PCR-based detection of the epibiotic fungus Atkinsonella hypoxylon associated with its host grass Danthonia spicata

Danthonia spicata (L.) Beauv., commonly known as poverty oatgrass, is a perennial bunch-type grass native to North America. Danthonia spicata is often managed as a turfgrass in areas of the United States where cool-season grasses are adapted, and it has potential for development as a low-input turfgrass option. Naturally occurring D. spicata plants occasionally exhibit "choke" of the flowering stems due to a proliferation of fungal hyphae by the ascomycete Atkinsonella hypoxylon (Peck) Diehl. (family Clavicipitaceae). Polymerase chain reaction (PCR) primers were designed to amplify a 116-bp A. hypoxylon-specific fragment within the 5.8S ribosomal RNA Internal Transcribed Spacer 1 (ITS 1) region. The primer set was then used to amplify the fragment from 24 A. hypoxylon isolates and 24 D. spicata terminal seed head DNA extractions collected from a greenhouse-maintained population that has never exhibited choke. The A. hypoxylon ITS 1 fragment was amplified from all fungal samples and all terminal seed head samples tested. The plant and fungal amplified fragments produced the same melt peak in high-resolution melt analysis, and sequence analysis of all terminal seed head samples showed highly significant blast hits with the ITS 1 sequence of A. hypoxylon GenBank Accession U57405.1 and the 24 A. hypoxylon isolates. These results provide evidence that all sampled D. spicata plants are associated with the fungus A. hypoxylon and provide a useful tool for the study A. hypoxylon and its association with D. spicata.