請用此 Handle URI 來引用此文件: http://hdl.handle.net/11455/31439
標題: 聚麩胺酸量產於枯草桿菌特殊功能生物殺菌劑發展之應用
The production of poly - (γ-glutamic acid) as a selective feature in biofungicide development of Bacillus subtilis
作者: 吳佳樺
Wu, Chia-Hua
關鍵字: Bacillus subtilis
枯草桿菌
poly -γ-glutamic acid
leek rust
聚麩胺酸
韭菜銹病
出版社: 植物病理學系所
引用: 何觀輝。2006。聚麩胺酸之結構特性與化學特性。化工資訊與商情 31:64-71。 柯勇、張德前。1999。葉菜類-韭菜。蔬菜病蟲害綜合防治專輯。30頁。 陳任芳。1990。韭菜病蟲害防治方法。花蓮區農業推廣簡訊7:14-15。 馮蘭香。1996。大蔥銹病。中國農作物病蟲害 上冊。中國農業出版社。1177-1178頁。 鄭安秀、林益昇。1995。台灣農家要覽農作篇(三)。行政院農業委員會。170-171頁。 Asaka, O., and Shoda, M. 1996. Biocontrol of Rhizoctonia solani damping-off of tomato with Bacillus subtilis RB14. Appl. Environ. Microbiol. 62: 4081-4085. Bais, H. P., Fall, R., and Vivanco, J. M. 2004. Biocontrol of Bacillus subtilis infection of Arabidopsis root by Pseudomonas syringae is facilitated by biofilm formation and surfactin production. Plant Physiol. 134:1-13. Buescher, J. M., and Margaritis, A. 2007. Microbial biosynthesis of poly glutamic acid biopolymer and applications in the biopharmaceutical, biomedical and food industries. Crit. Rev. Biotechnol. 27:1-19. Candela, T., and Fouet, A. 2006. Poly-γ-glutamate in bacteria. Mol. Microbiol.60:1091-1098. Doherty, M. A., and Preece, T. F. 1978. Bacillus cereus prevents germination of uredospores of Puccinia allii and the development of rust disease of leek,Allium porrum, in controlled environments. Physiol. Plant Pathol. 12:123-132. Emmert, E. A. B., and Handelsman, J. 1999. Biocontrol of plant disease: a (Gram-) positive perspective. FEMS Microbiol. Lett. 171:1-9. Eright, S. J. L., Linton, C. J., Edwards, R. A., and Drury, E. 1991. Isoamyl alcohol (3-methyl-1-butanol), a volatile anti-cyanobacterial and phytotoxic product of some Bacillus spp. Lett. Appl. Microbiol. 13:130-132. Ezzell, Jr J. W., and Welkos, S. L. 1999. The capsule of Bacillus anthracis, a review. Appl. Microbiol. 87:250. Fiddaman, P. J., and Rossall, S. 1994. Effect of substrate on the production of antifungal volatiles from Bacillus subtilis. J. Appl. Bacteriol. 76:395-405. Hölker, U., and Lenz, J. 2005. Solid-state fermentation - are there any biotechnological advantages? Curr. Opin. Microbiol. 8:301-306. Kim, H. S., Park, J., Choi, S. W., Choi, K. H., Lee, G. P., Ban, S. J., Lee, C. H., and Kim, C. S. 2003. Isolation and characterization of Bacillus strains for biological control. Microbiology. 41:196-201. Kloepper, J. W., Ryu, C. M., and Zhang, S. 2004. Induced systemic resistance and promotion of plant growth by Bacillus spp. Phytopathology. 94:1259-1266. Leejeerajumnean, A., Ames, J. M., and Owens, J. D. 2000. Effect of ammonia on the growth of Bacillus species and some other bacteria. Lett. Appl. Microbiol. 30:385-389. McLean, R. J., Beauchemin, D., Clapham, L., and Beveridge, T. J. 1990. Metal-binding characteristics of the gamma-glutamyl capsular polymer of Bacillus licheniformis ATCC 9945. Appl. Environ. Microbiol. 56: 3671-3677. Obst, M., and Steinbubüchel, A. 2004. Microbial degradation of poly (amino acid)s.Biomacromolecules 5:1166-1176. Okolo, B., Johnston, J. R., and Berry, D. R. 1987. Toxicity of ethanol, n-butanol and isoamyl alcohol in Saccharomyces cerevisiae when supplied separately and in mixtures. Biotechnol. Lett. 9:431-434. Park, C., Choi, J. C., Choi, Y. H., Nakamura, H., Shimanouchi, K., Horiuchi, T., Misono, H., Sewaki, T., Soda, K., Ashiuchi, M., Sung, M. H. 2005. Synthesis of super-high-molecular-weight poly-γ-glutamic acid by Bacillus subtilis subsp. chungkookjang. J. Mol. Catal., B Enzym. 