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Effects of extracts from Neem, Azadirachta Indica (A. Juss.), on aphids (Homoptera:Aphididae) with respect… Lowery, Donald Thomas 1992

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EFFECTS OF EXTRACTS PROM NEEM, AZADIRACHTA INDICA(A. JUSS.), ON APHIDS (HOMOPTERA:APHIDIDAE)WITH RESPECT TO THEIR CONTROLBYDONALD THOMAS LOWERYB.Sc., University of Guelph, 1980M.Sc.(Agr.), University of Guelph, 1985A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTORATE OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIES(Department of Plant Science)We accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIASeptember 1992Donald Thomas LoweryNational Libraryof CanadaCanadian Theses ServiceNOTICETHE QUALITY OF THIS MICROFICHEIS HEAVILY DEPENDENT UPON THEQUALITY OF THE THESIS SUBMITTEDFOR MICROFILMING.UNFORTUNATELY THE COLOUREDILLUSTRATIONS OF THIS THESISCAN ONLY YIELD DIFFERENT TONESOF GREY.Bibliothèque nationaledu CanadaService des theses canadiennesAVISLA QUALITE DE CETTE MICROFICHEDEPEND GRANDEMENT DE LA QUALITE DE LATHESE SOUMISE AU MICROFILMAGE.MALHEUREUSEMENT, LES DIFFERENTESILLUSTRATIONS EN COULEURS DE CETTETHESE NE PEUVENT DONNER QUE DESTEINTES DE GRIS.In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.Department of P/-d- Sce--The University of British ColumbiaVancouver, CanadaDate c2c& 1% /T92DE-6 (2/88)iiABSTRACTLaboratory and field trials with formulated neem seed oil (NSO)and neem seed extract (NSE) demonstrated that these materials arepotentially very effective as aphicides. Sprays of NSO to plantsin the laboratory resulted in significant reductions in numbersof green peach aphids, Myzus persicae (Suizer), on pepper andrutabaga; lettuce aphid, Nasonovia ribisnigri (Mosley), onlettuce; and strawberry aphid, Chaetosiphon fragaefolii(Cockerell), on strawberry. Under field conditions, NSO and NSEwere as effective or better than the commonly used botanicalinsecticide pyrethrum for the control of aphids on pepper,cabbage, and strawberry, but they were ineffective for thecontrol of aphids on lettuce.Leaf disk choice bioassays to assess the deterrency of neem toaphids produced variable results, and NSO at concentrations from1 to 2% were deterrent to only half the species tested.Differences in behavioral response of aphids to the variousvolatile and non—volatile components of neem likely explains, atleast in part, the contradictory and inconclusive results fromprevious studies. Deterrency of NSO to C. fragaefolii was lostafter 24 hours following applications to strawberry in thegreenhouse, suggesting that the antifeedant or repellent actionof neem is of limited value for the control of aphids or theplant viruses they transmit.In laboratory bioassays to determine the toxicity of neexn toiiiaphids, NSO applied to leaf disks at a concentration of 1.0%resulted in 94 to 100% mortality of second instar N. ribisnigri,M. persicae, and C. fragaefolii after nine days. The equivalentamount of purified AZA (40 ppm), considered to be the mostactive ingredient of neem, was as effective as NSO toward N.ribisnigri and M. persicae, but survival of C. fragaefolii wasunaffected by AZA even at twice that rate. Although survival ofadults was not reduced by exposure to NSO or AZA, survival ofoffspring from treated adult M. persicae and N. ribisnigri wasreduced significantly.The effective concentration of AZA resulting in 50% mortality(EC50) after nine days ranged from as low as 2.4 ppm for M.persicae on pepper, to 635 ppm for C. .fragaefolii on strawberry.The growth-disrupting effect of AZA towards M. persicae wasinfluenced by the host plant and the nymphal instar studied. Forexample, the EC50 for second instars on corn was more than 20times higher than on mustard cabbage, while fourth instars wereapproximately 20 times less sensitive than first instars. Inaddition to direct toxicity, nymphs exposed to NSO or AZA thatsuccessfully molted to adults were often smaller and possessedphysically deformed wings, legs, and stylets.Exposure of adult N. ribisnigri and M. persicae for three daysto 1.5% NSO applied to leaf disks resulted in the production of76% and 83% fewer live offspring, respectively, compared tocontrols over a one week period. The effective concentration ofAZA resulting in the production of 50% fewer offspring (EC50) foriveight species of aphids ranged from as low as 14.4 ppm for N.ribisnigri on lettuce to 616.4 ppm for Rhopalosiphuni padi (L.),the bird cherry—oat aphid, on corn. The decrease in aphidreproduction resulted from the production of fewer offspring andan increase in the number of embryos that failed to completedevelopment and were dead (embryonic) at birth.Laboratory studies indicated that exposure to neem reduced thesurvival of aphid predators, Coccinella undecimpunctata (L.), andEupeodes fumipennis (Thompson), and parasitoids, Aphidius sp.,but foliar applications of neem did not cause significant harm tonatural enemy populations in the field, demonstrating that neem—based insecticides are potentially compatible with integratedpest management programs. Neem provides effective control ofaphids, while causing little damage to beneficial insects, man,or the environment.VTABLE OF CONTENTSPageTitle Page iAuthorization iAbstract iiTable of Contents . VList of Tables viiiList of Figures xiiAcknowledgements xivGenerallntroduction 1Chapter 1.Laboratory and Field Evaluation of Neemfor the Control of Aphids 13Introduction 13Materials and Methods 17A. Laboratory studies 17B. Field studies 18C. Determination of azadirachtin content 21D. Statistical analysis 21Results and Discussion 23A. Control in the laboratory 23B. Control in the field 26Conclusion 41Chapter 2Antifeedant Activity of Neem towards Aphids 43Introduction 43Materials and Methods 47A. Plant material 47B. Leaf disk choice bioassays 47C. Statistical analysis 51Results and Discussion 52Conclusion 66viTABLE OF CONTENTS (Cont.)PageChapter 3Regulation of Aphid Growth and Development by NeemSeed Oil and its Active Ingredient Azadirachtin 69Introduction 69Materials and Methods 72A. Laboratory rearing conditions 72B. Determination of treatment rates 72C. Toxicity of NSQ and AZA 73D. Sub-lethal effect of NSO 75E. Statistical analysis 76Results and Discussion 77A. Toxicity of NSO and AZA 77B. Sub—lethal effects of NSO 92Conclusion 98Chapter 4Inhibition of Aphid Reproduction by Neem Seed Oiland its Active Ingredient Azadirachtin 100Introduction 100Materials and Methods 102A. Laboratory rearing conditions 102B. Test materials 102C. Inhibition of aphid reproduction 102D. Aphid dissections 104E. Statistical analysis 104Results and Discussion 106A. Inhibition of aphid reproduction 106B. Aphid dissections 113Conclusion 116Chapter 5Systemic Activity and Persistence of Neem 118Introduction 118Materials and Methods 124A. Systemic activity 124B. Persistence 127C. Statistical analysis 127viiTABLE OF CONTENTS (Cont.)PageChapter 5 (cont.)Results and Discussion 128A. Systemic activity 128B. Persistence 135Conclusion 139Chapter 6Effect of Neem on Natural Enemies of Aphids 142Introduction 142Materials and Methods 145A. Field studies 145B. Laboratory studies 146Results and Discussion 149A. Field studies 149B. Laboratory studies 152Conclusion 163General Discussion 165References 175Appendix: Scientific and Common Names of Aphids 189viiiLIST OF TABLESChapter 1 PageTable 1.1 Aphid control on intact plants in the laboratory following pre-infestation (Pre-I) andpost-infestation (Post-I) foliar applicationsof formulated neem seed oil (NSO) 24Table 1.2 Aphid control on strawberry in the fieldfollowing foliar applications of formulated neemseed oil (NSO) and neem seed extract (NSE) 27Table 1.3 Aphid control on lettuce in the field followingfoliar applications of formulated neem seed oil(NSO) and neem seed extract (NSE) 29Table 1.4 Aphid control on sweet pepper in the fieldfollowing foliar applications of formulated neemseed oil (NSO) and neem seed extract (NSE) 31Table 1.5 Control of aphids on cabbage in the fieldfollowing foliar applications of formulated neemseed oil (NSO) and neem seed extract (NSE) 33Chapter 2Table 2.1 Deterrency of neem seed oil (NSO) toward secondinstar strawberry aphid, Chaetosiphon fragae—foul (Cockerell). Effective concentrations ofNSO in leaf disk choice test bioassays resultingin 50% movement (EC50) from treated to untreatedleaf disks after 1 to 48 hours 53Table 2.2 Concentrations of neem seed oil (NSO) resultingin 50% deterrency (EC50) to several species ofaphids in 24 hour leaf disk choice testbioassays 56Table 2.3 Concentrations of neexn seed oil (NSO) appliedto leaf disks of strawberry resulting in 50%deterrency (EC50) to various instars ofstrawberry aphid, Chaetosiphon fragaefolii(Cockerell), in 24 hour choice test bioassays ... 58Table 2.4 Concentrations of neem seed oils (NSO) containing variable amounts of azadirachtin (AZA)applied to leaf disks of strawberry resultingin 50% deterrency (EC50) toward adult strawberryaphids, Chaetosiphon fragaefolii (Cockerell), in24 hour choice test bioassays 59ixLIST OF TABLES (cont.)Chapter 2 (cont.) PageTable 2.5 Persistence of the deterrent effect of neexn seedoil (NSO) toward adult strawberry aphid,Chaetosiphon fragaefolii (Cockerell). Choicetest bioassays beginning 0 to 4 days post-treatment, for leaf disks of strawberry held inpetri dishes 63Table 2.6 Persistence of the deterrent effect of neexn seedoil (NSO) toward adult strawberry aphid,Chaetosiphon fragaefolii (Cockerell). Choicetest bioassays beginning 1 to 48 hours post-treatment, for leaf disks from strawberrytreated with NSO and held in the greenhouse 64Chapter 3Table 3.1 Percent survival, days of survival, and numberof molts for second instar lettuce aphid (LA),green peach aphid (GPA) and strawberry aphid(SA) exposed to neem seed oil (NSO) andazadirachtin (AZA) applied to leaf disks oflettuce, pepper, and strawberry, respectively.... 78Table 3.2 Percent survival and days of survival for adultand fourth instar lettuce aphid (LA), greenpeach aphid (GPA) and strawberry aphid (SA)exposed to neem seed oil (NSO) and azadirachtinapplied to leaf disks of lettuce, pepper, andstrawberry, respectively 81Table 3.3 Nine day survival of first generation aphidsfrom adults exposed for three days to neem seedoil (NSO) or azadirachtin (AZA) 83Table 3.4 Effective concentration of azadirachtin (AZA)resulting in 50% mortality (EC50) after 9 daysfor aphids placed as second instars for 3 dayson treated leaf disks 86Table 3.5 Effective concentration of azadirachtin (AZA)resulting in 50% mortality (EC50) after ninedays for second instar M. persicae reared for3 days on treated leaf disks of various hostplants, and for different instars on pepper 88Table 3.6 Effective concentrations of neem seed oil (NSO)applied to second instar aphids by spraydroplets or with a microapplicator resultingin 50% mortality (EC50) after 9 days 91xLIST OF TABLES (cont.)Chapter 3 (cont) PageTable 3.7 Sublethal effect of formulated neem seed oil(NSO) on aphid development. Lengths of hindtibiae (TL) and third antennal segments (AL),and deformities of wings (W) and legs (L) ofadults that developed from third instars rearedfor three days on treated leaf disks 93Chapter 4Table 4.1 Reproduction of lettuce aphid (LA) on lettuce,green peach aphid (GPA) on pepper, and strawberry aphid (SA) on strawberry, followingexposure to neem seed oil (NSO) and azadirachtin(AZA) as adults or as fourth instars 107Table 4.2 Effective concentrations of azadirachtin (AZA)resulting in a 50% reduction (EC50) in thenumber of live offspring produced during aninitial 3 days on treated leaf disks, and afurther three days on untreated disks 109Table 4.3 Total number of embryos per aphid (>0.12 mm),and numbers in each of three size classes, fordissected adult lettuce aphids, Nasonoviaribisnigri (Mosley), exposed as fourth instarsto azadirachtin (AZA), neem seed oil (NSO), oremulsifier only as a control 114Chapter 5Table 5.1 Efficacy of neem seed extract (NSE) and neemseed oil (NSO) applied as a soil drench topotted plants for the control of aphids 129Table 5.2 Survival of second instar lettuce aphid,Nasonovia ribisnigri (Mosley), on lettucefollowing three days feeding on leaf disksurfaces treated with neem seed oil (facing),or on the opposite untreated surfaces(opposite) 133Table 5.3 Local systemic activity (translaminar) ofazadirachtin (AZA). Effective concentration ofAZA applied to the opposite sides of leaf disksresulting in 50% mortality (EC50) after ninedays for second instar aphids reared for threedays confined to the untreated surfaces 134xiLIST OF TABLES (cont.)Chapter 5 (cont.) PageTable 5.4 Persistence of the toxic effect of neem seed oil(NSO) to aphids. Survival (9 day) of secondinstar lettuce aphid, Nasonovia ribisnigri(Mosley), and green peach aphid, Myzus persicae(Sulzer), on leaf disks of lettuce and mustardcabbage, respectively, from plants treated withNSO and held in the greenhouse or outdoors, forbioassays beginning 0 to 9 days post-treatment.. 136Chapter 6Table 6.1 Effect of foliar applications of neem seed oil(NSO) and neem seed extract (NSE) to plants inthe field on numbers of aphid parasitoids(mummies) and predators (larvae and pupae) per280 plants and per 1,000 aphids 150Table 6.2 Percent survival to pupation and to adultemergence for second instar syrphid, Eupeodesfumipennis (Thompson), and coccinellid,Coccinella undecimpunctata (L.), larvae onM. persicae—infested canola treated with neemseed oil (NSO) 154Table 6.3 Percent survival to pupation and to adultemergence for second instar syrphid, Eupeodesfumipennis (Thompson), and coccinellid,Coccinella undecimpunctata (L.), larvaetopically treated with neem seed oil (NSO) 156Table 6.4 Parasitism of green peach aphid, Myzus persicae(Sulzer), by Aphidius sp. on caged mustardcabbage treated with formulated neem seed oil(NSO) 159Table 6.5 Percent emergence of adult parasitoids, Aphidiussp., from three age classes of green peachaphid, Myzus persicae (Sulzer), mummies dippedin solutions of neem seed oil (NSO) 161xiiLIST OF FIGURESchapter 1 PageFigure 1.1 Research plots at the Plant Science ResearchStation (Totem Field), U.B.C., for evaluatingthe efficacy of foliar neem sprays for thecontrol of aphids. Lettuce ‘Ithaca’immediately after transplanting to the field,and at pre-heading stage just prior todestructive sampling (post-spray) 19Figure 1.2 cabbage ‘Stonehead’ in the field one week afterthe final foliar application of 1.0% neem seedextract, or emulsifier only as a control 35Figure 1.3 Phytotoxicity of foliar applications of 1.0%and 2.0% formulated neem seed oil to strawberry‘Totem’ in the field compared to 1.0% neem seedextract and emulsifier only as a control 36Figure 1.4 Seeds of the Indian neem tree contain thehighest concentration of the limonoidazadirachtin (National Research council 1992).crushing of neem seeds produces neem seed(expellor) oil and neem cake 38Chapter 3Figure 3.1 Representative survival curves for second instargreen peach aphid, Myzus persicae (Sulzer),lettuce aphid, Nasonovia ribisnigri (Mosley),and strawberry aphid, Chaetosiphon .fragaefolii(cockerell), exposed to 40 ppm azadirachtin(AZA), 1.0% neem seed oil (NSO) containingapproximately 40 ppm AZA, or emulsifier onlyas a control 79Figure 3.2 compared to normal alatae, nymphal lettuceaphid, Nasonovia ribisnigri (Mosley), exposedto neem seed oil typically developed into alateadults with physically deformed wings, rangingfrom fully expanded but twisted or heldimproperly, incompletely expanded, to merestubs or lacking entirely 95Figure 3.3 compared to normal adult apterous lettuceaphids, Nasonovia ribisnigri (Mosley), apteraethat developed from nymphs exposed to neem seedoil were often more highly pigmented 96xiiiChapter 4Figure 4.1 Number of live and dead (embryonic) offspringper aphid per day produced by adult greenpeach aphid, Myzus persicae (Suizer), andlettuce aphid, Nasonovia ribisnigri (Mosley),following three days of exposure toazadirachtin (0-100 ppm) applied to leaf disksof pepper and lettuce, respectively 111Figure 4.2 Number of live and dead (embryonic) offspringper aphid per day produced by adult cotton ormelon aphid, Aphis gossypii Glover, potatoaphid, Macrosiphum euphorbiae (Thomas), birdcherry-oat aphid, Rhopalosiphum padi (L.), andstrawberry aphid, Chaetosiphon fragaefolii(Cockerell), following three days of exposureto azadirachtin (0-1,000 ppm) applied to leafdisks of cucumber, lettuce, corn andstrawberry, respectively 112Chapter 5Figure 5.1 Rearing system for evaluating the localsystemic activity of neem seed oil andazadirachtin whereby aphid feeding isrestricted to the lower untreated surfacesof leaf disks 125Chapter 6Figure 6.1 Dorsal and ventral view of adult eleven—spotladybeetle, Coccinella undecimpunctata L.,with physically deformed wings and elytrafollowing topical treatment of a second instarlarva with neem seed oil 157xivACKNOWLEDGEMENTSI wish to thank my supervisor, Dr. M.B. Isman, for hisassistance with this research project, and extend my thanks tothe other members of my committee, Dr. B.D. Frazer, Dr. R.S.Vernon and Dr. J.A. McLean.I would like to acknowledge Dr. J.T. Arnason for providingthe azadirachtin and di-hydro azadirachtin, Safer Ltd. forsupplying the neem materials, and Dr. G.W. Eaton and Dr. J.W.Hall for their advice with the statistical analysis. C.K. Changraciously allowed access to the aphid colonies and photographedthe insects which are displayed in this thesis. I want to thankDr. B.D. Frazer, Dr. J.R. Vockeroth and Dr. M. Mackauer foridentification of the aphid natural enemies utilized in thesestudies.The atmosphere in the laboratory was enlivened by the presenceof Dr. N.J. Smirle, Dr. 0. Koul, C.M. McCloskey, K.G. Craig, andothers, and I thank them for their lively discussion andsuggestions. I am particularly grateful to N.L. Brard for hercheerful disposition and technical assistance with the fieldresearch, plant and insect cultures, and HPLC analysis.Funding for this thesis research was provided by grants fromthe National Research Council (P..G.F.) and the Science Councilof British Columbia (G.R.E.A.T. Grant)This thesis is dedicated to my parents, and to R.M. DeYoungfor her patience and assistance.1GENERAL INTRODUCTIONDerivatives of the Indian neem tree (syn. Indian lilac,margosa tree, nirn), Azadirachta indica A. Juss. (syn. Meliaazadirachta L.) (Meliaceae) have traditionally been used in Asiaand Africa to protect crops, stored products, livestock, andhumans from the ravages of insects (Schmutterer 1987; Saxena1989; Koul et al. 1990). Modern entomological investigations ofthe insecticidal properties of neein and its principal activeingredient, the tetranortriterpenoid (limonoid) azadirachtin(AZA), began with the rediscovery of their potent antifeedanteffect toward the desert locust, Scthistocerca gregaria Forsk.(Pradhan et al. 1962). Shortly thereafter, extracts from theseeds of neem and pure AZA were both shown to possess multiplebiological activities toward insects from several orders (seeSchmutterer 1987, Saxena 1989, and Koul et al. 1990, forhistorical accounts). The diverse biological activities of neemand AZA include feeding and oviposition deterrence, repellency,growth disruption, reduced fitness, and sterility (Koul et al.1989; Saxena 1989; Isman et al. l990b; Schmutterer 1990a).Demonstrated activity has been reported against more than 200species of insects from several orders (Saxena 1989; Isman etal. 1990a), with the reported strength of the various effectsdepending on the concentration of the active principle and theinsect species tested (Schmutterer 1988; Schmutterer 1990a;Isman et al. 1991). Neem has demonstrated systemic activity2(Saxena 1987; Larew 1988; Osman & Port 1990), is active at lowconcentrations, has negligible toxicity to vertebrates (Ermel etal. 1987; Larson 1989; Jacobson 1989), and is rapidly degradedin the environment (Schmutterer 1988; Walter & Knauss 1990).The propagation, chemistry, mode of action, and utilization ofneem has been extensively studied during the last two decades,resulting in three international neem conferences (Rottach—Egern, Germany, 1980; Rauischholzhausen, Germany, 1983; Nairobi,Kenya, 1986), several national conferences (e.g. Locke & Lawson1990), books and book chapters (e.g. Schmutterer 1987; Arnasonet al. 1989; Jacobson 1989; National Research Council 1992),comprehensive neem reviews (e.g. Radwanski & Wickens 1981;Schmutterer 1985; Schmutterer 1987; Jacobson 1989; Saxena 1989;Koul et al. 1990), numerous journal articles, and a quarterlyneem newsletter.The modes—of—action of neem can be conveniently divided intobehavioural (antifeedant/repellent) and physiological (changesto the ecdysteroid—mediated control of insect growth,development, and reproduction) effects. The antifeedant actionof neem extracts and AZA has been well documented, particularlyfor orthopterans and larval coleopterans and lepidopterans(Butterworth & Morgan 1968; Rembold l989a; Saxena 1989; Isman etal. l990b; Isman et al. 1991). As stated by Jacobson et al.(1984), AZA is ‘possibly the world’s most fantasticallyeffective natural insect antifeedant yet known’. For example,AZA offered to desert locusts on sucrose—impregnated filter3paper completely inhibited feeding at rates as low as 10 to 40g/l (0.01 to 0.04 ppm) (Butterworth & Morgan 1968; Butterworth& Morgan 1971). Utilizing a bioassay based on the sensitivityof desert locusts to the phagodeterrent action of neem,Butterworth and Morgan (1968; 1971) were able to isolate andtentatively identify AZA. Neurophysiological and behaviouralstudies have demonstrated that deterrency results primarily froma contact gustatory effect, most likely resulting from thestimulation of deterrent neurons, or inhibition of stimulatoryneurons, of the maxillae (Simmonds & Blaney 1984; Schinuttererl990a). As well, reduced feeding has been observed followingtopical application or injection of neein extracts and AZA. Thissecondary, non—gustatory, effect may result from a reduction ingut motility, or from changes in hormonal titres; which, inturn, modify the insect’s behaviour (Mordue et al. 1985; Remboldl989a; Schmutterer 1990a). Neexn cannot be considered a generalantifeedant, however, and feeding of some insects does notappear to be influenced by neem or AZA. For example, whiledesert locusts are extremely sensitive to AZA, the migratorygrasshopper, Melanoplus sanguinipes (F.), readily consumed leafdisks of cabbage treated with AZA at rates as high as 500 ppm(Champagne et al. 1989).The repellent effects of neem have been investigated lessthoroughly, and many previous studies failed to distinguishfeeding deterrency from repellency (Saxena 1989). Adultsweetpotato whitefly, Bemisia tabaci (Gennadius), were repelled4by aqueous neem seed extracts applied to cotton in thelaboratory and greenhouse, resulting in fewer eggs beingdeposited on treated foliage (Coudriet et al. 1985). Similarly,fewer planthoppers and leafhoppers landed on rice plants sprayedwith neem oil (Saxena et al. 1981; Saxena 1989), and fewerfemale cotton bollworm moths, Heliothis armigera (Hubner),landed and oviposited on plants treated with neein seed kernelextract (Saxena & Rembold 1984). The repellent action of neemlikely results from the presence of volatile sulfur-containingcompounds (Balandrin et al. 1988). AZA has little volatility,and purified AZA was not repellent to cotton bollworm moths(Saxena & Rembold 1984).The most profound effect of neem results primarily fromchanges to hemolymph ecdysteroid titres, resulting frominterference with the neuroendocrine system (Sieber & Rembold1983; Dorn et al. 1987; Rembold et al. 1987b; Rembold 1989a).Histological studies demonstrated an increase in the stainablematerial of the neurosecretory cells of the pars intercerebralisof fifth instar migratory locusts, Locusta migratoria (L.),treated with AZA. Resulting changes in the ecdysteroid levels(Bidmon et al. 1987; Rembold 1989a; Barnby & Kiocke 1990)results in a cascade of various disruptive effects on insectgrowth, development, reproduction, and behaviour. Immatureinsects often die during molts, due to improper apolysis andecdysis (Schluter & Schultz 1984; Schmutterer l990a). Schauer(1984) demonstrated that during molts the exocuticle of pea5aphids, Acyrthosiphon pisum (Harris), exposed to neem extracts,began to detach from the body but could not be ruptured, or theskin ruptured but could not be cast off completely.Adult insects are generally less susceptible to the directtoxic effects of neem, but neem and AZA have been shown tonegatively influence the reproduction of female insects ofseveral orders (Steets 1976; Dorn et al. 1987; Rembold 1989b;Schmutterer l990a). The documented antifeedant action of neemcould contribute to a reduction in female reproductive rates,but inhibition of reproduction results primarily from changes toecdysteroid titres and interference with the hormonal regulationof reproduction (Bidmon et al. 1987; Dorn et al. 1987; Remboldet al. 1987b; Rembold 1989b).Histological investigations have demonstrated that, inaddition to other sensitive tissues, insects treated with AZAhave degenerate or improperly developed ovaries and fat bodies(Schiuter & Schultz 1984; Schmutterer 1985; Schiuter 1987).Depending on the stage of development, oocytes developabnormally and are resorbed, minimal amounts of yolk aredeposited, and eggs which are deposited often possess abnormalchorionic surfaces (Schulz & Schluter 1984; Schmutterer 1987;Rembold 1989a).The ovicidal effects of neem extracts or AZA have not beenstudied intensively, but eggs treated with AZA or neem extractsare generally not affected, and fertility appears to be normal(Schinutterer 1990a). For example, application of AZA to eggs of6red cotton bugs, Dysdercus koenigii F., did not reduce percentemergence (Koul l984b). However, eggs deposited by femalestreated with AZA or neem extracts often have poorly formedchorionic surfaces (Schulz & Schluter 1984), may be moresensitive to fungal attack, and are often less fertile(Schmutterer 1987).The vast majority of neern studies have involved insects withchewing mouthparts, while agricultural pests with piercing-sucking mouthparts have not been thoroughly investigated(Schmutterer l990b). Except for certain scale insects andmealybugs (Coccoidea), insects belonging to the order Homopteraare sensitive to neem products to a variable degree. Severalspecies of scale insect, including the Oriental yellow scale,Aonidiella orientalis (Maskell), are serious pests of neem trees(Schmutterer 1987; National Research Council 1992). Leafhoppers(Cicadoidea) and planthoppers (Fulgoroidea), on the other hand,are effectively controlled by extracts of neem applied to plantsin the laboratory and in the field.Aphids (Aphidoidea) are economically important pests that aredifficult to control because of their mobility, tremendousreproductive ability, and resistance to many syntheticpesticides. Studies directed against several species of aphidshave indicated that neem-based insecticides are potentiallyeffective natural control agents (Schauer 1984; Patel &Srivastava 1989; Schmutterer l990a) that may be suitable forinclusion in integrated pest management programs (Schmutterer71988; Saxena 1989; Hoelmer et al. 1990). Results from studiesinvolving aphids have often been inconclusive or contradictory,however, and some reports have stated that aphids are not goodcandidates for control with neem-based products (Schinutterer1990a; National Research Council 1992). There may be severalreasons for these variable results. The majority of earlierneem studies utilized crude neem extracts with undeterminedamounts of the most active ingredient. Studies havedemonstrated that the AZA content of neem extracts ranges fromundetectable levels to as high as 10,000 ppm (Ermel et al. 1987;Jones et al. 1989; Isman et al. 1991). The variation in AZAcontent likely results from a combination of factors, includingbiotype differences, climatic conditions, age of seeds, harvestand storage conditions, method of extraction, and type offormulation (Schmutterer & Zebitz 1984; Ermel et al. 1987).Also, AZA is rapidly degraded by the combined action of light,temperature, and pH, and neem extracts would rapidly lose theirpotency if not stored and handled properly (Larson 1987; Barnbyet al. 1989; Walter & Knauss 1990).The effectiveness of neem sprays may also be influenced by thehost plant of the insect (Schmutterer l990a). It has beendemonstrated previously that control of whitef lies with foliarapplications of neem is influenced by the host plant, and soildrenches of Nargosan-OTM, a registered neem—based insecticide(W.R. Grace and Co., Cambridge, Ma.), reduced the number