Azadirachta indica metabolites interfere with the host-endosymbiont relationship and inhibit the transmission of potato leafroll virus by Myzus persicae

The effects of neem (Azadirachta indica A. Juss) seed kernel extracts (NSKE) and azadirachtin on the ability of Myzus persicae (Sulz.) to transmit potato leafroll luteovirus (PLRV) was studied. Moreover, it was investigated whether treatments with these compounds would exert an effect on larval growth and mortality, and on the aphid intracellular symbionts. Endosymbiotic bacteria play an essential role in the performance of aphids, and in luteovirus transmission by aphids. NSKE and azadirachtin were offered to one-day-old M. persicae nymphs via a membrane feeding system. The neem metabolites displayed a 100% mortality at doses higher than 2560 ppm. At intermediate doses, ranging between 320 and 2560 ppm, larval growth and mortality were affected in a dose-dependent manner. The transmission of PLRV by M. persicae was inhibited by 55–90%. The endosymbiont population of the aphid was clearly affected by a treatment with neem metabolites as the release of their most abundant protein, Buchnera GroEL, into the haemocoel of the aphid was inhibited. Moreover, morphological aberrations on the bacterial endosymbionts were observed in aphids which fed on 2560 ppm of azadirachtin. At doses lower than 160 ppm of NSKE or azadirachtin, the endosymbiont population of M. persicae, and mortality, growth and feeding behaviour were similar to that of the untreated groups of aphids. However, PLRV transmission was still inhibited by 40–70%. The possible targets of the neem metabolites in the aphid are discussed.     

Insect growth regulating effects of neem extract and azadirachtin on aphids

Neem,Azadirachta indica (A. Juss.), seed oil (NSO) applied to leaf discs at a concentration of 1.0% resulted in 94% to 100% mortality of second instar nymphs of currant-lettuce aphid,Nasonovia ribis-nigri (Mosley), and green peach aphid,Myzus persicae (Sulzer), after nine days. The equivalent amount of pure azadirachtin (AZA) (≈40 ppm), the principle active ingredient of neem, was as effective as NSO. The survival of adult aphids was unaffected by NSO or AZA, but the survival of offspring from treated adultM. persicae andN. ribis-nigri was reduced significantly. The lethal concentration of AZA resulting in 50% mortality of second instar nymphs of nine species of aphids ranged from 2.4 ppm forM. persicae on pepper to 635.0 ppm for the strawberry aphid,Chaetosiphon fragaefolii (Cockerell), on strawberry. ForM. persicae, the growth regulating effect of AZA was influenced by the host plant and the nymphal instar treated.     

SYSTEMIC INSECTICIDAL ACTION OF AZADIRACHTIN, NEEM SEED AND CHINABERRY SEED EXTRACTS APPLIED AS SOIL DRENCHES TO POTTED PLANTS

Abstract  The systemic insecticidal activities of: 1) azadirachtin, a potent insect antifeedant and growth regulator isolated from seeds of the neem tree, Azadirachta indica , 2) a neem seed extract containing 0. 30 quantitative fraction of azadirachtin, and 3) a methanolic extract of seeds from the chinaberry tree, Melia azedarach , a close relative of neem were determined. Soil drenches of azadirachtin or the neem seed extract at 1 and 2. 5 mg to potted potato ( Solanum tuberosum ) and tomato ( Lycopersicon esculentum ), remarkably retarded the growth of Manduca sexta larvae and strongly disrupted the pupation of Leptinotarsa decemlineata larvae, respectively. However, five to ten times as much azadirachtin or neem seed extract was needed to reduce the amount of tomato and potato foliage consumed. On sorghum ( Surghum biocolor ), much higher quantity (≥25 mg) of azadirachtin or the neem seed extract was necessary to slow the population growth of Schizaphis graminum , and prolong the survival of treated plants. But methanolic extracts of chinaberry seeds ( M. azedarach ) from two sources (Guangdong, PRC and Hurricane, Utah, USA) at 150 mg per pot were ineffective in reducing aphid population growth and preventing plant damage.     

