請用此 Handle URI 來引用此文件: http://hdl.handle.net/11455/95770
標題: 蕈狀芽孢桿菌防治胡瓜猝倒病的功效與其殺死游走子的成分
Efficacy of Bacillus mycoides on controlling pythium damping-off of cucumber seedlings and its zoosporicidal components
作者: 彭玉湘
Yu-Hsiang Peng
關鍵字: 蕈狀芽胞桿菌
胡瓜苗猝倒病
生物防治
植物保護製劑
蕈狀芽孢桿菌菌落
營養源
抗生素
化學農藥
拮抗作用
表面素
脂胜肽
殺游走子
Bacillus mycoides
Cucumber seedling damping off
Biocontrol agent
Biopesticide
nutrient source
antibiotic
chemical pesticide
antagonist
surfactin
lipopeptide
zoosporicide
引用: 第一章 緒言 Aldrich, J., & Baker, R. 1970. Biological control of Fusarium roseum f. sp. dianthi by Bacillus subtilis. Plant Disease Reporter, 54(5), 446-448. Asaka, O. & Shoda, M. 1996. Biocontrol of Rhizoctonia solani damping-off of tomato with Bacillus subtilis RB14. Applied and Environmetal Microbiology, 62(11), 4081-4085. Bapat, S., & Shan, A. K. 2000. Biological control of fusarial wilt of pigeon pea by Bacillus brevis. Canadian Journal of Microbiology, 46(2), 125-132. Bargabus, R. L., Zidack, N. K., Sherwood, J. W., & Tavobsen, B. J. 2004. Screening for the identification of potential biological control agents that induce systemic acquired resistance in sugar beet. Biological Control, 30(2), 342-350. Braun-Kiewnick, A., Jacobsen, B. J., & Sands, D. C. 2000. Biological control of Pseudomonas syringae pv. syringae, the causal agent of basal kernel blight of barley, by antagonistic Pantoea agglomerans. Phytopathology, 90(4), 368-375. Buchanan, R. E., & Gibbons, N. E.(eds.) 1974. Bergeys’s Manual of Determinative Bacteriology, 8th eds. William and Wilkins Co., Baltimore, M. D. 1268 pp. Cawoy, H. M., Bettiol, Wagner, Fickers, Patrick, & Ongena, Marc mailto (2011). Bacillus-based biological control of plant diseases In M. Stoytcheva (Ed.), Pesticides in the Modern World - Pesticides Use and Management City : Rijeka ,Country : Croatia. InTech. Claus, D., & Berkeley, R. C. W. 1986. Genus Bacillus. Pages 1105-1139 In: Bergey’s Manual of Systematic Bacteriology vol. 2. P. H. Sneath (ed). Williams and Wilkins, Baltimore, U.S.A. Czaban, J., Księźniak, A., Wróblewska, B., & Paszkowski, W. 2004a. An attempt to protect winter wheat against Gaeumannomyces graminis var. tritici by the use of rhizobacteria, Pseudomonas fluorescens and Bacillus mycoides. Polish Journal of Microbiology, 53(2), 101-110. Czaban, J., Księźniak, A., & Perzynski, A. 2004b. An attempt to protect winter wheat against Fusarium culmorum by the use of rhizobacteria Pseudomonas fluorescens and Bacillus mycoides. Polish Journal of Microbiology, 53(3), 175-182. Dunleavy, J. 1955. Control of damping-off of sugar beet by Bacillus subtilis. Phytopathology, 45(3), 252-258. Di Franco, C., Beccari, E., Santini, T., Pisaneschi, G., & Tecce, G. (2002). Colony shape as a genetic trait in the pattern-forming Bacillus mycoides. BMC Microbiology, 2(1), 33. Di Franco, C. D., Santini, T., Pisaneschi, G., & Beccari, E. 2005. Insights into the genetic organization of the Bacillus mycoides cryptic plasmids pDx14.2 and pSin9.7 deduced from their complete nucleotide sequence. Plasmid, 54(3), 288-293. Fravel, D. R. (2005). Commercialization and Implementation of Biocontrol. Annual Reviews Phytopathology, 43(1), 337-359, Gardener, B. B. M., & Driks, D. 2004. Overview of the nature and application of biocontrol microbes: Bacillus spp. Phytopathology, 94(11),1244. Gueldner