Inheritance of resistance to the cowpea aphid in cowpea

Summary Inheritance of resistance to cowpea aphid, Aphis craccivora Koch, in three resistant cultivars of cowpea, Vigna unguiculata (L.) Walp, was studied. The parents, F₁ and F₂ population were grown in an insect-proof screenhouse. Each 3-day-old seedling was infested with 10 apterous adult aphids. Seedling reaction was recorded when the susceptible check was killed. The segregation data revealed that the resistance of ICV11 and TVU310 is governed by single dominant genes. All the F₂ seedlings of the cross ICV10xTVU310 were resistant, indicating that they have the same gene for resistance. However, the F₂ populations from the crosses ICV10xICV11 and ICV11xTVU310 segregated in a ratio of 151, indicating that the dominant genes in ICV11 and TVU310 are non-allelic and independent of each other. The resistance gene of ICV10 and TVU310 is designated as Ac₁ and that of ICV11 as Ac₂.     

Antimicrobial proteins from cowpea root exudates: inhibitory activity against Fusarium oxysporum and purification of a chitinase-like protein

Plants exude a variety of substances through their external surfaces and from germinating seeds, some of which have an inhibitory action against plant pathogens. The aim of this study was the investigation and characterization of defense proteins present in exudates from roots of cowpea seedlings (Vigna unguiculata (L.) Walp.). Root exudates were collected from seedlings that were grown hydroponically in three different media, including, 100 mM sodium acetate buffer pH 4.5, water pH 6.0 and 100 mM sodium phosphate buffer pH 7.5. The proteins from these exudates were analyzed by SDS–PAGE and SDS–Tricine–PAGE and the presence of antimicrobial proteins in the exudates was investigated by immunological and enzymatic assays. Results showed that roots from cowpea seedlings contained -1,3-Glucanases, chitinases and lipid transfer proteins (LTPs), all of which may potentially function as plant defense proteins. Immunolocalization of one of these proteins, chitinase, revealed its presence in the xylem cell wall vessel elements. These exudates also demonstrated an inhibitory effect on the growth of the fungus, Fusarium oxysporum, in vitro. The results suggest that plant roots may exude a variety of proteins that may function to repress the growth of root pathogenic fungi.     

Induction of an anionic peroxidase in cowpea leaves by exogenous salicylic acid

Two isoperoxidases were detected in cowpea (Vigna unguiculata) leaves. Treatment of the primary leaves with 10mM salicylic acid increased the total peroxidase activity contributed by the anionic isoform. To isolate both the anionic and cationic peroxidases the leaf crude extract was loaded on a Superose 12 HR 10/30 column followed by chromatography on Mono-Q HR 5/5. Both enzymes were stable in a pH range from 5 to 7. The optimum-temperatures for the cationic and anionic peroxidase isoforms were, respectively, 20-30 degrees C and 30 degrees C. The dependence of guaiacol oxidation rate varying its concentration at constant H(2)O(2) concentration showed, for both enzymes, Michaelis-Menten-type kinetic. Apparent K(m)(s) were 0.8 and 4.8 microM for the cationic and anionic isoperoxidases, respectively.     

Roots and leaves display contrasting oxidative response during salt stress and recovery in cowpea

In this study, we compare some antioxidative responses of leaves and roots associated to growth reduction in cowpea plants (Vigna unguiculata) during short-term salt stress and recovery. The salt treatment was imposed (200 mM NaCl) for six consecutive days and the salt withdrawal after 3 d. The salt treatment caused an almost complete cessation in the relative growth rate of both leaves and roots. Although NaCl withdrawal has induced an intense reduction in the Na(+) content from the leaves and roots, the growth recovery was slight, after 3 d. The leaf lipid peroxidation was increased in salt-stressed plants and slightly reduced in recovered plants after 3 d. Surprisingly, in the salt-stressed roots it decreased markedly after 3 d treatment and in the pre-stressed/recovered roots it was restored to levels near to the control. In leaves, catalase (CAT) activity showed a rapid and prominent decrease after 1 d of NaCl treatment and salt withdrawal had no effect on its recovery. In contrast, the root CAT activity was not changed by effects of both NaCl and salt withdrawal, over time interval. Leaf superoxide dismutase (SOD) activity did not change in all treatments, whereas in roots it significantly decreased after 3 d of salt treatment and recovered after NaCl withdrawal. Contrasting to the other enzymes, the guaiacol-peroxidase activity increased in leaves and roots, reaching almost 200% of control values and it significantly decreased in both organs from the pre-stressed/recovered plants. In conclusion, cowpea roots and leaves present distinct mechanisms of response to lipid peroxidation and CAT and SOD activities during salt stress and recovery. However, these responses and/or the oxidative damages caused by reactive oxygen species were not related with the growth reduction.     

