev, The Institute for Desert Research, Kiryat Sde-Boker 84993, Israel and Algarve University, Department of Agricultural Engineering, Rue de Santo Estevao, 32, 8700 Olhao, Faro, Portugal

Key words: bacteria, contamination, drip irrigation, effluent, maize, sprinkle irrigation

Abstract

Two drip irrigation systems for on-surface and subsurface effluent application were installed in sweet corn (Zea mays L. var. saccharatum) experimental fields. Effects of secondary effluent on yield and bacterial contamination in the soil and the corn plants were studied. Results were compared with those obtained for sprinkle irrigated plots. Bacterial contamination of the crop was maximal under sprinkle irrigation and minimal under drip application.

Introduction

The intense use of effluent for irrigation at­tracted public awareness of environmental pollu­tion and the impact on water quality (Asano and Mills, 1990; Blumenthal, 1989; Gamble, 1986; IAWPRC, 1991; Rose and Gerba, 1990; Smith, 1982). It was mainly due to sprinkle irrigation being the primary application method (Ward et a!., 1989). A possible remedy for this conflict is to use the treated wastewater with minimal pollution risk via subsurface and on-surface drip systems (Bresler, 1977; Chase, 1985; Oron and Karmeli, 1979; Oron et a!., 1986; Oron and DeMalach, 1987; Oron et a!., 1990). Secondary wastewater disposal and reuse is subject to various environmental and health criteria which are regularly defined for each region and pur­pose of utilization (Asano et a!., 1992; ISQW, 1981). Subsurface irrigation provides technologi­cal options and can as well satisfy environmental control constraints when effluent is disposed.

The advantages of subsurface application of effluent include the following: (a) reduced evapo­ration losses due to the dryness of the soil surface; (b) reduced generation of run-off; (c) better control of weeds due to their low germina-

tion rate; (d) improved traffic and maneuver conditions for the agricultural machinery equip­ment; (e) minimal environmental pollution due to restricted flow towards groundwater (con­trolled irrigation in upper soil layers only) and absence of aerosols generation. The effluent applied under on-surface drip irrigation is ex­posed to solar radiation which intensifies die­away of the contamination organisms.

Materials and methods

The hypothesis that subsurface drip application of wastewater for irrigation of processing and raw eaten vegetables and for application in public green entertainment sites, was examined last years in a series of fields experiments ( Oron et a!., 1990). The experiments were conducted in the commercial site of RM farm, south-east of the city of Beer-Sheeva, Israel. Secondary ef­fluent from facultative ponds have been applied for irrigation of a variety of crops, and was used in the experiments. Standards methods were applied for water analysis (APHA, 1981). Con­taminating organisms analysis was carried on in official public laboratories, utilizing conventional

590 Oron and Beltrao

Table 1. Constituents concentration range in the effluent applied for irrigation

procedures. Dissolved BOD 5 in the effluent applied is around 40 mg L -I and nutrients con­tent is sufficient to save artificial fertilization (Table 1). Effluent quality is monitored monthly.

Constituent 1988 (mg L - 1 )

Total COD 281-426 Ammonia (NH,) 33-47 P04 22-42 Na 218-263 Ca 95-105 Mg 36-41 K 28-33 TSS 90-121

*One sample only.

1989 (mg L - 1 )

199-436 29-64 32-39

227* 82* 49* 20* 60-149

Sweet corn (Zea mays L. var. saccharatum) was raised by conventional practices. The size of each plot was about 1000 m2 and it was divided into two replications. The corn was planted at row spacing of 0.96 m and one lateral drip tube serving one and two corn rows. Various subsur­face drip layouts were examined (Fig. 1). In order to cover the whole range of effluent application methods, an on-surface drip system,

"[t-il 2 11~---~ 2 11~----~2 111----i 2 1 : One lateral per row 2: One lateral per two rows

Depth (em]

0

10

20

30

... ; ;t- 1.::5 ... ; l+- 1.0 ... ; l+- 1.~

1+--17.0___.: :.--11.0 --+; l-11.o-: :..,._11.0 -t;

72.0

0 0 ON SUB

all values in meters

One lateral per row One lateral per two rows In on-surface sections In subsurface sections

ON

SUB

1.92 m -----------+ Fig. 1. Cross-section of soil profile with the position of the laterals.

and conventional sprinkle irrigation were ex­amined as well. In one additional plot high quality fresh water was applied by an on-surface drip system (control treatment). Fertilizers were injected in the control treatment at a rate similar to nutrient content in the applied effluent. Com­pensating emitters, with a flow rate of 2.3 L h - 1

were spaced 100 em apart on the laterals in all treatments and installed about 0.30 m below the soil surface . The experimental drip plots were irrigated twice a week and the sprinkle plot every ten days. The amount of effluent applied was determined using the data from class "A" pan data and was in the range of 6 to 9 mm I day. The pan coefficient was adjusted to the physio­logical stage of the plants development and was between 0.4 and 1.2 (Oron et al., 1988). Conse­quently, the irrigation shift for drip irrigation was 10 to 15 hours and for sprinkle irrigation (sprinklers of about 1500 L h - 1 and at spacing of 12 by 18 meters) around 12 hours . Total seasonal amount of effluent (water) applied was around 7000 m3 ha - I, were approximately 1300 m3 ha - 1

were applied by sprinkler irrigation for germina­tion prior to shifting to drip application. Accord­ingly, the seasonal amount of nitrogen applied was between 240 and 280 kg ha - I.

Results and discussion

The monitored parameters included bacteria concentration in the soil, on the leaves of the plants and on the grains of the cobs (for the analysis the husk was taken off). Generally, the corn yield in these experiments was better under lateral spacing of 0.96 m (Fig. 2). Bacteria and

0.96/FW 0.96/0N 0.96/SB 1.92/FW 1.92/0 N 1.92/SB

Tube Spacing, Location & Water Quality

1!311 Cob Wot WolgM ~Cob Dry Wolght

Fig. 2. Corn yield under various drip lateral locations.

Effluent for irrigation 591

Table 2. Total and fecal coliform count in the effluent applied at RM farm , Israel

Source Total coliform Fecal coliform (#100mL- ' ) (#lOOmL- 1 )

Fresh water Control head 0 0 Emitter outlet 0 0

Raw wastewater 5 X ]09 1 X 109

Treated wastewater Before filter of control head 3 X 105 7 X 104

After filter of control head 3 X 105 8 X 104

Emitter outlet 5 X 105 4 X 104

virus concentrations were monitored in the ap­plied effluent and the experimental plots (Table 2) .

Although sampling was conducted few days after irrigation termination , bacteria were de­tected in the various locations of the field and the plants . Maximal bacteria concentration was found under sprinkler irrigation on the grain cobs and the leaves (Fig. 3 and Table 3). High bacteria concentration was also found at soil depth of 0.30 m under subsurface application. Limited bacteria concentration was detected on the cobs under subsurface effluent application and fresh water irrigation, probably due to drift effect of aerosols from adjacent fields sprinkle irrigated with effluent. Minimal bacteria concen­tration on the cobs was detected in the on­surface drip effluent treatment. This last detec­tion can be attributed to rapid decay processes , due to direct exposure to solar radiation. These findings coincides with previous findings which

Ftosh Subsurfaco Onsurfaco Sptirtk.lo

Wa!er Quality and Emiuer Lee arion

Fig. 3. Contaminat ion of cobs' kernel. 1989.

592 Effluent for irrigation

Table 3. Bacterial contamination of the leaves, 1989

Date Treatment Fecal coliforms mean CPU-plant

6Aug. Freshwater <1.0 1989 Subsurface ND

On-surface ND Sprinkler 7.5 X 102

2 Sep. Freshwater ND 1989 Subsurface 4.2 X 103

On-surface 4.2 X 103

Sprinkler 8.6 X 103

emphasize the importance of humidity on the survival of pathogenic bacteria in the soil or on the plants ( Gerba et a!., 1975; Powelson et a!., 1990). Consequently the results indicate that drip irrigation with effluent is a promising technology to simultaneously satisfy water and nutrients demands and avoid pollution problems.

References

APHA 1981 Standard methods for the examination of water and wastewater. 15th edition. American Public Health Association, Washington, DC. 1115 p.

Asano T and Mills R A 1990 Planning and analysis for water reuse projects. J. Am. Water Works Association, 38-47.

Asano T, Richard D, Crites R Wand Tchobanoglous G 1992 Evolution of tertiary treatment requirements in California. Water Environ. Techno!. 4, 36-41.

Blumenthal U J, Strauss M, Mara D D and Cairncross S 1989 Generalized model of the effect of different control mea­sures in reduction health risks from wastewater reuse. Water Sci. Techno!. 21, 567-577.

Bresler E 1979 Trickle-drip irrigation: Principles and applica­tion to soil-water management. Adv. Agron. 29, 343-393.

Chase R G 1985 P application through a sub-surface trickle system. Proceedings of the IIIrd International Drip/Trickle Irrigation Congress, ASCE, 1. 393-400.

Gamble J 1986 A trickle irrigation system for recycling residential wastewater on fruit trees. HortScience 21, 28-32.

Gerba C P, Wallis G and Nelnick J L 1975 Fate of wastewater bacteria and viruses in soil., J. lrrig. Drain. Div. ASCE 101 (IR3), 157-174.

IAWPRC 1991 Study group on health related water micro­biology, bacteriophages as model viruses in water quality control. Water Res. 25, 529-545.

Fecal coliforms Fecal coliforms range-CPU no. of positive per plant samples

<1 1 of 1 ND Oof3 ND Oof3 <8.3 X 102 2of2

ND Oof 1 <1.2 X W' 1 of3 <1.2 X 104 2of2 <1.2 X 104 2 of2

ISQW 1981 Israel standards for quality of wastewater effluent to be reused for irrigation of agricultural crops. State of Israel, Israel Public Health Law No. 4263, paragraph 65.

Oron G and Karmeli D 1979 The flow regime in the root zone around a subsurface emitter. Israel J. Techno!. 17, 95-101.

Oron G, DeMalach Y and Bearman J E 1986 Trickle irrigation of wheat applying renovated wastewater. American Water Resources Association (AWRA), Water Resources Bulletin 22, 439-446.

Oron G and DeMalach Y 1987 Response of cotton to treated domestic wastewater applied through trickle irrigation. Irrig. Sci. 8, 291-300.

Oron G, DeMalach Y, Hoffman Z, Plazner N, Keren Y and Hartmann H 1988 Trickle irrigation of sweet corn applying renovated wastewater. Ben-Gurion University of the Negev, The Institute for Desert Research, Kiryat Sde­Boker, Israel. 64 p.

Oron G, DeMalach Y, Hoffman Z, Keren Y, Hartmann H and Plazner N 1990 Waste-water disposal by sub-surface trickle irrigation. Proc. 15th biennial conference of the IAWPRC, Kyoto, Japan. pp 2149-2158.

Oron G, De Malach Y, Hoffman Z and Manor Y 1992 Effect of effluent quality and application method on agricultural productivity and environmental control. Water Sci. Tech­no!. 26, 1593-1601.

Powelson D K, Simpson J R and Gerba C P 1990 Virus transport and survival in saturated and unsaturated flow through soil columns. J. Environ. Qual. 19, 396-401.

Rose J Band Gerba C P 1991 Assessing potential health risks from viruses and parasites in reclaimed water in Arizona and Florida, USA. Water Sci. Techno!. 23, 2091-2098.

Smith M A 1982 Retention of Bacteria, Viruses and Heavy Metals on Crops Irrigated with Reclaimed Water. Aus­tralian Water Resources Council, Canberra, 308 p.

Ward R L, Knowlton DR, Stober J, Jakubowski W, Mills T, Graham P and Camann D E 1989 Effect of wastewater spray irrigation on rotavirus infection rates in an exposed population. Water Res. 23, 1503-1509.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 593-596, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-014

Effect of salinity on the nutritional level of the avocado

E. LAHAV, R. STEINHARDT and D. KALMAR Agricultural Research Organization, The Volcani Center, Bet Dagan 50250, Israel

Key words: avocado, Persea americana Mill, rootstocks, salinity

Abstract

A long-term salinity experiment is conducted in an avocado plantation in the Western Galilee, Israel. The treatments are: four levels of salinity (90, 250 and 400 mg L -I and an adjustable concentration of 250-400 mg L -I of chloride in the irrigation water) and two water amounts (85% and 115% of the recommended amount). In addition, another treatment includes an increased level of nitrogen in the irrigation water. The experiment was conducted on two avocado cultivars ('Ettinger' and 'Hass') grafted on three rootstocks (two West-Indian and one Mexican). Only interim results of treatments and rootstocks effect on leaf nutrients are reported. As expected Cl and sometimes also Na levels increased in the leaves with increasing salinity levels. The chloride level in the West-Indian salt-resistant rootstocks was almost one third of that in the Mexican rootstock. Additional applied N did not affect N levels but significantly reduced Cl concentration in the leaves. This reduction occurred mainly when salinity was raised from medium to high levels. An interesting relationship was recorded between Mn levels and yield.

Introduction

Avocado trees are considered extremely sensi­tive to salinity in the soil solution. Damage attributed to both chloride and sodium has been reported (Kadman, 1963, 1964). During the last decade, the salinity of irrigation water in Israel has been increasing continuously. Therefore, the threshold for maximal salinity for avocado is being examined; a level is desired at which no significant damage to the avocado trees will be caused.

Avocado sensitivity to salts, characterized by leaf-tip burn, was already reported by Haas (1928). He also reported the relationship be­tween leaf-tipburn and chloride accumulation in the leaves and between Cl content and the irrigation and fertilization regime. Salinity dam­age to avocado trees increased when not enough water was supplied without sufficient leaching; it was also increased by lack of nitrate.

Oppenheimer (1947) has shown the differ-

ences among various horticultural avocado races, in their tolerance of NaCI. In general, West­Indian hybrids showed higher tolerance than Mexican varieties. The objectives of the present research are to measure the damage caused by salinity to avocado trees grafted on sensitive (Mexican) and resistant (West-Indian) avocado rootstocks and to study its possible reduction by means of appropriate irrigation and fertilization regimes. The following report concentrates ex­clusively on the effects of salinity on the nu­trients in the avocado tree (Steinhardt et a!., 1989, 1991).

Methods

The soil was a grumusol with 62% clay, 52% pore space, 5% lime and pH 7.5. Soil water contents at field capacity and wilting were: 39% and 25% (kg water/kg soil) respectively. The plantation was established in 1984 with 'Ettinger'

594 Lahav et al.

and 'Hass' trees grafted on either Mexican (sensitive to salinity) or West-Indian (resistant to salinity) rootstocks. The treatments, in a factori­al design comprised irrigation at two levels: 85% and 115% of the recommended water amount. Each irrigation treatment was combined with four levels of salinity (90, 250, and 400 mg L - 1

and a changing concentration of 250 -400 mg L -l) of Cl ion in the irrigation water. Salts were applied as NaCI and CaC1 2 • SAR never exceeded 4.

All treatments included continuous nitrogen fertilization in the form of (NH4 ) 2SO 4 and KN0 3 at a rate of 25 mg L - 1 • The ninth treat­ment included increased nitrogen application accompanying the low irrigation and medium­salinity level of 100 mg L - 1 N (as NH4N03 ).

Avocado leaves were sampled every summer as recommended (Lahav and Kadman, 1980), in order to assess the nutritional status of the trees.

Results and discussion

By the third year of the experiment a direct relationship had been found between chloride accumulation in the leaves and in the soil (Fig. 1 ). Trees grafted on Mexican rootstock accumu­lated five times more Cl than trees grafted on West-Indian rootstocks. The Cl contents in the Mexican rootstock undergoing the low-salinity treatment was almost equal to those in West­Indian rootstocks grown at high salinity levels. The Cl concentration was not found to be influenced by the water amount or the increased N application. Similar results were obtained during the fourth and fifth years but there was also some influence of the irrigation regimes. The increased water amount resulted in a small increase of Cl in the leaves on the Mexican rootstock. This effect is difficult to explain and needs further investigation; it might be attribut­able to lack of Cl leaching in the grumusol soil. The high clay content (above 60%) of this soil would cause excess of water and reduced aera­tion (Steinhardt et a!., 1989).

Unexpectedly, salinity increased Na levels, only slightly and only in leaves of trees grafted on one of the West-Indian (Ein Harod) root­stocks.

Chloride

(% d.w.) 0.9

t..tex:lc::o

"'

0.7 .... "' "' ....

0.5

Electrical conductivity (dS/m)

Fig. 1. Effects of salinity, rootstock and water amount (full symbols = surplus supply) on chloride concentration in the leaves.

Generally, nitrogen concentration was reduced in parallel with the increased salinity. This might be related to some damage caused to the roots, which affected their development and uptake ability. It seems that 'Hass' trees were suffi­ciently nourished since no deficiency in the major elements was found (Table 1 ). This is clear from the observation that the increased water amount combined with increased fertilizer application did not affect any of the nutrients. Even the increased N application did not in­crease N content in the leaves. However, the significant effect of the increased N on Cl con­centration in all rootstocks is remarkable. The increase in Cl uptake resulted from the increas­ing salinity from 90 to 250 mg L -I was reduced by almost half. Similar reductions of Cl in avocado leaves because of nitrogenous fertiliza­tion have been reported both in the plantation (Brom eta!., 1984) and in potted plants (Baret a!., 1987).

The rootstocks affected P, K, Zn and Fe in the leaves. Potassium levels were also affected by salinity; increased salinity caused increased K content. These effects may indicate a specific

Effect of salinity on the nutritional level of the avocado 595

Table 1. Effect of salinity and supplementary nitrogenous fertilization on the nutrient concentrations(% d.w.) of avocado leaves cv. 'Hass' (Avg. for 1988-1989) 1

Nutrient Rootstock Chloride concentration (g m - 3 )

90 250 250 + N3 250 + 400 400

Cl Ein Harod (W.I.) 0.12b 0.38, 0.19b 0.33, 0.41, Maagan Michael (W. I.) 0.13b 0.41, 0.24b 0.39, 0.50, Mexican 0.45d 1.05b 0.74, 1.07 b 1.27,

Na' Ein Harod (W. I.) 0.0058b 0.0123, 0.0120, 0.0100, 0.0099, Maagan Michael (W. I.) 0.0095 0.0073 0.0073 0.0091 0.0103 Mexican 0.0124 0.0126 0.0110 0.0148 0.0125

N Ein Harod (W. I.) 2.11, 2.06,b 2.05,b 1.97b 2.01,b Maagan Michael (W. I.) 2.07 2.12 2.09 2.02 2.12 Mexican 1.97b 1.97 b 2.01,b 1.96b 2.07,

p All rootstocks 0.108 0.110 0.101 0.108 0.106

K All rootstocks 6.9b 7.5, 7.6, 7.3, 7.5,

1 Values for the same rootstock followed by different letters differ significantly at the 0.05 level. 2 Sodium concentration for 1989 sampling only. 3 Values for supplementary nitrogen (100 mg L -I) and reduced irrigation only in contrast to the two irrigation treatments in all other analyses.

physiological defensive barrier against salinity, as found by Ben Hayyim (1987), who reported that citrus cells resistant to salinity contained more K than regular cells.

Some of the most interesting results relate to the manganese levels. There were significant differences among the rootstocks; trees grafted on Mexican rootstock contained much less Mn than those grafted on the West-Indians. Differ­ences in Mn levels according to rootstocks were reported by Reuveni and Raviv (1981).

800

;;;-• WEST-INDIAN "' "' ROOTSTOCK .... -.... ... MEXICAN "' 600 ....

ROOTSTOCK 5 "' • COPY(ll.l.) '=-

TREE Q

ol 400 0 WEST-INDIAN

+NITROGEN ;::

"' 6 MEXICAN > +NITROGEN ;::

< 200 _, 0 COPY(ll.l.) " +NITROGEN

,. " "'

0 0

A highly significant relationship was found between Mn concentration and productivity. (Fig. 2). Falcon ct a!. (1984) reported that reduction in the soil pH results in an increased Mn level in 'Hass' avocado leaves. It is therefore presumed that the difference in Mn uptake among the rootstocks and the influence on avocado productivity are connected with the rootstock ability to increase soil pH in the rhizosphere. Soil pH and Mn uptake are in­creased after fertilizing with ammonium fertiliz-

•

• I

0

• •• • .. 6 • ... 0

... ...... ... R~0.81

150 300 450 600

LEAF MANGANESE CONTENT

Fig. 2. 'Hass' 6 years cumulative yield vs. leaf manganese content (mg L I).

596 Effect of salinity on the nutritional level of the avocado

ers (Marschner, 1988). This might explain the increased Mn levels when supplementary nitro­gen was applied. There is no doubt that basic research is required in order to enable these observations to be used for rootstock selection and/ or to increase productivity m avocado plantations.

References

Bar Y, Kafkafi U and Lahav E 1987 Nitrate nutrition as a tool to reduce chloride toxicity in avocado. South Africa Avocado Grower's Assoc. Yrb. 10, 47-48.

Ben-Hayyim G 1987 Relationship between salt tolerance and resistance to polyethylene glycol-induced water stress in cultured citrus cells. Plant Physiol. 85, 430-433.

BromM, Sne M, Hausenberg I, Har Gani Hand Ben Yaacov A 1984 Avocado irrigation under various levels of salinity and nitrogen. Tel Isaak 1982/3 Rep. Agric. Res. Org. Bet Dagan, Israel. (Int. Report, In Hebrew).

Falcon M F, Fox R Land Trujillo E E 1984 Interactions of soil pH nutrients and moisture on phytophthora root rot of avocado. Plant and Soil 81, 165-176.

Haas A R C 1928 Relation of chlorine content to tipburn of avocado leaves. Yrbk. Calif. Avocado Soc. 1928: 57.

Kadman A 1963 The uptake and accumulation of chloride in avocado leaves and the tolerance of avocado seedlings under saline conditions. Am. Soc. Hortic. Sci. Proc. 83. 281-286.

Kadman A 1964 The uptake and accumulation of sodium in avocado seedlings. Am. Soc. Hortic. Sci. Proc. 84. 179-182.

Lahav E and Kadman A 1980 Avocado fertilization. Intern. Potash Inst. Bull. 6 Worblaufen-Bern, Switzerland. 123 p.

Marschner H 1988 Mechanisms of manganese acquisition by roots from soils. In Manganese in Soils and Plants. Eds. R D Graham, R J Hannam and N C Uren. Kluwer Academic Publishers, Dordrecht.

Oppenheimer Ch 1947 The acclimatisation of new tropical and subtropical fruit trees in Palestine. Bull. Agric. Res. Sta. Rehovoth No. 44, 184 p.

Reuveni 0 and Raviv M 1981 Importance of leaf retention to rooting of avocado cuttings. J. Am. Soc. Hortic. Sci. 106, 127-130.

Steinhardt R. Kalmar D and Lahav E 1991 Response of avocado trees to salinity and management of irrigation water. Res. Rep. for 1990/91. Agric. Res. Org. Bet Dagan, Israel. (In Hebrew).

Steinhardt R, Shalhevet J, Kalmar D and Lahav E 1989 Response of avocado trees to salinity and management of irrigation water. Interim report of the Akko experiment. Alon Hanotea 43, 853-865 (In Hebrew, with English summary).

M.A. C. Fragoso and M.L. van Beusichem (eds.) Optimization ofplant nutrition 597-601, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-069

Interaction of salinity and enhanced ammonium and potassium nutrition in wheat

A. SHAVIV and J. HAGIN Faculty of Agricultural Engineering, Technion, Israel Institute of Technology, Haifa, Israel

Key words: ammonium nutrition, potassium nutrition, salinity, Triticum aestivum L., wheat

Abstract

Wheat (Triticum aestivum L.) was grown to maturity in a pot experiment in a calcareous silty sand soil. N was applied at two levels as granulated N - P fertilizers, amended or not with nitrification inhibitors (1% and 5% DCD, 1% N-serve). Potassium as KCl was given at three levels of application. P was applied at a uniform rate. Two levels of salinity were obtained by using the soil as such (EC =

0.3 mmho/ em) and by adding NaCI to the same soil (EC = 2.4 mmho/ em). 1% DCD and 1% N-serve treatments gave significantly higher wheat grain yields and N-uptake than the other ones. Nitrate content of leachates indicated a prevalent nitrate nutrition in the treatment without nitrification inhibitors. The 5% DCD treatment showed a yield depression. In the lower N level treatments, a significant yield increase, generated by 1% DCD and N-serve was found in the salinized soil as compared to the non-saline soil. Soil salinity reduced N-uptake when nitrification inhibitors were not present. In treatments having the inhibitors, N-uptake was equal or greater in the salinized than in the non saline soil. An enhanced ammonium nutrition increased the P uptake.

Introduction

The beneficial effect of an enhanced ammonium nutrition, namely a ratio of ammonium to nitrate nitrogen of about 1 : 1 to 1: 2, on yields and on accumulation of reduced-nitrogen (protein) of several crops has been documented (Camberto and Bock, 1989; Hageman, 1984; Olsen 1986a; Shaviv et a!., 1987). Theoretical reasoning and experimental evidence indicate an improved beneficial effect by an enhanced application of potassium, due to its role in ammonium assimila­tion and transport (Hagin et a!., 1990; Olsen 1986b; Shaviv and Hagin, 1988).

There arc indications that an enhanced am­monium and potassium nutrition, under saline conditions, has even a stronger effect on yield than under normal soil conditions. Wheat grown in saline soil with regular nitrate-nitrogen nutri­tion may fail nearly completely, while with a

mixture of nitrate and ammonium nitrogen and additional potassium it may produce an accept­able yield (Shaviv et a!., 1990). A possible explanation for the effect of a mixed ammonium­nitrate nitrogen nutrition with an ample supply of potassium may be by the antagonistic effect of ammonium and potassium on sodium uptake and that of nitrate on chloride uptake (Bcn-Hayyim and Goffer, 1988; Hclal and Mengel, 1979).

A higher availability to plants of phosphates when applied with ammonium may be expected, because of the acidifying effect of ammonium nutrition (Shaviv et a!., 1990).

In the present greenhouse experiment, the effect of an enhanced ammonium and potassium nutrition on wheat yield and N accumulation, under moderate salinity conditions, was tested. The nutrients were applied in the form of granu­lated N-P fertilizers amended with nitrification inhibitors.

598 Shaviv and Hagin

Materials and methods

The experimental soil, classified as equivalent to Xeroftuvent, was collected from the Bessor area in the Negev. It is a calcareous (13.3% CaC0 3 )

silty sand, low in organic matter (0.22% C), with a pH of 8.0 and EC of 0.3 mmho/cm, both in a soil paste.

Part of the soil was salinized with NaCl, 4.0 g/ pot. The electrical conductivity of the salinized soil was 2.4mmhos/cm and the pH 7.7.

The soil was placed into 3 dm3 pots. Wheat (Triticum aestivum), cv. Beth Hashita was sown at the end of December 1990 and harvested when the grain was ripe at the beginning of May 1991. The grain was separated from the vegeta­tive parts and yields of both components were measured.

Grain obtained from each pot was analyzed separately. The material was digested according to Thomas et al. (1967). N and P concentrations were measured in an autoanalyzer and K in a flame photometer. Nutrients uptake was calcu­lated by multiplying concentration with yield. The vegetative parts from the four replicates were combined and analyzed. The uptake of nutrients by the vegetative parts is much smaller than by the grain. Therefore, these results are not presented.

Water was added frequently, by weight, to moisture field capacity. Three times during the growth period, on Jan. 22, Feb. 14 and Mar. 12, water was added in excess, to get about 200 mL leachate. The leachate was collected and ana­lyzed for its ammonium and nitrate nitrogen concentrations.

The basic fertilizer was a 10-10-0 granulated

fertilizer, supplied by Fertilizers and Chemicals Ltd. The fertilizer contained the N in an am­monium form. P was applied at a uniform rate of 0.52 g P/pot. At the lower N application level P was supplemented as SSP.

