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International audience; Pesticide contamination of the environment can result from agricultural practices. Persistence of pesticide residues is a threat to the soil biota including plant roots and beneficial microorganisms, which support an important number of soil ecosystem services. Arbuscular mycorrhizal fungi (AMF) are key symbiotic microorganisms contributing to plant nutrition. In the present study, we assessed whether AMF could indicate eventual side effects of pesticides when directly applied to field soils. We evaluated the ecotoxicological impact of a cocktail of three commonly used agricultural pesticides (fenhexamid, folpel, deltamethrin) on the abundance and composition of the AMF community in vineyard (Montagne de Saint-Emilion) and arable (Martincourt) soils subjected to different agricultural practices. The dissipation of applied pesticides was monitored by multiresidual analyses to determine the scenario of exposure of the AMF community. Diversity analysis before application of the pesticide cocktail showed that the AMF communities of vineyard soils, subjected to mechanical weeding or grass cover, and of the arable soil subjected to intensive agriculture, were dominated by Glomerales. Ribotypes specific to each soil and to each agricultural practice in the same soil were found, with the highest abundance and diversity of AMF being observed in the vineyard soil with a grass-cover. The abundance of the global AMF community (Glomeromycota) and of three taxa of AMF (Funneliformis mosseae, Claroideoglomus etunicatum/C. claroideum) was evaluated after pesticide application. The abundance of Glomeromycota decreased in both soils after pesticide application while the abundance of Claroideoglomus and F. mosseae decreased only in the arable soil. These results show that higher doses of pesticide exposure did not affect the global abundance, but altered the composition, of the AMF community. Resilience of the AMF community composition was observed only in the vineyard soil, where F. mosseae was the most tolerant taxon to pesticide exposure.

The impact of soil moisture content and temperature on isoproturon (3-(4-isopropylphenyl)-1,1-dimethyl-urea [IPU]) mineralization activity was assessed on an agricultural soil regularly exposed to this herbicide. Mineralization of 14C-IPU was monitored on soil microcosms incubated at different temperatures (10°C, 20°C, 28°C) and soil moisture contents (9%, 12%, 15, 18%, 21%, 24%). An increase in temperature and/or soil moisture significantly enhanced the maximum rate and percentage of IPU mineralization while it decreased the lag time before mineralization. The maximum rate and percentage of IPU mineralization respectively ranged from 0.18% day−1 and 9% for the lowest temperature and soil moisture content pair (10°C–9%) to 1.51% day−1 and 27.1% for the highest pair (28°C–24%). Statistics revealed a cross interaction of temperature and soil moisture content on the maximum rate of IPU mineralization. The optimum conditions for IPU mineralization, estimated from the double Gaussian model, were 25.8°C and 24% soil moisture content. The influence of fluctuations in soil moisture content on IPU-mineralization was investigated by subjecting the soil microcosms to drought stress. When IPU was added at the end of the drought stress, it had no statistical effect on IPU mineralization. However, when it was added before the drought stress, two mineralization phases were observed: (1) one corresponding to the drought stress for which mineralization was low and (2) another one observed after restoration of soil moisture content characterized by higher mineralization rate. It can be concluded that climatic fluctuations affect the activity of IPU mineralizing microbial community, and may lead to an increase in IPU persistence.

BACKGROUND: The diuron-mineralising ability of the microbiota of a Mediterranean vineyard soil exposed each year to this herbicide was measured. The impact of soil moisture and temperature on this microbial activity was assessed. RESULTS: The soil microbiota was shown to mineralise diuron. This mineralising activity was positively correlated with soil moisture content, being negligible at 5% and more than 30% at 20% soil moisture content. According to a double Gaussian model applied to fit the dataset, the optimum temperature/soil moisture conditions were 27.9 °C/19.3% for maximum mineralisation rate and 21.9 °C/18.3% for maximum percentage mineralisation. The impact of temperature and soil moisture content variations on diuron mineralisation was estimated. A simulated drought period had a suppressive effect on subsequent diuron mineralisation. This drought effect was more marked when higher temperatures were used to dry (40 °C versus 28 °C) or incubate (28 °C versus 20 °C) the soil. The diuron kinetic parameters measured after drought conditions were no longer in accordance with those estimated by the Gaussian model. CONCLUSION: Although soil microbiota can adapt to diuron mineralisation, its activity is strongly dependent on climatic conditions. It suggests that diuron is not rapidly degraded under Mediterranean climate, and that arable Mediterranean soils are likely to accumulate diuron residues. Copyright © 2010 Society of Chemical Industry

In the last 10 years, accelerated mineralization of Atrazine (2-chloro-ethylamino-6-isopropylamino-s-triazine) has been evidenced in agricultural soils repeatedly treated with this herbicide. Here, we report on the interaction between earthworms, considered as soil engineers, and the Atrazine-degrading community. The impact of earthworm macrofauna on Atrazine mineralization was assessed in representative soil microsites of earthworm activities (gut contents, casts, burrow linings). Soil with or without earthworms, namely the anecic species Lumbricus terrestris and the endogenic species Aporrectodea caliginosa, was either inoculated or not inoculated with Pseudomonas sp. ADP, an Atrazine-degrading strain, and was either treated or not treated with Atrazine. The structure of the bacterial community, the Atrazine-degrading activity and the abundance of atzA, B and C sequences in soil microsites were investigated. Atrazine mineralization was found to be reduced in representative soil microsites of earthworm activities. Earthworms significantly affected the structure of soil bacterial communities. They also reduced the size of the inoculated population of Pseudomonas sp. ADP, thereby contributing to the diminution of the Atrazine-degrading genetic potential in representative soil microsites of earthworm activities. This study illustrates the regulation produced by the earthworms on functional bacterial communities involved in the fate of organic pollutants in soils.