Neem seed oil inhibits aphid transmission of potato virus Y to pepper*

Transmission of potato vims Y to sweet pepper by the green peach aphid, Myzus persicae (Sulzer), was inhibited by foliar applications of 1.0% or 2.0% neem seed oil to infected source plants or to uninfected recipient plants. Neem seed oil interfered with virus acquisition and inoculation in a manner comparable to that of a commercial horticultural oil, while an oil-free neem seed extract did not reduce rates of transmission compared with controls. The finding that neem seed oil inhibits virus transmission, while oil-free neem seed extract does not, suggests that the presence of the oil rather than biologically active limonoids such as azadirachtin interfere with virus transmission. None of the treatments affected rates of infection when potato virus Y was transmitted mechanically, or the resulting virus titre and symptom expression. In addition to direct control of insect pests, formulated neem oils may help reduce or delay the spread of non-persistent plant viruses.     

Mycorrhizal inoculation in neem (Azadirachta indica) enhances azadirachtin content in seed kernels

In view of the high mycorrhizal dependency of neem trees (Azadirachta indica), an experiment was conducted to study if Arbuscular Mycorrhizal (AM) inoculation can enhance the azadirachtin content in seed kernels of trees grown in the field. Azadirachtin is an important active ingredient in neem seed kernels based on which a large biopesticide industry has emerged in India and few countries in Europe and the USA. Inoculation of neem seedlings in the nursery with Glomus fasciculatum and Glomus mosseae resulted in increased height, dry weight, root colonization and phosphorus (P) content. In a separate experiment, field-grown neem plants inoculated in the nursery and during transplantation with Glomus fasciculatum were evaluated after 5 years. No significant differences were found in the tree height, girth at breast height (GBH) and fruit yield but oil percentage, total triterpenoids and azadirachtin content in kernels increased significantly as a result of AM inoculation. A similar enhancement in azadirachtin was noted with P application. These results open up possibilities of producing quality neem seed with high bioactive ingredients through AM inoculation.     

Desensitization of fifth instar Spodoptera litura to azadirachtin and neem

The deterrence of azadirachtin, in its pure form and as a constituent of neem seed extract, to fifth instar Spodoptera litura (Fab.) larvae, was measured using cabbage, Brassica oleraceae (L.) var. capitata, leaf disc assays. Paired-choice assays, in which larvae could choose between feeding on a treated (1.3 ng azadirachtin per square cm leaf area) or an untreated leaf disc for 2 h, were conducted at 24 h intervals throughout the fifth instar. In addition, no-choice assays, in which larvae could feed on only one leaf disc (10 ng azadirachtin per square cm leaf area) for 1.5 h, were conducted consecutively over a six hour period at the beginning of the fifth instar. The effects of hunger and habituation on desensitization in our no-choice tests were partitioned. After repeated exposures, larvae became desensitized to pure azadirachtinal in both choice and no-choice tests, but did not desensitize to neem containing the same absolute amount of azadirachtin in choice tests. Hunger was responsible for approximately one third of the desensitization response in the no-choice tests. Sensitivity to azadirachtin was independent of age within the fifth instar.     

Evaluation of neem (Azadirachta indica) seed and ginger (Zingiber officinale) as potential control agents of yam (Dioscorea rotundata Poir.) tuber rot fungi

