Bull Environ Contam Toxicol (2013) 91:730–733 DOI 10.1007/s00128-013-1124-2

Degradation of Clodinafop Propargyl by Pseudomonas sp. Strain B2 Baljinder Singh

Received: 18 June 2013 / Accepted: 4 October 2013 / Published online: 12 October 2013 Ó Springer Science+Business Media New York 2013

Abstract Using clodinafop propargyl (CF) as a sole carbon, nitrogen and energy source, a CF-degrading bacterial strain was isolated from crop soil field. This strain was identified as Pseudomonas sp. strain B2 by 16S rRNA gene sequence analysis. 87.14 % CF was degraded out of initial provided 80 mg/L CF. Degradation of CF was accompanied by release of chloride ion. The optimal pH and temperature for the growth of B2 were 7.0 and 30°C, respectively in the mineral salts medium supplemented with CF. An actively growing culture of strain B2 degraded CF to clodinafop acid and 4-(4-Chloro-2-fluoro-phenoxy)phenol within 9 h, as determined by GC–MS analysis. A metabolic pathway for the degradation of CF by B2 has been proposed. Keywords Pseudomonas sp. strain B2  Clodinafop propargyl  Biodegradation  GC–MS

CF (prop-2-ynyl(R)-2-[4-(5-chloro-3-fluoro-2 pyridyloxy) phenoxy]propionate), is an important aryloxyphenoxypropionate herbicide. CF is used for post emergence control of annual grasses in cereals, including Avena, Lolium, Setaria, Phalaris and Alopecurus spp. (Tomloin 2006). CF is absorbed by the leaves and rapidly translocated to the growing points of leaves and stems. It interferes with the production of fatty acids needed for plant growth in susceptible grassy weeds (Hammami et al. 2011). CF acts by targeting the

B. Singh (&) Department of Biotechnology, Panjab University, Chandigarh 160014, India e-mail: [email protected]

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enzyme acetyl coenzyme-A-carboxylase, essential for lipid biosynthesis (Devine and Shimabukuro 1994). The widespread use of CF has resulted in the discharge of large amounts of the compound into the environment, which eventually reach the biosphere (Gherekhloo et al. 2010; Vazan et al. 2011). The half-lives of CF were 2.35–11.20 days in soil and it rapidly degraded to the acid derivative clodinafop as major metabolite in soil (Guan et al. 2013). Several studies have demonstrated that CF and its derivatives are toxic and carcinogenic to humans and other living organisms (Kashanian et al. 2008; Gui et al. 2011). Therefore, the degradation of CF in the environment is of great concern. Only a single study concerning the biodegradation of CF can be found in the literature. This might be because of its low persistence; the halflife in soil was reported to be 5 days, dependent on the soil type, pH, and microbial population (Roy and Singh 2006). They had reported 97.9 % CF-degradation without identifying its metabolites. In this study, we successfully isolated a Pseudomonas sp. That could use CF as the sole carbon, nitrogen and energy source. We named this strain Pseudomonas sp. strain B2. Degradation of CF by strain B2 in liquid culture was studied. Importantly, this is the first report of degradation of CF by genus Pseudomonas.

Materials and Methods Soil samples were collected from crop field area with a previous history of CF application, located in the city of Patiala, Punjab, India. Soil samples were held in sterile bottles and stored at 4°C until used. CF (99.4 % purity) was purchased from Sigma Aldrich (PESTANAL, Fluka analytical). All other chemicals used in this study were analytical grade or higher purity.

