In vitro interactions between Bradyrhizobium spp. and Tuber magnatum mycelium.

Tuber magnatum is the most expensive truffle, but its large-scale cultivation is still a challenge compared to other valuable Tuber species. T. magnatum mycelium has never been grown profitably until now, which has led to difficulties to studying it in vitro. This study describes beneficial interactions between T. magnatum mycelium and never before described bradyrhizobia, which allows the in vitro growth of T. magnatum mycelium. Three T. magnatum strains were co-isolated on modified Woody Plant Medium (mWPM) with aerobic bacteria and characterised through microscopic observations. The difficulties of growing alone both partners, bacteria and T. magnatum mycelium, on mWPM demonstrated the reciprocal dependency. Three bacterial isolates for each T. magnatum strain were obtained and molecularly characterised by sequencing the 16S rRNA, glnII, recA and nifH genes. Phylogenetic analyses showed that all nine bacterial strains were distributed among five subclades included in a new monophyletic lineage belonging to the Bradyrhizobium genus within the Bradyrhizobium jicamae supergroup. The nifH genes were detected in all bacterial isolates, suggesting nitrogen-fixing capacities. This is the first report of consistent T. magnatum mycelium growth in vitro conditions. It has important implications for the development of new technologies in white truffle cultivation and for further studies on T. magnatum biology and genetics.

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.

Widespread Bradyrhizobium distribution of diverse Type III effectors that trigger legume nodulation in the absence of Nod factor.

The establishment of the rhizobium-legume symbiosis is generally based on plant perception of Nod factors (NFs) synthesized by the bacteria. However, some Bradyrhizobium strains can nodulate certain legume species, such as Aeschynomene spp. or Glycine max, independently of NFs, and via two different processes that are distinguished by the necessity or not of a type III secretion system (T3SS). ErnA is the first known type III effector (T3E) triggering nodulation in Aeschynomene indica. In this study, a collection of 196 sequenced Bradyrhizobium strains was tested on A. indica. Only strains belonging to the photosynthetic supergroup can develop a NF-T3SS-independent symbiosis, while the ability to use a T3SS-dependent process is found in multiple supergroups. Of these, 14 strains lacking ernA were tested by mutagenesis to identify new T3Es triggering nodulation. We discovered a novel T3E, Sup3, a putative SUMO-protease without similarity to ErnA. Its mutation in Bradyrhizobium strains NAS96.2 and WSM1744 abolishes nodulation and its introduction in an ernA mutant of strain ORS3257 restores nodulation. Moreover, ectopic expression of sup3 in A. indica roots led to the formation of spontaneous nodules. We also report three other new T3Es, Ubi1, Ubi2 and Ubi3, which each contribute to the nodulation capacity of strain LMTR13. These T3Es have no homology to known proteins but share with ErnA three motifs necessary for ErnA activity. Together, our results highlight an unsuspected distribution and diversity of T3Es within the Bradyrhizobium genus that may contribute to their symbiotic efficiency by participating in triggering legume nodulation.

Multiple Gene Clusters and Their Role in the Degradation of Chlorophenoxyacetic Acids in Bradyrhizobium sp. RD5-C2 Isolated from Non-Contaminated Soil.

Bradyrhizobium sp. RD5-C2, isolated from soil that is not contaminated with 2,4-dichlorophenoxyacetic acid (2,4-D), degrades the herbicides 2,4-D and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). It possesses tfdAα and cadA (designated as cadA1), which encode 2,4-D dioxygenase and the oxygenase large subunit, respectively. In the present study, the genome of Bradyrhizobium sp. RD5-C2 was sequenced and a second cadA gene (designated as cadA2) was identified. The two cadA genes belonged to distinct clusters comprising the cadR1A1B1K1C1 and cadR2A2B2C2K2S genes. The proteins encoded by the cad1 cluster exhibited high amino acid sequence similarities to those of other 2,4-D degraders, while Cad2 proteins were more similar to those of non-2,4-D degraders. Both cad clusters were capable of degrading 2,4-D and 2,4,5-T when expressed in non-2,4-D-degrading Bradyrhizobium elkanii USDA94. To examine the contribution of each degradation gene cluster to the degradation activity of Bradyrhizobium sp. RD5-C2, cadA1, cadA2, and tfdAα deletion mutants were constructed. The cadA1 deletion resulted in a more significant decrease in the ability to degrade chlorophenoxy compounds than the cadA2 and tfdAα deletions, indicating that degradation activity was primarily governed by the cad1 cluster. The results of a quantitative reverse transcription-PCR analysis suggested that exposure to 2,4-D and 2,4,5-T markedly up-regulated cadA1 expression. Collectively, these results indicate that the cad1 cluster plays an important role in the degradation of Bradyrhizobium sp. RD5-C2 due to its high expression.

