Please use this identifier to cite or link to this item:
標題: 檬果炭疽病的生物防治菌鑑定與其防病之雛形醱酵營養配方設計
Identification for biocontrol agents of mango anthracnose and design of their pilot-scale fermenting nutrient recipes for the disease control
作者: 江嘉容
Chiang, Chia-Jung
關鍵字: biocontrol;生物防治;Bacillus licheniformis;fermentation;mango;mango anthracnose;Bacillus licheniformis;醱酵;檬果;檬果炭疽病
出版社: 植物病理學系所
引用: 石信德。2003。鏈黴菌 PMS-702 防治作物病害的功效與其抑菌主要代謝物治黴色基素之鑑定。國立中興大學植物病理學博士論文。151 pp。 安寶貞、呂理燊、莊再揚、高清文。1998。套袋與地面覆蓋對檬果炭疽病與蒂腐病之防治效果。植病會刊 7: 19-26。 行政院農業委員會。2008。民國 97 年農業統計年報。行政院農委會出版。352 pp。 孫守恭。2001。臺灣果樹病害。世維出版社。台中。429 pp。 莊再揚、安寶貞。1997。芒果炭疽病之生物防治。植保會刊 39: 227-240。 費雯綺、王玉美。2002。植物保護手冊 (果樹篇)。行政院農業委員會農業藥物毒物試驗所。台中。255 pp。 廖嘉信。1975。台灣檬果病害-蒂腐病。科學農業 2: 415-416。 蔡致謨。1961。檬果病蟲害之研究。植保會刊 3: 9-17。 鍾文全、黃振文。1994。應用微生物防治白花芥藍黑斑病。植保會刊 36: 117-130。 謝廷芳、黃振文、張志展、彭玉湘。2001。碳氮源影響拮抗細菌防治百合灰黴病的效應。植病會刊 10: 79-87。 Abd-Alla, M. A. and Haggag, W. M. 2010. New safe methods for controlling anthracnose disease of mango (Mangifera indica L.) fruits caused by Colletotrichum gloeosporioides. Am. Sci. 8: 361-367. Arguelles-Arias, A., Ongena, M., Halimi, B., Lara, Y., Brans, A., Joris, B., and Fickers, P. 2009. Bacillus amyloliquefaciens GA1 as a source of potent antibiotics and other secondary metabolites for biocontrol of plant pathogens. Microb. Cell Fact. 8: 63. Athukorala, S. N. P., Dilantha Fernando, W. G., and Rashid, K. Y. 2009. Identification of antifungal antibiotics of Bacillus species isolated from different microhabitats using polymerase chain reaction and MALDI-TOF mass spectrometry. Can. J. Microbiol. 55: 1021-1032. Baker, K. F. and Cook, R. J. 1974. Biological Control of Plant Pathogens. W. H. Freeman Co, San Francisco, California. 433 pp. Baysal, O., Caliskan, M., and Yesilova, O. 2008. An inhibitory effect of a new Bacillus subtilis strain (EU07) against Fusarium oxysporum f. sp radicis-lycopersici. Physiol. and Mol. Plant Pathol. 73: 25-32. Cappellini, R. A. and Ceponis, M. J. 1984. Postharvest losses in fresh fruits and vegetables. Pages 24-30 in: Postharvest Pathology of Fruits and Vegetables. Miller, W. G., Leveau, J. H. J. and Lindow, S. E. (eds). Agric. Exp. Stn., UC, Berkeley. Churchill, B. W. 1982. Mass production of microorganisms for biological control. Pages 139-156 in: Biological Control of Weeds with Plant Pathogens. Charudattan, R. and Walker, M. L. (eds). John Wiley and Sons, New York. Cook, R. J. and Baker, K. F. 1983. The Nature and Practice of Biological Control of Plant Pathogens. Amer. Phytopathol. Soc., St. Paul, Minnesota. 539 pp. Costa, E., Teixidó, N., Usall, J., Atarés, E., and Viñas, I. 2001. Production of the biocontrol agent Pantoea agglomerans strain CPA-2 using commercial products and by-products. Appl. Microbiol. Biotechnol. 56: 367-371. Christie, R., Atkins, N. E., and Munch-Peterson, E. 1944. A note on a lytic phenomenon shown by group B streptococci. Aust. J. Exp. Biol. Med. Sci. 22:197-200. De Jager, E. S., Hall, A. N., Wehner, F. C., and Korsten, L. 2001. Microbial ecology of the mango phylloplane. Microb. Ecol. 42: 201-207. Dharani-Aiyer, P. V. 2004. Effect of C: N ratio on alpha amylase production by Bacillus licheniformis SPT. Afr. J. Biotechnol. 10: 519-522. Dock, L. L., Nielsen, P. V., and Floros, J. D. 1998. Biological control of Botrytis cinerea growth on apples stored under modified atmospheres. J. Food Prot. 61: 1661-1665. Eckert, J. W. and Ogawa, J. M. 1985. The chemical control of postharvest diseases: subtropical and tropical fruits. Annu. Rev. Phytopathol. 