【作者】 屈海泳；

【导师】 魏胜林； 蒋立科；

【作者基本信息】 安徽农业大学 ， 果树学， 2003， 硕士

【摘要】 由灰葡萄孢（Botrytis cinerea Pers.ex Fr）所致的灰霉病（Botrytis cinerea Pers），是我国的草莓重要病害之一，不但发生普遍，且为害较重，还涉及其他园艺作物，也是世界性的重要的园艺作物病害，目前又出现单抗、双抗甚至多抗的灰霉病菌菌株，防治该类病害的任务艰巨。化学防治草莓灰霉病除带来环境污染和病原菌产生抗性以外，重要的是，草莓是一种鲜食果品，因对农药的残留问题特别敏感，给人的健康有心理影响。通过筛选和利用抗灰霉菌的有益微生物及其代谢产物的生物防治方法，正日益成为灰霉病控制中的一条重要而有效的途径。根据木霉良好的生防特性，采用木霉为出发菌株，通过离子束注入筛选出高效生物防治该类病害的菌株，是一条新的防治病害的有效途径。 木霉菌（Trichoderma spp．）属于半知菌类的丝孢菌纲，丛梗孢目，丛梗孢科，广泛存在于土壤、根围、叶围、种子和球茎等生态环境中。由于木霉菌的广泛适应性，广谱性及多机制性，一直是植病生防学家研究的重点对象，目前研究的热点是通过生物技术方法有目的地改造从自然界分离筛选到的木霉菌，以获得新型菌株，例如用紫外线照射诱变、原生质体融合和基因转化技术等来研制开发新型木霉菌株。 本文利用N⁺离子束注入生物产生能量沉积、动量的传递、质量沉积和电荷交换等四个方面的效应，通过在20kev的能量下，以30×2.6×10¹³N⁺／cm²、50×2.6×10¹³N⁺／cm²、70×2.6×10¹³N⁺／cm²、90×2.6×10¹³N⁺／cm²等四个不同剂量的离子束注入木霉，使木霉遗传物质发生突变，并对突变菌株在几丁质培养基上生长的菌落的大小、产孢量的多少、麸皮发酵液酶活性的大小和与灰霉菌对峙培养抑制草莓灰霉菌的抑制力的大小等四个方面进行筛选，优选出高拮抗草莓灰霉病菌的木霉突变菌株—T90-1。为了能使该突变菌株能更好的拮抗灰霉菌，从不同的培养基中筛选出能使T90-1产几丁质酶活性高的麸皮液体培养基，并对影响木霉发酵液几丁质酶活性的外界因素分析，结果表明发酵液在40℃，pH6.2，有Mg⁺⁺存在情况下，发酵液几丁质酶活性较高。 通过上述研究，并与原菌株及发酵液作用效果比较发现，T90-1与草莓灰霉菌对峙培养中，镜检T90-1产生附着丝、缠绕、穿透和溶解等多种拮抗机制来抑制灰霉菌的生长；T90-1无菌过滤后的发酵液稀释不同的倍数（50～200倍），防治草莓灰霉病均有防治效果，其中以50倍的发酵液防治效果较明显，当喷洒在灰霉菌丝上，8个小时完全降解灰霉菌丝壁，让原生质裸露出来杀死灰霉菌；用T90－l的抱子防治草毒灰霉病最佳使用时间，应在草荡发病前2天喷施的结果比发病后2天喷施感染指数降低达90％；T90．l的泡子和其无菌过滤后的发酵液喷施在草毒上后，通过同工酶凝胶电泳和紫外光分光度计检测酶的活性，表明能明显提高草萄的过氧化物酶（POD）和多酚氧化酶（PPO）的活性；通过在马铃薯培养基上连续8周的转接，每周检测其鼓皮发酵液的几丁质酶活性，在几丁质培养基产抱量的多少和生长率大小，统计表明离散度极小，差异不显著，而且 T90八的发酵液在常温下（25℃）存放 8周，每周检测发酵液几丁质酶活性，统计显示几丁质酶活性的标准差只有0．393％，变异度极小，几乎没有变化，有较长的货架期，说明＊90－1不仅有较强的抑制灰霉菌的作用，而且其遗传性稳定：＊90－1的无菌过滤后的发酵稀释液（100倍～200倍）对草毒保鲜不仅有较好的效果而且对桃灰霉菌、番茄灰霉菌、辣椒灰霉菌及黄瓜灰霉菌等几种园艺作物的灰霉病都有较强的抑制作用。 与其他同类方法相比，本文的创新点在于利用离子束注入木霉进行诱变，筛选出木霉突变菌株一T90d，并研究其鼓皮发酵液和菌丝抑制草毒灰霉菌机理的及测定其泡子和发酵液对草毒的过氧化物酶和多酚氧化酶的影响。同时该突变菌株遗传稳定性强，发酵液的货架期较长，初步展示了该菌的突变菌株作为生防制剂的广泛应用前景。更多还原

