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技術名稱
Project name
越南百香果無病毒苗繁殖體系之建立及以輕症疫苗防治百香果病毒之研發
Propagation system of virus-free passionfruit seedlings in Vietnam and development of mild strains for control of passionfruit virus
計畫單位
Project Operator
國立中興大學
National Chung Hsing University
展區位置
Booth location
生技與新藥
Bio-tech & New Drugs
技術說明
Technology Introduction
利用百香果病毒核酸分子檢測技術,檢測合格的百香果無病毒苗,經無性繁殖方式進行無病毒苗的原種保存,以嫁接方式繁殖無病毒百香果苗木,協助越南大量生產無病毒百香果苗木,目前年產400萬株苗木,對於越南百香果產業助益極大,也提升了他們的技術水平,使得越南百香果栽培面積五年(2013-2018)內由1500公頃增至一萬公頃,已成為亞洲最大百香果產國,其百香果果汁在世界市場的佔有率攀升到8%,且還在上升中。越南Nafoods公司已和興大簽訂十年技轉合約,總共240萬元,此為台灣技術輸出獲利的重要範例。另外也於2018年在越南成立「植物病毒檢測中心」(Nafoods-NCHU Plant Virus Certification Center),此中心為百香果苗場提供六種病毒核酸及血清檢測技術,以保證所出產的苗木均為無病毒苗,是Nafoods苗場最重要的核心設施。但百香果無病毒苗木在田間種植只能減少早期感染,生產過程還是會遇到病毒感染的嚴重問題,故本實驗室繼續積極研發輕症疫苗,利用交互保護方式來防治病毒感染,以確保百香果產業之永續發展。
百香果病毒性病害是全世界百香果栽培區生產百香果最重要的限制因子。目前在臺灣以及另一新興百香果產出國家越南發現東亞百香果木質化病毒 (East Asian passiflora virus, EAPV)以及另一鑒定的新百香果病毒(Passionfruit Vietnam potyvirus, PVNV) 的存在。此二病毒都是影響百香果產量的最主要的限制因子,且都屬於可被蚜蟲傳播的馬鈴薯Y屬病毒 (Potyvirus) 之成員,在百香果上都會造成木質化,畸形果及小果等病徵。目前,交互保護是有效的應用在防治potyvirus引起的百香果病毒病害的重要方法。由於本實驗室在經過十數年的交互保護機制研究中已發現數個與病毒致病力相關的重要功能區,包括兩個位於病毒HC-Pro蛋白N端之FFRG和FRNK功能區,另兩個則是分別位於HC-Pro蛋白中央及C端部分之功能區。本研究的首要目的是鑒定在臺灣以及越南的主要百香果病毒,並且開發此二病毒載體,並藉由改變病毒致病因子HC-Pro的基因功能區以獲得輕症弱病毒性之病毒突變株,應用於百香果之交互保護上,可產生免疫性狀以保護百香果之生產。先前的研究中,我們已建構EAPV之感染性病毒株,EAPV-TW全長度cDNA clone確實可在菸草及百香果上引起典型之病徵。故能進一步針對上述所提及之HC-Pro重要功能區進行點突變,利用圓葉菸草 (Nicotiana benthamiana)進行感染力及基因穩定性測試。篩選之後顯示,我們獲得EAPV-TW在HC-Pro功能區進行單點及雙點突變的多個不同感染力之病毒突變株 (EAPV-IF, -A, -B, -C; AB, -AC, -BC; -IFA, -IFB, -IFC)。經由接種測試結果顯示,雙點突變之EAPV-TW-AC及EAPV-TW-IFC在百香果造成無病徵之感染,並具有在溫室條件下完全對抗強係病毒的交互保護能力,此二輕症突變株可應用於進一步之百香果田間交互保護試驗。配合已建立的接穗嫁接方法,將此二病毒保持於接穗母株,即可大量繁殖,可避免大量接種苗木之問題。此策略可解決台灣及越南之EAPV之危害百香果外,亦可同樣的套用在越南病毒PVNV上,以獲得屬於越南專一性的弱系病毒疫苗,進而達到有效保護百香果免於病毒病害侵襲的創新栽培方法。