RC., Reilly CC., Pusey PL. Costello CF., Arrendale RF., Cox RH., Himmelsbach DS., Crumley FG., & Cutler HG. 1988. Isolation and identification of iturins as antifungal peptides in biological control of peach brown rot with Bacillus subtilis. Journal of agricultural and food chemistry, 36(2), 366-370. Guetsky, R., Dinoor, A., Elad, Y., & Shtienberg, D. 1998. Microorganism combinations for the biocontrol of gray mold (Botrytis cinerea) in strawberries. (Abstr.) Phytoparasitica, 26(2), 174. Guetsky, R., Elad, Y., Shtienberg, D., & Dinoor, A. 2001. Combining Biocontrol Agents to Reduce the Variability of Biological Control. Phytopathology, 91(7), 621-627 Hall, T. J. 1986. Effect of xylem-colonizing Bacillus spp. on Verticillium wilt in maples. Plant Disease, 70(6), 521-524. Jack, A. L. H., & Nelson, E. B. (2017). A seed-recruited microbiome protects developing seedlings from disease by altering homing responses of Pythium aphanidermatum zoospores. Plant and Soil, doi:10.1007/s11104-017-3257-2. Lin, W. G. 1987. Studies on the pathogenesis -related physiological and biological characteristics of Colletotrichum gloeosporioides on papaya. Master thesis, Department of Plant Pathology, National Chung-Hsing University, 63 pp. Liu, L., Kloepper, J. W., & Tuzun, S. 1995. Induction of systemic resistance in cucumber against bacterial angular leaf spot by plant growth-promoting rhizobacteria. Phytopathology, 85(8), 843-847. Montesinos, E. 2007. Antimicrobial peptides and plant disease control. FEMS Microbiologt Letters, 270(1), 1–11 Ongena M, Jacques P, Toure Y, Destain J, Jabrane A & Thonart P. 2005. Involvement of fengycin-type lipopeptides in the multifaceted biocontrol potential of Bacillus subtilis. Applied Microbiology and Biotechnology, 69(1), 29–38. Ongena M., & Jacques P. 2007. Bacillus lipopeptides: versatile weapons for plant disease biocontrol. Trends in Microbiology, 16(3), 115-125. Pruvost, O., & Luisetti, J. 1991. Attempts to develop a biological control of bacterial black spot of mangoes. Acta horticulturae, 291, 324-337. Pusey, P. L., Hotchkiss, M. W., Dulmage, H. T., Baumgardner, R. A., Zehu, E. I., Reilly, C. C., & Wilson, C. l. 1988. Pilot tests for commercial production and application of Bacillus subtilis (B-3) for postharvest control of peach brown rot. Plant Disease, 72(6), 622-626. Regnault-Roger, C. (2012). Trends for Commercialization of Biocontrol Agent (Biopesticide) Products. In J. M. Mérillon, & K. G. Ramawat (Eds.), Plant Defence: Biological Control (pp. 139-160). Dordrecht: Springer Netherlands. Stein, T. 2005. Bacillus subtilis antibiotics: structures, syntheses and specific functions. Molecular Microbiology, 56(4), 845–857. Tabassum, B., Khan, A., Tariq, M., Ramzan, M., Iqbal Khan, M. S., Shahid, N., & Aaliya, K. (2017). Bottlenecks in commercialisation and future prospects of PGPR. Applied Soil Ecology, 121(Supplement C), 102-117. Whipps, J. M. 2001. Microbial interactions and biocontrol in the rhizosphere. Journal of Experimental Botany, 52, 487-511. 第二章 篩選具有生物防治胡瓜苗猝倒病能力的蕈狀芽孢桿菌 鄭安秀。1995。瓜類育苗期病蟲害管理。瓜類作物保護技術研討會專刊 : 53-55。 