To test the nitrogen and nitrification effects, N was applied at two levels, 0.8 and 1.2 g N /pot. K as KCl was applied at a uniform level of 0.52 g K/pot. Nitrification was inhibited by additions of Dicyandiamide (DCD) 1% and 5% and N -serve 1%, calculated on applied N.

To test the potassium effect, N and P were applied at uniform levels, 0.52 g P/pot and 1.2 g N + 1% DCD/pot. K (KCl) was applied at three levels 0.26, 0.52 and 0.78 g g K/pot.

Statistical analysis was done by the SAS proce­dure.

Results

Yield of wheat grain is presented in Table 1. All treatments with 1% DCD and almost all with 1% N-serve gave significantly higher grain yields than those not receiving a nitrification inhibitor. It may be assumed, according to previous work (Shaviv and Hagin, 1988) and data presented in Table 5, that in the 1% DCD and 1% N-serve treatments both ammonium and nitrate were present in the soil solution, while in the treat­ment not receiving a nitrification inhibitor most of the ammonium was nitrified. Application of DCD at 5% generated a yield depression. In these treatments the plants had probably mainly an ammonium nutrition, although, a toxic effect of DCD cannot be overruled.

Interaction between salinity treatments and N-

Table 1. Yield of wheat grain (g/pot) in nitrification inhibitors and potassium amended soils

Nitrification K N (g/pot) N (g/pot) inhibitor (g/pot)

0.8 1.2 0.8 1.2 Non-saline soil Salinized soil

None 0.52 17.1 hi 17.2hi 17.7gb 18.2 def 1%DCD 0.52 18.7 bed 18.7 bed 19.3 b 18.9 be 5%DCD 0.52 16.7i 15.3 j 17.4 gh 14.1 k 1% N-serve 0.52 18.9 be 19.1 b 20.0 a 18.4 cde 1%DCD 0.26 19.1 b 18.1 def 1%DCD 0.78 18.0 efg 18.5 cde

Means having different letters are significantly (0.05 level) different.

Salinity in relation to ammonium and potassium nutrition 599

levels is significant. Comparison of the 0.8 g N treatments in saline and non-saline soils shows a significant yield increase in the salinized soil, generated by 1% DCD and N-serve. This indi­cates that with a limited supply of nitrogen, a mixed ammonium and nitrate nitrogen nutrition in the presence of potassium enhances wheat grain yield under moderate salinity conditions.

Yields obtained with the three levels of potas­sium application indicate a high level of plant available potassium in the soil. Exchangeable K is 11% of the CEC.

Results of N uptake by wheat grain are pre­sented in Table 2. Values of N uptake are an indication of protein yield. The highest N uptake was obtained with N-serve, but N uptake due to 1% DCD was statistically not different. The negative effect of 5% DCD observed in the yield results is repeated in N uptake results in the non-saline soil. The lowest N uptake was found in the treatment that did not receive a nitrifica­tion inhibitor, i.e. in which the plants had presumably mainly a nitrate nitrogen nutrition.

In treatments receiving the lower level of N

application without nitrification inhibitors, a sig­nificantly reduced N uptake was found in the salinized soil compared to that in the non saline soil. On the other hand, application of 1% N­serve or 5% DCD had the opposite effect.

At the lower level of K application, N uptake is significantly higher in the non-saline than in the salinized soil. At the higher level of K application, N uptake is significantly higher in the salinized soil than in the non saline.

The above results indicate that if an enhanced ammonium nitrogen nutrition is combined with an enhanced potassium application, under mod­erate saline conditions, wheat grain can accumu­late relatively large quantities of reduced N, meaning that it has a relatively high content of protein.

Potassium uptake by wheat grain is listed in Table 3. In the salinized soil, treatments receiv­ing 1% DCD or N-serve generate a significantly higher K uptake than those receiving 5% DCD or none. The results indicate that there is no antagonism between potassium uptake by wheat and an enhanced presence of sodium in the soil.

Table 2. N uptake by wheat grain (mgN/pot) in nitrification inhibitors and potassium amended soils

Nitrification K N (g/pot) N (g/pot) inhibitor (g/pot)

0.8 1.2 0.8 1.2 Non-saline soil Salinized soil

None 0.52 464 j 502ij 385 k 486j 1%DCD 0.52 558 h 684 bed 542 h 647f 5%DCD 0.52 493 j 560h 537 hi 608 g 1% N-serve 0.52 539 h 709 be 681 cd 719 b 1%DCD 0.26 765 a 617fg 1%DCD 0.78 663 d 763 a

Means having different letters are significantly (0.05 level) different.

Table 3. K uptake by wheat grain (mg K/pot) in nitrification inhibitors and potassium amended soils

Nitrification K N (g/pot) N (g/pot) inhibitor (g/pot)

0.8 1.2 0.8 1.2 Non-saline soil Salinized soil

None 0.52 81 ij 92 gh 88 hi 89h 1%DCD 0.52 96efg 97 defg 97 defg 107 c 5%DCD 0.52 92gb 81 ij 82 ij 77j 1% N-serve 0.52 94fgh 95 fgh 99def 102 cde 1%DCD 0.26 103 cd 103 cd 1%DCD 0.78 118 b 125 a

Means having different letters are significantly (0.05 level) different.

600 Shaviv and Hagin

Table 4. P uptake by wheat grain (mg P/pot) in nitrification inhibitors and potassium amended soils

Nitrification K N (g/pot) N(g/pot) inhibitor (g/pot)

0.8 1.2 0.8 1.2 Non-saline soil Salinized soil

None 0.52 55.4 ef 53.3 fgh 54.5 fg 58.4d 1%DCD 0.52 64.6a 61.1 c 58.2 d 62.3 be 5%DCD 0.52 54.6f 51.7 h 47.6 i 45.6 i 1% N-serve 0.52 52.0h 52.4 gh 58.4 d 58.4 d 1%DCD 0.26 57.1 de 58.9 d 1%DCD 0.78 57.4 de 63.7 ab

Means having different letters arc significantly (0.05 level) different.

Data of P uptake by wheat grain are presented in Table 4. The 1% DCD treatments generated significantly higher P uptake, than the other treatments.

Amounts of nitrate and ammonium leached out from pots in the first two leachings are given in Table 5. Those amounts were very small or nil in the third leachate and therefore they are not presented.

The result show clearly that the nitrification inhibitors were effective. Soil salinity by itself reduces the amount of nitrates in the soil solu­tion. Ammonium leached is approximately pro­portional to N applied.

Discussion and conclusions

Nitrate content of soil leachates proved that in the treatment without nitrification inhibitors ni­trate nutrition prevailed. Significantly higher wheat grain yields were obtained in treatments with 1% DCD and 1% N-serve than in the other ones. These results indicate that the 1% DCD and N-scrve treatments had a mixed ammonium­nitrate nutrition. The 5% DCD treatment showed a yield depression probably due to excess of DCD or/and of ammonium.

The comparison of the 0.8 g N treatments in saline and non-saline soils shows a significant

Table 5. Nitrate and ammonium (mg N/pot) leached from nitrification inhibitors and potassium amended soils

Nitrification N (g/pot) inhibitor

0.8 1.2 0.8 1.2

Nit. Amm. Nit. Amm. Nit. Amm. Nit. Amm.

Non-saline soil Salinized soil

1st leachate None 111 b 15 jk 140 a 36 h 70c 53 c 73 c 54e 1%DCD 3g 12 k 4fg 37 h 7 def 60 d 7 def 77b 5%DCD 2g 21 i 4fg 36 h 6 ef 46 f 6 ef 68 c 1% N-serve 8 de 16 j 8 de 38 h 9 de 42 g lOd 88 a

2nd leachate None 119b Oh 228 a 18 f 120 b 17 fg 111 c 32c 1%DCD 1 e 1h le 16 g Oe 9 i Oe 62 a 5%DCD Oe 3 j Oc 26 e Oe llh Oe 29 d 1% N-serve 1e lh 14 d 38 b Oe lOhi Oe 29 d

Means having different letters, separately under Nit and Amm, and under 1st and 2nd leachate, are significantly (0.05 level) different.

Salinity in relation to ammonium and potassium nutrition 601

yield increase in the salinized soil, generated by 1% DCD and N-serve.

Treatments with nitrification inhibitors, except those with 5% DCD, produced considerably higher N-uptakes, meaning a higher crude-pro­tein yield, than those without a nitrification inhibitor i.e. having only nitrate nutrition. These results emphasize the results obtained for grain yield. Soil salinity reduced N-uptakc where nitri­fication inhibitors were not present. In treat­ments having the inhibitors, N-uptake was equal or greater in the saline soil than in the non saline. This emphasizes again that a balanced ammonium-nitrate nutrition may alleviate the negative effect of moderate soil salinity.

Further, the results show that if an enhanced ammonium nitrogen nutrition is combined with an enhanced potassium application, under mod­erate saline conditions, wheat grain can accumu­late relatively large quantities of N, meaning a relatively high content of protein.

Leaching measurements indicate that soil salinity by itself may inhibit somewhat the nitrifi­cation process.

Acknowledgement

This work was partially supported by the Dead Sea Works Ltd., Beer Sheva, Israel.

References

Ben-Hayyim G and Goffer Y 1988 Effect of nitrogen source on salt tolerance of citrus cells in culture. Israel Agresearch 2, 139-152.

Camberato J J and Bock B R 1989 Response of grain sorghum to enhanced ammonium supply. Plant and Soil 13, 79-83.

Hageman R H 1984 Ammonium versus nitrate nutrition of higher plants. In Nitrogen in Crop Production. Ed. R D Hauck. pp 67-85. Am. Soc. Agron., Madison, WI.

Hagin J, Olsen S R and Shaviv A 1990 Review of interaction of ammonium-nitrate and potassium nutrition of crops. J. Plant Nutr. 13, 1211-1226.

Helal H M and Mengel K 1979 Nitrogen metabolism of young barley plants as affected by NaCl-salinity and potassium. Plant and Soil 51, 547-562.

Olsen S R 1986a The role of organic matter and ammonium in producing high corn yields. In The Role of organic Matter in modern Agriculture. Eds. Y Chen and Y Avnimelech. pp 29-54. Martinus Nijhoff Publishers, Dor­drecht.

Olsen S R 1986h Using soil and fertilizer chemistry to improve corn productivity. Potash and Phosphate Inst. Maximum Yield Corn Research Roundtable, St Louis, MO.

Shaviv A, Hagin J and Neumann P M 1987 Effects of a nitrification inhibitor on efficiency of nitrogen utilization by wheat and millet. Commun. Soil Sci. Plant Anal. 18, 815-833.

Shaviv A and Hagin J 1988 Interaction of ammonium and nitrate nutrition with potassium in wheat. Fert. Res. 17, 137-146.

Shaviv A, Hazan 0, Neumann P M and Hagin J 1990 Increasing salt tolerance of wheat by mixed ammonium nitrate nutrition. J. Plant Nutr. 13, 1227-1239.

Thomas R L, Sheard R Wand Moyer G R 1967 Comparison of conventional and automated procedures for nitrogen, phosphorus and potassium analysis of plant material using a single digestion. Agron. J. 59, 240-243.

Reprintedji'om Plant and Soi/154: 133-137, 1993.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 603-609, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-234

Response of wild subclovers to soil calcium in xeric and acid Spanish soils

J. PASTOR, A. MARTIN and S. OLIVER Centro de Ciencias Medioambientales, CSIC, 115 DpdQ Serrano St., 28006 Madrid, Spain

Key words: ecological profiles, plant macroelements (Ca, Mg, K, Na), plant microelements (Mn, Fe), soil calcium and aluminium, Trifolium subterraneum, var. brachycladum, var. subterraneum

Abstract

We studied nutrition in 123 field populations of Trifolium subterraneum, var. subterraneum and var. brachycladum Gib. et Belli, with respect to calcium and aluminium tolerance in low-fertility soils in western Spain, most of them situated in semi-arid environments. The observation of the ecological profiles allowed us to know the differences about the autoecological behaviour of the species and the two varieties with respect to soil exchangeable calcium and aluminium and percent of soil calcium carbonate. The response of populations of the two varieties of Trifolium subterraneum L. to the increase of soil exchangeable calcium directly affects the content of Mn and Na. Besides, we found significant differences for Mg and Fe contents in the populations of var. brachycladum. Differences in intervarietal behaviour were mainly related to populations that grew on soils with low and high exchangeable calcium levels, while the differences were small or absent at intermediate levels. There was a clear intervarietal difference with respect to the mean values of Na and Cain the plants. In soils with higher exchangeable calcium levels there were also differences between both varieties with respect to Mg and K plant content, and therefore for the divalent/monovalent ratio. Our study further showed that populations exposed to the same level of available nutrients in low-fertility soils have markedly different uptake capacities. The results confirm the existence of stress-tolerant populations growing in soils with exchangeable calcium levels lower than 3 meq. 100 g -t. The results obtained will help to clarify the use of nutrients by plant close taxa in stressful environments.

Introduction

Changes in Mediterranean agriculture increase the importance of pasture legumes. Within these autochthonous legumes, the subterranean clover is a very important species of ovine pastures. When it is adequately managed, it contributes to maintain the organic N stock in the soil, and due to its creeping features helps conserve it against erosive processes, specially in areas with dry continental mediterranean climate. This species contains groups of populations adapted to stressful conditions in many environ­ments (xericity, soil acidity and low nutrient

content) (Martin et a!., 1980; Pastor et a!., 1987).

Nutrient availability is an important selective factor operating in plant community develop­ment. Different concentrations of nutrients in soils modify species composition, while nutrient use efficiency is a factor contributing to species dominance. Plants in their native habitats vary less in their tissue nutrient concentration with variable soil nutrient conditions than do culti­vated plants, and infertile environments give rise to plant species with both slow growth and slow resource capture (Chapin, 1980).

Most of the studies on physiological adapta-

604 Pastor et al.

tion to soil mineral stresses of wild populations have been more successful in comparing growth response of plants and their morphological attri­butes to increasing mineral constraints than in comparing physiological traits (Wacquant et a!., 1992). These authors propose to confirm the existence of functional calcifuge and calcicole populations within one species (Dittrichia vis­cosa), and to test for a gradation in population differentiation in relation to the intensity of mineral stress in the habitats. They demonstrate the possibility of detecting nutritional differentia­tion of ecological significance among populations of this species.

Kruger (1987) examined correlation patterns between nutrition, plant forms and successional status for 33 species. These results indicate that the species exposed to the same amount of nutrient availability have markedly different up­take capacities, suggesting a great diversity of nutrition among the species within these infertile habitats; similar life-forms from different locali­ties tend to resemble each other in their foliar nutrients. Uptake capacities seem to be correlated with life-history strategies. des­pite that species with similar growth-form types and in the same habitat apparently may differ substantially in their uptake capacities. This fact coincides with the hypothesis presented in this study on mineral nutrition patterns of autochthonous populations of Trifolium subterraneum L. in a successionally mature status.

Rorison and Robinson (1984) considered some effects that may be attributed to a direct or indirect influence of Ca on plant distribution. According to these authors, changes in Ca + + and CaC0 3 activity is perhaps the most important edaphic determinant of plant distribution in temperate latitudes.

This study pretends to highlight the response of Trifolium subterraneum L. to soil exchange­able calcium when the species populations grow in their natural communities, and considers the whole range of soils in its distribution area in Western Spain. Our contribution is part of a series of studies devoted to determine essential ecological factors related to soil characteristics in the ecophysiological behavior of this species. We want to determine what exchangeable calcium levels in the upper soil layer influences the

mineral nutntiOn of Trifolium subterraneum close taxa characterized as calcifuge.

Material and methods

Plant material

Our studies are based on plant samples from 123 natural populations, chosen among more than 500 samples belonging to close taxonomic var­ieties of Trifolium subterraneum L., which were collected from the soils where they grow, for the range of values of their distribution area in Western Spain (between 36° 3' and 43° 32' latitude N and 2° 53' and 8° 38' longitude W, from Andalusia to Galicia).

The fresh plant material was classified (Kat­znelson, 1974). We conserve a collection of dry material composed of representative plants from each sampled locality. Thirty three of the plant populations considered in this study belong to Trifolium subterraneum L. var. brachycladum, while 90 belong to Trifolium subterraneum L. var. subterraneum.

The populations were grouped for their study in the following way for each variety: 1) popula­tions growing in soils with exchangeable calcium levels lower than 3.0 meq 100 g- 1 ; 2) with levels ranging from 3.0 to 9.0 meq 100g- 1 ; and 3) those with levels higher than 9.0 meq 100g- 1 •

In a plant nutrition study it is very important to indicate the humidity of the station where popu­lations develop. Var. brachycladum gathers the most xeric populations within the species, its preference for the driest places and its absence in humid stations is noticeable. On the other hand, var. subterraneum gathers the most mesic popu­lations within the species.

Plant analysis

Plant tops (leaves, shoots, flowers and fruits) were collected mainly at the phenological stages corresponding to the end of flowering and fructi­fication, adequate in order to establish com­parisons between the material coming from dif­ferent localities with respect to the nutrient concentration.

The type of selected sample was aimed at integrating the behaviour of the whole plant.

Some authors, such as Wacquant and Bouab (1985), suggested the possibility of using shoot plant analysis for further population screening when comparing the composition of sap and plant organs of different wild genotypes. It has been shown that the variation from one genotype to another observed in sap composition, was reflected by the whole plant and the shoot.

The material was washed up with distilled water and then dried at 80°C to a constant weight. One gram of dried, ground plant was dry-ashed. The powder was slowly incinerated until fumes disappeared, then it was placed in a muffle furnace at 450°C for 10 hours. The ashes were digested by heating with a 1: 1 HCI solution (macroelements). For analyzing the minor ele­ments the ashes were treated with a 111 I 8 mixture of HCI/HN0 3 Iwater. After filtration, Na, K and Ca were determined by flame-photo­metry, and Mg, Fe and Mn by atomic absorption spectrophotometry. The macroelements are given in meq per 100 g of dry matter, while the minor clements are expressed in mg kg - 1 .

Soil analysis

We collected close to 400 soil samples for analy­sis from the top 15 em around the Trifolium subterraneum (s.l.) roots.

The soil exchangeable calcium was extracted by percolation with 1N ammonium acetate solu­tion at pH= 7; the soil exchangeable aluminium was extracted also by percolation, using 1 N KCl solution. Calcium carbonate was analyzed by the acid neutralization method of Black (1965).

The calcium in the extracts was determined by flame photometry; the aluminium was deter­mined by titration of the NaOH obtained by adding NaF to the extract in order to form the fluoraluminate complex; the reaction lasted for 1 I 2-1 hour, the titration was carried out by means of a pH-meter with a 0.01 N HCl solution until a pH value of 8.3 remained constant for 2 minutes.

Data analysis

The ecological profiles of the species and of the varieties studied were calculated according to the methods pointed out by Gordon (1965), Guil­lerm (1971) and Gauthier et al. (1975).

Response of wild subclovers to soil calcium 605

The differences between the cation contents of plants were tested by variance analysis (F test).

Results and discussion

Soil calcium as an ecological factor

The soil exchangeable calcium is an important factor in the distribution of subterraneum clover taxa, specially for Trifolium subterraneum L. var. subterraneum and less for var. brach­ycladum. We note that climate related factors rather than those related to the soil are more 'active' in the distribution of var. brachycladum. Considering the soil factors, we found moderate­ly active the exchangeable calcium. With regards to the distribution of var. subterraneum, the edaphic factors are generally more 'active' than those related to climate, with exchangeable cal­cium being one of the most active (Pastor et al., 1987).

Table 1 shows the synthetic ecological profiles of the studied taxa for exchangeable calcium, percent of calcium carbonate and exchangeable aluminium.

The ecological width with respect to calcium for both varieties is consistent with that of the species, ranging from 0.4 to 36.0 meq 100 g - 1 of the fine soil fraction. In Western Spain there are specially suitable sites for the presence of Tri­folium subterraneum L., which have exchange­able calcium levels lower than 9.0 meq., while those with contents higher than 21.0 meq are not suitable. The species does not appear in soils with exchangeable calcium contents higher than 36.0 meq 100 g - 1 . Most of the studied popula­tions had an exchangeable calcium content in their habitats lower than 21.0 meq 100 g - 1 .

The populations that belong to var. brach­ycladum show a certain indifference to this factor, although they prefer soils with contents lower than 3 meq 100 g - 1 • The var. subter­raneum populations are better represented in soils with higher exchangeable calcium contents, thriving more in soils with an exchangeable calcium content ranging from 0.4 to 6.0 meq 100 g.

According to the above mentioned behaviour, we noted that the suitable soils for the species

606 Pastor et al.

Table 1. Synthetic ecological profiles of Trifolium subter-raneum L. and of the varieties brachycladum and subter-raneum in relation to soil exchangeable calcium and alu-minium (meq lOOg- 1 ) and soil CaC0 3 (%)

Factors and Trifolium subterraneum classes

sp. v ar. b rach ycladum var. subterraneum

Calcium < 3.0 VF VF VF

3.0- 6.0 F F 6.0- 9.0 F I 9.0-12.0 I

12.0-15.0 15.0-18.0 18.0-21.0 I I I 21.0-27.0 u u u 27.0-36.0 u u u

>36.0 A A A

Aluminium 0.0 u u u

tr.-0.2 F F F 0.2-0.4 F VF F 0.4-0.6 F VF F 0.6-0.8 F VF F 0.8-1.0 F VF F 1.0-1.6 VF I VF 1.6-2.4 VF VF

>2.4 VF VF

CaC03

0.0 VF VF VF tr.- 1.0 I u F 1.0- 2.0 u u 2.0- 3.0 u u u 3.0- 4.0 vu u vu 4.0-10.0 vu u vu

10.0-20.0 A A A >20.0 A A A

VF: Very favourable; F: Favourable; I: Indifferent; U: Unfavourable; VU: Very unfavourable; A: Absent; tr. traces.

are those that do not have any calcium carbon­ate, while those with levels higher than 1.0 percent are not suitable. The ecological width of var. brachycladum and var. subterraneum with respect to total soil calcium carbonate range between 0 for var. brachycladum and 0-2.0 for var. subterraneum.

The soil exchangeable aluminium levels are unsuitable for the two varieties in soils with little or no acid. The presence of populations of var. brachycladum is related to the range tr.-1.0 meq/ 100 g, but we should point out that they do not show any positive sensitivity to aluminium levels

higher than 1.0 meq/100 g. On the other hand, the presence rather than the abundance of var. subterraneum is positively related to soils with values higher than 1.0 meq (1.0-3.4 meq). Therefore we can state that the behaviour of varieties differs with respect to the soil exchange­able aluminium and that var. subterraneum is more tolerant to this element than var. brach­ycladum.

Effects of soil exchangeable calcium on plant composition

Table 2 shows the mean values of plant mineral nutrients and the results of the variance analysis for the soil exchangeable calcium levels where they grow. The response of the populations of the two Trifolium subterraneum L. varieties to the increase of soil exchangeable calcium directly affects the contents of Mn and Na, which are higher in the populations growing on soils with lower calcium levels than in those growing on soils with higher levels. The differences of the Mn contents are statistically significant for the populations of both varieties. The differences for Na contents are significant only for the popula­tions of var. brachycladum, due to the fact that they grow in more xeric sites, where the soil exchangeable Na levels are lower; this difference is very remarkable for soils with higher Ca contents. We noted that the soil exchangeable calcium level strongly interacts with sodium uptake in both varieties. The mean Na values for both varieties arc greater in the plants that grow with lower levels of soil exchangeable calcium. Mean Na values also favour the potassium up­take by plant populations of both varieties, with no statistically significant differences.

In a previous study Martin-Ramos et al. (1980) found that sodium was an important element in relation to the ecophysiological min­eral nutrition of the species. The relative low soil exchangeable sodium contents favour the potas­sium uptake by plants; this uptake decreases when Na soil level increases; the opposite is true for the sodium content in plants. In this study we found that sodium is really important for var. brachycladum. The exchangeable sodium and calcium of soils similarly affect the potassium content in plants.

Response of wild subclovers to soil calcium 607

Table 2. Effect of the soil exchangeable calcium levels on the mineral composition of the aerial parts of autochthonous populations of Trifolium subterraneum L. (Mn and Fe are expressed in mg kg-\ Ca, Mg, K and Na in meq. 100 g- 1 dry matter at 80°C)

Variety Calcium< 3 meq. Calcium 3-9 meq. Calcium> 9 meq. F (Snedecor)

brachycladum n = 11 n = 13

mean mean

Ca 72.7 64.8 Mg 22.7 22.5 K 48.9 55.8 Na 10.5 8.3 Ca + Mg + K + Na 154.8 151.3 Ca+Mg/K+Na 1.9 1.5 Mn 94.2 57.3 Fe 158.1 224.5

subterraneum n = 21 n = 41

mean men

Ca 60.3 64.0 Mg 21.6 22.4 K 40.6 45.9 Na 18.5 14.8 Ca+Mg+K+Na 141.0 147.2 Ca + Mg/K + Na 1.5 1.6 Mn 70.5 56.0 Fe 168.5 185.4

* p < 0.05 (significant). ** p < 0.01 (highly significant). *** p < 0.001 (very highly significant).

Besides, we found significant differences for Mg and Fe contents in the populations of var. brachycladum which generally grow on soils with lower AI contents; its presence is related to the range tr.-1.0 meq 100 g -I soil exchangeable alu­minium in soil, smaller content in general than the one supported by the populations of var. subterraneum (to 3.4 meq). A study of the mineral composition of the aerial part of Tri­folium subterraneum L. in relation to the level of soil exchangeable magnesium (Martfn-Ramos et a!., 1983) found that soil magnesium level affects the magnesium, copper and iron contents of the aerial part of plants in populations growing in pastures established on soils with exchangeable aluminium. These results agree with the ones found in this study for the var. subterraneum, which is positively related to soils with values of exchangeable aluminium higher than 1.0 meq (1.0-3.4 meq) (see Table 1). In this variety magnesium and iron contents in the plants are

n =9 (df2,30)

mean

58.6 3.10 17.5 5.43** 64.8 1.81

1.6 3.85* 142.5 0.98

1.2 2.64 29.8 9.26***

138.4 3.73*

n =28 (df2,87)

mean

68.1 2.20 24.3 1.64 52.0 2.73 12.1 1.64

156.5 2.65 1.5 0.23

38.6 10.86*** 177.7 0.30

higher when there is exchangeable aluminium in the soils; however this behaviour turns into the contrary for populations of the var. brach­ycladum in the case of higher levels of exchange­able calcium. The different behaviour of both varieties with respect to magnesium is clearly noted in soils with exchangeable calcium con­tents greater than 9.0 meq 100 g -I.

We noted as well the role of calcium in plants as a regulator of the amount of certain ions in their tissues. This may take place when the Ca level is low and is unbalanced in relation to the ionic environment as pointed out by Wallace et a!. (1966, 1968, 1971).

