Symbiotic functioning and bradyrhizobial biodiversity of cowpea (Vigna unguiculata L. Walp.) in Africa

Abstract

Background: Cowpea is the most important food grain legume in Sub-Saharan Africa. However, no study has so far assessed rhizobial biodiversity and/or nodule functioning in relation to strain IGS types at the continent level. In this study, 9 cowpea genotypes were planted in field experiments in Botswana, South Africa and Ghana with the aim of i) trapping indigenous cowpea root-nodule bacteria (cowpea "rhizobia") in the 3 countries for isolation, molecular characterisation using PCR-RFLP analysis, and sequencing of the 16S - 23S rDNA IGS gene, ii) quantifying N-fixed in the cowpea genotypes using the 15N natural abundance technique, and iii) relating the levels of nodule functioning (i.e. N-fixed) to the IGS types found inside nodules.

Results: Field measurements of N2 fixation revealed significant differences in plant growth, delta15N values, %Ndfa and amounts of N-fixed between and among the 9 cowpea genotypes in Ghana and South Africa. Following DNA analysis of 270 nodules from the 9 genotypes, 18 strain IGS types were found. Relating nodule function to the 18 IGS types revealed significant differences in IGS type N2-fixing efficiencies. Sequencing the 16S - 23S rDNA gene also revealed 4 clusters, with cluster 2 forming a distinct group that may be a new Bradyrhizobium species. Taken together, our data indicated greater biodiversity of cowpea bradyrhizobia in South Africa relative to Botswana and Ghana.

Conclusions: We have shown that cowpea is strongly dependant on N2 fixation for its N nutrition in both South Africa and Ghana. Strain IGS type symbiotic efficiency was assessed for the first time in this study, and a positive correlation was discernible where there was sole nodule occupancy. The differences in IGS type diversity and symbiotic efficiency probably accounts for the genotype x environment interaction that makes it difficult to select superior genotypes for use across Africa. The root-nodule bacteria nodulating cowpea in this study all belonged to the genus Bradyrhizobium. Some strains from Southern Africa were phylogenetically very distinct, suggesting a new Bradyrhizobium species.

Figure 1

UPGMA dendrogram derived from PCR-RFLP of bradyrhizobial DNA in cowpea nodules collected from South Africa, Botswana and Ghana, generated by HaeIII digestion of amplified rDNA products. Scale indicates % similarity.

Figure 2

Specific nodule activity for the 9 genotypes grown at A) Wa in Ghana, B) Taung in South Africa. Bars with dissimilar letters indicate significant differences at p ≤ 0.05. Numerals on the top of each bar represent the different IGS types (strains) that were found in the cowpea nodules from the particular genotype.

Figure 3

Phylogenetic relationship among 16S-23S rDNA IGS types of from cowpea nodules, reference strains and more closed isolates based upon aligned 16S-23S rDNA IGS region sequences constructed as rooted tree using neighbour-joining method. The bootstrap values (expressed as percentage of 1000 replications) shown at nodes are those greater than 70%.