Bitter gourd proteinase inhibitors: potential growth inhibitors of Helicoverpa armigera and Spodoptera litura

Proteinase inhibitors (PIs) from the seeds of bitter gourd (Momordica charantia L.) were identified as strong inhibitors of Helicoverpa armigera gut proteinases (HGP). Biochemical investigations showed that bitter gourd PIs (BGPIs) inhibited more than 80% HGP activity. Electrophoretic analysis revealed the presence of two major proteins (BGPI-1 and-2) and two minor proteins (BGPI-3 and-4) having inhibitory activity against both trypsin and HGP. The major isoforms BGPI-1 and BGPI-2 have molecular mass of 3.5 and 3.0 kDa, respectively. BGPIs inhibited HGP activity of larvae fed on different host plants, on artificial diet with or without added PIs and proteinases excreted in fecal matter. Degradation of BGPI-1 by HGP showed direct correlation with accumulation of BGPI-2-like peptide, which remained stable and active against high concentrations of HGP up to 3 h. Chemical inhibitors of serine proteinases offered partial protection to BGPI-1 from degradation by HGP, suggesting that trypsin and chymotrypsin like proteinases are involved in degradation of BGPI-1. In larval feeding studies, BGPIs were found to retard growth and development of two lepidopteran pests namely Helicoverpa armigera and Spodoptera litura. This is the first report showing that BGPIs mediated inhibition of insect gut proteinases directly affects fertility and fecundity of both H. armigera and S. litura. The results advocate use of BGPIs to introduce insect resistance in otherwise susceptible plants.     

Higher accumulation of proteinase inhibitors in flowers than leaves and fruits as a possible basis for differential feeding preference of Helicoverpa armigera on tomato (Lycopersicon esculentum Mill, Cv. Dhanashree)

Tomato (Lycopersicon esculentum, Mill; cultivar- Dhanashree) proteinase inhibitors (PIs) were tested for their trypsin inhibitory (TI) and Helicoverpa armigera gut proteinases inhibitory (HGPI) activity in different organs of the tomato plants. Analysis of TI and HGPI distribution in various parts of the plant showed that flowers accumulated about 300 and 1000 times higher levels of TI while 700 and 400 times higher levels of HGPI as compared to those in leaves and fruits, respectively. Field observation that H. armigera larvae infest leaves and fruits but not the flowers could be at least partially attributed to the protective role-played by the higher levels of PIs in the flower tissue. Tomato PIs inhibited about 50-80% HGP activity of H. armigera larvae feeding on various host plants including tomato, of larvae exposed to non-host plant PIs and of various larval instars. Tomato PIs were found to be highly stable to insect proteinases wherein incubation of inhibitor with HGP even for 3h at optimum conditions did not affect inhibitory activity. Bioassay using H. armigera larvae fed on artificial diet containing tomato PIs revealed adverse effect on larval growth, pupae development, adult formation and fecundity.     

In vivo and in vitro effect of Capsicum annum proteinase inhibitors on Helicoverpa armigera gut proteinases

Two proteinase inhibitors (PIs), CapA1 and CapA2, were purified from Capsicum annum Linn. Var. Phule Jyoti leaves and assessed for their in vitro and in vivo activity against Helicoverpa armigera gut proteinases (HGPs). Both the inhibitors exhibited molecular weights of about 12 kDa with inhibitory activity against bovine trypsin and chymotrypsin indicating presence of probable two-inhibitor repeats of PIN II family. CapA1 and CapA2 inhibited 60-80% HGP (azocaseinolytic) activity of fourth instar larvae feeding on various host plants while 45-65% inhibition of HGP activity of various instars (II to VI) larvae reared on artificial diet. The partial purification of HGP isoforms, their characterization with synthetic inhibitors and inhibition by C. annum PIs revealed that most of the trypsin-like activity (68-91%) of HGPs was sensitive to C. annum PIs while 39-85% chymotrypsin-like activity of HGPs was insensitive to these inhibitors. The feeding of C. annum leaf extracts and two purified PIs in various doses to H. armigera larvae for two successive generations through artificial diet demonstrated their potential in inhibiting larval growth and development, delay in pupation period and dramatic reduction in fecundity and fertility. This is the first report-demonstrating efficacy of C. annum PIs against insect gut proteinases as well as larval growth and development of H. armigera.     

