Isolation And Identification Of Functional Microorganism In Humus Organic Fertilizer And Study Of Microbial Agent

Excessive use of pesticides and fertilizers during agricultural production have caused a series of problems such as soil acidification and compaction,pollution of rivers and groundwater,pesticide residues in agricultural products.In recent years,the microbial agent have become research hotspots with advantages of green,safety,high efficiency and low pollution.It is an important way to establish resource-saving and environmentally friendly agriculture by replacing or reducing the use of traditional pesticides and fertilizers with functional microbial agent.In this study,the identification medium was used to isolate bacteria with starch-degrading,cellulose-degrading,biological nitrogen-fixing and biological antagonism ability from humus organic fertilizer.The single factor experiment and orthogonal experiment were used to study the growth characteristics,carbon source,nitrogen source,inorganic salt and mixed culture conditions of various functional strains.The wettable powder was selected as the agent formulation,and the carrier,wetting agent,dispersant,heat stabilizer and ultraviolet protective agent were screened.The pot experiment was used to preliminarily study the application effect of wettable powder on soil microorganisms and soil enzyme activities of tomato rhizosphere.In the meantime,the experiment was carried out by using the Oxford cup method and plate confrontation method to study biological antagonism of Bacillus methylotrophic BA18 against pathogenic fungi.The bacteriostatic metabolites of Bacillus methylotrophic BA18 were analyzed and identified by microbial metabolomics technology.The study results were as follows:?1?There were thirteen amylase-producing strains,twelve cellulase-producing strains and nine nitrogen-fixing strains were finally isolated.The high-yield amylase strains?BA18,BA16?,cellulase strains?CD4?,and nitrogenase strain?NF7?were identified as Bacillus methylotrophicus,Bacillus amyloliquefaciens,Paenibacillus taichungensis,Paenibacillus glucanolyticus respectively.?2?Based on the intergrowth relationship between cellulose-decomposing bacteria and nitrogen-fixing bacteria.The functional microorganisms CD4 and NF7 were selected for mixed culture.The results showed that the optimal medium formulation were as follows:molasses 20 g/L,tryptone powder 12 g/L,K₂HPO₄ 4 g/L,culture conditions were as follows:30°C,p H 7.5,130 r/min,according to the inoculation amount 12%,inoculation ratio 1:2?CD4:NF7?,the number of viable bacteria of mixed culture could reach 1.06×10⁸ CFU/m L.The mixed culture of two functional strains could meet the requirement of the number of viable bacteria of liquid bacterial fertilizer,which laid a good foundation for the development of compound microbial agent.?3?The formula of wettable powder were as follows:fermentation broth 70%,diatomaceous earth 10%,saponin powder 1.6%,sodium tripolyphosphate 14.4%,dipotassium phosphate 3%,xanthan gum 1%.The results of quality were as follows:wetting time of 35 s,suspension rate of 87.16%,p H value of 8.74,water content of 3.87%,live bacteria count of 3.22×10⁷CFU/g.The tomato pot experiment results showed that wettable powder?prepared into water suspension at a ratio of 1:100 and poured into the soil?affected the rhizosphere microorganisms and enzyme activities.The application of wettable powder could promote the growth of bacteria and actinomycetes in tomato rhizosphere soil.There was no significant difference in the total number of fungi in different treatment groups.At the 35th day,the activities of catalase,urease,and alkaline phosphatase in the rhizosphere soil of tomato in the wettable powder treatment group were higher than those in the fertilizer treatment group and sterile water treatment group.?4?The biological antagonistic characteristics of Bacillus methylotrophicus BA18 strain were studied.The results showed that the BA18 strain had obvious antagonistic effects on Gibberella saubinetii,Botrytis cinerea and Aspergillus niger,the supernatant and crude extracts had obvious inhibition on Gibberella saubinetii and Botrytis cinerea.The main antibacterial substances such as palmitic acid,azacitidine,3-hydroxybenzoate,myristic acid and salicylic acid were isolated and identified from the n-butanol extracts.The bacteriostatic protein related substances such as surfactin synthase subunit,polyketide synthase Pks N,polyketide synthase Pks L and flagellar associated protein were isolated and identified from the crude protein.