RESUMO
Soybean [Glycine max (L.) Merr.] has great economic and nutritional importance mainly due to its high protein content. All plant's N needs can be met by the symbiosis with elite Bradyrhizobium strains applied as inoculants to the seeds at sowing time; however, the increasing use of pesticides in seed treatments can impair the contribution of the biological nitrogen fixation. In this study, we report decreases in cell survival of two strains, B. japonicum SEMIA 5079 and B. elkanii SEMIA 587 in seeds inoculated and treated with StandakTop™, composed of the fungicides pyraclostrobin and thiophanate-methyl and the insecticide fipronil, the pesticides most used in soybean seed treatment in several countries. Cell death was enhanced with the time of exposure to the pesticides, and B. elkanii was less tolerant, with almost no detectable viable cells after 15 days. Change in colony morphology with smaller colonies was observed in the presence of the pesticides, being more drastic with the time of exposure, and attributed to an adaptive response towards survival in the presence of the abiotic stress. However, morphological changes were reversible after elimination of the stressing agent and symbiotic performance under controlled greenhouse conditions was similar between strains that had been or not exposed to the pesticides. In addition, no changes in DNA profiles (BOX-PCR) of both strains were observed after the contact with the pesticides. In two field experiments, impacting effects of the pesticides were observed mainly on the total N accumulated in grains of plants relying on both N2-fixation and N-fertilizer. Our data indicate that StandakTop® affects parameters never reported before, including colony morphology of Bradyrhizobium spp. and N metabolism and/or N remobilization to soybean grains.
Assuntos
Bradyrhizobium/crescimento & desenvolvimento , Glycine max/microbiologia , Praguicidas/efeitos adversos , Bradyrhizobium/efeitos dos fármacos , Bradyrhizobium/metabolismo , Viabilidade Microbiana/efeitos dos fármacos , Nitrogênio/metabolismo , Fixação de Nitrogênio/efeitos dos fármacos , Glycine max/crescimento & desenvolvimento , Glycine max/metabolismo , SimbioseRESUMO
The bacterial strains SEMIA 587 and 5019 (Bradyrhizobium elkanii), 5079 (Bradyrhizobium japonicum), and 5080 (Bradyrhizobium diazoefficiens) are recommended for soybean inoculants in Brazil. In several countries, the current regulations are insufficient to induce companies for improving the quality of their products, leading to low performance and subsequent abandonment of inoculant use. From 2010 to 2014, 1086 samples coming mainly from Argentina and the southern region of Brazil were analyzed for viable cells counting, strains identification, and purity analysis according to the SDA/MAPA no. 30/2010 Normative Instruction. Most products were imported and formulated in liquid carriers with 5.0 × 109 colony-forming units (CFU)/mL. The strains most frequently used were SEMIA 5079/5080. Only 2.21% of samples had contaminants. The guaranteed concentration of viable cells in inoculants mostly ranged from 4.1 × 109 to 5.0 × 109 CFU/mL or CFU/g. The most frequently found concentration was above 1.1 × 1010 CFU/mL or CFU/g, which was higher than the product guarantee. The inoculants used for soybean crop in Brazil have excellent quality, leading the country to the leadership in taking advantage of the biological nitrogen fixation benefits for a productive and sustainable agriculture.
