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Phylogenetic evidence of allopatric speciation of bradyrhizobia nodulating cowpea (Vigna unguiculata L. walp) in South African and Mozambican soils.

ABSTRACT: The legume host and soil environment play a major role in establishing effective symbiosis with diverse rhizobia for plant growth promotion and nodule formation. The aim of this study was to assess the morpho-physiology, distribution and phylogenetic position of rhizobia nodulating cowpea from South Africa and Mozambique. The results showed that the isolates were highly diverse in their appearance on yeast mannitol agar plates. The isolates tested also showed an ability to produce IAA at concentrations ranging from 0.64 to 56.46 μg.ml-1 and to solubilise phosphorus at levels from 0 to 3.55 index. Canonical correspondence analysis showed that soil pH and mineral nutrients significantly influenced bradyrhizobial distribution. Analysis of BOX-PCR placed the isolates in eight major clusters with 0.01 to 1.00 similarity coefficient which resulted in 45 unique BOX-types. Phylogenetic analyses based on 16S rRNA, atpD, glnII, gyrB and recA gene sequences showed distinct novel evolutionary lineages within the genus Bradyrhizobium, with some of them being closely related to Bradyrhizobium kavangense, B. subterraneum and B. pachyrhizi. Furthermore, symbiotic gene phylogenies suggested that the isolates' sym loci probably relates to the isolates' geographical origin. The results indicated that geographical origin did affect the isolates' phylogenetic placement and could be the basis for allopatric speciation.

SUBMITTER: Dabo M

PROVIDER: S-EPMC6531793 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Cowpea (Vigna unguiculata L. Walp.) is indigenous to Africa, and highly valued for its N2-fixing trait and the nutritional attributes of its grain and leaves. The species' ability to establish effective symbiosis with diverse rhizobial populations gives it survival and growth advantage in N-limited environments. To explore the functional diversity and phylogenetic positions of rhizobia nodulating cowpea in Africa, nodules were collected from various cowpea varieties grown in soils from the Guinea savanna and Sudano-sahelian agroecologies of Northern Ghana, and from the lowveld and middleveld areas of Mpumalanga Province in South Africa. Box-PCR profiling and multilocus sequence analysis revealed the presence of diverse microsymbionts responsible for cowpea nodulation across the study sites. BOX-PCR amplifications yielded variable band sizes, ranging from 618 bp to 5354 bp, which placed the isolates in six major clusters (Cluster A-F). Phylogenetic analysis based on 16S rRNA, atpD, glnII, gyrB, rpoB, nifH and nodC genes revealed the presence of diverse Bradyrhizobium sp. closely related to Bradyrhizobium daqingense, Bradyrhizobium subterraneum, Bradyrhizobium yuanmingense, Bradyrhizobium embrapense, Bradyrhizobium pachyrhizi, Bradyrhizobium elkanii and novel Bradyrhizobium species in the soils studied, a finding that could be attributed to the unique edapho-climatic conditions of the contrasting environments. The test isolates exhibited distinct symbiotic efficiencies, and also induced variable (p ≤ 0.001) photosynthetic rates, leaf transpiration, total chlorophyll and shoot biomass accumulation on cowpea (their homologous host). Canonical correspondence analysis showed that the distribution of these microsymbionts was influenced by the concentrations of macro- and micronutrients in soils. The pairwise genetic distances derived from phylogenies and nodule functioning showed significant (p < 0.05) correlation, which suggests that local environmental factors played a major role in the cowpea-Bradyrhizobium symbiosis.

Project description:BackgroundCowpea 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.ResultsField 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.ConclusionsWe 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.

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Phylogenetic evidence of allopatric speciation of bradyrhizobia nodulating cowpea (Vigna unguiculata L. walp) in South African and Mozambican soils.

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