Genomic identification and characterization of the elite strains Bradyrhizobium yuanmingense BR 3267 and Bradyrhizobium pachyrhizi BR 3262 recommended for cowpea inoculation in Brazil

ABSTRACT The leguminous inoculation with nodule-inducing bacteria that perform biological nitrogen fixation is a good example of an "eco-friendly agricultural practice". Bradyrhizobium strains BR 3267 and BR 3262 are recommended for cowpea (Vigna unguiculata) inoculation in Brazil and showed remarkable responses; nevertheless neither strain was characterized at species level, which is our goal in the present work using a polyphasic approach. The strains presented the typical phenotype of Bradyrhizobium with a slow growth and a white colony on yeast extract-mannitol medium. Strain BR 3267 was more versatile in its use of carbon sources compared to BR 3262. The fatty acid composition of BR 3267 was similar to the type strain of Bradyrhizobium yuanmingense; while BR 3262 was similar to Bradyrhizobium elkanii and Bradyrhizobium pachyrhizi. Phylogenetic analyses based on 16S rRNA and three housekeeping genes placed both strains within the genus Bradyrhizobium: strain BR 3267 was closest to B. yuanmingense and BR 3262 to B. pachyrhizi. Genome average nucleotide identity and DNA-DNA reassociation confirmed the genomic identification of B. yuanmingense BR 3267 and B. pachyrhizi BR 3262. The nodC and nifH gene analyses showed that strains BR 3267 and BR 3262 hold divergent symbiotic genes. In summary, the results indicate that cowpea can establish effective symbiosis with divergent bradyrhizobia isolated from Brazilian soils.

Phenotypic, genetic and symbiotic characterization of Erythrina velutina rhizobia from Caatinga dry forest

Abstract Erythrina velutina ("mulungu") is a legume tree from Caatinga that associates with rhizobia but the diversity and symbiotic ability of "mulungu" rhizobia are poorly understood. The aim of this study was to characterize "mulungu" rhizobia from Caatinga. Bacteria were obteined from Serra Talhada and Caruaru in Caatinga under natural regeneration. The bacteria were evaluated to the amplification of nifH and nodC and to metabolic characteristics. Ten selected bacteria identified by 16S rRNA sequences. They were tested in vitro to NaCl and temperature tolerance, auxin production and calcium phosphate solubilization. The symbiotic ability were assessed in an greenhouse experiment. A total of 32 bacteria were obtained and 17 amplified both symbiotic genes. The bacteria showed a high variable metabolic profile. Bradyrhizobium (6), Rhizobium (3) and Paraburkholderia (1) were identified, differing from their geographic origin. The isolates grew up to 45 °C to 0.51 mol L-1 of NaCl. Bacteria which produced more auxin in the medium with l-tryptophan and two Rhizobium and one Bradyrhizobium were phosphate solubilizers. All bacteria nodulated and ESA 90 (Rhizobium sp.) plus ESA 96 (Paraburkholderia sp.) were more efficient symbiotically. Diverse and efficient rhizobia inhabit the soils of Caatinga dry forests, with the bacterial differentiation by the sampling sites.

Draft genome sequence of Bradyrhizobium manausense strain BR 3351T, an effective symbiont isolated from Amazon rainforest

ABSTRACT The strain BR 3351T (Bradyrhizobium manausense) was obtained from nodules of cowpea (Vigna unguiculata L. Walp) growing in soil collected from Amazon rainforest. Furthermore, it was observed that the strain has high capacity to fix nitrogen symbiotically in symbioses with cowpea. We report here the draft genome sequence of strain BR 3351T. The information presented will be important for comparative analysis of nodulation and nitrogen fixation for diazotrophic bacteria. A draft genome with 9,145,311 bp and 62.9% of GC content was assembled in 127 scaffolds using 100 bp pair-end Illumina MiSeq system. The RAST annotation identified 8603 coding sequences, 51 RNAs genes, classified in 504 subsystems.