Assuntos
Inoculantes Agrícolas/isolamento & purificação , Bradyrhizobium/isolamento & purificação , Glycine max/microbiologia , Inoculantes Agrícolas/classificação , Inoculantes Agrícolas/genética , Inoculantes Agrícolas/crescimento & desenvolvimento , Bradyrhizobium/classificação , Bradyrhizobium/genética , Bradyrhizobium/crescimento & desenvolvimento , Brasil , Inocuidade dos Alimentos , Controle de Qualidade , Glycine max/químicaRESUMO
Harnessing the beneficial potential of plant growth-promoting rhizobacteria may be an alternative strategy to improve plant tolerance to drought stress. The effect of inoculation with Bradyrhizobium japonicum and Azospirillum brasilense either alone or in combination on the plant growth and drought tolerance of soybean [Glycine max (L.) Merrill.] was investigated in this study in greenhouse conditions. Treatments were arranged in a randomized block design in a 3 × 4 factorial: three irrigation regimes [100% of pot capacity-PC (well-watered control), 50% of PC (moderate stress) and 25% of PC (severe stress)] and four inoculation treatments [control (non-inoculated), inoculation with B. japonicum, inoculation with A. brasilense, and co-inoculation with B. japonicum and A. brasilense]. Leaf relative water content, cell membrane stability, root nodulation, plant growth, and morphophysiological indexes were recorded. The inoculation of soybean plants with B. japonicum and A.brasilense either alone or in combination improved leaf membrane stability under drought stress conditions when compared to non-inoculated plants; however, this lower damage to cell membranes was not sufficient to maintain the leaf water content of the plant under drought stress. Plants co-inoculated with B. japonicum and A.brasilense improved the root nodulation under severe drought conditions. Inoculation of B. japonicum and A. brasilense either alone or in combination reduced the pod abortion rate under moderate drought stress, but had no effect under severe drought stress. In summary, the co-inoculation of A. brasilense and B. japonicum alleviate adverse effects limited by drought stress to the growth of soybeans.Author: Please check and confirm that the authors [Elijanara Raissa Silva, Carlos Eduardo Silva Oliveira, Alan Mario Zuffo, Eduardo Pradi Vendruscolo] and their initials have been correctly identified and amend if necessary.The authors were correctly identified.
Assuntos
Azospirillum brasilense/fisiologia , Bradyrhizobium/fisiologia , Secas , Glycine max/crescimento & desenvolvimento , Glycine max/microbiologia , Estresse Fisiológico/fisiologia , Azospirillum brasilense/crescimento & desenvolvimento , Bradyrhizobium/crescimento & desenvolvimento , Folhas de Planta/microbiologia , Nodulação/fisiologia , Raízes de Plantas/microbiologia , Água/metabolismoRESUMO
Quorum-sensing (QS) mechanisms are important in intra- and inter-specific communication among bacteria. We investigated QS mechanisms in Bradyrhizobium japonicum strain CPAC 15 and Azospirillum brasilense strains Ab-V5 and Ab-V6, used in commercial co-inoculants for the soybean crop in Brazil. A transconjugant of CPAC 15-QS with partial inactivation of N-acyl-homoserine lactones (AHLs) was obtained and several parameters were evaluated; in vitro, CPAC 15 and the transconjugant differed in growth, but not in biofilm formation, and no differences were observed in the symbiotic performance in vivo. The genome of CPAC 15 carries functional luxI and luxR genes and low amounts of three AHL molecules were detected: 3-OH-C12-AHL, 3-OH-C14-AHL, and 3-oxo-C14-AHL. Multiple copies of luxR-like genes, but not of luxI are present in the genomes of Ab-V5 and Ab-V6, and differences in gene expression were observed when the strains were co-cultured with B. japonicum; we may infer that the luxR-genes of A. brasilense may perceive the AHL molecules of B. japonicum. Soybean symbiotic performance was improved especially by co-inoculation with Ab-V6, which, contrarily to Ab-V5, did not respond to the AHLs of CPAC 15. We concluded that A. brasilense Ab-V5, but not Ab-V6, responded to the QS signals of CPAC 15, and that the synergistic interaction may be credited, at least partially, to the QS interaction. In addition, we confirmed inter- and intra-species QS communication between B. japonicum and A. brasilense and, for Azospirillum, at the strain level, impacting several steps of the symbiosis, from cell growth to plant nodulation and growth.
