桃褐腐病菌对多菌灵抗性的AS-PCR检测技术

An allele-specific PCR for detecting the carbendazim resistance in Monilinia fructicola

由于杀菌剂的长期大量使用,植物病原菌对杀菌剂抗性问题日趋突出,严重影响病害防治的效果。褐腐病是一种在全世界范围内广泛发生和流行的重要果树病害。本文以桃褐腐病菌Monilinia fructicola为例,基于已知的多菌灵抗性机理,即靶标β微管蛋白基因TUB2的点突变E198A,建立了一种桃褐腐病菌对多菌灵抗性的等位基因专化性PCR检测技术。结果表明,碱基错配、内参引物、退火温度、dNTPs、Taq DNA聚合酶、专化性引物浓度及配比等因素均对检测效率有影响。经过对以上因素的优化,并对优化后的检测技术进行专化性及灵敏度评价,证实该检测技术能特异性地鉴别桃褐腐病菌M.fructicola对多菌灵抗性基因型E198A,且检测灵敏度可达到4.342 pg/μL病菌DNA,有望在实践中推广使用。

With the increasing use of fungicides,the fungicide resistance has become a serious problem that affects the effect of fungicides in practice.Brown rot caused by the ascomycete fungus Monilinia fructicola is an important fruit disease that occurs and spreads worldwide.In this study,based on the known resistance mechanism to carbendazim,i.e.,the point mutation E198A of the targetβ-tubulin gene(TUB2),an allele-specific PCR(AS-PCR)was developed to detect the carbendazim resistance in M.fructicola.The results showed that the detection efficiency was influenced by mismatch of nucleotides,internal reference gene primer,annealing temperature,dNTPs,Taq DNA polymerase,specific primer concentration and ratio etc.After these factors were optimized,the specificity and sensitivity of the developed AS-PCR were evaluated.The results showed that the AS-PCR could specifically identify the carbendazim-resistant genotype E198A at a sensitivity of 4.342 pg/μL of M.fructicola genomic DNA,which may be widely used in practice.