Culture-independent assessment of the diazotrophic Bradyrhizobium communities in the Pampa and Atlantic Forest Biomes localities in southern Brazil.

The isolation of rhizobial strains from the root and stem nodules remains a commonly used method despite its limitations as it enables the identification of mainly dominant symbiotic groups within rhizobial communities. To overcome these limitations, we used genus-specific nifD primers in a culture-independent assessment of Bradyrhizobium communities inhabiting soils in southern Brazil. The majority of nifD sequences were generated from DNA isolated from tropical-lowland pasture soils, although some soil samples originated from the Campos de Cima da Serra volcanic plateau. In the nifD tree, all the bradyrhizobial sequences comprised 38 clades, including 18 new clades. The sequences generated in this study were resolved into 22 clades and 21 singletons. The nifD bradyrhizobial assemblage contained Azorhizobium and α-proteobacterial methylotrophic genera, suggesting that these genera may have acquired their nif loci from Bradyrhizobium donors. The most common in the lowland pasture soils subclade III.3D branch comprises the isolates of mainly an American origin. On the other hand, subclade III.4, which was earlier detected in Brazil among Bradyrhizobium isolates nodulating native lupins, appears more common in the Campos de Cima da Serra soils. The second-largest group, Clade XXXVIII, has not yet been reported in culture-dependent studies, while another common group called Clade I represents a symbiovar predominating in Australia. The identification of the diverse nifD Clade I haplotypes in the tropical-lowland pastures infested by Australian Acacia spp implies that the introduction of these legumes to southern Brazil has resulted in the dissemination of their bradyrhizobial symbionts.

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.

Characterization of Retama sphaerocarpa microsymbionts in Zaida lead mine tailings in the Moroccan middle Atlas.

In the Moroccan Middle Atlas, the tailings rich in lead and other metal residues, in the abandoned Zaida mining district, represent a real threat to environment and the neighboring villages' inhabitants' health. In this semi-arid to arid area, phytostabilisation would be the best choice to limit the transfer of heavy metals to populations and groundwater. The aim of this work was to characterize the bacteria that nodulate Retama sphaerocarpa, spontaneous nitrogen fixing shrubby legume, native to the Zaida mining area, with great potential to develop for phytostabilisation. Forty-three bacteria isolated from root nodules of the plant were characterized. Based on REP-PCR and ARDRA, four strains were selected for further molecular analyzes. The 16S rRNA gene sequences analysis revealed that the isolated strains are members of the genus Bradyrhizobium, and the phylogenetic analysis of the housekeeping genes glnII, atpD, gyrB, rpoB, recA and dnaK individual sequences and their concatenation showed that the strains are close to B. algeriense RST89T and B. valentinum LmjM3T with similarity percentages of 89.07% to 95.66% which suggest that the newly isolated strains from this mining site may belong to a potential novel species. The phylogeny of the nodA and nodC genes showed that the strains belong to the symbiovar retamae of the genus Bradyrhizobium. These strains nodulate also R. monosperma, R. dasycarpa and Lupinus luteus.

Diversity and phylogenetic affinities of Bradyrhizobium isolates from Pampa and Atlantic Forest Biomes.

In this work, we investigated Bradyrhizobium strains isolated from soils collected from the rhizosphere of native and exotic legumes species inhabiting two ecoclimatic zones - asubtropical-lowland pasture (Pampa Biome) and a volcanic plateau covered by Araucaria Moist Forests (Atlantic Forest Biome). The rhizobial strains were isolated from the nodules of seven native and one exotic legume species used as rhizobium traps. Single-gene (recA, glnII, dnaK) and combined-gene MLSA analyses (dnaK-glnII-gyrB-recA-rpoB) revealed that nearly 85% of the isolates clustered in B. elkanii supergroup, while the remaining (except for two isolates) in B. japonicum supergroup, albeit, in most cases, separately from the type strains of Bradyrhizobium species. As a symbiotic gene marker, a portion of nifD gene was sequenced for 194 strains. In the nifD-tree, an American branch III.3D (104 isolates), was the most numerous among the isolates. A significant portion of the isolates clustered in American groups; subclade III.4 (40 strains), Clade VII (3 strains), and a new Clade XX (4 strains). Most of the remaining strains belonged to a pantropical III.3C branch (39 isolates). On the other hand, identification of isolates belonging, respectively, to Clade I and Clade II may result of spreading of the Australian (Clade I) and European (Clade II) bradyrhizobia following the introduction of their legume hosts. Our study indicated that the American groups predominated in the symbiotic Bradyrhizobium communities in southern Brazil. However, there is a significant component of exotic lineages, resulting from the dispersal of pantropical Fabaceae taxa and the introduction of exotic legumes.

