Influence of Molybdenum doses in inoculation and mineral fertilization in cowpea beans / Influência de doses de molibdênio na inoculação e na fertilização nitrogenada em feijão caupi

In some leguminous plants, associations with nitrogen-fixing microorganisms allow their nutrition with nitrogen (N) from the atmosphere. This process is known as Biological Nitrogen Fixation (BNF), where through nitrogenase enzymes, N2 is converted to an available form. This process can replace in part, or in total, nitrogen fertilizers. Cowpea bean is a legume species that is recognized for its high capacity to carry out BNF. In the last decades, studies have encouraged small farmers from north and northeast Brazil to use inoculants with rhizobia species since the results of researches have demonstrated that inoculation is an interesting strategy to improve cowpea production. Considering the specific function of molybdenum (Mo) in the N assimilation, different doses of Mo were tested in this study in order to find doses that could improve and enhance BNF. Therefore, this study aimed to compare nitrogen fertilization and BNF in the N assimilation by plants with different Mo doses. Inoculation was performed with the strains UFLA 03-84 and INPA 03-11B. Doses of Mo were applied in seeds and each pot contained five seeds. Thirty-five days after germination, the plants were analyzed for shoot dry matter and fresh matter, N contents and accumulation, as well as the Soil-Plant Analysis Development (SPAD) Index and nodulation in inoculated plants. The different doses of Mo and also the nodulation treatments did not show significant differences in the contents of N. Plants with N fertilization had significant higher shoot dry matter and root dry matter production, in addition to higher N foliar contents and N accumulation. Therefore, BNF was not as efficient as nitrogen fertilization in the evaluated experimental conditions using cowpea beans.

Em algumas plantas de leguminosas, as associações com microorganismos de fixação permitem a sua nutrição com N (nitrogênio) da atmosfera. Este processo é conhecido como Fixação Biológica de Nitrogênio (FBN), onde através de enzimas nitrogenase N2 é convertido na forma disponível. Este processo pode substituir em parte, ou no total, fertilizantes nitrogenados. O feijão-caupi é uma espécie de leguminosa que é reconhecida pela sua alta capacidade de FBN. Nas últimas décadas, estudos encorajaram os pequenos agricultores do norte e nordeste do Brasil a usar inoculantes com espécies de rizobio, uma vez que os resultados das pesquisas têm demonstrado que a inoculação é uma estratégia interessante para melhorar a produção dessa cultura. Considerando a função específica do molibdênio (Mo) na assimilação do N, diferentes doses de Mo foram testadas neste estudo, a fim de encontrar doses que possam melhorar e potencializar a FBN. A inoculação foi feita com as estirpes UFLA 03-84 e INPA 03-11B. As diferentes doses de Mo foram aplicadas na semente, cinco sementes por vaso. Trinta e cinco dias após a germinação, as plantas foram analisadas com relação à matéria seca aérea e radicular, teor de N e acúmulo, índice de análise de desenvolvimento solo-planta (SPAD) e nodulação nas plantas inoculadas. As diferentes doses de Mo não mostraram diferenças significativas no conteúdo de N, nem no processo de nodulação. As plantas com adubação nitrogenada tiveram mattera seca da parte aérea e de raízes superior as inoculadas, além de maiores teores foliares de N. Portanto, para feijão-caupi, a FBN não foi tão eficiente quanto o fertilizante nitrogenado nas condições experimentais avaliadas.

Changes induced by co-inoculation in nitrogen-carbon metabolism in cowpea under salinity stress

ABSTRACT To mitigate the deleterious effects of abiotic stress, the use of plant growth-promoting bacteria along with diazotrophic bacteria has been increasing. The objectives of this study were to investigate the key enzymes related to nitrogen and carbon metabolism in the biological nitrogen fixation process and to elucidate the activities of these enzymes by the synergistic interaction between Bradyrhizobium and plant growth-promoting bacteria in the absence and presence of salt stress. Cowpea plants were cultivated under axenic conditions, inoculated with Bradyrhizobium and co-inoculated with Bradyrhizobium sp. and Actinomadura sp., Bradyrhizobium sp. and Bacillus sp., Bradyrhizobium sp. and Paenibacillus graminis, and Bradyrhizobium sp. and Streptomycessp.; the plants were also maintained in the absence (control) and presence of salt stress (50 mmolL-1 NaCl). Salinity reduced the amino acids, free ammonia, ureides, proteins and total nitrogen content in nodules and increased the levels of sucrose and soluble sugars. The co-inoculations responded differently to the activity of glutamine synthetase enzymes under salt stress, as well as glutamate synthase, glutamate dehydrogenase aminating, and acid invertase in the control and salt stress. Considering the development conditions of this experiment, co-inoculation with Bradyrhizobium sp. and Bacillus sp. in cowpea provided better symbiotic performance, mitigating the deleterious effects of salt stress.

