Diversity of rhizobial Methylobacterium species associated with indigenous legumes in South Africa

Abstract

The genus Methylobacterium includes a variety of pink pigmented and cream white facultatively methylotrophic bacteria that are characterized by their ability to mainly utilize methanol as a carbon source. Methylobacterium includes only one known nitrogen fixing species (Methylobacterium nodulans), which was initially isolated from root nodules of the legume Crotalaria podocarpa, in Senegal. Additional Methylobacterium strains able to fix atmospheric nitrogen with members of Crotalaria and Listia legumes native to Southern Africa have since been isolated. The aim of this study thus was to investigate the taxonomic position and delineate the diversity of Methylobacterium isolates associated with Crotalaria and Listia species native to South Africa. This was achieved by employing housekeeping gene phylogenies and various phenotypic tests. Of the original 92 isolates investigated, 29 belonged to the genus Methylobacterium. Aligned sequences from the isolates, together with reference and outgroup sequences obtained from the National Center for Biotechnology Information (NCBI) database, were used for constructing phylogenetic trees. To confirm the phylogenetic results, phenotypic characterization tests were conducted. The phylogenetic analyses of the housekeeping genes of the Methylobacterium isolates grouped them into two clusters (A and B). Group A isolates were closely related to M. nodulans, while Group B formed a different cluster, grouping with a well-known Methylobacterium strain 4-46. From the results, it was clear that only isolates obtained from Crotalaria clustered in Group A with M. nodulans, whereas all Listia isolates and two Crotalaria isolates clustered in Group B. Results from this study showed that single phylogenies of 16S rRNA, recA and rpoB best delineated Methylobacterium isolates. Carbon utilization tests did not provide results that could be used for the separation of the Methylobacterium isolates according to the two assigned groups.