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.     

Production of azadirachtin from plant tissue culture: State of the art and future prospects

With increasing awareness towards environment-friendly and non-toxic pesticide azadirachtin obtained from neem tree (Azadirachta indica) is gaining more and more importance. Its broad-spectrum activity, peculiar mode of action. eco-friendly and non-toxic action towards beneficial organisms has offered many advantages over chemical pesticides. All currently use commercial formulations based on azadirachtin contains azadirachtin extracted from seeds of naturally grown whole plants which is labour intensive process depending upon many uncontrollable geographical and climatic factors. Plant tissue culture can be a potential process for the production, offering consistent, stable and controlled supply of this bioactive compound, However the research on tissue culture aspects of production are in preliminary stage and requires culture and process optimization for the development of a commercially viable process. This review states the present status and future challenges of plant tissue culture for azadirachtin production.     

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.     

Cytotoxic and antiproliferative effects induced by a non terpenoid polar extract of A. indica seeds on 3T6 murine fibroblasts in culture

Neem oil is a natural product obtained from the seeds of the tree Azadirachta indica. Its composition is very complex and the oil exhibits a number of biological activities. The most studied component is the terpenoid azadirachtin which is used for its insecticidal and putative antimicrobial properties. In this report we investigate the biological activity of partially purified components of the oil obtained from A. indica. We show that the semi-purified fractions have moderate to strong cytotoxicity. However, this is not attributable to azadirachtin but to other active compounds present in the mixture. Each fraction was further purified by appropriate extraction procedures and we observed a differential cytotoxicity in the various sub-fractions. This led us to investigate the mode of cell death. After treatment with the oil fractions we observed positivity to TUNEL staining and extensive internucleosomal DNA degradation both indicating apoptotic death. The anti-proliferative properties of the neem oil-derived compounds were also assayed by evaluation of the nuclear PCNA levels (Proliferating Cell Nuclear Antigen). PCNA is significantly reduced in cells treated with a specific fraction of neem oil. Finally, our results strongly suggest a possible involvement of the mitochondrial pathway in the apoptotic death.     

Production of biopesticide azadirachtin using plant cell and hairy root cultures

The extensive use of nondegradable chemical pesticides for pest management has developed serious environmental hazards. This has necessitated the urgent need to switch over to an alternative mode of biopesticide development for mass agriculture and field crop protection. Azadirachta indica A. Juss (commonly known as neem) houses a plethora of bioactive secondary metabolites with azadirachtin being the most active constituent explored in the sector of ecofriendly and biodegradable biopesticides characterized by low toxicity toward nontarget organisms. It has been reported that the highest content of azadirachtin and related limonoids is present in the seeds, available once in a year. Moreover, the inconsistent content and purity of the metabolites in whole plant makes it imperative to tap the potential of in vitro plant tissue culture applications, which would allow for several controlled manipulations for better yield and productivities. This review gives a summarized literature of the applied research and achievements in plant cell/hairy cultures of A. indica A. Juss mainly in context with the biopesticide azadirachtin and applications thereof.     

Efficacy of biorational insecticides against chilli thrips,Scirtothrips dorsalis (Thysanoptera: Thripidae), infesting roses under nursery conditions

We evaluated biopesticides based on entomopathogenic fungi, azadirachtin and horticultural oils for the management of the chilli thrips, Scirtothrips dorsalis Hood, an economically significant invasive pest in the United States. Insecticides were applied four times at 7‐ to 10‐day intervals against established S. dorsalis infestations on shrub roses KnockOut^®, Rosa x ‘Radrazz’ under simulated nursery conditions. When applied as stand‐alone treatments, Beauveria bassiana GHA (BotaniGard^® ES), Metarhizium brunneum F52 (Met‐52 EC), a horticultural oil (SuffOil‐X^®) and azadirachtin (Molt‐X^®) at label rates provided significant control, reducing populations of S. dorsalis by 48–71% compared with control over 4–6 weeks. Similar results were observed when the biopesticides were applied in rotation with each other. A conventional standard, spinosad (Conserve^® SC), was consistently the most effective treatment in these studies, reducing thrips populations by >95% overall. In another study, more effective control (87%–92%) was achieved in biopesticide rotation programmes that included spinosad, when compared with those that did not. Results also showed that these biopesticides can be tank‐mixed. However, there was no evidence that B. bassiana or M. brunneum combined with azadirachtin resulted in additive or synergistic control, as neither tank‐mix treatment improved control compared with azadirachtin alone. These findings highlight the potential use of biopesticides in rotation programmes with conventional insecticides to manage S. dorsalis on roses. Biopesticides evaluated in this study can be incorporated into an IPM programme for roses.     

