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题名: 兜兰属和杓兰属几种植物的菌根研究
作者: 袁莉
学位类别: 博士
答辩日期: 2008-05-30
授予单位: 中国科学院昆明植物研究所
授予地点: 昆明植物研究所
导师: 杨祝良
关键词: 兜兰 ; 杓兰 ; 兰科菌根 ; 菌根真菌 ; 胶膜菌 ; 专一性 ; 优势菌根真菌 ; 幼苗菌根化
学位专业: 植物学
中文摘要: 兜兰属(Paphiopedilum)和杓兰属(Cypripedium)都属于杓兰亚科,具有较近的亲缘关系,但是二属植物的分布、生境及生活习性却又存在着较大的差异。兜兰属多为常绿植物,地生或附生,分布于热带、亚热带地区;杓兰属植物冬季休眠,皆为地生,分布于北半球温带地区。在种子萌发、幼苗种植或成年植株异地栽培中,兜兰属植物较易成功,但杓兰属植物却存在较大困难。在兰科植物的整个生活史中,菌根都起着重要的作用,菌根形成是兰科植物进化史中的一个关键事件。本文对兜兰属和杓兰属6种植物的菌根结构和菌根真菌、半自然条件下杏黄兜兰菌根化及两种兜兰根部存在的一些“机会真菌”进行了研究。主要研究结果和结论如下: 1、对硬叶兜兰(P. micranthum)、杏黄兜兰(P. armeniacum)、长瓣兜兰(P. dianthum)、黄花杓兰(C. flavum)、紫点杓兰(C. guttatum)和西藏杓兰(C. tibeticum)根解剖结构进行对比观察,发现成年的兜兰和杓兰植株都有菌根,兜兰根被细胞多层,其中生长有大量真菌;杓兰植物没有根被,表皮由一层活细胞构成。在兜兰中,只有部分皮层细胞形成菌根;而在杓兰中,菌根真菌几乎侵染了所有的皮层细胞。在杓兰植物的根皮层中,存在着大量的淀粉粒,而兜兰植物的皮层却很少见到淀粉粒。根被有利于兜兰植物在干湿多变的生境中保持水分,菌根真菌侵染皮层的程度可能反应了落叶植物杓兰对菌根营养的需求程度大于常绿植物兜兰,皮层中的淀粉粒有助于杓兰度过休眠期。 2、运用真菌分离与直接扩增植物根中菌根真菌的ITS片和mtLSU片段相结合的方法,研究了上述六种兰花的菌根真菌。在200多个植物根的DNA样品中,得到真菌ITS序列175条,mtLSU序列80条。将这些序列与GenBank中的有关序列对比,结果表明大部分真菌(160条ITS序列,76条mtLSU序列)都属于胶膜菌科(Tulasnellaceae)真菌,只有少数为其它真菌(“机会真菌”),如镰刀菌(Fusarium solani)、枝孢霉(Cladosporium lignicola)和青霉(Penicillium sp.)等。首次发现兜兰属植物的菌根真菌和杓兰属植物菌根真菌一样,同属于胶膜菌科真菌,具有非常强的专一性。基于ITS序列构建的系统树显示,形成菌根的真菌可以聚为十一个分支,各分支可能代表了一个或两到三个亲缘关系较近的真菌物种。在与兜兰或杓兰形成菌根的多个分支真菌中,各种杓兰或兜兰都能与两个以上的分支的真菌形成菌根。不同真菌对于环境的适应能力不同,与多个分支的真菌形成菌根能增强兜兰或杓兰对于环境改变的适应能力。 3、基于ITS序列构建的系统树,首次发现在与一种兰花形成的多个分支真菌中,每种兰花一般都有一个或两个分支的真菌是其主要的菌根真菌,我们称之为优势菌根真菌。生长在相似小环境中的不同兜兰或杓兰具有一些相同的菌根真菌,但其优势菌根真菌却不相同。何种真菌能够成为优势菌根真菌,这在很大程度上是由植物本身决定的,环境因子影响次之。 4、营养充足的条件下,杏黄兜兰可以脱离菌根真菌而萌发并生长发育成幼苗。由种子萌发的杏黄兜兰无菌幼苗移栽到兰圃温室,在半自然条件下,经过一段时间的适应后,便长出新根。本研究发现幼苗的老根不形成菌根,但新根却能形成菌根,并首次对杏黄兜兰菌根化过程进行了跟踪,对菌根真菌进行了分子鉴定,发现其属于专一性很强的真菌(如 Clade 8 和 Clade 6)。首次发现硬叶兜兰成年植株从野外移栽到昆明和兴义的兰圃温室栽培一段时间后,优势真菌由原来的 Clade 11 变为 Clade 8(美孢胶膜菌 Tulasnella calospora)。美孢胶膜菌能和许多兰科植物形成菌根,但是我们发现其并不与杓兰属植物形成菌根。杓兰植物成年植株异地栽培后生长发育不良的原因之一可能是新环境中缺乏其专一性菌根真菌或环境不适宜于其菌根真菌的生长。兜兰成年植株异地栽培后优势菌根真菌的改变和半自然条件下杏黄兜兰菌根化的成功,在一定程度上解释了兜兰比杓兰容易栽培的原因,也为兰科植物试管苗快速菌根化和该科植物的迁地保护指明了方向。 5、除胶膜菌科真菌外,兜兰和杓兰的根部还存在一些“机会真菌”,这类真菌和兜兰或杓兰的关系尚不清楚。分别用硬叶兜兰与 4 种“机会真菌”及杏黄兜兰无菌苗与 7 种“机会真菌”共培养发现,其中部分真菌是兜兰的致病菌,部分对其生长无害。选择两种无害的“机会真菌”M2(Chaetomium sp.)和M7(Umbelopsis sp.)做了进一步的研究,发现它们都能促进硬叶兜兰的幼苗生长,M2能为硬叶兜兰提供一定的氮源,但M7促进幼苗生长的原因尚不清楚。兰科植物根部的真菌物种复杂多样,它们与其宿主植物的关系也许各有不同,在研究兰科植物与真菌相互作用时,不应忽视这些“机会真菌”。
英文摘要: The genera Paphiopedilum and Cypripedium both belong to the same subfamily Cypripedioideae of the family Orchidaceae. Although they are closely related to each other, there are great differences in ecological distributions, growth habitats and life forms between the two genera. The former is terrestrial or epiphytic, distributed in the tropics or sub-tropics, and usually evergreen, whereas the latter is terrestrial, deciduous, and distributed across most of temperate Northern Hemisphere, and has an “adult dormancy” period every year. The asymbiotic seed germination is much easier in Paphiopedilum than in Cypripedium, as is the case with seedlings plantation and the survival of adult plants transplanted from the wild. It is well