Bradyrhizobium ontarionense sp. nov., a novel bacterial symbiont isolated from Aeschynomene indica (Indian jointvetch), harbours photosynthesis, nitrogen fixation and nitrous oxide (N2O) reductase genes.

A novel bacterial symbiont, strain A19T, was previously isolated from a root-nodule of Aeschynomene indica and assigned to a new lineage in the photosynthetic clade of the genus Bradyrhizobium. Here data are presented for the detailed genomic and taxonomic analyses of novel strain A19T. Emphasis is placed on the analysis of genes of practical or ecological significance (photosynthesis, nitrous oxide reductase and nitrogen fixation genes). Phylogenomic analysis of whole genome sequences as well as 50 single-copy core gene sequences placed A19T in a highly supported lineage distinct from described Bradyrhizobium species with B. oligotrophicum as the closest relative. The digital DNA-DNA hybridization and average nucleotide identity values for A19T in pair-wise comparisons with close relatives were far lower than the respective threshold values of 70% and ~ 96% for definition of species boundaries. The complete genome of A19T consists of a single 8.44 Mbp chromosome and contains a photosynthesis gene cluster, nitrogen-fixation genes and genes encoding a complete denitrifying enzyme system including nitrous oxide reductase implicated in the reduction of N2O, a potent greenhouse gas, to inert dinitrogen. Nodulation and type III secretion system genes, needed for nodulation by most rhizobia, were not detected. Data for multiple phenotypic tests complemented the sequence-based analyses. Strain A19T elicits nitrogen-fixing nodules on stems and roots of A. indica plants but not on soybeans or Macroptilium atropurpureum. Based on the data presented, a new species named Bradyrhizobium ontarionense sp. nov. is proposed with strain A19T (= LMG 32638T = HAMBI 3761T) as the type strain.

Ensifer canadensis sp. nov. strain T173T isolated from Melilotus albus (sweet clover) in Canada possesses recombinant plasmid pT173b harbouring symbiosis and type IV secretion system genes apparently acquired from Ensifer medicae.

A bacterial strain, designated T173T, was previously isolated from a root-nodule of a Melilotus albus plant growing in Canada and identified as a novel Ensifer lineage that shared a clade with the non-symbiotic species, Ensifer adhaerens. Strain T173T was also previously found to harbour a symbiosis plasmid and to elicit root-nodules on Medicago and Melilotus species but not fix nitrogen. Here we present data for the genomic and taxonomic description of strain T173T. Phylogenetic analyses including the analysis of whole genome sequences and multiple locus sequence analysis (MLSA) of 53 concatenated ribosome protein subunit (rps) gene sequences confirmed placement of strain T173T in a highly supported lineage distinct from named Ensifer species with E. morelensis Lc04T as the closest relative. The highest digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values of genome sequences of strain T173T compared with closest relatives (35.7 and 87.9%, respectively) are well below the respective threshold values of 70% and 95-96% for bacterial species circumscription. The genome of strain T173T has a size of 8,094,229 bp with a DNA G + C content of 61.0 mol%. Six replicons were detected: a chromosome (4,051,102 bp) and five plasmids harbouring plasmid replication and segregation (repABC) genes. These plasmids were also found to possess five apparent conjugation systems based on analysis of TraA (relaxase), TrbE/VirB4 (part of the Type IV secretion system (T4SS)) and TraG/VirD4 (coupling protein). Ribosomal RNA operons encoding 16S, 23S, and 5S rRNAs that are usually restricted to bacterial chromosomes were detected on plasmids pT173d and pT173e (946,878 and 1,913,930 bp, respectively) as well as on the chromosome of strain T173T. Moreover, plasmid pT173b (204,278 bp) was found to harbour T4SS and symbiosis genes, including nodulation (nod, noe, nol) and nitrogen fixation (nif, fix) genes that were apparently acquired from E. medicae by horizontal transfer. Data for morphological, physiological and symbiotic characteristics complement the sequence-based characterization of strain T173T. The data presented support the description of a new species for which the name Ensifer canadensis sp. nov. is proposed with strain T173T (= LMG 32374T = HAMBI 3766T) as the species type strain.

Bradyrhizobium septentrionale sp. nov. (sv. septentrionale) and Bradyrhizobium quebecense sp. nov. (sv. septentrionale) associated with legumes native to Canada possess rearranged symbiosis genes and numerous insertion sequences.