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A selective minimal salt medium (MS) was prepared containing 40 mg/L CF as a sole source of carbon, nitrogen in addition to 4 g Na2HPO4.2H2O, 2 g KH2PO4 (0.025 %), MgSO4.7H2O (0.05 %), and 1 mL of trace element solution (0.1 g of ZnSO4.7H2O, 0.03 g of MnCl2.7H2O, 0.3 g of H3BO3, 0.2 g of CoCl2.6H2O, 0.01 g of CuCl2.2H2O, 0.02 g of NiCl2.6H2O, in 1 L of the solution). Five grams of soil sample were inoculated into Erlenmeyer flask (250 mL) containing 100 mL autoclaved water. Processed soil sample (0.5 mL) was spread on MS media plates and incubated at 30°C for 3 days until bacterial colonies became visible. Colonies grown on these plates were evaluated for their CF degrading capabilities. Single colony types were separated and subcultured on fresh plates to purity using identical growth conditions at each transfer, except that the CF concentration was increased stepwise from 40 to 120 mg/L. One strain, designated as B2, which possessed the highest CF-degrading ability and could utilise CF as the sole carbon source for growth, was purified and selected for further investigation. Strain B2 was classified by Gram staining, starch hydrolysis test, gelatin test, catalase test, NaCl, pH, temperature variation assay, and 16S rRNA analysis. Genomic DNA extraction from strain B2 was performed using the method described by Sambrook et al. (1989). Partial fragment of 16S rRNA gene of strain B2 was amplified by PCR with set of universal primers 27F (50 -AGAGTTTGATCCTGGCTCAG-30 ) and 1492R (50 -TACGGYTACCTTGTTACGACTT-30 ) following the PCR parameters as described by Singh et al. (2011). Solutions of CF were freshly prepared in methanol at concentration of 1 mg/mL. A 1 mL aliquot of CF solution was transferred into sterile 12 mL amber glass vials. The vials were left open in a fume hood to allow the solvent to evaporate. A 100 lL pre-grown single bacterial clone (OD600 = 0.5) was inoculated into each vial to give an initial CF concentration of 80 mg/L. The cultures were incubated at 30°C and 100 rpm on a rotary shaker for 12 h. In order evaluate the effect of temperature and pH, MS media containing 80 mg/L CF were incubated for 12 h at different temperatures (20, 25, 30, 35, 40, and 45°C) and under different pH conditions (5.0–10.0, in increments of 1.0 pH units). Uninoculated MS media was used as control. Each treatment was performed in three replicates, and the control experiment without microorganism was carried out under the same conditions. The chloride ion concentration was determined using Mohr method (Korkmaz). Two hundred microliters of a sample diluted so that the chloride concentration was up to 0.1 mM was added to 50 mL of 0.25 M potassium chromate. The reaction mixture was titrated with 0.1 M silver nitrate solution. Chloride ion concentrations were calculated by using volumetric analysis.

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During degradation, 1 mL sample was removed from each vial at regular intervals to measure the inoculants growth of the cell and CF concentration. To analyze CF and its metabolites sample aliquots (1 mL) were taken at regular intervals. The culture broth was centrifuged (10,000 rpm, 4°C for 10 min) and supernatant was acidified with 1 M H2SO4 to a pH \ 5. The solution was extracted with two 1 mL portions of hexane and derivatized by adding 0.1 mL diazoethane (Sigma Aldrich). Diazoethane is an ethylating agent that facilitates the simultaneous analysis of CF and clodinafop acid. Excess diazoethane was removed under a stream of cold nitrogen and the solution was dried over anhydrous Na2SO4. A cold stream of nitrogen was used to evaporate the hexane and to allow a solvent exchange to toluene. The final volume was adjusted to 2 mL with toluene. Samples were analysed by using high-pressure liquid chromatography (HPLC) on a reverse-phase C18 column at 258 nm. The mobile phase was acetonitrile/water (50:50 v/v) and flow rate was 1 mL/min. To calculate % degradation, peak areas were measured to quantify the CF. Extracts were analyzed using GC–MS-QP2010 Plus system (Shimadzu Corporation, Japan). GC column oven temperature was programmed for an initial hold of 1 min at 100°C; then temperature was increased at 10°C/min to 200°C; then up to 260°C at the rate of 15°C/min; followed up to 300°C at the rate of 3°C/min and then hold at 300°C for 2 min. The gas flow rate was 1 mL/min in splitless mode with injection temperature of 270°C. Conditions for MS measurements were: MS ion source at 200°C, MS interface temperature 250°C, electron impact ionisation (EI) at 70 eV. Chromatographic data were collected and recorded by GC–MS real time analysis software.

