Screening Of Soil Actinomycetes And Analysis Of Their Activity Against Fusarium Graminearum In The Sharp Pointed Long Bracted Fir Forest Of Sejila Mountain

As one of the important microbial resources,soil actinomycetes can produce complex,diverse,novel,and highly biologically active metabolites during their metabolism.Some have even been developed as antibiotics,which can inhibit viruses in soil,animals,plants,and humans.According to relevant statistics,among microbial resources,actinomycetes produce a relatively large proportion of agricultural antibiotics.Many important agricultural antibiotics that have been widely used come from the genus Streptomyces.More than 1000 species of Streptomyces have been found mainly in soil environments with good air permeability and low water content.Therefore,the study of soil actinomycete resources is of great significance for the further development of agriculture.In this study,soil samples were collected from 5 sample sites in the fast-pointed long-bracted fir forest of Sejila Mountain,and culturable soil actinomycetes were cultured and isolated.Using Fusarium graminearum as the main indicator bacteria,antibacterial activity screening was conducted,with a view to discovering actinomycete resources in soil microorganisms that have the effect of resisting plant pathogenic fungi.46 strains of soil actinomycetes were isolated through cultivation,and a cross confrontation experiment was conducted with the 46 strains of soil actinomycetes isolated using Fusarium graminearum as an indicator bacterium.A total of 8 strains with antibacterial activity were selected;Select different media to culture 8 strains of actinomycetes and observe the morphological characteristics of their colonies.The most suitable media for their growth has been found.Using the confrontation method alone often results in false positive results due to the reducibility of the sample to be tested.Therefore,the filter paper method is used to screen the antibacterial activity of these 8 strains after fermentation to ensure the reliability of the experimental results.The test results showed that the crude extracts from the fermentation broth of eight strains all had antibacterial activity.Using Rhizoctonia solani as an indicator bacterium,it was found that three of the eight strains screened had antibacterial activity against them,namely,ZLS22-037,ZLS22-006,and ZLS22-009.Therefore,16 S r DNA sequencing was performed on these three strains,and an evolutionary tree was constructed using NCBI database and MEGA software for strain identification and analysis.The results showed that the strains ZLS22-037,ZLS22-006,and ZLS22-009 may be Streptomyces griseus,Streptomyces azuleus,and Streptomyces citri.The diameter of the bacteriostatic ring of ZLS22-037 against Fusarium graminearum and Rhizoctonia solanacearum was above15 mm.Therefore,a single factor experimental design was used to screen the fermentation time,rotation speed,liquid loading,p H,carbon and nitrogen conditions of ZLS22-037 strain,in order to understand the fermentation conditions with better antibacterial activity among various factors,and to prepare for further research on the improvement of fermentation conditions and secondary metabolites of ZLS22-037 strain.The results showed that:(1)The antibacterial effect of ZLS22-037 showed a trend of first increasing and then decreasing with the increase of fermentation time,with the largest diameter of bacteriostatic ring at120 hours of fermentation time.At the initial stage of fermentation(24-72h),the quality of bacteriostatic secreted by strain ZLS22-037 is low,resulting in no or low bacteriostatic effect;At the late stage of fermentation(168-192 h),the bacteriostatic effect significantly decreased,possibly due to the basic depletion of nutrients in the fermentation broth,resulting in the decomposition and utilization of bacteriostatic substances secreted by ZLS22-037 strain.(2)As the rotational speed of the thermostatic shaking table increases,the diameter of the bacteriostatic ring of the fermentation broth of strain ZLS22-037 also increases.When the rotational speed reaches 210 r/min,the diameter of the bacteriostatic ring of strain ZLS22-037 is the largest,but if the rotational speed continues to increase,the diameter of the bacteriostatic ring of the fermentation broth decreases.(3)When the liquid content of ZLS22-037 is 40% of the bottle capacity,its bacteriostatic ring diameter is the largest.(4)When the p H of the fermentation medium is 6.4,the diameter of the bacteriostatic ring is the largest,and the average bacteriostatic ring diameter is 2.84 mm larger than that at p H 7.2.Therefore,this is the optimal fermentation p H.(5)Different carbon and nitrogen sources and the amount of the same carbon and nitrogen source have an impact on the growth and antibacterial activity of ZLS22-037 strain.Among the 10 carbon source treatments and 6 nitrogen source treatments tested,when 5g of sucrose was selected as the carbon source,the bacteriostatic circle was the largest,and when peptone(5 g)and potassium nitrate(10 g)were selected as nitrogen sources,the bacteriostatic circle diameter was close to 20 mm.Therefore,the optimal carbon source was to add 5 g of sucrose to the original culture medium,The best nitrogen source is to add 5 g peptone or 10 g potassium nitrate to the original culture medium.