23: 421-454. Fravel, D. R., Lewis, J. A., and Chittams, J. L. 1995. Alginate prill formulations of Talaromyces flavus with organic carries for biocontrol of Verticillium dahliae. Phytopathology 85: 165-168. Fu, G., Huang, S. L., Ye, Y. F., Wu, Y. G., Cen, L. Z., and Lin. S. H. 2010. Characterization of a bacterial biocontrol strain B106 and its efficacy in controlling banana leaf spot and post-harvest anthracnose diseases. Biol. Control 55: 1-10. Futagawa, M., Rimando, A. M., Tellez, M. R., and Wedge, D. E. 2002. pH Modulation of Zopfiellin Antifungal Activity to Colletotrichum and Botrytis. J. Agric. Food Chem. 50: 7007-7012. Garrett, S. D. 1965. Towards biological control of soil-borne plant pathogens. Page 571 in: Ecology of Soil-borne Plant Pathogens. Univ. Cali. Press, Berkeley. Govender, V. and Korsten, L. 2006. Evaluation of different formulations of Bacillus licheniformis in mango pack house trials. Biol. Control 37: 237-242. Govender, V., Korsten, L., and Sivakumar, D. 2005. Semi-commercial evaluation of Bacillus licheniformis to control mango postharvest diseases in South Africa. Postharvest Biol. Technol. 38: 57-65. Jackson, M. A. 1997. Optimizing nutritional conditions for the liquid culture production of effective fungal biological control agents. J. Ind. Microbiol. and Biotechnol. 19: 180-187. Johnson, G. I. and Coates, L. M. 1993. Postharvest diseases of mango. Postharvest News Inform. 4: 27-34. Janisiewicz, W. J. and Korsten, L. 2002. Biological control of postharvest diseases of fruits. Annu. Rev. Phytopathol. 40: 411-441. Kefialew, Y. and Ayalew, A. 2008. Postharvest biological control of anthracnose (Colletotrichum gloeosporioides) on mango (Mangifera indica). Postharvest Biol. Technol. 50: 8-11. Korsten, L. 1993. Biological Control of Avocado Fruit Diseases. PhD thesis. Univ. Pretoria, SA. Korsten, L., De Jager, E. S., De Villiers, E. E., Lourens, A., Kotze, J. M., and Wehner, F. C. 1995. Evaluation of bacterial epiphytes isolated from avocado leaf and fruit surfaces for biological control of avocado postharvest diseases. Plant Dis. 79: 1149-1156. Korsten, L. and Jeffries, P. 2000. Potential for biological control of diseases caused by Colletotrichum. Pages 266-295 in: Colletotrichum Host Specificity, Pathology and Host-Pathogen Interaction. Prusky, D., Freeman, S. and Dickman, M. B. (eds). APS Press St. Paul, MN. Koumoutsi, A., Chen, X. H., Anke, H., Liesegang, H., Hitzeroth, G., Franke, P., Vater, J., and Borriss, R. 2004. Structural and functional characterization of gene clusters directing nonribosomal synthesis of bioactive cyclic lipopeptides in Bacillus amyloliquefaciens strain FZB42. J. Bacteriol. 186: 1084-1096. McQuilken, M. P., Budge, S. P., and Whipps, J. M. 1997. Effects of culture media and environmental factors on conidial germination, pycnidial production and hyphal extension of Coniothyrium minitans. Mycol. Res. 101: 11-17. McQuilken, M. P., Gemmell, J., and Whipps, J. M. 2002. Some nutritional factors affecting production of biomass and antifungal metabolites of Coniothyrium minitans. Biocontrol Sci. Technol. 12: 443-454. Morin, L. 1992. Realizing the potential of bioherbicides. Plant Prot. Q. 7: 143-148. Mukherjee, S. K. 1997. Introduction: botany and importance. Pages 1-9 in: The Mango: Botany, Production and Uses. Litz, R. E. (ed). CAB International, Wallingford, U.K. Patino-Vera, M., Jimenez, B., Balderas, K., Ortiz, M. and Allende, R., Carrillo, A., and Galindo, E. 2005. Pilot-scale production and liquid formulation of Rhodotorula minuta, a potential biocontrol agent of mango anthracnose. J. Appl. Microbiol. 