Effects Of Rhizospheric And Rhizome Endophytic Microorganisms On Growth And Volatile Oil Accumulation Of Atractylodes Lancea Under High-temperature And Drought Stress

1 OBJECTIVEAtractylodes lancea is a perennial herb of the genus Atractylodes of the phylm Compositae.A.lancea is widely used as an ingredient of traditional Chinese clinical medicine.The volatile oil of A.lancea is considered to be the main active component to be accounted for its function of drying dampness,strengthening spleen,and its antibacterial and anti-inflammatory activities.A.lancea produced in the ‘Daodi’(geo-authentic)regions are of high medicinal values and high quality;the proportion of the four main volatile oil components contained in A.lancea is specific.Studies have shown that climatic factors play an important role in the quality of A.lancea in the geo-authentic regions.High temperature is one of the ecological restriction factors affecting the growth and development of A.lancea;precipitation is one of the major ecological factors affecting the composition of the volatile oil of A.lancea.In addition,some studies have shown that endophytic microorganisms also have significant regulatory effect on the composition and proportion of the four main volatile oil components of A.lancea;different microorganisms have differential regulatory effects on the components of the volatile oil.The microbe communities associated with medicinal plants vary due to geographical,climate factors and host plants.Hence,it can be deduced that the distinct microbial communities of the geo-authentic regions and the other regions may be an important biological factor causing the difference in the quality of A.lancea products.In order to explore the influence of the rhizospheric and endophytic microorganisms on the Daodi-ism and quality of A.lancea of different habitats,or under hightemperature stress or drought stress,in this research,comparative analyses on the microbial communities in the rhizosphere and in the rhizome of A.lancea from different habitats were performed.The core rhizome-associated microorganisms were screened and isolated from the rhizome of A.lancea from different habitats,and their biological functions were verified.The effects of soil microorganisms on the accumulation of the four major volatile compounds in A.lancea under high-temperature stress or drought stress were studied under laboratory contexts.2 METHODS2.1 Analyses on the differences of microbial communities in A.lancea rhizosphere and the rhizome endophytes of A.lancea from different habitats via high throughput sequencing and bioinformatics methodsThe conserved fragments of the rhizospheric and endophytic bacteria and fungi of A.lancea rhizome from different habitats were amplified using specific primers of bacterial 16 S and fungal ITS sequences,then put through highthroughput sequencing.The resulted microbial communities were compared to analyze the differential structure and species diversity of the core microbiomes of wild / cultivated A.lancea from different habitats,of different ecotypes and different chemotypes.PCo A and RDA/CCA were performed to analyze the environmental factors affecting the microbiome of A.lancea.The random forest model was used to detect the biomarker microbes of different A.lancea samples.Comparison of the microbial correlation networks of different A.lancea samples was performed via co-occurrence network analyses.Pearson correlation analyses were performed to identify the microbial genera associated with the accumulation of major volatile medicinal compounds.2.2 Isolation of the core endophytic microbe strains of A.lancea rhizome based on high-throughput sequencing and traditional microbial culturing methods,and confirmation of their functionsBased on the core endophytic microbial communities of A.lancea rhizome revealed via high-throughput sequencing,microbes of the core genera were targeted for isolation using the corresponding mediums.subsequently,the isolated endophytic microbe strains were used to inoculate A.lancea seedlings to verify their biological functions of promoting the growth,major volatile compound accumulation and inhibiting the pathogenic fungi.2.3 Studies on the effects of microorganisms on the growth and development of A.lancea and the composition of volatile oil under hightemperature or drought stressA.lancea seedlings inoculated with and without microorganisms were treated by high-temperature stress or drought stress in the laboratory,then,the phenotypes of A.lancea seedlings undergone different treatments were measured,and the concentration of mahor volatile compounds of the underground compartments of the A.lancea seedlings was detected via GC-MS.Thus,the effects of microorganisms on the biomass and the accumulation of the major volatile compounds of A.lancea were studied.Sequencing of 16 S and ITS amplicon was performed to analyze the differential rhizospheric and endophytic microbial communities of A.lancea root.Spearman/Pearson analyses were performed to reveal the correlations between microbial communities and accumulation of the four major volatile compounds.3 RESULTS3.1 The dominant factors affecting the microbial communities in the rhizosphere and rhizome of A.lancea from different habitats were differentThe rhizosphere bacteria of A.lancea from different habitats were mainly affected by abiotic factors such as the altitude,temperature and precipitation,and secondly by whether the host plant was wild or cultivated,and ecotype of the host.The difference of endophytic bacterial communities in the rhizome of A.lancea from different habitats