Isolation Of Antagonistic Bacteria Against Fusarium Graminearum And Cloning Of Antagonistic Genes

Fusarium head blight(FHB),which is mainly caused by Fusarium graminearum,is a devastating disease that leads to loss of yield and quality in wheat and barley.Recently,the biological control of FHB has become the hot spot in research.In this study,antagonistic bacteria against Fusarium graminearum were isolated,identified and the antagonistic related gene were cloned with the purpose of inhibiting the growth of Fusarium graminearum to control FHB.In this thesis,419 antagonistic bacteria against Fusarium graminearum were isolated from wheat samples and an antagonistic bacteria library was constructed.Among them,328 antagonistic bacteria were identified,and the results showed that 50%of the isolates belong to the genus Bacillus sp.,35%Pantoea sp.,5%Pseudomonas sp.,5%Stenotrophomonas sp.,and the remaining 5%of the non-common species.Pantoea agglomerans GH16 is another antagonistic bacterium against Fusarium graminearum which was isolated in our laboratory previously,and Burkholderia GH16 also can inhibit spore germination and mycelial growth of Fusariumgraminearum.In this study,strain GH16 was randomLy mutated using EZ-Tn5<R6Kyori/KAN-2>Tnp Transposome Kit.34 mutants which lost antifungal activity against Fusarium graminearum were obtained.The gene sequences flanking the transposon in the mutants were cloned and sequenced by using the methods of rescue cloning.At the same time,the complete genome sequences of GH16 were sequenced.The gene sequences flanking the transposon in the mutants were blasted in the Genbank and were compared with the complete genome sequence of GH16.The results showed that the damaged gene sequence by the transposon was a 981 bp gene in the No.16 mutant of GH16.The 981 bp gene is presumed to be a regulatory gene belonging to Lac I family which can regulate the expression of NRPS gene.Burkholderia sp.8B16 is an antagonistic bacterium against Fusarium graminearum which was isolated in our laboratory previously,and Burkholderia sp.8B16 can inhibit spore germination and mycelial growth of Fusarium graminearum.In this study,strain 8B16 was mutated by transposon insertion.Transposon of pSC123(suicide plasmid)was introduced into the genomic DNA of strain 8B16 by conjugation with E.coli DH5a(pSC123)as donor and strain 8B16 as recipient.65 mutants which lost antifungal activity against Fusarium graminearum were obtained.The gene sequences flanking the transposon in the mutants were cloned and sequenced by using the methods of I-PCR and thermal asymmetric PCR.At the same time,the complete genome sequences of 8B16 were sequenced.The gene sequences flanking the transposon in the mutants were blasted in the Genbank and were compared with the complete genome sequence of 8B16.The results showed that the damaged gene sequence by the transposon is Pyrrolnitrin synthase gene in the No.63 mutant of 8B16(named as PTB63).Thus we can conclude preliminaryily that Pyrrolnitrin is one of the antagonistic substances in 8B16.