A rapid PCR-based method for the detection of Magnaporthe oryzae from infected perennial ryegrass

Gray leaf spot caused by Magnaporthe oryzae is a serious disease of perennial ryegrass in the midwestern United States. Symptoms of gray leaf spot can be confused with those caused by other fungal diseases that also are common during periods of high temperatures and ample moisture. Because turf managers must select appropriate fungicides for remedial treatment, accurate and timely identification of the pathogen is essential for efficient and effective disease management. We developed and evaluated a polymerase chain reaction (PCR)-based method to detect M. oryzae in infected perennial ryegrass tissue. The method utilizes a commercially available kit that is used for isolation and amplification of plant DNA from leaf tissue. The kit protocol was modified and found to be reliable for the extraction of M. oryzae DNA from infected perennial ryegrass. Primers were designed to amplify a 687-bp fragment of the Pot2 transposon that is found in multiple copies in the genome of the pathogen. The protocol amplified amounts of purified DNA as low as 5 pg and consistently and specifically detected M. oryzae in single diseased leaf blades as well as in field samples of infected perennial ryegrass. The total time required for detection was approximately 4 to 8 h.