Protease inhibitors of pigeonpea (Cajanus cajan) and its wild relatives

Plant protease inhibitors have been implicated in defense against insect pests. Podborer and pod fly are major pests of developing seeds of pigeonpea ( Cajanus cajan L. Millsp.). Therefore, we studied the presence of protease inhibitors in seeds of pigeonpea and its wild relatives. Seed extracts were analyzed for protease inhibitor activities by caseinolytic assay, and the number of protease inhibitors determined by polyacrylamide gel electrophoresis. Besides trypsin and chymotrypsin inhibitors, seed extracts contained weak papain inhibitor(s) but no bromelain inhibitor. Treatment of seed extract with bromelain generated new active forms of trypsin inhibitors. The relative amounts of different trypsin inhibitors and the total trypsin inhibitor activity varied with different extraction media. Trypsin inhibitors were not detectable in pigeonpea leaves. The profiles of trypsin and chymotrypsin inhibitors in almost all the cultivars of pigeonpea analyzed were similar; however, those in wild relatives were quite variable.     

Identification of potent inhibitors of Helicoverpa armigera gut proteinases from winged bean seeds

Dry mature seeds of winged bean (Psophocarpus tetragonolobus L., DC.) (WB) contain several proteinase inhibitors. Two-dimensional gel analysis of WB seed protein followed by activity visualization using a gel-X-ray film contact print technique revealed at least 14 trypsin inhibitors (TIs) in the range of 28-6 kD. A total of seven inhibitors (WBTI-1 to 7) were purified by heat treatment and gel filtration followed by elution from preparative native gels. Based on their biochemical characterization such as molecular mass, pI, heat stability, and susceptibility to inactivation by reducing agents, WBTI-1 to 4 are Kunitz type inhibitors while WBTI-5 to 7 are classified as Bowman-Birk type serine proteinase inhibitors. Although Kunitz type TIs (20-24 kD) of WB have been reported, the smaller TIs that belong to the Bowman-Birk type have not been previously characterized. Seven major TIs isolated from WB seed were individually assessed for their potential to inhibit the gut proteinases (HGP) of Helicoverpa armigera, a pest of several economically important crops, which produces at least six major and several minor trypsin/chymotrypsin/elastase-like serine proteinases in the gut. WBTI-1 (28 kD) was identified as a potent inhibitor of HGP relative to trypsin and among the other WBTIs; it inhibited 94% of HGP activity while at the same concentration it inhibited only 22% of trypsin activity. WBTI-2 (24 kD) and WBTI-4 (20 kD) inhibited HGP activity greater than 85%. WBTI-3,-5,-6 and-7 showed limited inhibition of HGP as compared with trypsin. These results indicate that WBTIs have different binding potentials towards HGP although most of the HGP activity is trypsin-like. We also developed a simple and versatile method for identifying and purifying proteinase inhibitors after two-dimensional separation using the gel-X-ray film contact print technique.     

Morphological and chemical components of resistance to pod borer, Helicoverpa armigera in wild relatives of pigeonpea

Host plant resistance is an important component for minimizing the losses due to the pod borer, Helicoverpa armigera, which is the most devastating pest of pigeonpea. An understanding of different morphological and biochemical components of resistance is essential for developing strategies to breed for resistance to insect pests. Therefore, we studied the morphological and biochemical components associated with expression of resistance to H. armigera in wild relatives of pigeonpea to identify accessions with a diverse combination of characteristics associated with resistance to this pest. Among the wild relatives, oviposition non-preference was an important component of resistance in Cajanus scarabaeoides, while heavy egg-laying was recorded on C. cajanifolius (ICPW 28) and Rhynchosia bracteata (ICPW 214). Accessions belonging to R. aurea, C. scarabaeoides, C. sericeus, C. acutifolius, and Flemingia bracteata showed high levels of resistance to H. armigera, while C. cajanifolius was as susceptible as the susceptible check, ICPL 87. Glandular trichomes (type A) on the calyxes and pods were associated with susceptibility to H. armigera, while the non-glandular trichomes (trichome type C and D) were associated with resistance to this insect. Expression of resistance to H. armigera was also associated with low amounts of sugars and high amounts of tannins and polyphenols. Accessions of wild relatives of pigeonpea with non-glandular trichomes (type C and D) or low densities of glandular trichomes (type A), and high amounts of polyphenols and tannins may be used in wide hybridization to develop pigeonpea cultivars with resistance to H. armigera. Handling editor: Robert Glinwood     

