土壤稀有放线菌的选择性分离及其抗菌活性研究

Study on Selective Isolation and Antibiotic Activity of Rare Actinomycetes from Soil

在优化土壤稀有放线菌选择性分离方法的基础上,对从安徽、浙江、山东、陕西等地采集的31份土样中的稀有放线菌进行了选择性分离。结果表明:以改进的HV培养基为分离培养基,将土壤样品稀释100倍,添加2×10-5g/mL重铬酸钾+2×10-5g/mL萘啶酮酸+1×10-5g/mL卡那霉素作为抑制剂分离效果最好。以常见植物病原真菌和细菌为指示菌,从分离的417株放线菌中筛选出H32、H75、H223、H227、H238等5株可代谢抗菌活性物质的菌株。离体条件下,5株放线菌菌株发酵液对小麦根腐病菌Bipolarissorokiniana、玉米大斑病菌Exserohilumturcicum、烟草赤星病菌Alternariaalternate的菌丝生长和孢子萌发均有强烈的抑制作用,其中H223和H238菌株发酵液对病原真菌菌丝生长的抑制中浓度(EC50)值均小于10mL/L;H32菌株发酵液对小麦根腐病菌、玉米大斑病菌、烟草赤星病菌孢子萌发的EC50值分别为25.5、28.9和29.9mL/L。5株放线菌发酵液对枯草芽孢杆菌、大肠杆菌、金黄色葡萄球菌抑制作用显著,其中H223菌株发酵液抑制作用最强,抑菌圈达30mm以上。盆栽实验结果表明,5株放线菌发酵液对小麦白粉病的保护效果为73.45%～82.35%,治疗效果为67.74%～70.80%。

Based on an improved selective isolation techniques,31 soil sample collected from Anhui, Zhejiang, Shandong, Shaanxi et al provinces of China were screened. The result showed that when use the improved HV agar as the isolation medium, dilute the soil sample 100 times and use 2 × 10^-5 g/mL potassium dichromate plus 2 × 10 ^-5 g/mL nalidixic acid plus 1 × 10^ -5 g/mL kanamycin as inhibitor, the isolation was the best. All the fermentation of the screened 417 strains were tested against the plant pathogenic fungi, such as Bipolaris sorokiniana, Exserohilum turcicum, Altemaria alternata, and pathogenic bacteria, such as Bacillus subtilis, Escherichia coli, Staphylococcus aureus. Five of them have been proven to produce bioactive metabolites, which were labeled as H32, H75, H223, H227, H238, respectively. By means of mycelium growth rate, the fermentations of H223 and H238 showed the best inhibitive action, both EC50 value swere were less than 10 mL/L. Spore germination tests showed that the fermentation of H32 exhibited the strongest antibiotic activity against Bipolaris sorokiniana ,Exserohilum turcicum ,Alternaria alternata ,the EC50 values were 25.5,28.9,29.9 mL/L, respectively. Cup-plate tests indicated that the fermentations of the 5 strains had significant activities against B. subtilis, E. coli, S. aureus, and the inhibitive diameters of the fermentation of strain H223 to the 3 bacteria were all more than 30 mm. Pot tests on B. graminis exhibited that the protective efficacy of fermentations of the 5 strains were 73.45% - 82.35%, therapeutic efficacy were 67.74% - 70.80%.