Rapid on-site evaluation of the development of resistance to quinone outside inhibitors in Botrytis cinerea

Botrytis cinerea, a typical "high-risk" pathogenic fungus that rapidly develops resistance to fungicides, affects more than 1,000 species of 586 plant genera native to most continents and causes great economic losses. Therefore, a rapid and sensitive assay of fungicide resistance development in B. cinerea populations is crucial for scientific management. In this study, we established a Loop-mediated isothermal amplification (LAMP) system for the monitoring and evaluation of the risk of development of B. cinerea resistance to QoI fungicides; the method uses two LAMP assays. The first assay detects G143A mutants of B. cinerea, which are highly resistance to QoI fungicides. BCbi143/144 introns in B. cinerea are then detected by the second assay. HNB acts as a visual LAMP reaction indicator. The optimum reaction conditions of the LAMP assays were 61 °C for 50 min, and the detection limit of the LAMP assays was 100 × 10⁻⁴ ng/μl. We directly pre-treated the field samples by using All-DNA-Fast-Out to extract DNA within ten minutes, then performed the LAMP assay to achieve one-step rapid detection. In conclusion, we established a rapid and sensitive LAMP assay system for resistance risk assessment and for monitoring QoI-resistance of B. cinerea in the field.