Momordica charantia trypsin inhibitor II inhibits growth and development of Helicoverpa armigera

Abstract  Bitter gourd ( Momordica charantia L.) seeds contain several squash-type serine proteinase inhibitors (PIs), which inhibit the digestive proteinases of the polyphagous insect pest Helicoverpa armigera . In the present work isolation of a DNA sequence encoding the mature peptide of a trypsin inhibitor McTI-II, its cloning and expression as a recombinant protein using Pichia pastoris have been reported. Recombinant McTI-II inhibited bovine trypsin at 1: 1 molar ratio, as expected, but did not inhibit chymotrypsin or elastase. McTI-II also strongly inhibited trypsin-like proteinases (81% inhibition) as well as the total proteolytic activity of digestive proteinases (70% inhibition) from the midgut of H. armigera larvae. The insect larvae fed with McTI-II-incorporated artificial diet suffered over 70% reduction in the average larval weight after 12 days of feeding. Moreover, ingestion of McTI-II resulted in 23% mortality in the larval population. The strong antimetabolic activity of McTI-II toward H. armigera indicates its probable use in developing insect tolerance in susceptible plants.     

Complexity in specificities and expression of Helicoverpa armigera gut proteinases explains polyphagous nature of the insect pest

Helicoverpa armigera is a devastating pest of cotton and other important crop plants all over the world. A detailed biochemical investigation of H. armigera gut proteinases is essential for planning effective proteinase inhibitor (PI)-based strategies to counter the insect infestation. In this study, we report the complexity of gut proteinase composition of H. armigera fed on four different host plants, viz. chickpea, pigeonpea, cotton and okra, and during larval development. H. armigera fed on chickpea showed more than 2.5- to 3-fold proteinase activity than those fed on the other host plants. H. armigera gut proteinase composition revealed the predominance of serine proteinase activity; however, the larvae fed on pigeonpea revealed the presence of metalloproteases and low levels of aspartic and cysteine proteases as well. Gut proteinase activity increased during larval development with the highest activity seen in the fifth instar larvae which, however, declined sharply in the sixth instar. Over 90% of the gut proteinase activity of the fifth instar larvae was of the serine proteinase type, however, the second instar larvae showed the presence of proteinases of other mechanistic classes like metalloproteases, aspartic and cysteine proteases along with serine proteinase activity as evident by inhibition studies. Analysis of fecal matter of larvae showed significant increase in proteinase activity when fed on an artificial diet with or without non-host PIs than larvae fed on a natural diet. The diversity in the proteinase activity observed in H. armigera gut and the flexibility in their expression during developmental stages and depending upon the diet provides a base for selection of proper PIs for insect resistance in transgenic crop plants.     

Bioinsecticidal activity of Murraya koenigii miraculin-like protein against Helicoverpa armigera and Spodoptera litura

Miraculin-like proteins, belonging to the Kunitz superfamily, are natural plant defense agents against pests and predators, and therefore are potential biopesticides for incorporation into pest-resistant crops. Here, a miraculin-like protein from Murraya koenigii was assessed for its in vitro and in vivo effects against two polyphagous lepidopteran insect pests, Helicoverpa armigera and Spodoptera litura. M. koenigii miraculin-like protein (MKMLP) inhibited the trypsin-like activity and total protease activity of H. armigera gut proteinases (HGP) by 78.5 and 40%, respectively, and S.litura gut proteinases (SGP) by 81 and 48%, respectively. The inhibitor was stable and actively inhibited the proteolysis of both HGP and SGP enzymes for up to 72 h. Incorporation of MKMLP into artificial diet adversely affected the growth and development of pests in a dose-dependent manner. After 10 days of feeding on diets containing 200 μM MKMLP, larval weight was reduced to 69 and 44.8% and larval mortality was increased to 40 and 43.3% for H. armigera and S litura, respectively. The LC(50) of MKMLP was 0.34 and 0.22% of the diet for H.armigera and S. litura, respectively. These results demonstrate the efficacy of MKMLP as a potential plant defense agent against H. armigera and S. litura.     