Six bacterial strains isolated from root nodules of soybean plants that had been inoculated with root-zone soil of legumes native to Canada were previously characterized and 1) placed in two novel lineages within the genus Bradyrhizobium and 2) assigned to symbiovar septentrionale. Here we verified the taxonomic status of these strains using genomic and phenotypic analyses. Phylogenetic analyses of five protein encoding partial gene sequences as well as 52 full length ribosome protein subunit gene sequences confirmed placement of the novel strains in two highly supported lineages distinct from named Bradyrhizobium species. The highest average nucleotide identity values of strains representing these two lineages relative to type strains of closest relatives were 90.7 and 92.3% which is well below the threshold value for bacterial species circumscription. The genomes of representative strains 1S1T, 162S2 and 66S1MBT have sizes of 10598256, 10733150 and 9032145 bp with DNA G+C contents of 63.5, 63.4 and 63.8 mol%, respectively. These strains possess between one and three plasmids based on copy number of plasmid replication and segregation (repABC) genes. Novel strains also possess numerous insertion sequences, and, relative to reference strain Bradyrhizobium diazoefficiens USDA110T, exhibit inversion and fragmentation of nodulation (nod) and nitrogen-fixation (nif) gene clusters. Phylogenetic analyses of nodC and nifH gene sequences confirmed placement of novel strains in a distinct lineage corresponding to symbiovar septentrionale. Data for morphological, physiological and symbiotic characteristics complement the sequence-based results. The data presented here support the description of two new species for which the names Bradyrhizobium septentrionale sp. nov. (sv. septentrionale) and Bradyrhizobium quebecense sp. nov. (sv. septentrionale) are proposed, with 1S1T (=LMG 29930T=HAMBI 3676T) and 66S1MBT (=LMG 31547T=HAMBI 3720T) as type strains, respectively.

Strains of Bradyrhizobium cosmicum sp. nov., isolated from contrasting habitats in Japan and Canada possess photosynthesis gene clusters with the hallmark of genomic islands.

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.

Description and complete genome sequences of Bradyrhizobium symbiodeficiens sp. nov., a non-symbiotic bacterium associated with legumes native to Canada.

Four bacterial strains isolated from root nodules of soybean plants that had been inoculated with root-zone soil of either Amphicarpaea bracteata (Hog Peanut) or Desmodium canadense (Showy Tick Trefoil) growing in Canada, were previously characterized and placed in a novel lineage within the genus Bradyrhizobium. The taxonomic status of the novel strains was verified by genomic and phenotypic analyses. Phylogenetic analyses of individual and concatenated housekeeping gene sequences (atp D, gln II, rec A, gyr B and rpo B) placed all novel strains in a highly supported lineage distinct from named Bradyrhizobium species. Data for sequence similarities of concatenated housekeeping genes of novel strains relative to type strains of named species were consistent with the phylogenetic data. Average nucleotide identity values of genome sequences (84.5-93.7 %) were below the threshold value of 95-96 % for bacterial species circumscription. Close relatives to the novel strains are Bradyrhizobium amphicarpaeae, Bradyrhizobium ottawaense and Bradyrhizobium shewense. The complete genomes of strains 85S1MBT and 65S1MB consist of single chromosomes of size 7.04 and 7.13 Mbp, respectively. The genomes of both strains have a G+C content of 64.3 mol%. These strains lack a symbiosis island as well as key nodulation, nitrogen-fixation and photosystem genes. Data from various phenotypic tests including growth characteristics and carbon source utilization supported the sequence-based analyses. Based on the data presented here, the four strains represent a novel species for which the name B radyrhizobium symbiodeficiens sp. nov., is proposed, with 85S1MBT (=LMG 29937T=HAMBI 3684T) as the type strain.

Analysis of the Complete Genome Sequence of the Widely Studied Strain Bradyrhizobium betae PL7HG1T Reveals the Presence of Photosynthesis Genes and a Putative Plasmid.

Here, we present the complete genome sequence of the widely studied strain Bradyrhizobium betae PL7HG1T, isolated from a tumor on the roots of sugar beet. The genome consists of a 7.2-Mbp circular chromosome containing key photosynthesis genes but not genes for nodulation and nitrogen fixation. A putative plasmid was also detected.

Invasive Galega officinalis (Goat's rue) plants in Canada form a symbiotic association with strains of Neorhizobium galegae sv. officinalis originating from the Old World.

