| The plant disease characterized by pathogenic bacteria infection has been reported in numerous literatures since it has the great detriment to agriculture. The purpose of this study is to establish a standard for the soil sample collection according to index of altitude, soil type, ecological environment and surrounding plants, in doing so, the traditional plate dilution method was performed to separate actinomycetes in the collected soil samples. In the process of separation, the K2Cr2O7 was added into Gause NO.1 medium as the inhibitor. The results showed that 800 strains were separated from the soil samples. After the separation experiment, plate confrontation method was carried out for 322 strains using plant disease fungus as the indicator microorganism during the preliminary screening, the data of this section demonstrated that 221strains have the ability of antifungal bioactivity at least and the species number of anti-Magnaporthe grisea, anti-Verticillium dahliae, anti-Rhizoctonia solani, anti-Fusarium graminearum, anti-Botrytis cinerea Pers was 167, 119, 151, 125 and 130, respectively.In this study, there were strainⅡB 21 and strainⅡR21 have the strongly anti-Magnaporthe grisea bioactivity, by studies on morphology, cultural characteristics, 16S rDNA and phylogenetic tree, consequently, strainsⅡB21 andⅡR21 were identified as Streptomyces. The result of fermentation show that the fermentation liquor of strainsⅡB 21 andⅡR21 also have the ability of anti-Magnaporthe grisea. In order to improve the production of antimicrobial activity compound, we optimized the medium composition and the fermentation time. The result show that the mycelium medium and 30℃,180rpm are the suitable fermentation conditions.Studying on the fermention liquor having the anti-Magnaporthe grisea bioactivity at 24h,48h,72h,96h,120h,144h,168h and 192h during the fermention process by cylinder plate method, respectively, the result of antimicrobial diameter show that the production of secondary metabolies with antimicrobial activity will increase gradually with elongation of permention time concomitantly. The antimicrobial diameter of strainsⅡB 21 andⅡR21 reached the maximum 33.07mm,63.27mm respectively, and the ability will attenuate following the elongation of the permention time, and the optimal permention time is 120h.Correlative amount fermention liquor were treated at 4℃, room temperature, 30℃, 40℃, 50℃, 60℃, 70℃, 80℃, 90℃, 100℃for two hours and 121℃for 20 minutes , 30 minutes respectively, then measurement of the antimicrobial activity using cylinder plate method was carried out when the temperature coming down to room temperature. The result show that anti-Magnaporthe grisea bioactivity of fermention liquor changed a little after treated at 30℃~70℃for two hours, however, its bioactivity decrease significantly even loss its activity when the temperature above 90℃.Adjusting the pH value of permention liquor of strainsⅡB 21 andⅡR21 to pH2.0,pH3.0,pH4.0,pH5.0,pH6.0,pH8.0,pH9.0,pH10.0,pH11.0 with 6.0mol/L HCL and 6.0mol/L NaOH treated in 4℃for 24 hours, and then adjusting the pH value of permention liquor to bake. The part of this research show that the bioactive compounds were stable when the pH value between 5.0 and 8.0, which decreased significantly in strong acid or alkaline, even lost the bioactivity.Since the bioactive compounds of the permention liquor of strainsⅡB21 andⅡR21 were stable when temperature was between 30℃and 70℃or pH value between 5.0 and 8.0, we extracted the permention liquor with the same volume acetic ether, and then dehydrated with Anhydrous Sodium Fulfate for 24 hours, and obtained crude extracts of bioactive compounds through rotary evaporation, which were dissolved in Acetone and then stored at 4℃. The samples of crude extracts had fluorescence tape by TLC through n-butyl alcohol, ethanoic acid (volume ratio was 3:1:1), and the signal were obviously strong. |
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