Development of the Major Triterpenoids and Oil in the Fruit and Seeds of Neem (Azadirachta indica)

In order to learn the best time for harvesting Neem (Azadirachtaindica) seeds, the amount of the five major triterpenoids, togetherwith the oil content have been determined throughout a fruitingseason in six selected trees in Sri Lanka. The triterpenoidcontent and the relative proportions of the major compoundschanged little from the hard green fruit stage to mature seeds,while the oil content increased markedly with time. The highestcontent of azadirachtin (10 mg g^-1seed kernels) was recordedin newly ripened seeds. There was some loss of salannin andazadirachtin in storage after harvesting for up to 6 months. Azadirachtin; salannin; nimbin; neem seeds; neem fruit; seasonal change; neem oil; seed storage; Azadirachta indica ; Meliaceae     

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.     

Suppression of branches in Eucalyptus trees

The effect of neem oil, which acts as a suckericide in tobacco, on branch suppression in Eucalyptus tereticornis was assessed to help maximize stem biomass. Lateral branches of selected trees were pruned, and neem oil solutions at concentrations of either 80%, 40%, 20%, 10%, or 0% (untreated control) were applied to leaf axils of the pruned branches. Regeneration of branches was suppressed, and the magnitude of suppression was proportional to the concentration of neem oil. Compared to the control, the percentage reduction in branching at 80% neem oil was 41.6%. When regenerated branches were repruned and neem oil applied at either 100%, 80%, or 0% (control), the regenerating ability of these branches was severely repressed by 78% at 100% neem oil relative to the control. Apical shoots were also topped and treated at either 100% or 0% (control) neem oil to identify the principal suppressive component in neem oil. The principal component azadirachtin was tested at 375, 750, 1500, 3125, 6250, 12?500, 25?000, 50?000, and 100?000 ppm and 0 ppm as the control. Reduction in the coppicing shoot was as high as 85%. Azadirachtin was responsible for the suppression. By pruning the lateral branches with neem oil, wasteful consumption of photosynthates can be precluded and the stem biomass maximized.     

Neem secretory cells: developmental cytology and indications of cell autotoxicity

The neem tree (Azadirachta indica A.Juss.) contains a range of biologically active compounds—mainly triterpenoids produced in single secretory cells, which are distributed among all plant parts. Neem secretions are toxic to animal cells, triggering autolytic mechanisms that culminate in cell disruption. However, little is known about the self-toxicity of these secretions to the cells that produce them. We carried out an anatomical, histochemical, and ultrastructural investigation of neem’s single secretory cells in the shoot apex and in young leaves. We evaluated the morphological changes as possible evidences of stress reactions to their own secretions. The subcellular apparatus involved in synthesis and compartmentation was consistent with hydrophilic and lipophilic secretions. Polymorphic plastids devoid of thylakoids and abundant smooth endoplasmic reticulum in the later stages of differentiation are comparable with previous reports on neem cotyledons with regard to terpenoid synthesis. However, secretions were compartmentalized within autophagic vacuoles and periplasmic spaces instead of in terpenoid vesicles. Cellular swelling, increased vesiculation, dilatation of endoplasmic reticulum cisternae, mitochondrial hypertrophy in the cristolysis process, autolytic vacuoles, and vacuolar degeneration culminating in protoplast autolysis are all consistent with early indications of autotoxicity. The signaling stress reaction mechanism was expressed as cytoplasmic deposits of calcium salt and by the expression of a 70-kDa heat-shock protein. The morphological and histochemical changes in the secreting cells are comparable with those described in animal cells exposed to neem oil. Our data provide evidence of cell damage and signaling reactions linked to these cells’ own secretions before autolysis.

     

Effect of Neem and Selected Fungicides on Viability and Virulence of the Entomopathogenic Nematode Steinernema feltiae

Entomopathogenic nematodes are often used in conjunction with other pest management tactics and the lack of compatibility information is a major impediment in further expansion of their use. We evaluated the effects of different formulations of neem and selected fungicides commonly used in greenhouses on Steinernema feltiae which is used for the control of fungus gnats. Neem as pure oil at the field recommended concentrations (5- 10 mL L -1 ) had no effect on the viability and virulence of S. feltiae up to 120 h incubation. However the neem formulation, Nimbecidine and neem oil when mixed with a bactericidal soap (commonly used as a surfactant with neem oil) caused 13- 25% mortality of S. feltiae. This toxic effect was entirely due to the soap that alone caused about 24% mortality. Neither neem oil, Nimbecidine or soap had any effect on nematode virulence. The fungicide cinnamaldehyde (Cinnamate) was highly toxic, resulting in 100% nematode mortality after 4 h of incubation, followed by hydrogen dioxide/peroxyacetic acid mixture (ZeroTol) that caused 100% mortality after 120 h of incubation. Another fungicide, azoxystrobin (Abound) caused no nematode mortality. This investigation concludes that neem and the fungicide azoxystrobin (Abound) can be safely tank mixed at the field recommended concentrations with the infective juveniles of S. feltiae for application, but cinnamaldehyde (Cinnamate) and hydrogen dioxide/peroxyacetic mixture (ZeroTol), are incompatible. Also the surfactants that are usually recommended as 'tank-mix' applications can be toxic to the nematodes and should therefore be evaluated for compatibility prior to use.     