The toxic legume plant, Galega officinalis, is native to the Eastern Mediterranean and Black Sea regions. This legume is considered to be a noxious weed, and its establishment in Canada may have resulted from ornamental planting and/or field trials. In its native range, a highly specific nitrogen-fixing symbiosis with the bacterium, Neorhizobium galegae symbiovar (sv.) officinalis, is required for normal growth. In North America, nothing is known about the bacterial symbionts of G. officinalis. Our purpose was to determine the species and symbiovar identity of symbiotic bacteria associated with invasive plants of G. officinalis at five sites in the province of Ontario, Canada.  Sequence analysis of four housekeeping (16S rRNA, atpD, glnII, and recA) and two symbiosis (nodC and nifH) genes showed that all 50 bacterial isolates from root nodules of G. officinalis at the five Canadian sites were identical to strains of N. galegae sv. officinalis originating either from Europe or the Caucasus. Plant tests indicated that soils collected from four Canadian sites without a history of agriculture or presence of G. officinalis were deficient in symbiotic bacteria capable of eliciting nodules on this plant. Collectively our data support the hypothesis of anthropogenic co-introduction of G. officinalis and its specific symbiotic bacterium into Canada from the Old World. Factors that may limit the spread of G. officinalis in new environments are discussed.

Description and complete genome sequence of Bradyrhizobium amphicarpaeae sp. nov., harbouring photosystem and nitrogen-fixation genes.

A bacterial strain, designated 39S1MBT, isolated from a root nodule of a soybean plant that had been inoculated with root-zone soil of Amphicarpaea bracteata (hog peanut) growing in Canada, was previously characterized and placed in a novel lineage within the genus Bradyrhizobium. The taxonomic status of strain 39S1MBT was verified by genomic and phenotypic analyses. Phylogenetic analyses of individual and concatenated protein-encoding gene sequences (atpD, glnII, recA, gyrB and rpoB) placed 39S1MBT in a lineage distinct from named species. Data for sequence similarities of concatenated genes relative to type strains of named species supported the phylogenetic data. Average nucleotide identity values of genome sequences (84.5-91.7 %) were well below the threshold value for bacterial species circumscription. Based on these data, Bradyrhizobium ottawaense OO99T and Bradyrhizobium shewense ERR11T are close relatives of 39S1MBT. The complete genome of 39S1MBT consists of a single 7.04 Mbp chromosome without a symbiosis island; G+C content is 64.7 mol%. Present in the genome are key photosystem and nitrogen-fixation genes, but not nodulation and type III secretion system genes. Sequence analysis of the nitrogen fixation gene, nifH, placed 39S1MBT in a novel lineage distinct from named 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 amphicarpaeae sp. nov. is proposed with 39S1MBT (=LMG 29934T=HAMBI 3680T) as the type strain.

Complete Genome Sequence of Bradyrhizobium ottawaense OO99T, an Efficient Nitrogen-Fixing Symbiont of Soybean.

We present the complete genome sequence of Bradyrhizobium ottawaense strain OO99T, a nitrogen-fixing bacterium from root nodules of soybean. The genome consists of a single 8.6-Mb chromosome and includes a symbiosis island. Genes involved in symbiotic nitrogen fixation, stress response, resistance to antibiotics, and toxic compounds were detected.

Diversity of bacteria associated with corn roots inoculated with Canadian woodland soils, and description of Pseudomonas aylmerense sp. nov.

Bacteria associated with corn roots inoculated with soils collected from the Canadian woodlands were isolated and characterized. Genus-level identification based on 16S rRNA sequence analysis classified the 161 isolates in 19 genera. The majority (64%) of the isolates were affiliated with the genus Pseudomonas. Further analysis of the Pseudomonas isolates based on BLASTn and rpoD-rpoB-gyrB concatenated gene phylogeny revealed three unique clusters that could not be assigned to known species. This study reports the taxonomic description of one of the distinct lineages represented by two strains (S1E40T and S1E44) with P. lurida LMG 21995T, P. costantinii LMG 22119T, P. palleroniana LMG 23076T, P. simiae CCUG 50988T and P. extremorientalis LMG 19695T as the closest taxa. Both strains showed low ANIm (<90%) and genome-based DNA-DNA hybridization (<50%) values, which unequivocally delineated the new strains from the closest relatives. These findings were supported by multilocus sequence analysis (MLSA) and DNA fingerprinting. In addition, growth characteristics and biochemical tests revealed patterns that differed from the related species. Strains S1E40T and S1E44 are Gram-negative, aerobic, rod-shaped and motile by at least one flagellum; and grew optimally at 30 °C. The predominant polar lipid is phosphatidylethanolamine while the major respiratory quinone is ubiquinone-9. Based on phenotypic and genotypic data presented here, strains S1E40T and S1E44 represent a novel species for which the name Pseudomonas aylmerense sp. nov. is proposed. The type strain is S1E40T (= LMG 30784T = DOAB 703T = HAMI 3696T) with a G + C content of 61.6%.

