| In the soil ecological environment,plant roots could communicate and exchange substances with soil microorganisms through continuously releasing an enormous range of potentially valuable compounds into rhizosphere.And recently the field of plant biology has recognized the importance of root exudates in mediating these biological interactions including parasitism,symbiosis,infection,antagonism.In addition,plant root exudates could serve as chemical signal substances to regulate the activity of soil pathogenic microorganisms and induce the occurrence of soil-borne diseases through changing the soil micro-ecological environment.Therefore,explicating the relationship between root exudates and pathogenic microorganisms,and among plant,soil pathogenic microorganisms and micro-ecology mediated by root exudates is crucial for understanding the occurrence of soil borne diseases and the development of new green techniques.Bacterial wilt disease caused by Ralstonia solanacearum remains one of the most serious soil-borne diseases in tobacco cultivation and thereby seriously threatens the production of tobacco in China,and is also one of the most important factors in successive cropping obstacle.In recent years,based on triangle disease theory,the reason of tobacco bacteria wilt is almost always attributed to the R.solanacearum,tobacco plants and environmental factors.However,the role of plant biological characteristics in the occurrence of disease was neglected.So,to explore the role of root exudates in the triangle of disease was important to deeply understanding the cause of tobacco bacteria wilt.And it were also important to forming new ideas of prevention and developing green prevention and control.Organic acids represent dominant compounds of plant root exudates.They play an important role in the establishment and functioning of soil ecosystem in rhizosphere.And some organic acids are important semiochemicals in communication between plant and soil environment,and also can be served as growth factors and nutrient source for mostly microbes.But long-term releasing of some allelochemical organic acids could stimulate pathogenic microorganisms to propagate,and decrease soil immunity.Moreover,organic acids could alter the composition of microbial community and lead to the outburst of bacterial wilt.Over the past decade,although the relationship among organic acid,pathogenic microorganisms and some diseases has been reported,the research on interactions between organic acids from tobacco root exudates and R.solanacearum,and whether these organic acids could induce bacteria wilt are less studied.Accordingly,mechanism and experimental investigation on organic acids and R.solanacearum are conducted in this dissertation.Firstly,organic acids constituents of tobacco root exudates were identified and analyzed by using GC-MS.Furthermore,from the individual angle of R.solanacearum,several allelochemicals organic acids incuding cinnamic acid,lauric acid,phthalic acid,myristic acid,fumaric acid andbenzoic acid,have been individually assessed for their effects on the growth,swarming ability,chemotactic response and biofilm formation of R.solanacearum.The function of organic acids in the recruitment of R.solanacearum was evaluated under gnotobiotic conditions.In addition,from the perspective of soil microbial community,this dissertation showed the effect of tobacco root exudates and organic acids on soil microbial ecology and bacterial wilt based on the relationship among root secretion,soil microorganism and pathogenic microorganisms.Finally,this dissertation demonstrated the micro-ecological mechanism of soil immune and the occurrence of bacteria wilt under influence of tobacco root exudates and release of organic acids.1 Extraction and identification of organic acids compositions of root exudates from tobaccoIn order to study the organic acids compositions of root exudates from tobacco,the root exudates of tobacco Yunyan 87 transplanted about 40 days were collected by sand culture.Then the products of these fractions including acid,neutral,alkaline conditions were organic acids,esters and alkanes by using GC-MS,among which a total of 26 organic acids were identified.The highest levels of organic acids were found in acidic components,which contained 21 organic acids mainly including fumaric acid,succinic acid,3-hydroxy butyric acid,caproic acid,valeric acid,benzoic acid,cinnamic acid,myristic acid,palmitic acid,acetic acid,2-amyl alcohol pentadecane,2-acrylic acid,palmitic acid,3-hydroxy-4-benzoic acid,stearic acid,erucic acid.Moreover,esters and alkanes compounds were also detected in alkaline and neutral components,which contained 7 esters and 30alkanes.Esters compounds mainly includes benzoic acid ethyl ester,dimethyl phthalate,dioctyl adipate,2-butyl acrylate,butyl acetate,butyl acetate,butyl phthalate ester,ethyl butyrate,ethyl phthalate.Alkanes compounds mainly includes nonane,cyclohexane,cyclopentane,?E?