Optimization of Culture Medium and Flask Fermentation Conditions for Bacillus velezensis FX1 against Erwinia amylovora

doi:10.16409/j.cnki.2095-039x.2022.08.002

Abstract

Abstract: Bacillus velezensis FX1 was isolated and screened from the natural enzyme fermentation broth of fruits and vegetables in the pear production area of Kulle, Xinjiang, as a strain with strong antagonistic effect on the Erwinia amylovora. In order to improve the bacterial inhibitory activity and disease control effect, the medium components (carbon source, nitrogen source, inorganic salt) and shake flask fermentation conditions (temperature, rotation speed and initial pH) were screened and optimized by single-factor test, Plackett-Burman test and response surface method. The results showed that the optimal medium for FX1 was corn starch 10.0 g/L, yeast paste 25.1 g/L, KH₂PO₄ 0.5 g/L, MnSO₄ 0.001 g/L. The optimal fermentation conditions were initial pH 7.0, rotation speed 150 r/min, temperature 30.9 ℃, loading volume 100 mL/500 mL, and cultivation time 48 h. After optimization, the viable bacterial count of FX1 strain could reach 1.53×10⁹ CFU/mL and the diameter of inhibition circle could reach 26.6 mm, which increased 3.10 times and 1.32 times, respectively. Spraying of FX1 fermentation solution, sterile fermentation filtrate and crude extract of lipopeptides could significantly reduce the flower rot rate (P＜0.05), and the protective efficacy against fire blight could reach 68.53%, 71.47% and 76.30%, respectively, which were higher than that before optimization. The above results provide a basis for the development of Bacillus Bellais FX1 bacterial agent and its application in biological control of fire blight.

Key words: Erwinia amylovora, antagonistic bacteria, antagonistic substances, Plackrtt-Burman design, response surface analysis

CLC Number:

S476

Lü Tianyu, HE Xu, LUO Ming, HAN Jian, BAO Huifang, HUANG Wei. Optimization of Culture Medium and Flask Fermentation Conditions for Bacillus velezensis FX1 against Erwinia amylovora[J]. Chinese Journal of Biological Control, 2022, 38(6): 1553-1565.

