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Ann Microbiol (2016) 66:1355–1365 DOI 10.1007/s13213-016-1222-6 ORIGINAL ARTICLE Biodegradation of the fungicide propiconazole by Pseudomonas aeruginosa PS-4 strain isolated from a paddy soil 1 1,2 Praveen Satapute & Basappa Kaliwal Received: 4 January 2016 /Accepted: 26 May 2016 /Published online: 15 June 2016 Springer-Verlag Berlin Heidelberg and the University of Milan 2016 . . Abstract In India, propiconazole, a triazole group fungicide, Keyword Propiconazole Pseudomonas aeruginosa . . is broadly used against powdery mildew, rusts, and leaf spot 1,2,4-Triazole 2,4- Dichlorobenzoic acid 1-Chlorobenzene diseases of cereals and coffee. The toxicity of this fungicide is and CYP P450 monooxygenase known to affect the quality of the soil. Hence, in the present study, a bacterium isolated from contaminated paddy soil was used to study the degradation of propiconazole under in vitro Introduction conditions. The isolated bacterium was confirmed as Pseudomonas aeruginosa strain (PS-4) based on morpholog- Propiconazole (1-[[2-(2,4-dichlorophenyl)-4-propyl-1,3- ical and biochemical characteristics, and 16S rRNA gene se- dioxolan-2-yl]methyl]-1H-1,2,4-triazole) belongs to the tri- quencing. When the isolated bacterium was grown in mineral azole group of fungicides that inhibit demethylation. In salt medium amended with 10 μg/l propiconazole as a sole India, propiconazole is used extensively as a popular agro- carbon source, culture filtrates of the bacterium utilized up to chemical due to its wide spectrum of triazole action. This 8 μg/L of propiconazole after 72 h of incubation at 30 °C and fungicide is used as a foliar spray, and thus will drift and reach pH 7, as analyzed by HPLC. Degradation of propiconazole by the soil during application (Colson et al. 2003; Kim et al. the bacterium was also aided by the secretion of three metab- 2003;Z.H.Li et al. 2013). Triazole fungicides are toxic and olites—1,2,4-triazole; 2,4-dichlorobenzoic acid; and 1-chloro- persist in the soil for long periods of time, thus affecting soil benzene—as determined by their mass spectra. Furthermore, fertility and microflora (Elmholt 1992; Munier and Borde induction of monooxygenase activity and the CYP450 gene 2000). Remediation of fungicide toxicity has been a major was observed in the culture filtrate of strain PS-4, showing research concern, and application of traditional methods to evidence of their role in the degradation of propiconazole. reduce toxicity has many environmental side effects. These results revealed that PS-4 is an efficient candidate for Therefore, ecofriendly and feasible approaches such as micro- the reduction of contaminants present in the soil, thereby con- bial biodegradation are gaining importance. tributing to soil health and crop improvement. Microorganisms are most desirable biological tools, be- cause of their ability to resist various pesticides, and their metabolic capacity to degrade toxic compounds into non- Electronic supplementary material The online version of this article toxic forms. Hence, soil microorganisms are considered a (doi:10.1007/s13213-016-1222-6) contains supplementary material, key reservoir of biological activity with the potential to sig- which is available to authorized users. nificantly enhance environmental cleanup (Dong et al. 2008; Satapute et al. 2012; Kulkarni and Kaliwal 2014). Many * Basappa Kaliwal pesticide-degrading microorganisms have been reported be- firstname.lastname@example.org longing to various species of bacteria, fungi, algae and yeast. However, bacterial bio-remediation studies have been more Department of Microbiology and Biotechnology, Karnatak University, Dharwad, Karnataka, India successful because of the diversity of their metabolism and their ability to grow on complex carbon substrates. In addi- Department of Microbiology, Davangere University, Davangere, Karnataka, India tion, many genes involved in the metabolism of toxic 1356 Ann Microbiol (2016) 66:1355–1365 compounds have been identified. Additionally, cytochrome was done at Xcelris