臺灣博碩士論文加值系統

牛樟 (Cinnamomum kanehirai Hay.) 造林現今以扦插苗為主要苗木來源，但扦插苗最主要的缺點為缺乏主根，所以如何促進扦插苗根系生長以提升苗木品質是目前牛樟苗木培育技術最需解決之課題。近年來已證實暗色隔膜內生菌 (DSE) 具有促進牛樟根系生長的效益，並且DSE不需仰賴宿主進行繁殖，具有提供穩定接種源的應用潛力。因此本研究利用4株自牛樟根菌結合體分離出之菌株 (CkYC2、CkYC14、CkYL14及CkDB5) 以及嘉義林區管理處中興苗圃的二年生牛樟扦插苗為材料，探討這4株菌株分生鑑定及生化特性並於苗圃現場進行苗木接種試驗。希望能篩選出對牛樟扦插苗生長具有促進生長效益的DSE。另外，本研究亦利用苗木生長表現的影響程度係數來評估最佳效益菌株，並以其作為示範菌株設計專一性引子，希望能開發一更客觀之方法用作菌根驗收程序，以利確認接種作業是否落實。
試驗菌株rDNA-ITS序列經分生鑑定比對結果顯示，CkYC2菌株應屬於傘菌目 (Agaricales sp.)，CkYC14菌株應屬於Acrocalymma vagum，CkYL14菌株應屬於子囊菌門 (Ascomycota sp.)，CkDB5菌株應屬於子囊菌門 (Ascomycota sp.)；且上述菌株除了CkYC2外，其他3株菌株皆已有文獻報告證實為DSE。菌株生化特性結果顯示，試驗菌株皆具備漆氧化酵素之能力，且其中CkDB5菌株還具有分泌酪胺酸酵素及吲哚乙酸之能力。試驗苗木經培育7個月後結果顯示，牛樟扦插苗經菌株接種處理後在苗高淨生長量、苗木生物量及葉綠素濃度等項目數值均高於對照組，且其中以CkDB5菌株對苗木生長影響程度為最佳。因此本研究針對CkDB5菌株ITS區域序列設計引子並測試其專一性及靈敏度，測試結果顯示DB5-1R/DB5-1F的專一性及靈敏度 (0.096 ng μL-1) 良好。綜上所述，本研究證實DSE在苗圃現場對牛樟扦插苗的效益，其中以CkDB5菌株表現最為優異，並且該菌株之專一性引子已成功開發；希望未來可將此技術應用至驗收程序，以利牛樟扦插苗接種菌根菌技術落實至苗圃現場作業。

Cinnamomum kanehirae afforestation currently uses cutting as the main source of seedlings. However, the main disadvantage of cutting is the lack of taproot. Therefore, promoting the growth of roots to improve the quality of seedlings is the most important solution for the cultivation of Cinnamomum kanehirae. In recent years, it has been confirmed that the dark septate endophytes (DSE) has the beneficial effect of promoting the growth of the roots of Cinnamomum kanehirai. DSE does not need to rely on the host for propagation, and has the application potential of providing a stable inoculation source. In this study, the two-year-old cuttings and the four strains (CkYC2, CkYC14, CkYL14 and CkDB5) that were isolated from the root of Cinnamomum kanehiraewere used as materials. The classification, identification and biochemical characteristics of these four strains were investigated and on-site inoculation experiments were carried out. It is hoped that the DSE which is beneficial to the growth of Cinnamomum kanehirae cutting can be screened out. Futher more, the best performance strain was designed a specific primer. It is expected that to develop an objective method to confirm the presence of mycorrhizal fungus and to promote the application of them.
In the molecular analysis, the ITS sequence of strain CkYC2 was matched to Agaricales, strain CkYC14 was matched to Acrocalymma vagum, strain CkYL14 was matched to Ascomycota and strain CkDB5 was matched to Ascomycota. In addition to CkYC2, the above strains have been reported to be DSE. The results of biochemical characteristics revealed that the four strains could secrete laccase. Besides, CkDB5 strain could secrete tyrosinase and indoleacetic acid. The results of on-site inoculation experiments showed that the inoculation had the higher value of seedling height, seedling biomass and chlorophyll concentration than the control treatment. In addition, the specific primer (DB5-1R/DB5-1F) for the DNA sequence of the CkDB5 strain performed well to the specificity and sensitivity (0.096 ng μL-1) for the PCR reaction. In summary, this study confirms the application potential of DSE in forest nursery, and expects that mycorrhizal technology can be actively promoted and applied to forestry nursery.

摘要 I
Abstract II
謝誌 III
目錄 V
圖次 VII
表次 IX
Ⅰ、前言 1
Ⅱ、前人研究 3
(Ⅰ) 牛樟概述 3
(Ⅱ) 牛樟育苗造林情形 5
(Ⅲ) 菌根概述 7
(Ⅳ) 暗色隔膜內生菌概述 9
(Ⅴ) 暗色隔膜內生菌效益 13
(Ⅵ) 林木菌根應用於苗圃方法 17
(Ⅶ) 專一性引子於真菌鑑定研究之應用 19
Ⅲ、材料與方法 23
(Ⅰ) 試驗流程 23
(Ⅱ) 研究材料 24
(Ⅲ) 分泌吲哚乙酸能力測定 26
(Ⅳ) 分泌酵素能力測定 26
(Ⅴ) 接種源製備 28
(Ⅵ) 戳洞植菌法 28
(Ⅶ) 根菌結合體之染根觀察 29
(Ⅷ) 苗高生長量測量 29
(Ⅸ) 植株鮮、乾重測定 29
(Ⅹ) 葉綠素濃度測定 30
(XI)植體元素測定 30
(XⅡ) 培育介質性質測定 31
(XⅢ) 核酸抽取與電泳分析 32
(XⅣ) 引子設計及測試 33
(XⅤ) 統計分析 34
Ⅳ、結果 35
(Ⅰ) 菌株之分子生物鑑定 35
(Ⅱ) 菌株分泌吲哚乙酸分析 36
(Ⅲ) 菌株分泌酵素測定 38
(Ⅳ) 苗圃牛樟根菌結合體之染根觀察 41
(Ⅴ) 牛樟扦插苗之生長量調查分析 43
(Ⅵ) 牛樟扦插苗葉綠素濃度分析 48
(Ⅶ) 植體氮磷性質分析 50
(Ⅶ) PCR增幅反應 52
Ⅴ、討論 61
(Ⅰ) 試驗菌株特性分析 61
(Ⅱ) 苗圃牛樟根菌結合體之形態觀察 63
(Ⅲ) 牛樟扦插苗半合成接種試驗 64
(Ⅳ) 專一性引子對PCR增幅反應 67
Ⅵ、結論 70
Ⅶ、參考文獻 71

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