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The microbial degradation of azimsulfuron and its effect on the soil bacterial community

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The microbial degradation of azimsulfuron and its effect on the soil bacterial community
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  ORIGINAL ARTICLE The microbial degradation of azimsulfuron and its effect onthe soil bacterial community A. Valle 1 , G. Boschin 2 , M. Negri 1 , P. Abbruscato 1 , C. Sorlini 1 , A. D’Agostina 2 and E. Zanardini 3 1 Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche (DISTAM), Sezione MAAE, Universita ` degli Studi di Milano, Milan, Italy2 Dipartimento di Scienze Molecolari Agroalimentari (DISMA), Sezione di Chimica, Universita ` degli Studi di Milano, Milan, Italy3 Dipartimento di Scienze Chimiche ed Ambientali, Universita ` dell’Insubria, Como, Italy Introduction Sulfonylureas are widely used herbicides, as they are char-acterized by high crop selectivity, low application ratesand very low acute and chronic animal toxicity (Beyer et al.  1988; Brown 1990). Their mechanism of action isbased on the inhibition of acetolactate synthase, the key enzyme involved in branched-chain amino acid biosyn-thesis in plants and micro-organisms. Chemical hydrolysisand microbial degradation are considered the main mech-anisms of sulfonylurea herbicide transformation in soiland water (Joshi  et al.  1985; Blair and Martin 1988;Oppong and Sagar 1992; Li  et al.  1999; Brusa  et al.  2001;Kim  et al.  2003); however several studies indicate thatphotolysis may be an alternative degradation pathway (Pusino  et al.  1999; Scrano  et al.  1999; Vulliet  et al. 2002).Azimsulfuron, the active component of the com-mercial herbicide Gulliver   (Du Pont de Nemours,Cologno Monzese, Milan, Italy), is a postemergencesulfonylurea widely used in the weed control of ricefields. It is effective in controlling perennial weeds atvery low application rates (6–25 g of active ingredientper hectare), whereas higher concentrations are neededto control annual weeds (Shirakura  et al.  1995).Azimsulfuron, like other sulfonylureas, is a weak acid Keywords azimsulfuron, biodegradation, chemicaldegradation, microbial community,sulfonylurea herbicides. Correspondence A. Valle, DISTAM, Via Celoria 2, 20133 Milan,Italy. E-mail: anna.valle@unimi.it2005/0152: received 15 February 2005,revised 14 December 2005 and accepted 14December 2005doi:10.1111/j.1365-2672.2006.02937.x Abstract Aims:  Azimsulfuron is a recently introduced sulfonylurea herbicide useful incontrolling weeds in paddy fields. To date very little information is availableon the biodegradation of this pesticide and on its effect on the soil microbialcommunity. The aim of this work was to study its biodegradation both inslurry soil microcosms and in batch tests with mixed and pure cultures. Methods and Results:  Azimsulfuron was applied to forest bulk soil in order tostudy its effect on the structure of the bacterial soil community, as detectableby denaturant gradient gel electrophoresis (DGGE) analyses. Biodegradationand abiotic processes were investigated by HPLC analyses. In addition, amicrobial consortium was selected, that was able to use azimsulfuron as thesole energy and carbon source. One of the metabolites produced by the consor-tium was isolated and identified through LC-MS analyses. Cultivable bacteriaof the consortium were isolated and identified by 16S rDNA sequencing(1400 bp). Conclusions:  Azimsulfuron treatment seems to have the ability to cause chan-ges in the bacterial community structure that are detectable by DGGE analyses.It is easily biodegraded both in microcosms and in batch tests, with the forma-tion of an intermediate that was identified as 2-methyl-4-(2-methyl-2H-tetra-zol-5-yl)-2H-pyrazole-3-sulfonamide. Significance