Regeneration of plants from primary leaves of cowpea

Plants were regenerated from the in vitro cultured explants of primary leaves of cowpea (Vigna unguiculata L. Walp). Primary leaves, including the intact petiole, were excised from three-day-old seedlings and cultured on Gamborg's B5 basal medium containing 8×10^–7 M 2,4,5-trichlorophenoxyacetic acid, 1×10^–2 M L-glutamine and 1×10^–4 M adenine sulfate. Callus formed at the petiole end. Prolific shoot regeneration occurred when this callus was transferred to B5 basal medium containing 5×10^–6 M 6-benzyl-aminopurine (BAP). Regenerated shoots rooted in growth-regulator-free B5 basal medium and were established in soil.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - NAA 1-napthalene acetic acid - 2,4,5-T 2,4,5-trichloro-phenoxyacetic acid     

Molecular cloning of glutathione reductase cDNAs and analysis of GR gene expression in cowpea and common bean leaves during recovery from moderate drought stress

Two cDNAs of the enzyme glutathione reductase (GR; EC 1.6.4.2) encoding a dual-targeted isoform (dtGR) and a cytosolic isoform (cGR), were cloned from leaves of common bean (Phaseolus vulgaris L.). Moderate drought stress (Psi w=-1.5MPa) followed by re-watering was applied to common bean cultivars, one tolerant to drought (IPA), the other susceptible (Carioca) and to cowpea (Vigna unguiculata L. Walp) cultivars, one tolerant to drought (EPACE-1), and the other susceptible (1183). mRNA levels were much higher for PvcGR than for PvdtGR in all cases. Moderate drought stress induced an up-regulation of the expression of PvcGR in the susceptible cultivars. On the contrary, PvdtGR expression decreased. In the tolerant cowpea EPACE-1, GR gene expression remained stable under drought. During recovery from drought, an up-regulation of the two GR isoforms occurred, with a peak at 6-10h after re-hydration. This suggests that moderate drought stress may lead to a hardening process and acclimation tolerance. The role of GR isoforms in plant tolerance and capacity to recover from drought stress is discussed.     

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.     

In vitro somatic embryogenesis from cell suspension cultures of cowpea [Vigna unguiculata (L.) Walp]

We report, an efficient protocol for plantlet regeneration from the cell suspension cultures of cowpea through somatic embryogenesis. Primary leaf-derived, embryogenic calli initiated in MMS [MS salts (Murashige and Skoog 1962) with B5 (Gamborg et al. 1968) vitamins] medium containing 2,4-Dichlorophenoxyacetic acid (2,4-D), casein hydrolysate (CH), and l-Glutamic acid-5-amide (Gln). Fast-growing embryogenic cell suspensions were established in 0.5 mg l^–1 2,4-D, which resulted in the highest recovery of early stages of somatic embryos in liquid MMS medium. Embryo development was asynchronous and strongly influenced by the 2,4-D concentration. Mature monocotyledonary-stage somatic embryos were induced in liquid B5 medium containing 0.1 mg l^–1 2,4-D, 20 mg l^–1 l-Proline (Pro), 5 M Abscisic acid (ABA), and 2% mannitol. B5 medium was found superior for the maturation of somatic embryos compared to MS and MMS media. The importance of duration (5 d) for effective maturation of somatic embryos is demonstrated. A reduction in the 2,4-D level in suspensions increased the somatic embryo induction and maturation with decreased abnormalities. Sucrose was found to be the best carbon source for callus induction while mannitol for embryo maturation and maltose for embryo germination. Extension of hypocotyls and complete development of plantlet was achieved in half-strength B5 medium supplemented with 3% maltose, 2500 mg l^–1 potassium nitrate, and 0.05 mg l^–1 thidiazuron (TDZ) with 32% regeneration frequency. Field-established plants were morphologically normal and fertile. This regeneration protocol assures a high frequency of embryo induction, maturation, and plantlet conversion.     

Change in phenylalanine ammonia lyase activity and isozyme patterns of polyphenol oxidase and peroxidase by salicylic acid leading to enhance resistance in cowpea against <Emphasis Type="Italic">Rhizoctonia solani</Emphasis>

In this paper, we describe a study concerning the determination of phenylalanine ammonia lyase (PAL) activities leading to decline in disease progress caused by Rhizoctonia solani after application of salicylic acid (SA). Two applications of 1.4 mM SA (pH 6.5) followed by inoculation with Rhizoctonia solani resulted in a quantitative change in polyphenol oxidase (PPO), peroxidase (PRX) isoforms and increase in PAL activities from 4.38 to 19.48 unit g⁻¹ (FM) h⁻¹ in Bundel-1, UPC-4200 and IFC-902 cowpea genotypes. Increase in PAL activities was further observed specifically in UPC-4200 when plants were exposed with Rhizoctonia solani spores. Total soluble proteins did not change after SA treatment, however they were significantly increased in SA sprayed-inoculated UPC-4200 genotype. Of the ten detected isoforms of polyphenol oxidase, isoforms 7 and 10, and isoform 4 of peroxidase showed increased activities by SA application. The disease symptoms measured as areas under progress curve (AUDPC) indicated less A value in SA sprayed Bundel-1 and UPC-4200 genotypes over their controls.     

