- 學位論文
臺灣兩種主要柑橘類病毒之同步偵測、發病生態及交互作用之探討
Multiplex detection, disease ecology and interaction of Citrus exocortis viroid and Hop stunt viroid of citrus in Taiwan
摘要
柑橘鱗砧類病毒(Citrus exocortis viroid, CEVd)及啤酒花矮化類病毒(Hop stunt viroid, HSVd)為兩種台灣柑橘主要類病毒且逐漸成為柑橘產業上的潛在威脅。在台灣，兩病害雖然已在多年前被發現及紀載，但數十年來仍缺乏完整及詳細的本地研究及調查。本論文針對兩種柑橘主要類病毒的同步偵測、發病生態及交互作用此三大主題進行探討以期望在類病毒的基礎研究上做出貢獻。在同步偵測的部分：利用自行開發的同步反轉錄PCR及同步定量PCR之偵測技術，進行全台灣的柑橘園區，將近700個柑橘樣本中的兩類病毒感染率普查可得知HSVd (32.2%)的平均感染率略高於CEVd (30.4%)。值得注意的是兩種類病毒普遍以複合感染存在於田間，在特定柑橘品種中複合感染率達到55%。經由定量PCR分析顯示兩種類病毒在柑橘寄主的不同組織中呈現不均勻分布，此結果也推薦以枝條的樹皮作為類病毒檢測的標準採樣部位。基因多樣性比對顯示本文所定序的台灣各區的9條CEVd及8條HSVd中，分別存在10處與5處的主要突變熱點。而親緣樹狀圖分析則顯示與世界各地主要的兩種類病毒序列比較，台灣的CEVd與HSVd族群可分成3群與2群。除了兩種柑橘主要類病毒之外，本文也參考前人文獻並針對其他9種類病毒建立標準檢測流程，期望能為台灣未來防檢疫上做出貢獻及參考。在發病生態的部分：本文建構不同類型的類病毒之人工感染選殖株，並已成功的人工機械接種至Etrog citron Arizona 861-S枸櫞、Gynura aurantiaca紅鳳菜、Solanum lycopersicum cv. ‘Rutgers’番茄及Cucumis sativus黃瓜及Rangpur lime廣東檸檬等常用指示植物及感病之商用砧木。另外基於常用的番茄指示植物品種取得不易且價格昂貴，本文亦從29種台灣常用商用番茄品種中篩選出2種品種(蜜番茄’HV-206’及聖女番茄’V-191’)，其葉片可產生嚴重畸形、壞疽及綠島等病徵，可作為替代性的CEVd新的指示植物，其中CEVd在品種’V-191’中的複製含量較高顯示此品種更具有潛力成為新的指示植物。此外，類病毒是否經由種子傳播為一長期爭論的議題。本文檢測三種柑橘品種(紅肉柳丁、茂谷柑及檸檬)的種皮、種膜及種子，結果顯示雖然能少許偵測到CEVd或HSVd，但在帶病種子發芽長成的實生苗上卻無法偵測出類病毒的存在。此證據能證實CEVd與HSVd皆無法在柑橘寄主上經由種子所傳播。除此之外，類病毒是否入侵寄主的頂芽分生組織(shoot apical meristem, SAM)是近年來熱門的研究議題。本文以兩種受感染寄主(柑橘與番茄)的SAM組織切片，進行原位雜合技術(in situ hybridization)偵測顯示兩種類病毒具有相似的入侵區域並且僅會入侵至柑橘的SAM組織。此證據能幫助並改進目前的從頂芽分生組織中分離培養無毒種苗的作業要點上的盲點及缺失。在交互作用的部分：本文在雲林斗六一處柑橘田區(紅肉柳丁與茂谷柑)進行3年12季的長期類病毒族群動態調查，研究其環境溫度與類病毒之間關係及兩種類病毒之間的交互作用關係。結果顯示，除了被HSVd感染的紅肉柳丁之外，類病毒與溫度並無顯著的相關性。此結果說明氣溫並非類病毒生活史上關鍵的外在影響因子。另外經由定量PCR及多項統計分析可得知除了兩種類病毒存在於高溫下的紅肉柳丁內，其餘在兩種柑橘品種中的不同組織中皆具有顯著性的正相關。本文亦針對兩種類病毒在寄主組織及細胞內分布情形進行光學及穿透式電子顯微鏡的原位雜合研究，結果顯示兩種類病毒在兩種柑橘寄主(紅肉柳丁與茂谷柑)的四處不同組織(鱗砧部樹皮、根部、枝條樹皮及葉部)中皆具有相似的分布情形。經由分子技術、統計分析及組織細胞中分布的結果顯示，雖然複合感染的柑橘寄主上缺乏病徵加成的證據，CEVd與HSVd之間仍具有類病毒含量上正相關的關係且在寄主內具有相似的分布情形。
並列摘要
Two previously reported citrus viroids, Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd), represent main threats to the citrus industry in Taiwan. However, studies on these citrus viroids remain largely to be explored. To establish study about the current status of the citrus viroids in Taiwan, we focused on development of multiplex detection, disease ecology of the citrus viroids, and relationship between the two citrus viroids. In multiplex detections, a multiplex RT-PCR and a real-time RT-PCR were developed for detecting of CEVd and HSVd. Our field-survey assay of 689 citrus samples in Taiwan revealed that HSVd was slightly more prevalent than CEVd (32.2% vs. 30.4%). Furthermore, CEVd and HSVd commonly co-existed within the citrus cultivars (up to 55%). Results of the multiplex quantitative analysis suggest uneven distributions of both viroids within different plant tissues, of which twig bark appears to be the most appropriate and reliable material for viroid detections, thereby being used for quarantine inspections. The genetic diversity assay appeared the existence of 10 and five major mutation sites in CEVd and HSVd in Taiwan, respectively. A phylogenetic