Bradyrhizobium ottawaense sp. nov., a symbiotic nitrogen fixing bacterium from root nodules of soybeans in Canada

Sixteen strains of symbiotic bacteria from root nodules of Glycine max grown in Ottawa, Canada, were previously characterized and placed in a novel group within the genus Bradyrhizobium. To verify their taxonomic status, these strains were further characterized using a polyphasic approach. All strains possessed identical 16S rRNA gene sequences that were 99.79 % similar to the closest relative, Bradyrhizobium liaoningense LMG 18230T. Phylogenetic analysis of concatenated atpD, glnII, recA, gyrB, rpoB and dnaK genes divided the 16 strains into three multilocus sequence types that were placed in a highly supported lineage distinct from named species of the genus Bradyrhizobium consistent with results of DNA–DNA hybridization. Based on analysis of symbiosis gene sequences (nodC and nifH), all novel strains were placed in a phylogenetic group with five species of the genus Bradyrhizobium that nodulate soybeans. The combination of phenotypic characteristics from several tests including carbon and nitrogen source utilization and antibiotic resistance could be used to differentiate representative strains from recognized species of the genus Bradyrhizobium. Novel strain OO99T elicits effective nodules on Glycine max, Glycine soja and Macroptilium atropurpureum, partially effective nodules on Desmodium canadense and Vigna unguiculata, and ineffective nodules on Amphicarpaea bracteata and Phaseolus vulgaris. Based on the data presented, we conclude that our strains represent a novel species for which the name Bradyrhizobium ottawaense sp. nov. is proposed, with OO99T ( = LMG 26739T = HAMBI 3284T) as the type strain. The DNA G+C content is 62.6 mol%.

Sixteen strains of symbiotic bacteria from root nodules of Glycine max grown in Ottawa, Canada, were previously characterized and placed in a novel group within the genus Bradyrhizobium. To verify their taxonomic status, these strains were further characterized using a polyphasic approach. All strains possessed identical 16S rRNA gene sequences that were 99.79 % similar to the closest relative, Bradyrhizobium liaoningense LMG 18230 T . Phylogenetic analysis of concatenated atpD, glnII, recA, gyrB, rpoB and dnaK genes divided the 16 strains into three multilocus sequence types that were placed in a highly supported lineage distinct from named species of the genus Bradyrhizobium consistent with results of DNA-DNA hybridization. Based on analysis of symbiosis gene sequences (nodC and nifH), all novel strains were placed in a phylogenetic group with five species of the genus Bradyrhizobium that nodulate soybeans. The combination of phenotypic characteristics from several tests including carbon and nitrogen source utilization and antibiotic resistance could be used to differentiate representative strains from recognized species of the genus Bradyrhizobium. Novel strain OO99 T elicits effective nodules on Glycine max, Glycine soja and Macroptilium atropurpureum, partially effective nodules on Desmodium canadense and Vigna unguiculata, and ineffective nodules on Amphicarpaea bracteata and Phaseolus vulgaris. Based on the data presented, we conclude that our strains represent a novel species for which the name Bradyrhizobium ottawaense sp. nov. is proposed, with OO99 T (5LMG 26739 T 5HAMBI 3284 T ) as the type strain. The DNA G+C content is 62.6 mol%.
Soybean [Glycine max (L.) Merrill] is an economically important grain legume that can form a nitrogen-fixing association with species of soil bacteria belonging to the genus Bradyrhizobium. In a previous study (Tang et al., 2012), populations of symbiotic bacteria associated with soybeans at two field sites in eastern Canada were characterized on the basis of multilocus sequence analysis (MLSA) of six protein encoding (housekeeping) genes. Phylogenetic analyses resulted in the identification of several novel lineages within the genus Bradyrhizobium. One of these novel lineages was encountered at only one of the two field sites and consisted of a group of 16 strains. In this work, we further characterize these strains using a variety of genotypic and phenotypic methods, and, based on the results, a novel species, Bradyrhizobium ottawaense sp. nov. is proposed.
