Diversity of rhizobia nodulating Phaseolus vulgaris and Phaseolus coccineus in South Africa

Abstract

The association between root-nodulating bacteria and leguminous plants is advantageous due to their ability to alter atmospheric nitrogen into a useful form in a process known as biological nitrogen fixation (BNF). Research has shown that BNF is the most efficient way to supply the large amounts of nitrogen needed by plants to produce high-yielding crops. As a result, there have been numerous studies into the diversity and identity of the associated nitrogen-fixing bacterial symbionts. Recent advances in molecular microbiology together with the isolation of rhizobia from previously uninvestigated legumes have led to major revisions of rhizobial taxonomy, most notably the inclusion of bacteria from the ß-Proteobacteria in the genera Burkholderia and Ralstonia. In this study, the diversity of root nodule bacteria associated with Phaseolus coccineus and Phaseolus vulgaris species in South Africa was investigated. A selection of rhizobial isolates were characterised by SDS-PAGE of whole cell proteins and rep-PCR DNA fingerprint analyses. These results were supplemented by partial 16S rDNA sequencing of a select number of isolates to confirm their identity. Where isolates displayed unexpected genus associations, partial nodA sequencing was performed to determine whether these were incidental contaminants or true nodulators. Based on 16S rDNA sequence analysis, the majority of isolates investigated were fast-growers belonging to the genus Rhizobium. A few isolates showed close relationship to species of the ß-Proteobacteria genus, Burkholderia. Both the SDS-PAGE analyses and the combined rep-PCR analyses were able to resolve isolates down to strain level, but the comparison of the SDS-PAGE and 16S rDNA sequencing data confirmed that bacterial discrimination using SDS-PAGE is not useful at the genus level and higher, as isolates showing affinity to Burkholderia were mingled with isolates showing similarity to Rhizobium. These isolates were separate from the Rhizobium isolates in the combined rep-PCR dendrogram. While there were discrepancies between results obtained from SDS-PAGE and rep-PCR analyses, results from the combined rep-PCR analysis correlated with many of the results obtained in the SDS-PAGE analysis. Both geographic location and host plant species appear to have affected the grouping of isolates. Many clusters consisted of isolates from the same location or the same host plant species in both the SDS-PAGE dendrogram as well as the combined rep-PCR dendrogram. The nodA sequencing demonstrated that the majority of isolates tested contain the nodA gene indicating that they are capable of nodulation. There was a large strain diversity observed for the isolates of this study and a number of the root-nodulating bacteria of the Phaseolus spp. appear to constitute several novel nodulating genotypes.