Assuntos
Acil-Butirolactonas/metabolismo , Azospirillum brasilense/fisiologia , Bradyrhizobium/fisiologia , Glycine max/microbiologia , Interações Microbianas/fisiologia , Percepção de Quorum/fisiologia , Simbiose/fisiologia , Azospirillum brasilense/genética , Azospirillum brasilense/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Bradyrhizobium/genética , Bradyrhizobium/crescimento & desenvolvimento , Brasil , Técnicas de Cocultura , Regulação Bacteriana da Expressão Gênica , Percepção de Quorum/genética , Proteínas Repressoras/genética , Simbiose/genética , Transativadores/genética , Fatores de Transcrição/genéticaRESUMO
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)
Assuntos
Glycine max/microbiologia , Bradyrhizobium/crescimento & desenvolvimento , SimbioseRESUMO
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)
Assuntos
Glycine max/microbiologia , Bradyrhizobium/crescimento & desenvolvimento , SimbioseRESUMO
Soybean is an economically very important crop throughout the word and particularly in Argentina. Soybean yield may be affected by many factors such as the lack of some essential nutrients or pathogens attack. In this work we demonstrated that the co-inoculation of the native biocontrol bacterium Bacillus sp. CHEP5 which induces resistance against Cercospora sojina in soybean and the nitrogen fixing strain Bradyrhizobium japonicum E109, was more effective in reducing frog leaf spot severity than the inoculation of the biocontrol agent alone. Probably, this is related with the increase in the ability to form biofilm when both bacteria are growing together. Furthermore, Bacillus sp. CHEP5 inoculation did not affect Bradyrhizobium japonicum E109 symbiotic behavior and flavonoids composition of root exudates in pathogen challenged plants. These results suggest that co-inoculation of plants with rhizobia and biocontrol agents could be a strategy to improve soybean production in a sustainable system.
Assuntos
Ascomicetos/patogenicidade , Bacillus/crescimento & desenvolvimento , Agentes de Controle Biológico , Bradyrhizobium/crescimento & desenvolvimento , Glycine max/microbiologia , Fungos Mitospóricos/metabolismo , Doenças das Plantas/prevenção & controle , Raízes de Plantas/microbiologia , Bacillus/classificação , Doenças das Plantas/microbiologia , SimbioseRESUMO
Synergism between beneficial rhizobacteria and fungal pathogens is poorly understood. Therefore, evaluation of co-inoculation of bacteria that promote plant growth by different mechanisms in pathogen challenged plants would contribute to increase the knowledge about how plants manage interactions with different microorganisms. The goals of this work were a) to elucidate, in greenhouse experiments, the effect of co-inoculation of peanut with Bradyrhizobium sp. SEMIA6144 and the biocontrol agent Bacillus sp. CHEP5 on growth and symbiotic performance of Sclerotium rolfsii challenged plants, and b) to evaluate field performance of these bacteria in co-inoculated peanut plants. The capacity of Bacillus sp. CHEP5 to induce systemic resistance against S. rolfsii was not affected by the inoculation of Bradyrhizobium sp. SEMIA6144. This microsymbiont, protected peanut plants from the S. rolfsii detrimental effect, reducing the stem wilt incidence. However, disease incidence in plants inoculated with the isogenic mutant Bradyrhizobium sp. SEMIA6144 V2 (unable to produce Nod factors) was as high as in pathogen challenged plants. Therefore, Bradyrhizobium sp. SEMIA6144 Nod factors play a role in the systemic resistance against S. rolfsii. Bacillus sp. CHEP5 enhanced Bradyrhizobium sp. SEMIA6144 root surface colonization and improved its symbiotic behavior, even in S. rolfsii challenged plants. Results of field trials confirmed the Bacillus sp. CHEP5 ability to protect against fungal pathogens and to improve the yield of extra-large peanut seeds from 2.15% (in Río Cuarto) to 16.69% (in Las Vertientes), indicating that co-inoculation of beneficial rhizobacteria could be a useful strategy for the peanut production under sustainable agriculture system.
