Impact of repeated treatments of the pesticides chlorpyrifos or tebuconazole on soil microorganisms using alternative methods

International audience

Diuron mineralisation in a Mediterranean vineyard soil: impact of moisture content and temperature

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…

In vitro evolution of an atrazine-degrading population under cyanuric acid selection pressure: Evidence for the selective loss of a 47kb region on the plasmid ADP1 containing the atzA, B and C genes

International audience; The adaptation of microorganisms to pesticide biodegradation relies on the recruitment of catabolic genes by horizontal gene transfer and homologous recombination mediated by insertion sequences (IS). This environment-friendly function is maintained in the degrading population but it has a cost which could diminish its fitness. The loss of genes in the course of evolution being a major mechanism of ecological specialization, we mimicked evolution in vitro by sub-culturing the atrazine-degrading Pseudomonas sp. ADP in a liquid medium containing cyanuric acid as the sole source of nitrogen. After 120 generations, a new population evolved, which replaced the original on…

Depth matters : Effects of precipitation regime on soil microbial activity upon rewetting of a plant-soil system

International audience; Climate change is predicted to affect not only the amount but also the temporal distribution of rain. Changes in frequency and amplitude of rain events, i.e. precipitation patterns, result in different water conditions with soil depth, and likely affect plant growth and shape plant and soil microbial activity. Here, we used 18O stable isotope probing (SIP) to investigate bacterial and fungal communities that actively grew or not upon rewetting, at three different depths in plant-soil mesocosms previously subjected to frequent or infrequent watering for 12 weeks (equal total water input). Phylogenetic marker genes for bacteria and fungi were sequenced after rewetting,…

Genetic rearrangement of the atzAB atrazine-degrading gene cassette from pADP1::Tn5 to the chromosome of Variovorax sp. MD1 and MD2

International audience; We report the characterization of the rearrangement phenomena responsible for the movement of the atrazine-degrading atzA and B genes from pADP1::Tn5 to the chromosome of Variovorax sp. MD1 and MD2. Long PCRs and Southern blot analyses revealed that the two genes forming a gene cassette moved in a unique rearrangement event. It also revealed that the boundaries of the plasmid sequence inserted in the chromosome correspond to IS1071or to sequences close to IS1071. It suggests that this genetic rearrangement could result from the transposition of the composite transposon delimited by IS1071 insertion sequences and containing atzA and atzB genes. In addition, for MD1 an…

Transcriptomic and proteomic responses of Microbacterium sp. C448 exposed to sulfamethazine antibiotic

International audience; The Microbacterium sp. C448 was isolated from a soil regularly exposed to sulfamethazine(SMZ), for its ability to partly mineralise this antibiotic and other related sulfonamides.The aim of our study was to explore its metabolic adaptation towards exposure to SMZenvironmental (10 mg/L) and medicinal (250 mg/L) concentrations. Its transcriptomic andproteomic responses were analysed by focusing on the degradation regulon (sad genes) andresistance genes (folP and sul1).The transcriptomic and proteomic results were essentially congruent whatever theconcentrations tested. In culture conditions, exposure to the highest concentration of SMZ led tothe highest sad expression …

Utilisation des mécanismes d'adaptation microbienne pour caractériser les conséquences écologiques de l'interdiction d'utilisation du diuron en zone viticole

National audience

Effects of precipitation regime on soil bacterial and fungal activity upon rewetting of a plant-soil system using 18O-SIP: depth matters

EASPEBIOmEDOCT INRA; Climate change is predicted to affect not only the amount but also the temporal distribution of rain. Changes in frequency and amplitude of rain events, likely shape the activity of plants and soil microbes. Fluctuating water conditions will differ with soil depth between precipitation patterns, affecting plant growth and may result in differential microbial response upon rewetting. Our objective was to investigate, in plant-soil systems, the response of the metabolically active microbial communities to a rewetting event and to which extent this was modulated by 1) soil depth and 2) precipitation legacy. Wheat planted in soil mesocosms were subjected to frequent or infr…

Principaux processus impliqués dans l'atténuation naturelle des pesticides par les sols

