柑橘黃龍病之診斷、防治技術改進與新技術開發

Abstract

柑橘是台灣最重要的果樹之一，不僅種植面積廣，栽種品種亦繁多，但栽種柑橘時所伴隨的病害也是相當複雜，其中以柑橘黃龍病之危害最為嚴重，植株受感染後會影響其樹勢、生育，導致植株壽命減短、產量下降。目前柑橘黃龍病並無有效的防治方法，僅能透過健康種苗制度、拔除田間病株及防治媒介昆蟲來杜絕病害的擴散。台灣柑橘產業受柑橘黃龍病危害已超過70年，在前人的努力下已建立起相關的病害管理方法，但在執行時仍會遭遇部分問題，使得防治效果有所打折。有鑑於此，本論文將主要由目前防治黃龍病所面臨的三項困難出發，針對柑橘黃龍病的檢測與治療技術改進。柑橘系統性病害包含柑橘黃龍病 (Candidatus Liberibacter asiaticus=CLas造成)、柑橘萎縮病 (citrus tristeza virus=CTV造成)、柑橘破葉病 (citrus tatter leaf virus=CTLV造成) 與柑橘鱗砧病 (citrus exocortis viroid=CEVd造成) 是台灣柑橘產業的重要威脅。可靠的診斷方法對於系統性病害之病害管理十分重要。本論文開發出可同時偵測此四種病原之多合一反轉錄聚合酶連鎖反應 (multiplex reverse transcription-polymerase chain reaction，multiplex RT-PCR)，經最佳化反應條件後，可順利產生對各病原之專一增幅產物 (對CLas之295-bp，對CTV之468-bp，對CTLV之120-bp與對CEVd之196-bp)。靈敏度評估顯示此multiplex RT-PCR可偵測到相當低之病原量。與先前發表之單一 (反轉錄) 聚合酶連鎖反應相比，本次開發之multiplex RT-PCR對於田間樣品展示出更佳的檢測表現，田間調查顯示此四種系統性病原普遍存在於田間，且其中有約三成之田間樣品受到複合感染。本論文是台灣首次調查此四種重要柑橘系統性病害之發生情形，調查結果有助於改善目前柑橘病害管理。因此，此multiplex RT-PCR可應用於定期之病害調查與生產柑橘健康種苗。由於柑橘黃龍病菌在寄主體內具有菌量少且分布不均之特性，因此常靠實驗室操作之方法進行檢測，並無可於田間執行的精準檢測方法，使得開始啟動防治措施的時機不夠即時。因此，本研究旨在開發出一利用TaqMan探針之隔絕式恆溫聚合酶連鎖反應 (insulated isothermal polymerase chain reaction，iiPCR) 之快速且對使用者友善的柑橘黃龍病現地檢測系統 (on-site detection system)。透過簡單DNA萃取方法搭配可攜式POCKIT裝置，此現地檢測系統可於一小時內完成檢測。POCKIT裝置可執行PCR反應並且根據螢光訊號自動生成出定性結果。此TaqMan探針之iiPCR可偵測到單一copy之柑橘黃龍病菌，其靈敏度相當於即時定量PCR (real-time PCR)。後續之田間樣品測試顯示兩者之檢測結果完全一致，且該現地檢測系統在檢測柑橘黃龍病菌上也有良好表現。總結來說，本次開發出之現地檢測系統具有高專一性和靈敏度，將成為簡易、快速且有力之柑橘黃龍病田間診斷工具。自1976年在台灣嘗試以抗生素 (achromycin) 注射治療柑橘黃龍病以來，此方法雖有降低柑橘黃龍病嚴重度之效果，但抑制效果維持不長，因此需每年注射，使得農民因考量經濟成本而望之卻步。本研究按照蔡 (2007) 建立之操作方法，嘗試使用市面上農民可購得之抗生素—保美黴素 (鏈土黴素) 灌注於田間之柳橙與30年以上之雜柚植株，灌注鏈土黴素後之受感染柳橙與雜柚植株，其柑橘黃龍病發病狀況相比於對照組 (只有灌注水) 均有明顯下降。本研究進一步使用real-time PCR追蹤植株體內的CLas菌量，結果顯示植株內的CLas在灌注處理後也呈現大幅度減少 (在雜柚上CLas菌量最多下降1000倍，在柳橙上的CLas菌量下降可達10000倍)，可呼應上述病徵嚴重度下降之情況。此外，藉由分析收穫之雜柚果實可發現灌注處理後可減輕柑橘黃龍病對於果實之影響，增加果實品質。綜合而言，高壓灌注鏈土黴素對柑橘黃龍病有著良好之治療效果。

