Estimation of atrazine-degrading genetic potential and activity in three French agricultural soils

FEMS Microbiology Ecology 48 (2004) 425–435 www.fems-microbiology.org

Estimation of atrazine-degrading genetic potential and activity in three French agricultural soils Fabrice Martin-Laurent a,*, Laurent Cornet a, Lionel Ranjard a,
pez-Guti
errez a, Laurent Philippot a, Christophe Schwartz b, Juan-Carlos Lo R
emi Chaussod a, G
erard Catroux a, Guy Soulas a,1 a

INRA-CMSE, UMR 1229 INRA-Universit
e de Bourgogne, Microbiologie et G
eochimie des Sols, 17 rue Sully, BP 86510, 21065 Dijon Cedex, France b ENSAIA-INPL/INRA, Laboratoire Sols et Environnement UMR 1120, 2, avenue de la For^et de Haye, BP 172, F-54505 Vandoeuvre-les-Nancy, France Received 15 December 2003; received in revised form 28 January 2004; accepted 1 March 2004 First published online 2 April 2004

Abstract The impact of organic amendment (sewage sludge or waste water) used to fertilize agricultural soils was estimated on the atrazine-degrading activity, the atrazine-degrading genetic potential and the bacterial community structure of soils continuously cropped with corn. Long-term application of organic amendment did not modify atrazine-mineralizing activity, which was found to essentially depend on the soil type. It also did not modify atrazine-degrading genetic potential estimated by quantitative PCR targeting atzA, B and C genes, which was shown to depend on soil type. The structure of soil bacterial community determined by RISA fingerprinting was significantly affected by organic amendment. These results showed that modification of the structure of soil bacterial community in response to organic amendment is not necessarily accompanied by a modification of atrazine-degrading genetic potential or activity. In addition, these results revealed that different soils showing similar atrazine-degrading genetic potentials may exhibit different atrazine-degrading activities. Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Biodegradation; Atrazine; atz genes; Quantitative PCR; Soil bacterial community

1. Introduction Soil microorganisms are among the most diverse component of terrestrial ecosystems [1] where they transform the organic matter and contribute to carbon and nutrients fluxes [2]. They also play a key role in the quality of agricultural soils which is usually defined as the sustained capacity of the soil to produce healthy and nourishing crops, resist erosion and reduce the impact of environmental stresses on plants [3]. Soil microbiota * Corresponding author. Tel.: +33-3-80-69-34-06; fax: +33-3-80-6932-24. E-mail address: [email protected] (F. Martin-Laurent). 1 Present address: UMR Oenologie-Amp
elologie, Universit
e Victor Segalen Bordeaux 2, 351 cours de la lib
eration, 33405 Talence Cedex, France.

notably affects: (i) soil fertility (availability of plant nutrients) [4] and health (suppression of soil-borne plant disease) [5] and (ii) detoxifying ability (e.g., pesticides and xenobiotic compounds biodegradation) [6]. Soil microorganisms have long been regarded as ubiquitous, i.e., ‘‘everything is everywhere’’ [7]. This view led to the common assumption that soil microbial communities are black boxes often considered as passive catalysts for degradation, which is ultimately controlled by abiotic factors such as temperature, humidity and pH [7]. However, increasing evidence shows that abiotic variables are not universally suitable for describing major soil processes such as organic matter turnover and xenobiotics behaviour. It was suggested that the key to understanding soil functioning is the description of the composition and the biodiversity of soil microbial communities [8]. Although soil microorganisms have

0168-6496/$22.00 Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsec.2004.03.008

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F. Martin-Laurent et al. / FEMS Microbiology Ecology 48 (2004) 425–435

