Study On Rhizosphere Soil Microbiome Of Panax Notoginseng At Different Developmental Stages

Rhizosphere microbiome has been recognized as an important factor for plant nutrition,growth,and resistance against pathogen invasion.Plants could affect the diversity,composition,structure,and function of the rhizosphere microbiome Meanwhile,plant growth stages also affect the microbial community.However,it is not well understood how it is associated with the development stages of perennial medicinal plants.What is the profiles of diversity,composition,and potential functions of rhizosphere microbiomes during different development stages of P.notoginseng?What is the diversity,composition,and function of rhizosphere microbiomes at the 3YR harvest stage?What the relationship between the microbial communities and function and the yield of P.notoginseng?Today,there is a lack of relevant research on these issues.To study the temporal dynamics of microbiomes,rhizosphere soils of P.notoginseng were collected at five fields in Pingba(PB),Yanshan(YS)and Qiubei(QB)in Wenshan,Yunnan Province of China during two years growth.Three experimental sites in the region of Pingba,namely,Pingba A(PBA),Pingba B(PBB)and Pingba C(PBC),were used.Briefly,one-year-old seedlings were transplanted into each field and then cultivated in strict accordance with the Good Agricultural Practices(GAP).Then,the rhizosphere soil samples were collected at one of the three developmental stages of P.notoginseng in each year:(1)vegetative(V,May);(2)flowering(F,Jul),(3)root growth(R,Oct),namely 2-year V(2YV),2-year F(2YF),2-year R(2YR),3-year V(3YV),3-year F(3YF),and 3-year R(3YR).Thus,a total of 105 samples comprising 90rhizosphere soil samples at six developmental stages and 15 bulk soil samples in five fields were obtained.Here,we utilize amplicon to analyze the profiles of diversity,composition,and structure of rhizosphere microbiomes during different development stages of P.notoginseng.The function of rhizosphere microbiome at harvest stage was studied by metagenomic shotgun sequencing.Finally,the rhizosphere microbial communities and functional traits related to the yield of P.notoginseng were further analyzed.The main results are as follows:(1)Succession characteristics ofαlpha diversity of P.notoginseng during the developmental stagesRichness and Shannon indices were used to evaluate the alpha diversity of bacterial and fungal communities.Linear least-square regression with second-order term shows that theαlpha diversities of bacteria and fungi are parabolas with plant growth.The diversity of bacteria and fungi exhibited a tendency to increase and then decrease P.notoginseng growth.Compared to bulk soils,the bacterial and fungal diversity exhibits increasing trends in the rhizosphere soils of P.notoginseng.Interestingly,compared to 3YF stage,the richness and Shannon indices in soil bacterial and fungal communities at the 3YR stage are remarkably reduced.(2)The rhizosphere soil microbiome of P.notoginseng at 3YR was more active than that of other growth stagesWe used redundancy analysis(RDA)to preliminarily characterize the most influential factors to the variance of microbial communities at the developmental stages.Constrained analysis of principal coordinates showed distinct separation among samples of different stages,and samples of 3YR stage could be separated from others.Bray-Curtis dissimilarities between rhizosphere soils and BL samples increase with incremental growth.Differential abundance test of bacterial and fungal genus between each stages and the BL was carried out to analyze the enriched and depleted communities during the growth of P.notoginseng.More bacterial genera were enriched and depleted as plant growth,with159 and 128 genera showing consistent significant increase and decrease from 2YV stage to 3YR stage,respectively.In contrast,less fungal genera were enriched as plant growth,and there were 82 in 438 fungal genera maintaing stable abundance.Enrichment and depletion of fungal genus for each growth stages compared to last sampling time point.The enrichment and depletion of bacterial and fungal communities occur actively at the 3YR stage.At the 3YR harvest stage,the root biomass swelled rapidly and accompanied with bacterial enrichment,including Dyadobacter,Dysgonomonas,Burkholderia,Delftia,Lechevalieria and Hyphomicrobium.Some fungal genera,e.g.Cladosporium,Epicoccum,Leptosphaerulina,Phoma,Exophiala,Fusarium,and Ilyonectria,were also enriched at the 3YR stage.