Resistant Cowpea Research Articles

Association mapping of aphid resistance in USDA cowpea (Vigna unguiculata L. Walp.) core collection using SNPs

Cowpea aphid (Aphis craccivora) is a destructive insect pest of cowpea, as well as other legume crops including alfalfa, beans, chickpea, lentils, lupins and peanuts. The utilization of aphid resistance in cowpea breeding is one of the most efficient and environmental friendly methods to control aphids. Using marker-assisted selection will expedite cowpea breeding procedures, but it is limited by the lack of information on marker associations of the aphid resistance. The objective of this study was to identify single nucleotide polymorphism (SNP) markers associated with aphid resistance in cowpea and to conduct genetic diversity analysis for aphid resistant resources. In this study, 338 cowpea accessions from the cowpea core collection in United States Department of Agriculture (USDA) Germplasm Resources Information Network (GRIN), originally collected from 40 countries, were evaluated for their aphid resistance and 1047 SNPs identified from genotyping by sequencing (GBS) were used as the genotypic data. Single marker regression, general linear model, and mixed linear model in TASSEL were used for association analysis between the SNPs and the phenotypic data. Three clusters for the association panel of the 338 cowpea accessions were inferred by STRUCTURE analysis, in good agreement with the phylogenetic tree drawn by MEGA 6 based on the maximum likelihood model with the neighbor joining method. A genome-wide association study revealed that two SNP markers, C35011941_894 and Scaffold30061_3363, were strongly associated with aphid resistance across three models with the log of odds (LOD) value greater than 2.5. The results will provide useful information for selecting aphid resistance in cowpea molecular breeding.

English

Genetic linkage maps provide a genomic framework for quantitative trait loci identification applied in marker assisted selection breeding in crops with limited resources. It serves as a powerful tool to breeders for analysing the mode of inheritance of genes of interest and monitoring of the transmission of target genes from parents to progeny. Single nucleotide polymorphism (SNP) markers have a great influence on mapping research studies: for faster development of new varieties of crops due to its abundance and high number of polymorphism. Thus, this research work aimed at constructing genetic linkage map of cowpea using SNP markers. This will facilitate development of new varieties of cowpea. Single seed descent method of breeding was used to generate mapping population between IT-95K-193-12 and Ife-Brown. Fresh leaves from both parental lines and F1 hybrids were genotyped with 135 SNP markers. Data from125 polymorphic SNP markers were used to construct linkage map. Results revealed 12 linkage maps with an average of 10 markers to a chromosome and average marker distance of 3.02 cM between the markers. Constructed map provides basic information that could assist in genetic improvement of cowpea, most especially in developing brown blotch resistant cowpea varieties through detection of quantitative trait loci (QTL) responsible for brown blotch resistance in cowpea when utilizing marker assisted selection method. Key words: Cowpea, genetic linkage map, single nucleotide polymorphism (SNP) markers, brown blotch resistance.

Evaluation of F3 generations of the hybrid crosses between vegetable and field cowpeas and their parents for resistance to the cowpea bruchids (Callosobruchus maculatus (F))

