Agroecological transition: towards a better understanding of the impact of ecology-based farming practices on soil microbial ecotoxicology.

peer reviewedAlternative farming systems have developed since the beginning of industrial agriculture. Organic, biodynamic, conservation farming, agroecology and permaculture, all share a grounding in ecological concepts and a belief that farmers should work with nature rather than damage it. As ecology-based agricultures rely greatly on soil organisms to perform the functions necessary for agricultural production, it is thus important to evaluate the performance of these systems through the lens of soil organisms, especially soil microbes. They provide numerous services to plants, including growth promotion, nutrient supply, tolerance to environmental stresses and protection against pathogens. An overwhelming majority of studies confirm that ecology-based agricultures are beneficial for soil microorganisms. However, three practices were identified as posing potential ecotoxicological risks: the recycling of organic waste products, plastic mulching, and pest and disease management with biopesticides. The first two because they can be a source of contaminants; the third because of potential impacts on non-target microorganisms. Consequently, developing strategies to allow a safe recycling of the increasingly growing organic matter stocks produced in cities and factories, and the assessment of the ecotoxicological impact of biopesticides on non-target soil microorganisms, represent two challenges that ecology-based agricultural systems will have to face in the future

Development of a new functional marker for β-triketone herbicides exposure in agricultural soils

National audienceThe β-triketone herbicides are maize selective herbicides that have been largely applied in replacement of atrazine, banned in Europe in 2003. Their mode of action lays on the inhibition of the p-hydroxyphenylpyruvate dioxygenase (HPPD), a key enzyme of the carotenoid biosynthesis. In recent studies, we showed that within the soil bacterial community, many microorganisms possess a functional HPPD enzyme involved in tyrosine metabolism. These “non-target organisms” harbor the target of the β-triketone herbicides and consequently may be affected in response to its exposure. Within this context, the objective of our work is to check for the interest of hppd bacterial community as a marker of exposition and/or of impact sensitive to β-triketone herbicides. This will require the development of a molecular tool box to assess the abundance, activity and diversity of the hppd bacterial community in various arable soils exposed to β-triketone herbicides. The abundance and the activity of the hppd bacterial community will be monitored from the nucleic acids extracted directly from soils, and the diversity of the hppd community will be evaluated by a metagenomic study based on the high-throughput se- quencing of hppd amplicons. Our results will lead to the selection of a set of characteristic hppd sequences, allowing the development of hppd DNA chips to assess the ecotoxicological impact of β-triketone herbicides on soil microorganisms

DIAGSOL Development of a new functional marker for B-triketone herbicides exposure in agricultural soils

International audienceThe-triketone herbicides are maize selective herbicides that have been largely applied in replacement of atrazine, banned in Europe in 2004. Their mode of action lays on the inhi- bition of the p-hydroxyphenylpyruvate dioxygenase (HPPD), a key enzyme of the carotenoid biosynthesis.In recent studies, we showed that within the soil bacterial community, many microorganisms possess a functional HPPD enzyme involved in tyrosine metabolism. These ”non-target or- ganisms” harbor the target of the-triketone herbicides and consequently may be affected in response to its exposure. From this point of view, the bacterial community harboring the hppd gene might be a relevant marker to assess the ecotoxicological impact of _-triketone herbicides on soil bacterial functional diversity.Within this context, the objective of our work is to check for the interest of hppd bacterial community as a marker of exposition and/or of impact sensitive to _-triketone herbicides. This will require the development of a molecular tool box to assess the abundance, activity and diversity of the hppd bacterial community in various arable soils exposed to _-triketone herbicides. The abundance and the activity of the hppd bacterial community will be mon- itored from the nucleic acids extracted directly from soils, and the diversity of the hppd community will be evaluated by a metagenomic study based on the high-throughput se- quencing of hppd amplicons.Our results will lead to the selection of a set of characteristic hppd sequences, allowing the development of hppd DNA chips to assess the ecotoxicological impact of _-triketone herbicides on soil microorganisms

A new tool to assess the ecotoxicological impact of β-triketone herbicides on soil microbial communities

