Deterrent and insecticidal properties of bean seed (Phaseolus vulgaris L.) whole meal or protein extract incorporated into the diet of Callosobruchus maculatus (F.) (Coleoptera: Bruchidae)

Callosobruchus maculatus, a pest that causes serious damage to chickpea Cicer arietinum, cannot develop in the seeds of Phaseolus or Vigna spp. which contain lectins. The insecticidal activity towards C. maculatus in these seeds is attributed both to lectins with specific affinity to N-acetylglucosamine, the major component of insect chitin, and to alpha-amylase inhibitors (lectin-like proteins). The insecticidal properties of bean meal or bean protein extracts from different sources towards different pest species are variable and need to be experimentally evaluated. The main objective of this study was to determine through a feeding trial on artificial chickpea seed the potential of bean seed meal from a wild bean Vigna caracalla, four varieties of Phaseolus vulgaris, and of a protein extract of P. vulgaris seed, to alter different life history traits of C. maculatus. The chickpea weevil was set up on artificial chickpea seeds containing different amounts of bean meal to observe the effects on female oviposition, percentage of development to adulthood and juvenile development time. These traits were combined in a composite index measuring the alteration of the multiplication rate of C. maculatus fed on artificial seed. The activity of lectin-like extracts was observed on chickpea artificial seed spiked with bean seed extract. Incorporation of bean flour at a rate of 10 and 20% in chickpea artificial seed significantly decreased C. maculatus female fecundity, percentage of adult emergence, and greatly increased the development time. Feeding trials with protein extracts of P. vulgaris reduced fecundity and survival of C. maculatus. Incorporation of 10% V. caracalla bean seed meal in chickpea artificial seed, reduced the multiplication potential of C. maculatus by over 90% showing that bean seed lectin extracts are worthy of further investigation for post-harvest infestation control.     

Bioactivity of endosperm extracts from susceptible and resistant bean seeds against Callosobruchus maculatus

Due to their high nutritional value, seeds are often the target of herbivory. In response, plants have evolved a host of adaptations that physically and/or chemically reduce the likelihood of granivory. In grain legumes, the main chemical defences within the endosperm derive from anti-nutritional proteins such as lectins and protease inhibitors. However, less is known about the bioactivity of secondary metabolites found within seeds. Here, solvent extraction was used to identify the major classes of phytochemicals found within the endosperm of both resistant beans (Phaseolus vulgaris) and susceptible beans (Vigna unguiculata). Phenols and terpenoids were recovered from the resistant P. vulgaris seeds only, whilst glycosides and flavonoids were recovered in greater amounts from P. vulgaris in comparison to V. unguiculata. To assess the bioactivity of the extracts against the seed beetle, Callosobruchus maculatus, the extracts were incorporated into artificial seeds and key life-history traits of the beetle determined. The results show the endosperm of both resistant and susceptible beans to contain an ensemble of secondary metabolites that deter oviposition (antixenosis) by adult beetles and disrupt the survival and development of the larvae (antibiosis). However, no extract resulted in complete resistance, indicating that the phytochemicals function alongside other anti-herbivory mechanisms such as α-amylase inhibitors and protease inhibitors. Breeding programmes could potentially select for key phytochemical groups such as terpenoids or glycosides in cultivars in order to optimise the trade-off between resistance and agronomic quality by harnessing the anti-herbivory properties of secondary metabolites in conjunction with anti-nutritional proteins.     

Seed coat phytochemistry of both resistant and susceptible seeds affords some protection against the granivorous beetle Callosobruchus maculatus

The seed coat lies at the interface between the internal structures of the seed and the external environment and thus represents a key arena in the study of seed-herbivore interactions. Callosobruchus maculatus is a cosmopolitan pest of legume seeds, and under post-harvest conditions, females interact directly with the seed testa prior to laying their eggs. Here we investigate the effect of chemical extracts of the seed coat of the resistant Phaseolus vulgaris and the susceptible Vigna unguiculata beans on egg laying preferences and larval development of C. maculatus. Seed coat extracts contained phenolic, glycoside and alkaloid compounds. Upon re-incorporation of extracts into artificial host beans it was found that that several seed coat extracts from both the resistant and susceptible varieties reduced female oviposition and disrupted larval growth and development. However, none of the extracts assayed resulted in complete ovipositional or developmental failure suggesting that complete resistance in P. vulgaris is derived from other physical or chemical properties of the seed and/or seed coat that function either alone or synergistically. Further work is required to elucidate the importance of synergistic interactions between different physiological defence mechanisms on overall plant (seed) resistance.     

