Phylogeny and taxonomy of a diverse collection of Bradyrhizobium strains based on multilocus sequence analysis of the 16S rRNA gene, ITS region and glnII, recA, atpD and dnaK genes

Abstract

Phylogeny and taxonomy of a diverse collection of Bradyrhizobium strains based on multilocus sequence analysis of the 16S rRNA gene, ITS region and glnII, recA, atpD and dnaK genes were obtained with MLSA of glnII, recA, atpD and dnaK. Greatest variability was observed when the atpD gene was amplified, and five strains related to B. elkanii revealed a level of variability never reported before. Another important observation was that a group composed of strains USDA 110, SEMIA 5080 and SEMIA 6059, all isolated from soybean, clustered in all six trees with high bootstrap support and were quite distinct from the clusters that included B. japonicum USDA 6 T . The results confirm that MLSA is a rapid and reliable way of providing information on phylogenetic relationships and of identifying rhizobial strains potentially representative of novel species. INTRODUCTIONThe genus Bradyrhizobium was created to accommodate bacteria capable of establishing N 2 -fixing symbioses with several leguminous species and characterized by slow growth and an alkaline reaction in culture media containing mannitol as a carbon source (Jordan, 1982). Based on 16S rRNA gene sequences, the genus Bradyrhizobium forms a clade in the Alphaproteobacteria, along with oligotrophic soil and aquatic bacteria such as Rhodopseudomonas palustris, Rhodoplanes roseus, Nitrobacter winogradskyi and Blastobacter denitrificans and pathogens such as Afipia species, among others (Saito et al., 1998;Willems et al., 2001a;van Berkum & Eardly, 2002;Sawada et al., 2003). Symbiotic Bradyrhizobium strains have been isolated from Abbreviations: ITS, intergenic transcribed spacer; MLSA, multilocus sequence analysis.

Bradyrhizobium manausense sp. nov., isolated from effective nodules of Vigna unguiculata grown in Brazilian Amazonian rainforest soils

“…Although high percentages of 16S rRNA gene sequence similarity were obtained, previous reports have suggested that closely related species of the genus Bradyrhizobium are difficult to distinguish using 16S rRNA gene sequence analysis (Willems et al, 2001;Menna et al, 2009). Therefore, multi locus sequence analysis (MLSA) was performed with glnII (537 bp), recA (415 bp) and rpoB (429 bp) following previous reports (Vinuesa et al, 2005;Martens et al, 2008, Menna et al, 2009.…”

Bradyrhizobium stylosanthis sp. nov., comprising nitrogen-fixing symbionts isolated from nodules of the tropical forage legume Stylosanthes spp.

“…Highest similarity was observed with Bradyrhizobium huanghuaihaiense, followed by Bradyrhizobium arachidis (Table 2). Despite the 16S rRNA gene being broadly used in phylogenetic analyses, in some bacterial genera, such as Bradyrhizobium, the sequences are considered highly conserved, limiting species delineation Helene et al, 2015;Menna et al, 2009). However, it is noteworthy that this new group with the BR strains could be observed in the analysis of the 16 rRNA gene (Fig.…”

“…The higher variability of the ITS region in comparison with the 16S rRNA gene among species of the genus Bradyrhizobium allows a better delineation of species (Germano et al, 2006;Menna et al, 2009). In the ITS phylogram, the BR strains were clustered in a clade distinct from other bradyrhizobia, with a bootstrap support of 100 %, and showed higher similarity with Bradyrhizobium japonicum (Fig.…”

“…The genus includes root-nodulating bacteria, stem-nodulating bacteria with photosynthetic and nitrogen-fixing capacity, as well as non-symbiotic species, which occupy a variety of ecosystems (Eaglesham et al, 1990;Islam et al, 2008;Ormeño-Orrillo et al, 2013;Rivas et al, 2004). In the past few years, large biodiversity among strains of the genus Bradyrhizobium has been reported; however, the genus still encompasses few described species (Delamuta et al, 2012Germano et al, 2006;Helene et al, 2015;Hungria et al, 2015;Menna et al, 2009;Parker, 2015;Roma Neto et al, 2010).…”

“…A multilocus sequence analysis (MLSA) approach was also employed in our study, as there is an increasing number of reports showing the suitability of this method for the taxonomic classification of bradyrhizobia (Delamuta et al, 2012(Delamuta et al, , 2013Helene et al, 2015;Menna et al, 2009;Parker, 2015). In addition to providing a greater number of informative nucleotide sites, the effects of gene recombination and horizontal transfer are mitigated by the analysis of at least three concatenated protein-encoding gene sequences in the MLSA analysis (Gevers et al, 2005;Schleifer, 2009).…”