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研究生:王佑文
研究生(外文):Yu Wen Wang
論文名稱:拮抗性木黴菌Trichodermavirens抗生物質特性之研究
論文名稱(外文):Characterization of antagonistic substance from Trichoderma virens
指導教授:蔡竹固蔡竹固引用關係陳瑞祥陳瑞祥引用關係
指導教授(外文):Jwu-Guh TsayRuey-Shyang Chen
學位類別:碩士
校院名稱:國立嘉義大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2004
畢業學年度:90
語文別:中文
論文頁數:58
中文關鍵詞:木黴菌
外文關鍵詞:Trichoderma virens
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木黴菌 (Trichoderma spp.) 為存在於土壤中之腐生性真菌,對於許多植物病原真菌具防治效果。本研究所用之菌株Trichoderma virens FT-010和FT-023經玻璃紙抗生法測試發現其對 Botrytis spp.、Phytophthora parasitica、Sclerotium rolfsii 等植物病原真菌具有明顯的拮抗效果。進一步測試發現木黴菌FT-010及FT-023菌株具有產生幾丁質分解酵素、澱粉水解酵素、脂質分解酵素、蛋白分解酵素、果膠分解酵素及磷脂分解酵素等之能力,但無法測得纖維素分解酵素與尿素分解酵素之活性。經液體培養所得之培養濾液,利用抗生測試得知木黴菌FT-023於25- 30 ℃,pH 3-8時對Botrytis elliptica FB-037具有較明顯的抗生作用;而培養於PDB與YM等培養基中所得之培養濾液則具有較高的抗生活性。培養基氮素源 (NH4)2SO4 與 NH4NO3 的添加則對木黴菌FT-023分泌抗生物質有促進之效果,而碳素源的天家則對抗生物質的產生無明顯之影響。經酵素活性分析,發現具抗生活性之木黴菌FT-023與不具抗生活性之菌株FT-004其幾丁質分解酵素與 b-1,3-glucanase活性並無差異。將具抗生活性之木黴菌FT-023 菌株之培養濾液以等體積之氯仿萃取抗生素,經薄層色層分析(TLC)測試發現,木黴菌FT-023的培養濾液與gliotoxin具有相似結果,且經抗生測試確定此物質具有抗生活性。因此初步認為木黴菌FT-023的主要抗生物質可能是gliotoxin。進一步測試發現木黴菌FT-023菌株於溫度25-30 ℃、pH 4 —6的培養條件下,可以獲得產量較高的抗生物質。
Trichoderma spp. are well known biocontrol agents from soil, and have been used to control various phytopathogens. In this study, Trichoderma virens FT-010 and FT-023 were found to inhibit mycelial growth of Botrytis spp., Phytophthora parasitica, and Sclerotiun rolfsii. The enzyme activities of chitinase, amylase, lipase, protease, pectate lyase, and phospholipidic enzyme could be detected by solid medium enzyme test, but no cellulase and urease activity were detected. Using the culture filtrate of T. virens FT-023, the antagonistic activity on the mycelial growth of Botrytis elliptica FB-037 were higher when T. virens FT-023 incubated at 25-30 ℃, pH 3-8, and in the medium of PDB or YM. The addition of (NH4)2SO4 and NH4NO3 in Czapek-dox broth could improve the antagonistic activity of T. virens FT-023, but the carbon sources of medium were not shown. The chitinase and b-1,3-glucanase activity of antagonist T. virens FT-023 and non-antagonist Trichoderma sp. FT-004 was not significantly different. The antibiotic was extracted from T. virens FT-023 culture filtrate by chloroform, and was separated on TLC plate. The results showed that the antagonistic substance was similar to gliotoxin, and this compound had well mycelia growth inhibition of B. elliptica FB-037. Therefore the antibiotic substance production from T. virens FT-023 could be gliotoxin, and the activity were highest as incubated in the medium of pH 4-6 at 25-30℃.
