RFLP analysis of the rRNA operon of a Brazilian collection of bradyrhizobial strains from 33 legume species

Abstract

Genetic diversity in tropical rhizobial species is still poorly known. With the aim of increasing this knowledge, three ribosomal regions of 119 strains belonging to the official Brazilian culture collection of rhizobia and classified as Bradyrhizobium based on morphological and physiological characteristics in vitro were analysed by RFLP-PCR. The strains were isolated from 33 legume species, representing nine tribes and all three subfamilies; they all form very effective N2-fixing nodules and 43 of them are recommended for use in Brazilian commercial inoculants as the most effective for their hosts. For the 16S rRNA gene, type and reference strains of Bradyrhizobium japonicum fell into two major clusters, joined at a level of similarity of 50 %, which included 52 strains, 90 % of which were isolated from soybean. Two other clusters, joined at a similarity of 53 %, included reference strains of Bradyrhizobium elkanii, but not USDA 76T; furthermore, two other major clusters were identified and all strains were clustered at a final level of similarity of only 28 %. For the intergenic spacer (IGS) between genes coding for the 16S and 23S rRNA, strains were clustered at a final level of similarity of 27 %. Reference strains of B. japonicum fell into a major group with 51 strains, 84 % isolated from soybean, with a similarity of 59 %, while strains of B. elkanii fell into another major group, with a similarity of 55 %, clustering 44 strains, 59 % of which were isolated from hosts other than soybean. New clusters were also observed for the IGS region. The largest number of differences was detected in the analysis of the 23S rRNA gene, and 16 groups and isolated strains were joined at a very low level of similarity (16 %). In a combined analysis with the three ribosomal regions, the majority of strains isolated from soybean clustered with a similarity of 54 % with type and reference strains of B. japonicum, while most strains isolated from Brazilian indigenous legume species grouped with B. elkanii at a level of similarity of 46 %. All strains were clustered at a very low level of similarity (27 %), and at least two new clusters were clearly defined. These new clusters might be related to intraspecific differences or to novel subspecies, or even to novel species; indeed, strains from one of these clusters show higher 16S rRNA gene sequence similarity to members of the genus Burkholderia. The results obtained in this study emphasize the high level of diversity of symbiotic diazotrophic bacteria in the tropics that still remains to be determined.