Automatic, rapid screening of seed resistance in cowpea, Vigna unguiculata (L.) Walpers, to the seed beetle Callosobruchus maculatus (F.) (Coleoptera: Bruchidae) using acoustic monitoring

The biomonitor technique was investigated as a rapid and automatic method for measuring the resistance of cowpea varieties to the seed beetle Callosobruchus maculatus. This technique measures the activity of internally feeding insect larvae by counting ultrasonic emissions produced as they feed. Activity throughout the development of C. maculatus larvae in known susceptible and resistant cowpea varieties was recorded. This showed details of the development of each larval instar, and showed clear differences between the resistant and susceptible cowpeas. A rapid method for comparing cowpeas was proposed in which the activity of larvae was recorded for 24 h starting 14 days after oviposition. Using this method, significant differences in activity were apparent between one susceptible and two resistant cowpea varieties. Further comparisons using a randomised block experimental design also showed a clear difference in activity between one susceptible and one known resistant cowpea variety. This experimental protocol took 21 days. It is suggested that the biomonitor offers a promising method for screening seeds for resistance to insect pests with a reduction in the time and effort required over conventional bioassay methods.     

Resistance of Vigna unguiculata (cowpea) seeds to Callosobruchus maculatus is restricted to cotyledonary tissues

We have investigated the survival of Callosobruchus maculatus larvae when reared on resistant IT81D 1045 Vigna unguiculata seeds, whose resistance has been associated with variant forms of vicilins. Here, we present data which show that larvae of C. maculatus feeding on embryonic axis of resistant cowpea reach a mass of around 28 times higher than those feeding on cotyledonary tissues. Additionally, incorporation of 5–10% of embryonic axis flour on artificial seeds made of resistant flour restores toxicity of seeds to the bruchid. Vicilins purified from both susceptible and resistant embryonic axis had no deleterious effects either on insect development or on insect survival until a level of 4% of incorporation. In contrast, vicilins from resistant cotyledons show an LD₅₀ (50% lethal dose) and WD₅₀ (half weight dose) of 2%. Total vicilin contents of embryonic axes were around two times lower than in cotyledonary tissues, while proteolytic activities of all four proteinase classes were always higher in the former tissues. By 2D‐PAGE we visualized eight protein spots, which seem to be exclusively found on resistant cotyledons. Copyright © 2006 Society of Chemical Industry     

Effects of cowpea varietal susceptibility and low pressure on the mortality of life stages of Callosobruchus maculatus (Coleoptera: Bruchidae)

Low pressure creates a low-oxygen controlled atmosphere that can kill all developmental stages of Callosobruchus maculatus (F.) (Coleoptera: Bruchidae). This study investigated the interaction of low pressure with seed variety to determine the susceptibility of different cowpea varieties to developmental stages of C. maculatus. Laboratory studies were performed to determine exposure periods to low pressure (32.5 ± 1.0 mm Hg) necessary to kill all life stages of C. maculatus on different cowpea varieties. The results indicated that with the exception of eggs, differences in exposure periods required to achieve 100% mortality varied with life stage and variety. The larval stages were the most susceptible, and larvae required shorter exposure periods to low pressure when reared on C. maculatus resistant varieties (24-125B-3 and IT89KD-288) than on susceptible varieties (CB-3, CB-5, CB-46 and UC-27). Analysis of variance (ANOVA) showed a significant interaction between the life stages of C. maculatus, exposure time and cowpea varieties on mortality. Integration of low pressure applications with storage of cowpea varieties resistant to C. maculatus has the potential of providing an alternative pest management tool to fumigants, which are sometimes used in protecting cowpea in storage from infestation by C. maculatus.     