and Impact of the Study:  The study increases the knowledge onthe biodegradation of azimsulfuron and its effects on the soil microbiota. Journal of Applied Microbiology ISSN 1364-5072 ª  2006 The AuthorsJournal compilation  ª  2006 The Society for Applied Microbiology, Journal of Applied Microbiology  101  (2006) 443–452  443  and is more persistent at neutral and basic pH than inacidic conditions (having a half life of 70–120 days)(Barefoot  et al.  1996). In soil and water, indirect photo-lysis and microbial activity are considered the mostimportant azimsulfuron degradation mechanisms (Bare-foot  et al.  1996). As for the most sulfonylureas, azi-msulfuron hydrolytic degradation involves the cleavageof the sulfonylurea bridge, leading to the formation of a substituted pyrimidine and the corresponding sulfona-mide (Khan  et al.  1999). The highest levels of sorptionhave been observed in soil at low pH values and withhigh organic carbon and/or clay content (Pusino  et al. 2004). Inorganic soil colloids, such as smectite clay minerals and iron oxides, contribute to azimsulfuronsorption (Pinna  et al.  2004).Although very little information is available on themicrobiological degradation of azimsulfuron, it isknown that azimsulfuron is degraded more quickly inaerobic conditions and in the presence of flooded non-sterile soils rather than in sterile ones, thus suggestingthat microbial degradation plays a key role (Barefoot et al.  1995). No experimental data on the effects of azimsulfuron on microbial soil communities, biodegra-dation kinetics and metabolic pathways are known,although literature reports that at field rates(2–100 g ha ) 1 ), and even at higher concentrations, sul-fonylurea herbicides do not affect the total number of bacteria (El-Ghamry   et al.  2002), whereas they inhibitthe growth of nitrifiers and  Azotobacter   species (Burnetand Hodgson 1991; Allievi and Gigliotti 2001). Perucci and Scarponi (1996) reported transient negative effectsof rimsulfuron on the microbial biomass in soil.The main objectives of this work was to study thedegradation of azimsulfuron in a saturated soil, by assessing the biodegradation rate with respect to theabiotic degradation and to evaluate the structural chan-ges in the composition of the bacterial community. Forthis, an uncontaminated forest bulk soil, that has neverbeen in contact with sulfonylurea, was used on labscale microcosms (simulation of paddy soil conditions).Furthermore to avoid any photochemical processes thatare known to be very important for the azimsulfurondegradation, the microcosms were kept in the dark.This condition excludes the bacterial phototrophicmetabolism and their positive or negative interactionsbut up to now there is no evidence of a relevant roleof these physiological groups in the sulfonylurea degra-dation.In addition, we have evaluated the capability of a selec-ted soil microbial consortium to use azimsulfuron as asole energy and carbon source, as well as the number of cultivable bacterial strains composing the consortium andthe metabolite produced were investigated. Materials and methods Chemicals and solutions Azimsulfuron, N-[[(4,6-dimethoxy-2-pyrimidinyl)-2-amino]carbonyl]-1-methyl-4-(2-methyl-2H-tetrazol-5-yl) 1H-pyraz-ole-5-sulfonamide (99% purity), was provided by DuPontde Nemours Italiana (Cologno Monzese, Milan, Italy)(Fig. 1a).It was dissolved in 0 Æ 2 mol l ) 1 phosphate buffer(NaH 2 PO 4 , Na 2 HPO 4 ) pH 7 Æ 3 to obtain a 88-mg l ) 1 stock solution, which was used in the chemical stability and microbial degradation studies. An additional1000 mg l ) 1 solution was prepared for the enrichmentcultures. Both solutions were sterilized by filtrationthrough 0 Æ 22  l m filters (Millipore, Billerica, MA, USA)and stored at 4  C. The chemical stability of azimsulfu-ron was evaluated at pH 7 Æ 3 using a concentration