【Abstract】 There wre many kind of strawberry fungal diseases in China.Among all of fungal disease, strawberry Botrytis cinerea Pers, which was provoke by Botrytis cinerea Pers.ex Fr, was comparatively the most wide-spread and relatively the most harmful ones. Botrytis cinerea Pers.ex Fr can invade other horticultural plant else. What’s more,it has been single resistant, double resistant, even multi resistant. So controlling this kind of diseases is hardy undertaking. It is important that strawberry is direct eat and pesticide residue is very sensitivity. Through screening and utilizing profitable fastidious controlling Botrytis cinerea Pers is increasingly important means. Trichoderma has superior biocontrol character. Through ion beam influxing Trichoderma and sceening highly antagonizing Botrytis cinerea Pers is new and available way.Trichoderma belongs to Deuteromycotina - Hyphomycetes , Hyphomycetales -Hyphomycetaceae, Trichodermapers.ex Fr.It extensively exists in ecological environment of soil , rhizoplanex surface of leaf, seeds and bulbodium etc.In 1932,Wanding first discovered that Trichoderma lingnorm can parasitize many kind of pathogenic fungus in many plants. So he proposed that Trichoderma should be used in biologically controlling soil-borne disease of plant. The development work about biological control effects of Trichoderma was stared from this time. What’s more, Trichoderma has abroad adaptability, broad spectrum and multi- mechanism, so it is emphatically researched by many scholars who studied biological control of plant disease.Along with development of biotechnology, Trichoderma has been started to purposefully utilize biotechnology to transform and gain new type of strain, but it was only separated and selected from nature in the past. Now for example making use of ultraviolet radiation irradiation to get induction mutation, protoplast fusion and genie transformation technology, we could research and develop new-style Trichoderma strain.Optimizing mutagenic strain of Trichoderma that can high antagonize strawberry Botrytis cinerea Pers is T90-1 through ion beam implanting into Trichoderma to make genetic material generate mutation. Then the size of colony that mutagenic strain cultured on clitin cultivation medium, the number of producing spore, the measure of mill offals fermenting liquor and the value of inhibition to Botrytis cinerea Pers etc were four waysscreening.T90-1 and Botrytis cinerea Pers confront each other on cultivation medium . When T90-1 made contact with Botrytis cinerea Pers and subsequently the pathogen colonies were either overgrown or invaded by T90-1, leading pathogen to death. Zymolytic liquid of T90-1 can decompose mycelial wall that account for T90-1 excreteing chitinolytic enzymes during zymolysis.50X diluent of fermenting liquor can decompose mycelial wall of Botrytis cinerea Pers within 8 hours and let protoplast denudate.The spore and fermenting liquor of T90-1 can not only restrain Botrytis cinerea Pers but also increase disease resistance of strawberry. After spraying the spore and fermenting liquor of T90-1 on leaves of strawberry, enzyme activity of peroxidase and polyphenoloxidase were improved through analyzing isozyme of gel electrophoresis and ultraviolet spectrophotometer checking and measuring enzyme activity.T90-1 was continuously resetted on PDA cultivation medium eight weeks. Every week detecting the size of colony that mutagenic strain cultured on clitin cultivation medium , the number of producing spore, the measure of mill offals fermenting liquor, statistical results indicated that degree of separation is very little and odds is no markedness. Fermenting liquor was keeped in normal temperature(25) eight weeks and detected enzyme activity every week. Statistical results indicated that the standard deviation of enzyme activity is only 0.393%. So diversity is little and almost no variation.In addition, fermenting liquor of T90-1 had preferable effect on keeping strawberry fresh and could inhibite Botrytis ciner更多还原