Identification and Differentiation of Clavibacter michiganensis Subspecies by Polymerase Chain Reaction‐based Techniques

Abstract

For the identification and differentiation of the five subspecies of Clavibacter michiganensis, two different polymerase chain reaction (PCR)‐based techniques 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 designed for the identification of each subspecies. Using the designed primer pairs it was possible to identify each subspecies of C. michiganensis according to the amplification of a specific DNA fragment. No amplification products were obtained, when bacteria belonging to other genera were submitted to PCR under the same conditions. RAPD‐PCR conditions suitable for the differentiation of C. michiganensis subspecies were developed. RAPD typing was capable of distinguishing subspecies of C. michiganensis as well as strains within subspecies. Both genomic variation between subspecies and genetic polymorphisms between bacterial strains were identified as differences in the size and numbers of DNA fragments obtained.