Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/89322
標題: 台灣石竹新病毒病害之病原鑑定與特性分析
Identification and Characterization of the Causal Agent of a New Viral Disease on Hybrid Pink
作者: Meng-Chu Lo
羅明珠
關鍵字: 
NO
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摘要: 2012 年 12 月,在台灣中部發現雜交種石竹 '巴陵紫雲' (Dianthus hybrid'NCHU-1') 作物上疑似由病毒感染所引起之病害,病株葉片呈現褪綠及斑駁。利用已發表之石竹屬作物番茄叢矮病毒屬 (Tombusvirus)、康乃馨斑點病毒屬(Carmovirus)、壞疽病毒屬 (Necrovirus)、馬鈴薯 Y 病毒屬 (Potyvirus)、黃化病毒屬 (Closterovirus) 及康乃馨潛隱病毒屬 (Carlavirus) 之簡併式引子對 (degenerateprimer) 和胡瓜嵌紋病毒屬 (Cucumovirus) 之胡瓜嵌紋病毒 (Cucumber mosaicvirus, CMV) 之專一性引子對 (specific primers) 進行反轉錄聚合酶連鎖反應(Reverse transcription-polymerase chain reaction, RT-PCR) 結果顯示可用涵括馬鈴薯。Y 病毒屬之部分 NIb (nuclear inclusion protein b) 至部分鞘蛋白 (coat protein, CP)基因之簡併式引子對 (HRP 5 及 Pot 1) 增幅出一個大小約 700 bp 之 cDNA 片段。經選殖及定序後,所獲得的 cDNA 片段 (666 bp) 之基因序列於基因庫 (NCBIGenBank) 比對後,可知其與馬鈴薯 Y 病毒屬之康乃馨葉脈斑駁病毒 (Carnationvein mottle virus, CVMoV) 同源性最高,具有 96 - 97%之核苷酸序列相同度(nucleotide identity)。將罹病之雜交種石竹以奎藜 (Chenopodium quinoa) 進行三次單斑分離後,得到之病毒分離株暫名為康乃馨葉脈斑駁病毒台灣分離株(Carnation vein mottle virus Taiwan isolate, CVMoV-TW)。本研究利用全長鞘蛋白基因之選殖定序、病毒顆粒形態、病毒之生物學特性及血清學特性進行雜交種石竹病害與其致病原之診斷與鑑定,並製備病毒之專一性多元抗血清 (polyclonalantiserum),供日後偵檢之用。利用陰染法 (Negative staining) 及超薄切片法(Ultrathin sectioning) 於 CVMoV-TW 感染之奎藜病組織觀察到直徑約 12 - 15 nm、長約 750 nm 之長絲狀(filamentous) 病毒顆粒及風車狀 (Pinwheel) 之內含體(inclusion bodies)。純化之病毒顆粒懸浮液以蛋白質電泳 (sodium dodecylsulphate-polyacrylamide gel electrophoresis, SDS-PAGE) 分析後 可觀察到大小約 34,kDa 之鞘蛋白,將純化之鞘蛋白進行紐西蘭兔之免疫注射製備多元抗血清。將CVMoV-TW 感染之奎藜病葉及純化之病毒顆粒利用西方轉漬法 (western blotting)分析,使用 CVMoV-TW 之鞘蛋白多元抗血清可在大小約 34 kDa 位置有正反應。利用部分 NIb 基因之簡併式引子 (HRP 5) 搭配 oligo d(T) 引子進行 RT-PCR,可增幅出包含全長鞘蛋白基因之片段,經選殖定序後得全長鞘蛋白基因為 840 -nt。將鞘蛋白基因與基因庫 (NCBI GenBank) 上兩個 CVMoV 之分離株 (Accessionnumber: AY512554 和 AB017630) 比較,分別有 97.5%和 98.6%之核苷酸序列相同度 (nucleotide identity),以及 98.2%之胺基酸相同度 (amino acid identity)。在寄主範圍測試方面,總共測試了 12 個科 33 種植物,其中病徵為局部單班寄主者主要為藜屬 (Chenopodium) 之指示植物,在奎藜上接種葉及系統葉均可形成黃色單斑病徵;綠藜(Chenopodium murale) 及紅藜 (Chenopodium amaranticolor) 則只有接種葉可形成黃色單斑病徵。系統性寄主植物主要為石竹科 (Caryophyllaceae) 石竹(Dianthus) 之五彩石竹 (Dianthus chinensis)、西洋石竹 (Dianthus borbatus) 及康乃馨 (Dianthus caryophyllus),主要為形成褪綠和嵌紋或斑駁之病徵。將其回接(back-inoculation) 到健康之雜交種石竹寄主上 可產生與田間病株相似之系統性褪,綠及斑駁病徵。綜合以上各結果,可確定在台灣雜交種石竹上新病害為馬鈴薯 Y病毒屬之康乃馨葉脈斑駁病毒的一個分離株所造成。
