Pathogen Strains, Disease Ecology and Chemotherapy of Citrus Huanglongbing

自1951年柑橘黃龍病 (Huanglongbing = HLB) 在台灣北部首次發生，迅速蔓延成災威脅柑橘產業，經本系松本教授等 (1961) 並根據其病徵稱之為立枯病 (Likubin)。此病病原經國內外多方研究證實起因為不能培養之細菌 (Cadidatus Liberibacter asiaticus)，此菌初期只危害國內主要柑橘品種椪柑、桶柑、及柳橙，於1971後開始感染柚類，顯示此病菌系統近幾十年來發生演化。因此本論文研究利用四種鑑別柑橘品種行生物檢定，根據其病原性與毒性鑑別出目前台灣田間之黃龍病菌系統至少有四種系統，系統 I 僅對椪柑與柳橙具高病原性並產生典型黃龍病病徵，為原始系統 (wild strain)。系統 Ⅱ 對四種鑑別品種皆具高毒性，即對柚類與檸檬也感染，並可快速增殖其內。系統 Ⅲ 在椪柑與柳橙引起中度病徵，在文旦僅出現輕度病徵，然而卻無法感染檸檬。系統 Ⅳ 係弱毒系統，僅感染椪柑及柳橙，幾無病徵表現。經調查台灣田間病菌系統分佈，系統 Ⅱ 為目前分佈廣之最優勢系統族群，系統 Ⅲ 及 I 次之。 此外對台灣目前各柑橘產區內之萎黃植株中，黃龍病罹病率及其與柑橘萎縮病毒 (Citrus tristeza closterovirus ＝ CTV)、柑橘破葉病毒 (Citrus tatter leaf capillovirus = CTLV) 之複合感染情形很普遍。將逢機採集之病葉以聚合酶連鎖反應 (PCR) 偵測黃龍病菌 (Huanglongbing bacteria = HLBB)，酵素連結分析反應 (ELISA) 檢測CTV，反轉錄聚合酶連鎖反應 (RT-PCR) 偵測CTLV。結果顯示各柑橘品種之萎黃病株平均約3成感染黃龍病。其中屏東區之檸檬病株受HLB感染率最高，有52.8%，當中又有近40% 遭CTLV複合感染，但無CTV的複合感染。雲林縣與花蓮縣之柚類病株，則有30~45% 感染黃龍病，當中亦約40% 遭CTLV複合感染，而遭CTV的複合感染率低，僅3~8%。葡萄柚及明尼桔柚之黃龍病株則有90% 以上遭CTV複合感染。宜蘭縣金柑之萎黃病株，感染黃龍病比率最低，僅有10%，當中CTLV或CTV複合感染的比率皆超過60%。故黃龍病與病毒之複合感染於各地區之柑橘品種間有極顯著之差異。而看似健康之各品種柑橘株僅1.9% 受黃龍病菌潛伏感染，並只在文旦 (5%) 與檸檬株 (6.0%) 中發現，黃化病徵與黃龍病菌感染高度相關。 為瞭解黃龍病菌與柑橘病毒複合感染對柑橘植株的影響，以嫁接接種文旦與甜橙植株試驗後發現，受黃龍病菌與CTV或CTLV複合感染之植株其病徵加重而植株萎縮程度皆重於受到黃龍病單一病原感染株。而病原間之相互作用中，黃龍病菌可抑制CTV增殖速率，但黃龍病菌對CTLV無明顯抑制作用。 至於各柑橘經濟品種及各種砧木對黃龍病菌之抗感性資料。經病菌單獨接種試驗顯示椪柑、桶柑對黃龍病菌最為感病，發病速率與病徵進展最快。晚崙西亞次之，文旦、檸檬與金柑則相當耐病，病徵進展亦緩慢。砧木的抗病試驗顯示四類 (1) 感病型:台灣最普遍使用之酸橘、四季橘以及中國大陸常用之枸頭砧木為感病，自接種後第4個月起開始出現輕微黃龍病PCR訊號。(2) 中度耐病: Volkamer Lemon, Rangpur Lime, Rough Lemon, Citrus macrophylla及烏柑子感染黃龍病半年後出現葉片褪色或缺乏營養之黃萎病徵並可維持一年以上，病徵進展緩慢。(3) 高度耐病: Troyer citrange, Swingle citrumelo,，接種後一年後除葉片略縮小外，幾無黃化病徵出現，PCR偵測病原濃度一直維持在較低濃度狀態。(4) 抗病型: 木蘋果 (Limonia acidissima) 幾無 PCR訊號出現。 田間病株中以文旦、茂谷、紅江橙研究發現，病菌於植株之不同部位分佈極不平均，以果實最多，成熟葉與老葉次之，在根部與幼葉中測到少量病菌，在嫩芽最低，樹幹基部樹皮幾無。病原於四季之變化通常於春季四、五月與秋季九、十月之涼溫最多，高低溫之夏冬兩季病菌含量最低。一般在春季四、五月時木蝨具最高傳播力且經追蹤三年，感染病株之速率每年成倍數增加，種在鄰近黃龍病果園之無病苗果園，HLB之傳播由鄰近傳染源開始傳播，第一年傳播感染率少於1.9%，第二年增為5.5%，以後呈S線增加，並呈群落傳播 (gathering transmission)，至第三年達87/865 (12.7%) 進入對數期 (log phase)。 為此病與昆蟲防治管理，方便農民田間診斷管理，簡易的HLB碘試劑檢測套組(Iodine kit) 完成，並標準化: 以粗細度160~200C之砂紙片削取成熟葉上表面組織，放入PE封口袋，加1cc純水，揉成suspension，加1~2滴碘液 (KI 2.5%+I2 1.25%)，即呈紫黑色反應，1分內完成反應，目讀檢驗結果，此法廉價簡便，農民易使用。 更精確的即時定量PCR與簡易HLB核酸萃取法亦被利用於改進HLBB之PCR法: (1) 簡易HLB核酸抽取法之開發可大幅縮減核酸萃取時間與人力耗費。