Does Rhizobial Inoculation Change the Microbial Community in Field Soils? A| |Comparison with Agricultural Land-use Changes.

Although microbial inoculation may be effective for sustainable crop production, detrimental aspects have been argued because of the potential of inoculated microorganisms to behave as invaders and negatively affect the microbial ecosystem. We herein compared the impact of rhizobial inoculation on the soil bacterial community with that of agricultural land-use changes using a 16S rRNA amplicon ana-lysis. Soybean plants were cultivated with and without five types of bradyrhizobial inoculants (Bradyrhizobium diazoefficiens or Bradyrhizobium ottawaense) in experimental fields of Andosol, and the high nodule occupancy (35-72%) of bradyrhizobial inoculants was confirmed by nosZ PCR. However, bradyrhizobial inoculants did not significantly affect Shannon's diversity index (α-diversity) or shifts (ß-diversity) in the bacterial community in the soils. Moreover, the soil bacterial community was significantly affected by land-use types (conventional cropping, organic cropping, and original forest), where ß-diversity correlated with soil chemical properties (pH, carbon, and nitrogen contents). Therefore, the effects of bradyrhizobial inoculation on bacterial communities in bulk soil were minor, regardless of high nodule occupancy. We also observed a correlation between the relative abundance of bacterial classes (Alphaproteobacteria, Gammaproteobacteria, and Gemmatimonadetes) and land-use types or soil chemical properties. The impact of microbial inoculation on soil microbial ecosystems has been exami-ned to a limited extent, such as rhizosphere communities and viability. In the present study, we found that bacterial community shifts in soil were more strongly affected by land usage than by rhizobial inoculation. Therefore, the results obtained herein highlight the importance of assessing microbial inoculants in consideration of the entire land management system.

Diversity of soybean rhizobia in Northeast China and their application.

Biological nitrogen fixation is the main source of nitrogen in ecosystems. The diversity of soil rhizobia and their effects on soybeans need further research. In this study, we collected soybean rhizosphere samples from eight sites in the black soil soybean planting area in Northeast China. A total of 94 strains of bacteria were isolated and identified using the 16S rRNA and symbiotic genes (nodC, nifH) analysis, of which 70 strains were identified as rhizobia belonging to the genus Bradyrhizobium. To further validate the application effects of rhizobia, we selec-ted seven representative indigenous rhizobia based on the results of phylogenetic analysis, and conducted laboratory experiments to determine their nodulation and the impacts on soybeans. The results showed that, compared to the control without rhizobial inoculation, all the seven indigenous rhizobia exhibited good promoting and nodulation abilities. Among them, strains H7-L22 and H34-L6 performed the best, with the former significantly increasing plant height by 25.7% and the latter increasing root nodule dry weight by 20.9% to 67.1% compared to other indi-genous rhizobia treatments. We tested these two efficient rhizobia strains as soybean rhizobial inoculants in field experiments. The promoting effect of mixed rhizobial inoculants was significantly better than single ones. Compared to the control without inoculation, soybean yield increased by 8.4% with the strain H7-L22 treatment and by 17.9% with the mixed inoculant treatment. Additionally, there was a significant increase in the number of four-seed pods in soybeans. In conclusion, the application of rhizobial inoculants can significantly increase soybean yield, thereby reducing dependence on nitrogen fertilizer during soybean production, improving soil health, and promoting green development in agriculture in the black soil region of Northeast China.

Comparative genomic analysis of Bradyrhizobium strains with natural variability in the efficiency of nitrogen fixation, competitiveness, and adaptation to stressful edaphoclimatic conditions.

Bradyrhizobium is known for fixing atmospheric nitrogen in symbiosis with agronomically important crops. This study focused on two groups of strains, each containing eight natural variants of the parental strains, Bradyrhizobium japonicum SEMIA 586 (=CNPSo 17) or Bradyrhizobium diazoefficiens SEMIA 566 (=CNPSo 10). CNPSo 17 and CNPSo 10 were used as commercial inoculants for soybean crops in Brazil at the beginning of the crop expansion in the southern region in the 1960s-1970s. Variants derived from these parental strains were obtained in the late 1980s through a strain selection program aimed at identifying elite strains adapted to a new cropping frontier in the central-western Cerrado region, with a higher capacity of biological nitrogen fixation (BNF) and competitiveness. Here, we aimed to detect genetic variations possibly related to BNF, competitiveness for nodule occupancy, and adaptation to the stressful conditions of the Brazilian Cerrado soils. High-quality genome assemblies were produced for all strains. The core genome phylogeny revealed that strains of each group are closely related, as confirmed by high average nucleotide identity values. However, variants accumulated divergences resulting from horizontal gene transfer, genomic rearrangements, and nucleotide polymorphisms. The B. japonicum group presented a larger pangenome and a higher number of nucleotide polymorphisms than the B. diazoefficiens group, possibly due to its longer adaptation time to the Cerrado soil. Interestingly, five strains of the B. japonicum group carry two plasmids. The genetic variability found in both groups is discussed considering the observed differences in their BNF capacity, competitiveness for nodule occupancy, and environmental adaptation.IMPORTANCEToday, Brazil is a global leader in the study and use of biological nitrogen fixation with soybean crops. As Brazilian soils are naturally void of soybean-compatible bradyrhizobia, strain selection programs were established, starting with foreign isolates. Selection searched for adaptation to the local edaphoclimatic conditions, higher efficiency of nitrogen fixation, and strong competitiveness for nodule occupancy. We analyzed the genomes of two parental strains of Bradyrhizobium japonicum and Bradyrhizobium diazoefficiens and eight variant strains derived from each parental strain. We detected two plasmids in five strains and several genetic differences that might be related to adaptation to the stressful conditions of the soils of the Brazilian Cerrado biome. We also detected genetic variations in specific regions that may impact symbiotic nitrogen fixation. Our analysis contributes to new insights into the evolution of Bradyrhizobium, and some of the identified differences may be applied as genetic markers to assist strain selection programs.

