Direct amplification of nodD from community DNA reveals the genetic diversity of Rhizobium leguminosarum in soil

Sequences of nodD, a gene found only in rhizobia, were amplified from total community DNA isolated from a pasture soil. The polymerase chain reaction (PCR) primers used, Y5 and Y6, match nodD from Rhizobium leguminosarum biov ar trifolii, R. leguminosarum biovar viciae and Sinorhizobium meliloti. The PCR product was cloned and yielded 68 clones that were identified by restr iction pattern as derived from biovar trifolii [11 restriction fragment len gth polymorphism (RFLP) types] and 15 clones identified as viciae (seven RF LP types). These identifications were confirmed by sequencing. There were n o clones related to S. meliloti nodD. For comparison, 122 strains were isol ated from nodules of white clover (Trifolium repens) growing at the field s ite, and 134 from nodules on trap plants of T. repens inoculated with the s oil. The nodule isolates were of four nodD RFLP types, with 77% being of a single type. All four of these patterns were also found among the clones fr om soil DNA, and the same type was the most abundant, although it made up o nly 34% of the trifolii-like clones. We conclude that clover selects specif ic genotypes from the available soil population, and that R. leguminosarum biovar trifolii was approximately five times more abundant than biovar vici ae in this pasture soil, whereas S. meliloti was rare.