International audience; The effects of the herbicide, DNOC, 4,6-dinitro-o-cresol, a model pollutant, have been studied by comparing the potential of soil microbial communities present in individual soil aggregates or in larger soil microcosms as samples of soil aggregates to mineralise the herbicide 2,4D. We have shown that 2-3 mm soil aggregates vary widely in their 2,4-D mineralisation potential and that ageing or exposure to DNOC considerably simplified the distribution patterns of this capacity. The main factors of variation have been quantified and classified using a quasi-likelihood method derived from the Generalised Linear Model approach. Besides DNOC concentration and duration of exposure, an additional 'rank' factor reflecting a desiccation gradient of the aggregates on the microliter plates was found to have statistical significance. We concluded that it should be possible to derive an experimental approach, designated as 'functional profiling', with potential use to detect soil chemical contamination. Curves of 2,4-D mineralisation in individual soil aggregates could be classified according to three different types of kinetics, which were assumed to reflect heterogeneous spatial distribution, differences in microbial community composition and varying efficiency of the microbial consortia involved in 2,4-D degradation. Exposure to DNOC considerably simplified the distribution patterns of the different types of kinetics with one type, showing slow rate and low cumulative mineralisation, becoming predominant as ageing, concentration and duration of DNOC exposure increased. We argue on the possible use of 'kinetic profiling' as a sensitive bioindicator of soil quality. By comparison, in soil microcosms, 2,4-D mineralisation showed an extra mineralisation potential of 64% over individual aggregates in the control soil and exposure to DNOC was followed by concentration and time-dependent recovery of the 2,4-D mineralisation potential. It is likely that 2 g size soil microcosms gather a larger number of biochemical capacities which could complement each other to increase the potential of soil to mineralise xenobiotic compounds. (c) 2004 Elsevier Ltd. All fights reserved.

Using enrichment culture three isoproturon (IPU) mineralising bacterial isolates were isolated from a French agricultural soil mineralising up to 50% of the initially added 14C-ring labelled IPU within only eight days. These isolates showed similar metabolic (BIOLOG GN) and amplified rDNA restriction (ARDRA) profiles. Partial 16S rDNA sequencing revealed that they were identical and identified as Methylopila sp TES. This strain harbours a large plasmid (220 kb) putatively bearing essential IPU-degrading genes as demonstrated by a curing experiment. Methylopila sp. TES transformed IPU and its known metabolites to CO2 and biomass but did not degrade chlorotoluron, monolinuron, diuron and linuron.

The level of expression of highly conserved, plasmid-borne, and widely dispersed atrazine catabolic genes ( atz ) was studied by RT-qPCR in two telluric atrazine-degrading microbes. RT-qPCR assays, based on the use of real-time PCR, were developed in order to quantify atzABCDEF mRNAs in Pseudomonas sp. ADP and atzABC mRNAs in Chelatobacter heintzii . atz gene expression was expressed as mRNA copy number per 10 6 16S rRNA. In Pseudomonas sp. ADP, atz genes were basally expressed. It confirmed atrazine-degrading kinetics indicating that catabolic activity starts immediately after adding the herbicide. atz gene expression increased transitorily in response to atrazine treatment. This increase was only observed while low amount of atrazine remained in the medium. In C. heintzii , only atzA was basally expressed. atzA and atzB expression levels were similarly and significantly increased in response to atrazine treatment. atzC was not expressed even in the presence of high amounts of atrazine. This study showed that atz genes are basally expressed and up-regulated in response to atrazine treatment. atz gene expression patterns are different in Pseudomonas ADP and C. heintzii suggesting that the host may influence the expression of plasmid-borne atrazine-catabolic potential.

International audience; The mineralisation rate of atrazine measured in soil pre-treated with this herbicide, was significantly higher in the maize rhizosphere than in bulk soil. Maize rhizosphere was also shown to significantly increase microbial biomass C as compared with bulk soil. Ribosomal intergenic spacer analysis conducted on nucleic acids extracted directly from soil samples revealed that the structure of microbial communities observed in the rhizosphere was slightly different from that of bulk soil. The quantification of the relative amount of the gene atzC, which encodes an enzyme involved in atrazine mineralisation, was carried out on soil nucleic acids by using quantitative-competitive PCR assays. It revealed that atzC was present at a higher level in the rhizosphere than in bulk soil. In addition, the amount of atzC was transiently enhanced in both rhizosphere and bulk soils following atrazine treatment. These results suggest that the stimulation of atrazine mineralisation in the maize rhizosphere depends on the abundance of atrazine-degrading communities.

The large diversity of metabolic capacities and the high genetic plasticity of microorganisms allow them to degrade virtually all organic compounds of natural or anthropogenic (xenobiotics) origin including those that are sources of environmental pollution. Thus microorganisms are major actors to eliminate or alleviate pollutions in the environment. The natural attenuation processes due to microbial activities (biodegradation and/or biotransformation) as well as the possibilities of using microorganisms in preventive treatments and bioremediation – biostimulation, bioaugmentation, rhizostimulation, bioleaching, and bioimmobilization – are presented. The main methods for microbial treatment of pollution, the chemical structure and the origin of the major pollutants, as well as the mechanisms of degradation by microorganisms – on the basis of physiological, biochemical, and genetic approaches – are described. Examples of treatments are presented for urban wastewater (activated sludge, lagoons, and planted beds), solid wastes (aerobic treatment or composting, anaerobic treatment and methanization, discharges), gaseous effluents, pesticides, polychlorobiphenyls, and finally hydrocarbons and petroleum products in the marine environment.

A series of agricultural soils varying in their atrazine treatment history were sampled from 12 sites in France and two sites in Canada. The soils varied widely with respect to soil chemical, physical and microbiological (total microbial biomass, kinetics of C and N mineralization) properties. Soils treated with as few as two successive atrazine field applications mineralized [U- ring - 14 C]atrazine significantly more rapidly in 35 d laboratory incubations than did soils which had never received atrazine. Longer treatment history tended to favour more rapid mineralization in the so-called “adapted” soils. Up to 80% of the initially applied 14 C-atrazine was mineralized at the end of the incubations in these adapted soils. Of the properties tested, soil pH was the most significantly related to atrazine mineralized. In soils with pH lower than 6.5, less than 25% of the initial 14 C-atrazine was mineralized even after repeated application in field conditions. Atrazine retention in soil did not influence its mineralization rate. Both hydroxylated and dealkylated atrazine metabolites were detected, but no clear pattern of metabolite production could be determined. Large amounts of bound residues were formed in soils that mineralized little atrazine.