Differences among varieties of Trifolium subterraneum L. in uptake patterns

Table 3 shows the maximum and mm1mum values of mineral nutrient levels for the aerial plant parts of var. brachycladum and var. subter-

608 Pastor et al.

Table 3. Comparison of the mineral composition of aerial parts of autochthonous populations belonging to two varieties of Trifolium subterraneum L. from soils with three exchangeable calcium levels (Mn and Fe arc expressed in mg kg -I; Ca, Mg, K and Na in meg. 100 g dry matter at SOoC)

Exch. Ca < 3 meg Ca Mg K Na Ca+ Mg+K+Na Ca+ Mg/K + Na Mn Fe

Exch. Ca 3-9 meg. Ca Mg K Na Ca+Mg+K+Na Ca + Mg/K + Na Mn Fe

Exch. Ca > 9 meg. Ca Mg K Na Ca+ Mg+ K+Na Ca + Mg/K + Na Mn Fe

* p < 0.05 (significant); ** p < 0.01 (highly significant); *** p < 0.001 (very highly significant).

var. brachycladum

range

n = 11 48.6-115.9 18.1- 28.4 19.2- 97.2 1.3- 30.5

116.6-203.0 0.8- 4.8

36.0-220.0 77.0-225.0

n = 13 47.6- 82.0 14.8- 34.1 33.3-111.3 0.6- 21.7

133.1-182.1 0.6- 2.9

29.0-100.0 87.0-445.0

n=9 50.9- 66.5 12.8- 19.7 49.9- 88.2 0.5- 5.3

126.3-170.0 0.9- 1.4

20.0- 41.0 62.0-237.0

raneum populations. This table also includes the variance analysis that considers the variety as a factor in order to study the different mineral nutrient elements, for the different levels of soil exchangeable calcium. We used the following levels as a reference point for the separation of the populations into groups: the 'most fertile' soils were those with a content higher than 9.0 mcq 100 g -\ the 'low fertile' soils those with a content lower than 3.0 meq 100 g -\ and the group with 'intermediate soil fertility' was characterized by a content between 3.0 and 9.0mcq 100g- 1 .

The variance analysis shows statistically signifi­cant differences for Mg, Na, K, Ca, and the (Ca + Mg)/(K + Na) ratio in plants growing on

var. subterraneum F (Snedecor)

range

n = 21 (df 1,30) 39.0- 86.0 6.15** 15.2- 31.7 0.47 16.6- 92.1 1.20 6.1- 59.2 3.25

107.3-198.1 2.34 0.8- 3.2 1.74

31.0-132.0 2.70 75.0-362.0 0.14

n = 41 (df1,52) 35.3- 97.8 0.03 12.3- 34.1 0.002 7.7- 80.6 3.24 0.5- 44.4 3.59

97.1-218.0 0.35 0.8- 5.8 0.19

31.0-137.0 0.03 87.0-450.0 2.03

n =28 (df 1,35) 43.0- 99.2 4.52* 16.9- 43.6 12.12*** 23.0- 92.1 4.98* 0.8- 44.8 6.22*

119.5-207.5 2.54 0.8- 2.3 6.52*

11.0- 97.0 2.01 72.0-500.0 1.58

soils with exchangeable levels of Ca greater than 9 meq 100 g - 1 • These differences are only signifi­cant for the Ca content in populations growing on soils with Ca levels lower than 3 meq 100 g - 1 .

The populations of var. brachycladum that grow on more desaturated soils only present higher Ca values for the lower calcium exchangeable soils. This indicates that at those levels the former populations compete well for low calcium con­tents with other community plants, including populations of var. subterraneum. The popula­tions of this last variety, that thrive in infertile habitats, may be considered as stress-tolerant (within the variety), accepting the theory that stress induces a slight loss in tissue nutrient concentrations (Kruger, 1987). This fact also

confirms that the other populations are adapted to habitats with higher fertility in spite of being present with higher exchangeable aluminium levels. The effect of aluminium reported in nutritional differentiation in populations of Dit­trichia viscosa (Wacquant and Baus, 1992), in­creasing K and decreasing Ca and I or Mg ac­cumulation in plant, has been noted for other species (Asp et al., 1988; Bengtsson et al., 1988; Marschner, 1986; Sucoff et al., 1989).

This coincides with our results for var. brach­ycladum when it grows on soils with exchange­able calcium levels higher than 9.0 meq 100 g - 1 .

This means that the above-mentioned variety is adapted to low fertility soils, rather than to high aluminium contents in their habitats (according to their ecological preferences); this contrasts with the results obtained for var. subterraneum which is present in soils with higher exchange­able aluminium levels and shows preferences for higher calcium levels in the soils where the species thrives.

Acknowledgement

This study was financed by the Autonomous Community of Castilla-La Mancha.

References

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Bengtsson B, Asp H, Jensen P and Berggren D 1988 Influence of aluminium phosphate and calcium uptake in beech (Fagus sylvatica) grown in nutrient solution and soil solution. Physiol. Plant. 74, 299-305.

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Rev. Ecol. System. 11, 233-260. Gauthier B, Godron M, Hiernaux P and Lcpart J 1975 Un

type complementaire de profil ecologique. Le profil ecologique 'indice'. Can. J. Bot. 55, 2859-2865.

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Guillerm J L 1971 Calcul de !'information fournie par un profil ccologique et valeur indicatrice des especes. Oecol. Plant. 6, 209-225.

Response of wild subclovers to soil calcium 609

Katznelson J 1974 Biological flora of Israel. 5. The subterra­nean clovers of Trifolium subsect. Calycomorphum Katzn. Trifolium subterraneum L. (sensu latu). Israel J. Bot. 23, 69-108.

Kruger F J 1987 Responses of plants to nutrient supply in mediterranean-type ecosytsems. In Plant Response to Stress. pp 415-427. Eds. J D Tenhunen et al. NATO ASI Series, G 15, Springer-Verlag Berlin-Heidelberg.

Martin-Ramos A, Pastor J and Oliver S 1980 Sodium and potassium uptake by subclovers with respect to soil ex­changeable sodium. In II Congress of the Federation of European Societies of Plant Physiology, Santiago de Com­postela (Spain). pp 480-481.

Martin-Ramos A, Pastor J and Oliver S 1983 Absorci6n de nutrientes por el trebol subterraneo en relaci6n con el magnesia y aluminio de los suelos. In V Reunion Sociedad Espanola de Fisiologia Vegetal, Murcia (Spain). p 88.

Marschner H 1986 Mineral Nutrition of Higher Plants. Academic Press, London. 674 p.

Pastor J, Oliver S and Martfn-Ramos A 1980 Compor­tamiento diferencial de Trifolium subterraneum L., T. brachycalycinum Katz. et Morley y T. yanninicum Katz. et Morley, respecto a los faetores ecol6gicos en sus comunidades del Occidente de Espana. Pastas 10, 44-57.

Pastor J, Oliver S and Martin A 1987 Incidencia de los factores ambientales sobre los treboles subterraneos en las zonas de dehesa del Centro y Sudoeste de Espana. Seminario Internacional sobre dehesas y sistemas agrosil­vopastorales similares. Aetas: Madrid and Seville. MAB International Secretariat (UNESCO). 21p.

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Sucoff E, Buschena C and Bloom P 1989 Response of honey locust (Gleditsia triacanthos L.) to soil solution aluminium. Plant and Soil 113, 93-99.

Wacquant J P and Baus Picard J 1992 Nutritional differentia­tion among populations of the mediterranean shrub Dit­trichia viscosa (Asteraceae) in siliceous and calcareous habitats. Oeeologia 92, 14-22.

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J

Maximization of nutrient utilization in relation to environmental protection

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 613-617, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-333

Impact of intensive agriculture on resources and environment

KONRAD MENGEL Institute of Plant Nutrition, Justus-Liebig-University, Siidanlage 6, D-35390 Giessen, Germany

Key words: energy, environment, fertilizer, nitrogen, phosphate, slurry

Abstract

Crop production has a positive energy balance which even can be improved by an adequate fertilizer application. World phosphate reserves are limited and therefore fertilizer phosphate should be used with high efficiency. In intensive animal farms phosphate rates may exceed by far the demand of the crop and therefore result in a heavy waste of phosphate. The same applies for the application of nitrogen in the form of slurries. Nitrate may be leached out from agricultural land and pollute surface and groundwater and the denitrification product N20 is involved in the decomposition of ozone in the stratosphere. Also in future crop production needs fertilizers of which the application should be handled carefully in order to reduce pollution of the environment as much as possible.

Introduction

Since the earliest days mankind was continuously struggling with nature. Nature in whatever form it was met by man was strong and the struggle often was for survival. It is only quite recently in the long evolution process that man- due to his scientific and technical achievements- is in a position to intervene into nature and even to manipulate nature. Today man has very power­ful instruments of mechanical, chemical and biological kind, by which he can affect nature. The problem is that nature was not prepared during its evolutionary process for this new situation. Hence we must be very careful in using the above mentioned powerful instruments. The global problem is, clearly stated by the Club of Rome and confirmed by recent evaluations (Meadows et a!., 1992), that the resources de­cline and the environmental pollution increases in an exponential way. This situation also con­cerns agricultural production of which the major problems related to fertilizer application will be considered in the following.

Resources

Agricultural production and particularly crop production does not draw much on resources. On the contrary crop production contributes to the output of resources such as raw material and energy. In future this kind of resource product­ion will still gain in importance. Crop yields were remarkably increased during the last 100 years mainly by increasing application of mineral fer­tilizers (Beer, 1990). Although the production of fertilizers requires energy the energy output/ input ratio is higher than 1 for agricultural crops and even is still improved by competent fertilizer application (Lewis and Tatchell, 1979). Fertilizer application including liming and crop rotation are necessary measurements for maintaining an essential resource, namely the fertile soil which is today threatened by various hazards.

Plant nutrients used in heavy quantities as fertilizers arc nitrogen, phosphorus potassium and lime. Nitrogen fertilizer production is depen­dent on energy but the proportion required from total energy consumption of developed countries

614 Mengel

is in the order of 1% and therefore negligible. Lime and potassium reserves are still relatively high as compared with the reserves for phos­phate. According to Sheldon (1982) global known phosphate reserves will last still about 500 years if consumed at today's rate. This is no long time in man's history. If all the phosphate reserves are dissipated on agricultural land hard­ly any recycling will be possible. It should be emphasized that phosphorus is an essential ele­ment which cannot be substituted in its biological functions by other chemical elements.

There exist various measurements to use phos­phate fertilizers with a high efficiency. An im­portant one is to apply phosphate only in those cases where crop yields are improved. This depends on soils and crops. On representative arable sites in Germany no crop response to phosphate fertilizer application is probable if the level for available phosphorus is 70 mg P kg -I soil (Brune and Heyn, 1984) related to available P determined by the CAL-Method (Schuller, 1969). Further field trials in combination with soil tests for available phosphate are required for elaborating threshold data for available phos­phate for representative soil types, crops, and climatic conditions in order to improve phos­phate fertilizer efficiency.

Phosphate fertilizers can be rendered to un­available forms in soils. One of the most im­portant processes of this kind is the adsorption of phosphate to soil particles, especially to Al/Fe oxides/hydroxides. Adsorption is particularly strong at low pH which favours the formation of binuclear phosphate adsorption complexes (Haynes, 1984). Soil pH increase by liming may improve phosphate availability substantially and retard the process of irreversible adsorption (Barekzai and Mengel, 1985) at least in soils of the moderate climate with not too low a pH. In very acid tropical soils liming may promote the phosphate adsorption (Hauter, 1983) because of the formation of colloidal AI complexes which can fix phosphate strongly (Haynes, 1984). For moderate climate and soil conditions an approxi­mately neutral soil pH yields a fertilizer recovery in the range of 70 to 80% (Sturm and Isermann, 1978). Hence for an optimum fertilizer phos­phate efficiency a neutral pH is pertinent. Phos­phate efficiency depends also on soil structure

Table I. Excess (input- output) of plant nutrients of dairy farms on various soil types in the Netherlands, average data in kg, N, P, K ha- 1 year- 1 (after Spiertz, 1991)

Sand Clay Peat

N

486 466 462

p

32 33 30

K

125 78 94

since a good structure favours root growth and thus the capacity of plant roots to exploit soil phosphate (Keita and Steffens, 1989).

Excessive slurry application may lead to a waste of phosphate and an accumulation of phosphate even in deeper soil layers (Werner et a!., 1988). The problem is particularly severe in animal farms with high stocking rates as they exist in Holland, Belgian, and the northern part of Germany and France. In Table 1 input and output of major plant nutrients in dairy farms of the Netherlands are shown (Spiertz, 1991). The net input of phosphate is 30 kg P ha -I and hence in a range what is required by a good crop stand. Efficiency of fertilizer phosphate depends also on the phosphate type. Dissolution of phosphate rock in neutral and alkaline soils is generally poor and hence these fertilizer types should be mainly used on acid soils (Mengel, 1986).

Pollution

Pollution problems in intensive agriculture are mainly related to nitrogen fertilizers. The total quantity of industrially fixed N2 today amounts to 92 x 106 t N year- 1 which is about 60% of the biological annual fixation rate (Isermann, 1987). Nearly all of this industrially fixed nitrogen is used for fertilizer production and hence is brought into the soil. This nitrogen input repre­sents an interference in the natural N cycle the consequences of which are as yet little under­stood. In Table 2 the nitrogen flow from fertil­izer nitrogen to the consumption of nitrogen containing food, mainly proteins, is shown. The data are average values from West Germany for the year 1986 (Isermann, 1990). From the nitro­gen input into the soil, 218 kg N ha _,, only about 1 I 4 is present in the market products and are exported from the farm. A substantial amount of

Table 2. Nitrogen on its way from crop production to human consumption. Data originate from Germany for 1986 (after Isermann, 1990)

1. Total agricultural input (Fertilizer N, N of feeding concentrates, N of bioi. N2

fixation, sewage N) 2. N in agricultural market produces

Seeds, grains, vegetable Meet, milk, eggs

3. N in produces bought by men 4. N in food actually eaten 5. N remaining in human bodies

N (kg ha 1 year 1 )

23 28

218

51 28 22 0.07

the nitrogen input remains in the soil where it may be used for a following crop, partially it may be leached in form of nitrate and thus be a hazard for ground- and drinking water (Strebel et a!., 1985). Nitrate can also be denitrified (Schneider and Haider, 1992) and from the resulting molecules N 2 and N20, the latter is involved in the decomposition of ozone in the stratosphere.

The extent to which nitrogen is lost from the soil by nitrate leaching and/ or denitrification depends on agricultural practice, crop species and rotation and to a large extent on soil texture. Soils with medium to high CEC generally are less pervious than sandy soils and hence on these loamy soils the hazard of nitrate leaching is much lower than on sandy soils (Strebel et a!., 1985). In addition colloids of loamy soils may adsorb nitrogen containing macromolecules such as pep­tides (Loll and Bollag, 1983) and hence retard their microbial breakdown (Feng et a!., 1990). Soils with higher concentrations of 2: 1 clay minerals, especially vermiculite, may specifically adsorb NH: and thus protect it against nitrifica­tion which finally means against leaching and denitrification. The so-called fixed NH; is still available to plants (Yang et a!., 1992) and contributes to crop nutrition (Dressler and Mengel, 1985). Such loamy soils, particularly soils derived from loess may accumulate avail­able nitrogen in form of 'fixed NH;' but also in form of organic nitrogen (Hiitsch, 1991). De­nitrification is favoured in water logging soils, especially in fallow soils (Schneider and Haider, 1992) and when plenty of organic carbon IS

available to the microbes (Schloemer, 1991).

Intensive agriculture 615

Table 3. Agricultural NH 3 emission density of various Euro­pean countries (NH3 emission of agriculture divided by agricultural used land) after Isermann (1987)

Emission (kg N ha - 1 )

Netherlands 70 Belgium 55 Denmark 39 Norway 36 Germany 31 France 22 Great Britain 21 Ireland 20 Italy 20 Greece 10

From the nitrogen in market products noted in position 2 of Table 2 a substantial amount is present in animal produces. This nitrogen origi­nates from forage the farmer has produced and also from concentrates the farmer has bought and is mainly present in form of proteins. The efficiency of this protein nitrogen for the pro­duction of animal protein is not high, in the case of pork production in the range of 15%. For this reason relatively high amounts of nitrogen are excreted by the animals with the urine and faeces. This nitrogen present in farm yard ma­nures and slurry can be well used by crops provided the quantities applied per ha are ad­justed to the crop demand. In intensive animal farms this frequently is not the case. As can be seen from Table 1 the average output of nitrogen in intensive animal farms in Holland is in the order of 300 kg N ha _, which must have a strong polluting effect on surface water and ground­water (Isermann, 1991). Slurry application and also grazing particularly under the conditions of high stocking rates lead to a considerable emis­sion of NH3 into the atmosphere with deleteri­ous effects on ecology including forest trees (Roelofs et a!., 1988; Temmermann et a!., 1988). In Table 3 the agricultural emission density for some European countries is shown (Isermann, 1987).

Future prospects

Also in Europe agricultural production will play a major role in future and even gain in impor­tance. Production, however, has to take care

616 Mengel

that the environmental pollution is reduced to a minimum. Intensive production with high yield per ha or animal has been the target for genera­tions. Modern agriculture has to get familiar with the idea that production with a minimum of pollution is a further aim. In this respect a first important demand is to integrate animal pro­duction into crop production so that plant nu­trients excreted by animals are used with a high efficiency for crop production and pollution by nitrogen and dissipation of phosphate is avoided. In this context also the question is of relevance to which degree animal protein can be substi­tuted by plant protein. As already mentioned above animal protein production, especially meat protein, has a low efficiency. This is dem­onstrated by the following example: assuming a fertilizer nitrogen efficiency for soya protein production of 50%, the production of 1 t of soya protein requires about 400 kg of nitrogen. As­suming an efficiency of about 17% for pork protein production, 6 t of soya protein are re­quired for 1 t of pork protein. These 6 t of soya protein require about 2400 kg nitrogen. From this quantity 2200 kg nitrogen remain somewhere in the environment during the various steps of production. The problem is still more grotesque if one considers that in South America forests were cut in order to establish large soya bean fields of which the produces are exported to Western Europe where they are fed in intensive animal farms and here cause heavy pollution.

A further demand is to adjust nitrogen fertil­izer rates to the available nitrogen already in the soil. This requires reliable soil tests which not only should consider the inorganic soil nitrogen but also a fraction of organic nitrogen as was recently reported by Barekzai et al. (1991).

References

Barekzai A and Mengel K 1985 Alterung von wasserliislich­em Dungerphosphat bei verschiedenen Bodentypen. Z. Pflanzenernaehr. Bodenkd. 148, 365-378.

Barekzai A, Steffens D, Bohring J and Engels T 1992 Prinzip und Uberprufung des 'Giessener Modells' zur N-Dungeem­pfehlung bei Wintergetreide mit Hilfe der EUF-Methode. Agriobiol. Res. 45, 65-76.

Beer K 1990 Justus von Liebigs Werk 'Die organische Chemie in ihrer Anwendung auf Agricultur und Physio-

Iogie' ein Wegbereiter der wissenschaftlichen Dungung. Tagungsberichte d. Akademie d. Landwirtschaftswiss. der DDR 289, 15-22.

Brune H and Heyn J 1984 P-Dungeempfehlung fur die Praxis: Die Wirkung verschiedener P-Dunger-Formen in mehrjiihrigen Feldversuchen. DLG-Mitteiungen 2, 80-83.

Dressler A and Mengel K 1985 Bedeutung des peripheren spczifisch gebundenen NH; von Li:iss- und Alluvialbiiden fur die N-Dungerbedarfsermittlung. VDLUFA-Schriften­reihe, 16. Kongressband, pp 137-146.

Feng K, Dou H and Mengel K 1990 Turnover of plant matter in soils as assessed by electro-ultrafiltration and CaCI 2

extracts. Agribiol. Res. 43, 337-347. Hauter R 1983 Phosphatmobilisierung in Abhiingigkeit vom

pH des Bodens unter besonderer Berucksichtigung der Rhizosphiire. Ph.D. Thesis, Fac. Nutrition Justus Liebig University Giessen. pp 49-59.

Haynes R J 1984 Lime and phosphate in the soil-plant system. Adv. Agron. 37, 249-315.

Hutsch B 1991 EinfiuB differenzierter Bodenbearbeitung auf die Stickstoffdyamik im Boden in Abhiingigkeit von Bep­robungstermin und Standort, unter bcsonderer Berucksich­tigung von N-Freisetzung, Nitratverlagerung und Denitrifi­kation. Ph.D. Thesis, Agric. Fac. Justus-Liebig-University Giessen. pp 75-81.

Isermann K 1987 Environmental aspects of fertilizer applica­tion. In Ullmann's Encyclopedia of Industrial Chemistry. Vol A10. pp 400-409. VCH Verlagsgescllschaft Weinheim.

Isermann K 1990 Share of agriculture in nitrogen and phosphorus emissions into the surface water of Western Europe against the background of their eutrophication. Fertilizer Res. 26, 253-269.

Keita S and Steffens D 1989 EinfluB des Bodengefiiges auf Wurzelwachstum und Phosphataufnahme von Sommcr­weizen. Z. Pflanzenerniiehr. Bodenkd. 152, 345-351.

Lewis D A and Tatchell J A 1979 Energy in UK agriculture. J. Sci. Food Agric. 30, 449-457.

Loll M J and Bollag J-M 1983 Protein transformation in soil. Adv. Agron. 36, 351-382.

Meadows D, Meadows D and Randers J 1992 Beyond the Limits. Chelsea Green Publishing Co., Post Mills, VT.

Mengel K 1986 Umsatz im Boden und Ertragswirkung rohphosphathaltiger Dungemittel. z. Pflanzenerniiehr. Bodcnkd. 149, 67 4-690.

Roelofs J G M, Boxma A W and van Dijk H F G 1988 Effects of airborne ammonium on natural vegetation and forests. In Air Pollution and Ecosystems. Ed. P Mathy. pp 876-880. D. Reidel Publishing Company, Dordrecht, Bos­ton, Lancaster.

Schloemer S 1991 Denitrifikation cines gcmuscbaulich genut­zten Bodens in Abhiingigkeit von der Einarbeitung frischer Erntcrestc. Z. Pflanzenerniiehr. Bodenkd. 154, 265-269.

Schneider U and Haider K 1992 Denitrification- and nitrate leaching losses in intensively cropped watershed. Z. Pflan­zencrniiehr Bodcnkd. 155, 135-141.

Schuller H 1969 Die CAL-Methode, einc neue Methode zur Bestimmung des pflanzenverfugbaren Phosphates im Boden. Z. Pflanzenernaehr. Bodenkd. 123, 48-63.

Sheldon R P 1982 Phosphat- dcr unentbehrliche Rohstoff. Spektrum der Wissenschaft 8, 16-23.

Spiertz J H J 1991 Integrated agriculture in the Netherlands. In Quelles Fertilisations demain? Ed. COMIFER Forum, Strassburg. pp 52-63.

Strebel 0, Bottcher J and Duynisveld W H M 1985 Einftull von Standortbedingunngen und Bodennutzung auf Nit­ratauswaschung und Nitratkonzentration des Grundwas­sers. Landw. Forsch. Kongressband 1984. pp 34-44.

Sturm H and Isermann K 1978 Ubcrlcgungcn zur langfris­tigen Ausnutzung von Mineraldiinger-Phosphat auf Acker­boden. Landw. Forsch. Kongressband 35, 180-192.

Temmcrmann L de, Ronse A, van den Cruys K and Meeus­Verdine K 1988 Ammonium and pine tree dieback in

Intensive agriculture 617

Belgium. In Air Pollution and Ecosystems. Ed. P Mathy. pp 774-779. D. Reidel Publishing Company, Dordrecht, Boston, Lancaster, Tokyo.

Werner W, Fritsch F and Scherer H W 1988 Einfiull lang­jiihriger Giillediingung auf den Niihrstoffhaushalt des Bodens. 2. Mitteilung: Bindung und Loslichkeitskriterien der Bodenphosphate. Z. Pfianzenerniiehr. Bodenkd. 151, 63-68.

Yang X, Scherer H W and Werner W 1992 Fixation and release of ammonium by two typical paddy soils after incorporation of farmyard manure. Eur. J. Agron. 1, 29-35.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 619-622, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-207

Efficient management of nitrogen fertilization in modern cropping systems

A. AMBERGER Institute of Plant Nutrition, Technical University Munich, D-85350 Freising-Weihenstephan, Germany

Key words: cropping system, fertilization management, green and straw manuring, nitrification inhibitors, nitrogen losses, slurry, waste water

Abstract

Management of nitrogen fertilizing can be optimized in different ways: A clear calculation of the required amount of nitrogen for the expected yields IS necessary on an input-output basis. Nitrogen losses and agro-techniques to avoid them; -ammonia volatilization, by immediate incorporation of the inorganic or organic fertilizers into the

soil -denitrification, by optimizing soil structure -nitrate leaching, by straw manuring, late application of slurry and late incorporation of green

manure. Nitrification inhibitors (e.g. dicyandiamide, DCD); -when added to ammonium or urea fertilizers, they show economic advantages (lower labour costs)

and preserve fertilizer N from being translocated out of the rooting zone or leached out -as an additive to organic waste materials from plant and animal production or industrial processings

they inhibit nitrification. Efficient nitrogen management in cropping systems is a necessary demand both for economic and ecological reasons.

Introduction

The goal of nitrogen fertilizer management is an optimal feeding of plants, not only for economic benefits but also in order to avoid environmental pollution.

Results and discussion

I. The nitrogen requirements of crops must be calculated on basis of the expected yields. The amounts of nitrogen needed to produce one ton of a specific crop (e.g. small grains, potatoes) are well-known figures. 1. The first step is to determine the amount

of available nitrogen (N min) in the soil profile in spring (Wehrmann und Scharpf, 1987).

2. As a next step, nitrogen has to be sup­plemented for early growth up to 120 kg N /ha, followed by split applications later on, up to the calculated total fertilizer amount (Wehrmann und Scharpf, 1987).

3. The extent of net mineralization in the soil during the growing season is still an open question, because mineralization and im­mobilization are opposing processes de­pending on site and cropping conditions. For small grains- as experiments have demonstrated- mineralization is nearly equal to immobilization. However, for

620 Amberger

crops with slow initial growth (e.g. sugar beets, maize, potatoes) leaving the soil uncovered for a long period, mineraliza­tion exceeds immobilization by far (Gutser et al., 1989; Kuhlmann and Engels, 1992). Definite figures can be obtained from field experiments or from long-term ex­periences of the farmer on the respective site.

4. The nitrogen import from organic residues of plant or animal production (e.g. sugar beet leaves or animal slurry) must also be considered in this calculation according to their availability (Amberger, 1987b). Min­eral nitrogen fertilizers may be utilized generally by about 70%, depending on location, crops and agro-techniques, the remainder being immobilized or lost (Vilsmeier et al., 1989).

II. Nitrogen losses and agro-techniques to avoid them.

Nitrogen losses are not only an economic but also an environmental problem. 1. Ammonia volatilization predominately is a

matter of inappropriate fertilizer applica­tion (Amberger, 1989). Among mineral fertilizers, ammonium containing or am­monia liberating products are concerned. In case of urea, volatilization on the aver­age, can amount up to 20-30%, and even higher, with broadcast application. In­corporation into the soil minimizes volatile losses to only a few percent (Amberger, 1990 a,b; Dahler, 1990).

A very serious situation arises in the case of animal slurry which contains 60-70% of total N in form of ammonium-N. More than 90% of ammonia pollution of the air is caused by animal production (KTBL, 1990). Ammonia volatilization takes place already in the first hours after spreading and can amount up to 80% depending on air temperature and wind flow, soil type (high pH, compact soil structure), soil cover (stubbles of small grains), and also on the dry matter content of slurry- thick slurry infiltrates the soil very slowly (Amberger, 1991). For mini­mum losses, animal slurry therefore has to

be incorporated immediately after spread­ing (Rank et al., 1987; Rank, 1988). When slurry is applied to growing crops (e.g. small grains), the crop canopy and the reduced air flow decrease ammonia losses to about 10-20% (Huber und Amberger, 1989). In case of grassland, dilution of slurry with water is recommended.

2. Denitrification losses are a further prob­lem relevant mainly in heavy soils. Esti­mates of losses generally range between 15 and 20 kg N y - 1 (Amberger, 1987a), as­suming a high nitrate concentration in the soil, high temperature and moisture as well as high amounts of microbial decom­posable carbon. These losses can only be reduced by improving soil structure with appropriate agro-techniques.

3. Nitrate leaching not only means a substan­tial economic loss but also a hazard to ground and drinking water. It happens predominately in the fallow period during winter and early spring and can be as­sessed to 50-70 kg N ha - 1 y - 1 (Amberger, 1987 a,b) depending on site conditions and the cropping system. The main determi­nants are the amount of unused fertilizer N after harvest and the mineralization rate during autumn.

