小麦纹枯病菌对噻呋酰胺抗性机制研究

Thifluzamide, a SDHI fungicide, works wonders on inhibiting Rhizoctonia pathogens. Our previous studies showed that it has high efficacy against Rhizoctonia cerealis in vitro and its field control effect is better than that of common fungicides. It shows this fungicide has a good application prospect for controlling R. cerealis. R. cerealis produces no spores, is dikaryotic and don't have sexual stage. The resistance mechanism of which to thifluzamide is still unknown. By this project, we intend to obtain pure isolates of R. cerealis by separating single protoplast and induce resistant mutants by repeated exposure to fungicide. The difference of sequences of sdhA, sdhB, sdhC and sdhD between thifluzamide-sensitivive and thifluzamide-resistant isolates will be compared and analysised to identify the resistance target genes and mutations. The target gene will be cloned into pET32a+, and expressed in soluble form in E. coli. The expressed proteins will be purified. The in vitro binding characteristics of these proteins with thifluzamide in thifluzamide-sensitivive and thifluzamide-resistant isolates will be studied and compared by fluorescence quenching to illustrate the resistance mechanism of R. cerealis to thifluzamide. This study will provide some theory basis for the application of thifluzamide to control R. cerealis and research basis for screening new anti-fungus compounds based on the target gene in vitro.

噻呋酰胺是一种SDHIs杀菌剂，对丝核菌有特效。申请者前期研究结果表明噻呋酰胺对小麦纹枯病菌(Rhizoctonia cerealis)有很好的离体抑制活性，对病害的田间防治效果也优于常用杀菌剂，说明其在小麦纹枯病防治上具有良好的应用前景。R. cerealis无有性阶段，不产孢子，胞内双核，其对噻呋酰胺抗性机制未有报道。本研究拟采用单原生质体分离方法获得R. cerealis的纯系菌株，对纯系菌株进行药剂驯服，筛选噻呋酰胺抗性突变体，通过比较敏感和抗性菌株中琥珀酸脱氢酶四个亚基的核苷酸和氨基酸序列，明确抗药性靶标基因以及突变位点，再对靶标基因进行原核表达和蛋白纯化，采用荧光光谱法测定和比较敏感和抗性菌株中靶标蛋白与噻呋酰胺体外结合能力，阐明小麦纹枯病菌对噻呋酰胺的抗性机制，从而为科学制定该杀菌剂的应用策略提供理论依据，也为针对该靶标蛋白进行新药物的离体筛选提供研究基础。

【背景】：噻呋酰胺是一种SDHIs杀菌剂，对丝核菌有特效。本研究前期研究结果表明噻呋酰胺对小麦纹枯病菌(Rhizoctonia cerealis)有很好的离体抑制活性，对病害的田间防治效果也优于常用杀菌剂，说明其在小麦纹枯病防治上具有良好的应用前景。R. cerealis无有性阶段，不产孢子，胞内双核，其对噻呋酰胺抗性机制未有报道。.【研究结果】本研究拟通过药剂驯化和紫外诱变筛选噻呋酰胺抗性突变体，但只有紫外诱变的方法获得了20株噻呋酰胺抗性突变体。比较其琥珀酸脱氢酶基因的序列， 2株突变体在RCSdhB, RCSdhC 和 RCSdhD上无突变， 其它18株突变体在RCSdhB，RCSdhC 或 RCSdhD 上存在着突变位点。主要突变类型为RCSdhC基因的139位的组氨酸(H)突变为酪氨酸（Y）和RCSdhD的116位组氨酸(H)突变为酪氨酸(Y)。这些突变存在纯合突变或杂合突变。有一株突变体是RCSdhB的H246Y突变类型，该菌株对噻呋酰胺、啶酰菌胺和氟唑菌苯胺表现为中抗，对联苯吡菌胺表现为低抗；其它类型突变体对噻呋酰胺均表现为中抗，对啶酰菌胺、联苯吡菌胺和氟唑菌苯胺都表现为低抗。所有突变体对氟酰胺都表现为敏感。氟吡菌酰胺对小麦纹枯病菌无抑制效果。与亲本菌株相比，部分突变体在适合度上有轻微的下降，还有部分突变体与亲本菌株在适合度上无显著性差异。.【结论】小麦纹枯病菌对噻呋酰胺的抗性存在纯合和杂合抗性，抗性水平低到中等，抗性风险中等，抗性机制与RCSdhB，RCSdhC 和RCSdhD基因点突变相关。为了避免和延缓抗性的产生，我们在以后的应用中应该将该杀菌剂与其它药剂交替轮换使用。