(2) Real-time PCR的利用於早期定量測定寄主中之HLB或增殖量，因此測出最罹病性之椪柑株苗量最多，其次為桶柑、甜橙而抗性之柚及檸檬最低。 因此本試驗研究企圖從1972 (Su與Leu) 起開始施用之四環素治療，加以改進試驗。首先設計一抗生素灌注裝置可於實驗室內分析藥效，而後改進田間灌注方式，並開發出新法: 用加壓灌注器在80磅下，灌1000 ppm之四環素3次 (秋2春1)，可達完全化療效果，處理後不再發病且產生好品質柑果。注射後，剪定頂梢枯枝可增進治療效果，此法引起暫時性藥害包括葉脈褐化、葉片焦枯、大量落葉與新葉細長，於半年後恢復。(Huanglongbing = HLB) has been causing great damages to citrus industry in Taiwan since it first occurred in northern Taiwan in 1951. This psyllid-borne virus-like disease also known as Likubin, was proven to be caused by Candidatus Liberibacter asiaticus, an unculturable phloem-limited bacterium. In the previous time, this pathogen infected main citrus cultivars in Taiwan such as Ponkan mandarins, Tankan tangors and Liucheng sweet oranges whereas it did not infect pummelos before 1971. However, HLB has been able to infect all important citrus cultivars in the Southeast Asia countries including pummelos in the rent years. It implied that the evolution of pathogenicity occurred in these decades. This thesis was amed at differentiating the strains of HLB bacteria (HLBB) through the bioassays with four indicator hosts. Based on their different pathogenicity and virulence, four strains of HLBB were differtiated. The strain I showed pathogenicity on mandarins and sweet oranges by inducing typical HLB symptoms. Strain Ⅱ showed pathogenicity and high virulence on the all differential cultivars and multiplied fast in the all cultivars. Strain Ⅲ caused intermediate symptoms on mandarins and sweet oranges and mild symptoms on pummelos, and did not infect Eureka lemons. The strain Ⅳ is a mild strain, which could infecte mandarins and sweet oranges without symptoms. Strain Ⅱ commonly infected all citrus cultivars with wild distribution in Taiwan, and dominated over the strains Ⅲ and I in the field. The cultivated citrus trees infected by HLBB were commonly discovered in the field of Taiwan. Many citrus trees were infected simultaneously with HLBB, Citrus tristeza virus (CTV) and Citrus tatter leaf virus (CTLV). In the field survey, about 30% of the samples of citrus trees showing yellows symptoms were infected by HLB over the island. Eureka lemon samples collected