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马铃薯晚疫病菌对甲霜灵抗性的快速检测方法

引用本文：郭东锋,钟林宇,周挺,张玥,李林翰,周倩,陈凤平.马铃薯晚疫病菌对甲霜灵抗性的快速检测方法.植物保护学报,2023,50(1):214-223

DOI：10.13802/j.cnki.zwbhxb.2023.2021191

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作者	单位	E-mail
郭东锋	福建农林大学, 生物农药与化学生物学教育部重点实验室, 福州 350002 福建农林大学, 植物与微生物互相作用福建省高校重点实验室, 福州 350002
钟林宇	福建农林大学, 生物农药与化学生物学教育部重点实验室, 福州 350002
周挺	中国烟草总公司福建省公司, 福州 350003
张玥	福建农林大学, 生物农药与化学生物学教育部重点实验室, 福州 350002 福建农林大学, 植物与微生物互相作用福建省高校重点实验室, 福州 350002
李林翰	福建农林大学, 生物农药与化学生物学教育部重点实验室, 福州 350002
周倩	贵州省遵义市绥阳县林业局, 遵义 563300
陈凤平	福建农林大学, 生物农药与化学生物学教育部重点实验室, 福州 350002 福建农林大学, 植物与微生物互相作用福建省高校重点实验室, 福州 350002	chenfengping1207@126.com

中文摘要:为快速检测马铃薯晚疫病菌——致病疫霉Phytophthora infestans对甲霜灵的抗性,基于已知致病疫霉RPA190基因的T1145A变异引起的F382Y氨基酸点突变与甲霜灵抗性有关,通过设计4对特异性引物F382Y-F1/F382Y-R、F382Y-F2/F382Y-R、F382Y-F3/F382Y-R和F382Y-F4/F382Y-R建立等位基因特异性PCR(allele specific-polymerase chain reaction,AS-PCR)快速分子检测法,其中F382Y-R引物在4对引物里保持不变,并分析所建分子检测法的特异性和灵敏度。结果表明,正向特异性引物F382Y-F2、F382Y-F3和F382Y-F4的3'末端在致病疫霉抗甲霜灵菌株原有核苷酸突变位点1145A(对应引物为F382Y-F1)的基础上,再人工引入第1144位的核苷酸突变,将1144T分别突变成1144G、1144C和1144A,并优化退火温度和特异性引物与内参引物ITS1/ITS4比例,最终确定最适退火温度分别为54、60和58℃,特异性引物与内参引物最适浓度比例均为5∶1。以该3条引物对应的3对特异性引物和内参引物ITS1/ITS4组成的3组多重AS-PCR引物对甲霜灵敏感和抗性菌株具有良好的特异性,敏感菌株的扩增产物含1条879bp的内参片段,抗性菌株的扩增产物为1条879bp的内参片段和1条461bp的目的片段。3组AS-PCR检测体系均具有较高的灵敏度,其中引物F382Y-F4/F382Y-R对致病疫霉DNA的检测灵敏度达0.4pg/μL,引物F382Y-F2/F382Y-R和F382YF3/F382Y-R的检测灵敏度达4pg/μL。

中文关键词:致病疫霉甲霜灵快速检测技术等位基因特异性PCR

Rapid detection of metalaxyl resistance in potato late blight pathogen Phytophthora infestans

Author Name	Affiliation	E-mail
GUO Dong-feng	Key Laboratory of Biopesticides and Chemical Biology, Ministry of Education, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian Province, China Fujian University Key Laboratory for Plant-Microbe Interaction, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian Province, China
ZHONG Lin-yu	Key Laboratory of Biopesticides and Chemical Biology, Ministry of Education, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian Province, China
ZHOU Ting	Fujian Branch of National Tobacco Corporation, Fuzhou 350003, Fujian Province, China
ZHANG Yue	Key Laboratory of Biopesticides and Chemical Biology, Ministry of Education, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian Province, China Fujian University Key Laboratory for Plant-Microbe Interaction, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian Province, China
LI Lin-han	Key Laboratory of Biopesticides and Chemical Biology, Ministry of Education, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian Province, China
ZHOU Qian	Suiyang County Forestry Bureau, Zunyi 563300, Guizhou Province, China
CHEN Feng-ping	Key Laboratory of Biopesticides and Chemical Biology, Ministry of Education, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian Province, China Fujian University Key Laboratory for Plant-Microbe Interaction, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian Province, China	chenfengping1207@126.com

Abstract:In order to detect metalaxyl-resistant strains of potato late blight pathogen Phytophthora infestans rapidly, allele specific-polymerase chain reaction(AS-PCR) methods using four specific primers sets including F382Y-F1/F382Y-R, F382Y-F2/F382Y-R, F382Y-F3/F382Y-R and F382Y-F4/F382Y-R were established based on the known mechanism of metalaxyl resistance associated with amino acid point mutation of F382Y encoded by T1145A in RPA190 gene of P. infestans. The results showed that by using three forward primers F382Y-F2, F382Y-F3 and F382Y-F4, which were designed for point mutation 1145A plus an addition mutations 1144G, 1144C and 1144A, respectively, optimizing the annealing temperature to 54, 60 and 58℃, respectively, and setting the ratio of the three specific primers to referent primer set ITS1/ITS4 as 5∶1, the three multiple AS-PCRs using specific primers of F382Y-F2/F382Y-R, F382Y-F3/F382Y-R and F382Y-F4/F382Y-R, respectively plus referent primers ITS1/ITS4could distinguish specifically resistant strains from sensitive strains. The amplicons from the sensitive strains contained an internal reference fragment of 879 bp, and the amplicons from the resistant strains showed an internal reference fragment of 879 bp and a target fragment of 461 bp. The three multiple AS-PCR methods all had high sensitivity. Among them, the primers F38Y-F4/F382Y-R had the highest sensitivity in which the minimum detectable concentration of DNA template from P. infestans was 0.4 pg/μL, while the sensitivity of F382Y-F2/F382Y-R and F382Y-F3/F382Y-R was both 4 pg/μL.

keywords:Phytophthora infestans metalaxyl rapid detection technique allele specific-PCR(AS-PCR)

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