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.

Antioxidant response of cowpea co-inoculated with plant growth-promoting bacteria under salt stress

Abstract Soil salinity is an important abiotic stress worldwide, and salt-induced oxidative stress can have detrimental effects on the biological nitrogen fixation. We hypothesized that co-inoculation of cowpea plants with Bradyrhizobium and plant growth-promoting bacteria would minimize the deleterious effects of salt stress via the induction of enzymatic and non-enzymatic antioxidative protection. To test our hypothesis, cowpea seeds were inoculated with Bradyrhizobium or co-inoculated with Bradyrhizobium and plant growth-promoting bacteria and then submitted to salt stress. Afterward, the cowpea nodules were collected, and the levels of hydrogen peroxide; lipid peroxidation; total, reduced and oxidized forms of ascorbate and glutathione; and superoxide dismutase, catalase and phenol peroxidase activities were evaluated. The sodium and potassium ion concentrations were measured in shoot samples. Cowpea plants did not present significant differences in sodium and potassium levels when grown under non-saline conditions, but sodium content was strongly increased under salt stress conditions. Under non-saline and salt stress conditions, plants co-inoculated with Bradyrhizobium and Actinomadura or co-inoculated with Bradyrhizobium and Paenibacillus graminis showed lower hydrogen peroxide content in their nodules, whereas lipid peroxidation was increased by 31% in plants that were subjected to salt stress. Furthermore, cowpea nodules co-inoculated with Bradyrhizobium and plant growth-promoting bacteria and exposed to salt stress displayed significant alterations in the total, reduced and oxidized forms of ascorbate and glutathione. Inoculation with Bradyrhizobium and plant growth-promoting bacteria induced increased superoxide dismutase, catalase and phenol peroxidase activities in the nodules of cowpea plants exposed to salt stress. The catalase activity in plants co-inoculated with Bradyrhizobium and Streptomyces was 55% greater than in plants inoculated with Bradyrhizobium alone, and this value was remarkably greater than that in the other treatments. These results reinforce the beneficial effects of plant growth-promoting bacteria on the antioxidant system that detoxifies reactive oxygen species. We concluded that the combination of Bradyrhizobium and plant growth-promoting bacteria induces positive responses for coping with salt-induced oxidative stress in cowpea nodules, mainly in plants co-inoculated with Bradyrhizobium and P. graminis or co-inoculated with Bradyrhizobium and Bacillus.

Inoculação de diferentes doses de Bradyrhizobium por cobertura e seu efeito na cultura da soja / Inoculation by different doses of Bradyrhizobium by side dressing and its effect on Soybean / Inoculación de diferentes dosis de Bradyrhizobium por cubierta y su efecto en la Cultura de la soja

A soja é a cultura que mais cresceu no Brasil, com grande destaque para o agronegócio. Sendo o nutriente mais requerido, o nitrogênio, torna se necessário muitas vezes a utilização de fertilizantes nitrogenados e como uma alternativa sustentável, recomenda-se a inoculação com Bradyrhizobium realizando desse modo a fixação biológica de nitrogênio. O objetivo deste estudo foi avaliar os benefícios da inoculação de Bradyrhizobium na cultura da soja, pela pulverização em cobertura, na nodulação, no desenvolvimento das plantas e na produtividade. Foram conduzidos quatro tratamentos com design inteiramente casualizado e com quatro repetições, sendo tratamento 1 (controle) isento de pulverização, o segundo com pulverização de 500 mL ha­1, o terceiro de 1000 mL ha­1 e o quarto de 1500 mL ha­1 do inoculante com Bradyrhizobium. O número de sacas ha­1 com a inoculação de Bradyrhizobium por pulverização de 1500 mL ha­1 foi de 59 sacas. Reificouse ainda, aumentos significativos (p ≤ 0,05), sendo de 180,65 para número de nódulos, 4,44 g planta­1 para massa seca dos nódulos, 17,30 g planta­1 para massa seca das raízes e de 64,33 g planta­1 para massa seca da parte aérea em comparação com o tratamento controle, evidenciando o maior rendimento de soja para este tratamento. Conclui-se, portanto que a inoculação da soja com 1500 mL ha­1 de Bradyrhizobium em pulverização por cobertura, é a mais eficiente diante dos parâmetros testados no desenvolvimento e produção da soja.(AU)

