Characterization of rhizobia isolates obtained from nodules of wild genotypes of common bean

Abstract This study aimed to evaluate the tolerance to salinity and temperature, the genetic diversity and the symbiotic efficiency of rhizobia isolates obtained from wild genotypes of common bean cultivated in soil samples from the States of Goiás, Minas Gerais and Paraná. The isolates were subjected to different NaCl concentrations (0%, 1%, 2%, 4% and 6%) at different temperatures (28 °C, 33 °C, 38 °C, 43 °C and 48 °C). Genotypic characterization was performed based on BOX-PCR, REP-PCR markers and 16S rRNA sequencing. An evaluation of symbiotic efficiency was carried out under greenhouse conditions in autoclaved Leonard jars. Among 98 isolates about 45% of them and Rhizobium freirei PRF81 showed a high tolerance to temperature, while 24 isolates and Rhizobium tropici CIAT899 were able to use all of the carbon sources studied. Clustering analysis based on the ability to use carbon sources and on the tolerance to salinity and temperature grouped 49 isolates, R. tropici CIAT899 and R. tropici H12 with a similarity level of 76%. Based on genotypic characterization, 65% of the isolates showed an approximately 66% similarity with R. tropici CIAT899 and R. tropici H12. About 20% of the isolates showed symbiotic efficiency similar to or better than the best Rhizobium reference strain (R. tropici CIAT899). Phylogenetic analysis of the 16S rRNA revealed that two efficient isolates (ALSG5A1 and JPrG6A8) belong to the group of strains used as commercial inoculant for common bean in Brazil and must be assayed in field experiments.

Developing novel bacterial based bioformulation having PGPR properties for enhanced production of agricultural crops.

Plant growth promoting rhizobacteria (PGPR) are beneficial rhizobacteria which enhance plant growth as well as the productivity by a variety of mechanisms. PGPR were isolated from the rhizosphere region of som plants (Machilus bombycina King) maintained at the Central Muga Eri Research and Training Institute, Lahdoigarh, Jorhat. A bacterial based bioformulation was prepared and sprayed over the experimental crops including tomato (Solanum lycopersicum), cauliflower (Brassica oleracea var botrytis), chili (Capsicum annuum) and brinjal (Solanum melongena). Biochemical analysis was done on these PGPR treated crops as well as the untreated crops. The bioformulations prepared from Bacillus cereus (MTCC 8297), Pseudomonas rhodesiae (MTCC 8299) and Pseudomonas rhodesiae (MTCC 8300) was found to be the most effective in increasing the shoot height, number of leaves, early fruiting and total biomass content of the plants after treatment.

Efectividad de cepas rizobianas de frijolbajo diferentes regímenes de fósforo / Effectiveness of cowpea rhizobial strains under different phosphorus regimes

En Venezuela, el frijol representa una alternativa a la proteína animal, debido a su alto consumo y valor nutritivo, por ello se ha estimulado la implementación de programas para reactivar la economía de los pequeños y medianos productores, a fin de incrementar su producción y así tener mayor disponibilidad de proteína de alta calidad a bajo costo; de manera que, los estudios encaminados a mejorar su cultivo, son acertados. Se evaluó la efectividad de cepas rizobianas de crecimiento lento (cl) y rápido (cr) en frijol (Vigna unguiculata (L.) Walp.) cultivar TC9-6 en varios regímenes de fósforo (0, 20, 40 y 80 kgP2O5 ha-1), con un diseño experimental de bloques al azar con arreglo factorial. Las plantas se cultivaron en 4 kg de suelo de sabana 45 días y las cepas en caldo de levadura y manitol: 5 (cr: JV91) y 10 (cl: JV94) días. La inoculación (2 ml cada vez) fue aplicada a la siembra y 6 días más tarde. La utilización de fósforo (40-80 kgP2O5 ha-1) incrementó la nodulación (número, peso seco total e individual de nódulos) y favoreció la aparición de nódulos rojos; así mismo, acrecentó el peso de la materia seca, la altura, el número de hojas y la concentración de nitrógeno del vástago. Los valores fueron similares con ambos tipos de cepas (efectividad similar) y para las dos concentraciones (40-80 kgP2O5 ha-1), con las menores estimaciones para 0 y 20 kgP2O5 ha-1. De acuerdo con los resultados las concentraciones de 40 y 80 kgP2O5 ha-1 fueron las más favorables para el crecimiento y la nodulación de frijol.

