Extracellular plant DNA in Geneva groundwater and traditional artesian drinking water fountains

Chemosphere xxx (2009) xxx–xxx

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Chemosphere

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Extracellular plant DNA in Geneva groundwater and traditional artesiandrinking water fountains

John Poté a,b, Patrick Mavingui c, Elisabeth Navarro a, Walter Rosselli d, Walter Wildi b, Pascal Simonet a,Timothy M. Vogel a,*

a Environmental Microbial Genomics Group, Laboratoire AMPERE, Ecole Centrale de Lyon, Université de Lyon, 36 Avenue Guy de Collongue, 69134 Ecully, Franceb Forel Institute, University of Geneva, 10, Route de suisse, 1290 Versoix, Switzerlandc Université de Lyon, F-69622, Lyon, France; Université de Lyon 1; CNRS, UMR5557, Ecologie Microbienne, Franced WSL Swiss Federal Research Institute, Antenne Romande, 1015 Lausanne, Switzerland

a r t i c l e i n f o

Article history:Received 5 October 2008Received in revised form 16 December 2008Accepted 17 December 2008Available online xxxx

Keywords:Plant DNATransportGroundwaterGeneva basin

0045-6535/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.chemosphere.2008.12.048

* Corresponding author. Tel.: +33 4 72 18 65 14; faE-mail address: [email protected] (T.M. Vogel).

Please cite this article in press as: Poté, J.,(2009), doi:10.1016/j.chemosphere.2008.1

a b s t r a c t

DNA, as the signature of life, has been extensively studied in a wide range of environments. While DNAanalysis has become central to work on natural gene exchange, forensic analyses, soil bioremediation,genetically modified organisms, exobiology, and palaeontology, fundamental questions about DNA resis-tance to degradation remain. This paper investigated on the presence of plant DNA in groundwater andartesian fountain (groundwater-fed) samples, which relates to the movement and persistence of DNA inthe environment. The study was performed in the groundwater and in the fountains, which are consid-ered as a traditional artesian drinking water in Geneva Champagne Basin. DNA from water samples wasextracted, analysed and quantified. Plant gene sequences were detected using PCR amplification based on18S rRNA gene primers specific for eukaryotes. Physicochemical parameters of water samples includingtemperature, pH, conductivity, organic matter, dissolved organic carbon (DOC) and total organic carbon(TOC) were measured throughout the study. The results revealed that important quantities of plant DNAcan be found in the groundwater. PCR amplification based on 18S rDNA, cloning, RFLP analysis andsequencing demonstrated the presence of plant DNA including Vitis rupestris, Vitis berlandieri, Polygonumsp. Soltis, Boopis graminea, and Sinapis alba in the water samples. Our observations support the notion ofplant DNA release, long-term persistence and movement in the unsaturated medium as well as ingroundwater aquifers.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

The increase in global travel has increased the occurrence ofinvasive non-native plant species in temperate climates (Kolarand Lodge, 2001; Hails, 2002). At the same time, developmentand use of transgenic plants have increased the occurrence of exo-tic genes, if not plants, in agricultural applications. In both cases,concerns about the potential ecological and economic impact havebeen extensively debated (Mack and D’Antonio, 1998; Kolar andLodge, 2001). With invasive plants, most studies address ecologicaleffects, while with transgenic plants, most studies address the pos-sibility of gene transfer. Gene transfer from transgenic plants hasalso been the central issue in studies examining the potential forplant transgenes to be incorporated into soil bacteria genomes.