Assuntos
Arachis/microbiologia , Bacillus/fisiologia , Bradyrhizobium/fisiologia , Fungos/patogenicidade , Doenças das Plantas/microbiologia , Arachis/crescimento & desenvolvimento , Arachis/imunologia , Arachis/metabolismo , Ascomicetos/patogenicidade , Ascomicetos/fisiologia , Bacillus/genética , Bacillus/crescimento & desenvolvimento , Bacillus/isolamento & purificação , Biofilmes/crescimento & desenvolvimento , Agentes de Controle Biológico , Bradyrhizobium/genética , Bradyrhizobium/crescimento & desenvolvimento , Resistência à Doença , Fungos/fisiologia , Interações Hospedeiro-Parasita , Imunidade Inata , Doenças das Plantas/prevenção & controle , Microbiologia do Solo , SimbioseRESUMO
Bradyrhizobium diazoefficiens, a nitrogen-fixing endosymbiont of soybeans, is a model strain for studying rhizobial denitrification. This bacterium can also use nitrate as the sole nitrogen (N) source during aerobic growth by inducing an assimilatory nitrate reductase encoded by nasC located within the narK-bjgb-flp-nasC operon along with a nitrite reductase encoded by nirA at a different chromosomal locus. The global nitrogen two-component regulatory system NtrBC has been reported to coordinate the expression of key enzymes in nitrogen metabolism in several bacteria. In this study, we demonstrate that disruption of ntrC caused a growth defect in B. diazoefficiens cells in the presence of nitrate or nitrite as the sole N source and a decreased activity of the nitrate and nitrite reductase enzymes. Furthermore, the expression of narK-lacZ or nirA-lacZ transcriptional fusions was significantly reduced in the ntrC mutant after incubation under nitrate assimilation conditions. A B. diazoefficiens rpoN 1/2 mutant, lacking both copies of the gene encoding the alternative sigma factor σ54, was also defective in aerobic growth with nitrate as the N source as well as in nitrate and nitrite reductase expression. These results demonstrate that the NtrC regulator is required for expression of the B. diazoefficiens nasC and nirA genes and that the sigma factor RpoN is also involved in this regulation.
Assuntos
Proteínas de Bactérias/genética , Bradyrhizobium/metabolismo , Nitrato Redutase/metabolismo , Nitrito Redutases/metabolismo , Fator sigma/genética , Proteínas de Bactérias/metabolismo , Bradyrhizobium/genética , Bradyrhizobium/crescimento & desenvolvimento , Desnitrificação/fisiologia , Nitrato Redutase/genética , Nitrito Redutases/genética , Glycine max/microbiologiaRESUMO
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.
Assuntos
Inoculantes Agrícolas , Bradyrhizobium/fisiologia , Glycine max/microbiologia , Sementes/microbiologia , Aderência Bacteriana , Bradyrhizobium/crescimento & desenvolvimento , Quimiotaxia , Interações Microbianas , Fixação de Nitrogênio , Raízes de Plantas/microbiologia , Microbiologia do Solo , Especificidade da Espécie , SimbioseRESUMO
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
Assuntos
Glycine max/crescimento & desenvolvimento , Glycine max/metabolismo , Bradyrhizobium/crescimento & desenvolvimento , Bradyrhizobium/metabolismo , Inoculantes Agrícolas/metabolismo , Fixação de Nitrogênio , Aderência Bacteriana/fisiologia , Análise de SobrevidaRESUMO
Brazilian sugarcane has been shown to obtain part of its nitrogen via biological nitrogen fixation (BNF). Recent reports, based on the culture independent sequencing of bacterial nifH complementary DNA (cDNA) from sugarcane tissues, have suggested that members of the Bradyrhizobium genus could play a role in sugarcane-associated BNF. Here we report on the isolation of Bradyrhizobium spp. isolates and a few other species from roots of sugarcane cultivar RB867515 by two cultivation strategies: direct isolation on culture media and capture of Bradyrhizobium spp. using the promiscuous legume Vigna unguiculata as trap-plant. Both strategies permitted the isolation of genetically diverse Bradyrhizobium spp. isolates, as concluded from enterobacterial repetitive intergenic consensus polymerase chain reaction (PCR) fingerprinting and 16S ribosomal RNA, nifH and nodC sequence analyses. Several isolates presented nifH phylotypes highly similar to nifH cDNA phylotypes detected in field-grown sugarcane by a culture-independent approach. Four isolates obtained by direct plate cultivation were unable to nodulate V. unguiculata and, based on PCR analysis, lacked a nodC gene homologue. Acetylene reduction assay showed in vitro nitrogenase activity for some Bradyrhizobium spp. isolates, suggesting that these bacteria do not require a nodule environment for BNF. Therefore, this study brings further evidence that Bradyrhizobium spp. may play a role in sugarcane-associated BNF under field conditions.