A method to test compounds for feeding deterrence towards redlegged earth mite (Acarina: Penthaleidae)

A bioassay, based on a membrane sachet technique, has been developed to identify antifeeding compounds affecting the redlegged earth mite, Halotydeus destructor (Acari: Penthaleidae). The method consists of counting H. destructor numbers on membrane sachets in choice experiments, which was quicker and more efficient than weighing the mites. Five per cent aqueous glucose solution was used as a feeding stimulant, with Tween 80® at 5% concentration as a solubilising agent for water-insoluble compounds. (+)-Catechin, rutin, biochanin A, formononetin, chlorogenic acid, and gramine acted as feeding deterrents at 1% concentration. Quercetin (1 %) and azadirachtin (100 ppm) had no significant effect. At lower concentrations (0.01%), compounds showed antifeeding (gramine), phagostimulating (quercetin and chlorogenic acid), or no effects on mite numbers. Dose-dependent deterrent effects of plant extracts were demonstrated with the bioassay, which could be used for other mites.     

Differential thresholds of azadirachtin for feeding deterrence and toxicity in locusts and an aphid

Conclusions The importance of using azadirachtin in the field at levels causing primary antifeedancy may have been overemphasised in the past. Currently, it is recommended that commercial preparations of neem be applied at 50–100 ppm (a.i.). While this would undoubtedly have an antifeedant effect in many phytophagous insects it may also harm beneficial species (Schmutterer, 1990). By lowering the concentration of azadirachtin applied to the crop, food intake by insect pests with low chemoreceptor sensitivity to azadirachtin, e.g. aphids (Nisbet et al., 1993; 1994), would not initially be affected. However, secondary antifeedant effects, IGR and sterilant effects could rapidly manifest themselves and bring about crop protection by reducing insect pest populations without harming natural predator or parasitoid populations.     

Impact of azadirachtin on vine weevil (Coleoptera: Curculionidae) reproduction

Abstract 1 The dose–response of azadirachtin on vine weevil, Otiorhynchus sulcatus (Fabricius), reproduction is investigated by confining adults to feed on treated Taxus × media leaves, and by counting and evaluating development in the resulting eggs. 2 A dosage‐dependent reduction in oviposition is discovered for foliar surface residues of azadirachtin, with an EC₅₀ of 25–50 parts per million (p.p.m) and 99.2% inhibition of viable egg production with 100 p.p.m. 3 Switching weevils from treated to untreated foliage allows reproductive capability to be restored for weevils that cease egg laying after azadirachtin exposure of 50 p.p.m. Weevils that had already started laying eggs in untreated groups soon cease oviposition once switched to azadirachtin‐treated foliage. 4 A transovarial effect results in a decrease in the percentage of viable eggs as the azadirachtin concentration increases. 5 The amount of feeding on foliage does not appreciably decrease at these hormonally effective concentrations, and adult weevil mortality is only slightly greater in the azadirachtin‐treated groups. Therefore, the overall effect of azadirachtin on weevil populations in the field is difficult to assess, except by collecting weevils to determine whether they are able to lay viable eggs.     

