Plasmid Transfer from RSM2004 to CT0370

For filter crosses, parents were grown in liquid TY to mid-late log-phase. Then, 100 ^l of each culture were placed on a membrane filter and incubated overnight at 28°C on TY

plates. The mixed growth was suspended in 1 ml H2O by vortexing and serial dilutions were plated onto selective media. Sterile field soil was prepared by drying and milling, passing it through a 5 mm sieve and autoclaving it three times at 121°C for 1 hr with 24 hr intervals between sterilizations. Nonsterile conjugations were performed in fresh field soil (7.5% w/w H2O) previously passed through a 5mm sieve. Samples from late log phase cultures of CT0370 and RSM2004 were mixed, centrifuged, and resuspended in 500 |l/H2O. Then, 5 g of soil were added, the mixture was vortexed and incubated for 4 days at 28°C. Samples of parental cultures, and of soil after 2 and 4 days (resuspended as described for field soil) were diluted and plated onto selective media. To determine how plasmid transfer rate was affected by the presence of pea plants, and of introducing inoculant in peat granules, field soil in pots was mixed with peat inoculant to give RSM2004 and CT0370 levels of approximately 106 cfu g-1 soil, and peas were planted. Control pots contained the parents alone or strain CT0370 carrying pSym2004. Samples of the inoculant granules and of rhizo-sphere soil after one and two weeks were resuspended and plated as described for field samples. Root nodules were screened for GUS activity as described previously.

The highest transfer frequency of the pSym plasmid from RSM2004 to CT0370, i.e., 9 x 10-5 transconjugants per recipient, was observed in laboratory matings on filters. In sterile soil microcosms with 106 to 107 cfu of each parent strain per g soil, the highest frequency was 2.6 x 10-6 transconjugants per recipient cell; in nonsterile soil it was 2.3 x 10-7. In pot experiments with peat inoculant containing both parents, providing 5 x 106 cfu g-1 soil, no transconjugants were found, indicating that in field soil with only 102-103 RSM2004 and 104-105 CT0370 cfu g-1 soil, the frequency would most likely be too low to detect.

CT0370 colonies reisolated from soil were screened for the acquisition of other plas-mids from the soil population by subjecting them to PCR with primers designed to amplify two rhizobial plasmid replication origins identified in the field population.16,17 More than 1000 colonies were screened, pooled in groups of 10 for DNA extraction and tested by PCR. No positive results were obtained, although control reactions in which one colony of a positive field isolate was included did give the expected band.

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