The cultivation of soybean has presented the greatest increase in Brazil, with strong emphasis on agribusiness. Since nitrogen is the most required nutrient, nitrogen fertilizers are sometimes necessary. As a sustainable alternative, the inoculation of Bradyrhizobium is recommended, which can perform the biological fixation of nitrogen. The objective of this study was to evaluate the benefits of Bradyrhizobium inoculation in soybean crop, by spraying on its side dressing, nodulation, plant development and on productivity. Four treatments were carried out with a completely randomized design with four replications. The first treatment was left spray-free, the second was sprayed with 500 mL ha­1, the third with 1000 mL ha­1, and the fourth with 1500 mL ha­1 of the Bradyrhizobium inoculant. A total of 59 bags ha­1 were harvested with the application of the inoculation of Bradyrhizobium by spraying 1500 mL ha­1, and significant increases (p ≤ 0.05) were observed, namely 180.65 nodules, 4.44 g plant­1 for nodule dry matter, 17.30 g plant­1 for root dry matter, and 64.33 g plant­1 for shoot dry matter when compared to the control treatment, evidencing the higher soybean yield for this treatment. It can be concluded that the inoculation of soybean with 1500 mL ha­1 Bradyrhizobium in spray by side dressing is the most efficient in the tested parameters for the development and production of soybean.(AU)

La soja es la cultura que más creció en Brasil, con gran destaque para el agronegocio. Con el nutriente más requerido el nitrógeno, se hace necesario a menudo la utilización de fertilizantes nitrogenados y como una alternativa sostenible, se recomienda la inoculación con Bradyrhizobium realizando de ese modo la fijación biológica de nitrógeno. El objetivo de este estudio fue evaluar los beneficios de la inoculación de Bradyrhizobium en el cultivo de la soja, la pulverización en cobertura, la nodulación, el desarrollo de las plantas y la productividad. Se realizaron cuatro tratamientos con diseño completamente casualizado y con cuatro repeticiones, siendo tratamiento 1 (control) exento de pulverización, el segundo con pulverización de 500 mL ha-1, el tercero de 1000 mL ha-1 y el cuarto de 1500 mL ha-1 del inoculante con Bradyrhizobium. El número de sacas ha-1 con la inoculación de Bradyrhizobium por pulverización de 1500 mL ha-1 fue de 59 sacas Se observaron también aumentos significativos (p ≤ 0,05), siendo de 180,65 para número de nódulos, 4,44 g planta-1 para masa seca de los nódulos, 17,30 g planta-1 para masa seca de las raíces y de 64,33 g planta-1 para masa seca de la parte aérea en comparación con el tratamiento control, evidenciando mayor rendimiento de soja para este tratamiento. Se concluye, por lo tanto, que la inoculación de la soja con 1500 mL ha-1 de Bradyrhizobium en pulverización por cubierta es la más eficiente, frente a los parámetros probados en el desarrollo y producción de la soja.(AU)

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.

Seeds inoculation and nitrogen fertilization for cowpea production on latosol in the western amazon / Produtividade de feijão-caupi com inoculante e fonte mineral nitrogenada em latossolo da amazônia ocidental

The cowpea is an important food crop in the North and Northeast regions of Brazil, where the beans are consumed either green or ripe. Yet, considering its socio-economic importance and its tropical origin, cowpea yields are low in those regions, due to inadequate cultivation practices and incorrect soil management. Therefore, the objective of this study was to study the development of cowpea crop inoculated and fertilized with two different nitrogen (N) sources. The experiment was conducted in the municipality of Rorainópolis (RR). The experimental design was randomized blocks with four treatments and five replications. The plant material was 'BRS Guariba' cowpea cultivar, and the treatments were: (i) control (no nitrogen fertilization), (ii) seeds inoculated with Bradyrhizobium elkanii, (iii) urea as N source (60 kg N ha-1), and (iv) ammonium sulphate as N source (60 kg N ha-1). The following crop traits were evaluated: foliar macronutrients (N, P, K, Ca, Mg and S), green and dry weight of shoots, dry beans per plant, pod yield and bean yield. Urea and ammonium sulphate promoted high accumulation of nutrients in leaves. Ammonium sulphate also stood out regarding productivity traits, thus proving to be a viable N source for cowpea in the Amazon region. The nodulation with Bradyrhizobium elkanii wasn't efficient to replace the fertilization with nitrogen fertilizers for cowpea BRS Guariba.

