The Effect Of Epichlo(?) Endophyte On Phyllosphere Microbes And Leaf Metabolites In Achnatherum Inebrians

Endophytic and epiphytic environments of phyllosphere are important habitats for microbes.Epichlo(?) endophyte can coexist with other phyllosphere microbes in cold season grasses.Drunken horse grass(Achnerthum inbrains)is a perennial grass of Poaceae.It forms a mutually beneficial symbiont with Epichlo(?) endophyte,which has strong resistance to biotic and abiotic stress.In this study,the leaves of endophyteinfected(EI)and endophyte-free(EF)A.inbrains grown in the field,mature and without obvious diseases were used as experimental materials.Through traditional isolation and culture,Illumina sequencing technology and LC-MS detection,we explored the effect of Epichlo(?) endophyte on phyllosphere microbes and leaf metabolites,and the complex correlation between phyllosphere microbes and leaf metabolites.This present study is to further reveal the driving relationship among Epichlo(?) endophyte-phyllosphere microbes-leaf metabolites,and provide a theoretical basis for the scientific and rational utilization of A.inbrains resources.The main results obtained are as follows:(1)Through traditional isolation and culture,a total of 2180 bacterial strains were obtained from the phyllosphere of A.inebrians.Sequence alignment identified 4 phyla,22 genera and 47 species.The total number of colonies of culturable endophytic bacteria of EI plants were significantly(P<0.05)higher than that of EF plants.Epichlo(?)endophyte significantly(P<0.05)reduced the relative abundance of Pseudomonas in the culturable endophytic bacterial community,but significantly(P<0.05)increased its relative abundance in the epiphytic bacterial community.The interaction between Epichlo(?) endophyte and phyllosphere environment significantly(P<0.05)affected the diversity and richness of phyllosphere culturable bacterial communities.A total of 256 fungal strains were isolated from the phyllosphere of A.inebrians.Sequence alignment identified 2 phyla,18 genera and 24 species.Ascomycota was the most dominant taxa of phyllosphere culturable fungi community of A.inebrians.Epichlo(?) endophyte significantly(P<0.05)increased the relative abundance of Cladosporium;decreased the relative abundance of Alternaria.Epichlo(?) endophyte significantly(P<0.05)increased the diversity of phyllosphere culturable fungal communities of sterilized leaves.The diversity and richness of culturable fungal community of sterilized leaves were significantly(P<0.05)lower than those of non-sterilized leaves.Redundancy analysis(RDA)showed that the p H,organic carbon(OC),total nitrogen(TN)and total phosphorus(TP)contents of leaves affected the composition of phyllosphere culturable bacterial and fungal communities.(2)Through high-throughput sequencing,a total of 892,704 bacterial sequences and 1,018,368 fungal sequences were obtained from the phyllosphere of A.inebrians.The four main taxa in the phyllosphere bacterial community were Proteobacteria,Firmicutes,Actinobacteria and Bacteroidetes.The diversity and richness of the phyllosphere endophytic bacterial community were significantly(P<0.05)higher than those of the epiphytic bacterial community.Ascomycota was the most abundant phylum in the phyllosphere fungal community of A.inebrians,followed by Basidiomycota and Mortierellomycota.The richness of the phyllosphere endophytic fungal community was significantly(P<0.05)higher than that of the epiphytic fungal community,but there was no significant(P>0.05)difference in the diversity between the two communities.Epichlo(?) endophyte increased the diversity of phyllosphere fungal communities.(3)Through LC-MS/MS detection and analysis,414 metabolites were annotated from the leaves of A.inebrians,including 132 differential metabolites between EI and EF plants,which belonged to 24 classes.Under the infection of Epichlo(?) endophyte,purine derivatives,indole derivatives and phytoestrogens were up-regulated.12 classes of differential metabolites,including steroids,vitamins,antibiotics,curcuminoids,steroid hormones,flavonoids,protein-enzymes and transport proteins,were downregulated.(4)229 significant correlations between phyllosphere microbes and leaf metabolites were obtained by Spearman correlation analysis.Among them,there were115 significant(P<0.05)correlations between 61 differential metabolites and 9endophytic bacterial phyla.Firmicutes was positively correlated with indoleacetic acid and negatively correlated with vanilla.There were 50 significant(P<0.05)correlations between 50 differential metabolites and 10 epiphytic bacterial phyla.Actinobacteria was positively correlated with enterodiol and negatively correlated with benztropine and aldicarb.There were 39 significant(P<0.05)correlations between 34 differential metabolites and 8 endophytic fungal phyla.Mucoromycota and Zoopagomycota were positively correlated with deguelin(-).Ascomycota was negatively correlated with indoleacetic acid.There were 25 significant(P<0.05)correlations between 24 differential metabolites and 8 epiphytic fungal phyla.Ascomycota was positively correlated with methyl 4-hydroxybenzoate,and Basidiomycota was negatively correlated with methyl 4-hydroxybenzoate.