DGGE/TGGE as a tool for detection of 16S rDNA sequence variation in rhizobia and bradyrhizobia population of Northwest of Argentina (NWA

M. VERONCIA LOPEZ; O. MARIO AGUILAR

Sevilla, Spain

Congreso; Fourth European nitrogen fixation conference; 2000

Consejería de agricultura y pesca

  Denaturing Gradient Gel Electrophoresis (DGGE) and Temperature Gradient Gel Electrophoresis (TGGE) had been used in molecular microbial ecology to assess the genetic diversity of total bacterial communities or particular populations and to identify sequence variation in a number of genes from several different organisms. The aim of this work was to investigate the diversity of a collection of rhizobia isolates from NWA and reference strains by using the DGGE technique to resolve 16S rDNA PCR-products. Firstly, the alignment and comparison of 16S rDNA sequences of known species of rhizobia and bradyrhizobia were performed in order to identify the regions with high variability. A region of 260 bp that was described by Young et al was found to be more diverse and therefore exploited to design primers that were similar to those proposed by the authors. A GC-clamp was added in the 5’-end of forward primer. Secondly, we applied these primers in the study of reference strains and found DGGE-patterns that differentiated between related species R. etli and R. leguminosarum and also among others beans nodulating rhizobia such as R. tropici. Likewise, we found that the genus Bradyrhizobium and species of genus Rhizobium showed a significant difference in migration properties. This approach is applied to examine the degree of diversity in the native population of rhizobia isolated from wild beans Phaseolus vulgaris var aborigineus and from Phaseolus augusti found in the NWA. These legumes are nodulated by strains of genus Rhizobium and Bradyrhizobium respectively (Aguilar et al., 1998. App. Environ. Microbiol. 64,3520-3524 and López et al., Proceeding of the 12th International Congress on Nitrogen Fixation, Parana, 1999. p,203). We found that 16S rDNA-products from Rhizobium isolates were resolved by DGGE and some isolates yielded specific migration bands that differ of reference strains. However, results of analysis of most isolates are correlated with the date obtained by applying other approaches such as ARDRA and DNA fingerprinting.