| Banana bacterial soft rot is a devastating disease caused by Dickya zeae.In recent years,the disease broke out in Guangdong,Guangxi and Yunnan,which seriously threatened the local banana industry.The pathogen of this disease has strong adaptability and a wide range of hosts.Once the disease occurs in crops,it will often bring huge losses to agricultural production.Thus,a measure for controlling banana bacterial soft rot is urgently needed to explore in banana planting,and the research on the pathogenic mechanism of pathogens is an important premise to control the disease.In this study,the pathogenicity-related genes of D.zeae MS1 were screened by constructing a Tn5 random transposon mutant library,and the functions of some pathogenicity-related genes were determined by genetic methods such as gene knockout and complementation.The homology and genetic evolution of genes such as sucA,tus A,tus B,tus C,tus D,tus E,and mnm A in D.zeae strains isolated from different hosts were analyzed,and the virulence of some strains were evaluated.Meanwhile,the effects of carvacrol on the gene expression of sucA,tus A,tus B,tus C,tus D,tus E,and mnm A in D.zeae MS1 and the physiology and biochemistry of the pathogen were investigated.The results of this study are as follows:1.The Tn5 transposon mutant library of D.zeae MS1 was constructed,and14 mutants with lower virulence and 4 mutants with loss of pathogenicity were screened out.The result of transposon insertion site analysis of Tn5 mutant showed that four loss of pathogenicity mutants including XT20,C45,XT120 and Q63 were inserted into different positions of the sucA gene,respectively,which related to the tricarboxylic acid cycle.Lower virulence mutants including K83,T94 and Y56 were inserted into the tus B,tus C and mnm A genes,respectively,which related to the sulfur transport-related functions.2.The deletion mutant strain of sucA gene and its complementation strain in D.zeae MS1 were constructed.Phenotypic analysis of sucA gene showed that,deletion mutant strain of sucA gene reduced its growth ability,weakened the hypersensitive reactions(HR)in tobacco,lost the ability to secrete extracellular protease,significantly decreased the ability to secrete cellulase and pectinase,and lost its pathogenicity to banana seedlings.It indicated that the sucA gene in D.zeae MS1 was related to its pathogenicity.3.The deletion mutant of sulfur transport-related genes and its complementation strain in D.zeae MS1 were constructed.Phenotypic analysis of sulfur transport-related genes showed that the motility,biofilm formation,and virulence to banana seedlings of deletion mutant strains decreased significantly after deletion of the sulfur transport-related genes tus A,tus B,tus C,tus D,tus E and mnm A in D.zeae MS1,respectively.The results of transcriptome analysis showed that there were 785 genes that were significantly different at the transcriptome level between the gene deletion strain Δtus C and the wild-type strain MS1,of which 395 were significantly up-regulated and 390 were significantly down-regulated.The genes related to flagella,motility,chemotaxis,and type VI secretion system were significantly down-regulated.It was suggested that the genes related to sulfur transport play an important regulatory role in the pathogenic physiological process of D.zeae,and was speculated that these genes may affect the pathogenicity of the strain by regulating the expression of genes related to flagellar,chemotaxis and type VI secretion system.4.The homology of sucA gene and sulfur transport-related genes(tus A,tus B,tus C,tus D,tus E,mnm A)between strain MS1 and other D.zeae strains isolated from different host is more than 93%,indicating that the sucA gene and sulfur transport-related genes were relatively conserved in D.zeae species.The results of phylogenetic analysis of sucA,tus and mnm A genes from different hosts showed that strains CE1 from canna,PL65 from taro and JZL7 from clivia,were clustered in the same superior branch with banana host strains,while rice strains had a farther genetic distance,indicating that D.zeae presented a certain degree of genetic variation in the process of coevolution with different hosts.Strain CE1 from canna and strain MS1 from banana,which have close genetic relationship between hosts and strains,were not significantly different in whole genome,the secretory ability of extracellular degrading enzymes and their pathogenicity to different hosts.5.The minimum inhibitory concentration(MIC)of carvacrol to D.zeae MS1 was 0.1 mg/m L,and the minimum bactericidal concentration(MBC)was0.2 mg/m L.The expression of the gene sucA,tus A,tus B,tus C,tus D,tus E,and mnm A in D.zeae MS1 treated with carvacrol was tested using q RT-PCR.The results showed that gene tus A,tus B,tus C,tus D and tus E in D.zeae MS1 treated with carvacrol was downregulated,speculatng that these genes may be one of the target genes of carvacrol.The control efficacy of carvacrol at the concentration of 0.4 mg/m L against banana bacteria soft rot was 32.0% at the14 th day after carvacrol treatment in pot,indicating that carvacrol had a certain control efficacy on banana bacteria soft rot.When D.zeae strain MS1 was treated with carvacrol at the concentrations of 0(CK),MIC,2MIC and 4MIC,the intracellular ATP concentration in the bacteria was 0.15 μmol/L,0.030μmol/L,0.031 μmol/L and 0.023 μmol/L,the membrane potential mean fluorescence intensity(MFI)in the bacteria was 358.73 AU,277.40 AU,160.03 AU and 124.07 AU,respectively.Meanwhile,D.zeae strain MS1 treated with carvacrol,the soluble substances spilled out of the cells and the cell membrane structure was damaged,indicating that carvacrol could destroy the cell membrane structure of D.zeae strain MS1.Carvacrol at concentrations below the MIC could inhibit bacterial swimming and biofilm formation.In conclusion,mutants XT20,C45,XT120 and Q63 with loss of pathogenicity to banana and mutants K83,T94 and Y56 with significantly reduced virulence to banana were screened from the constructed Tn5 transposon mutant library of D.zeae MS1.The sucA gene and sulfur transport-related genes tus A,tus B,tus C,tus D,tus E and mnm A in D.zeae MS1 were related to its pathogenicity,and were relatively conserved in D.zeae species.Carvacrol has a certain control effect on banana bacterial soft rot.The bacteriostatic action of carvacrol on the D.zeae MS1 strain was associated with its effect on the energy metabolism and the permeability of cell membrane of the pathogen.It was preliminarily considered that these genes could be used as a target for screening chemical compounds to control banana bacterial soft rot.Carvacrol reduced the motility and the formation of biofilm of D.zeae MS1 by affecting the expression of genes tus A,tus B,tus C,tus D and tus E in D.zeae MS1,which leads to the decrease of pathogenicity of the pathogen.Carvacrol reduced virulence of D.zeae MS1 strain by affecting its motility and the formation of biofilm.The research results will provide a basis for elucidating the pathogenic mechanism of D.zeae and a new idea for the control of banana bacterial soft rot. |
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