Study On The Biocontrol Mechanisms Of Bacillus Subtillis 9407

Bacillus subtilis has been used as biological control agents to suppress different plant pathogens.B.subtilis 9407 is a beneficial bacteria against apple ring rot disease and isolated from healthy apples.In order to reveal the main biocontrol mechanisms of B.subtilis 9407,chemistry methods and molecular technologies were used to identify the primary antimicrobial compounds involed in its antimicrobial activities,determine the role of the primary antimicrobial compounds in biocontrol,elucidate the role of MecA in colonization and biocontrol,disclose the regulatory pathways of MecA on biofilm formation.The results of this study are not only beneficial to reveal the biocontrol mechanisms of beneficial Bacillus spp.,but also helpful to explore new way to increase the biocontrol activity of biological control agents.1.The role of fengycin produced by B.subtilis 9407 in the biocontrol of apple ring rot disease.To identify the primary antifungal compound of B.subtilis 9407 and elucidate the mechanisms by which B.subtilis 9407 inhibits B.dothidea growth,approximately 14,300 mutants were picked to detect their antifungal activity against B.dothidea YL1,and 34 mutants displayed antifungal activity that was clearly different from that of the wild-type strain were obtained.A mutant showed decreased in antifungal activity against B.dothidea YL1 was chosen for further study.Southern blot analysis,IPCR and transposon-flanking DNA sequencing revealed that the mariner transposon was inserted at a single location within ppsB.A markerless ppsB deletion mutant was constructed.Compared with the wild-type strain lipopeptide crude extracts from AppsB showed almost no inhibition of B.dothidea mycelial growth.Semipreparative reverse-phase high performance liquid chromatography(RP-HPLC)detection revealed that AppsB lost the ability to synthesize fengycin.These results suggest that fengycin is the major antifungal compound produced by B.subtilis 9407 against B.dothidea.Moreover,a biocontrol assay showed that the control efficacy of AppsB was reduced by half compared with the wild-type strain,indicating that fengycin plays a major role in controlling apple ring rot disease.2.The role of surfactin produced by B.subtilis 9407 in the biocontrol of bacterial fruit blotchB.subtilis 9407 showed strong antibacterial activity against A.citrulli and 61.7%biocontrol efficacy on melon plant under greenhouse conditions.To understand the biocontrol mechanism of B.subtilis 9407,identify the primary antibacterial compound and determine its role in controlling bacterial fruit blotch(BFB),a srfAB mutant(?srfAB)was constructed.The ?srfAB which was deficient in production of surfactin,not only showed no ability to inhibit growth of A.citrulli but also decreased biofilm formation and reduced swarming motility.Colonization assay demonstrated that B.subtilis 9407 could colonized on melon roots and leaves in a large population,while AsrfAB showed a 4-to 10-fold reduction in colonization of melon roots and leaves.Furthermore,a biocontrol assay showed that compared with the wild-type strain,?srfAB lost the biocontrol efficacy.We propose that surfactin via coordinating action of antibacterial activity and colonization to enhance biocontrol efficacy of B.subtilis 9407.3.The role of MecA in colonization of B.subtilis 9407 and the biocontrol of BFB.Studying on the genes involed in biofilm formation,elucidate the roles of them in colonization and biocontrol are beneficial to reveal the biocontrol mechanism of B.subtilis 9407.Approximately 5,000 mutants were picked to screened for alteration of the biofilm phenotype.About 32 such mutants were subsequently obtained and 17 genes were identified that were important for biofilm formation in B.subtilis 9407.The mutant that has a transposon insertion in the gene annotated as mecA showed dramatically decreased biofilm formation compared with the wild-type strain.However,there is no report on the relationship between MecA and colonization of biocontrol bacteria.To elucidate the role of MecA in colonization and biocontrol,mecA deletion mutant and its complementary strains were constructed.The AmecA(pBE2)showed decreased in biofilm formation,reduced in swarming motility,while,biofilm formation and swarming motility in AmecA(pBE2A)were restored.Colonization assay demonstrated that AmecA(pBE2A)and 9407(pBE2)colonized well on melon roots and leaves,while AmecA(pBE2)showed a 7-to 20-fold reduction in colonization.?mecA(pBE2),?mecA(pBE2A)and 9407(pBE2)showed strong antibacterial activity against A.Futhermore,a biocontrol assay showed that compared with ?mecA(pBE2A)and 9407(pBE2),AmecA(pBE2)lost the biocontrol efficacy against BFB.The results from this study indicating that MecA plays a major role in colonization of B.subtilis 9407 and biocontrol of BFB.4.The regulatory pathways of MecA on biofilm formation in B.subtilis 9407.In order to disclose the regulatory pathways of MecA on biofilm formation,strains with promoter lacZ reporter fusions were constructed.The activities of promoter lacZ reporter fusion showed that MecA positive regulates the expression of eps operon and negative regulates the expression of tapA-sipW-tasA operon.Previous evidence suggests that MecA negative regulates the expression of eps operon in B.subtilis 168.The dedicated mutants were constructed and biofilm phenotype showed that spoOA,sinl,sinR and abrB were involved in biofilm formation.Complementary strains with each of spoOA,sinl,sinR and abrB in ?mecA background restored biofilm fomation,indicating that spoOA,sinI,sinR and abrB were involved in the regulation pathways of MecA.The activities of promoter lacZ reporter fusion showed that MecA positive regulates the expression of spoOA and sinl,negative regulates the expression of sinR and abrB.Combined our experimental results and previous studies,we presented that MecA positive regulates the expression of via pathways of SinI/SinR and AbrB to regulate the biofilm formation,at the same time,MecA inhibits the expression of sinR,leading to derepression of matrix genes and promote the formation of biofilm,and furthermore MecA positive regulates the expression of abrB and then influence the expression of matrix genes.