烟叶霉变病拮抗菌的筛选、鉴定、培养条件优化及其防治效果研究

doi:10.3969/j.issn.1004-1524.20240815

浙江农业学报 ›› 2025, Vol. 37 ›› Issue (7): 1481-1491.DOI: 10.3969/j.issn.1004-1524.20240815

烟叶霉变病拮抗菌的筛选、鉴定、培养条件优化及其防治效果研究

周航¹(), 贾涛², 方蒋平³, 杨永锋⁴, 余洋洋¹, 裘垚¹, 陈思原¹, 冯魏¹, 张静远¹, 陈红丽¹^,^*()

1.河南农业大学烟草学院,河南郑州 450046
2.天昌国际烟草有限公司,河南许昌 461000
3.江苏中烟工业有限责任公司,江苏南京 210015
4.河南中烟工业有限责任公司技术中心,河南郑州 450016

收稿日期:2024-09-19 出版日期:2025-07-25 发布日期:2025-08-20
作者简介:周航(1999—),男,安徽滁州人,硕士研究生,研究方向为烟草加工与质量评价。E-mail:zhou813010@163.com
通讯作者: ^*陈红丽,E-mail:chenhonglili06@163.com
基金资助:
中国烟草总公司科技项目(110202202029)

Study on screening, identification, culture optimization, and biocontrol effects of antagonistic bacteria against tobacco leaf mildew

ZHOU Hang¹(), JIA Tao², FANG Jiangping³, YANG Yongfeng⁴, YU Yangyang¹, QIU Yao¹, CHEN Siyuan¹, FENG Wei¹, ZHANG Jingyuan¹, CHEN Hongli¹^,^*()

1. Tobacco College, Henan Agricultural University, Zhengzhou 450046, China
2. Tianchang International Tobacco Co., Ltd., Xuchang 461000, Henan, China
3. China Tobacco Jiangsu Industrial Co., Ltd., Nanjing 210015, China
4. Technology Center, China Tobacco Henan Industrial Co., Ltd., Zhengzhou 450016, China

Received:2024-09-19 Online:2025-07-25 Published:2025-08-20

摘要/Abstract

摘要： 为明确引起云南烟区仓储期烟叶霉变的病原菌,并筛选病原菌的拮抗菌株,本研究从霉变烟叶中分离病原菌,通过科赫法则验证、菌落培养特征和分子生物学对病原菌进行鉴定;采用平板稀释法从健康烟叶中筛选针对所得病原菌的拮抗菌株,对拮抗菌进行生理生化特性鉴定、培养条件优化,验证不同浓度下拮抗菌对烟叶霉变的防治效果。结果表明,从云南霉变烟叶中分离出2株致病菌,通过形态学和分子生物学鉴定为黑曲霉(Aspergillus niger)和黄曲霉(Aspergillus flavus);筛选获得霉变病原菌的拮抗菌贝莱斯芽孢杆菌(Bacillus velezensis)ZH-X10,扫描电镜(scanning electron microscope, SEM)观察显示,拮抗菌可使致病菌菌丝发生畸形、萎缩;菌株ZH-X10在pH值为7、35 ℃和NaCl质量分数为2%时发酵效果最好;菌株ZH-X10在浓度为10⁸ CFU·mL^-1时对黑曲霉和黄曲霉具有较好的防治效果,且此浓度为抑制霉菌生长的临界接种量。综上所述,贝莱斯芽孢杆菌ZH-X10对烟叶致霉菌具有良好的拮抗效果,可作为一种新型生防菌。

关键词: 烟叶, 仓储, 真菌, 霉变, 拮抗菌, 生物防治

Abstract:

To identify the pathogenic fungi responsible for moldy stored tobacco leaves in Yunnan tobacco-growing areas and screen antagonistic strains against these pathogens, this study isolated fungi from moldy tobacco leaves. Pathogens were identified through Koch’s postulates verification, cultural characteristics, and molecular biology. Antagonistic strains were screened from healthy tobacco leaves using the plate dilution method, followed by physiological-biochemical characterization and culture condition optimization. The biocontrol efficacy of antagonistic strains against tobacco mold at varying concentrations was validated. The results demonstrated that two pathogenic strains isolated from moldy tobacco leaves in Yunnan were identified as Aspergillus niger and Aspergillus flavus based on morphological and molecular characteristics. An antagonistic strain, Bacillus velezensis ZH-X10, was screened and found to induce mycelial deformation and shrinkage in pathogens, as observed via scanning electron microscopy (SEM). Optimal fermentation conditions for ZH-X10 were pH 7.0, 35 ℃, and NaCl mass fraction of 2%. A concentration of 10⁸ CFU·mL^-1 of ZH-X10 demonstrated effective control against A. niger and A. flavus, representing the threshold inoculation concentration for mold suppression. In conclusion, Bacillus velezensis ZH-X10 exhibited significant antagonistic activity against tobacco mold pathogens and showed potential as a novel biocontrol agent.

