Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/31417
標題: 鏈格孢屬真菌之蛋白質萃取液誘導白菜抗立枯病之效果評估
Evaluation for efficacy of total protein extracted from Alternaria tenuissima APR01 on inducing Chinese cabbage seedlings resistant to Rhizoctonia damping-off
作者: 謝子揚
Hsieh, Tzu-Yang
關鍵字: Alternaria tenuissima
鏈格孢屬真菌
mycelial extract
protein
Chinese cabbage seedlings Rhizoctonia damping-off
induced resistance
菌絲萃取物
蛋白質
誘導抗病
白菜幼苗立枯病
出版社: 植物病理學系所
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摘要: 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 µg/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.
URI: http://hdl.handle.net/11455/31417
其他識別: U0005-2008201005080400
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2008201005080400
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