RESUMO A simbiose Rhizobium-leguminosa desempenha um relevante papel na agricultura, entretanto não tem recebido suficiente atenção de estudos científicos no Equador. O objetivo deste artigo foi caracterizar a diversidade genética e fenotípica de isolados de Rhizobium associados com Phaseolus vulgaris do sul do Equador. A caracterização morfo-cultural, testes bioquímicos e análises fisiológicas foram realizados para autenticar e determinar a diversidade de isolados de bactérias Rhizobium. A diversidade genética foi determinada por por técnicas moleculares consistindo na extração de DNA genômico bacteriano, amplificação e sequenciamento parcial do gene 16SrRNA; e parâmetros de nodulação e fixação de nitrogênio de P. vulgaris sobre condições de estufa foram testados para determinar a diversidade fenotípica entre os isolados. Além disso, a produção de ácido indolacético foi avaliada por um método colorimétrico. A análise fisiológica da diversidade entre os isolados revelou o potencial de crescimento de diversos isolados em diferentes níveis de pH, salinidade e temperatura. O sequenciamento parcial do gene 16S rRNA mostrou o gênero Rhizobium em todas os locais de amostragem. De um total de 20 sequências alinhadas, 9 espécies de Rhizobium foram identificadas. A formação de nódulos e biomassa, bem como a fixação de nitrogênio mostraram um aumento nos parâmetros fenotípicos das plantas, os quais devem ser influenciados pela produção de IAA, especialmente pelas cepas R. mesoamericanum NAM1 e R. leguminosarum bv. viciae COL6. Estes resultados demonstram a eficiência de cepas diazotróficas simbióticas nativas para produção de inoculantes para leguminosas e fornece informações valiosas e uteis para a agricultura sustentável equatoriana. Neste sentido, este trabalho deve ser um elemento essencial para a realização de futuras pesquisas aplicadas relacionadas a cepas Rhizobium nativas e espalhar o uso de biofertizantes em campos equatorianos.

ABSTRACT Rhizobium-legume symbioses play relevant roles in agriculture but have not been well studied in Ecuador. The aim of this study was to characterize the genetic and phenotypic diversity of Rhizobium isolates associated with Phaseolus vulgaris from southern Ecuador. Morpho-cultural characterization, biochemical tests and physiological analyses were conducted to authenticate and determine the diversity of bacteria Rhizobium-like isolates. The genetic diversity of the isolates was determined by molecular techniques, which consisted of bacteria DNA extraction and amplification and sequencing of the 16S rRNA gene. The nodulation parameters and nitrogen fixation for P. vulgaris under greenhouse conditions were also assessed to determine the phenotypic diversity among isolates. Furthermore, bacteria indole-acetic-acid production was evaluated by the colorimetric method. Morpho-cultural and biochemical characteristic assessments demonstrated that Rhizobium-like bacteria was associated with the P. vulgaris nodules. The diversity among the isolates, as determined by physiological analyses, revealed the potential of several isolates to grow at different pH values, salinity conditions and temperatures. Partial sequencing of the 16S rRNA gene identified the Rhizobium genus in every sampling site. From a total of 20 aligned sequences, nine species of Rhizobium were identified. Nodule formation and biomass, as well as nitrogen fixation, showed an increase in plant phenotypic parameters, which could be influenced by IAA production, especially for the strains R. mesoamericanum NAM1 and R. leguminosarum bv. viciae COL6. These results demonstrated the efficiency of native symbiotic diazotrophic strains inoculants for legume production. This work can serve as the basis for additional studies of native Rhizobium strains and to help spread the use of biofertilizers in Ecuadorian fields.

Inoculation with selected Rhizobium strains of common bean (Phaseolus vulgaris L.) contributes to the agricultural yield of this legume, improves soil fertility and reduces the use of nitrogen fertilizers in sustainable agricultural systems. The research aimed to determine the effect in field of the native strain Rhizobium leguminosarum bv. viciae COL6 on growth, N fixation and agricultural yield in common bean cv. Percal. A field trial was carried out to evaluate the parameters of nodulation, biomass, nitrogen fixation, yield components and agricultural yield in common bean. In addition, soil analyzes were performed before and after experimentation. Prior to planting an inoculant was formulated with the strain COL6. The seeds of cv. Percal were mixed in 250 g inoculant per 10 kg of seed and it was sown manually in the soil. The results of growth, N fixation (total N %) and agricultural yield demonstrated the positive effect of inoculation with the bacterial inoculant comparable with chemical fertilizers. The increase in agricultural yield with bacterial inoculant application and chemical fertilization with respect to control was 62 and 64 %, respectively. The results of the soil analysis were variable with respect to the initial physical-chemical characteristics. The promising results of bacterial inoculant use and the effective stimulation in common bean, open the doors for its potential use in Ecuadorian soils and with that the desired potential yield can be reached.