analysis revealed that Taiwanese isolates of CEVd and HSVd were grouped in three and two clusters, respectively. To obtain single isolate of each viroid for disease ecology studies, different forms of infectious clones of CEVd and HSVd were established and successful inoculated to indicator plants, includings ‘Etrog citron’ Arizona 861-S (C. medica), Gynura aurantiaca, Solanum lycopersicum ‘Rutgers’ and Cucumis sativus, along with sensitive citrus rootstock of Rangpur lime (Citrus × limonia Osb.). To facilitate the availability and application of indicator plants for viroid detection, a parallel effort was directed toward developing alternative indicator plants for CEVd detection. Toward this end, 29 tomato commercial cultivars were screened by inoculations with two Taiwanese isolates of CEVd’s infectious RNAs. Only two tomato cultivars, Honey tomato (Xiaomi, ‘HV-206’) and Cherry tomato (‘V-191’), showed leaf distortion, and necrotic or green-island symptoms. Higher titers of both CEVd isolates also indicate that ‘V-191’ is a more susceptible and suitable indicator for CEVd detection. In disease transmission studies, CEVd and HSVd were detected only rarely on seed coats, seed membranes and inner seeds obtained from blood orange, Murcott mandarin, and lemon. The seedlings originating from infected seeds did not present detectable titers for the two viroids, suggesting that these two viroids do not exhibit seed-borne transmission in citrus hosts. In addition, two viroids showed similar patterns when invading shoot apical meristem (SAM) of tomato and citrus as shown by DIG-labeled in situ hybridization. The two viroids displayed the ability to invade into SAM and leaf primordia of citrus but only leaf primordia of tomato. The study provided useful information for improving of the experimental protocols designed for obtaining viroid-free meristem tissues for control of viroid diseases. For interactive studies, titers of the two viroids were examined by real-time RT-PCR in 17 citrus plants (including blood orange and Murcott mandarin) every 3 months (spring, summer, fall and winter) from 2011 to 2013. No correlation was found between temperature and titer of each viroid, except with HSVd-infected blood orange. This result suggests that temperature is likely not a critical environmental factor during the life cycles of the two viroids. Based on the results of real-time RT-PCR, statistical methods, significant positive correlations, the two viroids appeared in specific tissues of both orange cultivars, except for blood orange at high temperatures. At the cellular/subcellular levels, the two viroids showed similar localization patterns in four tissues and cells, as shown by in situ hybridization, fluorescence microscopy, and transmission electron microscopy. Our results demonstrate that the two viroids have a positive relationship and a similar infection pattern, while displaying titer enhancement and localization similarity with no symptom aggravation.