發展重要果樹病害「柑橘破葉病、番石榴果斑病與葡萄捲葉病」之接種與診斷鑑定技術以及防治策略之初探

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論文名稱: 發展重要果樹病害「柑橘破葉病、番石榴果斑病與葡萄捲葉病」之接種與診斷鑑定技術以及防治策略之初探
研究生姓名: 鄭淮嶸
指導教授姓名: 洪挺軒
出版年: 2017
學校名稱: 國立臺灣大學
系所名稱: 植物醫學碩士學位學程
關鍵字: 果樹病害;柑橘破葉病;番石榴果斑病;葡萄捲葉病;植物健康整合管理;Fruit tree diseases;Citrus tatter leaf virus;Guava fruit spot disease;Grapevine leafroll disease;Integrated plant health management
摘要: 果樹為臺灣農業生產作物中最豐富且重要之種類,農糧署2015年統計數據指出果品之生產種植面積占比22.7%,且臺灣地處亞熱帶氣候區,地形亦相當豐富,可栽種之果樹種類相當多元。果樹之最大生產限制因子為病害及蟲害,其中又以系統性病害及不明原因之危害,嚴重困擾農業生產者,影響其收益。本論文將以植物醫學實務案例分析之模式,針對柑橘破葉病、番石榴果斑病及葡萄捲葉病發展診斷鑑定技術及接種方法,並對其防治策略提出具整合性概念之健康管理建議,期能局部解決並探討此三項病害所衍生之產業問題。第一個案例探討柑橘破葉病毒 (Citrus tatter leaf virus,CTLV),其在臺灣發生歷史悠久,且潛伏感染之情況相當嚴重,雖目前已有無指定疫病蟲害之柑橘健康種苗可供選用,但CTLV可藉由機械及嫁接傳播,在柑橘園區防不勝防,又因柑橘類重要砧木枳殼及其雜交種對其相當感病,如大量使用於柑橘健康種苗之繁殖,將加劇CTLV之危害風險,目前針對CTLV已有快速檢測法可供應用。故本研究先就市售嫁接苗及健康種苗調查其帶毒率,進一步再調查臺灣柑橘重要產區之CTLV潛伏感染比率,結果發現市售柳橙及檸檬苗 (非合格的健康種苗) CTLV帶毒率達八成以上,而田間CTLV潛伏感染比率均較十年前上昇,採集的樣本中,柳橙、椪柑及桶柑已達一半以上之罹病率,甚至在使用柑橘健康種苗之園區亦有再感染之情況發生,顯示臺灣柑橘產業對CTLV的防治仍有很大的進步空間。未來學術上也有需要針對CTLV在柑橘商業品種實際危害之病原特性,應有更完整之探討。另一方面,本研究嘗試應用以金剛砂機械接種、刀片刻劃莖幹及樹皮切口法感染CTLV感染性選殖株之胞外轉錄病毒RNA於枳柚、柳橙、椪柑、檸檬及文旦柚等柑橘品種,期能在實生柑橘苗上檢視其病原性及病毒複製累積情形,然目前仍只能在指示植物白藜上造成感染,尚未成功感染其原木本植物寄主柑橘。第二個案例探討自2012年臺灣番石榴產區發生之果實不明原因紅色果斑病,果實外觀不佳直接造成販售價格大幅降低,農民深受其苦。受害之果實會產生深紅色圓形之凹陷病斑,此病斑不會腐爛或快速擴大,各方推測其成因不一,可能是寒害、植物生長必需元素缺乏、小型害蟲危害或是常見番石榴病害之初期病徵,本研究之首要目標便是釐清其成因,降低果斑之發生率。於2014年6月至2015年2月,在彰化縣溪州鄉一處栽植帝王拔及珍珠拔之果園,進行番石榴果斑病之防治試驗,自番石榴之官方建議殺菌劑清單中,選定三元硫酸銅為主要防治藥劑,搭配其他五種殺菌劑,及配合番石榴害蟲發生高峰期,選用殺蟲劑及殺蟎劑,建立適合連續採收作物之用藥處方籤,期能在不增高用藥成本之前提下,以適當的藥劑進行番石榴果斑病之防治,並於2014年12月及2015年1月採樣調查果實品質。防治試驗結果顯示果斑病感染情況大幅改善,果斑發生率在兩品種間分別降低約44.5%及37.4%,罹病嚴重指數亦下降51及45.6,顯著提升果實品質,為番石榴生產者提升收益,並將採收之果實送驗農藥殘留,其檢驗結果符合規範,本研究是為番石榴安全農業、健康管理實踐之成功案例。番石榴果斑病之成因,目前初步鑑定應是Mycosphaerella屬之真菌病原菌造成,然目前尚無法完成柯霍式準則,針對其發病生態及病原性尚無法做最後確認,但在調整藥劑處方籤之下,農民的難題已獲初步解決。第三個案例係在中國山東蓬萊地區之新栽植釀酒葡萄園區 (屬於臺灣、新加坡與中國的跨國企業) 發生疑似葡萄病毒之危害,每年當葡萄果串開始轉色成熟時,植株葉片會開始紅化並捲曲,果實成熟延緩並且糖度降低,為確認其病因是否為葡萄捲葉病毒 (Grapevine leafroll-associated virus,GLRaV) 所致,分別於2015年10月、2016年6月及2016年10月前往中國調查其葡萄植株罹病狀況,本研究採用事先在臺灣進行研發的葡萄病毒檢測技術,再前往病害發生區域提供相關植物醫學支援之模式,協助當地檢測葡萄病毒病害,以進一步輔助其研發無病毒葡萄組織培養苗,為防治葡萄捲葉病毒提出長期性之策略,目前已可應用反轉錄聚合酶鏈鎖反應進行第一型及第三型葡萄捲葉病毒之檢測。期能透過逐步改善現有葡萄病毒之檢測技術,未來得以再開發各種重要葡萄病毒與其他病原之整合式快速檢測方法。另一方面也同時檢視臺灣的情況,於2015年11月調查臺灣葡萄產區之葡萄捲葉病毒發生率,所幸目前檢測結果顯示在苗栗卓蘭、臺中新社、南投信義及水里地區所採得的葡萄樣本均無葡萄捲葉病毒之發生,未來在臺灣應以現行優良葡萄健康管理及生產體系為基礎,擬定釀酒葡萄之健康整合管理策略,為臺灣葡萄產業注入新活力,提升產業競爭力。綜合以上,本論文採取案例分析之模式,初步探討三項重要果樹病害之產業問題,以植物醫學解決問題的角度,逐一提出具體解決的方法或建議,擬定未來可研究之方向,強調植物醫師於農業之重要性。Fruit trees are the most important crops for the agriculture production in Taiwan. Many fruit varieties are cultivated in Taiwan geographically located in the tropical and subtropical areas with plenty of rain, high temperature and high moisture. The major problems for the fruit production in Taiwan are diseases and insect pests. In this thesis, we conducted three case studies of important fruit tree diseases based on the targets of plant medicine. The first case is citrus tatter leaf disease, which has occurred for a long time in Taiwan and considered to be an important citrus virus disease. The previous investigation demonstrated that high percentages of latent infection of CTLV commonly existed in the citrus orchards. CTLV can be transmitted by grafting and mechanical inoculation. It easily infected trifoliate rootstocks and commonly existed in the citrus-cultivated areas even though the pathogen-free nursery system has been promoted for many years in Taiwan. The rapid RT-PCR