Prod 2018-253e EA BIOmE INRA; National audience

Array of microbial indicators, a promise for a better monitoring of pesticide effects on stream biological quality

Visioconférence; National audience; Freshwater contamination by pesticide residues is a major and growing threat to aquatic communities, ecosystem functioning and ultimately human health worldwide. Typical pesticide contamination in agricultural landscapes is characterized by a cocktail of a large number of active compounds and their main transformation products, each of them found at very low and temporally fluctuating concentrations. This makes the quantification of pesticide residues in streams highly challenging and costly by means of grab chemical sampling. Accordingly, it makes also difficult to characterize the chronic exposure of aquatic communities in pesticide-contaminated streams…

Ecotoxicological impact of pesticides on natural microbial communities responsible for pesticide biodegradation in river sediments

International audience; The general use of pesticides in various crops allows ensuring the quality of the crop production but has environmental impact, pesticide residues being often found in water resources, notably river in the agrosystems. As a result of repeated exposure to pesticides microorganisms can adapt to their degradation being responsible for enhanced degradation, an environmental friendly process diminishing the persistence of pesticide residues in the environment. This process can occur in the soil but also in the sediment of the river regularly exposed to pesticide contamination. To our best knowledge the ecotoxicological impact of multiple pesticides contamination on the de…

Potential for microbial diuron mineralisation in a small wine-growing watershed: from treated plots to lotic receiver hydrosystem

BACKGROUND: Since biological degradation processes are known to be a major driver of the natural attenuation of pesticide residues in the environment, microbial communities adapted to pesticide biodegradation are likely to play a key environmental role in reducing pesticide exposure in contaminated ecosystems. The aim of this study was to assess the diuron-mineralising potential of microbial communities at a small-scale watershed level, including a diuron-treated vineyard (pollution source), its associated grass buffer strip (as a river protection area against pesticide runoff) and the lotic receiver hydrosystem (sediments and epilithon), by using radiorespirometry. RESULTS: Comparison of r…

Importance of genetic plasticity for microbial adaptation to pesticide biodegradation: in vitro evolution of Pseudomonas sp. ADP under atrazine or cyanuric acid selection pressure as case studies

International audience

Genetic plasticity of atrazine-degrader: evidence for the deletion of atzABC genes in pseudomonas sp. adp in response to cyanuric acid selection pressure

International audience; Several bacterial strains are known to metabolize s-triazines herbicides among which atrazine reported worldwidel to contaminate soil and water resources. The complete mineralization of atrazine to carbon dioxide and ammonium is catalyzed by 6 hydrolytic enzymes encoded by atzABCDEF genes located on the plasmid pADP1 in Pseudomonas ADP. The atzABC genes code for the upper pathway transforming atrazine to cyanuric acid, while the atzDEF genes code for the lower pathway transforming cyanuric acid to simple compounds. In order to study the genetic plasticity of the atrazine catabolic pathway, we performed an in vitro evolution study consisting in applying a continuous s…

Predicting pesticide biodegradation potential from microbial community composition: new tools for bioremediation

Bioaugmentation is receiving increasing attention as a green technology to treat contaminatedareas by inoculating specific biodegrading microorganisms. However, our understanding of therole of microbial community composition and structure in the expression of contaminantdegradation potential is yet to improve. It could help making wise choice for microorganisms –community or specific strain – to be inoculated in contaminated soils with consideration to theirindigeneous microbiota.Here we tried to predict the microbial degradation of two herbicides, glyphosate andisoproturon by means of penalized regression and machine learning methods routinely used ingenomic selection. To this end, we cond…

Développement d'indicateurs microbiens pour l'évaluation de l'impact des pesticides sur des fonctions écosystémiques terrestres et aquatiques

National audience

Evidence de l'adaptation des communautés microbiennes sédimentaires de rivière à la minéralisation du diuron: influence du ruissellement et de l'érosion des sols

International audience; Purpose Surface runoff and erosion are major drivers of pesticide transport from soils to rivers draining vineyard watersheds. A recent study showed that applications of diuron on vineyards and diuron dispersal could lead to microbial adaptation to diuron biodegradation from treated soils to the receiving hydrosystem. Given the limited knowledge on microbial adaptation to pesticide degradation in aquatic environments, we conducted a microcosm study designed to assess the impact of runoff and erosion processes on the adaptation of riverine-sediment microbial communities to diuron mineralization. Materials and methods The experimental laboratory set-up consisted in aqu…

