Genetic diversity of microsymbionts nodulating Trifolium pratense in subpolar and temperate climate regions

Rhizobia are soil-borne bacteria forming symbiotic associations with legumes and fixing atmospheric dinitrogen. The nitrogen-fixation potential depends on the type of host plants and microsymbionts as well as environmental factors that affect the distribution of rhizobia. In this study, we compared genetic diversity of bacteria isolated from root nodules of Trifolium pratense grown in two geographical regions (Tromsø, Norway and Lublin, Poland) located in distinct climatic (subpolar and temperate) zones. To characterize these isolates genetically, three PCR-based techniques (ERIC, BOX, and RFLP of the 16S-23S rRNA intergenic spacer), 16S rRNA sequencing, and multi-locus sequence analysis of chromosomal house-keeping genes (atpD, recA, rpoB, gyrB, and glnII) were done. Our results indicate that a great majority of the isolates are T. pratense microsymbionts belonging to Rhizobium leguminosarum sv. trifolii. A high diversity among these strains was detected. However, a lower diversity within the population derived from the subpolar region in comparison to that of the temperate region was found. Multi-locus sequence analysis showed that a majority of the strains formed distinct clusters characteristic for the individual climatic regions. The subpolar strains belonged to two (A and B) and the temperate strains to three R. leguminosarum genospecies (B, E, and K), respectively.