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研究生:劉賢玲
研究生(外文):Hsien-Ling Liu
論文名稱:臺灣蝴蝶蘭之病毒病因研究
論文名稱(外文):Etiological Study on Viruses Infecting Phalaenopsis Orchid in Taiwan
指導教授:蘇鴻基蘇鴻基引用關係
指導教授(外文):Hong-Ji Su
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:植物病理學研究所
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:2000
畢業學年度:88
語文別:中文
中文關鍵詞:蝴蝶蘭病毒病因蘭花嵌紋病毒齒舌蘭輪點病毒胡瓜嵌紋病毒黃圓斑
外文關鍵詞:PhalaenopsisvirusEtiological studyCymbidium mosaic virusOdontoglossum ringspot virusCucumber mosaic viruschlorotic ring spot
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蝴蝶蘭(Phalaenopsis spp.)為原產自亞洲地區之美麗氣生蘭,台灣東部、中南部蘭園與台糖公司引進組織培養技術大規模栽培,使蝴蝶蘭成為國內外重要觀賞花卉,切花、盆花皆有很大的市場。文獻記載且經證實於台灣蝴蝶蘭上發生之病毒有Cymbidium mosaic virus (CyMV)、Odontoglossum ringspot virus (ORSV)與Cucumber mosaic virus (CMV),但多數為零星之調查報告。利用八種指示植物進行生物檢定,於田間蝴蝶蘭蘭株上檢測出CyMV與ORSV之存在,且葉片具黃色條斑者多半可測得CyMV,嚴重發病時條斑會凹陷,且CyMV與ORSV覆合感染者葉片上黃色條斑較易有凹陷之情形。而ORSV單獨感染白花品系時成潛伏性無病徵,蝴蝶蘭對ORSV為抗病性。利用TENS RNA抽取法配合依據已知序列所設計出之二對引子對,可於無病徵蝴蝶蘭上以one-step RT-PCR法成功地檢測此二種病毒。以紫紅核酸染劑染色法得到細胞病理學上之ORSV及CyMV驗證。以源自田間蝴蝶蘭、嘉德麗亞蘭(Cattleya sp.)與蕙蘭(Cymbidium sp.)所得之CyMV與ORSV進行組織嫁接與機械接種試驗,證明此二種病毒於田間蝴蝶蘭與其他不同屬蘭花間可經由機械傳播之方式交互感染。ORSV培養溫度試驗發現,28℃下ORSV於白花品系蝴蝶蘭中之複製會受到短暫抑制,但一個月後病毒複製情形即趨近23℃之植株,而二者病徵表現均屬於潛伏性無病徵型(symptomless)。CyMV之溫度試驗則發現,此病毒之複製於28℃下植株中比23℃中好,於白花紅心品系蝴蝶蘭上之病徵表現屬於潛伏性無病徵型,於白花黃心品系中則於23℃下呈現潛伏性無病徵狀態,而在28℃下則表現顯著的黃萎條斑(chlorotic streak)病徵,證明蘭花品種間的差異與培養溫度對於CyMV於植株上之病徵表達有相當大之影響。以CMV單元抗體進行ELISA檢測,未發現蝴蝶蘭感染情形,而利用源自香蕉之四種不同CMV分離株進行重複接種,一個月至數個月後以ELISA檢測皆呈負反應,可能CMV系統不能感染蝴蝶蘭白花品系。此外,冬季低溫時在蝴蝶蘭組培實生苗上之疑為病毒發生所造成的黃圓斑新病害,經由生物檢定、CyMV與ORSV之RT-PCR偵測及CMV之ELISA檢測均測不到病毒。以各種機械接種法接種也未成功。以紫紅核酸染劑染色法觀察可見田間黃圓斑病株表皮細胞中看不到病毒引起之內涵體,但有菱形不著色之結晶體累積,且病斑部位維管束部分於螢光顯微鏡下可見異常之黃色螢光反應產生。罹病蝴蝶蘭葉片組織以葉切片浸展法(leaf dipping)電子顯微鏡觀察未發現病毒顆粒,組織超薄切片電子顯微鏡觀察發現葉綠體有osmiophilic bodies與澱粉累積,而細胞核與粒線體則無異常。此病在低溫易發生於組培實生苗,但病徵表現不穩定,老熟病株不再表現病徵。此病因似為無病毒感染狀。
Phalaenopsis spp., a beautiful orchid originated from Asia, become an important ornamental crop in Taiwan after applying tissue culture technique. Cymbidium mosaic virus (CyMV), odontoglossum ringspot virus (ORSV), and cucumber mosaic virus (CMV) were recorded as viral pathogen in Phalaenopsis spp. grown in Taiwan but most records were parts of survey rather than general reports. Bioassaying with 8 indicator plants, CyMV and ORSV were detected. CyMV was detected in most leaves with chlorotic streak. The streaks were usually depressed when the platn were infected with both CyMV and ORSV. White-flower Phalaenopsis cultivars infected only by ORSV were symptomless. With TENS RNA extraction method and one-step RT-PCR, the two primer pairs designed