黑曲霉拮抗菌Xenorhabdus bovienii 445筛选、鉴定及发酵优化

Screening,identification and optimization of fermentation conditions of antagonistic Xenorhabdus bovienii 445 against Aspergillus niger

【目的】从现有共生细菌资源中筛选对黑曲霉具有高拮抗作用的菌株,为寻求安全高效的生物保鲜材料提供更多选择。【方法】采用平板对峙法和琼脂扩散法,分别对现有昆虫病原线虫共生菌进行初筛和复筛,对筛选出的高效拮抗共生细菌进行生理生化以及16S rRNA序列进化分析鉴定,通过单因素试验和响应面分析法优化菌株培养条件,利用损伤接种法在红地球葡萄上验证对黑曲霉防治效果。【结果】分离筛选共获得20株拮抗共生菌,复筛得到1株抑菌效果显著的共生细菌(445),经生理生化及分子生物学分析为伯氏致病杆菌Xenorhabdus bovienii,GeneBank ID:OK560680,与菌株Xenorhabdus bovienii strain Xb139(MG995576.1)聚于同一分支,相似性达99.79%。获得445菌株抑菌活性的最优培养条件为接种量3.06%、pH 7.0、装液量100.15 mL/250 mL,抑菌圈直径为(29.67±0.28)mm,抑菌效价为10.59 cm/mL,比优化前提高39.16%。Xenorhabdus bovienii 445发酵液对黑曲霉具有较好防治效果,3 d后防效为63.50%。【结论】筛选得到1株高效拮抗黑曲霉的昆虫病原线虫共生细菌,其发酵液对黑曲霉有良好的抑制效果,具有较好的生防潜能。

【Objective】Entomopathogenic nematode symbiosis have broad antimicrobial spectrum and effective antibacterial effects,so screening strains with highly antagonistic effects against Aspergillus niger from the existing commensal bacteria resources will provide more choices for seeking safe and efficient biopreservation materials.【Methods】The existing entomopathogenic nematode symbiosis were preliminarily screened and re screened by plate confrontation method and agar diffusion method respectively,so as to screen the highly antagonistic symbiotic bacteria,identify the highly antagonistic symbiotic bacteria by physiological and biochemical analysis and 16S rRNA sequence evolution analysis,and optimize the strain culture conditions by single factor test and response surface analysis.The control efficiency for Aspergillus niger in Red Globe grape was verified by injury inoculation method.【Results】A total of 20 antagonistic symbiotic bacteria were obtained through preliminary isolation and screening,and a symbiotic bacterium(445)with significant antibacterial effect was obtained through re screening.Through physiological,biochemical and molecular biological analysis,it was identified as pathogenic bacterium Xenorhabdus bovienii,Genebank ID:OK560680 through physiological,biochemical and molecular biological analysis.It was clustered in the same branch as strain Xenorhabdus bovienii strain Xb139(MG995576.1),with a similarity of 99.79%.Through single factor test and response surface analysis,the optimal culture conditions for the antibacterial activity of strain 445 were as follows:inoculation amount of 3.06%,pH 7.0,liquid volume of 100.15 mL/250 mL.Under these conditions,the diameter of antibacterial circle was 29.67±0.28 mm,and the antibacterial titer was 10.59 cm/mL,which was 39.16%higher than that before optimization.In the normal temperature antiefficiency experiment of grapes,the fermentation broth of Xenorhabdus bovienii 445 had a better control effect on Aspergillus niger,and the control effect was 63.50%after 3 days.【Conclusion】In this study,entomopathogenic nematode symbiotic bacteria were used to control Aspergillus niger infection during grape storage for the first time,and a highly effective entomopathogenic nematode symbiotic bacteria against Aspergillus niger was screened.Its fermentation broth has good inhibitory effect on Aspergillus niger and good biocontrol potential,which provides new biocontrol materials and research ideas for biocontrol of postharvest pathogen Aspergillus niger.