from Pingtung area had a high percentage of HLB-infection at 52.8%. Approximately 40% of the HLBB-infected lemons were co-infected with CTLV and none CTV. Yellows pummelo samples collected from Yunlin and Hualien counties were infected at 30~45% by HLB and approximately 40% of HLBB-infected pummelos were co-infected with CTLV. Among HLBB-infected pummelo samples including Wentan, Peiyu, and Kao Pan, only 3~8% trees were co-infected by CTV. In HLBB-infected grapefruits and Minneola tangelos, 90% of samples were co-infection with CTV. Approximately 10% of yellows samples of kumquat collected from Yilan county were examined positive of HLBB, and 60% among the HLBB-infected kumquats were co-infected with CTV or CTLV. Therefore, HLB infection and complex infection with the viruses were significantly different among citrus cultivars. In latent infection of HLBB, only 1.9% healthy-looking pummelos and lemons were infected by HLBB. In general, HLBB-infection was closely correlated with yellowing symptoms. The studies on effect of CTV/CTLV on revealed that development of symptoms caused by HLBB, Mix-infection of the two pathogens (HLBB+CTV or HBB+CTLV) had a synergistic effect to induce most severe leaf symptoms and dwarfing. The interaction betwen HLBB and CTV/CTLV were found that HLBB interfered with the replication of CTV, but not CTLV. In the study of resistance/susceptibility of citrus cultivars to Huanglongbing disease, the seedlings of Sunki rootstock grafted with voriou virus-free scions graft-inoculated with HLBB-infected buds, Ponkan and Tonkan were the most susceptible for HLB produced most severe symptoms. Valenica sweet orange showed moderate susceptibility, and Wentan, Eureka lemon and kumquat were tolerant cultivars. According the disease index, the rootstock cultivars were categorized into 4 type including (1) susceptible type: Sunki, Calamondin and Kau Tou sour orange (C. aurantium) rootstocks were susceptible for HLB by producing mild HLB-symptoms and PCR detection 4 months after inoculation; (2) moderate tolerant type: Volkamer Lemon, Rangpur Lime and Rough Lemon. Citrus macrophylla and severinia boxifolia showing mild chlorosis six month