Bradyrhizobium sp. sv. retamae nodulates Retama monosperma grown in a lead and zinc mine tailings in Eastern Morocco.

The aim of this work was to characterize and identify some bacteria isolated from the root nodules of Retama monosperma grown in Sidi Boubker lead and zinc mine tailings. Very few root nodules were obtained on the root nodules of R. monosperma grown in these soils. The three bacteria isolated from the root nodules were tolerant in vitro to different concentrations of heavy metals, including lead and zinc. The rep-PCR experiments showed that the three isolates have different molecular fingerprints and were considered as three different strains. The analysis of their 16S rRNA gene sequences proved their affiliation to the genus Bradyrhizobium. The analysis and phylogeny of the housekeeping genes atpD, glnII, gyrB, recA, and rpoB confirmed that the closest species was B. valentinum with similarity percentages of 95.61 to 95.82%. The three isolates recovered from the root nodules were slow-growing rhizobia capable to renodulate their original host plant in the presence of Pb-acetate. They were able to nodulate R. sphaerocarpa and Lupinus luteus also but not Glycine max or Phaseolus vulgaris. The phylogeny of the nodA and nodC nodulation genes as well as the nifH gene of the three strains showed that they belong to the symbiovar retamae of the genus Bradyrhizobium. The three strains isolated could be considered for use as inoculum for Retama plants before use in phytoremediation experiments.

Genetic diversity and distribution of rhizobia associated with soybean in red soil in Hunan Province.

To explore the genetic diversity and distribution of rhizobia in the rhizosphere of soybean grown in red soil, we have collected 21 soil samples from soybean fields across seven counties in Hunan province, China. MiSeq sequencing of rpoB gene was used to determine the intra-species diversity of rhizobia existing in soybean rhizospheres. Soil chemical properties were determined by routine methods. The Principal Coordinates Analysis (PCoA) plot indicated a clear biogeographical pattern characterizing the soybean rhizosphere across different sites. The Mantel test demonstrated that biogeographical pattern was significantly correlated with the geographical distance (Mantel statistic R 0.385, p < 0.001). There were obvious differences in the rhizobial communities among northeastern eco-region, southeastern eco-region and western eco-region. In general, Bradyrhizobium diazoefficiens was the most abundant rhizobial species in the soybean rhizosphere. At an intermediate (10-400 km) spatial scale, the biogeographical pattern of rhizobial communities in soybean rhizosphere is associated with both soil properties and geographical distance. Redundancy analysis (RDA) showed that total potassium (TK), available potassium (AK), soil organic carbon (SOC), and available nitrogen (AN) were the main factors that influenced the α-diversity of rhizobial communities. Canonical correspondence analysis (CCA) showed that pH and exchangeable Ca and Mg had the greatest influence on the ß-diversity of the rhizobial communities in the soybean rhizosphere. These findings characterize the distribution pattern and its influencing factors of soybean rhizobia in rhizosphere in Hunan province, which may be helpful in selecting suitable strains or species as inoculants for soybeans in red soil regions.

qPCR assay targeting Bradyrhizobium japonicum shows that row spacing and soybean density affects Bradyrhizobium population.

The ability for a soybean plant to be efficiently nodulated when grown as a crop is dependent on the number of effective Bradyrhizobium japonicum that can be found in close proximity to the developing seedling shortly after planting. In Manitoba, the growing of soybean as a crop has increased from less than 500 000 acres in 2008 to over 2.3 million acres in 2017. Since the large increase in soybean production is relatively recent, populations of B. japonicum have not yet developed. In response to this, we developed a primer pair that can identify B. japonicum, and be used to determine the titre found in field soil. Their utility was demonstrated by being used to determine whether row spacing of soybean affects B. japonicum populations, as well as to follow B. japonicum populations in a soybean field over the course of a field season. The data show that plant density can affect B. japonicum populations. Moreover, evidence is presented that suggests plant development affects overall B. japonicum populations.