Compensatory proteolytic responses to dietary proteinase inhibitors from Albizia lebbeck seeds in the Helicoverpa armigera larvae

Plant proteinase inhibitors (PIs) have been shown to reduce the growth rates in larvae of numerous insect species. On the other hand, insects can also regulate their proteinases against plant PIs. In the present study, we report the compensatory activities of Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) gut proteinases against the PIs of Albizia lebbeck seeds. Total of ten proteinase inhibitor bands were detected in the seed extract of A. lebbeck. Bioassays were conducted by feeding H. armigera larvae on diet containing partially purified PIs from A. lebbeck seeds. Results show that larval growth and survival was significantly reduced by A. lebbeck PIs. We found that higher activity H. armigera gut proteinase (HGP) isoforms observed in the midgut of control larvae were inhibited in the midgut of larvae fed on test diet. Some HGP isoforms were induced in the larvae fed on PI containing test diet; however, these isoforms showed lower activity in the larvae fed on control diet. Aminopeptidase activities were significantly increased in the midgut of larvae fed on test diet. A population of susceptible and resistant enzymes was observed in the midgut of H. armigera, when fed on diet containing PIs from A. lebbeck seeds. Our initial observations indicate that H. armigera can regulate its digestive proteinase activity against non-host plant PIs, too. It is important to study the exact biochemical and molecular mechanisms underlying this phenomenon in order to develop PI-based insect control strategies.     

A Kunitz trypsin inhibitor from chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.) that exerts anti-metabolic effect on podborer (<Emphasis Type="Italic">Helicoverpa armigera</Emphasis>) larvae

Chickpea (Cicer arietinum L.) seeds contain Bowman–Birk proteinase inhibitors, which are ineffective against the digestive proteinases of larvae of the insect pest Helicoverpa armigera. We have identified and purified a low expressing proteinase inhibitor (PI), distinct from the Bowman–Birk Inhibitors and active against H. armigera gut proteinases (HGP), from chickpea seeds. N-terminal sequencing of this HGP inhibitor revealed a sequence similar to reported pea (Pisum sativum) and chickpea -l-fucosidases and also homologous to legume Kunitz inhibitors. The identity was confirmed by matrix assisted laser desorption ionization – time of flight analysis of tryptic peptides and isolation of DNA sequence coding for the mature protein. Available sequence data showed that this protein forms a distinct phylogenetic cluster with Kunitz inhibitors from Glycine max, Medicago truncatula, P. sativum and Canavalia lineata. The isolated coding sequence was cloned into a yeast expression vector and produced as a recombinant protein in Pichia pastoris. -l-fucosidase activity was not detectable in purified or recombinant protein, by solution assays. The recombinant protein did not inhibit chymotrypsin or subtilisin activity but did exhibit stoichiometric inhibition of trypsin, comparable to soybean Kunitz trypsin inhibitor. The recombinant protein exhibited higher inhibition of total HGP activity as compared to soybean kunitz inhibitor, even though it preferentially inhibited HGP-trypsins. H. armigera larvae fed on inhibitor-incorporated artificial diet showed significant reduction in average larval weight after 18 days of feeding demonstrating potent antimetabolic activity. The over-expression of this gene in chickpea could act as an endogenous source of resistance to H. armigera.     

