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.

Leguminosas forrageiras são importantes recursos na região semi-árida, devido a sua capacidade de adaptação a solos de baixa fertilidade e fixação de nitrogênio quando associados a bactérias diazotróficas. Aqui, aplicamos uma abordagem polifásica com o objetivo de caracterizar bactérias isoladas de nódulos obtidos de Clitoria ternatea e Stylosanthes capitata cultivadas em solos do semiárido brasileiro. O isolamento das bactérias produziu uma coleção de 126 isolados: 45 isolados de C. ternatea e 81 de S. capitata. Os testes de nodulação resultaram em apenas dez isolados que nodularam seu hospedeiro original: seis de C. ternatea e quatro de S. capitata. Esses dez isolados de nódulos de leguminosas tiveram suas características fenotípicas e genotípicos avaliadas. Todos os isolados cresceram em frutose, glicose, glutamato de sódio, maltose, xilose e sacarose. Testes metabólicos mostraram uma relação entre a tolerância ao sal e altas temperaturas; onde o isolado que tolerou a maior concentração de sal também tolerou a temperatura mais alta. Três isolados apresentaram atividade amilolítica e quatro foram capazes de produzir carboximetil celulolítico. A estreptomicina foi o antibiótico mais limitante para o crescimento bacteriano, e o ácido nalidíxico foi o menos limitante. Sete dos dez isolados eram produtores de IAA. O sequenciamento parcial do gene 16S rRNA permitiu identificar isolados como membros dos gêneros Bacillus (1), Bradyrhizobium (4), Leifsonia (3), Microvirga (1) e Rhizobium (1). Esses dados revelam diversidade fenotípicas e genotípicas de bactérias que habitam nódulos das forrageiras C. ternatea e S. capitata, e representam uma importante fonte microbiana para a prospecção de novos produtos biotecnológicos para promover melhor desenvolvimento das leguminosas forrageiras na região semiárida.

Forage legumes are important resources in semiarid regions due to their abilities to adapt to soils with low fertility and fix nitrogen when associated with diazotrophic bacteria. Here, we applied a polyphasic approach to characterize a set of legume nodule isolates obtained from Clitoria ternatea and Stylosanthes capitata cultivated in the soils of a semiarid region of Brazil. A total of 126 bacterial isolates were obtained: 45 isolates from C. ternatea and 81 isolates from S. capitata. Nodulation tests revealed only ten isolates that nodulated their original host: six isolates from C. ternatea and four isolates from S. capitate. These ten legume nodule isolates were phenotypically and genotypically characterized. All isolates grew in fructose, glucose, sodium glutamate, maltose, xylose, and sucrose. Metabolic tests showed a relationship between tolerance to salt and high temperatures, where isolates that tolerated the highest salt concentration also tolerated the highest temperature. Three isolates showed amylolytic activity, and four isolates showed carboxymethyl cellulolytic activity. Streptomycin was the most limiting and nalidixic acid was the least limiting antibiotic to bacterial growth. Seven out of ten isolates were indol-acetic acid producers. Additionally, 16S rRNA gene partial sequencing enabled the identification of members of the genera Bacillus (1), Bradyrhizobium (4), Leifsonia (3), Microvirga (1), and Rhizobium (1). These data reveal phenotypically and genotypically diverse bacteria inhabiting the nodules of the forage legumes C. ternatea and S. capitata represent an important microbial source to protect new biotechnological products and improve forage legumes in semiarid regions.

Abstract The strain BR 3267 is a nitrogen-fixing symbiotic bacteria isolated from soil of semi-arid area of Brazilian Northeast using cowpea as the trap plant. This strain is used as commercial inoculant for cowpea and presents high efficient in nitrogen fixation as consequence of its adaptation potential to semi-arid conditions. We report here the draft genome sequence of Bradyrhizobium sp. strain BR 3267, an elite bacterium used as inoculant for cowpea. Whole genome sequencing of BR 3267 using Illumina MiSeq sequencing technology has 55 scaffolds with a total genome size of 7,904,309 bp and C+G 63%. Annotation was added by the RAST prokaryotic genome annotation service and has shown 7314 coding sequences and 52 RNA genes.

