A PCR-Based Method for the Detection of Ophiosphaerella agrostis in Creeping Bentgrass.

Abstract

Dead spot is a relatively new disease of creeping bentgrass and hybrid bermudagrass that is incited by Ophiosphaerella agrostis. Initial symptoms are difficult to diagnose and clinicians generally rely on the presence of pseudothecia within infected tissue or isolation of O. agrostis on an artificial medium. The main goal of this study was to develop a polymerase chain reaction-based technique capable of quickly identifying O. agrostis within infected creeping bentgrass tissues. Oligonucleotide primers specific for O. agrostis were developed based on the internal transcribed spacer (ITS) rDNA regions (ITS1 and ITS2) of three previously sequenced isolates of O. agrostis. The 22-bp primers amplified a 445- or 446-bp region of 80 O. agrostis isolates collected from creeping bentgrass and bermudagrass in 11 states. Primers did not amplify DNA from other common turfgrass pathogens, including three closely related species of Ophiosphaerella. Selective amplification of O. agrostis was successful from field-infected creeping bent-grass samples and primers did not amplify the DNA of noninfected, field-grown creeping bent-grass or hybrid bermudagrass plants. Amplification of purified O. agrostis DNA was successful at quantities between 50 ng and 5 pg. The entire process, including DNA isolation, amplification, and amplicon visualization, may be completed within 4 h. These results indicate the specificity of these primers for assisting in the accurate and timely identification of O. agrostis and the diagnosis of dead spot in both bentgrass and bermudagrass hosts.