Variation in the response of Callosobruchus maculatus (F.) to a resistant veriety of cowpea

A Nigerian cultivar of the cowpea has previously been identified as being less suitable than conventional varieties for the development of a local strain of C. maculatus (F.). The survival and duration of development of the immature stages of three strains of C. maculatus in the dried seeds of this cultivar were compared with the performance of the same strains of beetle on a susceptible variety. In all beetle strains, fewer larvae survived to produce adults, and the duration of development was significantly extended, in the resistant cowpea cultivar. A Brazilian strain produced fewest adults and a Nigerian strain had the longest mean development period. Combining all the data, it appeared that a strain from the Yemen Arab Republic was least influenced and that from Brazil most adversely affected by the resistant cowpea. Evidence is presented indicating that factors in both the cowpea seeds and the beetles themselves may contribute to the poorer performance of bruchids in the resistant as compared to the susceptible variety.     

Seed coat has no value in protecting cowpea seed against attack by Callosobruchus maculatus (F.)

Experiments were conducted to compare the oviposition preference of the cowpea seed beetle Callosobruchus maculatus (F.) on 22 cowpea varieties, with and without seed coat. The cowpeas included five resistant, four moderately resistant and 13 susceptible varieties. Ten of the varieties had smooth seed coats while 12 were wrinkled. Mean numbers of eggs laid on smooth and wrinkled varieties were not significantly different. The survival of the bruchid was also assessed on intact and decorticated seeds of five varieties, comprising two resistant varieties “Kanannado” and “IT89KD-288”, a moderately resistant variety “IT93K-513-2” and two susceptible varieties “IT87K-941-1” and “IT89KD-374-57”. There was no correlation of resistance level with the number of eggs oviposited on cowpea varieties. Under limited and free-choice conditions, cowpea seeds with intact seed coats were preferred to decorticated seeds for oviposition. Adult emergence, mean development periods and pre-adult mortality were similar when seed coats were removed or left intact. It is concluded that the seed coat may not be a useful aspect to consider for breeding of bruchid resistance into cowpea varieties.     

Biochemical basis of insect resistance in inged bean (Psophocarpus tetragonolobus) seeds

Mature seeds of the inged bean (Psophocarpus tetragonolobus) are toxic to developing larvae of a range of cosmopolitan storage Bruchidae of economic importance, including the copea seed eevil, Callosobruchus maculatus. Insect feeding trials ere carried out in hich protein fractions from seeds of inged bean ere incorporated at a range of concentrations into artificial seeds, and their effects upon development of C maculatus determined. Both albumin and globulin fractions ere toxic to the developing larvae and their toxicity correlated ith their haemagglutinating activity. Assay of Psophocarpin A, B and C fractions demonstrated Psophocarpin B to be the most insecticidal and to contain the highest haemagglutinating activity. Purified basic seed lectin as highly insecticidal to C maculatus larvae, ith an LC₅₀ value of c. 3·5 g kg^?1. The physiological level of this protein in inged bean seeds is sufficient to account for their resistance to attack by C maculatus. inged bean trypsin inhibitor as also purified and tested in artificial seeds against C maculatus. Hoever, even at concentrations in excess of tice the physiological concentration it had no deleterious effects upon development.     

Seed coat tannins and bruchid resistance in stored cowpea seeds

Seeds of wild species and varieties of Vigna were screened for their tannins and α‐amylase inhibitor contents as defensive compounds against cowpea weevil. Seed coats contained condensed tannins that were positively correlated to their colour but not to their resistance against the insect. The α‐amylase inhibitors were present in different amount in cotyledons of all species analysed. Amongst the cultivated lines assayed, Vigna unguiculata TVu 2027, an accession identified as moderately resistant, was found to contain the higher amount of α‐amylase inhibitor. When wild species were considered, V luteola and V vexillata (two resistant species) showed the highest content of α‐amylase inhibitors. In addition, two cultivated accessions (Vita 7 and IT 84E‐1‐108) of cowpea seeds, both classified as susceptible accessions, showing a different degree of bruchid damage in storage, were also analysed. No α‐amylase inhibitory activity was found in cotyledons of undamaged Vita 7 seeds, while the seed coat tannin content was found to be 13 times higher in undamaged Vita 7 seeds than in IT 84E‐1‐108 infested seeds. These latter results support the hypothesis that seed coat tannins must also be considered in biochemical defence mechanisms, which can deter, poison or starve bruchid larvae that feed on cowpea seeds. Copyright © 2004 Society of Chemical Industry     