Rice bean (Vigna umbellata Thunb. Ohwi and Ohashi) protection against Callosobruchus maculatus F.” by presence of protein profile

To identify defence responses of rice bean against Callosobruchus maculatus, 10 genotypes of rice bean and a mungbean variety, PAU 911 as susceptible check were evaluated. The rice bean genotypes were found to restrain the growth and development of C. maculatus at different larval stages. To justify these results, seeds of different test genotypes were examined for the presence of plant protease inhibitors which inhibit insect proteases causing reduction in the availability of amino acids necessary for their growth and development. Trypsin, chymotrypsin and cysteine protease inhibitor content was significantly higher in rice bean genotypes which ranged from 299.99 to 342.97 TIU g⁻¹ seeds, 235.07 to 251.45 CIU/g seeds and 1060.70 to 1574.99 CPIU/g seeds, respectively as compared to mungbean variety, PAU 911 (235.52 TIU/g seeds, 217.91 CIU/g seeds and 499.99 CPIU/g seeds, respectively). The protein profile showed a polypeptide band of 25–30 kDa corresponding to presence of protease inhibitors in rice bean genotypes except PAU 911. The results indicate that protease inhibitors in different genotypes of rice bean are very effective against C. maculatus.     

Correlation between biophysical seed characteristics of rice bean,Vigna umbellata (Fabaceae: Faboideae: Phaseoleae) and the development of Callosobruchus maculatus (Coleoptera: Chrysomelidae: Bruchinae)

The bruchid, Callosobruchus maculatus is a serious pest of stored pulses. Four biophysical seed characteristics viz. Seed size, texture of seed surface, thickness of seed coat and seed hardness of 10 genotypes of rice bean, a wild pulse crop were investigated for assessing mechanism of resistance against C. maculatus. Oviposition preference and larval penetration in seed coat did not differ significantly among different test genotypes. Highest number of larvae which developed up to last larval/pupal instar were observed in susceptible check variety of mungbean, PAU 911 (30.00) and lowest in rice bean genotype, LRB 535 (0.83). Highest and lowest seed weight was noticed in rice bean genotype, LRB 529 (6.81 g) and PAU 911 (3.88 g), respectively. Almost similar seed coat thickness was recorded among different test genotypes. Maximum seed hardness was noted in genotype LRB 507 (6.45 kg) whereas minimum in PAU 911 (2.00 kg) among all test genotypes. Any remarkable correlation of biophysical seed characteristics of test genotypes with ovipositon, larval penetration and development of C. maculatus was not observed. Thus, biophysical seed characteristics of rice bean did not suggest to play a vital role in imparting resistance against C. maculatus.     

Oviposition preference and development of azuki bean weevil,Callosobruchus chinensis,on five different leguminous seeds

This study was conducted to determine the oviposition preference and development of azuki bean weevil, Callosobruchus chinensis (L.) (Coleoptera: Bruchidae) on five different leguminous seeds a) cowpea cultivar Yeonbundongbu, b) white kidney bean cultivar Hwanghyubilho, c) soybean cultivar Daepung, d) mung bean cultivar Seonhwanogdu, and e) azuki bean cultivar Hongeon. A multiple-choice test and a no-choice test were conducted to record the number of eggs laid, total developmental time, number of adults emerged, and adult longevity of C. chinensis on the five different leguminous seeds. Both choice and no-choice tests revealed cowpea seed as the most preferred oviposition substrate. Total developmental time from egg to adult stage ranged from 28 to 31 days; developmental time was shortest on the cowpea. Successful development of C. chinensis was not observed on soybean or on kidney bean. Rate of adult emergence was higher on azuki and mung bean seeds with longer adult longevity of those emerged from azuki bean and cowpea. Higher egg laying preference and shorter developmental time with longer adult longevity of the beetle on cowpea probably signifies importance of physical as well as chemical attributes of the seed as an oviposition substrate other than evolutionary history of the insect.     