Assuntos
Bradyrhizobium/classificação , Bradyrhizobium/isolamento & purificação , Endófitos/classificação , Endófitos/isolamento & purificação , Saccharum/microbiologia , Proteínas de Bactérias/genética , Bradyrhizobium/genética , Bradyrhizobium/crescimento & desenvolvimento , Brasil , Análise por Conglomerados , Meios de Cultura/química , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Endófitos/genética , Endófitos/crescimento & desenvolvimento , Técnicas Microbiológicas , Dados de Sequência Molecular , Tipagem Molecular , Nitrogenase/análise , Filogenia , Nodulação , Raízes de Plantas/microbiologia , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
Flagellar-driven bacterial motility is an important trait for colonization of natural environments. Bradyrhizobium japonicum is a soil species that possesses two different flagellar systems: one subpolar and the other lateral, each with a filament formed by a different set of flagellins. While synthesis of subpolar flagellins is constitutive, translation of lateral flagellins was detected in rhizobia grown with l-arabinose, but not with d-mannitol as sole carbon source, independently of whether bacteria were in liquid or semisolid medium. We characterized swarming of B. japonicum in semisolid medium and found that this motility was faster with l-arabinose than with d-mannitol. By using mutants with deletions in each flagellin set, we evaluated the contribution of each flagellum system to swarming in semisolid culture media, and in soil. Mutants devoid of either of the flagella were affected in swarming in culture media, with this impairment being stronger for mutants without lateral flagella. In sterile soil at 100% or 80% field capacity, flagellar-driven motility of mutants able to swim but impaired in swarming was similar to wild type, indicating that swimming was the predominant movement here.
Assuntos
Bradyrhizobium/fisiologia , Locomoção , Arabinose/metabolismo , Bradyrhizobium/crescimento & desenvolvimento , Bradyrhizobium/metabolismo , Meios de Cultura/química , Flagelos/fisiologia , Flagelina/genética , Flagelina/metabolismo , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Manitol/metabolismo , Microbiologia do SoloRESUMO
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.
Assuntos
Sequência de Bases , Bradyrhizobium/crescimento & desenvolvimento , Bradyrhizobium/isolamento & purificação , Fabaceae/crescimento & desenvolvimento , Amplificação de Genes , Técnicas In Vitro , Reação em Cadeia da Polimerase/métodos , Rhizobium/crescimento & desenvolvimento , Rhizobium/isolamento & purificação , MétodosRESUMO
Nitrogen-fixing bacteria of the Bradyrhizobium genus are major symbionts of legume plants in American tropical forests, but little is known about the effects of deforestation and change in land use on their diversity and community structure. Forest clearing is followed by cropping of bean (Phaseolus vulgaris) and maize as intercropped plants in Los Tuxtlas tropical forest of Mexico. The identity of bean-nodulating rhizobia in this area is not known. Using promiscuous trap plants, bradyrhizobia were isolated from soil samples collected in Los Tuxtlas undisturbed forest, and in areas where forest was cleared and land was used as crop fields or as pastures, or where secondary forests were established. Rhizobia were also trapped by using bean plants. Bradyrhizobium strains were classified into genospecies by dnaK sequence analysis supported by recA, glnII and 16S-23S rDNA IGS loci analyses. A total of 29 genospecies were identified, 24 of which did not correspond to any described taxa. A reduction in Bradyrhizobium diversity was observed when forest was turned to crop fields or pastures. Diversity seemed to recover to primary forest levels in secondary forests that derived from abandoned crop fields or pastures. The shifts in diversity were not related to soil characteristics but seemingly to the density of nodulating legumes present at each land use system (LUS). Bradyrhizobium community composition in soils was dependent on land use; however, similarities were observed between crop fields and pastures but not among forest and secondary forest. Most Bradyrhizobium genospecies present in forest were not recovered or become rare in the other LUS. Rhizobium etli was found as the dominant bean-nodulating rhizobia present in crop fields and pastures, and evidence was found that this species was introduced in Los Tuxtlas forest.
