Genotypic characterisation of indigenous soybean rhizobia by PCR-RFLP of 16S rDNA, rep-PCR and RAPD analysis

The taxonomy of nitrogen fixing bacteria that form symbiotic associations with leguminous plants has been deeply changed in recent years. The use of very sensitive and accurate molecular methods has enabled the detection of large rhizobial diversity, particularly among natural field populations of these soil bacteria. The aim of the present investigation was to identify and characterise the indigenous soybean rhizobia isolated from different soil types in eastern Croatia that are regularly used for agricultural production. The actual composition and genetic diversity of natural field population was studied by using different PCR fingerprinting methods such as 16S rDNA PCR-RFLP, rep-PCR and RAPD analysis. Eighteen rhizobial strains, isolated from soybean nodules, were characterised and compared with reference and/or type strains representing Bradyrhizobium japonicum, B. elkanii and Sinorhizobium fredii. Cluster analysis of combined RFLP patterns obtained with six restriction endonucleases revealed that all soybean isolates differ significantly from B. elkanii and S. fredii type strains, while they were closely related to B. japonicum type strains. However, a considerable level of genetic diversity was determined among B. japonicum isolates. PCR-RFLP of 16S rDNA clearly showed the existence of two divergent groups among indigenous bradyrhizobia. After identification at the species level, all isolates were further characterised by RAPD and rep-PCR. Both RAPD and rep-PCR generated highly specific and reproducible patterns that enabled accurate strain differentiation. Among B. japonicum strains a high level of diversity was found with these two fingerprinting methods. Dendrograms derived from RAPD, REP and ERIC profiles showed that all indigenous strains could be divided into three main groups. The grouping of strains was consistent with all methods used in this study.