Straw manuring (without additional fer­tilizer N) is an effective measure to pre­serve nitrate in the soil from leaching ( 0.1 ton of straw blocks about 1 kg N). How­ever, it has to be kept in mind that this biologically blocked nitrogen is not avail­able to the following crop, and therefore cannot enter into the fertilizer calculation (Amberger, 1991).

Slurry and nitrogen-rich waste water (e.g. from potato starch production, Am­berger and v. Tueher, 1989) when applied in autumn and incorporated into the soil, will be nitrified already within a few weeks, and are then readily leached out. The same is the case when green manure (or sugar beet leaves) is incorporated in early autumn (Schweiger, 1991). There­fore it is recommended to combine the nitrogen-rich slurry or waste water with straw poor in nitrogen, or to apply slurry

(20-30 m3 ha -I) as late as possible (during winter on slightly frozen unslopy soils with only a thin snow cover or spring) maybe with a nitrification inhibitor (Amberger, 1991; Vilsmcier und Amberger, 1987).

Catch crops or plant residues should also be incorporated as late as possible or- on sandy soils- better in early spring. The goal is to cover the soil as long as possible with crops preventing nitrate leaching. The utilization of nitrogen from green material by the following crop will be about 30% (Amberger, 1988; Gutser und Vilsmeier, 1988; Gutser, 1988).

III. Nitrification inhibitors (e.g. dicyandiamide, DCD) are also very effective instruments to control and optimize nitrogen management; they inhibit the first step of nitrification resulting in an accumulation of ammonium (Amberger, 1986).

Fertilizers on ammonium or urea basis can be amended with 8-10% DCD-N of total N, -trade names are Alzon, Basammon etc. When applied to small grains or leaf crops, they show convincing economic advantages (lower labour costs by concentration in two or even one single application) and produce the same or even higher yields with reduced amounts of nitrogen. Applied to leaf crops with slow early growth, they prevent translo­cation of nitrogen out of the rooting zone, ni­trate leaching and denitrification, especially in a rainy spring and in sandy soils (Klasse, 1991 ).

Dicyandiamide, when added in powder or liquid form to

-organic material from plant production, rich in nitrogen (e.g. vegetable residues, sugar beet leaves)

-waste water (e.g. from potato starch production)

- animal slurry inhibits nitrification and finally nitrate leach­ing, (Kolbl, 1987; Miiller-Wiesenfeld 1987; Schweiger, 1991) which is of great impor­tance especially for water catchment areas. A very important point is that the preserved ammonium N is fully available to the follow­ing crop contrary to the microbially blocked

Nitrogen management in crop systems 621

nitrogen with straw manuring and can be included in the fertilizer balance-sheet. Besides, DCD will be decomposed without further residues to NH3 , C02 and water, thus acting also as a slow-release N fertilizer.

References

Amberger A 1986 Potential of nitrification inhibitors in modern N fertilizer management. Z. Pflanzenernaehr. Bodenkd. 149, 469-484.

Amberger A 1987a Pllanzenerniihrung. UTB, Ulmer Verlag Stuttgart, 264 p.

Amberger A 1987b Utilization of organic wastes and its environmental implications. CIEC Symp. Braunschweig Proc. 4, 37-54.

Amberger A 1989 NH3 -Verluste aus der Anwendung organis­cher und anorganischer Diinger. VDLUFA-Schriftenreihe 30, 103-108.

Amberger A 1990a Harnstoffumsetzung imBoden und NH 3 -

Verluste. VDLUFA-Schriftenreihe 32, 243-248. Amberger A 1990b Ammonia emissions during and after

land spreading of slurry. In Odours and Ammonia Emis­sions from Livestock Farming. pp 126-131. Elsevier Ap­plied Science, London.

Amberger A 1991 Strategien zur Giilleanwendung in 6kologischer und 6konomischer Hinsicht. VDLUFA­Schriftenreihc 33, 105-110.

Amberger A and Von Tucher Th 1989 Einsatz von Kartoffel­fruchtwasser in der Landwirtschaft. Z. Kartoffelbau 40, 216-219.

Dahler H 1990 Laboratory and field experiments for estimat­ing ammonia losses from pig and cattle slurry following application. In Odours and Ammonia Emissions from Livestock Farming. pp 132-140. Elsevier Applied Science. London.

Gutser R 1988 Stickstoffmineralisation von Zwischenfriichten im Modellversuch. Kali Briefe 19, 213-223.

Gutser R and Vilsmeier K 1988 Mineralisation verschiedener Zwischenfriichte and N-Verwertung durch Pflanzen. Kali Briefe 19, 199-211.

Gutser R, Vilsmeier K, Teicher K and Beck Th 1989 Aussagekraft des N0 ,,-Stickstoffs fiir die N-Nachlieferung von Boden. VDLUFA-Schriftenreihe 30, 187-194.

Huber J and Amberger A 1989 NH3 -Verluste unter ver­schiedenen Anbaubedingungen. VDLUFA-Schriftenreihe 30, 109-115.

Klasse H J 1991 Versuchsergebnisse zur Wirkung stabilisier­ter Stickstoffdiinger auf die Nitratverlagerung bzw. -aus­waschung. In Stabilisierte Stickstoffdiinger, ein Beitrag zur Verminderung des Nitratproblems. Fachtagung Wiirzburg, BASF und SKW Broschiire. 157 p.

Kolbl Ch 1987 Modellversuche zur Wirkung von Nitri­fikationshemmstoffen auf den N-Umsatz von Ernteriick­stiinden des Gemiisebaues. Dip!. Arbeit TU Miinchen­Weihenstephan. 81 p.

622 Nitrogen management in crop systems

KTBL (Kuratorium fiir Technik und Bauwesen) 1990 Am­moniak in der Umwelt. Darmstadt. 45 Beitrage.

Kuhlmann H and Engels Th 1992 N-Freisetzung besser kalkulieren. DLG-Mitteilungen, 3, 38-40.

Miiller-Wiesenfeld G 1987 Wirkung von Didin auf Nitrat­auswaschung und Stickstoffverwertung gemiisebaulicher Riickstiinde. Dip!. Arbeit TV Miinchen-Weihenstephan. 78 p.

Rank M 1988 Untersuchungen zur Ammoniakverfliichtigung nach Giillediingung. Diss. TY Miinchen-Weihenstephan. 108 p.

Rank M, Huber J and Amberger A 1987 Model trials on the volatilization of ammonia following slurry application under controlled climate and field conditions. Proc. 4th Int. CIEC Symposium Braunschweig-Viilkenrode, pp 315-320.

Schweiger P 1991 Wege zur Minimierung des Nitrateintrages. In Stabilisierte Stickstoffdiinger, ein Beitrag zur Vermin­derung des Nitratproblems. Fachtagung Wiirzburg, BASF und SKW Broschiire. 157 p.

Vilsmeier K und Amberger A 1987 Zur nitrifikationshem­menden Wirkung von Dicyandiamid zu Giille in der Zeit zwischen Spiitherbst und Friihjahr. Z. Pflanzenernaehr. Bodenkd. 47-50.

Vilsmeier K, Amberger A und Gutser R 1989 Dynamik von Boden und Diingerstickstoff C'N) im Weihenstephaner Lysimeter. VDLUFA Schriftenreihe 28, 455-469.

Wehrmann J und Scharpf H C 1987 Sachgerechte Stick­stoffdiingung. AID-Heft, 1017.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 623-628, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-066

A review of internal cycling of nitrogen within trees in relation to soil fertility

P. MILLARD Macauley Land Use Research Institute, Craigiebuckler, Aberdeen, AB9 2QJ, UK

Key words: deciduous trees, evergreen trees, nitrogen, remobilisation, senescence

Abstract

The processes of internal cycling of N in trees are reviewed. Deciduous trees remobilise N from roots, trunks and twigs for spring growth of leaves, and withdraw leaf N during senescence. Evergreen trees have an additional retranslocation of N from the previous year's foliage to supply apical growing points during periods of flushing. The impact of nutrient supply on internal cycling has been studied at the ecosystem level, where N budget studies have concentrated on leaf senescence, demonstrating that enhanced soil fertility has either no effect or decreases the proportion of leaf N withdrawn. Remobilisation of N in the spring has seldom been quantified accurately under field conditions. Instead the use of 15N applied to young trees growing in sand culture has demonstrated that: (1) N remobilisation in the spring is dependent upon the amount of N in store and is unaffected by the current N supply; (2) internal cycling provides N for leaf growth in the spring before rapid root uptake of N occurs and (3) uptake of N in the autumn contributes directly to storage, while withdrawal of N from senescing leaves may provide a smaller proportion of the N subsequently recycled. These results are reviewed and discussed in relation to the internal cycling of N in both evergreen and deciduous trees.

Introduction

Nitrogen (N) is the nutrient most often limiting forest productivity in the northern hemisphere (Cole, 1981). N used by trees for growth can come from fertilisers, mineralisation of soil or­ganic matter, or atmospheric deposition which alone can provide up to 22 kg N ha -I year at some sites in western Europe (Skeffington, 1990). A major contribution also comes from N stored within the trees themselves, through the internal cycling of N. In deciduous trees such cycling comprises the remobilisation of N from perennial woody tissues for spring growth and the withdrawal of N from senescing leaves (Titus and Kang, 1982) and possibly roots (Ferrier and Alexander, 1991) (Fig. 1). Evergreen trees have a further component of retranslocation of N from older leaves (Fig. 2) to enhance the supply

to apical growing points during periods of flush­ing (Nambiar and Fife, 1987).

Internal cycling of N can be used by trees to maximise the availability of N in relation to carbon assimilation by the whole canopy (Field and Mooney, 1986) and contributes increasingly to seasonal growth as the tree ages (Miller and Miller, 1987).

In order to understand how trees will respond to changes in soil fertility it is necessary to understand the impact of nutrition on the pro­cesses of internal N cycling. These processes can be considered as the storage of N and the remobilisation of N from storage for further growth. Our understanding of these processes is reviewed and discussed in light of both eco­system studies and experiments on young trees that have used 15N in sand culture to quantify the effect of N supply on internal cycling.

624 Millard

Internal cycling of nutrients in deciduous trees

SPRING

R bl emo 11sation 1 upta e

storage

Twigs/trunk/roots Leaves WINTER SUMMER

storage

I uptake

AUTUMN

withdrawal

Fig. 1. The seasonal pattern of internal N cycling in decidu­ous trees, showing N storage (D) and flux of N (--> ).

WINTER

Internal nutrient cycling in evergreen trees

SPRING

Remobilisation

last year's foliage

Trunk/roots

storage

-

I uptake

AUTUMN

uptake I

storane

Foliage

storage

SUMMER

Fig. 2. The seasonal pattern of internal cycling in evergreen coniferous trees, showing N storage (D) and flux of N (-> ).

Nitrogen storage

The ability to store N is a characteristic feature of perennial plants. Millard (1988) considered that N is in store if 'it can be mobilised from one tissue and subsequently reused for growth or maintenance of another'. This definition was extended by Chapin et a!. (1990) to distinguish

between accumulation (luxury consumption), reserve formation and recycling. The importance of these definitions of N storage are twofold. Firstly, they emphasise the dynamic nature of storage (Millard, 1988). Secondly, they highlight that the ability of a plant to store N is not dependent upon its N status, which will only influence the amount of N held in store. During the internal cycling of N in deciduous trees there are two main periods of N storage, during the winter and the summer.

Perennial woody tissues store N in the winter, predominantly in the bark of twigs and trunk (Titus and Kang, 1982). Specific bark proteins have been isolated that are present in large quantities in winter and absent during the sum­mer (Nsimba-Lubaki and Penmans, 1986; Wetzel et a!., 1989). This reserve formation (as defined by Chapin et a!., 1990) is controlled by photo­period (Coleman et a!., 1992). Storage of N during the winter has also been reported in the roots of young trees (Taylor and May, 1967; Tromp, 1983; Millard and Proe, 1992). Free amino acids (principally arginine) were reported as N stores in roots of Morus alba (Suzuki and Kohno, 1984) and Malus domestica (Tromp, 1983), while in Acer pseudoplatanus seedlings that had a large tap root, N was stored in arginine-rich proteins (Millard and Proe, 1991 ). However, the importance of root storage of N in larger trees has not been determined.

The second period of N storage occurs during the summer (Figs. 1 and 2), when leaves are a dominant sink for N uptake (Millard and Neilsen, 1989; Pereira eta!., 1988). The majority of N in leaves is incorporated into proteins, the most abundant of which in C3 plants is Ribu­lose 1 ,5-bisphosphate carboxylase/ oxygenase (RUBISCO) (Stoddart and Thomas, 1982). It has been suggested that in annual plants RUBISCO has a role as a store for N (Huffaker and Miller, 1978; Millard and Catt, 1988), equiv­alent to the recycling defined by Chapin et a!. (1990). In M. domestica RUBISCO accounted for more than 90% of the soluble protein lost from leaves during senescence (Kang and Titus, 1980) and contributed between 32-48% of the N subsequently remobilised to support leaf growth the following year, depending on the N supply (Millard and Thomson, 1989). However, a role

for RUBISCO in reserve formation cannot be ruled out. Quick et al. (1991) reported that mutants of Nicotiana tabacum containing only 60% of the RUBISCO of wild types exhibited only a marginal inhibition of photosynthesis, suggesting that more of the protein was syn­thesised by the wild types than was needed to avoid a RUBISCO limitation to photosynthesis.

Remobilisation of stored nitrogen

Following the two main periods of N storage, remobilisation of N from storage in deciduous trees occurs predominantly in the autumn (dur­ing leaf senescence) and the spring (during leaf growth) (Fig. 1). Studies of large trees growing in the field have concentrated on N withdrawal from senescing leaves. Deciduous trees vary in their ability to withdraw N during leaf senesc­ence, ranging from 78% of the total leaf N in Quercus prinus (Ostman and Weaver, 1982) to 21% in Nothofagus truncata (Miller, 1963). The pattern of leaf demography is important in determining the ability to withdraw leaf N. Ecophysiological studies have demonstrated that in the arid conditions of Spain, the duration of the abscission period is closely correlated with the amount of leaf N withdrawn (Del Arco et a\., 1991). Site fertility can also affect the with­drawal, with a decrease in the proportion of leaf N reutilised in response to an increase in N supply reported by Flanagan and van Cleve (1983) and Boerner (1984). This is consistent with the observation that an increase in N supply to plants can delay the onset of senescence (Stoddart and Thomas, 1982), as observed in M. domestica by Millard and Thomson (1989) when contrasting amounts of N were supplied during the autumn. However, other workers have re­ported N withdrawal from leaves to be unaffect­ed by site fertility (Chapin and Kedrowski, 1983; Ostman and Weaver, 1982; Pereira et al., 1988; Staff, 1982). Nutrient retention in leaves of a range of species exhibiting contrasting leaf longevitics and efficiencies of nutrient withdraw­al during senescence was found by Escudero et al. (1992) to be prolonged on infertile sites. However, the effect of the efficiency of rcmobili-

Internal cycling of N in trees 625

sation of N on retention time was negligible compared with the effect of leaf longevity.

In coniferous, evergreen trees N is stored predominantly in needles and remobilised to support the growth of new foliage as evidenced by N budget studies (Aronsson and Elowson, 1980; Miller et a!., 1979; Nambiar and Fife, 1991; Rapp et a!., 1979). These studies have demonstrated that, in contrast to deciduous trees, remobilisation of N from the needles of evergreen trees can occur independently of senescence. Several studies have highlighted the importance of young needles as storage sites for N. Nambiar and Fife (1987) estimated that N from first year needles of P. radiata provided 30 and 57% of the N used for the growth of new needles of unfertilised trees and those that had received N additions in the previous year, re­spectively. Fertilisation increased the internal cycling of N from first year needles by increasing both their mass and the proportion of their N remobilised. Mead and Pritchett (1975) also reported that application of fertiliser to P. elliottii increased the amount of N remobilised from each first year needle during the second year of their experiment. While not all evergreen species remobilise leaf N independently of senescence (Jonasson 1989), it is likely that budget studies which estimated internal cycling as the difference in N content between green and senescent leaves will have underestimated the contribution made to the annual demand for N.

The form of the N stored in conifer needles is not known, although RUBISCO would again be a likely candidate. In a study of P. mariana, Greenway et a!. (1992) measured the seasonal N dynamics of different age classes of foliage and found no evidence that older needles served as nutrient storage sites. Instead, they suggested, on the evidence of gas exchange measurements, that the maintenance of long-lived foliage was due to their contribution to carbon assimilation.

The impact of N supply on remobilisation

Nitrogen budget studies have been used to quantify internal cycling in relation to site fertili­ty (e.g. Aronsson and Elowson, 1980; Miller et al., 1979; Nambiar and Fife, 1991) and stand age

626 Millard

(e.g. Helmisaari, 1992; Miller, 1981). A limita­tion of such studies is that they are relatively imprecise and have seldom allowed the processes of internal cycling to be measured directly. Remobilisation of N in the spring has either been assumed to be equal to the amount of N with­drawn from senescing leaves (e.g. Helmisaari, 1992) or calculated by net losses of N from older tissues (e.g. Miller et a!., 1979; Nambiar and Fife, 1987), often excluding roots. Such ap­proaches make it difficult to assess the impact of soil fertility on the efficiency of N remobilisation in the spring. The first method does not allow for N uptake in the autumn augmenting N storage directly. The second method is imprecise, because of the nature of measurements made and in some cases a failure to complete budgets for whole plants.

An alternative approach has been to use 15N to quantify internal cycling (e.g. Hill-Cottingham and Lloyd Jones, 1975; Weinbaum et a!., 1987). The use of sand culture techniques has allowed the nutrient supply to trees to be controlled precisely, and both N uptake by roots and remobilisation in the spring to be accurately measured (Millard and Neilsen, 1989; Millard and Thomson, 1989; Millard and Proe, 1991;

1992). Table 1 shows the results from such experiments, quantifying the remobilisation of N for spring leaf growth in a range of species. Young trees, typically three or four years old, were grown for one year and provided with 15N throughout. Internal cycling of N was quantified by the recovery of unlabelled N in the leaves over successive destructive harvests (e.g. Millard and Neilsen, 1989). Alternatively, trees were preconditioned with either a generous or poor 15N supply for a whole year and supplied un­labelled N in the second year. Recovery of 15N in the leaves grown in the second year quantified the remobilisation of N in the spring (e.g. Mil­lard and Proe, 1991).

The use of 15N in these experiments allowed several characteristics of remobilisation to be determined that would not have been possible from N budget studies. Firstly, in each species the amount of N remobilised for the spring growth of leaves was not affected by the current N supply (Table 1). In contrast, leaf growth was influenced. The generous N supply in each experiment produced a significantly greater leaf mass per tree than for those provided with a poor N supply. When Acer pseudoplatanus or Picea sitchensis had their growth preconditioned,

Table 1. The effect of current N supply on the spring remobilisation of N for leaf growth by a range of species. Values for the remobilisation of N (as measured using 15N), the leaf N content in the spring when remobilisation had finished, the maximum leaf mass per tree found in the growing season and the maximum leaf N content recovered during the growing season represent the mean and standard error of four replicates

Species N supply LeafN N remobil- Max leaf Max leaf content isation in mass of N content

Preconditioning Current in Spring spring tree -1 in the in summer summer

(mgN /tree) (mgN /tree) (g/tree) (mgN/tree)

Fraxinus excelsior Generous N 100 ± 17.1 65 ± 9.5 10.3 ± 0.9 315 ± 24.0 PoorN 53± 8.5 48± 7.7 5.1±0.7 118 ± 21.9

Betula pendula Generous N 121 ± 6.7 30± 3.3 10.6 ± 0.6 PoorN 44± 5.3 23 ± 4.8 4.4 ± 0.3

M a/us domestica a Generous N 118 ± 8.8 58± 5.1 8.3 ± 0.8 320± 7.8 PoorN 71 ± 4.4 62 ± 3.1 4.2 ± 0.9 71 ± 4.4

Acer pseudoplatanus b GenerousN Generous N 372 ± 23.6 238 ± 17.2 18.3 ± 2.1 382 ± 28.5 Generous N PoorN 283 ± 20.0 232 ± 20.0 9.4 ± 2.7 283 ± 20.0

PoorN Generous N 190 ± 30.2 46± 6.8 18.9 ± 1.3 317 ± 16.6 PoorN PoorN 88± 19.0 47± 11.0 8.3 ± 1.7 109 ± 21.1

Picea sitchensis' Generous N Generous N 167±27.9 121 ± 23.5 22.6 ± 1.3 600 ± 32.5 Generous N PoorN 116 ± 23.1 104± 3.1 12.8±2.1 162 ± 10.7

PoorN Generous N 70± 8.8 29 ± 3.4 29.0 ± 1.9 673 ± 30.7 PoorN PoorN 36± 3.9 28 ± 2.9 6.1 ± 1.0 78 ± 2.1

"Data from Millard and Neilsen (1989). bData from Millard and Proe (1991). 'Data relate to current season's foliage only.

their capacity for remobilisation of N in the spring of the second year was dependent only on theN supply the previous year (Table 1). There­fore, the efficiency of remobilisation of N in the spring was unaffected by the current N supply in both deciduous and evergreen trees.

Secondly, remobilisation of N tended to occur before the majority of root uptake occurred. The data in Table 1 show that in the spring (once remobilisation of N had finished), internal cy­cling had provided a large proportion of the total leaf N content. However, root uptake sub­sequently continued, so that later on in the summer the maximum total leaf N content was often greater than the amount of N supplied by internal cycling.

These findings demonstrate that fertilising trees will increase tree growth and the capacity for storage, but have little or no effect upon the efficiency of remobilisation of N for spring growth. In contrast, Nambiar and Fife (1991) reported that in Pinus radiata the retranslocation efficiency of N from needles during periods of flushing was increased by high soil fertility. However, they did not necessarily quantify all the N available for the growth of new foliage, since measurements of root N contents were not made in most of their experiments. In a study of P. sitchensis, Millard and Proe (1992) found that roots contributed a significant proportion of the N remobilised for needle growth, particularly in trees grown with a poor N supply. Both A. pseudoplatanus (Millard and Proe, 1991) and Fraxinus excelsior (Millard, unpublished) can also store significant amounts of N in their root systems during winter. However, there is less evidence for N being retranslocated from roots during their senescence. Split root experiments with young P. sitchensis trees have shown re­translocation of N from droughted roots (Ferrier and Alexander, 1991) and Meier et a!. (1985) concluded that there was significant retransloca­tion of N from the senescent roots of Abies amabilis. However, Aerts (1990) found no evi­dence for N withdrawal from senescent roots of a range of evergreen and deciduous heathland species.

Measurements of the processes of internal cycling using 15N have determined that: (1) N remobilisation in the spring is dependent on the size of the store and is unaffected by current N

Internal cycling of N in trees 627

supply (Table 1); (2) internal cycling provides N for leaf growth in the spring before rapid root uptake (Table 1; Millard and Neilsen, 1989; Millard and Proe, 1991) and (3) uptake of N in the autumn can contribute directly to storage (Millard and Proe, 1992), while N withdrawal from senescing leaves may provide a smaller proportion of the N subsequently recycled (Mil­lard and Proe, 1991). These findings support the view that internal cycling is not a mechanism for adapting to low soil fertility (Chapin and Ked­rowski, 1983). Instead, N retranslocation is a method of augmenting the N supply to the apical growing points during periods of flushing (Namb­iar and Fife, 1987; 1991).

Acknowledgements

This work was supported by the Scottish Office Agriculture and Fisheries Department.

References

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Growth, nitrogen uptake and internal cycling in Eucalyptus globulus seedlings in relation to nitrogen supply

P.O. CARVALH0 1 , M.C. CALDEIRA1 , P. MILLARD 2 and J.S. PEREIRA1

1 Department of Eng. Florestal, Instituto Superior de Agronomia, Tapada da Ajuda, 1399 Lisboa, Portugal; 2 Macaulay Land Use Research Institute, Craigiebuckler, Aberdeen, AB9 2QT UK

Key words: dry matter partitioning, eucalypt, nitrogen retranslocation, nitrogen use efficiency

Abstract

Eucalyptus globulus seedlings were grown in sand culture and supplied with a nutrient solution containing nitrogen labelled with 15N. We evaluated the effects of two different relative addition rates of N (0.12d- 1 and 0.04d- 1 ) on plant growth, biomass partitioning and the internal cycling of nitrogen. The increased relative growth rates with high rate on N supply resulted mostly from foliage growth. The treatments had no significant effect on root growth rates. Consequently the root/shoot ratio decreased with high N supply. The use of 15N as a tracer allowed the quantification of the N uptake by the different parts of the plants. Nitrogen was translocated from old foliage for the growth of new leaves, but there was no evidence that N was remobilised from roots. The remobilisation of previously existing N (unlabelled) for the growth of new tissues was relatively more important in 'low' than in 'high' N plants. As a consequence the nitrogen use efficiency (NUE) was markedly higher in 'low' than in 'high' N plants.

Introduction

Nitrogen is often a limiting factor for forest primary productivity. A major contribution for tree growth can come from N stored in their own tissues. In deciduous trees, N is stored over winter in perennial woody tissues and remobil­ised in the spring for new growth (Millard and Neilsen, 1989; Millard and Proe, 1991 ). Ever­green trees have a further component of internal N cycling, consisting of N stored in older leaves and remobilised to support new growth (Millard and Proe, 1992; Nambiar and Fife, 1987). Inter­nal cycling of N contributes increasingly to seasonal growth as a tree ages (Miller and Miller, 1987). Nitrogen use efficiency depends largely on the importance of internal cycling (Birk and Vitousek, 1986).

The objective of this experiment was to de­termine the effect of N supply on biomass

growth and partitioning and the internal cycling of nitrogen in Eucalyptus globulus seedlings.

Materials and methods

The effects of nitrogen supply on growth and distribution of N in Eucalyptus globulus seed­lings were studied in potted seedlings (standard nursery plants, with a height of 30 em, 4 months after sowing). The seedlings were transferred to 10 L pots filled with washed sand 2 weeks before the beginning of the experiment. The seedlings were grown in a glasshouse kept at a tempera­ture of 20°C day I 15°C night. The treatments consisted of watering to field capacity each plant every second day with complete nutrient solu­tions containing nitrogen as NH4N0 3 , double labeled with 15N at 4 atom percent enrichment. Nutrients other than N were in fixed weight

630 Carvalho et al.

proportions relative to N according to Ericsson (1989); K(64); P(13), S(8), Mg(9), Ca(9), Mn(0.4), Zn(0.03), Cu(0.03), B(0.2), Fe(0.7). N was supplied at two relative addition rates: 0.12 d - 1 ('high' N) and 0.04 d - 1 ('low' N) ac­cording to the exponential formula

N = N;(exp(RAR*t) -1)

where N = amount of nutrient supplied, N0 =

initial N content in the plants (7. 7 mg plant - 1);

RAR = relative addition rate ( d - 1), t = time of the nutrient supply (day) (Ingestad and Lund, 1979).

The experiment began on 4 of February 1991 and lasted until the 1st of April 1991. Five plants per treatment were periodically harvested at 9 day intervals and their biomass separated in the following components: new leaves and stems, (grown after the beginning of 15N application), old leaves, old stems and roots. The oven dry weight of all biomass components was obtained after drying at 80°C for 48 hours. Leaf area and leaf numbers were also determined.

The total N and 15N concentrations in samples were determined using an ANA-SIRA mass spectrometer. The 15N enrichment of organs was used as a tracer, to allow the uptake of N during the labelling period to be calculated (Millard and Neilsen, 1989). Since this labelled N was the only source of N available to the seedlings during the experiment, the difference between the total N content and the labeled N content of each organ (unlabelled N) gave a measure of internal cy­cling.