An evaluation of crosses between vegetable and field cowpea for resistance to the cowpea Bruchids, [ Callosobruchus maculatus (F)], was carried out at the Enugu State University of Science and Technology, Enugu southeastern Nigeria. There was a significant difference in the field infestation of the cowpea lines by C. maculatus . AD-36-WB had the highest percentage field infestation by the insect pest, whereas, AE—36-C X AN-16-D, and AN-14-D were free from the field infestation by the pest. Vegetable cowpea lines tested had some levels of resistance to the cowpea Bruchid and the gene responsible for the resistance were successfully transferred from vegetable cowpea to the susceptible field cowpea by cross-breeding techniques. No vegetable cowpea tested had complete resistance to the cowpea bruchid, however, they had lower susceptibility than the field cowpea tested. Reduction in the relative susceptibility of the cowpea to C. maculatus was achieved by cross breeding field cowpea with vegetable types. For instance, in crosses between AD-36-W (field cowpea) and AE-36-C (vegetable cowpea), AD-36-WB (field cowpea) and AN-14-D (vegetable cowpea), AD-36-W (field cowpea) and AE-36-F (vegetable cowpea), AD-36-WB (field cowpea) and AE-36-C (vegetable cowpea), AN-14-D and AE-36-F reduced susceptibility of AD-36-W and AD-36-WB to C. maculatus from 63.59% to 51.09%, 68.46% to 59.09%, 63.59% to 47.38% and 68.48% to 38.84% respectively. Also, gene responsible for susceptibility to the storage insect pest were successfully transferred from field cowpea to the vegetable cowpea through cross-breeding techniques. A cross between two vegetable types namely; AE-36-C and AN16-D had the least susceptibility level of 24.32%. There was a significant difference in mean number of eggs laid per cowpea line with AD-36-WB recording the highest mean number of 134.75 eggs and AE-36-F recording the least mean number of 4.75 eggs. There was also a significant difference in the percentage adults emergence per cowpea line with AD-36WB having the highest percentage of 68.46% adults, and AE-36-C X AN-16-D having the least percentage of 24.32% adults. The result of the experiment also showed a significant difference in mean longevity of female adults reared from cowpea lines with those reared from AD-36WB X An-14-D, and AE-36-C having the highest mean longevity of 14 days each whereas those from AD-36WXAE-36-C had the least mean longevity of 5.6 days. Also, there was a significant difference in mean testa thickness of the seeds tested with AE-36-C having the highest mean testa thickness of 0.20mm, whereas AD-36-WB had the least mean testa thickness of 0.02mm. However, there was no correlation between either testa thickness or moisture content and levels of infestation by the storage insect pest. Again, there was a significant difference between mean weight loss of seeds tested after infestation by C. maculatus , with AD-36WB recording the highest mean weight loss of 0.38g, whereas AE-36-C and AE-36-F, had the least mean weight loss of 0.07g. There was a positive correlation between the weight loss of the adult C. maculatus and the weight (g) of the seeds from which the insect pest was reared (r = 0.03). Key words: Cowpea lines, cross-breeding, Callosobruchus maculatus , resistance, susceptibility

A major QTL corresponding to the Rk locus for resistance to root-knot nematodes in cowpea (Vigna unguiculata L. Walp.).

Key messageGenome resolution of a major QTL associated with theRklocus in cowpea for resistance to root-knot nematodes has significance for plant breeding programs and R gene characterization. Cowpea (Vigna unguiculata L. Walp.) is a susceptible host of root-knot nematodes (Meloidogyne spp.) (RKN), major plant-parasitic pests in global agriculture. To date, breeding for host resistance in cowpea has relied on phenotypic selection which requires time-consuming and expensive controlled infection assays. To facilitate marker-based selection, we aimed to identify and map quantitative trait loci (QTL) conferring the resistance trait. One recombinant inbred line (RIL) and two F2:3 populations, each derived from a cross between a susceptible and a resistant parent, were genotyped with genome-wide single nucleotide polymorphism (SNP) markers. The populations were screened in the field for root-galling symptoms and/or under growth-chamber conditions for nematode reproduction levels using M. incognita and M. javanica biotypes. One major QTL was mapped consistently on linkage group VuLG11 of each population. By genotyping additional cowpea lines and near-isogenic lines derived from conventional backcrossing, we confirmed that the detected QTL co-localized with the genome region associated with the Rk locus for RKN resistance that has been used in conventional breeding for many decades. This chromosomal location defined with flanking markers will be a valuable target in marker-assisted breeding and for positional cloning of genes controlling RKN resistance.Electronic supplementary materialThe online version of this article (doi:10.1007/s00122-015-2611-0) contains supplementary material, which is available to authorized users.

