Please use this identifier to cite or link to this item:
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
引用: 引用文獻 王三太、林子凱。2005。小白菜。p.424-428。台灣農家要覽:農作篇 (二)。台灣農家要覽增修訂三版策劃委員會。行政院農業委員會出版。台北。926 pp。 王曉梅、于金萍、藏東初、韓兆勝、楊信東。2006。真菌蛋白的提取及其對玉米病害的抗性誘導。玉米科學 14 (6):138-140。 安寶貞。2001。植物病害的非農藥防治品-亞磷酸。植物病理學會刊 10: 147-164。 安寶貞、謝廷芳、蔡志濃、王姻婷、林俊義。2000。亞磷酸之簡便使用方法與防病範圍。植物病理學會會刊 9: 179 (摘要)。 吳文希。1988。Rhizoctonia solani 之性質。p. 1-74。植物土媒病原學 (立枯絲核菌之性質及防治)。國立編譯館。台北。260 pp。 李麗、邱德文、劉崢、楊秀芬、羅寬。2005。植物激活素對番茄抗病性的誘導作用。中國生物防治 21 (4):265-268。 武廣衍、邱德文、吳珍泉、武廣繁、趙勝英。2007。新型激活素對多種作物種子發芽的影響。華北農學報 22 (增刊):21-24。 邱德文。2004。微生物蛋白農藥研究進展。中國生物防治 20 (2):91-94。 郁宗雄。2001。蔬菜栽培。郁維強,郁維珍刊印。394 pp。 高景輝。2005。植物生理分析技術。初版。五南出版社。臺灣。168 pp。 張志剛、邱德文、楊秀芬、曾洪梅、袁京京、劉崢、楊曉萍、官春云。2007。細極鏈格孢菌蛋白激發子誘導棉苗基因表達差減文庫的構建及EST分析。棉花學報 19 (4):248-254。 陳甘澍、沈再發。1995。不結球白菜。P.317-322。台灣農家要覽:農作篇 (二)。農業委員會台灣農家要覽增修訂再版策劃委員會編。財團法人豐年社出版。台北。 698 pp。 陳梅、邱德文、劉崢、楊秀芬、曹克強。2006。植物激活素對菸草花葉病毒 RNA 複製及外殼蛋白合成的抑制作用。中國生物防治 22 (1):63-66。 黃振文、孫守恭、吳瑞香。1981。十字花科蔬菜黑斑病菌的選擇性培養基與存活。植保會刊 23 (4):279 (摘要)。 黃素清。2003。美國綠色化學挑戰總統獎案例(三)。永續產業發展雙月刊 11: P.21-22。 黃涵、洪立。1988。台灣蔬菜彩色圖說。行政院農業委員會、國立台灣大學園藝系編印。210 pp。 黃鴻章、黃振文、謝廷芳。2008。永續農業之植物病害管理。行政院農業委員會動植物防疫檢疫局、國立中興大學植物病理學系出版。臺中。319 pp。 楊正偉。2006。苦茶粕防治甘藍幼苗立枯病之效果與其有效成分之鑑定。國立中興大學植物病理學系碩士論文。49 pp。 臺灣植物病害名彙第四版編輯委員會。2002。台灣植物病害名彙。第四版。中華民國植物病理學會出版。台中。386 pp。 Abad, L. R., D’Urzo, M. P., Liu, D., Narasimhan, M. L., Reuveni, M., Zhu, J. K., Niu, X., Singh, N. H., Hasegawa, P. M., and Bressan, R. A. 1996. Antifungal activity of tabcco osmotin has specificity and involves plasma menbrane permeabilization. Plant Scicene 118: 11-23. Agrios, G. N. 2005. Plant Pathology. 5th ed. Academic Press. U.S.A. 922 pp. Angelova, Z., Georgiev, S., and Roos, W. 2006. Elicitation of plants. Biotechnol. Biotechnol. Eq. 20 (2): 72-83. Baillieul, F. Fritig, B., and Kauffmann, S. 1996. Occurrence among Phytophthora species of glycoprotein eliciting a hypersensitive response in tobacco and its relationships with elicitins. MPMI. 9 (3): 214-216. Baillieul, F., Genetet, I., Kopp, M., Saindrenan, P., Fritig, B., and Kauffmann, S. 1995. A new elicitor of the hypersensitive response in tobacco: a fungal glycoprotein elicits cell death, expression of defence genes, production of salicylic acid, and induction of systemic acquired resistance. Plant J. 8 (4): 551-560. Blein, J., Coutos-Thévenot, P., Marion D., and Ponchet, M. 2002. From elicitins to lipid-transfer proteins: a new insight in cell signalling involved in plant defence mechanisms. Trends Plant Sci. 7 (7): 293-296. Boudet, A. 1998. A new view of lignification. Trends Plant Sci. 3 (2): 67-71. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochem. 72: 248-259. Bridge, P. 1996. Protein extraction from fungi. Methods Mol. Biol. 59: 39-48. Bridge, P. D., Kokubun, T., and Simmonds, M. S. J. 2004. Protein extraction from fungi. Methods Mol. Biol. 244: 37-46. Broglie, K., Chet, I., Holliday, M., Cressman, R., Biddle, P., Knowlton, S., Mauvais, C. J., and Broglie, R. 1991 Transgenic plants with enhanced resistance to the fungal pathogen Rhizoctonia solani. Science 254 (5035): 1194-1197. Carver, T. L. W., Zhang, L., Zeyen, R. J., and Robbins, M. P. 1996. Phenolic biosynthesis inhibitor suppress adult plant resistance to Erysiphe graminisin oat at 20 ℃ and 10 ℃. Physiol. Mol. Plant Pathol. 