Results

The increase in total biomass in both treatments is shown in Figure 1. The exponential model was tested and the best fit gave relative growth rates (RGR) of 0.043 d- 1 in 'high' N plants and 0.023 d- 1 in 'low' N plants. The best exponential fit for the increase in total leaf area gave relative leaf area growth rates of 0.044 d- 1 in 'high' N plants and 0.021 d- 1 in 'low' N plants respective­ly. There was a close relationship between whole plant growth and foliage biomass and the number of leaves produced during the experi-

16

§14 -o- High N ~ Low N

-"§, 12

·~ 10

> .... 8 'C - 6 c ..!!!

'~------ -1 c.

iii -~ 0 0 10 20 30 40 50 60

Days after begining of treatment Fig. 1. Changes in total biomass in two groups of E. globulus seedlings grown in 10 L pots in the greenhouse, with two different addition rates of nutrients.

ment (Fig. 2). The leaf area partitioning, i.e., the relative increment in leaf area per unit increment in biomass (Potter and Jones, 1977) ca. 1 month after the beginning of the treatments was 135 and 91 cm2g -l in the 'high' Nand in the 'low' N plants, respectively.

Table 1 shows how N supply influenced the dry weight distribution in the seedlings by the end of the experiment. New leaves represent 43% of total dry weight in 'high' N plants against 21% in 'low' N plants. The proportions of the total plant biomass corresponding to roots in the 'high' N and 'low' N treatments were 0.17 ( ±0.02) and 0.30 ( ±0.03) respectively. Root growth however was not significantly different in either treatment, resulting in a significant (p < 0.05) decrease in the root/shoot ratio in the 'high' N plants after one month of treatment, as shown in Figure 3. Old leaves increased their mass from 0. 7 g to 1.6 g in both treatments accounting for only 0.12 ( ±0.02) and 0.27 (±0.04) of total biomass respectively, in 'high' and 'low' N treatments in the end of the experi­ment (Table 1). The percent distribution of total N in the different parts of the plants at the end of the experiment (Table 2) shows a pattern similar to the one in dry weight distribution. Most of the N in 'high' N plants was in new leaves ( 60%) whereas old leaves of 'low' N plants had the same percentage of N as in new leaves (ca. 30% ).

Internal cycling of nitrogen in Eucalyptus 631

~7 140 E! ~High N +Low N ~High N -.o·Low N

~6 "E 120 A ctl 8 > c.

]ls In 100. Q)

~4 > ctl 80

c: ..!!! o3 - 60 0 - ... .c: Q)

.Ql2 .c 40 Q)

__ ., E

3: 1 :J 20 - - + - - +- - -·- - - -1

.. - z > ... co 0

0 10 20 30 40 50 60 0 10 20 30 40 50 60

Days after begining of experiment Days after begining of experiment

Fig. 2. Changes in (A) dry weight of new leaves and (B) number of leaves per plant in E. globulus seedlings grown in 10 L pots in the greenhouse, with two different addition rates of nutrients.

Table 1. Effect of N supply on dry weight distribution at the end of the experiment as a fraction of total biomass, after 56 days growth

HighN LowN

New leaves 0.43 ± 0.02 0.21 ± 0.05 New stems 0.11 ±O.o2 0.04 ± O.Gl Old leaves 0.12 ± 0.02 0.27 ± 0.04 Old stems 0.15 ± 0.02 0.17 ± 0.02 Roots 0.17 ± 0.02 0.30 ± O.o3

0.8 ~High N ~Low N

0.7

.5:! 0.6 -ro ... 0.5 -0 _g 0.4

. ·J···· l···l···· .. j I

U)

:;:.- 0.3 0

~ 0.2

0.1

0 0 10 20 30 40 50 60

days after begining of treatment Fig. 3. Root/shoot ratios of E. globulus seedlings grown with different nutrient addition rates.

The whole plant and leaf N concentrations at different harvest occasions are shown in Table 3. The whole plant N concentration in 'high' N seedlings reached 3.5% of dry weight at the final

Table 2. Effect of N supply on the proportion of total plant N in the different biomass components at the end of the experiment, after 56 days of growth

HighN LowN

New leaves 0.60 ± 0.02 0.33 ± 0.05 New stems 0.09 ± 0.02 0.04 ± O.Gl Old leaves 0.10 ± 0.01 0.31 ± 0.04 Old stems 0.05 ± 0.01 0.06 ± 0.003 Roots 0.17±0.02 0.26 ± 0.03

Table 3. Whole plant and leaf N concentration (mg g- 1 DW) at different harvest occasions in E. globulus seedlings sup-plied with two different addition rates of nutrients. Values are the mean with the standard deviation in parenthesis

Days after the beginning of experiment

19 37 57

HighN Whole plant 12.6 23.1 30.4 35.4

( 1.87) (5.68) (0.76) ( 1.89) New leaves 51.3 55.3 48.6

(3.82) (2.81) (3. 95) Old leaves 20.7 28.9 31.1 28.6

( 4.08) (115) (2.33) (3.41)

LowN Whole plant 13.5 14.0 14.2 10.9

(1.59) (2.55) (1.49) ( 1.03) New leaves 32.9 30.1 16.1

(6.7) (1.34) (2.5) Old leaves 211 20.7 19.5 12.4

(2. 91) ( 4.08) (1.14) (1.5)

632 Carvalho et al.

harvest whereas in 'low' N seedlings it was much lower (1.1%) at the final harvest. The leaf concentrations of 'high' N seedlings were rather constant during the experiment. In the 'low' N seedlings the leaf N concentrations decreased in the last harvest.

The distribution of the unlabelled N in the seedlings was quantified at each harvest and expressed as a proportion of the total unlabelled N recovered in each tissue (Table 4 ). Nitrogen was translocated from the old foliage to support the growth of new leaves but there was no evidence that N was remobilised from roots. Remobilisation of N from old leaves occurred throughout the experiment in the 'high' N plants. In contrast, the recovery of unlabelled N in the leaves of 'low' N plants did not increase after 45 days. The amount of unlabelled N (i.e., nitrogen present in the tissues before the beginning of 15N supply) present in new leaves is of particular interest because it gives an estimate of how important remobilisation of N was to sustain growth. As shown in Table 5, only about 10% of the total N in the new leaves of 'high' N plants was unlabelled at the final harvest, compared with about 47% in the new leaves of the 'low' N plants.

The nitrogen use efficiency (NUE) was calcu­lated as the ratio of biomass produced/N uptake for a given period. The high rate of nutrient supply reduced the NUE for the whole experi-

Table 5. Amount of total N (i.e. labelled and unlabelled) and the proportions of unlabelled N relative to total N in the leaves of E. globulus seedlings grown in the greenhouse with two different addition rates of nutrients. Values are the mean with the standard deviation in parenthesis

Total N %Unlabelled N (mg plant- 1 )

HighN New leaves 287.46 0.10

( 47.47) (0.005) Old leaves 46.13 0.42

(8.81) (0.04)

LowN New leaves 21.8 0.47

( 4.01) (0.05) Old leaves 20.0 0.81

(2.86) (0.03)

ment from 100.5 g g N- 1 , in the 'low' N plants to 25.4 g g N- \ in the 'high' N plants.

Discussion

Biomass production was positively related to nitrogen supply. This resulted largely from the increase in the partitioning of biomass to new leaves, allowing for more light interception by the 'high' N plants as found in the same species under field conditions (Pereira et a!., 1989). There was also a significant increase in nitrogen content of 'high' N plants, especially in new

Table 4. Effect of N supply on the proportion of the total plant unlabelled N recovered in different biomass components throughout the experiment. Values are the mean with the standard deviation in parenthesis

Days after beginning of experiment

12 IY 28 37 45 57

HighN New 0 0.02 0.26 0.17 0.25 0.36 0.43

leaves (0.03) (0.05) (0.04) (0.04) (0.04) (0.02) Old 0.65 0.66 0.50 0.57 0.49 0.37 0.28

leaves (0.06) (0.05) (0.15) (0.04) (0.05) (0.04) (0.02) Roots 0.23 0.20 0.16 0.17 0.15 0.16 0.16

(0.05) (0.03) (0.06) (0.03) (0.02) (0.04) (0.01)

LowN New 0 0.01 0.06 0.15 0.23 0.26 0.27

leaves (0.01) (0.03) (0.06) (0.03) (0.07) (0.06) Old 0.68 0.67 0.61 0.54 0.49 0.37 0.43

leaves (0.03) (0.02) (0.05) (0.04) (0.02) (0.07) (0.05) Roots 0.20 0.21 0.21 0.22 0.20 0.25 0.20

(0.03) (0.02) (0.03) (0.03) (0.02) (0.03) (0.03)

leaves (Tables 2 and 3). This led to the increase in photosynthetic rates of these compared to 'low' N plants (Pereira et Ia., 1992a). As a result of this and of lower root/shoot ratios, the 'high' N plants had 20 to 45% higher net assimilation rates than 'low' N plants (data not shown). Higher values of net assimilation rate and of leaf area partitioning contributed to the higher growth rates in 'high' N plants (Patterson et al., 1978; Potter and Jones, 1977).

The proportion of total biomass in roots in the 'high' N treatment was approximately half that of 'low' N plants. A decrease in root/shoot ratio with high N supply is a common response that has been reported also for other eucalypt species (Cromer and Jarvis, 1990; Linder and Rook, 1984). However, constant partitioning of dry matter to roots and shoots with different N treatments was reported by Sheriff and Nambiar (1991) in E. globulus.

Recovery of the labelled 15N in the new growth during the course of the experiment allowed the uptake of N through the roots to be quantified directly. Any unlabelled N recovered in the new growth must have been remobilised from tissues present at the beginning of the experiment. Translocation of N to the new leaves occurred from the old foliage, but not from roots as found in the deciduous Acer pseudoplatanus (Millard and Proe, 1991). This difference may have been due to the fact that the eucalypt seedlings were already growing at the beginning of the experiment and roots may serve mainly as overwintering storage for N. On the other hand, foliage was found to be the pre­dominant source of N rcmobilised for the growth of new leaves in another evergreen species, Picea sitchensis (Millard and Proe, 1992).

In spite of the increase in plant growth, there was a strong decrease in NUE in 'high' N when compared to 'low' N plants. The values found for NUE are comparable to those found for other tree seedlings (e.g. Acer pseudoplatanus, Millard and Proe, 1991). The marked increase in NUE in 'low' N plants may be explained in part as a consequence of the remobilisation of previously absorbed N. The amount of remobilised N represented about 47% of the total N in the new leaves of the 'low' N plants at the final harvest against ca. 10% in the 'high' N plants in the

Internal cycling of nitrogen in Eucalyptus 633

same occasion (Table 5). Therefore, in relative terms, the internal cycling of N was more im­portant in the 'low' N than in the 'high' N plants. However, the recovery of a greater proportion of the plant unlabelled N in the new leaves of the 'high' N compared to 'low' N plants (Table 4) is probably the result of the rapid growth of foliage on branch stems towards the end of the experi­ment in response to high rate of N supply (data not shown).

Under field conditions we found only small differences in NUE in E. globulus trees grown with different nutrient supply rates (Pereira et al., 1992a; 1992b; Tome and Pereira, 1990). Generous nutrient supply rates resulted in a substantial increase in growth, mainly because of the great increase in foliage and light intercep­tion by the canopy. However, contrary to the present experiment there were only minor differ­ences in N concentrations in leaves (Tome and Pereira, 1990) and that resulted in small differ­ences in NUE independently of nutrient supply. Another important factor was the interaction of nutrition with water availability, since plants grown without water deficits in the summer had the highest values of NUE, independently of nutrient supply rate (Pereira et al., 1992a).

Acknowledgements

This research was supported by a grant from the Commission of the European Communities, con­tract Nr. STEP-CT90-0037-(SMA).

References

Birk E M and Vitousek P M 1986 Nitrogen availability and nitrogen use efficiency in loblolly pine stands. Ecology 67, 69-79.

Cromer R N and Jarvis P G 1990 Growth and biomass partitioning in Eucalyptus grandis seedlings in response to nitrogen supply. Aust. J. Plant Physiol. 17, 503-515.

Ericsson T 1989 Mineral nutrient requirement of Eucalyptus globulus seedlings. Internal report Swedish University of Agricultural Sciences. Dept. of Ecology.

Ingestad T and Lund A B 1979 Nitrogen stress in birch seedlings I. Growth technique and growth. Physiol. Plant. 45, 137-148.

Linder S and Rook D A 1984 Effects of mineral nutrition on carbon dioxide exchange and partitioning in trees. In

634 Internal cycling of nitrogen in Eucalyptus

Nutrition of Plantation Forests. Eds. G D Bowen and E K S Nambiar. pp 211-236. Academic Press, London.

Millard P and Neilsen G H 1989 The influence of nitrogen supply on the uptake and remobilisation of stored N for the seasonal growth of apple trees. Ann. Bot. 63, 301-309.

Millard P and Proe M F 1991 Leaf demography and the seasonal internal cycling of nitrogen in sycamore (Acer pseudoplatanus L.) seedlings in relation to nitrogen supply. New Phytol. 117, 587-596.

Millard P and Proe M F 1992 Storage and internal cycling of nitrogen in relation to seasonal growth of Sitka spruce. Tree Physiol 10, 33-43.

Miller H G and Miller J D 1987 Nutritional requirements of Sitka spruce. Proc. Roy. Edin. 93B, 75-83.

Nambiar E K S and Fife D N 1987 Growth and nutrient retranslocation in needles of radiata pine in relation to nitrogen supply. Ann. Bot. 60, 147-156.

Pereira J S, Linder S, Araujo M C, Pereira H, Ericsson T, Borralho N and Leal L 1989 Optimization of biomass production in Eucalyptus globulus plantations. A case study. In Biomass Production by Fast-Growing Trees. Eds. J S Pereira and J J Landsberg. pp 101-121. Kluwer, Dordrecht.

Pereira J S, Chaves M M, Carvalho P 0, Caldeira M C and Tome J 1992a Carbon assimilation growth and nitrogen supply in Eucalyptus globulus plants. In Whole Plant ?respective on Carbon Nitrogen Interactions. Eds. J Roy and E Granier. SPB Academic Pub, The Hague (In press).

Pereira J S, Chaves M M, Fonseca F, Araujo M C and Torres F 1992b Photosynthetic capacity of leaves of Eucalyptus globulus (Labill.) growing in the field with different nutrient and water supplies. Tree Physiol. 11, 381-389.

Patterson D T, Meyer C T and Quinby P C 1978 Effects of irradiance on relative growth rates, net assimilation rates and leaf area partitioning in cotton and three associated weeds. Plant Physiol. 62, 14-17.

Potter J R and Jones J W 1977 Leaf area partitioning as an important factor in growth. Plant Physiol. 59, 10-14.

Tome M and Pereira J S 1991 Growth and management of eucalypt plantations in Portugal. In Productivity in Per­spective. Third Australian Forest Soils and Nutrition Conference. Ed. P J Ryan. pp 147-157. Forestry Commis­sion of NSW, Sydney, Australia.

M.A. C. Fraf?oso and M.L. van Beusichem ( eds.) Optimization <~f plant nutrition 635-640, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-018

A method to optimize N-application in relation to soil supply of N, and yield of potato

D.K.L. MacKERRON, M.W. YOUNG and H.V. DAVIES Scottish Crop Research Institute, Invergowrie, Dundee, DD2 5DA, UK

Key words: fertilizer, optimum application, plant nitrogen, potato, soil nitrogen

Abstract

Mathematical models of crop growth can provide estimates of the potential yield of potato, and also the minimum, critical N-concentration required, [NJ, to attain that yield. Efficient use of nitrogen requires that the crop incorporates sufficient nitrogen to attain its potential yield and that excess uptake is avoided. Predictions of the rate of supply of nitrogen from the soil are imprecise and so it has been difficult to estimate accurately the required application of fertilizer-N. Our work has shown the feasibility of using the growing crop as a monitor of the rate of supply of N from the soil. Using a low initial application rate of N at planting and monitoring uptake rate, we can estimate the contribution from the soil, and couple that information with estimates of yield and the related [Ncl to give an estimate of the requirement for supplementary applied-N. The method can be seen, therefore, as a means to determine the size of a second or subsequent part of a split application of fertilizer. This approach avoids much of the uncertainty over the fate of applied nitrogen and should offer growers the double benefits of economic use of fertilizer and of minimizing leaching losses. Further, by tailoring applications of N-fertilizer to the crop's requirements the grower will be better able to ensure the quality considerations in his crop.

Introduction

Requirements for N-fertilizer differ greatly from field-to-field in ways that are difficult to predict. For example, the economic optimum level of N-application to potato has been found in the Netherlands to range between 0 and 400 kg ha-l (Neeteson and Wadman, 1987). The principal difficulty in estimating fertilizer requirement ahead of growing the crop is in determining the supply of mineral N from the soil organic matter. The best single estimator of the N available from the soil (and so of the fertilizer requirements, by difference from the total needed) is the amount of mineral N in the top 60 em of soil in spring (Greenwood et al., 1986). But, even this ac­counts for only 30% of the variation in optimum application rate (Neeteson and Zwetsloot, 1989).

Traditionally, recommended fertilizer rates

have been based on empirically-derived relations between application rates and yield. with arbit­rary adjustments for soil type and an allowance for the amount of soil mineral N in spring. However, the response curve of potato to ap­plied N presents an extended plateau over which commercial yields do not decline at higher appli­cations (e.g. Neeteson, 1989). Because of this there has been a tendency for growers to apply excess nitrogen and ignore the contribution from any organic manures that are added. Current concerns with environmental effects are causing us to re-examine this practice. Applications of fertilizer nitrogen should be adequate to ensure the potential growth of the crop, but low enough to minimize losses through leaching.

MacKerron et al. (1990a) showed that rela­tions could be derived between the rate of uptake of N in plant tissues and the final N-level

636 MacKerron et a!.

at the end of the growing season and suggested that it may be possible to estimate rate of uptake of N at an early stage in the season and compare it with the required total uptake for the antici­pated yield. Adjustments could then be made to the rate of supply above that from the soil and a base-level application of fertilizer by supple­mentary application of N.

This paper reports on two experiments per­formed in successive years to test the generality of the relations found earlier, and to provide an initial test of the method for calculating the optimum supplementary application of N fertil­izer.

Materials and methods

Two field experiments are described. Experi­ment A, performed in 1990, was designed to examine the relations, in two cultivars, between N-uptakc and total plant fresh weight, and between N-uptake rate and the final N-uptake. Experiment B, performed in 1991, was designed to test the theory and the feasibility of predicting the N requirement of a potato crop using the relations established from Experiment A. In both experiments irrigation was scheduled to maintain a soil moisture deficit not less than 30mm.

Experiment A

This comprised four replicate blocks (12 x 50 m) each containing six plots of 15 x 6 m (8 rows x 45 plants) for nitrogen treatments. Cultivar Cara was planted in one half of each plot and Maris Piper in the other. The nitrogen treatments were 0, 40, 80, 160, 80 + 80 (split application) and 240 kg N ha - 1 (NO, N1, N2, N3, N4 and N5 respectively). Nitrogen treatments were random­ized within blocks and cultivars were randomized within nitrogen treatment. The second applica­tion of N in treatment N4 was made on 28 June, soon after tuber initiation. At each of five harvest dates, an eight-plant sample (foliage and tubers) was lifted from each plot, cleaned, sepa­rated into lamina, stem (including petioles and mid-ribs), and tubers and then oven-dried at 80°C for 48 hours (72 h for tuber material).

Relations between N-uptake and total fresh weight were examined by regression analysis using the package GENST AT. The statistical significance of differences in slope were tested as interactions between treatment and the x-vari­ablc.

Experiment B

This was planted to cultivar Maris Piper and comprised four replicate blocks (12 x 50 m) each containing eight plots of 11 x 6 m (8 rows x 33 plants) for nitrogen treatments. The nitrogen treatments comprised two levels of base dressing ( 40 and 80 kg ha - 1) each followed by four levels of supplementary N (0, r-40, r, r + 40 kg ha - 1 )

where r = the recommended rate estimated as described later. Base nitrogen treatments were randomized within blocks and the second appli­cations were randomized within these. Harvests were taken on three occasions, 24 J une-1 July and 12-15 July, as described earlier, for analysis of N-uptake and calculation of fertilizer require­ment, and at the end of the growing season, 16-18 September, for determination of yields and final N-uptake.

In both experiments dried material was ground using a Retch mill with a 0.5-mm grid. The nitrogen concentrations of the tissues were de­termined using the Dumas combustion method, Kjeldahl digestion (Expt. A only), and near infra-red reflectance (NIR) (Young et al., 1993).

Calculation of the requirement for supplementary N

There are several mathematical models available that describe the development and yield of the potato. The most successful use functions of weather variables to drive growth and develop­ment (e.g. Jefferies and Heilbronn, 1991; Mac­Kerron and Waister, 1985). Although such models are successful in calculating yield for given weather conditions they face a major difficulty when applied to forecasting yield since weather forecasts for periods of weeks to months ahead are imprecise. We used long-term average weather for the site, therefore, to predict crop yield (MacKerron et al., 1990b). For Experiment

B we estimated that the crop would attain a potential yield of 60 t ha - 1 •

For any expected yield the required final concentration of nitrogen, [N, 1 is calculated following Greenwood et a!. (1985a). However, in Greenwood's models nitrogen concentration, [N], (Greenwood et a!., 1985a, b) is expressed on the basis of total plant dry weight and so [N, 1 is calculated in the sequence:

Yield::::? Tuber DWt (using tuber dry matter concentration, 20%)

Tuber DWt::::? Total DWt (using harvest index, 75%)

Total DWt=? [Nc1 (after Greenwood) where the symbol::::? represents 'leads to' or

'gives'.

Then the minimum final N-uptake necessary for the achievement of potential growth can be calculated (=total DWt x [Ncl) from which we can derive the required N concentration, [N,], expressed on a fresh weight basis, using a rela­tion, similar to that described by MacKerron et a1.(1990a), between final N-uptake and [N1. The necessary relations were derived from Experi­ment A, conducted in 1990.

In order to avoid an excessive use of fertilizer, the crop is planted with a base-dressing of N fertilizer that is less than the 'normal' recom­mendation for the crop and soil in question. Then, the necessary, supplementary application of nitrogen is estimated. The rate of N uptake and so the actual nitrogen concentration, [N.], is determined on a fresh-weight basis from two early harvests. That value of [N.1, is compared with the estimated [N,1. It is to be expected that [N .1 will be low since less fertilizer will have been applied. The difference in the concentra­tions ([N,1-[ N.]) must be made up by supple­mentary applications. The size of the supplement is determined from the response of [N.1 to N­application for that soil-type.

Results

Experiment A

In both cultivars, nitrogen was taken up by the plants throughout the growing season at rates

~400 m ..c

If 300 (!) ~

~ 200 a. :=l

a5 100 Ol

-~ z

Optimizing nitrogen application 637

cvar: Cara

50 100 Total Fresh Weight (t ha-1)

Fig. 1. The relation between total fresh weight and N-uptake for cultivar Cara, Experiment A, 1990. Labels indicate nitrogen treatments NO-NS. For sake of clarity, data points are given for treatments NO (e), N3 (+),and NS (*),only.

that declined as the season advanced and that differed between N-treatments. Nitrogen uptake was linearly related to plant fresh weight in both cultivars (Figs. 1, 2) indicating that [N], ex­pressed on a fresh-weight basis, was maintained throughout the season. The slope of the relation varied with nitrogen application rate (Table 1) as reported previously by MacKerron et a!. (1990a). The slope has units gN/kg FWt., and indicates the concentration, [N.J, of N in the crop. The slopes and, therefore, [N.] for the plants receiving 160 kg N ha - 1 (N3) and (80 + 80) kg N ha -I (N4) were not statistically sig­nificantly different in either Cara (p = 0.223) or Maris Piper (p = 0.963), indicating that the values of [N al and the N-uptake rates can be

~400 jg cvar: Maris Piper

E3oo (!) ~

~200 a. :J

a5 100 Ol 0

--~ z 00 50 100 Total Fresh Weight (t ha-1)

Fig. 2. The relation between total fresh weight and N-uptake for cultivar Maris Piper. Experiment A, 1990. Labels as in Figure 1.

638 MacKerron et al.

Table 1. Regression coefficients between total fresh weight (x) and N-uptake (y) of cultivars Cara and Maris Piper. Slope and SE have units of g N/kg FWt

Nitrogen treatment

NO N1

Cara Slope 1.04 1.01 S.E. O.D7 0.15

0.96 0.85 n 20 20

Maris Piper Slope 1.20 1.50 S.E. 0.14 0.21

0.88 0.86 n 20 20

altered by supplementary application of N, at least as late as shortly after tuber initia­tion.

Plotting final uptake of N against the regres­sion slope of total fresh weight and N-uptake, [N al, indicated that, as previously reported, there was a linear relation between the two variables (Fig. 3). The slopes of the relations for Cara and Maris Piper in 1991 were not sig­nificantly different from each other or from those reported by MacKerron et a!. (1990a). How-

~ 0.0035 u.. OJ 0.003

-'<:

z 0.0025

~ 0.002

~ 0.0015 Cl)

~ 0.001 (/)

.· 1984 Piper

.. ·• 1985 Cara

100 150 200 250 300 350 N Uptake by final harvest (kg ha.1)

Fig. 3. The relation between the concentration of nitrogen, [NJ, (the slopes from Figs. 1, 2), in each treatment and the N-uptake by final harvest. Each point corresponds to one N-treatment. e- Maris Piper, 1990, •- Cara, 1990. The relations reported earlier for Maris Piper by MacKerron et al. (1990a), are shown for interest ---84- Maris Piper, 1984, ---85- Maris Piper, 1985.

N2 N3 N4 NS

1.14 1.60 1.25 2.23 0.09 0.18 0.22 0.16 0.95 0.90 0.78 0.95

20 20 20 20

1.64 2.11 2.12 2.67 0.22 0.14 0.16 0.12 0.86 0.96 0.95 0.98

20 20 20 20

ever, the lines do not share the same inter­cepts.

Experiment B

The calculation of the required supplementary N was made using the relations established during the previous year in Experiment A. From the anticipated potential yield of 60 t ha -I we calcu­lated that the total N-uptake should be at least 226.1 kg ha- 1 for the potential to be realized. From the relation illustrated in Figure 3 we calculated that [N,] of such a crop would be 0.002048 kg N/kg FWt. The analyses of data on N-uptake and growth from the two early harvests indicated that the values of [N al were 0.001019 ± 0.000124 kg N /kg FWt and 0.001460 ± 0.000237 kg N/kg FWt, respectively, in the plants from the 40 kg N ha -I and 80 kg ha -I treatments. From the relation between [N al and applied-N from 1990, we calculated that the two supple­ments required were 174 kg N ha -I and 100 kg N ha-t in the two treatments respectively.

At the end of the growing season, by 16-18 September, N-uptake, total fresh weight and tuber yield were maximal at the calculated, 'recommended' supplemental rates, r, for both rates of base dressing (Figs. 4, 5). Standard errors were large but the results are consistent with the hypothesis that the optimum supple­ment was close to the rate calculated from the relations between [N] and plant fresh weight.

180 90"";-~ Base= 40 kg N ha·' m

..c m 160 80 _. ..c -Ol -o

.:s:. 140 70 a5 - >= <I)

.:s:. 120 60 -o m c 0.. m :J 100 50 ~ z

80 40 LL

0 r- r r+ sed Treatment

~ N Uptake m FWt [S] Yield Fig. 4. Nitrogen uptake, total fresh weight (haulm and tubers), and tuber yield for base dressing of 40 kg N ha- 1 , at four levels of supplementary N. 0 = no supplement, r = calculated optimum requirement, r- = (r- 40)kg N ha _,, r + = (r + 40 )kg N ha- 1 , sed = standard error of difference -relative to corresponding axis (p ~ 0.05).