It has been demonstrated that atrazine side-chain mineralisation could be substantially stimulated by addition of carbon-containing substrates such as cellulose, green manure, straw or sawdust in the presence of NH4+ nitrogen but poorly affected by amendments with glucose. Cellulose has the most beneficial effect. For that substrate it has been shown that (i) simultaneous application of the organic amendment and atrazine results in kinetics for side-chain dealkylation showing a lag phase which is reduced or even eliminated by preliminary incubation with the amendment, (ii) rate and extent of mineralisation of the ethylamino side chain are significantly accelerated by decreasing the C/N ratio of the amendment. By comparison, mineralisation of the isopropylamino side chain is not appreciably affected by a change in the value of the C/N ratio as far as atrazine is applied within a two- to three-week period following the organic treatment after which a small deficit in N supply has a definite beneficial effect on mineralisation. Cellulose and, to a lesser extent, straw induce a biphasic change in bacterial number with more numerous and/or active atrazine degraders being predominantly found in the later-developing bacterial community. The fungal microflora is relatively unaffected by all types of carbon substrates but glucose and, unexpectedly, by atrazine at high ratio of application. Activation of atrazine mineralisation seems to be a co-metabolic process which is kinetically controlled by the rate of release from polymerised C substrates of easily available and readily metabolisable low-molecular-weight co-substrates. Transient production of glucose as an end-product of cellulose depolymerisation might induce catabolic repression of dealkylation enzyme systems and be responsible for a lag in atrazine side-chain mineralisation. © 1998 Society of Chemical Industry

We investigated whether the chloroform fumigation extraction (CFE) method could be used to determine the microbial biomass in subsurface soils. The structure of microbial communities may vary over depth and the distribution of their cellular components may result in variations in the value of the calibration coefficient Kec. To verify whether this is so, we adopted a procedure based on in situ labelling of the microbial biomass using 14C-labelled carbon substrates. This procedure was evaluated under different operating conditions, by changing the chemical type (glucose or acetate), and the amount and duration of contact with the tracer substrates. Experimental data support two main conclusions. First, realistic values of the Kec coefficient can be obtained for subsoil microflora as long as they are determined under nutrient conditions similar to those prevailing in the natural environment. Second, a relationship exists between the value of the Kec coefficient and the depth in the soil profile.

A strain of Variovorax paradoxus degrading 2,4-dichlorophenoxyacetic acid (2,4-D) was isolated from the Dijon area (France) using continuous chemostat culture. This strain, designated TV1, grew on up to 5 mM 2,4-D and efficiently degraded the herbicide as sole carbon source as well as in presence of soil extracts. It also degraded phenol and 2-methyl, 4-chlorophenoxyacetic acid at 3 mM and 2,4-dichlorophenol at 1 mM. This organism contained a stable 200 kb plasmid, designated pTV1, which showed no similarity in its restriction pattern with the archetypal 2,4-D catabolic plasmid pJP4. However, pTV1 contained an 11 kb BamHI fragment which hybridized at low stringency with the 2,4-D degradative genes tfdA, tfdB and tfdR from pJP4. PTV1 partial tfdA sequence showed 77 % similarity with the archetypal tfdA gene sequence from Ralstonia eutropha JMP134. Tn5 mutagenesis confirmed the involvement of this gene in the 2,4-D catabolic pathway. © Rapid Science Ltd. 1998

cited By 26; International audience; Despite its importance in plant health and crop quality, the diversity of epiphytic bacteria on grape berries and other plantparts, like leaves and bark, remains poorly described, as does the role of telluric bacteria in plant colonization. In this study, we compare the bacterial community size and structure in vineyard soils, as well as on grapevine bark, leaves and berries. Analyses of culturable bacteria revealed differences in the size and structure of the populations in each ecosystem. The highest bacteria population counts and the greatest diversity of genera were found in soil samples, followed by bark, grapes and leaves. The identification of isolates revealed that some genera – Pseudomonas, urtobacterium, and Bacillus – were present in all ecosystems, but in different amounts, while others were ecosystem-specific. About 50% of the genera were common to soil and bark, but absent from leaves and grapes. The opposite was also observed: grape and leaf samples presented 50% of genera in common that were absent from trunk and soil. The bacterial community structure analyzed by -RFLP indicated similarities between the profiles of leaves and grapes, on the one hand, and bark and soil, on the other, reflecting the number of shared T-RFs. The results suggest an interaction between telluric bacterial communities and the epiphytic bacteria present on the different grapevine parts.

International audience; An integrated multicriteria analytical procedure for rapid, cost-effective characterisation of the biological status of soil bacterial community was developped. Commercially-available, light emission-based bioassays were selected for measuring cell density, activity, and diversity. All but Terminal Restriction Fragment Length Polymorphism (T-RFLP) were designed for multiwell-plate formats and high-throughput screening potential. Adenosine Tri Phosphate (ATP) was measured using a bioluminescence assay. Dehydrogenase activity (DHA) was measured on growing cells. Kinetic measurements of the formation of a coloured formazan derivative was used after nutrient broth addition to estimate initial cell concentrations by reference to Escherichia coli added as internal standard. Compared to conventional ATP and DHA determinations in soils, the procedures described here do not require extraction of ATP or formazan derivative from the soil matrix. Metabolic diversity was characterised using the Biolog™ system. T-RFLP was chosen for assessing bacterial community structure. The bioassays were performed on microbial preparations obtained after either direct dilution of soil suspensions or prior density-gradient separation of microbial cells from the soil matrix. Dilution maintains the original structure of native dominant microbial communities. Density-gradient separation of microbial cells is highly selective, drastically modifying metabolic (CLP Profiles) and species (T-RFLP patterns) diversity, as well as activity parameters.

Wine grapes are a primary source of microbial communities that play a prominent role in the quality of grapes prior to harvesting, as well as in the winemaking process. This study investigated the dynamics and diversity of the epiphytic bacteria on the grape berry surface during maturation. The quantitative and qualitative effects of conventional and organic farming systems on this microbial community were investigated, using both cultivation-dependent and independent approaches. Analyses of grape berry bacterial microbiota revealed changes in the size and structure of the population during the berry ripening process, with levels rising gradually and reaching their highest value when the berries were over ripe. As the season progressed to maturity, Gram-negative bacterial communities (mostly Pseudomonas spp.) declined whereas Gram-positive communities (mostly Micrococcus spp.) increased. The 16S rRNA gene sequences of cultured isolates were analysed and over 44 species were identified from 21 genera in the Proteobacteria, Actinobacteria, and Firmicutes phyla. Copper concentrations originating from phytosanitary treatments varied according to the vineyard and farming system. A negative correlation between copper concentrations and cell densities provided clear evidence that copper inhibited bacterial communities. The bacterial community structure was analysed by targeting the 16S rRNA genes, using PCR-DGGE on cultivable populations and T-RFLP on whole communities in cell suspension. The results suggest that the farming system has a clear impact on the bacterial community structure.