with known sequences were successfully to detect ORSV and CyMV in symptomless leaves. Both ORSV and CyMV infection were cytopathologically reconfirmed with Azure A staining method. Tissue inoculation and mechanical transmission tests with different isolates of ORSV (ORSV-Phal. & ORSV-Cym.) and CyMV (CyMV-Phal. & CyMV-Catt.) showed that interspecies infection of ORSV and CyMV could be caused by mechanical transmission. Growing at 28℃, ORSV replication in white-flower cultivars was shortly suppressed, then the replication increased after 1 month. The ORSV-infected plants grown at 28℃ or 23℃, were symptomless. Different cultivars and growing temperature affected symptom expression of CyMV in Phalaenopsis spp. CyMV in white-flower cultivar replcate better at 23℃ then at 28℃. CyMV-infected red-heart cultivar were symptomless at 23℃ or 28℃. Yellow-heart cultivar infected by CyMV showed apparent chlorotic streak at 28℃ while it stayed symptomless at 23℃. CMV infection was not detected by DAS-ELISA with monoclonal antibody (No. 7-1). Repeated inoculation tests with 4 different isolates / strains of CMV isolated from banana, were made, but every inoculated plantlet showed ELISA negative after 1 to 5 months. The new virus-like disease of chlorotic ring spot on leaves was studied by several methods (ex. bioassay, ORSV RT-PCR, CyMV RT-PCR, and CMV ELISA), but it revealed negative results of known viruses. The chlorotic ring spot could not be transmitted by mechanical transmission. Staining with Azure A, no specific cytoplasmic or nuclear inclusion was observed in epidermal cells of Phalaenopsis orchids showing chlorotic ring spot symptoms, but unstained diamond-shaped crystals were observed. No phloem-limited virus specific fluorescence was observed under UV microscope, but strong fluorescence was observed in sclerenchyma cells of vascular bundle tissue. No virus-like particle was observed under TEM observation with leaf dipping method. Cytopathological changes in mesophyll cells of thin section from chlorotic leaves of chlorotic spot diseased Phalaenopsis plants were observed. There were no cytopathological changes in mitochondria, nucleus, and cytoplasm, but deteriorated chloroplasts containing numerous osmiophilic bodies and starch grains with dispersed granae, were observed. The disease of chlorotic spot usually occurred to seed-born Phalaenopsis plantlet at low temperature, but the symptom expression was usually unstable. In view of the above-mentioned experiments, the causal agent of the chlorotic ring spot might not be a virus.