A variety of studies have shown that extracellular DNA releasedfrom organisms exists in surface water that is both fresh and mar-

ll rights reserved.

x: +33 478 43 37 17.

et al. Extracellular plant DNA2.048

ine, and in soil and sediment (DeFlaun et al., 1986; Ogram et al.,1987; Trevors, 1996; Blum et al., 1997; Paget et al., 1998; Willers-lev et al., 2003). The fate of DNA in the environment can be sum-marized as follows: (i) DNA release from organisms (plant,microorganisms, and animals) (ii) persistence of extracellularDNA; (iii) adsorption of extracellular DNA to the soil matrix; (iv)degradation of extracellular DNA by DNases; (v) extracellularDNA transforms competent soil microorganisms (binding of DNAonto soil components does not eliminate the ability of boundDNA to transform competent soil microorganisms); (vi) probabledispersal and vertical movement of an extracellular DNA in unsat-urated soil medium; and (vii) extracellular DNA used as a nutrientsource by soil and aquatic microorganisms. Although data aboutthe frequency or likelihood of each of these steps are available,the entire sequence is difficult to be monitored in the field.

When DNA was added to soil or groundwater aquifer micro-cosms, its stability and activity were associated with sorbed phases(Romanowski et al., 1993; Poté et al., 2003). DNA degradation rateshave been shown to be influenced by specific soil characteristics,and DNA adsorbed on clay or absorbed into soil organic matter is

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not as rapidly degraded by nucleases (Demanèche et al., 2001).DNA found in the surface water, especially in the presence of oxy-gen, has been considered unstable (Lindahl, 1993). Therefore, thecombination of DNA sorption to soil and its degradation in theaqueous phase would suggest that DNA cannot travel far inthe subsurface. However, our earlier laboratory work suggestedthat DNA degradation might be thwarted by the movement ofDNA away from degradative soil environments into the groundwa-ter (Poté et al., 2003, 2007).

For millions of years, DNA from plants has been naturally re-leased into the environment. This released DNA during the planttissue decomposition in the field may be degraded, stabilised onsoil particles and persist in the environment for a significant perioddepending on biotic and abiotic soil characteristics (Widmer et al.,1996; Paget et al., 1998). When extracellular DNA enters the soilenvironment, it might be dispersed by rain, irrigation-induced per-colation, runoff or groundwater (Ceccherini et al., 2007) beforebeing completely degraded.

DNA degradation due to physical, chemical, and biological pro-cesses reduces the probability of DNA survival ex vivo. Yet, exam-ples of DNA persistence have been reported when the DNA issequestered away from degradative elements (e.g. UV radiationhigh temperatures, and nucleases) such as when it is sorbed ontoclays or buried in bones. Thus, DNA is apparently either protectedby sorption or subjected to degradation in aqueous solutions (DeF-laun et al., 1986; Paul et al., 1987). Many recent studies (Lerat et al.,2005; Gulden et al., 2005; Zhu, 2006) have focused on what hap-pens to transgenes in the environment. However, there remains apaucity of information on the transport of DNA in subsurface soil,and no clear relationship between the persistence of plant DNAand groundwater characteristics has been established.

Poté et al. (2003) used extracted and purified DNA to study thetransport of antibiotic genes in saturated soil medium. Contact be-tween bacteria and the exogenous DNA was improved by mixingthe soil and DNA in a microcosm with the result that bacteria wereable to incorporate DNA in soil. Gulden et al. (2005) were able toquantify the amount of corn and soybean DNA released by theplant roots into leachate water during growth and early plantdecomposition in water. They showed that the detectable quanti-ties of plant target DNA were released into the soil environment,and were moved by water during growth and early decompositionof roots. Poté et al. (2007) simulated the natural processes of plantdecomposition in soil, DNA release, and vertical movement of re-leased DNA in vadose zone using unsaturated soil column. Thesestudies suggested that DNA could be transported in subsurface soiland can reach the groundwater.

The infiltration of DNA from the vadose zone to the groundwa-ter has not been completely evaluated. There is little known aboutthe identity and quantity of DNA in the groundwater. Here, we re-port on the widespread occurrence of plant DNA in groundwaterand traditional drinking water fountains in the Geneva ChampagneBasin.