genomics (Ahmedabad, India). The P450 monooxygenase, which constitutes a huge family of selected bacterial DNA was isolated using an Xcelgen protein haem thiolates capable of degradation of wide range kit, and DNA stock samples were quantified using a of toxic compounds, are extremely well characterised in bac- nanodrop spectrophotometer at 260 and 280 nm. teria (Degtyarenko 1999). Therefore, the purpose of present Simultaneously, DAN purity was checked by agarose investigation was to isolate and identify propiconazole- gel electrophoresis (Sambrook and Russell 2001). metabolising bacteria from contaminated paddy fields, and Bacterial 16S RNA gene fragments were amplified by to study the degradation mechanism of propiconazole under PCR from genomic DNA using 16S gene universal in vitro conditions. primers: 8 F and 1492R. Conditions of thermal cycling for PCR were, initial denaturation at 95 °C for 2 min in one cycle and final denaturation at 94 °C for 30 s, Materials and methods annealing at 52 °C for 30 s and extension at 72 °C for 90 s. The number of cycles for all three steps was Chemicals, media and soil sample 30, with a final extension at 72 °C for 10 min in one cycle. Further, the nucleotide sequence of the isolate Propiconazole of 94 % purity was obtained from the was checked by BLAST analysis using the NCBI server Nagarjuna Agrichem Co. (Srikakullam, India). Ethyl acetate (http://blast.ncbi.nlm.nih.gov/Blast.cgi), and a and acetonitrile used were of highest analytical and HPLC phylogenetic tree was constructed by the neighbor grades, respectively. Seubert’s mineral salts medium (MSM) joining method using MEGA 6 software (Tamura et al. (Seubert 1960)containing 10 μg/L propiconazole was used in 2013). the study. Soil was collected from a fungicide (upper layer 0– 10 cm)-contaminated paddy field in Dharwad, Karnataka, Degradation of propiconazole in soil India (15° 27′ 29 N, 75° 0′ 36E, 764 m altitude, reddish black soil). The physicochemical properties of the collected soil The physicochemical properties of soil were studied fol- sample were recorded. lowing the method of Tandon (2005). To determine the degradation rate of propiconazole in soil samples, two Isolation and screening of propiconazole-degrading different sets of experiments were conducted under in bacteria vitro conditions using different concentrations of propiconazole [commercial grade fungicide Tilt 25 EC −7 The soil was serially diluted up to 10 with sterile saline (http://www3.syngenta.com), at 0.05, 0.1 μg/kg of solution using 1 g sieved soil; 100 μL suspension of appro- technical grade]. All three concentrations of −5 −6 priate dilutions (10 and 10 ) was inoculated on mineral propiconazole were applied to soil as follows: one set salts agar (MSA) medium containing 10 μg/L propiconazole of sterile (controlled) and non-sterile (not controlled) as a sole source of carbon. After 7 days of incubation at 30 °C, soil samples (1000 g) were placed in 30 × 25 cm tray all the colonies that appeared on the plates were purified by and kept at 22 ± 2 °C and 64 ± 4 % humidity under the quadrant streaking method on nutrient agar plates. All laboratory conditions. Simultaneously, a similar set of strains were screened for their tolerance level to propiconazole sterile and not-sterilized soil samples were kept outside with different concentrations (10 μg/L, 20 μg/L and 30 μg/L) the laboratory (25 ± 2 °C, 68 ± 4 % relative humidity). in mineral salt medium, and controls without propiconazole The degradation rate of propiconazole in soil samples were maintained for all concentrations. All flasks were incu- was monitored immediately after the treatment, and bated at 30 °C on a rotary shaker at 120 rpm. The growth of all was repeated at 10-, 20-, 30- and 40-day intervals; the strains was observed regularly using a spectrophotometer half life of propiconazole (DT ) was also recorded. (Hitachi U2900) at 600 nm. Strains that showed luxuriant growth at all concentrations of propiconazole were selected Degradation of propiconazole in liquid medium by soil for further study. isolate PS-4 