山核桃内生细菌ZL34的鉴定、抑菌活性及发酵培养基优化

doi:10.16409/j.cnki.2095-039x.2024.02.042

摘要/Abstract

摘要： 茶藨子葡萄座腔菌Botryosphaeria dothidea引起的山核桃干腐病是目前山核桃林最为严重的病害之一。菌株ZL34是从山核桃根系中分离到的一株对茶藨子葡萄座腔菌有较强抑菌活性的内生细菌，为明确其分类地位、抑菌活性和优化发酵培养基，采用形态学观察、生理生化特征研究及分子生物学方法对菌株ZL34进行种属鉴定；采用平板对峙法和杯碟法测定了菌株ZL34的抗菌谱；采用插片法、生长速率法和琼脂平板法测定了菌株ZL34对茶藨子葡萄座腔菌的抑菌活性；采用正交试验优化其发酵配方。结果表明，菌株ZL34为贝莱斯芽胞杆菌Bacillus velezensis，其不仅对茶藨子葡萄座腔菌具有较强的抑菌活性，还对尖孢镰刀菌Fusarium oxysporum和金黄色葡萄球菌Staphylococcus aureus等多种病原真菌和细菌具有较强的抑菌活性。菌株ZL34发酵上清液可显著抑制茶藨子葡萄座腔菌菌丝的生长，其中5倍稀释液的抑制率为96.48%，但对孢子的萌发率无明显影响。受抑制的菌丝色素积累明显，其中气生菌丝表现为异常突起和膨大，形成孔洞，基内菌丝产生大量的分生孢子。筛选到由4%黄豆饼粉、4%玉米粉、0.5%尿素、0.6%(NH₄)₂HPO₄、0.35% CaCO₃和pH 7.0组成的发酵配方，发酵液活菌量为6.97×10⁹ CFU/mL，比优化前提高了44.79%，上清液20倍稀释液对茶藨子葡萄座腔菌的抑菌率为87.16%，比优化前提高了45.41%。本研究表明，贝莱斯芽胞杆菌ZL34对茶藨子葡萄座腔菌菌丝的生长有较强抑制作用，并建立了高产芽胞和高活性的发酵配方，为山核桃干腐病生防制剂的开发提供了研究基础。

关键词: 茶藨子葡萄座腔菌, 贝莱斯芽胞杆菌, 鉴定, 抑菌活性, 山核桃

Abstract: Canker disease of Carya cathayensis caused by Botryosphaeria dothidea is one of the most serious diseases affecting hickory plant in China. To effectively prevent and control the occurrence and development of this disease, we previously isolated an endophytic bacterium (strain ZL34) with strong antimicrobial activity against B. dothidea from the root of C. cathayensis. Taxonomic classification, antimicrobial characteristics of fermentation products, and optimization of fermentation medium of the strain ZL34 were investigated in this study. Firstly, based on its morphological and physiological characteristics combined with molecular biological analysis, the strain ZL34 was identified as Bacillus velezensis. Antimicrobial spectrum analysis revealed that, in addition to exhibiting strong antimicrobial activity against the canker disease pathogen B. dothidea, the fermentation supernatant of ZL34 also had significant antimicrobial effects against various pathogenic fungi and bacteria, including Fusarium oxysporum and Staphylococcus aureus. The fermentation supernatant of ZL34 significantly inhibited the growth of B. dothidea mycelia, with a 5-fold dilution inhibiting mycelial growth by 96.48%, although it had no noticeable effect on spore germination. The inhibited B. dothidea mycelia accumulated pigments markedly, showed abnormal hyphal protrusions and swelling, and a large number of conidia were produced from the substrate mycelia. Finally, we investigated the effect of medium components on the sporulation and active production of ZL34 through orthogonal design experiments, obtaining the optimal medium formulation. It was composed of 40 g/L soybean powder, 40 g/L corn flour, 5.0 g/L CO(NH₂)₂, 6.0 g/L(NH₄)₂HPO₄, 3.5 g/L CaCO₃ and pH 7.0. In the optimized medium, the viable bacteria count in the fermentation broth reached 6.97×10⁹ CFU/mL, a 44.79% increase compared to the medium before optimization. The 20-fold diluted supernatant had an antimicrobial rate of 87.16% against B. dothidea, a 45.41% increase compared to the supernatant before optimization. The above results indicated that B. velezensis ZL34 had strong inhibition activity against B. dothidea, and the medium formula of the strain was optimized with higher endospore production and antimicrobial activity. This research lays a solid foundation for the successful development of B. velezensis ZL34 into biological control agents against canker disease.

Key words: Botryosphaeria dothidea, Bacillus velezensis, identification, antimicrobial activity, Carya cathayensis

中图分类号:

S476

竺利红, 邱海萍, 项慧娟, 刘浩, 施跃峰, 袁玉伟. 山核桃内生细菌ZL34的鉴定、抑菌活性及发酵培养基优化[J]. 中国生物防治学报, 2024, 40(4): 894-904.

ZHU Lihong, QIU Haiping, XIANG Huijuan, LIU Hao, SHI Yuefeng, YUAN Yuwei. Identification, Antimicrobial Activity, and Optimization of Fermentation Medium of Endophytic Bacterium Strain ZL34 from Carya cathayensis[J]. Chinese Journal of Biological Control, 2024, 40(4): 894-904.

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