35:128-133. Pinton, R., Varanini, Z., and Nannipieri, P. 2001. The rhizosphere as a site of biochemical interactions among soil components, plants and microorganisms.Pages 1-17. In R. Pinton, Z. Varanini, P. Nannipieri (eds). The Rhizosphere:Biochemistry and Organic Substances in the Soil-Plant Interface. Marcel Dekker. New York. Priest, F. G. 1993. Systematics and ecology of Bacillus. Pages 3-16. In: Bacillus subtilis and Other Gram-positive Bacteria: Biochemistry, Physiology, and Molecular Genetics. A. L. Sonensheine, J. A. Koch, and R. Losick (eds). American Society of Microbiology. Washington. Ryu, C. M., Farag, M. A., Hu, C. H., Reddy, M. S., Wei, H. X., Paré, P. W., and Kloepper, J. W. 2003. Bacterial volatiles promote growth in Arabidopsis. Proc. Natl. Acad. Sci. U S A. 100:4927-4932. Ryu, C. M., Farag, M. A., Hu, C. H., Reddy, M. S., Wei, H. X., Kloepper, J. W., and Paré, P. W. 2004. Bacterial volatiles induce systemic resistance in Arabidopsis. Plant Physiol. 134:1017-1026. Shi, F., Xu, Z., and Cen, P. 2006. Efficient Production of Poly-γ-glutamic Acid by Bacillus subtilis ZJU-7. Appl. Biochem. Biotechnol. 133:271-281. Shih, I. L., and Van, Y. T. 2001. The production of poly-(γ-glutamic acid) from microorganisms and its various applications. Bioresour. Technol. 79:207-225. Sung, M. H., Park, C., Kim, C. J., Poo, H., Soda, K., and Ashiuchi, M. 2005. Natural and edible biopolymer poly-γ-glutamic acid: Synthesis, production, and applications. Chem. Rec. 5:352-366. Szczech, M., and Shoda, M. 2006. The effect of mode of application of Bacillus subtilis RB14-C on its efficacy as a biocontrol agent against Rhizoctonia solani. J. Phytopathol. 154:370-377. Wang, Q., Chen, S., Zhang, J., Sun, M., Liu, Z., and Yu, Z. 2008. Co-producing lipopeptides and poly-γ-glutamic acid by solid-state fermentation of Bacillus subtilis using soybean and sweet potato residues and its biocontrol and fertilizer synergistic effects. Bioresour. Technol. 99:3318-3323. Yang, C. W., Lin, T. C., and Huang, J. W. 2009. The effect of tea seed pomace on control of cabbage seedling damping-off caused by Rhizoctonia solani AG-4. J. Agri. & Fore. 58: 277-288. Yu, G. Y., Sinclair, J. B., Hartman, G. L., and Bertagnolli, B. L. 2002. Production of iturin A by Bacillus amyloliquefaciens suppressing Rhizoctonia solani . Soil Biol. Biochem. 34:955-963. 李雅惠。2002。拮抗性桿菌屬(Bacillus spp.)之分離、培養與抗生活性之改進以及病害防治之應用。國立中興大學植物病理學系碩士論文。79 頁。 何觀輝。2006。聚麩胺酸之結構特性與化學特性。化工資訊與商情 31:64-71。 邱燕欣。2004。拮抗性枯草桿菌 Bacillus subtilis WG-16 菌株於柑橘潰瘍病防治應用。國立中興大學植物病理學系碩士論文。92 頁。 梁瑩如。2008。生物殺菌劑Bacillus subtilis WG6-14做為產蛋雞益生菌及其排遺於植物病害管理應用之評估。國立中興大學植物病理學系碩士論文。88頁。 陳俊位、鄧雅靜、曾德賜。2009。功能性微生物製劑在有機作物栽培病害管理上之應用。功能性微生物製劑在有機作物栽培病害管理上之應用。有機農業產業發展研討會專輯--特刊96號。34頁。 Asaka, O., and Shoda, M. 1996. Biocontrol of Rhizoctonia solani damping-off of tomato with Bacillus subtilis RB14. Appl. Environ. Microbiol. 62: 4081-4085. Chung, W. C., Huang, J. W., and Huang, H. C. 2005. Formulation of a soil biofungicide for control of damping-off of Chinese cabbage (Brassica chinensis) caused by Rhizoctonia solani. Biol. Control. 