Whole genome sequencing of spotted stem borer, Chilo partellus, reveals multiple genes encoding enzymes for detoxification of insecticides.

Spotted stem borer, Chilo partellus, is the most important constraint for increasing the production and productivity of maize and sorghum, the two major coarse cereals in Asia and Africa. The levels of resistance to this pest in the cultivated germplasm are low to moderate, and hence, farmers have to use insecticides for effective control of this pest. However, there is no information on the detoxification mechanisms in C. partellus, which is one of the constraints for deployment of appropriate insecticides to control this pest. The ability to detoxify insecticides varies across insect populations, and hence, we sequenced different populations of C. partellus to identify and understand detoxification mechanisms to devise appropriate strategies for deployment of different insecticides for controlling this pest. Larval samples were sequenced from three different cohorts of C. partellus using the Illumina HiSeq 2500 platform. The data were subjected to identify putative genes that are involved in detoxification on insecticides in our cohort insect species. These studies resulted in identification of 64 cytochrome P450 genes (CYP450s), and 36 glutathione S-transferases genes (GSTs) encoding metabolic detoxification enzymes, primarily responsible for xenobiotic metabolism in insects. A total of 183 circadian genes with > 80% homolog and 11 olfactory receptor genes that mediate chemical cues were found in the C. partellus genome. Also, target receptors related to insecticide action, 4 acetylcholinesterase (AChE), 14 γ-aminobutyric acid (GABA), and 15 nicotinic acetylcholine (nAChR) receptors were detected. This is the first report of whole genome sequencing of C. partellus useful for understanding mode of action of different insecticides, and mechanisms of detoxification and designing target-specific insecticides to develop appropriate strategies to control C. partellus for sustainable crop production.

Label-free quantitative proteomics of Sorghum bicolor reveals the proteins strengthening plant defense against insect pest Chilo partellus.

BACKGROUND: Spotted stem borer- Chilo partellus - a Lepidopteran insect pest of Sorghum bicolor is responsible for major economic losses. It is an oligophagous pest, which bores through the plant stem, causing 'deadheart' and hampering the development of the main cob. We applied a label-free quantitative proteomics approach on three genotypes of S. bicolor with differential resistance/ susceptibility to insect pests, intending to identify the S. bicolor's systemic protein complement contributing to C. partellus tolerance. METHODS: The proteomes of S. bicolor with variable resistance to insect pests, ICSV700, IS2205 (resistant) and Swarna (susceptible) were investigated and compared using label-free quantitative proteomics to identify putative leaf proteins contributing to resistance to C. partellus. RESULTS: The multivariate analysis on a total of 967 proteins led to the identification of proteins correlating with insect resistance/susceptibility of S. bicolor. Upon C. partellus infestation S. bicolor responded by suppression of protein and amino acid biosynthesis, and induction of proteins involved in maintaining photosynthesis and responding to stresses. The gene ontology analysis revealed that C. partellus-responsive proteins in resistant S. bicolor genotypes were mainly involved in stress and defense, small molecule biosynthesis, amino acid metabolism, catalytic and translation regulation activities. At steady-state, the resistant S. bicolor genotypes displayed at least two-fold higher numbers of unique proteins than the susceptible genotype Swarna, mostly involved in catalytic activities. Gene expression analysis of selected candidates was performed on S. bicolor by artificial induction to mimic C. partellus infestation. CONCLUSION: The collection of identified proteins differentially expressed in resistant S. bicolor, are interesting candidates for further elucidation of their role in defense against insect pests.

Tryptophan fluorescence quenching as a binding assay to monitor protein conformation changes in the membrane of intact mitochondria.

