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.     

Comparisons of deterrency and toxicity of selected secondary plant compounds to an oligophagous and a polyphagous acridid

A comparison was made of the relationship between deterrence and toxicity of eight secondary compounds to two acridids. The grass specialist Locusta migratoria was compared with the polyphagous Schistocerca gregaria. L. migratoria was deterred by all compounds presented at natural concentrations to which S. gregaria showed a variety of behavioral responses. Overall, L. migratoria was significantly more susceptible to toxic effects of the chemicals when injected into the hemolymph than was S. gregaria and was more sensitive behaviourally to them. There was a significant correlation in both species between deterrency and toxicity of injected compounds, but little evidence of a relationship between deterrency and oral toxicity.

---

Résumé La comparaison a porté sur les relations entre répulsion et toxicité de huit substances secondaires chez deux acridiens: Locusta migratoria, spécialiste de graminées, et Schistocerca gregaria, polyphage. L. migratoria est repoussé par toutes les substances proposées, aux concentrations naturelles, pour lesquelles S. gregaria a présenté des comportements trés divers. Mais surtout, L. migratoria a été significativement plus sensible que S. gregaria aux effets toxiques de ces substances quand elles sont injectées dans son haemolymphe; S. gregaria a réagi à ces substances plus par des modifications de son comportement. Il y a une corrélation significative pour les deux espèces, entre la répulsion et la toxicité des substances injectées.

     

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.     

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.     

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.     

Species and age specificity of malathion towards the locusts Schistocerca gregaria and Locusta migratoria

The toxicity of malathion (2.5 % solution in kerosene) was determined by topical application to the hindwing base of mature adults of Schistocerca gregaria and Locusta migratoria migratorioides and of immature adults of S. gregaria. Mature adults of L. m. migrator ioides were slightly more resistant to malathion than those of S. gregaria. Immature adults of S. gregaria were approximately as susceptible as mature ones (at LD₈₉ level) when the criterion for toxicity was taken as the ultimate death of the dosed insects, but the symptoms of poisoning developed earlier and more strongly in the immature insects. Penetration of malathion was least in the mature L. m. migratorioides and highest in the immature S. gregaria. The higher rate of activation of malathion in the immature S. gregaria was, however, counterbalanced by a higher rate of hydrolysis. The toxicity reflected the rate of accumulation of malathion and/or mala-oxon inside the insect body.     

Insecticidal activity of mixtures of Bacillus thuringiensis crystals with plant oils of Sinapis alba and Azadirachta indica

The main purpose of this study was to determine the insecticidal activity of mixtures of Bacillus thuringiensis crystalline toxins and plant substances that could contribute to create a new bioinsecticide of high efficiency. We decided to determine the toxicity of crystals of B. thuringiensis MPU B9 isolate, azadirachtin (NeemAzal), mustard oil and their mixtures against lepidopteran pests: Cydia pomonella, Spodoptera exigua and Dendrolimus pini. The interaction of the components in mixtures against insects was evaluated and their utility in plant protection was assessed. The crystals and plant substances showed synergistic effect against all insect species. The occurrence of synergism between two components depended on the ratio of their concentration in mixtures. Slight amount of one of the components added to the mixture led to enhanced toxicity of the preparation. The supplementation of mustard oil with B. thuringiensis toxins from up to 0.3% of the mixture resulted in more than twofold higher observed mortality of L1 larvae of D. pini in comparison with expected mortality. The beneficial insecticidal effect of the mustard oil and crystals mixture was also noted when crystals comprised up to 1.3% and from 7 to 20% against L1 caterpillars of C. pomonella and L3 larvae of S. exigua, respectively. The insecticidal efficient effect was achieved when mixture of B. thuringiensis crystals and azadirachtin was used against the three species of lepidopteran pests. The combinations in which components interacted synergistically against caterpillars are potentially useful for making a new bioinsecticide for controlling lepidopteran pests in plant protection.     