strain Characterization of propiconazole-degrading bacterium To investigate the biodegradation of propiconazole, 100 mL MSM with the propiconazole (10 μg/L) as sole carbon source The bacterial isolate with highest tolerance to the differ- was placed in a 500 mL conical flask and inoculated with −6 ent concentrations of propiconazole was identified based 1 mLof PS-4straincontaining3×10 cfu/mL; MSM with on its colony morphology, gram staining and biochemi- the same propiconazole concentration but without the bacte- cally by the bioMérieux vitek2 (bioMérieux, Marcy- rial culture was used as a control. Treated and control flasks l’Étoile, France) system. Further, 16S rRNA sequencing were incubated at 30 °C on a rotary shaker at 120 rpm. In Ann Microbiol (2016) 66:1355–1365 1357 addition, DT of propiconazole was calculated according to Chemical analyses the obtained results. Propiconazole extraction from soil Effect of temperature and pH on biodegradation To analyze the degradation of propiconazole in the soil, 1 g of propiconazole soil was taken and mixed with 10 mL distilled water, and centrifuged at 10,000 g for 10 min (Eppendorf centrifuge); The effect of different temperatures (20 °C, 25 °C, 30 °C, the supernatant was extracted twice using ethyl acetate (1:1), 35 °C, 40 °C, 45 °C and 50 °C) and pH (5, 6, 7, 8 and 9) on and the residue dissolved in acetonitrile. Further, the percent- fungicide degradation was determined. The optimum temper- age of degradation was determined spectrophotometrically at ature and pH for the degradation of fungicide was determined 220 nm. In addition, sterile soil with applied Tilt fungicide spectrophotometrically at 220 nm, and bacterial cell density was examined by LC/MS-MS for its propiconazole degrada- was measured at 600 nm after 72 h of incubation. tion capacity . A sample aliquot of 10 μL was injected into an Agilent 1290 Infinity UHPLC system (http://www.agilent. com); 10 mM ammonium acetate in water (0.1 % FA) was Cytochrome P450 gene identification in PS-4 strain used as mobile phase A and acetonitrile (0.1%FA) as mobile phase B in a Shimpak ODS column 2.0 x 150 mm in size Genomic DNA of strain PS-4 was isolated, and CYP P450 gene (http://www.shimadzu.com).Thecolumnflowratewas ampl ified u sing t h e p rim e rs F: 5 ′ - adjusted to 0.2 mL/min for both the standard and samples. ACCACATGCTCAACCTCGAC-3 ′ and R: 5 ′ - Identification and quantification of the propiconazole was TCATTGGGCGATCCTCTCGAT-3′, which were designed based on its retention time and area. Mass spectra (Thermo from the CYP P450 gene of Pseudomonas aeruginosa Fisher-TSQ Vantage) conditions were Spray Voltage (LOCUS AP014839, 1426914 bp and accession AP014839). (positive) 4000 V,Spray Voltage (negative) 2800 V, The 50μL reaction mixture contained 1× DNA polymerase buff- Vaporizer temp 300 °C, sheath gas flow rate 20 Arb and er, 0.2 mM dNTP mix, 25 μM each forward and reverse primer Aux gas flow rate 10 Arb. (final concentration 0.5 μM), 1 U Taq DNA polymerase, and 50 ng template DNA. The reaction mixture was subjected to the Propiconazole extraction from biomass following PCR program (Applied Biosystems Life Technologies Veriti Thermal Cycler, 96 wells; https://www.thermofisher.com): To follow degradation of propiconazole by strain PS-4, initialdenaturationfor5minfollowedbythedenaturationat98°C 5 mL culture filtrate was withdrawn aseptically from for1min,followedby20cycles(98°Cfor30s,65°Cfor30sand liquid medium cultures at 12-h intervals and bacterial 72 °C for 90 s) and final extension at 72 °C for 5 min. The final cell growth was measured at 600 nm. To obtain culture amplified product was fractionated on a 1 % agarose gel using gel filtrates from bacterial suspensions, all samples were documentation (Uvitech, Firereader-v4; http://www.uvitec.co. subjected to centrifugation at 10,000 g for 15 min at uk), analyzed using a 5-kb ladder (Amnion Biosciences, 4 °C. The filtrate was extracted twice with ethyl acetate Bangalore, India) and sequencing was carried out using Applied (1:1) using the rotorflash evaporator (Buchirotavapor R Biosystems 