Influence de la biodégradation bactérienne des antibiotiques sur le devenir de ces substances et la dispersion des antibiorésistances dans les agrosystèmes

National audience; L’exposition environnementale des bactéries aux antibiotiques favorise la dispersion et le développement de capacités de résistance (antibiorésistance), engendrant un risque sanitaire majeur. Par ailleurs, il a récemment été mis en évidence dans des sols traités aux antibiotiques des bactéries capables d’utiliser ces substances pour leur croissance [1]. La dégradation bactérienne des antibiotiques pourrait être un moyen de réduire la pression de sélection positive qu’ils exercent sur la dispersion des antibiorésistances. Ma thèse se propose de vérifier cette hypothèse par l’exploration des scénarii de dispersion des capacités de résistance et de dégradation des antibiotiq…

Molecular analysis of the catechol-degrading bacterial community in a coal wasteland heavily contaminated with PAHs

International audience; A PCR-based molecular tool was developed to estimate the diversity of the catechol-degrading bacterial community in a coal wasteland heavily contaminated with PAHS. A degenerate primer pair specific to catA sequences was designed by multiple alignment of known sequences coding a key intermediate of the β-ketoadiapate pathway degrading catechol, namely catechol 1,2-dioxygenase. The specificity of this primer pair was assessed in 21 pure strains by PCR and sequencing. Comparison of the 16S rDNA and catA phylogenies revealed an absence of congruence between these two genes. The primer set was able to amplify catA sequences in DNA extracts from an industrial soil highly …

Le partage des tâches favorise la coexistence dans les communautés bactériennes dégradant l’atrazine

National audience; Les communautés microbiennes exercent un rôle primordial dans la dégradation des xénobiotiques1, et en particulier des pesticides. Dans le cas de l’herbicide atrazine, plusieurs études ont démontré que sa dégradation faisait intervenir un consortium, plutôt qu’une espèce isolée2,3,4. Cependant, on ne sait pas comment la mise en place de ces consortiums dégradants se fait dans la nature. La théorie de la Reine Noire formalise les conditions nécessaires pour le mise en place de dépendance entre espèces5 : un membre de la communauté, appelé « helper », fournit un bien commun à la communauté par la dégradation d’un composé complexe, alors que les autres, appelés « beneficiari…

Antibiotrophie : une fonction émergente favorisant l’implantation des bactéries antibiorésistantes dans les sols

National audience; La fonction antibiotrophique, permettant l’utilisation d’antibiotiques comme source nutritive, arécemment été découverte chez des bactéries antibiorésistantes environnementales. Pourvérifier si leur avantage sélectif en environnements pollués s’en trouvait augmenté, nous avonsconduit une expérience en microcosmes dans laquelle Microbacterium sp. C448 (C448),présentant des capacités de résistance et de dégradation vis-à-vis des sulfamides, a été inoculéedans quatre sols supplémentés ou non par l’antibiotique sulfaméthazine (SMZ) et/ou du lisier.Après un mois d’incubation, C448 n’a été détectée que dans les sols traités avec la SMZ,indiquant une faible compétitivité de la s…

Impact of a new bio-pesticide produced by Paenibacillus sp. strain B2 on the genetic structure and density of soil bacterial communities

The effect of paenimyxin, a new biopesticide produced by Paenibacillus sp. strain B2, on the density of soil bacterial communities was assessed by colony counting and by 16S rDNA and nirK quantitative polymerase chain reaction (PCR). Paenimyxin had a negative effect on the bacterial colony-forming unit (CFU) number, which was significantly reduced 2 and 4 days after treatment. The effect of paenimyxin on cultivatable bacteria was negligible 7 days after treatment. Approximately 107 16S rDNA sequences per gram of soil (dry weight) were detected by quantitative PCR in all samples. Paenimyxin did not affect the quantification of 16S rDNA or of the denitrifying bacterial community. In addition,…