Bargabus, R. L., Zidack, N. K., Sherwood, J. E., & Jacobsen, B. J. (2002). Characterisation of systemic resistance in sugar beet elicited by a non-pathogenic, phyllosphere-colonizing Bacillus mycoides, biological control agent. Physiological and Molecular Plant Pathology, 61(5), 289-298. Buyer, J. S. (1995). A Soil and Rhizosphere Microorganism Isolation and Enumeration Medium That Inhibits Bacillus mycoides. Applied and Environmetal Microbiology, 61(5), 1839-1842. Czaban, J., Ksiezniak, A., & Perzynski, A. (2004a). An attempt to protect winter wheat against Fusarium culmorum by the use of rhizobacteria Pseudomonas fluorescens and Bacillus mycoides. Polish Journal of Microbiology, 53(3), 175-182. Czaban, J., Ksiezniak, A., Wroblewska, B., & Paszkowski, W. L. (2004b). An attempt to protect winter wheat against Gaeumannomyces graminis var. tritici by the use of rhizobacteria Pseudomonas fluorescens and Bacillus mycoides. Polish Journal of Microbiology, 53(2), 101-110. Dal Cortivo, C., Barion, G., Visioli, G., Mattarozzi, M., Mosca, G., & Vamerali, T. (2017). Increased root growth and nitrogen accumulation in common wheat following PGPR inoculation: Assessment of plant-microbe interactions by ESEM. Agriculture, Ecosystems & Environment, 247(Supplement C), 396-408. Di Franco, C., Beccari, E., Santini, T., Pisaneschi, G., & Tecce, G. (2002). Colony shape as a genetic trait in the pattern-forming Bacillus mycoides. BMC Microbiology, 2(1), 33. Farr, D.F., & Rossman, A.Y., (2017). Fungal Databases. U.S. National Fungus Collections, ARS, USDA. from https://nt.ars-grin.gov/fungaldatabases/. Fravel, D. R. (2005). Commercialization and Implementation of Biocontrol. Annual Review Phytopathology, 43(1), 337-359. Glickmann, E., & Dessaux, Y. (1995). A Critical Examination of the Specificity of the Salkowski Reagent for Indolic Compounds Produced by Phytopathogenic Bacteria. Applied and Environmetal Microbiology, 61(2), 793-796. Guetsky, R., Shtienberg, D., Elad, Y., & Dinoor, A. (2001). Combining biocontrol agents to reduce the variability of biological control. Phytopathology, 91(7), 621-627. Hendrix, F. F., & Campbell, W. A. (1973). Pythiums as Plant Pathogens. Annual Review Phytopathology, 11(1), 77-98. Hopkins, W. G., & Hüner, N. P. A. (2008). Introduction to plant physiology: Wiley. Knaysi, G. (1945). A Study of Some Environmental Factors Which Control Endospore Formation by a Strain of Bacillus mycoides. Journal of Bacteriology, 49(5), 473-493. Lewis, I. M. (1932). Dissociation and Life Cycle of Bacillus mycoides. Journal of Bacteriology, 24(5), 381-421. Mohite, B. (2013). Isolation and characterization of indole acetic acid (IAA) producing bacteria from rhizospheric soil and its effect on plant growth. Journal of Soil Science and Plant Nutrition, 13(3), 638-649. Neher, O. T., Johnston, M. R., Zidack, N. K., & Jacobsen, B. J. (2009). Evaluation of Bacillus mycoides isolate BmJ and B. mojavensis isolate 203-7 for the control of anthracnose of cucurbits caused by Glomerella cingulata var. orbiculare. Biological Control, 48(2), 140-146. Tabassum, B., Khan, A., Tariq, M., Ramzan, M., Iqbal Khan, M. S., Shahid, N., & Aaliya, K. (2017). Bottlenecks in commercialisation and future prospects of PGPR. Applied Soil Ecology, 121(Supplement C), 102-117. Vejan, P., Abdullah, R., Khadiran, T., Ismail, S., & Nasrulhaq Boyce, A. (2016). Role of Plant Growth Promoting Rhizobacteria in Agricultural Sustainability—A Review. Molecules, 21(5), 573. Zhao, Y. (2010). Auxin Biosynthesis and Its Role in Plant Development. Annual Review of Plant Biology, 61(1), 49-64. 