been grouped into functional clusters, the ecological and process regulatory significance of species richness in soil communities remains obscure [1]. Indeed, the hypothetical interrelation between soil microbial biodiversity and soil ecological functioning is poorly documented. This underlines the need to develop approaches that allow explicit links to be established between the presence of specific microorganisms and the processes they catalyse [9]. In order to determine possible interrelations among (i) the functioning of specific soil microbial communities, (ii) the genetic potential and (iii) the soil microbial community structure, we have chosen to study atrazinedegrading communities in three French agricultural soils cropped with maize and yearly treated with atrazine at the dose of 1 kg ha
1 . Atrazine [2-chloro-4-(ethylamino)-6-(iso propyl-amino)-s-triazine] is one of the most widely used s-triazine-ring herbicides for controlling, through photosystem II inhibition, pre- and post-emergence broadleaf weeds in important crops such as maize (Zea mays) and sorghum (Sorghum sp.) [10]. This herbicide is moderately persistent in natural environments where it is slowly degraded to hydroxyatrazine by chemical processes [11] and partially hydroxylated to deethylatrazine (DEA) and deisopropylatrazine (DIA) by endogeneous bacterial mono-oxygenases [12]. As a result, atrazine and its two main metabolites DEA and DIA are environmentally prevalent s-triazines, frequently detected at concentrations exceeding the European Union limit of 0.1 lg l
1 of individual pesticide in drinking water [13]. Soils repeatedly treated with this herbicide show accelerated atrazine degradation accompanied by marked mineralization of s-triazine rings [12,14,15]. A great variety of atrazine-degrading bacteria have been isolated from soils that have been in contact with this chemical [16–19]. These bacteria commonly initiate atrazine degradation with a hydrolytic dechlorination, catalysed by ATZA [20] and followed by two amidohydrolytic reactions catalysed by ATZB [21] and ATZC [22], which together transform atrazine to cyanuric acid, which is then fully mineralized to CO2 and NH3 by three other hydrolases ATZD, E and F [23]. The atzA, B and C genes coding enzymes related to the amidohydrolase superfamily are widely dispersed, conserved and associated with transposable elements similar to IS1071 [24,25]. Atrazine-degrading microbes are phylogenetically diverse and show several catabolic gene combinations in which atz genes are not always clustered on the same plasmid suggesting that atrazine-degrading bacterial communities are still in evolution [17,25]. We report the evaluation of: (i) the activity of soil atrazine-degrading populations determined by radiorespirometry, (ii) the atrazine-degrading genetic potential of specific soil microbial communities estimated by quantifying atzA, B and C gene copy number by real-

time PCR conducted on DNA directly extracted from soil and (iii) the structure of soil microbial communities estimated by ribosomal intergenic spacer analysis (RISA) in French agricultural soils exposed to different agricultural practices (soil amendment with sewage sludge or waste water).

2. Materials and methods 2.1. Soil origin, treatment and properties Soil samples were collected from the agricultural soilsof Couhins (soil type: podzol), Pierrelaye (soil type: sandy neoluvisoil) and La Bouzule (soil type: redoxic neoluvisoil) cropped with corn and yearly treated with 1 kg ha
1 of atrazine. The soil of Couhins is characterized by a very high percentage of sand and a relatively low pH (Table 1). This field plot (Institut National de la Recherche Agronomique, INRA-Bordeaux, France) was continuously cropped with corn and treated for 19 years (from 1974 to 1993) as follows: none (N–P–K fertilizers only, with soil considered non-amended) (U), farmyard manure (10 tons of dry matter per ha each year) (FM), sewage sludge (10 tons of dry matter per ha each year) (SS10) and sewage sludge (100 tons of dry matter per ha every two year) (SS100). SS100 soil showed significantly higher CEC, microbial C biomass and contents of organic C and N than U, FM and SS10 soils. SS10 and SS100 soils amended with sewage sludge showed a Cu concentration 1.5 and 3.2 times higher than U soil, respectively, and a Zn concentration 7.0 and 26.0 times higher than U soil, respectively (Table 1). The soil of Pierrelaye is typical from the Seine alluvial valley presenting a high percentage of sand and a neutral pH (Table 1). This site was amended for 102 years with the municipal wastewater of Paris and used for monoculture of grain maize over the 30 last years. The site of Pierrelaye was divided in three different areas according to their level of contamination: weakly (WP), moderately (MP) or heavily (HP) polluted. CEC and organic C content were shown to be proportional to the level of contamination of the soil (HP > MP > LP). HP soil microbial C biomass was significantly higher than those of MP and LP. Pierrelaye soils were contaminated with heavy metals (Zn and Cu) in proportion to the level of soil pollution ([Cu/Zn]HP > [Cu/Zn]FP > [Cu/Zn]LP ). The soil of La Bouzule is typical from eastern part of France presenting a high percentage of silt and clay as well as a neutral pH (Table 1). This field experiment (Institut National Polytechnique de Lorraine, Nancy, France) previously cropped for 10 years with a winter wheat/rape rotation, was continuously cropped with corn since year 2002 and treated as follows: none (U), lightly dehydrated sewage sludge (LDSS), lightly dehy-

Redoxic neoluvisoil U LDSS LDCSS LDCSS + PAH La Bouzule2

Impact of soil amendment on soil physicochemical and biological properties of the agricultural soils of Couhins, Pierrelaye and La Bouzule. Soil treatments were: Couhins, (U) none (N–P–K fertilizers only, with soil considered non-amended), (FM) farmyard manure (10 tons of dry matter per ha each year), (SS10) sewage sludge (10 tons of dry matter per ha each year) and (SS100) sewage sludge (100 tons of dry matter per ha every two year); Pierrelaye, lightly polluted (LP), moderately polluted (MP) or heavily polluted (HP); La Bouzule, (U) none, (LDSS) lightly dehydrated sewage sludge, (LDCSS) lightly dehydrated composted sewage sludge and (LDCSS + PAH) lightly dehydrated composted sewage sludge amended with organic pollutants. 1 Ref. [48]. 2 This study, nd not determined. Values followed by the same letter did not significantly differ.