Intrinsic protein fluorescence is due to aromatic amino acids, mainly tryptophan, which can be selectively measured by exciting at 295 nm. Changes in emission spectra of tryptophan are due to the protein conformational transitions, subunit association, ligand binding or denaturation, which affect the local environment surrounding the indole ring. In this study, tryptophan fluorescence was monitored in intact mitochondria at 333 nm following excitation at 295 nm in presence of insecticides using spectrofluorometer. Methyl-parathion, carbofuran, and endosulfan induced Trp fluorescence quenching and release of cytochrome c when incubated with the mitochondria, except fenvalarate. Mechanism of insecticide-induced mitochondrial toxicity for the tested insecticides has been discussed. Reduction in the intensity of tryptophan emission spectra of mitochondrial membrane proteins in presence of an increasing concentration of a ligand can be used to study the interaction of insecticides/drugs with the intact mitochondria. Furthermore, this assay can be readily adapted for studying protein-ligand interactions in intact mitochondria and in other cell organelles extending its implications for pesticide and pharma industry and in drug discovery.

Antibiotics influence the toxicity of the delta endotoxins of Bacillus thuringiensis towards the cotton bollworm, Helicoverpa armigera.

BACKGROUND: The cotton bollworm, Helicoverpa armigera is one of the most important crop pests worldwide. It has developed high levels of resistance to synthetic insecticides, and hence, Bacillus thuringiensis (Bt) formulations are used as a safer pesticide and the Bt genes have been deployed in transgenic crops for controlling this pest. There is an apprehension that H. armigera might develop resistance to transgenic crops in future. Therefore, we studied the role of gut microbes by eliminating them with antibiotics in H. armigera larvae on the toxicity of Bt toxins against this pest. RESULTS: Commercial formulation of Bt (Biolep®) and the pure Cry1Ab and Cry1Ac toxin proteins were evaluated at ED50, LC50, and LC90 dosages against the H. armigera larvae with and without antibiotics (which removed the gut microbes). Lowest H. armigera larval mortality due to Bt formulation and the Bt toxins Cry1Ab and Cry1Ac was recorded in insects reared on diets with 250 and 500 µg ml-1 diet of each of the four antibiotics (gentamicin, penicillin, rifampicin, and streptomycin), while the highest larval mortality was recorded in insects reared on diets without the antibiotics. Mortality of H. armigera larvae fed on diets with Bt formulation and the δ-endotoxins Cry1Ab and Cry1Ac was inversely proportional to the concentration of antibiotics in the artificial diet. Nearly 30% reduction in larval mortality was observed in H. armigera larvae from F1 to F3 generation when the larvae were reared on diets without antibiotics (with gut microbes) and fed on 0.15% Bt or 12 µg Cry1Ab or Cry1Ac ml-1 diet, indicating development of resistance to Bt in the presence of gut microflora. However, there were no differences in larval mortality due to Bt, Cry1Ab or Cry1Ac across generations in insects when they were reared on diets with 250 µg of each antibiotic ml-1 diet (without gut microflora). CONCLUSIONS: The results suggested that antibiotics which eliminated gut microflora influenced the toxicity of Bt towards H. armigera, and any variation in diversity and abundance of gut microflora will have a major bearing on development of resistance to Bt toxins applied as foliar sprays or deployed in transgenic crops for pest management.

Constitutive and inducible resistance to Atherigona soccata (Diptera: Muscidae) in Sorghum bicolor.

Host plant resistance is one of the important components for minimizing the losses because of sorghum shoot fly, Atherigona soccata (Diptera: Muscidae) attack. Therefore, we studied the constitutive and inducible biochemical mechanisms of resistance to A. soccata in a diverse array of sorghum genotypes to identify lines with diverse mechanisms of resistance to this insect. Fifteen sorghum genotypes with different levels of resistance to A. soccata were evaluated. Methanol extracts of 10-d old damaged and undamaged sorghum seedlings were subjected to high-performance liquid chromatography analysis. Association between peak areas of the identified and unidentified compounds with parameters measuring A. soccata resistance was determined through correlation analysis. Amounts of p-hydroxy benzaldehyde and the unidentified compounds at RTs 24.38 and 3.70 min were associated with susceptibility to A. soccata. Genotypes exhibiting resistance to A. soccata were placed in four groups, and the lines showing constitutive and/or induced resistance to A. soccata with different combinations of biochemical factors potentially could be used for increasing the levels of resistance to A. soccata in sorghum.