Diverse forms of Pin-II family proteinase inhibitors from Capsicum annuum adversely affect the growth and development of Helicoverpa armigera

Novel forms of Pin-II type proteinase inhibitor (PIs) cDNAs (CanPIs) having three or four inhibitory repeat domains (IRD) were isolated from the developing green fruits of Capsicum annuum. Deduced amino acid (aa) sequences of the CanPIs showed up to 15% sequence divergence among each other or reported inhibitors (CanPI-1AF039398, CanPI-2AF221097). Amino acid sequence analysis of these CanPIs revealed that three IRD PIs have trypsin inhibitory sites, while four IRD CanPIs have both trypsin and chymotrypsin inhibitory sites. Four CanPIs, two having three IRD (CanPI-3AY986465 and CanPI-5DQ005912) and two having four IRD (CanPI-7DQ005913 and CanPI-9DQ005915), were cloned in Pichia pastoris to express recombinant CanPIs. Recombinant CanPIs inhibited 90% of bovine trypsin (TI), while chymotrypsin inhibition (CI) varied with the number of chymotrypsin inhibitory sites in the CanPIs. Recombinant inhibitors inhibited over 70% of the gut proteinase activity of Helicoverpa armigera. H. armigera larvae fed on recombinant CanPIs individually incorporated into artificial diet, showed 35% mortality; in addition, weight gain in H. armigera larvae and pupae was severely reduced compared to controls. Of the four CanPIs, CanPI-7, which has two sites for TI and CI, was the only one to have a consistently antagonistic effect on H. armigera growth and development. We conclude that among the four recombinant PIs tested, CanPIs containing diverse IRDs are best suited for developing insect-resistant transgenic plants.     

Organellar and Cytosolic Localization of Four Phosphoribosyl Diphosphate Synthase Isozymes in Spinach

We report on the efficacy of proteinase inhibitors (PIs) from three host plants (chickpea [Cicer arietinum], pigeonpea [Cajanus cajan], and cotton [Gossypium arboreum]) and three non-host (groundnut [Arachis hypogea], winged bean [Psophocarpus tetragonolobus], and potato [Solanum tuberosum]) in retarding the growth of Helicoverpa armigera larvae, a devastating pest of important crop plants. Enzyme assays and electrophoretic analysis of interaction of H. armigera gut proteinases (HGPs) with PIs revealed that non-host PIs inhibited HGP activity efficiently whereas host PIs were ineffective. In the electrophoretic assay, trypsin inhibitor activity bands were detected in all of the host and non-host plants, but HGP inhibitor activity bands were present only in non-host plants (except cotton in the host plant group). H. armigera larvae reared on a diet containing non-host PIs showed growth retardation, a reduction in total and trypsin-like proteinase activity, and the production of inhibitor-insensitive proteinases. Electrophoretic analysis of PI-induced HGP showed differential regulation of proteinase isoforms. Interestingly, HGP activity induced in response to dietary potato PI-II was inhibited by winged bean PIs. The optimized combination of potato PI-II and winged bean PIs identified in the present study and their proposed successive use has potential in developing H. armigera-resistant transgenic plants.     

Isolation and in vitro identification of proteinase inhibitors from soybean seeds inhibiting helicoverpa gut proteases

Helicoverpa armigera is a polyphagous pest damaging vast numbers of different crops leading to decrease in total production. Use of Bt transgenic to control H. armigera has worked well but has increased resistance against Bt in H. armigera and controversies about the Bt transgenic making it imperative to find another strategy to control attack. Soybean is a nonhost plant for H. armigera; reason could be laid in the defense system of the soybean. Proteinase Inhibitor (PIs) have been extensively studied for development of resistance against insect pest. Two cultivars developed by our university were investigated for the presence of proteinase inhibitors namely, MAUS-158 and MAUS-61. Partially purified inhibitors were showed inhibition of total protease activity of gut extract by 91.34±1.49 and 89.95±0.96% by MAUS-158 and MAUS-61, respectively. While inhibition of trypsin like proteases were found between 65 and 71% and inhibition of chymotrypsin like proteases ranges between 40 and 42%. The partial purification study shows stability of PIs up to 60°C. Soybean PIs are also showing more prominent inhibition pattern against trypsin than chymotrypsin.     

