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研究生:范敉晨
研究生(外文):Mi-Chen Fan
論文名稱:龍葵萃取物影響甘藍黑斑病菌發芽的效應與其抑制成分的鑑定
論文名稱(外文):Effect of black nightshade extracts on spore germination of Alternaria brassicicola and identification of its antifungal compounds
指導教授:黃振文黃振文引用關係
指導教授(外文):Jenn- Wen Huang
學位類別:碩士
校院名稱:國立中興大學
系所名稱:植物病理學系所
學門:農業科學學門
學類:植物保護學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:26
中文關鍵詞:龍葵甘藍黑斑病菌抑菌成份鑑定皂素植物源農藥degalactotigonin
外文關鍵詞:CabbageAlternaria brassicicolaSolanum nigrumsaponinsbotanical pesticidecompound identificationdegalactotigonin
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利用甘藍黑斑病菌(Alternaria brassicicola Schweinitz)檢測龍葵(Solanum nigrum L.) 70% 乙醇萃取物的抑菌活性,結果發現龍葵萃取物於500 ppm濃度下,可使處理過的孢子有33-42% 不發芽,且引起 100% 發芽孢子出現發芽管膨大的現象。此外,也以甘藍切離葉評估龍葵粗萃取物抑制黑斑病菌孢子侵入甘藍的效應,發現萃取物可以影響發芽孢子之分泌物對葉片的傷害;此外100 ppm 萃取物處理過的黑斑病菌孢子,其發芽管會出現膨大現象外,且會喪失感染甘藍葉片的能力。進一步,將乙醇萃取物分別以正己烷(n-hexane)、乙酸乙酯(ethyl acetate)、正丁醇(n-butanol)進行液相-液相萃取,獲得各溶劑萃取的濃縮成分,經抑菌試驗分析,證實乙酸乙酯與正丁醇萃取的物質具有抑菌的效果。隨後將正丁醇萃取的物質透過正相管柱層析(column chromatography, CC)進行初步分離後,再利用逆相管柱層析,分別以不同比例水與甲醇混合作為沖提劑,依極性不同逐次流洗,總共獲得18個分離部(Bu-11-01~Bu-11-18);其中Bu-11-13分離物以離心法得到1個白色粉狀化合物,其活性測試之最小抑制濃度為8 ppm。隨後利用核磁共振(1H NMR)及液相層析串聯式質譜儀 (LC/MS/MS) 分析,證明此化合物歸屬於皂素degalactotigonin。
Alternaria brassicicola isolates ABA-31 and ABB-16, the causal agents of black leaf spot of cabbage (Brassica oleracea L. Capitata Group) were used to evaluate antifungal activity of extracts from black nightshade (Solanum nigrum). The whole plant of black nightshade was air dried then grounded into powder; following subsequently extracted with 70% ethanol (EtOH) to obtain EtOH extract. The EtOH extracts presented a significant inhibitory activity to against 33-42% of spore germination of A. brassicicola ABA-31 and ABB-16, and make completely their germinated spores swollen at a concentration of 500 ppm. In addition, detached cabbage leaf was also used to analyze the efficacy of the extracts from S. nigrum on spore germination of the pathogen. The extracts were able to interrupt A. brassicicola ABA-31 and ABB-16 to release secretory substances in their spore germination fluids. Especially, it was found that germinated swelling spores of A. brassicicola ABA-31 and ABB-16 induced by 100 ppm of the extracts did not cause any symptoms on cabbage leaf surface. The EtOH extract was concentrated under reduced pressure and further divided into n-hexane, ethyl acetate and n-butanol soluble fractions by using liquid-liquid partition. All fractions and water residue were concentrated and evaluated their antifungal activities. Of the four extracts, only the n-butanol fraction exhibited potential antifungal activity by the suppression of conidial germination of A. brassicicola ABA-31 and ABB-16. To identify the active components, the n-butanol fraction was concentrated and subjected to column chromatography with silica gel using chloroform and methanol as eluents with decreasing polarity, and activity fraction then subjected to column chromatography with C18 gel using water and methanol as eluents with increasing polarity. Eighteen fractions were obtained(Bu-11-01~Bu-11-18)and they were freeze-dried and then tested individually for antifungal activities. Fraction Bu-11-13 was found to completely inhibit conidial germination of both isolates of A. brassicicola. A white powder was collected from fraction Bu-11-13 using centrifugal method and its minimum inhibitory concentration (MIC) was at a concentration of 8 ppm. Nuclear magnetic resonance (NMR) and liquid chromatography with tandem mass spectrometry detection (LC-MS/MS) analysis of the white powder compound was identified as degalactotigonin.
