| Recently,fusarium wilt disease,which is caused by Fusarium oxysporum f.sp.cucumerinum J.H.Owen(FOC),has lead to worldwide decrease of cucumber production.A plant growth promotion rhizobacteria Bacillus amyloliquefaciens SQR9 has been isolated from cucumber rhizosphere for the inhibition of FOC and growth promotion of cucumber plants.It is generally agreed that root exudates play important roles in processes in root-soil interactions.What is more,they might act as signals and mediate the communications between B.amyloliquefaciens SQR9,FOC and cucumber.It has been reported that low molecular organic compounds in root exudates are more likely to be signals involved in microbe-root interactions.In this study,we focused on the tripartite interaction between SQR9,FOC and cucumber and investigated the effects of inoculation of different microbes on root exudates composition by high throughput gas chromatography-mass spectrum analysis.Correlation between colonization and root exudates composition was calculated and the finaly results were verified by experiment.In an addition part,research on effect of SQR9 inoculation on cucumber root exudates showed that tryptophan in root exudates might be a major factor contributing to growth promotion.Results were summarized as follows.Root colonization of Bacillus amyloliquefaciens SQR9 was increased upon FOC infection.Analysis of the low molecular organic acid compounds in root exudates showed that citric acid and fumaric acid in the root exudates of cucumber infected by FOC were much higher than that of healthy cucumber.Further experiments indicated that citric acid was a chemoattractant of SQR9,while fumaric acid showed positive effect on biofilm formation.These results indicated that citric acid and fumaric acid should process positive effect in colonization of SQR9.Addition of B.amyloliquefaciens SQR9 with tryptophan as inoculant to the cucumber rhizosphere showed stronger promotion effect than SQR9 alone,suggesting that tryptophan could be an enhancer for growth promotion.By using a new designed split-root system,it was found that the secretion of tryptophan by cucumber root was strongly enhanced upon SQR9 inoculation,while transcriptional levels of the genes related to IAA synthesis of SQR9 on root of cucumber pre-inoculated by SQR9 were increased compared with that of control cucumber.Especially,the transcription of yhcX,a gene coding nitrilase that involved in IAA biosynthesis via indole-3-acetonitrile,was substantially increased to 20-fold higher.Growth of SQR9 was significantly increased by addition of exogenous root exudates from both regular cucumber and pre-inoculated cucumber;however,root exudates from pre-inoculated cucumber significantly stimulated IAA production of SQR9 but the regular root exudates did not.When the regular root exudates were modified by addition of external tryptophan to the concentration as pre-colonized cucumber root exudates,it exhibited similar stimulation of IAA production of SQR9 as the latter.In this part,results illustrated that the growth promotion effect on cucumber by SQR9 was not a unilateral biosynthesis of IAA,but a mutually beneficial interaction upon signal exchange.However,inoculation of the mutant(△yhcX)of SQR9,in which the yhcX was deleted,still showed growth promotion effect on cucumber,but lost the response to exogenous tryptophan.Therefore,other pathways independent on tryptophan or even IAA production were supposed to be involved.Anthranilate synthase play critical role in tryptophan synthesis in plant.Based on the blast results against PlantGDB,a specific tryptophan transporter was identified in cucumber but not in any other plants.Real-time PCR results showed that inoculation of SQR9 led to strong up-regulation of the tryptophan-specific transporter but not the anthranilate synthase.It was supposed that this transporter might be involved in tryptophan secretion.Nevertheless,the mechanism in this process is still unclear and need further investigation.To study the tripartite interaction between FOC,SQR9 and cucumber root,split-root system was used to avoid the direct interaction between FOC and SQR9 in rhizosphere thus a view of the indirect interaction through cucumber root exudates was practicable.By plate counting,pre-inoculation of both FOC and SQR9 to cucumber rhizosphere led to enhanced SQR9 colonization.Genes related to biofilm formation in SQR9 of the second inoculant in both SQR9 and FOC treatments were affected.The downstream gene eps and a positive regulator sinI were upregulated. The quantification of FOC performed by real-time PCR using specific primer pair indicated that both FOC and SQR9 pre-inoculation caused lower copy number of FOC on cucumber root.Detection of endogenous compounds by enzyme-linked immunosorbent assay showed that both SQR9 and FOC inoculation increased jasmonic acid content in root;SQR9 also increased salicylic acid in root and ethylene in stem.The increase of jasmonic acid and salicylic acid would cause systemic resistance thus be a reason fro decrease of colonization of FOC.To investigate the effect of root exudates on these colonization processes above,root exudates in the uninoculated side of the cucumber inoculated by SQR9,FOC,J12,and 168,as well as that from the regular cucumber were collected and frozen dried to powder.Further methoximation and trimethylsilylation derivatization were performed to transform compounds in root exudates into gasifiable and heat stable chemicals.By using high throughput GC-MS method,low molecular organic compounds were analyzed.Based on the MS spectrums,peaks were assigned by database and reported as peak height.Totally 110 chemicals were classified,including 8 amines,17 amino acids,20 sugars,7 sugar acids,13 sugar alcohols,9 fatty acids,18 organic acids and 18 others.Principal components analysis analysis showed that the treatments and control were separated with well repeat and significant difference between each other.Compared with control,SQR9 treatment caused most significant changes of the root exudates composition,as among the 110 chemicals,35 were significantly down regulated.Compared with control based on total peak height of each chemical group,amines,amino acids and the "others" group in SQR9 treatments were increased,while organic acids,fatty acids and sugars decreased.Amino acids and fatty acids in FOC treatments were increased while the "others" group decreased.Correlations between root exudates and colonization calculated based on spearman rank correlation algorithm showed that colonization of FOC was negatively correlated with amines and sugar acids.In addition,8 chemicals were found to be significantly correlated with colonization by SQR9 or FOC,respectively.In detail,colonization of FOC enjoyed positive correlations with raffinose and lyxitol,while negatively correlated with pentitol,glucose and shikimate.Colonization of SQR9 showed positive correlation with tryptophan and negative with maltitol and sorbitol.These 8 compounds were added to rhizosphere exogenously together with SQR9 or FOC for evaluate their effect on colonization individually.Results showed that colonization of FOC increased as the increase of concentration of raffinose or pentitol,while decreased as lytitol,glucose or shikimate rised.The trends were completely consistent with the correlation analysis.However,with the concentration of tryptophan,maltitol or sorbitol increased,colonization of SQR9 increased.This is consistent with the correlation for tryptophan but not for maltitol or sorbitol.Further research indicated that tryptophan enjoyed positive effect on biofilm formation but no significant effect on chemotaxis.However,The contents of pentitol,lyxitol,shikimate and maltitol in root exudates did not show significant difference between control and FOC or SQR9 treatments.Although these four compounds showed significant effect on colonization,they would not act as signal in this interaction.Effect of glucose and sorbitol on colonization showed contrary results with colonization in the split-root system,indicating these two chemicals did not play a critical role in colonization in nature.Finally,we confirmed raffinose a chemical involved in FOC colonization.Less raffinose would be secreted by cucumber challenged by FOC or with inoculation of SQR9,thus the colonization of FOC would be reduced;at the same time,secretion of tryptophan would be enhanced for recruiting SQR9 to against FOC or other stresses.In this study,the effect of rhizosphere interaction on root exudates was investigated,and the mechanism of increase of plant growth promotion of Bacillus amyloliquefaciens SQR9 mediated by tryptophan was explored.What is more,signals involved in root colonization of SQR9 and FOC were revealed. |
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