Cowpea resistance induced against Fusarium oxysporum f. sp. tracheiphilum by crustaceous chitosan and by biomass and chitosan obtained from Cunninghamella elegans

Pathogenic microorganisms increase the production of reactive oxygen species (ROS), and the response can cause oxidative damage and even death in a plant. Plant resistance inducers activate antioxidant enzymes such as catalase (CAT) and peroxidase (POX). The aims of this study were to evaluate the antimicrobial activity and the potential to induce the CAT and POX activities by the biopolymer chitosan in cowpea plants inoculated with the phytopathogen Fusarium oxysporum f. sp. tracheiphilum. The antimicrobial activity of crustaceous chitosan (CrCh) was tested in a laboratory assay and analyzed by optic and scanning electron microscopy. In a greenhouse experiment, the effectiveness of CrCh, fungal chitosan obtained from Cunninghamella elegans (C.eCh), and C. elegans biomass (C.eCh source) plus NPK biofertilizer, named bioprotector (C. elegans+NPKB), were evaluated in relation to the disease severity and the CAT and POX activities in cowpea plants infected with F. oxysporum f. sp. tracheiphilum. The higher rates of CrCh (4.0–6.0mgmL−1) showed the total fungicide ability, and the other concentrations (0.5–3.0mgmL−1) promoted a greater fungistatic effect and morphological changes on the growth of F. oxysporum f. sp. tracheiphilum during the antimicrobial test in vitro. Chitosan effectively protected cowpea plants against this pathogen. The higher chitosan concentrations (4.0 and 6.0mgmL−1) were directly related to CAT and POX activity, which controlled the ROS equilibrium for plant resistance and resulted in a significant reduction of the disease severity in cowpea. These findings are important for the establishment of a sustainable agriculture and to avoid the use of pesticides.

English

The obligate root parasitic weed Striga gesnerioides poses a severe constraint to cowpea productivity in the dry savannahs of West and Central Africa, where cowpea is a major crop. At least seven races of S. gesnerioides have been identified within the cowpea-growing regions of West and Central Africa, based on host differential response and genetic diversity analysis. Molecular markers linked to resistance to different races of S. gesneriodes have been identified. It was desirable to demonstrate the applicability and efficiency of such markers for use in marker-assisted selection (MAS) to fast-track the development of cowpea with resistance to S. gesnerioides. The objective of the study was to determine the suitability of two molecular markers in tracking resistance in cowpea to S. gesnerioides race SG3, the predominant race found in Nigeria. F2 mapping populations and recombinant inbred lines (RILs) derived from the cross involving IT97K-499-35 and a susceptible local landrace (Borno Brown), and another resistant parent B301 with the same susceptible land race (Borno Brown) were assayed using two linked markers. Genetic analysis showed that resistance to S. gesnerioides in cowpea is inherited as a single dominant gene. Two SCAR markers, 61RM2 and C42-2B were validated in the same F2 populations and subsequent recombinant inbred lines (RILs). The two markers were able to discriminate between resistance and susceptibility and the genotypic score was quite similar to the phenotypic score with the markers score showing greater efficiency in selection than phenotypic score. The 61RM2 had two bands in resistant cultivars and amplified a ~450 bp fragment with marker efficiency of 98% while C42-2B amplified a single ~250 bp fragment with marker efficiency of 96% in resistant cultivars and absent in susceptible cultivars. The genetic distance between 61RM2 and phenotypic score was 3.5 cM while that of C42-2B and phenotypic score was 8.5 cM. The two marker data set were significantly correlated with the phenotypic data (r=0.95). Based on the tight linkage with the resistant locus, 61RM2 was found to be a utility marker to initiate MAS in cowpea breeding for resistance to S. gesnerioides.   Key words: Cowpea, Striga, molecular marker, genetic distance, race-specific, obligate parasitic weed, Vigna unguiculata. &nbsp

Identification and confirmation of trailing-type vegetable cowpea resistance to anthracnose

