Generic identity of putative Rhizobial isolates as determined by PCR-RFLP of 165 rDNA and selected Phenotypic properties

Abstract

The taxonomy of root nodule bacteria is in a state of transition, primarily as a result of the increased isolation of rhizobia from legumes not investigated before and the advances made in molecular methods for classification of bacteria (Young, 1996). In order to ensure a stable taxonomy, it is important to include representative South African isolates. The first comprehensive study of indigenous rhizobia was done by Dagutat (1995). More than 300 putative rhizobia were isolated from diverse leguminous hosts and diverse geographic regions, and characterized with sodium dodecyl-sulphate polyacrylamide gel electrophoresis (SOS-PAGE) of whole cell proteins. Although a large group of these isolates showed similarity to previously described genera, possible new taxa were recognized as many isolates did not show any relatedness to authentic rhizobial strains included. The question arose whether these isolates were rhizobia capable of symbiotic nitrogen fixation or merely opportunistic endosymbionts. The aim of this study was to further characterize putative indigenous rhizobia at a genetic level with restriction fragment length polymorphism (RFLP) analysis of PCR-amplified 16S rDNA, and at a phenotypic level with the Biolog system (i.e. oxidation of 95 substrates), and to evaluate these methods as rapid, reliable tools for characterization of new rhizobial isolates. The collection of indigenous rhizobia was also expanded with the isolation of rhizobia from diverse leguminous hosts. Ninety six indigenous isolates and 35 reference strains were first characterized with SOS-PAGE of whole cell proteins, and representative strains were selected for further analysis with PCRRFLP and the Biolog system. Although most of the indigenous isolates investigated were slowgrowers belonging to the genus Bradyrhizobium, some isolates did show close relationship to species of the genera Rhizobium, Sinorhizobium and Mesorhizobium. Characterization of isolates with l 6S rONA PCR-RFLP analysis and the Biolog system showed good agreement with results obtained with SOS-PAGE. Close relationships between strains could be distinguished with SOSPAGE, whereas PCR-RFLP and Biolog analysis were limited in the distinction of closely related strains. PCR-RFLP and Biolog proved to be valuable tools for the differentiation of rhizobia at species and higher level.