【作者】 侯晓强；

【导师】 郭顺星；

【作者基本信息】 中国协和医科大学 ， 生药学， 2007， 博士

【摘要】 石斛为我国名贵中药材,石斛属多种植物均用作商品石斛药材。石斛来源植物复杂,通常以茎的结构特征作为鉴定依据,但茎的结构特征有限,很难区分所有的种类,这就要求有更多的形态特征来辅助鉴定,而根的解剖特点可作为重要的参考依据之一。目前,石斛药材的需求量的不断增加,而石斛属植物生境较为独特,自然繁殖率低,人工栽培难以大面积推广,导致天然石斛无序采集,自然资源锐减,部分石斛种濒临灭绝。石斛属植物具有兰科植物典型的菌根特征,在自然条件下生长的石斛种子萌发、植株营养生长、生殖生长均需要与菌根真菌共生。本文系统地研究了石斛属植物根和菌根的解剖学结构特征,对石斛属植物的内生真菌进行了分离培养,研究了石斛属植物内生真菌对金钗石斛和铁皮石斛的作用,并对能够促进金钗石斛苗生长的内生真菌进行了分类鉴定。对中国石斛属34种植物42个材料根的解剖结构进行了系统研究,石斛属植物根的结构为典型的单子叶植物根的结构,其从外到内依次为根被、外皮层、皮层、内皮层、中柱鞘、维管束和髓。首次发现了长距石斛、美花石斛和细茎石斛根的外皮层通道细胞外侧,有纤维体(Tilosome)结构的存在。提出了根解剖结构特征如根被最内层细胞的V型加厚、外皮层通道细胞外侧的纤维体、外皮层细胞的加厚方式、内皮层细胞的形状及髓的类型可以作为属下种间的鉴别主要特征;同时指出根被的层数、细胞的形状受石斛生长环境的影响较大,不易作为分类鉴定的特征;根被的形状与石斛的附生习性具有相关性,在根系附着基质的一侧,根被变窄,细胞受到挤压,细胞层数也略少于暴露的一侧。比较野生金钗石斛与人工栽培的壮苗和弱苗、野生铁皮石斛与人工栽培的壮苗和弱苗根的解剖结构发现,野生石斛与人工栽培的壮苗和弱苗在结构上的差异仅体现在根被的层数、厚度,维管束的数量上的变化,其结果为野生石斛高于人工栽培石斛,人工栽培的壮苗高于弱苗。首次采用聚类分析的方法,对石斛属25种植物根的解剖结构特征转化的数据进行分析,基于根的解剖结构特征建立了石斛属植物种间的系统发育树。该系统发育树将25种石斛划分为4个类群,这与中国植物志描述的石斛属植物属下组的划分具有一定的相关性。根据GeneBank数据中已知石斛属植物的rDNA ITS序列,对本实验中石斛属的19种石斛植物构建了分子系统发育树,考察了石斛属植物种间的分子亲缘关系。比较基于根的解剖结构特征的种间亲缘关系和分子亲缘关系,发现二者不具有相关性;分子亲缘关系与石斛属植物属下组的划分也存在分歧。通过对石斛属34种植物42个材料的菌根解剖结构的系统研究,发现菌根的主要特征为真菌从根被最外层细胞或根毛侵入根被组织,主要以菌丝的形式在根被组织中扩展,但仅观察到金钗石斛根中的真菌充满某一根被细胞的现象。菌根真菌从外皮层通道细胞进入皮层组织,在通道细胞中既可以菌丝形式直接穿过,也可围绕细胞核形成菌丝圈或菌丝团结构:皮层细胞中还可观察到有菌根真菌形成的疏松的菌丝团、衰败的菌丝团及二者共存的现象,菌丝团的形成是兰科植物菌根形成的典型特征。在一些皮层细胞中,疏松的菌丝团围绕着衰败的菌丝团存在。进一步的研究发现菌丝团在皮层中的分布位置与石斛属植物附生习性相关,根系附着基质一侧的皮层细胞中菌丝团密度较高。菌丝团主要在皮层中间的细胞中形成,而皮层最内侧1～2层细胞中,菌丝团没有分布或数量很少。菌根真菌一般不侵入内皮层和中柱,只有大苞鞘石斛和铁皮石斛的内皮层通道细胞和中柱鞘通道细胞中有菌丝团的存在,该现象在石斛属正常生长着的植物菌根研究中尚为首次发现。石斛属植物根的皮层中,菌根真菌的定殖率为0～80%,其中黑毛石斛高达70%～80%;而长距石斛等的皮层中没有菌根真菌的定殖。野生石斛、人工栽培的壮苗和弱苗的菌根结构存在差异,弱苗的皮层细胞中没有菌丝团结构或者仅存在衰败的菌丝团,其真菌的定殖率也低于野生石斛和人工栽培的壮苗。首次对石斛属34种植物42个材料中的内生真菌进行了系统分离和培养,共分离得到1313个内生真菌菌株,不同的植物其内生真菌的数量不同,内生真菌侵染率、分离率也不同。石斛中内生真菌的总侵染率分布在25.7%～61.1%:内生真菌分离率为0.29～1.18。石斛属植物内生真菌具有多样性特征,多样性指数分布在1.75-4.33之间。根据内生真菌的培养特征,可将山现频率高的菌株划分为10个形态类群,不同形态类群的菌株在石斛属植物中的分布不同,发生频度也存在差异。完成了本实验所得1313株内生真菌对石斛苗生长影响的筛选工作。筛选出了36个对金钗石斛苗有促生长作用的菌株、8个对铁皮石斛苗有促生长作用的菌株;对接种活性菌株的石斛苗根进行了侵染检测,发现内生真菌均可以侵入石斛苗根的根被组织,部分真菌可以穿过外皮层通道细胞进入皮层组织,其中菌株22-R-4和G08-R-17可以在石斛苗皮层中形成菌丝团结构。这些有促生长作用的真菌一般不能侵染石斛苗根的维管束,而能够侵染维管束的真菌不利于石斛苗的生长。内生真菌还能够影响石斛苗的化学成分含量。在琼脂培养基上,接种菌株22-R-4的金钗石斛苗总生物碱含量高于未接种组,接菌的铁皮石斛苗总多糖含量与未接种组相差不大。但在木屑支持物上,接种菌株22-R-4降低了金钗石斛苗总生物碱含量,也降低了铁皮石斛苗总多糖含量。免疫组织化学定位研究发现,接种菌株22-R-4的金钗石斛苗其根尖和茎尖IAA含量分别高于未接菌苗根尖和茎尖IAA的含量。对有促进金钗石斛苗生长的36个内生真菌进行了形态观察,其中18个菌株能够产生孢子,17个菌株鉴定到了种,分别属于14个属。对其余不产孢的18个菌株进行了rDNA ITS序列测定,并与GeneBank中序列相似性较高的已知菌株进行了比较分析,确定了序列相似性高的9个菌株的分类地位。更多还原

【Abstract】 Shihu is a rare traditonnal Chinese medicine which includes many species of medicinal plants in the genera of Dendrobium,and also there are many kinds of Shihu herbs for commercial use in China.Shihu’s identification usually was only by its structural characteristics of stem,and that will not be insufficient for the classification of the close species.So,it is very important to find root morphological characteristics to meet the identification need of different plants in Dendrobium.We are all known that the plants as Shihu are becoming extinct because of the need increasing of Shihu herbs,and the special habitat of this kind of plants as well as its low propagate rate,and