224.36a 430.28b 411.74b 383.52b nd nd nd nd 14.6a 15.2a 15.2a 16.9a

Sandy neoluvisoil LP MP HP Pierrelaye1

33.0

11.5

55.5

1.99a 2.01a 2.25a 1.71a

0.21a 0.21a 0.22a 0.18a

7.1a 7.1a 6.9a 7.2a

nd nd nd nd

110a 101a 125a 139a 278b 400c 42a 80b 154c 3.6a 5.2b 6.3c

Podzol Couhins

1

U FM SS10 SS100

8.0

75.0

17.0

1.30a 1.53a 3.33b

0.10a 0.12a 0.20b

7.4a 7.4a 7.4a

28.1a 51.6a 197b 730.7c 17.3a 16.8a 25.8b 55.8c 5.7a 6.3b 6.5b 5.7a 3.2a 3.9a 4.5b 7.3c 0.11a 0.15a 0.16a 0.39b 1.69a 1.86a 1.66a 3.25b 12.5 83.3

Silt Sand Clay

4.2

pH CEC Organic N (%) Organic C (%) Amount (%) of Soil type Treatment Soil

Table 1 Soil physicochemical properties of the three French agricultural soils

68.9a 79.7a 84a 125.5b

Zn (mg kg
1 of soil) Cu (mg kg
1 of soil)

Microbial C biomass (mg C kg
1 of soil)

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drated composted sewage sludge (LDCSS), lightly dehydrated composted sewage sludge added with organic pollutants (phenantrene 260 mg kg
1 dry matter, fluoranthene 250 mg kg
1 dry matter, pyrene 12.5 mg kg
1 dry matter and benzo(a)pyrene 7.3 mg kg
1 dry matter) (LDCSS + PAH). These organic amendments did not affect measured soil physicochemical properties. The microbial biomass C of LDSS, LDCSS and LDCSS + PAH soils of La Bouzule was 50% higher than the control (Table 1). Soil samples were collected from the 20-cm top layer of the experimental fields. Fresh soil samples were sieved (5-mm mesh) and stored less than one month at 4 °C until use. 2.2. Microbial biomass measurements Soil microbial biomass C was measured using the fumigation–extraction technique [26]. An automated UV–persulfate oxidation method was carried out with a Dohrman DC80 analyser [27]. Microbial biomass C was determined with the following formula: Microbial biomass C ¼ ðCfumigated extract
Cunfumigated extract Þ= Kc. A Kc factor of 0.38 was used to convert extractable C into microbial biomass C according to Nicolardot et al. [28]. 2.3. Atrazine mineralization The ability of indigenous soil microorganisms to mineralize atrazine was determined by radiorespirometry [29]. Soil samples (10 g equivalent dry weight) moistened to 80% of the water-holding capacity were treated with 1.7 kBq of 14 C uniformly (ring)-labelled atrazine [910 MBq mmol
1 , 98% radiochemical purity (Sigma)] to give the concentration of 1.5 mg kg
1 soil. They were incubated in the dark at 20 °C for 64 days. 14 CO2 resulting from the mineralization of 14 C-atrazine was trapped in 5 ml of 0.2 M NaOH solution and analysed by liquid scintillation counting using ACS II (Amersham) scintillation fluid. The Gompertz growth model modified to fit well second-order degradation ð
kðt
tiÞÞ kinetics [y ¼ ae
e þ ct] [30] was fitted to the atrazine mineralization data using inverse modelling (SigmaPlotÒ 4.0). Four parameters were determined: a, the plateau or maximum percentage of mineralization; ti, the abscissa of the inflexion point; k, the mineralization rate constant and c, the rate of 14 C turnover and cometabolic mineralization. Three replicated atrazine mineralization kinetics were realized per soil sample. 2.4. Soil DNA extraction Nucleic acids were extracted in triplicate from 250 mg of soil [31]. Briefly, samples were homogenized in 1 ml of extraction buffer (100 mM Tris (pH 8.0), 100 mM EDTA, 100 mM NaCl, 1% (w/v), polyvinylpyrrolidone

