ws14 4 part 1 rs komplett seitenzahl3 - trifolio-m · protection against the large pine weevil...

BIOLOGICAL CONTROL OF PLANT, MEDICAL AND VETERINARY PESTS

PROCEEDINGS OF THE 14TH WORKSHOP; WETZLAR, GERMANY, NOVEMBER 15TH - 16TH

2004

COMPILED AND PREPARED BY:

R. STRANG AND H. KLEEBERG

PUBLISHED BY:

TRIFOLIO-M GMBH, 35633 LAHNAU, DR.-HANS-WILHELMI-WEG 1

2009

DISTRIBUTION:

TRIFOLIO-M GMBH, DR.-HANS-WILHELMI-WEG 1, D-35633 LAHNAU, GERMANY

ISBN: 3-925 614-29-X

Contents

MODE OF ACTION OF STANDARDISED QUASSIA-EXTRACTS 5MARKUS HOLASCHKE, CHRISTINE KLICHE-SPORY

QUASSINOIDS IN BITTER WOOD EXTRACTS 15HUBERTUS KLEEBERG, CHRISTINE KLICHE-SPORY, ILONA SCHÄFER

TRIFOLIO S-FORTE – A NEW ADDITIVE FOR PLANT PROTECTION PRODUCTS 23WALTHER, N. AND DETZEL, P.

VETERINARY APPLICATIONS OF NEEM (AZADIRACHTA INDICA A JUSS) AND ITSPRODUCTS 27C.M. KETKAR

EFFECT OF NEEM DERIVED PRODUCTS ON GASTROINTESTINAL NEMATODES IN VITROAND IN VIVO IN SHEEP. 31VAN DER ESCH S.A , CARNEVALI F. AND AMICI A.

BIOLOGICAL ACTIVITIES OF NEEM OIL DERIVATIVES IN CULTURED MURINE FIBROBLASTS 41VINCENZO DI ILIO, NICOLETTA PASQUARIELLO, ANDREW S. VAN DER ESCH, MASSIMO CRISTOFARO,GIANFRANCO SCARSELLA, GIANFRANCO RISULEO

DEVELOPMENTAL, REGULATORY AND MARKETING EXPERIENCES WITH THEAZADIRACHTIN- BASED PLANT PROTECTION PRODUCT NEEMAZAL-T/S IN THENETHERLANDS 51VAN DEN ENDE, A.

EXPERIENCES IN HIGH QUALITY MANUFACTURING OF NEEMAZAL 57S. S. PILLAI, S. RADJENDIRANE AND G.R. SRIDHAR

THE POSSIBILITIES OF USING DIFFERENT NEEM PREPARATIONS FOR PEST CONTROL INVEGETABLES IN THE SUDAN 61EL SHAFIE, H.A.F., MUDATHIR, M. (KHARTOUM) & BASEDOW, TH. (GIESSEN)

BIO-EFFICACY OF NEEMAZAL® AND ITS FORMULATION AGAINST SPOTTED LEAF BEETLE,HENOSEPILACHNA VIGINTIOCTOPUNCTATA (FAB) 73R. SUDHAKARAN, LIZA JOZ AND D. SREENIVASA RAO

THE IMPACT OF AZADIRACHTIN A ON SOME FOREST PEST INSECTS 79H. MALINOWSKI, M. DOBROWOLSKI

ASSESSING THE EFFICACY OF NEEM FORMULATIONS AGAINST THE FEEDING ACTIVITY OFTHE LARGE PINE WEEVIL HYLOBIUS ABIETIS 87W BRYAN AND J R M THACKER

SIDE EFFECT OF SOME NEEM PRODUCTS ON NATURAL ENEMIES OF HELICOVERPATRICHOGRAMMA SPP. AND CHRYSOPERLA CARNEA 95NABIL EL-WAKEIL, NAWAL GAAFAR AND STEFAN VIDAL

LONG TERM STEM-APPLICATION IN VITICULTURE AND THE USE OF NEEMAZAL 107DÜKER, A.; KUBIAK, R.

EXPERIENCES WITH DIFFERENT NEEM PREPARATIONS IN THE CONTROL OF PESTATTACK AND AFLATOXIN FORMATION IN THE MEDICAL PLANT CASSIA SENNA (L.) ININDIA 119PHILIP MÜLLER & THIES BASEDOW

EFFECTS OF DIFFERENT PLANT POWDERS AND PLANT OILS ON PESTS OF STORED MAIZEIN ETHIOPIA 127TADESSE, ABRAHAM (ADDIS ABABA) & BASEDOW, THIES (GIESSEN)

A PROMISING BIOTECHNICAL APPROACH TO PEST MANAGEMENT OF DIABROTICAVIRGIFERA VIRGIFERA IN ILLINOINS MAIZE FIELDS UNDER KAIROMONAL SHIELDING WITHTHE NEW MSD TECHNIQUE 141HANS E. HUMMEL, JOHN T. SHAW, DETLEF F. HEIN

THE WESTERN CORN ROOTWORM DIABROTICA VIRGIFERA VIRGIFERA EN ROUTE TOGERMANY 151HANS E. HUMMEL, MARIO BERTOSSA, ALEXANDER WUDTKE, DETLEF F. HEIN, GREGOR UREK, SPELAMODIC, CHRISTIAN ULRICHS

EFFECT OF NEEMAZAL-T/S ON THE VEGETABLE PESTS IN ROMANIA 163MARIA CALIN, STOIAN LUCIAN, EDMUND HUMMEL

RESULTS OF TRIALS WITH NEEMAZAL-T/S IN SAXONIAN CABBAGE PRODUCTION 173KOEHLER, G.

REDUCTION IN THE SURVIVAL AND REPRODUCTION OF POLYPHAGOTARSONEMUS LATUS(BANKS) (ACARI: TARSONEMIDAE) ON CHILLI PEPPER TREATED WITH NEEMAZAL-T/S 177MADELAINE VENZON, MARIA CONSOLAÇÃO ROSADO, VANESSA DA SILVEIRA DUARTE, AMÉRICO IORIOCIOCIOLA JR

THE POTENTIAL OF A NEEM SEED EXTRACT (NEEMAZAL-T/S) FOR THE CONTROL OFCOFFEE LEAF PESTS 179MADELAINE VENZON, MARIA CONSOLAÇÃO ROSADO, MARCOS ANTÔNIO MATIELLO FADINI, AMÉRICOIORIO CIOCIOLA JR, ANGELO PALLINI

LETHAL AND SUBLETHAL EFFECTS OF NEEMAZAL ON MYZUS PERSICAE (HEMIPTERA:APHIDIDAE) AND ON ITS PREDATOR ERIOPIS CONNEXA (COLEOPTERA: COCCINELLIDAE) 181MADELAINE VENZON, MARIA CONSOLAÇÃO ROSADO, AMANDA FIALHO, DENISE ELIANE EUZÉBIO,AMÉRICO IORIO CIOCIOLA JR

EFFECT OF AZADIRACHTIN APPLIED SYSTEMICALLY THROUGH ROOTS OF PLANTS ONTHE GREENHOUSE WHITEFLY, TRIALEURODES VAPORARIORUM (WESTWOOD) 183ROMAN PAVELA

INSECTICIDAL ACTIVITY OF CERTAIN MEDICINAL PLANTS 195ROMAN PAVELA

EXPERIENCE OF NEEMAZAL-T/S AGAINST CABBAGE PESTS IN BELARUS IN 2004 199PRISHCHEPA, I.A, KOLYADKO, N.N., SHINKORENKO, E.G.

IMPACT OF SOME BIOPESTICIDES ON THE FEEDING ACTIVITY OF THE LARGE PINE WEEVIL(HYLOBIUS ABIETIS L.) (COLEOPTERA: CURCULIONIDAE) 203IVAR SIBUL AND ANGELA PLOOMI

PAPERS SUBMITTED AFTER THE 14TH WORKSHOP AND ACCEPTED FOR INCLUSION INTOTHE PROCEEDINGS:

INTEGRATED CONTROL OF BEMISIA TABACI IN EUPHORBIA PULCHERRIMA 213ELLEN RICHTER

EFFECTIVENESS OF NEEMAZAL-T/S APPLICATION AGAINST POTATO PESTS IN BELARUSIN 2004 219M.I. ZHUKOVA, CAND. OF AGR SCI., G.M. SEREDA, CAND. OF AGR.SCI

TREATMENT OF CONIFEROUS SEEDLINGS WITH NEEMAZAL–T/S: A POSSIBLE WAY OFPROTECTION AGAINST THE LARGE PINE WEEVIL HYLOBIUS ABIETIS (L.) 227NICOLAI OLENICI AND VALENTINA OLENICI

THE POTENTIAL OF EXTRACTS FROM MARINE ALGAE IN THE CONTROL OF BLATTELLAGERMANICA L., BLATTA ORIENTALIS L., MUSCA DOMESTICA L. AND AEDES AEGYPTI L. 239AL. VLADIMIRESCU, GABRIELA NICOLESCU, SANDA CHICIOROAGA, ANA ROSU

EFFECTIFENESS OF AZADIRACHTIN IN CONTROLLING THE HORSE-CHESTNUT LEAFMINER - CAMERARIA OHRIDELLA DESCHKA & DIMIĆ (LEP., GRACILLARIDAE ). 243GABRIEL ŁABANOWSKI, GRAŻYNA SOIKA & JUSZ ŚWIĘTOSŁAWSKI

EXPERIMENTS ON THE USE OF NEEM (A. INDICA)-DERIVED PRODUCTS FOR CONTROL OFTHE BLACK CHERRY APHID, MYZUS CERASI FAB. (HOM., APHIDIDAE) IN A CHERRYORCHARD IN BULGARIA 249RADEVA, K., NIKOLOV, P.

THE POTENTIAL OF NEEMAZAL BAITS IN THE CONTROL OF BLATTELLA GERMANICA L.AND MUSCA DOMESTICA L. 253GABRIELA NICOLESCU, AL. VLADIMIRESCU, ANA ROSU, VALERIA CIULACU-PURCAREA, SANDACHICIOROAGA

PRELEMINARY RESULTS OF USING OF NEEMAZAL-T/S AGAINST LARVAE OF THEBUFF-TIP MOTH (PHALERA BUCEPHALA L. LEPIDOPTERA, NOTODONTIDAE) IN THELABORATORY 257GNINENKO Y.

EXPERIMENTS ON THE USE OF THE MELIACEOUS PLANT PRODUCTS, NEEMAZAL-T/SAND NEEMPRO TEX, AGAINST THE EUROPEAN SPRUCE BARK BEETLE, IPSTYPOGRAPHUS (COL., SCOLYTIDAE) 261KREUTZ, J.; ZIMMERMANN, G.; VAUPEL, O.

EXPERIENCE WITH MODE OF ACTION OF NEEMAZAL-T/S AND TRIFOLIO S-FORTEFORMULATIONS AGAINST THE SPIDER MITE (TETRANYCHUS ATLANTICUS MCGREGOR)UNDER LABORATORY CONDITIONS 263S. YA. POPOV, E. HUMMEL

THE POTENTIAL OF USING BIOAGENTS AGAINST CHERRY FRUIT FLY AS A COMPONENTOF CHERRY AND CRAB-СHERRY PROTECTION SYSTEMS 273VASILIEVA, L. A.

5

MODE OF ACTION OF STANDARDISED QUASSIA-EXTRACTS

MARKUS HOLASCHKE1, CHRISTINE KLICHE-SPORY2

1INSTITUTE OF PHYTOPATHOLOGY AND APPLIED ZOOLOGY, JUSTUS-LIEBIG-UNIVERSITY GIEßEN, ALTERSTEINBACHER WEG 44, 35394 GIEßEN, GERMANY [email protected]

2TRIFOLIO-M GMBH, SONNENSTR. 22, 35633 LAHNAU, GERMANY [email protected]

Abstract

Here results of a newly developed standardised extract from the tropical tree Quassiaamara L. ex Blom are shown. The extract showed high and fast activity against severalaphid species belonging to the family of the Aphididae if taken up by the root system ofthe host plant. At a concentration of 10mg quassin/l the mortality rates after 24 hoursvaried from 48,6% for Aphis fabae to 96.9% for Sitobion avenae. The mortality forRhoplaosiphum padi and Brevicoryne brassicae were 60,6% and 91,2% respectively.Even at a concentration of only 1mg Quassin/l a mortality of 78,1% for Sitobion avenaecould be shown. Only Myzus persicae was more resistant and mortality reached only18.8% 12 days after treatment.

A promising effect of the Quassia-extract was determined in a field trial in oats. Wecould show a promising effect on Sitobion avenae which was also highly sensitiveagainst the extract in greenhouse trials. Results of the treatment were 50,8% mortalityat 10 g quassin/400 l and ha and 76.0% for 20 g Quassin respectively.

In addition on several crops ranging from cereals like wheat and oats to vegetables(tomato, cucumber, cabbage) tests for phytotoxicity were conducted. No signs of a toxicreaction (chlorosis or leaf deformation) or a significant decrease in freshweight weredetermined.

Introduction

The availability of environmental- and human health-friendly insecticides is still notadequate. One alternative for searching new active substances with insecticidalproperties is to test so-called botanicals, mainly plant extracts. In this work the efficacyof quassia-extracts against several aphid species was determined in greenhouse testsas well as in a field trial. Furthermore, tests to evaluate possible phytotoxic effects of theextract were carried out on different crop cultures.Extracts from the wood of the tree Quassia amara L. ex Blom have been used long andregularly before the development of synthetic insecticides because of their insecticidalcompounds. According to MCINDOO and SIEVERS (1917) BRANDE (1825) first mentionedBiological Control of Plant, Medical and Veterinary PestsR. Strang & H. Kleeberg (eds.)Copyright 2009 by Trifolio-M GmbH

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the insecticidal properties of Quassia amara. Quassia was introduced commercially intoEurope about the middle of the 18th century (FLÜCKINGER and HANBURY, 1879).ORMEROD reports in 1884 that the hop growers in England found quassia extracteffective against the hop aphid. Today, quassia extracts are mainly used in organicfarmed apple orchards to control the apple saw fly (Hoplocampa testudinea) (GRAF etal. 2002).CLARK (1937a and b, 1938) identified the bitter principles quassin, neoquassin andpicrasmin as insecticidal compounds of quassiawood. The main active compound isquassin. In average there is a content of 0,12% (0,10%-0,14%) of quassin in the drymatter of wood from Q. amara (EVANS and RAJ, 1991).

Materials and Methods

For testing the quassia extract in the greenhouse trials both for insecticidal tests as wellas for phytotoxic evaluation the chosen crop cultures are listed in the Table 1. The soilsubstrate was „Fruhstorfer Erde Type T“ which is fully fertilised. Before transferring theplants in pots they were grown in soil minimally fertilised („Fruhstorfer Erde Type P“).For testing the root-systemic uptake of the extract single plants were used in pots with adiameter of 10 cm. On each plant five aphids of L2-stage were transfered the day beforetesting and then checked daily.

Tested aphid species were as follows:Aphis fabae (black bean aphid)Brevicoryne brassicae (cabbage aphid)Myzus persicae (peach-potato aphid)Rhoplaosiphum padi (bird cherry aphid)Sitobion avenae (grain aphid)

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Table 1: Plant varieties used in experiments

The design of the field trial was completely randomised. The size of a single square was6 m x 6 m (36 m2). Each of the treatments including the untreated control werereplicated n=4 times. The measurement of the infestation was done by four individualcountings, which were separated at least by a distance of 0,50 m. Each individualcounting consisted of four single countings. A single counting consisted of one ear. Thefirst treatment was carried out on the 02/07/2004. The day before the averageinfestation was 34.03 aphids (S. aveane) per ear. Immediately after the spraying aheavy rain shower occurred. Because of that the treatment was repeated at day 4.Tested treatments were 10 g and 20 g quassin/ha and a untreated control. Tween 20was added with 0,05% as wettener to the quassin treatments. The water amount was400 l/ha. The variety was, like in the greenhouse tests, „Jumbo“. The amount ofquassin, which is contributed as main active substance, in the extract is 7,1 g/kg.

Results

The figures 1-7 show the results of root-systemic activity of quassia extract againstvarious aphid species. In the figures 1-7 one can see that all tested aphid speciesshowed a dose-dependant reaction after treatment with quassia-extract. B. brassicaeand S. avenae reacted very sensitive and efficacy reached more than 95% 24 hrs aftertreatment. With the lowest concentration used, 1 mg quassin l-1 the effect on these twospecies and A. fabae was more than 90% 72 hrs after the treatment. Concerning R.padi the highest efficacy was found for 10 mg quassin l-1 72 hrs after the treatment with 96.6%, the lowest effect was caused by using 1 mg quassin l-1 24 hrs after thetreatment with 27.3%. M. persicae reacted only very slightly and a long period after the

Common name Scientific name Variety

Oats Avena sativa Jumbo

Wheat Triticum aestivum Ritmo

Rapeseed Brassica napus ssp. napus Express

Tomato Lycopersicon esculentum Rheinlands Ruhm

Cabbage Brassica olera convar. acephala var.medullosa

Grüner Ring

Sweet pepper Capsicum annuum var. grossum Yolo Wonder B

Beans Vicia faba ssp. minor Scirocco

Cucumber Cucumis sativus Chinesische Schlange

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treatment. The highest efficacy was 34.4% 12 days after the treatment using 20 mgquassin l-1.

Figure 1: Root-systemic activity of quassia-extract against cabbage aphid (B. brassicae)

Figure 2: Root-systemic activity of quassia-extract against bird cherry aphid (R. padi)

0

20

40

60

80

100

1 2 3days after application

Effic

acy

in %

(H

ende

rson

-Tilt

on)

1 mg quassin/l 5 mg quassin/l 20 mg quassin/l

0

20

40

60

80

100



9

Figure 3: Root-systemic activity of quassia-extract against bean aphid (A. fabae)

Figure 4: Root-systemic activity of quassia-extract against grain aphid (S. avenae)

0

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40

60

80

100


Effic

acy

in %

(H

ende

rson

-Tilt

on)


0

20

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60

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100

1 2

days after application1 mg quassin/l 5 mg quassin/l 20 mg quassin/l

Effi

cacy

in %

(H

ende

rson

-Tilt

on)

10

Figure 5: Root-systemic activity of quassia-extract against peach-potato aphid (M. persicae)

In Fig. 6 and 7 the efficacy is shown when aphids were transfered one week after thetreatment. S. avenae still shows a high mortality reaching 89,3% for 5 mg quassin l-1

whereas R. padi is not effected very successful. 10 mg quassin l-1 causes 54,3%mortality.

Figure 6: Root-systemic activity of quassia-extract against grain aphid (S. avenae) one week afterapplication

0

10

20

30

40

6 9 12

days after application

Effic

acy

in %

(H

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7 8 9


Effic

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Figure 7: Root-systemic activity of quassia-extract against bird cherry aphid (R. padi) one week afterapplication

Field trial

In the field trial where S. avenae was the tested pest , calculation was done for only one(the 2nd) or both of the treatments. In Tables 2 and 3 the results are shown. For bothcalculations it is clear that there are only slight differences in the effectiveness of thetreatment/s. The variant with 10 g quassin. ha-1 shows almost no differences with anaverage efficacy of about 50% whereas the use of 20 g quassin ha-1 results in anefficacy of 84,1% at day 6 calculating 2 treatments respectively 77,5% calculating onlythe 2nd treatment. Figure 8 and Table 2 and 3 show the results of the field trial with S.avenae in oats.

0

20

40

60

80

7 8 9


Effic

acy

in %

(H

ende

rson

-Tilt

on)


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Figure 8: Development of the population of S. avenae after treatment with quassia-extract

Table 2: Efficacy in % (Henderson-Tilton) after 2 treatments

Table 3: Efficacy in % (Henderson-Tilton) after 2nd treatment

2nd treat.1st treat.

0

10

20

30

40

0 2 4 5 6 8days

Nr.

of S

. a

vean

e per

ea

control10g quassin/ha20g quassin/ha

day 5 day 6 day 8

10g quassin/ha 47,6 53,7 50,6

20g quassin/ha 70,7 84,1 83,1

day 5 day 6 day 8

10g quassin/ha 47,8 53,9 50,8

20g quassin/ha 58,3 77,5 76,0

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Table 4 shows the result of freshweight

Table 4: Freshweight [g] 14 days after spraying treatment with quassia-extract. Different letters in rowsindicate different significance levels (ANOVA, post-hoc Tukey)

Discussion

The overall results show that different aphid species show different reactions after atreatment with a quassia-extract. From a very sensitive reaction that S. avenae hasshown to an almost resistant reaction of M. persicae. Already Boucart (1913) describedgood effects of Q. amara extracts against cereal aphids. Şengonca and Brüggen (1991)received with a crude extract in laboratory trials an efficacy of more than 95% against S.avenae and R.padi. The method of Stoll (1987) was used producing the extract. Theresults show clearly that the active substances are transported via the roots into theplant and the phloem. Here it reaches within 24 hours the target aphids. Using only 1mg quassin.l-1 was an effective dosage against S. avenae and B. brassicae. Furthermore it was possible to treat the plants successfully against S. avenae even when theaphids were transfered to their host plants one week after the treatment. Only M.persicae could not be treated successfully. McIndoo and Sievers (1917) quote that theydid not find quassia active against Myzus persicae on eggplant. similar results werereported by Roark (1947) who found good efficacy against aphids in general exceptMyzus persicae. The results of the field trial shows that a sensitive species can also betreated by spraying. Şengonca and Brüggen (1991) found in trials with quassia-extractthat both S. avenae and R. padi could be successful treated. Finally a large variety ofcrop plants did not show any signs of phytotoxicity when sprayed with quassia-extract.

n control(100%)

16 mg quassin l-1

32 mg quassin l-1

wheat 20 6,63 a +37,9% b +38,7% b

oats 18 11,99 -3,1% -4,6%

rapeseed 20 35,93 ab +5,4% b -8,9% a

cabbage 20 26,01 -0,6% +8,2%

tomato 20 30,73 -1,4% +0,9%

cucumber 20 67,19 +9,4% +11,8%

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LiteratureBOUCART, E. (1913). Insecticides, fungicides, and weedkillers. London.

BRANDE, W. T. (1825). A Manual of Pharmacy. Quassia Lignum, 145-146. London.

CLARK, E. P. (1937a). Quassin I. The Preparation and Purification of Quassin andNeoquassin, with Information Concerning their Molecular Formulars. J. AmericanChem. Soc. 59, 927-931.

CLARK, E. P. (1937b). Quassin II. Neoquassin. J. American Chem. Soc. 59, 2511-2514.

CLARK, E. P. (1938). Quassin III. Picrasmin. J. American Chem. Soc. 60, 1146-1148.

EVANS, D. A. and RAJ, R. K. (1991). Larvicidal efficacy of Quassin against Culexquinquefasciatus. Indian J. Med. Res. 93, 324-327.

FLÜCKINGER F. A. and HANBURY, D. (1879). Pharmacographia. 2nd Edition. SimarubaeLignum Quassiae, 152-153. London.

GRAF, B.; HÖPLI, H. U. and HÖHN, H. (2002). The apple sawfly, Hoplocampa testudinea:egg development and forecasting of egg hatch. Ent. Exp. et Appl. 105, 55-60.

HOLLRUNG, M. (1898). Handuch der chemischen Mittel gegen Pflanzenkrankheiten.Verlag Paul Parey, Berlin; Hamburg.

MCINDOO, N. E. and SIEVERS, A. F. (1917). Quassia extract as a contact insecticide. J.agr. Res. 10, 497-531.

ORMEROD, E. A. (1884). Hop aphis, and Damson-Hop aphis (Phorodon) humuli,Schrank; and Aphis (Phorodon) Humuli, var. Mahaleb, Fonsc. Rpt. Observ. Injur.Insects 8, 43-56.

ŞENGONCA, ç. BRÜGGEN, K.-U. (1991). Untersuchungen über die Wirkung wäßrigerExtrakte aus Quassia amara (L.) auf Getreideblattläuse. J. Appl. Ent. 112,211-215.

STOLL, G. (1987). Naturgemäßer Pflanzenschutz mit hofeigenen Ressourcen in denTropen und Subtropen. Verlag J. Margraf, Aichtal.

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1 see for example: F.A.Flückinger. Lehrbuch der Pharmakognosie des Pflanzenreiches. Naturgeschichteder wichtigeren Arzneistoffe vegetabilischen Ursprunges. Berlin, London, Paris: Ed. Rudolf Gaertner,Williams & Norgate, Haar & Steinert, 1867, pp. 319-326.2 S.K. Chen, B.Y.Chen, in „Chinese Flora“ (‚Zhongguo Zhiwu Zhi‘), Ed.S.K.Chen, Science Press, Beijing,1997, Vol. 43(3), p 7-10.3 A. Bartels. Picrasma, das Bitterholz. Gartenpraxis Nr. 3/2002.

QUASSINOIDS IN BITTER WOOD EXTRACTS

HUBERTUS KLEEBERG, CHRISTINE KLICHE-SPORY, ILONA SCHÄFER

TRIFOLIO-M GMBH, SONNENSTR. 22, 35633 LAHNAU, GERMANY

Abstract:

Extracts from species of "Bitter Wood" (Quassia) (family: Simaroubaceae) are used inorganic farming against aphids and other pests. Main ingredients with insecticidalactivity in different species are quassin and neoquassin. The analytical determinationand standardisation of extracts focuses on these two substances. Further ingredientsare also subject to determination. The characteristics of quassia from different originhave been analysed with respect to its ingredients.

Introduction and Aim of our Studies

The wood species Quassia amara and Picrasma excelsa belong to the widelydistributed family of Simaroubaceae. The first occurs in the tropics of South and MiddleAmerica, the second one is a species from the Caribbean Islands. For both the mostlyused trivial name is bitter wood or just „Quassia“.Quassia amara has a height of 2-6 m,and grows in semi-shade. The quassinoids occur in the whole plant. Picrasma excelsais a tree that may reach 20 to 30 m in height. Descriptions of these plants can be foundquite early in the scientific European literature1.

Picrasma quassioides has its origin in China2. This species may be cultivated asneophyte in Central Europe as well, but is quite rare3.

Quassia caused our attention since a few years, as extracts may be used in organicfarming according to European and German regulations. Its main use is against theapple and plum saw fly, but also against different aphids. As the current regulatorystatus is supposed to change in the future, we are working on the possibility to developa plant protection agent on basis of quassia.

Biological Control of Plant, Medical and Veterinary PestsR. Strang & H. Kleeberg (eds.)Copyright 2009 by Trifolio-M GmbH

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1 F.E. Dayan, S.B. Watson, J.C.G. Galindo, A. Hernández, J. Dou, J.D.McChesney, S.O.Duke:Phytotoxicity of Quassinoids: Physiological Responses and Structural Requirements. PesticideBiochemistry and Physiology 65, (1999) pp. 15-24.V. De Feo, L. De Martino, E. Quaranta, C. Pizza. Isolation of Phytotoxic Compounds fromTree-of-Heaven (Ailanthus altissima Swingle). J. Agric. Food Chem. 2003, 51, 1177-1180.2 Lidert, Z., Wing, K., Insect Antifeedant and Growth Inhibitory Activity of forty-six Quassinoids on twoSpecies of Agricultural Pests. Journal of Natural Products, 1987, pp. 442-448.

Necessity for Qualification and Quantification of active ingredients(a.i.) in plant extracts

Species from the same plant family show high similarities in their appearance and alsosimilar other characteristics. Very often their exact botanical taxonomy is quitecomplicated. Plants from the same family produce closely related secondarymetabolites. In many cases, identical substances are produced. In case of difficultbotanical taxonomy the identification of secondary metabolites may also help in thedetermination.

Although chemical substances of related plants show closely related structures, as theirbiochemical pathway of syntheses differs in nuances only, this does not necessarilymean that the chemical behaviour of the substances is as close, too.

Therefore, it is of great importance to exactly know the plant or, at least, the chemicalingredients of a plant used in plant-protection agents.

It is, for example, quite often the case that genuine camomile is mixed up with otherspecies from the same family, without the afforded characteristic of the first one.In the case of “Quassia” it is of great importance to know about the offered wood oringredients. Literature also shows members of the family with ingredients, that haveunwanted phytotoxic1,2 effects, as can be seen in Figure 1. Holacanthone, a member ofthe quassinoid family, shows potent phytotoxicity against germination and growth ofbentgrass or lettuce, whereas Quassin and the closely related Neoquassin do not affectgermination. In general, it seems, that quassinoids containing an oxymethylene groupas holacanthone have cytotoxic or phytotoxic effects.

In order to have safety about the efficacy it is necessary to have knowledge about theidentity of ingredients. The quantity of active ingredients leads to statements about theirefficiency, providing the efficacy as being dose-dependent.

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1 see for example: Robins, J.R., Morgan, M.R.A., Rhodes, M.J.C., Furze, J.M. (1984). An enzyme-linkedimmuno-sorbent assay for quassin and closely related metabolites. Annal. Biochem. 136, 145-156.2 see for example: EUROPEAN COMMISSION, Scientific Committee on Food, 25 July 2002: Opinion ofthe Scientific Committee on Food on quassin. Lit. cited: Leung, A.Y., Fosters, S.(1996). Quassia. Encyclopedia of Common Natural Ingredients Used in Food,Drugs and Cosmetics. J. Wiley & Sons Inc., pp. 430-431. Within this literature no original source given.3 Sugimoto, N., Sato, K., Yamazaki, T., Tanamoto, K. in: Analysis of constituents in Jamaica quassiaextract, a natural bittering agent; Shokuhin Eiseigaku Zasshi. 2003 Dec. 44 (6) pp. 328-31. Abstract inEnglish, Article in Japanese.

Figure 1:

Preparation of Quassia Extracts and Identification of Ingredients

Quassia amara and Picrasma excelsa

According to information from literature about the species Quassia amara and Picrasmaexcelsa the main ingredients in Quassia amara are Quassin, Neoquassin and also18-Hydroxyquassin1, whereas Picrasma excelsa sometimes is described as containingNeoquassin and Picrasmin (Syn: Isoquassin), the latter quassinoid instead of quassin2.Picrasmin is a diastereomere form of Quassin, its C-14-epimer. Other authors indicatequassin as the major component of Jamaican bitter wood extracts3

Our aim was the qualitative and quantitative determination of the main ingredients fromwood samples of different origin in order to find out to which extend the amount ofingredients might vary. As Quassia amara and Picrasma excelsa might be substituted

O

O O

OCH3

H3COH

O

Quassin

1

2 10

14

16

1811

O

OH

OH

OH

OH

OH

H

OAcO

Holacanthone

18

by other species as well, we examined the specie Picrasma quassioides also, as thistree can be cultivated in non-tropical regions, for example in Europe.

We obtained wood samples from traders in Middle and South America, Germany, fromJamaica and the Netherlands (Picrasma quassioides). Out of these wood samples weprepared liquid extracts that were intended for HPLC analysis. In order to obtaincomparable extracts, we always used the same methodology for the preparation ofextracts from wood.

Where necessary the wood was dried until constant weight.

The wood samples were then cut up to a size that was suitable for extracting. Someamounts of wood were exactly weighed into an Erlenmeyer or round bottomed flask, asolvent mixture of methanol and water added and after at least two hours swelling timeat room temperature the flask was heated up to 70°C and shaken for two hours,. Afterthis extraction time the liquid extract was separated from the wooden residue by filteringthrough medium fast filter paper in vacuum, the residue was washed withmethanol/water. The procedure of heating and separating with methanol/water wasrepeated two times. The liquid phases were combined, concentrated and finallyconditioned for HPLC analysis. HPLC-analysis was performed by using Acetonitril/watermixtures as eluent at 254 nm with a RP-18 column from Knauer: Eurosper-100 C18, 5µm (Vertex).

Figure 1 shows the HPLC spectra of Picrasma excelsa, bitter wood from Jamaica.Figure 2 shows the HPLC-spectra from Costa Rican bitter wood, Quassia amara.

Figure 2: HPLC-spectrum from Jamaican bitter wood

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1 Dou, J., McChesney, D., Sindelar, R., Goins, D.K., Khan, I., Walker, L.; A New Quassinoid from CrudeQuassin-Extract of Quassia amara. Int. J. Pharmacognosy 1996, pp. 349-354.

Figure 3: HPLC from Costa Rican bitter wood

1: Quassin 5: Nigakilactone A2: Neoquassin 6: Parain or Isoparain3: Quassialacton 7: Quassialactol4: 12- or 18-Hydroxyquassin

In both species Quassin (1) and neoquassin (2) are the compounds of largest quantity,as can be seen in figures 2 and 3. Beside these, also the minor components 3 to 7 havebeen identified. Neoquassin splits into two signals, as the substance exists in twochemical forms in an equilibrium.

The identification of the different substances was performed by HPLC-MS/MS (Dr.Kirschbaum, Justus-Liebig-University of Giessen) and the results were compared withdata from literature1. In the case of quassin and neoquassin additionally 1H- and13C-NMR-spectra were taken and also compared with known data. Also, quassin andneoquassin were identified by comparison with commercially available standards.Whereas the substances 1,2,4,5,6 and 7 have been identified as natural products inbitter wood, quassialacton (3) has been described as a semi-synthetic product fromoxidation of quassialactol (7). As we found quassialacton only in the extracts from this

20

single wood sample from Costa Rica, its occurrence might be due to decomposition ofsecondary metabolites, as this wood was not directly dried after having been harvested.

During the past years we have analysed many samples of wood from different origins.Figure 4 gives an overview about the range of quassin contents we found in thesesamples. As can be seen in this figure, the range of quassin varies between 0 and 1mg/g wood, whereas neoquassin was found in amounts of one third to about threetimes more than quassin.

*: Origin and wood species as indicated by the supplier

Figure 4: Quassin and Neoquassin contents in different wood samples.

In all our research with Quassia amara and Picrasma excelsa it turned out that quassinand neoquassin are the substances with the largest quantity in wood samples.Therefore we suggest using one or even both of these substances as analytical"markers" for extracts of quassia. As neoquassin and quassin both show insecticidalactivity, this reduction upon two substances seems to be a well working and sufficientcompromise for the analytical description of such extracts. This method is equivalent tothe well known procedure in neem analysis, where Azadirachtin A is defined as amarker for analytical purposes.

Picrasma quassioides

In order to find an alternative for the use of Picrasma excelsa or Quassia amara, welooked for Picrasma quassioides, some species that can be grown in European regionswith mild climatic conditions. The wood from this tree could be of interest, in case itsamount of quassin and neoquassin was sufficient and also, if other substances ofunknown or unwanted characteristics would be neglectable because they could be

Origin* Wood* Batch Quassin mg/g Ratio Quassin/Neoquassin

Mexico Mex. Cuassia amaraChips

2002 0,001 about 1 : 1,5

Mexico Chapparo amargo 2003 0,08 not determined

unknown Quassia amara Type IS 0,08 1 : 0,6

Brazil Quassia amara A 0206052 0,13 1 : 1,6



Brazil Picrasma excelsa A 0203081 0,64 1 : 3,3

Costa Rica Quassia amara Harvest 01 0,79 1 : 0,7

Jamaica Picrasma excelsa - 0,88 1 : 1,6

Range up to 1mg/g 1:0,3 to 1:3,3

21

1 Sheng-Ping Yang; Jian-Min Yue.Five new Quassinoids from the Bark of Picrasma quassioides.Helvetica Chimica Acta 87 (2004) pp.1591-1600.

easily removed by simple extraction methods. Parts of the tree are used in Chinesetraditional medicine1.

Picture 1 shows some noticeable red twigs from a small tree of Picrasma quassioides.

Picture 1: Twigs from Picrasma quassioides

Figure 5: Extract from Picrasma quassioides, HPLC

2

1

22

The preparation of extracts for HPLC-analysis was performed analog to the preparationof extracts from other Quassia samples. The HPLC result may be seen in figure 5.

Substance 1 was identified as quassin. (The retention time (R.t) is not identical with thatof quassin in figures 1 and 2 because of some modification in the analytical program.)Signals 2 fit to the signals that are expected for neoquassin. The absolute amount ofquassin was about 0,13mg/g wood in Picrasma quassioides. Neoquassin was about0,59 mg/g.

The comparison of the HPLC-spectra from Picrasma quassioides, Picrasma excelsaand Quassia amara (figures 1,2 and 4) shows that far more substances in higherconcentration can be found in P. quassioides than in the other two species. Therefore,the substitution of P.excelsa and Q.amara against P. quassioides should be carefullyweighed up. Unknown toxic characteristics of such an extract may pose a problem.

23

TRIFOLIO S-FORTE – A NEW ADDITIVE FOR PLANT PROTECTIONPRODUCTS

WALTHER, N.1 AND DETZEL, P.2

1) TRIFOLIO-M GMBH, SONNENSTR. 22, D-35633 LAHNAU;

2) BETREUUNGSDIENST NÜTZLINGSEINSATZ BADEN E.V., AM VIEHMARKT, 76646 BRUCHSAL

World food production hugely depends on the employment of plant protection products(PPP). Not only conventional farming systems are dependent on these products. Organic plant protection also has become more and more important in organic farmingsystems to produce food on the highest quality level for acceptable prices. After about100 years of chemical crop protection, new active substances which fulfil all modernrequirements for registration as PPP, are hard to find. Additionally the manufacturingindustry has to face the problem, that the active ingredient (a.i.) must be formulated in astable product. Often compromises between a stable formulation and good performancein practice must be made. For this reason there is a demand for additives. They canincrease the performance of both chemical and organic PPP with low input of cost andwork by generating a multiple use.

Development and compatibility

Trifolio S-forte was developed in cooperation with the BetreuungsdienstNützlingseinsatz Baden e.V. Technical know how was combined with the pooledknowledge of practice-orientated field advisers. Following the EU-Directive EU/2091/91Trifolio S-forte can be use in organic farming systems.

Ingredients and mode of action

Trifolio S-forte contains 50% plant oils with 50% emulsifier based on renewable rawmaterial. Compared to other additives based on plant oil, Trifolio S-forte has a uniqueratio of its compounds in view of the fact that other plant oil based products mostlycome with an amount of more than 90% plant oil (see Table 1).

Despite the low amount of plant oil Trifolio S-forte has a good performance in holdingactive substances onto the leaf surface without building a sticky or fatty-looking layer.Particularly systemically acting substances can profit from the fact, that the spray filmwill not dry as fast as without the additive. Thus the a.i. has more time to diffuse into theleaf.

The high amount of emulsifier reduces the surface tension of the spray drops whichresults into a perfect thin layer spray coating. The received spray coat can be comparedBiological Control of Plant, Medical and Veterinary PestsR. Strang & H. Kleeberg (eds.)Copyright 2009 by Trifolio-M GmbH

24

to such produced by so called “Super-Spreader”. From this effect especially productswith active ingredients having a contact action benefit, because the PPP does reacheven hidden parts of the plant.

Table 1: Classification of Trifolio S-forte

Based on Syngenta 2004

The table clearly demonstrates the intermediate position of Trifolio S-forte between"Penetrators" and "Wetters". Trifolio S-forte covers two fields of use and thus providesthe advantages of both product groups combined in one product.

Further advantages of Trifolio S-forte are

Improves spray layer on the leaf surface

Avoid visible precipitates caused by polluting preparations like copper

Improves compatibility with sprout-suppressing substances like Chlormequat (CCC720).

Miscibility

According to our present knowledge Trifolio S-forte can be mixed with preparationscontaining the following active ingredients:

In organic farming Trifolio S-forte can be mixed with:

Cationic Wetter Frigate, MonfastOrganosilicones Break Thru S 240, Silwet Gold

Adhesive Polymers Bond, Greemax Wax CereNat E 30, Agrocer 010

Abamectin MancozebChlormequat Mancozeb + Metalaxyl MCopperhydroxid PropiconazolCyprodinil + Fludioxonil PymetrozinFenhexamid Spinosad

Plantoils Rako, Agrosom Net 5 Plantoils + Noninionic Wetter Trifolio S-forte

Wetter Nonionic Wetter Adhäsit, Agral

Rape seed oil, methylised

Actirob B Oils Mineral oil Para Sommer

Penetrator Non-Oily Lecithin Li 700 Protein derivatives ProNet-Alfa

Emulsifier Greemax

Mode of action Active Ingredients Product Name

Azadirachtin QuassiaBt-preparations SulphurCopper Virus-preparations

25

The following substances are not compatible with Trifolio S-forte:

Phytotoxicity Trifolio S-forte is perfectly safe for almost all plants. In orchards, pear trees can reactwith phytotoxic symptoms,even by drift deposition. In ornamentals some species, suchas Begonia spp., are showing incompatibilities.

Environmental behaviourAnother advantage of Trifolio S-forte is its environmentally friendly behaviour. Besidethe usual protection measures, no further product-related protection measuresconcerning water or human health must be followed. It is safe for beneficials and fullybiodegradable.

Azoxystobine TloylfluanidCaution is advised with Captan and Delan

27

VETERINARY APPLICATIONS OF NEEM (AZADIRACHTA INDICA AJUSS) AND ITS PRODUCTS

C.M. KETKAR

NEEM MISSION, 471, SHANWAR PETH, PUNE - 411 030. (M.S.) INDIA

Introduction

Azadirachta indica A Juss is an evergreen tree indigenous to and cultivated nearly allover India. Every part of the tree, the bark, the leaves, young branches, flowers, unripe& ripe fruits, oil and gum is reported to have medicinal properties.

A number of workers reported from time to time the various applications of neem formedicinal purposes, but ,prior to isolation of the active bitter principles in the pure state,these findings were mostly of empirical nature.

In view of curative properties attributed to folklore and traditional medicine to neem, ithas been subjected to chemical & therapeutic studies from about the beginning of the20th century.

Neem preparations have been used to treat blood disorders, hepatitis, eye diseases,cancer, ulcers, constipation, diabetes, indigestion, sleeplessness, stomach ache, boils,burns, cholera, gingivitis, malaria, measles, nausea, snakebite, rheumatism and syphilis(Jacobson, 1989).

Numerous formulations are used as antiseptics, astringents, emollients, febrifuges,anodynes, diurectics, parastificides, pediculicides, purgatives, sedetives, stomachics,and tonic (DUKE and WAYNE, 1981). Neem products with these reported activities areavailiable commercially (KOUL et, al, 1990, JACOBSON, 1989). Many of the biologicalactivities mentioned above are being substantiated by current research (JACOBSON,1989 VAN DER NAT et, al, 1991).

Small Ruminant Goat

India has about 117 million goats & they are raised in the country under traditionalsystems of management. Unfortunately the productivity of these goats is greatlyreduced by internal parasites known popularly as "worms" and to scientists asgastro-intestinal nematodes. Worm infestation reduces the nutritional benefit to theanimal from its feed intake, weakening it and preventing it from thriving and lowers ananimal's resistance making it more likely to become ill or diseased. Heavy worminfestation causes severe anemia and in some cases this can be fatal. If lactating goatsare affected, an inadequate supply of milk may lead to the death of kids.Biological Control of Plant, Medical and Veterinary PestsR. Strang & H. Kleeberg (eds.)Copyright 2009 by Trifolio-M GmbH

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Parasitic Worms

There are many different types of parasitic worms found around the world but the mostcommon one which causes serious problems in goats in many tropical areas areHaemonchus, Oesophagostomum, Trichuris and Moniezia spp. & they are bloodsucking parasites.

Material and Methods

Thereapeutic evaluation of indigenous plants extracts on Helminthiasis in goats werecarried out by M.V.S.C. Students of Dr. Punjabrao Deshmukh Krishi Vidyapeeth, Akola,M.S. India, Using neem seed extracts by MS. Thakare Minal Baburao, & Neem oil byMS Bhirangi seema Prabhakarrao.

Neem Seed extracts

Twenty-four goats, both males & females, between ages of 6 & 24 months harbouringmixed infections were selected by faecal Sample examination. These goats wererandomly divided into six equal groups. One additional group of four normal healthygoats was taken and these different groups were given different treatments. First groupwas normal healthy control. Second group was untreated control, third was treated withAzadirachta indica Seed extract, fourth with E. ribes Seed extracts, fifth withA-panciculata extract and sixth with mixture of extracts of equal quantity of Azadirachtaindica & E. ribes seeds. Each extract (20 ml of 20% solution) was given orally for threedays in the respective groups. A seventh group was treated with 15% closantel @15mg/kg. body weight orally.

Results

In clinical study when untreated group was compared with normal, the faecalexamination revealed that faecal egg contents of Haemonchus, Oesophagostomum,Trichuris and Moniezia spp. increased throughout the experimental period withpersistent diarrhoea with reduction in body weight.

The efficiency of neem seed extracts was 95.12% for Haemonchus spp. after 10days.100% for Oesophagotomum spp. Trichuris Spp. and Moneizia spp. after 30, 10 & 30days of treatment respectively. While the efficacy of E.ribes seed extract was 87.5%against Haemonchus spp and Oesophegostomum spp. after 20 and 2 days respectivelyand 100% against Trichuris spp. and Moneizia spp. after 2 and 30 days of treatment,respectively. Further extracts of A. paniculata was 93.75% effective againstHaemonchus Spp. after 10 days, 81.81% against Oesophagostonum spp. after 8 daysrespectively. Mixture of extract of Azadirachata indica & E. ribes reduced EPG counts93.33 and 87.5 percent against Haemonchus spp. and Oesophagostonum spp. after 8

29

to 10 days respectively. Closantel was 100% effective against Haemonchus andOesophagostomum spp after 4 and 20 days of treatment respectively. The decrease inEPG count resulted in subsiding diarrhoea and increasing ruminal motility with increasein body weights of treated animals.

In untreated group, haematobiochemical studies revealed macrocytic anemia indicatedby significant low levels of R.B.C. PCV and Hb associated with high levels of MCV,MCH and MCHC. While, eosinophilia indicated allergic reaction, hypoproteinaemia wasindicated by significant low levels of serum total proteins, albumin and globulin.Subsequenctly, immune response was indicated by higher levels of gamma globulinthan normal. These changes were due to presence of blood sucking helminth in thebody of the animals.

In all treated groups there was a reduction in anemia which was indicated by significanthigh levels of RBC, PCV and Hb with significant low levels of MCV in treated groupsthan untreated group. However, the levels were not restored to normal except in theseventh group, indicated presence of microcytic anemia after treatments.

The closantel treatment was found superior to all the other groups, as these bloodlevels were normalised in seventh group after treatment. Significant reduction in levelsof eosinophils in treated groups than untreated group, indicated reduction in allergicreaction and all these changes seem to be due to elimination of blood sucking parasitesdue to anthelmintic effect of given treatments. Similarly, cessation of loss of protein andreduction in immune response due to elimination of blood sucking parasites wereindicated by significant high levels of serum total proteins, albumin and globulin withsignificant low levels of gamma globulin in all treated than untreated groups.

Neem Oil

In this experiment, treatments were a) neem oil @ 1ml. per animal, b)10% C. papayaseed extract @ 50ml. per animal, c) 10% B. frondosa seed extract @ 50ml. per animalthen with mixture of 10ml. neem oil, + 10% B. frondosa seed extract 50ml. per animal15% closantel @ 15mg/kg body weight of goats orally for one day.

Conclusion

Neem seed extract & neem oil are both effective in controlling nematode infestations ingoats, and as this is a plant product, easily available in the villages, its use as ananathematic will benefit the poor goat owners.

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References

JACOBSON. M (Ed) (1989) 1988 Focus on photochemical Pesticides Vol. 1 the NeemTree CRC Press Boca Raton Fl. 178pp.

DUKE, J.A., WAYNE, K.K. (1981) Medical Plants of the world Vol.3. Computer Index.

KOUL, O., ISMAN, M.B., KETKAR, C.M. (1990) Properties and uses of neem,Azadirachta indica. Can. J. Bol. 68, 1-11.

VAN DER NAT, J.M. VANDER SLUIS, W.G., DE SILVA, K.T.D., LABADIE, R.P. (1991)Ethnopharma cognostical Survey of Azadirachte, indica A Juss (Meliaceae) J.Ethnopharmacol 35, 1-24.

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EFFECT OF NEEM DERIVED PRODUCTS ON GASTROINTESTINALNEMATODES IN VITRO AND IN VIVO IN SHEEP.

VAN DER ESCH S.A 1, CARNEVALI F.1 AND AMICI A.2

1ENEA CENTRO RICERCHE CASACCIA, UTS BIOTEC AGRO, ROMA,

2 DEPARTMENT OF ANIMAL PRODUCTION, UNIVERSITY OF TUSCIA, VITERBO

Abstract

Parasitic gastrointestinal (GI) nematodes are one of the most commonly acquiredinfections in the world, affecting both humans as animals. Parasitic helminths (whichincludes GI nematodes) also place a considerable constraint upon livestock industry,representing a major economic burden; one estimate (1993) suggested that £ 1.7 billionis spent annually on their control.

Several publications and anedoctical evidence have suggested that neem derivedproducts could be an alternative for the treatment of this infection.

In order to evaluate the veridicity of these claims on the efficacy of neem for GI controlwe have studied the effect of various neem derived extracts both in vivo as in vitro inadult sheep.

In vivo studies have been carried out with both a naturally infected flock and with anartificially infected flock. The GI nematodes identified in the natural infection andsubsequently used for the artificial infection were a pool of: Haemoncus contortus,Chabertia spp, Cooperia spp, Trichostrongylus vitrinus and Nematodirus spp. The neemderived products tested were neem leaf extract (NLE), dried neem leaf powder (NLP)and NeemAzal.

In vitro the neem derived products tested were NeemAzal, neem seed kernel powder(NSKP), dried neem leaf powder (NLP), neem leaf extract (NLE), neem seedssupercritical fluid extract (NSFE), neem oil (NO) and various controls.

Both in vitro as in vivo egg counts (EPG) and larval (LPG) counts were done with theMcMaster method.

In both the in vivo as in the in vitro experiments no significant effect on the EPG andLPG counts of any of the tested neem derived products has been observed. This seemsto imply that, under the experimental conditions used, the neem derived products testedin this study are of no efficacy for the control of GI nematodes infections in sheep.


32

Introduction

Helminthiasis is one of the most important animal diseases worldwide and ifuncontrolled can have dramatic effects on livestock mortality and productivity (1). Themassive use by farmers of conventional synthesised chemical drugs to control helminthparasites has lead to the alarming development of parasites’ resistance (2) tobroad-spectrum anthelmintics in all livestock species, not only in the so-calleddeveloping countries but also in Europe (3). Furthermore, there are increasing concernsabout the environmental impact (4) and the risk of chemical residues in edible animalproducts resulting from anthelmintic usage (5). For these reasons, scientific researchhas been intensified in pursuit of alternative parasite control methods in livestock, suchas nematode-trapping fungi (6), genetically resistant and/or tolerant breeds (7, 8),vaccines (9), and/or other simple management procedures (e.g. rotational grazing,alternate/mixed grazing) (10, 11). In the past twelve years, research into such biological,environmental and chemical control strategies, alone or in combination, has beendynamic, but due to the high costs and the extended time in development, substantialgoals are far from being achieved in a short term.

There is thus increasing interest in using new anthelmintics to fill intrinsic gaps in thepharmacopoeia, or gaps caused by evolving parasite resistance, and in assessing theireffectiveness and potential both for animal and human well-being. Plants and/or plantextracts can be more than a minor aspect of new methods for helminth control. Theirpossible use as anthelmintic is of particular importance when animal/human welfare,product safety and environmental protection are priorities in consumer perceptions anddemands.

One candidate plant to be used as a source of plant derived products in order to controlgastrointestinal nematodes is the neem tree (Azadirachta indica (A. Juss)). It has beenreported that cattle fed with neem leaves obtained a reduction of the total egg count(EPG) (12) while also at the Neem 2002 World Neem Conference a report was giventhat neem derived products had an anthelmintic effect. In order to verify thisexperiments were organised, both in vivo as in vitro, to experiment different neemderived products on their anthelmintic effects.

Materials & Methods

An experimental flock of 16 Appenninic sheep was used both for artificial infestation andas donors of infested faeces for the in vivo and in vitro experiments in which theanthelmintic effect of different Neem derived products was tested.

33

In vivo experiments

The in vivo experiments were performed by inducing an artificial infestation in adultsheep starting from spontaneously infested faecal material.

The GI nematodes identified in the natural infection and subsequently used for theartificial infection were a pool of: Haemoncus contortus, Chabertia spp, Cooperia spp,Trichostrongylus vitrinus and Nematodirus spp.

Artificial infection of animals (N = 16):

Almost three Kg. of faecal material was collected from 4 spontaneously infested sheepconfined in individual pens for 24 hours. The faecal material was pooled and EPG werecounted. As described below developed L3 stage larvae were recovered andresuspended in clean water in order to obtain 100.000 L3 stage /ml.

Sheep were infected by Os with 10 ml H2O containing 100.000 L3 stage. The operationwas repeated twice at one month intervals. Infestation course was monitored twice amonth by counting EPG in individual faecal material directly collected from the rectalampulla (3 samples in an 8 hour period). Sheep were introduced into the anthelminticprotocol when they presented infestation levels, for two consecutive examination, ofalmost 500 EPG (eggs per gram of faeces)

Neem derived products tested:

NeemAzal: Each animal received a daily dose of 10 ml olive oil containing 1.5g. ofNeemAzal (0.5 gr/day Aza A) for 4 days.

Neem Leaf Powder (NLP): Each animal received a daily dose of 70 g. NLP mixed withbentonite for 4 days.

Neem Leaf Extract (NLE): Each animal received a daily dose of 2.4 g. of NLE extractin 20 ml of H2O for 4 days.

Data collection:

At time -4, 0, 2, 7, 14 and 21 days after neem derived products treatments, faecalmaterial was directly collected from the rectal ampulla over a period of 8 hours (3samples taken from each animal). The daily samples of each sheep were pooled andEPG were counted and the hatching test was performed by counting the developedlarvae (larvae per gram faeces - LPG).

In vitro experiment

Three Kg. of faecal material was collected from 4 artificially infested sheep confined inindividual pens for 24 hours. This material was thoroughly mixed in order to obtain ahomogeneous distribution of the eggs within the faecal mass and EPG was determined.

34

In order to test the effect on the hatchability of the GI nematode eggs, the followingneem derived products were mixed with aliquots of 5 gr of faecal material. Treatmentswere done in triplicate

Neem derived products tested:

Two different experiments were done in vitro. Neem oil and neem seed kernel powder(NSKP) were present in both experiments.

Experiment A

SFE Neem Leaf Extract (6000, 600 and 60 ppm)

NeemAzal (5000, 500, 50 ppm Aza A)

Neem oil (10 and 1%)

Neem Seed Kernel Powder (20, 10 and 2%)

Experiment B

Neem leaf extract (NLE) (1000, 100 and 10 ppm)

Neem leaf powder (NLP) (20, 10 and 2%)

Neem oil (10 and 1%)

Neem Seed Kernel Powder (20, 10 and 2%)

In both experiments controls consisted of either untreated faecal material, samplestreated with 1 ml distilled H2O, samples treated with 2% absolute alcohol or samplestreated with 10% peanut oil.

Neem derived products used were: NeemAzal and Neem leaf powder (NLP) bothfurnished by Trifolio-M GmbH, (Germany). Neem oil (NO) and Neem leaf extract (NLE)both furnished by E.I.D.Parry (India) Ltd. The Neem seed kernel powder (NSKP) wasfurnished by Saroneem (Kenya) Ltd. The neem leaf supercritical fluid extract (NLSFE)was furnished by Nisarga Biotech Pvt. Ltd (India).

Counting methods:

The MacMaster method was used. The parameter used to measure faecal eggs (faecalegg count (FEC)) was the egg count per gram of faeces (EPG): 2.5 gr of faecessamples were mixed with 35 ml of flotation solution. Material was filtered and eggs werecounted in a 2-chamber MacMaster slide having 0.5 ml volume/chamber. Counts foreach sample were triplicated.

35

The parameter used to measure developed L3 stage larvae (faecal larvae counts (FLC))was the larvae count per gram of faeces (LPG): 5 gr. of faecal material, either from invivo treated animals or from in vitro treated faecal material, was incubated for 9 days at27 °C in 95% of humidity in the dark. Subsequently the material was suspended inwater for 48 hours during which time the L3 larvae migrate into the water. Afterdecantation the water level was restored to 35 ml. Migrated larvae were counted usingthe McMaster method in a 2-chamber MacMaster slide having 0.5 ml volume/chamber.Counts for each sample were triplicated.

Statistical analysis:

Individual data of faecal egg count (FEC expressed as EPG) and larvae (FLCexpressed as LPG) per gram of faeces (both expressed on wet and dry faeces weight)underwent a repeated measures analysis of variance (time 0, 2 , 7, 14 days etc. only forin vivo experiments) using the statistical software SAS® (1998), after a logarithmictransformation (Log10) in order to stabilise variance within groups. Differences betweenthe groups, within time, were tested with H test of hypothesis. Results are presented inthe figures as arithmetic means (±S.D.) and inferences were based on the transformeddata.

The percentage faecal egg and larvae reductions (FECR and FLCR, respectively) weredetermined by the method described by Coles et al. (1992) (13) using the formulapercentage reduction = 100 x (1 – T/C), where T and C are the arithmetic means in thetreated and control groups, respectively, on day 7 post treatment.

For the in vitro experiments analysis were performed using the SPSS 12.0 statisticalsoftware package. A model I, one-way analysis of variance was performed on theoriginal data, as they were normally distributed (data normal distribution wereinvestigated by mean of Shapiro-Wilk’s test). Pairwise comparisons were based onTamhan’s method because of non homogeneity of variance across groups; thesignificance of the differences between two means was evaluatad at p < 0.05 level.

Results

In vivo experiment.

The artificial infection had to be repeated twice at a 1 month interval in order to obtain asatisfactory infection level of 500 EGP as a mean over the whole flock (results notshown).