The 16 novel strains of symbiotic bacteria (Table S1, available in the online Supplementary Material) were obtained from root nodules of soybeans grown at a field site in Ottawa, Ontario (Tang et al., 2012). Bacteria were grown on yeast-extract mannitol (YEM) agar medium (Vincent, 1970) and pure cultures maintained in 20 % (w/v) glycerol at 280 u C. Bacterial cells were Gram-stain-negative (Powers, 1995), and, based on the Schaeffer-Fulton staining method (Hussey & Zayaitz, 2012), were non-spore-forming. Colonies on YEM agar were mucoid, beige, translucent, circular and measured ,1 mm in diameter after 7 days at 28 u C. Cell morphology was investigated using a transmission electron microscope (H-7000; Hitachi). Bacteria were cultured in stationary YEM broth for 2 days at 28 u C and stained with 1 % phosphotungstic acid (pH 7.0) (Hayat & Miller, 1990). All tested strains (OO99 T , OM9 and OO85) had rod-shaped cells, subpolar flagella and a cell size (Fig. S1) that is consistent with the characteristics of the genus Bradyrhizobium (Garrity et al., 2005). Cell motility was demonstrated using the semisolid medium puncture method (Shields & Cathcart, 2012) with bacteria grown on semisolid YEM agar medium. Production of an alkaline reaction on YEM agar containing bromothymol blue after 21 days at 28 u C (Bromfield et al., 2010) and mean generation times (12-13 h) of bacterial cultures in YEM broth (Itakura et al., 2008;Wittwer, 2014) (Table 1) were also typical of the genus Bradyrhizobium (Garrity et al., 2005;Vincent, 1970).
Almost full-length 16S rRNA gene sequences were generated using primers 16Sa and 16Sb (van Berkum & Fuhrmann, 2000) and sequence alignment was carried out using the Infernal secondary-structure-based aligner implemented in the Ribosomal Database Project program version 11.1 (Cole et al., 2014). Partial sequences of housekeeping (atpD, glnII, recA, gyrB, rpoB and dnaK) and symbiotic (nodC and nifH)  genes were generated for the 16 novel strains of the genus Bradyrhizobium as well as for reference taxa not available in public databases. Preparation of genomic DNA, amplification, primers, nucleotide sequencing, sequence alignment and editing was as described previously (Tang et al., 2012). The dnaK sequence of 'Bradyrhizobium retamae' Ro19 required designing a second forward sequencing primer (FdnaK_SC1: GAGCAGCAGATCCGGATTCA) in order to obtain the 39 end of the sequence. GenBank accession numbers of nucleotide sequences are given in Tables S1 and S2.
Bayesian phylogenetic analyses were carried out using MrBayes version 3.2.1 with default priors (Altekar et al. 2004). For each dataset, two concurrent analyses with four chains (three heated and one cold) were run for ten million generations with sampling every 2000 generations. Convergence was judged satisfactory when the average SD of split frequencies fell below 0.01 and the potential scale reduction factor statistics were approaching 1.0 (Ronquist et al., 2012). For each dataset, trees from the first 25 % of sampled generations were removed as burn in and a majority rule consensus tree was estimated based on pooled post-burn-in trees.
Best-fit substitution models were selected using the Bayesian information criterion implemented in jModelTest version 2 (Darriba et al., 2012). Maximum-likelihood (ML) phylogenetic analyses (Guindon et al., 2010) were carried out as previously described using 1000 non-parametric bootstrap replications to assess support (Tang et al., 2012). In all instances, tree topologies from Bayesian and ML analyses were similar; for brevity only the Bayesian trees are shown.
Consistent with a previous report (Wang et al., 2013), two major groups of species of the genus Bradyrhizobium were evident in phylogenetic trees of 16S rRNA gene sequences: one represented by Bradyrhizobium japonicum and the other by Bradyrhizobium elkanii (Fig. S2). All 16 novel strains had identical 16S rRNA gene sequences and were placed in the phylogenetic group represented by B. japonicum. The sequence similarities of novel strains varied between 98.44 (Bradyrhizobium denitrificans) and 99.79 % (Bradyrhizobium liaoningense) relative to type strains of the 15 species in the B. japonicum group (Table S3).