The diuron-mineralising ability of the microbiota of a Mediterranean vineyard soil exposed each year to this herbicide was measured. The impact of soil moisture and temperature on this microbial activity was assessed.The soil microbiota was shown to mineralise diuron. This mineralising activity was positively correlated with soil moisture content, being negligible at 5% and more than 30% at 20% soil moisture content. According to a double Gaussian model applied to fit the dataset, the optimum temperature/soil moisture conditions were 27.9 degrees C/19.3% for maximum mineralisation rate and 21.9 degrees C/18.3% for maximum percentage mineralisation. The impact of temperature and soil moisture content variations on diuron mineralisation was estimated. A simulated drought period had a suppressive effect on subsequent diuron mineralisation. This drought effect was more marked when higher temperatures were used to dry (40 degrees C versus 28 degrees C) or incubate (28 degrees C versus 20 degrees C) the soil. The diuron kinetic parameters measured after drought conditions were no longer in accordance with those estimated by the Gaussian model.Although soil microbiota can adapt to diuron mineralisation, its activity is strongly dependent on climatic conditions. It suggests that diuron is not rapidly degraded under Mediterranean climate, and that arable Mediterranean soils are likely to accumulate diuron residues.

In this paper we describe the principles of an analytical procedure based on radiorespirometric measurements that could be applied to the determination of residual concentrations of 2,4-D in the soil. Basically, it consists of comparing, in two soil samples, mineralization of 2,4-D, labeled at two different specific activities. The validity of the procedure depends on the concentration of herbicide, the location of the radioactive carbon within the molecule and the possibility to distinguish several phases in the evolution of 14CO2. The dosage has been proved more reliable at concentration levels that are consistent with agricultural practice than at lower concentrations. We also demonstrate that an appropriate mathematical treatment of the experimental data makes it possible to indentify the fraction of the mineralized radioactivity that specifically originates in the degradation of 2,4-D. This allows more realistic estimates of the amount of the parent molecule left unchanged in the soil by reducing interference by 14C-containing residuals resulting from microbial activity. We also demonstrate that carboxylic and methylenic carbon atoms of the side-chain are not assimilated with an equal efficiency. The absence of a true lag-phase indicates that the soil we have used contains a microbial population that degrades 2,4-D without requiring a preliminary period of adaptation.

International audience; Microbial cell concentration is a particularly important bioindicator of soil health and a yardstick for determining biological quotients which are likely to gain in ecological significance if they are calculated in relation to the viable, rather than total, microbial density. A dual-staining technique with fluorescent dyes was used for the spectrofluorimetric quantitative determination of the concentration of viable microbial cells present in three different soil types. This is a novel and substantially modified application of the dual-staining procedure implemented in the LIVE/DEAD™ BacLight® viability kit which has never been successfully applied to the quantification of naturally occurring soil microbial communities. Indigenous microbial cell concentrations were quantified using an internal standard, i.e. spiking environmental samples with suspensions containing different concentrations of live E. coli cells, and external calibration, by comparing fluorescence emission by indigenous bacteria and known concentrations of E. coli in nutrient saline. Two types of environmental samples were tested: bacterial preparations obtained by density gradient centrifugation and soil suspensions. In both cases, prior dilution of the sample was necessary to minimise fluorescence quenching by soil particulate matter. Spectrofluorimetric measurements of indigenous cell concentration in bacterial preparations were in close agreement with those found using epifluorescence microscopy. Limits of detection of 5 × 106 for the soil bacterial preparations and 8 × 107 for the soil suspensions were estimated. Deviations observed when soil suspensions are dealt with are likely due to the selection of a unique bacterial strain for standardisation and calibration. Thorough testing of a variety of reference bacteria and fungi is suggested to determine a more accurate average fluorescence enhancement per microbial cell or mass unit.

We tried to develop deterministic models for kinetics of 2,4-D breakdown in the soil based on the following considerations: (i) at low concentrations degradation results from maintenance consumption by a large fraction of the soil microbial population; (ii) at high concentration in addition to the maintenance consumption there is a growth-associated carbon incorporation by a small specific microbial population. Values for the biokinetic parameters are consistent with those commonly found in the literature. Comparison between observed and simulated curves suggests that a non-negligible part of the pesticidal carbon exists as microbial by-products.

We assessed the spatial variability of isoproturon mineralization in relation to that of physicochemical and biological parameters in fifty soil samples regularly collected along a sampling grid delimited across a 0.36 ha field plot (40 × 90 m). Only faint relationships were observed between isoproturon mineralization and the soil pH, microbial C biomass, and organic nitrogen. Considerable spatial variability was observed for six of the nine parameters tested (isoproturon mineralization rates, organic nitrogen, genetic structure of the microbial communities, soil pH, microbial biomass and equivalent humidity). The map of isoproturon mineralization rates distribution was similar to that of soil pH, microbial biomass, and organic nitrogen but different from those of structure of the microbial communities and equivalent humidity. Geostatistics revealed that the spatial heterogeneity in the rate of degradation of isoproturon corresponded to that of soil pH and microbial biomass.In field spatial variation of isoproturon mineralization mainly results from the spatial heterogeneity of soil pH and microbial C biomass.

