耐盐芽胞杆菌鉴定、发酵条件优化及其生防作用

doi:10.16409/j.cnki.2095-039x.2025.02.026

中国生物防治学报 ›› 2025, Vol. 41 ›› Issue (3): 571-584.DOI: 10.16409/j.cnki.2095-039x.2025.02.026

• 研究论文 • 上一篇

耐盐芽胞杆菌鉴定、发酵条件优化及其生防作用

廖永琴^1,2, 王楠^1,2, 施竹凤², 陈艳^2,3, 刘恩德^2,3, 普特², 何永宏¹, 杨佩文²

1. 云南农业大学植物保护学院, 昆明 650000;
2. 云南省农业科学院农业环境资源研究所, 昆明 650205;
3. 云南省昆明学院, 昆明 650214

收稿日期:2024-04-10 发布日期:2025-06-20
通讯作者: 何永宏, 杨佩文
作者简介:廖永琴，女，硕士研究生，E-mail： 279858953@qq.com;通信作者，杨佩文，研究员，E-mail： 398036877@qq.com;何永宏，副教授，E-mail： heyonghong700327@163.com。
基金资助:
云南省科技计划重大科技专项（202202AE090010）；云南省“兴滇英才”支持计划（XDYC-CYCX-2002-0071）;中央引导地方科技发展资金项目（202407AC110006）；云南省专家基层科研工作站—杨佩文专家工作站（嵩明县，陆良县）（云人社通〔 2022〕 17号）

Identification of Bacillus halotolerans, Optimization of Fermentation Conditions and Its Biocontrol Effect

LIAO Yongqin^1,2, WANG Nan^1,2, SHI Zhufeng², CHEN Yan^2,3, LIU Ende^2,3, PU Te², HE Yonghong¹, YANG Peiwen²

1. College of Plant Protection, Yunnan Agricultural University, Kunming 650000, China;
2. Agricultural Environment and Resources Institute, Yunnan Academy of Agricultural Sciences, Kunming 650205, China;
3. Yunnan University, Kunming 650091, China

Received:2024-04-10 Published:2025-06-20

摘要/Abstract

摘要： 为明确菌株YNK-FB0022在植物病害绿色防控方面的应用潜力，为该菌株产业化开发奠定基础。结合形态学和分子生物学对菌株YNK-FB0022进行分类地位鉴定；通过设计单因素和响应面优化试验，优化其发酵配方和发酵条件；利用48孔板测定菌株生物膜形成能力，结合扫描电镜观察菌株在番茄幼苗根系的定殖情况。盆栽试验测定菌株对番茄幼苗生长及对番茄枯萎病的防效效果，并测定番茄根系防御酶活。经鉴定YNK-FB0022为耐盐芽胞杆菌Bacillus halotolerans； YNK-FB0022最适培养配方为蔗糖15.00 g/L，酵母浸粉6.25 g/L、MgSO₄·7H₂O 8.75 g/L、蛋白胨10.00 g/L，发酵条件为温度30℃、pH 5.5、转速200 r/min、装液量200 mL；优化后生长量提升173.35%，抑菌率提升7.56%； 48孔板每孔可形成189 mg的生物膜，且可定殖于番茄根系表面，定殖量达到1.20× 10⁷ CFU/mm。盆栽试验表明，优化后的YNK-FB0022发酵液可显著提高番茄幼苗的株高、茎围、根长、根重、地上部位鲜重和地上部位干重等农艺性状，对番茄枯萎病有较好的防效，并显著提升番茄根系POD、SOD、PPO和CAT防御酶活。研究结果为耐盐芽胞杆菌YNK-FB0022生防菌剂的开发及其对番茄枯萎病的防治应用提供了理论依据。

