Phylogeny and distribution of Bradyrhizobium symbionts nodulating cowpea (Vigna unguiculata L. Walp) and their association with the physicochemical properties of acidic African soils

In the N₂-fixing symbiosis, the choice of a symbiotic partner is largely influenced by the host plant, the rhizobial symbiont, as well as soil factors. Understanding the soil environment conducive for the survival and multiplication of root-nodule bacteria is critical for microbial ecology. In this study, we collected cowpea-nodules from acidic soils in Ghana and South Africa, and nodule DNA isolates were characterized using 16S–23S rRNA-RFLP, phylogenetic analysis of housekeeping and symbiotic genes, and bradyrhizobial community structure through canonical correspondence analysis (CCA). The CCA ordination plot results showed that arrow of soil pH was overlapping on CCA2 axis and was the most important to the ordination. The test nodule DNA isolates from Ghana were positively influenced by soil Zn, Na and K while nodule DNA isolates from South Africa were influenced by P. The amplified 16S–23S rRNA region yielded single polymorphic bands of varying lengths (573–1298 bp) that were grouped into 28 ITS types. The constructed ITS-dendrogram placed all the nodule DNA isolates in five major clusters at low cut-off of approx. 0.1 Jaccard’s similarity coefficient. The phylogenetic analysis of 16S rRNA and housekeeping genes (glnII, gyrB, and atpD) formed distinct Bradyrhizobium groups in the phylogenetic trees. It revealed the presence of highly diverse bradyrhizobia (i.e. Bradyrhizobium vignae, Bradyrhizobium elkanii, Bradyrhizobium iriomotense, Bradyrhizobium pachyrhizi, and Bradyrhizobium yuanmingense) together with novel/unidentified bradyrhizobia in the acidic soils from Ghana and South Africa. Discrepancies noted in the phylogenies of some nodule DNA isolates could be attributed to horizontal gene transfer or recombination.     

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     

Potassium and magnesium relationship in cowpea (Vigna unguiculata (L.) Walp.)

Summary The effects of K and Mg application on dry matter yield and uptake of K, Mg and Ca in cowpea were studied in greenhouse at Haryana Agricultural University, Hissar (india). Dry matter yields of leaves, stems and roots increased by 17, 30 and 27 per cent over control due to application of 150 ppm K and 17, 16 and 26 per cent by 40 ppm Mg respectively. Potassium application has antagonistic effect on Ca concentration of leaves, stems and roots and synergistic on root Mg concentrations upto 25 ppm K. However, Mg had a synergistic effect on concentration of K upto 20 ppm Mg and antagonistic at 40 ppm Mg in all plant parts. Uptake of K, Mg and Ca increased by Mg application, but K increased only K uptake.     

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.     

Fast-growing bacteria from nodules of cowpea (Vigna unguiculata (L.) Walp.)

D akora , F.D. & V incent , J.M. 1984. Fast-growing bacteria from nodules of cowpea ( Vigna unguiculata (L.) Walp.) Journal of Applied Bacteriology 56 , 327–330.
First plating from nodules of cowpea frequently yielded fast-growing large colonies, either apparently uniform or associated with small colony forms typical of the expected slow-grower ( Bradyrhizobium ). Most cultures from single large colonies nodulated both cowpea and siratro ( Macroptilium atropurpureum ), but all such nodules revealed Bradyrhizobium alone or associated with a fast-growing form. Six of nine plants inoculated with a mixed inoculum of slow and fast forms had nodules occupied by both although in no case was the fast-grower able to secure solo invasion. Most of the fast-growing forms shared some internal antigens with Rhizo-biurn meliloti and/or R. trifolii ; none reacted with antiserum to Bradyrhizobium CB 756.     

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.     

Maternal inheritance of plant variegation in cowpea, Vigna unguiculata (L.) Walp.

A chimeric plant was observed in the F₂ generation of a cross between a mutant cultivar, Ife BPC, and a germplasm line, TVu 2, in cowpea, Vigna unguiculata (L.) Walp. The chimeric plant had four lateral branches, one of which was intensely variegated, while the others were mostly green with few white sectors. F₃ progeny from the intensely variegated branch of this plant were all variegated, while seed derived from the mostly green branches produced only green progeny. In subsequent generations, the descendants of the variegated branch bred true for the variegated trait, while those of the mostly green branches were also true-breeding for green colour. No pure-green or pure-white shoots were observed in any of the variegated plants examined in this study. Consequently, no pure-green or pure-white seedlings were produced from seeds harvested from the variegated plants. The results of reciprocal crosses between variegated and normal green plants indicate that variegation is inherited in a strictly uniparental maternal fashion. This is the first report of a cytoplasmically inherited mutation affecting foliage colour in cowpea. Received: 10 March 2000 / Accepted: 16 May 2000     

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.     