Yam is an important crop which serves as a source of income to small-holder farmers as well as a foreign exchange earner. Among the constraints faced by yam farmers are pests and diseases, especially deterioration caused by microbes during storage. Since over dependence on pesticides is being discouraged, neem seed and ginger extracts were evaluated as potential control agents against rot-causing fungi. The study was conducted in the Spanish laboratory, at the Faculty of Agriculture, University for Development Studies, Nyankpala Campus. Isolations were made from rotted yam tubers sampled from the Tamale Central market with potato dextrose agar (PDA) as the growth medium. Growth inhibition of the isolates was determined by growing pure cultures on PDA plates amended with 2?ml each of ethanol and aqueous extracts of neem seed and ginger as well as carbendazim. A pathogenicity test proved that Aspergillus flavus, A. niger, A. ochraceus and Penicillium digitatum isolated from the rotted tubers were responsible for the rot. A. niger had a significantly higher (p?<?0.05) occurrence (40%) than the others. Growth inhibitions by carbendazim and ethanol extracts of neem seed and ginger were comparable but significantly higher (p?<?0.05) than the aqueous extracts of neem seed and ginger as well as the control. However, the aqueous extracts of neem seed and ginger had a significantly higher (p?<?0.05) inhibition than the control. For instance, growth inhibition of A. niger by carbendazim, ethanol neem seed and ginger extracts were 70.5, 69.6 and 65.5%, respectively, while inhibition by aqueous neem seed and ginger extracts were 33.9 and 24.8%, respectively. Since aqueous extracts of neem seed and ginger significantly inhibited (p?<?0.05) growth of the rot-causing fungi, they can be used as surface protectants of stored tubers.     

Antifeedant effects of in vitro culture extracts of the neem tree,Azadirachta indica against the desert locust (Schistocerca gregaria (Forskål))

Callus and micropropagated shoots were initiated from leaf explants of the neem tree, Azadirachta indica A. Juss. A variety of whole plant and in vitro cell cultures from neem seedlings of Ghanian origin were tested for insect antifeedant compounds using the desert locust (Schistocerca gregaria (Forskål)). Feeding suppression occurred when whole extracts of seed, leaf, callus, suspension and shoot cultures were tested in no-choice feeding bioassays. Controls of sucrose, carrot callus and the plant growth medium showed no feeding deterrence. Azadirachtin, the main known antifeedant in neem seed kernels, was quantified from a seed extract by HPLC but was not detected in any of the other extracts. Antifeedancy was determined during batch growth of a suspension culture which had been in culture for 5 months; results indicated that antifeedants were still being formed and that levels increased after maximum biomass was attained.     

Host preference by Allothrombium pulvinum (Acari: Trombidiidae) larvae on aphids: Macrosiphum rosae,Aphis gossypii and Hyalopterus amygdali (Homoptera: Aphididae)

In this study aphid-plant association and its effect on host preference of parasitic Allothrombium pulvinum larvae was examined with multiple-choice tests. Host species selection, host size selection and superparasitism with mite larvae were studied with two-choice tests. Three aphid species were used: Macrosiphum rosae, Aphis gossypii and Hyalopterus amygdali. In multiple-choice tests, larvae of A. pulvinum showed no significant preference for any aphid-plant association when given M. rosae on rose, A. gossypii on cucumber and H. amygdali on apricot simultaneously. Two-choice tests showed that larval mites preferred H. amygdali to A. gossypii, but had no preference when offered a choice between A. gossypii and M. rosae or between H. amygdali and M. rosae. In host size selection and superparasitism tests, significantly more mites selected the larger host (M. rosae). Furthermore, parasitised H. amygdali were preferred to unparasitised ones. This revised version was published online in July 2006 with corrections to the Cover Date.     

Evaluation of neemAzadirachta indica seed extracts and oils as oviposition deterrents to noctuid moths

Applications of three concentrations of oil-free neem seed extracts (Azadirachta indica A. Juss; Meliaceae) to cabbage plants in cages did not deter oviposition by individuals of three species of noctuid moths,Trichoplusia ni, Peridroma saucia, andSpodoptera litura. The concentrations used corresponded to 10, 50, and 100 ppm of the main active ingredient, azadirachtin. The total number of eggs laid per female, female longevity, and median day of oviposition were not affected. Sprays of the neem oil-based insecticide Margosan-O ^R , and a 1% aqueous emulsion of a refined neem seed oil similarly had no effect on any of the parameters studied. However, a 1% crude oil emulsion significantly reduced the proportion of eggs laid byS. litura on treated plants. Our results suggest that literature reports of significant neem-based oviposition deterrence toS. litura are the result of compounds that are removed by higher levels of processing and thus not likely to be found in most commercial neem seed formulations. Sprays consisting of highly processed neem seed extracts, used at concentrations that provide larval control, are unlikely to be generally effective as oviposition deterrents to noctuid pests.     