Evidence for 2,4-D mineralisation in Mediterranean soils: impact of moisture content and temperature

BACKGROUND: The 2,4-D degradation ability of the microbiota of three arable Mediterranean soils was estimated. The impact of soil moisture and temperature on 2,4-D degradation was investigated. RESULTS: The microbiota of the three soils regularly exposed to 2,4-D were able rapidly to mineralise this herbicide. The half-life of 2,4-D ranged from 8 to 30 days, and maximum mineralisation of 14C-2,4-D ranged from 57 to 71%. Extractable 14C-2,4-D and 14C-bound residues accounted for less than 1 and 15% respectively of the 14C-2,4-D initially added. The highest amounts of 14C-2,4-D bound residues were recorded in the soil with the lowest 2,4-D-mineralising ability. Although all three soils were a…

Predicting pesticide biodegradation potential from microbial community composition : new tools for bioremediation

Bioaugmentation is receiving increasing attention as a green technology to treat contaminated areas by inoculating specific biodegrading microorganisms. However, our understanding of the role of microbial community composition and structure in the expression of contaminant degradation potential is yet to improve. It could help making wise choice for microorganisms – community or specific strain – to be inoculated in contaminated soils with consideration to their indigeneous microbiota. Here we tried to predict the microbial degradation of two herbicides, glyphosate and isoproturon by means of penalized regression and machine learning methods routinely used in genomic selection. To this end,…

Recurrent inoculation: a strategy for a better survival of Pseudomonas fluorescens strain in soil

A growing interest is shown for microbial inoculants as a promising alternative to agrochemicals for sustainable agricultural. However, biotic and abiotic stresses are known to affect their establishment and effectiveness, leading to inconsistent performance in field conditions. At the same time, a poor survival of the strain is often observed, which constitutes a main hamper for bacterial inoculant development. We hypothesize that recurrent inoculations would improve the survival of the strain in soil. For this purpose, we tested different scenarios of inoculation of the plant growth promoting rhizobacteria Pseudomonas fluorescens in soil microcosms: a single inoculation, two inoculations …

Nouveaux outils en bioremédiation : prédiction du potentiel de dégradation d’une communauté à partir de sa composition et création de communautés multidégradantes par coalescence

En bioremédiation, la bioaugmentation consiste en l’introduction de microorganismes dans un milieu contaminé afin d’augmenter et/ou de suppléer la population bactérienne en place, dans l’objectif de le dépolluer. Cette approche est cependant limitée par la difficulté de choisir des microorganismes efficients - communauté ou souche spécifique – à inoculer, en lien avec les conditions abiotiques et les microorganismes indigènes du milieu pollué.Dans ce travail nous avons cherché à prédire le potentiel microbien de dégradation de deux pesticides, le glyphosate et l’isoproturon, de communautés microbiennes telluriques en utilisant des méthodes statistiques issues de la prédiction génomique. Pou…

Evaluation de l’impact des pesticides sur des fonctions écosystémiques terrestres

Dans la cadre l’évaluation du risque environnemental menée pour obtenir l’autorisation de mise sur le marché, l’évaluation a prioride l’impact des pesticides sur les microorganismes du sol est estimée par deux tests OCDE mesurant la minéralisation de l’azote et du carbone. Ces deux tests globaux ne rendent pas compte des modifications potentielles d’abondance, de diversité et d’activité de la communauté microbienne des sols exposée aux pesticides. Dans ce contexte, le projet IMPEC visait à développer des indicateurs microbiens pour évaluer l’impact des pesticides sur des fonctions écosystémiques supportées par la communauté microbienne. Cette présentation aura pour objectif de présenter une…

Antibiotrophy: Key Function for Antibiotic-Resistant Bacteria to Colonize Soils—Case of Sulfamethazine-Degrading Microbacterium sp. C448

Chronic and repeated exposure of environmental bacterial communities to anthropogenic antibiotics have recently driven some antibiotic-resistant bacteria to acquire catabolic functions, enabling them to use antibiotics as nutritive sources (antibiotrophy). Antibiotrophy might confer a selective advantage facilitating the implantation and dispersion of antibiotrophs in contaminated environments. A microcosm experiment was conducted to test this hypothesis in an agroecosystem context. The sulfonamide-degrading and resistant bacterium Microbacterium sp. C448 was inoculated in four different soil types with and without added sulfamethazine and/or swine manure. After 1 month of incubation, Micro…