of 50 mg l ) 1 ; two different media were tested, M9 mineralsalts medium (Raffi  et al.  1991) plus 12 Æ 5% yeastextract and phosphate buffer (pH 7 Æ 3). Triplicate testsof 10 ml final volume were set up for each medium.Tests were incubated for 28 days at 4  C or 28  C andkept in the dark to exclude degradation by photolysis.From each test, small amounts of sample were takenperiodically for HPLC-UV analyses. The decrease of azimsulfuron peak area was monitored. Analytical techniques Before being analysed by HPLC, the samples from all thedegradation experiments (stock solutions, slurry micro-cosms, consortium and pure cultures) were filteredthrough 0 Æ 45  l m disposable nylon filters (Alltech, Milan,Italy). HPLC analyses were performed with a Hewlett-Packard HP-1050 (Agilent Technologies, Palo Alto, CA,USA) quaternary pump fitted with a Rheodyne injector(20  l l loop) and equipped with a HP-1050 VariableWavelength Detector (HPLC-VWD) (Agilent Technol-ogies). The column was a Lichrospher   100 RP-18(5  l m, 250  ·  4 Æ 6 mm, Merck, Darmstadt, Germany). Theanalyses were carried out using a linear gradient: from20 : 80  ¼  methanol : 1% acetic acid in water to 100%methanol over 50 min; the flow rate was 1 ml min ) 1 .Chromatograms were recorded at 254 nm and each analy-sis was repeated three times. The mean and the standarddeviation (SD) were calculated and normalized. Theresults are presented in the form  x   ± SD%. Significantdifferences in all the degradation experiments (slurry microcosms, consortium and pure cultures) were assessedby standard analysis of variance ( anova ), using the  ncss -Statistical Analysis System- pass  software. The null hypo-thesis assumed that during the experiment, the values of  Microbial degradation of azimsulfuron  A. Valle  et al. 444  Journal compilation  ª  2006 The Society for Applied Microbiology, Journal of Applied Microbiology  101  (2006) 443–452 ª  2006 The Authors  azimsulfuron residual concentration of both tests andcontrols showed random variations.HPLC-MS analyses were performed on a LC-MSD IonTrap Series 1100 system SL version (Agilent Technol-ogies). HPLC conditions were the same as describedabove. The percentage of acetic acid was 0 Æ 1%. The MSanalyses were performed with electrospray ionization(ESI), both in positive and negative modes. The columneluent was split in the ratio of 1 : 1 before entering theelectrospray interface. The operative conditions werecapillary voltage 3500 V, drying gas flow rate 8 l min ) 1 ,temperature 325  C, nebulizer gas pressure 60 psi andacquisition scan 50–520 mz ) 1 . 1 H-NMR (nuclear magnetic resonance) spectra wererecorded on a Bruker AMX-300 spectrometer usingCDCl 3  as solvent. Soil samples The forest soil was collected in Perego (Lecco, Italy) inOctober 2003, stored at 4  C and then sieved at 2 mmbefore use. It had the following characteristics: pHvalue 5 Æ 86, organic carbon content 1 Æ 62%, totalnitrogen content 0 Æ 18% and cation exchange capacity 12 Æ 56 cmol(+) kg ) 1 . The method reported by  Walkley and Black (1934) was used to determine the total organiccarbon, and the method reported by Kjeldahl (1883) wasused to determine the total nitrogen. The cation exchangecapacity was determined by the barium chloride andEDTA (pH 8 Æ 1) method (SISS 1985). Slurry microcosms Tests were performed in triplicate in 0 Æ 5 l bottles contain-ing 54 ml of M9 mineral salts medium, with 12 Æ 5% yeastextract and 16 ml of uncontaminated forest soil suspen-sion (soil : water  ¼  1 : 1 w/v). Bottles were incubated at28  C for 48 h for acclimatization. In addition to the testswith fresh soil, the experimental protocol included twodifferent control tests: biotic controls (three bottles) withfresh soil but without the addition of azimsulfuron; andabiotic controls (three bottles), using soil previously ster-ilized by gamma