Diversity of root nodulating bacteria associated with Cyclopia species

Abstract

In recent years, the rhizobial taxonomy changed significantly with the discovery of novel symbiotic associations between legumes and nodulating bacteria. This was aided by the focus shift from studying only agricultural crops to legumes indigenous to certain regions, ultimately to discover new inoculant strains and to uncover the secrets of the rhizobium¬legume symbiosis. In previous studies on the diversity of South African rhizobia, it has become clear that our country has a wealth of rhizobia. Cyclopia is a legume genus, which belongs to the fynbos biome of South Africa. Honeybush tea is a herbal infusion manufactured from the leaves and stems of certain Cyclopia spp. Commercial cultivation of this potentially new agricultural crop is now developed to protect the natural Cyclopia spp. populations from harvesting and ultimately extinction. Superior inoculant strains are necessary for these commercial seedlings. The diversity of root-nodulating strains isolated from 14 Cyclopia spp. was determined using 16S-23S IGS-RFLP and partial 16S rDNA base sequencing. Based on 16S-23S IGS-RFLP and partial 16S rDNA base sequencing most of the isolates, with the exception of seven strains, were found to belong to the genus Burkholderia. More extensive phylogenetic, symbiotic and phenotypic studies of selected strains were performed using near full-length 168 rDNA base sequencing, nodA base sequencing and substrate utilisation analysis. In the genus Burkholderia, the isolates belonged to the novel root-nodulating species Burkholderia tuberum and several novel, undescribed Burkholderia genotypes. However, no new Burkholderia species could formally be proposed, since DNA-DNA hybridisation analysis, which is a prerequisite for the description of new species could not be performed in our laboratory. The seven strains not affiliated with the Burkholderia genus belonged to two Bradyrhizobium genospecies, R tropici and a possibly new genus in the a-Proteobacteria. The nodA sequences of all the Cyclopia isolates corresponded to a large extent, indicating that different chromosomal genotypes harbour the same symbiotic genotype. All the isolates of the Cyclopia genus appear to be acid-tolerant, which is in agreement with the acidic nature of the soil from which the strains were isolated.