臺灣博碩士論文加值系統

木黴菌是重要生物製劑之一。近年來的溫室效應與土壤鹽鹼化會不利於木黴菌 ( Trichoderma spp.) 生長。我們以 45 ℃熱休克處理 6 小時或 2 ％ NaCl (約0.3 M)作為篩選條件。與正常生長條件的對照組菌絲直徑(~ 4.5cm ) 之相比 ( 生長率 )，當生長率大於60% 則屬於較耐熱或耐鹽之菌株。由300支木黴菌株篩選，其中 4 株兼具最佳耐鹽及最佳耐熱性的菌株，包括: ETS311、NTCg-Te S4-1、AW-MLR321、AW-MLR325。以 ITS 區域的序列和型態分類，鑑定結果顯示菌株 ETS311 為T.afroharzianum，其它皆為T.asperellum。在 0.5M NaC l(~3％) 環境下，菌株 NTCg-Te S4-1耐鹽性最佳，生長率為 67 ％，甚至能在 1 M NaCl ( ~6 % )的環境下生長。耐熱方面，以菌株NTCg-Te S4-1 最優秀，可在 36 ℃的環境下生長。對於 52 ℃ 6 小時的熱休則是ETS311具有最高的耐受性，熱休克後生長率 68 ％。冷熱交替對 AW-MLR325 的影響最小，經過 45 ℃與4 ℃的冷熱循環 2 次後，生長率約 67 ％。4 菌株對於不同的植物病原菌具不同程度的拮抗能力。其中以菌株AW-MLR325的抗菌能力最為優秀，可抑制 78 ％白娟菌的生長、56 ％立枯絲核菌的生長，在含有 2 ％ NaCl 之培養基上也有抑制菌絲生長的能力，仍可抑制 37 ％白娟菌絲的生長、25 ％立枯絲核菌的生長。進一步探討這些菌株對於小白菜耐鹽的影響。在培養土中添加 2 ％ NaCl做為實驗組別，與不添加 NaCl 之組別相比。結果顯示菌株 AW-MLR321 與 NTCg-Te S4-1皆對植株之鮮重與乾重有正面的影響。其中以 AW-MLR321 的影響最為顯著，鮮重增加 82％與乾重增加 28 ％。此外，菌株 AW-MLR325 與 NTCg-Te S4-1 可在鹽環境下幫助番茄種子發芽。相對於對照組，添加菌株 AW-MLR325 之番茄種子在 100 mM NaCl 環境下的發芽率從 40 ％提升至 74 ％，添加菌株 NTCg-Te S4-1 之番茄種子發芽率從 40 ％提升至 72 ％。研究結果顯示所篩選到的菌株具有應用的潛力。

Trichoderma spp., a saprophytic fungus, is commonly present in the bark, deciduous, decayed wood, tillage soil and animal excrement. Some strains with biological control function can be developed as a biological agent. In recent years, the growth of Trichoderma spp. in some regions is impeded due to the greenhouse effect and soil salinization. The heat-resistant or salt-tolerant strains were screened based on the growth ratio of the mycelial diameter,45 ℃ heat shock treatment or 2% NaCl to the control group(about 4.5cm). It was considered as heat or salt tolerance strains when the growth ratio was higher than 0.6. Among 300Trichoderma isolates, ~21% and 38% of isolates exhibited heat and salt tolerant ability, respectively. Four isolates with the best performance in both of salt tolerance and heat resistance were selected, including ETS311, NTCg-Te S4-1, AW-MLR321 and AW-MLR325. The growth of Trichoderma spp.is usually delayed as the temperature higher than 30oC. Among the tested strains, NTCg-Te S4-1 can grow continuously at 36℃. The growth of ETS311 mycelia was the least affected by heat shock at 52 ℃.AW-MLR321 continuously grew at 36℃ as well. The growth ratio of mycelial diameter of selected isolates,0.5M NaCl to the control, was higher than 50%.These strains have differently antagonism ability against Sclerotium rolfsii, Rhizoctonia solani, Alternaria brassicae and Colletotrichum higginsianum. The growth of anthraxwas inhibited the most efficiently by AW-MLR321. In addition, AW-MLR321 and AW-MLR325 have the best growth rate and sporulation capability. AW-MLR321 and AW-MLR325 and NTCg-Te S4-1 was identified as T. asperellum based on the sequence analyses of ITS region in genbank, as well as theirphenotypes.ETS311 wasidentified as T.afroharzianum .In the plant test, AW-MLR321 and NTCg-Te S4-1 had impact on Chinese cabbagein the saline. AW-MLR325 could help tomato seeds germination under saline condition. The results suggested that the selected strains couldbe applied to benefit plant growth under bio- or abio-stresspotentially.