Bradyrhizobium campsiandrae sp. nov., a nitrogen-fixing bacterial strain isolated from a native leguminous tree from the Amazon adapted to flooded conditions.

The nitrogen-fixing bacterial strain UFLA 01-1174T was isolated from nodules of Campsiandra laurilifolia Benth. originating from the Amazon region, Brazil. Its taxonomic position was defined using a polyphasic approach. Analysis of the 16S rRNA gene placed the strain in the Bradyrhizobium genus, the closest species being B. guangdongense CCBAU 51649T and B. guangzhouense CCBAU 51670T, both with 99.8% similarity. Multilocus sequence analysis (MLSA) of recA, gyrB, glnII, rpoB, atpD, and dnaK indicated that UFLA 01-1174T is a new species, most closely related to B. stylosanthis BR 446T (94.4%) and B. manausense BR 3351T (93.7%). Average nucleotide identity (ANI) differentiated UFLA 01-1174T from the closest species with values lower than 90%. The G + C content in the DNA of UFLA 01-1174T is 63.6 mol%. Based on this data, we conclude that the strain represents a new species. The name proposed is Bradyrhizobium campsiandrae, with UFLA 01-1174T (= INPA 394BT = LMG 10099T) as type strain.

Phylogenetic diversity analysis reveals Bradyrhizobium yuanmingense and Ensifer aridi as major symbionts of mung bean (Vigna radiata L.) in Pakistan.

The present study was carried out to evaluate the diversity of rhizobia associated with nodules of mung bean in Pakistan, because this information is necessary for inoculum development. Based on sequence analysis of 16S rRNA gene of thirty-one bacteria, 11 were assigned to genus Bradyrhizobium, 17 to Ensifer, and 3 to Rhizobium. Phylogenetic analyses on the basis of 16S-23S ITS region, atpD, recA, nifH, and nodA of representative strains revealed that B. yuanmingense is the predominant species distributed throughout different mung bean-growing areas. Among the fast-growing rhizobia, Ensifer aridi was predominant in Faisalabad, Layyah, and Rawalpindi, while E. meliloti in Thal desert. Sequence variations and phylogeny of nifH and nodA genes suggested that these genes might have been co-evolved with the housekeeping genes and maintained by vertical gene transfer in rhizobia detected in the present study. Host infectivity assay revealed the successful nodulation of host by rhizobia related to genera Bradyrhizobium, Ensifer and Rhizobium. Among all, Bradyrhizobium and Ensifer spp. inoculation exhibited a significantly higher number of nodules (11-34 nodules plant-1) and nitrogenase activity (nodule ARA 60-110 µmol g-1 h-1). Contrary to the previous studies, our data reveal that B. yuanmingense and E. aridi are predominant species forming effective nodules in mung bean in Pakistan. Furthermore, to the best of our knowledge, this is the first report showing the effective symbiosis of E. aridi, E. meliloti, and Rhizobium pusense with mung bean. The diversity of rhizobia in different habitats revealed in the present study will contribute towards designing site-specific inocula for mung bean.

Soybean tolerance to drought depends on the associated Bradyrhizobium strain.

We evaluated the effect of three different Bradyrhizobium strains inoculated in two soybean genotypes (R01-581F, drought-tolerant, and NA5858RR, drought-sensitive) submitted to drought in two trials conducted simultaneously under greenhouse. The strains (SEMIA 587, SEMIA 5019 (both B. elkanii), and SEMIA 5080 (B. diazoefficiens)) were inoculated individually in each genotype and then submitted to water restriction (or kept well-watered, control) between 45 and 62 days after emergence. No deep changes in plant physiological variables were observed under the moderate water restriction imposed during the first 10 days. Nevertheless, photosynthesis and transpiration decreased after the severe water restriction imposed for further 7 days. Water restriction reduced growth (- 30%) and the number of nodules (- 47% and - 58% for R01-581F and NA5858RR, respectively) of both genotypes, with a negative effect on N-metabolism. The genotype R01-581F inoculated with SEMIA 5019 strain had higher photosynthetic rates compared with NA5858RR, regardless of the Bradyrhizobium strain. On average, R01-581F showed better performance under drought than NA5858RR, with higher number of nodules (51 vs. 38 nodules per plant, respectively) and less accumulation of ureides in petioles (15 µmol g-1 vs. 34 µmol g-1, respectively). Moreover, plants inoculated with SEMIA 5080 had higher glutamine synthetase activity under severe water restriction, especially in the drought-tolerant R01-518F, suggesting maintenance of N metabolism under drought. The Bradyrhizobium strain affects the host plant responses to drought in which the strain SEMIA 5080 improves the drought tolerance of R01-518F genotype.

