Identification of an antagonistic strain <i>Bacillus amyloliquefaciens</i> E3 against <i>Dickeya zeae</i> and its antimicrobial activity

LIANG Liqiong; HUANG Shaoli; SHAO Hang; WANG Qingwei; WANG Xin; WANG Junxia; XU Linghui

doi:10.7671/j.issn.1001-411X.202009039

Journal of South China Agricultural University > 2021 > 42(4): 51-62. > DOI: 10.7671/j.issn.1001-411X.202009039

LIANG Liqiong, HUANG Shaoli, SHAO Hang, et al. Identification of an antagonistic strain Bacillus amyloliquefaciens E3 against Dickeya zeae and its antimicrobial activity[J]. Journal of South China Agricultural University, 2021, 42(4): 51-62. DOI: 10.7671/j.issn.1001-411X.202009039

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Identification of an antagonistic strain Bacillus amyloliquefaciens E3 against Dickeya zeae and its antimicrobial activity

Integrative Microbiology Research Centre, Guangdong Province Key Laboratory of Microbial Signals and Disease Control, South China Agricultural University, Guangzhou 510642, China

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Received Date: September 22, 2020
Available Online: May 17, 2023

Graphical Abstract

Abstract

Abstract

Objective
To isolate and identify Bacillus amyloliquefaciens strain which can inhibit the growth of Dickeya zeae EC1 from the rhizosphere soil of citrus plants, and analyze its antibacterial mechanism.

Method
The B. amyloliquefaciens E3 strain antagonizing the growth of D. zeae EC1 was screened from the rhizosphere soil by plate diffusion method. The E3 strain was classified and identified based on colony phenotype, physiological and biochemical characteristics of bacteria and 16S rDNA sequence analysis. Effects of E3 sterile culture medium on the seeds germination ability of rice infected by D. zeae EC1 was detected. The crude lipopeptides of E3 strain was extracted by hydrochloric acid precipitation combined with acetone extraction. The antimicrobial activity of crude lipopeptides against pathogenic fungi were determined by plate confrontation method. The antimicrobial activity of crude lipopetides against pathogenic bacteria were determined by agar diffusion method, and the minimum inhibitory concentration (MIC) was obtained. The crude lipopeptides were separated and purified by HPLC with Durashell C18 column. Liquid chromatography-mass spectrometry (LC-ESI-MS) analysis was used to identify the molecular weights of the antimicrobial substances and speculate its chemical composition.

Result
B. amyloliquefaciens E3 strain had an inhibitory effect against Fusarium solani, D. zeae MS1 and Ralstonia solanacearum, etc, indicating that it had broad-spectrum resistance. The sterile culture medium of B. amyloliquefaciens E3 strain inhibited the infection of germinating rice seeds by D. zeae EC1 and significantly increased the seed germination rate. The crude lipopeptides of E3 strain inhibited the growth of D. zeae EC1 with the MIC of 348.97 µg/mL. HPLC combined with LC-ESI-MS analysis showed that the main antibacterial substances secreted by E3 strain included surfactin, fengycin and iturin.

Conclusion
The B. amyloliquefaciens E3 strain is potential to be a biocontrol agent. It can antigonize a variety of plant pathogens such as D. zeae EC1, R. solanacearum and F. solani by secreting three kinds of lipopeptides including surfactin, fengycin and iturin. The results provide a theoretical basis for the application of B. amyloliquefaciens E3 in biological control.
- Dickeya zeae,
- Bacillus amyloliquefaciens,
- antibacterial activity,
- crude lipopeptides

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References (43)