Assessment of the antimetabolic effects of trypsin inhibitors from cowpea (Vigna unguiculata) and other legumes on development of the bruchid beetle Callosobruchus maculatus

Insect feeding trials were carried out whereby cowpea trypsin inhibitor (CPTI) was incorporated at various levels up to 10% in pelleted meals of both a susceptible variety of cowpea and another susceptible species of legume, the chickpea. The results confirmed that CPTI, prepared by affinity chromatography, is an effective antimetabolite against C. maculatus; furthermore, it is also effective when added to pelleted chickpea meals. Bioassays using soya bean trypsin inhibitor (SBTI) and lima bean trypsin inhibitor (LBTI) showed these protease inhibitors to be relatively ineffective. Methionine supplementation of meals of resistant cowpea resulted in increased adult survival from approximately 43 to 89% relative to the controls; addition of cysteine had a similar effect. Supplementation with both methionine and tryptophan, probably the next limiting amino acid in cowpea, did not increase adult survival any further. It is concluded that CPTI is an effective antimetabolite of C. maculatus and that this type of mechanism may be widespread. However, it relies upon a finely controlled balance within the host plant which has to make available sufficient nutrients for itself but insufficient to maintain predation since this example of resistance can be overcome by by-passing the limiting sulphur amino-acid block.     

Varietal effects of cowpea, Vigna unguiculata, on tolerance to malathion in Callosobruchus maculatus (Coleoptera: Bruchidae)

The impact of cowpea variety on the response of cowpea bruchid, Callosobruchus maculatus, to malathion was investigated. The interaction of six cowpea varieties (Adamawa Brown, Ife BPC, Ife Brown, Lilongwe, Ntcheu and NCRI-L25) with the geographical strains of C. maculatus (Brazil and Cameroon), temperature (23, 25, 27 °C) and insecticide concentration were considered. Cowpea variety (V) had an unpredictable effect on C. maculatus response to malathion. Bruchid populations produced by Ife BPC were the most susceptible to malathion while those yielded by NCRI-L25 were the most tolerant. Regardless of the cowpea variety, the Brazil strain showed higher tolerance than the Cameroon strain. There was significant effect of temperature (T) and insecticide concentration (C) on malathion tolerance in both strains (S). Likewise, there was significant impact of all two-way interactions on cowpea bruchid tolerance except V x C. Significant three-way interactions on C. maculatus tolerance to malathion was only observed in S × T × V and S × T × C. The predictability of changing one of the factors on the susceptibility of C. maculatus to insecticide was very low. This study suggests a need to take the insecticide tolerance of insect populations produced by novel varieties into account during plant breeding in addition to factors such as yield and resistance to insect and disease attack.     

Characterization and insecticidal properties of globulins and albumins from Luetzelburgia auriculata (Allemao) Ducke seeds towards Callosobruchus maculatus (F.) (Coleoptera: Bruchidae)

We evaluated the development of Callosobruchus maculatus growing in artificial seeds composed of Vigna unguiculata (cowpea) seed flour mixed with exogenous proteins from Luetzelburgia auriculata. Albumin and globulin fractions from Luetzelburgia auriculata were characterized in terms of protein content, amino acid composition and antimetabolic proteins (trypsin/chymotrypsin inhibitory, porcine pancreatic alpha-amylase inhibitory, lectin activity and presence of chitin-binding proteins). Both fractions were distinct in terms of protein content and diversity as determined by electrophoresis. Lectin activity was present only in the globulins. Neither fraction exhibited inhibitory activity towards porcine pancreatic alpha-amylase but trypsin inhibition was observed. Interestingly, chitin-binding proteins were detected in both protein classes. Albumins had a severe effect upon larval development and were detrimental to insect emergence (LD₅₀=0.4%) while globulins displayed slight toxicity upon larval development and no effect towards insect emergence. The presence of serine proteinase inhibitory activity and chitin binding proteins could explain, at least in part, the harmful effects on C. maculatus development while lectin activity and amino acid availability seem not to correlate with any deleterious effects. Luetzelburgia auriculata would be an interesting source of seed proteins to study behavior of C. maculatus upon infestation and genes coding for insecticidal proteins could become candidates for molecular biology programs devoted to producing transgenic seeds expressing resistance towards the beetle.     

