台灣重要柑橘品種複合感染柑橘黃龍病菌及柑橘萎縮病毒之研究

Abstract

柑橘黃龍病與柑橘萎縮病是柑橘產業最重要、最棘手的系統性病害。黃龍病 (HLB) 由靭皮部侷限細菌 (Candidatus Liberibacter asiaticus，簡稱Las) 所引起，萎縮病則由同樣寄生於靭皮部的 (Citrus tristeza virus，簡稱CTV) 所引起。過去研究發現田間常常發現有複合感染的情形，且因兩病原皆寄生於植株之韌皮部內，在侵染的過程中可能有交互作用產生。為了探討Las與CTV於台灣常見柑橘品種複合感染之影響，本論文實驗以嫁接方式將病原接種於尤利加檸檬、文旦柚、椪柑、甜橙及桶柑等不同品種之柑橘寄主，並分成複合感染、單獨接種Las或單獨接種CTV等實驗組及健康對照組，觀察接種病原一年期間內之株高變化與病徵發展，並且應用夾層免疫酵素聯結法 (ELISA) 、聚合酶連鎖反應 (PCR) 、即時定量聚合酶連鎖反應 (Real-time PCR) 等技術追蹤病原數量動態變化。試驗結果發現，不論是否有Las的存在，尤利加檸檬皆對CTV極感病，增殖速度快，可造成其矮化、小葉，生長不良，而黃龍病菌對之影響不大，數量較低；但CTV對文旦柚則影響小， Las對其病徵的產生則顯著，如葉步斑駁、葉脈木栓化等；而複合感染會使椪柑產生較單獨感染者更嚴重的病徵，表示兩病原有協同作用關係；桶柑為寬皮柑及甜橙之雜交種，而黃龍病菌對其危害程度較為關鍵。實驗中偵測病原方法以即時定量聚合酶連鎖反應之敏感度最高，其次為聚合酶連鎖反應、夾層免疫酵素聯結法。而為能提高田間柑橘植株複合感染的偵測效率，本論文亦開發Las/CTV雙目標單一步驟反轉錄聚合酶連鎖反應，具有高敏感度、經濟性及快速之優點。本論文之結果可對Las、CTV及柑橘寄主之間的關係有進一步的了解，有助於擬定一套針對黃龍病與萎縮病的共同防治策略。

Both citrus hualongbing (HLB) and tristeza are the most destructive diseases in many citrus-producing areas. HLB is caused by a fastidious bacterium tentatively named Candidatus Liberibacter asiaticus (Las) and tristeza is caused by Citrus tristza virus (CTV). Both Las and CTV inhabit phloem tissues, and the previous studies discovered that citrus plants were commonly co-infected with Las and CTV. They probably have interaction each other when they colonize in the phloem tissues. To investigate the interaction between Las and CTV in the different citrus hosts, Eureca lemon, Wentan pummelo, Ponkan mandarin, Liucheng sweet orange and Tankan hybrid mandarin were graft-inoculated with Las and CTV individually or simultaneously. The developed symptoms were observed and recorded, and the multiplicative fluctuations of pathogens were monitored by ELISA, PCR and Real-time PCR with a one-month interval after inoculation. The results showed that CTV is more important than Las in Eureca lemon plants, and CTV had relatively high titers and contributed more severe symptoms such as dwarf and atrophy. Las is more important than CTV in Wentan pummelo plants, and Las-infected Wentan plants produced typical HLB symptoms such as mottling and vein corking. Ponkan plants simultaneously co-infected with Las and CTV showed synergis-tic effects, and the symptoms were more severe than those infected by Las or CTV indi-vidually. Las contributed more severe symptoms in Tankan plants whereas CTV did not cause significant impact in them. For more efficient monitoring of Las and CTV in the co-infected plants, the 2-in-1 one-step RT-PCR was developed in this study to provide more sensitive, economic and rapid methods. The results presented in this thesis should bring detailed understanding associated with relationships among Las, CTV and citrus hosts, which will be helpful to suggest an integrated strategy for collaborative control of HLB and CTV.