3010XL capillary sequencer. The ABI’sBigDye® 210), and residues were dissolved in 3 mL HPLC grade Terminator v3.1 sequencing chemistry was used. In orderto carry acetonitrile. The percentage degradation of out pairwise/multiple sequence alignment, the ClustalW2 Tool propiconazole was monitored with a UV spectrophotom- (http://www.ebi.ac.uk/Tools/msa/clustalw2/) was used. eter (Hitachi U 2900) at 220 nm, and the degradation rate was confirmed by HPLC analysis. Further, to con- firm the chemical data, a 40-μL aliquot was injected Preparation of cell-free extracts for enzyme assay into an HPLC Agilent 1260 device equipped with quarternary pump, auto sampler and variable wavelength For extraction of cell-free filtrates, the method described by UV detector with a C18 column (diameter 150 × Talwar et al. (2014) was followed. Briefly, P. aeruginosa PS-4 4.6mm) with aparticlesizeof5 μm, and samples were cells were washed and grown in Tris–HCl buffer pH 6.8 eluted at 1.2 min/mL with the mobile phase amended with 10 μg/L propiconazole, sonicated (Sonics vibra acetonitrile:water (80:20). Identification of metabolites cell) for 5 min, and centrifuged. The supernatant was used for of propiconazole was based on the molecular weight enzyme assay and protein estimation. The activity of of the compound as determined by their mass spectra propiconazole monooxygenase was measured spectrophoto- (Shimadzu); the flow rate was 1 mL/min, injection tem- metrically, and determination of protein concentration was perature 250 °C—the temperature was programmed by done using NanoDrop (http://www.nanodrop.com/). the DI probe from 100 °C to 250 °C. 1358 Ann Microbiol (2016) 66:1355–1365 Table 1 Biochemical characteristics of the propiconazole-degrading bacterium Biochemical details 2 APPA -3 ADO -4 PyrA -5 IARL -7 dCEL - 9 BGAL - 10 H2S - 11 BNAG - 2 AGLTp + 13 dGLU + 14 GGt + 15 OFF - 17 BGLU - 18 dMAL - 19 dMAN - 20 dMNE + 21 BXYL - 22 BAIap + 23 ProA + 26 LIP + 27 PLE - 29 TyrA - 31 URE - 32 dSOR - 33 Sac - 34 dTAG - 35 dTRE - 36 CIT + 37 MNT + 39 5KG - 40 ILATk + 41 AGLU - 42 SUCT + 43 NAGA - 44 AGAL - 45 PHOS - 46 GlyA - 47 ODC - 48 LDC - 53 IHISa - 56 CMT + 57 BGUR - 58 O129R - 59 GGAA - 61 IMLTa + 62 ELLM - 64 ILATa - APPA Ala-Phe-Pro-aryllamidase, ADO adonitol, PyrA L-pyrrolydonyl-arylamidase, IARL L-arabitol, dCEL D-Cellobiose, BGAL β-galactosidase, H2S H2S production, BNAG β-N-acetyl-glucosaminidase, AGLTp glutamyl arylamidase pNA, dGLU D-glucose, GGt gamma-glutamyl-transferase, OFF fermentation/glucose, BGLU β-glucosidase, dMAL D-maltose, dMAN D- mannitol, dMNE D-mannose, BXYL β-xylosidase, BAIap β-alanine arylamidase pNA, ProA L-proline arylamidase, LIP lipase, PLE palatinose, TyrA tyrosine arylamidase, URE urease, dSOR D-sorbitol, Sac sucrose, dTAG D-tagatose, dTRE D-trehalose, CIT citrate, MNT malonate, 5KG 5-Keto-D-gluconate, ILATk L-lactate alkalinisation, AGLU alpha-glucosidase, SUCT succinate alkalinisation, NAGA β-N-acetyl-galactosaminidase, AGAL α-galactosidase, PHOS Phosphatase, GlyA glycine arylamidase, ODC ornithine decarboxylase, LDC lysine decarboxylase, IHISa L-histidine assimilation, CMT coumarate, BGUR β-glucoronidase, O129R O/129 resistance, GGAA Glu-Gly-Agr arylamidase, IMLTa L-malate assimilation, ELLM ellaman, ILATa L-lactate assimilation Statistical analysis strains, PS-4 was found to be the most promising strain in terms of its ability to grow on MSM, and was used for further All experimental data were determined in triplicate and biodegradation studies. expressed as means ± standard error. Statistical analyses of the data were performed using one-way ANOVA with SPSS Characterization of propiconazole-degrading bacterium version 20.0 software with advanced models (SPSS Japan, by bioMérieux Vitek2 analysis and 16S rRNA sequencing Tokyo, Japan). Differences between means were located using Tukey’stest (P <0.05). PS-4 strain is an aerobic Gram negative bacterium. Fully grown propiconazole-resistant colonies of strain PS-4 were circular in shape with raised elevation with an undulating margin. Results Microbial identification was by bioMérieux