Screening, Identification and Biocontrol Potential Analysis of an Antagonistic Strain against Ralstonia solanacearum

doi:10.13560/j.cnki.biotech.bull.1985.2024-0598

Abstract

Abstract:

【Objective】Tomato bacterial wilt caused by Ralstonia solanacearum（Rs）is main disease of tomato. In this study, the antagonistic strain against R. solanacearum and with growth promoting was screened from tomato rhizosphere, and its physiological and biochemical characteristic and biocontrol effect were investigated, which may provide a theoretical basis for the further development of biological control agents.【Method】Filter paper slice method was employed to screen the antagonistic strain against Rs. Physiological and biochemical characteristic and 16S rRNA sequences were analyzed to identify the antagonistic strain. Pot experiments were conducted to evaluate the biocontrol and growth promoting effects of the antagonistic strain on bacterial wilt and tomato, respectively. 16S rRNA gene amplicon sequencing and fluorescence quantitative PCR were used to investigate the effect of antagonistic strain on the bacterial community in tomato rhizosphere.【Result】An antagonistic strain A72 was screened out from the tomato rhizosphere. Strain A72 was identified as Bacillus siamensis by 16S rRNA gene sequence, and it had the ability to resolve phosphorus, release potassium, produce IAA, secrete extracellular hydrolases and siderophore, and form biofilms. The pot experiments showed that its control effect against tomato bacterial wilt was 63.80%, and it significantly increased the root length, plant height, dry weight, fresh weight and chlorophyll content of tomato plants. Application of strain A72 significantly decreased the density of R. solanacearum and altered the structure and composition of bacterial community in tomato rhizosphere.【Conclusion】Strain A72 has a good growth promotion effect on tomato seedlings and may effectively control tomato bacterial wilt.

Key words: antagonistic strain, Ralstonia solanacearum, rhizosphere bacterial community, Bacillus siamensis, biocontrol

MU Xue-nan, WU Tong, ZHENG Zi-wei, ZHANG Yue, WANG Zhi-gang, XU Wei-hui. Screening, Identification and Biocontrol Potential Analysis of an Antagonistic Strain against Ralstonia solanacearum[J]. Biotechnology Bulletin, 2025, 41(1): 276-286.

Figures/Tables 10

Fig. 1 Screening of strain A72 and phylogenetic tree analysis A: Inhibitory effect of strain A72 on R. solanacearum. B: Phylogenetic tree of strain A72 based on 16S rRNA gene sequence

Fig. 2 Physiological and biochemical characteristics and biofilm forming ability of B. siamensis A72 A: Determination of physiological and biochemical characteristics. B: Biofilm forming ability. The different lowercase letters indicate significant differences among treatments（P<0.05）. The same below

Table 1 Physiological and biochemical characteristics of B. siamensis A72

试验项目 Test item	结果 Result	试验项目 Test item	结果 Result
蛋白酶 Protease	+	固氮 Nitrogen fixation	-
淀粉酶 Amylase	+	解磷 Phosphorus resolving	+
纤维素酶 Cellulase	+	解钾 Potassium releasing	+
铁载体 Siderophore	+	IAA产率 IAA production/（µg·mL^-1）	35.17±1.91

Fig. 3 Symptoms of tomato seedlings with R. solanacearum inoculation in different treatments CK1: Water + Ralstonia solanacearum; CK2: sterile NB medium + Ralstonia solanacearum; CD: carbendazim + Ralstonia solanacearum; A72: biological control strains A72 + Ralstonia solanacearum

Table 2 Control effect of B. siamensis A72 on tomato bacterial wilt

处理 Treatment	接种青枯菌后16 d after inoculation with Rs		接种青枯菌后20 d after inoculation with Rs
处理 Treatment	病情指数Disease index	防治效果Control efficiency/%	病情指数Disease index	防治效果Control efficiency/%
CK1	22.50±2.50 a		35.00±5.00 b
CK2	25.83±1.44 a		48.33±5.77 a
CD	15.00±2.50 b	42.12±7.06 a	25.00±2.50 c	48.48±6.15 b
A72	11.67±2.89 b	55.15±8.40 a	17.50±2.50 c	63.80±2.78 a