In vivo and in vitro effect of Acacia nilotica seed proteinase inhibitors on Helicoverpa armigera (Hübner) larvae

Acacia nilotica proteinase inhibitor (AnPI) was isolated by ammonium sulphate precipitation followed by chromatography on DEAE-Sephadex A-25 and resulted in a purification of 10.68-fold with a 19.5 percentage yield. Electrophoretic analysis of purified AnPI protein resolved into a single band with molecular weight of approximately 18.6+1.00 kDa. AnPI had high stability at different pH values (2.0 to 10.0) except at pH 5.0 and are thermolabile beyond 80 degree C for 10 min. AnPI exhibited effective against total proteolytic activity and trypsin-like activity, but did not show any inhibitory effect on chymotrypsin activity of midgut of Helicoverpa armigera. The inhibition kinetics studies against H. armigera gut trypsin are of non-competitive type. AnPI had low affinity for H. armigera gut trypsin when compared to SBTI. The partially purified and purified PI proteins-incorporated test diets showed significant reduction in mean larval and pupal weight of H. armigera. The results provide important clues in designing strategies by using the proteinase inhibitors (PIs) from the A. nilotica that can be expressed in genetically engineered plants to confer resistance to H. armigera.     

Analysis of sequential accumulation of individual pigeonpea protease inhibitors during seed development

Seeds of pigeonpea are known to accumulate protease inhibitors (PIs), belonging to the Bowman-Burk inhibitor family. PIs are important for natural defense against phytophagous insect pests. Most insects attack crops at the early stages of seed development. Accumulation patterns of individual PIs and their relationship with each other were studied in developing seeds of 76 pigeonpea lines derived from BDN2 cultivar by ethyl methane sulfonate induced chemical mutagenesis. PIs extracted from these lines, collected between 10 and 40 days after flowering (DAF) and from mature seeds of BDN2 cultivar, were detected by using gel X-ray film contact print method. A total of 9 trypsin–chymotrypsin inhibitors were detected in mature seeds. All the nine PIs were capable of inhibiting proteases. Appearance of detectable levels of individual PIs started around 10 DAF. The PI-3 appeared early and was the most stable. It was accumulated in all the studied lines and was also detected in 84 % of the samples collected 10 DAF. In particular, 14 DAF, the individual PIs were accumulated and accumulation sequence was observed (PI-3, PI-2, PI-5, PI-7, PI-6, PI-4, PI-8, PI-9 and PI-1). Accumulation continued up to 40 DAF when seeds started hardening. Chemical mutagenesis could not produce any variation in the profile of individual PIs in the 76 studied lines in mature seeds. The process of accumulation of inhibitors is sturdy and mutagenesis fails to alter it. The robust mechanism is responsible for early appearance of PI-3. Early accumulated PIs in this study need further exploration for strengthening natural defense of pigeonpea against pests.     

Interaction of recombinant CanPIs with Helicoverpa armigera gut proteases reveals their processing patterns,stability and efficiency

Six diverse representative Capsicum annuum (common name: hot pepper; Solanaceae) protease inhibitor genes, viz CanPI‐5, ‐7, ‐13, ‐15, ‐19, and 22 comprising 1–4 inhibitory repeat domains (IRDs), were cloned and expressed in Pichia pastoris. The recombinant proteins were evaluated for their interactions with bovine trypsin, chymotrypsin, and Helicoverpa armigera gut proteases (HGP) using electrophoretic (native and denaturing) and mass spectrometric (MALDI‐TOF‐MS in combination with intensity fading assays) techniques. These techniques allow qualitative and semiquantitative analysis of multiple and processed IRDs of purified recombinant Capsicum annuum proteinase inhibitor (rCanPI) proteins. rCanPIs showed over 90% trypsin inhibition, varying chymotrypsin inhibition depending on the number of respective IRDs and over 60% inhibition of total HGP. rCanPI‐15 that has only one IRD showed exceptionally low inhibition of these proteases. Interaction studies of rCanPIs with proteases using intensity fading‐MALDI‐TOF‐MS revealed gradual processing of multi‐IRD rCanPIs into single IRD forms by the action of HGP at the linker region, unlike their interactions with trypsin and chymotrypsin. Intensity fading‐MALDI‐TOF‐MS assay showed that CanPI‐13 and ‐15, possessing single IRD and expressed predominantly in stem tissue are degraded by HGP; indicating their function other than defense. In vitro and in vivo studies on rCanPI‐5 and ‐7 showed maximum inhibition of HGP isoforms and their processed IRDs were also found to be stable in the presence of HGP. Even single amino acid variations in IRDs were found to change the HGP specificity like in the case of HGP‐8 inhibited only by IRD‐12. The presence of active PI in insect gut might be responsible for changed HGP profile. rCanPI‐5 and ‐7 enhanced HGP‐7, reduced HGP‐4, ‐5, ‐10 expression and new protease isoforms were induced. These results signify isoform complexity in plant PIs and insect proteases.     

