土壤中暗色真菌分离方法的研究

目的 寻找经济有效的土壤中暗色真菌的分离方法。方法 将一份含有暗色真菌的土壤样品悬液10倍比稀释成4个浓度，并配成含抗生素和不含抗生素2组，8支样品液分别接种于沙氏葡萄糖蛋白胨培养基（SDA）、马铃薯葡萄糖琼脂培养基（PDA）、孟加拉红培养基，各培养基含有0，100，200，300，500 mg/L 5个浓度的放线菌酮和100万U/L青霉素，50 mg/L氯霉素，200 mg/L链霉素，分别采用涂布法与倾注法培养，每种培养基共接种80个平皿。结果 三种培养基的分离效率分别为：PDA 70%，孟加拉红67.5%，SDA 3.75%。土壤液稀释100倍和培养基中放线菌酮质量浓度为200 mg/L时的分离效率最高，土壤液原液和稀释100倍时分离效率在PDA分别为50%和85%，两组比较，P ＜ 0.05；在孟加拉红培养基分别为45%和85%，两组比较，P ＜ 0.05。各培养基中无放线菌酮和放线菌酮质量浓度为200 mg/L时分离率分别为PDA 31.25%和87.5%，两组比较，P ＜ 0.05；孟加拉红培养基分别为37.5%和81.25%，两组比较，P ＜ 0.05。样本液中加抗生素和不加抗生素分离率比较，差异无统计学意义。各培养基涂布法与倾注法比较，总的分离率差异无统计学意义，但样品液中不加放线菌酮等抗生素时，倾注法比涂布法分离效率高，PDA分别为80%和40%，孟加拉红分别为80%和30%，各自比较差异均有统计学意义（P ＜ 0.05）。结论 改良的马铃薯、孟加拉红培养基是适合土壤中暗色真菌分离的培养基，培养基中放线菌酮浓度为200 mg/L为最适浓度，采用倾注平板法时方便、经济、有效。

Evaluation of methods for isolation of dematiaceous fungi from soil

Objective To seek a cost-effective method for isolation of dematiaceous fungi from soil.Methods Four concentrations of soil suspension samples were prepared by ten-fold serial dilution(1,1:10,1:100,1:1000).Then,each concentration of sample was divided into two parts:one part supplemented with antibiotics,including chloramphenicol,cycloheximide,penicilin and streptomycin,and the other free of antibiotics.Three culture media were prepared:Sabouraud's dextrose agar(SDA),potato dextrose agar (PDA)and rose bengal medium(RBM).Various concentrations(0,100,200,300,500 mg/L)of cyeloheximide were added into each culture medium along with defined concentration of penicillin(1000 u/mL)and streptomycin(0.2 g/L).In addition,chloramphenicol(50 mg/L)was added to PDA.The prepared soil samples were inoculated into these media by spread plate and pour plate,respectively.Results The total isolation rate of dematiaceous fungi was 70%(56/80)for PDA,67.5%(54/80)for RBM and 3.75%(3/80)for SDA.PDA and RBM were more efficient than SDA(both P＜0,05).Significant difference was observed in the isolation rate of dematiaceous fungi between the original and 1:100 diluted soil suspension samples in PDA(50%vs 85%,P＜0.05)and RBM(45%vs 85%,P＜0.05).An increased isolation rate was achieved in PDA(87.50%vs 31.25%)and RBM(81.25%vs 37.5%)with cycloheximide at 200 mg/L compared with those free of cycloheximide.No differenee was found in the isolation rate between antibiotic-supplemented and antibiotic-free soil samples.Although there was no diffefence in the total isolation rate between spead plate and pour plate,a higher isolation rate was observed by pour plate from antibiotic-free soil samples in PDA(80% vs 40%,P＜0.05)and RBM(80% vs 30%,P＜0.05)compared with spread plate.Condusions The modified PDA and RBM are suitable for the isolation of dematiaceous fungi from soil with the optimal concentration of cyeloheximide being 200 mg/L in the medium.Pour plate is more convenient and cost-effective than spread plate for the isolation of dematiaceous fungi from soil.