Variations in fatty acid composition of neem seeds collected from the Rajasthan state of India

Neem (Azadirachta indica) is a multipurpose tree native to the Indian subcontinent and South-East Asian countries. Products derived from neem have been used for centuries, particularly in India, for medicinal and pest-management purposes. Azadirachtin and neem oil are the two major commercially important products derived from the tree. The oil contains palmitic, stearic, oleic and linoleic acids in good proportion. Although there is growing demand for quality planting material for plantation of neem, efforts are lacking for the selection of neem trees based on their biochemical composition. In the present study, 60 Neem seed samples were collected from different provinances of the Rajasthan state in India. These samples were analysed by GLC to study the variability of fatty acid composition. Significant variability in individual fatty acids was observed. The palmitic acid ranged from 16 to 34%, stearic acid from 6 to 24%, oleic acid from 25 to 58% and linoleic acid from 6 to 17%. This variability can be exploited for selection of trees and for studying the genetic variability in neem. These selections can also be utilized for genetic improvement of the tree.     

Biodegradable polymer based encapsulation of neem oil nanoemulsion for controlled release of Aza-A

Azadirachtin a biological compound found in neem have medicinal and pesticidal properties. The present work reports on the encapsulation of neem oil nanoemulsion using sodium alginate (Na-Alg) by cross linking with glutaraldehyde. Starch and polyethylene glycol (PEG) were used as coating agents for smooth surface of beads. The SEM images showed beads exhibited nearly spherical shape. Swelling of the polymeric beads reduced with coating which in turn decreased the rate of release of Aza-A. Starch coated encapsulation of neem oil nanoemulsion was found to be effective when compared to PEG coated encapsulation of neem oil nanoemulsion. The release rate of neem Aza-A from the beads into an aqueous environment was analyzed by UV-visible spectrophotometer (214nm). The encapsulated neem oil nanoemulsion have the potential for controlled release of Aza-A. Neem oil nanoemulsion encapsulated beads coated with PEG was found to be toxic in lymphocyte cells.     

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.     

Comparative laboratory toxicity of neem pesticides to honey bees (Hymenoptera: Apidae), their mite parasites Varroa jacobsoni (Acari: Varroidae) and Acarapis woodi (Acari: Tarsonemidae), and brood pathogens Paenibacillus larvae and Ascophaera apis

Laboratory bioassays were conducted to evaluate neem oil and neem extract for the management of key honey bee (Apis mellifera L.) pests. Neem pesticides inhibited the growth of Paenibacillus larvae (Ash, Priest & Collins) in vitro but had no effect on the growth of Ascophaera apis (Olive & Spiltoir). Azadirachtin-rich extract (neem-aza) was 10 times more potent than crude neem oil (neem oil) against P. larvae suggesting that azadirachtin is a main antibiotic component in neem. Neem-aza, however, was ineffective at controlling the honey bee mite parasites Varroa jacobsoni (Ouduemans) and Acarapis woodi (Rennie). Honey bees also were deterred from feeding on sucrose syrup containing > 0.01 mg/ml of neem-aza. However, neem oil applied topically to infested bees in the laboratory proved highly effective against both mite species. Approximately 50-90% V. jacobsoni mortality was observed 48 h after treatment with associated bee mortality lower than 10%. Although topically applied neem oil did not result in direct A. woodi mortality, it offered significant protection of bees from infestation by A. woodi. Other vegetable and petroleum-based oils also offered selective control of honey bee mites, suggesting neem oil has both a physical and a toxicological mode of action. Although oils are not as selective as the V. jacobsoni acaricide tau-fluvalinate, they nonetheless hold promise for the simultaneous management of several honey bee pests.     

