枸杞内生真菌与宿主植物及炭疽菌互作的超微结构和细胞化学研究

More and more investigations revealed that many endophytic fungi have very great potential of biocontrol on plant diseases. Howere, it has been lack of understanding about interaction mechanism between endophytic fungi and host plant or pathogens. In this research, Lycium barbarum L. which was important sandy medicinal plant of Ningxia, will be chosen as a model. To trace the process of infection with green fluorescent protein (GFP), as a reporter gene, it is one of the most effective ways to study interaction between endophytic fungi and host plant. By light microscopy, scanning or transmission electron microscopy and immunocytochemical technique, we will elucidate the cytologic features in the process of infection and colonization of endophytic fungi from L. barbarum L. on host plant, and the effects of defence responses on defense enzyme system, cell wall modification and accumulation of reactive oxygen species (ROS) under adversity stress from Colletotrichum gloeosporioides Penz. On this basis, Ultrastructure and cytochemistry of hyphal interaction between endophytic fungi from L. barbarum L. and pathogen Colletotrichum gloeosporioides Penz.,and mycoparasitism, antifungal and synergistic interactions between cell wall degrading enzymes and antibiotics were investigated by electron microscopy and immunocytochemical technique. Further exploration is for the resistance mechanism of mutualistic symbiosis induced by endophytic fungi from L. barbarum L.. Through completion of this project, in theory, it was to enriches study on endophytic ecology and resource research about L. barbarum L. in the extreme environment. In practice, it was to explore a new way on biocontrol of chinese wolfberry anthracnose.

越来越多的调查研究表明，内生真菌对植物病害有很大的生防潜力，但在内生真菌侵染定殖过程中与宿主植物及病原菌互作的机制方面仍缺乏全面的了解和认识。本研究拟以宁夏重要的沙生药用植物枸杞为研究对象，利用绿色荧光蛋白标记对枸杞内生真菌在宿主植物中的侵染定殖行为进行示踪。采用光学显微镜、扫描电镜、透射电镜和免疫细胞化学技术，探寻内生真菌与宿主植物互作的细胞学特征，以及在枸杞炭疽菌胁迫下内生真菌与宿主植物互作中保护酶体系、细胞壁修饰、活性氧累积等防卫反应对枸杞炭疽菌侵染的影响。在此基础上，探寻内生真菌细胞壁降解酶、抗生素等在重寄生、抗生及协同抗菌中的作用，进一步揭示内生真菌与宿主植物互惠共生的抗病机制。项目的完成丰富了我国枸杞内生菌生态与资源的研究内容，为生产上实现枸杞病害生物防治探索一条新途径。

以宁夏枸杞为研究对象，在摸清其内生真菌及深色有隔内生真菌（Dark septate endophytes，DSE）侵染特征、多样性和分布的基础上，以枸杞胶孢炭疽菌（Colletotrichum gloeosporioides Penz.）为靶标菌，筛选高活性拮抗菌株，探究其抑菌、抗生和重寄生等生防潜力；筛选产几丁质酶和β-1,3-葡聚糖酶的菌株，探究其产酶条件；通过内生真菌对宿主的侵染定殖的光学显微和透射电镜的观察，示踪内生真菌的侵染行为，测定内生真菌对枸杞植株生长、抗病和防御酶活性的影响，初步揭示其抗病作用机制。以枸杞内生真菌NQ8GII4（Fusarium nematophilum）为供试菌株，利用PEG介导的遗传转化方法将质粒pFL2、pDL2、PKTN和rGNT转入原生质体中，获得GFP标记的重组子。优化菌龄、酶浓度、酶解时间、渗透压稳定剂对原生质体质量和产量的影响因素等条件，获得NQ8GII4菌株原生质体制备体系。对重组子的生物学特性和致病性进行检测，获得在生物学特性和致病性上与野生型菌株均无显著差异的GFP标记的内生真菌荧光菌株，用于对宿主侵染结构和侵染机制的研究。