臺灣博碩士論文加值系統

茶赤葉枯病為茶樹栽培過程中重要病害，全球已知有 16 種 Colletotrichum spp. 可引起茶赤葉枯病，台灣茶赤葉枯病目前僅被記錄由 C. camelliae 與 C. gloeosporioides 所引起。台灣近年來未能將茶赤葉枯病菌進行系統分類及鑑定，且目前尚未有合適的分子檢測技術可供台灣茶赤葉枯病進行快速分子檢測。本篇論文將對近年來所分離之台灣茶赤葉枯病菌進行形態與分子鑑定，並首次發現 C. fructicola 能在台灣引起茶赤葉枯病。在形態鑑定上，C. camelliae 之附著器多為不規則形，而 C. fructicola 則多為卵圓形。於30ºC 之培養條件下時，C. camelliae 菌絲生長較 C. fructicola緩慢，培養於 35ºC 的C. camelliae 之菌絲不生長而C. fructicola 菌絲仍能生長。培養於 pH 10 的培養基上，C. camelliae與C. fructicola 之菌絲生長長度具有顯著差異。在分子鑑定試驗中，使用 ApMat、GS、CAL、GAPDH、TUB2、ITS 等 6 個基因能將 C. camelliae 與 C. fructicola進行區分。從病原性測試的結果中得知，C. camelliae 的毒力明顯高於 C. fructicola。此外，本論文亦證實國際間發表之引子組 TB5/TBCG 對 Colletotrichum spp. 菌株具專一性，而 S576/S577 則不然； S572/S573 引子組可被用於檢測出 C. camelliae，惟 S572/S573 引子組對 C. camelliae 與 Botrytis cinerea 不具鑑別力。

Tea brown blight is the most important disease on tea plants. This disease caused by Colletotrichum spp, in which have been recorded 13 species in the world. C. camelliae and C. gloeosporioides as known, are the casual pathogens of tea brown blight in Taiwan. There is no research about the identification of tea brown blight in Taiwan, as well as no a molecular detection method have developed for the isolates causing tea brown blight in Taiwan. In this study, Taiwanese isolates of Colletotrichum spp. on tea plant were identified based on morphological and molecular phylogenetic analysis. In morphological identification, most of the appressoria of C. camelliae were irregular; as well as those of C. fructicola were ovoid and slightly obtuse at the tip in shape. In molecular identification, the phylogenetic tree based on six (ApMat、GS、CAL、GAPDH、TUB2、ITS) genes showed a distinguishing phylogenetic relationship between C. fructicola and C. camelliae. In the pathogenicity assay, the isolates of C. camelliae were more virulent than those of C. fructicola. In this study, the primer sets TB5/TBCG、S572/S573, and S576/S577 were used for the molecular detection of tea brown blight. Surprisingly, only one primer set, TB5/TBCG, can be used for specifically detect Colletotrichum spp.

目錄
摘 要....I
Abstract....II
謝誌....III
目錄....V
圖目錄....VIII
表目錄....X
附錄目錄....XI
壹、前言....1
貳、文獻回顧....3
一、茶的栽培重要性....3
二、茶赤葉枯病與其病原菌...3
三、茶赤葉枯病菌之分子檢測現況....4
四、研究目的....6
參、材料與方法....7
一、供試菌株....7
二、茶赤葉枯病菌之鑑定....7
(一) 形態鑑定....7
1. 菌落形態鑑定....7
2. 分生孢子形態鑑定....7
3. 附著器形態鑑定....7
(二) 環境條件適應性....7
1. 培養於不同溫度下對於茶赤葉枯病菌的生長影響....7
2. 培養於不同酸鹼值下對於茶赤葉枯病菌的生長影響....8
(三) 茶赤葉枯病菌之毒力評估....8
1. 傷口接種....8
2. 無傷口接種....8
(四) 分子鑑定....9
1. 序列比對....9
2. 單基因親緣樹分析....11
3. 多基因親緣樹分析....11
三、建立茶赤葉枯病菌之分子檢測技術....12
(一) 核酸樣品萃取與製備....12
1. 基因組 DNA (genomic DNA)....12
2. 標準 DNA (standard DNA)....12
(二) 茶赤葉枯病菌之專一性引子對....12
(三) 引子專一性測試....13
(四) 引子靈敏度測試....14
1. 基因體 DNA (genomic DNA)....14
2. 標準 DNA (standard DNA)....14
3. 分生孢子 (conidia)....14
(五) PCR 技術及電泳分析....14
1. PCR 技術....14
2. 電泳分析....15
(六) 即時定量聚合酶連鎖反應 (real-time PCR, qPCR)....15
(七) 茶赤葉枯病菌不同罹病程度組織之檢測流程....16
1. 病害分級....16
2. 帶菌組織之核酸萃取方法與流程....16
(八) 統計分析....16
肆、結果....17
一、茶赤葉枯病菌之鑑定....17
(一) 形態鑑定....17
1. 菌落形態鑑定....17
2. 分生孢子形態鑑定....17
3. 附著器形態鑑定....17
(二) 環境條件適應性....21
1. 不同溫度培養條件對於茶赤葉枯病菌的生長影響....21
2. 不同酸鹼值培養條件對於茶赤葉枯病菌的生長影響....26
(三) 茶赤葉枯病菌之毒力評估....32
(四) 分子鑑定....34
1. 序列比對....34
2. 單基因親緣樹分析....37
3. 多基因親緣樹分析....49
二、建立茶赤葉枯病菌之分子檢測技術....54
(一) 引子專一性測試結果....54
(二) 引子靈敏度分析....59
1. PCR 系統....59
2. Real-time PCR 系統....59
三、茶赤葉枯病菌不同罹病程度組織之檢測結果....67
伍、討論....70
一、形態鑑定結果之討論....70
二、環境條件適應性之討論....70
三、菌株毒力之討論....70
四、分子鑑定結果之討論....71
五、引子專一性結果之討論....71
六、PCR 與 qPCR 分子檢測技術結果之討論....71
陸、結論....73
柒、參考文獻....74
捌、附錄....79