Biological and biochemical diversity in different biotypes of spotted stem borer, Chilo partellus (Swinhoe) in India.

Because of variation in incidence and severity of damage by Chilo partellus (Swinhoe) in different geographical regions, it is difficult to identify stable sources of resistance against this pest. Therefore, the present studies were undertaken on biological attributes (damage in resistant and susceptible genotypes, survival and development) and biochemical profiles (amino acids and lipophilic compound) of C. partellus populations from eight geographical regions to understand it's population structure in India. There was a significant variation in biological attributes and biochemical profiles of C. partellus populations from different geographical regions. Based on virulence and biological attributes, similarity index placed the C. partellus populations in five groups. Likewise, lipophilic and amino acid profiling also placed the C. partellus populations in five groups. However, the different clusters based on biological and biochemical attributes did not include populations from the same regions. Similarity index based on virulence, biological attributes, and amino acids and lipophilic profiles placed the C. partellus populations in six groups. The C. partellus populations from Hisar, Hyderabad, Parbhani and Coimbatore were distinct from each other, indicating that there are four biotypes of C. partellus in India. The results suggested that sorghum and maize genotypes need to be tested against these four populations to identify stable sources of resistance. However, there is a need for further studies to establish the restriction in gene flow through molecular approaches across geographical regions to establish the distinctiveness of different biotypes of C. partellus in India.

Genetic regulation of diapause and associated traits in Chilo partellus (Swinhoe).

Diapause is an endocrine controlled arrested metabolic state to delay development or reproduction under unfavorable conditions. To gain an understanding of importance of diapause for ecological adaptation, it is important to study regulation of diapause in insects. We examined genetics of diapause in Chilo partellus by crossing the hibernating (HD), aestivating (AD), post-hibernating (PHD), post-aestivating (PAD), and nondiapause (ND) strains. Reciprocal crosses were also made to gain full understanding of diapause regulation and the maternal effects, if any. Data were recorded on fecundity, egg hatching, larval survival, diapause induction and termination, adult emergence, and morphometrics of larvae, pupae and adults in the parents (P1, P2), F1 hybrids, and the reciprocal crosses. Genetic analysis showed that AD strain is general combiner, which also improved egg hatching, larval survival, diapause termination, adult emergence and proportion of females in the progenies. Incidence of diapause was highest in HD × AD, whereas termination was greatest in PHD × AD. However, ND strain and its reciprocal crosses with other strains did not exhibit any noticeable developmental response associated with diapause. Specific combining ability analysis suggested that where PHD and AD strains exist together there will be likely reduction in diapause incidence, increased survival with greater fitness and faster multiplication of their progenies resulting in outbreak of C. partellus. Degree of dominance estimates revealed that diapause, developmental and morphometric traits in C. partellus are governed by over dominance gene effects, and mainly depend on parental diapause history.

Assessment of the suitability of Tinopal as an enhancing adjuvant in formulations of the insect pathogenic fungus Beauveria bassiana (Bals.) Vuillemin.

BACKGROUND: Biopesticides based on Beauveria bassiana (Bals.) Vuillemin hold great promise for the management of a wide range of insect pests. The conidia in the biopesticide formulation require an adjuvant to protect them from photoinactivation by sunlight. The suitability of Tinopal, an optical brightener used as sunscreen for baculovirus formulations, for use with B. bassiana was assessed. The aim was to study the effect of Tinopal on the growth and photoprotection of B. bassiana, and its effect on the susceptibility of insects to B. bassiana. RESULTS: Tinopal was found to have no adverse effect on the growth of B. bassiana. It was found to confer total protection (approximately 95% conidial germination at 10 g Tinopal L(-1)) from sunlight up to 3 h of exposure, and a better survival rate than controls even up to 4 h. Helicoverpa armigera Hübner larvae fed on diet with 5 g kg(-1) Tinopal were found to have reduced growth. The duration of the larval stage increased by 3-4 days in 1 and 5 g kg(-1) Tinopal treatments. Among the moths that emerged from larvae fed on diet with 5 g kg(-1) Tinopal, a significantly high number were malformed compared with controls. The larvae that were fed diet with Tinopal showed quicker and higher mortality and required a lower effective lethal dose (LC(50)) than the controls. Tinopal was found to have a synergistic effect with B. bassiana in causing insect mortality. CONCLUSIONS: Tinopal was found to be a suitable adjuvant for B. bassiana-based biopesticide formulations. It conferred tolerance to sunlight and caused stress in the insect, leading to a synergistic effect with B. bassiana.

