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.

RESUMO: Objetivou-se avaliar os efeitos de três soluções eletrolíticas enterais de manutenção com diferentes fontes de energia administradas em bovinos adultos por via nasorruminal em fluxo contínuo sobre parâmetros fisiológicos e hematológicos. Foram utilizadas seis novilhas holandesas em um delineamento crossover. Os animais foram submetidos a três tratamentos: Solução contendo Propionato de cálcio - 4g de NaCl, 0,5g de KCl, 0,3g de MgCl2 e 10g de propionato de cálcio para cada 1000mL (Osmolaridade mensurada: 299mOsm/L); Solução contendo Glicerol - 4g de NaCl, 0,5g de KCl, 0,3g de MgCl2, 1g de acetato de cálcio e 10mL de glicerol para cada 1000mL (Osmolaridade mensurada: 287mOsm/L); Solução contendo Propilenoglicol - 4g de NaCl, 0,5g de KCl, 0,3g de MgCl2, 1g de acetato de cálcio e 15mL de propilenoglicol para cada 1.000mL (Osmolaridade mensurada: 378mOsm/L). Foi realizado exame físico e colheita de sangue para os hemogramas imediatamente antes do início da hidratação e a cada três horas durante 12h de tratamento e mais uma colheita 12h após o final do período experimental, perfazendo seis colheitas ao total. Todas soluções promoveram ao longo das 12 horas de tratamento hemodiluição com redução nos valores de hemácias, concentração de hemoglobina e volume globular, sem, contudo, alterar o leucograma. A hidratação enteral em fluxo contínuo via nasorruminal, mostrou-se uma técnica bem tolerada pelos animais, como mínimos efeitos sobre o bem-estar, mesmo quando administrada por 12 horas, sendo, portanto, uma técnica indicada como uma opção à hidratação parenteral na terapia de manutenção de fluidos. As três soluções eletrolíticas aqui propostas são capazes de expandir significativamente a volemia.

ABSTRACT: The aim of this study was to evaluate the effects of three enteral electrolyte solutions, each with different energy sources, administrated as continuous flow on the physiological parameters and blood count of healthy Holstein heifers. Six Holstein heifers were used in a crossover design. All animals received all three treatments: solution with calcium propionate, 4g of NaCl, 0.5g of KCl, 0.3g of MgCl2, and 10g of calcium propionate diluted in 1000mL of water (measured osmolarity: 299mOsm/L); solution with glycerol, 4g of NaCl, 0.5g of KCl, 0.3g of MgCl2, 1g of calcium acetate, and 10mL of glycerol in 1000mL of water (measured osmolarity: 287mOsm/L); solution with propylene glycol, 4g of NaCl, 0.5g of KCl, 0.3g of MgCl2, 1g of calcium acetate, and 15mL of propylene glycol in 1000mL of water (measured osmolarity: 378mOsm/L). Physical evaluations and blood samples were collected immediately before the initiation of fluid therapy, at 3-hour intervals over the 12-hour period of fluid therapy, and 12 hours after the end of fluid therapy. Animals presented no signs of stress or discomfort. All solutions resulted in a significant decrease in erythrocyte concentration, hemoglobin concentration, and hematocrit, without affecting the leukogram. Enteral fluid therapy administered as continuous flow via the naso-ruminal route was well-tolerated by animals with minimal effects on animal welfare, even when administered for 12 hours. This technique is indicated as an alternative route for parenteral maintenance fluid therapy. Electrolyte solutions proposed here were able to significantly expand blood volume.

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.

