The toxicity and physiological effect of neem limonoids on Cnaphalocrocis medinalis (Guenée) the rice leaffolder

The effect of neem (Azadirachta indica) limonoids azadirachtin, salannin, deacetylgedunin, gedunin, 17-hydroxyazadiradione, and deaceytlnimbin on gut enzyme activity of the rice leaffolder larvae was investigated. When fed a diet of rice leaves treated with limonoids in bioassays, gut tissue enzymes—acid phosphatases (ACP), alkaline phosphatases (ALP), and adenosine triphosphatases (ATPase) activities of rice leaffolder (Cnaphalocrocis medinalis) larvae are affected. Azadirachtin was most potent in all experiments. Larvae that were chronically exposed to limonoids showed a reduction in weight (59-89%) and exhibited a significant reduction in ACP, ALP, and ATPase activities. These results indicate neem limonoids affects gut enzyme activities. These effects are most pronounced in early instars.     

Nimbolide is the Principal Cytotoxic Component of Neem-Seed Insecticide Preparations

Several neem-seed extracts, some used for preparing commercial azadirachtin-containing insecticides, are cytotoxic to N1E-115 murine neuroblastoma cells with IC₅₀ values of 20–200 μg extract ml⁻¹ culture medium. Bioassay-directed fractionation by reversed-phase HPLC shows that the toxicity to N1E-115 cells is associated primarily with a single minor component identified by isolation and NMR and MS as nimbolide with an IC₅₀ of 1·5 μg ml⁻¹ (3·2 μM ). The difference in quantity of nimbolide in seven neem extract sources generally correlates with their overall cytotoxicity. Three other limonoids (epoxyazadiradione, salannin and possibly deacetylsalannin) but not azadirachtin, nimbin and deacetylnimbin contribute in small part to the cytotoxicity. Reconstituted neem extract with only nimbolide removed is less cytotoxic than the original extract. It therefore appears that nimbolide is the principal cytotoxic component of the neem extracts examined and that such minor constituents may warrant consideration in safety evaluations.     

The effects of phytochemical pesticides on the growth of cultured invertebrate and vertebrate cells

A range of cultured cells of invertebrate and vertebrate origin was grown in the presence of a number of phytochemical pesticides to test the effect of the latter on cell proliferation. The main observation was that azadirachtin was a potent inhibitor of insect cell replication, with an EC50 of 1.5 x 10(10) M against Spodoptera cells and of 6.3 x 10(9) M against Aedes albopictus cells, whilst affecting mammalian cells only at high concentrations (> 10(-4) M). As expected, the other phytochemical pesticides, except for rotenone, had little effect on the growth of the cultured cells. Rotenone was highly effective in inhibiting the growth of insect cells (EC50:10(-8) M) but slightly less toxic towards mammalian cells (EC50:2 x 10(-7) M). Neem terpenoids other than azadirachtin and those very similar in structure significantly inhibited growth of the cell cultures, but to a lesser degree. The major neem seed terpenoids, nimbin and salannin, for example, inhibited insect cell growth by 23% and 15%, respectively.     

Bioactive compounds from neem tissue cultures and screening against insects

Hairy root cultures have been derived from neem (Azadirachta indica A Juss, Family Meliacceae) using Agrobacterium rhizogenes and have been studied for the production of compounds with antifeedant effects on insects. Six-week-old hairy root cultures were extracted, and HPLC yielded fractions ranging from polar to non-polar compounds. High antifeedancy levels against the desert locust were observed in fractions(F) 2, 3 and 4 whilst F1 and F5 were not significantly antifeedant. Interestingly F3 did not contain any of the well-known neem chemicals while F2 contained azadirachtin and 3-tigloylazadirachtol and F4 nimbin and salannin. © 1999 Society of Chemical Industry     

Microbiological and Chemical Analysis of Neem (Azadirachta indica) Extracts: New Data on Antimicrobial Activity

