ABSTRACT
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.
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.
Subject(s)
Bacteria/isolation & purification , Oryza/microbiology , Brazil , Bacteria/classification , Bacteria/genetics , Polymerase Chain ReactionABSTRACT
The study aimed to evaluate the contribution of biological nitrogen fixation (BNF), promoted by rhizobium strains, to development and grain yield of cowpea in Roraima. In the years 2005 and 2006 experiments were performed in cerrado and mata alterada areas, where were tested the strains INPA 03-11B, UFLA 3-84, BR3267 (recommended to the cowpea), the strains BR3299 and BR3262, two mineral nitrogen doses (50 and 80 kg ha-1 of N) and a control. The analyzed variables were: nodulation and cowpea plants dry matter production, and the grain yield in the harvest. It was observed, in the mean, that BR3262 strain provided a number and nodule dry mass significantly larger than the control, while among the recommended strains, this only occurred in a sporadic form with INPA 03-11B and BR3267. Besides, it was also observed that soil rhizobium population was determinant to plants nodulation in the experiments. Comparatively to the other strains, BR3262 together with BR3267, provided superior effectiveness in BNF to plant dry mass production. In relation to grain yield, the strain BR3267 and INPA 03-11B presented better resulted compared to UFLA 3-84, however, just the strains BR3262 provided grain yield (in the mean about 1700 kg h-1), equal to 50 kg ha-1 N dose and superior to the control in three of the four experiments performed, showing to be most suitable for cowpea inoculation in Roraima.
O estudo objetivou avaliar a contribuição da fixação biológica de nitrogênio (FBN) promovida por estirpes de rizóbio para o desenvolvimento e rendimento de grãos do feijão-caupi em Roraima. Nos anos de 2005 e 2006 foram conduzidos experimentos em área de cerrado e mata alterada, onde foram testadas as estirpes INPA 03-11B, UFLA 3-84, BR3267 (recomendadas à cultura), a estirpe BR3299 e BR3262, duas doses de nitrogênio mineral (50 e 80 kg ha-1 de N) e um controle. As variáveis avaliadas foram: nodulação e produção de massa seca da parte aérea de plantas de feijão-caupi e, o rendimento de grãos na colheita. Na média geral, foi observado que a estirpe BR3262 proporcionou número e massa de nódulos significativamente maiores ao controle, ao passo que entre as estirpes recomendadas, isto só ocorreu de forma esporádica com INPA 03-11B e BR3267. Além disso, também foi observado que a população de rizóbio do solo foi determinante à nodulação das plantas dos experimentos. Comparativamente as demais estirpes, BR3262 juntamente com BR3267, proporcionaram maior efetividade na FBN à produção de massa seca da parte aérea. Em relação à produtividade de grãos, as estirpes BR3267 e INPA 03-11B apresentaram melhores resultadas comparadas a UFLA 3-84, entretanto, apenas a estirpe BR3262 proporcionou rendimento de grãos (na média geral cerca de 1700 kg ha-1) igual à dose de 50 kg ha-1 de N e superior ao controle em três dos quatro experimentos conduzidos, mostrando ser a mais indicada para a inoculação do feijão-caupi em Roraima.