由薄葉牛皮消（＂Cynanchum Taiwaniamum＂ Yamazaki）所分離之胡瓜嵌紋病毒特性研究

薄葉牛皮消（Cynanchum taiwanianum Yamazaki）為蘿藦科台灣原生種植物，其地下塊莖可做為中藥材。2005年於台灣高雄縣內門鄉田間發現牛皮消植株葉片呈現疑似病毒性病害，受感染的植株葉片呈現嵌紋病徵，經電顯觀察結果發現，有球型病毒存在。內門鄉栽培區田間為88％，而網室栽培病徵變化調查，發現病徵表現與溫度有相關性，1－4月溫度較低病徵明顯，5－8月溫度較高病徵較不明顯。具嵌紋病徵之葉片汁液，經紅藜機械接種可成功分離出直徑約28nm之球型病毒粒子，命名為SW-1分離株；其熱不活化溫度為65℃，耐稀釋度為10-5，24℃及-80℃下，活性分別可維持96小時及12個月以上。每50克接種SW-1分離株的萬國士煙草病葉，經蔗糖梯度離心純化後，約可純化66.66mg之病毒。寄主範圍測試12科74種植物，其中葫蘆科、藜科、茄科等7科32種植物會被感染並表現病徵。以凝膠電泳分析估算病毒鞘蛋白分子量為26kDa。將純化病毒免疫注射於紐西蘭兔後，可製備出對應本病毒之力價 2048之專一性抗體；此抗體於西方轉漬反應（Western blotting）、瓊脂免疫雙向擴散反應（SDS-agar gel double diffusion test）、間接式酵素連結免疫吸附法（indirect ELISA）中，除可與同源抗原反應外，亦會與胡瓜嵌紋病毒（Cucumber mosaic virus）發生專一性反應。以反轉錄聚合酶連鎖反應（RT-PCR）選殖本病毒之鞘蛋白核酸，鞘蛋白基因定序共得全長889個核苷酸 (nucleotide, nt），其所對應轉譯含218個胺基酸；將鞘蛋白核苷酸與胺基酸序列，與已登錄於NCBI GenBank之胡瓜嵌纹病毒分離株比對，發現其與由番茄分離之胡瓜嵌紋病毒核苷酸序列有高達99％的相同度（D28780、AJ829779），胺基酸序列則與CMV（Y10886、AF523340）有100％之相似度。綜合以上結果，顯示本研究由薄葉牛皮消嵌紋病株分離之球型病毒應為胡瓜嵌纹病毒，此為薄葉牛皮消病毒病害之首次報導。

關鍵字

牛皮消病毒病；胡瓜嵌紋病毒；病毒鑑定

並列摘要

Taiwan swallowwort (Cynanchum taiwanianum Yamazaki) is a native herbal plant in Taiwan. It belongs to Asclepiadaceae and has economically cultivated at Neimen Township, Kaohsiung County, Taiwan, due to its tuber is used as herbal medicine. Taiwan swallowwort virus disease was first found at Neimen area in 2005. Isometric particles were observed in diseased leaves showing mosaic symptom by electron microscopy. A disease incidence of 88% was obtained in field survey at Neimen area. However, a fluctuation in disease symptoms relative to temperature could be observed at net house cultivation. An apparently disease symptom appeared during the low temperature period from January to April. While, mild disease symptoms were observed during high temperature period from May to August. An isometric viral particle of 28 nm in diameter was successfully isolated by mechanical inoculation from a diseased Taiwan swallowwort plant showing mosaic symptom to Chenopodium amaranticolor and named as SW-1. The thermal inactivation point of SW-1 and the dilution end point was 10-5. The longevity in vitro of SW-1 was 96 hours at 24℃and more then 12 months at -80℃. A purified viral preparation of 66.6 mg could be obtained from 50g SW-1 infected leaf tissue of Nicotiana tabacum cv. vam Hicks. The host range tests indicated that SW-1 could infect 32 plant species in 7 families such as Cucurbitaceae, Chenopodiaceae, Solanaceae and etc. among 74 plant species in 12 families inoculated mechanically. The molecular weight of coat protein of the virus was estimated at 26 KDa by electrophoresis in sodium dodecyl sulfate (SDS) polyacrylamide gel. An antiserum with a titer of 2048 was obtained by immunizing a New Zealand white rabbit with the purified virions. In Western blotting, indirect enzymelinked immunosorbent assay and SDS-agar gel double diffusion test, the antiserum against reacted strongly with its homologous antigens and Cucumber mosaic virus (CMV) control antigen. Furthermore, the gene of viral coat protein was cloned and sequenced. A sequence of 889 nucleotides encoding an open reading frame of 218 amino acid residues was obtained. An identity of 99% and 100%with CMV infecting tomato in nucleotide (D28780, AJ829779) and amino acid (Y10886, AF523340)levels respectively were evidenced. Based on these result a conclusion could be drawn that the virus (SW-1) from diseased Taiwan swallowwort is a isolated of Cucumber mosaic virus. To our knowledge, this is the first report of virus disease occurring in Taiwan swallowwort.

並列關鍵字

Taiwan swallowwort viral disease ； Cucumber mosaic virus ； virus identification

國際替代計量

由薄葉牛皮消（＂Cynanchum Taiwaniamum＂ Yamazaki）所分離之胡瓜嵌紋病毒特性研究

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