Detection of root-infecting fungi on cool-season turfgrasses using loop-mediated isothermal amplification and recombinase polymerase amplification

Abstract

Root-infecting fungal pathogens such as Gaeumannomyces avenae, Ophiosphaerella korrae, and Magnaporthiopsis poae cause extensive damage to amenity turfgrasses in temperate climates. The diseases they cause are difficult to diagnose by visual symptoms or microscopic inspection, and traditional polymerase chain reaction-based assays require large financial investments in equipment such as thermal cyclers and highly trained staff. The primary objective of this research was to develop fast and accurate detection assays for the three pathogens listed above that did not require the use of thermal cycling equipment. Loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) assays were developed for each pathogen based on known fungal cultures. The assays were tested on 27 samples received at the University of Wisconsin's Turfgrass Diagnostic Laboratory in 2016 and 2017 and both methods provided accurate diagnoses within about 30 min with minimal sample preparation. However, the RPA assays had lower levels of false positive contamination relative to the LAMP assays and are more likely to be effective in a field or diagnostic laboratory for improved turf root-pathogen detection.