A case for the standardization of protocols used in screening cowpea, Vigna unguiculata for resistance to Callosobruchus maculatus (Fabricius) (Coleoptera: Bruchidae)

Cowpea cultivars Ife Brown, Maiduguri-A, Maiduguri-B and TVu 2027, were screened for resistance to Callosobruchus maculatus (F.) attack using different seed and insect densities. The number of eggs laid on the seeds and most other demographic variables increased with increase in insect density (number of females per 40 seeds). Although the same trend was observed with increase in seed density (seeds per 8 females), this was less predictable. However, the number of eggs per seed decreased significantly as seed density increased, but increased with increase in female density. Adult emergence increased significantly with increase in both insect and seed density while the growth index did not vary. Significantly more eggs were laid on the seeds of TVu 2027 (the resistant control) than on all the other cultivars when 20 or 40 seeds were available to 8 females, or at the lowest insect density (2 females per 40 seeds). However, percentage adult emergence and growth were higher on Ife Brown (susceptible control) than on TVu 2027 and the two Maiduguri cultivars. Measurements of percentage adult emergence and growth index indicated clear cultivar differences at all seed and insect densities. Also, although the loss in weight of seed increased with increase in insect density and decreased significantly with increase in seed density, meaningful differences in resistance among test cultivars were not always apparent at all densities. Based on these findings, we propose using 40 seeds and 2 pairs of adult beetles in screening cowpea for resistance. In addition, we conclude that % adult emergence, growth index and % weight loss are the most reliable indicators for resistance of cowpea to damage by this insect. The need to standardize experimental procedures used for host plant resistance work on storage pests of cowpea is emphasized.     

Biochemical characterisation of α-amylase inhibitors from Achyranthes aspera and their interactions with digestive amylases of coleopteran and lepidopteran insects

BACKGROUND: Starchy seeds are an important food and a source of dietary ingredients in many countries. However, they suffer from extensive predation by bruchids (weevils) and other pests. α‐Amylase inhibitors are attractive candidates for the control of seed weevils, as these insects are highly dependent on starch as an energy source. RESULTS: A proteinaceous α‐amylase inhibitor from the seeds of Achyranthes aspera was identified, purified and characterised. In electrophoretic analysis, two prominent amylase inhibitor activity bands (AI1 and AI2) were detected. The inhibitor was purified 9.99‐fold with 1206.95 total amylase inhibitor units mg^?1 protein. The molecular weight of the purified inhibitor was around 6 kDa. The isolated α‐amylase inhibitor was found to be resistant to heat and proteolysis. Feeding analysis of Callosobruchus maculatus larvae on a diet containing seed powder of A. aspera revealed that survival of the larvae was severely affected, with the highest mortality rate occurring on the fifth day of feeding. The isolated inhibitor inhibited the majority of amylase isoforms of C. maculatus, Tribolium confusum and Helicoverpa armigera in electrophoretic analysis and solution assays. CONCLUSION: The information obtained in the present investigation could be useful for a genetic engineering approach that would make seeds resistant to storage pest infestations. Copyright © 2011 Society of Chemical Industry     

Cowpea resistance and bioactivity of two diatomaceous earths against Callosobruchus maculatus (F.) (Coleoptera: Chrysomelidae) on three cowpea varieties

