Bacillus malacitensis Z-5菌株合成抑菌物质培养基优化及成分鉴定
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摘要
【目的】Bacillus malacitensis Z-5菌株对棉花黄萎病有显著防治效果,产生抑菌物质是其发挥防治作用的主要因素。本研究旨在优化Z-5菌株产生抑菌物质的培养基组成,分析抑菌物质的成分及其合成相关基因。【方法】由单因素试验确定最佳碳源、氮源及无机盐,利用最速上升法确定碳源、氮源以及无机盐最优含量区域,根据Box-Behnken试验设计,以发酵上清液对棉花黄萎病菌大丽轮枝菌(Verticillium dahlia Kleb.)的拮抗活性为指标,筛选Z-5菌株产生抑菌物质的最优培养基组成。利用液相色谱-质谱联用(Liquid Chromatography-Mass Spectrometer,LC-MS)法鉴定抑菌物质,并对抑菌物质合成相关基因进行检测。【结果】Z-5菌株产生抑菌物质最佳培养基组成为2.68%(质量分数,下同)玉米粉、1.45%酵母粉、0.03%K_2SO_4、0.8%Na_2HPO_4·12H_2O、0.2% NaH_2PO_4·2H_2O(pH 7.2～7.4),抑菌圈直径为1.34 cm,与预测值相近,证实该模型可靠有效。抑菌物质主要为脂肽抗生素C14表面活性素B,C14～C17伊枯草菌素A,C14、C15、C17伊枯草菌素B等。聚合酶链式反应检测结果显示,Z-5菌株基因组含有合成脂肽抗生素的基因srf AB、ituC和ituD。【结论】本研究优化了Z-5菌株产生抑菌物质的培养基组成,明确了其抑菌物质为脂肽抗生素,为利用该抑菌物质进行棉花黄萎病菌的防治提供了理论参考和物质基础。
[Objective] Bacillus malacitensis Z-5 has a significant biocontrol effect on cotton Verticillium wilt mainly through its production of antifungal substances. This study aimed to optimize the culture medium's composition. Additionally, it aimed to identify the genes that produce the antifungal substances and determine the components of these antifungal substances.[Methods] Single factor tests were used to determine the optimal carbon and nitrogen sources, as well as the inorganic salt level.The most rapid rise method was used to determine the carbon and nitrogen sources and the inorganic salt's optimal content area.Box–Behnken response surface methodology was applied, and the composition of the medium that produced the optimum level of antifungal substances was determined using antifungal activity as the screening index. The composition of the antifungal substances and the related genes from the Z-5 strain were identified using liquid chromatography-mass spectrometry and polymerase chain reaction, respectively. [Results] The optimal medium for the generation of antifungal substances by the Z-5 strain consisted of 2.68%(mass fraction) corn flour, 1.45% yeast powder, 0.03% K_2SO_4, 0.8% Na_2HPO_4·12 H_2O, and 0.2%NaH_2PO_4·2 H_2O at pH 7.2 – 7.4. The antifungal zone was 1.34 cm in diameter, which was close to the predicted value,confirming that the model was reliable and effective. Antifungal substances were mainly lipopeptide antibiotics C14 surfactin B,C14–C17 iturin A, and C14, C15, and C17 iturin B. polymerase chain reaction revealed that srf AB, itu C, and ituD occur in the Z-5 genome. [Conclusion] This study optimized the culture medium for producing antifungal substances, which are composedof lipopeptide antibiotics, from the B. malacitensis Z-5 strain. The results provide a theoretical reference and material basis for the prevention of cotton Verticillium wilt using these antifungal substances.