Soybeans inoculated with root zone soils of Canadian native legumes harbour diverse and novel Bradyrhizobium spp. that possess agricultural potential.

An assessment was made of the evolutionary relationships of soybean nodulating bacteria associated with legumes native to eastern Canada to identify potential new sources of soybean inoculant strains. Short season soybeans were used to selectively trap bacteria from root zone soils of four native legume species. Screening of more than 800 bacterial isolates from soybean root nodules by analysis of recA gene sequences followed by analyses of selected genotypes using six core and two symbiosis (nodC and nifH) gene sequences permitted identification of diverse taxa that included eight novel and four named Bradyrhizobium species as well as lineages attributed to the genera Afipia and Tardiphaga. Plant tests showed that symbionts related to four named species as well as a novel Bradyrhizobium lineage were highly efficient with regard to nitrogen fixation on soybeans relative to an inoculant strain. A new symbiovar (sv. septentrionalis) is proposed based on a group of four novel Bradyrhizobium spp. that possess distinctive nodC and nifH gene sequences and symbiotic characteristics. Evidence is provided for horizontal transfer of sv. septentrionalis symbiosis genes between novel Bradyrhizobium spp., a process that rendered recipient bacteria ineffective on soybeans. Diverse lineages of non-symbiotic and symbiotic Bradyrhizobium spp. co-occured within monophyletic clusters in a phylogenetic tree of concatenated core genes, suggesting that loss and/or gain of symbiosis genes has occurred in the evolutionary history of the bacterial genus. Our data suggest that symbiont populations associated with legumes native to eastern Canada harbour elite strains of Bradyrhizobium for soybean inoculation.

Pseudomonas canadensis sp. nov., a biological control agent isolated from a field plot under long-term mineral fertilization.

The bacterial strain 2-92T, isolated from a field plot under long-term (>40 years) mineral fertilization, exhibited in vitro antagonistic properties against fungal pathogens. A polyphasic approach was undertaken to verify its taxonomic status. Strain 2-92T was Gram-reaction-negative, aerobic, non-spore-forming, motile by one or more flagella, and oxidase-, catalase- and urease-positive. The optimal growth temperature of strain 2-92T was 30 °C. 16S rRNA gene sequence analysis demonstrated that the strain is related to species of the genus Pseudomonas. Phylogenetic analysis of six housekeeping genes (dnaA, gyrB, recA, recF, rpoB and rpoD) revealed that strain 2-92T clustered as a distinct and well separated lineage with Pseudomonassimiae as the most closely related species. Polar lipid and fatty acid compositions corroborated the taxonomic position of strain 2-92T in the genus Pseudomonas. Phenotypic characteristics from carbon utilization tests could be used to differentiate strain 2-92T from closely related species of the genus Pseudomonas. DNA-DNA hybridization values (wet laboratory and genome-based) and average nucleotide identity data confirmed that this strain represents a novel species. On the basis of phenotypic and genotypic characteristics, it is concluded that this strain represents a separate novel species for which the name Pseudomonas canadensis sp. nov. is proposed, with type strain 2-92T (=LMG 28499T=DOAB 798T). The DNA G+C content is 60.30 mol%.

Draft Genome Sequences of Two Novel Pseudomonas Strains Exhibiting Differential Hypersensitivity Reactions on Tobacco and Corn Seedlings.

Two novel Pseudomonas strains (S1E40 and S3E12) isolated from corn roots are antagonistic to Rhizoctonia solani and exhibit differential hypersensitivity reactions on tobacco and corn seedlings. We report here the draft genome sequences of strains S1E40 and S3E12, consisting of 6.98 and 7.06 Mb with 6,150 and 6,129 predicted protein-coding sequences, respectively.

Draft Genome Sequence of Pseudomonas simiae Strain 2-36, an In Vitro Antagonist of Rhizoctonia solani and Gaeumannomyces graminis.

Pseudomonas simiae 2-36, isolated from a field plot under long-term mineral fertilization, exhibited strong in vitro antagonistic activities against Rhizoctonia solani and Gaeumannomyces graminis. We report here the draft genome sequence of Pseudomonas simiae 2-36, consisting of 6.4 Mbp with a 60.25% G+C content and 5,790 predicted protein-coding sequences.

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 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) ( = LMG 26739(T) = HAMBI 3284(T)) as the type strain. The DNA G+C content is 62.6 mol%.

Draft Genome Sequence of Pantoea ananatis Strain LMG 2665T, a Bacterial Pathogen of Pineapple Fruitlets.

We report the draft genome sequence of Pantoea ananatis LMG 2665(T), the bacterial causal agent of pineapple fruitlet rot.