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and 2% (w/v), sodium dodecyl sulfate) for 30 s at 1600 rpm in a mini-bead beater cell disrupter (Mikro-Dismembrator S, B. Braun Biotech International, Germany). After the removal of centrifuged soil and cell debris, the proteins were eliminated using sodium acetate precipitation. Then, nucleic acids were precipitated with cold isopropanol and washed with 70% ethanol. They were purified using a Sepharose 4B spin column. The integrity of the soil DNA was checked by electrophoresis on 1% agarose gel. DNA was quantified at 260 nm using a BioPhotometer (Eppendorf, Hamburg, Germany).

Eight-microliter aliquots were separated by electrophoresis on a native 6% acrylamide gel run for 17 h at 8 mA. Gels were stained with SYBR green II (Molecular Probes, Leiden, The Netherlands). RISA profiles were analysed with the One-D Scan 2.03 program (Scanalytics program) allowing the elaboration of matrices (presence–absence and relative intensity of each band). Principal component analysis (PCA) on covariance matrix was performed using the ADE-4 software [33].

2.5. Quantification of atzA, B and C gene copy number in soil

3.1. Atrazine-degrading communities

Quantitative PCR were carried out in a Smart Cycler (Cypheid, USA) using the Smart Kit for Sybr Green I according to Piutti et al. [17]. Twenty five nanograms of DNA extracted directly from soil was used as template for quantitative PCR carried out in presence of 0.625 lg of T4 Gene 32 product (Qbiogene, UK). The amplification conditions were as follows: 95 °C 600 s; 45 cycles of 15 s at 95 °C, 15 s at 60 °C, 15 s at 72 °C followed by 1 melting cycle measured from 60 °C to 95 °C by incrementing temperature of 0.2 °C/s. Gene-specific primers for the amplification of atzA, B and C genes were developed for this study, Af 50 -ACG GGC GTC AAT TCT ATG AC-30 , Ar 50 -CAC CCA CCT CAC CAT AGA CC-30 , Bf 50 -AGG GTG TTA GGT GGT GAA C-30 , Br 50 -CAC CAC TGT GCT GTG GTA GA-30 , Cf 50 -GCT CAC ATG CAG GTA CTC CA-30 and Cr 50 TCC CCC AAC TAA ATC ACA GC-30 . The specificity of these primers was checked by amplifying soil DNA samples, cloning and sequencing of PCR products (data not shown). Calibration of quantitative PCR was carried out using as template serial dilution of the appropriate cloned target sequence (from 101 to 108 copies). Calibration curves relating the log of the copy number of the target gene as a function of the Ct (cycle threshold) were developed, namely: log ðatzAÞ ¼
3:86  Ct þ 41:6 ðR2 ¼ 0:997Þ, log ðatzBÞ ¼
3:54  Ct þ 40:4 ðR2 ¼ 0:981Þ and log ðatzCÞ ¼
3:48  Ct þ 38:9 ðR2 ¼ 0:996Þ.

Atrazine mineralization kinetics was determined by radiorespirometry analysis. Representative kinetics of cumulative 14 C ring-labelled atrazine mineralization is given in Fig. 1. Three distinct groups of atrazine mineralization kinetics were observed in accordance with soil origin. The soils of Pierrelaye and Couhins showed atrazine mineralization kinetics typical from soil adapted to atrazine mineralization whereas the soil of La Bouzule poorly mineralized atrazine. Atrazine mineralization was very rapid for the soil of Pierrelaye reaching approximately 70% of the initially applied atrazine after only 10 days of incubation. For this soil no initial lag phase in 14 CO2 production was observed. On the contrary, in the soil of Couhins, a 20-day lag phase was observed, and atrazine mineralization reached approximately 60% of the initially applied atrazine only after 70 days of incubation. The mineralization kinetics observed for the soils of La Bouzule were almost linear suggesting the occurrence of co-metabolic degradation. Cumulative 14 CO2 evolutions at the end of the experiment were close to 30% of the initial radioactivity. Atrazine mineralization kinetics of the adapted soil samples (i.e., Pierrelaye and Couhins) were modelled by fitting the modified Gompertz model in order to determine kinetics parameters (Table 2). The model fit the experimental data well with correlation factors ranging from 0.997 to 0.999 (Table 2). In addition, all the modelled kinetics successfully passed the analysis of variance ðp < 0:0001Þ (data not shown). On one hand, for the experiment of Pierrelaye, the lightly polluted soil (LP) showed significantly lower a (maximum percentage of mineralization) and k (mineralization rate) values as well as a ti (abscissa of the inflexion point) value of the same order of magnitude compared to the moderately polluted soil (MP) and lower compared to the highly polluted soil (HP). This result suggests that atrazine mineralization efficiency is a positive function of organic amendment applied to this soil. On the other hand, for the experiment of Couhins, U, FM, SS10 and SS100 soils exhibited almost the same value averaging 75% of cumulative 14 CO2

