Quantification of viable cells of Clavibacter michiganensis subsp sepedonicus in digester material after heat treatment by TaqMan® BIO-PCR

With bacterial ring rot affected potato lots must be decontaminated under official control without causing a risk for the dissemination of the disease. Therefore, at the Bavarian State Research Center for Agriculture (LFL) in Freising a research project is conducted to investigate, which characteristics (temperature and retention period) during anaerobic digestion must be given to eliminate Clavibacter michiganensis subsp. sepedonicus. Most biogas plants are run at temperatures between 35 and 55 degrees C. Hence, at first a method was developed to study and describe thermosensitivity of the organism in digester material in the relevant temperature range. For this purpose the streptomycin resistant mutant Cms 4053 strep (NCPPB collection, UK) was used. Viable organisms could be exactly quantified after being incubated for ten minutes at different temperatures by combining an enrichment step on growing medium supplemented with streptomycin with real-time PCR (ABI Prism 7000) using TaqMan (R) probes (TaqMan (R) BIO-PCR). It could be shown, that the number of colony forming units (cfu) linearily declines between 35 and 55 degrees C with rising temperatures during equal incubation time periods. Every temperature increase of 8 degrees C reduced the number of cfu by a log(10) unit.