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BACKGROUNDMonitoring resistance to Plant Protection Products (PPPs) is crucial for understanding the evolution of resistances in bioagressors, thereby allowing scientists to design sound bioagressor management strategies. Globally, resistance monitoring is implemented by a wide range of actors that fall into three distinct categories: academic, governmental, and private. The purpose of this study was to investigate worldwide diversity in PPP resistance monitoring systems, and to shed light on their different facets.RESULTSA large survey involving 162 experts from 48 countries made it possible to identify and analyze 250 resistance monitoring systems. Through an in-depth analysis, the features of the different monitoring systems were identified. The main factor differentiating monitoring systems was essentially the capabilities (funding, manpower, technology, etc.) of the actors involved in each system. In most countries, and especially in those with a high Human Development Index, academic, governmental, and private monitoring systems coexist. Overall, systems focus far more on monitoring established resistances than on the detection of emerging resistances. Governmental and private resistance monitoring systems generally have considerable capacities to generate data, whereas academic resistance monitoring systems are more specialized. Governmental actors federate and enroll a wider variety of stakeholders.CONCLUSIONThe results show functional complementarities between the coexisting actors in countries where they coexist. We suggest PPP resistance monitoring might be enhanced if the different actors focus more on detecting emerging resistances (and associated benefits) and increase collaborative and collective efforts and transparency.

CONTEXTE - La sélection de génotypes debioagresseurs résistantsconstitue un des effetsnon intentionnels del’utilisation des produitsde protection des plantes(PPP). Le suivi et la gestion de ces génotypesreprésentent un enjeuagronomique majeurafin de préserver durablement l’efficacité des PPP.PRINCIPE DE GESTION - La résistance estle résultat de l’adaptation des bioagresseurs àla pression de sélectionexercée par les pratiquesde contrôle. Les élémentsde compréhension présentés dans cet articles’appliquent à tous lesPPP, qu’ils soient d’originesynthétique ou d’originenaturelle. Notre objectifest de sensibiliser leslecteurs à la nécessité dediversifier les leviers deprotection des cultureset de promouvoir des« bonnes pratiques » enphase avec la transitionagroécologique.

Avant d'entamer le dossier, voici un petit glossaire à l'usage des gestionnaires de la résistance aux produits phytopharmaceutiques.La résistance aux PPP est un phénomène souvent complexe, faisant appel à des notions de biochimie, génétique, sciences de l’évolution. De ce fait, pour apprécier au mieux les articles de ce dossier, il apparaît utile d’avoir sous la main un mémento donnant une définition scientifique des termes les plus courants.

National audience

Réseau de Réflexion et de Recherche sur la Résistance aux Pesticides (R4P) : www.r4p-inra.frCe site met à disposition des outils et des informations sur la résistance aux produits de protection des plantes. Ces informations peuvent intéresser les professionnels impliqués dans la prévention et la gestion des résistances, des enseignants ou étudiants, et quiconque intéressé par cette thématique.Ring for Research and Reflection on Resistance to Pesticides (R4P) :www.r4p-inra.frThis site provides tools and information on resistance to plant protection products. This information may be of interest to professionals involved in the prevention and management of resistance, teachers or students, and anyone interested in this topic.