later; (3) highly tolerant: Troyer citrange, Swingle citrumelo showing mild stunt and low PCR signal; (4) resistant: woody apple (Limonia acidissima), no symptom and PCR signal. The distribution of HLBB in infected trees of pommelo, tangor and sweet orange in the field. The population of HLBB was most highly in the fruit; mature leaves came to the second; roots and young leaves harbored less; the bark of trunk was found no HLBB. Seasonal dynamic of HLBB population was high in late spring (April to May) and autumn (September and October), and low population in hot summer and cold winter. In general, the highest transmission rate of HLBB by vector psyllides was laid in spring (April and May), and increased at double times yearly. The pathogen-free citrus seedlings transplanted in an orchards near by HLB-diseased Eureka lemon orchard became infected by HLBB at 1.9％ in the first year, starting from the trees grown in edge of new orchard close to the inoculum source. The transmission rate increased to 5.5％ by the end of second year, and 12.7％ after the 3rd year coming to log phase. The HLB transmission by psyllid in PF-citrus trees revealed S curve in gathering pattein. In order to facilitate in disease management and IPM of psyllid, on iodine kit for HLBB detection with 80％ accuracy was developed. The standardized method was as follows: scratch the upper surface of disease leaf with a piece of sand paper (160~200C), then put the sand-paper piece, into a small PE bug, add 1 ml of pure water and rub the sand-paper piece in water for washing off the tissue debris into water. Finally a drop of iodine solution (KI, 2.5％＋I2, 1.25％) was dropping into the suspension in bug for coloring reaction with starch color-turning to purple black within one minute showed positive of HLBB infection. This method was cheap and simple, so that it is easy to be used by farmers. The real-time PCR and simple method of Nucleic acid extraction were applied to improvement of PCR method for HLBB detection: (1) Simplified extraction of HLBB-DNA with two buffer solation was adopted successfully to detect HLBB by short time and cost. (2) Real-time PCR was used for quntily detection of HLBB