[Objective] Bacillus malacitensis Z-5 has a significant biocontrol effect on cotton Verticillium wilt mainly through its production of antifungal substances. This study aimed to optimize the culture medium's composition. Additionally, it aimed to identify the genes that produce the antifungal substances and determine the components of these antifungal substances.[Methods] Single factor tests were used to determine the optimal carbon and nitrogen sources, as well as the inorganic salt level.The most rapid rise method was used to determine the carbon and nitrogen sources and the inorganic salt's optimal content area.Box–Behnken response surface methodology was applied, and the composition of the medium that produced the optimum level of antifungal substances was determined using antifungal activity as the screening index. The composition of the antifungal substances and the related genes from the Z-5 strain were identified using liquid chromatography-mass spectrometry and polymerase chain reaction, respectively. [Results] The optimal medium for the generation of antifungal substances by the Z-5 strain consisted of 2.68%(mass fraction) corn flour, 1.45% yeast powder, 0.03% K_2SO_4, 0.8% Na_2HPO_4·12 H_2O, and 0.2%NaH_2PO_4·2 H_2O at pH 7.2 – 7.4. The antifungal zone was 1.34 cm in diameter, which was close to the predicted value,confirming that the model was reliable and effective. Antifungal substances were mainly lipopeptide antibiotics C14 surfactin B,C14–C17 iturin A, and C14, C15, and C17 iturin B. polymerase chain reaction revealed that srf AB, itu C, and ituD occur in the Z-5 genome. [Conclusion] This study optimized the culture medium for producing antifungal substances, which are composedof lipopeptide antibiotics, from the B. malacitensis Z-5 strain. The results provide a theoretical reference and material basis for the prevention of cotton Verticillium wilt using these antifungal substances.
引文
[1]杜威世,杜雄明,马峙英.棉花黄萎病抗性基因SSR标记研究[J].西北农林科技大学学报(自然科学版), 2004, 32(3):20-24.Du Weishi, Du Xiongming, Ma Zhiying. Studies on SSR markers of resistance gene of Verticillium wilt in cotton[J]. Journal of Northwest Sci-Tech University of Agriculture and Forestry(Natural Science Edition), 2004, 32(3):20-24.
[2]朱荷琴.棉花主要病害研究概要[J].棉花学报, 2007,19(5):391-398.Zhu Heqin. A summary of researches on main cotton diseases[J].Cotton Science, 2007, 19(5):391-398.
[3] Rowe R C, Powelson M L. Potato early dying:Management challenges in a changing production environment[J]. Plant Disease, 2002, 86(11):1184-1193.
[4] Xiao C L, Subbarao K V. Relationships between Verticillium dahlia inoculum density and wilt incidence, severity, and growth of cauliflower[J]. Phytopathology, 1998, 88(10):1108-1115.
[5] Uppal A K, Hadrami A E I, Adam L R, et al. Pathogenic variability of Verticillium dahlia isolates from potato fields in Manitoba and screening of bacteria for their biocontrol[J]. Canadian Journal of Plant Pathology, 2007, 29(2):141-152.
[6]张洪涛,于频频,艾山江·阿布都拉,等.棉花黄萎病高效拮抗菌XJUL-6的筛选鉴定及其特性研究[J].微生物学报, 2007,47(6):1084-1087.Zhang Hongtao, Yu Pinpin, Abudula H, et al. Characteristics and identification of an antagonistic XJUL-6 against cotton Verticillium wilt[J]. Acta Microbiologica Sinica, 2007, 47(6):1084-1087.
[7] Zhang D D, Guo X J, Wang Y J, et al. Novel screening strategy reveals a potent Bacillus antagonist capable of mitigating wheat take-all disease caused by Gaeumannomyces graminis var. tritici[J]. Letters in Applied Microbiology, 2017, 65:512-519.
[8]严婉荣,赵廷昌,肖彤斌,等.生防细菌在植物病害防治中的应用[J].基因组学与应用生物学, 2013, 32(4):533-539.Yan Wanrong, Zhao Tingchang, Xiao Tongbin, et al. Applications of biocontrol bacteria in plant disease control[J]. Genomics and Applied Biology, 2013, 32(4):533-539.
[9] Cook R J, Bruckart W L, Coulson J R, et al. Safety of microorganisms intended for pest and plant disease control:A framework for scientific evaluation[J]. Biological Control, 1996, 7(3):333-351.
[10] Falardeau J, Wise C, Novitsky L, et al. Ecological and mechanistic insights into the direct and indirect antimicrobial properties of Bacillus subtilis lipopeptides on plant pathogens[J].Journal of Chemical Ecology, 2013, 39(7):869-878.
[11] Liu J J, Hagberg I, Novitsky L, et al. Interaction of antimicrobial cyclic lipopeptides from Bacillus subtilis influences their effect on spore germination and membrane permeability in fungal plant pathogens[J]. Fungal Biology, 2014, 118:855-861.
[12] Hamdache A, Lamarti A, Aleu J, et al. Non-peptide metabolites from the genus Bacillus[J]. Journal of Natural Products, 2011,74(4):893-899.
[13] Wise C, Novitsky L, Tsopmo A, et al. Production and antimicrobial activity of 3-hydroxypropionaldehyde from Bacillus subtilis strain CU12[J]. Journal of Chemical Ecology, 2012, 38(12):1521-1527.
