感染大王仙丹之番茄嵌紋病毒分離株之鑑定與特性分析

蔡佳眞(J. C. Tsai) ； 陳滄海(T. H. Chen) ； 張清安(C. A. Chang)

《植物病理學會刊》 17卷2期 (2008/06) Pp. 143-155

https://doi.org/10.6649/PPB.200806_17(2).0005

摘要

王仙丹(Ixora duffii cv. 'Super King)，爲台灣普遍栽培之觀賞植物，其繁殖主要以無性扞插方式爲之。1994年吾等於屏東內埔地區發現部份大王仙丹葉片出現疑似病毒所引起之嵌紋病徵。經調查此類病徵可於田間全年發現，但以夏季多雨季節中所新生之枝條與葉片上最爲明顯。以電子顯微鏡檢查其葉片汁液發現有硬桿狀病毒顆粒存在，但利用傳統磷酸緩衝液研磨病組織並無法成功接種至其他植物。本研究修改Chagas等人所報告之緩衝液配方，成功將上述嵌紋病徵葉片以機械接種法接種於紅藜(Chenopodium amaranticolor)及奎藜(C. quinoa Willd.)引發局部病斑反應，並將單一病斑重複接種分離獲得一分離株(BNL)。電子顯微鏡觀察證實BNL分離株具硬桿狀病毒顆粒，與田間大王仙丹病葉中所觀察者類似。測定BNL分離株之熱不活化溫度爲65-70%，耐稀釋度爲10^(-9)-10^(-10)，冷凍(-70℃)及室溫(24℃)下保存逾14個月仍能維持感染力。將BNL機械接種於19科69種植物，有7科34種會受到感染，茄科植物中多數煙草與番茄均可引發系統陸感染造成嵌紋病徵。本研究以奎藜接種的病葉爲材料，經高低速交替離心及硫酸絕密度梯度離心獲得純化之病毒顆粒，其鞘蛋白基本單位(coat protein subunit)之分子量經電泳分析估算約爲17.5 kDa。以純化之BNL病毒顆粒所製備之專一性抗體於免疫擴散反應(SDS-immunodiffusion, test)、西方轉漬(western blotting)及間接酵素連結免疫分析法（indirect或ELISA）下與同源BNL抗原及Tomato mosaic virus (ToMV)抗原可產生強烈反應，但對於Odontoglossum ringspot、virus (ORSV)與Cucumber green mottle virus (CGMMV)等tobamoviruses抗原則反應微弱。利用ToMV之專一性引子對(P10113/P10114)可由感染BNL之奎藜及純化病毒之全量RNA經反轉錄聚合酶連鎖反應(RT-PCR)增幅出一段符合預期之480 bP核酸產物。此產物經解序後證實其核苷酸序列與已登錄之ToMV鞘蛋白核苷酸序列相同度達到97.7-99.0%，胺基酸序列相同度亦高達97.5-98.8%，顯示本研究自大王仙丹所分離之BNL應爲ToMV之一種分離株。利用RT-PCR及西方轉漬法可由田間呈現嵌紋病徵之大王仙丹病葉與幼根組織中穩定偵測到ToMV-BNL之訊號，顯示ToMV確實可感染大王仙丹並於其組織內呈現系統性感染。此乃證實大王仙丹爲ToMV之自然界寄主(natural host)之首度報導。

關鍵字

大王仙丹 ； 病毒鑑定 ； 番茄嵌紋病毒

並列摘要

Ixora (Lxora duffii cv. 'Super King'), originated from west pacific Caroline Islands. is now widely cultivated as ornamental shrubs in Taiwan. A virus-like disease exhibiting mosaic symptom on the leaves of Ixora plants was first observed in 1994 at Nei Pu. Pingtung County, but its etiology remained unclear until 2004 that we successfully transferred the mosaic Ixora tissue to Chenopodium, quinoa by mechanical inoculation. After seven successive single lesion transfers, a virus isolate designated as BNL was obtained. Rigid rod shaped virus particles approximately 300 nm in length were observed in BNL-infected tissue under electron microscopy. Physical properties of BNL were characterized as thermal inactivation point, 65-70℃; dilution end point, 10^(-9)-10^(-10); and longevity in vitro at both 24 and -70℃. more than 14 months. Host range tests of BNL isolate revealed that it could infect at least 34 species among 7 plant families by mechanical inoculation. Most tobacco species and tomato were infected inducing systemic mosaic symptoms by this isolate. Virus particles of BNL isolate was successfully purified from infected C. quinoa leaves and used for antiserum production. Analysis of the purified virion its SDS-polyacrylamide gel electrophoresis showed that it contained a protein subunit with a relative molecular mass of 17.5 kDa. Antiserum produced against BNL reacted strongly with its homologous antigen and to Tomato mosaic virus (ToMV), but weakly with two other tobamoviruses, i.e. Odontoglosum ringspot virus and Cucumber green mottle mosaic virus in indirect ELISA and SDS-immunodiffusion test. By the use of a ToMV-specific primer pair reported by Jan et al, an expected 483 bp of DNA fragment was consistently amplified from BNL-inoculated C. quinoa and purified virions its reverse transcription polymerase chain reaction (RTPCR). Sequence analyses confirmed that the amplicon shared 97.7-99.0% and 97.5-98.8% identity to those of published ToMV CP gene in nucleotide and amino acid sequences, respectively, indicating BNL isolate is a strain of ToMV. In the experiments of RT-PCR, Southern and Western blotting tests, BNL was found residing in the leaf and root tissues of lxora but with relatively low concentrations, confirming that lxora is a natural host for ToMV. To our knowledge, this represents the first record for the natural infection of ToMV in lxora spp.

並列關鍵字

Ixora ； Virus identification ； Tomato mosaic virus