向日葵黃化斑點病毒之分離與鑑定

Isolation and Identification of a Virus Causing Sunflower Chlorotic Spots in Taiwan

廖吉彥( J. YLiao) ； 張清安( C. A Chang) ； 陳金枝( C. C Chen) ； 鄧汀欽( T. C Deng)

《植物病理學會刊》 10卷4期 (2001/12) Pp. 173-180

https://doi.org/10.6649/PPB.200112_10(4).0004

摘要

本試驗由埔里地區向日葵栽培園發現部分植株之葉片出現過去國內文獻未曾紀錄之黃化斑點及全葉黃化等疑似病毒所造成之病徵。病株葉片粗汁液接種奎藜會形成局部斑點，經三次重複單斑篩選後獲得一個病毒分離株(SF-1)。於電子顯微鏡下以陰染法觀察罹病葉滴(leaf dip)可發現長絲壯病毒顆粒，大小約770×13nm。觀察罹病組織之超薄切片於細胞質內可發現束狀(bundled shaped)及風車狀(pinwheel)之圓柱內含體(Cylindrical inclusions)。以奎藜接種葉為材料經由高低速交替離心及硫酸銫平衡離心可獲得純化之病毒試料，利用膠體電泳分析後確定SF-1之鞘蛋白乃由單一種蛋白基本單位(Subunit protein)所組成，其分子量估計為35kDa。將純化之病毒試料注射於白兔後製備出對應SF-1之專一性抗體。此抗體於瓊脂擴散反應(SDS-immunodiffusion test)、西方轉漬反應(Western blotting test)及間接型免疫酵素分析(Indirect ELISA)下均只與SF-1同源抗原反應，而不與健康向日葵及22種Potyvirus屬病毒抗原產生反應。但SF-1抗原卻能與購自Agdia Co.之Potyvirus-specific monoclonal antibody產生正反應，顯示SF-1應屬一種未曾記載於台灣之Potyvirus屬病毒，而且試驗結果發現SF-1之各項生物特性與國外文獻所曾紀錄可以感染向日葵之幾種potyviruses均有所差異，故推測SF-1可能是一種新紀錄之向日葵病毒。因此根據SF-1感染向日葵於葉片上所造成之特殊黃化斑點病徵命名其為向日葵黃化斑點病毒(Sunflower chlorotic spot virus,簡稱SCSV)。

關鍵字

向日葵 ； 向日葵黃化斑點病毒 ； 鑑定 ； Potyvirus

並列摘要

Sunflower (Helianthus annuus) plants exhibiting chlorotic spot, yellowing and stunting symptoms were found in Pu-Li area in central part of Taiwan. Local lesions were induced on Chenopodium quinoa by inoculation with crude sap of diseased sunflower plants, and a pure virus isolate (SF-l) was obtained by single lesion isolation procedure and the culture was propagated and maintained in C. quinoa or Nicotiana benthamiana. Besides sunflower, C. quinoa and N. benthamiana, SF-I could only be transmitted mechanically to C. amaranticolor among 26 plant species tested. Flexuous filamentous particles about 770 x 13 nm in size were consistently observed in leaf dips by electron microscopy. Ultra-thin sectioned diseased tissue revealed bundled-shaped, scrolls and laminated aggregated cylindrical inclusions in the cytoplasm of infected cells. The virus was successfully purified from inoculated leaves of C. quinoa by differential and isopycnic centrifugation in cesium sulfate. By analyzing the purified virus sample in SDS-PAGE, the virus was found consisting of one species of coat protein subunit and the molecular weight was estimated to be 35 kDa. An antiserum was prepared by immunizing the purified SF-1 virions in a New Zealand white rabbit. In SDS-immunodiffusion, indirect ELISA, and Western blotting tests, antiserum against SF-l reacted strongly with its homologous antigens from infected tissues of C. quinoa and sunflower, but not to healthy control and 22 different potyviruses antigens. Reciprocally, antisera against five different potyviruses did not react with SF-I antigen in SDS-immunodiffusion test. However, a potyvirus group-specific monoclonal antibody (Agdia Co.) did react with SF-l in indirect ELISA. On the basis of these results, SF-I should reasonably recognized as a species of Potyvirus. This is the first record of virus disease of sunflower in Taiwan. Comparing with the characteristics of several potyviruses documented in the literature, SF-l is probably an unique virus species infecting sunflowers for none of the reported viruses shares similar properties. Consequently, we designated SF-l as Sunflower chlorotic spot virus based on its specific symptoms on sunflower plants.

並列關鍵字

sunflower ； Helianthus annuus ； Potyvirus ； inclusion body