The influence of azadirachtin on the feeding behaviour of cereal aphids and slugs

The probing behaviour of Rhopalosiphum padi (L.) and Sitobion avenae (F.) and the feeding behaviour of several slug species, (Deroceras reticulatum (Müller), Arion distinctus Mabille, Agriolimax caruanae Pollonera, Maximus sp.) were assessed on seedlings of winter barley (Hordeum marinum) treated with different concentrations of azadirachtin. Settling behaviour of both aphid species was strongly biased towards the untreated seedlings or those treated with low concentrations of azadirachtin. Concentrations of <500 ppm were effective with topical application and probing activity was reduced for at least 4 days after application. Systemic activity of azadirachtin against cereal aphids was also demonstrated. Feeding behaviour of the slug species, as seen by the amount of leaf eaten compared to the controls, was not affected by the presence of azadirachtin at those concentrations which deterred aphids from feeding. The relevance of these results to crop protection is discussed.     

Genetic, biochemical, and behavioral uniformity among populations of Myzus nicotianae and Myzus persicae

Prior to designation as distinct species, an appellation presently in question, the tobacco aphid, Myzus nicotianae Blackman (Homoptera: Aphididae), was classified as a tobacco-feeding form of the green peach aphid, Myzus persicae (Sulzer). In this study, RAPD polymorphisms distinguished members of the Myzus persicae complex (M. persicae and M. nicotianae) from three outgroup Myzus species (M. cerasi (F.), M. hemerocallis Takahashi, and M. varians Davidson). Polymorphisms within the complex did not separate populations on the basis of host association (tobacco versus other host plants) or geographic origin (collections from the United States, Europe, and Japan). Similarly, while GC-MS analysis of cuticular hydrocarbon profiles revealed both developmental and inter-populational differences within the M. persicae complex, it did not separate populations of tobacco feeding aphids from those collected off non-tobacco hosts. Finally, with the exception of their responses to a choice between lettuce and collards, the host preference behavior of a green peach aphid population, a red tobacco aphid population, and a green tobacco aphid population was indistinguishable in host preference experiments. These results add to a growing body of evidence suggesting M. nicotianae and M. persicae are conspecific.     

Potential of Margosan-O, an azadirachtin-containing formulation from neem seed extract, as a control agent for spruce budworm, Choristoneura fumiferana

The effects on spruce budworm larvae, Choristoneura fumiferana (Clem.), produced by ingestion of Margosan-O, a commercially available neem seed extract formulation containing 0.3% azadirachtin, were investigated. Bioassays with the test material were conducted using various instars of spruce budworm larvae, with either artificial diet, cut branches of balsam fir, Abies balsamea (L.) Mill., or small growing balsam fir trees as substrates. The dose-response data on feeding reduction, developmental retardation, and mortality (LC50, LC95, and LD50) suggest that Margosan-O has promise as a control agent for spruce budworm in an integrated pest management program.     

Effect of some crude and azadirachtin-enriched neem (Azadirachta indica) seed kernel extracts on larvae of Aedes aegypti

Bioassays against larvae of Aedes aegypti were conducted with neem seed kernel extracts obtained by extraction with water and organic solvents. Permanent exposure of fourth instar larvae to treated water resulted in a conspicuous growth-disrupting effect, mainly during imaginal development. The effectiveness of the extracts increased with decreasing polarity of the solvents used for extraction. Three neem seed kernel extracts caused an extreme prolongation of the larval period when first instar larvae were continuously exposed to treated water until adult emergence. The time necessary for lethal action of neem seed kernel extracts to set in was similar to that reported for some synthetic IGR's.