The bruchid, Callosobruchus maculatus (F.) is the most important pest of stored cowpea in tropical climates. Experiments were conducted to evaluate the susceptibility of three cowpea varieties and the bioactivity of SilicoSec® and FossilShield® against this insect pest under ambient laboratory conditions at the contents 0, 0.5, 1, 1.5 and 2 g/kg of cowpea. The Dobie susceptibility index for cowpea genotypes was estimated to compare their relative tolerance to this insect pest. Adult mortality was assessed after 1, 2, 4 and 6 days of exposure followed by the evaluation of the F₁ progeny. Population increase, percentage seed damage and weight loss were assessed after three months of storage. The results showed that the CRPS and TN5-78 varieties were highly susceptible to C. maculatus, while Lori variety was moderately susceptible to the weevil. SilicoSec® and FossilShield® caused a significant and increasing bruchid mortality according to the dose rates and periods of exposure. Malathion 5% caused complete mortality after one day exposure time. SilicoSec® was more effective than FossilShield® on the three varieties of cowpea. The greatest dose of SilicoSec® and FossilShield® (2 g/kg) caused 100% mortality on all three varieties; 4 days of exposure in the Lori variety was the shortest exposure time leading to complete mortality. Regarding LD₅₀ and LD₉₅ values, it was observed that C. maculatus adults are more susceptible to SilicoSec® than to FossilShield®.The two formulations of diatomaceous earth (1.5 g/kg) reduced the F₁ progeny by more than 70%, and produced less than 10% of seed damage and weight loss for the three varieties of cowpea. The varietal resistance and the use of diatomaceous earth against C. maculatus in storage could represent an alternative to Malagrain® for the protection of the cowpea.     

Trehalose and proline failed to enhance cold tolerance of the cowpea weevil,Callosobruchus maculatus (F.) (Col.: Bruchidae)

The cowpea weevil, Callosobruchus maculatus (F.) (Col.: Bruchidae) is a cosmopolitan field-to-store pest ranked as the major post-harvest pest of cowpea in tropical regions. The cold tolerance of an insect species can vary as a result of abiotic features including food resources. In this study, C. maculatus larvae were fed with proline and trehalose (10, 20, and 40 mmol) treated cowpea seeds to determine the effects of these potential cryoprotectants on the supercooling (SCP) and cold hardiness of the upcoming adult beetles. The SCPs of the control, proline-fed and trehalose-fed adults non-significantly changed from −18.2 °C for the control to −17.2 °C for trehalose-fed adults. The cold hardiness (24 h at 0, -5.0, −7.5, −10.0, and −12.5 °C) of the adults was almost the same for control and treatments. Median lethal times (LT₅₀; lethal time for 50% mortality) were 6.3, 6.0, and 5.4 h, respectively. Moreover, feeding the larvae with proline and trehalose-treated seeds did not affect the proline and trehalose contents of the adult beetles. Our results showed that C. maculatus could not tolerate subzero temperatures well above their SCP, indicating that this species might be a chill-susceptible insect.     

PURIFICATION AND PARTIAL CHARACTERIZATION OF A PROTEINASE INHIBITOR FROM TEPARY BEAN (PHASEOLUS ACUTIFOLIUS) SEEDS

In a qualitative screening of 36 accessions of tepary beans seeds, all the accessions inhibit the activity of bovine trypsin and trypsin-like proteinases from the insect P. truncatus, and the majority of them inhibit α-amylase activity of several important insect pests. A protein proteinase inhibitor was purified from accession L-242-45, using fractional precipitation, gel filtration, ion exchange chromatography and reverse-phase HPLC. The protein showed an apparent molecular weight of 7,100 by PAGE. However, contrary to other inhibitors previously reported, the inhibitory activity was only present in the trimeric form. The protein was characterized as a serine-proteinase inhibitor that recognized trypsin, chymotrypsin and trypsin-like proteinases, but it also recognized aspartic acid proteinases from different insects. It contained no carbohydrate residues and showed a high stability at 96C at low pH.     

