台灣澎湖地區感染香瓜茄(Solanum muricatum)之番茄嵌紋病毒(Tomato mosaic virus)鑑定與發生調查

Abstract

香瓜茄屬於茄科 (Solanaceae) 蔬菜，從澎湖地區栽種的香瓜茄植株，單斑分離得到一純系病毒株，機械接種可感染的寄主包括番杏、紅藜、綠藜、奎藜、千日紅、菠菜、日日春、鹿角萵苣、百日草、落葵、小葉碎米薺、南瓜、蒲瓜、胡麻、草胡椒、金魚草、辣椒、甜椒、萬國士菸草、圓葉菸草、番茄、茄子、龍葵、香瓜茄、馬鈴薯等11科25(品)種植物。電子顯微鏡觀察純化的病毒為300-310 x 18-20 nm的桿狀粒子，經電泳分析病毒鞘蛋白 (coat protein, CP) 分子量約為19 kDa，並用以製備抗血清。經SDS-免疫擴散反應、酶聯抗體免疫分析 (enzyme-linked immunosorbent assay, ELISA)、及西方轉漬 (western blotting) 等血清反應，初步鑑定所分離的病毒是菸草嵌紋病毒屬 (Tobamovirus) 的一種。進一步鑑定則利用該屬病毒共通的CP基因序列引子對 (TMVmpf / TMV3nr)，進行反轉錄聚合酶鏈式反應 (reverse transcription-polymerase chain reaction, RT-PCR)，擴增出800 bp的DNA片段，解序後登錄於GenBank資料庫，其Accession No.為JQ085379。經核酸序列比對及親緣分析，與所有供試番茄嵌紋病毒(Tomato mosaic virus, ToMV) 分離株的CP基因核苷酸序列相同度都達99%以上，與同屬的菸草嵌紋病毒 (Tobacco mosaic virus, TMV) 分離株則只有75%，因此鑑定本病毒為ToMV之一分離株，屬於Clade I基因型。利用本株病毒抗體以ELISA進行ToMV在香瓜茄的發生調查，結果顯示澎湖經濟栽培的香瓜茄植株及果實中皆可檢測出ToMV；在台灣本島生產的香瓜茄扦插用枝條與播種用種子皆未測出病毒。本報告為ToMV感染國內香瓜茄植株的首篇紀錄。 Pepino (Solanum muricatum) is a kind of solanaceous vegetables. We have isolated a virus from the cultivated pepino plants in Penghu, an archipelago situates in Taiwan Strait. The virus isolate, designated as ToMV-Penghu, was obtained after three successive local-lesion isolations on Chenopodium quinoa. By mechanical inoculation, ToMV-Penghu could infect Tetragonia tetragonioides, Chenopodium amaranticolor, C. quinoa, C. murale, Gomphrena globosa, Spinacia oleracea, Catharanthus roseus, Lactuca sativa, Zinnia violacea, Basella alba, Cardamine flexuosa, Cucurbita pepo, Lagenaria siceraria, Sesamum indicum, Antirrhinum majus, Capsicum annuum, Nicotiana benthamiana, N. tabacum, Solanum lycopersicum, S. melongena, S. muricatum, S. nigrum, and S. tuberosum in the experiment. Rod-shaped particles with the size of 300-310 x 18-20 nm can be observed in purified preparation of ToMV-Penghu by electron microscopy. The relative molecular weight of viral coat protein was estimated as about 19kDa by SDS-PAGE. The protein was applied to prepare the specific rabbit polyclonal antiserum against ToMV-Penghu. By serological reactions in SDS-immunodiffusion, enzyme-linked immunosorbent analysis (ELISA) and western blot, the ToMV-Penghu was identified as a species of Tobamovirus. For further identification, a pair of primers (TMVmpf / TMV3nr) spanning the tobamovirus coat protein (CP) gene were adopted for reverse transcription polymerase chain reaction (RT-PCR) and that resulted in producing an 800-bp DNA fragment. After cloning and sequencing, this nucleotide sequence was deposited in the GenBank database as Accession No. JQ085379. Comparing with the CP gene of other tobamoviruses in the Genebank by nucleotide sequence alignment, JQ085379 shared more than 99% of identities with tested ToMV isolates but only 75% with the TMV isolates. Therefore, ToMV-Penghu is identified as an isolate of Tomato mosaic virus (ToMV) and belongs to Clade I genotype as tested by phylogenetic analysis. The incidence of ToMV in pepino plants was surveyed with specific antibody against ToMV-Penghu by ELISA. ToMV could be commonly detected in plants and fruits of economically cultivated pepino collected in Penghu but not in pepino twigs or seeds collected in Taiwan. This is the first report of a ToMV infecting pepino plants in Taiwan.