References

[1] Heitefuss R. Fire blight, history, biology, and management[J]. Journal of Phytopathology, 2012, 160(7-8): 440-440.
[2] 王俊, 高建诚, 巴音克西克, 等. 利用电加热自动消毒修枝剪阻断梨火疫病田间传播[J]. 植物检疫, 2022, 36(2): 25-28.
[3] Zhao Y Q, Tian Y L, Wang L M,et al. Fire blight disease, a fast-approaching threat to apple and pear production in China[J]. Journal of Integrative Agriculture, 2019, 18(4): 117-122.
[4] Doolotkeldieva T, Bobushova S, Schuster C, et al. Isolation and genetic characterization of Erwinia amylovora bacteria from Kyrgyzstan[J]. European Journal of Plant Pathology, 2019, 155(2): 677-686.
[5] Jock S, Wensing A, Pulawska J, et al. Molecular analyses of Erwinia amylovora strains isolated in Russia, Poland, Slovenia and Austria describing further spread of fire blight in Europe[J]. Microbiological Research, 2013, 168(7): 447-454.
[6] Myung I S, Yun M J, Lee Y H, et al. First report of fire blight caused by Erwinia amylovora on Chinese quince in South Korea[J]. Plant Disease. 2016, 100(12): 2521-2521.
[7] Kim W S, Hildebrand M, Jock S, et al. Molecular comparison of pathogenic bacteria from pear trees in Japan and the fire blight pathogen Erwinia amylovora [J]. Microbiology, 2001, 147(11): 2951-2959.
[8] Tancos K A, Cox K D. Effects of consecutive streptomycin and kasugamycin applications on epiphytic bacteria in the apple phyllosphere[J]. Plant Disease, 2017, 101(1): 158-164.
[9] 热孜亚·麦麦提, 盛强, 白雪莹, 等. 新疆梨火疫病菌对噻霉酮的敏感性及抗药性分析[J].果树学报, 2022, 39(9): 1669-1677.
[10] Sharifazizi M, Harighi B, Sadeghi A. Evaluation of biological control of Erwinia amylovora, causal agent of fire blight disease of pear by antagonistic bacteria[J]. Biological Control, 2017, 104: 28.
[11] Vahid F M, Samaneh G, Akbar S, et al. Characterization of paenibacillus polymixa N179 as a robust and multifunctional biocontrol agent[J]. Biological Control, 2020, 104-505.
[12] Gusberti M, Klemm U, Meier M.S. A comparison of different fire blight control methods in switzerland with respect to biosafety, efficacy and durability[J]. Environ Res Public Health, 2015, 12: 11422-11447.
[13] Bahadou S A, Ouijja A, Karfach A, et al. New potential bacterial antagonists for the biocontrol of fire blight disease in Morocco[J]. Microbial Pathogenesis, 2018: 7-15.
[14] Steven E L, Glenn M, Rachel E. Interactions of antibiotics with Pseudomonas fluorescens strain A506 in the control of fire blight and frost injury to pear[J]. Phytopathology, 1996, 86(8): 841-848.
[15] Zeller W, Wolf B. Studiesonbiological control of fire blight[J]. Acta Horticulturae. 1996, 0(411): 341-346.
[16] Vanneste J L, Yu J, Bonn W G. Biological control of fire blight using Erwinia herbicola EH252 and Pseudomonas fluorescens A506 separately or in combination[J]. Acta Horticulturae, 1996, 1(411): 351-354.
[17] Steven E L, Glenn M, Rachel E. Interactions of antibiotics with Pseudomonas fluorescens Strain A506 in the control of fire blight and frost injury to pear[J]. Phytopathology, 1996, 86(8): 841-848.
[18] 徐琳赟, 古丽孜热·曼合木提, 韩剑, 等. 香梨内生拮抗细菌的筛选及对梨火疫病的生防潜力[J]. 西北植物学报, 2021, 41(1): 132-141.
[19] 鲁晏宏, 郝金辉, 罗明, 等. 梨火疫病拮抗菌筛选及温室防效测定[J]. 微生物学通报, 2021, 48(10): 3690-3699.
[20] 王俊, 高建诚, 巴音克西克, 等. 利用蜜蜂传播生防菌防治梨火疫病[J]. 植物检疫, 2022, 36(1): 9-12.
[21] 张德锋, 高艳侠, 王亚军, 等. 贝莱斯芽孢杆菌的分类、拮抗功能及其应用研究进展[J]. 微生物学通报, 2020 47(11): 3634-3649.
[22] 沙月霞, 隋书婷, 曾庆超, 等. 贝莱斯芽孢杆菌E69 预防稻瘟病等多种真菌病害的潜力[J]. 中国农业科学, 2019, 52(11): 1908-1917.
[23] Cui LX, Yang C D, Wei L J, et al. Isolation and identification of an endophytic bacteria Bacillus velezensis 8-4 exhibiting biocontrol activity against potato scab[J]. Biological Control, 2020, 141.
[24] 贺旭. 梨火疫病菌拮抗细菌抑菌物质分析及发酵条件优化.[D]. 乌鲁木齐: 新疆农业大学, 2022.
[25] 朱海云, 马瑜, 柯杨, 等. 抗猕猴桃细菌性溃疡病蜡样芽孢杆菌MA23培养基及发酵条件优化[J]. 中国农学通报, 2021, 37(7): 112-118.
[26] 李术娜, 姜军坡, 朱莹, 等. 大丽轮枝菌拮抗细菌枯草芽孢杆菌12-34菌株发酵产抗菌蛋白的条件优化[J]. 植物保护, 2009, 35(3): 51-56.
[27] 谭峥,王雨, 杨欢, 等. 贝莱斯芽胞杆菌HN-2发酵上清液抑制三种黄单胞菌的生物活性研究[J]. 中国生物防治学报, 2020, 36(3): 414-420.
[28] 王屈祎, 钟倩, 闫如玉, 等. 淀粉酶产生菌的筛选、鉴定及其发酵条件优化[J]. 微生物学通报, 2022, 49(1): 173-188.
[29] 陶永梅, 潘洪吉, 黄健, 等. 新型生防微生物因子贝莱斯芽孢杆菌Bacillus velezensis的研究与应用[J]. 中国植保导刊, 2019, 39(9): 26-33.
[30] 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]. Front Microbiol, 2019, 10: 1279-1280.
[31] 任建雯, 罗云艳, 冯印印, 等. 贝莱斯芽孢杆菌RJW-5-5的分离鉴定及细菌素、抗菌肽基因簇挖掘[J]. 微生物学通报, 2021, 48(3): 742-754.
[32] 陈龙, 吴兴利, 闫晓刚, 等. 贝莱斯芽孢杆菌的分类、次级代谢产物及应用[J]. 家畜生态学报, 2020, 41(1): 1-8.
[33] 何亚文, 李广悦, 谭红, 等. 我国生防微生物代谢产物研发应用进展与展望[J]. 中国生物防治学报, 2022, 38(3): 537-548.
[34] 何明川, 王志江, 谢永辉, 等. 烟草黑胫病拮抗菌的筛选、鉴定及发酵条件优化[J]. 中国生物防治学报, 2022, 38(2): 428-439.
[35] 赵朋超, 权春善, 金黎明, 等. 氮源和碳源对解淀粉芽孢杆菌Q-426 抗菌脂肽合成的影响[J]. 中国生物工程杂志, 2012, 32(10): 50-56.
[36] Jin H, Zhang XR, Li KP, et al. Direct bio-utilization of untreated rapeseed meal for effective iturin A production by Bacill subtilisus in submerged fermentation[J]. PLoS, ONE, 2014, 9 : 111-171.
[37] Abdel-Mawgoud A M, Aboulwafa M M, Hassouna N A. Characterization of surfactin produced by Bacillus subtilis isolate BS5. Appl Biochem Biotechnol, 2008, 150(3): 289-303.
[38] 黄君. 海洋枯草芽孢杆菌HS-A38产抗菌脂肽类物质发酵条件的优化[D]. 大连: 大连工业大学, 2018.