罹病康乃馨之Carnation mottle virus的分離與鑑定

Isolation and Characterization of Carnation mottle virus from Carnation

陳慶忠 ； 林靜宜 ； 柯文華 ； 詹富智

《植物病理學會刊》 12卷3期 (2003/09) Pp. 199-208

https://doi.org/10.6649/PPB.200309_12(3).0008

摘要

於彰化縣北斗鎮、田尾鄉及永靖鄉田間栽種之康乃馨(Dianthus caryophyllus L.)及康乃馨雜交品系”小可愛(Kooij Echo kgr, Holland)”發現類似感染病毒而引起植株上方分蘗心葉附近葉片產生黃化 壞疽斑點或黃化現駁等徵狀。上述病組織初步以陰染法於電子顯微鏡觀察到一種直徑約32 - 33 nm之球形病毒顆粒。以雙向免疫擴散反應及酵素聯結免疫分析等血清學方法分析顯示上述康乃馨所分離之病毒分離株與康乃馨斑駁病毒(Carnation mottle virus, CarMV)血清呈現正反應。超薄切片罹病組織以電子顯微鏡鏡檢於葉肉細胞之細胞質內可觀察到病毒顆粒呈分散或結晶狀排列,大小與陰染法之球形病毒顆粒相似。以電泳分析(SDS-polyacrylamide)此康乃馨病毒分離株得到一分子量約40kDa之鞘蛋白。進一步針對CarMV鞘蛋白基因保守區域設計專一性引子對,利用反轉錄聚合?連鎖反應(RTPCR)增幅CarMV康乃馨分離株鞘蛋白基因並進行選殖及定序,將所得之1047-nt(nucleotides)鞘蛋白基因核酸序列與基因庫(GenBank)中已發表之各CarMV分離株鞘蛋白基因序列進行比對分析,經比對後發現與CarMV各分離株間具95.6-97.9%之核酸序列相同度(nucleotide identity)及98.0 - 99.1%的胺基酸序列相同度(amino acid identity)。綜合以上試驗結果分離自台灣康乃馨引起黃化斑駁病徵之病毒鑑定為CarMV的一分離株,同時CarMV感染康乃馨在台灣亦為首次記錄。

關鍵字

康乃馨 ； 康乃馨斑駁病毒 ； CarMV ； 鑑定

並列摘要

Virus cultures were isolated from leaves of carnation (Dianthus caryophyllus L.) and mini-carnation [Dianthus caryophyllus hybrid Kooij (Echo kgr)] showing viral disease-like symptoms of yellow necrotic spots and yellow mottle beginning on upper leaves in the fields in central Taiwan. Isometric particles about 32-33 nm in diameter were found in crude saps of diseased tissues. Similar particles were also observed in the cytoplasm of the mesophyll cell in ultrathin sections prepared from infected D. caryophyllus and D. chinensis. SDS-polyacrylamide gel electrophoresis showed that the virus contains one structural polypeptide about 40 kDa. Serological tests with double immunodiffusion test and double antibody sandwich-enzyme-linked immunosorbent assay showed that the virus reacted positively with an antiserum prepared against Carnation mottle virus (CarMV). The coat protein (CP) gene of the virus was amplified by reverse transcription-polymerase chain reaction (RT-PCR), cloned, and sequenced using primers designed from a conserved sequence of CarMV. Comparisons of the 1047 -nucleotide CP gene with those of 23 CarMV isolates available in GenBank showed that the virus shared 95.6-97.9% nucleotide identity and 98.0-99.1% amino acid identity. Our results demonstrate that the virus infecting carnation is an isolate of CarMV. This is the first report of CarMV infecting carnation in Taiwan.

並列關鍵字

carnation ； Carnation mottle virus ； CarMV ； identification