Search

4-hydroxyphenylpyruvate dioxygenase (HPPD) is the molecular target of β-triketone herbicides in plants. This enzyme, involved in the tyrosine pathway, is also present in a wide range of living organisms, including microorganisms. Previous studies, focusing on a few strains and using high herbicide concentrations, showed that β-triketones are able to inhibit microbial HPPD. Here, we measured the effect of agronomical doses of β-triketone herbicides on soil bacterial strains. The HPPD activity of six bacterial strains was tested with 1× or 10× the recommended field dose of the herbicide sulcotrione. The selected strains were tested with 0.01× to 15× the recommended field dose of sulcotrione, mesotrione, and tembotrione. Molecular docking was also used to measure and model the binding mode of the three herbicides with the different bacterial HPPD. Our results show that responses to herbicides are strain-dependent with Pseudomonas fluorescens F113 HPPD activity not inhibited by any of the herbicide tested, when all three β-triketone herbicides inhibited HPPD in Bacillus cereus ATCC14579 and Shewanella oneidensis MR-1. These responses are also molecule-dependent with tembotrione harboring the strongest inhibitory effect. Molecular docking also reveals different binding potentials. This is the first time that the inhibitory effect of β-triketone herbicides is tested on environmental strains at agronomical doses, showing a potential effect of these molecules on the HPPD enzymatic activity of non-target microorganisms. The whole-cell assay developed in this study, coupled with molecular docking analysis, appears as an interesting way to have a first idea of the effect of herbicides on microbial communities, prior to setting up microcosm or even field experiments. This methodology could then largely be applied to other family of pesticides also targeting an enzyme present in microorganisms.

The emergence of pesticides of natural origin appears as an environmental-friendly alternative to synthetic pesticides for managing weeds. To verify this assumption, leptospermone, a natural β-triketone herbicide, and sulcotrione, a synthetic one, were applied to soil microcosms at 0× (control), 1× or 10× recommended field dose. The fate of these two herbicides (i.e. dissipation and formation of transformation products) was monitored to assess the scenario of exposure of soil microorganisms to natural and synthetic herbicides. Ecotoxicological impact of both herbicides was explored by monitoring soil bacterial diversity and activity using next-generation sequencing of 16S rRNA gene amplicons and soil metabolomics. Both leptospermone and sulcotrione fully dissipated over the incubation period. During their dissipation, transformation products of natural and synthetic β-triketone were detected. Hydroxy-leptospermone was almost completely dissipated by the end of the experiment, while CMBA, the major metabolite of sulcotrione, remained in soil microcosms. After 8 days of exposure, the diversity and structure of the soil bacterial community treated with leptospermone was significantly modified, while less significant changes were observed for sulcotrione. For both herbicides, the diversity of the soil bacterial community was still not completely recovered by the end of the experiment (45 days). The combined use of next-generation sequencing and metabolomic approaches allowed us to assess the ecotoxicological impact of natural and synthetic pesticides on non-target soil microorganisms and to detect potential biomarkers of soil exposure to β-triketones.

International audience; Sharka disease, caused by Plum pox virus (PPV), induces several changes in Prunus. In leaf tissues, the infection may cause oxidative stress and disrupt the photosynthetic process. Moreover, several defense responses can be activated after PPV infection and have been detected at the phytohormonal, transcriptomic, proteomic, and even translatome levels. As proposed in this review, some responses may be systemic and earlier to the onset of symptoms. Nevertheless, these changes are highly dependent among species, variety, sensitivity, and tissue type. In the case of fruit tissues, PPV infection can modify the ripening process, induced by an alteration of the primary metabolism, including sugars and organic acids, and secondary metabolism, including phenolic compounds. Interestingly, metabolomics is an emerging tool to better understand Prunus–PPV interactions mainly in primary and secondary metabolisms. Moreover, through untargeted metabolomics analyses, specific and early candidate biomarkers of PPV infection can be detected. Nevertheless, these candidate biomarkers need to be validated before being selected for a diagnostic or prognosis by targeted analyses. The development of a new method for early detection of PPV-infected trees would be crucial for better management of the outbreak, especially since there is no curative treatment.

