蟹爪蘭X病毒與紅龍果X病毒之分子特性與偵測

Abstract

紅龍果(pitaya)屬於仙人掌科(Cactaceae)三角柱屬(Hylocereus spp.)的攀緣性肉質植物，為近年來新興栽種之熱帶果樹之一。根據研究報告顯示 Potexvirus屬之仙人掌X病毒(Cactus virus X, CVX)為國內已知唯一感染紅龍果的病毒，且普遍存在於栽種的紅龍果植株中。2006年本實驗室對陽明山觀光果園的紅龍果進行田間調查，利用CVX抗血清和專一性引子對，同時以indirect-ELISA和RT-PCR檢測所採集樣品，結果發現每一個樣品都對CVX抗血清呈現正反應。在RT-PCR結果中，編號39號紅龍果樣品無法增幅出CVX的專一性RT-PCR產物，卻產生約150 bp的非專一性片段。將此150 bp的片段純化、定序及比對後，發現與Potexvirus屬之蟹爪蘭X病毒(Zygocactus virus X, ZVX)有最高之序列相同度。將39號紅龍果樣品組織液接種至奎藜(Chenopodium quinoa)，7至8天後接種葉出現褪綠病斑。因此以奎藜進行三次單斑分離，獲得一病毒分離株稱為P39。另外編號37號紅龍果樣品RT-PCR檢測結果中，除了CVX之預期片段外，亦產生一條300 bp的非專一性片段。經純化、解序與比對後發現此序列並非CVX病毒序列，但與多種potexviruses有高相同度。將37號紅龍果樣品組織液接種至紅藜(C. amaranticolor)，約5至8天後接種葉出現帶黃暈的局限性病斑。因此利用紅藜進行三次單斑分離，獲得一非CVX的病毒分離株稱為P37。我們分別對P37和P39病毒株進行寄主範圍之測試及病毒全長度基因體的解序，並利用已解完之序列設計專一性引子，以RACE增幅5’端的基因體片段，並選殖和定序，以確定5’端的序列。P37和P39分離株全長度分別為6677個及6624個核苷酸，基因體結構與potexvirus相同，應為Potexvirus屬的成員。將P37和P39分離株各個基因與其它已發表全長序列的Potexvirus屬病毒進行序列比對及類緣分析，發現P39與ZVX的RdRp與CP基因之胺基酸序列相同度分別達97%和96%，故可確認P39為一新發現可感染紅龍果之ZVX分離株，並命名為ZVX-P39。此為台灣ZVX的首次報導，亦為ZVX感染紅龍果之新記錄。從序列分析結果可知P37病毒株與Schlumbergera virus X (SVX)的序列相同度最高，兩者的CP核苷酸與胺基酸序列相同度為68%及82%。根據第八版ICTV報告對Potexvirus屬病毒種的規定，P37病毒株應為一種未曾報導的感染紅龍果的新potexvirus，因此命名為紅龍果X病毒(Pitaya virus X, PiVX)。分子類源分析結果顯示，感染仙人掌科植物的potexvirus都屬於同一個分群，顯示彼此的類緣關係較密切。為了解台灣紅龍果受到CVX、PiVX和ZVX病毒之感染及分布情況，針對這三種病毒分別設計專一性引子對，並加入可檢測粒線體nad5 mRNA的引子對，作為RT-PCR的反應內在對照組，成功開發紅龍果病毒的multiplex RT-PCR檢測方法。我們重新檢驗本實驗室於2006年採集自北部陽明山紅龍果樣品之植物全RNA，並且對採集自台中縣和屏東縣紅龍果樣品進行檢測。結果顯示台灣紅龍果普遍受到CVX侵染，北、中、南部三地的感染比例分別為100%、85.7%和100%；而在北部及南部紅龍果之ZVX感染比例為47.5%和84%，中部則無ZVX感染情形；而PiVX目前只在北部紅龍果園檢驗出，其感染比例為12.5%，尚未在其他兩區發現。本研究發展的快速且準確度高之multiplex RT-PCR法，除了可用於田間樣品檢測外，期望未來能協助紅龍果健康種苗的生產。