Abstract The strain BR 3262 was isolated from nodule of cowpea (Vigna unguiculata L. Walp) growing in soil of the Atlantic Forest area in Brazil and it is reported as an efficient nitrogen fixing bacterium associated to cowpea. Firstly, this strain was assigned as Bradyrhizobium elkanii, however, recently a more detailed genetic and molecular characterization has indicated it could be a Bradyrhizobium pachyrhizi species. We report here the draft genome sequence of B. pachyrhizi strain BR 3262, an elite bacterium used as inoculant for cowpea. The whole genome with 116 scaffolds, 8,965,178 bp and 63.8% of C+G content for BR 3262 was obtained using Illumina MiSeq sequencing technology. Annotation was added by the RAST prokaryotic genome annotation service and shown 8369 coding sequences, 52 RNAs genes, classified in 504 subsystems.

A biodiversidade de rizóbio de solos do Vale do São Francisco é desconhecida e pode ser estudada utilizando feijão-caupi como planta-isca. Este trabalho teve por objetivo verificar a diversidade de bactérias diazotróficas que nodulam feijão-caupi em solos do Submédio do Vale do São Francisco por meio da caracterização morfológica e genotípica. Sete amostras de solos (A1, A2, A3, A4, C1, C2 e MC) foram coletadas para a captura de bactérias, utilizando cinco cultivares de feijão-caupi (IPA 206, BRS Pujante, BRS Marataoã, Canapu Roxo e Sempre Verde), perfazendo um fatorial 5 x 7 com três repetições. Aos 30 dias após a emergência das plantas, os nódulos foram coletados e as bactérias isoladas e analisadas quanto às características culturais em meio YMA. Foram obtidos 581 isolados, que se agruparam em 49 grupos morfológicos. Desse total, 62,3 % formaram colônias em até três dias, 33,4 % cresceram a partir do sexto dia e 4,3 % cresceram entre quatro e cinco dias após a incubação. Quanto à formação de ácido e álcalis, 63 % acidificaram o meio de cultura, 12 % alcalinizaram e 25 % mantiveram o pH do meio neutro. As maiores diversidades foram encontradas nas amostras A3 e nos isolados associados às cultivares Canapu Roxo e BRS Pujante. Escolheram-se 38 isolados representativos para a caracterização genotípica, em que foram obtidos quatro grupos a partir da análise de restrição do 16s rDNA. Este agrupamento teve forte correlação com o local de origem dos isolados, mostrando 13 deles com perfil eletroforético distinto das estirpespadrão de rizóbios utilizadas no estudo.

The biodiversity of rhizobium in soils of the São Francisco Valley is unknown and can be studied using cowpea as trap plants. The objective of this study was to verify the diversity of diazotrophic bacteria that nodulate cowpea in soils of the lower half of the São Francisco River Valley by morphological and genotypic characterization. Seven soil samples (A1, A2, A3, A4, C1, C2 and MC) were collected to capture bacteria associated to five cowpea cultivars (IPA 206, BRS Pujante, BRS Marataoã, Canapu Roxo, and Sempre Verde), in a 5x7 factorial design with three replications. Thirty days after plant emergence, the nodules were collected and the bacteria isolated and analyzed in relation to their growth characteristics in YMA medium. The 581 isolates were grouped in 49 morphologic groups. Of this total, 62.3 % formed colonies in up to three days, 33.4 % grew from the 6th day on, and 4.3 % began to grow 4 to 5 days after incubation. Regarding the formation of acids and alkalis, 63 % acidified the medium, 12 % made it alkaline and 25 % maintained the medium at neutral pH. The highest diversity was observed in the A3 sample and in isolates associated with the cultivars Canapu Roxo and BRS Pujante. Thirty-eight representative isolates were chosen for the genotypic characterization, clustered in four groups based on the restriction analysis of 16s rDNA. This grouping was strongly correlated with the sampling site; 13 rhizobium isolates had an electrophoretic profile distinct from the standard rhizobium strains used in this study.