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 Cowpea is a very important crop to Brazilian Semi-Arid mainly small family-based farmers. Rhizobia inoculation is a practice, easy to use, and cheap technology that increases cowpea productivity. The aim of this study was to evaluate the efficiency of two new rhizobia isolates in greenhouse and field as well as classify them taxonomically. To bacterial identification the 16S rRNA gene of ESA 17 and ESA 18 isolates were sequenced. The greenhouse test was conducted with pots containing 3 L of soil and the bacterial isolates evaluated were ESA 17, ESA 18, BR 3267 or BR 3262 strains. A field experiment was implemented on a Vertisol in Juazeiro, Bahia State, to evaluate the cowpea growth and productivity. In this experiment, the peat-based inoculants with ESA 17, ESA 18, BR 3267 or UFLA 3-84 were used in 2 cowpea cultivars. Both bacteria were identified as Bradyrhizobium, but related to different species. ESA 17 was related to B. japonicum and ESA 18 was closer to B. pachyrhizi. At greenhouse, both isolates increased cowpea nitrogen content in the shoots due to the presence of very efficient nodules. In the field, the isolate ESA 18 inoculated at BRS Pujante cultivar induced higher production than observed for the absolute control, and for BR 17 Gurguéia cultivar, the ESA 17 and BR 3267 stood out both by inducing high production and grain protein content. The results indicate that both isolates can be evaluated in network experiments aiming at official recommendation for new bacteria to cowpea inoculant in Brazil.

ABSTRACT Legume-rhizobia symbiosis is a cross-kingdom association that results in large amounts of nitrogen incorporated in food webs. For the Brazilian semi-arid region, data on genetic variability and symbiotic efficiency of Papilionoidae rhizobial communities are very scarce. The aim of this study was to evaluate the genetic variability and the symbiotic efficiency of eight rhizobial isolates obtained from “mulungu” (Erythrina velutina Willd.) nodules. For 16S rRNA gene sequencing, the genomic DNA was extracted using a commercial kit, amplified with universal primers, and subjected to sequencing reactions. For the isolate ESA 71, PCR amplifications for nodC and nodA genes were attempted. Rhizobial efficiency was assessed by two greenhouse experiments. The first assay was carried out under gnotobiotic conditions, with sterile sand as a substrate; the second experiment was conducted in a non-sterile soil. For both experiments, the inoculation treatments consisted of a single inoculation of each isolate, in addition to a treatment with Bradyrhizobium elkanii BR 5609 as a reference strain. Furthermore, two non-inoculated control treatments, supplied and not supplied with mineral N, were also evaluated. Bacterial identification indicated that both α and β-rhizobia could be found in “mulungu” root nodules. Three isolates where classified within the Rhizobium genus, four bacteria belonged to Bradyrhizobium and one isolate clustered with Burkholderia. Positive amplification of an intragenic fragment of the nodA gene using a primer set to β-rhizobia could be found for ESA 71 (Burkholderia). All bacterial isolates were effective in colonizing “mulungu” roots. In the first experiment, all inoculated treatments and N fertilization increased the N concentration in “mulungu” shoot tissues. For total N in the shoots, the isolates ESA 70, ESA 72, and ESA 75 stood out. In the non-sterile substrate experiment, the isolates ESA 70, ESA 71, ESA 72, and ESA 75, together with the reference strains, induced increases in the shoot N concentration and total accumulation compared to the absolute control. The results indicate that “mulungu” is able to establish associations with efficient α and β-rhizobia in Brazilian semi-arid soils.

O objetivo desse estudo foi avaliar as características fenotípicas dos isolados de nódulos de mulungu (Erythrina velutina Willd.) e determinar seu fingerprinting molecular por meio de Box-PCR. Todas as bactérias foram avaliadas pelas seguintes características fenotípicas: tempo de crescimento, alteração de pH, cor da colónia e produção de muco. As bactérias isoladas hábeis em renodular o hospedeiro original também foram avaliadas em relação à tolerância ao aumento da salinidade e da temperatura de incubação, capacidade de crescimento utilizando diferentes fontes de carbono, resistência intrínseca aos antibióticos e biossíntese de auxina “in vitro”. O fingerprint molecular foi estabelecido usando a técnica Box-PCR e os isolados foram agrupados pelos perfis obtidos. Dos 22 isolados obtidos, oito foram hábeis em renodular o hospedeiro original. Entre eles, alguns foram tolerantes a 1% de NaCl e a 39° C e todos metabolizaram as fontes maltose, frutose, glicose, sacarose e arabinose, resistiram à rifampicina e produziram auxina. As bactérias isoladas mostraram baixa similaridade genética entre elas e as estirpes de referência, o que indica a grande variabilidade genética dos isolados. Os resultados deste trabalho constituem o primeiro relato sobre bactérias capazes de nodular esta espécie endémica. O estudo mais aprofundado destas bactérias poderá revelar a existência de isolados tolerantes a stresses ambientais e adequados a serem utilizados como futuros inoculantes de mulungu.

