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Effects Of Rhizosphere/endophytic Microbial Communities And Growth-promoting Microorganisms On Host Growth And Metabolism Of Codonopsis Pilosula

Posted on:2024-08-26Degree:DoctorType:Dissertation
Country:ChinaCandidate:T T MengFull Text:PDF
GTID:1520307148973699Subject:Renewable Energy and Environmental Engineering
Abstract/Summary:PDF Full Text Request
As a low-cost succedaneum for Panax ginseng,Codonopsis pilosula is used for regulating immune and endocrine system,regulating blood glucose level,protecting cardiovascular system and nervous system,etc,and has gradually become a new hot spot in drug research and development.With the continuous application of various chemicals,the quality of C.pilosula decreased seriously.Plant growth-promotion bacteria can not only effectively promote the growth of crops,but also increase crop yield to a certain extent.Moreover,it also has the advantages of green,low-carbon,environmental protection,no pollution and pollution-free,which has been paid more and more attention.Therefore,it is very important to know the growth habit of C.pilosula and to explore a kind of functional bacterial fertilizer to promote the growth.Based on this,firstly,perennial C.pilosula root and rhizospheric soil samples were collected.The differences in growth and development,metabolite accumulation,antioxidant defense enzymes,metabolic enzyme activities,generation of signaling molecules,soil physicochemical factors,endophytic and rhizospheric microbial communities of C.Codonopsis in different years were carried out,respectively,to illustrate the growth habits and rules of C.pilosula,and clarify the driving effect of soil environmental factors on the microbial assembly and function of C.pilosula roots.Two growth-promoting bacterial strains were isolated from C.codonopsis roots,and identified as Pseudomonas fluorescens RHBA17and Geobacillu sp.RHBA19,respectively,based on polyphase taxonomic methods and molecular biology techniques.The growth promoting abilities of the two strains in different environments were evaluated.Meanwhile,the phylogenetic topology and key genetic characteristics were characterized by whole genome sequencing,and the growth promoting properties of the two strains were analyzed at the genetic level.The seeds were soaked with two kinds of bacterial agents and then pot experiment was performed to verify the microbial effects on the growth and development of C.pilosula,and to explore the antioxidant defense enzymes,metabolic enzyme activities,generation of signaling molecules and other properties of C.pilosula.The purpose of this study is to reveal the interaction mechanism between endophytes and plants,and to provide a theoretical basis for improving the yield and quality of C.pilosula.Specific research results were as follows:1.The root samples of C.pilosula with different growth periods were studied.The results showed that with time went by,the root length,weight and diameter increased,and also the contents of reducing sugar,chlorogenic acid and lobetyolin increased in C.pilosula.And it was noticed that the root growth and metabolite accumulation in the first two years were higher than those in the third year.The indexes of two-year-old sample were higher than those of one-year-old and three-year-old samples.The activities of phenylalanine ammonia lyase(PAL),sucrose synthase(SS)and sucrose phosphate synthase(SPS)in carbon and nitrogen metabolism enzymes were significantly higher than those in one-year-old and three-year-old samples.The contents of five signal molecules in the roots of C.pilosula displayed different trends,in which the content of superoxide anion(O2-)increased year by year,while the content of jasmonic acid(JA)and nitric oxide(NO)decreased year by year or decreased then stabilized,the content of salicylic acid(SA)showed a stable trend at first and then increased,and the content of H2O2 had no significant change.2.The rhizospheric and non-rhizospheric soil samples of C.pilosula collected in different years were analyzed.The results proved that except for organic matter(SOM),other physical and chemical factors were significantly different among the sample groups(P<0.05).Further analyses of the diversities of non-rhizospheric soil,rhizospheric soil and endophytic microbial community demonstrated that the number of microbial OTUs from the three different sources collected in the second year were higher than those collected in the first and third year.The richness of microorganisms in soil was higher than that of endophytic microorganisms,and the richness of fungi decreased with the sampling position varying from non-rhizospheric soil to root.The fitting curve analysis of soil nutrients and microbial dominant species showed that soil total nitrogen(TN)and soil phosphatase(PHP)were positively correlated with Fusarium oxysporum.The correlation analysis between C.pilosula and environmental factors confirmed that TN,available potassium(AK)and WC