In Venezuela, cowpea is an alternative to animal protein due to its high consumption and nutritious value, so it has stimulated the implementation of programs to reactivate the small and medium producers economy, in order to increase its production and to have major high quality protein availability at low cost; so that, the studies carry on to improve its cultivation, are well-aimed. The effectiveness of slow (sg) and fast (fg) growing rhizobial strains was evaluated in cowpea (Vigna unguiculata (L.) Walp) cultivar TC9-6 at various phosphorus regimes (0, 20, 40 and 80 kgP2O5 ha-1): randomized block design with factorial arrangement. Plants were cultivated in 4 kg savannah soil: 45 days, and the strains in yeast and mannitol broth: 5 (fg: JV91) and 10 (sg: JV94) days. The inoculation (2 ml each time) was applied at sowing time and 6 days later. Phosphorus utilization (40-80 kgP2O5 ha-1) increased nodulation (nodule number, total and individual dry weight) and favoured nodule red colour appearance; also, incremented shoot dry matter weight, height, leaves number and nitrogen concentration. Values were similar with both strain types (similar effectiveness) and to the two doses (40-80 kgP2O5 ha-1), with lower estimations to 0 and 20 kgP2O5 ha-1. Accordingly with the results, the doses of 40 and 80 kgP2O5 ha-1 were the most favourable to cowpea growth and nodulation.

Caracterización fenotípica y genotípica de doce rizobios aislados de diversas regiones geográficas de Venezuela

Phenotypic and genotypic characterization of twelve rhizobial isolates from different regions of Venezuela. Rhizobial taxonomy and systematics have progressed substantially, nevertheless, few studies have been developed on venezuelan species. This study evaluated the phenotypic and genetic variation between 12 venezuelan indigenous rhizobial isolates and 10 international referential strains, by phenotypical traits and DNA molecular markers. In this regard, a PCR-RFLP of the 16S rDNA gene, the presence of large plasmids, metabolic assays in solid media, salinity resistance, pH and temperature growth conditions, and intrinsic antibiotic resistance were assayed. In reference to the phenotypic attributes, we recognized three main groups: A group I, which comprised all the strains metabolizing between 67.5%-90% of the C and N sources. They were also acid-tolerant, as well as acid producers, capable of growing at 40ºC and in high salinity conditions (2-2.5% NaCl). With regard to the antibiotic sensitivity, this group was susceptible to a 30% of the antibiotic assayed. Strains belonging to Group II exhibited a lower salt tolerance (0.1-1.5%NaCl), as well as a lower acid tolerance, since they grew well at pH values equal or higher than 5.0. This group appeared to be resistant to all of the antibiotics assayed and only metabolized between 52.5%-82.5% of the C and N sources. Group III was represented by a single bacterial strain: it has a extremely low salt tolerance (0.1% NaCl). This strain grew at a pH equal or higher than 5.6, was susceptible to 50% of the antibiotics assayed and metabolized 72% of the C and N sources. On the basis of a PCR- RFLP of the 16S rDNA, three groups were also obtained. Members of the group A showed a close resemblance to Rhizobium tropici CIAT 899 and Sinorhizobium americanum CFN-EI 156, while Group B was closely related to Bradyrhizobium spp. Group C, was also represented by only one isolate. The Trebol isolate, was the only one strain able to form nodules and does not appear to be related to any of the referential rhizobial strains, suggesting a possible symbiotic horizontal gene transfer. Finally, in this work, there are evidences of a genetic diversity in the venezuelan rhizobial strains. A different geographical origin is perhaps an important factor affecting the diversity of the indigenous rhizobia in this study. Rev. Biol. Trop. 59 (3): 1017-1036. Epub 2011 September 01.