Antifertility effects of neem (Azadirachta indica) oil by single intrauterine administration: a novel method for contraception

A novel use of neem (Azadirachta indica) oil, a traditional plant product, for long-term and reversible blocking of fertility after a single intrauterine application is described. Female Wistar rats of proven fertility were given a single dose (100 microliters) of neem oil by intrauterine route; control animals received the same volume of peanut oil. Whereas all control animals became pregnant and delivered normal litters, the rats treated with neem oil remained infertile for variable periods ranging from 107 to 180 days even after repeated matings with males of proven fertility. The block in fertility was, however, reversible as half of the animals regained fertility and delivered normal litters by five months after treatment, without any apparent teratogenic effects. Unilateral administration of neem oil in the uterus blocked pregnancy only on the side of application whereas the contralateral uterine horn treated with peanut oil had normally developing foetuses; no sign of implantation or foetal resorption was noted in the neem-oil-treated horn. The ovaries on both sides had 4-6 corpora lutea indicating no effect of treatment on ovarian functions. The animals treated with neem oil showed a significant leukocytic infiltration in the uterine epithelium between days 3 and 5 post coitum, i.e. during the pre-implantation period. Intrauterine application of neem oil appears to induce a pre-implantation block in fertility; the possible mechanisms of the antifertility action are discussed.     

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.     

Field evaluation of neem and canola oil for the selective control of the honey bee (Hymenoptera: Apidae) mite parasites Varroa jacobsoni (Acari: Varroidae) and Acarapis woodi (Acari: Tarsonemidae)

Neem oil, neem extract (neem-aza), and canola oil were evaluated for the management of the honey bee mite parasites Varroa jacobsoni (Oudemans) and Acarapis woodi (Rennie) in field experiments. Spraying neem oil on bees was more effective at controlling V. jacobsoni than feeding oil in a sucrose-based matrix (patty), feeding neem-aza in syrup, or spraying canola oil. Neem oil sprays also protected susceptible bees from A. woodi infestation. Only neem oil provided V. jacobsoni control comparable to the known varroacide formic acid, but it was not as effective as the synthetic product Apistan (tau-fluvalinate). Neem oil was effective only when sprayed six times at 4-d intervals and not when applied three times at 8-d intervals. Neem oil spray treatments had no effect on adult honey bee populations, but treatments reduced the amount of sealed brood in colonies by 50% and caused queen loss at higher doses. Taken together, the results suggest that neem and canola oil show some promise for managing honey bee parasitic mites, but the negative effects of treatments to colonies and the lower efficacy against V. jacobsoni compared with synthetic acaricides may limit their usefulness to beekeepers.     

Effects of neem oil on feeding behaviour and development of the pear sawfly, Caliroa cerasi

Neem oil deterred feeding by pear sawfly, Caliroa cerasi L., larvae (Hymenoptera: Tenthredinidae), both in choice and in no-choice cherry leaf disk bioassays. Deterrence was greater in the choice tests, with 50% inhibition occurring at 0.49% aqueous neem oil compared with 1.11% in the no-choice tests. Antifeedant activity towards pear sawfly larvae is slightly less than has been observed for Lepidoptera, but is higher than deterrence to other insects such as aphids. Topical application of neem oil to sawfly larvae resulted in reduced feeding, increased mortality, and a trend towards slower development. Most larval mortality after neem treatment resulted from incomplete subsequent moults. The potential utilization of neem insecticides for control of pear sawfly in tree fruit pest management is discussed.     

Mouse sperm-egg interaction in vitro in the presence of neem oil

In vitro evidence is presented showing toxicity of neem oil on sperm-egg interaction in mouse. Cumulus oophorus-enclosed ova, inseminated with capacitated spermatozoa, were cultured in 1 ml of in vitro fertilization (IVF) medium and overlayered by 1 ml of different concentrations of neem oil (1, 5, 10, 25, 50 and 100%) for IVF duration of 4h. At the end of incubation, ova were allowed to grow in neem oil-free culture medium and assessed for fertilization, first cleavage (2-cell formation) and blastocyst formation in vitro at 4–14h, 24h and 108h post-insemination respectively. The study showed that the presence of neem oil at concentrations of 10, 25 and 50% caused inhibition of IVF in a dose-dependent manner. The toxic effect of exposure of 25 and 50% neem oil was further carried over to the first cleavage of the resulting fertilized ova and the toxic effect of 5, 10, 25, and 50% was carried over to the blastocyst formation from the resulting fertilized ova when grown in neem-oil free culture medium. A total of 94.1% inhibition of 2-cell formation and 100% inhibition of blastocyst formation from the inseminated ova was observed in 50 and 25% neem oil-treated groups respectively. Neem oil at 100% concentration caused 100% degeneration of ova at 1h of sperm-ova coculture. The study showed a direct toxic effect of neem oil on sperm-egg interaction in vitro and encourages research investigations of this herbal product as a pre-coital contraceptive.     