Effect of maize plants on colonisation of cowpea plants by bean flower thrips,Megalurothrips sjostedti

The effect of non-host maize plants on colonisation of cowpea byMegalurothrips sjostedti (Trybom) (Thysanoptera; Thripidae) was investigated. There were no differences in population density and activity ofM. sjostedti on sole cowpea crop and mixed cowpea/maize crop during the colonisation phase (i.e. 10–30 days after emergence of the plants. However, subsequentlyM. sjostedti numbers were lower in the mixed than in the sole crop, suggesting that maize did not interfere with colonisation of cowpea crop by thrips. In a choice situation, higher numbers ofM. sjostedti oriented towards, and settled on, sole cowpea plants than on those mixed with maize. Olfactory tests indicated that fewer thrips oriented towards a cowpea/maize mixed odour source. When equal numbers of thrips were introduced into the centre of sole- and mixed-cropped cowpea plots, the thrips became randomly distributed in each plot. Fewer thrips were recovered from the mixture than from the sole crop. It is concluded that, although non-host plant odours do not reduce thrips colonisation they interfere with host plant utilisation.     

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     

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.     

Identification and pathogenic characterization of endophytic <Emphasis Type="Italic">Fusarium</Emphasis> species from cowpea seeds

Isolates of Fusarium were obtained and identified from seeds of cowpea, Vigna unguiculata (L.) Walp., by means of blotter tests and slide cultures. Species were differentiated according to the morphology of the macroconidia, microconidia and their arrangement in chains or false heads, the size and type of conidiophore, and the presence or absence of chlamydospores. The species were identified as F. semitectum, F. equiseti, F. oxysporum, F. solani, F. anthophilum, F. sporotrichioides, F. moniliforme, and Fusarium sp. Among the species, F. semitectum was the most frequently detected. None of these species were pathogenic when inoculated in susceptible cowpea cultivar (BR 17- Gurgueia). But, an isolate of F. oxysporum f. sp. tracheiphilum used as a standard of comparison for pathogenicity (control) induced symptoms of yellowing, vascular wilting, and death of a susceptible cowpea cultivar under the same environmental conditions.     

Characterization of two cDNAs for novel drought-inducible genes in the highly drought-tolerant cowpea

Ten cDNAs for drought-inducible genes were isolated using differential screening of a cDNA library prepared from 10-hr dehydrated cowpea plants,Vigna unguiculata (S. Iuchi, K. Yamaguchi-Shinozaki, T. Urao, T. Terao, K. Shinozaki; Plant Cell Physiology, 1996 in press). Two of the cDNA clones, designated CPRD12 and CPRD46, were sequenced and characterized. The CPRD12 and CPRD46 cDNAs encode putative proteins related to nonmetallo-short-chain alcohol dehydrogenase (CPRD12) and chloroplastic lipoxygenase (CPRD46). Northern blot analysis revealed that these genes are induced by high-salinity stress and exogenous abscisic acid, but not by cold stress. The CPRD46 gene is also responsive to heat stress and methyl jasmonate and salicylic acid. Genomic Southern blot analysis suggested that CPRD12 constitutes a small gene family, but that CPRD46 is a single copy gene. We discuss the possible functions of these two CPRD gene products under drought stress.     

Partitioning of biologically fixed nitrogen in cowpea during pod development

Two days after exposure of roots to¹⁵N labeled N₂, partitioning of biologically fixed N into leaves, stems, peduncles, pods, roots and nodules was measured in the early pod development stage of cowpea (Vigna unguiculata (L.). The experimental objective was to determine the quantity of biologically fixed N that is incorporated into vegetative tissue before being mobilized to pods. For the three varieties of cowpea included in the experiment a maximum of 50% of the N, biologically fixed two days earlier, was contained in the pods. The remaining N was distributed throughout the vegetative portion of the plant with at least 30% in stems and leaves which indicates that much of the newly fixed N must cycle through a N pool in these tissues before reaching the pods.     

