穿龙薯蓣内生真菌分离鉴定及其抑菌活性研究

目的 分离纯化穿龙薯蓣内生真菌，并对分离得到的内生真菌进行抑菌活性筛选及分类鉴定。方法 采用组织分离法纯化穿龙薯蓣内生真菌，采用载片培养法、挑片培养法、插片培养法，对照《真菌鉴定手册》和《半知菌纲手册》进行初步鉴定；以金黄色葡萄球菌Staphylococcus aureus、白色葡萄球菌Staphylococcus albus、柠檬色葡萄球菌Staphylococcus citreu、四联球菌Micrococcus tetragenus、大肠杆菌Escherichia coli、伤寒杆菌Salmonella typhi、铜绿色假单胞菌Pseudomonas aeruginosa、变形杆菌Bacterium termo、肺炎杆菌Bacillus endocarditidis capsulatus、福氏痢疾杆菌Shigella flexneri、乙型副伤寒杆菌Bacterium paratyphosum B作为受试对象，检测内生真菌菌株发酵液以及菌丝提取物的抑菌活性，应用分子生物学方法对筛选出的活性菌株进行分类鉴定。结果 从穿龙薯蓣叶片中分离得到52株内生真菌，根据形态学特征将10株产孢子的内生真菌初步鉴定为2个目、3个科、9个属；其中有3株内生真菌的菌丝提取物具有广谱抗菌活性，结合形态学和分子生物学特征鉴定C37菌为赤霉属Gibberella真菌，C30和C50菌为链格孢属Alternaria真菌。结论 穿龙薯蓣叶片中存在具有抑菌活性的内生真菌，具有开发抑菌生物制剂的潜力。

Isolation, identification and antimicrobial activity of endophytic fungi from Dioscorea nipponica

Objective To isolate endophytic fungi from the fresh leaves of Dioscorea nipponica Makino, and to determine their antimicrobial activities. Methods Endophytic fungi from the fresh leaves of Dioscorea nipponica Makino were isolated by tissue isolation method, and the preliminary identification was carried out by slide plate culture, tissue culture, and plug culture. After isolating the endophytic fungi, the antimicrobial activity from the isolated endophytic fungi was detected using filter paper method with Staphylococcus aureus, Staphylococcus albus, Staphylococcus citreu, Micrococcus tetragenus, Escherichia coli, Salmonella typhi, Pseudomonas aeruginosa, Bacterium termo, Bacillus endocarditidiscapsulatus, Shigella flexneri, Bacterium paratyphosum B indicator microorganisms. Results A total of 52 strains of the endophytic fungi were isolated. According to morphological characteristics, 10 endophytic fungi producing spores were identified as two orders, three families, and nine genera. The extracts of fungal mycelium had the antibiotic activity of strains of C37, C30, and C50. With the identification by morphology and molecular biology, strain C37 was determined to belong to the genus Gibberella, and C30, C50 to Alternaria. Conclusion The antibacterial activity of these endophytes in the leaves of Dioscorea nipponica Makino could be exploited for the development of new antibacterial biological agents.