臺灣博碩士論文加值系統

香蕉為世界生產量最高水果之一，高居臺灣農產品出口第三名。香蕉除了常受香蕉黃葉病 (Panama disease) 的危害之外，胡瓜嵌紋病毒 (cucumber mosaic virus, CMV)、香蕉萎縮病毒 (banana bunchy top virus, BBTV)、香蕉條紋病毒 (banana streak virus, BSV) 及香蕉苞葉嵌紋病毒 (banana bract mosaic virus, BBrMV) 所造成的蕉株矮化、生長不良、不結果或果實及葉片嵌紋、畸形等影響也不容小覷。其中以CMV及BBTV對臺灣香蕉產業影響較大，而BSV和BBrMV也都曾出現病例，雖未猖獗但具有潛在的風險。臺灣香蕉的繁殖方式以組織培養苗 (組培苗) 為主，為避免香蕉病毒藉由組培苗來傳播，農委會防檢局於106年特別訂定「香蕉種苗病害驗證作業須知」，全力推動香蕉健康種苗制度。然而此制度必需仰賴正確而有效率的病毒檢測方法，確保繁殖過程無四種病毒感染。過去針對四種病毒分別已研發四種不同的檢測方法，若無整合，整個驗證過程繁複而缺乏效率。有基於此，本論文旨在研發一套有效率的檢驗方法-「香蕉病毒之四合一反轉錄聚合酶連鎖反應法 (4-in-1 multiplex RT-PCR) 」，將四種病毒的檢測合而為一，取代傳統上需針對不同病毒利用不同方式來檢測，此法將可降低檢測所耗費的成本與時間，也能夠應付國內外大批組培苗訂單及引種進口時的檢疫工作，提升作業流程效率。此外，本論文也利用此檢測法進行臺灣香蕉田間病毒感染情況調查，於屏東、高雄、台南、嘉義、雲林、南投、花蓮、台東等地的香蕉園區進行採樣，採樣結果發現，臺灣因推動健康組培苗已將病毒病害明顯降低，CMV及BBTV兩者在臺灣較常見的病毒，感染率分別只有7.7 %及13.9 %，而BBrMV只在香蕉研究所中自國外引進的Saba蕉園及在2018年有在田間採集到一例過，BSV在採樣中並無發現。此次研究的普遍性調查，證實此法能應用於各式各樣的香蕉品種及不同的株齡，皆能有效地檢測出病毒的存在。又因香蕉是國際貿易上最重要的水果作物，CMV、BBTV、BSV與BBrMV是各香蕉生產國家都共同面臨的產業限制因子，本項技術有製成商品化套組的潛力而推展至國際上。

Banana is one of the most important fruits in the world and ranks the third of export crop in Taiwan. In addition to Panama disease caused by Fusarium oxysporum f.sp. cubense (Foc), the banana plants also face with the threat from different viruses such as cucumber mosaic virus (CMV), banana bunchy top virus (BBTV), banana streak virus (BSV) and banana bract mosaic virus (BBrMV). These four viruses cause various symptoms including stunting, foliar chlorosis or mottling, poor fruit production and decline. CMV and BBTV are the most widespread and serious in Taiwan among four viruses, but BSV and BBrMV have potential impact for several new promoting banana and plantain cultivars. The main propagative method of banana is the tissue culture in Taiwan. To avoid the banana viruses transmitted by vegetative propagation of tissue culture, Bureau of Animal and Plant Health Inspection and Quarantine has established “the certificate instruction of banana diseases for banana seedlings” to promote the pathogen-free nursery system of banana. This system needs accurate and efficient detection methods for those four viruses to verify the seedlings without virus infection. However, the whole procedure of complete virus detection is complicate and time-consuming when the four virus detections have to be conducted individually. This study is dedicated to develop a 4-in-1 multiplex RT-PCR (reverse transcription – polymerase chain reaction) to simultaneously detect four viruses in one assay for saving time and budget. Most banana-producing countries have the same problem caused by these four viruses. Thus, this 4-in-1 virus detection has the internationally commercialized potential for the quarantine and inspection. The developed multiplex RT-PCR was applied to investigate the distribution and disease-severity of four banana viruses in Taiwan. The banana samples collected from different banana-cultivated areas such as Pingtung, Kaohsiung, Tainan, Chiayi, Yunlin, Nantou, Taitung and Hualien showed that the infectious percentages of CMV and BBTV are 7.7 % and 13.9 %, respectively. BBrMV was detected only in few samples (Saba cultivar) collected from Taiwan Banana Research Institute (TBRI), one from Pingtung and one from Yunlin. The BSV-infected sample has not yet been found in the field. This devised multiplex RT-PCR technique could be successfully and efficiently applied in the investigations of four banana viruses in different banana cultivars.