Bradyrhizobium hipponense sp. nov., isolated from Lupinus angustifolius growing in the northern region of Tunisia.

Strain aSej3T was isolated from a root nodule of a Lupinus angustifolius plant growing in Bizerte, Tunisia. 16S rRNA gene analysis placed this strain within the genus Bradyrhizobium. Multilocus sequence analysis (MLSA) including three housekeeping genes (glnII, gyrB and recA) grouped aSej3T together with Bradyrhizobium rifense CTAW71T, Bradyrhizobium cytisi CTAW11T, Bradyrhizobium ganzhouense RITF806T, Bradyrhizobium lupini USDA 3051T and Bradyrhizobium canariense BTA-1T. MLSA with five housekeeping genes (dnaK, glnII, gyrB, recA and rpoB) revealed that this strain shares less than 93.5 % nucleotide identity with other type strains. Genome sequencing and inspection revealed a genome size of 8.83 Mbp with a G+C content of 62.8 mol%. Genome-wide average nucleotide identity and digital DNA-DNA hybridization values were below 87.5 and 36.2 %, respectively, when compared to described Bradyrhizobium species. Strain aSej3T nodulated L. angustifolius plants under axenic conditions and its nodC gene clustered within the genistearum symbiovar. Altogether, the phylogenetic data and the chemotaxonomic characteristics of this strain support that aSej3T represents a new species for which we propose the name Bradyrhizobium hipponense sp. nov. with the type strain aSej3T (=DSM 108913T=LMG 31020T).

Long-term monoculture reduces the symbiotic rhizobial biodiversity of peanut.

Long-term monoculture (LTM) decreases the yield and quality of peanut, even resulting in changes in the microbial community. However, the effect of LTM on peanut rhizobial communities has still not been elucidated. In this study, we isolated and characterized peanut rhizobia from 6 sampling plots with different monoculture cropping durations. The community structure and diversity index for each sampling site were analyzed, and the correlations between a peanut rhizobium and soil characteristics were evaluated to clarify the effects on peanut rhizobial communities. The competitive abilities among representative strains were also analyzed. A total of 283 isolates were obtained from 6 sampling plots. Nineteen recA haplotypes were defined and were grouped into 8 genospecies of Bradyrhizobium, with B. liaoningense and B. ottawaense as the dominant groups in each sample. The diversity indexes of the rhizobial community decreased, and the dominant groups of B. liaoningense and B. ottawaense were enriched significantly with extended culture duration. Available potassium (AK), available phosphorus (AP), available nitrogen (AN), total nitrogen (TN) and organic carbon (OC) gradually increased with increasing monoculture duration. OC, TN, AP and AK were the main soil characteristics affecting the distribution of rhizobial genospecies in the samples. A competitive nodulation test indicated that B. liaoningense presented an excellent competitive ability, which was congruent with its high isolation frequency. This study revealed that soil characteristics and the competitive ability of rhizobia shape the symbiotic rhizobial community and provides information on community formation and the biogeographic properties of rhizobia.

Phylogenetic distribution and evolutionary dynamics of nod and T3SS genes in the genus Bradyrhizobium.