The main objective of this work was to characterize an atrazine-mineralizing community originating from agrochemical factory soil, especially to elucidate the catabolic pathway and individual metabolic and genetic potentials of culturable members. A stable four-member bacterial community, characterized by colony morphology and 16S rDNA sequencing, was rapidly able to mineralize atrazine to CO 2 and NH 3 . Two primary organisms were identified as Arthrobacter species (ATZ1 and ATZ2) and two secondary organisms (CA1 and CA2) belonged to the genera Ochrobactrum and Pseudomonas, respectively. PCR assessment of atrazine-degrading genetic potential of the community, revealed the presence of trzN, trzD, atzB and atzC genes. Isolates ATZ1 and ATZ2 were capable of dechlorinating atrazine to hydroxyatrazine and contained the trzN gene. ATZ2 further degraded hydroxyatrazine to cyanuric acid and contained atzB and atzC genes whereas ATZ1 contained atzC but not atzB. Isolates CA1 and CA2 grew on cyanuric acid and contained the trzD gene. Complete atrazine degradation was a result of the combined metabolic attack on the atrazine molecule, and complex interactions may exist between the community members sharing carbon and nitrogen from atrazine mineralization. Scientific relevance: Despite numerous reports on atrazine degradation by pure bacterial cultures, the pathways and the atrazine-degrading gene combinations harboured by bacterial communities are only poorly described. In this work, we characterized a four-member atrazine-mineralizing community enriched from an agrochemical factory soil, which was capable of rapidly metabolizing atrazine to CO 2 . This study will contribute towards better understanding of the genetic potential and metabolic activities of atrazine-degrading communities, which are generally considered to be responsible for atrazine mineralization in the natural environment.

In the last 10 years, accelerated mineralization of Atrazine (2-chloro-ethylamino-6-isopropylamino-s-triazine) has been evidenced in agricultural soils repeatedly treated with this herbicide. Here, we report on the interaction between earthworms, considered as soil engineers, and the Atrazine-degrading community. The impact of earthworm macrofauna on Atrazine mineralization was assessed in representative soil microsites of earthworm activities (gut contents, casts, burrow linings). Soil with or without earthworms, namely the anecic species Lumbricus terrestris and the endogenic species Aporrectodea caliginosa, was either inoculated or not inoculated with Pseudomonas sp. ADP, an Atrazine-degrading strain, and was either treated or not treated with Atrazine. The structure of the bacterial community, the Atrazine-degrading activity and the abundance of atzA, B and C sequences in soil microsites were investigated. Atrazine mineralization was found to be reduced in representative soil microsites of earthworm activities. Earthworms significantly affected the structure of soil bacterial communities. They also reduced the size of the inoculated population of Pseudomonas sp. ADP, thereby contributing to the diminution of the Atrazine-degrading genetic potential in representative soil microsites of earthworm activities. This study illustrates the regulation produced by the earthworms on functional bacterial communities involved in the fate of organic pollutants in soils.

International audience; Sixty-six atrazine-degrading bacterial communities utilizing atrazine as sole N source and citrate as principal C source were isolated from unplanted and maize planted soils treated with atrazine. Ribosomal intergenic spacer analysis (RISA) fingerprints revealed that the genetic structure of atrazine-degrading bacterial communities was modified in the maize rhizosphere. To assess the underlying microbial diversity, 16S rDNA sequences amplified from each bacterial community were cloned. Libraries containing 660 16S rDNA clones were screened by restriction fragment length polymorphism (RFLP) analysis. In all, 63 clone families were identified. Rarefaction curves did not reach a clear saturation, indicating that the analysis of a greater number of clones would have revealed further diversity. Recovered 16S rDNA sequences were related to Actinobacteria, Bacteroidetes and Proteobacteria. The four dominant RFLP families were highly similar to Variovorax paradoxus, Burkholderia cepacia, Arthrobacter sp. and Bosea sp. The composition of most of the atrazine-degrading bacterial communities consisted of 2–7 different bacterial species. Various atrazine-degrading gene compositions were observed, two of these atzABCDEF, trzND and atzBCDEF, trzN being largely dominant. The first was more frequently detected in bacterial communities isolated from the maize rhizosphere whereas the second was more frequently detected in communities isolated from bulk soil. Monitoring of atrazine-degrading activity showed that 76% of the bacterial communities degraded up to 80% of the initially added atrazine within 15 days of culture. Altogether our results indicate that the maize rhizosphere has an impact on the genetic structure, the diversity and atrazine-degrading gene composition of the atrazine-degrading communities.

International audience; 14C-ring-labelled isoproturon mineralisation was investigated in a French agricultural soil previously exposed to isoproturon. 50 different soil samples collected every 2 m along a transect of 100 m in length were treated one or two times with isoproturon under laboratory conditions and analysed by radiorespirometry. 94% of the soil samples showed a high ability to mineralise isoproturon with a relatively low variability in the cumulative percentage of mineralisation ranging from 30 to 51% of the initially added radioactivity for the samples treated once with the herbicide. About 45 to 67% of the initially added radioactivity was transformed into 14CO2 in soil samples treated twice with isoproturon. Fifty-one to 30% of the radioactive pesticide formed bound residues 120 days after the first isoproturon treatment. The radioactive compound incorporated in the microbial biomass ranged from 3 to 4% of the initially added radioactivity. The methanol-extractable radioactivity was negligible and in the majority of soil samples no metabolites could be detected by high performance liquid chromatography analyses. However, in one soil sample showing low ability to mineralise isoproturon, the mono-demethyl isoproturon derivative represented about 12% of the methanol-extractable fraction. After the second isoproturon treatment, the rate of isoproturon mineralisation was enhanced in most soil samples and the number of soil samples showing a low isoproturon mineralisation capacity decreased. A significant relationship (correlation coefficient, 0.89) between the logarithm of the mineralisation rate (ln k) and the soil pH was found, with a particularly strong positive effect on isoproturon mineralisation for pH > 6.5.

International audience

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.

The possibility to improve atrazine degradation in soils by bioaugmentation was studied. The atrazine-mineralizing strain, Chelatobacter heintzii Cit1, was inoculated in four sterile and four non-sterile soils, at varying inoculum densities. Two soils, which had shown enhanced atrazine mineralization, were used to determine which inoculum density was capable of restoring their original mineralizing capacity after sterilization. The two other soils, with intermediate and low capacity to mineralize atrazine, were used in order to demonstrate that atrazine mineralization in such soils could be improved by inoculation. Mineralization kinetics were fitted using the Gompertz model. In the case of soils adapted to atrazine mineralization, inoculation of C. heintzii did not accelerate the rate of atrazine mineralization, which was essentially performed by the indigenous microflora. However, with soils that did not mineralize atrazine, the introduction of 10 4 cfu g −1 resulted in a 3-fold increase of atrazine mineralization capacity.

