|
|
|
| Analysis of Antifungal Substances Produced by Bacillus subtilis Isolate Against Fusarium oxysporum |
| QIAO Xin-Lei, FAN Xue-Rui, HUO Xiao-Yi, ZHANG Xue-Chao, XIAO Jia-Wen, ZHANG Dong-Dong* |
| College of Life Science, Hebei Agricultural University/Hebei Provincial Engineering Research Center of Agricultural Wastes Resource Utilization, Baoding 071000, China |
|
|
|
|
Abstract Bacillus subtilis Z-5 has significant antagonistic effect against many plant pathogenic fungi. In this study, antagonistic effect of strain Z-5 on Fusarium oxysporum was detected by confrontation culture. The activities of protease, amylase, cellulase and siderophore in the sterile filtrate were detected by the method of transparent circle. The antifungal substances were extracted by acid precipitation, and the antagonistic effect of the extract against F. oxysporum was detected by inhibition zone method. The extract was separated by high performance liquid chromatography(HPLC) and the activities of the separated components were detected by inhibition zone method. The structures of antifungal substances were identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS) and mass spectrometry/mass spectrometry(MS/MS). The results showed that strain Z-5 presented significant antagonistic activity against F. oxysporum, and the sterile filtrate had significant protease, amylase and cellulase activities as well as the siderophore. The extracts of the sterile filtrate showed significant antagonistic effect against F. oxysporum and three antifungal components were obtained, which were identified as lipopeptide antibiotics C14 iturin A, C15 iturin A and C14-C18 fengycin A. The detection of antifungal substances from B. subtilis strain Z-5 laid the foundation for the study of its antifungal mechanism and the effective use of biocontrol agents.
|
|
Received: 29 January 2021
|
|
|
|
Corresponding Authors:
* zhangdongcumt@163.com
|
|
|
|
[1] 陈晓萌, 王亚杰, 李佳, 等. 2018. 枯草芽胞杆菌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, 45(3): 511-519.
(Chen X M, Wang Y J, Li J, et al.2018. Characterization of antifungal substances of Bacillus subtilis MG-4 against Gaeumannomyces graminis var. tritic causing wheat take-all[J]. Journal of Plant Protection, 45(3): 511-519.)
[2] 李嘉维, 徐兰依, 王冬霜, 等. 2019. 枣炭疽病菌拮抗芽孢杆菌的筛选鉴定及其抑菌物质分析[J]. 园艺学报, 46(12): 2406-2414.
(Li J W, Xü L Y, Wang D S, et al.2019. Screening and identification of Bacillus antagonist against anthracnose of Jujube and analysis of its antifungal substances[J]. Acta Horticulturae Sinica, 46(12): 2406-2414.)
[3] 刘新月, 李凡, 陈海如, 等. 2008. 致病性尖孢镰刀菌生物防治研究进展[J]. 云南大学学报(自然科学版), 30(S1): 89-93.
(Liu X Y, Li F, Chen H R, et al.2008. Advances in biocontrol of pathogenic Fusarium oxysporum[J]. Journal of Yunnan University (Natural Science Edition), 30(S1): 89-93.)
[4] 林宝英, 谭志琼, 张容意. 2013. 枯草芽孢杆菌B25抗菌物质初步研究[J]. 广东农业科学, 40(1): 82-84.
(Lin B Y, Tan Z Q, Zhang R Y.2013. Preliminary study of antimicrobial substance produced by Bacillus subtilis B25[J]. Guangdong Agricultural Science, 40(1): 82-84.)
[5] 刘雅琴, 陈海魁, 孔令全. 2010. α-淀粉酶产生菌的分离筛选与诱变选育[J]. 畜牧与饲料科学, 31(9): 1-3.
(Liu Y Q, Chen H K, Kong L Q.2010. Separation and mutagenesis of alpha-amylase producing Bacillus[J]. Animal Husbandry and Feed Science, 31(9): 1-3.)
[6] 刘颖, 徐庆, 陈章良. 1999. 抗真菌肽LP-1的分离纯化及特性分析[J]. 微生物学报, 39(5): 441-447.