International audienceThe β-triketone herbicides are post-emergence maize selective herbicides that have beenintroduced on the market, in replacement of atrazine, banned in Europe in 2004. Qualified as “eco-friendly”, since they are based on natural phytotoxin properties, these herbicides target an enzymeinvolved in carotenoid biosynthesis called 4-hydroxyphenylpyruvate dioxygenase (HPPD) encoded bythe hppd gene. The inhibition of this enzyme provokes bleaching symptoms, necrosis and death ofweeds.The hppd gene is not only find in eukaryotes such as plants, animals and humans but also inprokaryotes such as fungi, yeasts and bacteria. In recent studies, we showed that, within the soil bacterialcommunity, many of them possess a functional HPPD enzyme involved in tyrosine metabolism1–3.However, although soil microorganisms are classified as "non-target organisms" according to EUregulation for authorization of pesticides, they may harbor the target of the β-triketone herbicides andconsequently might be affected in response to its exposure. From this point of view, the bacterialcommunity harboring the hppd gene might be a relevant bioindicator to assess, a priori, the possiblerisks incurred by the soil ecosystem in response to an exposition to β-triketone herbicides. Thisbioindicator could also be helpful to assess, a posteriori, the ecotoxicological impact of β-triketoneherbicides on soil bacterial diversity and abundance.Within this context, the aim of our work is to check for the interest of hppd bacterial communityas a bioindicator of exposition and/or of impact sensitive to β-triketone herbicides. This will require thedevelopment of a molecular toolbox to assess the abundance, and diversity of the hppd bacterialcommunity in various arable soils exposed to β-triketone herbicides. The abundance of the hppdbacterial community will be monitored from the nucleic acids extracted directly from soils. Moreover,the diversity of the hppd community will be evaluated thanks to high-throughput sequencing of hppdamplicons obtained from the DNA and RNA extracted from the soils. Our results will lead to theselection of a set of characteristic hppd sequences, allowing the development of hppd DNA chips toassess the ecotoxicological impact of β-triketone herbicides on soil bacterial diversity.1. Romdhane, S. et al. Evidence for photolytic and microbial degradation processes in the dissipationof leptospermone, a natural β-triketone herbicide. Environ. Sci. Pollut. Res. 1–12 (2017).doi:10.1007/s11356-017-9728-42. Romdhane, S. et al. Isolation and characterization of Bradyrhizobium sp. SR1 degrading two β-triketone herbicides. Environ. Sci. Pollut. Res. Int. 23, 4138–4148 (2016).3. Calvayrac, C. et al. Isolation and characterisation of a bacterial strain degrading the herbicidesulcotrione from an agricultural soil. Pest Manag. Sci. 68, 340–347 (2012)

Plant mediates soil water content effects on soil microbiota independently of its water uptake

International audienceThe on-going climate change impacts soil water distribution and availability, ranging from highly limited during droughts to excessive during rainfalls. While effects of water availability were documented for soil microbiota and plant growth, it remains unclear if the soil microbiota is directly impacted by soil water content (SWC) or via the plant presence and its own response to water fluctuations. Uncoupling these effects is challenging, since the plant alters SWC via its water-uptake and transpiration. We aimed to identify a potential effect of the plant on the soil microbiota, independent of its water uptake. We studied soil microbiota in two contrasting soils exposed to three water levels, either under the presence or absence of Brassica juncea (drought-sensitive, water-logging-tolerant) or Brachypodium distachyon (drought-tolerant, water-logging-sensitive). Using an automatic high-throughput watering system, we accurately maintained SWC levels to compensate plant water uptake and evapotranspiration, thus enabling the detection of plant effects independent of its water uptake. Plant traits were measured and the bacterial and fungal communities with or without plants were analysed. We highlighted effects of the plant on the microbiota under varying SWC, independently of plant water uptake. These effects were likely instigated by the physiological state of the plant due to SWC, and were dependant on the plant species and the soil considered. While bacterial communities were more sensitive to SWC than fungal communities, we found that the fungal community in the clayey soil was directly affected by SWC, and could opportunistically interact with a drought sensitive plant like Brassica juncea

