丁二醇诱导匍匐翦股颖抗病性及其ISR机理研究

Creeping Bentgrass, a high quality species of turfgrass with beautiful look, low and frenquently mowing resistance, shallow root system, strong need of water supplement, is sensitive to diseases caused by fungus. In this research, the varieties of Creeping Bentgrass- Penn A-1, Penna-4 and Putter will be chosen as doner materials. Butanediol, a new type of agent, which is environmental-friendly and can induct disease resistance in plant, will be applied to those materials by root to produce disease resistance. With the help of Laser confocal Scanning Fluorescence Microscopy and physiology and biochemistry measurement, we will observe and analyze the changes of cell structure, pectin, xylogen, cellulose, hemicellulose after disease resistance is inducted in creeping Bentgrass, probe into the change rules in materials such as active oxygen, phytoalexin, antioxygen-ascorbic acid and GSH, related enzyme of defence system for antioxidant such as CAT, SOD, APOX and GR, the key enzyme such as PAL, CHS and CHI used for benzedrine metabolism and their products-phenols and flavonoid, to verify the effect of disease resistance made by BDO in creeping bentgrass. The gene expression level correlated disease resistance will be detected by PCR, which will expound the correlation between the gene expression level and phenotype of disease resistance, physiological response respectively. The mechanism of disease resistance induction based on ISR way and triggered by BDO in creeping bentgrass will be revealed systematically in cellular, physiology and molecular level, and it provides theoretical basis for follow-up study on the development of the agent for resistance induction as a new commercial product and on the application technology. So the project has its scientific significance and application value.

匍匐翦股颖美观、耐修剪，但根系浅，喜水，易感病。本项目选用匍匐翦股颖品种Penn A-1、Penn A-4和普特为材料，拟将环保的新型抗病性诱导剂丁二醇BDO，经根部施入材料后使之产生抗病性。采用荧光扫描共聚焦显微技术和生理生化测定法观察和分析诱导后匍匐翦股颖细胞结构以及果胶、木质素、纤维素、半纤维素变化；探讨细胞内活性氧、植保素、抗氧化剂（抗坏血酸和GSH）、氧化防御体系相关酶CAT、SOD、APOX和GR、苯丙胺代谢关键酶PAL、CHS和CHI及产物-酚类和黄铜类物质变化规律，探明BDO对匍匐翦股颖抗病性诱导效应；以RT-PCR技术分析诱导后材料抗病基因表达水平，阐明抗病基因表达量与其抗性表型和生理响应的相关性。通过项目实施，从细胞、生理和分子水平上系统揭示BDO引发的基于ISR方式的匍匐翦股颖抗病性诱导机理，为后续研发该诱抗剂新产品和应用技术提供理论支撑，具有重要科学意义和应用价值。

植物根际益生菌产生的挥发性化合物，可直接或间接影响植物的生长发育。人工合成的细菌挥发性化合物类似物，如丁二醇（2, 3-丁二醇和2R,3R-丁二醇），可诱导植物产生抗病性。本研究以常用草坪草匍匐翦股颖（Agrostis stolonifera）为试验材料，利用2, 3-丁二醇和2R,3R-丁二醇处理其幼苗后接种立枯丝核菌（Rhizoctonia solani），探索丁二醇对匍匐翦股颖抗褐斑病能力的诱导效应，并在此基础上揭示丁二醇诱发的基于ISR方式的匍匐翦股颖抗病性诱导机理。取得的主要研究结果有：（1）250 µmol/L的2, 3-丁二醇和100 µmol/L的2R, 3R-丁二醇能够有效诱导匍匐翦股颖对褐斑病的抗性，且2, 3-丁二醇的作用效果优于2R, 3R-丁二醇；（2）2, 3-丁二醇处理可提高匍匐翦股颖叶片SOD、APX和GR活性，增加ASA/DHA、GSH/GSSG比值，以清除感病植株细胞内积累的活性氧；（3）2,3-BD诱导可促使匍匐翦股颖叶片细胞结构发生变化，如上表皮细胞显著增大、叶绿体数量明显增加和皮层组织加厚等，同时2,3-BD诱导可增加叶片内木质素、纤维素、半纤维素和HRGP含量的积累，促进叶片木质化过程，加强植物细胞壁强度、以抵御病原菌的入侵；（4）2, 3-丁二醇可在增强苯丙烷代谢系统关键酶PAL、4CL及CHI活性的同时，促进苯丙烷代谢途径抗菌物质总酚、黄酮的合成，以及植保素合成相关的物质绿原酸和肉桂酸含量；（5）2, 3-丁二醇处理可引起匍匐翦股颖叶片内抗性相关转录因子（bHLH，C2C2-CO和NAC）的上调表达，以及抗病相关的基因，例如茉莉酸信号基因，LRR转膜蛋白激酶，PR5受体激酶，NBS-LRR结构域包含的R-基因也显著上调表达，即丁二醇可通过改变植物基因在诱导系统抗性（ISR）通路上的转录水平来诱导植物产生抗病性。.本研究筛选出了2, 3-丁二醇诱导匐翦股颖抗病性的有效浓度，并从细胞、生理和分子水平上系统揭示了2, 3-丁二醇诱导匍匐翦股颖抗褐斑病的作用机理（基于 ISR 方式），为后续研发该诱抗剂新产品和应用技术，草坪草抗病新品种的培育奠定了一定的理论基础，具有重要科学意义和应用价值。