ofleafhoppers on marigold and chrysanthemum, but not on zinnia8(Knodel-Montz et al. 1985). As mentioned previously, neem hassystemic activity, and penetration of the leaf cuticle ortranslocation of the active component within plants likelyvaries between species. Differences in systemic activity willbe a particularly important factor for the control of phloexnfeeding insects such as aphids. Different formulations,application rates, and spray coverage, will also influence thedegree of systemic activity. Schauer (1984) demonstrated thatthe addition of lecithin II and combinations of lecithin II withsesame oil and dimethyl-sulfoxide (DMSO) improved the efficacyof a tertiary-methyl-butyl-ether extract of neem for the controlof A. pisum and black bean aphids, Aphis fabae Scopoli, onbroadbean. Neem formulations and the use of additives requiresfurther study (Schmutterer 1990b).Studies of the effects of neem on aphids with respect to theircontrol could help answer several questions pertaining to thepotential usefulness of this plant-based ‘organic’ pesticide.Research is required to widen the spectrum of neem—sensitiveagricultural pests. Certain pest groups have largely beenneglected in the past, specifically phloem-feeding insect pests(Saxena et al. 1981; Schmutterer l990b). Moreover, it is wellknown that good or even excellent control of insects in thelaboratory, particularly with insect growth regulators (IGR’s),does not guarantee effective control under field conditions(Schmutterer & Hellpap 1989).In the present studies, control of several economically9important aphid pests of vegetables and strawberries withproperly formulated neem seed oil (NSO) and an ethanolic neemseed extract (NSE) was evaluated in the laboratory and in thefield. Additionally, these studies were intended to provideinformation on the variability in the degree of control betweenaphid species on various host plants. In an effort to determineif the host plant of an aphid influences the efficacy of neem,throughout these investigations an attempt was made to evaluatethe effects of neem on a single species of aphid reared on morethan one host plant. Differences in sensitivity to neem for agroup of closely related insects has seldom been evaluated.Aphids are the most important vectors of plant viruses (Watson& Plumb 1972). Several investigations have shown that foliarneem sprays may help prevent the transmission of plant virusesby aphids (Hunter & Ullman 1992), planthoppers, and leafhoppers(Saxena et al. 1981; Saxena 1986). Reduced viral transmissionby aphids could result from the reported antifeedant andrepellent actions of neem (Saxena 1986; Hunter & Uliman 1992),or NSO may interfere with the attachment or removal of virusparticles from aphid mouthparts in a manner similar to otherhorticultural (stylet) oils (Vanderveken 1977; Lowery et al.1990). A series of experiments were conducted to evaluate therepellent and antifeedant activities of neem against severalspecies of aphids. In addition to its potential impact on virustransmission, the antifeedant activity of neem might alsocontribute to the control of aphid populations in the field.10To determine the means by which neem reduces aphidpopulations, the effects of neem extracts and AZA on aphidgrowth, development, and reproduction were assessed in aseries of controlled laboratory experiments. In addition todirect toxicity, the long-term fitness-reducing effects ofneem products on insects could be of special interest forfuture pest control. These include the loss or reduction inability to fly, male impotence, reduced size, and inabilityto transmit or recognize pheromones (Lindquist et al. 1990;Schmutterer 1990b). For these studies, adult sizes and thepresence of abnormalities were recorded to determine thefitness—reducing effects of neem on aphid populations.Additionally, the growth and development of first generationoffspring from adults exposed to neem was evaluated todetermine if neem had an impact on subsequent generations ofthese viviparous insects.Because aphids are phloem feeders, they may be useful fordetermining differences in systemic activity of neem betweenvarious plants. Proper formulations and application methodsare required to maximize penetration of AZA and the subsequentcontrol of aphids. Characterization and standardization ofneem materials has too often been lacking in the past (Ermel etal. 1987; Isman et al. 1990a; Isman et al. 1990b). The currentstudies utilized properly formulated neem seed extractscontaining quantified amounts of the most active ingredient,AZA. Failure to define the active ingredient in the various11extracts has led to mixed and contradictory results in the past(Larson 1989).Finally, aphids serve as a food source for numerous predatoryand parasitic insects, and field and laboratory studies of theeffects of neem on natural enemies of aphids could determinethe potential usefulness of neem in integrated pest management(1PM) or biological control programs. Many aphid problems aresecondary in nature, with outbreaks and damaging populationlevels occurring after broad—spectrum synthetic insecticideshave reduced or eliminated natural enemies that normally helpto keep aphid populations in check. Neem sprays targeted forother insects may cause less damage to the predators andparasitoids of aphids, which would indirectly prevent aphidpopulation increases. According to Schmutterer (1990b) thereis a wide open field for research aimed at the integration ofneem products into 1PM systems. Neein materials appear to beonly slightly harmful to natural enemies of pest insects(Saxena 1989; Hoelmer et al. 1990; Schmutterer 1990a), andneem materials applied to rice fields did not affect predatoryminds or spiders (Saxena 1989). In a separate field trial,parasitization of larval rice leaffolders, Cnaphalocrocismedinalis (Guenee), was higher in rice fields sprayed weeklywith neem oil than in unsprayed fields, and adult parasitoidsemerged normally (Saxena et al. 1981).12The specific objectives of this thesis research are:1) to determine if neem is efficacious for the controlof aphids in the laboratory and in the field;2) to determine if neem is an effective aphid feedingdeterrent;3) to determine the effects of neem on aphid growth,development, and reproduction;4) to determine the systemic activity and persistence of foliarneein applications;5) to determine the effect of neem on natural enemies ofaphids, in order to assess neem’s potential compatibilitywith 1PM or biological controlprograms.13CHAPTER 1LABORATORY AND FIELD EVALUATION OF NEEMFOR THE CONTROL OF APHIDSINTRODUCTIONFor centuries, farmers in India and other parts of Asia haveprotected crops using the natural feeding deterrent derived fromthe leaves and fruits of neem (Saxena 1989). To prepare asimple aqueous extract, ground neem seeds are added to water andallowed to stand for 5 to 6 hours, after which time thesuspension would be filtered through coarse cloth to removelarge particles (Schmutterer & Heilpap 1989; National ResearchCouncil 1992) . The effectiveness of these crude extracts wasoften highly variable, however, due to differences in AZAcontent of the starting material and the rapid degradation ofAZA by the action of ultraviolet light and pH of the spraysolutions (Walter & Knauss 1990).An alternative starting material for neem—based pesticides isthe thick viscous brown oil produced when neem seeds are crushed(expeller oil). Neem seed oil (NSO) may be a favoured startingmaterial for several reasons. Neem oils may contain otheractive ingredients, and natural antioxidants and UV—absorbingsubstances contained in the oil may retard the degradation ofAZA (Larson 1989; Isman et al. 1990b). As well, the oil itself14may be biologically active against small, soft-bodied insectsand help prevent the transmission of plant viruses.Several companies in India have recently started marketingneem products based on seed—extracts (e.g. RepelinTM andWellgroTM), partially purified extracts (e.g. NeeMarkT), andoils (e.g. NimbosolTM and BiosolTM). In the United States,Margosan—OTM has received registration on non—food crops, andregistration on food crops is pending (Larson 1989). Still,there is a dearth of scientific publications regarding theefficacy of neem products under field conditions, and currentlyone of the most important steps in neem research is theimplementation of laboratory results to practical cropprotection (Dreyer 1987).Most published field studies of the efficacy of neem have beenconducted in tropical or sub—tropical countries. In the Sudan,several pests of potato, including the cotton or melon aphid,Aphis gossypii (Glover), were effectively controlled with weeklyapplications of aqueous neem seed extracts, resulting in yieldincreases of 0.5 tonnes/ha (Siddig 1987). Weekly applicationsof neem seed kernel extracts were as effective as the currentlyregistered synthetic insecticide deltamethrin (DecisTM) for thecontrol of lepidopteran larvae on Chinese cabbage, cabbage,cauliflower, and tomato, in Mauritius (Fagoonee 1987).Similarly, in a series of field trials in Togo, foliar neemsprays were superior to the microbial insecticide Bacillusthuringiensis (DipelTM) for control of diamondback moth,15Plutella xylostella (L.), and cabbage webworm, Hellula undalis(F.), on cabbage (Dreyer 1987). The antifeedant action of neemseed kernel suspensions prevented damage to tobacco in India bythe tobacco caterpillar, Spodoptera litura F., and it is nowrecommended for the protection of seedlings in nurseries (Joshiet al. 1984). Neem is increasingly popular among tobaccogrowers in India due to its effectiveness and low cost incomparison to synthetic insecticides. Use of neem seed kernelextracts in India is five times less expensive than any of thesynthetic insecticides recommended for the protection of tobaccocrops against the tobacco caterpillar, and it is a materialwhich is readily available, safe to handle, and non-polluting(Joshi et al. 1984).Except for leafhoppers and planthoppers, relatively few fieldtrials have been directed against insects that feed on thevascular system of plants. Rice plants sprayed weekly withultra—low volumes of neem oil effectively controlled populationsof green leafhoppers, Nephotettix virescens (Distant), reducedthe incidence of rice viral diseases, and increased yields(Saxena et al. 1981; Saxena 1989). Applications of neem alsocontrolled numbers of whitef lies on zucchini squash andleafhoppers on eggplant (Dreyer 1984; Dreyer 1987).Unfortunately, the preceding examples involving the use of neemfor the control of pests under field conditions did notdetermine the AZA content of the spray materials, which makes itdifficult to compare results from different trials. Also, these16tests were conducted under tropical or sub—tropical conditions,and results may differ substantially under temperate conditions.In the present study, the efficacy of formulated NSO and anethanolic neem seed extract (NSE) was evaluated in laboratoryand field experiments for the control of aphids. Pyrethrumtreatments were included for comparative purposes, as it is themost commonly used botanical insecticide worldwide. Unlike mostprevious aphid control studies, neem formulations used in thesetrials contained determined concentrations of the most activeprinciple, AZA. It is well known that good or even excellentcontrol of insects with insect growth regulators in thelaboratory does not automatically translate to equally effectivecontrol under field conditions (Schinutterer & Hellpap 1989).Most studies to date have focused on control of aphids underlaboratory conditions. Therefore, foliar applications tostrawberry, Fragaria X ananassa Duch.; head lettuce, Lactucasativa L.; cabbage, Brassica oleraceae L.; and sweet greenpepper, Capsicum annuum L.; for the control of several speciesof economically important aphids provides an accurate assessmentof neem’s performance under temperate North American fieldconditions.17MATERIALS AND METHODSLaboratory Studies. Lettuce ‘Ithaca’; sweet pepper‘California Wonder’; rutabaga ‘Laurentian’, Brassicanapobrassicae (L.); and strawberry ‘Totem’; were grown inplastic pots (10 cm diameter) containing a mixture of sandy loamsoil and peatmoss (4:1). Pots were placed in a greenhouse withsupplemental lighting supplied by sodium vapor lamps (Poot P1-780, P.L. Light Systems Canada, Grimbsy, Ont.) (l,5OO fc), orin growth chambers illuminated with incandescent and fluorescentlights under a photoperiod of 16:8 (L:D). Plants werefertilized bi-weekly with soluble 20:20:20 (nitrogen:phosphorous:potassium) (Peter’s Professional, W.R. Grace, Fogelsville,Pa.) and irrigated as required.At four to six weeks of age, when the plants had acquiredseveral true leaves, the foliage was treated with formulated NSO(0.5%, 1.0% or 2.0%), at approximately 10 ml/plant, or with ablank formulation of emulsifier only (1.06 mi/L, Mazon BSF19,Mazer Chemicals, Inc., Gurnee, Ii.) as a control. Plants weresprayed from all sides with a hand-held mist sprayer(Continental E-Z Sprayer Plant and Garden Sprayer, ContinentalIndustries, Brampton, Ont.) to completely cover the foliage toincipient runoff. Plants were then enclosed in glass cylinders,12.5 cm X 10 cm diameter, which fit snugly inside the upper rimof the pots, while the upper end was covered with fine meshcloth to provide ventilation.18With the use of a moistened, fine squirrel—hair brush, tenadult aphids were transferred to each plant either 48 hr priorto spraying (post-infestation spray), or immediately after theplants had dried (pre-infestation spray). Green peach aphids,Myzus persicae (Sulzer), were transferred to rutabaga andpepper, while lettuce aphids, Nasonovia ribisnigri (Mosley), andstrawberry aphids, Chaetosiphon fragaefolii (Cockerell), weretransferred to lettuce and strawberry, respectively (seeAppendix for list of aphid scientific names). Followingtreatment, the individually—enclosed plants were placed in agrowth chamber at 20°C and a photoperiod of 16:8 (L:D) for oneweek, after which time aphids were counted. Each treatment wasapplied to six plants, and the entire experiment was conductedon two separate occasions.Field Studies. During 1989 and 1990, plots of lettuce‘Ithaca’, pepper ‘California Wonder’, cabbage ‘Stonehead’, andstrawberry ‘Totem’, were established at the Plant Science FieldStation (Totem Field), U.B.C. (Figure 1.1). Individual plots oflettuce, cabbage, and pepper, consisted of two rows of sevenplants, with aphid counts taken from the center ten plants.Pepper and lettuce plants were spaced 0.2 m within rows and 0.8m between rows, while cabbages were spaced 0.3 m within rows and1.0 in and 1.25 m between rows in 1989 and 1990, respectively.Adjacent plots were separated by untreated guard rows, and theends of plots were separated by 1.5 m of bare soil. Strawberryplots consisted of single rows of ten plants spaced 0.25 in19Figure 1.1. Research plots at the Plant Science ResearchStation (Totem Field), U.B.C., for evaluating the efficacy offoliar neem sprays for the control of aphids. (a) LettuceIthaca immediately after transplanting to the field, and (b)at pre—heading stage just prior to destructive sampling (post—spray).II—., 1-— r—20apart, with 1.0 in between adjacent rows. Lettuce wastransplanted on 22 Sept., 1989, and 14 Sept., 1990; cabbage on16 July, 1989, and 17 July 1990; and pepper on 25 July and 4Sept., 1990; while strawberries were planted as bare—root stockon 25 May, 1990. Both lettuce and pepper were transplanted fromthe greenhouse at approximately four weeks of age, and cabbageat five weeks of age. Lettuce and strawberry plots were weededby hand, while trifluralin (TreflanM) was applied to plots ofpepper and cabbage (2.0 L/ha) prior to transplanting for thecontrol of weeds. Each experiment was designed as randomizedcomplete blocks (RCBD) with four replicates per treatment, andall experiments were conducted twice for each crop.Except for cabbage, treatments consisted of formulated NSO orNSE (0.5%, 1.0%, or 2.0%), pyrethrum (0.5 g/L), a mixture of NSEor NSO (1.0%) and pyrethrum (0.5 g/L), or emulsifier only (1.06mi/L, Mazon BSF19) as a control. Cabbages were treated with asingle rate of NSO and NSE (1.0%), pyrethrum (0.5 g/L), andthree rates of NSO or NSE (0.5, 1.0, and 2.0%) mixed withpyrethrum (0.25, 0.5, and 1.0 g/L), as well as emulsifier onlyas a control. NSO 1% and NSE 1% formulations both containedapproximately 20 ppm AZA. Except for cabbages, foliar sprayswere applied twice at weekly intervals using commercial gardensprayers (Chapin Model 1101, R.E. Chapin Manufacturing Works,New York) at rates of 0.25 L/plot. Cabbages were sprayed weeklyfor three weeks at a rate of 0.4 L/plot. The desired volume wasapplied by four or five passes per row, which adequately covered21leaf surfaces. Treatments were first applied to lettuce on 3Oct., 1989, and 28 Sept., 1990; to pepper on 5 and 18 Sept.,1990; to cabbage on 23 Aug., 1989, and 9 Aug., 1990; and tostrawberry on 3 July and 7 Aug., 1990.Natural aphid populations on strawberry and lettuce weresupplemented with artificial infestations of five adult C.fragaefolii or N. ribisnigri per plant, respectively, one weekprior to the first sprays. One week after the final spray,plants were carefully harvested, foliar weights recorded, andaphid numbers per plant assessed. Counts of M. persicae and A.gossypii on pepper; C. fragaefolii and Fimbriaphis fimbriataRichards on strawberry; M. persicae and the cabbage aphid,Brevicoryne brassicae (L.) on cabbage; and N. ribisnigri and acomplex of aphids, consisting mostly of the foxglove aphid,Aulacorthum solani (Kalt.), and the potato aphid, Macrosiphumeuphorbiae (Thomas), on lettuce were recorded separately.Determination of Azadirachtin Content. The AZA content offormulated NSO and NSE solutions, provided by Safer Ltd.(Victoria, B.C.) was determined using reverse—phase gradienthigh performance liquid chromatography (HPLC) (Isman et al.1990a). The HPLC system consisted of a Waters model 640chromatograph (Millipore Canada Ltd., Waters ChromatographyDiv., Mississauga, Ont.) with a model 490 multiwave UV detector.A comparative standard of purified (>95%) azadirachtin wassupplied by J.T. Arnason (University of Ottawa, Ottawa, Ont.).Statistical Analysis. Aphid numbers per plant were22transformed to remove dependency of the variances on the means(Little & Hills, 1978). M. persicae counts on pepper in thelaboratory and on pepper and cabbage in the field weretransformed by 1og0(x+1.0), B. brassicae numbers bylog10(x+1.0), all other field counts by 1og(x+1.O); while theremaining laboratory counts were transformed by square root(x+0.5). Plant weights were not transformed. For clarity, theuntransformed mean numbers of aphids per plant are shown in alltables.Transformed values were subjected to analysis of variance(ANOVA) and separation of means was determined by Tukey’smultiple range tests (Wilkinson 1990). Orthogonal contrastswere used to compare controls with the three neem treatmentrates; with those that were significantly different (p<0.05)subsequently subjected to regression analysis for thedetermination of effective concentrations required to reduceaphid populations by 50% (EC50). The two field trials on eachcrop were analyzed separately due to differences in plant size,time of season, and other variable conditions.23RESULTS ND DISCUSSIONControl in the Laboratory. In the laboratory, with theexception of pre- and post-infestation treatments of 0.5% NSO tostrawberry, foliar applications of NSO significantly reducedaphid numbers on pepper, rutabaga, lettuce and strawberry at alltreatment rates (p<0.05) (Table 1.1). NSO effectively reducedaphid numbers in a dose-dependent manner, with EC50 valuesranging from 1.4% for C. fragaefolii on strawberry, pre—infestation spray, to as low as 0.2% for M. persicae on pepper,pre— and post-infestation sprays. These values correspond toapproximately 28.0 ppm AZA and 4.0 ppm AZA, respectively, basedon HPLC analysis of the formulated NSO. A 1% rate of neem, thestandard concentration used in these trials, reduced M. persicaenumbers on pepper and rutabaga an average 95.4% and 77.7%,respectively, compared to their controls; while numbers of N.ribisnigri and C. fragaefolii were 74.5% and 44.6% lower,respectively, on plants treated with NSO at the same rate.Based on the 95% confidence limits, comparisons of the slopes(b) of the regression equations indicates that applications ofNSO 48 hr following aphid infestation (post-infestation) werenot significantly different (p>0.05) from applicationsimmediately prior to the infestation (pre-infestation) for anyof the species tested. These results suggest that contacttoxicity of neem does not contribute significantly to thereduction in aphid numbers. Otherwise, adults and offspring24Table 1.1. Aphid control on intact plants in the laboratory followingpre-infestation (Pre-I) and post-infestation (Post-I) foliar applications offormulated neem seed oil (NSO)Mean no. aphidsa/plant (SEM)Species M. persicae M. persicae N. ribisnigri C. fragaefolii/host /pepper /rutabaga /lettuce /strawberryTreatment Pre-I Post-I Pre-I Post-I Pre-I Post-I Pre-I Post-IControl lll.5a 130.2a 145.9a l50.8a 71.8a 73.5a 5l.3a(18.9) (16.4) (8.6) (16.1) (6.6) (7.8) (4.6) (8.4)NSO 0.5% ll.7b l6.2b 68.4b 93.6b 36.7b 54.3ab 37.9a(3.1) (2.7) (7.0) (14.8) (5.7) (5.0) (4.3) (8.8)NSO 1.0% 6.3b 4.7c 21.6c 15.5c 45.7b 21.6bc(1.4) (1.1) (4.8) (7.0) (2.9) (3.3) (3.7) (6.5)NSO 2.0% 0.8c l.ld 9.7d 3.ld 16.3c 6.3c 20.7c 8.5c(0.4) (0.6) (2.4) (1.7) (2.4) (1.3) (3.1) (4.3)EC50(%NSO) 0.2 0.2 0.6 0.6 0.9 0.5 1.1 1.40.94 0.96 0.99 0.97 0.91 0.97 0.91 0.97bb—1.46 —1.74 —8.17 —9.73 —4.26 —5.59 —3.36 —3.87(95% C.I.) (±0.68) (±0.44) (±1.87) (±2.23) (±1.58) (±1.04) (±1.10) (±1.59)Means within a column followed by the same letter are not significantlydifferent (p>O.O5; Tukey’s multiple range test).aMYZUS persicae (Sulzer), green peach aphid; Nasonovia ribisnigri (Mosley),lettuce aphid; Chaetosiphon fragaefolii (Cockerell), strawberry aphid.bslope of the regression equation.25produced during the first two days should have suffered fromgreater rates of mortality resulting from contact with dropletsof NSO when they were on the plants at the time of treatment.However, 50% mortality of second instar aphids topically treatedwith NSO ranged from 1.7 to 5.3% NSO (Table 3.6), indicatingthat contact toxicity should contribute to the control ofaphids. Aphids on intact plants may have been sheltered fromthe spray droplets to a large extent. According to Schmutterer(1990a), neem possesses relatively weak contact toxicity.Sprays of Margosan—OTM did not have to contact larval greenhousewhitef lies, Trialeurodes vaporarium (Westwood), to provideadequate control (Lindquist et al. 1990).Foliar applications of NSO resulted in significantly bettercontrol of M. persicae on pepper than on rutabaga at all testedrates (Table 1.1). Estimated EC50 values for M. persicae onpepper are 0.2% NS0 for both pre- and post-infestation sprays,while for this aphid on rutabaga the equivalent EC501 are both0.6% NSO. The different levels of control for this aphid onpepper as compared with rutabaga indicates that theeffectiveness of neein is influenced by the host plant of theaphid. It has been demonstrated previously that control ofwhitef lies with neem is influenced by the species of host plant(Schmutterer 1990a), and soil drenches with Margosan—OTM reducedthe number of adult leafhoppers on marigold and chrysanthemum,but not on zinnias (Knodel-Montz et al. 1985). The cause ofthis difference is currently unknown, but variability in the26systemic activity of neem may vary with the species of plant.Control in the field. Applications of NSO effectively reducedC. fragaefolii and F. finibriata numbers on strawberry. AverageEC50 values for C. fragaefolii in the field (0.75%) (Table 1.2)are comparable to those established in the laboratory (1.25%)(Table 1.1). Compared to the controls, 1% NSO reduced C.fragaefolii numbers in the field by 55.2% and in the laboratoryby 44.6%. The generally poorer control in the laboratory mighthave resulted from the inability of the aphids to move off thecaged plants. In the current study, laboratory trials indicatedthat 1% NSO applied to leaf disks deterred feeding of C.fragaefolii, but not N. ribisnigri or M. persicae (Table 2.2).A 1% NSE spray provided significantly better control of C.fragaefolii during the second trial (p<0.05), but in all otherinstances 1% NSE and 1% NSO were equally effective for thecontrol of aphids on strawberry.Pyrethrum significantly reduced F. .fimbriata numbers onstrawberry by more than 50% (p<0.05) (Table 1.2), but theaddition of pyrethrum to 1% NSO did not improve efficacycompared to 1% NSO alone (p>0.05). Pyrethrum sprays wereineffective, however, for the control of C. fragaefolii(p>O.05), and numbers of this aphid on strawberry treated with amixture of pyrethrum and 1% NSO were not different from numberson plants treated with 1% NSO alone (p>0.05) (Table 1.2). Thelarger size of the strawberry plants during the second triallikely accounts for the poorer control of C. fragaefolii and F.27Table 1.2. Aphid control on strawberry in the field following foliarapplications of formulated neem seed oil (NSO) and neem seed extract (NSE)Mean no. aphidsa/plant (SEM) Plant weight (g)C. Eragaef0111 F. fimbriataTreatment Trial 1 Trial 2 Trial 1 Trial 2 Trial 1 Trial 2Control 21.4a ll.9a 5.7a 4l.2ab 134.2a(1.8) (2.5) (0.7) (1.5) (1.3) (6.3)NSO 0.5% 13.7b 3.4bc 2.3b 7.9ab 41.Oab l20.3a(1.5) (0.8) (0.5) (1.5) (1.5) (7.2)NSO 1.0% 6.Ocd 8.9a 0.4c 3.8c 33.7cd 120.6a(0.9) (1.5) (0.1) (1.0) (1.4) (4.2)NSO 2.0% 3.6d 2.3c 0.4c l.9c 30.4d l26.8a(0.6) (0.5) (0.2) (0.4) (1.4) (2.6)NSE 1.0% 6.5cd 4.lbc 1.Obc 3.3c 45.2a l15.7a(0.8) (1.0) (0.3) (0.7) (1.7) (8.0)Pyrethrum l6.2ab 8.2a 4.3bc 43.2ab l30.2a(1.5) (1.2) (0.5) (0.7) (2.1) (5.2)NSO 1.0% ÷ 7.9c 6.3ab 0.6c 2.4c 37.lbc l14.8aPyrethrum (1.5) (1.3) (0.3) (0.6) (1.3) (4.4)EC50(%NSO) 0.6 0.9 0.4 0.60.94 0.27 0.81 0.92bb—1.49 —1.01 —1.52 —1.27(95% C.I.) (±0.38) (±0.79) (±0.60) (±0.44)Means within a column followed by the same letter are not significantlydifferent (p>O.O5; Tukey’s multiple range test).achaetosiphon fragaefolii (Cockerell), strawberry aphid; Fimbriaphisfimbriata Richards.bSlope of the regression equation.28fimbriata during this time. Lindquist et al. (1990)demonstrated that control of greenhouse whitef lies on poinsettiawith Margosan—OTM was not affected by droplet size or spraydensity, but only dosage. They were able to show that goodcoverage of both the top and bottom leaf surfaces improvedefficacy.Although applications of NSO reduced N. ribisnigri numbers onlettuce in the laboratory in a dose-dependent relationship withan average EC50 of 0.7% (Table 1.1), neither NSE nor NSO wereeffective for the control of this aphid in the field (p>0.05)(Table 1.3). The aphid complex on lettuce was also not affectedby applications of neem. Pyrethrum significantly reduced N.ribisnigri populations in the first trial (p<0.05), but amixture of 1% NSO with pyrethrum did not suppress aphid numbersfurther (p>0.05) (Table 1.3). Neem sprays might not havecontrolled aphids on lettuce in the field due to theenvironmental degradation of AZA (Schmutterer 1990a; Walter &Knauss 1990), particularly from the action of rain andultraviolet light, but changes in plant growth or leaf cuticlethickness cannot be overlooked. Lettuce plants began to formheads more rapidly in the field than in the laboratory, whichmay have shielded the aphids from the neem sprays. Temperatureswere relatively cool during the lettuce field trials, withaverage mean daily temperatures of 11.5°C (2.6 cm precip.) and10.6°C (5.1 cm precip.) during the first and second trials,respectively (Env. Can., Vancouver Int’l Airport, Vancouver,29Table 1.3. Aphid control on lettuce in the field following foliarapplications of formulated neem seed oil (NSO) and neem seed extract (NSE)Mean no. aphidsa/plant (SEM) Plant weight (g)N. ribisnigri Aphid complexTreatment Trial 1 Trial 2 Trial 1 Trial 2 Trial 1 Trial 2Control 25.4a 4.9abc 0.4a 3.7b 24.7a 41.Oabc(3.1) (0.9) (0.2) (0.6) (1.5) (2.4)NSO 0.5% 16.9a 4.6abc 0.5a 5.3ab 34.4bc(1.9) (0.9) (0.2) (0.8) (2.0) (1.9)NSO 1.0% 14.9a 3.5abc 0.4a 3.Ob 21.8a 33.3c(2.0) (0.6) (0.2) (0.4) (1.9) (1.8)NSO 2.0% 21.8a 5.Oab 0.4a 2.8b 20.2a 33.7c(2.7) (1.0) (0.1) (0.4) (1.8) (1.9)NSE 1.0% 5.8a 6.4a 43.3ab(1.1) (0.8) (2.3)Pyrethrum 7.Ob 1.7c 3.6b 24.Oa 38.7abc(0.9) (0.3) (0.1) (0.8) (1.9) (1.9)NSO 1.0% + 5.2b 2.4bc 0.2a 1.5c 25.5a 44.6aPyrethrum (0.8) (0.5) (0.1) (0.3) (1.7) (2.7)EC50(%NSO) n.s. n.s. n.s.Means within a column followed by the same letter are not significantlydifferent (p>O.O5; Tukey’s