第三章 影響蕈狀芽孢桿菌生育的營養源及化學藥劑 許如君。2017。農用藥劑分類及作用機制檢索(第二版)。行政院農委會動物植物防疫檢疫局。https://www.baphiq.gov.tw/files/web_articles_files/baphiq/16394/16213.pdf 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(2): 79-87. 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(2): 117-130 Cawoy, H. M., Bettiol, Wagner, Fickers, Patrick, & Ongena, Marc mailto (2011). Bacillus-based biological control of plant diseases In M. Stoytcheva (Ed.), Pesticides in the Modern World - Pesticides Use and Management City : Rijeka ,Country : Croatia. InTech. Dakora, F. D., & Phillips, D. A. (2002). Root exudates as mediators of mineral acquisition in low-nutrient environments.. Plant and Soil, 245(1), 35-47. Di Franco, C., Beccari, E., Santini, T., Pisaneschi, G., & Tecce, G. (2002). Colony shape as a genetic trait in the pattern-forming Bacillus mycoides. BMC Microbiology, 2(1), 33. Fravel, D. R. (2005). Commercialization and Implementation of Biocontrol. Annual Review Phytopathology, 43(1), 337-359. Gause, G. (1939). Some physiological properties of dextral and of sinistral forms in Bacillus mycoides flügge. Biology Bulletin Woods Hole, MA, 76(3), 448 - 465. Knight, B. C. J. G., & Proom, H. (1950). A Comparative Survey of the Nutrition and Physiology of Mesophilic Species in the Genus Bacillus. Microbiology, 4(3), 508-538. Schneider-Poetsch, T., Ju, J., Eyler, D. E., Dang, Y., Bhat, S., Merrick, W. C., et al. (2010). Inhibition of Eukaryotic Translation Elongation by Cycloheximide and Lactimidomycin. Nature chemical biology, 6(3), 209-217. Soufiane, B., & Côté, J.-C. (2013). Bacillus weihenstephanensis characteristics are present in Bacillus cereus and Bacillus mycoides strains. FEMS Microbiology Letters, 341(2), 127-137. Thakore, Y. (2006). The biopesticide market for global agricultural use. Industrial Biotechnology, 2(3), 194-208. Walker, T. S., Bais, H. P., Grotewold, E., & Vivanco, J. M. (2003). Root Exudation and Rhizosphere Biology. Plant Physiology, 132(1), 44-51. Yánez-Mendizábal, V., Viñas, I., Usall, J., Torres, R., Solsona, C., & Teixidó, N. (2012). Production of the postharvest biocontrol agent Bacillus subtilis CPA-8 using low cost commercial products and by-products. Biological Control, 60(3), 280-289. Zheng, Y., Chen, F., & Wang, M. (2013). Use of Bacillus-Based Biocontrol Agents for Promoting Plant Growth and Health. In D. K. Maheshwari (Ed.), Bacteria in Agrobiology: Disease Management (pp. 243-258). Berlin, Heidelberg: Springer Berlin Heidelberg. 第四章 蕈狀芽孢桿菌之抑菌成分分析 Cawoy, H. M., Bettiol, Wagner, Fickers, Patrick, & Ongena, Marc mailto (2011). Bacillus-based biological control of plant diseases In M. Stoytcheva (Ed.), Pesticides in the Modern World - Pesticides Use and Management City : Rijeka ,Country : Croatia. InTech. Chaurasia, B., Pandey, A., Palni, L. M. S., Trivedi, P., Kumar, B., & Colvin, N. (2005). Diffusible and volatile compounds produced by an antagonistic Bacillus subtilis strain cause structural deformations in pathogenic fungi in vitro. Microbiological Research, 160(1), 75-81. Chen, H., Wang, L., Yuan, C., Zheng, Z., & Yu, Z. (2008). Isolation and identification of lipopeptides produced by Bacillus subtilis using high performance liquid chromatography and electrospray ionization mass spectrometry. Se Pu, 26(3), 