detection method of CTLV developed previously was used for the field survey in several citrus production area in Taiwan. The results showed that over 80% of Liucheng sweet oranges and Eureka lemons (not qualified and certificated pathogen-free seedlings) were infected with CTLV. Compared with the survey results demonstrated ten years ago, the present infectious percentages of CTLV were much higher. Over 50% of collected samples from the orchards of Liucheng sweet orange, Ponkan mandarin and Tankan mandarin were infected by CTLV. These data revealed that the control of CTLV in Taiwan should be further improved. On the other hand, we try to infect different citrus cultivars such as Citrumelo, Liucheng orange, Ponkan mandarin, Eureka lemon and Wentan pomelo with in vitro transcripts which was synthesized from infectious clone of CTLV-Pk. Unfortunately, even though we used three different inoculum techniques, the transcripts only successfully infected Chenopodium quinoa but not citrus hosts. The second case was the fruit spot disease with unknown agents recently emerged on the fruits of guava (Psidium guajava) since 2012 in Taiwan. The diseased fruits produced dark-red, round, and sunken limited spots with 1 - 5 mm in diameter without rotting. We assumed that microorganisms were the most possible causal agent, therefore, we developed the new prescription of chemicals containing tribasic copper sulfate (main ingredient) and other five fungicides for controlling this putative disease of guava. The field trials were conducted in the field with two cultivars of P. guajava located in Changhua County from June 2014 to February 2015. The results showed that the incidence of the fruit spots on the two guava cultivars treated with our prescription was reduced 51% and 45.6% respectively than the control. Our developed prescription showed the apparent effect of control for this fruit spot disease of guava, which may provide a temporary method for the guava growers to reduce their economic losses. The third case is the study associated with suspected grapevine virus diseases occurring in Shandong, China. Since 2015, there were more and more diseased grapevines showing the leaf curl symptom in the newly planted vineyards. We observed and recorded the symptoms since October 2015, and started to identify the causal agent. The detection methods based on RT-PCR assays for the Grapevine leafroll-associated viruses (GLRaVs) were gradually developed and then applied in the detections of the grapevine samples in Shandong. The results indicated that those diseased grapevines were proven to be infected by Grapevine leafroll-associated virus 1 and 3. The detection methods were subsequently used in the preparation of virus-free tissue-cultured seedlings of grapevine through the monitoring of GLRaVs. We also tested the vineyards in Taiwan with the rapid detection method of GLRaVs. The results showed that all grapevine samples collected in Taiwan showed negative for GLRaVs so far. We may attempt to develop the wine-making grapevine industry especially when the GLRaVs were hardly discovered in Taiwan. These three case studies presented as above were expected to immediately resolve partial disease problems occurring in the citrus, guava and grapevine cultivations, which might highlight the important role of plant medicine.
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