香蕉條紋病毒快速檢測方法之建立與臺灣香蕉種原條紋病之調查

Development of a rapid detection method for Banana streak virus and the survey of banana streak disease on Musa spp. germplasm collection in Taiwan

黃偲佳(Sz-Jia Huang)

指導教授 ： 葉信宏

共同指導教授 ： 蘇鴻基

國立臺灣大學/生物資源暨農學院/植物病理與微生物學研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.01912

摘要

香蕉條紋病毒 (Banana streak virus, BSV) 為Caulimoviridae科、Badnavirus屬的雙股DNA病毒，一般游離狀呈桿菌型病毒顆粒，具有蟲媒傳染性而引起條紋病徵。部分BSV基因體可嵌入所有包含B基因型拔蕉 (Musa balbisiana) 的基因體中，稱之為內源類反轉錄病毒序列 (endogenous pararetroviral sequence, EPRV)。此類嵌入型 (integrated form) 病毒並不會在香蕉上引起病徵，但在大量組織培養繁殖時，EPRV會活化成游離型 (episomal form)，而造成嚴重的病害。BSV之演化系統複雜，目前共有6個系統被報導。先前的偵測方法僅針對單一系統的香蕉條紋病毒進行偵測，且EPRV序列亦會干擾PCR偵測游離型病毒，造成偽陽性 (false positive) 結果，故建立區分嵌入型和游離型BSV的快速檢測方法為目前急欲發展的目標之一。過去本實驗室利用反轉錄聚合酶鏈鎖反應實驗 (reverse transcription polymerase chain reaction, RT-PCR)，已針對臺灣香蕉種原品種中發現過之BSV-Mysore (common) 及OL系統，開發出可用來偵測並區分EPRV與游離型BSV的方法。本實驗針對香蕉條紋病毒六個系統的序列相似區域 (common region) 設計出適當的引子對進行RT-PCR。從序列比對後的結果設計出六對引子對，初步以RT-PCR測試篩選出最適合的引子對—5747F/6720R (6S) 引子對。經過試驗調整RT-PCR之最佳偵測條件後，對臺灣香蕉研究所保有的兩百多個國內外香蕉種原進行全面的檢驗，結果觀察到BSV典型病徵者其RT-PCR結果皆為正反應，並確定約11.35%的種原受到BSV的感染，且發現之前臺灣未報導之新BSV-GF和BSV-GD系統。部分樣本並進一步以蟲傳試驗配合先前用過的Mysore和OL專一性引子對進行RT-PCR，以探討6S引子對的可用性，結果更證實6S的可靠性及其偵測除了Mysore和OL之外的其他BSV系統之可用性。此外，我們也在種原編號119、123、222和232的組織培養苗上發現到嵌入型BSV活化的現象。此快速偵測方法建立後，可提供更準確的香蕉條紋病毒之偵測，有利於今後對此病毒病害的檢疫及防疫。

關鍵字

香蕉條紋病毒 ； 內源類反轉錄病毒序列 ； 嵌入型 ； 游離型 ； 反轉錄聚合酶鏈鎖反應

並列摘要

BSV belonging to the genus Badnavirus in the family Caulimoviridae, is a double-stranded DNA virus. Part of the BSV genome usually becomes endogenous pararetroviral sequences (EPRVs) that can be found in all banana varieties containing genome derived from Musa balbisiana. It indicated an integration event happened in Musa balbisiana. EPRV does not cause symptoms on bananas, but it will sometimes become active (episomal form) during vegetative propagation such as tissue culture and cause severe symptoms on plants. Currently 6 different strains of BSV have been reported. Despite several methods have been developed for the detection of BSV including the enzyme-linked immunosorbent assay (ELISA), the immunocapture polymerase chain reaction (IC-PCR) and the multiplex IC-PCR (M-IC-PCR) etc., these methods can not detect all strains of BSV. Besides, the presence of EPRV leads to false positive results by PCR based methods to detect the episomal form of BSV. A reverse transcription PCR (RT-PCR) method has been developed for BSV strains, Mysore and OL, reported in Taiwan, to detect and to differenciate the EPRV and episomal form of BSV in our laboratory. In this study we designed several pairs of specific primer from the common regions shared by BSV strains to perfrom RT-PCR, and primer pairs were designed. One primer pair, 5747F/6720R (6S), was found to be the most effective in BSVstrain detection. After optimizing the detection conditions of 6S, we screened more than 200 of Musa spp. germplasm collected in Taiwan Banana Research Institute (TBRI), and about 11.35% of germplasm is infected by BSV. BSV strains, GF and GD, previously not reported in Taiwan were found in our survey. Some BSV positive samples (detected by RT-PCR with 6S primer pair) have been further selected for insect transmission. The result confirmed the availability of 6S primer pairs, and showed that 6S can be used to detect other BSV strains besides Mysore and OL. In addition, we also found the activation of integrated BSV in germplasm accession, including 119, 123, 222 and 232, during tissue culture propagation. The developed rapid detection method can help the management of BSV.

並列關鍵字

Banana streak virus ； EPRV ； integrated form ； episomal form ； RT-PCR

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