豇豆微斑駁病毒之鑑定與生物特性分析

Identification and Biological Characteristics of Cowpea Mild Mottle Virus in Taiwan

鄭櫻慧(Ying-Huey Cheng) ； 陳金枝(Chin-Chih Chen) ； 簡蘭懿(Lan-Yi Chien) ； 張清安(Chin-An Chang)

《台灣農業研究》 70卷1期 (2021/03) Pp. 67-79

https://doi.org/10.6156/JTAR.202103_70(1).0006

摘要

採集自嘉義縣新港鄉的長豇豆及南投縣草屯鎮的敏豆疑似感染病毒樣本，經過分離得到Calavirus屬的豇豆微斑駁病毒（cowpea mild mottle virus; CpMMV）的長豇豆分離株（CpMMV-V）及敏豆分離株（CpMMV-P），以機械接種回接長豇豆及敏豆均可以感染並引起與田間相似葉片斑駁的病徵。2分離株接種其他豆科作物時，以植株全株來看無明顯矮化或發育不良，但從葉片可觀察到斑駁與黃化斑點等輕微病徵。採自病株種子以出芽測試未測得CpMMV藉由種子傳播的現象，電子顯微鏡則可觀察到長桿狀病毒顆粒。抽取植物之總量RNA，以potyviruses簡併式引子（HRP5/dT）進行反轉錄聚合酵素連鎖反應，意外擴增出約1 Kb的DNA產物，定序分析並與登錄於GenBank上的序列比對，此段DNA含括豇豆微斑駁病毒的部分鞘蛋白基因至基因體3'端。再次以專一性引子擴增、選殖及定序分析完整鞘蛋白基因，長豇豆分離株之鞘蛋白基因（JX070669）與敏豆分離株（JX020701）之鞘蛋白基因具有88.9%核苷酸相同度與97.6%胺基酸相同度，與網站上其他CpMMV鞘蛋白基因比較，核苷酸相同度介於98.7－75.7%，胺基酸相同度介於97.9－91.3%之間。部分純化病毒懸浮液再以sodium dodecyl sulfate polyacrylamide gel electrophoresis（SDS-PAGE）純化病毒的鞘蛋白，經免疫白兔製備抗血清，其抗體可應用於西方漬染法及酵素結合免疫吸附分析法（enzyme-linked immunosorbent assay; ELISA）檢測。因此，乃進行田間偵測調查，發現CpMMV常見危害台灣中部地區栽培之敏豆。由於粉蝨為台灣常見害蟲，其媒介傳播之CpMMV除了危害長豇豆及敏豆以外，是否危害其他豆科作物尚待更多調查。

關鍵字

豇豆微斑駁病毒 ； 鞘蛋白 ； 抗血清

並列摘要

Sample plants of asparagus bean and French bean bearing putatively viral symptoms were found in vegetable gardens in Chiayi and Nantou Counties, Taiwan. The symptomatic plants were mechanically inoculated to Chenopodium quinoa and two virus isolates, cowpea mild mottle virus (CpMMV)-V and CpMMV-P, were isolated after 3 times of single lesion isolation. The virus sources were established by inoculating local lesions to C. quinoa or Nicotiana benthamiana. By back-inoculation, plants of asparagus bean and French bean developed symptoms similar to those found in the field. When two isolates were inoculated on other legume crops, there was no obvious dwarfing or stunting in the whole plant, but some mild symptoms such as mottle and yellow spots can be seen on leaves. CpMMV has not been detected to be transmitted through seeds by growing out test. Total RNAs extracted from diseased asparagus bean and French bean used as templates, and reverse transcription-ploymerase chain reaction were performed with degenerate primers for potyviruses (Hrp5/Pot1). Unexpectedly, an 1 Kb of DNA product was amplified. Comparing with gene sequences on GenBank, sequences of this amplified DNA includes part of the 3'end of the coat protein gene of CpMMV. The complete coat protein gene was cloned with specific primers based on the reported sequences and then for sequence analysis. The coat protein gene of CpMMV-V (JX070669) and CpMMV-V (JX020701) shared 88.8% and 97.6% sequence identities of nucleotide and amino acid, respectively. Comparing with other coat protein genes of CpMMV, the nucleotide sequence identity is 98.7-75.7%, and the amino acid sequence identity is 97.9-91.3%. Electron micrographs showed that carlavirus-like particles were found in the leaf dips of infected asparagus bean and French bean samples. The coat protein obtained by partially purified viral particles and through sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used for antiserum preparation. The antiserum was available for Western blot and enzyme-linked immunosorbent assay (ELISA), especially are suitable for field detection and investigation. Therefore, we found that CpMMV is commonly infected French bean in middle Taiwan. In addition to asparagus bean and French bean, whether CpMMV affects other leguminous crops needs more investigation.

並列關鍵字

Cowpea mild mottle virus (CpMMV) ； Coat protein ； Antisera