Vitek2 (biochemi- cal analysis) tests (Table 1), and showed positive reactions for Isolation and screening of propiconazole-degrading catalase, oxidase and citrate. The 16S rRNA sequence obtained bacteria was 850 bp in length, and was identified as P. aeruginosa PS-4 strain. The closest relative were first determined based on the Twenty-seven (PS-1 to PS-27) strains were isolated from pad- similarity of their 16S rRNA sequences obtained by a direct dy soil and the bacterium with most potential for utilizing blast search of NCBI GenBank. The results shown that PS-4 propiconazole was screened in MSM with the propiconazole sequence showed closest matches with those of soil microor- as sole carbon source. Based on the growth of the isolated ganisms that play a vital role in pesticide degradation. The Fig. 1 Phylogenetic connections based on 16S rRNA gene sequences among Pseudomonas aeruginosa PS-4 and similar strains retrieved from NCBI GenBank constructed through the neighbour joining (NJ) method Ann Microbiol (2016) 66:1355–1365 1359 Table 2 Physicochemical properties of soil magnesium (1032 mg/kg) zinc (1.368 mg/kg), iron (1.15 mg/kg), manganese (1.15 mg/kg) and copper Nutrition Available nutrition in soil (1.718 mg/kg) (Table 2). The rate of degradation of pH 7.945 propiconazole in sterile soil placed outside the laborato- Electric conductivity dS/m 0.291 ry was found to be 21.95 % to 49.85 % (Fig. 2a), Organic carbon (mg/kg) 9500 whereas, 21.29 % to 39.65 % (Fig. 2b) of propiconazole Nitrogen (mg/kg) 90.21 was degraded in controlled soil kept inside the labora- tory for 40 days. In contrast to this, the rate of loss of Phosphorus (mg/kg) 63.44 Potassium (mg/kg) 476.8 fungicidal activity in the non-sterile soil is low, at 25.37 % to 41.02 % (Fig. 2c) in soil placed outside Sulfur (mg/kg) 15.465 the laboratory, and 16.33 % to 32.27 % (Fig. 2d)deg- Calcium (mg/kg) 11880 radation was observed in not controlled soil placed in- Magnesium (mg/kg) 1032 side the laboratory for 40 days, respectively. A high Zinc (mg/kg) 1.368 (27°C) and low (22°C) temperature was recorded during Iron (mg/kg) 1.15 the experimental set up. The optimum half life of DT Manganese (mg/kg) 1.15 was occurred on day 40 in the sterile soil placed outside Copper (mg/kg) 1.718 the laboratory. Degradation of propiconazole in liquid medium by soil isolate strain PS-4 sequence of this organism was deposited in the NCBI GenBank under the accession number KM923901. A phylogenetic tree The isolated P. aeruginosa PS-4 was investigated for its constructed with microbes classified as potent agents in biore- propiconazole degradation ability. The results show that mediation is presented in Fig. 1. 8 μg/L propiconazole was degraded after 3 days of incubation when compared to its respective control without PS-4 strain. (Fig. 3); the half life DT of Degradation of propiconazole in soil propiconazole in MSM was found to be 34 h. The physicochemical properties of paddy soil revealed reddish black soil, with pH 7.9 and electric conductivity Effect of temperature and pH on biodegradation of 291 dS/m. It was also noted that the soil contained of propiconazole organic carbon (9500 mg/kg), nitrogen (90.21 mg/kg), phosphorus (63.44 mg/kg), potassium (476.8 mg/kg), It was observed that 80 % of degradation was achieved sulfur (15.465 mg/kg), calcium (11,880 mg/kg), at 30 °C and pH 7, which indicates the mesophillic Fig. 2a–d Degradation of ab propiconazole in soil. a Sterile soil placed out side the laboratory. 