Table 1.
Cycle threshold (Ct) of quantitative real-time polymerase chain reaction for the detection of Clavibacter michiganensis subsp. michiganensis (107 CFU mL-1) treated with propidium monoazide (PMA) and PMAxx (an improved PMA).
Fig 1.
Specificity and sensitivity of PMAxx-qPCR assay in detecting viable cells of Clavibacter michiganensis subsp. michiganensis (Cmm).
Viable or heat-killed Cmm cells at various concentrations were treated with 20 μM PMAxx, followed by DNA extraction and qPCR detection. Ct: threshold cycle of qPCR. CFU: colony forming unit. PMA: propidium monoazide. Columns and bars represent mean values and standard deviations. Means followed by different letters are significantly different (P < 0.05).
Fig 2.
Correlation between bacterial population on agar plates and threshold cycle (Ct) of PMAxx-qPCR for culturable Clavibacter michiganensis subsp. michiganensis (Cmm).
The final concentration of PMAxx used in bacterial cells treatment was 20 μM. The X-axis indicates the Ct value of PMAxx-qPCR and the Y-axis indicates the lg CFU mL-1 of bacterial cells.
Fig 3.
Detection of VBNC cells of Clavibacter michiganensis subsp. michiganensis (Cmm) by PMAxx-qPCR and flow cytometry (FCM).
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.
Fig 4.
Detection of culturable, viable but non-culturable (VBNC) and dead cells of Clavibacter michiganensis subsp. michiganensis (Cmm) by PMAxx-qPCR from artificially inoculated tomato seed.
Ten tomato seeds were soaked in log phase (A), copper-induced VBNC (B) and heat-killed (C) Cmm cells suspension (108 CFU mL-1) by vacuum infiltration. After inoculation, the seed was broken by a ball mill and diluted 20-fold with 0.85% NaCl solution, followed by treatment with or without PMAxx at a final concentration of 20 μM. DNA was extracted after PMAxx treatment and used for qPCR assay. Cycle threshold (Ct) of qPCR was separated using multiple range test, and means labeled with different letters were significantly different (P < 0.05).