广谱拮抗放线菌S5-1的鉴定、发酵工艺优化及发酵粗提物抑菌活性评价

Identification, Optimization of fermentation process of an antagonistic actinomycete S5-1 and evaluation of antibacterial activity of the fermented crude extract

摘要:

【背景】如何有效防治植物病害、实现农作物安全生产、保护农业生态环境，是农业可持续发展中亟待解决的问题之一。【目的】开展放线菌S5-1在植物病害生物防治中的应用潜能和代谢活性物质研究，可为植物病害的生物防治提供有效菌源，具有重要的实践意义。【方法】采用平板对峙法和发酵液制备带药平板法检测抑菌活性；通过形态和生理生化特征及16S rRNA基因序列分析确定分类地位；利用单因素试验和正交试验优化发酵工艺条件、测评发酵粗提物抑菌活性。【结果】菌株S5-1对10种植物病原菌具有较强的抑菌活性，菌株S5-1被鉴定为毒三素链霉菌(Streptomyces toxytricini)，在发酵培养基为TSB、初始pH 6.5、250 mL三角瓶中装液量100 mL、接种量10%、28 ℃、发酵7 d的发酵液抑菌相对效价最高。发酵粗提物能有效抑制烟草疫霉(Phytophthora parasitica)和灰霉菌(Botrytis cinerea)的菌丝生长、孢子萌发，并抑制烟草疫霉对烟草叶片和灰霉菌对圣女果果实的侵染，发酵液粗提物适宜pH 6.0−7.0，超过37 ℃易失活，‒20 ℃储存30 d、紫外光处理不超过6 h的条件下仍能保持稳定的抑菌活性。【结论】对菌株S5-1的开发利用将有助于植物病害的绿色防控，为拮抗放线菌的生防应用提供优良菌株资源，同时为新型抑菌活性药物的研究和应用提供实验基础。

关键词:拮抗放线菌;抑菌谱;分类鉴定;发酵条件优化;毒三素链霉菌

Abstract:

[Background] How to effectively prevent and control plant diseases and achieve safe crop production without causing damage to the agro-environment is a key problem to be solved in the sustainable development of agriculture. [Objective] The strains and active metabolites of Streptomyces strains were screened out to provide the strain resources and approaches for the biocontrol of plant diseases, which is of great practical significance for the research on the potential application of strain S5-1 in the biocontrol of plant diseases. [Methods] The antimicrobial activity was examined by the plate confrontation method and the plate culture method with the fermentation broth of the strain. The strain was identified based on the morphological and physicochemical characteristics and 16S rRNA gene sequences. Furthermore, the single factor and orthogonal experiments were carried out to optimize the fermentation conditions, and the antimicrobial activity of the fermented crude extract was examined. [Results] The strain S5-1 had strong inhibitory activities on ten plant pathogens and was identified as Streptomyces toxytricini. The fermentations conditions were optimized as follows: TSB medium, initial pH 6.5, medium loading capacity of 100 mL/250 mL, inoculation amount of 10%, and fermentation at 28 ℃ for 7 d. The fermented crude extract inhibited the mycelial growth and spore germination of Phytophthora parasitica and Botrytis cinerea. Furthermore, it reduced the infection of the two pathogens in tobacco leaves and cherry tomatoes. The extract was stable at pH 6.0–7.0 and lost activity at the temperature above 37 ℃. In addition, the storage at ‒20 ℃ for 30 d and ultraviolet irradiation not exceeding 6 h caused slight changes in the extract activity. [Conclusion] The development and utilization of strain S5-1 contribute to the green prevention and control of plant diseases and provide excellent strain resources of actinomycetes for biocontrol. This study provide an experimental basis for the research and application of new antimicrobial agents.

Key words:antagonistic actinomycetes;antimicrobial spectrum;identification;fermentation condition optimization;Streptomyces toxytricini