Performance of bean bruchids Callosobruchus maculatus and Zabrotes subfasciatus (Coleoptera: Bruchidae) reared on resistant (IT81D-1045) and susceptible (Epace 10) Vigna unguiculata seeds: relationship with trypsin inhibitor and vicilin excretion

Callosobruchus maculatus (Cm) and Zabrotes subfasciatus (Zs) were reared on resistant (IT81D-1045) and on susceptible (Epace 10) cowpea seeds. The emergence of adult insects, total developmental period (TDP) and excretion of trypsin inhibitor and vicilin were determined for both bruchid populations. Parameter evaluation showed that the Zs populations emerged from both seeds had no significant differences in emergence and TDP. The Cm population raised from resistant seeds had lower emergence (5.6+/-1.3%) and delayed TDP (46+/-1.25 days) than those emerged from susceptible seeds. The excretion of defense proteins showed that Zs reared in resistant seeds excreted 1.7 times more trypsin inhibitor, but this did not affect emergence or TDP. Furthermore, Cm population emerged from resistant seeds excreted 7 times higher vicilin and 0.4 times less trypsin inhibitor than that emerged from susceptible seeds. These results indicate that vicilins from resistant seeds are involved to significantly longer TDP (46 days) and also drastic reduction of insect emergence ( approximately 5%) of C. maculatus.     

Reproduction and oviposition preferences of Zabrotes subfasciatus stocks reared from two host plant species

The reproductive activity of a specialist bruchid (Zabrotes subfasciatus Boh., Coleoptera) was studied in two beetle stocks obtained from the same area. The first was harvested on the insect's wild host plant, Phaseolus lunatus L. The second was collected on a cultivated host plant, P. vulgaris L.Within each of the two insect stocks, fecundities and ovarian productions were similar whether females were offered seeds of the host plant on which they completed their larval development or an alternative host. When given the choice of host plants, females of both stock oviposited preferentially on P. vulgaris. The substrate used for larval development did not influence the choice of subsequent oviposition sites.There appears to be no behavioural or physiological barriers preventing a single population of Z. subfasciatus from reproducing on both wild and cultivated host plants where they grow together.

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Reproduction et choix du lieu de ponte de Zabrotes subfasciatus en présence de plantes hôtes sauvages et cultivées

Résumé L'activité reproductrice d'un insecte spécialiste (Zabrotes subfasciatus) est étudiée sur deux lots d'individus provenant de la même zone. Le premier a été récolté sur une plante hôte sauvage de l'insecte, Phaseolus lunatus L., et le deuxième sur une plante hôte cultivée, P. vulgaris L.La fécondité, la production ovarienne et les préférences d'oviposition des femelles en présence de graines de P. lunatus ou de P. vulgaris ou en présence de graines des deux plantes hôtes sont analysées séparément pour chaque lot.En présence de graines de l'une ou de l'autre plante hôte (absence de choix du lieu de ponte) les fécondités et productions ovariennes des femelles de chacun des lots sont comparables. Par contre, lorsqu'on leur donne le choix de pondre sur les deux plantes hôtes, les femelles pondent préférentiellement sur P. vulgaris. Le substrat utilisé pour le développement larvaire n'influence pas le choix du lieu de ponte.Il n'y a donc pas d'obstacle physiologique ou comportemental empêchant Z. subfasciatus de se reproduire sur ces deux plantes hôtes, dans le contexte écologique d'où proviennent les insectes étudiés ici.

     

The ability of Zabrotes subfasciatus to attack cowpeas

The biological and behavioural responses of two strains of Zabrotes subfasciatus (Boh.) to beans and cowpeas were compared. One strain was collected from beans in Colombia and the other from cowpeas in Uganda. The two strains irrespective of their geographical or nutritional histories laid eggs on both types of seed and the distribution over time of egg laying for the two strains was found to be similar on each commodity. Both strains fed and developed on the two types of seed and no significant differences were found between the numbers of progeny developing on each.Competition between Z. subfasciatus and Callosobruchus maculatus was investigated using replacement series experiments at high and low population densities. Competition coefficients were calculated for the two species and at both densities neither species had a clear competitive advantage over the other. It is concluded that Z. subfascicatus should be regarded as a potential cosmopolitan pest of stored cowpeas.