O feijão-caupi é uma das principais alternativas alimentares para as populações das regiões norte e nordeste do Brasil, sendo consumido na forma de grãos verdes ou maduros. Apesar da importância social e da origem tropical, a espécie apresenta baixa produtividade na região amazônica, incluindo o estado de Roraima, devido à baixa qualidade agronômica e manejo incorreto do solo. O objetivo do trabalho foi caracterizar o desenvolvimento da cultura do feijão-de-corda com duas diferentes fontes de nitrogênio (N) tratadas com inoculante. O experimento foi realizado no município de Rorainópolis (RR). O delineamento foi em blocos casualizados, com quatro tratamentos e cinco repetições. Foi utilizada a cultivar de feijão-caupi 'BRS Guariba'. Os tratamentos foram: testemunha (ausência de adubação nitrogenada), inoculante Bradyrhizobium elkanii, 60 kg ha-1 de N provenientes da uréia e 60 kg ha-1 de N da fonte sulfato de amônio. Foram avaliados os teores foliares de macronutientes (N, P, K, Ca, Mg e S), peso da massa verde e seca da parte aérea, número de grãos secos por planta, produção de vagens e produção de grãos. Em relação ao acúmulo de nutrientes a uréia e sulfato de amônio foram as fontes que apresentaram desempenho superior. Em relação as características produtivas, o sulfato de amônio foi o que mais se destacou, sendo uma fonte viável de suprimento de N para o feijão-caupi na região amazônica. A nodulação com Bradyrhizobium elkanii não foi eficiente para substituir a adubação com fertilizantes nitrogenados para o feijão-caupi BRS Guariba.

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.

Draft genome sequence of Bradyrhizobium sp. strain BR 3262, an effective microsymbiont recommended for cowpea inoculation in Brazil

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.

Draft genome sequence of Bradyrhizobium sp. strain BR 3267, an elite strain recommended for cowpea inoculation in Brazil

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.

Formononetin associated with phosphorus influences soybean symbiosis with mycorrhizal fungi and Bradyrhizobium / Formononetina associada com fósforo influencia a simbiose de soja com fungos micorrízicos e Bradyrhizobium

Arbuscular mycorrhizal fungi play an important role on nutrient supply to plants, specially P. However, the availability of inoculants for large-scale usage in agriculture is still limited because these organisms are obligatory symbionts. The use of symbiosis stimulants such as flavonoids can be an alternative to improve the beneficial effects of mycorrhiza for plant nutrition. The aim of this study was to evaluate the effect of the isoflavonoid biostimulant formononetin (7-hydroxy, 4'-methoxy isoflavone) in combination with three levels of phosphorus fertilization on mycorrhizal colonization, nodulation, and productivity of soybean, under field conditions. A 3 x 4 factorial scheme (levels of P: 0, 60 and 120 kg ha-1 P2O5 and doses of formononetin: 0, 25, 50 and 100 g ha-1) was used with five replicates. The following parameters were quantified at full bloom: plant height, shoot dry weight, nodule number, nodule dry weight, mycorrhizal colonization, and shoot N and P concentrations. Productivity was also evaluated at the end of the crop cycle. Formononetin stimulated mycorrhizal colonization at lower levels of P (0 and 60 kg ha-1), with colonization increasing from 56 to 64%. When applied with 60 kg ha-1 P2O5, formononetin increased soybean productivity to values similar to those observed when 120 kg ha-1 de P2O5, was applied. At doses above 50 g ha-1, formononetin applied to the seeds can reduce the need of P fertilization by 50%.