(Liu Y, Xu Q, Chen Z L.1999. Purification and characterization of antifungal peptide LP-1[J]. Acta Microbiologica Sinica, 39(5): 441-447.
[7] 王亚杰, 高宇, 陈晓萌, 等. 2018. 解淀粉芽胞杆菌防治番茄果实灰霉病及其抑菌物质分析[J]. 园艺学报, 45(7): 1296-1304.
(Wang Y J, Gao Y, Chen X M, et al.2018. Biocontrol ability of Bacillus amyloliquefaciens against gray mold on tomato fruit[J]. Acta Horticulturae Sinica, 45(7): 1296-1304.)
[8] 谢安娜, 徐浩飞, 张志林, 等. 2020. 致病镰刀菌的研究进展[J]. 湖北工程学院学报, 40(6): 37-41.
(Xie A N, Xü H F, Zhang Z L, et al.2020. Research development of Fusarium[J]. Journal of Hubei Engineering University, 40(6): 37-41.)
[9] 余贤美, 郑服丛, 林超, 等. 2009. 土壤产嗜铁素拮抗细菌CAS15的分离鉴定[J]. 植物保护学报, 36(2): 129-135.
(Yu X M, Zheng F C, Lin C, et al.2009. Isolation and identification of siderophore producing bacteria CAS15 from the soil[J]. Acta Phytophylacica Sinica, 36(2): 129-135.)
[10] 张冬冬, 雷白时, 朱宝成, 等. 2014. 小麦全蚀病生防芽孢杆菌对玉米秸秆的降解及抗菌谱检测[J]. 河北农业大学学报, 37(2): 65-70.
(Zhang D D, Lei B S, Zhu B C, et al.2014. Detection of corn stover degradation and antimicrobial spectrum of biocontrol Bacillus spp.strains against wheat take-all diseases[J]. Journal of Agricultural university of Hebei, 37(2): 65-70.)
[11] Baysal O, Lai D, Xu H H, et al.2013. A proteomic approach provides new insights into the control of soil-borne plant pathogens by Bacillus species[J]. PLOS ONE, 8: 1-12.
[12] Bright J J, Claydon M A, Soufian M, et al.2002. Rapid typing of bacteria using matrix-assisted laser desorption ionisation time-of-flight mass spectrometry and pattern recognition software[J]. Journal of Microbiological Methods, 48(2/3): 127-138.
[13] Caldeira A, Arteiro J, Coelho A, et al.2011. Combined use of LC-ESI-MS and antifungal tests for rapid identification of bioactive lipopeptides produced by Bacillus amyloliquefaciens CCMI 1051[J]. Process Biochemistry, 46: 1738-1746.
[14] Che J, Liu B, Ruan C, et al.2015. Biocontrol of Lasiodiplodia theobromae, which causes black spot disease of harvested wax apple fruit, using a strain of Brevibacillus brevis FJAT-0809-GLX[J]. Crop Protection, 67: 178-183.
[15] Falardeau J, Wise C, Novitsky L, et al.2013. Ecological and mechanistic insights into the direct and indirect antimicrobial properties of Bacillus subtilis lipopeptides on plant pathogens[J]. Journal of Chemical Ecology, 39: 869-878.
[16] Ghose T.1987. Measurement of cellulase activities[J]. Pure and Applied Chemistry, 59: 257-268.
[17] He L, Chen W, Liu Y.2006. Production and partial characterization of bacteriocin-like pepitdes by Bacillus licheniformis ZJU12[J]. Microbiological Research, 161: 321-326.
[18] Howard KL, Boyer GL.2007. Adduct simplification in the analysis of cyanobacterial toxins by matrix-assisted laser desorption/ionization mass spectrometry[J]. Rapid Communications in Mass Spectrometry, 21(5): 699-706.
[19] Kurabachew H, Wydra K.2013. Characterization of plant growth promoting rhizobacteria and their potential as bioprotectant against tomato bacterial wilt caused by Ralstonia solanacearum[J]. Biological Control, 67: 75-83.
