Detection of the low-germination-rate resting oospores of Pythium myriotylum from soil by PCR

AIMS: To establish a sensitive and specific polymerase chain reaction (PCR)-based method for detecting Pythium myriotylum in soils. METHODS AND RESULTS: Oospores of P. myriotylum were separated from large soil particles by flotation in sucrose solution. The thick-walled oospores were disrupted by vortex with sea sand and its DNA was extracted by the Cetyl trimethyl Ammonium Bromide (CTAB) method. The recovered DNA was verified by PCR amplification of a 150-bp target sequence of P. myriotylum. Samples of 10 g of soil were assayed; thus, the detection limit by PCR-based method was 10 oospores per gram soil. The method was successfully applied for the detection of P. myriotylum in soils collected in March, prior to planting of ginger crops. CONCLUSIONS: A PCR-based method for detecting P. myriotylum from soil was achieved. SIGNIFICANCE AND IMPACT OF THE STUDY: The PCR method has allowed us to monitor the presence of P. myriotylum in soil prior planting season as a way of reducing or eliminating disease.