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研究生:賴柏蓉
研究生(外文):Po-Jung Lai
論文名稱:台灣萵苣萎凋病菌生理小種分子鑑定技術之開發
論文名稱(外文):Development of molecular identification for races of Fusarium oxysporum f. sp. lactucae from Taiwan
指導教授:張碧芳張碧芳引用關係
學位類別:碩士
校院名稱:國立中興大學
系所名稱:植物病理學系所
學門:農業科學學門
學類:植物保護學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:71
中文關鍵詞:分子檢測分子標誌生理小種萎凋病萵苣萵苣萎凋病菌隨機增幅多型性核酸
外文關鍵詞:Fusarium oxysporum f. sp. lactucaeFusarium wiltracelettuce (Lactuca sativa)molecular detectionmolecular markerrandom amplified polymorphic DNA
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台灣自 1996 年於雲林縣西螺鎮萵苣 (Lactuca sativa L.) 栽植田中分離到對萵苣具有專一性的萵苣萎凋病菌(Fusarium oxysporum f. sp. lactucae,簡稱 Fola)後,萵苣萎凋病現已成為台灣萵苣夏季生產的主要限制因子之一。鑑定Fola 生理小種 (race),將有助於田間健康管理之用,因此本研究將台灣目前已報導過之生理小種與未知生理小種之 Fola 菌株以及由日本引進之三型生理小種之標準菌株,利用土壤混菌法進行接種試驗,結果顯示 SB1-1、Fo-2、Fo-18、LFO11-13、LFO32-14、LFO106-1 及 LFO106-3 鑑定為 race 1,而 744085及未知生理小種之 Fo-10 與 Fo-40 菌株經接種鑑定為 race 3,由於 Fo-10 與 Fo-40菌株無法利用前人已發表分別針對 race 1 與 race 2 之專一性引子對 (FLA0101 F/R 和 Hani3’/Hanilatt3rev 與 FLA0201 F/R) 增幅出特定核酸 (DNA) 條帶,並且利用隨機增幅多型性核酸 (random amplified polymorphic DNA, RAPD) 技術分析後,得知由這兩株菌株所增幅出之核酸條帶與其它菌株差異甚大,但與接種結果為 race 3 之菌株核酸條帶相似,顯示台灣可能已出現尚未被報導過之 race 3。而為能達到快速檢測萵苣萎凋病菌之目的,對國外已發表之 Fola 專一性引子對 (FLA0001 F/R、FLA0101 F/R 及 Hani3’/Hanilatt3rev) 進行最佳化條件測試,更改後之 PCR 條件可將檢測之靈敏度提升十至一百倍;另外,本研究透過不同隨機引子 (OP-2, OP-3, OP-5, OP-6 及 OP-7) 對萵苣萎凋病菌株核酸進行 RAPD 分析,可從中篩選出對各生理小種具專一性之特異性片段,可進而統整出一檢索表做為快速區分未知萵苣萎凋病菌生理小種之依據,目前本研究利用上述 RAPD 技術,再輔以經最佳化條件後之國外檢測系統,已能快速鑑定出 Fola 菌株各不同生理小種,此技術之開發將免除繁複費時之接種測試,並大幅節省該病原菌之檢測時程。

Fusarium wilt of lettuce (Lactuca sativa L.), caused by the vascular wilt pathogen Fusarium oxysporum (Schlechtend.: Fr.) f. sp. lactucae (Fola) was found in Yunlin lettuce field in Taiwan since 1996, become one of the major factors restricting the stable production of lettuce on summer in Taiwan. Identification of pathogenic races of Fola is fundamentally necessary for lettuce growers to choose appropriate cultivars for production and for breeders to develop Fola-resistant cultivars. In our study, Fola isolates in Taiwan and reference isolates from Japan were subjected to inoculation test for race identification. The results showed that isolates SB1-1 (race 1 reference isolate), Fo-2, Fo-18, LFO11-13, LFO32-14, LFO106-1, and LFO106-3 were identified as race 1; 744085 (race 3 reference isolate), Fo-10, and Fo-40 isolates, with their races unknown, were identified as race 3 in this study. Furthermore, primer sets of FLA0101 F/R and Hani3’/Hanilatt3rev, and FLA0201 F/R were unable to amplified the DNA bands by polymerase chain reaction (PCR) specific to race 1 and race 2, respectively, in the DNA samples of Fo-10 and Fo-40. The random amplified polymorphic DNA (RAPD) patterns also showed differences between Fo-10 and Fo-40, and the other Fola isolates tested. Combining the results of inoculation tests and PCR assay, our results suggested that a new race of Fola may have occurred in Taiwan. In addition, we use the published primer sets (FLA0001 F/R, FLA0101 F/R, and Hani3’/Hanilatt3rev) to test the specificity and sensitivity for Fola isolates. The sensitivity can be increased by 10 to 100 folds by modified PCR conditions. Besides, RAPD analysis of the representative isolates of each race could be a useful mean for rapid and unambiguous race identification of Fola. The RAPD patterns shown in this study could differentiate race 1, race 2, and race 3 of Fola isolates using different random primers (OP-2, OP-3, OP-5, OP-6, and OP-7) that amplified DNA bands specific to race 1, race 2, and race 3, individually. These bands could be organized as a schematism to differentiate races of Fola isolates in Taiwan. In this study, we can differentiate different races of Fola by using RAPD and PCR technique with the combinations of different random primers and primer sets, to reduce the time and efforts for Fola identification and detection.