壹、前言-------------------------------------------------------1
貳、前人研究---------------------------------------------------4
參、材料與方法-------------------------------------------------8
(一) 健康植物之準備------------------------------------------8
(二) 罹病材料之來源與保存------------------------------------8
(三) 細胞病理學觀察------------------------------------------9
1. 紫紅核酸染劑染色觀察法
2. 螢光顯微鏡觀察法
3. 穿透式電子顯微鏡觀察法
(1) 葉切片浸展法(Leaf Dipping)
a. Uranyl acetate (UA)染色法
b. Potassium phosphotungstate (PTA)染色法
c. Ammonium molybdate (AM)染色法
(2) 超薄切片(Ultra-thin section)
a. 樣品塊製備
b. 超薄切片
c. 超薄切片染色
d. 切片觀察與照相
(四) 溫度與病徵發展-----------------------------------------13
1. 田間黃圓斑罹病株發病與溫度之關係
2. ORSV及CyMV接種植株發病與溫度之關係
(五) 機械接種法---------------------------------------------14
1. 由蝴蝶蘭病株病汁接種至指示植物~摩擦接種法(rubbing)
2. 由指示植物病毒回接至蝴蝶蘭
(1) 摩擦接種法(rubbing)
(2) 穿刺接種法(puncturing)
a. 組織穿刺接種法
b. 汁液穿刺接種法
(3) 刀片切割接種法(slicing)
3. 蝴蝶蘭組織嫁接法
4. 由香蕉嵌紋病株CMV接種至指示植物 ~摩擦接種法(rubbing)
5. 由指示植物中病毒接種至菸草~摩擦接種法(rubbing)
(六) 反轉錄聚合酉每鍊鎖反應(RT-PCR)偵測齒舌蘭輪斑病毒
(ORSV)與蘭花嵌紋病毒(CYMV)-----------------------------17
1. RNA抽取法
(1) RNeasyTM Plant Mini Kit (QIAGEN)
(2) LiCl法
(3) Guanidine法
(4) Total nucleic acid extraction法
(5) Tris-Guanidine法
(6) TENS法
2. ORSV RT-PCR引子對(OR-608)設計
3. CyMV RT-PCR引子對(Cy-690)設計
4. ORSV與CyMV RT-PCR之進行
5. PCR產物電泳膠體分析
(七) 南方雜合分析法-----------------------------------------23
1. 核酸探針之製備
(1) Gel extraction (QIAquickTM Gel Extraction Kit)
(2) 核酸探針之標誌
a. 聚合酉每鍊鎖反應法(PCR-labeling)
b. 切口移位法(Nick-translation)
2. PCR產物電泳膠體分析
3. 核酸轉印(Transfer)
4. 雜合反應(Hybridization)
5. Post-washing
6. 呈色反應偵測(Detection)
(八) 直接酵素連結免疫反應法(DAS-ELISA)偵測胡瓜嵌紋病毒(CMV)-27
肆、結果------------------------------------------------------29
(一) RNA抽取法之調整----------------------------------------29
1. RNA抽取法之選擇
2. RNA沈澱時間之調整
(二) 齒舌蘭輪點病毒ODONTOGLOSSUM RING SPOT TOBAMOVIRUS (ORSV)
-------------------------------------------------------30
1. 病徵觀察
2. 病原鑑定
(1) 生物檢定
(2) RT-PCR檢測
(3) 細胞病理觀察
3. 接種試驗
(1) 接種源試驗
(2) 分離株差異性試驗
4. 溫度變化與發病情形
※RT-PCR產物之再確定
5. 蝴蝶蘭品系對ORSV之抗性
(三) 蘭花嵌紋病毒CYMBIDIUM MOSAIC POTEXVIRUS (CYMV)---------35
1. 病徵觀察
2. 病原鑑定
(1) 生物檢定
(2) RT-PCR檢測
(3) 細胞病理觀察
3. 接種試驗
4. 溫度與發病之影響
(1) CyMV分離株接種株之培養溫度試驗
(2) CyMV蝴蝶蘭分離株(CyMV / Phal.)之培養溫度試驗
(四) 胡瓜嵌紋病毒CUCUMBER MOSAIC CUCUMOVIRUS (CMV)----------39
1. 田間蝴蝶蘭DAS-ELISA檢測
2. 細胞病理觀察
3. 接種試驗
(1) CMV系統之鑑別
(2) CMV接種
(3) 接種植株病毒偵測
(五) 新似病毒病(VIRUS-LIKE DISEASE):黃圓斑病---------------42
1. 病徵觀察
2. 病原鑑定
(1) 生物檢定
(2) 細胞病理觀察
a. 紫紅核酸染劑染色觀察
b. 螢光顯微鏡觀察
c. 電子顯微鏡觀察
3. 接種試驗
4. 溫度變化與發病情形
伍、討論------------------------------------------------------47
陸、中文摘要--------------------------------------------------51
柒、英文摘要--------------------------------------------------53
捌、參考文獻--------------------------------------------------55
玖、圖表說明--------------------------------------------------62
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