生姜青枯病菌拮抗芽孢杆菌的筛选、鉴定及发酵条件优化

Screening, identification and optimization of fermentation conditions for Bacillus antagonistic against Ralstonia solanacearum

  • 摘要: 【目的】筛选出对生姜青枯病病原菌具有较好拮抗效果的芽孢杆菌,并优化其发酵条件,旨在为丰富生姜青枯病的生防芽孢杆菌菌种资源及生防制剂的进一步开发应用打下理论基础。【方法】以生姜根际土壤为研究对象,以生姜青枯病菌为靶标,通过稀释涂布法和抑菌圈法从中分离并筛选出能高效拮抗生姜青枯病菌的芽孢杆菌;通过形态学观察、生理生化测定和分子生物学鉴定,确定拮抗菌株的分类地位;采用牛津杯法测定拮抗菌株对生姜青枯病菌的抑菌活性,并结合单因素和正交试验对其发酵条件进行优化。【结果】从生姜根际土壤中分离出1株对生姜青枯病菌具有较强拮抗作用的菌株HC-5,其抑菌圈直径达0.71 cm。综合形态学特征、生理生化特性及分子鉴定结果,将菌株HC-5鉴定为贝莱斯芽孢杆菌(Bacillus velezensis)。单因素和正交试验结果显示,菌株HC-5的最适发酵培养基组分为糯米粉5.0 g/L、酵母浸粉7.0 g/L、硫酸锌11.0 g/L,最适发酵条件为培养温度35℃、培养时间36 h、接种量1.0%、转速180 r/min、初始pH 5.5、装液量100 m L。优化后菌株HC-5无菌上清液对生姜青枯病菌的抑菌圈直径增大至3.03 cm,相比于LB和发酵培养基条件下分别增大76.2%和62.9%。【结论】菌株HC-5对生姜青枯病菌具有良好的拮抗效果,经发酵条件优化后其无菌上清液对生姜青枯病菌的抑菌活性显著增强。菌株HC-5具有开发成生姜青枯病生防菌剂的潜力。

     

    Abstract: 【Objective】This study aimed to identify Bacillus strains that showed effective antagonistic activity against the ginger bacterial wilt causative pathogen Ralstonia solanacearum, and to optimize the fermentation conditions of these strains, thereby providing theoretical foundation for enriching the resources of biocontrol Bacillus strains and further developing and applying biocontrol agents for ginger wilt disease. 【Method】Ginger rhizosphere soil samples were research object,ginger R. solanacearum strains were targets, dilution coating method and antibacterial circle method were employed to screen strains exhibiting potent antagonistic effects against R. solanacearum. Antagonistic strains were then classified based on morphological observations, physiological and biochemical testing, as well as molecular biological identification. The antibacterial activity of the selected strains against R. solanacearum was quantified using the Oxford cup method. Subsequently, both single-factor and orthogonal experiments were conducted to refine their fermentation conditions. 【Result】Strain HC-5, isolated from the ginger rhizosphere soil and exhibiting strong antagonistic properties against R. solanacearum formed an inhibition zone of 0.71 cm in diameter. Integrating morphological features, physiological and biochemical characteristics and molecular identification results, HC-5 was classified as Bacillus velezensis. The results of single factor and orthogonal experiments showed that, optimal growth medium for strain HC-5 was established to contain glutinous rice flour(5.0 g/L), yeast extract(7.0 g/L), and zinc sulfate(11.0 g/L). The best fermentation conditions were determined as temperature of 35 ℃, fermentation duration of 36 h, inoculation amount of 1.0%, rotation speed of 180r/min, initial pH of 5.5 and liquid volume of 100 mL. The diameter of the antibacterial zone of the sterile supernatant of the optimized strain HC-5 against R. solanacearum increased to 3.03 cm, which was 76.2% and 62.9% larger than under LB and fermentation medium conditions, respectively. 【Conclusion】Strain HC-5 demonstrates a substantial antagonistic effect against R. solanacearum. The enhanced antibacterial activity of its sterile supernatant following fermentation condition optimization suggests its potential as a biocontrol agent in combating ginger bacterial wilt.

     

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