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標題: 可機械傳播感染東方型甜瓜新德里捲葉病毒之鑑定及特性
Identification and characterization of a mechanically transmissible Tomato leaf curl New Delhi virus infecting oriental melon
作者: 簡瑞哲
Chien, Rui-Che
關鍵字: Cucumis melo L.
Tomato leaf curl New Delhi virus
出版社: 植物病理學系所
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摘要: 2007年於東方型甜瓜銀輝栽培種 (Cucumis melo L. var. Sliver Light) 之栽培園發現疑似病毒感染的罹病株,其新葉產生嵌紋並嚴重捲曲皺縮的病徵,於田間一共採集了三個罹病樣本。從中我們得到一個病毒分離株Silver Light-1 (SL-1),並以機械接種方式在圓葉菸草與東方型甜瓜銀輝栽培種上產生與田間罹病作物相同的病徵。萃取田間罹病植株葉片總量DNA後,利用針對begomovirus DNA-A與DNA-B及satellite DNA-β所設計之簡併式引子對 (degenerate primers) 進行聚合酶鏈鎖反應 (PCR) 的檢測,於所有感病之材料中都能增幅出預期大小的病毒DNA-A與DNA-B條帶,分別為1.5及2.5 kb,將此二DNA片段選殖且定序並由所得之序列設計能夠增幅全長度DNA-A與DNA-B之專一性引子對。由SL-1病毒分離株中獲得的DNA-A全長度為2739核苷酸 (nucleotide, nt),其序列上發現有8個open reading frames (ORFs),其中三個 (AV1 to AV3) 於病毒股上,另五個 (AC1 to AC5) 則位於病毒互補股;全長度DNA-B同樣經選殖且解序後得全長序列為2673 nt,序列中有兩個ORFs,其中nuclear shuttle protein位於病毒股,而movement protein則位於病毒互補股。利用GenBank資料庫中報導可感染葫蘆科作物之begomoviruses作序列比對分析,結果顯示此SL-1 DNA-A與DNA-B全長與在泰國危害胡瓜之新德里蕃茄捲葉病毒 (Tomato leaf curl New Delhi virus, ToLCNDV) 序列相同度最高,分別為97% (DNA-A) 及93% (DNA-B) 的核苷酸序列相同度。基於序列上的比對結果,顯示造成東方型甜瓜銀輝栽培種病徵之病原為ToLCNDV的一個分離株,我們將其命名為ToLCNDV-OM (ToLCNDV-oriental melon isolate)。寄主範圍測試當中,從茄科、藜科、葫蘆科和豆科4個植物科別內的38種植物中,可利用ToLCNDV-OM之病毒粗汁液以機械接種方式產生病徵的葫蘆科作物有東方型甜瓜、胡瓜、矮南瓜、越瓜、扁蒲、絲瓜以及茄科作物中的圓葉菸草及番茄 (亞蔬 CLN 1558A)。這是首次於東方型甜瓜上報導有關於ToLCNDV的危害,也是第一次發現ToLCNDV可以利用機械接種傳播病害的報導。
Mosaic, leaf curl, and puckering symptoms were observed in Cucumis melo L. var. Silver Light plants in the field during 2007 spring season. Three samples were collected from symptomatic plants. A virus culture SL-1 was isolated and established in the systemic hosts, Nicotiana benthamiana and C. melo L. plants via mechanical inoculation. Viral DNA was extracted from diseased oriental melon plants with leaf curl from field and polymerase chain reaction (PCR) with reported primers was used to detect the presence of begomoviral DNA-A, DNA-B, and associated satellite DNA. Begomoviral DNA-A and DNA-B were detected in all three diseased samples. The PCR-amplified 1.5 kb viral DNA-A and 2.5 kb DNA-B from one positive sample were cloned and sequenced. Base on the assemblled sequence, specific primers were designed for cloning the full-length DNA-A and DNA-B of the isolate SL-1, which were determined as 2739 and 2673, repectively, nucleotides (nt) in length. Sequence analyses were conducted using DNAMAN Sequence Analysis Software. The DNA-A contained the conserved nanonucleotides-TAATATTAC and eight open reading frames, including three in virus sense (AV1 to AV3) and five in virus complementary sense (AC1 to AC5). The DNA-B contained two open reading frames, including the nuclear shuttle protein in virus sense and the moving protein in the virus complementary sense. BLASTn analysis and sequence comparison with those available in the GenBank showed that isolate SL-1 has highest nucleotide identity of 97% and 93%, respectively, with the DNA-A and DNA-B of Tomato leaf curl New Delhi virus (ToLCNDV) from Thailand. Our results indicate that the virus causing leaf curl disease on oriental melon is an isolate of ToLCNDV and designated as ToLCNDV-OM (ToLCNDV-oriental melon isolate). In addition, 38 plants species from 4 families (Solanaceae, Chenopodiaceae, Cucurbitaceae, Leguminosae) were mechanically inoculated with ToLCNDV-OM for host reaction assay. Systemic symptoms were observed on plants of C. melo L., C. sativus L., Luffa aegyptiaca, Cucurbita pepo, Lagenaria siceraria, C. melo Linn, N. benthamiana, and Lycopersicum esculentum. To our knowledge, this is the first report of ToLCNDV causing a severe disease on oriental melon plants and can be transmitted through mechanical inoculation.
其他識別: U0005-1708200719535200
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