180 Base = 80 kg N ha·' 90"";-~

m '";- ..c m 160 80 _. ..c -Ol -o

.:s:. 140 70 Q) - >= <I)

.:s:. 120 60 -o m c 0.. m :J 100 50 ~ z

80 40 LL

0 r- r f+ sed Treatment

~ N Uptake m FWt [S] Yield

Fig. 5. Nitrogen uptake, total fresh weight (haulm and tubers), and tuber yield for base dressing of 80kg N ha- 1 , at four levels of supplementary N. 0, r, r-, r+, same as in Figure 4.

Discussion

The test (Experiment B) of the proposed meth­od for determining the optimum application of N gave recommendations for the supplements that differed by more than the original applications of fertilizer, so that the total application on the treatment with the low base dressing was rather higher than would normally be recommended. This almost certainly reflects the lapse of time while early growth was made and the uptake rates were being established, followed by the

Optimizing nitrogen application 639

need to take up the required nitrogen at a faster rate if the requirement were to be met in the time available. Where the base application was at around half of the 'normal' rate, the sum of base and supplementary applications was close to normal. This emphasises the importance of giv­ing an adequate initial dressing for early growth.

Total uptake of N in the two 'recommended' N-treatments was rather different, being much lower in the plants given a base-dressing of only 40 kg ha -I. Those plants did not take up all the fertilizer that was available, regardless of any soil mineralization. In contrast, the plants given the higher base-dressing took up the equivalent of all the applied N fertilizer. This result shows the importance of giving the supplementary applica­tions early in the growing season.

In deriving an estimated value for potential yield we used long-term average weather and a simulation model of potato growth and yield (Jefferies and Heilbronn, 1991). In more general applications, where either a suitable simulation model or records of long-term average weather are not available, or where yields are not ex­pected to reach the potential values it may be better to chose yield levels for a particular farm or field on the basis of past experience. The annual variation in potential yields is generally less than 20% (MacKerron, 1985). Even allow­ing for extreme conditions the eventual, true values of yield will probably lie within 20% of such estimates.

The work reported here has shown that the growing crop can be used to monitor the rate of supply of N from the soil whether from applied fertilizer or mineralized organic matter. That information can be coupled with values for expected yield and the related [N cl to give an estimate of any requirement for further applied­N. The method can be seen, therefore, as providing a rational basis for using split applica­tions of fertilizer and as a means to determine the size of a second or subsequent part of a split application. This approach avoids much of the uncertainty over N levels in soil and if de­veloped, should enable a closer match between application and requirement for N. The method should offer growers the double benefits of economic use of fertilizer and of minimizing environmental impact through leaching losses.

640 Optimizing nitrogen application

Initial examination of this proposed method for optimizing the application of fertilizer N has confirmed that the method can work but has also, shown the urgent need for timeliness i~ estimation of actual rates of N-uptake, the need not to have the initial rate of application too low, and the need for a suitable method of applica­tion. The principal weakness in the method is the need to use a relation between applied N and response of uptake.

Acknowledgements

This work has been supported by the Potato Marketing Board, Great Britain and the Scottish Office Agriculture and Fisheries Department.

References

Greenwood, D J, Neeteson J J and Draycott A 1985a Response of potatoes to N fertilizer: Quantitative relations for components of growth. Plant and Soil 85, 163-183.

Greenwood D J, Neeteson J J and Draycott A 1985b Response of potatoes to N fertilizer: Dynamic model. Plant and Soil 85, 185-203.

Greenwood, D J, Neeteson J J and Draycott A 1986 Quantitative relationships for the dependence of growth rate of arable crops on their nitrogen content, dry weight and aerial environment. Plant and Soil 91, 281-301.

Jefferies R A and Heilbronn T D 1991 Water-stress as a constraint on growth in the potato crop. I. Model develop­ment. Agric. For. Meteorol. 53, 185-196.

MacKerron D K L and Waister P D 1985 A simple model of potato growth and yield. I. Model development and sensitivity analysis. Agric. For. Meteorol. 34, 241-251.

MacKerron D K L 1985 A simple model of potato growth and yield. II. Validation and external sensitivity. Agric. For. Meteorol. 34, 285-300.

MacKerron D K L, Davies H V, Marshall B and Millard P 1990a Optimum nitrogen supply and yield- the crop as an indicator. Abstracts 11th Triennial Conference of the European Association for Potato Research, Edinburgh, 8-13 July 1990. pp 135-136.

MacKerron D K L, Greenwood D J, Marshall B, Rabbinge R and Schober B 1990b Forecasting systems for the potato crop. Proceedings 11th Triennial Conference of the Euro­pean Association for Potato Research, Edinburgh. 8-13 July 1990. pp 85-106.

Neeteson J J 1989 Evaluation of the performance of three advisory methods for nitrogen fertilization of sugar beet and potatoes. Neth. J. Agric. Sci. 37, 143-155.

Neeteson J J and Wadman W P 1987 Assessment of economi­cally optimum application rates of fertilizer N on the basis of response curves. Fertilizer Res. 18, 37-52.

Neeteson J J and Zwetsloot H J C 1989 An analysis of the response of sugar beet and potatoes to fertilizer nitrogen and soil mineral nitrogen. Neth. J. Agric. Sci. 37, 129-141.

Young M W, Davies H V and MacKerron D K L 1993 Comparison of techniques for nitrogen analysis in potato crops. In Optimization of Plant Nutrition. Eds. M L Van Beusichem and M A C Fragoso. Kluwer Academic Pub­lishers, Dordrecht.

Reprinted from Plant and Soi/154: 139-144, 1993.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 641-649, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-122

Nitrogen fertilization of potato and maize in relation to yield, quality of the production and risks to the environment

J.P. GOFFART and J. GUIOT Agronomical Research Centre, Experimental Station for Crop Husbandry, 5030 Gembloux, Belgium

Key words: maize, organic manure, potato, soil mineral nitrogen

Abstract

With regard to the nitrogen fertilization of main crops, one of the major issues is now the research into the appropriate balance between crop yield, quality of the production and reduction of the soil mineral N content at harvest time. To value for these three objects the effects of non-adapted or excessive N fertilizer rates in potato and maize crop, three trials were carried out for both crops in the southern part of Belgium (loamy region) during two consecutive years (1990 and 1991). The experimental sites mainly differed by the management of organic manures applied. For each trial, different N rates of ammonium­nitrate fertilizer ranging from 0 to 250 or 300 kg N ha-t were tested. Optimal N fertilizer rates for yield were highly variable according to climatic conditions and type or amounts of residual organic manures. The large potato tubers proportion still increased with excessive N rates for yield while dry matter content decreased but not significantly. Excessive rates also contributed to increasing the mineral N content in the soil profile, but especially for cropping systems including large amounts of organic manures. The management of the intercropping period before potato and maize crops also turned to be of major importance in avoiding the leaching of nitrate ions, released from soil and manures, to soil layers that will have poor chance to be reached by the root system of the cropped species, which is especially true for the potato crop.

Introduction

In several western European countries, impor­tant amounts of organic amendments (farmyard manure, cattle or pig slurries) are usually applied before sowing or planting of silage maize and potato crops, both head of rotation (Sluijsmans et a!., 1978). During the months following such important applications, variable amounts of min­eral N will be released in the arable soil layer, from which one unknown but highly variable part of nitrate N will be submitted to leaching to deeper soil layers during the winter period, while another part is used by the following cropped species (Powlson eta!., 1989). In such cropping systems, the risks of nitrate N losses and of subsequent ground water pollution will be great­ly increased in the case of N fertilizer doses

applied in excess to the crops, with respect to optimal yield and quality of the production (Prins et a!., 1988).

In the last ten years, in Belgium, potato and silage maize crops areas have considerably ex­tended. In 1990 these two crops respectively represented 7.3 and 18.5% of the total arable area used for main crops, i.e. 48,300 and 123,000 ha, corresponding nearly to increases of 30% and 40% as compared to 1980 (Van Heeke, 1992). Increases were particularly important in the loamy region, one of the top production areas in Belgium. Owing to environmental con­cern, i.e. the risk of pollution of several major groundwater tables located in that region, the following requirements are essential: 1) a search for cultural practices leading to a serious reduc­tion of nitrate leaching during the winter period

642 Goffart and Guiot

preceding potato and maize crops: 2) the demon­stration of the agronomical and environmental consequences of excessive N fertilizer rates ap­plied to these crops included in cropping systems with high amounts of organic amendments; 3) the valuing of the existence of nitrogen fertiliza­tion practices leading to maximal yield but also to specific quality parameters for potato tubers according to their use, and finally to low soil mineral N residues at harvest.

With respect to these points, research has been recently initiated on both crops at our Experimental Research Station. This paper pre­sents and discusses the results of field trials realized in 1990 and 1991 on potato and silage maize crops located on deep loamy soils where contrasted amounts of organic manures had been applied. In each trial, the effects of increasing N fertilizer doses were tested on yield, quality of

the potato tubers and soil mineral N content at harvest.

Materials and methods

Description of the field trials

The field trials were located in the loamy soil area of Belgium. Their cultural and soil charac­teristics are described for potato and silage maize trials in Tables 1 and 2, respectively. For each tested crop, the trials essentially differ with respect to the management of organic manures (farmyard manure, slurry or green manure) the year before sowing or planting. For each potato trial, N fertilizer rates of ammonium-nitrate ranging from 0 to 250 or 300 kg ha -I were tested, while for maize crop the tested N rates

Table 1. Cultural and arable soil layer (30 em) characteristics for the potato trials

Locality and year

Liege 1990 Gembloux 1991 Liege 1991

Preceding crop Winter wheat with Winter barley with Winter wheat with straw exported straw exported straw exported

Organic amendments 25 t ha - 1 farmyard 25 t ha - 1 farmyard 25 t ha - 1 farmyard before potato crop manure (every three manure for the first manure and rye grass

years) time in ten years as green manure Potato cultivar Bintje Bintje Bintje Planting date 04/03/90 04/09/91 04110/91 Harvest date 09/28/90 09105191 09/26/91 Plantation density 32,000 28,500 31,000 (seeds tubers number ha- 1)

Seeds tuber size (em) 2R/35 28/35 35/40 P-K fertilizers (kg ha - 1) 60-230 80-150 124-280 Soil type (1) Aba Aba Aba pH H 20 (2) 7.3 7.4 pH KCl (2) 7.2 7.3 Oxydable C (%) (3) 1.5 1.2 N content (%o) ( 4) 1.1 1.2 C/N ratio 14.7 10.0 Exchangeable P (mg 100 g- 1) (5) 19.5 26.8 Exchangeable K (mg 100 g- 1 ) (5) 28.0 32.1 Exchangeable Ca (mg 100 g- 1 ) (5) 255 322 Exchangeable Mg (mg 100 g- 1 ) (5) 13.4 12.4

(1) Soil series from the 'Carte des sols de Belgique' (Avril, 1987): first letter A (loamy soil); second letter b (good soil drainage); third letter a (Alfisol). (2) Soil pH values determined in a soil/water or KCl 1N solution ratio of 115 after 24 h contact. (3) Methode of Walkley and Black (1934). ( 4) Kjeldahl procedure. (5) Exchangeable P, K, Ca and Mg have been extracted by the procedure of Egner et al. (1960) and their concentration measured by spectrophotometry, or colorimetry for P.

N fertilization of potato and maize 643

Table 2. Cultural and arable soil layer (30 em) characteristics for the silage maize trials

Locality and year

Mons 1990 Gembloux I 1991 Gembloux II 1991

Preceding crop Winter wheat with Witloof chicory Winter barley with straw exported straw exported

Organic amendments 30 t ha ·I farmyard No amendment 30 tha-I farmyard before maize crop manure manure

30,000 L ha -I pig slurry Maize cultivar Cuzco Aviso Tamango Sowing date 05/02/90 04/25/91 04/14/91 Harvest date 09/20/90 10/05/91 10/06/91 Sowing density 110,000 116,000 116,000 (seeds number ha - 1 )

Germination rate 90% 92% 80% P-K fertilizers (kg ha -I) 80-120 100-150 None Soil type ( 1) Ada Aba Ada pHH,O (2) 6.4 7.3 pHKCl (2) 5.4 7.1 Oxydable C (%) (3) 1.1 1.1 N content (%o) (4) 1.0 1.2 C/N ratio 11.0 9.2 Exchangeable P (mg 100 g 1 ) ( 5) 14.6 28.9 Exchangeable K (mg 100 g- 1 ) (5) 19.8 32.5 Exchangeable Ca (mg 100 g -I) (5) 150 266 Exchangeable Mg (mg 100 g- 1 ) (5) 12.8 27.1

(1) Soil series from the 'Carte des sols de Belgique' (Avril, 1987): first letter A (loamy soil); second letter b (good soil drainage) or d (imperfect soil drainage); third letter a (Alfisol). For (2), (3), (4), (5) see Table 1.

ranged from 0 to 200 kg ha -I. Experimental plots were replicated six times and distributed in a complete randomized bloc design.

Assessment of quality parameters for potato tubers

For each N fertilizer rate, dry matter content (DM) and total proteinic matter content (TPM) of tubers equal to or bigger than 60 mm in size were assessed on tubers samples of 10 kg per experimental plot. DM values were determined by drying cutted tubers under IR rays, and TPM by extraction with trichloracetic acid (10%) followed by mineralization in the presence of H 2S0 4 and H 3P0 4 •

Assessment of soil mineral N content

Soil mineral N content (NH4 ions+ N0 3 ions) was assessed on 10 soil samples before crop sowing or planting, but also at harvest in ex­perimental plots kept bare as well as in cropped

plots replicated four times and randomly dis­persed. This was realized by coring soil samples on a 1.5 m soil profile, cut in 0.15 m soil layer. Details on soil sampling, soil mineral N extrac­tion and measurement procedures are described by Guiot et al. (1992).

Results

N fertilizer rate and yield

Potato crop In the trial located in Gembloux in 1991, charac­terized by low organic manure applications (Table 1), the best fresh tuber yield was reached for a N fertilizer rate of 200 kg ha -J (Fig. 1) but it is not significantly different from that with 100 kg N ha - 1 . On the other hand, in the case of frequent and important farmyard manure appli­cations as described in the two trials located near Liege in 1990 and 1991 (Table 1), no significant differences in yield were found for N fertilizer

644 Goffart and Guiot

55 .-------------=---=---- -----, N-lertilizer

~ ra<o(kgha·' 1 rates ranging from 0 to 250 kg N ha- t (Fig. 1) . In the same way, yield of potato tubers equal to or bigger than 60 mm in size still increases with the N fertilizer rate, except for the trial in Gembloux where it starts to decrease for values higher than 150 kg ha -t (Fig. 1).

2 40 ~ 35

~ 30 >.. 25

i 20 .2: tS

~ 10 ol: 5

0

.:-.. c.

~ E ~

" 15

il ;::

Utge 1990 Gembloux 1991

locality and year of trial

Li6ge 1991

• 0

li!:il 100

0 150

0200

121 250

Fig. 1. Relation between N fertilizer rate and fresh tuber yield of potato. Corresponding yields of tubers equal to a bigger than 60 mm in size are represented by the horizontal line inside each bar. Results of three trials realized in 1990 and 1991 (see description in Table 1). (LSD for p = 0.95 are as follows: Liege 1990: 3.9; Gembloux 1991: 2.0; Liege 1991: 2.1).

20 N·fertlllzer

Maize crop

rato{l<gha · 1 )

In 1990, in the situation including regular farmyard manure applications (Table 2), no differences appeared for the different N fertilizer rates (Fig. 2) but dry matter yield was also strongly reduced to values of about 10 tha - t due to the very dry weather conditions during May, July and August (Vandiepenbeek, 1990). In 1991, maximal dry matter yield was reached with a 100 kg ha- t N fertilizer rate, in the absence of organic manure applications (Gembloux I, Fig. 2) . It contrasts with the zero N fertilizer rate leading to dry matter yields similar to those observed for higher rates (Fig. 2) in the situation of Gembloux II, the latter being characterized by frequent and important farmyard manure and slurry applications (Table 2).

IS Ill 0

10 ~ 100

0 150

Ill 200

lAO!'\$ 19'90 Gembtoux I- t991 Gembloux II • 1991

Locality and year of trial

Fit?- 2. Relation between N fertilizer rate and dry matter yield of maize crop. Results of three trials realized in 1990 and 1991 (see description in Table 2) . (LSD for p = 0.95 are as follows: Mons 1990: 1.2; Gembloux I 1991: 1.4; Gembloux II 1991: 1.9) .

N fertilizer rate and quality parameters of potato tubers Results of Table 3 show that DM of tubers equal to or bigger than 60 mm in size regularly de­creases while N fertilizer rate increases, but not

Table 3. Relation between N fertilizer level and dry matter content (DM, in % ) or total proteinic matter (TPM, in % if OM) of potato tubers equal to or bigger in size than 60 mm (cultivar: Bintje). Results of three trials realized in 1990 and 1991

N ferti lizer rate (kg ha _, )

0

100

150

200

250

300

Locality and year

Liege 1990

%DM ±s

24.4 a (1) ±0.7 23.6 a ±0.6 24.0 a ± 0.4 23.7 a ± 0.2 23.2 a ± 0.4 23.1 a ±0.5

%TPM ±s

10.5 a ± 0.8 11.2 a

± 0.2 10.7 a

± 0.7 11.5a ±0.6 11.4 a

± 0.5 11.4 a

± 0.3

Liege 1991

%DM %TPM ± s ± s

23.5 a 8.6 a ± 1.1 ± 0.5 23.1 a 10.1 b ± 0.9 ± 1.0 22.4 a 11.2 b

± 0.6 ± 0.4 21.9 a 11.3 b

± 0.1 ± 0.3 21.2 a 11.2 b ± 1.1 ± 0.8

( 1) Mean values followed by same letter are not significantly different for p = 0.95 (NK test).

Gembloux 1991

%DM %TPM ± s ±s

23.2 a 5.R a ± 0.9 ± 0.3 22.4 a 7.9 b ± 1.5 ±0.1 22.3 a 8.6 c ± 0.5 ± 0.2 21.6 a 9.4d ± 0.8 ± 0.2 20.6 a 10.2e

± 0.6 ± 0.2

significantly and not in a wide range (differences between values varying only from about 1 to 3% ). TPM of tubers increases in the same time that N rate increases, but only significantly in the trial with low organic manure applications (Gem­bloux, 1991).

N fertilizer rate and soil mineral N content at harvest

Potato crop From the measures realized in the 0 to 60 em soil layer in Gembloux (situation with low level of organic manure applications, Table 1), it was shown that soil mineral N contents at harvest time range from 12 to 18 kg ha - 1 while N fertilizer rates increase from 0 to 300 kg ha -I (Table 4). Such values are extremely low com­pared to those ranging from around 40 to 120 kg ha- 1 for N fertilizer rates ranging from 0 to 250 kg ha -I (Table 4) and observed in the two other trials characterized by important and regu­lar organic amendments applications (Table 1). The differences for mineral N soil contents between harvest and planting times (Table 4) also indicate that the mineralization process during the growth period in the upper soil layer,

N fertilization of potato and maize 645

has led to values of 154 and 187 kg ha -I in the trials of Liege in 1990 and 1991, where important organic manures were regularly applied. Such values are 1.5 to 2 times higher than the value of 109 kg ha -I in the trial of Gembloux in 1991.

In the 60 to 150 em soil layer and for the three trials, the soil mineral N contents for all the N fertilizer rates are similar to the contents as­sessed at planting time (Table 4), suggesting a limited N uptake by the roots system of the potato plants in such soil depths. It must also be noted that, at planting time, the mineral N content in the 60 to 150 em soil layer was the lowest in the trial of Liege in 1991 where ryegrass had been cropped as green manure during the autumn preceding the potato crop. In this case, the value in the 0 to 60 em soil layer is six times higher than that observed in the 60 to 150 em soil layer, while in the other trial of Gembloux in 1991 both values are quite similar.

Maize crop With regard to the soil mineral N contents in the 0 to 60 em soil layer, the values for the trial at Gembloux I in 1991 (with no organic amend­ments applied, Table 2), ranging from 4 to 32 kg ha - 1 with N fertilizer rates ranging from 0 to

Table 4. Mineral N contents in the soil profile at harvest of potato crop for different N fertilizer rates and evolution of soil mineral N content during the growing period. Results of three trials realized in 1990 and 1991 (see description in Table 1)

N fertilizer rate (kg ha - 1 )

0 100 150 200 250 300

( 1 )b (2) (2) -(1)

Locality and year

Liege 1990 Liege 1991

0-60' 60-150 0-150 0-60

Soil mineral N content (kg ha -I) at harvest

44' 45 89 40 75 49 124 62

68 91 43 134 68

123 127 42 169

Evolution of soil mineral N content (kg ha -I) 63 46 109 88

217 61 278 275 +154 + 15 +169 +187

60-150

19 21 27 22 25

15 24 +9

0-150

59 83 95 90

148

103 299

+196

" Soil mineral N content is affected by variation coefficients ranging from 10 to 20%. b ( 1) Soil mineral N content at sowing time.

(2) Soil mineral N content at harvest time (soil kept bare and without N fertilizer). (2) -( 1) Evolution of soil mineral N content in bare soil.

'Soil layer (em).

Gembloux 1991

0-60

12

16 18 18

46 155

+109

60-150

41

46 51 52

49 45 -4

0-150

53

62 69 70

95 200

+105

646 Goffart and Guiot

Table 5. Mineral N content in the soil profile at harvest of silage maize crop for different N fertilizer rates and evolution of mineral N soil content during the growing period. Results of three trials realized in 1990 and 1991 (see description in Table 2)

N fertilizer Locality and year level (kg ha - 1 )

Mons 1990 Gembloux I-1991 Gembloux II-1991

0-60' 60-150 0-150 0-60 60-150 0-150 0-60 60-150 0-150

Soil mineral N content (kg ha 1 ) at harvest 0 9' 24 33 4 11 15 27 77 104

100 52 18 70 11 23 34 51 70 121 150 10 22 32 99 70 169 200 109 18 127 32 31 63 129 74 203

Evolution of soil mineral N content (kg ha - 1 )

(1 )" 55 28 83 39 31 70 78 103 181 (2) 168 39 207 100 28 128 205 80 285 (2) -(1) + 113 +11 +124 +61 -3 +58 +127 -23 +104

' Soil mineral N content is affected by variation coefficients ranging from 10 to 20%. "(1) Soil mineral N content at sowing time.

(2) Soil mineral N content at harvest time (soil kept bare and without N fertilizer). (2) -(1) Evolution of soil mineral N content in bare soil.

'Soil layer (em).

200 kg ha -I (Table 5), appear to be extremely low compared to those observed in the trials located at Mons in 1990 (soil mineral N contents ranging from 0 to 109 kg ha-l) and at Gembloux II in 1991 (values ranging from 27 to 129 kg ha- 1), both characterized by regular and im­portant organic manure applications (Table 2). In that soil layer, the increase of mineral N soil content during the growing season was also two times higher in the last two trials (113 and 127 kg ha -I, respectively) than in the trial of Gembloux I (61 kg ha- 1 ) (Table 5).

In the 60 to 150 em soil layer, the mineral N soil contents in the trial at Mons in 1990 were low at harvest time and similar to those assessed at sowing time. This situation was probably due to dry climatic conditions before but also during the growing period (Vandiepenbeek, 1990) re­ducing the nitrate leaching. The values observed for Gembloux I in 1991 were also rather low, probably due in this case to relatively low amounts of mineralized nitrogen and also to a good N uptake by the root system in that soil layer, at least for the lowest N fertilizer rates (Table 5).

On the other hand, three to four times higher soil mineral N contents (around 70 kg ha -I) were observed at harvest time in the 60 to 150 em soil layer in the trial of Gembloux II in 1991 (Table

5). It has to be related to the important amounts of soil mineral N content ( 103 kg ha - 1) already present in that soil layer at sowing time, from which at least 23 kg ha - 1 was estimated, during the growing period, to be leached under 1.5 m in the bare soil (Table 5). Total rainfalls higher than 200 mm were indeed recorded during June and July 1991 (Vandiepenbeek, 1991). From the similar values of soil mineral N contents ob­served in cropped plots for all the N fertilizer levels (from 70 to 77 kg ha-l) compared to the values in bare soil at harvest time (80 kg ha - 1), it could be deduced that the N uptake by the roots was highly reduced in the 60 to 150 em soil layer, leaving high amounts of soil mineral N at high risk of later leaching.

Discussion

For potato and silage maize crops, the experi­ments and results presented describe the ag­ronomical and environmental consequences that arose in Belgian loamy soils from excessive applications of animal wastes and subsequent soil mineral N release.

Depending on the intensity and frequency of organic manure applications, mineral N supply by manures and N release during the autumn-

winter period preceding potato and silage maize crops have led at sowing or planting time to contrasted amounts of nitrate N in the soil profile. Especially in the case of important or­ganic manure applications, the presence of min­eral N residues in the 60 to 150 em soil layer at sowing or planting time indicates that good management of the intercropping period before potato and maize crop is required to avoid the leaching of nitrate nitrogen in soil layers that have a really poor chance to be reached by the root system of cropped species such as maize and potato. This is particularly important for the potato crop of which the root system's maximal extension is restricted to 60 em if considering active N uptake. This is in agreement with the shallow rooting of potato described by Lesczynski and Tanner (1976) and its uniform roots density generally limited to the depth of 40 to 60 em after which rooting is less intensive (Stalham, 1989). The maize root system seems to be able to reach deeper soil layers but essentially only in the presence of low mineral N amounts in the upper soil layers, confirming the findings of Blanchar and Caldwell (1966) on the influence of high amounts of mineral nitrogen on the limita­tion of the root system extension of maize plant. The presence of a ryegrass cropped as green manure before the winter period seems to have considerably reduced the mineral N content at planting time in the 60 to 150 em soil layer, and then appeared as a solution to avoid nitrate leaching during the intercropping period. This hypothesis was already studied by other scientists (Christian et a!., 1992).

Soil mineral N supply and its distribution in the soil profile at sowing or planting time will normally contribute to the recommended N fertilizer rate to be applied according to the crop N requirement as stated for other arable crops such as winter wheat (Guiot et a!., 1983; Vai­dyanathan et a!., 1991), with respect to low soil nitrate N residues at harvest. In cropping sys­tems including low organic manure applications, the results indicate that for potato as well as for maize crops, N fertilizer rates leading to maximal yield also lead to low soil mineral N contents at harvest, at least in the 0 to 60 em soil layer. Such soil mineral N contents are in agreement with the low values observed, but only on a 1.5 m soil

N fertilization of potato and maize 647

profile, in the loamy soils area in Belgium after a winter wheat crop preceded by a sugar beet crop with leaves exported (Goffart et a!., 1992). For the potato trial realized at Gembloux in 1991, the significantly increasing values of TPM in tubers, related to the increasing N fertilizer rates, also suggest the good N uptake by the crop. Lorenz and Steffens (1992) have also shown that careful slurry and N fertilizer applica­tions to potato and maize crops, leading to optimum N levels, also lead to soil mineral N content at harvest in the 0 to 90 em soil layer no higher than in unfertilized treatment. However, Wadman et a!. (1989) have observed increasing soil mineral nitrogen residue after potato harvest even at optimal N supply by slurry and N fertilizer.

In contrast, in cropping systems including large and frequent applications of organic ma­nures, such as farmyard manure and slurry during the year preceding the sowing of these two crops, the yield response to the nitrogen of the fertilizer is hampered. It can be restricted to N fertilizer levels lower than 100 kg ha -J or even absent in the case of important amounts of farmyard manure and pig slurry, leading at harvest to very high mineral N residues in the 0 to 60 em soil layer, and even in the 60 to 150 em layer, probably related to early applications of manures. Recommended N fertilizer rates must then absolutely be lowered in such cropping systems, as stated in other studies (MAFF, 1985; Neeteson and Zwetsloot, 1989).

