利用光學顯微鏡、電子顯微鏡及血清學技術診斷洋桔梗壞疽病毒

Diagnosis of Lisianthus Necrosis Virus Infection by Light and Electron Microscopy and Serological Assays

陳慶忠( C. C Chen) ； 曹淑麗( S. L Tsao) ； 徐惠迪( H. T Hsu)

《植物病理學會刊》 10卷3期 (2001/09) Pp. 105-114

https://doi.org/10.6649/PPB.200109_10(3).0002

摘要

洋桔梗壞疽病毒（Lisianthus necrotic virus,LNV）為晚近在中部地區洋桔梗上新發現及鑑定之球形病毒。本試驗利用光學顯微鏡、電子顯微鏡及三種血清學技術，探討以其偵測及診斷LNV之可行性。利用橙綠蛋白質染劑（Calcomine orange-Luxol brilliant green BL）染色罹病洋桔梗葉片表皮，以光學顯微鏡可觀察到細胞質內含有不規則束狀類似內含體之構造。醋酸鈾（uranyl acetate）溶液陰染罹病葉片粗汁液或純化懸浮液於電子顯微鏡下，可觀察到直徑約32 nm之球形病毒；超薄切片罹病葉片及花瓣於細胞質內可觀察到類似之病毒顆粒或病毒聚集之情形。以LNV兔子抗血清及老鼠單元抗體進行西方轉漬反應（Western blot ）可偵測到單一鞘蛋白基本單位（coat protein subunit），分子量約為38 kDa。相同之抗血清進行間接酵素連結免疫分析（indirect ELISA）或三層酵素連結免疫分析（TAS ELISA），LNV罹病植物組織粗汁液全抗原之最終稀釋倍數為１０-4或１０-5。於２６℃，LNV罹病葉粗汁液機械接種於洋桔梗幼苗，以直接組織轉漬法（direct tissue blot,DTB）於接種後１２小時即能偵測到新複製之LNV抗原，而indirect ELISA則於接種後４８小時始能偵測之，顯然以DTB偵測新複製之LNV抗原的時間較indirect ELISA為早。

關鍵字

洋桔梗壞疽病毒；診斷方法

並列摘要

Lisianthus necrotic virus (LNV) is a spherical virus recently found and identified in certain imported lisianthus seedlings in central Taiwan. In this study, diagnostic procedures, including light and electron microscopy (TEM) and serological techniques, were compared for detection and identification of LNV in virus-infected plants. Light microscopy showed the presence of cytoplasmic inclusion-like structures with boundless shapes in epidennal cells of leaves of infected lisianthus when stained with Calcomine orange-Luxol brilliant green BL. TEM of leaf dip preparations of infected plants or purified samples of LNV stained with 2% uranyl acetate revealed the presence of spherical particles measuring about 32 nm in diameter. Silmilar particles or virions aggregation were observed in cytoplasm of leaves and petals of infected plants in ultra-thin sections. In Western blot analysis using rabbit antisera or mouse monoclonal antibodies, a single capsid protein (about 38 kDa, MWr) band was detected in crude leaf extracts of infected plants and purified virus preparation. In indirect ELISA using rabbit antisera or mouse monoclonal antibodies, the dilution endpoints of LNV antigens in leaf extracts of infected plants were about 10-4 or 10-5. Similar dilution endpoints of LNV antigens were also obtained when and mouse antibody was used in TAS ELISA. As early as 12 hr after inoculation, localization of newly synthesized LNV antigen was visualized by direct tissue blot immunoassay, whereas the LNV antigen could not be detected by ELISA test until 48 hr after inoculation.

並列關鍵字

diagnosis methods ； Lisianthus necrosis virus