32:287-294. Doherty, M. A., and Preece, T. F. 1978. Bacillus cereus prevents germination of uredospores of Puccinia allii and the development of rust disease of leek, Allium porrum, in controlled environments. Physiol. Plant Pathol. 12:123-132. Eddy, B. P. 1961. The Voges-Proskauer reaction and its significance: A review. J. appl.Bact. 24:27-41. Fernando, W. G. D., Ramarathnam, R., Krishnamoorthy, A. S., and Savchuk, S. C. 2005. Identification and use of potential bacterial organic antifungal volatiles in biocontrol. Soil Biol. Biochem. 37:955-964. Kunioka, M. 1997. Biosynthesis and chemical reactions of poly(amino acid)s from microorganisms. Appl. Microbiol. Biotechnol. 47:469-475. Okolo, B., Johnston, J. R., and Berry, D. R. 1987. Toxicity of ethanol, n-butanol and iso-amyl alcohol in Saccharomyces cerevisiae when supplied separately and in mixtures. Biotechnol. Lett . 9:431-434. Park, C., Choi, J. C., Choi, Y. H., Nakamura, H., Shimanouchi, K., Horiuchi, T., Misono, H., Sewaki, T., Soda, K., Ashiuchi, M., Sung, M. H. 2005. Synthesis of super-high-molecular-weight poly-γ-glutamic acid by Bacillus subtilis subsp. chungkookjang. J. Mol. Catal., B Enzym. 35:128-133. Ping, L., and Boland, W. 2004. Signals from the underground: bacterial volatiles promote growth in Arabidopsis. Trends Plant Sci. 9:263-266. Ryu, C. M., Farag, M. A., Hu, C. H., Reddy, M. S., Wei, H. X., Paré, P. W., and Kloepper, J. W. 2003. Bacterial volatiles promote growth in Arabidopsis. Proc. Natl. Acad. Sci. U. S. A. 100:4927-4932. Ryu, C. M., Farag, M. A., Hu, C. H., Reddy, M. S., Wei, H. X., Kloepper, J. W., and Paré, P. W. 2004. Bacterial volatiles induce systemic resistance in Arabidopsis. Plant Physiol. 134:1017-1026. Saksena, N., and Tripathi, H. H. S. 1987. Effect of organic volatiles from Saccharomyces on the spore germination of fungi. Acta. Microbiol. Acad. Sci. Hung. 34:255-157. Shi, F., Xu, Z., and Cen, P. 2006. Efficient production of poly-γ-glutamic acid by Bacillus subtilis ZJU-7. Appl. Biochem. Biotechnol. 133:271-281. 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. Shih, I. L., and Van, Y. T. 2001. The production of poly-(γ-glutamic acid) from microorganisms and its various applications. Bioresour. Technol. 79:207-225. Sung, M. H., Park, C., Kim, C. J., Poo, H., Soda, K., and Ashiuchi, M. 2005. Natural and edible biopolymer poly-γ-glutamic acid: Synthesis, production, and applications. Chem. Rec. 5:352-366. Szczech, M., and Shoda, M. 2004. Biocontrol of Rhizoctonia damping-off of tomato by Bacillus subtilis combined with Burkholderia cepacia. J. Phytopathol. 152:549-556. Szczech, M., and Shoda, M. 2006. The effect of mode of application of Bacillus subtilis RB14-C on its efficacy as a biocontrol agent against Rhizoctonia solani. J. Phytopathol. 154:370-377. Troy, F. A. 1973. Chemistry and biosynthesis of the poly(γ-D-glutamyl) capsule in Bacillus licheniformis, I. Properties of the membrane-mediated biosynthesis reaction. J. Biol. Chem., 248:305-316. Wang, Q., Chen, S., Zhang, J., Sun, M., Liu, Z., and Yu, Z. 2008. Co-producing lipopeptides and poly-γ-glutamic acid by solid-state fermentation of Bacillus subtilis using soybean and sweet potato residues and its biocontrol and fertilizer synergistic effects. Bioresour. Technol. 99:3318-3323.