2.6. Ribosomal intergenic spacer analysis The 16S–23S intergenic spacer of the bacterial rDNA was amplified in a final volume of 50 ll from 50 ng of soil DNA with 1 lM of 38r (50 -CCG GGT TTC CCC ATT CGG-30 ) and 72f (50 -TGC GGC TGG ATC TCC TT-30 ) universal primers [32] using 2.5 U of Taq DNA polymerase (Appligene Oncor, France). PCR were carried out in a PTC 200 gradient cycler (MJ Research, Waltham, MA) with the following conditions: 5 min at 94 °C, 35 cycles of 1 min at 94 °C, 1 min at 55 °C and 2 min at 72 °C, plus an additional 15-min cycle at 72 °C.

3. Results activity

of

soil

microbial

F. Martin-Laurent et al. / FEMS Microbiology Ecology 48 (2004) 425–435

A

Table 2 Parameters of atrazine mineralization kinetics obtained for the soil of Couhins U, FM, SS10 and SS100 and for the soil of Pierrelaye LP, MP or HP after fitting the modified Gompertz growth model

Soil of Couhins

% cumulative14CO2

80 60

% cumulative14CO2

Treatment

r2

a

k

ti

Couhins

U FM SS10 SS100

0.999 0.997 0.998 0.999

70.71a 70.19a 75.48a 76.70a

0.04a 0.04a 0.04a 0.1b

35.70a 36.67a 38.24a 29.33b

Pierrelaye

LP MP HP

0.999 0.997 0.998

61.15a 64.47a 62.68a

0.61a 0.85b 0.78b

2.95a 3.15a 4.67b

SS10 SS100

20

FM 0

B

Soil

U

40

0

20

429

40 Time (day)

60

80

r2 refers to R2 value of non linear regression, a to the maximum percentage of mineralization, k to the mineralization rate, ti to the abscissa of the inflexion point. Values are means (n ¼ 5). Considering each parameter values followed by different letters are significantly different (p < 0:05). Values followed by the same letter do not significantly differ.

Soil of Pierrelaye 80

60

40

LP

20

FP

MP

not tremendously affect the maximum percentage of atrazine mineralization. 3.2. Atrazine-degrading potential of soil microbial communities

0 0

C

20

40 Time (day)

80

So il of La Bouzule

80

% cumulative14CO2

60

U LDSS

60

LDCSS LDCSS+PAH

40 20 0 0

20

40

60

80

Time (day) Fig. 1. Kinetics of degradation of 14 C ring-labelled atrazine in [panel A] the soil of Couhins (U) non-amended, amended with (FM) farmyard manure, (SS10) sewage sludge (10 ton ha
1 year
1 ) and (SS100) sewage sludge (100 ton ha
1 year
2 ); [panel B] the soil of Pierrelaye amended with (LP) lightly polluted, (MP) moderately polluted or (HP) highly polluted waste water; [panel C] the soil of La Bouzule (U) nonamended, (LDSS) amended with lightly dehydrated sewage sludge, (LDCSS) amended with lightly dehydrated composted sewage sludge and (LDCSS + PAH) amended with lightly dehydrated composted sewage sludge added with polyaromatic hydrocarbon.

emitted in 70 days. The rate of mineralization of atrazine (k) of SS100 soil was higher than those found in the control, FM and SS10 soils. Therefore, the application of organic amendment to the three agricultural soils did