Programmed cell death during development of cowpea (Vigna unguiculata (L.) Walp.) seed coat

The seed coat develops primarily from maternal tissues and comprises multiple cell layers at maturity, providing a metabolically dynamic interface between the developing embryo and the environment during embryogenesis, dormancy and germination of seeds. Seed coat development involves dramatic cellular changes, and the aim of this research was to investigate the role of programmed cell death (PCD) events during the development of seed coats of cowpea [Vigna unguiculata (L.) Walp.]. We demonstrate that cells of the developing cowpea seed coats undergo a programme of autolytic cell death, detected as cellular morphological changes in nuclei, mitochondria, chloroplasts and vacuoles, DNA fragmentation and oligonucleosome accumulation in the cytoplasm, and loss of membrane viability. We show for the first time that classes 6 and 8 caspase‐like enzymes are active during seed coat development, and that these activities may be compartmentalized by translocation between vacuoles and cytoplasm during PCD events.     

Cellular compartmentation of ureide biogenesis in root nodules of cowpea (Vigna unguiculata (L.) Walp.)

Cowpea (Vigna unguiculata (L.) Walp.) nodules have been investigated by means of cytochemical and immunocytochemical procedures at the ultrastructural level in order to assess the role of the uninfected cells in ureide biogenesis. Uricase activity in the nodules was shown by cytochemical methods to be localized exclusively in the numberous large peroxisomes confined to the uninfected cells; the small peroxisomes in the infected cells did not stain for uricase. Uricase was also localized in the peroxisomes of uninfected cells by immunogold techniques employing polyclonal antibodies against nodule-specific uricase of soybean. There was no labeling above background of any structures in the infected cells. The results indicate that the uninfected cells are essential for ureide biogenesis in cowpea. Although tubular endoplasmic reticulum, the presumptive site of allantoinase, increases greatly in the uninfected cells during nodule development, it virtually disappears as the nodules mature. The inconsistency between the disappearance of the tubular endoplasmic reticulum from older nodules and the high allantoinase activity reported for older plants remains to be explained.Abbreviations DAB 3,3-diaminobenzidine - ER endoplasmic reticulum - GARG goat anti-rabbit immunoglobulin G - IgG immunoglobulin G - kDa knodalton - Mr apparent molecular mass - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Interaction between Cu toxicity and P deficiency in soil-grown cowpea (Vigna unguiculata (L.) Walp.)

Anthropogenic contamination with Cu is an important issue and it is necessary to understand how Cu toxicity influences the uptake/acquisition of nutrients by plants. An experiment was conducted with soil-grown cowpea (Vigna unguiculata (L.) Walp.) to investigate the interaction between Cu toxicity and P deficiency. Plant performance was related to the activity of Cu²⁺ at the outer surface of the root plasma membrane, {Cu²⁺} ₀ ^o , which was calculated from properties of the soil solution. The addition of Cu to the soil was found to reduce growth of plant shoots by inducing Cu toxicity, which was associated with a reduction in the shoot tissue Fe concentration. The critical value (50% reduction in shoot growth) determined for {Cu²⁺} ₀ ^o in this soil-based experiment (3.8 μM) corresponds well to values determined previously. Importantly, regression analyses indicated that although the alleviation of P deficiency improved overall growth, the P-status of the plant did not influence the apparent toxicity of the Cu. This result was unexpected, given that Cu inhibits the growth of roots hairs; these being important for the uptake of immobile nutrients such as P. This study advances our understanding of Cu toxicity and its impact upon nutrient uptake.     

Recovery of nodulated cowpea plants (Vigna unguiculata (L.) Walp.) from waterlogging during vegetative growth

Summary Waterlogging pot-grown cowpea plants for eight days (24–32 from sowing) during the period of rapid vegetative growth when symbiotic nitrogen fixation rates are also increasing at maximum rate had effects on anatomical, morphological and physiological attributes of the legume system viz reduced nodule production and increased nodule cortication, stimulated adventitious root formation at, or near, the water table but killed older submerged roots and nodules, caused the main stem to become hypertrophic just above the water line, accelerated senescence of the lowermost leaves and delayed branch formation. After re-draining the rooting medium nodule growth and symbiotic fixation activity occurred at the expense of rapid recovery by the host plant. The physiological basis for these findings in relation to identifying cowpea cultivars more tolerant of waterlogged soils is discussed.One of a series of papers describing work undertaken in a collaborative project with the International Institute of Tropical Agriculture, Nigeria, sponsored by the U.K. Ministry of Overseas Development.     

In vitro plant regeneration via organogenesis of cowpea [Vigna unguiculata (L.) Walp.]