摘要: 枯草桿菌群(Bacillus subtilis group)細菌為生物殺菌劑發展上極為重要的生物資源,亦為多種農工業發展上應用極為普遍之有益菌,近年來,利用此一種群成員量產由麩胺酸以γ-amide linkage合成之高分子量聚麩胺酸 (poly -γ-glutamic acid , γ-PGA ),其具有多種特殊特性,為目前產業發展上甚被看好的生技產品。有關γ-PGA的應用性,在農業方面以土壤保水力與土壤結構的改善、植物生長有機氮源的提供以及植物生長的促進最受到重視。本研究旨在瞭解此一種群菌株之γ-PGA產生能力、最適化產出方法、對植物生長之促進性以及對病原危害之應用性,期能作為相關生物製劑量產技術改進之參考。利用HSP1、TKS1-1、TLB7-7、OF3-16、WG6-14、SP4-17及natto2等拮抗性枯草桿菌群菌株作為供試菌株,於添加麩酸一鈉(MSG)的modified SYM、523 medium與medium E等液態培養後,以TLB7-7菌株獲得最高產量,而以two step culture system(菌株培養於SYM 18小時後,再培養於modified medium E,以SYM/E作為簡稱)培養供試菌株,則以TLB7-7菌株產出的γ-PGA量最高,每毫升可達12 mg左右,並以SDS PAGE電泳分析,其分子量大於95kD。以澆灌方式處理測試含與不含γ-PGA的培養菌液對土壤保水能力的影響,以處理後10天為例,處理含6 mg/ml γ-PGA的培養菌液的土壤,其殘餘水量較只澆灌水之對照組多約150%,顯示所添加菌液中之γ-PGA與保水力的提升明顯有關。至於病害防治效果,含γ-PGA之培養液於稀釋100倍下,每週二次澆灌毛豆與甘藍幼苗,皆能明顯促進根部的生長,其中毛豆根的鮮重皆明顯比對照組重61%,甘藍根的鮮重則比對照組重46.2-80.6%。至於病害防治的應用,以澆灌與噴灑方式施用γ-PGA培養菌液能有效防治甘藍猝倒病與由Puccinia allii (DC.) Rudolphi引起的韭菜銹病。以人工方式接種104 cfu/ml銹孢子懸浮液於韭菜葉片,隨即噴灑γ-PGA培養菌液能有效地抑制銹孢子的感染,而在接種銹孢子24小時後,再噴灑培養菌液,其抑制效果亦非常顯著。進一步以玻片測試方法觀察γ-PGA培養液對銹孢子的影響情形,γ-PGA培養液能明顯抑制銹孢子發芽,並造成發芽管膨大畸形,此抑制情形與γ-PGA培養液濃度呈正相關,顯示γ-PGA培養液藉由抑制銹孢子的發芽與發芽管形成,達到降低韭菜銹病的感染。另外將供試菌株培養於黃豆粉固態培養基,檢測γ-PGA產量、菌體產量及對AG4拮抗能力,試驗結果則以WG6-14菌株與SP4-17菌株所獲得的表現最好。除了γ-PGA的生產之外,此兩隻菌株所產生的揮發性代謝物質能有效地促進植物生長。收集SP4-17菌株SYM/E培養液、WG6-14與SP4-17菌株之固態產物所產生的揮發性代謝物質,以GC/MS分析此些揮發性代謝物質,均含有2,3-butanediol,而2,3-butanediol為已被研究證實能促進植物的生長的揮發性物質。培養產物所產生的揮發性代謝物質與植物生長的促進性被證實具有相關性,而WG6-14與SP4-17菌株之固態培養產物,其揮發性代謝物質亦具抑制AG4菌絲生長,且抑制效果與濃度具有相關性,當揮發性代謝物質到達某一濃度後具有殺菌效果。在未來配方改進上,如何由培養基質切入,使γ-PGA、揮發性氣體代謝物之生長促進性得以與既有的抗生活性結合,發揮其統合功效將為繼續努力之重點。
The Bacillus subtilis group bacterium is renowned for its importance as natural resources for biofungicides, and many other agro-industry applications. The bacterium has attracted great attention recently for its use in producing poly-γ-glutamic acid (γ-PGA) which has multi-function and is now one of the most favored biotech products. In agriculture, the major application of γ-PGA which has attracted most attention includes improvement of soil structure and water holding capability, the supplementation of organic nitrogen, and the promotion of plant growth. The main objective of this investigation is to explore the production of γ-PGA by the antagonistic bacilli known with potential in biofungicide application, to optimize the production, and to learn its significance in regarding to the growth promotion and disease control effectiveness. The ultimate goal is to provide some useful data bases for strengthening the market value of targeted biological fungicide. Using HSP1, TKS1-1, TLB7-7, OF3-16, WG6-14, SP4-17, and natto2 antagonistic Bacillus subtilis group bacterium as test strains, production of γ-PGA was compared on monosodium glutamate (MSG) supplemented SYM and 523 medium, and on medium E wherein L-glutamate was substituted by MSG. Examination on γ-PGA production on these 3 media indicated that TLB7-7 was among the tested strains which gave the highest yield. By a two step culture system that the test strains were allowed to grow in SYM for 18 hrs before transferred to MSG substituted Medium E (abbreviated as SYM/E in the following), the γ-PGA yield of TLB7-7 reached approximately 12 mg/ml. And the results of SDS-PAGE also indicated the produced γ-PGA has molecular weight greater than 95kD. By drenching application, the effect of culture broth with and without γ-PGA production on water holding capability of soil in plastic pots were compared in greenhouse. At 10 days after treatment, the residual water content of soil treated with