【作者】 曹荣花；

【导师】 高克祥； 刘晓光；

【作者基本信息】 山东农业大学 ， 植物病理学， 2008， 硕士

【摘要】 内生真菌Choiromyces aboriginum Mü1W1C6、Stachybotrys elegans Mt2W1C1和Cylindrocarpon sp 5/97-12 Mt2W3C4是从德国康斯坦茨波登湖区芦苇中分离的优势菌株。本文通过研究三株内生菌与多种植物病原菌的拮抗活性及对温室植物病害的防治,为进一步明确其生防机制奠定了良好的理论基础,同时也为研究内生真菌与病原物和寄主植物之间的互作,为Mt2W1C1、Mt2W3C4和Mü1W1C6应用于植物病害生物防治并提高其生防效果提供了理论依据。研究内容及结果如下:1.平皿对峙试验。结果表明,Mü1W1C6,Mt2W1C1和Mt2W3C4对11种植物病原菌菌丝的生长有不同程度的抑制作用,有四种类型是型互作用:(a)菌落接触后成僵持状态,都不再生长;(b)僵持状态后内生菌部分覆盖病原菌的菌落;(c)僵持状态后内生菌的菌落会完全覆盖病原菌菌落;(d)两菌落生长均不受影响。显微观察表明内生菌Mü1W1C6、Mt2W1C1和Mt2W3C4单独培养时没有寄生现象发生,而当和病原菌对峙培养时,内生菌可以贴生和缠绕病原菌的菌丝、侵入到寄主菌丝体内部生长、甚至可以穿透寄主菌丝。病原真菌由于内生真菌的寄生作用,菌丝往往表现为变形、干瘪甚至消解等现象。2.细胞壁降解酶的测定。内生真菌经SMCS培养基以胶体几丁质做碳源,纤维素培养基以微晶纤维素(CM)、羧甲基纤维素(CMC)、立枯丝核菌(R. solani)或瓜果腐霉(P. aphanidermatum)的细胞壁做碳源,诱导测定了几丁质酶、β-1,3-葡聚糖酶、滤纸酶(FPA)、β-葡萄糖苷酶(BG)、内切-1,4-β-D-葡聚糖酶(EG)和外切-1,4-β-D-葡聚糖酶(CBH)的活性。结果显示β-1,3-葡聚糖酶活性最高,最佳培养基为SMCS培养基,立枯丝核菌的细胞壁也是很好的碳源;纤维素酶和几丁质酶活均较低。3.内生真菌在温室对部分植物病害的防治试验。选取室内拮抗活性较好的两个菌株Mü1W1C6和Mt2W1C1,在温室采取根部接菌法,将内生菌接种于黄瓜、番茄、菜豆、小麦和玉米幼苗根部,发现Mü1W1C6对蔬菜灰霉病、黄瓜苗期猝倒病和白绢病有很好的防效:Mü1W1C6在菜豆上对灰霉病的防效达48.94%,对黄瓜猝倒病防效达88.49%,对黄瓜白绢病的防效为53.83%;而Mt2W1C1虽对灰霉病有一定的生防潜力,但对种子出苗率及秧苗生长有不良影响。4.内生真菌Mü1W1C6与病原菌在植物体内的互作。光学显微镜和电子显微镜观察了Mü1W1C6与瓜果腐霉P. aphanidermatum在黄瓜根部的侵染互作,结果显示,Mü1W1C6可成功定殖于黄瓜苗的根部,提前接种Mü1W1C6 48 h可有效抑制瓜果腐霉P. aphanidermatum在黄瓜根部组织细胞内的扩展和定殖。更多还原

【Abstract】 New dominant strains of fungal endophytes Choiromyces aboriginum Mü1w1c6, Stachybotrys elegans Mt2W1C1 and Cylindrocarpon sp 5/97-12 Mt2W3C4 were isolated from common reed in Boden Sea of Kanstanz, Germany. By studing antagonistic activity between three endophytes and several phitopathogenic fungi, and by biocontrol activty on some diseases in greenhouse, this study made a good theory foundation for knowing of biocontrol mechanism.At the same timethis study made theory foundation for studying interactions among endophytic fungi, phytopathogenic fungi and plant, and also for making use of Mü1W1C6、Mt2W1C1 and Mt2W3C4. The major congtent and results are as follows:1. Dual-culture experiment.The results showed that Mü1W1C6, Mt2W1C1 and Mt2W3C4 can parasitize hyphae of eleven phytopathogenic fungi. There are four modes of interactions during : (a) Deadlock with mycelial contact for ever; (b) Partial replacement after initial deadlock at a distance; (c) Endophytic fungus complete replacement phytopathogenic fungi after initial deadlock with mycelial contact. (d) No influence on each other. Microscopic observation indicated that there was no mycoparasitism when Mü1W1C6、Mt2W1C1 or Mt2W3C4 grew alone. However, they can attached, coiled and even entried into hyphae of phytopathogenic fungi in dule cultures, and causing distortion, witheredly even dispels and so on.2. Assay of cell wall-degrading enzymes.Activities of cell wall-degrading enzymes including chitinases,β-1,3-glucanases, filter paper activity (FPase),β-glucosidases (BG), exdo-1,4-β-D-glucanase (EG), exo-1,4-β-D-glucanase (CBH) were induced and assayed, when endophytic fungi grew in colloidal chitin culture,basal medium supplemented with either microcrystalline cellulose powder, carboxymethy1 cellulose (CMC), cell wall preparation (CWP) of P. aphanidermatum or R. solani. The result showed that activity ofβ-1,3-glucanases was higher than others. The best culture is SMCS, and CWP of R. solani is good carbon. Activities if ellulose and chitinases were relatively lower. 3. In vivo effects of biocontrol agents on some diseases in greenhouse.Aggressive isolates Mü1W1C6 and Mt2W1C1 with high antagonism, were used to control disease on cucumber, tomato, bean, wheat and corn through root inoculation. Promising control efficacy was obtained at the absent of Mü1W1C6, including bean grey mould (48.94%) and cucumber damping-off (88.49) as well as sclerotinia rot (53.83). Though Mt2W1C1 has potential biocontrol activity on bean grey mould, it affects seeds from germination and throughout development.4. Interactions between endophytic fungi Mü1w1c6 and phytopathogenic fungi in planta.The interactions between Mü1w1c6 and the phytopathogenic fungi P. aphanidermatum and cucumber root were investigated under light and electron microscopy. It showed that Mü1w1c6 can successfully colonize the root of cucumber seedlings. If pre-inoculation of C. aboriginum Mü1w1c6 48h, Mü1w1c6 can significantly hinder the infection and colonization of the phytopathogenic P.aphanidermatum in the root of cucumber seedlings. It can successfully protect cucumber seedlings from damping-off caused by P. aphanidermatum.更多还原