Screening And Mechanism Study Of Endophytic Fungi Of Epimedium Koreanum Based On Growth-promoting Effect

Objective:The purpose of this study is to use the model plant Arabidopsis thaliana to explore the growth-promoting effect and mechanism of endophytic fungi in Epimedium koreanum Nakai,to find endophytic fungi with significant growth-promoting effect,and to provide scientific theoretical guidance for the development of microbial preparations and the improvement of the quality of medicinal materials.Methods:In this study,endophytic fungi were isolated from the leaves of Epimedium koreanum Nakai by tissue culture method.The nitrogen fixation and phosphorus solubilization ability,siderophore production ability and indole acetic acid secretion of the isolated endophytic fungi were investigated,and the functional strains with growth promoting effect were preliminarily screened.Using Arabidopsis thaliana as a model plant,the strains with the most significant growth-promoting effect on Arabidopsis thaliana were screened.Through in-depth analysis of metabolomics,the molecular basis of endophytic fungi from Epimedium koreanum Nakai promoting the growth of Arabidopsis thaliana was explored.Results:（1）A total of 15 strains of endophytic fungi were isolated in this experiment.Functional screening experiments showed that 15 strains of endophytic fungi had no obvious ability to dissolve phosphorus,but all had nitrogen fixation ability.In the siderophore production experiment,11 strains with siderophore production ability were obtained,of which Y8 had the strongest ability.Three strains of Y₆,Y₈and Y₁₁were screened by high performance liquid chromatography.They had the ability to secrete IAA,of which Y₈had the strongest ability and Y₆had the weakest ability.Through the preliminary screening of 15 strains of endophytic fungi,it was found that Y₆,Y₈and Y₁₁had better growth promoting effect than others.（2）The three strains were identified by molecular biology and morphology as endophytic fungi of the genus Anthrax,which were Y₆（Colletotrichum changpingense）,Y₈（Colletotrichum endophytum）and Y₁₁（Colletotrichum cobbitiense）.（3）The co-culture experiment of Arabidopsis showed that Y₈strain had the most significant effect on the growth indexes of Arabidopsis such as the length of taproot and the number of lateral roots,followed by Y₆and Y₁₁;The three strains have significant effects on the activities of POD,SOD,CAT,F3H and other enzymes of Arabidopsis,of which Y₈has the strongest effect.The three strains have no significant effects on the soluble sugar content and chlorophyll content of Arabidopsis.（4）Using metabonomics to study the growth promotion mechanism of Y8 strain on Arabidopsis thaliana,the following conclusions were obtained:a total of 475 identifiable differential metabolites were obtained,including 291 up-regulated differential metabolites and 184 down-regulated differential metabolites.The KEGG enrichment pathway analysis revealed 10 significantly enriched metabolic pathways,including vitamin B6 metabolism,tyrosine metabolism,phenylalanine biosynthesis,nicotinic acid and nicotinamide metabolism Flavonoids and flavonol biosynthesis,starch and sucrose metabolismβ-Down-regulated pathways such as alanine metabolism,pyrimidine metabolism,phosphate and hypophosphate metabolism and flavonoid biosynthesis,among which,Y₈strain plays a role in enhancing the plant’s ability to resist invasion and promoting plant growth and development by increasing the expression of ferulic acid and sinacyl coenzyme A in Arabidopsis plants.Conclusion:In this study,the function screening experiment and Arabidopsis interaction experiment showed that the growth promotion effect of Y₈strain was the most obvious.Through metabonomics analysis,the growth promotion mechanism of Y₈strain was analyzed,and 10 metabolic pathways with significant changes were initially screened,which laid the foundation for subsequent molecular biology research such as transcriptomics and genomics,and also provided new research ideas for quality improvement of medicinal plants,It is of positive significance for the sustainable development of the endangered genuine medicine industry.