irradiation (2  ·  10 5 rad h ) 1 ). Becausethis sterilizing procedure is always dependent on theradiation dose and the type of soil, the three abioticcontrols were repeatedly checked by cultural methods forcomplete absence of micro-organisms. All samples werekept in the dark to avoid photolysis. After acclimatizationin the incubator, the stock solution of azimsulfuron wasadded to the tests and the abiotic controls to the finalconcentration of 20 mg l ) 1 . The same volume of phos-phate buffer (0 Æ 2 mol l ) 1 ) was added to the biotic con-trols. The experiment lasted 4 weeks during which testswere kept in static conditions at 28  C and samples for SNNOCH 3 NNNNNNCH 3  OCH 3 NHOOCONHH 3 C (a)(c)(b) SNNNNNNCH 3 NH 2 OOH 3 C Figure 1  (a) Chemical structure of azimsulfu-ron; (b) Intermediate degradation product:2-methyl-4-(2-methyl-2H-tetrazol-5-yl)-2H-pyrazole-3-sulfonamide; (c) HPLC-UV chroma-togram of 28th day sample; test performedwith the selected consortium in mineral saltsmedium M9 with 100 mg l ) 1 of azimsulfuron.A. Valle  et al.  Microbial degradation of azimsulfuron ª  2006 The AuthorsJournal compilation  ª  2006 The Society for Applied Microbiology, Journal of Applied Microbiology  101  (2006) 443–452  445  microbial counts, chemical analyses and DNA extractionwere collected on days 0, 7, 14, 21, and 28. Cultural media and microbial cultivations In order to count total heterotrophic bacteria and fungi,tenfold serial dilutions of soil slurry samples were plated,respectively, on plate count agar, PCA (Oxoid, UK) andRosa Bengala Cloramphenicol (Oxoid) standard laborat-ory media.Enrichment cultures were performed in 90 ml of M9mineral salts medium with the addition of 100 mg l ) 1 of azimsulfuron, using 10 g of forest soil as an initial inocu-lum. After nine transfers into fresh medium, anazimsulfuron degrading consortium was selected.A variety of standard cultural media, such as trypticsoy agar, TSA (Oxoid),  Pseudomonas  selection agar, PSA(Oxoid), Luria–Bertani agar, LBA (Oxoid), R2A (Oxoid)and M9 mineral salts medium agar with and without0 Æ 01% w/v of azimsulfuron were used to isolate bacterialstrains from the azimsulfuron degrading consortium. Degradation tests using selected consortium To study the degradation of azimsulfuron by the previously selected consortium, a new experiment was performed.Triplicate tests were set up in 90 ml of M9 mineral saltsmedium with the addition of 100 mg l ) 1 of azimsulfuronand inoculated with 10 ml of the selected consortium(10 6 cells ml ) 1 of 9th enrichment culture transfer deter-mined by cultural methods). In addition, triplicate controlswithout inoculum were performed and azimsulfuron deg-radation was monitored by HPLC-UV analyses at weekly intervals. The experiment lasted 34 days.Similarly, the biodegradability of metabolite B by theselected consortium was assessed at the concentration of 100 mg l ) 1 in 50 ml of M9 mineral salts medium. Testsand controls (without microbial inoculation) wereperformed in triplicate and microbial degradation wasmonitored weekly by HPLC-UV analyses for 34 days.In addition, all the bacterial strains isolated from theconsortium were tested for azimsulfuron degradation inpure culture. Triplicate tests and controls (without inocu-lum) were performed. In this case, 10 ml of pure culture(10 6 cells ml ) 1 ) was inoculated in 90 ml of M9 mineralsalts medium with the addition of 100 mg l ) 1 of azimsulfuron, the experiment lasted for 48 days. All theexperiments were kept in the dark to avoid photolysis. Preparation and identification of the product B Azimsulfuron (50 mg) was dissolved in 8 ml meth-anol : water (1 : 1, v/v) and 1 ml 6 mol l ) 1 HCl wasadded. After 60 days at a temperature of 20–22  C, thecrude reaction mixture was concentrated under vacuumand the residue was purified by flash chromatography onsilica gel (Merck) using hexane : ethyl acetate (4 : 6, v/v)as eluent. The main metabolite eluted was named com-pound B, its structure was characterized by HPLC-ESI-MS and NMR as described above (Fig. 1b). 