國際重要香蕉檢疫病毒之診斷平台建立與感染技術之建構

Abstract

香蕉種類繁多，為全球重要的果樹及糧食作物。香蕉病毒病害常造成葉片褪綠、植株矮化、生長勢下降及產量降低，目前尚無有效藥劑可根除病毒感染，僅能藉由移除植株及重新種植健康蕉苗降低其危害，因此健康種苗制度對香蕉產業極為重要。臺灣香蕉產業長期推動無病毒組織培養苗，使主要病毒病害如香蕉萎縮病毒（banana bunchy top virus，BBTV）與胡瓜嵌紋病毒（cucumber mosaic virus，CMV）之發生率已大幅降低。然而，過去臺灣對香蕉苞葉嵌紋病毒（banana bract mosaic virus，BBrMV）與香蕉輕微嵌紋病毒（banana mild mosaic virus，BanMMV）兩個具國際檢疫重要性之香蕉病毒研究甚少，直至 2017–2023 年間田間調查始發現兩種病毒於臺灣存在。BBrMV 曾於世界多個香蕉產區造成嚴重減產，因而備受產業界重視，臺灣目前僅見零星田間感染。為研發快速簡易的檢測技術，本研究首先以融合瘤（hybridoma）技術製備 BBrMV 單株抗體，成功獲得具高專一性及高靈敏性之單株抗體 E9C11，可應用於健康種苗生產及病毒檢測。田間調查顯示，目前 BBrMV 尚未於臺灣主要香蕉栽培區形成穩定的感染趨勢。另一方面，為研究不同香蕉品種對 BBrMV 之感病性，本研究建構臺灣 BBrMV F92 分離株之全長基因體感染性選殖株，並發現病毒基因體 3’ 端 poly(A) tail 長度對建立穩定系統性感染具有關鍵影響。利用農桿菌法接種 BBrMV 並評估臺灣主要栽培品種之感病性，結果發現「玫瑰蕉」（AA）表現出最高感病性，感染後出現嚴重葉片畸形及假莖倒伏；相較之下，「北蕉」（AAA）多僅呈現輕微病徵。除 BBrMV 外，本研究亦首次發現 BanMMV 於臺灣之感染，並完成 GF-3 分離株之全長基因體解序及感染性選殖株建構。BanMMV 單獨感染 Cavendish 之「寶島蕉」（AAA）時僅造成極輕微條紋或無明顯病徵，且與 BBrMV 複合感染於短期內未產生明顯病徵協力作用，說明其在田間不易被察覺之原因。最後，針對 BanMMV 基因高度變異之特性，本研究重新設計專一性引子（CP10-4 / CP-R3，set 6），搭配 random hexamer 反轉錄之 RT-PCR 檢測流程，可提升檢測之穩定性。田間樣本回溯性檢測顯示，BanMMV 確實存在於臺灣部分商業栽培田區，但尚未於 Cavendish（AAA）品系中檢測到感染。外鞘蛋白基因序列分析顯示，BanMMV 具有極高遺傳多樣性，單一植株內病毒序列歧異度最高可達 24.55%。總括而言，本研究建立 BBrMV 與 BanMMV 兩個國際重要香蕉檢疫病毒之快速檢測技術及人工感染平台，為臺灣香蕉種原保存、健康種苗生產、植物檢疫及未來抗病育種研究提供重要技術基礎，裨益全面性香蕉病毒檢疫與防疫體系之建立。

Bananas are diverse and are important fruit and food crops worldwide. Banana viral diseases often cause leaf chlorosis, stunting, reduced plant vigor, and yield losses. Currently, there are no effective agents to eradicate viral infections; their damage can only be reduced by removing infected plants and replanting with healthy seedlings. Therefore, a pathogen-free planting material system is essential for the banana industry. Taiwan's banana industry has long promoted pathogen-free tissue-cultured seedlings, significantly reducing the incidence of major viral diseases such as banana bunchy top virus (BBTV) and cucumber mosaic virus (CMV). However, research in Taiwan on banana bract mosaic virus (BBrMV) and banana mild mosaic virus (BanMMV), two banana viruses of international quarantine importance, has been limited. Field surveys conducted between 2017 and 2023 revealed the presence of both viruses in Taiwan. BBrMV has caused severe yield losses in several banana-producing regions worldwide, but only sporadic infections have been detected in Taiwan. To develop a rapid and simple detection method, a highly specific and sensitive monoclonal antibody, E9C11, was produced using hybridoma technology for healthy planting material production and virus detection. Field surveys indicated that BBrMV has not established a stable population in major banana-growing areas of Taiwan. To evaluate cultivar susceptibility, a full-length infectious clone of the Taiwanese BBrMV isolate F92 was constructed, and the length of the 3’ poly(A) tail was found to be critical for stable systemic infection. Using Agrobacterium-mediated inoculation, banana cultivar ‘Rose’ (AA) exhibited the highest susceptibility, with severe leaf deformation and pseudostem collapse, whereas the Cavendish cultivar ‘Pei-Chiao’ (AAA) developed only mild symptoms. This study also reports the first detection of BanMMV in Taiwan and the complete genome sequencing and infectious clone construction of the GF-3 isolate. BanMMV alone caused only very mild streaks or no obvious symptoms in banana cultivar ‘Formosana’ (AAA), while co-infection with BBrMV caused no obvious synergistic symptoms during the short-term observation period, explaining why BanMMV may remain unnoticed in the field. Given the high genetic variability of BanMMV, a specific primer set, CP10-4/CP-R3 (Set 6), was designed for an RT-PCR protocol using random hexamers for reverse transcription, improving detection stability. Retrospective testing of field samples confirmed BanMMV in some commercial banana plantations in Taiwan, but no infections were detected in Cavendish (AAA) cultivars. Coat protein gene analysis revealed high genetic diversity, with sequence divergence reaching 24.55% within a single plant. In summary, this study established rapid detection methods and artificial infection platforms for BBrMV and BanMMV, two internationally important banana quarantine viruses, providing a technical foundation for banana germplasm preservation, healthy planting material production, plant quarantine, and future disease-resistance breeding, as well as comprehensive banana virus quarantine and disease management in Taiwan.