关键词: 耐盐芽胞杆菌, 单因素试验设计, 响应面优化, 番茄枯萎病, 防御酶活性

Abstract: The potential application of strain YNK-FB0022 in the sustainable management of plant diseases was assessed, providing a foundation for its future industrial development. The strain was characterized through both morphological and molecular biological techniques to determine its taxonomic classification. Single-factor and response surface methodology were employed to optimize its fermentation medium and conditions. The biofilm-forming capacity of the strain was evaluated using a 48-well plate assay, while scanning electron microscopy was utilized to examine its colonization on the root surface of tomato seedlings. Pot experiments were performed to assess the impact of the strain on tomato seedling growth, its efficacy in controlling tomato wilt disease, and the activity of defense-related enzymes in the tomato roots.The strain YNK-FB0022 was identified as Bacillus halotolerans. The optimal fermentation medium for YNK-FB0022 was composed of 15.00 g/L sucrose, 6.25 g/L yeast extract, 8.75 g/L MgSO₄·7H₂O, and 10.00 g/L peptone, with fermentation conditions set at 30℃, pH 5.5, 200 rpm, and a liquid volume of 200 mL. Following optimization, the strain exhibited a 173.35% increase in growth, and its antibacterial activity improved by 7.56%. In the 48-well plate assay, each well formed 189 mg of biofilm, and the strain successfully colonized the root surface of tomato seedlings, with a colonization density of 1.20×10⁷ CFU/mm. Pot trials demonst rated that the optimized YNK-FB0022 fermentation broth significantly enhanced several agronomic traits of tomato seedlings, including plant height, stem diameter, root length, root weight, as well as the fresh and dry weight of aerial parts. Moreover, the broth exhibited substantial protective effects against tomato wilt disease and significantly increased the activity of root defense enzymes, such as POD, SOD, PPO, and CAT. These findings provide a theoretical basis for the development of the Bacillus halotolerans YNK-FB0022 as a biocontrol agent and its application in the management of tomato wilt disease.

Key words: Bacillus halotolerans, single-factor experiment, response surface optimization, tomo wilt disease, defense enzym activity

中图分类号:

S476

廖永琴, 王楠, 施竹凤, 陈艳, 刘恩德, 普特, 何永宏, 杨佩文. 耐盐芽胞杆菌鉴定、发酵条件优化及其生防作用[J]. 中国生物防治学报, 2025, 41(3): 571-584.

LIAO Yongqin, WANG Nan, SHI Zhufeng, CHEN Yan, LIU Ende, PU Te, HE Yonghong, YANG Peiwen. Identification of Bacillus halotolerans, Optimization of Fermentation Conditions and Its Biocontrol Effect[J]. Chinese Journal of Biological Control, 2025, 41(3): 571-584.