The protection of barley seedlings from attack bySchistocerca gregaria using azadirachtin and related analogues

Barley seedlings were sprayed with azadirachtin or analogues and the resulting crop protection againstSchistocerca gregaria was examined. Any change of the parent azadirachtin molecule resulted in less effective insect control and hence more crop damage. Crop protection by azadirachtin and dihydroaza-dirachtin resulted from both antifeedancy and toxicity, 3-tigloylazadirachtol was more effective by direct toxicity after significant ingestion and bromoethoxyazadirachtin showed extremely low antifeedant and insecticidal properties.S. gregaria was shown to be very sensitive to change in the azadirachtin molecule.     

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.     

Selective inhibitors of germination of legume seeds in activated sludge compost

Water extracts of the compost produced from activated sludge and coffee residue were found to be selectively inhibitory to seed germination of some legumes. Germination rate of white clover (Trifolium repens L.), red clover (Trifolium pratense L.) and alfalfa (Medicago sativa L.) seeds were reduced to 2, 29 and 73% of the control, respectively, by water extracts of the compost (20 g l^–1). However, the extracts did not show any inhibition to seed germination of sorghum (Sorghum bicolor Moench), African millet (Eleusine coracana Gaertn.), and Komatsuna (Brassica rapa L.) at the same concentration. The inhibitors in the compost extracts were separated by ion-exchange chromatography and reverse-phase high performance liquid chromatography (HPLC) and the inhibitory activities of seed germination were tested with white clover seeds. Five inhibitors were isolated and identified as 3,4-dichlorophenylacetic acid (3,4-DCP), 3,4-dichlorobenzoic acid (3,4-DCB), 3,4,5-trichlorophenylacetic acid, 3,4,5-trichlorobenzoic acid and mono-2-ethylhexylphthalate by ¹H-, ¹³C-NMR spectroscopy and mass spectrometry. The inhibitory activities of some authentic chemicals of the inhibitors and the related compounds were compared. The results indicated that the main inhibitor in the compost could be 3,4-DCB, which was contained at the concentration of 6.58 mg kg^–1 compost and showed the strongest inhibitory effect on seed germination of white clover among the tested compounds.     

An endophytic fungus from <Emphasis Type="Italic">Azadirachta indica</Emphasis> A. Juss. that produces azadirachtin

Azadirachtin A and its structural analogues are a well-known class of natural insecticides having antifeedant and insect growth-regulating properties. These compounds are exclusive to the neem tree, Azadirachta indica A. Juss, from where they are currently sourced. Here we report for the first time, the isolation and characterization of a novel endophytic fungus from A. indica, which produces azadirachtin A and B in rich mycological medium (Sabouraud dextrose broth), under shake-flask fermentation conditions. The fungus was identified as Eupenicillium parvum by ITS analysis (ITS1 and ITS2 regions and the intervening 5.8S rDNA region). Azadirachtin A and B were identified and quantified by LC-HRMS and LC-HRMS², and by comparison with the authentic reference standards. The biosynthesis of azadirachtin A and B by the cultured endophyte, which is also produced by the host neem plant, provides an exciting platform for further scientific exploration within both the ecological and biochemical contexts.     

Statistical medium optimization for enhanced azadirachtin production in hairy root culture of Azadirachta indica

Azadirachtin, a well-known biopesticide, is a secondary metabolite extracted from the seeds of Azadirachta indica. In the present study, azadirachtin was produced in hairy roots of A. indica, generated by Agrobacterium rhizogenes-mediated transformation of leaf explants. Liquid cultures of A. indica hairy roots were developed with a liquid-to-flask volume ratio of 0.15. The kinetics of growth and azadirachtin production were established in a basal plant growth medium containing MS medium major and minor salts, Gamborg’s medium vitamins, and 30 g l⁻¹ sucrose. The highest azadirachtin accumulation in the hairy roots (up to 3.3 mg g⁻¹) and azadirachtin production (∼44 mg l⁻¹) was obtained on Day 25 of the growth cycle, with a biomass production of 13.3 g l⁻¹ dry weight. To enhance the production of azadirachtin, a Plackett–Burman experimental design protocol was used to identify key medium nutrients and concentrations to support high root biomass production and azadirachtin accumulation in hairy roots. The optimal nutrients and concentrations were as follows: 40 g l⁻¹ sucrose, 0.19 g l⁻¹ potassium dihydrogen phosphate, 3.1 g l⁻¹ potassium nitrate, and 0.41 g l⁻¹ magnesium sulfate. Concentrations were determined by a central composite design protocol and verified in shake-flask cultivation. The optimized medium composition yielded a root biomass production of 14.2 g l⁻¹ and azadirachtin accumulation of 5.2 mg g⁻¹, which was equivalent to an overall azadirachtin production of 73.84 mg l⁻¹, 68% more than that obtained under non-optimized conditions.     

