一株香蕉枯萎病拮抗菌HQB-1的分离鉴定及其发酵条件优化
|
摘要
【背景】香蕉枯萎病是由尖孢镰刀菌古巴专化型(Fusarium oxysporum f. sp. cubense,Foc)引起的一种真菌毁灭性土传病害,近年来施用生防菌被认为是一种有效的防治手段。【目的】从香蕉根际土壤中分离筛选具有良好防效的生防菌,并通过优化培养基及发酵条件,提高生防菌数量及抑菌效率。【方法】以福建省漳州蕉园中根际土壤为样品,以香蕉枯萎病致病菌(4号生理小种)为指示菌,通过稀释涂布、平板对峙法筛选得到一株具有较强抑菌活性的拮抗菌株HQB-1。通过形态观察、生理生化检测及16SrRNA基因序列分析,初步鉴定其种属,并采用单因素试验及正交设计优化菌株的发酵培养基及发酵条件。【结果】初步鉴定HQB-1菌株为Burkholderiastagnalis;最适培养基为:牛肉膏5.0 g/L,酵母浸粉10.0 g/L,NaCl 5.0 g/L;最佳发酵条件为:温度27°C,pH 7.0,转速200 r/min,接种量1%,培养时间36 h。【结论】使用该条件培养获得的有效活菌数及抑菌率较优化前明显提高,其中OD600由优化前的1.251提高至1.881,抑菌率由优化前的9.18%提高至34.60%。
[Background] Banana wilt is a fungal devastating soil-borne disease caused by Fusarium oxysporum f. sp. cubense(Foc). In recent years, the application of biocontrol bacterium is an effective means of prevention and control. [Objective] Biocontrol bacteria with good control effect were isolated and screened from the rhizosphere soil of banana, and the number of biocontrol bacteria and antibacterial efficiency were improved by optimizing the medium and fermentation conditions. [Methods] The rhizosphere soil was collected from orchard in Zhangzhou, Fujian province. The pathogen of Foc Tropical Race 4(Foc 4) was used as indicator fungus. An antagonistic strain named HQB-1 with strong antifungal activity was isolated by dilution coating and plate confrontation method. Strain HQB-1 was identified by morphological observation, physiological and biochemical experiments and 16 S rRNA gene sequence analysis. The fermentation conditions were optimized by single factor experiment and orthogonal experiment design. [Results] Strain HQB-1 was identified as Burkholderia stagnalis, the optimized medium was beef extract 5.0 g/L, yeast extract 10.0 g/L, NaCl 5.0 g/L, and the optimal culture temperature at 27 °C, pH 7.0, rotation speed 200 r/min, inoculation amount 1%, and the culture time 36 h. [Conclusion] The number of effective viable cells and the inhibition rate were, improved significantly than before. The OD600 was increased from 1.251 to 1.881, and the inhibition rate was from9.18% to 34.60%.
[Background] Banana wilt is a fungal devastating soil-borne disease caused by Fusarium oxysporum f. sp. cubense(Foc). In recent years, the application of biocontrol bacterium is an effective means of prevention and control. [Objective] Biocontrol bacteria with good control effect were isolated and screened from the rhizosphere soil of banana, and the number of biocontrol bacteria and antibacterial efficiency were improved by optimizing the medium and fermentation conditions. [Methods] The rhizosphere soil was collected from orchard in Zhangzhou, Fujian province. The pathogen of Foc Tropical Race 4(Foc 4) was used as indicator fungus. An antagonistic strain named HQB-1 with strong antifungal activity was isolated by dilution coating and plate confrontation method. Strain HQB-1 was identified by morphological observation, physiological and biochemical experiments and 16 S rRNA gene sequence analysis. The fermentation conditions were optimized by single factor experiment and orthogonal experiment design. [Results] Strain HQB-1 was identified as Burkholderia stagnalis, the optimized medium was beef extract 5.0 g/L, yeast extract 10.0 g/L, NaCl 5.0 g/L, and the optimal culture temperature at 27 °C, pH 7.0, rotation speed 200 r/min, inoculation amount 1%, and the culture time 36 h. [Conclusion] The number of effective viable cells and the inhibition rate were, improved significantly than before. The OD600 was increased from 1.251 to 1.881, and the inhibition rate was from9.18% to 34.60%.
