【作者】 张春英；

【导师】 戴思兰；

【作者基本信息】 北京林业大学 ， 园林植物与观赏园艺， 2008， 博士

【摘要】 杜鹃花科(Ericaceae)大多数植物的根系在自然生境中形成一种特殊类型的内生菌根共生体,即杜鹃花类菌根(也称为欧石南类菌根)(Ericoid mycorrhizas,ERM).ERM对于改善植物营养吸收、增强对重金属离子的抗性、缓解环境因子的胁迫等方面具有重要的作用。人工接种ERM真菌对某些杜鹃花科植物的生长有明显促进作用。为了发掘和利用菌根的潜能,满足生态建设和园艺生产的需要,国际园艺学界对ERM进行了相关研究,特别是近年来这一问题受到广泛关注。我国杜鹃花科植物分布广泛,但对ERM研究几乎空白,尚未见到相关正式报道。云锦杜鹃(Rhododendron fortuneiL.)是原产我国的杜鹃花科杜鹃花属大叶常绿杜鹃花种类之一,观赏价值高且抗逆性突出,为珍贵的育种资源,因而在国际园艺史上占有重要地位。但是以该植物为代表的大叶常绿杜鹃花的引种栽培,一直是困扰我国园林园艺界的难题。本研究以云锦杜鹃为试验材料首次对我国ERM及其菌根真菌多样性进行分析,获得了大量的ERM真菌资源和显著的幼苗接种效应,将对推动我国ERM的研究、菌根技术在园林中的应用及杜鹃花引种栽培和繁育等技术问题的解决具有重要的意义。论文通过对云锦杜鹃菌根显微结构、分离真菌的形态及其遗传多样性、接种效应等方面的探索性研究,获得了以下主要结论:1.利用扫描电镜、锥虫兰染色压片和树脂半超薄切片法首次对自然生境中云锦杜鹃的毛根菌根侵染率和显微结构进行研究。结果显示:毛根表面不形成根毛结构,而是包裹着一层或致密或疏松的菌丝。菌根真菌的侵染一般发生在营养根和生长根的成熟根段,总侵染率比较高,都在65%以上。菌根与毛根解剖结构类似,由1层表皮细胞、1-2层皮层细胞和狭小中柱组成,表皮细胞内具有典型的ERM菌丝体结构。云锦杜鹃菌根90%以上含有菌丝体的表皮细胞壁加厚不明显。菌根表皮细胞内有两类形态差异明显的菌丝复合体:一类为粗菌丝形成紧密菌丝圈和另一类为细菌丝形成松散菌丝圈。2.作者对Pearson & Read(1973)建立的杜鹃花科植物根系内生真菌分离技术进行了优化,首次改良马丁氏培养基并成功应用于ERM真菌的分离,提高了真菌分离的效率。试验从4个云锦杜鹃自然分布地的根系中分离了280个菌株,开始了我国ERM真菌资源多样性的研究。通过对菌落形态、菌丝体显微特征、rDNA-ITS片段的RFLP图谱分析,把280个菌株分为17个类型(14个RFLP类型和3个未能进行分子鉴定的类型)。根据rDNA-ITS序列比对和Bootstrap检验结果:云锦杜鹃菌根分离真菌的主体仍是子囊菌,其rDNA-ITS序列与已知分离菌株序列相似性为89%—99%,14个RFLP类型中,有7个类型(即50%的类型)与现已经发现的杜鹃花类菌根真菌亲缘关系密切,分别为Oidiodendron maius Barron、Cryptosporiopsis ericae Siglersp.nov和5种Epacrid root endophyte同源或近缘种;2个类型为黑色有隔内生菌类(Dark Septate Root Endophytes,DSE),即Phialocephala类菌株;3个为未知类型的子囊菌类,1个为枝状枝孢霉类,1为未知菌种类型。研究还发现:同一地带的4个云锦杜鹃群落菌根分离真菌都具有丰富的多样性,大部分菌种类型在不同样地间具有一致性,70%以上的菌株类型为2个以上的样地共有;但不同样地的菌株类型也表现了明显的趋异性,华顶山和幕阜山森林公园主导菌株为Phialocephala fortinii wang &Wilcox类,而四明山森林公园和黄山风景区为Oidiodendron maius Barron类菌株。3.将分离到的真菌菌落接种云锦杜鹃幼苗,锥虫兰染色压片镜检幼苗毛根内是否具有ERM菌丝体,作为初步判断ERM真菌的标准。结果显示:17个菌株类型中,9个类型33个菌株接种苗毛根内检测到ERM结构,8个类型没有观察到ERM结构。初步确定9个菌株类型为ERM真菌,其中5个类型为其他ERM植物的菌根真菌同源或近缘类型,分别为Oidiodendron maius Barron及4个不同的Epacris root associatedfungus类菌株,2个类型,即Cladosporium cladosporioides和Fungal sp.R27近缘菌株,为本研究首次分离的新ERM真菌类型,Cryptosporiopsis ericae Sigler sp.nov1和Phialocephala fluminis Shearer,JL Crane & MA Mill.近缘类型是2个首次合成ERM的菌株类型。4.研究首次对多种类型的ERM菌株的接种效应进行研究。通过14个类型的菌株接种效应的统计分析表明:不论是否为ERM真菌菌株,绝大多数对接种幼苗的生长没有伤害作用;对云锦杜鹃接种幼苗具有明显促进作用的菌株都是确认的ERM真菌,其中大部分为其他ERM植物分离菌根真菌的同源种或近缘种。其中首次发现5个类型(3个Epacris root associated fungus、C.ericae、Fungal sp.R27的近缘菌株)的菌株能显著地增加接种幼苗的生物量。不同类型ERM菌株或同一类型不同菌株在接种效应上差异很大,接种苗干重的增加率多在20-250%之间,最高可达400%。5个ERM菌株在不同氮源培养基上纯培养和幼苗接种试验表明:不同的ERM菌株能够吸收单一有机氮源,并且能帮助寄主氮营养的吸收从而促进幼苗生物量的增加。菌株在不同氮源培养基上接种效应差异显著,5个菌株接种苗在有机氮BSA、精氨酸培养基上生物量增加显著高于氨态氮和对照培养基上的幼苗。以上研究结果表明:菌根在云锦杜鹃的生长发育中占有重要地位,菌根的形成对其幼苗的生长具有关键作用。接种菌根真菌,实现幼苗的早期菌根化,有希望成为解决大叶常绿杜鹃花繁育中幼苗生长缓慢、死亡率高等问题的一个良策。研究获得的菌种资源和技术方法,为优良菌剂的研制及菌根技术的应用奠定了基础,同时为探索植物与真菌之间的共生机制建立了良好的模式。因此,本研究实践价值和科学意义深远。更多还原