Os Fungos micorrízicos arbusculares desempenham papel importante no fornecimento de nutrientes para as plantas, especialmente P. No entanto, a disponibilidade de inoculantes com esses fungos, para o uso em larga escala na agricultura é ainda limitada, porque estes organismos são simbiontes obrigatórios. O uso de estimulantes simbióticos, como os flavonóides, podem ser uma alternativa para melhorar os efeitos benéficos da micorrrização na nutrição das plantas. O objetivo neste estudo foi avaliar o efeito do isoflavonóide bioestimulante formononetina (7-hidroxi, 4'-metoxi isoflavona) em combinação com três níveis de adubação fosfatada sobre a colonização micorrízica, a nodulação e a produtividade da soja, em condições de campo. Um esquema fatorial 3 x 4 (níveis de P: 0, 60 e 120 kg ha-1 de P2O5 e doses de formononetina: 0, 25, 50 e 100 g ha-1) foi utilizado, com cinco repetições. Os seguintes parâmetros foram quantificados em plena floração: altura da planta, matéria seca da parte aérea, número e matéria seca de nódulos, colonização micorrízica, e concentrações de N e P na parte aérea das plantas. A produtividade também foi avaliada no final do ciclo da cultura. A Formononetina estimulou a colonização micorrízica em níveis mais baixos de P (0 e 60 kg ha- 1), com aumentos de 56-64%. Quando aplicado com 60 kg ha-1 de P2O5, a formononetina aumentou a produtividade da soja, alcançando valores semelhantes aos observados quando foi aplicado 120 kg ha-1 de P2O5. Em doses acima de 50 g ha- 1, a formononetina aplicada na semente pode reduzir a necessidade de fertilização fosfatada em 50%.

Interrogantes en la tecnología de la inoculación de semillas de soja con Bradyrhizobium spp / Queries related to the technology of soybean seed inoculation with Bradyrhizobium spp

With the aim of exploiting symbiotic nitrogen fixation, soybean crops are inoculated with selected strains of Bradyrhizobium japonicum, Bradyrhizobium diazoefficiens or Bradyrhizobium elkanii (collectively referred to as Bradyrhizobium spp.). The most common method of inoculation used is seed inoculation, whether performed immediately before sowing or using preinoculated seeds or pretreated seeds by the professional seed treatment. The methodology of inoculation should not only cover the seeds with living rhizobia, but must also optimize the chances of these rhizobia to infect the roots and nodulate. To this end, inoculated rhizobia must be in such an amount and condition that would allow them to overcome the competition exerted by the rhizobia of the allochthonous population of the soil, which are usually less effective for nitrogen fixation and thus dilute the effect of inoculation on yield. This optimization requires solving some queries related to the current knowledge of seed inoculation, which are addressed in this article. I conclude that the aspects that require further research are the adhesion and survival of rhizobia on seeds, the release of rhizobia once the seeds are deposited in the soil, and the movement of rhizobia from the vicinity of the seeds to the infection sites in the roots

Con el fin de aprovechar la fijación simbiótica de nitrógeno, el cultivo de soja se inocula con cepas seleccionadas de Bradyrhizobium japonicum, Bradyrhizobium diazoefficiens o Bradyrhizobium elkanii (conjuntamente referidas como Bradyrhizobium spp.). El método más común de hacerlo es la inoculación en semillas, ya sea que esta se realice en el momento previo a la siembra o que se utilicen semillas preinoculadas o pretratadas mediante el tratamiento profesional de semillas. La metodología de inoculación no debe limitarse a recubrir las semillas con rizobios vivos, sino que también debe optimizar las chances de esos rizobios para infectar las raíces y nodular. Para ello los rizobios inoculados deben estar en una cantidad y un estado tales que les permitan superar la competición ejercida por los rizobios de la población alóctona del suelo, los cuales usualmente son menos eficaces para la fijación de nitrógeno y así diluyen el efecto de la inoculación sobre el rendimiento. Esta optimización requiere resolver algunos interrogantes, que son abordados en el presente artículo. Concluyo que los aspectos que requieren más investigación son la adhesión y supervivencia de los rizobios en las semillas, la liberación de los rizobios una vez que las semillas se depositan en el suelo y el movimiento de los rizobios desde las inmediaciones de las semillas hasta los sitios de infección en las raíces

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.

