Development of a PCR-Based Assay for the Detection of Sclerotinia Homoeocarpa From Creeping Bentgrass (Agrostis stolonifera) Seed.

Dollar spot caused by Sclerotinia homoeocarpa is the most economically important disease of creeping bentgrass.  The disease occurs in aggregated patterns that spread in the stand over time.  This suggests that one mechanism of dollar spot initiation is from infested seed during turfgrass stand establishment. To determine if S. homoeocarpa-infested seed is a mode of infestation in putting greens, a rapid seed assay would be useful. Therefore, the objective of this study was to develop a polymerase chain reaction (PCR)-based assay for the detection of S. homoeocarpa in creeping bentgrass seed.  Two species-specific primer sets were obtained for validation, SHelf1F/SHelf1R and JP4/JP5. The newly developed SHelf1F/Shelf1R primers were found to be more sensitive and specific for amplifying DNA from S. homoeocarpa than the previously established JP4/JP5 primers.  Using SHelf1F/Shelf1R, a subsequent seed assay protocol was developed.  The first step in the seed assay protocol was to use a beadbeater to grind 0.1 g of creeping bentgrass seed that were placed in a micro-centrifuge tube along with glass beads and cooled in liquid nitrogen between grinding cycles.  A commercial DNA extraction kit (DNEasy; Qiagen, Inc.) was used to extract genomic seed DNA.  The seed DNA was combined with extracted S. homoeocarpa DNA and the SHelf1F/Shelf1R primers were found to amplify 1 ng of S. homoeocarpa DNA in 20 ml of seed DNA.  Additionally, after mixing 0.1 g of sterilized creeping bentgrass seed with 1,5, and 10 S. homoeocarpa-infected seed, the SHelf1F/Shelf1R primers were found to amplify DNA from the sample with 5 infected seed.  Using the SHelf1F/Shelf1R primers it is possible to detect minute quantities of S. homoeocarpa DNA mixed with bulk extractions of creeping bentgrass seed DNA.