16S rDNA amplification and denaturing gradient gelelectrophoresis (DGGE) Aliquots (2 ml) both of the overnight 10 ml broth cul-tures in LB medium and of slurry tests were centrifugedat 20 817  g   for 5 min. The pellets were resuspended in540  l l of 10 mmol l ) 1 Tris–HCl buffer pH 8 supplemen-ted with 1 mmol l ) 1 EDTA pH 8 (TE buffer pH 8) and50  l l lysozyme (50 mg ml ) 1 ) and incubated at 37  C for20 min. Then 11 Æ 5  l l of sodium dodecylsulfate 25% and6  l l proteinase K (20 mg ml ) 1 ) were added. After 1 h at37  C the samples were supplied with 100  l l of 5 mol l ) 1 NaCl and 80  l l of cetyl-tri-methylammonium bromide25%, then incubated at 65  C for 10 min. The DNAwas extracted twice with phenol : chloroform : isoamylalcohol (25 : 24 : 1, v/v/v), then precipitated in ethanolafter centrifuging at 20 817  g   for 30 min, dried and resus-pended in MilliQ RNase free water (Qiagen, Milan, Italy).DNA samples extracted from the slurry and the consor-tium were used as templates to amplify the V3 region of the 16S rDNA fragments corresponding to the nucleotidepositions of 341–518 bp of the  Escherichia coli  16S rDNAnumbering (Brosius  et al.  1981), using an adjoining40-bp guanine–cytosine (GC) clamp with a forward primerfor Eubacteria GC341 F (Muyzer  et al.  1993) and reverseuniversal primer 518R (Neefs  et al.  1990). The PCR reac-tion was performed in 50  l l final volume with 1 ·  PCR buffer (Sigma-Aldrich, Milan, Italy), each deoxynucleosidetriphosphate at the concentration of 0 Æ 2 mmol l ) 1 , eachprimer at the concentration of 0 Æ 2  l mol l ) 1 and 2 U of  Taq  DNA polymerase (Sigma-Aldrich). Initial denatura-tion at 95  C for 5 min was followed by 16 cycles consist-ing of denaturation at 95  C for 1 min, annealing at 63  Cfor 1 min and extension at 72  C for 1 min. Then a fur-ther 14 cycles consisting of denaturation at 95  C for1 min, annealing at 55  C for 1 min and extension at72  C for 1 min. Finally, there was an extension step at72  C for 10 min. PCR was performed using the thermalcycler (PCR Express Hybaid, Franklin, MA, USA).The GC-clamped products were separated on 10%(w/v) polyacrylamide gels with 30–60% urea/formamidedenaturing gradient. To compare the different DGGEgels, a custom-made marker was prepared by assem-bling various bacterial strains isolated from the samesoil. Microbial degradation of azimsulfuron  A. Valle  et al. 446  Journal compilation  ª  2006 The Society for Applied Microbiology, Journal of Applied Microbiology  101  (2006) 443–452 ª  2006 The Authors  Denaturing gradient gels were cast and run using theD-Code system (Bio-Rad, Hercules, CA, USA). Gels elec-trophoresed at 60  C at a constant voltage of 200 V for4 Æ 5 h were stained with SYBR green nucleic acid gel stain(Fluka, St. Louis, MO, USA) and visualized by UV trans-illumination. The images were acquired with the GelDoc2000 System (Bio-Rad, Milan, Italy). The patterns wereanalysed assuming that the number of bands representedthe number of different organisms and species (Yin  et al. 2000). This represents an approximation, as many bac-teria harbour several rDNA operons per cell that may thus give more than one band. The band intensity wastaken to represent the abundance of each phylotypes. TheShannon’s Index was used to calculate the diversity index: H   ¼  ) P ( n i /  N  ) ln ( n i /  N  ), where  n i  is the area of eachband peak and  N   the sum of all the band peak areas (Yin et al.  2000). Significance was determined based on tripli-cate readings using an  anova . Moreover DGGE profileswere analysed by reamplifying, cloning and sequencingthe bands of interest. Bands were excised from gels