multiple range test).aNasonovia ribisnigri (Mosley), lettuce aphid.bNon_significant regression (p>O.O5).30B.C.). These cool and wet conditions might have reduced theeffectiveness of neem, partly by decreasing aphid feeding(Schmutterer & Hellpap 1989).Except for the second trial involving A. gossypii, NSE reducedaphid numbers on pepper in a dose—dependent manner, withestimated EC50 values ranging from 0.6% to 1.7% (Table 1.4).Although both neem materials contained approximately the sameconcentration of AZA (2,000 ppm) 1% NSO was significantlybetter than 1% NSE (p<0.05) for the control of M. persicae onpepper. Numbers of A. gossypii were less, but not significantlyso (p>0.05), on pepper treated with 1% NSO compared to 1% NSE.Because of this difference, EC50 values established in thelaboratory based on NSO applications (Table 1.1) are notcomparable to those from the field. As compared to theirrespective controls, however, NSO 1% reduced M. persicae numberson pepper in the laboratory an average 95.4% (Table 1.1) and inthe field 80.9% (Table 1.4). Although slightly better in thelaboratory, NSO effectively controlled M. persicae on pepper inthe laboratory and in the field.Pyrethrum did not reduce populations of A. gossypil incomparison to the control (p>0.05), while M. persicae numberswere significantly higher on pyrethrum-treated peppers (p<0.05).The increase in M. persicae numbers may have resulted from adeleterious effect on natural enemies (Radcliffe 1972), or froma direct physiological stimulation of aphid reproduction, asdemonstrated for other insecticides (Lowery and Sears l986a;31Table 1.4. Aphid control on sweet pepper in the field following foliarapplications of formulated neem seed oil (NSO) and neem seed extract (NSE)Mean no. aphidsa/plant (SEM) Plant weight (g)N. persicae A. gossypiiTreatment Trial 1 Trial 2 Trial 1 Trial 2 Trial 1 Trial 2Control 25.Ob l0.Ob 96.9a 20.2ab 5.8ab(4.3) (2.0) (22.2) (4.2) (4.4) (0.3)NSE 0.5% 8.8c 14.9b 23.Ob 39.Oa 5.4ab(1.1) (3.5) (2.3) (8.0) (4.4) (0.3)NSE 1.0% 7.2c 7.4b 18.7bc ll.9b 68.Oa 4.7b(0.8) (1.4) (3.5) (2.1) (4.4) (0.3)NSE 2.0% 3.ld 4.3c 9.5c 17.Ob 55.2a (0.9) (1.1) (3.5) (3.4) (0.3)NSO 1.0% 3.3d 3.4c 13.5c 9.Ob 52.9a 5.8ab(0.5) (0.8) (2.6) (1.5) (4.2) (0.3)Pyrethrum 50.Oa 24.5a 48.2a l9.3ab 68.7a 5.Oab(6.4) (3.4) (5.8) (3.4) (4.8) (0.3)NSE 1.0% + 10.Oc 4.8bc 15.Obc 8.9b 68.Oa 5.5abPyrethrum (1.6) (0.7) (1.8) (1.7) (4.8) (0.3)EC50(%NSE) 0.6 1.7 0.5R2 0.96 0.28 0.85—0.67 —0.31 —1.89(95% C.I.) (±0.21) (±0.31) (±0.57)Means within a column followed by the same letter are not significantlydifferent (p>O.O5; Tukey’s multiple range tests).aMYZUS persicae (Sulzer), green peach aphid; Aphis gossypii Glover, cottonaphid.DNon...significant regression (p>O.O5).cslope of the regression equation.32Lowery and Sears 1986b). Not surprisingly, mixtures of 1% NSEand pyrethrum were not superior to 1% NSE alone (p>O.0S).Although plants were much smaller and spray coverage would,therefore, have been better during the second trial, neem sprayswere less effective for the control of M. persicae and A.gossypil in comparison with the first trial (Table 1.4).Weather conditions were similar during the two trials (16.6°C,4.26 cm precip., and 16.1°C, 5.0 cm precip. for the first andsecond trials, respectively), but the pepper plants grew verylittle during the second trial, likely due to the shorterinterval of time from transplanting and from the relativelyhigher density of aphids. Although plants were approximately 10times larger during the first trial, aphid numbers were only 2.5to 5 times higher compared to the second trial. Reduced growthof the plants or higher aphid densities may have limited thesystemic activity of neem.Neem was very effective for the control of both M. persicaeand B. brassicae on cabbage (Table 1.5). For M. persicae, 1%NSO and 1% NSE reduced populations from 86 to 91%, while thesame treatments reduced numbers of B. brassicae 87 to 100%compared to controls. Numbers of M. persicae on cabbage treatedwith pyrethrum were not significantly different from thecontrols (p>0.05). Populations of B. brassicae, however, weresignificantly lower (p<0.05) on pyrethrum-treated cabbagecompared to controls, with an average reduction of 64.5% for thetwo trials. As for the previous field trials, mixtures of NSO33Table 1.5. Control of aphids on cabbage in the field followingfoliar applications of formulated neena seed oil (NSO) and neem seedextract (NSE)Mean no. aphidsa/plant (SEM)N. persicae B. brassicae Plant wt. (g)Treatment Trial Trial Trial Trial Trial Trial1 2 1 2 1 2Control 7.8a 6.7a l.5a 27.3a 996ab 864a(1.3) (1.1) (0.4) (4.6) (7.1) (7.8)NSO 1.0% 0.6b 0.0b 0.6b 1060a 861a(0.6) (0.6) (0.0) (0.7) (6.3) (7.1)NSO 0.5% + 3.4ab 0.3b 3.9b 883b 798aPyrethrum (0.9) (0.5) (0.3) (1.2) (6.0) (7.0)NSO 1.0% + 2.4bc 0.5b 0.2b 4.4b 928ab 770aPyrethrum (0.8) (0.4) (0.5) (1.1) (6.9) (7.2)NSO 2.0% + 1.Oc 0.4b 0.Ob 924ab 830aPyrethrum (0.6) (0.4) (0.0) (0.6) (6.9) (7.7)Pyrethrum 7.4a 5.9a 0.9b 3.Ob 849b 886a(1.3) (1.1) (0.8) (0.6) (6.7) (8.1)NSE 1.0% ——— 0.9b ——— 3.4b ——— 920a(0.6) (1.7) (7.6)NSE 0.5% + ——— 0.7b --- 2.6b ——— 977aPyrethrum (0.5) (1.9) (8.0)NSE 1.0% + ——— 0.6b ——— 0.8b ——— 891aPyrethrum (0.4) (0.2) (8.1)NSE 2.0% + ——— 0.2b ——— 0.7b ——— 812aPyrethrum (0.3) (1.3) (7.2)Means within a column followed by the same letter are not significantly different (p>0.O5, Tukey’s multiple range test).aMyzus persicae (Suizer), green peach aphid; Brevicoryne brassicae(L.), cabbage aphid.34or NSE (1%) with pyrethrum were not more effective than eithermaterial used alone (p>0.05). NSO applied alone or incombination with pyrethrum was not more efficacious than NSEapplied alone or mixed with pyrethrum (p>O.O5) (Table 1.5).During the first trial, combinations of NSO and pyrethrumreduced M. persicae numbers in a dose-dependent manner, butthere were no differences between treatment rates for NSO or NSEwith pyrethrum for the remaining trials (p>0.05).Neexn sprays did not result in visible phytotoxicity to pepper,cabbage (Figure 1.2), or lettuce, and NSO or NSE treatments didnot result in cabbage or pepper plants that were significantlysmaller than the controls (p>0.05) (Table 1.4, Table 1.5). NSEapplied at a rate of 1% did not reduce lettuce weights (p>0.05)(Table 1.3), and although plants treated with 0.5%, 1%, or 2%NSO often weighed slightly less than the controls, thesedifferences were not significant (p>O.05). Applications of NSO,but not NSE, resulted in visible burning of strawberry foliageduring both trials (Figure 1.3). For the first trial, 1% and 2%NSO treatments reduced plant weights significantly (p<0.05)(Table 1.2) compared to controls, and while strawberries treatedwith a mixture of pyrethrum and 1% NSO were slightly smallerthan controls, the difference was not significant (p>0.05).Strawberry plants were damaged by herbicide drift (paraquat)during the second trial and it would be inappropriate,therefore, to utilize the plant weights from this trial as ameasure of phytotoxicity. Generally, NSE, pyrethrum, or a35Figure 1.2w Cabbage Stoneheade in the field one week afterthe final foliar application of (a) lO% neexu seed extract, or(b) emulsifier only as a control.4—. -..5 ‘4Fp -L______. - _Ld36Figure 13. Phytotoxicity of foliar applications of (a) 1O%and (b) 2O% formulated neein seed oil to strawberry eTote& inthe field compared to (c) LO% neem seed extract and (d)emulsifier only as a control037mixture of the two, did not cause a reduction in weights ofstrawberry, lettuce or pepper as compared with the control.Formulated neem oils have proven to be phytotoxic to tomato,potato, onion, and white cabbage (Schmutterer 1990b), and thereis a need to determine the sensitivity of crop plants toformulated NSO and NSE.In spite of concerns about the phytotoxicity of neem oils,they may be useful starting materials for the development ofcommercial insecticides (Figure 1.4). Neem oils may containother active ingredients, and natural antioxidants and UV—absorbing substances may help protect AZA (Figure 1.4) fromenvironmental degradation (Larson 1989; Isman et al. 1990b).Oil—based formulations may be superior to extracts for thecontrol of aphids (Schmutterer 1990a) and provide some controlof insect-transmitted plant viral diseases (Saxena 1989). Neemoils containing over 4,000 ppm AZA are available (Isman et al.1990b) and the oils could be enriched with high AZA extracts.These oil-based materials with higher AZA content could beapplied at lower rates, which would retain some of the benefitsassociated with oils while reducing the risk of phytotoxicity.The aphicidal properties of neem have not been extensivelystudied, particularly under field conditions, and it isdifficult to compare the results from these trials with previousreports. Singh et al. (1988) determined the 24 hr contacttoxicity of aqueous, ethanolic, and hexane extracts of neem seedkernels toward adult mustard aphids, Lipaphis erysimi Kalt., but38ACOOMeazadirachtinFigure 14. Seeds of the Indian neein tree contain the highestconcentration of (a) the limonoid azadirachtin (NationalResearch Council 1992). Crushing of neem seeds produces (b)neem seed (expellor) oil and (c) neem cake.B39their extracts contained only trace amounts of AZA, and theactivity was attributed to salannin, a derivative of salannin,and a non—terpenoid fraction. Neem and AZA possess only weakcontact toxicity, and mortality of adult insects is generallyvery limited (Schmutterer 1990a; Price & Schuster 1990). Inanother study, 1% NSO applied to detached leaves of cowpearesulted in 96.7% and 43.3% mortality of third-instar and adultcowpea aphids, Aphis craccivora Koch, respectively, 24 hoursafter exposure (Patel & Srivastava 1989). The AZA content ofthe oil was not determined, and it was unlikely to have beenresponsible for the observed mortalities. Studies involvingseveral aphid species demonstrated that NSO or AZA applied toleaf disks resulted in few aphid deaths during the first two orthree days, regardless of treatment rates, and completeexpression of mortality occurred only after six to ten days at17°C (Chapter 3). Similarly, neem seed kernel extracts appliedto broadbean killed first-instar A. pisum and A. fabae caged onthe treated plants, but little mortality occurred during thefirst two days after treatment, and many of the mortality curveswere still increasing relative to controls even eight days aftertreatment (Schauer 1984). Rather than direct toxic effects,aphid deaths resulted from the disruption of nymphal molts,which has been demonstrated previously for several other insects(Schmutterer 1990a; Isman et al. 1991).According to Schmutterer (1990a) aphids are only slightlyinfluenced by oil-free extracts of neem. An unidentified aphid40population on tobacco was not affected by an enriched,formulated NSE, but the addition of neem oil reduced aphidnumbers 80% (Schmutterer 1985). In the current trials, NSO wassuperior to NSE for the control of M. persicae, but not A.gossypil, on pepper (Table 1.4), while NSE was as effective asNSO against F. firnbriata and C. fragaefolii on strawberry (Table1.2), and M. persicae and B. brassicae on cabbage (Table 1.5).As well, aqueous neem seed extracts reduced numbers of A.gossypii on potato in the Sudan by 26% to 75% (Siddig 1987).Many earlier studies utilized crude extracts with undeterminedAZA contents, and there is a need for standardization of neemproducts (Larew 1988; Schmutterer & Helipap 1989; Isman et al.1990b). Failure to define the active ingredient(s) in thevarious extracts has led to mixed and contradictory results inthe past (Larson 1989).41CONCLUSIONThis study has demonstrated that neem—based products arepotentially very efficacious against several species of aphidsin the laboratory and in the field. Generally, NSO and NSEprovided levels of control equal to or better than the currentlyused botanical insecticide pyrethrum, and there was little addedbenefit when the two materials were applied as a mixture. Inprevious studies, combinations of NSO with various insecticidesdid not result in superior control of insects on rice ormustard, compared to applications of NSO alone (Sontakke 1989;Mani et al. 1990). In the present study, the level of controlwith NSO and NSE was variable, however, likely due todifferences in aphid susceptibility and changes in temperatureand rainfall. As well, the host plant of the aphid appears toinfluence the degree of control, and greater attention should bepaid to spray coverage and the systemic activity of neem.Therefore, improvements in neem formulations and use of sprayadditives, such as those utilized by Schauer (1984) to improvepenetration of the leaf cuticle, requires further study.Finally, aphids often increase in number following applicationsof broad—spectrum synthetic insecticides, and the effect of neemon predators and parasitoids should be studied more fully todetermine the usefulness of neem in integrated control programs(Schmutterer l990b). Results of the current study (Chapter 6)indicated that sprays of NSQ and NSE resulted in minimal damage42to numbers of natural enemies of aphids on plants in the field,demonstrating that neem is potentially compatible with 1PMprograms. Neem is a promising new material for the control ofaphids, and when targeted for the control of other primarypests, it may also reduce the incidence of secondary aphidoutbreaks.43CHAPTER 2ANTIFEEDANT ACTIVITY OF NEEM TOWARDS APHIDSINTRODUCTIONAphids damage plants due to their direct removal of water andnutrients, or from the transmission of plant diseases duringfeeding. Antifeedants are compounds which when perceived,reduce or prevent insect feeding (Saxena et al. 1981). Theyconstitute important natural barriers against insect attack forsome plants, and because they are generally pest—specific, non—target organisms are generally unaffected. Antifeedants offer anovel approach to vector and disease management by renderingplants unattractive or unacceptable (Saxena & Khan 1981).However, the use of chemical repellents or deterrents for thecontrol of aphid—borne viruses has so far received littleattention, despite possible environmental and safety advantagesover synthetic insecticides (Gibson et al. 1982). As an exampleof the potential use of repellents for the control of vectorsand plant viruses, dodecanoic acid and polygodial, from marshpepper, Polygonum hydropiper, decreased settling of M. persicaeon treated chinese cabbage (Gibson et al. 1982). Polygodialreduced the acquisition and subsequent transmission of beetyellows virus (BYV) and potato virus Y (PVY); while dodecanoicacid decreased the acquisition of BYV, cauliflower mosaic virus,44and potato leaf roll virus, but not PVY. Neem-based aphicidesmay be directly useful for the control of aphid populations, andthe reported antifeedant activity may help prevent the spread ofaphid-transmitted plant diseases.Neem has almost legendary insect repellent and antifeedantproperties, acquired over its long historical use in manycountries of Asia and Africa (Saxena 1986). The majority ofantifeedant studies have involved phytophagous insects withchewing mouthparts, particularly orthopterans and larvallepidopterans and coleopterans, and it was the use of aqueousneem extracts for the protection of crops from destruction bydesert locusts that led to the ‘rediscovery’ of neexn by Pradhanet al. in 1962. Later, a bioassay based on the antifeedantactivity of neem to this insect led to the isolation anddetermination of the most active ingredient, AZA (Butterworth &Morgan 1968; Butterworth & Morgan 1971). Subsequently, theantifeedant activity of neem seed extracts has been reported forvarious insect pests, such as the variegated cutworm, Peridromasaucia Hubner, (Isman et al. l990a); cabbage webworm,Crocidolomia binotalis Zell. (Fagoonee & Lauge 1981); striped,Acalymma vittatum (F.), and spotted, Diabrotica undecimpunctataBarber, cucumber beetles (Reed et al. 1982); green riceleafhoppers (Saxena & Zhan 1985); and brown rice planthoppers,Nilaparvata lugens (Stal) (Saxena et al. 1981), among manyothers. However, the effective concentrations necessary tosignificantly deter feeding varies markedly between insect45pests. In leaf disk choice bioassays, the concentration of AZArequired to reduce feeding by 70% ranged from 1 ppm for thirdinstar fall armyworm, Spodoptera frugiperda Smith, on lima beanto 250 ppm for second instar Colorado potato beetle,Leptinotarsa decemlineata L., on potato (Wood 1990).Previous studies of the antifeedant action of neem extracts orAZA against aphids have produced contradictory results. A neem—based product, RD-RepelinTM was highly repellent to A. pisum atconcentrations from 1 to 10% (Hunter & Uliman 1992). Aphidsavoided contact with treated leaves, abandoned treated leavesmore often, and spent a greater amount of time wandering aboutthe test arena. In another study, AZA applied topically orsystemically to wheat seedlings at a concentration of 500 ppmdeterred settling and probing by bird cherry-oat aphid,Rhopalosiphum padi (L.), and grain aphid, Sitobion avenae (F.)(West & Mordue 1992). Electronic recordings of the feeding ofM. persicae on seedlings of Nicotiana clevelandii Graydemonstrated that AZA applied systemically at rates of 500 or1,000 ppm reduced the time aphids fed on phloem to one thirdthat of controls (Woodford et al. 1991). It should be notedthat these studies utilized very high rates of AZA that would beof little practical importance. Contrary to these results,Griffiths et al. (1989) demonstrated that extracts of neem seedsfailed to deter settling or probing of M. persicae. Similarly,electronic recordings of alate M. persicae feeding on iceburglettuce treated with Margosan-OTM indicated only a slight46reduction in the total amount of time aphids probed the treatedplants, but there was no difference in the amount of timesalivating, walking, or ingesting phloem compared to controls(Braker et al. 1991).In an attempt to clarify the antifeedant action of neem towardaphids, feeding deterrency of NSO, AZA, and the volatile neemcomponent di—n—propyl disulfide, was assessed against severalspecies of aphids in leaf disk choice bioassays. Deterrency ofneem may contribute to the control of aphids observed in thelaboratory and in the field (Chapter 1), and a reduction infeeding could be useful for the control of aphid-borne plantdiseases.47MATERIALS AND METHODSPlant Material. Lettuce ‘Ithaca’; sweet pepper ‘Californiawonder’; strawberry ‘Totem’; broadbean ‘Windsor long pod’, Viciafaba L.; and mustard cabbage ‘Pak—choi’, Brassica chinensis L.;were grown in the greenhouse as outlined in Chapter 1. Leafdisks were removed from young, fully expanded leaves of six toeight week old plants, except for strawberry, which wasmaintained in a perennial manner.Leaf Disk Choice Bioassays. Test conditions for the leaf diskchoice bioassay used in this study were adapted from a methodfor rearing individual apterous M. persicae, or groups of alateM. persicae, on leaf disks of potato (Lowery & Sears l986b;Lowery & Boiteau 1989). Aphids were placed two per disk on fourleaf disks (20 mm diameter) cut from leaves with a cork borerand placed in a petri dish (9 by 50 mm) with ten small holes inthe tight-fitting lid. Dishes were then placed upside down onseveral layers of moist paper towels (KimwipesTM) lining thebottom of clear plastic containers, which were held in a growthchamber (17±2°C) under constant, indirect fluorescent light.For all choice bioassays, two leaf disks treated with the testmaterial and two disks treated with emulsifier only as a controlwere allowed to dry and then arranged alternately in each dishwith their edges barely touching. Because aphids are verysensitive to the condition of their host plant (van Emden 1972),the four disks in each dish were removed from the same leaf or48leaflet. At concentrations of 0%, all four disks were dipped inemulsifier only, and one pair of opposite disks was randomlyassigned to the 0.0% NSO treatment rate. All test solutionscontained the emulsifier Triton X-l00 (1.25 milL) (BDHChemicals, Toronto, Ont.), unless otherwise stated. Thedeterrency of the test materials was determined by theproportion of aphids on the treated disks relative to the totalnumber of aphids on treated and untreated disks.To determine how rapidly neem affected aphid settling,deterrency of a 40% emulsifiable concentrate (EC) NSO (l,600ppm AZA) to adult C. fragaefolii was assessed 1, 3, 6, 24 and 48hr after treatment of the leaf disks. At each time interval,deterrency of NSO was tested at five concentrations (0.0, 0.375,0.75, 1.5 and 3.0%). At each concentration, control disks weredipped in emulsifier (Mazon BSF19) at a concentration equivalentto that used in the corresponding NSO treatment. Six replicates(dishes), each with eight aphids per dish, were used for eachtime interval and concentration.The deterrent activity of neem toward C. fragaefolii, F.fimbriata, and an unidentified Chaetosiphon species (most likelyC. thomasi (Hille Ris Lambers), on strawberry; A. pisum onbroadbean; M. persicae on pepper; and N. ribisnigri on lettuce;was determined in the manner outlined above, except for thefollowing changes. A NSO containing approximately 4,000 ppm AZAwas tested at rates ranging from 0 to 2% (0.0, 0.25, 0.5, 1.0,2.0%) with the position of the aphids assessed after 24 hr. The49entire experiment was replicated twice, resulting in 12replicates for each aphid species and concentration of NSO. Inaddition to adults, experiments were also conducted with firstand third instar C. fragaefolii to determine if aphids respondeddifferently with age. Again, the entire experiment wasreplicated twice, so that 1,440 aphids were involved in thestudy with C. .fragaefolii instars.The possible component of NSO responsible for the deterrentactivity was investigated in a series of three experiments. Inthe first one, choice bioassays with adult C. fragaefolii wereconducted for seven NSO’s containing AZA at concentrationsranging from <50 (undetectable) to 6,877 ppm at rates of 0 to 2%(0.0, 0.25, 0.5, 1.0, and 2.0%). Experiments were replicatedtwice, resulting in 12 replicates for each concentration ofevery oil. The deterrent activity of purified AZA was evaluatedin the same manner at concentrations up to 500 ppm, with thehighest concentration serially diluted by 50% to produce fiveconcentrations (0.0, 62.5, 125.0, 250.0 and 500.0 ppm). M.persicae, N. ribisnigri, and C. fragaefolii were used as thetest aphid species, and each concentration was replicated sixtimes for each species. The repellent activity of the majorvolatile component, di-n-propyl disulfide (Pfaltz and Bauer,Inc., Waterbury, Ct.), to adult C. fragaefolii was assessed atconcentrations up to 10,000 ppm (0, 10, 100, 1,000,and 10,000ppm). Six replicates were tested at each concentration.To be of any practical benefit, the repellent/deterrent action50of NSO would have to persist for several days. Persistence ofthe deterrent effect of NSO (4,000 ppm AZA) toward adult C.fragaefolii was determined for treated leaf disks held in petridishes for one to four days, and for leaf disks from intactstrawberry leaflets dipped in NSO up to 48 hr followingtreatment. For the first trial, leaf disks were treated withNSO at five concentrations (0.0, 0.25, 0.5, 1.0 and 2.0%), asoutlined previously. Leaf disk choice bioassays were thenconducted with adult C. fragaefolii beginning 0 to 4 daysfollowing treatment of the disks. For the second trial, oneleaflet of an intact strawberry leaf was dipped in NSO (1.0 or2.0%), while one of the remaining two leaflets was dipped inemulsifier only as a control. At a concentration of 0.0%, bothleaflets were dipped in emulsifier only, and one was designatedas the NSO treatment. Pairs of treated and control leaf disksfrom an individual leaf were then utilized in choice testsbeginning 0 to 48 hr after the leaf material had dried. On eachday, 12 replicates were used at each concentration for leafmaterial held in the growth chamber, while treatments applied tostrawberries in the greenhouse were replicated 10 times in asingle trial.The AZA content of the NSO’s was determined using reverse—phase gradient high performance liquid chromatography (HPLC) asoutlined in Chapter 1. Stability of the purified AZA solutions,which were stored at 4°C in the dark, was assessed periodicallyby HPLC, and new solutions were made every two to three months51as required.Statistical Analysis. The proportion of aphids on the treateddisks relative to the total number of aphids was transformed byarcsin qx to normalize the variances (Neter et al. 1985).Transformed values were subjected to analysis of variance(ANOVA) (Wilkinson 1990), and linear regression analysis.Inverse prediction (Neter et al. 1985) was used to determine theeffective concentration required to deter 50% of the aphids(i.e. when 25% of the aphids remained on the treated disks).The coefficient of determination (R2) was partitioned, with thevalues shown being equivalent to those from regression based onthe transformed treatment means. Treatment concentrations weretransformed, lne(x+l.O), as required, to improve linearity; andconcentrations in experiments where rates exceeded 100 ppm werescaled to improve precision.For the final choice test bioassay to evaluate the persistenceof NSO applied to intact plants in the greenhouse, followingANOVA, Fisher’s least significant difference test was used todetermine differences between treatment means (Wilkinson 1990).52RESULTS AND DISCUSSIONDeterrency of NSO (40 EC) toward adult C. fraqaefolii did notdiffer for choice feeding bioassays lasting from 1 to 48 hr,indicating that the deterrent effect occurred rapidly, withinthe first hour (Table 2.1). EC50 values decreased slightly asthe duration of the tests increased from 1 to 24 hr, but theslopes of the regression equations did not differ significantly(p>0.05) for any of the bioassays. Multiple regression analysisinvolving both oil concentration and time also demonstrated thatduration of the bioassay (time) was not significant (p=O.968).The antifeedant activity of neem seed extracts to desertlocusts occurred after the insects examined test filter papersimpregnated with the neem solutions (Butterworth & Morgan 1971).Although neem deterred feeding of locusts in a very rapidmanner, it could not be termed repellent, as the insects crawledupon and tasted the treated papers. On the other hand, feweradult sweetpotato whitef lies landed on cotton treated with neemseed extract, (Coudriet et al. 1985), demonstrating that neemwas repellent to this insect. Results from choice testsindicated that fewer A. pisum contacted excised leaves ofbroadbean treated with RD—RepelinTM (Hunter & Ullman 1992).Purified AZA applied to wheat seedlings at concentrations of 250or 500 ppm reduced probing activity and increased locomotoryactivity of S. avenae and R. padi during the first 25 minutes ofa feeding observation trial (West & Mordue 1992).53Table 2.1. Deterrency of neem seed oil (NSO) toward secondinstar strawberry aphid, Chaetosiphon fragaefolii (Cockerell).Effective concentrations of NSO in leaf disk choice testbioassays resulting in 50% movement (EC50) from treated tountreated leaf disks after 1 to 48 hoursTime (hr) EC50 (%NSO) Slope (b) (SEb) R2 p(reg.)1 3.3 —0.187a (0.024) 0.951 0.0273 3.1 —O.l73a (0.066) 0.694 0.0286 2.8 —0.183a (0.062) 0.743 0.01524 2.0 —0.280a (0.063) 0.869 0.00448 4.8 —0.158a (0.036) 0.864 0.049Slopes of regression equations for proportions of aphids onNSO-treated leaf disks vs. me concentration NSO (n=30) followedby the same letter are not significantly different (p>0.05,based on 95% confidence intervals.54Results of the current leaf disk choice bioassays demonstratethat NSO deterred C. fragaefolii in less than one hour, but theresults cannot be used to distinguish between repellent andantifeedant activity. Careful observation of M. persicae, N.ribisnigri, and C. fragaefolii on NSO-treated leaf disks, usinga 10 power magnifying lens, indicated that NSO was not repellentto aphids. During the 20 minutes of observation, all aphidsattempted to probe the treated leaf material at least once.Probing behaviour of M. persicae and N. ribisnigri on leaf diskstreated with NSO did not differ compared to controls. C.fragaefolii probed less and spent more time off strawberry leafdisks treated with NSO compared to controls, but the differenceswere not significant (p>0.05) (data not shown). After feedingfor a variable amount of time, a few adult C. fragaefoliiexposed to NSO behaved in an agitated manner, and rapidly movedoff the leaf disks. These observations suggest that NSO isacting as a feeding deterrent to aphids rather than as arepellent. The responsiveness of aphids to volatile substancesis generally weak and variable, and most studies have concludedthat aphids discriminate between suitable hosts followingingestion of plant fluids (Papaj & Rausher 1983). Aphidsinvariably attempt to probe the surface of whatever substratethey land on (Gibson & Plumb 1977), and it is during these shorttest probes that a small amount of fluid is ingested and‘tasted’ with the precibarial chemosensillae (Backus 1988).Choice test bioassays involving six species of aphids showed55that they were not equally deterred by NSO over the range ofconcentrations tested (Table 2.2). NSO was deterrent to C.fragaefolii, A. pisum, and Chaetosiphon sp. (regression p<0.05),but it was ineffective against F. fimbriata, M. persicae, and N.ribisnigri (regression p>0.05). The effective concentration ofNSO required to deter feeding (settling) of the first threeaphid species ranged from 1.2 to 2.1%, which corresponds to anAZA content of 48 to 84 ppm. Slopes of the regression equationsdid not differ for C. fragaefolii, A. pisum, or Chaetosiphon sp.