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研究生:李淑錚
研究生(外文):Shu-Cheng Li
論文名稱:梔子果實粗萃物促進木黴菌生長及產孢之研究
論文名稱(外文):The Effects of Gardenia Fruit Crude Extracts on Promoting Mycelial Growth and Sporulation of Trichoderma spp.
指導教授:羅朝村羅朝村引用關係
指導教授(外文):Chaur-Tsuen Lo
學位類別:碩士
校院名稱:國立虎尾科技大學
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:2015
畢業學年度:103
語文別:中文
論文頁數:147
中文關鍵詞:木黴菌梔子產孢
外文關鍵詞:Trichoderma spp.Gardenia jasminoidessporulation
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木黴菌可抑制病原菌也可促進植物生長,是一個被公認可發展成優良生物製劑的重要微生物之一。雖然木黴菌可生長在各種醣類物質,但隨著供應基質的不同,其產生的生物質量則會有明顯的差異。為有效增加木黴菌產量及縮短其培養時間,以利於開發生物製劑之生產成本,本試驗擬從20種中草藥粗萃物中,測試與篩選可促進木黴菌孢子發芽或生長之物質;結果顯示,其中一種由梔子(Gardenia jasminoides) 粗萃之物質可明顯促進木黴菌菌株編號T2 (Trichoderma harzianum)、T3 (T. virens) 及 T1-1 (T. harzianum)等三菌株孢子發芽之效果。相較於對照組,在1% glucose + 1% L-Asparagine培養液中培養10小時,添加1000 ppm梔子粗萃物之處理組對三株木黴菌,皆可使之發芽率高達90%以上 (如T1-1菌株,處理組孢子發芽率為90.33%,對照組為48.67%;T2菌株,處理組孢子發芽率為90.17%,對照組為50%;T3菌株,處理組孢子發芽率為93.67%,對照組為51.83%)。在未添加營養物質之無菌水中培養14小時,添加1000 ppm梔子粗萃物之處理組,三株木黴菌之發芽率亦皆高於對照組 (T1-1菌株,處理組孢子發芽率為73.5%,對照組為52.67%; T2菌株,處理組孢子發芽率為65.5%,對照組為48.33%;T3菌株,處理組孢子發芽率為79%,對照組為51.17%)。為能了解梔子粗萃物促進木黴菌生長之機制,首先假設其可能扮演碳或氮源角色。經分別與不同的碳源、氮源做比較測試,結果顯示Water agar (WA)含有1000 ppm梔子以及0.1% TritonX-100培養9天,菌絲生長長度皆高於WA含有碳源/氮源以及0.1% TritonX-100,測試之碳源為: 葡萄糖(Glucose) 、蔗糖(Sucrose) 、阿拉伯糖(Arabinose) 、乳糖(Lactose) 、麥芽糖(Maltose) 、甘露糖醇(Mannitol) 、木糖(Xylose) 、果糖(Fructose) ;氮源為: 硝酸納(Sodium nitrate) 、氯化銨(Ammonium chloride) 、硝酸鈣(Calcium nitrate) 、醋酸銨(Ammonium acetate) 、硝酸銨(Ammonium nitrate)。經與複合式營養比較,如Czpek''s agar 、 Potato dextrose agar (PDA) 、 Malt extract agar (MEA)等,結果顯示3株菌在含有1000 ppm梔子之WA處理組,菌絲長度皆高於Czpek、PDA及MEA下培養,顯示除具有複合式培養基之營養外,仍具有其他可能之角色。在縮短培養時間方面,則進行產孢量測試,即將農業廢棄物如稻稈、甘蔗渣分別浸泡於1000 ppm梔子粗萃物中1天,滅菌後,再接種木黴菌一毫升之孢子懸浮液 (濃度105 spores/ml),結果顯示梔子粗萃物與稻稈或蔗渣共培養與未添加之對照組相比較,各菌株分別可縮短2-4天不等。

