Monitoring Ectotrophic Root-Infecting Fungi Associated with Bermudagrass Putting Green Roots Using Quantitative Multiplex Assays

Abstract

Bermudagrass decline (BD) is a destructive root rot disease of hybrid bermudagrass putting greens in the southeastern United States. Previous studies reported a single ectotrophic root-infecting (ERI) fungus, Gaeumannomyces graminis, associated with BD. Recently, three novel ERI fungi, Gaeumannomyces sp., Magnaporthiopsis cynodontis, and Candidacolonium cynodontis, have also been associated with BD. Current diagnostic methods for identification can be inconclusive and inaccurate for distinguishing these ERI fungal species. Here, we present the development and application of novel real-time PCR assays designed to detect four selected ERI fungi associated with BD. The distribution of these ERI fungi in hybrid bermudagrass samples collected from putting greens and fairways throughout the southeastern United States was also assessed. In addition, we established a reliable and rapid protocol to simultaneously detect these selected fungi directly from bermudagrass root tissue. The limit of detection for each assay was as low as 1 pg/µl of DNA per reaction, proving the sensitivity of the method to detect low quantities of fungal DNA when present in bermudagrass roots. The presence of the selected ERI fungi was detected in 36 of 37 bermudagrass root samples, with one fungus having a wider distribution among the samples tested and two fungi with greater DNA quantities when compared with the bermudagrass roots. The novel assays provide a reliable method for accurate detection of the four selected ERI fungi. The acute sensitivity of each assay makes it a viable tool for early detection of ERI fungi in hybrid bermudagrass roots.