Crotalaria zanzibarica is an exotic and widely distributed leguminous plant in Taiwan. The relationship between C. zanzibarica and its rhizobial symbionts has been suggested to contribute to its successful invasion. A rhizobial strain (designed as CzR2) isolated from the root nodules of C. zanzibarica and cultivated in standard YEM medium displayed pleomorphism, with cells ranging between 2 and 10 �gm in length and some branching. In the present study, we identified this rhizobial strain, investigated the causes of pleomorphism, and examined the nodules formed. The results of a multilocus sequence analysis of the atpD, dnaK, glnII, gyrB, recA, and rpoB genes revealed that CzR2 belongs to Bradyrhizobium arachidis, a peanut symbiont recently isolated from China. Cells of the strain were uniformly rod-shaped in basal HM medium, but displayed pleomorphism in the presence of yeast extract, mannitol, or fructose. These results indicate that the morphology of CzR2 in its free-living state is affected by nutrient conditions. Several highly pleomorphic bacteroids enclosed in symbiosomes were frequently detected in FM and TEM observations of sections of the indeterminate nodules induced by CzR2; however, no infection thread was identified. Flow cytometric analyses showed that CzR2 cells in YEM medium and in the nodules of C. zanzibarica had two or more than two peaks in relative DNA contents, respectively, suggesting that the elongated cells of CzR2 in its free-living state occur due to a cell cycle-delayed process, while those in its symbiotic state are from genomic endo-reduplication.

Seven slow-growing rhizobia isolated from effective nodules of Arachis hypogaea were assigned to the genus Bradyrhizobium based on sharing 96.3-99.9 % 16S rRNA gene sequence similarity with the type strains of recognized Bradyrhizobium species. Multilocus sequence analysis of glnII, recA, gyrB and dnaK genes indicated that the seven strains belonged to two novel species represented by CCBAU 51649T and CCBAU 53363T. Strain CCBAU 51649T shared 94, 93.4, 92.3 and 94.9 % and CCBAU 53363T shared 91.4, 94.5, 94.6 and 97.7 % sequence similarity for the glnII, recA, gyrB and dnaK genes, respectively, with respect to the closest related species Bradyrhizobium manausense BR 3351T and Bradyrhizobium yuanmingense CCBAU 10071T. Summed feature 8 and C16 : 0 were the predominant fatty acid components for strains CCBAU 51649T and CCBAU 53363T. DNA-DNA hybridization and analysis of phenotypic characteristics also distinguished these strains from the closest related Bradyrhizobium species. The strains formed effective nodules on Arachis hypogaea, Lablab purpureus and Aeschynomene indica, and they had identical nodA genes to Bradyrhizobium sp. PI237 but were phylogenetically divergent from other available nodA genes at less than 66 % similarity. Based in these results, strains CCBAU 51649T (= CGMCC 1.15034T = LMG 28620T) and CCBAU 53363T (= CGMCC 1.15035T = LMG 28621T) are designated the type strains of two novel species, for which the names Bradyrhizobium guangdongense sp. nov. and Bradyrhizobium guangxiense sp. nov. are proposed, respectively.

A multilocus sequence typing (MLST) method based on allelic variation of seven chromosomal loci was developed for characterizing genotypes (GT) within the genus Bradyrhizobium. With the method, 29 distinct multilocus GT were identified among 190 culture collection soybean strains. The occupancy of 347 nodules taken from uninoculated field-grown soybean plants also was determined. The bacteroid GT were either the same as or were closely related to GT identified among strains in the culture collection. Double-nodule occupancy estimates of 2.9% were much lower than values published based on serology. Of the 347 nodules examined, 337 and 10 were occupied by Bradyrhizobium japonicum and B. elkanii, respectively. The collection strains within the species B. japonicum and B. elkaniialso were compared with Bradyrhizobium cultures from other legumes. In many cases, the observed GT varied more according to their geographic origin than by their trap hosts of isolation. In other cases, there were no apparent relationships with either the legume or geographic source. The MLST method that was developed should be a useful tool in determining the influence of geographic location, temperature, season, soil type, and host plant cultivar on the distribution of GT of Bradyrhizobium spp.

The use of ftsA gene sequences for taxonomic studies of the genus Bradyrhizobium bacteria was assessed. The ftsA gene codes for an actin-like protein involved in prokaryotic cell division. Up to now, this gene has not been used as a phylogenetic marker for analysis of bacteria establishing root nodule symbiosis with Fabaceae plants. In this study, the ftsA gene sequences obtained for bradyrhizobia forming N2 fixing symbiosis with four Genisteae tribe plants growing in Poland and most of the type strains of the genus Bradyrhizobium species were analyzed and evaluated as molecular markers for phylogenetic studies of these bacteria for the first time. The ftsA gene sequences of all bradyrhizobial strains with completely or partially sequenced genomes, available in the GenBank database, were also included into the analysis. The phylogeny of the ftsA gene was compared to the phylogenies of other chromosomal genes commonly used in the studies of Bradyrhizobium bacteria. The results showed that the phylogenies of ftsA and the core genes recA and glnII were congruent, making the ftsA gene useful as a phylogenetic marker. Analysis of the ftsA gene sequences revealed a single-nucleotide polymorphism unique to Bradyrhizobium japonicum strains, and the potential use of this SNP for identification of this species was discussed.

Twenty-three bacterial strains isolated from root nodules of Arachis hypogaea and Lablab purpureus grown in five provinces of China were classified as a novel group within the genus Bradyrhizobium by analyses of PCR-based RFLP of the 16S rRNA gene and 16S-23S IGS. To determine their taxonomic position, four representative strains were further characterized. The comparative sequence analyses of 16S rRNA and six housekeeping genes clustered the four strains into a distinctive group closely related to the defined species Bradyrhizobium liaoningense, Bradyrhizobium yuanmingense, Bradyrhizobium huanghuaihaiense, Bradyrhizobium japonicum and Bradyrhizobium daqingense. The DNA-DNA relatedness between the reference strain of the novel group, CCBAU 051107(T), and the corresponding type strains of the five mentioned species varied between 46.05% and 13.64%. The nodC and nifH genes of CCBAU 051107(T) were phylogenetically divergent from those of the reference strains for the related species. The four representative strains could nodulate with A. hypogaea and L. purpureus. In addition, some phenotypic features differentiated the novel group from the related species. Based on all the results, we propose a new species Bradyrhizobium arachidis sp. nov. and designate CCBAU 051107(T) (=CGMCC 1.12100(T)=HAMBI 3281(T)=LMG 26795(T)) as the type strain, which was isolated from a root nodule of A. hypogaea and had a DNA G+C mol% of 60.1 (Tm).