(p>0.05), however, indicating that NSO was equally deterrent tothese three species.Previous studies with aphids often produced contradictory orinconclusive results. For example, Hunter and Ullman (1992)demonstrated that neem was highly repellent to A. pisum atconcentrations of 1%, while Griffiths et al. (1989) reportedthat neem extracts failed to deter settling and feeding of M.persicae. In light of the current findings, these opposingresults can best be explained by differences in behaviouralresponse to neem for the two species.A high degree of variation in the behavioural response ofinsects to neem extracts and AZA has been documented previously.Concentrations of AZA applied to leaf disks necessary to reducefeeding by 70% ranged from 1 ppm for third instar fall armywormto 250 ppm for second instar Colorado potato beetle (Wood 1990).The concentrations necessary to effectively deter feeding variessignificantly between insect pests, and very high levels are56Table 2.2. Concentrations of neem seed oil (NSO) resulting in50% deterrency (EC50) to several species of aphids in 24 hourleaf disk choice test bioassaysAphid speciesa/ EC50 S1ope(b)’ (SEb) R2 p(reg.)host plant (%NSO)C. fragaefolii/ 1.2 —0.206a (0.027) 0.950 <0.001strawberryA. pisum/ 1.7 —0.153a (0.037) 0.848 0.003broadbeanChaetosiphon sp./ 2.1 —0.126a (0.015) 0.961 0.026strawberryF. fimbriata/ fl.S.C ——— 0.006 0.801strawberryM. persicae/ n.s. --- --- 0.560 0.128pepperN. ribisnigri/ n.s. ——— ——— 0.099 0.888lettuceastrawberry aphid, Chaetosiphon fragaefolii (Cockerell); peaaphid, Acyrthosiphon pisum (Harris); unidentified Chaetosiphonspecies; Fimbriaphis fimbriata Richards; green peach aphid,Myzus persicae (Suizer); lettuce aphid, Nasonovia ribisnigri(Mosley).bSlopes of the regression equations for proportions of aphidson NSO-treated leaf disks vs. concentration NSO (n=60) followedby the same letter are not significantly different (p>0.05,based on 95% confidence intervals).cNon_significant regression (p>0. 05).57required against some species. In choice test bioassays, a0.01% hexane extract of neem seeds was deterrent to Californiared scale, Aonidiella aurantii (Maskell), while concentrationsof 0.1% and 1.0% were required to deter yellow scale, Aonidiellacitrina (Coquillet), and citrus mealybug, Planococcus citri(Risso), respectively (Jacobson et al. 1978). Similarly,significantly fewer female brown plant hoppers and whitebackedplanthoppers, Sogatella furcifera (Horvath), arrived on riceplants treated with ultra—low volumes of NSO at concentrationsfrom 5 to 50%, but NSO was not repellent to female greenleafhoppers (Heyde et al. 1984). Feeding by all three speciesof homopterans decreased with increasing concentrations of NSO,however, suggesting that the antifeedant and repellentactivities of neem are independent. The same may hold true foraphids.Although the deterrency of NSO was variable for differentaphid species, there was no difference in the response betweenfirst instar, third instar, and adult C. fragaefolii (Table2.3). EC50 values were almost identical, and slopes of theregression equations were not significantly different (p>0.05).Deterrency of NSO to adult C. fragaefolii was clearly not duesolely to the presence of AZA. EC50 values for oils with AZAcontents ranging from <50 to 6,877 ppm were very similar, andslopes of the regression lines did not differ significantly(p>O.OS) (Table 2.4), indicating that some other component(s) ofthe oil was responsible for the observed activity. Also, the58Table 2.3. Concentrations of neem seed oil (NSO) applied toleaf disks of strawberry resulting in 50% deterrency (EC50) tovarious instars of strawberry aphid, Chaetosiphon fragaefolii(Cockerell), in 24 hour choice test bioassaysInstar EC50(%NSO) Slope(b) (SEb) R2 p(reg.)first 1.1 —0.234a (0.041) 0.916 <0.001third 1.2 —0.186a (0.023) 0.957 <0.001adult 1.2 —O.206a (0.027) 0.950 <0.001Slopes of the regression equations for proportions of aphidson NSO-treated leaf disks vs. concentration NSO (n=60) followedby the same letter are not significantly different (p>0.05,based on 95% confidence intervals).59Table 2.4. Concentrations of neem seed oils (NSO) containingvariable amounts of azadirachtin (AZA) applied to leaf disks ofstrawberry resulting in 50% deterrency (EC50) toward adultstrawberry aphids, Chaetosiphon fragaefolii (Cockerell), in 24hour choice test bioassaysOil [AZA] EC50 Slope(b) (SEb) R2 p(reg.)%NSO (AZA ppm)<50 0.96 (———) —0.413a (0.122) 0.791 <0.0011,084 0.89 (9.6) —0.34la (0.015) 0.994 <0.0012,500 0.80 (20.0) —O.406a (0.025) 0.989 <0.0014,000 0.98 (39.2) —0.370a (0.073) 0.895 <0.0014,700 1.04 (48.9) —0.353a (0.035) 0.972 <0.0016,877 0.86 (59.1) —0.459a (0.067) 0.939 <0.001Slopes of regression equations for proportions of aphids onNSO-treated leaf disks vs.‘econcentration NSO (n=60) followedby the same letter are not significantly different (p>0.05,based on 95% confidence intervals).60NSO containing no detectable amounts of AZA retained itsdeterrency to C. fragaefolii.Choice test bioassays with various extractives from neem seedsdemonstrated that several components were responsible for theantifeedant activity against a number of insects, includingCalifornia red scale, yellow scale, citrus inealybug, and woollywhitefly, Aleurothrixus floccosus (Maskell) (Jacobson et al.1978). Purified AZA applied to leaf disks of cabbage was notdeterrent to M. persicae at rates up to 100 ppm (R2=0.296,regression p=0.198) (data not shown). AZA was deterrent to C.fragaefolii on strawberry (R2=0.986, regression p=0.00l),however, with an estimated EC50 of 119.5 ppm. At aconcentration of 100 ppm, 27% (±10.5%) of adult C. fragaefoliihad settled on the AZA-treated leaf disks after 24 hr, comparedto 49% (±9.9%) at 0 ppm AZA (data not shown). These resultsindicate that AZA may be responsible for some of the deterrencyof NSO to aphids. For example, a 2% solution of NSO containing6,877 ppm AZA would result in the application of approximately137.5 ppm AZA, which exceeds the EC50 of purified AZA. However,based on the AZA content of this oil, the EC50 corresponds to59.1 ppm AZA (0.86% NSO) (Table 2.4), or approximately one halfthe EC50 of pure AZA (119.5 ppm). Clearly, other components ofNSO contribute to the antifeedant or repellent activity of neemto aphids.Purified AZA applied to cotton leaves deterred feeding ofSpodoptera littoralis (Boisd.) and Earias insulana (Boisd.)61larvae at concentrations as low as 10 ppm, while the neemcomponent salannin was deterrent at rates as low as 100 ppm(Meisner et al. 1981). Schwinger et al. (1984) demonstratedthat the bioactive neem components salannin and 3—deacetylsalannin were as effective as AZA for the prevention offeeding by Mexican bean beetles, Epilachna varivestis Mulsant,with 100% inhibition of feeding occurring at concentrationsaround 0.01% for all three compounds. In their studies,azadiron, azadiradion, 14-epoxyazadiradion, gedunin, nimbinen,6—deacetylnimbinien, and melianone were effective deterrents atconcentrations 10 to 100 times higher. In light of the largenumber and types of triterpenoids isolated from neem (see Joneset al. 1989), deterrency of NSO likely results from the combinedactivities of the numerous compounds.Saxena and Rembold (1984) determined that the volatilecomponent of neem seeds repelled adult female cotton bollwormsprior to contact, while neem seed oil was not repellent, but didinhibit oviposition on contact. AZA neither repelled the mothsnor deterred oviposition. It is likely that the numerousvolatile and non—volatile components of neem work in concert,producing several behavioural responses that differ in magnitudebetween insect species.Studies of the persistence of the deterrent activity of NSOdemonstrated that the antifeedant effect of NSO to C..fragaefolii lasted for at least four days when treated leafdisks of strawberry were held in a growth chamber under low62light conditions (Table 2.5). Although the regression line wasnot significant for the choice test bioassay conducted threedays after treatment of the disks (p=0.292), slopes of theremaining regression lines did not differ significantly fortests conducted on days 0 to 4 (p>0.05) . Estimated EC50 valuesranged from 1.2% NSO for trials conducted immediately after theleaf material had dried (day 0) to 1.9% one day later. Undergreenhouse conditions, however, deterrency of NSO 1% and NSO 2%disappeared after 12 and 24 hr, respectively (Table 2.6). At 24hr post-treatment, fewer adult C. fragaefolii had settled ondisks treated with NSO 1% or 2% than on disks treated withemulsifier only, but the differences were not significant(p>0.05). The elevated light intensity and warmer, drierconditions in the greenhouse likely accounts for the rapidreduction in antifeedant activity as compared to material heldin the growth chamber.As for most natural plant products, neexn materials are readilydegraded in the environment (Ermel et al. 1987; Barnby et al.1989; Walter & Knauss 1990). For example, feeding of fallarmyworm on corn plants sprayed with 20 to 100 ppm AZA wassignificantly reduced for up to seven days when plants were heldin the laboratory. After 72 hours in the field, however, AZAresidues no longer provided significant feeding deterrence evenat rates as high as 600 ppm (Wood 1990).The rapid decline in the antifeedant activity of NSO observedin the present trials might suggest the action of volatile63Table 2.5. Persistence of the deterrent effect of neem seedoil (NSO) toward adult strawberry aphid, Chaetosiphonfragaefolii (Cockerell). Choice test bioassays beginning 0 to 4days post-treatment, for leaf disks of strawberry held in petridishesDay EC50(%NSO) Slope(b) (SEb) p(reg.)0 1.2 —0.206a (0.027) 0.950 0.0011 1.9 —O.123a (0.033) 0.821 0.0032 1.7 —0.134a (0.010) 0.983 0.0013 ——— ——— 0.084 0.2924 1.6 —0.112a (0.094) 0.320 0.020Slopes of the regression equations for proportions of aphidson NSO—treated leaf disks vs. concentration NSO (n=60) followedby the same letter are not significantly different (p>0.05,based on 95% confidence intervals).aNon....significant regression, p>0. 05.64Table 2.6. Persistence of the deterrent effect of neem seedoil (NSO) toward adult strawberry aphid, Chaetosiphonfragaefolii (Cockerell). Choice test bioassays beginning 1 to48 hours post—treatment, for leaf disks from strawberry treatedwith NSO and held in the greenhouseProportion of aphids on NSO-treated disks (SD)Treatment 0 hr 3 hr 6 hr 12 hr 24 hr 48 hrNSO 0.0% 0.47a 0.40a 0.42a 0.45a 0.47a 0.49a(0.09) (0.19) (0.07) (0.10) (0.11) (0.11)NSO 1.0% 0.30b O.12b 0.18b 0.34ab 0.33a 0.47a(0.16) (0.11) (0.16) (0.12) (0.17) (0.15)NSO 2.0% 0.06c 0.21b 0.llb 0.27b 0.34a 0.45a(0.04) (0.13) (0.06) (0.18) (0.21) (0.09)At each time interval, means followed by the same letter arenot significantly different (p>0.05, Fisher’s least significantdifference test).65compounds. For example, the volatile repellent polygodial isunstable, and its activity toward M. persicae is rapidly lostunder field conditions (Gibson et al. 1982). Leaf disk choicebioassays with di-n-propyl disulfide, the major volatilecomponent of neem (Balandrin et al. 1988), however, producednegative results (data not shown). Di-n-propyl disulfideapplied to leaf disks of strawberry at concentrations up to10,000 ppm did not deter C. fragaefolii, and no dose-dependenteffect was evident (R2=0.075, p=0.108). The presence ofvolatile organosulfur compounds might partially explain theinsect repellent effect of neem leaves and seeds (Balandrin etal. 1988), but purified di-n-propyl-disulfide, which comprisesapproximately 76% of the volatile components of neem, does notaccount for the deterrency of NSO toward C. fragaefolii.Volatile components of neem have been implicated in otherstudies as active feeding deterrents toward insects. Forexample, based on electronic monitoring, the odour of NSO (6 to12.5%) permeating test arenas disrupted normal feeding of greenleafhoppers on rice (Saxena & Khan 1986).66ConclusionNeem extracts have been shown to be repellent and/orantifeedant to several homopteran pests, including leafhoppersand planthoppers on rice (Heyde et al. 1984; Islam 1984; Saxena1984; Saxena & Boncodin 1988), sweetpotato whitefly on cotton(Coudriet et al. 1985), Asiatic citrus psyllid, Diaphorinacitri, on citrus (Chiu 1984), and A. pisum on broadbean (Hunter& Ullman 1992). According to Wood (1990), however, thecommercial significance of the antifeedant activity of neem maybe limited, as there appears to be considerable variationbetween insect species in their sensitivity to AZA as anantifeedant, and high concentrations are required to deterfeeding by many species. AZA does not appear to be a generalinhibitor of insect feeding (Butterworth & Morgan 1971).Additionally, there is some evidence that antifeedant activitiesmay be short lived under field conditions.Results of the current investigations involving severalspecies of aphids concur with previous reports. A NSOcontaining 4,000 ppm AZA was deterrent to C. fragaefolii, A.pisum, and Chaetosiphon sp., while feeding of F. fimbriata, M.persicae, and N. ribisnigri was not altered at rates that wouldbe useful for the control of aphids in the field (l.0% NSO)(see Chapter 1). Systemic applications of AZA reduced the totalamount of time M. persicae fed on Nicotiana clevelandii Gray,but only at rates of 500 to 1,000 ppm (Woodford et al. 1991).67Similarly, West and Mordue (1992) found that AZA applied tobarley at concentrations exceeding 250 ppm reduced probing by R.path and S. avenae for up to four days, but effective deterrencyto these aphids would require applications of 12.5% NSO based onthe AZA content of the neem materials used for the control ofaphids on plants in the laboratory and in the field (Chapter 1).Margosan—OTM contains higher amounts of AZA, approximately 3,000ppm (Larson 1989), but applications exceeding 8% would still berequired to effectively deter feeding of R. padi and S. avenae.Margosan—OTM is recommended at a rate of approximately 1%, or 30ppm AZA (Larson 1989; Lindquist et al. 1990).Deterrency of NSO may have contributed to the control of C.fragaefolii in the field (Chapter 1). Regardless of the AZAconcentration, C. fragaefolii was deterred by NSO’s atconcentrations of approximately 1%. The generally poorercontrol of this aphid on caged plants in the laboratory (avg.EC50=l.25% NSO, Table 1.1), compared to control in the field(avg. EC500.75% NSO, Table 1.2), may have resulted from theinability of the aphids to move away from the treated plants.It is questionable whether the deterrent action of NSO would beuseful for the prevention of virus transmission by this aphid,due to the rapid decline in activity under greenhouse conditions(Table 3.6). Deterrency of NSO would likely decline even morerapidly under field conditions.The degree of variability between the species investigated inthis study may help explain some of the conflicting results68regarding the deterrent activity of neem toward aphids. Aswell, these results indicate that the usefulness of neexn—basedproducts for the control of aphids and the diseases theytransmit would, therefore, have to be evaluated separately foreach aphid species.69CHAPTER 3REGULATION OF APHID GROWTH AND DEVELOPMENTBY NEEM SEED OILAND ITS ACTIVE INGREDIENT AZADIRACHTININTRODUCTIONSome of the reported biological activities of neem extracts orAZA include feeding and ovipositional deterrence, repellency,growth disruption, reduced fitness, and sterility (Schmutterer1985; Rembold 1989b; Koul et al. 1990). Of these activities,disruption of development (molting failure) is considered to bethe most profound effect (Saxena 1989; Wood 1990). Generally,concentrations of neem or AZA required for phagodeterrencyconsiderably exceed those which disrupt development (Barnby &Kiocke 1987; Rembold 1989a). For example, AZA added toartificial diet completely inhibited molting of Rhodniusprolixus Stal. at concentrations as low as 1 ppm (Souza Garcia &Rembold 1984), while topical or oral treatment of migratorylocusts resulted in 100% molting failure at a rate of 2 Lg/ginsect weight (Sieber & Rembold 1984). Complete expression ofthe IGR effects of neem are often delayed, but they do notappear to be reversible.In one of the earliest studies of the growth-disruptingeffects of neem, Ruscoe (1972) demonstrated that combined oral70and topical treatment with AZA affected the development ofdiamondback moth, cabbage white butterfly, Pieris brassicae, andtobacco budworm, Heliothis virescens, larvae, as well as nyxnphalcotton stainer, Dysdercus fasciatus. Although the degree ofinhibition varies between species, the growth-disrupting actionof AZA has been recorded for insects in all orders tested todate (Rembold 1984). Molting of insects requires precise coordination between diverse physiological, developmental,biosynthetic, and behavioural systems (Truman 1990). AZAdisrupts the hormonal regulation of the molting process byinterfering with the release and metabolism of ecdysteroids(Bidmon et al. 1987; Rembold 1989b). Examination of thehemolymph ecdysteroid titres of insects topically or orallytreated with AZA demonstrated that peaks of the molting hormoneecdysone were delayed and declined less rapidly compared tocontrols ( Sieber & Rembold 1983; Bidmon et al. 1987; Barnby &Klocke 1990). Prolonged ecdysone levels may also preventrelease of eclosion hormone, so that treated insects undergoapolysis but not ecdysis (Sieber & Rembold 1983; Souza Garcia &Rembold 1984). Resorption of molting fluid is incomplete, newcuticle is not properly formed or sclerotized, and insectsexposed to AZA are unable to shed the old cuticle.In addition to direct toxicity, exposure to neein may result inlong-term chronic effects that significantly reduce theviability of insects (Schmutterer 1985). For example, neemtreatments delayed the emergence of greenhouse whitef lies, and71adults were smaller and developed abnormally (Lindquist et al.1990). Immature insects exposed to neem or AZA often developinto adults that possess physical abnormalities, particularly oftheir wings (Rembold et al. 1982; Schluter & Schulz 1984).These chronic effects may be important in the long—term controlof insect populations.In the current study, the effects of purified AZA and NSO onthe development and growth of several species of aphids weredetermined in a series of laboratory investigations. Studieswere conducted to evaluate the toxicity of neem and assessdifferences in susceptibility between aphid species. The effectof neem on survival of offspring from treated parental aphidswas also evaluated. For M. persicae, the effectiveconcentrations of AZA resulting in 50% mortality (EC50) weredetermined for the four nymphal instars, and for second instarsreared on various host plants. In addition to direct toxicityresulting from molting failure, as a measure of reduced fitness,sizes and physical abnormalities of adult aphids exposed asnymphs to NSO was evaluated. Comprehensive studies of thisnature involving several aphid species on various hosts couldhelp determine how neem controls aphid populations in the field,and explain some of the observed variability (Chapter 1) in thelevel of control.72MATERIALS AND METHODSLaboratory Rearing Conditions. Aphids were reared in petridishes as outlined for the leaf disk choice bioassays (Chapter2), except that only three treated leaf disks were placed ineach dish. Aphids were reared for three days on disks dipped inthe test solutions and then moved to untreated disks for theremainder of the study, with new leaf disks supplied every threedays.Lettuce ‘Ithaca’; sweet pepper ‘California wonder’; strawberry‘Totem’; corn ‘Sunny vee’, Zea mays L.; broadbean ‘Windsor longpod’; mustard cabbage ‘Pak—choi’; and cucumber ‘Marketmore’,Cucumis sativus L., were grown in the greenhouse as outlinedpreviously (Chapter 1). Disks were removed from young, fullyexpanded leaves of six to eight week old plants, except forcorn, which was more suitable for aphid colonization during thetasselling stage.Determination of Treatment Rates. Test materials consisted ofNSO and AZA, and unless otherwise stated, solutions wereemulsified with Triton-X 100 (1.25 ml/L) in distilled water.The amount of solution applied to the leaf disks was quantifiedusing3H2-22,23-dihydro-AZA (supplied by J.T. Arnason,University of Ottawa, Ottawa, Ont.). Measured quantities ofsolution were applied to leaf disks, and after air—drying thedisks were placed in vials (20 ml) containing 5 ml of ethanoland acetone (50:50) for 12 hr. After the vials had been73vigorously shaken, a 150 jl aliquot was measured in a liquidscintillation counter (LS5000 TA, Beckman Inst. Inc., Fullerton,Ca.), and the efficiency of recovery determined by comparison toa standard curve based on measured quantities of3H2—dihydro—AZAadded directly to the scintillation cocktail. Leaf disks werethen dipped in solution, and application rates determined.The AZA content of the NSO was determined by HPLC, as outlinedin Chapter 1.Toxicity of NSO and AZA. The effect of NSO on survival ofsecond instar, fourth instar, and adult M. persicae on pepper,C. fragaefolii on strawberry, and N. ribisnigri on lettuce wascompared with purified AZA. Treatments consisted of NSOcontaining approximately 4,000 ppm AZA (0.5, 1.0 or 1.5%),purified AZA (40 or 80 ppm), or a blank formulation as acontrol. Survival of aphids was recorded daily for three dayson treated leaf disks, and a further six days on untreated leafdisks. As well, to determine if neem had an effect onsubsequent generations, survival of first generation offspringfrom exposed adult (parental) aphids was recorded for nine dayson untreated disks. For each species and instar, aphids werereared two per dish, with six dishes per treatment, and theentire experiment was replicated two or three times. Offspringwere reared up to ten per dish.The effective concentration of AZA resulting in 50% mortality(EC50) was determined for second instar M. persicae on pepper;N. ribisnigri and M. euphorbiae on lettuce; A. pisum on74broadbean; R. padi and the rose—grain aphid, Metopolophiumdirhodum (Walker), on corn; A. gossypii on cucumber; and C.fragaefolii and F. fimbriata on strawberry. Based onpreliminary trials, mortality after nine days (initial threedays on treated disks) was recorded for aphids on disks dippedin AZA at concentrations of 0 to 10 ppm, 0 to 100 ppm, or 0 to1,000 ppm. The highest concentration was serially diluted by50%, providing six treatment rates for each range ofconcentrations tested. For each species, aphids were rearedfive per dish, with five dishes for each concentration.Depending on the degree of variability, each trial wasreplicated once or twice.Differences in susceptibility between instars of M. persicaewas determined by exposing first to adult instars to AZA appliedto leaf disks of pepper as outlined above. Additionally, secondinstar M. persicae were reared on mustard cabbage, lettuce,broadbean, and corn, to determine the influence of the hostplant on toxicity of neem to this aphid.The contact toxicity of neem was determined by application offormulated NSO or AZA to the dorsal abdomen of second instar N.ribisnigri using a microapplicator (Model M, InstrumentationSpecialties Co., Lincoln, Nb.). The approximate weight of theinstar was assessed prior to the experiment, and a fine 5 1syringe (7105 series syringe, Hamilton Co., Reno, NV.) was usedto apply 0.036 j.l solution per insect (0.42 l/mg). AZA and NSOwere tested at rates up to 1,000 ppm and 5.0%, respectively.75Second instar M. persicae, N. ribisnigri, A. gossypii, C.fragaefolii, R. padi, and F. fimbriata were also sprayed withNSO (1.25, 2.5, or 5.0%), or with a blank formulation as acontrol. For each species, sixty aphids were treated at eachconcentration in groups of ten per petri dish, and then reared(5 per dish) for nine days on untreated leaf disks as outlinedpreviously. For treatment, aphids were placed on filter paperin petri dishes (8.5 cm diameter) with fluon (Northern ProductsInc., Woonsocket, RI.) painted around the upper edge to preventescape. The spray tower consisted of a plexiglass tube (15.5 by8.8 cm diameter) with the nozzle from a perfume bottle (JulianoParfum, Fairfield, Ct.) fastened at the top. A short length ofplastic tube connected the nozzle to a syringe (5 ml), which wasused to deliver 1.0 ml as a fine mist (8.4 il/cm2). Contacttoxicity trials were conducted with NSO, containingapproximately 2,000 ppm AZA, formulated by Safer Ltd. (1.06ml/L, Mazon BSF19).Sub—lethal Effect of NSO. To determine the sub-lethal effectof neem on aphid development, third instar M. persicae, N.ribisnigri and C. fragaefolii exposed for three days to NSO asoutlined previously were reared to maturity, eight per dish, onleaf disks of mustard cabbage, lettuce, and strawberry,respectively. Morphological measurements of lengths of hindtibiae and third antennal segments were made at 40X fromunmounted aphids in 70% ethanol using a micrometer eyepiece.Measurement of these scierotized parts provides a measure of76aphid size (Dixon 1985, Lowery & Boiteau 1989). As well,deformities in the wings and legs of adults were observed andtabulated. Treatments consisted of 0.5% NSO (zlO ppm AZA) or ablank formulation as a control. A variable number of disheswere used to produce a sufficient number of adult aphids.Statistical Analysis. Proportions of surviving aphids weretransformed by arcsin ifx to normalize the variances (Neter etal. 1985), and the data was analyzed as outlined in Chapter 2.Morphological differences, based on untransformed micrometermeasurements (42 divisions 1 mm), were determined by 95%confidence intervals. Differences in survival of second,fourth, and adult C. fragaefolii, N. ribisnigri, and M. persicaeexposed to NSO and AZA were determined by chi-square analysis(Zar 1984)77RESULTS AND DISCUSSIONToxicity of NSO and AZA. Except for second instar M. persicaeon leaf disks of pepper treated with 0.5% NSO, no second instarM. persicae or N. ribisnigri survived for nine days on diskstreated with any concentration of NSO or AZA (Table 3.1). At0.5% NSO, approximately 8% of M. persicae survived for ninedays. Representative survival curves for these two speciesexposed to 40 ppm AZA or 1.0% NSO containing approximately 40ppm AZA (Figure 3.1), were virtually identical, demonstratingthat AZA accounts for most of the observed mortality for thesetwo species. Survival of controls after nine days wasapproximately 67% for N. ribisnigri and M. persicae. For C.fragaefolii, however, mortality rates were significantly higher(p<O.05) on disks treated with 1.0% NSO, but not on diskstreated with 40 ppm AZA (p>0.05) compared to controls (Table3.1) (Figure 3.1). Survival of C. fragaefolii on disks treatedwith 0.5, 1.0 or 1.5% NSO was one third that of controls (8.3%),but it was not different for aphids on disks treated with 40 ppm(25%) or 80 ppm AZA (33%) compared to controls (25%) (p>0.O5)Survival of controls was less than optimal for C. fragaefolii,but these results suggest that some component of NSO other thanAZA is responsible for the observed mortality.Although AZA is often considered to be the most bioactivecomponent of neem (Ermel et al. 1987; Isman et al. 1991), neemextracts contain several other limonoids with demonstrated IGR78Table 3.1. Percent survival, days of survival, and number of moltsfor second instar lettuce aphid (LA), green peach aphid (GPA) andstrawberry aphid (SA) exposed to neem seed 01a (NSO) and azadirachtin(AZA) applied to leaf disks of lettuce, pepper, and strawberry,respectivelyPercent Survival Days of Survival Mean No. MoltsTreatment LA GPA SA LA GPA SA LA GPA SAControl 66.7a 66.7a 25.Oa 6.7a 4.9a 4.3a 2.4a 2.Oa l.4aNSO 0.5% 0.Ob 8.3b 8.3b 2.4b 2.8b 0.4b 0.9b l.OaNSO 1.0% 0.Ob 0.Ob 8.3b 2.5b 2.2b 1.8a 0.5b 0.5c 0.6aNSO 1.5% 0.Ob 0.Ob 8.3b l.9b l.9a 0.2b O.5c 0.3aAZA 40ppm 0.Ob 0.Ob 25.Oa 2.4b l.9b 4.6a 0.5b 0.5c l.2aAZA Boppm 0.Ob 0.Ob 33.3a 2.2b 2.Ob 4.3a 0.3b 0.4c l.OaContrasts: bCont. vs.NSO + AZA 0.0]. 