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Cowpea (Vigna unguiculata) is recognized as a promiscuous legume in its symbiotic relationships with rhizobia, capable of forming associations with a wide range of bacterial species. Our study focused on assessing the diversity of bacterial strains present in cowpea nodules when inoculated with soils from six indigenous lands of Mato Grosso do Sul state, Central-Western Brazil, comprising the Cerrado and the Pantanal biomes, which are known for their rich diversity. The DNA profiles (BOX-PCR) of 89 strains indicated great genetic diversity, with 20 groups and 23 strains occupying single positions, and all strains grouped at a final similarity level of only 25%. Further characterization using 16S rRNA gene sequencing revealed a diverse array of bacterial genera associated with the cowpea nodules. The strains (number in parenthesis) were classified into ten genera: Agrobacterium (47), Ancylobacter (2), Burkholderia (12), Ensifer (1), Enterobacter (1), Mesorhizobium (1), Microbacterium (1), Paraburkholderia (1), Rhizobium (22), and Stenotrophomonas (1), split into four different classes. Notably, only Ensifer, Mesorhizobium, Rhizobium, and Paraburkholderia are classified as rhizobia. Phylogenetic analysis was conducted based on the classes of the identified genera and the type strains of the closest species. Our integrated analyses, combining phenotypic, genotypic, and phylogenetic approaches, highlighted the significant promiscuity of cowpea in associating with a diverse array of bacteria within nodules, showcasing the Brazilian soils as a hotspot of bacterial diversity., Competing Interests: Declarations. Conflicts of interests: The authors declare that they have no competing or conflicting interests, or ethical conflicts., (© 2025. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

The phosphate (P)-solubilizing potential of rhizobia isolated from active root nodules of Brazilian native Mimosa and Desmodium was assessed. Out of the 15 strains selected, five Paraburkholderia isolated from Mimosa spp. grown in rocky outcrops stood out. The Ca 3 (PO 4 ) 2 -solubilizing efficiency of these strains ranged from 110.67 to 356.3 mgL -1 , with less expressive results for FePO 4 and Al(H 2 PO 4 ) 3, that might be attributed to the low solubility of these two P compounds. Paraburkholderia strains CNPSo 3281 and CNPSo 3076 were the most efficient siderophore producers (44.17 and 41.87 µMol EDTA) and two of the top FePO 4 solubilizers. Acidification of the culture media was observed for all the strains and P sources. Regarding Ca 3 (PO 4 ) 2 solubilization , the main organic acids detected were glucuronic (an important component of rhizobia exopolysaccharides) and gluconic acids. Genomic analysis of P. nodosa CNPSo 3281 and CNPSo 3076 along with other phosphate-solubilizing Paraburkholderia species of the genus pointed out a conserved gene organization of phoUBR, pstSCAB, ppk and ppx. Greenhouse experiment revealed that P. nodosa CNPSo 3281 and CNPSo 3076 promoted maize growth under low P. Our results indicate the relevance of native rhizobia as multifunctional plant-associated bacteria and the rocky outcrops ecosystems as hotspots for bioprospection., (© 2024. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

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., Competing Interests: The authors declare no conflict of interest.

The rhizobia-Desmodium (Leguminosae, Papilionoideae) symbiosis is generally described by its specificity with alpha-rhizobia, especially with Bradyrhizobium. Our study aimed to isolate rhizobia from root nodules of native D. barbatum, D. incanum, and D. discolor, collected in remnants of the biomes of Atlantic Forest and Cerrado in protected areas of the Paraná State, southern Brazil. Based on the 16S rRNA phylogeny, 18 out of 29 isolates were classified as Alphaproteobacteria (Bradyrhizobium and Allorhizobium/Rhizobium) and 11 as Betaproteobacteria (Paraburkholderia). Phylogeny of the recA gene of the alpha-rhizobia resulted in ten main clades, of which two did not group with any described rhizobial species. In the 16S rRNA phylogeny of the beta-rhizobia, Paraburkholderia strains from the same host and conservation unity occupied the same clade. Phenotypic characterization of representative strains revealed the ability of Desmodium rhizobia to grow under stressful conditions such as high temperature, salinity, low pH conditions, and tolerance of heavy metals and xenobiotic compounds. Contrasting with previous reports, our results revealed that Brazilian native Desmodium can exploit symbiotic interactions with stress-tolerant strains of alpha- and beta-rhizobia. Stress tolerance can highly contribute to the ecological success of Desmodium in this phytogeographic region, possibly relating to its pioneering ability in Brazil. We propose Desmodium as a promising model for studies of plant-rhizobia interactions., (© 2023. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

