Development of TaqMan Real-Time PCR Protocols for Simultaneous Detection and Quantification of the Bacterial Pathogen Ralstonia solanacearum and Their Specific Lytic Bacteriophages

Abstract

Ralstonia solanacearum is the causal agent of bacterial wilt, one of the most destructive diseases of solanaceous plants, affecting staple crops worldwide. The bacterium survives in water, soil, and other reservoirs, and is difficult to control. In this sense, the use of three specific lytic R. solanacearum bacteriophages was recently patented for bacterial wilt biocontrol in environmental water and in plants. To optimize their applications, the phages and the bacterium need to be accurately monitored and quantified, which is laborious and time-consuming with biological methods. In this work, primers and TaqMan probes were designed, and duplex and multiplex real-time quantitative PCR (qPCR) protocols were developed and optimized for the simultaneous quantification of R. solanacearum and their phages. The quantification range was established from 10⁸ to 10 PFU/mL for the phages and from 10⁸ to 10² CFU/mL for R. solanacearum. Additionally, the multiplex qPCR protocol was validated for the detection and quantification of the phages with a limit ranging from 10² targets/mL in water and plant extracts to 10³ targets/g in soil, and the target bacterium with a limit ranging from 10³ targets/mL in water and plant extracts to 10⁴ targets/g in soil, using direct methods of sample preparation.

Keywords: bacterial wilt; duplex; enumeration; identification; multiplex; phage.

Figure 1

Graphical summary of the methodology developed for the detection and quantification of the Ralstonia solanacearum phages vRsoP-WF2, vRsoP-WM2, and vRsoP-WR2, and the target bacterium in environmental water, soil, and plant material using duplex and multiplex qPCR.

Figure 2

Nucleotide alignment of the sequences of Ralstonia solanacearum phages vRsoP-WF2, vRsoP-WM2, and vRsoP-WR2 in Geneious Prime software. Primer and probe sequences of the three phages (A), and primer and probe sequences specific to phage vRsoP-WR2 (B).

Figure 3

Quantification range of real-time duplex PCR. Amplification curves of 10-fold serial dilutions from 10⁸ to 10 PFU/mL for vRsoP-WF2, vRsoP-WM2, and vRsoP-WR2 phages (A), and standard curve (B). Amplification curves of 10-fold serial dilutions from 10⁸ to 10² CFU/mL for Ralstonia solanacearum (C), and standard curve (D). Standard curves were obtained with mean values of three replicates for each of the ten-fold serial dilutions.

Figure 4

Real-time multiplex PCR. Amplification curves of serial dilutions from 10⁶ to 10² PFU/mL of vRsoP-WF2, vRsoP-WM2, and vRsoP-WR2 phages ((A)—red), amplification curves of serial dilutions from 10⁶ to 10³ CFU/mL of Ralstonia solanacearum ((B)—blue), amplification curves of serial dilutions from 10⁶ to 10³ CFU/mL of vRsoP-WR2 phage ((C)—green), and amplification curves of serial dilutions from 10⁶ to 10² CFU/mL of vRsoP-WF2, vRsoP-WM2, and vRsoP-WR2 phages, and from 10⁶ to 10³ CFU/mL of bacteria ((D)—red + green + blue). Dilutions of phages and the bacterium were performed in river water and direct sample preparation was used. Amplifications were performed in triplicate.