99: 540-550. Peighami-Ashnaei, S., Sharifi-Tehrani, A., Ahmadzadeh, M., and Behboudi, K. 2009. Interaction of different media on production and biocontrol efficacy of Pseudomonas fluorescens P-35 and Bacillus subtilis B-3 against greymould of apple. J. Plant Pathol. 91: 65-70. Peppler, H. J. 1982. Yeast extract. Pages 293-321 in: Fermented Foods. Rose, A. H. (ed.). Academic Press, London, UK. Ramarathnam, R., Bo, S., Chen, Y., Dilantha Fernando, W. G., Xuewen, G., and De Kievit, T. 2007. Molecular and biochemical detection of fengycin and bacillomycin D-producing Bacillus spp., antagonistic to fungal pathogens of canola and wheat. Can. J. Microbiol. 53: 901-911. Rivas, R., Velazquez, R. R. E., Zurdo-Pineiro, J. L., Mateos, P. F., and Molina, E. M. 2004. Identification of microorganisms by PCR amplification and sequencing of a universal amplified ribosomal region present in both prokaryotes and eukaryotes. J. Microbiol. Methods 56: 413-426. Rush, A. B. 1789. An account of the bilious remitting fever as it appeared in Philadelphia in the summer and autumn of the year 1780. Pages 104-117 in: Medical Inquires and Observations. Benjamin Rush, M. D. (ed.). Prichard and Hall Philadelphia. Simmons, J. S. 1926. A culture medium for differentiating organisms of typhoid-colon aeorgenes groups and for isolation of certain fungi. J. Inf. Dis. 39: 209-214. Smith, T. J., Hillier, A. J., and Lee, G. J. 1975. The nature of the stimulation of the growth of Streptococcus lactis by yeast extract. J. Dairy Res. 42: 123-138. Thaniyavarn, J., Roongsawang, N., Kameyama, T., Haruki, M., Imanaka, T., Morikawa, M., and Kanaya, S. 2003. Production and characterization of biosurfactants from Bacillus licheniformis F2.2. Biosci. Biotechnol. Biochem. 67: 1239-1244. Terry, L. A. and Joyce, D. C. 2004. Elicitors of induced disease resistance in postharvest horticultural crops: a brief review. Postharvest Biol. Technol. 32: 1-13. Vivekananthan, R., Ravi, M., Saravanakumar, D., Kumar, N., Prakasam, V., and Samiyappan, R. 2004. Microbially induced defense related proteins against postharvest anthracnose infection in mango. Crop Prot. 23: 1061-1067. Yang, R., Han, Y. C., Li, G. Q., Jiang, D. H., and Huang, H. C. 2008. Effects of ambient pH and nutritional factors on antifungal activity of the mycoparasite Coniothyrium minitans. Biol. Control 44: 116-127.
Colletotrichum gloeosporioides引起的檬果炭疽病,是一個重要的貯藏病害,也是造成檬果在貯藏期損失的主要原因之一。目前防治檬果炭疽病之方法中,最常用的方法是以施用化學藥劑為主,但由於抗藥性菌株的出現及環保意識的抬頭,亟須找尋替代的防治方法。本研究主要目的在於篩選有效防治檬果炭疽病的拮抗細菌,並調製培養拮抗細菌的培養基配方,祈能研製微生物植物保護製劑,用於防治檬果炭疽病。自雲林及南投分離細菌後,經由切離葉生物分析、溶血測試及 16S rDNA 分子鑑定,結果發現 Paenibacillus macerans EC-21-02、Bacillus licheniformis EC-31-02 和 EC-34-01 以及 Brevibacillus aqri NA-27-01 和 NN-13-01 等菌株可將檬果炭疽病之罹病度由 100% 降至 0%,且它們均不具有溶血反應與不屬於人體之伺機性病原。設計 25 種不同組成配方對於拮抗菌抑菌的影響,試驗結果發現將 EC-34-01、NA-27-01 及NN-13-01 分別培養於 SY+raffinose pentahydrate、SY+molasses 及 Yeast+NaCl 三種不同之培養基配方中,其醱酵液對於 C. gloeosporioides MG-2 之菌絲抑制率最佳;進一步將 EC-34-01、NA-27-01 及NN-13-01 培養於 Surimi-Molasses (SM) 及 Soybean meal-Surimi-Molasses (SSM) 培養基中,其獲得的醱酵液以切離葉評估防病之功效,結果發現 EC-34-01 培養於 SSM 培養基之 100 稀釋醱酵液可使罹病度由 100% 降至 14% 左右,其中又以培養在酸鹼值 8.0 之 SSM 醱酵液的效果最為優異。利用生理生化測試、Biolog 鑑定系統及脂肪酸鑑定系統鑑定,進一步佐証 EC-34-01 菌株的身分外,隨後利用 27 種不同的專一性引子對偵測 B. licheniformis EC-34-01 可能產生的代謝產物,結果推測其擁有可產生 4’-phosphopantetheinyl transferase、Acyl-homoserine lactonasem、Bacillomycin A、Bacillomycin D、Iturin C、Iturin A、Bacilysin、Bacillaene、Difficidin、Bacillibactin、Fengycin、Macrolactin、Surfactin 及 Pleiotropic regulator 等 14 種物質之基因序列。為提昇 B. licheniformis EC-34-01 在 SSM 培養基之醱酵液的防病效果,發現醱酵液中加入 5% 酒精之 500 倍稀釋液可使罹病度由 100% 降至 10% 左右,具有良好防治病害的效果。將 B. licheniformis EC-34-01 醱酵液之粗萃取液進行薄層生物分析,只有在酸鹼值 2 的環境下所萃取之粗萃取液可對 C. gloeosporioides MG-2 產生抑制圈,且具有抑制炭疽病菌菌絲生長與孢子發芽之效果。EC-34-01 之粗萃取液中的主要抑菌成分之 Rf 值約 0.78,歸屬於低極性分子且為耐熱之胜肽類。