2. Materials and methods

2.1. Study area

The research was performed in the Geneva basin (WesternSwitzerland) called ‘‘Champagne Genevoise”, which is character-ised by an intense agricultural land use mainly with wheat, sweetcorn, grapes and vegetables. No transgenic plants are cultivated inthis area. In this part of the Alpine forestland, marly and sandy bed-rock has been eroded during the last glacial period by glacial andfluvial processes. Surface sediments are glacial till, glacio-lacus-trine and lacustrine shale and fluvio-glacial sand and gravel

Please cite this article in press as: Poté, J., et al. Extracellular plant DNA(2009), doi:10.1016/j.chemosphere.2008.12.048

(Moscariello et al., 1998). The main aquifers are located in thesecoarse clastic deposits (Fig. 1). A shallow aquifer, covered by afew meters of colluvial gravel and sand and 50 cm of soil, feedsthe traditional village fountains. Groundwater comes from the lo-cal infiltration of rain water. Rainfall is about 1000 mm per yearand falls mainly in autumn and spring (Ebener, 2000).

2.2. Water sampling

Groundwater was sampled from a monitoring well in the shal-low aquifer described above using the ‘‘Service Cantonal de Géolo-gie” sampler (Geneva, Switzerland). At this location, thegroundwater was at a depth of 3.2 m (Fig. 2). In addition, tradi-tional drinking water sources from this aquifer were sampled twicefrom different village fountains in the Champagne Genèvoise areaduring high and low water conditions. Water was sampled at theexit of the pipes that fed the fountains. For each sampling site,water samples were taken by filling three sterile 2 L plastic bottles.While sampling water at each site, three sterile plastic bottles(controls) containing 2 L of distilled water were kept open to theair throughout the period of sampling in order to test possible air-borne DNA contamination, such as pollen. After sampling, all thesamples were stored in an icebox at 4 �C, and were immediatelytransported to the laboratory for analysis within 24 h.

2.3. Physicochemical parameter analysis

Physicochemical parameters including pH, conductivity, dis-solved organic carbon (DOC) and total organic carbon (TOC) weredetermined. The electrical conductivity, the temperature and thepH were measured using a Multi 350i (WTW). The DOC and theTOC measurements were performed using Shimadzu High Temper-ature Total Organic Carbon Analyzer (5000 GmbH, Switzerland) onacidified samples (200 lL of 2 M HCl in 30 mL sample).

2.4. DNA extraction from water samples

Before DNA extraction, water samples were concentrated 200times using Kühner Lyophilisateur (Adolf Kühner, Birsfelden, Swit-zerland) at �45 �C and by Speed Vac plus SC 110A (Fisher Bioblock,Kirch France) at 20 �C. This did not affect the quantity nor the qual-ity of DNA (DeFlaun et al., 1986; Paul et al., 1987). The concen-trated water was stored at �20 �C until DNA extraction.

The concentrated water samples were divided into three frac-tions in order to extract different DNA compartments. DNA in frac-tion 1 (total DNA) was extracted using Ultraclean soil DNA Kit (MoBio Labs, Solana Beach, CA 92075). Adsorbed DNA (fraction 2) anddissolved DNA (fraction 3) were extracted following the proceduredescribed previously (Poté et al., 2007).

Five millilitres of concentrated water were centrifuged at13000g for 15 min at 10 �C. Extracellular dissolved DNA in super-natant was precipitated in absolute ethanol, rinsed, and resus-pended in sterile water following the method described byFrostegärd et al. (1999). The pellet was used to extract adsorbedDNA following the method described above for the extraction oftotal DNA. The possibility of plant cells occurring in this fractioncannot be completely excluded.

In order to remove interference from humic acids, extractedDNA was subsequently precipitated in absolute ethanol (Norma-pur, Prolabo), rinsed with 70% v/v ethanol in water, and then resus-pended in sterile water. Following this, the DNA suspension wasthen passed through purification Microspin S-400 (Pharmacia Bio-tech) to remove all traces of humic acids (Poté et al., 2003). Thepurified DNA was kept at �20 �C until used. The concentration ofrecovered DNA water samples was quantified spectrophotometri-cally (OD260).

in Geneva groundwater and traditional artesian ... Chemosphere

Fig. 1. General profile and lithological description of sample sites in the Geneva basin.