Legume seed vicilins (7S storage proteins) interfere with the development of the cowpea weevil (Callosobruchus maculatus (F))

Vicilins (7S storage proteins) isolated from the seeds of the legumes Vigna unguiculata (cowpea), Vigna angularis (adzuki bean), Canavalia ensiformis (jack bean), Glycine max (soybean), Phaseolus vulgaris (common bean) and Phaseolus lunatus (lima bean) were shown to be immunologically related and to bind to a chitin matrix. The effect of the isolated vicilins on the development of the cowpea weevil Callosobruchus maculatus was examined. Vicilins from all non-host seeds, including those of the C maculatus-resistant cowpea line IT81D-1045, strongly inhibited larval development (ED₅₀ of 1·07±0·32% to 2·22±0·64%). Vicilins from the C maculatus-susceptible cowpea CE-31 and adzuki bean seeds were the exception with ED₅₀ of 6·25±0·75% and 5·40±1·54%, respectively. These results correlate well with the host range of C maculatus and are possibly a reflection of the low digestibility of vicilins by insect midgut proteinases in addition to the ability they show in binding to chitin-containing structures like the ones found in the bruchid midguts. © 1998 SCI.     

Characterization of new sources of mungbean (Vigna radiata (L.) Wilczek) resistance to bruchids, Callosobruchus spp. (Coleoptera: Bruchidae)

The bruchid beetles Callosobruchus chinensis (L.) and C. maculatus (F.) are destructive pests of stored mungbean (Vigna radiata (L.) Wilczek). The development of resistant mungbean varieties to manage bruchids is a major breeding objective. In the present study, we investigated the characteristics of resistance to C. chinensis and C. maculatus in two new resistant mungbean accessions V1128 and V2817, and two previously reported resistant accessions V2709 and V2802, compared to the susceptible mungbean cultivar, KPS1. Evaluation for resistance to both bruchid species using whole and decorticated seeds revealed that V1128 and V2817 were free from damage, V2709 and V2802 showed partial damage with low or moderate number of bruchids emerging from seeds, and KPS1 showed complete damage with the highest number of bruchids emerged. Comparison of the seeds harvested at seed filling, early maturity and full maturity stages revealed that the percentage of damaged seeds from resistant accessions was lower at all stages compared with KPS1. V1128 and V2817 showed complete resistance against both bruchids regardless of when their seeds were harvested, while resistance in V2709 and V2802 were most pronounced at full maturity, and KPS1 was totally damaged at all times tested. These results suggest that the chemical factor(s) conferring resistance is synthesized as early as the seed filling stage. Evaluation of resistance using artificial seeds showed that increasing the percentage of resistant seed powder adversely affected bruchid growth and development. The number of adults emerging from seeds and number of damaged seeds decreased while adult developmental period increased as the proportion of resistant seed powder increased. The weight of emerging male and female adults of C. maculatus was lighter than those from the seeds containing susceptible seed powder alone. However, C. chinensis adults were not affected by the same test. The results suggest that biochemical(s) in cotyledon tissue are responsible for the resistance and the seed coat had no protective role against the bruchids. Although all four resistant accessions evaluated are useful for mungbean breeding, V1128 and V2817 show complete resistance to both C. chinensis and C. maculatus. Thus, these two new resistant sources may be the most effective for breeding purposes.     

