应用特异等位PCR快速鉴定灰霉病菌抗苯莱特与乙霉威菌株

RAPID IDENTIFICATION OF BENOMYL AND DIETHOTHFENCARB RESISTANCE STRAINS OF BOTRYTIS CINEREA USING AN ALLELE-SPECIFIC PCR (ASP)

根据灰霉病菌Botrytis cinerea对苯并咪唑类（苯莱特）及N－苯氨基甲酸酯类（乙霉威）杀菌剂的抗性与其β－微管蛋白的198和200位密码子的单碱基突变紧密相关的原理，建立了一种同时检测灰霉病菌对这两类杀菌剂抗性表型的特异等位聚合酶链式反应（ASP,Allele-specific Polymerase Chain Reaction）方法，即将突变的单碱基作为正引物的3'末端，与之对应的未突变的单碱基作为负引物的3'末端， 自行设计了LP4～LP8等五个等位点特异性引物，应用包括这五个引物在内的七组引物，对生测为BenSNPCR、BenHRNPCS和BenHRNPCR菌株进行了ASP扩增，结果表明：该方法可以检测出相应抗药表型的菌株，并能有效地检测田间灰霉病菌的抗药性，从而该方法克服了生物检测方法费时和准确性差等缺点。该方法还能鉴定出生测法确定为同一表型如BenHRNPCR 中的BenSNPCR异核菌丝，或灰霉病菌侵染寄主后所产生的回复突变，因此其方法的灵敏度极高，可应用于灰霉病菌抗药性分子生态学机理研究和田间抗性菌株的快速鉴定。由于未发现BenMRNPCR型菌株，其适用性有待进一步验证。

The resistance of Botrytis cinerea to benzimidazole and diethofencarb had a close relation with codon 198 and 200 mutation of β-tubulin protein. On the basis of the mutations, a method relied on allele-specific PCR(ASP) was established. The method could detect the resistance phenotype of Botrytis cinerea to the two fungicides together. Several positive and negative primers were designed using codon mutation at positive primers' terminal 3' base and codon normal at negative primers' terminal 3' base. The isolates of BenSNPCR phenotype and BenHRNPCS phenotype were amplified using the primers. Therefore, the composite fragments were received, with which the phenotype of an unknown strain could be identified.