| Melalecuca bracteata leaves are rich in essential oils,fragrant,and have a strong inhibitory effect on plant pathogens.In the early stage,the research group purified and isolated the active ingredient with Quorum Sensing inhibition effect from the essential oil of Melalecuca bracteata(M.bracteata EO),which was identified as methyl eugenol.In order to study the mechanism of inhibiting pathogenic bacteria by M.bracteata EO and methyl eugenol(ME),the whole genome of pathogenic bacteria was sequenced by the combination of third-generation Pac Bio and second-generation Illumina technologies.Therefore,the physiological and biochemical characteristics,transcriptome and metabolome of M.bracteata EO and ME treated soft rot pathogen were studied in this paper,and the mechanism of inhibiting pathogenicity and virulence genes of pathogen under low concentration of M.bracteata EO and ME was discussed.It will provide theoretical basis and working basis for further study on the mechanism of ME inhibiting QS and pathogenicity of plant pathogenic bacteria,and guide the production and development of M.bracteata EO.The main results are as follows:1.In this study,Dickeya fangzhongdai Onc5,a pathogen causing soft rot of Oncidium ‘Gower Ramsey’,was selected as the research object.In order to reveal its pathogenicity to a variety of plants and clarify the genetic components and genomic heterogeneity of the determinants of pathogenicity,the structure and function of its genome were analyzed by bacterial whole genome sequencing.Comparative genome analysis was performed on strains of Dickeya fangzhongdai.The total genome length of Dickeya fangzhongdai Onc5 was 4967236bp,the N50 value was 22287 bp,and the average GC content was 56.76%.A total of 8 gene islands and 6 trusted CRISPR were predicted.A total of 4190 protein-coding genes were predicted,and 75 tRNAs,22 rNAs and 31 sRNAs were found in chromosomes.Through CAZy analysis,we further screened the results of Dickeya fangzhongdai Onc5 CAZy database,and a total of 268 related genes were screened out,which are important factors for the invasion of plant pathogens.In addition,we compared Dickeya fangzhongdai Onc5 genome with Pathogen Host Interactions(PHI),and the results showed that Dickeya fangzhongdai Onc5 contains 79 genes involved in pathogen host interaction reported by Dickeya spp.and 25 genes related to Pectobacterium spp.reported.Genomic comparative analysis showed that Dickeya fangzhongdai ND14 b,Dickeya fangzhongdai PA1 and Dickeya fangzhongdai Onc5 had more specific genes,and Circos diagram analysis showed that,D.fangzhongdai Onc5 had a strong correlation with D.fangzhongdai ND14 b,while D.fangzhongdai Onc5 had a low correlation with other strains.It is helpful to understand the toxicity mechanism and invasiveness of the plant pathogen,and provide the basis for elucidating the biological control mechanism of the plant pathogen.2.M.bracteata EO and ME could inhibit the QS induction system of Dickeya fangzhongdai Onc5.In this study,the effects of EO and ME on colony movement(including Swimming and Swarming),biofilm content and formation,C6-HSL signaling molecules and plant cell wall degradation enzymes regulated by bacterial soft rot strain QS were detected.The results show: Melaleuca essential oil and methyl eugenol at subinhibitory concentrations(20‰,10‰,5‰,2.5‰,and1.25‰)could significantly interfere with the Swarming movement of Onc5 and inhibit the formation of biofilms,without affecting the growth of Dickeya fangzhongdai Onc5.Fluorescence microscopy(normal mode)observed that the loose state of biofilm was concentration-dependent.Quantitative detection of M.bracteata EO and ME(10 ‰)inhibition of biofilm in inhibition rate were 57% and 70% respectively.At the subinhibitory concentration(20 ‰),the signal molecule C6-HSL was detected by biological assay.The results showed that M.bracteata EO and ME could completely inhibit the production of C6-HSL,and no plant cell wall degradation enzyme regulated by quorum sensing system was detected.The content of C6-HSL was decreased at other subinhibitory concentrations,but there was no effect on plant cell wall degradation enzymes.These results provide a feasible basis for further research and development of melaleuca essential oil in the field of plant derived pesticides.3.M.bracteata EO and ME weaken the pathogenicity of Dickeya fangzhongdai Onc5,and transcriptome and metabonomics were used to analyze the main pathogenic factors of inhibiting pathogenic bacteria at low concentration,which were flagella system and biofilm,which were consistent with the results of physiological characteristics.In order to promote the development and application of M.bracteata EO,transcriptome and metabolomics analysis of the inhibition of M.bracteata EO and ME at low concentration(10‰)were studied,which not only revealed the mechanism of low concentration bacteriostatic,but also helped to develop M.bracteata EO into economic biological pesticide.Transcriptome analysis showed that 220 genes were significantly up-regulated and 167 genes were significantly down-regulated in M.bracteata EO treatment group compared with control group.In ME treatment group,420 genes were significantly up-regulated and 328 genes were significantly down-regulated.T1-vs-T2 difference gene significantly increases for 228,significantly lower gene in 71.The significantly enriched GO term corresponding to the down-regulated differentially expressed genes in different groups was analyzed to form the enrichment string diagram.The results showed that the decomposition and metabolism of arabinose under M.bracteata EO treatment had the greatest influence,corresponding to the gene ara A related to the mutual transformation of pentose and glucuronate in KEGG.The pathogen pentose pathway under ME treatment was also inhibited,as well as flagellum assembly related genes,which act as virulence and regulatory factors during host infection,including flagellin gene fli O,peptidoglycan hydrolase gene flg J,flagellum biosynthetic protein gene fliQ,flhB and flgN.Metabonomics analysis found that M.bracteata EO treatment compared with control group,a total of 184 kinds of different metabolites.Compared with the control group,there were 248 kinds of differential metabolites under ME treatment.The enrichment pathways under M.bracteata EO and ME treatments both contained metabolic pathways and ABC transporters.The enriched pathways under essential oil and methyl eugenol treatment contained both metabolic pathways and ABC transporters.Transcriptome and metabolome association analysis results showed that the genes enriched most in M.bracteata EO and ME treatment groups under positive and negative ion modes were related to ABC transporter,the most correlated genes with metabolites were ABC transporter,various amino acids and lipids.In conclusion,the genes and metabolites affected by M.bracteata EO and ME at low concentrations were most correlated with ABC transporters,various amino acids(mainly lysine),sugars and lipids related to the pathogen biofilm and flagellar system. |
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