摘要
为明确灰葡萄孢Botrytis cinerea对腐霉利的抗性现状,于2017—2018年采用单孢分离法从浙江省5个地区的草莓大棚共分离获得200个菌株。通过区分剂量法测定了其对腐霉利的抗性,对抗药性菌株的分子机制进行了分析,并根据抗药性分子机制,建立了B.cinerea腐霉利高抗基因型的环介导等温扩增(loop-mediated isothermal amplification,LAMP)检测技术。结果表明:浙江省草莓B.cinerea群体对腐霉利的抗性频率高达71.5%,以低抗菌株为主。抗药性菌株的BcOS1基因上存在3种类型的突变:第Ⅰ类为BcOS1基因第365位密码子由ATC突变为AGC,导致编码的氨基酸由异亮氨酸(Ile,I)突变为丝氨酸(Ser,S);第Ⅱ类为BcOS1基因第365位密码子由ATC突变为AAC,导致编码的氨基酸由异亮氨酸(Ile,I)突变为天冬酰胺(Asn,N)。这两类单点突变均导致B.cinerea对腐霉利表现低或中水平抗性。第Ⅲ类包含两个连锁的突变位点,第369位和第373位密码子分别由CAG和AAC突变为CCG和AGC,导致氨基酸分别由谷氨酰胺(Gln,Q)和天冬酰胺(Asn,N)突变成脯氨酸(Pro,P)和丝氨酸(Ser,S),使得B.cinerea对腐霉利表现高水平抗性。本研究所建立的LAMP检测技术可在63℃恒温条件下,在50 min内完成对腐霉利高抗菌株Q369P的检测,最低检测限为10×10^(−3) ng/μL,灵敏度是常规PCR的10倍。本研究结果可为腐霉利在草莓灰霉病防治上的科学使用及抗药性治理提供理论依据和技术手段。
To clarify the resistance of Botrytis cinerea associated with strawberry to procymidone in Zhejiang Province,a total of 200 isolates were collected from strawberry greenhouses in five regions of Zhejiang Province by the single spore isolation method from 2017 to 2018.The resistance of B.cinerea population to procymidone was determined by distinguishing dosage method and the molecular mechanism of resistance was further studied.According to the molecular mechanism of resistance,a loop mediated isothermal amplification(LAMP)technique was developed.The results showed that the total resistance frequency to procymidone was 71.5%,while most resistance isolates were of low resistance.There were three types of mutations in BcOS1 gene of procymidone-resistant B.cinerea.TypeⅠhad the mutation at codon 365 from ATC to AGC,which lead to the amino acid change from isoleucine(Ile,I)to serine(Ser,S).The typeⅡhad the mutation at codon 365 from ATC to AAC,which resulted in the amino acid change from isoleucine(Ile,I)to asparagine(Asn,N).These two types of mutations caused low or moderate resistance to procymidone.Type Ⅲ isolates were highly resistant which contained mutations at codon 369 and 373,which mutated from CAG and AAC to CCG and AGC,respectively,and resulted in amino acids change from glutamine(Gln,Q)and asparagine(Asn,N)to proline(Pro,P)and serine(Ser,S).A LAMP detection technique was developed to detect the highly resistant phenotype(Q369P)in 50 minutes at 64℃.The detection limit of this assay was 10×10^(−3) ng/μL,and the sensitivity was 10 times as high as that of the conventional PCR.This study can provide theoretical basis and technical means for the further resistance management and scientific application of procymidone for the control of B.cinerea.
作者
郑远
沈瑶
汪汉成
戴德江
沈颖
吴鉴艳
张传清
ZHENG Yuan;SHEN Yao;WANG Hancheng;DAI Dejiang;SHEN Ying;WU Jianyan;ZHANG Chuanqing(College of Agriculture and Food Science,Zhejiang Agriculture&Forestry University,Hangzhou 311300,China;Station for the Plant Protection&Quarantine and Control of Agrochemicals,Zhejiang Province,Hangzhou 310004,China;Guizhou Academy of Tobacco Science,Guiyang 550081,China)
出处
《农药学学报》
CAS
CSCD
北大核心
2021年第1期90-96,共7页
Chinese Journal of Pesticide Science
基金
国家自然科学基金(31501679)
浙江省重点研发项目(2015C02015).
关键词
灰葡萄孢
腐霉利
抗药性机制
环介导等温扩增
抗性检测
Botrytis cinerea
procymidone
resistance mechanism
loop-mediated isothermal amplification
resistance detection
作者简介
郑远,男,硕士研究生,E-mail:785118940@qq.com;共同通信作者:汪汉成,男,博士,研究员,主要从事杀菌剂生物学研究,E-mail:xiaobaiyang126@hotmail.com;通信作者:张传清,男,博士,教授,主要从事杀菌剂生物学与植物病害治理研究,E-mail:cqzhang9603@126.com。
