1999/2000 FINAL RESEARCH REPORT : WINETECH

FRUIT TYPE : Grapes PROJECT NUMBER: WW18/12

ORGANIZATION : Nietvoorbij RESEARCHER : C.C. Mullins

PROJECT TITLE : An assessment of symbiotic nitrogen fixation by leguminous cover crops and

its contribution to the nitrogen balance in a vineyard-cover crop system.

OBJECTIVE OF PROJECT:

The assessment of the uptake of symbiotically fixed nitrogen by legume cover crops for sustainable

vineyard management.

OBJECTIVES OF CURRENT YEAR:

To evaluate different legume cover crops for their ability to enhance the N nutrition of the vine on a sandy

soil and to determine the nitrogen contribution made by legume cover crops as measured by 15N natural

abundance methodologies.

UPDATED FINDINGS:

Throughout this study it has become evident that, among the legumes, grazing vetch has the greatest

potential to improve the soil N status of the vine in the Olifants River Region as it supplies the highest

aboveground N. Nitrogen supply from legume cover crops are synchronized with vine N demand, yet

adequate moisture has to be supplied in order to gain the full benefit of legumes with regard to N supply.

This research has shown that cover cropping is beneficial to the nitrogen economy of the vine.

ADVANTAGES TO INDUSTRY:

Potential to promote cost-effective, environmentally friendly and sustainable viticultural practices for

resource limited producers. Legume cover crops could supplement nitrogen fertilization and thereby

reduce the application of chemical fertilizers and input costs.

RECOMMENDATIONS FOR THE FOLLOWING YEAR:

The project has been completed and the results will be communicated in article form.

AGRICULTURAL RESEARCH COUNCIL

LANDBOUNAVORSINGSRAAD

Nietvoorbij Centre for Vine and WineNietvoorbij Sentrum vir Wingerd en Wyn

p/bag p/sak X5026 - Stellenbosch - 7599 - rsa

1999/2000 FINAL RESEARCH REPORT : WINETECH

PROJECT NUMBER WW 18/12

PROJECT TITLE An assessment of symbiotic nitrogen fixation by leguminous cover

crops and its contribution to the nitrogen balance in a vineyard-

cover crop system.

PROJECT LEADER C.C. Mullins

COMMENCEMENT DATE : 1997COMPLETION DATE : 2000

No part of this document may be reproduced or distributed in any form without the express writtenpermission of ARC-lnfruitec/Nietvoorbij.

Telefoon 021 - 809 3100Int. 27 21 809 3100

Telefaks 021 - 809 3002Int. 27 21 809 3002

Telephone

Telefax

021 - 809 3100Int. 27 21 8093100

021 - 809 3002Int. 27 21 809 3002

1999/2000 FINAL RESEARCH REPORT

PROJECT NUMBER PROJECT LEADER CO-WORKERS

WW18/12 C.C. Mullins P.J.E. Louw

F.D. Dakora

J.C. Fourie

PROJECT TITLE

An assessment of symbiotic nitrogen fixation by leguminous cover crops and its contribution to the

nitrogen balance in a vineyard-cover crop system.

ACKNOWLEDGEMENTS

Winetech is acknowledged for partial funding of this project.

PROBLEM BEING ADDRESSED / AIM OR HYPOTHESIS

It is known that leguminous cover crops can contribute towards the nitrogen balance of a system.

However, how much is contributed and when, are questions that remain unresolved. The aim of the

project is to determine the quantity of nitrogen fixed and at what stage most fixed nitrogen is contributed to

the vine.

RESEARCH NEED BEING ADDRESSED

Winetech Technical Committee, 1996: 3.1 Nitrogen fertilization. (Document in Afrikaans only).

LONG-TERM OBJECTIVES

• Evaluate different legume cover crops for their ability to enhance the N nutrition of the vine on a

sandy soil.

• Determine the nitrogen contribution made by legume cover crops as measured by 15N natural

abundance methodologies.

• Examine the effect of cover cropping on the soil nitrogen status of the vineyard.

• Assess the effect of cover cropping on the nitrogen nutrition of the vine.

RESULTS AND DISCUSSION

The results of this project have been used for my masters dissertation. All data collected from 1997-

1999 has been presented and discussed in the thesis. Addendum A is a copy of the dissertation.

CONCLUSIONS

As chemical inputs of N fertilizers have to be reduced to limit environmental pollution, it has become

imperative to seek more environmentally friendly and cost-effective alternatives. Legume cover crops

have been known to improve the N status of the adjacent crop. The main objective of this project was to

recommend a legume cover crop species, in terms of its N contribution, for use in vineyards in the Olifants

River region.

For legume cover crops to benefit vineyards in terms of N provision, it is essential that N release by

legumes coincides with vine N demand. This occurs from budbreak to veraison and from harvest to leaf-

fall. This synchrony was observed in this study.

Although N fertilizer application and winter rainfall was reduced in the second season, 515N-values in vine

leaves were lower compared to the previous year. As a low 515N-value denotes a high uptake of

biologically fixed N, this indicated that the proportion of fixed N uptake to fertilizer N uptake gradually

increased in the vine with time. Throughout this study it has become evident that the use of grazing vetch

in vineyards in the Olifants River Region could be successfully used for the provision of N and will result in

the reduction of synthetic fertilizer inputs. The omission of a green cover as described in the control plot

had significantly lower leaf nitrate reductase activity, emphasizing the importance of cover crops in

viticulture.

A high fluctuation in mineral N levels were observed, yet during the decomposition period (January-

February) legume cover crop treatments were higher in nitrogen. A decline in soil N was observed in the

second season due to shortage of irrigation water and low rainfall. Grazing vetch decomposes rapidly

when mechanically tilled at budbreak. This practice can therefore not be recommended to producers. The

die-back treatment had a high dry material on the soil surface than the before budbreak treatments.

Nitrogen fixation proceeded effectively in all treatments for both seasons. Grazing vetch and to a lesser

extent Seradella emena provided an excellent source of aboveground N.

The following conclusions have been drawn from this study:

(i) N supply from legume cover crops are synchronized with vine N demand,

(ii) Grazing vetch is suitable for use as a green cover in vineyards in the Olifants River Region as

it supplies the highest aboveground N.

(iii) Mechanical tillage of cover crops result in a short-term release of N and this practice is

inefficient,

(iv) Adequate moisture has to be supplied in order to gain the full benefit of legumes with regard

to N supply.

SUMMARY OF 1999/2000-RESEARCH REPORT

PROJECT NUMBER PROJECT LEADER CO-WORKERS

WW18/12 C.C. Mullins P.J.E. Louw

F.D. Dakora

J.C. Fourie

PROJECT TITLE

An assessment of symbiotic nitrogen fixation by leguminous cover crops and its contribution to the

nitrogen balance in a vineyard-cover crop system.

The aim of this investigation was to evaluate different legume cover crops for their ability to enhance the N

nutrition of the vine on a sandy soil. The study site was located in Lutzville, in the Olifants River region and

was conducted on a six-year old Sauvignon blanc vineyard grafted onto Ramsey rootstock.

The uptake and assimilation of nitrogen by grapevines were monitored monthly in a legume-based cover

cropping system over two seasons during 1997-1999. An in vivo nitrate reductase assay was conducted

on fresh vine leaves during the growing season as it is an indicator of newly absorbed nitrogen. The

uptake of biologically fixed nitrogen was measured by determining the natural abundance of 15N in vine

leaves. The uptake and assimilation of biologically fixed N coincided with the annual demand for nitrogen

by the vine, which occurs from budbreak to the first stage of berry development and after harvest. Nitrate

reductase activity in vines intercropped with legumes was relatively higher than the cereal and control

counterparts, despite the reduction of nitrogen fertilization in these treatments. The omission of a green

cover in the control plot had significantly lower leaf nitrate reductase activity, emphasizing the importance

of cover crops in viticulture. The proportion of fixed N uptake to fertilizer N uptake gradually increased in

the vine with time, as &15N values of vine leaves were lower in the second season. Results from soil

analysis revealed that the soil nitrogen (total and inorganic) was low and differences in soil nitrogen levels

are expected to be more distinct in the longer term.

The N content and biomass of legumes were determined to evaluate various legume cover crop species.

