Development and application of a rapid detection method for Clarireedia spp. using loop-mediated isothermal amplification (LAMP) assay

Dollar spot, caused by the ascomycete fungus, Clarireedia spp., is a disease that seriously damages most grass species, especially those grown on golf courses. Early detection and intervention are essential for the effective management of this disease. In this study, Clarireedia spp. was detected in a faster, more efficient, and specific way than current conventional methods by using a loop-mediated isothermal amplification (LAMP) system that was developed based on the β-tubulin gene. When hydroxynaphthol blue (HNB) was added, Clarireedia spp. DNA samples changed color from violet to sky blue after amplification, but those without DNA (negative control) did not. The LAMP assay was specific for Clarireedia spp. and did not amplify other common fungal pathogens or closely related species. The sensitivity of the LAMP method was 100 times greater than conventional PCR. In 219 potentially infected samples, 212 (96.80%) were confirmed positive by LAMP, 214 (97.72%) by tissue isolation, and 135 (61.64%) by PCR. All results validated that the rapid, sensitive, and reliable LAMP system could be conveniently applied for the detection of Clarireedia spp., as well as provide an expedient reference for the control and management of dollar spot.