Nodules of Medicago spp. Host a Diverse Community of Rhizobial Species in Natural Ecosystems

Biological nitrogen fixation by rhizobia-nodulated legumes reduces the dependence on synthetic nitrogen fertilizers. Identification of locally adapted rhizobia may uncover economically valuable strains for sustainable agriculture. This study investigated the diversity and symbiotic potential of rhizobia associated with three Medicago species from Eastern Romania's ecosystems. Phenotypic screening ensured that only rhizobial species were retained for molecular characterization. 16S rDNA sequencing clustered the isolates into four distinct groups: Sinorhizobium meliloti, Sinorhizobium medicae, Rhizobium leguminosarum, and Mesorhizobium spp. The chromosomal genes (atpD, glnII, recA) and nifH phylogenies were congruent, while the nodA phylogeny grouped the Mesorhizobium spp. isolates with R. leguminosarum. Medicago sativa was the most sampled plant species, and only S. meliloti and R. leguminosarum were found in its nodules, while Medicago falcata nodules hosted S. meliloti and Mesorhizobium spp. Medicago lupulina was the only species that hosted all four identified rhizobial groups, including S. medicae. This study provides the first report on the Mesorhizobium spp. associated with M. falcata nodules. Additionally, R. leguminosarum and two Mesorhizobium genospecies were identified as novel symbionts for Medicago spp. Comparative analysis of Medicago-associated rhizobia from other studies revealed that differences in 16S rDNA sequence type composition were influenced by Medicago species identity rather than geographic region.