口試委員審定書……………………………………………………………………...ⅰ
誌謝……………………………………………………………………………….…..ⅱ
中文摘要……………………………………………………………………………...ⅲ
英文摘要……………………………………………………………………………....ⅴ
壹、 前言………………………………………………………………………………1
貳、 前人研究…………………………………………………………………………4
一、 cucumber mosaic virus (CMV)……………………..………………………4
二、 banana bunchy top virus (BBTV)…………………..………………………6
三、 banana streak virus (BSV)...……………………………………………….10
四、 banana bract mosaic virus (BBrMV)………………...…………………….12
五、 多合一檢測方法…………………………………………………………..14
參、 材料與方法……………………………………………………………………..16
一、 病毒來源…………………………………………………………………..16
二、 病毒保存…………………………………………………………………..16
A. cucumber mosaic virus (CMV)…………………...…………...............16
B. banana bunchy top virus (BBTV)...………………..………….............16
C. banana streak virus (BSV)……………...……………….…………….17
D. banana bract mosaic virus (BBrMV)………………..…..…….............17
三、 專一性引子對設計………………………………………………………..17
cucumber mosaic virus (CMV)…………..……………………………….17
banana bunchy top virus (BBTV)...……………………………………….18
banana streak virus (BSV)..……………………………………………….18
banana bract mosaic virus (BBrMV)…………..………………………….19
banana actin-1……………………………………………………………..19
四、總核酸萃取法……………………………………………………………..20
五、去氧核醣核酸酶 (DNase Ⅰ) 處理………………………………………..21
六、引子對最佳化條件測試…………………………………………………..21
七、一步驟四合一反轉錄聚合酶連鎖反應 ( One-step 4-in-1 multiplex RT-PCR)……………………………………………………………………….21
八、兩步驟四合一反轉錄聚合酶連鎖反應 ( Two-step 4-in-1 multiplex RT-PCR)………………………………………………………………………22
九、四合一產物再確認-TA cloning…………………………………………..22
十、四種香蕉病毒田間感染情形…………………………………………….23
肆、結果……………………………………………………………………………..24
一、 引子對最佳化條件測試…………………………………………………..24
二、 引子對偵測極限 (One-step RT-PCR)……………………………………24
三、 引子對偵測極限 (Two-step RT-PCR)……………………………………24
四、 與國際已發表之引子對比較……………………………………………..25
五、 引子對最佳混合比例……………………………………………………..25
六、 四合一引子對 (4-in-1 primers) 最佳化條件測試………………………26
七、 四合一引子對 (4-in-1 primers) 測定不同病毒排列組合………………26
八、 核酸萃取品質 (internal control)………………………………………….26
九、 DNase I處理後結果………………………………………………………27
十、 實驗室自行接種及吸芽移植的香蕉病徵………………………………..27
十一、田間香蕉病徵………………………………………………………......28
十二、田間病毒感染情況……………………………………………………..28
伍、討論……………………………………………………………………………..30
陸、參考文獻………………………………………………………………………..37
柒、圖………………………………………………………………………………..46
捌、表………………………………………………………………………………..67
玖、附錄……………………………………………………………………………..85

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