Shoot regeneration via organogenesis was achieved from axenic cowpea [Vigna unguiculata subsp. unguiculata L. (Walp.) Verde.] hypocotyls and cotyledons of advanced breeding lines and varieties. Cotyledons and embryos were excised from green immature pods. The apical parts of the embryos were removed and the hypocotyls were transferred to regeneration media. Cotyledons and hypocotyls were tested on media with gradients of several hormonal and putrescine combinations. Cowpea cotyledons and hypocotyls exhibited a pattern of shoot formation that occurred in three distinct phases. Multiple shoots developed within 45 days from the wounded region of the primary hypocotyl and cotyledons in different media containing a high cytokinin concentration. The induced plant explants were then grown for 20 days in low-intensity light (10 μmol m^–2 s^–1) on the same medium and numerous shoot buds emerged de novo from the upper part of the hypocotyl and the wounded part of the cotyledons. These buds had no apparent vascular connection with the parent tissues. The plant regeneration capability of this procedure was tested with several cowpea genotypes, five of which (83D-442, 86D-1010, 93K-624, Vita 3 and Ife Brown) responded positively with shoot development and were able to form roots and whole plants. Some somaclonal variation was observed. Received: 14 June 1996 / Revision received: 14 December 1996 / Accepted: 25 January 1997     

Phylogenetic multilocus sequence analysis of indigenous slow-growing rhizobia nodulating cowpea (Vigna unguiculata L.) in Greece

Cowpea (Vigna unguiculata) is a promiscuous grain legume, capable of establishing efficient symbiosis with diverse symbiotic bacteria, mainly slow-growing rhizobial species belonging to the genus Bradyrhizobium. Although much research has been done on cowpea-nodulating bacteria in various countries around the world, little is known about the genetic and symbiotic diversity of indigenous cowpea rhizobia in European soils. In the present study, the genetic and symbiotic diversity of indigenous rhizobia isolated from field-grown cowpea nodules in three geographically different Greek regions were studied. Forty-five authenticated strains were subjected to a polyphasic approach. ERIC-PCR based fingerprinting analysis grouped the isolates into seven groups and representative strains of each group were further analyzed. The analysis of the rrs gene showed that the strains belong to different species of the genus Bradyrhizobium. The analysis of the 16S-23S IGS region showed that the strains from each geographic region were characterized by distinct IGS types which may represent novel phylogenetic lineages, closely related to the type species of Bradyrhizobium pachyrhizi, Bradyrhizobium ferriligni and Bradyrhizobium liaoningense. MLSA analysis of three housekeeping genes (recA, glnII, and gyrB) showed the close relatedness of our strains with B. pachyrhizi PAC48^T and B. liaoningense USDA 3622^T and confirmed that the B. liaoningense-related isolate VUEP21 may constitute a novel species within Bradyrhizobium. Moreover, symbiotic gene phylogenies, based on nodC and nifH genes, showed that the B. pachyrhizi-related isolates belonged to symbiovar vignae, whereas the B. liaoningense-related isolates may represent a novel symbiovar.     

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.     

Effect of root temperature on floral morphology in cowpea (Vigna unguiculata (L.) Walp.) cv K 2809

Summary When pot-grown cowpea plants experienced a large diurnal variation in root temperature (33°C day–19°C night) during reproductive growth, floral morphology was markedly affected. Petals were constricted in such a way as to restrict self-pollination hence seed yields were drastically reduced. These abnormal flowers were very similar to those mediating mechanical male sterility in cowpea, a genetically inherited, recessive, outcrossing mechanism identified in field populations.     

Photoperiodic Regulation of Flowering in Cowpeas (Vigna unguiculata (L.) Walp.)

To test the proposition that photoperiodic controls synchronizethe flowering of cowpeas, Vigna unguiculata (L.) Walp. [V. sinensis(L.) Savi], the day-length requirements for floral initiationand for flowering were investigated in several short-day accessions.No evidence was found of different critical photoperiods atdifferent stages of development, but exposure to only 2–4short days was required for floral initiation compared withabout 20 for development to open flowers. Pod setting was increasedafter exposure to even one short day more than the number requiredfor flower opening. Floral buds at higher nodes appeared to require fewer shortdays for development to flowering than buds at the lower nodes,and displayed faster rates of development. Inflorescence budsdid not resume development if they were exposed to 15 or morelong days following inflorescence initiation. Thus, any tendencytowards synchronous flowering in cowpeas is not due to the criticalday-length for flower development being shorter than that forflower initiation, but could be the result of cumulative photoperiodicinduction of plants and the more rapid development of later-formedflowers. Vigna unguiculata (L.) Walp., cowpeas, flower initiation, flower development, fruit set, photoperiodism