台灣阿里山櫻花簇葉病之PCR檢測與鑑定

櫻花簇葉病，日本俗稱天狗巢病，有別於一般植物菌質體所造成之簇葉病，此病由真菌 Taphrina wiesneri (Ráthay) Mix 引起，屬於子囊菌，於花季結束後藉由子囊孢子傳播，因此傳播速度快，受感染櫻花枝條出現腫脹且分枝密度增加、葉片粗短而扭曲及花苞數量減少等現象，病株樹勢逐年減弱，最終導致櫻花樹死亡，為日本觀賞櫻花之重要病害。針對櫻花簇葉病，日本雖已設計專一性引子對 TwITSF/ TwITSR 進行聚合酶鏈鎖反應 (PCR) 檢測，但日方所設計之引子對產物大小僅有123bp大小，定序後能取得之序列資訊相當有限，且產物片段小易與反應殘留之引子對分子混雜造成判讀上的不便，而本研究設計之引子對所增幅的產物大小為323bp，並位於足以作為區別生理小種的多變異ITS區域，所取得之序列資訊較為充足，在未來進行生理小種或是親緣鑑定上具有一定優勢。義大利亦針對 T. wiesneri 進行研究，興日本研究相同，皆發現此病原可以菌絲體狀態於葉片、枝條與花苞內在櫻花樹上過冬，待隔年春天櫻花抽芽時進入有性世代，產孢後繼續感染櫻花樹。近年台灣阿里山亦發現櫻花樹簇葉之現象，因此本研先以日本所設計之專一性引子對，進行 PCR 檢測及定序，證實台灣阿里山櫻花受T. wiesneri感染。為欲進一步確認病原來源，本研究針對病原 rDNA ITS (internal transcribed spacer) 區域設計產物長度較長之專一性引子對 Tw-F/ Tw-R，定序後再進行序列比對，希望透過較大片段DNA序列，來解明台灣和日本櫻花簇病病原是否同源，經增幅反應片段定序結果為100%相似度，也證實造成台灣阿里山櫻花簇葉病與日本為同一來源，此病極有可能由日本傳入國內。隨機採樣阿里山地區之櫻花枝條與櫻花苗木進行PCR檢測，發現阿里山工作站地區櫻花樹與櫻花苗木發生簇葉病之潛伏感染情形嚴重，除需加強防治外，亦需加速苗木之進口檢疫檢測。本研究設計之專一性引子對除具備較日本發表之引子對更高的靈敏度可用於檢測，較長的產物長度亦提供定序所需資源，檢測與定序並進，即時發現病原可能產生的變異，並進行必要之隔離。

Cherry Clump Witches' Broom Disease, commonly known in Japan as Witches' Broom Disease (Tengu Nest Disease), differs from typical clump witches' broom diseases caused by phytoplasmas. This disease is caused by the fungus *Taphrina wiesneri* (Ráthay) Mix, which belongs to the phylum Ascomycota. It is disseminated by ascospores after the flowering season, leading to rapid spread. Infected cherry branches exhibit symptoms including swelling, increased branching density, shortened and distorted leaves, and reduced number of flower buds. The disease gradually weakens the vigor of infected trees over successive years, eventually resulting in the death of cherry trees, making it a major destructive disease affecting ornamental cherry trees in Japan.

Although Japan has developed a specific primer pair TwITSF/TwITSR for polymerase chain reaction (PCR) detection, the amplicon size of this primer pair is only 123 bp. The sequence information obtained after sequencing is relatively limited, and the small amplicon is prone to contamination with residual primer molecules, causing inconvenience in result interpretation.

The primer pair designed in this study amplifies a 323 bp fragment located in the highly variable ITS (Internal Transcribed Spacer) region, which is sufficient for distinguishing physiological races. The obtained sequence information is more abundant, offering certain advantages for future physiological race identification and phylogenetic analysis.

Italy has also conducted studies on *T. wiesneri*. Consistent with the Japanese research, it was found that the pathogen overwinters in cherry trees as mycelia within leaves, branches and flower buds. It enters its sexual generation when cherry trees sprout in the following spring, producing spores to continue infecting cherry trees.

In recent years, cherry clump witches' broom symptoms have also been observed in Alishan, Taiwan. This study first used the Japanese-developed specific primer pair for PCR detection and sequencing, confirming that cherry trees in Alishan, Taiwan were infected by *T. wiesneri*.

To further confirm the origin of the pathogen, this study designed a longer amplicon-specific primer pair Tw-F/Tw-R targeting the rDNA ITS region of the pathogen, and performed sequence alignment after sequencing. The aim was to clarify whether the pathogens causing cherry clump witches' broom disease in Taiwan and Japan are homologous by analyzing the longer DNA fragment sequence. The sequencing results of the amplified fragment showed 100% similarity, confirming that the pathogen causing the disease in Alishan, Taiwan shares the same origin as that in Japan, indicating that the disease was most likely introduced into Taiwan from Japan.

Random sampling of cherry branches and nursery stock in the Alishan area for PCR detection revealed that the latent infection rate of clump witches' broom disease in cherry trees and nursery stock at the Alishan Workstation area was severe. This necessitates strengthened prevention and control measures, as well as accelerated import quarantine and detection of nursery stock.

The specific primer pair designed in this study has higher detection sensitivity than the Japanese-published primer pair, and the longer amplicon size provides sufficient resources for sequencing. Combining detection and sequencing allows timely detection of potential pathogen variants and implementation of necessary isolation measures.