(3)The variations in co-occurrence network structure of rhizosphere microbiomes across developmental stages of P.notoginsengThe MDS based on graphlet degree distribution distances shows that bacterial and fungal communities significantly determine the disparate networks(Anosim,R=0.97,P<0.001).The number of nodes and edges in both bacterial and fungal networks constructed from different developmental stages show an upward trend,and then a downward trend.Modularity and average path length of the rhizosphere bacterial and fungal networks decrease more than that of bulk soils.As the plants grow,modularity and average path length of the bacterial networks decrease slightly,while that of fungal networks increase.(4)The diversity,composition and function of P.notoginseng rhizosphere soil microbiome at harvest stageThe diversity,composition,and function of rhizosphere microbiome in the soils of P.notoginseng plants with diferent yields were investigated using metagenomic sequencing.The alpha diversity of the rhizosphere microbiome showed the difference in the sample sites.Shannon index at phylum,class,order,and family levels were higher in the soils of YS and QB than that in the soils of PBA,PBB,and PBC.PCo A was performed based on metagenomic sequencing using the Bray–Curtis metric to visualise the diference in microbial communities among soil samples,thereby revealing signifcant diference in the microbial community in the rhizosphere soils using adonis test analysis(Adonis:R~2=0.42,P=0.001).At the phylum level,the relative abundance of Actinobacteria was significantly higher in YS and QB than in PBA,PBB,and PBC.In total,1,330,812 genes were hit in the KEGG databases and were assigned to4436 KEGG orthology(KO)functional categories.The KOs were mainly involved in 6 KEGG level 1 pathways and 43 KEGG level 2 pathways.At KEGG level 1,the relative abundance of cellular processes,environmental information processing,genetic information processing,and human diseases were signifcantly higher in rhizosphere soils of PBA than PBB,PBC,YS,and QB(P<0.05).At KEGG level 1,the relative abundance of biosynthesis of other secondary metabolites,drug resistance,environmental adaptation,glycan biosynthesis and metabolism,metabolism of cofactors and vitamins,membrane transport,and signal transduction were signifcantly higher in rhizosphere soils of PBA than PBB,PBC,YS,and QB(P<0.05).The relative abundance of lipid metabolism was signifcantly lower in rhizosphere soils of QB than PBA,PBB,PBC,and YS(P<0.05).PCo A was performed based on KO functional categories using the Bray–Curtis metric,and an adonis test(Adnois:R~2=0.62,P=0.001)showed signifcant diference in sample sites.The KOs involved in known plant-microbe and microbe-microbe interactions,such as bacterial secretion systems,blagella assembly,bacterial chemotaxis,two-component systems,plant-pathogen interaction,and ABC transporters,showed high relative abundance in the rhizosphere soil of P.notoginseng with discrepant yields.(5)There was a correlation between rhizosphere soil microbiome and P.notoginseng yieldPearson correlation analysis was conducted between the abundance of rhizosphere microbial community and functional traits and the yield of P.notoginseng at harvest stage.At the genus level,one hundred and twenty-seven bacterial gerera were correlated with the yield of P.notoginseng(Pearson?s,|R|>0.5),of which 63 were positive and 64were negative.Acidothermus,Aquicella,Caenimonas,Crossiella,Hydrogenispora,Lysinibacillu,Sporichthya and Sporosarcina were significiantly correlated with P.notoginseng yields(P<0.05).Fifty-seven fungal genera were correlated with the yield of P.notoginseng(Pearson?s,|R|>0.5),of which 26 were positive and 31 were negative.Aspergillus,Leptosphaeria,and Preussia were significantly correlated with P.notoginseng yield(P<0.05).A total of 85 functional traits were significantly(P<0.05)correlated with P.notoginseng yields,among 12and 73 functional traits were positively and negatively correlated with yields,respectively.Functional traits,likely steroid biosynthesis and MAPK signaling pathway were positively correlated with P.notoginseng yields.In contrast,functional traits,such as bacterial secretion system,ABC transporters,metabolism of xenobiotics by cytochrome P450,and drug metabolism-cytochrome P450,were negatively associated with yields.Our data show that developmental stage is a significant driving force affecting the rhizosphere soil microbiomes,and succession characteristics of bacterial and fungal diversity were similar and parabolic patterns during the developmental stages,reflecting the adaptability of the plant microbial communities to the changes in the plant development.The variation of microbiomes is active at the 3YR stage.The transplantation of P.notoginseng reduces the stability of the network.Microbial community and functional traits are closely related to P.notoginseng yield at the harvest stage.