【作者】 韩翔；

【导师】 檀根甲；

【作者基本信息】 安徽农业大学 ， 植物病理学， 2004， 硕士

【摘要】 通过对安徽省瓜蒌炭疽病标样的采集，经过室内分离、纯化培养得到8个菌株，然后采用同工酶电泳技术对分离到的8个瓜蒌炭疽菌菌株进行辅助分类与鉴定，详细研究了各种环境因子和营养物质对瓜蒌炭疽菌生长的影响及其分泌的胞外酶种类和活性，并对该病的化学和生物防治效果进行了探讨。本论文的主要研究结果包括：1 瓜蒌炭疽病病原菌的鉴定根据形态特征、培养性状、致病力及寄主范围的测定结果，按照Sutton的分类系统分种检索，将引起安徽省瓜蒌炭疽病的病原菌初步鉴定为Colletotrichum orbiculare分离到的8个菌株间在PDA培养基上的培养性状存在差异。分离到的8个瓜蒌炭疽菌菌株对瓜蒌的致病力表现出一定的差异。其中S5致病力最强。S9的致病力次之，其余的6个菌株通过Duncan’s新复极差测验显示致病力间无显著差异。未针刺处理组未发病，可能由于瓜蒌果实表皮较厚而无法造成有效侵染。瓜蒌炭疽菌在人工创伤接种条件下能够侵染包括珙桐科、木兰科、大麻科、葫芦科、悬铃木科、黄杨科、桑科、旋花科、蔷薇科、茄科等10科13种植物。2 瓜蒌炭疽菌生理生态的研究2.1瓜蒌炭疽菌越冬能力病原菌在0℃～15℃范围内的病菌均可安全越冬。-5℃中未能分离出瓜蒌炭疽菌。在土壤含水量为0-25%范围时，病菌均能安全越冬。瓜蒌炭疽菌在种子内部和种子表面均能安全越冬。瓜蒌病果、病叶、病种子等是引起田间发病的主要初侵染来源，且对越冬条件要求不严格。2.2 环境因子对瓜蒌炭疽菌的影响瓜蒌炭疽菌菌丝生长适温在21℃-30℃之间，其中以28℃最适菌丝生长。低温对菌丝无致死作用，只能抑制其生长，恢复常温后菌丝仍具生长能力，而高温则可丧失菌丝的生活力。孢子在10℃以下和40℃时均不能萌发，而在15℃-36℃之间均可萌发，且孢子萌发率差异不显著，均在96%以上。各菌株在RH50%—100%范围中，菌落速率差异不显著。在不同的RH条件下炭疽菌分生孢子萌发率均较高（>70%），差异不显著。在PDA中培养时，各菌株在pH4-12之间均可生长，以pH5时菌落直径最大。从各菌株液体培养的菌丝干重来看，以pH5-7菌丝干重处于高峰值，为生长的最适范围。各菌株在pH4-11之间的孢子萌发率差异不显著，从pH10开始逐渐下降，到pH12时孢子几乎不萌发。各菌株在不同的光照条件下的菌落直径差异不显著，说明瓜蒌炭疽菌菌丝生长对<WP=9>光照条件要求不严格。2.3 营养物质对瓜蒌炭疽菌的影响加入碳源促进各菌株的生长。但瓜蒌炭疽菌对不同碳源的利用程度不同。其中对葡萄糖、果糖和蔗糖的利用率最大，而对淀粉、甘露醇、半乳糖的利用较差，对乳糖几乎不能利用。各菌株在不同碳源和对照培养基上的孢子萌发率均较高，差异不显著。瓜蒌炭疽菌对有机氮中各类氨基酸（精氨酸、赖氨酸除外）、牛肉浸膏、酵母浸汁和无机氮中的硝态氮利用较好，对无机氮中的铵态和亚硝酸态氮几乎不能利用。对有机氮中的尿素的利用率还有待研究，因为同对照相比，尿素在平板中培养时的菌落直径很小，但在液体培养时菌丝干重较重，两者差异很大。不同生长因子(维生素)对炭疽菌菌丝生长有一定的影响，但促进作用不很明显，仅VB6作用后，菌落直径同对照组一样，其余各种维生素对菌丝生长促进作用不大。各种维生素的不同浓度之间对菌丝生长也存在着差异。以3μg/ml对炭疽菌的生长促进作用最大。维生素对瓜蒌炭疽菌的具体作用还不明确。2.4培养基优化的研究采用二次通用旋转组合设计方法对瓜蒌炭疽菌菌株S1和S9的培养基最佳组合进行了研究，建立了具有较好预测性能的培养基反应模型，并用试验数据验证了该模型的可靠性，利用该模型对培养基最优组合的各种重要单因子要素对菌丝生长量反应规律及交互作用进行了探讨，并对影响两个菌株生长的因子进行了比较，结果表明：选择的三个因子（pH、葡萄糖、组氨酸）能够较好的反映出对各菌株生长影响的情况。三个因子对S1与S9菌落直径影响大小顺序为：pH>葡萄糖>组氨酸，说明了pH对两菌株在平板上平铺生长的影响最大，组氨酸影响最小；三个因子对S1菌丝干重影响大小为：葡萄糖>组氨酸>pH，对S9菌丝干重影响大小为：葡萄糖>pH>组氨酸，说明了葡萄糖在两菌株液体培养过程中起的作用最大，而pH与组氨酸对两菌株的作用则表现出一定的差异。两因子交互作用对S1菌落直径的影响的大小顺序为：葡萄糖与组氨酸互作>pH 与葡萄糖互作>pH与组氨酸互作。对S9菌落直径的影响的大小顺序为：葡萄糖与组氨酸互作>pH与组氨酸互作>pH与葡萄糖互作。说明了两菌株在对各因子互作效应中的表现存在着一定的差异。2.5同工酶电泳分析对瓜蒌炭疽菌的8个菌株和其他4株分别分离自苹果、玉米、西瓜和山茱萸的炭疽菌进行了八种同工酶聚丙烯酰胺凝胶电泳的比较研究。这八种同工酶分别是：酯酶、多酚氧化酶、过氧化物酶、苹果酸脱氢酶、乙醇脱氢酶、谷氨酸脱氢酶、半乳糖脱氢酶和可溶性蛋白。对同工酶酶谱资料的聚类分析，在0.64的相似性水平上将12个菌株归为3组，a组包括：S1、S2、S3、S4、S5、S9、S10、XG八个菌株；b组包括：S6、PG和SZY四个菌株；c组包括：YM一个菌株；结果表明供试12个炭疽菌菌株<WP=10>内酯酶、苹果酸脱氢酶更多还原

