Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/54845
標題: 開發果瓜實蠅及新發生植物疫病蟲害防疫技術
Development of Management Technology for the Control of Fruit, Melon and New Plant Diseases and Other Pests
作者: 林益昇
關鍵字: 植物保護類
應用研究
摘要: 1.應用基因轉殖防治東方果實蠅之技術開發: 本計畫主要在於利用基因轉殖技術開發防治東方果實蠅(Bactrocera dorsalis (Hendel))的策略。前期計畫已利用轉基因子(transposon),piggyBac,成功地將一個外來基因,DsRed(紅色螢光蛋白),轉殖入果實蠅體內,證實轉基因在東方果實蠅進行的可行性,故而繼續進行後續研究。本期計畫係延續前期計畫,繼續執行以下各項內容:(1)轉機因在果實蠅體內穩定性之觀察。繼代飼養DsRed轉基因果實蠅迄今已歷經十五代,經測試外來基因DsRed在蟲體內的表現仍然穩定,此結果對後續開發防治策略具重要參考價值故仍繼續觀察;(2)完成啟動子(promoter)轉殖與活性專一性測試。已選定果實蠅卵黃原蛋白(vitellogenin)基因上游之基因調節區序列1.4 kb和2.7 kb具較高活性的片段,其後接一綠色螢光蛋白並構築入轉基因載體piggyBac上,將之轉殖入果實蠅體內以確認此啟動子的專一性;(3)依據第二項之試驗結果確定選用啟動子之片段即可構築、轉殖並建立攜帶可調控致死基因表現機制之族群,於實驗室甚或溫室中測試以轉基因果實蠅防治野生東方果實蠅(滅雌)之效果。 2.瓜類作物重要病害之防治: (1)洋香瓜黑點根腐病之生態與防治:洋香瓜黑點根腐病菌 (Monosporascus cannonballus)子囊孢子被認為是在土壤中的存活單位及最初感染源,卻又不易發芽,故其生態行為至今尚未瞭解。本研究利用網篩法發現其在田間土壤中存活的子囊孢子數不超過 5.2 (ascospores/g soil),因而發展出循環沈殿法。在子囊孢子發芽方面,利用 Stanghellini 等人研發的土壤試管法僅能觀察到已發芽的子囊孢子對植株根部之侵入率,未能反應準確的發芽率,因此本研究新研發包埋子囊孢子試管法,試驗結果顯示子囊孢子的發芽受到時間、植株根圈效應與土壤等多因子的影響,越靠近根表的子囊孢子越早發芽,且發芽率越高,在距離根表 2 mm 內子囊孢子發芽率可高達 83% 以上。本研究將進一步利用循環沈殿法與包埋子囊孢子試管法進行本病原菌在土壤中的生態學研究。另外,目前本病害尚無有效的防治藥劑與方法可供推廣使用,經多年研究與篩選,終於成功利用舌狀靠接法於田間防治本病害,進一步將探討其可能的機制,與改善洋香瓜的果實品質。 (2)胡瓜萎凋病生物防治:從花蓮抑病土篩選獲得具有生物防治胡瓜萎凋病潛力的拮抗菌株Fo-366,在溫室利用種子粉衣接種、育苗介質混菌接種以及剪胚軸接種等三種方法,將拮抗菌導入胡瓜幼苗內,觀察其對胡瓜萎凋病病勢發展之影響。結果發現,以剪胚軸接種法的防治效果最為顯著,發病度僅21%,未處理拮抗菌之發病度達83%。此外,進行拮抗微生物對各種蔬菜之纏據測試,發現除胡瓜外,對四季豆及長豇豆皆有不錯之纏據效果,初步溫室試驗對於萎凋病亦有延遲發病之情形,未來將進行更詳細的測試。另外,亦要進行將Fo-366施用於田間的可行信評估。此外,利用GUS基因融合系統進一步探討Fo-366與病原菌之間的關連性及抑病機制。 3.雌性雙性基因干擾對東方果實蠅生育力之影響: 東方果實蠅(Bactrocera dorsalis Hendel)是台灣主要經濟害蟲之一。遭到果實蠅危害的水果,嚴重地降低水果的品質與產量。雖然每年政府投入大量人力物力,但仍無法有效控制東方果實蠅的危害。近年來對昆蟲性別控制機制之研究獲得許多進展,基於這些昆蟲生理資料之累積,國際上也逐漸探討發展新的蟲害防治策略之可行性。本研究計畫用doublesex ( dsx )基因為實驗切入點,已決定東方果實蠅雙性基因之結構,再証實了其對東方果實蠅雌性發育之功能。從果蠅之研究資料顯示,雌性和雄性雙性蛋白都是轉錄控制因子,能夠決定與性別相關蛋白之表現與否,雌性雙性蛋白能夠活化卵黃蛋白的表現。本實驗室目前已由東方果實蠅基因庫中獲得雙性 cDNA序列,並以核醣核酸干擾( RNA interference ) 確認了其對果實蠅雌性發育之影晌。本實驗將探尋控制雌蠅發育之方法,並配合研發中之果實蠅轉殖技術,提出針對東方果實蠅的『性別專一性』防治策略。去年已開始轉殖東方果實蠅之研發工作,在今年的計劃中將繼續針對轉殖東方果實蠅体內dsx dsRNA之表現及其對雌蟲生育能力的干擾能力加以評估。若能確認轉殖東方果實蠅中外源dsx dsRNA之作用,此法將會是一個具有專一性的新防治方法。 4.台灣梨樹梨衰弱病之病原與其媒介昆
1.應用基因轉殖防治東方果實蠅之技術開發: The aim of this study is to develop a strategy using transgenic technique to control the oriental fruit fly Bactrocera dorsalis (Hendel). In the previous study, we have developed the processes for germline transformation and successfully transformed the fly with the transposon, piggyBac, carrying the red florescent gene, DsRed. We continue the study in this project and keep work on the following contains: (1) Examine the stability of transgene in the transgenic fly: Up to now, the DsRed-transformed fly has been continuously reared for 15 generations, and the performance of the transgene is still very stable. This character will still observed. (2) Finish the activity analyzing of the vitellogenin promoter: A vitellogenin promoter-driven EGFP (an enhanced green florescent protein, as an indicator of gene expression) was constructed onto the piggyBac and further transformed the fly. (3) Start to construct the tetracycline regulatory system: This experiment is base on the result of the secondary assay; we start to construct the regulatory system that is able to conditionally control the expression of a lethal gene in females, and further evaluate the efficiency of this transgenic population on controlling the wild type oriental fruit fly. 2.