[14] De Vrieze M, Pandey P, Bucheli T D, et al. Volatile organic compounds from native potato-associated Pseudomonas as potential anti-oomycete agents[J]. Frontiers in Microbiology, 2015,6:1295. https://doi.org/10.3389/fmicb.2015.01295
[15] Chen X H, Vater J, Piel J, et al. Structural and functional characterization of three polyketide synthase gene clusters in Bacillus amyloliquefaciens FZB42[J]. Journal of Bacteriology, 2006,188:4024-4036.
[16]陈晓萌,王亚杰,李佳,等.枯草芽孢杆菌MG-4抑制小麦全蚀病菌物质及其性质分析[J].植物保护学报, 2018, 45(3):511-519. https://doi.org/10.13802/j.cnki.zwbhxb.2018.2017033Chen Xiaomeng, Wang Yajie, Li Jia, et al. Characterization of antifungal substances of Bacillus subtilis MG-4 against Gaeumannomyces graminis var. tritic causing wheat take-all[J].Journal of Plant Protection, 2018, 45(3):511-519.
[17] Bais H, Fall R, Vivanco J. Biocontrol of Bacillus subtilis against infection of Arabidopsis roots by Pseudomonas syringae is facilitated by biofilm formation and surfactin production[J].Plant Physiology, 2004, 134:307-319.
[18] Stein T. Bacillus subtilis antibiotics:Structures, syntheses and specific functions[J]. Molecular Microbiology, 2005, 56:845-857.
[19] Guo Q G, Dong W X, Li S Z, et al. Fengycin produced by Bacillus subtilis NCD-2 plays a major role in biocontrol of cotton seedling damping-off disease[J]. Microbiological Research,2014, 169(7/8):533-540. https://doi.org/10.1016/j.micres.2013.12.001
[20] Bie X M, Lu Z X, Lu F X. Identification of fengycin homologues from Bacillus subtilis with ESI-MS/CID[J]. Journal of Microbiological Methods, 2009, 79(3):272-278.
[21]张冬冬,李术娜,郭晓军,等.一株棉花黄萎病拮抗芽孢细菌的分离鉴定及活性检测[J].棉花学报, 2012, 24(4):358-362.Zhang Dongdong, Li Shuna, Guo Xiaojun, et al. Isolation, identification and activity of an antagonistic spore bacterium against cotton Verticillium wilt[J]. Cotton Science, 2012, 24(4):358-362.
[22]张冬冬,刘涛,高同国,等.棉花黄萎病拮抗细菌Z-5菌株的定殖能力检测[J].棉花学报, 2013, 25(6):510-516.Zhang Dongdong, Liu Tao, Gao Tongguo, et al. Detection of the colonization ability of antagonistic bacteria strain Z-5 against cotton Verticillium wilt[J]. Cotton Science, 2013, 25(6):510-516.
[23] Xiao Z J, Liu P H, Qin J Y. Statistical optimization of medium components for enhanced acetoin production from molasses and soybean meal hydrolysate[J]. Applied Microbiology and Biotechnology, 2007, 74(1):61-68.
[24] Caldeira A T, Arteiro J M S, Coelho A V, et al. Combined use of LC-ESI-MS and antifungal tests for rapid identification of bioactive lipopeptides produced by Bacillus amyloliquefaciens CCMI 1051[J]. Process Biochemistry, 2011, 46:1738-1746.
[25] Yu G Y, Sinclair J B, Hartman G L, et al. Production of iturin A by Bacillus amyloliquefaciens suppressing Rhizoctonia solani[J]. Soil Biology&Biochemistry, 2002, 34:955-963.
[26] Waseem R, Yang X, Wu H, et al. Isolation and characterization of fusaricidin-type compound-producing strain of Paenibacillus polymyxa SQR-21 active against Fusarium oxysporum f. sp.Nevium[J]. European Journal of Plant Pathology, 2009, 125:471-483.
[27] Mora I, Cabrefiga J, Montesinos E. Antimicrobial peptide genesin Bacillus strains from plant environments[J]. International Microbiology, 2011, 14(4):213-223.
[28] Joshi R, Mc Spadden Gardener B B. Identification and characterization of novel genetic markers associated with biological control activities in Bacillus subtilis[J]. Phytopathology, 2006,96(2):145-154.