引文
[1]Hwang SC,Ko WH.Cavendish banana cultivars resistant to Fusarium wilt acquired through somaclonal variation in Taiwan[J].Plant Disease,2004,88(6):580-588
[2]Ploetz RC.Fusarium wilt of banana is caused by several pathogens referred to as Fusarium oxysporum f.sp.cubense[J].Phytopathology,2006,96(6):653-656
[3]Wang ZZ.Advances in research on banana wilt and its control[J].Plant Quarantine,2006,20(3):198-200(in Chinese)王振中.香蕉枯萎病及其防治研究进展[J].植物检疫,2006,20(3):198-200
[4]Di Pietro A,Huertas-González MD,Gutierrez-Corona JF,et al.Molecular characterization of a subtilase from the vascular wilt fungus Fusarium oxysporum[J].Molecular Plant-Microbe Interactions,2001,14(5):653-662
[5]Huang JF,Zhang FB,Pang YW,et al.Inhibition of banana Fusarium wilt by two biocontrol agents[J].Microbiology China,2017,44(4):835-844(in Chinese)黄建凤,张发宝,逄玉万,等.两株香蕉枯萎病拮抗细菌的筛选及抑菌机理[J].微生物学通报,2017,44(4):835-844
[6]Zhang N,Wu K,He X,et al.A new bioorganic fertilizer can effectively control banana wilt by strong colonization with Bacillus subtilis N11[J].Plant and Soil,2011,344(1/2):87-97
[7]Jung BK,Hong SJ,Park GS,et al.Isolation of Burkholderia cepacia JBK9 with plant growth-promoting activity while producing pyrrolnitrin antagonistic to plant fungal diseases[J].Applied Biological Chemistry,2018,61(2):173-180
[8]Waweru B,Turoop L,Kahangi E,et al.Non-pathogenic Fusarium oxysporum endophytes provide field control of nematodes,improving yield of banana(Musa sp.)[J].Biological Control,2014,74:82-88
[9]Zhang XF,Xiang L,Wang YF,et al.Identification of Penicillium oxalicum A1 strain and antagonistic effects on four species of Fusarium pathogen of apple[J].Acta Horticulturae Sinica,2016,43(5):841-852(in Chinese)张先富,相立,王艳芳,等.草酸青霉A1菌株的鉴定及对苹果4种镰孢病菌的拮抗作用[J].园艺学报,2016,43(5):841-852
[10]Yu L,Ling N,Zhang N,et al.Isolation,identification and biocontrol effect of antagonistic bacteria against Fusarium oxysporum f.sp.cubense[J].Journal of Nanjing Agricultural University,2012,35(4):81-86(in Chinese)俞鲁,凌宁,张楠,等.香蕉枯萎病拮抗菌的筛选鉴定及其生防效果[J].南京农业大学学报,2012,35(4):81-86
[11]Lugtenberg B,Kamilova F.Plant-growth-promoting rhizobacteria[J].Annual Review of Microbiology,2009,63(1):541-556
[12]Hao LH,Sun PX,Jiang ZB,et al.Liquid fermentation conditions of Bacillus subtilis[J].Journal of Shanghai Jiaotong University(Agricultural Science),2006,24(4):380-385(in Chinese)郝林华,孙丕喜,姜振波,等.枯草芽孢杆菌(Bacillus subtilis)液体发酵条件[J].上海交通大学学报:农业科学版,2006,24(4):380-385
[13]Hong P,An GD,Hu MY,et al.Optimizing fermentation condition for Bacillus amyloliquefaciens HF-01[J].Chinese Journal of Biological Control,2013,29(4):569-578(in Chinese)洪鹏,安国栋,胡美英,等.解淀粉芽孢杆菌HF-01发酵条件优化[J].中国生物防治学报,2013,29(4):569-578
[14]Dong XZ,Cai MY.Common Bacterial System Identification Manual[M].Beijing:Science Press,2001:166-171(in Chinese)东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001:166-171
[15]Zhao B,He SJ.Microbiology Experiment[M].Beijing:Science Press,2002:26-120(in Chinese)赵斌,何绍江.微生物学实验[M].北京:科学出版社,2002:26-120
[16]Saitou N,Nei M.The neighbor-joining method:a new method for reconstructing phylogenetic trees[J].Molecular Biology and Evolution,1987,4(4):406-425
[17]Sijam K,Dikin A.Biochemical and physiological characterization of Burkholderia cepacia as biological control agent[J].International Journal of Agriculture&Biology,2005,7(3):385-388
