Study Of Antibiotic Actinomycetes And Its Antagonistic Substance Against Botrytis Cinerea

Actinomycete is one kind of very important microbiotic populations for agriculture production. Its secondary metabolites are the major sources of agricultural antibiotics. In this study, an antagonistic actinomycete strain S-091for tomato grey mold pathogen Botrytis cinerea was isolated from soil samples and its biological classification, antimicrobial activity evaluation, antimicrobial mechanisms, active component(s) isolation and purification and molecular structure characterization have been conducted.Only S-091among112actinomycete strains isolated from33soil samples collected from different regions across China was found to be with strong suppression activity against Botrytis cinerea. Besides, it shows significantly antagonistic activities against several plant fungal pathogens. S-091was identified with classic actinomycete classification principles combined molecular identification methods. Its mycelium is smooth with coiled spore-bearing mycelium and the spores are oval and finely echinulate on the surface. Based on morphological characteristics, cultivation, physiological and biochemical properties and16s rDNA sequence analysis, S-091was identified as Streptomyces ahygrocopicus var. wuyishan.With single-factorial evaluation and orthogonal experimental design, the fermentation conditions for S-091were selected and optimized. Based on the results, we found that starch content affects most significantly the S-091antibiotic productivity than peanut cake. Among the tested microelements, KC1is a key factor for S-091antibiotic productivity. The optimum medium was composed of1%peanut cake,4.5%starch soluble,0.4%KC1,0.2%yeast extract and0.1%NH4NO3. The optimum fermentation conditions are temperature at32℃,140r·min-1and medium pH at7.0. It is suitable for250mL triangular flask to contain100-125mL of fermentation medium.The suppression mechanisms of S-091against Botrytis cinerea was conducted through microscopic investigation. When mycellium and spores of Botrytis cinerea were treated with the crude extract of S-091broths, the cell contents of the fungal pathogen were concentrated, and the mycellium ceased growth and the spores could not germinate. The cells at the growth point of mycellium were unmormally swelled after treatment. There were no any evidences that cell wall and cell membrane of Botrytis cinerea were broken and its cell contents outflowed ater treatment with the crude extract of S-091broths. Additionally, conductivity detection experiment showed that there is no electrolyte leakage of the pathogen cells treated with the crude extract.The physicochemical property of antibiotics from fermented broth of the strain S-091were studied. The results showed that the antibiotics from fermented broth was stability to visible light, ultraviolet light and heat, but lost antibiotic activity in acidic conditions of pH value less than5. The test result of organic solvent extraction to fermented broth showed that the active ingredients of fermentation liquid were insoluble in ethyl acetate and other organic solvents. The deposit when it was under pH value5was not antagonistic activity to pathogen, and the deposit from fermented broth when it was added5times acetone was not antagonistic activity to pathogen either. The active ingredients of fermentation liquid were tested by pH paper chromatography, Czech eight solvent system and Betina solvent system paper chromatography and paper electrophoresis, the results showed that active ingredients were composed by two or more components from fermented broth, and the molecular polarity of active components was nutral or large, one part of which was alkaline, the other part was strongly alkaline.Using polymer adsorbents, silica gel column chromatograph and combined with other methods, the antibiotic crude extract of S-091broths was isolated and then purified with HPLC to produce the pure compound of the active component. It is an amorphic white powdery material. Through mass spectrometry and nuclear magnetic resonance spectrum analysis, the active component is confirmed as one kind of tetraene macrolide antibiotics with molecular weight of695and its chemical structure formula C35H53NO13, and it was named (1S,3R,5R,7E,13E,15E,17E,19E,21S,23R,24S,25R)-21-((2R,3S,4S,5S,6R)-4-amino-tetrahydr o-3,5-dihydroxy-6-methyl-2H-pyran-2-yloxy)-12-ethyl-1,3,5,25-tetrahydroxy-11-methyl-9-ox o-10,27-dioxa-bicyclo[21.3.1]heptacosa-7,13,15,17,19-pentaene-24-carboxylic acid. It was found to be tetramycin A through literature retrieval, which has not been reported to be used as an agricultural antibiotic.