343-347. Hendrix, F. F., & Campbell, W. A. (1973). Pythiums as Plant Pathogens. Annu Rev Phytopathol, 11(1), 77-98. Meena, K. R., & Kanwar, S. S. (2015). Lipopeptides as the Antifungal and Antibacterial Agents: Applications in Food Safety and Therapeutics. BioMed Research International, 2015, 9, doi:10.1155/2015/473050. Najafi, A. R., Rahimpour, M. R., Jahanmiri, A. H., Roostaazad, R., Arabian, D., & Ghobadi, Z. (2010). Enhancing biosurfactant production from an indigenous strain of Bacillus mycoides by optimizing the growth conditions using a response surface methodology. Chemical Engineering Journal, 163(3), 188-194. Ongena, M., & Jacques, P. (2008). Bacillus lipopeptides: versatile weapons for plant disease biocontrol. Trends in Microbiology, 16(3), 115-125. Du, Yi-Chen, Jen, Jen-Fon, Huang, Jenn-Wen, Liu, & Yung-Chuan. (2011). Rapid determination of surfactin in microbial fermentation broths by salt-assisted homogeneous liquid-liquid extraction coupled with HPLC-UV. 2nd Dalian International Symposium and Exhibition on Chromatography and Related Techniques (2nd DISEC), Dalian Institute of Chemical Physics, Chinese Academy of Sciences. 第五章 結論 黃靜淑。2008。Bacillus mycoides 防治甘藍幼苗病害之效果評估。國立中興大學植物病理學系碩士論文。70 pp.。 楊玉婷、陳枻廷。2015。農用生物製劑產業發展與有機。農業農業生技產業季刊44:25-34 Dennis, P. G., Miller, A. J., & Hirsch, P. R. (2010). Are root exudates more important than other sources of rhizodeposits in structuring rhizosphere bacterial communities? FEMS Microbiology Ecology, 72(3), 313-327, doi:10.1111/j.1574-6941.2010.00860.x. Ishag, A. E. S. A., Abdelbagi, A. O., Hammad, A. M. A., Elsheikh, E. A. E., Elsaid, O. E., Hur, J. H., & Laing, M. D. (2016). Biodegradation of Chlorpyrifos, Malathion, and Dimethoate by Three Strains of Bacteria Isolated from Pesticide-Polluted Soils in Sudan. Journal of Agriculture and Food Chemistry, 64(45), 8491-8498. Jafari haghighi, B., Alizadeh, O., & Hedayati Firoozabadi, A. (2011). The role of Plant Growth Promoting Rhizobacteria (PGPR) in sustainable Agriculture. Advances in Environmental Biology, 5(10), 3079-3083. Ongena, M., & Jacques, P. (2008). Bacillus lipopeptides: versatile weapons for plant disease biocontrol. Trends in Microbiology, 16(3), 115-125. Perry, L. G., Weir, T. L., Prithiviraj, B., Paschke, M. W., & Vivanco, J. M. (2006). Root Exudation and Rhizosphere Biology: Multiple Functions of a Plant Secondary Metabolite. In F. Baluška, S. Mancuso, & D. Volkmann (Eds.), Communication in Plants: Neuronal Aspects of Plant Life (pp. 403-420). Berlin, Heidelberg: Springer Berlin Heidelberg. Sharef, Ibrahim B., Abdelbagi, Azhari O., Elsheikh, Elsiddig A. E., & Ahmed, Abd Elaziz S. (2013). Biodegradation of Pendimethalin by Three strains of Bacteria Isolated from Pesticide-Polluted Soils. University of Khartoum Journal of Agricultural Sciences, 21(2), 233-252. Tyagny-Ryadno, M. (2009). The relations of Bacillus mycoides with ammonification, nitrification, and soil fertility. The Journal of Agricultural Science, 23(3), 335-358. Vejan, P., Abdullah, R., Khadiran, T., Ismail, S., & Nasrulhaq Boyce, A. (2016). Role of Plant Growth Promoting Rhizobacteria in Agricultural Sustainability—A Review. Molecules, 21(5), 573. Walker, T. S., Bais, H. P., Grotewold, E., & Vivanco, J. M. (2003). Root Exudation and Rhizosphere Biology. Plant Physiology, 132(1), 44-51.