-3-Dodecene,2,6,10-trimethyl-Dodecane,1-Pentadecene,?Z?-4-Tetradecene,1-Nonadecene.2 Determination of the R.solanacearum growth rate and biofilm formation in organic acidsThe growth rates of R.solanacearum were measured under different concentration conditions at12 and 24 h post incubation?hpi?,and the results showed that R.solanacearum grew in M63 and NB medium when supplemented with different concentrations of 50-200?M organic acids.At 12 and 24hpi,the growth of R.solanacearum with all of the treatments was similar,and no significant differences were observed,suggesting that these organic acids had no effect on the growth of R.solanacearum.To investigate the metabolic response of R.solanacearum to organics,the persistent levels of cinnamic and myristic acid in M63 and NB medium were determined by HPLC and GC.The results showed that the levels of cinnamic and myristic acids changed only slightly throughout the rest of the incubation period.The retention rates of cinnamic and myristic acids were 87-95%and 85-90%12,24 and 36 hpi,respectively.These results confirmed that these organic acids cannot serve as carbon sources for R.solanacearum.Crystal violet staining in 96-well microtiter plates.and RT-PCR was used for the quantification of biofilms and the expression of relative genes.Twenty-four hours after inoculation,benzoic acid,myristic acid,cinnamic acid and fumaric acid could significantly increase the biofilm biomass.What is more,biofilm biomass was relevant to the concentration of four organic acids.Benzoic acid and myristic acid at 50 to 200?M significantly increased the biofilm biomass,showing enhancements of60%-116.70%and 55.30%-61.40%compared with the control.In addition,100-150?M cinnamic acid and fumaric acid also could promote the biofilm biomass.We further investigated the expression of biofilm-related genes following the addition of organic acids.The results showed that the expression of the biofilm-related genes epsE,lecM,and benzoic acid at 50 to 150?M markedly induced the expression of epsE,lecM,with increases of 1.75,5.38,4.88-fold and 4.25,1.48,1.97-fold compared with the control.Moreover,200?M fumaric and 100?M myristic acid also markedly induced the expression of genes epsE,with increases of 4.13-5.25-fold compared with the control.Overall,benzoic acid,myristic acid,cinnamic acid and fumaric acid could be severed as signaling chemicals for biofilm and promote the higher mRNA expression of the motility-related genes epsE and lec M in some concentrations.3 Effect of organic acids on the characteristic of interaction between R.solanacearum and tobacco3.1 Effect of organic acids on the infection characteristic of R.solanacearumThe swarming assay and a modified capillary assay was performed to assess R.solanacearum swarming motility in the presence of organic acids.The results showed that all six organic acids at concentrations ranging from 50 to 200?M induced swarming motility.At various concentrations,myristic acid exerted marked effects on swarming motility,and the motility diameters obtained at concentrations ranging from 50 to 200?M reached nearly 4.22-,4.25-,4.37-and 3.89-fold higher values compared with those of the control,respectively.Furthermore,cinnamic and fumaric acids also markedly stimulated motility compared with the other treatments.Chemotaxis of R.solanacearum induced by organic acids were qualitatively and quantitatively analyzed.The results demonstrated that all six organic acids could induce chemotaxis at 150?M.Cinnamic acid,myristic acid and fumaric acids,at a concentration of at 150?M,significantly attracted R.solanacearum into capillary tubes,and the resulting populations of R.solanacearum were significantly higher than those obtained with the other treatments,reaching values of 7.07×107 CFU m L-1,6.77×107 CFU mL-1and 5.70×107 CFU m L-1 respectively.In addition,Cinnamic acid,myristic acid and fumaric acids also could up-regulate