Trichoderma spp. were known as good biological agents; they not only inhibit pathogens but also promote the growth of plants. Although Trichoderma can be grown in a variety of carbohydrates, with different supply matrix, the biological quality of the product will be significantly different. To effectively increase the biomass production of Trichoderma and shorten its incubation time for reducing the development and production costs of biological agents, this study intends to screen the crude extracts from 20 kinds of Chinese herbs for promoting the mycelial growth and spore germination of Trichoderma strains. The results show, gardenia (Gardenia jasminoides) crude extracts with 1000 to 2000 ppm can significantly promote spore germination of Trichoderma strains T2 (Trichoderma harzianum), T3 (T. virens) and T1-1 (T. harzianum) as compared to the control group cultured in 1% glucose + 1% L-Asparagine solution for 10 hours. The spore germination of T1-1 strain , T2 strain, and T3 strain treated with 1000 ppm of gardenia crude extract was 90.33%, 90.17% and 93.67%, respectively, and treated with control group was 48.67%, 50% and 51.83%, respectively. Similarily, cultured in sterile water for 14 hours, treatment with 1000 ppm gardenia crude extract had, was higher germination rate of three strains of Trichoderma above than that of untreated control groups. To understand the mechanism of gardenia crude extract for promoting the mycelial growth of Trichoderma spp. The hypothesis that it may play a role in carbon or nitrogen source, and complex medium was tested. The carbon sources, and nitrogen sources were compared with 1000 ppm gardenia for mycelial growth of different Trichoderma strains in water agar. The carbon sources included Glucose, Sucrose, Arabinose, Lactose, Maltose, Mannitol, Xylose, and Fructose. The nitrogen sources were Sodium nitrate, Ammonium chloride, Calcium nitrate, Ammonium acetate, and Ammonium nitrate. The complex media were Czpek''s agar, Potato dextrose agar (PDA), and Malt extract agar (MEA). The results showed that the mycelial growth of three strains of Trichoderma spp. in water agar containing 1000 ppm gardenia crude extract were growth faster than that of with different tested carbon source, and nitrogen source or complex medium alone. In shortening the incubation time tests, rice straw, sugarcane bagasse were soaked in 1000 ppm gardenia crude extract for 24 hours. The medium were sterilized, and then inoculated with 105 spores / ml of Trichoderma strain. The results indicated that sporulation of various Trichoderma strains in rice straw and sugarcane bagasse with 1000 ppm gardenia crude extract was faster than that of with untreated control. The incubation time at the same spore concentration per dry gram was shorten 2-4 days.

摘要............................................i
Abstract............................................iii
誌謝............................................v
目錄............................................vi
表目錄............................................xi
圖目錄............................................xii
第一章 前言............................................1
第二章 文獻回顧............................................3
2.1木黴菌介紹............................................3
2.1.1木黴菌分類............................................3
2.1.2木黴菌型態............................................5
2.1.3木黴菌生物防治功能............................................5
2.1.4木黴菌的商品化條件與量產............................................7
2.1.4.1木黴菌的商品化條件............................................7
2.1.4.2 木黴菌之培養與量產............................................ 8
2.2中草藥與微生物的關係............................................9
2.3梔子介紹............................................10
2.3.1梔子的成分............................................11
2.3.2梔子的應用............................................11
2.4農業廢棄物............................................12
2.4.1稻草廢棄物............................................14
2.4.2甘蔗廢棄物............................................14
2.5相關專利檢索............................................16
第三章 材料與方法............................................17
3.1實驗材料............................................17
3.1.1菌株來源及保存............................................17
3.1.2中草藥來源............................................17
3.2實驗方法............................................18
3.2.1木黴菌菌株活化及培養............................................18
3.2.2中草藥萃取物製備............................................18
3.2.3有益木黴菌發芽或生長之中藥草篩選............................................19
3.2.4梔子成分之萃取............................................19
3.2.5梔子粗萃物對木黴菌孢子發芽測試............................................20
3.2.6不同濃度梔子粗萃物對木黴菌菌株生長之測試............................................20
3.2.7添加梔子(1000 ppm)與各種不同濃度的碳源、氮源對木黴菌菌株生長之比較............................................21
3.2.8添加梔子(1000 ppm)與複合式營養對木黴菌生長之影響 與比較 ............................................22
3.2.9梔子粗萃物中可促進木黴菌發芽或生長之主要有效成分............................................22
3.2.10梔子粗萃物與稻稈或蔗渣共培養與未添加之對照組相比較進行產孢量測試............................................23
3.2.11木黴菌鑑定方法............................................24
第四章 結果............................................31
4.1促進木黴菌發芽或生長之中藥草篩選............................................31
4.2梔子粗萃物對木黴菌孢子發芽測試............................................32
4.3添加梔子(1000 ppm)與各種不同濃度的碳源對木黴菌生長 之比較 ............................................32
4.4添加梔子(1000 ppm)與各種不同濃度的氮源對木黴菌生長 之比較 ............................................34
4.5測試添加梔子(1000 ppm)與複合式營養對木黴菌生長之比較 ............................................35
4.6梔子粗萃物與梔子主要成分對木黴菌發芽或生長之比較............................................35
4.6.1梔子水萃物對孢子發芽測試............................................35
4.6.2梔子純物質對孢子發芽之影響測試............................................36
4.7梔子粗萃物與稻稈共培養與未添加之對照組相比較進行產孢量測試 ............................................37
4.8梔子粗萃物與蔗渣共培養與未添加之對照組相比較進行產孢量測試 ............................................38
4.9木黴菌T1-1之菌種鑑定............................................39
第五章 討論............................................40
參考文獻............................................116
附錄............................................129
Extended Abstract............................................136
簡歷(CV)............................................147



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