recognized that mycorrhiza plays a vital role in the orchid life, and the development of mycorrhizal relationship is also a crucial event in the evolution of the Orchidaceae. In this thesis, studies on mycorrhizal structure and mycorrhizal fungi of three Paphiopedilum species and three Cypripedium species, seedling mycorrhization of P. armeniacum in semi-natural conditions, and some “opportunistic fungi” in the roots of two Paphiopedilum species were carried out. The main results are summarized as follows: 1. The root cross-sections of P. micranthum, P. armeniacum, P. dianthum, C. flavum, C. guttatum and C. tibeticum were observed, and mycorrhizae were found in adult plants of all six orchid species. The epidermis of Paphiopedilum root zones was a multiseriate velamen filled with many hyphae while roots of Cypripedium had a simple epidermis of living cells. In the roots of Paphiopedilum, mycorrhizal infection was confined to a proportion of cortex cells, while Cypripedium exhibited a pattern of dense infection. Lots of starch granules were found in the cortex of Cypripedium but seldom in Paphiopedilum. The velamen cells benefit Paphiopedilum plants by holding water from their habitats where soil water supplies sometimes changed dramatically. The density of mycorrihzal infection might indicate that deciduous Cypripedium plants need more mycotrophy than evergreen Paphiopedilum species, and starch granules in the cortex helped Cypripedium over the winter. 2. Mycorrhizal fungi of six orchids were studied by the method of isolation of mycobionts combined with molecular sequencing (ITS and mtLSU) of fungi directly from the mycorrhizae. 175 ITS and 80 mtLSU fungal sequences from over 200 DNA samples of orchid roots were obtained. It was found that most of them (160 ITS sequences and 76 mtLSU sequences) belonged to the family Tulasnellaceae, while only a few were other fungi (“opportunistic fungi”), such as Fusarium solani, Cladosporium lignicola, Penicillium sp., etc. The phylogenetic analysis revealed for the first time that Paphiopedilum mycorrhizal fungi were members of the fungal family Tulasnellaceae, just the same as those in Cypripedium, having a strong specificity. The phylogenetic tree constructed from ITS sequences of mycorrhizal fungi from Cypripedium species and Paphiopedilum species could be divided into 11 clades, and each clade might represent one or two or even three related fungal species. Among all the mycorrhizal fungal clades, every orchid studied was associated with more than two fungal clades. The ability to be associated with several fungi improves the orchid adaptation to the environment variations. 3. Based on the phylogenetic tree constructed from ITS sequences, it was found for the first time that each orchid had one or two main fungal clades among several fungal clades associated, which we called “dominant mycorrhizal fungi”. Different species of Paphiopedilum or Cypripedium having similar habitats might have some identical mycorrhizal fungi, yet the dominant mycorrhizal fungi for each orchid species were not the same. Which fungi could be the dominant ones are determined by the plant, yet influenced with the habitats. 