【作者】 王艳娜；

【导师】 王贵禧； 朴春根；

【作者基本信息】 中国林业科学研究院 ， 森林培育， 2007， 博士

【摘要】 梨果实轮纹病是造成梨果实烂果的主要原因，由于轮纹病菌是在幼果期潜伏侵染，在果实成熟及贮运期间发病，控制和防范都有很大困难。目前对于轮纹病菌造成果实腐烂的机制以及果实对病原菌的抵抗机制并不十分明确。控制轮纹病害的首要任务是要认识轮纹病害的发病机理，这其中包括病原真菌对果实的致病性和果实对病原菌的抗病性。本研究从形态结构、生理状态、生化水平上研究轮纹病菌与梨果实的互作关系。探寻病原菌产生毒害物质的种类及产生条件；果实的主要抗病性物质及其在发育和贮藏期间的变化。为阐述梨轮纹病的发病机理提供理论依据，也为其它果实病害的研究提供可借鉴的研究方法。1．研究了不同发育期套袋处理、不同发育期解袋暴露处理、不同发育期涂抹和刺伤接种轮纹病菌分生孢子悬液处理的鸭梨果实采后不同时期的发病规律。结果表明，鸭梨轮纹病菌主要侵染期为盛花期后50～90d，但盛花期110d以后仍然可以侵染，而盛花期后53d内不易感染轮纹病菌；轮纹病病原菌更容易通过伤口侵染；在采后梯度降温贮藏条件下，鸭梨轮纹病的集中发病期为采后40～60d；接种孢子悬液使果实的发病期提前、发病率提高。2．梨皮组织具有荧光性物质，采用组织切片和荧光显微镜观察，皮孔特异荧光性孔底母细胞可能对防治轮纹病菌从皮孔进入有关，此外角质层、石细胞单宁细胞为鸭梨果实的抗病原菌结构。刺伤果肉的扫描电镜观察结果显示，孢子以果肉为生长基质快速萌发并在果肉细胞间隙生长繁殖，48h菌丝已经产生大量菌丝缠绕在果肉细胞表面。透射电镜结果表明，孢子接种24～36h后菌丝已进入果肉细胞内部，被侵染果肉出现质壁分离、细胞质降解等现象。利用β-1，3-葡聚糖免疫标记单克隆抗体进行的免疫细胞化学的研究结果表明，胞壁沉积物还有β-1，3-葡聚糖，侵入果肉细胞的菌丝壁也存在β-1，3-葡聚糖。3．利用HPLC测定果实生长期、贮藏期和发病前后果实中有机酸的含量和种类，在离体条件下测定不同有机酸对轮纹病菌生长的抑制作用。成熟鸭梨中主要含有柠檬酸、苹果酸、莽草酸。50mg／100g以上的苹果酸和柠檬酸、60mg／100g以上的乳酸，10mg／100g以上琥珀酸、8mg／100g的奎宁酸均能显著抑制病原菌的生长，且抑菌培养基pH值多在5.0以下。病原菌侵染诱导乳酸和琥珀酸的产生。4．鸭梨在幼果期与抗病性相关的酚类物质主要有没食子酸、绿原酸、咖啡酸，随成果实发育成熟这些酚类含量逐渐下降。果实在不同时期对病原菌的抵抗能力与果实中酚类物质变化关系密切。贮藏期酚类物质含量随着果实生理代谢总体呈下降趋势，对抵抗病原菌扩展能力减弱。10mg／100g的没食子酸、1mg／100g的绿原酸、5mg／100g的单宁酸、0.05mg／100g的香豆酸添加到PSA培养基能够显著抑制病原菌菌落生长；添加浓度高于7 mg／100g没食子酸，40mg／100g绿原酸，0.5mg／100g单宁酸，1mg／100g儿茶酸和1mg／100g的香豆酸的能够显著抑制菌丝的生长。5．本文研究了鸭梨果实受轮纹病原菌侵染后及其在不同的生长期、贮藏期防御酶活性的变化。结果表明，对鸭梨健康果实接种轮纹病菌后过氧化物酶（POD）、过氧化氢酶（CAT）、超氧化物歧化酶（SOD）活性均显著高于对照，说明这些酶对鸭梨果实抵抗轮纹病密切相关。鸭梨果实在幼果期活性较高的酶主要是POD、CAT；在近成熟期与果实抗病性相关酶主要是SOD，SOD活性在盛花后75d快速升高，果实成熟时达到最大值；而鸭梨果实在贮藏期间与抗病性相关的这3种酶活性总体呈下降趋势，对抵抗病原扩展能力减弱。果实在不同时期对病原菌的抵抗能力与果实中防御酶活性变化密切相关。6．低温和病原菌的胁迫下，ATP含量显著降低，抵御逆境条件下生理变化。在病原菌侵染过程中为了保持细胞内pH稳定，调节酸的种类，接种病原菌24h内H⁺-ATPase活性显著上升，而后逐渐下降；而Ca²⁺-ATPase在病原菌侵染过程中活性先升高后降低，细胞内Ca²⁺浓度过高或维持时间过长，促进果肉细胞坏死。7．为了筛选鸭梨轮纹病最优毒素产生条件，从鸭梨轮纹病斑分离纯化的轮纹病菌，在不同条件下进行培养并进行生物测定，选择健康成熟的鸭梨果实，采用针刺法对粗毒素的致病力进行测试。最终确定有利于病菌产毒的温度为30℃、pH值为7.5、培养时间为10d、培养基为PS+少量的梨汁，黑暗及静置培养条件。粗毒素中蛋白含量很高，占69.1％，其次是糖27.2％。蛋白质为单链多肽蛋白。更多还原