0.01 0.27 0.01 0.01 0.08Cont. vs. NSO 0.01 0.01 0.07 0.01 0.01 0.03Cont. vs. AZA 0.01 0.01 0.92 0.01 0.01 0.37NSO vs. AZA 0.90 0.10 0.02 0.78 0.03 0.11Means within a column followed by the same letter are not significantly different (p>O.O5, Tukey’s multiple range test, chi-square testsfor percent survival).Lettuce aphid, Nasonovia ribisnigri (Mosley); green peach aphid,Myzus persicae (Sulzer); strawberry aphid, Chaetosiphon .fragaefolii(Cockerell).9Contains approximately 4,000 ppm AZA based on HPLC analysis.b values for orthogonal comparisons.79i:: 1::60 60• DCl, Cl,*40 *4020 200 I I 0 I I0 1 2 3 4 5 6 0 1 2 3 4 5 6 7DAYS AFTER TREATMENT DAYS AFTER TREATMENT100 C8060 — ControlNSO 1 .0%D40 •AZA 40ppm+20‘4-. . 4-..4.0 I I I I • -0 1 2 3 4 5 6 7DAYS AFTER TREATMENTFigure 3.1. Representative survival curves for second instar(a) green peach aphid, Myzus persicae (Sulzer), (b) lettuceaphid, Nasonovia ribisnigri (Mosley), and (c) strawberry aphid,Chaetosiphon fragaefolii (Cockerell), exposed to 40 ppmazadirachtin (AZA), 1.0% neem seed oil (NSO) containingapproximately 40 ppm AZA, or emulsifier only as a control.80activity (Schwinger et al. 1984; Jacobson 1990). As well,Balandrin et al. (1988) demonstrated that di-n-propyl disulfide,the major volatile constituent of neein seeds, was toxic to thirdinstar mosquito larvae and first instar lepidoptera larvae atconcentrations ranging from 66 to 1,000 ppm. Based on resultsfrom this study, however, NSO and NSE did not possess anyfumigant activity toward aphids, and di-n-propyl disulfideapplied to leaf disks at concentrations as high as 10,000 ppmwas not toxic to second instar M. persicae or N. ribisnigri(data not shown). Singh et al. (1988) attributed the toxicityof aqueous, ethanolic, and hexane extracts of neem seed kernelsto adult L. erysimi to the limonoid salannin, as their extractscontained only trace amounts of AZA. In the current study, NSOand AZA were not toxic to adult aphids, and survival did notdiffer (p>0.05) from controls at any of the rates tested (Table3.2). For the three species combined, survival of adult aphidsfor NSO, AZA, and control treatments averaged 75%, 80%, and 77%,respectively.Survival of fourth instar N. ribisnigri was not significantlydifferent for NSO as compared with AZA (p>0.05) (Table 3.2).From 0% to 16.7% N. ribisnigri survived 12 days on lettucetreated with NSO or AZA, compared to 100% survival of controls.Although survival of fourth instar C. fragaefolii was slightlylower on leaf disks treated with NSO, survival did not differfor any treatments (p>0.05) (Table 3.2). Days of survival wasnot a very useful measure of aphid performance in these trials,81Table 3.2. Percent survival and days of survival for adult and fourthinstar lettuce aphid (L9, green peach aphid (GPA) and strawberry aphid (SA)exposed to neem seed oil (NSO) and azadirachtin (AZA) applied to leaf disksof lettuce, pepper, and strawberry, respectivelyAdult (9 day) Fourth Instar (12 day)Surv. (%) Surv. (d) Surv. (%) Surv. (d)Treatment LA GPA SA LA GPA SA LA SA LA SAControl 75.0 91.7 70.8 5.0 5.8 8.1 lOO.Oa 62.5 12.0 7.8NSO 0.5% 66.7 100.0 75.0 4.8 6.0 7.8 8.3b 62.5 4.1 7.6NSO 1.0% 83.3 75.0 45.8 5.4 5.6 6.3 0.Ob 50.0 3.8 4.1NSO 1.5% 87.5 91.7 70.8 5.7 6.0 7.3 8.3b 50.0 2.7 6.3AZA 4oppm 87.5 91.7 70.8 5.1 6.0 7.3 l6.7b 66.7 6.0 6.8AZA Boppm 75.0 83.3 75.0 5.2 5.2 7.8 8.3b 66.7 3.6 8.5Contrasts: bCont. vs.NSO + AZA 0.47 0.76 0.16 0.01 0.49Cont. vs. NSO 0.41 0.94 0.12 0.01 0.30Cont. vs. AZA 0.65 0.44 0.35 0.01 0.95NSO vs. AZA 0.67 0.26 0.45 0.11 0.23Means within columns are not significantly different (p>O.O5, Tukey’smultiple range test, chi—square test for percent survival), except forsurvival of fourth instar lettuce aphid, where control treatment isdifferent from all others.Lettuce aphid, Nasonovia rihisnigri (Mosley); green peach aphid, Myzuspersicae (Sulzer); strawberry aphid, Chaetosiphon fragaefolii (Cockerell).acontains approximately 4,000 ppm AZA based on HPLC analysis.b values for orthogonal comparisons.82as complete nymphal mortality only occurred after several days.Differences would have become increasingly pronounced over time,but the distinction was not clear even after nine days.Although adults exposed to NSO or AZA did not suffer higherrates of mortality, offspring from treated M. persicae and N.ribisnigri (but not C. fragaefolii) generally had higher ratesof mortality compared to controls (Table 3.3). Although notsignificantly different (p>0.05), survival of offspring fromadult M. persicae exposed to NSO was more than 50% lower thancontrols at all of the tested rates. No offspring from eitherAZA treatment survived, but because of the small number ofreplicates and variability in survival rates, AZA treatmentswere not significantly different from NSO treatments (p>0.05).AZA treatments were significantly different from controls(p<0.05). Based on orthogonal contrasts, survival of aphids inthe controls was significantly different from the NSO treatments(p<0.00l) and from the AZA treatments (p<0.0l), while AZAtreatments and NSO treatments did not differ (p=0.058). Itshould also be noted that although survival of offspring in theAZA treatments appears to be poorer, the average concentrationof AZA in the NSO treatments was only 40 ppm, compared to 60 ppmfor purified AZA.For N. ribisnigri, all rates of NSO resulted in significantly(p<0.05) lower survival of offspring compared to the control,with from 47 to 83% fewer offspring surviving for nine days(Table 3.3). All rates of NSO resulted in higher mortality83Table 3.3. Nine day survival of first generation aphids fromadults exposed for three days to neem seed oil (NSOa) orazadirachtin (AZA)Aphid Species” (n)CTreatment M. persicae N. ribisnigri C. fragaefoliiControl 42.Oa (177) (195) 59.4a (82)NSO 0.5% l6.4ab (79) 46.9b (105) 69.5a (79)NSO 1.0% 10.Oab (27) 37.4b (69) 52.8a (68)NSO 1.5% 19.Oab (28) (46) 46.5a (67)AZA 4oppm 0.Ob (39) 49.6ab (139) 55.8a (75)AZA BOppm 0.Ob (26) 59.3a (85) 62.5a (62)Means within a column followed by the same letter are notsignificantly different (p>0.05; chi-square tests).acontains approximately 4,000 ppm AZA based on HPLC analysis.bMyzus persicae (Sulzer), green peach aphid; Nasonoviaribisnigri (Mosley), lettuce aphid; Chaetosiphon fragaefolii(Cockerell), strawberry aphid.cNumbers of offspring reared for each treatment.84compared to AZA 80 ppm (p<0.05), but not AZA 40 ppm (p>0.05).Average survival of offspring from AZA-treated adults was 38%lower than controls, but they were not significantly (p>0.05)different. Based on orthogonal contrasts, aphid survival onleaf disks treated with AZA was poorer than controls (p=0.Ol),and AZA treatments did not differ from NSO treatments (p=0.067).Mortality of C. fragaefolii did not differ from controls for anyrate of NSO or AZA (p>0.05) (Table 3.3).Due to the variability in survival of first generation aphidsfrom treated parents, more detailed studies are warranted tofully assess the impact of neem on subsequent generations. Itis clear, however, that exposure of adult aphids to neexn affectsthe fitness of their offspring. In preliminary trials to assessthe resistance of aphids to neem, plants infested with M.persicae and N. ribisnigri were sprayed with NSO 2.0% in anattempt to reduce the aphid populations by approximately 90%(see Chapter 1). No aphids survived, however, after transferone week later to newly treated plants. Similar trials wereconducted with NSO at concentrations as low as 0.25%, but in noinstance did aphids survive for more than a few generations, andthe experiment was subsequently discontinued. According toSchmutterer (l990a), control of aphids often requires repeatedapplications. Improved control may result from several factors,including the magnification of deleterious effects fromgeneration to generation. Viviparous paedogenetic insects, suchas aphids, may be particularly sensitive to the long—term action85of neem.Biological properties of aphids, such as fecundity, size, anddevelopmental rate, are partly determined while the embryo isdeveloping within the mother (Adams & van Emden 1972), andviviparous reproduction would likely expose the developingembryos to the detrimental effects of neem. Neem applied toinsect eggs has little effect on viability (Schmutterer 1985),but eggs deposited by treated adults may have damaged chorions,less yolk, and be more susceptible to fungal infections (Schulz& Schluter 1984). Eggs may fail to develop properly, and beless viable. To my knowledge, the trans—generational effect ofneem on survival of viviparous insects has not been reportedpreviously. Reproduction of adults treated with NSO or AZA wasdramatically reduced (see Chapter 4), and these results are,therefore, based on a relatively small sample size. Thisphenomenon may be important for the long—term reduction in aphidpopulation growth, and it should be studied more fully.Susceptibility of aphids to AZA was variable, with EC50 values(9 day) for nine species of aphids reared for an initial threedays on leaf disks dipped in AZA ranging from as low as 2.4 ppmfor M. persicae on pepper, to 635 ppm for C. fragaefolii onstrawberry (Table 3.4). These results support the earlierfindings that second instar C. fragaefolii are sensitive to NSO,but less so to AZA (Table 3.1 and Figure 3.1). The bioactivityof neem to insects varies from species to species (Schmutterer1988; Schmutterer l990a), likely due to several factors. The86Table 3.4. Effective concentration of azadirachtin (AZA)resulting in 50% mortality (EC50) after 9 days for aphidsplaced as second instars for 3 days on treated leaf disksAphid speciesa Host plant EC50b (95% C.I.) R2 n(ppm AZA)M. persicae pepper 2.4 (±0.5) 0.878 30N. ribisnigri lettuce 3.1 (±0.6) 0.905 30M. euphorbiae lettuce 8.0 (±1.2) 0.938 60A. pisum broadbean 44.7 (±6.4) 0.746 60M. dirhodum corn 67.6 (±3.6) 0.948 60F. fimbriata strawberry 69.1 (±2.3) 0.981 60R. padi corn 87.9 (±4.5) 0.963 60A. gossypii cucumber 90.6 (±15.0) 0.807 30C. fragaefolii strawberry 635.0 (±28.4) 0.952 30aGreen peach aphid, Myzus persicae (Sulzer); lettuce aphid,Nasonovia ribisnigri (Mosley); potato aphid, Macrosiphumeuphorbiae (Thomas); pea aphid, Acyrthosiphon pisum (Harris);rose-grain aphid, Metopolophium dirhodum (Walker); Fimbriaphisfimbriata Richards; bird cherry-oat aphid, Rhopalosiphum padi(L.); cotton or melon aphid, Aphis gossypii Glover; strawberryahid, Chaetosiphon fragaefolii (Cockerell).From linear regression analysis and inverse prediction.87interspecific variability observed in the current study could beintrinsic to the aphids, or movement of the active components ofneeni into leaves might vary between plant species. Onstrawberry, the EC50 for C. fragaefolii is more than nine timesthat of F. fiinbriata, while for the pairs of aphids on lettuceand corn the EC501s, though significantly different (p<0.05),are similar in magnitude (Table 3.4).Differences in the amount of solution applied to leaf disks ofthe various plant species would not account for much of theobserved variability. Based on radioisotope measurements, theamount of solution applied to lettuce, corn, cabbage, pepper,broadbean, cucumber and strawberry was approximately 14.5, 12.1,14.0, 18.3, 20.0, 20.4, and 27.2 J.Ll per disk, respectively. Infact, there does not appear to be any correlation of EC50 valueswith the amount of solution applied to each host species. Thehighest value (635 ppm AZA) was recorded for C. fragaefolii onstrawberry, which received the highest dosage.Susceptibility of M. persicae to AZA is clearly dependent onthe host plant. The EC50 for second instar M. persicae on corn(43.3 ppm) was more than 20 times higher than on mustard cabbage(1.8 ppm), and the intermediate values for this species onlettuce and broadbean also differed significantly (p<0.05)(Table 3.5). Combined with the previous findings, these resultsdemonstrate that sensitivity to AZA is highly variable foraphids, likely depending both on the species of aphid and on itshost plant. In previous studies, foliar applications of NSO88Table 3.5. Effective concentration of azadirachtin (AZA)resulting in 50% mortality (EC50) after nine days, for secondinstar M. persicae reared for 3 days on treated leaf disks ofvarious host plants, and for different instars on pepperInstar/Host EC50a (95% C.I.) R2(ppm AZA)second/mustard cabbage 1.8 (±0.5) 0.870 30second/lettuce 10.4 (±1.4) 0.940 60second/broadbean 15.4 (±1.9) 0.945 60second/corn 43.3 (±5.0) 0.838 60first/pepper 1.8 (±0.4) 0.872 60second/pepper 2.4 (±0.5) 0.878 30third/pepper 3.6 (±0.5) 0.879 60fourth/pepper 37.4 (±15.2) 0.814 60adult/pepper >1,000 --- --- 30aFrom linear regression analysis and inverse prediction.Myzus persicae (Suizer), green peach aphid.n89were more efficacious for the control of M. persicae on pepperthan on rutabaga at all the tested rates (0.5 to 2.0%) (Table1.1). As well, although NSO effectively controlled N.ribisnigri in the laboratory (Table 1.1), foliar applicationswere ineffective under field conditions (Table 1.3), perhapsresulting from changes in leaf cuticle thickness or plantgrowth. It has been demonstrated previously that control ofwhitef lies with foliar applications of neem is influenced by thehost plant (Schmutterer 1990a), and efficacy of Margosan-OTM wasinfluenced by the host plant of the leafhoppers (Jacobson 1990).EC50 values for M. persicae exposed to AZA increased fromfirst (1.8 ppm) to fourth (37.4 ppm) instar (Table 3.5), andthere was no toxicity to adults even at the highest rate of1,000 ppm. The EC50 for first instar M. persicae did not differsignificantly from that of second instars (p>0.05), and whilevalues for first and third instars differed significantly(p<0.0S), they were of similar magnitude. Price and Schuster(1990) demonstrated that earlier instars of sweetpotato whiteflywere more susceptible to Margosan—O1 than later instars, whilethere was little mortality to eggs or adults. Toxicity of AZAoccurs primarily during aphid molts, and little mortality occursduring the first two or three days (Figure 3.1). The largeincrease in the EC50 value for fourth instar M. persicae, ascompared to earlier instars (Table 3.5), may result from fourthinstars molting to adults rather than to another nymphal instarthat is still susceptible to the molt-disrupting effect of AZA.90Neem and AZA interfere with the titres of the molting hormoneecdysone (Rembold et al. 1984; Barnby & Kiocke 1990), preventingproper ecdysis and apolysis (Schiuter & Schultz 1984;Schmutterer 1990a). In the present study, aphids were observedto swell (apolysis) in preparation for ecdysis, but they eitherfailed to molt, or could not escape the exuvium. Studies bySchauer (1984) demonstrated that during molts the old nymphalskin of A. pisum on broadbean treated with neem seed kernelextract began to detach from the body but could not be ruptured,or the skin ruptured but could not be cast off completely.The EC50 values for second instar aphids sprayed withformulated NSO ranged from 1.7% for F. fimbriata to 5.3% for A.gossypii (Table 3.6), corresponding to 36 to 116 ppm AZA. TheEC50 values for aphids sprayed with NSO did not vary as greatly(3.2 fold) compared to those for aphids reared on leaf diskstreated with AZA (265 fold) (Table 3.4), which supports theearlier hypothesis that the host plant of the aphid isresponsible for a substantial amount of the observed variabilityin susceptibility of aphids to neem.For N. ribisnigri, the EC50 for NSO applied as spray droplets(5.0%) was significantly higher (p<0.05) than, but comparableto, the EC53 for NSO applied with a microapplicator (2.9%)(Table 3.6). The EC50 value for topical applications ofpurified AZA (704 ppm) is substantially higher, however, beingequivalent to 32.1% NSO based on the amount of AZA in the oil.The greater toxicity of NSO as compared to AZA might have91Table 3.6. Effective concentrations of neem seed oil (NSO)aapplied to second instar aphids by spray droplets or with amicroapplicator resulting in 50% mortality (EC50)b after 9 daysEC50Aphid speciesc NSO%(95% C.I.) (AZAppm) R2Spray dropletsF. fimbriata 1.66 (±.15) (36) 0.989C. fragaefolii 2.57 (±.89) (56) 0.756R. padi 2.79 (±.21) (61) 0.984N. ribisnigri 4.99 (±.25) (109) 0.989M. persicae 5.04 (±.56) (110) 0.933A. gossypii 5.30 (±.25) (116) 0.987Microappi icatorN. ribisnigri 2.88 (±.410) (63) .896N. ribisnigri (32.1) 70d (±17) .990acontains approximately 2,000 ppm AZA based on HPLC analysis.bFrom linear regression analysis and inverse prediction.cFimbriaphis fimbriata Richards; strawberry aphid, Chaetosiphonfragaefolii (Cockerell); bird cherry-oat aphid, Rhopalosiphumpadi (L.); lettuce aphid, Nasonovia ribisnigri (Mosley); greenpeach aphid, Myzus persicae (Sulzer); cotton or melon aphid,Aphis gossypii Glover.0Treated with purified AZA.92resulted from the presence of other active components, the oilmight have helped AZA penetrate the aphid cuticle, or it mighthave physically smothered the aphids. Alternatively, theemulsifiers and other additives in the NSO formulation mighthave improved the penetration of neem. Wetting agents increasethe penetration of water soluble substances through insectcuticle, and mixtures may penetrate faster than individualcomponents would on their own (Borror et al. 1981). Theformulation of NSO used in these studies, was also utilized inprevious laboratory and field trials, at rates of 0.5 to 2.0%,for the control of aphids (Chapter 1), indicating that contacttoxicity would contribute somewhat to the control of aphids onintact plants. According to Schmutterer (1990a) neem possessesrelatively weak contact toxicity. However, topical applicationsof neem extracts or AZA have been extensively used with insectsfrom several orders to study the various physiological effectsof neem (i.e. Koul 1984a; Ladd et al. 1984). For Homopterans,contact toxicity has been observed for citrus psyllid (Chiu1984), planthoppers, leafhoppers (Saxena 1987), and aphids(Goyal et al. 1971; Pandey et al. 1987).Sub-lethal Effects of NSO. Third instar M. persicae and N.ribisnigri exposed to 0.5% NSO that successfully molted toadults were generally smaller in size than controls (Table 3.7).Alate N. persicae had significantly shorter hind tibiae andthird antennal segments compared to controls (p<0.05), whileonly the tibiae of apterae were significantly shorter. For N.93Table 3.7. Sublethal effect of formulated neem seed oil (NSO)on aphid development. Lengths of hind tibiae (TL) and thirdantennal segments (AL), and deformities of wings (W) and legsCL) of adults that developed from third instars reared for threedays on treated leaf disksSpeciesa/ TL (mm) AL (mm) deformitiesmorph Treatment (95% C.I.) (95% C.I.) W(%) L(%) nfri. persjcae/ Control 1.07 0.42 0.0 0.0 33alatae (±.028) (±.026)M. persicae/ NSO 0.5% 0.95 0.37 92.6 18.5 26alatae (±.024) (±.018)M. persicae/ Control 0.83 0.32 --- 0.0 47apterae (±.025) (±.011)M. persicae/ NSO 0.5% 0.76 0.31——— 9.3 42apterae (±.027) (±.014)N. ribisnigri/ Control 1.46 0.58 1.1 1.1 95alatae (±.027) (±.009)N. ribisnigri/ NSO 0.5% 1.40 0.56 78.3 26.0 65alatae (±.035) (±.011)N. ribisnigri/ Control 1.29 0.52 --- 0.0 35apterae (±.050) (±.019)N. ribisnigri/ NSO 0.5% 1.28 0.50——— 0.0 24apterae (±.089) (±.034)aGreen peach aphid, Myzus persicae (Sulzer); lettuce aphid,Nasonovia ribisnigri (Mosley).94ribisnigri, tibiae and antennal segments of both adult morphswere consistently shorter in length than those of controls, butonly the antennal segments of alatae were significantlydifferent (p<O.05). Lengths of antennae and legs are correlatedwith aphid body size (Dixon 1985), indicating that exposure ofnymphs to neem can result in smaller adults.In addition to their smaller size, adults from treated nymphsoften had deformed wings and hind tibiae (Table 3.7).Approximately 93% of alate M. persicae and 78% of N. ribisnigrihad abnormal wings, ranging from incomplete expansion or minortwisting to small vestiges or complete lack of wings (Figure3.2). In addition to these obvious abnormalities, wings oftreated aphids were often held improperly or dragged on thesubstrate, and few of the treated aphids would be able to fly.Alatae appear to be more sensitive to developmentalabnormalities than apterae, as the legs of 18.5% of alate M.persicae and 26% of N. ribisnigri were deformed, while theaverage for apterae of both species was only 4.7% (Table 3.7).Deformed legs were usually mildly to severely twisted, but asmall number were lacking distal segments. Very commonly,treated adult apterae were darker in colour (Figure 3.3) anddark spots were often visible beneath the cuticle of theirabdomen. These ‘black bodies’ have been described previouslyfor Mexican bean beetles (Schluter & Schulz 1984) and for aphids(Schauer 1984) exposed to neem or AZA. According to Schiuterand Schulz (1984) cells of Mexican bean beetles undergoing high95Figure 32. Compared to (a) normal alatae, nymphal lettuceaphid, Nasonovia ribisnigz-i (Mosley), exposed to neem seed oiltypically developed into alate adults with physically deformedwings, ranging from (b) fully expanded but twisted or heldimproperly, (c) incompletely expanded, to (d) mere stubs orlacking entirely.II‘a_i - -.‘• •.96Figur 33 Compared to (a) normal adult apterous lettuceaphids, Nasonovia ribisnigri (Mosley), (b) apterae thatdeveloped from nymphs exposed to neem seed oil were often morehighly pigmented.— 4:1, :¶• %t97rates of mitosis, such as wing buds, are particularly sensitiveto AZA. Mitosis may proceed normally, but cells fail to formorgans or tissues and eventually degenerate.Neem may cause numerous fitness—reducing effects which couldbe important for the long—term control of insect populations(Schmutterer 1987; Schmutterer 1990a). Aphid mobility might bereduced, limiting migration and the transmission of viraldiseases of plants. Greenhouse whitef lies exposed to neem weresubstantially smaller and slower to emerge as adults (Lindquistet al. 1990); adult blowfly, Calliphora vicina, from larvaetreated with AZA, possessed malformed wings, legs, and probosci(Bidmon et al. 1987); and planthoppers and leafhoppers on ricetreated with NSO developed abnormally and often had malformedwings (Heyde et al. 1984). In addition to loss or reduction offlight, other reported sub—lethal effects of neem extracts orAZA include reduced fecundity and sterility (Chapter 4), failureto perceive or transmit pheromones, ovipositional and feedingdeterrency (Chapter 2), and repellency (Schmutterer 1987; Koulet al. 1989; Saxena 1989; Schmutterer 1990a).98CONCLUS IONIt has been suggested that aphids are poor candidates forcontrol with neem—based products (National Research Council1992), as they are generally not very susceptible to foliarapplications of neem (Schmutterer & Heilpap 1989). Foliarapplications of NSO or neem seed extract have been shown,however, to effectively control M. persicae and A. gossypii onpepper; M. persicae and B. brassicae on cabbage; C. fragaefoliiand F. finibriata on strawberry (Chapter 1); A. gossypil oncotton (Siddig 1987); A. pisum and A. fabae on broadbean(Schauer 1984); and A. craccivora on cowpea (Patel andSrivastava 1989).The present study has demonstrated that nine species of aphidsare susceptible to the IGR effect of NSO or its principle activeingredient AZA. Susceptibility varies with aphid species,instar, and host plant; and while AZA appears to be responsiblefor most of the observed activity, other components of NSO maybe important for the control of certain species (e.g. C.fragaefolii, Table 3.1, Figure 3.1). In addition to this sourceof variability, most earlier studies were conducted with crudeextracts containing unknown concentrations of AZA, which mayhave contributed to some of the contradictory reports in thepast (Larson 1989).Although activity results primarily from ingestion of neem,properly formulated materials possess some contact toxicity, but99not fumigant action. Depending on temperature, at least ninedays may be required for the complete expression of the IGReffects, and first generation offspring from treated adults havepoorer survival rates. Treated nymphs that successfully moltedto adults often possessed abnormal wings and legs, and thesefitness—reducing effects may be important for the control ofaphid populations over longer periods of time (Lindquist et al.1990; Schmutterer 1990a). Although deterrency (Chapter 2) andreduced fecundity (Chapter 4) contribute significantly to thecontrol of aphid populations, growth disruption anddevelopmental abnormalities appear to be the most importantfactors for the control of aphid populations with neexn.100CHAPTER 4INHIBITION OF APHID REPRODUCTION BY NEEM SEED OILAND ITS ACTIVE INGREDIENT AZADIRACHTININTRODUCTIONExtracts from the seeds of neem and purified AZA have beenshown to negatively influence the reproduction of female insectsfrom several orders (Rembold 1989a; Schmutterer 1987). Thedocumented antifeedant action of neem (Chapter 2) (Saxena 1989;Schmutterer 1990a) could result in an indirect reduction infemale reproductive rates, but inhibition of reproductionprimarily is a consequence of changes to ecdysteroid titres andinterference with the endocrine control of reproduction (Bidmonet al. 1987; Rembold et al. 1987; Rembold l989a). Histologicalinvestigations have demonstrated that, in addition to othersensitive tissues, insects treated with AZA have degenerate orimproperly developed ovaries and fat bodies (Schluter & Schultz1984; Schmutterer 1985; Schluter 1987). Depending on the stageof development, oocytes develop abnormally and are resorbed,minimal amounts of yolk are deposited, and eggs which aredeposited often possess abnormal chorionic surfaces (Schulz &Schluter 1984; Schmutterer 1987; Rembold 1989a).The ovicidal effects of neem extracts or AZA have not beenstudied intensively, but the fertility of treated eggs appears101to be normal (Schmutterer 1990a). For example, application ofAZA to eggs of red cotton bugs did not reduce percent nymphalemergence (Koul 1984a). However, eggs deposited by femalestreated with AZA or neem extracts often have poorly formedchorionic surfaces (Schulz & Schluter 1984), may be moresensitive to fungal attack, and are often less fertile(Schmutterer 1987).In this study, the fecundity-reducing effects of purified AZAand NSO toward several species of aphids were examined todetermine if reduced production of offspring contributes to thecontrol of aphid populations. Few previous studies havecompared the sterilizing effects of neein extracts with those ofpure AZA, or determined the degree of variability within closelyrelated insect taxa. As well, the effects of neem on thereproduction of viviparous insects have not been investigated toany extent.102MATERIALS AND METHODSLaboratory Rearing Conditions. A moistened, fine squirrel—hair paintbrush was used to transfer aphids to three disks (20mm diameter) cut from leaves with a cork borer, and reared insmall self—sealing petri dishes (50 X 9 mm) as outlinedpreviously (Chapter 1, Chapter 2). Aphids were reared (17±2°C)for three days on leaf disks dipped in test solutions, and thenmoved to untreated disks for the remainder of the study, withnew leaf disks supplied every three days.Leaf disks utilized in the bioassays were removed from Lettuce‘Ithaca’, sweet pepper ‘California wonder’, strawberry ‘Totem’,corn ‘Sunny vee’, and cucumber ‘Marketmore’, grown in thegreenhouse as outlined previously (Chapter 1).Test Materials. NSO, containing approximately 4,000 ppm AZAbased on HPLC analysis (Chapter 1), and purified AZA wereemulsified with Triton-X 100 in distilled water. Applicationrates were determined as outlined previously (Chapter 3).Inhibition of Aphid Reproduction. The effect of NSO and AZAon reproduction of N. ribisnigri on lettuce, M. persicae onpepper, and C. fragaefolii on strawberry, was evaluated byrearing aphids, two per dish, for three days on leaf disksdipped in solutions of NSO (0.5%, 1.0%, and 1.5%), purified AZA(40 or 80 ppm), or emulsifier (Triton-X 100) only as a control.Adults were reared for a further six days on untreated disks,while aphids exposed as fourth instars were reared for a further103nine days. Aphid survival and numbers of live and deadoffspring were recorded daily, and the average reproductive rate(live offspring/aphid/day) was calculated for each replicate(dish). For aphids treated as fourth instars, calculations werebased