Insect growth regulating and antifeedant effects of neem extracts and azadirachtin on two aphid species of ornamental plants

Leaf disc choice test bioassay demonstrated that formulated neem seed extracts were highly deterrent and growth regulatory to rose aphid,Microsiphum rosae (L.) and Chrysanthemum aphid,Macrosiphoniella sanbornii (Gillete). Effective concentrations to produce 50% feeding deterrence was 0.80 and 0.84% respectively for 2nd instar nymphs irrespective of bioassay duration. The disruption of aphid feeding was related to the presence of azadirachtin concentration in the extract. The toxicity on contact from the leaf surface or via topical application due to azadirachtin was significantly different and topical treatment was at least 7 times more effective for both species. Thus growth regulatory effects of azadirachtin were influenced by the host plant and the stage of treatment. Field evaluation with formulated neem extracts revealed the effect to be more of growth regulatory nature thereby showing that azadirachtin is a physiological toxin for aphid species. Neem seed extracts reduced the population of aphid on respective host plants significantly, EC₅₀ values being 0.88 and 0.96% forM. rosae andM. sanbornii respectively.     

Behavioural and neurophysiological responses ofSpodoptera littoralis to azadirachtin and a range of synthetic analogues

The antifeedant activity of azadirachtin and 56 azadirachtin analogues, including 22,23-dihydroazalirachtin, against larvae ofSpodoptera littoralis was investigated using behavioural and electrophysiological bioassays. None of the analogues was as active as azadirachtin, although many showed significant antifeedant activity at high concentrations. The majority of the analogues stimulated a dose-dependent response from a neurone in the medial styloconic maxillary sensilla which correlated with the behavioural activity. Methylation of the hydroxy substitutions on the azadirachtin molecule usually resulted in a decrease in antifeedant activity, as did the addition of bulky groups to the dihydrofuran ring.     

Metabolism of phenolic compounds in <Emphasis Type="Italic">Vitis riparia</Emphasis> seeds during stratification and during germination under optimal and low temperature stress conditions

We studied the alterations in phenolic compounds in grape seeds during their stratification and germination under optimal conditions (+25 °C) and at low temperature (+10 °C). Biological materials in the study were seeds of Vitis riparia. Phenolic compounds were extracted from defatted seeds using 80 % methanol or 80 % acetone. The content of total phenolics was determined with the Folin-Ciocalteau reagent, while the content of tannins was determined by vanillin assay and the protein (BSA) precipitation method. The RP-HPLC method was used to determine phenolic compounds (phenolic acids, catechins) in the extracts. High amounts of tannins, catechins, gallic acid and lesser amounts of p-coumaric acid were found in the seeds. The content of total phenolics in acetone extracts was higher than that obtained using methanol. The amounts of phenolic acids and tannins found in V. riparia seeds after stratification were much lower. It may confirm a possible role of these compounds in dormancy of V. riparia seeds. After 72 h of low temperature treatment, inhibition of grape root growth and biochemical changes in seeds were detected. The chilling stimulated increased accumulation of some phenolic compounds (free gallic acid and catechins) in the seeds. These substances can protect plants against some abiotic stressors.     