Bioefficacy and mode of action of rocaglamide from Aglaia elaeagnoidea (syn. A. roxburghiana) against gram pod borer,Helicoverpa armigera (Hübner)

Abstract: Rocaglamide, a highly substituted benzofuran, was isolated and identified as the main biologically active component in Aglaia elaeagnoidea (syn. A. roxburghiana) for gram pod borer Helicoverpa armigera (Hübner). Addition of rocaglamide to an artificial diet retarded the growth of neonate larvae in a dose‐dependent manner with EC₅₀ values of 0.76 p.p.m. These values compared favourably with azadirachtin (EC₅₀ = 0.23 p.p.m.). However, azadirachtin was apparently more potent than rocaglamide in inducing growth inhibition via oral administration to these first stadium larvae. The candidate compound was found to have LD₅₀ and LD₉₅ values of 0.40 and 1.02 μg per larva, respectively, in topical application against third instar larvae 96 h post‐treatment. However, these values for azadirachtin were 8.16 and 25.8 μg per larva for the same period. This shows that azadirachtin was less effective against third instar H. armigera larvae in inducing acute toxicity via topical treatment in comparison with rocaglamide. However, severe morphological larval deformities were observed in such azadirachtin‐treated larvae during the process of ecdysis. The cytotoxic nature of rocaglamide was established by evaluating dietary utilization and the results did not implicate any antifeedant effect but the toxicity‐mediated effect due to reduced efficiency of conversion of ingested food. It was obvious that feeding deterrence is not the primary mode of action but a centrally mediated effect, which could be due to the induced cytotoxicity at non‐specific cellular levels.     

The effect of neem allelochemicals on nutritional physiology of larval Spodoptera litura

Neem allelochemicals azadirachtin, salannin, nimbinene and nimbin were administered to different larval instars of the tobacco armyworm, Spodoptera litura orally in artificial diet, topically or via injection. Nutritional analyses revealed strong antifeedant and growth regulatory effects of azadirachtin which were independent of each other. While salannin and nimbinene induced concentration dependent feeding deterrence only; nimbin was inactive to the 1000 ppm level against this insect species. One of the causes of the reduced growth rate of azadirachtin treated insects was due to an increase in the costs associated with growth. This was relative to a drastic reduction in the activity of gut trypsin. Salannin and nimbinene, however, did not interfere with the trypsin activity of the gut. These results and those from nutritional studies suggest that salannin and nimbinene have no toxicity mediated effects on S. litura larvae and antifeedant activity is a result of the effects on deterrent and other chemoreceptors. The fact that azadirachtin directly or indirectly inhibits the secretion of trypsin by the enzyme-secreting cells of the gut is discussed.     

Volumetric feedback and the control of meal size in Schistocerca gregaria

The role and nature of volumetric feedbacks in the regulation of meal size was investigated for Schistocerca gregaria. Feedback from the anterior region of the crop was found to be important and denervation led to hyperphagia. Additional feedbacks were found from more posterior gut regions. Cannulating agar or paraffin into the mid- and hindgut caused a decrease in the size of a following meal. This effect was removed by cutting the ventral nerve cord anterior to the terminal abdominal ganglion. Increasing the blood volume by injections of isotonic saline into the haemocoel was also found to decrease subsequent meal size. This effect was not mediated via the gut or body wall stretch receptors and possible mechanisms are discussed. The results for S. gregaria are compared with those obtained previously from Locusta migratoria.

---

Résumé L'étude de la nature et du rôle des feed backs volumétriques dans la régulation de la taille du repas a été effectuée chez S. gregaria. Un feed back, originaire de la région antérieure du jabot, s'est révélé important et la dénervation a provoqué l'hyperphagie. Des feed backs supplémentaires ont été décelés dans des régions plus postérieures du jabot. De l'agar ou de la paraffine canulés dans la jabot moyen et postérieur ont provoqué une diminution de la taille du repas suivant. Ces effets sont supprimés avec la section de la corde nerveuse centrale avant le ganglion abdominal terminal. L'augmentation du volume sanguin par des injections de solution saline isotonique a aussi diminué la taille du repas suivant. Cet effet n'est pas transmis via des récepteurs de tension du jabot ou de la paroi du corps; des mécanismes envisageables sont discutés. Ces résultats avec S. gregaria sont comparés à ceux obtenus antérieurement avec Locusta migratoria.

     

Dosage reduction to improve the selectivity of deltamethrin between aphids and coccinellids in cereals