Assuntos
Bradyrhizobium/crescimento & desenvolvimento , Bradyrhizobium/genética , Ecossistema , Chuva , Rhizobium etli/crescimento & desenvolvimento , Microbiologia do Solo , Árvores , Clima Tropical , Agricultura , Proteínas de Bactérias/genética , Bradyrhizobium/classificação , Bradyrhizobium/isolamento & purificação , DNA Ribossômico/genética , Agricultura Florestal , México , Filogenia , RNA Ribossômico 16S/genética , Rhizobium etli/classificação , Rhizobium etli/genética , Rhizobium etli/isolamento & purificação , Análise de Sequência de DNA , SimbioseRESUMO
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.
Assuntos
Bradyrhizobium/crescimento & desenvolvimento , Fungos , Fabaceae/crescimento & desenvolvimento , Micorrizas/crescimento & desenvolvimento , Micorrizas/isolamento & purificação , Fixação de Nitrogênio , Valor Nutritivo , Amostras de Alimentos , Métodos , MétodosRESUMO
Bradyrhizobium elkanii SEMIA587 is a symbiotic nitrogen-fixing bacterium of the group commonly called rhizobia, which induce nodule formation in legumes, and is widely used in Brazilian commercial inoculants of soybean. In response to flavonoid compounds released by plant roots, besides Nod factors, other molecular signals are secreted by rhizobia, such as proteins secreted by type III secretion systems (T3SSs). Rhizobial T3SSs are activated by the transcription regulator TtsI, which binds to sequences present in the promoter regions of T3SS genes via a conserved sequence called the tts box. To study the role of the T3SS of B. elkanii SEMIA587, ttsI was mutated. Protein secretion and flavonoid induction analysis, as well as nodulation tests, were performed with the wild-type and mutant strains. The results obtained showed that B. elkanii SEMIA587 secretes at least two proteins (NopA and NopL, known rhizobial T3SS substrates) after genistein induction, whilst supernatants of the ttsI mutant did not contain these Nops. Unusually for rhizobia, the promoter region of the B. elkanii SEMIA587 ttsI gene contains a tts box, which is responsive to flavonoid induction and to which TtsI can bind. Nodulation tests performed with three different leguminous plants showed that the B. elkanii SEMIA587 ttsI mutant displays host-dependent characteristics; in particular, nodulation of two soybean cultivars, Peking and EMBRAPA 48, was more efficient when TtsI of B. elkanii was functional.
Assuntos
Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos , Bradyrhizobium/efeitos dos fármacos , Flavonoides/farmacologia , Regulação Bacteriana da Expressão Gênica , Glycine max/microbiologia , Fatores de Transcrição/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sequência de Bases , Bradyrhizobium/genética , Bradyrhizobium/crescimento & desenvolvimento , Bradyrhizobium/metabolismo , Sequência Conservada , Elementos Facilitadores Genéticos , Flavonoides/metabolismo , Genisteína/metabolismo , Genisteína/farmacologia , Isoflavonas/metabolismo , Isoflavonas/farmacologia , Dados de Sequência Molecular , Mutação , Regiões Promotoras Genéticas , Análise de Sequência de DNA , Glycine max/metabolismo , Simbiose , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
Several bacterial isolates were recovered from surface-sterilized root nodules of Arachis hypogaea L. (peanut) plants growing in soils from Córdoba, Argentina. The 16S rDNA sequences of seven fast-growing strains were obtained and the phylogenetic analysis showed that these isolates belonged to the Phylum Proteobacteria, Class Gammaproteobacteria, and included Pseudomonas spp., Enterobacter spp., and Klebsiella spp. After storage, these strains became unable to induce nodule formation in Arachis hypogaea L. plants, but they enhanced plant yield. When the isolates were co-inoculated with an infective Bradyrhizobium strain, they were even found colonizing pre-formed nodules. Analysis of symbiotic genes showed that the nifH gene was only detected for the Klebsiella-like isolates and the nodC gene could not be amplified by PCR or be detected by Southern blotting in any of the isolates. The results obtained support the idea that these isolates are opportunistic bacteria able to colonize nodules induced by rhizobia.
