| Atractylodes lancea,a member of the genus of Atractylodes of the Composite family,is a perennial root herb.The dry rhizome of A.lancea is a famous medicine known as Cangzhu,which can expel wind,clear cold,eliminate dampness,and strengthen spleen.Cangzhu can treat abdominal fullness and distention,diarrhoea,edema,rheumatism,and digestive disorders.The quality of A.lancea strongly depends on the area in which the herb is produced,and A.lancea grown in the Maoshan area of the Jiangsu Province is the geo-authentic herb.The active pharmaceutical ingredients in A.lancea are volatile oils,including sesquiterpenoids(?-caryophyllene,zingiberene,P-sesquiphellandrene,caryophyllene oxide,hinesol,?-eudesmol,and atractylone)and polyacetylenes(atractylodin).In recent years,the geo-authentic A.lancea has become endangered due to habitat destruction and overexploitation as well as the low rate of fruit setting and low growth rate of rhizome.However,the market demand for A.lancea is growing gradually,leading to the increasing gap between supply and demand.Although cultivation can relieve the pressure on the demand of wild herb,the cultivation of A.lancea usually faces several problems,such as plant diseases and continuous cropping obstacles.Furthermore,the content and diversity of active pharmaceutical ingredients in cultivated A.lancea is considerably decreased compared to that of the wild herb.The changes of active pharmaceutical ingredients in medicines will result in the decreased medicinal efficacies of existing Chinese patent medicines and causes tremendous impact on the traditional Chinese medicine industry,which is a main problem in Chinese medicine cultivation.Our previous studies have shown that there are rich resources of endophytes in A.lancea and several endophytic fungi can promote the volatile oil accumulation in planta and improve the medicine quality.However,the promoting efficiencies of reported endophytes on the volatile oil accumulation in A.lancea cannot meet the requirement of medicine cultivation.Because of the strong antibacterial activity of volatile oils,we suppose whether there are endophytic bacteria in A.lancea.Moreover,the effects of endophytic bacteria on the growth and volatile oil accumulation in A.lancea are worthy to be studied.Therefore,this paper has isolated and screed out an endophytic bacterium ALEB7B by comparing with reported endophytes.Endophytic bacterium ALEB7B has been identified as Pseudomonas fluorescens,which can increase the volatile oil content in A.lancea by 78%.A.lancea contains diverse volatile oil components and P.fluorescens ALEB7B can efficiently enhance the volatile oil accumulation.Therefore,the interaction between A.lancea and P.fluorescens ALEB7B is a good model to elaborate the mechanisms underlying the endophytic bacteria enhancing the plant ecological adaptability and promoting the secondary metabolite accumulation in host plant from multiple angles.Firstly,we isolate endophytic bacteria from the geo-authentic A.lancea grown in the Maoshan area of the Jiangsu Province.In order to analyze the diversity of endophytic bacteria in A.lancea,the obtained endophytic bacteria are subjected to Amplified Ribosomal DNA Restriction Analysis(ARDRA)and BOX-PCR.Based on the ARDRA fingerprints,the 45 subculturable endophytic bacterial strains are classified into 14 clusters.The BOX-PCR is used to study the diversity of the endophytic bacteria in the main ARDRA cluster,showing that the endophytic bacteria in the same cluster are not same,indicating a broad genetic diversity in the same genera.The 16S rRNA fragments of representative strains in different clusters are sequenced and analyzed,showing that the isolated endophytic bacteria belonged to Pantoea,Microbacterium,Curtobacterium,Agrobacterium,Pseudomonas,and Bacillus.Among them,Pseudomonas was the most dominant genera.After being screened on specific media,ten endophytic bacteria can grow normally on Burk's medium,indicating their capacity to fix nitrogen.Nineteen and fifteen bacteria can respectively grow normally on National Botanical Research Institute Phosphate and Mengjinna Organic Culture Medium,indicating their capacity to dissolve insoluble calcium phosphate and lecithin.Twenty-four bacteria can grow on silicate medium,indicating their capacity to dissolve insoluble potassium feldspar.Forty-three endophytic bacteria can produce and release indole acetic acid(IAA)from tryptophan.This part firstly reveals the diversity of endophytic bacteria in A.lancea and their plant growth-promoting potentials.Then,we screened out an endophytic bacterium ALEB7B from the strains isolated in the previous part,using the reported endophytes that can promote the volatile oil accumulation in A.lancea as standards,including Gilmaniella sp.AL12,A.strictum AL16,and Acinetobacter sp.ALEB16.According to the sequence