Introgression of Shoot Fly (Atherigona soccata L. Moench) Resistance QTLs into Elite Post-rainy Season Sorghum Varieties Using Marker Assisted Backcrossing (MABC).

Shoot fly (Atherigona soccata L. Moench) is a serious pest in sorghum production. Management of shoot fly using insecticides is expensive and environmentally un-safe. Developing host-plant resistance is the best method to manage shoot fly infestation. Number of component traits contribute for imparting shoot fly resistance in sorghum and molecular markers have been reported which were closely linked to QTLs controlling these component traits. In this study, three QTLs associated with shoot fly resistance were introgressed into elite cultivars Parbhani Moti (= SPV1411) and ICSB29004 using marker assisted backcrossing (MABC). Crosses were made between recurrent parents and the QTL donors viz., J2658, J2614, and J2714. The F1s after confirmation for QTL presence were backcrossed to recurrent parents and the resultant lines after two backcrosses were selfed thrice for advancement. The foreground selection was carried out in F1 and BCnF1 generations with 22 polymorphic markers. Forty-three evenly distributed simple sequence repeat markers in the sorghum genome were used in background selection to identify plants with higher recurrent parent genome recovery. By using two backcrosses and four rounds of selfing, six BC2F4 progenies were selected for ICSB29004 × J2658, five BC2F4 progenies were selected for ICSB29004 × J2714 and six BC2F4 progenies were selected for Parbhani Moti × J2614 crosses. Phenotyping of these lines led to the identification of two resistant lines for each QTL region present on chromosome SBI-01, SBI-07 and SBI-10 in ICSB 29004 and Parbhani Moti. All the introgression lines (ILs) showed better shoot fly resistance than the recurrent parents and their agronomic performance was the same or better than the recurrent parents. Further, the ILs had medium plant height, desirable maturity with high yield potential which makes them better candidates for commercialization. In the present study, MABC has successfully improved the shoot fly resistance in sorghum without a yield penalty. This is the first report on the use of MABC for improving shoot fly resistance in post-rainy season sorghum.

Influence of cytoplasmic male sterility on expression of different mechanisms of resistance in sorghum to Atherigona soccata (Diptera: Muscidae).

Atherigona soccata (Rondani) (Diptera: Muscidae) is one of the most important pests of sorghum, Sorghum bicolor (L.) Moench, in Asia, Africa, and the Mediterranean Europe. Exploitation of cytoplasmic male sterility (CMS) for hybrid production has resulted in considerable narrowing of the genetic base and may increase the vulnerability of this crop to insect pests. Therefore, we studied the expression of different mechanisms of resistance in sorghum to A. soccata in CMS (A) and maintainer (B) lines of 12 genotypes under field and greenhouse conditions. The CMS lines of A. soccata-resistant genotypes were preferred for oviposition (78.5 versus 71.5% plants with eggs) and suffered greater deadheart incidence (47.6 versus 41.6%) than the corresponding maintainer lines, whereas such differences were not apparent in CMS lines belonging to the susceptible genotypes (92.7 versus 92.3% plants with eggs and 75.6 versus 74.6% deadhearts) under multichoice field conditions. Similar differences also were observed under controlled conditions in the greenhouse. The larval period (9.0 versus 8.8 d) and pupal mortality (18.4 versus 13.4%) were greater on maintainer lines than that on the CMS lines in the resistant group. The male and female pupal weights, fecundity, and antibiosis index were greater on the CMS than on the maintainer lines. The maintainer lines showed better recovery resistance than the CMS lines, but no such differences were observed in tiller deadhearts. The differences in susceptibility to A. soccata were greater in the A. soccata resistant CMS and maintainer lines than in the CMS and maintainer lines belonging to susceptible genotypes. Conversion of A. soccata-resistant genotypes into alternate less susceptible cytoplasmic backgrounds may be undertaken for developing sorghum hybrids with stable resistance to A. soccata.

