Hans E. Hummel 1,2. Justus-Liebig-University. Institute of Crop Production and Plant Breeding II Giessen, Germany

Transcription

1 Neem as a natural resource in sustainable plant protection: extraction, purification, characterization of azadirachtin and use of neem oil in Diabrotica pest management Hans E. Hummel 1,2 1 Justus-Liebig-University Institute of Crop Production and Plant Breeding II Giessen, Germany 2 Illinois Natural History Survey Ecological Entomology Champaign, USA

2 Homage to H. Schmutterer, * April 11, 1926

3 Agenda 1. Azadirachtin preparation by solvent extraction 2. Purification of neem extracts by various chromatographic techniques including CCC 3. Analytical and biological characterization and quantification 4. Bioassays of azadirachtin in various insect and microbial model systems 5. Neem analysis by immunological methods via ELISA 6. Application of crude neem oil in pest management of the western corn rootworm in Illinois

4 Introduction

5 Basics Neem and azadirachtin containing products are botanical biorationals Prominent features of biorationals are: - some specificity - low environmental persistence, thus reduced ecotoxicological impact - reduced likelyhood of developing insect resistance due to less focussed selection pressure - sustainability, annually renewable resource - compatibility with natural agents, thus combinable with biological and biotechnical approaches - alternative to conventional pesticides in niche markets such as organic farming - improvement of local and regional infrastructure with benefits for new enterprise and incentives for the poor - not only agricultural and silvicultural, but also medical benefit neem acts slowly and will not produce 100 % kill (no immediately dead insects and therefore no classical insecticide)

6 Office International Research, Educ. & Development, Virginia Tech. Blacksburg, Virg., USA D.F. Hein (1994), Giessen, Germany

7 Caterpillars of gypsy moth (Lymantria dispar) on oak leaf above: three weeks after neem treatment of leaf below: untreated control Schmutterer and Huber 2005

8 Incomplete moulting of Pieris brassicae from larva to pupa after neem treatment resulting in damage to imaginal wing discs. Schmutterer and Huber 2005

9 Neem effect on Epilachna varivestis (Col.: Coccinellidae); above left: pupal adu above right: damaged elytra, and below: damaged wings; Hein, D.F. 1994

10 Macroscopic effects of neem and azadirachtin - antifeedant - modifier of growth and development - subtle biological effects at the behavioral level, including mate finding and food plant location - general fitness reduction of insects

11 Examples of macroscopic effects observable in: Lepidoptera gypsy moth, Lymantria dispar large white cabbage butterfly, Pieris brassicae Spodoptera frugiperda, S. littoralis Coleoptera Orthoptera Heteroptera Hemiptera Diptera Mexican bean beetle, Epilachna varivestis Colorado potato beetle, Leptinotarsa decemlineata Japanese beetle, Popilia japonica house crickets Linden bug, Dysdercus fasciatus Locusta migratoria, Schistocerca gregaria cherry fruit fly, Ceratitis capitata

12 Neem effects on neurosecretory cells in the insect brain

13 Mode of action of azadirachtin at the molecular level Rembold (2002)

14 Azadirachtin preparation by solvent extraction

15 Extraction of neem seeds for obtaining AZT extract (~10 % azadirachtin) Hummel et al. (1997)

16 Analytical and biological characterization and quantification

17 azadirachtin A C 35 H 44 O 16 marrangin C 35 H 44 O 15

18 Thin layer chromatographic characterization of neem extracts Hein, D.F. (1998)

19 Quantitative HPLC analytics of neem ingredients Hein, D.F. (1998)

20 NMR spectra of marrangin and azadirachtin A for unequivocal identification Azadirachtin spectrum below, Marrangin spectrum above Kalinowski et al. (1993)

21 FAB-mass spectra of purified CCC-fractions from neem seed extracts

22 Multilayer dual coil column assembly countercurrent chromatograph of Dr. Y. ITO, N. I. H., Lab of Biophysical Chemistry, Bethesda, Md., USA

