基于高通量解析和可培养技术的白蔹内生真菌类群分析及分离菌株的抗痤疮致病菌活性

Taxonomic analysis of fungal endophytes from Ampelopsis japonica by high-throughput sequencing and culture-dependent methods and the activity of the isolated strains against acne-associated pathogens

摘要:

【背景】药用植物蕴含丰富的内生真菌类群资源，而且内生真菌可产生与宿主相关的次级代谢产物，具有多种生物活性，是活性化合物生产的潜在经济来源。【目的】分析药用植物白蔹根部内生真菌的物种多样性，并筛选出具有抗痤疮致病菌活性的目标菌株。【方法】基于高通量和组织分离法分析白蔹内生真菌的类群结构多样性；通过琼脂扩散法对内生真菌代谢产物的抗痤疮致病菌活性进行筛选；以微量肉汤稀释法测定代谢产物的最小抑菌浓度(minimum inhibitory concentration, MIC)和最小杀菌浓度(minimum bactericidal concentration, MBC)。【结果】高通量测序结果显示，白蔹根部内生真菌可注释到8门22纲45目73科93属，根部的优势属为Tainosphaeria (20.86%)和镰刀菌属(Fusarium, 15.38%)。基于组织分离法共从白蔹根部分离获得83株内生真菌，隶属于12个属，其中青霉属(Penicillium, 24.10%)、木霉属(Trichoderma, 14.46%)、背芽突霉属(Cadophora, 13.25%)、镰刀菌属(Fusarium, 12.05%)为主要类群。17株代表性菌株的抗痤疮致病菌活性检测结果显示对痤疮丙酸杆菌和金黄色葡萄球菌具拮抗活性的菌株分别占总数的11.76%和23.53%，其中内生真菌Fusarium oxysporum BLR17的发酵液对2种病原菌均具有明显的抑菌效果，其发酵液的乙酸乙酯粗提物对痤疮丙酸杆菌和金黄色葡萄球菌的MIC值和MBC值均分别为7.81 μg/mL和62.5 μg/mL。【结论】白蔹内生真菌具有较为丰富的多样性，也蕴含着一定比例的抗痤疮活性菌种资源，其中Fusarium oxysporum BLR17有望成为抗痤疮先导化合物筛选的资源菌株，值得进一步研究。

Abstract:

[Background] Medicinal plants harbor abundant fungal endophytes, producing host-related secondary metabolites with a variety of biological activities. [Objective] To analyze the diversity of endophytic fungi in the roots of medicinal species Ampelopsis japonica (Thunb.) Makino and isolate strains producing chemicals with anti-acne activity. [Methods] The taxonomic diversity of fungal endophytes was analyzed based on high-throughput sequencing and tissue isolation. The activity of metabolites of the endophytes against acne-associated pathogens was tested with agar diffusion method and the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined by broth microdilution.[Results] The endophytic fungi from the roots of A. japonica belonged to 93 genera, 73 families, 45 orders, 22 classes, and 8 phyla. The dominant genera were Tainosphaeria (20.86%) and Fusarium (15.38%). With the tissue isolation method, 83 fungal strains (12 genera) were isolated, among which Penicillium (24.10%), Trichoderma (14.46%),Cadophora (13.25%), and Fusarium (12.05%) dominated. The results of anti-acne bacteria activities of 17 representative strains showed that the strains with activities against Propionibacterium acnes and Staphylococcus aureus accounted for 11.76% and 23.53%, respectively. The fermentation broth of the endophytic Fusarium oxysporum BLR17 displayed obvious inhibitory effect on both pathogens. The MIC of its crude ethyl acetate extract against P. acnes and S. aureus was 7.81 μg/mL and 62.5 μg/mL, respectively, and the MBC was 7.81 μg/mL and 62.5 μg/mL, separately. [Conclusion] The endophytic fungi of A. japonica had high diversity and contained a certain proportion of anti-acne species resources. F. oxysporum BLR17 is expected to be a resource strain for screening the anti-acne bioactive compounds, which is worthy of further study.