La asociación de gramíneas silvestres con bacterias diazotróficas en los biomas brasileños es poco conocida. El aislamiento y caracterización de las bacterias asociadas con gramíneas silvestres puede contribuir a entender la ecología de las diazotróficas y bacterias con aplicaciones biotecnológicas. En este estudio, caracterizamos aislamientos bacterianos de diazotróficas de Oryza glumaepatula recolectadas en Cerrado y zonas boscosas de la Amazonía en el estado de Roraima, Brasil. Plantas sanas de O. glumepatula fueron recolectadas en cinco zonas boscosas y siete en Cerrado. Las plantas de Cerrado fueron recolectadas en septiembre 2008, mientras que las del bosque en Junio 2008 y Abril 2009. Las plantas y el suelo adherido a las raíces se transfirieron a macetas y se cultivaron durante 35 días en condiciones de invernadero. Durante la cosecha, los brotes y las raíces se trituraron por separado en una solución salina, la suspensión se diluyó en serie y se inocularon en placas Petri que contenían medio Dyg. Todas las colonias de bacterias se purificaron en el mismo medio. Se evaluó la capacidad diazotrófica de cada bacteria en condiciones microaerofílicas en medio semisólido BMGM. Además, los aislamientos bacterianos que formaron películas se evaluaron también mediante amplificación por PCR para el gen nifH. La diversidad de bacterias nifH+ se analizó por Huella Genética utilizando la Reacción en Cadena de la Polimerasa. Para las cepas seleccionadas, la capacidad de promover el crecimiento de O. sativa como modelo de planta también se evaluó. Se obtuvo un total de 992 cepas bacterianas. Cincuenta y un bacterias fueron capaces de formar películas en el medio semisólido y 38 amplificaron positivamente el fragmento 360bp del gen nifH. De los 38 aislamientos de nifH+, 24 fueron obtenidos de los brotes, mientras que 14 se originaron a partir de las raíces. Los perfiles de PCR-Box mostraron que los aislamientos bacterianos obtenidos en este estudio presentaron una baja similitud con las cepas de referencia pertenecientes a Herbaspirillum, Azospirillum y el género Burkholderia. La capacidad promotora del crecimiento fue confirmada por al menos cinco aislamientos. Para esta bacteria, la raíz y brote mostraron resultados de crecimiento mayores en comparación con los observados en las plantas inoculadas con las cepas de referencia. Estos resultados indican que O. glumaepatula es colonizada por una muy diversa comunidad diazotrófica en la Amazonia brasileña. Se están llevando a cabo otras investigaciones para esclarecer la taxonomía de estas cepas y sus mecanismos para promover el crecimiento.

The association of wild grasses with diazotrophic bacteria in Brazilian biomes is poorly understood. The isolation and characterization of bacteria associated with wild grasses can contribute to understand the diazotrophic ecology as well as to identify bacteria with biotechnological applications. In this study, we isolated and characterized diazotrophic bacterial isolates from Oryza glumaepatula collected in Cerrado and Forest areas of the Amazon in Roraima State, Brazil. Healthy O. glumepatula plants were collected at five sampling sites at Forest and seven at Cerrado, respectively. The plants were collected at the Cerrado areas in September 2008 while the Forest plants were collected in June/2008 and April/2009. The plants and the soil adhering to the roots were transferred to pots and grown for 35 days in greenhouse conditions. During the harvest, the shoots and the roots were crushed separately in a saline solution; the suspension was diluted serially and inoculated in Petri dishes containing Dyg’s medium. All distinct bacterial colonies were purified in the same medium. The diazotrophic capacity of each bacterium in microaerophilic conditions was assessed in semisolid BMGM medium. In addition, the pellicles forming bacterial isolates were also evaluated by PCR amplification for nifH gene. The diversity of nifH+ bacteria was analyzed by Box-PCR fingerprinting. For selected strains, the growth promoting capacity of O. sativa as a model plant was also evaluated. A total of 992 bacterial isolates were obtained. Fifty- one bacteria were able to form pellicles in the semisolid medium and 38 also positively amplified the 360bp nifH gene fragment. Among the 38 nifH+ isolates, 24 were obtained from the shoots, while 14 originated from the roots. The Box-PCR profiles showed that the bacterial isolates obtained in this study presented a low similarity with the reference strains belonging to the Herbaspirillum, Azospirillum and Burkholderia genus. The growth- promoting ability was confirmed for at least five isolates. For these bacteria, the root and shoot growing results showed higher increases when compared to those observed in plants inoculated with the evaluated reference strains. These results indicate that O. glumaepatula is colonized by a high diverse diazotrophic community in the Brazilian Amazon. Further investigations are now being carried out to determine the taxonomic positions of these isolates and their growth promoting mechanisms.

This study evaluated 26 pigeonpea rhizobial isolates according to their cultural characteristics, intrinsic antibiotic resistance, salt and temperature tolerance, carbon source utilization and amylolytic activity. The cultural characterization showed that the majority of them presented the ability to acidify the YMA. Among the 27 isolates evaluated, 25 were able to grow when incubated at 42° C and 11 showed tolerance to 3% (w/v) of NaCl in YMA medium. The patterns of carbon sources utilization was very diverse among the isolates. It was observed the capacity of three strains to metabolize all the carbon sources evaluated and a total of 42% of the bacterial isolates was able to grow in the culture medium supplemented with at least, six carbon sources. The carbon sources mannitol (control) and sucrose were metabilized by all isolates evaluated. The profile of intrinsic resistance to antibiotics showed that the isolates were mostly resistant to streptomycin and ampicillin, but susceptible to kanamycin and chloranphenicol. High amylolytic activity of, at least, four isolates was also demonstrated, especially for isolated 47.3b, which showed the highest enzymatic index. These results indicate the metabolic versatility of the pigeonpea rhizobia, and indicates the isolate 47.3b to further studies regarding the amylase production and characterization.

