The influence of the symbiotic plasmid pRL1JI on the distribution of GM rhizobia in soil and crop rhizospheres, and implications for gene flow

The distribution of two genetically modified Rhizobium leguminosarum strains was investigated in the field. One, RSM2004, released in 1987, carries a Tn5 marker on its conjugative symbiotic plasmid (pSym). The second, CT0370, released at the same site in 1994, has a gusA gene integrated into its chromosome but no pSym. Plate counts indicated that the CT0370 population became established at a higher level than RSM2004. However, when peas, alfalfa and barley were grown, RSM2004 was found to outnumber CT0370 on all roots and by 100-fold on pea. Although the transfer of pSym from RSM2004 to CT0370 could be detected on plates and in microcosm studies with high inoculum densities, no transfer was detected in the field. Subsequent transfer of pSym from RSM2004 to CT0370 demonstrated that it conferred an advantage in the rhizosphere. In addition to increasing host fitness, plasmids may transfer, or mobilise other genetic elements, to other bacteria. This is more likely in sites such as the rhizosphere, where cells are active and numbers are high. The distribution of pSym and other genetic elements associated with rhizobia, in bacterial sub-populations from the soil and roots of the different plants, was investigated using PCR. The genetic elements studied were: ISRm3, an insertion element from Sinorhizobium meliloti; pSB102, a broad host range mer plasmid; the Rhizobium nodC gene (carried on pSym) and plasmid replication origins repCI and repCII. As expected, ISRm3 was detected in rhizoflora cultured from alfalfa but not the other plants. The mer gene was ubiquitous but the transfer region of pSB102 was not detected. The nodC and both repC primers amplified products from all the plants, giving further evidence for the occurrence of plasmids originating from Rhizobium in the rhizoflora of non-host plants. Despite the abundance of elements associated with transferable plasmids in rhizobia, none was detected in either inoculant strain.