andwere eluted in water. Recovered DNA was reamplifiedusing 341F and 518R primers as described above. Prod-ucts were cloned into pGEM  T (Promega, Milan, Italy)and were sequenced using SP6 forward and T7 reverseprimers. After screening, the clones were sequenced usingthe BigDye Sequencing Kit on an ABI PRISM 310 GeneticAnalyser (Applied Biosystems, Perkin-Elmer, Foster city,CA, USA) sequencer according to manufacturer’s instruc-tions. Sequence homology was determined by   blastn searches to detect the most similar sequences in GenBank,EMBL, DDBBJ and PDB databases. Identification of isolates A large portion (approximately 1400 bp) of the 16S rRNAgene was amplified using the universal primers ( E. coli 16S rDNA numbering), forward 8F (5 ¢ -AGAGTTTGATC-CTGGCTCAG-3 ¢ ) and reverse 1492R (5 ¢ -GGTTACCTTG-TTACGACTT-3 ¢ ) (Crump  et al.  1999). PCR productswere purified using QIAquick PCR Purification Kit (Qi-agen, Milan, Italy). Sequencing of PCR products andsequence homology were performed as described above.The results of   blastn  were crosschecked by analysing thephylogenetic position of the sequences using the Ribo-somal Subunit Database and  phylip  program. Finally, thesequences were submitted to GenBank. Results Chemical stability of azimsulfuron The chemical stability of azimsulfuron was investigated at4 and 28  C in a M9 mineral salts medium, containing yeast extract and a phosphate buffer at pH 7 Æ 3. Azi-msulfuron was stable in both conditions and tempera-tures; a slight degradation (13 ± 1 Æ 6%) was observed only in the tests with the M9 mineral salts medium at 28  C onthe 17th day and no further degradation occurred untilthe end of the experiment. Moreover, no degradationproducts were detected by HPLC-UV analyses. Slurry microcosms An experiment in slurry microcosms was carried out inorder to evaluate the effects of azimsulfuron on the micro-bial community of an uncontaminated forest soil andto estimate the microbial degradation rate in comparisonwith abiotic degradation. No significant differenceswere detected for the heterotrophic bacteria counts inthe treated samples and controls, being 1 Æ 1  · 10 7 ± 2 Æ 1 CFU gdw  ) 1 of soil. Fungi were also monitoredduring the experiment because it is well known that they play an important role in the degradation of herbicides insoil. In this study, they were detected only at the begin-ning of the study (6 Æ 14  ·  10 4 ± 1 Æ 6 CFU gdw  ) 1 ) andthereafter no further fungal growth was observed. Thiswas probably due more to the slurry experimentalconditions than to a negative effect of azimsulfuron. Theresidual concentration of azimsulfuron was regularly monitored by HPLC analyses in all samples (Fig. 2,expressed as residual percentages). In the forest soil micro-cosms, the residual concentration of azimsulfuron after28 days was 1 Æ 51 mg l ) 1 , corresponding to a total degrada-tion of 87 ± 4 Æ 73%. The decrease was gradual and highly significant ( P   ¼  0 Æ 001) by   anova . On the contrary,azimsulfuron was not significantly degraded ( P   ¼  0 Æ 43) inthe abiotic control microcosms with a reduction of  0204060801001200 7 14 21 28Time (days)     R  e  s   i   d  u  a   l  a  z   i  m  s  u   l   f  u  r  o  n   (   %   ) Figure 2  Azimsulfuron degradation in slurry microcosms – abioticcontrol (sterilized sample) in white bars and test (not sterilized) in greybars. Forest soil slurry microcosms were treated with 20 mg l ) 1 of azi-msulfuron; tests were performed in triplicate. Azimsulfuron residualconcentrations were displayed in percentages for days 0, 7, 14, 21and 28. Errors bars represent SD.A. Valle  et al.  Microbial degradation of azimsulfuron ª  2006 The AuthorsJournal compilation  ª  2006 The Society for Applied Microbiology, Journal of Applied Microbiology  101  (2006) 443–452  447
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