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2.5. PCR amplification

Aliquots of the extracted DNA were used for PCR amplification.The presence of plant sequences in water samples was tested usingPCR amplification based on 18S primers F889 (5’-GAATGGCTCAT-TAAATCAGTTA-3’) and F890 (5’-CTTGATTAATGAAAACATCCTTG-3’), which were complementary to a part of the plant 18S gene. Arange of photosynthetic eukaryotes could be responsible for thesignal. PCR was carried out in 50 lL reaction volume containing1X PCR buffer, 1.5 mM MgCl2, 200 lM of each dNTP, 200 nM ofeach primer, 0.5 units Taq DNA polymerase (Invitrogen, France),and a template DNA (100 ng). PCR was performed in a Biometrathermocycler (BIOLABO, France) in the following conditions: aninitial denaturation at 94 �C for 5 min; 35 cycles of denaturation(94 �C for 45 s), annealing (55 �C for 45 s), extension (72 �C for45 s); and a final extension for 5 min at 72 �C. PCR products wereseparated and visualized using electrophoresis on 0.8% agarosegel stained with ethidium bromide (10 lg mL�1, Sigma, USA).

2.6. Cloning, RFLP analysis and sequencing

The PCR products were cloned in a pCR�2.1-TOPO vector usingTOPO-TA cloning Kit (Invitrogen, UK) to characterise the planteukaryote species in water samples. The recombinant plasmidswere chemically transformed into Escherichia coli DH5a chemicallycompetent cells by following the instructions of manufacturers.The clones were selected on LB medium supplemented with kana-

Please cite this article in press as: Poté, J., et al. Extracellular plant DNA(2009), doi:10.1016/j.chemosphere.2008.12.048

mycin (50 lg mL�1), IPTG (40 lg mL�1) and X-GAL (60 lg mL�1).The clones were subcultured again on the same medium and usedfor plasmid extraction by using QIAprep Spin Miniprep Kit (QIA-GEN, Courtaboeuf, France).

The insert fragments were released by EcoRI digestion, and werefurther analyzed by Restriction Fragment Length Polymorphism(RFLP) using HaeIII and RsaI endonucleases. Fragments with dis-tinct RFLP patterns were submitted to sequencing at Genome Ex-press (Meylan, France).

Sequences were submitted to NCBI data bank and comparedusing the BLASTN algorithm (Altschul et al., 1990). On the basisof the results of database analyses, sequences were aligned withrepresentative bacterial sequences from GenBank(www.ncbi.nlm.nih.gov/blast) using ClustalX (Thompson et al.,1997). Phylogenetic trees were constructed by using the Neigh-bor-Joining method (Saitou and Nei, 1987) and visualized withNjPLOT (Perriere and Gouy, 1996). All the sequences described inthis study have been submitted to EMBL database under accessionnumbers AJ866532 to AJ866543.

3. Results

3.1. Water characterisation

The chemical characteristics of water samples are reported inTable 1. The water temperature was about 14 �C, and the pH wasabout 7.5. The high values of conductivity (1314 lS cm�1), DOC

in Geneva groundwater and traditional artesian ... Chemosphere

Fig. 2. Location of groundwater and traditional village fountain sampling sites in the Geneva basin.

Table 1Physicochemical parameters of water samples.

Number on map (Fig. 2) Fountain designs Temp. (�C) pH Conductivity (lS cm�1) DOC ± sd (mg L�1) TOC ± sd (mg L�1)

1 Passeiry a 12.9 7.5 821 1 ± 0.09 1.1 ± 0.06b 13.4 7.5 713 1 ± 0.05 1.3 ± 0.05

2 Meurons a 14.1 7.7 704 1 ± 0.05 6 ± 0.12b 15.2 7.3 685 1.3 ± 0.02 1.5 ± 0.03

3 Epeisses a 13.9 7.6 619 0.15 ± 0.005 0.3 ± 0.003b 13.4 7.7 585 0.24 ± 0.001 0.5 ± 0.006

P4 Groundwater a 13.9 7.9 1314 18.6 ± 0.4 29.3 ± 0.07

a: Sampled October 2001.b: Sampled October 2002.sd: Standard deviation value determined from three replicates.DOC: dissolved organic carbon, TOC: total organic carbon.

