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研究生:陳盈諠
研究生(外文):Chen, Yinghsuan
論文名稱:以分子生物方法調查台灣亞高山生態系台灣冷杉與台灣鐵杉之外生菌根真菌多樣性
論文名稱(外文):Ectomycorrhizal Fungal Diversity of Abies kawakamii and Tsuga chinensis var. formosana in a Subalpine Forest of Taiwan Determined by Molecular Analysis
指導教授:汪碧涵汪碧涵引用關係
指導教授(外文):Wang, Pihan
口試委員:曾顯雄吳聲華
口試委員(外文):Tzean, SheanshongWu, Shenghua
口試日期:2013-01-30
學位類別:碩士
校院名稱:東海大學
系所名稱:生命科學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:122
中文關鍵詞:跨宿主胸高直徑樹齡菌根形態變性梯度凝膠電泳合歡山高海拔
外文關鍵詞:Multi-hostdiameter at breast height (DBH)tree ageectomycorrhizal morphotypedenaturing gradient gel electrophoresis (DGGE)Mt. He-Huanhigh elevation
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林木外生菌根真菌多樣性及群落組成的研究以歐美的溫帶森林為主,且多採子實體調查,但子實體調查受出菇物候限制,未必能如實呈現宿主的外生菌根真菌多樣性。本研究以變性梯度凝膠電泳 (DGGE) 與 rDNA ITS 序列分析,調查台灣亞高山生態系的台灣冷杉與台灣鐵杉菌根之外生菌根真菌多樣性,探討各胸徑林木的菌根菌群落組成與各形態菌根的外生菌根真菌。由合歡山的台灣冷杉與台灣鐵杉混合林,分別選取面積 100 m2 的台灣冷杉和台灣鐵杉樣區各一個,樣區內為單一樹種與玉山箭竹混生。每個樣區選取四個不同徑級樣木,收集菌根樣本,經變性梯度凝膠電泳分析,發現各胸徑林木之外生菌根真菌遺傳多樣性差異不大,胸徑大小相近的樹木菌根菌群落組成相似度較高。將 24 個主要 DGGE 條帶進行基因選殖及定序,共獲得 6 科 25 種外生菌根真菌,台灣冷杉和台灣鐵杉各有 24 種和 23 種外生菌根真菌,有 22 種菌跨兩種宿主共生,相似度分析 (ANOSIM) 顯示,二樹種的外生菌根菌群落組成沒有顯著差異。此外,分別在台灣冷杉和台灣鐵杉的最大樣樹採取八份菌根樣本,以基因選殖和定序分析六種形態菌根共 36 個單一根尖樣本,共比對出 13 種外生菌根真菌核酸序列,DGGE 測得其中的 6 種。在六種菌根形態中,四種形態測得 1~2 種外生菌根真菌,有兩種菌根形態分別測得 4 種和 6 種菌。其中一種蠟蘑 (Laccaria sp. 1) 由台灣冷杉的二種菌根形態測得;皮氏紅菇 (Russula peckii) 與兩種宿主均可形成 2 種以上菌根形態。本研究以兩種方法共測得 32 種外生菌根真菌,其中 29 種外生菌根真菌沒有子實體記錄,只有李逵鵝膏 (Amanita liquii)、正紅菇 (Russula vinosa) 和皮氏紅菇的子實體記錄 (高明脩,2012),而有子實體記錄的辣乳菇 (Lactarius piperatus)、松塔牛肝菌 (Strobilomyces strobilaceus) 和蠟傘 (Hygrophorus sp.) 未在菌根樣本中測得。本研究獲得結論如下:一、台灣亞高山森林生態系針葉樹擁有高多樣性的共生菌。二、此針葉混合林以跨宿主的外生菌根真菌為主,且兩種林木的外生菌根真菌群落組成無顯著差異;三、各徑級林木的外生菌根真菌豐度未見差異,群落組成則隨樹徑、樹齡而變化;單株林木有近 20 種外生菌根真菌;四、子實體調查與菌根核酸分析並行,更能呈現實際的外生菌根真菌多樣性;五、四種菌根形態測得 1 或 2 種外生菌根真菌,二種菌根形態分別測得 4 或 6 種菌種;六、蠟蘑 (Laccaria sp.1) 和皮氏紅菇 (Russula peckii) 能由宿主不同形態的菌根中測得。
Studies of ectomycorrhizal (ECM) fungal communities and diversity on forest trees are mainly restricted to fruitbody surveys in Europe and North America. Fruitbody surveys, however, are limited by fruiting phenology and may not reveal true ECM fungal diversity. I used denaturing gradient gel electrophoresis (DGGE) and analysis of internal transcribed spacer sequences of ribosomal DNA to investigate ECM fungal diversity from ectomycorrhizae of Abies kawakamii and Tsuga chinensis var. formosana. I examined ECM community composition on trees with different diameters breast height (DBH) and ECM fungi of different ectmoycorrhizal morphotypes. In a mixed forest of Abies kawakamii and Tsuga chinensis var. formosana in the subalpine ecosystem at Mt. He-Huan, I chose a 100 m2 plot for each tree species. In each plot, I selected four conspecific trees with varying DBHs. From each tree, I collected ectomycorrhizae samples and analyzed their DNA. The DGGE patterns showed similar ECM genetic diversity among trees with different DBH, but ECM community composition had the highest similarity on trees with similar DBH. Cloning and sequencing of 24 major DGGE bands identified 25 ECM fungal