The toxicity of the pyrethroid insecticide deltamethrin to a cereal aphid and a coccinellid beetle predator was assessed. Deltamethrin gave effective aphid control in winter wheat at dose-rates of 6.25, 3.13 and 1.56g a.i./ha. The direct exposure of adultCoccinella septempunctata L. (Coleoptera: Coccinellidae) to spray drops was estimated at a range of positions in a cereal crop canopy from volumetric analysis of fluorescent tracer deposits. These measurements were used to calculate exposure to deltamethrin at the three experimental dose-rates. Observations of coccinellid beetle distribution through a cereal crop canopy permitted a realistic range of direct contact doses tobe calculated and the toxic effects of these levels of exposure to be predicted from laboratory dose-response data. Estimated beetle mortalities from direct exposure were 19, 8 and 3% at the three experimental dose-rates.In situbioassays with adultC. septempunctata which exposed beetles continuously to deltamethrin residues on flag leaves, resulted in 100, 94 and 39% mortality respectively at these dose-rates during the 10 days after spray application. Additionalin situ bioassays exposed beetles to deltamethrin residues on flag leaves for 24 h and then transferred surviving beetles to the soil under the cereal crop canopy for a further 9 days. This resulted in 89, 69 and 29% beetle mortality respectively at the three dose-rates. Mortality predictions combining both direct contact and residual exposure were made for the three dose-rates.to determine the maximum impact of summer sprays of deltamethrin on adult coccinellid populations in cereals. These worst case predictions suggested that a reduction in dose-rate by as much as three quarters of the recommended application rate in UK cereals may be necessary to preserve approximately 60% of adultC. septempunctata in the crop over the 10 days after a deltamethrin spray application. The methodology described may be appropriate for estimating selective dose-rates for key enemies in a range of crops.     

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.     

Chemical and biological properties of wheat soil in response to paddy straw incorporation and its biodegradation by fungal inoculants

A field experiment was conducted to evaluate the relative contribution of organic fertilizers (paddy straw, microbial inoculants and vermicompost) and inorganic fertilizers (urea and superphosphate) in improving pH, C, N, humus, microbial biomass, dehydrogenase, phosphatase, cellulase, β-glucosidase and xylanase activities of soil under wheat crop. Vermicompost fertilization resulted in highest microbial biomass, available phosphorus, and nitrogen content of wheat soil. It was also found effective in minimizing the alkalinity of soil compared to other treatments as indicated by pH change. However incorporation of paddy straw in conjunction with N₆₀P₆₀ and T. reesei inoculation resulted in maximum dehydrogenase, alkaline phosphatase and highest humus content of soil. Mixed inoculation of A. awamori and T. reesei did not prove effective in improving the soil biochemical properties in comparison to single inoculation of T. reesei. Results showed that in situ incorporation of paddy straw in combination with N₆₀P₆₀ and T. reesei inoculation can be used as an effective measure for valuable disposal of paddy straw and to improve the soil health by reducing mineral fertilization.     

不同杀虫剂对西花蓟马的室内毒力及田间药效

【目的】西花蓟马是一种世界性的危险性入侵害虫,筛选防治西花蓟马的有效药剂可为生产中科学用药提供依据。【方法】通过室内毒力测定和田间药效试验,测定了2种化学药剂和8种生物药剂对西花蓟马成虫的致死中浓度(LC_(50))和药后不同时期的防治效果。【结果】室内毒力测定依据LC_(50)值将各药剂对西花蓟马的敏感性由高到低依次排序为乙基多杀菌素(0.1958 mg·L~(-1))、印楝素(0.9399 mg·L~(-1))、苦参碱(1.2483 mg·L~(-1))、阿维菌素(1.8096 mg·L~(-1))、高效氯氰菊酯(4.4458 mg·L~(-1))、藜芦碱(10.7628 mg·L~(-1))、鱼藤酮(18.1898 mg·L~(-1))、吡虫啉(46.3964 mg·L~(-1))、松脂酸钠(131.5214 mg·L~(-1))、苏云金杆菌(446.2318 mg·L~(-1));田间药效试验发现,乙基多杀菌素和吡虫啉防治西花蓟马均表现出较强的速效性和持效性,药后1～14 d的防效分别可达84%和73%以上;其次是藜芦碱,药后1～14 d的防效可达48.15%～61.37%;高效氯氰菊酯的防效较低,药后14 d的防效为46.22%;生物药剂阿维菌素、苏云金杆菌、苦参碱、印楝素的速效性均较低,但防效随施药后时间的延长而逐渐上升;鱼藤酮持效性最低。【结论】乙基多杀菌素和吡虫啉可推荐为生产中防治西花蓟马的首选药剂,植物源药剂藜芦碱和生物药剂阿维菌素可作为交替使用药剂。     

Systemic antifeedant effects of azadirachtin on the peach-potato aphidMyzus persicae

