Deciphering Salvia Miltiorrhiza Associated Microbiome And Regulating Mechanism Of Tanshinone Biosynthesis By Their Dominant Species

Danshen(Salvia miltiorrhiza)is an important rhizomatous Chinese medicinal material,the biosynthesis of tanshinone and salvianolic acid was regulated by its associated microbiome.However,there were few reports on the structure and assembly rules of S.miltiorrhiza seed and root associated microbiome,and the studies on regulating its secondary metabolism by dominant species of associated microbiome were also not systematic enough.Here,by the amplicon and mtagenome sequencing methods,this theis aims to analyze the core microbiome of S.miltiorrhiza seed,and to study the structure,assembly rule and population dynamics of root-associated microbiome,and to compare the structural differences and functional characteristics of rhizosphere and root endosphere of S.miltiorrhiza and S.miltiorrhiza Bge.f.alba.Then the regulate mechanism of tanshinone biosynthesis by some dominant species were studied by the co-culture system of sterile tissue culture seedlings,which provided theoretical basis for revealing the roles of microbe on the quality formation of S.miltiorrhiza.The main results are as follows:i.The dominant genus of the core microbiome of S.miltiorrhiza seeds were including the genus Pantoea,Pseudomonas and Alternaria,which enriched many functions closely related to terpene metabolism and is a gene reserve for secondary metabolic regulation of S.miltiorrhiza.ii.An endophytic fungus of S.miltiorrhiza seed,Phoma herbarum D603 has the abilities to produce phytohormone IAA,phosphorus-soluble and siderophore production,which could colonized among intercellular space of root tissues,promote its growth and root development,then to promote the synthesis and increase the content of medicinal components in root by stimulating up-regulation of key genes,such as DXS,HMGR,GGPPS,CPS,KSL and CYP76AH1.iii.The same S.miltiorrhiza plants grown in different soils,there were nine bacterial genera,such as Steroidobacter,Phenylobacterium,Pseudomonas,and five fungal genera,such as Ceratobasidium,Leptosphaerulina,Glomus,were co-enriched in its rhizosphere microbiome.Across the life cycle of S.miltiorrhiza,the relative abundance of Gammaproteobacteria and Dothideomycetes in rhizosphere microbiome decreased sharply,Alphaproteobacteria did not change much,and the abundance of Betaproteobacteria,Acidobacteria,Actinabacteria and Sordariomycetes were also increased significantly,each stage has its own unique dominant taxa.The shared bacterial genera among spermsphere and rhizosphere of S.miltiorrhiza included Pseudomonas,Novsphingobium and Sphingomonas,while the shared fungal genera were Phoma,Cladosporium and Fusarium.iv.S.miltiorrhiza rhizosphere enriched in Wallemia and Mortierella,while Sphingomonas,Cladosporium and Wardomyces were found to be more abundant in the rhizosphere of S.miltiorrhiza Bge.f.alba.In the endosphere of S.miltiorrhiza root were enriched genera Ohtaekwangia,Glomus,Funneliformis,Rhizophagus and Claroideoglomus,while S.miltiorrhiza Bge.f.alba endosphere were found more genera Cellvibrio,Sphingobium,Steroidobacter and Olpidium.By the metagenome analysis,we found the rhizosphere microbiome structure of S.miltiorrhiza and S.miltiorrhiza Bge.f.alba were different,but they had the same or similar important functional characteristics,and there are some functional differences at the same time.These two Salvia rhizosphere both enriched functional traits were involved in membrane transport-related trait ABC transporters,metabolism-related carbon metabolism,amino acid synthesis and metabolism,fatty acid metabolism,and functional features associated with plant-microbe and microbe-microbe interactions,such as flagellar assembly,bacterial chemotaxis,and two component systems.v.Cladosporium tenuissimum DF11,a representative strain of dominant genus in S.miltiorrhiza rhizosphere microbiome,could colonize in the intercellular space of root tissue,by regulating the up-regulated expression of key genes,such as HMGR,DXS,DXR,GGPPS,CPS and CYP76AH1 in tanshinone synthesis pathway,the to promote the biosynthesis of tanshinone and increase its content in root.vi.Some rhizosphere cultivable dominant species,such as,Acremonium sp.DF2,Mucor circinelloides DF20,Aspergillus terreus DF23 and Penicillium steckii DF33 were all could produce IAA and colonized in the intercellular space of root tissue,and promote the tanshinone accumulation by regulating the up-regulated expression of key genes such as,DXS,DXR,HMGR,GGPPS,CPS,KSL and CYP76AH1.The expression levels of most genes reached the response peak at the first or second week after colonization,and the promotion effects of these strains on the biosynthesis of cryptotanshinone and tanshinone IIA were significant.In this study,by analyzing the structure of danshen seed and root associated microbiome,the dominant species,assembly rules and population dynamics of danshen associated microbiome were preliminarily deciphered,and the structural differences and functional characteristics of root associated microbiome of S.miltiorrhiza and S.miltiorrhiza Bge.f.alba were revealed.It was also found that the dominant species of S.miltiorrhiza seed and rhizosphere associated microbiome could colonize in the root of S.miltiorrhiza seedling,and promote the synthesis and accumulation of tanshinone by stimulating the up-regulation expression key genes,thus improving the quality of S.miltiorrhiza medicinal materials.These results provided the theoretical guidance for elucidating the important role of S.miltiorrhiza associated microbiome in the regulation of secondary metabolism and quality formation,as well as the high quality cultivation and the origin of geoherbalism of S.miltiorrhiza.