【Abstract】 Most members of family Ericaceae have a distinctive form of the symbiosis referred to as ericoid mycorrhiza (ERM), which appear to be able to mobilize nutrients from organic matter and alleviate certain environmental stresses, such as readily available metals, poor or very free drainge and high or tow temperatures et al., and so facilitate the establishment and survival of Ericaceae. And some ERM fungal strains were found beneficial for the growth of Ericaceous plants in vitro or nurseries. In order to understand and utilize the mycorrhizal potentials, and satisfy the requirement of ecology constructing and horticulture producing, ericoid mycorrhiza has attracted abroad attention in overseas. In China, various ericaceous species are distributed widely in various types of forest vegetation. However, no fungal isolates have been reported from the roots of those plants. Rhododendron fortunei L., a broadleaf rhododendron, is originated and distributed widely in China. The research focus on the diversity of ericoid mycorrhizal fungi isolated from the hair roots of R. fortunei in natural habitats in China. The main experimental results are as follows:The microstructure and colonial rate of mycorrhizal root of R. fortunei were observed with electron microscope scanning and fresh mount of hair root stained with trypan blue and resin sectioning. The results showed that mean percent root cell colonization of hair roots from natural sites were over 65%; the hair roots consisted of one layer of epidermis cells and 1-2 layers cortical cells surrounding the stele, they did not form root hair and were covered with loose or compact fungal mantle; the presence of the mycorrhizal fungus in epidermal cells did not appear to disrupt the primary tissue structure of the root, except that mature epidermal contain ericoid mycorrhizal hyphal coils; the fungal complex were mostly observed in epidermal cells and the cell wall of 90% epidermal cells with fungus were not obviously thickened; as judged by the size and structure of the fungal complex, at least two types of fungi appeared to form mycorrhizal coils in epidermal cells. One type was a thick mycelium fungus which formed tight coils, the other was a thin mycelium fungus formed loose coils.We successfully modified the method of isolation of ericoid mycorrhizal fungi according to Pearson & Read (1973) from the ericaceous hair roots. Firstly Martin agar medium was modified and introduced to ERM fungi isolation. So the contamination rate of bacterium and actinomycetes were greatly decreased. In total, 280 slow-growing fungal isolates were obtained from hair roots of R. fortunei from 4 natural habitats. The isolates were initially grouped as 17 types based on the results of ITS-RFLP analysis and the colonial characters. ITS sequences were obtained for representative isolates from each RFLP type and compared phylogenetically with sequences for known ericoid mycorrhizal endophytes and selected ascomycetes. Most of fungal isolates were also ascomycetes, and the sequence similarity is 89%-99%. 