the expression of the chemotaxis-related genes cheA,cheW,and cheY and the motility-related genes motA,motB,and filA,which was in line with the result determined by capillary assay.Thus,we subsequently evaluated the effects of these organic acids on R.solanacearum tobacco root colonization under hydroponic conditions.Among the six organic acids,myristic and cinnamic acids more significantly increased the population of R.solanacearum that colonized the roots of tobacco plants and myristic acid and cinnamic acidshowed an apparent effect on recruitment ability,resulting in 18.67-15.08-fold greater colonization than the control treatment.Overall,Cinnamic,myristic and fumaric acids could be served as semiochemical attractants to induce the colonization and infection of R.solanacearum.3.2 Effect of organic acids on the pathogenesis of tobacco bacterial wiltWe determined the effects of organic acids on bacterial wilt disease progression through an inoculation assay under climate-controlled conditions and field.The result showed that organic acids could accelerate disease progression on tobacco in pot experiment.Fourteen days after inoculation,tobacco plants were treated with myristic acid,cinnamic acid and fumaric acid at a concentration of150?M significantly reduced the survival rate by 10%,20%and 20%,respectively,which is significantly lower than that of the control plants 50%.Then the survival rate of tobacco plant treated with myristic acid and cinnamic acid decreased to 10%after inoculated eighteen days.Moreover,based on the disease severity?DS?and disease index?DI?,the AUDPCs obtained with the myristic acid,cinnamic acid and fumaric acid treatments were significantly higher than those of the control,with DS values of 583.34,488.23 and 486.65,respectively,and DI values of 476.25,420.41and 391.25,respectively.In field experiment,myristic acid,cinnamic acid and fumaric acid treatments could promote the occurrence degree of bacteria wilt,most notably cinnamic acid.And the root colonization of R.solanacearum reached 1.07×104 CFU/g after treated by cinnamic acid in early infected stage,which almost twice time than that of control.Then,incidence and disease index of tobacco plants after treated thirteen and nineteen by cinnamic acid were reached to 21.19%,3.01and 29.42%,6.40 respectively,which significantly higher than control 10.55%,1.32 and 17.68%,3.67 respectively.Therefore,myristic acid,cinnamic acid and fumaric acid could promote the root colonization of R.solanacearum and accelerate the early infection and increase the incidence of the disease,particular cinnamic acid.4 The mechanism of TRE-OA degradation by soil microbeThe dynamic changes of myristic acid,cinnamic acid in soil environment were determined by exogenous applications of these organic acids.And the results demonstrated that degradation of myristic acid,cinnamic acid were reached over 94%,95%and 84%,93%,which were cultivated 14days in natural soil.However,persistent levels of myristic acid,cinnamic acid were reached much higher than 80%and 60%in sterile soil.These results showed that some soil microbe could efficiently degrade TRE-OA and prevent the accumulation of organic acids in soil.Moreover,the study further identified and characterized the function of Trichoderma harzianum T-22 in degrading cinnamic acid.And the result demonstrated that T.harzianum could efficiently degrade cinnamic acid at a concentration of 50-150?M,and degradation rate was 100%in PDB culture medium.And the severity of tobacco bacterial wilt was decreased after application of T.harzianum.Sixteen days after inoculation,the incidence and disease index of tobacco plants treated by T.harzianum were significantly reduced by 27.22%and 25.52 on the average respectively,compared to the treatments with cinnamic acid?75?g/g,150?g/g?.5 Effect of tobacco root exudates on soil microbial communityPersistent levels of myristic acid,cinnamic acid were determined at incubation in soil.The result showed that myristic acid,cinnamic acid were not persistent,and decomposition speed of myristic acid and cinnamic acid was quickly at incubation 3-7 days.Then effects of tobacco root exudates on soil microbial communities were determined by using the technology of 16S rDNA.The result showed that the tobacco root exudates selectively inhibited or stimulated certain communal bacterial,actinomycetes and fungal species,with decreasing a relative abundance of the bacterial taxa Actinobacteria and Sphingomonadaceae and increasing a relative abundance of Acidobacteria,Acidobacteriaceae compared and analyzed from different levels of phylum,family separately.Relative abundance of Gemmatimonas reached highest level cultured for 7 days.Furthermore,dominant bacterial community after tobacco exudates treatment was analyzed by using Lefsize.And along with increase of tobacco root exudates and incubation time,the population of dominant soil microbial upgraded firstly and then descending.The population decreased in the following order:7d?13?