Analysis of the Growth-promoting Effects and Active Components of Volatile Organic Compounds Produced by Bacillus velezensis NZ-4

doi:10.13560/j.cnki.biotech.bull.1985.2025-0135

Abstract

Abstract:

Objective In previous research, five Bacillus strains were identified to secret volatile substances with antifungal effects. This study aims to further elucidate the growth-promoting function of volatiles produced by biocontrol Bacillus strains and to identify their active volatile compounds. The goal is to obtain the strains that demonstrate both antifungal and plant-promoting properties, and to develop the volatile natural products with growth-promoting effects. Method The best growth-promoting strains were comprehensively evaluated using the pot fumigation method, selective medium assay and an entropy calculation method. The effect of volatile substances secreted by the strains on the contents of three endogenous hormones,cytokinin, gibberellin, and auxin in potato leaves,was determined by high-performance liquid chromatography (HPLC). The expressions of related genes (StCKX1, StLAX1, and StGA2ox1) were analyzed using real-time quantitative PCR. The specific components of VOCs produced by the strain were detected using two-dimensional gas chromatography-mass spectrometry (GC×GC-MS), and identified by the NIST/EPA/NIH database. Three pure compounds with the highest relative contents were then procured, and the plant growth-promoting effects of these substances on plants were tested in vitro. Result The heights and root lengths of plants increased by 40.01% and 15.15%, respectively, after fumigation with volatiles from Bacillus velezensis NZ-4. B. velezensis NZ-4 was also capable of solubilizing phosphorus, fixing nitrogen, and producing siderophore. It received the highest comprehensive score (0.947 6) in the evaluation using the entropy method and was identified as the optimal growth-promoting strain. The levels of cytokinin, gibberellin, and auxin represented increases of 1.09-fold, 0.87-fold, and 1.89-fold, compared to the control group.The expressions of the StGA20ox1 and StLAX1 genes increased by 3.61-fold and 4.71-fold, respectively. GC×GC-MS analysis revealed that 2-heptanone, 3-ethyltoluene, and 2-nonanone were the most abundant compounds. A concentration of 1 μg/μL of 2-heptanone and 100 ng/μL of 3-ethyltoluene significantly promoted plant growth, while 2-nonanone had no significant effect. Conclusion The VOCs produced by B. velezensis NZ-4 promote the growth of potato, with 2-heptanone and 3-ethyltoluene identified as the active volatile compounds. These VOCs have potential for development into novel natural products with growth-promoting properties.

Key words: plant growth promotion, Bacillus velezensis NZ-4, 2-heptanone, 3-ethyltoluene

SHI Yan-hua, LI Shuo, GAO Yu-zhu, ZHENG Bao-kun, ZHU Jie-hua, ZHANG Dai, YANG Zhi-hui. Analysis of the Growth-promoting Effects and Active Components of Volatile Organic Compounds Produced by Bacillus velezensis NZ-4[J]. Biotechnology Bulletin, 2025, 41(8): 300-310.