[29]陈志谊.芽孢杆菌类生物杀菌剂的研发与应用[J].中国生物防治学报, 2015, 31(5):723-732. https://doi.org/10.16409/j.cnki.2095-039x.2015.05.012Chen Zhiyi. Research and application of bio-fungicide with Bacillus spp.[J]. Chinese Journal of Biological Control, 2015,31(5):723-732.
[30]乔俊卿,陈志谊,梁雪杰,等.枯草芽孢杆菌Bs916在番茄根部的定殖[J].江苏农业学报, 2015, 31(6):1278-1283. https://doi.org/10.3969/j.issn.1000-4440.2015.06.013Qiao Junqing, Chen Zhiyi, Liang Xuejie, et al. Colonization of Bacillus subtilis Bs916 on tomato root[J]. Jiangsu Journal of Agricultural Science, 2015, 31(6):1278-1283.
[31]林东,徐庆,刘忆舟,等.枯草芽孢杆菌SO113分泌蛋白的抑菌作用及抗菌蛋白的分离纯化[J].农业生物技术学报, 2001,9(1):77-80.Lin Dong, Xu Qing, Liu Yizhou, et al. The antibacterial effect of the secreted peptide from Bacillus subtilis SO113 and separation and purification of the antibacterial peptides[J]. Journal of Agricultural Biotechnology, 2001, 9(1):77-80.
[32]张冬冬,姜军坡,朱宝成.棉花黄萎病生防芽孢杆菌Z-5菌株发酵培养基的优化[J].棉花学报, 2014, 26(1):10-17.Zhang Dongdong, Jiang Junpo, Zhu Baocheng. Optimization of fermentation conditions of a biocontrol bacterial isolate Bacillus Z-5 against cotton Verticillium wilt[J]. Cotton Science, 2014,26(1):10-17.
[33] Zhang D D, Gao T G, Li H Y, et al. Identification of antifungal substances secreted by Bacillus subtilis Z-14 that suppress Gaeumannomyces graminis var. tritici[J]. Biocontrol Science and Technology, 2017, 27(2):237-251.
[34] Ongena M, Jacques P, TouréY, et al. Involvement of fengycin-type lipopeptidesin the multifaceted biocontrol potential of Bacillus subtilis[J]. Applied Microbiology and Biotechnology, 2005, 69:29-38.
[35] Yang L R, Quan X, Xue B G, et al. Isolation and identification of Bacillus subtilis strain YB-05 and its antifungal substances showing antagonism against Gaeumannomyces graminis var.tritici[J]. Biological Control, 2015, 85:52-58. https://doi.org/10.1016/j.biocontrol.2014.12.010
[36] Ye Y F, Li Q Q, Fu G, et al. Identification of antifungal substance(iturin A2)produced by Bacillus subtilis B47 and its effect on southern corn leaf blight[J]. Journal of Integrative Agriculture, 2012, 11(1):90-99.
[2]朱荷琴.棉花主要病害研究概要[J].棉花学报, 2007,19(5):391-398.Zhu Heqin. A summary of researches on main cotton diseases[J].Cotton Science, 2007, 19(5):391-398.
[3] Rowe R C, Powelson M L. Potato early dying:Management challenges in a changing production environment[J]. Plant Disease, 2002, 86(11):1184-1193.
[4] Xiao C L, Subbarao K V. Relationships between Verticillium dahlia inoculum density and wilt incidence, severity, and growth of cauliflower[J]. Phytopathology, 1998, 88(10):1108-1115.
[5] Uppal A K, Hadrami A E I, Adam L R, et al. Pathogenic variability of Verticillium dahlia isolates from potato fields in Manitoba and screening of bacteria for their biocontrol[J]. Canadian Journal of Plant Pathology, 2007, 29(2):141-152.
[6]张洪涛,于频频,艾山江·阿布都拉,等.棉花黄萎病高效拮抗菌XJUL-6的筛选鉴定及其特性研究[J].微生物学报, 2007,47(6):1084-1087.Zhang Hongtao, Yu Pinpin, Abudula H, et al. Characteristics and identification of an antagonistic XJUL-6 against cotton Verticillium wilt[J]. Acta Microbiologica Sinica, 2007, 47(6):1084-1087.
[7] Zhang D D, Guo X J, Wang Y J, et al. Novel screening strategy reveals a potent Bacillus antagonist capable of mitigating wheat take-all disease caused by Gaeumannomyces graminis var. tritici[J]. Letters in Applied Microbiology, 2017, 65:512-519.