目錄
中文摘要----------------------------------------------------------------------------------.1
英文摘要-----------------------------------------------------------------------------------2
前言-----------------------------------------------------------------------------------------3
材料與方法------------------------------------------------------------------------------10
一、供試菌株---------------------------------------------------------------------10
二、玻璃紙抗生測試------------------------------------------------------------10
三、細胞外水解酵素之測定---------------------------------------------------10
四、木黴菌培養濾液(culture filtrate)之製備與抗生測試-------------14
五、溫度對木黴菌FT-023培養濾液抗生活性之影響--------------------14
六、酸鹼值對木黴菌FT-023培養濾液抗生活性之影響-----------------15
七、培養基對木黴菌FT-023培養濾液抗生活性之影響-----------------15
八、碳素源對木黴菌FT-023培養濾液抗生活性之影響-----------------16
九、氮素源對木黴菌FT-023培養濾液抗生活性之影響-----------------16
十、培養濾液之濃縮與酵素濃度測定---------------------------------------16
十一、培養濾液濃縮液抗生活性之測試------------------------------------18
十二、抗生物質之萃取與薄層色層分析------------------------------------18
十三、抗生物質種類之鑑別---------------------------------------------------19
十四、氯仿抽出物之抗生活性測試------------------------------------------19
十五、培養溫度對木黴菌FT-023抗生物質生成之影響-----------------19
十六、酸鹼值對木黴菌FT-023抗生物質生成之影響--------------------20
十七、培養時間對木黴菌FT-023抗生物質生成之影響-----------------20
十八、熱處理對木黴菌FT-023抗生物質活性之影響--------------------20
十九、玻璃紙抗生測試平板中抗生物質之測定---------------------------20
二十、氯仿抽出物之玻璃紙抗生測試---------------------------------------21
結果---------------------------------------------------------------------------------------22
一、玻璃紙抗生測試------------------------------------------------------------22
二、細胞外水解酵素之測定---------------------------------------------------22
三、溫度對木黴菌FT-023抗生活性之影響--------------------------------22
四、酸鹼值對木黴菌FT-023抗生活性之影響-----------------------------25
五、培養基對木黴菌FT-023抗生活性之影響-----------------------------25
六、碳素源對木黴菌FT-023抗生活性之影響-----------------------------29
七、氮素源對木黴菌FT-023抗生活性之影響-----------------------------29
八、酵素活性之測定及其抗生活性------------------------------------------29
九、利用薄層色層分析(TLC)鑑別木黴菌抗生物質種類-------------33
十、氯仿抽出物之抗生活性測試---------------------------------------------33
十一、培養條件對木黴菌FT-023抗生物質生成之影響-----------------36
十二、熱處理對木黴菌FT-023培養濾液中抗生物質活性之影響-----36
十三、玻璃紙抗生測試平板中木黴菌抗生物質之含量------------------36
十四、氯仿抽出物之玻璃紙抗生測試---------------------------------------41
討論---------------------------------------------------------------------------------------44
參考文獻---------------------------------------------------------------------------------48
圖目錄
圖一、培養溫度對木黴菌FT-023拮抗Botrytis elliptica FB-037之影響---26
圖二、培養基酸鹼值對木黴菌FT-023拮抗Botrytis elliptica FB-037生長之影響------------------------------------------------------------------------------27
圖三、培養基對木黴菌FT-023拮抗Botrytis elliptica FB-037之影響------28
圖四、碳素源對木黴菌FT-023拮抗Botrytis elliptica FB-037之影響------30
圖五、氮素源對木黴菌FT-023拮抗Botrytis elliptica FB-037之影響------31
圖六、木黴菌培養濾液之氯仿抽出物之薄層色層分析結果------------------34
圖七、木黴菌FT-010與FT-023氯仿抽出物對Botrytis elliptica FB-037菌絲生長之影響------------------------------------------------------------------35
圖八、 培養溫度對木黴菌FT-023抗生物質生成之影響---------------------37
圖九、培養基酸鹼值對木黴菌FT-023抗生物質生成之影響-----------------38
圖十、培養時間對木黴菌FT-023抗生物質生成之影響-----------------------39
圖十一、熱處理對木黴菌FT-023氯仿抽出物抗生活性之影響--------------40
圖十二、玻璃紙抗生測試平板中木黴菌抗生物質之含量---------------------42
表目錄
表一、利用玻璃紙抗生法測試不同木黴菌菌株對植物病原真菌生長之拮抗作用------------------------------------------------------------------------------23
表二、木黴菌菌株細胞外水解酵素種類之測定結果---------------------------24
表三、木黴菌菌株FT-010與FT-023幾丁質分解酵素及 b-1,3-葡聚糖分解酵素活性之比較及其拮抗Botrytis elliptica FB-037之效果----------32
表四、木黴菌FT-023氯仿抽出物對Botrytis elliptica FB-037之玻璃紙抗生測試------------------------------------------------------------------------------43
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