目的 分離純化穿龍薯蕷內(nèi)生真菌，并對分離得到的內(nèi)生真菌進(jìn)行抑菌活性篩選及分類鑒定。方法 采用組織分離法純化穿龍薯蕷內(nèi)生真菌，采用載片培養(yǎng)法、挑片培養(yǎng)法、插片培養(yǎng)法，對照《真菌鑒定手冊》和《半知菌綱手冊》進(jìn)行初步鑒定；以金黃色葡萄球菌Staphylococcus aureus、白色葡萄球菌Staphylococcus albus、檸檬色葡萄球菌Staphylococcus citreu、四聯(lián)球菌Micrococcus tetragenus、大腸桿菌Escherichia coli、傷寒桿菌Salmonella typhi、銅綠色假單胞菌Pseudomonas aeruginosa、變形桿菌Bacterium termo、肺炎桿菌Bacillus endocarditidis capsulatus、福氏痢疾桿菌Shigella flexneri、乙型副傷寒桿菌Bacterium paratyphosum B作為受試對象，檢測內(nèi)生真菌菌株發(fā)酵液以及菌絲提取物的抑菌活性，應(yīng)用分子生物學(xué)方法對篩選出的活性菌株進(jìn)行分類鑒定。結(jié)果 從穿龍薯蕷葉片中分離得到52株內(nèi)生真菌，根據(jù)形態(tài)學(xué)特征將10株產(chǎn)孢子的內(nèi)生真菌初步鑒定為2個目、3個科、9個屬；其中有3株內(nèi)生真菌的菌絲提取物具有廣譜抗菌活性，結(jié)合形態(tài)學(xué)和分子生物學(xué)特征鑒定C37菌為赤霉屬Gibberella真菌，C30和C50菌為鏈格孢屬Alternaria真菌。結(jié)論 穿龍薯蕷葉片中存在具有抑菌活性的內(nèi)生真菌，具有開發(fā)抑菌生物制劑的潛力。

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.

國家自然科學(xué)基金資助項目（No.81373902）