臺灣博碩士論文加值系統

搜集台灣各地鏈格孢屬 (Genus Alternaria) 真菌共計 12 菌株，分別以液態培養 5-7 天後，取得它們的菌絲體，經由液態氮研磨，並以三胺基甲烷 (Tris) 緩衝液進行萃取，再以Bradford 蛋白質定量法，逐一調整濃度至 2 &;micro;g/ml BSA (bovine serum albumin) 對應的吸光值。 然後利用它們的萃取物分別噴佈於白菜幼苗，結果發現 APR01、ACO01 等菌株之萃取液，可減輕白菜幼苗立枯病的發生率，其中尤以 APR01 的萃取物的防治效果最佳達 33.33%。 催芽後的白菜種子，浸泡於各萃取物後，發現 ATA01、ABR03、ALY01、AOR06 及 APR01 等 5 株菌株的萃取物具有促進白菜幼苗胚根與側根發育的效果，其中 APR01 萃取物促進根部延長的效果最佳，可達36.56%。 依據 APR01 的產孢方式與孢子型態，並以其 ITS 序列佐證，筆者將 APR01 鑑定為 Alternaria tenuissima。 在溫室試驗中，將A. tenuissima APR01 的萃取物噴佈於白菜幼苗一次或三次後，結果處理組的白菜鮮重比對照未處理組者分別高 38.3% 或 48%。 此外，將立枯絲核菌的菌絲塊接種於白菜之葉片上，發現處理過 APR01 萃取物之葉片病斑擴展速度顯著低於對照組。 測試 APR01 菌株的總蛋白質萃取液對立枯絲核病菌菌絲生長的影響，發現 APR01 萃取物不具抑菌之功效。 進一步，分析處理過 APR01 萃取物之白菜植體的 PAL (phenylalanine ammonia lyase) 與 POD (peroxidase) 酵素的活性，結果發現處理 APR01 萃取物及接種過立枯絲核菌的白菜，其 PAL 及 POD 的活性顯著提升，顯然 APR01 萃取物中，存在著誘導白菜抗病的組成因子。 分析 APR01 萃取物中活性成分之特性，發現 APR01 的萃取物經 SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) 後，於電泳膠片上呈現條帶；若將其以熱處理至 50 ℃ 時，即失去防病功效，顯示 APR01 萃取物中的活性成分，屬於一種蛋白質。 利用超過濾 (ultra-filtrated) 的方式，分離 APR01 的萃取物，發現分子量在 10 KDa 以下的組成，不具有防病功效。

Twelve isolates of Alternaria spp. were isolated from various crops in Taiwan. Those fungi were respectively grown in liquid culture media for 5-7 days and harvested their mycelia. The mycelial samples were pulverized in liquid nitrogen, and then homogenized with precooled Tris-buffer, estimated the quantity of their total protein by the Bradford method, bovine serum albumin (BSA) solution was used as a standard, the absorbance value equal to 2 µg BSA/ml. Spraying the mycelial extracts obtained from Alternaria spp. on Chinese cabbage seedlings inoculated with Rhizoctonia solani AG-4, APR01-TP and ACO01-TP, were able to reduce 33.33% disease incidence of Rhizoctonia damping-off of Chinese cabbage seedlings. Dipping germinated Chinese cabbage seeds with each of mycelial extracts obtained from 12 isolates of Alternaria spp., ATA01-TP, ABR03-TP, ALY01-TP, AOR01-TP and APR01-TP was respctively able to promote the extension of Chinese cabbage roots as well as to enhance development of their lateral roots. Especially, the mycelial extract of APR01 was more effective in stimulating the growth of plants at 36.56%. APR01 was identified as Alternaria tenuissima according to its conidial morphology, conidigenous cells, and ITS sequencing. In greenhouse tests, spraying the mycelial extract from A. tenuissima APR01 once or thrice on Chinese cabbage seedlings grown on soil infested with R. solani AG-4 RST-04, the fresh weight of Chinese cabbage seedlings were heavier up to 38.3% and 48% compared to untreated control. Inoculating mycelial discs of R. solani AG-4 RST-04 on Chinese cabbage leaves after sparying mycelial extract of A. tenuissima APR01, the expansion of the lesion size were slower compared to the control. It was proved that the mycelial extract of A. tenuissima APR01 could not inhibit mycelial growth of R. solani AG-4 RST-04. To estimate the activity of phenylalanine ammonia lyase (PAL) and peroxidase (POD) in Chinese cabbage seedlings after spraying the mycelial extract of A. tenuissima APR01 and inoculating R. solani AG-4 RST-04, the enzyme activity of the PAL and POD in treated plants were higher than the control. These results suggested that there were some elements existed in mycelial extract of isolate APR01 were able to induce Chinese cabbage seedlings resistant against the pathogen. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed for mycelial extract of A. tenuissima APR01 and the bands were displayed on polyacrylamide gel stained with CBR. Mycelial extract of A. tenuissima APR01 was heated at 50 ℃ water bath and lost its biological activity. The effective component of the mycelial extract of A. tenuissima APR01 was not ultra-filtrated in the membrane tubing with molecular weight cut-off of 10,000. Therefore, the major activity of mycelial extract of A. tenuissima APR01 is a protein with > 10 kilodalton of molecular weight.

