Diversity Analysis And Isolation And Identification Of Fungi From Feces Of White Feathered Broilers At Different Days Of Age

White feather broiler has the advantages of high nutritional value and high feed conversion rate.It is an important breed in China’s livestock and poultry industry.Intestinal flora plays an important role in immune regulation and nutrition metabolism of the body.The advent of high-throughput sequencing technology has enabled people to have a deeper understanding of intestinal flora.The current research mainly focuses on intestinal bacteria,and there is relatively little research on intestinal fungal flora.However,intestinal fungi also play an important role in maintaining intestinal microecological balance.In this paper,the feces of white feathered broilers of different ages were collected at different stages of the growth cycle of white feathered broilers,and the fecal fungal flora of white feathered broilers was studied by high-throughput sequencing technology,bioinformatics technology and culturing omics technology.The results of this study are as follows:1.Diversity analysis of chicken fecal fungal floraIn this experiment,fecal samples of eight days old(1,3,5,7,14,21,35 and 42 d)in the growth cycle of broilers were selected,and the composition and distribution of fecal fungal flora of white feather broilers at different days old were analyzed by ITS amplicon sequencing technology combined with bioinformatics technology.The results showed that at the phyla level,five species of fungi with relative abundance greater than 1%were annotated,including Ascomycota,Basidiomycota,Chytidiomycota,Glomeromycota and Zygomycota;At the family level,there are 9 species of fungi with relative abundance greater than 1%,including Saccharomycetales＿family＿Incertae＿sedis,Trichosporonaceae,Trichocomaceae,Glomeraceae,Nectriaceae,Sclerotiniaceae,Ophiocordycepitaceae,Mortierellaceae,Mucoraceae;At the genus level,9 fungal genera with relative abundance greater than 1%were annotated,including Candida,Trichosporon,Aspergillus,Penicillium,Glomus,Loderomyces,Ophiocordyceps,Mortierella,Mucor;Ascomycota is the dominant phylum,and its relative abundance in each ages sample is more than 60%,Saccharomycetales＿family＿Incertae＿sedis is the dominant family.αdiversity analysis showed that the diversity and richness of fecal fungal flora of broilers decreased from 1d group to 3 d group,reached the lowest in 3 d group,increased from 3 d group to 14 d group,reached the highest in 14 d group,decreased from 14 d group to 35 d group,and then increased to 42 d group.βdiversity analysis showed that there were significant differences among the groups from the 1 d to the 42 d(R~2>0.2,P=0.001).By using Kruskal Wallis test to calculate the P value,the differences of the top 20 fungal genera in the feces of broilers at eight days of age were analyzed.The results showed that the abundance of 12 fungal genera in the feces of broilers at eight days of age was significantly different(P<0.05).2.Interaction analysis of bacteria and fungi in broiler fecesThrough the correlation analysis of bacteria and fungi in broiler faeces,the interaction network was constructed.It was found that the interaction network in 1 d group was more complex than that in 3 d,5 d,7 d,14 d,21 d,35 d and 42 d groups.The interaction between fecal bacteria and fungi was more negatively correlated than positively correlated in 1 d group.The interaction between fecal bacteria and fungi in 3 d,5 d,7 d,14 d,21 d,35 d and 42 d groups were more positively correlated than negatively correlated.3.Isolation and identification of fecal fungi from broilersThe fecal samples of broilers on 21 d and 35 d group were isolated and cultured by cultomics.A total of 142 fungi belonging to 3 phyla,8 classes,8 orders,9 families,11 genera and 19 species were successfully identified by ITS1 and ITS2 sequencing.At the phylum level,Ascomycota(80.99%)accounts for the largest proportion,followed by Basidiomycota(18.81%),Chytridiomycota(0.70%);At the genus level,Diutina is the most isolated and cultured genus;At the species level,a total of 19 fungi were obtained,including the potential probiotic Rhodotorula mucilaginosa and 8 potential new fungal strains.In addition,the number of fecal mycophyla of35 d broilers group cultured was more than that of 21 d broilers group.Among them,Aspergillus,Cutaneotrichoporon,Fusarium,Diutina,Rhodotorula,Meyerozyma and Trichosporon were all cultured in the feces of broilers on 21 d and 35 d group.4.10 Genomic analysis of 10 strains of chicken fecal fungiAmong the fecal fungi successfully isolated from white feather broilers,10 fecal fungi from Ascomycetes,which accounted for the largest proportion,were selected for genome second generation sequencing.The results showed that 10 strains of fungi came from 4 genera,including3 strains of Aspergillus fumigatus,3 strains of Candida parapsilosis,3 strains of Diutina catenulata and 1 strain of Fusarium foetens.Bioinformatics technology was used to analyze the related genes annotated.COG function annotation showed that 10 strains of broiler fecal fungi annotated genes related to the transportation and metabolism of more carbohydrates,amino acid transportation and metabolism.KEGG function annotation showed the annotation of carbohydrate metabolism related genes was the most in 10 strains of broiler fecal fungi.GO functional annotation results showed that the genes related to cells,cell components,organelles,cellular processes and metabolic processes were more annotated in the 10 broiler fecal fungi strains.To sum up,this study analyzed the distribution rule of fecal fungi flora of broilers at different ages,preliminarily established the intestinal fungi strain bank of broilers,completed the preliminary prediction of the gene function of broiler fecal fungi,and provided theoretical and data reference for further research on intestinal fungi of broilers in the future.