| The results of existing epidemiological investigations show that the global incidence of Superficial fungal infections(SFIs)is as high as 20%to 25%,which has become one of the important diseases affecting human health.At present,the drug resistance rate of superficial pathogenic fungi is getting higher and higher,and the clinical effect of existing drugs is not good,so it is necessary to develop new antifungal drugs as soon as possible.Anemarrhena asphodeloides is a traditional Chinese herbal medicine,which has many pharmacological effects,such as antifungal,anticancer,anti-inflammatory and antioxidant.Experimental results showed that A.asphodeloides has trong inhibitory effect on a variety of common superficial pathogenic fungi,and its rhizome water extract also has the ability of biosynthesis of silver nanoparticles(AgNPs)and gold nanoparticles(Au NPs).Due to the gradual decrease of wild A.asphodeloides,it is necessary to find its alternative resources.Compared with other microbial communities,due to long-term co-evolution with the host,medicinal plant endophytic fungi can produce not only the same or similar active substances as the host plants,but also a variety of new secondary metabolites.AgNPs are widely used in dermatology because of their good antibacterial and anti-inflammatory properties.Some studies have shown that medicinal plant endophytic fungi can bioconjugation their secondary metabolites with their synthetic nanoparticles to form a nanomedicine complex.This biosynthetic nanocomposite has a good application prospect in the biomedical industry,especially in targeted drug delivery and preparation of nanomedicine against multiple drug-resistant microorganisms.Compared with chemical and physical methods,biosynthetic AgNPs are more safe and environmentally friendly,lower toxicity and better biocompatibility,so they are concerned by researchers.However,there are no reports on the biological diversity of endophytic fungi of A.asphodeloides and their biosynthetic nanomaterials against superficial pathogenic fungi.Therefore,this study will systematically analyze the diversity of endophytic fungi in roots,stems and leaves of medicinal plants A.asphodeloides and the antibacterial activity of their secondary metabolites against superficial pathogenic fungi,so as to provide a new strain resource bank for new drug development.At the same time,the endophytic fungi with the potential for the synthesis of AgNPs were screened,and the antifungal activity and cytotoxicity of the AgNPs were evaluated.The purpose of this study is to lay a foundation for the search for potential drugs for dermatomycosis and the biosynthesis of AgNPs by medicinal plant endophytic fungi.The specific research contents and results of this paper are as follows:(1)The diversity of endophytic fungi of A.asphodeloides was analyzed by high-throughput sequencing technique and traditional isolation and culture method.The results of high-throughput sequencing showed that the diversity of endophytic fungi in A.asphodeloides leaves was the highest,followed by stems,and the diversity in roots was the lowest.Setophoma,Aspergillus,Mortierella,Chaetomium and Fusarium are the dominant genera in A.asphodeloides.The dominant genera in the roots were Setophoma,Ilternarsa,Aspergillus and Fusarium,the dominant genera in the stems were Mortierella,Chaetomium,Fusarium and Emericellopsis,the dominant genera in the leaves were Aspergillus,Colletotrichum,Fusarium and Ilternarsa,and the dominant genera Fusarium was detected in roots,stems and leaves.The genera with the highest relative abundance in roots,stems and leaves were Setophoma,Mortierella and Aspergillus.There were some differences in the distribution of dominant genera in different tissue parts of A.asphodeloides.The results of isolation and culture showed that 23 genera and 438 strains of endophytic fungi were isolated from the roots,stems and leaves of A.asphodeloides.The total colonization rate and isolation rate of A.asphodeloides endophytic fungi were 73.56% and 97.33% respectively,and the total diversity index was 1.33.Although the dominant groups in the roots,stems and leaves of A.asphodeloides are all Fusarium,the genera Sarocladium,Setophoma,Leptosphaeria,Periconia and Dictyocheirospora are only distributed in the roots,Clonostachys is only distributed in the stems,while Ilternarsa,Colletotrichum,Cercospora,Cladosporium and Arthopyrenia are isolated only in the leaves.(2)The results of the antifungal experiment on the fermentation broth of 31 representative strains showed that: 96.77%,100.00%,100.00%,3.23% and 3.23% of the isolated endophytic fungi had inhibitory activity against Microsporum canis,Trichophyton rubrum,Trichosporon mucoides,Malassezia furfur and Candida albicans,respectively.All the endophytic fungi tested had inhibitory effect on at least one kind of superficial pathogenic fungi,and 96.77% of the strains were active against 3 species(M.canis,T.rubrum,T.mucoides).The inhibition rate of the fermentation broth of strain Aspergillus sp.ZML33 to M.canis,T.rubrum and T.mucoides was higher than 70%.The fermentation broth of strain Periconia sp.ZMR35 not only had strong activity against M.canis(55.12%),T.rubrum(57.49%)and T.mucoides(49.52%),but also inhibited the growth of M.furfur.(3)In this study,the extracellular synthesis of AgNPs by endophytic fungi Clonostachys sp.ZMS27 was found for the first time.It was characterized by UV–Visible spectrophotometry(UV-Vis),Transmission electron microscope(TEM),Scanning electron microscope(SEM),X-ray energy dispersive spectroscopy(EDS)and Fourier transform infrared spectrometer(FT-IR).The biosynthetic AgNPs(ZMS27-AgNPs)showed no toxicity to two kinds of normal skin cells in the concentration range of1.5625-25 μg/m L,and showed different inhibitory effects on five kinds of superficial pathogenic fungi.The minimum inhibitory concentration(MIC)against M.canis was the lowest(1.5625 μg/m L),and its antifungal effect was better than that of broad-spectrum antifungal agents voriconazole(2μg/m L)and commercial AgNPs(200 μg/m L),which was consistent with that of silver nitrate(1.5625 μg/m L).However,silver nitrate showed obvious cytotoxicity to mouse fibroblast cell line NIH-3T3 at 1.5625μg/m L.The MIC value of ZMS27-AgNPs against C.albicans was consistent with that of MFC(25 μg/m L),indicating that ZMS27-AgNPs is not only an antifungal agent but also a fungicide.In summary,this study found for the first time that A.asphodeloides endophytic fungi have rich species diversity,and there are differences in the distribution of fungi in different tissue parts.Among the isolated endophytic fungi,there was a high proportion of strains with antagonistic activity against superficial pathogenic fungi,in which endophytic fungi Clonostachys sp.ZMS27 had the ability to synthesize AgNPs.At the same time,it was found that the synthesized AgNPs had strong antifungal activity against superficial pathogenic fungi and had no toxicity to normal skin cells in a certain concentration range.This study is of great significance for finding new antifungal drugs and comprehensively excavating the endophytic resources of medicinal plants. |