摘要 i
ABSTRACT ii
表目錄 vii
圖目錄 viii
前言 1
材料與方法 5
一、 供試植物 5
二、 供試菌株來源 5
三、 真菌體內蛋白質的抽取 5
四、 鏈格孢屬真菌之蛋白質萃取液的十二烷基磺酸鈉聚丙烯醯胺膠體電泳分析 (SDS-PAGE) 7
五、 磷酸回溶緩衝液 (PBS lysis buffer) 對白菜生長之影響 7
六、 促進白菜根部發育的蛋白質萃取液篩選 7
七、 降低白菜幼苗立枯病發生率之蛋白質萃取液篩選 8
八、 不同蛋白質萃取液濃度對植株生育的影響 8
九、 APR01鏈格孢菌的鑑定 8
(一)、 培養特性及形態 8
(二)、 ITS 序列比對 9
十、 Alternaria tenuissima APR01 之蛋白質萃取液抑菌活性測試 10
十一、 Alternaria tenuissima APR01 蛋白質萃取液防治白菜立枯病的效果評估 10
十二、 噴佈蛋白質萃取液的次數對白菜幼苗生育的影響 10
十三、 Alternaria tenuissima蛋白質萃取液對立枯絲核菌危害白菜葉片的影響 11
十四、 苯丙胺酸氨裂解酵素 (phenylalanine ammonia lyase, PAL) 之活性定 11
十五、 過氧化酵素之測定 11
十六、 Alternaria tenuissima APR01 蛋白質萃取液對熱處理的安定性測定 12
十七、 Alternaria tenuissima APR01 蛋白質萃取液有效成分之分子量分析 12
結果 14
一、 磷酸回溶緩衝液 (PBS lysis buffer) 對白菜生長之影響 14
二、 促進白菜根部發育的蛋白質萃取液篩選 14
三、 降低白菜幼苗立枯病發生率之蛋白質萃取液篩選 14
四、 不同蛋白質萃取液濃度對植株生育的影響 14
五、 APR01鏈格孢菌的鑑定 15
(一)、 培養特性及形態 15
(二)、 ITS序列比對 15
六、 Alternaria tenuissima APR01 之蛋白質萃取液抑菌活性測試 15
七、 Alternaria tenuissima APR01 蛋白質萃取液防治白菜立枯病的效果評估 16
八、 噴佈蛋白質萃取液的次數對白菜幼苗生育的影響 16
九、 Alternaria tenuissima蛋白質萃取液對立枯絲核菌危害白菜葉片的影響 16
十、 酵素活性測定 16
十一、 Alternaria tenuissima APR01 蛋白質萃取液對熱處理的安定性測定 17
十二、 Alternaria tenuissima APR01 蛋白質萃取液有效成分之分子量分析 17
討論 18
引用文獻 23
圖表 31
附錄 46

表目錄
表 一、本研究之 Alternaria spp. 菌株來源 31
表 二、Alternaria tenuissima APR01 蛋白質萃取液防治白菜立枯病的效果評估 32
表 三、白菜幼苗處理 Alternaria tenuissima APR01 的總蛋白質萃取液 12 小時後接種Rhizoctonia solani AG-4 RST-04 對植株之防禦反應相關酵素活性之影響 33
表 四、Alternaria tenuissima APR01 總蛋白質萃取液中不同分子量蛋白對於白菜立枯病發生的影響 34

圖目錄
圖 一、不同稀釋倍率的磷酸回溶緩衝液對白菜種子發芽率之影響。 35
圖 二、不同稀釋倍率的磷酸回溶緩衝對白菜種子發芽率之影響。 36
圖 三、不同 Alternaria spp. 菌株的總蛋白質萃取液對於白菜主根與側根生育的影響。 37
圖 四、不同 Alternaria spp. 菌株之總蛋白質萃取液防治白菜幼苗立枯病的效果。 38
圖 五、不同 Alteraria tenuissima APR01 的總蛋白質萃取液之施用濃度對白菜幼苗鮮重的影響。 39
圖 六、Alternaria tenuissima APR01，其分生孢子產生方式及分生孢子之形態。 40
圖 七、Alternaria tenuissima APR01 的總蛋白質萃取液對 Rhizoctonia solani AG-4 RST-04 菌絲生長的影響。 41
圖 八、處理不同次數的 Alternaria tenuissima APR01 的總蛋白萃取液對種植於Rhizoctonia solani AG-4 RST04 病土中的白菜幼苗鮮重之影響。 42
圖 九、白菜處理過 Alternaria tenuissima APR01總蛋白質萃取液後 12 小時，於葉片表面接種 Rhizoctonia solani AG-4 RST-04 經過 48-180 小時，其病斑擴展的情形。 43
圖 十、白菜處理過 Alternaria tenuissima APR01總蛋白質萃取液後 12 小時，於葉片表面接種 Rhizoctonia solani AG-4 RST-04 經過 96-168 小時，其病斑擴展的情形。 44
圖 十一、溫度處理對 Alternaria tenuissima APR01 總蛋白萃取液 (APR01-TP) 防治白菜幼苗立枯病發生率的影響。 45

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