Fig. 4 Composition of bacterial communities in the tomato rhizosphere in different treatments A: Principal coordinate analysis of bacterial communities based on Bray-Curtis distance（PCoA）. B: Relative abundance（%）of all bacterial phyla in different treatments（Student's t test, P<0.05）. C: Relative abundance（%）of bacterial genera in different treatments. D: Heat map of cluster analysis of bacterial genera in different treatments

Fig. 5 Analysis of differential abundance and linear discriminant analysis（LEfSe）of rhizosphere bacterial genera in different treatments A: Analysis of differential abundance rhizosphere bacterial genera in different treatments（Wilcoxon Kruskal-Wallis, P<0.05）. B: Linear discriminant analysis of rhizosphere bacterial genera in different treatments（LDA≥2.5）

Fig. 6 Analysis of correlation between rhizosphere bacterial genera and disease index, and absolute abundances of Bacillus and Ralstonia solanacearum in tomato rhizosphere A: The correlation between rhizosphere bacteria and disease index based on Pearson correlation coefficient. B: Absolute abundance of rhizosphere Bacillus. C: Absolute abundance of rhizosphere Ralstonia solanacearum. The corresponding value of the heat map is Pearson correlation coefficient r, where r > 0 is positive correlation and r < 0 is negative correlation. *, **, and *** indicate significant differences at P < 0.05, P < 0.01, and P < 0.001, respectively

Fig. 7 Growth promotion effect（A）and evaluation of comprehensive ability of strain A72 on tomato seedlings（bar=10 cm） CSCK1: Water; CSCK2: sterile NB medium; CSA72: bacterial suspension

