| The metabolites of symbiotic bacteria associated with entomopathogenic nematodes own much bioactivity such as antifungal, antibacterial, insecticidal, antivirus and anticancer. Until now, more than 11 class 40 kinds of structurally diverse secondary metabolites are isolated and/or known from different Xenorhabdus strains. The chemical structure of these metabolites is diverse, which make it become a kind of novel bio resource with high potential for development and application. Previous study of Research and Development Center of Biorational Pesticides of Northwest A&F University found that, fermentation broth of Xenorhabdus nematophila YL001 has a good inhibitory effect on many kinds of plant pathogens fungi, and isolated 1 kinds of cyclic peptide compounds with good inhibitory activity against Botrytis cinerea from YL001 fermentation. In order to further research the antimicrobial activity of the YL001, on the basis of previous studies, the bioassay-guided method was used to extract, isolate and identify the active components of the extracellular products of YL001. On this basis, using HPLC analysis method to preliminary study the factors, fermentation conditions, bacteria(fermentation method and medium initial p H) and cpx R gene mutation, which may influence on antimicrobial components. The results obtained are listed as following:1. After YL001 fermentation supernatant was adsorbed by D101 macro porous resin, the total extract was eluted with methanol. The results showed that, the total extract of ethylacetate, n-butanol extract and water relative had biological activity to Bacillus subtilis. By bioassay-guided method,combined with silica gel column chromatography, gel column chromatography and crystallization separation technology,five compounds were separated from YL001 extracellular metabolites ethyl acetate extract and n-butanol extract of active components.The chemical structures of 3 compounds were identified as : C1 :Benzylideneacetone(BZA),C3:Nematophin,C5:Tryptamine. By using 732 cation exchange resin, the activity of extracellular products of YL001 was preliminarily separated. After treated with different p H(p H 2.0, 3.0, 4.0, 5.0, 6.0), respectively, samples were passed through a cation exchange resin, and then the column was eluted with 0.5mol/L ammonia water in sequence. The eluate was concentrated with the five extracts. The antimicrobial activities of extracellular metabolic products from p H2.0 to p H6.0 of YL001, were tested in vitro and in vivo. The bioassay results indicated that,at p H2.0~4.0,the extracts gave the highest activities. Using LC-MS analysis the active substances relative content of the five extracts under the condition of different p H had obvious differences. LC-MS analysis showed that active substances content of the five extracts under the condition of different p H was different, at p H2.0~4.0, the cationic resin has a greater adsorption on the main antimicrobial substances.2. The minimum inhibitory concentration of 7 kinds of bacteria of nematophin and benzylideneacetone were detected by broth dilution technique. The results showed that, two compounds of nematophin and benzylideneacetone have certain antibacterial activity. Nematophin and benzylideneacetone displayed highly antibacterial activity against gram-positive bacteria, but week antibacterial activity against gram negative ones. The nematophin and benzylideneacetone, has good inhibitory effect on Staphylococcus aureus and its MIC were 2.5 μg/m L and 12.5 μg/m L. Under the condition of laboratory, inhibitive activities of 2 kinds of fungicides to the pathogenic fungus were tested by means of mycelium growth rate method, respectively. The bioassay results indicated that, Nematophin has the better control effects to Botrytis cirerea, Thanatephorus cucumerisat, Valsa mali at 100μg/ml. Among then, the inhibition of Botrytis cirerea was the highest, which was 88.63%. On the basis of the above results, the toxicity of Nematophin on Botrytis cirerea, Thanatephorus cucumerisat, Valsa mali was determined, and the EC50 values were 22.765 mg/L, 36.236 mg/L and 30.227 mg/L. Tissue experiment showed that the therapeutic of nematophin(250 mg/L、500 mg/L、750 mg/L、1000 mg/L)were 24.41%~55.38, was lower than the control(67.98%); The protective effect of nematophin(250 mg/L、500 mg/L、750 mg/L、1000 mg/L) were 48.71%~76.79%, lower than the control(84.07%).3. Using HPLC analysis, this paper preliminary study the factors, different strains, different fermentation conditions(fermentation method and culture medium initial p H) and cpx R gene mutant,which may influence yield of nematophin. The results showed that, Xenorhabdus nematophila can produce nematophin, Xenorhabdus bovienii can not produce nematophin; Concentration of Nematophinis different from strains, the yield of nematophin was the highest in YL001, which was 115.23 μg/m L; The concentration of nematophin was improved with the increase of initial p H4.5 to p H8.5, the concentration was highest at p H8.5, which was 155.08 μg/m L; The concentration of Nematophin was improved with fermentation condition. In the 5 L auto-fermenter, rotary speed 150 rpm, ventilator capacity 2.5 L/min, the concentration of Nematophin of YL001 was improved with the increase of initial p H, and the concentration was highest at p H8.5, increasing by 79.85% than the Erlenmeyer flask, which was 206.97 μg/m L.In conclusion, the antibacterial activity of YL001 metabolites was complex. Especially, the antibacterial activity components of n-butanol fraction and the other compounds were worth further research and isolation; the fermentation method and the medium initial p H can affect the yield of the main antibacterial substances. Yield of nematophin in YL001 wild strains was 115.23 μg/m L, which was 1.7 times of cpx R mutant strains(67.84 μg/m L). Cpx R positively regulates the producti on of nematophin. |
PDF Full Text Request