[8]严婉荣,赵廷昌,肖彤斌,等.生防细菌在植物病害防治中的应用[J].基因组学与应用生物学, 2013, 32(4):533-539.Yan Wanrong, Zhao Tingchang, Xiao Tongbin, et al. Applications of biocontrol bacteria in plant disease control[J]. Genomics and Applied Biology, 2013, 32(4):533-539.
[9] Cook R J, Bruckart W L, Coulson J R, et al. Safety of microorganisms intended for pest and plant disease control:A framework for scientific evaluation[J]. Biological Control, 1996, 7(3):333-351.
[10] Falardeau J, Wise C, Novitsky L, et al. Ecological and mechanistic insights into the direct and indirect antimicrobial properties of Bacillus subtilis lipopeptides on plant pathogens[J].Journal of Chemical Ecology, 2013, 39(7):869-878.
[11] Liu J J, Hagberg I, Novitsky L, et al. Interaction of antimicrobial cyclic lipopeptides from Bacillus subtilis influences their effect on spore germination and membrane permeability in fungal plant pathogens[J]. Fungal Biology, 2014, 118:855-861.
[12] Hamdache A, Lamarti A, Aleu J, et al. Non-peptide metabolites from the genus Bacillus[J]. Journal of Natural Products, 2011,74(4):893-899.
[13] Wise C, Novitsky L, Tsopmo A, et al. Production and antimicrobial activity of 3-hydroxypropionaldehyde from Bacillus subtilis strain CU12[J]. Journal of Chemical Ecology, 2012, 38(12):1521-1527.
[14] De Vrieze M, Pandey P, Bucheli T D, et al. Volatile organic compounds from native potato-associated Pseudomonas as potential anti-oomycete agents[J]. Frontiers in Microbiology, 2015,6:1295. https://doi.org/10.3389/fmicb.2015.01295
[15] Chen X H, Vater J, Piel J, et al. Structural and functional characterization of three polyketide synthase gene clusters in Bacillus amyloliquefaciens FZB42[J]. Journal of Bacteriology, 2006,188:4024-4036.
[16]陈晓萌,王亚杰,李佳,等.枯草芽孢杆菌MG-4抑制小麦全蚀病菌物质及其性质分析[J].植物保护学报, 2018, 45(3):511-519. https://doi.org/10.13802/j.cnki.zwbhxb.2018.2017033Chen Xiaomeng, Wang Yajie, Li Jia, et al. Characterization of antifungal substances of Bacillus subtilis MG-4 against Gaeumannomyces graminis var. tritic causing wheat take-all[J].Journal of Plant Protection, 2018, 45(3):511-519.
[17] Bais H, Fall R, Vivanco J. Biocontrol of Bacillus subtilis against infection of Arabidopsis roots by Pseudomonas syringae is facilitated by biofilm formation and surfactin production[J].Plant Physiology, 2004, 134:307-319.
[18] Stein T. Bacillus subtilis antibiotics:Structures, syntheses and specific functions[J]. Molecular Microbiology, 2005, 56:845-857.
[19] Guo Q G, Dong W X, Li S Z, et al. Fengycin produced by Bacillus subtilis NCD-2 plays a major role in biocontrol of cotton seedling damping-off disease[J]. Microbiological Research,2014, 169(7/8):533-540. https://doi.org/10.1016/j.micres.2013.12.001
[20] Bie X M, Lu Z X, Lu F X. Identification of fengycin homologues from Bacillus subtilis with ESI-MS/CID[J]. Journal of Microbiological Methods, 2009, 79(3):272-278.
[21]张冬冬,李术娜,郭晓军,等.一株棉花黄萎病拮抗芽孢细菌的分离鉴定及活性检测[J].棉花学报, 2012, 24(4):358-362.Zhang Dongdong, Li Shuna, Guo Xiaojun, et al. Isolation, identification and activity of an antagonistic spore bacterium against cotton Verticillium wilt[J]. Cotton Science, 2012, 24(4):358-362.
[22]张冬冬,刘涛,高同国,等.棉花黄萎病拮抗细菌Z-5菌株的定殖能力检测[J].棉花学报, 2013, 25(6):510-516.Zhang Dongdong, Liu Tao, Gao Tongguo, et al. Detection of the colonization ability of antagonistic bacteria strain Z-5 against cotton Verticillium wilt[J]. Cotton Science, 2013, 25(6):510-516.
