Isolation&Characterization Of Trifluralin-Resistant Novel Species TFL-3 And Trifluralin-Degrading Strain TFL-5

Trifluralin,a selective pre-emergence dinitroaniline herbicide,is the second largest selective herbicide used in the world.It is widely applied to control a variety of annual gramineous grasses and certain dicotyledonous grasses in the fields of cotton,soybean and wheat because of its safety,high efficiency and broad spectrum.Trifluralin is not easy to migrate in the soil ang has a long half-life period.Trifluralin shows high toxicity to aquatic organisms,especially have an increasing side effect on fish.In this study,two strains were isolated from the sediment of Yangtze River,named TFL-3 and TFL-5,which have considerable tolerance to trifluralin and capable of degrading trifluralin respectively,and the biological characteristics of two strains are studied.Strain TFL-3 were Gram-reaction-negative,rod-shaped and non-motile.Cells dimensions were about 0.5-0.7 ?m×1.2-8.0 ?m,colonies of strain on LB agar plate were orange yellow and slimy.Strain TFL-5 were Gram-reaction-negative,rod-shaped and non-motile.Cells dimensions were about 0.3-0.8 ?m×1.0-1.3 ?m,colonies of strain on LB agar plate were yellow and circular with entire margins.The phylogenetic analysis of 16S rRNA gene sequences indicated that strain TFL-3 belonged to the genus Chitinophaga and formed a subclade with C barathri YLT18T,C.niabensis JS13-10T and C.cymbidii R156-2T,and strain TFL-3 showed the highest sequence similarity with C.barathri YLT18T(94.93%).Meanwhile,The phylogenetic analysis of 16S rRNA gene sequences indicated that strain TFL-5 belonged to the genus Dyadobacter and formed a subclade with D.jiangsuensis L-1T,and strain TFL-5 showed the highest sequence similarity with D.endophyticus 65T(97.45%).Thus,on the basis of phylogenetic analysis,strain TFL-3 and TFL-5 were identified as Chitinophaga sp.and Dyadobacter sp.respectively?The results of trifliralin degradation properties by strain TFL-5 showed that the optimal temperature and pH was 30? and 7.5 respectively.Two metabolic products of strain TFL-5 were detected by HPLC-MS/MS during the degradation of trifluralin.The[M+H]+ was m/z 306.1487 and m/z 276.1650,and the metabolic products of strain TFL-5 were identified as 2-nitro-6-amino-N,N-dipropyl-4-trifluoromethyl aniline and 2,6-diamino-N,N-dipropyl-4-trifluoromethyl aniline respectively.A partial metabolic pathway of trifluralin in strain TFL-5 was proposed.Firstly,one of the nitro-group of trifluralin was reduced to amino-group through nitroreduction to generate 2-nitro-6-amino-N,N-dipropyl-4-trifluoromethyl aniline,which was subsequently transformed to 2,6-diamino-N,N-dipropyl-4-trifluoromethyl aniline through nitroreduction at the other nitro-group.A comprehensive and systematic research of physio-biochemical characteristics,chemical composition of cells and genetic features of strain TFL-3 were performed.It showed that the optimum temperature was 30?,optimum pH 7.0 and optimum NaCl concentrations 0-1.0%for strain TFL-3 growth.Oxidase and catalase activity were both positive.The hydrolysis of gelatin and tween 40 were detected,but chitin and urea were not hydrolyzed.Cells were resistent to kanamycin,streptomycin,ampicillin,spectinomycin,neomycin,amikacin and gentamycin.Voges-Proskauer test was positive,production of indole and H2S are negative,cells were positive for fermentation of D-glucose,D-melibiose,L-arabinose and amygdalin,Nitrate reduction and hydrolysis of L-arginine and L-trypophane were negative.Strain TFL-3 utilized dextrin,D-maltose,D-trehalose,D-cellobiose,gentiobiose,sucrose,D-turanose,a-D-lactose,D-melibiose,?-methyl-D-glucoside,D-salicin,N-acetyl-D-glucosamine,N-acetyl-D-galactosamine,D-glucose,D-mannose,D-fructose,D-galactose,L-fucose,D-rhamnose,glycyl-L-proline,L-aspartic acid,L-glutamic acid,L-serine,pectin,D-galacturonic acid,D-glucuronic acid glucuronamide,acetic acid,acetoacetic acid and aztreonam as sole carbon sources for growth.The major compositions of fatty acids were iso-C,5:o(35.7%),C16:i?5c(22.0%)and summed feature 3(20.24%).MK-7 was detected as the predominant respiratory ubiquinone.The major polar lipids of strain TFL-3 contained phosphatidylethanolamine(PE),unknown aminolipid(AL),unknown glycolipid(GL)and unknown lipid(L).The DNA G+C content of strain TFL-3 was 50.4 mol%.Analysis of the characteristic above together,strain TFL-3 was identified as a novel species of genus Chitinophaga,and named Chitinophaga sedimenti sp.nov.