Anthracnose, caused by Colletotrichum lindemuthianum, is the most destructive disease in cowpea worldwide, leading to complete crop loss with no recommended management strategy. The few resistance sources available belong to the bush type grain cowpea (ssp. cylindrica) and usage of these lines in resistance breeding invites extensive backcrossing. Recently, from farmers’ fields of Malappuram district of Kerala state in India, a trailing-type vegetable cowpea (ssp. sesquipedalis), Arimbra Local has been reported to possess resistance to this disease. The present study confirmed the resistance through controlled artificial inoculation and the basis of resistance was analyzed through protein profiling and defense enzyme analyses. Protein profiling has revealed that the resistant varieties are capable to maintain a high level of RuBisCo, the main photosynthetic enzyme, consistently, whereas the susceptible varieties had shown a drastic fall in the level of this enzyme soon after fungal infection. Further, the defense enzymes such as peroxidase and phenylalanine ammonia-lyase were also related to the level of resistance. Hence, Arimbra Local is recommended for the crop improvement programs in trailing-type vegetable cowpea, intended for enhancing the resistance to anthracnose.

Genetic mapping and legume synteny of aphid resistance in African cowpea (Vigna unguiculata L. Walp.) grown in California.

The cowpea aphid Aphis craccivora Koch (CPA) is a destructive insect pest of cowpea, a staple legume crop in Sub-Saharan Africa and other semiarid warm tropics and subtropics. In California, CPA causes damage on all local cultivars from early vegetative to pod development growth stages. Sources of CPA resistance are available in African cowpea germplasm. However, their utilization in breeding is limited by the lack of information on inheritance, genomic location and marker linkage associations of the resistance determinants. In the research reported here, a recombinant inbred line (RIL) population derived from a cross between a susceptible California blackeye cultivar (CB27) and a resistant African breeding line (IT97K-556-6) was genotyped with 1,536 SNP markers. The RILs and parents were phenotyped for CPA resistance using field-based screenings during two main crop seasons in a ‘hotspot’ location for this pest within the primary growing region of the Central Valley of California. One minor and one major quantitative trait locus (QTL) were consistently mapped on linkage groups 1 and 7, respectively, both with favorable alleles contributed from IT97K-556-6. The major QTL appeared dominant based on a validation test in a related F2 population. SNP markers flanking each QTL were positioned in physical contigs carrying genes involved in plant defense based on synteny with related legumes. These markers could be used to introgress resistance alleles from IT97K-556-6 into susceptible local blackeye varieties by backcrossing.Electronic supplementary materialThe online version of this article (doi:10.1007/s11032-015-0254-0) contains supplementary material, which is available to authorized users.

Enhanced Synthesis of Antioxidant Enzymes, Defense Proteins and Leghemoglobin in Rhizobium-Free Cowpea Roots after Challenging with Meloydogine incognita.

The root knot nematodes (RKN), Meloydogine spp., particularly Meloidogyne incognita and Meloidogyne javanica species, parasitize several plant species and are responsible for large annual yield losses all over the world. Only a few available chemical nematicides are still authorized for RKN control owing to environmental and health reasons. Thus, plant resistance is currently considered the method of choice for controlling RKN, and research performed on the molecular interactions between plants and nematodes to identify genes of interest is of paramount importance. The present work aimed to identify the differential accumulation of root proteins of a resistant cowpea genotype (CE-31) inoculated with M. incognita (Race 3) in comparison with mock-inoculated control, using 2D electrophoresis assay, mass spectrometry identification and gene expression analyses by RT-PCR. The results showed that at least 22 proteins were differentially represented in response to RKN challenge of cowpea roots mainly within 4–6 days after inoculation. Amongst the up-represented proteins were SOD, APX, PR-1, β-1,3-glucanase, chitinases, cysteine protease, secondary metabolism enzymes, key enzymes involved in ethylene biosynthesis, proteins involved in MAPK pathway signaling and, surprisingly, leghemoglobin in non-rhizobium-bacterized cowpea. These findings show that an important rearrangement in the resistant cowpea root proteome occurred following challenge with M. incognita.

