Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/31503
標題: 引起台灣茄科作物兩種新病毒病害之病原鑑定
Identification of the causal agents of two new viral diseases on Solanaceae plants in Taiwan
作者: 黃志鴻
Huang, Chih-Hung
關鍵字: Tomato spotted wilt virus (TSWV)
番茄斑萎病毒
Capsicum chlorosis virus (CaCV)
Antiserum
Identification
番椒黃化病毒
血清
病毒鑑定
出版社: 植物病理學系所
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摘要: 2009年7月在南投清境之甜椒 (Capsicum annuum cv. Andalus) 園發生大面積疑似由病毒感染而造成植株矮化、葉片呈黃化、黃化斑點、輪斑和壞疽病徵,利用可能感染茄科作物之不同病毒屬 (degenerate primer) 簡併式引子進行反轉錄聚合酶連鎖反應 (RT-PCR),結果在使用廣效性番茄斑萎病毒屬 (Tospovirus) 部分複製酶基因 (L gene) 之簡併式引子對 (gL3637 and gL4435c) 可增幅出約800 bp的DNA片段,經選殖及定序,定序結果所獲得的819 bp之基因序列與基因庫 (GenBank) 比對後,發現與番茄斑萎病毒 (Tomato spotted wilt virus, TSWV) 具有94.5-97.6%之核酸序列相同度 (nucleotide identity)。將甜椒病葉進行單斑分離後得到一甜椒病毒分離株TwPep1。將感染TwPep1的病葉利用西方轉漬法 (western blotting) 分析,使用TSWV之核鞘蛋白 (nucleocapsid protein, NP) 抗血清可在29 kDa位置有正反應;將感染TwPep1之圓葉菸草 (Nicotiana benthamiana) 粗汁液藉由陰染可在電子顯微鏡下可觀察到大小約為80-100 nm之球型病毒顆粒。於鄰近的甜椒園區中的病株分離獲得第二個病毒分離株TwPep2,使用番茄斑萎病毒之專一性引子對進行TwPep1及TwPep2之核鞘蛋白基因進行增幅後選殖及定序,將兩個病毒分離株之777 nt (nucleotide) 核鞘蛋白基因進行比對結果顯示兩者具有99.7%之核酸序列相同度及99.6%胺基酸序列相同度 (amino acid identity),將這兩株病毒分離株序列與基因庫中已發表的21個TSWV核鞘蛋白基因序列進行比對,與這些分離株間具98.3-99.1%之核酸序列相同度及98.5-99.6%的胺基酸序列相同度。將TwPep1進行回接試驗,並以磷酸緩衝液接種作為負對照,甜椒在接種8~10天後可產生與田間罹病株相似的黃化及黃化斑點之病徵。將回接成功之甜椒罹病組織進行超薄切片,也可發現約80-100 nm相似的球形病毒顆粒。藉由比對TwPep1與不同番茄斑萎病毒分離株間有98.5-99.6%的胺基酸序列相同度及回接試驗結果,證實引起台灣甜椒上新病害為番茄斑萎病毒的一個分離株所造成。 2009年12月在台中霧峰之番茄 (Solanum lycopersicum cv. Known-you 301) 園發生疑似由病毒感染而造葉片黃化及壞疽病徵,利用可能感染茄科作物之不同病毒屬簡併式引子進行反轉錄聚合酶連鎖反應,結果在使用廣效性番茄斑萎病毒屬部分複製酶基因之簡併式引子對 (gL3637 and gL4435c) 可增幅出約650及800 bp的DNA片段,選殖約800 bp之片段進行定序,定序結果所獲得的816 bp之基因序列與基因庫 (GenBank) 比對後,發現與番椒黃化病毒 (Capsicum chlorosis virrus, CaCV) 具有87%之核酸序列相同度。將番茄病葉進行單斑分離後得到一番茄病毒分離株TwTom1。將感染TwTom1的病葉利用西方轉漬法分析,使用CaCV之核鞘蛋白抗血清可在30 kDa位置有正反應;將感染TwTom1之奎藜粗汁液藉由陰染可在電子顯微鏡下可觀察到大小約為80-100 nm之球形病毒顆粒。使用番椒黃化病毒 (CaCV)之專一性引子對進行TwTom1核鞘蛋白基因的增幅後選殖及定序,將病毒分離株之828 nt核鞘蛋白基與基因庫中已發表的26個CaCV核鞘蛋白基因序列進行比對,具85-98.1%之核酸序列相同度及92-100%的胺基酸序列相同度。將TwTom1進行回接試驗,並以磷酸緩衝液接種作為負對照,番茄在接種約20天後產生與田間病株相似的壞疽病徵。藉由比對TwTom1與不同CaCV分離株間具有92-100%的胺基酸序列相同度及回接試驗結果,證實引起台灣番茄上新病害為番椒黃化病毒的一個分離株所造成。
A virus TwPep1 was isolated from leaves of a sweet pepper plant (Capsicum annuum cv. Andalus) showing viral disease-like symptoms of stunting, chlorosis, chlorotic spots, necrotic spots and ring spots in the fields in central Taiwan in July, 2009. A partial RNA-dependent RNA polymerase gene (L gene) of Tospovirus was amplified by a reverse transcription- polymerase chain reaction (RT-PCR) with degenerate primers designed from major viruses of Solanaceae plants, and was then cloned and sequenced. This 819-nucleotide partial L gene sequences was compared with sequences of tospoviruses available in GenBank and the result revealed that TwPep1 isolate shared 94.4-97.7% of nucleotide identity with Tomato spotted wilt virus (TSWV). A 29 KDa protein of TwPep1 isolate reacted positively with antiserum against the N protein of TSWV using western blot. A second isolate, TwPep2 was isolated from leaves of a symptomatic sweet pepper plant in the field nearby. The nucleocapsid (NP) gene of the TwPep1 and TwPep2 were amplified by RT-PCR using primers for the N gene of TSWV. After the amplified 777-nucleotide NP gene fragments