摘要......i
Abstract......ii
誌謝......iv
目錄......v
表目錄......viii
圖目錄......ix
第一章前言......1
第二章文獻回顧......2
2.1木黴菌......2
2.1.1木黴菌起源及分類......2
2.1.2木黴菌應用......2
2.1.3木黴菌用於生物防治的作用方式......3
2.2非生物逆境......4
2.2.1溫度......4
2.2.2鹽分......5
2.3專利檢索......7
第三章材料與方法......11
3.1實驗儀器及藥品......11
3.1.1實驗藥品及材料......11
3.1.2實驗儀器設備......11
3.1.3菌種來源......11
3.2菌種培養及活化......11
3.3篩選耐熱與耐鹽菌株......12
3.4鹽濃度對菌株的影響......12
3.5溫度對菌株生長的影響......12
3.5.1菌絲耐熱測試......12
3.5.2熱休克時間對菌絲生長影響......12
3.5.3菌絲耐高溫測試......13
3.5.4溫度變化對菌絲的影響......13
3.6菌株產孢能力測試......13
3.7菌種功能性試驗......13
3.7.1可溶性澱粉分解功能試驗......13
3.7.2溶磷功能性測試......14
3.7.3幾丁質分解酵素定性測試......14
3.7.4纖維素分解測試......14
3.8木黴菌鑑定方法......15
3.8.1型態鑑定......15
3.8.2分子鑑定......15
3.9病原菌對峙實驗......17
3.10熱與鹽對菌株分泌蛋白的影響......17
3.11木黴菌對植物的影響......18
3.11.1木黴菌對小白菜生長的影響......18
3.11.2木黴菌對於番茄種子發芽的影響......18
3.12耐熱菌株的幾丁質酶熱穩定度測試......18
3.12.1誘導菌株產生胞外幾丁質酶......18
3.9.2胞外幾丁質酶之耐熱測試......19
第四章結果......20
4.1耐熱耐鹽木黴菌之篩選......20
4.2.鹽濃度對菌絲生長......20
4.3溫度對菌株影響......21
4.3.1 菌株是否能在高溫生長......21
4.3.2高溫對菌絲生長影響......21
4.3.3熱休克時間對於菌絲生長的影響......21
4.3.4冷熱交替對於菌絲生長影響......22
4.4熱與鹽對菌株產孢能力的影響......22
4.5菌種功能性試驗......23
4.6病原菌對峙實驗......23
4.7.菌種鑑定......23
4.8熱與鹽對菌株分泌蛋白的影響......24
4.9木黴菌對植物的影響......24
4.9.1木黴菌對小白菜生長的影響......24
4.9.2木黴菌是否能幫助番茄種子發芽......25
4.10耐熱菌株產生胞外幾丁質酶之耐熱測試......25
第五章討論......26
第六章結論......29
參考文獻......50
附錄一rDNA ITS1-5.8S-ITS2區域之序列......56
附錄二、1.菌株ETS311於不同鹽濃度之生長......60
附錄二、2.菌株ETS311於不同溫度之生長......61
附錄三、1.菌株NTcg-Te-S4-1於不同鹽濃度之生長......62
附錄三、2.菌株NTcg-Te-S4-1於不同溫度之生長......63
附錄四、1.菌株AW-ML R321於不同鹽濃度之生長......64
附錄四、2.菌株AW-ML R321於不同溫度之生長......65
附錄五、1.菌株AW-ML R325於不同鹽濃度之生長......66
附錄五、2.菌株AW-ML R325於不同溫度之生長......67
附錄六鹽環境下立枯絲核菌與木黴菌之平板對峙......68
附錄七鹽環境下白娟菌與木黴菌之平板對峙......69
附錄八黑斑菌與木黴菌之平板對峙......70
附錄九炭疽菌與木黴菌之平板對峙......71
附錄十木黴菌對於番茄種子發芽的影響......72
Extended Abstract......73

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