摘要: 從台灣中南部不同作物田間分離獲得20株蕈狀芽孢桿菌(Bacillus mycoides),隨後分別施用於塑膠杯水耕栽培胡瓜幼苗,並接種胡瓜猝倒病菌(Pythium aphanidermatum) Kpa及Potap1菌株後,選獲BM02及NP02兩菌株均有防治胡瓜苗猝倒病的效果。進一步,於溫室試驗分別將蕈狀芽孢桿菌菌株的細菌懸浮液包覆處理胡瓜種子進行播種培育,或以BM02及NP02之豆漿(SM)發酵液澆灌處理胡瓜苗,再以病原菌游走子接種胡瓜苗,保濕3天後,結果顯示BM02及NP02兩菌株亦均具顯著降低猝倒病發生之功效,其中尤以NP02菌株降低30%植株死亡率效果最佳。另外,評估蕈狀芽孢桿菌對於胡瓜生長試驗,發現將蕈狀芽孢桿菌包覆胡瓜種子與澆灌SM發酵液,均可促進胡瓜幼苗植株的發育。進一步,將胡瓜苗進行植體分析,發現蕈狀芽孢桿菌有助於提升胡瓜苗吸收大量元素,其中處理過NP02的植株含有較高濃度的氮、鉀、鈣及鎂,惟磷含量較低;BM02菌株有助於植株吸收氮及鎂元素,惟硫元素的含量較低。微量元素吸收方面,NP02菌株處理組的鐵與錳元素顯著高於BM02組及對照組。蕈狀芽孢桿菌BM02及NP02菌株培養在添加色胺酸的LB培養液後,檢測發酵液結果發現兩株均具生產植物生長素(IAA)的能力。 利用蕈狀芽孢桿菌BM02與NP02兩菌株生長在培養基平板的菌落特徵,評估蕈狀芽孢桿菌菌落之生長溫度範圍,結果顯示BM02菌株最適於生長在28-32℃,NP02菌株則在28-36℃。探討BM02及NP02菌株菌落生長的營養需求及化學藥劑的影響,發現兩菌株均以營養瓊脂(NA)為最佳生長培養基,惟馬鈴薯葡萄糖瓊脂(PDA)卻抑制菌落生長。採用改良式查氏培養基(modified Czapek’s medium)作為合成培養基的基礎配方,篩選單一種碳氮素營養源,發現果膠鹽及鳥胺酸為兩菌株菌落生長的最佳碳及氮素化合物。綜合最佳碳氮素營養源的合成培養基,選用果膠鹽搭配5種氮素化合物,分別培養BM02及NP02菌株,結果以果膠鹽與鳥胺酸之組合配方,可使BM02與NP02菌落生長速度最佳。在NA培養基平板上試驗抗生素及化學農藥對蕈狀芽孢桿菌的影響,發現各抗生素中除環己醯亞胺(cycloheximide)外,其餘皆可產生抑菌透明圈。化學農藥處理試驗中,四氯異苯腈、鋅錳乃浦及巴拉刈等3種處理有呈現抑菌透明圈,惟鋅錳乃浦處理組於培養24小時後,透明圈有逐漸縮小的趨勢。進一步,評估27種農藥包括殺菌劑、殺蟲劑及除草劑種類對蕈狀芽孢桿菌生長的影響,結果發現測試的除草劑均不利於BM02及NP02菌落生長。在測試其他殺菌劑及殺蟲劑的結果中,顯示BM02與NP02菌株可在14-15種農藥的培養基上生長。 採用兩培養皿對扣法評估蕈狀芽孢桿菌BM02及NP02菌株抑制胡瓜苗猝倒病菌Kpa及Potap1菌株的抑制功效,結果顯示BM02及NP02菌株皆可產生氣體抑制Kpa及Potap1菌株菌絲生長;其中BM02菌株抑菌效果優於NP02菌株,尤其BM02培養於TAS培養基的抑菌效果最佳。此外,將蕈狀芽孢桿菌與猝倒病菌共同培養馬鈴薯葡萄糖瓊脂(PDA)、馬鈴薯蔗糖瓊脂(PSA)、馬鈴薯萃取物瓊脂(PA)及V8蔬菜汁瓊脂(V8A)等4種培養基,結果發現BM02菌株在PA培養基抑制病原菌Kpa菌株及於V8A培養基抑制Potap1菌株的效果最佳;NP02菌株則在PSA抑制Kpa菌株及於V8A抑制Potap1菌株的效果最佳。將胡瓜苗猝倒病菌培養於96孔微量培養盤,每孔槽內添加接種過猝倒病菌的15%(v/v)V8蔬菜汁培養液150μl,在30℃光照12小時的定溫箱中培養4天,取菌絲團以無菌水清洗後,浸於無菌水中2-4小時,菌絲開始產生孢囊釋放出游走子。蕈狀芽孢桿菌於SM、SD31及CCD51等3種天然物培養之發酵液均具有抑制游走子產生的功效,尤其NP02在SM及CCD51之培養液可完全抑制游走子產生。將10種胺基酸與糊精組合的AMT-D1及果膠鹽與單一種胺基酸組合的 AMT-P1與AMT-P2培養蕈狀芽孢桿菌之發酵液,均有降低病原菌產生游走子之效果,其中BM02的AMT-P1發酵液及NP02菌株AMT-P1與AMT-P2發酵液亦均可完全抑制病原菌產生游走子。利用羊血培養基檢測法,證明蕈狀芽孢桿菌具產生脂胜肽(lipoepetides)代謝物的潛力。進一步,採用SHLLME-HPLC萃取及層析技術,分析蕈狀芽孢桿菌有無產生表面素(Surfactin)及伊枯草菌素(Iturin A)的脂胜肽代謝物的能力,結果證明蕈狀芽孢桿菌生長於PSG-C的發酵液可抑制猝倒病菌產生游走子及抑制病害發生。進一步採用表面素層析技術分析發酵液的萃取物,發現蕈狀芽孢桿菌NP02菌株可產生類似surfactin A的脂胜肽代謝物。收集NP02菌株產生脂胜肽代謝物的分餾物及市售表面素標準品,分別稀釋不同濃度並與猝倒病菌Kpa及Potap1游走子進行抑菌活性分析,結果顯示100 ppm的NP02分餾物與750ppm的表面素濃度均可抑制猝倒病菌產生游走子達99%以上。此外,利用脂胜肽(表面素與伊枯草菌素)層析技術分析蕈狀芽孢桿菌之SM發酵液的萃取物,結果發現兩種脂胜肽區域均有多個波峰出現。BM02及NP02菌株有3個表面素代謝物波峰出現,其中有類似表面素surfactin A及surfactin DE的波峰,另一個滯留時間則出現在Surfactin B與C之間;至於在伊枯草菌素代謝物區域中,BM02菌株之代謝產物出現3個近似A1、A3及A6波峰,NP02菌株的代謝物則有5個波峰的滯留時間與伊枯草菌素近似。綜合上述試驗結果證實蕈狀芽孢桿菌BM02及NP02菌株具有防治胡瓜苗猝倒病及促進胡瓜苗發育的功效,並產生具有抑菌活性的脂胜肽代謝物,確是一種值得發展成為植物保護製劑的菌種。
Twenty isolates of Bacillus mycoides Flügge were obtained from crop rhizophere soils in central and southern Taiwan. Using inoculation method of cucumber seedlings cultured in the hydroponic plastic beaker, B. mycoides isolates BM02 and NP02 were able to effectively control the Pythium damping-off caused by Pythium aphanidermatum isolates Kpa and Potap1. The greenhouse experiments indicated that both cucumber seeds coated with cell suspension of each isolate and seedlings drenched with each fermentation broth of