The antimicrobial effects of extracts of neem seed (Azadirachta indica A. Juss.) were investigated using microbial growth inhibition assays. A laboratory-prepared neem seed extract along with a commercially available formulated product, were characterized using HPLC, and shown to be effective against a range of bacteria in an agar diffusion assay. The active ingredient,i.e., the unformulated seed extract of the commercial product, also showed activity and this was further investigated in a biochromatogram, using the sensitive bacteriumBacillus mycoides. Results showed antibacterial activity as three discrete inhibition zones that did not correspond to the R_f of the major neem metabolites, azadirachtin, nimbin and salannin. This suggests that these compounds were not antibacterial. The colony radial growth rates of the fungal pathogens that cause ‘take-all’ and ‘snow mould’ disease were both significantly affected when the commercial, unformulated, neem seed extract was incorporated into the growth medium. Experiments in liquid culture suggested that the effect was fungistatic. Conidial germination of the commercially important obligate pathogenSphaerotheca fuliginea (powdery mildew) was reduced to 11%. The results show that neem seed extracts possess antimicrobial activity with notable effects on some fungal phytopathogens. This Work demonstrates that neem seed extracts have potential for controlling both microbial and insect pests. http://www.phytoparasitica.org posting Sept. 16,2001.     

Identification of antifungal compounds from the seed oil ofAzadirachta Indica

Evaluation of the activity of the cold expeller neem oil(Azadirachta indica A. Juss.) and the fractions derived through solvent partitioning, againstDrechslera oryzae, Fusarium oxysporum andAlternaria tenuis showed that the active antifungal fraction is a mixture of tetranortriterpenoids. Further, testing the triterpenoidal mixture derived from the 90% methanol (MeOH) extract of neem oil against 13 phytopathogenic fungi revealed that various species were inhibited to different degrees. Direct preparative High Performance Liquid Chromatography (HPLC) of the active fractions and subsequent bioassay of the semi-pure fractions indicated that the active fractions contained major compounds such as 6-deacetylnimbin, azadiradione, nimbin, salannin and epoxyazadiradione. Pure azadiradione, nimbin, salannin and epoxy-azadiradione did not have appreciable activity. However, when these terpenoids were mixed and bioassayed, they showed antifungal activity, indicating possible additive/synergistic effects.     

Response ofSpodoptera littoralis (Boisd.) andEarias insulana (Boisd.) larvae to azadirachtin and salannin

The effect of azadirachtin and salannin, two triterpenoids isolated from seeds of neem (Azadirachta indica A. Juss), on the feeding response ofSpodoptera littoralis (Boisd.) andEarias insulana (Boisd.) larvae, was investigated. Styropor (foamed polystyrene) lamellae were painted on both sides with a mixture of 5% sucrose with different concentrations of either azadirachtin or salannin dissoled in methanol-water (3∶7). Azadirachtin strongly suppressed feeding inS. littoralis larvae even at 0.001%, whereas salannin showed some antifeedant activity at 0.005% and above. Larvae ofE. insulana were deterred from feeding on azadirachtin-treated lamellae even at 0.005%, whereas salannin was effective only at 0.01% and above. Azadirachtin applied on cotton leaves deterred larvae ofS. littoralis from feeding at all concentrations ranging between 0.001 and 0.02%.     

Activity of neem (Azadirachta indica A. Juss) seed kernel extracts against the mustard aphid,Lipaphis erysimi

An aqueous, an ethanolic and a hexane extract obtained from neem(Azadirachta indica A. Juss) seed kernels were tested for insecticidal activity against the mustard aphid,Lipaphis erysimi Kalt. The hexane extract, which exhibited a much higher activity than the two other extracts, had an LC50 of 0.674%. When the hexane extract was partitioned with ethanol, the ethanol-soluble fraction had an LC₅₀ of 0.328%, whereas the ethanol-insoluble part showed no activity even at 1%. Column chromatography of the ethanol-soluble fraction yielded eight compounds: nimbin, epinimbin, desacetylnimbin, salannin, desacetyl-salannin, azadirachtin and two unidentified compounds — a salannin derivative and a nonterpenoid. Of these, only five could be tested. Nimbin and epinimbin exhibited no toxicity at 0.3%, whereas salannin, a salannin derivative and the non-terpenoid gave LC₅₀ values of 0.055, 0.096 and 0.104%, respectively.     

