台灣七種柑橘類病毒快速偵測方法之建立及應用

Abstract

類病毒為不具有蛋白質外鞘且無蛋白編碼之單股環狀RNA分子 (non-coding single-stranded circular RNA)，大小為249至401個核酸，並藉由分子間氫鍵形成桿狀或具分枝之二級結構。儘管如此，類病毒為具有感染力並可造成多種植物病害的病原菌。柑橘是重要的經濟果樹作物之一，其受類病毒的危害甚廣；曾在臺灣發生而被報導的柑橘類病毒有柑橘鱗砧類病毒 (Citrus exocortis viroid)以及啤酒花矮化類病毒 (Hop stunt viroid)。國外尚有柑橘曲葉類病毒 (Citrus bent leaf viroid, CBLVd)、柑橘矮化類病毒 (Citrus dwarf viroid, CDVd)、柑橘樹皮裂紋類病毒 (Citrus bark cracking viroid, CBCVd)、柑橘類病毒第五型 (Citrus viroid V, CVd-V)及柑橘類病毒第六型 (Citrus viroid VI, CVd-VI)等柑橘類病毒病害之報導。反轉錄聚合酶連鎖反應 (RT-PCR)為目前普遍運用於柑橘類病毒之偵測技術，而國內外田間病害報導常發現作物受柑橘類病毒複合感染，又現行偵測技術中針對複合感染的柑橘植株，僅能分別進行檢測，費時且繁瑣，故本論文研究目標為改良柑橘類病毒偵測技術，簡化現行檢測流程並運用於台灣田間柑橘類病毒之偵測調查。本研究目前已完成台灣各地的類病毒檢測，包括新竹、彰化、斗六、嘉義、屏東、宜蘭、花蓮及台東等地，偵測結果發現台灣除了柑橘鱗砧類病毒 (CEVd)以及啤酒花矮化類病毒 (HSVd)外，可能存在另外三種柑橘類病毒，柑橘曲葉類病毒 (Citrus bent leaf viroid, CBLVd)、柑橘矮化類病毒 (Citrus dwarf viroid, CDVd)、柑橘樹皮裂紋類病毒 (Citrus bark cracking viroid, CBCVd)。進一步利用或得到的田間類病毒序列進行類緣分析顯示，台灣島內的CEVd可分為兩群，且該兩群與已知之分類群並無顯著相似性; 相反的，其他于台灣存在的其他柑橘類病毒則分散性的分佈于世界各地之分類型內，顯現這些柑橘類病毒可能為近年來由世界各地傳至台灣島內。

Viroids consist of solely non-coding ssRNA (249-401 bp) and frequently form secondary structure by intramolecular interactions. They are known as the smallest plant pathogens and infect a wide variety of crops. In spite of its small and simple property, plant viroids cause a great deal of agricultural loss. Citrus is one of the most important crops, of which numerous cultivars are susceptible to several viroids. In Taiwan, two plant viroids have been reported, namely Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd). Nevertheless, Citrus bent leaf viroid (CBLVd), Citrus dwarf viroid (CDVd), Citrus bark cracking viroid (CBCVd), Citrus viroid V and VI (CVd-V and VI) are also reported globally to be able to infect citrus crops worldwide. The molecular biology technique reverse transcription polymerase chain reaction (RT-PCR) is commonly employed to detect the citrus viroid. Complex infections can generally be found in citrus crops. Due to the fact that a single plant may be infected by multiple viroids simultaneously, the present detection method for citrus viroids is time-consuming and laborious. Thus, the purpose of this study aims to improve the detection technique for multiple citrus viroids in a time-saving and cost-effective manner, thereby facilitating the field survey and inspection of citrus viroids. The study concludes a survey of citrus viroids over various locations in Taiwan, including Hsinchu, Changhwa, Douliu, Chiayi, Pingtung, Yilan, Hualien and Taitun. Our result suggests that rather than CEVd and HSVd, additional three viroids, CBLVd, CDVd and CBCVd, might also exist in the citrus fields of Taiwan. In addition, via phylogenetic analyses, we suspected that CEVd exists in Taiwan for a long time to form two specific clades, whereas other 3 detectable citrus viroids might be imported in recent years from all over of the world.