49 (2): 121-141. Chen, C., Belanger, R. R., Benhamoun, N., and Paulitz, T. C. 2000. Defense enzymes induced in cucumber roots by treatment with plant growth-promoting rhizobacteria (PGPR) and Pythium aphanidermatum. Physiol. Mol. Plant Pathol. 56: 13-23. Chen, C. Y., and Huang, H. E. 1997. Salicylic acid-induced resistance of lily leaves against Botrytis elliptica. Plant Pathol. Bull. 6: 76-82. Collopy, P. D., Largeteau-Mamoun, M. L., Romaine, C. P., and Royse, D. J. 2001. Molecular phylogenetic analyses of Verticillium fungicola and related species causing dry bubble disease of the cultivated button mushroom, Agaricus bisporus. Phytopathology 91 (9): 905-912. Cordelier, S., Ruffray, P., Fritig, B., and Kauffmann, S. 2003. Biological and molecular comparison between localized and systemic acquired resistance induced in tobacco by a Phytophthora megasperma glycoprotein elicitin. Plant Mol. Biol. 51: 109-118. Cvikrová, M., Malá, J., Hrubcová, M., and Eder, J. 2006. Soluble and cell wall-bound phenolics and lignin in Ascocalyx abietina infected Norway spruces. Plant Sci. 170: 563-570. De Ascensao, A. R. D. C. F., and Dubery, I. A. 2000. Panama disease: cell wall reinforcement in banana roots in response to elicitors from Fusarium oxysporum f. sp. cubense race four. Biochem. Cell Biol. 90 (10): 1173-1180. Dong, H., Delaney, T. P., Bauer, D. W., and Beer, S. V. 1999. Harpin induces disease resistance in Arabidopsis through the systemic acquired resistance pathway mediated by salicylic acid and the NIM1 gene. Plant J. 20: 207-215. Durrant, W. E., and Dong, X. 2004. Systemic acquired resistance. 42: 185-209. Ebel, J., and Cosio, E.G. 1994. Elicitors of plant defense responses. Int. Rev. Cytol. 148: 1-36. Farmer, E. E., and Helgeson, J. P. 1987. An extracellular protein from Phytophthora parasitica var. nicotianae is associated with stress metabolite accumulation in tobacco callus. Plant Physiol. 85: 733-740. Greenberg, J. T. 1997. Programmed cell death in plant-pathogen interactions. Annu. Rev. Plant Physiol. Plant Mol. Biol. 48: 525-45 Hammerschmidt, R. 1999. Induced disease resistance: how do induced plants stop pathogens? Phisol. Mol. Plant Pathol. 55: 77-84. Harkin, J. M., and Obst, J. R. 1973. Lignification in trees: indication of exclusive peroxidase participation. Science 180: 296-298 Heller, W. E., and Gessler, C. 1986. Induced systemic resistance in tomato plants against Phytophthora infestans. J. Phytopathol. 