Isolation and Characterization of Gut Bacterial Proteases Involved in Inducing Pathogenicity of Bacillus thuringiensis Toxin in Cotton Bollworm, Helicoverpa armigera.

Bacillus thuringiensis toxin proteins are deployed in transgenic plants for pest management. The present studies were aimed at characterization of gut bacterial proteases involved in activation of inactive Cry1Ac protoxin (pro-Cry1Ac) to active toxin in Helicoverpa armigera. Bacterial strains were isolated from H. armigera midgut and screened for their proteolytic activation toward pro-Cry1Ac. Among 12 gut bacterial isolates seven isolates showed proteolytic activity, and proteases from three isolates (IVS1, IVS2, and IVS3) were found to be involved in the proteolytic conversion of pro-Cry1Ac into active toxin. The proteases from IVS1, IVS2, and IVS3 isolates were purified to 11.90-, 15.50-, and 17.20-fold, respectively. The optimum pH and temperature for gut bacterial protease activity was 8.0 and 40°C. Maximum inhibition of total proteolytic activity was exerted by phenylmethane sulfonyl fluoride followed by EDTA. Fluorescence zymography revealed that proteases from IVS1, IVS2, and IVS3 were chymotrypsin-like and showing protease band at ~15, 65, and 15 kDa, respectively. Active Cry1Ac formed from processing pro-Cry1Ac by gut bacterial proteases exhibited toxicity toward H. armigera. The gut bacterial isolates IVS1, IVS2, and IVS3 showed homology with B. thuringiensis (CP003763.1), Vibrio fischeri (CP000020.2), and Escherichia coli (CP011342.1), respectively. Proteases produced by midgut bacteria are involved in proteolytic processing of B. thuringiensis protoxin and play a major role in inducing pathogenicity of B. thuringiensis toxins in H. armigera.

Insecticidal activity of a novel fatty acid amide derivative from Streptomyces species against Helicoverpa armigera.

Helicoverpa armigera, an important pest causes serious damage to grain legumes. The main objective of this study was to isolate and identify the metabolite against H. armigera from a previously characterised Streptomyces sp. CAI-155. The culture filtrate of CAI-155 was extracted using Diaion HP-20 and the active fractions were fractionated on Silica and C18 column chromatography. The C18 active fraction was further fractionated on Silica gel 60 F254 thin layer chromatography (TLC). The most active fraction (Rf 0.64) purified from TLC led to the identification of a novel metabolite N-(1-(2,2-dimethyl-5-undecyl-1,3-dioxolan-4-yl)-2-hydroxyethyl)stearamide by spectral studies. The purified metabolite showed 70-78% mortality in 2nd instar H. armigera by diet impregnation assay, detached leaf assay and greenhouse assay. The LD50 and LD90 values of the purified metabolite were 627 and 2276 ppm, respectively. Hence, this novel metabolite can be exploited for pest management in future.

Capsicum annuum proteinase inhibitor ingestion negatively impacts the growth of sorghum pest Chilo partellus and promotes differential protease expression.

