黄瓜枯萎病拮抗放线菌的筛选、鉴定及发酵条件优化
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摘要
【背景】黄瓜枯萎病是由尖孢镰刀菌(Fusarium oxysporum f. sp. cucumerinum)黄瓜专化型引起的土传真菌性病害,严重制约着黄瓜产业的发展。【目的】从河西走廊敦煌地区盐碱土壤中分离筛选出一株对黄瓜枯萎病病菌有良好拮抗效果的放线菌菌株,探究其分类地位及其最优发酵条件。【方法】采用稀释平板涂布法分离放线菌,平板对峙法、抑制菌丝生长速率法筛选拮抗菌株,通过培养特征、生理生化试验及16SrRNA基因序列分析确定其分类地位,利用单因素试验和正交试验方法确定其最优发酵配方及培养条件。【结果】菌株16-3-10鉴定为链霉菌属(Streptomyces sp.)菌株,最优发酵配方(g/L):小米10.0,乳糖20.0,蛋白胨1.0,NaCl 5.0,CaCO3 6.0,最优发酵条件:培养温度28°C,装瓶量50/250 mL,培养3 d,起始pH 10.0,抑菌率达82.50%,比优化前增加153.43%。【结论】菌株16-3-10对黄瓜枯萎病病菌具有显著的拮抗效果,有较好的应用前景。
[Background] Cucumber Fusarium wilt is a soil-borne fungal disease caused by Fusarium oxysporum f. sp. cucumerinum, which seriously restricts the development of cucumber industry.[Objective] Screening and identification antagonistic actinomycete strains with strongly inhibitory activity against Fusarium oxysporum f. sp. cucumerinum was isolated from saline-alkali soil in Hexi corridor. We identified the antagonistic actinomycete strain and optimized the fermentation conditions. [Methods] The method of dilution plate coating was applied to isolate actinomycetes; The methods of growth plate-confrontation and speed rate were applied to screen antagonistic actinomycetes; The morphological characteristics, physiological and biochemical characteristics, and 16 S rRNA gene sequence of strain16-3-10 were analyzed the taxonomic status. The conditions of fermentation and medium composition were optimized through single factor and orthogonal experiment. [Results] The result of identification revealed that strain 16-3-10 can be classified as Streptomyces sp., the optimum culture conditions of strain16-3-10 were with a medium was as follows: millet 10.0 g/L, lactose 20.0 g/L, peptone 1.0 g/L, NaCl5.0 g/L, CaCO3 6.0 g/L at 28 °C, liquid volume 50 m L in 250 mL flask, initial pH of 10.0 for 3 d, and inhibition rate of strain 16-3-10 to pathogen reached to 82.50% under this condition. [Conclusion] The result indicates that 16-3-10, strongly inhibitory activity against Fusarium oxysporum f. sp. cucumerinum,might act as a potential candidate for the application.
[Background] Cucumber Fusarium wilt is a soil-borne fungal disease caused by Fusarium oxysporum f. sp. cucumerinum, which seriously restricts the development of cucumber industry.[Objective] Screening and identification antagonistic actinomycete strains with strongly inhibitory activity against Fusarium oxysporum f. sp. cucumerinum was isolated from saline-alkali soil in Hexi corridor. We identified the antagonistic actinomycete strain and optimized the fermentation conditions. [Methods] The method of dilution plate coating was applied to isolate actinomycetes; The methods of growth plate-confrontation and speed rate were applied to screen antagonistic actinomycetes; The morphological characteristics, physiological and biochemical characteristics, and 16 S rRNA gene sequence of strain16-3-10 were analyzed the taxonomic status. The conditions of fermentation and medium composition were optimized through single factor and orthogonal experiment. [Results] The result of identification revealed that strain 16-3-10 can be classified as Streptomyces sp., the optimum culture conditions of strain16-3-10 were with a medium was as follows: millet 10.0 g/L, lactose 20.0 g/L, peptone 1.0 g/L, NaCl5.0 g/L, CaCO3 6.0 g/L at 28 °C, liquid volume 50 m L in 250 mL flask, initial pH of 10.0 for 3 d, and inhibition rate of strain 16-3-10 to pathogen reached to 82.50% under this condition. [Conclusion] The result indicates that 16-3-10, strongly inhibitory activity against Fusarium oxysporum f. sp. cucumerinum,might act as a potential candidate for the application.