Grazing vetch had the highest above-ground N. &15N values differed according to the various legume plant

components. The roots and shoots were depleted of 15N indicating the active fixation of nitrogen while the

nodules were enriched in 15N. This was due to the inherent characteristic of the species used. The lower

rainfall in the latter season resulted in a decline in legume biomass production. 815N-values of legumes

were lower in the second season indicating that nitrogen fixation proceeded even more successfully

regardless of climatic conditions, proving the long-term benefits of using cover crops. In general grazing

vetch showed the greatest potential for improving the N status of the vine.

OPSOMMING VAN 1999/2000-NAVORSINGSVERSLAG

PROJEKNOMMER PROJEKLEIER MEDEWERKERS

WW18/12 C.C. Mullins Dr. F. Dakora (UCT)

P.J.E. Louw

J.C. Fourie

PROJEKTITEL

'n Ondersoek na simbiotiese stikstofbinding deur peulplantdekgewasse en die bydrae daarvan tot die

stikstofbalans in 'n wingerd-dekgewassisteem.

Die doel van hierdie projek was om die geskiktheid van verskillende dekgewasbestuurspraktyke te oordeel

op grond van hul vermoe om stikstofvoeding in die wingerd te verbeter op 'n sandgrond te Lutzville.

Die opname en assimilasie van stikstof deur die wingerd is maandeliks gemonitor in 'n

peuldekgewassisteem oor twee seisoene gedurende 1997-1999. 'n In vivo nitraat-reduktase

bepalingstoets is uitgevoer op vars wingerdblare gedurende die groeiseisoen omdat dit 'n aanduiding is

van pasopgeneemde stikstof. Die opname van biologiesgebinde stikstof het oorgeengestem met die

jaarlikse N-aanvraag deur die wingerd, wat plaasvind van bot tot die eerste fase van korrelontwikkeling en

na oes. Nitraat-reduktase aktiwiteit in wingerdblare, tussenverbou met peuldekgewasse, was relatief hoer

as die graan en kontrole behandelings, ten spyte van 'n vermindering in stikstofbemesting in hierdie

behandelings. Die skoonbewerkte kontrole het 'n betekenisvolle laer nitraat-reduktase aktiwiteit getoon.

Dit beklemtoon die rol van dekgewasse in wingerdbou. Die verhouding van gebinde N-opname tot

bemesting N-opname het geleidelik toegeneem in die wingerd want 815N-waardes in wingerdblare het

gedaal in die tweede seisoen. Stikstof in die grond (totaal en anorganies) was laag en verskille word

verwag om meer opvallend te wees op die langtermyn.

Die N-inhoud en biomassa van die peulplant is bepaal om die peulplantdekgewasse te evalueer.

Weiwieke het die hoogste bogrondse N getoon. &15N waardes het verskil volgens die plantkomponente.

Die wortels en lote was laag in 15N - 'n aanduiding van die aktiewe binding van stikstof terwyl die knoppies

verryk was in 15N. Dit was kenmerkend van die spesies onder studie. Die lae reenval in die laaste

seisoen het 'n afname in biomassa produksie veroorsaak. Ten spyte van die lae biomassa produksie het

stikstofbinding wel plaasgevind (&15N waardes van peulplante was laer in die tweede seisoen). Dit was 'n

aanduiding dat stikstofbinding meer suksesvol voortgegaan het ongeag die klimatiese toestande. Dit

bewys die langtermynvoordele van dekgewasse. In die algemeen het weiwieke die grootste potensiaal

om die N status van die wingerd te verbeter. Hierdie dekgewas toon groot potensiaal om as koste-

effektiewe en omgewingsvriendelike alternatief te dien vir die toediening van N-bemesting en sal van groot

nut wees vir hulpbron-beperkte produsente.

ADDENDUM A

Contribution of Nitrogen Fixation by Cover Crop Legumes to the

Nitrogen Economy of a Vine-based Cropping System

by

Carmen Mullins

Botany Department

University of Cape Town

Presented for the Degree of Master of Science

ABSTRACT

The escalating cost of fertilizer N manufacture and concern over soil erosion has renewed

interests in legumes and their role in cropping systems. Winter legume cover crops may provide

significant quantities of fixed N while conserving soil and water resources and sustaining or

improving soil productivity. Poor N content of sandy soils has prompted research into the use of

winter legume cover crops in vineyards. The aim of this investigation was to evaluate different

legume cover crops for their ability to enhance the N nutrition of the vine on a sandy soil. The

study site is located in Lutzville, in the Olifants River region. The study was conducted on a six-

year old Sauvignon blanc vineyard grafted onto Ramsey rootstock. The following legume cover

crops were used: Vicia villosa v. dasycarpa, Medicago truncatula v. Parabinga, Medicago

truncatula v. Paraggio, Ornithopus sativa v. Emena. The cereals used in this study were: Secale

cereale and Avena sativa v. Saia. All treatments were replicated three times in a randomized

block design. A control in which weeds were mechanically controlled in the working row was

included.

The uptake and assimilation of nitrogen by grapevines were monitored monthly in a legume-

based cover cropping system over two seasons during 1997-1999. An in vivo nitrate reductase

assay was conducted on fresh vine leaves during the growing season as it is an indicator of

newly absorbed nitrogen. The uptake of biologically fixed nitrogen was measured by determining

the natural abundance of 15N in vine leaves. The uptake and assimilation of biologically fixed N

coincided with the annual demand for nitrogen by the vine, which occurs from budbreak to the

first stage of berry development and after harvest. Nitrate reductase activity in vines intercropped

with legumes was relatively higher than the cereal and control counterparts, despite the reduction

of nitrogen fertilization in these treatments. The omission of a green cover in the control plot had

significantly lower leaf nitrate reductase activity, emphasizing the importance of cover crops in

viticulture. The proportion of fixed N uptake to fertilizer N uptake gradually increased in the vine

with time as 61SN values of vine leaves were lower in th second season. Results from soil

analysis revealed that the soil nitrogen (total and inorganic) was low and differences in soil

nitrogen levels are expected to be more distinct in the longer term. The N content and biomass of

legumes were determined to evaluate various legume cover crop species. Vicia villosa v.

dasycarpa had the highest above-ground N. &15N values differed according to the varios legume

plant components and nodules were found to be markedly positive. The lower rainfall in the latter

season resulted in a decline in legume biomass production. &15N-values of legumes were lower

in the second season indicating that nitrogen fixation proceeded even more successfully

regardless of climatic conditions, proving the long-term benefits of using cover crops. In general

showed the greatest potential for improving the N status of the vine.

CONTENTS

1 GENERAL INTRODUCTION 1

1.1 Nitrogen dynamics in agricultural soils 2

1.1.1 Influence of soil type 4

1.2 The assimilation of nitrogen in higher plants 4

1.2.1 Regulation of nitrate reductase activity 6

1.3 Nitrogen nutrition of vines 6

1.4 The role of cover cropping systems in sustainable agriculture 8

1.5 The use of legumes as cover crops 10

1.6 Management of cover cropping systems 14

1.7 References 16

2 GENERAL MATERIALS AND METHODS 23

2.1 Experimental design and fertilizer regimes 23

2.1.1 Nitrogen fertilizer regimes during 199-1997 23

2.2 Plant analysis 26

2.2.1 In vivo nitrate reductase assay 26

2.2.2 15N natural abundance 27

2.3 Soil Analysis 28

2.3.1 Inorganic nitrogen 28

2.3.2 Total soil nitrogen 29

2.3.3 Stastical analysis 29

2.4 References 30

3 VINE UPTAKE AND ASSIMILATION OF NITROGEN IN A COVER

CROPPING SYSTEM IN LUTZVILLE 31

3.1 Introduction 31

3.2 Materials and Methods 33

3.2.1 In vivo nitrate reductase activity of vine leaves 33

3.2.2 Determination of &15N natural abundance of plant material 33

3.3 Results and Discussion 34

3.3.1 Nitrate Reductase Activity 34

3.3.2 &15N natural abundance of vine plant material 35

3.4 Conclusion 48

3.5 References 49

4 THE INFLUENCE OF COVER CROPPING MANAGEMENT PRACTICES

ON THE SOIL NITROGEN STATUS OF A VINEYARD 51

4.1 Introduction 51

4.2 Materials and Methods 53

4.2.1 Soil analysis 53

4.2.1.1 Inorganic nitrogen 53

4.2.1.2 Total soil nitrogen 53

4.3 Results and Discussion 54

4.4 Conclusion . 72

4.5 References 73

5 THE CONTRIBUTION OF BIOLOGICALLY FIXED NITROGEN BY

LEGUMINOUSCOVER CROPS TO THE NITROGEN STATUS OF A

SAUVIGNON BLANC VINEYARD IN THE OLIFANTS RIVER REGION 75

5.1 Introduction 75

5.2 Materials and Methods 76

5.3 Results and Discussion 78

5.3.1 Biomass production 78

5.3.2 &15N signature of legume components harvested in the field 78

5.3.3 Legume N content 79

5.4 Conclusion 87

5.5 References 88

6 SUMMARY AND CONCLUSIONS 91

CHAPTER 1

GENERAL INTRODUCTION

At present the terrestrial input of nitrogen from biological nitrogen fixation (BNF) is estimated to

be in the range of 139 to 170 x 106 ton N per year (Marschner, 1997b). The increase in both

the costs of fossil energy and worldwide demand for nitrogen fertilizer used for food production

are major reasons for renewed interest in BNF as an alternative or at least as a supplement to

the use of chemical nitrogen fertilizer. Legumes were an important component of crop rotations

before World War II. In the post-war years, however, inexpensive and abundant fertilizer N

diminished the role of N-fixing plants in cropping systems (Hoyt and Hargrove, 1986). The

escalating cost of fertilizer N manufacture and concern over soil erosion has renewed interests

in legumes and their role in cropping systems. Winter legume cover crops may provide

significant quantities of fixed N while conserving soil and water resources and sustaining or

improving soil productivity.

Poor N content of sandy soils has prompted research into the use of winter legume cover crops

in vineyards. The aim of this investigation was to evaluate different legume cover crops for

their ability to enhance the N nutrition of the vine on a sandy soil. The study site is located in

Lutzville, in the Olifants River region (Fig 1.1). The study was conducted on a six-year old

Sauvignon blanc vineyard grafted onto Ramsey rootstock. Lutzville is a winter rainfall semi-arid

region. This is a class V climatic region (Winkler, 1962) at 31°36' latitude. Annual precipitation

is less than 200mm (Conradie and Myburgh (unpublished), 1999) of which only 40mm falls

during the growing season, resulting in an irrigation requirement of 675mm for this period (Van

Zyl, 1981). The soil is deep, red, calcareous (Hutton: Maitengwe, according to MacVicar & Soil

Survey Staff, 1977), representative of the so-called 'Karoo-soils' of this area. This study used a

subset of treatments from a larger study designed to measure the effects of several cover crop

management practices on the chemical and physical properties of the soil, including water

usage and vine performance.

Rietpoort

Nuwerus

,Bitterfontein

Nieuwoudville

Citrusdal

Fig. 1.1: Location of study site

1.1 Nitrogen dynamics in agricuitural soils

Nitrogen entering the soil system is subjected to various chemical and biological

transformations (Table 1.1). Knowledge of N dynamics in soil is therefore essential for

obtaining high use efficiency of fertilizer-N. Results of studies using the stable isotope 15N have

shown that from 20 to 40% of the N applied as fertilizer remains behind in the soil in organic

forms after the first growing season. Only a small portion of this immobilized N (

Nitrate (N03~) and ammonium (NH4+) are the major sources of inorganic nitrogen taken up by

the roots of higher plants. Levels of exchangeable NH4+ and NO3 vary from day to day and

from one season to another and depend on a variety of environmental factors mentioned

below:

• Seasonal variation: Levels of exchangeable NH4+ and NO 3 are greatly affected by

temperature and rainfall. The amounts found in the surface layer of soils of the temperate

humid climatic zone are lowest in winter because of leaching, rise in spring as

mineralization of organic matter commences, decrease in summer through consumption by

plants, and increase once again in the fall when plant growth ceases and crop residues

start to decay. The level in summer is higher than that in spring.

• Mineralization and immobilization: Biological turnover leads to the interchange of NH4+ and

NO3 with N of the organic matter. Accordingly mineral N levels represent a delicate

balance between mineralization and immobilization and are affected by the activities of soil

microorganisms and the C/N ratios of plant residues.

• Growing plants: Plants exert a depressing effect on mineral N levels in the soil. In addition

to direct uptake, NH4+ and NO3 levels may be altered by immobilization and denitrification

in the root zone, and possibly by inhibition of nitrification by root exudation products

(Harmsen and Kolenbrander, 1965);.

• Leaching of nitrate: Nitrate is the form of N that is most mobile and that is subject to

leaching and movement into water supplies. The magnitude of nitrate is difficult to estimate

and depends on a number of variables, including quantity of nitrate, amount and time of

rainfall, infiltration and percolation rates, evapotranspiration, water-holding capacity of the

soil and presence of growing plants. Leaching is generally greatest during cool seasons

when precipitation exceeds evaporation; downward movement in summer is restricted to

periods of heavy rainfall.

• Volatilization of NH3: Rapid changes in NH4+ can occur as a consequence of chemical

volatilization of NH3. Losses are greatest on saline soils, especially when NH4+-forming

fertilizers are used. Only slight losses occur in soils with pH values less than 7.0, but

losses increased markedly as the pH increases. For any given soil, losses increase when

neutral or alkaline soils containing NH4+ in the surface layer are dried out. The presence of

adequate moisture reduces volatilization, even from alkaline soils.

• Losses of nitrate through denitrification: Significant loss of NO3'-N can and does occur as a

consequence of denitrification. Under anaerobic conditions, such as occur frequently in

soils following a heavy rain, NO3' can be volatilized quantitatively in a comparatively short

time, particularly when energy is available in the form of organic residues.

• Buildup of NH4 and NO3by fertilizer applications: In soils of humid and semi-humid

regions, any fertilizer N added in excess of plant or microbial needs will be lost through

leaching and/or denitrification. Thus mineral forms of N seldom carry over from one season

to the next. However, where leaching and denitrification are minimal, such as in soils of

arid and semi-arid regions, some carry-over occurs and repeated annual applications of N

fertilizer can lead to a buildup of NO3 in the soil profile.

1.1.1 Influence of soil type

On sandy soils relatively high proportions of fertilizer N may be assimilated by soil microbes

due to the high mass flow rates of nitrate to plant roots (Peschke et a/., 1984). The fertilizer N

thus immobilized in early summer may be mineralized in autumn and may later be leached by

winter rains and will therefore be lost by the system. This may be one reason why sandy soils

are generally poor in organic nitrogen.

In acid soils the mineralization of organic nitrogen is retarded or even blocked (Kuntze and

Bartels, 1979). Soil temperature and soil moisture influence N mineralization (Honeycutt,

1991). Kladiviko and Keeney (1987) found a six to seven fold higher N mineralization rate at

35°C than at 10°C. High mineralization rates are observed in summer and lowest in winter

(Weller, 1983). Dry periods in summer cause a drastic reduction in N mineralization.

Lochmann and co-workers (1989) reported that the net N immobilization (microbial assimilation)

was highest in spring while in summer net mineralization dominated. There is evidence that

soil texture has a strong impact on N mineralization. N mineralization in sandy soils is more

rapid than in soils with a higher clay content. Soils with a higher clay content are able to store

organic nitrogen in the form of adsorbed polypeptides and thus may add to the potential of

mineralizable soil N. In sandy soils in which the proteins of decomposing biomass are hardly

adsorbed, mineralization will occur in late summer followed by nitrate leaching in winter. There

is more organic N in no-till treatments than ploughed treatments. The input of proteins (green

manure) induces a flush of N mineralization within a few weeks, the input of organic C (straw)

an assimilation (immobilization) of inorganic nitrogen.

1.2 The assimilation of nitrogen in higher plants

As mentioned earlier, nitrate and, to a lesser extent, ammonium are the principal sources of

nitrogen that are available to higher plants under normal field conditions; hence the nitrate

assimilation pathway is the major point of entry of inorganic nitrogen into organic compounds.

Most of the ammonium has to be incorporated into organic compounds in the roots, whereas

nitrate is readily mobile in the xylem and can also be stored in the vacuoles of roots, shoots and

storage organs (Marschner, 1997a). In order to be incorporated into organic structures and to

fulfil its essential functions as a plant nutrient, nitrate has to be reduced to ammonia.

The reduction of nitrate to ammonia is mediated by two enzymes: nitrate reductase and nitrite

reductase. Nitrate reductase (NR) is located in the cytoplasm and regulates the two-electron

reduction of nitrate to nitrite. Nitrite reductase (NiR) is situated in the chloroplast and

transforms nitrate to ammonia in a six electron reduction. Despite the spatial separation of NR

and NiR, nitrite rarely accumulates, in intact plants under normal conditions.

In higher plants NR is a complex enzyme containing two identical subunits (i.e. it exists as a

dimer). Each subunit can function separately in reduction of nitrate and contains 3 prosthetic

groups: flavine adenine dinucleotide (FAD), cytochrome 557 (cytj and a molybdenum cofactor

(MoCo).

In vines, a major part of nitrate is reduced in the leaves (Perez and Kliewer, 1978). The nitrate

content in the different plant organs evolves from nitrate absorption and nitrate transfer

(Mengel, 1986). The following factors determine the nitrate quantity in grapevines or in specific

tissue:

1. absorption by the roots

2. nitrate reductase activity of the roots

3. the transport rate to the next place of reduction

4. nitrate reductase activity in the leaves

5. production of the biomass in the vines and in the individual organs

The translocation of nitrate-N is through the xylem with a partial reduction of nitrate in the roots.

Nitrate is transported in the xylem from the roots, and thus their starting point for amino acid

biosynthesis is glutamine/glutamate (Ireland, 1990). Developing seeds or fruits, which are very

active in amino acid biosynthesis, will receive most of their nitrogen in the form of amino acids

supplied by the phloem. The age of the tissue also affects nitrogen flow: young leaves

consume all of the incoming nitrogen for growth, mature leaves re-export (in the phloem) much

of the nitrogen they receive to the growing apex or developing fruit, as do senescing leaves,

which also convert a lot of their proteins and other nitrogenous molecules to transport

compounds for export.

Regulation of nitrate reductase activity

NR is regulated by several different modes exerted at different levels, namely: enzyme

synthesis and degradation, reversible inactivation and concentration of substrate and effectors.

Nitrate induces the de novo synthesis of the enzyme. The steady state level of nitrate

reductase is determined by the rate of degradation (turnover) as well as by the rate of

synthesis. A newly synthesized nitrate reductase protein has a half-life of only a few hours in

the cell. Thus when the nitrate is withdrawn, the level of nitrate rapidly decreases. The

enzyme has a half-life of only a few hours (Beevers and Hageman, 1983) and in plants

receiving no nitrate it is merely absent (Li and Gresshof, 1990). NR can be induced within a

few hours by addition of nitrate (Oaks et a/., 1972) and suppressed by certain amino acids

(Bretelerand Smit, 1974; Oaks, 1991).

NR activity in photosynthetic tissues generally varies on a diurnal basis, with peak rates during

the light period and lowest rates at the end of the dark period. Diurnal rhythm in nitrate

reduction may reflect fluctuations in carbohydrate level (Aslam and Huffaker, 1984) and in the

corresponding supply of reducing equivalents and carbon skeletons. However, besides these

coarse regulations various mechanisms of fine regulations exist, on the level of enzyme

modulation in carbon partitioning or direct modulation of the NR by enzyme phosphoryiation

(Kaiser and Spill, 1991). In the light-dark transition this activation of NR occurs within a few

minutes and thus accumulation of nitrite is prevented (Riens and Heldt, 1992). In light,

transpiration increases and nitrate enters the leaf in the xylem stream. Alternatively, light may

cause the release of nitrate from the vacuolar storage pools within the leaf. In photosynthetic

tissues in the light, the light reactions in the chloroplasts are probably the major source of

electrons to nitrate assimilation.

During the ontogenesis of an individual leaf, a typical pattern is observed in NR activity.

Maximum activity occurs when the rate of leaf expansion is maximal. Thereafter, the activity

declines rapidly. Thus in fully expanded leaves, NR activity is usually very low and often the

nitrate levels are correspondingly high (Santoro and Magalhaes, 1983).

1.3 Nitrogen nutrition of vines

Grapevines have a lower fertilization requirement than other horticultural crops (Christensen et

a/., 1978). This is due to the well-distributed root system of vines, facilitating the uptake of

nutrients from a large soil volume, as well as a long growing season, which stretches over eight

months in a warm climate country like South Africa (Conradie and Saayman, 1989). Under

these climatic conditions, the grapevine utilizes about ca. 3.7kg N for the production of one ton

of fresh grapes, with 1.6kg being in the clusters and 2.1kg in the vegetative growth (Conradie,

1991).

In a study conducted in Germany in the winegrowing area of Rheingau, Lohnertz (1991)

demonstrated that fertilization with amounts ranging from 0 to 70 kgN/ha is sufficient for the

cultivar Riesling. From this study it was evident that increasing nitrate content in different soils,

does not lead to an increase in production. After several years of omitting N fertilization, the

number of bunches per vine were only slightly lower (this was not statistically). This could

indicate that even a visible nitrogen shortage in the variety Riesling has no influence on the

number of influorescences.

Problems created by excess N include excessive vigour, poor bud fruitfulness, excessive berry

drop, bud necrosis, delayed crop maturity, and increased levels of stem necrosis disorder,

bunch rot and leafhopper activity (Peacock et a/., 1991). Luxuriant growth and high N

fertilization are not compatible with quality production. On the other hand, N deficiencies lead

to a reduction in crop size as well as a low N concentration in the must, which can cause

problems such as lagging fermentation in the cellar and consequently inferior wine quality

(Saayman, 1981). It is therefore necessary to adjust the application rates of N fertilizers in order

to ensure optimum efficiency of plant uptake, while at the same time reducing losses of N to the

environment.

Nitrogen uptake of the grapevine from the soil begins after budburst. The internal cycle of N in

the vine makes the plant independent of the N supply of the soil solution, especially in the

period from budburst to unfolding leaves. It sees therefore necessary to omit all actions (tillage,

fertilization), which mobilize soluble nitrogen in the soil before budburst, to prevent leaching

losses. Table 1.2 describes the seasonal uptake of N in vines under South African conditions.

The peak annual demand for nitrogen in grapevines occurs from budbreak through the first

stage of berry development (Peacock et al., 1991). N uptake is greatest from budbreak to

veraison and from harvest to leaf-fall. N applications should be timed to maximize levels of

nitrate to the root zone during these periods. Vines depend heavily on stored forms of N to

support spring growth and post harvest fertilization is more effective in increasing stored N.

Fertilization may not be necessary when high levels of nitrate are present in the irrigation water

or when legume cover crops are grown.

Table 1.2: Different phases in the N-nutrition cycle of the grapevine (taken from

Conradie, 1991).

Phase in I

nutrition cycle

II

III

IV

Growth Stage

Budbreak to end of bloom

1. End of bloom to end of

rapid shoot growth

2. End of rapid shoot

growth to veraison

Veraison to harvest

Harvest to start of leaf-fall

Period of berry

developmenta

II

III

Specific

characteristics for N-

uptake

New growth partially

dependent on reserve N

accumulated during

previous season(s)

Active root uptake.

Amount of "new" N

sufficient to supply

demand of new growth.

Leaves and bunches

both important sinks for

N.

Root uptake may stop.

Bunches main sink for

N. Redistribution from

roots, shoots and leaves

to bunches.

Active root uptake.

Redistribution from

shoots and leaves to

permanent structure.

According to Winkler et a/., (1974).

1.4 The role of cover cropping systems in sustainable agriculture

Cover crops play a multitude of roles in modern crop management systems (Doran and Smith,

1991):

1. They provide cover and protect the soil from wind and water erosion.

2. They serve as sinks for plant nutrients that might otherwise be lost by volatilization or

leaching.

3. They provide weed control through competition and allelopathy.

4. They assist in control of disease and insects by increasing crop diversity.

5. They act as a source of supplemental N (legumes) and slow release nutrients.

Legume cover crops fix N symbiotically from the atmosphere and supply it to the succeeding

cover crop. Nonlegume cover crops, in contrast, are effective in reducing nitrate leaching from

the soil during winter months by absorbing large amounts of available N through their extensive

root systems. Cover crops, therefore, affect the cycling of N in the root zone and the N status of

the grapevine (Tan and Crabtree, 1990, Van Huyssteen et al., 1984). Mineralized N from

incorporated cover crop residues can then become available to the grapevine during the

summer months N and contribute to more efficient nitrogen cycling. Shipley and co-workers

(1992) reported a residual level of 94 kg N ha"1 in the soil profile (0-80cm) in autumn following

corn harvest in a cover cropping system. By April of the following spring, cereal rye had

recovered 45% of the residual fertilizer N while crimson clover recovered only 8% of the

fertilizer N, indicating the ability of rye to capture greater amounts of N compared to crimson

clover. Kuo et al. (1997) reported that rye and annual rye grass were ineffective in enhancing

soil inorganic N levels. However, they were more effective than the N-fixers hairy vetch,

Austrian winter pea and canoia in increasing soil organic N accumulation because of a higher

biomass potential and a larger input of biomass C. The two principal elements regulating soil

biological activity, and hence nutrient cycling, are carbon and nitrogen. Winter annual cover

crops can improve the soil C and carbohydrate concentrations due to the magnitude of the C

inputs from the respective cover crops. Large C:N ratios result in N mobilization and reduced N

availability to the succeeding crop. The decomposition rate at which cover crop residues are

incorporated into the soil affects soil organic N and C concentrations. Ladd et al. (1981)

showed that the value of legumes as a source of N was due not so much to their capacity to

provide relatively large amounts of immediately available N, but their ability to maintain or

increase soil organic N levels in the long-term, thereby insuring adequate supply of N by slow

decomposition of the stable organic N.

Cover crops can also provide a vegetative cover in erosion-prone areas in winter and improve

physical, chemical and biological properties of the soil (Hargrove, 1986). Louw and Bennie

(1991) showed that the presence of a straw mulch or cover crop was an effective way of

preventing crust formation runoff and erosion from vineyard soils. Cover crops are effective in

reducing weeds in strawberry, decreasing insect populations in orchards and vineyards and

curtailing pathogen severity in lettuce. Cover crop growth and C and N contributed by them

depends on species, length of the growing season, climate and soil conditions. The choice of a

10

cover crop depends on whether the primary purpose is to supply N to the succeeding crop and

reduce fertilizer application, or to improve soil properties.

In a vineyard cover crop trial in Oudtshoorn it was observed that a mulched vineyard soil

conserved water in comparison with a bare soil due to limited evaporation losses (Van

Huyssteen et a/., 1984). Water extraction by weeds on the bare soil also contributed to water

loss. However, cover crops allowed to complete their growth cycle after budburst of the

vineyard wasted so much water - 75mm in 36 days - that this practice could not be

recommended in a dry area with an uncertain water supply. According to the results of this

experiment subtract 100kg N/ha when vetch is used as a cover crop and apply 50kg N/ha

additionally when Wimmera is being sown.

Water conservation in cover crop soils occur due to the following reasons:

1. Reduced evaporation due to a mulch effect

2. Increased infiltration and retention of precipitation

3. Loss of water by transpiration from the cover crop canopy

4. Altered water usage by the summer crop if its growth is affected

5. Mulch serves as barrier to water vapour movement

6. Reduction in solar radiation from shading

7. Insulation of the soil surface

Soil surface structure usually degrades in early spring due to prolonged wet conditions and a

lack of surface cover during winter. Caron et al. (1992) found that cover crops reduce seasonal

variation in aggregate stability when compared to bare fallow. Hermawan and Bomke (1997)

supported this conclusion and showed that improved aggregate stability with cover crops was

related to increasing organic carbon in the soil. Winter cover crops such as annual ryegrass,

may protect aggregate breakdown and surface soil structure during winter rainfall and resulted

in better structure after spring tillage operations when compared to the bare soil. When grown

over winter months, most studies, however, have only found significant benefits after several

years of cover crops (Kuo and Sainju, 1994).

1.5 Use of legumes as cover crops

Legumes normally produce organic matter higher in N content than grasses. As a result of this,

organic matter originating from legumes usually decomposes at a faster rate than that from

grasses. The greatest benefit from green manuring of a legume cover crop is obtained if the

crop is turned under at the stage of 50% flowering or before it produces seed. Grasses usually

produce more biomass than legumes under adequate fertility levels. Since grass herbage is

usually lower in N content than legumes, it decomposes more slowly. Crops planted soon after

large amounts of grass herbage have been incorporated into the soil may require additional

nitrogen fertilizer because some nitrogen will be required by soil organisms that are

decomposing the grass cover crop.

Legumes accumulate N from different sources: seed-N, soil-N, fertilizer-N and atmospheric-N.15N natural abundance methodology has been used to estimate N2 fixation by legumes. The N

released from roots is the main route of N-transfer; however, the presence or inoculation of

mycorrhizas (particularly VAM) may stimulate the transfer of N, probably through connection of

mycorrhiza hyphae between component crops (Frey and Schuepp, 1992).

The natural abundance of the stable isotope, 15N, in soils is commonly found to be greater than

in the N2 of the atmosphere (Cheng et al., 1964; Delwiche and Steyn, 1970; Shearer et a/.,

1974) and may increase with depth in the soil profile (Steele et al., 1981). In contrast, the

natural abundance of 15N in plants, which utilize biologically fixed atmospheric N2, is

significantly lower than in the N of the soil in which they grow. This difference has been used

for the detection of N2-fixing plants (Virginia et al., 1981) and for estimating the proportions of

plant N obtained from soil or atmosphere (Amarger et al., 1979).

Under conditions of near-optimal nitrogen supply, the allocation of carbon and nitrogen is

directed towards the shoot, whereas under conditions of extremely low nitrogen supply, it is

towards the root (Van der Werf et al 1993c). In general, fast-growing species produce more

biomass in the short run than slow-growing species, irrespective of the nitrogen supply

(Berendseefa/., 1992).

1.6 Management of cover cropping systems

The capacity of winter cover crops to serve as an effective source of nutrients depends on

climate, growth stage and quality of the cover crop, soil and cropping characteristics and tillage

management practices (Doran and Smith, 1991). With legume cover crops, the potential for

significant N contributions may not be realized unless the legume N becomes available during

the periods of high demand by the subsequent crop. Cover crop management alternatives

aimed at synchronizing N availability to crop demand are important to foster adequate N-use

efficiency relationships in cropping systems. If it is assumed that the C:N ratio of a cover crop

can govern decomposition and N release, then management techniques that modify this

characteristic may provide the flexibility to manage cover crop derived N in a more efficient

12

manner. If mineralization of organic N does not correspond to plant N growth requirements,

yields will be depressed in the absence of other available N sources (Burket, 1997; Griffin and

Hesterman, 1991).

In the vineyards of the coastal region of the Western Cape, clean cultivation is generally

maintained, consisting of the following: In autumn a cover crop such as oats (Avena spp.),

barley (Hordeum spp.) or Rye (Lolium spp.) is sown in alternate rows. During June/July, a

moderately deep furrow or trench (150-300mm) is ploughed using a trenching plough in the

rows without a cover crop. Vine prunings and manure are ploughed under, and at the same

time the cover crop is disced in. The strip remaining underneath the vines is then ploughed,

while the surviving weeds are hoed by hand towards the middle of the row. Thus a clean hard

soil surface underneath and between the vines is obtained. The inter-row spaces are disced

two or four times during the growing season to keep the vineyard free from weeds. Depending

on the depth of the trenches, this system of cultivation is referred to as the deep (250-300mm)

or shallow (150-200mm) trench furrow system. In cases where a trenchfurrow is not ploughed,

all inter row spaces are sown to a cover crop which is disced into the soil before budburst after

which one to three additional cultivations may be necessary.

Due to the danger of leaching losses, N fertilization is usually applied in three installments -

during budding, after flowering and post-harvest (Saayman, 1982). If a legume cover crop is

grown during winter, about 25kg N/ha (the equivalent of 90kg LAN/ha) is fixed and this quantity

can be omitted from the spring fertilization. A non-leguminous cover crop, on the other hand,

requires about 30 kg N/ha (about 100 kg LAN/ha), half of which should be applied as

topdressing. Cover crops provide cover and protect the soil from wind and water erosion (Doran

and Smith, 1991). Cover cropping is therefore recommended on poor soils and in regions with

insufficient rainfall (Meyer and Cuinier, 1997).

During the growing season of a vineyard from the beginning of September to about the end of

March - growing weeds and/or cover crops compete with the vine for moisture and nutrients

(Fourie, 1988). Van Huyssteen and Weber (1980) showed that a vineyard under dryland

conditions must be free of weed competition during the growing season of the vines. These

weeds must be eliminated as competitors before budburst. Practices in which weeds and/or

cover crops are only controlled, but not killed, will result in a serious decrease in vegetative

growth and yield. Although winter weeds and cover crops in year of normal rainfall are not

regarded as deleterious to the dormant vineyard, the weeds must be destroyed before the

vineyard buds so as to eliminate harmful competition. Weeds that grow in summer are

13

potentially the most harmful and could compete with the vineyard for nitrogen to such an extent

that fermentation problems could arise.

The following management practices are commonly employed in weed control programs:

• Mechanical control in the working rows and chemical control on the ridges

• Full surface chemical control

• Biological control by means of a cover crop that is destroyed by spraying a post-emergence

herbicide just before budding.

Grbic and Zorsic (1963) and also Nedelthcev (1965), compared yield and berry quality of vines

from differently cultivated soils. They found that when using herbicides, summer cultivation of

the vineyards can be reduced to absolute minimum or even completely omitted, with no

adverse effects on quality and yield. Quidet and co-workers (1967) could also not find any

differences in yield between mechanically clean cultivated and herbicide treated vineyards on

different soil types in France. These examples indicate that mechanically clean cultivation may

be redundant.

The N fixed by the nodules of legume cover crops is transported to the stems and leaves of the

growing legume to form proteins, chlorophyll and other N-conaining compounds. The fixed

nitrogen only becomes available to the next crop until the legume decomposes. Annual

legumes that are allowed to flower and mature will transport a large portion of their biomass

nitrogen into the seeds or beans. Once the legume has stopped actively growing, the N-fixing

symbiosis ends. In annual legumes this occurs at the time of flowering; no additional N gain will

occur after that point. Unless it is intended for a legume to reseed itself, it is a general practice

to kill the legume cover crop in the early- to mid-blossom stage. At this point, the maximum N

would have been obtained and residue composition can commence.

The management and climatic events following the death of the legume greatly affects the

amount and timing of N release from the legume to the soil. Bacteria immediately decompose

the cover crop. The result can be a very rapid and large release of nitrate into the soil within a

week of the green manure's demise. This N release is more rapid when covers are ploughed

down than when left on the surface. As much as 140 Ib. N/A has been measured 7-10 days

after plough down of hairy vetch (Sarrantonio and Scott, 1988). Green manures that are less

protein rich (N-rich) will take longer to release N. Those that are old and fibrous or woody are

generally left for hard-working but somewhat sluggish fungi to convert slowly to humus over the

years, gradually releasing small amounts of nutrients. Temperature, soil moisture, soil pH and

soil fertility affect microbial decomposers.

14

1.6.1 Effect of tillage on soil N status

In agriculture, the purposes of tillage operations are to prepare seedbeds and rootbeds,

incorporate amendments, control weeds and pests, enhance infiltration, and control erosion

(Schafer and Johnson, 1982). Tillage leads to a rapid decline in soil organic nitrogen (Campbell

and Souster, 1982; Collins et al., 1992) and a decline occurs when a soil is left fallow

(Campbell and Souster, 1982; Elliot, 1986). Van Huyssteen and Weber (1980) showed that in

vineyards, soil parameters such as pore volume distribution, compaction indices, activity of

microorganisms and availability of plant nutrients, were found to be more favourable in

minimum tillage treatments than on conventionally tilled plots. Previous studies have shown

that higher levels of organic C, nitrate N and extractable Ca, Mg and K are observed in the soil

when crop residues remain on the surface (no-till) as compared with situations in which

residues are ploughed under (Lai, 1976). Fields with winter cover crops had higher soil organic

C, total N and exchangeable Ca and Mg than fields without cover crops (Lewis and Hunter,

1940; Wilson et al., 1982). They also found the inclusion of winter legumes and covers resulted

in higher soil pH, organic C, total N and exchangeable cations than those cropped to winter

grasses.

Tillage speeds up organic matter decomposition by exposing more surface area to oxygen,

warming and drying the soil and breaking residue into smaller pieces with more surfaces that

can be attacked by decomposers. The resulting loss of organic matter causes the breakdown

of soil aggregates and the poor structure often seen in overtilled soil. Frequent tillage can

result in the deterioration of the soil structure. However, when conducted in early spring, tillage

could increase surface evaporation (Slowifiska-Jurkiewicz, 1994). This reduces the water

content and improves soil aggregation. (Perfect et al., 1990). The effects of tillage on soil

aggregation may also be controlled by the presence of cover crops.

For many decades shoot prunings and winter cover crops were buried in vineyards as a

cultivation practice. In this process the soils have been regularly tilled in order to provide a

bare surface of pulverized soil (Van Huyssteen, 1987). Although meant to create favourable

surface soil conditions, research has showed that under South African conditions excessive

mechanical cultivation can damage pore continuity to the deeper layers. Conservation tillage

and cover crops are important management tools for improving short-term erosion control, and

increasing long-term soil N reserves and N mineralization and enhancing soil productivity

(Ebelhar et al., 1984; Frye et al., 1985; Wood ef al., 1991). However, a long-term study

conducted in southern Brazil (Amado et al., 1998) showed that the use of legumes in corn

15

cropping systems increased N soil reserves, regardless of tillage system. Increased soil N from

legume cover crops induced increase in corn yield. It was evident from this study that

combined use of legume cover crops and no-tillage, by promoting increased soil N and crop N

uptake, is and efficient management practice to promote soil sustainability.

Conventional ploughing and aggressive disking can cause a rapid decomposition of green

manures, which could provide a premature release of N in the cropping season. No-till systems

will have a reduced and more gradual release of N, but some of that N may be vulnerable to

gaseous loss, either by ammonia volatilization or by denitrification. Studies have shown that,

regardless of the tillage system employed, decomposing legumes release a pulse of available

mineral N at two to five weeks after killing of the cover crop in Spring (with herbicide or tillage),

followed by a gradual decline over the growing season. Thus, depending on management, soil

and climatic conditions, N from legume cover crops may not be more efficiently used than N

from legume cover crops may not be more efficiently used than N from fertilizer.

Legume cover crops play an important role in sustainable agricultural practices. Its use can

lead to a potential reduction in N fertilizer costs for small-scale farmers. It is therefore the aim

of this study to determine the most effective cultivation practice for managing the legume cover

crop that can contribute the maximal amount of nitrogen at the most suitable time, i.e. the

release of nitrogen by the cover crop is synchronized with peak periods of nutrient demand by

the vine.

Chapter 3 assesses the effect of cover cropping on the nitrogen nutrition of the vine.

Chapter 4 examines the effect of cover cropping on the soil nitrogen status of the vineyard.

Chapter 5 determines the nitrogen contribution made by legume cover crops as measured by15N natural abundance methodologies.

16

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Schafer RL, Johnson CE. 1982. Changing soil condition - the soil dynamics of tillage. In: Predicting

Tillage Effects on Soil Physical Properties and Processes. ASA Special Publication No. 44,

(P.W. Unger and D.M. Van Doren Jr. eds.),pp. 13-28.

21

Sarrantonio M, Scott TW. 1988. Tillage effects on availability of nitrogen to corn following a winter

green manure crop. Soil Science Society of America Journal 52, 1661-1668.

Shipley PR, Meisinger JJ, Decker AM. 1992. Conserving residual corn fertilizer nitrogen with winter

cover crops. Agronomy Journal 84, 869-876.

Shearer GB, Khol DH, Commoner B. 1974. The precision of determinations of nitrogen-15 in soils,

fertilizers and shelf chemicals. SoU Science 118, 308-316.

Slowifiska-Jurkiewicz A. 1994. Changes in the structure and physical properties of soil during spring

tillage operations. Soil Tillage Research 29, 397-407.

Steele KW, Wilson AT, Saunders WMH. 1981. Nitrogen isotope ratios in surface and sub-surface

horizons of New Zealand improved grassland soils. New Zealand Journal of Agricultural

Research 24, 167-170.

Tan S, Crabtree GD. 1990. Competition between perrennial ryegrass sod and 'Chardonnay" wine

grapes for mineral nutrients. Horticultural Science 25, 533-535.

Van der Werf A, Enserink T, Smit B, Booij R. 1993. Allocation of carbon and nitrogen as a function of

the internal nitrogen status of a plant: modeling allocation under non-steady-state conditions.

Plant and Soil 155/156, 183-186.

Van Huyssteen L. 1987. Benefits of minimum tillage of vineyards,. In Research highlights 1987: Plant

Production (L.L. Lotter ed.). Dept. of Agric & Water Supply, Pretoria, pp. 115-116.

Van Huyssteen L, Weber HW. 1980. The effect of conventional and minimum tillage practices on some

soil properties in a dryland vineyard. South African Journal of Enology and Viticulture 1, 35-45.

Van Huyssteen L, Van Zyl JL, Koen P. 1984. The effect of cover crop management on soil conditions

and weed control in a Colombar vineyard in Oudtshoorn. South African Journal of Enology and

Viticulture 5, 7-17.

Vance CP, Griffith S.M. 1990. The molecular biology of N metabolism. In: Plant Physiology,

Biochemistry and Molecular Biology, ed. D.T. Dennis and D.H. Turpin. Addison-Wesley

Longman, London, pp. 373-388.

22

Virginia RA, Jarell WM, Kohl DH, Shearer G.B. 1981. Symbiotic nitrogen fixation in Prosopis

(Legumiosae)-dominated desert ecosystems. In: Curernt Perspectives in Nitrogen Fixation

(A.H. Gibson and W.E. Newton, Eds)p. 483. Australian Academy of Science Canberra.

Weller F. 1983. Stickstoffumsatz in einigen obstbaulich genutzten Boden Sudwestdeutschlands.

Zeitschrift fuer Pfianzenernaehrung und Bodenkunde 146, 261-270.

Wilson GF, Lai R, Okigbo BN. 1982. Effect of cover crops on soil structure and on yield of subsequent

arable crops grown under strip tillage on an eroded Alfisol. Soil Tillage Research. 2, 233-250.

WinklerAJ, Cook JA, Kliewer WM, Lider LA. 1974. General Viticulture. 710pp. University of California

Press, Berkeley and Los Angeles.

23

Chapter 2

GENERAL MATERIALS AND METHODS

The following techniques were used to achieve the objectives of this study:

(i) Quantification of the contribution of fixed nitrogen by legume cover crops:

• 15N natural abundance of field- and glasshouse-grown legumes and

reference crop

• Growth measurement of legume plant components

(ii) Determination of the effect of cover cropping on the nutritional status of the vine:

• In vivo nitrate reductase assay of leaf blades

• Total amino acid content of vines

• 15N natural abundance of vine leaves

(iii) Monitoring the effect of cereal and legume cover crops on the nitrogen levels of the soil:

• Soil inorganic nitrogen levels

• Total soil nitrogen determination by Kjeldahl method

2.1 Experimental design and fertilizer regimes

The study was conducted on a six-year old Sauvignon blanc vineyard grafted onto Ramsey

rootstock. The trial was situated at the ARC-Nietvoorbij experimental farm near Lutzville. Six

leguminous and cereal cover crops, with a total of fifteen treatments, were replicated three times

in a randomised block design (See Table 2.1). Vines were spaced one and a half metres apart

within rows, and three metres apart between rows. A control in which weeds were mechanically

controlled in the working row was included. Each treatment row consisted of 6-8 vines and was

surrounded by a buffer row intercropped with Secale cereale L. All sample collections were taken

from these 6-8 vines.

2.1.1. Nitrogen fertilizer regimes during 1997-1999

All non-legume treatments, including the control, received a topdressing of N fertilizer (Fig. 2.1).

Vines in all legume treatments received a berm application of fertilizer and soil samples were

always taken in the middle of the cover crop row where no N was applied. In 1997 all treatments

received 42 kg N/ha after harvest and after budbreak (near flowering), except the two grazing

vetch treatments which were mechanically tilled and chemically controlled at budburst including

24

Seradella Emena which was chemically controlled before budburst received 21 kg N/ha after

harvest. In 1998 all treatments received 42 kg N/ha in April and 28 kg N/ha in May.

25

Table 2.1 : Cultivation management on a vineyard-cover crop system in Lutzville

Cover Crops* Cultivation management of cover crops

Secale cereale L.

(Rye)

Chemically controlled before vine budburst

Allowed to die-back

Avena sativa v. Saia

(Oats)

Chemically controlled before vine budburst

Allowed to die-back

Mechanically tilled before vine budburst

Vicia villosa v. dasycarpa

(Grazing vetch)

Mechanically tilled before vine budburst

Chemically controlled before vine budburst

Allowed to die-back

Medicago truncatula v. Parabinga Chemically controlled before vine budburst

(Parabinga medic) Allowed to die-back

Medicago truncatula v. Paraggio

(Paraggio medic)

Chemically controlled before vine budburst

Allowed to die-back

Ornithopus sativa v. Emena

(Seradella Emena)

Chemically controlled before vine budburst

Allowed to die-back

Control - no cover crop Mechanically tilled in row, chemical control under vine

"Common names are indicated in parentheses

26

vine

legume cover crop stand

cereal cover crop stand

fertilizer placement

Fig 2.1 : Diagrammatic representation of fertilizer placement in a cover cropping trial in

Lutzville

2.2 Plant analysis

2.2.1 In vivo Nitrate Reductase Assay

The nitrate reductase assay was conducted monthly during the growing season to assess the

assimilation of biologically fixed and synthetic nitrate by the vine. Approximately six mature vine

leaves from each treatment were collected, placed on ice and analyzed immediately. Each

replicate was sampled separately (due to the diurnal variation of nitrate reductase activity) and on

the same side of the vine. The leaves were chopped and the exact weight of 0.5-1.0g was

recorded. Each treatment was duplicated. The method as described by Hageman and Hucklesby

(1971) was followed and adapted for field conditions as the laboratory was situated 440km from

the trial site and stability of the nitrate reductase enzyme would therefore not be feasible. The

leaves were transferred to a McCartney bottle which contained 10ml of phosphate buffer (0.1 M

KH2PO4/K2HPO4 at pH 7.7, 0.1M KNO3 and 1% v/v isopropanol) and was wrapped in foil in order

to exclude light. The bottle was shaken well for 2-3 minutes and flushed with nitrogen gas for 1

27

minute to displace oxygen in order to prevent deactivation of the enzyme. The mixture was

incubated for 30 minutes at room temperature. One ml of the extract, 2ml of 1% sulphaniiamide

in 1M HCI and 2ml 0.01% NED (napthyl-ethylene-diamine-dihydrochloride) was added to a

cuvette. The absorbance was measured at 540nm on a Spectronic 20 spectrophotometer.

Sodium nitrite concentrations of 0.01, 0.02, 0.04, 0.06 and 0.08 µmol were used as standards.

Activity of the enzyme was expressed as |imol/g fresh weight/hr.

2.2.2 15N Natural abundance

All 15N methods used for field studies depend upon the sources of nitrogen for plant growth, viz.

soil nitrogen, fertilizer nitrogen and atmospheric N2 being of different isotopic composition

(Bergersen and Turner, 1983). The determination of 15N required the use of a mass

spectrometer, which separates ions in a highly evacuated atmosphere on the basis of their

mass/charge ratios and determines their relative proportions. This reaction is done in an

evacuated container and the N2 produced is introduced into the mass spectrometer. Double-

collector instruments, which have high precision, are used when studying variations in natural 15N

abundance.

All plant material prepared for isotope ratio analysis was oven-dried to constant weight at 75°C

and milled to a fine uniform powder. A subsample was weighed (1mg for legume material and

2.5mg for non-legume material) and transferred to tin capsules, whereafter each sample was

analysed on-line in a NA 1500 NC (CHN analyser) connected to a Conflo device MAT 252.

Vines: Vine leaf material was collected from each treatment monthly during the growing season of

the vine to obtain evidence for the transfer of legume-fixed N2 to vines.

Legumes: Nitrogen fixation by legume cover crops was studied by using the natural abundance of15N. Legume cover crop material (roots, shoots and nodules) was sampled annually from the field

during winter to quantify the contribution of legumes to the N status of the vineyard. Four

replicates of the four cover crop species were grown in the glass-house. The plants were

watered with distilled water thrice a week prior to germination. Thereafter the plants were fed

300ml of half-strength Hoagland's N-free nutrient solution thrice a week and received a flushing of

distilled water after every tenth feeding. All potted legumes were harvested prior to flowering and

divided into its various components (i.e. roots, shoots and nodules). All plant parts were oven-

dried, weighed to determine biomass production and ground for 15N determination.

