感染紅龍果之仙人掌病毒X之鑑定與分佈調查

Detection and Incidence of Cactus Virus X on Pitaya in Taiwan

廖吉彥( J. Y Liao) ； 張清安( C. A Chang) ； 顏昌瑞( C. R Yan) ； 陳昱初( Y. C Chen) ； 鄧汀欽( T. C Deng)

《植物病理學會刊》 12卷4期 (2003/12) Pp. 225-234

https://doi.org/10.6649/PPB.200312_12(4).0002

摘要

紅龍果(Hylocereus undatus Britt. ＆ Rose)為近年自國外引進而逐漸風行全台之醬肉果樹，目前只有報告仙人掌病毒X (Cactus virus X, CVX) 可以感染紅龍果。本研究調查台灣各地及金門地區紅龍果園，發現有疑似病毒感染病徵之植株分佈頗為普遍，依病徵型態可歸納為退綠斑點型、斑駁型、壞疽型及黃化型等四型。其中以斑駁型發生較廣。本研究由宜蘭所獲得之斑駁型紅龍果罹病株上經單斑分離獲得一病毒分離株(EL1)，以機械接種方式EL1可感染雞冠花造成系統性黃化斑點，於千日紅、綠藜、奎藜及紅藜則產生局部壞疽病斑。電子顯徵鏡觀察EL1病毒顆粒為長絲狀，大小約490-510×13nm。將EL1繁殖於奎藜，利用硫酸銫等密度平衡離心可獲得純化之病毒顆粒，並用以作為免疫用抗原製備出專一性抗血清。以電泳分析(SDS-PAGE)純化之病毒顆粒，其鞘蛋白基本單位(subunit protein)分子量約為30kDa。所製備之抗血清於西方轉漬、SDS-免疫擴散反應及間接型酵素連接抗體法等免疫分析反應中均與其同源抗原產生明顯反應，而不與健康對照抗原反應。為釐清EL1與CVX之關係，根據已知之CVX鞘蛋白核甘酸序列設計引子對，利用反轉錄聚合酵素(RT-PCR)可由EL1組織所抽取之全量RNA中增幅出預期之1.2kbp之DNA片段。此片段經選殖與解序後證實屬於CVX 3'端基因體序列，包括部分ORF2、ORF3、ORF4及ORF5 (coat protein)。取其中部分ORF4及完整ORF5片段共864個核甘酸(對應288個氨基酸，Access No. AY241392)與CVX(Access No. AF308158)相比對，分析發現EL1鞘蛋白基因之核甘酸序列與CVX相同度達95.3%，而兩者氨基酸相似度為92.8%。經上述各項特性之比對，證實EL1係CVX之一種系統。依罹病株不同部位及不同病徵組織，以間接法酵素連接免疫分析(ELISA)偵測，得知新生側芽及初期病徵(退綠斑點及斑駁)，可測得較高ELISA反應值。經各地調查及採樣偵測，屏東縣紅龍果園CVX發生率約90%以上，金門縣約50%，其餘地區約60-70%。紅龍果品種保存園中CVX普查結果，國姓黃皮(A13)等20個品種植株仍未被CVX所感染，標示為健康母本，進而加以隔離，以供日後發展健康種苗繁殖體系之用。

關鍵字

紅龍果 ； 仙人掌病毒X ； 病徵 ； 抗血清 ； 比對 ； 調查

並列摘要

Pitaya (Hylocereus undatus) plants showing viral symptoms of chlorotic spots, mottling, necrosis and yellowing were found in orchards around Taiwan. From diseased pitaya plant which a virus isolate was obtained through series of single lesion isolation. By host range test, electron microscope observation, immunosorbent electron microscopy, coat protein electrophoresis, Western blotting, and RNA sequencing, the virus was identified as an isolate of Cactus virus X (CVX). The virus systemically infected Celosia argentea and caused chlorotic spots, but locally infected Gomphrena globosa, Chenopodium amaranticolor, C. quinoa, C. murale caused necrotic lesions. Electron microscopy of diseased tissue showed filamentous virus particles with 490-510 × 13 nm. The virus was purified from inoculated leaves of C. quinoa by isopycnic centrifugation in cesium sulfate and injected in rabbit for antiserum production. In SDS-PAGE, the virus was found consisting of one species of coat protein subunit and its molecular weight was estimated to be 30 kDa. In SDS-immunodiffusion, indirect ELISA and Western blot, the antiserum was found to specifically with its homologous antigen. Total of 864 nucleotides from the 3’-end region of this virus were sequenced and compared with the Cactus virus X from in GenBank (Access No. AF308158) showing the nucleotide identity index of the whole CP to be 95.3%, and the amino acid similarity index to be 92.8%. Using indirect ELISA to detect the CVX on different tissues of pitaya revealed that virus titers are higher in young shoots or parts with initial symptoms (chlorotic spots and/or mottling) than any other tissues tested. The incidence of CVX occurrence on pitaya orchard was surveyed around the Taiwan area and was found to be above 90% in Pintung, 50% in Kinman and from 60-70% in other areas. To find the CVX-free materials, a census was conducted to examine all the plants preserved in pitaya variety collection orchard located in National Pingtung University of Science and Technology. From this orchard a total of 77 samples were indexed out of which only 20 pitaya plants of 20 varieties were considered to be healthy. All of the healthy plants were separately multiplied for setting up the healthy pitaya seedling propagation system.

並列關鍵字

Pitaya ； Cactus virus X ； symptom ； indirect ELISA ； incidence