were cloned and sequenced, the sequences showed 99.7% of nucleotide identity and 99.6% of amino acid identity between TwPep1 and TwPep2. The NP gene sequences of these two Taiwanese isolates shared 98.3-99.1% of nucleotide identity and 98.5-99.6% of amino acid identity with those of 21 TSWV isolates available in GenBank. The TwPep1 isolate was back-inoculated onto three sweet pepper plants for pathogenicity test while three other plants were inoculated with phosphate buffer as controls. The inoculated sweet pepper plants showed symptoms of chlorosis and chlorotic spots which were similar to those observed in the field 8~10 days post-inoculation. Isometric particles about 80-100 nm in diameter in crude saps of TwPep1-infected Nicotiana benthamiana leaves was detected by electron microscopic examination. Similar particles were also observed in the ultrathin sections of symptomatic tissues prepared from back-inoculated leaves of sweet pepper plants. Based on the results of high nucleotide and amino acid identities shared between TwPep1 isolate and other TSWV isolates and their pathogenicity test, it is concluded that the causal agent of the new sweet pepper disease in Taiwan was indeed an isolate of TSWV. A virus TwTom1 was isolated from leaves of a tomato plant (Solanum lycopersicum cv. Known-you 301) showing viral disease-like symptoms of chlorosis and necrosis in the fields in central Taiwan in December, 2009. A partial RNA-dependent RNA polymerase gene (L gene) of Tospovirus was amplified by RT-PCR with degenerate primers designed from major viruses of Solanaceae plants, and it was then cloned and sequenced. This 816-nucleotide partial L gene sequences was compared with those of tospoviruses available in GenBank. The result revealed that TwTom1 isolate shared 87% of nucleotide identity with Capsicum chlorosis virus (CaCV). A 30 KDa protein of TwTom1 isolate reacted positively with antiserum against the N protein of CaCV using western blot. The nucleocapsid (NP) gene of the TwTom1 was amplified by RT-PCR using primers for the N gene of CaCV. After the amplified 828-nucleotide NP gene fragment of TwTom1 were cloned and sequenced, the sequences showed 85-98.1% of nucleotide identity and 92-100% of amino acid identity with those of 26 CaCV isolates available in GenBank. The TwTom1 isolate was back-inoculated onto three tomato plants for pathogenicity test while three other plants were inoculated with phosphate buffer as controls. The inoculated tomato plants showed symptoms of chlorosis which was similar to those observed in the field 20 days post-inoculation. Isometric particles about 80-100 nm in diameter in crude saps of TwTom1-infected Chenopodium quinoa leaves was detected by electron microscopic examination. Based on the results of high nucleotide and amino acid identities shared between TwTom1 isolate and other CaCV isolates and the pathogenicity test, it is concluded that the causal agent of the new tomato disease in Taiwan is indeed an isolate of CaCV.
URI: http://hdl.handle.net/11455/31503
其他識別: U0005-3007201016172400
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-3007201016172400
Appears in Collections:植物病理學系

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