B. mycoides isolates BM02 and NP02 were also effective in controlling cucumber seedling damping-off. Especially, the isolate NP02 could reduce more than 30% of damping-off disease. Both cucumber seed coated with cell suspension and its seedlings drenched with each fermentation broth of B. mycoides isolates BM02 and NP02 could also enhance the growth of cucumber seedlings. Both isolates of B. mycoides increased fresh weight and height of cucumber seedlings by 25 % compared to the control. Foliar analyses of the treated cucumber seedlings indicated that B. mycoides had a stimulatory effect on nutrient uptake of cucumber seedlings in the greenhouse. NP02 promoted plant to uptake macro-element nutrients including N, K, Ca and Mg, except for P. BM02 promoted uptake of macro-element nutrients including N and Mg. Cucumber seedlings could uptake more concentrations of micro-element nutrients including Fe and Mn stimulated by NP02 (177ppm and 72 ppm) than by BM02 (115ppm and 27 ppm) compared to untreated control (130ppm and 18 ppm). In addition, B. mycoides isolates BM02 and NP02 were cultured in LB (Luria-Bertani-Miller) broth with tryptophan and showed the ability of producing auxin (IAA). NP02 produced the concentration of IAA at 36.2ppm more than BM02 at15.9 ppm. This study indicated that B. mycoides isolates BM02 and NP02 were more effective in controlling cucumber damping-off and promoting the growth of cucumber seedlings. It is suggested that isolates BM02 and NP02 of B. mycoides are potential biocontrol agents for developing biopesticide. Due to unique colony form, the colony size of Bacillus mycoides isolates BM02 and NP02 grown on the solid agar were used to evaluate their physiological conditions and sensitivities to pesticides. The optimal growth temperatures were at 28-32℃for BM02 and at 28-36℃ for NP02. Four different recipe media were used to evaluate the colony growth of isolates BM02 and NP02. The results showed that nutrient agar (NA) was the best for the colony growth of both isolates. However, colony growth of isolate BM02 could not grow on potato dextrose agar (PDA). Moreover, 31 carbon sources and 38 nitrogen sources were respectively added into modified Czapeck’s media without carbon and nitrogen compounds. The results indicated that colony growth of B. mycoides isolates BM02 and NP02 cultured on the solid media with polygalacturonic acid salt (PolyGA salt) and ornithine was significantly enhanced. In addition, three peptones from natural source were more suitable for colony growth of B. mycoides isolates BM02 and NP02. The combinations of PolyGA salt and five nitrogen sources were evaluated the colony growth of B. mycoides isolates BM02 and NP02. The result indicated that combination of PolyGA salt and ornithine was the best for the growth of isolates BM02 and NP02 in modified Czapeck’s medium. The sensitivity of B. mycoides isolates BM02 and NP02 to antibiotics and chemical