Widespread occurrence of Sinorhizobium meliloti strains with a type IV secretion system

Improving symbiotic nitrogen fixation would reduce reliance on synthetic fertilizers, but establishment of effective bacterial strains is hampered due to competition by indigenous, less effective rhizobia. This study investigated the origins, diversity, and competitiveness of Sinorhizobium meliloti (syn. Ensifer meliloti) strains isolated from root nodules of alfalfa (lucerne; Medicago sativa L.) grown in soils that had not been in alfalfa cultivation for over 30 years. Sets of PCR primers were developed to identify Sinorhizobium spp. and to identify strains with virD₄, the defining gene for a type IV secretion system (T4SS), which has been implicated in increasing strain competitiveness for nodule colonization of M. truncatula, an annual species closely related to alfalfa. A collection of S. meliloti isolates was made from nodules of field-grown plants and from seeds used for establishing field plots that had been inoculated with rhizobia by the manufacturer prior to packaging. Diversity among strains was examined by repetitive element palindromic PCR (rep-PCR) DNA fingerprinting using the BOXA1R primer. Strains originating from the seed inoculant could not be detected in nodules, even in the first year of alfalfa establishment, which were occupied exclusively by indigenous rhizobia originating from soil. Field strains were very diverse within and among field sites, and genetically distinct from inoculant strains. Approximately 33% of field strains were positive for virD₄, a component of the T4SSb gene cluster, which putatively mobilizes effector proteins across the bacterial cell envelop into the host cell. Strains with virD₄, the presence of which suggests that they have a functional T4SS, had more rapid nodulation kinetics than did those lacking virD₄. These results provide additional support for the role of the S. meliloti T4SS in competitiveness for nodule occupancy.