content in citrus plants i.e. highest content of HLB in most susceptible Ponkan tree and lower cont in Tankan, Valencia and Wentan Pummelo in decending order, and lowest content in Eureka lemon resistant to the pathogen. In order to improve the current transfusion method of tetracycline for chemotherapy of HLB since 1972 (Su and Leu), infusion devise was designed for screening antifiotics, and transfusion trials of antibiotic formula and application numbers in the field were made. Three applications (Autum 2, Spring 1) of 1000 ppm achromycin with air-pressured injector at 80 lbs. showed best curing efficacy of disease pummelo trees. No occurrence of HLB appeared in the recovered pummel trees which produced nomal fruits with good qulity. Pruning upper die-back improved the therapeutic efficacy of treated trees. This improved therapy procedure caused temporary phytotoxity including mild vein browning, slender leaves, leaf scoaching and defotiation soon followed by nomal growth.口試委員審定書 i 致謝 ii 中文摘要 iii 英文摘要 vi 第一章 緒論 1 第二章 黃龍病菌之病原性系統鑑別 3 一、 前言 3 二、 材料與方法 4 三、 結果 6 四、 討論 8 第三章 黃龍病發病生態之研究 14 一、 前言 14 二、 材料與方法 18 三、 結果 26 (一)台灣地區柑橘黃龍病與萎縮病毒、破葉病毒田間複合感染之研究 26 (二)柑橘黃龍病菌與萎縮病毒交互作用 30 (三)黃龍病與柑橘破葉病毒之交互作用探討 39 (四)柑橘黃龍病各寄主對黃龍病之抗感性試驗 47 1. 台灣重要經濟柑橘品種對黃龍病之抗感性試驗 47 2. 世界常用之柑橘砧木品種抗感性測試 51 3. 非柑橘類之黃龍病寄主植物黃龍病之感病性試驗 60 (五)黃龍病菌於植物各部位之分佈及罹病樹中病原菌四季之消長變化 63 (六) 田間健康種苗之黃龍病再感染試驗 67 (七)黃龍病新偵測法之研發 69 1. 黃龍病菌簡易核酸製備法與PCR偵測法 69 2. 黃龍病與澱粉分解酵素之關係及碘液檢測試劑套組之開發 73 3. 即時定量聚合酶連鎖反應(Real-time PCR)之研發 87 四、 討論 94 第四章 黃龍病之化學治療 105 一、 前言 105 二、 材料與方法 106 三、 結果 109 四、 討論 114 引用文獻 142 圖目錄 圖2.1 黃龍病於台灣流行傳染區域 12 圖2.2 四種黃龍病菌系統於四種鑑別品種所引起之病徵 13 圖3.2.1 紅江橙以人工嫁接接種病原一年後之萎縮情況 35 圖3.2.2 紅江橙接種不同病原一年後之葉部病徵圖 36 圖3.2.3 西施柚以人工嫁接接種病原一年後之萎縮情況 37 圖3.2.4 西施柚接種一年之葉部病徵 38 圖3.3.1 柳橙實生苗以人工嫁接接種病原一年後之萎縮情況 43 圖3.3.2 柳橙接種一年之葉部病徵圖 44 圖3.3.3 白柚實生苗以人工嫁接接種病原一年後之萎縮情況 45 圖3.3.4 白柚接種一年之葉部病徵圖 46 圖3.4.1 台灣各商用柑橘品種受黃龍病菌感染後，對其病徵及生長勢之影響 50 圖3.4.2 各砧木接種黃龍病菌一年之葉部病徵 56 圖3.4.3 黃龍病菌對各砧木品種生長勢之影響 58 圖3.4.4 受黃龍病菌感染的日日春病徵圖 62 圖3.6.1 無病健康苗於田間再感染實驗 68 圖3.7.1 簡易核酸萃取法應用於各品種柑橘品種之HLB PCR偵測 70 圖3.7.2 三種簡易組織磨萃法之PCR增幅效果比較 71 圖3.7.3 以簡易核酸萃取法萃取媒介昆蟲木蝨之總核酸進行PCR分析 72 圖3.7.4 電子顯微鏡觀察黃龍病葉組織細胞變異情形 81 圖3.7.5 澱粉分解酵素活性測試 81 圖3.7.6 eppendoff 檢測套組試驗 82 圖3.7.7 以親水性之nylon