Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/54479
標題: 開發瓜果實蠅及新發生植物疫病蟲害防疫技術
Development of Management Techology for the Control of Fruit, Melon Flies and New Plant Diseases and Other Pests
作者: 林益昇
葉文斌
路光暉
齊心
楊曼妙
楊恩誠
張誠
林長平
關鍵字: 應用研究
Monosporascus cannonballus
植物保護類
洋香瓜黑點根腐病
包埋子囊孢子試管法
洋香瓜
胡瓜萎凋病
無病原性尖鐮胞菌
拮抗細菌
生物防治
雙性基因
西方點墨法
原卵黃蛋白
東方果實蠅
核酸干擾
梨衰弱病
植物菌質體
聚合酵素連鎖反應
媒介昆蟲
瓜實蠅
生命表
整合性防治
梨木蝨
細胞色素I基因
核醣體間區
AFLP指紋
族群變異
卵黃原蛋白基因
東方果實蠅
基因轉殖
IATM
Muskmelon
Fusarium Wilt of Cucumber
Non-pathogenic Fusarium oxysporum
Antagonistic Bacteria
Biological Control
Doublesex Gene
Western Blot
Vitellogenin
Bactrocera dorsalis (Hendel)
RNA Interference
Pear Decline
Phytoplasma
Polymerase Chain Reaction (PCR)
Insect Vector
Bactrocera cucurbitae
Life Table
Integrated Control
Pear Psyllid
Cytochrome Oxidase I
Intergenic Spacer
AFLP
Population Variation
Vitellogenin Gene
Oriental Fruit Fly
Transgenesis
摘要: 1. 應用基因轉殖防治東方果實蠅之技術開發:本年度目標:確認卵黃原蛋白結構基因調節區域,並開始利用重組載體之轉殖進行東方果實蠅蟲體調節位置及調節機制的功能分析。 2. 瓜類作物重要疫病之防治: a. 本年度目標:探討影響病原菌子囊孢子發芽的因子與瞭解黑點根腐病的發病臨界濃度。 b. 本年度目標:去年在花蓮土壤及其他植株或土壤中獲得多株細菌性的拮抗菌,以各種的施用方法,單獨或混合這些菌株與尖鐮胞菌在溫室及田間試驗觀察其防病效果,並篩選出拮抗菌之最佳導入方法及可能的防治機制。設計田間防治試驗,將無病原性尖鐮胞菌混合細菌施用於胡瓜病田中,觀察與計錄其發病情形。研發以剪除胚軸後導入拮抗菌的導入法,使拮抗菌能以內生菌模式存活於胡瓜植株之中,成為一人工的抗病品種。 3. 雙性基因核酸干擾與東方果實蠅防治:本年度目標: (1) 使用piggybac轉移子構築雌性doublesex RNAi表現載體。 (2) 發展並培育doublesex RNAi轉殖東方果實蠅。 4. 台灣梨樹梨衰弱病之病原與其媒介昆蟲之探討:本年度目標: (1) 完成梨衰弱病罹病株採樣及DNA抽取,以獲得研究所需之梨衰弱病菌質體之DNA檢體。 (2) 進行由共同主持之研究單位由梨園中所採集提供之韌皮部刺吸式同翅目昆蟲(木蝨、飛蝨、葉蟬及蚜蟲等)體內菌質體之PCR偵測。 (3) 完成媒介昆蟲體內植物菌質體之16S rDNA全長序列定序。 (4) 針對已研發之台灣梨衰弱病PCR專一性引子對完成其靈敏度之測定,並擬定病原菌質體檢測技術如multiplex PCR、nested PCR、RFLP-PCR及booster PCR等之檢測流程。 (5) 配合上述PCR檢測技術,挑選PCR檢測呈現正反應之確定罹病組織從事超薄切片,以穿透式電子顯微鏡(TEM)觀測,確認植物菌質體於植物組織中的分布情形及觀察其形態構造,藉以獲得直接之切片證據。 (6) 尋求國內昆蟲專家配合,針對本研究中偵測出梨衰弱病菌質體之媒介昆蟲種類,進行傳毒試驗之設計,以確定田間主要自然傳播之昆蟲媒介。 5. 以生命表為基礎之瓜實蠅整合性防治:本年度目標:由於瓜實蠅之際主植物種類多,本年度擬再進行瓜實蠅生命表研究,於實驗室內研究以木瓜飼育時之生命表,以了解其族群在木瓜上之增長潛能。為了解其田間生命表,擬繼續調查田間族群密度。本年度亦擬繼續試驗不同年齡成蟲對誘引劑的反應。比較cue-lure與雌蟲對雄蟲之引誘效果,並嘗試研究雌性誘引劑。 6. 台灣梨園木蝨遺傳變異及分子鑑定:本年度目標:持續進行各地之田間採樣,分別進行形態特徵與分子分析。形態部分將比較翅脈、頰錐與生殖器等各項特徵之變異,釐清台中東勢、和平與新竹尖石之有待確認物種之界定。分子生物部分將分析粒線體COI基因及核醣體ITS區段之DNA序列,並應用AFLP DNA指紋技術,分析各地族群之間的遺傳變異。 1. 應用基因轉殖防治東方果實蠅之技術開發:預計在本年度完成卵黃原蛋白基因啟動子有效活性長度之確立,取得致死基因,完成致死基因表現調控系統之構築,以及進行蟲體轉殖效果測試與評估調控系統對致死基因表現的調控能力。 2. 瓜類作物重要疫病之防治: a. 瞭解子囊孢子在土壤中的生態行為與影響其發芽的因子,並探討其應用在田間防治上的可能性。 b. 獲得作物萎凋病之拮抗菌株及生物防治方法。 3. 雙性基因核酸干擾與東方果實蠅防治:研究東方果實蠅雙性(doublesex)基因之作用機制,發展出以核酸干擾為基礎之滅雌方法。 4. 台灣梨樹梨衰弱病之病原與其媒介昆蟲之探討:針對台灣梨衰弱病病害之病原及其媒介昆蟲進行探討,以獲得植物菌質體之16S rDNA全長序列及16S-23S rDNA spacer序列,與國外梨衰弱病菌質體之序列進行比對分析,以確定其分類地位。並利用電子顯微切片直接進行梨衰弱病病原菌之觀察,以獲得電顯切片之直接證據。確定在台灣中部梨園內梨衰弱病菌質體媒介昆蟲之種類,並配合其他分子檢測技術如multiplex PCR、nested PCR、RFLP-PCR及booster PCR等,發展快速靈敏之檢測技術,此外亦可配合進口接穗與害蟲之防疫檢疫篩檢。將本研究成果應用於台灣梨樹衰弱病發病生態之研究,完成台灣梨樹衰弱病之檢測流程及防治策略之制定。 5. 以生命表為基礎之瓜實蠅整合性防治:生命表研究為蟲害防治之學術理論基礎,依據瓜實蠅之室內生命表與田間生命表可較精確了解瓜實蠅之族群增長潛能。食物之質與量對壽命與繁殖率之研究,則可以了解田間期望壽命之變異性。生命表研究配合分散速率、誘引劑效率之研究預期能了解瓜實蠅發生猖獗與不易防治之原因,並能以科學基礎擬定經濟有效之防治策略。 6. 台灣梨園木蝨遺傳變異及分子鑑定:本研究從昆蟲系統分類學及遺傳結構為著眼點,釐清台灣地區梨
1. 應用基因轉殖防治東方果實蠅之技術開發: In this study, based on the procedures for embryonic microinjection set up previously, we have successfully transfected the oriental fruit fly, Bactrocera dorsalis (Hendel), with atransposable element (piggyBac); and we further selected and established a stable line of transgenic B. dorsalis which are able to display red fluorescence (DsRed). These results demonstrate that the transgenesis of the oriental fruit fly is successful and we are able to work on insect transgenesis in our laboratory. The objectives of this year research are mainly focused on: first, using in vitro transfection assay or in vivo