Anthyllis vulneraria and Lotus corniculatus on calamine heaps form nodules with Bradyrhizobium liaoningense-related strains harboring novel in Europe symbiotic nifD haplotypes

Abstract

Calamine tailings, a habitat known for high concentrations of zinc and lead, are inhabited by a limited number of plants. The objective of this project was a phylogenetic characterization of Bradyrhizobium strains that form effective symbiosis with Anthyllis vulneraria L. and Lotus corniculatus L. (Fabaceae) spontaneously growing on old calamine tailings in Southern Poland using a multilocus sequence analysis (MLSA) approach based on amplification of selected housekeeping dnaK, glnII, gyrB, recA, and rpoB, and symbiotic nifD genes. The isolates were also tested for their metal tolerance and nitrogenase activity. The analyses have shown enhanced growth of Bradyrhizobium strains in the presence of lead (Pb) or zinc (Zn), unlike uninoculated plants which died within three weeks. In 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 Loteae tribe. In nifD gene tree, Bradyrhizobium isolates clustered within a group referred to as Clade XVII, encompassing Fabaceae symbionts that originate from different geographical areas but also non-nodulating Bradyrhizobium diazotrophs. Summing up, this study revealed that in these heavy metal-contaminated soils L. corniculatus and A. vulneraria are infected by Bradyrhizobium strains harboring symbiotic nifD gene haplotypes phylogenetically unrelated to Clade II strains that predominate in Europe among bradyrhizobia nodulating Genisteae and Loteae spp.