Shift of Sensitivity in Botrytis cinerea to Benzimidazole Fungicides in Strawberry Greenhouse Ascribing to the Rising-lowering of E198A Subpopulation and its Visual On-site Monitoring by Loop-mediated Isothermal Amplification

摘要

Grey mold disease results from Botrytis cinerea, a classical “high-risk” plant pathogenic fungus in meaning of resistance development to fungicides, and its management depends largely on the frequent applications of fungicides. The evolution of resistance to benzimidazole chemicals during 2008 and 2016 was monitored continuously in strawberry greenhouses located in Zhejiang province. Results showed that extensive applications of the mixture of carbendazim and diethofencarb caused the rapid spread of Ben MR subpopulation. The withdraw of this mixture lead to the sharply decrease of Ben MR and re-dominance of Ben HR isolates of B. cinerea with the E198A mutation in β-tubulin gene. The LAMP primers, based on the E198A point mutation, were designed to detect the E198A genotype specifically. HNB (Hydroxynaphthol blue), a metalion indicator, acted as a visual LAMP reaction indicator that turned the violet colored into a sky-blue color. The detection limit of concentration of DNA was 100 × 10⁻² ng/μL and this LAMP assay could be applied to detect the E198A genotype with 100% accuracy in strawberry greenhouses of three Province and was more rapid and easier to operate. In summary, we establish a simple and sensitive on-field LAMP assay which can be adopted to determine within 1.5 h whether the benzimidazoles or the mixture of a benzimidazole fungicide and diethofencarb is suitable for management of B. cinerea.