transformation assay to analyze different constructs, which contain various regions of the 3-kb frame of the upstream regulatory and promoter regions of the yolk protein gene, to identify the minimal requirement for the regulation of specific expression of an exogenous gene; second, to acquire the cDNA of an appropriate lethal gene to construct recombinant plasmid for transformation of B. dorsalis embryos. By the end of this year project, we expect to start to work on the transformation of B. dorsalis embryos with a regulated exogenous gene and establish the methods to distinguish the transgenic and nontransgenic insects. We will continuously investigate and develop the pest control strategy based on our current results. 2. 瓜類作物重要疫病之防治: a. The phenomenon of root rot/vine decline of muskmelon due to the infection of (Monosporascus cannonballus) is known in many regions around the world. This disease can be very severe, capable of destroying the entire crop in many fields. To date, disease management in the world has been mainly based on methyl bromide fumigation of the soil prior to planting. Since methyl bromide use will be prohibited in the near future, there is an urgent need to develop alternative strategies for disease management. The land use is very intensive and continuous cropping of melon is common in Taiwan. The fungus may be widespread and persistent in soils that crop rotation is of limited value. There is no suitable fungicide to be used to control this disease in fields either, although fluaznam was the most effective one against the fungus among many fungicides tested in vitro. Therefore, grafting melon plants onto resistant rootstocks was an approach studied in Japan, Israel, Greece and some Asian and European countries. However, muskmelon is usually grafted onto the same species, but very rarely onto pumpkin and white gourd rootstocks, since fruit shape and the taste of plants grafted onto pumpkin, show a remarkable deterioration. The pathogen's ascospores are unable to geminate on agar media. The biology and ecology of this disease are still not clear so far. It is difficult to complete the Koch's Rules to do the identification work. Thus, in this project, we propose to study the host range and ecological behavior of the pathogen in soils, and to develop a suitable grafting method and suitable rootstocks for melon plants to control this disease in greenhouse and field. b. Fusarium wilt is one of the most prevalent and damaging disease of cucumber wherever cucumber are grown intensively. The pathogen, Fusarium oxusporum f. sp. Cucurmerinum, is a soil inhabitant. The fungus is so widespread and so persistent in soils that seedbed sterilization and crop rotation are of limited value. Resistant variety of cucumber to this fungus is not available in Taiwan. Moreover, it is very difficult to control by chemicals either. Therefore, in the last several years biological control of fusarial wilt has given encouraging results. Control may involve prior inoculation of plants with formae specialis of F. oxysporum not pathogenic to each crop or the use of antagonistic Pseudomonads.
URI: http://hdl.handle.net/11455/54479
其他識別: 94農科-13.2.1-檢-B3
文章連結: http://grbsearch.stpi.narl.org.tw/GRB/result.jsp?id=1162930&plan_no=94%E8%BE%B2%E7%A7%91-13.2.1-%E6%AA%A2-B3&plan_year=94&projkey=PG9410-2446&target=plan&highStr=*&check=0&pnchDesc=%E9%96%8B%E7%99%BC%E7%93%9C%E6%9E%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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