Study On Identification, Culture And Antimicrobial Activity Of Entomopathognic Bacteria

The metabolites of symbiotic bacteria associated with entomopathogenic nematodes own much bioactivity such as antifungal, antibacterial, insecticidal, nematicidal, antivirus and anticancer in the field of medicine, public health and agriculture, which make it become a kind of novel bioresource with high potential for development and broad prospects for application. Previous study found that strains YL001, YL002 and TB obtained from the entomopathogenic nematodes screened from Yangling, Shaanxi Province and Taibai Moutain showed obvious antimicrobial activity. In order to determine the taxonomy and systemic evolutional status of the strains, and lay basis for further study, development and application of the bioresource, we studied their taxonomic status, culture and antimicrobial activity of the metabolites. The main results and conclusions of the thesis are as follows:1. The 16S rRNA complete sequences of strains YL001, YL002 and TB were amplified by PCR, cloned and measured. The homology comparison and clustering analysis of the 16S rRNA sequences of the strains were conducted by comparison with the known strains in GenBank. In constructed phylogenetic tree, strains YL001, YL002 and TB formed a monophyletic clade with strains of X. nematophilus with sequence homology higher than 99%, and located at different branch. Based on previous research, we can conclude that strains YL001, YL002 and TB belong to the different strain of X. nematophila.2. All the three strains exhibited antagonism effect on tested 26 plant pathogenic fungi, and different strains showed different antagonism effect, but all showed high effect on P. capsici. The extracellular metabolite of different strains exhibited a greater difference in inhibitory effect on pathogen mycelium, but all exhibited high inhibitory rate (>90%) on mycelium growth of P. capsici, B. cinerea, S. sclerotiorum and B. sorokiniana at ten times diluted liquid. The strains all showed strongest inhibitory effect on S. aureus. Tissue experiment showed that the efficacy of the strains on B. cinerea all higher than 65%, and different strains showed different therapeutic and protective effect on B. cinerea, among which, YL002 showed best therapeutic effect (77.31%) and TB showed best protective effect (75.30%). Potted plant experiment showed that the efficacy of the strains on B. cinerea, P. capsici and P. cubensis all higher than 60%, among which, YL001 showed best therapeutic effect (78.21%) on P. cubensis, YL002 showed best protective effect (77.73%) on B. cinerea and TB showed best protective effect (76.13%) on P. capsici; The efficacy of strain TB to B. graminis was higher than 60%, which was better that the other strains; only strain TB showed some protective effect on E. turctcum.The methanol extracts of the three strains all showed strong effect on mycelium growth of tested pathogens, among which, YL001 showed strongest effect on P. capsici, YL002 showed strongest effect on B. cinerea and TB showed strongest effect on S. sclerotiorum, with EC50 of 35.11, 42.16 and 18.95 mg/L, respectively. The methanol extracts all showed strongest effect on spore germination of tested pathogens, and all exhibited highest inhibitory effect on E. turctcum with EC50 of 28.03, 36.77and 26.40 mg/L. At 1000 mg/L, the efficacy of methanol extracts to B. cirerea (tissue experiment) were all higher than 70% and to B. cirerea and P. capsici were all higher than 65% (potted plant experiment), different strains showed different therapeutic and protective effect on B. cirerea and P. capsici.At 1000 mg/L, the inhibitory rates of ethanol extracts of the three strains on mycelium growth of P. capsici were all higher than 80%. Tissue experiment showed that ethanol extracts of different strains showed different efficacy to B. cirerea, among which, strain YL002 showed strongest therapeutic effect (79.16%) and protective effect (61.66%). Potted plant experiment showed that strain YL002 showed best therapeutic effect on B. cirerea, P. capsici and P. cubensis with 82.19, 70.75 and 81.12%, respectively, and strain TB showed best protective effect with 74.40, 65.87 and 78.92%, respectively.3. Study on the culture characteristics of strains YL001, YL002 and TB showed that the strains growth was in accordance with the general discipline of bacteria growth; The changing trends of glucose and amido nitrogen of the strains were similar, which decreased sharply at first and then kept stable; The optimum fermentation period was 54, 66 and 66h, respectively.4. Statistical methods such as Plackett–Burman design and response surface methodology were employed synthetically to optimize the fermentation medium and conditions together so as to improve antibiotic activity of X. nematophila TB. The optimum composition of the fermentation medium (g/L): peptone 25.60, glucose 5.00, NaCl 5.00, K₂HPO₄ 2.50, and the optimum fermentation condition: fermentation time 54.07 h,initial pH 7.59, inoculation volume 9.95%, inoculation age (OD₆₀₀: 2.00), medium volume (100/250 mL),rotary speed 150 rpm,temperature 25°C. The optimum composition of the industrial fermentation medium (g/L): bran 10.00, cotton cake powder 24.20, bean cake powder 6.41, NaCl 5.00, K₂HPO₄ 5.00, and the optimum fermentation condition: fermentation time 72 h, inoculation age (OD₆₀₀: 2.00), medium volume (50/250 mL), initial pH 7.96, inoculation volume 10.02%, rotary speed 150 rpm. After optimization, the antibiotic activity of strain TB improved by 73.52% and 82.58%, respectively. 5. Experiments of different culture mode on cell growth and antibiotic activity of strain TB showed that 70L fermenter was optimum for the cell growth with DCW of 16.70 g/L, which improved by 25.85 and 41.41% than that of 250 mL flask and 5L fermenter, respectively. Flask was optimum for the antibiotic production with antibiotic activity of 358.3 U/mL, which improved by 10.83 and 14.36% than that of 5L and 70L fermenter.6. The antimicrobial components of the extra and intra cellular metabolite of strain TB was separated based on activity tracking using B. cirerea and B. subtilis as the target pathogens. One compound with antimicrobial activity was separated from the intracellular metabolite, and its structure was determined initially, which was bis (1-hydroxy-2-methoxye thy) phthalate. Five compounds were separated from extracellular metabolite, and their structures are under identification. Activity experiments indicated that 4#and 5# showed stronger inhibitory effect on mycelium growth of B. cirerea, which was 69.1 and 72.8%, respectively; 4#, 5# and 6#showed stronger inhibitory effect on B. subtilis with the inhibition zone of 10.9, 11.6 and 10.2 mm, respectively.