culture broth containing 6 mg/ml γ-PGA was 150% higher than that treated with only water. The effectiveness of γ-PGA in strengthening water holding capability of soil was clearly demonstrated. For greenhouse growing vegetable soybean and cabbage plants drenching treated with 100x diluted γ-PGA containing broth culture twice a week, significantly promoted plant growth were observed. The fresh weight of vegetable soybean roots was significantly increased 61% while that of cabbage was increased 46.2-80.6%, as compared to that of control plants treated with only water. As for disease control, the water and spray application of these γ-PGA containing culture broths were found to be effective in controlling cabbage damping off disease and leek rust disease (Puccinia allii (DC.) Rudolphi). For pot grown leek plants artificially inoculated with urediniospore suspension at 104 spore/ml, the simultaneous application of the γ-PGA containing broth culture of the bacilli strains nearly completely inhibited the infection of the urediniospores. The inhibitory effect of rust infection was still evident even the culture broth was applied 24 hours after urediniospores inoculation. The reduced rust infection was due evidently to the inhibition of spore germination and germ tube development. On a glass slide system, the application of the culture broth was found leading to greatly reduced germination, and the germ tube growth was greatly reduced and deformed. The inhibitory effect on spore germination appeared to be concentration dependent. Moreover, the effectiveness was found better by broth culture rather than filtrates obtained by Millipore membrane filtration indicating the involvement of secreted and spore-contained antibiotic in the antifungal function observed. In addition to broth culture system, the γ-PGA production of tested strains was also explored by solid state fermentation system. Among the 7 tested strains, WG6-14 and SP4-17 performed the best in the regard of γ-PGA productions and antagonistic effectiveness against Rhizoctonia solani AG4. Along with γ-PGA production, the 2 strains were also found to be the best in the production of volatile compounds which appeared to be effective in promoting plant growth. During the course of broth culture of SP4-17 and solid fermentation of SP4-17 and WG6-14, volatile compounds were collected from the air space of the culture container. By GC/MS, the production of 2,3-butanediol, the compound known to be of plant growth promoting effectiveness was identified. Although the correlation of the identified volatile compound and the observed plant growth promoting effect remain to be illustrated, the volatile compounds collected from WG6-14 and SP4-17 strains during the solid-state fermentation process were found to be inhibitory to the growth of Rhizoctonia solani AG4. The inhibitory effect observed was clearly dose dependent. What worth to mention is that at high dose application, the volatile compounds appeared to kill the tested fungus in 24 hours. The combined effect of γ-PGA and plant growth promoting / fungicidal metabolites shown in this study appeared to indicate a valuable way to the industrial formulation improvement of the targeted biofungicide.
URI: http://hdl.handle.net/11455/31439
其他識別: U0005-2308201010483000
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2308201010483000
顯示於類別:植物病理學系

文件中的檔案:
沒有與此文件相關的檔案。


在 DSpace 系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。