This study aims to evaluate the phenotypical characteristics of bacterial isolates from mulungu (Erythrina velutina Willd.) nodules and determinate their Box-PCR fingerprinting. All bacteria were evaluated by the following phenotypic characteristics: growth rate, pH change, colony color and mucus production. The bacterial isolates able to re-nodulate the original host were also evaluated regarding its tolerance to increased salinity and different incubation temperatures, ability to growth using different carbon sources, intrinsic antibiotic resistance and “in vitro” auxin biosynthesis. The molecular fingerprints were set up using the Box-PCR technique and the isolates were clustered by its profiles. Among the 22 bacterial isolates obtained, eight were able to re-nodulate the original host. Among the nodule inducing isolates, some were tolerant to 1% of NaCl and 39° C and all of them metabolized the maltose, fructose, glucose, sucrose and arabinose, were resistant to rifampicin and produced auxin. The bacteria showed low genetic similarity among them and reference strains, which indicates the great genetic variability of the isolates. The results of this work are the first reports about the bacterial isolates able to nodulate this endemic specie. A more deep study of these bacteria may reveal the existence of isolates tolerant to environmental stresses and suitable as a future mulungu inoculant.

As espécies vegetais que ocorrem naturalmente na Caatinga brasileira apresentam diversas adaptações às condições adversas que podem ser exploradas para aumentar a produção vegetal. Entre essas estratégias adaptativas para o enfrentamento da baixa disponibilidade hídrica, a tolerância à dessecação pode ser destacada. Até o momento, a associação entre essas espécies e os microrganismos benéficos do solo não é bem entendida. Os objetivos deste trabalho foram caracterizar bactérias diazotróficas de Tripogon spicatus e avaliar a capacidade de promoção do crescimento delas em plantas de arroz. Dezesseis isolados bacterianos foram estudados quanto a seu posicionamento taxonômico por meio do sequenciamento parcial do gene 16S rRNA, potencial diazotrófico, produção de ácido indol acético (AIA) e solubilização de fosfato de cálcio in vitro, metabolismo de nove diferentes fontes de C em meio semissólido, tolerância a diferentes concentrações de NaCl a pHs e resistência intrínseca a nove antibióticos. A habilidade em promover o crescimento vegetal foi avaliada utilizando o arroz (Oryza sativa) como planta-modelo. Dentre os 16 isolados avaliados, oito foram classificados como pertencentes à família Enterobacteriaceae, relacionados aos gêneros Enterobacter e Pantoea. Seis outros isolados foram relacionados a Bacillus e outros dois a Rhizobium e Stenotrophomonas. A avaliação da incorporação do N ao meio semissólido demonstrou o potencial diazotrófico de todos os isolados. Duas bactérias produziram mais AIA do que a estirpe BR 11175T de Herbaspirillum seropedicae. Isolados bacterianos foram capazes de formar película em meio semissólido suplementado com concentrações de NaCl de até 1.27 mol L-1. A resistência intrínseca a antibióticos e o metabolismo de diferentes fontes de C indicaram um perfil fisiológico muito variável. Sete isolados foram capazes de aumentar o crescimento das plantas de arroz, mais eficientemente do que a estirpe de referência Ab-V5 de Azospirillum brasilense. Os resultados indicaram o potencial de T. spicatus como uma espécie nativa fonte de bactérias promotoras de crescimento vegetal.

Plant species that naturally occur in the Brazilian Caatinga(xeric shrubland) adapt in several ways to these harsh conditions, and that can be exploited to increase crop production. Among the strategic adaptations to confront low water availability, desiccation tolerance stands out. Up to now, the association of those species with beneficial soil microorganisms is not well understood. The aim of this study was to characterize Tripogon spicatusdiazotrophic bacterial isolates from the Caatingabiome and evaluate their ability to promote plant growth in rice. Sixteen bacterial isolates were studied in regard to their taxonomic position by partial sequencing of the 16S rRNA gene, putative diazotrophic capacity, in vitro indole-acetic acid (IAA) production and calcium phosphate solubilization, metabolism of nine different C sources in semi-solid media, tolerance to different concentrations of NaCl to pHs and intrinsic resistance to nine antibiotics. Finally, the ability of the bacterial isolates to promote plant growth was evaluated using rice (Oryza sativa) as a model plant. Among the 16 isolates evaluated, eight of them were classified as Enterobacteriaceae members, related to Enterobacter andPantoeagenera. Six other bacteria were related toBacillus, and the remaining two were related toRhizobiumand Stenotrophomonas.The evaluation of total N incorporation into the semi-solid medium indicated that all the bacteria studied have putative diazotrophic capacity. Two bacteria were able to produce more IAA than that observed for the strain BR 11175Tof Herbaspirillum seropedicae.Bacterial isolates were also able to form a microaerophilic pellicle in a semi-solid medium supplemented with different NaCl concentrations up to 1.27 mol L-1. Intrinsic resistance to antibiotics and the metabolism of different C sources indicated a great variation in physiological profile. Seven isolates were able to promote rice growth, and two bacteria were more efficient than the reference strainAzospirillum brasilense, Ab-V5. The results indicate the potential of T. spicatus as native plant source of plant growth promoting bacteria.

