Research Of The Regulation Between Antimicrobial Substance And Phenotype In Bacillus Amyloliquefaciens And Identification And Application Of The Bacillus Spp.strains

Some species of Bacillus bacteria,such as B.amyloliquefaciens,are of the most important plant growth promoting rhizobacteria(PGPR).They can produce various antibacterial substances,such as antibiotics,promoting substances and extracellular proteases.These substances play the important role in biocontrol function of Bacillus.Therefore,the regulation mechanism study of antibacterial substances synthesized and the function of these substances in biocontrol phenotypes,such as biofilm formation and swarming,can provide a theoretical basis for the application of Bacillus spp.In addition,the isolation and biocontrol effect of Bacillus from different geographical environment provided the bacterial resources for the research and exploitation of Bacillus bio-fertilizers and biopesticides.The main contents include:1.The Rap-Phr system in Bacillus consisted of aspartyl phosphatase and phosphatase regulator plays a key role in antibacterial substance systhesized.The analysis of B.amyloliquefaciens genome showed that the homology between RapAl,Rap A3,RapA4 and Rap from B.subtilis 168 is 50%or less,and the function of these Rap are unknown.Heterogeneous expression,phenomenon experiment(surfactin yield,sporulation efficiency and genetic competence)and were used to study the plasmid-borne Rap-Phr system.Phenomenon experiment results showed that expression of rapA1,rapA3,rap A 4 in OKB105 strongly suppressed its surfactin production,sporulation efficiency and genetic competence.Interesting,co-expression of phr or addition of synthesized Phr pentapeptide in vitro,only PhrA4 could compensate for the supressive effects caused by rapA4.The result of qRT-PCR showed that rapA4 decreased the transcriptional level of the sporulation-related gene spoIIE and surfactin synthesis-related gene srfAA,meanwhile,the transcriptional levels of those genes could be rescued by co-expressed phrA4 and addition of PhrA4 pentapeptide in vitro.Meanwhile,when rapA4 and rapA4phrA4 integrated into the genome of FZB42,phenomenon experiment results showed that expression of rapA4 in FZB42 strongly suppressed its surfactin production,sporulation efficiency and genetic competence.In addition,co-expression of phrA4 in vitro could compensate for the supressive effects caused by rapA4.2.B.amyloliquefaciens FZB42 could produce lipopeptides(surfactin,fengycin and bacillomycin D)and polyketides(bacillaene,difficidin and macrolactin).These substances could inhibit fungal or bacteria,and play an important role in the prevention and treatment of plant diseases.What is the function of these substance in bacteria swarming and biofilm formation?Corresponding research focused on surfactin so far.Our study conducted with a variety of antibacterial substances mutant strains from FZB42.The results for swarming of FZB42 showed that surfactin mutant was impaired in swarming motility.Difficidin and macrolactin mutant were not impaired in swarming motility,moreover,their swarming motility were strengthened.The biofilm formation results of FZB42 derivative mutants showed that surfactin,difficifin and macrolactin deficiency mutants significantly reduced biofilm formation ability,surfactin deficiency got the most significantly decresed results.The results of qRT-PCR showed that srfAA mutant repressed the transcriptional level of the swarming-related gene cheB and the sfp mutant advanced the transcriptional level of the swarming-related gene swrA and flgB.All the results indicated that surfactin plays an important role in swarming and biofilm formation of FZB42.3.The excellent strains resources are the basis of biological pesticides,so it's necessary to carry out the isolation and identification research work of strains.The mainly purpose of this study is to screen the Bacillus spp.with high antimicrobial activity from the rhizosphere soil of Malaysia.The lipopeptides produced by the Bacillus strains were identified,and biocontrol activities of these strains were evaluated.The analysis of antagonistic activity was used to screen the Bacillus spp.with strong antagonistic activities against Xanthomonas oryzae pv.oryzae or Sclerotinia sclerotiorum.The phylogenetic tree based on the sequences of 16S rDNA and gyrB gene was used to identify the isolated Bacillus spp.strains.The MALDI-TOF-MS was used to analysis the lipopeptide compound produced by the isolated strains.The greenhouse expriments were performed to evaluate the biocontrol effect on rice bacterial leaf blight of Bacillus spp.strains.The results of antagonistic activities showed that six isolated Bacillus strains(D15,W3,Klu7,YM16,D1 and PLZ26)have significant inhibitory activities against Xanthomonas oryzae pv.oryzae or Sclerotinia sclerotiorum,among these strains,D15 and W3 have inhibitory activities to both of them.Phylogenetic tree indicated that these six isolates were identified to be most closely related to Bacillus amyloliquefaciens(D15 and W3),B.pumilus(Klu7),B.cereus(YM16)and B.thuringiensis(D1 and PLZ26).Lipopeptide compound analysis via MALDI-TOF-MS showed that PLZ26 and Klu7 produced Surfactin,strains D15 and W3 produced IturinA.The evaluation of the biocontrol effect showed that PLZ26 has good control effect on bacterial blight of rice.This study provided the bacterial resources for the research and exploitation of biopesticides.