DIAGSOL : Développement d’un outil microbien pour évaluer l’exposition des sols agricoles aux herbicides β-tricétones

International audienceEn 2011, en France, plus de 2 millions d’hectares de champs de maïs ont été traités avec desherbicides β-tricétones. Qualifiés de « respectueux de l’environnement » en raison d’une efficacité àfaible dose, de récentes études ont démontré un effet transitoire des β-tricétones sur la diversitébactérienne des sols (Romdhane et al., 2016). L’utilisation récurrente des β-tricétones pourrait, àterme, perturber la diversité bactérienne et les fonctions écosystémiques des sols qu’elle soutient.Il est essentiel d’évaluer l’exposition des microorganismes des sols aux β-tricétones.L’enzyme 4-hydroxyphénylpyruvate dioxygénase (4-HPPD), cible de ces herbicides chez les plantesadventices, est retrouvée chez de nombreuses bactéries. De ce fait, le gène hppd pourrait constituerun biomarqueur d’exposition. Afin d’explorer cette perspective, des couples d’amorces spécifiquesont été dessinés à l’aide des 1200 séquences hppd disponibles dans GenBank. Leur spécificité a étévalidée par le clonage et le séquençage d’amplicons générés à partir d’ADN bactérien extrait d’un sol.Des essais de qPCR visant à quantifier l’abondance des séquences hppd dans les sols ont étédéveloppés. Une expérience en microcosmes de sol exposés ou non à différentes doses desulcotrione est en cours. La dissipation de la sulcotrione dans le temps sera calculée et l’impactécotoxicologique de cet herbicide sur les bactéries sera estimé en quantifiant l’évolution del’abondance et de la diversité des séquences hppd. Nous prévoyons de réitérer cette approche enmicrocosmes à partir du DECANO®, produit formulé de la sulcotrione. Ces résultats seront présentésà la session EcotoxicoMic

Gene of non-target microorganisms : a new tool to monitor the exposure of soil microbial communities to b-triketone herbicides ?

International audienceβ-triketone herbicides are among the most used herbicides in corn crop. These herbicides inhibit the 4-hydroxyphenylpyruvate dioxygenase (4-HPPD), lead to bleaching and prevent the growth of broadleaf weeds. This enzyme is not only found in plants but in all living organisms, including microorganisms where it plays a role in the tyrosine degradation pathway. Thus, microorganisms classified as “non-target organisms” by current EU regulation for pesticide authorization, might be impacted by β-triketones, with consequences on microbial function supporting soil ecosystem services. Since microorganisms have been proposed by EFSA as key-drivers to protect soil ecosystem services, we suggest a novel approach consisting in considering the hppd bacterial community as a biomarker sensitive to the exposure to β-triketone residues.With this objective, we developed a toolbox to monitor the abundance, the composition, the diversity and the activity of the hppd bacterial community. Each method was tested in a lab-to-field experimental design following the tiered-approach recommended by EFSA to conduct pesticide environmental risk assessment (ERA). Under lab conditions, soil microcosms not exposed (control) or exposed to x1 or x10 the agronomical dose of sulcotrione (active ingredient) or Decano® (one of the commercial formulation of sulcotrione) were studied. Under field conditions, samples were collected in corn crop exposed to β-triketones. Soil samples were also collected from a non-treated corn field (not exposed control). Analytical chemistry was applied to all samples to search for β-triketone residues and to estimate the scenario of exposure of soil microorganisms. Nucleic acids (DNA/RNA) were extracted from soil samples to measure the abundance (quantitative PCR), the expression (quantitative RT-qPCR), the composition (α-diversity) and the diversity (β-diversity) (NGS) of the hppd bacterial community. Our results will be presented to the audience with the aim to identify the better proxy of the hppd bacterial community that could be used as a biomarker to reflect the exposure of soil microbial community to β-triketone residues.This explanatory work might be extended to other pesticides targeting other enzymes that are also present in so call non-target organisms such as sulfonylureas inhibiting acetohydroxy acid synthase (AHAS)

