Design of a 16S rRNA-Directed Oligonucleotide Probe for Bradyrhizobium Tropical Strains

Abstract

The root nodule bacteria of the genera Rhizobium and Bradyrhizobium comprise a diverse group of microorganisms commonly found in tropical soils, where they are mainly represented by potentially new species (Zhang et al. 1991; Moreira et al., 1993). These bacteria may interact with the roots of leguminous plants, inducing the formation of nodules in which the fixation of atmospheric nitrogen occurs (Hirsch, 1992). Considering the ecological importance of these nitrogen-fixers to major ecosystems and their role as potential nitrogen suppliers in sustainable agriculture, it is of great relevance to evaluate and monitor the rhizobia diversity in natural communities. Direct DNA extraction from soil and the use of specific molecular probes and PCR primers provide better tools for assessing microbial diversity in environmental samples, overcoming several limitations of traditional isolation and culturing methods (Pace, 1996). Following this rationale, a 21-mer oligonucleotide probe for Bradyrhizobium tropical strains was designed based on the alignment and comparison of 16S rRNA sequences available in the RDP database (Ribosomal Databases Project). The CHECK_PROBE software, provided by the RDP, was used to confirm the specificity of the potential probes against the complete RDP 16S rRNA dataset. Probe specificity was further evaluated in hybridization experiments using Bradyrhizobium reference strains, other phylogenetically related taxa and Bradyrhizobium strains isolated from trees and shrubs of Brazilian rainforests, cerrado and Amazonia. The oligonucleotide probe was shown to be specific to the soybean nodulating strains of B. elkanii and to the tropical bradyrhizobia isolates used. These results offer a new tool to be used in the detection of tropical Bradyrhizobium strains directly in soil samples and studies of the occurrence and diversity of these organisms in different environments.