Study On Classification Of Plant Miniature Fungi And Secondary Metabolites In Shenzhen Area

The classification and identification of fungi have dynamic renewal characteristics.As a microbial resource with great potential,fungi need to be excavated and fully utilized.Shenzhen area has a specific geographical and ecological environments suitable for the growth of fungi.Humid climate,extensive vegetation coverage,the number of potential plant fungal species cannot be underestimated.However,according to the literature search and investigation,there is less work on identification and classification of fungi in this area,the list of fungi is still incomplete,the development and utilization of fungal resources are relatively shallow.Therefore,it is planned to study the classification,species diversity and biological activities of plant microfungi in shenzhen.The research of this project had certain scientific significance for the identification and classification of plant microfungi in shenzhen,it can clarify plant microfungi,species diversity,fill in the relevant list of fungi,explore the types and activities of fungal secondary metabolites.It provided relevant research data for the development and utilization of fungal resources.In this study,samples of leaves,branches and rotten bark of plant fungi distributed in different areas of shenzhen were collected.The structure of fungi fruiting bodies,ascus,ascospores,conidia and other structure were observed under a dissecting microscope and light microscope.From the above observation,the morphological characteristics of different fungi was understood intuitively.An average value was obtained by measuring the size of the above structure through a camera composite microscope for accurate description.Genomic DNA extraction and PCR experiments were carried out to obtain the bidirectional sequences of ITS(Internal Transcribed Spacer,ITS),LSU(28S r DNA),SSU(18S r DNA),and TEF-1(Transcription elongation factor,TEF).After finishing a series of data such as BLAST sequence alignment,Bioeidt sequence editing,Mafft data analysis,Alteralignment data alignment,and Clustal X file conversion by raxml GUI v.1.3,the maximum likelihood method evolution tree was obtained,Mr Bayes v.3.0b4 obtained the bayesian method evolution tree,and PAUP v.4.0 BETA obtained the maximum parsimony method evolution tree.The calculated values of the three phylogenetic trees were integrated into the maximum likelihood method phylogenetic tree as the ultimate fungal classification basis.Fig Tree v.1.4.0 software was used to view the phylogenetic tree related information.The identified new fungal strains were fermented and cultured to obtain fermentation broth,and the information of main metabolites was obtained by liquid chromatography-mass spectrometry.Principal Component Analysis(PCA)and Orthogonal Partial Least Squares Analysis(OPLS-DA)were used to calculate the differential metabolite information of the new strains in pairs.The data were enriched by KEGG(Kyoto Encyclopedia of Genes and Genomes)data and Annotate the differential metabolite enrichment pathway.The experimental results showed that 87 of the 107 samples had fungal fruiting bodies,and 52 samples were successfully obtained after monospore separation.The fungal samples were located in the order of the Pleosporales,Botryosphaericeae,Pestalotiopsis,Trichosphaeria,Aureobasidium,Phomopsis,Leptosphaeria,Pyrenochaeta,Aspcrgillus,Dictyosporium and Lichen.Three new fungal strains 49 A were named Phaeosphaeria chinensis,number 51 was named Melanomma chinensis,number 52 was named Dictyosporium shenzhenense.The secondary metabolites included phenolic acids,alkaloids,lignans and coumarins,terpenes,etc.The secondary metabolites of Phaeosphaeria chinensis were mostly concentrated in the degradation pathway of aromatic compounds.The fungal secondary metabolites of Melanomma chinensis were highly enriched and significantly enriched in the phenylalanine synthesis and decomposition pathway.The strain Dictyosporium shenzhenense contained significant enrichment of glutamic acid and degradation pathways.The secondary metabolites of the three fungi may all have anti-MRSA activity.This study conducted preliminary research and exploration on the species and morphology of some plant microfungi in shenzhen,supplementing the directory of plant microfungi in the region and showing the diversity of fungal species.Secondly,3 new plant microfungi strains were named and identified by bioinformatics methods;thirdly,secondary metabolites,differential compounds,and pathway enrichment of 3 new fungi strains were explored;finally,the secondary metabolites of 3 new fungal strains and their anti-MRSA activity were initially explored by paper diffusion method,which provided experimental data for the study of plant microfungi.