References

[1]	冯成玉. 水稻细菌性基腐病发生情况与研究进展[J]. 中国稻米, 2009(4): 21-23. doi: 10.3969/j.issn.1006-8082.2009.04.007
[2]	刘琼光, 张庆, 魏楚丹. 水稻细菌性基腐病研究进展[J]. 中国农业科学, 2013, 46(14): 2923-2931. doi: 10.3864/j.issn.0578-1752.2013.14.008
[3]	PU X M, ZHOU J N, LIN B R, et al. First report of bacterial foot rot of rice caused by a Dickeya zeae in China[J]. Plant Disease, 2012, 96(12): 1818.
[4]	张蝶. 浙江省水稻细菌性基腐病发生因素及化学防治研究[D]. 金华: 浙江师范大学, 2016.
[5]	SANSINENEA E, VACA J, ELENA ROJAS N, et al. A wide spectrum of antibacterial activity of secondary metabolites from Bacillus amyloliquefaciens ELI149[J]. Bioscience Journal, 2020, 36(1): 235-244.
[6]	BEN ABDALLAH D, TOUNSI S, GHARSALLAH H, et al. Lipopeptides from Bacillus amyloliquefaciens strain 32a as promising biocontrol compounds against the plant pathogen Agrobacterium tumefaciens[J]. Environmental Science and Pollution Research, 2018, 25(36): 36518-36529.
[7]	STEIN T. Bacillus subtilis antibiotics: Structures, syntheses and specific functions[J]. Molecular Microbiology, 2005, 56(4): 845-857. doi: 10.1111/j.1365-2958.2005.04587.x
[8]	WU L M, WU H J, CHEN L N, et al. Difficidin and bacilysin from Bacillus amyloliquefaciens FZB42 have antibacterial activity against Xanthomonas oryzae rice pathogens[J]. Scientific Reports, 2015, 5(1). doi: http://doi.org/10.1038/srep12975.
[9]	LIU Y F, CHEN Z Y, NG T B, et al. Bacisubin, an antifungal protein with ribonuclease and hemagglutinating activities from Bacillus subtilis strain B-916[J]. Peptides, 2007, 28(3): 553-559. doi: 10.1016/j.peptides.2006.10.009
[10]	TAO Y, BIE X M, LV F X, et al. Antifungal activity and mechanism of fengycin in the presence and absence of commercial surfactin against Rhizopus stolonifer[J]. Journal of Microbiology, 2011, 49(1): 146-150. doi: 10.1007/s12275-011-0171-9
[11]	CHO S J, LEE S K, CHA B J, et al. Detection and characterization of the Gloeosporium gloeosporioides growth inhibitory compound iturin A from Bacillus subtilis strain KS03[J]. FEMS Microbiology Letters, 2003, 223(1): 47-51. doi: 10.1016/S0378-1097(03)00329-X
[12]	CAO H Y, JIAO Y, YIN N, et al. Analysis of the activity and biological control efficacy of the Bacillus subtilis strain Bs-1 against Meloidogyne incognita[J]. Crop Protection, 2019, 122: 125-135. doi: 10.1016/j.cropro.2019.04.021
[13]	ZHAO P, QUAN C, WANG Y, et al. Bacillus amyloliquefaciens Q-426 as a potential biocontrol agent against Fusarium oxysporum f. sp spinaciae[J]. Journal of Basic Microbiology, 2014, 54(5): 448-456. doi: 10.1002/jobm.201200414
[14]	ARGUELLES-ARIAS A, ONGENA M, HALIMI B, et al. Bacillus amyloliquefaciens GA1 as a source of potent antibiotics and other secondary metabolites for biocontrol of plant pathogens[J]. Microbial Cell Factories, 2009, 8: 63.
[15]	ONGENA M, JACQUES P. Bacillus lipopeptides: Versatile weapons for plant disease biocontrol[J]. Trends in Microbiology, 2008, 16(3): 115-125. doi: 10.1016/j.tim.2007.12.009
[16]	CAWOY H, MARIUTTO M, HENRY G, et al. Plant defense stimulation by natural isolates of Bacillus depends on efficient surfactin production[J]. Molecular Plant-Microbe Interactions, 2014, 27(2): 87-100. doi: 10.1094/MPMI-09-13-0262-R
[17]	ZHOU J, CHENG Y, LV M, et al. The complete genome sequence of Dickeya zeae EC1 reveals substantial divergence from other Dickeya strains and species[J]. BMC Genomics, 2015, 16(1): 571. doi: 10.1186/s12864-015-1545-x
[18]	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: 267-294.
[19]	布坎南, 等. 伯杰细菌鉴定手册[M]. 北京: 科学出版社, 1984: 729-758.
[20]	REDBURN A C, PATEL B K. Phylogenetic analysis of Desulfotomaculum thermobenzoicum using polymerase chain reaction-amplified 16S rRNA-specific DNA[J]. FEMS Microbiology Letters, 1993, 113(1): 81-86. doi: 10.1111/j.1574-6968.1993.tb06492.x
[21]	刘勇, 毛爱军, 李辉, 等. 枯草芽孢杆菌群β-甘露聚糖酶基因克隆及同源性分析[J]. 基因组学与应用生物学, 2009, 28(5): 845-850. doi: 10.3969/gab.028.000845
[22]	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. doi: 10.7506/spkx1002-6630-201515025
[23]	张睿佳, 胡杰, 陆建忠, 等. 不同成熟度对水稻种子萌发的影响[J]. 上海农业科技, 2017(5): 62-63. doi: 10.3969/j.issn.1001-0106.2017.05.029
