夜香花嵌紋病毒百香果分離株分子特性及廣效性分子檢測技術之開發應用

Molecular Characterization of the Telosma mosaic virus Passionfruit Isolates and the Development and Application of Its Wide-Spectrum Molecular Detection Techniques

李文立(Wen-Li Lee) ； Chinnapan Thanarut ； 徐智政(Chih-Cheng Hsu) ； 李國基(Guo-Chi Lee) ； 江芬蘭(Fen-Lang Chiang) ； 陳金枝(Chin-Chih Chen)

《台灣農業研究》 69卷1期 (2020/03) Pp. 46-64

https://doi.org/10.6156/JTAR.202003_69(1).0004

摘要

本研究以細菌生合成系統誘導表現之夜香花嵌紋病毒（Telosma mosaic virus; TeMV）鞘蛋白（coat protein; CP）作為抗原，製備出具有廣效檢測TeMV、East Asian passiflora virus（EAPV）、Cowpea aphid borne mosaic virus （CABMV）、Bean yellow mosaic virus（BYMV）及Soybean mosaic virus（SMV）等馬鈴薯Y屬病毒之多元抗體。針對台灣百香果TeMV-DC6分離株對應其全長度基因體核酸序列，共解得10,022個核苷酸，於GenBank登錄序號為MN316594。2017-2019年期間，利用所研製之TeMV多元抗體與引子對，調查台灣田間百香果之TeMV發生率大約32.1-35.8%；而由泰國採集之17個葉片嵌紋與果實木質化的樣品，TeMV檢出率為100%。台灣及泰國百香果田間所收集之TeMV分離株，與泰國已發表之百香果TeMV（AM409188）之間的CP胺基酸序列，相同度均高達90%以上；但不同地理位置所分離到的百香果TeMV分離株在類緣演化上具有差異性，TeMV-DC6等9個台灣分離株自成一類群，與17個泰國分離株有所區隔。依據台灣和泰國TeMV分離株設計3組引子對，以RT-PCR法檢測兩地之田間百香果TeMV罹病組織，在受測67個台灣分離株和17個泰國分離株中，台灣引子對（TwTe650u/TwTe650d）可檢出所有受測的台灣分離株及9個泰國分離株；而泰國引子對（ThaTe430u/ThaTe430d）僅檢出12個泰國分離株，且無法檢出全部的台灣分離株；由台灣泰國廣效性引子對（TwTha840u/TwTha840d）則可檢出全部受測的台灣和泰國分離株。本研究結果顯示，目前台灣和泰國百香果之TeMV發生概況，並首度完成台灣分離株TeMV-DC6之全長度基因體核酸序列之定序，釐清台灣與泰國TeMV不同分離株之類緣差異性，並開發可廣效性檢測台灣和泰國百香果TeMV分離株之引子對，可提升對不同來源TeMV之檢測效率。

關鍵字

百香果 ； 夜香花嵌紋病毒 ； 多元抗體 ； 全長度核酸序列 ； RT-PCR廣效性檢測

並列摘要

In this study, a polyclonal antibody against Telosma mosaic virus (Ab-TeMV) was prepared from the bacteria expressed TeMV coat protein gene. Ab-TeMV had a broad-spectrum detection efficiency for the potyviruses of TeMV, East Asian passiflora virus (EAPV), Cowpea aphid borne mosaic virus (CABMV), Bean yellow mosaic virus (BYMV) and Soybean mosaic virus (SMV). The full-length genomic nucleotide sequences of TeMV-DC6, an isolate infecting passiflora in Taiwan, was determined (GenBank accession number MN316594). The incidence of TeMV on passiflora in fields of Taiwan during 2017-2019 was 32.1 to 35.8% by serological and molecular detection reagents of TeMV prepared in this study. Moreover, only TeMV was positively detected from those 17 virus-infected passiflora samples (showed symptoms of leaf mosaic and woodiness fruit) collected from Thailand. The CP amino acid sequences of those TeMV isolates collected from Taiwan and Thailand's passifloa fields shared higer than 90% identities with that of the known TeMV (AM409188) in GenBank. However, the phylogenetic analysis indicated that TeMV isolates of passiflora from different geographical locations are different in phylogenetic evolution, because 9 Taiwan isolates are closely related and separated from those 17 Thailand isolates. Three sets of primer pairs were designed based on the aligned nucleotide sequences of Taiwan and Thailand TeMVs, and used in reverse transcription-polymerase chain reaction (RT-PCR). Among the 67 Taiwan isolates and 17 Thailand isolates, the Taiwan primer pair (TwTe650u/TwTe650d) can amplify the expected amplicons from all of the tested Taiwan isolates and 9 out of the 17 Thailand isolates. The Thailand primer pair (ThaTe430u/ThaTe430d) only detected 12 out of the 17 Thailand isolates and could not detect all of the Taiwan isolates. The Taiwan-Thailand primer pair (TwTha840u/TwTha840d) could detect all of the tested isolates. In this report, the incidences of TeMV in Taiwan and Thailand were clarified and the full-length genomic sequence of Taiwan isolate DC6 was first completed. The phylogenetic differences between Taiwan and Thailand TeMV isolates were demonstrated, and the primer pairs were developed for the wide-spectrum detection of those passiflora TeMV isolates in Taiwan and Thailand to improve the detection efficacy of TeMV.

並列關鍵字

Passiflora sp. ； Telosma mosaic virus (TeMV) ； Polyclonal antibody ； Full-length sequence ； Broad-spectrum detection by RT-PCR