The inhibition of digestive enzymes by extracts of field bean (Vicia faba).

Protease inhibitor activity was determined for a wide range of differing field bean (Vicia faba) varieties and genotypes. Although a two-fold variation was observed under the assay conditions used, the highest values were approximately five times less active in inhibiting trypsin than either soya or navy beans. No correlation was found between protein content and protease inhibitor activity. Water extracts from the testa from coloured-flower varieties inhibited the activity of α-amylase, lipase and trypsin. The addition of polyvinylpyrrolidone reactivated these enzymes to varying degrees. No inhibitory activity was found in similar extracts from white-flowered varieties. It was concluded that the enzyme inhibition exhibited by extracts of the seed coat of coloured varieties was due to the presence of tannins.     

α-Amylase inhibitor levels in seeds generally available in Europe

The α-amylase inhibitor (αAI) levels in 18 seeds, which are generally available throughout Europe, have been determined. Kidney beans, haricot beans, pinto beans and runner beans had high contents of αAI (2–4 g equivalent kg^?1 seed meal). Butter beans, blackeyed peas, chickpeas, field beans and sweet lupinseeds contained 0.1–0.2 g inhibitor equivalent kg^?1 seed meal. No αAI activity was detected in samples of adzuki bean, lentils, mung beans, peas, soya beans, sunflower seeds or winged beans. The αAI activity present in whole kidney beans was relatively heat-resistant. However, it could be completely abolished by aqueous heat treatment of fully imbibed beans at 100°C for 5–10 min.     

Evaluating the combination of a parasitoid and a predator for biological control of seed beetles (Chrysomelidae: Bruchinae) in stored beans

The two seed beetle species Acanthoscelides obtectus and Callosobruchus maculatus are among the economically most important pests of stored legume seeds world-wide. Insect natural enemies for biological control were considered in IPM strategies to control bean weevils in store. In this contribution, the control effect by a combination of the parasitoid Anisopteromalus calandrae and the predator Xylocoris flavipes on both seed beetle species was evaluated in a laboratory study on black eyed beans Vigna unguiculata. The combination of parasitoid and predator was most effective against both species of seed beetles. However, in C. maculatus the combination was not significantly more effective compared to A. calandrae alone, suggesting a poor effect on adults and no effect on eggs and larvae by X. flavipes. In the case of A. obtectus, the suppressive effect by the combination of the parasitoid and the predator was high. For C. maculatus, germination of black eyed beans was approximately three times higher when both natural enemies were present. The combination of A. calandrae and X. flavipes is considered a promising component for integrated control of A. obtectus.     

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.     

Host specificity in the Bruchidae—VII. The effect of carbohydrate composition on varietal resistance of garden peas to Callosobruchus chinensis L.

Sources of resistance to Callosobruchus chinensis (L.) are found in commercial Pisum sativum L. seeds. Resistance differs in wrinkled peas compared to smooth pea varieties. Wrinkled pea starch is high in amylose, and of poor nutritional value to C. chinensis. In smooth varieties, starch composition is not a limiting factor. Raffinose and total sugar content is linearly correlated to susceptibility. The insect may be utilizing seed amylase activity, in addition to its own α-amylase, for initial digestion of starch granules in the intact seed.     

Impregnated bags for safer storage of legume grains in West and Central Africa

The use of jute bags impregnated with aqueous extracts from two insecticidal plants, Chenopodium ambrosioides and Lantana camara, to reduce damage to stored legume seeds by two major bruchid insect pests, Acanthoscelides obtectus and Callosobruchus maculatus, was compared with a direct seed-treatment method using plant powders. In the first method bags were treated with a 10% concentration of the aqueous plant extract and filled with bean or cowpea seeds, whereas in the second method seeds were thoroughly mixed with leaf powder from the same plants at the rate of 4% (w/w). The bag-treatment method significantly reduced seed damage compared with the untreated control after 6 months of storage (4- to 6-fold decrease in percentage seed damage). Even though relatively low (? 20%), damage by the two insect species controlled by this method was significantly higher than the damage recorded in the seed-treatment method. Orthogonal contrast analysis across test insects showed a significant seed damage difference between treatments with C. ambrosioides and those with L. camara, the latter having the lowest damage profile. The biosafety implication of the use of plant extract-impregnated bags for the protection of food grains against damage by storage bruchid pests is highlighted.     