In Tables 1 and 3 the treatment, respectively with neem leaf powder (NLP) andNeemAzal, had no effect on the faecal egg counts (FEC) in vivo. A high variability existsamong the different sampling times (-4, 0, 4, 7. 14 and 21 days) but no significant

36

differences between control animals and treated animals could be observed. The faecalegg count reduction (FECR), as determined by the formula of Coles et al. (1992)indicates respectively a value of –50 and –4 for NLP and NeemAzal treatments. Thesevalues of FECR actually indicate an increase of the egg counts on day 7 for the treatedanimals. But as there did not exist a statistical significant difference between the EPGand LPG values these differences are not significant either.

Also the development of L3 larvae, observed in the faecal material from in vivo treatedsheep, is not impaired by the in vivo treatment. (values of FLCR 5 and 16 respectivelyfor the NLP and NeemAzal treatment considered on day 7) (See Tables 2 and 4).

The data concerning the Neem Leaf Extract (NLE) are not presented here as the resultswere identical to the other two neem derived products (NLP and NeemAzal) shown, i.e.no significant reduction in vivo was observed both on FEC as on FLC.

In vitro:

Two experiments are presented in Table 5. In both experiments the major significantreduction was obtained when 10% neem oil was applied (54% reduction for bothexperiment A and B). But if these results are compared to the control sample containing10% peanut oil the difference isn’t significant anymore. The NSKP (neem seed kernelpowder) also had significant effects on the development of the L3 larvae in bothexperiments but no dose response was observable (among the different concentrationsno significant differences exist) and the level of inhibition (between 40 – 50%) isn’tconsidered sufficient to be considered effective.

Nor NeemAzal (5000, 500 and 50 ppm) neem leaf supercritical fluid extract (NLSFE)(6000, 600 and 60 ppm) neem leaf extract (NLE) (1000, 100 and 10 ppm) neem leafpowder (NLP) (20%, 10% and 2%) had any significant effect on the FLCR.

Table 1: In vivo:arithmetic means and SD of faecal egg counts (FEC) on days 0, 4, 7, 14 and 21 oncontrol sheep and sheep treated with neem leaf powder after artificial infection with GInematodes.

No significant differences were detected at the level of P<0.05aPercent reduction in FEC using formula Coles et al. (1992) determined on day 7bDay of treatment

Treatment No. of sheep Arithmetric mean (± SD) of FEC expressed asEPG

FECRa

0b 4 7 14 21Control 3 655 (±

371)375 (±25

364 (±129)

606 (±134)

518 (±247)

Neem leaf extract 7 487 (±542)

420 (±472)

549 (±652)

346 (±473)

399 (±490)

-50

37

Table 2: In vivo. Arithmetic means and SD of L3 larvaec (FLC) on days 0, 4, 7, 14 and 21 on controlsheep and sheep treated with neem leaf powder after artificial infection with GI nematodes.

No significant differences were detected at the level of P<0.05aPercent reduction in LPG using formula Coles et al. (1992) determined on day 7bDay of treatmentcHatched in vitro after sample and FEC count

Table 3d: In vivo: arithmetic means and SD of faecal egg counts (FEC) on days –4, 0, 4, 7, 14 and 21 oncontrol sheep and sheep treated with NeemAzal after artificial infection with GI nematodes.

No significant differences were detected at the level of P<0.05dFootnotes see Table 1

Table 4e: In vivo: arithmetic means and SD of L3 larvaec (FLC) on days –4, 0, 4, 7, 14 and 21 on controlsheep and sheep treated with NeemAzal after artificial infection with GI nematodes.

No significant differences were detected at the level of P<0.05eFootnotes see Table 2

Treatment No. of sheep Arithmetric mean (± SD) of FLC expressed asLPG

FLCRa

0b 4 7 14 21Control 3 431 (±

368)219 (± 7) 354 (±

173)450 (±270)

320 (±295)

Neem leaf extract 7 437 (±387)

321 (±271)

338 (±382)

599 (±686)

274 (±295)

5

Treatment No. ofsheep

Arithmetric mean (± SD) of FEC expressed as EPG FECRa

-4b 0 4 7 14 21Control 3 408 (±

478)560 (±561)

250 (±178)

422 (±409)

373 (±352)

434 (±341)

NeemAzal

8 487 (±433)

596 (±638)

431 (±426)

450 (±465)

389 (±315)

418 (±387)

-4

Treatment No. of sheep Arithmetric mean (± SD) of FLC expressed as LPG FLCRa

-4 0b 4 7 14 21Control 3 184 (±

274)107 (±65)

259 (±222)

343 (±228)

335 (±333)

287 (±341)

NeemAzal 8 193 (±134)

239 (±212)

239 (±191)

289 (±210)

434 (±435)

297 (±242)

16

38

Table 5: In vitro. Effect of different neem derived products on larval development (L3 stage) in vitro.

* p < 0.05

Discussion

Plants and/or plant extracts with alleged and /or suspected anthelmintic properties havebeen widely used in traditional human (ayurvedic) and veterinary (i.e. ethno-veterinary)medicine for many years, or naturally grazed/browsed by herbivorous as pasture plants(14). Evidence exists that neem derived products could play a useful role forhelminthiasis control in livestock (12, 13). For this reason a systematic study has beenundertaken to verify if neem derived products are effective in the control ofgastrointestinal nematodes in sheep. The experimental design considered both the invivo as the in vitro effects. Assessment on the adult worms was not directly undertaken(i.e. scarifying the animals and assessing the presence of adult worms in the intestines)but indirectly through the amount of GI nematode eggs produced. The in vitro

Treatment Expriment Larvae count (LPG)(Mean ± SD)

FLCR

Control A 204,55 ± 60,96Control + H2O (1 mL) 185,11 ± 46.44 10Control + Peanut Oil (10%) 116,66 ± 51.26* 56Control + C2H5OH (2%) 185,11 ± 76,13 10SFE neem leaves 1000 ppm 142,33 ± 58,27 31SFE neem leaves 100 ppm 204,55 ± 5219 0SFE neem leaves 10 ppm 169,55 ± 42,65 18NeemAzal 5000ppm 143,88 ± 28,32 31NeemAzal 500 ppm 127,16 ± 42.4 38NeemAzal 50 ppm 150,5 ± 63,49 27Neem oil 10% 95,66 ± 35,66* 54Neem oil 1% 135,33 ± 29,44* 34Neem seed kernel powder 20% 122,11 ± 34,89* 41Neem seed kernel powder 10% 103, 83 ± 29,52* 50Neem seed kernel powder 2% 128,33 ± 43,81* 38

Control B 206,5 ± 62,64Control + H2O (1 mL) 184,33 ± 44,6 10Control + Peanut Oil (10%) 131,83 ± 44,05 56Control + C2H5OH (2%) 201,83 ± 71,99 0Neem leaf extract 1000 ppm 198,42 ± 38,13 4Neem leaf extract 100 ppm 170,33 ± 19,99 28Neem leaf extract 10 ppm 171,50 ± 29,98 28Neem leaf powder 20% 176,17 ± 19,27 25Neem leaf powder 10% 298,67 ± 68,79 -44Neem leaf powder 2% 284,67 ± 105,36 -37Neem oil 10% 95.66 ± 35,23* 54Neem oil 1% 135,33 ± 29,44 34Neem seed kernel powder 20% 113,16 ± 31,28* 46Neem seed kernel powder 10% 103,83 ± 29,52* 50Neem seed kernel powder 2% 123,85 ± 38,16 41

39

experiments were designed in order to assess the possibility to control GI nematodesinfestation through the treatment of the larval development stage.

Recent harmonisation on anthelmintic efficacy guidelines in monogastric animals haveindicated that for a drug to be considered to be efficacious, a 90% reduction in faecalegg count (FEC) or total worm count (TWC) should be achieved (15). However, theeffect of plants is unknown so in our study we adopted the same criteria as adopted in(14), a reduction of 70% in FEC or FLC was used as a cut-off point for biologicalsignificance.

In this work the neem derived products tested in vivo (NeemAzal, neem leaf powder(NLP) and neem leaf extract (NLE)) didn’t demonstrate any significant reduction of therelative GI nematode egg counts and larval (L3) counts on day 7 of the treatment. Thetreatment effect was all the same followed for 21 days and no significant reductionscould be observed.

The only reliable report of the control of GI nematodes in vivo known to us (12)observed a significant reduction in EPG in a herd of heifers supplemented in their dietwith cement blocks containing 10 – 30% dried neem leaves (ad libitium) over a 90 dayperiod. It has been reported (personal communications) that neem oil seems to have aneffect on GI nematodes. The experimental results presented here seem to exclude thepossibility that neem derived products could be effective for the control of GInematodes. All the same it would be worthwhile to further study the effects of neem oil(NO) in vivo, as our experimental data have only considered the neem oil in our in vitrotests. In our experiments the presence of the oil (either peanut or neem oil) has amechanical effect rather than a biological effect on the inhibition of larval developmenttill the L3 stage. All the same it can not be excluded at this stage that neem oil mighthave a significant biological effect in vivo.

Bibliography

Perry B.D. and Randolph T.F. (1999) Improving the assessment of the economic impactof parasitic diseases and of their control in production animals. Vet. Parasitol. 84,145-168

Waller P.J. (1997) Anthelmintic resistance. Vet. Parasitol. 72 (Issues 3-4), 145-168

Vercruysse J. and Dorny P. (1999) Integrated control of nematode infections in cattle: Areality? A need? A future? Int. J. Parasitol. 29, 165-175

Lumaret J.P. and Errouissi F. (2002) Use of anthelmintics in herbivores and evaluationof risks for the non target fauna of pastures. Vet. Res. 33 547–562

40

Imperiale F, Sallovitz J, Lifschitz A, Lanusse C. (2002) Determination of ivermectin andmoxidecin residues in bovine milk and examination of the effects of theseresidues on acid fermentation of milk. Food Addit. Contam.; 19(9): 810-8.

Larsen M (1999) Biological control of helminths. Int. J. Parasitol. 29, 139-146

Silvestre A, Leignel V, Berrai B,Gasnier N, Humbert JF, Chartier C, Cabaret J. (2002)Sheep and goat nematode resistance to anthelmintics: pro and cons amongbreeding management factors. Vet. Res. 33, 465 - 480

Stear M.J, Strain S, Bishop S.C, (1999) Int. J. Parasitol. 29, 51-56

Dalton J.P, Brindley P.J, Knox D.P, Braidy C.P, Hotez P.J, Donnelly S, O’Neill S.M,Mulcahy G, Loukas A. (2003) Helminth vaccines: from mining genomicinformation for vaccine targets to systems used for protein expression. Int. J.Parasitol. 33, 621 – 640

Niven P, Anderson N, Vizard A. L, (2002) The integration of grazing management andsummer treatments for the control of trichostrongylid infections in Merinoweaners. Aust Vet J. ;80(9):559-66.

Schreurs N. M, Molan A. L, Lopez-Villalobos N, Barry T. N, McNabb W. C. (2002)Effects of grazing undrenched weaner deer on chicory or perennialryegrass/white clover pasture on the viability of gastrointestinal nematodes andlungworms. Vet Rec.;151(12):348-53.

Pietrosemoli, S., Olavez, R., Montilla, T., Campos, Z., (1999) Empleo de hojas de Neem(Azadirachta indica A. Juss) en control de nematodes gastrointestinales debovinos a pastoreo. Rev. Fac. Agron (LUZ). 16 Suppl 1: 220 – 225

Coles, G.C., Bauer, C., Borgsteede, F.H., Geerts, S., Klei, T.R., Taylor, M.A., Waller,P.J., (1992) World association for the advancement of veterinary parasitology(WAAVP) methods for the detection of anthelmintic resistance in nematodes ofveterinary importance. Vet. Parasitol. 44, 35 - 44

Githiori J.B, Höglund J, Waller P.J, Leyden Baker R, (2003) Evaluation of anthelminticproperties of extracts from some plants used as livestock dewormers bypastoralist and smallholder farmers in Kenya against Heligmosomoidespolygyrus infections in mice. Vet. Parasitol. 118: 215 – 226

Vercruysse, J., Holdsworth, P., Letonja, T., Conder, G., Hamamoto, K., Okana, K.,Rehbein, S., (2002) International harmonisation of anthelmintic efficacyguidelines (Part. 2) Vet. Parasitol. 103, 277 - 297

41

BIOLOGICAL ACTIVITIES OF NEEM OIL DERIVATIVES IN CULTUREDMURINE FIBROBLASTS

VINCENZO DI ILIO1,4, NICOLETTA PASQUARIELLO2, ANDREW S. VAN DER ESCH1,MASSIMO CRISTOFARO1,3, GIANFRANCO SCARSELLA4, GIANFRANCO RISULEO2

1BIOTECHNOLOGY BIOLOGICAL CONTROL AGENCY, V. DEL BOSCO, 10 – 00060 SACROFANO, ROMA,ITALY;

2DIP.TO GENETICA E BIOLOGIA MOLECOLARE, UNIVERSITÀ DI ROMA “LA SAPIENZA”, P.LE A. MORO, 5 –00185, ROMA, ITALY;

3ENEA C.R. CASACCIA, V. ANGUILLARESE, 301 – 00060 S. M. GALERIA, ROMA, ITALY;

4 DIP.TO BIOLOGIA CELLULARE E DELLO SVILUPPO, UNIVERSITÀ DI ROMA “LA SAPIENZA”, P.LE A. MORO,5 – 00185, ROMA, ITALY;

Abstract

Neem oil is a natural product obtained from the seeds of the tree Azadirachta indica. Itscomposition is very complex and the oil exhibits a number of biological activities. Themost studied component is the terpenoid azadirachtin which is used for its antimicrobialand insecticidal properties. In this report we investigate the biological activity of partiallypurified components of the oil obtained from A. indica. We show that the semi-purifiedfractions have moderate to severe cytotoxicity. However this is not attributable toazadirachtin but to other active principles present in the mixture. Each fraction wasfurther purified by appropriate extraction procedures and we observed a differentialcytotoxicity in the various sub-fractions.

Keywords: Neem oil, Cytotoxicity, Antiproliferative effect, Apoptosis, PCNA

1. Introduction

Neem oil is a very interesting natural product with diverse potential biological activities(1-3). The oil is made up by a very complex mixture of substances obtained from theseeds of Azadirachta indica (A. Juss, neem tree). This organic mixture is obtaineddirectly from seeds and its production depends on a series of variables connected to theplant (e.g. nature of the soil and climatic conditions). The Ayurvedic medicinetraditionally utilizes decoctions and infusions obtained from seeds, leaves and rootswhich show antipyretic, antiseptic and antiviral properties (4). The natural insecticidalproperties of neem extracts are currently exploited in agriculture for pest control sincethese extracts affect feeding, growth, reproduction and metamorphosis of many insectspecies (3, 5). Biological Control of Plant, Medical and Veterinary PestsR. Strang & H. Kleeberg (eds.)Copyright 2009 by Trifolio-M GmbH

42

These empirical applications find a scientific support in more recent studies; in particularterpenoids, abundant in neem extracts, are endowed with diverse biological activities(6). Azadirachtin, the best characterized component of neem extracts, is a potentinhibitor of insect cell proliferation, but can also affect mammalian cells even though athigher concentrations (7 and references therein). Other terpenoids, similar in structureto azadirachtin, are able to inhibit the growth of mammalian cell cultures to a lesserdegree (8, 9). The action of azadirachtin on insect cultured cells can be attributed, atleast partly, to the inhibition of the polymerization of tubulin (7). Recent works showedthat different neem extracts exhibit a strong cytotoxic activity, but also anti-inflammatory,antibiotic and antiviral properties. Moreover, a possible usage as an anti-tumor drug hasbeen envisaged (4). Many plants, in fact, produce secondary metabolites that haveclear anti-tumor properties which can be used as an alternative therapy for many typesof hyper-proliferative pathologies. Taxol, camptothecin and colchicine (10 - 14) arepossibly the best characterized among these natural compounds. These play a majorrole in cancer therapy when the standard treatments fail. The “rescue therapy” in thesecases is difficult because most tumors show cross-resistance to compounds exhibitingthe same mode of action. Natural substances in general have a different bioactivity andare able to operate on different cellular pathways (12). Some authors attribute the effectof neem extracts to the terpenoid azadirachtin, although this conclusion is disputable. Infact, the high variability of content of this specific compound and the highly complexformulation of neem industrial preparations, do not allow definitive conclusions (15).

Toxicity studies conducted in vitro on mammalian cells, provide a useful informationregard to critical cellular targets, but reports on the toxicity of neem derived compoundson mammalian cell lines are still conflicting (16, 17).

The work reported here is focused on the characterization of the effects of the neem oilcomponents on cultured mammalian cells i.e. a mouse stabilized fibroblast line (3T6)used as a model system in many works from our laboratory (see, for instance 18 – 20).We observed a cytotoxic effect induced by the application of the oil extracts. This urgedus to investigate the mode of cell death. Our results clearly demonstrate that cells die byapoptosis with a possible involvement of the mitochondrial pathway.

2. Materials and Methods

2.1 Cell Cultures and cytotoxicity assays

The stable murine fibroblasts 3T6 line was used throughout the work. The cultures wereroutinely maintained at 37 °C in 5% carbon dioxide atmosphere. Culture medium wasDMEM-10% newborn serum supplemented with glutamine (50 mM f.c.) and

43

penicillin-streptomycin (10000 U/ml each). Cells were sub-cultured every second day.Fresh cultures were set up after 15 to 20 passages.

The neem components (see below) were assayed by administration to cells plated 24hours earlier. Treatments were continued for 24 hours, if not otherwise specified, in thepresence of 2% ethanol (f.c.) that was added also to controls. Each treatment groupwas carried out in duplicate.

2.2 Phytochemical compounds

Aflatoxin-free neem oil was supplied by Trifolio-M GmbH (Lahnau – Germany). The oilwas extracted with 12 volumes of methanol. The alcoholic polar fraction (MEX) wasused in the assays. Methanol was removed by drying at low temperature (Savant SpeedVac) and the pellet was resuspended in ethanol at 100 mg/ml. The fraction MEX wasalso treated at 90 °C for 24 hrs to destroy azadirachtin and other thermally-unstablecompounds and the pellet resuspended as above (ethanol at 100 mg/ml). This fractionis indicated as HT-MEX. Serial dilutions of MEX and HT-MEX were used asexperimental solutions.

Pure azadirachtin (chromatographic grade courtesy of Trifolio-MGmbH) was used incontrol assays since the content of azadirachtin in the above alcoholic extracts isestimated to range between 0.05 and 0.30 µg/ml.

MEX was fractionated by solid phase chromatography using Supelclean LC-Florisil®

SPE tubes, to obtain fractions “A” to “E”. The column was eluted with petroleum etherand produced fraction “A” and “B”; petroleum ether and ethyl acetate 3:2 (fraction “C”);ethyl acetate (fraction “D”); methanol (fraction “E”). A further fraction “D” was insolublein the aqueous culture medium and was therefore disregarded. Fractions “A” to “E” werealso treated at 90°c for 24 hrs as described above.

Fraction “C” was divided into six additional sub-fractions. Fraction “C” was batch elutedby chromatographic silica column. The elution solvent was hexane/ethyl acetate (1/1,v/v). Eluates were analyzed by thin layer chromatography in the same solvent and eachbatch was pooled according to their rate front. Sub-fractions “f”, “g”, “h”, “i”, “l”, “m”, arethus homogenous pools derived from fraction “C”.

2.3 Estimation of cell viability

After 24 hrs of treatment viable cells were assessed by the MTT assay (21). Theabsorbance of the formazan blue was measured at 570 nm. A standard calibrationcurve was carried out to ensure correlation between the absorbance and the actualnumber of viable

44

Results of the experiments with MTT were analyzed by one way ANOVA and MultipleRange Tests LSD with significance level 0,05 using SPSS for Windows (22), with aminimum of six replicates.

3. Results

3.1 Assessment of the cytotoxicity of neem components

Preliminary experiments were carried out to assay the biological activity and propertiesof the whole oil. However these trials produced conflicting results because of the its lowsolubility in the aqueous culture medium and the different commercial origin of thematerial. Both methanol extracts, MEX and HT-MEX, showed high cytotoxicity; on theother hand pure azadirachtin, at two different concentrations produced no effects oncultured cells (Fig. 1). In addition the bioactivity of the MEX is strongly concentrationdependent (Fig. 2); a significant difference between control and all treated samples wasdetected. No differences were monitored between control and samples treated atconcentrations lower than 100 µg/ml. These results suggest an activity thresholdbetween 10 and 100 µg/ml.

Fractions “A” to “E” also have a concentration dependent activity (Fig. 3). Heating doesnot seem to cause modifications in the biological activity of any fraction, althoughfraction “C” seems to be the most active in the inhibition of cell growth already at 20µg/ml. Statistical differences were observed at 200 µg/ml. Fractions “A” and “C” show adifferential cytotoxicity, while fractions “B” and “E” are more similar to the controlalthough still significantly different from a statistical point of view. Fraction “A” is theflow-through of MEX in the first elution step, therefore it behaves like the whole MEX.

Exposure to sub-fractions “f” to “m” induced toxicity on 3T6 cells. A relevant differenceexists between 20 and 200 µg/ml treatments (Fig. 4). The number of viable cellsstrongly decreases and fraction “h” is the most effective to inhibit 3T6 cell growth,although at 200 µg/ml also fractions “g” and “i” gave similar results.

Cell viability was in all cases assessed by the MTT assay. The decrease ofmitochondrial formazan accumulation indicates cell death and it is considered adiagnostic of mitochondrial damage, for a review see (23).

45

Figure 1. Toxicity of MEX, HT-MEX and Azadirachtin A.

Average number (+ S.D.) of viable cells after 24 h (grey bars) and 48 h (white bars)treatment. MEX and HT-MEX are significantly different from the control (F=39,61;p<0.01; d.f.=29). LSD tests confirmed that no significant differences were foundbetween the treatment with these two components, nor between control andAzadirachtin at both concentration (columns labeled by the same letter are notsignificantly different at the LSD test significance level α<0.05).

Figure 2. Toxicity of MEX and HT-MEX at various concentrations.

Means (+ S.D.) of viable cells referred as percentage to the control after 24 h treatmentwith native MEX (grey bars) and heated (white bars) HT-MEX. Bioactivity of both MEXand HT-MEX is strongly dose dependent. Statistically significant differences were found

46

between control and treatment groups, both for MEX (F=35,01; p<0.01; d.f.=14) andHT-MEX (F=29,66; p<0.01; d.f.=14). LSD tests confirmed a highly significant differencebetween control and treated samples, while no statistical differences were detectedbetween control and samples treated at concentrations lower than 100 µg/ml (columnslabeled by the same letter are not significantly different at the LSD test significance levelα<0.05).

Figure 3. Toxicity of fraction A rhrough E

Means (+ S.D.) of viable cells referred as percentage to the control after 24 h treatmentwith native MEX (grey bars) and heated (white bars) HT-MEX fractions. Heating doesnot cause modifications in the biological activity of any fraction. no significantdifferences were found between heated and native ones at the two testedconcentrations (20 and 200 µg/ml) (respectively F=0,002; p>0.05 d.f.=29) and F=0,52;p>0.05; d.f.=29). Although fraction “C” seems to be the most active to inhibit cell growth,at 20 µg/ml, no significant differences were found among the single fractions both fornative (F=0,76; p>0.05; d.f.=14) and heated ones (F=0,26; p>0.05; d.f.=14). On thecontrary, at 200 µg/ml statistical differences were observed as a consequence of alltreatments (native F=22,36; p<0.01; d.f.=14; heated F=8,33; p<0.01; d.f.=14). LSD testevidenced as Fractions “A” and “C” show difference, while “B” and “E” fractions aremore similar to the control although still significantly different (columns labeled by thesame letter are not significantly different at the LSD test significance level α<0.05).

47

Figure 4. Toxicity of sub-fraction “f” through “m”

Number of viable cells after 24 h treatment with sub-fractions obtained from native MEXat the concentration of 20 µg/ml (grey bars) and 200 µg/ml (white bars). Data shown ahighly significant difference among the treatment groups both at 20 µg/ml (F = 282,99;p<0.01; d.f.=27) and at 200 µg/ml (F = 609,16; p<0.01 d.f.=27). In particular LSD testconfirmed that sub-fraction “h” is the most effective to control 3T6 cells in ourexperimental system, although at 200 µg/ml also sub-fractions “g” and “i” gave similarresults (columns labeled by the same letter are not significantly different at the LSD testsignificance level α< 0.05; groups treated with 200 µg/ml are marked with asterisk).

4. Discussion

Neem oil has many potential biological activities. The composition of this natural andcomplex mixture is variable and highly unpredictable. As a matter of fact, the nature ofits compounds depends on a series of variables connected, for instance, to the natureof the soil and climatic conditions. The traditional Ayurvedic medicine commonly utilizesdecoctions and infusions obtained from seeds, leaves and roots which show therapeuticproperties. Also, the insecticidal action of neem extracts is exploited for pest control. Inthis work we present a characterization of neem components and of partially purifiedfractions. In a series of preliminary experiments we assayed the biological activity of thewhole neem oil. Due to its very low solubility in the culture medium the results of theexperiments were inconsistent. The different commercial origins of the experimentalmaterial played also a role in the poor reproducibility of the results. Throughout thiswork we used an aflatoxin-free oil as guaranteed by the supplier. Therefore all effectsthat we monitored on cells are due to intrinsic features of neem oil fractions. We

48

proceeded with a first very gross purification by extracting the oil with methanol to obtainthe fraction called MEX. This first extraction eliminates from the oil the water insolublelipid component. Heating the MEX at high temperature for 24 hours produces anothercrude fraction, the HT-MEX, that is deprived of heat sensitive terpenoids, among whichis azadirachtin A. Treatment of 3T6 cells with the crude fractions shows that bothextracts have a moderate to quite severe cytotoxic effects also, we detected nodifference after heat inactivation. Pure azadirachtin, on the contrary, does not affect cellviability in our experimental conditions, but shows a hardly interpretable and paradoxicalgrowth stimulatory effect. This result is in apparent contrast with literature data showingthat this compound is toxic to insect cells in culture. As a matter of fact also otherauthors showed that also the murine fibroblast line L929 is resistant to azidirachtin (9),therefore this data could be valid only for mammalian cells.

The effect is highly concentration-dependent both for MEX and HT-MEX and thecytotoxicity threshold seems to be between 10 and 100 µg/ml. We attempted a furtherpurification of the bioactive MEX by column chromatography and obtained five differentfraction named “A” to “E”; fraction “D” was water-insoluble and was therefore excludedfrom the study. Fraction “C” exhibits the most interesting activity comparable with wholeMEX. Fraction “A”, that represents the flow through of the loaded material, has acomposition virtually identical to the whole MEX and thus behaves in the same fashion.The results obtained with fraction “C” confirm that the critical cytotoxic concentration isbetween 10 and 100 µg/ml as already observed with whole MEX and HT-MEX. Fraction“C” was further purified to produce six new sub-fractions (“f” through “m”); sub-fraction“h” is the one endowed with the most relevant bioactivity at low concentration oftreatment (20 µg/ml). However, at higher concentration (200 µg/ml) also other fractionspresent a distinctive cytotoxic effect; this suggests that our purification procedurepermits the isolation of a limited set of components of the whole neem oil principallyresponsible for the negative control of cell growth. Interestingly, both fraction “C” andsub-fraction “h” represent a population of neem components eluted in a medium polarityrange.

The cytotoxic effect of these partially purified neem components was studied by theMosmann assay that is considered diagnostic of mitochondrial malfunction. A damageof this organelle could be involved in the activation of the apoptotic pathway.

In conclusion, we report here a study on the biological activity of neem oil components:this oil is normally considered a by-product of more valuable components. However, theresults of the present work show that our partially purified mixtures have anantiproliferative action on mammalian cells. The results of the MTT assays wouldsuggest a mitochondrial involvement and the triggering of apoptosis, but further studiesare necessary to elucidate this point. Furthermore, an important development of this

49

work, is the evaluation of the differential activity of the neem components towardsprimary and tumor cells; also the complete chemico-physical characterization of thepurified compound(s) endowed with this activity, is a goal of fundamental interest.Experiments are in progress in our laboratory to address these specific points.

Acknowlegedments

This work was supported by grants from BBCA (V.D.I) and MIUR (G.R).

References

Mulla, M. S. and Su, T. (1999) Activity and biological effects of neem products againstarthropods of medical and veterinary importance. J. Am. Mosq. Control Assoc.15, 133-152.

Connell, E. B. (1991) Barrier contraceptives, spermicides, and periodic abstinence.Curr. Opin. Obstet. Gynecol. 3, 477-481

Schmutterer, H. (1990) Properties and potential of natural pesticides from the neemtree, Azadirachta indica. Annu. Rev. Entomol. 35, 271-297.

Brahmachari, G. (2004) Neem, an omnipotent plant: a retrospection. Chembiochem. 5,408-21.

Di Ilio, V., Cristofaro, M., Marchini, D., Nobili, P., and Dallai, R. (1999) Effects of a neemcompound on the fecundity of Ceratitis capitata (Wiedemann) (Diptera:Tephritidae). J. Econ. Entomol. 92, 76-82.

Eppler, A. (1995) Effect of neem on viruses and organism, in The neem Tree(Schmutterer, H., ed.), VCH Publications, Weinheim, New York, pp. 93-106.

Salehzadeh, A., Akhkha, A., Cushley, W., Adams, R.L.P., Kusel, J.R., and Strang,R.H.C. (2003) The antimitotic effect of the neem terpenoid azadirachtin oncultured insect cells. Ins. Biochem. Mol. Biol. 33, 681-689

Rembold, H. and Annadurai, R.S. (1993) Azadirachtin inibits proliferation of Sf9 cells inmonolayer culture. Z. Natureforsh 48, 495-499

Salehzadeh, A., Jabbar, A., Jennens, L., Ley, S.V., Annadurai, R.S., Adams, R. andStrang, R.H.C. (2002) The effects of phytochemical pesticides on the growth ofcultured invertebrate and vertebrate cells. Pest Manag. Sci. 58, 268-276.

Wani, M.C., Taylor, H.L., Wall, M.E., Coggon, P. and Mc Phail, A.T. (1971) Plantantitumor agents. VI. The isolation and structure of taxol, a novel antileukemicand antitumor agent from Taxus brevifolia. J. Am. Chem. Soc. 93, 2325-2327.

Wall, M.E., Wani, M.C., Cook, C.E., Palmer, K.H., Mc Phail, A.T. and Sim, G.A. (1966)Plant antitumor agents. I. The isolation and structure of camptothecin, a novel

50

alkaloidal leukaemia and tumor inhibitor from Camptotheca acuminata. J. Am.Chem. Soc., 88, 3888-3890.

Wall, M.E. and Wani, M.C. (1996) Camptothecin. Discovery to clinic. Ann. N.Y. AcadSci. 803, 1-12.

Wall, M.E. and Wani, M.C. (1995) Camptothecin and taxol: discovery to clinic -thirteenth Bruce F. Cain Memorial Award Lecture. Cancer Res. 55, 753-60.

Lee, K.H. (1999) Novel antitumor agents from higher plants, Med Res Rev. 19, 569-596.

Sidhu, O.P., Kumar, V. and Behl, H.M. (2003) Variability in neem (Azadirachta Indica)with respect to Azadirachtin content. J. Agric. Food Chem. 51, 910-915.

Cohen, E., Quistad, G.B. and Casida, J.E. (1996) Cytotoxicity of nimbolide,epoxyazadiradione and other limonoids from neem insecticide. Life Sci. 58, 1075-1181.

Akudugu, J., Gäde, G. and Böhm, L. (2001) Cytotoxicity of azadirachtin A in humanglioblastoma cell lines. Life Sci. 68, 1153-1160.

Bresin, A., Iacoangeli, A., Risuleo, G. and Scarsella, G. (2001) Ubiquitin dependentproteolysis is activated in apoptotic fibroblasts in culture. Mol. Cell. Biochem.220, 57-60

Campanella, L., Delfini, M., Ercole, P., Iacoangeli, A. and Risuleo, G. (2002) Molecularcharacterization and action of usnic acid: a drug that inhibits proliferation ofmouse polyomavirus in vitro and its main target is rna transcription. Biochimie84, 329-334

Pastore, D., Iacoangeli, A., Galati, G., Izzo, L., Fiori, E., Caputo, M., Castelli, M. andRisuleo G. (2004) Variations of Telomerase Activity in Cultured MouseFibroblasts upon proliferation of Polyomavirus. Anticancer Research 24, 791-794

Mosmann, T. (1983) Rapid colorimetric assay for cellular growth and survival:application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55-63.

SPSS (1993) Statistical Procedures of the Social Sciences, SPSS-X user’s guide, 6thed. SPSS Inc., Chicago,.

Eckert, A., Keil, U., Kressmann, S., Schindowski, K., Leutner, S., Leutz, S. and Muller,W.E. (2003) Effects of EGb 761 Ginkgo biloba extract on mitochondrial functionandoxidative stress. Pharmacopsychiatry 36, 15-23.

51

DEVELOPMENTAL, REGULATORY AND MARKETING EXPERIENCESWITH THE AZADIRACHTIN- BASED PLANT PROTECTION PRODUCTNEEMAZAL-T/S IN THE NETHERLANDS

VAN DEN ENDE, A.

ASEPTA B.V., CYCLOTRONWEG 1, POSTBUS 33, 2600 AA DELFT, THE NETHERLANDS, E-MAIL: [email protected].

Asepta B.V. has been involved in the development and registration of NeemAzal-T/S inThe Netherlands since the early nineteen nineties. The focus of the development ofNeemAzal-T/S was on Integrated Pest Management cropping systems as well asBiological cropping systems in topfruit, horticulture and floriculture.

In the Netherlands NeemAzal-T/S, despite being a biological crop protection compound,was regarded by the registration authorities as a regular crop protection compound.NeemAzal-T/S therefore was evaluated in the same way as done for synthetic cropprotection products. It only was due to properties intrinsic to the active substanceAzadirachtin-A that the registration process was relatively easy as a result of what ahigher Tier approach was not always needed and some data could be waived.Furthermore the political willingness to register a biological compound has helped tospeed up the registration procedure.

In order to achieve a registration several trials have been carried out to prove theefficacy of the product in topfruit, floriculture and horticulture under Dutchcircumstances.

The work on the control of the Dysaphis plantiginea and Lygus pabulinus in apples,where different application timings were tested and a comparison was made with thecommercial standard imidacloprid, showed good efficacy of NeemAzal-T/S.

Table 1. Treatment list of tests in apple 1998.

Green bud(10 Apr)

Pink bud(24 Apr)

End of petal fall(15 May)

Control = O water water waterAsepta NeemAzal T/S = A 3 l/ha - -Asepta NeemAzal T/S = B 3 l/ha 3 l/haAsepta NeemAzal T/S = C - 3 l/ha -Admire (70% imidacloprid)+ wetter = D

- - 0.1 kg/ha +0.1%


52

Fig. 1. Number of Dysaphis plantiginea per leaf (300 leaves / plot)

Fig. 2. Percentage infestation by Disaphis plantiginea (30 shoots/plot; 10 leaves)

Fig. 3. Number of shoots damaged by Lygus pabulinus (50 shoots / plot)

Fig. 4. Number of damaged fruit (21 Sept; 500 / plot)

2.2

0.23

0

0.5

1

1.5

2

2.5

5 June

OABCD----------------LSD (P< 0.05)

12.5

4.21.7

0.5

4.2

1.2

0

5

10

15

20

Shoot Leaf

OABCDLSD (p<0.05)

25.5

1116.5

20.515

42

05

101520253035404550

28 May

OABCD--------------LSD (P<0.05)

05

10152025303540

D. plantigineea % L. pabulinus %

OABCD--------------LSD (P<0.05)

53

Table 2. Treatment list of tests in apple 1999

Fig. 5. Number of Dysaphis plantiginea per leaf (300 leaves /plot)

Fig. 6. Number Dysaphis platiginea infested shoots (30 / plot)

Fig. 7. Number of Lygus pabulinus damaged shoots (50 / plot)

Pink bud(14 Apr)

Blossom start(23 Apr)

Fruit 6-10 mm(20 May)

Control = O water water waterAsepta NeemAzal T/S = A 3 l/ha - -Asepta NeemAzal T/S = B - 3 l/ha -Admire + wetter = C - - 0.1 kg/ha + 0.1%

1,95

0,42

0,0025 0,00250,34

0

0,5

1

1,5

2

2,5

3

Aphid /leaf (03 June)

OABC--------------LSD (p<0.05)

1,50,25 0,25

2,86

11,25

0

2

4

6

8

10

12

3 June

OABC---------------LSD (P<0.05)

8.75

2.51.75 2.25

3.3

0

2

4

6

8

10

02 June

OABC---------------LSD (p<0.05)

54

Fig. 8. Number of fruits damaged (14 October; 700 / plot)

Furthermore the effect on the population level of Forficula auricularia in apple orchardswas investigated in 2004 and shown to be absent.

Table 3. Treatment list of tests in apple 1998

Fig. 9. Number of Forficula auricularia per assessment unit (18 June)

In floriculture in greenhouses the efficacy against Trialeurodes vaporariorum wasvariable. The levels of efficacy of the commercial standard pyridaben never wasreached.

Table 3. Treatment list of tests in hibiscus 2000.

93,5

13,2514,54,5

112,25

14,53,75

27,37

6,04

0

20

40

60

80

100

Dysaphis plantiginea Lygus pabulinus

OABC---------------LSD (P<0.05)

Object Pink bud (13 Apr) Full BlossomControl = O water waterAsepta NeemAzal T/S = A 3 l/ha -Asepta NeemAzal T/S = B 3 l/ha 3 l/ha

05

1015202530354045

Larvae Adults

OAB----------------LSD (p<0.05)

Object RateControl waterAsepta NeemAzal T/S 0.25%Aseptacarex (pyridaben 157 g/l) 0.07%Admiral (pyriproxifen 100 g/l) 0.025%

55

Fig. 10. Number of Trialeurodes vaporariorum larvae per leaf (50 leaves / plot)

Table 4. Treatment list of tests in hibiscus 2001.

Fig. 11. Number of Trialeurodes vaporariorum larvea and pupae per leaf (50 / plot)

The efficacy against Tetranychus urticae was more consistent and on levelscomparable to the commercial standard pyridaben.

Fig. 12. Number of Tetranychus urticae per leaf (50 / plot)

0

10

20

30

40

50

21June

28June

5 July 12July

19July

26July

Control

Asepta NeemAzalT/SAseptacarex

Admiral

---------------

LSD (P<0.05)

Object RateControl waterAsepta NeemAzal T/S 0.25%Aseptacarex (pyridaben 157 g/l) 0.07%

01020304050607080

20June

27June

4 July 11July

18July

25July

Control


----------------

LSD (P<0.05)

0

1

2

3

4

5

6

20June

27June

4 July 11July

18July

25July

Control


----------------

LSD (P<0.05)

56

Against Clepsis spectrana in alstroemeria (Amaryllidaceae) NeemAzal-T/S showed agood effect.

Fig. 13. Number of plants damaged by Clepsis spectrana in Alstoemeria in 2004.

It turned out that in Dutch greenhouse circumstances in general multiple consecutiveapplications are needed. The development stage of the pest population in whichapplication of NeemAzal-T/S is started is critical to its success. NeemAzal-T/S performsbest when used in situations where population dynamics are not too intense, such asspring and fall. The above mentioned is supported with experiences from commercialuse of NeemAzal-T/S after a registration was gained in June 2003. FurthermoreNeemAzal-T/S has proven not always to be innocuous on specific rose varieties grownin the greenhouse.

0

5

1015

20

25

30

8July

16July

20July

26July

03-Aug

Control

AseptaNeemAzal T/S0,25%

57

EXPERIENCES IN HIGH QUALITY MANUFACTURING OF NEEMAZAL

S. S. PILLAI, S. RADJENDIRANE AND G.R. SRIDHAR

BIO-PRODUCTS DIVISION, E.I.D. PARRY (INDIA) LTD.CHENNAI, INDIA

E.I.D. Parry (India) Limited (Parry), is a part of Murugappa Group, a businessconglomerate based in Chennai, in South India, currently having a sales of over US$1.2 Billion. The Group is in diversified businesses like Farm Inputs, Abrasives, SteelTubes, Bicycles, Insurance, Plantations, Nutraceuticals, Sugar, Sanitaryware andBio-Products.

Parry, the flagship company of Murugappa Group, is nearly 218 year old and this is oneof the oldest companies in India. The company is in the business of Sugar,Sanitaryware and Bioproducts.

Bio-Products business of E.I.D. Parry started with a collaboration agreement withTrifolio-M GmbH of Germany in the year 1992, for the process of extraction of anAzadirachtin rich concentrate – NeemAzal, from Neem Seed Kernels. Parry up-scaledthe process in to a commercial technology and set up a commercial plant. Thecommercial plant for the production of NeemAzal in India established in 1998 has thecapacity produce 7000 Kgs of 100% Azadirachtin. NeemAzal and its range of productsare being marketed in 23 countries around the world, with the main markets being USA,Europe and India.

Factors influencing quality of NeemAzal

Quality of Neem Seeds:

The Raw material for the production of NeemAzal is Neem Seeds. The quality ofNeemAzal is significantly affected due to the quality of the Neem seeds, both in terms ofthe Azadirachtin content as well as fungal contamination. The procurement of Neemseeds is carried out by a team of experienced people. The process starts with theproper education of the villagers who collect the seeds and trade in them. They aretaught the skills to ensure that the fruits are de-pulped using simple device and driedbefore sending them to the procurement centers. Over the years, Parry has been ableto identify the geographical locations in India, from where seeds with comparativelyhigher Azadirachtin content can be procured and maximizes procurement from theselocations.

Once the raw seeds are brought to cleaning centers operated by Parry, they arewashed with water and dried to bring the moisture levels to less than 10%. The seeds,Biological Control of Plant, Medical and Veterinary PestsR. Strang & H. Kleeberg (eds.)Copyright 2009 by Trifolio-M GmbH

58

which contain approximately 20 % extraneous matter is cleaned through a series ofprocesses. A typical analysis of the yield in the cleaning process is given in Table 1below:

Table 1. Typical analysis of raw neem seed

Storage of Neem Seeds:

The cleaned seeds need to be stored in specially designed storage facilities to ensurethat the azadirachtin content does not deteriorate. The design of the facility alsoensures that there is no opportunity for fungal contamination.

De-cortication to kernels:

Dried and cleaned Neem seeds are decorticated using a specially designed equipmentto get unbroken kernels, which are used for the extraction process

NEEMAZAL EXTRACTION PROCESS

The process consists of a three-step extraction process followed by, removal ofaflatoxins and vacuum drying.

1. SOLID-LIQUID EXTRACTION:

‘Solid-liquid extraction’ is the first step in which neem seed kernel is extracted withSolvent 1. Kernel and Solvent 1 are mixed in a reactor and continuous circulation iscarried-out to extract azadirachtin and other active ingredients in to the medium. Thisprocess takes 8 to 12 hours time to reach a saturation point. This extract is filtered andthe neem kernel removed after filtration process is dried and put though a millingprocess to extract oil and residual cake. The Solvent 1, containing azadirachtin, is takenfor further process.

Details in %Raw neem seed 100Dried and Clean neem seed 80Neem fruits 5.5Over size other seeds 0.1Dust 3.5Stones 1.5Moisture 9.4

59

2. LIQUID – LIQUID EXTRACTION:

To the filtered extract which contains azadirachtin and other active ingredients, Solvent2 is added for Liquid – Liquid extraction. After separation of Solvent, in to which theactive ingredients are extracted in preference over Solvent 1, the extract is centrifugedto remove suspended solids before evaporation.

3. EVAPORATION & CONCENTRATION:

Solvent 2 containing the active ingredients, collected from the centrifuge, is subjected toevaporation in two stage evaporators where evaporation takes place under vacuum,controlled temperature and in a closed system. This ensures that the active ingredientsare not affected due to high temperature.

4. PURIFICATION & REMOVAL OF AFLATOXIN:

The concentrate containing azadirachtin is precipitated in Solvent 3 to remove impuritiesand pure azadirachtin settles at the bottom. This is once again re-dissolved in Solvent 2and subjected through a proprietary process for removal of aflatoxins. The resultantextract is dried under vacuum at controlled temperature without affecting the product.The final dried powder is brownish yellow in color containing minimum of 35%azadirachtin and other active ingredients of Neem Seed Kernels. This product isNeemAzal and the typical analysis of is given in Table 2.

Table 2. TYPICAL ANALYSIS OF NEEMAZAL

*PCM – Partially Characterized Matter

CERTIFICATIONS:

The NeemAzal production facility of Parry is certified by Institute fur Markotokolige,(IMO) Switzerland as suitable for organic cultivation.

The facility has also obtained the ISO 14001:1996 Environmental Management SystemStandard approval from DET NORSKE VERITAS.

Ingredients in %Aza A 30.9Aza B 6.17Salannin 2.90Nimbin 0.81Other limonoids 21.63Fats & Lipoids 4.60PCM* 31.50Water <1.00

61

THE POSSIBILITIES OF USING DIFFERENT NEEM PREPARATIONS FORPEST CONTROL IN VEGETABLES IN THE SUDAN

EL SHAFIE, H.A.F., MUDATHIR, M. (KHARTOUM) & 1BASEDOW, TH. (GIESSEN)1 INSTITUTE OF PHYTOPATHOLOGY AND APPLIED ZOOLOGY, GIESSEN UNIVERSITY, EXPERIMENTALSTATION, ALTER STEINBACHER WEG 44, D-35394 GIESSEN, GERMANY

Abstract

Studies were conducted in solanaceous crops (potatoes, aubergines and tomatoes), inokra (Abelmoschus esculentus, Malvaceae), and in onions (Liliaceae). Preparationsused were: 1. Neem Kernel Water Extract (NKWE) of local material; in tomato, okra andonions: sesame oil was added. 2. NeemAzal T/S (NA). 3. Neem Oil (NO) (in potatoesand aubergines). In potatoes and aubergines, Jacobiasca lybica, Bemisia tabaci andAphis gosypii proved to be best controlled by NA. NO and NKWE were less effective.While NA increased yield significantly in all experiments, NO and NKWE did this in onlyone of three experiments. In tomatoes, NA exerted a significant negative effect on B.tabaci, NKWE on A. gossypii. The agromyzid fly Liriomyza trifolii was controlled by NAand NKWE plus sesame oil. NKWE plus sesame oil increased the yield of tomatoessignificantly. In okra, A. gossypii, B. tabaci, Earias vitella (Lep., Noctuidae) andPodagrica puncticollis (Col., Chrysomelidae) were controlled by NA and NKWE plussesame oil, which resulted in increased yield. In onion, Thrips tabaci was not affectednegatively by neem preparations.

1. Introduction

SCHMUTTERER (1969) has shown that crops in the Sudan are prone to the attack bymany pests. Their control with synthetic insecticides bears problems, e.g. insecticideresistance in pests (CAHILL et al. 1995). It is therefore important to find alternativeproducts for pest control. Since neem trees are abundant in the Sudan (BADI et al.1989), the use of neem preparations may be of advantage (SCHMUTTERER 1995). Wehave tested therefore different neem preparations in five vegetable crops in the Sudanfor pest control. A report on the results (EL SHAFIE & BASEDOW 2003; MUDATHIR &BASEDOW 2004) is given here.


62

2. Study sites

The studies took place from summer 1997, to winter 1999/2000 in North Khartoum(Shambat), Sudan, at the Khartoum University demonstration farm. The fields of thestation were situated near to the river Nile and had silty clay-loam soil, with a pH of 7.

At Khartoum, the summer season lasts from April to October, with temperatures mostly31-35°C and a maximum of 42°C. It includes a rainy period, July and August (70 mmprecipitation). The winter season lasts from November to March, mostly at 21-29°C anda minimum of 16°C. Of the most common vegetables, potato is grown in the winterseason, while okra, onion and tomato are grown in the summer. Egg plant can be grownin either season.

Growing, fertilising and irrigation (with Nile water, every fourtnight) of the crops studiedfollowed local practice. Nitrogen was applied as urea (46% N), at 240 to 300 kg ha-1

(110 to 138 kg N. ha-1), depending on the crop in two applications three weeks apart.

3. Methods

3.1 experiments with potatoes and aubergines (El Shafie & Basedow 2003)

The following preparations were applied weekly: NeemAzal-T/S® (NA), neem kernelwater extract (NKWE), Neem Oil with emulsifier Rimulgan® (NO), Fenvalerate(Sumicidin®; Sumitomo) (synthetic pyrethroid). Neem kernels were obtained as localfresh material, while NO and NA came from Germany (Neem-Handel, D-64347Griesheim, and Trifolio-M, D-35633 Lahnau, respectively). NKWE was prepared using50 g fresh neem seeds (ground) per litre as a water extract filtered through fine gauzebefore spraying. In the first season, no fresh neem seeds were available and NKWEwas not used. Concentrations of the other preparations, at 20 l ha-1 with a rotatingatomiser (ULVA + Sprayer Micron Sprayers Ltd., Bromyard, Herts, U.K.) at therecommended dosages (table 1). The main active ingredient of the neem preparationsis azadirachtin A (AzaA), an antihormone for insects (Schmutterer 1995). TheAzaA-content of NA and NO was 1% and 0.024%, respectively. The azadirachtinA-content of the local fresh seeds was not measured but was estimated to be 0.2 to0.5% (average 0.32%) (Ermel 1995).

Bemisia tabaci Genn. (Hom., Aleyrodidae), Aphis gossypii Glov. (Hom., Aphididae) andJacobiasca lybica (de Berg) (Hom., Cicadellidae) were present on both crops (Siddig1987).

Two field experiments (winter season) with potato (cv. "Alpha"), and three experiments(two in winter, one in autumn), with eggplant (cv. "Long Purple") were conducted with 5x 5 m plots, with four replicates. Insect counts were started two weeks before the first

63

treatment. Twenty plants per plot were examined for insect attack on each of ten days,with five leaves per plant. The assessment of yield depended on the crop studied. Inpotatoes, one m² per plot was harvested at the end of the season. In egg plant, fruitshad to be harvested in the centre of the plots during the whole fruiting season for five toten weeks, every three to seven days. A cumulative value per unit area was calculatedin the end of the season, as tons per feddan (1 feddan = 0.42 ha).

Insect attack was calculated as cumulative insect-days (CID) of seven dates (Ruppel1983). Statistical analysis was conducted with the SPSS-programme, selecting theappropriate tests.

Table 1. The preparations, their ingredients, and their concentrations used in all trials with ULVA +sprayer

1) We are grateful to Dr. B. RUCH, Lahnau, for the analysis.

2) according to ERMEL 1995

3.2 Experiments with tomatoes, okra, and onions (Mudathir & Basedow 2004)

On plots of 6 x 7 m, with 3 replicates, experiments were conducted in Okra, 1997-99,Onion and in Tomato, 1998. Preparations used were local Neem Kernel Water Extract(NKWE, with addition of sesame oil), NeemAzal-T/S (NA), and for comparisonFenvalerate (Sumicidin®) (partly). Applications were done weekly, 8 times per growingseason. Also harvest and insect counts were conducted weekly, in okra and tomato. Inonions, the bulbs were harvested in the end of the experiments.

Preparation Mostimportantactiveingredient

a. i. (g.l-1) Rate ofapplication(l ha-1)

a. i. (g.ha-1) in20 l of waterha-1

NeemAzal-T/S (NA) Azadirachtin A 10 2 20

Neem Oil +Rimulgan®

(emulsifier) (NO)

Azadirachtin A 0.241) 0.08 0.2

Local neem kernelwater extract (NKWE)

Azadirachtin A 0.1 to0.25(estimated)2)

20 2 to 5

Sumicidin® 20 % EC Fenvalerate 200 0.7 140

64

4. Results

4.1 Potatoes

In potatoes, two field experiments were conducted. The findings in potatoes are seen inTable 2 and fig. 1. With weekly treatments, NA gave the same results as Fenvalerate, inpest attack as in yield. The effects of NO were not significant, while NKWE had a lowereffect than NA.

4.2 Aubergines

In aubergines, three field experiments were possible. Fig. 2 and 3 show that the attackby Bemisia tabaci and Jacobiasca lybica were reduced by weekly treatments by NAnearly as well as by Fenvalerate, but also significantly by NO and NKWE.

The yields are shown in fig. 4. NA-treatments gave the highest yields, followed byFenvalerate. NO and NKWE gave significant yield increase only in one experiment,each.

65

Table 2. Cumulative insect-days (per 100 leaves following seven counts) in field experiments in potato,Khartoum North. Weekly applications with VLV-technique. For treatment concentrations used,see Table 1. Figures in a column followed by different letters are significantly different (p < 0.05,Tukey). Data in parenthesis indicate the per cent reduction of insect numbers compared withwater control

Period Treat- Cumulative insect-days(winter) ment Jacobiasca lybica Bemisia tabaci Aphis gossypii1998/1999 Water 34,792 ± 1,585.2a

(100 %)2,739.4 ± 90.6a(100 %)

508,729.5 ± 45,756.3a(100 %)

NA 5,047.5 ± 82.7b(- 85.5 %)

812.2 ± 107.1bc(- 70.3 %)

109,538.2 ± 2,465.5b(- 78.5 %)

NO 12,100.6 ± 1,626.5c(- 65.2 %)

1,067.7 ±1786.7c(- 61.0 %)

808,783.7 ± 1,797.8c(- 60.0 %)

Fenvale-rate

4,881.4 ± 263.5b(- 86.0 %)

748.5 ± 157.9(- 72.7 %)

63,046.9 ± 2,310.2b(- 87.6 %)

1999/2000 Water 21,784.8 ± 1,334.0a(100 %)

7,566.7 ± 656.7a(100 %)

407,797.8 ± 9,475.3a(100 %)

NA 4,009.4 ± 350.7b(- 81.60 %)

1,773.2 ± 237.1b(- 76.50 %)

109,447.2 ± 3,278.5c(- 73.2 %)

NO 9,273.8 ± 768.9c(- 57.4 %)

2,709.3 ± 410.1c(- 64.2 %)

164.850.5 ± 2.923.5d(- 59.6 %)

NKWE 12,000.3 ± 1,171.4d(- 44.9 %)

3,304.9 ± 295.5c(- 56.3 %)

174,860.8 ± 1,581.5d(- 57.1 %)

Fenvale-rate

3,905.7 ± 76.4b(- 82.1 %)

1,673.7 ± 172.8b(- 77.9 %)

64,570.5 ± 2,694.2b(- 84.2 %)

Mean re-duction

NA(- 77.59)

- 83.6 - 73.4 - 75.8

(%) NO(- 61.23)

- 61.3 - 62.6 - 59.8

NKWE(- 52.78)

- 44.9 (n = 1) - 56.3 (n = 1) - 57.1 (n = 1)

Fenvale-rate(-81.73)

- 84.0 - 75.3 - 85.9

66

Fig. 1. The yield of potatoes in two field experiments with different weekly treatments (withVLV-technique) at Khartoum North. For concentrations used, see Table 1. Columns of one yearwith different letters are significantly different (p < 0.05, Tukey)

4.3 Okra, tomatoes and onions

The results are shown in Table 3 and 4.

In okra, Aphis gossypii (Hom. Aphididae), Bemisia tabaci (Hom., Aleyrodidae), Eariasvittella (Lep., Noctuidae) and Podagrica puncticollis (Col., Chrysomelidae) could besuccessfully controlled by both neem preparations. Also the yields in neem treatmentsproved to be higher than in untreated and in the Sumicidin-treatment.

In tomato, the attack by Aphis gossypii and Bemisia tabaci was significantly reduced bySumicidin. The neem preparations showed varying effects: NA reduced significantly B.tabaci, and NKWE A. gossypii. Liriomyza trifolii (Dipt., Agromyzidae) could be controlledby the neem preparations as well as by Sumicidin. The yield of tomatoes wassignificantly increased by the NKWE-treatment, only.

In onion, Thrips tabaci can be controlled by Sumicidin, but not by neem, as anexperiment in 1998 showed.

potato yield

01234567

W ater NA NO NKW E Fenval.

w eekly treatm ents

yiel

d (t

fedd

an-1

)

1998/991999/00

a a

bb

a ac

b

b

67

Fig. 2. Cumulative insect-days (CID) of B. tabaci on eggplant with different weekly treatments byVLV-technique, Khartoum-North, Sudan, 1998-2000. For concentrations used, see Table 1.

Columns of one year with different letters are significantly different (p < 0.05, Tukey)

5. Discussion

The findings reported are confirmed by other authors. So, for Liriomyza trifolii: KNAUSS

& WALTER (1995) and for homopterous pests: LOWERY et al. (1993); BASEDOW et al.(2002). On the basis of the results obtained here, it can be concluded, that neempreparations do have the potential partly to replace the use of synthetic insecticides inthe Sudan. Before coming to further conclusions, two points have to be considered inthis connection: 1. The availability of neem trees in Sudan, in relation to the growingarea of vegetables, and 2. The necessary work for preparing Neem Kernel WaterExtract (NKWE).

The number of neem trees in the Sudan is not exactly known, but estimated at morethan one million (BADI et al. 1989, FÖRSTER & MOSER 2000). The annual yield of seedsof a neem tree averages ca. 20 kg (SCHMUTTERER 1995), but also lower yields of 5 kgper tree have been reported (FÖRSTER & MOSER 2000). The area under cultivation ofvegetable in Sudan is estimated at 36,000 hectare (BAUDOIN 1994). If it is assumed,that one kg of neem Seed is needed per hectare, there is no doubt that the neem seedavailable in Sudan is sufficient: If only 5 kg seed per each of one million trees areassumed in the Sudan, per year 5,000 t seed would be available.

B e m is ia ta b a c i, e g g p la n t

0

1 0 0

2 0 0

3 0 0

4 0 0

5 0 0

6 0 0

7 0 0

8 0 0

W a te r N A N O N K W E F e n va l.

CID

(10 p

lants

, 7 d

ates)

1 9 9 8 /9 9 1 9 9 9 1 9 9 9 /0 0

a

cd

b

a

c cd

b

a

b b b b

68

Additional to the work when a commercial insecticide is used, five steps are necessaryin case of neem seed: Harvesting/collecting of neem seed; depulping and drying it;storing it properly (since the harvesting period of neem fruit in the Sudan does notcoincide with the need in the field); decortication; finally grinding (HELLPAP & DREYER

1995). So, the use of NKWE is not cheap, when all necessary procedures are regarded(though – in contrast to imported products – it only consumes local resources).Considersing the labour involved, it can be expected that only a limited number ofSudanese farmers will use NKWE.

Fig. 3. Cumulative insect-days (CID) of J. lybica on eggplant with different weekly treatments byVLV-technique, Khartoum-North, Sudan, 1998-2000. For concentrations used, see Table 1.Columns of one year with different letters are significantly different (p < 0.05, Tukey)

J . ly b ic a , e g g p la n t

0

1 0 0 0

2 0 0 0

3 0 0 0

4 0 0 0

5 0 0 0

6 0 0 0

W a te r N A N O N K W E F e n v a l.CID - 1

0 plan

ts, 7

dates

1 9 9 8 /9 9 1 9 9 9 1 9 9 9 /0 0

a

c

bb

a

b

c c

b

a

cd

e

b

69

Fig. 4. Effects of weekly treatments (with VLV-technique) of different preparations on aubergine yield inthree subsequent seasons at Khartoum North, Sudan. For concentrations used, see Table 1.Columns in one season with different letters are significantly different (p < 0.05, Tukey)

Under the precondition that chemical companies carry on developing safer insecticides,it can be said finally, that it is important for sustainable agriculture to have manypossibilities of plant production, and of plant protection. The mixture of these will givethe optimal output and biodiversity (BASEDOW 2002). Alternating use of neempreparations and synthetic insecticides could also reduce the danger of risinginsecticide resistance in pests. The effects of neem preparations on E. vittella give hintsto the use of neem also in cotton, against Earias insulana (Boisd.), the Spiny Bollworm.

EL SHAFIE & BASEDOW (2003) in potatoes and eggplants (with ULV-technique), showedthat neem preparations can be used also in these crops, being harmless to beneficialinsects. So neem kernels, available in Sudan, can be used there successfully to replacethe use of synthetic insecticides, in vegetable production (at least partly). The control ofE. vittella gives hints to the possible use of neem preparations against Earias insulanain cotton.

The problem of the application of neem preparations in the Sudan will remain one of theavailability and of the price.

e g g p la n t y ie ld

02468

1 01 21 4

W a te r N A N O N K W E F e nva l.

w e e k ly tre a tm e n ts

yield

(t fed

dan

-1)

1 9 9 8 /9 9 1 9 9 9 1 9 9 9 /0 0

b db

aa

aa

b

a

b c bc

b

70

Table 3. The attack of okra, tomato and onion by pests, in the field experiments at Khartoum-North,1998-99. Active ingredient applied per ha increased with the spraying volume during theseason, to cover greater plants. Figures in one line followed by different letters are significantlydifferent at p = 0.05 (LSD)

Crop Pest species,average attack

Year Control(water)

NeemKernelWaterExtract

(2.5-5.0 gAzaA/ha)

plus SesameOil

NeemAzal-T/S(6-12 g

AzaA/ha)

Sumicidin20 EC

(50-100 gFenvalerate/

ha)

Okra Aphis gosypii,no./leaf

1998 20.6b 9.0a 8.0a -

1999 420.4c 273.3b 127.5a 478.1cBemisia tabaci,no./leaf

1998 11.6b 3.1a 4.5a -

Earias vittella,no. infestedplants

1998 10.5b 1.1a 1.6a -

Podagricapuncticollis, no.damagedleaves

1998 4.5b 0.4a 1.4a -

Tomato Aphis gosypii,no./leaf

1998 10.1b 5.9a 8.6b 4a

Bemisia tabaci,no./leaf

1998 4.8b 3.3b 1.6a 1.5a

Liriomyza trifolii,no. of leavesattacked

1998 3.3b 1.3a 1.8a 1.1a

Onion Thrips tabaci,no./plant

1998 39.3b 31.5b 30.9b 18.9a

71

Table 4. Cumulative yield (kg/125 m²) of Okra and Tomato, and final yield of onions (kg/12 m²) in thefield experiments at Khartoum-North, in the seasons 1997-99. Active ingredient applied per haincreased with the spraying volume during the season, to cover greater plants. Figures in oneline followed be different letters are significantly different at p = 0.05 (LSD)

References

BADI, K. H., AHMED, A. E. H. & BAYOUMI, A. A. M. S. (1989): The forest of Sudan.Ministry of Agriculture, Khartoum, Sudan.

BASEDOW, TH. (2002): Konventionelle Landwirtschaft (in ihrer gegenwärtigenAusprägung) oder ökologische Landwirtschaft? – Für die maximale Biodiversitätsind beide erforderlich. (Conventional Agriculture (in its present form) orEcological Agriculture? - For the maximal biodiversity both are necessary).Gesunde Pflanzen 54, 177-182.

BASEDOW, TH., OßIEWATSCH, H. R., BERNAL VEGA, J. A., KOLLMANN, S., EL SHAFIE, H.A. F. & NICOL, C. M. Y. (2002): Control of aphids and whiteflies (Homoptera:Aphididae and Aleyrodidae) with different Neem preparations in laboratory,greenhouse and field: effects and limitations. Journal of Plant Diseases andProtection 109, 612-123.

BAUDOIN, W. O. (1994): An approach towards the production of healthy vegetables inAfrica. In: DABROWSKI, Z. T. (Ed.): Integrated vegetable crop management in theSudan. ICIPE Science Press, Nairobi, Kenya, 6-11 (ISBN 9290640871).

CAHILL, M., BYRNE, F.J., GORMAN, K., DENHOLM, I. & DEVONSHIRE, A.U. (1995):Pyrethroid and organophosphate resistance in tobacco whitefly Bemisia tabaci(Homoptera: Aleyrodidae). Bull. Ent. Res. 85, 181-187.

ERMEL, K. (1995): Azadirachtin content of neem seed kernels from different regions ofthe world. In: SCHMUTTERER, H. (Ed.) The Neem Tree. Source of unique naturalproducts for integrated pest management, medicine, industry and otherpurposes. Weinheim, New York, Basel, Cambridge, Tokyo (VCH), 89-92.

Crop year Control(water)

Neem Kernel WaterExtract (2.5-5.0 g

AzaA/ha) plusSesame Oil

NeemAzal-T/S(6-12 g

AzaA/ha)

Sumicidin20 EC (50-100 gFenvalerate/ha)

Okra 1997 28.3a 73.9b 69.4b -1998 37.8a 58.0b 51.0b -1999 29.0a 36.9b 41.4b 28.8a

Tomato 1998 39.0a 68.2b 54.5a 49.3aOnion 1998 4.0a 4.0a 4.1a 4.8a

72

EL SHAFIE, H.A.F. & BASEDOW, TH. (2003): The efficacy of different neem preparationsfor the control of insects damaging potatoes and eggplants in the Sudan. CropProtection 22, 1015-1021.

FÖRSTER, P. & MOSER, G. (2000): Status report on global neem usage. UniversumVerlagsanstalt, D-65175 Wiesbaden, pp. 8-28. GTZ, Eschborn, Germany.

HELLPAP, C. & DREYER, M. (1995): The smallholder’s homemade products. In:SCHMUTTERER, H. (Ed.) The Neem Tree. Source of unique natural products forintegrated pest management, medicine, industry and other purposes. Weinheim,New York, Basel, Cambridge, Tokyo (VCH), 367-375.

KNAUSS, J.F. & WALTER, J.F. (1995): Control of pests of ornamentals in greenhouses inthe U.S.A. with ‘Margosan-O’. In: SCHMUTTERER, H. (Ed.) The Neem Tree.Source of unique natural products for integrated pest management, medicine,industry and other purposes. Weinheim, New York, Basel, Cambridge, Tokyo(VCH), 437-445.

LOWERY, D. T., ISMAN, M.B. & BRARD, N.L. (1993): Laboratory and field evaluation ofNeem for the control of aphids (Homoptera: Aphididae). J. Economic Entomol.86, 864-870.

MUDATHIR, M. & BASEDOW, TH. (2004): Field experiments on the effects of neemproducts on pests and yields of Okra (Abelmoschus esculentus), Tomato(Lycopersicum esculentum), and Onion (Allium cepa) in the Sudan. Mitteilungender Deutschen Gesellschaft für Allgemeine und Angewandte Entomologie 14,407-410.

SCHMUTTERER, H. (1969): Pests of Crops in Northeast and Central Africa. Fischer,Stuttgart und Portland, 296 S.

SCHMUTTERER, H. (Ed.) (1995): The Neem Tree. Source of unique natural products forintegrated pest management, medicine, industry and other purposes. Weinheim,New York, Basel, Cambridge, Tokyo (VCH), 695 S.

Ruppel, R. F., 1983. Cumulative Insect-Days as an index of crop protection. Journal ofEconomic Entomology 76, 375-377.

Siddig, A. S., 1987. A proposed pest management program including neem treatmentfor combating potato pests in the Sudan. In: Schmutterer, H. and Ascher, K. R.S. (Eds.): Natural pesticides from the neem tree and other tropical plants Proc3rd International Neem Conference (Nairobi, 1986) pp. 449-459, GTZ Eschborn,Germany.

.

73

BIO-EFFICACY OF NEEMAZAL® AND ITS FORMULATION AGAINSTSPOTTED LEAF BEETLE, HENOSEPILACHNA VIGINTIOCTOPUNCTATA(FAB)

R. SUDHAKARAN, LIZA JOZ AND D. SREENIVASA RAO

BIO-PRODUCTS DIVISION, EID PARRY INDIA LTD., R&D CENTRE, 145, DEVANAHALLI ROAD, OFF. OLDMADRAS ROAD, BANGALORE 560 049, KARNATAKA, S. INDIA

Abstract

The activity of azadirachtin-rich neem seed kernel extract NeemAzal®, its formulationsand pure Azadirachtin-A (Aza-A) was compared in the laboratory against the grubs ofspotted leaf beetle Henosepilachna vigintioctopunctata (Fab) (Coccinellidae:Coleoptera). Antifeedancy, growth inhibition, mortality and adult emergence activitieswere evaluated. The EC50 (antifeedancy and growth inhibition), LD50 and LT50 valueswere calculated using Probit analysis. The comparative activity of Aza-A, NeemAzal®

and its formulations was discussed and presented.

Keywords: Henosepilachna vigintioctopunctata, Azadirachtin-A, NeemAzal®,Antifeeding Index, Growth Inhibition, Mortality, LD50, LT50 and EC50

Introduction

Spotted leaf beetle, Henosepilachna vigintioctopunctata (Fabr.), is a major pest ofvegetables. It has been reported attacking several Solanaceous and Cucurbitaceouscrops like brinjal, tomato, potato, gourds, melon and cucumber (Bhalla and Pawar,1977; Hill, 1987). Both the adults and grubs feed voraciously on the leaves. The leavesare eaten between the veins, sometimes being completely stripped to the midribs, this isthe typical symptom of the infestation (Hill, 1987). Bio-pesticides have gainedimportance today due to the various problems of synthetic pesticides like residues, pestresistance, pest resurgence etc. The effect of azadirachtin and related compoundsagainst this pest was investigated and reported to have growth-disturbing andantifeedant effects (Schmutterer, 2002).

The present study was undertaken to investigate the virtuosity of NeemAzal® and itsformulations against the 3rd instar larvae of H. vigintioctopunctata for antifeedancy,mortality, pupation and adult emergence. The comparative efficacy of these productswith azadirachtin was also studied.


74


The test samples, NeemAzal® (Aza-A 33.72 %), the neem seed extract, and itsformulations NeemAzal® F 5 % and NeemAzal®-T/S 1 % were provided by E.I.D. Parry(I) Ltd., Thyagavalli. Aza-A (99.4% pure) was isolated and its purity was established inthe laboratory. The test solutions were prepared on the basis of Aza-A concentrationand by dissolving the required quantity of the samples in distilled water in standardvolumetric flasks.

The insects used for the bioassay were reared in the laboratory at R&D Centre, EIDParry (I) Ltd., Bangalore. The insects collected from the brinjal field were used for theinitiation of culture and were reared in sterilized cages and containers. The massrearing was done using self-cultivated brinjal leaves.

The leaf disc method was followed in this study which was used to study the activity ofazadirachtin earlier with lepidopteran larvae (Ramachandran et al., 1989). According tothe method, tender brinjal leaves were cut into circular discs of 50 mm dia. using a leafperforator after washing the leaf thoroughly with distilled water. The leaf discs weredipped in different test solutions for 1 min and later air dried for 30 min and placed overmoist filter paper in petri dishes (9 cm). Leaf discs dipped in water were used asuntreated control. Freshly moulted third instar grubs of Henosepilachna beetles werecollected from the stock culture and preconditioned for 6 hrs without food. One grub wasreleased into the each petri dishes containing the treated leaves and allowed to feed for48 hrs. After 48 hrs of treatment, fresh untreated leaves were given until pupation ordeath. Three replications with four grubs under each treatment were maintained.

Antifeeding index was calculated after 48 hrs of feeding. The treated and untreated leafdiscs were subjected to leaf analysis using leaf analysis software (Marcatus ImageAnalysis Ver 1.2). The Antifeeding Index (AFI) was calculated by the following formulaas adopted by Mathur and Nigam (1993).

Area consumed in untreated control - area consumed in treatedAntifeeding Index (AFI) =___________________________________________ X 100

Area consumed in untreated control

Growth inhibition (GI %) was measured by weighing the larvae on of 0th, 2nd and 5 Daysof Treatment (DAT). The weight gained by the larvae on these days was calculated bysubtracting the initial weight from final weight. GI (%) was calculated using formulagiven below

75

Mean of difference in weight in control - Mean of difference in weight in Treated%GI=___________________________________________________________ X 100

Mean of difference in weight in control

Mean of each replicate is calculated separately and the GI % of the treatment is arrivedby calculating the mean of the replications.

Mortality was recorded at intervals of 2, 5 and 7days after treatment. The grubs havingno activity when touched were considered as dead. Moribund larvae or incompletedevelopment also considered as dead. The mortality was corrected using the methodadopted by Regupathy and Dhamu (2001) as follows.

Mortality in treatment - Mortality in untreated

Corrected mortality (%) =__________________________________________X 100

100 - Mortality in untreated

The percentage of pupation and adult emergence was also recorded. The aboveexperiments were carried out at ambient laboratory conditions. The data on growthinhibition, antifeedancy and mortality were subjected to Probit analysis (Finney, 1971) todetermine EC50 for antifeedancy and growth inhibition and LD50 and LT50 values.

Results and Discussion

The antifeeding index is positively correlated with the concentration of Aza-A in all thetest samples. As the concentration increases the antifeedancy also increased and amaximum of 95.58 % was observed with NeemAzal®-T/S at a dose equivalent to 23ppm of Aza-A whereas pure Aza-A gave 83.82 % at 30 ppm. The EC50 for antifeedancywas found to be 2.95 ppm for Aza-A but was observed low for NeemAzal®-T/S (1.26ppm Aza-A), NeemAzal® F (1.54 ppm Aza-A) and NeemAzal® (2.29 ppm Aza-A). Krauset al. (1993) reported 13 ppm of Aza-A as EC50 for antifeeding activity to Epilachnaverivestis. Ascher (1981) found that antiffedant effect was prominent in the 4th instarlarvae when aqueous emulsions of methanolic neem seed kernel extracts sprayed onbean plants. Both the larvae and adults showed reduction in the feeding when neem oilis treated to the host plants in the laboratory (Tewari and Moorthy, 1985).

In case of growth inhibition (GI), all the samples have shown higher activity at doses of10-15 ppm of Aza-A. Interestingly, the GI has decreased with increase in Aza-Aconcentration (25 and 30 ppm), which may be due to the higher antifeedant activity.EC50 for growth inhibition was observed to be high at 0.57 ppm for Aza-A, compared to0.039, 0.025 and 0.26 ppm of Aza-A equivalent of NeemAzal®-T/S, NeemAzal® F andNeemAzal® respectively. Rembold (1989) reported that EC50 value for growth inhibition

76

was 1.66 ppm when the Aza A is given through feeding to adults of E. verivestis. Steets(1975) reported that when the grubs and adults of epilachna beetles were treated withazadirachtin and methanolic leaf extract of neem, a significant reduction in the feedingand disturbance in the growth and metamorphosis. Schwinger et al. (1984) reportedboth antifeedant and growth disturbing effect of neem against the 4th instar grubs ofEpilachna beetle. The EC90 values for both antifeedancy and growth inhibition whichwere determined in the present study also indicated the same trend of activity.

The mortality was cent per cent at doses equivalent to 10-20 ppm of Aza-A for all thetest samples whereas it was recorded as slightly lower for Aza-A (25-30 ppm). LD50 forNeemAzal® T/S was found to be 0.80 ppm azadirachtin-A, whereas NeemAzal®,NeemAzal® F and Aza-A have recorded 1.20, 1.49 and 2.23 ppm respectively (Table 1).The time that required to effect 50 % mortality (LT50) was determined at a single dose of25 ppm Aza-A for all the samples which was found to be 5.25, 4.79, 3.23 and 4.01 daysfor Aza-A, NeemAzal®-T/S, NeemAzal® F and NeemAzal® respectively.

Table 1. Efficacy of NeemAzal®, its formulations and Aza-A against spotted leaf beetle, H.vigintioctopunctata against antifeedancy, growth inhibition and mortality

Though the larval mortality was observed low for NeemAzal® and its formulations atlower doses of Aza-A (1 and 5 ppm), the subsequent pupation was hampered and amaximum of 25 % of larvae only were pupated. Higher pupation of 58.3 % wasobserved for pure Aza-A at these concentrations. In addition to this a delay of 4-8 dayswas observed in pupation with all the test samples compared to untreated control. AgainNeemAzal® and its formulations have shown longer delay than that of pure Aza-A(Table 2). Furthermore, lower doses of Aza-A resulted in deformed pupae and adults. Similar results were reported by Rembold (2002), that doses of 1.25 and 0.25 ppm ofAza-A would interfere with larval-pupal and pupal-adult moulting process respectively.

In the case of adult emergence, it was observed at doses equivalent of 1 ppm of Aza-A.All the samples except NeemAzal® and pure Aza-A have inhibited adult emergence

Parameters NeemAzal® NeemAzal®-T/S NeemAzal® F Aza-AAntifeedancy-Median effective concentration

EC50 – 2 DAT 2.29 ppm 1.26 ppm 1.54 ppm 2.95 ppmEC90 – 2 DAT 36.58 ppm 18.65 ppm 23.73 ppm 66.37 ppm

Growth Inhibition - Median effective concentrationEC50 – 5 DAT 0.26 ppm 0.039 ppm 0.025 ppm 0.569 ppmEC90 – 5 DAT 2.13 ppm 6.92 ppm 15.95 ppm 59.69 ppm

Mortality Median Lethal dose

LD50 – 7 DAT 1.20 ppm 0.80 ppm 1.49 ppm 2.23 ppmLD90 – 7 DAT 26.47 ppm 22.90 ppm 10.08 ppm 12.94 ppm

Median Lethal timeLT50 – 25 ppm 4.01 days 4.79 days 3.23 days 5.25 daysLT50 – 20 ppm 4.13 days 4.25 days 3.51 days 4.97 days

77

(Table 2). Any survived pupae and adults were observed to be severely malformed, withshriveled wings, and were often unable to complete eclosion.

Table 2. Efficacy of NeemAzal®, its formulations and Aza-A against spotted leaf beetle, H.vigintioctopunctata against pupation and adult emergence

Thus, the above studies evidenced that Aza-A imparts antifeedance and affects thegrowth and metamorphosis of spotted leaf beetle at different doses. Higher activitieswere exhibited for NeemAzal® and its formulations when compared to pure Aza-Aalone, which could be attributed to the presence of other limonoids and active principlesof neem. Thus, effective control of these insects would be achieved with NeemAzal®

and its formulations.

References

Ascher, K.R.S. 1981. Some physical (solubility) properties and biological (Sterilant forEpilachna varivestis females) effects for a dried methanolic neem (Azadirachtaindica) seed kernel extract. In: Proc. 1st Int. Neem Conf. (Rottach-Egern,Germany, 1980) Eds. Schmutterer, H., Ascher, K.R. S. and Rembold, H. pp,63-74.

Bhalla, O.P. and Pawar. A.D. 1977. A survey study of insect and non-insect pests ofEconomic Importance in Himachal Pradesh. Tiku, KitabMahal. 192, D.N. road,Bombay. p.80.

Finney, 1971, Probit Analysis Cambridge University Press. p. 333.

Hill, D.S. 1987. Agricultural insect pests of the tropics and their control. II edition.Cambridge University Press. New York. p. 438.

Parameters /Dose NeemAzal® NeemAzal®-T/S NeemAzal® F Aza-APupation (%)

30 ppm 0 8.33 (18 DAT) 0 025 ppm 8.33 (18 DAT) 0 0 020 ppm 0 0 0 015 ppm 0 0 0 010 ppm 0 0 0 8.33 (17 DAT)5 ppm 16.67 (20 DAT) 0 0 16.66 (17 DAT)1 ppm 25.00 (19 DAT) 16.67 (16 DAT) 16.67 (16 DAT) 58.33 (17 DAT)Untreated control 91.67 - 100 (12 DAT)

Adult emergence (%)30 ppm 0 0 0 025 ppm 0 0 0 020 ppm 0 0 0 015 ppm 0 0 0 010 ppm 0 0 0 8.33 (22 DAT)5 ppm 0 0 0 16.66 (21 DAT)1 ppm 16.67 (21 DAT) 0 0 41.67 (22 DAT)Untreated control 91.67 - 100 (16-18 DAT)

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Kraus, W., Bokel, M., Schwinger, M. Vogler, B., Soellner, R., Wendisch, D., Steffens, R.and Wachendorff, U. 1993. The chemistry of Azadirachtin and other insecticidalconstituents of Meliaceae. In: Phytochemistry and Agriculture. Eds. Van Beek,T., Breteler, H. Oxford University Press, Oxford, UK, pp. 18-39.

Mathur, Y. K. and Nigam. S. 1993. Insecticide, Antifeeding and juvenilizing effects ofneem (Azadirachtia indica A. Juss) oil against Corcyra cephalonica Staint. andEpilachna vigintioctopunctata F. In : Neem and Environment. Ed. Singh et al.Vol. 1. pp. 335- 342.

Ramachandran, R., S. N.Mukherjee and R. N. Sharma. 1989. Effects of fooddeprivation and concentration of azadirachtin on the performance of Achoeajanata and Spodoptera litura on young and mature leaves of Ricinus communis.Entomol Exp. Appl. 51: 29-35.

Regupathy, A and Dhamu, K. 2001. Statistics work book for Insecticide Toxicology.Tamil Nadu Agricultural University. pp. 15-22.

Rembold, H. 1989. Azadirachtins, their structure and mode of action. In. Insecticides ofPlant origin. Eds. Arnason, J.T., Philogene, B.J.R., Morand, P. ACS Symp. Ser.387, American Chemical Society, Washington, DC, USA, pp. 150-163.

Rembold, H. 2002. Growth and Metamorphosis. In: The Neem Tree, Azadirachta indicaA. Juss and other meliaceous Plants. Ed. Schmutterer, H. II edition. NeemFoundation, Mumbai. pp. 237-254.

Schmutterer, H. 2002. The Neem Tree, Azadirachta indica A. Juss and othermeliaceous Plants. II edition. Neem Foundation, Mumbai.

Schwinger, M., Ehhammer, B. and Kraus. W. 1984. Methodology of the Epilachnavarivestis bioassay of antifeedants demonstrated with some compounds fromAzadirachta indica and Melia azadarach. In : Natural pesticides from the neemtree and other tropical plants. Proc. 2nd Int. Neem Conf. (Rauischhilzhausen,Germany) Eds. Schmutterer, H., Ascher, K. R.S. pp. 181-198.

Steets, R. 1975. Die wirkung von Rohextrakten aus den Meliaceen Azadirachta indicaand Melia azederach auf verschiedene insektenarten. Z. angew. Entomol. 77:306, 312.

Tewari, G.C. and Moorthy. P.N.K. 1985. Plant extracts as antifeedants againstHenosepilachna vigintioctopunctata (Fabricius) and their effect on its parasite.Ind. J. Agric. Sci., 55 (2): 120 – 124.

79

THE IMPACT OF AZADIRACHTIN A ON SOME FOREST PEST INSECTS

H. MALINOWSKI, M. DOBROWOLSKI

FOREST PROTECTION DEPARTMENT, FOREST RESEARCH INSTITUTE, SEKOCIN-LAS, 05-090 RASZYN,POLAND

Abstract

In the presented research the impact of Azadirachtin A on some forest pest insects wasassessed. The preparation NeemAzal-T/S (Trifolio-M GmbH, Germany) in 6concentrations of active compound was applied to larvae of nun moth (Lymantriamonacha L.), pine moth (Dendrolimus pini L.) and pine sawflies (Neodiprion sertiferGeoffr.). The feeding activity, larval weight and mortality, number of pupae and adultswere measured after 7 (pine moth), 5 (nun moth) and 3 days (sawfly) of feeding oflarvae on Scots pine (Pinus silvestris L.) needles treated with preparations. Next alllarvae were fed on untreated pine shoots until the pupation and emergence of adultsoccurred. Generally the effect on feeding activity was dependent on the concentration ofAzadirachtin A. The highest decrease in feeding was observed in the case of nun mothlarvae (80-95% decrease compared to the control). The lowest effect occurred in thecase of N. sertifer - 60-85% reduction of feeding. Dramatic reduction in feeding wascorrelated with sharp decrease in the body weight of larvae and with very high mortality.The same was in the case of pupation and emergence of adult insects. The number ofpupae decreased inversely with the concentration of Azadirachtin A, and only in thecase of the lowest concentrations of the active compound did imagos emerge. Theconclusion is that the concentration of Azadirachtin A for practical control of these forestpest insects may be at the level of 0,0001%, but this has to be confirmed by fieldevaluation.

Introduction

Polish forests dominated by Scots pine stands are often threatened by outbreaks ofdangerous insect pests like nun moth or pine moth. The total area of insecticide controlof insect populations exceeds tens of thousands of hectares per year. In some years itreaches hundreds of thousands of hectares. Additionally, the chemical control isconcentrated in some forest complexes and repeated there many times. Such a highintensity of using of insecticides in forests results in many disadvantageous changes inthe forest environment. The main problem in this situation is the relative low selectivityof insecticides used and following this decline of many animal species which are playingBiological Control of Plant, Medical and Veterinary PestsR. Strang & H. Kleeberg (eds.)Copyright 2009 by Trifolio-M GmbH

80

regulatory role in ecosystems. Searching for new insecticides which do not cause suchstrong side effects is the main task for today.

Botanical insecticides obtained from neem tree (Azadirachta indica ) seem to be verypromising compounds for controlling forest insect pests; hence, the idea of this projectand the choice of insect species used in the investigation. The aim of the study was toassess different effects of azadirachtin A on the larvae of three serious forest pests.

Materials and methods

As an insecticide for tests we used Azadirachtin A obtained from Trifolio-M GmbH(Germany) as NeemAzal-T/S containing 10 g/l of active compound. The insecticide wasused as water emultions at six concentrations: 0.1%, 0.01%, 0.001%, 0.0001%,0.00001%, 0,000001%.

Biological tests were carried out on nun moth (Lymantria monacha L., Lepidopt.) andpine moth (Dendrolimus pini L., Lepidopt.) second instar caterpillars and on sawfly(Neodiprion sertifer Geoffr., Diptera) second instar larvae.

2 years old shoots of Scots pine were treated by dipping for 10 seconds in watersolutions of Azadirachtin A and after that they were dried. 15 or 20 larvae of testedspecies were put on them. Then larvae on needles were reared in Petri dishes inconstant conditions (Sanyo Incubator, T=25ºC, RH=75%, L:D=16:8).

Two to five repetitions of every concentration were made. The number of repetitionsdepended on insect species.

When the treated food had been consumed, the new untreated shoots were deliveredfor breeding. Breeding was continued until the last pupation occured or last larva died inthe dish.

The same routine was used for controls treated with pure water. The followingparameters were assessed: body and excrement weight, mortality of larvae and pupae,number of emerging pupae and adults.

Results and disscussion

We observed very strong antyfeedant effect in all studied pests (Fig. 1, 2, 3). Weight ofpine moth excrements decreased strongly in two highest concentrations but at 1 ppm concentration this parameter reached the similar value as the excrement weight in thelowest concentration after one month. Surprisingly in the lowest concentration theweight of excrements was even higher than in control. In the case of nun moth theobserved decrease in the excrements weight was similar in every examinedconcentration.

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The difference between control and other variants in excrement weight was noticedalready five days after exposition and it increased during subsequent days. In pine moththis difference was lower than in other species. In sawfly, at all concentrations, thedecrease in this parameter was very marked and only in the lowest concentrationfeeding it was continued longer than six days after exposition.

The impact of azadirachtin on examined pests resulted in high mortality of larvae (Fig.4). The highest mortality was noticed in the sawfly variant. After twelve days of theexperiment all larvae in all concentration variants were dead.

In the case of Lepidoptera caterpillars we noticed 100% mortality in two highestconcentrations and very high mortality in 10 ppm concentration. The 1 ppmconcentration caused mortality lower than 50% but in two lowest concentrations weobserved the differences between these two pests. Nun moth caterpillars were moresusceptible and the mortality was observed in all variants whereas pine moth larvae didnot die in the lowest concentration.

The strong decrease in feeding resulted in retarded developement of larvae. This wasobserved in all tested pests, but the longest delay of larval growth was noticed in thepine moth variant (Fig. 5). The larval development was delayed at every concentration besides the highest one where all larvae died after 34 days. The delay was the highestat 10 ppm but larvae which formed cocoons after 75 days of experiment didn't formpupae and they all died at this stage. At 1 ppm the period of developement was shorterand body weight was similar to the control one but all larvae died after they formedcocoons.

Pupation of pine moth was observed in the lowest two concentrations but weight andpercent of formed pupae was apparently lower than in control. The time ofdevelopement in these two concentrations was longer than in control too.

After all the emergence of adults in pine moth and in nun moth in two lowestconcentrations was observed. In the case of nun moth about 10-20% of pupae passedinto the adult stage in relation to the concentration. In compare 50% of adults emergedfrom pupae in the control variant. The very similar results were obtained in pine mothexperiment. We didn't notice any female in both pests so we couldn't say if thesterilisation effect occurred. We didn't notice any morphological abnormalities too.

The explanation which mode of azadirachtin action was responsible for observed effectsis very difficult but it can be said that very quick and strong decrease in food uptake wasthe result of so-called "secondary antifeedant" effect or anorexia. Only in the case ofsawfly we observed the primary or gustatory effect in two highest concentrations. In thiscase the treated food was not eaten until the end of experiment. In other concentrationand pest variants all treated food was eaten progressively. All disturbances in

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developement, elongation of moulting periods and inability of pupae forming are widelyknown effects of changes in hormone balance made by azadirachtin in many insects.

First conclusion derived from this research is that the azadirachtin A causes strongphysiological effect in all tested species. It is observed at every concentration ofcompound used in experiment.

The very important conclusion from the practical point of view is that even in mediumconcentrations of azadirachtin the uptake of needles is not broken completely down. Itmeans that damages in foliage of tress are growing until the last larva stops eatingbefore pupation or last moulting. This slow mode of action of azadirachtin may bestrongly criticised by some foresters. In the case of high population density thedecrease in this parameter maybe not appropriate to reduce damages to the safetylevel. On the other side the natural ability of trees for regeneration and the presence ofweakened pest larvae as a good food for parasites and predators gives chances of thedevelopment of the natural regulatory response to the outbreak.

It may be suggested that the effective field dose of azadirachtin A used against forestpests could be at the range between 0.0001 and 0.00001%. However, this must betested in the field experiments

Fig. 1. The influence of azadirachtin on the feeding of the 2nd instar larvae of pine moth (Dendrolimuspini)

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Fig. 2. The influence of azadirachtin on the feeding of the 2nd instar larvae of nun moth (Lymantriamonacha)

Fig. 3. The influence of azadirachtin on the feeding of the 2nd instar larvae of pine sawfly (Neodiprionsertifer)

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Fig. 4. The influence of azadirachtin on the mortality of nun moth (Lymantria monacha), pine moth(Dendrolimus pini) and pine sawfly (Neodiprion sertifer) 2nd instar larvae

Fig. 5. The influence of azadirachtin A on the body weight of pine moth 2nd instar larvae

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Fig. 6. The influence of azadirachtin on the pupation and the weight of pine moth (Dendrolimus pini)

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ASSESSING THE EFFICACY OF NEEM FORMULATIONS AGAINST THEFEEDING ACTIVITY OF THE LARGE PINE WEEVIL HYLOBIUS ABIETIS

W BRYAN AND J R M THACKER

DEPARTMENT OF BIOLOGICAL SCIENCES, UNIVERSITY OF PAISLEY, SCOTLAND, PA1 2BE, UK, EMAIL: [email protected]

Introduction

The large pine weevil (Hylobius abietis) is the principal noxious pest in reforestationareas throughout the UK and Scandinavia. Attracted to their breeding grounds byvolatile chemicals released from recently felled trees, adult H. abietis invade restocksites and lay their eggs in and around tree stumps. The young stages of H. abietis feedbelow the ground cause no economic damage, however the adults, above the ground,feed on the stems of young seedlings of the newly planted crop (Fig. 1). Feeding by H.abietis scars the phloem of seedlings, and girdling of the stem base will rapidly killthem.

With populations recorded of up to 125,000 insects per hectare, losses of seedlings canrange from 30-100% in sites where no plant protection is provided. Feeding damagegenerally occurs when the adults are active between March and October, though it isalso recognised that there are two distinct feeding peaks for H. abietis. That said,feeding is often difficult to predict. As a consequence since 2004 prophylacticprotection is routinely provide as a combination of pre-planting and post plantingtreatments using the synthetic pyrethroid alpha-cypermethrin.

Pre-planting treatment is carried out at a centralised treatment facility using theelectrodyn spray system and is said to provide up to 6 months protection. Following

The adults feed on the

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The larvae utilise conifer stumps as a

breeding substrateFig. 1.


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planting, ‘top up’ treatments are applied in the field using a knapsack sprayer; thisprovides protection lasting up to 6 weeks (Fig. 2). Neem formulations are currentlybeing assessed as plant protection products for forestry use primarily due to theuncertain future of the pyrethroid insecticides but also due to the ongoing concerns ofoperator exposure to these chemicals and the growing concerns for the environment.

Methods

Field trials were established in the spring of 2003 and 2004 to assess the efficacy ofthree neem formulations, NeemAzal-T/S (10,000ppm AZA), NeemAzal-T (50,000ppmAZA) and Bugban (Neem Seed Oil). Trials were situated in restock sites in LochArdForest (exp. 1-4) and Forest of Ae (exp. 5), central and southern Scotland, respectively,where H. abietis were expected to be present. All the trials were set out in arandomised block design using standard planting methods. Three year old Sitka spruce(Picea sitchensis) seedlings of approximately 30-40cm in height, which where of seedorigin Queen Charlotte Islands, Washington, were used. Unless otherwise stated,treatments were targeted to treat the main stem 0-15cm upwards from the root collar. Two controls were used for each experiment 1) untreated control 2) synthetic chemicalof current use: either Alpha-cypermethrin or Permethrin (Permit ® (12% a.i.).

Exp. 1(a). The objective of this experiment was to assess the efficacy of theazadirachtin formulations NeemAzal-T/S and NeemAzal-T, against the syntheticchemical Alpha-cypermethrin. Three concentrations of the neem formulations were

Time of treatment Method ofapplication

Active Ingredient Premixed Doseapplied

Pre-planting Electrodyn Alpha-cypermethrin(6%)

0.1ml Alpha C 6ED

Post planting Knapsacksprayer

Alpha-cypermethrin(0.1%)

10ml Alphaguard100EC

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used, these were: undiluted, 50% dilution and 25% dilution (25% chemical: 75%dilutant); the dilutant used was water. Applications of treatments were madepre-planting using a paint brush to apply 5ml per plant. The synthetic chemical (AlphaC6EC) was applied pre-planting using an electrostatic application system at a dose of0.1ml per plant. Exp. 1(b). Procedures where exactly the same as those in exp. 1(a)and planted beside 1(a) for comparison but application of all treatments (includingsynthetic chemical (Alphaguard 100EC)) was applied post planting using a forestry spotgun fitted with a narrow cone nozzle and calibrated to deliver a 10ml dose per plant. Exp. 2. The objective of this experiment was to assess the efficacy of undilutedazadirachtin formulation NeemAzal-T/S applied at two different treatment lengths, 15cmand 30cm at a dose of 10 and 20ml respectively. All treatments were appliedpre-planting; NeemAzal was applied with a paint brush whilst the permit treatment wasapplied using an electrostatic application system at a dose of 0.1ml per plant. Exp. 3 (a& b). The objective of these two experiments was to assess the dose response ofBugban. Treatments used were; A-1000g/l (no dilution), B- 800g/l, C- 600g/l, D- 400g/l,E- 200g/l, F- permethrin (exp. 4) /alpha-cypermethrin (exp. 5) & G- untreated control(exp. 4)/vegetable oil (exp. 5), the dilutant used was vegetable oil. Exp. 3(a). Applications of treatments were made pre-planting, the treatments were applied using apaint brush at a dose of 10ml per plant, the synthetic chemical (AlphaC 6EC) wasapplied using an electrostatic application system at a dose of 0.1ml per plant. Exp. 3(b). Due to the consistency of treatments A & B application had to be applied by paintpad before planting. The remaining treatments were applied post planting using aforestry spot gun fitted with a narrow cone nozzle and calibrated at 10ml dose perplant.

Two types of assessment were used 1) percentage bark removed (0-15cm from rootcollar) which was recorded regularly throughout the season between May andNovember (graphs 1-4) and 2) an assessment at the end of each feeding peak (Julyand November), to assess the seedlings chances of survival (graph 5).

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Results

Fig. 1 (a)

Fig. 1 (b)

Fig. 1 (a & b) Efficacy of azadirachtin formulations NeemAzal-T and NeemAzal-T/S applied at threedifferent dilutions (a) before planting with assessment at wk 12 and (b) after planting withassessment at wk 16. Each trial consisted of eight treatments with twelve plants per plot andeach plot was replicated five times n = 480 (plant no). Arcsine transformed data shows meanpercent bark removed with respect to treatment. Error bars signify 95% CL and analysis ofvariance (ANOVA single factor) demonstrates a statistically significant difference betweentreatments for both graphs Fig. 1(a) (P=2.47 x 10¯17) and Fig. 1(b) (P= 1.58 x 10¯17).

Fig. 1(a). After a period of 12 weeks, pre-planting application of NeemAzal-T at thethree concentrations and NeemAzal T/S undiluted and at 50% dilution were able toreduce feeding damage significantly in comparison to that of the untreated control.

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NeemAzal-T applied at 50% dilution showed that feeding was reduced to a levelstatistically comparable to that of the protection provided by the alpha-cypermethrininsecticide. The untreated control and NeemAzal-T/S treatment at dilution 25% havethe same value at 38% damage which is significantly higher than that ofalpha-cypermethrin (9%). The efficacy of azadirachtin formulations appears to increasewith the increasing concentration of azadirachtin. Although protection is notcomparable to the synthetic chemical control, when applied post-planting the onlytreatment which reduced feeding significantly lower than that of the untreated controlwas using NeemAzal-T at 25% dilution (value 26%). All other neem treatments whenapplied post planting showed an approximate value of 33% bark removed, indicatinglittle reduction in feeding damage in comparison to the untreated control (37%).

Fig. 2. Efficacy of NeemAzal-T/S applied undiluted before planting at two different treatment lengths. Arcsine transformed data showing mean percent bark removed at 26 wks with respect totreatment. Error bars signify 95% CL Analysis of variance (ANOVA single factor) demonstratesa statistically significant difference between treatments (P= 7.854 x 10¯6). The trial consisted offour treatments with nine plants per plot, each plot was replicated four times n = 144.

Fig. 2 illustrates the effects of NeemAzal-T/S when applied at different treatment lengths(15 & 30cm) against the feeding damage of H. abietis. The graph indicates that therewas no significant difference in protection provided by applying treatments at differentlengths. Both NeemAzal-T/S treatments reduced feeding damage to a level comparableto that of the protection provided by the synthetic chemical and were significantly lowerthan that of the untreated control.

Fig. 3(a) demonstrates that all of the chemical treatments received significantly lessdamage than that of the untreated control. Treatments A (100%) through to D (40%)dilution produced less damage than that of the chemical control although treatment C

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(60%) was not able to reduce the damage significantly. In fig. 3(b) the letters areincluded to show treatment differences. Pairs of treatments with no letter in common aresignificantly different from each other (assuming a 5% significance level for each pairwise comparison). All treatments except E (20%) exceeded the untreated control (G)and treatments A (100%), B (80%), C (60%) and D (40%) were all comparable to F(permit).

Fig. 3(a)

Fig. 3(b)

Fig. 3 (a & b). Dose response of neem oil at five different concentrations. Arcsine transformed datashowing (3a) mean percent bark removed and (3b) mean percent survival at 8 wks withrespect to treatment (A)1000g/l (100%), (B) 800g/1l (80%), (C) 600g/1l (60%),(D) 400g/1l (40%)(E) 200g/1l (20%) (F) synthetic chemical and (G) Control. Error bars signify 95% CL. Fig. 3(a)The trial consisted of seven treatments with twelve plants per plot, each plot was replicated fivetimes n = 420. (P= 1.16 x 10¯38). Fig. 3 (b). The trial consisted of 36 plants per plot with fivereplicates per treatment, n=1260. (P=0.017).

Discussion

Pre-planting application of Azadirachtin formulations NeemAzal-T and NeemAzal-T/S(concentrations 50,000ppm – 5,000ppm AZA) were able to reduce the feeding of H.abietis significantly for a period of 12wks compared to the untreated control. When

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applied post-planting and assessed at 16wks, treatments NeemAzal–T 25%(12,500ppm) was the only treatment able to reduce damage to a level significantly lowerthan that of the untreated control. Despite increasing the dose of formulations appliedfor post planting treatment visual observations after treatment noted the unevenapplication of formulations. The data highlights that the mode of application is critical indetermining the efficacy of neem insecticides.

The application of NeemAzal-T/S undiluted and applied at 10ml was able to reducefeeding damage to a level comparable to the synthetic chemical permethrin after aperiod of 26weeks (Fig. 2). However, the site management where this trial was plantedinvolved mounding the ground, which not only benefits the growth of the plant but isalso said to reduce feeding damage by H. abietis. It is therefore suggested that thecombined effects of the mounding and the neem contributed to the lower levels offeeding over such a long period.

Dilutions of neem oil produce a reduction in feeding damage of H. abietis atconcentrations down to 40% neem oil after a period of 8 weeks. As neem oil is anundwefined mixture of active ingredients with little azadirachtin A, further research isrequired to assess the effects of the different active ingredients within the oil asinsecticides. It is apparent that azadirachtin formulations are able to reduce the feedingdamage of H. abietis with the efficacy increasing with an increase in azadirachtincontent. Whether this is economic for forestry use is, as yet, unknown.

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SIDE EFFECT OF SOME NEEM PRODUCTS ON NATURAL ENEMIESOF HELICOVERPA TRICHOGRAMMA SPP. AND CHRYSOPERLACARNEA

NABIL EL-WAKEIL1&2, NAWAL GAAFAR 1&2 AND STEFAN VIDAL2

1) PESTS& PLANT PROTECTION DEPT., NATIONAL RESEARCH CENTRE, CAIRO, EGYPT

2) INSTITUTE OF PLANT PATHOLOGY, GEORGE- AUGUST UNIVERSITY, 37077 GOETTINGEN, GERMANY [email protected]

ABSTRACT

The present work was conducted to evaluate the side effect of neem products asnatural insecticide on some of natural enemies of Helicoverpa armigera Hüb.(Trichogramma spp. and Chrysoperla sp.). The work is divided into two parts, laboratoryand greenhouse experiments. The laboratory experiments dealt with the side effect ofNeem products; NeemAzal-T/S, NeemAzal Blank (composition: all formulation additiveswithout NeemAzal technical), NeemAzal PC 05 and NeemAzal PC 05 Blank onTrichogramma spp and C. carnea (Stephen). Neem concentrations; 2, 1, 0.5 and 0.25%were arranged in a completely randomized block design and each treatment wasreplicated at least 6 times. Parasitism and emergence rates of T. pretiosum (Riley) andT. minutum (Riley) and predation rates of C. carnea and its longevity were investigated.The greenhouse experiments dealt with side effect of Neem products on parasitismrates of T. pretiosum and T. minutum on 3 different cotton cultivars. There was noserious side effect on parasitism and emergence rates of Trichogramma spp., and onefficiency of Chrysoperla. Similarly, neem products achieved a good control of H.armigera in laboratory and greenhouse. Therefore, neem products are recommendedfor controlling Helicoverpa and are compatible with mass release of Trichogramma andChrysoperla. Future field experiments are suggested to further elucidate the effects ofneem on natural enemies of H. armigera.

Keywords: Neem, Helicoverpa, Trichogramma, Chrysoperla, laboratory andGreenhouse

INTRODUCTION

Studying side effects of pesticides on beneficial arthropods is very important to test thesuitable techniques and standard methods which are urgently needed for soundIntegrated Pest Management (IPM) (Kakakhel & Hassan 1998). Despite the sensitivityof insects of most orders to azadirachtin, neem products are selective, as they do notBiological Control of Plant, Medical and Veterinary PestsR. Strang & H. Kleeberg (eds.)Copyright 2009 by Trifolio-M GmbH

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harm important natural enemies of pests (Isman & Port ,1990). They are also non-toxicto warm-blooded animals. Neem-seed extracts have, therefore, a considerable potentialfor integrated pest control measures (Schmutterer, 1988). Neem has systemic activity, itis active at low concentrations, and degrades rapidly in the environment (Schmutterer,1988, 1990). The side-effect test revealed that NeemAzal-T/S is harmless to numerousof natural enemies. The test was based on evaluation of the survival of the larvae, theirdevelopment and pupation and the fecundity and fertility of the resulting adults(Natarajan 1990; Salem & Matter 1991; Kaethner 1991; Kienzle & Zebitz 1996).

The results of laboratory experiments conducted to study the effect of neemformulations compared with insecticides on Trichogramma species, based on rate ofparasitization and emergence of adults from parasitized eggs, revealed that neemproducts were safer compared to synthetic insecticides (Klemm & Schmutterer ,1993;Lakshmi et al., 1997; Zhang MinLing, 1997; Dodan & Roshan 1999; Markandeya &Divakar 1999; Thakur & Pawar 2000; Prabal & Parameswaran 2001). Raguraman &Singh (1999) tested neem seed oil at concentrations of 5.0, 2.5, 1.2, 0.6, and 0.3% foroviposition deterrence, feeding deterrence, toxicity, sterility and insect growth regulatoreffects against T. chilonis Ishii. Neem seed oil at 0.3% deterred oviposition(parasitization) by the parasitoid. Neem seed oil also deterred feeding at or above 1.2%concentration. In feeding toxicity tests, neem seed oil at 5% concentration caused 50%mortality to both males and females.

Schuster & Stansly (2000) tested Azatin EC on two species of green lacewings; theyfound that the neem product was not toxic to eggs, larvae and adults topically orresidually. On the other hand NeemAzal-T/S (formulation dried residues on glasspanes) was harmful to larvae of the lacewing C. carnea causing mortality and difficultiesto molt (Schmutterer 1997; Hermann et al., 1997; Srinivasan & Babu 2000). Thelongevity of treated adults by some neem products ranged between 18.66 and 20.66 d,while it was 23.66 d in controls. Fecundity was also affected slightly by all neemproducts (599.66 to 741.66 eggs) as against 874.66 eggs in controls, (Karuppuchamy etal., 1998; Deole et al., 2000)

Results of using parasitoids and predators in integrated pest management of H.armigera in cotton fields, to investigate egg parasitism, toxicity of insecticides toparasitoids and predators, revealed that egg parasitism in the laboratory by T. chiloniswere 75.6% (Raja et al., 1998). Nimbecidine (a neem product) tested against T. chilonisand C. carnea resulted in zero mortality. The integrated pest management components(T. chilonis, C. carnea, and nimbecidine) gave a good control to H. armigera (Reddy &Manjunatha 2000). Therefore, we will study the side effects of neem products (1) onparasitism rates of T. pretiosum (Riley) and T. minutum (Riley) in the laboratory and

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greenhouse, (2) on emergence rates of T. pretiosum and T. minutum in the laboratory(3) on predation rates and longevity of C. carnea in the lab.

MATERIAL AND METHODS

Insects: Helicoverpa eggs used throughout this study were obtained from a culture inthe laboratory. Larvae were reared on a modified diet according to (Shorey and Hala,1965), at a regime of 27 oC, 70% RH and a photoperiod of 16:8 (L:D). Trichogrammapretiosum and T. minutum were provided by Institute of Biological control (BBA),Darmstadt, and were reared on Sitotroga eggs. C. carnea was ordered from ÖREBio-protect Company (Germany), and maintained in a climatic cabinet at 25±2°C and70% RH and 16: 8 h L: D).

Plants: Three different cotton cultivars (Giza 89, Giza 86 and Alex 4) were cultivated incontrolled greenhouse, and used in the greenhouse experiments.

Neem products: Trifolio–M GmbH provided NeemAzal-T/S, NeemAzal blank,NeemAzal PC 05 and NeemAzal PC 05 blank. Four concentrations were used; 2, 1,0.5& 0.25 %

Effect of Neem products on parasitism of Trichogramma spp. on Helicoverpaeggs in lab

Our aim was to study whether or not Trichogramma parasitoids could parasitize ontreated eggs if the farmers spray the neem products before releasing theTrichogramma. 20 Helicoverpa eggs were sprayed with concentrations of neem productand left for 20 minutes to dry. After that, were exposed to Trichogramma species (20parasitoid females / 20 Helicoverpa eggs) into glass vials 40 ml. After 4-6 days, theblack eggs were recorded to calculate parasitism rates. Replications were 6 vials foreach neem concentrations.

Side effect on emergence rates of Trichogramma spp from treated parasitizedeggs in lab

Our aim was to study whether or not Trichogramma wasps could emerge from treatedeggs if the farmers spray the neem products after parasitisation. Ten parasitizedHelicoverpa eggs were sprayed with 4 concentrations of neem products and left for20-30 minutes to dry and put in glass viasl (40ml). After 4-5 days, the emergedparasitoids were counted to calculate the emergence rates. Replicates were 6 for eachconcentration.

Side effect on parasitism of Trichogramma spp. on Helicoverpa eggs on 3 cottoncultivars

Ten Helicoverpa eggs were sprayed with 4 concentrations of each neem product andleft to dry, then attached on 1st, 3rd and 5th leaves on cotton plants. These plants were

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placed in wood cages in the greenhouse, and then exposed to Trichogramma spp., atratio 2 parasitoid females/ host eggs. After 24 hours, these eggs were collected andincubated at 27°C, RH 70% and L: D 16:8. After 4-5 days, the black eggs were countedto calculate the parasitism rates. Three plants were used as replications for each cottoncultivar / treatment.

Side effect on predation rates of Chrysoperla on Helicoverpa egg in thelaboratory

Ten treated Helicoverpa eggs were sprayed with 4 concentrations of neem products.After drying, the treated eggs were placed in glass vials (50ml) with Chrysoperla larvaeto study effect of neem on predation rates on Helicoverpa eggs. Daily, Helicoverpa eggswere investigated and calculated the consumed eggs and added new enough eggs(treated eggs) for Chrysoperla larvae until Chrysoperla pupation. Replications were 6vials for each neem concentration.

Statistical analysis: Statistical analyses were conducted using analysis of variance(ANOVA), and following with Tukey test to compare means of treatments with control,significant differences were noticed for P < 0.05 for all trials using Program SYSTAT 8(Wilkinson et al..1998). The percentage data were arcsine transformed prior to analysis.

RESULTS

Effect of Neem products on parasitism of Trichogramma spp. on Helicoverpaeggs in lab

In Fig 1A, NeemAzal-T/S reduced parasitism rates of T. pretiosum as follows: 50, 60,66.7 and 80 % at concentrations o,f respectively, 2, 1, 0.5 and 0.25%. NeemAzal-PC 05reduced parasitism rates to 66.7, 70.8, 71.7 and 83.3 % in a similar dose-relatedmanner. While parasitism rates were reduced in NeemAzal Blank to 70, 75.8, 86.7 and95%. It is no obvious side effect on parasitism rates with NeemAzal PC05 Blank; thelater reduced the parasitism rates to 88.3, 91.8, 95.8 and 96.7 %, while parasitism rateswere 100% at control.

Side effect of neem on T. minutum is in the same trend with T. pretiosum as shown inFig (1B). NeemAzal-T/S had side effect on parasitism rates of T. minutum on H.armigera eggs; parasitism rates were reduced to 40, 43.3, 51.7 and 80 % (2, 1, 0.5 and0.25% cons.). NeemAzal PC 05 reduced the parasitism rates to 55, 75, 77.5 and 90.8 %on 4 consequent cons respectively. While NeemAzal Blank reduced the parasitism ratesas follows: 70, 84.2, 90.8 and 95.8%. Parasitism rates in NeemAzal PC05 Blanktreatment were reduced to 92.5, 77.4, 95.7 and 97.5 % at four cons, compared to 98.3%at control (Fig. 1B).

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Side effect on emergence of Trichogramma spp from treated parasitized eggs inthe lab

In Fig 2A, the results indicated that the emergence rates of T. pretiosum from treatedparasitized Helicoverpa eggs by NeemAzal-T/S were 78.3, 90, 93.3 and 100% for 2, 1,0.5 and 0.25% concentrations respectively. NeemAzal PC 05 reduced the emergencerates to 80, 90, 91.7 and 100% at the same concentrations. Both blanks had the leasteffect on emergence rates; they were was 80, 91.7, 100 and 100 % in NeemAzal Blank,and were 95, 95, 100 and 100% in NeemAzal PC05 Blank at succeeding concentrationscompared to 100% on control.

The emergence rates of T. minutum from treated parasitized Helicoverpa eggs byNeemAzal-T/S were 65, 80, 83.3 and 93.3% for 2, 1, 0.5 and 0.25% cons respectivelyas shown in Fig (2B). NeemAzal PC 05 reduced the emergence rates to 78.3, 81.7,88.3 and 96.7% at the same concentrations. Emergence rates in NeemAzal Blanktreatment were 80, 86.7, 90 and 90 %, and in NeemAzal PC05 Blank were 83.3, 88.3,

Fig. 2 Effect of neem products on emergence rates of Trichogramma spp.from parasitized Helicoverpa eggs

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Fig . 1 Effect of neem products on parasitism rates of Trichogramma spp.on Helicoverpa eggs in the laboratory

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93.3 and 100% at subsequent concentrations compared to 100% on control treatment(Fig. 2B).

Side effect on parasitism rates of T. pretiosum on Helicoverpa eggs on 3 cottoncultivars

The results indicated that NeemAzal-T/S reduced the parasitism rates to 50, 48.9, 71.1and 73.3% at 2, 1, 0.5, 0.25% cons on Giza 89 cultivar respectively (Fig. 3A). While onGiza 86 cultivar parasitism rates were reduced to 48.8, 51.1, 53.3 and 83.3%, and oncultivar Alex 4; they were 42.2, 53.3, 54.4 and 87.7% compared to 96.6, 93.3 and97.7% on control plants for succeeding cotton cultivars (Giza 89, Giza 86 and Alex 4).NeemAzal PC 05 reduced the parasitism rates to 70, 67.8, 70 and 80% on Giza 89cultivar, and reduced to 65.3, 58.9, 80 and 78.8% on Giza 86 cultivar, while reduced to84.4, 64.4, 82.2 and 88.8 % on Alex 4 cultivar on succeeding concentrations; 2, 1, 0.5and 0.25%. Neem blanks achieved a less side effect on T. pretiosum. NeemAzal Blankreduced the parasitism rates to 81.1% on Giza 89 and to 80.7% on Giza 86 and to84.4% on Alex 4. NeemAzal PC05 Blank reduced the parasitism rates to 93.3% on Giza89 and to 90% on Giza 86 and to 97.7% on Alex 4, compared to 95.6, 93.3 and 98.7%on control plants for succeeding cotton cultivars Giza 89, Giza 86 and Alex 4 (Fig. 3A).

Side effect on parasitism rates of T. minutum on Helicoverpa eggs on 3 cottoncultivars

In Fig. (3B) The results indicated that NeemAzal-T/S reduced the parasitism rates, to40, 55.4, 77.8 and 81.3 % (at 2,1, 0.5, 0.25% concentrations.) on Giza 89 cultivar, whileparasitism rates were reduced to 43.3, 55.5, 61.1 and 76.6% on Giza 86 and reduced to58.8, 68.8, 78.8 and 87.7% on Alex 4 cultivar compared to 93.3, 92.2 and 93.3% oncontrol plants for successive cotton cultivars Giza 89, Giza 86 and Alex 4. NeemAzalPC 05 reduced the parasitism rates to 82.2, 82.2, 74.4 and 83.3% on Giza 89, reducedto 80, 78.8, 81.1 and 75.5% on Giza 86, and reduced to 90, 85.5, 85.5 and 86.6% onAlex 4 on succeeding concentrations; 2, 1, 0.5 and 0.25%. Neem blanks resulted in alower side-effect on T. minutum. NeemAzal Blank reduced the parasitism rates to74.4% on Giza 89, and to 75.5% on Giza 86 and to 84.4% on Alex 4. Parasitism rateswere reduced by NeemAzal PC05 Blank to 86.7% on Giza 89, reduced to 82.2% onGiza 86 and reduced to 87.7% on Alex 4, compared to 93.3, 92.2 and 93.3% on controlplants of these cultivars Giza 89, Giza 86 and Alex 4 (Fig. 3B).

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With respect to the cotton cultivars, there was no significant difference in parasitismrates between them. Parasitism rates on these cultivars could be arranged as thefollowings, Alex 4 had the highest rates of parasitism, then Giza 89 and in the last stagecoming Giza 86, this may be refereed to different chemical components in each cultivarfor attracting Trichogramma wasps.

Effect of Neem products on predation rates of Chrysoperla on Helicoverpa egg inthe lab

Data in Fig 4 revealed that the neem products did not affect three larval instars ofChrysoperla. The Helicoverpa consumed eggs by Chrysoperla larvae differed betweenneem products and concentrations, which were lower in high concentrations comparedto low concentrations. On other hand, longevity in high concentrations was greater than at low concentrations. The results indicated that no significant difference in parasitisedeggs and also no side effect of neem treatments compared to control.

Fig. 3 Effect of neem products on parasitism rates of Trichogramma spp.on Helicoverpa eggs in the greenhouse

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DISCUSSION

The results indicated that there is a side effect of neem products on parasitism rates ofT. pretiosum and T. minutum in 2% concentration. Our results are corresponding withKhorkhordin & Mironova (1996) and Maheshkumar et al., (2000), who pointed onlyslight effect of neem formulations at high concentrations on the degree of parasitismand the adults emergence of the egg parasite T. japonicum. The results also related toRaja et al., (1998) and Thakur & Pawar (2000); their results revealed that neem-basedpesticides and bio pesticides are slightly toxic to egg parasitoid. It will be effective to useneem products in controlled concentrations in IMP programs combining with testednatural enemies species, which have shown a level of resistance to this biopesticide.

The results of emergence rates indicated that neem products had no serious effects onemergence rates of T. pretiosum and T. minutum from treated Helicoverpa eggs. Thesefinding are in the same line with Markandeya & Divakar, (1999) who reported thatemergence of T. chilonis from the parasitized eggs was not affected. Treating host eggswith the neem formulation any time after parasitization did not affect emergence of theparasitoid. This confirms that using neem products with the pest's natural enemies willnot adversely affect the predator population.

Fig. 4 Effect of neem products on effecincy of Chrysoperla larvae in preying Helicoverpa eggs

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Parasitism rates in the greenhouse experiment indicated that the effect of tested neemproducts on T. pretiosum and T. minutum did not reduce the efficiency ofTrichogramma in controlling H. armigera except at 2% concentration. Our results areconsistent with Raguraman& Singh, (1999) and Reddy& Manjunatha, (2000) andMaheshkumar et al., (2000), who confirmed that there is only a slight effect of neemproducts at high concentrations on the parasitism rates of Trichogramma species andthe crops produced the highest yield in the treated plots. It is possible to use Neemproducts in IMP program in combination with Trichogramma to keep cotton productionorganically. Therefore, future experiments are suggested to further elucidate the effectsof neem on H. armigera and Trichogramma in the field.

The results suggest that neem products have no deleterious effect on predaciousefficiency of C. carnea under laboratory conditions, These findings agree with theresults obtained by Kienzle & Zebitz (1996) and Vogt et al., (1997) and Hermann et al.,(1997) who reported no negative effect of NeemAzal T/S and NeemAzal-F on C. carneaefficacy. The only side effect of neem products was on longevity of larval instars ofChrysoperla. These results are similar with Srinivasan & Babu (2000) they found thatlongevity of treated C. carnea was affected by neem products compared to control. It isvery useful to use Neem products compatibility with C. carnea to control H. armigera tomaintain on organic cotton production to protect our environment from bad effects ofpesticides.

In conclusion, the neem products tested on Trichogramma species and C. carneashowed slightly adverse activity at high concentrations. Thus, due to its shortpersistence, neem formulations with moderate concentrations could be considered apromising active ingredient to use in IPM programs, more compatible to Trichogrammaspecies and C. carnea than synthetic insecticides. In fact, most of the chemicals testedby other researchers had a long residual toxicity on these biocontrol agents (Kakakhel &Hassan (1998) and Deole et al., 2000). Therefore, also considering that laboratoryconditions are very strict and that in field conditions less toxic effects are likely, the useof neem formulations seems to be advisable. However, a short delay between thetreatment and the introduction of the biocontrol agents is suggested for a successfulcombination of the use of neem and of Trichogramma species and C. carnea.

ACKNOWLEDGMENTS

My deeply thank to Dr. Kleeberg for providing us the Neem products for this study, andalso to all those who contributed in any way to this work but are not mentioned here.

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REFERENCES

Deole, S. A., S.N Bodhade,L.B. Mahajan , V.Y. Deotale & B.K. Sharnagat (2000)Residual toxicity of some pesticides used in cotton pest management against achrysopid, C. carnea. J. Soils and Crops, 10: 279-281.

Dodan, D. S.& L. Roshan (1999) Integrated management of neck blast and stem borerin scented rice. Haryana Agric. Uni. J. Res., 29: 47-49.

Hermann, P., C. P. W .Zebitz& J. Kienzle (1997) Effects of different NeemAzal-formulations on larvae of Chrysoperla carnea in lab. and semi-field. Practiceoriented results on use and production of neem-ingredients and pheromones.ED: Kleeberg H.& C. P. W. Zebitz. Proceedings 5th Workshop Wetzlar,Germany, 22-25 Jan.1996,1997:183-188.

Isman, M. Z. & G. R. Port. (1990) Systemic action of neem seed substances againstPieris brassicae. Entomologia Exp. et Appli., 54: 297-300.

Kaethner, M.(1991) No side effects of neem on the aphidophagous predatorsChrysoperla carnea and Coccinella septempunctata. Anz. Schaed, Pflanz,Umwel, 64: 97-99.

Kakakhel, S.A & S.A. Hassan (1998) the side effects of pesticides on the egg parasitoidTrichogramma cacoeciae Marchal, acute dose response. Pesticides andbeneficial organisms. IOBC bulletin, 21: 61-69

Karuppuchamy, P., G.Balasubramanian, & P. C. S. Babu (1998) Seasonal incidenceand management of aphid, Aphis punicae on pomegranate. Madras Agric. J., 85:224-226.

Khorkhordin, E.G. &M.K. Mironova (1996) Effects of NeemAzal on beneficial insects.

Practice Oriented Results on use and production of Neem ingredients and pheromonesIV. Eds. Kleeberg & Hummel (1996) PP: 157-163.

Kienzle, J. & C.P.W. Zebitz (1996) Impact of NeemAzal on the arthropods fauna in an

Organic apple orchard. Practice Oriented Results on use and production of Neemingredients and pheromones IV. Eds. Kleeberg & Hummel (1996) PP: 165-169.

Klemm, U. & H. Schmutterer (1993) Effects of neem preparations on Plutella xylostella L. and its natural enemies of the genus Trichogramma. Zeit.

Pflanzenkrankheiten und -schutz, 100: 113-128.

Lakshmi, V. J., G. Katti, N. V. Krishnaiah & T. Lingaiah (1997) Laboratory evaluation of

commercial neem formulations vis-a-vis insecticides against egg parasitoid,Trichogramma japonicum. J. Bio. control, 11: 29-32.

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Maheshkumar, K.,N.V. Krishnaiah, T. Lingaiah, I.C. PASLU, & K. Krishnaiah (2000) Effects of some commercial neem-based insecticides against Nilaparvata

lugens, Sogatella furcifera and Nephotettix virescens. In: Kleeberg, H. andZebitz, C. P. W. (Eds.). Practice oriented results on use and production of neemingredients and pheromones IX. pp.23-33, Germany.

Markandeya, V.& B.J. Divakar (1999) Effect of a neem formulation on fourbioagents.Plant Prot. Bull. (Faridabad), 51: 28-29.

Natarajan, K.( 1990) Natural enemies of Bemisia tabaci Gennadius and effect ofinsecticides on their activity. J. Bio. control, 4: 86-88.

Prabal S. & S. Parameswaran (2001) Contact toxicity of chemical and bio-pesticidesagainst Cnaphalocrocis medinalis guenee and Trichogramma chilonis Ishii. J.Appl. Zoo. Res., 12: 86-87.

Raguraman, S. & R. P. Singh (1999) Biological effects of neem (Azadirachta indica)seed oil on an egg parasitoid, Trichogramma chilonis. J. Econo. Entomo, 92:1274-1280.

Raja, J., B. Rajendran & C.M. Pappiah (1998) Management of bhendi fruit borer, Eariasvittella (F.).Advances in IPM for horticultural crops. Proceedings of the FirstNational Symposium on Pest Management in Horticultural Crops: environmentalimplications and thrusts, Bangalore, India, 15-17 October 1997. 1998: 118-120.

Reddy, G. V. P. & M. Manjunatha (2000) Laboratory and field studies on the integratedpest management of Helicoverpa armigera in cotton, based on pheromone trapcatches threshold level. J. Appl. Entomo., 124: 213- 221.

Salem, S. A.& M. M. Matter (1991) Relative effects of neem seed oil and Deenate onthe

cotton leafworm, Spodoptera littoralis Boisd. and the most prevalent predators in cottonfields at Menoufyia Governorate. Bull. Fac. Agric., Cairo Uni., 42: 941-952.

Schmutterer, H., (1997) Side effects of neem (Azadirachta indica) products on insectpathogens and natural enemies of spider mites and insects. J. Appl. Entomo.,121: 121-128.

Schmutterer, H. (1990) Properties and potential of natural pesticides from the neemtree, Azadirachta indica. Annu. Rev. Entomo., 35: 271-297.

Schmutterer, H. (1988) Potential of azadirachtin-containing pesticides for integratedpest control in developing and industrialized countries. J. Insect Physio., 34:713-719.

Schuster, D. J. & P.A. Stansly (2000) Response of two lacewing species to biorationaland broad-spectrum insecticides. Phytoparasitica, 28: 297-304.

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Shorey HH, Hala RL (1965) Mass rearing of some noctuid species on a simple artificialmedium. J. Econo. Entomo., 58: 522-544.

Srinivasan, G.& P. C. S. Babu (2000) Effect of neem products on predatory greenlacewing, Chrysoperla carnea (Chrysopidae; Neuroptera). Pest. Res. J., 12:123-126.

Thakur, J. N. & A.D. Pawar (2000) Comparative toxicity of different insecticides againstTrichogramma chilosis Ishii. J. Bio. Control, 14: 51-53.

Vogt, H., U. Handel & E. Vinuela (1997) Field investigations on the efficacy ofneemAzal-T/S against Dysaphis plantaginea and its effects on larvae ofChrysoperla carnea. Practice oriented results on use and production ofneem-ingredients and pheromones. Proceedings 5th Workshop Wetzlar,Germany, 22-25 Jan. 1996, 1997:105-114.

Wilkinson, L., M.A.Hill, & E.Vang (1998) SYSTAT: STATISTICS; version 8 Edition.Evanston, IL: SYSTAT ;INC. 1086 pp. (ISBN-56827-222-7).

Zhang MinLing (1997) Effects of 14 insecticides on adults, larvae, eggs, and pupae ofTrichogramma confusum. Natural Enemies of Insects, 19: 11-14.

107

LONG TERM STEM-APPLICATION IN VITICULTURE AND THE USE OFNEEMAZAL

DÜKER, A.; KUBIAK, R.

DIENSTLEISTUNGSZENTRUM LÄNDLICHER RAUM (DLR) – RHEINPFALZ, ABTEILUNG AGRARÖKOLOGIE

Introduction

The main disadvantage of application by means of spraying and sprinkling devices isthat the plant protective agents enter the environment. This results in residue depositsin the vicinity of bodies of water and the outskirts of towns and cities (Fig. 1).

Fig 1: Potential deposits in the vicinity of water bodies (left) and the outskirts of towns and cities (right)

Furthermore, the application of pesticides by helicopter in the steep areas of Germanviticulture is forbidden, on behalf of this administration is sometimes carried outlaboriously with a knapsack sprayer. In such problematic locations, the direct xylem application of plant protection agents canrepresent an environmentally useful alternative.

The Problem is the entry of plant protection agents in to theenvironment

Successful plant protection in viticulture requires several treatments per year. The maindisadvantage of application by means of spraying and sprinkling devices is that theplant protection agents enter the environment. This takes place via drift (1), volatilisation(2), drip down (3), infiltration (4) and run off (5) (Fig 2).


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Fig 2: Pathways of plant protect to products into the environment

Could we find a way to prevent this entry into the environment?

Stem-application an ecological alternative?

Since the mid-1970s single point-applications in plantations and forestry have beentested.

But: Single point-applications aren’t transferable to viticulture!So: There’s a necessity of a new special system for viticulture!

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Stem-application method for viticulture

Fig 3: The vision of a stem-application method for viticulture

The direct stem application of plant protective (Fig. 3) agents into the xylem is anenvironmentally useful method of plant protection. With this method, the appliedchemicals are entered directly via the stem into the target organs of the respectiveplant, without affecting the environment (volatilisation, drift into the soil and groundwater, etc.). Furthermore, the use of stem application results in a reduction of theamount of plant protective agents required, whereby the ecological advantages of thismethod are furthermore amplified by economic advantages.

The problem of stem-application systems for long time

Grapevines in foliate state – intact transpiration stream

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Installation injures xylem; air enters xylem-vessels and leats to air embolism

Parenchyma cells grow into vessels (thyloses)

Running times of the systems : 1-26 days only

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A way to prevent embolism during the installation of stem-applicationsystems

The following figure shows:

1.) Transpiration stream (foliated plants)2.) Vessel emptying in autumn3.) air filled vessels in winter4.) Vessel refilling in spring (root pressure!)5.) Transpiration stream (leaf expansion)

The entry of air into the vascular tracts during the assembly of stem application systemsonto foliate (transpiring) vines results in embolism. This means that the maximumlongevity of such systems is limited to one month. In winter, however, when the vinesare leafless the xylem vessels are already filled with air – an ideal time for assembly.The application systems are then put into operation simultaneously with the springrefilling of the vascular tracts through the vine's own root pressure. Using this method, afirst stem application prototype with a longevity of over one year was achieved as earlyas in 1999.

No embolism and running times of the system : 4-15 months !!

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Up-to-date-System

Within the current Federal Ministry of Education and Research-funded projectpractise-oriented systems are being constructed and tested with the engineeringsupport of the co-operation partner, the University of Kaiserslautern (Department ofMechanical and Process Engineering, Chair of Turbo Machinery and PositiveDisplacement Pumps).

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Uptake of stem-applicated agents into the leaves

Due to the low lateral distribution in the vine xylem, several injection ports are requiredper plant to ensure sufficient supply. By means of a four-day Eosin application via threeneedles, it was possible to turn red the leaves of a container-grown grapevine. Theautoradiogram of leaves from a container-grown grapevine treated with 14C-markedSpiroxamine furthermore indicates the uniform distribution of the xylem-applied agent inthe intercostal areas of the laminae.

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1DÜKER, A.; KUBIAK, R.; HÖFER, V.; 2006: Stem Application of Plant Protective Agents in Viticulture.Shaker Verlag, Aachen, Germany.

Effect of stem-applied NeemAzal U1

Material and method (container-grown grapevines)

The first experiment related to effect of xylem-applied NeemAzal U was carried out onseveral-week-old nursery plant grapevines of the type Müller-Thurgau. The infusionswere applied gravimetrically using the application System described below.

Three 1.1 -mm needles (one needle directly below the shoot, one to the left and oneto the right of it) were assembled onto each experimental grapevine and controlplant.

The concentration of the applied solution was 0.3 g NeemAzal U / l water. On anaverage, the grapevines absorbed 8.1 +/- 1.0 ml of the infusion. Water was applied tothe control plants.

The average of the measurements from five repetitions was calculated for each plant.The L3 of the Cabbage Moth (Mamestra brassicae) was used as the testorganism. Pursuant to instructions given by Hummel (TRIFOLIO, personalcommunication), two temporally spaced blocks were tested. The larvae were put on fourand eight days, respectively, after the xylem application of NeemAzal U. The larvae wereapplied individually in leaf cages (7 cm2).

Estimates were carried out daily (both cumulative and in percentages) from day fourafter the application, whereby the cage was then placed on a different leaf of the samegrapevine.

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Results and discussion (container-grown grapevines)

Fig. 4 shows that the feeding damage of the control plants was larger than thedamage to the experimental grapevines treated with NeemAzal U. During the firstexperimental block (days 4-7) the percentage of injury sustained by the control plantswas two to three times higher than the damage detected in the experimentalgrapevines.

The significance of the different levels of feeding damage in this block(estimation days 4-7) was confirmed using tests following Dunnett, Tuckey andScheffe.

Thus in this case, the xylem-applied NeemAzal U had a protective effect against

caterpillar damage.

Fig. 4 Feeding damage {Mammestra brassicae, L3) to experimental grapevines and controls.

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No significant protective effect could be determined for the measurements of the secondexperimental block (estimation days 8-12), however. The effect of NeemAzal U hadprobably already diminished at this point - a further indication of the importance of along-term, permanent stem application systems in viticulture that can be usedwhenever required.

Material and method (field-grown grapevines)

The continued experiments with NeemAzal U took place on 18-year-old field-growngrapevines of the type Riesling.

In order to rule out distortions of the results by other plant protection agents, plantprotection was terminated as early as nine weeks before the experiment began.

The stem application solution was prepared according to Hummel‘s instructions(TRIFOLIO, personal communication). First a NeemAzal U suspension (10 g NeemAzalU / l water) was prepared and stirred for 30 mins. It was then left to stand for 10 mins,after which a sediment was formed. The sediment was removed and the solution wasused for the application.

The yielded NeemAzal U solution was then applied to four grapevines. Pure water wasapplied to four further grapevines, which served as control plants.

Each application took place via two ChemJet® tree injectors (Fig. 5) per grapevine.The capacity of a ChemJet® tree injector is 20 ml. A 4 mm hole was drilled into thenarrow side of each grapevine stem to mount the injector. The injectors were thenscrewed into the holes. Once the ChemJet catches had been released the applicationcommenced.

Fig. 5 ChemJet® tree injector.

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Five leaves (from different shoots) were harvested daily (days 0, 1, 2, 3, 4, 5, 6, 7, 8,9, 10 and 11) from each grapevine. These leaves were offered as food to L4 TurnipMoth caterpillars (Agrotis segetum) and renewed daily. The feeding damage wasestimated daily.

Fresh larvae were first applied on day 0, then on day 4 and day 8. Before being

used in the experiment the caterpillars were adapted to grapevine leaves for

three days.

Results and discussion (field-grown grapevines)

Fig. 6 demonstrates that the feeding damage sustained by the leaves of the field-growngrapevines treated with NeemAzal U was slightly lower than the feeding damagesuffered by the leaves of the control plants. Application of the T-Test resulted in a 95%probability of significant differences. Nonetheless, the protective effect was very low inthis case.

This fact was discussed at the "14th Workshop of Biological Control of Plant, Medicaland Veterinary Pests" in Wetzlar. It was pointed out that the above-mentionedprocedure to prepare the applied solution had already been improved. In this case theagent probably did not enter the solution completely, thus blocking the infusion port.This meant that only some of the agent reached the grapevine leaves where it coulddevelop its protective effect (Kleeberg, TRIFOLIO, personal communication).

Fig. 6 Feeding damage (Agrotis segetum, L4) to experimental grapevines and controls.

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EXPERIENCES WITH DIFFERENT NEEM PREPARATIONS IN THECONTROL OF PEST ATTACK AND AFLATOXIN FORMATION IN THEMEDICAL PLANT CASSIA SENNA (L.) IN INDIA

PHILIP MÜLLER & THIES BASEDOW

INSTITUTE OF PHYTOPATHOLOGY AND APPLIED ZOOLOGY, JUSTUS-LIEBIG-UNIVERSITY,

EXPERIMENTAL STATION, ALTER STEINBACHER WEG 44, D-35394 GIESSEN, GERMANY

Abstract

Ephestia elutella (Hübner) (Lep., Pyralidae) attacks the seed in the pods of Cassiasenna L. (Caesalpiniaceae). By this attack the production of aflatoxins mainly byAspergillus flavus Link is promoted.

Though Ephestia-larvae were susceptible to NeemAzal-T/S in the laboratory, fieldexperiments did not result in a reduction of the Ephestia-attack in Cassia senna,presumably due to their hidden life within the pods. So no reduction in aflatoxin-contentsof the seed was achieved.

Fresh seed pods of Cassia senna, when dipped into neem-leaf-water-extract (NLWE) orNeemAzal-T/S (NA) and kept in petri dishes for four days, in comparison with watertreatment showed a reduction in their aflatoxin content. NLWE was more effective thanNA. But with the concentrations used, aflatoxin-contents were not suppressed below thedesired level.

The results presented are discussed concerning there value for future developments inthe production of Cassia senna.

Introduction

The occurrence of toxic and carcinogenic aflatoxins is an unwanted phenomenon,especially in medical plants. While in most countries of the world aflatoxin contents of 5to 50 µg/kg are tolerated (HANSEN 1993), within the EU since 1999/2000/2001 only 4µg/kg are tolerated. Cassia senna L. (Caesalpiniaceae) is a perennial plant ofsemi-deserts (Bruneton 1995) with a tolerance to salt (Mahmoud 1985), distributed inthe arid and semiarid zones of Asia, Arabia and Africa. (IWU 1990, AL-YAHYA et al.1987, GUPTA 1974, SHIRAH & KAGEL 1985). The leaves and the fruits are classicalAnthrachinon-drugs. When being dried, Sennosids are formed (STOLL & BECKER 1950,LEMLI & CUVEELE 1978), which, in the intestines, are transformed to laxatives(GOODMAN & GILMAN 1970).Biological Control of Plant, Medical and Veterinary PestsR. Strang & H. Kleeberg (eds.)Copyright 2009 by Trifolio-M GmbH

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The possibilities of reducing the aflatoxin contents in the production of Cassia sennawere studied in India for five years, when also neem preparations were used (MÜLLER

2004). A part of the results of these studies is shown and discussed here.

Methods

With the applied HPLC-method aflatoxins B1, B2, G1, G2, Ba, B2, G1 AFB1, AFB2,AFG1 and AFG2 (resp.) were identified. In Cassia senna, the far outweighing part ofaflatoxin consisted of the highly toxic AFB1. The other toxins, AFB2, AFG1 and AFG2

were only found at minor concentrations.

For identifying the insect damaging seeds of Cassia senna, lepidopteran larvae, whichhad frequently fallen out of the harvested pods, were collected and reared in thelaboratory until the adult stage. Adults and larvae were sent for identification to Berlin(Federal Biological Centre, Institute for Stored Product protection).

Laboratory experiments were mostly conducted in petri dishes, with at least fourreplications per treatment.

One field experiment was conducted on a field of 6,000 m², 20 km west of the southIndian town Tirunelveli, Tamil Nadu, with "Black Cotton Soil" (Vertisol). Plots were 20 x20 m, with three replicates.

3. Results

3.1 Contaminated parts of the plant

Only in fruits of C. senna aflatoxin concentrations were found to exceed critical limits. Incontrast, leaves of Indian C. senna were free of aflatoxin to a large extent (table 1). Iffound, concentrations were 100 times less than in pods (table 2).

Table 1: Number of samples of fresh parts of C. senna-plants, collected in the field, loaded with aflatoxin

Part of the plant N Samples withaflatoxin (%)

Samples withoutaflatoxin (%)

Leaves 30 0 (0) 30 (100)

Flowers 30 0 (0) 30 (100)

Green Fruits 30 7 (23) 23 (77)

Fruits dried on the plant 30 21 (70) 9 (30)

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Table 2: Formation of aflatoxins (in µg/kg B1, B2, G1, G2) on fresh leaves or fruits of C. senna, in Petridishes in the laboratory.

3.2 Production of aflatoxin and insect damage

The study of different intensities of insect damage (table 3) showed, that fruits withoutinsect damage had hardly any aflatoxin content, while the aflatoxin content of damagedseeds proved to be high.

Table 3: The load of Aflatoxin B1 in fresh C. senna fruits in dependence on the size of damage done byinsects

3.3 Identification of the insect species attacking seeds of C. senna

The 200 larvae collected at the drying place yielded 60 adult micro-lepidopterans. Larvaand adults, when having been sent to Berlin for identification, turned out to be a storedproduct pest in Europe:

Ephestia elutella (Hübner) (Lepidoptera; Pyralidae).

This species was mainly responsable in Tamilnadu for the infection of pods of C. sennawith Aspergillus fungi, and lastly for the production of aflatoxins before the harvest.

3.4 Experiments to control the attack by Ephestia elutella with NeemAzal-T/S

3.4.1 Tests in the laboratory (table 4) From table 4 it can be seen, that the treatmentwith NeemAzal-T/S prevented the appearance of pupae/adults of E. elutella, in thelaboratory. Pupae in brackets were malformed (see table 4).

Sample C. senna folia C. senna fructus1 18,1 668.42 9,3 1.124,53 6,1 2.057,2Ø 11,8 1.283,4

Insectdamage

n Sampleswith aflatoxin

Samples withoutaflatoxin

Average of aflatoxinconcentration (µg/kg)

No 10 0 10 n.n.

Small 10 4 6 30

Medium 9 7 2 117

Large 8 8 0 410

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Table 4: Development of larvae of Ephestia elutella of different size on fruits of C. senna treated or notwith NeemAzal T/S, in petri dishes

3.4.2 Results of a field experiment

There was no significant difference in insect attack between plots sprayed withNeemAzal-T/S and untreated plots. But watering had a positive effect on the insectattack (Table 5).

Table 5. The attack (%) of pods of C. senna pods by E. elutella, after different treatments

3.5 Studies in the laboratory of the effect of different neem preparations on theaflatoxin content of the pods of Cassia senna

It was tested, whether neem preparations can stop or reduce the production of aflatoxin.Two different neem preparations were used, NeemAzal-T/S (NA), and a Neem leafwater extract (NLWE): 100 g of fresh neem leaves were chopped, and 1 l of boilingwater added. Two hours later, after repeated stirring, the solution, cooled down, wassieved and used immediately.

Differently treated undamaged fresh pods of C. senna (12 g each) were kept for fourdays in closed petri dishes to allow the fungi to grow, and were dried then. Prior to thefour days, the pods of eleven samples, each, had been dipped in water, in NA-solutionor in NLWE.

The results are shown in fig. 1. The neem preparation did reduce the aflatoxin contentof the Cassia pods. NLWE was more effective in this respect than NA. But bothpreparations, used in the concentrations given, did not lower the aflatoxin content to thedesired low level.

Size of larvae, and treatment of fruits Larvae Pupae Adults

Larvae > 7 mm, fruits untreated 20 16 14

Larvae 4 – 7 mm, fruits untreated 20 11 9

Larvae > 7 mm, fruits treated with NeemAzal T/S 20 (6) 0

Larvae 4 – 7 mm, fruits treated with NeemAzal T/S 20 0 0

Harvest period Untreated NeemAzal-T/S WateredFirst 5.8 4.3 5.7Second 8.0 6.0 14.0Third 1.8 2.2 3.7Average 5.2 4.2 7.8

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Fig. 1: Box Wishker Plots with Maxima-, Quartil- and Minima-concentrations of Aflatoxins B1, B2, G1,G2 of 11 samples of C. senna fruit, which had been kept in petri dishes, after treatment withwater, NA or NLWE

Discussion

Control of Ephestia elutella

It seems very difficult to control E. elutella, due to its hidden life in the pods of C. senna.Though the larvae are susceptible to azadirachtins, the are not met by the applications,like the codling moth, Cydia pomonella (SCHMUTTERER 2002). E. elutella apparentlydoes not attack Cassia senna in a great extent. Therefore, to reduce the aflatoxincontent of seed products of C. senna, it seems to be important, to find safe methods tosort out attacked seeds.

The possibilities of using neem products to reduce aflatoxin production

It is known that Neem Leaf extracts can reduce the aflatoxin production by Aspergillusflavus and A. parasiticus (BHATNAGAR et al. 1990, ZERINGUE & BHATNAGAR 1990),attributed to C3- to C9-Alkenals (ZERINGUE & BHATNAGAR 1994). The timing isimportant, here, since the start of the secondary metabolism in the fungi has to bestopped (BHATNAGAR et al. 1993).

16809

25491649

25683777

1591

4146

6012

149

8117

3878

4523

0

2.000

4.000

6.000

8.000

10.000

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14.000

16.000

18.000

NLWE NeemAzal T/S water

Afla

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µg/

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This observation should be followed up in future. But it was also interesting thatNeemAzal-T/S has a certain effect. So also neem seed apparently contains a certainamount of effective ingredients.

References

AL-YAHYA, M. A., MOSSA, J. S., AL-BADAR, A. A., TARIQ, M. & AL-MESHAI, I. A. 1987.Phytochemical and Biological Studies on Saudi Medicinal Plants: Part 12. AStudy on Saudi Plants of Family "Leguminosae". Int. J. Crude Drug. Res. 25, 65– 71.

BHATNAGAR, D., ZERINGUE, H.J. & MCCORMICK, S.P. 1990. Neem leaf extracts inhibitaflatoxin biosynthesis in Aspergillus flavus and A. parasiticus. In: Neem'spotential in pest management programs. Proceeding of the USDA workshop,April 16 to 17, 1990, Beltsville, Md. USDA/ARS publication 86 (Washington,D.C.), 118-127.

BHATNAGAR, D., COTTY, P.J. & CLEVELAND, T.E. 1993. Preharvest aflatoxincontamination. Molecular strategies for the control. ACS-symposium-series 528,272-292.

BRUNETON, J. 1995. Pharmacognosy, Phytochemistry, Medicinal Plants. Paris LavoisierPublishing.

GOODMAN L. S., & GILMAN A. 1970. The Pharmacological Basis of Therapeutics. FourthEdition, 1024 S.

GUPTA, R. 1974. Wild Occurring Senna (Cassia senna L. Vahl.) from Kutch, Gujarat.Curr. Sci. 43(3): 89.

HANSEN, T. J. 1993. Quantitative testing for mycotoxins. Am. Assoc. Cereal Chem. 38,346 348.

IWU, M. M. 1990. Handbook of African Medicinal Plants. Boca Raton: CRC Press. p.143-144.

LEMLI, J., CUVEELE, J. 1978. Umwandlung der Anthronderivate während des Trocknensder Blätter von Cassia angustifolia und Rhamnus frangula. Planta Med. 33, 293.

MAHMOUD, A. 1985. Germination of Cassia senna from Saudi Arabia. Journal of AridEnvironments 9, 39-49.

MÜLLER, P. 2004. Anbaubegleitende Untersuchungen zu Vorkommen and Vermeidungvon Aflatoxinen und Insektenbeschädigungen in der Produktion derArzneipflanze Cassia senna L. in Indien. Diss. FB 9, Univ. Giessen.

SCHMUTTERER, H. (Ed.) 2002. The Neem Tree Azadirachta indica A. Juss and othermeliaceous plants. Sources of unique natural products for integrated pest

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management, medicine, industry and other purposes. 2nd Ed. Mumbai (NeemFoundation).

SHIRAH, H. & KAGEI, K. 1985. Studies on the Cultivation of Senna Cassia senna L. (1).The Germination of Seed Growth Flowering and Podding. Shoyakugaku Zasshi,39(2): 111-117.

STOLL, A. & BECKER, B. 1950. Sennosides A and B, the active principle of senna.Fortsch. Chem. org. Naturist 7, 248.

ZERINGUE, H.J & BHATNAGAR, D. 1990 . Inhibition of aflatoxin production in Aspergillusflavus infected cotton bolls after treatment with neem (Azadirachta indica) leafextracts. Journal of the American Oil Chemists' Society, 67 (4): 215-216.

ZERINGUE, H.J. & BHATNAGAR, D. 1994. Effects of neem leaf volatiles on submergedcultures of aflatoxigenic Aspergillus parasiticus. Applied and EnvironmentalMicrobiology, 60 (10): 3543-3547.

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EFFECTS OF DIFFERENT PLANT POWDERS AND PLANT OILS ONPESTS OF STORED MAIZE IN ETHIOPIA

TADESSE, ABRAHAM (ADDIS ABABA) & BASEDOW, THIES (GIESSEN)

INSTITUTE OF PHYTOPATHOLOGY AND APPLIED ZOOLOGY, JUSTUS-LIEBIG-UNIVERSITY,

EXPERIMENTAL STATION, ALTER STEINBACHER WEG 44, D-35394 GIESSEN, GERMANY

Abstract

All experiments were conducted in the laboratory at Bako (Western Ethiopia) in2000/2001, with Sitophilus zeamais. Due to the situation in Ethiopia at that time(drought and lack of maize), no field studies with the substances were possible. Plantpowders: Leaf powder of Chenopodium ambrosioides and leaf and seed powder ofAzadirachta indica were tested at different rates. Though all treatments gave significantdifferences when compared with the untreated control, Ch. a. induced the highestmortality in the weevils, exerting a long-term effect. Neem seed powder proved to bemore effectic than neem leaf powder. Plant oils: Seed oils of Azadirachta indica(Meliaceae), Zea mais (Poaceae), Helianthus annuus and Guizotia abbyssinica(Asteraceae), and Sesamum indicum (Pedaliaceae) were tested at different rates.Though Neem oil contained Azadirachtins, its effect was not stronger than that of theother oils. At 10 ml oil kg-1, weevil mortality was 100 % after one week in all oils. Noweevil progeny was observed, and grain damage was significantly lower than in theuntreated check.

1. Introduction

In Ethiopia, where a high percentage of farmers is illiterate (TADESSE & BASEDOW

2004), it seems important to replace the use of synthetic insecticides in stored productprotection by natural non-toxic substances, if possible (TADESSE & BASEDOW 2005). Inthe years 2000/2001 at Bako, Western Ethiopia, Laboratory studies were conductedwith Sitophilus zeamais to find out candidate substances for this purpose. We reporthere on the results obtained.

2. Materials and Methods

A continuous mass rearing of S. zeamais was established to have enough insects forthe experiments. The temperature averaged 24°C (range 22° to 27°), the relativehumidity ranged from 45 % in January to March, in the dry season to 85 % inJuly/August, when it rained. The other months lay in between.Biological Control of Plant, Medical and Veterinary PestsR. Strang & H. Kleeberg (eds.)Copyright 2009 by Trifolio-M GmbH

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The sources of materials used were different. Fresh seed of Neem (Azadirachta indicaA. Juss) (Meliaceae) was obtained from Dire Dawa and Melka Werer, both in theeastern part of Ethiopia. Neem leaf was obtained from the Nazareth Research Center(NRC). Chenopodium ambrosioides L. (Chenopodiaceae) ("Mexican Tea") wascollected from along road sides in Addis Ababa. C. ambrosioides is an occasional weedat high elevations (2400 m above sea level) (STROUD & PARKER 1989). It is a stronglyaromatic hairy annual or perennial herb, native to tropical America (SU, 1991). Neem seedwas dried, decorticated and ground using mortar and pestle. Neem leaf and Mexicantea were sun dried under shade and ground to fine powders. Oils of maize (Zea maysL.), sesame (Sesamum indicum L.), noug or niger seed, Guizotia abyssinica L. (Cass.)and sunflower (Helianthus annuus L.) were purchased from a supermarket in AddisAbeba. Neem oil was obtained from Niem-Handel, D-64579 Gernsheim, Germany.Pirimiphos-methyl (Actellic) 2% was purchased in Addis Ababa. Oils and botanicalswere kept in a refrigerator while the other materials were kept in a dry and cool placeuntil use.

Three botanical powders were tested (Mexican Tea Powder, Neem leaf- and Neemseed-powder). Oils of five plants (neem, maize, sunflower, noug and sesame) werecompared with each other and with pirimiphos-methyl treated and untreated checks.

150 g of clean maize grain (12.6% moisture content) were put in each of several glassjars with lids allowing ventilation. The different rates of each material were weighed andadded in the corresponding glass jars and shaken well in order to have a uniformmixture. Fifty randomly picked adult maize weevils of mixed age were introduced ineach jar, including the untreated check following three to five days after treatment. Eachexperiment was arranged in a completely randomised design with four replications.

Mortality of introduced weevils was counted and recorded at an interval of five days, forsix weeks. At the last day, all of the introduced weevils were removed and the numberof dead and live ones was recorded. The grain was kept at the same condition forprogeny emergence and inspected frequently starting from the appearance of the firstprogeny weevil. The number of dead and live progeny weevils was recorded untilemergence was completed. After 115 to 130 days, the number and weight of damagedand undamaged grain in each jar were recorded. The percentage of grain weight losseswere calculated using the count and weigh method (ADAMS & SCHULTEN 1978).

ANOVA was conducted, after square root transformation where necessary, using thecomputer program SPSS 10.

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3. Results

3.1 The effects of plant powders at selected rates

Mexican tea powder (MTP), neem leaf powder (NLP), and neem seed powder (NSP),each at different rates were evaluated in three different experiments at differentconcentrations. But also here, only the results of an experiment with selected rates shallbe shown. The effects of MTP, NLP and NSP at selected rates on the mortality of adultweevils at different dates after infestation are presented in Fig. 1. NLP at the rate of 5 %did not differ significantly from the untreated check in the percentage of dead weevilsafter six days of infestation. The percentages of dead weevils in both rates of MTP weresignificantly (P < 0.01) greater than that of the NLP and NSP treatments after six daysof infestation. Furthermore, the difference between the MTP and the syntheticinsecticide treatments in the amount of dead weevils was not significant (Fig. 1). After12 to 30 days, when MTP and PMM (pirimiphos-methyl used as chemical standard) hadinduced 100% mortality, both rates of NLP and NSP had a lower efficacy (Fig. 1).

The effects of the different botanicals at different rates on the percentage mortality ofweevils re-infested after 91 days of treatment are presented in Fig. 2. The higher rate ofMTP caused mortality of 22.7% and 76.7%, within two and four days after re-infestation,respectively, and these figures were significantly (P < 0.05) higher than that of the otherbotanicals and rates. NSP at 3% was the next best among the botanicals causing55.3% mortality within four days after re-infestation. The lower rate of MTP, the higherrate of NLP and the lower rate of NSP did not differ significantly from each other but allof them differed significantly from the untreated check. However, the lower rate of NLPdid not differ significantly from the untreated check following four days after infestation.

After six days of re-infestation, all rates of the MTP and the NSP were as effective asthe synthetic insecticide, all of which were significantly superior to both rates of NLP.There was no significant variation between the NLP rates in the percentage of deadweevils but both of them differed significantly from the untreated check. All of thebotanicals and their rates inflicted as high mortality as the synthetic insecticide following10 days after infestation, in the laboratory (Fig. 2).

3.2 The effect of plant oils on maize weevil populations

Neem oil (NMO), maize oil (MZO), noug/niger seed oil (Guizotia abbyssinica) (NGO),sesame oil (SSO), and sunflower oil (SFO) were evaluated each at 2.5, 5, 7.5, 10 and12.5 ml kg-1 maize grain. The experimental series showed that from 5 ml oil per kg ofmaize weevil mortality increased. We show here only a comparison of all oils tested at10 ml per kg maize. The amounts of dead adult weevils, progeny emerged and

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damaged maize grain in different plant oil treatments at the concentration of 10 ml/kgmaize are presented in Table 1. All oils caused significantly (P < 0.01) higher mortalitythan the untreated check. The rate of mortality following one week after infestation inthe different oil treatments was 100%. Moreover, no progeny weevils emerged from theoil treatments (Table 1). The percentages of damaged grain after 137 and 338 days ofinfestation were significantly (P < 0.01) higher in the untreated check than that of the oiltreatments in which almost no grain was damaged at both dates (Tables 1 and 2). Theamount of damaged grain in the untreated check after the later date of infestation was100% (Table 2). Moreover, the amounts of grain weight losses in the untreated checkwere significantly higher than that of the oil treatments at both dates.

All oil treatments reduced seed germination significantly more than the untreated checkfollowing 185 days after treatment. The NMO caused significantly higher loss of seedgermination than the SFO. However, both oils did not differ significantly from the otheroil treatments in the amount of germinated seeds (Table 2).

Fig. 1. The effects of different botanicals on the percentage mortality of maize weevils at different dates

after infestation (dai) in the laboratory. T-beams represent standard errors. The rate of mortalities at 20

and 30 dai were similar and not shown in the graph. For significant differences see text

0

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4. Discussion

4.1 Mexican tea powder (MTP)

The significantly high mortality of adult weevils caused by the different rates of the MTPtreatments five days after infestation is in accordance with previous reports. ETICHA &TADESSE (1999) found that MTP applied at the rate of 10 % caused 92 % mortality of adultmaize weevils seven days after infestation. At the end of the experiment (four weeks), 100% mortality was recorded in both the botanical and the synthetic insecticide treatments.TAPANDJOU et al. (2002) tested powder and essential oil obtained from dry ground leavesof Chenopodium ambrosioides at rates ranging from 0.8 to 6.4 % against six insect pestsincluding the granary and the maize weevils on wheat and maize and reported that thehighest dosage of 6.4 % induced 100 % mortality of both species two days after treatment,although mortality of the larger grain borer was only 44 %. NOVO et al. (1997) evaluatedcrude extracts in ethanol, hexane and chloroform of this and other plants for their repellenteffects against Tribolium castaneum and found Chenopodium ambrosioides in chloroformwas the second best. The insecticidal activity of the essential oil from Chenopodiumambrosioides against several stored grain insect pests has been reported by SU (1991)who indicated that the oil was highly toxic to Callosobruchus maculatus and Lasiodermaserricorne, moderately toxic to Sitophilus oryzae and slightly toxic to Tribolium castaneum.According to TAPANDJOU et al. (2002), essential oils were more effective than the groundleaf treatments except in the case of Callosobruchus maculatus and Sitophilus zeamaiswhich were least affected. A terpene peroxide ascaridole (DELOBEL & MALONGA 1987) andothers are reported to be responsible for the toxic properties of MTP. TAPANDJOU et al.(2002) reported the chemical constituents of the leaf oil to be alpha-terpinen (37.6%),cymol (p-cymen) (50%), cis-ß-farnesen (1.4%), ascaridole (3.5%) and carvacrol (3.3%)and suggested the toxicity to be related to compounds present in larger quantities thanascaridole. We have observed reduced performance against the granary weevil on wheatwhen lower rate of old material was used (TADESSE & BASEDOW 2003). The inhibition ofprogeny emergence from grain treated with MTP is also in line with the findings of ETICHA

& TADESSE (1999). TAPANDJOU et al. (2002) also reported that progeny production wascompletely suppressed in the Sitophilus species and the larger grain borer at the dosageranging from 1.6 to 6.4 %. This may be due to the death of insects before oviposition orthe effect of the treatment on either the egg or on other development stages of the insect.Unfortunately, MTP, like the other botanicals could not be tested in our field trials: Due tolack of maize and stores priorities had to be set.

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4.2 Neem leaf powder (NLP)

Hellpap & Dreyer (1995) reported that, in contrast to neem seeds, neem leaves do notcontain azadirachtins, but presumably other triterpenoids with insecticidal properties(SCHMUTTERER 2002). GETU (1999) reported only 15 % survival of adult Sitotrogacerealella six hours after infestation on grain treated with 10 % NLP, and it did not differsignificantly from NSP at 10% and PMM at 10 ppm. ETICHA & TADESSE (1999) reportedmortality rates of 64 and 91 % in 10 % NLP treatment after one and four weeks ofinfestation, respectively, although in another experiment a mortality of only 20 % wasfound using the same rate. The source of the material, method of preparation and age ofthe material before use could contribute to the difference in effectiveness of neemproducts. TANZUBIL (1987), after evaluating neem leaf at 2, 5 and 10 % together with othermaterials for effectiveness against the cowpea weevil, reported that the leaf powdertreatments provided a reasonable level of protection up to the 12th week only, after whichseed damage and subsequent weight loss became serious. Although emergence ofprogeny weevils was delayed and the number emerged decreased significantly with therate of the NLP applied, no treatment prevented progeny emergence. PEREIRA &WOHLGEMUTH (1982) tested NLP at different rates (0.5 to 8%) against six species ofstored grain pests on maize and found that the productivity of Sitophilus zeamais wasnot reduced while the fecundity of Sitophilus oryzae was reduced three fold at the 8%treatment.

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Fig. 2. The effects of different botanical treatments on the percentage mortality of weevils reinfestedafter 91 days of treatment in the laboratory at Bako. T-beams represent standard errors. Forsignificant differences see text

4.3 Neem seed powder (NSP)

NSP rates of 2 % caused equal amounts of adult weevil mortality as higher rates, followingfive days after infestation. This indicates that addition of NSP at rates higher than 2 % hasno added benefit as far as adult mortality is concerned. PEREIRA & WOHLGEMUTH (1982)evaluated NSP at rates ranging from 0.5 to 8 % against six species of stored grain pestson maize and found it to be toxic to the adults of Sitophilus oryzae followed byCryptolestes ferrugineus, Rhizopertha dominica and Sitophilus zeamais. With Sitophilusoryzae, there was 99 % mortality at 4 % NSP treatment by the third day of exposure.ETICHA & TADESSE (1999) found adult weevil mortality rates of 94 % and 98 %,respectively, within four weeks after infestation on maize grain treated with NSP at 5 %,protection of the grain lasting for about 49 weeks. SIDDIG (1980) tested the effect ofNSP on Trogoderma granarium Everts infesting wheat in the Sudan and found thatdamage was significantly reduced when wheat was treated with powdered neem seedsat doses of 1, 2 or 4 %, the effect increasing as the dose of neem increased. This effectwas maintained in one experiment for 207 days and in a second experiment for 490days after treatment. According to KETKAR & KETKAR (1993), neem products in storageoccasionally retain their activity for periods of one year or longer. PEREIRA &WOHLGEMUTH (1982) found that even after three months storage of the treated maize,

0

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MTP2

.5

MTP2

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NLP20

NSP2NSP

3PM

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neem seed reduced the adult populations of Sitophilus oryzae, Sitophilus zeamais,Rhizopertha dominica and Ephestia cautella. However, after seven months, only thepopulation of Rhizopertha dominica was significantly reduced. NSP has been foundeffective against almost all stored grain pests. The insecticidal and repellent propertiesof neem kernels has been attributed to the triterpenoid Azadirachtin and other relatedcompounds (SCHMUTTERER 2002; GOVENDACHARI et al., 2000).

Table 1. The effect of different plant oils on adult weevil mortality, progeny emergence and maize graindamaged at different dates after infestation (dai) in the laboratory at Bako. Means followed bythe same letter within a column are not significantly different from each other at 5% (DMRT).NMO = neem oil, MZO = maize oil, NGO = noug/niger seed oil (Guizotia abbyssinica), SSO =sesame oil and SFO = sunflower oil.

Untreated

Plant oils(10 ml kg-1)

Percent dead adult weevils(days after infestation, dai)

Weevilprogenyemerged (no.)

Amount of graindamaged (%)

3 dai 7 dai 106 dai 137 dai

0.00 ± 0.00 b 1.85 ± 1.19 b 99.00 ± 0.00 a 25.20 ± 5.16 aNMO 95.64 ± 4.49 a 100.00 ± 0.00 a 0.00 ± 0.00 b 0.00 ± 0.00 bMZO 100.00 ± 0.00 a 100.00 ± 0.00 a 0.00 ± 0.00 b 0.07 ± 0. 07 bSFO 96.25 ± 0.72 a 100.00 ± 0.00 a 0.00 ± 0.00 b 0.00 ± 0.00 bNGO 97.50 ± 2.50 a 100.00 ± 0.00 a 0.00 ± 0.00 b 0.14 ± 0.14 bSSO 98.75 ± 1.25 a 100.00 ± 0.00 a 0.00 ± 0.00 b 0.00 ± 0.00 b

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Table 2. The percentages of damaged grain, grain weight losses and germination of maize treated withdifferent plant oils against the maize weevil in the laboratory at Bako, at different days afterinfestation (dai). Means followed by the same letter(s) within a column are not significantlydifferent from each other at 5% (DMRT).

Untreated

4.4 Plant oils

It was interesting to note that all of the vegetable oils showed the same effect as theNMO which contains Azadirachtin. The complete mortality of adult maize weevils in thedifferent plant oil treatments tested in this study is in accordance with the reports ofother authors who have proven that oil coating is effective in controlling insect pests ofstored grains regardless of the source of oil. IVBIJARO et al. (1985) found that oils ofcoconut and groundnut admixed with maize grains at dosage rates of 5 and 10 ml kg –1

killed 67 to 100 % Sitophilus oryzae within 24 hr. At 1 ml oil kg –1, mortality of adult weevilsreached 100 % in seven days. YUN-TAI & BURKHOLDER (1981) found that oils ofcottonseed, soybean, maize and groundnut at 5 to 10 ml kg –1 suppressed Sitophilusgranarius in wheat. According to IVBIJARO (1984), groundnut oil at rates of 5 to 20 ml kg –1

completely controlled infestation of stored maize by Sitophilus zeamais and Sitophilusoryzae. OBENG-OFORI (1995) evaluated several plant oils including MZO at differentdosages as grain protectants against Cryptolestes pusillus and Rhizopertha dominica inmaize and sorghum, and found that grain treated with 10 ml kg-1 of the different oilsinduced 100 % mortality within 24 h. DEY & SARUP (1993) studied oils from several plantsincluding neem and sesame at different rates against stored maize insects and found thathigher mortality occurred one day after treatment of the grain irrespective of the type of oil.TEMBO & MURFITT (1995) found that oils of groundnut, rape seed and sunflower at 10ml kg-1 caused considerable mortality (60.0 to 80.0%) within 14 days and there was littledifference between the three oils in their effect. Application of NMO at a lowconcentration of 0.1 % (w/w) to wheat grain reduced egg laying by Sitotroga cerealella

Plant oils (10 mlkg-1)

Percentdamaged

maize grain(338 dai)

Percent grain weight losses Percent seedgermination

(185 dai)

137 dai 338 dai

100.00 ± 0.00 a 2.47 ± 0.74 a 100.00 ± 0.00 a 81.00 ± 5.25 aNMO 0.47 ± 0.27 b 0.00 ± 0.00 b 0.01 ± 0.01 b 59.00 ± 4.12 cMZO 0.00 ± 0.00 b 0.07 ± 0.07 b 0.00 ± 0.00 b 63.00 ± 6.40 bcSFO 0.00 ± 0.00 b 0.00 ± 0.00 b 0.00 ± 0.00 b 78.47 ± 3.21 bNGO 0.00 ± 0.00 b 0.00 ± 0.00 b 0.00 ± 0.00 b 67.83 ± 6.60 bcSSO 0.22 ± 0.22 b 0.00 ± 0.00 b 0.02 ± 0.02 b 65.00 ± 1.91 bc

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as effectively as 5% malathion dust treatment (SAXENA 1995). IVBIJARO et al. (1985)showed that plant oils at different rates significantly reduced oviposition, grain damageand grain weight losses in maize. DON-PEDRO (1989) found oils from maize, sunflower,sesame, groundnut, palm and coconut being effective against eggs and early stagelarvae of Sitophilus zeamais. Despite their different properties, origin and purity, all ofthe oils tested were equally effective in reducing progeny development.

However, the mode of action of vegetable oils in the protection of treated seeds iscomplex and not clear (YUN-TAI & BURKHOLDER, 1981; OBENG-OFORI, 1995). Themechanism of protection may be due to physical properties of the oil (SINGH et al. 1978;OBENG-OFORI 1995), oils providing a physical barrier to insect respiration resulting insuffocation (HEWLETT 1975; SCHOONHOVEN 1978; CREDLAND 1992) and may alsocontain insecticidal or repellent compounds, including fatty acids and other compoundsproduced by chemical breakdown of the oil after application. According to HILL &SCHOONHOVEN (1981), the insecticidal action of palm and vegetable oils was due totriglyceride fractions. HEWLETT (1975) suggested that the most likely mechanism ofaction is blockage of the tracheal system by the oils and the beetle dies of anoxia. Theprotection of grains by oils could therefore be due to both physical and chemical factors(OBENG-OFORI 1995). The oils could also act as antifeedants or modify the storagemicro-environment, thereby discouraging insect penetration and feeding (OBENG-OFORI

1995). Concerning insect eggs, it was suggested that the activity of plant oils againstinsect eggs was by a general physical property of the oil coating rather than a specificchemical action leading to a reduction in oviposition of bruchid beetles on treated grains(SINGH et al. 1978; MESSINA & RENWICK, 1983). DON-PEDRO (1989) observed nosignificant effect on oviposition in no choice experiments but in two way choiceexperiments, groundnut oil at 14 ml kg-1 significantly deterred oviposition and hesuggested it to be possibly due to a repellent effect. The repellent effects of plant oils havealso been reported by YUN-TAI & BURKHOLDER (1981) and AKOU-EDI (1984).

The observed reductions in seed germination in the oil treatments is in accordance withthe results of YUN-TAI & BURKHOLDER (1981) who reported adverse effects of vegetableoils on seed germination.

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5. Conclusion

In the end, it can be concluded that a lot of alternative/traditional substances ormeasures exist in Ethiopia, which can reduce the attack of stored maize by insect pestssignificantly. They could replace the use of synthetic insecticides successfully.

References

ADAMS, J. M. & SCHULTEN, G. G. M. (1978): Losses caused by insects, mites andmicroorganims. In: HARRIS, K.L. & LINDBLAD, C.J. (Eds.) Post harvest grain lossassessment methods. Washington, D.C. (American Association of CerealChemistry), 83-93.

AKOU-EDI, D. (1984): Effects of neem seed powder and oil on Tribolium confusum andSitophilus zeamais. In: SCHMUTTERER, H. & ASCHER, K.R.S. (Eds.): Naturalpesticides from the neem tree (Azadirachta indica A. Juss) and other tropicalplants. Proceedings of the Second International Neem Conference,Rauischholzhausen, Germany, 25-28 May, 1983. GTZ (Eschborn, Germany),445-451.

CREDLAND, P. F (1992): The structure of bruchid eggs may the ovicidal effects of oils.Journal of Stored Products Research 28, 1-9.

DELOBEL, A. & MALONGA, P. (1987): Insecticidal properties of six plant species againstCaryeon serratus (Ol.) (Coleoptera: Bruchidae). Journal of Stored ProductsResearch 23, 173-176.

DON-PEDRO, K. N. (1989): Mechanism of action of some vegetable oils against Sitophiluszeamais Motsch. (Coleoptera: Curculionidae). Journal of Stored ProductsResearch 25, 217-223.

ETICHA, F. & TADESSE, A. (1989): Effeects of some botanicals and other materials againstthe maize weevil (Sitophilus zeamais Motsch.) on stored maize. In: Maizeproduction technology for the future: Challenges and opportunities. Proceedings ofthe Sixth Eastern and Southern Africa Regional Maize Conference, 21.-25. Sept.1998, Addis Ababa (CYMMIT and EARO), 101-104.

GETU, E. (1999): Use of botanical plants in the control of stored maize grain insect pests inEthiopia. In: Maize production technology for the future: Challenges andopportunities. Proceedings of the Sixth Eastern and Southern Africa RegionalMaize Conference, 21.-25. Sept. 1998, Addis Abeba (CYMMIT and EARO),105-108.

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GOVINDACHARI, T.R., SURESH, G., GOPALAKRISHNAN, G. & WESLEY, S. D. (2000): Insectantifeedant and growth regulating activities of neem seed oil - the role of majortetranortriterpenoids. Journal of applied Entomology 124, 287-291.

HELLPAP, C. & DREYER, M. (1995): Neem products for pest management, practical resultsof neem application against arthropod pests, and probability of development ofresistance. In: Schmutterer, H. (Ed.), The Neem Tree, Azadirachta indica A. Jussand other meliaceous plants. Source of unique natural products for integrated pestmanagement, medicine, industry and other purposes. VCH (Weinheim, Germany),367-375.

HEWLETT, P. S. (1975): Lethal action of a refined mineral oil on adult Sitophilus granariusL. Journal Stored Product Research 11, 119-120.

HILL, J. & SCHOONHOVEN, H. V. (1981): Effectiveness of vegetable fractions in controllingMexican bean weevil on stored beans. Journal of economic Entomology 74,478-479.

IVBIJARO, M.F., LIGAN, C. & YOUDEOWE, A. (1985): Control of rice weevils, Sitophilusoryzae (L.), in stored maize with vegetable oils. Agricultural Ecology andEnvironment 14, 237-242.

KETKAR, C. M. & KETKAR, M. S. (1993): Versatile neem - a source for plant protection. In:CHARI, M. S. & RAMAPRASAD, G. (Eds.): Botanical pesticides in integrated pestmanagement. Proceedings of a National Symposium, Indian Society of TobaccoScience, Rajamundri, 5331105, India, 118-124.

MESSINA, F.J. & RENWICK, J.A.A. (1983): Effectiveness of oils in protecting storedcowpeas from the cowpea weevil (Coleoptera: Bruchidae). Journal of economicEntomology 76, 634-636.

NOVO, R.J., VIGLIANCO, A. & NASSETTA, M. (1997): Repellent activity of different plantextracts on T. canstaneum (Herbst). Agriscientia 14, 31-36.

PEREIRA, J. & WOHLGEMUTH, R. (1982): Neem (Azadirachta indica A. Juss) of west Africanorigin as a protectant of stored maize. Zeitschrift für Angewandte Zoologie 94,208-214.

OBENG-OFORI, D. (1995): Plant oils as grain protectants against infestations ofCryptolestes pusillus and Rhizopertha dominica in stored grain. Entomologiaexperimentalis et applicata 77, 133-139.

SAXENA, R. C. )1995): Pests of stored products. In: Schmutterer, H. (Ed.), The Neem Tree,Azadirachta indica A. Juss and other meliaceous plants. Source of unique naturalproducts for integrated pest management, medicine, industry and other purposes.VCH (Weinheim, Germany), 418-432.

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SCHMUTTERER, H. (Ed.) (2000): The Neem Tree, Azadirachta indica A. Juss and othermeliaceous plants. Source of unique natural products for integrated pestmanagement, medicine, industry and other purposes. 2nd Edition. Mumbai, India:Neem Faundation.

SCHOONHOVEN, A. V. (1978): Use of vegetable oils to protect stored beans from bruchidattack. Journal of economic Entomology 71, 254-256.

SINGH, S. R., LUSE, R. A., LEUSCHNER, K. & NANGJU, D. (1978): Groundnut oil treatmentfor control of Callosobruchus maculatus during cowpea storage. Journal of StoredProduct Research 14, 77-80.

STROUD, A. & PARKER, C. A. (1989): Weed identification guide for Ethiopia. Rome (FAO).

SU, H. (1991): Toxicity and repellancy of chenopodium oil to four species of stored productinsects. Journal of Entomological Science 26, 76-80.

TADESSE, A. & BASEDOW, TH. (2003): Comparative evaluation of SilicoSec, Mexican teapowder and neem oil against the granary weevil, Sitophilus granarius L., onwheat in the laboratory. Mitteilungen der Deutschen Gesellschaft für allgemeineund angewandte Entomologie 14, 343-346.

TADESSE, A. & BASEDOW, TH. (2004): A survey of insect pest problems and storedproduct protection in maize in Ethiopia in the year 2000. Journal of PlantDiseases and Protection 111, 257-265.

TADESSE, A. & BASEDOW, TH. (2005): LABORATORY AND FIELD STUDIES ON THE EFFECT

OF NATURAL CONTROL MEASURES AGAINST INSECT PESTS IN STORED MAIZE IN

ETHIOPIA. - J. PLANT DISEASES AND PROTECTION 112, IN PRESS.

TEMBO, E. & MURFITT, R.F.A. (1995): Effect of combining vegetable oil withpirimiphos-methyl for protection of stored wheat against Sitophilus granarius (L.).Journal of Stored Product Research 31, 77-81.

YUN-TAI, Q. & BURKHOLDER, W.E., Protection of stored wheat from the granary weevil byvegetable oils. Journal of economic Entomology 74, 502-505, 1981.

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A PROMISING BIOTECHNICAL APPROACH TO PEST MANAGEMENT OFDIABROTICA VIRGIFERA VIRGIFERA IN ILLINOINS MAIZE FIELDSUNDER KAIROMONAL SHIELDING WITH THE NEW MSD TECHNIQUE

HANS E. HUMMEL1,2, JOHN T. SHAW2, DETLEF F. HEIN1

1 JUSTUS-LIEBIG-UNIVERSITY, PROFESSORSHIP FOR ORGANIC AGRICULTURE, KARL-GLÖCKNER-STR. 21C,35394 GIEßEN, GERMANY; E-MAIL: [email protected]

2 ILLINOIS NATURAL HISTORY SURVEY, CENTER FOR ECOLOGICAL ENTOMOLOGY, CHAMPAIGN, ILLINOIS,61820, USA; E-MAIL: [email protected]

Zusammenfassung

Umweltgerechter und nachhaltiger Pflanzenschutz erfordert eine Vielzahl verschiedenerStrategien zum Management von Schadinsekten auf ihren Wirtspflanzen. Insekten- undpflanzeneigene Signalstoffe sowie ihre synthetischen Analoga bieten einen nahezuunerschöpflichen Vorrat spezifischer Lockwirkungen und Interventionsmöglichkeiten an,der bisher leider nur unzureichend genutzt wird. Kürzliche Entdeckungen sowohl neuerKairomon-Lockstoffe als auch neuer Verfahrensschritte für das Management deswestlichen Maiswurzelbohrers Diabrotica virgifera virgifera LeConte (Coleoptera:Chrysomelidae) (D.v.v.) erweitern das Spektrum anwendbarer Management-Optionenfür diesen schwer bekämpfbaren Schädling im nordamerikanischen Maisanbau. Seitseiner Einschleppung nach Europa (ČAMPRAG & BAČA 1995) wird D.v.v. aberneuerdings auch zunehmend zum Problemschädling an Mais in Europa. Die neue"MSD"-Technik besteht aus einer Kombination von Massenabfang ("mass trapping"),Abschirmung ("shielding") und Umlenkung ("diversion") der Blattkäfer. Fallen hoherFangkapazität, die das Kairomon 4-Methoxyzimtaldehyd (MCA) und Cucurbitacin-Pulver als fraßfördernden Stoff enthalten, können bei Aufstellung als "Fallenzaun"relativ geringer Dichte die Käfer so umlenken, dass zwischen den beiden Seiten diesesZauns eine unsichtbare Geruchs-Barriere entsteht. Diese führt zu einer meßbaren undsignifikanten Verminderung der Käferzahl zwischen MCA behandelten Feldabschnittengegenüber ihren unbehandelten Kontrollen. In Maisfeldern der Standorte Urbana undChampaign des US-Staates Illinois ließen sich während der Monate August undSeptember 2003 und 2004 nach MCA-Behandlung gegenüber Kontrollen deutliche undsignifikante Verminderungen von D.v.v. an Hand dreier Kriterien nachweisen: 1.Käferzahlen auf Maispflanzen innerhalb des "MSD-Feldes", 2. Käferzahlen inSexuallockstoff-Fallen im MSD Feld und 3. Zahlen abgelegter D.v.v.-Eier im Boden desMSD Feldes nehmen ab. Der beobachtete Effekt läßt sich nicht allein auf diePopulationsverminderung infolge hoher Abfangzahlen zurückführen. Es gibt darüberBiological Control of Plant, Medical and Veterinary PestsR. Strang & H. Kleeberg (eds.)Copyright 2009 by Trifolio-M GmbH

142

hinaus einen Abschirm- und Umlenkeffekt, dessen sinnes- undverhaltensphysiologische Mechanismen zusätzliche künftige Erforschungen erforderlichmachen.

Abstract

Environmentally compatible and sustainable plant protection requires novel approachesto pest management characterized by minimal emphasis on toxicants. Classicaltoxicants traditionally dominated economic entomology for half a century. But worldwideproblems with environmental pollution and with increasing resistance levels in all majorpesticide classes and in many key insect species including Diabrotica virgifera virgifera(D.v.v.) strongly advocate a rethinking and a change in management paradigms used.Soft, minimally invasive, biological, biotechnical and cultural approaches should replacehard pesticides which are in favor up to now. Fortunately, pheromones, kairomones,plant attractants, better traps, new plant varieties and cultural methods like croprotation, in short more sophisticated methods are now available as pressure for findingand exploring novel strategies increases. Facing this situation, a new biotechnicalapproach of population reduction of D.v.v., called "MSD" technique, is introduced. MSDis characterized as an approach combining mass trapping, shielding and deflecting ofadult insects along an invisable odor barrier of synthetic kairomone which diminishesthe flux of insects across a high capacity trap line baited with kairomone , thus reducingthe population number and its reproductive success within the shielded area. In thecase of D.v.v. in Zea mays fields, effects realized by the MSD technique have beenmeasured simultaneously by a number of independent criteria during the summers of2003 and 2004 at 2 different locations in Illinois maize fields of up to one half hectaresize. Results observed are statistically significant and cannot be explained by masstrapping alone. There is also an additional shielding and deflection, in short “diversion”effect whose basic sensory and behavioral mechanisms calls for future exploration.

Keywords: Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), Zeamays, 4-methoxycinnamaldehyde (MCA), kairomone, diversion technique

Introduction

The leaf beetle Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae) (D.v.v.), alsocalled the western corn rootworm, is endemic to the New World and ranks among thetop ten insect pests in worldwide grain production. D.v.v. causes annual damages of 1billion US Dollars and is a notoriously difficult insect pest to control and manage, asentomological history of the past 50 years amply demonstrates (METCALF 1986).Considering recent emphasis on environmentally compatible and sustainablemanagement strategies, entomologists and practitioners are encouraged to pay

143

increased attention to novel approaches such as biotechnial methods which today arecharacterized by preferential use of signal compounds. Fortunately, both insect andplants provide a wide variety of such natural resources. In the case of D.v.v., sexpheromones and plant kairomones as specific attractants and management tools arerelatively well investigated through numerous contributions by GUSS et al. (1982),METCALF & METCALF (1992) and METCALF (1994) and many recent publications on theadvance and spread of D.v.v. within Europe (BERGER 1995–2004, HUMMEL 2003,HUMMEL et al. 2005 a and b, VIDAL et al. 2005).

Principle of MSD method: In this paper, the plant kairomone 4-methoxycinnamaldehyde(MCA), a specific attractant for D.v.v., is being used as a tool within the newly proposed"MSD" strategy. It combines a two pronge approach consisting of the well known masstrapping with the novel shielding and deflecting, called in short "diversion" andintroduced here for the first time (see fig. 1). An invisible “curtain” or “fence” of MCAvapor released from a MCA trap line establishes a behavioural barrier which the flyingbeetles cannot easily pass without being 1. either caught in one of the high capacitytraps or 2. being diverted elsewhere. The net effect is a significant reduction in adultpopulation density and oviposition within the MCA treated field as compared to anuntreated control field. These effects can be experimentally measured by 1. adult beetlecounts on maize plants, 2. by counts in independent monitoring traps baited with theD.v.v. sex pheromone, and 3. by egg counts taken in soil samples.


1. Fields: In 2003, a very late planted maize field of 2 x 0.27 ha size has been used. Itwas located on University of Illinois South Farm close to the intersection of Philo andCurtis Road in Southeast Urbana. A 2 m wide access path divided the two field sectionsserving as control and treatment, respectively. In 2004, a maize field of 0.27 ha size andlocated at First Street and Windsor Road within South Farm of Illinois Natural HistorySurvey at Champaign was used and divided into two sections of 2 x 0.12 ha with a0.03 ha maize buffer zone in between. The two field sites at Urbana and Champaignhad a distance of 4 miles from each other.

2. Traps and lures: For the MSD technique, two types of high capacity D.v.v. traps wereemployed. The first, depicted in fig. 2, was developed and built by the authors in 2003. Itwas replaced in 2004 by the commercially available Uni-trap (see fig. 2), formerly knownas Vario trap and purchased from Trifolio-M, D-35633 Lahnau.

144

Fig. 1: Field of Zea mays under treatment with the MSD strategy. A trap line of high capacity trapscombined with kairomone release stations surrounds the treatment section and reducesimmigration of beetles from surrounding areas across the barrier zone.

Fig. 2: IRC-high capacity trap (left) and Uni-trap (right) used for both mass trapping and diversion ofD.v.v. in Illinois maize fields during the growing seasons of 2003 and 2004, resp. Mass trappedbeetles are collected in bottom containers where they can be counted, sexed, and weighed.

Both trap types were baited with MCA as a medium range D.v.v. attractant described byMETCALF (1994). Beetles attracted to this kairomonal bait could feed on a substrate ofbitter cucurbitacin powder known to keep the beetles compulsorily attracted while

145

poisoning them with a tiny amount of carbaryl insecticide mixed in for quick knockdowninto a bottom container, from which the dead beetles could be removed, weighed orcounted. The specific properties and features of the traps will be described in aseparate report by Hummel and Shaw. MCA was purchased from Lancaster Co.,Frankfurt, catalog item 5467 in a purity of 98 %+, while D.v.v. sex pheromone camefrom Trifolio-M, D-35633 Lahnau. The bitter cucurbitacin kairomones in the powderoriginate from Cucurbita foetidissima (buffalo gourd). In combination with the D.v.v. sexpheromone 8-methyldecane-2-olpropanoate (Guss et al. 1982), the Metcalf traps(HUMMEL 2003, HUMMEL et al. 2003) were also useful as tools for monitoring andcomparing population densities within the control and treatment sections of the field.

Evaluation of eggs in soil samples: After harvest of the maize plants in October, soilcore samples of 1 kg each were obtained , prepared and washed in the corn rootwormegg separator and extraction machine described by SHAW & HUMMEL (2003).Recovered eggs were identified by their chorion sculpturing features.

Arrangement of "trap curtains" around perimeter of treated fields: A line of 14 traps wasestablished in 2003 with an individual trap distance of 20 m from each other. In 2004,between each of the 16 traps an individual distance of 10 m was selected. Traps werefastened at ear height of maize plants, monitored and rebaited twice a week. Traps in2003 were baited with 10 mg of MCA formulated with Tween-80 and Triton X-100 as a0.5 % emulsion (see HUMMEL & SHAW 2004) and soaked into a small piece of syntheticsponge. Uni-Traps used in 2004 received 10 mg of solid MCA on a piece of filter paper.

Evaluation of D.v.v. populations: Beetle counts on plants and in central monitoring trapswere recorded on a daily basis to see fluctuations. For counting beetles on maizeplants, between 25 and up to 75 plants per field were selected at random once to twicea day. Not only beetles on or below the leaf surface, but also those hiding in the cornear tip and in the whorls were counted.

Results and discussion

Immediately obvious are the significantly reduced beetle counts within the treated fieldsection as depicted in Fig. 3a and 3b for the years 2003 and 2004. These counts havebeen taken on a daily basis, sometimes twice a day, from a representative sample ofplants selected along a central line of the maize field and at least 10 m away from anylarge capacity trap. Differences between control and treatment are significant.

Less obvious but nevertheless significant are differences in daily beetle counts inkairomone (year 2003, 16 beetles per trap per day in treatment vs. 47 beetles per trapper day in control) and pheromone (year 2004) baited center traps (fig. 4). The centraltrap location was chosen for making sure that the average beetle population was

146

measured and not preferentially and primarily the influence of nearby mass trappingstations drawing beetles away from their immediate vicinity on the field perimeter.

More hidden but by far the most precisely measurable and practically significant effectsare those on egg counts in the soil. Both in the fall of 2003 and 2004, egg numbers insamples taken from the treated sections were lower than in the untreated controlsection. While the differences in 2003, due to high variance, could not be statisticallysecured (17 eggs in treatment vs. 93 in control), in 2004 they were very highlysignificant (2 eggs in treated plot vs. 60 eggs in control plot) (see tab. 1). Thus, inconclusion, the MCA trapping fence surrounding the treated section has a clear effecton both beetle population and oviposition. For the practicing farmer, both effects aremeaningful: Flying beetles like to feed on maize silk.

Fig. 3a and b: D.v.v. population counted on maize plants along central field line in Urbana 2003 (upper section) and Champaign 2004 (lower section).

17.8

19.8

21.8

23.8

25.8

27.8

29.8

31.8

2.9

4.9

6.9

8.9

10.9

12.9

0

1

2

3

*

*

**

**

****

** treatment control

beet

les

per p

lant

date in 2003

2.8

4.8

6.8

8.8

10.8

12.8

14.8

16.8

18.8

20.8

22.8

24.8

26.8

28.8

30.8

0

1

2

3

4

5

*****

****

*

**

*

*****

* treatment control

beet

les

per p

lant

date in 2004

147

Fig. 4: D.v.v. numbers counted in sex pheromone baited Uni-traps in 2004. Asterisk symbols stand forlevel of statistical significance.

Tab. 1: Effects of MSD technique on D.v.v. egg counts* in soil samples of maize fields, South Farm,Champaign, Illinois, USA, Oct. 2004

*take 5 cylindrical soil core samples from within the plant rows and 5 samples from between rows, each15.2 cm deep x 10.16 cm diameter = 0.473 litres = 1 kg; wash and extract eggs in machine of SHAW &HUMMEL (2003); count eggs under microscope;Binomial test for egg presence in soil: highest significance (p<0.001) for counts in control vs. treatment,no significance for counts taken from “between” vs. “within” rows.

By clipping pollen conducting tubes, the rate of kernel formation in the developing cornears may be reduced. As to reduced oviposition, fewer eggs hatching in the cominggrowing season will immediately benefit the growing maize plants and will reduce theneed for preventive or actual treatments. On the cost side, investments for traps andpoles purchased may not be cheap but once purchased may last for many seasons.Treatment costs for the MCA kairomone and cucurbitacin powder are quite moderate.

6.8

8.8

10.8

12.8

14.8

16.8

18.8

20.8

22.8

24.8

26.8

28.8

30.8

0

10

20

30

40

50

60

70

***

*

**

**

***

*

***

***

*****

*

treatment control

num

ber o

f bee

tles

per

pher

omon

e tra

p

date in 2004

Number of eggs counted insouth field with

“shielding/deflecting” and masstrapping

north field without“shielding/deflecting” = untreated

control

within rows 00020

311585

betweenrows

00000

43809

sum total 2 60

148

MCA costs for one month for ten rebaitings per trap are in the range of ten cents. Moreimportant will be the cost factor for labor. However, a number of measures for costreduction through future advances in MSD technology are imaginable if not likely.

Acknowledgements

The authors gratefully acknowledge permission to use maize fields in the south farmsection of the University of Illinois and the Illinois Natural History Survey during thegrowing seasons of 2003 and 2004. Dr. M. Hollenhorst of the computing center of JLUGießen provided help in the statistical evaluation of data. The Schwarz Foundation,D-74172 Heilbronn-Neckarsulm, kindly supported field experiments in 2004.

References

BERGER, H.K., ed. (1995–2004). IWGO Newsletters Vol. XIV–XXV reporting onadvances within the Diabrotica subgroup of IWGO.

ČAMPRAG, D., BAČA, F. (1995). Diabrotica virgifera virgifera (Coleoptera:Chrysomelidae): a new pest of maize in Yugoslavia. Pest. Sci. 45(3): 291–292.

GUSS, P.L., TUMLINSON, J. H., SONNET, P.E., PROVEAUX, A.T. 1982. Identification of afemale produced sex pheromone of the western corn rootworm Diabroticavirgifera virgifera. J. Chem. Ecol. 8: 545–556.

HUMMEL, H.E. (2003). Introduction of Diabrotica virgifera virgifera into the old world andits consequences: a recently aquired invasive alien pest species of Zea maysfrom North America. Comm. Appl. Biol. Sci., Ghent University, 68(4a): 45–57.

HUMMEL, H.E., BAČA, F., ERSKI, P. (2003). Orientation disruption of Diabrotica virgiferavirgifera in maize by a liquid MCA formulation released from paper squares in theBanat region of Serbia and Montenegro. Comm. Appl. Biol. Sci., GhentUniversity. 68(4a): 99–104.

HUMMEL, H.E., SHAW, J.T. (2004). Orientation disruption of the corn rootworm maizepests Diabrotica virgifera virgifera and D. barberi within odor spaces permeatedwith the kairomone mimetic MCA. Mitt. Dtsch. Ges. allg. angew. Ent. 14:171–175.

HUMMEL, H.E., UREK, G., MODIC, S., HEIN, D.F. (2005a). Monitoring presence andadvance of the alien invasive western corn rootworm beetle in eastern Sloveniawith highly sensitive Metcalf traps. . Mitt. Dtsch. Ges. allg. angew. Ent., DresdenSymposium 2005 (submitted)

HUMMEL, H.E., WUDTKE, A., BERTOSSA, M., HEIN, D.F., ULRICHS, CH. (2005b) Thewestern corn rootworm Diabrotica virgifera virgifera en route to Germany. Mitt.Dtsch. Ges. allg. angew. Ent., Dresden Symposium 2005 (submitted)

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METCALF, R.L. (1986). Foreword. pp. VII-XV In: KRYSAN, J.L., MILLER, T.A. (eds.)Methods for the Study of Pest Diabrotica. Springer, New York.

METCALF, R.L., METCALF, E.R. (1992). Plant Kairomones in Insect Ecology and Control.Chapman and Hall, New York.

METCALF, R.L. (1994). Chemical Ecology of Diabroticites. pp. 153–169 In: JOLIVET,P.H., COX, M.L., PETITPIERRE, E. (eds.). Novel Aspects of the Biology ofChrysomelidae. Kluwer Academic Publishers. Amsterdam.

SHAW, J.T., HUMMEL, H.E. (2003). A monitoring trap for Diabrotica virgifera virgifera andD. barberi adults lured with a poisoned cucurbitacin bait. Comm. Appl. Biol. Sci.,Ghent University 68(4a): 67–72.

Vidal, S., Kuhlmann, U., Edwards, C.R. (eds.) (2005). Western corn rootworm. Ecologyand Management. CABI Publishing, Wallingford.

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THE WESTERN CORN ROOTWORM DIABROTICA VIRGIFERAVIRGIFERA EN ROUTE TO GERMANY

HANS E. HUMMEL1,2, MARIO BERTOSSA3, ALEXANDER WUDTKE4, DETLEF F. HEIN1,GREGOR UREK5, SPELA MODIC5, CHRISTIAN ULRICHS4

1 JUSTUS-LIEBIG-UNIVERSITY, PROFESSORSHIP FOR ORGANIC AGRICULTURE, KARL-GLÖCKNER-STR. 21C,35394 GIEßEN, GERMANY; E-MAIL: [email protected]

2 ILLINOIS NATURAL HISTORY SURVEY, CENTER FOR ECOLOGICAL ENTOMOLOGY, CHAMPAIGN, ILLINOIS,61820, USA;

3 SWISS FEDERAL AGRICULTURAL RESEARCH STATION, CENTRO DI CADENAZZO, CH-6594 CONTONE,SWITZERLAND; E-MAIL: [email protected]

4 URBANER GARTENBAU, HUMBOLDT-UNIVERSITÄT ZU BERLIN, LENTZEALLEE 75, 14195 BERLIN; E-MAIL:[email protected]

5 AGRICULTURAL INSTITUTE OF SLOVENIA, HACQUETOVA 17, 1000 LJUBLJANA, SLOVENIA; E-MAIL:[email protected]

Zusammenfassung

Der westliche Maiswurzelbohrer, Diabrotica virgifera virgifera LeConte (Coleoptera:Chrysomelidae) (D.v.v.) ist einer der wichtigsten Maisschädlinge in Nordamerika. Seitseiner Einschleppung nach Serbien und seinem Nachweis bei Belgrad durch Baca imJahr 1993 breitet er sich schnell über Südosteuropa und von dort zunehmend nachZentraleuropa aus. Bis 2004 war Deutschland zwar noch frei von D.v.v., ist aber außeran seiner Nordost- und Nordflanke von Ländern mit nachweislichen D.v.v. Populationenumgeben. Außer stets möglichen Einschleppungen durch Flugzeuge gibt es dreiHauptrichtungen für das terrestrische Vordringen auf deutsches Staatsgebiet. Vondiesen ist die südlich-nördliche Stoßrichtung von der Lombardei in Norditalien über denTessin und die Nordschweiz bis nach Südbaden die wahrscheinlichste. Sie folgt einemsehr gut ausgebauten Straßen- und Schienennetz durch die Schweiz mithochentwickelten Verteilungszentren für Güter und Dienstleistungen, womit die aktiveMitwirkung des Menschen als Verbreitungsvektor des Schädlings unterstrichen wird.Kürzlich erlassene Fruchtfolge-Vorschriften im Schweizer Kanton Tessin konnten dieAusbreitung nach Norden zwar bremsen, aber nicht verhindern. In Anbetracht seinesjüngsten Vordringens bis an die deutschen Grenzen dürfte die Einschleppung vonD.v.v. auf deutsches Staatsgebiet bevorstehen und als längerfristig unvermeidlichgelten. Im Gegensatz zur Südschweiz ist die Population von D.v.v. in Slovenieninsgesamt noch recht gering. Jedoch ist die Zunahme in dessen Ostprovinzen imSommer 2004 unverkennbar gestiegen und kann angesichts relativ kurzer und schnellerBiological Control of Plant, Medical and Veterinary PestsR. Strang & H. Kleeberg (eds.)Copyright 2009 by Trifolio-M GmbH

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Transportwege über Klagenfurt, Villach und Salzburg eine weitere Einfallspforte nachSüdostbayern eröffnen.

Abstract

The western corn rootworm Diabrotica virgifera virgifera LeConte (D.v.v.) is one of themost important maize pests in North America. Ever since its invasion into Europe andits detection near Belgrade airport by Baca in 1993 it quickly spread all oversoutheastern Europe and is now advancing towards central Europe. Up until summer2004 considered free of D.v.v., Germany is, with the exception of its northern andnortheastern borders, surrounded by countries with proven D.v.v. infestations. Inaddition to simultaneous spot introductions by airplanes, three main routes for terrestrialintroduction into Germany are likely: 1. from south to north via Lombardy (Italy) throughSwitzerland to the State of Baden-Wuerttemberg in the southwest; 2. from south east tonorthwest via Croatia, Slovenia, Austria into the State of Bavaria; and 3. from Belgiumand the Netherlands in southeasterly direction to the state of Northrhine-Westfalia. Ofthese, route 1 is so far the most successful. It follows the well established network ofroad and rail connections through Switzerland and underscores the active role mankindand its technology plays as an active distribution vector for D.v.v. Mandatory croprotation in the Swiss State of Ticino did slow down but could not prevent the northboundadvance of D.v.v. in 2004. Considering the recent discovery of D.v.v. near the SouthGerman border, its introduction into German territory is only a matter of time and maybe ecologically unavoidable. In Slovenia, another relatively small transit state, the D.v.v.population density is still much lower than in Switzerland but with significantly increasingtrend during 2004 and with special emphasis in its southeastern provinces. Consideringits relatively short distance to southeastern Bavaria and the well developed transalpinerail and road system, Slovenia as a transit state may provide another access route forD.v.v. of lesser but still significant importance to Germany.

Keywords: Diabrotica virgifera virgifera LeConte, Zea mays, Metcalf trap, pheromone,kairomone, alien invasive species

Introduction

Diabrotica virgifera virgifera is the subject of many publications (for reviews see KRYSAN

& MILLER 1986, HUMMEL 2003, VIDAL et al. 2005). The beetle is a major maize pest inNorth America causing annual damage and treatment costs of about 1 billion US Dollars(METCALF 1986). D.v.v. today ranks on place 9 of the most important pests in worldgrain agriculture (UNGER & BAUFELD 2004). Its introduction into Germany and its furtherdistribution from this central location could cause harvest losses on 11.5 million ha ofmaize fields with a total annual loss figure of 25 million Euro. Figures for the total of

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Europe could be ten times higher. Trapping studies were performed during July andAugust of 2004 both in Switzerland and Slovenia. Both European regions arecharacterized as relatively small countries with a well developed traffic and roadinfrastructure. Both have proven D.v.v. infestations and both provide a transit functionfor transfer of these infestations to territories north of the Alpine mountain chain andbeyond into Germany. While a D.v.v. population in Slovenia is just developing andexpanding from selected spots discovered in 2003 (UREK & MODIC 2004, UREK et al.2005), the population found after 2000 in Southern Switzerland (BERTOSSA et al. 2001,DERRON 2003, BERTOSSA 2004) is now already well established and expanding to thenorth up to Basel and into French territory immediately adjacent to German borders.

The well developed infrastructure of transport routes by airplanes, trains, and freewaysthrough Switzerland is considered the prime import direction of D.v.v. to the north. Verysoon after its first detection at the airport of Agno, the Swiss plant protection officeestablished traps for monitoring of advance and spread of D.v.v. Our research in 2003and mainly in 2004 dealt with the population pressure building up along distributionroute 1 which is defined as the connecting line from the Lombardy region in northernItaly to urban centers of northern Switzerland. Route 2 runs from Hungary and Austria(CATE 2004, 2005) along Danube valley into Bavaria, while route 3 is connectingnorthern France, Belgium, and the Netherlands with areas in northwestern Germany.The latter two paths, also supported by model calculations of Baufeld & Enzian (2005),are not considered here any further although their future importance should by nomeans be neglected. Another south easterly path connecting Zagreb, Ljubljana,Klagenfurt, Villach, Salzburg to Eastern Bavaria via tunnels or passes acrossKarawanken and Austrian Tauern mountains should receive some attention.

In this investigation we established a series of monitoring traps of the Metcalf typebaited with two different lures. We placed these traps at strategic locations within the 3zones A, B and C, and X of Switzerland and in the eastern provinces of Slovenia (seefig. 1, fig. 4, and tab. 1) and report the usefulness of these traps. Our results suggestthat in coming years the transfer and import of D.v.v. into southwest Germany is to beexpected if it has not silently and inadvertently taken place already.

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Metcalf traps (see fig. 2) have been used. Their properties are described by LEVINE &METCALF (1988), NOVAK et al. (2001), HUMMEL (2003), HUMMEL et al. (2003), HUMMEL

& SHAW (2004). Lures for baiting these traps have been described by GUSS et al. (1982)and by METCALF (1994). Traps in Switzerland and Slovenia were monitored for a total ofsome 7 weeks starting on July 18 and ending on Sept 1, 2004. Reading of results withsimultaneous exchange of lures occurred once to twice per week. Three different zonesof decreasing population were verified in Switzerland as previously defined byBERTOSSA et al. (2001) and DERRON (2003). In addition, a likely transition zone couldbe identified in the relatively long but narrow mountain valley roads connectingBellinzona with villages situated north on the way to mountain passes of St. Gotthard,St. Bernardino and beyond.

Fig. 1: Above: outline of Switzerland with new D.v.v. infestations 2003 north of the Alpine chain aremarked with circles. Location Winterthur with new infestation of 2004 is marked with a square.Below: Zones A – C with D.v.v. infestation in Ticino; A: area around Chiasso; B: area aroundLugano; C: area around Magadino plain. Grisons is situated to the right (geographically east) ofTicino.

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Fig. 2: Metcalf sticky cup trap on Zea mays plant.

The southern Zone A is located immediately behind the border crossing of Chiassobetween Italy and Switzerland and includes Novazzano with its extensive railyard androad connections. To the north, within zone B, Breganzona with airport Agno is situated.It is considered to be the original starting point of D.v.v. within the canton Ticino in theyear 2000. Geographically further to the north, but separated by the relatively shallowMt. Ceneri pass, zone C follows. Its 2.000 ha area includes the entire Magadino plain, afertile area with a high percentage of maize agriculture, but also characterized by thepresence of the canton capital Bellinzona. A dense system of major transit traffic linesfor handling the transfer of goods and services from Italy to Northern Switzerland,France and Germany and beyond are running through this area. A small network of 7trapping stations is located there. It consists of traps at Mt. Ceneri, Camorino, Contone,Magadino sports center, Gordola airport, Gudo, and at Cadenazzo Research Center.Each trapping station usually consists of a pair of trapping sites, a minimum of 20 mapart, with two Metcalf traps baited with 0.1 mg of D.v.v. sex pheromone8-methyl-decane-2-olpropanoate and 10 mg of the kairomone4-methoxycinnamaldehyde (MCA) and situated just a few rows inside the perimeter ofmaize fields in order to protect them from wind gusts and view.

Results

The Metcalf traps turned out to be a very sensitive tool for monitoring and comparingthe weekly abundance of beetles within the 3 zones. In its attraction power, the Metcalftrap, in combination with the sex pheromone lure, was sometimes superior, both intrapping rate and very early catching date, to the PAL trap mostly used by otherauthors. The claim of sensitivity and early documentation of beetle presence can be

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substantiated by experiments undertaken in 2004 both in southern Switzerland and ineastern Slovenia.

Comparing Metcalf traps baited with different lures (sex pheromone vs. kairomoneMCA), the sex pheromone bait expectably attracts beetle numbers 2 to 10 times higherthan the kairomone MCA does. Data in support of this notion for southern Switzerland in2004 are given in fig. 3. It should be noted, however, that MCA bait, in spite of lowernumbers, can attract both males and females and thus give a more precise status of thechanging composition of a D.v.v. population at the trapping site at any one time.

Table 1 lists averaged numbers for D.v.v. densities per trap per week in the zones A toC of the canton Ticino. It also lists some additional observations for the connectingroutes along the sites S. Vittore and Lostallo which are located within the cantonGrisons along the major transfer route to Northern Switzerland via S. Bernardino passand merging into the Upper Rhine valley with connections to the Lake Constance area.

Experiments with Metcalf traps performed in Eastern Slovenia at the sites Vitan,Vodranci, Gibina, Gibina border crossing, and Pince (see fig. 4) established asubstantially expanding and advancing D.v.v. population in the border area near toCroatia, Hungary, and Austria (UREK & MODIC 2004, UREK et al. 2005).

Discussion and conclusions

Fig. 1 and tab. 1 seen in perspective, a stepwise population decline becomes apparentas one moves northward from zone A to C within the canton Ticino. However, there is astriking discontinuity at the transition from Ticino region C to Grisons zone X. This iseasily explainable, however, as a consequence of omitted crop rotation. With croprotation, the locations of San Vittore and Lostallo would probably show D.v.v. densitiesof 20-30 beetles /trap/ week. These figures would fit quite well into a population gradientdecreasing steadily in northerly direction.

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Tab. 1: Standardized beetle counts of Diabrotica virgifera virgifera per trap and week dated from July toSeptember 2004 in the Swiss cantons Ticino and Grisons

1 Locations Camorino and Contone near the main freeways, railyards, and train stations2 Locations Magadino Sports Center, Gordola, and Gudo more remote from major traffic flux3 Location Cadenazzo Research Station, maize research plots, similarly remote as location 2

Ticino zone Metcalf trap withpheromone as lure

Metcalf trap withMCA as lure

remarks

A(location

Novazzano)63 38 with crop rotation

B(location

Breganzona)40 7 with crop rotation

CMagadino plain

(near 1)24 4 with crop rotation

CMagadino plain

(remote 2)3 0.4 with crop rotation

CMagadino plain

(remote 3)2 0.3 with crop rotation

Grisons zone XSan Vittore 56 3.5 without crop rotation

(maizemonoculture)

Lostallo 73.8 15.2 without crop rotation(maize

monoculture)

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Fig. 3: Comparison of trapping efficacies of Metcalf traps baited with sex pheromone lure and MCA atvarious locations in 2004 at the Magadino plain of southern Switzerland.

Fig. 4: Trapping locations of D.v.v. in Eastern Slovenia during 2004.

However, without crop rotation, the actually measured density is of course higher by afactor of 2 to 3. Accordingly, the chance of northbound transportation of D.v.v. byhuman activity increases as well. This in turn translates into faster import into thenorthern provinces of Switzerland and, in consequence, to the relatively open German(and French) border areas adjacent to the north. In hindsight, the specimen found in2004 at Winterthur (SCHAUB & BERTOSSA 2005) some 30 km from the German border

Camorino Contone Magadino Gordola Gudo0

25

50

75

100

125

150

175

200nu

mbe

r of b

eetle

s

location

Phero MCA

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may be considered the “wave front” of D.v.v. spilling northward from southern regions.The next and larger wave of 2005 may reach German territory. Additional and parallelimportation of D.v.v. by air plane to Zürich international and some regional airports northof the Alps may further speed up the appearance of D.v.v. as an invasive pest.Independent model calculations by BAUFELD & ENZIAN (2005) reinforce ourexperimental findings of 2003 and 2004 in the Val Blenio (Ticino) and Moesa Valley(Grisons). The last maize field at the village of Olivone and Campo Blenio at about 800m elevation is only some 20 km away from Lukmanier pass and about 65 km away fromthe next villages and maize fields in the Upper Rhine (Vorderrhein) Valley of Grisons. In2003, the field at Olivone had a noticeable and surprising D.v.v. population whichproved that D.v.v. can do quite well at these elevations and could give rise to beetlestravelling north if given the chance to do so. Similarly, Lostallo in the Moesa valley issome 15 km from San Bernardino pass and about the same distance of some 15 kmfrom the Hinterrhein Valley and the villages Nufenen and Spluegen where more maizefields can be found. Such distances can be covered by trucks, campers, and passengercars within an hour, and survival of dispersing beetles with the inclination and ability tooviposit may not be a major hurdle. In their calculations for Switzerland, BAUFELD &ENZIAN (2005) list some of the locations cited above as medium to high risk areas forD.v.v. attack. There is but a trivial 100 km distance linking these valley areas south andnorth of the Alpine chain, and it is easy to imagine that D.v.v. can overcome suchobstacles within a few years only, notably with the involuntary help of man and histransalpine traffic and trade connections. Similar model calculations, so far not reported,may apply to the situation of Slovenia and its northbound traffic connection into Austriaand Germany. Progress of D.v.v. along the Danube valley from northern Hungarythrough Burgenland into lower Austria and from there to the west towards Bavaria isanother route which will be taken by D.v.v. in the future. A short comment on data by P.CATE (2005) shall suffice: He reports rapid spreading and multiplication being observedin the area of Vienna. In the north of Austria the annual increase in range observed from2003 to 2004 was approximately 40 km inland.

Considering the high sensitivity, versatility, and low cost of the Metcalf trap, its futureuse for monitoring low populations of D.v.v. is recommended. It can prove presence orabsence of beetles at a quite early time in their flight cycle and thus is a powerful helpfor deciding on pest management actions to be taken in the immediate future. Trappingresults reinforce the previous findings of BERTOSSA et al. (2001) of 3 geographicallydistinguishable population levels in Zone A (highest), B (medium) and C (low). Withinthese areas, there is some expectable variation in population density both in space andtime. It is, however, noteworthy that those trapping locations which are situated next toor near to major freeways, train tracks, railyards, and distribution centers showed a

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significantly higher density of beetles (Camorino and Contone) than those situated moreremotely, such as those at Magadino sports center, Gordola, Gudo, and the CadenazzoResearch Station. We conclude that trade routes with lots of traffic are also a majormeans of beetle transport and spread, thus identifying man and his technology as majorvectors of the rapid D.v.v. spread observed (HUMMEL et al. 2005). Given thismechanism, transfer by traffic and commerce seems to be unavoidable except underthe most tedious, time consuming, costly and thus impracticable phytosanitarymeasures imaginable.

Mandatory crop rotation starting in 2003 in the entire Ticino area could retard but noteliminate the advance of D.v.v. also because important populations migrate fromLombardy (I) to the Ticino region every year. In the neighboring cantonal region ofGrisons where crop rotation is neither mandatory nor enforced, there is an increase inpopulation pressure. From the standpoint of D.v.v., this increase happens to occur atthe strategically most favorable route along one of the major traffic routes to NorthernSwitzerland, Southern Germany, and Eastern France. Thus, regional, even nationalprecautions against D.v.v. are not longer sufficient. Europe as a unit, it seems, needs awell coordinated and dense system of quarantine measures. Germany situated at thevery center of Europe will have a very responsible future role to play in implementingpreventive measures.

Acknowledgements

The authors are grateful for helpful cooperation with R. Brunetti of the Ticino plantprotection office. Also, generous funding by Schwarz Foundation during the summer of2004 was essential.

References

BAČA, F. (1993). New member of the harmful entomofauna of Yugoslavia Diabroticavirgifera virgifera LeConte (Chrysomelidae). IWGO Newsletter XII (12), p. 21.

BAUFELD, P., ENZIAN, S. (2005). Maize high-risk areas and potential yield losses due towestern corn rootworm (Diabrotica virgifera virgifera) damage in selectedeuropean countries. pp. 285–302, specifically pp. 293 and 296. In: VIDAL S. et al.loc. cit.

BERTOSSA, M., DERRON, J., COLOMBI, L., BRUNETTI, R. (2001). Update of monitoring ofDiabrotica virgifera virgifera LeConte in Switzerland in 2001. pp. 169–173 In:Veneto Agricoltura (ed.) Proceedings of XXI IWGO Conference and VIIIth

Diabrotica Subgroup Meeting. Legnaro, Padova, Venice, Italy, Oct. 27 – Nov. 3,2001.

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BERTOSSA, M. (2004). Effect of containment strategies against Diabrotica virgiferavirgifera in Switzerland. Abstract 10th Diabrotica subgroup meeting Jan. 14–16,2004, Engelberg, Switzerland.

CATE, P.C. (2004). Present distribution of western corn rootworm (Diabrotica virgiferavirgifera) in Austria. Abstract 10th Diabrotica subgroup meeting Jan. 14–16,2004, Engelberg, Switzerland.

CATE, P. (2005). Results of the 2004 monitoring program for WCR in Austria. Abstracts11th Diabrotica subgroup meeting, Feb. 14–17, 2005, Bratislava, SlovakRepublic.

DERRON, J. (2003). Monitoring of Diabrotica in Switzerland in 2003. IWGO News Letter,Vol. XXV, Nr. 2, 10/04 (http:// www.iwgo.org/diabrotica-meeting-2004/index.htm)

GUSS, P.L., TUMLINSON, J. H., SONNET, P.E., PROVEAUX, A.T. (1982). Identification of afemale produced sex pheromone of the western corn rootworm Diabroticavirgifera virgifera. J. Chem. Ecol. 8: 545–556.

HUMMEL, H.E. (2003). Introduction of Diabrotica virgifera virgifera into the old world andits consequences: a recently acquired invasive alien pest species of Zea maysfrom North America. Comm. Appl. Biol. Sci, Ghent University, 68(4a), 45–57.

HUMMEL, H.E., BAČA, F., ERSKI, P. (2003). Orientation disruption of Diabrotica virgiferavirgifera in maize by a liquid MCA formulation released from paper squares in theBanat region of Serbia and Montenegro. Comm. Appl. Biol. Sci., GhentUniversity. 68(4a): 99–104.

HUMMEL, H.E., SHAW, J.T. (2004). Orientation disruption of the corn rootworm maizepests Diabrotica virgifera virgifera and D. barberi within odor spaces permeatedwith the kairomone mimetic MCA. Mitt. Dtsch. Ges. allg. angew. Ent. 14:171–175.

HUMMEL, H.E., WUDTKE, A., HEIN, D.F., ULRICHS, Ch., BERTOSSA, M., UREK, G. (2005).The western corn rootworm en route to the German Federal Republic. Abstracts11th Diabrotica subgroup meeting, Feb. 14–17, 2005, Bratislava, SlovakRepublic.

KRYSAN, J.L., MILLER, T.A. (eds.) (1986). Methods for the Study of Pest Diabrotica.Springer, New York.

LEVINE, E., METCALF, R.L. (1988). Sticky attractant traps for monitoring corn rootwormbeetles. Ill. Nat. Hist. Survey reports no. 279, Sept. 1988, 2 pages.

METCALF, R.L. (1986). Foreword. pp. VII-XV In: KRYSAN, J.L. & MILLER, T.A. (eds) loc.cit.

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METCALF, R.L. (1994). Chemical Ecology of Diabroticites. pp. 153–169. In: JOLIVET,P.H., COX, M.L., PETITPIERRE, E. (eds.). Novel Aspects of the Biology ofChrysomelidae. Kluwer Academic Publishers. Amsterdam.

NOVAK, R.J., METCALF, R.L., LAMPMAN, R.L., HUMMEL, H.E. (2001). Innovative PestManagement of Corn Rootworms: the use of Kairomone-Impregnated Baits,pp.60–72. In: MULLA, Mir S., (ed.) Biopesticides: Biotechnology, Toxicity,Efficacy, Safety, Development and Proper Use. Proc. 2nd InternationalConference on Biopesticides. Compact Print, Thailand. ISBN 974-229-056-3.

SCHAUB, L., BERTOSSA M. (2005). Evaluation of control strategies through monitoring inSwitzerland. Abstract 11th Diabrotica subgroup meeting Feb. 14–16, 2005,Bratislava SK.

UNGER, J.-G., BAUFELD, P. (2004) Die Ausbreitung des westlichen Maiswurzelbohrers.Mais 2: 40–43.

UREK, G., MODIC, S. (2004). First report on western corn rootworm (Diabrotica virgiferavirgifera LeConte) in Slovenia. Abstract 10th Diabrotica subgroup meeting Jan.14–16, 2004, Engelberg, Switzerland, p. 34. See also IWGO Newsletter XXV,no. 2, Oct 2004, pp. 19–20.

UREK, G., MODIC, S. KNAPIC, M., ČERGAN, Z. (2005). Spreading of WCR in Slovenia in2004. Abstracts 11th Diabrotica subgroup meeting, Feb. 14–17, 2005, Bratislava,Slovak Republic.

VIDAL, S., KUHLMANN, U., EDWARDS, C.R. (eds.) (2005). Western corn rootworm.Ecology and Management. CABI Publishing, Wallingford.

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EFFECT OF NEEMAZAL-T/S ON THE VEGETABLE PESTS IN ROMANIA

MARIA CALIN*, STOIAN LUCIAN*, EDMUND HUMMEL**

* VEGETABLE RESEARCH - DEVELOPMENT STATION BACĂU, CALEA BÂRLADULUI, NR. 220, COD 600 388,BACĂU, ROMANIA, TEL. 4(0) - 234 - 544963, FAX 4(0) - 234 - 517370, [email protected],WWW.ARTELECOM.NET/LEGUMEBAC

** DEPARTMENT OF ENTOMOLOGY, TRIFOLIO-M GMBH, DR.-HANS-WILHELMI-WEG 1, 35633 LAHNAU -GERMANY, TEL: +49 - 64 41-20 977 25, FAX: +49 - 64 41-20 977 50, WWW.TRIFOLIO-M.DE

Abstract

During June - October 2004, vegetable field and greenhouse experiments wereperformed in Vegetable Research-Development Station Bacau - Romania, in order toevaluate the effect of natural insecticide NeemAzal-T/S on: cabbage aphid -Brevicorynebrassicae (L), in cabbage; black bean aphid - Aphis fabae Scopoli in bean; Coloradobeetle - Leptinotarsa decemlineata Say, in eggplant; glasshouse or potato aphid -Macrosiphum euphorbiae (Thomas) and potato aphid - Aulocorthum solani (Kaltenbach)in tomato and the cucumber aphid - Aphis gossypii Glov. in cucumber. The experimentswere carried out in a research field. The treatments evaluated were NeemAzal-T/S0.5% and compared with untreated control. NeemAzal-T/S - 0.5% showed a very goodefficacy, betwen 91,7 - 96,5%, after 4 to 7 days of treatments for cabbage, bean, eggplant and tomato pests and 75.95 % for cucumber aphids.

After two treatments, the time of mortality was shorter for 12 - 30 hours, compared tothe first treatment.

Introduction

Vegetable crops are a special ecosystem of complex interrelationships between plants,animals and cultural operation (Baicu and Săvescu, 1986). The damage of pests invegetable crops increased in last years (Calin, 2004). Natural means are of primaryinterest for organic growers as control measures against vegetable pests. There are aseries of promising data for the practical use of Azadiractins in control of vegetablepests (Manger, 2000, Meadow et al., 2000, Hellesaar et al., 2000, Almie, 2000). Wetested the formulation NeemAzal-T/S, which is based on an extract of neem seedkernels. It is a product of Trifolio-M GmbH, Germany and contains 1% Azadirachtin A,as the main active ingredient.

The preliminary evaluation of the practical, use of NeemAzal-T/S in Romania againstvegetable pests is analyzed in this paper. Biological Control of Plant, Medical and Veterinary PestsR. Strang & H. Kleeberg (eds.)Copyright 2009 by Trifolio-M GmbH

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During June - October 2004, vegetable field experiments were performed in theVegetable Research-Development Station Bacau - Romania, in order to valuate theeffect of the natural insecticide NeemAzal-T/S on: cabbage aphid -Brevicorynebrassicae (L), in cabbage; black bean aphid - Aphis fabae Scopoli in bean; Coloradobeetle - Leptinotarsa decemlineata Say, in eggplant; glasshouse or potato aphid -Macrosiphum euphorbiae (Thomas) and potato aphid - Aulocorthum solani (Kaltenbach)in tomato as well as the cucumber aphid - Aphis gossypii Glov. in cucumber.

The trials: 04 - 103 in cabbage, 04 - 108 in tomato, 04 - 111 in climbing bean, 04 - 125in cucumber, used aphides as target pests and, 04 - 121 in eggplant used Coloradobeetle as the target pest. The trials were conducted by V.R.D.S. Bacau in open fields,except 04 - 125 in cucumber, which was performed in the greenhouse.

NeemAzal-T/S was applied in 0.5 % concentration and compared with untreated controlplants.

The application were made on:

- 30 June 1 treatment of cabbage aphids Brevicoryne brassicae L. in cabbage;

- 13 August, first treatment of black bean aphid - Aphis fabae Scopoli in bean; Coloradobeetle - Leptinotarsa decemlineata Say, in eggplant; glasshouse or potato aphid -Macrosiphum euphorbiae (Thomas) and potato aphid - Aulocorthum solani (Kaltenbach)in tomato;

- 26 August, second treatment of black bean aphid - Aphis fabae Scopoli in bean;Colorado beetle - Leptinotarsa decemlineata Say, in eggplant or potato aphid -Macrosiphon euphorbiae and potato aphid - Aulocorthum solani (Kaltenbach) in tomato;

- 27 September first treatment of cucumber aphid - Aphis gossypii Glov. in cucumber.

The observations were made before the application and 3, 5 and 7 days afterapplication. Observations were made on:

- all adult and non-adult stages (larvae + pupae) of cabbage aphids were counted on 50randmly chosen leaves per plot;

- all adult and non-adult stages (larvae + pupae) of black bean aphids and glasshouseaphid, potato aphid and cucumber aphid were counted on 10 randomly chosen plantsper plot;

- ten larvae of Colorado beetle 3 instar (L3) per plant

We determined the efficiency of NeemAzal-T/S against pests, in % of mortality.

Assessments were also made on phytotoxicity, crop development and visible residues.

165

Test variants/concentrations were:

1. NeemAzal-T/S - 0.5 %

2. Untreated control

Treatment technique: spraying till run-of (600 l/ha in cabbage, 800 l/ha in egg plant andcucumber, 1 000 l/ha in tomato, 1 720 l in climbing bean) with Guarany sprayer;

Crop and cultivar: cabbage, Gloria variety; tomato, Siberian and Ace Royal varieties,egg plant, Contesa vaiety, cucumber, 9C line.

Soil type: alluvial medium advanced;

Plot size: 5 m x 1,4 m (40 plants per plot).


The efficacy of the application of NeemAzal-T/S (0.5 %) against vegetable pests isshown in Table 1.

Table 1 The efficiency of NeemAzal-T/S (0.5 %) against vegetable pests

Plot Target pest NeemAzalconcentra

tion inwater

Sprayvolume(l/ha)

Efficacy(%)

Theinsecticidal

effects

04 - 103 Control cabbge aphid, in cabbageNA-T/S Brevicoryne brassicae 0.5 % 600 96.5 Very good

04 - 108 Control aphids in tomatoNA-T/S Macrosiphon

euphorbiaeAulocorthum solani

0.5 % 1 000 93.4 Very good

04 - 111 Control black bean aphid in climbing bean NA-T/S Aphis fabae 0.5 % 1 720 95.1 Very good

04 - 121 Control Colorado beetle in egg plantNA-T/S Leptinotarsa

decemlineata0.5 % 800 94.2 Very good

04 - 125 Control cucumber aphid in cucumberNA-T/S Aphis gossypii 0.5 % 800 75.95 Good

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The trial 04 - 103, Control of cabbage aphid in cabbage

A high population density of cabbage aphides was determined at the beginning of thetest (fig. 1).

Fig. 1 Average number of aphides per leaf

The population decreased strongly 3, 5 and 7 days after treatment with NeemAzal-T/S -0.5% (fig. 1). Treatment was significantly different to the untreated content. Theefficiency of NeemAzal-T/S - 0.5 % was 96.5 % (table 1). The insecticide effect ofNeemAzal-T/S was very good against cabbage aphids. The surviving larvae in theNeemAzal-T/S were very small and they caused minimal damage.

In the untreated plot virginoparae formed colonies which developed progressively,spreading to leaves at the heart of plants. Winged individuals emerged when thepopulation had reached a maximum (in middle of June) and infestations spread to newplants. Colonies were then diminished progressively by predators and parasites. In thelast of June (time of treatment) was a high population density of cabbage aphides andthe summer high temperature and zoophagous induced the very small decrease ofaphides number per leaf, but this was insignificant (fig. 1).

0

5

10

15

20

25

30

35

40

45

0 3 5 7No. days after treatment

No.

aph

ides

per

leaf

NeemAzal-T/S - 0,5%Untreated

167

The trial 04 - 108 Control aphids in tomato

A high population density of aphides was determined in the middle of August Fig. 2 inthe tomato cultures (see Fig 2).

Fig. 2 Average number of aphids per leaf

It can be seen from Fig. 2 that the population decreased strongly after 3, 5 and 7 daysafter treatment of NeemAzal-T/S (0.5%). The treatment was significantly different to theuntreated control. The efficiency of NeemAzal-T/S - 0.5 % was 93.4 % (table 1). Theinsecticidal effect of NeemAzal-T/S was very good against glasshouse and potatoaphids. The surviving aphids in the NeemAzal-T/S plot were small. But because wingedindividuals spread infestations to new plants the application was repeatet for a secondtreatment.

In the untreated plot, aphid colonies increase rapidly from early August. Virginoparaewere present on leaves and flower or leaf peduncles. The form of potato aphid wasgreen, which typically infested lower leaves of tomato. Virginoparae formed colonieswhich developed progressively, spreading to leaves of the plants. Winged individualsemerged when the population had reached a maximum (in the middle of August) andspread infestations to new plants. At the beginning to middle of August the colonieswere diminished progressively by predators and parasites. (fig. 2).

The trial 04 - 111 Control black bean aphid in climbing bean

A high population density of aphids was determined in August (see fig. 3) in the climbingbean cultures.

The number of aphides decreased strongly 3, 5 and 7 days after treatment of

0

2

4

6

8

10

12

14

16


No.

aph

ides

per

leaf

NeemAzal-T/S -0,5%Untreated

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NeemAzal-T/S - 0.5% (fig. 3). Treatment was significantly different to the untreated. Theefficiency of NeemAzal-T/S - 0.5 % was 95.1 % (see table 1). The insecticidal effect ofNeemAzal-T/S was very good against black bean aphid. The surviving aphides in theNeemAzal-T/S plot were very small.

Fig. 3 Average of aphids per plant

But because winged individuals spread infestations to new plants the treatment wasrepeated.In the untreated, from last July onwards, winged virginoparae colonize numerous beanplants, depositing apterous nymphs on the underside of leaves or at the stems. Aphidcolonies increase rapidly until mid-August (fig. 3).

The trial 04 - 121 Control Colorado beetle in egg plant

A low population density of Colorado beetle was determined in August (fig. 4) in the eggplant cultures. The number of larvae decreased 3, 5 and 7 days after treatment ofNeemAzal-T/S - 0.5% (fig. 4).

Results after treatment were significantly different to the untreated plot. The efficiency ofNeemAzal-T/S - 0.5 % was 94.2 % (table 1). The insecticide effect of NeemAzal-T/Swas very good for control of L3 Colorado beetle.

In the untreated plot, the population of second generation of Colorado beetle was verylow. For experiment we infested 10 plants per plot with 10 larvae per plant. In thissituation the populations were diminished progressively by predators and parasites. (fig.4).

0

50

100

150

200

250

300

0 3 5 7No days after treatment

No.

aph

ides

per

pla

nt


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The trial 04 - 125 Control cucumber aphid in cucumber

Because the climatic condition was unfavorable for cucumbers (low temperatures andheavy rains) the cucumber cultures in open field did not survive in this year.

Fig. 4 Larvae of Colorado beetle per leaf

A high population density of aphides was determined in September (fig. 5) in the greenhouse cucumber culture and we used it for NeemAzal-T/S test. The number of aphidesdecreased 3, 5 and 7 days after treatment of NeemAzal-T/S - 0.5% (table 1 and fig. 5).

Fig. 5 Afides per plant

0

2

4

6

8

10

12

0 3 5 7

No. days after treatment

No.

larv

ae p

er le

af

NeemAzal-T/S -0,5%Untreated

050

100150200250300350400450500


No.

aph

ides

per

pla

nt


170

Treatment was significantly different to the untreated. The efficiency of NeemAzal-T/S -0.5 % was 75.95 %.

Conclusions

The trial 04 - 103, Control cabbge aphid, in cabbage

A high population density of cabbage aphides was determined at the end of June.

The population decreased strongly 3, 5 and 7 days after treatment with NeemAzal-T/S -0.5% (fig. 1). Treatment was significantly different to the untreated control. Theefficiency of NeemAzal-T/S - 0.5 % was 96.5 %. The insecticidal effect ofNeemAzal-T/S was very good against cabbage aphid. The surviving larvae in theNeemAzal-T/S were very small and they caused minimal damage.

The trial 04 - 108 Control aphids in tomato

The population decreased strongly 3, 5 and 7 days after treatment of NeemAzal-T/S -0.5%. Treatment was significantly different to the untreated control. The efficiency ofNeemAzal-T/S - 0.5 % was 93.4 %. The insecticidal effect of NeemAzal-T/S was verygood against glasshouse and potato aphid. The surviving aphides in the NeemAzal-T/Splot were small.

The trial 04 - 111 Control black bean aphid in climbing bean

Treatment was significantly different to the untreated control. The efficiency ofNeemAzal-T/S - 0.5 % was 95.1 %. The insecticidal effect of NeemAzal-T/S was verygood against black bean aphid. The surviving aphides in the NeemAzal-T/S plot werevery small.

The trial 04 - 121 Control Colorado beetle in egg plant

The number of larvae decreased 3, 5 and 7 days after treatment of NeemAzal-T/S -0.5%. Treatment was significantly different to the untreated control. The efficiency ofNeemAzal-T/S - 0.5 % was 94.2 %. The insecticidal effect of NeemAzal-T/S was verygood for control of L3 Colorado beetle.

The trial 04 - 125 Control cucumber aphid in cucumber

Treatment was significantly different to the untreated control. The efficiency ofNeemAzal-T/S - 0.5 % was 75.95 %.

171

References

Almie M van den Berg, 2000, The effects of botanical pesticides on diamondback moth.Practice Oriented Results on Use and Production of Neem - Ingredients andPheromones, 177 - 178.

Baicu T., 1989, Câteva recomandări privind organizarea experienţelor, înregistrarea şiprelucrarea datelor experimentale în protecţia plantelor. Testarea miojloacelor deprotecţie a plantelor, vol. Xl.

Călin Maria 2004, Dăunătorii polifagi ai plantelor legumicole şi combaterea lor Înagricultură biologică. Ed. Gad Print, Bacău, 60 pp.

Hellesaar K., Metspalu L., Joudu J., kuusik A., 2000, Diverse effects of NeemAzal - T/Srevelead by preimaginal stages of colorado potato beetles, Leptinotarsadecemlineta Say. . Practice Oriented Results on Use and Production of Neem -Ingredients and Pheromones, 79 - 83.

Manger W., 2000, Results of NeemAzal-T/S against white flies in practice, PracticeOriented Results on Use and Production of Neem - Ingredients andPheromones, 43 - 49.

Meadow et al., 2000, The effect of Neem extracts on the turnip root fly and the cabbagemoth. Practice Oriented Results on Use and Production of Neem - Ingredientsand Pheromones, 55 - 60.

