New Tools in the Pathologist's Tool Box: Diagnostic Assays for the Identification of Rhizoctonia and Rhizoctonia-Like Diseases of Turfgrass.

The fungal genus Rhizoctonia and its relatives are responsible for several important turfgrass diseases, including brown patch (R. solani), yellow patch (Ceratobasidium cereale), brown ring patch (Waitea circinata), and sheath and leaf spot (Rhizoctonia zeae).  Adequate disease management and control is dependent on correct pathogen identification; however, there remains no accurate, reliable and quick method for diagnosing these fungi.  In this study, two assays were developed for the rapid identification of Rhizoctonia and related species from turfgrass: 1) a real-time PCR assay and 2) an isothermal PCR assay. In the real-time PCR assay, an 11-46 nucleotide difference in the ITS1 probe region served to uniquely distinguish between W. circinata, R. solani and C. cereale. Between 6-25 nucleotide differences in the ITS2 probe region allowed for discrimination of the various subgroups of W. circinata responsible for brown ring patch, basal leaf blight, and the two causal agents of Rhizoctonia leaf spots (R. zeae and R. oryzae). Multiplexing was performed within both regions to allow for simultaneous pathogen discrimination. In the isothermal PCR assay, a fluorescently labeled (FAM) phosphorylated probe containing an internal D-spacer and two recombinase polymerase amplification primers (including a biotinlyated reverse primer) were designed within the ITS2 region for diagnosis of W. circinata. The fungus was accurately detected with a single incubation step performed at 37°-39°C using a simple sandwich lateral flow ‘dip stick’ with anti-FAM and anti-biotin antibodies, eliminating the need for expensive laboratory equipment. The assays developed in this study allow for the accurate and timely detection of several important Rhizoctonia and closely related species, can be implemented by diagnosticians and pathologists with relative ease, and will serve as a foundation for designing additional assays for pathogen detection.