Results and Discussion Standard isolation and enrichment techniques yielded a bacterial isolate, designated as strain B2 capable of degrading CF and selected for further studies. B2 could degrade 87.14 % of 80 mg/L CF in 9 h. When grown on LB agar, cells of this strain are non-spore-forming, gram negative, motile, and globular- or globular-rod-shaped. The nucleotide sequence coding for the 16S rRNA of strain B2 (1,081 bp) was deposited in the GenBank database with accession number KF254765. BLASTN and phylogenetic analysis of 16S rRNA gene sequence revealed that strain B2 belonged to Pseudomonas sp. (99 % similarity). HPLC chromatograms of control and test reactions were recorded and CF peak was observed after retention time 2.779 min. Limits of detection (LODs) were calculated using a peak-to peak height signal to noise ratio of 3:1, at the lowest calibration concentration of analyte. LOD for CF was 2 ng/L. The major metabolites, clodinafop acid and 4-(4-Chloro-2-

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Fig. 1 Degradation of CF by strain B2. a Effect of concentration on CF degradation. b A time course study of CF degradation in MS medium supplemented with 80 mg/L CF. Data are presented as mean

and standard error of three independent observations. Some error bars are not present because they are smaller than the diameter of the symbol

fluoro-phenoxy)-phenol peaks were observed at 4.519 and 1.874 min respectively. In order to evaluate the effect of CF concentration on microbial growth, strain B2 was cultivated in MS at CF concentrations 40, 80 and 120 mg/L. The degradation of CF by strain B2 could be affected by substrate concentration (Fig. 1a). Substrate concentration is one of the factors governing the diffusive mass transfer of pollutants to microbes. Therefore, at a higher initial pollutant concentration, more of the pollutant is able to enter the cells as a result of faster diffusion and up-take by the cells. Ma et al. (2013) reported that higher intracellular pollutant concentration would result in higher degradation rate, but this is not consistent with our observation, which showed that higher CF concentration resulted in slower degradation. The optimum concentration at which strain B2 showed the maximum growth was 80 mg/L (Fig. 1a). At a higher concentration of CF (120 mg/L), it was observed that strain B2 could maintain a gradual degradation rate to final 55 % degradation of the initial amount of CF after 9 h of incubation (Fig. 1a). This limited growth at higher concentrations of CF could result from higher toxicity, meaning that CF uptake was limited. The effects of pH and temperature on the biodegradation of CF were also investigated. When the pH was between 7.0 and 8.0, more than 80 % of 80 mg/L CF could be degraded by B2 within 9 h. The optimum temperature for the biodegradation of CF was 30–35°C. However, CF biodegradation decreased when the temperature dropped to 20°C or rose to 40°C, indicating that lower and higher temperatures were not beneficial for the biodegradation of CF by B2. The growth of strain B2 on CF and its ability to degrade CF is shown in Fig. 1b. With CF as the carbon, nitrogen and energy source, strain B2 produced a typical sigmoidal growth curve consisting of a relatively very short lag phase

and an exponential phase of approximately 9 h, followed by a abrupt transition to the stationary phase (Fig. 1b). The OD600 showed a steady increase in bacterial mass. Simultaneously, HPLC analysis showed a substantial reduction in the levels of CF. Results demonstrated an initial CF degradation rate and the biomass formation were detected. After incubation of 9 h, 87.14 % CF (80 mg/L) initially added to the MS medium was degraded by strain B2 (Fig. 1b). Further no further degradation was observed. Uninoculated controls showed no change in CF concentration was observed in cultures that were not inoculated. Only trace amounts of 4-(4-Chloro-2-fluoro-phenoxy)-phenol) were detected during the early stages of growth (1–2 h), high concentrations of this metabolite in the growth medium during the log and stationary phases (14–30 h) suggested that 4-(4-Chloro-2-fluoro-phenoxy)-phenol was the major degradation product. This was in agreement with previous observations by Smith-Grenier and Adkins (1996). They reported the degradation of diclofop-methyl by Chryseomonas luteola and Sphingomonas paucimobilis and formation 4-(2,4-dichlorophenoxy)phenol as metabolites. The formation of phenol as metabolite during growth of strain B2 in MS medium provided an indication that it might be due to esterase activity as reported previously (Hou et al. 2011). However, on the basis of the structures of the metabolites, the initial degradation of the compound is suggested to take place via cleavage of the ester bond. The presence of metabolite, [4-(4-chloro-2-fluorophenoxy) phenol], supported this suggestion. Other possible breakdown product, including clodinafop acid was also observed. The release of chloride ion from the ring of CF during the biodegradation pathway was of the particular interest for this work. During the reaction amounts of chloride ion (2.1 ± 0.3 mg/L) were