Effects of neem on adults of Eretmocerus warrae (Hym., Aphelinidae), a parasitoid of Bemisia tabaci (Horn., Aleyrodidae) in tropical horticulture systems

Journal of Plant Diseases and Protection - Side effects of two commercial neem products, NeemAzal- T/S (1% azadirachtin) as foliar application and NeemAzal- U (17% azadirachtin) as soil application...     

Effect of azadirachtin-derived decalin (perhydronaphthalene) and dihydrofuranacetal (furo[2, 3-fo]pyran) fragments on the feeding behaviour of Spodoptera littoralis

Decalin and dihydrofuranacetal fragments related to those in azadirachtin exhibited antifeedant activity against larvae of the African leaf-worm Spodoptera littoralis. Boisd. All the decalin fragments tested were methoxy (C11) derivatives of azadirachtin. The most active decalin fragment had a ketone substitution at C7. Overall, the compounds were more active when tested in combinations of one decalin fragment and one dihydrofuranacetal fragment than when tested singly. Although some of these combinations did show significant levels of antifeedant activity, sometimes coupled with a synergistic effect, they were not as active as either azadirachtin or dihydroazadirachtin.     

Wirkung von Neem-Produkten auf höhere Pflanzen und ihre mögliche Nutzung zur Reduzierung der Seitentriebe von Kulturpflanzen sowie zur Unkrautbekämpfung – eine Bestandsaufnahme

The neem tree (Azadirachta indica A. Juss) offers many possibilities of usage. In plant protection mostly the insecticidal activity of neem products is important. In different parts of the plant several biologically active substances occur. One is the active ingredient azadirachtin, which already is commercially used as an insecticide against insect pests. Some active ingredients of neem may be systemically taken up by the treated plants. Often a rapid decomposition occurred which was enhanced by UV radiation. Therefore formulated neem products often need a UV blocker to elongate their activity. After the application of neem products within plant protection sometimes phytotoxic effects could be observed on different plant species. This phytotoxicity can be used to reduce suckers on tobacco plants and recently also on eucalyptus plants. The existing trials to use neem products for weed control mostly concentrated on the direct treatment of some parasitic weeds like Cuscuta, Orobanche and Striga as well as on a few other weeds (e.g. Echinochloa crus-galli). Mostly the growth of the weeds which were often repeatedly treated with high dosages was reduced, but many plants remained still alive. At present the potential of neem products for an effective weed control is not sufficiently investigated. As a consequence of some already known insecticidal and microbiocidal activities of many neem products also side effects on parts of the epigeic and soil fauna and on the nitrification should be considered after usage for weed control.     

Insect growth regulator effects of azadirachtin and neem oil on survivorship, development and fecundity of Aphis glycines (Homoptera: Aphididae) and its predator, Harmonia axyridis (Coleoptera: Coccinellidae)

BACKGROUND: Aphis glycines Matsumura, an invasive insect pest in North American soybeans, is fed upon by a key biological control agent, Harmonia axyridis Pallas. Although biological control is preferentially relied upon to suppress insect pests in organic agriculture, approved insecticides, such as neem, are periodically utilized to reduce damaging pest populations. The authors evaluated direct spray treatments of two neem formulations, azadirachtin and neem seed oil, under controlled conditions for effects on survivorship, development time and fecundity in A. glycines and H. axyridis. RESULTS: Both azadirachtin and neem seed oil significantly increased aphid nymphal mortality (80 and 77% respectively) while significantly increasing development time of those surviving to adulthood. First-instar H. axyridis survival to adulthood was also significantly reduced by both neem formulations, while only azadirachtin reduced third-instar survivorship. Azadirachtin increased H. axyridis development time to adult when applied to both instars, while neem oil only increased time to adult when applied to first instar. Neither neem formulation affected the fecundity of either insect. CONCLUSIONS: Results are discussed within the context of future laboratory and field studies aimed at clarifying if neem-derived insecticides can be effectively integrated with biological control for soybean aphid management in organic soybeans.     

