菸草微綠嵌纹病毒引起之辣椒新病害及其感染性選殖株之建構

Abstract

辣椒(Capsicum annuum. L)是一種常見的蔬菜烹飪原料，為茄科番椒屬，一年生或多年生的草本植物，原產於南美洲的秘魯，十五世紀時由哥倫布帶入歐洲，約明代時傳入中國，目前幾乎世界各地均有分佈。辣椒作物生長喜歡溫暖乾燥的環境，目前國內主要的栽種地區為屏東縣、彰化縣、南投縣、雲林縣及嘉義縣等地，是高經濟蔬菜；田間栽種的辣椒普遍有病毒複合感染的情形，造成重大威脅，依資料記載可感染番椒屬的病毒多達25種。本實驗室於2003年自田間發現辣椒疑似病毒感染的植株，經單斑分離、電顯觀察發現為桿狀病毒感染造成危害，由指示植物接種測試發現與之前曾報導的Pepper mild mottle virus (PMMoV)、 Tobacco mosaic virus (TMV)、 Tomato mosaic virus (ToMV)皆不同，應該為另一種桿狀病毒（tobamovirus）。利用poly (A) polymerase 在病毒基因體RNA的3’端加上poly A tail，並以RACE的策略進行選殖，成功得到選殖株，經解序、序列比對，發現選殖的分離株所帶的片段位於Tobacco mild green mosaic virus (TMGMV)的中間部份。TMGMV感染力強會對大部份的茄科作物造成危害，對歐美等地區的菸草危害甚鉅，但在台灣卻是首次在辣椒葉上被發現，此分離株命名為TMGMV-HP。為進一步研究，從已知序列設計專一性引子對分別增幅3’與5’的片段並選殖，再以GCG程式連接各片段而得到完整的TMGMV-HP序列。與已發表的18種tobamovirus的全長核酸序列進行比對，發現與其它感染茄科植物的桿狀病毒具有較高的相同度，又與TMGMV Japanese分離株有98%以上的相同度，另一方面為了解此病毒在台灣的感染情形，利用ORSV 抗血清進行ELISA並配合RT-PCR對田間植物進行檢測，發現由國外引進種子在台灣種植的辣椒感染機會較高，極有可能為種皮傳毒，除了深入調查外也建議加強檢疫工作的準備。同時，TMGMV在感染寄主時多與其它的桿狀病毒進行複合式感染，推測彼此間的交互關係是一種有意義的現象。為了深入研究，目前已完成TMGMV全長度cDNA選殖株的構築，先以植物接種試驗進行篩選，再接種菸草原生質體測試其感染力，總共獲得五個具感染力的cDNA選殖株。目前的工作著重於建構一個可供多方面應用的病毒載體，使後續的研究可以更完備，應用層次更多元化 。

Chili pepper (Capsicum annuum. L) belonging to the genus Capsicum in the family Solanaceae is a herbaceous plant that can be grown annually or a perennially (at tropic areas). Chili pepper was originated from Peru, South America and carried to the Europe by Christopher Columbus in the fifteenth century. Today, pepper is globally present and has becomes an important agricultural produce in Taiwan. Pingtung, Changhua, Nantou, Yunlin, and Chiayi counties are the main pepper growing districts in Taiwan. More than 25 viruses have been reported to infect pepper and have made virus disease one of limiting factors of pepper production. Virus-like yellowing and mild necrosis were observed on the chili pepper plants in the NTU experimental field. By the successive single-lesion isolation on Chenopodium amaranticolor, a viral isolate of a virus was obtained. Transmission electronic microscopy revealed numerous rigid rod-like particles about 300 nm long, that strongly suggesting that isolated virus was belong to the tobamovirus. According to the literature, tobamoviruses isolated from peppers in Taiwan included Pepper mild mottle virus (PMMoV), Tobacco mosaic virus (TMV), and Tomato mosaic virus (ToMV). To identify this unknown tobamovirus, first, molecular cloning this viral genome was conducted a poly (A) tail was added to the 3′ end of the purified viral RNA and the RACE strategy was perform to obtain independent clones and then sequenced. The result of blast search against the NCBI database indicated that the clones contained a partial sequence of Tobacco mild green mosaic virus (TMGMV). To our knowledge, this is the first report of TMGMV identified in Taiwan and this isolate was named as TMGMV-HP. To further cloning and characterization, the entire genome of the TMGMV-HP revealed that it was highly homologous to tobamovirus infecting solanaceous plants and showed 98% identity with the TMGMV-Japanese isolate. Also, a full-length cDNA clone of TMGMV was constructed and followed by successive infectivity tests, five infectious cDNA clones were obtained. An efficient TMGMV-based viral vector is still under construction. To determine the prevalence of TMGMV in Taiwan, samples collected from different sources were detected by ELISA and RT-PCR. The result suggested that mixed infection among TMGMV and other tobamoviruses occurred occasionally.