Abstract
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.
Highlights
Bacterial wilt and canker of tomato (Solanum lycopersicum) is caused by the Gram-positive bacterium Clavibacter michiganensis subsp. michiganensis (Cmm) [1,2]
The Cycle threshold (Ct) value of dead cells showed little change when the concentration of propidium monoazide (PMA) and PMAxx was higher than 10 μM, and most values were greater than 34.00, except the values for dead cells treated with 30 μM PMA or 10 μM PMAxx (Table 1)
We have demonstrated an improved and effective strategy to detect Cmm in the viable but nonculturable (VBNC) state, which cannot be otherwise detected by using conventional bio-PCR, and confirmed that the new photoactivatable dye PMAxx had higher sensitivity to differentiate viable and dead cells and less false binding to the DNA of viable cells compared to PMA
Summary
Bacterial wilt and canker of tomato (Solanum lycopersicum) is caused by the Gram-positive bacterium Clavibacter michiganensis subsp. michiganensis (Cmm) [1,2]. Bacterial wilt and canker of tomato (Solanum lycopersicum) is caused by the Gram-positive bacterium Clavibacter michiganensis subsp. Michiganensis (Cmm) [1,2] It is a devastating disease and has caused serious economic losses [3,4]. Disease symptoms include wilting of the whole plant, cankers and necrosis on stems and petioles as well as the reduced quantity and quality of fruit yield [5,6]. Bio-PCR improves the accuracy in which culturable bacteria can grow on selective agar media prior to PCR. This method is currently recommended for detecting Cmm in tomato seed samples [13]. The bio-PCR protocols have been developed by the International Seed Testing Association (ISTA), the European and Mediterranean Plant Protection Organization (EPPO) and other international organizations [4]
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