瓜類作物重要病害之防治: (1) The ecology and grafting management of root rot/vine decline of muskmelon: Monosporascus cannonballus, a root-infecting ascomycete, is the causal agent of root rot/vine decline of muskmelon (Cucumis melo L.) worldwide. Ascospores, which are produced one or rare two per ascus, was proved to be the survival structure in soil and performed as primary inoculum for root infection. Due to the failure of ascospore germination on artificial media, the records on the ecological behavior of M. cannonballus ascospores are rare. In this study, sieve method (SM) was used to quantify the ascospore density in different field soils, and turned out less than 5.2 ascospores/g soil. So, we developed a new method of circulating sucrose and sedimentation tank method (CSSTM). Furthermore, soil tube method (STM) developed by Stanghellini et al was employed in this study to assess the effect of plants on the ascospore germination. However, this method could only reveal the penetration rate of germinated ascospores to the root system, not reflect the real ascospore germination rate. Therefore, the immersed ascospore tube method (IATM) was developed to observe the germination of ascospores on agar medium, and showed that germination rates of ascospores were more than 83% at 21 and 28 days after treatment where the ascospores apart from root surface were less than 2 mm. We will use CSSTM and IATM in studying the soil biological behaviors of M. cannonballus. Until today, this disease was not an effective and used extensively method in management. After many years in study, tongue approach grafting was a tactic effectively in managing this disease. Further, we will study the disease resistance mechanisms and improve the fruit quality of grafted muskmelon. (2) Biological Control of Fusarium Wilt of Cucumber: A nonpathogenic Fusarium oxysporum (Fo-366) was inoculated into cucumber seedlings by using the hypocotyl cutting inoculation method. The inoculated seedlings could control Fusarium wilts of cucumber and asparagus bean effectively in greenhouse. Furthermore, we will evaluate the possibility putting the use of Fo-366 in field.And in order to study the connection and mechanism between Fo-366 and pathogen, GUS gene fusion system will be adopted. 3.雌性雙性基因干擾對東方果實蠅生育力之影響:Oriental fruit fly (Bactrocera dorsalis Hendel) leads to a huge economic loss every year. Great efforts are applied by government to redu
URI: http://hdl.handle.net/11455/54845
其他識別: 95農科-13.2.1-檢-B3
文章連結: http://grbsearch.stpi.narl.org.tw/GRB/result.jsp?id=1272298&plan_no=95%E8%BE%B2%E7%A7%91-13.2.1-%E6%AA%A2-B3&plan_year=95&projkey=PG9507-3572&target=plan&highStr=*&check=0&pnchDesc=%E9%96%8B%E7%99%BC%E6%9E%9C%E7%93%9C%E5%AF%A6%E8%A0%85%E5%8F%8A%E6%96%B0%E7%99%BC%E7%94%9F%E6%A4%8D%E7%89%A9%E7%96%AB%E7%97%85%E8%9F%B2%E5%AE%B3%E9%98%B2%E7%96%AB%E6%8A%80%E8%A1%93
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