O objetivo deste trabalho foi avaliar a contribuição de estirpes de rizóbio, eficientes na fixação de nitrogênio, sobre a produção de grãos de novas cultivares de feijão-caupi, indicadas para cultivo no Semiárido brasileiro, no Submédio do Vale do Rio São Francisco. Dois experimentos foram implantados nos perímetros irrigados de Mandacaru (Juazeiro, BA) e Bebedouro (Petrolina, PE). Os tratamentos consistiram da inoculação isolada de cinco estirpes de rizóbio - BR 3267, BR 3262, INPA 03-11B, UFLA 03-84 (Bradyrhizobiumsp.) e BR 3299T (Microvirga vignae) -, além de um tratamento com nitrogênio e de um controle sem inoculação ou aplicação de N. As seguintes cultivares de feijão-caupi foram avaliadas: BRS Pujante, BRS Tapaihum, BRS Carijó e BRS Acauã. Utilizou-se o delineamento experimental de blocos ao acaso, com quatro repetições. As plantas inoculadas apresentaram produtividade de grãos similar à observada em plantas adubadas com 80 kg ha-1 de N. As cultivares BRS Tapaihum e BRS Pujante destacaram-se quanto à produtividade e ao teor de proteínas nos grãos, quando inoculadas, o que mostra seu potencial para cultivo na região do Submédio do Vale do São Francisco.

The objective of this work was to evaluate the contribution of efficient nitrogen-fixing rhizobial strains to grain yield of new cowpea cultivars, indicated for cultivation in the Brazilian Semiarid region, in the sub-medium of the São Francisco River Valley. Two experiments were set up at the irrigated perimeters of Mandacaru (Juazeiro, state of Bahia) and Bebedouro (Petrolina, state of Pernambuco). The treatments consisted of single inoculation of five rhizobial strains - BR 3267, BR 3262, INPA 03-11B, UFLA 03-84 (Bradyrhizobiumsp.), and BR 3299T(Microvirga vignae) -, besides a treatment with nitrogen and a control without inoculation or N application. The following cowpea cultivars were evaluated: BRS Pujante, BRS Tapaihum, BRS Carijó, and BRS Acauã. A randomized complete block design, with four replicates, was used. Inoculated plants showed similar grain yield to the one observed with plants fertilized with 80 kg ha-1 N. The cultivars BRS Tapaihum and BRS Pujante stood out in grain yield and protein contents when inoculated, showing their potential for cultivation in the sub-medium of the São Francisco River Valley.

O objetivo deste trabalho foi avaliar a resposta da macrofauna edáfica, carbono da biomassa microbiana, respiração do solo, quociente metabólico e carbono lábil de solo fumigado em Neossolo Flúvico. O delineamento experimental foi em locos ao acaso com cinco tratamentos (quatro combinações de coquetéis vegetais e testemunha) e quatro repetições, e subseqüente plantio de melão na região semi-árida de Juazeiro, Bahia, Brasil, entre os meses de agosto de 2006 a agosto de 2007. Foram realizadas quatro coletas de solo na profundidade de 0-10 cm aos: seis dias antes do plantio do coquetel vegetal, 117; 215 e 354 dias após plantio do coquetel vegetal. Os resultados obtidos indicam que todos os atributos não apresentaram diferença significativa para os tratamentos com coquetel vegetal, mas diferiram significativamente em função da época da coleta. Maiores teores de carbono da biomassa microbiana e carbono lábil de solo fumigado foram obtidos aos 117 dias após o plantio. Carbono da biomassa microbiana correlacionou-se inversamente à respiração do solo e ao quociente metabólico, que aumentaram a partir de 215 dias após plantio. Aos 354 dias após plantio, a macrofauna edáfica apresentou maior densidade, riqueza, diversidade e uniformidade, tendo formigas e besouros sido os animais mais abundantes.

The goal of this work was to evaluate the response of edaphic macrofauna, soil microbial biomass carbon, soil basal respiration, metabolic quotient and labile carbon of fumigated soil in a Fluvic Neosol. The experimental design was in randomized blocks, with five treatments (four green manure mix and one control) and four replications, and subsequent planting of melon (Cucumis melo L.) under semi-arid conditions in Juazeiro, Bahia State, Brazil, from August 2006 to August 2007. Soil samples were collected at a depth of 0-10 cm at 6 days before, and 117; 215 and 354 days after green manure mix planting. The results indicate that none of the attributes presented a significant difference for the treatments, but changed significantly in relation to the time of sample collection. The higher levels of soil microbial biomass carbon and labile carbon of fumigated soil were measured 117 days after planting. Soil microbial biomass carbon was inversely correlated with soil basal respiration and metabolic quotient, which increased as of 215 days after planting. At 354 days after planting, the edaphic macrofauna presented the highest density, richness, diversity and uniformity. Ants and beetles were most abundant in the areas studied.

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.