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Résumé Le Zabrotes subfasciatus est un parasite cosmopolite des haricots stockés (Phaseolus vulgaris). Il s'est installé dans des zones restreintes en tant que parasite des pois sauvages (Vigna unguiculata). Des spécimens de Z. subfasciatus ont été recueillis dans des haricots infestés en Colombie et dans les pois sauvages infestés d'Ouganda afin de comparer leurs réactions biologiques et leur comportement vis-à-vis des haricots et des pois sauvages. Les deux variétés, indépendamment de leurs passés géographique ou alimentaires, ont pondu des oeufs sur les deux types de graines et al répartition dans le temps de la ponte des oeufs s'est révélée être similaire pour les deux variétés sur chaque denrée. Les deux variétés se sont nourries et développées sur les deux types de graines, et aucune différence significative entre les deux variétés n'a été constatée dans l'importance de la progéniture se développant sur chacun des deux types de graines.Une concurrence entre le Z. subfasciatus et le Callosobruchus maculatus (un parasite cosmopolite courant des pois sauvages) a été étudiée en effectuant des expériences en série de substitutions. Des mélanges d'adultes des deux espèces ont été mis sur des pois sauvages, les proportions des deux espèces étant différentes dans les divers traitements. Les coefficients de concurrence, basés sur l'importance de la progéniture de chaque espèce provenant des divers mélanges, ont été calculés et il a été constaté qu'aucune des espèces n'avait un advantage de concurrence évident par rapport à l'autre.On en a conclu que le Z. subfasciatus devait être considéré comme un parasite cosmopolite éventuel des pois sauvages stockés.

     

Genetic transformation of cowpea (Vigna unguiculata L.) and stable transmission of the transgenes to progeny

Cowpeas are nutritious grains that provide the main source of protein, highly digestible energy and vitamins to some of the world's poorest people. The demand for cowpeas is high but yields remain critically low, largely because of insect pests. Cowpea germplasm contains little or no resistance to major insect pests and a gene technology approach to adding insect protection traits is now a high priority. We have adapted features of several legume and other transformation systems and reproducibly obtained transgenic cowpeas that obey Mendelian rules in transmitting the transgene to their progeny. Critical parameters in this transformation system include the choice of cotyledonary nodes from developing or mature seeds as explants and a tissue culture medium devoid of auxins in the early stages, but including the cytokinin BAP at low levels during shoot initiation and elongation. Addition of thiol-compounds during infection and co-culture with Agrobacterium and the choice of the bar gene for selection with phosphinothricin were also important. Transgenic cowpeas that transmit the transgenes to their progeny can be recovered at a rate of one fertile plant per thousand explants. These results pave the way for the introduction of new traits into cowpea and the first genes to be trialled will include those with potential to protect against insect pests.     

Expression of the Arcelin 1 gene from Phaseolus vulgaris L. in cowpea seeds (Vigna unguiculata L.) confers bruchid resistance

Persistent decrease in the productivity of cowpea (Vigna unguiculata L.) has been partly due to attack by bruchids including Zabrotes subfasciatus and Callosobruchus maculatus. Resistance to these insects in Phaseolus vulgaris L. has been shown to be associated with arcelins, a family of seed proteins encoded by a multigenic family of lectins on the APA locus. In this work, we report the construction of an expression vector containing Arc1 gene isolated from P. vulgaris and introduced into cowpea as a strategy to confer resistance to insect attack. Following transformation and selection, feeding experiments in which C. maculatus and Z. subfasciatus were fed with transgenic (L3 and L5) and non-transgenic (control) grains showed that introduced gene protected the transgenic line. Significant differences (p < .05 and p < .01) were found in the number of eggs laid, the number of emerging insects and the loss of grain mass in L3, compared with control, for both insects. Similar observations were made in L5 with the exception of the number of laid eggs. The strategy here described may form the basis for the development of a cowpea variety tolerant to bruchids in a crop cultivated by farmers throughout Latin America and Africa.     