目次
中文摘要……………………………………………………………………………….1
英文摘要……………………………………………………………………………….2
前言…………………………………………………………………………………….4
材料與方法…………………………………………………………………………….8
供試菌株來源與培養……………………………………………………….….....8
抽取總量 DNA…………………………………………………………………...8
(一) 微波爐法…………………………………………………………………..…8
(二) 傳統抽取法…………………………………………………………………..9
萵苣萎凋病菌之生理小種鑑定…………………………………………………10
(一) 供試植株來源………………………………………………………………10
(二) 接種原製備及病菌土製作…………………………………………………10
(三) 土壤中病原菌密度之測定…………………………………………………11
(四) 生理小種之鑑定及其接種方式……………………………………………11
萵苣萎凋病菌之分子檢測………………………………………………………12
(一) 以聚合酶連鎖反應 (Polymerase Chain Reaction, PCR) 進行專一性測試…………………………………………………………………………….12
(二) 以聚合酶連鎖反應進行靈敏度測試……………………...…………….…13
(三) 南方雜合分析………………………………………………………………13
(四) 以 Hani3’ / Hanilatt3rev 進行 PCR 最佳化條件測試………...……...…14
(1) 不同黏合溫度測試……………………………………………………..14
(2) 不同鎂離子濃度測試…………………………………………………..15
(3) 不同聚合循環次數測試………………………………………………..15
(4) 靈敏度測試……………………………………………………………..16
(五) 利用 RAPD 技術分析萵苣萎凋病菌之核酸指紋圖譜…………………………………………………………………………….16
(六) 病原菌之標的 DNA 的選殖與定序……………………………………...17
(七) 定序結果分析………………………………………………………………18
結果…………………………………………………………………………………...19
萵苣萎凋病菌之生理小種鑑定…………………………………………………19
萵苣萎凋病菌之分子檢測………………………………………………………19
(一) 以聚合酶連鎖反應進行專一性測試………………...……………………19
(二) 以聚合酶連鎖反應進行靈敏度測試………...……………………………20
(三) 將 Hani3’ / Hanilatt3re 引子進行 PCR 之最佳化條件測試…...………21
(1) 不同黏合溫度之測試…………………………………………………..21
(2) 不同二價鎂離子濃度之測試…………………………………………..21
(3) 不同聚合循環次數之測試…………………………………………….21
(4) 靈敏度測試…………………………………………………………….22
(四) 利用 RAPD 技術分析萵苣萎凋病菌之核酸指紋圖譜…………………22
(五) 病原菌之標的 DNA 的選殖與定序……………………………………..23
討論…………………………………………………………………………………...24
引用文獻……………………………………………………………………………...28
圖表……………………………………………………………………………………33

圖表目次
表一、本研究所採用之菌株………………………………………………………33
表二、供試萵苣萎凋病菌菌株之生理小種鑑定……………………………………37
圖一、分子標誌 FLA0001 對萵苣萎凋病菌之專一性 PCR 測試……………39
圖二、分子標誌 FLA0101 對萵苣萎凋病菌之專一性 PCR 測試……………41
圖三、分子標誌 Hani3’ / Hanilatt3rev 對萵苣萎凋病菌之專一性 PCR 測試...43
圖四、分子標誌 FLA0201 對萵苣萎凋病菌之專一性 PCR 測試……………45
圖五、利用 FLA0001 F/R、FLA0101 F/R 及 Hani3’ / Hanilatt3rev F/R 三組引子
對對萵苣萎凋病菌 LFO 106-2 菌株核酸進行 PCR 靈敏度測試…47
圖六、以專一性引子對 Hani3’ / Hanilatt3rev 對萵苣萎凋病菌測試不同黏合溫度之 PCR 結果…………………………………………………………49
圖七、以專一性引子對 Hani3’ / Hanilatt3rev 對萵苣萎凋病菌 LFO 106-2 菌株核酸測試不同 Mg2+ 終濃度之 PCR 結果……………………………51
圖八、以專一性引子對 Hani3’ / Hanilatt3rev 對萵苣萎凋病菌 LFO 106-2 菌株核酸測試不同聚合循環次數之 PCR 結果……………………………52
圖九、利用最佳化條件進行 FLA0001 F/R、FLA0101 F/R 及
Hani3’ / Hanilatt3re 引子對之 PCR 靈敏度測試……………..……………53
圖十、利用隨機引子 OP-1 分析萵苣萎凋病菌之核酸指紋圖譜………………55
圖十一、利用隨機引子 OP-2 分析萵苣萎凋病菌之核酸指紋圖譜……………57
圖十二、利用隨機引子 OP-3 分析萵苣萎凋病菌之核酸指紋圖譜……………59
圖十三、利用隨機引子 OP-5 分析萵苣萎凋病菌之核酸指紋圖譜……………61
圖十四、利用隨機引子 OP-6 分析萵苣萎凋病菌之核酸指紋圖譜……………63
圖十五、利用隨機引子 OP-7 分析萵苣萎凋病菌之核酸指紋圖譜……………65
圖十六、利用OP-2、OP-3、OP-5、OP-6、OP-7 等隨機引子進行 PCR 所增幅出之特異性片段,繪製出可區分萵苣萎凋病菌菌株各生理小種之檢索表…67
附錄一、本研究使用之引子序列及其 PCR 反應程序……………………………69
附錄二、本研究使用之隨機引子……………………………………………………70
附錄三、萵苣萎凋病菌核酸以隨機引子 OP-1 進行 PCR 增福產生 399 bp 之片段………………………………………………………………………………71


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