[18]Ren JH,Li H,Liu H,et al.Influence of Burkholderia pyrrocinia JK-SH007 on the microbial population and functional diversity of microbial communities in the rhizosphere soil of poplar[J].Scientia Silvae Sinicae,2016,52(5):126-133(in Chinese)任嘉红,李浩,刘辉,等.吡咯伯克霍尔德氏菌JK-SH007对杨树根际微生物数量及功能多样性的影响[J].林业科学,2016,52(5):126-133
[19]Li H,Ren JH,Ye JR.Optimization of biocontrol agent Burkholderia pyrrocinia strain JK-SH007 fermentation by response surface methodology[J].Chinese Journal of Biotechnology,2013,29(2):243-246(in Chinese)李浩,任嘉红,叶建仁.响应面法优化生防菌吡咯伯克霍尔德氏菌JK-SH007的发酵工艺[J].生物工程学报,2013,29(2):243-246
[20]Jiang Y,Huang LL,Chen CQ,et al.Screen,identification and optimized fermentation condition of an actinomycete strain against pathogenic fungus Fulvia fulva[J].Acta Microbiologica Sinica,2007,47(4):622-627(in Chinese)姜云,黄丽丽,陈长卿,等.一株拮抗番茄叶霉病菌的放线菌筛选、鉴定及发酵条件研究[J].微生物学报,2007,47(4):622-627
[21]Han YZ,Deng Z,Zhang B,et al.Optimization of fermentation conditions for production of antifungal peptides by Bacillus amyloliquefaciens H15 and comparison of extraction methods for antifugal peptides[J].Food Science,2015,36(15):135-141(in Chinese)韩玉竹,邓钊,张宝,等.解淀粉芽孢杆菌H15产抗菌肽的发酵条件优化和提取方法比较研究[J].食品科学,2015,36(15):135-141
[22]Liu JL,Xue YR,Liu CH.Optimization of shake flask-fermentation conditions for Iturin A production by endophytic Bacillus amyloliquefaciens CC09[J].Microbiology China,2014,41(1):75-82(in Chinese)刘京兰,薛雅蓉,刘常宏.内生解淀粉芽孢杆菌CC09产Iturin A摇瓶发酵条件优化[J].微生物学通报,2014,41(1):75-82
[23]Zhang JD,Yang Q.Optimization of solid-state fermentation conditions for Trichoderma harzianum using an orthogonal test[J].Genetics and Molecular Research,2015,14(1):1771-1781
[24]Liu J,Li GK,Sui Y.Optimization of culture medium enhances viable biomass production and biocontrol efficacy of the antagonistic yeast,Candida diversa[J].Frontiers in Microbiology,2017,8:2021
[2]Ploetz RC.Fusarium wilt of banana is caused by several pathogens referred to as Fusarium oxysporum f.sp.cubense[J].Phytopathology,2006,96(6):653-656
[3]Wang ZZ.Advances in research on banana wilt and its control[J].Plant Quarantine,2006,20(3):198-200(in Chinese)王振中.香蕉枯萎病及其防治研究进展[J].植物检疫,2006,20(3):198-200
[4]Di Pietro A,Huertas-González MD,Gutierrez-Corona JF,et al.Molecular characterization of a subtilase from the vascular wilt fungus Fusarium oxysporum[J].Molecular Plant-Microbe Interactions,2001,14(5):653-662
[5]Huang JF,Zhang FB,Pang YW,et al.Inhibition of banana Fusarium wilt by two biocontrol agents[J].Microbiology China,2017,44(4):835-844(in Chinese)黄建凤,张发宝,逄玉万,等.两株香蕉枯萎病拮抗细菌的筛选及抑菌机理[J].微生物学通报,2017,44(4):835-844
[6]Zhang N,Wu K,He X,et al.A new bioorganic fertilizer can effectively control banana wilt by strong colonization with Bacillus subtilis N11[J].Plant and Soil,2011,344(1/2):87-97
[7]Jung BK,Hong SJ,Park GS,et al.Isolation of Burkholderia cepacia JBK9 with plant growth-promoting activity while producing pyrrolnitrin antagonistic to plant fungal diseases[J].Applied Biological Chemistry,2018,61(2):173-180
[8]Waweru B,Turoop L,Kahangi E,et al.Non-pathogenic Fusarium oxysporum endophytes provide field control of nematodes,improving yield of banana(Musa sp.)[J].Biological Control,2014,74:82-88
[9]Zhang XF,Xiang L,Wang YF,et al.Identification of Penicillium oxalicum A1 strain and antagonistic effects on four species of Fusarium pathogen of apple[J].Acta Horticulturae Sinica,2016,43(5):841-852(in Chinese)张先富,相立,王艳芳,等.草酸青霉A1菌株的鉴定及对苹果4种镰孢病菌的拮抗作用[J].园艺学报,2016,43(5):841-852