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RESULTS OF TRIALS WITH NEEMAZAL-T/S IN SAXONIAN CABBAGEPRODUCTION

KOEHLER, G.

SAXON STATE INSTITUT FOR AGRICULTURE, SÖBRIGENER STR. 3A, 01327 DRESDEN, GERMANY,[email protected]

In five Saxonian enterprises of the ecological agriculture 1997 the preparationNeemAzal-T/S were tested in small plots of cabbage. Four enterprises were verycontent with the result. In a fifth enterprise due to logistic difficulties, the treatment wasbegun too late. This enterprise was not included into the evaluation.

The treatments against Cabbage earth Fleas (Phyllotreta sp. , Col., Chrysomelidae)were successful. With smaller infestation degrees of the pest 2 l/ha was sufficient,strong infestation needed a treatment with 3l/ha (Tab. 1).

Table 1. Infestation of crop with Cabbage earth Fleas

NeemAzal-T/S has a very good effect against Cabbage Aphid (Brevicoryne brassicae)in White Cabbage (fig. 1), Red Cabbage, cauliflower, Broccoli, Savoy Cabbage andTurnip Cabbage. Depending upon infestation degrees up to three treatments werenecessary (0,5% or 3 l/ha in 500 l solution).

Crop Date of % of demaged plantsapplication observation (>10% leaf area)

Broccoli 27.6. 23.6. 1015.7. 0

White Cabbage 19.8. 7.8. 204.9. 0

Brussels sprout 19.8. 7.8. 804.9. 0

2l/ha 3l/haRed Cabbage 15.5. 12.05. 100 100

26.05. 20 09.06. 0 0

Savoy Cabbage 12.6. 09.06. 10 1026.06. 0 0

Turnip Cabbage 19.8. 12.08. 20 2027.08. 0 0


174

Fig. 1 Infestation of White Cabbage with Cabbage Aphid (1997)

On Brussels Sprout the treatment was not successful in all areas (fig. 2), because onthe late infestation of crop with pest could not be reacted in the late summer andautumn (harvest work etc).

Fig. 2 Infestation of Brussels Sprot with Cabbage Aphid, 1997

In area 1 and 2 the treatments was successful, because on beginning of infestation theplants were treated twice in an interval of 10 days; additionaly, in area 1 with increase ofnew infestation NeemAzal-T/S was used again. In area 3 the interval between first andsecond treatments with nearly 3 weeks was too far and so the result was not

175

satisfactory at the end. In the areas 4 and 5 a second treatment was necessary too.Also the infestation of Brussels Sprout Fruit was dependent on the management of theenterprises and not on treatments of NeemAzal-T/S; in the future the application mustbe better organize in the late summer/autumn.

The effect of NeemAzal-T/S against caterpillars on this crop was satisfying only on 50 %of the area. The problem was mainly the treatment strategy. A preliminary small trial testin Saxonian Plant Protection department showed that with five treatments correspondthe current infestation good results can be obtained. A regular control of the existenceof pest is necessary. That was not in every case achievable during practice testing.Could be only realized three to four treatments also to far interval. With late infestationin the Brussels Sprout with catterpillars showed up the same problem as in case withCabbage aphid and must be better planed by the enterprises.

The results of this praxis trials were evaluated of this four ecological enterprises aspositive, because damage was acceptable and infestation of crop and yield losses weresubstantially smaller in 1997 than 1996.

NeemAzal-T/S should absolutely be available for the ecological vegetable growing. Theeffect against Cabbage Moth is very good and a high effect against caterpillars can beattained by better application management of the enterprises. Besides it must beconsidered that other products in ecological agriculture available against catterpillars(Bacillus thuringiensis, Trichogramma evanescens), needed more intensive monitoringfor evaluation of the infestation of plant with pests and more exact termination oftreatments. As an advantage of NeemAzal-T/S must be rated additionally the effectsagainst several sucking and biting pest.

177

REDUCTION IN THE SURVIVAL AND REPRODUCTION OFPOLYPHAGOTARSONEMUS LATUS (BANKS) (ACARI:TARSONEMIDAE) ON CHILLI PEPPER TREATED WITH NEEMAZAL-T/S

MADELAINE VENZON1, MARIA CONSOLAÇÃO ROSADO1, VANESSA DA SILVEIRA DUARTE1,AMÉRICO IORIO CIOCIOLA JR2

1) AGRICULTURE AND LIVESTOCK RESEARCH ENTERPRISE OF MINAS GERAIS (EPAMIG), VILA GIANETTI46, 36570 000 VIÇOSA, MINAS GERAIS, BRAZIL

2) AGRICULTURE AND LIVESTOOK RESEARCH ENTERPRISE OF MINAS GERAIS (EPAMIG), AFONSO RATO1301, 38001 970 UBERABA, MINAS GERAIS, BRAZIL

The broad mite Polyphagotarsonemus latus is one of the most serious pests attackingchilli pepper (Capsicum frutescens) in Brazil. In conventional production systems, itscontrol has been done exclusively with chemical acaricides. However, in organicsystems these products are not allowed and the use of botanical extracts may representan alternative for pest control. The extract of the neem tree, Azadirachta indica, hasgreat potential for use in integrated and biological pest management programs. To testthe effect of neem seed kernel extract on the survival and reproduction of P. latus alaboratory experiment was carried out. Chilli pepper seedlings (h = 6 cm) were sprayedwith aqueous solution of neem seed extract (NeemAzal-T/S, 1% of azadirachtin) atconcentrations of 0.5%, 1%, 1.5% and 2.0%, with an aqueous solution of abamectin1.8% (Vertimec 18 CE) and with water. After plants had dried, 10 adult females of P.latus were transferred to each plant. The final population of mites on each plant (adults,eggs and juveniles) was evaluated after six days and the instantaneous rate of increase(ri) for each treatment was calculated. All mites on plants treated with abamectin diedbefore the end of the experiment without ovipositing. Thus, it was not possible tocalculate the ri for this treatment. The instantaneous rate of increase (ri) of P. latusdecreased linearly with increasing neem concentration (F = 25.2; P < 0.00004; r2 =0.51; y = - 0.226x + 0.287). The decline of P. latus population, headed it towardextinction, was obtained when mites were exposed to plants treated with neem seedextract at concentration higher than 1.3% (ri = - 0,007).

Keywords: broad mite, Capsicum frutescens, Azadiracta indica, population growth


179

THE POTENTIAL OF A NEEM SEED EXTRACT (NEEMAZAL-T/S) FORTHE CONTROL OF COFFEE LEAF PESTS

MADELAINE VENZON1, MARIA CONSOLAÇÃO ROSADO1, MARCOS ANTÔNIO MATIELLOFADINI1, AMÉRICO IORIO CIOCIOLA JR2, ANGELO PALLINI3


2) AGRICULTURE AND LIVESTOOK RESEARCH ENTERPRISE OF MINAS GERAIS (EPAMIG), AFONSO RATO1301, 38001 970 UBERABA, MINAS GERAIS, BRAZIL;

3) FEDERAL UNIVERSITY OF VIÇOSA (UFV), 36570 000 VIÇOSA, MINAS GERAIS, BRAZIL

The effects of a neem seed extract (NeemAzal-T/S) on two pests attacking coffeeleaves, the coffee leaf miner (Leucoptera coffeella) and the coffee red mite(Oligonychus ilicis), and on the predatory mite Iphiseiodes zuluagai were evaluated.Greenhouse cage experiments were carried out to evaluate the repellence of neem onthe oviposition of L. coffeella. Females of L. coffeella oviposited on coffee seedlingstreated with 0.1 g/l of azadirachtin, but mine development stopped when leaves witheggs or larvae of L. coffeella were treated with 0.025 to 0.1 g/l of azadirachtin. No adultsemerged from neem treated leaves. Survival of O. ilicis was shorter on leaves treatedwith neem, but longer than on ethion treated leaves. The instantaneous rate of increase(ri) of O. ilicis decreased linearly with increasing neem concentration and negativevalues of ri were obtained with concentration above 0.065 g/l of azadirachtin. Survival ofthe predatory mite I. zuluagai was not affected by contacting neem-treated leaf oringesting prey-fed neem. The potential of NeemAzal-T/S to control two important pestsoccurring in coffee plantations was demonstrated in this study, especially as it was notlethal to an important predator commonly found in coffee agroecosystems in Brazil.

Keywords: Coffee arabica, Leucoptera coffeella, Oligonychus ilicis, Iphiseiodeszuluagai, Azadiracta indica


181

LETHAL AND SUBLETHAL EFFECTS OF NEEMAZAL ON MYZUSPERSICAE (HEMIPTERA: APHIDIDAE) AND ON ITS PREDATORERIOPIS CONNEXA (COLEOPTERA: COCCINELLIDAE)

MADELAINE VENZON1, MARIA CONSOLAÇÃO ROSADO1, AMANDA FIALHO1, DENISEELIANE EUZÉBIO1, AMÉRICO IORIO CIOCIOLA JR2


2) AGRICULTURE AND LIVESTOOK RESEARCH ENTERPRISE OF MINAS GERAIS (EPAMIG), AFONSO RATO1301, 38001 970 UBERABA, MINAS GERAIS, BRAZIL

The effects of neem seed kernel extract on an important chilli pepper pest, the greenpeach aphid Myzus persicae and on the predator Eriopis connexa were assessed inlaboratory. Chilli pepper seedlings were sprayed with an aqueous solution of neem seedextract (NeemAzal-T/S, 1% of azadiractin) at concentrations of 0.25%, 0.5 %, 0.75%and 1.0%, with acephate (0.75 g/l) (Orthene 750 BR) and with water. After plants haddried, 5 females of M. persicae were transferred to treated plants and after six daysplants were checked for aphids. Aphid population on plants treated with neem and withacephate were significantly lower than on water treated plants. There was no significantdifference among neem concentrations. The effect of neem on the predator E. connexawas evaluated by placing adults and larvae on pepper plants infested with aphids afterthey have been treated with neem (NeemAzal-T/S) at 0.5% (adults), at 0.25 and 0.5%(larvae). Insecticides (metamidophos, 0.06 g/L, and acephate, 0.75 g/l) and water wereused as control. There was no significant difference in the E. connexa adult mortalityand reproduction when plants were treated with neem and with water, but they weresignificantly different from plants treated with metamidophos (100 % mortality). Mortalityof E. connexa larvae five days after treatment did not differ between neem treatedplants (20% of mortality at 0.25 and 22% of mortality at 0.5%) and water treated plants(0 % of mortality); but these treatments differ from insecticide treated plants (100 %mortality). Even though, only 10% of neem exposed larvae at the two concentrationsreached pupation and there was no adult formation; 100% of larvae on water treatedplant succeed in pupate and 86% became adults. Thus, despite of potential ofNeemAzal-T/S in reducing aphid population and the lack of lethal effects in E. connexa,the product can cause sublethal effects on predator larvae.

Keywords: Green peach aphis, predator Capsicum frutescens, Azadiracta indica


183

EFFECT OF AZADIRACHTIN APPLIED SYSTEMICALLY THROUGHROOTS OF PLANTS ON THE GREENHOUSE WHITEFLY,TRIALEURODES VAPORARIORUM (WESTWOOD)

ROMAN PAVELA

RESEARCH INSTITUTE OF CROP PRODUCTION, DRNOVSKÁ 507, 161 06 PRAHA 6 – RUZYNE

EMAIL: [email protected]

Abstract

The effect of low concentrations of azadirachtin applied systemically through roottissues of tomato plants (Lycopersicon lycopersicum L.) cv. Vilma on the developmentof population of the greenhouse whitefly (Trialeurodes vaporariorum Westwood (Hem.:Aleyrodidae) was studied. The concentrations of azadirachtin A tested were: 80, 20, 5and 0.5 ppm. The reduction of adults, nymphs and eggs of greenhouse whiteflyincreased significantly with increasing concentrations.

Keywords: Azadirachtin; systemic action; mortality; greenhouse whitefly; Trialeurodesvaporariorum; botanical insecticides; NeemAzal-U.

1. Introduction

Hydroponic and soilless cultivation systems of plant production are used worldwide togrow flower, foliage, bedding and vegetable crops. Plants are grown using nutrientsolutions with or without solid substrates for root growth. Hydroponic systems withoutsubstrates include the nutrient film technique (NFT), the deep flow technique (DFT),trough culture and slow sand filtration (Schwarz, 1995). The nutrient solution can eitherbe re-circulated in a closed system or drained after one use in an open system. Thesesystems have become popular over the last 20 years all over the world for the growth ofhigh-value crops in glasshouses (Savvas et al, 2002; Mine et al. 2003).

Tomatoes can be cultivated quite well in a hydroponic system as can vegetables orornamental species (Chatzivassiliou et al., 2000). The greenhouse whitefly,Trialeurodes vaporariorum (Westwood), is an important pest of horticultural crops ingreenhouses and an increasing pest problem on outdoor crops such as strawberry,raspberry, pepper, cucumber, tomato, lettuce, citrus, bean and cotton. It causeseconomic damage to crops by ingestion of plant sap, contamination of crop productswith honeydew which forms a substrate for the development of sooty molds andtransmission of plant virus diseases (Omer et al., 1992; Johnson et al., 1992; Liu et al.,1993).Biological Control of Plant, Medical and Veterinary PestsR. Strang & H. Kleeberg (eds.)Copyright 2009 by Trifolio-M GmbH

184

In the 1960's, the greenhouse whitefly was successfully controlled with broad-spectrumpesticides; however, in the early 1970's growers were confronted with seriousresistance problems (2). These problems were solved mainly by the introduction of theparasitic wasp Encarsia formosa Gahan. However, biocontrol with Encarsia was notalways successful, especially at lower (<16°C) and higher (>30 °C) temperatures, thusrequiring additional control measures; other biocontrol agents and botanicalsinsecticides.

One promising natural insecticide of interest is an extract from seeds of the neem tree(Azadirachta indica JUSS.) as it is effective on many pests and is biodegradable(Isman, 1999). The primary active ingredient of most neem-based pesticides isazadirachtin, a limonoid compound with an excellent insecticidal activity against manyphytophagous pests. Azadirachtin affects insect growth, feeding, and reproduction(Mordue et al., 1998, Walter, 1999). Neem extracts have minimal toxicity to non-targetorganisms such as parasitoids, predators, and pollinators (Lowery and Isman, 1994,Naumann and Isman, 1996) and degrade rapidly in the environment (Isman, 1999).

Nevertheless, the rapid degradability is a disadvantage for foliar applications. For thisreason optimal timing of applications with higher concentrations of azadirachtin arenecessary (Pavela and Holý, 2003). Therefore, new types of applications, such as treeinjection (Helson at al., 2001, Naumann et al., 1994, Wannker et al., 1997) and soilapplications (Basedow et al., 2002, Opender et al, 1995, Sundaram, 1996) resulting inuptake by the roots system are desirable. Such is the tree injection or uptake of theneem-insecticide by the root system.

The roots of plants take up dissolved azadirachtin and build it into their tissue as anatural metabolite (Sundaram, 1996, Pavela et al., 2004).

The effect of azadirachtin, taken up by the root system, on aphids is not clear. Currentresearch shows that azadirachtin applied on the plant surface affects aphids (Dimetryand Schmidt, 1992, Lowery and Isman, 1994, Nisbet et al., 1994, Pavela et al. 2002). Inour study we investigated the effects of various concentrations of azadirachtin appliedhydroponically through the roots of plants on the mortality, and fecundity of greenhousewhitefly.

185

2. Material and methods

2.1. Plant material

The seeds of a tomato (Lycopersicon lycopersicum L.) cv. Vilma were obtained fromseed market.

2.2. Insect

The greenhouse whitefly Trialeurodes vaporariorum (West.) (Hemiptera: Aleyrodidae)adults were reared from the greenhouse colony (from Research Institute of CropProduction). The colony were maintained in an greenhouse insectary at 22 ± 3°C, 60 –90 % RH and 16:8 L:D.

2.3. Chemicals

NeemAzal-U: The insecticidal test formulation produced of Trifolio-M GmbH, Germany,contains 17 % azadirachtin A as its main active ingredient.

2.4. Hydroponic cultivation

Healthy tomato seeds were surface sterilized in 0.5% sodium hypochloride solution for2 minutes, rinsed with sterile distilled water and then germinated on sterile moist gauzeat 30°C in dark for 72 h. The germinated seedlings were sown in a seedling tray inwhich the holes contained rockwool previously sterilized at 121°C for 20 min. Theseedling tray was placed in a nutrient solution sterilized by filtration. After 20 dayscultivation in a greenhouse, seedlings as high as 10 cm with four to five leaves weretransplanted to a bigger seedling tray in a hydroponic cultivation system.

Each hydroponic flowerpot used in this experiment had separate circle with nutrientsolution. The nutrient solution was based upon the recipe of Gunes et al (1998). Thevolume of store tanks was ten liters. Plants were fixed in rockwool, kept with nutrientsolution or nutrient solution with adding of NeemAzal-U (for experiment). This solutionwas changed every 10 – 15 days (it depend on pH and amount of salts). The amount ofsalts and pH was measured once a week. Plants were placed in greenhouse.

Precultivating of experimental plants and also whole experiment ran under theseconditions:

long day conditions,

temperature was kept at 25 - 28°C in the daytime and 13–16°C at the night,

relative humidity was kept at 50-85%.

186

2.5. Experiment

The biological tests had started at the beginning of plant flowering. Afterward, the plantswere completely isolated and infected with 25 adults of T. vaporariorum , though we didnot specify exact age of them. Those adults have been left for ten days in a plant cage,before starting the experiment.

Treatments

In every 10 litre tank was prepared new nutrient solution mixed with 5, 1.5, 0.3 and 0.03g of NeemAzal-U, which means that each nutrient solution contained 85, 20,5 and 0.5ppm of active ingredient Azadirachtin. Those nutrient solutions (containing Azadirachtin)were filled in the store tanks of hydroponic flowerpots. In experiment we used onlynutrient solutions as control.

The plants we leaved in nutrient solutions:

whole time of experiment (1 application )

whole procedure was repeated after 10 days from first application (2 applications ).

Evaluation was made 20 days after first application. Then, the number of adults,nymphs and eggs were found out. Plants were cut and in the laboratory all adults werekilled with ethyl acetate. By using the microscope all adults were calculated per eachplant and which were 10 leaves taken and at each of them was determined the numberof eggs and nymphs.

Data analyses and statistics

Data for development, longevity and fertility were subject to analysis of variance(One-way ANOVA) and treatment differences were determined by Tukey´s test.Differences among means were considered significant at a probability level of fivepercent (P≤0.05).

3. Results and discussion

The systemic application of low concentrations of azadirachtin to the tomato plantscaused a significant increase in a reduction of population of greenhouse whitefly.

The figure 1 represents the azadirachtin effect on the decrease of the number of adultsT. vaporariorum if the plants were left in nutrient solutions whole time of experiment (1treatment) and whole procedure was repeated after 10 days from first application ( 2applications ).

Significantly lower number of adults (Fig.1) was found on plants treated with the dosesof 80 and 20 ppm vs. control (P<0.05), if the plants were left in nutrient solutions whole

187

time of experiment. The number of adults was reduced (Fig. 4) by 96 and 94 % vs.control of concentrations of 80 and 20 ppm, respectively.

But the number of L2 and L3-5 mines was significantly lower on the higher two dosesthan on the control or dose 0.08 g/cm The number of adults was significantly (P≤0.05)reduced compared to control only at the concentrations 80 and 20 ppm.

The azadirachtin effect on decreasing number of adults is shown in Fig.1. If thisapplication was made only once at the beginning of the experiment, than the number ofadults was significantly smaller in comparison with the control only with concentration80 and 20 ppm. The adults were reduced more than 90 % in comparison with thecontrol (Fig.1). If there was done a change of the nutrient solution (2 applications), thanthe number of adults was significantly lower in all applied concentrations. The adultswere reduced from 70 to 100 % in comparison with the control (Fig.2).

Though in the experiment with 1 application were not the numbers of adults significantlylower in concentration 5 and 0.5 ppm (Fig. 1), nevertheless were found the numbers ofnymphs (Fig. 2) and eggs (Fig. 3) significantly lower in all tested concentrations. Thenymphs were reduced from 62 to 93 % in comparison with the control (Fig. 4).

If the azadirachtin procedure was repeated after 10 days from first application (2applications), than was found significant effect to the decreasing numbers of nymphs(Fig. 2) and eggs (Fig. 3). The reduction of nymph and egg numbers (Fig. 5) was similarto only 1 azadirachtin application.

Fig. 1: Average number (± S.E.) of adults of Trialeurodes vaporariorum after treatmend of NeemAzal-Uin water hydroponics. (Tukey HSD test, P<0.05)

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Fig. 2: Average number (± S.E.) of nymphs of Trialeurodes vaporariorum after treatmend ofNeemAzal-U in water hydroponics. (Tukey HSD test, P<0.05)

Fig. 3: Average number (± S.E.) of eggs of Trialeurodes vaporariorum after treatmend of NeemAzal-Uin water hydroponics. (Tukey HSD test, P<0.05)

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Fig.4. Effect of azadirachtin on reduction of population T. trialeurodes. Average percentage reductionof eggs, nymphs and/or adults (1 treatment) vs. control

Fig.5. Effect of azadirachtin on reduction of population T. trialeurodes. Average percentage reductionof eggs, nymphs and/or adults (2 treatments) vs. control

A female whitefly may lay up to 300 eggs during her lifetime, and live as long as 42days at 18°C and 8 days at 27°C. After hatching (8th -12th day), the eggs undergo 4stages or instars before becoming adults. The first instar or larval stage hatches in 5–10days. They are flat and scale-like, and crawl around for a short while before becomingimmobile. The second and third instars or larval stages are followed by the fourth instaror pupal stage, from which the adult emerges. On average, the whitefly completes itslife cycle in 35 days at 18 °C and 18 days at 30 °C (Campos et al., 2003).

0102030405060708090

100

%

80 ppm 20 ppm 5 ppm 0.5 ppm

eggsnymphsadults

0102030405060708090

100

%

80 ppm 20 ppm 5 ppm 0.5 ppm

eggsnymphsadults

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In our experiment was made the first azadirachtin application into the water ten daysafter the introduction of adults to the experimental plants. It is probably, that the activesubstance had implicated firstly the first two instars, which are the sensitivest. Byreason that the adults were not removed they could continue with laying eggs. Evolutionof next larvae, which were hatched from the later eggs could not be implicate with thefull dose of azadirachtin and a part of them could evolve to the adults (especially inconcentrations 5 and 0.5 ppm).

If we used this low concentrations of azadirachtin again (5 and 0.5 ppm), the mortality oflarvae has increased. This mortality had shown itself with a low number of adults.Generally it came to significant reduction of the population T. vaporariorum.

Basedow et al. (2002) has shown „systemic" effects of AzadirachtinA after soiltreatment. When pots with a peat-sand mixture, in which seedlings of Vicia faba weregrowing, were supplied with 10 ml of Neem Kernel Water Extract (containing 14 mgAzaA), a transport of AzaA to the broad bean leaves was observed with a maximumafter five days, after which the AzaA-content of the leaves slowly declined. On otherpots, populations of Aphis fabae increased until the fourth day in treated and untreated,and then started to decline in treated pots, while populations in untreated pots grew fast.This experimental observation shows that plants can take up AzaA by the roots andtransport it, so that it can affect sucking insects.

Pavela et al. (2004) referred in bioassays with azadirachtin applied systemically throughthe roots of rape plants showed significant differences in mortality and fecundity of B.brassicae feeding on the plants. It is more probable, that by the repeated using ofnutrient solutions with the input of azadirachtin could decrease the fecundity of adultsthe next generation so much that the population of T. vaporariorium could be under theeconomical treshold.

If the active ingredients are taken up by the root system and then transported by thevascular bundles throughout the plant, low concentrations of azadirachtin or botanicalinsecticides containing azadirachtin might be more effective than foliar applications forcontrol of pest species. In practice this could have a great influence on the cost and thenumber of applications of pesticide necessary for control of crop pests.

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4. References:

BASEDOW, T., OSSIEWATSCH, H.R., BERNAL VEGA, J.A., KOLLMANN, S., EL-SHAFIE,H.A.F., NICOL, C.M.Y. (2002) Control of aphids and whiteflies (Homoptera:Aphididae and Aleyrodidae) with different Neem preparations in laboratory,greenhouse and field: effects and limitations. Zeitschrift fur Pflanzenkrankheitenund Pflanzenschutz 109 (6): 612-623.

CAMPOS, O.R., CROCOMO, W.B., LABINAS, A.M. (2003) Comparative of the WhiteflyTrialeurodes vaporariorum (West.) (Hemiptera: Aleyrodidae) on Soybean andBean Cultivars. Neotr. Entomol. 32 (1): 133-138.

CHATZIVASSILIOU E.K, LIVIERATOS I., JENSER G., KATIS N.I. (2000) Ornamental plantsand thrips populations associated with tomato spotted wilt virus in Greece.Phytoparasitica 28: 257–264.

DE CLERCQ, R. (1975) Recherches sur les manifestations de la mouche blanche(Trialeurodes vaporariorum Westwood) dans des serres de la region de Malinesen 1974 et sur les moyens de lutte contre cet insecte. Rev. Agric. 6:1511-1517.

DIMETRY, N., SCHMIDT, G.H. (1992) Efficacy of NeemAzal-TS and Margosan-O againstthe bean aphid Aphis fabae Scop. Anz. Schaedlingskd PflanzenschutzUmweltschutz 65: 75-79.

GUNES, A., ALPASLAN M., INAL, A. (1998) Critical nutrient concentrations andantagonistic and synergistic relationships among the nutrients of NFT-grownyoung tomato plants. J. Plant Nutr. 21: 2035–2047.

HELSON, B.V., LYONS, B.D., WANKER, K.W., SCARR, T.A. (2001) Control of coniferdefoliators with neem-based systemic bioinsecticides using a novel injectiondevice. Can. Ent. 133: 729-744.

ISMAN, M.B. (1999) Neem and related natural products. In: F.R. Hall and J.J. Menn(eds.), Biopesticides: Use and Delivery. Humana, Totowa, NJ.: 139-153.

JOHNSON, M.W., CAPRIO, L.C., COUGHLIN, J.A., ROSENHEIM, J.A. AND WELTER, S.C.(1992). Effect of Trialeurodes vaporiariorum (Homoptera: Aleyrodidae) on yieldof French market tomatoes. J. Econ. Entomol. 85 (6): 2370–2376.

KOUL, O. (1998) Effect of neem extract and azadirachtin on fertility and fecundity ofcabbage aphid, Brevicoryne brassicae (L.) Pest. Res. J. 10: 258-261.

LIU, T.X., OETTING, R.D., BUNTIN, G.D. (1993). Population dynamics and distribution ofTrialeurodes vaporariorum and Bermisia tabaci (Homoptera: Aleyrodidae) onpoinsettia following applications of three chemical insecticides. J. Entomol. Sci.28 (1): 126–135

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LOWERY, D.T., ISMAN, M.B. (1995) Toxicity of neem to natural enemies of aphids.Phytoparasitica 23: 297-306.

LOWERY, D.T., ISMAN, M.B. (1996) Inhibition of aphid (Homoptera:Aphididae)reproduction by neem seed oil and azadirachtin. J. Econ. Ent. 89: 602-607.

LOWERY, D.T., ISMAN, M.B. (1994) Insect growth regulating effects of neem extract andazadirachtin on aphids. Ent. Exp. Appl. 72: 77-84.

Y. MINE, R. SAKIYAMA, Y. YAMAKI, M. SUEMATSU AND H. SAKA (2003) Influence ofripening state of filters on microbe removal efficiency of slow sand filtration usedto disinfect a closed soilless culture system. J. Jpn. Soc. Hortic. Sci. 72:190–196.

MORDUE (LUNTZ) A.J., SIMMONDS, M.S.J., LEY, S.V., BLANEY, W.M., MORDUE, W.,NASIRUDDIN, M, NISBET A.J. (1998) Action of azadirachtin, a plant allelochemical,against insects. Pest. Sci. 54: 277-284.

NAUMANN, K., ISMAN, M.B. (1996) Toxicity of neem (Azadirachta indica A. JUSS) seedextracts to larval honeybees and estimation of dangers from field application.Am. Bee J. 136: 518-520.

NAUMANN, K., RANKIN, L.J., ISMAN, M.B., (1994) Systemic action of neem seed extracton mountain pine beetle (Coleoptera : Scolytidae) in lodgepole pine. J. Econ.Ent. 87:1580-1585.

NISBET, A.J., WOODFORT, J.A.T., STRANG, R.H. (1994) The effects ofazadirachtin-treated diets on the feeding behaviour and fecundity of thepeach-potato aphid, Myzus persicae. Ent. Exp. Appl. 71: 65-72.

SCHWARZ, D. (1995). Soilless Culture Management. Springer, Berlin, Heidelberg, pp.33–91.

SAVVAS, D., MANOS, G., KOTSIRAS A., SOUVALIOTIS S. (2002), Effects of silicon andnutrient-induced salinity on yield, flower quality and nutrient uptake of gerberagrown in a closed hydroponic system. J. Appl. Bot. 76: 153–158.

OMER, A.D., LEIGH, T.F., GRANETT, J. (1992). Insecticide resistance in field populationsof greenhouse whitefly (Homoptera: Aleyrodidae) in the San Joaquin valley(California) cotton system. J. Econ. Entomol. 85 (1): 21–27.

OPENDER, K., SHANKAR, J.S., KOUL, O. (1995) Systemic uptake of azadirachtin intoRicinus communis and its effects on Spodoptera litura larvae. Indian J. Exp. Biol.33: 865-867.

PAVELA, R., BARNET, M., BELANGER, A., BROSSEAU, M. (2002) Effectiveness of newplant insecticides obtained from neem-tree (Azadirachta indica juss.) againstcabbage aphid (Brevicoryne brassicae). Veg. Crops Res. Bull. 56: 95-102.

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PAVELA, R. AND HOLY, K. (2003) Effects of azadirachtin on larvae of Lymantria dispar,Spodoptera littoralis and Mamestra brassicae. Hortic. Veg. Growing 22: 434-441.

PAVELA, R., BARNET, M., KOCOUREK, F. (2004) Effect of Azadirachtin AppliedSystemically through Roots of Plants on the Mortality, Development andFecundity of the Cabbage Aphid (Brevicoryne brassicae). Phytoparasitica32(3):286-294

STARK, J.D., RANGUS, T.M. (1994) Lethal and sublethal effects of the Neem insecticideformulation, ‘Margosan-O’, on the pea aphid. Pest. Sci. 41: 155-160.

SUNDARAM, K.M.S., (1996) Root uptake, translocation, accumulation and dissipation ofthe botanical insecticide, azadirachtin, in young spruce trees. J. Envir. Sci.Health 31: 1289-1306.

WALTER, J.F. (1999) Commercial experience with neem products. In: F.R. Hall and J.J.Menn (eds.), Biopesticides: use and deliver. Humana, Totowa, NJ.:155-170.

WANNER, K.W., HELSON, B.V., KOSTYK, B.C. (1997) Foliar and systemic applications ofneem seed extract for control of spruce budworm, Choristoneura fumifera(CLEM) (Lepidoptera: Tortricidae), infesting black and white spruce seedorchards. Can. Ent. 129: 645-655.

ZHANG, S., SONG, W., HUANG, Z. (2002) Study on environmental control technology ofsome unlimited growth tomato in greenhouse by deep flow technique. J. ChinaAgric. Univ. 7: 34–38.

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INSECTICIDAL ACTIVITY OF CERTAIN MEDICINAL PLANTS

ROMAN PAVELA

RESEARCH INSTITUTE OF CROP PRODUCTION, DRNOVSKA 507, 161 06 PRAHA 6, RUZYNE, CZECHREPUBLIC

Abstract

The methanol extracts of eight species of medicinal plants were tested for insecticidalactivity in third instar larvae of Egyptian cottonworm (Spodoptera littoralis). All extractsshowed a certain degree of larval toxicity. The extracts of Ocimum basilicum, Origanummajorana and Salvia officinalis appeared to be highly toxic.The extracts significantlyaffected the growth indexes (RGR, ECI, ECD).

Keywords: Insecticidal activity; Spodoptera littoralis; Origanum majorana; Ocimumbasilicum; Cnicus benedictus; Marrubium vulgare; Hyssopus officinalis; Salviasplendens; S. officinalis; Melissa officinalis

Table 1 Plant material tested for toxicity against S. littoralis larvae

Plants. Reported in Table 1.

Uses in traditional medicine. The plants have a wide range of application in medicine,both internally and externally, according to the Czech [1] and EuropeanPharmacopoeias [2, 3].

Previously isolated classes of constituents. The chemicals of these plants are welldocumented and include flavonoids, terpenoids as well as monoterpenes andsesquiterpenes [2-4].

Plants Plant stage Plant material Collection dateMass yield

(g)Origanum majorana L.

(Lamiaceae) Vegetative Aerial part 20 July 2002 42.3Ocimum basilicum L.

(Lamiaceae) Flowering Aerial part 20 July 2002 65.8Cnicus benedictus L.

(Asteraceae) Pre-flowering Aerial part 29 July 2002 35.9Marrubium vulgare L.

(Lamiaceae) Flowering Leaves 20 July 2002 68.3Hyssopus officinalis L.

(Lamiaceae) Flowering Aerial part 24 July 2002 50.5Salvia splendens Sellow

(Lamiaceae) FloweringLeaves and

flower 15 August 2002 35.3S. officinalis L.(Lamiaceae) Flowering Aerial part 26 July 2002 48.2

Melissa officinalisL.(Lamiaceae) Flowering Aerial part 27 August 2002 69.3


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Tested material. Methanol extracts ( yields from 250 g of plant material in Table 1).

Studied activity. Larval mortality. Larvae were reared on an artificial diet containingvarious amounts of the tested compounds in MeOH: water (2 ml: 750ml to obtain 1000g diet). Five concentrations were tested: 0 (control), 0.5, 1, 5 and 10 % (w/v). The dietwas changed every 2 days. Surviving larvae received the diet until the pupal stage.Mortality was recorded for both larvae and pupae. Each experiment was performed on25 larvae with 5 replications. Growth index. Larvae were reared for 7 days on anartificial diet containing 0.5 % (w/v) of the test compound prepared as above.

Each treatment was performed with 5 larvae with 10 replications. The surviving larvaewere fed the diet until pupation of the surviving insects; the remaining diet and theproduced excrements were weighed, and the rate of mortality was determined.

Criteria for evaluation of the insecticidal activity. Larval mortality and LC50 ; relativegrowth rate (RGR) [5]; efficiency of conversion of ingested food (ECI) [5]; efficiency ofconversion of digested food (ECD) [5].

The data were elaborated by analysis of variance (ANOVA). Tukey test at P ≤ 0.05.Probit analysis was used to determine LC50, and the corresponding 95% confidenceintervals.

Used insect. Experiments were made on third instar larvae (10-12 mg) of S. littoralis(Boisduval) (Lepidoptera: Noctuidae), maintained on artificial diet for 30 generations.The larvae were kept under 16:8 light:dark photoperiodic regime at constanttemperature of 25°C and 70% r.h.

Results. Reported in Tables 2 and 3.

Table 2 Index of relative growth rate (RGR), efficiency of conversion of ingested food (ECI) andefficiency of conversion of digested food (ECD) by S. littoralis larvae exposed to 0.5 % (w/v)plant extract in an artificial diet

*Values followed by a common letter don't differ significantly from P<0.05

Plants RGR (S.E.)* ECI (S.E.)* ECD (S.E.)*O. majorana 3.28 (0.92)bc 6.32 (1.57)a 0.19 (0.05)cO. basilicum 3.75 (0.67) bc 4.66 (0.58)a 0.26 (0.04)bc

C. benedictus 4.93 (0.94)bc 4.14 (0.76)a 0.31 (0.05)bM. vulgare 3.26 (0.35)c 5.99 (1.19)a 0.20 (0.04)c

H. officinalis 5.47 (0.33)b 4.88 (0.97)a 0.25 (0.05)bcS. splendens 5.23 (0.99)bc 4.60 (1.33)a 0.30 (0.08)bS. officinalis 3.76 (1.52)bc 6.32 (1.33)a 0.22 (0.08)bcM. officinalis 4.24 (0.98)bc 4.96 (1.38)a 0.26 (0.07)bc

Control 17.45 (3.28)a 2.27 (0.46)b 0.54 (0.03)a

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Table 3 Mortality of S. littoralis larvae and pupae and LC 50 index of larvae exposed to plant extracts inan artificial diet

* 95% ci – denotes confidence interval

Conclusion

The increasing number of investigations on plant-insect chemical interactions [6] in thelast few decades unveiled the potential of utilizing secondary plant metabolites, orallelochemicals, as pest control agents. This interest in botanical insecticides resultedfrom the need to provide an alternative in IPM programs to the synthetic insecticides,whose adverse effects on agro ecological systems are well known.

PlantsConcentration

s % (w/v)

Larvalmortality

(%)

Pupalmortality

(%)

Totalmortality

(%)LC 50

(95% ci)*O. majorana 10 77.8 10.6 88.4 5 75.5 11.8 87.3 0.36 1 62.2 5.5 67.7 (0.30-0.42) 0.5 51.1 3.3 54.4 O. basilicum 10 88.8 5.5 94.3 5 82.2 9.7 91.9 0.17 1 75.6 10.3 85.9 (0.11-0.22) 0.5 75.6 0.0 75.6 C. benedictus 10 48.8 40.2 89 5 37.7 40.8 78.5 13.79

1 28.9 42.1 71(9.91-15.60)

0.5 21.4 30.7 52.1 M. vulgare 10 64.4 16.3 80.7 5 42.2 18.8 61 5.38 1 28.8 31.1 59.9 (4.21-5.98) 0.5 20 5.6 25.6 H. officinalis 10 77.8 12.6 90.4 5 53.3 20 73.3 1.78 1 35.5 10.1 45.6 (1.66-1.82) 0.5 28.9 5.5 34.4 S. splendens 10 53.3 30.3 83.6 5 44.5 40.5 85 7.71 1 42.2 20.1 62.3 (7.0-8.29) 0.5 31.1 9.3 40.4 S. officinalis 10 97.7 2 99.7 5 71.1 9.9 81 0.47 1 64.5 15.3 79.8 (0.26-0.99) 0.5 57.8 12.6 70.4 M. officinalis 10 62.2 3.3 65.5 5 44.5 3.3 47.8 3.74 1 44.4 0.0 44.4 (2.12-5.62) 0.5 31.1 0.0 31.1 Control 0 0 0 0

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This study could also contribute to assess the possibility of using medicinal plants aspotential insecticides. All of the plant extracts were toxic to the larvae S. littoralis.Nevertheless, differences among individual extracts were found. Three of the plantextracts were highly toxic to the larvae: extract from O. basilicum, O. majorana, S.officinalis (LC50 0.17, 0.36, 0.47, respectively).

As could be expected, a high degree of relationships exists among the weight increase,the quantity of diet ingested, and the quantity of excrements produced during the wholeassay period. This shows the basically antifeedant character of the extracts tested [7].

Mortality occurred progressively throughout the entire assay. The results demonstrateda cumulative mortality [5], as the larvae that reached the pupal stage exhibited highermortality.

Acknowledgements

The study was supported by Czech Ministry of Agriculture, Project No MZE-M01-03-04.

References

Korbelář J, Endris Z. editors. Naše rostliny v lékařství. Praha: Avicenum,1981.

Blaschek EW, Hänsel R, Keller K, Reichling J, Rimpler H, Schneider G. editors. HagersHandbuch der Pharmazeutischen Praxis 3. Berlin, Heidelberg and New York:Springer-Verlag, 1998.

Hänsel R, Keller K, Rimpler H, Schneider G. editors. Hagers Handbuch derPharmazeutischen Praxis 6. Berlin, Heidelberg and New York : Springer-Verlag,1994.

Bruneton J. Pharmacognosy Phytochemistry Medicinal Plants. Paris: Lavoiser, 1999.

Miller JR, Miller TA. Insect-Plant Interactions. New York: Springer-Verlag, 1988.

Prakash A, Rao J. Botanical Pesticides in Agriculture. Boca Raton, Florida: CRC Press,2000.

Schmutterer H. The Neem Tree. Weinheim, Germany: VCH, 1995.

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EXPERIENCE OF NEEMAZAL-T/S AGAINST CABBAGE PESTS INBELARUS IN 2004

PRISHCHEPA, I.A, KOLYADKO, N.N., SHINKORENKO, E.G.

INSTITUTE OF PLANT PROTECTION, NATIONAL ACADEMY OF SCIENCES OF BELARUS, EMAIL:[email protected]

Introduction

The ecological situation developed in Belarus on the foreground puts forward thenecessity of search and development of new effective preparations for vegetable cropprotection adequate to modern requirements of ecological safety.

Recently the attention of scientists and practical men is directed to studying theopportunity of accessible natural raw material use containing in its structure substancespossessing the insecticide properties and substantiation of the mechanism of theiraction on noxious pest species. Especially the actual intention is the creation on theirbasis the ecologically safe preparations allowing to influence actively the pestpopulations at the cost of their number and harmfulness decrease and also promotingthe preservation of the environment and getting of qualitative ecological non-pollutingproduction.

In a complex of harmful entomofauna formed in cabbage crops during vegetation thedominant species are leaf-biting phytophages from Lepidoptera, like cabbage cutworm(Barathra brassicae L), cabbage moth (Plutella maculipennis Curt.), cabbage and turnipwhite butterfly ( Pieris brassicae L., P.rapae L.); from sucking insects: cabbage aphid(Brevicoryne brassicae L.). To protect the crop against pests the “Catalogue…….”recommends the use of mainly chemical synthetic preparations for repeated insecticidetreatments during vegetation. In this connection studying of a new insecticideNeemAzal-T/S (Trifolio-M GmbH, Co., Germany) possessing the selective action ondifferent stages of phytophage development on white head cabbage against a complexof pest species (whitefly, looper, moth , aphid) ) is rather actual.

Conditions and method of carrying out researches

The preparation testing was carried out in white head cabbage crops in the State–farm-Agrocompany “Rassvet” Minsk region.

Experimental plot area - 25 m2, experiment repetitions – 4 . The agronomical practicesof cultivation and taking care after crops correspond to the technologies used in theRepublic. Cabbage crops treatment by NeemAzal-T/S was done by scheme:Biological Control of Plant, Medical and Veterinary PestsR. Strang & H. Kleeberg (eds.)Copyright 2009 by Trifolio-M GmbH

200

Spraying of plants was done with the help of knapsack sprayer. Working solution rateuse – 500 l/ha. For carrying out the researches the methods stated in the books“Methods of the experimental business in vegetable and melon growing“ under Belika’sEd. (1992), “The forecast of occurrence and record of pests and agricultural cropdiseases number“ under Kosov’s V.V., Polyakova’s I.Ya. Ed. (1958) were used. Numberof pests were recorded before treatment and in 7 days after spraying.

The biological efficiency from the insecticide application was calculated based on pestnumber decrease in the experimental variants in comparison with the control.

Number of Cabbage aphids was determined by classes of plant colonization (I-colonyfrom 1-5 aphids, II –5-15, III- 15-30, IV - >30) with calculation of colonies number per 1plant.

The economic efficiency as estimated by total yield and head marketable quantity fromtotal plot area. During researches carrying out of monitoring on changes of pestpopulations and their entomophages, evaluation of general condition of cabbage plants,their growth and development was accomplished.

The obtained data were processed by Dospekhov B.N. method of dispersive analysis(1985).

Results of researches

2004 vegetative period was characterized by a long spring with a significant cold snapin the III ecade of April – 1 decade of May. Sharp downturn of temperature has delayedgrowth and development of plants for 2-3 weeks. The developed weather conditionsrendered essential influence on harmful entomofauna formation in white head cabbageplantings of medium and early varieties.

Among pests in cabbage crops the first to be registered were cabbage aphid alateindividuals, the larvae hatching of which took place on July,16. By the III decade of Julybeginning, the phytophage population density has made 73-95 larvae per 25 plants at30% colonization. In the III decade the hatching of leaf-biting pest caterpillars started.

No Experiment variant Preparationapplicationrate, l/ha

Pests Method and time oftreatment

Number oftreatments

1. NeemAzal-T/S 2,5 Cabbage aphid,cabbagecutworm,cabbage moth,white butterfly,turnip whitebutterfly

Spraying of plantsduring vegetation at 1-2instar caterpillars oraphid self-settlers at thebeginning of the firstlarvae hatching .Working solution rateuse – 500 l/ha

12. NeemAzal-T/S 1,5 1

3. Control (without treatment)

- - 1

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Their number in the experimental plot has made depending on species: turnip whitebutterfly –5-17 caterpillars, cabbage moth –6-12, cabbage cutworm – 10-17individuals/25 plants with 10-12% plant colonization.

The treatment of crops with the insecticides was done on July, 27. By this time theagrocoenosis of the experimental plot was simultaneously represented by all the listedleaf-biting and sucking pest species. Extremely favorable weather conditions at day oftreatment and the next days (warm without rainfall) promoted obtaining the reliable dataon the tested insecticides toxicity. It is necessary to note that in butterfly populationprevailed, mainly, the I-III instar caterpillars what has allowed to carry out thecomparative evaluation of the toxic NeemAzal-T/S action on different phytophagegroups.

Butterfly caterpillar inspections under laboratory conditions which were placed (20individuals in every repetition) on NeemAzal-T/S treated cabbage leaves showed thatcaterpillars turned out to be less mobile, their feeding activity has sharply decreased,the process of transition from one larval stage of development to the other one hasstopped and, as a result, 100 % kill took place.

It is determined that NeemAzal-T/S preparation at the rate of 2,5 l/ha is of highinsecticide activity in relation to butterfly caterpillars. Turnip white butterfly death on the7-th day after treatment has made 92,6%; cabbage moth –96,9%, cabbage cutworm –94,6% (Table 1).

Table 1. Evaluation of NeemAzal-T/S preparation efficiency against a complex of white head cabbagepests (State-farm- Agrocompany “Rassvet”, Minsk region, cv. Mara, 2004)

In the variant with NeemAzal-T/S preparation application at the rate of 2,5 l/ha cabbagehead damage by a pest complex has made 6,5, in the control -33,1%. Standardproduction output 93,5 and 66,9, accordingly (see table 2).

Conclusions

Under laboratory and field conditions it is proved that the preparation NeemAzal–T/Spossesses well defined insecticide properties. The evaluation of NeemAzal-T/Sbiological efficiency in cabbage showed that the most toxic to butterfly caterpillars (white

Experiment variant

Applicationrate of a

preparationL/ha

Yield,centner/ha

Yieldincrease,

centner/ha

Headdamageby pests,

%

Standardproduction

output

NeemAzal-T/S 2,5 391 73 6,5 93,5NeemAzal-T/S 1,5 343 25 10,1 89,7Control 318 0 33,1 66,9 SED05

202

butterfly, moth, cutworm) at early stages of their development (I-III instars) was theapplication at the rate of 2,5 l/ha. It´s efficiency depending on insect species and theirdensity fluctuated from 92,6 to 96,8%. Single treatment of cabbage plants with theinsecticide decreases aphid number for 72,7%.

NeemAzal-T/S preparation does not render the negative influence on cabbage plantgrowth and development and can be applied at any stage of the crop growing.

Table 2. Yield increase and production output after application of NeemAzal-T/S

The results of study allow to recommend a biological insecticide NeemAzal-T/S for theplant protection under climatic conditions of Belarus against a complex of biting andsucking pests on white head cabbage at the rate of 2,5 l/ha.

Experimentvariant

Applicationrate of a

preparationL/ha

Yield,centner/

ha

Yieldincrease,

centner/ha

Headdamageby pests,

%

Standardproduction

output

NeemAzal-T/S 2,5 391 73 6,5 93,5NeemAzal-T/S 1,5 343 25 10,1 89,7Control 318 0 33,1 66,9 SED05

203

IMPACT OF SOME BIOPESTICIDES ON THE FEEDING ACTIVITY OF THELARGE PINE WEEVIL (HYLOBIUS ABIETIS L.) (COLEOPTERA:CURCULIONIDAE)

IVAR SIBUL1 AND ANGELA PLOOMI2

1FACULTY OF FORESTRY, ESTONIAN AGRICULTURAL UNIVERSITY, KREUTZWALDI 5, 51014 TARTU,ESTONIA, E-MAIL: [email protected];

2INSTITUTE OF PLANT PROTECTION, ESTONIAN AGRICULTURAL UNIVERSITY, KREUTZWALDI 64, 51014TARTU, ESTONIA

Abstract

In the present study we compared the efficiency of different dilutions of somepreparations – NeemAzal Paint (2,5% azadirachtin), NeemAzal-T/S (1% azadirachtin;dilutions of 20%, 40%), NeemAzal Blank Formulation, NeemAzal Paint Blank,TRF-002-Paint (0,05% quassin, dilutions of 20%, 40%) and TRF-002-Paint Blank (0%quassin) (Trifolio-M GmbH, Germany) on the maturation feeding of the large pineweevil, in laboratory and field experiments.

In laboratory choice feeding tests comprised assaying the effects of biopesticides on H.abietis feeding activity during 24–96 h by confining male and female weevils in Petridishes with Scots pine twigs. Field trials were established in a fresh clear-cutting area inan intensively managed forest area. Both field and laboratory studies indicated that alltested preparations had a significant deterrent and antifeedant influence on the weevilfeeding. In laboratory choice feeding tests males were more sensitive to azadirachtinthan females. In contrary, preparations containing quassin depressed significantly thefeeding activity in females. In laboratory conditions compared to the other preparationsNeemAzal-T/S had stronger and long-term influence to the pine weevil feeding.

NeemAzal Paint, NeemAzal Paint (40%, vegetable oil dilution), NeemAzal Paint Blank,TRF-002-Paint (20% and 40% water dilutions) and TRF-002-Paint Blank effectedsignificantly (p<0.05) the feeding behaviour of pine weevil in forest conditions.

Keywords: antifeedants, azadirachtin, biopesticides, feeding activity, pine weevil,quassin

Introduction

The large pine weevil, Hylobius abietis, is the major insect species-affectingreforestation in Estonia. This insect is also a well-known problem in reforestation areasin boreal coniferous forests extending from Northern and Western Europe to EasternBiological Control of Plant, Medical and Veterinary PestsR. Strang & H. Kleeberg (eds.)Copyright 2009 by Trifolio-M GmbH

204

Siberia and Japan (Eidmann, 1974; Gourov, 2000). The adults feed on the bark of Scotspine (Pinus sylvestris L.) and Norway spruce (Picea abies (L.) Karst.) seedlings duringthe first few years after cutting a conifer stand. They inflict significant economicaldamage by destroying a large proportion of seedlings (Örlander and Nilsson, 1999;Hannerz et al., 2002). This problem is largely dealt with by treating seedlings withchemical insecticides. The growing threat of environmental contamination and aprohibition on the use of chemical insecticides in forests in many countries haveencouraged the conducting of experiments with various biological insecticides, pestcontrol products directly obtained from plants (Sibul et al., 2001, 2002; Thacker et al.,2003; Månsson and Schlyter, 2004). The demonstrated efficacy of the seed kernelextract of Azadirachta indica A. Juss (bioactive ingredients are azadirachtin and otherlimonoids), has stimulated research and development of other botanical insecticides.Some botanical products have proved their use, these include nicotine (from Nicotianatabacum L.), quassin (from Quassia amara L. and Picrasma excelsa Lindl.), and ryania(from Ryania speciosa Vahl.) (Isman, 1997). In the present study, the effects of somebiopesticides on the forest pest Hylobius abietis (L.) feeding behaviour were testedunder laboratory and field conditions.

Material and methods

Insect

For laboratory experiments H. abietis adults were collected in May and June 2003 frompitfall traps (25 x 40 x 50 cm) in a fresh clear-cutting area in an intensively managedforest area in the forest district of Räpina, Southern Estonia. The adult weevils werepreserved in plastic boxes with moistened moss, with access to food (Scots pine twigs)and water, in a refrigerator at +6 to +8 ºC. Before the experiments, the weevils werekept in similar boxes at room temperature (+22±1 ºC) 12 h without food, but with accessto water. The gender of the weevils was determined.

Tested biopesticides

The effects of different biopesticides were determined: NeemAzal Paint (2,5%azadirachtin), NeemAzal-T/S (1% azadirachtin), NeemAzal-T/S Blank Formulation (0%azadirachtin), NeemAzal Paint Blank (0% azadirachtin), TRF-002-Paint (0,05%quassin), TRF-002-Paint Blank (0% quassin). The diluent (water or vegetable oil) andtype of condition is mentioned in table 1. All tested preparations are produced byTrifolio-M GmbH (Germany).

205

Table 1. Trade name, active ingredient, % of a.i., diluent, rate of application, type of condition ofbiopesticides used in laboratory and field experiments in Estonia during 2003-2004.

Laboratory test

The laboratory test were carried out in the laboratories of the Faculty of Forestry,Estonian Agricultural University, Estonia in November 2003. Laboratory experimentscomprised assaying the effects of the biopesticides on weevil feeding activity byconfining weevils in 9,5 cm diameter Petri dishes with Scots pine twigs of 8 cm long and5–7 mm in diameter. The ends of the twigs dipped in melted wax and after the pinetwigs were dipped inside the pesticide. Treated and untreated twigs were placed inindividual moistened paper sleeves (to prevent contact between twigs) within Petri dish.One weevil was confined with two twigs that were either both untreated (control), bothtreated (no-choice), or with only one twig treated (choice). Twelve replicate Petri disheswere used per treatment. The Petri dishes were exposed to natural light conditions(8L:16D) in temperature +22±1 ºC, 65±5 % RH. The gender of the beetles wasestablished for the estimation of feeding differences between sexes. An area estimate

No. Trade name Active ingredient(a.i.)

% ofa.i.

Diluent Rate ofapplication

Type ofcondition

1 NeemAzalPaint

azadirachtin 2,5 vegetableoil

20 ml/l laboratory

2 NeemAzalPaint

azadirachtin 2,5 water 20 ml/l laboratory

3 NeemAzalPaint

azadirachtin 2,5 vegetableoil

40 ml/l field

4 NeemAzal-T/S azadirachtin 1 water 20ml/l laboratory5 NeemAzal-T/S azadirachtin 1 water 20ml/l field6 NeemAzal-T/S azadirachtin 1 water 40ml/l field7 NeemAzal

BlankFormulation

– 0 – – laboratory

8 NeemAzalBlankFormulation

– 0 – – field

9 NeemAzalPaint Blank

– 0 – – laboratory

10 NeemAzalPaint Blank

– 0 – – field

11 TRF-002-Paint quassin 0,05 water 10ml/l laboratory12 TRF-002-Paint quassin 0,05 water 20ml/l laboratory13 TRF-002-Paint quassin 0,05 water 20ml/l field14 TRF-002-Paint quassin 0,05 water 40ml/l field15 TRF-002-Paint

Blank– 0 – – laboratory

16 TRF-002-PaintBlank

– 0 – – field

17 Control (water) – – – laboratory/field

206

of weevil feeding on cambial tissue was made with the help of the transparent mm2

paper after 24 h, 48 h, 72 h and 96 h for each twig. The mean feeding area of weevil perdifferent periods and their standard deviation were calculated. The significance ofdifferences was controlled by the Student-t test at 0.05 level.

Field experiment

Field trials were established in a fresh clear-cutting area in an intensively managedforest area in the forest district of Räpina, South Estonia. In total, 360 four-year-oldNorway spruce seedlings were planted in grids with 2 m spacing using randomisedblock (every of 40 blocks contained all differently treated seedlings and an untreatedcontrol seedling) design for each experiment on 3 June 2004. After planting differentemulsions were applied around the root collar of the seedling trees using a paintbrush.The biopesticides was changed by dilutions of water and vegetable oil. Trees weretreated with 5 ml of the biopesticides from 0–10 cm above the root collar. Controlseedlings were left untreated. The biopesticides and their concentrations used in thisexperiment are shown in table 1. Feeding damage to the planted seedlings wereassessed weekly from the beginning of June to the end of August 2004 by using visualpercentage scoring system with 10% increments. A score of 0% indicated no feedingdamage and a score 100% indicated that all of the bark had been removed within theassessment zone. Damage was assessed at 0–10 cm above the root collar. The meanof trees damaged by weevils per variant and their standard deviations were calculatedin different periods. In addition, the survival percentage of differently treated anduntreated control plants was calculated in the end of the experiment.


In laboratory choice feeding tests males were more sensitive to azadirachtin thanfemales (Fig. 1A, B). A similar behaviour of male beetles was observed in previousanalogous experiments with plant extracts (Luik et al., 1998; Sibul et al., 2001, 2002). Incontrary, preparations containing quassin depressed significantly the feeding activity infemales. Quassin and other quassinoids have shown promising activity as antifeedantsagainst aphids (Powell et al., 1997; Polonsky et al., 1989). However, insufficient workhas been conducted regarding insecticidal effects of quassin on important forest pests.The effects of neem formulations have already been assayed on several insect pests ofdifferent classes to evaluate them in IPM.

In no-choice situations compared to the other preparations NeemAzal-T/S had strongerand long-term influence to the male than female pine weevil feeding. The feedingactivity was significantly depressed probably because of the high concentration of activevolatile ingredients.

207

A

B

Figure 1.Mean (± SD) feeding area (mm2) of females (A) and males (B) of Hylobius abietis during 96hours on pine twigs treated with different biopesticides.