[24]	LI S B, XU S R, ZHANG R N, et al. The antibiosis action and rice-induced resistance, mediated by a lipopeptide from Bacillus amyloliquefaciens B014, in controlling rice disease caused by Xanthomonas oryzae pv. oryzae[J]. Journal of Microbiology and Biotechnology, 2016, 26(4): 748-756. doi: 10.4014/jmb.1510.10072
[25]	朱弘元, 康健, 范昕, 等. 解淀粉芽孢杆菌B15产脂肽的分离鉴定及抑菌机理[J]. 江苏农业科学, 2016, 44(5): 186-189.
[26]	张玉胜, 谭亚男. 油菜核盘菌拮抗菌的筛选及鉴定[J]. 生命科学仪器, 2009, 7(8): 27-28.
[27]	陈华, 王丽, 袁成凌, 等. 高效液相色谱-电喷雾质谱法分离和鉴别枯草芽孢杆菌产生的脂肽类化合物[J]. 色谱, 2008(3): 343-347. doi: 10.3321/j.issn:1000-8713.2008.03.016
[28]	CHEN M C, WANG J P, ZHU Y J, et al. Antibacterial activity against Ralstonia solanacearum of the lipopeptides secreted from the Bacillus amyloliquefaciens strain FJAT-2349[J]. Journal of General and Applied Microbiology, 2019, 126(5): 1519-1529. doi: 10.1111/jam.14213
[29]	周海亮. 水稻细菌性基腐病病原学及检测技术的研究[D]. 武汉: 华中农业大学, 2012.
[30]	VATER J, GAO X, HITZEROTH G, et al. "Whole cell"--matrix-assisted laser desorption ionization-time of flight-mass spectrometry, an emerging technique for efficient screening of biocombinatorial libraries of natural compounds-present state of research[J]. Combinatorial Chemistry& High Throughput Screening, 2003, 6(6): 557-567.
[31]	AERON A, KHARE E, JHA C K, et al. Revisiting the plant growth-promoting rhizobacteria: Lessons from the past and objectives for the future[J]. Archives of Microbiology, 2020, 202(4): 665-676. doi: 10.1007/s00203-019-01779-w
[32]	GOUDA S, KERRY R G, DAS G, et al. Revitalization of plant growth promoting rhizobacteria for sustainable development in agriculture[J]. Research in Microbiology, 2018, 206: 131-140. doi: 10.1016/j.micres.2017.08.016
[33]	KHAYALETHU N, LESIBA K L, MOLEMI E R, et al. The mode of action of bacillus species against Fusarium graminearum, tools for investigation, and future prospects[J]. Toxins, 2019, 11(10). doi: http://doi.org/10.3390/toxins11100606.
[34]	严婉荣, 肖敏, 陈圆, 等. 解淀粉芽孢杆菌抗菌脂肽研究进展[J]. 北方园艺, 2018(7): 162-167.
[35]	FAN B, WANG C, SONG X, et al. Bacillus velezensis FZB42 in 2018: The gram-positive model strain for plant growth promotion and biocontrol[J]. Frontiers in Microbiology, 2018, 9: 2491. doi: 10.3389/fmicb.2018.02491
[36]	CHOWDHURY S P, UHL J, GROSCH R, et al. Cyclic lipopeptides of Bacillus amyloliquefaciens subsp. plantarum colonizing the lettuce rhizosphere enhance plant defense responses toward the bottom rot pathogen Rhizoctonia solani[J]. Molecular Plant-Microbe Interactions, 2015, 28(9): 984-995. doi: 10.1094/MPMI-03-15-0066-R
[37]	向亚萍, 周华飞, 刘永锋, 等. 解淀粉芽孢杆菌B1619脂肽类抗生素的分离鉴定及其对番茄枯萎病菌的抑制作用[J]. 中国农业科学, 2016, 49(15): 2935-2944. doi: 10.3864/j.issn.0578-1752.2016.15.008
[38]	FIRA D, DIMKIC I, BERIC T, et al. Biological control of plant pathogens byBacillus species[J]. Biotechnology Journal, 2018, 285: 44-55. doi: 10.1016/j.jbiotec.2018.07.044
[39]	ZERIOUH H, ROMERO D, GARCIA-GUTIERREZ L, et al. The iturin-like lipopeptides are essential components in the biological control arsenal of Bacillus subtilis against bacterial diseases of cucurbits[J]. Molecular Plant-Microbe Interactions, 2011, 24(12): 1540-1552. doi: 10.1094/MPMI-06-11-0162
[40]	CAO Y, PI H L, CHANDRANGSU P, et al. Antagonism of two plant-growth promoting Bacillus velezensis isolates against Ralstonia solanacearum and Fusarium oxysporum[J]. Scientific Reports, 2018, 8(1). doi: http://doi.org/10.1038/S41598-018-22782-Z.
[41]	HADIZADEH I, PEIVASTEGAN B, HANNUKKALA A, et al. Biological control of potato soft rot caused by Dickeya solani and the survival of bacterial antagonists under cold storage conditions[J]. Plant Pathology, 2019, 68(2): 297-311. doi: 10.1111/ppa.12956
[42]	CHEN J, LIU T, WEI M, et al. Macrolactin a is the key antibacterial substance of Bacillus amyloliquefaciens D2WM against the pathogen Dickeya chrysanthemi[J]. European Journal of Plant Pathology, 2019, 155(2): 393-404. doi: 10.1007/s10658-019-01774-3
[43]	LI J, HU M, XUE Y, et al. Screening, identification and efficacy evaluation of antagonistic bacteria for biocontrol of soft rot disease caused by Dickeya zeae[J]. Microorganisms, 2020, 8(6975). doi: 10.3390/microorganisms8050697.

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Identification of an antagonistic strain Bacillus amyloliquefaciens E3 against Dickeya zeae and its antimicrobial activity

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