A Rapid PCR-Based Method for the Detection of Magnaporthe oryzae from Infected Perennial Ryegrass

The fungus Magnaporthe oryzae Couch (anamorph = Pyricularia oryzae Cavara) (previously known as Magnaporthe grisea (Herbert) Barr) (4) causes gray leaf spot disease of perennial ryegrass (Lolium perenne L.) (12). In recent years, this disease has become a serious but sporadic problem for turfgrass managers in the mid-Atlantic and midwestern United States (9,12,16, 20,22). The disease can cause blighting and death of large areas of turf and occurs most frequently on perennial ryegrass managed for athletic fields and golf courses. Diagnosis of gray leaf spot and identification of M. oryzae in diseased perennial ryegrass samples can be difficult and timeconsuming, depending on clinical expertise, the condition of the sample, the extent of symptoms, and the concomitant presence of other fungal pathogens. With extended periods of favorable environmental conditions, leaf spot symptoms may be short-lived, because infected leaf blades rapidly collapse and become necrotic (12,19). Leaf spots and blight also are associated with other summer diseases that result in little or no lasting turf damage. Because the summer leaf spot diseases of perennial ryegrass require different management strategies, an accurate and timely diagnosis is important for effective and efficient disease management. Current diagnostic protocols for gray leaf spot may include incubation of turf specimens for at least 24 h and microscopic examination of leaves and leaf tissues for the presence of characteristic conidia produced by M. oryzae. Therefore, numerous factors can reduce the reliability and timeliness of the diagnosis by these conventional methods. Polymerase chain reaction (PCR) is a common molecular procedure that has been developed to detect or identify a number of plant pathogens (1–3,5,11,13– 15,17). Implementation of these methods varies, but an increasing number of diagnostic laboratories have the ability and resources to utilize PCR methods for disease identification. PCR-based methods are very reliable, timely, and species-specific when appropriate oligonucleotides are selected as primers. They are especially useful for detecting pathogens that incite sporadic disease outbreaks or may be easily confused with less damaging pathogens at the start of the epidemic. In this research, we designed primers to PCR-amplify regions of the Pot2 transposon (EMBL Accession No. Z33638). Pot2 has been reported in and is believed to be present only in isolates of M. grisea and M. oryzae from various hosts (7,8,10). Significantly, the Pot2 transposon has been found in all of approximately 50 perennial ryegrass isolates of M. oryzae tested (M. Farman, personal communication) and, therefore, is more specific than other potentially useful sequences, such as the internal transcribed spacer region of rDNA. Pot2 is believed to have originated in an ancestor of these fungi, before the evolution of host specificity, and was estimated to be present in 100 copies in the M. oryzae genome (10). A recent study of perennial ryegrass isolates of M. oryzae based on repetitive probes, including Pot2, reported that the pathogen population that infects perennial ryegrass has limited genetic diversity (6). The presence of multiple copies of the Pot2 sequence in the genome facilitates PCR detection of the pathogen in infected tissue. The main objective of this research was to develop an accurate and rapid method to detect M. oryzae in symptomatic perennial ryegrass samples. For this protocol to be successful, the PCR primers had to selectively amplify a region of the Pot2 transposon in isolates of M. oryzae from perennial ryegrass, without amplifying DNA from perennial ryegrass itself or other pathogens. The protocol represents a modification of a commercially available kit for plant DNA isolation and detects the pathogen in less than 8 h.