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

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 10(8) to 10 PFU/mL for the phages and from 10(8) to 10(2) 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 10(2) targets/mL in water and plant extracts to 10(3) targets/g in soil, and the target bacterium with a limit ranging from 10(3) targets/mL in water and plant extracts to 10(4) targets/g in soil, using direct methods of sample preparation.