RFLP analysis of the rRNA operon of a Brazilian collection of bradyrhizobial strains from 33 legume species

Abstract

Genetic diversity in tropical rhizobial species is still poorly known. With the aim of increasing this knowledge, three ribosomal regions of 119 strains belonging to the official Brazilian culture collection of rhizobia and classified as Bradyrhizobium based on morphological and physiological characteristics in vitro were analysed by RFLP-PCR. The strains were isolated from 33 legume species, representing nine tribes and all three subfamilies; they all form very effective N 2 -fixing nodules and 43 of them are recommended for use in Brazilian commercial inoculants as the most effective for their hosts. For the 16S rRNA gene, type and reference strains of Bradyrhizobium japonicum fell into two major clusters, joined at a level of similarity of 50 %, which included 52 strains, 90 % of which were isolated from soybean. Two other clusters, joined at a similarity of 53 %, included reference strains of Bradyrhizobium elkanii, but not USDA 76 T ; furthermore, two other major clusters were identified and all strains were clustered at a final level of similarity of only 28 %. For the intergenic spacer (IGS) between genes coding for the 16S and 23S rRNA, strains were clustered at a final level of similarity of 27 %. Reference strains of B. japonicum fell into a major group with 51 strains, 84 % isolated from soybean, with a similarity of 59 %, while strains of B. elkanii fell into another major group, with a similarity of 55 %, clustering 44 strains, 59 % of which were isolated from hosts other than soybean. New clusters were also observed for the IGS region. The largest number of differences was detected in the analysis of the 23S rRNA gene, and 16 groups and isolated strains were joined at a very low level of similarity (16 %). In a combined analysis with the three ribosomal regions, the majority of strains isolated from soybean clustered with a similarity of 54 % with type and reference strains of B. japonicum, while most strains isolated from Brazilian indigenous legume species grouped with B. elkanii at a level of similarity of 46 %. All strains were clustered at a very low level of similarity (27 %), and at least two new clusters were clearly defined. These new clusters might be related to intraspecific differences or to novel subspecies, or even to novel species; indeed, strains from one of these clusters show higher 16S rRNA gene sequence similarity to members of the genus Burkholderia. The results obtained in this study emphasize the high level of diversity of symbiotic diazotrophic bacteria in the tropics that still remains to be determined. INTRODUCTIONThe Leguminosae (known as the Fabaceae in the USA) is one of the largest families of plants, with over 18 000 species classified into around 650 genera, representing approximately one-twelfth of all known flowering plants and occupying nearly all terrestrial biomes (Polhill & Raven, 1981). Many species within this family are capable of establishing symbioses with a group of bacteria collectively called rhizobia, of which the most important feature is the capacity...

Soybean [Glycine max (L.) Merrill] rhizobial diversity in Brazilian oxisols under various soil, cropping, and inoculation managements

“…Gels were stained with ethidium bromide, visualized under UV radiation, and photographed. As in previous studies by our group (Germano et al 2006), the profiles obtained were confirmed in triplicate.…”

Bradyrhizobium iriomotensesp. nov., Isolated from a Tumor-Like Root of the LegumeEntada koshunensisfrom Iriomote Island in Japan

“…Although many members of mimoseae have been studied for rhizobial symbionts, [15][16][17] this legume, in spite of its availability in the tropics, is unreported in this regard. In the present study, polyphasic analysis, including phenotypic and molecular taxonomic approaches, revealed that the isolated strain represents a novel species of the genus Bradyrhizobium, for which we propose the name Bradyrhizobium iriomotense sp.…”

Caracterização da região espaçadora 16-23S rDNA para diferenciação de estirpes de rizóbios utilizadas na produção de inoculantes comerciais no Brasil

“…(EARDLY, et al, 2005;RASHID et al, 2009). Pela análise do perfil de restrição individual com cada enzima de restrição, existe um indicativo de que o maior poder discriminatório poderia ser atribuído à Dde I. Contudo, conforme também salientado em outros estudos (GERMANO et al, 2006), o posicionamento taxonômico correto só é possível com a análise de várias enzimas de restrição. Recomenda-se, portanto, o uso de mais de uma endonuclease de restrição para a caracterização das estirpes de rizóbios.…”

“…Dados de sequências da região espaçadora entre os genes 16S-23S rDNA têm se acumulado nos últimos anos, e o alinhamento de sequências disponíveis em bancos de dados mostra que pode ocorrer variação considerável intra e infra-específica, tanto no tamanho como na sequência de bases (LAGUERRE, et al, 1996;CHUEIRE, et al, 2003;GERMANO, et al, 2006), evidenciando que esta região pode ser útil na diferenciação de estirpes de rizóbios recomendadas para produção de inoculantes.…”