Multilocus sequence analysis (MLSA) of Bradyrhizobium strains: revealing high diversity of tropical diazotrophic symbiotic bacteria

Symbiotic association of several genera of bacteria collectively called as rhizobia and plants belonging to the family Leguminosae (=Fabaceae) results in the process of biological nitrogen fixation, playing a key role in global N cycling, and also bringing relevant contributions to the agriculture. Bradyrhizobium is considered as the ancestral of all nitrogen-fixing rhizobial species, probably originated in the tropics. The genus encompasses a variety of diverse bacteria, but the diversity captured in the analysis of the 16S rRNA is often low. In this study, we analyzed twelve Bradyrhizobium strains selected from previous studies performed by our group for showing high genetic diversity in relation to the described species. In addition to the 16S rRNA, five housekeeping genes (recA, atpD, glnII, gyrB and rpoB) were analyzed in the MLSA (multilocus sequence analysis) approach. Analysis of each gene and of the concatenated housekeeping genes captured a considerably higher level of genetic diversity, with indication of putative new species. The results highlight the high genetic variability associated with Bradyrhizobium microsymbionts of a variety of legumes. In addition, the MLSA approach has proved to represent a rapid and reliable method to be employed in phylogenetic and taxonomic studies, speeding the identification of the still poorly known diversity of nitrogen-fixing rhizobia in the tropics.

Resposta da co-inoculação de bactérias promotoras de crescimento em plantas e Bradyrhizobium sp. em caupi / Co-inoculation response of growth promoting bacterias in plants and Bradyrhizobium sp. in cowpea

Para a região árida e semi-árida do Nordeste brasileiro, o cultivo do caupi (Vigna unguiculata [L.] Walp.) vem se destacando por ser uma cultura adaptável as condições da região. Devido à capacidade do caupi de, em simbiose com o rizóbio, realizar a FBN, a co-inoculação rizóbio e bactérias promotoras de crescimento em plantas (BPCP's) poderá optimizar a fixação do N2 atmosférico dependendo da combinação e compatibilidade das estirpes envolvidas. O objetivo do trabalho foi avaliar a resposta da co-inoculação de BPCP's e Bradyrhizobium sp. como alternativa para optimizar a performance simbiótica e o desenvolvimento do caupi em condições de casa-de-vegetação. O experimento foi instalado em casa de vegetação, utilizando como substrato areia lavada e autoclavada em vasos de Leonard. Os tratamentos foram: 22 estirpes de BPCP's comparadas com a estirpe padrão (BR 3267), no caupi cv. "IPA 206". A colheita foi efetuada aos 35 dias após o plantio (DAP) e foram avaliados as seguintes variáveis: altura de planta (AP) aos 15, 25 e 35 dias, comprimento da raiz (CR), matéria seca da parte aérea (MSPA), matéria seca da raiz (MSR), matéria seca do nódulo (MSN), nodulação específica (NE) e nitrogênio acumulado na parte aérea (Nac). Os resultados mostram que a cultura do caupi respondeu significativamente a inoculação de estirpes de Bacillus, Brevibacillus e Paenibacillus co-inoculados com a estirpe de Bradyrhizobium sp. (BR 3267). A interação dos micro-organismos estudados não influenciou as variáveis, nodulação específica e matéria seca dos nódulos no caupi cv. IPA 206. A estirpe de Paenibacillus graminis (MC 04.21) co-inoculada com Bradyrhizobium sp. (BR 3267) foi superior em relação às demais estirpes avaliadas na produção de nitrogênio acumulado na matéria seca da parte aérea, proporcionando uma melhor performance simbiótica.

In the arid and semi-arid northeastern Brazil, the cultivation of cowpea has stood out for being an adaptable culture conditions in the region. Because of the ability of cowpea, in symbiosis with rhizobia carry out the FBN, the co-inoculation Rhizobium and growth promoting bacteria in plants can optimize the fixation of atmospheric N2 depending on the combination and compatibility of the strains involved. The objective of this study was to evaluate the response of the inoculation BPCP's and Bradyrhizobium sp. as an alternative to optimize performance and development of symbiotic cowpea under conditions of green-house. The experiment was conducted in a greenhouse, using as substrate washed and sterilized sand in Leonard jars. The treatments were: 22 strains BPCP's compared with the strain (BR 3267), on cowpea cv. IPA 206. At harvest 35 days after planting (DAP) and the following variables were evaluated: plant height (AP) 15, 25 and 35 days, root length (CR), shoot dry matter (MSPA), root dry matter (MSR), nodule dry matter (MSN), specific nodulation (NE) and nitrogen accumulated in shoots (Nac). The results show that the culture of cowpea responded significantly to inoculation of strains of Bacillus, Brevibacillus and Paenibacillus co-inoculated with the strain of Bradyrhizobium sp. (BR 3267). The interaction of microorganisms studied non-influenced the variables specific nodulation and dry matter of nodules in cowpea cv. IPA 206. The strain of Paenibacillus graminis (MC 04/21) coinoculated with Bradyrhizobium sp. (BR 3267) was higher compared to other strains tested in the production of nitrogen accumulated in shoot dry matter, providing a better performance symbiotic.