Effects of neem seed derivatives on behavioral and physiological responses of the Cosmopolites sordidus (Coleoptera: Curculionidae)

Both in a choice and multi-choice laboratory tests, fewer adults of the banana root borer, Cosmopolites sordidus (Germar), settled under the corms of the susceptible banana "Nakyetengu" treated with 5% aqueous extract of neem seed powder or cake or 2.5 and 5% emulsified neem oil than on water-treated corms. Feeding damage by larvae on banana pseudostem discs treated with 5% extract of powdered neem seed, kernel, or cake, or 5% emulsified neem oil was significantly less than on untreated discs. The larvae took much longer to locate feeding sites, initiate feeding and bore into pseudostem discs treated with extract of powdered neem seed or kernel. Few larvae survived when confined for 14 d on neem-treated banana pseudostems; the survivors weighed two to four times less than the larvae developing on untreated pseudostems. Females deposited up to 75% fewer eggs on neem-treated corms. In addition, egg hatching was reduced on neem-treated corms. The higher the concentration of neem materials the more severe the effect.     

The effect of neem (Azadirachta indica A. Juss) oil on oviposition, development and reproductive potentials of Sesamia calamistis Hampson (Lepidoptera: Noctuidae) and Eldana saccharina Walker (Lepidoptera: Pyralidae)

Abstract 1 The effect of different treatments of neem oil (0, 0.075, 0.1 and 0.15 mL/plant) and their persistence (0, 5 and 10 days after application; DAA) on the oviposition behaviour and the bionomics of the noctuid Sesamia calamistis and the pyralid Eldana saccharina were tested in laboratory and greenhouse experiments. 2 For most variables, no difference was found between DAA, showing that the treatments had a long‐term effect, and pooled analyses across DAA were performed. Compared with the control (0 mL/plant), mean reductions due to neem in numbers of egg batch and eggs laid were 70 and 88% for S. calamistis and 50 and 49% for E. saccharina, respectively, but no differences were found among neem concentrations. 3 For both species, larval and pupal development time was shortest in the control and longest with the highest oil concentration. Immature survival, larval weight and fecundity were highest in the control and similar in the neem treatments. 4 No differences were found in sex ratios. Egg viability was highest in the control (approximately 87%) and lowest (72%) with the highest oil concentration. As a result of lower fecundity and longer developmental time, on average, the intrinsic rates of increase in the neem treatments were 30% lower than in the control. 5 In view of the low oviposition rates, immature survival, fecundity and egg viability in the neem treatments, and the relatively high persistence of neem oil, it can be expected that the reduction in densities of the two borers species in the field will be considerable.     

Bacillus thuringiensis and neem seed oil (Azadirachta indica) effects on the potato tuber moth Phthorimaea operculella zeller in the field and stores

A comparative evaluation for the efficacy of Bacillus thuringiensis and neem seed oil on Phthorimaea operculella has been carried out in the field and store. These two preparations were almost equally effective on the potato tuber moth infestation. The percentage of infestation was reduced through successive application of either preparations in the field up to harvest. No synergism was observed upon using combination of the two preparations. In the store, neem seed oil (500 ppm) was highly protective and was as effective as sevin. A combination of both neem and B.t. (Delfin) significantly protects the tubers. This suggests the possible use of either neem seed oil or B.t. in combating the insect pest in the field or during storage.     

Growth and insect assays of Beauveria bassiana with neem to test their compatibility and synergism

Beauveria bassiana is being used as a biopesticide for many insect pests. Neem oil (azadirachtin) is an eco-safe popular botanical pesticide. A biopesticde with a neem compatible isolate of B. bassiana will enable their simultaneous use in pest management. A sample of 30 isolates of B. bassiana from culture collections was screened for compatibility with a commercial formulation of neem oil (Margoside®) at the field recommended dose (0.3%, v/v). Compatibility was tested in vitro through germination and growth assays. In all isolates, conidial germination was delayed but not significantly decreased by neem. In the growth assays, 23 isolates were found compatible with neem. In the neem sensitive isolates, growth was decreased but not totally inhibited. The effect of combined treatment with B. bassiana and neem in comparison to single treatments with either of them on Spodoptera litura Fabricius was tested in laboratory bioassays. The combined treatment was found to have synergistic effect on insect mortality when a B. bassiana isolate compatible with neem was used, while, with an isolate sensitive to neem, an antagonistic effect was observed.     

Oil body formation in Euphorbia tirucalli L. cell suspension cultures

The culture of cells of Euphorbia tirucalli L. resulted in the formation of intracellular particles which osmium tetroxide staining and electron microscopy indicated to be oil bodies. Chemical analysis (TLC, HPLC, GLC and GC-MS) disclosed steroids, triterpenoids, and diterpenoids as the major oil components of the cultured cells.     

Management of root lesion nematode, Pratylenchus delattrei in crossandra using oil cakes

Selected oil cakes, neem, castor and mahua, were tried independently and in combination with a chemical nematicide (carbofuran 3G) for the management of Pratylenchus delattrei in crossandra under glass house conditions. The neem oil cake was effective compared to other oil cakes used and there was a synergistic effect when the neemcake was coupled with carbofuran 3G in the management of Pratylenchus delattrei. The treatment resulted in better establishment of seedlings, and with increased plant bio-mass and flower yield.