116 (4): 323-328. Hendrix, J. W. 1964. Sterol induction of reproduction and stimulation of growth of Pythium and Phytophthora. Science 144 (3621): 1028-1029. Jones, J. 2001. Harpin. Pestic. Outlook 12: 134-135. Jones, J. D. G., and Dangl, J. L. 2006. The plant immune system. Nature 444 (16): 323-329. Kamoun, S., Young, M., Glascock, C. B., and Tyler, B. M. 1993. Extracellular protein elicitors from Phytophthora: host-specificity and induction of resistance to bacterial and fungal phytopathogens. Molecular Plant-Microbe Interactions 6 (1): 15-25. Keen, N. T. 1975. Specific elicitors of plant phytoalexin production: determin- ants of race specificity in pathogens? Science 187 (4171): 74-75. Keizer, D. W., Schuster, B., Grant, B. R., and Gayler, K. R. 1998. Interactions between elicitins and radish Raphanus sativus. Planta 204: 480-489. Kitajima, S., and Sato, F. 1999. Plant pathogenesis-related proteins: molecular mechanisms of gene expression and protein function. J. Biochem. 125: 1-8. Kloepper, J. W., Tuzun, S., and Kuc, J. A. 1992. Proposed definitions related to induced disease resistance. Biocontrol Sci. Technol. 2 (4): 349-351. Lagrimini, L. M., Gingas, V., Finger, F., Rothstein, S., and Liu, T. T. Y. 1997. Characterization of antisense transformed plants deficient in the tobacco anionic peroxidase. Plant Physiol. 114 (4): 1187-1196. Li, J., Zhang, Z. G., Ji, R., Wang. Y. C., and Zheng, X. B. 2006. Hydrogen peroxide regulates elicitor PB90-induced cell death and defense in non-heading Chinese cabbage. Physiol. Mol. Plant Pathol. 67: 220-230. Low, P. S., and Merida, J. R. 1996. The oxidative burst in plant defense: function and signal transduction. Physiol. Plant. 96: 533-542. Montesano, M., Brader, G., and Palva, E. T. 2003. Pathogen derived elicitors: searching for receptors in plants. Mol. Plant Pathol. 4 (1): 73-79. Métraux, J. P., Nawrath, C., and Genoud, T. 2002. Systemic acquired resistance. Euphytica 124: 237–243. Niederman, T. Genetet, I., Bruyere, T., Gees, R., Stintzi, A., Legrand, M., Fritig, B., and Mosinger, E. 1995. Pathogenesis related PR-1 proteins are antifungal. Plant Physiol. 108: 17-27. Nimchuk, Z., Rohmer, L., Chang, J. H., and Dangl, J. L. 2001. Knowing the dancer from the dance: R-gene products and their interactions with other proteins from host and pathogen. Curr. Opin. Plant Biol. 4: 288-294. Nürnberger, T. 1999. Signal perception in plant pathogen defense. Cell. Mol. Life Sci.ence 55: 167-182. Nürnberger, T., Nennstiel, D., Hahlbrock, K., and Scheel, D. 1995. Covalent cross-linking of the Phytophthora megasperma oligopeptide elicitor to its receptor in parsley membranes. Biochemistry 92: 2338-2342. Nürnberger, T., and Scheel, D. 2001. Signal transmission in the plant immune response. Trends Plant Sci. 6: 372-379. Oostendorp, M., Kunz, W., Dietrich, B., and Staub, T. 2001. Induced disease resistance in plants by chemicals. Eur. J. Plant Pathol. 107: 19-28. Ricci, P., Bonnet, P., Huet, J-C., Sallantin, M., Beauvais-Cante, F., Bruneteau, M., Billard, V., Michel, G., and Pernollet J-C. 1989. Structure and activity of proteins from pathogenic fungi Phytophthora eliciting necrosis and acquired resistance in tobacco. Eur. J. Biochem. 