Hybrid pink plants (Dianthus hybrid 'NCHU-1') with symptoms of chlorosis and mottle were observed in central Taiwan in December 2012. Preliminary tests by reverse- transcription polymerase chain reaction (RT-PCR) have been performed with the degenerate primers designd for Caryophyllaceae-infecting viruses, including the genera of Tombusvirus, Carmovirus, Necrovirus, Potyvirus, Closterovirus, and Carlavirus , as well as the specific primers for Cucumber mosaic virus (CMV). A cDNA band of about 700 bp in size was amplified by using the potyvirus degenerate primers (HRP 5 and Pot 1). The amplifies cDNA was 666-nucleotide long and consisted of 3' part of nuclear inclusion B (NIb) and 5' part of coat protein (CP) genes of a potyvirus. Sequence analysis revealed that it shared 96-97% nucleotide identity with two isolates of Carnation vein mottle virus (CVMoV). A virus culture, designated as CVMoV-TW, has been isolated from the symptomatic leaves after successive single lesion isolations and was maintained on Chenopodium quinoa for further applications. Back inoculation of the virus to hybrid pink plants indicated the pathogenicity of CVMoV-TW to its original host. Among 33 species plants in 12 families had been tested, the virus caused local infections mainly on plants of Chenopodium spp. while systemic infections on Dianthus spp. The virus, with a filamentous particle of about 720-850 x 12-15 nm in size, can be observed in the leaf-dip preparations of diseased leaf tissue and in the purified preparation of the virus as well. Pinwheel-type inclusion bodies which are typical indication of potyvirus infection also can be observed in the infected cells of diseased hybrid pink and inoculated Chenopodium quinoa. SDS-PAGE had revealed that the viral coat protein of CVMoV-TW consisted of a peptide subunit with a relative molecular weight of about 34 kDa. Polyclonal antiserum againt CVMoV-TW has been prepared by immunizing rabbits with the purified coat protein of the virus in this study. The titer and its specificity of the antiserum have been tested by double diffusions, indirect ELISA, and western blotting tests indicating the antiserum had wide applicability to serological detection of CVMoV-TW. The full length coat protein gene of CVMoV-TW has been cloned and sequenced. It contains 840 nucleotides and encodes a coat protein subunit of 280 amino acids. The CP gene shared 97.5-98.6% nucleotide sequence identity and 98.2% amino acid identity to that of two CVMoV isolates (Accession number: AY512554 and AB017630). The work of sequencing the full length genome of CVMoV is under processing and is about to be completed. Hence, the CVMoV-TW isolated from hybrid pink in December 2012 has been confirmed as the causal agent of the chlorosis and mottle disease of hybrid pink. This is the first report of the occureence of the disease in Taiwan.
URI: http://hdl.handle.net/11455/89322
文章公開時間: 10000-01-01
Appears in Collections:植物病理學系

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