| Isoproturon is one of the representative varieties of substituted urea herbicides,which is widely used in agricultural production because of its high herbicidal activity and broad herbicidal spectrum.However,due to heavy use of isoproturon and its long half-life in the soil,it has brought many environmental problems.It was found that the improper use of isoproturon can cause phytotoxicity to crops and lead to reduced production,and isoproturon as well as its metabolites can also affect human health.Therefore,how to effectively remove the residue of isoproturon in farmland soil has become an urgent problem to be solved.The degradation of microorganisms is an important way to degrade isoproturon.In the present study,an isoproturon-degrading strain Sphingobium sp.YBL2,that was isolated by our lab previously,was used as the tested strain to optimize its liquid fermentation conditions and develop its solid microbial agent.At the same time,wheat was selected as the tested crop due to its sensitivity to isoproturon.The colonization of strain YBL2 in wheat rhizosphere,the effect of strain YBL2 on the removal of isoproturon,and the microbial ecological effect of strain YBL2 in the degradation of isoproturon were investigated.The research results of this study can provide theoretical basis and technical support for the bioremediation of isoproturon-contaminated soil.1.Optimization of liquid fermentation conditions of strain YBL2By optimizing the liquid fermentation conditions of strain YBL2 with the combined methodologies of Placket-Burman and Box-Behnken Design,The best medium formula was obtained and as follows:9.38 g/L yeast extract/(NH4)2SO4(mol/mol,4/1),10 g/L glucose,2 g/L K2HPO4,0.5 g/L KH2PO4,2 g/L NaCl,0.6 g/L MgSO4.The best cultivation conditions are:inoculum amount of 4%.32.5℃.180 rpm.pH 7.0 and liquid volume 35 mL/250 mL.After 18 hours of fermentation.the number of viable YBL2 was 2.23×109 CFU/mL.which was 1.28 times the original unoptimized conditions of 1.75×109 CFU/mL.The optimized parameters of the shake flask were used to cultivate YBL2 on a 7.5 L fermentor,and the number of viable YBL2 was 9.96×109 CFU/mL,which was 4.47 times that of the shake flask culture.2.Development of solid microbial agent of strain YBL2Rice bran,vermiculite,corn stalk powder,biochar,chicken manure,earthworm manure,pig manure,and straw fertilizer were selected as carriers to develop the solid microbial agents of YBL2.When the initial inoculation amount of strain YBL2 was 0.92×108 CFU/g,after storage at room temperature for 180 days,the four solid microbial agents using rice bran,chicken manure,pig manure and corn stalk powder as carriers all met the quality standards for the industry of agricultural microbial agents,among which the highest number of viable bacteria using rice bran as a carrier was highest,being 6.76×108 CFU/g.Finally,these four solid microbial agents were used in the degradation of isoproturon in soil,the results showed that the rice bran bacteria agent had the fastest degradation rate for isoproturon,it could not be detected by the 4th day.while the degradation rates of the chicken manure,pig manure and corn stalk powder bacteria agents were 94.28%,81.83%and 69.58%,respectively.3.GFP labeling of strain YBL2 and its colonization in wheat rhizosphereIn order to study the colonization of the strain YBL2 in the wheat rhizosphere,we first labeled the gfp gene on the plasmid No.4 of strain YBL2 and named it YBL2-gfp.The degradation of isoproturon by strains YBL2-gfp and YBL2 and their growth were studied,the results showed that the strain YBL2-gfp was similar to YBL2 in its ability to degrade isoproturon and utilize’ it for growth,indicating that-the introduction of the gfp gene did not affect the growth of strain YBL2-gfp and its ability to degrade isoproturon.According to the subculture experiment,the genetic stability of the strain YBL2-gfp was 95.93%after 10 consecutive passages,which indicated that the plasmid harboring marker gene gfp had a higher genetic stability in the strain YBL2-gfp.The colonization of strain YBL2-gfp on the root surface of wheat was observed by confocal laser scanning microscopy(CLSM).The results showed that strain YBL2-gfp mainly colonized the surface of the meristematic zone,elongation zone and mature