Rasgos fenotípicos y marcadores moleculares de ADN se utilizaron para investigar la variación fenotípica y genética entre 12 aislados rizobianos venezolanos y 10 cepas de referencia internacionales. Para ello, se realizó un PCR-RFLP del gen rDNA 16S, la presencia de plásmidos grandes, análisis metabólicos en medios sólidos, resistencia a la salinidad, condiciones del crecimiento a diferentes pH y temperaturas y la resistencia intrínseca a antibióticos. En referencia a las cualidades fenotípicas, se diferenciaron tres grupos principales, un grupo I que abarcó a todas aquellas cepas que metabolizaban entre 67.5% y 90% de las fuentes de C y de N. También eran tolerantes a la acidez y productoras de ácido, capaces de crecer a 40ºC y a altas condiciones de salinidad (NaCl 2-2.5%). Con respecto a la sensibilidad a antibióticos, este grupo era susceptible a un 30% de los antibióticos empleados. Las cepas que pertenecen al grupo II exhibieron una tolerancia salina más baja (0.1- 1.5%NaCl), así como una menor tolerancia a la acidez, puesto que crecieron bien en valores de pH iguales o mayores a 5.0. Este grupo era resistente a todos los antibióticos probados y metabolizaban solamente entre 52.5%-82.5% de las fuentes de C y de N. Una sola cepa bacteriana representó al grupo III, con una baja tolerancia salina (0.1% NaCl). Este aislado creció a un pH mayor o igual a 5.6, era susceptible a 50% de los antibióticos probados y metabolizó el 72% de las fuentes de C y de N. Al tener como base el PCR-RFLP del 16S rDNA, se diferenciaron también tres grupos. Los miembros del grupo A demostraron una estrecha relación con Rhizobium tropici CiAT 899 y Sinorhizobium americanum CFN-Ei156, mientras que el grupo B está estrechamente vinculado a Bradyrhizobium spp. El grupo C, está representado por solo un aislado. El aislado Trebol, fue la única cepa capaz de formar nódulos y no aparece relacionado con ninguna cepa de referencia, y sugiere una transferencia horizontal de genes simbióticos. Finalmente, en este trabajo se evidencia una diversidad genética en las cepas rizobianas venezolanas. El origen geográfico diverso de estas cepas, quizás sea un factor importante que influencie la diversidad de los rizobios indígenas utilizados en este estudio.

Mycelial colonization by bradyrhizobia and azorhizobia.

This study examines mycelial colonization of common soil fungi by bradyrhizobia and an azorhizobial strain, resulting in the forming of biofilms. The effects of the fungal exudates on a bradyrhizobial strain have also been investigated. Bradyrhizobia gradually colonized the mycelia for about 18 days, after which the biofilm structures collapsed with the release of the rhizobial cell clusters to the culture medium. The azorhizobial strain showed differential colonization of the mycelia. In general, there were no considerable mycotoxin effects of the fungal exudates on the bradyrhizobial strain used, instead the rhizobial strain utilized the exudates as a source of nutrition. This study indicates that the present microbial association with biofilm formation has important implications in the survival of rhizobia under adverse soil conditions devoid of vegetation. Further, it could have developed an as yet unidentified nitrogen fixing system that could have contributed to the nitrogen economy of soils.

Serological distribution of Bradyrhizobium japonicum from Brazilian "cerrados" areas under soybean cultivation

Foi conduzido um estudo sobre ocorrência de sorogrupos de Bradyrhizobium japonicum em nódulos de soja cultivar Doko. O experimento foi conduzido em vasos com solos coletados em vários locais dos Cerrados, sendo a soja semeada sem inoculaçäo O sorogrupo 566 foi o dominante em 13, dentre 20 areas de cultivo de soja, que, aparentemente, nunca haviam sido inoculadas com estirpes desse serogrupo. Resultados semelhantes foram encontrados na área experimental do CPAC, onde essa estirpe nunca havia sido inoculada, mas que apresentou plantas de soja com até 94 por cento dos nódulos ocupados por essa estirpe. Em outro estudo, objetivou-se acompanhar as flutuaçöes sazonais da ocorrência dos serogrupos na soja em uma área no campo, através de amostragens mensais de solo que eram distribuidos em vasos e semeados com soja näo inoculada. Apesar de ocorrerem flutuaçöes significativas nas percentagens de ocorrência dos serogrupos, näo houve alteraçöes na ordem de dominância das estirpes durante os meses em que foi conduzido o experimento