species in six families. Abies kawakamii and Tsuga chinensis var. formosana had 24 and 23 ECM fungi respectively. Twenty-two ECM fungi were in symbiosis with both tree species. Analysis of similarity (ANOSIM) shows there was no significant difference in ECM community composition between tree species. On the other hand, six ectomycorrhizal morphotypes were observed in sixteen ectomycorrhizae samples, eight samples each from the largest Abies and Tsuga trees. Restriction fragment length polymorphism analysis, cloning, and sequencing of 36 root tips revealed 13 ECM species, including six species detected in the DGGE analysis. I detected 1–2 ECM fungal species from each of four morphotypes. The remaining two morphotypes had 4 and 6 ECM species. Laccaria sp. 1 formed 2 morphotypes with Abies kawakamii. Russula peckii formed more than 2 morphotypes with both tree species. By combining these two methods, I identified 32 ECM species, 29 of which have no fruitbody record. Amanita liquii, Russula vinosa and Russula peckii were identified in a fruitbody survey conducted in the same area (Kao, 2012). Lactarius piperatus, Strobilomyces strobilaceus and Hygrophorus sp. have fruitbody records, but were not detected in this study. In summary, 1) dominant ECM fungi were in symbiosis with both tree species in this mixed conifer forest as there is no significant difference in ECM community composition between the two tree species; 2) ECM genetic diversity among trees with different DBH were similar, but ECM community composition varied with tree DBH and age; 3) for most ectomycorrhiza morphotypes, each type was formed by 1–2 ECM fungi, but a few morphotypes had 4–6 ECM fungi; 4) Laccaria sp. 1 and Russula peckii formed different ectomycorrhiza morphotypes on the same host. Use of both fruitbody survey and ectomycorrhizae analysis may give a better view of ECM fungal diversity. Combining fruitbody survey with ectomycorrhizae analysis demonstrated that this subalpine ecosystem in Taiwan had high ECM fungal diversity.
中文摘要 I
Abstract III
前言 1
一、 何謂外生菌根真菌 1
二、 外生菌根真菌在森林生態系的重要性 3
三、 研究外生菌根真菌群落的方法 5
四、 外生菌根真菌的文獻回顧 9
4.1 外生菌根真菌在世界上的分布 9
4.2 不同尺度下的外生菌根真菌的多樣性 11
4.3 影響外生菌根真菌的因子 13
4.4 國內文獻回顧 17
五、 研究目的 19
材料與方法 20
一、 研究地點 20
二、 實驗設計 21
2.1 四個徑級的台灣冷杉和台灣鐵杉外生菌根菌群落組成 21
2.2 採樣頻度測定 22
2.3 各菌根形態的外生菌根真菌組成 22
三、 菌根採樣方法與樣本處理 23
四、 菌根DNA的萃取 24
五、 聚合酶鏈鎖反應和瓊脂凝膠電泳 25
六、 限制內切酶片段多型性分析 26
七、 變性梯度膠體電泳 26
八、 PCR產物純化 27
九、 基因選殖 28
十、 定序與序列排列比對 29
十一、 統計與分子親緣關係分析 30
結果 31
一、 樣本數的決定 31
二、 四個徑級台灣冷杉與台灣鐵杉外生菌根真菌的遺傳多樣性 31
三、 台灣冷杉與台灣鐵杉之外生菌根真菌群落組成比較 32
四、 各徑級台灣冷杉的外生菌根真菌多樣性與群落組成 33
五、 各徑級台灣鐵杉的外生菌根真菌多樣性與群落組成 34
六、 台灣冷杉和台灣鐵杉的菌根形態 35
七、 六種菌根形態的外觀描述 36
八、 六種菌根形態型的外生菌根真菌組成 37
九、 紅菇科與絲膜菌科的親緣樹分析 38
討論 41
一、 台灣冷杉與台灣鐵杉混合林的外生菌根真菌豐度 41
二、 子實體調查與菌根核酸分析結果比較 41
三、 本研究外生菌根真菌在世界的地理分布和功能群分類 42
四、 宿主物種與外生菌根真菌多樣性 44
五、 樹齡、樹徑與外生菌根真菌多樣性 46
5.1 外生菌根真菌遺傳多樣性 46
5.2 優勢菌種之外生菌根真菌豐度 47
六、 一棵樹的外生菌根真菌多樣性 48
七、 各形態菌根之共生菌種 49
八、 單一菌根根尖的外生菌根菌多樣性 51
九、 變性梯度凝膠電泳分析之探討 52
十、 多源基因體學的可行性 52
結論 54
參考文獻 56
表目錄 72
圖目錄 73
附錄目錄 74
表 75
圖 87
附錄 100
個人資料 113

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