Atrazine-degrading genetic potential of soil microbial communities was determined by real-time PCR quantification of the copy number of atzA, B and C genes. The copy number of 16S rDNA sequences was determined for each soil DNA sample in order to verify the efficiency of DNA extraction/amplification (data not shown). Specific quantitative PCR protocols were developed for each targeted gene according to Piutti et al. [17]. The results of the quantification of the copy number of atzA, B and C sequences determined on DNA extracted directly from the soils of Couhins, Pierrelaye and La Bouzule are presented in Fig. 2. The atzA, B and C genes were detected in all tested soil DNA samples in quantities varying from 5.0  103 to 8.0  104 copies per gram of soil. For the soil of Couhins, atzB was detected in significantly (p < 0:05) higher density in FM soil (23.06  4.10  103 atzB g
1 of soil) than in SS10 (8.70  2.6  103 atzB g
1 of soil). atzC was detected in significantly (p < 0:05) higher density in SS10 soil (18.63 3.90  103 atzC g
1 of soil) than in SS100 (11.57 1.89  103 atzC g
1 of soil) (Fig. 2A). Similar densities of atzA (in mean 11.50  3.20  103 atzA g
1 of soil) were detected in U, FM, SS10 and SS100 soil samples (Fig. 2A). For the soil of Pierrelaye, the atzA, B and C genes showed very similar pattern of quantification. atz genes were detected in significantly (p < 0:05) higher number in the lightly polluted soil (LP) than in the moderately (MP) and highly (HP) polluted soil (Fig. 2B). It is noteworthy that the density of atz sequences detected in the soil of Pierrelaye which exhibited the highest

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amount of atz sequences depend on the atz gene and on the soil type considered. 3.3. Structure of soil microbial communities

Fig. 2. Quantitative PCR analysis of atzA, B and C sequences from DNA samples extracted from [panel A] the soil of Couhins U, FM, SS10 and SS100; [panel B] the soil of Pierrelaye LP, MP or HP; [panel C] the soil of La Bouzule U, LDSS, LDCSS and LDCSS + PAH.

atrazine-degrading capability among the soils tested here was higher that those of the soil of Couhins and La Bouzule. For the soil of La Bouzule, atzA and atzB genes exhibited similar pattern of detection being detected in lower densities in U and LDSS than in LDCSS and LDCSS-PAH soil samples (Fig. 2C). On the contrary, atzC was quantified in significantly higher amount in U and LDSS than in LDCSS and LDCSS-PAH samples. These results showed that: (i) the amount of atz A, B, C sequences quantified seems soil type specific and that (ii) the impact of organic amendment on the

The structure of soil microbial communities was evaluated by applying Ribosomal Intergenic Spacer Analysis (RISA) on DNA extracted directly from soil. RISA reveals the length polymorphism of the 16S–23S intergenic spacer of bacterial ribosomal operon. RISA conducted on DNA extracted directly from soil samples of Couhins, Pierrelaye and La Bouzule produced relatively complex fingerprints (20–35 bands per lane) and in most cases, very similar among the replicates of a treatment, illustrating the relatively good reproducibility of DNA extraction, amplification and separation (Fig. 3, panels IA, IB and IC). The three different soils could be easily distinguished by comparison of their RISA fingerprint. Furthermore, based on the number and intensity of the bands observed, RISA fingerprints were compared by pairwise analysis using principal component analysis, which allowed to ordinate microbial communities associated with the various treatments on the plane defined by the two first principal components and to compare the magnitude of changes induced by treatment (Fig. 3, panels IIA, IIB and IIC). For the soil of Couhins, the first principal component explained 45.3% of the variances in the data and 15.2% was explained by the second component (Fig. 3, panel IIA). The factorial map showed that ordination on PC1 allowed to differentiate the microbial communities according to the treatment applied to the soil. U and FM were similar and different from S10 and S100. For the soil of Pierrelaye, the first principal component explained 30% of the variances in the data and 21 % was explained by the second component (Fig. 3, panel IIB). The factorial map revealed that ordination on PC1 allowed for discrimination of the microbial communities of LP and MP with HP, which is related to the level of organic amendment applied to the soil. For the soil of La Bouzule, the first principal component explained 30% of the variances in the data and 20% was explained by the second component (Fig. 3, panel IIC). The factorial map revealed that ordination on PC1 only allowed to discriminate the microbial communities found in U, LDSS from LDCSS and LDCSS-PAH soil samples.

4. Discussion Three agricultural soils – Couhins (South West of France), Pierrelaye (Center of France) and La Bouzule (East of France) – belonging to three different soil types – Podzol, Neoluvisoil and Redoxic Neoluvisoil – cropped with maize, repeatedly treated with atrazine and amended with organic matter were studied to estimate

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431

Fig. 3. [Panel I]: RISA fingerprints of PCR products amplified with 16S–23S rDNA universal primers (38r and 72f ) from DNA extracted from [panel IA] the soil of Couhins U, FM, SS10 and SS100; [panel IB] the soil of Pierrelaye LP, MP or HP; [panel IC] the soil of La Bouzule U, LDSS, LDCSS and LDCSS + PAH. [Panel II]: PCA ordination of the genetic structure of the whole soil bacterial communities found in [panel IIA] the soil of Couhins U, FM, SS10 and SS100; [panel IIB] the soil of Pierrelaye LP, MP or HP; [panel IIC] the soil of La Bouzule U, LDSS, LDCSS and LDCSS + PAH.