La inoculación con cepas seleccionadas de Rhizobium sobre frijol común (Phaseolus vulgaris L.) contribuyen al rendimiento agrícola de esta leguminosa, mejoran la fertilidad del suelo y reducen la utilización de fertilizantes nitrogenados en sistemas agrícolas sostenibles. La investigación tuvo como objetivo determinar el efecto en campo de la cepa nativa Rhizobium leguminosarum bv. viciae COL6 sobre el crecimiento, fijación de N y rendimiento agrícola en frijol común cv. Percal. Se realizó un ensayo en campo donde se evaluaron los parámetros de nodulación, biomasa, fijación de nitrógeno, componentes del rendimiento y rendimiento agrícola. Además, se realizaron análisis del suelo antes y después de la experimentación. Antes de la siembra se formuló un inoculante con la cepa bacteriana. Las semillas de cv. Percal se mezclaron en 250 g del inoculante para 10 kg de semilla y se sembraron manualmente en el suelo. Los resultados concernientes al crecimiento, fijación de N (% N total) y rendimiento agrícola, demostraron el efecto positivo de la inoculación con la cepa nativa, comparable con la fertilización química. El aumento en el rendimiento agrícola con la aplicación del inoculante bacteriano y la fertilización química respecto al control fue de 62 y 64 % respectivamente. Los resultados de los análisis de suelo fueron variables en proporción a las características físico-químicas iniciales. Los resultados promisorios del uso del inoculante bacteriano y la estimulación efectiva en frijol común, abren las puertas para su uso potencial en suelos del Ecuador y con ello, permiten que se puedan alcanzar los rendimientos potenciales deseados.

The development of biofertilizers based on Rhizobium strains for common bean (Phaseolus vulgaris L.) requires studies about bacterial interaction with target cultivars. For these reason, the aim of this paper was to determine the effect of the inoculation of Rhizobium native strains, isolated from southern Ecuador, on P. vulgaris cultivar Mantequilla. An assay was performed in greenhouse. It were evaluated the parameters of nodulation, biomass, nitrogen fixation and efficiency of the symbiosis. All inoculated strains were able to nodulate bean seedlings. The total number of nodules, nodular biomass and plant biomass, were favourably affected by inoculation of Rhizobium strains. The best results were obtained with R. mesoamericanum NAM1, R. leguminosarum COL 6 and R. etli PIN 1 strains. The experimental evidences shows the potential of native strains of Rhizobium for it use as biofertilizers because they are able to raise the rates of nitrogen fixation in common bean in southern Ecuador.

El desarrollo de biofertilizantes a base de cepas de Rhizobium para su aplicación en el cultivo de frijol común (Phaseolus vulgaris L.) requiere del estudio previo de la interacción bacteriana con los cultivares de interés. Por ese motivo esta investigación tuvo como objetivo determinar el efecto de la inoculación de cepas nativas de Rhizobium, aisladas del sur de Ecuador, sobre P. vulgaris cultivar Mantequilla. Se realizó un ensayo en casa de cultivo, donde se evaluaron parámetros de nodulación, biomasa, fijación de nitrógeno y la eficiencia de la simbiosis. Todas las cepas inoculadas fueron capaces de nodular las plántulas de frijol. El número de nódulos totales, la biomasa nodular y la biomasa de las plantas, se afectaron favorablemente con la inoculación de diferentes cepas de Rhizobium. Los mejores resultados se obtuvieron al utilizar las cepas R. mesoamericanum NAM1, R. leguminosarum COL6 y R. etli PIN 1. Las evidencias experimentales mostraron el potencial de cepas nativas de Rhizobium para su utilización como biofertilizantes al elevar las tasas de fijación de nitrógeno en frijol común en el sur de Ecuador.

The present work was carried out with the aim to determine the effect of Rhizobium isolates previously characterized and genetically identified on morphological and physiological parameters and the nitrogen fixation of common bean genotypes. The phenotypic analysis of isolates carried out under controlled conditions, where the nodulation, morphological and physiological parameters and the nitrogen fixation of the genotypes ICA Piajo and BAT-304 were evaluated. The results showed that the isolated strains have the ability for abundant nodule formation in roots plant of both genotypes analyzed. The remarkable beneficial effect was for R. etli specie and the isolates from Santa Clara and Camajuaní-1, as well as the isolated of R. pisi from Camajuaní-2. The genotypic variability showed the close positive correlation among these strains and the genotype BAT-304 compared with ICA Pijao.

El presente trabajo se llevó a cabo con el objetivo de determinar el efecto de aislados de Rhizobium previamente caracterizados e identificados genéticamente sobre los parámetros morfológicos, fisiológicos y la fijación de nitrógeno de genotipos de frijol común. El análisis fenotípico de los aislados se condujo en condiciones controladas, evaluándose los parámetros de nodulación, morfológicos, fisiológicos y la fijación de nitrógeno de los genotipos ICA Pijao y BAT-304 de frijol común. Los resultados obtenidos demuestran que las cepas aisladas de Rhizobium tienen habilidad para formar una cantidad abundante de nódulos en las raíces de las plantas de frijol de los genotipos analizados. Se destacó el efecto beneficioso de la especie R. etli y los aislados procedentes de Santa Clara y Camajuaní-1, así como el aislado R. pisi de Camajuaní-2. La variabilidad genotípica demostró la estrecha correlación positiva entre esas cepas y el genotipo BAT-304 en comparación con ICA Pijao.