Identification and differentiation of Clavibacter michiganensis subspeciesby polymerase chain reaction-based techniques

For the identification and differentiation of the five subspecies of Clavib acter michiganensis, two different polymerase chain reaction (PCR)-based te chniques were employed: amplification with subspecies-specific primers and amplification with random primers (RAPD). Based on the sequence data of the intergenic spacer region between 16S and 23S rRNA genes, primers were desi gned for the identification of each subspecies. Using the designed primer p airs it was possible to identify each subspecies of C. michiganensis accord ing to the amplification of a specific DNA fragment. No amplification produ cts were obtained, when bacteria belonging to other genera were submitted t o PCR under the same conditions. RAPD-PCR conditions suitable for the diffe rentiation of C. michiganensis subspecies were developed. RAPD typing was c apable of distinguishing subspecies of C. michiganensis as well as strains within subspecies. Both genomic variation between subspecies and genetic po lymorphisms between bacterial strains were identified as differences in the size and numbers of DNA fragments obtained.