灰葡萄孢琥珀酸脱氢酶抑制剂类杀菌剂抗性的等位基因特异性PCR快速检测

Rapid allele-specific PCR detection of Botrytis cinerea strains with resistance to succinate dehydrogenase inhibitors

摘要:

【背景】灰葡萄孢(Botrytis cinerea)是引起植物灰霉病的病原菌，严重威胁着各种重要经济作物的产量。化学防治是最有效的一种防治措施，其中，琥珀酸脱氢酶抑制剂类(succinate dehydrogenase inhibitor, SDHI)杀菌剂是防治灰霉病的一类常用杀菌剂，但目前灰葡萄孢已对其产生抗性。【目的】建立一种能快速检测对SDHI杀菌剂产生抗性的灰葡萄孢菌株的方法。【方法】以SdhB^P225F点突变为材料，通过设计特异性引物，优化PCR体系退火温度和内参引物浓度、评价等位基因特异性PCR (allele-specific PCR, AS-PCR)的灵敏度和特异稳定性，最后以快速提取的田间菌株的DNA为模板进行AS-PCR检测，并将检测结果与测序结果进行比对。【结果】AS-PCR引物对P225F-3F/P225F-R在退火温度为58 ℃，内参引物浓度为2.5 μmol/L时可稳定区分抗性和敏感菌株，灵敏度为10 fg/μL，粗提田间菌株的AS-PCR检测结果与测序结果一致。【结论】该AS-PCR检测方法操作简单、灵敏度高、特异性强，可为田间灰葡萄孢对SDHI抗性菌株的快速检测提供一种较为可行的方法。

Abstract:

[Background] Botrytis cinerea is a pathogen causing gray mold, posing a serious threat to the yields of various important economic crops. Chemical control is an effective prevention and control measure, and succinate dehydrogenase inhibitor (SDHI) are commonly used fungicides for controlling gray mold. However, B. cinerea has developed resistance to SDHI. [Objective] To establish a method for rapidly detecting B. cinerea strains with resistance to SDHI. [Methods] We designed specific primers based on the point mutation SdhB^P225F and optimized the annealing temperature and internal reference primer concentration of the PCR system. We then evaluated the sensitivity, specificity, and stability of allele-specific PCR (AS-PCR). Finally, AS-PCR detection with rapidly extracted DNA from field strains as templates was conducted, and the detection results were compared with sequencing results. [Results] The AS-PCR with primers P225F-3F/P225F-R stably distinguished between resistant and sensitive strains at an annealing temperature of 58 ℃ and an internal reference primer concentration of 2.5 μmol/L, with the sensitivity of 10 fg/μL. The AS-PCR detection results of strains in the field were consistent with the sequencing results. [Conclusion] The AS-PCR detection method has simple operation, high sensitivity, and strong specificity, serving as a feasible method for rapid detection of B. cinerea strains with resistance to SDHI in the field.