Evaluation of the ecotoxicological impact of pesticides on the diversity and the abundance of soil microorganisms

Utilisation du potentiel de biodégradation de sédiments de rivière comme indicateur d’une diminution du niveau d’exposition a l’herbicide diuron suite a son interdiction d’utilisation

National audience; Les communautés microbiennes présentent une forte capacité à répondre rapidement à des changements environnementaux et à s’y adapter de manière transitoire ou irréversible. En cas d’exposition chronique à des polluants organiques, les processus d’adaptation microbienne peuvent ainsi conduire à l’amélioration des capacités de biodégradation de ces composés. Cette fonctionnalité concerne principalement les microorganismes hétérotrophes (essentiellement les bactéries et les champignons) capables de dégrader les polluants (totalement ou partiellement) pour les utiliser comme une source d’énergie et de nutriments pour leur croissance. Plusieurs études réalisées en milieu terre…

Recurrent inoculation: a strategy for a better survival of Pseudomonas fluorescens strain in soil

A growing interest is shown for microbial inoculants as a promising alternative to agrochemicals for sustainable agricultural. However, biotic and abiotic stresses are known to affect their establishment and effectiveness, leading to inconsistent performance in field conditions. At the same time, a poor survival of the strain is often observed, which constitutes a main hamper for bacterial inoculant development. We hypothesize that recurrent inoculations would improve the survival of the strain in soil. For this purpose, we tested different scenarios of inoculation of the plant growth promoting rhizobacteria Pseudomonas fluorescens in soil microcosms: a single inoculation, two inoculations …

Fitness drift of an atrazine-degrading population under atrazine selection pressure.

International audience; Pseudomonas sp. ADP harbouring the atrazine catabolic plasmid ADP1 was subcultured in liquid medium containing atrazine as sole source of nitrogen. After approximately 320 generations, a new population evolved which replaced the initial population. This newly evolved population grew faster and degraded atrazine more rapidly than the initial population. Plasmid profiles and Southern blot analyses revealed that the evolved strain, unlike the ancestral strain, presented a tandem duplication of the atzB gene encoding the second enzyme of the atrazine catabolic pathway responsible for the transformation of hydroxyatrazine to N-isopropylammelide. This duplication resulted …

Antibiotics favor the establishment of antibiotrophic bacteria in agricultural soil microbial communities, but are not always sufficient to enhance antibiotic-degradation: manure spreading can help.

International audience; During the past decades, environmental concentrations of antibiotics have largely increased,resulting in a risk of ecosystem disturbance. However, because their composition is often rich innitrogen and carbon, antibiotics are of nutritional interest for microorganisms, as long as theirbiocidal character is not considered. Antibiotic-degrading bacteria have therefore emerged amongststrains that were resistant to antibiotics. Called antibiotrophs, they are able to use selectedantibiotics as nutritive sources for their growth. While several antibiotrophs have been isolated fromdifferent agroecosystems, little is known about their ecology. In particular, their dispersal …

Analyse et modélisation des mécanismes de transfert de produits phytosanitaires aux échelles de la parcelle agricole et du bassin versant. Le cas d'un système hydrologique en milieu méditerranéen cultivé

National audience

Evidence of atrazine mineralization in a soil from the Nile Delta: Isolation of Arthrobacter sp. TES6, an atrazine-degrading strain

International audience; The s-triazine herbicide atrazine was rapidly mineralized (i.e., about 60% of C-14-ring-labelled atrazine released as (CO2)-C-14 within 21 days) by an agricultural soil from the Nile Delta (Egypt) that had been cropped with corn and periodically treated with this herbicide. Seven strains able to degrade atrazine were isolated by enrichment cultures of this soil. DNA fingerprint and phylogenetic studies based on 165 rRNA analysis showed that the seven strains were identical and belonged to the phylogeny of the genus Arthrobacter (99% similarity with Arthrobacter sp. AD38, EU710554). One strain, designated Arthrobacter sp. strain TES6, degraded atrazine and mineralized…