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Zusammenfassung Zehn mit Wasser und verschiedenen organischen Lösungsmitteln hergestellte Niem-Samen-Extrakte wurden auf ihre Wirkung auf Larven der Gelbfiebermücke Aedes aegypti untersucht. Dauerhaltung der Viertlarven in behandeltem Wasser führte zu einer beträchtlichen wachstumshemmenden Wirkung, die hauptsächlich während der Imaginalentwicklung in Erscheinung trat. Der Wirkungsgrad der Extrakte steigerte sich im biotest mit abnehmender Polarität der bei der Extraktion verwendeten Lösungsmittel. Drei Extrakte (ANSKE, AZT-VR-K-E und MTB/H₂O-K-NR-E) deren Wirkung auch auf Erstlarven untersucht wurde, verursachten erhebliche Entwicklungsverzögerungen wenn die Larven bis zum Erscheinen der Imagines behandeltem Wasser ausgesetzt waren. Die von den Extrakten verursachte Mortalität trat zu einem ähnlichen Zeitpunkt ein, wie er für einige synthetische Insektenwachstumshemmer berichtet wird.

     

Influence of growth medium on antifungal activity of neem oil (<Emphasis Type="Italic">Azadirachta indica</Emphasis>) against <Emphasis Type="Italic">Lagenidium giganteum</Emphasis> and <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis>

The inhibition of mycelial growth of Lagenidium giganteum by neem oil was lower than that of Metarhizium anisopliae in PYG and Emerson’s YpSs agar media. However, neem oil did not inhibit the mycelial growth of L. giganteum in sunflower seed extract agar medium, but did it inhibit the mycelial growth of M. anisopliae. The minimum inhibitory concentration of neem oil for L. giganteum was higher than that for M. anisopliae. The minimum fungicidal concentration of neem oil in PYG medium was lower than in YpSs for both fungi. The spores of L. giganteum grown in SFE medium could be used with neem oil for vector control.     

Comparative bioactivity of selected extracts from Meliaceae and some commercial botanical insecticides against two noctuid caterpillars, <Emphasis Type="Italic">Trichoplusia ni</Emphasis> and <Emphasis Type="Italic">Pseudaletia unipuncta</Emphasis>

Plant-derived extracts and phytochemicals have long been a subject of research in an effort to develop alternatives to conventional insecticides but with reduced health and environmental impacts. In this review we compare the bioactivities of some plant extracts with those of commercially available botanical insecticides against two important agricultural pests, the cabbage looper, Trichoplusia ni and the armyworm, Pseudaletia unipuncta. Test materials included extracts of Azadirachta indica (neem), A. excelsa (sentang), Melia volkensii, M. azedarach (Chinaberry) and Trichilia americana, (all belonging to the family Meliaceae) along with commercial botanical insecticides ryania, pyrethrum, rotenone and essential oils of rosemary and clove leaf. Most of the extracts and botanicals tested proved to be strong growth inhibitors, contact toxins and significant feeding deterrents to both lepidopteran species. However, there were interspecific differences with T. ni generally more susceptible to the botanicals than the armyworm, P. unipuncta. All botanicals were more inhibitory to growth and toxic (through feeding) to T. ni than to P. unipuncta, except for M. azedarach which was more toxic to P. unipuncta than to T. ni. Athough, pyrethrum was the most toxic botanical to both noctuids, A. indica, A. excelsa, and M. volkensii were more toxic than ryania, rotenone, clove oil and rosemary oil for T. ni. As feeding deterrents, pyrethrum was the most potent against T. ni, whereas A. indica was the most potent against the armyworm. Based upon growth inhibition, chronic toxicity, and antifeedant activity, some of these plant extracts have levels of activity that compare favorably to botanical products currently in commercial use and have potential for development as commercial insecticides.     

