Isolation&Characterization Of Dicamba-Degrading Strain Ndbn-20(Sphingomonas Sp.)and Study Of Its Biological Feature

Dicamba is a kind of low toxicity selective herbicide with high efficiency and broad spectrum.It is mainly used to control annual and perennial broadleaf weeds in corn and grain fields.Dicamba demethylase,in the process of dicamba-degrading by microorganism,is the targeted enzyme which is applied to product dicamba-resistance genetically modified crops.It has significant application in the field of actual production.The purpose of this paper is to isolate one dicamba-degrading strain which has demethylase that had never been reported.Studying its evolution status,biological characteristics,dicamba-degrading characteristics and the dicamba-demethylation mechanism in order to provide the theory basis for further studying of the dicamba-degrading mechanism and cloning related gene.Strain Ndbn-20(16S rDNA GenBank Accession No:KP064570)which can utilize dicamba was isolated from compost.Ndbn-20 was identified as Sphingomonas sp.finally according to its physiological and biochemical characteristic and the similarity analysis of its 16S rDNA sequence.The optimal condition for the growth of strain Ndbn-20 was pH 7.0 and 30 ?.Ndbn-20 can growth well when the concentration of NaCl between 0.5-3%.The aeration was related positively to strain growth.The optimal condition of the degrading of dicamab for strain Ndbn-20 was the same as its growth and remain positively to initial inoculum size.Strain Ndbn-20 can absolutely mineralize 100 mg·L-1 dicamba in 72 h and degrade 200 mg·L-1 dicamba in 108 h.Using HPLC and LC-MS/MS to determine the intermediate metabolites of dicamba after degrading by strain Ndbn-20,the results show that its main intermediate metabolite is DCSA which generated by dicamba-demethylation.Ndbn-10,another strain isolated from activated sludge which collected from dicamba manufacturer by our laboratory,has been proven to be DMO positive.Primers were designed according to the reported dicamba monooxygenase gene(DMO)from Pseudomonas sp..Amplified DMO gene fragments from Ndbn-20 and Ndbn-10 at the same time.As a result,the DMO gene sequence could not amplified from Ndbn-20 total genomic DNA.Meanwhile,the cell-free extract of strain Ndbn-20 displayed demethylase only when tetrahydrofolic acid(THFA)was added,but not NADH,and no demethylase was detected in the cell-free extract of strain Ndbn-20 under anaerobic condiction with or without addition of THFA.Above all,these results suggest that the dicamba-converting enzyme in strain Ndbn-20 was an aerobic THFA-dependent demethylase,which was different from the NADH-dependent DMO which has been reported.