Historical Aspects: The Hormone Herbicides Biological Aspects of Pesticide Degradation in Soils Pesticide Degradation in Natural Environments Keywords: biodegradation; biodegradation of pesticides; bioremediation; cometabolism; soil bioremediation (pesticides); soils; white rot fungi in bioremediation

We report the development of quantitative competitive (QC) PCR assays for quantifying the 16S, 18S ribosomal and atzC genes in nucleic acids directly extracted from soil. QC-PCR assays were standardised, calibrated and evaluated with an experimental study aiming to evaluate the impact of atrazine application on soil microflora. Comparison of QC-PCR 16S and 18S results with those of soil microbial biomass showed that, following atrazine application, the microbial biomass was not affected and that the amount of 16S rDNA gene representing 'bacteria' increased transitorily, while the amount of 18S rDNA gene representing fungi decreased in soil. In addition, comparison of atzC QC-PCR results with those of atrazine mineralisation revealed that, in response to atrazine treatment, the amount of atzC gene increased transitorily in soil pre-treated with atrazine, suggesting that accelerated atrazine biodegradation in soil could be due to a transient increase in the size of the atrazine mineralising community.

International audience; We have studied the structural effects of application to the soil of a potentially detrimental herbicide, 4,6-dinitroorthocresol (DNOC) by analysing amplified ribosomal DNA restriction analysis (ARDRA) and terminal restriction fragment length polymorphism (T-RFLP) signatures of 16S rDNA fragments of culturable bacterial communities isolated from diluted soil suspensions. This approach has the potential to reveal changes induced by stressing the soil microflora with DNOC. This paper shows that, whereas only few changes of the ARDRA and T-RFLP profiles result from ageing of the soil, treatment of the soil with DNOC induces major modifications of these profiles. Therefore, for the practical purpose of pesticide registration, ARDRA and T-RFLP analysis performed on the dominant culturable fraction of the soil bacteria, implemented using conventional gel electrophoresis, offers the means of a routine, simple and meaningful test for detecting some of the changes affecting the structure of the soil microflora in response to pesticide application.

We analysed the ability of soil units of millimetre size to mineralise a herbicide, 2,4-D, using incubations of individual aggregates (2–7 mm diameter) and 6×6×6 mm 3 cubes dissected from soil cores, under standard conditions. Mineralisation of 14 C-ring labelled 2,4-D was measured using a barite paper trap and a Phosphorimager to record the evolved 14 C-CO 2 from these very small soil samples. We found a large variability of 2,4-D mineralisation potential between aggregate size classes, between individual aggregates of the same size and between the different dissected cubes from a given core. We explained this variability by an uneven distribution of the degrading microorganisms at this scale, and to a lesser extent, an uneven distribution of C, necessary for co-metabolism. Furthermore, we found that in a soil core, the dissected cubes with a large mineralisation potential were not randomly distributed, but rather organised into centimetre sized hot spots.

A greenhouse study was conducted to investigate the ability of four crops (wheat, corn, oilseed rape and soybean) to influence the degradation of bentazone, diclofop-methyl, diuron, isoproturon and pendimethalin in soil. The present study showed that microbial biomass-carbon was significantly higher in planted soils than in bulk soil, especially with wheat and corn, after several cropping cycles. The biomass in corn and soybean planted soils was adversely affected by bentazone but recovered after three cropping cycles. In wheat-planted soils, diclofop-methyl application resulted in persistent increase of the amount of microbial biomass. Bentazone did not show accelerated degradation even after five successive treatments, differing from diclofop-methyl, for which two applications were sufficient to enhance significantly its rate of degradation. Enhanced degradation of diclofop-methyl was even more pronounced in wheat-planted soil. The rates of mineralisation of diuron, isoproturon and pendimethalin were not affected after the first cropping cycle, but were significantly increased in planted soils after five cropping cycles. The results confirm that plants may promote pesticide degradation in soil by stimulating biodegradation processes. In the case of diclofop-methyl, stimulation of accelerated degradation was observed.

In a previous study, we isolated a collection of atrazine-degrading bacteria from various soils. The aim of this study was to localise the atrazine-degrading genes in these 25 atrazine-degrading strains. In the case of the Gram-negative strains of Chelatobacter heintzii, six to seven plasmids were observed. The atzABC and trzD genes were located on two or three plasmids with variable molecular masses. For the Gram-positive strains of Arthrobacter crystallopoietes, the atzBC genes were located on a single plasmid of 117 kb. The organisation of atrazine-degrading genes seems to be highly variable between the strains studied. We have shown by a specific PCR the occurrence of IS1071-like sequences in all strains carrying atzBC genes, while these sequences seem to be absent in strains carrying only the atzA gene. Thus, we show a strong correlation between the presence of these insertion sequences and the presence of some genes involved in atrazine degradation.

International audience; An experiment was carried out in the greenhouse in order to compare atrazine mineralization in bulk soil and maize rhizosphere at different development stages. After 4, 8 and 12 weeks, we have (1) measured the soil microbial biomass C, (2) characterized the C substrate utilization profiles of the culturable microflora, and (3) analyzed atrazine mineralization. Microbial growth was stimulated in planted soil and different C substrate utilization patterns were obtained in bulk and rhizosphere soils during the first 2 months. During this period, laboratory tests for atrazine biodegradation revealed a lower mineralization potential in bulk than in planted soil. Atrazine mineralization was stimulated to a greater extent after atrazine application in the greenhouse but again the presence of plants had a favorable effect. After 12 weeks of cropping, the atrazine mineralization potential decreased in planted soil with or without prior atrazine application.

The impact of three different soil DNA extraction methods on bacterial diversity was evaluated using PCR-based 16S ribosomal DNA analysis. DNA extracted directly from three soils showing contrasting physicochemical properties was subjected to amplified ribosomal DNA restriction analysis and ribosomal intergenic spacer analysis (RISA). The obtained RISA patterns revealed clearly that both the phylotype abundance and the composition of the indigenous bacterial community are dependent on the DNA recovery method used. In addition, this effect was also shown in the context of an experimental study aiming to estimate the impact on soil biodiversity of the application of farmyard manure or sewage sludge onto a monoculture of maize for 15 years.

