Pathogenic response of seed mycoflora associated with cowpea,Vigna unguiculata

Cowpea, Vigna unguiculata (L.) Walp. (Fabaceae), has always been an important grain legume in tropical countries and a veritable source of dietary protein for the teeming population of human and livestock. The crop is often attacked and deteriorated by a number of pathogens; many of these are seed-borne fungi, which produce an adverse effect on the growth and development of plants. In this study, the effects of fungi on germinability and seedling health were determined, and the pathogenicity was established. Eleven fungal species were isolated from cowpea seeds which were used as inocula on cowpea. All the tested fungi show a reduction in the germination rate of the cowpea plants, and different types of disease symptoms including reduction in shoot and root lengths were noted for different pathogenic fungi. The symptoms included seed rot, seedling rot, yellowing of leaves, deformation of leaves, etc. Many phytopathogenic fungi penetrate deep into the seeds. These fungi produce enzymes, toxins and metabolites which adversely affect the plant growth and produce characteristic symptoms and cause deterioration of this important protein-rich crop.     

Development of transgenic imazapyr-tolerant cowpea (Vigna unguiculata)

Key message

Here we present the development of cowpea lines tolerant to a herbicide from imidazoline class (imazapyr). Plants presented tolerance to fourfold the commercial recommended dose for weed control.

Abstract

Cowpea is one of the most important and widely cultivated legumes in many parts of the world. Its cultivation is drastically affected by weeds, causing damages during growth and development of plants, competing for light, nutrients and water. Consequently, weed control is critical, especially using no-tillage farming systems. In tropical regions, no-till farming is much easier with the use of herbicides to control weeds. This study was conducted to evaluate the possibility of obtaining transgenic cowpea plants resistant to imidazolinone, which would facilitate weed control during the summer season. The biolistic process was used to insert a mutated acetohydroxyacid synthase coding gene (Atahas) which confers tolerance to imazapyr. The transgene integration was confirmed by Southern blot analysis. Out of ten lines tested for tolerance to 100 g ha^?1 imazapyr, eight presented some tolerance. One line (named 59) revealed high herbicide tolerance and developmental growth comparable to non-transgenic plants. This line was further tested for tolerance to higher herbicide concentrations and presented tolerance to 400 g ha^?1 imazapyr (fourfold the commercial recommended dose) with no visible symptoms. Line 59 will be the foundation for generating imidazolinone-tolerant cowpea varieties, which will facilitate cultivation of this crop in large areas.     

Alarming increase in the incidence of Cucumber mosaic virus in cowpea (Vigna unguiculata (L.) Walp.) in northern Nigeria

Cowpea plays a key nutritional role in the diet of the Nigerian people. Viral diseases are a major limitation to cowpea production worldwide, and thus, constant viral surveillance is crucial for monitoring and management purposes. In this study, cowpea leaf samples from fields in three northern Nigeria states, Kano, Kaduna and Niger, were tested to determine the status of six common viruses previously reported in these cowpea-producing states following the release of virus-resistant varieties. Cowpea aphid-borne mosaic virus (CABMV), Blackeye cowpea mosaic virus (BICMV), Cowpea mottle virus, Southern bean mosaic virus and Cucumber mosaic virus (CMV) were detected. Cowpea yellow mosaic virus, which was previously reported in all three states, was not detected in any of the samples tested, while CMV that was previously regarded as unimportant to cowpea production in Nigeria had the highest incidence in all three states, and the overall highest incidence of 58.8%, while CABMV had the lowest incidence (7.5%). CMV was also present in seven of the ten mixed infection combinations detected. Dual infection of CMV and BICMV, which often results in cowpea stunt, the most devastating cowpea disease in the USA, was the most frequently detected mixed infection (28.1%) and was detected in all three states. This observed elevation in CMV infection in cowpea must be closely monitored and swiftly managed to avert possible devastating crop yield losses.     

