| Fluorescent Pseudomonads producing 2,4-diacetylphloroglucinol (2,4-DAPG) have been taken as one of the promising candidates in biological control due to the antibiosis activity of 2,4-DAPG on a broad spectrum of organisms and this kind of bacteria could suppress certain root and seedling diseases caused by soil-borne pathogens. Most of the 2,4-DAPG producer also showed strong colonization ability and adaptability. Pseudomonas fluorescens 2P24 is a well characteristic biocontrol agent isolated from wheat take-all decline soil in China. This strain produces several antifungal compounds, such as 2,4-diacetylphloroglucinol (2,4-DAPG), hydrogen cyanide (HCN), siderophores and proteinase. In this thesis, the biological mechanism was exploited and the biosynthsis and regulation gene of 2,4-DAPG was furter investigated.2,4-DAPG producers were identified from the biocontrol pseudomonads, furthermore, the candidates with high antagonisis, broad antimicrobe spectrum and good colonization were re-selected after the dual test on pathogens causing milk vetch root rot and tomato bacterial wilt and the biocontrol assays against these two diseases. The strains 2P24 and CPFIO were final lupicked for genetic studys since both of them could well perform on plant disease control( up to 50%) and root colonization as well.The 2,4-DAPG biosynthetic locus was cloned base on the screening on 2P24 genomic library. The determination system of 2,4-DAPG expression was constructed after the insertion of a promoterless LacZ gene in 2,4-DAPG biosynthetic locus. Tn5-mediated chromosomal integration was performed and the mutants with the reduced expression of the lacZ gene were selected out. Futher anaylsis showed that the insertion mutation on phlF gene contributed to reduced expression of the 2,4-DAPG biosynthetic locus.PhlH and phlG, two genes located upstream of 2,4-DAPG biosynthetic locus were cloned. Mutagenesis and function complement proved that phlH could partially regulate the expression of 2 ,4-DAPG genes in transcriptional level, while the genetic analysis of phlG showed a more complex phynotype. An in-frame deletion of phlG do not produce the 2,4-DAPG, but still retained the antifungal activity in dual cultural test. Futher research is needed for clarification of the relationship between the phlG gene and 2,4-DAPG production. With the same fusion technique, the post-transcriptional regulation of 2,4-DAPG by GacS/GacA two components system was determined as well.HPLC was employed to determine the 2,4-DAPG produced by CPF10 and 2P24 in different carbon resources, nitrogen resources, micro-elements, different culture time and temperature. It indicated that for both of the strains, different carbon resource could lead to different amount of 2,4-DAPG production. With sucrose as the carbon resource, CPFIO could produce more antibiotics while 2P24 less or undetectable. However, when glucose as the carbon resource, 2P24 could give higher 2,4-DAPG production when it was undetectable in CPFIO. These observations were consistent with the result from plate dual culture test. As far as the effect of temperature on 2,4-DAPG , 2P24 produced the highestantibiotics under the temperature as low as 12℃. Most micro-elements lead to the neutral effect on 2,4-DAPG production, unless some ones, such as Zn 2+,Mo2+, Mg2+ could increase the production.2,4-DAPG biosynthetic locus was intergrated into the chromosome of wild type Pseudomonads strains P32 (2,4-DAPG'), CPF-10 and 2P24, respectively. The HPLC result showed that P32, the 2,4-DAPG minus strain could obtain the ability to produce the antibiotics and the 2,4-DAPG yield were significant increased in the 2,4-DAPG plus strains CPF-10 and 2P24. Both the plate dual cultural test on pathogens causing tomato bacterial wilt, wheat take-all and cotton damping-off and the green house experiment on tomato bacterial wilt and wheat take-all indicated that the 2,4-DAPG contributed to the high plant protection effect. There was no significant difference on root colonization between engineered bacteria and wild type. It i...
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