Table 2DNA concentration in ground and drinking water samples.

Fountains Total DNA(lgL�1 ± sd)

Adsorbed DNA(lgL�1 ± sd)

Dissolved DNA(lgL�1 ± sd)

Passeiry a – – –b 12.3 ± 0.7 5.7 ± 0.3 2.8 ± 0.6

Meurons a 17.1 ± 2 9.4 ± 1.5 1.9 ± 0.8b 11.6 ± 0.9 6.4 ± 0.6 3.2 ± 0.5

Epeisses a 8.5 ± 0.9 3.3 ± 0.5 1.7 ± 0.6b 6.2 ± 0.8 2.9 ± 0.3 1.4 ± 0.2

Groundwater 23.8 ± 4 11.6 ± 2 5.3 ± 2

– : Analysis not performed.a : Sampled in October 2001.b : Sampled in October 2002.sd : Standard deviation value determined from three replicates.

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and TOC were measured in the groundwater. DOC and TOC concen-trations were 18.6 and 29.3 mg L�1, respectively.

3.2. DNA concentration in water samples

The amount of recovered DNA in water samples was deter-mined using spectrophotometer at OD260. The DNA concentrationin water samples are reported in Table 2. The amount of recoveredDNA in the groundwater was almost the same order of magnitudeas in the water fountains. No great difference in DNA concentrationwas observed as a function of sampling condition. The estimatedconcentration of DNA in groundwater was 23.8, 11.6 and 5.3 lg L�1

for total DNA, adsorbed DNA and dissolved DNA, respectively. DNAconcentration in water fountains ranged from 6.2 to 17.1, from 3.3

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to 9.4 and from 1.4 to 3.2 lg L�1 for total DNA, adsorbed DNA anddissolved DNA, respectively. No DNA was detected in the controlsamples.

3.3. Quality of DNA in water samples

DNA degradation in water samples was measured qualitativelyby comparing the DNA size distribution on the agarose gel with astandard DNA ladder. The genomic analysis of agarose gel electro-phoresis showed the similar profile for total and adsorbed DNArecovered from all water samples. No noticeable degradation wasobserved. In addition, the smear, which indicates nucleic acid deg-radation, was observed for all dissolved DNA extracted from watersamples (Fig. 3).

3.4. Detection of plant genes in water samples

Screening for plant DNA using PCR amplification with 18S rRNAgene primers specific for eukaryotes was used to detect the plantDNA in water samples. The expected 1 Kb length of 18S eukaryoterRNA gene fragment was positive for the groundwater and the arte-sian fountain drinking water samples. The RFLP analysis of thecloned 18S rRNA gene PCR products of groundwater and fountainwater showed considerable eukaryote polymorphism and diversity(Table 3). The control samples did not show any PCR amplification.

Fig. 3. Gel signature of DNA extracted from groundwater and artesian drinkingfountain water. Lane 1: molecular weight standard (Smart Ladder); Lane 2: totalDNA from groundwater; Lane 3: sorbed DNA from groundwater; Lane 4 : total DNAfrom fountain of Meurons, October 2001; Lane 5: total DNA from fountain ofMeurons October 2002; Lane 6: total DNA from fountain of Meurons, October 2002;Lane 7: extracellular DNA from groundwater; Lane 8: extracellullar DNA fromfountain of Meurons October 2002.

Table 3Plant profiles analysed by RFLP.

Fountains Plant DNAprofile type

Number of clones testedby RFLP for their profile type

Passeiry a – –b B, B, D, D 4

Meurons a C, D 2b A, A, A, D 4

Epeisses a D, E, E 3b A, A, A, A 4

Groundwater A, A, A, B 4

– : Analysis not performed.a : Sampled in October 2001.b : Sampled in October 2002.A, B, C, D indicate the type of profile observed.