alignments of 16S rRNA,rpoD,and gyrB genes,endophytic bacterium ALEB7B is identified as P.fluorescens.P.fluorescens ALEB7B can secrete IAA.After the inoculation of P.fluorescens ALEB7B,the endogenous IAA content in A.lancea significantly increases,indicating that the IAA secreted by P.fluorescens ALEB7B may participate in inducing the volatile oil accumulation in planta.Then we confirm the influences of exogenous IAA on the volatile oil accumulation in A.lancea.As the concentration of IAA increasing,the root number of A.lancea plantlets increases.When the concentration is 0.5 mg L-1,IAA significantly promotes the volatile oil accumulation in A.lancea.This result indicates that P.fluorescens ALEB7B may promote the plant root development and increase the nutrient uptake,which will provide the material and energy basis for plant secondary metabolite synthesis.Moreover,1-naphthoxyacetic acid,the specific inhibitor of IAA influx carrier AUX1/LAX on plant cell membrane,reverses the increased accumulations of IAA and volatile oils induced by bacterial inoculation,indicating that the IAA secreted by P.fluorescens ALEB7B is absorbed by plants through AUX1/LAX and then promotes the plant root development,increases the material and energy accumulation,and promotes the plant secondary metabolism.Thirdly,we study the influences of P.fluorescens ALEB7B on ecological adaptability of A.lancea.P.fluorescens ALEB7B can antagonize Athelia rolfsii SY4,which causes the southern blight of A.lancea.The inoculation of P.fluorescens ALEB7B can significantly decrease the morbidity of southern blight in A.lancea.In situ observation using scanning electron microscopy shows that P.fluorescens ALEB7B can steadily colonize in A.lancea.And the carbon and nitrogen source utilizations of P.fluorescens ALEB7B are similar with A.rolfsii SY4.Therefore,P.fluorescens ALEB7B can decrease the colonization rate of A.rolfsii SY4 in planta by the niche competition.It is noteworthy that this part firstly reports that P.fluorescens ALEB 7B can release abundant volatiles that can inhibit the growth of A.rolfsii,when it is grown on the Luria-Bertani medium.Among the bacterial volatiles,the dimethyl disulfide plays the essential roles in antagonizing A.rolfsii SY4.Moreover,the cell-free fermentation broth of P.fluorescens ALEB 7B can also inhibit the growth of A.rolfsii SY4,among which the 2-piperidone plays the essential roles.Other strategies used by P.fluorescens ALEB7B antagonizing the A.rolfsii SY4 include the production of antibiotics(such as 2,4-diacetylphloroglucinol),the secretion of cell wall-degrading enzymes(such as amylase,xylanase,cellulase,and pectinase),and the secretion of siderophore.This part provides an antagonistic bacterium with practical convenience and ecologically amity,which can be used in the disease control in cultivation of A.lancea.Because P.fluorescens ALEB7B can release abundant volatiles that have pathogen antagonistic ability,we propose that volatiles released by P.fluorescens ALEB7B may affect the growth and volatile oil accumulation in A.lancea.This part demonstrates that P.fluorescens ALEB7B can release several nitrogenous volatiles,such as formamide and N,N-dimethyl-formamide,which significantly promote the growth of non-infected A.lancea.And the bacterial nitrogenous volatiles cannot be detected in the A.lancea-P.fluorescens symbiont,indicating the nitrogenous volatiles or their precursors may have been consumed by the host plant.Moreover,the main bacterial volatile benzaldehyde significantly promotes volatile oil accumulation in non-infected A.lancea via activating plant defense responses.And the benzaldehyde can be detected in the A.lancea-P.fluorescens symbiont,which can affect the uninfected plants around.This part firstly demonstrates that the interaction between plant and endophytic bacterium is not limited to the commonly known physical contact,extending the ecological functions of endophyte in the phytosphere and deepening the understandings about the symbiotic interactions.Previous parts demonstrate that P.fluorescens ALEB7B can produce various metabolites,including exoenzymes,antibiotics,and volatiles.All these metabolites have potential to be bacterial elicitors.Moreover,P.fluorescens is the gram negative bacterium,which can synthesize and secrete several protein elicitors.This part demonstrates that extracellular protein and polysaccharide elicitors can significantly induce the expression of HMGR gene and promote the sesquiterpenoid production in A.lancea.Further study demonstrates that bacterial elicitors can induce the burst of reactive oxygen species(ROS)in planta,therefore,we speculate that the burst of ROS induced by P.fluorescens ALEB7B and its elicitors can oxidize the sesquiterpenoids.Because the elicitors cannot increase the synthesis precursors of sesquiterpenoids,they only