the genetic potential and the activity of specific atrazinedegrading soil microbial communities. Sewage sludge or waste-water amendments have for a long time been used in agriculture as a valuable source of plant nutrients and

organic matter [34]. Only the field plot of Couhins amended for 15 years with high quantity of sewage sludge (i.e., 100 tons/ha every two years) and the HP soil of Pierrelaye amended for 102 years with municipal

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wastewater showed changes in some soil physicochemical parameters such as organic C. However, although organic amendment increases soil fertility, it also contributes to soil contamination with heavy metals, which are widespread and highly persistent pollutants in soil environment [35,36]. The quantification of Cu and Zn content revealed a strong contamination of the soils of Couhins amended with farmyard manure or sewage sludge, and of the field plots of Pierrelaye, in proportion to the amount of the waste-water applied. Several studies have showed that heavy metals decrease soil microbial biomass [37]. In the present study, we showed that soil microbial C biomass is either unaffected by waste-water application (soil of Pierrelaye) or increased after amendment with sewage sludge (soils of La Bouzule and of Couhins). It is noteworthy that the impact of soil amendment with organic matter on soil microbiota is difficult to monitor since numerous abiotic and biotic factors need to be considered. In addition, the control soil, which should be the same soil but lacking the amendment, is in reality hardly ever achieved (i.e., case of the field plot of Pierrelaye). When available, the control often differs in other properties from the amended soil enabling to establish the impact of the amendment [35]. The impact of organic amendment on atrazine-degrading activity, catalysed by specific microbial communities genetically able to degrade atrazine, was estimated. As a result of (i) repeated atrazine application, which favours the adaptation of soil bacterial communities [12] and of (ii) maize cultivation, which stimulates atrazine-degrading bacterial communities [15,38], the soils of Couhins, La Bouzule and Pierrelaye were adapted to atrazine biodegradation as demonstrated by the kinetics of atrazine mineralization and/or the presence of genes found in soils showing relatively high atrazine-degrading activity. On the basis of the comparison of atrazine-mineralizing activity, the three soils could easily be differentiated: (i) the soil of Pierrelaye mineralized up to 75% of the atrazine initially added after only 15 days of incubation, (ii) the soil of Couhins mineralized 70% of the atrazine within 60 days after a 20 day lag phase and (iii) while the soil of La Bouzule mineralized only 30% of the atrazine over 72 days of incubation. It is noteworthy that soils regularly treated with atrazine over the past two decades (i.e., Couhins and Pierrelaye) were strongly adapted to atrazine-biodegradation whereas soil only recently treated with atrazine (i.e., La Bouzule) was lightly adapted. Pesticide fate in soil is governed by interactions between retention, transformation and transport processes [10]. The soils of Couhins and Pierrelaye presented similar physico-chemical properties except that they differed in pH. This may explain the lag phase observed for the soil of Couhins since atrazine accelerated biodegradation has been reported to occur mainly in soils with a

pH > 6:5 [14]. The high amount of clay contained in the soil of La Bouzule may reduce the bioavailability of atrazine and may explain the relatively low level of atrazine-mineralization observed for this soil despite the presence of atz genes. Sorption of pesticide on organic matter determines pesticide bioavailability [6] and modification of soil organic matter through organic matter application has been shown to affect atrazine sorption and thus microbial catabolism of atrazine [39,40]. Modelling of atrazine mineralizing kinetics only revealed two significant differences: (i) the soil of Couhins amended with 100 tons of sewage sludge per hectare every two years mineralized atrazine more rapidly than the control soil with a 150% increase of atrazine mineralization rate and a 20% decrease of the ti parameter and (ii) the soils of Pierrelaye amended with either lightly and moderately polluted waste water mineralized atrazine more rapidly than soil amended with highly polluted waste water (i.e., 35% decrease of atrazine ti). However, in both cases these differences in atrazine mineralization rate (k) and in the parameter ti did not modify significantly the value of the plateau (a) reflecting the total amount of atrazine degraded. It therefore seems that high organic soil amendment affects mainly the rate of atrazine mineralization (k) but do not modified the total amount of atrazine mineralized (a). The quantification of atzA, B and C copy number in DNA samples extracted directly from adapted soils revealed that the atrazine-degrading community was averaging 104 copy of atz gene per g of soil. This result is in accordance with previous results obtained by 14 C-most-probable-number technique [41], quantitative-competitive PCR [42] and quantitative PCR [17]. It also confirms that atzA, B and C genes coding enzymes involved in atrazine catabolism are widespread in atrazine-contaminated environment [24]. It is noteworthy that the soil of Pierrelaye showing the highest atrazine-degrading activity also exhibited the highest density of atz gene sequences and the gene density reflecting the type of the organic treatment. The soil of Couhins and La Bouzule exhibited similar atrazine-degrading genetic potential ranging from 1.0 to 2.0  104 atz copy number per gram of soil. However, their atrazine-degrading activities were different; the soil of Couhins was characterized by a 20-day lag phase and a maximal percentage of atrazine mineralization reaching 60% over a 60-day incubation period, while the soil of La Bouzule did not show a lag phase but reached a maximal percentage of atrazine mineralization of only 40%. This observation suggests that the expression of atrazine-degrading genetic potential is affected by soil physicochemical properties, which modify pesticide fate and biodisponibility as well as microbial diversity and activity [6,10,14]. Indeed, for each atrazine-degrading microbe the expression of its degrading genetic potential depends on several levels of regulation (i.e., transcrip-