National audience; β-triketone herbicides are among the most used herbicides in corn crop to control broadleaf weeds.These herbicides inhibit the 4-hydroxyphenylpyruvate dioxygenase (4-HPPD) and lead to bleaching anddeath of weeds. This enzyme is not only found in plants but in all living organisms, includingmicroorganisms where it takes part to the tyrosine degradation pathway. Thus, microorganismsclassified as “non-target organisms” by current EU regulation for pesticide authorization, might beimpacted by β-triketones, with possible domino effect on microbial functions supporting soilecosystem services (Thiour-Mauprivez et al. 2019). Since microorganisms have been proposed by EFSAas key-drivers to be monitored to better protect soil ecosystem services, we tested the hypothesis thathppd bacterial community can constitute a biomarker of exposure to β-triketones. Within this context,we developed a toolbox to monitor the abundance, the diversity and the activity of the hppd bacterialcommunity.Abundance and diversity of hppd bacterial community were tested in a lab-to-field experimentaldesign following the tiered-approach recommended by EFSA to conduct pesticide environmental riskassessment (ERA). Under lab conditions, soil microcosms not exposed (control) or exposed to x1 or x10times the agronomical dose of sulcotrione (active ingredient) or Decano® (one of the commercialformulation of sulcotrione) were studied. Under field conditions, samples were collected in corn cropexposed to β-triketones or not (control). Analytical chemistry was applied to all samples to study thedissipation of β-triketone, search for residues and estimate the scenario of exposure of soilmicroorganisms. Nucleic acids (DNA/RNA) were extracted from soil samples. Home-made degeneratedprimers, specific to the hppd gene of soil bacteria, allowed us to measure the abundance (quantitativePCR), the composition (α-diversity) and the diversity (β-diversity) (NGS) of the hppd bacterialcommunity (Thiour-Mauprivez et al. 2020). Finally, the inhibition of the activity of the 4-HPPD wasassessed on pure bacterial strains under wet lab conditions to measure EC50 of β-triketones.Our poster will be presented to the audience with the aim to identify the better proxy of the hppdbacterial community that could be used as a biomarker to reflect the exposure of soil microbialcommunity to β-triketone residues. As a perspective my work might be extended to other pesticidestargeting other enzymes that are also present in so call non-target organisms such as sulfonylureasinhibiting acetohydroxy acid synthase (AHAS).

National audience; The present work aimed to design a degenerate primer pair to target a large part of the hppd soil bacterial community, possibly affected by herbicides. We validated these primers by qPCR and high-throughput sequencing analysis of soil samples.

International audience; Conventional agriculture still relics on the general use of agrochemicals (herbicides, fungicides and insecticides) to control various pests (weeds, fungal pathogens and insects), to ensure the yield of crop and to feed a constantly growing population. The generalized use of pesticides in agriculture leads to the contamination of soil and other connected environmental resources. The persistence of pesticide residues in soil is identified as a major threat for in-soil living organisms that are supporting an important number of ecosystem services. Although authorities released pesticides on the market only after their careful and thorough evaluation, the risk assessment for in-soil living organisms is unsatisfactory, particularly for microorganisms for which pesticide toxicity is solely considered by one global test measuring N mineralization. Recently, European Food Safety Authority (EFSA) underlined the lack of standardized methods to assess pesticide ecotoxicological effects on soil microorganisms. Within this context, there is an obvious need to develop innovative microbial markers sensitive to pesticide exposure. Biomarkers that reveal direct effects of pesticides on microorganisms are often viewed as the panacea. Such biomarkers can only be developed for pesticides having a mode of action inhibiting a specific enzyme not only found in the targeted organisms but also in microorganisms which are considered as "non-target organisms" by current regulations. This review explores possible ways of innovation to develop such biomarkers for herbicides. We scanned the herbicide classification by considering the mode of action, the targeted enzyme and the ecotoxicological effects of each class of active substance in order to identify those that can be tracked using sensitive microbial markers.

The photolytic behaviour of tembotrione, a new chemical herbicide intended for foliar application in corn, was investigated under unnatural and extreme photochemical exposure in aqueous solutions in the laboratory. It appeared that degradation was dependent on pH and occurred more rapidly under acidic and neutral conditions, leading predominantly to the formation of a xanthenedione type compound by intramolecular cyclisation with loss of HCl. Trace amounts of benzoic acid by-products appeared also during UV-C irradiation (λ=254 nm) of the parent compound. Results were comparable to those obtained with sulcotrione, another β-triketone herbicide. These extreme irradiation conditions clearly accelerated the phototransformation of sulcotrione vs. simulated sunlight irradiation. Furthermore, the photolysis of the degradation by-products, resulting from either photolysis, hydrolysis or biotic pathways of the two active ingredients, was also carried out. The benzoic acid by-products appeared more stable to photolysis than their parent molecules. Xanthenedione derivatives were degraded more rapidly with several differences depending on the pH value.

