Abstract
Strains of the genus Bradyrhizobium associated with agronomically important crops such as soybean (Glycine max) are increasingly studied; however, information about symbionts of wild Glycine species is scarce. Australia is a genetic centre of wild Glycine species and we performed a polyphasic analysis of three Bradyrhizobium strains—CNPSo 4010T, CNPSo 4016T, and CNPSo 4019T—trapped from Western Australian soils with Glycine clandestina, Glycine tabacina and Glycine max, respectively. The phylogenetic tree of the 16S rRNA gene clustered all strains into the Bradyrhizobium japonicum superclade; strains CNPSo 4010T and CNPSo 4016T had Bradyrhizobium yuanmingense CCBAU 10071T as the closest species, whereas strain CNPSo 4019T was closer to Bradyrhizobium liaoningense LMG 18230T. The multilocus sequence analysis (MLSA) with five housekeeping genes—dnaK, glnII, gyrB, recA and rpoB—confirmed the same clusters as the 16S rRNA phylogeny, but indicated low similarity to described species, with nucleotide identities ranging from 93.6 to 97.6% of similarity. Considering the genomes of the three strains, the average nucleotide identity and digital DNA–DNA hybridization values were lower than 94.97 and 59.80 %, respectively, with the closest species. In the nodC phylogeny, strains CNPSo 4010T and CNPSo 4019T grouped with Bradyrhizobium zhanjiangense and Bradyrhizobium ganzhouense, respectively, while strain CNPSo 4016T was positioned separately from the all symbiotic Bradyrhizobium species. Other genomic (BOX-PCR), phenotypic and symbiotic properties were evaluated and corroborated with the description of three new lineages of Bradyrhizobium. We propose the names of Bradyrhizobium agreste sp. nov. for CNPSo 4010T (=WSM 4802T=LMG 31645T) isolated from Glycine clandestina, Bradyrhizobium glycinis sp. nov. for CNPSo 4016T (=WSM 4801T=LMG 31649T) isolated from Glycine tabacina and Bradyrhizobium diversitatis sp. nov. for CNPSo 4019T (=WSM 4799T=LMG 31650T) isolated from G. max.
Highlights
Balanced levels of N in soil are required to obtain high productivities and healthy plants
We report a polyphasic study with three lineages of Bradyrhizobium isolated from Glycine clandestina, G. tabacina and G. max that resulted in the description of three new species
In the multilocus sequence analysis (MLSA) with five housekeeping genes (Fig. 2), strains CNPSo 4010T and CNPSo 4016T remained in a consistent cluster with 99 % bootstrap support, with B. yuanmingense CCBAU 10071T as the closest species with 96.4 and 96.1 % nucleotide identity, respectively (Fig. 2, Table 2)
Summary
All phylogenies of single housekeeping genes clustered the strains from this study in clades separated from all described Bradyrhizobium species. In the MLSA with five housekeeping genes (Fig. 2), strains CNPSo 4010T and CNPSo 4016T remained in a consistent cluster with 99 % bootstrap support, with B. yuanmingense CCBAU 10071T as the closest species with 96.4 and 96.1 % nucleotide identity, respectively (Fig. 2, Table 2). The nodC gene analysis revealed that CNPSo 4010T from G. clandestina clustered in a well-s upported clade with B. zhanjiangense CCBAU 51778T isolated from nodules of Arachis hypogaea in southeast China [53], CNPSo 4019T from G. max clustered with 100 % bootstrap support with B. ganzhouense RITF806T isolated from nodules of Acacia melanoxylon grown in China [54]; whereas CNPSo 4016T, isolated from G. tabacina, occupied an isolated position (Fig. 3). Comparing the core 16S rRNA and housekeeping genes with the nodC gene phylogenies, a different evolutionary pattern
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