Protein metabolism in Spodoptera litura (F.) is influenced by the botanical insecticide azadirachtin

Azadirachtin, as a botanical insecticide, affects a wide variety of biological processes, including reduction of feeding, suspension of molting, death of larvae and pupae, and sterility of emerged adults in a dose-dependent manner. However, the mode of action of this toxin remains obscure. By using proteomic techniques, we analyzed changes in protein metabolism of Spodoptera litura (F.) induced by azadirachtin. Following feeding 4th instar larvae of Spodoptera litura (F.) with an artificial diet containing 1 ppm azadirachtin until pupation, 48 h old pupae were collected and protein samples prepared. Total soluble protein content was measured and the results showed that azadirachtin significantly influenced protein level. Moreover, the proteins were separated by 2-DE (two-dimensional polyacrylamide gel electrophoresis) and 10 proteins were significantly affected by azadirachtin treatment when compared to an untreated control. Six of these proteins were identified with peptide mass fingerprinting using MALDI-TOF-MS after in-gel trypsin digestion. These proteins are involved in various cellular functions. One identified protein may function as an ecdysone receptor, which regulates insect development, and reproduction. It is suggested that the botanical insecticide azadirachtin affects protein expression and the azadirachtin-related proteins would be essential for a better understanding of the mechanisms by which neem toxins exert their effects on insects.     

The production of azadirachtin by in-vitro tissue cultures of neem,Azadirachta indica

Callus produced from leaves of a Ghanaian strain of the neem tree, Azadirachta indica A. Juss has been shown to produce the natural insecticide azadirachtin when grown in a defined medium. The azadirachtin was isolated by standard procedures of solvent partition and column chromatography monitored by supercritical fluid chromatography. Biological activity was monitored with antifeedant tests using the desert locust (Schistocerca gregaria Forsk.). The azadirachtin was identified by chromatography on three independent chromatographic systems (SFC, HPLC & TLC) and two thin-layer colour tests. It has 100% antifeedant activity at < 0.04mg litre^?1. The yield of azadirachtin was 0.0007% based on dry weight of callus.     

Growth-inhibiting activity of azadirachtin onCorcyra cephalonica

The growth-inhibiting properties of azadirachtin, a bioeffective triterpenoid compound isolated from neem (Azadirachia indica A. Juss) seeds, was evaluated on the rice moth,Corcyra cephalonica Staint., a serious pest of stored products. By topical application of various azadirachtin doses (0.5 to 10 μg/larva) in methanol to last-instar spinning stage larvae, development was inhibited. The effect was dose-dependent; at higher doses, many of the insects remained in the larval stage (55% with 10 μg/larva). Disturbance of both larval-pupal and pupal-adult molting is discussed and interpreted as interference with the morphogenetic hormone pool size.     

Effects of neem (Azadirachta indica) extracts onLeptocorisa chinensis under choice and no-choice conditions

Two extracts from neem (Azadirachta indica A. Juss. (Meliaceae)) seeds, azadirachtin and oil, and a mixture of neem oil and abamectin, were tested on second-instar nymphs of the rice bugLeptocorisa chinensis (Dallas) (Hemiptera: Alydidae). To clarify the effect of spraying coverage on bioefficacy of test materials, experiments were conducted under choice and no-choice conditions in field cages. In a choice test, treatment with the mixture of neem oil and abamectin was most effective in reducing the survival ofL. chinensis, followed by azadirachtin at 60 ppm, 30 ppm and 3% neem oil, whereas all treatments except neem oil caused 100% mortality within 3 weeks in a no-choice test. When second-instar nymphs had choices of treated and untreated plants within a treatment, no differences in yield and sum of dead and stained grains were found between those two choices, indicating that nymphs neither caused significant reduction in yield nor reduced the quality of untreated plants. Regardless of treatment, the difference in overall yield between treated and untreated plants under choice conditions was not statistically significant (P>0.05). Our results indicate that neem-based formulations, used alone or in combination with abamectin, have the potential to be integrated into the existing programs to control the rice bug. http://www.phytoparasitica.org posting Aug. 28, 2005.     