参考文献

[1] 李志田, 蔡淑琳, 王合, 等. 浑圆链霉菌QH-16鉴定及抑菌活性产物的结构研究[J]. 中国生物防治学报, 2021, 37(1): 156-164.
[2] 王超, 郭坚华, 席运官, 等. 拮抗细菌在植物病害生物防治中应用的研究进展[J]. 江苏农业科学, 2017, 45(18): 1-6.
[3] Lim S, Junqueira A, Uchida A, Purbojati R W, et al. Genome sequence of Bacillus velezensis SGAir0473, isolated from tropical air collected in Singapore[J]. Genome Announcements, 2018, 6(27): e00642-18.
[4] Sarwar A, Brader G, Corretto E, et al. Qualitative analysis of biosurfactants from Bacillus species exhibiting antifungal activity[J]. PLoS ONE, 2018, 13(6): e0198107.
[5] 仇月, 孙守民, 李鑫荣, 等. 贝莱斯芽胞杆菌SDTB038与化学药剂协同防治草莓枯萎病的研究[J]. 中国生物防治学报, 2021, 37(5): 989-996.
[6] Wang F, Xiao J, Zhang Y, et al. Biocontrol ability and action mechanism of Bacillus halotolerans against Botrytis cinerea causing grey mould in postharvest strawberry fruit[J]. Postharvest Biology and Technology, 2021, 174: 111456.
[7] Feng Z, Xu M, Yang J, et al. Molecular characterization of a novel strain of Bacillus halotolerans protecting wheat from sheath blight disease caused by Rhizoctonia solani Kühn[J]. Frontiers in Plant Science, 2022, 13: 1019512.
[8] 申云鑫, 施竹凤, 李铭刚, 等. 贝莱斯芽胞杆菌SH-1471发酵条件优化及其番茄枯萎病的防治效果[J]. 微生物学报, 2024, 64(1): 220-237.
[9] Prieto P, Schiliro E, Maldonado-Gonzalez M M, et al. Root hairs play a key role in the endophytic colonization of olive roots by Pseudomonas spp. with biocontrol activity[J]. Microbial Ecology, 2011, 62(2): 435-445.
[10] Sreekumar G, Soundarajan K. Enhanced biomass production study on probiotic Bacillus subtilis SK09 by medium optimization using response surface methodology[J]. African Journal of Biotechnology, 2010, 9(47): 8078-8084.
[11] 汪晶晶, 曹雪梅, 李欢, 等. 抗菌海洋解淀粉芽胞杆菌GM-1-2菌株摇瓶发酵培养基和发酵条件优化[J]. 江苏农业科学, 2018, 46(8): 291-295.
[12] Buchanan R E, Gibbons N E. 伯杰细菌鉴定手册[M]. 北京: 科学出版社, 1984.
[13] 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.
[14] Walsh P S, Metzger D A, Higuchi R. Chelex 100 as a medium for simple extraction of DNA for PCRbased typing from forensic material[J]. BioTechniques, 1991, 10(4): 506-513.
[15] Kim O S, Cho Y J, Lee K, et al. Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species[J]. International Journal of Systematic and Evolutionary Microbiology, 2012, 62: 716-721.
[16] 廖永琴, 王楠, 申云鑫, 等. 三株酚酸降解菌的筛选与鉴定及其生物活性[JOL]. 微生物学通报, 1-23[2024-01-11].
[17] Li J, Zhao Q, Wuriyanghan H, et al. Biocontrol bacteria strains Y4 and Y8 alleviate tobacco bacterial wilt disease by altering their rhizosphere soil bacteria community[J]. Rhizosphere, 2021, 19: 100390.
[18] 徐靖, 牛邦彦, 张亚南, 等. 芽胞杆菌属Bacillus分类学研究进展[J]. 中国土壤与肥料, 2022(12): 225-237.
[19] 刘雪停, 夏彦飞, 李身, 等. 杨山牡丹生防内生细菌的分离及鉴定[J]. 江西农业学报, 2022, 34(2): 62-68.
[20] 许世洋, 范雨轩, 汪学苗, 等. 辣椒镰孢根腐病防病促生细菌的筛选及其效应[J]. 微生物学报, 2022, 62(7): 2735-2750.
[21] 赵远征,徐利敏,彭静文, 等. 一株马铃薯内生生防细菌YB9的分离与鉴定[JOL]. 分子植物育种, 1-9[2024-01-12].
[22] Arrebol AE, Jacobs R, Korsten L. Iturin A is theprincipal inhibitor in the biocontrol activity of Bacillus amyloliquefaciens PPCB004 against postharvest fungal pathogens [J]. Journal of Applied Microbiology, 2010, 108(2): 386395.
[23] Kim P I, Chung K C. Production of an antifungal protein for control of Colletotrichum lagenarium by Bacillus amylolique faciens MET0908[J]. FEMS Microbiology Letters, 2004, 234(1): 177-183.
[24] 程亮亮, 叶磊, 王文凯, 等. 小麦纹枯病拮抗菌HB-10的筛选及其发酵条件优化[J]. 中国生物防治学报, 2024, 40(2): 435-447.
[25] 李术娜, 姜军坡, 朱莹, 等. 大丽轮枝菌拮抗细菌枯草芽胞杆菌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.
[26] 王俊芳, 张飘丹. 枯草芽孢杆菌G10合成表面活性素的发酵条件优化[J]. 中国酿造, 2024, 43(3): 157-162.
[27] Lin X, Ye J, Mu C. Research on the fermentation conditions of Bacillus subtilis B-332[J]. Agricultural Science & Technology, 2013, 14(1): 81.
[28] Barahona E, Navazo A, Martinez-granero F, et al. Pseudomonas fluorescens F113 mutant with enhanced competitive colonization ability and improved biocontrol activity against fungal root pathogens[J]. Applied and Environmental Microbiology, 2011, 77(15): 5412-5419.
[29] Niu, B, Paulson, J N, Zheng X, et al. Simplified and representative bacterial community of maize roots[J]. Proceedings of the National Academy of Sciences USA, 2017, 114: E2450-E2459.
[30] Sheng Y X, Shi Z F, Zhao J Y, et al. Whole genome sequencing provides evidence for Bacillus velezensis SH-1471 as a beneficial rhizosphere bacterium in plants[J]. Scientific Reports, 2023, 13(1): 20929.
[31] 邹强, 牛新湘, 刘萍, 等. 拮抗葡萄灰霉病贝莱斯芽胞杆菌的生长特征及对相关防御酶活性的影响[J]. 食品工业科技, 2023, 44(15): 126-133.
[32] 陈刘军, 俞仪阳, 王超, 等. 蜡质芽胞杆菌AR156防治水稻纹枯病机理初探[J]. 中国生物防治学报, 2014, 30(1): 107-112.