affected the bacterial community in non-rhizospheric and rhizospheric soil samples,while AK,available phosphorus(AP),TN,total phosphorus(TP),WC and SOM affected the root endophytic bacterial community,WC,total potassium(TK),TN,urease(SUC)and microbial biomass nitrogen(MBN)affected the fungal community.Symbiotic network analysis stated that the bacterial richness in the endosphere,non-rhizospheric and rhizospheric soils were the same.The results of partial least square path analysis(PLS-PM)denoted that soil physicochemical and enzymatic factors affected a series of physiological and biochemical processes such as growth,development and stress resistance of C.pilosula,and had significant effects on microbial community structure in rhizosphere and root.3.Two growth-promoting functional strains RHBA17 and RHBA19 were isolated from the roots of C.pilosula by plate marking method.The results of polyphasic taxonomy proved that RHBA17 and RHBA19 were classified as Pseudomonas fluorescens and Geobacillu sp.,respectively,based on the comparisons of phenotypic,biochemical characteristics and chemical compositions with the standard reference strains.Both strains had strong ability to fix nitrogen,combat drought,produce IAA,siderophores,xylanase and generate biofilm.Furthermore,they could still perform certain plant growth-promoting ability even under extreme conditions like low temperature(4℃),high temperature(45℃),high salt concentration(9%Na Cl),strong acid(p H 4.0)and strong alkali(p H 11.0)environments.In detail,the highest siderophores productivities of RHBA17 and RHBA19 were 36.78%and92.23%,the highest IAA productivities were 19.36 mg/L and 202.31 mg/L,and the strongest biofilm productivities were 1.18 and 2.25,respectively.4.The genes related to plant-bacterium interaction,biocontrol and plant growth-promoting traits of P.fluorescens RHBA17 and Geobacillus sp.RHBA19 were explored based on whole genome analysis.The annotations of GO and KEGG reflect the different emphasis of P.fluorescens RHBA17 and Geobacillus sp.RHBA19 in promoting growth.RHBA17 plays key roles mainly at the level of regulatory response and secondary metabolite synthesis,while RHBA19 has stronger primary metabolic ability and functions in enhancing carbon and nitrogen cycle.The annotations based on CAZy database displayed that both RHBA17 and RHBA19 contained rich carbohydrate-active enzymes,including degrading enzymes and redox enzymes.Most of the enzymes were reported to be beneficial to antagonizing pathogenic fungi,colonizing in plants and promoting plant defense,but there were significant differences in carbohydrate active enzyme components between RHBA17and RHBA19.The analysis of biosynthetic gene cluster revealed that RHBA17 has higher potential to produce secondary metabolites.Particularly,as to the biosynthesis of antibacterial products,RHBA17 and RHBA19 contain gene clusters for the synthesis of 2,4-diacetylphloroglucinol and progenin,respectively,with different antibacterial spectra.5.By employing potting technique,the growth and development,metabolite accumulation,antioxidant defense system,metabolic enzyme activities and generation of signal molecules of C.pilosula treated respectively with sterile water(CK),single culture(Geobacillu sp.RHBA19)(G)and mixed cultures(Geobacillu sp.RHBA19 and P.fluorescens RHBA17)(M)were analyzed and compared.The results found that compared with CK,group G and group M could significantly increase root length,root fresh weight,chlorophyll content and root activity.The root length in the treatments increased by 7.69%and 19.71%,the root fresh weight increased by 12.92%and 13.37%,the total chlorophyll content increased by 67.44%and 49.44%,and the root activity increased by 12.91%and35.70%,respectively.Both the treatments could promote the accumulation of polysaccharides and lobetyolin in various tissues of C.pilosula.Compared with CK,the content of polysaccharides in root,stem and leaf of group G increased by 19.1%,5.9%and 31.9%,respectively,and the content of lobetyolin increased by 58.4%,52.8%and 69.2%,respectively.Similarly,in group M,the content of polysaccharides increased by 13.3%,10.1%,39.1%,respectively,and the content of lobetyolin increased by 66.0%,95.5%,38.7%,respectively.In addition,both bacterial treatments increased the activities of antioxidant enzymes and the contents of non-antioxidant enzymes(CAT,SOD,POD,MDA),as well as the contents(P<0.05)of proline(Pro)and soluble proteins(SP)in various tissues of C.pilosula.In addition both bacterial treatments significantly increased the activities of enzymes related to carbon and nitrogen metabolism(sucrose synthase,sucrose phosphate synthase,acid converting enzyme,phenylalanine deaminase,and nitrate reductase),as well as the content of signaling molecules(NO and H2O2)in C.pilosula.
Keywords/Search Tags:Codonopsis pilosula (Franch.) Nannf., Microbial communities, Growth-promoting microorganisms, Whole genome, Pot experiment
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