Biology of Ophiosphaerella agrostis, epidemiology of dead spot, and a molecular description of the pathogen

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Ophiosphaerella agrostis is a newly described pathogen that incites dead spot disease of creeping bentgrass (Agrostis stolonifera) putting greens throughout the eastern United States. The objectives of this research project were to: 1) monitor environmental conditions and develop a predictive model for disease progress and pathogen dispersal under field conditions; 2) determine the effect of various fertilizers on dead spot recovery in bentgrass; and 3) develop molecular methods to diagnoses the disease and determine genetic variation among O. agrostis isolates. Field studies showed that the appearance of dead spot infection centers was influenced predominantly by air (greater than or equal to 18 degrees Centigrade) and soil (greater than or equal to 20 degrees Centigrade) temperature, but other factors including low relative humidity (less than or equal to 80%), shortened periods of leaf wetness (less than or equal to 14 hours), and high levels of solar radiation (greater than or equal to 230 W m to the minus 2) were associated with the development of dead spot epidemics. Patch diameter of O. agrostis infection centers and pseudothecia production generally increased at a linear rate between mid-June and early to mid-August. In a growth chamber study, ascospores were released from pseudothecia following a sharp decrease in relative humidity. Field studies revealed that ascospore release occurred primarily at dawn and dusk or during precipitation events. During precipitation events, ascospores may be forcefully discharged during the entire event, but most ascospores are released within the first 10 hours after precipitation begins. O. agrostis successfully over-wintered in bentgrass leaf sheaths, crowns, roots, and stolons, but little or no reactivation of the disease occurred in the second year. Plots treated with ammonium sulfate and isobutylidene diurea were among the fastest and slowest to recover from dead spot, respectively. In the second year (2003), dead spot recurred in plots treated since 2002 with KNO3, Ca(NO3)2, urea, and a complete fertilizer (20-20-20), but disease symptoms did not recur in plots receiving ammonium sulfate. Species-specific primers capable of detecting O. agrostis at very low concentrations (5 pg DNA) were developed and can be used to assist in diagnosing dead spot. Amplified fragment length polymorphism (AFLP) DNA fingerprinting resulted in placement of isolates (n=77) into three distinct clades that were greater than or equal to 69% similar.