由草海桐黃化輪斑病株所分離之胡瓜嵌紋病毒特性之研究

Characterization of Cucumber mosaic virus isolated from sea lettuce (Scaevola sericea Vahl) showing chlorotic ringspot symptom in Taiwan

翁容淑(Weng Jung Shu)

指導教授 ： 陳滄海

國立屏東科技大學/農學院/植物醫學系所/碩士(2009年)

https://doi.org/10.6346/NPUST.2009.00300

摘要

草海桐(Scaevola sericea Vahl)為草海桐科(Goodeniaceae)常綠灌木，生長迅速，適應力強，抗風、耐鹽、寒、旱性佳。分佈於亞、澳洲及太平洋熱帶島嶼，亦為台灣全島常見之海濱植物，乃濱海地區重要之防風、定砂植物，亦可栽培供觀賞。2007年首先於台灣屏東沿海地區發現草海桐葉片呈現疑似病毒病害之黃化輪斑病徵，田間病害發生率為23-100%。經周年性病徵觀察，發現季節與病徵變化有相關性，一年中以5月~11月其病徵較嚴重明顯，12月~4月病徵較輕微不明顯。田間病葉經由奎藜(Chenopodium quinoa)作單斑分離，可分離出一種球形病毒編號為SR-1，病毒粒子直徑大小約為30 nm。病毒熱不活化溫度為55℃，耐稀釋度為10-4，室溫(25℃)及-80℃下，活性分別可維持29天及12個月以上。電顯下觀察奎藜單斑病組織切片，於葉肉組織細胞質內可觀察到病毒粒子聚集形成內含體結晶構造及細胞胞器病變。寄主範圍測試17科63種供試植物，其中以番杏科、莧科、蘿摩科、菊科、藜科、十字花科、豆科和茄科共8科16種植物會被感染分別出現局部性黃斑或系統性嵌紋輪斑等病徵。種子媒介傳播率達27%。蚜蟲傳播測試發現可經由桃蚜(Myzus persicae Sulzer)與棉蚜(Aphis gossypii Glover)以非持續型方式傳播。以菸草病葉100 g為材料經高低速交替離心及蔗糖密度梯度離心，可獲得純化病毒收量約22 mg。進一步以電泳分析估算病毒鞘蛋白分子量約為26 kDa。將純化病毒免疫紐西蘭白兔後製備以環形介面法測得對應本病毒抗體力價達1024；此抗體於西方轉漬反應(western blotting)、間接式酵素免疫吸附分析法(indirect Enzyme Linked Immunosorbent Assay, ELISA)和瓊膠免疫擴散反應(SDS- agar gel double diffusion test)可與抗原產生同源反應。進一步以反轉錄聚合酶連鎖反應(RT-PCR)選殖本病毒之鞘蛋白基因核酸，所解得核苷酸序列(nucleotide, nt)共657個核苷酸，與已登錄NCBI GenBank之Cucumovirus序列比對，結果顯示與感染中國四季豆的胡瓜嵌紋病毒(CMV)分離株(accession number EF 424708) 具97%之相同度，胺基酸序列則與胡瓜嵌紋病毒(Cucumber mosaic virus)之CaW、Ag、Danshen、Vi、Sr、CaM和K3a(accession number EF424778、EF424775、AY600989、EF424 784、EF424781、EF4247810 EF424781)等分離株有99%之相似度，將SR-1病毒株與CMV IA、IB和II系統之核苷酸序列以Neighbor-joining比對結果其親緣關係歸為Cucumber mosaic virus之IB亞群。綜合以上結果，顯示本研究由草海桐黃化輪斑病毒所分離之球形病毒應為胡瓜嵌紋病毒的草海桐分離株，此為台灣地區首次有關草海桐病毒病害之研究報導。

關鍵字

草海桐 ； 病毒病害 ； 胡瓜嵌紋病毒 ； 病毒鑑定

並列摘要

Sea lettuce (Scaevola sericea Vahl) is a perennial shrub of the family Goodeniaceae which is resistant to wind, cold, drought and tolerant to salt. Sea lettuce is widely grown in Asia, Australia and the Pacific tropical island, and also as a general seashore coast and landscaping plant in Taiwan. A previously undescribed virus disease that showed chlorotic ringspot on leaves of sea lettuce was found in 2007 at Pingtung, Taiwan. The field survey indicated that a disease incidence of 23-100% was found in Mioli, Kaohsiung and Pintung, Taiwan. An isometric virus of 30 nm in diameter was successfully isolated by mechanical inoculation from diseased sea lettuce showing chlorotic and ringspot symptom. The inactivation temperature of the virus was 55℃, diluted end point was 10-4, and the longevity in vitro was 29 days at 25℃ and more than 12 months at -80℃. Irregular crystalline of isometric virus particles were found in cytoplasm and the aggregation of virus
particles was also observed in chloroplasts of virus infected Chenopodium quinoa leaf tissues in ultrathin section by electron microscopy. Host range test indicated that the virus can infect 16 plant species belonging to Aizoaceae, Amaranthaceae, Asclepiadaceae, Asteraceae, Chenopodiaece, Cruciferae, Leguminosae, and Solanaceace among 63 plant species in 17 families tested. The disease incidence was about 27% by seed transmission, and Myzus persicae Sulzer and Aphis gossypii Glover were able to transmit the virus in a non-persistent manner. The purified virus preparation was obtained with a yield of 22 mg/100 g from infected tobacco leaves. The molecular weight of capsid protein of the virus was estimated 26 kDa by sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE). An antiserum with a titer 1024 was obtained by immunizing a white rabbit with purified virions. In western blotting, indirect ELISA, and SDS-agar gel double diffusion test, the antiserum against the virus isolated from sea lettuce reacted with its homologus antigen. In order to further characterize the virus at the molecular level, the coat protein (CP) gene of sea lettuce virus isolate was cloned, sequenced and analyzed. A DNA fragment sequence of 657 nucleotides (nts) was amplified and the percent age of nucleotide identity of the CP gene was 97% comparing to Cucumber mosaic virus (CMV) isolated from Ph (accession number EF424708). Similarly, percent identities of amino acid sequence of CP gene to those of fifteen CMV isolates revealed 99% with seven isolates of Caw, Ag, Danshen, Vi, Sr, CaM and K3a (accession number EF424778、EF424775、AY600989、EF424 784、EF424781、EF4247810 and EF424781). The virus could be classified as Cucumber mosaic virus IB subgroup by Neighbor-Joining analysis based on amino acid sequences of CP In conclusion, the results indicated that the virus isolated from sea lettuce showing chlortic ringsot symptom is a IB strain of CMV and is first reported for in Taiwan.

並列關鍵字

Sea lettuce ； viral disease ； Cucumber mosaic virus ； virus identification

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