Rhizobia inhabiting nodules and rhizosphere soils of alfalfa: A strong selection of facultative microsymbionts

Alfalfa (Medicago sativa L.) is an important leguminous forage crop, which is hypothesized to select compatible rhizobial partners from rhizosphere to form nitrogen-fixing nodules. Here we investigated the diversity of rhizobia from both nodules and rhizosphere of alfalfa from farmlands and natural ecosystems along the Yarlung Zangbo River valley in the Tibetan Plateau. 581 nodule isolates were characterized using BOX-A1R PCR fingerprinting, sequences of rpoB and nodulation gene nodC. They belong to four rpoB haplotypes of Sinorhizobium meliloti (rpoB-I, 473 isolates; rpoB-II, 95; rpoB-III, 1; rpoB-IV, 10), Sinorhizobium medicae (1 isolate) and Rhizobium sp. (1 isolate). Re-inoculation experiments of the 32 representative strains suggested a great variation in their symbiotic performance on alfalfa. High-throughput sequencing of rpoB for 34 rhizosphere samples uncovered that the predominant rpoB-I in nodules also dominated among fourteen rpoB haplotypes of S. meliloti in rhizosphere soils. In addition to S. meliloti, more than 40 rhizobial species documented as microsymbionts of other legumes were also identified. Although a very low level of nucleotide diversity for S. meliloti was found among sampling sites, multivariate statistical analysis demonstrated a significant differentiation of total rhizobial community between farmlands and natural ecosystems. Moreover, edaphic conditions, especially pH and nitrogen content, were revealed as important deterministic factors shaping the observed beta-diversity of rhizobial community. Taken together, these results demonstrated that the rhizospheric pool of rhizobial germplasms was significantly affected by land tillage compared to that of natural ecosystems, while a predominant population of S. meliloti was selected by alfalfa.