台湾麒麟花病毒病害及由其分离之大戟轮斑病毒特性研究.DOC

台灣麒麟花病毒病害及由其分離之大戟輪斑病毒特性研究Crown-of-Thorns mosaic disease and Euphorbia ringspot virus isolated from Crown-of-Thorns (Euphorbia milii Desm.) in Taiwan 研究生：蘇文勝 Sue Wen-Sheng 指導教授：陳滄海 Chen THai 【摘要】 麒麟花(Euphorbia milii)為一種無性繁殖之盆栽、花壇或綠籬觀賞植物。西元1995年首度在台灣高屏地區發現田間麒麟花葉片出現嚴重嵌紋之病徵。田間周年性調查，田間以嵌紋病徵為主。全年可見其嵌紋病徵會因季節變化而有輕微嚴重程度不同。以電顯觀察麒麟花病葉粗汁液，可發現有絲狀粒子的病毒粒子存在。經由機械接種至奎藜可功分離出一種粒子長800-840 nm微彎曲之長絲狀病毒，為台灣地區首次麒麟花病毒病害之報導。病毒熱不活化溫度為55-60℃；耐稀釋度為10-5~10-6；室溫(24℃)下活性可維持14-15天，冷凍(-74℃)保存可維持15個月；電顯觀察奎藜單斑病組織超薄切片可於細胞質中見到potyviruses所誘導形成之風車狀(pinwheel)及薄板狀(laminated aggregates)內含體；而在嵌紋麒麟花病組織切片中，亦可觀察到細胞核、葉綠體等細胞胞器之病變。機械接種13科66種植物，僅藜科的奎藜、紅藜及綠藜等三種藜科植物受感染，葉部呈現局部斑點病徵。以藜病葉為材料經高低速交替離心及硫酸銫等密度梯度離心可獲得純化病毒收量為1.15 mg，其鞘蛋白分子經電泳分析估算約為26 kDa；以西方轉漬反應（Western blotting）亦能反應出相同正反應。進一步以核苷酸（nucleotide, nt）序列，與已登錄於GenBank之前11個親緣關係較近之potyviruses進行基因比對，發現其核苷酸序列與麒麟花（accession number AY697300）上之大戟輪斑病毒有98.2％之相同度為最高，胺基酸序列則有97.2％之相度，3端非轉譯區(3-NR)則有85.5％之相同度。綜合以上結果顯示本研究自麒麟花所分離之病毒應為國外發表之感染麒麟花的大戟輪斑病毒Euphorbia ringspot virus，此為國內外首次發現並成功自麒麟花分離大戟輪斑病毒的報告。關鍵字：麒麟花、、、病毒鑑定Crown-of-Thorns (Euphorbia milii Desm) was planted at Chang-Hua, KaohSiung, Pingtung county, Taiwan as potting, bedding and ornamental plant. A previously undescribed virus disease that caused severe mosaic on leaves of Crown-of-Thorns plants was found in 1995 at Pingtung, Taiwan. The first discernible symptom observed is irregular light mosaic. Later, the mosaic advanced and making the whole leaf mosaic. A flexuous filamentous virus of 800-840 nm in particle size, was successfully isolated by mechanical inoculation from disease Crown-of-Thorns showing leaf mosaic symptom. It is the first report of Crown-of-Thorns virus disease in Taiwan. The thermal inactivation point of the virus was 55-60℃, dilution end point was 10-5 to 10-6 and the longevity in vitro was 14-15 days at 24℃ and more than 15 months at -74℃. Pinwheel and Laminated aggregates inclusion bodies typical of potyvirus infection were found in local lesion of Chenopodium quinoa and mosaic leaf of Crown