四季海棠為矮南瓜黃化嵌紋病毒新寄主之研究

Identification of Begonia as a New Host for Zucchini Yellow Mosaic Virus

四季海棠（ Begonia semperflorens Link&Otto.）是一種常見的海棠科（Begoniaceae）多年生草本盆花植物。本研究於中興大學溫室所栽植的四季海棠植株上發現疑似病毒感染所引起的病徵，葉片出現黃色輪紋狀病斑，病斑密集處呈黃化區塊。機械接種於奎藜（Chenopodium quinoa） 7-10天後接種葉產生黃化單斑。單離的病毒回接於四季海棠10天後，新葉產生與原始病株類似之黃化輪紋的病徵，證實該單離的病毒分離株確實為造成四季海棠輪紋之病原。病組織粗汁液經電子顯微鏡鏡檢，可觀察到大小約為750×13 nm的長絲狀病毒顆粒，且在寄主植物細胞內觀察到典型potyviruses的病毒內含體。經血清學及分子檢測技術，包括酵素連結免疫吸附分析反應（enzyme-linked immunosorbent assay，ELISA）、西方轉漬法（western blot）和反轉錄聚合鏈鎖反應（reverse transcription-polymerase chain reaction，RT-PCRR）等方法檢測，證實該病毒分離株為矮南瓜黃化嵌紋病毒（ZYMV），故以ZYMV-Bego為其代稱。ZYMV-Bego的寄主範圍與本土的矮南瓜分離株ZYMV-TW-TN3 （AF127929）類似，惟在數種測試的個別寄主上引起的病徵較ZYMV-TW-TN3所引起者輕微。本研究結果顯示四季海棠為ZYMV的天然寄主之一，此發現為一新記錄。以ZYMV-SG （AJ316228）序列為參考設計引子對，ZYMV-Bego的核酸序列除3'poly（A）外已完成解序（9591 nt）。將所得的核酸序列與NCBI資料庫已登錄的ZYMV病毒株比對後可發現，ZYMV-Bego的核酸序列與其他ZYMV的核酸相同度介於92-98%之間。比較病毒的每一個單一開放讀架（open reading frame，ORF）的序列後可知，序列差異度最大者為P1基因，最小者為NIa-Pro基因。

"A begonia (Begonia semperflorens) potted plant with chlorotic ringspot symptoms on leaves was found in Taichung. When the ringspot symptoms severely developed, lots of ringspots would tend to fuse into a chlorotic region. Mechanical inoculation with this sample on Chenopodium quinoa, numerous chlorotic local lesions appeared after 7-10 days. Back inoculation of this virus isolate to healthy begonia induced chlorotic ringspot symptoms on new leaves similar to those formed on original diseased plant after 14 days. Electronic microscopy techniques were used to identify the causal agent of this symptom, and flexuous virus particles with 750 × 13 nm in length were observed. Subsequent ELISA and western blotting studies revealed that this virus isolate was serologically related to Zucchini yellow mosaic virus (ZYMV). Western blotting results also showed that coat protein of the causal virus is similar to ZYMV in their molecular weight of 33 kDa. Based on these findings, we recognized the causal virus of begonia ringspot symptom was an isolate of ZYMV and hence designated it as ZYMV-Bego. Host range test and symptom comparison between ZYMV-Bego and ZYMV-TW-TN3 showed that ZYMV-Bego is probably a mild strain of ZYMV. ZYMV primers designed based on known sequences of ZYMV-SG were used to sequence the genome of ZYMV-Bego (Acce. No. AM422386). Based on the alignment results, this virus was confirmed as a ZYMV isolate that is similar to ZYMV-TW-TN3 and ZYMV-SG which were found in Asia. In brief, this study provided evidence showing ZYMV was able to induce ringspot symptoms on begonia, representing a new host record for ZYMV."