Screening,Identification And Degradation Characteristics Of Epoxiconazole Degrading Bacteria

As a new type of absorbent triazole containing fluorobactericide,epoxiconazole can inhibit the synthesis of ergot sterol,hinder the formation of the cell wall of pathogenic bacteria,and has a good prevention and treatment effect on powdery mildew and stripe rust of cereal crops.With the widespread application of triazole fungicides in agricultural production,the environmental residue problem has attracted people's attention.Microorganisms play an important role in the repair of pesticide residues in the environment,and screening out microorganisms that can efficiently degrade epoxiconazole in the environment is of great significance for reducing pesticide pollution.In this study,strain F1was isolated and screened from the surrounding soil of the pesticide plant for first time.The strain identification was carried out through the morphology,physio-biochemical characteristics and 16S r RNA sequence analysis.The environmental conditions for the degradation of epoxiconazole by strain F1 was optimized.Then,the bacterium was labeled with green fiuorescent protein,and the transformant strain F1-GFP was obtained.Finally,the effect of strain F1 on the remediation of epoxiconazole contaminated soil was studied in the soil-vegetable system,which provided the microbial resources and scientific basis for the remediation of epoxiconazole contaminated soil in the future.The main findings are as follows:1.Using a single carbon source separation method and a flate streak method,eight degradation strains F1?F8 with epoxiconazole as the only carbon source were isolated and screened from the soil,and the degradation rate of epoxiconazole of strain F1 was 90.7%after 6 days of culture under the condition of 30?and p H value of 7.0,therefore,efficient degradation strain F1 was selected as the research object of this project.Through observation of the colony morphology,physio-biochemical characteristics and 16S r RNA sequence similarity and phylogenetic analysis,the genetic relationship of strains F1 was closest to Pseudomonas kunmingensis HL22-2,and the preliminary identification of strain F1 was Pseudomonas sp..2.The degradation characteristics of epoxiconazole degrading bacteria were studied by single factor experiment and orthogonal experiment.The results showed that the degradation rate is up to 90.4%After 6 days of cultivation,with the initial concentration of epoxiconazole was 20 mg/L,the temperature was30?,the p H value was 6.0?8.0,the OD₆₀₀ value of inorganic salt medium was0.10 by adding the bacterial suspension.Furthermorethe,increase of inoculation amount can significantly increase the degradation rate of epoxiconazole.The result of orthogonal experiment shows that the major factor affecting the degradation rate of epoxiconazole for strain F1 is temperature>p H>substrate concentration>inoculation amount in ture,and the optimal combination of degradation characteristics of epoxiconazole is 30?,Ph=7.0,inoculation amount 0.50,substrate concentration 20 mg/L.3.The three-parent grafting method was used to label the epoxiconazole degrading bacteria with GFP,and it was verified that they could be colonized in Brassica chinensis L..By mixing and cultivating the donor bacteria E.coli TG1/p PROBE-p Tet^r OT containing GFP gene,the helper bacteria DH5?/p RK2013 and the recipient bacteria strain F1 with different antibiotic resistance in a volume ratio of 1:1:1,the GFP gene in the donor bacteria was induced to transfer into the target strain F1.Strain F1 and transformant F1-GFP showed no significant difference in growth kinetics and degradation performance of epoxiconazole.The labeled strain F1-GFP were inoculated in Brassica chinensis L.,which Observed by fluorescence microscope,obvious green fluorescence could be observed in the root tissues of Brassica chinensis L.by fluorescence microscopy,indicating that the labeled strain F1-GFP can colonize in the tissues of B.chinensis L..4.By carrying out the greenhouse pot experiment,the effects of inoculation strain F1 on the degradation,residue and B.chinensis L.absorption of epoxiconazole were studied in epoxiconazole contaminated soil.The results indicate that the degradation rate of epoxiconazole in soil was accelerated with the increase of the amount of inoculated bacteria from 2%to 8%.Under the condition of 8%inoculation amount,the degradation rate can reach 92.1%in only6 days.In the soil-pakchoi system,compared with the non-inoculated control group,the residual amount of epoxiconazole in the soil was reduced by26.5%?63.7%after inoculation,and the residual amount of epoxiconazole in the root part of the plant was reduced by 17.3%?28.7%,the residual amount of epoxiconazole in the stem and leaf part of the plant was reduced by 24.3%?51.9%,the biomass of stem,leaf and root increased significantly(P<0.05),with an increase of 42.8%and 50.8%respectively.These results show that the degradation rate of epoxiconazole in soil can be improved by exogenous adding of epoxiconazole degrading bacteria,so as to reduce epoxiconazole residue in soil and reduce the absorption of epoxiconazole by B.chinensis L..The results indicated that the strain F1 had a certain application potential in the remediation of epoxiconazole contaminated soil.