The capacity of 12 soils to degrade atrazine was studied in laboratory incubations using radiolabelled atrazine. Eight soils showed enhanced degradation of this compound. Twenty-five bacterial strains able to degrade atrazine were isolated by an enrichment method from 10 of these soils. These soils were chosen for their wide range of physico-chemical characteristics. Their history of treatment with atrazine was also variable. The genetic diversity of atrazine degraders was determined by amplified ribosomal restriction analysis (ARDRA) of the 16S rDNA gene with three restriction endonucleases. The 25 bacterial strains were grouped into five ARDRA types. By sequencing and aligning the 16S rDNA genes, the isolates were shown to belong to the Gram-negative species Chelatobacter heintzii, Aminobacter aminovorans, Stenotrophomonas maltophilia and to the Gram-positive genus Arthrobacter crystallopoietes. These species were not described previously as being capable of atrazine degradation. Most Gram-negative bacteria could mineralise (14)C ring labelled atrazine and carried the atzA, atzB, atzC and trzD genes. Gram-positive strains could convert atrazine to cyanuric acid and carried only the atzB and atzC genes. In this study, we describe the atrazine degradation capacities and corresponding genes in bacterial species that were not known as atrazine degraders. We report for the first time the occurrence of the trzD gene in these atrazine-mineralising bacteria and we demonstrate the potential use of colony hybridisation to isolate bacteria involved in atrazine degradation.

Because the primary factor controlling the persistence of pesticides in soil is microbial transformation, its mathematical modelling deserves attention. Soil persistence and leaching models are very sensitive to the parameters governing sorption and degradation. As a first part of this paper, we will present currently used rate laws for pesticide degradation in soils, essentially based on first-order reaction kinetics. We will discuss how recent knowledge on pesticide microbiology and practical observations like accelerated and stimulated degradation can be included in the description of the degradation process. We discuss the influence of spatial variability and parameter uncertainty on the distribution of the model output. In the last part of the paper, we suggest methodological approaches for studying and describing temperature and moisture effects and for obtaining more realistic values for the first-order rate constant or other biokinetic parameters, which control persistence of pesticides in soils.

The aim of the present work was to study the occurrence, distribution and diversity of 1,2-dichlorocatechol dioxygenase genes among 2,4-dichlorophenoxyacetic acid degrading bacteria. Phylogenetic relationships between the 31 strains or isolates were evaluated by amplified ribosomal DNA restriction analysis of the 16S rDNA gene. All the strains could be assigned to the β or γ subdivisions of the Proteobacteria. tfdC genes were detected by PCR amplification using degenerated primers. Two specific probes were produced from Ralstonia eutropha strain JMP134 and from a soil isolate strain PLAE6 which was grouped with Variovorax paradoxus. Sequence analysis of the probes revealed that they were homologous to the tfdC genes of JMP134 located on plasmid pJP4 and to the tfdC gene of Pseudomonas putida strain PaW85 located on plasmid pEST4011. The localization and the copy number of tfdC genes were determined by hybridization of plasmid profiles and genomic DNA restriction fragment length polymorphism profiles with the two probes. Most of the strains were found to bear tfdC genes on plasmids ranging from 78 to 532 kb; two strains without any plasmids were also found to hybridize with the probes, revealing a chromosomal localization of catabolic genes. Sequence analysis of the PCR products from different strains confirmed that four different classes of chlorocatechol 1,2-dioxygenase genes were present in the strains and isolates studied.

Among 15 soils with different cropping practices, seven which had an history of repeated atrazine applications showed accelerated degradation of this herbicide. By contrast, grassland or agricultural soils with no recorded atrazine application, at least for the last three years, had a low degradation potential. No direct relation was found between the rate of atrazine mineralisation and the size of the microbial biomass. In adapted soils, the amounts of extractable residues were lowered and the very high percentages of radioactivity from [ring-14C]atrazine recovered as [14C]carbon dioxide demonstrated that N-dealkylation and deamidation were the only processes for micro-organisms to derive carbon and energy for heterotrophic growth. Kinetics of microbial 14C accumulation revealed that atrazine ring carbon could be incorporated by direct oxidative condensation with structural components of the bacterial or fungal cell whereas side-chain carbon was preferentially used for biosynthesis of new protoplasmic cell material, as confirmed by the high turnover rate of radiolabelled microbial components. From the determination of the Michaelis–Menten parameters, Vm and Km in the presence of different selective biocides, it was possible to conclude that fungi were probably less active in atrazine degradation than bacteria and that over years the microbial atrazine-degrading community showed an increased efficiency. © 1999 Society of Chemical Industry

The ability of denaturing gradient gel electrophoresis (DGGE) technique to resolve 16S rDNA products generated from two different collections of bacteria using universal 16S primers was investigated. Alignments of 16S rDNA sequences of known species of rhizobia and methanotrophs were performed in order to determine the genetic variations within a 200 bp product obtained with PCR primers which amplify the 16S rRNA encoding genes from Eubacteria. Theoretical DNA melting curves were obtained with the Melt87 program and found to correlate with the ability to resolve fragments by DGGE. In the case of the rhizobia, the inability of DGGE analysis to resolve the PCR products from closely related species was in accordance with the low polymorphism observed amongst the sequences in the amplified area. In the case of the methanotrophs, the PCR products were surprisingly difficult to resolve given the high degree of sequence polymorphism of the amplified area in some distantly related species. The difference in sequence divergence within the two groups members allowed therefore to scale the resolution ability of the DGGE technique.