Temporal and tissue localization of a cowpea (Vigna unguiculata) cystatin

A cowpea ( Vigna unguiculata L. Walpers cv. Pitiúba) cystatin was analysed to determine its localization during development and germination of cowpea seeds, using western blotting with a specific antiserum. The pattern of immunoreactive proteins changed during development, with the major reactive bands present in dried seeds being mobilized after a 62-h period of imbibition. Immunohistochemical analysis revealed that cowpea cystatin is distributed in both embryonic axes and cotyledons with the highest level being present in the outer cell walls of the adaxial surface of the cotyledons.     

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.     

Occurrence of latent virus infection in visually-rated cowpea (Vigna unguiculata L. Walp) seedlings

Abstract

The presence of latent infections was studied in five cowpeas varieties. Seeds of the varieties were planted and the seedlings inoculated with antigens from Cucumber mosaic virus (CMV) genus Cucumovirus, Bean common mosaic virus (BCMV) genus Potyvirus (Blackeye cowpea mosaic virus strain), Southern bean mosaic virus (SBMV) genus Sobemovirus and Cowpea mottle virus (CPMoV) genus Carmovirus seven days after planting. Seedlings expressing symptoms were rouged at two weeks after inoculation, while asymptomatic ones were subjected to serological indexing to detect the presence/absence of latent infection. Protein A-sandwich enzyme-linked immunosorbent assay (PAS ELISA) was employed for the serological detection of CMV, SBMV and CPMoV, while antigen-coated plate (ACP) ELISA was used to detect BCMV in the asymptomatic plants. Cowpea seedlings without virus symptoms but with positive serological reactions were considered as being latently infected. All of the inoculated TVu 1272 and SuVita-2 plants showed symptoms consistent with CMV and CPMoV infections, respectively. The rate of CMV latent infection was high in TVu 1179 (14.5%), low in SuVita-2 (1.3%) but not recorded in TVu 1272.     

Field resistance in cowpea, Vigna unguiculata to the cowpea moth, Cydia ptychora

Field evaluation of 26 selected cowpea cultivars showed that several of them had good levels of resistance to Cydia ptychora; TVu 946, Adzuki, ‘Vita 5’ and Igbirra were the most resistant. Percentage seed damaged by the larvae was positively correlated with days to 50% flowering (r= 0.39*), days to 50% pod ripening (r= 0.48*) and weight of 100 seeds (r= 0.56**). All four cultivars were early maturing, had semi-determinate or determinate plant types and carried their pods above the leaf canopy. No single morphological character could be related to the degree of seed damage but some association with phenological characteristics was indicated. Earliness, determinacy and uniform pod maturity were important plant characters associated with the expression of field resistance. Even if these characters are not causative, they provide a rational basis for screening of a large germplasm to identify or synthesise materials with greater resistance to this moth.     

Water relations of three cowpea cultivars (Vigna unguiculata,L.)

The effects of soil water stress imposed at different growth stages of three cowpea varieties, namely, Adzuki (an erect type), Ife Brown (semi erect) and New Era (a spreading type) on growth, floral abscission and yield were investigated in the greenhouse. Stomatal density, aperture and behaviour as influenced by soil water potential was also evaluated. Root system development, grain yield, soil water content and potentials were determined in a field experiment. Soil moisture stress significantly reduced the growth and yield (34–46%) of the three cowpea cultivars. Although grain yield reduction was highest (36.8%) when stress was imposed at flowering/podding stage for Ife Brown, the variety New Era showed no reduction in yield. Floral abortion which increased with a decrease in matric potential may also be linked with imperfect aeration conditions. The choice of these cultivars for intercropping purposes should take cognisance of depth of rooting, lateral root spread, and root density, which affect water extraction at different soil depths, leaf area index, stomatal density, aperture and behaviour to decreased soil water potential all of which differed widely amongst the cultivars.     