BACKGROUND: Chilo partellus is an important insect pest infesting sorghum and maize. The larvae internalize in the stem, rendering difficulties in pest management. We investigated the effects of Capsicum annuum proteinase inhibitors (CanPIs) on C. partellus larvae by in-vitro and in-vivo experiments. METHODS: Recombinant CanPI-7 (with four-Inhibitory Repeat Domains, IRDs), -22 (two-IRDs) and insect proteinase activities were estimated by proteinase assays, dot blot assays and in gel activity assays. Feeding bioassays of lab reared C. partellus with CanPI-7 and -22 were performed. C. partellus proteinase gene expression was done by RT-PCR. In-silico structure prediction of proteinases and CanPI IRDs was carried out, their validation and molecular docking was done for estimating the interaction strength. RESULTS: Larval proteinases of C. partellus showed higher activity at alkaline pH and expressed few proteinase isoforms. Both CanPIs showed strong inhibition of C. partellus larval proteinases. Feeding bioassays of C. partellus with CanPIs revealed a dose dependent retardation of larval growth, reduction of pupal mass and fecundity, while larval and pupal periods increased significantly. Ingestion of CanPIs resulted in differential up-regulation of C. partellus proteinase isoforms, which were sensitive to CanPI-7 but were insensitive to CanPI-22. In-silico interaction studies indicated the strong interaction of IRD-9 (of CanPI-22) with Chilo proteinases tested. CONCLUSIONS: Of the two PIs tested, CanPI-7 prevents induction of inhibitor insensitive proteinases in C. partellus so it can be explored for developing C. partellus tolerance in sorghum. GENERAL SIGNIFICANCE: Ingestion of CanPIs, effectively retards C. partellus growth; while differentially regulating the proteinases.

Detached leaf assay to screen for host plant resistance to Helicoverpa armigera.

The noctuid Helicoverpa armigera (Hübner) is a major insect pest of chickpea Cicer arietinum L., pigeonpea Cajanus cajan (L.) Millsp., peanut Arachis hypogaea L., and cotton Gossypium spp., and host plant resistance is an important component for managing this pest in different crops. Because of variations in insect density and staggered flowering of the test material, it is difficult to identify cultivars with stable resistance to H. armigera across seasons and locations. To overcome these problems, we standardized the detached leaf assay to screen for resistance to this pest in chickpea, pigeonpea, peanut, and cotton under uniform insect pressure under laboratory conditions. Terminal branch (three to four fully expanded leaves) of chickpea, first fully expanded leaf of cotton, trifoliate of pigeonpea, or quadrifoliate of peanut, embedded in 3% agar-agar in a plastic cup/jar of appropriate size (250-500-ml capacity) infested with 10-20 neonate larvae can be used to screen for resistance to H. armigera. This technique keeps the leaves in a turgid condition for approximately 1 wk. The experiments can be terminated when the larvae have caused > 80% leaf damage in the susceptible check or when differences in leaf feeding between the resistant and susceptible checks are maximum. Detached leaf assay can be used as a rapid screening technique to evaluate germplasm, segregating breeding materials, and mapping populations for resistance to H. armigera in a short span of time with minimal cost, and under uniform insect infestation. It also provides useful information on antibiosis component of resistance to the target insect pest.

Quantitative genetic analysis of agronomic and morphological traits in sorghum, Sorghum bicolor.

The productivity in sorghum is low, owing to various biotic and abiotic constraints. Combining insect resistance with desirable agronomic and morphological traits is important to increase sorghum productivity. Therefore, it is important to understand the variability for various agronomic traits, their heritabilities and nature of gene action to develop appropriate strategies for crop improvement. Therefore, a full diallel set of 10 parents and their 90 crosses including reciprocals were evaluated in replicated trials during the 2013-14 rainy and postrainy seasons. The crosses between the parents with early- and late-flowering flowered early, indicating dominance of earliness for anthesis in the test material used. Association between the shoot fly resistance, morphological, and agronomic traits suggested complex interactions between shoot fly resistance and morphological traits. Significance of the mean sum of squares for GCA (general combining ability) and SCA (specific combining ability) of all the studied traits suggested the importance of both additive and non-additive components in inheritance of these traits. The GCA/SCA, and the predictability ratios indicated predominance of additive gene effects for majority of the traits studied. High broad-sense and narrow-sense heritability estimates were observed for most of the morphological and agronomic traits. The significance of reciprocal combining ability effects for days to 50% flowering, plant height and 100 seed weight, suggested maternal effects for inheritance of these traits. Plant height and grain yield across seasons, days to 50% flowering, inflorescence exsertion, and panicle shape in the postrainy season showed greater specific combining ability variance, indicating the predominance of non-additive type of gene action/epistatic interactions in controlling the expression of these traits. Additive gene action in the rainy season, and dominance in the postrainy season for days to 50% flowering and plant height suggested G X E interactions for these traits.