Biochemical Basis for Bruchid Resistance in Cowpea, Chickpea and Soybean Genotypes

Biochemical Basis for Bruchid Resistance in Cowpea, Chickpea and Soybean Genotypes

English

Transgenic cowpeas are those genetically engineered with Cry1Ab gene for protection against the lepidopteran pest Maruca vitrata that could inflict upto 80% yield loss in severe infestation. Two transgenic events TCL-709 and TCL-711 were crossed to three non-transgenic lines IT97K-499-35, IT93K-693-2 and IT86D-1010 to generate six set of F1 hybrids. The study was designed and conducted to evaluate the performance of transgenic cowpeas and hybrids derived from them under natural field conditions. The transgenics, non-transgenics and the hybrids were planted with intra and inter-row spacing of 30 and 75 cm respectively in a completely Randomized Block Design during the 2012 cowpea growing season at Confined Field Trial site of Institute for Agricultural Research of Ahmadu Bello University Samaru-Zaria. The plot sizes consisted of 2 rows of 5 m long. The trial was kept weed-free by constant manual weeding. The various generations of  IT97K-499-35 x TCL-709 were infested six times with first instar Maruca larvae at four day interval at flower initiation stage while the remaining crosses were exposed to natural infestation of Maruca. The data were recoreded on various characters and the results revealed that the cowpea lines containing the cry1Ab gene produced enough toxin to kill and inhibit the feeding of Maruca on cowpea plants. The result on infestation of Maruca larvae scored as number of damaged pods per plant gave a clear proof of the achievement of the goal of the genetic transformation of cowpea lines with Cry1Ab gene. As the Cry1Ab gene conferred a high degree of resistance to cowpea pod borer, the transgenic cowpea lines should be used as a precious insect-resistant line to be employed in traditional breeding programs to develop Maruca resistant cowpea varieties.   Key words: Bacillus thuriengiensis, confined field trial, Cry1Ab gene, transgenic cowpea, Maruca vitrata.

VuDREB2A, a novel DREB2-type transcription factor in the drought-tolerant legume cowpea, mediates DRE-dependent expression of stress-responsive genes and confers enhanced drought resistance in transgenic Arabidopsis.

VuDREB2A exists in cowpea as a canonical DREB2-type transcription factor, having the ability to bind dehydration-responsive elements in vitro and confer enhanced drought resistance in transgenic Arabidopsis. Cowpea (Vigna unguiculata L. Walp) is an important cultivated legume that can survive better in arid conditions than other crops. But the molecular mechanisms involved in the drought tolerance of this species remain elusive with very few reported candidate genes. The Dehydration-Responsive Element-Binding Protein2 (DREB2) group of transcription factors plays key roles in plant responses to drought. However, no DREB2 ortholog has been reported from cowpea so far. In this study, we isolated and characterized a gene from cowpea, namely VuDREB2A, encoding a protein of 377 amino acids exhibiting features of reported DREB2-type proteins. In cowpea, VuDREB2A transcript accumulation was highly induced by desiccation, heat and salt, but slightly by exogenous abscisic acid (ABA) treatment. We also isolated the VuDREB2A promoter and predicted stress-responsive cis-elements in it using Arabidopsis microarray data. The E. coli-expressed VuDREB2A protein showed binding to synthetic oligonucleotides with Dehydration-Responsive Elements (DREs) from Arabidopsis, in electrophoretic mobility shift assays. Heterologous expression of VuDREB2A in Arabidopsis significantly improved plant survival under drought. In addition, overexpression of a truncated version of VuDREB2A, after removal of a putative negative regulatory domain (between amino acids 132-182) led to a dwarf phenotype in the transgenic plants. Microarray and quantitative PCR analyses of VuDREB2A overexpressing Arabidopsis revealed up-regulation of stress-responsive genes having DRE overrepresented in their promoters. In summary, our results indicate that VuDREB2A conserves the basic functionality and mode of regulation of DREB2A in Arabidopsis and could be a potent candidate gene for the genetic improvement of drought resistance in cowpea.

