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.
ABSTRACT
The distribution of three Ophiosphaerella spp. that cause spring dead spot (SDS) of bermudagrass was studied by systematically sampling two golf courses in Oklahoma and one in Kansas. O. herpotricha was isolated from all three locations and was the most abundant species. It was the only SDS pathogen found at Jenks, Oklahoma. O. korrae was isolated from Afton, Oklahoma, and Independence, Kansas, whereas O. narmari was only detected in samples from Afton. This is the first report of all three Ophiosphaerella species on bermudagrass at the same location. Amplified fragment length polymorphism (AFLP) marker analysis was used to investigate inter- and intraspecific genetic diversity of Ophiosphaerella isolates from North America and Australia. A majority of the O. herpotricha and O. narmari isolates from Afton were distinct haplotypes, suggesting that sexual recombination was occurring within the population. Conversely, the presence of multiple isolates of O. herpotricha and O. narmari with the same haplotype also indicated that asexual propagation was occurring. The genetic diversity among O. herpotricha isolates from Afton was not distinctly different from that of isolates collected throughout the southern United States. In contrast, O. narmari isolates from Afton were distinct from those collected in Australia. The genetic diversity in O. korrae was markedly different than that in the other Ophiosphaerella spp. The population at Afton was dominated by just a few haplotypes, and these were nearly identical to isolates collected from bermudagrass and Kentucky bluegrass throughout western, central, and northern North America. However, O. korrae isolates collected in the southeastern United States were only distantly similar to other North American isolates.