The genome sequences of two nitrogen-fixing type strains of the Rhizobium tropici group were obtained: Rhizobium calliandrae CCGE524 T and R. mayense CCGE526 T . Genomic analyses confirmed their taxonomic position and identified three complete sequences of the repABC genes, indicative of three plasmids, one of them carrying symbiotic genes., Competing Interests: The authors declare no conflict of interest.

The genomes of five elite strains identified as growth promoters of lowland rice (Oryza sativa L.) in Brazil were sequenced. They ranged in size from 3,695,387 bp to 5,682,101 bp, encompassing genes of saprophytic ability and stress tolerance. Genome taxonomy enabled their classification as Priestia megaterium, Bacillus altitudinis, and three putative new species of Pseudomonas, Lysinibacillus , and Agrobacterium ., Competing Interests: The authors declare no conflict of interest.

Bradyrhizobium is a heterogeneous bacterial genus capable of establishing symbiotic associations with a broad range of legume hosts, including species of economic and environmental importance. This study was focused on the taxonomic and symbiovar definition of four strains - CNPSo 4026 T , WSM 1704 T , WSM 1738 T and WSM 4400 T - previously isolated from nodules of legumes in Western Australia and South Africa. The 16S rRNA gene phylogenetic tree allocated the strains to the Bradyrhizobium elkanii supergroup. The multilocus sequence analysis (MLSA) with partial sequences of six housekeeping genes - atpD , dnaK , glnII , gyrB , recA and rpoB - did not cluster the strains under study as conspecific to any described Bradyrhizobium species. Average nucleotide identity and digital DNA-DNA hybridization values were calculated for the four strains of this study and the closest species according to the MLSA phylogeny with the highest values being 95.46 and 62.20 %, respectively; therefore, both being lower than the species delineation cut-off values. The nodC and nifH phylogenies included strains WSM 1738 T and WSM 4400 T in the symbiovars retamae and vignae respectively, and also allowed the definition of three new symbiovars, sv. cenepequi, sv. glycinis, and sv. cajani. Analysis of morphophysiological characterization reinforced the identification of four novel proposed Bradyrhizobium species that are accordingly named as follows: Bradyrhizobium cenepequi sp. nov. (CNPSo 4026 T =WSM 4798 T =LMG 31653 T ), isolated from Vigna unguiculata ; Bradyrhizobium semiaridum sp. nov. (WSM 1704 T =CNPSo 4028 T =LMG 31654 T ), isolated from Tephrosia gardneri ; Bradyrhizobium hereditatis sp. nov. (WSM 1738 T =CNPSo 4025 T =LMG 31652 T ), isolated from Indigofera sp.; and Bradyrhizobium australafricanum sp. nov. (WSM 4400 T =CNPSo 4015 T =LMG 31648 T ) isolated from Glycine sp.

Since early studies, the history of prokaryotes taxonomy has dealt with many changes driven by the development of new and more robust technologies. As a result, the number of new taxa descriptions is exponentially increasing, while an increasing number of others has been subject of reclassification, demanding from the taxonomists more effort to maintain an organized hierarchical system. However, expectations are that the taxonomy of prokaryotes will acquire a more stable status with the genomic era. Other analyses may continue to be necessary to determine microbial features, but the use of genomic data might be sufficient to provide reliable taxa delineation, helping taxonomy to reach the goal of correct classification and identification. Here we describe the evolution of prokaryotes' taxonomy until the genomic era, emphasizing bacteria and taking as an example the history of rhizobia taxonomy. This example was chosen because of the importance of the symbiotic nitrogen fixation of legumes with rhizobia to the nitrogen input to both natural ecosystems and agricultural crops. This case study reports the technological advances and the methodologies used to classify and identify bacterial species and indicates the actual rules required for an accurate description of new taxa., Competing Interests: The authors declare that they have no conflicts of interest regarding the publishing of this paper., (Copyright © 2022 Luisa Caroline Ferraz Helene et al.)

