Rapid Detection and Quantification of Viable Cells of Pectobacterium brasiliense Using Propidium Monoazide Combined with Real-Time PCR

Abstract

Pectobacterium brasiliense (Pbr) has caused significant economic losses in major vegetable production areas in Northern China by causing bacterial soft rot in cash crops such as potatoes and cucumbers. This study aimed to establish a PMA-qPCR detection method for Pbr by screening specific and sensitive primers based on the glu gene and the conserved region of the 23S rRNA gene. Based on the optimized PMA pretreatment conditions, a standard curve was designed and constructed for PMA-qPCR detection (y = -3.391x + 36.28; R² = 0.99). The amplification efficiency reached 97%, and the lowest detection limit of viable cells was approximately 2 × 10² CFU·mL^-1. The feasibility of the PMA-qPCR method was confirmed through a manually simulated viable/dead cell assay under various concentrations. The analysis of potato tubers and cucumber seeds revealed that nine naturally collected seed samples contained a range from 10² to 10⁴ CFU·g^-1 viable Pbr bacteria. Furthermore, the system effectively identified changes in the number of pathogenic bacteria in cucumber and potato leaves affected by soft rot throughout the disease period. Overall, the detection and prevention of bacterial soft rot caused by Pbr is crucial.

Keywords: PMA-qPCR; Pectobacterium brasiliense; bacterial soft rot; detection; viable cell.

Figure 1

Alignment indicating the positions of the designed Pectobacterium brasiliense primers and probes on the 16-23S intergenic spacer region of ribosomal RNA and the tRNA-Glu nucleotide sequence in comparison to other Pectobacterium species.

Figure 2

qPCR solubility curve (A) and electrophoresis graph of amplification products (B) for Pbr and other species strains. Notes: M: 2000 bp Maker. The strain numbers are listed in Table 1.

Figure 3

qPCR amplification curves (A) and standard curves (B) for the copy numbers of gradient-diluted standard plasmids. (A) The amplification curves represent the copy number in the range from 2.24 × 10⁷ copies·μL⁻¹ to 2.24 × 10¹ copies·μL⁻¹. Dilution gradients are indicated by the number range from 1 to 7. Note: No.1: 2.24 × 10⁷ copies·μL⁻¹, No.2: 2.24 × 10⁶ copies·μL⁻¹, No.3: 2.24 × 10⁵ copies·μL⁻¹, No.4: 2.24 × 10⁴ copies·μL⁻¹, No.5: 2.24 × 10³ copies·μL⁻¹, No.6: 2.24 × 10² copies·μL⁻¹, No.7: 2.24 × 10¹ copies·μL⁻¹. (B) The logarithms of the plasmid copy numbers were plotted against the Ct values, and the regression line equation and correlation coefficient (R²) are displayed. The error bars represent the standard deviations of three replicate reactions.

Figure 4

Ct values of viable or dead cells of the Pbr strain treated with different concentrations of PMA. Note: ^a, 10⁶ CFU·mL⁻¹; ^b, 10⁷ CFU·mL⁻¹; and ^c, 10⁸ CFU·mL⁻¹. The data have been presented as the means of three replicates ± standard deviations.

Figure 5

Effects of soaking in warm broth on the mortality of Pbr in seeds. The data are presented as the means of three replicates ± standard deviations.

Figure 6

Dynamic lesions and number of viable cells of Pbr in cucumber and potato leaves from 0 to 48 h after infection with bacterial soft rot disease. (A) Dynamics of cucumber leaves over 48 h. (B) Dynamics of potato leaves over 48 h. (C) Number of viable Pbr bacteria on cucumber and potato leaves detected via PMA-qPCR. The data are presented as the means of three replicates ± standard deviations.