Isolation of Fungi Associated with Fairy Rings on Golf Putting Greens and Identification Using Analysis of the ITS1 and ITS2 rDNA Regions.

Because isolation of basidiomycetes from the soil is difficult, identification of fungi associated with fairy rings on putting greens is based solely on basidiocarp morphology. Basidiocarps often do not develop or do not reach maturity due to turf management practices, making this method of identification problematic. Puffballs and soil blocks were collected from 5 bermudagrass and 4 bentgrass greens in FL, HI, NC, and SC exhibiting fairy ring symptoms. Four isolates from soil blocks and twenty isolates from puffballs were recovered by plating on Leonian medium amended with 2 μg ml^-1 benomyl, 50 μg ml^-1 chloramphenicol, 50 μg ml^-1 streptomycin, and 50 μg ml^-1 tetracycline. Genomic DNA was extracted, and the ITS1 and ITS2 regions of rDNA were amplified and sequenced using the basidiomycete-specific primer set ITS1f/ITS4b. Phylogenetic analyses, including construction of a parsimony tree and Bremer partition support calculations, were performed on the 24 collected isolates and 6 downloaded sequences from Genbank. Parsimony analysis revealed only one tree, having high bootstrap and Bremer supports at each node. Nineteen of the unknown puffball isolates and all four soil isolates clustered with Genbank accessions of either Vascellum pratense (Pers.) Kreisel, or Lycoperdon pusillum Batsch, not with Lycoperdon perlatum Pers., which is often cited as a prevalent cause of fairy rings in golf course putting greens in the Southeast. One of the unknown puffball isolates grouped in a separate haplotype from all others. Two isolates from the same site, one isolated from the soil and another from a puffball, had identical ITS sequences. Puffballs with a true capillitium had ITS sequences most similar to Lycoperdon pusillum, while puffballs without capillitial threads had sequences most similar to Vascellum pratense. Specific primers for both Vascellum pratense and Lycoperdon pusillum are being evaluated for use on genomic DNA extracted directly from fairy ring infested soils.