Dynamique des antibiotiques et des antibiorésistances dans les agrosystèmes : Réponse écotoxicologique et adaptation des communautés microbiennes terrestres

National audience; La contamination environnementale par les antibiotiques favorise la dispersion de bactéries antibiorésistantes, ainsi que le développement de bactéries dégradantes pouvant les utiliser pour leur croissance. Propices pour réduire la contamination environnementale et donc la dispersion de l’antibiorésistance, elles représentent toutefois un danger potentiel envers l’efficacité des antibiotiques avec l’éventualité de transmissions de leur capacité aux microorganismes pathogènes. Ainsi, ma thèse a pour but d’explorer les scénarii de dispersion des capacités de résistance et de dégradation dans le contexte agricole de l’épandage de lisier porcin contaminé en antibiotique sulfa…

Adaptation des communautés microbiennes autotrophes et hétérotrophes aux changements spatio-temporels d'exposition aux pesticides en cours d'eau

National audience

Metabolic response of Microbacterium sp. C448 exposed to environmental and medicinal concentrations of sulfamethazine antibiotic

International audience

Impact écotoxicologique de pesticides sur les communautés microbiennes naturelles responsables de la biodégradation de pesticides dans les sédiments de rivière

National audience; L’utilisation quasi généralisée de pesticides dans de nombreuses cultures permet d’assurer une certaine qualité de la production végétale mais elle a des conséquences environnementales causant la contamination des eaux de surface, notamment les rivières circulant dans les agrosystèmes. Suite à l’exposition répétée aux pesticides, une fraction de la microflore est capable de s’adapter pour les dégrader conduisant à la mise en place de la biodégradation accélérée, un processus intéressant du point de vue environnemental parce qu’il diminue la persistance des pesticides dans l’environnement. La biodégradation accélérée peut avoir lieu dans les sols mais également dans les sé…

Mécanismes d'évolution de pseudomonas sp. adp sous pression de sélection exercée par l'atrazine

L’atrazine est un pesticide qui est largement utilisé dans le monde depuis une quarantaine d’années. Son application répétée aaboutit non seulement à la contamination des eaux mais également à l’apparition de bactéries telluriques capables d’utiliserce xénobiotique comme source d’azote pour leur croissance. La caractérisation des gènes atz responsables de la dégradationde cette molécule a révélé qu’ils étaient largement dispersés et très conservés au sein du monde bactérien traduisant ainsid’une évolution et d’une dispersion récente de ces gènes, probablement concomitantes avec l’application d’atrazine dansl’environnement

Freshwater sediment pesticide biodegradation potential as an ecological indicator of microbial recovery following a decrease in chronic pesticide exposure

International audience; Water resources and aquatic ecosystems are an essential concern of any policy for sustainable development. In the context of the European Water Framework Directive, which aims to achieve good chemical and ecological status of waters, the herbicide diuron was banned in France in December 2008. In lotic ecosystems, benthic microbial assemblages are considered as useful potential indicators of ecological status because they integrate the effects of multiple anthropogenic disturbances and have strong capacities to adapt to novel environmental conditions. Field studies have thus revealed that in situ diuron exposure can induce microbial adaptation leading to an increase i…

Long-term impact of 19 years' farmyard manure or sewage sludge application on the structure, diversity and density of the protocatechuate-degrading bacterial community

International audience; Impact of long-term biosolids application on soil-living micro-organisms key players of ecosystemic services is scarcely reported. Here, the impact of the 19 year-long application of farmyard manure (FM) and sewage sludge (SS) organic fertilisation regimes on the protocatechuate-degrading bacterial (pca) community was estimated by comparison to a mineral fertilisation regime (U). The structure, diversity and density of the pca community were determined using pcaH, a gene encoding the protocatechuate 3,4-dioxygenase. Ten years after the last application, the structure of the pca community in soils amended with 55100 (100 t/ha/2 years) and to a lesser extent with FM (1…

Processes involved in the adaptation of soil microbiota to enhance biodegradation of pesticides: atrazine-degrading community as a study case

International audience