Development and Application of a LAMP Assay for Detecting E198A-Type MBC-Resistant Clarireedia monteithiana

The E198A (GAG→GCG) mutation at codon 198 of the β-tubulin gene confers high levels of resistance to methyl benzimidazole carbamate (MBC) fungicides in Clarireedia monteithiana worldwide. Early detection of E198A-type resistant populations is therefore essential for effective dollar spot management. In this study, we developed a rapid loop-mediated isothermal amplification (LAMP) assay for detecting MBC-resistant C. monteithiana. Optimal amplification occurred at 64°C for 60 min, and positive reactions were readily identified by a distinct purple-to-sky-blue color change using hydroxynaphthol blue (HNB) as the indicator. The selected primer set specifically distinguished E198A isolates from wild-type isolates, F200Y mutants, and closely related fungal species. The assay exhibited a detection limit of 2 × 10⁴ copies, reflecting a 100-fold higher sensitivity than conventional PCR. In host inoculation assays, the LAMP method achieved an accuracy of 95.83-96.78% across two experimental groups under near-field conditions, whereas PCR produced successful amplification for only 124 of 140 samples (88.57%). Collectively, these results demonstrate that the developed LAMP assay provides a rapid, sensitive, and robust tool for monitoring E198A-type MBC-resistant isolates and offers substantial value for the early detection and management of dollar spot disease.