Botrytis cinerea is a plant pathogen that exhibits prominent light responses including the formation of the reproduction structures (photomorphogenesis), secondary metabolites/ pigments, and antioxidant enzymes. A complex regulatory network of photoreceptors, transcription factors (TFs) and chromatin modifiers is supposed to initiate, transmit, and fine-tune the responses to different wavelengths of light on the transcriptional level that finally leads to the observable phenotypes. As the formation of the reproduction structures is strictly regulated by light in this fungus - conidia are formed in the light, sclerotia in the dark - the output can be easily monitored. The GATA-type TFs BcWCL1 (as part of the White Collar complex (WCC)) and BcLTF1 are important regulators as their deletions result in light-independent conidiation (“always conidia”) due to the deregulation of BcLTF2 (Schumacher et al. 2014; Canessa et al. 2013; Cohrs et al. 2016). Study of light-induced gene (LIG) expression in both deletion mutants highlighted the role of the TFs in activating EARLY and in repressing LATE LIG expression, including bcltf2 encoding the master regulator of conidiation and further LTFs that may function downstream or in parallel with BcLTF2 in regulating the conidiation process. As the group of LIGs also contained genes that are induced by various stresses in a BcSAK1-dependent manner, the phosphorylation status of the stress-activated MAPK BcSAK1 was studied and shown to increase after exposure to light. Deletion of bcsak1 impairs LIG expression suggesting that BcSAK1 functions as a co-activator of the WCC in inducing EARLY genes, and as a component releasing the LATE promoters (e.g. bcltf2) from repression by BcLTF1 and the WCC resulting in the expression of the conidiation genes. This preliminary model is in agreement with the observations that the deletion of BcSAK1 results in a “never conidia” phenotype (Segmüller et al. 2007), and that the exposure of the wild type to heat and osmotic stress bypasses the requirement of light for conidiation.

Botrytis cinerea is a necrotrophic, polyphageous plant pathogen, that causes gray mold disease and can infect over 1000 plant species including several agronomically important crops (grapevine, strawberry, tomatoes …). Fungicides remain the most effective means to combat this disease. However B. cinerea rapidly adapts to fungicides. Presently, the phenylpyrrole fludioxonil is one of the most efficient fungicides against B. cinerea. Therefore deciphering the response to fludioxonil in B. cinerea is crucial. Fungi rapidly adapt to their environment involving to signalling pathways like those of mitogen activated protein kinases (MAPKs). In Botrytis cinerea, the fungicide fludioxonil activates the Sak1 and Bmp3 MAPKs, which are respectively involved in osmoregulation, cell wall integrity, development and pathogenicity. In order to trace the transduction of fludioxonil to the MAPK pathways, we have started a phosphoproteomic approach and the functional analysis of key genes. A phosphoproteomic pilot project of the wild-type strain subjected to fludioxonil, lead to the identification of “fludioxonil” candidate signalling proteins such as the phosducin-like protein PhnA. In eukaryotes, phosducin allows the dimerization between the subunits Gβ and Gγ in the G-protein signalling pathway. In the phytopathogenic fungi Cryphonectria parasitica and Fusarium graminearum, phosducin is involved in pigmentation, sporulation, and pathogenicity while in Aspergillus nidulans this protein is involved in growth, development and mycotoxin production. We have initiated the functional analysis of phnA in B. cinerea. Its deletion revealed that phnA is involved in vegetative growth, pathogenicity, and in development. This functional analysis of phnA highlighted it as a new pathogenicity factor in B. cinerea. The role of PhnA-phosphorylation for its activity and the link to fludioxonil, G-protein, and cAMP signalling is under investigation by site-directed mutagenesis of the identified phosphorylation site.

Adaptation to changing environmental conditions takes place rapidly in all living organisms: cells must respond to a wide variety of signals. Through successive protein phosphorylation and de-phosphorylation steps, signal transduction cascades mediate between the perception of the extracellular conditions and the adaptation of intracellular processes. Botrytis cinerea is a necrotrophic, polyphageous plant pathogen, that causes gray mold disease and can infect over 1000 plant species including several agronomically important crops (grapevine, strawberry, tomatoes …). Fungicides remain the most effective means to combat this disease. However B. cinerea rapidly adapts to fungicides. Presently, the phenylpyrrole fludioxonil is one of the most efficient fungicides against B. cinerea. Therefore deciphering the response to fludioxonil in B. cinerea is crucial. In Botrytis cinerea, the fungicide fludioxonil activates the Sak1 (Hog1-like) and Bmp3 (Slt2-like) MAPKs, which are respectively involved in osmoregulation, cell wall integrity, development and pathogenicity. In order to trace the transduction of fludioxonil to the MAPK pathways, we have undertaken a comparative proteomics and phosphoproteomics study of the B. cinerea osmosensing pathway challenged by the fungicide fludioxonil. We compared mutants of the sensor histidine-kinase Bos1 and of the MAPK Sak1 to the parental wild-type. Strains were exposed (or not) to fludioxonil for 15 min during exponential in vitro growth. Shotgun proteomics revealed considerable differences in protein content among the strains, but no treatment effect. These results indicate a strong transcriptional and/or translational regulation under Bos1 and Sak1 control, respectively, under standard conditions. One of the most relevant result is the abundance of 263 proteins controlled by Bos1 and Sak1 independently. Proteins of the oxidative stress response are under negative control of Bos1, while some proteases are positively regulated. In addition, proteins involved in translation are specifically regulated by Bos1. It appears that Bos1 is important in different biological processes. Sak1, on its turn, controls the abundance of proteins involved in oxidative stress response, early secretome, protein folding, primary metabolism and secondary metabolism. Quantitative phosphoproteomics is ongoing and it will permit to verify a clear response to the fludioxonil treatment as well as (de)phosphorylation events controlled independently by Bos1 or Sak1, or by both kinases

