Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/1/2006
Publication Date: 8/1/2006
Citation: Kaminski, J., Dernoeden, P., Oneill, N.R., Whetzel, H.C. 2005. A pcr-based method for the detection of Ophiosphaerella agrostis in creeping bentgrass. Plant Disease. 89:980-985. Interpretive Summary: A disease affecting bentgrass was discovered on golf courses in three states in 1998 and since has been isolated from creeping bentgrass in at least 11 states. The disease is caused by a newly described species of the fungus Ophiosphaerella. Species in the genus Ophiosphaerella belong to a very important class of fungi, many of which are serious agricultural pests that attack many crops. The symptoms in turfgrass initially appear as small, reddish-brown spots that enlarge to about 8 cm in diameter throughout the summer months, killing the grass. Initial symptoms are difficult to diagnose and often are mistaken for damage caused by other common turfgrass diseases and pests such as dollar spot, copper spot, Microdochium patch, and black cutworms. It is important to accurately identify pathogens so that appropriate disease control methods can be implemented. Due to slow recovery of the turfgrass and the limited ability to manage the disease curatively, early identification of dead spot is critical. Laboratory identification is difficult and may take several weeks. We developed and tested a rapid molecular technique to positively identify the specific fungus, Ophiosphaerella agrostis, within infected creeping bentgrass tissues and within commercially available grass seed. The development of a molecular species-specific tool may also be useful to the turfgrass industry in determining the origin of the disease and preventing its spread.
Technical Abstract: Dead spot is a relatively new disease of creeping bentgrass and hybrid bermudagrass and is incited by Ophiosphaerella agrostis. Initial symptoms are difficult to diagnose and clinicians generally rely on the presence of pseudothecia for isolation of O. agrostis on an artificial medium. The main goal of this study was to develop a PCR-based molecular technique capable of quickly identifying O. agrostis within infected creeping bentgrass tissues. Oligonucleotide primers specific for O. agrostis were developed based on the ITS1 and ITS2 regions of three previously sequenced isolates of O. agrostis. The 22 base-pair (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 bentgrass samples and primers did not amplify the DNA of asymptomatic, field-grown creeping bentgrass or hybrid bermudagrass plants. Amplification of purified O. agrostis DNA was successful at quantities between 50 nanograms and 5 picograms. The entire process including DNA isolation, amplification and amplicon visualization may be completed within 4 hours. 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.