RAPID IDENTIFICATION OF MEDICAGO NODULATING STRAINS BY USING 2 OLIGONUCLEOTIDE PROBES COMPLEMENTARY TO 16S RDNA SEQUENCES

Symbiotic bacteria associated with the Medicago genus are separated in to two closely related species named Sinorhizobium meliloti and Sinorh izobium medicae. To discriminate rapidly between these two bacterial s pecies, two 15-base DNA probes, 16Smfs and 16Smed, were designed from the alignment of 16S rDNA sequences to differentiate S. meliloti from S. medicae. Their specificities were evaluated by dot-blot hybridizati on experiments on 25 reference strains representing 13 species of Rhiz obium and Sinorhizobium, and by comparison with all 16S rDNA sequences available in the GenBank data base. No cross-reaction was found with 16Smed, which was thus considered species specific for S. medicae. By contrast, as expected according to the 16S rDNA sequence alignment, th e labeled 16Smfs probe cross-hybridized with the DNAs of S. meliloti, Sinorhizobium fredii, and Sinorhizobium saheli but not with the DNA of S. medicae. Since S. saheli and S. fredii do not nodulate Medicago, 1 6Smed and 16Smfs can be routinely used to characterize the two Sinorhi zobium species nodulating Medicago from pure cultures or from Medicago root nodules. Fifty strains isolated from eight annual Medicago speci es were then characterized by using colony hybridizations. Sinorhizobi um meliloti was more frequently obtained (>80% isolates) than was S. m edicae. Both Sinorhizobium species seemed to be trapped by annual Medi cago and no plant-host specificity was detected.