Differences in calcium efficiency between cowpea (Vigna unguiculata (L.) Walp.) cultivars

The cowpea (Vigna unguiculata (L.) Walp.) cultivars TVu 354 and Solojo were grown in solution culture at 10 to 1000 M Ca supply. The Ca supply did not vary by more than 10% during the experiment. The pH value was kept constant within 0.1 units at 4.0 by automatic titration. The cultivar TVu 354 proved to be much more Ca-efficient than Solojo. At 10 M Ca supply Solojo died, whereas TVu 354 was hardly affected in dry matter production. The differences in Ca efficiency were independent of the P supply. They could not be explained by differences in Ca uptake or Ca concentrations in the plant tissue. Short-term studies using ⁴⁵Ca, both in the dark and in the light, indicated better transport of Ca from the roots to the shoots and within the shoots to the younger leaves in the Ca-efficient cultivar TVu 354. However, the main reason for the differences between the cultivars in sensitivity to low Ca supply were differences in the Ca requirement of the plant tissue to maintain tissue organization and function. Sequential fractionation of the freeze-dried leaf tissue with hot water, 0.5 M NaCl, 1 M CH₃COOH, and 2 M HCl did not reveal cultivar differences in Ca binding state. The results clearly show that considerable genetic potential in tolerance to low Ca supply exists in cowpea. However, a better understanding of the physiological/biochemical reasons for low internal Ca requirement is needed.     

Development of PCR-based chloroplast DNA markers that characterize domesticated cowpea (Vigna unguiculata ssp. unguiculata var. unguiculata) and highlight its crop-weed complex

In 1992, Vaillancourt and Weeden discovered a very important mutation for studying cowpea evolution and domestication. A loss of a BamHI restriction site in chloroplast DNA characterized all domesticated accessions and a few wild (Vigna unguiculata ssp. unguiculata var. spontanea) accessions. In order to screen a larger number of accessions, primers were designed to check this mutation using PCR RFLP or direct PCR methods. Using these new primers, 54 domesticated cowpea accessions and 130 accessions from the wild progenitor were screened. The absence of haplotype 0 was confirmed within domesticated accessions, including primitive landraces from cultivar-groups Biflora and Textilis, suggesting that this mutation occurred prior to domestication. However, 40 var. spontanea accessions distributed from Senegal to Tanzania and South Africa showed haplotype 1. Whereas this marker could not be used to identify a precise center of origin, it did highlight the widely distributed cowpea crop-weed complex. Its very high frequency in West Africa could be interpreted as a result of either genetic swamping of the wild/weedy gene pool by the domesticated cowpea gene pool or as the result of domestication by ethnic groups focusing primarily on cowpea as fodder.     

Resistance to the cowpea weevil (Callosobruchus maculatus) larva in pods of cowpea (Vigna unguiculata)

Thirty cowpea, Vigna unguiculata (L.) Walpers, cultivars were evaluated as intact pods to determine if any possessed resistance to the cowpea weevil, Callosobruchus maculatus (Fabr.). Pod resistance was measured as pre-establishment larval mortality (PreM); those larvae dying after egg hatch but before penetrating into the seeds, and as post-establishment within-seed mortality (PostM); those larvae dying after penetrating into the seeds. Among the 30 varieties examined, PreM ranged from 57.9% to 99.4% and PostM ranged from 6.7% to 82.6%. Ten varieties exhibited total intact pod mortality (mortality from egg hatch to adult emergence from the seed) greater than 95%.Physical measurements were made of several pod and seed characteristics to ascertain whether the observed pod resistance was due to seed factors, pod-wall factors, or to interactions between the pod and seeds. Although resistance to breakage (from handling plus pod dehiscence) is a necessary characteristic for overall pod protection against C. maculatus; other factors are also important. Among the other pod and seed characteristics measured to identify major resistance factors, seed coat thickness was the one most highly correlated with pod resistance. Our results suggest that interactions between pod-wall and seed coat characteristics play a large role in pod resistance of cowpeas to C. maculatus.     