Citrus is one of the most important crops in Taiwan with the widest planting areas and the most production. After first found citrus Huanglongbing (HLB) in 1951, this disease severely affected the citrus industry in Taiwan. Some strategies including healthy citrus seedling certification, controlling insect vectors, and removing diseased plants are established for HLB management. However, the control effect isn’t identical to expectation. Therefore, the major goal of this study was to increase the control effectiveness (or extend the control effective period). The citrus systemic diseases including citrus Huanglongbing (caused by Candidatus Liberibacter asiaticus, CLas), citrus tristeza (caused by citrus tristeza virus, CTV), citrus tatter leaf (caused by citrus tatter leaf virus, CTLV) and citrus exocortis (caused by citrus exocortis viroid, CEVd) are the threats to citrus production in Taiwan. Reliable diagnostic methods are important for the management of these systemic diseases. In this study, we developed a multiplex reverse transcription-polymerase chain reaction (RT-PCR) assay to detect four pathogens simultaneously. Herein, the specific amplicons from each pathogen (295-bp for CLas, 468-bp for CTV, 120-bp for CTLV, and 196-bp for CEVd) were successfully produced by the optimized multiplex RT-PCR described here. The sensitivities evaluation showed that low titers of pathogens could be detected by this multiplex RT-PCR. Compared with published simplex (RT-) PCR, the detection of field samples by the multiplex RT-PCR developed in this study showed a better performance. The detections by multiplex RT-PCR revealed that these 4 citrus systemic pathogens were commonly found in fields and about 30% of field samples were mix-infected. To our knowledge, this is the first study of a survey of the 4 important citrus systemic diseases in Taiwan and it gives information for improving the disease management. Therefore, the multiplex RT-PCR assay provides a useful method for the routine diseases survey and the production of pathogen-free citrus plants. Due to the low amount of CLas and uneven distribution in plant hosts, the detection of CLas is mainly by PCR-based methods. There is no reliable method for HLB diagnosis in the field. This study was aimed to develop a rapid and user-friendly on-site detection system for CLas using the TaqMan probe-based insulated isothermal polymerase chain reaction (iiPCR) assay. The CLas-specific on-site detection system could be completed within one hour by simple DNA extraction coupled with a portable POCKIT device, which can perform PCR amplification and automatically provide qualitative results derived from fluorescence signals. The sensitivity of the TaqMan probe-iiPCR assay could be as low as single copy of CLas, comparable to a real-time PCR method. Further testing of the field citrus samples showed 100% agreement between the TaqMan probe-iiPCR assay and the real-time PCR method, and the on-site detection system also demonstrated a great performance of CLas detection. With high specificity and sensitivity, the on-site detection system developed in this study becomes a simple, rapid and powerful tool for detecting CLas in fields. The antibiotic (achromycin) injection for curing the HLB was first conducted in Taiwan in 1976. The severity of HLB could be reduced by this method; however, the inhibition period was short (only for 1 year). Therefore, for maintaining the curing efficacy, continuous injections of antibiotics were needed. In this study, the curing efficacy of a commercial antibiotic (streptomycin + oxytetracycline) was tested in Liucheng sweet orange and hybrid pomelo trees in fields. After pressure transfusion, the HLB severities of treated trees (both in Liucheng sweet orange and hybrid pomelo) were reduced. The CLas titers in tested trees were also monitored by real-time PCR. The quantification results showed that the amount of CLas was 1000-fold decreased in treated hybrid pomelo and 10000-fold decreased in treated Liucheng sweet orange. In addition, the quality analysis of tested hybrid pomelo fruits demonstrated that the fruit quality (including fruit weight and brix) of treated trees was increased. In summary, the pressure transfusion of streptomycin + oxytetracycline shows a great therapy effect on HLB-affected trees.