- 學位論文
櫻花簇葉病分子檢測技術建立與檢疫應用
Development of Molecular Detection Technology and Quarantine Applications of Cherry Blossom Tree Witch's Broom Disease
摘要
櫻花簇葉病是由子囊菌Taphrina wiesneri (Rathay) Mix引起的真菌性病害，受感染的櫻花樹會出現分枝密度增加、葉片粗短扭曲及花苞減少的病徵，嚴重影響櫻花觀光產業。近年來本病在日本發生情形日趨嚴重，由於T. wiesneri具有潛伏感染且不易分離培養的特性，櫻花樹受到侵染後約一年半才會顯現病徵，因此Komatsu等人(2010)解析T. wiesneri之internal transcribed spacer (ITS)序列，設計出增幅產物為123 bp的引子對(TwITS-F/TwITS-R)，開發了PCR快速檢測技術，以利健康種苗檢測，防止病害繼續傳播。近年臺灣山區也發現疑似罹患簇葉病的櫻花樹，本研究意欲探索兩個領域：其一為釐清台灣的櫻花簇葉病是否為T. wiesneri所引起，其二為開發出靈敏度與專一性更高之檢測技術，以供病害溯源、病害調查、病害防治成效評估及種苗檢測與檢疫之用。在釐清病原菌的研究方面，採集台灣的罹病櫻花組織進行DNA萃取，利用引子對(TwITS-F/TwITS-R)進行PCR擴增，結果顯示確實有T. wiesneri存在；繼之以形態及分子鑑定確認分離自台灣地區櫻花樹罹病組織之真菌為T. wiesneri，將此T. wiesneri進行純培養後所得之孢子懸浮液以噴灑或針刺進行接種試驗，或是將野外採集之櫻花罹病枝條進行嫁接，再利用分生技術檢測，得知T. wiesneri在寄主內增生與擴展，因此推論其可能為引起櫻花簇葉病的病原，此部分預期可於日後本研究接種之櫻花樹顯現病徵時得到驗證。在開發高靈敏度與專一性之檢測技術方面，本研究解析台灣T. wiesneri菌株的ITS序列，開發出一組增幅產物為326 bp的新引子對(Tw-F/Tw-R)，其偵測T. wiesneri之DNA的靈敏度達1.5 pg/ul，而且與LAMP或qPCR相較，有更高的專一性。此研究並開發出一組增幅產物80 bp的引子對(TWrt-F/TWrt-R)用於進行即時定量聚合酶連鎖反應(quantitative real-time PCR(qPCR)，其靈敏度可達15fg/ul。環型恆溫核酸增幅法(loop-mediated isothermal amplification，LAMP)的開發是以T. wiesneri lys2 gene for aminoadipate reductase (1672 bp)設計所得的三組primers進行試驗，其中效果最佳之第二組的靈敏度達15 pg/ul。將本研究開發之PCR與qPCR檢測技術實際應用於田間調查，結果如下：南部的阿里山吉野櫻採樣區，2012年5月時成株感染率50%，苗木33%；2013年5月檢測50棵櫻花樹之感染率為100%；2013年10月感染率為14%，推測可能是同年6月施用化學藥劑之防治效果。中部的清境山櫻花採樣區，有病徵的部位檢測率為100%，無病徵但鄰近病徵部位也有50%左右輕微感染的情形。北部的陽明山山櫻花採樣區並未觀察到任何有病徵的櫻花樹，也未檢測出T. wiesneri。本研究新開發之qPCR技術冀能應用於檢疫偵測及病害潛伏期之帶菌量檢測，LAMP技術冀能應用於田間快速篩檢，而PCR技術則適用於每年度的病害抽樣監測。由於T. wiesneri侵染寄主速度較為緩慢，建議剪除已受感染的病枝，搭配本研究所開發之檢測技術進行監測，或可有效防止病原菌繼續擴散。
並列摘要
Witch’s broom disease of cherry blossom tree, commonly known as Tengu nest disease in Japan, is caused by the ascomycetous fungus Taphrina wiesneri (Rathay) Mix. With typical symptoms including swollen and hyperplasic branches, stubby and deformed leaves, and reduced buds, the disease poses big threat to tourism industry. Recently, the disease infection in Japan has increased alarmingly. Trying to prevent the spread of disease, Komatsu et al (2010) designed a specific primer pair TwITSF/TwITSR (amplicon 123 bp) for PCR detection of T. wiesneri in host plants. In recent years, the symptoms of witch’s broom disease of cherry blossom tree had been observed in some mountain areas in Taiwan. In this study, we first detected the existence of T. wiesneri in symptomatic cherry trees using the TwITSF/TwITSR primers. We successfully isolated and pure-cultured the pathogen, which then confirmed to be T. wiesneri based on its morphological characteristics and ITS sequences. To test the pathogenicity, we inoculated the plants by spraying with sporidial suspension, stabbing with sporidia, and grafting with diseased scions. Combined with our newly-developed detection techniques, we provided evidence of the proliferation and migration of our T. wiesneri isolates in host branches. The detection techniques developed in this study include the conventional PCR (primer pair Tw-F/Tw-R, amplicon 326 bp), the quantitative real-time PCR (qPCR, primer pair TWrt-F/TWrt-R, amplicon 80 bp), and the loop-mediated isothermal amplification (LAMP, primer set 2) techniques. The sensitivity tests showed that the detection limits for PCR, qPCR, and LAMP can reach 1.5 pg/ul, 15 fg/ul, and 15 pg/ul, respectively. The specificity tests using Botryosphearia dothidea, a common parasite in cherry blossom trees, indicated that conventional PCR with Tw-F/Tw-R has the highest specificity. False-positive signals can appear during the final cycles of the LAMP and qPCR reactions, suggesting that the reaction time should not be set for too long. In addition, we applied the PCR and qPCR techniques for cherry witch’s broom disease detection in field investigation. It was found that in Alishan of southern Taiwan, T. wiesneri was detected in 50% adult trees and 33.3% seedlings in the samples collected in May 2012. In May 2013, Taiwan Forestry Research Institute randomly tested 50 cherry blossom trees and found that 100% of them were infected. However, in Oct 2013, the infection rate has decreased to 14%, which might be due to the effective disease control by applying fungicides in June 2013. In CingJing mountain area of central Taiwan, T. wiesneri was clearly detected in all the symptomatic host tissues; minor infection was also found in some symptomless parts nearby the symptomatic tissues. The cherry blossom trees in Yangmingshan of northern Taiwan did not show any symptoms, and the detection results were all negative. The growth of T. wiesneri may have been inhibited under the relatively higher temperatures in Yanmingshan. With regard to the research and management of witch’s broom disease of cherry blossom tree, the highly-sensitive qPCR technique can be used for quarantine detection as well as the quantification of T. wiesneri in host during the latent infection period; the LAMP technique can be applied for rapid on-site screening in the field; the PCR will be suitable for annual sampling and disease monitoring. Since T. wiesneri multiplies and migrates slowly in the host, early detection and removal of infected branches would help prevent the occurrence and spread of the disease.