Key words: tobacco leaf, storage, fungus, mold, antagonistic bacteria, biological control

中图分类号:

S432.44

周航, 贾涛, 方蒋平, 杨永锋, 余洋洋, 裘垚, 陈思原, 冯魏, 张静远, 陈红丽. 烟叶霉变病拮抗菌的筛选、鉴定、培养条件优化及其防治效果研究[J]. 浙江农业学报, 2025, 37(7): 1481-1491.

ZHOU Hang, JIA Tao, FANG Jiangping, YANG Yongfeng, YU Yangyang, QIU Yao, CHEN Siyuan, FENG Wei, ZHANG Jingyuan, CHEN Hongli. Study on screening, identification, culture optimization, and biocontrol effects of antagonistic bacteria against tobacco leaf mildew[J]. Acta Agriculturae Zhejiangensis, 2025, 37(7): 1481-1491.

图/表 12

图1 ZZ-1和ZZ-2的菌落培养特征

Fig.1 Cultivation characteristics of ZZ-1 and ZZ-2 colonies

图2 菌株ZZ-1和ZZ-2的系统发育树

Fig.2 Phylogenetic trees of strains ZZ-1 and ZZ-2

图3 接种黑曲霉和黄曲霉后烟叶的发病情况 CK,接种无菌水;A,接种黑曲霉;B,接种黄曲霉。

Fig.3 Infection symptoms of tobacco leaves inoculated with Aspergillus niger and Aspergillus flavus CK, Inoculated with sterile water; A, Inoculated with Aspergillus niger; B, Inoculated with Aspergillus flavus.

图4 拮抗菌株的初筛效果 A,黑曲霉;B,黄曲霉。

Fig.4 Initial screening effect of antagonistic strains A, Aspergillus niger; B, Aspergillus flavus.

图5 拮抗菌株ZH-X10的复筛结果 A,黑曲霉对照;B,黄曲霉对照;C,黑曲霉复筛;D,黄曲霉复筛。

Fig.5 Rescreening results of antagonistic strain ZH-X10 A, Aspergillus niger control; B, Aspergillus flavus control; C, Aspergillus niger re-screening; D, Aspergillus flavus re-screening.

图6 拮抗菌ZH-X10对黑曲霉和黄曲霉菌丝生长的影响 A,黑曲霉对照;B,拮抗菌ZH-X10处理的黑曲霉;C,黄曲霉对照;D,拮抗菌ZH-X10处理的黄曲霉。

Fig.6 Effect of antagonistic strain ZH-X10 on hyphae growth of Aspergillus niger and Aspergillus flavus A, Aspergillus niger control; B, Aspergillus niger treated with antagonistic strain ZH-X10; C, Aspergillus flavus control; D, Aspergillus flavus treated with antagonistic strain ZH-X10.

图7 基于16S rRNA序列的菌株ZH-X10系统发育树

Fig.7 Phylogenetic tree of strain ZH-X10 based on 16S rRNA

图8 菌株ZH-X10的生长曲线

Fig.8 Growth curve of the strain ZH-X10

图9 pH值对菌株ZH-X10生长的影响

Fig.9 Effects of pH value on the growth of strain ZH-X10

图10 温度对菌株ZH-X10生长的影响

Fig.10 Effect of temperature on the growth of strain ZH-X10

图11 NaCl质量分数对菌株ZH-X10生长的影响

Fig.11 Effect of NaCl mass fraction on the growth of strain ZH-X10

图12 ZH-X10菌悬液对烟叶的防霉效果 CK,对照组,接种无菌水;T1~T4,分别接种106、107、108、109 CFU·mL-1的ZH-X10菌悬液。

Fig.12 Control effect of ZH-X10 suspension against mold in tobacco leaves CK, Control group, inoculated with sterile water; T1-T4 were inoculated with ZH-X10 bacterial suspension of 106,107,108,109 CFU·mL-1, respectively.

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烟叶霉变病拮抗菌的筛选、鉴定、培养条件优化及其防治效果研究

Study on screening, identification, culture optimization, and biocontrol effects of antagonistic bacteria against tobacco leaf mildew

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