on the number of offspring produced during days three totwelve, as all fourth instars should have inolted to adultswithin the initial three day period. For each species andinstar, six replicates were used for each treatment, anddepending on the degree of variability, the entire experimentwas replicated once or twice.The effective concentration of AZA resulting in a 50%reduction in reproduction (EC50) was determined for eightspecies of aphids reared as adults for three days on treatedleaf disks, and a further three days on untreated disks. Inaddition to the three species listed above, EC50ts weredetermined for M. euphorbiae on lettuce, F. fimbriata onstrawberry, A. gossypli on cucumber, and M. dirhodum and R. pathon corn. Based on preliminary trials, numbers of live and deadoffspring were recorded for aphids on disks dipped in AZA atconcentrations of 0 to 100, or 0 to 1,000 ppm. The highestconcentration was serially diluted by 50%, producing sixtreatment rates for each range of concentrations tested. N.ribisnigri, M. persicae, and M. euphorbiae were reared two perdish, and the remaining species, five per dish. Aphids wereinspected and new leaf disks were added every three days. Foreach species, five dishes (replicates) were used for each104concentration, and depending on the degree of variability, theentire experiment was replicated once, twice, or three times.Adult survival and numbers of offspring were used to calculateseparate reproductive rates for the initial three days on thetreated disks, and the final three days on untreated disks. Aswell, numbers of offspring that died prior to birth (embryonic)were recorded for the entire experiment.Aphid Dissections. Adult N. ribisnigri exposed for three daysto AZA (40 ppm), NSO (1.0%), or emulsifier only as a control,were preserved in 70% ethanol and dissected to determine whetherdecreased production of offspring resulted from reducedproduction of embryos (decreased oocyte maturation), or was dueto increased mortality of developing embryos. Unmounted aphidswere carefully dissected in distilled water at a magnificationof 80X, and embryos larger than 5 micrometer units (zO.l2 mm)measured and counted. Abnormal appearance of embryos,discoloration, or other abnormalities were also noted. To makedissections easier, the bottoms of watchglasses were coated witha thin layer of clear silicon sealant and allowed to dry priorto use. The silicon helped to hold the aphids in place andreduced streaming of the solution.Statistical Analysis. Untransformed reproductive rates weresubjected to analysis of variance (ANOVA), and Tukey’s multiplerange test was used to determine differences between treatmentmeans (Wilkinson 1990). orthogonal contrasts were used toprovide further information on differences between control, AZA,105and NSO treatments. Linear regression analysis and inverseprediction were used to determine the effective concentrationsrequired to reduce aphid reproduction by 50% (EC50), as outlinedpreviously (Chapter 2).106RESULTS AND DISCUSSIONInhibition of Aphid Reproduction. Except for adult N.ribisnigri treated with the lowest rates of AZA and NSO,exposure of adult or fourth instar M. persicae and N. ribisnigrito AZA or NSO resulted in the production of significantly(p<0.05) fewer offspring at all of the tested rates (Table 4.1).Reproduction of C. fragaefolii exposed as adults or fourthinstars, however, was not affected by NSO or AZA at any of theconcentrations tested. The reduction in the number of offspringproduced did not differ (p>0.05) for aphids exposed to 40 ppmAZA compared to NSO 1.0% containing the equivalent amount ofAZA, indicating that this triterpenoid constituent of neem isprimarily responsible for the observed decrease in reproduction.For N. ribisnigri exposed as adults, an orthogonal comparison ofAZA and NSO treatments did indicate that NSO was more effectivethan AZA (p=0.042), but the difference was only marginallysignificant.Compared to treatment of adults, reproduction of N. ribisnigriwas suppressed to a greater extent when fourth instars wereexposed to NSO or AZA (Table 4.1). Adults treated as fourthinstars produced from 87% to nearly 100% fewer offspring,compared to controls, over the range of concentrations tested;while treatment of adults resulted in 32% to 76% feweroffspring.Few studies have directly compared AZA with NSO or neem seed107Table 4.1. Reproduction of lettuce aphid (LA) on lettuce,green peach aphid (GPA) on pepper, and strawberry aphid (SA) onstrawberry, following exposure to neem seed 01a (NSO) andazadirachtin (AZA) as adults or as fourth instarsMean no. of fspring/aphidL/day (SD)Treated adults Treated fourthsTreatment LA GPA SA LA SAControl l.48a 2.36a 0.65a 1.04a 0.87a(0.67) (0.72) (0.21) (0.56) (0.28)AZA 4oppm l.Olab 0.58b 0.62a 0.lOb 0.Bla(0.55) (0.44) (0.25) (0.16) (0.34)AZA Boppm 0.68b 0.44b 0.58a 0.08b O.68a(0.36) (0.23) (0.18) (0.12) (0.28)NSO 0.5% 0.92ab l.lOb 0.67a 0.02b 0.83a(0.47) (0.52) (0.15) (0.03) (0.35)NSO 1.0% 0.50b 0.43b 0.57a 0.14b 0.62a(0.35) (0.39) (0.27) (0.23) (0.49)NSO 1.5% 0.35b 0.40b 0.60a 0.Olb 0.67a(0.19) (0.49) (0.25) (0.03) (0.49)Contrasts CControl vs.AZA + NSO 0.001 0.001 0.573 0.001 0.176Control vs. AZA 0.001 0.001 0.537 0.001 0.298Control vs. NSO 0.001 0.001 0.649 0.001 0.164AZA vs. NSO 0.042 0.523 0.800 0.764 0.703Means within a column followed by the same letter are notsignificantly different (p>0.05, Tukey’s multiple range test).(n=12, except n=6 for GPA and fourth instar LA (2 aphids/dish)).acontains approximately 4,000 ppm AZA based on HPLC analysis.bLettuce aphid, Nasonovia ribisnigri (Mosley); green peachaphid, Myzus persicae (Suizer); strawberry aphid, Chaetosiphonfragaefolii (Cockerell).Cp values for orthogonal comparisons.108extracts, but several previous studies have demonstrated thatneem extracts and AZA are both potent inhibitors of insectreproduction. For example, purified AZA injected into adultfemale large milkweed bugs, Oncopeltus fasciatus (Dallas),resulted in a dose—dependent decrease in fecundity (Dorn et al.1987). Doses as low as 4 ng per bug reduced fecundity 15%,while injection of 8 .tg per bug resulted in complete sterility.Similarly, AZA injected into newly eclosed adult red cottonbugs, Dysdercus koenigii F., caused a reduction in female weightand oviposition rates, and resulted in infertile eggs (Koull984b). Adult female Colorado potato beetles, Leptinotarsadecemlineata Say, reared on potato leaves treated with aqueousor enriched neem seed extract deposited 88 and 87% fewer eggs,respectively, compared to controls over their entirereproductive period of nearly three months (Schmutterer 1987).The sterilizing effect of neem was, in fact, much stronger, asthe vast majority of the eggs produced by the beetles exposed toneem resulted from two females in each group which refused tofeed on the treated leaves and, therefore, produced normalnumbers of eggs.The effective concentrations of AZA resulting in 50% fewerlive offspring (EC50) during the last three days of the studyvaried from as low as 14.4 ppm for N. ribisnigri on lettuce to616.4 ppm for R. padi on corn (Table 4.2). AZA acted veryrapidly, with suppression of aphid reproduction occurring duringthe first three days. Because there was little effect during109Table 4.2. Effective concentrations of azadirachtin (AZA)resulting in a 50% reduction (EC50) in the number of liveoffspring produced during an initial 3 days on treated leafdisks, and a further three days on untreated disksday 1—3 day 4—6Aphida/host EC50 (ppm) R2 EC50 (ppm) R2 nN. ribisnigri/ 76.3a 0.797 14.4a 0.904 60lettuceM. euphorbiae/ 115.3b 0.678 50.2b 0.548 30lettuceM. persicae/ n.s.’ -—— 59.6bc 0.808 60pepperF. fimbriata/ n.s. ——— 73.8c 0.760 30strawberryC. fragaefolii/ 475.6c 0.743 102.5d 0.863 30strawberryA. gossypii/ 235.3d 0.904 175.9e 0.870 30cucumberM. dirhodum/ n.s. ——— 506.5f 0.907 30cornR. padi/ n.s. ——— 616.4f 0.870 90cornEC50 values within a column followed by the same letter arenot significantly different based on 95% confidence intervals(p>0.05)8Lettuce aphid, Nasonovia ribisnigri (Mosley); potato aphid,Macrosiphurn euphorbiae (Thomas); green peach aphid, Myzuspersicae (Sulzer); Fimbriaphis fimbriata Richards; strawberryaphid Chaetosiphon fragaefolii (Cockerell); cotton or melonaphid, Aphis gossypii Glover; rose-grain aphid, Metopolophiumdirhodum (Walker); bird cherry-oat aphid, Rhopalosiphum padi(L.).‘Non—significant linear regressions (p>0.O5).110the first day, and the number of offspring produced was variablefor the next one to two days, many of the regression equationswere non—significant or non-linear. However, AZA did inhibitthe reproduction of four species of aphids in a linear dose—dependent manner during the first three days, with resultingEC501s ranging from 76.3 ppm for N. ribisnigri to 475.6 ppm forC. fragaefolii (Table 4.2). The rapid sterilizing effect ofneem was also demonstrated for adult female Colorado potatobeetles fed potato leaves treated with purified extract of neemfruit (Steets 1977). Oviposition by treated beetles wasarrested within one day after treatment.In addition to a decrease in the number of live offspring,aphids treated with AZA produced a large number of deadoffspring (Figure 4.1 and Figure 4.2). These almost fully-developed offspring (here termed embryonic) were very dark incolour, appendages were not free of the body, and they wereclearly non-viable at birth. These distinctive embryonicoffspring occurred very rarely in controls (<1.0%), while at thehighest rate of AZA (100 or 1,000 ppm) embryonic offspringaccounted for 6.3 to 75.5% of the total number of offspringproduced. The increase in numbers of embryonic offspring doesnot account for all of the decrease in live offspring, though,as total numbers of offspring declined even more dramatically.Concentrations of AZA that inhibited the production of livenymphs by 50% are of approximately the same magnitude as thoseresulting in 50% mortality of second instar aphids,111II_____6.25 12.5 25 50 100Azadirachtin concentration (ppm)live off./aph id/day dead off./aph id/dayFigure 4.1. Number of live and dead (embryonic) offspring peraphid per day produced by adult (a) green peach aphid, Myzuspersicae (Sulzer), and (b) lettuce aphid, Nasonovia ribisnigri(Mosley) following three days of exposure to azadirachtin (0-100ppm) applied to leaf disks of pepper and lettuce, respectively(see Table 4.2 for statistical analysis). -A0.8 -0.6 -0.4 -0.2 -00.0 6.25 12.5 25 50 1001. 4.2. Number of live and dead (embryonic) offspring peraphid per day produced by adult (a) cotton or melon aphid, Aphisgossypii Glover, (b) potato aphid, Macrosiphum euphorbiae(Thomas), (c) bird cherry-oat aphid, Rhopalosiphum padi (L.),and (d) strawberry aphid, Chaetosiphon .fragaefolii (Cockerell),following three days of exposure to azadirachtin (0-1,000 ppm)applied to leaf disks of cucumber, lettuce, corn and strawberry,respectively (see Table 4.2 for statistical analysis).21.50.6A1000B—.1.5 I I C260 500 10000. 125 250 500Azadirachtin concentration (ppm)live oIt./aphid/day dead ott/aphid/day1000 62.5 126 260 600Azadirachtln concentration (ppm)live ott/aphid/day lED dead otf./aphidlday113demonstrating that reduced reproduction would also contributesignificantly to the control of aphids in the laboratory and inthe field. Concentrations of AZA resulting in 50% mortalityranged from 2.4 to 635.0 ppm (Table 3.4), compared to 14.4 to616.4 ppm for 50% inhibition of adult reproduction (Table 4.2).Furthermore, reproduction was affected to a greater extent whenaphids were exposed as fourth instars (Table 4.1).Aphid Dissections. Adult N. ribisnigri exposed to AZA (40ppm) or NSO (1.0%) contained from 35 to 43% fewer embryoscompared to controls (p<0.05) (Table 4.3). Embryos larger than0.53 mm and from 0.12 to 0.34 mm were significantly fewer(p<0.05) for NSO and AZA treatments compared to controls, butthere was no difference in the number of embryos between 0.34and 0.53 mm in length (p>0.05). Based on orthogonal contrasts,the total number of embryos and the number in each size classdid not differ (p>0.05) for NSO and AZA treatments, againindicating that AZA is likely responsible for the sterilizingactivity of neem toward aphids. NSO and AZA treatments reducedthe number, but not the ultimate size of embryos, as there wasno difference in length for the largest embryos in eachtreatment (p<0.05) (Table 4.3).The dissection of adult N. ribisnigri suggests that fewer liveoffspring are produced primarily because of reduced oocytematuration, but a significant number of embryos may also die atmaturity. The discoloration and death of the previouslydescribed embryos occurred just prior to birth. Embryos began114Table 4.3. Total number of embryos per aphid (>0.12 mm), andnumbers in each of three size classes, for dissected adultlettuce aphids, Nasonovia ribisnigri (Mosley), exposed as fourthinstars to azadirachtin (AZA), neem seed 011a (Nso), oremulsifier only as a controlMean no. embryos (SEM)Treatment >0.53mm 0.53-0.34mm 0.34-0.12mm TotalControl 5.1 (0.79) 5.3 (0.33) 12.0 (1.43) 22.5 (2.12)AZA 4oppm 2.3 (0.44) 5.7 (0.39) 6.7 (1.10) 14.7 (1.41)NSO 1.0% 3.5 (0.55) 4.4 (0.67) 4.9 (1.58) 12.8 (2.16)Contrasts : bControl vs.AZA + NSO 0.013 0.762 0.005 0.005AZA vs. NSO 0.194 0.121 0.413 0.531Based on 10 aphids per treatment.aContains approximately 4,000 ppm AZA based on HPLC analysis.bp values for orthogonal comparisons.115to darken just before they reached maximum size, and at maturitytheir appendages remained tightly bound to their bodies as theyfailed to escape from the embryonic membrane (follicularepithelia). Discoloration or other abnormalities were notobserved for younger embryos.Histological examinations of the ovaries of Mexican beanbeetles, Epilachna varivestis Mulsant, fed bean leaves treatedwith inethanolic neem seed extract demonstrated that neein—treatment resulted in cytopathological alterations andresorption of developing oocytes (Schulz & Schluter 1984). Fewoocytes reached the stage of vitellogenesis, and minimal amountsof yolk were deposited. Although egg production was drasticallyreduced, hatchability of eggs from large milkweed bugs injectedwith AZA was close to normal (Dorn et al. 1987), suggesting thattrophic cells or young oocytes are more sensitive to neem thanare developing embryos. It is difficult to compare viviparousreproduction of aphids with results from studies with oviparousinsects, however, and further studies of the effect of neem onaphid oocyte and embryo development are warranted.116CONCLUS IONThe fecundity of homopterous insects is strongly influenced bytreatment with neem extracts or AZA (Schmutterer l990a), but theeffect on reproduction of viviparous insects has not beenthoroughly investigated. Reproduction of adult A. pisum placedas first instars on broadbean plants sprayed with 0.002%methanolic neem seed extract produced 1.6 offspring per femaleper day, compared to 7.1 offspring per female per day foruntreated controls (Schauer 1984). Sweetpotato whitef liesconfined to cotton treated with neem seed extract (2%) depositedmore than 80% fewer eggs compared to controls up to seven daysfollowing treatment, but reduced oviposition could have resultedfrom the repellent action of neein. Treatment of eggs reducedhatchability by approximately 29%, and resulted in 90% mortalityof nymphs by day 20 (Coudriet et al. 1985).The present studies have demonstrated that NSO and AZAeffectively inhibited aphid reproduction, due to both a decreasein the maturation of oocytes and to increased mortality ofmature embryos. As for mortality of second instar aphids(Chapter 3), inhibition of aphid reproduction was variable.Effective concentrations of AZA resulting in 50% inhibition ofadult reproduction ranged from as low as 14.4 ppm for N.ribisnigri, to 616.4 ppm for R. padi (Table 4.2). Control ofaphids with foliar applications of neem would result primarilyfrom decreased rates of reproduction and increased levels of117mortality. Due to their tremendous reproductive ability, even amodest decrease in the production of offspring might allownatural enemies to effectively control aphid populations. Theimpact of neem materials on aphid reproduction should beinvestigated more fully. In addition to an immediate effect onrates of aphid reproduction, exposure to neem may contributesignificantly to the control of aphid populations over longerperiods of time.118CHAPTER 5SYSTEMIC ACTIVITY AND PERSISTENCE OF NEEMINTRODUCTIONThe systemic activity of neem would be advantageous for thecontrol of insects, such as aphids, that feed from the vascularsystem of plants. However, except for a small number ofantifeedant studies, the systemic activity of neem towardphloem-feeding insects has not been studied in any detail.Systemic uptake and translocation of active ingredients wouldalso improve the efficacy of neem, otherwise insects in thefield will chose untreated plant parts (Klocke et al. 1989). Aswell, movement of AZA and other bioactive components of neeminto leaf tissues may help reduce the rate of photo-degradation.Although there are sporadic anecdotal reports regarding thesystemic activity of neem, few published experimental studieshave specifically addressed this topic (Isman et al. 1991).For the purposes of this study, systemic activity can bedivided into two processes: local systemic activity,(translaminar movement) involving penetration of the leafsurface and movement throughout the leaf; and translocation fromone part of the plant to another via phloem or xylem. Theuptake of neem by plant roots and translocation to untreatedparts has been documented in a few previous studies. For119example, soil drenches of NSE or AZA protected bean seedlingsfrom feeding by desert locusts for up to 25 days (Gill & Lewis1971), while 0.1 and 0.4% NSE solutions applied as drenches topotted chrysanthemum reduced emergence of adult vegetableleafminer, Liriomyza trifolii (Burgess), by 89 and 100%,respectively (Larew et al. 1985). While studies of this natureindicate that neem materials are effectively taken up by rootsand translocated in the xylem, the degree of movement of neem inphloem is controversial. According to Schmutterer (1987), onlysmall amounts of neem are translocated in phloem. NSE appliedonly to the upper or to the lower leaves of chrysanthemum didnot move in high enough concentrations to protect the untreatedleaves from leafminers, suggesting limited phloem transport(Larew 1988). NSE applied to individual leaves of chrysanthemumand bean did, however, move to adjacent untreated leaves.Although movement in phloem may be limited, applications ofAZA (100 ppm) to leaves of corn provided partial protectionagainst corn rootworm, Diabrotica virgifera (Leconte), andradio-labelled di-hydro-AZA was detected in roots of corn 24hours after application to leaves (Xie et al. 1991). Finally,feeding of leafhoppers on phloem of rice seedlings systemicallytreated with neem decreased in a dose—dependent manner, whilethe amount of time salivating and feeding on xylem increased(Saxena & Khan 1985; Saxena & Boncodin 1988). Neem likelyoccurred in the phloem of these plants at sufficiently highconcentrations to deter feeding.120In order to provide a better understanding of the systemicaction of neem toward aphids, the current study investigated theuptake and translocation of neem from roots to untreatedfoliage, as well as the local systemic activity (translaminar).Studies of this nature are of practical importance for thecontrol of aphids, as they help determine appropriateapplication methods and spray coverage. As well, littleattention has been devoted to improvements in formulations orthe use of additives to enhance penetration of leaf surfaces.In conjunction with other techniques, aphid—based bioassayscould be utilized to quantify differences in the systemicactivity of formulated neem materials.Like many other natural products, persistence of neem extractsappears to be limited under field conditions (Schmutterer1990a), as AZA is rapidly degraded by elevated temperatures, UVlight, and acidic and alkaline conditions (Larson 1987;Schmutterer 1987; Barnby et al. 1989). Based on mortality offourth instar diamondback moth larvae, liv irradiation of anenriched neem seed extract reduced the growth regulatingactivity by approximately 46% after 24 hours. The AZA contentof a methanolic extract of neem seeds exposed to UV lightdeclined by approximately 65% after 14 hours based on HPLCanalysis (Ermel et al. 1987). However, the rate at which AZAdegrades is likely influenced by the extraction method andcomposition of the solution. For example, HPLC analysis of thematerials used in the current study demonstrated that purified121AZA emulsified in distilled water with Triton-X 100 was stablefor at least two months when solutions were refrigerated in thedark, but AZA in methanol degraded almost completely within twoto three weeks under the same storage conditions (data notshown). AZA degrades more slowly when the natural anti—oxidantsand UV-protectants contained in NSO are present (Larson 1987;Isman et al. 1991). Margosan—OTM,which retains some neem oilas a natural surfactant and sunscreen, has been formulated withadditives to improve stability, providing a shelf-life of atleast two years. The rapid degradation of neem solutions bysunlight limits its potential use as a crop protectant, butimproved formulations incorporating sunshields and antioxidantscould be used to prolong its activity (Saxena 1987).The persistence of neem should not be confused with delayedactivity (Schmutterer 1990a). Mortality, resulting from failureto molt or pupate, can occur many days after immature insectsare exposed to neem or AZA; and in certain instances, the periodof time between molts can be lengthened significantly. Forexample, the intermolt period for fourth and fifth instarmigratory locusts injected with AZA (0.6 to 2.0 j.Ll/g) within twodays after molting lasted from 8 to 60 days compared with 6 to 9days for controls (Sieber & Rembold 1983). Similarly, whilemost fifth instar migratory grasshoppers fed AZA at rates from15 to 25 g/g insect weight died in a failed attempt to molt,some nymphs lived for up to 60 days, compared to an intermoltperiod of S or 9 days for controls (Champagne et al. 1989).122Prolonged intermolts of locusts treated with AZA was alwaysassociated with molting disruption and death. In the currentstudy, increased rates of mortality for second instar aphids onleaf disks dipped in AZA or NSO did not occur during the first 2or 3 days, and at least 6 to 9 days was required for mortalityto be fully expressed (Chapter 3). The growth regulatingactivity of neem does not afford quick kill, and increasedmortality is typically observed 3 to 15 days followingtreatment, depending on the target insect and the timing ofapplication (Wood 1990).It is also necessary to separate persistence of the deterrentor repellent activities of neem and AZA (behavioral effects)from persistence of the growth—regulating and reproductiveeffects (physiological effects). The deterrent activity of neemtoward aphids persists for only a short period of time (Chapter2). Under greenhouse conditions, deterrency of NSO to C.fragaefolii disappeared in less than 24 hours followingapplications to strawberry at concentrations of 1 or 2% (Table2.6). Similarly, after 72 hours in the field, residues of AZAapplied to corn (600 mg/l) were no longer deterrent to feedingof the fall armyworm (Wood 1990). Based on the results of leafdisk choice tests, feeding by spotted cucumber beetles on leafdisks dipped in 0.1% solutions of AZA was almost completelyinhibited immediately following treatment, but activity was lostafter 22 hours when leaf disks were maintained in the laboratory(Reed et al. 1982).123The antifeedant activity of neem—based materials appears to beshort-lived under field conditions (Wood 1990). The generallyrapid reduction in the deterrent action of neein extracts or AZAcan perhaps best be explained by the combined loss of volatilecompounds, the photo—degradation of active compounds on leafsurfaces, and movement of materials into leaves. In comparisonto the behavioral effects of neein, physiological effects persistfor a longer period of time (Schmutterer 1990a).Neem controls aphids primarily from increased nymphalmortality (Chapter 3) and a reduction of adult reproduction(Chapter 4). Therefore, persistence of the toxic effect of neemto second instar aphids was investigated for treated plants heldoutdoors and in the greenhouse. Earlier studies of this naturehave produced variable results. Mortality of third instarMexican bean beetles was not different from controls when larvaewere fed leaves from bean plants sprayed with a partiallypurified neem seed kernel extract (100 ppm) beginning six daysafter treatment (Steets 1976). On the other hand, neem seedextract (500 ppm) applied to oak leaves in the field wasrelatively persistent. Twelve days after treatment, residueseffectively prevented successful molting of second instar gypsymoth, Lymantria dispar (L.) (Skatulla & Meisner 1975). Longerperiods of residual activity may simply result from higherinitial application rates, but the concentration of the activeingredients, particularly AZA, was not determined in thepreceding examples, precluding direct comparison.124MATERIALS AND METHODSSystemic activity. For studies of the uptake of NSO and NSEfrom soil, plants in 10 cm plastic pots were grown in growthchambers as outlined previously (Chapter 1). At four to sixweeks of age, plants were individually caged and artificiallyinfested with adult aphids (10/plant). Two days later, potswere drenched (50 mi/pot) with NSE (1, 2, or 4%), NSO (2%), oremulsifier only as a control. The volume was sufficient toallow small amounts of solution to leach out of most pots. M.persicae were transferred to pepper ‘California wonder’ andmustard cabbage ‘Pak choi’, N. ribisnigri to lettuce ‘Ithaca’,C. fragaefolii to strawberry ‘Totem’, R. padi to corn ‘Sunnyvee’, and A. fabae to broadbean ‘Windsor long pod’. Followingtreatment, caged plants were maintained in growth chambers(20°C) for one week, when numbers of aphids per plant weredetermined. Each treatment was applied to 5 pots, and theentire experiment was replicated twice.The rearing system utilized for studies of the translaminarmovement of NSO and AZA was modified from the system outlined inChapter 2. Aphids were allowed access to the lower surface(abaxial) of leaf disks (27 mm diameter) placed over openings(18 mm diameter) in the lids of the self-sealing petri dishes.A piece of parafilm ( 75 X 75 mm) was placed over each leafdisk, and a petri dish lid held them both snugly in place(Figure 5.1). The parafilm prevented the rapid desiccation of125petri dish lidparafilmleaf disk (27 mm dia.)petri dish with hole (18 mm dia.)petri dish lid with 10 small holesFigure 5.1. Rearing system for evaluating the local systemicactivity of neem seed oil and azadirachtin whereby aphid feedingis restricted to the lower untreated surfaces of leaf disks.126the leaf disks and allowed for the attachment of the additionallid to what is normally considered to be the bottom of the petridish.In an initial trial, solutions of NSO (1 or 2%), or aformulated control, were applied (35 tl/disk) with a micro-pipette to either the upper (adaxial) or lower surfaces of leafdisks excised from lettuce plants grown in the greenhouse.After the disks had dried, second instar N. ribisnigri (8/dish)were allowed to feed on the treated (facing), or untreated(opposite), leaf surface for three days, after which time theywere transferred to untreated leaf disks for a further six daysas outlined previously (Chapter 3). Each treatment wasreplicated five times, and the entire experiment was repeatedonce.The effective concentration of AZA resulting in 50% mortality(EC50) for second instar N. ribisnigri on lettuce, M. persicaeon pepper, A. pisum on broadbean, R. path on corn, and C.fragaefolii on strawberry, was determined for AZA applied to theadaxial (opposite) surfaces of leaf disks as outlined above. Inall other respects, the experimental procedure was the same asthat outlined for mortality of second instar aphids on leafdisks dipped in solutions of AZA (Chapter 3), such that theresults of the two experiments could be compared. Leaf diskswere treated with AZA at six concentrations ranging from 0 to10, 0 to 100, or 0 to 1,000 ppm, and each rate was applied tofive dishes. For each species, the entire experiment was127replicated once or twice.Persistence. Persistence of NSO applied to potted lettuce andmustard cabbage was evaluated for treated plants held in thegreenhouse and outdoors. Plants were reared in the greenhouseas