0

50

100

150

200

NeemAzalPaint 20%(vegetable

oil emulsion)

NeemAzalPaint 20%

(wateremulsion)

NeemAzalT/S 20%

(wateremulsion)

NeemAzalPaint Blank

NeemAzalBlank

Formulation

TRF-002-Paint 10%

(wateremulsion)

TRF-002-Paint 20%

(wateremulsion)

TRF-002-Paint Blank

Treatments

Mea

n fe

edin

g ar

ea, m

m2 ±

SD

ControlTreated

0

50

100

150

200

NeemAzalPaint 20%

(vegetable oilemulsion)

NeemAzalPaint 20%

(wateremulsion)

NeemAzalT/S 20%

(wateremulsion)

NeemAzalPaint Blank

NeemAzalBlank

Formulation

TRF-002-Paint 10%

(wateremulsion)

TRF-002-Paint 20%

(wateremulsion)

TRF-002-Paint Blank

Treatments

Mea

n fe

edin

g ar

ea, m

m2 ±

SD

ControlTreated

208

NeemAzal Paint, NeemAzal Paint (40%, vegetable oil dilution), NeemAzal Paint Blank,TRF-002-Paint (20% and 40% water dilutions) and TRF-002-Paint Blank effectedsignificantly (p<0.05) the feeding behaviour of pine weevil in forest conditions. Damagelevel to seedlings treated with NeemAzal Paint was considerably low during the firstthree weeks, then damage increased (Fig. 2). Survival percentage of differently treatedand untreated control plants in autumn were 67,7 – 96,7 and 47,5, respectively (Fig 3).

Figure 2.The amount (%) of damaged four-year-old spruce seedlings treated with different biopesticidesand untreated seedlings by Hylobius abietis on fresh clear-cut area through time.

Figure 3.Survival percentage of differently treated and untreated (control) four-year-old spruce seedlingsin autumn.

0

20

40

60

80

100

2.06.2004 12.06.2004 22.06.2004 2.07.2004 12.07.2004 22.07.2004 1.08.2004 11.08.2004

Time

The

amou

nt o

f dam

aged

see

dlin

gs, %

Control

NeemAzal Paint 40%(vegetable oil emulsion)

NeemAzal T/S 20% (wateremulsion)

NeemAzal T/S 40% (wateremulsion)

NeemAzal Paint Blank

NeemAzal Blank Formulation

NeemAzal Paint

TRF-002-Paint 20% (wateremulsion)

TRF-002-Paint 40% (wateremulsion)

TRF-002-Paint Blank

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Control NeemAzalPaint 40%

(vegetable oilemulsion)

NeemAzalT/S 20%(water

emulsion)

NeemAzalT/S 40%(water

emulsion)

NeemAzalPaint Blank

NeemAzalBlank

Formulation

NeemAzalPaint

TRF-002-Paint 20%

(wateremulsion)

TRF-002-Paint 40%

(wateremulsion)

TRF-002-Paint Blank

Treatment

Mea

n pe

rcen

t of s

eedl

ing

surv

ival

209

From the point of view of ecological pest control the most promising are theplant-produced compounds which are acting as repellents, inhibitors, or antifeedants asthey only disorientate insects but do not directly kill them. What kind of compounds actsas repellents or deterrents depends also on insect species (Schmutterer, 1992; Bernaysand Chapman, 1994). Botanical insecticides are mostly non-persistent, but in thepresent test NeemAzal Paint, TRF-002-Paint 40% had long-term effect.

Both field and laboratory studies indicated that all tested preparations had a significantdeterrent and antifeedant influence on the weevil feeding.

References

Bernays, E.A., Chapman, R.F. 1994. Host-plant selection by phytophagous insects.Chapman and Hall, New York, 141.

Eidmann, H.H. 1974. Hylobius Schönh. In: W. Schwenke (ed.), Die ForstschädlingeEuropas. 2. Bd. Käfer. Verlag Paul Parey, Hamburg, berlin, 272–293.

Gourov, A. 2000. Hylobius species (Coleoptera: Curculionidae) from Siberia and thedistribution patterns of adults feeding in Scots pine stands. EntomologicaFennica, 11, 57–66.

Hannerz, M., Thorsén, Åke., Mattsson, S., Weslien, J. 2002. Pine weevil (Hylobiusabietis) damage to cuttings and seedlings of Norway spruce. Forest Ecology andManagement, 160, 11–17.

Isman, M.B. 1997. Neem and Other Botanical Insecticides: Barriers toCommercialization. Phytoparasitica, 25 (4), 339–344.

Luik, A., Sibul, I., Voolma, K. 1998. Taimetõmmised männikärsaka küpsussöömamõjutajatena. (Influence of plant extracts on the maturation feeding of the largepine weevil, Hylobius abietis L.). Metsanduslikud uurimused, 29, 146–154. (InEstonian with English summary).

Månsson, P.E., Schlyter, F. 2004. Hylobius pine weevils adult host selection andantifeedants: feeding behaviour on host and non-host woody scandinavianplants. Agricultural and forest Entomology, 6, 165–171.

Polonsky, J., Bhatnagar, S.C., Griffiths, D.C., Pickett, J.A., Woodcock, C.M. 1989.Activity of quassinoids as antifeedants against aphids. Journal of ChemicalEcology, 15, 993–998.

Powell, G., Hardie, J., Pickett, J.A. 1997. Laboratory evaluation of antifeedantcompounds for inhibiting settling by cereal aphids. Entomologia Experimentaliset Applicata, 84, 189–193.

210

Schmutterer, H. 1992. Higher plants as sources of novel pesticides. In: Insecticides:mechanism of action and resistance. Intercept Ltd., Andover, 3–5.

Sibul, I., Luik, A., Voolma, K. 2001. Possibilities to influence maturation feeding of largepine weevil, Hylobius abietis L., with plant extracts and neem preparations. In:Practice oriented results on the use of plant extracts and pheromones in pestcontrol. Proc. of the Intern. Workshop, Tartu, Estonia, January 24–25, 2001,112–119.

Sibul, I., Voolma, K., Luik, A., Ploomi, A. 2002. Influence of some natural insecticides onthe feeding activity of the large pine weevil, Hylobius abietis (L.) (Coleoptera,Curculionidae). Proc. of the Sci. Intern. Conf. “Plant Protection in the BalticRegion in the Context of Integration to EU”, Kaunas, 2002, 107–110.

Thacker, J.R.M., Bryan, W.J., McGinley, C., Heritage, S., Strang, R.H.C. 2003. Fieldand laboratory studies on the effects of neem (Azadirachta indica) oil on thefeeding activity of the large pine weevil (Hylobius abietis L.) and implications forpest control in commercial conifer plantations. Crop Protection, 22, 753–760.

Örlander, G., Nilsson, U. 1999. Effect of reforestation methods on pine weevil (Hylobiusabietis) damage and seedling survival. Scandinavian Journal of ForestResearch, 14, 341–354.

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PAPERS SUBMITTED AFTER THE 14TH WORKSHOP AND ACCEPTEDFOR INCLUSION INTO THE PROCEEDINGS

213

1Paper submitted: 06.04.2005

INTEGRATED CONTROL OF BEMISIA TABACI IN EUPHORBIAPULCHERRIMA1

ELLEN RICHTER

BBA, INSTITUTE FOR PLANT PROTECTION IN HORTICULTURE, MESSEWEG 11/12, D-38104 BRAUNSCHWEIG,GERMANY

Introduction

The white fly species Bemisia tabaci (Gennadius) is a common pest in the production ofpoinsettia (Euphorbia pulcherrima Willd. ex Klotsch). Biological control of B. tabaci isdifficult because the standard beneficial organism the parasitoid wasp, Encarsiaformosa Gahan parasites larvae of B. tabaci to a lower extend than its regular hostTrialeurodes vaporariorum Westwood. In mixed populations the wasp prefers thespecies T. vaporariorum. In the last years there was a notable lack of efficacy ofEncarsia formosa in poinsettia production in Germany. As one possible reason areduction in the quality of E. formosa was discussed. This assumption was rejected. Inprevious trials it was shown that E. formosa is very efficient in parasitising B. tabaci andthat it is capable of building large populations. One reason for the decrease in efficacyhas been side effects of different pesticides. Additionally, there are difficulties with thisparasitoid accepting the new host B. tabaci in the greenhouse.

In this study a reliable biological method to control B. tabaci is being developed andeconomically improved. For that reason selective insecticides were implemented. Firstthey were tested for use in plant cutting production for their efficacy and phytotoxicity.Afterwards, one efficient pesticide was chosen for implementation in a greenhouse trial.

Material and methods

Implementation of insecticides - treatment of cuttings

In all investigations the cultivar “Cortez Red” was used. The cuttings where taken fromstock plants infested with B. tabaci. Forty four cuttings where dipped into each pesticidesolution for one minute. The pesticide concentrations conformed, unless otherwisenoted, with the registered spraying concentration. After dipping, the cuttings wereplanted in 6 cm pots and in groups of 11 placed into plastic “mini greenhouses”. After 3to 5 weeks, depending on the climatic conditions, the plants were inspected forinfestation with B. tabaci larvae and for empty pupal cases.


214

Implementation of insecticides – greenhouse treatment

For these trials four greenhouse cabins were used as plots with 250 poinsettia plants ineach. Cuttings were taken from infested stock plants. The cuttings used in plot 3 and 4were dipped into an azadirachtin A solution before they took root. In the greenhousethese plots were sprayed when catches of white flies exceeded 10 insects per stickytrap. Trial layout:

To monitor flight activity of B. tabaci three yellow sticky traps in each cabin wereexamined weekly. To monitor population development of B. tabaci the most infested leaffrom 25 plants was collected weekly from each cabin and the number of L2-L4 larvae,parasited larvae and empty cases of parasitised larvae were counted. When beneficialrelease ended (November 4) and at the end of the trial four weeks later (December 2),25 plants were examined for total infestation.


Implementation of insecticides - treatment of cuttings

In cutting-producing nurseries, stock plants are often already be infested with B. tabaci.However, clean cuttings are often necessary for further cultivation in the same nurseryand especially when cuttings are to be sold. Eggs and young larvae of B. tabaci onthese cuttings can develop during the period of root taking under atomised spraying.Treatments with pesticides are difficult during this period. Particularly the infestedunderside of the leaves is not wettable. To reduce the initial infestation, the cuttingswere fully dipped into solutions of different insecticides directly after being cut off.Efficacy and phytotoxicity of the different pesticides were examined (Table 1).

1 Untreated control without beneficials “control”2 Encarsia formosa - 1. week 4, then 2 wasps/plant

weekly“Encarsia 2x”

3 Encarsia formosa - 1 wasp/plant weekly “Encarsia Neem”4 Eretmocerus mundus - 1 wasp/plant weekly “Eretmocerus

Neem”

215

Table 1. Infestation of poinsettia cuttings with Bemisia tabaci larvae after dipping in different insecticideswith subsequent root taking in plastic “mini greenhouses”

Numbers are the totals from 40 leaves.

The results show clearly that a dipping is very well suited to reduce the initial infestation,but the efficacy of the various insecticides varied greatly. The active ingredientsabamectin (Vertimec), fenazaquin (Magister 200 EC), spinosad (Conserve),azadirachtin (NeemAzal-T/S) and mineral oil were highly effective. A few activeingredients caused phytotoxic symptoms: insecticidal soap and isopropanol, in higherconcentrations rape oil and azadirachtin A as well as insecticides with themicroorganisms Verticillium lecanii, Beauveria bassiana and Poecilomycesfumosoroseus. The reason for the latter is still unclear, it is possibly the formula of thespore carrying solution.

Implementation of insecticides – greenhouse treatment

Cuttings were taken and dipped at the end of May, potted four weeks later and thenseparated to the four greenhouse cabins. The infestation with B. tabaci larvae on thedipped and the untreated cuttings was recorded. On the untreated cuttings (“control”and “Encarsia 2x”) nearly 25 larvae per plant were found, whereas the dipping reducedthe infestation to 5 larvae per plant. In the two greenhouses with treated cuttings(“Encarsia Neem” and “Eretmocerus Neem”) the insecticide NeemAzal-T/S was sprayedwhen catches on the sticky traps exceeded ten white flies.

As shown in Figure 1 population increase of the white flies took some time. From thebeginning of August a clear increase of the population was observed on the sticky trapsin the treatment “control”. With one little exception which occurred in the beginning of

Experiment 1 Experiment 2 Experiment 3active ingredient number active ingredient number Active ingredient numberwater 171 water 369 water 472rape oil 1 % 130 insecticidal soap 1

%325 indoxacarb 0.014

%314

plant strengthener 98 plant strengthener 248 teflubenzuron0.05%

254

insecticidal soap 4%

89 Nematodes2500/ml

208 buprofezin 0.03 % 230

nematodes 2500/ml 57 Fenazaquin 0.1 % 137 pymetrozine 0.08%

227

azadirachtin 0.5% 27 azadirachtin 0.5 % 42 isopropanol 35 % 109fenazaquin 0.5 % 0 rape oil 2 % 12 spinosad 0.08 % 65

abamectin 0.06 % 12 azadirachtin 2.5% 2Fenazaquin 0.5% 14 mineral oil 2 % 1

216

September, trap catches were high. From the beginning of August until well intoOctober, a clear increase of the B. tabaci population on the plants could be observed.From this point in time the population remained static because of the weatherconditions.

During the cultivation period the poinsettia in treatments “Encarsia Neem“ and“Eretmocerus Neem” showed only minor infestations. They had to be treated withAzadirachtin only twice in “Encarsia Neem“ and three times in “Eretmocerus Neem“(Figure 2).

Figure 1.Catches of Bemisia tabaci on yellow sticky traps in greenhouse cabins after release of differentparasitoid wasps in 2004.

In “Encarsia 2x” the wasps showed their immense potential to control B. tabaci. In spiteof the high initial infestation they were able to reduce the infestation in eight weeks tothe same level the treated plants showed (Figure 3). They kept this low level during thewhole period. At the end of the investigation, there was nearly no difference betweenthe varying treatments. All treated plants were marketable (Table 2).

From these and previous investigations it can be reasoned that the standard amount ofEncarsia formosa released against Trialeurodes vaporariorum (one wasps per threeplants) is not sufficient to control B. tabaci successfully. As a sufficient amount onewasp per plant is recommended. At costs of 1 Cent per E. formosa and 1.2 Cent per E.mundus with 17 releases as well as 0.2 Cent per plant for the NeemAzal-T/S treatment,the “Encarsia Neem” option is economically favourable. Probably costs can be reducedfurthermore by reducing the frequency of wasp releases in practice.

0

100

200

300

400

500

600

700

800

29.06

.

06.07

.

14.07

.

22.07

.

28.07

.

04.08

.

11.08

.

17.08

.

25.08

.

01.09

.

08.09

.

15.09

.

22.09

.

30.09

.

07.10

.

13.10

.

20.10

.

28.10

.

04.11

.

control Encarsia 2x Eretmocerus Neem Encarsia Neem

NeemAzal-T/S treatments

217

Figure 2.Effect of different biocontrol strategies on the infestation with Bemisia tabaci in poinsettia duringthe production period.

Table 2: Final infestation with larvae of Bemisia tabaci on poinsettia and costs of control (beneficialwasps and insecticidal treatments per plant).

Acknowledgements

Many thanks to the company Sautter&Stepper for providing the beneficials and the gooddiscussions and to all assistants for helping to count thousands of Bemisia tabaci larvae.

0

5

10

15

20

25

23.06

.

30.06

.

07.07

.

14.07

.

22.07

.

28.07

.

04.08

.

11.08

.

17.08

.

01.09

.

25.08

.

08.09

.

15.09

.

21.09

.

30.09

.

07.10

.

13.10

.

20.10

.

28.10

.

control Encarsia 2x Eretmocerus Neem Encarsia NeemNum

ber o

f Bem

isia

taba

ci la

rvae

per

leaf

.

NeemAzal-T/S treatments

Treatment / number of white flies

“control” “Encarsia 2x” “EncarsiaNeem”

“EretmocerusNeem”

white flies, 4.11.2004 180.2 1.6 0.3 0.8white flies, 2.12.2004 283.8 0.7 0.0 0.5costs/plant - 36 Cents 17.4 Cents 20.1 Cents

219


EFFECTIVENESS OF NEEMAZAL-T/S APPLICATION AGAINST POTATOPESTS IN BELARUS IN 20041

M.I. ZHUKOVA, CAND. OF AGR SCI., G.M. SEREDA, CAND. OF AGR.SCI

INSTITUTE OF PLANT PROTECTION, NATIONAL ACADEMY OF SCIENCES OF BELARUS

INTRODUCTION

Pests play an important role in the phytosanitary stress in potato crops in Belarus. Aspecial danger for potato growing in the Republic, especially in its southern region, iscreated by Colorado Potato Beetle (Leptinotarsa decemlineata Say.). High ecologicaladaptability and biological vigour give an opportunity for the phytophage to easily adaptto changing habitat conditions.

In 2002, based on the expert evaluation of Plant Protection Service data on biologicalefficiency of insecticides against the pest, in the Vitebsk area, this index varied from32% to 88%; in the Central Region (Minsk, Mogiliov, Grodno areas) from 40,0% to97,5%; and from 29,0% to 98% in the Southern region (Brest and Gomel areas). Thesame pattern was observed in 2003. Examples of “attenuation” of pyrethroid grouppreparation efficiency were observed in Agricultural Production Cooperative (APC) “Giant” Bobruisk region, Mogiliov area. Thus, using Fastak,10% EC (0,10 l/ha), ColoradoPotato larvae decreased from the initial pest density of more than 60 indiv./plant by 83%the 3rd day after treatment, and 53% on the 7-th day, and using Fury 10 EW, 10% a.e.(0,07 l/ha) the figures were 72,0% and 52,0%, respectively. As is well known, dryweather conditions and high temperatures can contribute to the decreasedeffectiveness of insecticide treatments. However, such a phenomenon is also possibleas a result of decreased sensitivity of some Colorado Potato Beetle populations topyrethroid group preparations, which have been applied continuously in Belarus since1982.

In this context, it is important to use against this most dangerous potato pest,insecticides with other mechanisms of action.

MATERIALS AND METHODS

Testing of NeemAzal preparation “Trifolio-M GmbH” Co., (Germany) against Coloradopotato beetle was carried out on the Agricultural and Production Cooperative (APC)“Tschemyslitsa”, Minsk region, Minsk area on medium-maturing potato variety Scarb.The plot soil was soddy-podzolic loamy with a humus content of 3,88%, soil pH of 6,35.


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Prior to planting the potatoes, the field carried a crop of winter rye. After harvesting therye, the fields were ploughed in autumn. In spring, 80 tons of peat – manure compost,(N100N50K90), per ha were applied, the cultivation was accomplished with ridging, andpotatoes planted at a density of 60 thousand plants per ha with an inter-row distance of70 cm.

The measures taken to tend the crops included three inter-row treatments by cultivatorridge maker, KGO-3; the herbicide Dabizin WP application at the rate of 0,70 kg/ha,working solution rate use of 200 l/ha; three times fungicide spraying against late blight(1- Acrobat MC, WP: 2,0 kg/ha, Ridomil Gold MC, WP: 2,5 kg/ha, 3- Penncozeb, WP:1,6 kg/ha). Protection against Colorado potato beetle was done as indicated below.

For NeemAzal testing, the experimental plot area was 30 m2, 4 repetitions, plot locationrandomized. As a positive control or bench-mark of the effectiveness of thephytochemical treatment, Banocol 50% WP was used. This is a pesticide based on ananalogue of nereis toxin and is a neurotoxin.

The scheme included the following variants:

1. Control –without treatment;

2. NeemAzal, 2,5 l/ha –2 times;

3. NeemAzal, 1,5 l/ha –2 times;

4. Bancol, 50% WP (standard), 0,2 kg/ha

5. Bancol, 50% WP (standard), 0,2-0,25 kg/ha.

Spraying was done by the method of general surface treatment of growing plants usinga knapsack sprayer with the working solution rate use of 500 l/ha, for this, the first treat-ment ( June 23,2004), at the end of potato blossoming by mass appearance of the I andII instar larvae, the second one (August 3) in 10 days after the first. One should notice,that the number of treatments was predetermined by phytosanitary situation on Colo-rado Potato Beetle in 2004 season.

The pest number was noted before treatment and after 1, 3, 7, 14 and 21 days on spe-cific, labelled potato plants, counting larvae by instars, from 1 to 4.

At 14 and 21 days after the first treatment, the leaf surface damage was evaluated byquartiles: 0-25%=I, 25-50%=II, 50-75%=III, > 75=IV.

At the same time, in each of the experimental variants, the aphid number was deter-mined on 100-leaf samples.

RESULTS

It was clear (Table 1) that the lower than average temperatures and rainfall of the 2004growth period in the NeemAzal testing zone limited potato beetle development.

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During May, June and the first half of July both were below the normal averages.

Table 1. Agrometeorological conditions during the NeemAzal insecticide testing period.(Data from Minskhydrometeorological station )

Potato field colonization by the pest before treatment was of a margin character with theaverage larvae density 27,7 indiv./1 colonized plant, and most (> 90% ) of the larvaewere in instars I and II (Table 2).

Table 2. Initial Colorado Potato Beetle larvae number before treatment with NeemAzal or Bancol.Record date – July 23, 2004

The results (Table 3) show that the studied plant-derived insecticide NeemAzal–T/Sunder phytosanitary situation conditions (colonization, number, Colorado potato beetlepopulation structure) soon after treatment showed clear differences in comparison withBancol used as a bench mark. Already in a day after Bancol application at the rate of

Treatment Larvae number by ages,indiv/plant

Total numberof indiv./plant

L1 L2 L3 L41 Control-without treatment 15,9 7,7 2,6 0 26,22 NeemAzal,2,5 l/ha –2 times 16,9 8,7 3,1 0 28,73 NeemAzal, 1,5 l/ha –2 times 12,2 16,7 1,7 0,3 30,94 Bancol, 50% WP (standard)

– 0,2 kg/ha 10,2 15,8 2,3 0,1 28,4

5 Bancol, 50% WP (standard)–0,2-0,25 kg/ha 12,7 7,2 3,6 0,9 24,4

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0,2 kg/ha, pest death reached 95,8-98,0% by larvae number compared to the controlplants, on which the numbers had risen to 35,5 indiv./plant. These numbers did notsignificantly change on days 3 and 7 after treatment.

In contrast to the synthetic insecticide, NeemAzal–T/S acts as a stomach poison withlow contact action, and so did not produce a quick action on Colorado potato beetlelarvae. However, considering the results of phytophage records in 1 and 3 days afterthe first treatment, the most effective NeemAzal was at the rate of 2,5 l/ha: the I, II, IIIand IV–instar larvae number per recorded plant were 5,8; 6,7; 2,8; 0,4 and 2,4; 5,3, 4,8;1,0, compared to the control 8,3; 19,7; 5,7; 1,8 in 1 day, giving a reduction of 56%, and3,5; 9,3; 7,2; 5,2 individuals in 3 days. In a variant with NeemAzal application at the rateof 1,5 l/ha the I-IV –instar larvae number in 3 days after the first treatment has made asit is seen from the table 3, 3,5; 8,1; 7,0; 0,8 individuals per 1 recorded plant, a reductionof 17%.

Table 3. Colorado potato beetle larvae number immediately after treatment by NeemAzal or Bancol.

As is known, the mechanism of NeemAzal action lies in blocking the hormonal systemresponsible for moulting hormone ecdysone in larvae after oral uptake of the activeingredient with the plant mass or sap. Colorado potato beetle larvae records 7 daysafter the first treatment showed their significant effect both in the variant with NeemAzalapplication at the rate of 2,5 l/ha (12,1 indiv./plant) and 1,5 l/ha (11,5 indiv./plant) by thetotal larvae number 17,3 per plant in the control. However, the most voraciousIII-IV-instar larvae number by NeemAzal application was 1,6-2,1 times less than in the

Treatment Application rate, l,kg/ha

Larvae number (indiv./plant)

L1 L2 L3 L4 TotalIn a day after the first treatmentControl without treatment 8,3 19,7 5,7 1,8 35,5NeemAzal 2,52,5 5,8 6,7 2,8 0,4 15,7NeemAzal 1,51,5 11,6 13,8 3,7 0,4 29,5Bancol, 50% w.p.(standard)

0,2 0,1 1,0 0,3 0,1 1,5

Bancol, 50% w.p.(standard)

0,2→0,25 0,1 0,4 0,2 0,0 0,7

In 3 days after the first treatmentControl without treatment 3,5 9,3 7,2 5,2 25,2NeemAzal 2,52,5 2,4 5,3 4,8 1,0 13,5NeemAzal 1,51,5 3,5 8,1 7,0 0,8 19,4Bancol, 50% w.p.(standard)

0,2 0,1 0,4 0,2 0,3 1,0


0,2→0,25 0,0 0,0 0,1 0,3 0,4

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control where this parameter has made 14,6 individuals per one recorded plant (Table4).

The presence of Colorado Potato Beetle larvae on treated plants 7 days afterinsecticide application indicated the necessity of repeated treatment, which was carriedout 10 days after the first.

As the pest number decreased till singular individuals in 14 and 21 days after the firsttreatment (or on the 3 and 10-th day after the second) in the control variant inconnection with the pupation of the main part of the elder age larvae, the efficiency ofthe protective action of the insecticide used was evaluated by degree of leaf surfacedamage.

Table 4. Change of age structure of Colorado Potato Beetle larval population under NeemAzal treatment.

As it is seen from data presented in Fig.1. leaf surface damage in the control variant (1)21 days after the first treatment exceeded the 50% level, compared to 20% in theNeemAzal treatments (2 and 3). Treatment with Banocol (4 and 5), however, reducedthe leaf area destruction to 10%. Based on the dynamics of Colorado potato beetlelarvae number judged by record dates one can come to the conclusion that a share ofthe first treatment influence on the decrease of phytophage harmfulness was moresignificant.

Treatment Application rate,l, kg/hа Larvae number, indiv./bush

L1+L2 L3+L4 totalIn 7 days after the first treatment

Control without treatment 2,7 14,6 17,3NeemAzal 2,52,5 3,3 8,8 12,1NeemAzal 1,51,5 4,6 6,9 11,5Bancol, 50% w.p.(standard)

0,2 0,6 1,7 2,3


0,2→0,25 0,1 0,8 0,9

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Fig.1. Efficiency of NeemAzal-T/S and Banocol judged by leaf area destruction. (Treatment variants are:1 = Control, 2 and 3= NeemAzal-T/S, 3 and 4= Banocol)

The results of plant productivity evaluation prove the protective action of NeemAzalpreparation in the decrease of Colorado potato beetle destruction. Under 2004conditions the use of the tested insecticide gave an opportunity to preserve up to 25,7%yield (Table 5), a rate similar to that produced by the nereis toxin analogue.

NeemAzal influence on aphids as virus infection vectors was evaluated by the conditionof wingless population. Judged by aphid decrease in a certain period of time in thecontrol variant (105 individuals per record unit before treatment and 37, 6, 15,7 - in3,7,14, 21 days after the first treatment, accordingly) the wingless population condition,perhaps, in a larger degree was determined by agrometeorological situation: the largestrate of precipitation was in the III decade (10 day period) of July while evaluatingNeemAzal-T/S insecticidal action – 104 mm or 325% higher than usual for the period(Table 1). The character of wingless aphid dynamics number under NeemAzal influencefollowing different programs of treatments (2 times at the rates of 2,5 and 1,5 l/ha) as itis seen from Table 5 data, is the same as the control. Clearly, the unusual weatherconditions overwhelmed any differential effects of the pesticides.

As a final observation, it can be said that there was no phytotoxicity of the testedpreparations in relation to growing plants.

0102030405060

dam

aged

leaf

ar

ea,%

1 2 3 4 5

14 days 21 days

variant after the first treatment

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Table 5. Economic efficiency of NeemAzal insecticide for potato protection against Colorado potatobeetle

Table 6. Number of aphids on potato plants with different treatments

*) in 3 days after the second treatment

**) in 10 days after the second treatment

CONCLUSIONS

It was determined by researches that the preparation NeemAzal-T/S containingsubstances from the limonoid group, namely azadirachtin A (1%) and azadirachtinsB,V,G, D etc. (0,5%), other neem substances (2,5%), vegetable oil (51%) and vegetablesurfactant (45%), does not produce a quick insecticidal action on Colorado PotatoBeetle larvae both at the levels of treatment used against the younger instar larvae.

The protective effect is seen, first of all, in the decrease of the pest feeding activity: theleaf surface damage in 21 days after the first NeemAzal treatment has not reached 20%at that time in the control variant the given parameter exceeded 50% level.

Control Withouttreatment

251 - -

NeemAzal 2,52,5 338 87 25,7NeemAzal 1,5 1,5 337 86 25,5Bancol,50% w.p. (standard) 0,2 347 96 27,7Bancol,50% w.p. (standard) 0,2 0,25 343 92 26,8SED05 47,98

Treatment Applicationrate, l(kg)/ha Yield

centner/ha Preservedcentner/ha centner/ha

Treatment Application rate,l(kg)/ha Aphids number, indiv./100 leaves

Beforetreatment In days after treatment

3 7 14 21Control Without treatment 105 37 6 15 7

NeemAzal 2,52,5 89 25 2 6* 2**NeemAzal 1,51,5 122 62 14 9* 3**

Bancol,50%w.p.(standard) 0,2 67 23 8 12 4

Bancol,50%w.p. (standard) 02,0,25 124 34 5 11* 7**

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Overall, however, judged by the final economic efficiency, the azadirachtin-containingplant protection product gave the same improvement over the control as the nereis-toxinanalogue: namely 25.7%.

The conditions in the growing season on 2004 when the experiment was done, wereunusually bad for insects, which showed a lower-than-average population growth.

To explore fully the possible use of azadirachtin-containing pesticides for protection ofthe potato crop in Belarus, it will be necessary to repeat the trials on a wider scale andin more clement weather conditions.

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TREATMENT OF CONIFEROUS SEEDLINGS WITH NEEMAZAL–T/S: APOSSIBLE WAY OF PROTECTION AGAINST THE LARGE PINEWEEVIL HYLOBIUS ABIETIS (L.)1

NICOLAI OLENICI AND VALENTINA OLENICI

FOREST RESEARCH AND MANAGEMENT INSTITUTE, EXPERIMENTAL STATION FOR NORWAY SPRUCESYLVICULTURE, CALEA BUCOVINEI 73 BIS, 725100 CAMPULUNG MOLDOVENESC, ROMANIA, E-MAIL:[email protected]

Abstract

The paper presents three laboratory experiments aimed to establish how longNeemAzal-T/S can protect the seedlings against the weevils, and if the effectivelifespan of insecticide could be prolonged by mixing the emulsion with the adjuvantNu-Film 17. In choice tests with mature as well as with young, immature insects, theefficiency of protection against the damages caused by the large pine weevil wasestablished treatment with 5 %, 10 % and 20 % NeemAzal-T/S (having 1 % azadirachtinA), as well as with a mixture of 20 % NeemAzal-T/S and 10 % Nu-Film 17. The insectfeeding was affected by all treatments and the magnitude of effect was increasing withthe concentration of insecticide, but it was also influenced by the gender andphysiological state of the weevils. In mature, post oviposition insects, females weremore sensitive to insecticide than males, and the opposite was true in young, immatureweevils. The best results were achieved with 20 % NeemAzal-T/S and 10 % Nu-Film 17against the male young weevils. A very good protection (efficacy higher than 75 %) ispossible only a few days after treatment, but sufficient protection is achieved after twoweeks too, because most of the wounds are superficial, without severe impact on theplants. Treating the seedlings with 20% NeemAzal-T/S (or higher concentrations) everytwo weeks, during the periods with high rates of feeding, could keep damages andseedling losses at a normal level. The efficiency could be improved and the periodbetween treatments prolonged by mixing NeemAzal-T/S with Nu-Film. Fieldexperiments are necessary to validate our conclusions.

Keywords: coniferous seedlings protection, NeemAzal-T/S, large pine weevil, Hylobiusabietis, Nu-Film 17.

Introduction

In Romania, as in many other countries, the Large Pine Weevil (Hylobius abietis) is themain pest of coniferous plantations which are replanted shortly after wood harvesting.Biological Control of Plant, Medical and Veterinary PestsR. Strang & H. Kleeberg (eds.)Copyright 2009 by Trifolio-M GmbH

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Without control measures it can damage 50-80 % of the seedlings and sometimes theplantations have been totally destroyed. In order to avoid the damage, we haveelaborated a methodology for the quantification of risk of infestation, and recommendappropriate protective measures according to the degree of risk (Olenici & Olenici,2003). In the most cases with a high risk of attack, a postponement of planting for 2years after clear cutting is accepted, but during the 3rd or the 4th season (according tothe length of generation development of the pest) the risk is still high enough to requireother protection measures. One of them is to catch the weevils using traps baited withalpha-pinene and ethanol (Olenici & Olenici, 2004), a procedure that is already widelyused in Poland (Stocki, 2000), but the young weevils emerging in July-September arenot attracted to this kind of stimulus (Nordenhem & Eidmann, 1991; Malphettes et al.,1994; Zumr et al., 1995; Olenici & Olenici, 2002a). For such situations, spraying ofseedlings with synthetic pyrethroids like deltamethrin, cypermethrin etc. is usually theonly possible protective measure. The pyrethroids are also used to treat the seedlingsbefore planting. Due to the low persistence of this kind of insecticide, we have tried toimprove the efficiency of the treatment by combining the insecticides with an adjuvant(Nu-Film 17) and the results are very good (Olenici & Olenici, 2002b), but in a few yearssuch insecticides will no longer be allowed for use in Europe. Therefore, starting fromthe results published by Luik (2000) and Thacker & Bryan (2003a), we began to studythe use of NeemAzal-T/S for the protection of coniferous seedlings against the largepine weevil in 2004.

Using 5 % and 10 % water emulsions of this plant protection product, Luik (2000), noteda significant reduction of weevil feeding, especially in mature males, for a period of 72hours after treatment. However, the published data indicate a clear tendency of declineof efficiency, although the observation period was very short compared to the feedingperiod of the insects in the field. On the other hand, Thacker & Bryan (2003) usedundiluted neem extract and obtained a very good protection for the three-week period ofobservation, but the temperature was quite low, reducing weevil feeding and increasingthe lifespan of insecticide. In addition, the use of undiluted extract could be tooexpensive and possibly toxic for plants, especially if the treatment is applied by dippingthe seedlings into insecticide or by spraying. Therefore, the aims of our first experimentswere to establish how long NeemAzal-T/S can protect the seedlings against the weevils,and if the effective lifespan of insecticide could be prolonged by mixing the emulsionwith the adjuvant Nu-Film 17.


We have conducted three laboratory experiments with NeemAzal-T/S (1 % azadirachtinA) from Trifolio-M GmbH (Table 1). In the first experiment we have used basal stem

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segments (8 cm long, without needles) of 4 year-old Norway spruce seedlings, but inthe other two experiments, twigs (6 cm long) of Scots pine from a single tree, have beenused. All the pieces selected were free of wounds and exfoliation of the outer layer. Allwere about the same diameter (7-9 mm). Cutting of segments took place shortly afterthe harvesting of twigs and their ends were dipped into melted wax to prevent thedessication as well as to avoid a very high release rate of terpenes from the cut barkand wood. After that, the twig segments were treated with water emulsion ofNeemAzal-T/S, by immersing them in the liquid for 10 seconds. Control twigs weredipped in water. The twigs were then allowed to dry at room temperature for 4-5 hoursin vertical position, before placing them in glass jars with weevils.

Table 1. Summary of the laboratory experiments with NeemAzal-T/S

During the observation period, the twigs remained in vertical position, on a moistenedlayer of peat and sand in glass jars of 300-400 ml capacity. A treated segment (or inExperiment III, two treated segments) and a control segment were placed in each jar,about 4 cm apart. In the experiments I and II each treatment had a separate controlsegment, but in the experiment III only one.

One weevil was confined in each jar. In experiments I and II, we used mature weevilsthat had been collected from the field in June and supplied with fresh Scots pine twigsuntil the beginning of the experiments. For the 3rd experiment, we used young,immature weevils, obtained from Norway spruce trap billets. Each weevil was used inonly one experiment.

The jars were held in laboratory, at room temperature (about 18-22oC), in natural lightregime. Jars were covered with plastic fiber mesh, allowing a free movement of air.

The area of bark removed from the twigs was measured periodically. Each time theareas of outer and inner bark (phloem) removed were quantified separately. The meanarea of removed bark was calculated for each observation time and the significance ofdifferences was controlled by Student-t test.

Experiment Period ofobservation

Weevils Vegetal material Emulsion concentrations and number of replications

I 14-26.072004

mature males stem segmentsof spruceseedlings

5 % and 10 % NeemAzal; 15 replications

II 3.08-7.092004

mature malesand females

twig segmentsof Scots pine

10 % and 20 % NeemAzal;15 replications

III 16-25.092004

immaturemales andfemales

twig segmentsof Scots pine

20 % NeemAzal and 20%NeemAzal + 10% Nu-Film;20 replications

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Results

In the experiment I, 5 days after treatment, the total area of bark removed by theweevils on stem segments treated with 5 % NeemAzal was 41.5 % smaller than thateaten on the control segments, whereas on those treated with 10 % NeemAzal the areawas only 31.2 % smaller than that consumed on the controls (Table 2). However, thereduction in area of the deep wounds (with bark and phloem removed), was 28.2% and69 % in the case of the treatment with 5% and 10 % NeemAzal-T/S, respectively.

Seven days later, the differences between the mean area of bark removed from thetreated segments and their control decreased to 8.6 % for 5% NeemAzal, and 15 % for10% NeemAzal-T/S. Apparently, it means that weevils caused about the same damageirrespective of the treatment, but after 12 days from the beginning of the experiment theaverage area of deep wounds was respectively 38.5 % and 46.7% less than that oncontrol segments. The differences between the means were not statistically significant,because of very high variability of the data.

Results from the second experiment (Table 3) indicate that the weevils gnawed lessbark and phloem on twigs treated with 20% NeemAzal and their control pairs, than ontwigs with 10 % NeemAzal and corresponding controls, regardless of the weevil’s sexand the observation time. It means that the higher concentration (20 %) was moreeffective than the lower one (10 %), but the differences between the treatmentsvanished gradually over the following month.

On the other hand, one can see that when considering the total area of removed bark(Table 4), the treatment was more effective against the female weevils, except the first 5days. The males gnawed on both kinds of twigs, either treated or untreated, at least thesame quantity of bark as females had gnawed, or more. In addition, during the first 5days, they caused mainly deep wounds on treated twings, and shallow wounds oncontrol ones. At the end of the experiment, the shallow wounds represented more than50 % of removed bark area on both treated and untreated twigs. By contrast, thefemales caused mainly deep wounds on twigs treated with 10 % NeemAzal and on theircontrols, but shallow wounds on the counterparts with 20 % NeemAzal.

The efficiency of the treatments was quite low even 2-5 days after the application ofinsecticide (Table 4) and, in most cases, the differences between the average valueswere not statistically significant because of very high variability of the results.

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Table 2. Bark area removed by the weevils in experiment I (mean ± standard deviation (mm2)

Notes: Mean results bearing the same superscript within each column are not significantly different asjudged by Student-t test at the 95 % level of probability.

Table 3. Bark area removed by the weevils in experiment II (mean ± standard deviation, (mm2)

Notes: 1) For each concentration, the means bearing the same small superscript within each row are notsignificantly different. 2) For each date, the means bearing the same capital superscript within eachcolumn are not significantly different (Student-t test at the 95 % level of probability).

10 % 12.1a ±20.2

2.7a ±4.4

14.8a ±19.7

43.7a ±45.4

5.6a ±7.5

49.3a ±44.3

control for10 %

12.9a ±12.7

8.7a ±18.8

21.5a ±26.2

47.4a ±40.3

10.5a ±21.5

58.0a ±45.5

5 % 7.7a ±14.3

8.9a ±22.8

16.5a ±34.1

50.8a ±78.4

7.2a ±16.5

57.5a ±89.0

Controlfor 5 %

15.8a ±19,5

12.4a ±22.7

28.2a ±23.9

51.3a ±65,1

11.7a ±15.2

62.9a ±66.9

Treatment 19.07.2004 26.07.2004Type of wound Type of wound

shallow deep total shallow deep total

Datum Weevils Type ofwound

Treatment

10 %NeemAzal

Control for10%

NeemAzal

20%NeemAzal

Control for20%

NeemAzal5.08 males shallow 6.6a ± 9.3 31.0b ± 37.4 5.3a ± 11.6 23.0a ± 30.2

deep 15.4a ± 18.7 13.3a ± 13.7 10.0a ± 13.3 15.2a ± 12.9total 22.0aA ± 17.2 43.6bA ± 37.3 15.3aA ± 21.1 38.2bA ± 31.6

females shallow 6.2a ± 19.0 15.4a ± 19.1 7.7a ± 11.7 7.2a ± 7.9deep 24.2a ± 27.5 25.9a ± 19.4 5.0a ± 6.9 13.6a ± 15.6total 30.3aA ± 31.4 41.2aA ± 25.4 12.7aA ± 14.2 20.8aA ± 15.4

8.08 males shallow 16.6a ± 14.3 52.0b ± 54.9 12.5a ± 18.0 43.6a ± 62.9deep 28.0a ± 31.0 21.4a ± 21.2 20.0a ± 19.2 35.7a ± 35.1total 44.5aA ± 31.4 73.4aA ± 54.0 32.6aA ± 31.4 79.3aA ± 94.4

females shallow 12.5a ± 38.1 23.7a ± 25.2 15.0a ± 20.9 17.8a ± 16.6deep 34.9a ± 42.5 31.2a ± 20.2 10.7a ± 9.7 18.4a ± 19.6total 47.4aA ± 54.2 54.9aA ± 33.5 25.7aA ± 22.8 36.2aA ± 30.1

7.09 males shallow 110.1a ±56.1

218.3b ±145.7

102.1a ±112.3

159.6a ±103.3

deep 93.1a ± 66.3 136.1a ± 74.7 96.1a ± 58.3 111.1a ± 79.9total 203.2aA ±

48.1354.4bA ±

162.1198.2aA ±

124.4270.6aA ±

159.5

females shallow 69.3a ± 94.6 109.8a ± 85.8 62.3a ± 60.8 117.1a ±142.2

deep 74.8a ± 51.4 133.3a ±136.3

41.6a ± 30.5 60.9a ± 33.0

total 144.1aA ±113.4

243.1bA ±137.6

104.0aB ±63.1

178.0aA ±141.2

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Results from the experiment III (Table 5 and fig. 1-2), when we used young weevils anda concentration of 20 % NeemAzal, revealed that treatment efficiency was dependenton the weevil’s sex, the males being more affected than females. A high efficiency(more than 75 %) was achieved only during the first 3 days for females, and 5 days formales. After that, the weevils consumed about the same quantity of bark and phloem onboth treated and untreated twigs, so that two weeks after the treatment the femaleweevils damaged equally the treated and untreated twings. The adjuvant Nu-Film 17increased the efficiency of treatment and this was more obvious with increasing the timeelapsed from the beginning of treatment.

Table 4. The efficiency (%) of treatments in the second experiment, expressed as reduction of totaldamaged area and of the area of deep wounds area, respectively

Treatment Datum Total removed bark area Deep wounds areaMales Females Males Females

10 % NeemAzal 5.08 49.5 26.5 -15.8 6.68.08 39.4 13.7 -30.8 -11.97.09 42.7 40.7 31.6 43.9

20 % NeemAzal 5.08 59.9 38.9 34.2 63.28.08 58.9 29.0 44.0 41.87.09 26.8 41.6 13.5 31.7

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Table 5. Bark area removed by the weevils in experiment III (mean ± standard deviation, mm2)

Notes: 1) Means bearing the same small superscript within each row are not significantly different asjudged by Student-t test at the 95 % level of probability. 2) For each feeding time, the means bearing thesame capital superscript within each column are not significantly different (the same test).

As in the other experiments, the shallow wounds were predominant, especially incontrol twigs.

females 105.9aA ± 98.8 89.3aA ± 61.0 123.5aA ± 70.6300 males 91.4aA ± 56.0 72.9abA ± 51.0 134.3bA ± 85.7

females 125.7aA ± 102.1 115.6aA ± 71.9 132.7aA ± 73.3324 males 99.1aA ± 59.0 81.4aA ± 53.5 142.5bA ± 89.2

females 144.8aA ± 110.9 122.4aA ± 77.1 143.4aA ± 69.1

Feeding time(hours)

Weevils Treatment

20% NeemAzal 20% NeemAzal +10% Nu-Film

Control

36 males 6.7aA ± 11.7 1.8aA ± 5.7 36.2bA ± 31.7females 11.4aA ± 20.3 8.4aA ± 18.9 44.3bA ± 34.2







204 males 55.7aA ± 34.6 51.2aA ± 40.5 108.7bA ± 69.9females 85.1aA ± 93.1 65.7aA ± 59.6 112.7aA ± 66.9

228 males 67.6aA ± 70.5 56.1aA ± 44.9 113.9bA ± 70.5females 98.0aA ± 95.2 77.1aA ± 58.6 117.7aA ± 70.4

252 males 77.5aA ± 48.1 64.1aA ± 46.1 123.0bA ± 76.2

234

Fig. 1. Weevil feeding dynamics in the 3rd experiment, showing the differences between males (top)and females (bottom) as well as the extent of deep and shallow wounds.

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235

Discussion

The laboratory experiments remove the uncontrolled vagaries of the conditions existingin the field. Therefore, good results from laboratory can never assure similar results inthe field. This is the reason why we have tried to simulate in laboratory experiments thefield conditions. However, in these experiments, the absence of rain and direct sunlightprobably reduced insecticide decompostion, and thus exaggerated the effects of thedegree of control.

Fig. 2. The evolution of treatment efficiency when using young beetles and 20 % NeemAzal-T/S

Under the described conditions, considered as optimum for feeding (Christiansen &Bakke, 1968; Havukkala & Selander, 1976), we tested the protection efficiency againstthe weevil damage starting with low concentrations of NeemAzal-T/S (5 % and 10 %),and with male weevils, which had previously been reported to be more sensitive thanfemales (Luik, 2000). Both treatments decreased the attack, and the effect was strongerwith the higher concentration, confirming the results of Luik (2000), but the efficacy wasquite low and of short duration, in the light of the very long feeding period of Hylobiusabietis. In addition, the results were highly variable. This observation was true for all ofour experiments and could be a result of using well-fed insects and moistenedsubstratum, as a combination of starvation and thirst reduce variance regarding theparameters of feeding (Schlyter et al., 2004). In addition, the variability from the firstexperiment could be due to variable attraction of seedlings for weevils, but such a thingis normal in field condition.

The results from the second experiment confirmed that the increase of insecticideconcentration from 10 % to 20 % led to an increase in treatment efficacy, but it still

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236

remained quite low, even in the first days after treatment. However, the effect was stilldetectable after one month. It seems that the males were more sensitive than females,but only during the first 5 days and only if we take into consideration the total area ofbark removed by weevils. If we consider the deep wounds, it is obvious that femaleswere more strongly affected than males. In addition, they gnawed less bark and phloemthan males. Our results contrast with the results already published (Luik, 2000), as wellas with our results from the third experiment, and suggests that the reaction of weevilsto azadirachtin depends not only on the sex, but also on the physiological state ofweevils. The second experiment took place in August, with beetles collected from thefield in June and maintained in environmental condition. That means that for femalesthe oviposition period had passed (Lekander et al., 1985) and this could explain the lowfeeding intensity, because after ceasing egg-laying, the feeding rate of the femalesdeclines to the same level as in the males (Bylund et al., 2004). On the other hand, theweevils used in the 3rd experiment were immature, and we can infer that also during thematuration feeding, before the oviposition, the females consume more food than males.

The average rate of feeding was 8.9-10 mm2 Norway spruce bark per weevil per day inthe first experiment (only mature males), 8.3-16.4 mm2 Scots pine bark in the secondone (mature males and females), and 23.9-30.4 mm2 Scots pine bark in the 3rdexperiment. These results show that young weevils have a higher rate of feeding andconsequently the damage caused by this category of insects could be very important ina shorter time than in cases when population is represented predominantly by maturepost-oviposition insects, if the insecticide protective effect disappeared.

The addition of Nu-Film in insecticide emulsion increased the efficiency of the treatment,but it is difficult to say that the effect is due to a longer lifespan of insecticide or due tophysical characteristics and high concentration of the adjuvant. Other tests, includinglower concentration of Nu-Film, should be conducted to answer this question.

In our experiments, the shallow wounds made up a higher proportion, especially oncontrol twigs. On treated twigs, shallow wounds became prevalent only gradually. Itseems that insects avoided to gnaw superficially on treated twigs so long as theirsurface had high quantity of azadirachtin. This contrasts with the results reported byMånsson & Schlyter (2004), who found large superficial damages in several speciesthat probably possess antifeedant substances but in the inner bark.

Because we had no no-choice situations, it is impossible to say how much the feedingbehavior of weevils was altered by the presence of insecticide in nearness of untreatedtwigs, but it can be seen that, at the end of the observation periods, the average area ofdeep wounding was quite small on both treated and untreated twigs, and such damagesare normally not dangerous for 3-4 year old seedlings. It means that treating theseedlings with 20% NeemAzal (or higher concentrations) every two weeks during the

237

periods with high rates of feeding (depending on the age of clear cutting area), thedamages and seedling losses could be kept at an aceptable level. Our results suggestthe the efficiency could be improved and the period between treatments prolonged bymixing NeemAzal-T/S with Nu-Film. Studies on the economics of such treatmentsshould be conducted to establish if the repeated treatments with or without Nu-Film areacceptable or too expensive. Good results have been obtained in field tests by Metspaluet al. (2003) and Thacker et al. (2003b), but they used 20 % NeemAzal-T (with 5 %azadirachtin) and undiluted neem oil with 30 % azadirachtin, respectively.

Conclusion

Water emulsion of NeemAzal-T/S in concentration of 5-20 % affected the feedingactivity of the large pine weevil. The magnitude of the effect was dependent onconcentration, as well as on the sex and physiological state of the weevils. Highefficiency was achieved only during a few days after treatment, but after 2-4 weeks thedeep wounds were still small. It seems that the efficacy could be increased mixing theinsecticide with Nu-Film. To achieve an adequate protection of the seedlings, a wateremulsion of 20 % or more NeemAzal should be used every two weeks during theperiods with high rates of weevil feeding.

References

Christiansen, E., Bakke, A., 1968: Temperature preference in adults of Hylobius abietisL. (Coleoptera: Curculionidae) during feeding and oviposition. Zeitschrift frangewandte Entomologie 62: 83-89.

Havukkala, I., Selander, J., 1976 : Reactions of the large pine weevil, Hylobius abietis L.(Col., Curculionidae), to various light and humidity stimuli during three stages ofits life cycle. Ann. Ent. Fenn. 42 : 54-62.

Luik, A., 2000: Die Wirkung von NeenAzal –T/S auf den Fortpflanzungsfrass desgrossen braunen Rüsselkäfers (Hylobius abietis L.). In Kleeberg, H., Zebitz,C.P.W. (eds.): Practice oriented results on use and production of Neemingredients and pheromones VIII. Druck & Graphic, Giessen, Germany. pp.33-37.

Månsson, E. P., Schlyter, F., 2004: Hylobius pine weevils adult host selection andantifeedants: feeding behaviour on host and non-host woody scandinavianplants. Agricultural and Forest Entomology 6: 165-171.

Metspalu, L., Luik, A., Hiiesaar, K., Kuusik, A., Sibul, I., 2003: On the influence of neempreparations on some agricultural and forest pests. In Kleeberg, H., Zebitz,

238

C.P.W. (eds.): Practice oriented results on use and production of Neemingredients and pheromones IX. Druck & Graphic, Giessen, Germany. pp. 85-91.

Malphettes, C. B., Fourgeres, D., Saintonge, F. X., 1994: Untersuchungen ber dieSexualentwicklung der mit Kairomonenfallen gefangenen Weibchen desGrossen Braunen Rsselkfers. Anz. Schdlingskde, Pflanzenschutz, Umweltschutz67: 147-155.

Nordenhem, H., Eidmann, H.H., 1991: Response of the pine weevil Hylobius abietis L.(Col.. Curculionidae) to host volatiles in different phases of its adult life cycle.J.Appl. Ent. 112. 353-358.

Olenici. N., Olenici. V. 2002a: Use of synthetic attractants in monitoring of large pineweevil [Hylobius abietis (L.)] populations. Revista Pădurilor 4: 11-23.

Olenici, N., Olenici, V., 2002b: Protection of conifer seedlings against pine weevil(Hylobius abietis) feeding by dipping into Supersect 10EC and Nu-Film 17 beforeplanting. Bucovina Forestieră 1-2: 25-32.

Olenici, N., Olenici, V., 2003: Differentiation of protective measures against the largepine weevil (Hylobius abietis) attack in coniferous cultures according to the riskof attack. Revista Pădurilor 6: 6-9.

Olenici, N., Olenici, V., 2004: Conifer seedling protection from attack of pine weevil(Hylobius abietis L.) by using of pitfall traps baited with synthetic attractants.Revista Pădurilor 4: 18-23.

Schlyter. F., Marling. E. Löfqvist. J.. 2004: A new microassay for antifeedants inHylobius pine weevils (Coleoptera). Journal of Pest Science 77 (4): 191 – 195.

Stocki. J. S. 2000: The use of pheromones and pheromone traps in forest protection inPoland in the years 1980-1997. In: Kleeberg. H.. Zebitz. C.P.W. (eds.). Practiceoriented results on the use and production of Neem ingredients and pheromonesVIII. Druck & Graphics, Giessen, pp.128-133.

Thacker. J.R.M. Bryan. W.J. 2003a: Use of neem in plant protection in temperateforestry. The Science & Application of Neem, Glasgow, pp. 15-18.

Thacker. J.R.M., Bryan. W.J., McGinley. C. Heritage. S. Strang. R.H.C. 2003b: Fieldand laboratory studies on the effects of neem (Azadirachta indica) oil on thefeeding activity of the large pine weevil (Hylobius abietis L.) and implications forpest control in commercial conifer plantations. Crop protection 22: 753-760.

Zumr. V., Star. P. Dostalkova. I. 1995: Comparison of seasonal responses of Hylobiusabietis (L.) (Col.. Curculionidae) to chemical and natural lures in baited pitfalltraps. Anz. Schdlingskde., Pflanzenschutz, Umweltschutz 68: 166-168.

239


THE POTENTIAL OF EXTRACTS FROM MARINE ALGAE IN THECONTROL OF BLATTELLA GERMANICA L., BLATTA ORIENTALIS L.,MUSCA DOMESTICA L. AND AEDES AEGYPTI L.1

AL. VLADIMIRESCU*, GABRIELA NICOLESCU*, SANDA CHICIOROAGA*, ANA ROSU**

*MEDICAL ENTOMOLOGY LABORATORY, CANTACUZINO INSTITUTE, BUCHAREST

SPL. INDEPENDENTEI 103, P.O. BOX 1-525, 70100 BUCHAREST, ROMANIA

E- MAIL: [email protected]

** PLANT BIOTECHNOLOGY CHAIR, FACULTY OF BIOTECHNOLOGY, BUCHAREST

BD. MARASTI 59, POSTAL CODE 7133, BUCHAREST, ROMANIA

Summary

The effects of the action of 5 extracts from Romanian marine algae in ethanol 96 %(green alga Enteromorpha intestinalis (L.) Link and 4 red algae: Callithamnioncorymbosum (Smith) Lyngb., Ceramium rubrum (Huds) C. Ag., C. elegans (Roth) Ducl.and Polysiphonia denudata (Dillw) Grev ex. Harv.) against Blattella germanica L., Blattaorientalis L., Musca domestica L. and Aedes aegypti L. nymphs or larvae wereinvestigated.

The extracts induced significant mortality and retarded growth and development intreated insects.

The results showed the need for future laboratory investigations regarding the potentialof these seaweeds as sources of allelochemicals against medically important insects.

Introduction

Marine algae are rich sources of important compounds for human health and industry.About 30 species of seaweeds have been proved to be effective for the control ofmedically important insects and the list is not yet completed.

Seaweeds as well as other plant species (Nicolescu et al. 2000) represent a naturalsource to obtain cheap and biodegradable allelochemicals to be used against insects.There is a need to select the species containing such active compounds and to developreliable procedures to extract and process them.

Our study aimed to investigate the action of the extracts from 5 Romanian seaweeds(green alga Enteromorpha intestinalis (L.) Link and 4 red algae: Callithamnion


240

corymbosum (Smith) Lyngb., Ceramium rubrum (Huds) C. Ag., C. elegans (Roth) Ducl.and Polysiphonia denudata (Dillw) Grev ex. Harv.) against Blattella germanica L. andBlatta orientalis L. nymphs, and Musca domestica L. and Aedes aegypti L. larvae.


Extracts

The algae were collected at Saturn and Venus seaside resorts on the Black Sea. Thesewere washed with tap water and extracted with 10 volumes of 96 % ethanol (w/v). Theextracts were decanted from residues.

Bioassays

1 g of ground sugar mixed with 1ml of algal extract was offered, after drying, as food for10 days to 50 first instar nymphs of Blattella germanica or to 10 nymphs of Blattaorientalis of the same instar. The control was given 1g of ground sugar dried after theabsorption of 1 ml of ethanol. The sugar was replaced by normal food (powdered meat,brewer’s yeast, wheat bran, milk powder) after 10 days.

50 two-day old larvae of Musca domestica were placed on to 100 g of normal breedingmedium (wheat bran, milk powder, brewer’s yeast and water) with 1 ml of algal extractin ethanol, to complete their development and emerge as adults.

Groups of 25 late third and early fourth instar larvae of Aedes aegypti were placed in100 ml of breeding medium (distilled water), to which was added 1 ml of algal extract inethanol.

The breeding medium in controls contained 1 ml of solvent only.

The mortality of the insects, the emergence of adults and / or the hatching of theprogeny were recorded.


The results of the action of seaweed extracts against B. germanica and B. orientalis areshown in Table 1.

241

Table 1: The results of the ten days treatments with seaweed extracts on B. germanica and B. orientalisfirst instar nymphs.

The action of seaweed extracts against nymphs of cockroaches induced effectsdetected only over a long period of time.

There was little evidence of direct toxicity, but the mortality in the treated insects,especially in B. orientalis, was significantly higher than in controls at the end of theobservation period. This phenomenon could be the consequence of the effect of growthand development inhibition induced by the algal extracts. In addition, a significantslowing-down growth and development of the treated insects was observed.