Figures/Tables 11

References 49

[1]	Thomloudi EE, Tsalgatidou PC, Baira E, et al. Genomic and metabolomic insights into secondary metabolites of the novel Bacillus halotolerans Hil4, an endophyte with promising antagonistic activity against gray mold and plant growth promoting potential [J]. Microorganisms, 2021, 9(12): 2508.
[2]	Li H, Guan Y, Dong YL, et al. Isolation and evaluation of endophytic Bacillus tequilensis GYLH001 with potential application for biological control of Magnaporthe oryzae [J]. PLoS One, 2018, 13(10): e0203505.
[3]	Zhang D, Yu SQ, Zhao DM, et al. Inhibitory effects of non-volatiles lipopeptides and volatiles ketones metabolites secreted by Bacillus velezensis C16 against Alternaria solani [J]. Biol Control, 2021, 152: 104421.
[4]	Morath SU, Hung R, Bennett JW. Fungal volatile organic compounds: a review with emphasis on their biotechnological potential [J]. Fungal Biol Rev, 2012, 26(2/3): 73-83.
[5]	Arrebola E, Sivakumar D, Korsten L. Effect of volatile compounds produced by Bacillus strains on postharvest decay in Citrus [J]. Biol Control, 2010, 53(1): 122-128.
[6]	Lemfack MC, Nickel J, Dunkel M, et al. mVOC: a database of microbial volatiles [J]. Nucl Acids Res, 2014, 42(D1): D744-D748.
[7]	Asari S, Matzén S, Petersen MA, et al. Multiple effects of Bacillus amyloliquefaciens volatile compounds: plant growth promotion and growth inhibition of phytopathogens [J]. FEMS Microbiol Ecol, 2016, 92(6): fiw070.
[8]	Wang Y, Li YX, Yang JL, et al. Microbial volatile organic compounds and their application in microorganism identification in foodstuff [J]. Trac Trends Anal Chem, 2016, 78: 1-16.
[9]	Stahl PD, Parkin TB. Microbial production of volatile organic compounds in soil microcosms [J]. Soil Sci Soc Am J, 1996, 60(3): 821-828.
[10]	Zhang HM, Kim MS, Krishnamachari V, et al. Rhizobacterial volatile emissions regulate auxin homeostasis and cell expansion in Arabidopsis [J]. Planta, 2007, 226(4): 839-851.
[11]	Meldau DG, Long HH, Baldwin IT. A native plant growth promoting bacterium, Bacillus sp. B55, rescues growth performance of an ethylene-insensitive plant genotype in nature [J]. Front Plant Sci, 2012, 3: 112.
[12]	Xie XT, Zhang HM, Paré PW. Sustained growth promotion in Arabidopsis with long-term exposure to the beneficial soil bacterium Bacillus subtilis (GB03) [J]. Plant Signal Behav, 2009, 4(10): 948-953.
[13]	Liu HW, Brettell LE. Plant defense by VOC-induced microbial priming [J]. Trends Plant Sci, 2019, 24(3): 187-189.
[14]	van Agtmaal M, Straathof AL, Termorshuizen A, et al. Volatile-mediated suppression of plant pathogens is related to soil properties and microbial community composition [J]. Soil Biol Biochem, 2018, 117: 164-174.
[15]	Zou CS, Li ZF, Yu DQ. Bacillus megaterium strain XTBG34 promotes plant growth by producing 2-pentylfuran [J]. J Microbiol, 2010, 48(4): 460-466.
[16]	Meldau DG, Meldau S, Hoang LH, et al. Dimethyl disulfide produced by the naturally associated bacterium Bacillus sp B55 promotes Nicotiana attenuata growth by enhancing sulfur nutrition [J]. Plant Cell, 2013, 25(7): 2731-2747.
[17]	Zhao DY, Jiao JH, Du BH, et al. Volatile organic compounds from Lysinibacillus macroides regulating the seedling growth of Arabidopsis thaliana [J]. Physiol Mol Biol Plants, 2022, 28(11-12): 1997-2009.