This study presents a novel approach for assessing the risk of agrochemicals in soil microcosms through the use of non-targeted metabolomics. The metabolome of treated soils was extracted and tested through LCMS profiling in order to generate an "Environmental Metabolic Footprint" (EMF). A dynamic characterization of pollution biomarkers was obtained through a multivariate statistical analysis of EMF data, where our results show the possible evolution towards a state of resilience. The EMF methodology was applied to two β-triketone herbicides in soil microcosms: one natural, leptospermone, and one synthetic, sulcotrione. In spite of a four-fold higher application dose, leptospermone exhibited a lower resilience time than did sulcotrione (ca. 30 days vs ca. 45 days respectively).

The ecotoxicological impact of leptospermone, a β-triketone bioherbicide, on the bacterial community of two arable soils was investigated. Soil microcosms were exposed or not (control, 0×) to leptospermone applied at 1× or 10× recommended dose. Leptospermone was moderately adsorbed to both soils (i.e.: Kfa  1.2 and Koc  140 ml.g-1). Dissipation of leptospermone was lower in sterilized than in unsterilized soils suggesting that it was mainly influenced by biotic factors. Within 45 days, leptospermone disappeared almost entirely from one of the two soils (i.e. DT50 < 10 days), while 25% of the leptospermone remained in the other. The composition of the microbial community assessed by qPCR targeting 11 microbial groups was found to be significantly modified in soil microcosms exposed to leptospermone. Pyrosequencing of 16S rRNA gene amplicons showed a shift in the bacterial community structure and a significant impact of leptospermone on the diversity of the soil bacterial community. Changes in the composition as well as in the α- and β-diversity of microbial community were transient in the soil able to fully dissipate the leptospermone. They were persistent in the soil where leptospermone remained. To conclude the bacterial community of the two soils was sensitive to leptospermone and its resilience was observed only when leptospermone was fully dissipated.

BACKGROUND: The dissipation kinetics of the herbicide sulcotrione sprayed 4 times on a French soil was studied using a laboratory microcosm approach. An advanced cultivation-based method was then used to isolate the bacteria responsible for biotransformation of sulcotrione. Chromatographic methods were employed as complementary tools to define its metabolic pathway. RESULTS: Soil microflora was able quickly to biotransform the herbicide (DT50 ≈ 8 days). 2-Chloro-4-mesylbenzoic acid, one of its main metabolites, was clearly detected. However, no accelerated biodegradation process was observed. Eight pure sulcotrione-resistant strains were isolated, but only one (1OP) was capable of degrading this herbicide with a relatively high efficiency and to use it as a sole source of carbon and energy. In parallel, another sulcotrione-resistant strain (1TRANS) was shown to be incapable of degrading the herbicide. Amplified ribosomal restriction analysis (ARDRA) and repetitive extragenic palendromic PCR genomic (REP-PCR) fingerprinting of strains 1OP and 1TRANS gave indistinguishable profiles. CONCLUSION: Sequencing and aligning analysis of 16S rDNA genes of each pure strain revealed identical sequences and a close phylogenetic relationship (99% sequence identity) to Pseudomonas putida. Such physiological and genetic properties of 1OP to metabolise sulcotrione were probably governed by mobile genetic elements in the genome of the bacteria. Copyright © 2011 Society of Chemical Industry

