Characteristics And Mechanisms Of Dichlorvos Degradation By Trichoderma Atroviride Strain T23

Dichlorvos(dichlorovinyl dimethyl phosphate,C₄H₇Cl₂O₄P,DDVP)is a highly effective,broad-spectrum,moderate toxic organophosphorus insecticide,and widely used in crop production,food storage pest control and even the treatment of parasitic infections in livestock and poultry.DDVP residues have caused widespread contamination of water bodies,agricultural soils,crops,aquatic products,livestock and poultry products,etc.,posing a serious threat to agricultural products and human health.Fungi can degrade organic pollutants in the environment by chemically modifying or affecting the bioavailability of compounds due to their mycelial network.Rapid formation and simultaneous remediation of multiple contaminants in complex environments are of increasing interest.As a naturally widespread,multifunctional fungus,Trichoderma is ecologically adaptable with the ability to degrade many environmental pollutants.In this study,we used DDVP as a target,studied Trichoderma atroviride strain T23 degrading DDVP on the histological,enzymatic and molecular levels,revealed its enzymatic mechanism,functional gene and expression pattern and enriched the underlying theory of fungal biodegradation of organophosphorus pesticides.Specific findings were as follows.(1)Tolerance of T.atroviride T23 to DDVP stressThe effects on the metabolites of T.atroviride T23 were analyzed under 300?g/mL dichlorvos stress.The analysis showed that the differential metabolites were mainly concentrated on amino acid metabolism,amino saccharide and nucleotide sugar metabolism,glyoxylic acid and dicarboxylic acid metabolism,and tricarboxylic acid cycle,which associated with primary metabolism,as well as riboflavin metabolism,methane metabolism,pantothenic acid and Co A biosynthetic pathway which associated with secondary metabolism.The relative levels of tolerance-related betaine and its precursor indoles were significantly increased at 24 h,with the lower DDVP concentration the relative content of tolerance-related compounds began to decline,confirming that the T.atroviride T23 adapted to DDVP stress by adjusting the levels of compounds in tolerance-related metabolic pathways.(2)Enzymatic degradation of DDVP by T.atroviride T23Adsorption of dichlorvos by Trichoderma viride T23 mycelia was detected by SEM-EDS,however,no characteristic element Cl of DDVP was detected.The degradation of DDVP by living and non-living mycelia was identified by GC-FPD,and it was found that the degradation ability of non-living mycelia to DDVP was consistent with that of DDVP hydrolyzed,which was significantly lower than that of living mycelia.Dichlorvos can induce the expression of intracellular and extracellular enzymes of T.atroviride T23.When the concentration of DDVP reached to 500 g/mL,the degradation rate of DDVP by intracellular and extracellular enzymes were 54.88%and 14.48%,respectively,and the intracellular enzyme activity was always higher than that of extracellular enzyme.The activity of lactonase,arylesterase and peroxidase(POD)in T.atroviride T23 increased firstly and then decreased induced by DDVP,and the content of paraoxnase1 was also increased.The changes of above enzyme activities were positively correlated with the degradation content of DDVP,which provided information for the identification of the degradation products of DDVP and the screening of DDVP degradation enzymes.(3)Identified metabolites and proposed degradation pathways of T.atroviride T23for the degradation of DDVPThe main products of DDVP degraded by T.atroviride T23 were identified and the pathways of degradation of DDVP were proposed.The degradation products of DDVP were ethanol,acetic acid,dichloroacetaldehyde,2,2-dichloroethanol,2,2,2-trichloroethanol,dichloroacetic acid,ethyl dichloroacetate,2,2-dichloroethyl acetate,dimethyl phosphate,trimethyl phosphate,(Z,E)-2-chlorochloridyl phosphate dimethyl ester identified by GC-MS,and PO₄^3-and Cl^-were identified by IC.It was assumed that there were two main degradation pathways:Pathway I,DDVP firstly was transformed to dimethyl phosphate and dichloroacetaldehyde through P—O bond cleavage,and then,dimethyl phosphate was gradually metabolized to PO₄^3-,CO₂ and H₂O,and dichloroacetaldehyde was oxidized to dichloroacetic acid or reduced to 2,2-dichloroethanol.Some of the 2,2-dichloroethanol was further converted to 2,2,2-trichloroethanol and the residues was dechlorinated to ethanol.Pathway II,DDVP generated a dechlorination reaction to produce(Z)-2-vinyl chloride phosphate dimethyl ester or(E)-2-vinyl chloride dimethyl phosphate,which produced by dehydration of 2-chloroacetaldehyde to trimethyl phosphate and gradually converted to PO₄^3-.Degradation of DDVP residues in natural water using T.atroviride T23 revealed that the composition of the degradation products was similar to that of the degradation products under laboratory conditions.The degradation products of T.atroviride T23 to DDVP residual in the natural water sample were consistent with the composition of the degraded products under the laboratory conditions.The half-life of DDVP in the sterilized natural water sample after treatment of T.atroviride T23 was 54.6 h slightly lower than that of the unsterilized natural water sample(61.7h).