[18]	Hao HT, Zhao X, Shang QH, et al. Comparative digital gene expression analysis of the Arabidopsis response to volatiles emitted by Bacillus amyloliquefaciens [J]. PLoS One, 2016, 11(8): e0158621.
[19]	Jiang CH, Xie YS, Zhu K, et al. Volatile organic compounds emitted by Bacillus sp. JC03 promote plant growth through the action of auxin and strigolactone [J]. Plant Growth Regul, 2019, 87(2): 317-328.
[20]	Ryu CM, Farag MA, Hu CH, et al. Bacterial volatiles promote growth in Arabidopsis [J]. Proc Natl Acad Sci USA, 2003, 100(8): 4927-4932.
[21]	Ramírez V, Munive JA, Cortes L, et al. Long-chain hydrocarbons (C21, C24, and C31) released by Bacillus sp. MH778713 break dormancy of mesquite seeds subjected to chromium stress [J]. Front Microbiol, 2020, 11: 741.
[22]	Cordovez V, Carrion VJ, Etalo DW, et al. Diversity and functions of volatile organic compounds produced by Streptomyces from a disease-suppressive soil [J]. Front Microbiol, 2015, 6: 1081.
[23]	Effah E, Holopainen JK, McCormick AC. Potential roles of volatile organic compounds in plant competition [J]. Perspect Plant Ecol Evol Syst, 2019, 38: 58-63.
[24]	强然, 张岱, 杨喆, 等. 解淀粉芽胞杆菌L19对马铃薯枯萎病菌的抑制及植株的促生作用 [J]. 园艺学报, 2024, 51(4): 875-892.
	Qiang R, Zhang D, Yang Z, et al. Inhibition of potato Fusarium wilt and growth promotion by Bacillus amyloliquefaciens L19 [J]. Acta Horticulturae Sinica, 2024, 51(4): 875-892.
[25]	李铮, 王金辉, 丁丽丽, 等. 贝莱斯芽孢杆菌菌株NZ-4生防潜能及基因组学分析 [J]. 江苏农业科学, 2023,51(2): 117-125.
	Li Z, Wang JH, Ding LL, et al. Biocontrol potential and genomic analysis of Bacillus velezensis strain NZ-4 [J]. Jiangsu Agricultural Sciences, 2023, 51(2): 117-125.
[26]	张岱. 枯草芽胞杆菌ZD01次级代谢产物对马铃薯早疫病菌的抑制机理 [D]. 保定: 河北农业大学, 2023.
	Zhang D. The antifungal mechanism of secondary metabolites produced by Bacillus subtilis ZD01 against Alternaria solani [D]. Baoding: Hebei Agricultural University, 2023.
[27]	高学策, 张岱, 魏笑薇, 等. 马铃薯黑痣病生防芽胞杆菌的筛选及其次级代谢产物的抑菌特性 [J]. 西南农业学报, 2023, 36(9): 1942-1949.
	Gao XC, Zhang D, Wei XW, et al. Screening of biocontrol Bacillus against potato black scurf and antimicrobial characteristics of its secondary metabolites [J]. Southwest China Journal of Agricultural Sciences, 2023, 36(9): 1942-1949.
[28]	朱明明, 张岱, 赵冬梅, 等. 马铃薯黑痣病生防芽孢杆菌的筛选与鉴定 [J]. 江苏农业科学, 2018, 46(14): 97-101.
	Zhu MM, Zhang D, Zhao DM, et al. Screening and identification of antagonistic Bacillus against potato black scurf [J]. Jiangsu Agric Sci, 2018, 46(14): 97-101.
[29]	Zhao J, Zhou ZJ, Bai XF, et al. A novel of new class II bacteriocin from Bacillus velezensis HN-Q-8 and its antibacterial activity on Streptomyces scabies [J]. Front Microbiol, 2022, 13: 943232.
[30]	宁楚涵, 李文彬, 张晨, 等. 定殖植物根内和根围放线菌的分离鉴定及其体外抑菌促生效应 [J]. 微生物学报, 2019, 59(10): 2024-2037.
	Ning CH, Li WB, Zhang C, et al. Isolation and identification of antagonizing and growthpromoting Actinobacteria colonized in plant roots and rhizosphere [J]. Acta Microbiol Sin, 2019, 59(10): 2024-2037.
[31]	刘泽平, 王志刚, 徐伟慧, 等. 水稻根际促生菌的筛选鉴定及促生能力分析 [J]. 农业资源与环境学报, 2018, 35(2): 119-125.
	Liu ZP, Wang ZG, Xu WH, et al. Screen, identification and analysis on the growth-promoting ability for the rice growth-promoting rhizobacteria [J]. J Agric Resour Environ, 2018, 35(2): 119-125.