[20] Liu J, Hagberg I, Novitsky L, et al.2014. Interaction of antimicrobial cyclic lipopeptides from Bacillus subtilis influences their effect on spore germination and membrane permeability in fungal plant pathogens[J]. Fungal Biology, 118: 855-861.
[21] Loper J E, Henkels M D.1997. Availability of iron to Pseudomonas fluorescens in rhizosphere and bulk soil evaluated with an ice nucleation reporter gene[J]. Applied and Environmental Microbiology, 63(1): 99-105.
[22] Manjula K, Kishore G, Podile A.2004. Whole cells of Bacillus subtilis AF1 proved more effective than cell-free and chitinase-based formulations in biological control of citrus fruit rot and groundnut rust[J]. Canadian Journal of Microbiology, 50: 737-744.
[23] Schwyn B, Neilands J.1987. Universal chemical assay for the detection and determination of siderophores[J]. Analytical Biochemistry, 160: 47-56.
[24] Sun Z, Yuan X, Zhang H, et al.2013. Isolation, screening and identification of antagonistic downy mildew endophytic bacteria from cucumber[J]. European Journal of Plant Pathology, 137: 847-857.
[25] Tanaka K, Fukuda M, Amaki Y, et al.2017. Importance of prumycin produced by Bacillus amyloliquefaciens SD-32 in biocontrol against cucumber powdery mildew disease[J]. Pest Management Science, 73(12): 2419-2428.
[26] Velho R, Medina L, Segalin J, et al.2011. Production of lipopeptides among Bacillus strains showing growth inhibition of phytopathogenic fungi[J]. Folia Microbiologica, 56(4): 297-303.
[27] Waseem R, Yang X, Wu H, et al.2009. 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, 125(3): 471-483.
[28] Wise C, Novitsky L, Tsopmo A, et al.2012. Production and antimicrobial activity of 3-hydroxypropionaldehyde from Bacillus subtilis strain CU12[J]. Journal of Chemical Ecology, 38: 1521-1527.
[29] Xie S, Zang H, Wu H, et al.2018. Antibacterial effects of volatiles produced by Bacillus strain D13 against Xanthomonas oryzae pv. Oryzae[J]. Molecular Plant Pathology, 19(1): 49-58.
[30] Xu T, Li Y, Zeng X, et al.2017. Isolation and evaluation of endophytic Streptomyces endus OsiSh-2 with potential application for biocontrol of rice blast disease[J]. Journal of the Science of Food and Agriculture, 97(4): 1149-1157.
[31] Yang J, Liu H, Zhu G, et al.2008. Diversity analysis of antagonists from rice associated bacteria and their application in biocontrol of rice diseases[J]. Journal of Applied Microbiology, 104: 91-104.
[32] Yang L, Quan X, Xue B, et al.2015. Isolation and identification of Bacillus subtilis strain YB-05 and its antifungal substances showing antagonism against Gaeumannomyces graminis var. tritici[J]. Biological Control, 85: 52-58.
[33] Ye Y, Li Q, Fu G, et al.2012. Identification of antifungal substance (Iturin A2) produced by Bacillus subtilis B47 and its effect on southern corn leaf blight[J]. Journal of Integrative Agriculture, 11(1): 90-99.
[34] Yu G, Sinclair J, Hartman G, et al.2002. Production of iturin A by Bacillus amyloliquefaciens suppressing Rhizoctonia solani[J]. Soil Biology & Biochemistry, 34: 955-963.
[35] Zhang D, Gao T, Li H, et al.2017a. Identification of antifungal substances secreted by Bacillus subtilis Z-14 that suppress Gaeumannomyces graminis var. tritici[J]. Biocontrol Science and Technology, 27(2): 237-251.
[36] Zhang D, Guo X, Wang Y, et al.2017b. 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, 65(6): 512-519.
[37] Zhang X, Chen X, Qiao X, et al.2021. Isolation and yield optimization of lipopeptides from Bacillus subtilis Z-14 active against wheat take-all caused by Gaeumannomyces graminis var. tritici[J]. Journal of Separation Science, 2021. DOI: 10.1002/jssc.201901274. |
|
|
|