183: 555-563. Ross, A. F. 1961a. Localized acquired resistance to plant virus infection in hypersensitive hosts. Virology 14 (3): 329-339. Ross, A. F. 1961b. Systemic acquired resistance induced by localized virus infections in plants. Virology 14 (3): 340-358. Rotem, J. 1994. The genus Alternaria: Biology, Epidemiology, and Pathogenesity. APS press. U.S.A. 326 pp. Ryals, J. A., Neuenschwander, U. H., Willits, M. G., Molina, A., Steiner, H. Y., and Hunt, M. D. 1996. Systemic acquired resistance. Plant Cell 8: 1809-1819. Shiau, F. L., Chung, W. C., Huang, J. W., and Huang, H. C. 1999. Organic amendment of commercial culture media for improving control of Rhizoctonia damping-off of cabbage. Can. J. Plant Pathol. 21: 368-374. Simmons, E. G. 2007. Alternaria : An Identification Manual. 1st ed. Centraalbureau voor Schimmelcultures. Netherlands. 900 pp. Smith, J. A., and Hammerschmidt, R. 1988. Comparative study of acidic peroxidases associated with induced resistance in cucumber, muskmelon and watermelon. Physiol. Mol. Plant Pathol. 33 (2): 255-261. Sticher, L., Mauch-Mani, B., and Métraux, J. P. 1997. Systemic acquired resistance. Annu. Rev. Phytopathol. 35: 235-270. Taiz, L., and Zeiger, E. 2002. Secondary metabolites and plant defense. p. 283-308. Plant Physiology, 3rd ed. Sinauer Associates Inc. U.S.A. 690 pp. Takenaka, S., Nishio, Z., and Nakamura, Y. 2003. Induction of defense reactions in sugar beet and wheat by treatment with cell wall protein fractions from the mycoparasite Pythium oligandrum. Phytopathology 93: 1228-1232. Tu, C. C., Hsieh, T. F., and Chang, Y. C. 1996. Characterization of Rhizoctonia isolate, disease occurrence and management in vegetable crop. Plant Pathol. Bull. 5: 69-79. van Loon, L. C. 1997. Induced resistance in plants and the role of pathogenesis-related proteins. Eur. J. Plant Pathol. 103: 753-765. Wang, Y. C., Hu, D. W., Zhang, Z. G., Ma, Z. C., Zheng, X. B., and Li, D. B. 2003. Purification and immunocytolocalization of a novel Phytophthora boehmeriae protein inducing the hypersensitive response and systemic acquired resistance in tobacco and Chinese cabbage. Physiol. Mol. Plant Pathol. 63: 223–232. Way, H. M., Kazan, K., Mitter, N., Goulter, K. C., Birch, R. G., and Manners J. M. 2002. Constitutive expression of a phenylalanine ammonia-lyase gene from Stylosanthes humilis in transgenic tobacco leads to inhanced disease resistance but impaired plant growth. Physiol. Mol. Plant Pathol. 60 (6): 275-282. Wei, Z. M., Laby, R. J., Zumoff, C. H., Bauer, D. W., He, S. Y., Collmer, A., and Beer, S. V. 1992. Harpin, elicitor of the hypersensitive response produced by the plant pathogen Erwinia amylovora. Science 257 (5066): 85-88. Wu, W. S. 1978. Mycological investigations of the cultivated plants at highland farm (II). Mem. Coll. Agric. Nat. Taiwan Univ. 18(1): 124-128. Zhang, Y., Yang, X., Liu, Q., Qiu, D., Zhang, Y., Zeng, H., Yuan, J., and Mao, J. 2009. Purification of novel protein elicitor from Botrytis cinerea that induces disease resistance and drought tolerance in plants. Microbiol. Res. 165(2): 142-151.
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.
其他識別: U0005-2008201005080400
Appears in Collections:植物病理學系

Show full item record
TAIR Related Article

Google ScholarTM


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.