(4)Expression of T.atroviride T23 cytochrome P450 genes and the function of TaCyp548-2 on the transformation of DDVP intermediate product39 cytochrome P450 genes with complete open reading frames were cloned from T.atroviride T23 genome,which encoded cytochrome P450 proteins were distributed in 29 families under 21 clans.There were four cytochrome P450 genes associated with primary metabolism,14 associated with secondary metabolism,and 21 associated with degradation of xenobiotics.The P450 genes associated with degradation of xenobiotics were analyzed by q RT-PCR in the presence of DDVP induced at 0 h,2 h,6 h,24 h,and48 h,and the changes in expression of 21 genes showed seven expression patterns;After 24 h induce by 100?g/mL,300?g/mL,500?g/mL and 1000?g/mL DDVP respectively,relative expression of eight P450 genes from family of TaCyp548,TaCyp620,TaCyp52,TaCyp528,TaCyp504 were at least 1-fold up-regulated.By homologous cloning of TaCyp548-2,which was 1,911 bp in length,contained four introns of 125,75,and 61 and 57 bp,encoding 530 amino acids,whose expression was induced by DDVP.Deletion of TaCyp548-2 will significantly reduce the content of 2,2-dichloroethanol,an intermediate product of DDVP,to convert to the final product 2,2-dichloroethyl acetate.The microsomes of T23,?TaCyp548-2,and co-TaCyp548-2induced by DDVP were carried out in vitro assays for fatty acid oxidation,and it was found that TaCyp548-2 belonged to the?-hydroxylase which was responsible for fatty acid oxidation and positively regulated the formation of low low-molecular-weight organic acids such as acetic acid,propionic acid,isobutyric acid,dibutyric acid,etc.These low-molecular-weight organic acids can promote the futher transformation of DDVP intermediate product 2,2-dichloroethanol.(5)The paraoxonase1-like gene of T.atroviride T23 and its function in hydrolyzing DDVPThe paraoxonase1-like gene,TaPon1-like,was cloned from T.atroviride T23.It contains 1,384 bp open reading frame,which encodes 438 amino acids with the typical6 propeller hydrolase domain.The expression of TaPon1-like is consistently up-regulated induced by DDVP.The TaPon1-like knockout strain KO1 and the complementation strain CO1 were constructed by the ATMT method.At 24 h,the residue of DDVP treated with KO1 was 124.88?g/mL and 130.30?g/mL higher than that of the wild type strain T23 and the complementation strain CO1,respectively,demonstrating that TaPon1-like could catalyze the transformation of DDVP.The TaPon1-like was expressed in E.coli Origami B(DE3),and the recombinant reTaPon1-like was obtained by GST-tag purification resin,which can transform DDVP to dimethyl phosphate and dichloroacetic acid.This result indicated that TaPon1-like is a gene encoding an enzyme catalyzing the hydrolysis of P—O bond.Re TaPon1-like degraded DDVP in the optimum reaction temperature range of 20-35°C.Ca²⁺and Zn²⁺can significantly increase the enzyme activity by 489.7%and 134.4%,respectively.Mg²⁺,Na⁺,Ba²⁺and Mn²⁺increased enzyme activity by 19.9%to 76.5%,but Cu²⁺inhibited the enzyme activity.reTaPon1-like has a broad spectrum of hydrolyzed substrates,besides being hydrolytically active against p-oxapropyl compounds(chlorpyrifos,mevinphos,malathion and omethoate),it is also active against aromatic compounds(phenyl acetate,p-nitrophenyl acetate and p-nitrophenyl butyrate)and lactones(3,4-dihydrocoumarin).The enzyme had a high affinity for DDVP,with a K_mranging from 0.23 to 1.58 mmol/L.The k_cat ranged from 6.1 to 3261.1 s^-1,where the K_mfor DDVP was 0.23 mmol/L,204.3 s^-1 for k_cat,and k_cat/K_m was 8.88×10⁵ s^-1 M^-1.In conclusion,the main enzymatic and molecular mechanisms of the degradation of DDVP by T.atroviride T23 were systematically investigated in this study.The characteristics of enzymatic degradation of DDVP by T.atroviride T23 were revealed and the major degradation pathway of DDVP was proposed based on the DDVP degradation-released products.For the first time,we cloned paraoxonase1-like gene TaPon1-like,which encoded enzyme and was responsible for hydrolysis of the P—O bond of DDVP.Moreover,we investigated a diverse expression pattern of cytochrome P450 under DDVP stress,and discovered TaCyp548-2 positively regulated the production of extracellular low-molecular-weight organic acids and promoted the conversion of DDVP intermediates.In sum,the study revealed the major biological degradation process from the hydrolysis of P—O bonds to transformation of DDVP degradation intermediates by T.atroviride T23.