Characterization of Burkholderia species associated with root nodules of legumes indigenous to South Africa

Abstract

Bukholderia species are commonly isolated from the root nodules of Mimosoid legumes of the subfamily Mimosoideae. In contrast, few studies have reported the association between Burkholderia species and legumes of other subfamilies within the Leguminoseae family. This is also true for the legumes indigenous to South Africa, where only one previous study reported the possible existence of novel species of Burkholderia nodulating legumes within the Papilionoid subfamily. In this previous study, 69 isolates were obtained from the root nodules of legumes in the genera Hypocalyptus, Virgilia, Cyclopia and Podalyria, which are all indigenous to the Cape Floristic Region. The aim of this study was to characterize the 69 isolates for identification and classification using a polyphasic approach. For this purpose the DNA sequence information and phylogenetic analyses of three housekeeping genes (rpoB, atpD and gyrB) were used. The results indicated that one of the genes (gyrB) occurred in multiple copies in the genomes of the bacteria examined, and were thus of limited use for delineating species. However, analyses of rpoB and atpD suggested that these genes were highly informative and separated the isolates into 25 groups (SA 1 to SA 25), similar to what was found previously. The set of isolates examined in this study was also subjected to phenotypic tests, which were achieved by determining their colony sizes, growth abilities on various media, Gram stain, cell size and growth in the presence of varying salt concentrations. These tests showed that the isolates all represented Gram negative rods, with similar cell size within each species group. The growth on different media also did not allow unambiguous separation of the different groups, although the use of API 20NE and Biolog GN2 revealed numerous phenotypic differences among the isolates and groups of isolates. Cluster analysis of these data based on UPGMA separated the isolates into groups that broadly corresponded to those identified using DNA sequence information. Therefore, both the phylogenetic and phenotypic methods used in this study, confirmed these isolates as belonging to the genus Burkholderia. At least ten new species (each represented by 2 or more isolates) could be distinguished with a high degree of certainty and will be formally described once relevant DNA-DNA hybridization data becomes available from future studies.