兰科作为被子植物第二大科，其进化程度最高，包含很多药用植物与名贵花卉，具有较高的商业价值。兰科植物种子细小，无胚乳，在自然条件下必须与合适的真菌建立共生关系，才能完成种子萌发，部分兰科植物整个生活史离不开菌根真菌为其提供营养。兰科菌根是一种典型的菌根共生关系，且研究表明兰科植物与其菌根真菌之间存在一定的专一性关系， 但两者之间专一性机制相比于丛枝菌根与外生菌
根作用机制研究报道较少。本文以药用石斛种子接菌共生萌发为例，研究石斛属种子与菌根真菌共生萌发的专一性， 初步探究两者之间的共生机制，为深入揭示兰科种子共生萌发机制提供可参考数据；同时获得更多的活性菌根真菌用于石斛属植物的保护和栽培中，对兰科植物种质资源保护有重要意义。 本论文的研究结果归纳如下：
一、铁皮石斛和霍山石斛内生真菌的分离和鉴定
利用植物组织块、单菌丝团分离法获得铁皮石斛（Dendrobium officinale）根部内生真菌 358 株，鉴定 292 株；分离获得霍山石斛（D. huoshanense）根部内生真菌23 株，鉴定 20 株。所鉴定的真菌分类上属于 65 个属，其中镰刀菌属（Fusarium）（25.00%）占比最多，分离获得典型的兰科菌根真菌如胶膜菌类真菌（Tulasnelloid
fungi） 46 株，经形态学与分子生物学鉴定合并后为 29 株，系统发育分析将其分为9 个分支；蜡壳菌类真菌（Sebacinoid fungi） 20 株，经形态学与分子生物学鉴定合并后为 11 株，系统发育分析将其分为 4 个分支。
二、菌根真菌促进药用石斛种子萌发的活性评价
结合分离的菌根真菌的系统发育关系，我们挑选了 16 株真菌（11 株 Tulasnellasp., 3 株 Sebacina spp., 1 株 Athelia sp., 1 株未鉴定）分别与铁皮石斛种子共培养，结果发现 11 株菌根真菌（6 株 Tulasnella sp., 3 株 Sebacina spp., 1 株 Athelia sp., 1 株未
鉴定）有较好的促铁皮石斛种子萌发活性，但活力不同， 5 株 Tulasnella spp. 只能促进种子萌发到 2 级阶段。 20 株真菌（13 株 Tulasnella spp., 7 株 Sebacina spp.）分别与霍山石斛种子共培养，结果表明其中 9 株菌根真菌（5 株 Tulasnella spp., 4 株
Sebacina spp.）有较好的促种子萌发活性， 而 5 株 Tulasnella spp.不能促进种子萌发，暗示实验室条件下，该两种石斛与菌根真菌之间在种-种相互作用的水平上表现出较低专一性。此外，本实验首次成功实现实验室条件下霍山石斛种子共生萌发达到幼苗阶段。
