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Abstract

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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 (108 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 103 to 107 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.

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Figure legend:

Fig 3. Detection of VBNC cells of Clavibacter michiganensis subsp. michiganensis (Cmm) by PMAxx-qPCR and flow cytometry (FCM). Picture credit: (doi: 10.1371/journal.pone.0196525)

Viable and non-culturable (VBNC) cells of Cmm was induced by 50 μM CuSO₄ in 0.85% NaCl. The bacterial cells were collected at 0 h, 3 h, 1 d, 3 d, 6 d, 10 d, 15 d, 20 d and 30 d, and used for viability detection by PMAxx-qPCR and flow cytometry method, while the culturability was determined on LB plates. Each data point represents the mean of two biological replicates. * indicates that the means were significantly different (P < 0.05) at the corresponding time point.

Learn more:

In this study, the effectiveness of PMAxx™ q-PCR dye was evaluated through Clavibacter michiganensis subsp. michiganensis (Cmm) detection.  Learn more about PMAxx™ q-PCR dye and other viability PCR dyes & kits.

DOI:

doi: 10.1371/journal.pone.0196525[/vc_column_text][/vc_column][vc_column][/vc_column][/vc_row][vc_row][vc_column][vc_column_text css=”.vc_custom_1598017023330{margin-bottom: 0px !important;}”]

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