outlined previously, and at approximately six weeks of age,half the plants were moved outdoors to be hardened off. Atapproximately eight weeks of age, they were thoroughly sprayedto runoff with NSO (1 or 2%) or a control solution of emulsifieronly. Beginning 0, 3, 6, and 9 days after treatment, secondinstar N. ribisnig-ri and M. persicae were reared (8/dish) fornine days on leaf disks (3/dish) from lettuce and mustardcabbage, respectively, held outdoors and in the greenhouse. Theaphid rearing conditions were outlined previously (Chapter 3),except that aphids were reared on leaf disks from treated plantsfor the duration of the trial. For each concentration andspecies of aphid, treatments were replicated six times forplants held in the greenhouse, and 10 times for plantsmaintained outdoors.Statistical Analysis. Aphid numbers per plant weretransformed by log(X+l.O) prior to ANOVA and Tukey’s meanseparation procedures. Proportions of aphids surviving ninedays were arcsine transformed and analyzed as outlinedpreviously (Chapter 3).128RESULTS AD DISCUSSIONSystemic Activity. Control of aphids on plants drenched withsolutions of neem was variable. Drenches of NSE reduced numbersof M. persicae on pepper and R. padi on corn in a dose—dependentmanner. At the highest concentration, NSE reduced numbers of M.persicae and R. padi approximately 79 and 67%, respectively,compared to controls. Generally, there was no difference inaphid counts for plants drenched with 2% NSO as compared to 2%NSE, and numbers of M. persicae on pepper and R. padi on corndid not differ for these two treatments (p>O.05) (Table 5.1).However, even at the highest concentration tested (4%), NSE didnot reduce numbers of M. persicae on mustard cabbage, N.ribisnigri on lettuce, C. fragaefolii on strawberry, or A. fabaeon broadbean compared to controls (p>O.05) (Table 5.1).Although NSE did not reduce numbers of M. persicae on mustardcabbage at any treatment rate, 2% NSO did result in a modest,but significant (p<O.05), reduction of about 37%.It is not surprising that drenches of neem did not reducenumbers of C. fragaefolii on strawberry, as previous studies haddemonstrated that very high rates of AZA (635 ppm) were requiredfor 50% mortality of second instar C. .fragaefolii on treatedleaf disks (Table 3.4). The highest rate of NSE (4%) containedonly about 80 ppm AZA. Differences in susceptibility to AZAcannot account for the variability in the levels of control forthe other aphids, however, suggesting that the uptake and129Table 5.1. Efficacy of neem seed extract (NSE) and neem seedoil (NSO) applied as a soil drench to potted plants for thecontrol of aphidsNo. aphids/plant (SD)Aphid speciesa/ Treatmenthost plant Control NSE 1% NSE 2% NSE 4% NSO 2%M. persicae/ 90.8a 62.4ab 19.2d 54.5bcpepper (12.3) (20.7) (12.9) (4.6) (20.9)M. persicae/ 1l2.5a 115.6a 116.3a 100.8ab 71.2bmustard cabbage (20.9) (21.1) (30.1) (29.1) (25.1)N. ribisnigri/ 67.6a 60.6a 62.2a 40.8a 6l.6alettuce (9.3) (22.8) (19.5) (15.6) (13.6)C. fragaefolii/ 49.6ab 48.Oab 38.9a 43.4ab 55.7bstrawberry (6.7) (8.1) (8.7) (5.3) (9.7)R. padi/ 165.6a 99.Bab 55.3b 64.lbcorn (33.3) (19.5) (28.1) (25.3) (27.5)A. fabae/ 147.Oa l59.Oa 150.5a 144.7a 135.labroadbean (47.2) (24.0) (39.8) (24.5) (42.6)For each aphid species, numbers within a row followed by thesame letter are not significantly different (p>0.O5, Tukey’smultiple range test).aGreen peach aphid, Myzus persicae (Sulzer); lettuce aphid,Nasonovia ribisnigri (Mosley); strawberry aphid, Chaetosiphonfragaefolil (Cockerell); bird cherry-oat aphid, Rhopalosiphumpadi (L.); black bean aphid, Aphis fabae (L.).130translocation of the active components of neem varies dependingon the host plant. For example, effective concentrations of AZAresulting in 50% mortality for second instar M. persicae on leafdisks of mustard cabbage and on pepper, and N. ribisnigri onlettuce, did not differ significantly (Table 3.4, Table 3.5),but drenches of neem only reduced numbers of M. persicae onpepper (Table 5.1). In a similar manner, Margosan-OTM appliedas a drench at concentrations from 0.17 to 0.33% reduced thenumber of adult leafhoppers on chrysanthemum and marigold, butnot on zinnia (Knodel-Montz et al. 1985). Apparently, plantsvary in their ability to absorb and translocate the activecomponents of neem.Results from other studies of the translocation of neem varyeven for closely related plants. For example, larvae of cabbagewhite butterflies, Pieris brassicae (L.), failed to survive onpotted cabbage grown in soil treated with neem seed powder (10to 80 gm/Kg) for up to 15 days (Osman & Port 1990), and NSEtaken up by the roots of canola controlled diamondback mothlarvae for up to 10 days (Tan & Sudderuddin 1978). On the otherhand, applications of NSE applied via hydroponic solutions onlyaffected M. persicae on kale at concentrations that were clearlyphytotoxic to the plants (Griffiths et al. 1978). In thecurrent study, drenches of NSE and NSO also failed to control M.persicae on mustard cabbage (Table 5.1). It is difficult todraw definitive conclusions regarding differences in thesystemic activity of neem from these various studies, however,131as they utilized not only different plants, but also differenttest insects, neexn solutions, and potting media. Because AZAis a relatively large, highly oxygenated molecule, it mayreadily bind to the organic matter in soils. The composition ofthe potting media could, therefore, strongly influence theavailability of AZA to the plants. For example, AZA applied insolution to the bare roots of cantaloupe plants controlledfeeding by striped and spotted cucumber beetles, but there wasno effect when AZA was applied to the potting soil (Reed et al.1982).Interestingly, results from this study indicate that drenchesof NSE and NSO were more effective than anticipated for thecontrol of R. padi on corn. The EC50 for AZA applied to leafdisks of corn was estimated to be 87.9 ppm (Table 3.4), whiledrenches of 4% NSE (approx. 80 ppm AZA) and 2% NSO (approx. 40ppm AZA) reduced R. padi numbers on corn approximately 67 and62%, respectively (Table 5.1). Translocation of neem has beendocumented for rice (Saxena et al. 1978; Heyde et al. 1984;Saxena et al. 1984; Saxena & Boncodin 1988; Kareem et al. 1989),corn (Klocke et al. 1989; Xie et al. 1991), and wheat (West &Mordue 1992), perhaps indicating that the active components ofneem are translocated more effectively by graminaceous plants incomparison to other plant families.The translaminar movement of neem, following application toleaf disks of lettuce, was very good. For 1 and 2% NSO, nineday survival was not significantly different for second instar132N. ribisnigri which fed for three days on the opposite untreatedleaf surface, as compared to those that fed on the treated(facing) surface (p>0.05) (Table 5.2). An orthogonal contrastof aphid survival on treated compared to untreated leaf surfaceswas not significant (p=0.205).Effective concentrations of AZA applied in a local systemicmanner resulting in a 50% reduction in survival of second instaraphids ranged from as low as 7.2 ppm for N. ribisnigri onlettuce, to 629.1 ppm for C. fragaefolii on strawberry (Table5.3). Generally, these values are comparable to the EC501s forsurvival of the same species on leaf disks dipped in AZAsolutions (Table 3.4). The EC50’s for AZA applied systemicallywere, on average, 1.4 times higher than the EC501s for leafdisks dipped in AZA, indicating that the translaminar movementof AZA is not a limiting factor for the control of aphids. Thedesign of these experiments does not, however, allow for thedetermination of differences in the penetration of leafsurfaces, and additional studies are required to assess movementof AZA across the cuticle and epidermis of leaves.According to Larew (1988) neem moves readily into leaves, butthe degree of movement is influenced by the plant species. Onthe other hand, in comparison to treatment of abaxial surfaces,control of greenhouse whitef lies was poor when Margosan—OTM wasapplied to adaxial leaf surfaces of poinsettia, indicating poortranslaminar movement (Lindquist et al. 1990). Systemicactivity of neem varies between host plants (Larew 1988;133Table 5.2. Survival of second instar lettuce aphid,Nasonovia ribisnigri (Mosley), on lettuce following three daysfeeding on leaf disk surfaces treated with neem seed oil(facing), or on the opposite untreated surfaces (opposite).Proportion of aphids surviving nine days (SD)Treatment Control NSO 10% NSO 2.0%Facing 0.80a (0.20) 0.lOb (0.10) 0.03b (0.07)Opposite 0.90a (0.15) 0.05b (0.12) 0.05b (0.12)Contrast: Facing vs. opposite, p=0.205Numbers followed by the same letter are not significantlydifferent (p>O.O5, Tukey’s multiple range test).134Table 5.3. Local systemic activity (translaminar) ofazadirachtin (AZA). Effective concentration of AZA applied tothe opposite sides of leaf disks resulting in 50% mortality(EC50) after nine days for second instar aphids reared for threedays confined to the untreated surfacesAphid speciesa/ EC50b (95% CI)b R2 p(reg.) nhost plant [AZAppin]N. ribisnigri/ 7.2 (+0.40) 0.850 <0.001 30lettuceM. persicae/ 8.8 (+0.31) 0.878 <0.001 30pepperA. pisurn/ 11.9 (+0.30) 0.855 <0.001 30broadbeanR. padi/ 454.0 (+0.51) 0.918 <0.001 60cornC. fragaefolii/ 629.1 (+1.32) 0.854 <0.001 60strawberryaLettuce aphid, Nasonovia ribisnigri (Mosley); green peachaphid, Myzus persicae (Suizer); pea aphid, Acyrthosiphon pisuraHarris; bird cherry—oat aphid, Rhopalosiphum padi (L.);strawberry aphid, Chaetosiphon .fragaefolii (Cockerell).bFrom linear regression analysis and inverse prediction.135Jacobson 1990; National Research Council 1992), and it is anarea of research that requires further investigation (Wood1990). Differential penetration of leaf surfaces may helpexplain some of the observed variability in the toxicity of neemto aphids. Schauer (1984) increased the efficacy of neemextracts to A. pisum on broadbean with the addition of sesameoil, dimethyl-sulfoxide, or lecithin II. These materials areknown to improve penetration of leaf surfaces, stronglysuggesting that movement of neem into leaves is a limitingfactor for the control of phloem-feeding insects. Improving thesystemic activity of neem with the use of additives should beinvestigated more fully.Persistence. Residues of NSO applied to mustard cabbageoutdoors and in the greenhouse persisted for at least nine days(Table 5.4). Mortality was significantly higher than controls(p<0.05) for second instar M. persicae reared on leaf disks frommustard cabbage treated with 1 or 2% NSO based on bioassaysbeginning 0, 3, 6, and 9 days after treatment of the plants.After nine days, residues of 2% NSO resulted in 31 and 76%higher mortality compared to controls, for mustard cabbage heldin the greenhouse and outdoors, respectively. Mortality of M.persicae on leaf material from plants treated with 1% NSOdiffered significantly from 2% NSO only for plants held outdoorsfor nine days (p>0.05).Foliar applications of 1 and 2% NSO to lettuce in thegreenhouse resulted in higher mortality of N. ribisnigri for up136Table 5.4. Persistence of the toxic effect of neem seed oil(NSO) to aphids. Survival (9 day) of second instar lettuceaphid, Nasonovia ribisnigri (Mosley), and green peach aphid,Myzus persicae (Suizer), on leaf disks of lettuce and mustardcabbage, respectively, from plants treated with NSO and held inthe greenhouse or outdoors, for bioassays beginning 0 to 9 dayspost-treatmentDays N. ribisnigri M. persicaeafterTreatment Control NSO 1% NSO 2% Control NSO 1% NSO 2%Greenhouse0 88.3a 13.3b 6.7b 93.3a 15.Ob O.Ob3 81.7a 1O.Ob 16.7b 88.3a 20.Ob 0.Ob6 86.7a 25.Ob 6.7b 93.3a 43.3b 48.3b9 86.7a 36.7b 20.Oc 96.7a 86.7b 66.7bOutdoors0 95.Oa 60.Ob 31.7b 85.Oa 33.8b 5.Ob3 98.3a 28.8b 22.5b 80.Oa 18.8b 6.3b6 98.3a 86.7ab 75.Ob 8l.3a 35.Ob 18.8b9 90.Oa 83.3ab 65.Ob 95.3a 60.Ob 22.5cFor each aphid species, numbers within a row (day) followed bythe same letter are not significantly different (p>O.05, Tukey’smultiple range test).137to nine days, providing an average reduction in survival of 67%after nine days compared to controls (Table 5.4). Treatments of1% NSO to potted lettuce outdoors did not control N. ribisnigriafter 6 or 9 days, and although mortality of N. ribisnigri onlettuce treated with 2% NSO was higher than controls 6 and 9days post—treatment (p<0.05), survival was only 26% lower, onaverage, compared to controls. The relatively short residualactivity of NSO applied to lettuce held outdoors might helpexplain why N. ribisnigri was effectively controlled in thelaboratory (Table 1.1), but not in the field (Table 1.3).Results from the current trials with aphids produced residualtimes that are comparable to those from previous studies. NSEapplied to greenhouse-grown lima bean killed larvae of vegetableleafminers for up to seven days, with effectiveness decliningsteadily from day 0 (86% mortality) to day 7 (44% mortality)(Webb et al. 1983). In the greenhouse, NSE (0.2 and 2.0%)applied to cotton effectively reduced oviposition and adultemergence of sweetpotato whitefly after seven days, but therewas little residual activity after 14 days (Coudriet et al.1985). Following applications of NSE (100 ppm) to beans grownoutdoors, the growth-regulating activity to third instar Mexicanbean beetles disappeared after six days, but rates ten timeshigher were still highly effective for a longer period of time(Steets 1976). Obviously, under identical test conditions theresidual activity of neem will depend largely on the initialapplication rate. Depending on environmental conditions, the138residual activity of neem extracts or AZA on leaf surfacespersists for approximately 4 to 8 days (Schmutterer 1988), whilesystemic effects may persist for at least 7 to 10 days(Schinutterer l990a).139CONCLUS IONGill and Lewis (1971) were the first to demonstrate thesystemic activity of neem, whereby potted bean seedlingsdrenched with NSE or AZA were protected from feeding by desertlocusts. Since then, systemic applications of various neemmaterials have been shown to control several insect pests, mostoften following uptake by roots. Based on mortality of nymphalaphids, for the plants utilized in these studies, the localsystemic activity of NSO and AZA appears to be sufficiently high(Table 5.2, Table 5.3). The distinction should be made,however, that these and previous studies were not designed tocompare penetration of leaf surfaces for different host plants.The observed variability in levels of aphid control on varioushost plants could partly reflect differences in the rate ofmovement of neem into leaves. Control of aphids might not befeasible on plants that possess leaves that are relativelyimpermeable to neem.Uptake and translocation of NSO and NSE applied as drenchesvaried with the host plant and may be of limited importance forthe control of aphids (Table 5.1). AZA may bind with soilparticles, which would require higher rates of application, butneem materials may still be useful for the control of aphids onplants grown hydroponically or in artificial media.The systemic activity of neem is still poorly understood(Schmutterer 1988; Wood 1990), and penetration of leaf surfaces,140translaminar movement, and translocation of the activecomponents of neein, requires additional study. A betterunderstanding of these processes might help explain the variablelevels of control observed in the past, particularly for phloem—feeding insects such as aphids. Proper neem formulations andthe use of additives to improve penetration of leaf surfacesshould be given greater attention. For example, the reportedsuperiority of NSO as compared to oil-free extracts for thecontrol of aphids (Schmutterer 1990a; National Research Council1992) possibly results from improved systemic activity due tothe presence of the oil (Schauer 1984; Larson 1989). Neem seedextracts might dry faster on leaf surfaces, which would limitthe uptake of the active components of neem. In the presentstudy, however, properly formulated NSO and NSE were botheffective for the control of aphids (Chapter 1).In the current study, persistence of the toxic effect of NSOtoward N. ribisnigri was limited to about three days for lettuceheld outdoors, while residues remained active for up to ninedays when plants were grown in the greenhouse (Table 5.4). NSOtreatments (1 or 2%) were active against M. persicae for atleast nine days for mustard cabbage grown outdoors and in thegreenhouse. In addition to environmental conditions and initialapplication rates, persistence of neem appears to be affected bythe host plant of the insect. Lettuce leaves are somewhattransparent, which might provide less protection from UVdegradation, but poorer penetration of the leaf surface might141also have reduced the initial concentrations within the plant.Studies of the persistence of neern are of practical importancefor the control of aphids in the laboratory and in the field.Persistence of 6 to 9 days is likely adequate under mostconditions. Weekly applications may be required when aphids arerapidly colonizing treated plants, but neem could be appliedless frequently if prolonged periods of time are required beforeaphid numbers reached damaging levels.The systemic activity of neem will also influence thefrequency of sprays. Improving the penetration of leaf surfacesshould increase the effectiveness of neem and lengthen the sprayinterval. As for many other systemic insecticides, however, theefficacy of neem is likely influenced by temperature, moisture,plant growth, and insect infestation levels; which likelyexplains the generally poorer control of M. persicae and A.gossypli on pepper during the second field trial (Table 1.4).Under certain conditions, effective control may require morefrequent applications of neem at higher concentrations.142CHAPTER 6EFFECT OF NEEM ON NATURAL ENEMIES OF APHIDSINTRODUCTIONContinuous use of broad—spectrum insecticides has resulted inthe development of resistant pests, resurgence of target insectpopulations, and secondary pest outbreaks. All of theseconsequences can be at least partially related to the disruptionof natural enemy populations (Hsieh & Allen 1986), and there is,therefore, a need for effective, bio—degradable pest controlmaterials with greater selectivity (Saxena 1987).Implementation of integrated pest management (1PM) programs,which, in addition to other control strategies, aims to maximizethe utilization of natural enemies, has been limited by a lackof selective pesticides. Although there are exceptions,pesticides that kill phytophagous pests while sparing adultbeneficials have mainly been observed for growth—regulatingcompounds (Schmutterer 1987).The current level of interest in pesticides derived from theneem tree is partly due to their reported selectivity towardphytophagous insects (Hoelmer et al. 1990). According to Larson(1990), Margosan—OTM is non—toxic to mammals, earthworms,predators, and honeybees. In general, neem products arethought to possess medium- to broad-spectrum activity toward143phytophagous insects, while being relatively benign tobeneficial insects, such as pollinators, predators, andparasitoids (Saxena 1987; Schmutterer 1988; National ResearchCouncil 1992). The selectivity of neem is believed to resultprimarily from a general lack of contact toxicity and the needfor ingestion (Saxena 1989; Schmutterer 1990a). However, areview of the current literature reveals that few data areavailable regarding the impact of neem on beneficial insects,other than direct contact toxicity studies (Hoelmer et al.1990)Laboratory experiments may be useful in determining the long—term or sublethal effects of neem on predatory and parasiticinsects, such as reduced growth, mobility, and reproduction. Anassessment of the effects of neem on aphid natural enemies underfield conditions is important, as results may differsubstantially from those obtained in the laboratory. Forexample, coccinellid and neuropteran larvae fed prey insectstreated with Margosan—OTM had lower rates of survival underlaboratory conditions, while the same material was essentiallynon-toxic to predators and parasitoids of A. gossypii and thesweetpotato whitefly in greenhouse trials (Hoelmer et al. 1990).Natural enemies of aphids are relatively mobile, and they maymove from areas treated with neem, or avoid consuming treatedprey. As an example, parasitoids attacked and parasitizedsignificantly fewer neem-treated whitefly nymphs, with thedeterrent effect diminishing over time (Hoelmer et al. 1990).144Exposure of beneficials to neem will also be minimized due tothe relatively short residual activity of neem (see Chapter 5).In the current study, the effect of neem on aphidophagousinsects was evaluated in the laboratory and in the field.Because aphids serve as a food source for a large number ofpredatory and parasitic insects, the effect of neem on non—target organisms is particularly important for the management ofaphid populations. Suitability for use in 1PM programs could beone of the most important properties of neem—based insecticides.145MATERIALS AND METHODSField Studies. The effect of neem on populations of aphidpredators and parasitoids was determined during the 1990 fieldtrials for the control of aphids with foliar applications of NSOand NSE (Chapter 1). All known members of the Aphidiidae andAphelinidae pupate inside or below the hardened skin of the deadhost, and counts of these aphid mummies may be used to estimatethe relative abundance of parasitoids (Mackauer & Kambhampati1988). Numbers of larval and pupal predators (syrphids,neuropterans, coccinellids, and cecidomyiids) and aphid mummies(without emergence holes) per plant were assessed during thefinal destructive counts. Numbers of predators and parasitoidson lettuce (2 trials), strawberry (2 trials), pepper (2 trials),and cabbage (1 trial), treated with NSO (1.0%), NSE (1.0%),pyrethrum (0.5 g/L), or emulsifier only as a control (1.06 ml/L,Mazon BSF19), were included in the final analysis, as thesetreatments were applied to all crops.Data were analyzed for natural enemy numbers per plot (10plants), with the various host plants serving as replicatedblocks. Total numbers of predators per plot and mummies perplot were transformed 1og0(X+l.0) and log(X+l.0), respectively,prior to ANOVA and Tukey’s mean separation tests.Predators and parasitoids may respond numerically to increasesin aphid numbers (density dependent), such that higher numbersof prey are correlated with higher numbers of natural enemies146(e.g. Stary 1970; Frazer 1988). Therefore, predator andparasitoid numbers per 1,000 aphids were also analyzed asoutlined above.Laboratory Studies. The effect of neem on an aphidparasitoid, Aphidius sp. (Aphidiidae), a hoverfly, Eupeodesfumipennis (Thompson) (Syrphidae), and the eleven-spotladybeetle, Coccinella undecimpunctata (L.) (Coccinellidae), wasinvestigated in a series of laboratory studies. E. fumipennisand Aphidius sp. were collected from natural infestations oncabbage in the Plant Science Greenhouses, U.B.C., on 27 April,and 13 June, 1991, respectively; and C. undecimpunctata wascollected at Totem Field, U.B.C., on 12 September, 1991.Colonies of all three species were maintained on canola ormustard cabbage plants infested with M. persicae. Larvae ofspecific ages were reared from eggs in standard petri dishes (15mm x 90 mm diameter), with aphids supplied as required. A smallhole (13 mm diameter) plugged with a cork allowed access andpermitted some air exchange. Adult C. undecimpunctata usuallyoviposited on the cork, and it was a preferred site for larvae.Petri dishes were sealed with strips of parafilm, and a filterpaper (90 mm diameter) was placed on the bottom of each dish.The effect of foliar neem sprays on survival of E. fumipennisand C. undecimpunctata was evaluated for larvae rearedindividually on neem—treated canola ‘Tobin’, Brassica campestrisL., infested with M. persicae as outlined previously (Chapter1). Early second instar larvae were transferred to canola147treated with NSO (0.0, 0.5, 1.0, or 2.0%) (10 mi/plant) for oneweek, at which time larvae were removed from the plants. E.fumipennis were then reared in groups of up to six individualsin plastic containers (13.5 X 9.0 X 21 cm) until they completedtheir development, while C. undecimpunctata were reared, two perdish, in petri dishes as outlined above. While in thecontainers or dishes, E. fumipennis and C. undecirnpunctatalarvae were supplied untreated M. persicae every one to twodays, and numbers of pupae and adults were recorded for theduration of the trial. Each treatment was applied to sixplants, and the entire experiment was replicated three times.The effect of neem on survival of E. fumipennis and C.undecimpunctata was also determined for larvae topically treatedwith NSO. Treatments (0, 1, 2, or 4% NSO) were applied using amicro—applicator to the dorsal abdomen of larvae, as outlinedpreviously for topical treatment of aphids (Chapter 3). At eachconcentration, 26 early second instar C. undecimpunctata and 23E. fumipennis were treated with 0.2 .Ll per larvae (approximately0.4 and 0.36 al/mg, respectively). Larvae were reared, two perdish, in petri dishes as outlined above, with survival topupation and to adult emergence assessed every two to threedays.The effect of foliar neem sprays on parasitism of M. persicaein the laboratory was determined under the conditions outlinedpreviously. Individual adult Aphidius females were placed ontreated mustard cabbage, and nine days later the number of148mummies and aphids per plant were assessed. As well, samples ofmummies were collected and placed in small petri dishes toevaluate adult emergence. Each treatment (0.0, 0.5, 1.0, and2.0% NSO) was applied to six plants, and the experiment wasreplicated a second time.Finally, M. persicae mummies were attached ventrally to cleartape and dipped in solutions of NSO (0.0, 2.5, and 5.0%) todetermine if exposure to neem reduced emergence of adultAphidius. After the mummies dried, they were placed in smallself-sealing petri dishes and held in the laboratory at roomtemperature. Prior to treatment, mummies were sorted into threeage classes: mature mummies, somewhat grey in colour; tancoloured mummies of medium age; and newly formed opaque mummies.Emergence of Aphidius adults from old, medium, and young mummiesoccurred approximately 0 to 4, 2 to 6, and 5 to 7 days aftertreatment, respectively.Numbers of mummies per plant and per 100 aphids were subjectedto ANOVA and Tukeys mean separation tests. Numbers of mummiesand parasitism rates were transformed by 1og0(X+l.0) andlog(X+l.O), respectively, prior to analysis. Chi-squareanalysis was used to determine differences in survival rates andadult emergence for predators and parasitoids exposed to neem.149RESULTS AND DISCUSSIONField Studies. Foliar applications of 1% NSO and 1% NSEsignificantly reduced the total number of aphid predatorscompared with controls (p<O.05) (Table 6.1). Numbers of larvaland pupal syrphids, cecidoxnyiids, coccinellids, andneuropterans, were consistently lower on plants treated with NSOand NSE, compared with controls, and populations of allpredators were 56% and 38% lower on plants treated with NSO andNSE, respectively. Pyrethrum sprays also reduced predatornumbers significantly (p<0.05) compared to controls, and therewas no overall difference between neem and pyrethrum treatments(p>0.05). Absolute counts of parasitized aphids (mummies), onthe other hand, did not differ significantly (p>0.05) for anytreatments (Table 6.1).In relation to their prey, natural enemies of aphids were notadversely affected by applications of neem. Generally, numbersof syrphids, cecidomyiids, coccinellids, and neuropterans per1,000 aphids were higher on plants treated with neem compared topyrethrum or to controls, but overall differences in predatornumbers were not significantly different for any treatment(p>0.05) (Table 6.1). The number of mummies per 1,000 aphidswas significantly higher for NSO and NSE treatments than forcontrols (p<0.05), being, on average, nearly 3.7 times higher.Numbers of predators on plants sprayed with neem may have beenlower as a result of lower aphid populations. Numbers of150Table 6.1. Effect of foliar applications of neem seed oil(NSO) and neem seed extract (NSE) to plants in the field onnumbers of aphid parasitoids (mummies) and predators (larvae andpupae) per 280 plants and per 1,000 aphidsPredatorsa ParasitoidsTreatment Syrph. Cecc. Cocc. Neur. Total TotalNo./280 plantsControl 137 184 78 14 413a 85aNSO 1.0% 92 74 12 2 180b 88aNSE 1.0% 71 118 61 5 255b lOlaPyrethrum 64 143 34 4 245b 93aNo./1,000 aphidsControl 13.7 18.4 7.8 1.4 41.3a 8.5aNSO 1.0% 34.2 27.5 4.5 0.7 66.9a 