The treated B. orientalis nymphs exhibited the same size as the fourth instar nymphsafter 135 days, when the control insects were already adults.

A decrease of the fertility of the adults obtained from treated nymphs was observed inB. germanica. The F1 progeny of the adults obtained from the treated nymphsrepresented only 8.1 % compared to the control progeny in the case of C. rubrumextract and 17 % in the case of C. elegans extract.

The treatment against M. domestica larvae with E. intestinalis extract resulted in a 20 %emergence of adults in comparison with 52 % in the control, and the C. elegans extractentirely suppressed the emergence of adult flies.

The extracts from C. rubrum and P. denudata induced a 100 % mortality of A. aegyptilarvae after 48 hours.

The red algae tested by us proved to be active against medically-important insects. Thisis in accordance with previous results obtained in experiments with other red seaweedextracts from Plocamium telfairaiae (Watanabe et al. 1989, 1990), Pl. cartilagineum(Gonzaletz 1975) and different species of Laurencia (Minott 1988, Watanabe et al.

Seaweedspecies

Mortality (%)

Blattella germanica Blatta orientalis10 days 80 days 10 days 150 days

Enteromorphaintestinalis

32 70 - -

Callithamnioncorymbosum

32 74 10 80

Ceramiumrubrum

30 74 10 80

Ceramiumelegans

25 68 20 100

Polysiphoniadenudata

25 66 0 90

Control 20 42 0 10

242

1989, Williams 1991). Algae belonging to Rhodomelaceae and Ceramiaceae familiesseem to contain important compounds such as polyhalogenated monoterpenes actingagainst pest insects.

While the action of E. intestinalis extracts has already been demonstrated againstmosquito larvae (Thangam and Kathiresan 1992), our results demonstrated the effectsof the extracts from this green alga also against cockroaches and flies.

The extracts of marine algae seem to act as toxicants and also as inhibitors both ofgrowth and development and reproductive capacity of the medically important insects.There is the need for future laboratory investigations on the potential of seaweeds assources of effective allelochemicals against insects.

References

GONZALETZ A.G., ARTEGO J.M., MARTIN J.D., RODRIGUEZ M.L., FAYOS J., RIPOLLIS

M.M. 1975. Two new polyhalogenated monoterpenes from the red algaPlocamium cartilagineum. Phytochem. 17, 947

MINOTT A.D. 1988. Chamigranes from Jamaican Laurencia and an approach tochamigrane synthesis. Ph. D. Thesis University of West Indies Mona, Jamaica,299

NICOLESCU G., CIULACU-PURCĂREA V., VLADIMIRESCU A., HOANCĂ D., ROŞU A.,CHICIOROAGĂ S. 2000. Screening of some Romanian plants for their activityagainst medically important insects. Rom. Arch. Microbiol. Immunol., 59, 3: 227 -236

THANGAM T.S., KATHIRESAN K. 1992. Mosquito larvicidal activity of seaweed extractsagainst Aedes aegypti and Culex quinquefasciatus. International Pest Control35, 4: 94-95

WATANABE K., MIYAKADO M., OHNO N., OKUDA A., YANAGI K., MORIGUCHI K. 1989. Apolyhalogenated insecticidal monoterpene from the red alga Plocamiumtelfairiae. Phytochem. 28, 77

WATANABE K., UMEDA K., MIYAKADO M. 1989. Isolation and identification of threeinsecticidal principies from the red alga Laurencia nipponica. Agric.Biol.Chem.,53, 2513

WATANABE K, UMEDA K., KURITA Y., TAKAYAMA C., MIYAKADO M. 1990. Pesticide.Biochem. and Physiol., 37, 275 - 286.

WILLIAMS L.A.D. 1991. Acaricidal activity of five marine algae extracts on femaleBoophilus microplus (Acari -Ixodidae). Fla. Entomologist 74: 3, 15501

243


EFFECTIFENESS OF AZADIRACHTIN IN CONTROLLING THEHORSE-CHESTNUT LEAF MINER - CAMERARIA OHRIDELLADESCHKA & DIMIĆ (LEP., GRACILLARIDAE ).1

GABRIEL ŁABANOWSKI*, GRAŻYNA SOIKA* & JUSZ ŚWIĘTOSŁAWSKI**

*RESEARCH INSTITUTE OF POMOLOGY AND FLORICULTURE, 96-100 SKIERNIEWICE, POLAND

**R & D LABORATORY “PESTINOVA”, 43-602 JAWORZNO, POLAND

Abstract

Results presented in this paper provide the evidence that azadirachtin applied as aninjection treatment gave an excellent and long-term efficacy against the horse leafmineron Aesculus hippocastanum.

Keywords: Cameraria ohridella, Aesculus hippocastaneum, azadirachtin control

Introduction

The horse-chestnut leaf miner appeared in Macedonia in 1985 and was described byDeschka and Dimić (1986), but in Poland it was noted for the first time on July 7, 1998in Wojsławice n/Wrocław (ŁABANOWSKI and SOIKA 1998). Up to this time it spreadrapidly throughout Europe and it is common in all parts of Poland causing prematurebrowning and leaf-drop.

NeemAzal-T/S (1% azadirachtin) applied in the concentration of 0.3% by a large volumesprayer with the assistance of the fire brigade, showed only moderate efficacy and wasrestricted to the first generation (LEHMANN, 2003 and 2004). First trials with treeinjections using insecticides – NeemAzal-T (5% azadirachtin) or NeemAzal-U (16%azadirachtin) at dose 0.15 g or 0.25 g of active ingredient per 1 cm of trunk diametergave effective control of the first, second and third generation of Cameraria ohridella forat least 23 weeks (PAVELA & BÁRNET, 2004).

The aim of the investigations presented here was the indication of optimal time forapplication of azadirachtin and determination of the most effective formulation.


The research was carried out in 2003-2004 on old specimens of horse chestnut(Aesculus hippocastaneum) in Kalwaria Zebrzydowska n/Kraków (treatments on April10, 2003 and April 5,2004), Łódź (treatments on March 19, 2004 and April 30, 2004)


244

and Oporów n/Kutno (treatments on April 3, 2003 and March 16, 2004). Azadirachtinwas applied as four different formulations: NeemAzal-T/S, NeemAzal-U and Treex Bio020 SL at the same volume, namely, 20 ml formulation per 100 cm of trunkcircumference, but in the different concentrations of active ingredient (Table 1). Twotechniques of application were tested in experiments: a) by drilling and b) by “Chemjet”method. In the drilling method, holes were drilled in the trunks, approximately 1 m abovethe ground at 45ş angle, at every 15 cm, 30 cm or 45 cm around the circumference ofthe trunk. They had a diameter of 8 mm and penetrated 7 cm into the xylem. In the“Chemjet” method 1- 4 holes were drilled, depending of trunk circumference,approximately 1 m above the ground at 90 ş angle. They had a diameter of 5 mm andpenetrated 4 cm into e xylem. Compounds were introduced into xylem initially under apressure of 5 bar. The efficacy of azadirachtin compounds was compared with Gel 20PC and Confidor 200 SL containing imidacloprid. Effectiveness of azadirachtin in thehorse-chestnut leafminer control was estimated in a sample of 40 leaves per tree, first inthe year of application (July or August), and then one year later. At each date leavesfrom untreated trees were also collected. In the laboratory, large mines on leaves, (over0.5 cm length) were counted. Data was evaluated statistically by analysis of variance.

Table 1. Characteristics of compounds injected to trunk trees

* - the dose is given as water suspension – 1 g wettable powder per 10 ml water

Results

In 2003, in Kalwaria Zebrzydowska, NeemAzal-T/S applied in holes drilled every 15 cmof trunk circumference showed good activity against the horse-chestnut leafminer at thehigh level. Efficacy of NeemAzal-T/S was very similar to Gel 20 PC, both in the year of

Trade name andmanufacturecompany

Active ingredient Dose in ml or g per 100 cm oftrunk circumference

of compound of activeingredient

NeemAzal-T/S –Trifolio-M GmbHNeemAzal – U –Trifolio-M. GmbHTreex Bio 020 SL –R & D PestinovaGel 20 PC – BestPestConfidor 200 SL –Bayer CropScience

Azadirachtin A, 1%

Azadirachtin A, 16%

Azadirachtin A+B 20 g/l+ UV filter

Imidachloprid, 2.5%

Imidachloprid, 200 g/l

20,0

20,0*

20,0

18,0

20,0

0,2

0,32

0,4

0,45

4,0

245

application and the following year. (Table 2). In the next year, the same high level ofefficacy was obtained with Treex Bio 020 SL applied by Chemjet in 3 or 4 holes drilledat the distance of over 80 cm. The results of Bioneem 020 SL and Confidor 200 SL inthe reduction of large mines were very similar.

Table 2. Efficacy of azadirachtin in the horse-chestnut leafminer control in Kalwaria Zebrzydowska.

Means within the same column followed by the same letter are not significantly different (P=0.05)according to Duncan’s multiple t-test.

In Oporów, in 2003, NeemAzal-T/S was applied with the same method, at the samedose and at the similar period as in Kalwaria Zebrzydowska, but the results weredifferent. NeemAzal-T/S showed no activity against the horse-chestnut leafminer in theyear of application, nor in the following year (Table 3). Probably the young trees (85 cmof trunk circumference) in contrast to older trees (150 cm of trunk circumference) haddifficulties in translocating the compound to leaves due to its very viscous formulation.In Oporów, in 2004 Treex Bio 020 SL was applied at the dose 20 ml per 1 m of trunkcircumference, but holes were drilled every 15 cm and 30 cm. In both cases the resultsin the year of application were very satisfactory, similar to Confidor 200 SL. However,one year later Treex Bio 020 SL did not show such a good activity against the horse-chestnut leafminer and the results were much poorer than Confidor 200 SL (Table 3).

Method and period ofapplication

Circumfof treetrunk incm

Date of observation

August 24, 2003(T+136 days)

August 13, 2004(one year later)

Number ofmines/leaf

Reductionof minesin (%)

Numberofmines/leaf

Reductionof minesin (%)

Drilling method, holes every15 cm– April 10, 2003NeemAzal T/S – 3 ml/holeGel 20 PC – 2,7 g/holeUntreated trees

150,0220,0

-

4,2 a6,6 a

130,5 b

96,894,9

-

1,3 a2,2 a

37,9 b

96,694,2

-Date of observation

July 7, 2004 (T+93days)

2005(not yet sampled )

Chemjet method, 3-4holes/trunk– April 5, 2004Treex Bio 020 SL – 20 ml/1 mConfidor 200 SL – 20 ml/1 mUntreated trees

262,7243,3

-

3,32 a2,01 a49,5 b

93,395,9

-

---

---

246

Table 3. Efficacy of azadirachtin in the horse-chestnut leafminer control in Oporów n/Kutno

Means within the same column followed by the same letter are not significantly different (P=0.05)according to multiple Duncan’s t-test.

In Łódź, in 2004, the results obtained with azadirachtin as NeemAzal T/S and Treex Bio020 SL applied using drilling method in earlier period (March 19) were good andcomparable with Gel 20 PC. However, reduction of large mines in the year ofapplication obtained with Treex Bio 020 SL was a little higher than it was by NeemAzalT/S (Table 4). The decrease of dose of compounds containing azadirachtin from 20 mlper 1 m of trunk circumference (3 ml of compound per hole drilled every 15 cm trunkcircumference) to 10 ml (3 ml of compound per hole drilled every 30 cm) or even to 6, 7ml (3 ml of compound per hole drilled every 45 cm) did not result in significant increase


Circumf.of treetrunk incm

Date of observation

June 13, 2003 (T+ 70days)

August 8, 2004(one year later)

Number ofmines/leaf

Reduction ofmines in %

Number ofmines/leaf

Reductionof minesin %

Drilling method, holesevery 15 cm – April 3,2003NeemAzal-T/S – 3 ml/holeGel 20 PC – 2, 7 g/holeUntreated trees

85,0270,0

-

90,2 b5,6 a

58,3 b

0,090,4

-

61,3 b4,4 a

49,1 b

0,091,0

-

Date of observationJuly 8, 2004 (T+82

days)August 11, 2005(one year later)

Drilling method, holesevery 15 cm – March 16,2004Treex Bio 020 SL – 3ml/holeConfidor 200 SL – 3ml/holeDrilling method, holesevery 30 cm – March 16,2004Treex Bio 020 SL – 6ml/holeConfidor 200 SL – 6ml/holeUntreated trees

195,0175,0

240,0230,0

-

1,1 a0,3 a

1,2 a1,5 a

112,5 b

99,099,7

98,998,7

-

31,60,0

27,8

247

of leaf damage. Similar results were obtained with NeemAzal T/S and Gel 20 PC oneyear after application (Table 4).

Both azadirachtin compounds – NeemAzal-U and Treex Bio 020 SL applied by drillmethod in later period (April 30), also gave good results in the horse-chestnut leafminercontrol, in both the year of application and the following year (Table 4).

Table 4. Efficacy of azadirachtin in the horse-chestnut leafminer control in Łódź

Remark: means within the same column followed by the same letter are not significantly different(P=0.05) according to multiple Duncan’s t-test.


Circumf.of trunktrees incm

Date of observation

July 8, 2004 (T+82days)

July 29, 2005(one year later)

Number ofmines/leaf

Reductionof mines in(%)

Number ofmines/leaf

Reductionof mines in(%)

Drilling method, holes every 15cm – March 19, 2004 NeemAzal-T/S – 3 ml/holeTreex Bio 020 SL – 3 ml/holeGel 20 PC – 2, 7 g/holeDrilling method, holes every 30cm – March 19, 2004NeemAzal-T/S – 3 ml/holeTreex Bio 020 SL – 3 ml/holeGel 20 PC – 2, 7 g/holeDrill method, holes every 45 cm– March 19, 2004NeemAzal-T/S – 3 ml/holeTreex Bio 020 SL – 3 ml/holeGel 20 PC – 2,7 g/holeUntreated trees

130,0111,7187,7

123,3173,7130,0

202,5135,0116,7

-

2,9 ab0,5 a0,9 a

8,4 b2,3 ab1,0 a

3,4 ab1,3 a0,4 a29,5 c

90,298,397,0

71,592,296,6

88,595,698,6

-

2,5 b-

0,8 a

2,8 b-

1,6 ab

4,8 b-

0,4 a40,0 c

93,8-

98,0

93,0-

96,0

88,0-

99,0-

Date of observationJuly 8, 2004 (T+82

days)July 29, 2005

(one year later)Drilling method, holes every 15cm – April 30, 2004NeemAzal-U – 3 ml/hole Treex Bio 020 SL – 3 ml/holeGel 20 PC – 2,7 g/holeChemjet method, 1-2 holes/trunk – April 30, 2004NeemAzal-T/S – 20 ml/1 mTreex Bio 020 SL – 20 ml/1 mConfidor 200 SL – 20 ml/1 mUntreated trees

131,0135,0185,0

80,570,492,8

-

3,5 b0,0 a3,5 b

26,7 c0,5 ab1,7 b29,5 c

88,1100,088,1

9,598,394,2

-

1,3 b0,7 a1,9 b

2,1 b0,2 a0,4 a40,0 c

96,898,395,3

94,899,599,0

-

248

Compounds applied by Chemjet to the young trees (trunk circumferences less than 100cm) in one or two holes drilled every 80 cm or less of trunk circumference gave goodresults except NeemAzal-T/S in the year of application. It was affected by the very slowtransportation of NeemAzal-T/S in xylem. The time of injection for this compound wastwice as long as Treex Bio 020 SL or Confidor 200 SL, both formulations are solubleliquids. One year after application all products showed very high efficacy against thehorse-chestnut leafminer.

Conclusions

All the three formulations containing azadirachtin applied as injection treatment, bothdrilling and Chemjet methods in March-April protected trees against the horse-chestnutleafminer in the year of application, and they also showed very good activity one yearafter treatment.

Azadirachtin dose of 20 ml of product per 1 m of trunk circumference, independently ofapplication method, is enough to control the horse-chestnut leafminer effectively.

References

LEHMANN M., 2003. Bekämpfung der Kastenienminiermotte mit NeemAzal-T/S.Nachrichtenbl.Deut. Pflazenschutzd., 55(10): 237-239.

LEHMANN M., 2004. Control of the horse chestnut leaf miner with NeemAzal T/S.Abstracts 1st International Cameraria Symposium – Cameraria ohridella andother invasive leaf-miners in Europe. IOCB Prague, March 24-27, 2004: 25.

ŁABANOWSKI G., SOIKA G., 1998. Szrotówek kasztanowcowiaczek zagrażakasztanowcom w Polsce. Ochrona Roślin, 12: 12.

PAVELA R., BÁRNET M., 2004. Systemic applications of bioinsecticides for control ofAesculus hippocastanum against Cameraria ohridella. Abstracts 1st InternationalCameraria Symposium – Cameraria ohridella and other invasive leaf-miners inEurope. IOCB Prague, March 24-27, 2004: 39.

249


EXPERIMENTS ON THE USE OF NEEM (A. INDICA)-DERIVEDPRODUCTS FOR CONTROL OF THE BLACK CHERRY APHID, MYZUSCERASI FAB. (HOM., APHIDIDAE) IN A CHERRY ORCHARD INBULGARIA1

RADEVA, K.*, NIKOLOV, P.**

* AGROBIOCONSULT, BULGARIA,

** NSPP, BULGARIA

Introduction:

The Black Cherry Aphid is a serious pest in commercial cherry production, especially inyoung cherry orchards and seed-beds. It spoils the buds, the young leaves and thedeveloping annual shoots in early spring. Especially serious is the damage on theannual shoots, which bend, bear no fruit and suffer frost-damage in winter.

Knowing the type and timing of damage caused by the aphid, we have studied thepossibilities of the use of the neem-derived products and their place in the technologyfor integrated pest management in the production of cherries.

Materials and Methods:

The observations and the trials were carried out during the period 2001-2003 in thevillage of Peshtera, region of Kiustendil. The chosen cherry orchard was established in1997-1999 in two stages. It has an extremely favourable location. The rows are wellcultivated and the inter-row space is grassed and well-maintained. For the purpose ofthe studies reported here, the orchard was divided into two sections, each of whichreceived different treatments (“Variants”). Variant I, with an area of 2000m2 was treatedwith the neem products (1% and 2% neem oil, and NeemAzal-T/S) and the other part(Variant II) was treated conventionally (preparations approved for use by the stateauthorities, detailed below in Table 2).

Before starting the experiments, in the year 2000, pest infestation of the orchard wasmerely observed, in order to determine the key pests and to estimate whether thecherry aphid really represented as serious a problem as the owners claimed.

After this the trials started in the year 2001 as described above. Sprayings of variousplant protection products in both experimental variants (I and II) were carried outsimultaneously.


250

In both variants we used yellow strips (“Celaflor” type) for catching of the aphid andquantification. Apart from the coloured strips we made observations also of themovement and of finding of damaged annual shoots. We also observed young annualshoots.

Although the aphid was the main subject of the experiments, we also madeobservations of the Cherry Fly (Rhagoletis cerasi) which were trapped using strips of“Rebel” type.

The effectiveness of the various treatments was determined according to the Abbottformula.

Results:

The results of the measures to protect plants in both variants are shown in detail inTables 1 and 2.

The number of insect-damaged annual shoots and the density of the colonies of theblack cherry aphid are also indicated.

As may be seen from the tables, in Variant I, 2 treatments were carried out against theaphid with the neem products early in 2001, with an interval of 10 days. Thesetreatments reduced sharply the density of the pest and there was no need of thirdtreatment.

In this variant we observed also an extremely low infestation level of the Cherry Fly too,which meant that no specific treatment was required for this pest.

The density of the aphid started to increase again in the beginning of August, when wedid a new spraying. Subsequent observations failed to find an increase of pest density.

In Variant II we did treatments against the aphid involving the use of syntheticinsecticides. In this version, unlike the Variant I, the density of the cherry fly requiredone more treatment in order to protect the crop. In the beginning of August we carriedout one more vegetation treatment against the cherry aphid, reporting its appearanceand its autumn multiplication.

The plan for neem-products observation went on also with their application for winterspraying with neem-oil (1% and 2%) directed against hibernating eggs of the blackcherry aphid. For the conventional variant we used the winter oil (Parasomer) also in1% and 2%.

During 2002 and 2003 we continued the work with this plan by observing the need forspraying depending on the density and the development of the pest. As it may be seenfrom Variant II, during each of the three years it was necessary to spray against thecherry fly too.

251

Table 1 Damaged annual shoots and density of the aphids for the period 2001-2003. (Treatment VariantI)

Table 2 Damaged annual shoots and density of the aphids for the period 2001-2003. (Treatment VariantII)

Neem-productsPlant Protection

ProductDate of

treatmentNumber ofdamaged

annual shoots

Number ofaphid per

colony (±SD)

Effectivness(%)

NeemAzal–T/S 23.04.2001 32 65±4 92NeemAzal–T/S 03.05.2001 49 83±2 94.6NeemAzal–T/S 02.08.2001 24 22±5 99

Neem-oil 2% 07.01.2002 - - -Neem-oil 1% 16.03.2002 - - -NeemAzal–T/S 29.04.2001 14 33±2 100NeemAzal–T/S 10.08.2002 4 16±2 100

Neem oil 2% –Trifolio-S forte

11.02.2003 - - -

Neem oil 1% 19.02.2003 - - -NeemAzal–T/S 18.04.2003 3 12±4 -

Conventional protectionPlant Protection

ProductDate of

treatmentNumber ofdamaged

annual shoots

Number ofaphid per

colony(±SD)

Effectivness(%)

Bi-58 23.04.2001 35 69±3 94Karate 2.5EK 03.05.2001 52 79±2 91Decis 2.5EK 29.05.2001 Treatment against cherry flyKarate 2.5EK 02.08.2001 29 42±6 87

Parasomer 2% 07.01.2002 - - -Parasomer 1% 16.03.2002 - - -Pyrimor 29.04.2002 39 74±2 83Karate 2.5EK 12.05.2002 34 46±2 86Fastak 26.05.2002 Treatment against cherry flyFastak 10.08.2002 11 44±3 92

Parasomer 1% 11.02.2003 - - -Parasomer 2% 19.02.2003 - - -Fastak 18.04.2003 24 48+4 92Decis 2.5EK 22.05.2003 Treatment for cherry flyBi-58 06.08.2003 12 22+3 88

252

Conclusions:

The application of neem-products was highly effective against the Cherry Aphid over thewhole growing season. As an incidental benefit, we also observed the reduction inCherry Fly population also. This pest was inadequately controlled by conventionalsynthetic insecticides (Variant II). The long-term effect of the neem-derived productswas due to their preventing normal larval development and their sterilizing effect.

Another feature of the results was that fewer treatments were necessary in Variant I toreduce greatly the level of infestation, an observation which has both economic as wellas ecological benefits.

The results we obtained exceeded our expectations regarding the effectiveness of theneem plant protection products in achieving orchard protection against the two pestsstudied.

Based on all this, we strongly recommend this application strategy in the technologiesfor integrated and biological production of cherries with the application of theneem-products.

253


THE POTENTIAL OF NEEMAZAL BAITS IN THE CONTROL OFBLATTELLA GERMANICA L. AND MUSCA DOMESTICA L.1

GABRIELA NICOLESCU*, AL. VLADIMIRESCU*, ANA ROSU**, VALERIACIULACU-PURCAREA*, SANDA CHICIOROAGA*

*MEDICAL ENTOMOLOGY LABORATORY, CANTACUZINO INSTITUTE, BUCHAREST – ROMANIA,SPL.INDEPENDENTEI 103, P.O. BOX 1-525, 70100 BUCHAREST, E-MAIL: [email protected]

**PLANT BIOTECHNOLOGY CHAIR, FACULTY OF BIOTECHNOLOGY, BUCHAREST – ROMANIA, BD.MARASTI59, COD 7133

Summary

Different volumes (1.0, 0.5 and 0.25 ml) of NeemAzal (Trifolio-M GmbH, Germany)mixed with 1 g ground sugar were given as food for 7 days to Blattella germanica firstinstar nymphs, and for 3 days to newly emerged Musca domestica adults.

The mortality (96 - 100 %) of treated nymphs of B. germanica was observed over 28 -40 days, the survival being shorter at higher dosages. The growth and developmentwere slowed-down, the treated insects remaining as second instar nymphs whileuntreated ones reached the fifth instar.

Mortality of 22 - 38 % in M. domestica treated females were observed after 12 days. NoF1 adult survived among treated females.

The results show the potential of NeemAzal to be used in baits against flies andcockroaches.

Introduction

NeemAzal is a concentrated and standardized extract from the neem seeds(Azadirachta indica A. Juss.) containing a group of terpenoids named Azadirachtins asactive compounds against different insect species. NeemAzal-T/S is a plant protectionformulation which contains 1 % Azadirachtin A (Tro et al. 1998).

Our study aimed to investigate the possibility of using NeemAzal-T/S in baits againstsome medically-important insects such as adult house fly (Musca domestica L.), andGerman cockroach (Blattella germanica L.) nymphs.


254


B. germanica

1 g of ground sugar mixed with different volumes of NeemAzal-T/S (3 ml, 1.5 ml and0.75 ml) was offered as food for 7 days to the first instar nymphs of B. germanica. Thecontrol was given 1 g of untreated ground sugar. The treated sugar was replaced bynormal food (powdered meat, brewer’s yeast, wheat bran, milk powder) after 7 days.The insects were observed until the total extinction of the treated ones.

M. domestica

50 adults (sex ratio 1:1) of Musca domestica one day after emergence were placed for 3days in the presence of 1 g of ground sugar mixed with different volumes of NeemAzal(3 ml, 1.5 ml and 0.75 ml). The control was given ground sugar only. After 3 days, allthe insects were fed with regular food (powdered milk and sugar). When the flies were12 days old, the surviving females were placed for one hour in the presence of smallquantities of larval breeding medium (wheat bran, brewer’s yeast, milk powder andwater) to lay eggs. In every experiment the eggs were placed to breed in 100 g of thesame medium. The mortality of the insects, the emergence of the adults and / or thehatching of progeny and its development were observed.


The action of NeemAzal-T/S against nymphs of German cockroaches resulted in theextinction of the treated insects before their emergence as adults (Table 1)

Table1. The mortality of B. germanica nymphs observed after a treatment of 7 days with NeemAzal- T/Sin food supplied to newly hatched first instar nymphs.

The intervals of reaching the 100 % mortality of the nymphs were in direct relation to thequantities of neem preparation administered; thus, the higher dose induced 100 %nymphal mortality in 28 days and the lower one in 42 days.

The pattern of nymphal mortality during the experiments showed two peaks: the firstone covered a period of 8 - 10 days (including the 7 days of treatment) with 92 %mortality at higher dose and 32 - 34 % at two lower doses; after a period of low mortality

Cumulative mortality (%) after different intervals (days)NeemAzal-T/S

doses (ml) 10 days 20 days 28 days 31 days 40 days 42 days3 92 92 100 - - -

1.5 34 48 78 98 100 -0.75 32 44 70 92 98 100

Control 0 0 2 2 4 4

255

(0 - 12 %) between the 10th - 20th day, the second peak of mortality was recordedbetween the 21st and 31st day, with values of 46 - 50 % of the mortality at the two lowerdoses. In the experiment with 3ml of NeemAzal-T/S, after the high initial peak ofmortality, only 8 % of the nymphs survived and they died at the end of the period of 28days. Although they survived for 28 days, these nymphs were still in the first instar whilethe control nymphs reached the third instar. Also, the nymphs treated with 1.5 and 0.75ml of NeemAzal were still in the first or second instar when they died after 40 andrespectively 42 days, while the control nymphs had already reached the fifth instar.

The analysis of these data suggests that the first peak of nymphal mortality could beessentially the result of the direct toxicity of neem preparation and the level of mortalitywas correlated to the increase of the dose.

The subsequent mortality recorded during a quite long period of time after thetreatments could be rather the result of the typical insect growth regulatory effectsmaterialized in slowing-down the growth, delayed moulting and inability to completeecdysis. But the few surviving nymphs, especially those which remained very small insize even after about 40 days, all these effects could be at least partly due to theantifeedancy effects already observed in many insect species (Mordue (Luntz) etBlackwell, 1993).

The action of NeemAzal-T/S in food on B. germanica nymphs is comparable to theaction of a synthetic analogue of juvenile hormone observed in some of our previousinvestigations on the same species (Nosec et al. 1977, Cristodorescu et al. 1977, 1978).

The action of different doses of NeemAzal-T/S against M. domestica adults produced amortality of 28 - 52 % during a period of 12 days, their levels being in directlyproportional to the concentration of the active ingredient (Table 2).

Table 2: The mortality after 12 days of adult flies treated after their emergence with NeemAzal-T/S infood for 3 days.

The mortality in females were 2.7 - 3.7 times higher than in males, probably due to theingestion of greater quantities of treated food by the females with developing eggs inspite of likely antifeedancy effect.

Mortality ( %)NeemAzal-T/S

doses (ml)Females Males Total

3 38 14 521.5 30 10 400.75 22 6 28

Control 0 0 0

256

The treated females laid small batches of eggs compared to the controls, and a verysmall number of larvae hatched from them (about 10 - 15 larvae in every experiment incomparison with several hundred in the control). They were quite inactive, and after 3 -4 days no movements at all were observed.

About 10 dead and abnormally chitinized larvae of 1 - 6 mm length were recovered ineach experiment in comparison with several hundred of normal puparia in the control(from which several hundred of adult flies subsequently emerged).

It is obvious that NeemAzal-T/S has a strong action on reproductive capacity of treatedadult flies, resulting in reduced number of eggs laid, very low rate of larval hatching andalso the total failure of these F1 larvae to grow and develop.

The results of our investigation have shown the potential of NeemAzal-T/S to be usedas food baits in the control of B. germanica and M. domestica.

References

Cristodorescu G., Nosec I., Tacu V. (1977). The action of juvenile hormone analogue, 1- (4`- ethylphenoxy) - 6,7 - epoxy - 3,7 - dimethyl - 2 - octene on the last nymphalinstar of Blattella germanica (Note II). Arch. Roum. Path. exp. Microbiol., 36, 1,73 – 78

Cristodorescu G., Nosec I., Tacu V. (1978). The action of juvenile hormone analogue, 1- (4`-ethylphenoxy) - 6,7 - epoxy - 3,7 - dimethyl - 2 - octene in Blattellagermanica by continuous treatment (Note II). Arch. Roum. Path. exp. Microbiol.,37, 1, 47 – 53

Mordue Luntz A. J., Blackwell A. (1993). Azadirachtin: an update. J. Insect. Physiol., 39,903 – 924

Nosec I., Cristodorescu G., Tacu V. (1977). The action of juvenile hormone analogue, 1- (4`- ethylphenoxy) - 6,7 - epoxy - 3,7 - dimethyl - 2 - octene on the Blattellagermanica nymphal instars (Note I). Arch. Roum. Path. exp. Microbiol., 36, 1, 67– 72

Tro R., Bernauer – Jacob V., Hummel Ed., Kleeberg H. (1998). Azadirachtin A: Contentand Bio – efficacy in Hair treated with NeemAzal Formulations. In PracticeOriented Results on Use and Production of Neem – Ingredients andPheromones VII., H. Kleeberg (ed.) ISBN: 3 – 925614-22-2, 9 - 20

257


PRELEMINARY RESULTS OF USING OF NEEMAZAL-T/S AGAINSTLARVAE OF THE BUFF-TIP MOTH (PHALERA BUCEPHALA L.LEPIDOPTERA, NOTODONTIDAE) IN THE LABORATORY1

GNINENKO Y.

RUSSIAN RESEARCH INSTITUTE OF SILVICULTURE AND MECHANIZATION OF FORESTRY. PUSHKINO,MOSKOW REGION, RUSSIA. EMAIL: [email protected]

Introduction

The caterpillars of P. bucephala were first observed in a large infestation in southRussia in 2nd half of the 20th century. Subsequent outbreaks of its population were lessserious (Gninenko, 2002). P. bucephala L. is perennially present now in most regions ofNorth Asia and West, Central and East Europe and is a noted pest of oak in both inforest and public recreation areas. (Zashev, Keremidchiev, 1968; Zlatanov, 1971;Marushina et al, 1971).

In West Siberia and North Kazakhstan P. bucephala L. lives mainly on birch and it is onthese trees that it has occasional outbreaks (Fedoryak, 1974). There have, however,been few studies on the biology and ecology of this moth in the Siberian part of itsnatural habitat (Nikitina, 1989).

Normally, the bacterial insecticide Lepidocide has been employed against caterpillars P.bucephala. In 2006 there was started a small preliminary study with NeemAzal-T/S, abotanical plant protection product produced by Trifolio-M GmbH, Germany. Thispreparation is made from Indian Neem tree Azadirachta indica, and has already beenwidely tested in plant protection (Suri, Mehrotra, 1994, Kaul, Sharma, 1999, Hummel etal, 2006, Gopinathan et al 2007).

Methods and Materials

The eggs of P. bucephala L. were collected in the Moscow region, and caterpillars werereared under laboratory conditions. On 11th August 2002 specially prepared branches oflime (linden) tree (Tilia spp) were placed in water vessels and were sprayed withsuspension of NeemAzal-T/S containing 1% azdirachtin, the active ingredient. Controlswere sprayed only with water. After drying, every branch was infected with 20caterpillars of P. bucephala L. in 3rd instar, and placed into an insect breeding cage.


258

The feeding activity of the caterpillars was assessed three days after application bycollection and weighing of excrements, and mortality was assessed 3 days later, whenno fresh excrement was found.

The leaves of lime branches collected were slightly infected by leafminer Phylonorycterissikii (Lepidoptera, Gracillaridae) too, and it was impossible to eliminate it. Thus theexperiments were done with both pests, because the mortality of miner caterpillars wasevaluated as well.

Results and Discussion

NeemAzal-T/S did not produce an immediate antifeedant effect. On the contrary, duringthe first three days after application the larvae in treated variants ate more leaf massthan in the control, and so produced a greater mass of excrement (Table 1).

Table 1. Feeding activity of caterpillars P. bucephala L. estimated by weight of excrement.

Only from the fourth day after treatment of NeemAzal-T/S was the feeding activity ofcaterpillars drastically reduced, and the first dead larvae were observed. By the sixthday of the experiment, all larvae in NeemAzal treatments had died. There were nodeaths among the control insects (Table 2).

Table 2. Mortality of caterpillars P. bucephala L. on 6th day after NeemAzal-T/S application

NeemAzal-T/S is an insecticide without contact action. By insect larvae it inhibits ofchitin synthesis and so the mode of action is similar to Dimilin. In the trials was foundwhen caterpillars stopped feeding initially they appeared to be paralyzed, then theirbody length diminished. Mortality started 1-2 days after paralysis, but at the same timethere were no chitin breaks.

Assessment of efficacy of NeemAzal-T/S against lime-tree leafminer P. issikii showedthat most caterpillars in mines were dead on 6th day after treatment (Table 3). However,

Treatment Mass of excrements (% of control)3 days 4 days 5 days 6 days

NeemAzal(1) 117,86 65,61 28,01 0NeemAzal (2) 134,96 48,94 31,2 0Control 100 100 100 100

Treatment Number of caterpillars in cage Dead caterpillars, %NeemAzal (1) 20 100NeemAzal (2) 20 100Control 20 0

259

a significant proportion of caterpillars in control died as well, which may indicate that P.issikii caterpillars are very sensitive to foliage condition changes.

Table 3. Mortality of caterpillars of leafminer P. issikii in mines after NeemAzal-T/S application

ConclusionsIn a laboratory experiment, NeemAzal-T/S showed a high insecticidal effect againstcaterpillars P. halera bucephala L. from 4th day after application. It may berecommended for registration field trials against leaf and needle-eating pests in Russia.Experiments with leaf miner P. issikii should be continued.

Reference

Gninenko Yu.I. Lunka serebristaya (Phalera bucephala L., Lepidoptera: Notodontidae) vbereznyakah Zapadnoy Sibiri I Severnogo Kazakhstana. // Nauka za gorata v..3-4, 2002. - pp. 95-101 (in Russian).

Gopinathan M.C. Neem research, product development and commercialization at globallevel – an Indian experience. // BioEco 2007, Bioresourse and Biodiversity. June27-28, 2007, Tianjin, P.R. China 2007/ - pp. 21 – 40.

Fedoryak V.E.. Lunka serebristaya – jpasnyy vreditel’ lesov Severnogo Kazachstana //Zashchita rsateniy, 1973, №3. – p. 50. (in Russian).

Hummel E. Vozmozhnost’ primenenia rastitel’nogo insrkticida NeemAzal-T/S vzashchite rasteniy. // Zashchita rasteniy. Strategiya I taktika zashchity rasteniy.vol. 30, t. 1, Minsk, 2006. – pp. 515-517. (in Russian)

Kaul B.K., Sharma P.K. Efficacy of Neem based insecticides against the major insectpests of rice in the hill of Himachal Pradesh (India)/ // Journal Entom. Res., 1999,23 (4), p. 337.

Marushina N.G. Lunka serebristaya (Phalera bucephala L.) v Rostov region // Voprosyzashchity lesa, t. 38, М., MLTI, 1971.- pp. 83 – 95. (in Russian).

Nikitina S.I. Biologia lunki serebristoy (Lepidoptera, Notodontidae) na yuge ZapadnoySibiri.// Fauna, ekologiya I zoogeografiya pozvonocznych I czlenistinogich.“Nauka”Novosibirsk, 1998. – pp. 161 – 167. (in Russsian).

Suri R.K., Mehrotra A. Neem (Azadiracta indica A. Juss) a wonder tree. D.K. PublishingHouse. A House of Forestry, Environment & Scientific Books, 1994, Bhopal.

Treatments Number of mines on foliage Proportion of dead caterpillars( %)NeemAzal(1) 15 93,3NeemAzal(2) 14 100,0Control 16 62,5

260

Zashev B., Keremedchiev M. – Atlas na gorskite nasekomi. Sofia, 1968. – 273 pp. (inBulgarian).

Zlatanov S. Nasekomni vrediteli na daba. // BAN, Sofia 1971, - 250 pp. (in Bulgarian).

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EXPERIMENTS ON THE USE OF THE MELIACEOUS PLANT PRODUCTS,NEEMAZAL-T/S AND NEEMPRO TEX, AGAINST THE EUROPEANSPRUCE BARK BEETLE, IPS TYPOGRAPHUS (COL., SCOLYTIDAE)1

KREUTZ, J.*; ZIMMERMANN, G.*; VAUPEL, O.**

* FEDERAL BIOLOGICAL RESEARCH CENTRE FOR AGRICULTURE AND FORESTRY, INSTITUTE FORBIOLOGICAL CONTROL, DARMSTADT, GERMANY.

** HESSIAN FOREST SERVICE CENTRE FOR FOREST MANAGEMENT, INFORMATION AND FOREST RESEARCH,HANN. MÜNDEN, GERMANY.

Introduction

The European spruce bark beetle, Ips typographus, is considered to be one of the mostdestructive pest insects of spruce in Europe. In the past, hot and dry summers andheavy storms causing falling of trees, often resulting in a rapid increase of populationsof this pest in spruce forests. There are various control measures in use, i.e. (1) fellingof infested trees and elimination of the bark, (2) use of pheromone traps and (3)chemical treatment of logs and branches. In addition, biological control methods like theuse of entomopathogenic fungi, e.g. Beauveria bassiana, or of microsporidia, e.g.Gregarina typographi and Chytridiopsis typographi, are under investigation.


As a new control measure, we investigated the effect of the two neem products,NeemAzal-T/S and NeemPro Tex, on bark beetle populations in spruce logs ingreenhouse experiments. Extracts from seeds of the neem tree, Azadirachta indica, arewell-known natural insecticides. The main active ingredient is azadirachtin, whichaffects the insect growth, the feeding, the behaviour and the reproduction. In ourexperiment, a spruce log (50 cm length, 20 cm diameter) was sprayed each with 1%NeemAzal-T/S and 1% NeemPro Tex. In the control, the logs were sprayed with wateronly. After drying, each log was colonised with 110 bark beetles, freshly captured inpheromone traps, and then stored in wooden and glass cages (58 x 58 x 35 cm) at25°C, 50% RH and a photoperiod of 16:8 L:D, in a greenhouse. After 7 weeks, the barkof all logs was removed, and the beetle galleries, and the number of living and deadlarvae, pupae and adults of I. typographus were examined. The experiment wasrepeated five times.


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Results

The results showed that both Neem products had a significant effect on the formation ofbreeding galleries of I. typographus as well as on the number of living individualscompared to the control. In NeemAzal-T/S, the number of bore-holes was reduced by58%, the breeding chambers by 39%, the mother and larval galleries by 40% and 63%,respectively and the pupal chambers by 93%. There also was a significant reduction of62% in the lengths of the remaining mother galleries compared to the control. Thenumber of adults and pupae in the galleries were reduced by about 80% and the larvaeby 100%. The mortality of adult beetles after the treatment was 79%, and significantlyhigher than in the control (18%). In the NeemPro Tex treatment, similar effects and aslightly higher efficacy were noticed with a significant reduction in the number andlength of the breeding galleries and the number of living individuals compared to thecontrol logs.

The results demonstrate that both neem products were highly effective against theEuropean spruce bark beetle, I. typographus after spraying of spruce bark. Furtherinvestigations on the treatment of bark in trees and the injection of neem products underpractical conditions are suggested.

263


EXPERIENCE WITH MODE OF ACTION OF NEEMAZAL-T/S ANDTRIFOLIO S-FORTE FORMULATIONS AGAINST THE SPIDER MITE(TETRANYCHUS ATLANTICUS MCGREGOR) UNDER LABORATORYCONDITIONS1

S. YA. POPOV*, E. HUMMEL**

* RUSSIAN STATE AGRARIAN UNIVERSITY – MOSCOW TIMIRYAZEV AGRICULTURAL ACADEMY, DEPT. OFCHEMICAL PLANT PROTECTION, MOSCOW, RUSSIA, EMAIL: [email protected]

** TRIFOLIO-M LTD., LAHNAU, GERMANY, EMAIL: [email protected]

Introduction

The Atlantic Spider Mite (Tetranychus atlanticus McGregor), one of the most dangerouscosmopolitan species of spider mites present in the Euro-Asiatic continent, in NorthAmerica and the Near East, has been found on grass plants in many regions of Russia,in Hungary and Bulgaria (Popov, 1988, 1994). Russian acarologists (Mitrofanov et al.,1987; Popov, 1988; 1994) define this spider mite as a separate species, while WesternEuropean and American taxonomists consider it identical to the Turkestan Spider Mite(Tetranychus turkestani (Ugarov and Nikolski), although the two species can bedistinguished with ease according to morphologic characteristics.. The biologicalparameters of T. atlanticus and T. turkestani populations are very similar to theTwo-spotted Spider Mite (Tetranychus urticae Koch).

NeemAzal-T/S is a biological preparation with insecticidal and acaricidal activity,extracted from fruits of Indian Neem Tree (Azadirachta indica) growing in India whichhas already been used in plant protection throughout the world (Suri, Mehrotra, 1994;Kaul, Sharma, 1999; Hummel et al., 2006; Gopinathan et al., 2007 This paper reportsthe results of laboratory trials aimed at investigating the mode of action and efficacy ofNeemAzal-T/S and Trifolio S-forte against the mite T. atlanticus.


In experiments during 2006 one application of NeemAzal-T/S (“NATS”) and therecently-developed Trifolio S-forte (“TSf”) were applied separately and in combination(“NATS + TSf”) in the 0,5% spray solution against different developmental stages of T.atlanticus in 4-10 replications (leavess). In different tests, one variant and control had48-50 females or 80 – 282 juvenile individuals.


264

The spider mites were placed on leaf cultures of bean, strawberry and currant in Petridishes and the neem formulations were applied by means of an "Automatic PotterSpray Tower" (Burkard, UK) with approximately 80 drops of spray solution/cm2, which isequivalent of 500 l water/ha. In the control variant (“C”) only water was used. After theapplication the test plant cultures were placed in the climatic chamber maintained at 25or 27±1°C, 65-80% RH and 16:8 h (L:D) photoperiod to observe of development of themites during next 10-12 days. The biological effect of the treatment was based oncumulative values of mortality compared to controls.

Experiment 1. Topical effect on the eggs

For the experiment on egg-laying, females of T. atlanticus were allowed to lay eggs for24 h on leaf cultures (Fig. 1). Before treatment, no differences were found in the fourtest variants. The numbers of eggs laid were: “C”: 145; “NATS”: 159; TsF: 269;NATS+TsF: 194.

Figure 1. Passive (from leaf to leaf culture) of new leaf cultures with Spider mite from the rearing.

Experiment 2. Topical effect on the juvenile stage (larvae and nymphs)

For this trial every strawberry leaf culture was infected with spider mite one day beforethe treatment passively (leaf with mites to leaf) (Fig.1) with 12-20 individuals, so thataltogether “C”:152; "NATS": 180: "TSf": 282: "NATS+TSf": 252, individuals were found.

Experiment 3. Contact/enteric effect on the juvenile postembryonic stages aftertreatment of plants only.

In this test the contact/enteric effect of the preparations against postembryonic stages ofSpider mite was examined. The leaf bean cultures only were treated. After drying of thespray solution on leaves (1 h) the mobile juvenile individuals of mites were

265

infected/colonized as described in the experiment 1. At the beginning of the experimentthe leaf cultures had: “C”: 166; “NATS”: 124; “NATS+TsF: 91 mobile individuals.

Experiment 4. Translaminar effect on the females after treatment of plants only

In this experiment detached currant leaves, the most viable among other plants (S.Ya.Popov, 1981), were used. This method of spider mites cultivation on currant originallywas developed by S.Ya. Popov.

In the trial only the upper side of currant detached leaves was treated. The leaves,lower side uppermost, were placed on the dry surface of a vessel containing water (Fig.2). The upper surface of the container had holes for the petioles, so that leaves werekept fresh throughout the experiment. On top of each leaf was placed a plastic plate,with holes bored to act as pest “cages”. Into each of these cages was placed 5 femalemites, by means of a fine brush. Application of petroleum jelly to the edges of theseholes prevented any escape of the mites. At the beginning of the experiment a total of48 – 50 females in each variant and control were used. The insects were observed overa period of 13 days.

Figure 2. Translaminar test method

Experiment 5. Topical effect on the females only

Female mites were first directly treated with spray solution on filter paper and thereaftertransferred by brush into cages on the untreated strawberry leaves. The numbers usedwere: “C”: 38; “NATS”: 37; “TSf”: 36: “NATS+TSf”: 35 individuals.

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Experiment 1. Topical effect on the eggs

The first dead eggs were found 3 days after application, and after that the mortalityincreased dramatically (Fig. 3). The mortality of eggs on the 5-th and 9-th dayrespectively reached: in variant “NATS” 19 and 58,3%, in variant “TSf” 27% and 76,5%;but the highest mortality was in the combination of both preparations: 23% and 83,4%.Other species, such as Whitefly, do not show this ovicidal effect of neem-containingformulations. (von Elling et al, 2002).

During the observation period it was also noted that as well as the ovicidal effect thepreparations also killed the unborn larvae in the eggs. so that at the seventh day in“NATS” 23 eggs as well as 44 dead larvae were found, and 2 days later the numberswere 37 and 58 respectively. Altogether in this variant at the 9/10 day observation38.9% of the larvae in the "egg stage", in "TSf" were already killed 29.3% and"NATS+TSf" 49,7%. In control cultures during the nine-day observation period only 5dead eggs from 145 were found, and there were no dead larvae.

Figure 3. Mortality of the eggs after topical treatments.

Experiment 2. Topical effect on the juvenile stage (larvae and nymphs)

Biological effects of the applied preparations presented in Fig. 4, showed a mortality oflarvae and nymphs of the Atlantic spider mite above the control already 1 day after

Experiment 1

0

20

40

60

80

100

1 3 5 7 9

Day after treatment

Mor

talit

y, %

C (145 eggs) NATS (159 eggs) TSf (269 eggs) NATS+TSf (194 eggs)

267

treatment. The mortality in all variants was steadily increased, until at the 10th day in“NATS” it was 61%, in “TSf”, 56,5% and in the combination, 85,6%. In the controlpopulation in the same time only 1 individual from 152 was dead.

Figure 4. Mortality of the juvenile stage after topical treatment.

Experiment 3. Contact/enteric effect on the postembryonic juvenile developmentstages after treatment on plants only.

The results of this experiment showed (Fig. 4) that leaf application did not result in ahigh contact/enteric effect on this pest. Mortality of spider mites in all variants at 10th

day after treatment was not more than 40%. In similar trials from Thoeming et al. (2003)with cut leaf petiole inserted into the tube with neem solution (0,1-0,05%) againstanother cell sucking pest, Frankliniella occidentalis (Thysanoptera, Thripidae), themortality was only 50,6%. In case of phloem-sucking pests like aphids, however,Schliephake (1977) found a clear effect of leaf application on vitality of the aphids.

Experiment 2

0

20

40

60

80

100

1 3 4 6 8 10

Day after treatment

Mor

talit

y, %

C (152 ind.) NATS (180 ind) TSf (282 ind.) NATS+TSf (252 ind.)

268

Figure 5. Mortality at the juvenile postembryonic stages after leaf treatment.

Experiment 4. Translaminar effect on the females after treatment of plants only

Because NeemAzal-T/S inhibits the production of the hormone ecdysone in juvenileinsects, it was not expected to be effective against adult mites. As anticipated, thereduction in adult numbers was low, with all the treatments showing mortalities quitesimilar to each other, at less than 50% by the end of the experimental period.

Experiment 3

0

20

40

60

80

100

1 3 6 8 10 12

Day after treatment

Mor

talit

y, %


269

Figure 6. Mortality of the adult females

Experiment 5. Topical effect on the females only

The effect of NeemAzal-T/S on the females mites was not high. At 8-th day afterapplication (Fig. 7) only 47,2% of females on “NATS” were dead. The best results wereshown by the combination of formulations (NATS+TSf) at 64,8%, while the efficacy of"TSf" was under 40%. This means that the neem formulations had a poor contactefficacy for females. The limited mortality observed can probably be attributed to anenteric effect.

Experiment 4

0

20

40

60

80

100

1 2 3 6 7 8 9 10 13

Day aftrer treatment

Mor

talit

y, %


270

Figure 7. Mortality of the adult females after topical treatment.

Summary

1. In all formulation variants the neem preparations showed clear topical andcontact/enteric and low translaminar effects against T. atlanticus.

2. The eggs and juvenile stages of Spider mite react sensitively to all preparations andtheir combination after topical application; the biological effectiveness of (“NATS+TSf”)in 9/10 days after treatment for eggs was 83.4%, for larvae and nymphs, 85.6%. Foregglaying females this effect was somewhat lower.

3. One application of NeemAzal-T/S and additive Trifolio S-forte showed the best effectagainst the Spider mite.

4. On the basis of the results of this experiment it is necessary to consider that the highacaricidal activity of NeemAzal-T/S is influenced by the formulation and preparation onall development stages of Spider mite.

Literature:

Mitrofanov V. I., Strunkova Z.I, Livschitz Z.I. 1987. Opredelitel´ tetranihovyh kletschejfauni SSSR i sopredel´nyh stran. Duschanbe: Donisch. 224 s. (in Russian).

Popov S.Ya. 1981. Tablici vizhivaniya i biologicheskie parametri populyacii pautinnogokleshca Tetranychus turkestani Ug. et Nik. (Life tables and biological parameters

Experiment 5

0

20

40

60

80

100

1 2 4 6 8

Day after treatment

Mor

talit

y, %


271

of Spider mite population Tetranychus turkestani Ug. et Nik.) // Izvestiya TSKhA.Issue 1. P.124-133) (in Russian).

Popov S.Ya. 1988. Vidovoj sostav rastitel´nojadnih kletschej v zakritom grunte(Phytophagous mites in the greenhouse) // Zashchita rastenii. N 1. P. 46-48 (inRussian).

Popov S.Ya. 1994. K identifikacii mestoobitanij pautinnih kletschej (Acariformes,Tetranychidae) po biologicheskim pokazateljam (On the identification of localitiesof spider mites (Acariformes, Tetranychidae) using biological characteristics) //Zoologicheskii Zhurnal. V. 73: 7-8. P. 31-41 (in Russian).Hummel E. 2006.Vozmozhnost’ primenenija rastitel’nogo insekticida NeemAzal-T/S v zashchiterastenij. // Zashchita rasteniy. Strategiya I taktika zashchity rasteniy. V. 30, t. 1,Minsk, 2006. – S. 515-517 (in Russian)

Gopinathan M.C. 2007. Neem research, product development and commercialization atglobal level – an Indian experience. // BioEco 2007, Bioresourse andBiodiversity. June 27-28, 2007, Tianjin, P.R. China 2007/ - pp. 21 – 40.

Kaul B.K., Sharma P.K. 1999. Efficacy of Neem based insecticides against the majorinsect pests of rice in the hill of Himachal Pradesh (India)/ // Journal Entom.Res., 1999, 23 (4), p. 337.

Schliephake, E. 1997. Die Wirkung von NeemAzal-auf das Saugverhalten derErbsenblattlaus Acyrthisiphon pisum (Hom., Aphididae) an Ackerbohne. In: H.Kleeberg, C.P.W. Zebitz. Practice Oriented Results on Use and Production onNeem-Ingredients and Pheromones. Proceedings of the 6th Workshop;Hohensolms, Germany, Feb. 10-14, 1997: 123-127.

Thoeming G, Borgemeister C, Sétamou M, Poehling H-M. 2003. Systemic Effects ofNeem on Western Flower Thrips, Frankliniella occidentalis (Thysanoptera:Thripidae). Journal of Economic Entomology 96: 817-825.

Von Elling, K., Borgemeister, C., Setamou, M., Poehling, H.-M. 2002. Effect ofNeemAzal-T/S, a commercial neem product, on different developmental stage ofthe common greenhouse whitefly Trialeurodes vaporariorum Westwood (Hom.,Aleyrodidae). J. Appl. Ent. 126, 40-45.

273


THE POTENTIAL OF USING BIOAGENTS AGAINST CHERRY FRUITFLY AS A COMPONENT OF CHERRY AND CRAB-СHERRYPROTECTION SYSTEMS1

VASILIEVA, L. A.

ALL-RUSSIAN RESEARCH INSTITUTE OF BIOLOGICAL PLANT PROTECTION, KRASNODAR, RUSSIA.

Research on cherry fruit fly (Rhagoletis cerasi) biology and ecology has been conductedin the All-Russian Research Institute (Krasnodar, Russia) throughout the period2001-2007. Based on the research results, methods of integrated pest management arebeing developed at the Institute to protect cherry and crab-cherry orchards against thispest. A special emphasis within this integrated pest management system is placed onmore flexible tactics of conducting protective operations based on the combination ofchemical and biological control methods by using predominantly biocontrols dependenton the situation of fruit cherry outbreak in an industrial orchard. Taking into account thefacilities available on farms, the most appropriate approach would be to replace chemicalinsecticides with available formulations of bioproducts having lower toxicity and shorterwaiting time. To date, there are no bioproducts for controlling cherry fruit fly in theRussian market of pest management agents because research conducted in this field isinsufficient. Therefore, it is necessary to study the effects of naturally-derived productson cherry fruit fly populations and the damage caused by them, and to expand the rangeof commercially-available bioproducts that could be applied against this pest.

During our field experiments in May-June, 2007, the following bioproducts were testedfor their effectiveness against cherry fruit fly in commercial crab cherry orchards:Fitoferm (producer: OOO NBC „Fambiomed“), NeemAzal-T/S (Trifolio-M, Germany),Quassia-MD (Trifolio-M, Germany) and their compositions with chemical insecticides.The experimental design is presented in the Table 1. According to the results of ourexperiments these products showed rather high biological effectiveness against cherryfruit fly that was comparable with chemical insecticides (Table 2). During the experimentyellow glue traps were used for monitoring the pest population dynamics and maturity ofeggs in cherry fruit fly females caught in the traps. The numbers of flies trapped eachday was rather high in all experimental treatments, from 1 to 10 specimen per trap. Asmall number of females having mature eggs (0.5%) had been trapped already at thestart of our experiment, but their quantity did not increase during the experimentalperiod. At the end of the experiment, however, when the activity of the appliedbioproducts ceased, the number of females with mature eggs reached about 50%.


274

By analyzing the data obtained from the traps, and taking into account both the previousyear’s damage caused to cherry and crab-cherry orchards and possibility of cherry fruitfly migration from neighbouring orchards, control tactics have been developed andapplied by the Institute’s scientists. During mass outbreaks chemical controls are mosteffective against this pest. But after the number of overwintering puparia in the soilwithin the circles surrounding the tree trunks has been reduced, the fruit infestation isgenerally limited to migrating insects, and in this case it is more desirable to use moreenvironmentally-friendly protective methods. According to our data, the intensive flymigration starts in two or three weeks after the start of the imago flight. If chemicalinsecticides, which, as a rule, have the waiting time of 20 days, are applied, fruit have tobe harvested before this waiting time expires. Bioproducts have a much shorter waitingtime (from 2 to 7 days). Therefore, if the effectiveness of bioproducts is adequate andcomparable to chemical insecticides, we have good reason to replace highly toxicchemical products with the more benign biological ones.

Variants area 1st application(time of

application andpreparation)

2nd application(time of

application andpreparation)

Number oflarvae/fruit

on tree

Number oflarvae /fall fruit

on the soil

1. Untreatedcontrolprofessional(0,5 ha)

26.05 – Actelik 1.06 – Fitoverm(Aversektin C)

4 / 290 44 / 290

1a Untreatedcontrol (privatearea)

30% offruit

infected

60% of fruitinfected

2 Quassia-MD1,5 kg /1600 Lwater/ha (1га)

23.05 1.06 – Fitoverm(Aversektin C)

0 / 320 15 / 210

3 NeemAzal-T/S,2,5 л/1600water /ha(1 ha)

23.05 1.06 3 / 520 2 / 350

ws14 4 part 1 rs komplett seitenzahl3 - trifolio-m · protection against the large pine weevil...

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