基于表型组学的嘧菌酯对烟草赤星病菌的作用机理及其抗性机理研究

Alternaria alternate (Fries) (A. alternate) Keissler causes leaf brown spot of tobacco (Nicotiana tabacum L.) in most tobacco-growing regions of the world. The impact of this pathogen on tobacco is enormous, including poor leaf production, declining leaf quality and sustainable development problem of tobacco industry. More attention should be paid to solve the problem. During the last 20 years, many experiments were conducted to control the Alternaria diseases with strobilurins azoxystrobin, as well as chemical using technique, development of resistance, mode of action and mechanisms of resistance. However, to our best of knowledge, no report of the above projects was conducted based on phenomics. Phenomics is a novel technology with typical advantages, and is widely used in microorganism researches. More than 1000 cell phenotypes could be tested by a standard set of assays on 96-well plates. Based on the biological activities of azoxystrobin to A. alternate in our previous study, the objective of this study is to test the phenotypes of A. alternate, especially phenotypes of the pathogen when inhibited by azoxystrobin during mycelial growth and germination of spores, and to test the phenotypes of isolates of A. alternate resistant to azoxystrobin. Results of this study will explain the mode of action and resistant mechanisms of azoxystrobin from phenomics, and will provide useful information for tobacco brown spot management.

链格孢引起的烟草赤星病是国内外最重要的烟草病害之一，不仅直接导致产量损失，还会严重影响烟叶品质，严重威胁着烟草生产，亟待解决。近年来，国内外采用甲氧基丙烯酸酯类杀菌剂嘧菌酯对链格孢引起的病害进行了大量的防治技术、病原菌抗药性监测、作用机理和抗药性机理方面的研究，但从微生物表型组学上对其进行生物活性、作用机理和抗药性机理的研究却未见报道。在项目前期开展嘧菌酯对赤星病病原菌生物活性研究的基础上，本申请拟采用目前微生物表型组学研究中广泛使用和具有独特优势的高通量表型芯片技术，开展烟草赤星病菌的表型组学分析；开展嘧菌酯抑制作用下，菌丝生长和孢子萌发过程中赤星病菌的表型组学分析；同时开展赤星病菌嘧菌酯抗药性突变体的表型组学分析。项目的完成将从表型组学水平上为嘧菌酯对链格孢的作用机理及其抗性机理提出一个合理的解释，对指导赤星病的化学防治具有重要的理论意义。

烟草赤星病是国内外最重要的烟草病害之一，导致产量损失并影响烟叶品质。项目采用微生物表型芯片技术研究了赤星病菌的表型组学；研究了嘧菌酯作用下，菌丝生长和孢子萌发过程中赤星病菌的代谢表型组学；同时研究了赤星病菌嘧菌酯抗性突变体（菌株6-5 (cyt b未突变，抗性倍数25.28) 和6-11 (cyt b基因L244 F和V259A突变， 52.91倍)与亲本菌株在代谢表型上的差异。结果表明，赤星病能够代谢24.74%的供试碳源、85.26%氮源、97.14%硫源、89.83%磷源。高效代谢的碳、氮源为碳水化合物和氨基酸类。它具有较强的渗透压和pH适应能力，pH适应范围为3.5-10，它具有较强的脱羧酶的活性，无脱氨酶活性。菌丝生长阶段，嘧菌酯（1、10和100 mg/L）对赤星病菌的碳、氮、磷、硫的代谢，及渗透压和pH代谢表型抑制较弱。低浓度嘧菌酯（1、10 mg /L）不影响赤星病菌的pH代谢表型。100 mg/L水杨肟酸与10 mg/L与100 mg/L嘧菌酯协同下，均增强了对赤星病菌代谢的抑制，病原菌仅分别能够代谢5.26%和2.1%的碳源、18.16%和8.61%的氮源，磷、硫源和生物合成途径的代谢均被完全抑制，同时失去脱羧酶活性。孢子萌发阶段，10 mg/L与200 mg/L嘧菌酯均抑制了赤星病菌的代谢，它分别能代谢12.1%与3.15%的碳源、67.8%与3.12%的氮源、15%与0%磷源、及13.8%与0%的硫源；仅有55.79%与6.32%的渗透压代谢表型，失去了脱羧酶活性。在100 mg/L水杨肟酸与嘧菌酯（10 mg/L、100 mg/L）协同下，赤星病菌的碳、氮、磷、硫、生物合成途径、渗透压及pH环境的代谢均被完全抑制。大部分被抑制的碳、氮源均发生在糖酵解和三羧酸循环这2个生化途径。突变体6-11与亲本菌株的代谢表型基本一致，但在氨基酸氮源代谢上略有差异，它不能代谢糖酵解和尿素循环中的氮源D-甘露糖胺和L-鸟氨酸。突变体6-5不能代谢糖酵解过程中的D-甘露糖胺和嘌呤代谢过程中的3,5-环磷酸鸟苷，且不能在pH 10.0的环境下生长。赤星病菌对D-甘露糖胺代谢途径的变化可能与其对嘧菌酯抗性有关。项目从代谢表型组学上揭示了嘧菌酯对烟草赤星病菌的作用机理和抗药性机理。研究结果对指导赤星病的化学防治具有重要的理论意义。