引起洋桔梗黃化斑駁、嵌紋與矮化病徵之Tobamovirus鑑定

Abstract

A virus LV-3 was isolated from leaves of lisianthus (Eustoma rusellianum) showing viral disease-like symptoms of stunting, yellowing mottle and yellow spots in the fields of central Taiwan. Rigid rod particles similar to tobamoviruses about 300 18 nm in diameter were found in the crude saps of infected lisianthus leaves by electron microscope. Similar particles were also observed from the viruses purified from Nicotiana tabacum leaves infected with LV-3. SDS-polyacrylamide gel electrophoresis analysis of purified LV-3 virions showed that this virus contains one structural polypeptides, with around 18 kDa relative molecular weight, reacting positively to Tomato mosaic virus (ToMV) antiserum in Western assay. Serological tests with SDS-double diffusion tests and indirect ELISA showed that the lisianthus virus LV-3 is also positively reactive to antisera prepared to ToMV and Tobacco mosaic virus (TMV). The coat protein (CP) gene of LV-3 was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and then cloned and sequenced with primers designed from the conserve sequence of ToMV and TMV. Comparison of the 480 nucleotides CP gene region with those of ToMV available in GenBank revealed 84.6-99.4% nucleotide identity and 91.2-99.4% amino acid identity. Similar results (68.8-99.2% nucleotide identity and 78.1-99.4% amino acid identity) were obtained when GenBank TMV data were compared with the CP gene. Taken together, our results demonstrate that LV-3 isolated from lisianthus is a tobamovirus. Furthermore, this is the first report of lisianthus as a host of Tobamovirus in Taiwan. 自台灣中、南部所栽培之洋桔梗(Eustoma russellianum Don. Griseb)採集呈現矮化、黃化斑駁、葉片黃綠間雜嵌紋及黃化斑點疑似病毒感染引起之異常植株，初步以醋酸鈾陰染罹病株葉片粗汁液並經由電子顯微鏡鏡檢觀察到一面桿狀，人小約300 18 nm之類似病毒顆粒，經單斑分離得到一洋桔梗病毒分離株(LV-3)。由感染LV-3之菸草葉片(Nicotiana tabacum)亦可純化出類似之病毒顆粒。將純化之LV-3病毒顆粒以SDS-PAGE電泳分析後，顯示該病毒具有一鞘蛋白(coat protein)，分子量約為18 kDa。將分析後所得之蛋白分子另以西方轉漬法(Western blotting)分析，得知該蛋白與番茄嵌紋病毒(Tomato mosaic virus, ToMV)之抗血清有正反應。進一步以雙向免疫擴散反應和間接酵素連結免疫法藉以分析洋桔梗病毒分離株LV-3與其他tobamoviruses的抗血清反應關係，結果顯示洋桔梗病毒LV-3與菸草嵌紋病毒(Tobacco mosaic virus, TMV)及ToMV的抗血清均有反應。進一步針對ToMV及TMV鞘蛋白基因保守區域設計專一性引子對，利用反轉錄聚合醉連鎖反應(RT-PCR)增幅LV-3鞘蛋白基因並進行選殖及定序，將所得之480-nt (nucleotides)鞘蛋白基因核酸序列與基因庫(GenBank)中已發表之各ToMV及TMV分離株鞘蛋白基因序列進行比對分析，經比對後與ToMV各分離株間具84.6-99.4%之核酸序列相同度(nucleotide identity)及91.2-99.4%的胺基酸序列相同度(amino acid identity)，而與TMV各分離株間為61.8-99.2%之核酸序列相同度及69.8-99.4%的胺基酸序列相同度。綜合以上試驗結果初步鑑定引起台灣洋桔梗黃化嵌紋與矮化病徵的LV-3為一tobamovirtls，此外本研究亦為台灣首次發現Tobamovirus屬病毒可感染洋桔梗之報告。