MLSA of at least five housekeeping genes is used as a reliable method to define phylogenetic relationships and to identify novel lineages within the genus Bradyrhizobium (Rivas et al., 2009;Tang et al., 2012). The Bayesian phylogenetic tree of six concatenated housekeeping gene sequences (length 3210 bp) for the 16 novel strains and reference taxa is shown in Fig. 1 To further analyse genetic differences between novel strains, we generated random amplified polymorphic DNA (RAPD) fingerprints for representative strains OO99 T , OM9 and OO85 and reference taxa using four random primers (P1, P2, P3 and P5) and amplification methods described by Sikora et al. (2002). An example of the fingerprint profiles generated by one of the primers (P1) is shown in Fig. S3. A dendrogram based on the combined character matrix of fingerprint profiles generated by the four primers was reconstructed using UPGMA and the Dice coefficient implemented in GelCompare II software version 5.10 (Applied Maths). The three representative strains were readily distinguished and were placed in a single cluster separate from reference taxa (Fig. S4), consistent with their assignment to distinct STs, based on MLSA of protein encoding genes.
DNA-DNA hybridization experiments were performed as described by Willems et al., (2001) Wayne et al., (1987). The G+C mol% content of DNA for the three strains, determined by HPLC (Mesbah et al., 1989), varied between 62.3 and 63.5 % which is within the range of DNA G+C content for the genus Bradyrhizobium.
Analysis of accessory genes encoding symbiotic functions (nod and nif) can provide useful information on the host range and specificity of symbiotic bacteria (Laguerre et al., 2001). The phylogenetic tree of partial nodC sequences of novel strains and reference taxa is shown in Fig. S5. All 16 novel strains had identical nodC sequences and were placed in a group with five named species of the genus Bradyrhizobium that are symbiotic with soybeans. Sequence similarities of novel strains relative to these five species were ¢99.86 % (Table S3). Analyses of nifH gene sequences ( Fig. S6 and Table S3) provided almost identical results.
Plant tests were carried out using Leonard jars as described by Tang et al., (2012). The results (Table S5) show that representative strain, OO99 T , elicited effective nitrogen fixing nodules (fix+) on Glycine max, Glycine soja and Macroptilium atropurpureum, partially effective nodules (fix±) on Desmodium canadense and Vigna unguiculata, and ineffective nodules (fix-) on Amphicarpaea bracteata and Phaseolus vulgaris.
For analysis of fatty acids, strains were grown on YEM agar at 28 u C. Bacteria were harvested and fatty acids extracted as described by Sasser (1990). Fatty acid identification was done using the Sherlock Microbial Identification System (MIDI) version 6.0 and the RTSBA6 database. Fatty acid profiles of three novel strains and reference taxa are shown in Table S6. The fatty acid profile of OO99 T was characteristic of the genus Bradyrhizobium with fatty acids 16 : 0 (12.83 %) and summed feature 8 (76.99 %) predominating (Tighe et al., 2000). Five fatty acids (16 : 0, 16 : 1v5c, 18 : 0 and summed feature 8) were common to all strains tested whereas seven (12 : 0, 14 : 0, 17 : 1v8c, 18 : 1v5c, 19 : 0 cyclo v8c and summed feature 3) were detected in only some strains.
Phenotypic characteristics were further investigated using a variety of tests. Utilization of carbon and nitrogen sources was tested using YNB and YCB (Becton Dickinson) basal liquid media as described by Chahboune et al., (2011) except that bromothymol blue was omitted. Cultures were incubated at 28 u C for 20 days on a rotary shaker when bacterial growth (turbidity) was recorded visually and using a Spectronic 21 UV spectrophotometer (Milton Roy) at 660 nm.

Description of
The type strain, OO99 T (5LMG 26739 T 5HAMBI 3284 T ), was isolated from an effective nodule of Glycine max in Ottawa, Ontario, Canada. The DNA G+C content of the type strain is 62.6 mol%.