Pitaya (Hylocereus spp.), a climbing succulent plant in the family Cactaceae, is cultivated as new tropical fruit. According to the literatures, Cactus virus X (CVX) is the only known and widespread virus in the field grown pitaya plants in Taiwan. In 2006, our laboratory surveyed the pitayas in the Yangmingshan orchard by indirect ELISA using the antiserum specific against CVX and one tube multiplex RT-PCR. The ELISA data showed that every sample reacted positively to the CVX antiserum. In the result of multiplex RT-PCR, No. 39 pitaya sample produced a 150-bp nonspecific fragment without the expected CVX cDNA fragments. This 150-bp fragment was sequenced and analyzed, and it showed the highest sequence identity with a potexvirus, Zygocactus virus X (ZVX). The plant sap of No. 39 pitaya sample was inoculated to Chenopodium quinoa and chlorotic spots appeared on the inoculated leaves at 7-8 days post inoculation (dpi). A virus isolate named P39 was obtained after three successive single lesion isolations in C. quinoa. In addition to the CVX specific fragments, No. 37 sample also generated a 300-bp nonspecific RT-PCR product. After sequencing the 300-bp cDNA fragment, the result indicated that it was not the sequence of CVX but had high sequence identity with many potexviruses. The plant sap of No. 37 sample was inoculated to C. amaranticolor and necrotic lesions with yellow halo appeared on the inoculated leaves of C. amaranticolor at 5-8 dpi. After three successive single lesion isolations in C. amaranticolor, a non-CVX virus isolate was obtained and named P37. To further characterize the biological and molecular features of P37 and P39 isolates, host range test and cloning and sequencing the full-length viral genomes were performed. The 5’terminal sequences of both isolates were determined by sequencing the RACE products. The full-length genomes of P37 and P39 isolates have 6677 and 6624 nucleotides, respectively. Both of them have the same genome organization of the genus Potexvirus and must be potexviruses. The complete genomic sequences of P37 and P39 isolates were compared with those of other potexviruses. The amino acid sequence identities of RdRp and CP genes between P39 and ZVX are 97% and 96%, respectively. Therefore, P39 isolate is considered as a new ZVX isolate and designated as ZVX-P39. This is the first ZVX identification report in Taiwan and also the first ZVX infecting pitaya report. According to sequence analyses, P37 isolate and Schlumbergera virus X (SVX) had the highest sequence identity. Their nucleotide and amino acid sequence identities of CP gene are 68% and 82%, respectively. Based on the species demarcating criteria of the genus Potexvirus in the 8th ICTV report, P37 isolate is a new pitaya-infecting potexvirus and thus named Pitaya virus X (PiVX). The results of phylogenetic analyses of ZVX-P39 and PiVX-P37 with other completely sequenced potexviruses demonstrated that all potexviruses infecting plants in the family Cactaceae belong to the same cluster and have a close relationship. In order to understand the infection and distribution of CVX, PiVX and ZVX in pitaya plants in Taiwan, we developed a multiplex RT-PCR method for field survey. We designed specific primers for each virus species, and also the specific primer of mitochondria nad5 mRNA to amplify the cDNA fragment as internal control of multiplex RT-PCR. Pitaya samples collected from Taipei city, Taichung and Pingtung counties were detected by multiplex RT-PCR. The result showed that CVX appeared in 100%, 85.7% and 100% of tested pitayas from north, middle and south parts, respectively. ZVX was detected in 47.5% and 84% of samples in Taipei and Pingtung. Only the pitaya samples from Yangmingshan orchard in Taipei were tested positive for PiVX, and the infection was 12.5%. This rapid and accurate detection method of multiplex RT-PCR can be used for field survey as well as the certification program of pitayas.