Symbiotic Paraburkholderia have been increasingly studied in the past 20 years, especially when associated with Mimosa; however, studies with native/endemic species are still scarce. In this study, thirty strains were isolated from root nodules of native Mimosa paranapiacabae and M. micropteris in two locations of the Campos Gerais. The BOX-PCR fingerprinting revealed high genomic diversity, and the 16S rRNA phylogeny clustered the strains in three distinct groups (GI, GII, GIII), with one strain occupying an isolated position. Phylogenetic analysis with four concatenated housekeeping genes (atpD + gltB + gyrB + recA) confirmed the same clusters of 16S rRNA, and the closest species were P. nodosa BR 3437 T and P. guartelaensis CNPSo 3008 T ; this last one isolated from another Mimosa species of the Campos Gerais. The phylogenies of the symbiotic genes nodAC and nifH placed all strains in a well-supported branch with the other species of the symbiovar mimosae. The phylogenetic analyses indicated that the strains represent novel lineages of sv. mimosae and that endemic Mimosa coevolved with indigenous Paraburkholderia in their natural environments., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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 1744 T , WSM 1736 and WSM 1737; (2) WSM 1791 T and WSM 1742; and (3) WSM 1741 T , 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 1744 T (=CNPSo 4013 T =LMG 31646 T ), WSM 1791 T (=CNPSo 4014 T =LMG 31647 T ) and WSM 1741 T (=CNPSo 4020 T =LMG 31651 T ) designated as type strains, respectively.

The symbioses between legumes and nitrogen-fixing rhizobia make the greatest contribution to the global nitrogen input via the process of biological nitrogen fixation (BNF). Bradyrhizobium stands out as the main genus nodulating basal Caesalpinioideae. We performed a polyphasic study with 11 strains isolated from root nodules of Chamaecristafasciculata , an annual multi-functional native legume of the USA. In the 16S rRNA gene phylogeny the strains were clustered in the Bradyrhizobium japonicum superclade. The results of analysis of the intergenic transcribed spacer (ITS) indicated less than 89.9 % similarity to other Bradyrhizobium species. Multilocus sequence analysis (MLSA) with four housekeeping genes ( glnII , gyrB , recA and rpoB ) confirmed the new group, sharing less than 95.2 % nucleotide identity with other species. The MLSA with 10 housekeeping genes ( atpD , dnaK , gap , glnII , gltA , gyrB , pnp , recA , rpoB and thrC ) indicated Bradyrhizobium daqingense as the closest species. Noteworthy, high genetic diversity among the strains was confirmed in the analyses of ITS, MLSA and BOX-PCR. Average nucleotide identity and digital DNA-DNA hybridization values were below the threshold of described Bradyrhizobium species, of 89.7 and 40 %, respectively. In the nifH and nodC phylogenies, the strains were grouped together, but with an indication of horizontal gene transfer, showing higher similarity to Bradyrhizobium arachidis and Bradyrhizobium forestalis . Other phenotypic, genotypic and symbiotic properties were evaluated, and the results altogether support the description of the CNPSo strains as representatives of the new species Bradyrhizobiumfrederickii sp. nov., with CNPSo 3426 T (=USDA 10052 T =U686 T =CL 20 T ) as the type strain.

Information about the symbionts of legumes of the Caesalpinioideae subfamily is still limited, and we performed a polyphasic approach with three Bradyrhizobium strains-CNPSo 3448 T , CNPSo 3394 and CNPSo 3442-isolated from Chamaecrista fasciculata , a native legume broadly distributed in the USA. In the phylogenetic analysis of both the 16S rRNA gene and the intergenic transcribed spacer, the CNPSo strains were clustered within the Bradyrhizobium japonicum superclade. Multilocus sequence analysis with six housekeeping genes- glnII , gyrB , recA , rpoB , atpD and dnaK -indicated that Bradyrhizobium diazoefficiens is the closest species, with 83 % of nucleotide identity. In the genome analyses of CNPSo 3448 T , average nucleotide identity and digital DNA-DNA hybridization results confirmed higher similarity with B. diazoefficiens , with values estimated of 93.35 and 51.50 %, respectively, both below the threshold of the same species, confirming that the CNPSo strains represent a new lineage. BOX-PCR profiles indicated high intraspecific genetic diversity between the CNPSo strains. In the analyses of the symbiotic genes nodC and nifH the CNPSo strains were clustered with Bradyrhizobium arachidis , Bradyrhizobium forestalis , Bradyrhizobium cajani , Bradyrhizobium kavangense and Bradyrhizobium vignae , indicating a different phylogenetic history compared to the conserved core genes. Other physiological (C utilization, tolerance to antibiotics and abiotic stresses), chemical (fatty acid profile) and symbiotic (nodulation host range) properties were evaluated and are described. The data from our study support the description of the CNPSo strains as the novel species Bradyrhizobiumniftali sp. nov., with CNPSo 3448 T (=USDA 10051 T =U687 T =CL 40 T ) designated as the type strain.

