| Pseudomonas fluorescens is plant growth-promoting rhizobacteria (PGPR). Most P. fluorescens isolated from plant rhizosphere can play an important role on biological control of plant diseases and stimulation plant growth. Therefore, it is potential for P. fluorescens to suppress plant diseases. Whether P. fluorescens colonize the roots, leaves and other parts of the plants well is one of key factors in controlling plant diseases efficiently. Formation of biofilm is an important colonization strategy for bacteria in various environments. Micro-colony formation on surface of plant root is regarded as a form of biofilm. However, biofilm formation of P. fluorescens, effect of genes involved in biofilm on root colonization and role of theses genes in biological control ability were few reported before. This study was focused on several areas, including isolation and identification of P. fluorescens with high antagonistic activity to soil-borne plant diseases, exploitation potentiality of P. fluorescens in biological control of plant diseases, study on mechanism of biofilm formation of P. fluorescens and roles in root colonization and biological control of soil-borne root pathogens.A total 102 of fluorescent, rod shaped bacterial strains with monotrichous or lophotrichous flagella were isolated from 488 soil samples collected from Shandong, Hainan, Yunnan and Xinjiang Provinces. one of these strains, named TC222, showed antagonistic activity to plant pathogens, e.g. Gaeumannomyces graminis, Magnaporthe grisea, Drechslera sorakiniana, Alternaria brassicae, Botrytis cinerea, Phomopsis asparagi, Fulvia fulva, Colletotrichum lindemuthianum Sacc. et Magn and Phytophthora parasitica var nicotianae. Strain TC222 was finally identified to be Pseudomonas fluorescens according to amplified ribosomal DNA restriction analysis (ARDRA) and physiological and biochemical characteristics. Colonization experiment showed that P. fluorescens could colonize the rhizosphere of wheat efficiently. P. fluorescens TC222 showed a potential prospect in biological controlling for plant diseases. Moreover, ARDRA analysis provided a convenient method for P. fluorescens isolation and identification.In order to study the mechanism of biofilm formation of P. fluorescens TC222, pJBA28 carrying mini-Tn5 transposon was reconstructed. The Escherichia coli origin of replication p15A was inserted into transposition region of pJBA28 to produce the suicide transposon vector pJB15A. Target genomic DNA with origin of replication p15A can replicate in Escherichia coli host strain after self-ligation. The transposon vector pJB15A provided a novel and convenient tool for clone and isolation important functional genes of P. fluorescens. Tn5-based transposon mutagenesis to P. fluorescens TC222 was performed. Approximately 10,000 kanamycin-resistant transposon insertion mutants were obtained.Biofilm formation mutants are often found to have defective or altered motility. The motility phenotype was exploited to identify Pseudomonas fluorescens biofilm formation mutants. A total of 8 mutants exhibited two distinct motility patterns compared with the wild-type strain was screened and was named as Lip1, Lip2, Lip3, Fla4, Fla5, Hyp6, Hyp7 and Hyp8. All the mutants having similar growth rates as the wild-type strain suggested that the varied motility was not caused by change of growth.The eight genotype mutants were tested for the ability to form biofilm on polystyrene (hydrophobic) and borosilicate glass (hydrophilic). Mutants Hyp6, Hyp7 and Fla have reduced ability to form biofilm on both surfaces. However, mutant Hyp8 shows increased attachment to both substrata and mutant Lip1 shows reduced biofilm formation on hydrophilic surface but increased biofilm formation on hydrophobic surface. To obtain a better understanding of the effect of the mutation on the root colonization and antagonistic activity, experiments of root colonization and disease suppression were performed. The five genotype mutants showed impaired root colonization, defective competitive growth and decreased antagonistic activity to some extent. The flanking DNA of the Tn5 insertion of eight mutants was sequenced and homology analysis was performed. BLAST analysis revealed that the transposon-interrupted genes have the highest identities with gene encoding dTDP-4-dehydrorhamnose reductase (lipA) involved in lipopolysaccharide biosynthesis in Lip1, Lip2 and Lip3, gene encoding flagellar biosynthesis protein FliR (flaR) involved in flagellar biosynthesis in Fla4 and Fla5, gene encoding a probable bacteriophage signal peptide protein (bspA) in Hyp6, gene encoding a putative Mig-14 like protein (epsB) in Hyp7 and gene encoding soluble pyridine nucleotide transhydrogenase (pyrA) in Hyp8. These data suggested that all the genes involved in biofilm formation and affected colonization to some extent. Moreover, epsB,bspA and pyrA were found firstly to be involved in biofilm formation in P.fluorescens. These genes provided insight to direct further work into mechanism of biofilm formation.To determine whether the genes are really involved in biofilm formation, lipA and epsB were selected to complement the corresponding mutants. Biofilm formation of the mutants was partially restored by pQMV-lipA and pQMV-epsB, respectively. The data confirmed that the both genes lipA and epsB were involved in biofilm formation. Semi-quantitative RT-PCR analysis showed that the epsB and lipA gene can be transcripted in both the complemented strains and the wild-type strain. However, the epsB and lipA were failed to detect the transcripts in their respective mutant. In order to prove the epsB to lie in potential exopolysaccharide biosynthesis operon, RT-PCR showed that epsB, upstream gene epsA and downstream gene epsC all located in the same transcriptional unit. Co-transcriptional analysis data explained why the biofilm of the complemented strains couldn't be restored to the level of the wild-type strain.Moreover, lipA and epsB gene were expressed in E. coli BL21 respectively. They both could be expressed about 30kD protein in the precipitation.
|
PDF Full Text Request