台灣中南部瓜類作物與粉蝨中雙生病毒之監測與其基因體變異性分析

Abstract

洋香瓜等瓜類作物極具經濟價值，然近年來迭遭數種新興病毒病害之大規模感染，造成農民的重大損失。其中由菸草粉蝨(Bemisia tabaci species complex)所傳播的南瓜捲葉病毒Squash leaf curl virus (SqLCV，雙生病毒科)於台灣中、南部地區洋香瓜上造成捲葉病的肆虐，在2008 下半年造成洋香瓜捲葉病害的大流行，甚而導致全園廢耕的嚴重經濟損失，成為洋香瓜產業的主要限制因子之一。亟需開發方便有效的方法，以監測SqLCV 於粉蝨與作物中的帶毒與感染狀況，預作適當之防治工作。本研究研發利用滾環式擴增法(Rolling Circle Amplification，RCA) 檢測雙生病毒環狀DNA 的技術，持續於雲林、嘉義、與台南等縣市之主要瓜類作物栽培區進行SqLCV 之監測。發現SqLCV 普遍存在於田間瓜類作物與粉蝨族群中，但基因體變異性極大。且帶毒率與感染率隨作物種類而不同，其中以西瓜最低，洋香瓜最高。為進一步了解雙生病毒基因體在不同時空環境中的變異性，本研究分析比對自2007 年至2010 年不同時間點於台灣中南部分屬不同地理位置的洋香瓜栽培區所採集的SqLCV 基因體序列，並分別比對來自於洋香瓜葉片或粉蝨體內所選殖之SqLCV 基因體序列，發現在不同時空環境下經常發生變異的核苷酸位置較常出現於基因間隙區域(intergenic region)與外鞘蛋白(coat protein)編碼區域中。而其他基因體區域相對較為穩定。不同的地理環境與瓜類作物對於雙生病毒的基因體序列亦有影響，此基因體序列之變異性亦可作為用於追蹤雙生病毒來源與設計有效病毒防治策略的參考工具。 Cucurbits are economically important crops in Taiwan. However, several emerging viral diseases have devastated the production of cucurbits and caused great losses in recent years. Squash leaf curl virus (SqLCV, Geus Begomovirus , Family Geminiviridae), transmitted by whiteflies (Bemisia tabaci species complex) is among the most severe viral pathogens in cucurbits. The development of efficient and convenient methods for the monitoring of SqLCV in cucurbits is in need. In this study, modified rolling circle amplification (RCA) method to detect the circular DNA genome of SqLCV was developed and applied in the survey of SqLCV in whiteflies and cucurbit crops in the major production areas in southern and central Taiwan. The results revealed the common and persistent presence of SqLCV among various cucurbit crops and whitefly populations. The disease incidences and viruliferous ratios varied widely among different crops and whitefly populations, with watermelon and muskmelon being the lowest and highest, respectively. The spatial and temporal effects on the variations of SqLCV genomic sequences were further analyzed using samples collected from various localities in Taiwan over the period of 2007 to 2010. Most genomic variations were identified in intergenic region and coat protein coding sequences, while the Rep coding sequences were highly conserved. These results provide applicable references for the tracing of origins of SqLCV isolates and the design of effective disease management measures for SqLCV.