贝莱斯芽胞杆菌NZ-4挥发性有机物的促生作用及其活性成分分析

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

摘要/Abstract

摘要：

目的实验室前期筛选得到能够分泌具有抑菌作用挥发性物质的5株芽胞杆菌菌株，进一步明确生防芽胞杆菌挥发性物质的促生功能，并鉴定其挥发性活性组分，以期获得具有抑菌和促生双重功能的优良菌株，研发具有促生作用的挥发性天然产物。方法采用盆栽熏蒸法、选择培养基测定和熵值计算法综合评定出最佳促生菌株。通过高效液相色谱技术检测其分泌的挥发性物质对马铃薯中细胞分裂素、赤霉素和生长素含量的影响，并采用实时荧光定量PCR分析挥发性物质对马铃薯内源激素相关合成基因（StCKX1、StLAX1和StGA2ox1）表达量的影响。通过全二维气相色谱-质谱（two-dimensional gas chromatography mass spectrometry, GC×GC-MS）对菌株挥发性成分进行检测，利用NIST/EPA/NIH数据库鉴定成分。根据峰面积比例，购买相对含量最高的3种纯品，体外检测其对植株的促生作用。结果贝莱斯芽胞杆菌NZ-4分泌的挥发性物质熏蒸后马铃薯促生作用最为显著，植株株高和根长分别增加了40.01%和15.15%；NZ-4具有溶磷、固氮和产生铁载体的能力；熵值法综合评价结果中NZ-4菌株得分最高，为0.947 6。因此，确定NZ-4为最优促生菌株。经贝莱斯芽胞杆菌NZ-4挥发性物质熏蒸后，马铃薯叶片中细胞分裂素、赤霉素和生长素的含量较对照组分别增加了1.09倍、0.87倍和1.89倍，StGA20ox1和StLAX1基因的表达量较对照提升了3.61倍和4.71倍。GC×GC-MS结果表明，2-庚酮、3-乙基甲苯和2-壬酮相对含量最高；浓度为1 μg/μL的2-庚酮和100 ng/μL的3-乙基甲苯能够显著促进植株的生长，2-壬酮对植株的生长没有明显作用。结论贝莱斯芽胞杆菌NZ-4产生的挥发性物质对马铃薯具有良好的促生作用，明确了2-庚酮和3-乙基甲苯为挥发性活性成分，NZ-4菌株产生的挥发性有机物具有开发新型天然产物的潜力。

关键词: 促生, 贝莱斯芽胞杆菌菌株NZ-4, 2-庚酮, 3-乙基甲苯

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

石艳华, 李朔, 高玉珠, 郑保坤, 朱杰华, 张岱, 杨志辉. 贝莱斯芽胞杆菌NZ-4挥发性有机物的促生作用及其活性成分分析[J]. 生物技术通报, 2025, 41(8): 300-310.

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.

图/表 11

参考文献 49

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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	+	+	+	+