Diversity Analysis And Isolation And Identification Of Intestinal Fungi From Microtus Brandti

Microtus brandtiis widely distributed,mainly in Russia,Mongolia and northeast China,and is the main carrier of plague and other zoonoses.However,there are still many unknown microorganisms in the intestine,and the fungi carried in the intestine are unknown.Therefore,we studied the intestinal fungi of 26 wild Voles in Uzhumqinqi,eastern Inner Mongolia by high-throughput sequencing technology combined with culture omics.The specific results are as follows:Sequencing results of ITS(Internal Transcribed Spacer)amplicon showed that: At the phylum level,a total of 4 species of fungi were identified based on 1% threshold,namely Ascomycota,Basidiomycota,Mucoromycota and Chytridiomycota.Ascomycota and basidiomycota were the dominant phyla in both male and female groups.At order level,the proportion of Mucorales in male Microtus brandti group was significantly higher than that in female.At the genus level,the proportion of Circinella in male Microtus brandti group was significantly higher than that in female.The results of difference analysis showed that there were19 species affected by significant differences in the male Microtus brandti group,and the sequence was as follows: Mucoromycetes,Mucorales,Mucoromycota,Circinella,Lichtheimiaceae,Circinella Nodulosa,Aspergillus＿candus,Penicillium,Condenascus,Condenascus tortuosus,Apiotrichum,Cephalotrichum Asperulum,Agaricomycetes,Penicillium bilaiae,Didymella,Gamsia,Beauveria Felina,Beauveria,Cordycipitaceae,and only one species,Phaeosphaeriaceae,was significantly affected in the female Microtus brandti group.The results of fungal phenotypic prediction showed that saprophytic fungi accounted for about 60%,followed by symbiotic and pathophytic fungi.The intestinal fungal flora of Microtus brandti was studied by using ITS sequencing and traditional culture omics.A total of 589 fungal strains were isolated,and 194 fungal strains were identified,belonging to 3 phyla,10 classes,16 orders,21 families,34 genera,61 species.The most isolated fungal species were Meyerozyma athensensis(n= 50,25.77%),Aspergillus glaucus(n=16,8.25%)and Preussia persica(n=10,5.15%).It was also proved that the ITS amplicon analysis and culture omics could be used to verify each other,and culture omics could be used to culture more species that could not be annotated by ITS sequencing technology.Among 194 fungal strains,13 strains had less than 98.5 percent similarity to known strains,which might be potential new strains.In addition,some of the functional fungi isolated from the experiment can be used for the study of specific fields,such as Coniochaeta,Pichia guilliermondii,Rhodotorula mucilaginosa,etc.Among the isolated intestinal fungi,20 strains of fungi from ascomycota,which accounted for the largest proportion of intestinal core bacteria,were sequenced in the second generation.The results showed that the 20 isolates were from 7 genera,including 3 strains of Candida,5 strains of Aspergillus,1 strain of Trichoderma,1 strain of Penicillium,1 strain of Trichoderma,1 strain of Fusarium and 8 strains of Meyerozyma.A total of 11 strains were identified to species level,including 8 strains of Montessori,2 strains of Aspergillus cinerea and1 strain of Candida.Bioinformatics analysis was used to analyze the related functional genes.CAZy gene annotation showed that the number of genes related to glycoside hydrolase and glycosyltransferase was the largest among 20 fungi strains.KEGG functional annotation showed that protein metabolism,carbohydrate metabolism and transcription-related genes were the most abundant,and there were more genes related to human disease pathways.In conclusion,this study constructed the intestinal fungi library for the first time,and completed the preliminary prediction of the intestinal fungi function of M.brandti,which has important scientific value and significance.