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24 Raw azadirachtin purification on the CCC-1000 instrument (Ito & Conway 1996) Hummel et al. 1997

25 Analytical MLCCC with high separation power for very closely related azadiracht and marrangin Triplet coil CCC-1000, 1,000 rpm, 180 ml coil volume, solvent system: n-hexane : EtOAc : MeOH : water 4 : 5 : 4 : 5, elution rate 2 ml/min.; aza A and mar concentration as determined by analytical HPLC Hummel et al. 1997

26 Crystals of azadirachtin from MLCCC grown on glass surface. Size of aggregates 1-2 mm.

27 Bioassays of azadirachtin in various insect and microbial model systems

28 Probit/log diagram for determination EC 50 values of feeding activity in E. varivestis larvae after azadirachtin A application

29 Sensitive immunological determination of azadirachtin

30 Sequence of steps for antibody preparation toward highly sensitive azadirachtin detection at the ppb level Azadirachtin Azadirachtin NaH, CH 3 OH 3-desacetyl-azadirachtin reacting with succinic anhydrid, pyridine, diisopropylcarbodiimide-3- hemisuccinylazadirachtin 7-hemisuccinyl-azadirachtin reacting with succinic anhydride, pyridine Conjugation to bovine serumalbumine BSA Keyhole limpet hemocyanine (KLH) and tyramine Immunization of rabbits Screening of antisera with 2-dimensional titration ELISA coating antigen Antibody binding study (summarized from Schuetz et al. 1997, J. Agr. Food Chem. 45(6), )

31 Calibration curves for BSA adducts

32 Standard curve for free azadirachtin A and shortly hydrolyzed azadirachtin using #7056 antiserum and 2a-BSA conjugate (0,2 µg/l) as coating antigen

33 Field application of neem oil in pest management

34 Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae) Photo: S. Dinnesen (2006)

35 `Metcalf sticky trap with TIC bait

36 Reduction by neem treatments by D.v.v. beetles attracted t TIC baited traps 1 in Zea mays 2,3 Control plot 4 high conc. neem treatment 5 low conc. neem treatment 6 Total no. beetles trapped, sum of Aug. 6 19, (22,1 %) 150 (50,3 %) 7 1: Lampman and Metcalf (1987) 2: Hummel (1989) 3: field of Zea mays at university of Illinois, South Farm, Urbana, Ill., USA in August : control treated with water spray only 5: treated with neem oil containing 34.3 ppm azadirachtin, applied as emulsion in water 6: treated with neem oil containing 11.3 ppm azadirachtin, applied as emulsion in water 7: actual attraction level observed in pecent after respective treatment, with no. of beetles in control plots set as 100 %

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38 Discussion of neem effects Neem oil effects observed can be summarized from results of three different experiments: 1) Neem oil repels D. v. virgifera from treated maize plants to a significant degree. 2) Neem oil reduces the beetles fitness for take off and flight when disturbed. 3) Beetles within the treated field sections have reduced olfactory acuity and orientation ability towards point sources baited with attractants.

39 GENERAL CONCLUSIONS Neem is one of the few natural resources whose scientific development is far advanced (3 symposium proceedings volumes by H. Schmutterer et al. 1981,1984,1987; 1 comprehensive monograph by H.Schmutterer, Wiley 1995; Neem Foundation, Mumbai 2002; 14 neem workshops by H. Kleeberg, Lahnau ; 5 to primary references sources in the open literature ) Neem is therefore ready for worldwide application and distribution No further delays: Plant trees, improve on storage, transport systems and easy access to world markets Thank you for your kind attention!

40 Acknowledgements Schwarz-Foundation, Heilbronn-Neckarsulm Prof. Dr. H. Schmutterer, JLU Giessen Dr. Y. Ito, NIH, Bethesda, Md., USA Dr. H.-O. Kalinowski, JLU Giessen Prof. Dr. B.D. Hammock, UC Davis, California Prof. Dr. G. Leithold, JLU Giessen EU-Commission, Brussels, AZTEC (AIR2-CT )