Variation in polar-group content in lipids of cowpea (Vigna unguiculata) Cell cultures as a mechanism of haloadaptation

Callus and cell suspension cultures of cowpea (Vigna unguiculata) were induced with 2,4-dichlorophenoxyacetic acid and grown at different NaCl concentrations. The cell biomass yield and its total lipid content decreased with increasing salinity. However, while the hexose content in lipids was higher, the amount of lipid phosphorus was significantly lower in both agar and cell suspension cultures. Ion-transport rates with artificial membranes prepared with different lipid fractions showed that lipids from cells grown in a saline medium were less permeable to Na⁺ and to Cl^- than those grown in a non-saline medium. Also the permeability of membranes prepared with glycolipids was lower than those prepared with phospholipids and whole lipids. Apparently, the increase of hexose/phosphorus ratio in membrane lipids is induced in response to the halo-adaptation process.     

Isolation and characterization of trypsin inhibitors from cowpea (Vigna unguiculata)

The trypsin inhibitor fraction from cowpea (Vigna unguiculata) has been purified and characterized. Although the total trypsin inhibitor as purified by affinity chromatography on immobilised trypsin was shown to be heterogeneous by gel electrophoresis and isoelectric focusing as well as by function, it was relatively homogeneous in MW (ca 17 000) on gel filtration. The total trypsin inhibitor was divided into inhibitors active against trypsin only and active against trypsin and chymotrypsin by affinity chromatography on immobilised chymotrypsin. The ‘trypsin-only’ inhibitor was the major component of the total trypsin inhibitor. It was shown by isoelectric focusing and gel electrophoresis to contain several isoinhibitors. Determination of the combining weight of this inhibitor and investigation of the complexes formed with trypsin by gel filtration indicated the presence of two protease binding sites per inhibitor molecule. The chymotrypsin/trypsin inhibitor was also shown to be composed of several isoinhibitors. On the basis of gel electrophoresis and gel filtration in dissociating and non-dissociating media both inhibitors were considered to be dimeric molecules with the subunits linked by disulphide bonds; this implies that the ‘trypsin-only’ inhibitor has one binding site per subunit.     

In vitro regeneration of Vigna unguiculata (L.) Walp. cv. Blackeye cowpea via shoot organogenesis

An in vitro regeneration system was developed in cowpea [Vigna unguiculata (L.) Walp.] Blackeye. Among several explants studied, shoot initiation response was observed from shoot apices of 3–5-day-old seedlings. The optimal medium for maximum shoot initiation comprised MS salts, B₅ vitamins, 8.88 μM N ⁶-benzylaminopurine, 1 gl^-1 casein hydrolysate, 342 μM L-glutamine, 3% sucrose, 0.3% phytagel, adjusted to pH 5.8. A shift in pH from 5.8 to 7.0 had no effect on shoot initiation and on number of shoots per explant. The highest shoot initiation frequency (77%) was obtained using this preferred medium, reaching a maximum of eight shoots per explant. For shoot elongation, 14 μM gibberellic acid was supplemented in the shoot initiation medium. Presence of indolebutyric acid in the rooting medium had no effect on root induction. The regenerated plants were fertile and developed normally.     

Factors responsible for genotypic manganese tolerance in cowpea (Vigna unguiculata)

In experiments with 29 cowpea genotypes considerable variation in Mn tolerance could be found. Ranking according to Mn tolerance was almost the same in sand and water culture. Mn tolerance is not related to greater vigour or exclusion of Mn from uptake and translocation, but depends mainly on the internal tolerance to excess Mn especially in the leaf tissue.Growth depression by Mn excess is characterized by local accumulatiòn of Mn, deposition of Mn oxides, and typical macro-symptoms on the older leaves (brown spotsclorosisshedding of the leaves). Autoradiographic studies with⁵⁴Mn and extraction of the leaves with methanol and H₂O indicate a causal relationship between Mn tolerance and the more homogenous distribution of Mn in the tissue. In tolerant genotypes local accumulation and deposition of Mn is inhibited or retarded.Mn applied to the petioles of fully expanded leaves induces the same toxicity symptoms on the leaf blades as Mn absorbed by the roots. There is a good agreement between the rankings of the different genotypes for Mn tolerance according to the depression of shoot dry matter production by Mn excess in long term pot experiments and the appearance of toxicity symptoms after application of Mn to the petioles.The regulation of Mn tolerance at the leaf tissue level allows a quick and non-destructive screening of large numbers of genotypes for Mn tolerance.     

