Methods of Identification and Distribution of Gaeumannomyces Spp. within Ultradwarf Bermudagrass Greens.

Take all root-rot and bermudagrass decline are detrimental diseases of ultradwarf bermudagrass (UDB) greens in the southeastern US. These diseases are caused by a complex of ectotrophic root-infecting (ERI) fungal pathogens including Gaeumannomyces spp. Due to sterility in culture, fungal identification can be challenging. Rapid identification of multiple ERI pathogens would be desirable. Multiplex qPCR was developed to facilitate the identification of ERI pathogens associated with UDB roots and determine their distribution within three UDB greens. A fishnet grid system was established for sampling greens at a local golf course using ArcGIS. Aerification cores were collected within 2.4 m² of each centroid. Composite root samples were prepared for genomic DNA extraction and stored at −20ºC. Initially, we standardized an assay to detect Gaeumannomyces spp. in samples using qPCR. Total genomic DNA was isolated from axenic fungal cultures of Gaeumannomyces spp. One specific primer and two LNA-probes were designed based on the sequence of the MCM7 gene of two Gaeumannomyces spp. Specific primers and LNA-probes were also developed based on the sequence of the TEF-1 gene of UDB root to serve as a positive control. A 10-fold serial dilution of the fungal and plant DNA was used to determine the sensitivity of the assay, resulting in amplification efficiencies of 104% to 108% for each Gaeumannomyces spp. and 105% for UDB. The lowest detectable concentration of both Gaeumannomyces spp. was to 1×10^-3 µg/µL of pure DNA. To determine the specificity of the assay, a panel composed of eight different fungal species was applied, resulting in no amplification from any of the non-target fungi tested. As a next step for this study, we plan to employ multiplex qPCR using the UDB roots to identify, quantify, and determine the distribution of Gaeumannomyces spp. throughout the selected putting greens.