The Relationship Between Endophytic Fungi And Differential Metabolites Formation Of The Stems And Roots In Ephedra Sinica

As a common Chinese medicine,Ephedra sinica stapf is one of the basic source plants of Mahuang and Mahuanggen in the Chinese Pharmacopoeia.The biggest trait of E.sinica is its stem and root has opposite efficacy.The stem is mostly used for sweating and dispersing cold,while the root is a potent antiperspirant.This phenomenon of "same origin,disparate efficacy" is closely related to the difference in chemical composition in stem and root.In the course of long-term co-evolution,stable mutually beneficial symbiotic relationship was established between endophytic fungi and host plant.The endophytes got highly diverse and special function,and they can greatly affect the metabolite composition of the host plant.They distributed differently in different tissues of the host and play important role in physiology and metabolism of different tissues.Thus,this paper systematically investigated the composition and distribution of endophytic fungal communities,comprehensively analyze the differences of metabolite composition in the stem and root of E.sinica using metagenomics and metabolomics techniques,and then analyze and explore the relationship between endophytic fungi and the differential metabolites.The findings list as follows:1.The ITS2 barcode was first used to identify the species of wild E.sinica collected from Datong,Shanxi Province.By comparing the similarity between the ITS2 sequence of E.sinica and Gen Bank record using the BLAST method,the collected plant materials were identified as E.sinica according to the similarity of 100%.2.The composition and diversity of endophytic fungal communities were analyzed in the stem and root of wild E.sinica collected in May and September,based on Illumina Miseq high-throughput sequencing data.The results showed that there were significant differences in the endophytic fungal community composition between the stem and root of E.sinica.Through species classification analysis,the dominant endophytic fungi in the stem of E.sinica collected in September were Phyllosticta,Pleosporales_spp.,Alternaria and Allophaeosphaeria,while the dominant fungi in root were Talaromyces,Aporospora,Aspergillus and Phaeoacremonium;The dominant endophytic fungi in the stem of E.sinica collected in May were Fusarium,Didymellaceae_spp.,Botrytis and Helvellosebacina,while the dominant fungi in root were Hypocreales_spp.,Eurotiomycetes_spp.It was analyzed of the distribution of 39 dominant fungal OTU in each group through fungal OTU data.3.A total of 14 primary metabolites and 22 secondary metabolites were identified in the stem and root of E.sinica collected in May and September based on NMR spectrum and metabolomics.Through multivariate data analysis,a total of 17 differential metabolites were obtained.Among these metabolites,ephedrine,pseudoephedrine and methylephedrine were only found in stem,whereas ephedrannin A,mahuannin A,mahuannin F,mahuannin H and afzelechin were found only in root.Furthermore,the root of E.sinica collected in September contained more arginine,serine,?-glucose and sucrose,and less isoleucine,valine,threonine,glutamate,aspartate,proline,catechin and fumarate,as compared with the stem.The root of E.sinica collected in May contained more arginine,serine,?-glucose and sucrose,and less isoleucine,leucine,valine,threonine,alanine,glutamate,aspartate and catechin,as compared with the stem.The stem of E.sinica collected in September contained more ephedrine,methylephedrine,glutamate,proline and sucrose,and less leucine,valine,aspartate,serine,pseudoephedrine and ?-glucose,as compared with the stem in May.The root of E.sinica collected in September contained more sucrose,arginine,ephedrannin A,mahuannin A,mahuannin F and mahuannin H,and less leucine,proline,?-glucose,?-glucose and afzelechin,as compared with the root in May.4.The correlation was analyzed between 39 dominant fungi endophytic fungal OTUs and 23 differential metabolites.The results indicated that 21 fungal OTUs were significantly or highly significantly correlated with these differential metabolites.Of these,ephedrine,pseudoephedrine and methylephedrine exhibited significantly positive correlations with OTU15762,OTU24893,OTU27552(Helvellosebacina),OTU30383(Alternaria),OTU31159(Botrytis),OTU34134,and significantly negative correlations with OTU30157(Phaeoacremonium),OTU30521(Aporospora);ephedrannin A,mahuannin A,mahuannin F and mahuannin H exhibited significantly positive correlations with OTU30099(Pezizales_sp.),OTU30157(Phaeoacremonium),OTU30236(Aspergillus),OTU30521(Alternaria),OTU30668(Talaromyces),OTU30901,and significantly negative correlations with OTU48,OTU87,OTU206,OTU30383(Alternaria).The results indicate that these endophytic fungi can promote or inhibit the formation of metabolites in the stem and root of E.sinica.This study mainly explored the correlated relationships between endophytic fungi and the formation of differential metabolites in wild E.sinica using current “multi-omics” tools.The results provide a data for further understanding of endophytic fungi on the formation of differential metabolism between the stem and root of E.sinica,and provide new ideas for further development of functional endophytic fungi and regulation of Ephedra quality.