The effects of α-amylase inhibitors on insect storage pests: Inhibition of α-amylase in vitro and effects on development in vivo

Protein α-amylase inhibitors were prepared from wheat and their effects tested against insect storage pests both in vitro against the insect α-amylases and in vivo in insect feeding trials. Inhibitor fraction A was found to inhibit porcine pancreatic α-amylase but not insect α-amylases, whereas fractions B, C and D (0.28) did not inhibit porcine pancreatic α-amylase but were strong inhibitors of digestive α-amylases from larvae of Tribolium confusum, a storage pest of wheat products, and Callosobruchus maculatus, a storage pest of legume seeds. Fraction D, which was a single polypeptide of M_r 13 000 was the most effective inhibitor in vitro. It would appear that the degree of inhibition by the wheat α-amylase inhibitor preparations can be correlated with the presence of the M_r 13 000 (0.28) polypeptide since the purer this polypeptide the stronger was the inhibition; fraction A which contained two polypeptides of M_r 60 000 and 58 000 caused no inhibition. The effects of fractions B and C on larval development were determined in insect feeding trials. With C. maculatus both fractions were toxic, their relative effectiveness being directly paralleled by their effectiveness observed in vitro. Only fraction C was tested against T. confusum in feeding trials. Despite this fraction being equally effective against both pests in vitro it had very little effect upon larval development of T. confusum in vivo, thus suggesting that this organism is able to detoxify the wheat α-amylase inhibitors. As far as the authors are aware, this is the first time that the effects of identified inhibitor fractions have been monitored both in vitro and in vivo. The results, in contrast to previous proposals, suggest that selecting wheat varieties for high α-amylase inhibitory activity may not be a very reliable criterion in selecting for insect resistance.     

Identification,biochemical characterization and biological role of defense proteins from common bean genotypes seeds in response to Callosobruchus maculatus infestation

Common bean is a legume of significant socioeconomic importance and is cultivated worldwide. This crop is affected by several pests and diseases, which cause considerable economic losses and reduce yield. In recent years, several studies have demonstrated the role of proteins and peptides with activity against a wide range of insects and pathogens. The objective of this work was to identify defense proteins, such as antimicrobial peptides, protease and amylases inhibitors in common bean genotypes and evaluate the relationship of these proteins with Phaseolus vulgaris seed resistance to Callosobruchus maculatus infestation. Nineteen common bean genotypes were subjected to protein extraction, pH 5.4, and precipitation with ammonium sulfate at 70% saturation. The obtained extracts were separated by tricine gel electrophoresis. Experiments were carried out with natural seeds of common beans and artificial seeds (Vigna unguiculata seeds covered with seed coats of common beans) to evaluate the rate of oviposition and development of the insect species Callosobruchus maculatus. Lipid-transfer proteins were identified in nine genotypes whereas defensins were present in all genotypes. The inhibitory activity of α-amylases and trypsin and fungal development were determined in crude extracts (50 μg mL⁻¹). The results also indicated that the extracts from all bean genotypes inhibited the activity of human salivary α-amylase and C. maculatus larval α-amylase. Except for the extracts of four genotypes, all other extracts inhibited trypsin activity. None of the extracts from the evaluated bean genotypes inhibited the growth of tested fungi. Natural seeds from all genotypes did not inhibit insect oviposition, however, the larvae did not survive after feeding on these seeds. Artificial seeds containing seed coat flour of all genotypes inhibited the oviposition of C. maculatus, indicating that the seed coat was also repellent to insect.     

Detection and partial characterisation of subtilisin inhibitors in legume seeds by isoelectric focusing

Seed extracts of ten legume species were subjected to isoelectric focusing in acrylamide gels and inhibitors of subtilisin, three other bacterial proteases, trypsin and chymotrypsin were detected by a negative-staining technique based on the chromogenic substrate, acetyl-D, L-phenylalanine-2-naphthylester. In addition to complex patterns of trypsin and chymotrypsin inhibitor zones, most legume seeds examined exhibited one major and one minor inhibitor of subtilisin, and the other bacterial proteases. However, in cowpea and black gram only the major, and in lentil and soya bean only two minor subtilisin inhibitor zones were detected. Isoelectric points of the subtilisin inhibitors range from 4.4 to 5.9. The inhibitor patterns obtained by isoelectric focusing of extracts mixed with bacterial protease confirmed that inhibitors of the bacterial proteases represent a new type of inhibitor different from the well characterised trypsin and chymotrypsin inhibitors.