[32]	Schwyn B, Neilands JB. Universal chemical assay for the detection and determination of siderophores [J]. Anal Biochem, 1987, 160(1): 47-56.
[33]	朱喜安, 魏国栋. 熵值法中无量纲化方法优良标准的探讨 [J]. 统计与决策, 2015, 31(2): 12-15.
	Zhu XA, Wei GD. Discussion on the excellent standard of dimensionless method in entropy method [J]. Stat Decis, 2015, 31(2): 12-15.
[34]	罗梦娜. 生长调节剂对小麦和玉米内源激素的影响 [D]. 杨凌: 西北农林科技大学, 2019.
	Luo MN. Effect of growth regulator on endogenous hormones in wheat and maize [D]. Yangling: Northwest A & F University, 2019.
[35]	李葵秀, 罗崇玉, 傅琪景, 等. 马铃薯StDRO2基因的生物信息学分析 [J]. 西南农业学报, 2021, 34(4): 679-688.
	Li KX, Luo CY, Fu QJ, et al. Bioinformatics analysis of potato StDRO2 gene [J]. Southwest China J Agric Sci, 2021, 34(4): 679-688.
[36]	Syed-Ab-Rahman SF, Carvalhais LC, Chua ET, et al. Soil bacterial diffusible and volatile organic compounds inhibit Phytophthora capsici and promote plant growth [J]. Sci Total Environ, 2019, 692: 267-280.
[37]	Ghazala I, Chiab N, Saidi MN, et al. Volatile organic compounds from Bacillus mojavensis I4 promote plant growth and inhibit phytopathogens [J]. Physiol Mol Plant Pathol, 2022, 121: 101887.
[38]	He AL, Zhao LY, Ren W, et al. A volatile producing Bacillus subtilis strain from the rhizosphere of Haloxylon ammodendron promotes plant root development [J]. Plant Soil, 2023, 486(1): 661-680.
[39]	Rath M, Mitchell TR, Gold SE. Volatiles produced by Bacillus mojavensis RRC101 act as plant growth modulators and are strongly culture-dependent [J]. Microbiol Res, 2018, 208: 76-84.
[40]	Fincheira P, Venthur H, Mutis A, et al. Growth promotion of Lactuca sativa in response to volatile organic compounds emitted from diverse bacterial species [J]. Microbiol Res, 2016, 193: 39-47.
[41]	Byrne ME. Making leaves [J]. Curr Opin Plant Biol, 2012, 15(1): 24-30.
[42]	Dudareva N, Negre F, Nagegowda DA, et al. Plant volatiles: recent advances and future perspectives [J]. Crit Rev Plant Sci, 2006, 25(5): 417-440.
[43]	Zou XL, Ning JQ, Zhao XJ, et al. Bacillus velezensis LY7 promotes pepper growth and induces resistance to Colletotrichum scovillei [J]. Biol Control, 2024, 192: 105480.
[44]	Tahir HAS, Gu Q, Wu HJ, et al. Plant growth promotion by volatile organic compounds produced by Bacillus subtilis SYST2 [J]. Front Microbiol, 2017, 8: 171.
[45]	Kwon YS, Ryu CM, Lee S, et al. Proteome analysis of Arabidopsis seedlings exposed to bacterial volatiles [J]. Planta, 2010, 232(6): 1355-1370.
[46]	Ren FF, Liu N, Gao B, et al. Identification of Stutzerimonas stutzeri volatile organic compounds that enhance the colonization and promote tomato seedling growth [J]. J Appl Microbiol, 2024, 135(10): lxae248.
[47]	Wu YC, Zhou JY, Li CG, et al. Antifungal and plant growth promotion activity of volatile organic compounds produced by Bacillus amyloliquefaciens [J]. Microbiologyopen, 2019, 8(8): e00813.
[48]	Cole LK, Blum MS. Antifungal properties of the insect alarm pheromones, citral, 2-heptanone, and 4-methyl-3-heptanone [J]. Mycologia, 1975, 67(4): 701-708.
[49]	Zhu M, Chen Y, Zhao NH, et al. Multiple olfactory pathways contribute to the lure process of Caenorhabditis elegans by pathogenic bacteria [J]. Sci China Life Sci, 2021, 64(8): 1346-1354.

