TYPING OF RHIZOBIA BY PCR DNA-FINGERPRINTING AND PCR-RESTRICTION FRAGMENT LENGTH POLYMORPHISM ANALYSIS OF CHROMOSOMAL AND SYMBIOTIC GENE REGIONS - APPLICATION TO RHIZOBIUM-LEGUMINOSARUM AND ITS DIFFERENT BIOVARS

Characterization of 43 strains of Rhizobium leguminosarum biovars vici ae, trifolii, and phaseoli was performed by two methodologies based on PCR amplification, i.e., PCR DNA fingerprinting of interrepeat sequen ces and restriction fragment length polymorphism (RFLP) analysis of PC R-amplified chromosomal and symbiotic gene regions, Groupings generate d by PCR DNA fingerprinting with either extragenic palindromic repetit ive primers or two different single random primers were correlated wit h similar levels of resolution, Although less discriminating, PCR-RFLP analysis of intergenic spacer between genes coding for 16S and 23S rR NA (16S and 23S rDNA) yielded intraspecific polymorphisms. The classif ication of strains was independent of the biovar status and was in agr eement with those obtained by PCR DNA fingerprinting. Intrabiovar vari ation within symbiotic gene regions was detected by PCR-RFLP analysis of nifDK and nodD gene regions, but the strains were grouped according to their biovar, The rDNA intergenic spacer and nif primers were veri fied to be universal for rhizobial species by testing of various refer ence strains, whereas the nod primers designed in this study were biov ar or species specific for R. leguminosarum and Rhizobium etli. Classi fications of R. leguminosarum strains by the PCR-based methods were co rrelated with those previously obtained by conventional total DNA rest riction profile comparisons and RFLP analysis using chromosomal and sy mbiotic gene probes, Ranges of discriminating powers were also equival ent between the two approaches, However, the PCR-based methods are muc h less time-consuming and are therefore more convenient.