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Sequencing and blast analysis indicate the presence of differentplant species including Vitis rupestris (Viridiplantae – VitaceaAF321272); Vitis berlandieri � Vitis vinifera (Viridiplantae – VitaceaeAF321263); Polygonum sp. Soltis (Caryophyllidae – PolygonaceaeAF206996) Sinapis alba (Rosidae – Brassicaceae X17062); and Boopisgraminea (Asteridae – Calyceraceae AF107583). In addition, DNAfrom microalgae, and Phytophthora megasperma (OomycetesX54265) was also detected (Fig. 4).

4. Discussion

Nucleic acids are ubiquitous in many environments, includingfresh water, marine water column and sediments, soil and terres-trial subsurface (DeFlaun et al., 1987; Paul et al., 1987; Trevors,1996; Blum et al., 1997). Extracellular DNA is naturally releasedfrom the organisms including plants, animals and microorganisms(Paget et al., 1998). The release of DNA from plants, animals andmany microorganisms occurs by cell lysis after the death of organ-isms (Paget and Simonet, 1994). In an aquatic environment, theamount of extracellular DNA is the result of complex interactions,involving production, release and degradation of DNA (Paul et al.,1987). Therefore, freshwater and oceans constitute a large reser-voir of extracellular DNA. This DNA is a constituent of both totaland dissolved DNA. Extracellular dissolved DNA includes a solublefraction (free DNA) and DNA adsorbed to organic or inorganic par-ticles (Poté et al., 2007). The concentration of total DNA in surfacewater has been estimated to range from 2 to 90 lg L�1 and from0.03 to 30 lg L�1 for dissolved DNA (Lorenz and Wackernagel,1994; DeFlaun et al., 1987; Paul et al., 1987).

Here, we investigated the detection, quantification and charac-terisation of DNA in groundwater and fountain water of the Genevabasin. The results of this study demonstrate that the large quantitiesof DNA can also be found in groundwater and traditional artesiandrinking water of Geneva basin. The amount of recovered DNA fromall water samples included prokaryotes and eukaryotes DNA frac-tions. Although the quantity of total and adsorbed DNA recoveredwas significant, no great difference was observed between the quan-tity of total DNA recovered from groundwater and artesian fountainwater. The concentration of total DNA in groundwater was about24 lg L�1, and ranged from 6.2 to 17.1 lg L�1 in the water fountains.The concentration of adsorbed DNA on soil particles or colloids wasabout 12 lg L�1 in groundwater, and ranged from 3.3 to 9.4 lg L�1

in fountain water. Again, no significant difference was observed be-tween the amounts of adsorbed DNA recovered. In addition, the con-centration of dissolved (extracellular) DNA was relatively low. Themaximum concentrations were 5.3 and 3.2 lg L�1 in groundwaterand fountain water, respectively (Table 2).

The DNA degradation in water samples was measured qualita-tively. Genomic analysis by agarose gel electrophoresis showedthe degradation (presence of a smear) of dissolved DNA in all watersamples (Fig. 3). The presence of a smear on agarose gel does notmean that all DNA molecules are degraded, but it does mean thatDNA molecules have been partially degraded. Numerous experi-ments have demonstrated the importance of soil components inprotecting extracellular DNA against nuclease degradation (Khan-na and Stotzky, 1992; Pietramellara et al., 2007). The persistenceof DNA in soil is thought to be due to DNA adhesion to soil compo-nents resulting in its protection against nuclease activities (Pagetand Simonet, 1994). Thus, while the majority of DNA adsorbsstrongly onto soil particles and the small non-adsorbed fractionis rapidly degraded by soil nucleases, a small quantity remainsavailable to be washed into the subsurface soil by rainwater(Poté et al., 2003, 2007).

