港口馬兜鈴(Aristolochia zolligeriana Miq.,簡稱馬兜)為鳳蝶幼蟲之取食植物,晚近於特有生物保育中心試驗區中發現所栽植之部份馬兜鈴植株葉片出現系統性嵌紋病徵,經接種試驗、血清反應法及核酸分析鑑定結果,證實為胡瓜嵌紋病毒(cucumber mosaic cucumovirus, CMV)感染所引起。本研究利用CMV之局部病斑寄主,奎藜(Chenopodium quinoa Willd.),進行單斑分離及接種,獲得一分離株(Azl)做為本研究後續試驗表病毒分離株。將Azl接種於健康之馬兜鈴實生幼苗,約30天後其葉片會出現與田間罹病株相同之嵌紋徵。接種於Nicotiana benthaminana Domin 及N.tabavum cv.Xanthi 等煙草品種,亦可引起系統性嵌紋病徵。將接種Azl之N.benhtaminana葉片材料進行除色退綠處理濃縮後,以電顯觀察可以發現大小約為30nm之球型病毒顆粒,其外型及大小均與CMV顆粒之特性類似。於ELISA反應中,田間表現嵌紋病徵之馬兜鈴及Az1分離株之接種材料均與對照CMV抗血清產生強烈正反應。於SDS-免疫擴散反應中上述材料均與CMV抗血清產生專一性沈澱反應,且與對照標準CMV抗原產生無法分辨差異之同源性反應(identical reaction )。利用對應CMV之專一性引子對,分別以田間馬兜鈐病株葉片、感染Azl之煙草與馬兜鈴之總量核酸為模版,進行反轉錄-聚合脢鏈鎖反應(RT-PCR),均可獲得一大小約486 bp PCR核酸片段,其大小與以CMV核酸為模板所增幅之預估值相符。而這些RT-PCR增幅產物均可與非放射性標定之對照CMV核酸探針產生雜配反應(hybridization)。上述材料若以tomato aspermy cucumovirus (TAV)之專一性引子對進行RT-PCR時均未能獲得任何增幅產物。將Azl分離株之486 bp PCR 核酸產物選殖pCR II載體並進行核甘酸序列分析,結果亦顯示其與已知CMV之相關核甘酸序列相符,經比對後發現二者之相同度高垟92~93%。而序列範內對應之鞘蛋白3'端60個氨基酸序列,則與對應之CMV已知列達98%之相同度。綜合上述鑑定結果,本研證明CMV為引起港口馬兜鈴生嵌紋病之致病病原,港口馬兜鈴則為CMV之記錄寄主。
The Aristolochia zolligeriana Miq. (AZO), a native species of Aristolochiaceae in Taiwan, is the major victual plant of the larva of Papilio spp. butterfly in nature. During artificial cultivation of AZO, some plants exhibiting systemic mosaic symptoms were found in one of the experiment plantation in Taiwan Endemic Species Research Institute. A virus isolate (Az1) was obtained from the diseased AZO plants by three successive local-lesion selections on Chenopodium quinoa and subsequently propagated in Nicotiana benthamiana. The same mosaic symptom as that on the original diseased plants was reproduced on healthy AZO seedlings by inoculation with Azl, indicating that Azl should be the causal agent of AZO mosaic disease. Besides AZO, Az1 could infect N. benthamiana and N. tobacum cv. Xanthi and induce systemic mosaic symptoms. By electron microscopy, isometric particles with approximately 30nm in diameter was observed in negatively stained sample obtained from clarified viral concentrate (CVC) procedure described y Christie et al. (1987). In ELISA test, field collected tissue from diseased AZO, Az1-noculated tissues of N.