Strains of the genus Bradyrhizobium associated with agronomically important crops such as soybean ( Glycine max ) are increasingly studied; however, information about symbionts of wild Glycine species is scarce. Australia is a genetic centre of wild Glycine species and we performed a polyphasic analysis of three Bradyrhizobium strains-CNPSo 4010 T , CNPSo 4016 T , and CNPSo 4019 T -trapped from Western Australian soils with Glycine clandestina , Glycine tabacina and Glycine max , respectively. The phylogenetic tree of the 16S rRNA gene clustered all strains into the Bradyrhizobium japonicum superclade; strains CNPSo 4010 T and CNPSo 4016 T had Bradyrhizobium yuanmingense CCBAU 10071 T as the closest species, whereas strain CNPSo 4019 T was closer to Bradyrhizobium liaoningense LMG 18230 T . The multilocus sequence analysis (MLSA) with five housekeeping genes- dnaK , glnII , gyrB , recA and rpoB -confirmed the same clusters as the 16S rRNA phylogeny, but indicated low similarity to described species, with nucleotide identities ranging from 93.6 to 97.6% of similarity. Considering the genomes of the three strains, the average nucleotide identity and digital DNA-DNA hybridization values were lower than 94.97 and 59.80 %, respectively, with the closest species. In the nodC phylogeny, strains CNPSo 4010 T and CNPSo 4019 T grouped with Bradyrhizobium zhanjiangense and Bradyrhizobium ganzhouense , respectively, while strain CNPSo 4016 T was positioned separately from the all symbiotic Bradyrhizobium species. Other genomic (BOX-PCR), phenotypic and symbiotic properties were evaluated and corroborated with the description of three new lineages of Bradyrhizobium . We propose the names of Bradyrhizobium agreste sp. nov. for CNPSo 4010 T (=WSM 4802 T =LMG 31645 T ) isolated from Glycine clandestina , Bradyrhizobium glycinis sp. nov. for CNPSo 4016 T (=WSM 4801 T =LMG 31649 T ) isolated from Glycine tabacina and Bradyrhizobium diversitatis sp. nov. for CNPSo 4019 T (=WSM 4799 T =LMG 31650 T ) isolated from G. max .

Floristic surveys performed in "Campos Gerais" (Paraná, Brazil), an ecotone of Mata Atlântica and Cerrado biomes, highlights the richness and relative abundance of the family Fabaceae and point out the diversity and endemism of Mimosa spp. Our study reports the genetic diversity of rhizobia isolated from root nodules of native/endemic Mimosa gymnas Barneby in three areas of Guartelá State Park, an important conservation unit of "Campos Gerais". Soils of the sample areas were characterized as sandy, acid, poor in nutrients and organic matter. The genetic variability among the isolates was revealed by BOX-PCR genomic fingerprinting. Phylogeny based on 16S rRNA gene grouped the strains in a large cluster including Paraburkholderia nodosa and P. bannensis, while recA-gyrB phylogeny separated the strains in two groups: one including P. nodosa and the other without any described Paraburkholderia species. MLSA confirmed the separate position of this second group of strains within the genus Paraburkholderia and the nucleotide identity of the five concatened housekeeping genes was 95.9% in relation to P. nodosa BR 3437 T . Phylogram based on symbiosis-essential nodC gene was in agreement with 16S rRNA analysis. Our molecular phylogenetic analysis support that Paraburkholderia are the main symbionts of native Mimosa in specific edaphic conditions found in South America and reveal the importance of endemic/native leguminous plants as reservoirs of novel rhizobial species.