【Abstract】 Pear ring rot,caused by Botryosphaeria. berengeriana f. sp. piricola, is an economicallyimportant disease in China. Because pathogenic germs infect the host pear latently at growthstage, and induce rot during the postharvest period, it is difficult to control the disease. Up tonow, it is still not clearly that the mechanisms of fruit decay and defence reaction due to M.Kawatsukaii Hara. It is necessary to know the mechanisms of ring rot disease, which includedhow the pathogen developing in pear fruit and cause the decay, and how the fruit resistant todisease development. We are planning to study fruit-pathogenic fungus interactions frommorphological level, physiological level, and biochemical level. We expect that we could findthe compounds and there producing condition of pathogenic fungus toxins; that the resistantsubstance and its role and content change during growth and storage. This will provide theorybasis for pathogenesis of pear ring rot and feasible way to control the post-harvest disease.1. In order to make sure the latent infection and rotting stage of the ring rot disease causedby Physalospora piricola, the ’Ya’ pear fruit were bagged and un-bagged at different days afterblossom, and the smearing and inoculation with P. piricola conidio-spores were also conductedregularly during the growth of fruit. The results showed that the main infection time of P.piricola to the pear fruit was from 50d to 90d after blossom, the infection was also occurredafter 110d of blossom, but it was not infected before 50d of blossom. The pathogen was easilyinfected the fruit through tiny wounds. The rotting fruit was mainly took place at the stage of40～60d after harvest at the step-cooling storage condition. The fruit inoculated with sporesolution at growth stage was more easily decayed than the control.2. As fluorescent substances exist in skin of pears, we successfully made a anatomicalobservation of pear fresh tissues under fluorescent microscope. Fluorescent mother cell atbottom of peel lenticel had function to defense agaist pathogen, in addition, cuticle, sclereid,tannin cells were defensive structure, also. Surface of the smearing and inoculation with P.piricola conidio-spores flesh were observed by scanning electron microscopy （SEM）;Conidiospore germination and appressorium formation completed 24h after inoculation, themycelium developed more lushly and mass hyphae surround the pulp cells after 48h after inoculation. The ultrastructure of flesh were observed by transmission electron microscopy（TEM） showed the hyphae were invaded into pulp cell during 24～36h after inoculation. Usingimmunogold labeling with monoclonal antibody againstβ-1,3-glucan and secondary antibody,β-1,3-glucan were localized in cell wall appositions and fungal cell walls.3. The use of HPLC method enable us to successfully study the component and content oforganic acids. Citric acid, malic acid, shikimic acid were main composition of total acid. Thepathogen growth was inhibited with higher concentration of 50mg/100g Citric acid,50mg/100g malic acid, 60mg/100g lactic acid, 10mg/100g succinic acid, 8mg/100g quinic acid,pH level lower than 5.0. Pathogen invading the flesh induce some new kind of organic acids,such as lactic acid and succinic acid.4. The main defense phenolics with high content at the young stage of pear fruit werephenolics, chlorogenic acid,gallic acid and caffeic acid, which decreased at premature stage.Pear fruit disease resistance reduced with the content of these phenolics all declined duringstorage. The defense resistance of fruit at different stage was closely related to the change of itsphenolics. The pathogen growth was inhibited with higher concentration of 1mg/100gchlorgenic acid, 10mg/100g gallic acid, 5mg/100g gallotannic acid 0.05mg/100g coumaricacid in PSA culture medium,and 7mg/100g gallic acid, 40mg/100g chlorgenic acid,0.5mg/100g gallotannic acid, 1mg/100g catehin 1mg/100g coumaric acid.5. The activities of POD,SOD and CAT in ’Ya’ pear fruit inoculated with Botryosphaeriaberengriana and sound pear fruit in growth and storage were studied. The results showed thatmetabolic defense induced by the pathogen including the activities of POD, SOD and CAT, andthe activities of these enzymes markedly increased, which suggested that those enzymes wereclosely-related to pear fruit against B. berengriana infection. The main defense enzymes withhigh activity at the young stage of pear fruit were POD and CAT, the activities of these enzymeat rapid growing stage and pre-mature stage of fruit decreased sharply, except PAL with peakactivity at premature stage of fruit. The main defense enzymes with high activity at thepre-mamre stage of pear fruit were SOD, The activity of SOD increased rapidly to highest levelafter 75 days after bolssom, and the activities of these enzymes all declined during storage.6. In order to defense low temperature and pathogen stress physiological effect, ATPcontents observably decreased. H⁺-ATPase could modulate pulp cell pH level and organic acid concentration, thus 24 hours after inoculation the activity of H⁺ -ATPase markedlyincrease, then decline constantly. Ca²⁺-ATPase activity increase rapidly and then decreasedalong with time of inoculation. Higher content of Ca²⁺ resulted in fresh dead and decayed.7. The best conditions for phytotoxin production of pear ring rot pathogen were studied.The results showed that toxicity of toxin produced in condition such as, pH value 7.5, 30℃, 10days after duration of culturing, culture medium containing PS and a little pear juice, stillcultivation under darkness. The composition of the also be determined. The ratio of protein andsugar was 69.1% and 29.6% respectively. The species of protein was single chain andpolypeptide.更多还原