【作者】 刘艳兵；

【导师】 林英任；

【作者基本信息】 安徽农业大学 ， 微生物学， 2008， 硕士

【摘要】 本论文采用目前世界上较新颖、先进的分类系统和多性状综合分类方法,根据形态解剖学、个体发育学、生态及生物学等特征,对采自安徽天堂寨国家森林公园、黄山风景区和牯牛降自然保护区的斑痣盘菌科（Rhytismataceae）3属的若干种进行了分类学研究。同时,采用9个重复性好的随机引物对散斑壳属（Lophodermium Chevall.）15个菌株进行ISSR-PCR扩增反应,通过ISSR分析结果结合表型性状的比较和分析,确定了部分形态学特征以及寄主种类差异在该类真菌种水平分类上的重要性,进一步明确了散斑壳属若干种内和种间的遗传多样性和亲缘关系。以Darker（1967）的分类系统为基础,同时参考Korf（1973）、Cannon&Minter（1986）、Kirk et al.（2001）等人的若干补充和修订,对采自安徽天堂寨国家森林公园、黄山风景区、牯牛降自然保护区以及保藏于安徽农业大学森保教研室（AAUFP）的标本进行鉴定、分类。共查明隶属于齿裂菌属（Coccomyces）、散斑壳属（Lophodermium）和斑痣盘菌属（Rhytisma）3属的8个种,其中包括2个新种,即棒囊齿裂菌（Coccomycesclavatus sp.nov.）和尖孢散斑壳（Lophodermiun acutisporum sp.nov.）;并且对6个国内已知种进行了补充研究,追加了这些种的新寄主和地理新分布。以组织分离法对采自天堂寨国家森林公园的阔叶树生斑痣盘菌活材料进行分离培养,共获得隶属于齿裂菌属（Coccomyces）、皮下盘菌属（Hypoderma）、散斑壳属（Lophodermium）和斑痣盘菌属（Rhytisma）的8号分离株,接菌于MA培养基上,定期观察、记载各菌株的菌落形状、大小、色泽、结构、气生菌丝和潜生菌丝状况、生长速度、分泌色素与否以及是否产生分生孢子等特征。利用氯化苄法对采自天堂寨国家森林公园的散斑壳属（Lophodermium Chevall.）15个菌株进行DNA提取。通过对模板用量、随机引物用量、Mg²⁺离子用量、dNTP用量、Taq酶用量和反应退火温度及时间、延伸温度及时间、循环次数等参数进行优化,建立了该类菌物ISSR-PCR反应的最佳体系,即在15μl反应体积中,DNA模板（45ng/μl）2.0μl,随机引物（10μM）0.6μl,MgCl₂（25mM）1.5μl,dNTPs（10mM）0.3μl,Taq酶（5U/μl）0.3μl,10×PCR buffer 1.5μl,ddH₂O 8.8μl。扩增程序为:94℃预变性2min;每个循环94℃变性1min,特定温度下退火60s,72℃延伸2min30s,共35个循环;循环结束后72℃延伸10min。利用筛选出9条重复性好、条带清晰的引物,使用优化的ISSR-PCR反应参数及程序对散斑壳属15个菌株的DNA进行ISSR分子标记。9个引物共扩增出154个位点,其中多态性位点数150个,占总数的97.40%。扩增位点数最多的是引物S45,有21个位点数;扩增位点数最少的是S12,有14个位点数,扩增出的片断长度大多在100-2000bp之间。采用UPGMA方法构建聚类树状图,遗传距离在7.08处,15个菌株被划分为6个类群,其聚类结果与表型性状分类极其相近。通过ISSR分析结果结合表型性状的比较和分析,确定了子囊果埋生位置,子座、子囊、子囊孢子、侧丝等形态学特征以及寄主种类差异等表型性状在该类菌物分类上的重要性。更多还原

【Abstract】 Some members of three genera in Rhytismataceae which collected from the south mountains of Anhui province were systematically studied with the most novelty and advanced poly-character integrated taxonomy method which is based on the characters of morphology,developmental biology,ecology and distribution.Fifteen Lophodermium strains were charaterized by ISSR-PCR reaction with 9 random primers whose gel electroersis diagrams can be amplified repeatedly and clearly.By the comparison of ISSR analytic results and the phenotypic characters,indicated the importance of some morphological characters such as the embedded position of ascomata,stromata,asci, ascospores,paraphyses and the category of hosts on the classification of species level. Relationships of some intraspecific and interspecific strains of Lophodermuim on angiosperms were affirmed.The main result is as follows:Some samples from the south mountains of Anhui province and Anhui Agricultural University Forest Protection（AUUFP） systematically studied with the techniques of modern classification.Morphology structure and development biology of these fungi were roundly examined according to taxonomic principles of Darker（1967）,Kor（1973）,Cannon & Minter（1986）,Kirk et al.（2001）.Among them,Coccomyces clavatus sp.nov,and Lophodermiun acutisporum sp.nov.were new species,and 6 known species of China were supplemented and studied in new hosts and new geography distribution.The living materials of fungi of Rytismataceae from Tiantangzhai National Forest Park were separated and cultivated by tissue isolation techniques.Eight strains of Coccomyces,Hypoderma rubi,Lophodermium and Rhytisma were obtained.The strains were cultured on MA culture medium,and then regularly observed and noted the shape, size,luster,structure,mycelium,the rate of growth,and if which produce pigment and conidiomata.Template DNA of 15 Lophodermium strains from Tiantangzhai National Forest Park were extracted by Benzyl Chloride.The ISSR-PCR amplified system was optimized from aspects of the dosage of template DNA,prime,Mg²⁺,dNTPs and Taq enzyme,and anneal temperature and time,elongation temperature and time,cycle number.The amplified diagrams were quite well.The final reaction system（15μl） included ddH₂O 8.8μl, 10×Buffer 1.5,Mg²⁺(25mmol.L^-1) 1.5μl,dNTPs(10mmol.L^-1) 0.3μl,primer(10umol.L^-1) 0.6ul,Taq enzyme(5U.μl^-1)0.3μl,template DNA(45ng.μl^-1) 2μl.Its amplified procedure was pre-denature at 94℃for 2min.cycles 35 times,and every cycle included denature at 94℃for 1min,anneal for 60s,elongation at 72℃for 150s.And the latest cycle elongation time is 10min.The DAN of 15 strains of Lophodermium were amplified with 9 collected primers by the optimal ISSR-PCR process,154 fragments were obtained,150 polymorphic ones among them,accounting for 97.4%.The most sites,21 ones,were amplified by S45,the second ones were 20.Fourteen sites were the least,amplified by Primers S12.The length of most fragments ranged from 100bp to 2000bp,too.From the clustering tree map which were established by the method of UPGMA.The result demonstrates that 15 Lophodermuim strains on angiosperms are clustered into 6 groups in genetic distance 7.08. By the comparison of ISSR analytic results and the phenotypic character,we can confirm the importance of some morphological character such as the embedded position of ascomata,stromata,asci,ascospores,paraphyses and the category of hosts on the classification of Lophodermium.更多还原