三、促种子萌发活性不同的菌根真菌的植物细胞壁降解酶活性比较分析通过平板法初步定性分析上述促种子萌发活性不同的菌株的胞外水解酶活力。结果表明，本次测试的蜡壳菌类真菌在 PDA-ABTS 培养基上均分泌漆酶，而胶膜菌类真菌不分泌漆酶，两者之间分泌漆酶的能力具有显著性差异（p<0.05） ；根据 41株真菌分别在刚果红培养基和蛋白初筛培养基上分泌果胶酶、蛋白酶能力（EI）按照单因素方差分析中 SNK 法对不同组别进行差异显著性分析（p<0.05） ，得出测试真菌菌株共分为 17 和 12 个组，组间差异性不明显，绝大多数 EI 值均为 0-2 之间。平板法酶活性分析表明，本次分离所得的胶膜菌类真菌均不分泌漆酶，但其中一些胶膜菌促进石斛属种子萌发至 5 级阶段，而有些胶膜菌不能促进种子萌发。 8株蜡壳菌类真菌分泌果胶酶的能力有显著差异（P<0.05），但均能促进霍山石斛种子萌发至 5 级阶段， 9 株胶膜菌分泌果胶酶能力没有显著性差异，但只有其中 3 株胶膜菌促进石斛种子萌发，其余 6 株不能促进石斛种子萌发。分泌蛋白酶能力较强的菌株 NC-1、 NC-2 却不能促进霍山石斛种子萌发，不分泌蛋白酶的菌株 WX-1 促进霍山石斛种子萌发至 4 级阶段。因此不同菌根真菌在不同专性诱导胞外酶分泌的培养基上产漆酶、果胶酶、蛋白酶的能力差异与其促进石斛种子萌发活性之间没有直接联系，推测自然条件下促进石斛种子萌发很可能是多种水解酶共同作用的结果，或真菌在纯培养状态下分泌细胞壁降解酶的能力与共生状态下分泌相关酶的能力不同。
四、两种真菌与铁皮石斛种子共生萌发的比较转录组学分析
采用促种子萌发活力相同的两种真菌 Tulasnella sp.和 Sebacina sp.分别与铁皮石斛种子共生萌发，通过比较分析共生萌发组之间以及共生与无菌萌发的样品间的转录组数据，鉴定参与共生萌发过程的植物来源以及真菌来源的关键候选基因和蛋白。研究结果鉴定了 1003 个共差异表达的植物基因，即相对于无菌萌发而言， 2 种真菌分别作用于铁皮石斛种子，在整个共生萌发过程中均能诱导植物表达的差异基
因，这些基因很可能是参与共生过程的核心基因。这些差异表达的基因主要富集在植物病原互作（plant-pathogen interaction）、 植物激素信号转导（plant hormone signaltransduction）、 淀粉和蔗糖代谢（starch and sucrose metabolism）、 苯丙烷合成途径
（phenylpropanoid biosynthesis pathway）等四条主要代谢途径，表明这些途径在种子共生萌发过程中发挥重要作用。真菌来源的基因表达分析结果鉴定了 260 个基因（Sebacina sp. 49 个， Tulasnella211 个）在铁皮石斛种子共生过程中上调表达，这些基因分别编码小分泌蛋白（Smallsecretory protein, SSP）、脂酶（Lipases）、蛋白酶（Proteinase）、碳水化合物活性酶（Cazymes），表明这些基因很可能参与了兰科菌根共生过程。从中挑选出了 48个差异基因，后续将通过 qRT-PCR 验证，初步揭示真菌与石斛种子共生萌发的互作机制。
通过本文的研究，我们初步明确了实验室条件下铁皮石斛种子萌发与菌根真菌的专一性关系；新获得了多个促种子萌发的活性菌株，丰富了功能菌株的多样性；为利用菌根共生技术实现兰科植物的资源再生和保护提供重要的真菌资源。同时鉴定了一组与铁皮石斛种子萌发的关键候选基因，为深入研究真菌与兰科菌根共生机制提供可参考的数据。
 