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.     

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.     

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.     

Storage of mung bean (Vigna radiata [L.] Wilczek) and pigeonpea grains (Cajanus cajan [L.] Millsp) in hermetic triple-layer bags stops losses caused by Callosobruchus maculatus (F.) (Coleoptera: Bruchidae)

Dry mung bean and pigeonpea grains that had sustained some insect damage but fumigated before the start of the experiment were stored in triple-layer hermetic bags (Purdue Improved Crop Storage (PICS™ bags) or woven polypropylene (PP) bags for 6 months. Some of the bags were artificially infested with cowpea bruchid Callosobruchus maculatus (F.) (PICS1, PP1) while others were not (PICS0, PP0). In an additional trial, PP bags containing the grains were treated with Actellic Super^® dust before being artificially infested (PP1Ac). Moisture content, live adult C. maculatus count, grain damage, weight loss, and seed germination were determined on a monthly basis. At six months, moisture contents of grain stored in PICS and PP bags remained below 12%. Storage in PICS bags halted multiplication of C. maculatus, and the initial damage level and weight of grains did not change. Conversely, in the PP bags, C. maculatus populations increased massively and seed damage reached 71.8 ± 1.9%, 76.9 ± 0.4%, and 60.3 ± 0.6% corresponding to weight losses of 14.5 ± 0.1%, 16.5 ± 0.2% and 12.5 ± 0.1% in PP0, PP1 and PP1Ac, respectively, in mung beans. With the pigeonpeas, seed damage reached 55.1 ± 0.6%, 95.7 ± 0.4% and 75.8 ± 0.9%, corresponding to weight losses of 13.0% ± 0.3%, 26.2 ± 0.2% and 13.5 ± 0.1%, in PP0, PP1 and PP1Ac, respectively. PICS bags are an effective tool for preserving mung beans and pigeonpeas against C. maculatus attack, and their performance is superior to that of Actellic Super^® dust.     

Resistance screening of 14 Cajanus cajan (L.) Millsp. cultivars to Callosobruchus maculatus (F.) (Coleoptera:Bruchidae)

Pigeonpea, Cajanus cajan, is a rich source of human dietary protein and carbohydrate as well as an income generator for farmers especially in developing countries. However, a major limiting factor to the viable production and post-harvest preservation of pigeonpea is infestation by the cowpea weevil, Callosobruchus maculatus. An experiment to screen for resistance in 14 newly developed C. cajan cultivars from The University of the West Indies (UWI) was conducted. The experiment compared oviposition preference based on eight parameters, namely, testa thickness, testa hardness, seed sphericity, surface area, total adult emergence, larval + pupal mortality, seed hardness and surface roughness. Four of the pigeon pea cultivars screened were resistant and could be ranked in the order of decreasing resistance as: A16 = A66PL1> A112 > A196PL2. Seed surface roughness, seed testa hardness and seed grain hardness were all found to play a major role in oviposition deterrence with seed roughness being the most effective deterrent. The results suggest that breeding for pigeonpea cultivars with rougher seed coats, harder seed testa and harder grains could enhance pigeonpea seed resistance to C. maculatus by reducing infestation and damage while in storage.     

