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万佛山国家森林公园斑痣盘菌科的种群结构研究及散斑壳属两近似种的分子确认

Studies on Population Structure of Rhytismataceae from Wanfoshan National Forest Park and Its Sequence Analysis of Two Related Lophodermium Species

【作者】 杨中周

【导师】 林英任;

【作者基本信息】 安徽农业大学 , 森林保护学, 2010, 硕士

【摘要】 本文采用目前世界上较先进的菌物学分类技术和多形状综合分析方法,从形态解剖、个体发育、生态及地理分布、性状培养等方面,对万佛山国家森林公园的斑痣盘菌科成员进行较系统的分类研究。同时对散斑壳属的两个近似种(松针散斑壳Lophodermium pinastri和乔松散斑壳L. pini-excelsae)13个菌株的rDNA-ITS序列进行PCR扩增与测定,并与GenBank中已有的相关序列进行同源性分析;利用MEGA邻接法(neighbour-joining method , NJ )构建系统发育树。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,进一步弄清了此二种的种内及种间的遗传多样性和亲缘关系。以Darker(1967)、Korf (1973)、Cannon & Minter(1983、1986)、Johnston(1986)、Spooner(1991)、Kirk et al. (2008)等人提出的若干分类原则为主要依据,对源于该区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、皮下盘菌属Hypoderma、小鞋孢盘菌属Soleella、特里尔盘菌属Terriera等5属11种,其中1个新纪录属Terriera,1个新种T.huangshanensis,1个新纪录种T.minus;并对9个国内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。编制了二叉式分种检索表。特里尔盘菌属Terriera与散斑壳属Lophodermium的区别在于前者唇缺,子座覆盖层有一强度碳化、无明显结构的延伸物,其下方多具一淡色的薄壁丝组织薄层,覆盖层与基部层毗邻处具一近无色或淡灰褐色的矩胞组织或角胞组织,侧丝常于顶部膨大、分枝,有时相互交织或粘结而形成囊层被。其与皮下盘菌属Hypoderma的区别在于后者唇通常发育良好,侧丝顶部罕膨大,子囊孢子一般椭圆形、圆柱形至棍棒状。生于毛枝格药柃落叶上的黄山特里尔盘菌T. huangshanensis Z. Z.Yang, Y. R. Lin & C. L. Hou以其纵缝两侧各有一强烈碳化的无明显结构的延伸物;覆盖层与基部层毗邻处为不发达的角状细胞组织;唇缺等特征而划入Terriera属。T. huangshanensis在本属成员中很特别,因为它的子囊同期成熟,子实层成熟以后往往突破覆盖层,甚至向上扩展以至超出覆盖层顶部,故将其定为新种。结果显示:在形态解剖学方面,L. pinastri除子座基部层、线纹及分生孢子器开口特征外,其余重要特征与L. pini-excelsae具有很大的相似性。L. pinastri的ITS序列的G+C平均含量(51.5%)大于L. pini-excelsae的G+C平均含量(50.1%)。从系统发育树可以看出,L. pinastri与L. pini-excelsae的13个菌株构成了两个独立进化的分支I和II,分支I由A,B两个小的分支组成,分支A由寄生在欧洲赤松和同为欧洲本土树种的欧洲黑松上的7株L. pinastri菌株组成。在分支B中,寄生在赤松和千头赤松上的L. pinastri菌株优先聚在一起。进化分支II由分别分离自日本五针松和华山松上2个L. pini-excelsae菌株组成,日本五针松和华山松系单维管束松亚属五针松组植物,而进化分支I中的寄主为双维管束松亚属油松组植物。由此可以看出,寄主对种内遗传多样性也产生较重要影响,产地与种内遗传差异无明显关联。ITS序列分析可以作为区分这两个近似种的一种有效手段。

【Abstract】 Members of Rhytismataceae which collected from the Wanfoshan National forest park 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, and culture characters etc. At the same time, rDNA-ITS sequences of 13 strains were amplified and determinated and the genetic homology between these sequences and other related sequences in GenBank were analyzed for the two related species of Lophodermium, L. pinastri and L. pini-excelsae. A phylogenetic tree of these strains was constrcuted by MEGA, using neighbour-joining method. Results indicated the importance of some morphological characters and the category of hosts on the classification of species level. Genetic diversity and relationships of some intraspecific and interspecific strains of two related species were further affirmed.Some samples of Rhytismataceae from the park were systematically studied according to the taxonomic principles of Darker(1967)、Korf(1973)、Cannon & Minte(r1983、1986)、Johnston(1986)、Spoone(r1991)、Kirk et al.(2001). 11 species among 5 genera were identified. The 5 genera were Coccomyces, Lophodermium, Hypoderma, Soleella and Terriera. Among the taxa, Terriera was a newly recorded genus from China, T.huangshanensis was a new species, and T.minus was a new record for China. The studies of 9 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.Terriera is distinguished from Lophodermium by the absence of lip cells, the extension comprising strongly carbonized tissue with no obvious cellular structure, the excipulum closely adhering to sides of the covering stroma, the subhymenium with a region of near-colorless or ficelle, hyaline, textura prismatica or textura angularis at the edge of the ascoma, and the paraphyses sometimes swelling at the apex and agglutinating to form a dark epithecium. Hypoderma is similar to Terriera, but differs in the usually well-developed lip cells, the paraphyses swelling rarely at the apex, and the ellipsoidal, cylindrical to clavate ascospores. T.huangshanensis on leaves of Eurya muricata var. huiana (Kobuski) Hu & L.K. Ling, is placed in the genus Terriera on the basis of the presence of strongly carbonized extension adjacent to both sides of the ascoma opening, poorly developed angular cells in the tissue at the marginal parts of the ascoma and the absence of lip cells. T. huangshanensis is miraculous within the genus because of the asci synchronously ripening and the hymenium often overtopping the extension of the covering stroma when mature.The results showed that important characteristics of L. pinastri were remarkably similar to those of L. pini-excelsae, except those of stroma basic layer, zone lines and opening of conidiomata. G+C content of L. pinastri(51.5%) was more than that of L. pini-excelsae(50.1%). The phylogenetic tree indicated that 13 strains of L. pinastri on diploxylon pines and L. pini-excelsae on haploxylon pines were sharply classified into clade I and clade II respectively; clade I was comprised of clade A and clade B; 7 strains of L. pinastri from Pinus. sylvestris and P. nigra, which were the European native trees, formed clade A; clade B consisted of 4 strains of L. pinastri from P. densiflora and P. densiflora cv.‘Umbraculifera’; clade II was composed of 2 strains of L. pini-excelsae from P. parviflora and P. armandii respectively. It can be seen from this that hosts had a definite impact on intraspecific genetic diversity and origin was not significantly associated with intraspecific genetic difference. ITS sequence analysis was an effective means to distinguish the two related species.

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