pesticides was evaluated by using paper disc method. Among 7 antibiotics and 13 chemical pesticides tested, 6 antibiotics and 3 chemical pesticides, which included streptomycin, neomycin, kanamycin, ampicillin, cholortetracycline, chloramphenicol, paraquat, chlorothalonil, and mancozeb, inhibited B. mycoides isolates BM02 and NP02 for 12hrs on NA plates. In advance, B. mycoides isolates BM02 and NP02 were cultured on NA medium with each of 27 chemical pesticides. Three herbicides completely inhibited the colony growth of B. mycoides isolates BM02 and NP02. Among 17 fungicides and 5 insecticides, isolate BM02 could grow on NA medium with each of 15 chemical pesticides. However NP02 could grow on NA medium with each of 14 chemical pesticides. Bacillus mycoides isolates BM02 and NP02 were effective in controlling cucumber damping off. Volatile metabolites released from B. mycoides isolates BM02 and NP02 on different culture media showed antagonistic to Pythium aphanidematum isolates Kpa and Potap1. Especially, the inhibitory activity of volatile metabolites produced from isolate BM02 grown on tryptic soy agar (TSA) was more stronger. NP02 was dually cultured in potato sucrose agar (PSA) and V8 juice agar (V8A) and showed suppressive effect on the pathogen. Moreover, the experiments indicated that the lipopeptides produced by B. mycoides were able to inhibit zoospore production by P. aphanidermatum. Several natural matter media and synthetic media were used to culture B. mycoides isolates BM02 and NP02 and evaluated for inhibiting P. aphanidermatum. Co-application of fermentation broths of B. mycoides and P. aphanidermatum was markedly effective in inhibiting zoospore release and causing zoospore lysis. PSG-C fermentation broths of B. mycoides completely inhibited zoospore production by P. aphanidermatum and protected detached cucumber leaves from water soak lesion. The fermentation broths of B. mycoides were analyzed by salt-assisted homogeneous liquid-liquid micro-extraction coupled with high-performance liquid chromatography (SHLLME-HPLC). The results showed one isoform fraction of surfactin was extracted and eluted from PSG-C fermentation broths, 3 isoform fractions of surfactin and 3-5 isoform fractions of iturin A were extracted and eluted from SM fermentation broths of B. mycoides. Moreover, comparison of zoospores released from sporangia of P. aphanidermatum suppressed by lipopeptide fractions extracted from the culture broth of B. mycoides isolate NP02 and commercial surfactin was conducted. Inhibitory activity (over 99%) of lipopeptide fraction from B. mycoides isolate NP02 at 100 ppm was greater than commercial surfactin (750 ppm). This study indicated that B. mycoides could produce surfactin/ lipopeptide metabolites to inhibit zoospore production and lyse zoospores.
URI: http://hdl.handle.net/11455/95770
文章公開時間: 2021-02-08
顯示於類別:植物病理學系

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