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tional, translational and post-translational regulations) influenced by the perception of biotic and abiotic factors specific for each soil environment [43]. In addition, atrazine-degrading bacteria hosting atz genes affected their pattern of expression, thereby modulating the expression of atrazine-degrading genetic potential [44]. The impact produced by soil organic amendment on atrazine-degrading genetic potential corresponded to that observed on atrazine-degrading activity for the experimental plot of Pierrelaye. However, for the two other experimental plots considered, the atrazine-degrading activity was either slightly or not affected by organic amendment and the atrazine-degrading potential estimated as the copy number of atz sequences, only showed small variations in response to organic amendment. These variations were inconsistent since quantification of atz genes from soil DNA extracts by real-time PCR only allowed to confidently discriminate samples different of one order of magnitude. These results, however, indicate that both atrazinedegrading activity and atrazine-degrading genetic potential were not tremendously affected by soil organic amendment. To further assess the impact of organic amendment on soil microbial communities Ribosomal Intergenic Spacer Analysis (RISA) which has previously been demonstrated to be relevant and sensitive enough to study bacterial communities associated with different microscale environment [45] or vegetation cover [46] was applied. RISA fingerprints generated from soil DNA were relatively complex and well reproducible among replicates indicating the absence of variability in the soil samples studied and/or extraction and PCR amplification biases due to some soil physicochemical properties (O.C., N, pH) [31]. RISA analysis revealed that the structure of soil bacterial community was modified in response to soil amendment with either sewage sludge or waste-water. The strongest alterations of the structure of the soil bacterial community were recorded for the long-term field experiments with the soils of Pierrelaye and Couhins for which the different samples were strongly differentiated along the first principal component (PC1). For the experimental plot of La Bouzule, which was only established for only five years, the structure of soil microbial community was not modified in response to organic amendment. RISA confirmed previous data showing that long-term organic amendment modified the global structure of the soil bacterial community [34,36,47]. This data set seems to further indicate that long-term application of organic amendment which furnishes C and N nutrients and exposes soil microbiota to heavy metal led in turn to change the microbial community structure. Though soil organic amendment modified importantly soil microbial community structure, it did not tremendously alter both atrazine-degrading activity and genetic potential. It may

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be possible that RISA provides only a skewed view of the global structure of soil microbial communities revealing primarily the numerically dominant phylotypes, while phylotypes specific for atrazine biodegradation, which represent only a small part of the total bacterial community, remained hidden. These results, however, question the importance of bacterial community structure for the expression of a microbial function in complex environments, such as soil, particularly in the case of a redundant function, such as atrazine-degrading activity. In addition, this study demonstrates that the genetic potential estimated by real-time PCR quantification of atz sequences reflects in some cases the measured atrazine-degrading activity of adapted soil bacterial communities but also that soils exhibiting similar atrazine-degrading genetic potential showed different atrazine-degrading activities. Further work will aim to study the functioning of atrazine-degrading bacterial strain and/or bacterial community in order to elucidate key factors involved in the expression of atrazine-degrading genetic potential.

Acknowledgements This work was supported by the Ministere de l’Am
enagement du Territoire et de l’Environnement: MATE (contract of research No. PE01/042001/031) and GESSOL (contract of research No. PA31/A014994). We thank Michel Schiavon for help in sampling and analysing the soil of La Bouzule. We also thank Monique Lin
eres and Isabelle Lamy for giving us access to the field experiments of Couhins and Pierrelaye, respectively.

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