引文
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[2]Liang Y,Zhang GY,Wang C,et al.Identification and biocontrol effect of a strain of actinomycete antagonistic to wilt disease of cucumber[J].Acta Pedologica Sinica,2013,50(4):810-817(in Chinese)梁银,张谷月,王辰,等.一株拮抗放线菌的鉴定及其对黄瓜枯萎病的生防效应研究[J].土壤学报,2013,50(4):810-817
[3]Chen F,Wang M,Zheng Y,et al.Quantitative changes of plant defense enzymes and phytohormone in biocontrol of cucumber Fusarium wilt by Bacillus subtilis B579[J].World Journal of Microbiology and Biotechnology,2010,26(4):675-684
[4]Lan JL,Su MX,Ge CB,et al.Relativity between the distribution of Fusarium oxysporum and the course of disease of wilt disease of Watermelon[J].Chinese Agricultural Science Bulletin,2007,23(12):302-305(in Chinese)蓝江林,苏明星,葛慈斌,等.尖孢镰刀菌的分布与西瓜枯萎病病程的相关性[J].中国农学通报,2007,23(12):302-305
[5]An DD,Chen YM,Li J,et al.Anti-microbial activity of endophytic actinomycetes isolated from Ammodendron bifolium and their antagonism to endophytic bacteria[J].Chinese Journal of Applied Ecology,2010,21(4):1021-1025(in Chinese)安登第,陈玉梅,李进,等.银沙槐内生放线菌抗菌活性及其与内生细菌的拮抗关系[J].应用生态学报,2010,21(4):1021-1025
[6]Li R,Shen ZZ,Sun L,et al.Novel soil fumigation method for suppressing cucumber Fusarium wilt disease associated with soil microflora alterations[J].Applied Soil Ecology,2016,101:28-36
[7]Jiang Y,Duan SR,Tang SK,et al.Rare actinomycete isolation method[J].Microbiology China,2006,33(1):181-183(in Chinese)姜怡,段淑蓉,唐蜀昆,等.稀有放线菌分离方法[J].微生物学通报,2006,33(1):181-183
[8]Niu SQ,Geng H,Yan WR,et al.Screening and identification of actinomycetes on Astragalus membranaceus root rot and optimization of fermentation conditions[J].Acta Phytophylacica Sinica,2016,43(6):943-950(in Chinese)牛世全,耿晖,阎薇茹,等.黄芪根腐病生防放线菌筛选鉴定及其优化培养[J].植物保护学报,2016,43(6):943-950
[9]Actinomycetes Classification Group,Institute of Microbiology,Chinese Academy of Sciences.Streptomyces Identification Manual[M].Beijing:Science Press,1975:162-185(in Chinese)中国科学院微生物研究所放线菌分类组.链霉菌鉴定手册[M].北京:科学出版社,1975:162-185
[10]Rong LY,Yao T,Zhao GQ,et al.Screening of siderophore-producing PGPR bacteria and their antagonism against the pathogens[J].Plant Protection,2011,37(1):59-64(in Chinese)荣良燕,姚拓,赵桂琴,等.产铁载体PGPR菌筛选及其对病原菌的拮抗作用[J].植物保护,2011,37(1):59-64
[11]Wang H,Wang JJ,Xu S,et al.Screening and growth promoting characteristics of efficient organophosphate-degradation bacteria[J].Acta Microbiologica Sinica,2017,57(5):667-680(in Chinese)王欢,王敬敬,徐松,等.有机磷降解菌的筛选及其促生特性[J].微生物学报,2017,57(5):667-680
[12]Li ZG,Luo YM,Teng Y.The Research Method of Soil and Environmental Microorganism[M].Beijing:Science Press,2008:52-61(in Chinese)李振高,骆永明,腾应.土壤与环境微生物研究法[M].北京:科学出版社,2008:52-61
[13]Zhao SL,Ren FE,Liu JL,et al.Screening,identification and optimization of fermentation conditions of an antagonistic actinomycetes strain to Setosphaeria turcica[J].Acta Microbiologica Sinica,2012,52(10):1228-1236(in Chinese)赵淑莉,任飞娥,刘金亮,等.玉米大斑病生防放线菌的筛选鉴定及发酵条件优化[J].微生物学报,2012,52(10):1228-1236