耐盐芽胞杆菌鉴定、发酵条件优化及其生防作用

Identification of Bacillus halotolerans, Optimization of Fermentation Conditions and Its Biocontrol Effect

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 10

编辑推荐

Metrics

本文评价

联系我们

[1]	申云鑫, 李铭刚, 施竹凤, 赵江源, 王楠, 李者芬, 杨明英, 陈齐斌, 杨佩文. 贝莱斯芽胞杆菌SH-1471可湿性粉剂研制及其对番茄枯萎病的防治效果[J]. 中国生物防治学报, 2023, 39(4): 904-914.
[2]	李梦玮, 陈晓霞, 补欢欢, 廖礼彬, 李婧怡, 张楠楠, 石福孙. 花椒根腐病拮抗真菌的分离鉴定及其生防潜力探究[J]. 中国生物防治学报, 2023, 39(1): 176-183.
[3]	肖姬玲, 熊毅, 梁志怀, 杨科. 尖孢镰刀菌F-1对草莓灰霉病防控效果及机制初探[J]. 中国生物防治学报, 2022, 38(4): 840-845.
[4]	郭景红, 韩兴, 甄丹妹, 孟庆芳, 康占海, 王冰, 李亚宁, 刘大群. 玫瑰黄链霉菌Men-myco-93-63诱抗粗蛋白对黄瓜的诱抗作用[J]. 中国生物防治学报, 2020, 36(2): 272-279.
[5]	唐彤彤, 孙星, 董元华, 刘勤. 对番茄枯萎病具防效的海洋源赤杆菌YH-07的发酵条件探究[J]. 中国生物防治学报, 2018, 34(5): 753-761.
[6]	孙润红, 徐俊蕾, 杨丽荣, 张洁, 夏明聪, 武超, 薛保国, 吴坤. 枯草芽胞杆菌YB-05对小麦全蚀病菌胞内酶活的影响[J]. 中国生物防治学报, 2018, 34(1): 156-162.
[7]	张亮, 盛浩, 袁红, 赵兰凤, 李华兴. 荧光假单胞菌PEF-5#18防控番茄枯萎病的定殖机理[J]. 中国生物防治学报, 2017, 33(5): 658-666.
[8]	王璐瑶, 李兴东, 段天凤, 杨晓云, 刘永峰, 陈志谊. 解淀粉芽胞杆菌B1619防控设施番茄枯萎病田间使用技术研究与示范[J]. 中国生物防治学报, 2017, 33(4): 512-518.
[9]	张亮, 王改兰, 段建南, 赵兰凤, 李华兴. 广谱生防菌对番茄枯萎病的防病效果及其机理[J]. 中国生物防治学报, 2015, 31(6): 897-906.
[10]	刘邮洲1 , 陈志谊1 , 梁雪杰1 , 朱剑花2 . 番茄枯萎病和青枯病拮抗细菌的筛选、评价与鉴定[J]. , 2012, 28(1): 101-108.