Monday, November 5, 2007
74-7
Isolation and Identification of the Basidiomycete Fungi Causing Fairy Rings in Golf Course Putting Greens.
Identification of the fungi responsible for causing fairy ring symptoms rests solely on basidiocarp formation, which often does not occur. Samples from the soil and thatch layers of four bentgrass and two bermudagrass golf course putting greens exhibiting fairy ring symptoms were collected. Several soil isolation techniques were evaluated, including traditional soil block and soil sprinkle methods, and the use of toothpicks or other wooden substrates as baits. A method involving spore washing and the use of a lignin-benomyl-guaiacol selective media was also attempted. Puffballs associated with symptoms on two of the sites were also collected, and mycelia were isolated on modified Leonian media. Isolated mycelia were examined for the presence of clamp connections or identifiable asexual or sexual structures, and genomic DNA was extracted from putative basidiomycetes. PCR amplification of rDNA regions ITS1, 5.8S rRNA, and ITS2 was performed using the universal fungal primers ITS5 and ITS4 and the basidiomycete-specific primers ITS1f and ITS4b. Primers ITS1f and ITS4b exclusively amplified isolates with clamp connections and isolates from puffball tissue, yielding an 800 – 900 bp fragment that was sequenced and used for phylogenetic analysis. No basidiomycetes were isolated from 5 of the 6 sampled soils, including the sites where puffballs were associated with symptoms. A sterile white fungus with clamp connections was routinely isolated from one bermudagrass site with all of the methods attempted. However, three isolates collected at different times had different ITS sequences, showing similarity to Marasmius, Laetisaria, and Sphaerobolus respectively. Three puffball isolates collected in North Carolina showed high ITS sequence similarity to Vascellum pratense, and two puffball isolates collected in South Carolina showed high ITS sequence similarity to Lycoperdon pusillum. These species have been associated with fairy rings in Japan , and species-specific primers were previously designed for identification and diagnosis.
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