[14]Xue YY,Fan WZ,Zhang SW,et al.Screening,identification and biocontrol effect of antagonistic actinomycetes against the pathogen of Cytospora sp.for apple tree[J].Chinese Journal of Applied Ecology,2016,27(10):3379-3386(in Chinese)薛应钰,范万泽,张树武,等.苹果树腐烂病菌拮抗放线菌的筛选、鉴定及防效[J].应用生态学报,2016,27(10):3379-3386
[15]Niu SQ,Li WJ,Li HY,et al.Screening of actinomyces on antagonism to Fusariun spp.isolated from saline-alkali soils in Hexi corridor[J].Journal of Northwest Normal University(Natural Science),2017,53(2):94-98(in Chinese)牛世全,李渭娟,李海云,等.河西走廊盐碱土壤中抗马铃薯干腐病放线菌的筛选鉴定[J].西北师范大学学报:自然科学版,2017,53(2):94-98
[16]Shirling EB,Gottlieb D.Methods for characterization of Streptomyces species[J].International Journal of Systematic and Evolutionary Microbiology,1966,16(3):313-340
[17]Xu LH,Li WJ,Liu ZH,et al.Actinomycete SystematicPrinciple,Methods and Practice[M].Beijing:Science Press,2007:40-46(in Chinese)徐丽华,李文均,刘志恒,等.放线菌系统学-原理、方法及实践[M].北京:科学出版社,2007:40-46
[18]Xu P,Li WJ,Xu LH,et al.A microwave-based method for genomic DNA extraction from actinomycetes[J].Microbiology China,2003,30(4):82-84(in Chinese)徐平,李文均,徐丽华,等.微波法快速提取放线菌基因组DNA[J].微生物学通报,2003,30(4):82-84
[19]Stach JEM,Maldonado LA,Ward AC,et al.New primers for the class Actinobacteria:application to marine and terrestrial environments[J].Environmental Microbiology,2003,5(10):828-841
[20]Himaman W,Thamchaipenet A,Pathom-Aree W,et al.Actinomycetes from Eucalyptus and their biological activities for controlling Eucalyptus leaf and shoot blight[J].Microbiological Research,2016,188-189:42-52
[21]Demain AL,Sanchez S.Microbial drug discovery:80 years of progress[J].The Journal of Antibiotics,2009,62(1):5-16
[22]Li WJ,Xu P,Xu LH,et al.Actinomycete resources in extreme environments[J].Microbiology China,2003,30(4):125-127(in Chinese)李文均,徐平,徐丽华,等.极端环境中的放线菌资源[J].微生物学通报,2003,30(4):125-127
[23]Guan TW,Zhang LL,Zhao ZY,et al.Soil actinomycetes in Tarim Basin desert saline-alkali habitat[J].Journal of Microbiology,2007,27(1):80-84(in Chinese)关统伟,张利莉,赵震宇,等.塔里木盆地荒漠盐碱生境土壤放线菌的初步研究[J].微生物学杂志,2007,27(1):80-84
[24]Gurovic MSV,Olivera NL.Antibacterial producing actinomycetes from Extra Andean Patagonia[J].Journal of Arid Environments,2017,144:216-219
[25]Cheng QY,Ye L,Xing YY,et al.Fermentation conditions and characteristics of anti-bacteria active substance by marine actinomycete WB-F5[J].Chinese Journal of Natural Medicines,2007,5(2):142-145(in Chinese)程沁园,叶亮,邢莹莹,等.海洋放线菌WB-F5发酵条件的优化及抑菌活性产物[J].中国天然药物,2007,5(2):142-145
[26]Zhu HJ,Ouyang XY,Zhou Q,et al.Isolation,identification and optimizing fermentation conditions of an antagonistic strain against Colletotrichum acutata[J].Acta Phytopathologica Sinica,2012,42(4):418-424(in Chinese)朱宏建,欧阳小燕,周倩,等.一株辣椒尖孢炭疽病菌拮抗菌株的分离鉴定与发酵条件优化[J].植物病理学报,2012,42(4):418-424
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