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

Abstract

Characterization of 43 strains of Rhizobium leguminosarum biovars viciae, trifolii, and phaseoli was performed by two methodologies based on PCR amplification, i.e., PCR DNA fingerprinting of interrepeat sequences and restriction fragment length polymorphism (RFLP) analysis of PCR-amplified chromosomal and symbiotic gene regions. Groupings generated by PCR DNA fingerprinting with either extragenic palindromic repetitive primers or two different single random primers were correlated with similar levels of resolution. Although less discriminating, PCR-RFLP analysis of intergenic spacer between genes coding for 16S and 23S rRNA (16S and 23S rDNA) yielded intraspecific polymorphisms. The classification of strains was independent of the biovar status and was in agreement with those obtained by PCR DNA fingerprinting. Intrabiovar variation 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 verified to be universal for rhizobial species by testing of various reference strains, whereas the nod primers designed in this study were biovar or species specific for R. leguminosarum and Rhizobium etli. Classifications of R. leguminosarum strains by the PCR-based methods were correlated with those previously obtained by conventional total DNA restriction profile comparisons and RFLP analysis using chromosomal and symbiotic gene probes. Ranges of discriminating powers were also equivalent between the two approaches. However, the PCR-based methods are much less time-consuming and are therefore more convenient.

Ecology and evolution of bacterial microdiversity

“…trifolii, viciae and phaseoli strains. This grouping disagreed with the clusters obtained using PCR-RFLP of nod and nif genes which were in accordance with the biovar classi¢cation [95,96]. Since nod and nif genes are found on the Sym plasmid which is exchanged between soil microbial populations, and speci¢c interactions may occur between chromosomal and plasmidic markers [97], it is highly probable that the biovar classi¢cation system of R. leguminosarum is based at least partially on features that are subject to gene transfer.…”

Bradyrhizobium elkanii,Bradyrhizobium yuanmingenseandBradyrhizobium japonicumare the main rhizobia associated withVigna unguiculataandVigna radiatain the subtropical region of China

“…Amplified 16S rDNA restriction analysis (ARDRA) and restriction fragment length polymorphism (RFLP) of 16S--23S rRNA gene internal transcribed spacer (ITS) Primers P1 and P6 and DNA extracted from each strain were used to amplify the 16S rRNA gene as described previously (Tan et al, 1997). The PCR products were digested separately with MspI, HinfI, HaeIII and AluI (Laguerre et al, 1996). The ITS fragments were amplified with primers FGPS1490 and FGPL132 0 according to the procedure of Laguerre et al (1996) and digested separately with MspI, HaeIII and AluI.…”

Assessment of core and accessory genetic variation in Rhizobium leguminosarum symbiovar trifolii strains from diverse locations and host plants using PCR-based methods

“…The common chromosomal lineage of the species Rh. leguminosarum and host-specific selection of symbiosis genes in the various symbiovars results in the development of communities containing individual isolates with closely related core genomes but divergent plasmid differing in host specificity and conversely isolates with divergent chromosome but closely related symbiosis plasmids (Laguerre et al 1996;Tian et al 2010;Provorov et al 2012). There is a considerable variation in host specificity within each symbiovar, not all rhizobial isolates that can nodulate a host will be 'effective' (able to fix N), and some will be more efficient than others.…”