the difficults to cultivate Dendrobium plants under artificial conditions.Dendrobium plants have mycorrhizal characteristics of Orchidaceae,which means that the plans must be formed mycorrhiza with symbiotic fungi in all their life cycle from seed germination,development of seedlings.The anatomical characteristics of roots and orchid mycorrhiza of 34 species of plants in Dendrobium,which included the isolation of endophytic fungi,its evaluation to promoting growth effectives on D.nobile and D.candidum,and identifications of effective fungi,were systematically studied in this thesisThe root structures of Dendrobium were typical structures of monocotyledons,which was consisted of velamina,exodermis,cortex,endodermis,pericycle,vascular cylinder and pith from outside to inside.The V-thickened cells in innermost layer of velamen,tilosomes,type of thickened wall in exodermis,shape of ceil in endodermis and ratio of cortex diameter and stele radius could be used as specific character for the identification of the species Dendrobium species.Since the number of layers in velamina was easy to be influenced by the growing environment,it might not be properly to be used as a character for phylogeny identification in Dendrobium.The shapes of velamina are correlated to the epiphytic habit of Dendrobium roots.Velamina are frequently narrower on the side of a root attached to a substrate than that on the exposed side.Comparative anatomy between the wild Dendrobium and the seedlings showed that number of layers in velamina, diameter of velamina and number of vascular cylinder in Dendrobium seedlings are less than that in the wild one.Phylogenetic assessment of anatomical features data showed that some species are cluster in a dendrogram,which was congruent with Dendrobium section.This results was not consistent with the phylogenetic cladogram of the rDNA ITS sequence data.Moreover,the molecular cladogram of molecular data was inconsistent with the classification of section in Dendrobium genera.The anatomical characters of orchid mycorrhiza were observed with the light microscope.The hyphae penetrated into the velamina from the outmost cells or root hair cells and existed in the intercellular or intracellular of velamina.The hyphae colonized in the intracellular space cell and formed hyphal coil in velamina of D.nobile.The root exodermis of Dendrobium was comprised of a series of thick-walled dead cells and few thin-walled passage cells at irregular intervals.The hyphae in velaman cells only penetrated the thin-walled passage cells and formed hyphal peloton structure surrounding the nucleus.Once across the exodermis layer through the passage cells,the mycorrhizal fungal hyphae entered the thin-walled cortical cells and formed typical orchid mycorrhizal structure(peloton) therein.The peloton included the vigorous peloton and the digested one.Two types of peloton could co-exist in one cortex cell.Distribution of peloton in the cortex was related with the epiphytic habit of Dendrobium roots.Pelotons were frequent distribution on the side of a root attached to a substrate than on the exposed