Effect of monoterpenoids on oviposition and mortality of Callosobruchus maculatus (F.) (Coleoptera: Bruchidae) under hermetic conditions

Monoterpeniods have been shown to cause mortality in certain stored-product insect pests. The current report investigated the prospects of using monoterpenoids as oviposition deterrents of the cowpea beetle, Callosobruchus maculatus (Fabricius), as well as in the management of populations of the beetle. The monoterpenoids investigated include E-anethole, estragole, S-carvone, linalool, L-fenchone, geraniol, γ-terpinene and DL-camphor, and at the concentrations of 66.7, 33.3, 16.7, 8.33 and 0 μL/L. Exposure of the life stages of the beetle, which included eggs, young larvae (first instar), 4th instar, pupae and adults to different concentrations of the monoterpenoids over 24 h period caused varying levels of mortality. The stages of the beetle that were the least susceptible to the monoterpenoids were the 4th instar, and the pupae, which required high concentrations of the monoterpenoids to achieve 99% mortality. The adults and the eggs exhibited the highest susceptibility to the monoterpenoids. Mated C. maculatus females that were offered cowpea seeds upon treatment with low doses (8.33 μL/L) of the monoterpenoids did not lay eggs, while control female beetles offered untreated seeds laid several eggs. However, mated C. maculatus females laid eggs on cowpea seeds treated with monoterpenoids 3 weeks before to the day of experimentation. The monoterpenoids did not exhibit residual toxicity to the cowpea beetles. These monoterpenoids could be further investigated for the postharvest management of seed beetles of grain legumes.     

The short-chain fatty acids as potential protective agents against Callosobruchus maculatus infestation

The cowpea weevil, Callosobruchus maculatus, is one of the most common pests of stored legumes. Its occurrence adversely affects the quality of stored beans, making them unfit for consumption, resulting in substantial financial losses. The aim of this study was to investigate the potential insecticidal properties of the volatile fatty acids (VFAs) (C1 – C5) and their influence on the insect’s physiology and behavior. All VFAs in concentrations equal to 4  μl and 8  μl showed fumigant toxicity significantly higher from the control. The strongest effect was observed in the case of propionic and valeric acid in volume of 4  μl and 8  μl, where mortality was close to 100%. Except for butyric acid, all acids showed a significant repellent effect. Additionally, all VFAs significantly decreased the number of infested beans and influenced the locomotor activity. Of all tested acids only the formic acid did not affect the oxygen consumption of the insects. As the studied VFAs have noteworthy properties against C. maculatus, they could be considered as promising agents in new strategies for stored products pest management.     

Biochemical basis of insect resistance in Vigna unguiculata

The bruchid beetle Callosobruchus maculatus (F.) causes extensive damage to seeds of the cowpea, Vigna unguiculata (L.) (Walp.), when this important tropical foodstuff is stored. A variety of cowpea resistant to attack by this pest has been described. In the present work seeds of a number of cowpea varieties, including the resistant one, were tested for the presence of a physical resistance to C. maculatus, in terms of repulsion of oviposition or of failure of larvae to enter the seeds. No evidence to suggest the presence of a physical resistance was found. When seeds of cowpea varieties were tested for the presence of various antimetabolic secondary compounds, only inhibitory activity against trypsin and, to a much lesser extent, chymotrypsin, could be detected. The resistant variety of cowpea contained a significantly higher level of inhibitors, about twice as much as any other variety. A proteinase inhibitor active against trypsin was purified from cowpea varieties by affinity chromatography on trypsin-Sepharose. The purified inhibitor was shown to inhibit chyraotrypsin also, in such proportions as to account for chymotrypsin inhibition by seed extracts. The inhibitor was shown to consist of a number of isoinhibitors by gel electrophoresis and isoelectric focusing, but no qualitative differences in the inhibitor between varieties could be detected. The antimetabolic nature of the cowpea trypsin inhibitor was confirmed by insect feeding trials in which various protein fractions were added to a basic meal and the effect on larval survival noted. The albumin proteins of cowpea (containing the trypsin inhibitors) at a level of 10% were toxic to larvae of C. maculatus whereas the globulin fractions were not. Further, if cowpea trypsin inhibitor was removed from the albumin proteins they ceased to be toxic. When purified cowpea trypsin inhibitor was added to the basic meal it was shown that a level slightly less than that found in the resistant variety of cowpea caused complete mortality of larvae, whereas lower levels had lesser or no effect. It is concluded that this example of insect resistance in the cowpea is due to an elevated level of trypsin inhibitor.