Diverse members of Bradyrhizobium diazoefficiens, B. japonicum, and B. ottawaense were isolated from the roots of field-grown sorghum plants in Fukushima, and classified into "Rhizobia" with nodulated soybeans, "Free-living diazotrophs", and "Non-diazotrophs" by nitrogen fixation and nodulation assays. Genome analyses revealed that B. ottawaense members possessed genes for N 2 O reduction, but lacked those for the Type VI secretion system (T6SS). T6SS is a new bacterial weapon against microbial competitors. Since T6SS-possessing B. diazoefficiens and B. japonicum have mainly been isolated from soybean nodules in Japan, T6SS-lacking B. ottawaense members may be a cryptic lineage of soybean bradyrhizobia in Japan.

The taxonomic status of two previously characterized Bradyrhizobium strains (58S1T and S23321) isolated from contrasting habitats in Canada and Japan was verified by genomic and phenotypic analyses. Phylogenetic analyses of five and 27 concatenated protein-encoding core gene sequences placed both strains in a highly supported lineage distinct from named species in the genus Bradyrhizobium with Bradyrhizobium betae as the closest relative. Average nucleotide identity values of genome sequences between the test and reference strains were between 84.5 and 94.2 %, which is below the threshold value for bacterial species circumscription. The complete genomes of strains 58S1T and S23321 consist of single chromosomes of 7.30 and 7.23 Mbp, respectively, and do not have symbiosis islands. The genomes of both strains have a G+C content of 64.3 mol%. Present in the genome of these strains is a photosynthesis gene cluster (PGC) containing key photosynthesis genes. A tRNA gene and its partial tandem duplication were found at the boundaries of the PGC region in both strains, which is likely the hallmark of genomic island insertion. Key nitrogen-fixation genes were detected in the genomes of both strains, but nodulation and type III secretion system genes were not found. Sequence analysis of the nitrogen fixation gene, nifH, placed 58S1T and S23321 in a novel lineage distinct from described Bradyrhizobium species. Data for phenotypic tests, including growth characteristics and carbon source utilization, supported the sequence-based analyses. Based on the data presented here, a novel species with the name Bradyrhizobium cosmicum sp. nov. is proposed with 58S1T (=LMG 31545T=HAMBI 3725T) as the type strain.

Vegetable soybean (Glycine max [L.]) is mainly consumed in Asian countries, but has recently attracted attention worldwide due to its high nutritional value. We aimed to identify the indigenous rhizobia of vegetable soybean in Yao City, Osaka Prefecture, Japan, and to clarify the relationships between the rhizobial community and soil environmental factors. Soil samples were collected from 12 vegetable soybean cultivation fields under two different conditions (six greenhouses and six open fields) in Yao City with different varieties of vegetable soybean. A total of 217 isolates were obtained from the nodules and clustered into nine operational taxonomic units (OTUs) with 97% homology based on the 16S-23S rRNA internal transcribed spacer (ITS) region. A phylogenetic analysis showed that OTUs were closely related to Bradyrhizobium liaoningense, B. ottawaense, B. elkanii, and other Bradyrhizobium species and were dominant in this order. B. liaoningense was widely found in sampled sites and accounted for 50.7% of all isolates, while B. ottawaense was mostly limited to open fields. This rhizobial community differed from Japanese soybean rhizobia, in which B. diazoefficiens, B. japonicum, and B. elkanii were dominant. These results imply the characteristic differences among host plants or regional specialties. A non-metric multidimensional scaling (NMDS) analysis revealed the significant impact of soil pH and the contents of Ca, Mg, Mn, total nitrogen (TN), and total carbon (TC) on the distribution of rhizobia. B. liaoningense was detected in soils with a neutral pH, and high TN and low Mn contents increased its abundance. The present study provides novel insights into Japanese rhizobia and potentially novel resources for sustainable agriculture.

Four bacterial strains isolated from root nodules of soybean plants that had been inoculated with root-zone soils of legumes native to Canada were previously identified as a novel Bradyrhizobium lineage consisting of symbiovars (sv.) glycinearum and septentrionale. Our purpose was to verify the taxonomic status of these strains using phylogenetic, genomic and phenotypic analyses. Multiple phylogenetic analyses including analysis of 51 full-length ribosome protein subunit (rps) gene sequences confirmed placement of the novel strains in a highly supported lineage distinct from named Bradyrhizobium species with B. japonicum USDA 6T as the closest relative. The results of genomic and phylogenomic analyses based on digital DNA–DNA hybridization and genome blast distance phylogeny showed that novel strains in comparisons with type strains of closest relatives were below the established threshold (70 %) for species delineation. Moreover, the novel strains were divided into two subspecies clusters based on the established threshold of 79 %. The genomes of strains 144S4T, 323S2, 1S5 and 38S5T have sizes of 11 399 526, 11 474 152, 10580853 and 10 530 141 bp with DNA G+C contents of 63.1, 63.0, 63.4 and 63.3 mol%, respectively. These strains possess symbiosis islands harbouring key nodulation, nitrogen-fixation and type III secretion system genes as well as abundant insertion sequences and between two and four putative plasmids. Strains 144S4T and 323S2 (sv. glycinearum) are effective with regard to nitrogen fixation in symbiotic association with soybeans whereas strains 1S5 and 38S5T (sv. septentrionale) are ineffective. Data for morphological, physiological and symbiotic characteristics complement the sequence-based results. The data presented here support the description of a new species and two new subspecies for which the names Bradyrhizobium barranii sp. nov. subsp. barranii subsp. nov. (sv. glycinearum) and Bradyrhizobium barranii sp. nov. subsp. apii subsp. nov. (sv. septentrionale) are proposed with strain 144S4T (=LMG 31552T=HAMBI 3722T) as the species type strain and type strain of subsp. barranii subsp. nov., and strain 38S5T (=LMG 31556T=HAMBI 3721T) as the type strain of subsp. apii subsp. nov.