side.In cortex,peloton mainly colonized the middle layers cells and hardly existed in innermost 1～2 layer cells. Mycorrhizal fungi usually could not infect the endodermis,pericycle and vascular cylinder.But we observed the infected passage cells in the endodermis and pericycle of D.candidum and D. wardianium.Colonized ratio of the peloton in cortex is from 0 to 80%,but D.williamsonii is the special one with a ratio of 70～80%,D.longicornu doesn’t form the peloton in cortex.Comparative anatomy of roots between the wild Dendrobium and the cultivated seedlings showed that colonized ratio of peloton in cortex of the wild Dendrobium is more than that of the cultivated seedlings.The digested pelotons existed only in the cortex of the weak cultivated seedling.Pure fungal cultures were successfully obtained using tissue isolation method.1313 endophytic strains were isolated from 43 samples of Dendrobium.Fungal colonization rate and fungal isolation rate are different in each Dendrobium plant.The endophytie fungi existed in Dendrobium plants have the character of diversity,and the coefficient of diversity was from 1.75 to 4.33.Based on the morphological characteristics,these fungi were divided into ten distinct morphotypes.The relative frequency of these morphotypes was different in each species of Dendrobium.Interaction between each fungus and seedlings was studied by co-culture.36 strains had significant positive effects on the growth of seedlings of D.nobile in various growth parameters including fresh weight,dry weight,stem diameter,number of new roots,length of longest root, number of new buds,as well as height of highest bud.And 8 strains inoculated to D.candidum seedlings greatly enhanced seedlings’ plant height,stem diameter,new roots number and biomass. Anatomical features of the inoculated roots with different fungi were observed.Each inoculated fungi could infect the velamina of seedlings.However,some of which could penetrate the cortex cells through passage cells.Fungi 22-R-4 and G08-R-17 could form the pelotons in the cortex cells respectively.The fungi with effectives for growth promotion of plants could not infect the endodermis and vascular bundle sheath,but which was exception for other fungi with harmful to seedlings.Endophytic fungi inoculated to seedlings could affect the content of chemical compositions of plants.The isolate 22-R-4 not only could promote the growth of D.nobile and D. candidum,but also enhanced the content of alkaloid for the first plant and polysaccharide for the second one on MS co-culture medium.The isolate 22-R-4 inoculated to D.nobile seedlings also affected the content and distribution of auxin in the seedlings.Combined with classic morphologic classification,Saccardo and Hughes-Tabaki-Barron fungal classification system,17 of 36 effective strains were identified which were subjected to 14 genera. Ten species of fungi without conidiophore were identified by sequencing the PCR-amplified rDNA ITS1-5.8S-ITS2(internal transcribed spacer ITS) regions.In comparison with similar taxa,the sequence was grouped together with the known strains.更多还原