50 b a b Sterile soil (controlled) placed b 30 b under laboratory conditions. c a Tilt 30 Tilt b c a c Non-sterile soil situated outside 20 0.05 µg/kg b 0.05 µg/kg the laboratory. d Non-sterile soil a b 0.1 µg/kg 0.1 µg/kg 10 c (not controlled) placed inside the a b a a a laboratory. Values are means ± SE 0 0 0 10203040 0 10203040 of three independent replicates for Days Days each incubation period. Means cd followed by the different letter(s) 50 40 are significantly different from a 40 a each other according to Tukey’s a b test (P <0.05) 30 b b Tilt 20 b c Tilt c a 20 b 0.05 µg/kg c 0.05 0.1 µg/kg 10 µg/kg a a a a 010 20 30 40 0 10203040 Days Days Propiconazole degradation Propiconazole degradation (%) (%) Propiconazole degradation Propiconazole degradation (%) (%) 1360 Ann Microbiol (2016) 66:1355–1365 110 1.2 Fig. 3 Growth (▲)and degradation of propiconazole (■) 1.1 by Pseudomonas aeruginosa PS- 4 and uninoculated controls (●)in MSM amended with 10 μg/L 0.9 propiconazole. Values are means of three replicates ± standard error 0.8 (SE) 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0 0 122436 4860 72 Time (h) nature of bacterium and hydrogen ion balance during identified in other strains of P. aeruginosa and the resulting the degradation of propiconazole by PS-4 strain. phyllogram and dendrogram showed that the gene segment However, 52.44 % degradation was found at 20 °C (amplicon) was most closely related to P. aeruginosa and 27.14 % at 50 °C (Fig. 4). Similarly, degradation VRFPA04, complete genome (Fig. 7). of propiconazole was found to be 44.6 % at pH 5 and 26 % at pH 9 (Fig. 5). Extraction of cell-free filtrates for enzyme assay Cytochrome P450 gene identification in strain PS-4 The extracts of cell-free solution showed monooxygenase ac- tivity and catalyse propiconazole, yielding major three metab- The presence of the CYP P450 gene in P. aeruginosa PS-4 olites: 1,2,4-triazole; 2,4-dichlorobenzoic acid; and 1-chloro- was confirmed and identified with a product size of 1.3 kb benzene. The enzyme and specific activity was found to be (Fig. 6). Blast analysis showed that the sequence of the am- −1 −1 0.241 ± 12 μmol min and 0.310 ± 0.3 μmol min mg plified gene had high homology with cytochrome P450 genes 80 b 20 25 30 35 40 45 50 56789 Temperature ºC pH range Fig. 4 Effect of temperature on the degradation of propiconazole; data Fig. 5 Effects of pH on propiconazole degradation; data are from 72 h are from 72 h samples from different degradation experiments at different samples from different degradation experiments. Values are means ± SE temperatures. Values are means ± SE of three independent replicates. of three independent replicates. Means with different letters are Means with different letters are significantly different from each other significantly different from each other according to Tukey’stest (P < according to Tukey’stest(P <0.05) 0.05) % Degradation % Degradation % Degradation Absorbance at 600nm Ann Microbiol (2016) 66:1355–1365 1361 filtrate. Three possible products of propiconazole were iden- tified based on molecular weight of the compounds, namely 1, 2,4-triazole; 2,4-dichlorobenzoic acid; and 1-chlorobenzene, which are possible metabolites involved in the propiconazole degradative pathway (Fig. 10). Discussion Superior, safe and affordable food for a constantly increasing population is the basic agricultural target of any nation (Babu et al. 2015). Pesticides are used widely as crop protection products to combat losses caused by pests and diseases. However, the harmful effects of these pesticides on human health and the environment is well known. In the last few decades, researchers have established that microbial degrada- Fig. 6 Agarose gel (1 %) electrophoresis stained with ethidium bromide. tion can have beneficial effects on soil fertility and crop Lanes: 1 5kb ladder, 2 amplification of CYP P450 gene (1.3 kb) growth. Several degradation studies have shown that bacteria metabolise toxic compounds under in vitro conditions (Cain and Mitchell 1996; Mitchell and Cain 1996; Shetti and −1 protein , respectively. The concentration of protein in the cell Kaliwal 2012; Abraham and Silambarasan 2013). −1 free extracts was found to be 0.969 mg mL . In the current study, a low rate of degradation and persistence of propiconazole was observed after 30 days under all conditions Chemical analyses tested. Interestingly, soil samples placed outside the laboratory showed a good rate of degradation compared to soil placed inside Based on the liquid chromatography mass spectroscopy se- thelaboratory.Therefore,microbialdegradationofpropiconazole lected reaction monitoring (LC/MS/SRM), propiconazole was