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題名 | 蕙蘭嵌紋病毒(CymMV)與齒舌蘭輪斑病毒(ORSV)快速檢定技術=Rapid Indexing Methods for CymMV (Cymbidium mosaic virus) and ORSV (Odontoglossum ringspot virus) in Orchids |
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作者 | 楊佐琦; 莊佳茹; 蕭芳蘭; 孫永偉; 陳駿季; 蕭吉雄; Yang, Tso-chi; Chuang, Chia-ju; Shiau, Fang-lan; Sun, Yung-wei; Chen, Junne-jih; Hsiao, Chi-hsiung; |
期刊 | 植物種苗 |
出版日期 | 20041200 |
卷期 | 6:4 2004.12[民93.12] |
頁次 | 頁7-30 |
分類號 | 435.431 |
語文 | chi |
關鍵詞 | 蕙蘭嵌紋病毒; 齒舌蘭輪斑病毒; 生物檢定法; 光學顯微鏡病毒內含體診斷法; 酵素聯結抗體免疫測定法; 反轉錄-聚合酶連鎖反應技術; Cymbidium mosaic virus; CymMV; Odontoglossum ringspot virus; ORSV; Bioassay; Virus inclusion; ELISA; RT-PCR; |
中文摘要 | 以間接法酵素聯結抗體免疫測定法(I-ELISA)調查我國蝴蝶蘭病毒之發生以蕙蘭嵌紋病毒(Cymbidium mosaic virus, CymMV)之單獨感染最高,約為27.9%,齒舌蘭輪斑病毒(Odontoglossum ringspot virus, ORSV)單獨感染最低,約為5.7%,而CymMV與ORSV複合感染約19.1%,無此二種病毒者佔47.2%。文心蘭病毒之發生亦以CymMV單獨感染最高約為14.2%,ORSV單獨感染最低,約為8.1%,而CymMV與ORSV複合感染僅為3.4%,無此二種病毒者佔74.3%,顯示我國健康母株普遍存在。生物檢定法則以汁液摩擦接種至紅藜(Chenopodium amaranticolor)、奎藜(C. quinoa)、千日紅(Gomphrena globosa)與望江南(Cassia occidentalis)等指示植物,視其局部病斑發展判定。從二種病毒複合感染之材料中單離病毒,經試驗結果發現在紅藜、奎藜黃色局部病斑、千日紅之褐色輪狀病斑中,ORSV單獨感染者最高分別佔69.5%、95.7%、93.8%,ORSV與CymMV複合感染者次之分別佔30.5%、4.3%、6.2%,且皆無CymMV單獨感染。至於望江南之針狀小褐點病斑中,全部只有CymMV單獨感染之存在。光學顯微鏡病毒內含體診斷法,ORSV在細胞質中形成之披針狀結晶型內含體,於微波爐(720瓦特)中加熱20秒鐘後被橙綠蛋白質染劑染成綠色,但以紫紅核酸染劑染色必須於微波爐中加熱20秒鐘後再行加熱20秒鐘,內含體方可染成紫紅色。CymMV則在細胞壁附近形成似帶狀之內含體,以微波爐中加熱20秒鐘後被橙綠蛋白質染劑染成綠色,亦被紫紅核酸染劑染成紫紅色。發展適宜CymMV 與ORSV 之酵素聯結抗體免疫測定法(ELISA)血清檢定法,取樣點包含花、葉及根等多部位組織。應用反轉錄-聚合?連鎖反應技術(RT-PCR),以一組引子對配合一步驟RT-PCR之應用,可同時偵測出條帶約為534 bp之CymMV及290 bp之ORSV條帶,除可節省一倍量之試劑支出及縮短一倍之檢驗時間,全部流程可縮短於4小時內完成。 |
英文摘要 | Surveys conducted in 2001-2003 by indirect enzyme-linked immunosorbent assay (I-ELISA) showed that Cymbidium mosaic virus (CymMV) is the predominant virus infecting commercial Phalaenopsis and Oncidium in Taiwan. Of the 1103 Phalaenopsis samples tested, 27.9% were infected with CymMV, 5.7% with Odontoglossum ringspot virus (ORSV), and 19.1% were with both viruses, respectively. Of the 148 Oncidium samples tested, 14.2% were infected with CymMV, 8.1% with ORSV and 3.4% with both viruses, respectively. These results also indicate that healthy Phalaenopsis and Oncidium plants are existed in Taiwan through virus certification program. To make sure virus infectivity, bioassay plants of Chenopodium amarnticolor, C. quinoa, Gomphrena globosa, and Cassia occidentalis were inoculated onto respectively. ORSV were mostly found in chlorotic local lesions or brown ringspot on C. amaranticolor (69.5%), C. quinoa (95.7%) and G. globosa (93.8%). Moreover, 25.9, 3.5, and 6.2% of local lesions were infected by both CymMV and ORSV. However, no CymMV singly infected samples was found on these plants. In addition, C. occidentalis was found to be a good indicator plant for CymMV since all of tiny necrotic lesions were singly infected with CymMV. ORSV induces spindle-shaped paracrystalline inclusions in the epidermal cells stained to olive green by orange-green combination after heating in microwave (720 watts) for 20 seconds but unable to be stained by azure A under the same condition. Replaced by heat treatment for 20 seconds twice, the heating method can improve the staining efficacy of ORSV by azure A. Moreover, CymMV induces laminate inclusions adjacent to cell walls when stained by O-G combination or azure A respectively upon heating for 20 seconds in a microwave. The I-ELISA in this study offers a feasible, fast and reliable indexing method for orchid viruses but sampling requires more various parts of a plant including corolla, leaves, and roots in order to avoid misleading results. One pair of primers and one-step reverse-transcription polymerase chain reaction (RT-PCR) was applied to detect bands approximately at 534 or 290 bp for CymMV and ORSV respectively. Meanwhile, we have set up a time-saving and labor-saving protocol to detect CymMV and ORSV in both Phalaenopsis spp. and Oncidium spp. |
本系統之摘要資訊系依該期刊論文摘要之資訊為主。