Pod surface exudates of wild relatives of pigeonpea influence the feeding preference of the pod borer, Helicoverpa armigera

Wild relatives of crops are an important source of resistance genes against insect pests. However, it is important to identify the accessions of wild relatives of crops with different mechanisms of resistance to broaden the basis and increase the levels of resistance to insect pests. Therefore, we studied the feeding behavior of pod borer, Helicoverpa armigera, which is the most damaging pest of pigeonpea, in relation to biochemical characteristics of the pod surface exudates in a diverse array of germplasm accessions belonging to 12 species of pigeonpea wild relatives. Feeding by H. armigera larvae was significantly lower on the unwashed or water-, methanol-, or hexane-washed pods of Canajus sericeus, C. scarabaeoides, Flemingia bracteata, F. stricta, and Rhynchosia aurea than those of C. acutifolius, C. albicans, C. cajanifolius, C. lineatus, D. ferruginea, P. scariosa, R. bracteata, and the cultivated pigeonpea, C. cajan genotypes, ICPL 87, and ICPL 332, although there were a few exceptions. The methanol-washed pods of wild relatives were less preferred for feeding by the H. armigera larvae than the unwashed pods, but the hexane-washed pods were preferred more than the unwashed pods. The results suggested that methanol extracted the phagostimulants from the pod surface, while hexane removed the antifeedants. The high-performance liquid chromatography (HPLC) finger printing of methanol and hexane pod surface extracts showed qualitative and quantitative differences in compounds present on the pod surface of different wild relatives of pigeonpea. Some of the peaks in HPLC profiles were associated with feeding preference of the third-instar larvae of H. armigera. There was considerable diversity in wild relatives of pigeonpea as revealed by principal component analysis based on HPLC fingerprints of pod surface extracts in methanol and hexane, and H. armigera feeding on the pods. Wild pigeonpea accessions with low amounts of phagostimulants and high amounts of antifeedants may be used for introgression of resistance genes into the cultivated pigeonpea to develop varieties with broad-based resistance to H. armigera. There is considerable diversity among the wild relatives of pigeonpea, and the accessions with resistance to pod borer. These can be used to broaden the basis and increase the levels of resistance to H. armigera.     

Characterization of two midgut proteinases of Helicoverpa armigera and their interaction with proteinase inhibitors

Two serine proteinases from the midgut of Helicoverpa armigera have been partially purified and characterized. One proteinase, HGP-1, was capable of hydrolyzing a synthetic substrate of elastase and was inhibited by elastatinal. The second proteinase, HGP-2, was inhibited by a trypsin inhibitor. Molecular weights of HGP-1 and HGP-2 were approximately 26.0 and 29.0kDa, respectively. Both the proteinases exhibited alkaline pH optima in the range of 10-11. Furthermore, interaction of HGP-1 and HGP-2 with proteinase inhibitors (PIs) from host and non-host plants was studied. HGP-1 was not only insensitive to a PI from chickpea (host) but was also able to degrade it. The same PI from chickpea was able to inhibit over 50% activity of HGP-2. On the contrary, PIs from potato (non-host) showed strong inhibition of both, HGP-1 and HGP-2 and also demonstrated protection of chickpea seed proteins from digestion by both the HGPs. These results could provide important clues in designing strategies for sustainable use of plant PIs in developing insect-tolerant transgenic plants.