International audience; In this study, a bacterial strain able to use sulcotrione,a β-triketone herbicide, as sole source of carbon and energy was isolated from soil samples previously treated with this herbicide. Phylogenetic study based on16S rRNA gene sequence showed that the isolate has 100 % of similarity with several Bradyrhizobium and was accordingly designated as Bradyrhizobium sp. SR1. Plasmid profiling revealed the presence of a large plasmid (>50 kb) in SR1 not cured under nonselective conditions. Its transfer to Escherichia coli by electroporation failed to induce β-triketone degrading capacity,suggesting that degrading genes possibly located on this plasmid cannot be expressed in E. coli or that they are not plasmid borne. The evaluation of the SR1 ability to degrade various synthetic (mesotrione and tembotrione) and natural (leptospermone) triketones showed that this strain was also able to degrademesotrione. Although SR1 was able to entirely dissipate both herbicides, degradation rate of sulcotrione was ten times higher than that of mesotrione, showing a greater affinity of degrading-enzyme system to sulcotrione. Degradation pathway of sulcotrione involved the formation of 2-chloro-4-mesylbenzoic acid (CMBA), previously identified in sulcotrione degradation, and of a new metabolite identified as hydroxy-sulcotrione.Mesotrione degradation pathway leads to the accumulation of-methylsulfonyl-2-nitrobenzoic acid(MNBA) and 2-amino-4 methylsulfonylbenzoic acid(AMBA), two well-known metabolites of this herbicide. Along with the dissipation of β-triketones, one could observe the decrease in 4-hydroxyphenylpyruvate dioxygenase(HPPD) inhibition, indicating that toxicity was due to parent molecules, and not to the formed metabolites. This is the first report of the isolation of bacterial strain able to transform two β-triketones.

International audience; Pseudomonas sp. 1OP, previously isolated from a French agricultural soil, has been described as the first sulcotrione degrading bacteria. Different conditions of initial pH and herbicide concentration in liquid culture were tested to evaluate the growth performances of the isolate and its degrading capacity, with sulcotrione as the sole carbon and/or energy source. Maximal growth rate (μmax) was obtained under initial neutral conditions and with initial concentration of sulcotrione close to 180 μM, and was described, during the exponential phase, by a sigmoidal curve which could be easily fitted to the modified Gompertz equation. Complementary studies carried on the CMBA by-product and on another β-triketone herbicide showed the relative specificity of the strain against sulcotrione. The sulcotrione degrading phenotype of Pseudomonas sp.1OP was shown to be lost under non-selective conditions. Plasmid-Eckardt modified method, consecutively applied for plasmid profiling, showed that this strain carries one large plasmid (>12 kb) bearing putative genes involved in sulcotrione degradation, as demonstrated by curing experiment.

The photolytic behaviour of tembotrione, a new chemical herbicide intended for foliar application in corn, was investigated under unnatural and extreme photochemical exposure in aqueous solutions in the laboratory. It appeared that degradation was dependent on pH and occurred more rapidly under acidic and neutral conditions, leading predominantly to the formation of a xanthenedione type compound by intramolecular cyclisation with loss of HCl. Trace amounts of benzoic acid by-products appeared also during UV-C irradiation (λ=254 nm) of the parent compound. Results were comparable to those obtained with sulcotrione, another β-triketone herbicide. These extreme irradiation conditions clearly accelerated the phototransformation of sulcotrione vs. simulated sunlight irradiation. Furthermore, the photolysis of the degradation by-products, resulting from either photolysis, hydrolysis or biotic pathways of the two active ingredients, was also carried out. The benzoic acid by-products appeared more stable to photolysis than their parent molecules. Xanthenedione derivatives were degraded more rapidly with several differences depending on the pH value.

The dissipation kinetics of the herbicide sulcotrione sprayed 4 times on a French soil was studied using a laboratory microcosm approach. An advanced cultivation-based method was then used to isolate the bacteria responsible for biotransformation of sulcotrione. Chromatographic methods were employed as complementary tools to define its metabolic pathway.Soil microflora was able quickly to biotransform the herbicide (DT(50) ≈ 8 days). 2-Chloro-4-mesylbenzoic acid, one of its main metabolites, was clearly detected. However, no accelerated biodegradation process was observed. Eight pure sulcotrione-resistant strains were isolated, but only one (1OP) was capable of degrading this herbicide with a relatively high efficiency and to use it as a sole source of carbon and energy. In parallel, another sulcotrione-resistant strain (1TRANS) was shown to be incapable of degrading the herbicide. Amplified ribosomal restriction analysis (ARDRA) and repetitive extragenic palendromic PCR genomic (REP-PCR) fingerprinting of strains 1OP and 1TRANS gave indistinguishable profiles.Sequencing and aligning analysis of 16S rDNA genes of each pure strain revealed identical sequences and a close phylogenetic relationship (99% sequence identity) to Pseudomonas putida. Such physiological and genetic properties of 1OP to metabolise sulcotrione were probably governed by mobile genetic elements in the genome of the bacteria.

