贝莱斯芽孢杆菌ZHX-7的分离鉴定及抑菌促生效果

doi:10.13560/j.cnki.biotech.bull.1985.2023-0152

摘要/Abstract

摘要：

为获得高效拮抗花生病害的生防微生物，以花生冠腐病菌为指示菌，采用稀释涂布平板法和平板对峙法，从山东莱西采集的花生连作田的病、健株根际土壤样品中筛选到1株高效生防菌株，编号为ZHX-7。结合形态学特征和16S rDNA、gyrB序列分析，确定ZHX-7为贝莱斯芽孢杆菌（Bacillus velezensis）。ZHX-7的菌悬液、无菌发酵液和挥发性气体对花生白绢病菌、基腐病菌、网斑病菌和轮斑病菌菌丝生长的抑制均达到极显著的水平，平板对峙试验表明ZHX-7的菌悬液对花生白绢病菌、基腐病菌、网斑病菌和轮斑病菌的抑菌率分别为64.40%、70.37%、77.43%和65.32%。在以ZHX-7无菌发酵液稀释10倍的PDA培养基上，花生白绢病菌、基腐病菌、网斑病菌和轮斑病菌菌丝的生长抑制率分别为67.81%、52.16%、23.52%和69.93%。双皿对扣试验表明ZHX-7的挥发性气体对花生白绢病菌、基腐病菌、网斑病菌和轮斑病菌的抑菌率分别为86.82%、35.40%、81.70%和44.19%。ZHX-7可产生蛋白酶、纤维素酶和淀粉酶等次级代谢产物；此外，在温室盆栽条件下，ZHX-7有菌发酵液明显促进了花生的生长，同对照相比，鲜质量、干质量分别增加31.24%和26.47%，并且ZHX-7有菌发酵液明显提高了花生对白绢病的抗性，防效达到51.76%。综上所述，本研究筛选获得的一株贝莱斯芽孢杆菌ZHX-7，能够抑制多种花生病原真菌，同时促进花生生长，是一株具有宽广拮抗谱的多功能菌株，具有较好的生防应用潜力。

关键词: 贝莱斯芽孢杆菌, 花生病害, 分离鉴定, 抑菌率, 生物防治, 促生作用, 白绢病菌

Abstract:

In order to obtain highly effective biocontrol microorganism for controlling peanut diseases, using Aspergillus niger as the indicator, a highly effective biocontrol strain ZHX-7 was screened from soil samples by agar disk dilution and dual-culture assay. The soil samples were collected from the rhizosphere of diseased and healthy peanut plants in continuous cropping field in Laixi, Shandong province. The strain ZHX-7 was identified as Bacillus velezensis based on the morphological characteristics, 16S rDNA and gyrB sequence analysis. The bacterial suspension, sterile fermentation broth and volatile gas of ZHX-7 significantly inhibited the growth of Sclerotium rolfsii, Fusarium neocosmosporiellum, Phoma arachidicola and Alternaria alternata. The antagonistic experiment results showed that the inhibition rates of ZHX-7 suspension against the growth of S. rolfsii, F. neocosmosporiellum, P. arachidicola and A. alternata were 64.40%, 70.37%, 77.43% and 65.32%, respectively. In the sterile fermentation broth of ZHX-7 diluted 10 times with PDA medium, the inhibition rates of ZHX-7 against the growth of S. rolfsii, F. neocosmosporiellum, P. arachidicola and A. alternata were 67.81%, 52.16%, 23.52% and 69.93%, respectively. The double dish test showed that the inhibition rates of ZHX-7 volatile gas against the growth of S. rolfsii, F. neocosmosporiellum, P. arachidicola and A. alternata were 86.82%, 35.40%, 81.70% and 44.19%, respectively. ZHX-7 produced secondary metabolites such as protease, cellulose and amylase. In addition, under the condition of pot culture in greenhouse, ZHX-7 fermentation broth significantly promoted the growth of peanut. Compared with the control, the fresh mass and dry mass increased by 31.24% and 26.47%, respectively. ZHX-7 fermentation broth improved the resistance of peanut to S. rolfsii, and the control effect reached 51.76%. In conclusion, a strain of B. velezensis ZHX-7 screened in this study may inhibit a variety of peanut pathogenic fungi and promote peanut growth at the same time. It is a multifunctional strain with broad antagonistic spectrum against peanut diseases, and high potential for biocontrol application.

Key words: Bacillus velezensis, peanut diseases, isolation and identification, antibacterial rate, biological control, growth-promoting effect, Sclerotium rolfsii

李莹, 宋新颖, 何康, 郭志青, 于静, 张霞. 贝莱斯芽孢杆菌ZHX-7的分离鉴定及抑菌促生效果[J]. 生物技术通报, 2023, 39(12): 229-236.

LI Ying, SONG Xin-ying, HE Kang, GUO Zhi-qing, YU Jing, ZHANG Xia. Isolation and Identification of Bacillus velezensis ZHX-7 and Its Antibacterial and Growth-promoting Effects[J]. Biotechnology Bulletin, 2023, 39(12): 229-236.

图/表 7

图1 ZHX-7对冠腐病菌的拮抗效果（A）及其菌落形态（B）

Fig. 1 Antagonistic effect of ZHX-7 against A. niger（A）and its colony morphology（B）

图2 基于16S rDNA和gyrB序列的ZHX-7系统发育分析

Fig. 2 Phylogenetic analysis of ZXH-7 based on 16S rDNA and gyrB sequences

图3 ZHX-7菌悬液对4种病原真菌拮抗活性的测定 A：ZHX-7对4种花生病原菌的拮抗效果图；B：病原菌的菌落生长情况统计；C：ZHX-7对4种花生病原菌的抑菌率统计。**在0.01水平差异极显著。下同

Fig. 3 Determination of antagonistic activity of ZHX-7 bacterial suspension against four pathogens A: Antagonistic effect of ZHX-7 on four peanut pathogens. B: Statistics of colony growth of pathogens. C: Statistics of inhibition rate of ZHX-7 against four peanut pathogens. ** indicates extremely significant differences at 0.01 level. The same below

图4 ZHX-7无菌发酵液对4种病原真菌生长的影响

Fig. 4 Effects of ZHX-7 fermentation broth on the growth of four pathogens

图5 ZHX-7挥发性气体对4种病原真菌的抑菌作用

Fig. 5 Inhibitory effects of ZHX-7 volatiles against four pathogens

图6 菌株ZHX-7次级代谢产物胞外酶的情况

Fig. 6 Detection of ZHX-7 secondary metabolite extracellular enzyme activity

表1 ZHX-7对花生生长及白绢病的防治效果

Table1 Control effect of ZHX-7 on peanut growth and peanut Sclerotium blight

处理Treatment	鲜质量Fresh mass/g	干质量Dry mass/g	死亡率Mortality/%	病情指数Disease index	防效Efficacy/%
CK	10.47±0.74 A	1.48±0.10 A	56.67±5.77 A	85.42±3.15 A	/
ZHX-7	13.74±0.96 B	1.88±0.11 B	18.33±2.89 B	41.25±3.31 B	51.76±2.13

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