Mango anthracnose, caused by Colletotrichum gloeosporioides, is not only an important postharvest disease but also one of the reasons causing crop losses. Up to now, the use of fungicides is the main approach to control mango anthracnose, however, due to the emergence of fungicide-resistant pathogens and the rise of environmental awareness, the search for alternative methods is important. The main purpose of this study is to search and formulate a medium for antagonistic bacteria to control mango anthracnose, and in turn develop it into a biopesticide to control the disease. The antagonistic bacteria obtained from Nantou and Yunlin were identified and evaluated by detached leaf bioassay, hemolysis test and molecular identification – 16S rDNA. Results indicated that five strains, including Paenibacillus macerans EC-21-02, Bacillus licheniformis EC-31-02 and EC-34-01, Brevibacillus aqri NA-27-01 and NN-13-01 were able to reduce the severity of mango anthracnose from 100% to 0%, did not produce hemolysis reactions and were not opportunistic pathogens for humans. Twenty-five cultural media containing different components were designed. The fermented broths of EC-34-01 cultured in SY+raffinose pentahydrate, NA-27-01 in SY+molasses and NN-13-01 in Yeast+NaCl did show to be the most effective in inhibiting mycelial growth of the pathogen. Then fermented broths of EC-34-01, NA-27-01 and NN-13-01 cultured in Surimi-Molasses (SM) and Soybean meal-Surimi-Molasses (SSM) media were analyzed by bioassay method of detached mango leaf. One hundredfold dilution of fermented broth of EC-34-01 cultured in SSM medium could reduce 86% anthracnose severity compared to the control, and found SSM at pH 8.0 was the most optimal for culturing EC-34-01 in controlling mango anthracnose. B. licheniformis EC-34-01 was reconfirmed via physiological and biochemical characteristics assays, Biolog MicrologTM computer software and the analysis of fatty acid methyl esters microbial identification system. In addition, twenty-seven specific primers were used to detect gene sequences in Bacillus licheniformis EC-34-01 with fourteen kinds of ability for producing 4’-phosphopantetheinyl transferase、Acyl-homoserine lactonasem、Bacillomycin A、Bacillomycin D、Iturin C、Iturin A、Bacilysin、Bacillaene、Difficidin、Bacillibactin、Fengycin、Macrolactin、Surfactin and Pleiotropic regulator. After fermentation, five hundredfold dilution of EC-34-01 fermented broth with 5% ethanol was effective in reducing disease severity level from 100% to 10%. Analysis of the crude extracts of EC-34-01 fermented broths by inhibitory activity assay showed that the crude extract obtained from the condition at pH 2 did not only show inhibitory zone for C. gloeosporioides MG-2 but also inhibited its mycelial growth and conidial germination. The Rf value of bioactive compounds of crude extract of EC-34-01 fermented broth was approximately 0.78, indicating the compounds belong to the low polarity and heat-tolerance of peptides.
其他識別: U0005-2507201116494700
Appears in Collections:植物病理學系

Show full item record

Google ScholarTM


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