Fig. 8 Effects of B. siamensis A72 on soil physicochemical properties

References 47

[1]	李志丹, 黄奇, 林刿, 等. 利迪链霉菌 M01 对番茄生长、青枯病发病率及根际细菌群落组成的影响[J]. 微生物学通报, 2023, 50(6): 2508-2518.
	Li ZD, Huang Q, Lin G, et al. Effects of Streptomyces lydicus M01 on growth, bacterial wilt incidence, and rhizosphere bacterial community composition of tomatoes[J]. Microbiology China, 2023, 50(6): 2508-2518.
[2]	吴思炫, 高复云, 张锐澎, 等. 番茄青枯病生物防治的研究进展[J]. 应用生态学报, 2023, 34(9): 2585-2592. doi: 10.13287/j.1001-9332.202309.028
	Wu SX, Gao FY, Zhang RP, et al. Research progress in biological control of tomato bacterial wilt[J]. Chin J Appl Ecol, 2023, 34(9): 2585-2592. doi: 10.13287/j.1001-9332.202309.028
[3]	Jiang GF, Wei Z, Xu J, et al. Bacterial wilt in China: history, current status, and future perspectives[J]. Front Plant Sci, 2017, 8: 1549. doi: 10.3389/fpls.2017.01549 pmid: 28955350
[4]	Rivera-Zuluaga K, Hiles R, Barua P, et al. Getting to the root of Ral-stonia invasion[J]. Semin Cell Dev Biol, 2023, 148/149: 3-12.
[5]	王梅, 尹显慧, 龙友华, 等. 几种杀菌剂对番茄青枯病菌的毒力测定及田间药效[J]. 江苏农业科学, 2015, 43(4): 151-153.
	Wang M, Yin XH, Long YH, et al. Toxicity determination and field efficacy of several fungicides against tomato bacterial wilt[J]. Jiangsu Agric Sci, 2015, 43(4): 151-153.
[6]	陈远松, 朱晓伟, 龚翔宇, 等. 分子标记在我国番茄抗病育种的应用研究进展[J]. 浙江农业学报, 2017, 29(8): 1415-1420. doi: 10.3969/j.issn.1004-1524.2017.08.25
	Chen YS, Zhu XW, Gong XY, et al. Research advance of molecular marker-assisted selection in tomato disease resistance breeding in China[J]. Acta Agric Zhejiangensis, 2017, 29(8): 1415-1420. doi: 10.3969/j.issn.1004-1524.2017.08.25
[7]	徐欣韵, 王宁, 丁佳, 等. 番茄青枯病拮抗菌的定向筛选及其抗病促生机制研究[J]. 微生物学报, 2021, 61(10): 3276-3290.
	Xu XY, Wang N, Ding J, et al. Isolation and identification of antagonistic bacteria against tomato bacterial wilt and the mechanisms in disease prevention and plant growth promotion[J]. Acta Microbiol Sin, 2021, 61(10): 3276-3290.
[8]	Chowdhury SP, Dietel K, Rändler M, et al. Effects of Bacillus am-yloliquefaciens FZB42 on lettuce growth and health under pathogen pressure and its impact on the rhizosphere bacterial community[J]. PLoS One, 2013, 8(7): e68818.
[9]	Yang BY, Zheng MZ, Dong WP, et al. Plant disease resistance-related pathways recruit beneficial bacteria by remodeling root exudates upon Bacillus cereus AR156 treatment[J]. Microbiol Spectr, 2023, 11(2): e0361122.
[10]	Ahmed W, Dai ZL, Zhang JH, et al. Plant-microbe interaction: mining the impact of native Bacillus amyloliquefaciens WS-10 on tobacco bacterial wilt disease and rhizosphere microbial communities[J]. Microbiol Spectr, 2022, 10(4): e0147122.
[11]	秦菁菁, 曹璐, 付威, 等. 拮抗两种辣椒病害的菌株鉴定及生防潜力评价[J]. 微生物学通报, 2024, 51(7): 2450-2462.
	Qin JJ, Cao L, Fu W, et al. Identification and biocontrol potential of a strain against two pepper diseases[J]. Microbiology China, 2024, 51(7): 2450-2462.
[12]	王琦, 陈秀玲, 王傲雪. 一株具有促生作用的生防细菌YN-2A的分离、鉴定及全基因组测序分析[J]. 微生物学通报, 2024: 1-18.
	Wang Q, Chen XL, Wang AX. A biocontrol bacterium YN-2A with growth-promoting effect: isolation, identification, and genome sequencing[J]. Microbiology China, 2024: 1-18.
[13]	Andreote FD, Pereira E Silva MC. Microbial communities associated with plants: learning from nature to apply it in agriculture[J]. Curr Opin Microbiol, 2017, 37: 29-34. doi: S1369-5274(17)30014-0 pmid: 28437663
[14]	沈萍, 陈向东. 微生物学实验[M]. 4版. 北京: 高等教育出版社, 2007.
	Shen P, Chen XD. Microbiological experiment[M]. 4th ed. Beijing: Higher Education Press, 2007.
[15]	杨革. 微生物学实验教程[M]. 北京: 科学出版社, 2004.
	Yang G. Microbiology experiment course[M]. Beijing: Science Press, 2004.