A Fosmid Cloning Strategy for Detecting the Widest Possible Spectrum of Microbes from the International Space Station Drinking Water System

In this study, fosmid cloning strategies were used to assess the microbial populations in water from the International Space Station (ISS) drinking water system (henceforth referred to as Prebiocide and Tank A water samples). The goals of this study were: to compare the sensitivity of the fosmid cloning strategy with that of traditional culture-based and 16S rRNA-based approaches and to detect the widest possible spectrum of microbial populations during the water purification process. Initially, microbes could not be cultivated, and conventional PCR failed to amplify 16S rDNA fragments from these low biomass samples. Therefore, randomly primed rolling-circle amplification was used to amplify any DNA that might be present in the samples, followed by size selection by using pulsed-field gel electrophoresis. The amplified high-molecular-weight DNA from both samples was cloned into fosmid vectors. Several hundred clones were randomly selected for sequencing, followed by Blastn/Blastx searches. Sequences encoding specific genes from Burkholderia, a species abundant in the soil and groundwater, were found in both samples. Bradyrhizobium and Mesorhizobium, which belong to rhizobia, a large community of nitrogen fixers often found in association with plant roots, were present in the Prebiocide samples. Ralstonia, which is prevalent in soils with a high heavy metal content, was detected in the Tank A samples. The detection of many unidentified sequences suggests the presence of potentially novel microbial fingerprints. The bacterial diversity detected in this pilot study using a fosmid vector approach was higher than that detected by conventional 16S rRNA gene sequencing.

Triple inoculation with Bradyrhizobium, Glomus and Paenibacillus on cowpea (Vigna unguiculata) [L. ] walp. )development. (au)

The use of microorganisms to improve the availability of nutrients to plants is of great importance to agriculture. This study aimed to evaluate the effect of triple inoculation of cowpea with arbuscular mycorrhizal fungi (AMF), plant growth-promoting bacteria (PGPB) and rhizobia to maximize biological nitrogen fixation (BNF) and promote plant growth. The experiment was conducted in a greenhouse using cowpea plants (Vigna unguiculata L. Walp cv. IPA 206). The treatments included inoculation with strains of Bradyrhizobium sp. (BR 3267 and EI - 6) individually and as a mixture, an absolute control (AC) and mineral nitrogen control (NC), all combined with the presence or absence of native AMF (Glomus etunicatum) and PGPB (Paenibacillus brasilensis - 24) in a 5x2x2 factorial design. All treatments were replicated three times. Contrasts were performed to study the treatment of variables. Inoculation with Bradyrhizobium sp. (BR 3267 and EI - 6) and G. etunicatum favored nitrogen acquisition and phosphorus availability for the cowpea plants. Inoculation with P. brasilensis - 24 increased colonization by Bradyrhizobium sp. and G. etunicatum and promoted cowpea growth, while the nitrogen from symbiosis was sufficient to supply the plants nutritional needs.

Doses de inoculante e nitrogênio na semeadura da soja em área de primeiro cultivo / Doses of inoculant and nitrogen at sowing of soybean in first cultivation area

A inoculação da cultura da soja com bactérias do gênero Bradyrhizobium, é uma prática amplamente difundida, devido aos ótimos resultados alcançados em produtividade, porém, algumas dúvidas ainda surgem em relação à necessidade de aplicação de nitrogênio mineral na semeadura. Neste sentido, este trabalho teve como objetivo avaliar a resposta da cultura da soja a doses de inoculante e nitrogênio mineral na semeadura em solo de primeiro ano de cultivo. Os experimentos foram realizados no Campo Experimental da Universidade do Estado de Mato Grosso, Campus de Alta Floresta, MT, nos anos agrícolas de 2006/2007 e 2007/2008. O delineamento experimental foi em blocos ao acaso, num esquema fatorial 2x4, com quatro repetições. Os fatores consistiram de inoculação das sementes com Bradyrhizobium japonicum (3 e 6 mL kg-1 de sementes), e adubação nitrogenada na semeadura (0, 10, 20 e 40 kg de N ha-1). As seguintes variáveis foram avaliadas: altura de plantas, altura de inserção da primeira vagem, número de vagens por planta, número de grãos por vagem, massa de 100 grãos e produtividade de grãos. Nos dois anos agrícolas não houve diferença significativa para as doses de inoculante aplicadas em primeiro ano de cultivo com soja. Para a aplicação de nitrogênio na semeadura da cultura da soja houve efeito quadrático para altura de plantas e de inserção da primeira vagem e linear crescente para número de vagens por planta no segundo ano de cultivo, porém, sem incremento significativo na produtividade.

