【背景】粘细菌是一类具有社会性行为的高等原核生物，能产生丰富、多样、新颖的具有生物活性的次级代谢产物，具有很大的研究开发价值。【目的】从土壤样品中筛选出对致病疫霉(Phytophthora infestans)具有拮抗作用的粘细菌，并对其次级代谢产物进行研究。【方法】对分离到的一株拮抗P. infestans菌株，结合形态观察和16S rRNA基因序列分析确定其分类地位，并通过单因素分析与正交优化相结合的方法对菌株的发酵参数进行研究。采用滤纸片法对其浓缩发酵液中活性物质的稳定性及抗菌活性进行检测，并通过薄层色谱法(thin layer chromatography，TLC)与高效液相色谱法(high performance liquid chromatography，HPLC)等初步分离后，将具有抗P. infestans活性的组分利用液相色谱-质谱联用技术(HPLC-MS)进行检测，最后通过离体叶片法测定活性物质对马铃薯晚疫病的防病作用。【结果】从土壤样品中分离得到的菌株B25-I-3对P. infestans表现出较强的拮抗活性，经鉴定为橙色粘球菌(Myxococcus fulvus)。其拮抗P. infestans的活性物质主要存在于胞外，产活性物质的最优发酵条件为：摇床转速180 r/min，接种量10%，培养温度30 °C，发酵周期7 d。该菌株产生的活性物质耐受蛋白酶K以及紫外线与自然光照射，对温度耐受性极强，而且易于在低温下保存，对枯草芽孢杆菌(Bacillus subtilis)、金黄色葡萄球菌(Staphylococcus aureus)、大肠杆菌(Escherichia coli)、酿酒酵母(Saccharomyces cerevisiae)、立枯丝核菌(Rhizoctonia solani)、尖孢镰刀菌(Fusarium oxysporum)、向日葵核盘菌(Sclerotinia sclerotiorum)均有不同程度的抑制作用。其对P. infestans的拮抗组分中含有N-(3-氨基-2-羟丙基)-N-甲基硫酸二酰胺[N-(3-amino-2-hydroxypropyl)-N-methylsulfuric diamide]与甲基(2R)-2-叠氮基-3-羟基-2-甲基丙酸酯[methyl(2R)-2-azido-3-hydroxyl-2-methylpropanoate]，该活性物质能明显抑制P. infestans侵染马铃薯叶片且对不同品种的叶片均无损伤作用。【结论】研究为M. fulvus B25-I-3活性物质的分离鉴定及抗马铃薯晚疫病生物农药的研发提供了基础数据。
[Background] Myxobacteria are a group of higher prokaryotes with social behaviors. They can produce rich, diverse, and novel secondary metabolites with biological activity, and have great research and development value. [Objective] To screen and identify culturable myxobacteria resistant to the pathogen of potato late blight from soil samples and study secondary metabolites of the isolates. [Methods] Myxobacteria were isolated from soil samples by rabbit feces inducing method. The strain resistant to the pathogen of potato late blight was identified by morphological observation and the 16S rRNA gene sequence analysis. Then, the fermentation parameters were studied by the combination of univariate analysis and orthogonal optimization. The stability and antibacterial activity of the active substances in the concentrated fermentation broth were detected by the filter paper method. The active substances were separated by TLC and HPLC, and the components with antibiotic activity were detected by HPLC-MS. The anti-disease effect of the active substances on potato late blight was determined by the in vitro leaf method. [Results] Strain B25-I-3 isolated from the soil sample showed strong antagonistic activity against P. infestans and was identified as Myxococcus fulvus. The antibiotic substances against P. infestans were mainly present in the extracellular matrix. Its optimal fermentation conditions were: shaking speed 180 r/min, inoculum size 10%, incubation temperature 30 °C, incubation time 7 d. The active substances produced by strain B25-I-3 were highly tolerant to the treatment of protease K, ultraviolet and natural light, temperature, and were easily preserved at low temperatures. The antibacterial and antifungal activity analysis showed the active substances exhibited different degrees of antagonism against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Saccharomyces cerevisiae, Rhizoctonia solani, Fusarium oxysporum, Sclerotinia sclerotiorum. The antagonistic components of strain B25-I-3 contained N-(3-amino-2-hydroxypropyl)-N-methylsulfuric diamide and methyl(2R)-2-azido-3-hydroxyl-2-methylpropanoate. The results of disease control on potato leaves showed that the active substances could significantly inhibit the infection of P. infestans on the detached potato leaves of different varieties without harming. [Conclusion] This study provides basic data for the isolation and identification of active substances and the development of biopesticide against potato late blight.