[16]	章梦婷, 王二兴, 张雅婷, 等. 黄瓜根际促生菌Bacillus subti-lis S1的分离鉴定与促生抗病[J]. 微生物学通报, 2024, 51(6): 2141-2157.
	Zhang MT, Wang EX, Zhang YT, et al. Isolation and characterization of Bacillus subtilis S1 capable of inducing resistance against powdery mildew and promoting cucumber growth from cucumber rhizosphere[J]. Microbiology China, 2024, 51(6): 2141-2157.
[17]	张盈盈, 安晓霞, 马春晖, 等. 解磷细菌与磷肥耦合提高苜蓿生长及光合性能[J]. 中国草地学报, 2023, 45(11): 43-51.
	Zhang YY, An XX, Ma CH, et al. The coupling of phosphate solubilizing bacteria and phosphate fertilizer to improve the growth and photosynthetic performance of alfalfa[J]. Chin J Grassland, 2023, 45(11): 43-51.
[18]	Wu X, Xie Y, Qiao J, et al. Rhizobacteria strain from a hypersaline environment promotes plant growth of Kengyilia thoroldiana[J]. Microbiology, 2019, 88(2): 220-231.
[19]	Rajawat MVS, Singh S, Tyagi SP, et al. A modified plate assay for rapid screening of potassium-solubilizing bacteria[J]. Pedosphere, 2016, 26(5): 768-773.
[20]	冯路遥, 赵江源, 施竹凤, 等. 森林根际土壤细菌的分离、鉴定及生物活性筛选[J]. 生物技术通报, 2024, 40(1): 294-307. doi: 10.13560/j.cnki.biotech.bull.1985.2023-0586
	Feng LY, Zhao JY, Shi ZF, et al. Isolation, characterization and bioactivity screening of forest inter-root soil bacteria[J]. Biotechnology Bulletin, 2024, 40(1): 294-307.
[21]	张东艳, 刘晔, 吴越, 等. 花生根际产IAA菌的筛选鉴定及其效应研究[J]. 中国油料作物学报, 2016, 38(1): 104-110.
	Zhang DY, Liu Y, Wu Y, et al. Isolation and identification of IAA-producing strains from peanut rhizosphere and its promoting effects on peanut growth[J]. Chin J Oil Crop Sci, 2016, 38(1): 104-110.
[22]	Lee SM, Kong HG, Song GC, et al. Disruption of firmicutes and actinobacteria abundance in tomato rhizosphere causes the incidence of bacterial wilt disease[J]. ISME J, 2021, 15(1): 330-347.
[23]	刘明艳, 马嘉晗, 李瑜, 等. 16S rRNA基因高变区V4和V3-V4及测序深度对油藏细菌菌群分析的影响[J]. 微生物学通报, 2020, 47(2): 440-449.
	Liu MY, Ma JH, Li Y, et al. Influence of 16S rRNA gene V4 and V3-V4 sequencing and sequencing depth on unraveling bacterial communities inhabiting oil reservoirs[J]. Microbiol China, 2020, 47(2): 440-449.
[24]	Mori K, Iriye R, Hirata M, et al. Quantification ofBacillus species in a wastewater treatment system by the molecular analyses[J]. Biotechnol Bioprocess Eng, 2004, 9(6): 482-489.
[25]	Schönfeld J, Heuer H, Van Elsas JD, et al. Specific and sensitive detection of Ralstonia solanacearum in soil on the basis of PCR amplification of fliC fragments[J]. Appl Environ Microbiol, 2003, 69(12): 7248-7256.
[26]	努尔凯麦尔·木拉提, 杨亚杰, 帕尔哈提·阿布都克日木, 等. 小麦叶绿素含量测定方法比较[J]. 江苏农业科学, 2021, 49(9): 156-159.
	Nuerkaimaier MLT, Yang YJ, Paerhati ABDKRM, et al. Comparison of determination methods of chlorophyll content in wheat[J]. Jiangsu Agric Sci, 2021, 49(9): 156-159.
[27]	张韫. 土壤·水·植物理化分析教程[M]. 北京: 中国林业出版社, 2011.
	Zhang Y. Course of soil, water and plant physical and chemical analysis[M]. Beijing: China Forestry Publishing House, 2011.
[28]	牛义岭, 商丽敏. 番茄青枯病的发生及防治[J]. 现代农业科技, 2023(12): 100-102, 108.
	Niu YL, Shang LM. Occurrence and control of tomato bacterial wilt[J]. Mod Agric Sci Technol, 2023(12): 100-102, 108.
[29]	Bonaterra A, Badosa E, Daranas N, et al. Bacteria as biological control agents of plant diseases[J]. Microorganisms, 2022, 10(9): 1759.
[30]	Yin JK, Zhang ZL, Zhu CC, et al. Heritability of tomato rhizobacteria resistant to Ralstonia solanacearum[J]. Microbiome, 2022, 10(1): 227.
[31]	Li CY, Hu WC, Pan B, et al. Rhizobacterium Bacillus amyloliq-uefaciens strain SQRT3-mediated induced systemic resistance controls bacterial wilt of tomato[J]. Pedosphere, 2017, 27(6): 1135-1146.
[32]	韦中. 生物有机肥防控土传番茄青枯病的效果及其机制研究[D]. 南京: 南京农业大学, 2012.
