Polyphasic taxonomy of rhizobia associated with legumes occuring in South Africa

Abstract

The advantageous association between rhizobia and leguminous plants has motivated numerous studies into the diversity and identity of the associated bacterial symbionts. This, as well as developments in molecular microbiology, has led to major revisions of rhizobial taxonomy. Previous investigations of the rhizobia, associated with various leguminous plants from South Africa, concluded that most of the indigenous strains were related to the genus Bradyrhizobium (Dagutat, 1995; Kruger, 1998). The other rhizobial genera represented, albeit to a lesser extent, were Rhizobium, Sinorhizobium and Mesorhizobium. The major shortcoming of these investigations was the lack of sufficient genotypic characterisation. Since the completion of these initial investigations, a new rhizobial genus and several new species have been described. These developments and the additional isolation of rhizobia, from previously uninvestigated legumes, necessitated a more detailed analysis of the indigenous rhizobia. The aim of this study was therefore to study the diversity of the indigenous strains, focusing particularly on genotypic traits. A selection of indigenous rhizobia was characterised by partial 16S rDNA sequencing, restriction fragment length polymorphism (RFLP) of the 16S-23S intergenic spacer (IGS) region, partial nifH sequencing and nodC RFLP. Based on 16S rDNA sequencing, most of the isolates could be assigned to a specific genus, most being related to the genus Bradyrhizobium. A group of isolates was also related to the genus Methylobacyterium. The IGS- RFLP analyses were sufficiently discriminatory to indicate additional variation among isolates which showed little or no 16S rDNA sequence variation. The nifH phylogenetic groupings correlated well with those obtained by 16S rDNA sequencing. However, nodC RFLP indicated that the indigenous rhizobia carry diverse range nodC genotypes, with only a few showing host-specific associations. In the absence of sequence data of these nodC genotypes, their origin and correspondence to known nodC genes of other rhizibial genera, remain uncertain. Considering the results obtained here and the phenotypic characteristics determined previously, several novel Bradyrhizobium and Mesorhizobium strains were identified, however, their specific status should be validated by DNA homology studies.