Culture-Based and Molecular Detection of Sclerotinia Homoeocarpa in Commercial Creeping Bentgrass Seed.

Sclerotinia homoeocarpa, the causal agent of dollar spot disease of turfgrass, is nearly ubiquitous in amenity turfgrass settings. Despite this, information pertaining to the primary source of inoculum for this pathogen is limited. A number of factors, including the limited genetic diversity of cool-season isolates of S. homoeocarpa and spatial distribution of dollar spot epidemics, have led to the hypothesis that commercially produced turfgrass seed serves as a source of primary inoculum for this disease. This hypothesis was tested by obtaining seed from multiple lots of six creeping bentgrass (Agrostis stolonifera L.) cultivars. Culture-based detection was performed by individually plating 1000 seeds from two lots of each cultivar on media selective for S. homoeocarpa. The experiment was replicated three times with only a single isolation of S. homoeocarpa. This indicates that viable isolates of S. homoeocarpa are present on commercial seed, but at such low incidence that culture-based detection is impractical. A nested PCR method based on primers specific for the S. homoeocarpa elongation factor 1-α gene was developed for molecular detection. Chromosomal DNA was extracted from ten 50mg subsamples of each seed lot. Two μl of chromosomal DNA served as the starting template for the primary PCR reaction and one μl of the primary PCR product was used in the nested PCR reaction. Products of both primary and nested reactions were run on electrophoresis gels and positive bands were excised and sequenced. Two of five seed lots tested with this method thus far have been found positive for the presence of S. homoeocarpa DNA. Additionally, sequence data have confirmed all positive samples as S. homoeocarpa, demonstrating specificity of the primary and nested primer sets. At a 1.5 lb seeding rate, the number of positive subsamples in the two contaminated seed lots translates to 3000-8000 infected seeds per 1000 ft². This research provides novel insights into S. homoeocarpa epidemiology and introduces a tool that may be adapted and utilized by the seed production industry.