Apple bitter rot is a serious agricultural disease caused by Colletotrichum gloeosporioides. In recent years, carbendazim-resistant C. gloeosporioides strains bearing an E198A point mutation in the β-tubulin gene (GAG to GCG) have emerged, threatening global apple production. As such, rapidly detecting the presence of this E198A mutation in C. gloeosporioides isolates is essential in order to monitor the spread of this pathogen and to prevent outbreaks of disease. Herein, we developed a simple loop-mediated isothermal amplification (LAMP) approach to detecting the E198A mutation in C. gloeosporioides isolates from ‘Gala’ apple samples. This optimized LAMP protocol was sufficient to establish the E198A genotype of a given isolate following a 60 min incubation at 63 °C by using four specific primers. The results of this reaction could be interpreted visually based on a fluorescent yellow-green color change upon the addition of the SYBR Green I dye, and were additionally confirmed via gel electrophoresis. Importantly, this LAMP assay was capable of rapidly and reliably detecting apples that were infected with carbendazim-resistant isolates harboring this E198A mutation. In conclusion, this LAMP assay in this study can rapidly, specifically, and sensitively detect cases of apple bitter rot caused by C. gloeosporioides isolates harboring the E198A mutation.

苹果苦腐病是由胶孢炭疽菌引起的一种严重的农业病害。近年来，出现了在β-微管蛋白基因（GAG 到 GCG）中带有 E198A 点突变的耐多菌灵的C. gloeosporioides菌株，威胁着全球苹果生产。因此，快速检测C. gloeosporioides分离物中这种 E198A 突变的存在对于监测这种病原体的传播和防止疾病爆发至关重要。在此，我们开发了一种简单的环介导等温扩增 (LAMP) 方法来检测C. gloeosporioides中的 E198A 突变从“Gala”苹果样品中分离出来。这种优化的 LAMP 方案足以通过使用四种特异性引物在 63°C 下孵育 60 分钟后确定给定分离物的 E198A 基因型。该反应的结果可以根据添加 SYBR Green I 染料后的荧光黄绿色变化直观地解释，并通过凝胶电泳另外确认。重要的是，这种 LAMP 检测能够快速、可靠地检测出感染了带有这种 E198A 突变的多菌灵抗性分离株的苹果。总之，本研究中的 LAMP 检测可以快速、特异性、灵敏地检测由带有 E198A 突变的C. gloeosporioides分离株引起的苹果苦腐病病例。