Bradyrhizobium are abundant soil bacteria and the major symbiont of legumes. The recent availability of Bradyrhizobium genome sequences provides a large source of information for analysis of symbiotic traits. In this study, we investigated the evolutionary dynamics of the nodulation genes (nod) and their relationship with the genes encoding type III secretion systems (T3SS) and their effectors among bradyrhizobia. Based on the comparative analysis of 146 Bradyrhizobium genome sequences, we identified six different types of T3SS gene clusters. The two predominant cluster types are designated RhcIa and RhcIb and both belong to the RhcI-T3SS family previously described in other rhizobia. They are found in 92/146 strains, most of them also containing nod genes. RhcIa and RhcIb gene clusters differ in the genes they carry: while the translocon-encoding gene nopX is systematically found in strains containing RhcIb, the nopE and nopH genes are specifically conserved in strains containing RhcIa, suggesting that these last two genes might functionally substitute nopX and play a role related to effector translocation. Phylogenetic analysis suggests that bradyrhizobia simultaneously gained nod and RhcI-T3SS gene clusters via horizontal transfer or subsequent vertical inheritance of a symbiotic island containing both. Sequence similarity searches for known Nop effector proteins in bradyrhizobial proteomes revealed the absence of a so-called core effectome, i.e. that no effector is conserved among all Bradyrhizobium strains. However, NopM and SUMO proteases were found to be the main effector families, being represented in the majority of the genus. This study indicates that bradyrhizobial T3SSs might play a more significant symbiotic role than previously thought and provides new candidates among T3SS structural proteins and effectors for future functional investigations.

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.

Bradyrhizobium archetypum sp. nov., Bradyrhizobium australiense sp. nov. and Bradyrhizobium murdochi sp. nov., isolated from nodules of legumes indigenous to Western Australia.

The genus Bradyrhizobium is considered as the probable ancestor lineage of all rhizobia, broadly spread in a variety of ecosystems and with remarkable diversity. A polyphasic study was performed to characterize and clarify the taxonomic position of eight bradyrhizobial strains isolated from indigenous legumes to Western Australia. As expected for the genus, the 16S rRNA gene sequences were highly conserved, but the results of multilocus sequence analysis with four housekeeping genes (dnaK, glnII, gyrB and recA) confirmed three new distinct clades including the following strains: (1) WSM 1744T, WSM 1736 and WSM 1737; (2) WSM 1791T and WSM 1742; and (3) WSM 1741T, WSM 1735 and WSM 1790. The highest ANI values of the three groups in relation to the closest type strains were 92.4, 92.3 and 93.3 %, respectively, below the threshold of species circumscription. The digital DNA-DNA hybridization analysis also confirmed new species descriptions, with less than 52 % relatedness with the closest type strains. The phylogeny of the symbiotic gene nodC clustered the eight strains into the symbiovar retamae, together with seven Bradyrhizobium type strains, sharing from 94.2-98.1 % nucleotide identity (NI), and less than 88.7 % NI with other related strains and symbiovars. Morpho-physiological, phylogenetics, genomic and symbiotic traits were determined for the new groups and our data support the description of three new species, Bradyrhizobium archetypum sp. nov., Bradyrhizobium australiense sp. nov. and Bradyrhizobium murdochi sp. nov., with WSM 1744T (=CNPSo 4013T=LMG 31646T), WSM 1791T (=CNPSo 4014T=LMG 31647T) and WSM 1741T (=CNPSo 4020T=LMG 31651T) designated as type strains, respectively.

Efficient Nitrogen-Fixing Bacteria Isolated from Soybean Nodules in the Semi-arid Region of Northeast Brazil are Classified as Bradyrhizobium brasilense (Symbiovar Sojae).

Soybean (Glycine max L.) is an important legume that greatly benefits from inoculation with nitrogen-fixing bacteria. In a previous study, five efficient nitrogen-fixing bacterial strains, isolated from nodules of soybean inoculated with soil from semi-arid region, Northeast Brazil, were identified as a new group within the genus Bradyrhizobium. The taxonomic status of these strains was evaluated in this study. The phylogenetic analysis of the 16S rRNA gene showed the high similarity of the five strains to Bradyrhizobium brasilense UFLA03-321T (100%), B. pachyrhizi PAC48T (100%), B. ripae WR4T (100%), B. elkanii USDA 76T (99.91%), and B. macuxiense BR 10303T (99.91%). However, multilocus sequence analysis of the housekeeping genes atpD, dnaK, gyrB, recA, and rpoB, average nucleotide identity, and digital DNA-DNA hybridization analyses supported the classification of the group as B. brasilense. Some phenotypic characteristics allowed differentiating the five strains and the type strain of B. brasilense from the two neighboring species (B. pachyrhizi PAC48T and B. elkanii USDA 76T). The nodC and nifH genes' analyses showed that these strains belong to symbiovar sojae, together with B. elkanii (USDA 76T) and B. ferriligni (CCBAU 51502T). The present results support the classification of these five strains as Bradyrhizobium brasilense (symbiovar sojae).