The Antagonistic Mechanism Of Bacillus Amyloliquefaciens SQR9 Agaginst Soil-borne Pathogens And In Situ Rhizosphere Antibiotic Gene Expression

With the extensive use of chemical pesticides,environmental pollution caused by pesticide residues has become an emergency to be dealing with.Researchers have shed light on biocontrol technology,using biocontrol agents and microbial organic fertilizers in biocontrol of plant disease which shows great prospect in agricultural application.Bacillus amyloliquefaciens is considered as an important group of plant growth-promoting rhizobacteria?PGPR?and has been widely used in agriculture.As one of the important members of the PGPR family,it secretes a variety of antibiotics,as it is one of the important members of the PGPR family.A laboratory-stored bacterial strain isolated from healthy cucumber rhizosphere,named Bacillus amyloliquefaciens SQR9 was used in this study.SQR9 had great biocontrol activity against soil-borne Fusarium Wilt of cucumber,and the fermentation liquid of SQR9 had obvious promoting effect on the growth of cucumber.This study investigated the antagonistic spectrum of Bacillus amyloliquefaciens SQR9 against a range of soil-borne pathogens in vitro.Diffierent NRPS genes and their cooresponding compounds were detected when confronted with different fungal pathogens.Key genes involved in the biocontrol progress of SQR9 against Rastonia solanacearum were also been screened.Antibiotic genes of SQR9 were also detected in situ when SQR9 was added into tomato rhizosphere.Following findings were achieved:1.SQR9 showed antagonistic activity against Verticillium dahlia kleb,Sclerotinia sclerotiorum,Fusarium oxysporum,Fusarium solani,Rhizoctonia solani and Phytophthora parasitica.Gene clusters bmyDABC?fenEDCBA?srfAA-AD and dhbEDCBA was found in SQR9 which were responsible for the systhese of three NRPS genes:bacillomycin D,fengycin,surfactin and a siderophore bacillibactin.SQR9 altered its spectrum of antifungal compounds production and transcription of antifungal genes responding to different fungal pathogen.Single mutants of these genes were constructed/used,supernatants were used to futher verify the role of NRPSs compounds in the antagonistic activities of SQR9 when confronted with six soilborne fungal pathogens.The supernatants showed significant difference?P<0.05?,which represent the fact that SQR9 regulated the production of antibiotics when fighting against different fungal pathogens.Bacillomycin D was the major LP produced when SQR9 was confronted with Fusarium oxysporum.Fengycin contributed to the antagonistic activity against Verticillium dahlia kleb,Fusarium oxysporum,Fusarium solani and Phytophthora parasitica.Surfactin participated in the antagonistic process against Sclerotinia sclerotiorum,Rhizoctonia solani and Fusarium solani.Bacillibactin was up-regulated when SQR9 was confronted with all tested fungi.These results provide a new understanding of specific cues in bacteria-fungi interactions and provide insights for agricultural applications.2.SQR9 showed antagonistic activity against Rastonia solanacearum?RS?in vitro.Gene cluster mlnA-??dfnAYXB-M?baeB-E,acpK,baeG-NR and bacA-E,ywfG was found in SQR9 corrosponding for the synthesis of macrolactin,bacillane and bacilysin which were reported to participate in the antagonistic process of SQR9 against bacterial pathogen.Besides the bmyDABC,fenEDCBA,srfAA-AD and dhbEDCBA,8 genes were considered to be involved in the antagonistic process of SQR9 against RS.As phosphopantetheinyl transferase?Sfp?was responsible for the synthese of compounds we were about to study for their role in antagonism,sfp gene was knocked out.Besides,we knocked out bac gene which is not affected by Sfp,by evaluating the supernatant of sfp and bac mutants,we found out that bacilysin was not involved in the antagonistic process of SQR9 against RS.sfp mutant lost antagonistic activity against RS,which lead us to the 7 genes we analyzed in SQR9.Single mutants of NRPS genes:bamD,fenA,srfA,dhb,bac and PKS genes:dfn,mln,bae were constructed,by testing the antagonistic activity of each mutant against RS,we discovered that srf and dfn mutants weakened their activity against RS,while other 5 mutants remains the same antagonistic activity as SQR9 wild type.Srf,dfn double mutant lost 72.8%antibiotic ability compared with SQR9 wild type,showed the fact that surfactin and difficidin were the active compounds when SQR9 fighting against RS.3.Long-term tomato planting soil was used;sterilized soil and non-sterilized were designed as two main treatments.Add 1×10⁸ CFU·g^-1 SQR9-GFP and 1×10⁶ CFU·g^-1 SQR9-GFP to the soil separately,using blank soil with tomato growing in as control.Using real-time PCR,construct standard curve of functional genes,NRPS genes:bam,fen,srf,dhb,bac,and PKS genes:dfn,mln,bae were detected in the rhizosphere of tomato in their transcriptional level.Results showed that soil samples in treatment of 1×10⁸ CFU·g^-1 cell was detected with 10⁷-10⁸ copies·g^-1 of SQR9 specific gene with in the 30 days we tested.Transcriptional data of SQR9 specific gene in non-sterilized soil showed no significant difference?P?0.5?compared with that in sterilized soil.In 7,15 and 30 days,transcriptional data of SQR9 specific gene in sterilized soil SQR9 specific gene was 1.98,2.08,1.85 times higher than that in non-sterilized soil,respectively.Several functional genes detected in the soil showed significant difference in their transcriptional level.As a surfactant,surfactin participate in the biofilm formation process.Surfactin was also reported to trigger systemic resistance of plants.Srf gene was the most highly transcriptional type among all the genes tested;in the 30th day,srf gene detected 10⁹ copies·g^-1.This result futher provide the fact that surfactin is an important compound produced by SQR9 in the rhizosphere.As the only siderophore synthesis gene discovered in SQR9 genome,dhb gene was10⁷-10⁸ copies·g^-1 in the 1×10⁸ CFU·g^-1 cell treatment during the 30 days,which shows that SQR9 could produce bacillibactin for Fe3+ in the rhizosphere when surviving in the rhizosphere.Conclusion:Bacillus amyloliquefaciens SQR9 use different antibiotic strategy when confronted with different soil-borne pathogens;72.8%of the antagonistic activity in SQR9 fighting against Rastonia solanacearum were contributed by surfactin and difficidin;SQR9 produce anomous surfactin in situ,due to the reason that surfactin perticipate in root colonization and induced resistance of plant,the result provided evidence that SQR9 obtained highly potential as a biocontrol PGPR strain.