Inhibitory Effect Of 5-8 On Rice Blast And Regulation Mechanism Of RpoS On Antibiotic Synthesis In Biocontrol Bacterium FD6

The results showed that the B.velezensis strain 5-8 from the antagonistic bacteria stored in our laboratory significantly reduced the development of rice blast,and the control effect could be up to more than 90%.Strain 5-8 had broad antifungal spectrum and displayed strong antagonistic ability to many plant pathogenic fungi.Different concentrations of crude extracts showed different antimicrobial activities.Approximately 50%of spores treated with 1.5 mg/mL crude compounds extract germinated,while the germination of spores was completely inhibited when the concentration of crude extract was added 2 mg/mL.The results of PI staining showed that 1 mg/mL crude extract could not damage the cell membrane,and the damage rate of cell membrane was 34%when 2 mg/mL crude extract was added.In conclusion,B.velezensis 5-8 could inhibit the germination of conidia of Magnaporthe grisea and destroy the integrity of cell membrane,suggesting that biocontrol agent from strain 5-8 has a good application prospectIn order to clarify the effect of RpoS on the biocontrol ability of FD6,we examined the characteristics of biocontrol related factors in the rpoS deletion background.The results showed that rpoS negatively regulates bacterial motility,the PLT and 2,4-DAPG synthesis,and has a positive regulatory effect on the biofilm formation of FD6.To further determine the regulatory mechanism of rpoS on the synthesis of 2,4-DAPG and PLT,the transcriptional fusion(6522-phl and 6522-plt)and translation fusion structures(6013-phl and 6013-plt)constructed.Theβ-galactosidse activities showed that the phlA and pltA β-galactosidase activities increased significantly and reached the highest level at the late stage in both transcriptional and translational levels.The results of qRT-PCR showed that the expression levels of 2,4-DAPG-and PLT-related genes significantly increased in rpoS deletion mutant compared with the wild-type FD6,in which the expression levels of pltL increased-47 fold.The results showed that rpoS negatively regulated the synthesis of antibiotics 2,4-dapg and PLT at transcription and translation levels.RpoS usually binds the-10 promoter region of the target gene,and the conserved binding motif is CTA[A/T/G/C][A/T/G/C][A/T/G/C]T.This motif is used to search for genes related to antibiotic synthesis.Similar binding motifs were found in the promoter regions of phlA,phlG,pltR,pltL and pltF.To verify this result,the bacterial single hybridization experiments confirmed that RpoS could directly bind the promoter regions of phlA,phlG,pltR,pltL and pltF.RpoS fused with His tag protein were successfully expressed when the pET22b(+)was used as the expression vector and BL21 as the host strain.Most parts existed in the form of soluble protein with a molecular weight of 44 kDa.When eluted with 200 mM and 250 mM imidazole concentration eluents,high purity RpoS protein was obtained.The concentration of concentrated RpoS protein was 846 ng/μL.Biotin-labeled promoter region probes of antibiotic-related genes were used to perform the EMSA experiments,and the results further verified that RpoS could directly bind the promoter regions of the above-mentioned genes.In conclusion,the global regulatory factor RpoS in Pseudomonas FD6 negatively regulates the synthesis of 2,4-DAPG and PLT by directly binding the phlA,phlG,pltR,pltL and pltF.The results provide a theoretical basis for improving the disease control effect of biocontrol agent.In conclusion,the global regulation factor RpoS of biocontrol FD6 negatively regulates 2,4-DAPG and PLT synthesis by directly combining phlA,phlG,pltR,pltL and pltF,and the results provide a theoretical basis for improving the biocontrol effect of biocontrol bacteria.