Phenolic Acid Degradation Mechanism Of Growth Promoting Bacteria GLY-P2 And Its Alleviating Effect On Cucumber Autotoxicity

Continuous cropping obstacles are an important issue in protected vegetable cultivation.The accumulation of phenolic acid is one of the main causes of continuous cropping obstacles in cucumber-planted soil.Ferulic acid（FA）and p-Hydroxybenzoic acid（PHBA）are the main phenolic acids in continuous cropping soil.Therefore,the application of FA and PHBA-degrading bacteria in the soil can help alleviate the autotoxicity of cucumber continuous cropping obstacles.In this study,a strain GLY-P2 that can degrade both FA and PHBA was isolated from cucumber rhizosphere soil.After physiological and biochemical analysis and molecular identification,the strain was a new species of Streptomyces,named Streptomyces sp.GLY-P2.GLY-P2 strain has ACC deaminase activity,can secrete indole acetic acid,can produce cellulase,protease andβ-glucanase,and which has plant growth characteristics.GLY-P2 had better FA and PHBA degradation ability under the conditions of initial concentration of mixed phenolic acid of FA and PHBA of 0.2g/L,temperature of 40°C and p H7.Protocatechuic acid,vanillin and vanillic acid were found to be intermediate products produced by the strain’s metabolism of FA and PHBA.During the degradation of FA and PHBA,superoxide dismutase（SOD）,catalase（CAT）,ascorbate peroxidase（APX）and dehydroascorbate reductase（DHAR）in GLY-P2 cells antioxidant enzyme activity.To elucidate the phenolic acid degradation mechanism of the GLY-P2 strain,the strain was subjected to genome sequencing.The genome of the GLY-P2 strain consists of a closed circular chromosome with a total genome length of 10,311,688 bp,a GC content of 71.2%,and 9,409coding genes.There are 4 prephages,36 gene islands,and 362 secreted protein genes.Based on this,the comparative transcriptome sequencing analysis was performed.A total of 1360significantly differently expressed genes（DEGs）were found,of which 790 genes were up-regulated and 570 genes were down-regulated.The results of GO functional annotation and functional enrichment analysis of DEGs shows that the up-regulated genes were mainly concentrated in protein metabolism,metabolism of organic nitrogen compounds,N-acetyltransferase activity,N-acyltransferase activity,and RNA binding.Through KEGG annotation analysis,a total of 98 KEGG metabolic pathways were obtained,and 48 DEGs were found to participate in the biodegradation of chemicals containing a benzene ring structure.Ferulyl-Co A synthetase gene,Protocatechuate 3,4-dioxygenase including twelve FA metabolizing enzyme genes were significantly up-regulated.In addition,based on the results of the transcriptome analysis of GLY-P2,the annotation function of the genome,and the KEGG pathway of benzoic acid degradation,eleven PHBA metabolic enzyme genes of this strain were also found.The results of real-time quantitative PCR shows that FA and PHBA significantly increased the expression of those metabolic enzyme genes.Therefore,this study proposes the degradation pathway of FA and PHBA as follows:FA is catalyzed to form vanillin by ferulyl-Co A synthetase（fcs）and enoyl-Co A hydrolase/aldolase（ech）,and then oxidized to Vanillic acid,demethylated to form protocatechuic acid;PHBA is decomposed into protocatechuic acid by 4-hydroxybenzoate-3-monooxidase（pob A）;both protocatechuic acid produced by FA and PHBA metabolism is gradually degraded by theβ-ketoadipate metabolic pathway,and finally acetyl-Co A is formed to enter the tricarboxylic acid cycle.In order to analyze the effect of GLY-P2 strain on alleviating continuous cropping obstacles,this strain was inoculated into soil containing FA and PHBA mixed phenolic acid and cucumber planted.The results show that under the mixed phenolic acid conditions of FA and PHBA,GLY-P2 can reduce the content of FA and PHBA in the soil,improve plant growth,enhance the activities of antioxidant enzymes such as SOD,CAT,APX,DHAR,etc,increased the content of antioxidants such as ascorbic acid（ASA）and reduced glutathione（GSH）in the leaves,increased the expression of genes such as Cu/Zn-SOD,Mn-SOD,CAT and APX,making The contents of dialdehyde（MDA）,hydrogen peroxide（H₂O₂）,and superoxide anion（O₂.^-）of leaves decreased by 64.59%,18.16%,and 58.56%,respectively.After analyzing the composition of bacterial communities in the rhizosphere soil of cucumber based on 16S r DNA high-throughput sequencing technology,it was found that the FA and PHBA significantly increased the relative abundance of 28 genera and significantly reduced the relative abundance of 27 genera.Under the conditions of FA and PHBA,GLY-P2 changed the richness,diversity and community composition of soil microbial communities.The relative abundance of 19genera increased significantly,and the relative abundance of 38 genera decreased significantly.It also affects the co-occurrence pattern of microflora and network topology properties.In addition,GLY-P2 increased the activities of soil enzymes such as phosphatase,catalase,urease,and sucrase,and significantly increased the content of hydrolyzed nitrogen,available phosphorus,available potassium,and organic carbon in rhizosphere soil.In order to analyze the phenolic acid degradation mechanism of GLY-P2 strain in soil and promote plant growth characteristics,In this study,the metabolic enzyme genes in the FA degradation pathway were cloned,the engineered bacteria were superexpressed one by one.After measuring the FA degradation rate of each engineered bacteria,it was found that the rate of FA degradation by engineered bacteria that overexpressed the vanillin dehydrogenase gene（vdh）not only was significantly higher than that of other engineered bacteria,but also the FA degradation rates of the strains were 1.2,1.3,1.01,and 0.92 times than it at 60,72,84,and 96hours respectively,so vdh was identified as the rate-limiting enzyme for the FA degradation pathway in the GLY-P2 strain.The ACC deaminase gene（acd S）of GLY-P2 strain and the rate-limiting enzyme gene（pca IJ）of PHBA degradation pathway in GLY-P2 were also cloned,the two genes were overexpressed one by one in GLY-P2 to obtain engineering bacteria GLY-P2A and GLY-P2P,respectively.In addition,the tandem co-expression vector of acd S,vdh,and pca IJ genes was constructed,and engineered bacteria GLY-P2FP that overexpressed vdh and pca IJ genes,and engineered bacteria GLY-P2AFP that overexpressed acd S,vdh,and pca IJ genes were obtained.After inoculating engineering bacteria in soil containing FA and PHBA mixed phenolic acids and planting cucumber,engineering bacteria GLY-P2FP,GLY-P2A,and GLY-P2AFP significantly increased plant growth,increased the antioxidant enzyme activity and the content of antioxidants in cucumber,reduced the level of active oxygen in the leaves,and enhanced the soil enzyme activity and the N,K,P,C cycle compared with the original strain GLY-P2.This indicates that the vdh,pca IJ and acd S genes of GLY-P2 were involved in the phenolic acid degradation and plant growth promotion of the strain in soil.Therefore,GLYP2alleviated the stress of FA and PHBA on cucumber by degrading the content of FA and PHBA in the soil,secreting ACC deaminase and other plant growth-promoting substances,activating plant antioxidant enzyme activity and inducing soil enzyme activity and thereby changing the soil physical and chemical properties and microbial community structure.This provides theoretical support for the application of GLY-P2 in cucumber continuous cropping obstacles.