32.7bNSE 1.0% 21.1 33.2 18.1 1.5 73.9a 30.ObPyrethrum 8.3 18.6 4.4 0.5 31.8a l2.lbFor absolute counts and numbers per 1,000 aphids, means withina column followed by the same letter are not significantlydifferent (p>0.05, Tukey’s multiple range test).aLarval and pupal Syrphidae, Cecidomyiidae, Coccinellidae, andNeuroptera.151cecidomyiid eggs on Brussel’s sprouts were almost proportionalto numbers of M. persicae, and numbers of adult coccinellids oncorn were highly correlated with aphid densities (Wright & Laing1980; Nijveldt 1988). Due to the decrease in aphid numbers,counts of predators per plant may have over—estimated thedamaging effect of NSO and NSE. However, although an increasein host density is usually followed by an increase in numbers ofpredators and parasitoids, the relationship is often variableand difficult to determine (Stary 1970; Coderre 1988).Therefore, numbers per 1,000 aphids may not have provided anaccurate measure of neem’s impact on natural enemies. Neemsprays may have resulted in some mortality to immaturepredators, but the overall effect on predator populationsappears to be minimal.The effect of neem on entomophagous insects has beeninvestigated in only a few previous studies, and results havegenerally not been presented in peer—reviewed journals. Ingreenhouse trials, rates of parasitism of A. gossypil andgreenhouse whitefly on plants treated up to four times withMargosan—OTM were comparable to controls and significantlyhigher than other pesticide treatments (Hoelmer et al. 1990).Predacious coccinellids survived field applications of NSO thatsuccessfully controlled corn leaf aphid, Rhopalosiphum maidis(Fitch), and sorghum aphid, Melanaphis sacchari (Zehntner)(Srivastava & Parmar 1985). Similarly, applications of NSO tofield plots of rice, beginning 20 to 100 days after152transplanting, controlled leafhoppers and planthoppers, but didnot affect numbers of predatory spiders or mind bugs (Saxena etal. 1981). Parasitism of rice leaf folder larvae, Cnaphalocrocismedinalis (Guenee), was significantly higher on rice treatedwith neem, perhaps because treated larvae were unable to foldthe leaves properly. Endoparasitic hymenoptera pupated andemerged normally from leaffolder larvae fed neem—treated leavesof rice (Schmutterer et al. 1983).Results of the current trials indicate that neem sprays arenot seriously detrimental to natural enemy populations underfield conditions, particularly in comparison with syntheticinsecticides. Generally, natural enemies are more susceptibleto insecticides than are plant pests, and toxicity favouringaphids in comparison to natural enemies is well—documented (Horn& Wadleigh 1988). Elimination of predators and parasitoidsfollowing the use of broad—spectrum insecticides results fromdirect toxicity, starvation, and emigration. Based on a reviewof the available literature, Schmutterer (1988) concluded thatimportant enemies of insect pests, such as spiders, earwigs,ants, and some parasitic hymenoptera, are only slightly or notat all harmed by neem, and in some cases are even favoured.However, additional studies are required to evaluate thepossible long—term effects of neexn on the survival, behaviour,and fitness of predators and parasitoids (Hoelmer et al. 1990).Laboratory Studies. NSO applied to canola infested with M.persicae in the laboratory had a negative impact on the survival153of E. fumipennis and C. undecimpunctata (Table 6.2). For E.fumipennis, survival to pupation and to adult emergencedecreased with increasing concentrations of NSO. Pupation ratesfollowing exposure to NSO were 34 to 100% lower than controls,while successful emergence of adults ranged from 6.3% (0.5% NSO)to 0.0% (2.0% NSO), compared to 58.3% for controls. C.undecirnpunctata were even more sensitive to the growth—disrupting effect of NSO. At the lowest concentration tested(0.5%), only 1 of 24 larvae pupated (4.2%), and it subsequentlydied in the pupal stage (Table 6.2).Greater mortality in the laboratory, relative to the field,may have resulted from more thorough spray coverage and theinability of predators to select untreated prey, or move tountreated plant parts. Also, predator populations in the fieldare composed of several species which may differ in sensitivityto neem. Alternatively, applications of neem to plants in thefield may have had a stronger negative effect on aphid predatorsthan indicated by the data. Neem may have prolonged larvaldevelopment, or prevented pupal eclosion to adults, resulting inelevated counts. For example, Margosan—OTM had little effect onlarvae of horn flies, Haematobia irritans (L.), stable flies,Stomoxys calcitrans (L.), house flies, Musca domestica L., orfruit flies, which resulted in normal pupation. However, adultemergence was almost completely prevented (Miller et al. 1989;Stark et al. 1990). Under laboratory conditions, coccinellidand lacewing larvae have shown some growth—regulating effects154Table 6.2. Percent survival to pupation and to adultemergence for second instar syrphid, Eupeodes fumipennis(Thompson), and coccinellid, Coccinella undecimpunctata (L.),larvae on M. persicaea_infested canola treated with neem seedoil (NSO)E. fumipennis C. undecimpunctataTreatment pupation emergence (n) pupation emergence (n)% % % %Control 66.7a 58.3a (24) 50.Oa 41.7a (24)NSO 0.5% 43.Bab 6.3b (16) 4.2b 0.Ob (24)NSO 1.0% 33.3bc 4.2b (24) 0.Ob 0.Ob (24)NSO 2.0% 0.Oc O.Ob (6) 0.Ob 0.Ob (24)Means within a column followed by the same letter are notsignificantly different (p>0.05, Chi-square analysis).aMyzus persicae (Suizer), green peach aphid.155from neem picked up by prey insects, but in greenhouse trialsneem was essentially non-toxic (Hoelmer et al. 1990; NationalResearch Council 1992).Topical treatment of early second instar larvae with NSO atrates as high as 4% did not result in higher rates of mortalityfor C. undecimpunctata or E. fumipennis (Table 6.3), and almostall adults appeared normal. One C. undecimpunctata larvatreated with 4% NSO developed into an adult with deformed andinflated forewings and unfoldable hindwings (Figure 6.1). Thephysical appearance of this individual closely resemblesprevious descriptions of abnormal adult Mexican bean beetlesthat developed from larvae reared on bean leaves treated withneem seed kernel extract (Steets 1976). The phytophagousMexican bean beetle has been thoroughly investigated because ofits sensitivity to neem (Schinutterer 1987). Predaceouscoccinellids, however, are believed to be less sensitive toneem. For example, residues of Margosan—OTM applied to hibiscusleaves or to glass vials were not toxic to coccinellidpredators, Delphastus pusillus and Scyrnnus sp., of whitefly eggsor aphids (Hoelmer et al. 1990). As well, adult femalessupplied whitefly eggs treated with Margosan-OTM reproducednormally.According to Schmutterer (l990a), syrphid larvae are verysensitive to neem. Third instar larvae of the hoverflyEpisyrphus balteatus were mostly killed following treatment withan enriched neem seed kernel extract synergized with sesame oil156TreatmentControl 87.OaNSO 1% 69.6aNSO 2% 73.9aNSO 4% 73.9aMeans within a columnsignificantly different82.6a (23) 88.5a 69.2ab69.6a (23) 53.8b 42.3a65.2a (23) 69.2ab 61.5ab65.2a (23) 80.8a 76.9bfollowed by the same letter are not(p>O.05, chi—square analysis).Table 6.3. Percent survival to pupation and to adultemergence for second instar syrphid, Eupeodes fuinipennis(Thompson), and coccinellid, Coccinella undecimpunctata (L.),larvae topically treated with neem seed oil (NSO)E. fumipennis C. undecimpunctatapupation emergence (n) pupation emergence (n)% % % %(26)(26)(26)(26)157Figure 61 (a) Dorsal and (b) ventral view of adult eleven—spot ladybeetle, Coccinella undecimpunctata (L), withphysically deformed wings and elytra following topical treatmentof a second instar larva with neem seed oiL—-c3a---‘“. ‘I’.158(Schauer 1985). In the current study, E. fumipennis and C.undecimpunctata were both sensitive to NSO applied to canola(Table 6.2), but neither predator was sensitive to NSO appliedtopically (Table 6.3). Aphid nymphs were more sensitive to thecontact activity of NSO compared to predatory larvae. Theestimated concentration of NSO resulting in 50% mortality of N.ribisnigri was approximately 2.9% (Table 3.6), while mortalityof E. fumipennis and C. undecimpunctata topically treated withNSO did not differ from controls even at rates as high as 4%.The greater sensitivity of aphids to the contact activity ofneem might have resulted from differences in cuticlepermeability, as aphids are relatively soft-bodied in comparisonto most other insects.In the laboratory, NSO applied to mustard cabbage had littleeffect on rates of parasitism of M. persicae by Aphidius sp.(Table 6.4). Increasing concentrations of NSO reduced numbersof aphid mummies per plant in a dose-dependent manner, with thehighest concentration (2% NSO) resulting in 99% fewer mummiescompared to controls. However, aphid populations decreased in asimilar manner, so that rates of parasitism (mummies/lOO aphids)did not differ for any treatment. Parasitism rates wereconsistently lower at all rates of NSO, but the differences werenot statistically significant (p>0.05). Increasingconcentrations of NSO did, however, reduce emergence of adultparasitoids in a dose-dependent manner (Table 6.4), but thedifferences between rates of NSO were not significant (p>O.O5).159Table 6.4. Parasitism of green peach aphid, Myzus persicae(Suizer), by Aphidius sp. on caged mustard cabbage treated withformulated neem seed oil (NSO)Treatment Mummies/plant Mummies/lOO aphids Emergence (%)Control 66.8a (22.1) 1O.4a (2.1) 56.4aNSO 0.5% 3.3b (1.1) 2.4a (1.3) 20.ObNSO 1.0% 1.2b (0.4) 3.3a (6.4) 13.3bNSO 2.0% O.6b (0.3) 6.5a (7.9) 0.ObMeans within a column followed by the same letter are notsignificantly different (p>0.05, Tukey’s multiple range test;chi—square analysis for emergence).160Eclosion to adults for Aphidius exposed to NSO on intact plantsranged from 20% (0.5% NSQ) to 0% (2.0% NSO), compared to 56.4%for controls.Emergence of Aphidius adults from newly formed (young) mummiesdipped in solutions of NSQ was almost completely inhibited atconcentrations of 2.5 and 5.0% (Table 6.5). Results for the twoolder classes of mummies were very similar, with increasingconcentrations of NSO resulting in decreasing adult emergence.Eclosion to adults for mummies dipped in 0.0, 2.5, and 5.0% NSOaveraged approximately 83, 50, and 22%, respectively, for thesetwo classes combined, and all treatments were significantlydifferent (p<0.05).In previous studies, spraying of aphid mummies containinglarvae or pupae of braconid parasitoids, Dieraetiella rapae andAphidius cerasicola, with neem seed kernel extract did notprevent normal adult emergence (Schauer 1985). As well, eggs ofSpodoptera litura sprayed with neem seed kernel suspensionsprior to, or following, parasitism by Telenomus remus Nixon didnot affect adult emergence (Joshi et al. 1982). Dipping ofaphid or whitefly mummies in Margosan-OTM (0.67%) did not affecttwo species of aphid parasitoid, or two species of whiteflyparasitoid, but emergence of a third whitefly parasitoid,Eretmocerus californicus, was one half that of controls (Hoelmeret al. 1990). Whitefly mummies parasitized by E. californicushave a ventral opening, which may have allowed the oily solutionto enter the protective mummies and physically smother the161Table 6.5. Percent emergence of adult parasitoids, Aphidiussp., from three age classes of green peach aphid, Myzus persicae(Suizer), mummies dipped in solutions of neem seed oil (NSO)Adult Emergence (%)Treatment Young Mid MatureControl 20/24 = 83.3a 70/80 = 87.5a 22/28 = 78.6aNSO 2.5% 0/24 = 0.Ob 40/80 = 50.Ob 14/28 = 50.ObNSO 5.0% 1/24 = 4.2b 18/80 = 22.5c 6/28 = 21.4cMeans within a column followed by the same letter are notsignificantly different (p>0.05, chi-square analysis).162larvae or pupae.The oil—based formulations used in the current study may alsohave physically smothered the developing parasitoids, buttreatments were also applied at higher rates (up to 5% NSO) thanmost previous studies. Emergence of Aphidius was significantlyreduced, also, for mummies collected from plants treated withNSO at rates useful for the control of aphids in the laboratoryand in the field (0.5 to 2.0%) (Table 6.4). Compared to moredeveloped mummies, Aphiclius developing within newly formedmummies were more sensitive to NSO (Table 6.5), indicating thatparasitoids may be harmed by neem to a greater extent followingexposure at an earlier stage of development. Older parasitoidswithin protective mummies might not be affected as greatly, anddipping of mummies may underestimate the effect of neem on theseinsects.Compared to synthetic insecticides, exposure to neem wouldlikely be less harmful to parasitoids. Exposure of M. persicaemummies to field rates of diazinon and malathion was estimatedto inhibit emergence of Diaeretiella rapae (M’Intosh) by 100 and82%, respectively, while acephate and permethrin significantlyreduced survival of emerging D. rapae adults (Hsieh & Allen1986). Hymenoptera, in general, are highly susceptible to mostchemical insecticides (Horn & Wadleigh 1988). Limited toxicityto adults, and to larvae and pupae within aphid mummies, wouldgive neem a decided advantage over synthetic pesticides for thepreservation of parasitoids.163CONCLUS IONResults of the current study demonstrated that applications ofneem to plants in the laboratory negatively affected thesurvival of aphid predators and parasitoids (Table 6.2, Table6.4), and foliar sprays of NSO and NSE to plants in the fieldreduced the absolute number of predators, but not parasitoids(Table 6.1). However, numbers of natural enemies in relation toaphid numbers were as high as or higher than controls,indicating that neem is not very damaging to aphidophagousinsects in the field. The lack of damage to predator andparasitoid populations in the field, as compared to thelaboratory, might be due to poorer spray coverage, avoidance oftreated plant parts or prey, or reinfestation of treated plotsby natural enemies. As well, sensitivity of natural enemies toneem likely varies with the species and age of the insect.The effect of neem on natural enemies of insects has not beeninvestigated in any detail, and results should be interpretedwith caution. The short-term toxicity of neem applied to aspecific growth stage has often been used to evaluate effects onbeneficial insects, and many reports are anecdotal in nature.As Schmutterer (1987) points out, in contrast to bioassays withconventional pesticides, bioassays involving neem last for arelatively long time, sometimes for weeks, due to the delayedeffects of AZA and related compounds. In addition to directmortality, the possible long-term effects of neem on the164behaviour and fitness of predators and parasitoids needs to bestudied in greater detail (Hoelmer et al. 1990).In spite of the above concerns, neem appears to be suitablefor inclusion in 1PM programs. Desirable properties of neemthat likely contribute to the preservation of natural enemies,in addition to mainly an oral route of toxicity, includesystemic activity, limited persistence, antifeedant andrepellent activities, and a general lack of toxicity to adultinsects. The use of neem—based products offers an approach topest control that is less damaging than other methods tobeneficial insects.165GENERAL DISCUSSIONAphids are undoubtedly the most important insect pests ofagriculture in the temperate climatic zones (Minks & Harrewijn1989). In the past, control of aphids and the plant virusesthey transmit has depended almost exclusively on the use ofbroad—spectrum synthetic insecticides. As a result of thewidespread and often indiscriminate use of these chemicals, atleast 20 species of aphids are now resistant to insecticides,mainly organophosphates, but several species are also resistantto organochlorines, carbamates, and pyrethroids (van Lenteren1990). As an increasing number of synthetic pesticides arebeing removed from use, and pests are becoming more resistant tothe remaining ones, the search for novel botanical insecticidesthat are less likely to cause damage to man and the environmenthas hastened (Saxena 1989; Locke & Lawson 1990). Extracts fromthe Indian neem tree, which affect insects in a multitude ofways, are among the most promising new botanical materials.The utility of neem extracts was demonstrated in the presentstudy in that several economically important species of aphidson strawberry, cabbage, rutabaga, and pepper were effectivelycontrolled in the laboratory and in the field. For example,compared to controls, applications of 1% NSO reduced numbers ofM. persicae on sweet pepper and cabbage in the field by 66 to91%, comparable to the 77 to 96% reduction in numbers of M.persicae following sprays of 1% NSO to pepper and rutabaga in166the laboratory.Foliar applications of neem may not be efficacious for thecontrol of aphids under certain conditions. For example, whileapplications of NSO reduced numbers of N. ribisnigri on lettucein the laboratory in a dose—dependent manner, it was ineffectivefor the control of this species in the field. The reasons forthis difference are unclear at the present time, but failure tocontrol N. ribisnigri in the field may have resulted from therapid environmental degradation of the active compounds, fromcooler temperatures, or from changes to the host plant. Studiesof the persistence of neem indicated that residual activity ofNSO lasted for at least nine days when lettuce was reared in thegreenhouse, but activity was lost after three days when plantswere held outdoors. On the other hand, the toxic effect of NSOto M. persicae persisted for at least nine days followingapplications to mustard cabbage grown outdoors or in thegreenhouse. The current studies demonstrated conclusively thattoxicity of NSO or AZA was influenced by the host plant of theaphid.In the current investigation, effective aphid control wasachieved, at least in part, by the use of formulated materialshaving known amounts of the most active ingredient, AZA. Toensure that results are reliable and to allow for comparisonsbetween studies, there is a need for greater standardization ofneem products (Schmutterer & Helipap 1989; Isman et al. 1990b).The AZA content of neem extracts varies tremendously, and, at167the very least, concentrations of this limonoid should bereported in future studies (Ermel et al. 1987; Isman et al1990a). Failure to control aphids, as well as other insects, inthe past may have resulted from the use of poorly formulatedmaterials having little or no active ingredients.Increased efficacy of neem materials can likely be achievedwith the use of additives to protect AZA from UV light and toimprove penetration of leaf surfaces. Degradation of NSO bysunlight was reduced by adding 1% carbon particles or 2% liquidlatex (Saxena 1987), while toxicity of neem extracts to nymphalA. pisum and A. fabae on intact plants was greatly increasedwith additives that improved systemic activity (Schauer 1985).Control of aphids may also be enhanced with the use ofinsecticide synergists. As an example, addition of thesynergist piperonyl butoxide improved the toxicity of NSE andMargosan—OTM to larval Colorado potato beetles (Lange &Feuerhake 1984; Zehnder & Warthen 1990). Research to improveformulations and application methods is particularly importantfor the control of phloem—feeding insects, as the activecomponents of neem must enter the plant to be fully effective.Results from this study indicate that the active components ofneem move readily throughout leaves, but penetration of leafsurfaces may vary with the species of plant, and possibly withenvironmental conditions. On the other hand, neem applied as adrench is generally not taken up from soil and translocated toleaves in high enough concentrations to effectively reduce aphid168numbers. There are exceptions, however, and applications toroots may be efficacious in the greenhouse for plants grownhydroponically or in artificial media.Except where virus diseases are a concern, control of aphidsis usually required only at relatively high population levels(van Lenteren 1990). Applications of neem can be used in thesesituations to maintain numbers below economic injury levels.For the majority of crops, neem will likely prove to be aneffective aphicide when used alone, or in conjunction with othercontrol strategies. However, compared to synthetic neurotoxins,neem is slow—acting and rarely results in complete suppressionof aphid populations, making it unsuitable for use in certainsituations. Neem would be a poor candidate for crops that havevirtually a zero tolerance for aphid infestations, such as leafyproduce sold on the fresh market.Because neem is somewhat selective toward phytophagous insects(Schmutterer 1987; Saxena 1989), it may be suitable forinclusion in 1PM programs, where aphid control can be enhancedby the activity of predators and parasitoids. In the currentstudy, although exposure to neem decreased the survival ofAphidius, C. undecinipunctata, and E. fumipennis under laboratoryconditions, foliar treatments appeared to cause only minimaldamage to natural enemy populations in the field. Parasitoidpopulations, as indicated by numbers of aphid mummies, were notsupressed by foliar applications of NSO or NSE, and parasitismrates were significantly higher on plants treated with neem169compared to controls. Numbers of predators were lower in neemplots than in controls, but numbers relative to aphidpopulations were not significantly different. Although neem’ssuitability for inclusion in 1PM programs promises to be one ofits most important assets, considerably more research in thisarea is required.Control of insects with neem results from several combinedactivities, including feeding deterrency, inhibition of growth,and reduced reproduction. Although the antifeedant andrepellent activities of neem are well documented for insectsfrom several orders, and many insects respond to neem or AZA atvery low concentrations (Pradhan et al. 1962; Butterworth &Morgan 1971; Saxena 1989; Schmutterer 1990a), deterrency toaphids appears to be of limited importance. At practicalapplication rates (i.e. 0.5 to 2.0% NSO, or 10 to 40 ppm AZA)NSO was only deterrent to half the species tested.Additionally, activity toward C. fragaefolii was lost in lessthan one day following applications of 1 or 2% NSO to plants inthe greenhouse. The components of neem responsible for thedeterrency to aphids are currently unknown, but activity likelyresults from the combined action of several terpenoid and non—terpenoid compounds. Although the antifeedant or repellentactivity of neem appears to be of limited value for the controlof aphids, it may contribute to the control of certain speciesand the viruses they transmit.Undoubtedly, control of aphids results primarily from the170physiological effects of neem. Aphid nymphs exposed to NSO orAZA failed to molt, while treated adults, or treated nymphs thatsuccesfully molted to adults, produced substantially feweroffspring. The toxic and sterilizing effects of neem towardaphids were similar in magnitude, but sensitivity was highlyvariable between species. For example, the concentration of AZAresulting in 50% mortality of second instar nymphs ranged fromas low as 1.8 ppm for M. persicae on mustard cabbage, to 635 ppmfor C. fragaefolii on strawberry; while 50% fewer offspringwere produced by adults exposed to AZA at concentrations rangingfrom 14.4 ppm for N. ribisnigri on lettuce to 616.4 ppm for C.fragaefolii on strawberry. Decreased production of offspringmost likely resulted from reduced maturation of oocytes, butincreasing concentrations of AZA also caused developing embryosto die just prior to birth.Exposure to neem also results in several sub—lethal andfitness-reducing effects that may be important for the controlof populations over longer periods of time. Adult N. ribisnigriand M. persicae that developed from nymphs exposed to NSO oftenpossessed physical abnormalities, particularly to their legs andwings. Deformities to the wings of alates ranged from minortwisting to complete absence. Inability of aphids to dispersewould increase their susceptibility to predation, and limit thespread of plant diseases.Survival of nymphs from adult aphids treated with NSO or AZAwas significantly lower than controls, most likely because171viviparous reproduction exposes developing embryos to the activecomponents of neem. Exposure to neem may cause additional subacute effects that would contribute to the management of aphidpopulations. For example, AZA fed to last-instar R. prolixusnegatively affected the cellular and humoral defense mechanisms,resulting in a decreased ability to prevent infection by thebacteria Enterobacter cloacae (Azambuja et al. 1991), and thereare reports that neem limits the growth and functioning of theobligatory endosymbionts of leafhoppers (R.C. Saxena, personalcommunication). Control of aphids with neem might, therefore,be enhanced by the activity of naturally occurring or appliedpathogenic organisms.AZA accounts for most of the observed activity of NSO, butthere are important exceptions. For example, AZA applied toleaf disks of strawberry at a concentration of 40 ppm did notresult in higher mortality of second instar C. fragaefoliicompared to controls, while NSO containing the equivalent amountof AZA resulted in 67% higher mortality. Similarly, NSO appliedto strawberry at concentrations of 1 to 2% effectivelycontrolled C. fragaefolii, but very high concentrations of AZA(635 ppm) were required for 50% mortaltiy of this species ontreated leaf disks. In addition to several other limonoids,neem extracts contain numerous other bioactive compounds,including fatty acids, sterols, phenols, flavonoids, glycosides,and organosulfur compounds (Balandrin et al. 1988; Jones et al.1989; Klocke et al. 1989; Koul et al. 1990). For this reason,172studies involving AZA may underestimate the effect of neem oninsects, as the response to individual components appears tovary for certain species.Exposure of insects to neem results in numerous behaviouraland physiological disturbances, and resistance to neem is,therefore, remote. Two genetically distinct lines ofdiamondback moth exposed to neemn seed kernel extracts over 42generations showed no signs of resistance, while exposure to thepyrethroid insecticide deltamethrin (DecisTM) over the sameperiod of time resulted in resistance levels 20 to 35 timeshigher (Vollinger 1987). The widespread use of neem is unlikelyto result in pest resurgences and the need for increasinglyhigher rates of application.New approaches are required to properly evaluate neemextracts, as effects on insects are often subtle and delayed.Acute, short—term bioassays previously utilized for studies ofsynthetic neurotoxins are inappropriate for investigations ofneem. Additionally, due to a higher degree of variability,investigations should ideally involve several closely relatedinsects on different hosts. Results have often beencontradictory and inconclusive in the past, and because neemn isa natural product, materials have to be evaluated, handled, andapplied with greater care in comparison to syntheticinsecticides.Novel botanical insecticides from neem represent a moreenlightened and selective approach to the management of insect173populations. They offer an effective means of pest control thatis compatible with other control strategies, while causingminimal harm to man, beneficial organisms, or the environment.The current investigation has hopefully added to our knowledgeand assisted in the evaluation and future registration of neem—based materials for the control of aphids in Canada andthroughout the world.174REFERENCESAdams, J.B. & H.P. van Emdem. 1972. The biological propertiesof aphids and their host plant relationships, pp. 47-104. 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USDA ARS-86.189APPENDIXSCIENTIFIC AND COMMON NAMES OF APHIDSAcyrthosiphon pisum (Harris) pea aphidAphis craccivora Koch cowpea aphidAphis fabae Scopoli black bean aphidAphis gossypii Glover cotton or melon aphidAulacorthurn solani (Kalt.) foxglove aphidBrevicoryne brassicae (L.) cabbage aphidChaetosiphon fragaefolii (Cockerell) strawberry aphidChaetosiphon spFimbriaphis fimbriata Richards (no common name)Lipaphis erysimi Kalt mustard aphidMacrosiphum euphorbiae (Thomas) potato aphidMetopolophium dirhodum (Walker) rose-grain aphidMyzus persicae (Suizer) green peach aphidNasonovia ribisnigri (Mosley) lettuce aphidRhopalosiphum padi (L.) bird cherry-oat aphidSitobion avenae (F.) english grain aphid


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