Effect of some biorational insecticides on Spodoptera eridania in organic cabbage

BACKGROUND: To reduce pest attack, several biorational products are allowed for use on organic vegetables in Brazil. This study investigated eight biorational products applied singly or in combination against Spodoptera eridania Cramer in field plots of cabbage intercropped with coriander. The treatments were applied once a week over a 5 week period, beginning 34 days after transplanting. The evaluations consisted of counting the larvae of S. eridania on the day before and 7 and 21 days after spraying. The damage to leaves and cabbage head, the commercial weight of head and the percentage of head losses were evaluated.RESULTS: Leaf injury in plots treated with Beauveria bassiana and neem oil (Dalneem) yielded scores of 1.3 and 2.5 (scale ranging from 0 to 4) respectively, in comparison with a score of 3.6 from untreated plots. Head weight losses were 6.1, 5.3 and 4.9% with an aqueous extract of neem leaves, neem oil and B. bassiana respectively, compared with 24.6% lost from untreated plots.CONCLUSION: Dalneem, B. bassiana and the extract of neem leaves at 20% exhibited the best performance for control of S. eridania.     

The commercial use of azadirachtin and its integration into viable pest control programmes

With the continued robust growth of the global biopesticide market, azadirachtin is uniquely positioned to become a key insecticide to expand in this market segment. In the USA the actual or impending cancellation of some organophosphate and carbamate insecticides that have either lost patent protection or are not being re-registered in many markets because of the Food Quality Protection Act of 1996, has opened new opportunities for biopesticides and reduced-risk pesticides in general. The broad-spectrum activity of azadirachtin at low use rates (12·5 to 40 g AI ha^-1) coupled with the insect growth regulator activity (in all larval/nymphal instars including the pupal stage) and unique mode of action (ecdysone disruptor), make azadirachtin an ideal candidate for insecticide resistance, integrated pest control and organic pest control programmes. Azadirachtin has been exempted from residue tolerance requirements by the US Environmental Protection Agency for food crop applications. Azadirachtin exhibits good efficacy against key pests such as whiteflies, leafminers, fungus gnats, thrips, aphids and many leaf-eating caterpillars. Azadirachtin has minimal to no impact on non-target organisms, is compatible with other biological control agents and has a good fit into classical Integrated Pest Management programmes. The world's largest azadirachtin extraction facility has been fully commissioned in India to process over 10,000 tonnes neem seeds per annum. This will ensure the wide availability of azadirachtin technical grade material in the future. © 1998 Society of Chemical Industry.     

Comparison of the impact of a neem seed-kernel extract formulation, “Margosan-O” and chlorpyrifos on non-target invertebrates inhabiting turf grass

Several laboratory studies have indicated that neem seed-kernel extracts, or the neem insecticide azadirachtin, are active against certain types of pestiferous insects but do not harm several types of beneficial arthropods. The abundance of several groups of invertebrates inhabiting a turf grass ecosystem was determined in 1991 after application of a neem-based insecticide formulation, “Margosan-O®” (MO; W. R. Grace & Co., Columbia, Maryland, USA) containing 3.0 g litre^?1 azadirachtin, and chlorpyrifos. MO was less detrimental than chlorpyrifos to most of the invertebrates studied. However, oribatid mites were more sensitive to MO than to chlorpyrifos. Sminthurid and non-sminthurid collembola were also susceptible to MO, although less so than to chlorpyrifos. MO had no significant effect on non-oribatid mites and spiders. Environmental implications of using selective versus broad-spectrum insecticides in agriculture are discussed.     

Behandlung von Reis mit Neem und Diatomeenerde zur Bek?mpfung der vorratssch?dlichen Coleopteren Sitophilus oryzae und Tribolium castaneum

The commercial available diatomaceous earth Fossil Shield® and the neem product Neem Azal-T/S® are tested as single treatments and in combination as controls for the stored product pests Sitophilus oryzae (L.) and Tribolium castaneum (Herbst). The diatomaceous earth, applied in concentrations of 0.5 g, 1 g and 2 g kg^-1 rice, reduced numbers of surviving beetles significantly (over a period of 3 weeks). A single treatment with the neem product, in concentrations of 0.01 g, 0.1 g, 0.2 g and 1 g azadirachtin kg^-1 rice, increased the mortality rate for both species significantly. The combination of neem and diatomaceous earth (1 g diatomaceous earth with 0.2 g or 1 g azadirachtin kg^-1 rice) was more effective than the single treatment in reducing numbers of surviving beetles.