Effect of neem on the growth and development of the obliquebanded leafroller, Choristoneura rosaceana

Neem, Azadirachta indica A. Juss. (Meliaceae), seed oil (NSO) added to meridic diet at concentrations as low as 0.016% reduced pupation and prevented adult eclosion of obliquebanded leafroller, Choristoneura rosaceana (Harris) (Lepidoptera: Tortricidae). At a rate of 0.0016%, NSO reduced the fitness of C. rosaceana, resulting in longer developmental times, lower adult eclosion rates, and reduced egg production compared with controls. The neem insecticide Margosan-O ^TM produced comparable results based on concentrations of the most biologically active constituent, azadirachtin. Pupation was completely inhibited at concentrations of 0.25% and 1.0% for larvae exposed at 5th or 6th instar, respectively; rates as low as 0.016% reduced pupal weights and adult eclosion rates. For larvae transferred to treated diet at 5th instar, physical abnormalities in the wings of adults occurred at a rate of 0.004% NSO and increased with increasing treatment rates. NSO at concentrations as high as 2.0% was not antifeedant to neonate larvae, based on 24 and 48 h choice test bioassays, when incorporated into a meridic diet.     

Efficacy of neem pesticides on whorl larva,stem-borer and panicle insect pests of sorghum in Nigeria

Two field trials were conducted from 2003 to 2005 on the effects of neem pesticides on whorl larva, stem-borer and panicle pests of sorghum. In the first trial (2003–2004) the effect of neem seed granules (NSG) and carbofuran (furadan 3G®) inserted into the sorghum whorl at 30, 40 and 50 days after sowing (DAS) were tested on whorl larva, stem borer and grain yield. Results showed that NSG and carbofuran significantly (P ≤ 0.01) reduced whorl larva feeding, leaf puncturing and chaffy panicle, and significantly (P ≤ 0.01) increased grain yield compared with untreated check. In the second trial, (2004–2005), aqueous neem seed extract (ANSE), neem seed oil (NSO) and deltamethrin (Decis 12 EC®) were sprayed at anthesis and grain filling stages. Results also showed that the three pesticides significantly (P ≤ 0.01) reduced insect damage and also significantly (P ≤ 0.01) increased grain yield by 28.3% (deltamethrin), 19.4% (NSO) and 17.9% (ANSE) above the yield of check. It is suggested that neem pesticides may be suitable as alternatives to synthetic pesticides for the management of sorghum whorl larva, stem borers and panicle insect pests in the Nigerian Sudan savannah.     

Increased neem extract content enhances drying survival of co-encapsulated Saccharomyces cerevisiae and decreases relative release of azadirachtin

Plant extracts from the neem tree can be applied as a bioinsecticidal compound in ‘attract-and-kill’ co-formulations to control soil-dwelling insect pests. Since insects are attracted to the bioinsecticide, the attract-and-kill approach benefits from a reduced dose of insecticide needed. Here, we demonstrate that a powdery neem seed kernel extract in combination with corn starch significantly enhances the drying survival of co-encapsulated CO₂-releasing Saccharomyces cerevisiae. Additionally, an increased content of the neem extract was shown to slow down the relative release of azadirachtin A. Both findings are of high relevance for the development of formulations in the field of biological pest control.     

Variability of azadirachtin inAzarirachta indica (neem) and batch kinetics studies of cell suspension culture

Seeds of neem were collected from different parts of India and analyzed for their azadirachtin content by High Performance Liquid Chromatography (HPLC). In order to assess the effects of genotypic and geographical variation on azadirachtin content in cell cultures, callus development was attempted in the seeds containing high and low concentration of azadirachtin. The concentration of azadirachtin in callus cultures was significantly affected by the explant source. Seed kernels with higher azadirachtin content produced higher azadiractin content in callus cultures and lower azadirachtin content was seen in callus cultures produced from seed kernels with low azadiractin content. The protocol for development of elite stock culture ofAzadirachta indica was established with the objective of selecting a high azadirachtin-producing cell line. The highest azadirachtin-producing cell line was selected and the effects of different media and illumination conditions on growth and azadirachtin production were studied in shake flask suspension culture. Detailed batch growth kinetics was also established. These studies provided elite starter culture and associated protocols for cultivation ofA. indica plant cell culture in the bioreactor.