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Prof. Dr. H.E. Hummel Organic Agriculture

„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. Hummel1,2

1Justus-Liebig-UniversityOrganic Agriculture

Institute of Crop Production and Plant Breeding IIGiessen, Germany

2Illinois Natural History SurveyEcological Entomology

Champaign, USA


Homage to H. Schmutterer, * April 11, 1926


Agenda

1. Azadirachtin preparation by solvent extraction

2. Purification of neem extracts by various chromatographic techniquesincluding 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 cornrootworm in Illinois


Introduction


BasicsNeem and azadirachtin containing products are botanical biorationalsProminent 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)


Office International Research, Educ. & Development, Virginia Tech. Blacksburg, Virg., USA

D.F. Hein (1994), Giessen, Germany


Caterpillars of gypsy moth (Lymantria dispar) on oak leaf –above: three weeks after neem treatment of leafbelow: untreated control

Schmutterer and Huber 2005


Incomplete moulting of Pieris brassicae from larva to pupa after neem treatmentresulting in damage to imaginal wing discs.

Schmutterer and Huber 2005


Neem effect on Epilachna varivestis (Col.: Coccinellidae); above left: pupal adult;above right: damaged elytra, and below: damaged wings;

Hein, D.F. 1994


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


Examples of macroscopic effects observable in:

Lepidoptera gypsy moth, Lymantria disparlarge white cabbage butterfly, Pieris brassicaeSpodoptera frugiperda, S. littoralis

Coleoptera Mexican bean beetle, Epilachna varivestisColorado potato beetle , Leptinotarsa decemlineataJapanese beetle, Popilia japonica

Orthoptera house cricketsHeteroptera Linden bug, Dysdercus fasciatusHemiptera

Locusta migratoria, Schistocerca gregariaDiptera cherry fruit fly, Ceratitis capitata


Neem effects on neurosecretory cells in the insect brain


Mode of action of azadirachtin at the molecular level

Rembold (2002)


Azadirachtin preparation by solvent extraction


Extraction of neem seeds for obtaining „AZT“ extract (~10 % azadirachtin)

Hummel et al. (1997)


Analytical and biological characterization and quantification


azadirachtin AC35H44O16

marranginC35H44O15


Thin layer chromatographic characterization of neem extractsHein, D.F. (1998)


Quantitative HPLC analytics of neem ingredients

Hein, D.F. (1998)


NMR spectra of marrangin and azadirachtin A forunequivocal identification

Azadirachtin spectrum below, Marrangin spectrum above

Kalinowski et al. (1993)


FAB-mass–spectra of purified CCC-fractionsfrom neem seed extracts


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


Raw azadirachtin purification on the CCC-1000 instrument (Ito & Conway 1996)

Hummel et al. 1997


Analytical MLCCC with high separation power for very closely related azadirachtinand 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


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


Bioassays of azadirachtin in various insect and microbial model systems


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


Sensitive immunological determination ofazadirachtin


Sequence of steps for antibody preparation toward highly sensitiveazadirachtin detection at the ppb level

Azadirachtin

3-desacetyl-azadirachtin

succinic anhydrid, pyridine, diisopropylcarbodiimide-3-hemisuccinylazadirachtin

Azadirachtin

NaH, CH3OHreacting with succinic

anhydride, pyridinereacting with

7-hemisuccinyl-azadirachtin

Conjugation to bovine serumalbumine BSAKeyhole 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), 2363-2368)


Calibration curves for BSA adducts


Standard curve for free azadirachtin A and shortly hydrolyzedazadirachtin using #7056 antiserum and 2a-BSA conjugate

(0,2 µg/l) as coating antigen


Field application of neem oilin pest management


Diabrotica virgifera virgifera LeConte(Coleoptera: Chrysomelidae)

Photo: S. Dinnesen (2006)


`Metcalf´ sticky trap with TIC bait


Reduction by neem treatments by D.v.v. beetles attracted toTIC baited traps1 in Zea mays 2,3

150(50,3 %)7

298

75 (22,1 %)

340Total no. beetlestrapped, sum of Aug. 6 – 19, 1988

low conc. neemtreatment6

high conc. neemtreatment5

Control plot4

1: Lampman and Metcalf (1987)2: Hummel (1989)3: field of Zea mays at university of Illinois, South Farm, Urbana, Ill., USA in August 19884: control treated with water spray only5: treated with neem oil containing 34.3 ppm azadirachtin, applied as emulsion in water6: treated with neem oil containing 11.3 ppm azadirachtin, applied as emulsion in water7: actual attraction level observed in pecent after respective treatment, with no. of beetles in control plots set as 100 %


Discussion of neem effects

Neem oil effects observed can be summarized fromresults of three different experiments:

1) Neem oil repels D. v. virgifera from treated maizeplants 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 havereduced olfactory acuity and orientation abilitytowards point sources baited with attractants.


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 1990-2006;• 5 to 10 000 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 !


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-CT94-1343)

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