【作者】 于红梅；

【导师】 林英任；

【作者基本信息】 安徽农业大学 ， 森林保护学， 2008， 硕士

【摘要】 本文采用较先进的菌物学分类技术,从形态解剖、个体发育、生态及地理分布、性状培养等方面,对生长在石栎属植物上的斑痣盘菌科中一些种进行较系统的分类研究。同时采用ISSR技术对皮下盘菌属的23个菌株和齿裂菌属的12个菌株进行种内及种间的遗传多样性分析研究,进一步弄清了各分类群之间的系统发育关系。以Darker（1967）、Korf（1973）、Cannon & Minter（1983、1986）、Johnston（1986）、Spooner（1991）、Kirk et al。（2001）等人提出的若干分类原则为主要依据,对源于皖南山区和大别山区生于石栎属植物上的斑痣盘菌科菌物进行分类研究。共查明隶属于齿裂菌属（Coccomyces）、散斑壳属（Lophodermium）2属7种,其中包括等径散斑壳（Lophodermium isodiametum）、拟尖丝齿裂菌（Coccomyces mucronatoides）2个新种;对5个已知种进行了补充研究,使其形态结构、个体发育等特征得以完善,同时追记了它们的寄主新记录和地理新分布。对各种均提供了形态解剖学特征的描述与图解、寄主及生境的记载,并对其相近类群、寄生性与致病性等加以讨论。编制了各属的二又式分种检索表。分别对皮下盘菌属（Hypoderma）的23个分离株和齿裂菌属（Coccomyces）的12个菌株进行了DNA提取,ISSR-PCR扩增体系的优化,并对提取的DNA进行ISSR-PCR扩增反应。采用单因素循环法,寻找一个适合本试验的ISSR-PCR反应体系,以提高其稳定性与重复性。笔者从模板浓度、引物浓度、Mg²⁺浓度、dNTPs浓度、Taq酶用量、退火温度及时间、延伸时间、循环次数等方面对齿裂菌属和皮下盘菌属两属的ISSR反应体系进行了优化,最终确定的ISSR反应体系（15μl）含:10×Taq酶缓冲液为1.5μL,DNA模板为8ng/μL,MgCl₂为2.5mmol/L,dNTP为0.15mmol/L,引物浓度为0.4μmol/L,Taq酶为1.0U,ddH₂O为9.0μL。扩增程序:94℃预变性2min,接着进行35个循环:94℃变性1min,48～54℃退火（根据不同的引物退火温度来定）1min,72℃延伸2.5min。循环结束后72℃延伸10min。筛选出8个随机引物,使用优化的ISSR-PCR反应参数及程序对齿裂菌属12个菌株的DNA进行扩增,共扩增出134个位点,其中多态性位点数为129个,占总数的96.26%。扩增位点最多的是引物S45,有20个位点数;扩增位点数最少的则是引物S13,只有13个位点数。扩增出的片断长度大多在150-2000bp之间。采用UPGMA方法构建聚类树状图,通过表型性状分类和ISSR分析结果比较,二者能较好地统一起来。按优化的ISSR-PCR反应体系对皮下盘菌23个菌株的DNA进行扩增,8个随机引物共扩增出134个位点,其中多态性位点数为127个,占总数的94.78%。扩增位点最多的是引物S42,有21个位点数;扩增位点数最少的是引物S31、S32、S46,仅有15个位点数。扩增片断长度也是位于150-2000bp之间。使用UPGMA法构建聚类树状图,在遗传相似系数0.58处,23个菌株被分成4类,该结果表明悬钩子皮下盘菌种内遗传差异体现在子囊果形状、大小及埋生深度、侧丝顶端形状、子囊和子囊孢子大小,以及寄主、着生部位和分布区域方面;皮下盘菌属的种问差异主要表现在形态学特征、寄主及着生部位等方面。更多还原

【Abstract】 Systematics of part of species of Rhytismataceae on Lithocarpus spp.was studied from morphology,development biology,ecology,distribution,culture characters etc.with the techniques of modern mycology.And phylogenetic relationships of intraspecies and similar interspecies of 23 Hypoderma strains and 12 Coccomyces ones were studied.It is clear further to the phylogenetic relationships among these groups.Some samples of Rhytismataceae on Lithocarpus spp.from the mountains of south and west Anhui and were systematically studied according to the taxonomic principles of Darker（1967）、Korf（1973）、Cannon & Minter（1983、1986）、Johnston（1986）、Spooner （1991）、Kirk et al.（2001）.7 species among 2 genera were identified.Among the taxa there were 2 new species including Lophodermium isodiametum sp.nov.,Coccomyces mucronatoides sp.nov.;The studies of 5 known species involved in adding characters of morphology,development biology,new record of host and geographical distribution. Morphological characteristics were described and illustrated,and hosts and distribution were recorded for all the species,and their similar groups,parasititism and nosogenesis were discussed.Dichotomous key of the species was compiled.The DNA of 12 Coccomyces strains and 23 Hypoderma ones were respectively extracted.The ISSR-PCR reaction system was optimized,and the DNA from intraspecies and interspecies were amplified.It is necessary to find a ISSR reaction system suitable for this experiment by the method of the single factor screening in order to promote the stability and repetition.The reaction system of Coccomyces and Hypoderma were optimized in the quantity of primers,template,Mg²⁺,dNTPs,Taq DNA polymerase and annealing temperature,annealing time,elongation and cycle numbers.The optimal ISSR-PCR reaction system included 2.5μl 10×Taq Buffer,8ng/μl template,0.15mmol/L dNTPs,9.0μl ddH₂O,2.5mmol/L Mg²⁺,1.0UTaq DNA polymerase,0.4μmol/L random primers in the system of 15μl.The amplification process as follow:94℃beforehand denaturalization for 2 minutes,35 cycles;94℃denaturalization for one minute, 48～54℃（determined according to the annealing temperture of different primer） annealing for 60 seconds,72℃elongation for 2.5 minutes,after the last circulation,then 72℃elongation for 10 minutes.The DAN of 12 strains of Coccomyces were amplified with 8 collected random primers by the optimal process,134 fragments were obtained,129 polymorphic ones among,accounting for 96.26%.The most sites,20 ones,was amplified by Primer S45.The least ones,13 ones,was amplified by Primer S13.The length of most amplified fragments ranged from 150bp to 2000bp.The phenotype was well consistent with the results of ISSR based on the clustering tree map which were established by the method of UPGMA.The DAN of 23 strains of Hypoderma were amplified with 8 collected random primers using the reaction process,134 fragments were obtained,127 polymorphic ones among them,accounting for 94.78%.The most sites,21 ones,were amplified by S42.Fifteen sites were the least,amplified by Primers S31、S32、S46.The length of most fragments ranged from 150bp to 2000bp,too.From the clustering tree map which were established by the method of UPGMA,23 strains were classified into 4 sorts at the genetic similarity coefficient of 0.58.By the synthetical comparison of ISSR analytical results and the phenotypic characteristics,the genetic difference of intraspecific strains of Hypoderma rubi was embodied in the size,shape and deep of ascomata,the shape of paraphyses,the size of asci and ascospores,and the difference of hosts and regions.The interspecific difference of Hypoderma is mainly embodied in morphological characteristics,host kinds and inhabiting position.更多还原