28

Weeds: In order to calculate the P-value it was necessary to identify suitable reference plants and

to determine the &15N-values of glass-house grown legume cover crops. Weeds growing in and

around the vineyard and which were present during cover crop growth were identified and

evaluated for use as a reference plant. Dimorphotheca pluvialis, Senecio arenarius and

Raphanus raphanistrum were the predominant weeds growing with the cover crops. The above-

mentioned weeds were also collected during August 1998. Assumptions inherent in 15N methods

are that: (i) the reference plant lacks the ability to fix N2 and the 15N/14N ratio measured in its

products of growth is the same as plant-available soil mineral N and (ii) the legume and non-N2-

fixing reference plant explore a soil N pool of identical 15N/14N abundance (Peoples and Herridge,

1990)

All 15N data was expressed in terms of parts per thousand (515N or %o):

&15N %o = 1000 x (atom% 15Nsample - atom% 15Nstandard) / atom%

15Nstandard,

where the standard is usually atmospheric N2 (0.3663 atom%). The proportion (P) of plant N

derived from N2fixation was calculated using the expression (Bergersen et al., 1985):

P = (&15 N ref. - &15N Ieg.)/(&15N ref. - B)

in which it is assumed that the 15N composition of the non-fixing reference plant (ref.) integrates

the isotopic composition of plant-available soil N and that the 15N composition of the legume (leg.)

is due to N assimilated from the same soil source plus the proportion derived from atmospheric

N2. The value B is the &15N of the plants grown in glasshouse culture with atmospheric N2 as the

sole source of nitrogen.

2.3 Soil analysis

Soil samples were collected monthly throughout 1997-1999 from the middle of the cover crop row

at each fourth vine at depths 0-300mm, 300-600mm and 600-900mm. All soil samples were air-

dried, ground and sieved prior to analysis.

2.3.1 Inorganic nitrogen

Exchangeable NH4+ and NO3 were extracted from soil by adding 60ml 2N KCI to 10g of soil. The

mixture was shaken for 1 hour and then filtered. The inorganic N content of the KCI extract was

measured on an autoanalyser. The filtrate was analyzed for NH4+ by the indophenol blue

29

reaction. The indophenol blue method of determining NH4+ depends on the fact that the phenolic

compound, salicylate, reacts with NH3 in the presence of an oxidising agent, in this case,

hypochlorite to form a coloured complex under alkaline pH conditions. The addition of sodium

nitroprusside as a catalyst in the reaction between salicylate and NH3 increased the sensitivity of

the method about 10-fold. The maximum sensitivity and accuracy of the indophenol blue method

is attained when spectrometric measurements of the intensity of the coloured complex are made

at 636 nm. NO3 in the extract was reduced to NO2 by passage through a column of copperized

Cd in an NH4CI matrix, and the resulting NO2" was quantified by a modified Griess-llosvay

method.

2.3.2 Total Soil N

The Kjeidahl procedure was employed for the determination of total nitrogen in the soil. Total

Kjeldahl N includes ammonium, amines, and other organic forms which can be converted to

ammonium forms during the digestion. This procedure does not measure all of the nitrates in the

tissue. The method of Bremner and Mulvaney (1982) was adhered to with a few minor

adjustments. The digestion was performed by heating 10g of soil with 30ml of concentrated H2S04

and selenium. Digestion of the sample promoted the oxidation of organic matter to NH4+-N. The

NH4+-N in the digest was determined by collecting the NH3 liberated by distillation of the digest

with 10N NaOH. The distillate was collected in 4% boric acid indicator solution and 0.1 M HCI.

The total N content was expressed in parts per million.

2.4 Statistical Analysis

Statistical analysis was performed by SAS and the differences between means were determined

by LSD at 0.05.

30

REFERENCES

Bergersen FJ, Turner GL. 1983. An evaluation of 15N methods for estimating nitrogen fixation in

a subterranean clover - perennial ryegrass sward. Australian Journal of Agricultural Research

34,391-401.

Bergersen FJ, Turner GL, Gault RR, Chase DL. 1985. The natural abundance of 15N in an

irrigated soybean crop and its use for the calculation of nitrogen fixation. Australian Journal of

Agricultural Research 36,411-423.

Bremner JM, Mulvaney CS. Nitrogen - Total. In: Methods of Soil Analysis, Agro. Monogr. 9, pt.

2, Second Edition (A.L Page, ed.), pp. 595-624. American Society of Agronomy, Madison,

Wisconsin.

Hageman RH, Huclesby DP. 1971. Nitrate reductase from higher plants. Methods in Enzymology

23, 491-503.

Peoples MB, Herridge DF. 1990. Nitrogen fixation by legumes in tropical and subtropical

agriculture. Advances in Agronomy 44, 155-223

31

CHAPTER 3

VINE UPTAKE AND ASSIMILATION OF NITROGEN IN A COVER CROPPING SYSTEM IN

LUTZVILLE

3.1 Introduction

In contrast with annual crops, grapevines have a relatively low nutritional requirement for nitrogen

(Saayman, 1982). This is attributed to:

(i) the small quantities of nitrogen that are naturally taken up by the vine;

(ii) the vine's well developed rooting system that can readily obtain its required nutrients,

even in poor soil;

(iii) a period of about 8 months during which nutrients can be assimilated;

(iv) the fact that approximately 90% of the annual growth is returned to the soil as leaves and

shoots and because 90% of that portion which is removed annually (the crop) consists of

carbon dioxide and water.

Conradie (1980) showed that the aerial growth of the vine requires ca. 3.7kg N for the production

of one ton of fresh grapes, with 1.6kg being in the clusters and 2.1kg in the vegetative growth.

Nitrogen uptake is greatest from budbreak to veraison and from harvest to leaf-fall. Nitrogen

fertilizer applications are therefore usually timed to maximize levels of nitrate in the root zone

during these periods. However, with the escalating cost of fertilizer, there is a need to examine

alternative sources of mineral nutrients for use in vineyards. A safe and beneficial alternative is

the use of legume cover crops.

Legume cover crops fix N symbiotically from the atmosphere and supply it to the succeeding

crop. Nonlegume cover crops, in contrast, are effective in reducing nitrate leaching from the soil

during winter months by absorbing large amounts of available N through their extensive rooting

systems. Cover crops, therefore, affect the cycling of N in the root zone and the N status of the

grapevine (Tan and Crabtree, 1990; Van Huyssteen et a/., 1984). Mineralized N from

incorporated cover crop residues can then become available to the grapevine during the summer

months and contribute to more efficient nitrogen cycling.

The N fixed by the nodules of legume cover crops is transported to the stems and leaves of the

growing legume to form proteins, chlorophyll and other N-containing compounds. The fixed

nitrogen only becomes available to the next crop until the legume decomposes. Annual legumes

that are allowed to flower and mature will transport a large portion of their biomass nitrogen into

32

the seeds or beans. Once the legume has stopped actively growing, the N-fixing symbiosis ends.

In annual legumes this occurs at the time of flowering. Unless it is intended for a legume to

reseed itself, it is a general practice to kill the legume cover crop in the early- to mid-blossom

stage. At this point, the maximum N would have been obtained and residue decomposition can

commence.

The management and climatic events following the death of the legume greatly affects the

amount and timing of N release from the legume to the soil. With legume cover crops, the

potential for significant N contributions may not be realized unless the legume N becomes

available during the periods of high demand by the subsequent crop. Cover crop management

alternatives aimed at synchronizing N availability to crop demand are important to foster

adequate N-use efficiency relationships in cropping systems. If mineralization of organic N does

not correspond to plant N growth requirements, yields will be depressed in the absence of other

available N sources (Burket, 1997; Griffin and Hesterman, 1991).

Although the idea of mulch tillage is common amongst producers and consultants, there are

problems in selecting a suitable species. The aim of this investigation was to evaluate different

legume cover crops for their ability to enhance the N nutrition of the vine on a sandy soil and to

determine whether the release of nitrogen by the cover crop is synchronized with peak periods of

nutrient demand by the vine.

The real criterion of success or failure of a soil cultivation measure is not the change

accomplished in the soil per se but the response of the crop (Van Huyssteen and Weber, 1980).

The 15N natural abundance method has been used in this study to determine the fate of fixed

nitrogen from the soil and its consumption by the vine. There are two stable nitrogen isotopes of

nitrogen, 15N and 14N. Soil nitrogen frequently contains a slightly higher percentage of 15N than

does nitrogen in the atmosphere. The&15N signature of fixed N is distinctly different from that of

soil N (Unkovich, 1996), and can therefore be used to differentiate between soil N and biologically

fixed N in vine plants. In most biochemical reactions, through isotope discrimination, the lighter of

the two isotopes is favoured slightly over the heavier. Thus, during N2 fixation these two

phenomena result in the fixed nitrogen having a slightly lower 15N.

Seasonal fluctuations of nitrate reductase activity in grapevine leaves and roots correspond to

periods of maximal root growth (Hunter and Ruffner, 1997). The determination of leaf nitrate

reductase activity therefore provides valuable information of nitrate assimilation in vines and was

used as an indicato