wascarriedoutforcompleteremediationby isolatingtheresistant detected at m/z 342.13 (Fig. 8a). The degradation rate of bacteria. The population of isolated Pseudomonas aeruginosa propiconazole in Tilt-applied soil was measured based on (PS-4)strainfrompaddyfieldswasfoundtobemostpredominant the area count, the compound eluted at retention time in the propiconazole-contaminatedpaddy soil. Moreover, theiso- 8.69 min, and it was confirmed that 49 % of degradation lated PS-4 strain utilized propiconazole as a sole source of carbon was observed (Fig. 8b). Note that culture filtrates of PS-4 and energy for growth in MSM, resulting degradation of 8 μg/L strain analyzed by HPLC expressed different peak patterns. propiconazole under the optimal conditions of pH 7 and 30 °C Whereas the propiconazole was eluted at retention time within 3 days. This is in good agreement with the findings of 2.811 min, there was a significant decrease in the earlier researchers who demonstrated the degradation of triazole propiconazole peak (Fig. 9), indicating degradation of fungicides;thelatterreportedthatdegradationoffungicidesunder propiconazole. The results obtained from mass spectra were different environmental conditions was found to be used to predict possible metabolites accumulated in the culture more eco-friendly, efficient and useful for cleaning up Fig. 7 Phyllogram of CYP P450 gene generated by multiple sequence alignment using ClustalW2 1362 Ann Microbiol (2016) 66:1355–1365 RT: 8.68 NL: 1.06E5 AA: 600462 341.90 159.00 Propiconazole Standard 7 RT: 8.68 NL: 4.50E4 AA: 261079 341.90 159.00 Sample Day 0 RT: 8.69 AA: 182085 NL: 3.18E4 341.90 159.00 50 Sample Day 20 RT: 8.69 AA: 163497 NL: 2.98E4 341.90 159.00 Sample Day 30 RT: 8.69 NL: 2.70E4 AA: 139774 341.90 159.00 Sample Day 40 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 Time (min) Fig. 8 a Determination of molecular weight of propiconazole detected at m/z 342.13. b Liquid chromatography mass spectroscopy selected reaction monitoring (LC-MS/MS) chromatograms of propiconazole degradation of the sterile soil sample (Tilt 25 EC) of polluted areas (Nelson et al. 1973;Baileyand Coffey monooxygenase metabolizes propiconazole by yielding 1985;Oltmanns etal. 1989). (1-[[2-(2,4-dichlorophenyl)-4-methyl-1,3-dioxolan-2- In our study, Pseudomonas aeruginosa PS-4 strain was yl]methyl]-1H-1,2,4-triazole) (m/z = 313). Further, found to be the most efficient while utilising propiconazole monooxygenase activities give rise to 2,4-dichlorobenzoic ac- as the sole substrate. The results also show that CYP P450 id (m/z = 193) by the partial fragmentation of dioxolane ring Relative Abundance Ann Microbiol (2016) 66:1355–1365 1363 confirmed based on the formation of the above-mentioned metabolites and CYP P450 monooxygenase activity. Also, the results obtained can be used to construct a pathway of propiconazole degradation by the P. aeruginosa PS-4 strain. These results show for the first time that a degradative path- way for the propiconazole by P. aeruginosa PS-4 acts mainly via propiconazole CYP P450 monooxygenase activity. The previous experiment showed varying degrees of deg- radation of propiconazole, yielding 1-[[2(2,4- dichlorophenyl)-2-(1,2,4-triazole-1-yl) ketone, 1-(2,4- dichlorophenyl)-2-(1,2,4-triazole-1-yl) ethanol and 1[[2(2,4- dichlorophenyl)-4-hydroxypropyl-1,3-dioxolane-2- yl]methyl]1H-1,2,4-triazole. Also, propiconazole dissolution in paddy soil under anaerobic conditions was found to be minimal, which indicates that temperature and aeration will play an important role in the degradation of propiconazole (Kim et al. 2002). Similarly, the degradation rate of flutriafol, epoxiconazole, propiconazole, triadimefon and triadimenol fungicides also increases with increased temperature (Bromilow et al. 1999). Similarly, Chlorpyrifos degradation in soil was enhanced by an increase in temperature (Racke et al. 1994). Even though the application of triazole fungicides in agriculture is extensive, very few studies have been con- ducted on microbial degradation of these pesticides. Previously, propiconazole biodegradation was