Differentiation of Clavibacter michiganensis subsp. sepedonicus using PCR melting profile and variable number of tandem repeat methods.

The potato phytopathogen Clavibacter michiganensis subsp. sepedonicus (Cms) is a causative agent of bacterial ring rot, which is a serious threat to crops. In EU member countries, Cms is subject to quarantine and has to be combated. The knowledge about the transmission of C. michiganensis strains is limited due to a lack of methods which could be used for epidemiological analysis. In this study, PCR melting profile (PCR MP) and variable number tandem repeat methods were used in Cms epidemiological analysis for the first time. PCR MP was based on the melting temperature analysis of BamHI restriction fragments of chromosomal DNA. Respectively, for the variable number tandem repeat (VNTR) method, six loci were identified and used in the differentiation of Cms isolates. PCR MP was used for 93 Cms isolated in Poland. Both PCR MP and VNTR methods were used for the differentiation of 47 Cms strains in this collection. Both these methods were found to be useful for the epidemiological analysis of Cms. SIGNIFICANCE AND IMPACT OF THE STUDY: The potato phytopathogen, Clavibacter michiganensis subsp. sepedonicus (Cms), is a serious threat to crops and lead to significant economic losses. The only way to control and eliminate the disease caused by this pathogen is the use of certified seed potato and strict quarantine of infected fields. Here, for the first time, two molecular typing methods (PCR melting profile (PCR MP) and variable number tandem repeat (VNTR)) were evaluated in respect of their potential in differentiation of Cms isolates. As a result, we obtained characteristic profiles of DNA fragments (PCR MP) and numeric patterns (VNTR), which enable the intraspecies genotyping of Cms strains confirming the effectiveness of PCR MP and VNTR methods in differentiation of Cms strains.