4. The seeds of P. armeniacum could germinate freely and develop into seedlings in the absence of mycorrhizal fungi when sufficient nutrients provided. In semi-natural conditions, P. armeniacum seedlings developed from asymbiotic seed germination could be successfully transplanted to an orchid nursery in a greenhouse. For a certain period of time after transplantation, new roots were found to be mycorrhized while the old ones not. We traced the process of P. armeniacum seedling mycorrhization and identified mycorrhizal fungi to be its specificity fungi (such as Clade 8 and Clade 6) by molecular method for the first time. It was newly found that the dominant mycorrhizal fungi of P. micranthum switched from fungal Clade 11 to Clade 8 (Tulasnella calospora) when transplanted from the wild to the orchid greenhouse in Kunming or Xingyi. T. calospora is the mycorrhizal fungus of Paphiopedilum and many other known orchids, but not Cypripedium. The reason why adult Cypripedium plants grew poorly when transplanted might be due to the shortage of their specificity mycorrhizal fungi in the new environments or due to the new environments incompatible with their fungal partners. The dominant mycorrhizal fungi switch in P. micanthum when transplanted and successful mycorrhization of P. armeniacum seedlings in semi-natural conditions may be responsible for the easiness of Paphiopedilum plantations as compared with Cypripedium, and also throw some light on the axenic seedlings mycorrhization and conservation of the family. 5. Apart from Tulasnellaceae, there existed some “opportunistic fungi” in the roots of Cypripedium and Paphiopedilum. The relationships between orchids and them are still largely unknown. By co-cultivations of four “opportunistic fungi” with axenic P. micranthum seedlings, and seven with P. armeniacum seedlings, it was found that some of the fungi caused death of the seedlings while others not. Two harmless opportunistic fungi, M2 and M7, identified as Chaetomium sp. and Umbelopsis sp. respectively by ITS sequences, were selected to perform a further study. Data showed that both fungi stimulated the seedling growth. It was found that M2 could supply seedlings with some nitrogen nutrients. However, the reason for M7 remains unclear. The fungi living in the orchid are very diverse and abundant, and their relationships with their host plants are much more complicated. To understand their mutual actions between orchid and fungi, the opportunistic fungi should not be neglected.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.kib.ac.cn/handle/151853/278
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兜兰属和杓兰属几种植物的菌根研究.袁莉[d].中国科学院昆明植物研究所,2008.20-25
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