Genotyping Icelandic isolates of rhizobia based on rDNA-RFLP

SUMMARY Simple PCR amplification using universal rhizobial oligonucleotide primers for intergenic spacer (IGS) regions of 16S-23S ribosomal genes (rDNA) was able to discriminate species, biovars and strains of rhizobial isolates. Six isolates of Rhizobium and Bradyrhizobium species from Iceland were analysed in comparison with four isolates from Scandinavia and Siberia. The rhizobia in pure cultures were isolated from nodules of leguminous species in the genera Trifolium, Lathyrus, Lupinus, Astragalus and Thermopsis. The amplified DNA fragments were between 400 and 1400-bp long and all the isolates could be dif- ferentiated except one pair of samples. The discrimination of isolates corresponded reasonably well to the rhizobial classification and was independent of their geographic origin. Further analysis was per- formed by fractionation of the PCR amplified products with restriction endonucleases. Twelve restric- tion enzymes were used, one at a time. Six of these (i.e. EcoRI, HaeIII, MboI, TaqI, PstI and PvuII) were able to identify all isolates except the pair that could not be differentiated by the PCR amplification. This pair was likely to be of the same genotype. The use of single enzyme digestion provided a simple means to verify rhizobial identification, whereas combinations of two or more restriction enzymes, used simul- taneously, maximised resolution of the restriction fragment length polymorphism (RFLP) profiles. The genetic distance analysis based on rDNA-RFLP profiles showed two clusters: one of these included essentially R. leguminosarum bv. trifolii and the other, which was a more diverse group, included other species. The rDNA-RFLP used in this study identified rhizobial species and strains, and detected mis- identification of some isolates.