Genotypic, phenotypic and symbiotic characterization of Bradyrhizobium strains isolated from Amazonia and Minas Gerais soils

Abstract

The genus Bradyrhizobium stands out among the genus of legume-nodulating nitrogen-fixing bacteria because it predominates among efficient microsymbionts of forest species, forage and green manure, as well as grain species. Due to this importance, the aim of this study was to characterize symbiotic, phenotypic and genetically Bradyrhizobium strains isolated from different soils of Brazilian ecosystems. For it was performed nodulation test in soybean, test of resistance to different antibiotics and tolerance to different levels of salinity and phylogenetic analysis of five housekeeping genes (atpD, dnaK, gyrB, recA, and rpoB) with 50 strains isolated from soils of Amazon region and Minas Gerais state. From the results obtained in the phylogenetic analysis, a group of five strains (GII) was selected for further studies in order to identify the taxonomic position of these strains. In this second part of the work it was performed tests of tolerance at different temperatures and pH values, solubilization of inorganic phosphates and DNA:DNA hybridization. Regarding the 50 strains analyzed, eleven strains were able to nodulate soybean and, in general, the strains were resistant to most of the tested antibiotics. In the salinity test bacterial growth was observed for all strains up to a concentration of 0,5% NaCl and only the strains UFLA03-142, UFLA03-143, UFLA03-145 and UFLA03-146 grew at 1% NaCl. Phylogenetic analysis highlighted strains with the potential to be new species of bacteria Bradyrhizobium. Phylogenetic analysis of concatenated sequences of four housekeeping genes atpD, recA, dnaK and gyrB generated a group formed by the five strains that were different from Bradyrhizobium species already described, confirming the results found in the first part of the work. The type strain of the specie B. liaoningense was closest to the group with 96.12% similarity. The delimitation of the species was performed by DNA:DNA hybridization test, of which results were shown below 70%. The strains of this group (GII) grow at pH 4 to 9, at temperatures 15 and 28ºC, salinity equal to 0,5% NaCl and shown variable behavior in regarding resistance to antibiotics. The strains do not have the capacity to solubilize inorganic phosphates, not nodulate soybean, and nodulate cowpea. Based on genetic, phenotypic and symbiotic analyses demonstrated in this work, we verified the existence of strains with high potential to be new species of Bradyrhizobium. It was proposed that the strains belonging to GII should be included in a new species called Bradyrhizobium amazonense sp. nov., with UFLA03-150T as the type strain.