membrane所測試結果 82 圖3.7.8 以嫌水性nylon membrane測試 83 圖3.7.9 砂紙＋封口袋套組測試試驗 83 圖3.7.10 病葉刮取位置試驗 84 圖3.7.11 碘液檢測各柑橘品種 85 圖3.7.12 六種田間常見之黃化褪色病徵型 86 圖3.7.13 TaqMan primer/probe系統對台灣各經濟品種內黃龍病菌之專一性試驗 91 圖3.7.14 以系列稀釋黃龍病樣本之總DNA分析TaqMan系統之靈敏度 92 圖3.7.15 絕對定量曲線之建立 93 圖4.1 抗生素裝置 128 圖4.2 改良式之耐高壓灌注器與可移動式之充電電鑽、鑽頭 129 圖4.3 四種不同病指數之白柚經三次高劑量灌注後恢復情形 130 圖4.4 白柚病株經四環黴素與青黴素混合液灌注三次後，一年半後病徵消失，樹勢恢復圖 132 圖4.5 白柚病株灌注四環黴素與Harpin混合液 (1000 ppm + 100 ppm)，樹勢完全恢復，生長極茂盛 133 圖4.6 單灌注Harpin (100 ppm) 柚病株，並無法完全抑制黃龍病徵之發展 134 圖4.7 重度發病多年之文旦老病株以四環黴素與青黴素混合液三次灌注後恢復圖 135 圖4.8 以濃縮抗生素溶液藥療重度發病之雜柚大病株，經各配方3次灌注 (秋2春1) 1年半後康復情形 137 圖4.9 嘉義茂谷黃龍病株經各藥劑配方高濃度抗生素溶液三次 (秋2春1) 灌注後1年半恢復情形 139 圖4.10 柚病株灌注後生產果實恢復情形 140 圖4.11 各品種柑橘抗生素灌注後產生之葉部藥害病徵 141 表目錄 表2.1 柑橘黃龍病病菌系統之鑑別 9 表2.2 各黃龍病菌系統之增殖速率與病徵潛伏期 10 表2.3 各黃龍病菌系統於台灣各地之分佈情形 11 表3.1.1 各主要柑橘產區之黃化衰弱病株感染HLB及其與CTV、CTLV複合感染之比率 28 表3.1.2 各柑橘品種植株潛伏感染HLB、CTV及CTLV之比率調查 29 表3.2.1 柑桔黃龍病病原菌與萎縮病毒於紅江橙交互作用對複製速率之影響 32 表3.2.2 柑橘黃龍病菌與萎縮病毒於西施柚交互作用對複製速率探討 33 表3.2.3 黃龍病與萎縮病毒感染對紅江橙病徵及生長勢影響試驗 34 表3.2.4 黃龍病與CTV感染對西施柚病徵及生長勢之影響 34 表3.3.1 黃龍病菌、破葉病毒單獨與複合感染於柳橙實生苗之增殖動態 40 表3.3.2 黃龍病菌、破葉病毒單獨與複合感染於白柚實生苗之增殖動態 41 表3.3.3 黃龍病菌與破葉病單獨與複合感染對柳橙實生苗病徵及生長勢影響試驗 42 表3.3.4 黃龍病與破葉病單獨及複合感染白柚實生苗病徵及生長勢之影響試驗 42 表3.4.1 黃龍病菌於台灣各重要柑橘品種內之增殖速率與所引起之病徵嚴重度 48 表3.4.2 黃龍病對台灣商用柑橘品種生長勢之影響 49 表3.4.3 各種砧木對黃龍病之抗感性試驗 53 表3.4.4 黃龍病對各品種砧木生長勢之影響 55 表3.4.5 受黃龍病菌感染的菟蕬子與日日春之PCR檢測 61 表3.5.1 黃龍病菌於紅江橙病株各部位分佈及四季消長變化 64 表3.5.2 黃龍病菌於茂谷病株各部位分佈及四季消長變化 65 表3.5.3 黃龍病菌於白柚病株各部位分佈及四季消長變化 66 表3.7.1 以碘液染色分析病與健葉內之澱粉分解酵素活性 77 表3.7.2 砂紙粒度大小對碘液染色效果與澱粉懸浮力試驗 77 表3.7.3 碘液濃度對澱粉、病葉殘體染色力及抗還原力比較 77 表3.7.4 黃龍病葉之澱粉含量於室溫降解速率 78 表3.7.5 黃龍病葉之澱粉含量於4℃降解速率 78 表3.7.6 比較碘試劑及PCR偵測黃龍病株澱粉累積與病原菌複製情形 78 表3.7.7 以碘試劑及PCR進行田間黃化病株之普測 79 表3.7.8 以TaqMan法評估台灣各經濟品種接種一年後於成熟葉之菌濃度 89 表3.7.9 以TaqMan法評估世界各砧木品種接種一年後於成熟葉之菌濃度 90 表4.1 以抗生素灌輸裝置測試四環黴素與青黴素對黃龍病菌之藥效 117 表4.2 以浸泡法測試四環黴素與青黴素對黃龍病菌之有效濃度 118 表4.3 抗生素藥害測定，經灌輸法處理過接枝以嫁接組織存活度表示 119 表4.4 抗生素藥害測定，經浸泡法以1個月後之嫁接病組織存活度表示 119 表4.5 對20年生重度發病文旦株一次加壓灌注之藥效試驗 120 表4.6 不同發病程度之30年生白柚植株抗生素灌三次灌注之藥效試驗 121 表4.7 斗六 20年生重度黃龍病文旦病株田間三次灌注之藥效試驗 122 表4.8 對20年生重度發病大株雜柚之田間三次灌注之藥效試驗(嘉義分所) 123 表4.9 重度發病文旦大株，高濃度抗生素溶液田間三次灌注之藥效試驗(花蓮) 124 表4.10 抗生素灌注時機與次數對藥效試驗 125 表4.11 不同發病程度之茂谷病株三次濃縮抗生素溶液灌注之藥效試驗 126 表4.12 三次灌注後之白柚園果實品質調查 127 表4.13 雜柚病株三次灌注後之果實品質調查 127 表4.14 茂谷病株三次灌注後之果實品質調查 12