臺灣博碩士論文加值系統

Anthraquinones存在於許多藥用植物及微生物所分泌之代謝物中，其功能包括通便、抗細菌、抗真菌、抗病毒及抗癌等作用。本研究從Trichoderma harzianum菌株ETS 323純化出兩種anthraquinones的衍生物chrysophanol與pachybasin，兩者為已知的化合物，但第一次由T.harzianum菌株ETS 323純化出，希望能了解此兩化合物及T.harzianum菌株ETS 323在作物病害生物防治所扮演的角色。將 T.harzianum菌株ETS 323在PDA上培養 6天後，以無菌水沖洗，以獲得孢子懸浮液 (106 spores/mL)，並將此孢子懸浮液接種於甘蔗渣中培養8天，再以乙酸乙酯 (EA)萃取後，經矽膠薄層色層分析 (silica gel TLC)得到分離條件，再以矽膠管柱層析 (silica gel column chromatography)純化，分別得到黃色粉末與橘黃色粉末，其結構經X光單晶繞射 (x-ray diffraction)確定橘黃色粉末為chrysophanol；以核磁共振 (NMR)知道黃色粉末為pachybasin。此兩化合物分別用以測試是否可影響植物種子萌芽率與側根數目，另外亦檢測對T. harziznum菌株ETS 323、T.virens與植物病原菌 (A.brassicicola, P.aphanidermatum, R.solani, P.capsici及E.carotovora)之生長及孢子萌芽之影響。在十種作物測試之結果經Ducan’s test分析顯示，以chrysophanol處理後之辣椒、甜椒與茄子之種子萌芽率相較於對照組有顯著性增加；至於芥藍、油菜與蕃茄的側根數亦有相對性顯著增加；而pachybasin則僅可增加油菜與蕃茄的側根數。在植物病原菌之生長及孢子萌芽實驗上，chrysophanol與pachybasin僅會抑制R.solani菌絲生長速度。總結來說，本研究可謂首次記載可從T.harziznum 菌株ETS 323中純化出pachybasin與chrysophanol兩物質，並發現可改善植物種子萌芽率、增加部份作物之側根數目及可抑制R.solani菌絲生長速度。往後應可更廣泛測試其他植物種子及土棲性病原菌之影響，以冀望能進一步了解其在生物防治的相關角色。

Anthraquinones existed in many herb plants and metabolites of microorganisms. Their biological activities include laxative, antibacterial, antifungal, antiviral and anticancer. In this study, two kinds of anthraquinones derivatives, chrysophanol and pachybasin were isolated from Trichoderma harzianum strain ETS 323. Though both of them are known compounds, they were first isolated from T.harzianum strain ETS 323. We were hoping to understand the function of the two compounds on biological control of plant diseases. The spores of T.harzianum strain ETS 323 were collected for 6 days inculation on PDA and inoculated on sugarcane bagasse. After 8 days sugarcane bagasse was extracted with ethyl acetate. The extract was analyzed by thin layer chromatography to test the appropriate condition for silica gel column chromatography. There were two compounds, yellow material and orange material, obtained by silica gel column chromatography. The structure of orange material was identified as chrysophanol by the X-ray diffraction. The structure of yellow material was identified as pachybasin by NMR. Then the effects on germination and number of lateral root of plant seeds, number of spores germination and growth of T.harzianum strain ETS 323, T.virens and plant pathogenic fungi and bacteria. ( A.brassicicola, P.aphanidermatum, R. solani, P.capsici, E.carotovora) were tested. For plant seeds germination, the result showed that pepper, sweet pepper and eggplant and number of lateral root of broccoli, rape and tomato were promoted when treating with chrysophanol by using Ducan’s test. Pachybasin promoted number of lateral root of rape and tomato. In T.harzianum strain ETS 323, T.virens and pathogenic fungi and bacteria, chrysophanol and pachybasin inhibited mycelium germination of R.solani, but not others. In summary, the study first reported chrysophanol and pachybasin from T. harzianum strain ETS 323. They improved some plant seeds germination rate and number of lateral root. Also they inhibited mycelium germination of R. solani. In the future, other plant seeds and locally born phytopathogenic fungi and bacteria will be extensively and systemic and more knowledge of biological control.