Effect of high soil temperatures on nodulation of cowpea, Vigna unguiculata

Cowpeas, inoculated with one of five effective strains of Rhizobium isolated from African soils, were grown at root temperatures of 30 ^oC continuously or at 36, 38, 40, 42 and 44 ^oC for 5 h/day and returned to glasshouse ambient for the intermediate period. Growth was best at 30 and 36 ^oC; above 40 ^oC growth was poor and no nodules formed. At 40 ^oC two strains failed to nodulate. Symbiotic performance was not dependent only on nodule production as nodule efficiency varied inversely with temperature. The number of nodules formed by strain R5000 after exposure of inoculated seed or seedlings to 40,42 or 44 ^oC for 5 h/day on each of 3 or 6 days depended on the age of plant and the duration of exposure to stress. When exposed to 42 or 44 ^oC during the first 3 days after sowing nodulation was reduced from 18 to 1–3 nodules/plant even after a further 40 days growth at ambient (30 day, 20 ^oC night). Nodulation was unaffected when 10–15-day-old seedlings were exposed to the same conditions. Numbers of strain R5000 on seed declined rapidly following three daily exposures of 5 h at 39 and 42 ^oC; at 45 ^oC less than three bacteria survived on each seed. Other rhizobia of the cowpea group varied greatly in their toleration of high temperatures, some survived well at 45 ^oC whereas others behaved like R5000.     

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.     

Allozyme diversity of cultivated cowpea Vigna unguiculata (L.) Walp.

A survey of allozyme variation in cultivar-groups of cowpea [Vigna unguiculata (L.) Walp.] was undertaken by examining 21 enzyme systems encoded by 36 loci in 271 accessions representing the five cultivar-groups. Very low levels of variation were found within accessions, which is typical of self-pollinating species. Little variation was also found among accessions. Compared with other legume crops, V. unguiculata is depauperate in allozyme variation. We found an average of 1.61 alleles per locus with 42% of the loci polymorphic and a total heterozygosity of 0.061. Of the variation present, 90% was found within cultivar-groups, while 10% was among cultivar-groups. Data analyses revealed continuous variation among cultivar-groups and geographic regions with the accessions failing to segregate into discrete morphophysiological or geographic clusters. However, evolved cultivar-groups (cv.-gr. Melanophthalmus and cv.-gr. Sesquipedalis) appear to be less diverse than their putative primitive cultivar-group progenitors. Due to the lack of availability of critical material, no clear center of origin can be established. However, the data presented suggest that Northeast Africa could be a possible center of domestication. Received: 18 February 1999 / Accepted: 4 November 1999     

The genome of cowpea (Vigna unguiculata [L.] Walp.)

Cowpea (Vigna unguiculata [L.] Walp.) is a major crop for worldwide food and nutritional security, especially in sub‐Saharan Africa, that is resilient to hot and drought‐prone environments. An assembly of the single‐haplotype inbred genome of cowpea IT97K‐499‐35 was developed by exploiting the synergies between single‐molecule real‐time sequencing, optical and genetic mapping, and an assembly reconciliation algorithm. A total of 519 Mb is included in the assembled sequences. Nearly half of the assembled sequence is composed of repetitive elements, which are enriched within recombination‐poor pericentromeric regions. A comparative analysis of these elements suggests that genome size differences between Vigna species are mainly attributable to changes in the amount of Gypsy retrotransposons. Conversely, genes are more abundant in more distal, high‐recombination regions of the chromosomes; there appears to be more duplication of genes within the NBS‐LRR and the SAUR‐like auxin superfamilies compared with other warm‐season legumes that have been sequenced. A surprising outcome is the identification of an inversion of 4.2 Mb among landraces and cultivars, which includes a gene that has been associated in other plants with interactions with the parasitic weed Striga gesnerioides. The genome sequence facilitated the identification of a putative syntelog for multiple organ gigantism in legumes. A revised numbering system has been adopted for cowpea chromosomes based on synteny with common bean (Phaseolus vulgaris). An estimate of nuclear genome size of 640.6 Mbp based on cytometry is presented.     