The electrical penetration graph (EPG) method was used to analyse the feeding behaviour of apterous, adultMyzus persicae (Sulzer) (Homoptera: Aphididae) onNicotiana clevelandii (Gray) seedlings, treated systemically with azadirachtin. A preliminary experiment showed that the effects of tethering aphids for EPG recording were minimal. The percentage of the 9 h recording period devoted to non-penetration activities, and to stylet pathway patterns increased as the azadirachtin concentration in the root treatment increased. The number of probes initiated, and the numbers of sieve tube penetrations also increased with increased azadirachtin concentration. The mean time elapsing between the initiation of the first probe to reach a sieve element and contact with this tissue was not significantly altered by azadirachtin treatment. However, azadirachtin treatment significantly reduced the percentage of probes that reached sieve elements and increased non-penetration activity before and after the first perid of ingestion from the sieve elements. The percentage of the recording period spent in the EPG pattern associated with sieve tube penetration was significantly reduced by an azadirachtin concentration of 300 ppm, and the duration of each individual penetration was significantly reduced by an azadirachtin concentration of 100 ppm. When the total EPG was split into 3 h periods, significant interactions were seen between time period and azadirachtin concentration for the duration of non-penetration, pathway, and sieve tube penetration patterns.     

Transgenic tomato plants expressing an <Emphasis Type="Italic">Arabidopsis</Emphasis> thionin (<Emphasis Type="Italic">Thi2.1</Emphasis>) driven by fruit-inactive promoter battle against phytopathogenic attack

Tomato is one of the most important crop plants; however, attacks by pathogens can cause serious losses in production. In this report, we explore the potential of using the Arabidopsis thionin (Thi2.1) gene to genetically engineer enhanced resistance to multiple diseases in tomato. Potential thionin toxicity in fruits was negated by the use of a fruit-inactive promoter to drive the Thi2.1 gene. In transgenic lines containing RB7/Thi2.1, constitutive Thi2.1 expression was detected in roots and incidentally in leaves, but not in fruits. Disease assays revealed that the transgenic lines that were tested conferred significant levels of enhanced resistance to bacterial wilt (BW) and Fusarium wilt (FW). Further studies indicated that BW disease progression in transgenic lines was delayed by a systemic suppression of bacterial multiplication. By adopting a safe genetic engineering strategy, the present investigation is another step forward demonstrating thionin practicality in crop protection.     

Application of infrared thermography to the study of behavioural fever in the desert locust

Infrared (IR) thermography is used increasingly to estimate body temperature in small ectotherms such as insects. We used the thermal behaviour of an agricultural pest, the desert locust, Schistocerca gregaria, as a case study to demonstrate the application of this method to thermal biology. During microbial infection, the desert locust uses environmentally derived heat to elevate its body temperature. This ‘behavioural fever’ delays onset of disease caused by a fungal biopesticide. Understanding the thermal biology of S. gregaria is therefore a prerequisite for the development of a more effective mycoinsecticide. To accurately use IR thermography as a method of temperature measurement, IR data must first be calibrated with body temperature. Here, we identify two major factors which affect the IR data output and hence need to be incorporated into a camera calibration: (1) emissivity, predominantly determined by colour, and (2) the internal temperature of the camera. We demonstrate the limitations of thermocouple-based methods of temperature measurement in comparison to IR thermography. The detail provided by the large data sets revealed for the first time an early onset of fever in S. gregaria during infection with Metarhizium acridum viz. 20–25 h post-inoculation in comparison to the 48 h demonstrated previously.     

印楝素A与印楝素B对粉纹夜蛾BTI-Tn-5B1-4的细胞毒性

为了明确印楝素A和B活性差异的机理,本研究比较了印楝素A和印楝素B对粉纹夜蛾Trichoplusia ni离体培养胚胎细胞系BTI-Tn-5B1-4的毒性。结果表明：印楝素A与印楝素B对BTI-Tn-5B1-4细胞具有良好的增殖抑制活性,处理后3 d,其IC₅₀值分别为2.9 μg/mL和9.85 μg/mL,印楝素A的细胞毒力显著高于印楝素B。倒置显微镜观察发现,印楝素A和印楝素B处理可导致细胞变形,贴壁能力下降,并出现明显空泡,印楝素A的影响明显高于印楝素B。流式细胞仪检测结果表明,印楝素可导致BTI-Tn-5B1-4细胞体积显著膨大,印楝素A处理细胞体积增大程度显著高于印楝素B;印楝素可以明显影响BTI-Tn-5B1-4细胞膜电位,1.25 μg/mL印楝素A和印楝素B处理后3 d,细胞DiBAC4（3）荧光强度分别增加88.12%和55.37%,印楝素A的影响显著高于印楝素B。荧光显微镜观察发现,印楝素对BTI-Tn-5B1-4细胞核具有明显影响,印楝素B的影响明显高于印楝素A,印楝素B处理后,细胞核受损细胞数更多,受损程度更严重。结果显示印楝素A和印楝素B的细胞作用机理存在差异,本研究从细胞学水平解释了印楝素的生长发育抑制作用机理。     

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.