引物名称 Primer name	序列 Sequence （5′-3′）
qStLAX1-F	GGTTTGGCATTTAATTGTAC
qStLAX1-R	CAGATTCGGTAGTTGTGA
qCKX1-F	AAGTTCAGTTCTGAAGGAAAAGT
qCKX1-R	AAAAACTTCTTGTGCAAAGTCATG
qStGA20ox1-F	CGGCCCAACAAGCATCTAAG
qStGA20ox1-R	AAGCCATGACTCCGACG
Stactin-F	GGTATTGTGCTGGATTCTGG
Stactin-R	CGTTCAGCACTAGTGGTGAA

引物名称 Primer name	序列 Sequence （5′-3′）
qStLAX1-F	GGTTTGGCATTTAATTGTAC
qStLAX1-R	CAGATTCGGTAGTTGTGA
qCKX1-F	AAGTTCAGTTCTGAAGGAAAAGT
qCKX1-R	AAAAACTTCTTGTGCAAAGTCATG
qStGA20ox1-F	CGGCCCAACAAGCATCTAAG
qStGA20ox1-R	AAGCCATGACTCCGACG
Stactin-F	GGTATTGTGCTGGATTCTGG
Stactin-R	CGTTCAGCACTAGTGGTGAA

菌株编号 Strain No.	株高 Plant height （cm）	茎粗 Stem diameter （cm）	根长 Root length （cm）	根体积 Root volume （cm³）	地上鲜重 Aboveground fresh weight （g）	地下鲜重 Belowground fresh weight （g）	地上干重 Aboveground dry weight （g）
Control	16.32±2.09d	1.83±0.26b	30.62±1.60b	8.67±1.63b	5.17±0.59b	6.77±1.30b	0.48±0.05b
L19	21.32±1.61c	2.58±0.15a	32.46±5.10ab	10.00±2.53a	8.53±1.29a	7.66±0.46a	0.54±0.07a
G32	21.38±1.07bc	2.44±0.21a	33.72±2.60ab	12.00±3.35a	8.51±0.81a	7.33±0.94a	0.54±0.06a
NZ-4	22.85±1.87a	2.32±0.37a	35.26±2.65a	12.00±2.76a	8.55±1.33a	7.97±2.07a	0.54±0.10a
HN-Q-8	21.04±1.06abc	2.46±0.11a	29.04±4.50ab	10.67±2.73a	8.01±0.69a	6.98±0.99a	0.53±0.05a
ZD01	22.81±1.52ab	2.40±0.21a	31.75±1.31ab	9.33±2.07a	8.75±1.16a	7.33±1.16a	0.55±0.09a

菌株编号 Strain No.	株高 Plant height （cm）	茎粗 Stem diameter （cm）	根长 Root length （cm）	根体积 Root volume （cm³）	地上鲜重 Aboveground fresh weight （g）	地下鲜重 Belowground fresh weight （g）	地上干重 Aboveground dry weight （g）
Control	16.32±2.09d	1.83±0.26b	30.62±1.60b	8.67±1.63b	5.17±0.59b	6.77±1.30b	0.48±0.05b
L19	21.32±1.61c	2.58±0.15a	32.46±5.10ab	10.00±2.53a	8.53±1.29a	7.66±0.46a	0.54±0.07a
G32	21.38±1.07bc	2.44±0.21a	33.72±2.60ab	12.00±3.35a	8.51±0.81a	7.33±0.94a	0.54±0.06a
NZ-4	22.85±1.87a	2.32±0.37a	35.26±2.65a	12.00±2.76a	8.55±1.33a	7.97±2.07a	0.54±0.10a
HN-Q-8	21.04±1.06abc	2.46±0.11a	29.04±4.50ab	10.67±2.73a	8.01±0.69a	6.98±0.99a	0.53±0.05a
ZD01	22.81±1.52ab	2.40±0.21a	31.75±1.31ab	9.33±2.07a	8.75±1.16a	7.33±1.16a	0.55±0.09a

处理 Treatment	溶解无机磷 Soluble inorganic phosphorus	溶解有机磷 Dissolved organic phosphorus	固氮 Nitrogen fixation	铁载体 Siderophore
L19	+	+	-	-
G32	-	+	+	+
NZ-4	+	+	+	+
HN-Q-8	+	+	-	+
ZD01	+	+	+	+