Separation And Purification Of Active Metabolites Produced By Streptomyces Felleus YJ1 And The Biocontrol Of Sclerotinia Sclerotiorum By Its Crude Extracts

Rape is one of the most important oil crops in our country and even in the world, which limited by Sclerotinia stem rot mainly. Exploiting the antagonistic actinomycetes to control the disease is a safe and effective method. So, it is important for the coming out of new antibiotics against Sclerotinia sclerotiorum to study the active secondary metabolites of antagonistic actinomycetes further. In the study, Streptomyces felleus YJ1 with strong antagonistic activity against S. sclerotiorum screened by preliminary research was selected as the study subject, testing the distribution and physicochemical property of its active metabolites and separating it through a series of methods and technologies, then testing the virulence and antimicrobial spectrum of crude extracts and its control effects against S. sclerotiorum in the indoor pots test, providing the experimental basis for the further development and utilization of antagonistic actinomycetes. The main results were as follows:1. Distribution and physicochemical property test of active metabolites produced by actinomycete YJ1Through a series of tests in the part, it was sure that active metabolites were widely distributed in the fermentation supernatant and mycelia, inhibition rates were 91.15%,86.95% respectively. However, the mycelia soak had poor stabilities in the pH and thermal conditions, which was not conducive for the further extraction. Therefore, we only selected the supernatant to research in the next step, the mycelia part could be abandoned directly. Meanwhile, active substances in the supernatant had good pH and thermal stabilities, inhibition rates reached 88.51% or more, but only under acidic conditions, high temperature would influence its activities, so we should avoid heating the active metabolites in acidic conditions. Through comparing the extraction effects of dichloromethane, ethyl acetate and n-butanol for active substances under different pH conditions, we ensured that n-butanol with pH10 was the best extraction condition. Moreover, results of the solubility tests showed that, active metabolites had excellent solubility in the alcohol, indicating that the polarity of active substances was medium to large.2. Separation and purification of active metabolites produced by actinomycete YJ1Pretreatment, extraction, thin layer chromatography (TLC), silica gel column chromatography and high performance liquid chromatography (HPLC) analysis of the active metabolites were included in the part. The research results were:(1) In the pretreatment, adjusting pH of the fermentation broths to 5 to remove impurities; (2) The solvent system was determined as n-butanol:methanol= 5:1(v/v) by TLC; (3) Silica gel column chromatography adopted gradient elution, eluent were n-butanol and methanol; (4) Reaction conditions for HPLC analysis:chromatographic column:Kromasil 100-5C18 250 X 4.6mm; mobile phase:acetonitrile:H2O=80:20(v/v); current speed:0.8mL/min; detection wavelength:210nm; column temperature:25℃.3. Virulence and antimicrobial spectrum assays of the crude extractsCrude extracts virulence assay results showed that the EC50 was 297.3719μg/mL, EC90 was 1312.1999μg/mL, the correlation coefficient(r) was 0.9742. According to the EC90, preparing standard solution of the crude extracts, then testing its inhibition effects against 15 kinds of plant pathogenic fungi, using broths of no bacterium as stock solution control. The results showed that, fermentation broths and its crude extracts both had good inhibition effects in different extent against Alternaria alternate, Alternaria mali, Alternaria solani, Alternaria solani, Botrytis cinerea, Colletotrichum orbiculare, et. al, and the inhibition rates were all above 60%. Antibacterial spectrums were also basically identical, but the inhibition rates of the broths were generally a little higher than the crude extracts’.4. The biocontrol of Sclerotinia sclerotiorum by actinomycete YJ1 fermentation broths and its crude extractsThe indoors trials indicated that actinomycete YJ1 fermentation broths and its crude extracts could both limit the expansion of disease spots in some degree through seeds soaking, root irrigation, spraying leaves and in vitro tests. Seeds soaking with fermentation broths and its crude extracts, the highest control effects were respectively 85.24%,65.04%, and root irrigation’s highest control effects were respectively 59.54%,57.80%, whereas 60.00%,55.65% when spraying leaves. In vitro tests, the control effects of being inoculated immediately after spraying were the best, reaching 68.07%,55.42% respectively.