Germination-Arrest Factor (GAF): 3. Determination that the herbicidal activity of GAF is associated with a ninhydrin-reactive compound and counteracted by selected amino acids

A novel, naturally-occurring herbicide (Germination-Arrest Factor, GAF), produced by Pseudomonas fluorescens WH6 and several related isolates of rhizosphere bacteria, irreversibly arrests germination of the seeds of a wide range of graminaceous species, including a number of important grassy weed species. GAF activity has been shown previously to be associated with a hydrophilic, low molecular weight compound that contains an acid group. In the present study, thin-layer chromatography (TLC) of extracts of WH6 culture filtrate demonstrated that GAF activity migrates on TLC plates with a particular ninhydrin-reactive compound. This compound was found to be present in GAF-producing P. fluorescens isolates and absent in P. fluorescens strains that lack the ability to produce GAF. Treatments, including mutagenesis, which resulted in the loss of GAF activity in culture filtrates from P. fluorescens WH6 were shown to result in the disappearance of this ninhydrin-reactive compound from extracts of WH6 culture filtrates or in alteration of its appearance on TLC chromatograms. The ninhydrin-reactivity of GAF indicates that it probably contains an amino group, as well as the acid group previously demonstrated, and suggests that GAF may be a small peptide or amino acid analog. Biological investigations motivated by this conclusion demonstrated that the effects of GAF in inhibiting the germination of seeds of annual bluegrass (Poa annua L.) could be counteracted by treatment with alanine or glutamine and, to lesser extent, by several other amino acids, suggesting that this compound may act by interfering with some aspect of amino acid metabolism or function.     

Susceptibility of the eggs of the field slug Deroceras reticulatum to contact with pesticides and substances of biological origin on artificial soil

The toxicity of 14 substances, including a number of pesticides, to the eggs of the pest slug Deroceras reticulatum was determined in laboratory experiments. Eggs were kept in contact with a precisely defined artificial soil to which a range of concentrations of the test substances had been applied. Mortality of the eggs was assessed every 24 h and the median lethal doses (LD₅₀) were determined. The herbicides bromoxynil, ioxynil and pyridate + bromoxynil, the insecticides thiocyclam, diflubenzuron and azadirachtin, the molluscicides metaldehyde and methiocarb, and other compounds such as carvone, iron‐EDDHA, saponin, and an extract of Pongamia pinnata, killed the eggs after periods of exposure ranging from 2 to 14 days, depending on the compound and the dose. Only two compounds, the insecticides imidacloprid and teflubenzuron, failed to kill the eggs of D. reticulatum at any of the doses tested. Values of LD₅₀ below 0.01 mg a.i. cm^?2 were obtained for the herbicides bromoxynil, ioxynil and pyridate + bromoxynil, and for the biological pesticide azadirachtin. The feasibility of slug egg control in different contexts is discussed.     

Compatibility of flonicamid and a formulated mixture of pyrethrins and azadirachtin with predators for soybean aphid (Hemiptera: Aphididae) management

The invasive soybean aphid, Aphis glycines Matsumura, is an important pest in North American soybean production. Predators can play an important role in suppressing A. glycines. However, current A. glycines management practices rely primarily on broad-spectrum insecticides, which can adversely affect natural enemy populations. An alternative is the use of selective insecticides that control the targeted pest species, while having a reduced impact on natural enemies. In greenhouse and laboratory assays, we tested the effects of lambda-cyhalothrin, two rates of flonicamid, which is currently not registered for use in soybean, and a formulated mixture of pyrthrins and azadirachtin on A. glycines and its natural enemies, Chrysoperla rufilabris (Burmeister), Orius insidiosus (Say) and Hippodamia convergens (Guerin-Meneville). All insecticides significantly reduced A. glycines populations. Lambda-cyhalothrin was highly toxic to the natural enemies tested. Flonicamid showed the lowest toxicity to natural enemies, but the high rate did decrease survival of O. insidiosus. The mixture of pyrethrins and azadirachtin was toxic to larvae of C. rufilabris and adult O. insidiosus. Moreover, the mixture of pyrethrins and azadirachtin increased the developmental time of C. rufilabris. These results indicate potential for flonicamid and the mixture of pyrethrins and azadirachtin to increase compatibility between chemical and biological controls.