SPEGESTAD INRA; Organisateurs du congrès. Journées d’Échanges sur les Résistances aux Produits de Protection des Plantes 2017

Evolved resistance to fungicides is a major problem limiting our ability to control agricultural, medical and veterinary pathogens and is frequently associated with substitutions in the amino acid sequence of the target protein. The convention for describing amino-acid substitutions is to cite the wild type amino acid, the codon number and the new amino acid, using the one letter amino acid code. It has frequently been observed that orthologous amino acid mutations have been selected in different species by fungicides from the same mode of action class, but the amino acids have different numbers. These differences in numbering arise from the different lengths of the proteins in each species. The purpose of the current paper is to propose a system for unifying the labelling of amino acids in fungicide target proteins. To do this we have produced alignments between fungicide target proteins of relevant species fitted to a well-studied “archetype” species. Orthologous amino acids in all species are then assigned numerical “labels” based on the position of the amino acid in the archetype protein.

National audience; Multidrug resistance (MDR) is a common trait developed by many organisms to counteract chemicals and/or drugs used against them. The basic MDR mechanism is relying on an overexpressed efflux transport system that actively expulses the toxic agent outside the cell. In fungi, MDR (or PDR) has been extensively studied in Saccharomyces cerevisiae and Candida albicans. Plant pathogenic fungi are also concerned by this phenomenon. MDR strains were detected in septoria leaf blotch (Zymoseptoria tritici) field populations since 2008. These strains are cross-resistant to fungicides with different modes of action due to active fungicide efflux. In a previous study, we identified the MFS1 gene overexpressed in all tested MDR field strains (1). This gene encodes a major facilitator membrane transporter whose inactivation abolished the MDR phenotype in two resistant isolates (MDR6 and MDR7). To identify the mutation(s) responsible for MDR phenotype we applied bulkprogeny sequencing to crosses involving MDR6 and MDR7 strains. This analysis enabled us to identify a 519 bp insert in the MFS1 promoter in both strains. The insert, a reminiscence of a recent retrotransposition event, is responsible for MFS1 overexpression and the MDR phenotype. Genotyping of various field strains revealed that at least one additional mutation is responsible for the MDR phenotype.

National audience; Fungi rapidly adapt to their environment involving signalling pathways, e.g. those under the control of mitogen activated protein kinases (MAPKs). The fungicide fludioxonil, one of the most efficient fungicides especially against the grey mould agent, Botrytis cinerea, activates the MAPKs involved in osmoregulation, cell wall integrity, development and pathogenicity in this necrotrophic pathogenic fungus. In order to understand the perception and transduction of the fludioxonil signal and, more globally, the stress signalling pathway, a comparative phosphoproteomic analysis using fludioxonil was performed. In a preliminary study approximately hundred differentially phosphorylated proteins were identified in B. cinerea. Among those the phosducin-like protein protein PhnA. Since phosducins are involved in the G-protein signalling pathway, we wanted to understand the involvement of PhnA in fludioxonil signalling and the link to the MAPKs. We have therefore started its functional analysis by gene knock-out. The phnA deletion mutant did not produce necrosis in planta, and displayed reduced in vitro growth. Moreover it has strongly reduced conidia and sclerotia production. Thus phnA is involved in vegetative growth, in development and is a new pathogenicity factor in B. cinerea. We have identified serine 66 in the PhnA protein to be phosphorylated after exposure to fludioxonil. Through the construction of PhnAS66A and PhnAS66D mutants we are currently investigating the role of the phosphorylation status in PhnA function. In addition, the transduction of the fludioxonil signal to the MAPKs Sak1 and Bmp3 will be analysed..

