請用此 Handle URI 來引用此文件: http://hdl.handle.net/11455/95780
標題: 小麥赤黴病生物防治策略之研發
Development of Biocontrol Strategies for Fusarium Head Blight of Wheat
作者: 楊翰祁
Han-Chi Yang
關鍵字: 小麥赤黴病
生物防治
反應曲面法
新月毒素生合成
Fusarium head blight
biological control
response surface methodology
trichothecene biosynthesis
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摘要: 小麥赤黴病在世界各地的產區造成嚴重的危害,除了造成直接的經濟損失外,赤黴病菌另會產生新月毒素污染穀粒。許多台灣農民以友善環境的耕作方式生產小麥,但缺乏有效防治小麥赤黴病的方法,本研究欲開發生物防治策略,包含拮抗微生物之開發及建立可篩選降低新月毒素生合成之拮抗微生物的平台,期融入台灣小麥的生產體系。本研究拮抗微生物有二,一來自台灣農業試驗所提供之保存菌株,另分離小麥花藥之拮抗微生物,依菌落型態又可分為類芽孢桿菌型與黃色黏稠型,經過孢子發芽抑制測試後分別選擇黃色黏稠型菌株I-1-4,與類芽孢桿菌型菌株I-2-9進行後續試驗,分別以PDB及LB作為基礎培養基,進行工業生產原料的替換與篩選。為鑑定2菌株的種名,菌株I-1-4以多位點基因分析法鑑定為Pantoea sp.;菌株I-2-9則以專一性引子對鑑定為Bacillus amyloliquefaciens。經一系列之營養源篩選,菌株I-1-4以蔗糖、馬鈴薯萃取物與葵花油組成的配方有較佳的拮抗能力;菌株I-2-9以脫脂奶粉、酵母粉、蔗糖與氯化鈉組成之配方有較佳的拮抗效果。為瞭解合適的培養條件與成分配置,進行環境因子與培養基成分影響之分析,經過二因子水準分析發現,菌株I-1-4的配方中,酵母萃取物與馬鈴薯萃取物顯著影響拮抗能力;菌株I-2-9的配方則以綠豆粉顯著影響拮抗能力。以二因子水準分析的配方進行溫室試驗,結果顯示在接種病原菌20天後,施用菌株I-2-9的10倍稀釋培養液,可降低58%的罹病度以及40%的罹病率,而施用菌株I-1-4的25倍稀釋培養液則在接種病原菌20天後,可降低67%的罹病度以及73%的罹病率,顯示其具有開發為生物製劑之潛力,因此進一步以最陡上升分析及中央合成設計分析優化上述顯著影響成分,中央合成設計分析獲得之迴歸方程式顯示,菌株I-1-4稀釋5倍之濾液可達到83.3%孢子發芽抑制率;菌株I-2-9以20倍稀釋的培養液可達到77.95%的孢子發芽抑制率。在建立偵測新月毒素生合成的平台方面,利用新月毒素生合成路徑第一個酵素TRI5的啟動子與GFP基因結合後之載體,進行PEG媒介基因轉殖後,產生9個轉殖株,南方墨點分析證實上述載體均成功插入轉殖株的TRI5基因座,並於誘導TRI5表現之培養基中,可見到明顯的GFP表現,於即時聚合酶鏈鎖反應顯示,野生型r-p-1的TRI5基因表現與轉殖株TPG-1及TPG-6的GFP表現,分別於新月毒素誘導及非誘導培養基中,有相同趨勢的表現情形,期未來可以轉殖株作為新月毒素生合成的偵測平台,篩選能夠降低新月毒素產生之拮抗微生物。
Fusarium head blight (FHB) is a devastating disease worldwide. Besides the direct economic loss, the pathogens produce trichothecene that contaminates kernels. The environmentally-friendly cultural practice is used to produce wheat in Taiwan, which lacks of an effective method in the control of FHB. This study aimed to develop a biocontrol agent and establish a platform to screen antagonists which can reduce trichothecene biosynthesis. The sources of antagonistic strains were the collection of Taiwan Agricultural Research Institute and the isolates from wheat anther. The antagonists were divided into two colony types, Bacillus-like colony and yellow mucoid colony. The strain I-2-9 with Bacillus-like colony and strain I-1-4 with yellow mucoid colony highly inhibited the spore germination of FHB pathogens in LB and PDB, respectively. Strain I-1-4 was identified as Pantoea sp. by multilocus sequence analysis while strain I-2-9 was identified as Bacillus amyloliquefaciens by species-specific primers. The strains and media were used as the basis for nutrient screening. The formula of strain I-1-4 composed of sucrose, potato extract and sunflower oil displayed a high antagonistic ability. The formula of strain I-2-9 composed of skim milk, yeast powder, sucrose and NaCl had a better antagonistic ability. For optimizing the formula, the environmental conditions and significance of ingredients were evaluated. The two-level factorial designed assay showed that yeast extract and potato extract in I-1-4 medium, and mung bean in I-2-9 medium significantly influenced antagonistic ability. A greenhouse test was carried out with the best medium composition in the two-level factorial design assay. The 10-fold dilution of I-2-9 reduced the disease severity by 58% and disease incidence by 40%. The 25-fold dilution of I-1-4 reduced the disease severity by 67% and disease incidence by 73%. The two strains showed the potential to develop as biocontrol agents. Moreover, the steepest ascent assay and central composited design were used to optimize the formula. Accroding to the regression equation obtained from central composite design. The 5-fold dilution filtrate of the strain I-1-4 could reach 83.3% spore inhibition rate. The strain I-2-9 could reach 77.95% spore inhibition rate with 20-fold dilution culture. On the other hand, to select antagonists that can suppress trichothecene biosynthesis, 9 fungal transformants were gernerated by PEG-mediated transformation, which contained the TRI5 promoter::GFP construct and significantly expressed GFP in a TRI5-inducing condition. The real-time PCR showed that TRI5 of wild type r-p-1 had a corresponding effect on GFP expression of transformants TPG-1 and TPG-6. The transformants can be used as a detection platform for screening antagonists which reduce the biosynthesis of trichothecene.
URI: http://hdl.handle.net/11455/95780
文章公開時間: 2020-08-23
顯示於類別:植物病理學系

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