Quantification of viable cells of <i>Clavibacter michiganensis</i> subsp. <i>michiganensis</i> using a DNA binding dye and a real‐time PCR assay

Abstract

Viable cells of Clavibacter michiganensis subsp. michiganensis (CMM), the causal agent of bacterial canker of tomato, were discriminated from the dead cells by quantitative real‐time polymerase chain reaction (PCR), after the bacterial solution was treated with the DNA binding dye ethidium monoazide (EMA). The primers and TaqMan probe, based on the 16S‐23S rDNA spacer sequences, were highly specific for CMM at the subspecies level. The detection limit of the direct real‐time PCR was 10 3 colony forming units per mL (cfu mL −1 ) in samples and with an apparent sensitivity of 2 cfu of target cells in PCR reaction solution. Application of this method allows for selective quantification of viable cells of CMM and facilitates monitoring the pathogen in tomato seeds.