>14d?6?>3d?5?>28d?4?>0d?3?.Moreover,Biolog-ECO was adopted to determine the functional diversity of soil microorganisms.The result showed that soil microorganisms could significantly utilize carbon sources after incubated 7d treated by root exudates,especially amine and polymer compounds.6 Microbial impacts of bacteria wilt influenced by cinnamic acid in the rhizosphere of tobaccoLaboratory experiment and pot experiment were conducted to investigate effects of cinnamic acid treatments on occurrence of tobacco bacteria wilt and rhizosphere microbial communities by adding exogenous organic acid.The results showed that cinnamic acid?75?g/g,150?g/g?also deduced the diversity and richness of microbial fungal community,and domiant genus fell to 6 kinds from 14 kinds.In addition,the proportion of Fusarium was increased to 79.93%and 69.96%.Furthermore,cinnamic acid changed the structure and composition of rhizosphere bacterial and fungal communities with increasing the relative abundances of fugal taxa Nectriaceae,Pseudeurotiaceae,Hypocreaceae,Dothideomycetes,Aspergillus,Phoma,Fusarium and the relative abundances of bacterial taxa Proteobacteria,Acidobacteria,Burkholderiaceae,Ralstonia and with decreasing the relative abundances of bacterial taxa Actinobacteria,Thaumarchaeota.In addition,cinnamic acid significantly decreased microbial activity?AWCD value?and functional diversity,in particularly phenolic acid,carboxylic acid,amine and other carbon sources.Further pot experiment by using the soil treated by cinnamic acid demonstrated that cinnamic acid?75?g/g,150?g/g?decreased soil immune activity and aggravated the occurrence degree of disease.So,cinnamic acid could disrupt the balance of microbial populations,increasing the abundance of pathogenic microorganisms,decreasing the proportion of beneficial microorganisms and leading to the occurrence of bacterial wilt.To sum up,from the perspective of interaction of R.solanacearum and tobacco,the effect of organic acids on the characteristic of interaction between R.solanacearum and tobacco was analyzed.And this dissertation confirmed that cinnamic,myristic and fumaric acids could serve as semiochemical attractants,rather than nutrition to induce the colonization,biofilm formation and infection of R.solanacearum.Furthermore,from the interactions with organic acids and soil microorganism,we elucidated that tobacco root exudates and organic acids could significantly change the microbiological structure and functional characteristics,caused the imbalance of beneficial and pathogenic microorganisms,decreased microbial activity?AWCD value?,in particularly carbohydrate,amine and other carbon sources,and lead to the occurrence of bacterial wilt.We also demonstrated that soil microbe could efficiently degrade TRE-OA and prevent the accumulation of organic acids in soil.Furthermore,this dissertation elucidated that cinnamic acid and T.harzianum were two key factors,which deeply affected the occurrence of bacterial wilt.On one hand,cinnamic acid could served as semiochemical attractants to induce R.solanacearum and changed the microbiological structure,deceased the abundance of beneficial bacteria in soil and aggravated the occurrence degree of disease.One the other hand,T.harzianum maintained dynamic equilibrium between the releasing of organic acid and microbial community stability by degradation of soil organic acids,and decreased the occurrence degree of disease.Consequently,the conclusion of this dissertation is important for deeply understanding the role of organic acids in the occurrence of bacteria wilt and also contributes to form new ideas on developing green and bio-control techniques.More importantly,our study lays a theoretical foundation for control bacteria wilt and plant health maintained based on micro-ecological theory. |
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