Widely Targeted Metabolomic Analysis Of Ginseng Endophytes And Their Effect On The Adventitious Roots Of Ginseng

Ginseng(Panax ginseng C.A.Meyer)is a perennial plant belonging to the genus Panax of the Araliaceae family,has a long history of medicinal use as a traditional and valuable Chinese herb.The main active ingredients in ginseng are ginsenosides.Endophytes are microorganisms that live in host plant tissues for some or all of the time in symbiosis with the host plant,and their presence does not cause visible lesions in the host plant.In this study,ginseng endophytes were isolated and purified from 11 ginseng samples collected at different growth stages,from which endophytic with ginsenosides-producing capacity were selected.They were then identified by morphological and molecular biology methods,and their biological properties and optimal fermentation conditions were investigated.Finally,the metabolites were analyzed using a widely targeted metabolomic approach and prepared as microbial elicitors to investigate the effects of different elicitors concentrations and duration of action on the adventitious roots of ginseng.The specific results were as follows:1.By comparing the effects of different isolation methods and different medium agar concentrations on the isolation of ginseng endophytes,the best isolation method,tissue milling,was determined,and the optimal medium agar concentration was 9 g/L.143 ginseng endophytes,including 106 endophytic bacteria and 37 endophytic fungi,were isolated from 11 ginseng samples using this method.2.The 143 ginseng endophytes were cultured in liquid and the products were extracted using the ODS method.The LC-MS method was used to determine the presence of ginsenoside Rg2(133.36 ng/L)in the culture solution of strain PDA-1.The culture solution of strain PDA-8contained ginsenoside Rg2,Rg3 and Re at 436.96 ng/L,1,169.04 ng/L and 1,626.00 ng/L respectively.3.The results of morphological and molecular biological identification of strains PDA-1and PDA-8 showed that strain PDA-1 was a fungus of the Fusarium sp.,presumably Fusarium oxysporum;strain PDA-8 was a fungus of the Ilyonectria sp.,presumably Ilyonectria robusta.4.The results of biological characterization showed that strain PDA-1 reached the stabilization stage in liquid culture for 30 h.The optimal medium formulation was 200 g potato,20 g glucose,5 g peptone,1 g KCl,1 L water,p H adjusted to 7.0.The optimal culture temperature was 25°C and the optimal light condition was total darkness.Strain PDA-8 liquid culture 84 h to reach the stable stage,its optimal medium formula is potato 200 g,galactose 20 g,yeast extract 5 g,Ca Cl2 1 g,water 1 L,p H adjusted to 8.0.optimal culture temperature 20℃,optimal light conditions for alternating light and dark.5.The response surface methodology was used to optimize the fermentation conditions of the two strains.The optimal fermentation conditions for strain PDA-1 were determined to be1.9% inoculum,110 rpm shaker speed,p H 7.2,incubation temperature 26.6°C and incubation time 58 h.The yield of ginsenoside Rg2 was 193.915 ng/L which was significantly higher than that before optimization.The optimal fermentation conditions for strain PDA-8 were determined to be 2% inoculum,170 rpm shaker speed,p H 7.9,incubation temperature 22°C and incubation time 122 h.The yield of ginsenoside Rg3 was 1,591.47 ng/L which was significantly higher than that before optimization.6.391 metabolites were identified from strains PDA-1 and PDA-8 using a widely targeted metabolomic approach,which included not only a wide range of terpenoids and precursors,but also a variety of metabolites that may affect basal and secondary metabolic synthesis in ginseng.7.The ginseng adventitious roots were treated with lyophilized bacterial aqueous extracts of ginseng endophytes PDA-1 and PDA-8 as elicitors.The results showed that both elicitors could promote the accumulation of ginseng adventitious root biomass and significantly increase the content of saponins in ginseng adventitious roots by regulating the expression of key enzyme genes in the ginsenoside synthesis pathway at suitable treatment concentrations and action times.