圖目錄
圖 1、茶赤葉枯病菌生長於 PDA 上的形態。由左至右依序為菌落正反面、分生孢子、附著器。(a)-(d) C. camelliae，(e)-(h) C. fructicola。比例尺=10 μm。....18
圖 2、分生孢子與附著器之長寬量測數據。....19
圖 3、Colletotrichum camelliae (M7, P6, T21, T46) 與 C. fructicola (H29, M31, M63, T22) 培養於不同溫度培養條件下 7 天後之 (a) 菌絲直徑、(b) 產孢數量、(c) 菌落照。....22
圖 4、Colletotrichum camelliae (M7, P6, T21, T46) and C. fructicola (H29, M31, M63, T22) 培養於不同溫度條件下1、3、5、7 天，每兩天之平均生長速率。....24
圖 5、Colletotrichum camelliae (M7, P6, T21, T46) 與 C. fructicola (H29, M31, M63, T22) 培養於不同酸鹼值條件下 7 天後之 (a) 菌絲直徑、(b) 產孢數量、(c) 菌落照。....28
圖 6、Colletotrichum camelliae (M7, P6, T21, T46) and C. fructicola (H29, M31, M63, T22) 培養於不同酸鹼值下 1、3、5、7 天，每兩天之平均生長速率。....30
圖 7、接種不同茶赤葉枯病菌之病勢發展圖。....33
圖 8、利用貝葉氏譜系分析法分析 ApMat 序列。....38
圖 9、利用貝葉氏譜系分析法分析 CAL 序列。....40
圖 10、利用貝葉氏譜系分析法分析 GAPDH 序列。....42
圖 11、利用貝葉氏譜系分析法分析 GS 序列。....44
圖 12、利用貝葉氏譜系分析法分析 ITS 序列。....46
圖 13、利用貝葉氏譜系分析法分析 TUB2 序列。....47
圖 14、利用貝葉氏譜系分析法分析 ApMat, GS, CAL, GAPDH, TUB2, ITS 等 6 個基因序列。....50
圖 15、利用貝葉氏譜系分析法分析 ApMat 與 CAL 等 2 個基因序列。....52
圖 16、本研究中所採用的 3 組引子對基因組 DNA 樣本之 SYBR green-based real-time PCR 靈敏度分析結果。....62
圖 17、本研究中所採用的 3 組引子對標準 DNA 樣本之 SYBR green-based real-time PCR 靈敏度分析結果。....64
圖 18、本研究中所採用的 3 組引子對分生孢子樣本之 SYBR green-based real-time PCR 靈敏度分析結果。....66

表目錄
表 1、親緣樹分析定所使用之引子對....10
表 2、茶赤葉枯病菌形態鑑定之測量結果....20
表 3、茶赤葉枯病菌與炭疽病菌之分子鑑定結果....35
表 4、PCR 及 real-time PCR 所使用之專一性引子對....55
表 5、本試驗所使用病原菌及 PCR 引子專一性測試結果....56
表 6、本研究中所採用的 3 組引子對基因組 DNA 樣本之 PCR 及 SYBR green-based real-time PCR 靈敏度分析結果....61
表 7、本研究中所採用的 3 組引子對，標準 DNA 樣本之 PCR 及 SYBR green-based real-time PCR 靈敏度分析結果....63
表 8、本研究中所採用的 3 組引子對分生孢子樣本之 PCR 及 SYBR green-based real-time PCR 靈敏度分析結果....65
表 9、茶赤葉枯病不同罹病級數之分子檢測結果....69

附錄目錄
附錄 1、茶赤葉枯病病徵與茶赤葉枯病菌。....79
附錄 2、本研究所使用 Colletotrichum 供試菌株....80
附錄 3、茶赤葉枯病之病徵分級。....81

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