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Fig. 2 Proposed pathway of CF degradation by Pseudomonas sp. strain B2. 1 CF, 2 acid metabolite, clodinafop acid, 3 4-(4-Chloro-2-fluoro-phenoxy)phenol, 4 phenol

released from initial provided 80 mg/L CF in 9 h. Therefore, it is possible that the chloride ion release leads to catabolism of the pyridyl moiety in CF. In summary, the results indicate that strain B2 is capable of rapidly hydrolyzing the ester bond of CF to produce clodinafop acid, which in turn may either be directly hydrolyzed to form 4-(4-Chloro-2-fluoro-phenoxy)-phenol (Fig. 2). Future experiments using 14C-labelled compounds will assist in clarifying this point. At this time, it is difficult to ascertain the full degradation potential of strain B2. References Devine MD, Shimabukuro RH (1994) Resistance to acetyl coenzyme a carboxylase inhibiting herbicides. In: Powles SB, Holtum JAM (eds) Herbicide resistance in plants: biology and biochemistry. CRC, Boca Raton, pp 141–169 Gherekhloo J, Rashed MH, Nassiri M, Zand E, Ghanbari A (2010) Investigating the retention, absorption and translocation of herbicide in two Phalaris minor diclofop-methyl resistant populations. In: Proceedings of the 3rd Iranian Weed Science Congress, pp 388–391 Babolsar, Iran Guan W, Ma Y, Zhang H (2013) Dissipation of clodinafop-propargyl and its metabolite in wheat field ecosystem. Bull Environ Contam Toxicol. doi:10.1007/s00128-013-09974 Gui W, Dong Q, Zhou S, Wang X, Liu S, Zhu G (2011) Waterborne exposure to clodinafop-propargyl disrupts the posterior and ventral development of zebrafish embryos. Environ Toxicol Chem 30(7):1576–1581

Hammami H, Hassan M, Mohassel R, Aliverdi A (2011) Surfactant and rainfall influenced clodinafop-propargyl efficacy to control wild oat (Avena ludoviciana Durieu.). Aus J Crop Sci 5(1):39–43 Hou Y, Tao J, Shen W, Liu J, Li J, Cao H, Cui Z (2011) Isolation of the fenoxaprop-ethyl (FE)-degrading bacterium Rhodococcus sp. T1, and cloning of FE hydrolase gene feh. FEMS Microbiol Lett 323:196–203 Kashanian S, Askari S, Ahmadi F, Omidfar K, Sirous G, Tarighat FA (2008) In vitro study of DNA interaction with clodinafoppropargyl herbicide. DNA Cell Biol 27:581–586 Korkmaz D Precipitation titration: ‘‘Determination of Chloride by the Mohr Method’’, available online at: http://academic.brooklyn. cuny.edu/esl/gonsolves/tutorials/writing-a-Lab-Report/xPrecipit ation%20Titration%20edited%203.pdf Ma J, Xu L, Jia L (2013) Characterization of pyrene degradation by Pseudomonas sp. Strain Jpyr-1 isolated from active sewage sludge. Bioresour Technol 140:15–21 Roy S, Singh SB (2006) Effect of soil type, soil pH, and microbial activity on persistence of clodinafop herbicide. Bull Environ Contam Toxicol 77:260–266 Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. CSH Laboratory Press, Cold Spring Harbor Singh B, Kaur J, Singh K (2011) Biodegradation of malathion by Brevibacillus sp. strain KB2 and Bacillus cereus. World J Microbiol Biotechnol 28(3):1133–1141 Smith-Grenier LL, Adkins A (1996) Degradation of diclofop-methyl by pure cultures of bacteria isolated from Manitoban soils. Can J Microbiol 42:227–233 Tomloin CDS (2006) The pesticide manual, vol 14. BCPC, UK Vazan S, Oveisi M, Baziar S (2011) Efficiency of mesosulfuronmethyl and clodinafop-propargyl dose for the control of Lolium perenne in wheat. Crop Protection 30:592–597

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