induce the conversions among different sesquiterpenoid components.However,P.fluorescens ALEB7B can increase the contents of sesquiterpenoid synthesis precursors,such as acetyl coenzyme A and pyruvic acid.Therefore,ALEB7B can simultaneously increase the accumulations of oxygen-free and oxygenous sesquiterpenoids.Furthermore,the extracellular protein and polysaccharide elicitors induce accumulations of different oxygenous sesquiterpenoids,indicating that different bacterial elicitors may activate different signaling pathways in inducing the volatile oil accumulations in A.lancea.Identification of components shows that bacterial extracellular polysaccharide is composed of mannose,glucose,and galactose.However,mannose,glucose,and galactose all have no effects on the volatile oil accumulation in A.lancea,indicating that there might be other components in bacterial extracellular polysaccharide that can induce the sesquiterpenoid synthesis.After isolation and purification,a solute-binding protein is obtained from bacterial extracellular protein,which can significantly promote the accumulations of caryophyllene oxide,hinesol,?-eudesmol,and atractylone.This part demonstrates that the volatile oil accumulation in A.lancea efficiently promoted by P.fluorescens is the synergistic effects of multiple elicitors.And different bacterial elicitors may activate different signaling pathways and induce different sesquiterpenoid component syntheses.This part reveals another mechanism underlying P.fluorescens efficiently promoting the volatile oil accumulation.To further demonstrate whether the ROS induced by P.fluorescens ALEB7B and its elicitors can directly oxidize sesquiterpenoids,we conduct the follow-up experiments.After the colonization of P.fluorescens ALEB7B,oxidative burst and oxygenous sesquiterpenoid accumulation in A.lancea occur synchronously.Treatment with exogenous hydrogen peroxide(H2O2)or singlet oxygen induces oxidative burst and promotes oxygenous sesquiterpenoid accumulation in planta.Conversely,pretreatment of plantlets with the ROS scavenger ascorbic acid significantly inhibits the oxidative burst and oxygenous sesquiterpenoid accumulation induced by P.fluorescens ALEB7B.In vitro oxidation experiments show that several oxygenous sesquiterpenoids,such as hinesol,caryophyllene oxide,humulene epoxide II,and spathulenol,can be obtained from direct oxidation caused by H2O2 or singlet oxygen.Summarily,this part demonstrates that endophytic bacterium-induced ROS can directly oxidize oxygen-free sesquiterpenes and increase the oxygenous sesquiterpenoid content and diversity in A.lancea,indicating the formation of some oxygenous sesquiterpenoids results from direct oxidation by ROS in plants after they encountering biotic of abiotic stresses.This part not only provides a novel viewpoint on the mechanisms of diverse oxygenous terpenoid syntheses in multiple medicinal plants,but also is essential to further understand interactions between endophytes and plants.Finally,ROS induced by endophytic infection not only can directly oxidize the sesquiterpenes,but also can act as signal molecules and then activate the downstream hormone signaling,which can promote the volatile oil accumulation in A.lancea.This part demonstrates that P.fluorescens ALEB7B firstly activates H2O2 signaling inA.lancea,which then simultaneously activates gibberellin(GA)and ethylene(ET)signaling.Subsequently,ET activates abscisic acid(ABA)signaling.GA and ABA increase the expressions of the HMGR and DXR genes,which encode key enzymes involved in sesquiterpenoid biosynthesis,leading to the increased activities of corresponding enzymes and then the accumulation of volatile oils.Specific ROS and hormone signaling cascades induced by P.fluorescens ALEB7B may contribute to high-efficiency volatile oil accumulation in A.lancea.Illustrating the regulation mechanisms underlying P.fluorescens ALEB7B-induced oxygenous sesquiterpenoid biosynthesis not only provides the theoretical basis for the inducible synthesis of terpenoids(such as artemisinin,paclitaxel,menthol,and glycyrrhizin)in many medicinal plants,but also further reveals the complex and diverse interactions among different plants and their endophytes.In conclusion,this paper uses the interaction between A.lancea that contains rich volatile oil compositions and P.fluorescens ALEB7B that can efficiently enhance the volatile oil accumulation in planta as the model,elaborating the mechanisms underlying the endophyte enhancing the plant ecological adaptability and promoting the secondary metabolite accumulation in host plant from multiple angles.This study provides not only theoretical bases for using endophytes in medicinal material production,but also potential technical supports for cultivation of high-quality medicines. |
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