西瓜蔓枯病菌啶酰菌胺抗性突变体的生物学特性研究及其Sdh B基因位点检测

Study on biological characteristics and resistance molecular mechanism of Didymella bryoniae to boscalid

为评价西瓜蔓枯病菌对啶酰菌胺的抗性风险,了解其抗性机理,室内通过药剂驯化方法获得2株啶酰菌胺的抗性突变体XF21-3和YC60-1,测定了抗性突变体的生物学特性,并通过对Sdh B基因片段的测序比对,分析了西瓜蔓枯病菌对啶酰菌胺的抗性机理。生物测定结果表明:啶酰菌胺对2株抗性突变体的EC_(50)值分别为108和124μg/m L,抗性倍数(RR)分别为1 007和1 347,均为高抗菌株;抗性突变体的菌丝生长速率和产孢量均大于亲本菌株,但其致病性与亲本菌株无显著差异,对外界环境渗透压的敏感性低于亲本菌株;此外,啶酰菌胺与萎锈灵、戊唑醇、乙霉威及醚菌酯之间均不存在交互抗性,但与噻呋酰胺之间存在交互抗性。Sdh B基因片段测序及比对结果表明,高抗性突变体中Sdh B亚基277位上的氨基酸所对应的碱基由CAC突变为TAC,即由组氨酸(His)突变为酪氨酸(Tyr)。研究表明,西瓜蔓枯病菌在药剂选择压力下容易形成啶酰菌胺的抗性群体,且抗性突变体的离体适合度高于亲本菌株,此外,啶酰菌胺与同类型杀菌剂噻呋酰胺之间存在交互抗性,因此认为西瓜蔓枯病菌对啶酰菌胺具有中等抗性风险;同时进一步验证了Sdh B亚基277位上的氨基酸突变(His→Tyr,CAC→TAC)是西瓜蔓枯病菌对啶酰菌胺产生抗性的原因。

The resistance risk of Didymella bryoniae against boscalid was valuated and the resistance mechanism was investigated. By using fungicide adaptation in door, the boscalid-resistant mutants XF21-3 and YC60-1 were obtained, and their biological characteristics were investigated. The partial Sdh B gene sequences of resistant mutants were sequenced to study the molecular mechanism of resistance of D. bryoniae to boscalid. Results showed that the EC50 values of two resistant mutants, XF21-3 and YC60-1, were 108 and 124 μg/mL, with corresponding resistance ratio of 1 007 and 1 347, respectively. The mycelial growth rate and sporulation of two resistant mutants were better than that of their corresponding parents. There is no significant difference in pathogenicity between resistant mutants and their parents. However, resistant mutants showed reduced sensitivity to the high osmotic pressure compared to parental strains. Besides, the resistant mutants had cross-resistance with thifluzamid and had no cross-resistance with carboxin, diethofencrab, tebuconazole, and kresoxoim- methyl. The comparison of Sdh B gene sequencing showed that resistant mutants harbored the point mutation at position 277 in Sdh B subunit with amino acid changing from histidine to tyrosine. The findings indicated that it is easy for D. bryoniae to produce boscalid-resistance under higher selection pressure, and the in vitro fitness for resistant mutants was higher than that of sensitive strains. The results also showed that boscalid was a medium resistance risk fungicide, because the boscalid-resistant mutants showed cross-resistance between boscalid and thifluzamid. Finally, the point mutation at codon 277 （His→Tyr, CAC→TAC） in Sdh B may confer resistance ofD. bryoniae to boscalid.