Virus-free passionfruit seedlings were examined and selected by specific PCR-based molecular detection method. These certified virus-free passionfruit seedlings were propagated through grafting or cutting method and have been transferred to Vietnam for the high demand of local passionfruit industries. The total passionfruit plantation area in Vietnam have increased from 1500 hectares to 10000 hectares in the past five years and still increasing. Through the technological transfer from National Chung Hsing Univerity, Vietnam has become the largest passionfruit plantation and exportation country in Asia, with the hold of 8% of world passionfruit juice production.
This virus-free certification technology and strategy have been successfully transferred to Nafoods company, in Vietnam by a ten year agreement with the technology transfer fee 2.4 million Taiwan dollars. This technology transfer will serve as a good example for the future collaboration between Taiwan and Vietnam, also to other Asean countries. In April 2018 , an office of ‘Nafoods-NCHU Plant Virus Certification Center’ was set up which provides the methods of PCR based detection and serological ELISA detection in order to provide the certified virus-free passionfruit seedlings of Nafoods passionfruit nurserys. However, the virus-free passionfruit does not solve the virus infection problem under the open field conditions, thus the development of mild strain virus to cross protect the passionfruit from severe virus infection are under process in order to solve the serious virus infection problem.
Passionfruit is now an important and valuable fruit for juice processing in Taiwan and South East Asian countries, especially, Vietnam. However, several passionfruit diseases associated with viruses limit its production in different geographic areas. Recently, East Asian passiflora virus (EAPV) and a new emerging passionfruit potyvirus, Passionfruit Veitnam potyvirus (PVNV) have been found damaging passionfruit in Taiwan and Vietnam, thus, seriously limited the production of passionfruit. EAPV and PVNV are both aphid-transmitted potyviruses that cause severe woodiness, distortion and size-reduction fruits on infected passionfruit (Passiflora spp.) plants cultivated in Taiwan and Vietnam. For effective control of potyvirus infecting passionfruit plants, cross-protection is a feasible approach. In the past decade, we have identified several functional motifs, FFRG and FRNK motifs located at the N-terminal region and two other essential motifs at the central and C-terminal regions of HC-Pro gene, which determine potyvirial pathogenicity. Our goal is to clarify the molecular identity of the new emerging PVNV and develop the viral vector of both EAPV and PVNV. Moreover, through the manipulation of viral pathogenicity by modifying the essential motifs o HC-Pro we attempt to obtain useful attenuated mutants for cross protection. Recently, the EAPV-TW infectious clone was constructed, and the aforementioned functional motifs of HC-Pro was modified through single or double-point mutations, to generate attenuated mutants. The virulence and genetic stability of the obtained mutants were assayed on Nicotiana bethamiana plants. Cross protection assay showed that double-point mutants, EAPV-TW-AC and EAPV-TW-IFC, have great potential to be used for cross-protection. This strategy will further apply on PVNV to obtain the protective mild strain of PVNV for control of passionfruit virus disease in Vietnam. For practical application, the protective scionst-stock will be used to graft on the virus free root stock plants. Thus, a novel and effective approach for solving the virus problems of passionfruit will be established.
科學突破性
Scientific Innovation
本技術在越南成立「植物病毒檢測中心」, 協助大量生產無病毒百香果苗木。 利用植物病毒交互保護技術不但可替代基因轉植作物的使用,且可成爲其他作物在病毒病害的一個非基改防治典範。輕症病毒導入接穗母株之運用,將同時讓所有嫁接苗攜帶輕症病毒且不增加成本,此將成為防治百香果病毒之世界首例。

The established of office ‘Nafoods-NCHU Plant Virus Certification Center’ provides several passionfruit viruses detection methods in order to assist the Vietnam companies to produce certified virus-free passionfruit seedlings. The cross-protection strategy will protect the plants against the severe virus infection which is an alternatives strategy in replacement of transgenic resistant strategy.
產業應用性
Industrial application
本技術為中興大學提供專業技術的輸出及產業在國外的布建,也提供無病毒苗繁殖體系以協助越南的種苗公司百香果種苗的生產。本技術所研發的輕徵疫苗可以於田間不同作物上達到交互保護效果,避免農作物受到病毒病害而產量損失嚴重。此技術之經驗將可作為其他重要經濟作物或新興作物病毒病害防治之借鏡。

The virus-free certification technology has been transferred to Nafoods company, in Vietnam by a ten-year agreement with the technology transfer fee 2.4 million Taiwan dollars to NCHU. This technology transfer will serve as a good example for the future collaboration between Taiwan and Vietnam, also to other Asean countries. Our cross protection assay indicates that the mild virus vaccine have great potential to be used for cross-protection in Taiwan and Vietnam.
聯絡人
Contact Person
黃雅鈴
電子信箱
Mail