【Abstract】 Mongolian snakegourd (Trichosanthes kirilowii) is an important traditional Chinese medicine. Trichosanthin (TCS) can be extracted from the root of Mongolian snakegourd, which can inhibit the propagation of HIV. Along with the enlargement of culture area of Mongolian snakegourd, its anthracnose caused by Colletotrichum sp. has been a serious problem and it can bring numerous yield losses. However, so far its pathogen is unknown for us. In the dissertation, eight isolates of Colletotrichum were obtained from diseased fruit and foliage of Mongolian snakegourd, single-spore-cultured, grown on potato dextrose agar (PDA) from Qian’shan county, Anhui province, China P.R. Then, Polyacrylamicide gel electrophoretic analysis of isozymes of 8 isolates of Colletotrichum from Trichosanthes Kirilowii and 4 isolates of four other apple、watermelon、corn and dogwood were studied as an accessorial method for the taxonomy of anthracnose pathogens. The effects of diverse environmental factors and nutriments on the growth of Mongolian snakegourd anthracnose pathogens, the variety and activity of extracellular enzyme secreted by Mongolian snakegourd anthracnose pathogens were studied. Also the effects of the fungicides and biocontrol stain B-24 to control this disease were studied. The experimental results were summarized as follows:1. The Identification of Mongolian snakegourd Anthracnose Pathogen According to the experimental results of cultural and morphological characteristics, pathogencity and host range test, meanwhile, consulted the classification criteria of Colletotrichum edited by Sutton (1980), the pathogen of Mongolian snakegourd anthracnose was identified as Colletotrichum orbiculare (Berk. &Mont.) Arx primarily. The cultural characteristics were some differences among eight isolates from diverse locations on PDA medium.The results of pathogenicity test to Mongolian snakegourd showed some difference among eight isolates. Isolate S5 had the strongest pathogenicity to Mongolian snakegourd, while isolate S9 showed the weakest pathogenictiy among eight isolates. The pathogenicity of other six isolates showed no apparent differences by means of Duncan’s test. Mongolian snakegourd didn’t be infected by unwounded inoculation.The host range of C.orbiculare was investigated by inoculating several dozens of plants with the methods of conidial suspension wounded and unwounded inoculation. The results indicated that Mongolian snakegourd anthracnose pathogen could infect 13 kinds of plants from 10 families by means of wounded inoculation. These plants included Camptotheca acuminata, Magnolia grandiflora, Humulus scandens, Citrullus lanatus, Platanus hispanica, Broussonetia popyrifera, Euonymus japonica, Ipomoea batatas, Cucurbita pepo, Luffa cylindrical, Malus pumila, Cucumis sativa, Lycopericon <WP=12>esculentum,etc.2. Study on the physiological and ecological characteristics2.1 The Overwinter Survival Ability of Mongolian snakegourd Anthracnose PathogenMongolian snakegourd anthracnose pathogen could overwinter safely at the range from 0℃ to 15℃. Due to the low temperature, C. orbiculare could not be isolated from diseased organism. When the water content in soil was at the range from 0% to 25%, C.orbiculare could overwinter safely indoor, too. C.orbiculare was isolated from the outer and inner of the overwinter seeds. So, the diseased fruits, diseased leaves and diseased seeds were proved to be the major initial effective sources of anthracnose. C.orbiculare on Mongolian snakegourd was not strict with the overwinter conditions.2.2 The Effect of Diverse Environmental Factors on the Growth of Colletotrichum orbiculareThe results showed as follows: the growth optimum temperature ranged from 21℃ to 30℃.The mycelia grew best at 28℃. Low temperature such as 0℃,inhibited hyphal growth and conidia germination only, but was not the deadly line. C.orbiculare could not survive at high temperature such as 40℃. Conidia couldn’t germinate under 10℃ or above 40℃. The percentage of conidia germination of diverse isol更多还原