| In this thesis, a strain of actinomycete (No. 19G-317) isolated from the soil of natural conservation area of Sanjiangyuan Nature Reserve was chose as the main research object, the antifungal activity, the biology characteristics, taxonomic status, fermentation condition optimization, isolation and identified of bioactivity compounds from strain 19G-317 metabolites were studied. The main results and conclusions were outlined as follows:(1) The fungicidal activities of the fermentation product of actinomycete strain 19G-317 were evaluated by mycelium growth rate, in vivo inhibition and potted plant experiment against plant pathothic fungi and bacteria.The bioassay results showed that both of fermentation broth, extract product of mycelium and fermentation broth not only have stronger bioactivity against 22 tested plant pathogenic fungi, but also have certain inhibition activity against 4 tested plant pathogenic bacteria. The mycelium growth inhibition rate of extract product of actinomycete (No. 19G-317) mycelium were over 90 % to 8 tested plant pathogenic fungi; meanwhile, the mycelium growth inhibition rate of extract product of actinomycete (No. 19G-317) fermentation broth were over 90 % to 11 tested plant pathogenic fungi under the concentration of 1 mg/mL; The results of spore germination test showed that the inhibition rate of the fermentation broth and the extract product of mycelium and the fermentation extraction were 97.30 %,98.99 % and 100.00 % to Botrytis cinerea under the concentration of 5 mg/mL. Tissue experiment showed that the efficacy of the fermentation broth, the extract product of mycelium and the fermentation extraction were 72.17 %,76.95 % and 88.63 % against Botrytis cinerea under the concentration of 10 mg/mL. Furthermore, the control efficacy of the extract product of mycelium and the fermentation extraction were all 100% against Sclerotinia sclerotiorum under the concentration of 10 mg/mL. Potted plant experiment showed that the fermentation products exhibited a protective efficacy of 23.80 %,38.60 %,63.35 % and a therapeutic efficacy of 34.24 %,35.17 %,43.80 % against Phytophthora capsici, Botrytis cinerea and Blumeria graminis, the extract product of mycelium were 43.80 %,47.59 %,59.43 % and 42.74 %,44.57 %,45.09%; The efficacy of the fermentation broth,the extract product of mycelium were 30.60 % and 49.19 %, 45.08 % and 60.42 % against Gaeumannomyces graminis and Rhizoctonia cerealis.(2) The biology characteristics and passage stability of the strain 19G-317 were determined. The results showed that the growth of the strain 19G-317 need more oxygen, that is, it was aerobic microorganism; the favorite PH value for the strain growth ranged from 6.8 to 8.4, and the optimum PH was 7.6; The osmotic pressure for colony growth of the strain was much broader, it can grow constantly on the concentration of NaCl ranged from 0.5～20 g/L. However, the strain was sensitive to ultraviolet radiation, the colony growth become weaker after 1h ultraviolet radiation; Furthermore, the stability result showed that heredity character of the strain 19G-317 was stable.(3) Based on morphological characteristics, growth characteristics, biochemical and physiological characteristics and 16S rDNA sequence of the strain 19G-317, it was identified as Streptomyces flavus sp. The 16s rDNA sequences have been deposited in GenBank with the accession numbers EU872020.(4) Statistical methods such as Single factor, Plackett–Burman design and response surface methodology were employed synthetically to optimize the fermentation medium and conditions of actinomycete strain 19G-317. The optimum composition of the fermentation medium (g/L): Maltose 50.8 g/L,Tryptone 10.5 g/L,NaCl 2.5 g/L,CaCO3 2.0 g/L;and the optimum fermentation condition: medium volume 125mL/250mL,rotary speed 195 r/min,temperature 28℃,inoculation volume 7 %,initial PH 7。After optimization, the antibiotic activity of strain 19G-317 improved by 22.67 % and 38.30 % against Botrytis cinerea and Bacillus subtilis, respectively.(5) Stability test of fermentation products of strain 19G-317 had been carried out to acid, alkali, temperature and ultraviolet radiation. The fermentation products of strain 19G-317 were stable to temperature, but it was not stable to acid, alkali and ultraviolet radiation. The inhibition rate of fermentation products of strain 19G-317 against Botrytis cinerea and Bacillus subtilis continued to adjust downwards, in line with the rising temperature and extended handling time. The inhibition rate of fermentation products of strain 19G-317 against Botrytis cinerea and Bacillus subtilis under acidic or alkali conditions were lower obviously than under neutral conditions. After 45 min ultraviolet radiation, the inhibition rate of fermentation products of strain 19G-317 against Botrytis cinerea and Bacillus subtilis decreased by 30.83 %,8.7 % than that 5 min ultraviolet radiation. Furthermore, study on the culture characteristics of strain 19G-317showed that the strains growth was in accordance with the general discipline of microorganism growth.(6) 14 compounds were isolated from fermentation products of strain 19G-317 and the chemical structures of 8 compounds were identified. Those chemicals include 1-methyl-9-hydroxymethylphenazin (A),β-Sitosterol (C), stearic acid(D) ,Ethyl-hexadecanoate(H), 4- alkene -1,11-(1,5-pentadiol)- Decalactone(J), 5- alkene -1,18- Octadecane diacid, dimethyl ester(K), 3,4,6- triacetic acid -1- cyclohexene(L), cyclo-(Ala-Phe)(M).The bioassay results of the chemicals for the actinomycete strain 19G-317 showed that the main bioactivity components belongs to middle or high polarity compounds, the further bioassay indicated that three compounds(M,N,O) have stronger inhibitory effect on Botrytis cinerea and Bacillus subtilis, the inhibition zone diameter of three compounds were over 11mm against Bacillus subtilis, the inhabition rate were over 59.67 % against mycelium growth of Botrytis cinerea, and over 77.9 % against spore germination of Botrytis cinerea. |
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