由茉莉花（Jaminum sambac）分離之一種新potyvirus之特性研究

Characterization of A New Portyvirus Isolated from Jasmin (Jasminum sambac (L.) Ait) in Taiwan

林宜螢(Y. Y. Lin) ； 陳滄海(T. H. Chen) ； 張清安(C. A. Chang)

《植物病理學會刊》 13卷1期 (2004/03) Pp. 69-84

https://doi.org/10.6649/PPB.200403_13(1).0008

摘要

1995年筆者等在高屏地區首度發現田間茉莉花葉片出現嚴重黃化嵌紋之病徵，病徵初期呈現不規則黃斑，最後漸擴大至整個葉片黃化，經調查主要栽培區的田間病徵約可區分為無病徵感染、不規則黃斑及嚴重黃化嵌紋等三種。以電顯陰染觀察茉莉花病葉粗汁液，發現其內至少含有三種長度不一絲狀粗子的病毒病原，乃一複合感染之病害。經由機械接種可以由紅藜及奎藜成功分離出一種粗子長720-750 nm微彎曲之長絲狀病毒，此為台灣地區的首次的茉莉花病毒病害之報導。病毒熱不活化溫度為50-55°C；耐稀釋度為10^(-3)-10^(-4)；室溫（24°C）下活性可維持2-3天，冷凍（-70°C）保存可維持10個月；電顯觀察紅藜單斑病組織超薄切片可於細胞質中見到potyviruses所誘導形成之典型風車狀（pinwheels）及薄板狀（laminated aggregates）內含體；而在黃化嵌紋茉莉花病組織之切片中，亦可觀察到細胞核、葉綠體、粒線體等細胞器的病變。機械接種12科56種供試植物，僅藜科3種植物段到感染蚜媒傳播並未成功。奎藜病葉為材料經高低速交替離心及硫酸銫密度梯度離心可獲得純化病毒，其鞘蛋白分子量經電泳分析估算約為32 DA；將純化病毒注射於白兔後製備出對應本病毒之專一性抗體。此抗體於西方轉漬反應（Western blotting）、間接式酵素連結免疫吸附法（Indirect ELISA）、瓊脂雙向擴散反應（SDS-agar gel double diffusion test）下均只與其同源抗原反應，並不與其它14種potyviruses之抗原反應。進一步以反轉錄聚合酶連鎖反應（RT-PCR）選殖本病毒之核酸，將所解得之核苷酸（nucleotide, nt）序列，與已登錄於GenBank之20種potyviruses基因比對，親緣最接近之病毒與本病毒CP及3’-NCR區域中之核苷酸序列相同度分別低於69及54%；CP之胺基酸序列相同度則低於69%。綜點上述病毒形態大小、基本物理特性、細胞病理學、血清學反應及核酸序列比對等種種證據，一致顯示本研究中所分離之病毒為一種新種potyvirus，亦全世界首次成功地由感染茉莉植株中分離出病毒，因此特將此由台灣地區茉莉花黃化嵌紋病所分離之新種potyvirus命名為茉莉病毒T〔Jasmin virus T, JaVT〕。

關鍵字

茉莉花 ； poryvirus ； 病毒鑑定 ； 茉莉病毒T

並列摘要

A previously undescribed virus disease that caused severe yellow mosaic on leaves of jasmine plants wad found in 1995 at Kaohsiung and Pingtung. Taiwan. The first discernible symptom observed is irregular yellow spot. Later, the spot advanced and making the whole leaf yellowing. Three major symptom types, i.e. symptomless virus infection, irregular yellow spot and severe yellow mosaic were evidenced in the field. The virus disease is a complex disease, more than three filamentous viruses were observed in crude saps of field jasmine. A flexuous filamentous virus of 720-750 nm in particle size, was successfully isolated by mechanical inoculation from diseased jasmine showing yellow mosaic symptom. It is the first report of jasmine virus disease in Taiwan. The thermal inactivation point of the virus was 50-55°C, dilution end point was 10^(-3) to 10^(-4) and the longevity in vitro was 2-3 days at 24°C and more than 10 months at -70°C. Pinwheel-shaped and laminated inclusion bodies typical of potyvirus infection were found in local lesion of Chenopodium amaranticolor. Cellular organelles alterations of nuclei, chloroplast, mitochondria in virus-infected leaf of jasmine showing yellow mosaic symptom were also observed by electron microscopy. It had a very narrow host range restricted to three species in chenopodiaceous plants among 56 tested plant species belonging to 12 families. Attempts to transmit the virus by aphids using C. quinoa and C. amaranticolor as source plants failed. Protein with a molecular weight of 32 kDa was detected where purified virus was denatured with sodium dodcyl sulfate (SDS) and analyzed by electrophoresis in polyacrylamide gels (SDS-PAGE) and by western blotting. An antiserum with a titre of 512 was obtained by immunizing a white rabbit with the purified virions. In Western blotting, indirect ELISA, SDS-agar gel double diffusion test, the antiserum against the jasmine virus isolate reacted strongly with its homologous antigens, but not to antigens of fourteen other potyviruses. In order to further characterize the virus at the molecular level, CP genes of jasmine virus isolate was cloned, sequenced and analyzed. A sequence of 1289 nucleotides (nts) was found and the percent of nucleotide identities of the CP gene and the NCR were less than 69% and 54%, comparing to twenty potyviruses, respectively. Similarly, percent identities of CP amino acid sequence to those of twenty other known potyviruses were all below 69%. The virus was identified as a new potyvirus species on the basis of its particle morphology and size, physical properties, cytopatholoy, serological characteristics and percent of nucleotide and amino acid sequences identity, and a name, Jasmin virus T, JaVT, was previsionally proposed. It is the first report about successful virus isolation from jasmine in the world.

並列關鍵字

Jasmin ； Potyvirus ； Virus identification ； Jasmin virus T