was slight.The rhizosphere fungal communities of A.lancea from different habitats were mainly affected by whether the host plant was wild or cultivated,then by the altitude,temperature and precipitation.The endophytic fungal communities of A.lancea from different habitats were mainly affected by A.lancea ecotype.3.2 Specific bacterial and fungal communities were formed around the rhizome of A.lanceaAt the phylum level,the rhizosphere bacterial communities of A.lancea from different habitats were mainly consisted of the phyla Proteobacteria,Actinobacteria,Firmicutes,Acidobacteria and Chloroflexi,which varied due to the geographic distribution.Compared with the rhizosphere,the relative abundance of endophytic Bacteroidetes was higher,while the relative abundance of endophytic Acidobacteria and Chloroflexi was lower,forming specific endophytic bacterial communities.The rhizosphere fungal communities of A.lancea were mainly consisted of Ascomycota,Basidiomycota and unclassified＿k＿fungi;the major endophytic fungal communities were Ascomycota and unclassified＿k＿fungi.A.lancea had a core rhizome endophytic microbiome consisted of stably and specifically co-existing microbes,in which the core bacteria were of the genera Rhodococcus,Ralstonia,Burkholderia-caballeronia-paraburkholderia,Sphingomonas and Pseudomonas;while the core fungi were of the genus g＿unclassified＿p＿ascomycota.3.3 The core rhizospheric and endophytic bacterial and fungal communities varied between wild and cultivated A.lanceaThe relative abundance of the beneficial rhizospheric and endophytic nitrogen fixing bacteria genus Bradyrhizobium of wild A.lancea was higher than cultivated A.lancea.The relative abundance of the pathogenic fungal genera Fusarium and Paraphoma in the rhizosphere of cultivated A.lancea was higher than wild A.lancea;the beneficial fungal genera Trichoderma and Penicillium were present in the rhizosphere of the wild but not the cultivated A.lancea.In addition,there were more complex negative interactions in the cooccurrence network of the rhizospheric bacterial and endophytic fungal communities of the cultivated A.lancea,which could likely disturb the stability of the communities.This might be an important factor that causes the outbreak of diseases and pests on cultivated A.lancea.3.4 The biological functions of the microbe strains isolated from A.lancea rhizome were variedA total of 128 bacterial strains and 22 fungal strains were isolated from the rhizome of A.lancea,including some core rhizome-associated microorganisms.The biological functions of different microbe strains were varied.Endophytic bacteria had strong biocontrol effect on A.lancea pathogenic fungi,and could promote the accumulation of hinesol and β-eudesmol.Endophytic fungi had a strong promoting effect on the growth of A.lancea,and could promote the accumulation of atractylon and atractylodin.3.5 Microorganisms improved the tolerance of A.lancea to hightemperature or drought stressHigh-temperature or drought stress inhibited the growth of A.lancea and affected the composition of microbial communities composition in the rhizosphere and root of A.lancea plantlets.Soil microorganisms could promote the growth of A.lancea root and the accumulation of major volatile compounds,and improve the tolerance of A.lancea against abiotic stress.3.6 Exogenous microorganisms and the selectivity of A.lancea root had great impact on its rhizospheric and endophytic bacterial communitiesThe rhizospheric and endophytic bacterial communities of A.lancea under high-temperature stress was mainly affected by exogenous microorganism inoculation and the selectivity of roots,then by the high-temperature stress.Rhodococcus,Ralstonia and Burkholderia-caballeronia-paraburkholderia were enriched in A.lancea root,which consisted with the findings in cultivated A.lancea.3.7 Drought indirectly inhibited the accumulation of major volatile compounds by changing the rhizospheric and endophytic microbial communities of A.lanceaDrought stress indirectly inhibited the concentration of the major volatile compounds by affecting the composition of rhizospheric and endophytic microbial communities of A.lancea,indicating that the composition of volatile components of A.lancea was jointly regulated by biotic and abiotic factors,in which microorganisms played a very important connecting role.4 CONCLUSIONSThis study revealed differential rhizospheric and endophytic microbial communities of A.lancea from different habitats.The dominant factors causing the differences of microbial communities in the rhizosphere and rhizome of A.lancea were different,and the biological functions of microorganisms isolated from the rhizome of A.lancea were varied.Endophytic bacteria had strong biocontrol effect on A.lancea pathogenic fungi,and could promote the accumulation of hinesol and β-eudesmol.Endophytic fungi had a strong promoting effect on the growth of A.lancea,and could promote the accumulation of atractylon and atractylodin.Under high-temperature or drought stress,microorganisms could improve the tolerance of A.lancea to abiotic stress,promote the growth of A.lancea and the accumulation of major volatile compounds.Drought stress could indirectly inhibit the accumulation of major volatile compounds in A.lancea root by affecting the microbial communities.The above findings added to the advances of researches on the A.lancea microbiome,and revealed the patterns of effects of the microorganisms on the volatile compound accumulation in A.lancea.These results provided scientific guidance for the application of microorganisms in A.lancea agriculture.