柑橘黃龍病與萎縮病對台灣各主要柑橘品種之罹病率調查與診斷方法之改良

Abstract

柑橘為台灣重要經濟果樹，約有四萬農戶賴以維生。台灣栽培的柑橘大都是自大陸華南地區引進，如西元1701年引進文旦，1789年引入桶柑，1812年引入椪柑，1930年引進柳橙。早期栽培種以椪柑、桶柑及柳橙為主，至1991年，椪柑、桶柑及柳橙佔栽培總面積之 85%，其他柑種所佔比率仍屬少量。自政府積極推動少量多樣化及品種更新後，台灣栽培之柑橘品種逐年增加，至2004年已呈現品種多樣化，而椪柑、桶柑及柳橙所佔比率隨之降為63%，麻豆文旦之總栽種面積亦高達18%。 柑橘黃龍病(Huanglongbing, HLB)及萎縮病毒(Citrus tristeza closterovirus , CTV)普遍感染多種柑橘品種，在台灣與其他熱帶、亞熱帶柑橘產區嚴重危害寬皮柑及橙類，影響其產量與品質。此類病害是系統性病害，可全身性感染寄主，經無性繁殖之種苗傳播，在田間亦經媒介昆蟲傳播或由工具機械傳染而蔓延成災。耐熱型之黃龍病於1950年傳入台灣，蘇等稱此病為立枯病，發病後縮短柑橘樹齡10年以下，很快蔓延全島，此病在東南亞及南北美洲地區亦發生流行，包括印度、美國、巴西、泰國、日本等國。 本論文針對台灣目前主要柑橘產區之萎黃植株，包括柳橙、椪柑、檸檬以及文旦之黃龍病罹病率及其與柑橘萎縮病毒之複合感染情形進行研究。將逢機採樣之病葉以聚合酶連鎖反應(PCR)偵測HLB，反轉錄聚合酶連鎖反應 (RT-PCR)偵測CTV。結果顯示台灣各柑橘品種平均約六成感染率，其中屏東縣之檸檬感染率為38%具黃龍病，而與CTV的複合感染情形則未發現，與2007蔡等研究結果相同；產於台南縣、嘉義縣、新竹縣、花蓮縣和台東縣之柚類，則有46-63%感染黃龍病，而與CTV的複合感染情形則未發現；產於嘉義縣與雲林縣之柳橙，有高達92-96%感染黃龍病，而其中有25-58%與CTV複合感染；產於台中縣與嘉義縣之椪柑，有37%-83%感染黃龍病，而其中有0-17%與CTV複合感染;。黃龍病與萎縮病毒之複合感染於各地區種植之柑橘品種間有極顯著之差異，2014年統計之感染率與2007蔡等人之統計資料也有部分差異。 1984年蘇等人曾以生物檢定法調查台灣各柑橘品種均可發現感染，近年來林等發展反轉錄聚合酵素連鎖反應(RT-PCR)檢定法能更敏感且準確的偵測病原。由於各病原檢測技術之進步，本研究得以分子診斷技術取代過去之生物檢定法，大量檢定目前台灣各重要柑橘產區之病株感染HLB之比率，亦針對柑橘萎縮病毒之複合感染情形做一調查。本研究亦針對診斷方法之改良，發展多重聚合酶連鎖反應 (Multiplex PCR) 檢定法，能以更敏感且更省時的方法一次偵測多種不同病原，對於大量檢定各重要柑橘產區之病株提供方便又省時的改良方法。

Citrus is one of the most important fruit crops worldwide. Citrus huanglongbing (HLB) and Citrus Tristeza Virus (CTV) are two of the most serious and destructive diseases for citrus. Because of lacking high effective bactericides and resistant citrus varieties for the diseases, many countries mainly relies on uprooting and burning diseased trees from quarantine areas; or enhancement of quarantine regulations in non-quarantine area to prevent the invasion of both diseases. The cultivated citrus trees infected by HLB bacteria (HLBB) were commonly discovered in the field of Taiwan. Many citrus trees were infected simultaneously with HLB bacteria and Citrus tristeza virus (CTV). In this study, we aimed at surveying of morbidity of citrus huanglongbing and tristeza diseases on several important citrus cultivars all over Taiwan and improving their diagnostic method. In the field survey, about 34% of citrus trees showing yellowing symptoms were infected by HLB , about 33% were infected by CTV and approximate 8% were co-infected with both pathogens. Sweet orange samples collected from Yunling area had a highest percentages of both HLB-infection at 71% and 79% infection for CTV. As a result, approximate 58% of the HLB bacteria-infected sweet orange were co-infected with CTV. Ponkan samples collected from Taichung had a lower percentage of HLB-infection at 4% , 33% infection for CTV and none co-infection. 38% of Eureka lemon samples collected from Pintung were infected by HLB bacteria, 46 % of pummelo samples from Tainan were infected and both of them tolerated by CTV infection. In Chiayi, the main citrus industry in Taiwan, sweet orange samples had a high percentage of CTV-infection at 88%, 33% for HLB-infection and 25% for co-infection; Ponkan samples were infected by HLB bacteria at 29%, 54% by CTV and approximate 17% for co-infection; 63% of pummelo samples were infected by HLB bacteria and none by CTV. In this study, we aimed to improve the diagnostic method. We developed a multiplex polymerase chain reaction detection system using unique primer sets specific for Candidatus Liberibacter asiaticus (Las) and also Citrus Tristeza Virus. The two fragments ( 226 for Las and 655 for CTV) were simultaneously amplified using a single PCR reaction and Reverse Transcript-PCR. Consequently, the results of the multiplex PCR were compared with simplex PCR and RT-PCR for detection of each pathogen respectively, it matched. We reported the discovery of co-infection of Las and CTV in the field citrus plants in Pingtung, Tainan, Chiayi, Yunlin, Taichung, Taiwan. Our rapid, sensitive , specific duplex assay should be useful for diagnosis of HLB and CTV especially for quarantine purpose, identification of Las and CTV, and certification and plant improvement program for obtaining HLB-free or CTV-free planting materials. This method should be applicable for confirmation of visual method upon routine symptom.

Keywords: Candidatus Liberibacter asiaticus (Las), Citrus Tristeza Virus, Multiplex PCR. Detection, HLB, CTV