In the determination of the optimal N fertilizer level, qualitative parameters must also be taken into account, especially for the potato tubers. For processing into French fries or crisps, high DM contents (20 to 25%) are required, but nitrogen decreases DM content (Perrenoud, 1983). In this respect, N fertilizer levels leading to maximal yield and to low soil mineral N contents in the soil at harvest could also lead to the required quality values. But with respect to industrial use of tubers as fries, there is also the need for growers to reach a high ratio of large tuber sizes with low DM content (essentially to reduce the susceptibility of tubers to superficial blemishes). As shown by the results, increasing N fertilizer levels in terms of maximal yield leads to higher rates of large tuber size with lower DM

648 Goffart and Guiot

contents. According to our results and with respect to low soil mineral N contents at harvest, such excessive rates are only allowed in cropping systems that include low organic manure applica­tions. However, increasing N fertilizer rate is not the only way to increase tuber size. The presence of a N /K ratio of 1/2 in the soil is also a major factor to take into account with respect to tuber size (Gravoueille, 1987; Schippers, 1968) and high K fertilizer rates also reduce the suscep­tibility of tubers to superficial blemishes (Van der Zaag and Meijers, 1970) and also to internal blackening (Perrenoud, 1983). In this respect, K supply by farmyard manure and slurry should also be taken into account. The effect of K on tuber size can in particular explain the decreas­ing yields of large tubers from the N fertilizer rate of 150 kg ha _, in the trial at Gembloux in 1991, due to the limited K fertilizer rate (150 kg ha- 1 ) (Table 1).

It can be concluded that where organic ma­nures are applied, adequate allowance for their nitrogen value should be taken into account by farmers, especially that from slurries applied under optimum conditions (Chambers and Smith, 1992; Wadman and Neeteson, 1992). However, according to our field experience, in spite of the agronomical and environmental problems described, Belgian farmers are making little or no allowance for the nitrogen supplied from organic manures especially for the nitrogen fertilization of potato and silage maize, as far­mers in other countries do (Chambers et al., 1991 ). Soil (Neeteson and Zwetsloot, 1989) and plant (Van Loon et al., 1987) analysis and modelling (Neeteson et al., 1987) have useful roles to play in this respect.

Acknowledgements

We are grateful to the following people or services for their help in the accomplishment of this study. Service des Ingenieurs agronomes de l'Etat, Ministere de I' Agriculture, Belgium, for financial support; Station de Chimie et de Physique Agricoles (Dir.: Dr E Fran<;ois), CRA Gembloux, Belgium, for soil chemical analysis; Station de Haute Belgique (Dir: R Biston), CRA Gembloux, Belgium, for chemical analysis

on potato tubers; Centre Independant de Promo­tion Fourragere (CIPF), UCL, Louvain-la­Neuve, Belgium, for technical assistance in maize trials.

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MAFF 1985 Fertilizer Recommendations 1985-1986. Minis-

try of Agriculture Fisheries and Food, Reference Book 209, HMSO, London, 25 p.

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Neeteson J J, Greenwood D J and Draycott A 1987 A dynamic model to predict yield and optimum nitrogen fertilizer application rate for potatoes. Proc. Fertil. Soc. 262, 31 p.

Pcrrenoud S 1983 Potato: fertilizers for yield and quality. International Potash Institute Bulletin, 8, 84 p.

Powlson D S, Poulton P R, Addiscott T M and McCann D S 1989 Leaching of nitrate from soils receiving organic and inorganic fertilizers continuously for 135 years. In Nitrogen in organic Wastes applied to Soils. Eds. A Hansen and K Hendriksen. pp 334-345. Academic Press, London.

Prins W H, Dilz K and Neeteson J J 1988 Current re­commendations for nitrogen fertilization within the E.E.C. in relation to nitrate leaching. Proc. Fertil. Soc. 276, 27 p.

Schippers P A 1968 The influence of rates of nitrogen and potassium application on the yield and specific gravity of four potato varieties. Eur. Potato J. 12, 251-263.

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Statham M A 1989 Growth and water use of potato variety. Record on contrasting sites. Ph.D. Thesis, University of Cambridge.

Vaidyanathan LV, Shepherd M A and Chambers B J 1991 Mineral nitrogen arising from soil organic matter and

N fertilization of potato and maize 649

organic manures related to winter wheat production. In Advances in Soil Organic Matter Research- The Impact on Agriculture and the Environment. Ed. W Wilson, Royal Society of Chemistry, Special Publication 90, 315-327.

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M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 651-656, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-068

Controlled supply of fertilizers for increasing use efficiency and reducing environmental damage

A. SHAVIV Faculty of Agricultural Engineering, Technion, Israel Institute of Technology, Haifa, Israel

Key words: controlled release, environmental damage, nutrient use efficiency

Abstract

An approach aiming at increasing nutrient use efficiency and reducing environmental damage by means of sophisticated fertilizers or fertilization practices is presented. The following main factors affecting use efficiency and pollution are considered: -economic aspects of nutrient losses; -stress conditions imposed on plants at different growth stages; - stress imposed by deficiencies originating from poor fertilization management; -environmental factors affecting plant nutrients interactions; -accumulation of pollutants in the environment or in plants; and -soil degradation induced by improper fertilization. Measures that offer a control over the factors listed above are addressed: -synchronization of nutrients supply with their demand (controlled supply); -provision of beneficial nutrient compositions based on synergistic effects in the rhizosphere or in plant tissues. Matching nutrient supply with plant demand and provision of predetermined 'beneficial' compositions by means of controlled or slow release fertilizers (CRF's or SRF's) or by applying ammonium rich fertilizers amended with nitrification inhibitors (NI's) is addressed. Basic questions related to development of such fertilizers and the need to assess criteria and guidelines for that purpose are emphasized.

Introduction

Total world consumption of fertilizer N, P, and K 1990/91 was 78, 16, and 22 million tons per annum, respectively (FGW, 1991). The demand for fertilizers in the future is expected to expand with world population growth and the need for food (FGW, 1991; Jenkinson, 1990). The pro­jected increase in fertilizers use will occur mainly in developing countries where food production needs to be urgent) y increased (Bockman et al., 1990). The 'developed market economies' use relatively large amounts of N and other fertiliz­ers per unit area (Stangel, 1984 ). Among those economies, Western Europe countries seem to take the lead (FAO, 1982).

Nitrogen recovery may often be in the range of 30 to 80% of the applied amount, depending

on factors such as: crop, soil, climate, manage­ment of N and water supplies (Bock and Hergert, 1991; Dilz, 1988; Keeny and Follett, 1991). The unrecovered N is mainly due to leaching and gaseous losses (Bock and Hergert, 1991; Hauck, 1984). Bock and Hergert (1991) showed that N removed from fields in harvested corn grain in the US tripled during the last 40 years. However, the rate of N fertilizer applica­tion to corn increased more than 15 times during that period, indicating an alarming situation from an environmental and resource conserva­tion points of view. Such observations lead to the inevitable conclusion that continuous efforts and measures have to be taken for reducing possible losses of nutrients to the environment, particu­larly in those regions where large amounts of N are applied.

652 Shaviv

According to Aldrich (1984) the measures to be taken for increasing N use efficiency and reducing pollution should include: crop factors; general management practices; and optimum N application practices (application rates and tlmmg, N forms, manuring). The present paper focuses on the contributions of advanced application methods of fertilizers and slow or controlled release fertilizers (SRF or CRF) to reducing environmental damage and increas­ing nutrients use efficiency. Nevertheless, one should also consider possible interactions with crop factors and other management practices mentioned by Aldrich (1984). The basic con­cept guiding Aldrich's approach is that 'the best way to minimize, to the extent feasible, the nitrates in surface and ground­water is to focus on practices that favor high crop yields'. Nowadays, focus should be shifted toward practices which favor 'optimal' yields where its definition is modified in such a manner that it accounts for adverse effects on the environment in addition to economic considerations (Pollet and Walker, 1989; Wilson, 1988).

It is well recognized that crop yields obtained in solution cultures are significantly greater than those obtained in natural soils. This is attributed to several factors such as: improved supply of water, nutrients and oxygen and the possibility to control the pH, temperature and ionic strength of the solution. The introduction of irrigation systems by which water and fertilizer supplies could be matched to plant demand (e.g. trickle/ drip irrigation) significantly increas­ed yield potentials (Randall et a!., 1985). At the same time the control of environmental pollution was improved as well. In fact most of the agricultural lands rely on less controll­able water supply, the least controllable being rainfed agriculture. In such cases more efficient supply of nutrients may be achieved by either split applications or by the use of more sophisticated fertilizers (e.g. CRF, SRF).

In order to assess the possibilities of increasing fertilizer use efficiency and those of reducing environmental damage (by fertilization) one has to consider the factors affecting both. A brief description of such factors is presented below and followed by suggested measures to be taken

for reducing environmental damage and for increasing fertilizer use efficiency.

Factors affecting fertilizer use efficiency

Nutrient losses

These may occur via physical processes such as leaching (N0 3 and other mobile nutrients), volatilization (NH3 , N2 0 etc.), and surface runoff; via biological transformations (fixation, demineralization, de-nitrification); or via chemi­cal reactions such as precipitation, decomposi­tion or fixation. The potential for losses due to fixation lies in the fact that the fixed nutrient may be released/mineralized in an uncontrolled manner and when it is not needed. The rate at which nutrients are lost by such processes in soil is considered proportional to their excess supply over plants demand due to the concentration dependence (Shaviv and Mikkelsen, 1993).

Deficiency stress

Such effects may occur due to unbalanced supply of nutrients (competition between ions) or due to deficiency of micro-nutrients. Deficiencies may be observed even with ample supply of nutrients due to poor management of their application (e.g., bad timing).

Functional stress or toxicity

Osmotic effects due to temporal surplus supply of nutrients may cause injury at germination or development stages of young plants. Excess supply of certain ions (N03 , NH4 ) can induce specific toxicity.

Interactions of nutrients with environmental factors

Nutrients use efficiency can be greatly affected through their interactions with soil salinity (Kafkafi, 1987) or with water stress (shortage or excess).

Factors affecting environmental pollution and damage

Accumulation of pollutants

Due to mismanagement of fertilizer supply, pollutants or toxic materials like nitrates, ni­trites, nitrozoamines (Bockman et a!., 1990; Nelson, 1984) can be found in plant tissues. They accumulate in water sources or soil (Smith eta!., 1990); or accumulate as toxic gases, ammonia or nitrous oxides. The latter may directly affect various ecosystems and indirectly increase the exposure of humans and animals to excessive ultraviolet radiation (Newbould, 1989).

Soil degradation and damage

Very high local salt concentrations and drastic pH changes may be induced by improper fertili­zation (Hauck, 1984). These, in turn, may result in: displacement of exchangeable cations (e.g. Ca, Mg); deterioration of soil hydraulic prop­erties; and accelerated dissolution of soil miner­als. In addition, excess N or P can induce crust formation due to accelerated algal growth (Givol, 1991). Such a situation is enhanced under temporal excess of water supply to soil surface (e.g. after irrigating soils with low infil­tration rate). Soil compaction may also occur if the land is exposed to repeated fertilizer applica­tions by heavy machinery.

Suggested measures

The following measures are suggested as a pos­sible solution to some of the fertility and en­vironmental problems associated with the above listed factors: synchronizing fertilizer supply with its demand by plants and simultaneous provision of beneficial compositions of nutrients.

Synchronizing N and other nutrients supply with plants demand

The general pattern of N, K and P demand during the growth of field crops is sigmoidal (Brown et a!., 1984). Any fertilizer or applica­tion method may offer means for optimizing yields and reducing pollution if it is synchronized

Controlled supply of fertilizers 653

with such a pattern (Wilson, 1988). In some cases the optimal response may be obtained by a release pattern matching some luxury consump­tion (Hauck, 1985). Realization of synchronized supply in rainfed agriculture or even in surface irrigated fields, by common fertilization prac­tices, would be impractical in most cases.

A controlled supply of N could, assumably, be achieved by using CRF's or SRF's. However, that may prove to be uneconomical. It is also doubted whether most of the SRF's have release patterns compatible with plant demand. Alter­natively, inhibition of nitrification may serve as a slow release system. This may be particularly efficient with localized ammonium sources in combination with nitrification inhibitors (NI) like band application, nests, large or super granules (Amberger, 1989; Hauck, 1984, 1985). Two major advantages are expected from such an approach: 1. -a microsite is formed, reducing nitrification

by the presence of both relatively high con­centrations of ammonium and NI;

2. -nitrate formation (with time!) has a sigmoi­dal shape (Darrah eta!., 1987; Shaviv, 1988). In other words, the release of the labile N form (No;) from the microsites into the active root zone is expected to be sigmoidal too.

Optimal conditions for plant development

Synchronization of N application with its de­mand should improve the physical conditions for plants growth. The two major reasons for that are: a. improved germination due to the rela­tively low levels of fertilizer supplied at this stage; and b. improved growth conditions of plants. Those are sensitive to salinity (e.g. if an excess of fertilizer is supplied) or are sensitive to excess of specific ions such as NO; or NH;. These effects however have not been studied extensively enough, regarding the potential ben­efits expected from the use of SRF's or am­monium in combination with NI's.

Optimal composition of nutrients

The supply of beneficial nutrient compositiOns according to plants preference offers a great potential for increasing the use efficiency of

654 Shaviv

nutrients and hence it reduces pollution hazards. A mixed ammonium-nitrate nutrition increases dry matter and protein yields of cereals com­pared to the sources alone (Bock, 1986). A positive interaction between K supply and mixed ammonium-nitrate supply has been shown as well (Hagin et al., 1990; Lips et al., 1987). Table 1 demonstrates both the effect of obtaining higher protein yields with mixed ammonium/ nitrate nutrition and its enhancement by addition of adequate amounts of K (Shaviv and Hagin, 1989).

Furthermore, the presence of ammonium in soil solution is known to increase P availability in neutral and basic soils, which is mainly caused by rhizosphere acidification (Marschner, 1986; Nye, 1986). A similar effect is reported in regard with increasing Fe availability by ammonium or potas­sium induced rhizosphere acidification (Barak and Chen, 1984; Shaviv and Hagin, 1988b). Table 2 summaries experimental results with different soils in which mixed ammonium/nitrate nutrition induced higher accumulation of P as compared to nitrate alone (Shaviv et al., 1987; Shaviv and Hagin, 1988a; Shaviv and Hagin, 1989). A pre-requisite for achieving the above

Table 1. Effect of applied NH4 /NH 3 ratio (percent) on accumulation of reduced-N in wheat grain and stover and its interaction with K. Pot experiment with a sandy silt (2 g N pot- 1 )

Kappl. N-NO,/N-NH 4 ratio (g pot)

100/0 75/25 50150

0.63" 0.91 1.02 0.50 0.71 1.01 1.13 1.00 0.75 1.04 1.28 LSD (0.05) 0.07

"g N/pot

mentioned effects is to maintain the desired proportions of ammonium-nitrate and other nu­trients in soil and preferably in the same micro­site. Controlled release fertilizers or frequent fertigation (e.g. by drip irrigation) is mentioned by Hauck (1984) as possible means for this purpose. Ammonium sources with NI's is men­tioned as well, but are considered somewhat less precise. Yet, from a practical point of view it may prove sufficient enough for some crops.

Controlled and slow release fertilizers

The use of CRF's or SRF's is restricted to ash­crops, nurseries, turfs etc. (Hauck, 1985; Shaviv and Mikkelsen, 1993). The main reason is their high cost. However, it is well recognized that CR or SR fertilizers offer a good control over N, P and K timing of supply, maintenance of desired forms of nutrients, and a reliable management of losses and pollution (Aldrich, 1984; Hauck, 1984; Shaviv and Mikkelsen 1993). Little atten­tion, however, has been given to the mode of action of CRF's under various conditions and to release patterns and their relevance to crops (Shaviv and Mikkelsen, 1993). The possibility of reducing environmental stress by using such materials is often mentioned, yet, efforts have to be done to quantify such effects in practice.

A recent literature survey of CR materials (Shavitt, 1990) covering agriculture, phar­maceutics and food, has shown that the state of published knowledge regarding CRF's is far behind that of existing in pharamaceutics or food. It is believed that the adoption of recent developments in the manufacturing of CRF's is likely to bring a breakthrough in their use in horticulture and even extensive agriculture.

Table 2. Effect of applied ratio (percent) of N-NH 4 /N-N0 3 on P accumulation in wheat and millet (mg P/pot) as obtained with four different soils in pot experiments. Level of P in all cases was 1.0 g/pot

N-NH,/N-NO, Loamy sand Sandy loam Clay Sandy silt applied Wheat Wheat Wheat

Wheat Millet

0/100 56c 38b 94c 118c 80c 2S/7S 79ab 48a 118b 151a 95ab SO/SO 89a 53 a 146a 139ah 106a

Different letters denote differences at significance level of O.OS.

Use of ammonium sources with nitrification inhibitors

The possibility to apply ammonium sources with NI's is well known and well documented (Am­berger, 1989; Hauck, 1985). The possibility of reducing nitrification rate by formation of micro­sites with high ammonium concentrations is also known for many years. Efforts were made re­cently to model the effect of high ammonium concentration on its rate of oxidation in order to make it predictable under various conditions (Darrah et al., 1987; Ned an, 1990; Shaviv, 1988). However, the amplification of the effect of high (local) ammonium concentration on nitrification rate by amending this source with a NI is still far from realization in practice. Re­cently obtained results are nevertheless promis­ing (Nedan, 1990; Shaviv and Nedan, 1992). They demonstrate that the amount of NI needed for reducing the rate of nitrification can be significantly reduced when an ammonium fertil­izer is granulated with a NI. Table 3 shows increases in the accumulation of reduced-N (pro­tein) in wheat grain as a result of amending an N-P fertilizer by co-granulation with small amounts of a NI (DCD or N-serve). It is noteworthy that 1% DCD in the fertilizer was sufficient for preventing leaching of mineral N from the pots. Hauck (1984) already mentioned the possibility to reduce amounts of applied NI by band or other localized applications, just because of the higher NI concentrations formed on site. Little, if any, effort was done to model synergism between high ammonium concentra-

Table 3. Accumulation of reduced N (g N/pot) in wheat grain and leaching of mineral N from pots as affected by: a. addition of NI (DCD, or NS = N-serve) to a N-P fertilizer (10-10-0) consisting of (NH 4 ) 2S04 and superphosphate; and b. band application vs. bulk mixing

N Bulk mixed Band appl.

Grain-N Leach. Grain-N Leach.

N-P 0.50e 0.19a 0.67cd 0.19a N-P +5%NS 0.73b O.Old 0.77ab 0.03cd N-P +l%NS 0.64d 0.15ab 0.74b 0.07c N-P +5%DCD 0.64d O.Oid 0.76ab O.Old N-P+l%DCD 0.7lbc O.Old 0.79a O.Old

Different letters denote differences at significance level of 0.05. Applied 0.75 g N/pot

Controlled supply of fertilizers 655

tion and amendment with a NI. By doing so one may provide a tool for predicting N transforma­tions under various conditions in the field, thus enabling better management of N supply and also improved control of ammonium-nitrate ratios.

Conclusion

The potential exists to reduce environmental damage and maintain or increase nutrient use efficiency at the same time by using CRF's or bio-amended fertilizers. Yet, there is still a long way before such sophisticated agro-techniques are implemented in extensive agriculture. Sys­tematic and intensive research is needed aiming at the realization of benefits from advanced fertilization methods. Introduction of environ­mentally sound practices of nutrient supply and a better understanding of the factors which affect them should lead to: - increases of dry matter and protein yields due

to maintenance of optimal NH4 /N0 3 /K pro­portions in soil;

- beneficial conditions for germination and plant growth;

- increased bio-availability of P due to synergis­tic effects between P and K or N forms due to their co-placement in soil;

-increased micro-nutrients availability due to synergistic interactions in the rhizosphere;

- reduction of machinery and labor needs for split applications;

-better control over damage to soil (compac­tion, erosion, weathering);

- reduced environmental pollution induced by accumulation of toxic materials in ground water, plant tissues or in the atmosphere;

- improved conservation of natural resources due to reduced losses of nutrients.

References

Aldrich S R 1984 Nitrogen management to minimize adverse effects on the environment. In Nitrogen in Crop Pro­duction. Ed. R D Hauck. pp 663-673. ASA Publication, Madison, WI.

Amberger A 1989 Research on dicyandiamide as nitrification

656 Controlled supply of fertilizers

inhibitor and future outlook. Comm. Soil Sci. Plant Anal. 20, 1933-1956.

Barak P and Chen Y 1984 The effect of potassium on iron chlorosis in calcareous soils. J. Plant Nutr. 7, 125-133.

Bock B R 1984 Efficient usc of nitrogen in cropping systems. In Nitrogen in Crop Production. Ed. R D Hauck. pp 273-294. ASA Publication, Madison, WI.

Bock B R and Hergert G W 1991 Fertilizer nitrogen management. In Managing Nitrogen for Ground Water Quality and Farm Profitability. Eds. R F Follet et al., pp 139-164. SSSA, Madison, WI.

Bockman 0 C, Kaarstad 0, Lie 0 H and Richards I 1990 Agriculture and Fertilizers. Agricultural Group, Norsk Hydro, Oslo. 245 p.

Brown D A and Scott S H 1984 In Roots, Nutrient and Water Influx, and Plant Growth. ASA Special Pub. No. 49. pp 101-136. Madison, WI.

Darrah P R, Nye PH and WhiteR E 1987 The effect of high solution concentration on nitrification rates in soil. Plant and Soil 97, 37-45.

Dilz K 1988 Efficiency of uptake and utilization of fertilizer nitrogen by plant. In Nitrogen Efficiency in Agricultural Soils. Eds. D S Jenkinson and K A Smith. pp 1-26. Elsevier Applied Science, London.

Follet R F and Walker D J 1989 Ground water concerns about nitrogen. In Nitrogen Management and Ground­water Protection. Ed. R F Follet. pp 1-22. Elsevier, Amsterdam.

Food and Agricultural Organization 1982 Current fertilizer situation and outlook. Commission on Fertilizers, Seventh Session. Agriculture Organization of the United Nations, Rome, Italy.

FWG- Fertilizer Working Group 1991 Nitrogen, Phosphate, and Potash Forecasts. Technical Paper no. 144. World Bank, Washington DC.

Givol M 1991 Controlled release fertilizers interaction with plants and soils. M.Sc. Faculty of Agric. Eng., Technion, Israel.

Hagin J, Olsen S R and Shaviv A 1990 Review of interaction of ammonium-nitrate and potassium nutrition of crops. J. Plant Nutr. 13, 1211-1226.

Hauck R D 1984 Technological approaches to improving the efficiency of nitrogen fertilizer use by crop plants. In Nitrogen in Crop Production. Ed. R D Hauck. pp 551-560. ASA Publication, Madison, WI.

Hauck R D 1985 Slow-release and bioinhibitor-amended nitrogen fertilizers. In Fertilizer Technology and Use. Ed. 0 P Engelstad. pp 293-322. SSSA Publication. Madison, WI.

Jenkinson D S 1990 An introduction to the global nitrogen cycle. Soil Use Manage. 6, 56-61.

Kafkafi U 1987 Plant nutrition under saline conditions. Fertil Agric 5.

Keeney D R and Follett R F 1991 Managing nitrogen for groundwater quality and farm profitability: Overview and introduction. In Managing Nitrogen for Groundwater Quality and Farm Profitability. Eds. R F Follett, D R Keeney and R M Cruse. pp 1-7. SSSA, Madison, WI.

Lips H S, Soares M I M, Kaiser J J and Lewis 0 A 1987 K+ modulation of nitrogen uptake and assimilation in plants. In Inorganic Nitrogen Metabolism. Eds. Ullrich, Aparicio and Palacios pp 233-239. Springer Verlag.

Marschner H 1986 Mineral Nutrition of higher Plants. Academic Press, London. 674 p.

Nedan S 1990 Control of nitrification rate in soil by am­monium and inhibitors. M.Sc. Thesis. Faculty of Agric. Eng., Technion, Israel.

Nelson D W 1984 Effect of nitrogen excess on quality of food and fiber. In Nitrogen in Crop Production. Ed R D Hauck. pp 643-661. ASA, Madison, WI.

Newbould P 1989 The use of fertiliser in agriculture. Where do we go practically and ecologically? Plant and Soil 115, 297-311.

Nye P H 1986 Acid-base changes in the rhizosphere. Adv. Plant Nutr. 2, 129-153.

Randall G W, Wells K L and Hanaway J J 1985 Modern techniques in fertilizer application. In Fertilizer Technolo­gy and Use. Ed. 0 P Engelstad. pp 521-560. SSSA Publication. Madison, WI.

Shavit U 1990 A model of solute efflux from controlled release devices. M.Sc. Thesis. Fac. of Agric. Eng., Techn­ion, Israel.

Shaviv A 1988 Control of nitrification rate by increasing ammonium concentration. Fert. Res. 17, 177-188.

Shaviv A, Hagin J and Neumann P 1987 Effects of nitrifica­tion inhibitor on efficiency of nitrogen utilization by wheat and millet. Commun. Soil Sci. Plant Anal. 18, 815-833.

Shaviv A and Hagin J 1988a Interaction of ammonium and nitrate nutrition with potassium in wheat. Fert. Res. 17, 137-146.

Shaviv A and Hagin J 1988b Correction of lime-induced chlorosis by application of iron and potassium sulphate. Fert. Res. 13, 161-167.

Shaviv A and Hagin J 1989 Ammonium, nitrate, nitrification inhibitor and potassium solutions for wheat. J. Fert. Issues 6, 44-49.

Shaviv A and Nedan S 1992 Mixed N-P granulated fertilizer amended with nitrification inhibitors: Less pollution and higher efficiency. 7th Nitrogen Workshop, Edinburgh.

Shaviv A, Nedan S and Hagin J 1992 Regulation of NH 4 -

N 0 3 proportions in soil for increasing N fertilizers use efficiency and reducing pollution. CIEC Symposium 1990, Cyprus.

Shaviv A and Mikkelsen R L 1993 Slow release fertilizers for a safer environment maintaining high agronomic use efficiency. Review article In Controlled Release Fertilizers. Fert. Res. (In Press).

Smith S J, Schepers J S and Porter L K 1990 Assessing and managing nitrogen losses to the environment. Adv. Soil Sci. 14, 1-45.

Stangel P J 1984 World nitrogen situation-trends, outlook, and requirements. In Nitrogen in Crop Production. Ed. R D Hauck. pp 23-54. ASA Publication, MI.

Wilson F N 1988 Slow release- True or False? A case for control. Proceedings of Fertiliser Society, UK. No. 268, 34 p.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 657-663, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-123

Local resource management in computer aided farming: A new approach for sustainable agriculture

E. SCHNUG 1 , D.P. MURPHY\ S.H. HANEKLAUS 1 and E.J. EVANS 2

1/nstitute for Soil Science and Plant Nutrition (F AL), Bundesalle 50, D-3300 Braunschweig, Germany; 2Department of Agriculture, University of Newcastle upon Tyne, Newcastle, NEl 7RU, UK

Key words: equifertile, farming by soil, geostatistics, GIS, GPS, LORIS, machinery, pedocell, soil variation, substainable agriculture

Abstract

Investigations of spatial vanatlon in soil characteristics and crop yield are reported. The results presented indicate that where average soil pH was 6.1 on a 32 ha site in Northumberland, England, applications of CaO in excess of 0.5 t ha-l were required on only 20% of the area giving a 60% reduction in typical lime applications. The use of the Global Positioning System in implementing spatially variable applications is advocated, and a concept for the development of machinery systems that apply crop inputs on a site specific basis is reported. A computer based Local Resource Information System (LORIS) for conventional spreadsheet programmes is proposed. This enables the handling, interpolation and interpretation of spatially distributed data.