Pigeonpea genotypes influence parasitization preference and survival and development of the Helicoverpa armigera larval parasitoid, Campoletis chlorideae.

Studies were undertaken to identify pigeonpea, Cajanus cajan (L.) Millspaugh and the wild relative of pigeonpea, Cajanus scarabaeoides (L.) (accession ICPW 125,) genotypes that are hospitable to the pod borer, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) larval parasitoid, Campoletis chlorideae Uchida (Hymenoptera: Ichneumonidae) for the management of this pest in pigeonpea based cropping systems. Percentage parasitization of the H. armigera larvae by the C. chlorideae females was greater under no-choice conditions than under multi-choice conditions because of forced parasitization under no-choice conditions. Lowest parasitization was recorded on the wild relative, ICPW 125, which may be due to long nonglandular hairs and low survival of H. armigera larvae. Parasitization of H. armigera larvae was greater under no-choice, dual-choice and/or multi-choice conditions on ICPL 87, ICPL 87119 and ICPL 87091, which are susceptible to H. armigera, than on the pod borer-resistant genotypes ICPL 332WR, ICPL 84060 and ICPB 2042; while survival and development of the parasitoid was better on H. armigera larvae fed on ICPL 87, ICPL 87119, LRG 41, ICP 7035 and ICPL 87091 than on ICPL 332WR, ICPL 84060, ICPB 2042 and ICPW 125. The genotypes ICPL 87, ICPL 87119, LRG 42 and ICPL 87091 that are hospitable to C. chloridae, are better suited for use in integrated pest management to minimize the losses due to H. armigera in pigeonpea.

Influence of oxalic and malic acids in chickpea leaf exudates on the biological activity of CryIAc towards Helicoverpa armigera.

Efforts are being made to express toxin genes from the bacterium, Bacillus thuringiensis (Bt) in chickpea for minimizing the losses due to the pod borer, Helicoverpa armigera. However, there is an apprehension that acidic exudates in chickpea leaves may influence the protoxin-toxin conversion in the insect midgut, and thus, reduce the efficacy of Bt toxins. Therefore, we studied the influence of organic acids (oxalic acid and malic acid) present in the trichome exudates of chickpea on the biological activity and binding of Bt δ-endotoxin Cry1Ac to brush border membrane vesicles (BBMV) of the pod borer, H. armigera. Oxalic and malic acids in combination at concentrations present in chickpea leaves did not influence the biological activity of Bt toxin Cry1Ac towards H. armigera larvae. Amounts of Cry1Ac protein in the midgut of insects reared on diets with organic acids were similar to those reared on artificial diet without the organic acids. However, very high concentrations of the organic acids reduced the amounts of Cry1Ac in the midgut of H. armigera larvae. Organic acids in the artificial diet also increased the excretion of Cry1Ac in the fecal matter. Organic acids reduced the amount of protein in the BBMV of insects reared on diets with Cry1Ac, possibly because of reduced size of the larvae. Oxalic and malic acids at concentrations present in chickpea leaves did not affect the biological activity of Cry1Ac, but it will be desirable to have high levels of expression of Cry1Ac toxin proteins in chickpea for effective control of the pod borer, H. armigera.

In vivo inhibition of Helicoverpa armigera gut pro-proteinase activation by non-host plant protease inhibitors.