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.

The elite strain INPA03-11B approved as a cowpea inoculant in Brazil represents a new Bradyrhizobium species and it has high adaptability to stressful soil conditions.

The strain INPA03-11BT, isolated in the 1980s from nodules of Centrosema sp. collected in Manaus, Amazonas, Brazil, was approved by the Brazilian Ministry of Agriculture as a cowpea inoculant in 2004. Since then, several studies have been conducted regarding its phenotypic, genetic, and symbiotic characteristics under axenic and field conditions. Phenotypic features demonstrate its high adaptability to stressful soil conditions, such as tolerance to acidity, high temperatures, and 13 antibiotics, and, especially, its high symbiotic efficiency with cowpea and soybean, proven in the field. The nodC and nifH phylogenies placed the INPA strain in the same clade as the species B. macuxiense BR 10303T which was also isolated from the Amazon region. The sequencing of the 16S rRNA ribosomal gene and housekeeping genes, as well as BOX-PCR profiles, showed its potential as a new species, which was confirmed by a similarity percentage of 94.7% and 92.6% in Average Nucleotide Identity with the closest phylogenetically related species Bradyrhizobium tropiciagri CNPSo1112T and B. viridifuturi SEMIA690T, respectively. dDDH values between INPA03-11BT and both CNPSo 1112T and SEMIA690T were respectively 58.5% and 48.1%, which are much lower than the limit for species boundary (70%). Therefore, we propose the name Bradyrhizobium amazonense for INPA03-11BT (= BR3301 = SEMIA6463).

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.

Genetic and physiological diversity of white Spanish broom (Chamaecytisus albus) endophytes.

Chamaecytisus albus (Spanish broom) is a legume shrub that can be found in only one natural locality in Poland. This specimen is critically endangered; therefore, different actions focusing on protection of this plant in the natural habitat are undertaken, and one of them involves studies of the population of Chamaecytisus albus bacterial endophytes, which in the future could be used as bioprotectants and/or biofertilizers. A collection of 94 isolates was obtained from Spanish broom nodules, and the physiological and genetic diversity of these strains was studied. A few potentially beneficial traits were detected, i.e. secretion of cellulases (66 isolates), production of siderophores (60 isolates), phosphate solubilization (25 isolates), and production of IAA (58 isolates), indole (16 isolates), or HCN (3 isolates). Twenty-nine of the 94 tested isolates were able to induce the development of root nodules in plants grown in vitro and can therefore be assumed as Chamaecytisus albus symbionts. Genome fingerprinting by BOX-PCR, as well as gyrB and nodZ gene sequencing revealed a great genetic diversity of specimens in the collection. The symbiotic isolates were classified in different clades, suggesting they could belong to different species, however, most of them revealed sequence similarity to Bradyrhizobium genus.

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.

Nutrients cause consolidation of soil carbon flux to small proportion of bacterial community.

Nutrient amendment diminished bacterial functional diversity, consolidating carbon flow through fewer bacterial taxa. Here, we show strong differences in the bacterial taxa responsible for respiration from four ecosystems, indicating the potential for taxon-specific control over soil carbon cycling. Trends in functional diversity, defined as the richness of bacteria contributing to carbon flux and their equitability of carbon use, paralleled trends in taxonomic diversity although functional diversity was lower overall. Among genera common to all ecosystems, Bradyrhizobium, the Acidobacteria genus RB41, and Streptomyces together composed 45-57% of carbon flow through bacterial productivity and respiration. Bacteria that utilized the most carbon amendment (glucose) were also those that utilized the most native soil carbon, suggesting that the behavior of key soil taxa may influence carbon balance. Mapping carbon flow through different microbial taxa as demonstrated here is crucial in developing taxon-sensitive soil carbon models that may reduce the uncertainty in climate change projections.

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.

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.

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.

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.

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.

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).

The promiscuity of Phaseolus vulgaris L. (common bean) for nodulation with rhizobia: a review.

Phaseolus vulgaris L. (common bean) is a legume indigenous to American countries currently cultivated in all continents, which is nodulated by different rhizobial species and symbiovars. Most of species able to nodulate this legume worldwide belong to the genus Rhizobium, followed by those belonging to the genera Ensifer (formerly Sinorhizobium) and Pararhizobium (formerly Rhizobium) and minority by species of the genus Bradyrhizobium. All these genera belong to the phylum alpha-Proteobacteria, but the nodulation of P. vulgaris has also been reported for some species belonging to Paraburkholderia and Cupriavidus from the beta-Proteobacteria. Several species nodulating P. vulgaris were originally isolated from nodules of this legume in American countries and are linked to the symbiovars phaseoli and tropici, which are currently present in other continents probably because they were spread in their soils together with the P. vulgaris seeds. In addition, this legume can be nodulated by species and symbiovars originally isolated from nodules of other legumes due its high promiscuity, a concept currently related with the ability of a legume to be nodulated by several symbiovars rather than by several species. In this article we review the species and symbiovars able to nodulate P. vulgaris in different countries and continents and the challenges on the study of the P. vulgaris endosymbionts diversity in those countries where they have not been studied yet, that will allow to select highly effective rhizobial strains in order to guarantee the success of P. vulgaris inoculation.