Varietal Susceptibility of Improved Cowpea Vigna unguiculata (L.) (Walp.) Cultivars to Field and Storage Pests

<p>Cowpea, <em>Vigna unguiculata</em> is widely cultivated in the tropics and subtropics as food for man and livestock. The crop is highly prone to pests and diseases which limit its production. Resistant cowpea varieties have become increasingly important in the management of both field and storage pests. Three improved cowpea varieties and one local variety were screened for their susceptibility to field and storage pests. The experiment was conducted in a randomized complete block design with four treatments and three replications. The different pests infesting the plants were identified and counted. Aphids and thrips were scored on a scale of 0-5, depending on the levels of infestations. Natural enemies’ numbers, leaf damage, pod damage and yield were assessed. <em>Callosobruchus maculatus</em> infestations during storage and weight reduction were determined over a six week period. Aphids and <em>M. sjostedti</em> score, <em>M. virtrata</em> and <em>Empoasca</em> sp. numbers did not differ significantly; however, significantly more pod sucking bubs attacked the local variety than the improved varieties. Natural enemies’ numbers on the various cultivars did not differ significantly. Yield was however significantly better in the improved varieties. The local variety suffered the largest weight reduction (20.91%) during storage while Nhyira suffered the least weight reduction of 4.23%. The use of improved varieties and application of pests’ control measures will increase cowpea production in Ghana.</p>

Identification and mapping of a powdery mildew resistance geneVu-Pm1in the Chinese asparagus bean landrace ZN016

Powdery mildew (PM), caused by the fungus Erysiphe polygoni D.C, is one of the most common and serious diseases of cowpea (Vigna. unguiculata L. Walp.)/asparagus bean (Vigna. unguiculata ssp. sesquipedialis). Deployment of scab-resistant varieties is the major strategy to curb this disease. ZN016, a Chinese asparagus bean landrace, is highly resistant to powdery mildew. To identify the resistance genes in ZN016, an F7:8 recombinant inbred line (RIL) population derived from the cross between ZN016 and a susceptible cultivar Zhijiang 282 was evaluated for PM resistance in the open field in 2008 and 2009. Phenotypic analysis indicated that ZN016 harbors a single PM resistance gene. Using a SNP and SSR based genetic map constructed with this population, the PM resistance gene was mapped to a locus on LG9 flanked by SSR markers CLM0305 and CLM0260, with a genetic distance of 2.0 cM and 5.0 cM, respectively. To our knowledge, this is the first powdery mildew resistance gene reported in cowpea, which was temporarily designated as Vu-Pm1. The identification of Vu-Pm1 opens a door for dissecting resistance mechanisms and applying marker-assisted selection for the development of cultivars with PM resistance in cowpea.

Proteomics changes during the incompatible interaction between cowpea and Colletotrichum gloeosporioides (Penz.) Penz and Sacc

Anthracnose represents an important disease of cowpea [Vigna unguiculata L. (Walp.)] caused by the hemibiothrophic fungus Colletotrichum gloeosporioides that drastically reduces cowpea field production. In this study we investigated some biochemical aspects underlying the incompatible interaction between a resistant cowpea genotype and C. gloeosporioides using a proteomic approach. Analyses of two-dimensional gel electrophoresis patterns and protein identification indicate C. gloeosporioides infection-dependent cowpea leaf proteome changes associated with metabolism, photosynthesis, response to stress, oxidative burst and scavenging, defense signaling, and pathogenesis-related proteins. Moreover the C. gloeosporioides responsive proteins interaction network in cowpea revealed the interconnected modulation of key cellular processes involving particularly antioxidants proteins, photosynthetic apparatus forming proteins and proteins of the energetic metabolism that interact with each other suggesting that their expression changes are also important for resistance of cowpea to C. gloeosporioides.