Introduction

To date, farmers have managed their land on a field by field basis with fertilisers and pesticides applied according to what is assessed as the typical requirements. The enlargement of fields has increased the within field variation in soil type, fertility and pest incidence giving rise to inappropriate fertiliser and pesticide applications over large areas. Within field variability in soil resources has been the subject of investigations over the last ten years (e.g. Robert, 1989), and the development of technology to manage this variability is expected to make a major impact on crop production (Aschcman, 1993). Any attempt to address the management of this variation in research or in applied agriculture presents two fundamental problems: the identifi­cation of position and the processing of large data sets. This paper presents a concept for dealing with spatial variation in agronomic re­sources and reports on an examination of spatial

variation in soil pH and crop yield. The objective is to develop a farm resource information system that can be used to optimise the use of ag­ronomic inputs.

Integrated machinery management - a concept

A cyclical machinery management programme centered on the processing of spatially variable data in an ecological data base describing ag­ronomic resources is envisaged. Yield data col­lected at harvest are combined with data quan­tifying spatial variation in soil resources, inci­dences of weeds, diseases and pests. The pro­cessing of these data together results in digital maps of the application of crop inputs. The efficacy of the resultant variable programmes may be monitored further by continued mapping of crop yield.

The processing of the data must first identify two strategies for crop management. Where it is

658 Schnug et al.

determined that crop yield is limited by un­controlled factors such as water availability, inputs are adapted to local productivity. If, however, the factors determining yield can be technically influenced (e.g. nutrient supply), inputs are adjusted so that crop potential is fully realised.

The research and development work reported conducted in collaboration with agricultural en­gineers in Denmark has focused on four areas: -The identification of position -Data collection -Data processing -Implementation.

The identification of position

Accurate position finding is a pre-reqms1te for the management of spatial variation in ag­ronomic resources; it is necessary to be able to locate the position, digitally, in a precise and unambiguous way. An accuracy of about 10m is required in intensive crop production systems. The satellite aided Global Positioning System (GPS) with differential assistance (DGPS) en­ables position on the earth's surface to be iden­tified accurately within 10m in terms of the global grid system, i.e. longitude and latitude. This system satisfies requirements for the man­agement of agronomic resources; it is used in many industries according to established stan­dards, identifies position digitally, is accurate and it operates independent of surface features. The equipment is very robust, compact and inexpensive.

Data collection

A wide range of agronomic factors vary in space: yield potential, soil type, soil pH, nutrient con­tent, incidence of disease, pests and weeds. Ideally, a computer data base should enable the grower's knowledge of soil, weeds and pests to be combined with data collected at harvest or by soil survey. This data collection phase may already have been done in the case of soil type or may be done automatically in the case of yield (Schnug et a!., 1993).

The first step in establishing a data base of agronomic resources is to map the boundaries,

slope and aspect of each field. Some of the normal agricultural operations provide ideal op­portunities for accurate logging of terrain related data. The plough or other cultivator fitted with a GPS receiver and sensors to measure slope may be a particularly effective tool for collecting such data because it is operated slowly and sys­tematically over the whole cropped area.

O'Callaghan (1988) proposed that the deter­mination of the spatial variability in crop yield at the point of harvest would serve as a biological indicator of factors affecting productivity and provide a valuable diagnostic tool. Recent de­velopments in agricultural and electronic en­gineering enable this monitoring using a combi­nation of grain flow meters, global positioning and data logging with the result that yield map­ping may now be carried out automatically.

Many characteristics that affect crop perform­ance such as clay content and drainage, the incidence of weeds and pests are easily assessed in the field and the resultant data may be geo­coded on the site using hand-held GPS receivers. Weed species, especially grassweeds, occur in patches that are stable in time (Stafford and Miller, 1992). The incidence of pests and dis­eases may be linked to soil texture, for example, yield losses resulting from infections of Cercos­porella Herotrichoides (eyespot) are more com­mon on heavy soils (Glynne, 1942). Similarly, many farms are already mapped for soil type and the digitising of these maps can provide valuable data for interpreting yield variations. In Ger­many, for instance, all farms were mapped for selected parameters (e.g. texture and soil type) between 1930 and 1944 by the 'Reich­sbodenschaetzung'. Mapping chemical charac­teristics such as pH, potassium and phosphorus levels is more problematic because of the cost of analysis. However, once satisfactory levels have been established, applications of P and K may be made according to crop yield (Mengel and Kirby, 1987) as determined by automatic yield mapping.

Data processing

The mapping process generates very large data sets. Recent developments in computing enable these data sets to be handled quickly and effi-

ciently revealing interactions between crop per­formance and associated agronomic variables prior to the determination of strategies for the application of inputs. Techniques used in Geog­raphic Information Systems (GIS) are of some relevance here, however these data have some particular requirements. First, the data are quan­titative, and second, software programmes that are widely available and 'user friendly' are re­quired if this technology is to be adopted in agriculture.

Implementation

None of this technology is of any practical use if it is not converted into variable input applica­tions. Prototype sprayers and spreaders equipped with GPS receivers and units to control flow rates are now being developed and have been successfully field tested in Denmark in 1992.

Very rapid progress has been made in the development of the technology exploited by this concept. The research reported here has concen­trated on establishing how this technology is best exploited at farm level.

Methods

Yield mapping

In 1990 in Schleswig-Holstein, Germany, a Class Dominator combine fitted with a Claydon yield­o-meter system (Searcy et a!., 1989) was equipped with a SEL Globos 2000 GPS receiver. This yield metering system works on a volu­metric basis using a chamber fitted with a paddle wheel that intercepts the grain before entering the grain tank. The paddle wheel is engaged when the chamber is full and disengaged before it is empty so that the sections of the paddle wheel are always completely filled. The number of revolutions recorded is a very accurate mea­sure of the total volume of grain harvested. The combination of the rate of grain flow as de­termined by the proportion of time during which the paddle wheel is revolving, grain bulk density and forward speed is used to calculate spot yield throughout the field. In 1991, in Cleveland,

Computer aided farming 659

England, a MF38 combine harvester fitted with a 'Flow Control' yield monitoring system was equipped with a 'Shipmate' GPS receiver. This monitor relies on the attenuation of gamma rays by the flow of the grain. Geo-coded spot yield readings were logged every two seconds.

With both systems, the data were transfered via a 'chipcard' to an IBM compatible computing system. Erroneous yield and/ or positioning val­ues were screened out. The yield values for a regular 30 x 30 m grid were calculated from the 20 nearest data points by kriging, a geostatistical procedure (Webster, 1990), using SURFER.

Yield data were also recorded by hand at an intensively monitored site in Northumberland, England. Winter wheat and oilseed rape were grown in two 16 ha fields and harvested with a Claas Dominator combine fitted with a Claydon yield-o-meter. The combine position spot yield readings were recorded every 20 m with 5 m between harvester tracks. More than 3500 yield data were recorded and positioned in these fields. The yield values for a 30 x 30m grid were calculated by kriging using the 20 nearest data points.

Determination of spatial variation in pH

In 1991, soil and plant tissue samples were taken in a 20 x 40 m grid in the winter wheat and oilseed rape crops refered to above ( 498 samples in total). The soil was sampled by bulking 10 samples taken within a 10 radius of the centre of the sampling position. Soil pH was measured in a suspension of soil in water (1: 2.5). These sample points were digitised as for crop yield and the data were interpolated and synchronised with the yield data by kriging.

Data analysis

Data processing was performed using Rockworks (Rockware, 1991) and GEO-EAS, (Englund and Sparks, 1991). The data for pH and relative crop yield were subjected to boundary line analysis to identify the optimum pH for the site. The points with below optimum pH were identified and their lime requirement estimated in terms of CaO.

660 Schnug et al.

Results

Yield mapping

The GPS was operating for more than 85% of the run time with an estimated error of less than 20 metres for 95% of the operational time. Figure 1 shows an example of the GPS moni­tored tracks in a 80 ha field in Germany together with the resultant yield map. The GPS as it was then operating combined with post processing proved to be sufficiently accurate for yield map­ping.

Both yield metering systems are designed principally to measure total grain yield within an area, however, the data collected have contrast­ing statistical characteristics reflecting the differ­ence in the way yield is monitored. The 'Flow Control' system installed in the MF38 machine

m

'

'

measures spot yield directly from the mass of uninterrupted grain flow combined with the harvester's forward speed. With the Claydon yield-o-meter system, fluctuations in grain flow are buffered by the volumetric mechanism. These differences were reflected in the statistical analysis of the data. With the MF38 machine, the data from a crop of wheat with a mean yield of 11.1 t ha - 1 had a standard deviation of 2.2. 2% of the data indicated a yield of less than 4 t ha - 1 •

The data from the Claydon yield-o-meter system were less variable; data with a mean of 9.1 t ha - 1

and a range of 6 to 12.3 had a standard deviation of 0.6.

Soil pH, crop yield and lime requirement

The map of soil pH is given in Figure 2. pH varied from 5.4 to 6.8. Liming acid mineral soils

0,. 1<4t22l West (m)

7 371 445 ~ 5f' Moll 7~2 117 n1 M61o.JIIIIl118812G

' 1--

ll,.; ~ -r-;' ~ l~ ) rr r- . \~ ~- ~ ~~ l3'-

&!'-" R:> 'fi~ II': 0JJ ~ 1: Dl" ~~ r,2; v . ~r I\: ;,;

;: ~2: ~'=- ·~::

v~ ~ . . o::-. ~( Ill ,

J~~ .~G b~ ~ I( ' "-..l_2-..J ro ~ !\ ' 1-- ;)

0 74 '" 223 217371 44~ $lO "'- "21171111 0

West (m)

Fig. 1. GPS monitored harvester tracks in a field of wheat and the resultant map of relative yield, (Pronsdorf, Germany, 1991 ).

'2-~ 0.1 .Q

.~ ~ 0.05

&

Fig. 2. Soil pH map and the semivariogram.

Distance (r;~)

to a pH of around pH 5.5 is required to prevent phytotoxic levels of aluminium (van Lierop, 1990) while liming to pH higher than 6.5 in­creases the risk of manganese deficiency (Farley and Draycott, 1973). Thus, this map shows that some parts of these fields had a pH low enough to cause yield losses and other areas had a sufficiently high pH to result in manganese deficiency. Analysis of leaf tissue of rapeseed taken at the start of flowering had manganese concentrations below 15 mg kg - 1 and manganese deficiency has been identified as a limitation to crop yield in the region (Schnug and Evans, 1992). The processing of the kriged yield data together with the kriged pH data produced the scatter plot shown in Figure 3. A boundary line was fitted (Webb, 1972). This shows that yields in excess of the 120% of the mean yield were confined to points in the fields where pH was greater than 5.55 and less than 6.05. This con­firms the view that liming to pH 5.5 or above is all that is necessary for optimal yields in many crops (van Lierop).

The semivariograms for crop yield and soil pH accompany the maps in Figures 2 and 3. In both, the semivariance is small at short distances and increases as distance between sampling points increases until a sill is reached. Spherical models were fitted to the data and the variogram ranges were estimated to be 97 and 143m for soil pH and seed yield respectively. When the semivariance for pH and yield are compared, it is evident that the semivariance for pH is greatly affected by short distances over 0-20 m. This

120

~ e.___ 100 u (ij ·:;:., 80 Q) > B (ij 60 a:

40

5

Po~l1001iaJ app<oach -

Computer aided farming 661

- ': ... \::···. ~ .-··<: :':' .... ··

5.5

•,, I ' '•

6

pH

6.5 7

·2998 +1460')( + -22-4.9')1"2 + 11.37"x~3 --··· -208.9 + 477"oos(1'x) + -1 12.5'sin(1'x) + ·179.5'cos(2'x) + 86.8"sin(2')() + 28.4'cos{3'x) t ·27"sin(3""x)

Fig. 4. The border line analysis of soil pH and crop yield.

very steep increase in semivariance over short distances has been observed by others (Webster and Oliver, 1990). The more gradual increase in semivariances for yield may reflect the yield sampling proceedure used - the Claydon yield-o­meter is expected to have buffered short distance yield variability.

The further processing of these data using the optimum pH as determined by boundary line analysis resulted in the lime application map given in Figure 5. Applications in excess of 0.5 t CaO ha - 1 were confined to less than 20% of the area giving a 60% reduction in typical lime applications.

0.13

D <:·.12 0 0 0 0

"' >-.9 0.11

~ ~ 01 oO

~ 0.09

0.08 0 50 10Cl 15<1 200 25<1 300

D1!>!::J.nce (m)

Fig. 3. Map of relative crop yield and the semivariogram.

662 Schnug et al.

Fig. 5. The lime application map. The lime requirement is expressed in tonnes of CaO per hectare.

Conclusions

The studies reported here demonstrate clearly the benefits of exploiting knowledge of spatial variability in soil characteristics. The monitoring of soil pH on a 20 x 40 m grid enabled large savings in lime inputs. The environmental bene­fits of the extension of this approach to enable the application of crop nutrients and pesticides according to local requirements and productivity are obvious. However, this variability in soil pH has implications not only for the application of lime or chalk; soil pH is an important factor determining the environmental impact of the disposal of sewage sludge on agricultural land (Sauerbeck, 1991), and sewage sludge on soils with a pH lower than 5 is forbidden in Germany. Sewage sludge disposal is normally carried out by contractors using high output expensive ma­chinery. The extra cost involved in conducting spatially variable applications of sewage is margi­nal compared with the high cost of the equip­ment used.

Establishing the costs and profitability of vari­able rate application programmes has proved difficult because comparisons between conven­tional and variable rate applications are outside the scope of standard field experimentation tech­niques. Wollenhaupt and Buchholz (1993) con­cluded, from a review of field studies conducted in the U.S.A., that the variable rate applications of fertilisers may produce small economic bene-

fits for the farmer when the costs of data collec­tion and analysis are considered; however great­er benefits for the environment cannot be quan­tified.

The management of spatial variability relies on the availability of an inexpensive positioning system that uses robust, compact and widely accepted technologies. The GPS system satisfies these requirements and very rapid developments in this technology with other industries will provide agriculture with accurate and inexpen­sive positioning equipment.

A systematic approach to Local Resource Management in agriculture commences with yield mapping. It is proposed that yield mapping should be carried out over a number of years so that consistently high and low yielding areas can be identified to determine equifertiles (Schnug et al., 1993). Knowledge of equifertiles may pro­vide opportunities for limiting soil sampling for rapidly changing variables such as mineral nitro­gen to permanent monitoring points located within each equifertile. The GPS system offers the opportunity to relocate these points easily with way point navigation facilities.

The mapping process generates very large data sets. Recent developments in computing enable these data sets to be handled quickly and effi­ciently revealing interactions between crop per­formance and associated agronomic variables prior to the determination of strategies for the application of inputs. The studies reported here were carried out in conjunction with software development. LORIS, a Local Resource In­formation System (Schnug and Junge, 1993) enables the handling and interpolation of spatial­ly distributed data, their transfer and processing into standard files.

This technology also facilitates new ap­proaches to research into crops' responses to nutrients. In accordance with Liebig's law, a crop's response to a nutrient examined with conventional field trials may be affected by some other factor limiting crop yield within the trial plots. The combination of yield maps and maps of soil resources provides enormous oppor­tunities for overcoming this limitation to identify critical nutrient levels within the multivariate environment.

These concepts rely on the efficient capturing

of the spatially distributed data. Investigations into the use of micro-computers to handle the data are required to provide the crop manager with a convenient tool to examine spatial vari­ability in agronomic resources. It is essential that the manufacturers' equipment and developments are compatible. More reliance will be placed on exploiting soil resources as increasing emphasis is placed on the production of crops with minimum economic and environmental costs. If the de­velopments reported here are realised at farm level, the grower will be provided with a new tool that facilitates the full exploitation of the inherent productive capacity of the land.

References

Ascheman R E 1992 Managing variability- some practical field applications. Proc. Int. Workshop on Soil Specific Crop Management. Eds. P C Robert, R H Rust and W E Larson. pp 79-86. University of Mineapolis, MN.

Englund E and Sparks A 1991 GEO-EAS 121 Geostatistical Environmental Assessment Software- User's Guide. En­vironmental monitoring systems laboratory office of re­search and development. U.S. Environmental Protection Agency, Las Vegas, NV. 140 p.

Farley R F and Draycott A P 1973 Manganese deficiency of sugar beet in organic soil. Plant and Soil 38, 235-244.

Glynne M D 1942 Cercosporel/a Herpotrichoides Fron., causing eyespot of wheat in Great Britain. Ann. Appl. Bioi. 29, 254-264.

Mengel K and Kirby E A 1987 Principles of plant nutrition. International Potash Institute, Bern, Switzerland. 687 p.

O'Callaghan J R 1988 Engineering Applications and De­velopments. In Towards an Agro-Industrial Future. Proc. of the 6th. Royal Show International Symposium. pp 45-50. The Royal Agricultural Society of England, Stoneleigh, Warwickshire, UK.

Robert P C 1989 Land evaluation at farm level using soil

Computer aided farming 663

survey information systems. In Land Qualities in Space and Time. Eds. J Bouma and K Bregr. PUDOC, Wagen­ingen, The Netherlands.

Rockware 1991 Rockworks Gridding and Contouring Soft­ware. RockWare Incorporated, Wheat Ridge, CO. 231 p.

Sauerbeck D 1991 Plant, clement and soil properties govern­ing uptake and availability of heavy metals derived from sewage sludge. Water, Air, Soil Pol.. Special Vol. 57-58, 227-237.

Schnug E and Evans E 1992 Symptomatologic von Man­ganmangels an Raps. Raps 10, 43-45.

Schnug E and Junge R 1993 Strukturierung des Inter­pretationsmoduls und Konzeption des LORIS (Local Re­source Information System) fiir Anwendung im 'Computer Aided Farming' (CAF). KTBL- Schriftenreihe.

Schnug E, Murphy D and Haneklaus S 1993 Importance, evaluation and application of equifertiles to CAF ( Compu­ter Aided Farming). Landbaufiirschung Volkenre (In press).

Schnug E, Murphy D, Evans E, Haneklaus S and Lamp J 1992 Yield mapping and application of yield maps to computer aided local resource management. Eds. P C Robert, R H Rust and W E Larson. pp 87-93. University of Minnesota, MN.

Searcy S W, Schueller J K, Bae Y H, Borgelt S C and Stout S C 1989 Mapping of spatial variable yields during combin­ing. Trans. ASAE 32, 826-829.

Stafford J V and Miller P C H 1992 Spatially Selective Field operations Annu. Rep. Silsoe Res. Inst. 1991-92 pp 28-29. Silsoe Research Institute Silsoe, Bedfordshire UK.

Van Lierop 1990 Soil pH and lime requirement determi­nation. In Soil Testing and Plant Analysis, 3rd Edition. Ed. R L Westerman. Soil Science Society of America Book Series No. 3. The Soil Science Society of America, WI.

Webb R A 1972 Use of boundary line in the analysis of biological data. J. Hortic. Sci. 47, 309-319.

Webster R and Oliver M A 1990 Statistical Methods in Soil and Land Resource Survey. Oxford University Press, Oxford, UK. 316 p.

Wollenhampt N C and Buchholyz D D 1993 Profitability of farming by soils. Eds. Proc. Int. Workshop in Soil Specific Crop Management. University of Minnesota, MN. pp 199-212.

M.A.C. Fragoso and M.L. van Beusichem (eds.), Optimization of Plant Nutrition, 665-671, 1993. © 1993 Kluwer Academic Publishers. PLSO IAOPN-210

Suitability for agricultural use of sediments from the Maranhao reservoir, Portugal

R. FONSECA\ F.J.A.S. BARRIGA1 and W.S. FYFE2

1Department of Geology, FCUL, Campo Grande, P 1700 Lisbon, Portugal; 2Department of Geology, University Western Ontario, London, Ontario, Canada N6A 5B7

Key words: agricultural productivity, Maranhao reservoir, mineralogy, Portugal, sediments, sediment geochemistry

Abstract

Bottom sediments of the Maranhao reservoir were the subject of a fertility experiment. The samples were subjected to chemical and physical studies and to a study of growth rates with tulips. Comparing our results with natural soils, we conclude that these sediments could be good agricultural soils on their own, or even fertilizers for low quality soils. The suitability for agricultural use may eventually solve the classic problem of dam reservoir filling with sediments, if and when sediment removal from the reservoir becomes economically feasible.

Introduction

Hydroelectricity is one of the less polluting energy sources, but dams are a problem in the natural sediment transport cycle. In fact, they represent barriers to much needed sediment transport and accumulation in coastal zones. The reservoirs themselves lose value as they become filled with sediment. On the other hand, the amount and nature of the sediments that ac­cumulate in reservoir bottoms result not only from natural processes, but also from over­erosion in drainage areas related to factors such as agriculture, deforestation, and related deserti­fication.

The present study is a first report on a continu­ing study aimed at determining the suitability of the finer fractions of reservoir sediments for agricultural use, to reverse the negative effect of both (1) scarcity of sediment transport to coastal areas, and (2) excessive, agriculture-related ero­sion. Thus we have studied the bottom sediments of the Maranhao reservoir to test their fertility,

through chemical and physical analysis and a comparative growth study with tulips. The Maranhao reservoir is an interesting subject for this kind of research because of the remarkable geological diversity of its drainage basin.

The Maranhao reservoir, situated in Alentejo, South Portugal (Fig. 1), belongs to the hydro­logical system of the Sorraia river (Tagus Basin) and is the largest surface water reserve of Alto Alentejo (Brogveira, 1989). The surface area is about 19.6km2 , the volume 205·106 m3 , the maximum depth is 55 m and the average depth 16m (Brovgeira, 1989; Vale and Gonc;alves, 1986). In the Maranhao drainage basin (area 2282 km2 ) there occurs a Cenozoic sedimentary cover over Paleozoic and Precambrian forma­tions of the Variscan Foldbelt. This basement includes a large diversity of metasediments (shales and pelitic schists, greywackes, quar­tzites, conglomerates, carbonate rocks), metavol­canic sequences ranging in composition from acid to basic rocks and an intrusive massif (several granitic rocks with different geochemical

666 Fonseca and Barriga

MARANHAO RESERVOIR

DAM

• silty clay/clayey silt

~silt Wsalld-.silt-clay

liTI sandy silt

~sand

D coarse sand-gravel/silty coarse sa11d

I§ sillY 9ri1Yel/9ravel lkm

Fig. 1. Textural facies map of bottom sediments and location of the Maranhao reservoir.

features and a few mafic and intramafic intrusive bodies). The Cenozoic sedimentary cover is mainly detrital.

Methods

Chemical and physical analysis

Surface sediments were collected from a regular sampling net with 60 points in February-March 1990 with a modified VanVeen grab. Sampling depths varied from a minimum of 1.5 m to a maximum of 35m. The sediments were subject­ed to most of the chemical and physical studies routinely used for the evaluation of soil fertility (according to Santos, 1991): grain-size, organic matter content, pH, available major (P, K) and micronutricnts (Fe, Mn, Zn, Cu, B and Mo) and sediment water content (field capacity, wilting point and plant-available water capacity). We have also performed detailed mineralogical and geochemical studies: total elemental analyses and clay minerals identification, characterization and semi-quantification. These are very impor­tant in the evaluation of the fertility of sedi­ments. The difference between total elemental and plant available abundances provides a mea­sure of existing reserve nutrients, potentially usable by plants, provided that these elements

are contained in minerals susceptible to altera­tion in the soil environment. The clay miner­alogy study is particularly important, because clays, together with organic matter, constitute the most active part of soils (Bear, 1964; Santos, 1991). The nature of clays is fundamental knowl­edge regarding the soil capacity for nutrient and water retention, and buffer properties. The min­eralogical and geochemical studies are also im­portant in view of the geological diversity of the drainage basin that feeds the Maranhao bottom sediment.

All the sediments were subjected to grain-size, total elemental and organic matter content anal­ysis. According to grain-size distribution sedi­ments were classified and mapped (Fig. 1). Six­teen representative samples of the various mor­phological characteristics of the reservoir were selected for the other complementary studies. These analyses were also performed on commer­cial potting soil (AS soil used as reference, see section on agricultural experiments below). The methods used were:

Grain-size analysis Separation by grain size classes (Wentworth Lane scale, see Pettijohn, 1975) by wet sieving (gravel-sand-silt clay: Buller and McManus, 1979), dry sieving (grain-size sand distribution) and measurement with a laser sedimentometer (silt clay). Representation of

sand, silt and clay proportions in a Shepard (1954) triangular diagram (Pettijohn, 1975) and subsequent classification.

Clay minerals characterization Extraction of clay fractions and preparation of oriented slides by a procedure described by Thorez (1976) and Prates (1986). Samples were subjected to the "classical" treatments- normal (untreated and dried at room temperature), ethylene glycol solvation, heating to 550°C (Lucas, 1979; Thorez, 1976) and submitted to X-ray diffraction analysis. Clay minerals identification, characteri­zation and semi-quantification were based on Lucas (1959), Thorez (1976) and Prates (1986).

Organic matter Samples were frozen soon after collection at -20°C, and the organic matter content was determined by oxidation with K2 Cr2 0 7 and titration with FeS04 (LNEC, Portugal, standard E201, 1967).

pH PH was measured by a potentiometer in a water-sediment suspension- pH(H20).

Total elemental geochemistry Total abundance of selected elements (P, K, Fe, Mn, Zn, Cu, B, Mo) were determined by IC and DC Plasma at the Bondar-Clegg Laboratories, Canada. Total nitrogen was measured by the Kjeldahl tech­nique at the LQARS (Laborat6rio Quimico Agricola Rebelo da Silva, Lis boa; Silva et a!., 1975).

Available nutrients and available water cap­acity Selected elements were determined at the LQARS: P and K (Egner-Riehm method), Fe, Cu, Mn and Zn (Lakanen method), B (boiling water method) and Mo (thiocinate method). Plant-available water capacity (AW) was esti­mated as a difference between field capacity (FC) and wilting point (WP); these were de­termined by the percentage of soil water held at a water potential less than -1/3 bar and -15 bar respectively (Donahue eta!., 1983).

Agricultural experiments We have conducted some fertility tests with tulips in a greenhouse, with a few representative Maranhao samples and with one sample of commercial potting soil (AS)

Maranhao reservoir sediments and agriculture 667

used as a reference. This AS soil is a very rich turfy loam which has the ideal conditions for the plantation of bulbs (Dumoncel, 1969).

We have selected tulips for agricultural experi­ments for several reasons: (1) fast growth and growth easily quantified by a great diversity of development parameters; (2) economic rentabili­ty; (3) plantation feasibility in the most advan­tageous season to our work; (4) quick and accurate reproduction measurement (to be re­ported elsewhere). It should also be mentioned that good tulip flowering, with commercial inter­est, requires clay-sandy soils rich in organic matter and available K, and pH= 6-8 (Castano, 1977; Dumoncel, 1969; Sousa Veloso et a!., 1981).

The more clayey and silty samples were mixed with pure quartz sand at various ratios (85%-90% in the more clayey sediments and 80%-85% in the more silty ones) and the sandy sediments were used without any additional sand mixture. The pure silica sand used in these experiments can be regarded as chemically inert and is used to give physical support to the growing plant and by allowing free drainage, to ensure an adequate aeration of the tulips root system.

We have performed experiments on 25 pots (Maranhao sediments: 16; reference soil AS: 9), with 2 tulips in each, under the same external conditions (temperature, humidity, luminosity). We have done weekly measurements of a few growth parameters: plant and corolla maximum height, flower opening, flowering period, period of time since plantation until rise, period of time since plantation until grown-up plant and growth rate.

Results

Chemical and physical analysis

Representative chemical and physical parameters of the Maranhao sediments and reference soil AS are presented in Figure 2, and/ or described in the text below. To test the fertility level of the sediments, each parameter was compared with the corresponding medium interval as defined for various mineral soils and crops after Bear