| Huperzia serrata,a perennial fern plant with slow growth,short stature,and medicinal value,grows in shaded and damp habitats such as forests.Its metabolism produces a bioactive alkaloid called Huperzine A(Hup A),a natural and potent acetylcholinesterase inhibitor that has shown certain therapeutic effects for Alzheimer’s disease.However,the wild resources of H.serrata are scarce and cannot meet the growing market demand.The long-term symbiotic relationship between endophytic fungi and their hosts allows some strains to produce bioactive substances that are identical or similar to those of the host.This study provides new insights into the diversity,composition,and correlation with Hup A production of endophytic fungi in different ecological regions of H.serrata through comparative analysis.It contributes to further research on the fungal community of H.serrata and its interaction with Hup A production,helping to clarify the ecological functions of endophytic fungi.Endophytic fungi were isolated in large quantities using tissue separation methods,and identified using morphological and molecular biology techniques.Hup A-producing strains were selected through HPLC detection,laying the foundation for the industrial application of endophytic fungi fermentation to produce Hup A.Strains that can prevent and control gray mold were screened through plate confrontation culture,fermentation broth paper diffusion culture,and fermentation broth disease prevention screening experiments,providing microbial resources for its biological control.The primary findings and conclusions of this study are summarized as follows:(1)High-throughput sequencing technology was used to analyze the diversity and taxonomic composition of endophytic fungi in H.serrata from two ecological regions,Chengdu and Qixian Lake.A total of 1,434,573 valid sequences were obtained,which were divided into 2,521 operational taxonomic units(OTUs),covering 9 phyla,40 classes,102 orders,228 families,and 430 genera.In the Chengdu and Qixian Lake samples,1,829 and 1,288 OTUs were detected,respectively,and 140 and 102 OTUs were shared between the two ecological regions.As for the phylum classification level,Ascomycota(54.34%)and Basidiomycota(41.51%)were dominant fungal groups.The unidentified Ascomycota genus had the highest content in the leaves and stems,but the lowest content in the roots.The Sebacina genus was the second most abundant genus in Chengdu,but was almost undetected in Qixian Lake.The clustering heatmap showed that root samples from both regions clustered together,while stem and leaf samples clustered together.Alpha diversity indicated that the diversity of endophytic fungi in root tissue was significantly lower than that in stem and leaf tissues,while beta diversity indicated that the fungal communities in stem and leaf tissues were more similar to each other than to those in root tissues.These results indicate that plant tissue and ecological regions have an impact on the structure and diversity of endophytic fungal communities in H.serrata,which is important for understanding the mechanisms of plant-microbe interactions and the biodiversity of ecosystems.(2)Based on the Pearson correlation analysis,five genera(Ascomycota_unclassified,Cyphellophora,Fungi_unclassified,Sporobolomyces,and Trichomeriaceae_unclassified)showed a significant positive correlation with Hup A content,suggesting the potential of endophytic fungi from these five genera to produce Hup A as an alternative to H.serrata.Meanwhile,the results of interspecies correlation analysis indicated a positive correlation among multiple endophytic fungi and the endophytic fungi from these five genera,which provides the information for exploring efficient endophytic fungi to produce Hup A.(3)More than 120 endophytic fungi were isolated from the stems of H.serrata plants,among which 43 strains exhibited inhibitory activity in the ACh E inhibition assay.These active strains were further identified based on morphology and molecular biology,and two strains,Colletotrichum siamense S-1-2 and L-2-15,were selected for their ability to produce Hup A as determined by HPLC analysis.These findings lay the foundation for the industrialization of Hup A production through the efficient cultivation of endophytic fungi from H.serrata.(4)Through plate confrontation culture experiments,it was found that five endophytic fungi could inhibit the growth of gray mold.Further screening of fermentation broth by filter paper diffusion and tomato disease resistance testing showed that Colletotrichum gloeosporioides S-7-4,Alternaria alternata L-11-7,and Phyllosticta capitalensis S-3-7 had significant inhibitory effects on botrytis cinerea.This provides a fungal resource for obtaining biocontrol agents that can inhibit gray mold. |
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