誌謝............................................I
摘要.....................................................II
Abstract.................................................IV
目錄.....................................................VI
圖目錄...................................................XI
表目錄..................................................XVI
一.緒論...................................................1
1-1研究緣起 ..............................................1
1-2研究目的...............................................2
二.文獻回顧...............................................3
2-1真菌簡介...............................................3
2-1-1真菌分類.............................................4
2-2木黴菌簡介.............................................5
2-3病原菌簡介.............................................6
2-3-1 Erwinia carotovora..................................6
2-3-2 Alternaria brassicicola ............................7
2-3-3 Pythium aphanidermatum .............................8
2-3-4 Phytophthora capsici................................9
2-3-5 Rhizoctonia solani ................................10
2-4 Anthraquinones簡介...................................11
2-5薄層色層分析法 (Thin Layer Chromatography)簡介........14
2-5-1原理................................................14
2-5-2 Rf (Rentention factor).............................14
2-5-3步驟................................................15
2-6矽膠管柱層析法 (Silica gel column chromatography)簡介.15
2-6-1步驟................................................15
2-7變異數分析 (Analysis of variance) ....................16
三.材料與方法............................................17
3-1菌種、植物種子與甘蔗渣來源............................17
3-2化學藥品來源..........................................17
3-3菌種培養..............................................18
3-4甘蔗渣處理............................................18
3-5固態培養..............................................18
3-6純化方法..............................................19
3-6-1萃取................................................19
3-6-2薄層色層分析 (Thin Layer Chromatography)............19
3-6-3矽膠管柱層析 (Silica gel column chromatography) ....19
3-6-4 高效液相層析 (High Performance Liquid Chromatography) .........................................20
3-6-5吹氨氣之薄層色層分析................................20
3-6-6吹氨氣之矽膠管柱層析................................20
3-7結構鑑定 .............................................20
3-7-1氫譜核磁共振 (1H-NMR)...............................20
3-7-2氣相層析質譜儀 (GC-Mass Spectrometry)...............21
3-7-3 X光單晶繞射儀 (X-RAY/CCD) .........................21
3-7-4 1D與2D-NMR ........................................21
3-8紫外光-可見光譜測定...................................22
3-9 代謝物對T.harzianum菌株ETS 323孢子萌芽率影響之實驗...22
3-10代謝物對T.harzianum菌株ETS 323、T.virens及植物病原菌
生長影響之實驗...........................................23
3-10-1 T.harzianum菌株ETS 323, T.virens及R.solani培養方法.......................................................23
3-10-2 P.aphanidermatum, P.capsici及A.brassicicola
培養方法 ................................................24
3-10-3 E.carotovora培養方法..............................24
3-11代謝物對植物種子萌芽率及側根數影響之實驗.............25
四.結果..................................................27
4-1甘蔗渣的處理與固態培養結果............................27
4-2薄層色層分析結果......................................28
4-3高效液相層析分析結果..................................30
4-4吹氨氣之薄層色層分析..................................30
4-5氫譜核磁共振..........................................31
4-6氣相層析質譜..........................................32
4-7以X光單晶繞射儀鑑定橘黃色粉末之結構...................32
4-8以1D與2D-NMR鑑定黃色粉末之結構........................33
4-8-1 1H-NMR圖譜.........................................33
4-8-2 13C-NMR圖譜........................................34
4-8-3 DEPT圖譜...........................................34
4-8-4 1H,1H-COSY圖譜.....................................34
4-8-5 HMBC與HSQC圖譜.....................................35
4-9紫外光-可見光譜圖 ....................................35
4-10 T.harzianum菌株ETS 323孢子萌芽實驗結果..............36
4-11代謝物對T.harzianum菌株ETS 323, T.virens及病原菌
生長抑制影響..........................................38
4-11-1 對T.harzianum菌株ETS 323與T.virens之影響..........38
4-11-2 對E.carotovora之影響..............................39
4-11-3 對A.brassicicola, P.aphanidermatum與P.capsici之影
響.................................................40
4-11-4 對R.solani之影響..................................42
4-11-5 以控制組、NP-40、pachybasin及chrysophanol處理
之菌種每天平均生長速度...................................42
4-12代謝物對植物種子萌芽率及側根數影響之結果.............43
五.討論..................................................45
六.結論..................................................50
七.參考文獻..............................................52
八. 附錄.................................................56

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