undertaken and was achieved successfully with amendment of glucose in the degradation medium by Pseudomonas putida. However, the degradation products have not been reported Fig. 9 HPLC elution profile of propiconazole extracted immediately as is the case in our study (Sarkar et al. 2009). Interestingly, after the addition of compound to a MSM and b degradation basidiomycete fungi were found to be efficient for the degra- metabolites observed at day 3 dation of propiconazole and tubeconazole, but the degradation pathway has not been studied (Woo et al. 2010). Chlorobenzene degradation by P. putida through 3- and complete cleavage of the1,2,4-triazole ring. Also, a car- boxylic group and one chlorine atom was fragmented from 2, chlorocatechol by the meta cleavage pathway involving the activity of catechol 2,3-dioxygenase (Mars et al. 1997)strong- 4-dichlorobenzoic acid by the action of propiconazole CYP P450 monooxygenase activity by yielding 1-chlorobenzene ly supports our experimental results. Some investigations specified that microorganisms will not degrade propiconazole, (m/z = 113). Thus, the metabolism of propiconazole was Fig. 10 Proposed pathway for degradation of propiconazole by Cl Cl Cl Pseudomonas aeruginosa strain O Monooxygenase PS-4 Cl Cl Cl O O OH N N 2,4-dichlorobenzoic acid 1-((2-(2,4-dichlorophenyl)-4- 1-((2-(2,4-dichlorophenyl)-4 propyl-1,3-dioxolan-2-yl)methyl) -methyl-1,3-dioxolan-2-yl)methyl) -1H-1,2,4-triazole -1H-1,2,4-triazole Cl O - O O 1-chlorobenzene acetaldehyde pyruvate Monooxygenase CYP P450 1364 Ann Microbiol (2016) 66:1355–1365 because of its strong adsorption to soil organic matter uniqueness of our study lies in the fact that the degradation (Kloskowski et al. 1987;Ekler 1988; Taylor and Spencer pathway for the propiconazole metabolism by the bacteria is 1990). Although several reports on the degradation of proposed for the first time. triazoles in soil are available, microbial degradation studies Acknowledgements The authors are thankful to the Department of have been vastly under represented. A few reports on Biotechnology (DBT), Ministry of Science and Technology, tubaconazole and other fungicide degradation by bacteria iso- Government of India, Delhi, for providing the instrumentation facility lated from contaminated soil have also been reported (Nicole (Centrifuge, UV spectrophotometer and rotorflash evaporator) (BT/PR/ et al. 2009; Megadi et al. 2010; Elhussein et al. 2011). 4555/INF/22/126/2010 dated 30 September 2010). The authors are also thankful to the UGC-UPE fellowships and Post Graduate Department of Moreover, Pseudomonas fluorescence was found to degrade Studies in Microbiology and Biotechnology, Karnatak University 10 % to 70 % tubeconazole in the culture medium with a time Dharwad for providing the laboratory facilities. gap of 6–21 days. In addition, Pseudomonas chrysosporium also showed some tubeconazole degradation (Obanda and Compliance with ethical standards Shupe 2009). Recently Vaz et al. (2015) reported the efficacy Conflict of interest The authors do not have any conflict of interest of a Pseudomonas sp. that has the ability to degrade connected to the manuscript. Paclobutrazol fungicide, a trizole fungicide well known for its longer persistence in soil. Ethical approval This article does not contain any studies related to human participants or animals. Here, we have demonstrated for the first time that P. aeruginosa PS-4 strain degrades propiconazole via the meta- bolic activity of CYP P450, involving three metabolites name- References ly 1,2,4-triazole; 2,4-dichlorobenzoic acid; and 1-chloroben- zene. Microbial degradation via CYP P450 monooxygenase is Abraham J, Silambarasan S (2013) Biodegradation of chlorpyrifos and its efficient and acts as a precursor for the degradation of many hydrolyzing metabolite 3,5,6-trichloro-2-pyridinol by toxic compounds. CYP P450 monooxygenase has been Sphingobacterium sp. JAS3. 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Annals of Microbiology – Springer Journals
Published: Jun 15, 2016
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