We evaluated 22 different host and non-host plant protease inhibitors (PIs) for in vivo inhibition of Helicoverpa armigera gut pro- and proteinases, and their biological activity against the pod borer, H. armigera, the most important pest of agriculture and horticultural crops worldwide. In vitro activation of H. armigera gut pro-proteinases (HaGPPs) in larvae fed on non-host plant PIs showed significant in vivo inhibition of HaGPPs activation in solution as well as in gel assays. The larvae fed on diet incorporated with Datura alba ness PIs showed highest inhibition of HaGPPs, followed by Psophocarpus tetragonolobus. Non-host plant PIs from Pongamia pinnata, Mucuna pruriens, Capsicum annuum, and Nigela sativa showed maximum inhibitory potential towards HaGPs in vivo, and also exhibited moderate level of inhibition of pro-proteinases. However, some of non-host plant PIs, such as those from Penganum harmala and Solanum nigrum, and the principal host plant PIs, viz., Cicer arietinum and Cajanus cajan did not inhibit HaGPP activity. Pro-proteinase level increased with the growth of the larvae, and maximum HaGPP activity was observed in the fifth-instars. Larvae fed on diets with D. alba ness PIs showed greater inhibition of HaGPPs as compared to the larvae fed on diets with P. tetragonolobus. Low concentrations of partially purified HaGPs treated with gut extract of larvae fed on D. alba ness showed that out of 10 proteinase isoforms, HaGPs 5 and 9 were activators of pro-proteinases. Larval growth and development were significantly reduced in the larvae fed on the non-host plant PIs, of which D. alba ness resulted in highest stunted growth of H. armigera larvae. The in vivo studies indicated that non-host plant PIs were good candidates as inhibitors of the HaGPs as well as HaGPPs. The PIs from the non-host plants can be expressed in genetically engineered plants to confer resistance to H. armigera.

Homodimeric alkaline phosphatase located at Helicoverpa armigera midgut, a putative receptor of Cry1Ac contains alpha-GalNAc in terminal glycan structure as interactive epitope.

Helicoverpa armigera causes massive yield loss of various crops globally. Bacillus thuringiensis coded Cry1Ac toxin is effective against the pest. Through the present investigation, an alkaline phosphatase type putative receptor of the toxin was identified in the target insect gut BBMV. Lectin-ligand immunoblot assay detected the presence of alpha-GalNAc residue at the nonreducing terminal of the glycan structure in the membrane bound HaALP protein which mediates the toxin-receptor interaction.

Phenolic compounds on the pod-surface of pigeonpea, Cajanus cajan, mediate feeding behavior of Helicoverpa armigera larvae.

A methanol extract of the pod surfaces of Cajanus cajan, a feeding stimulant for fifth-instar Helicoverpa armigera, was shown to contain four main phenolic compounds. Three of these were identified as isoquercitrin, quercetin, and quercetin-3-methyl ether, by comparing UV spectra and HPLC retention times with authentic standards. The fourth compound was isolated by semipreparative HPLC and determined to be 3-hydroxy-4-prenyl-5-methoxystilbene-2-carboxylic acid (stilbene) by NMR spectroscopy and mass spectrometry. Quercetin, isoquercitrin, and quercetin-3-methyl did not affect the selection-behavior of fifth-instar H. armigera. However, larvae were deterred from feeding on glass-fiber disks impregnated with the stilbene. Furthermore, larvae exposed to quercetin-3-methyl ether consumed significant amounts of both disks. In a binary-choice bioassay, a combination of quercetin-3-methyl ether and the stilbene on one disk and pure quercetin-3-methyl ether on the other disk resulted in increased consumption of both glass-fiber disks by larvae. In contrast, consumption was reduced if the combination was presented to larvae on one disk with purified stilbene on the other disk. Cajanus cajan cultivars that varied in their susceptibility to H. armigera were surveyed forthe presence of the four phenolic compounds. An absence of quercetin and higher concentrations of isoquercitrin than the cultivated variety characterized pod surface extracts of pod-borer-resistant cultivars. In addition, the ratio of the stilbene to quercetin-3-methyl ether was greater in the pod-borer-resistant cultivars. These findings are discussed in relation to the identification of chemical characters that can be used for crop improvement.