山核桃内生细菌ZL34的鉴定、抑菌活性及发酵培养基优化
Identification,Antimicrobial Activity,and Optimization of Fermentation Medium of Endophytic Bacterium Strain ZL34 from Carya cathayensis
竺利红 1邱海萍 1项慧娟 2刘浩 3施跃峰 1袁玉伟1
作者信息
- 1. 浙江省农业科学院植物保护与微生物研究所,杭州 310021
- 2. 杭州市淳安县石林镇人民政府,杭州 311701
- 3. 天津科技大学生物工程学院,天津 300222
- 折叠
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.
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.
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Key words
Botryosphaeria dothidea/Bacillus velezensis/identification/antimicrobial activity/Carya cathayensis引用本文复制引用
基金项目
浙江省公益技术研究农业项目(2015C32065)
浙江省农业科学院高层次人才引进与持续培养项目()
出版年
2024
国家科技期刊平台
NSTL
万方数据