Departamento de Microbiología, Facultad de Ciencias, Universidad de Málaga, 29071 Málaga, Spain; and Departamento de Microbiología, Instituto de Hortofruticultura Subtropical y Mediterránea, "La Mayora" Universidad de Málaga-Consejo Superior de Investigaciones Científicas, Campus de Teatinos, 29071 Málaga, Spain.
Plant Dis. 2019 Jul;103(7):1515-1524. doi: 10.1094/PDIS-12-18-2256-RE. Epub 2019 May 6.
Powdery mildew, caused by the fungus , is one of the most economically important diseases affecting cucurbit crops in Spain. Currently, chemical control offers the most efficient management of the disease; however, isolates resistant to multiple classes of site-specific fungicides have been reported in the Spanish cucurbit powdery mildew population. In previous studies, resistance to the fungicides known as methyl benzimidazole carbamates (MBCs) was found to be caused by the amino acid substitution E198A on β-tubulin. To detect MBC-resistant isolates in a faster, more efficient, and more specific way than the traditional methods used to date, a loop-mediated isothermal amplification (LAMP) system was developed. In this study, three sets of LAMP primers were designed. One set was designed for the detection of the wild-type allele and two sets were designed for the E198A amino acid change. Positive results were only obtained with both mutant sets; however, LAMP reaction conditions were only optimized with primer set 2, which was selected for optimal detection of the E198A amino acid change in -resistant isolates, along with the optimal temperature and duration parameters of 65°C for 75 min, respectively. The hydroxynaphthol blue (HNB) metal indicator was used for quick visualization of results through the color change from violet to sky blue when the amplification was positive. HNB was added before the amplification to avoid opening the lids, thus decreasing the probability of contamination. To confirm that the amplified product corresponded to the gene, the LAMP product was digested with the enzyme and sequenced. Our results show that the LAMP technique is a specific and reproducible method that could be used for monitoring MBC resistance of directly in the field.
白粉病由真菌引起，是在西班牙影响葫芦科作物的最重要的经济疾病之一。目前，化学控制是管理该疾病最有效的方法；然而，在西班牙葫芦白粉病种群中已经报道了对多种类别的位点特异性杀菌剂具有抗性的分离物。在之前的研究中，发现对称为甲基苯并咪唑氨基甲酸酯（MBC）的杀菌剂的抗性是由β-微管蛋白上的氨基酸取代 E198A 引起的。为了比迄今为止用于检测 MBC 抗性分离物的传统方法更快、更有效和更具体地检测到 MBC 抗性分离物，开发了环介导等温扩增（LAMP）系统。在这项研究中，设计了三组 LAMP 引物。一组用于检测野生型等位基因，两组用于检测 E198A 氨基酸变化。只有使用两组突变体才能获得阳性结果；然而，仅优化了引物组 2 的 LAMP 反应条件，该引物组 2 用于最佳检测 E198A 氨基酸变化在 -抗性分离物中，以及最佳温度和持续时间参数分别为 65°C 75 分钟。羟萘酚蓝（HNB）金属指示剂用于通过在扩增阳性时从紫色变为天蓝色来快速可视化结果。在扩增之前添加 HNB 可避免打开盖子，从而降低污染的可能性。为了确认扩增产物与 基因相对应，用酶 消化 LAMP 产物并测序。我们的结果表明，LAMP 技术是一种特异性和可重复的方法，可直接在田间监测 的 MBC 抗性。