摘要..............................................i
Abstract..........................................ii
目錄..............................................iv
表目錄........................................... vi
圖目錄.......................................... vii
壹、前言..........................................1
貳、材料與方法....................................4
一、菌種來源及保存................................4
二、供試植物來源..................................4
三、孢子懸浮液之製備..............................4
四、萃取物抑制孢子發芽試驗........................5
五、甘藍切離葉保濕培養方法........................5
六、甘藍黑斑病菌之孢子發芽液 (Spore germination fluid, SGF) 的製作.................................................5
七、龍葵成份之萃取.....................................5
(一) 不同產地龍葵之最適萃取時間測試....................5
(二) 不同產地龍葵之最佳萃取溶劑比例評估..................6
(三) 龍葵萃取物回收量之統計分析..........................6
八、龍葵粗萃取液最小抑制濃度之測試.......................6
九、龍葵粗萃取液對病原菌侵入甘藍葉片之影響...........7
十、龍葵粗萃取液抑制孢子侵入甘藍葉片之效應...........7
(一) 龍葵萃取物抑制或毒殺甘藍黑斑病孢子之測試............7
(二) 龍葵萃取物對孢子發芽液與發芽膨大之孢子的影響........8
(a)傷口處理之效應......................................8
(b)龍葵粗萃取液對孢子發芽液之影響......................8
(c)龍葵粗萃取液對發芽管膨大之孢子侵入甘藍葉片的影響....8
十一、龍葵萃取物有效成分之鑑定...........................9
(一) 龍葵粗萃取液之製備..................................9
(二) 龍葵粗萃取液之液相-液相萃取分割....................9
(三) 萃取物之分離.......................................10
(四) 核磁共振光譜分析...................................11
(五) 液相層析串聯式質譜儀分析 (LC/MS/MS)..............................................11
叁、結果................................................12
ㄧ、最適萃取條件建立....................................12
(一) 不同產地龍葵之最適萃取時間測試.....................12
(二) 不同產地龍葵之最佳萃取溶劑比例評估.................12
(三) 龍葵萃取物回收量之統計分析......................................................13
二、葵粗萃取液最小抑制濃度之測試......................................................13
三、龍葵粗萃液對病原菌入侵甘藍葉片之影響......................................................13
四、龍葵粗萃取液抑制孢子侵入甘藍葉片之效應......................................................13
(一) 龍葵萃取液抑制或毒殺甘藍黑斑病孢子之測試...........13
(二) 龍葵萃取物對孢子發芽液與發芽膨大之孢子的影響.......14
(a)傷口處理之效應.....................................14
(b)龍葵粗萃取液對孢子發芽液之影響.....................14
(c)龍葵粗萃取液對發芽管膨大之孢子侵入甘藍葉片的影響...14
五、龍葵萃取物有效成分之鑑定............................15
(一) 龍葵萃取物製備.....................................15
(二) 龍葵粗萃取液之液相-液相萃取分割...................15
(三) 活性成分分離.......................................15
(四) 1H NMR分析.........................................16
(五) 液相層析串聯式質譜儀分析 (LC/MS/MS)................16
肆、討論................................................17
伍、引用文獻............................................22
陸、圖表................................................27
柒、附錄................................................50
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