Assuntos
Arachis/microbiologia , Bradyrhizobium/crescimento & desenvolvimento , Gammaproteobacteria/classificação , Gammaproteobacteria/crescimento & desenvolvimento , Nodulação , Nódulos Radiculares de Plantas/microbiologia , Arachis/crescimento & desenvolvimento , Argentina , Bradyrhizobium/classificação , Bradyrhizobium/genética , Gammaproteobacteria/genética , Dados de Sequência Molecular , Oxirredutases/genética , Filogenia , Análise de Sequência de DNA , Microbiologia do SoloRESUMO
GSH appears to be essential for proper development of the root nodules during the symbiotic association of legume-rhizobia in which the entry of rhizobia involves the formation of infection threads. In the particular case of peanut-rhizobia symbiosis, the entry of rhizobia occurs by the mechanism of infection called 'crack entry', i.e. entry at the point of emergence of lateral roots. We have previously shown the role of GSH content of Bradyrhizobium sp. SEMIA 6144 during the symbiotic association with peanut using a GSH-deficient mutant obtained by disruption of the gshA gene, encoding gamma-glutamylcysteine synthetase (gamma-GCS), which was able to induce nodules in peanut roots without alterations in the symbiotic phenotype. To investigate the role of the peanut GSH content in the symbiosis, the compound L-buthionine-sulfoximine (BSO), a specific inhibitor of gamma-GCS in plants, was used. There were no differences in the plant growth and the typical anatomic structure of the peanut roots when the plants grew in the Fahraeus medium either in presence or in absence of 0.1 mM BSO. However, the GSH content was reduced by 51% after treatment with BSO. The BSO-treated plants inoculated with wild-type or mutant strains of Bradyrhizobium sp. showed a significant reduction in the number and dry weight of nodules, suggesting that GSH content could play an important role in the nodulation process of root peanut with Bradyrhizobium sp.
Assuntos
Arachis/metabolismo , Glutationa/metabolismo , Nodulação/fisiologia , Simbiose/fisiologia , Arachis/efeitos dos fármacos , Arachis/microbiologia , Bradyrhizobium/crescimento & desenvolvimento , Butionina Sulfoximina/farmacologia , Inibidores Enzimáticos/farmacologia , Glutamato-Cisteína Ligase/antagonistas & inibidores , Glutamato-Cisteína Ligase/metabolismo , Glutationa/fisiologia , Nodulação/efeitos dos fármacos , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/metabolismo , Raízes de Plantas/microbiologia , Simbiose/efeitos dos fármacosRESUMO
We previously showed the important role of glutathione (GSH) in the protection mechanism against different stresses, such as acid pH, saline, and oxidative stress, using a GSH-deficient mutant of Bradyrhizobium sp. (peanut microsymbiont). In this work, we studied the role of GSH in the protection mechanism against methylglyoxal (MG) toxicity. MG is a naturally occurring toxic electrophilic compound, and it has been shown that GSH is involved in the detoxification of MG in Escherichia coli. One recognized component of this detoxification process is the formation of a GSH adduct, which in turn transports potassium (K(+)) out of bacterial cells. Our results showed that growth of wild-type strain Bradyrhizobium sp. SEMIA 6144 was not affected at a MG concentration of 0.5 mM in the yeast extract-mannitol culture medium. However, a reduction of growth, at concentrations of 1.5 and 2.5 mM MG and reaching complete growth inhibition at 3.0 mM MG, was observed. In wild-type strain, intracellular GSH content decreased, and intracellular K(+ )content was unchanged, whereas GSH-deficient mutant SEMIA 6144-S7Z was unable to grow at 1.5 mM MG. The addition of external GSH to the incubation medium did not restore the growth rate either in wild-type or mutant strains. Our findings showed that GSH has not proven to be protective against the cell-growth inhibiting activity of MG. Therefore, the response of Bradyrhizobium sp. growth to MG is different from that reported in E. coli and other Gram-negative bacteria.