| Insects are characterized by many species,large number and wide range of distribution.There are abundant and diverse microbial resources in insects.Systematic isolation of endophytic fungi from insects,which can not only enrich microbial resources,but lay a foundation for finding many types of novel active metabolites.In this study,an efficient isolation and identification system for endophytic fungi was established with Lepidoptera.A large number of endophytic fungi were isolated,identified and analyzed from different areas of Qinling Mountains.Last,the study of secondary metabolites was carried on isolated endophytic fungus.In order to lay a foundation for the discovery of antibacterial active compounds with novel structures,the secondary metabolites of high active strain were isolated and the monomer compounds were preliminarily analyzed.The main research contents are as follows.1.The optimal pretreatment method for Lepidoptera was tissue shearing method,and the most suitable medium for endophytic fungi was Rose Bengal medium.Optimized the conditions for obtaining endophytic fungal DNA template by mechanical wall-breaking combined with microwave method,any mycelium was placed in a 2mL centrifuge tube with 100 ?L 10 × Buffer TE,broken mechanically for 1min at 40 Hz,and then cracked for 3mins at 700 W in the microwave.Last,the supernatant was taken as the DNA template.The results can provide a more efficient method for this study.2.Isolation,identification and analyzation of endophytic fungi in Lepidoptera from Qinling mountains.Using efficient systematic method,a total of 169 strains were isolated from Lepidoptera in Qinling different areas,and classified into 22 genera(6 classes,9 orders,14 families).Cladosporium was the dominant genus,and its separation frequency was 20.12%.The study found that 53 strains were isolated from Lepidoptera in Huyi county,xi'an city,and classified into 14 genera(2 special genera).55 strains were isolated from Lepidoptera in Fengxian county,Baoji city,and classified into 17 genera(4 special genera).35 strains were isolated from Lepidoptera in Liuba county,Hanzhong city,and classified into 14 genera(2 special genera).And 26 strains were isolated from Lepidoptera in Zhashui county,Shangluo city,and classified into 9 genera.The statistical analysis showed that endophytic fungi of Lepidoptera in Qinling were diverse,and the diversity of endophytes was the most abundant and the community structure was relatively stable in the samples from Fengxian county,Baoji city,the similarity of endophytes between the four different regions was moderate.3.Research on the active metabolites of endophytic fungi.The results of antimicrobial activity showed that among the tested 169 srains,67 strains were active,more than 77.61% of the strains with moderate antibacterial activity,92.54% showed the inhibitory activity on Staphylococcus aureus,34.33% displayed an inhibition on Escherichia coli,22.39% had inhibition on Candida albicans,41.79% had inhibitory activity against at least two or more indicator strains,of which the strain(KC-50)Talaromyces funiculosu has the best antibacterial activity and the broadest antibacterial spectrum.There were 20 monomer compounds were isolated from the metabolites of the Talaromyces funiculosu,and analyzed by HPLC-MS,which laid a foundation for the discovery of new structure antibacterial monomer compounds.The results of this study indicated that the endophytic fungi of the Lepidoptera of Qinling were abundant and the metabolites were active about antibiosis.The Talaromyces funiculosu was isolated from insects for the first time.This study established an efficient isolation and identification system about endophytic fungi of Lepidoptera,enriched the endophytic fungi of Qinling insects,and laid a foundation for the discovery of new structure antibacterial active monomer compounds. |
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