English

Cowpea is popular in Eastern Kenya where it is attractive to farmers because of its high economic value and the belief that it does not require many external inputs. Farmers are however discouraged to grow the crop in this region due to massive attack by a parasitic weed Alectra vogelii (Benth). Yield losses due to A. vogelii have being estimated to range from 50 to 100% in Mbeere, Kitui and Makueni districts. No single method however is available to farmers in these regions in control of the parasitic weed. Combining several control methods, as in the management of Striga spp in Western Kenya should be a sustainable option. Field studies were conducted in 2010 and 2011 at Kenya Agricultural Research Institute (KARI), Kiboko farm to determine the response of 143 cowpea genotypes to Alectra infestation. The aim for the study was to identify resistant genotypes that could be used in breeding programme. Significant differences were observed amongst cowpea genotypes in days to first Alectra emergence, number of Alectra shoots emerged at 6, 8, 10 and 12 week after planting and grain yield. Cowpea genotypes Kir/Nya-005 and Mbe/Mach-022 showed complete resistance to Alectra while Ken-Kunde, M66 and K80 (all commercial varieties) supported the highest number of Alectra shoots. Grain yield loss in the three susceptible varieties was 80, 79 and 50% respectively. On the other hand, Sia/Cia-004, Mbe/Mach-014 and Kib-006 had high grain yields despite the high number of Alectra shoots present. There was a strong correlation (r = -0.57) between grain yield and number of Alectra shoots emerged at 12 weeks after planting. A significant negative (r = -0.37) correlation was also obtained between pod number per plant and number of emerged Alectra shoots at 12 weeks after planting. This negative correlation proves the high accumulation dry matter in the cowpea roots at the expense of the pods thus decreasing grain yield. This information showed that there is sufficient genetic variability in the cowpea genotypes studied, which can be exploited in breeding improved cowpea varieties for resistance to A. vogelii in Kenya. A great progress towards developing improved cowpea variety that meets farmer’s preferences with durable resistance to A. vogelii can be achieved if the genes from the resistant and tolerant local cowpea cultivars identified in this study could be introgressed into the adapted susceptible improved varieties. This will increase the potential impact of adoption of resistant cowpea varieties in the zones. Key words: Cowpea, Alectra vogelii, Resistance/tolerance and grain yield.

Effects of initial infestation levels on damage to resistant and susceptible cowpea by Callosobruchus maculatus (Fabr.)

Cowpea cultivars Ife Brown and IT84S-2246-4 were screened for resistance and susceptibility to Callosobruchus maculatus (Fabr.) infestation using different insect densities. The number of eggs laid on the seeds; number of seed bearing eggs and seed without eggs; mean number of eggs per seed; number of F1 progeny; number of seed with and without holes; mean number of seed with hole; and developmental time increased as insect population increased. Significantly, more eggs were laid on the seed of Ife Brown (Susceptible cowpea variety) than on IT84S-2246-4. However, low adult emergence and high development period were observed on IT84S-2246-4, indicating clear cultivar difference at all insect population. The assessment of the first filial generation (F1 progeny) of the two varieties showed further that oviposition reduced in the insect that emerged from IT84S-2246-4 (resistant variety) when reared on Ife Brown cowpea seeds. Irrespective of cowpea varieties used in the experiment, egg laid and adult emergence by C. maculatus significantly increased with an increase in insect density.

Variant vicilins from a resistant Vigna unguiculata lineage (IT81D-1053) accumulate inside Callosobruchus maculatus larval midgut epithelium

It has been demonstrated that variant vicilins are the main resistance factor of cowpea seeds (Vigna unguiculata) against attack by the cowpea beetle Callosobruchus maculatus. There is evidence that the toxic properties of these storage proteins may be related to their interaction with glycoproteins and other microvillar membrane constituents along the digestive tract of the larvae. New findings have shown that following interaction with the microvilli, the vicilins are absorbed across the intestinal epithelium and thus reach the internal environment of the larvae. In the present paper we studied the insecticidal activity of the variant vicilins purified from a resistant cowpea variety (IT81D-1053). Bioassays showed that the seeds of this genotype affected larval growth, causing developmental retardation and 100% mortality. By feeding C. maculatus larvae on susceptible and IT81D-1053 derived vicilins (FITC labelled or unlabelled), followed by fluorescence and immunogold cytolocalization, we were able to demonstrate that both susceptible and variant forms are internalized in the midgut cells and migrate inside vesicular structures from the apex to the basal portion of the enterocytes. However, when larvae were fed with the labelled vicilins for 24h and then returned to a control diet, the concentration of the variant form remained relatively high, suggesting that variant vicilins are not removed from the cells at the same rate as the non-variant vicilins. We suggest that the toxic effects of variant vicilins on midgut cells involve the binding of these proteins to the cell surface followed by internalization and interference with the normal physiology of the enterocytes, thereby affecting larval development in vivo.