As the second largest family of angiosperms, Orchidaceae has the highest degree of evolution, contains many medicinal plants and precious flowers, and has high commercial
value. Orchid seeds have no endosperm, so they establish a symbiotic relationship with suitable fungi in order to complete seed germination under natural conditions. Some orchids must depend on mycorrhizal fungi to provide nutrients to complete the entire life history. Orchid mycorrhiza is one of the most concerned mycorrhizal type among seven major mycorrhizal types. Studies have shown that there is a certain specific relationship between orchids and mycorrhizal fungi, but the specific symbiosis mechanism is still unclear. This article starts with the specificity of symbiotic germination between
Dendrobium sp. seed and different fungi, and then to explore the seed symbiotic specificity mechanism between Dendrobium officinale with two different mycorrhizal fungi. The research results of this paper are summarized as follows:

1. Isolation and identification the endophytic fungi of D. officinale and D. huoshanense
358 strains of endophytic fungi from the roots of D. officinale were obtained by the method of tissue block and single hyphae cluster, and 292 strains were identified; 23 strains
of endophytic fungi from the roots of D. huoshanense were isolated and 20 strains were identified. The identified fungi belong to 65 genera in taxonomy, of which Fusarium spp.
(25.00%) accounts for the most. A total of 46 strains of typical orchid mycorrhizal fungi such as Tulasnella fungi were divided into 29 strains after morphological and molecular
identification. After constructing a phylogenetic tree, they were divided into 9 clades; A total of 20 strains of typical orchid mycorrhizal fungi such as Sebacina fungi were divided
into 11 strains after morphological and molecular identification. After constructing a phylogenetic tree, they were divided into 4 clades.

2. Evaluation of mycorrhizal fungi in promoting the germination of medicinal Dendrobium seeds
Combining the classification status of mycorrhizal fungi, 16 mycorrhizal fungi (11 strains of Tulasnella and 3 strains of Sebacina, 1 strain of Athelia and 1 strain identified)
were selected for co-cultivation with D. officinale seeds, and it was found that 11 mycorrhizal fungi had better seed promotion, and the germination-promoting activities of
those fungi were different. 5 strains of mycorrhizal fungi could not promote seeds germination. At the level of species-species interaction, the seeds of D. officinale symbiotic germinate with a variety of mycorrhizal fungi, and the degree of specificity between them is low. 20 mycorrhizal fungi (13 strains of Tulasnella, 7 strains of Sebacina) co-cultured with seeds of D. huoshanense respectively, and germination-promoting activities of those fungi were different. 5 mycorrhizal fungi could not promote seed germination, and the mycorrhizal fungi 12825 isolated from D. moniliforme has the best effect on promoting germination, and the non-mycorrhizal fungus does not promote the germination of seeds. At the level of species-species interaction, the seeds of D. huoshanense can be symbiotic germinated with a variety of mycorrhizal fungi, and the degree of specificity between them is low. This experiment successfully realized the symbiotic germination of D. huoshanense seeds and reached the seedling stage in the laboratory condition for the first time.

3. Comparative analysis of plant cell wall degrading enzyme activities of mycorrhizal fungi with different seed germination activities

Preliminary qualitative analysis of the extracellular hydrolase activity of the abovementioned strains with different promoting seed germination activities was carried out by the plate method. The results showed that the orchid mycorrhizal fungi Sebacina sp. can secrete laccase, while the Tulasnella fungi do not secrete laccase There is no significant difference in the ability of secreting pectinase, protease between most mycorrhizal fungi and non-mycorrhizal fungi. Under the same culture conditions, there is no direct relationship between the ability of produceing enzyme of fungi (laccase, pectinase, and protease) on the specific media with obligate induction of extracellular enzyme secretion and their activities to promote the germination of Dendrobium seeds. It is speculated that the symbiotic germination of D. officinale seeds probabaly depend many kinds of degrading enzymes of many fungi under natural conditions, or the ability of fungi secreting plant cell wall degrading enzymes in a pure culture state is different from the ability to secrete related enzymes in a symbiotic state.

4. Comparative transcriptomic analysis the symbiotic germination between D. officinale seeds and two kind of different fungi.
By comparative transcriptomic analysis of symbiotic germination of D. officinale seeds inoculated with two different fungal species, the co-expressed and differentially expressed
genes of the fungi were screened. We focus on the gene expression encoding small secretory proteins, lipases, proteases, plant cell wall degrading enzymes of different fungi
during the process of symbiosis with D. officinale. The results showed that, 1003 genes were differentially expressed across all symbiotic samples after inoculated two fungi. The subsequent qRT-PCR verification will be carried out to verify the expression of these genes.
In this study, we have preliminarily clarified the specific relationship between the seed germination between D. officinale and mycorrhizal fungi under laboratory conditions;
A number of strains with promoting germination were isolated from the roots of D.officinale. Moreover, a set of key candidate genes for germination of D. officinale seeds have been identified, which provides reference data for further study of the symbiosis mechanism between orchid and mycorrhiza fungi.