| ObjectiveIn recent years,the nonstandard use of antibacterial drugs has led to the prevalence of multiple drug resistance around the world.It is urgent to develop novel resistant antibacterial drugs.The secondary metabolites of microorganisms in special habitats are the focus of research and development of lead drugs.The lichens of the lichen genus Thamnolia spp.are distributed in high-altitude areas,and the unique nature of the habitat endows them with good antibacterial activity substances,making them an important resource for developing natural antibacterial activity substances.However,the slow growth rate and difficulty in artificial cultivation of lichens in the genus Thamnolia spp.have to some extent limited the development and utilization of the pharmaceutical field.In essence,the antibacterial activity of lichens is mainly attributed to their symbiotic fungi.Conducting research on lichen fungi not only saves lichen resources,but also allows for large-scale production of antibacterial drugs in the future.Therefore,this article will isolate endophytic fungi from the lichens of the genus Thamnolia spp.,screen strains with strong antibacterial activity,and further clarify the basis of their antibacterial activity substances,providing a theoretical basis for innovative drug research and development,and providing a scientific basis for further development of Cangshan lichens.MethodsThe main results are as follows:(1)In this experiment,three methods of UV fluorescence method,Chemical chromogenic reaction method and Thin-layer chromatography method were used to accurately identify Thamnolia vermicularis and Thamnolia subuliformis,and a rapid and economical identification method was selected.At the same time,the microbial diversity of Thamnolia vermicularis and Thamnolia subuliformis samples was analyzed,and the similarities and differences between the two in the composition of microbial community were further explored;(2)The endophytic fungi of Thamnolia vermicularis and Thamnolia subuliformis were isolated and purified by tissue block method.The in vitro antibacterial activity of endophytic fungal fermentation broth against pathogenic bacteria was screened by plate punching method,and the highly active strains were screened for antibacterial activity.In order to further understand the active strain,the mycelium morphology observation and molecular biology identification were carried out;(3)In order to clarify the material basis of antibacterial activity of active strain Coprinopsis sp.TsF016,MRSA was used as the antibacterial object,and the metabolites of active strain Coprinopsis sp.Ts-F016 were preliminarily explored by silica gel column chromatography,thin-layer chromatography and UHPLC-TOF-MS,which provided the basis for the development of subsequent experiments.Results(1)UV fluorescence method,Chemical chromogenic reaction method and Thinlayer chromatography method could accurately identify the samples of Thamnolia vermicularis and Thamnolia subuliformis.Each method has its own advantages and disadvantages and researchers could choose based on their actual needs.In addition,there are differences in the composition of microbial communities between Thamnolia vermicularis and Thamnolia subuliformis.Ascomycetes occupy an absolute advantage in Thamnolia vermicularis samples,and the abundance is as high as 70% ～90%.Compared with Thamnolia vermicularis,the relative abundance of basidiomycetes is higher in the Thamnolia subuliformis,suggesting that although Thamnolia vermicularis and Thamnolia subuliform is belong to the lichen of the genus Thamnolia spp,the fungal community structure is also different.The results of antibacterial activity test of methanol extracts of Thamnolia vermicularis and Thamnolia subuliform showed that different strains showed different sensitivity to methanol extracts of Thamnolia vermicularis and Thamnolia subuliform.In comparison,the antibacterial activity of methanol extracts of Thamnolia vermicularis was stronger and wider.Therefore,the genus Thamnolia spp has good antibacterial activity and the potential for further development.(2)46 strains of endophytic fungi were isolated from the lichens of the genus Thamnolia spp,and the antibacterial activity of 46 strains of endophytic fungi was preliminarily screened.8 strains of endophytic fungi showed antibacterial activity.Among them,an endophytic fungus numbered 202109-Ts-F016 isolated from the lichens of the genus Thamnolia spp had a strong and wide range of antibacterial activity against a variety of pathogenic bacteria,especially against MRSA.Therefore,the secondary metabolites of the strain have the potential to be developed as anti-super bacterial drugs.(3)The metabolites of the active strain Coprinopsis sp.TsF016 were analyzed by silica gel column chromatography combined with thin-layer chromatography.The antibacterial effect of component E on MRSA was verified.It was speculated that the main chemical components were terpenoids,polyketides and aromatic compounds(phenolic acids).ConclusionUV fluorescence method can be applied to quickly and accurately identify Thamnolia vermicularis and Thamnolia subuliform with extremely similar external morphology.This method is simple to operate and can ensure the integrity of the lichen body,which is a prerequisite for rapid subsequent experiments.Among the 46 strains of endophytic fungi obtained,one strain,numbered 202109-Ts-F016,has a good antibacterial effect and a wide range of metabolites.MRSA is extremely sensitive to its metabolites.After molecular biology identification and morphological observation,this strain is an undetermined new species of Coprinopsis sp.,A preliminary separation of the metabolites of the active strain Coprinopsis sp.Ts-F016 was carried out through silica gel column chromatography combined with thin layer chromatography.Eight components were obtained,after activity verification,the antibacterial active ingredient only exists in component E,and a single antibacterial active compound in this active component needs further research. |
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