Developmental pattern formation of somatic embryos induced in cell suspension cultures of cowpea [Vigna unguiculata (L.) Walp]

The sequence of events in the functional body pattern formation during the somatic embryo development in cowpea suspensions is described under three heads. Early stages of somatic embryogenesis were characterized by both periclinal and anticlinal cell divisions. Differentiation of the protoderm cell layer by periclinal divisions marked the commencement of somatic embryogenesis. The most critical events appear to be the formation of apical meristems, establishment of apical-basal patterns of symmetry, and cellular organization in oblong-stage somatic embryo for the transition to torpedo and cotyledonary-stage somatic embryos. Two different stages of mature embryos showing distinct morphology, classified based on the number of cotyledons and their ability to convert into plantlets, were visualized. Repeated mitotic divisions of the sub-epidermal cell layers marked the induction of proembryogenic mass (PEM) in the embryogenic calli. The first division plane was periclinally-oriented, the second anticlinally-oriented, and the subsequent division planes appeared in any direction, leading to clusters of proembryogenic clumps. Differentiation of the protoderm layer marks the beginning of the structural differentiation in globular stage. Incipient procambium formation is the first sign of somatic embryo transition. Axial elongation of inner isodiametric cells of the globular somatic embryo followed by the change in the growth axis of the procambium is an important event in oblong-stage somatic embryo. Vacuolation in the ground meristem of torpedo-stage embryo begins the process of histodifferentiation. Three major embryonic tissue systems; shoot apical meristem, root apical meristem, and the differentiation of procambial strands, are visible in torpedo-stage somatic embryo. Monocotyledonary-stage somatic embryo induced both the shoot apical meristem and two leaf primordia compared to the ansiocotyledonary somatic embryo.     

Identification and survival of the causal organism of leaf smut disease of cowpea in Nigeria

Protomycopsis phaseoli (Ramak and Subram) is the causal agent of the cowpea leaf smut disease in Nigeria and not Entyloma vignae as claimed by some authors. This pathogen formed dark ash-grey to sooty-black lesions of 3–10 mm in diameter, while young lesions had yellow haloes. P. phaseoli produced dark reddish-brown chlamydospores that are globose to oval measured 23.8 μm, thick-walled and rugose. The chlamydospores germinated and produced globose vesicles. The pathogen grew on potato dextrose agar only when the leaf tissue was dipped in acidified water (1% H₂SO₄). The organism was slowly growing at 24–28 °C with snow white colour. Chlamydospores of P. phaseoli in infected cowpea leaves survived longer when buried in the soil for five months than when they were left on the soil surface for the same period at temperatures (26–27 °C) and humidity (70–82%) prevailing in Ibadan. Destruction of leaf debris before crop emergence, long period of rotation and no tillage cropping are suggested to prevent the onset and spread of leaf smut disease of cowpea. This revised version was published online in June 2006 with corrections to the Cover Date.     

Response of cowpea to variations in planting density and nutrient level

A greenhouse study was carried out using cowpea (Vigna unguiculata (L.) Walp.) grown in Perlite^® and inoculated with Nitragin^® to investigate the concentration of plant nutrients and planting density required for optimum biomass production. Five concentrations (full, 0.5, 0.2, 0.1 and 0.05 strength) of Bisseling's nutrient solution and five planting densities (one to five plants per pot) were tested in a factorial randomized Graeco-Latin square design. Growth was determined as fresh and dry weights of leaves, stems, petioles, roots, flowers and pods, and whole plant.Optimum biomass production was found at 0.5 strength nutrient solution and a density of one plant per pot. Plants were more sensitive to higher planting density than to alterations of nutrient level. Over a twenty-fold range of nutrient supply, whole plant biomass yield varied at most by 44%, whereas increasing planting density from one to five plants per pot decreased biomass production by as much as 77%. There is a decrease in the shoot/root ratio as nutrient level decreases. The data suggests a potential for higher seed production at the higher densities and lowest nutrient levels, but this data was inconclusive.     

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.     

Effects of water deficit on N2(C2H2) fixation in cowpea and groundnut

The effect of water deficit on nodulation, N₂ fixation, photosynthesis, and total soluble sugars and leghemoglobin in nodules was investigated in cowpea and groundnut. Nitrogenase activity completely ceased in cowpea with a decrease in leaf water potential ( _leaf) from –0.4 MPa to –0.9 MPa, while in groundnut it continued down to –1.7 MPa. With increasing water stress, the acetylene reduction activity (ARA) declined very sharply in cowpea, but ARA gradually decreased in groundnut. Even with mild water stress ( _leaf of 0.2 MPa), nodule fresh weight declined 50% in cowpea partly due to a severe nodule shedding whereas nodule fresh weight declined in groundnut only when _leaf decreased by 1.0 MPa. No nodule shedding was noticed even at a higher stress level in groundnut. Photosynthesis and stomatal conductance were also more stable in groundnut than in cowpea under water stress. There was a sharp increase in total soluble sugars and leghemoglobin in the nodules of groundut with water stress, but no definite trend could be found in cowpea.