The 2,4-dichlorophenoxyacetic acid (2,4-D) degrading potential of an agricultural gleyic luvisol from the Dijon area (France) was assessed. 8% of the soil aggregates tested were shown to degrade chain labelled [14C]2,4-D. Mineralization was observed either immediately or after a 4 week lag period. The diversity of the 2,4-D degrading microflora of three aggregates was further investigated. 16S rRNA genes from 68 isolates were therefore amplified, which led to the discrimination of 17 distinguishable RFLP patterns. Instability of the side chain 2,4-D degrading capabilities was observed amongst 25% of the isolates. None of the unstable 2,4-D degraders amplified when the tfdA and tfdB primer sets were used. These strains presented different 16S patterns by the RsaI restriction enzyme and therefore belonged to various microbial species. Amongst the stable 2,4-D degrading isolates, two strains which were isolated from different aggregates did amplify using both tfdA and tfdB primer sets. These strains presented identical tfdA and tfdB PCR-RFLP patterns which were of the A. eutrophus JMP134 (pJP4) and Burkholderia sp RASC type, respectively, but showed different 16S rDNA PCR-RFLP patterns demonstrating that the degrading genes were distributed amongst various bacterial groups prior to any enrichment. Six strains amplified using the tfdB primer set but not using the tfdA primer set. These strains presented either a Burkholderia sp. RASC tfdB pattern or an undescribed new tfdB PCR-RFLP pattern but all shared a common 16S PCR-RFLP pattern. A third group contained stable degraders in which the presence of tfdA and tfdB-like genes could not be detected. These strains presented 12 distinct 16S PCR-RFLP patterns which differed from those observed in the first two groups of stable degraders. Therefore our results demonstrate the involvement of diverse bacterial species and genes in 2,4-D degradation within a soil.

International audience; Twenty-five 2,4-dichlorophenoxyacetic acid (2,4-D) degrading bacteria from geographically diverse locations and presenting various degrees of similarity or no similarity to the tfdA and tfdB genes from Alcaligenes eutrophus JMP134 were analysed by PCR-RFLP (restriction length fragment polymorphism). Primers for the 2,4-D etherase gene were derived by sequence alignment of the tfdA genes from A. eutrophus JMP134 and Burkholderia sp. RASC. Primers for the 2,4-dichlorophenolhydroxylase gene were based on the tfdB gene sequence from A. eutrophus JMP134 by taking codon degeneration and variations in amino acid residue sequences into consideration. PCR amplification using the tfdA primer set produced fragments of 0.3 kb from 17 strains which showed varying degrees of similarity to the tfdA gene probe from A. eutrophus JMP134. Significant variations in the gene sequences were confirmed by PCR-RFLP analysis. DNA amplification using the tfdB primer set produced a 1.1 kb fragment from 19 strains. Amongst them, two did not show any similarity to the tfdB gene probe. The size and restriction pattern of the products obtained from A. eutrophus JMP134 were in accordance with the expected size calculated from the A. eutrophus tfdA and tfdB gene sequence and their theoretical PCR-RFLP patterns. Some strains which did not amplify using the tfdA primer set did however amplify with the tfdB primer set. These results suggest the independent evolution of these two genes in the construction of the 2,4-D metabolic pathway. Our tfdA and tfdB primer sets could be used for the detection of similar sequences in bacteria and soils. Moreover, PCR-RFLP patterns could also be used to select subsets of strains for sequencing to study the phylogeny of the tfdA and tfdB genes.

Summary Recent advances in measuring soil microbial biomass by chloroform fumigation–extraction (CFE) and microbial heterotrophic activity interpreted by quantitative concentration–activity relations (QCAR) have renewed interest in assessing side–effects of agricultural chemicals on soil microorganisms. We have studied the effects of a herbicide, 4,6-dinitroorthocresol (DNOC), taken as a test chemical, on the rate of microbial carbon turnover and the size of the soil microbial biomass. We used the CFE technique in combination with in-situ labelling of the soil biomass. Exposure of prelabelled soil samples to the herbicide resulted in a significant increase in 14CO2 production during which the radioactive carbon content of the microflora decreased exponentially without apparent reduction in the size of the biomass. The extra production of 14CO2 by DNOC-treated soil over control, or carbon-enriched soil, is the expression of an increased rate of endogenous metabolism to compensate for shortage in energy caused by a decoupling of ATP generation at the oxidative phosphorylations level by the DNOC. To assess the influence of DNOC on soil microbial communities we also compared the advantages of short-term respirometric tests with those resulting from application of heterotrophic activity measurements in connection with QCAR. Both procedures detected modifications in the metabolic behaviour of soil microorganisms when faced with chemical stress. Short-term respirometric tests showed that DNOC causes a decrease in the respirometric activity of the soil microflora. Measuring heterotrophic activity also makes it possible to interpret microbial responses in terms of changes in the physiological traits of the microbial communities. DNOC provokes an apparent enrichment in microorganisms with a smaller saturation constant, Km and, as a consequence, a greater affinity for carbon substrates.

In an agricultural soil profile, the availability of soil organic C for microbial activity decreased from 0 to 100 cm depth in winter, spring and summer. Availability was defined as the ratio of respiration rate to total organic C, or the ratio of microbial biomass C to total organic C. The amount and availability of organic C in various soil fractions was measured. Fractions consisted of aqueous extracts from throughout the profile, and particle-size and density fractions from the 0–20 and 80–100 cm layers. The fractions were incubated with soil inoculum for 24 or 28 days. Availability of organic C in soluble fractions was measured by the decrease in dissolved organic C. Availability of organic C in insoluble fractions was measured by the increase in inorganic C. Availability of organic C in all fractions was similar at each depth, except for the weakly-adsorbed and clay fractions, in which organic C was less available at depth. Availability was least in the silt fractions. Approximately half of the organic C mineralized during incubations originated from the clay fraction in both the 0–10 and 80– 100 cm layers. Of the remainder, at the surface a higher proportion was in the silt and light (>50 μm, d

We tried to assess the dynamic behaviour of the soil microbial community that degrades 2,4-dichlorophenoxyacetic acid (2,4- d ) either by comparing the kinetics of mineralization of 14 C-2,4- d applied at concentrations between 0.16 and 20 mg kg −1 or by determining the 2,4- d mineralization potential that develops in soils amended with the herbicide in the same range of concentrations. For that purpose a mixing procedure that allows determination of the rate of herbicide mineralization independently of its residual concentration was applied. Kinetics of mineralization of 14 C-2,4- d revealed that in the soil at least two different microbial groups are responsible for the degradation of the herbicide. They probably differ in their tolerance to the bioactivity of the herbicide as well as their capacity to metabolize it. The time variations of the 2,4-D mineralization potential showed a delayed period of 2,4- d degradation that was attributed to a possible shift in the degrading species. Exhaustion of labile C-sources was thought to be responsible for the strengthening of the role of microorganisms currently living from the consumption of the more recalcitrant aromatic structures of the soil organic matter.