As espécies de mucuna são muito utilizadas como adubos verdes, e poucas informações estão disponíveis a respeito dos rizóbios nativos capazes de nodulá-las. O objetivo deste trabalho foi avaliar a diversidade e a capacidade simbiótica de isolados bacterianos de nódulos de mucuna-cinza (Mucuna pruriens (L.) DC.) e mucuna-anã (Mucuna deeringiana (Bort.) Merr.). As bactérias foram isoladas de nódulos de mucunas cinza e anã cultivadas em vasos com solos de um sistema de produção agroecológica. Foram isoladas 160 bactérias, sendo 80 de mucuna-anã e 80 de mucuna-cinza, que foram autenticadas e selecionadas para avaliação da capacidade simbiótica. A diversidade dos isolados foi avaliada por meio das características culturais em meio de cultura YMA e da técnica de análise de restrição do produto de PCR do gene 16S rDNA. A inoculação de cinco isolados em mucuna-cinza e dois em mucuna-anã apresentou elevada biomassa da parte aérea. A maioria dos isolados apresentou crescimento rápido e acidificou o meio de cultura. A análise de restrição demonstrou que as bactérias isoladas apresentam baixa similaridade com estirpes de referência, sugerindo a existência de isolados pertencentes a novos grupos, capazes de nodular as mucunas anã e cinza.

Mucuna species are widely used as green manure, and little information is available about rhizobia that can nodulate them. The objective of this study was to evaluate the diversity and ability of bacteria isolated from symbiotic nodules of velvet bean (Mucuna pruriens (L.) DC.) and Mucuna deeringiana (Bort.) Merr. From the nodules of either mucuna species, grown in pots with soils from an agroecological system, 80 bacteria were isolated. The selected bacteria were classified and used to assess the symbiotic capacity. The diversity assessment of the isolates was based on cultural characteristics on YMA medium and the technique of restriction analysis of PCR products of 16S rDNA. The inoculation of Mucuna pruriens with five and Mucuna deeringiana with two isolates increased the aboveground biomass. Most isolates grew rapidly and acidified the culture medium. Restriction analysis showed low similarity of the reference strains with the bacterial isolates, suggesting the existence of isolates belonging to new groups that are able to nodulate the two mucuna species.

O objetivo deste trabalho foi avaliar a estrutura metabólica e genética de comunidades bacterianas em Latossolo de cerrado sob vegetação nativa ou cultivado em sistema de rotação soja/milho sob preparo convencional e plantio direto. Foram utilizadas microplacas EcoPlate para determinar o perfil e a diversidade metabólica das comunidades bacterianas, e eletroforese em gel com gradiente desnaturante (DGGE) para avaliar a estrutura genética. O teste estatístico de Mantel foi utilizado para avaliar a relação entre a estrutura metabólica e a genética. A comunidade bacteriana sob vegetação nativa apresentou perfil metabólico diferente do encontrado em solos cultivados. No solo cultivado com soja sob preparo convencional, o padrão de utilização das fontes de carbono diferenciou-se dos demais tratamentos. Com base nos resultados de DGGE, a comunidade bacteriana sob vegetação nativa apresentou 35% de similaridade com as de áreas cultivadas. Foram formados grupos distintos de comunidades bacterianas do solo entre as áreas sob preparo convencional e plantio direto. Houve correlação significativa de 62% entre as matrizes geradas pelas microplacas EcoPlate e pela DGGE. Variações no perfil metabólico estão relacionadas às variações na estrutura genética das comunidades bacterianas do solo.

The objective of this work was to evaluate the metabolic and genetic structure of bacterial communities in a cerrado Oxisol under native vegetation or cultivated in a soybean/maize rotation system under conventional tillage and no tillage. EcoPlate microplates were used to determine the metabolic profile and diversity of bacterial communities, and denaturing gradient gel electrophoresis (DGGE) to assess the genetic structure. The Mantel statistic test was used to evaluate the relationship between the metabolic and the genetic structure. The bacterial community under native vegetation showed a different metabolic profile than that of cultivated soils. In the area cultivated with soybean under conventional tillage, the carbon source utilization pattern differed from the other treatments. Based on the DGGE results, the bacterial community under native vegetation showed 35% similarity to the ones found under cultivation. Distinct groups of soil bacterial communities were formed between the areas under conventional tillage and no tillage. A significant correlation of 62% was observed between matrices generated by EcoPlate microplates and by DGGE. Variations in the metabolic profile are related to changes in the genetic structure of soil bacterial communities.

