Antimicrobial Activity On Agricultural Pathogenic Fungus From Marine-derived Actinomycetes Secondary Metabolites

The ocean has a vast environment,accounting for about 70%of the earth's area.Unique conditions such as high salinity,high pressure,low temperature,low oxygen,weak light,and even no light have led to a large number of biological populations which different from the land.Microorganisms of marine origin can produce secondary metabolites with novel structure and good activity due to their unique environment in the ocean.The secondary metabolites of actinomycetes account for 70%of tens of thousands of microbial-derived bioactive substances.They are currently widely used in the pharmaceutical and agricultural fields.For instance,the drugs and products are mainly used in the market for antibacterial,antibacterial,and antitumor,which have potential research and development value.This article conducts preliminary research on actinomycetes in the seafloor sediments of the Bohai Bay of China and Arctic regions.The main research contents and conclusions are as follows:1.Actinomycetes were isolated from the sediments of Bohai Bay and the Arctic using 13kinds of isolation medium.75 strains of actinomycetes were isolated from 8 samples of wet soil,and 81 strains of 8 samples were isolated from dry soil.After screening colony characteristics,42 strains of actinomycetes were successfully amplified by PCR.The 42strains of actinomycetes belong to 14 genera,of which Streptomyces is the dominant species.The 16S rRNA gene sequences of strains B200012 and B200099 had the highest similarity rates with the effective strains Agromyces iriomotensis IY0720^T?AB546308?and Pseudolysinimonas kribbensis MSL13^T?EF466129?,which were 98.15%and 98.13%,respectively.They may be potential new species.2.These 156 strains of actinomycetes were cultured on a small scale by fermentation.The fermentation broth was extracted with ethyl acetate,and the antibacterial activity was screened by the paper method.E.coli,Staphylococcus aureus,Candida albicans,and two strains of agricultural pathogenic fungi?Phomopsis asparagi?Fusarium oxysporum f.sp.niveum?were selected as test bacteria.The results showed that 79 strains of actinomycetes had antibacterial effects on at least one test strain,and only one actinomycete had effect on five test bacteria.All the tested bacteria have antibacterial effect.Among them,50 strains had anti-Staphylococcus aureus activity,which were the largest proportion.3.Based on the HPLC chromatogram and antibacterial activity of actinomycetes,the Arctic-derived B200036 was identified as the target strain.Meanwhile,we optimized the culture conditions based on the OSMAC strategy.The optimum pH was determined to be 7.8and the optimum temperature was 37°C.Under the optimum pH and temperature fermentation conditions,the effects of adding trace amounts of lanthanum chloride heptahydrate or L-phenylalanine on the production of active substances by actinomycetes were investigated.As a result,it was found that the antibacterial activity of metabolites against pathogenic bacterium was significantly enhanced in YMG medium supplemented with L-phenylalanine and ISP2 medium supplemented with L-phenylalanine.It is shown that the diameter of the bacteriostatic circle can reach 37 mm and 26 mm,respectively.In order to verify the effectiveness of the optimal fermentation conditions on the actual field,cucumbers were applied to the fermentation broth of the strain under the optimal fermentation medium conditions in the field.We then observe the field control effect of strain fermentation broth on cucumber downy mildew and cucumber powdery mildew.The results showed that the maximum effect of fermentation broth on cucumber downy mildew was 31.61%,and that of cucumber powdery mildew was 82.15%.4.The separation of active metabolites were using modern separation techniques such as silica gel column chromatography,ODS reversed-phase column chromatography,and preparative HPLC.Finally,2 monomer compounds were obtained.The structure of the compound was determined to be bipyridine alkaloid by nuclear magnetic resonance spectroscopy,and comparison with literature data.5.Perform active re-screening on the monomer compound.The results showed that the MIC values of Compound 1 against Fusarium oxysporum f.sp.niveum and Asparagus stem blight were both 8?g·mL^-1,and the MIC values of Compound 2 were greater than 128?g·mL^-1.