Simple and sensitive BIO-PCR detection of potato blackleg pathogens from stem, tuber, and soil samples

A BIO-PCR was developed for the detection of potato blackleg pathogens, including Pectobacterium wasabiae (Pw), P. atrosepticum (Pa), P. carotovorum subsp. brasiliense (Pcb), and Dickeya dianthicola (Ddi). The method was based on the semi-selective incubation of pathogens using pectate-based liquid enrichment medium (LEM) and PCR detection of blackleg pathogens (triplex PCR for Pw, Pa, and Ddi and simplex PCR for Pcb with previously reported primers). Incubation in LEM for 24–120 h at 25 °C was effective in detecting the pathogens from plants (stems and tubers) and the soil around the roots. The detection limit by BIO-PCR from the tuber sample (0.6 g) in 10 ml medium was 2–8 colony-forming units (cfu) for four pathogens, whereas that from soil (10 g) in 100 ml medium was 2 × 102, 3 × 103, 6 × 101, and 2 × 101 cfu for Pw, Pa, Pcb, and Ddi, respectively. All four blackleg pathogens could also be detected from symptomatic stems, progeny tubers, and the soil around the stem base of diseased plants by BIO-PCR. This method is a promising technique for the efficient detection of potato blackleg pathogens not only for seed inspection but also for epidemiological research.