O objetivo deste trabalho foi avaliar a produção e o comportamento reológico de exopolissacarídeos (EPS) produzidos por rizóbios isolados de nódulos de guandu [Cajanus cajan (L.) Millsp.], e a similaridade desses isolados, pela técnica de análise de restrição do DNA ribossomal amplificado. As bactérias foram cultivadas em meio YM líquido e os EPS foram obtidos por precipitação com etanol gelado a partir do caldo de cultivo. Em seguida, eles foram recuperados por centrifugação, secados a vácuo, pesados e ressuspensos em água para avaliações reológicas. Os três isolados avaliados apresentaram diferenças na produção e na eficiência relativa da produção de EPS, com destaque para o isolado 8.1c. Os EPS demonstraram comportamento não newtoniano e pseudoplástico, porém também apresentaram diferenças na viscosidade aparente em uma mesma taxa de cisalhamento. Na taxa de cisalhamento de 1 s-1, os três isolados foram diferentes, enquanto na taxa de cisalhamento de 40 s-1, os isolados 53.5 e 30.6a2 foram iguais e diferiram do isolado 8.1c. A similaridade dos isolados foi condizente com os resultados de reologia dos EPS, em que bactérias que sintetizaram EPS com menor viscosidade aparente apresentaram maior similaridade

The objective of this work was to evaluate the production and the rheological behavior of exopolysaccharides (EPS) produced by pigeonpea [Cajanus cajan (L.) Millsp.] rhizobia isolates, as well as to evaluate the similarity of these isolates through amplified ribosomal DNA restriction analysis technique. The bacteria were cultured in YM liquid media and the EPS were precipited with cold ethanol from culture broth. After that, they were vacuum dried, weighted, stored and resuspended in water to rheological evaluations. The three evaluated isolates showed differences on the production and productivity of EPS, and the isolate 8.1c stood out, with higher relative efficiency. The evaluated EPS showed non-newtonian and pseudoplastic behavior, however they also presented differences as to the apparent viscosity at the same shear rate. At the shear rate of 1 s-1 all isolates were different, while at the shear rate of 40 s-1 the isolates 53.5 and 30.6a2 were the same, differing from the isolate 8.1c. Genetic similarity of the isolates was in agreement with the rheology results, where bacteria that synthesized EPS with lower apparent viscosity presented the higher similarity

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.

O objetivo deste trabalho foi avaliar o efeito da solarização e da biofumigação sobre a comunidade microbiana do solo, por meio da atividade da enzima beta-glicosidase e do perfil do 16S rDNA, determinado com PCR-DGGE. A solarização do solo, com cobertura de plástico, foi feita por períodos de dois, quatro e seis meses, e a biofumigação foi realizada pela incorporação de 2 e 5% (v/v) de cama-de-frango ao solo. Logo após a retirada da cobertura de plástico e aos 30 dias após a remoção, a atividade da beta-glicosidase foi menor em relação ao tratamento não solarizado. Aos 60 dias, não foram mais observadas diferenças entre os tratamentos. A adição de cama-de-frango a 5% estimulou a atividade da beta-glicosidase. O perfil da estrutura da comunidade bacteriana foi influenciado pelo tempo de solarização, independentemente da época da retirada da cobertura de plástico. Não foi observado efeito da adição de cama-de-frango ao solo, no perfil da comunidade. A solarização afeta a atividade da beta-glicosidase, mas esses efeitos não são mais detectáveis após 60 dias da retirada da cobertura de plástico, diferentemente do que foi observado em relação à estrutura da comunidade bacteriana por PCR-DGGE. A biofumigação estimula a atividade da beta-glicosidase, mas não afeta o perfil da comunidade microbiana.

The objective of this work was to evaluate the effects of solarization and biofumigation on the soil microbial communities, by means of beta-glucosidase activity and 16S rDNA PCR-DGGE analyses. Solarization with a plastic covering of the soil took place over two, four and six months, and the soils were biofumigated by the addition of 2 and 5% (v/v) of chicken litter to the soil. Right after the plastic cover removal and after 30 days, beta-glucosidase was lower than in the nonsolarizated control. After 60 days, there were no longer significant differences in beta-glucosidase activity between treatments. The addition of 5% chicken litter stimulated beta-glucosidase activity. Bacterial community profile was influenced by solarization time, regardless of time of plastic cover removal. There was no effect of chicken litter amendments over the bacterial community structure. Solarization affects beta-glucosidase activity but, after 60 days, its effects are no longer detectable, differently of the observed data regarding soil bacterial community structure by PCR-DGGE. Biofumigation stimulates beta-glicosidase activity, but it doesn't affect the bacterial community structure.