Expression of multiple proteins using full-length and deleted versions of cowpea mosaic virus RNA-2

The use of multiple copies of vectors based on either full-length or deleted versions of cowpea mosaic virus RNA-2 for the production of heteromeric proteins in plants was investigated. Co-infiltration of two full-length RNA-2 constructs containing different marker genes into Nicotiana benthamiana in the presence of RNA-1 showed that the two foreign proteins were efficiently expressed within the same cell in inoculated tissue. Furthermore, the proteins were co-localized to the same subcellular compartments, an essential prerequisite for heteromer formation. However, segregation of two separate RNA-2 molecules, and therefore expression of the two proteins, was observed on systemic spread of the recombinant viruses. Thus, efficient assembly of heteromeric proteins is likely to occur only in inoculated tissue. To determine the optimum approach for expression in inoculated tissue, the heavy and light chains of the blood group-typing immunoglobulin G (IgG) C5-1 were inserted into full-length and deleted versions of RNA-2, and the constructs were agroinfiltrated in the presence of RNA-1. The results obtained showed that full-size IgG molecules accumulated using both approaches, but that the levels were significantly higher when deleted RNA-2 vectors were used. The levels were also greatly enhanced by the inclusion of an endoplasmic reticulum retention signal at the C-terminus of the heavy chain. As the potential benefit of using full-length RNA-2 constructs, the ability to spread systemically, appears to be irrelevant to the production of heteromeric proteins, the use of deleted versions of RNA-2 is clearly advantageous, particularly as they offer the benefit of biocontainment.     

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.     

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.     

A mosaic disease of cowpea (Vigna sinensis Endl.)

A mosaic disease of cowpea ( Vigna sinensis Endl.), prevalent in the bean-growing regions of China, is described.
The characteristic symptoms of the disease are conspicuous mosaic patterns, deformation and distortion of leaves and stunting of the plant.
The cowpea mosaic virus is transmitted by Aphis rumicis L., Macrosiphum pisi Kalt. and Aphis gossypii Glov. It is also transmitted by artificial juice inoculation. The virus is seed-bome. It withstands ageing m vitro for 3 days at 220 C. Its inactivation temperature is approximately 62° C. its tolerance to dilution is about 1: 3000.
Cowpea mosaic virus infects lima bean and Adzuki bean in addition to cowpea.
The relation of this virus to cowpea mosaic virus described by McLean and asparagus-bean mosaic virus described by Snyder is discussed.     

The replication of cowpea mosaic virus

Cowpea mosaic virus (CPMV) is the type member of the comovirus group, which contains 14 different plant viruses that have the same structural organization of genomic RNAs and virions and use the same mechanism for expression of the viral RNAs. The combined structure and organization of the two CPMV genomic RNAs is strikingly similar to that of the single genome of animal picornaviruses. This suggests a common ancestry and similar replication mechanisms. CPMV is by far the best-studied comovirus and we shall limit this review to some recent data on this virus. For additional general information the reader is referred to other recent reviews on CPMV and comoviruses^1,2.     

Scab disease of cowpea (Vigna unguiculata) caused by Sphaceloma a species of the fungus

Scab disease of cowpea (Vigna unguiculuta) was shown to be caused by Sphaceloma sp. It affects all above ground parts of the plant. The first symptom of the disease, appearing within 3 to 6 days of inoculation, is puckering of the lamina. Spots on mature leaves are white with or without brown margins. Typical scab lesions on petiole, stem, peduncle and pod are white turning dark brown when chlamydospores are formed and are oblong-elongate except for pod lesions that are ovoid. The most destructive phase is scab of the flowering axis which causes flower and, or, pod abortion or completely prevents flower formation. Inoculation of asparagus pea (V. sesquipedalis) with a cowpea isolate of Sphaceloma sp. produced symptoms similar to those on cowpea. Inoculated hyacinth bean (Lablab niger) produced atypical mild lesions. The following legumes were not affected when artificially inoculated with the fungus: black gram (Phaseolus mungo), green gram (P, aureus), French bean (P. vulgaris), Lima bean (P. lunatus), groundnut (Arachis hypogaea), and soyabean (Glycine max). The characteristics of the fungus on potato dextrose agar are described.