International audience; The behaviour of sulcotrione, a recently introduced triketone herbicide, in various soil types was studied under laboratory conditions. In particular, degradation and sorption processes were examined on Ghent and Perpignan soils. Kinetics showed that the degradation of sulcotrione was influenced by biotic and/or abiotic factors. Half-lives ranged between 45 and 65 days. Among the degradation compounds identified were 1,3-cyclohexanedione (CHD) and 2-chloro-4-mesyl benzoic acid (CMBA), previously described as hydrolysis products, and, under special conditions, a derivative of phenylheptanoic acid (PHD). This new degradation product suggested that sulcotrione could follow two possible pathways in the soil, as in water. During the sorption study, a moderate retention of sulcotrione and CMBA relative to CHD and PHD, which were highly adsorbed whatever the soil type, was reported. Experiments carried out under the same conditions for sulcotrione and mesotrione, another triketone herbicide recommended in maize culture, made it possible to compare the two triketones and to conclude that they exhibited relatively similar behaviour in the soil, i.e. that their leaching potential needs to be properly addressed and risks evaluated.

visioconférence, France, 6-9 octobre 2020; International audience

Dans le cadre de la procédure d’évaluation des substances prévue par le Règlement REACH n°1907/2006 (articles 44 à 48), les États Membres évaluent chaque année des substances jugées prioritaires, dans le but de clarifier une ou des préoccupation(s) émanant de la fabrication et/ou de l’utilisation de ces substances et qui pourrai(en)t entrainer un risque pour la santé humaine et/ou pour l’environnement. Ces substances sont inscrites sur le plan d’action continu communautaire (CoRAP), publié sur le site internet de l’Agence européenne des produits chimiques (ECHA) avec une courte description des préoccupations initialement identifiées pour chacune des substances. Dans la majorité des cas, ces préoccupations initiales sont liées aux propriétés de danger potentiel, en combinaison avec une utilisation susceptible de conduire à une dispersion environnementale ou des usages générant une exposition pour les consommateurs.Chaque année, les États membres de l’Union européenne et des pays de l’Espace économique européen (à savoir la Norvège, l'Islande et le Liechtenstein) évaluent les substances inscrites au CoRAP dans le but de clarifier la ou les préoccupation(s) initiale(s) émanant de la fabrication et/ou l’utilisation de ces substances et qui pourraient représenter un risque pour la santé humaine et/ou pour l’environnement. Le CoRAP en 2018 incluait deux substances dont l’évaluation a été confiée à l’Anses.Les États membres peuvent cibler leur évaluation sur la préoccupation initiale, mais peuvent aussi l’élargir à tout ou partie des propriétés de la substance. A l’issue des 12 mois d'évaluation, l’État membre évaluateur peut demander des informations supplémentaires aux déclarants des substances, si ces données additionnelles sont jugées nécessaires pour clarifier une ou des préoccupation(s). Dans ce cas, un projet de décision est soumis aux États membres et aux déclarants pour commentaires, puis ce projet de décision peut être discuté au Comité des États Membres (CEM) de l’ECHA en cas d’avis divergents entre les États membres. Alternativement, il peut être conclu qu'aucune donnée supplémentaire n'est nécessaire pour clarifier le risque. Dans ce cas, un document de conclusion est rédigé. Le cas échéant, si des préoccupations sont confirmées, la rédaction d’une analyse de la meilleure option de gestion des risques (RMOA) peut être proposée.Les deux substances évaluées en 2018 par la France ont été les suivantes: - trichlorure de n-butylétain (n°CAS 1118-46-3) - dioxyde de titane (n°CAS 13463-67-7)(reproduction d'un tableau non intégré sur cette notice)Ces deux substances sont enregistrées auprès de l’ECHA dans le cadre de l’application du règlement REACH, ce qui signifie que des industriels ont déposé des dossiers d’enregistrement contenant les données requises en fonction de leur bande de tonnage de production et/ou d’importation.Le présent avis a pour objet de résumer les principales étapes de l’analyse des deux substances évaluées par l’Anses en 2018 et les décisions ou conclusions issues de cette expertise.