洋桔梗贅脈捲葉病毒之瓜類分離株特性分析

雙生病毒科其中的豆類金黃嵌紋病毒屬在世界各地皆造成作物嚴重的損失。台中改良場於2016年，在其南瓜試驗田間發現一新型病毒病害，根據其豆類金黃嵌紋病毒屬所通用之引子對增幅的基因片段，其比對結果與洋桔梗贅脈捲葉病毒(lisianthus enation leaf curl virus)之基因片段有百分之九十八的相似度。為了確認該病害是否為洋桔梗贅脈捲葉病毒的新寄主紀錄，本研究設計背對背引子對，使用聚合酶連鎖反應增幅病毒全幅序列，並進一步建構感染性選殖株(infectious clone)，本研究所取得之病毒全幅序列與現今NCBI資料庫所發表的洋桔梗贅脈捲葉病毒BG-1和BG-9 (genebank accession: LC091539.1和LC091538.1)分別有90.8% 和 91.7%的序列相似度。建構出的1.75倍長度之病毒序列轉入pJL89二元載體並利用農桿菌接種至圓葉菸草與番茄植株，接種後21 天（於菸草）及30天（於蕃茄）後，能觀察到捲葉之病徵，且能透過聚合酶連鎖反應增幅到病毒序列。而農桿菌接種至原先寄主南瓜 (Cucurbita pepo 栽培種：阿成)於六十天後測得微弱的病毒訊號並出現微弱的病毒病徵。

關鍵字

柯霍氏法則；豆類金黃嵌紋病毒屬；親緣性分析；感染性選殖株；協力作用

並列摘要

Viruses in the genus Begomovirus, within the family Geminiviridae, cause serious diseases on crops around the world. In 2016, a viral disease was found in pumpkins in experimental farm of Taichung District Agricultural Research and Extension Station. Partial genome was amplified by universal primers designed to amplify begomovirus, and the genome sequence revealed that it shares 98% sequence identity with lisianthus enation leaf curl virus (LELCV). To characterize the LELCV found in pumpkins, we designed back to back primers to amplify the whole genome of LELCV by PCR. The full-length genome of LELCV were sequenced, and it shares 90.8% and 91.7% sequence identity with two previously published LELCV isolates (accession number LC091539.1 and LC091538.1), respectively. The 1.75-folds genome of LELCV was cloned to pJL89 binary vector to generate pJL89LELCV infectious clone. Nicotinamide benthemiana and tomato (Solanum lycopersicum cv. Bi碧玉) infiltrated with agrobacterium carrying pJL89LELCV showed upward leaf curling symptoms after 21 and 30 days post inoculation (dpi), respectively. The virus can be detected by PCR in both inoculated tobacco and tomato. Infiltration of agrobacterium carrying pJL89LELCV in pumpkin (Cucurbita pepo cv. 阿成) show mild symptoms at 60 dpi, and LELCV can be detected by PCR.