National audience

National audience; The Botrytis species complex responsible for grey mould disease on grapevine is composed of two species: Botrytis cinerea the major one (about 90%) and Botrytis pseudocinerea. Despite their genetic polymorphism, these species cannot be morphologically distinguished. However, they do differ in their response to several fungicides, especially to the sterol biosynthesis inhibitor fenhexamid. While B. cinerea is sensitive to this hydroxyanilide, B. pseudocinerea is naturally resistant. Enzyme assays showed that in B. pseudocinerea the fenhexamid target enzyme, the sterol 3-ketoreductase was less sensitive to fenhexamid. In addition, a synergic effect between fenhexamid and sterol 14Α- demethylation inhibitors (DMIs) known to inhibit Cyp51, a cytochrome P450 monooxygenase was observed in B. pseudocinerea. These results could suggest detoxification of fenhexamid by cytochromes P450. The cyp684 gene showing the strongest similarity to cyp51 among all B. cinerea cytochrome P450 genes was found strongly overexpressed in the presence of fenhexamid in B. pseudocinerea. In this work, we studied separately the effect of B. pseudocinerea erg27 polymorphism, erg27 encoding 3-ketoreductase, and of the recently identified cytochrome P450 gene, cyp684, on resistance to fenhexamid. The objective is to determine their respective implication in resistance. Experiments were conducted by exchange between erg27B.cinerea and erg27B.pseudocinerea in B. cinerea and by cyp684 deletion in B. pseudocinerea. In parallel, metabolization studies are conducted to identify metabolites and test their activity on Botrytis spp.

National audience; La résistance multidrogues (MDR) est en progression dans les populations françaises de Z. tritici, depuis sa première détection en 2008. Ce mécanisme original est lié à un efflux accru de fongicides, induit par la surexpression du transporteur membranaire MgMFS1. Le phénotype MDR est déterminée par une insertion de 519 bp dans la région promotrice de MgMFS1, mais également, pour certaines souches, par au moins un autre mécanisme, à identifier. Le mécanisme MDR, associé à des modifications de cible de fongicides, notamment Cyp51 (cible des triazoles), confère de forts niveaux de résistance croisée. Ces résultats posent plus généralement la question de l’efficacité résiduelle des différentes spécialités fongicides si les souches MDR venaient à se généraliser.

International audience; The Botrytis species complex responsible for grey mould disease on grapevine is composed of two species: Botrytis cinerea the major one (about 90%) and Botrytis pseudocinerea. Despite their genetic polymorphism, these species cannot be morphologically distinguished. However, they do differ in their response to several fungicides, especially to the sterol biosynthesis inhibitor fenhexamid. While B. cinerea is sensitive to this hydroxyanilide, B. pseudocinerea is naturally resistant. Enzyme assays showed that in B. pseudocinerea the fenhexamid target enzyme, the sterol 3-ketoreductase was less sensitive to fenhexamid. In addition, a strong synergism between fenhexamid and sterol 14α-demethylation inhibitors (DMIs) known to inhibit Cyp51, a cytochrome P450 monooxygenase was observed in B. pseudocinerea. These results could suggest detoxification of fenhexamid by cytochromes P450. The cyp684 gene showing the strongest similarity to cyp51 among all B. cinerea cytochrome P450 genes was found strongly overexpressed in the presence of fenhexamid in B. pseudocinerea. In this work, we studied separately the effect of B. pseudocinerea erg27 polymorphism, erg27 encoding 3-ketoreductase, and of the recently identified cytochrome P450 gene, cyp684, on resistance to fenhexamid, respectively by erg27 gene, and cyp684 inactivation. In parallel, metabolization studies are conducted to identify metabolites and test their activity on Botrytis spp.