Response of soil bacterial and hppd communities to tembotrione herbicide

International audienceHerbicides used in agriculture aim to prevent weed growth but are known to end up in contactwith soil microorganisms, thus defined as non-target organisms. Tembotrione, a recentlymarketed β-triketone herbicide, is known to inhibit the 4-HydroxyPhenylPyruvateDioxygenase(4-HPPD) in weeds. This enzyme is also found in numerous soil microorganisms, such as somePGPR and symbiotic bacteria, that play a key role in maintenance of ecosystem services.In this study, one of the major concerns is to assess whether tembotrione could have toxiceffects on soil microorganisms and could disturb soil microbial community dynamic andstructure. To investigate the possible impacts of this herbicide on these communities, a soilmicrocosm approach was performed using 1-fold or 10-fold the recommended tembotrioneagronomical dose (RAD).Soil samples were collected at day 0, 3, 7, 14, 24, 40, and 55 to determine the followingendpoints: (i) dissipation of the active ingredient in soil, (ii) bacterial diversity with highthroughput sequencing of the 16S rDNA, (iii) hppd community diversity with high throughputsequencing of the hppd gene using degenerated primers designed by our own. Moreover, aculturable approach was applied to soil microcosm samples to study antibiotic resistance (ABR)of isolated soil bacteria, and to screen for HPPD+ strains.Whatever the treatment applied, tembotrione was fully dissipated from soil after 24 days at1*RAD with a DT50 of 7 days, and after 55 days at 10*RAD with a DT50 of 15 days. Interestingly,among the culturable strains isolated from the microcosms, 29 presenting ABR, were alsoHPPD+. Further studies are currently performed to characterize their sensitivity to β-triketonesherbicides, and their resistance to antibiotics. Finally, microbial and hppd diversity analyseswith sequencing data are still ongoing at the time of writing this abstract, but results should beavailable by October 2022

Gene of non-target microorganisms : a new tool to monitor the exposure of soil microbial communities to b-triketone herbicides ?

International audienc

Gene of non-target microorganisms : a new tool to monitor the exposure of soil microbial communities to b-triketone herbicides ?

International audienceβ-triketone herbicides are among the most used herbicides in corn crop. These herbicides inhibit the 4-hydroxyphenylpyruvate dioxygenase (4-HPPD), lead to bleaching and prevent the growth of broadleaf weeds. This enzyme is not only found in plants but in all living organisms, including microorganisms where it plays a role in the tyrosine degradation pathway. Thus, microorganisms classified as “non-target organisms” by current EU regulation for pesticide authorization, might be impacted by β-triketones, with consequences on microbial function supporting soil ecosystem services. Since microorganisms have been proposed by EFSA as key-drivers to protect soil ecosystem services, we suggest a novel approach consisting in considering the hppd bacterial community as a biomarker sensitive to the exposure to β-triketone residues.With this objective, we developed a toolbox to monitor the abundance, the composition, the diversity and the activity of the hppd bacterial community. Each method was tested in a lab-to-field experimental design following the tiered-approach recommended by EFSA to conduct pesticide environmental risk assessment (ERA). Under lab conditions, soil microcosms not exposed (control) or exposed to x1 or x10 the agronomical dose of sulcotrione (active ingredient) or Decano® (one of the commercial formulation of sulcotrione) were studied. Under field conditions, samples were collected in corn crop exposed to β-triketones. Soil samples were also collected from a non-treated corn field (not exposed control). Analytical chemistry was applied to all samples to search for β-triketone residues and to estimate the scenario of exposure of soil microorganisms. Nucleic acids (DNA/RNA) were extracted from soil samples to measure the abundance (quantitative PCR), the expression (quantitative RT-qPCR), the composition (α-diversity) and the diversity (β-diversity) (NGS) of the hppd bacterial community. Our results will be presented to the audience with the aim to identify the better proxy of the hppd bacterial community that could be used as a biomarker to reflect the exposure of soil microbial community to β-triketone residues.This explanatory work might be extended to other pesticides targeting other enzymes that are also present in so call non-target organisms such as sulfonylureas inhibiting acetohydroxy acid synthase (AHAS)