【作者】 王建华；

【导师】 王惠； 黄丽丽；

【作者基本信息】 西北农林科技大学 ， 应用化学， 2012， 硕士

【摘要】 苹果树腐烂病是苹果树体上重大病害之一，它是由苹果黑腐皮壳菌（Valsa maliMayabe et Yamada）引起一种苹果树枝干皮层腐烂病害。该病菌可以侵染苹果树的主枝、主干、果实等多个部位，使苹果树主枝干枯死，树势衰弱。近年来，该病在我国各苹果主产区普遍发生，严重降低了苹果产量和品质，给苹果产业带来了严重的经济损失。其原因与病原菌侵染致病的毒性因子及致病机理不明关系极大。因此，本项目从研究苹果树腐烂病菌分泌的致病毒素入手探索苹果树腐烂病菌侵染致病的物质基础，利用抑制莴苣种子芽生长及烟草叶片接种法检测生物活性；GC-MS法分析发酵液中的有机酸成分；用HPLC法检测发酵液中各种成分对根皮苷的降解情况，分析降解根皮苷的主要成分；用大孔树脂吸附、乙酸乙酯萃取、硅胶柱层析、薄层层析等手段，对发酵液进行进一步的提取分离，并用波谱法对所得毒素成分进行化学结构的初步鉴定。对明确病菌毒素在病害发展中的作用机制，指导该病害的田间防治具有重大意义。初步生测结果表明，20d发酵液对莴苣种子芽生长的抑制率达到57.91%。发酵原液、发酵产生的蛋白类物质、去蛋白后的发酵液对烟草叶片均有致病作用。GC-MS结果显示发酵液中含有7种有机酸，分别是2-羟基丙酸、羟基乙酸、丙二酸、琥珀酸、羟基丁二酸、棕榈酸、十八烷酸，其相对含量分别为4.42%、0.56%、0.56%、59.10%、5.87%、3.66%、0.65%。以根皮苷为碳源发酵培养病原菌10d后每100mL发酵液中菌丝干质量为48mg。同时验证了苹果腐烂病菌及其发酵液对苹果树根皮苷的降解作用，病原菌对根皮苷的降解率为92.46%，发酵液对根皮苷的降解率为99.88%。为了进一步明确苹果树腐烂病菌降解根皮苷的主要成分，对发酵液分离得到的蛋白质部分、有机酸以及去蛋白后的发酵液分别进行了根皮苷的降解实验。结果发现，分离得到的蛋白类物质（1mg/mL）与根皮苷作用5d，根皮苷的降解率为90.4%，有机酸类对根皮苷的降解率仅为1.6%，去蛋白剩余发酵液对根皮苷的降解率为5.9%。有机酸与酶共同作用对根皮苷的降解率为98.7%。根皮苷的降解率随蛋白液质量浓度和作用时间的增加而增大。表明苹果腐烂病菌对根皮苷起降解作用的主要物质为其代谢产生的蛋白质，其可能为细胞壁降解酶类。有机酸可以使酶活性加强。采用活性跟踪法对除蛋白剩余发酵液液进行进一步的提取分离，得到一个小分子毒素成分，初步推测该化合物可能为：2-乙基-3-羟甲基-4-异丙氧基-苯甲酸。更多还原

【Abstract】 Apple valsa canker is one of the major diseases of apple tree, which is caused by ValsaMali Mayabe et Yamada and leads to decay of apple tree stem cortex. The pathogen can infectthe apple tree branches, trunks, fruits and other multiple parts. It makes the apple trees weakand branches dead. In recent years, the disease occurs widespreadly in the main appleproducing areas of our country, severely reducing the apple output and quality, leading seriouseconomic loss to apple industry. The reason has greatly related to the unclear pathogenicvirulence factors and its pathogenic mechanism.Therefore, the project started from the pathotoxins secreted from pathogen duringinfecting. Inhibition of lettuce seed bud growth and tobacco leaf inoculation method was usedfor the detection of biological activity. GC-MS was used for the analysis of organic acidscomposition in fermentation broth. Using HPLC assay for the detection of phlorizindegradation by various components in fermented liquid and finding out which is the maincomposition leading the degradation of phlorizin. Using macroporous adsorption resin, ethylacetate extraction, silica gel column chromatography, thin layer chromatography and othermeans for further extraction and separation of fermentation liquid. Spectroscopic method wasused for chemical structure identification of the toxin component. It has great significance tomake clear the pathogenic mechanism and guide field disease control.The bioassay results indicated that: the inhibition rate on lettuce seed bud growth by20d’fermentation liquid reached57.91%and fermentation liquid, proteins, residul fermentationliquid all have pathogenic role on tobacco leaves. GC-MS results showed that the filtratecontained7kinds of organic acids and respectively was2-hydroxy propionic acid, glycolicacid, malonic acid, succinic acid, malic acid, palmitic acid, octadecanoic acid. Their relativecontents were4.42%,0.56%,0.56%,59.10%,5.87%,3.66%,0.65%. The mycelium dryweight was48mg per100mL in10day’s fermentation liquid using phlorizin as carbonsource. The degradation rate of phlorizin by the pathogen was92.46%after10days’ reaction,while the rate reached99.88%by starch fermentation filtrate. Proteins of1mg/mL candegrade90.4%of phlorizin after5days’ treatment. Organic acids and residual liquid, on theother hand, could merely degrade1.6%and5.9%of phlorizin, respectively. The phlorizindegradation rate is98.7%by organic acids and proteins. And the phlorizin degradation rate increased as the proteins concentration and reaction time increased. Proteins, which possiblywere cell wall degradation enzymes, produced by pathogen play an important role in phlorizindegradation. The organic acids can strengthen enzymes’ activity. The activity tracking methodwas used for further extraction and separation of the proteins free residual fermentation liquidand a small molecular toxin component was got. Its spectral character indicated that thecompound’s chemical structure may be:2-ethyl-3-methyl-4-isopropyl-benzoic acid.更多还原