Department of Plant Pathology, Zhejiang Agriculture and Forest University, Lin'an, 311300, China.
Institute for the Control of Agrochemicals of Zhejiang Province, Hangzhou, 310020, China.
Sci Rep. 2017 Oct 24;7(1):13861. doi: 10.1038/s41598-017-13317-z.
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 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.
灰葡萄孢菌（Botrytis cinerea）是一种典型的“高风险”致病真菌，它对杀菌剂的耐药性迅速发展，影响到来自各大洲的 586 个属的 1000 多种植物，造成巨大的经济损失。因此，快速灵敏地检测灰葡萄孢菌种群中杀菌剂抗性的发展对于科学管理至关重要。在本研究中，我们建立了一种环介导等温扩增（LAMP）系统，用于监测和评估灰葡萄孢菌对 QoI 类杀菌剂产生抗性的风险；该方法使用两种 LAMP 检测。第一种检测方法检测到灰葡萄孢菌的 G143A 突变体，这些突变体对 QoI 类杀菌剂具有高度抗性。然后，通过第二种检测方法检测 B. cinerea 的 BCbi143/144 内含子。HNB 作为 LAMP 反应的可视化指示剂。LAMP 检测的最佳反应条件为 61°C 50min，检测限为 100×10ng/μl。我们直接使用 All-DNA-Fast-Out 在十分钟内预处理田间样本，然后进行 LAMP 检测，实现一步快速检测。总之，我们建立了一种快速灵敏的 LAMP 检测系统，用于评估灰葡萄孢菌的抗性风险，并用于监测田间 QoI 抗性。