Initial screening for plant DNA, using PCR with 18S rRNA geneprimers specific for eukaryotes, was positive for all extracted

in Geneva groundwater and traditional artesian ... Chemosphere

Pythium-AJ238653

Phytophthora-X54265

Epeisses398

A.thaliana-X16077

S.alba-X17062

Epeisses5

Epeisses2

84

B.graminea-AF107583

Meurons278

C.uvifera-U42798

Polygonumsp-AF206996

Epeisses1

Groundwater2

Passeiry1

Epeisses4

Meurons3

80

91

96

75

A.glabrata-L37581

E.pulchrerrima-U42535

A.altissima-AF206842

V.berlandieri-AF321263

V.rupestris-AF321272

Groundwater1

Meurons1

Groundwater3

100

80

99

81

75

100

0.02

Fig. 4. Phylogenetic tree containing most similar plants and the 18s rRNA gene from groundwater and village fountains. Village fountain DNA is represented by the villagename.

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DNA. The positive PCR amplification confirms the release andmovement of plant DNA under unsaturated soil conditions as wellas the possibility that the plant DNA can reach the groundwateraquifer. Some of the detected plants, in particular V. and V. ber-landieri, are perennial plants, and S. alba is frequency cultivatedin this area. The residues of these plants can decompose (Kögel-Knabner, 2002; Poté et al., 2005) and release their DNA into thesoil. Some plants such as P. sp. Soltis, and B. graminea are foundin the research area (widespread), also these plants are not the ma-jor components of local flora and are considered as weeds. Othersplants are scarce. The DNA released during the plant decomposi-tion in soil may or may not be itself in a degraded state. Ceccherini

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et al. (2003) demonstrated that the majority of plant DNA is de-graded within the plant tissues before being released. Thus, DNAcan reach the groundwater in a free dissolved state (whether de-graded or not), or adsorbed onto organic or mineral soil colloids.Extracellular dissolved DNA stems mainly from the gentle washingof the soil, and probably represents the DNA that is not tightly ad-sorbed to the soil (Frostegard et al., 1999), and thus is relativelymobile. Its presence suggests that if DNA is recently released, thisDNA can be transported by rapid rainwater infiltration to thegroundwater.

The convective vertical transport by water in unsaturated soildepends on the mineral and organic composition and the rate of

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water flow which is namely a function of grain-size distribution,porosity and stratification of the solid matrix of soil. The litholog-ical profile of sampled area (Fig. 1) is probably quite permeable torainwater transporting DNA and other organic compounds to thevadose zone.

5. Conclusion

The present research discussed the presence of plant DNA ingroundwater and artesian fountain drinking water. Our observa-tions support the notion of long-term survival and movement ofplant DNA in vadose zone, as DNA avoids at least complete degra-dation. The results indicate that DNA can be released from plantsand may infiltrate into agricultural soil and reach the groundwatertable. Also, as a consequence of infiltration of DNA from agricul-tural land into groundwater, drinking water of traditional foun-tains of the Champagne Genevoise contains eukaryote DNA. Inthe Geneva basin, no transgenic plants are under cultivation. Thepresence of DNA in groundwater demonstrates the possibility ofplant DNA reaching drinking water supplies. The deliberate or acci-dental release of genetically engineered microorganisms or antibi-otic resistance genes from transgenic plants into environmentcould be a potential source of biological contamination of groundor surface water (Alvarez et al., 1996; Poté et al., 2003). The resultsof this study may be of particular interest in the perspective of theuse of new organisms in agriculture and scientific research. The in-crease in global travel has increased the occurrence of invasivenon-native plant species in temperate climates (Kolar and Lodge,2001). Thus, the occurrence of one such invasive plant both inthe fields and in its DNA in the groundwater demonstrates the ex-tent to which DNA can be disseminated.

Acknowledgements

The authors thank Dr. Gabriel Del Los Cobos, service cantonal deGéologie de Genève for water samples. This research was sup-ported in part by the European Community 5th RTD program‘‘Quality of Life and Management of Living Resources”, project:TRANSBAC QLK3-CT-2001-02242 and by Ernst and LucieSchmidheiny Foundation, Geneva, Switzerland.

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