Anthyllis vulneraria and Lotus corniculatus on calamine heaps form nodules with Bradyrhizobium liaoningense-related strains harboring novel in Europe symbiotic nifD haplotypes. A Multilocus Sequence Analysis (MLSA) of heavy metal tolerant Bradyrhizobium isolates

Six Bradyrhizobium strains were isolated from root nodules of Anthyllis vulneraria and Lotus corniculatus (Fabaceae) plants that grew on old zinc mine tailings in southern Poland. The isolates formed effective nodules on their original legume hosts in soil substrate containing high concentrations of lead and zinc. Moreover, they exhibited enhanced growth in a liquid medium supplemented with lead or zinc. Phylogenetic affinity of the isolates was assessed following a multilocus sequence analysis (MLSA) approach, based on amplification of portions of dnaK, glnII, gyrB, recA, and rpoB, and symbiotic nifD gene. In the housekeeping gene trees, all heavy metal-resistant bradyrhizobia clustered close to B. liaoningense, a species which until now has not been recorded among Bradyrhizobium symbionts of the Loteae tribe. In the nifD gene tree, the Bradyrhizobium isolates clustered within a group referred to as Clade XVII, encompassing legume symbionts that originate from different geographical areas but also non-nodulating Bradyrhizobium diazotrophs. Thus, this study revealed that in the heavy metal-contaminated soils in southern Poland L. corniculatus and A. vulneraria plants are infected by Bradyrhizobium strains harboring symbiotic nifD gene haplotypes phylogenetically unrelated to Clade II - the dominant group in Europe encompassing bradyrhizobia nodulating Genisteae and Loteae species.