Development, fecundity and egg dispersion of Zabrotes subfasciatus

Zabrotes subfasciatus Boh. (Coleoptera: Bruchidae) is the smallest of the bruchids commonly infesting stored legume seeds, yet its wild and cultivated hosts, Phaseolus lunatus and P. vulgaris, have large seeds. It is demonstrated that the maximum fecundity of females is around 55 eggs which are aggregated onto some of the available hosts. About 80% of the eggs normally hatch and development at 27°C and 70% relative humidity takes around 34 days. The sex ratio of emerging adults is slightly biased towards males. About 75% of the larvae in a seed produce adults at low and moderate initial densities and up to 20 adults can emerge from a single seed. Adult weight is not influenced by the initial larval density in the seed but there is a strong correlation between the weight of females at emergence and their fecundity. These results are considered in the light of existing knowledge of Z. subfasciatus, much of which is apparently contradictory or inconsistent. Many of these difficulties are resolved and it is demonstrated that the behaviour and bionomics are well adapted to the normal situation in which the beetle is found and that the differences between this species and other bruchids are explicable in this context.     

Effects of aluminium on tap-root elongation of soybean (Glycine max), cowpea (Vigna unguiculata) and green gram (Vigna radiata) grown in the presence of organic acids

The role of fulvic, malic, and oxalic acids in alleviating the toxic effects of aluminium (Al) on tap-root elongation of soybean cv. Fitzroy, cowpea cv. Vita 4, and green gram cv. Berken was studied. Treatments consisted of a factorial combination of four Al concentrations (0, 12.5, 25 and 50 µM as Al(NO₃)₃·9H₂O) and two concentrations either of malic or oxalic acid (0, 50 µM) or fulvic acid (0, 65 mg L^-1 of organic carbon). The free monomeric Al in solution was determined using a pyrocatechol violet procedure which distinguishes between monomeric and organically complexed Al. Fulvic acid completely alleviated the toxic effect of Al at all concentrations on soybean and cowpea and at concentrations <25 µM on green gram. The non-toxic Al-fulvate complex remained in solution. Both malic and oxalic acid, at the concentrations tested, failed to alleviate Al toxicity on any species; a much higher proportion of the added Al remained in monomeric form in the presence of these acids.     

Silicon promotes nodule formation and nodule function in symbiotic cowpea (Vigna unguiculata)

Applying silicon in the form of metasilicic acid (H₄SiO₃) or silicic acid (H₄SiO₃) to Bradyrhizobium -infected, hydroponically grown cowpea seedlings resulted in a significant ( P 0.05) increase in the number of nodules, nodule dry matter, and nitrogen fixed on a per plant basis. Total dry matter of plants increased with silicon supply, and the differences were significant ( P 0.05) at the higher silicon concentrations. Cowpea plants cultured in sand were also assessed for their response to silicic acid. Nodule number and nodule mass increased with silicon supply to sand cultured plants, though nitrogen fixation was unaltered. Although silicon is not essential for growth of cowpea, it is important for nodule formation and nodule functioning in hydroponically grown plants. Consequently, data collected and conclusions drawn from earlier glasshouse experiments, which have excluded silicon from nutrient solutions, may be flawed. Future studies on nodulation and nitrogen fixation using legumes in liquid culture must therefore include silicon as a nutrient element.     

Nodulation and symbiotic nitrogen fixation of cowpea (Vigna unguiculata (L.) Walp)

Summary Rhizobium strains CIAT 301, CIAT 79 and SLM 602 were tested and found effective in the nodulation and nitrogen fixation of cowpea cv. MI-35 (Vigna unguiculata (L.) Walp) plants in growth chamber experiments. Fresh weight of nodules increased with plant age initially and stabilized in 20–30 days from planting, followed by a secondary flush of nodule growth after 30 days. Apparent nitrogen fixation per gram nodule fresh weight reached a maximum in 20–30 days after planting and then decreased, even though a flush of new nodules was produced.     