zone of wheat roots,while the number of colonies on the lateral roots and root cap of wheat was small.Moreover,no colonization of strain YBL2-gfp was observed inside the wheat root system.The quantitative analysis by plating count method revealed that strain YBL2-gfp(the initial inoculum size was 2.71×108 CFU/g dry soil)could colonize at least for 12 days on the wheat roots surface,and at least 20 days in the thizosphere and non-rhizosphere soils.During the entire cultivation period,the number of YBL2-gfp reached 104-107 CFU/g wheat roots and 105-108 CFU/g rhizosphere soil,and the number of YBL2-gfp in the rhizosphere soil was higher than that of the non-rhizosphere soil(105-108 CFU/g dry soil).4.Degradation on isoproturon in wheat rhizosphere by strain YBL2-gfp and the related ecological effectStrain YBL2-gfp(1.05×107 CFU/g dry soil)was added to the wheat planting soil with isoproturon(0.177 and 2 mg/kg dry soil)by soil mixing,and the degradation efficiency of isoproturon and the wheat growth indexes were measured.The results indicated that in wheat rhizosphere soil supplemented with isoproturon at 2 mg/kg dry soil,the natural degradation rate within 21 days was 15.69%.While isoproturon could not be detected in rhizosphere soil after the inoculation of strain YBL2-gfp,and wheat growth indicators have also been significantly improved,almost restoring to the control level,which indicated that the addition of strain YBL2-gfp could significantly remove isoproturon residues in wheat rhizosphere soil,and relieve wheat phytotoxicity.Through the method of fluorescence quantitative PCR,we quantitatively determined the key gene(pdmA)responsible for isoproturon degradation in strain YBL2-gfp,so as to indirectly quantify strain YBL2-gfp in wheat rhizosphere.the results showed that when the initial density of strain YBL2-gfp in wheat rhizosphere was 9.86×107 copies/g dry soil,the strain density would gradually decrease over time.By the 21st day.the number of strains YBL2-gfp decreased to(2.19-4.51)×107 copies/g dry soil.and the number of strain YBL2-gfp was the highest in the soil added with 2 mg/kg dry soil isoproturon.which was 2.1 times and 1.6 times that of the addition of strain YBL2-gfp but without the addition of isoproturon and the soil supplemented with 0.177 mg/kg dry soil isoproturon treatment.respectively.The high-throughput sequencing was used to further explore the dynamic changes of bacterial and fungal community structure in rhizosphere soil samples.The results showed that the addition of strain YBL2-gfp alone(Y)had no significant effect on the richness and diversity of bacterial community in the soil.however.it would significantly reduce(P<0.05)the richness and diversity of fungal community.Compared with the untreated soil sample(CK).the application of different concentrations of isoproturon(IL or IH)had no significant effect on the richness and diversity of bacterial community in the soil:while in the fungal community,most isoproturon treatments would reduce(P<0.05)the richness and diversity of the fungal community in the soil.Compared with the treatment with isoproturon,most strain YBL2-gfp+isoproturon treatments(ILY or IHY)had no significant effect on the richness and diversity of bacterial community,but would reduce the richness and diversity of fungal community.Species difference analysis indicated that under isoproturon treatment,regardless of bacterial or fungal communities,the genera with significant differences are low-abundance species.In a short period of time,the increase in isoproturon concentration would promote or inhibit certain bacterial genera,while almost all fungal genera would be inhibited as the isoproturon concentration increased.In addition to the concentration-dependent effect of isoproturon,the bacterial genera Anaeromyxobacter and Microvirga also showed time-dependent responses along the incubation time.The addition of strain YBL2-gfp could also promote or inhibit the growth of certain bacterial and fungal genera,and the bacterial genera Sphingobium、Steroidobacter.Massilia、Pseudomonas、Microvirga and Bradyrhizobium and fungal genera Mycoarthris、Staphylotrichum、Trichosporon and Pseudocoleophomas all showed a dependent effect on incubation time. |
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