感染佛手瓜之南瓜捲葉菲律賓病毒初探

Abstract

佛手瓜（Sicyos edulis）是葫蘆科的多年生植物，原產於南墨西哥與中美洲，並已被引入多國，其中也包括臺灣。佛手瓜的栽培需要溫暖的氣候，在臺灣，人們經常會將發芽的整顆果實直接種在露天田區的畦面上，在栽培過程中會遭受各種病蟲害的侵擾，特別是病毒的威脅，目前在臺灣佛手瓜的病毒病中，僅有南瓜捲葉菲律賓病毒Squash leaf curl Philippines virus（SLCuPV）的感染紀錄。儘管佛手瓜是重要的夏季蔬菜之一，臺灣對於SLCuPV感染佛手瓜的研究仍然相對有限，但我們需要更深入了解 SLCuPV、佛手瓜、環境與傳播此病毒的昆蟲之間的關係，方能制定更有效且適合的整合管理策略。本研究旨在優化和標準化SLCuPV PCR與qPCR檢測技術，並設計針對ToLCNDV具有高度專一性與敏感度的引子對。透過這些技術工具，我們評估佛手瓜田間的感染情形，進行了詳細的分析並驗證觀察結果，同時研究罹病率、氣候與田間操作之間的關聯。此外，我們對SLCuPV PCR檢測結果為陽性的葉片進行病徵的分類與計數。最後，我們使用qPCR評估不同植物組織、均質方法和萃取方法對病毒量的影響，並評估感染植株的實生苗其中SLCuPV的傳播率和病毒量。研究結果顯示，經由PCR，新植田的罹病率較低，且翻耕可有效降低罹病率，但持續效果有限。根據肉眼觀察，春天的罹病率較秋天低，但PCR檢測結果顯示罹病率仍相當高，且無病徵型的植株在新植田與春天為主要病徵型態，因此它們成為田間潛在的病毒來源，而ToLCNDV則尚未被發現於佛手瓜田間；經由qPCR，我們證實植物組織、均質方法與萃取方法對病毒量皆有顯著影響，使用者可依據環境限制選擇因地制宜的方法使用；在生長測試中，有22.5%的實生苗可檢測到SLCuPV，且即使在種植後8週，仍能檢測到極低量的病毒。但是，這種現象是否在佛手瓜作物中普遍存在，與實生苗中的SLCuPV 是否能透過昆蟲傳播和感染健康植株，還有待進一步的研究。 本研究首次建立完整感染佛手瓜的SLCuPV之PCR與qPCR檢測系統，並首次使用絕對定量的SYBR green qPCR系統進行SLCuPV的檢測，更重要的是，這是首次發現SLCuPV具有實生苗傳播的特性，這些研究結果將有助於揭示SLCuPV實生苗傳播的機制和影響，並對佛手瓜的栽培提供重要的參考依據。

Chayote, a perennial plant of the Cucurbitaceae family, is native to southern Mexico and Central America, and has been introduced to various countries, including Taiwan. Chayote cultivation requires a warm climate. In Taiwan, people often directly sow the entire germinated fruits in open field beds. In general, chayote suffers from various pests and diseases, particularly from viral threats. However, only Squash leaf curl Philippines virus (SLCuPV) has been currently confirmed to infect chayote in Taiwan. Despite being an important summer vegetable, there is limited research on SLCuPV infecting chayote in Taiwan. However, there's a need for a deeper understanding of the relationship between SLCuPV, chayote, the environment, and the insects transmitting this virus to develop more effective and integrated management strategies. This study is aiming to optimize and standardize SLCuPV PCR and qPCR protocols, and to design primer pairs with high specificity and sensitivity for detecting ToLCNDV. Using these tools, we evaluated infection in chayote fields, conducted detailed analysis, and validated the phenomena observed in the fields, while also researching the relationship between incidence rates, climate, and field operations. Besides, we classified symptom types of chayote leaves which were positive in SLCuPV PCR. Finally, we used qPCR to assess the impact of different plant tissues, homogenization methods, and extraction methods on the viral quantity, and evaluate the transmission rate and viral quantity of SLCuPV in infected seedlings in grow-out test. In our results, via PCR, the disease incidence was lower in new plant fields, and replanting effectively reduced the disease, but the sustained effect was limited. According to visual observation, disease incidence was lower in spring than in autumn. However, PCR testing results showed a relatively high incidence rate, with asymptomatic plants in newly planted fields and spring being the primary symptom type, thus they become potential virus sources in fields. Besides, ToLCNDV has not yet been detected in chayote fields. Through qPCR, we confirmed that plant tissue, homogenization method, and extraction methods have a significant impact on the viral quantity. Users can select appropriate methods based on environmental constraints. In the grow-out test, SLCuPV could be detected in 22.5% seedlings, and even after 8 weeks of planting, a minimal amount of the virus could still be detected. However, whether this phenomenon is prevalent in chayote and whether SLCuPV in seedlings can be transmitted and infect healthy plants through insects remains to be further researched. This study is the first to establish a complete PCR and qPCR detection system for SLCuPV infections in chayote and the first to use the absolute quantitative SYBR green qPCR system for SLCuPV detection. More importantly, this is the first discovery of the SLCuPV transmission through seedlings. These research results will help reveal the transmission mechanism and impact of SLCuPV in seedlings and provide valuable reference for chayote cultivation.