Study On The Preparation And Application Of Solid State Microbial Agent With Streptomyces Alboflavus TD-1

In recent years,due to the long-term over-reliance on chemical fertilizers in China’s agriculture,the soil has been slabbed and the micro-ecology of the soil has been badly hit,in which a large number of beneficial microbiological flora have been destroyed.Microorganisms are capable of releasing secondary metabolites during normal growth,enhancing plant resistance,stimulating crop root growth and reducing crop yield loss.There are specific microorganisms within the microbial agents that can ensure the supply of plant nutrients through their life activities,produce growth hormones and inhibit the formation of harmful microorganisms.Therefore,the application of high quality microbial mycorrhizal agents in soil can provide a new direction to solve the current problems of soil ecological quality and crop yield in agriculture.In this thesis,a strain of Streptomyces alboflavus TD-1 with independent intellectual property rights is taken as the research object.Firstly,the single factor optimization experiment of liquid fermentation medium and fermentation conditions is carried out,and the ISP4 medium which is conducive to the spore propagation of S.alboflavus TD-1 is selected.The optimal liquid culture conditions are determined as follows:the cultivation time is 6 days,the p H is 6,the cultivation temperature is 30℃,the inoculation amount is 10 m L/100 m L,and the bottling amount is 100 m L/250 m L.The components of the medium were optimized by response surface methodology.Taking the amount of S.alboflavus TD-1 in each gram of dry substrate as the response value,three significant factors of soluble starch,sodium chloride and manganese chloride are screened.The optimal fermentation conditions as follows:soluble starch is0.8005g,Na Cl is 1.2338g and Mn Cl₂is 0.1200 g.The maximum response is predicted to be 0.72 g/g dry substrate.The actual response value is 0.68 g/g dry substrate,which is closed to the predicted value.Secondly,through the optimization of solid-state fermentation substrate and fermentation conditions,the optimal solid-state fermentation process with S.alboflavus TD-1 is:low value rice 25 g,cassava powder 1 g,cottonseed cake powder 2 g,bran 2g,water content 30%（w/w）,K₂HPO₄ 0.50%（m/v）,Mg SO₄ 0.30%（m/v）,Fe SO₄ 0.02%（m/v）,Na Cl 0.40%（m/v）,Mn Cl₂ 0.10%（m/v）,Co Cl₂ 15 mg/m L,1 g chitin,p H is 5,and the inoculation amount of seed liquid is 5 m L/30 g,the cultivation temperature is30℃for 8 days.After fermentation,the effective number of live bacteria per gram of fermentation substrate are 1.067×10⁹ as measured by the national standard of Pesticide Microbial Agent.In order to improve the stability of solid agent with S.alboflavus TD-1,the process conditions for the addition of some additives were optimized in the thesis,and the suitable types of additives for solid agent with S.alboflavus TD-1 are derived as follows:carrier is soap clay,protectant is humic acid,dispersant is PEG1000,and wetting agent is saponin powder.After orthogonal optimization,the best combination of additives is soap clay 30%（w/w）,humic acid 1%（w/w）,PEG1000 2.5%（w/w）,soap powder 4%（w/w）,and the initial fungicide compound mixed evenly that the best formulation of S.alboflavus TD-1 fungicide,the suspension rate is measured at 25.19%,the wetting time is 149.35 s and the effective number of live bacteria are per gram 2.25×10⁸.The growth experiment of potted chive seedlings is carried out by the fermentation solution with S.alboflavus TD-1 and mycelia of the liquid fermentation obtained by compounding with other microbial strains,and it can be concluded that the treatment group with the application of mycelia with S.alboflavus TD-1 has a significant effect of promoting growth on the plant height of chives,which increases by 21.21%compares with the control group,while the treatment group with the addition of mycelia with both S.alboflavus TD-1 and Bacillus subtilis meanwhile,the preliminary investigation of this thesis finds that spraying S.alboflavus TD-1 fermentation solution on the leaves of tomato plants has a certain effect on the control of gray mold,and the diameter of the spots on the treated leaves is significantly smaller than the diameter of the spots on the leaves of the control group,and the control effect can reach 63.49%.Finally,the pot experiment shows that the application of 3%（m/v）of solid agent with S.alboflavus TD-1 in soil can increase the activity of disease-resistant defense kinases related to tomato leaves such as PAL,POD and SOD to a certain extent,502.12U,55.21 U and 34.79 U,respectively.