A wounding-induced PPO from cowpea (Vigna unguiculata) seedlings

Polyphenol oxidases (PPO) are induced in cowpea plants by wounding. The highest activity levels were detected 48h after this stimulus in both wounded and neighbor-to-wounded unifoliates of cowpea seedlings; the increase of activity was in the order of 13 to 15-fold, respectively, in comparison to control unifoliates. Multiple molecular forms of active PPO (Mrs 58, 73 and congruent with220kDa) were detected by partially denaturing SDS-PAGE. Wounding-induced cowpea PPO were extracted and purified through (NH(4))(2)SO(4) precipitation and ion-exchange chromatography. The effects of substrate specificity, pH, thermal stability and sensitivity to various inhibitors - resorcinol, EDTA, sodium azide and tropolone - of partially purified soluble PPO were investigated. Purified wounding-induced cowpea PPO (wicPPO) showed the highest activities towards 4-methylcatechol (K(m)=9.86mM, V(max)=24.66 EU [DeltaAmin(-1)]) and catechol (K(m)=3.44mM, V(max)=6.64 EU [DeltaAmin(-1)]); no activity was observed towards l-tyrosine, under the assay conditions used. The optimum pH for wound-induced cowpea PPO was 6.0 with 4-methylcatechol as substrate. The enzyme was optimally activated by 10 mM SDS and was highly stable even after 5 min at 80 degrees C. The most effective inhibitor was tropolone, whereas addition of 10mM of resorcinol, EDTA and sodium azide were able to reduce PPO activities by 40%, 15% and 100%, respectively.     

Toxic effects of Ni2+ on growth of cowpea (Vigna unguiculata)

Despite the importance of Ni-polluted soils throughout the world, comparatively little is known about the activity of Ni²⁺ required to reduce plant growth and the effects that Ni²⁺ toxicity has on the plant. Cowpea (Vigna unguiculata (L.) Walp. cv Caloona) was grown in dilute nutrient solutions to investigate the effect of Ni²⁺ activity on shoot and root growth. A Ni²⁺ activity of 1.4 μM was found to cause a 10% reduction in the relative fresh mass of the root and shoots. The primary site of Ni²⁺ toxicity was the shoots, with the younger leaves displaying an interveinal chlorosis (possibly a Ni-induced Fe deficiency) at Ni²⁺ activities ≥1.7 μM. Lateral root formation was inhibited in the two highest Ni²⁺ treatments (3.3 and 5.1 μM), and the roots growing at the highest Ni²⁺ activity were short and stubby and brown in color. However, no other symptoms of toxicity were observed on the roots at lower Ni²⁺ activities.     

Isolation and characterization of polymorphic microsatellite markers in Zabrotes subfasciatus Boheman (Coleoptera: Bruchidae)

Bruchid beetles of the genus Zabrotes (Coleoptera: Bruchidae) are important worldwide pests of legume seeds. Samples of Zabrotes subfasciatus and Z. sylvestris were collected from seeds of two Phaseolus species throughout Mexico to assess the role of host plant variation and plant domestication on the evolution of host use in this species. For this purpose six polymorphic microsatellite loci were isolated and characterized for the bruchid Z. subfasciatus. Cross‐species amplification tests were performed on Z. sylvestris and revealed that three loci amplified successfully and were polymorphic in this closely related species.     

Genetic tranformation of cotyledon explants of cowpea (Vigna unguiculata L. Walp) using Agrobacterium tumefaciens

Mature de-embryonated cotyledons with intact proximal end of Vigna unguiculata were cultured on B5 basal medium containing varying concentrations of BAP. Thirty-six percent of the explants produced shoots on B5 medium supplemented with 8× 10^–6 M BAP. Cotyledon explants were pre-incubated for 24 h, inoculated with A. tumefaciens pUCD2614 carrying pUCD2340, co-cultivated for 48 h and transferred to hygromycin-B (25 mg/l) containing shoot induction medium. Approximately 15–19% of the explants produced shoots on the selection medium. The elongated shoots were subsequently rooted on B5 basal medium containing hygromycin. The transgenic plants were later established in pots. The presence of hpt gene in the transgenic plants was confirmed by Southern blot hybridization.Abbreviations BAP 6-Benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - hpt hygromycin phosphotransferase - IAA Indole-3-acetic acid - NAA 1-naphthaleneacetic acid