The inoculation of soybean crop with Bradyrhizobium bacteria is a widespread practice, due to excellent results achieved in productivity, however, some doubt still arise in relation the need of application of mineral nitrogen at sowing. In that mean, this work aimed to evaluate the response of the doses at inoculant and mineral nitrogen in the sowing of crop soybean in area of first cultivation. The experiments were carried in the experimental field of the University of the Estate of Mato Grosso, Campus of Alta Floresta - MT, Brazil, in the growing seasons of 2006/2007 and 2007/2008. The experimental design was randomized blocks, in a factorial escheme 2 x 4, with four replications. The factors consisted in the inoculation of seeds with Bradyrhizobium japonicum (3 e 6 mL kg-1 of seeds), and nitrogen fertilizer at sowing (0, 10, 20 e 40 kg ha-1). The following variables were evaluated: plant height, insertion height of first pod, number of pod per plant, number of grain per pod, mass of 100 grain and grain productivity. In the two growing seasons there was no significant difference for doses of inoculants applied in first year of cultivation with soybean. To nitrogen application at sowing of soybean crop there was quadratic effect to plant height and of insertion height of first pod and growing linear to number of pod per plant in the second year of cultivation, however, without significant increase the productivity

Caracterización de cepas de Rhizobium y Bradyrhizobium (con habilidad de nodulación) seleccionados de los cultivos de fríjol caupi (Vigna unguiculata) como potenciales bioinóculos / Characterization of Rhizobium and Bradyrhizobiu´s strains (with ability of nodulation) selected from bean (Vigna unguiculata) cultures as a potentials bioinoculants

Los suelos de Latinoamérica son ácidos y deficientes en nitrógeno; por esta razón es necesario explorar nuevas alternativas con el fin de modular el desarrollo de plantas, especialmente las leguminosas. El uso del potencial de las bacterias para inducir nodulación y fijar el nitrógeno en las plantas leguminosas se ha estudiado como una opción con potencial impacto. En este estudio se analizó la diversidad de cepas de rhizobios aislados del fríjol Vigna unguiculata (fríjol caupí) cultivado en el norte del departamento de Bolívar (Colombia). Se identificaron aislados capaces de crecer mejor en ambientes hostiles (los cuales tienen un uso potencial como bioinoculantes). Se describe además un acercamiento a la taxonomía de estas bioespecies. Se caracterizaron 52 cepas de rizobios (basados en sus características morfológicas, requerimientos de cultivo, metabólicas, resistencia a metales y antibióticos y de autenticación). De acuerdo con sus propiedades de crecimiento, el 63,5 por ciento fueron de cepas de lento, mientras que el 36,5 por ciento, de rápido crecimiento. Prevalecieron las de lento crecimiento con un 63,5 por ciento del total sobre un 36,5 por ciento de rápido crecimiento. Los aislados fueron caracterizados de acuerdo con sus patrones de asimilación de carbohidratos, y se encontraron microorganismos de los géneros Rhizobium, Bradyrhizobium y Mesorhizobium (12 cepas identificadas como potenciales bioinoculantes).

Latin America´s soils are acidic and nitrogen-deficient, for this reason is necessary to explore new alternatives in order to modulate in the development of plants, specially the leguminous plants. The use of bacteria´s potential to induce nodulation and to fix nitrogen in leguminous plants has been studied as an option with potential impact. In our study, we analyzed the diversity of rhizobial isolated of the bean Vigna unguiculata (beans cowpea) cultured in the north of Bolivar department (Colombia). We identified strains able of growing in hostile environment (which have a potential use like bioinoculants). Additionally, we described an approach to taxonomy of these biospecies. We reached the characterization of 52 rhizobial strains (based on their morphologic, requires of culture, metabolic, resistance to metals and antibiotics and authentication characteristics). According with their growing properties, 63.5 percent were slow growth strains while the 36.5 percent were of rapid growth. The isolates were characterized according to their assimilation carbohydrate´s pattern, finding microorganism as Rhizobium, Bradyrhizobium and Mesorhizobium (12 of which were identified as potential bioinoculants).