[23] Xiao Z J, Liu P H, Qin J Y. Statistical optimization of medium components for enhanced acetoin production from molasses and soybean meal hydrolysate[J]. Applied Microbiology and Biotechnology, 2007, 74(1):61-68.
[24] Caldeira A T, Arteiro J M S, Coelho A V, et al. Combined use of LC-ESI-MS and antifungal tests for rapid identification of bioactive lipopeptides produced by Bacillus amyloliquefaciens CCMI 1051[J]. Process Biochemistry, 2011, 46:1738-1746.
[25] Yu G Y, Sinclair J B, Hartman G L, et al. Production of iturin A by Bacillus amyloliquefaciens suppressing Rhizoctonia solani[J]. Soil Biology&Biochemistry, 2002, 34:955-963.
[26] Waseem R, Yang X, Wu H, et al. Isolation and characterization of fusaricidin-type compound-producing strain of Paenibacillus polymyxa SQR-21 active against Fusarium oxysporum f. sp.Nevium[J]. European Journal of Plant Pathology, 2009, 125:471-483.
[27] Mora I, Cabrefiga J, Montesinos E. Antimicrobial peptide genesin Bacillus strains from plant environments[J]. International Microbiology, 2011, 14(4):213-223.
[28] Joshi R, Mc Spadden Gardener B B. Identification and characterization of novel genetic markers associated with biological control activities in Bacillus subtilis[J]. Phytopathology, 2006,96(2):145-154.
[29]陈志谊.芽孢杆菌类生物杀菌剂的研发与应用[J].中国生物防治学报, 2015, 31(5):723-732. https://doi.org/10.16409/j.cnki.2095-039x.2015.05.012Chen Zhiyi. Research and application of bio-fungicide with Bacillus spp.[J]. Chinese Journal of Biological Control, 2015,31(5):723-732.
[30]乔俊卿,陈志谊,梁雪杰,等.枯草芽孢杆菌Bs916在番茄根部的定殖[J].江苏农业学报, 2015, 31(6):1278-1283. https://doi.org/10.3969/j.issn.1000-4440.2015.06.013Qiao Junqing, Chen Zhiyi, Liang Xuejie, et al. Colonization of Bacillus subtilis Bs916 on tomato root[J]. Jiangsu Journal of Agricultural Science, 2015, 31(6):1278-1283.
[31]林东,徐庆,刘忆舟,等.枯草芽孢杆菌SO113分泌蛋白的抑菌作用及抗菌蛋白的分离纯化[J].农业生物技术学报, 2001,9(1):77-80.Lin Dong, Xu Qing, Liu Yizhou, et al. The antibacterial effect of the secreted peptide from Bacillus subtilis SO113 and separation and purification of the antibacterial peptides[J]. Journal of Agricultural Biotechnology, 2001, 9(1):77-80.
[32]张冬冬,姜军坡,朱宝成.棉花黄萎病生防芽孢杆菌Z-5菌株发酵培养基的优化[J].棉花学报, 2014, 26(1):10-17.Zhang Dongdong, Jiang Junpo, Zhu Baocheng. Optimization of fermentation conditions of a biocontrol bacterial isolate Bacillus Z-5 against cotton Verticillium wilt[J]. Cotton Science, 2014,26(1):10-17.
[33] Zhang D D, Gao T G, Li H Y, et al. Identification of antifungal substances secreted by Bacillus subtilis Z-14 that suppress Gaeumannomyces graminis var. tritici[J]. Biocontrol Science and Technology, 2017, 27(2):237-251.
[34] Ongena M, Jacques P, TouréY, et al. Involvement of fengycin-type lipopeptidesin the multifaceted biocontrol potential of Bacillus subtilis[J]. Applied Microbiology and Biotechnology, 2005, 69:29-38.
[35] Yang L R, Quan X, Xue B G, et al. Isolation and identification of Bacillus subtilis strain YB-05 and its antifungal substances showing antagonism against Gaeumannomyces graminis var.tritici[J]. Biological Control, 2015, 85:52-58. https://doi.org/10.1016/j.biocontrol.2014.12.010
[36] Ye Y F, Li Q Q, Fu G, et al. Identification of antifungal substance(iturin A2)produced by Bacillus subtilis B47 and its effect on southern corn leaf blight[J]. Journal of Integrative Agriculture, 2012, 11(1):90-99.