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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 has been extensively studied in Saccharomyces cerevisiae and Candida albicans, but also plant pathogenic fungi are concerned by this phenomenon. In this study, we confirmed a drug efflux based resistance mechanism in two isolates of the septoria leaf blotch agent Mycosphaerella graminicola. Using next generation sequencing approaches, we could demonstrate that the MgMFS1 gene encoding a membrane transporter of the major-facilitatorsuperfamily was strongly overexpressed in both isolates. The mutation(s) responsible for the MDR phenotype was/were mapped by a bulk-progeny-sequencing approach to a 38.5 kb genomic fragment on chromosome VII, covering also the MgMFS1 gene. Inactivation of the MgMFS1 gene the MDR6 strain abolished the MDR phenotype showing the involvement of this transporter in drug resistance. However fine mapping is necessary to identify the mdr mutation(s) leading to MgMFS1 overexpression.

Antifungal compounds such as ergosterol biosynthesis inhibitors are widely used to control crop diseases. Among them, one of the most recent, the hydroxyanilide fenhexamid, is efficient principally against Botrytis cinerea, the major causal agent of grey mould. Fenhexamid is a new type of ergosterol biosynthesis inhibitor affecting the sterol C4 demethylation processes due to its specific interaction with one of the four proteins of the enzymatic complex, the 3-ketoreductase. Our regular monitoring conducted on French vineyards allowed the identification of the first isolates of B. cinerea with acquired resistance. Two types of resistant isolates named HydR3- and HydR3+ were distinguished by their resistance level. This acquired resistance is due to point mutations in the erg27 gene leading to target modifications. These modifications induce a reduced in affinity of fenhexamid towards its target, the 3-ketoreductase. Because of their high resistant level, the HydR3+ strains have to be considered relative to the risk of resistance phenomenom occurrence in vineyards. Fitness studies conducted in vitro on isogenic mutants showed altered "overwintering" capacities of HydR3+ mutants suggesting that they probably do not impact fenhexamid's field efficacy. While B. cinerea's acquired resistance could be explained only by target modifications, as in most cases of fungicide resistance, the situation is different for the related species Botrytis pseudocinerea naturally resistant to fenhexamid. We show that erg27 polymorphism only slightly contributes to resistance whereas fenhexamid detoxification by a cytochrome P450 named cyp68.4 is the major mechanism responsible for the resistance. This is the first case of a functional validation of fungicide detoxification involved in resistance.

Oral - Session 1 : Interactions Moléculaires; absent

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National audience; Due to the development of resistant srains, the practical control of Botrytis cinerea remains a problem. Little is known about its respiratory chain and this paper describes the preparation and the properties of B cinerea mitochondria with the aim of testing new antifungal compounds. NADH, succinate and α-ketoglutarate as substrates provided good oxygen consumption. Respiratory control ratios were high and showed that electron transport was well coupled to oxidative phosphorylation. The phosphorylation efficiencies (ADP/O) obtained with all 3 substrates were compatible with theoretical values. The addition of cytochrome c did not greatly enhance the oxygen consumption due to ascorbate: the quality of the outer mitochondrial membrane was good enough to prevent externally added cytochrome c from interacting easily with the respiratory chain. These observations indicate that the mitochondria isolated sustain a high level of structural and functional integrity. This study showed also that they were devoid of the alternative pathway resistant to antimycin A and cyanide, but that they exhibited a rotenone-insensitive respiratory route. The action and the effective doses of oligomycin, carbonylcyanide m-chlorophenylhydrazone (CCCP) and carboxyatractyloside on B cinerea mitochondria were similar to those observed with plant mitochondria. At 10 μM, carboxin completely inhibited succinate oxidation in mitochondria from B cinerea but failed to have any effect on NADH and α-ketoglutarate oxidation. This toxicity level was comparable to that reported by other authors with members of the fungal class Basidiomycetes.; Du fait de l’apparition de phénomènes de résistance aux produits anti-Botrytis, la lutte contre la pourriture grise demeure un problème. La chaîne respiratoire de B cinerea, dont on connaît peu de chose, pourrait constituer une cible intéressante pour découvrir des produits de remplacement. Cet article décrit une méthode simple pour préparer des mitochondries de B cinerea et précise leurs caractéristiques. Le NADH, le succinate et l’α-cétoglutarate sont des substrats qui stimulent fortement la consommation d’oxygène. Les contrôles respiratoires sont élevés et prouvent que le transport des électrons est bien couplé à la phosphorylation oxydative. Les rapports ADP/O obtenus avec les 3 substrats sont compatibles avec les valeurs théoriques. L’addition de cytochrome c n’augmente pas notablement la consommation en oxygène provoquée par l’ascorbate : la qualité de la membrane externe des mitochondries est suffisante pour empêcher le cytochrome c de réagir sur la chaîne respiratoire. Ces observations montrent que les mitochondries isolées présentent une bonne intégrité tant sur le plan structural que fonctionnel. Cette étude montre aussi qu’elles ne possèdent pas une voie alternative résistante à l’antimycine A et au cyanure mais présentent une voie insensible à la roténone. Les effets et les niveaux d’activité de l’oligomycine, du carbonylcyanide m-chlorophénylhydrazone (CCCP) et du carboxyatractyloside sur les mitochondries de B cinerea ont été similaires à ceux généralement observés avec les mitochondries végétales. A 10 μmol.l-1, la carboxine inhibe complètement l’oxydation du succinate par les mitochondries de B cinerea mais n’a aucun effet sur les oxydations du NADH et de l’α-cétoglutarate. Ce niveau de toxicité est comparable à celui relevé par d’autres auteurs avec des champignons appartenant à la classe des Basidiomycètes.