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Clavibacter michiganensis  subsp.  michiganensis  ( Cmm ) is a seed-borne pathogen that causes bacterial canker disease of tomato.  Cmm  is typically detected in tomato seeds using quantitative real-time polymerase chain reaction (qPCR) combined with culture-based isolation. The viable but nonculturable (VBNC) state of  Cmm  may result in the underestimation or false negative detection of the pathogen. In the present study, propidium monoazide (PMA) and its improved structure PMAxx were used to pretreat  Cmm  prior to DNA extraction, followed by qPCR. Both PMA and PMAxx could bind to the chromosomal DNA of dead bacterial cells and therefore block DNA amplification by PCR. This effect, however, does not occur in living bacterial cells, as the chemicals cannot penetrate through the undamaged cell membrane. Both viable and dead  Cmm  cells were treated with PMA and PMAxx at various concentrations. With this treatment, the range of the cell population was determined for effective detection. PMAxx showed a better discrimination effect than PMA on the viable and dead cells of  Cmm  and was therefore used throughout the present study. VBNC cells of  Cmm  (10 8  CFU mL -1 ) was induced by 50 μM copper sulfate, which was detected at different sampling times up to a month by using both PMAxx-qPCR and flow cytometry assays. The optimal PMAxx concentration was 20 μM for detecting membrane-intact  Cmm  cells. High specificity and sensitivity were obtained at  Cmm  concentrations ranging from 10 3  to 10 7  CFU mL -1 . The accurate and robust results of PMAxx-qPCR were confirmed by flow cytometry method to detect viable  Cmm  cells. Furthermore, the PMAxx-qPCR assay was successfully used in detecting VBNC  Cmm  cells in tomato seeds with as few as 10 seeds per set.

Detection of Clavibacter michiganensis subsp. michiganensis in viable but nonculturable state from tomato seed using improved qPCR