PCR-RFLP for Detection of Fusarium graminearum Genotypes with Resistance to Phenamacril
PCR-RFLP for Detection of Fusarium graminearum Genotypes with Resistance to Phenamacril
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PCR-RFLP检测非那丙烯利抗性禾谷镰刀菌基因型
DOI：
10.1094/pdis-06-20-1156-re
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发表时间：
2021
期刊：
影响因子：
4.5
通讯作者：
Yi-Ping Hou
中科院分区：
文献类型：
--
作者：
Jiao-Sheng Li;Luo-Yu Wu;Hui Zhang;Xiu-Shi Song;Jian-Xin Wang;Ming-Guo Zhou;Yi-Ping Hou
非那普利是一种氰基丙烯酸酯类杀菌剂，对主要由禾谷镰刀菌和镰刀菌引起的小麦赤霉病有很好的防治效果。亚洲人。以往的研究表明镰刀菌肌球蛋白5基因密码子的突变是导致镰刀菌肌球蛋白5基因突变的主要原因。在体外实验室实验中赋予对非那普利的抗性。本研究利用PCR限制性片段长度多态性（RFLP）技术，检测了杀菌剂驯化诱导禾谷镰刀菌3种常见突变（A135 T，GCC → ACC; S217 L，TCA → TTA; E420 K，GAA → AAA）。用合适的引物对扩增肌球蛋白5基因A135 T突变为841 bp、S217 L突变为802 bp、E420 K突变为1,649 bp的PCR产物。用限制性内切酶KpnI、TasI和DraI分别对A135 T、S217 L和E420 K突变型的苯那吖啶敏感和耐药菌株进行鉴定，KpnI将密码子A135 T突变型的苯那吖啶耐药菌株的841 bp PCR产物酶切为256和585 bp两个片段。TasI将密码子突变为S217 L的非那吖啶耐药菌株的802 bp PCR产物酶切为461、287和54 bp三个片段。相比之下，TasI酶切非那吖啶敏感菌株的802-bp PCR产物仅产生515和287 bp的两个片段。DraI酶切具有密码子突变E420 K的非那吖啶抗性菌株的1，649-bp PCR产物，使其产生932和717 bp的两个片段，而非那吖啶敏感菌株的PCR产物不被酶切。通过分析PCR产物酶切片段的电泳图谱，确定3种耐药突变基因型。采用PCR-RFLP方法对48株经杀菌剂驯化培养的苯啶酰菌胺抗性菌株进行了鉴定，并与常规方法（菌丝生长法）进行了比较。PCR-RFLP方法检测F.与常规方法相比，禾谷氨酰胺对苯那普利的耐药率为95.12%。生物信息学分析表明，PCR-RFLP方法也可用于亚洲镰刀菌A135 T和E420 K密码子突变的检测。
Phenamacril is a cyanoacrylate fungicide that provides excellent control of Fusarium head blight (FHB) or wheat scab, which is caused predominantly byFusarium graminearumandF. asiaticum. Previous studies revealed that codon mutations of themyosin-5gene ofFusariumspp. conferred resistance to phenamacril in in vitro lab experiments. In this study, PCR restriction fragment length polymorphism (RFLP) was developed to detect three common mutations (A135T, GCC to ACC at codon 135; S217L, TCA to TTA at codon 217; and E420K, GAA to AAA at codon 420) inF. graminearuminduced by fungicide domestication in vitro. PCR products of 841 bp (for mutation of A135T), 802 bp (for mutation of S217L), or 1,649 bp (for mutation of E420K) in themyosin-5gene were amplified by appropriate primer pairs. Restriction enzymeKpnI,TasI, orDraI was used to distinguish phenamacril-sensitive and -resistant strains with mutation genotypes of A135T, S217L, and E420K, respectively.KpnI digested the 841-bp PCR products of phenamacril-resistant strains with codon mutation A135T into two fragments of 256 and 585 bp. In contrast,KpnI did not digest the PCR products of sensitive strains.TasI digested the 802-bp PCR products of phenamacril-resistant strains with codon mutation S217L into three fragments of 461, 287, and 54 bp. In contrast,TasI digestion of the 802-bp PCR products of phenamacril-sensitive strains resulted in only two fragments of 515 and 287 bp.DraI digested the 1,649-bp PCR products of phenamacril-resistant strains with codon mutation E420K into two fragments of 932 and 717 bp, while the PCR products of phenamacril-sensitive strains was not digested. The three genotypes of resistance mutations were determined by analyzing electrophoresis patterns of the digestion fragments of PCR products. The PCR-RFLP method was evaluated on 48 phenamacril-resistant strains induced by fungicide domestication in vitro and compared with the conventional method (mycelial growth on fungicide-amended agar). The accuracy of the PCR-RFLP method for detecting the three mutation genotypes ofF. graminearumresistant to phenamacril was 95.12% compared with conventional method. Bioinformatics analysis revealed that the PCR-RFLP method could also be used to detect the codon mutations of A135T and E420K inF. asiaticum.