| Plant-associated microbiomes play an important role in plant growth promotion,protection against diseases and biotic or abiotic stresses,and plant metabolism regulation.Astragalus membranaceus(Fisch.)Bunge var.mongholicus(A.mongholicus)is a traditional medicine with a high pharmaceutical value,and the synthesis of its main medicinal ingredients astragalosides and flavonoids is regulated by its associated microbiome.However,there are few studies on the structure,distribution patterns and community assembly of A.mongholicus associated microbiome,especially the study on which microbiota regulates the medicinal ingredients remained unclear.Therefore,in our study,high-throughput sequencing technology was used to analyze the associated microbial communities of the samples collected from three typical A.mongholicus planting areas(Dangchang County in Gansu Province,Zizhou County in Shaanxi Province,Hunyuan County in Shanxi Province)in China.High-performance liquid chromatography-mass spectrometry(LC-MS)was applied to determine the content of medicinal ingredients.Our aims were to decipher the composition,structure and assembly rules of microbial communities and the correlation relationships between associated microbiome and medicinal ingredients.Meanwhile,the greenhouse pot experiment was carried out to explore the effects of A.mongholicus root-associated microbiome on the quality of A.mongholicus,which provided the theoretical basis for revealing the role of microbes in the quality formation and the technical support for large-scale cultivation of A.mongholicus.The main findings are as follows:1.The complex relationships among the root-associated microbiome,medicinal ingredients and soil physicochemical properties of A.mongholicus in different cultivation years were investigated.The study found that the relative abundance of Proteobacteria and Ascomycota showed the variation trend of increase first and then decrease as the cultivation year increased.Redundancy analysis(RDA)found that cultivation year had a greater impact on bacterial composition in bulk soil and rhizosphere,but the bacterial microbiome in the roots remained relatively stable,and not affected by soil properties and cultivation years.While fungal community composition was mainly affected by soil physicochemical properties.Spearman correlation revealed that the root-associated bacteria had a stronger correlation with the accumulation of medicinal ingredients.The abundance of Stenotrophomonas in the rhizosphere was positively correlated with astragalosides content,while Phyllobacterium and Inquilinus in roots were positively related to Calycosin(CA)content.Additionally,the other dominant species in the rhizosphere,such as Sphingopyxis and Lysobacter were also positively related to the astragalosides content.2.The biogeographic distribution patterns of bacterial and fungal communities in the rhizosphere differed from those in bulk soil.The results showed that soil physicochemical properties and spatial distance drove the rhizospheric microbial community composition.Soil p H and available potassium(AK),were respectively the key factors affecting the bacterial and fungal communities in bulk soil,while total phosphorus(TP)and total nitrogen(TN)affecting microbical community in the rhizosphere.The rhizosphere networks had fewer nodes and edges,a lower density but higher modularity and greater positive links compared to the bulk soil networks,suggesting that a less complex topology and niche sharing were features of the A.mongholicus rhizosphere network.The distance-decay pattern showed that the bacterial and fungal communities in the bulk soil and rhizosphere displayed somewhat similarity.Stochastic processes mainly shaped the microbial community structures in both compartments.Spearman correlation analysis revealed that the keystone bacterial species in rhizosphere were positively associated with the Astragalosides I(Ast I)content,but negatively correlated with the flavonoids content.3.The results demonstrated that sampling sites had a greater effect on the endophytic communities especially leaf endophytic bacteria and root endophytic fungi than plant compartments.The content of medicinal ingredients also displayed location effects.The network complexity and connectivity of the root network were higher than those of stem and leaf networks.Spearman correlation analysis and random forest(RF)model suggested that endophytic bacteria were closely correlated with medicinal ingredient content,especially the keystone species in the pivotal modules,the abundance of Bacillus and Arthrobacter positively related with the astragalosides content,while the abundance of Devosia and Flavobacterium showed a positive correlation with the flavonoids content.4.The results demonstrated that the composition and structure of rhizosphere bacteria and fungi were affected by inoculation of soil suspension,especially for rhizosphere bacteria.In addition,the inoculation of soil suspension also impacted the plant physiological indexes,the accumulation of medicinal ingredients,and soil physicochemical properties.The cooccurrence pattern of rhizosphere microbiome indicated that the network complexity and stability in the treatment with inoculation of YL soil suspension was higher.In different inoculation treatments,the rhizosphere bacterial community assembly process was deterministic,dominated by homogeneous selection,while the fungal community assembly process was stochastic,dominated by dispersal limitation.Random forest model revealed that rhizosphere bacteria were related to the accumulation of medicinal ingredients and plant growth.Structural equations model indicated that the content of secondary metabolites was directly influenced by the inoculation of soil suspension,soil type,and rhizosphere bacterial composition.5.The root-associated microbial communities were analyzed before and after transplanting during the planting process,and the results revealed that the root-associated bacterial community after transplanting was not only affected by soil type and growth stage,but also influenced by the bacterial community initially colonizing the roots of seedlings,which also impacted the growth and content of medicinal ingredients in A.mongholicus.The co-occurrence network of root-associated communities in different soils were distinctive,which suggested that soil type affected the microbial interactions.The impact of bacterial community in the rhizosphere and roots on the secondary metabolites showed selective preference.Rhizospheric bacteria enriched in SYL soil could promote the synthesis of flavonoids,while endophytic bacteria in the root enriched in SGF soil could promote the astragalosides content.The source track of microbiome related to medicinal ingredients in rhizosphere and roots showed that they came from the bacteria that colonized the roots of seedlings,indicating that the root-associated bacteria showed the priority and genetic effects.The re-inoculation experiments of synthetic bacterial community further proved that the keystone species colonized the roots in the early stage can promote the increase of the medicinal ingredients in A.mongholicus.In this study,we analyzed the structure,assembly rules and co-occurrence networks of A.mongholicus associated microbiome,and correlation relationships of their secondary metabolism,which clarified the influencing factors of A.mongholicus associated community assembly,and laid a theoretical foundation for the interaction between A.mongholicus and microbiome.Simultaneously,we confirmed the important role of A.mongholicus associated microbiome in the regulation of secondary metabolism.The dominant strains from the A.mongholicus microbial communities that could promote the accumulation of flavonoids and astragalosides provide the microbial resource for improving the quality of A.mongholicus. |