7 types of 14 RFLP types are related to known ericoid mycorrhizal endophytes, such as Oidiodendron maius、Epacris microphylla root associated fungus、Epacrid root endophyte sp.. Cryptosporiopsis ericae et al.; 2 types are Phialocephala-like strains; 2 types are unclassified ascomycetes; 1 type is similar to Cladosporium cladosporioides; 1 type is unknown fungal strains. Among the fungal strains, over 70% types were Simultaneously isolated from the hair roots of 2 natural sites. The fungal isolate types are also different among 4 natural sites. Phialocephala fortinii-like strains are the dominant fungal isolates from Huading and Mufu mountain forest parks, while Oidiodendron maius strains are dominant in the isolates from the other two Rhododendron fortunei habitats.In order to verify ericoid mycorrhiza status, fungal strains of each type were inoculated on the seedlings of R. fortunei in vitro. During the inoculation experiment the most seedlings remained healthy with well-developed root system. Based on the results of fresh mount of hair roots stained with trypan blue, 9 types, 33 fungal strains, were initially identified as ERM fungi with typical ericoid mycorrhizal coils in epidermal cells of hair roots; 8 types were not detected fungal coils in the hair roots of inoculated seedlings. Among the 9 types putative ERM strains, 5 types were identified as Oidiodendron maius and different Epacris root associated fungus which are ERM fungi from other ERM plants; 3 types were confirmed as Cladosporium cladosporioides, Cryptosporiopsis ericae and Phialocephala ftuminis; 1 fungal type was unknown strains.Most ERM fungal strains showed obviously beneficial to growth of the seedlings, normally the biomass of colonized seedlings increased by 20-250%, the highest being 400%. Some Epacris root associated fungus, C. ericae and unknown fungal isolates were firstly shown significantly positive effects on the colonized seedlings. At the same time, 5 ERM fungal strains were tested for the ability to provide their host with access to inorganic and organic N. Regardless of which mycorrhizal fungus colonized the roots and which nitrogen source was provided; mycorrhizal plants grew better than nonmycorrhizal plants. There were, however, differences among the fungi in use of the N source. Colonized seedlings on nitrate and organic nitrogen grew much better than on ammoniacal nitrogen and control medium, especially on BSA medium.In conclusion, we suggested that the presence of ericoid mycorrhizal associations play an important role in success of R. fortunei at natural habitats and the growth of the seedlings at artificial conditions. In vitro or nursery inoculating seedlings with ERM strains maybe a good applied technique to resolve some problems in Rhododendron propagation and cultivation.更多还原