Application of Cry1Ab/Ac Bt strip for screening of resistance for Maruca vitrata in cowpea

Maruca vitrata is a significant constraint to cowpea production in most cowpea growing areas of sub-Saharan Africa. Yield losses caused by M. vitrata in these regions are estimated in millions of tons annually and the prevalence of M. vitrata infestation is steadily increasing. Recombinant DNA technology have led to development of some cowpea lines with Maruca resistance as well as other important agronomic traits but it is time-consuming and difficult to screen for the resistant trait especially in the segregating populations using conventional screening techniques, which will lead to delay in the development of Maruca resistant cowpea varieties. The use of allele-based selection tool will make it easier to select plant traits and reduce the time needed to develop new Maruca resistant cowpea varieties. In this study, the efficacy of using Cry1Ab/Ac Bt strip for detecting Maruca resistant transgene in transgenic cowpea was systematically investigated for the first time through field derived progenies. The results show that the Cry1Ab/Ac Bt strip was effective for detecting the presence of the resistant gene in cowpea genome. Maruca resistant plants were successfully screened from the segregating cowpea plants and the genetics of the gene was monitored. The Cry1Ab/Ac Bt strip was found to be suitable for genetic analysis of the Maruca resistant transgene in cowpea. This study has demonstrated the precision of using Cry1Ab/Ac Bt strips as a screening tool of transgenic lines containing Cry1Ab gene, this has an importance in the hybridization programme where genotypes having cry gene can be distinguished at seedling stage at lesser time, with the potential of putting the breeding process on a fast track and increase the efficiency of breeding activities. Keywords: Bacillus thuriengiensis , Cry1Ab/Ac Bt strips, transgenic cowpea, Maruca vitrata . African Journal of Biotechnology Vol. 12(40), pp. 5869-5874

English

Maruca vitrata is a significant constraint to cowpea production in most cowpea growing areas of  sub-Saharan Africa. Yield losses caused by M. vitrata in these regions are estimated in millions of tons annually and the prevalence of M. vitratainfestation is steadily increasing. Recombinant DNA technology have led to development of some cowpea lines with Maruca resistance as well as other important agronomic traits but it is time-consuming and difficult to screen for the resistant trait especially in the segregating populations using conventional screening techniques, which will lead to delay in the development of Maruca resistant cowpea varieties. The use of allele-based selection tool will make it easier to select plant traits and reduce the time needed to develop new Maruca resistant cowpea varieties. In this study, theefficacy of using Cry1Ab/Ac Bt strip for detecting Maruca resistant transgene in transgenic cowpea was systematically investigated for the first time through field derived progenies. The results show that the Cry1Ab/Ac Bt strip was effective for detecting the presence of the resistant gene in cowpea genome. Maruca resistant plants were successfully screened from the segregating cowpea plants and the genetics of the gene was monitored.  The Cry1Ab/Ac Bt strip was found to be suitable for genetic analysis of the Maruca resistant transgene in cowpea. This study hasdemonstrated the precision of using Cry1Ab/Ac Bt strips as a screening tool of transgenic lines containing Cry1Ab gene, this has an importance in the hybridization programme where genotypes having cry gene can be distinguished at seedling stage at lesser time, with the potential of putting the breeding process on a fast track and increase the efficiency of breeding activities.   Key words: Bacillus thuriengiensis, Cry1Ab/Ac Bt strips, transgenic cowpea, Maruca vitrata.