Genome sequence of Bradyrhizobium embrapense strain CNPSo 2833T, isolated from a root nodule of Desmodium heterocarpon

Abstract Bradyrhizobium embrapense CNPSo 2833T is a nitrogen-fixing symbiont of the legume pasture Desmodium. Its draft genome contains 8,267,832 bp and 7876 CDSs. The symbiotic island includes nodulation and nitrogen fixation genes resembling the operon organization of B. japonicum. Several CDSs related to secretion proteins and stress tolerance were also identified.

Genetic diversity of Rhizobia isolates from Amazon soils using cowpea (Vigna unguiculata) as trap plant

The aim of this work was to characterize rhizobia isolated from the root nodules of cowpea (Vigna unguiculata) plants cultivated in Amazon soils samples by means of ARDRA (Amplified rDNA Restriction Analysis) and sequencing analysis, to know their phylogenetic relationships. The 16S rRNA gene of rhizobia was amplified by PCR (polymerase chain reaction) using universal primers Y1 and Y3. The amplification products were analyzed by the restriction enzymes HinfI, MspI and DdeI and also sequenced with Y1, Y3 and six intermediate primers. The clustering analysis based on ARDRA profiles separated the Amazon isolates in three subgroups, which formed a group apart from the reference isolates of Bradyrhizobium japonicum and Bradyrhizobium elkanii. The clustering analysis of 16S rRNA gene sequences showed that the fast-growing isolates had similarity with Enterobacter, Rhizobium, Klebsiella and Bradyrhizobium and all the slow-growing clustered close to Bradyrhizobium.

Gene probe designing for evaluation of the diversity of Bradyrhizobium japonicum isolates

Many researchers consider the use of different probes for hybridization assays as suitable for studying the genetic diversity of nitrogen fixing bacteria. In this study for asessing genetic diversity among Bradyrhizobium japonicum isolates, two different probes [sucA and topA] chosen from the chromosomal genome of Bradyrhizobium strain USDA 110 were designed, evaluated by DNAMAN software and implemented in the DNA/DNA hybridization of eighteen isolates. Hybridization patterns of the sucA and topA probes showed that all B. japonicum isolates were clustered into 4 and 5 groups, respectively. It was also found that the sequences of these genes among the isolates were different, thus making them suitable for studying the genetic diversity of rhizobial bacteria. The sequence diversit of topA gene was more variable than that of the sucA gene

Crescimento e competiçäo entre duas estirpes de Bradyrhizobium japonicum em caldo e em um inoculante turfoso: eficiência na fixaçäo do dinitrogênio e competiçäo por sitios de nodulaçäo / Growth and competition between two strains of bradyrhizobium japonicum in broth and in a peat-based inoculant: dinitrogen fixation efficiency and competition for nodulation sites

Resumo: Este trabalho relata uma pesquisa sobre o crescimento e competiçäo em emio líquido, em inoculante em veículo turfoso e por sítios de nodulaçäo entre duas estirpes de Bradyrhizobium japonicum recomendadas para produçäo de inoculante de soja. Foi avaliada também a eficiência na fixaçäo do nitrogênio. As estirpes SEMIA 587 e SEMIA 5019 foram cultivadas em mistura ou isolante em um fermentador de bancada e em frascos em aitador. Taxa de cresciemtno máximo e menor tempo de geraçäo ocorreu a 28 graus C e 259 rpm. Caldo semeado com igual concentraçäo de ambas as estirpees após 120 horas apresentava concentraçäo de 69 (por cento) da 587 e 31 (por cento) 5019 devido a menor taxa de crescimento e maior tempo turfoso estéril a mistura das estirpes na concentraçäo citada écom concentraçäo 1:1, após 180 dias de incubaçäo apresentava somente 10,2 (por cento) e 34,7 (por cento) da estirpe 5019 respectivamnete. A concentraçäo celeular aumentava por 30 dias e declinava após drasticamente. Em experimento em vasos Leonard a inoculaçäo da mistura das estirpes resultou maior ocupaçäo dos nódulos pela estirpe 587. Esta estirpe também apresentou fixaçäo do nitrogênio mais alta (au)