	Wei Z. Study on the effect and mechanism of bio-organic fertilizer in controlling soil-borne tomato bacterial wilt[D]. Nanjing: Nanjing Agricultural University, 2012.
[33]	王芳, 于璐, 齐泽铮, 等. 大豆镰刀菌根腐病拮抗菌的筛选及生防效果[J]. 生物技术通报, 2024, 40(7): 216-225. doi: 10.13560/j.cnki.biotech.bull.1985.2024-0070
	Wang F, Yu L, Qi ZZ, et al. Screening and biocontrol effect of antagonistic bacteria against soybean root rot[J]. Biotechnology Bulletin, 2024, 40(7): 216-225. doi: 10.13560/j.cnki.biotech.bull.1985.2024-0070
[34]	曾婉宁, 王彦譞, 王繁珍, 等. 暹罗芽孢杆菌Y-54对番茄叶霉病的生防作用[J]. 河南农业科学, 2024, 53(3): 103-109.
	Zeng WN, Wang YX, Wang FZ, et al. Biocontrol effect of Bacillus Siamese Y-54 on tomato leaf mold[J]. J Henan Agric Sci, 2024, 53(3): 103-109.
[35]	曹云娥, 吴庆, 张美君, 等. 瓜类枯萎病生防菌WQ-6的筛选鉴定、发酵工艺优化及防效研究[J]. 园艺学报, 2020, 47(6): 1072-1086. doi: 10.16420/j.issn.0513-353x.2019-0925
	Cao YE, Wu Q, Zhang MJ, et al. Screening, identification and optimization of fermentation conditions of biocontrol strain WQ-6 to melon Fusarium wilt disease[J]. Acta Hortic Sin, 2020, 47(6): 1072-1086.
[36]	王位, 薛鸣, 雷婷越, 等. 番茄青枯病拮抗菌的筛选及防治效果[J]. 海南大学学报: 自然科学版, 2024, 42(1): 30-36.
	Wang W, Xue M, Lei TY, et al. Screening and control effect of antagonistic bacteria against tomato bacterial wilt[J]. Nat Sci J Hainan Univ, 2024, 42(1): 30-36.
[37]	Rooijakkers SHM, van Strijp JAG. Bacterial complement evasion[J]. Mol Immunol, 2007, 44(1-3): 23-32. pmid: 16875737
[38]	Passari AK, Mishra VK, Leo VV, et al. Phytohormone production endowed with antagonistic potential and plant growth promoting abilities of culturable endophytic bacteria isolated from Cleroden-drum colebrookianum Walp[J]. Microbiol Res, 2016, 193: 57-73. doi: S0944-5013(16)30306-8 pmid: 27825487
[39]	Chen Y, Yan F, Chai YR, et al. Biocontrol of tomato wilt disease by Bacillus subtilis isolates from natural environments depends on conserved genes mediating biofilm formation[J]. Environ Microbiol, 2013, 15(3): 848-864. doi: 10.1111/j.1462-2920.2012.02860.x pmid: 22934631
[40]	Durairaj K, Velmurugan P, Park JH, et al. Potential for plant biocontrol activity of isolated Pseudomonas aeruginosa and Bacillus stratosphericus strains against bacterial pathogens acting through both induced plant resistance and direct antagonism[J]. FEMS Microbiol Lett, 2017, 364(23): 10.1093/femsle/fnx225.
[41]	Deng XH, Zhang N, Li YC, et al. Bio-organic soil amendment promotes the suppression of Ralstonia solanacearum by inducing changes in the functionality and composition of rhizosphere bacterial communities[J]. New Phytol, 2022, 235(4): 1558-1574.
[42]	Kwak MJ, Kong HG, Choi K, et al. Rhizosphere microbiome structure alters to enable wilt resistance in tomato[J]. Nat Biotechnol, 2018, 36(11): 1117.
[43]	王晓楠, 冯晓晓, 施斌, 等. 内生细菌ZN-S10的鉴定及其对番茄青枯病菌的抑菌作用[J]. 浙江农业学报, 2023, 35(11): 2636-2644. doi: 10.3969/j.issn.1004-1524.20230864
	Wang XN, Feng XX, Shi B, et al. Identification of Bacillus velezen-sis ZN-S10 and its antification effect on tomato bacterial wilt[J]. Acta Agric Zhejiangensis, 2023, 35(11): 2636-2644.
[44]	Weller DM, Raaijmakers JM, Gardener BB, et al. Microbial populations responsible for specific soil suppressiveness to plant pathogens[J]. Annu Rev Phytopathol, 2002, 40: 309-348. pmid: 12147763
[45]	Wen T, Zhao ML, Liu T, et al. High abundance of Ralstonia sola-nacearum changed tomato rhizosphere microbiome and metabolome[J]. BMC Plant Biol, 2020, 20(1): 166.
[46]	Li JY, Zhao QQ, 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.
[47]	Zhang ZY, Li J, Zhang ZQ, et al. Tomato endophytic bacteria composition and mechanism of suppressiveness of wilt disease(Fusar-ium oxysporum)[J]. Front Microbiol, 2021, 12: 731764.