Studies On The Co-metabolism Of Nicotinic Insecticidal Imidacloprid By Pseudoxanthomonas Indica CGMCC 6648

Bacterial strains which can transform the neonicotinoid pesticide Imidacloprid(IMI)were screened and isolated and its IMI degradation mechanism was studied.A new imidacloprid(IMI)degrading bacterium named as z9 isolated from our stock culture and deposited under the accession number of CGMCC 6648 displayed the highest IMI degradation.Strain CGMCC 6648 was identified with Pseudoxanthomonas indica by 16S rDNA gene analysis.HPLC analysis showed that the degradation of IMI by P.inidca CGMCC 6648 produced two metabolites which were identified as olefin and 5-hydroxy IMI by liquid chromatography-mass spectrometry(LC-MS)and nuclear magnetic resonance analysis.The other neonicotinoid insecticide Thiacloprid(THI),Imidaclothiz(IMT)and Acetamiprid(AAP)could be transformed by P.indica CGMCC 6648 to form the metabolite 4-hydroxy THI,5-hydroxy IMT and N-demethylation product IM-2-1 identified by LC-MS analysis,respectively.P.indica CGMCC 6648 transforms IMI via a co-metabolism mechanism.Carbohydrates and organic acids have significant effects on the degradation of IMI.The degradation rate of control groups without co-metabolism substrates was 5%while the experiment group with 100 mmol/L glucose was 37.4%in 2 d.P.indica CGMCC 6648 degraded 70.1%of IMI(1.22 mmol/L)and formed 0.93 mmol/L 5-hydroxy IMI and 0.05 mmol/L olefin IMI in 6 d and in the presence of 100 mmol/L glucose.The half-life of IMI degradation was 3.6 d.P.indica CGMCC 6648 transforms IMI via a co-metabolism mechanism.Different carbohydrates have significant effects on 5-hydroxy IMI formation,whereas different organic acids have substantial effects on olefin IMI production Lactose is the best co-substrate for IMI degradation and 5-hydroxy IMI formation with 0.77 mmol/L degraded and 0.67 mmol/L formed in 48 h,respectively.Pyruvate is the best co-substrate for olefin IMI formation with 0.17 mmol/L produced in 96 h for all carbon sources tested.Pyruvate significantly stimulates the conversion of 5-hydroxy IMI to olefin IMI,whereas glucose slightly inhibits this reactionIn addition,P.indica CGMCC 6648 has some plant-growth promoting activities,it can produce ammonia,iron carrier,salicylic acid and so on.Whereas,it doesn’t produce prussic acid and indoleacetic acid,and it also doesn’t have the ability to dissolve phosphorus.The genomic DNA of P.indica CGMCC 6648 was examined in Illumina Hiseq2000 platform,and 754 Mb of data were achieved.The genomic DNA of P.indica CGMCC 6648 has a length of 4304970 bp,contained 3935 genes,which includes seven monooxygenase genes.Two monooxygenase genes of P.indica CGMCC 6648 which have higher homology with Stenotrophomonas maltophilia were cloned and expressed into E.coli Rosetta.The two monooxygenase genes had been successfully expressed in Escherichia coli Rosetta.Objective proteins are soluble;however they did not transformed IMI when compared with the control without harboring the monooxygenase gene.