[10]Yu L,Ling N,Zhang N,et al.Isolation,identification and biocontrol effect of antagonistic bacteria against Fusarium oxysporum f.sp.cubense[J].Journal of Nanjing Agricultural University,2012,35(4):81-86(in Chinese)俞鲁,凌宁,张楠,等.香蕉枯萎病拮抗菌的筛选鉴定及其生防效果[J].南京农业大学学报,2012,35(4):81-86
[11]Lugtenberg B,Kamilova F.Plant-growth-promoting rhizobacteria[J].Annual Review of Microbiology,2009,63(1):541-556
[12]Hao LH,Sun PX,Jiang ZB,et al.Liquid fermentation conditions of Bacillus subtilis[J].Journal of Shanghai Jiaotong University(Agricultural Science),2006,24(4):380-385(in Chinese)郝林华,孙丕喜,姜振波,等.枯草芽孢杆菌(Bacillus subtilis)液体发酵条件[J].上海交通大学学报:农业科学版,2006,24(4):380-385
[13]Hong P,An GD,Hu MY,et al.Optimizing fermentation condition for Bacillus amyloliquefaciens HF-01[J].Chinese Journal of Biological Control,2013,29(4):569-578(in Chinese)洪鹏,安国栋,胡美英,等.解淀粉芽孢杆菌HF-01发酵条件优化[J].中国生物防治学报,2013,29(4):569-578
[14]Dong XZ,Cai MY.Common Bacterial System Identification Manual[M].Beijing:Science Press,2001:166-171(in Chinese)东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001:166-171
[15]Zhao B,He SJ.Microbiology Experiment[M].Beijing:Science Press,2002:26-120(in Chinese)赵斌,何绍江.微生物学实验[M].北京:科学出版社,2002:26-120
[16]Saitou N,Nei M.The neighbor-joining method:a new method for reconstructing phylogenetic trees[J].Molecular Biology and Evolution,1987,4(4):406-425
[17]Sijam K,Dikin A.Biochemical and physiological characterization of Burkholderia cepacia as biological control agent[J].International Journal of Agriculture&Biology,2005,7(3):385-388
[18]Ren JH,Li H,Liu H,et al.Influence of Burkholderia pyrrocinia JK-SH007 on the microbial population and functional diversity of microbial communities in the rhizosphere soil of poplar[J].Scientia Silvae Sinicae,2016,52(5):126-133(in Chinese)任嘉红,李浩,刘辉,等.吡咯伯克霍尔德氏菌JK-SH007对杨树根际微生物数量及功能多样性的影响[J].林业科学,2016,52(5):126-133
[19]Li H,Ren JH,Ye JR.Optimization of biocontrol agent Burkholderia pyrrocinia strain JK-SH007 fermentation by response surface methodology[J].Chinese Journal of Biotechnology,2013,29(2):243-246(in Chinese)李浩,任嘉红,叶建仁.响应面法优化生防菌吡咯伯克霍尔德氏菌JK-SH007的发酵工艺[J].生物工程学报,2013,29(2):243-246
[20]Jiang Y,Huang LL,Chen CQ,et al.Screen,identification and optimized fermentation condition of an actinomycete strain against pathogenic fungus Fulvia fulva[J].Acta Microbiologica Sinica,2007,47(4):622-627(in Chinese)姜云,黄丽丽,陈长卿,等.一株拮抗番茄叶霉病菌的放线菌筛选、鉴定及发酵条件研究[J].微生物学报,2007,47(4):622-627
[21]Han YZ,Deng Z,Zhang B,et al.Optimization of fermentation conditions for production of antifungal peptides by Bacillus amyloliquefaciens H15 and comparison of extraction methods for antifugal peptides[J].Food Science,2015,36(15):135-141(in Chinese)韩玉竹,邓钊,张宝,等.解淀粉芽孢杆菌H15产抗菌肽的发酵条件优化和提取方法比较研究[J].食品科学,2015,36(15):135-141
[22]Liu JL,Xue YR,Liu CH.Optimization of shake flask-fermentation conditions for Iturin A production by endophytic Bacillus amyloliquefaciens CC09[J].Microbiology China,2014,41(1):75-82(in Chinese)刘京兰,薛雅蓉,刘常宏.内生解淀粉芽孢杆菌CC09产Iturin A摇瓶发酵条件优化[J].微生物学通报,2014,41(1):75-82
[23]Zhang JD,Yang Q.Optimization of solid-state fermentation conditions for Trichoderma harzianum using an orthogonal test[J].Genetics and Molecular Research,2015,14(1):1771-1781
[24]Liu J,Li GK,Sui Y.Optimization of culture medium enhances viable biomass production and biocontrol efficacy of the antagonistic yeast,Candida diversa[J].Frontiers in Microbiology,2017,8:2021
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |