The grey mould fungus Botrytis cinerea causes losses of commercially important fruits, vegetables and ornamentals worldwide. Fungicide treatments are effective for disease control, but bear the risk of resistance development. The major resistance mechanism in fungi is target protein modification resulting in reduced drug binding. Multiple drug resistance (MDR) caused by increased efflux activity is common in human pathogenic microbes, but rarely described for plant pathogens. Annual monitoring for fungicide resistance in field isolates from fungicide-treated vineyards in France and Germany revealed a rapidly increasing appearance of B. cinerea field populations with three distinct MDR phenotypes. All MDR strains showed increased fungicide efflux activity and overexpression of efflux transporter genes. Similar to clinical MDR isolates of Candida yeasts that are due to transcription factor mutations, all MDR1 strains were shown to harbor activating mutations in a transcription factor (Mrr1) that controls the gene encoding ABC transporter AtrB. MDR2 strains had undergone a unique rearrangement in the promoter region of the major facilitator superfamily transporter gene mfsM2, induced by insertion of a retrotransposon-derived sequence. MDR2 strains carrying the same rearranged mfsM2 allele have probably migrated from French to German wine-growing regions. The roles of atrB, mrr1 and mfsM2 were proven by the phenotypes of knock-out and overexpression mutants. As confirmed by sexual crosses, combinations of mrr1 and mfsM2 mutations lead to MDR3 strains with higher broad-spectrum resistance. An MDR3 strain was shown in field experiments to be selected against sensitive strains by fungicide treatments. Our data document for the first time the rising prevalence, spread and molecular basis of MDR populations in a major plant pathogen in agricultural environments. These populations will increase the risk of grey mould rot and hamper the effectiveness of current strategies for fungicide resistance management.

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Drug transporters are membrane proteins that provide protection for organisms against natural toxic products and fungicides. In plant pathogens, drug transporters function in baseline sensitivity to fungicides, multidrug resistance (MDR) and virulence on host plants. This paper describes drug transporters of the filamentous fungi Aspergillus nidulans (Eidam) Winter, Botrytis cinerea Pers and Mycosphaerella graminicola (Fückel) Schroter that function in fungicide sensitivity and resistance. The fungi possess ATP-binding cassette (ABC) drug transporters that mediate MDR to fungicides in laboratory mutants. Similar mutants are not pronounced in field resistance to most classes of fungicide but may play a role in resistance to azoles. MDR may also explain historical cases of resistance to aromatic hydrocarbon fungicides and dodine. In clinical situations, MDR development in Candida albicans (Robin) Berkhout mediated by ABC transporters in patients suffering from candidiasis is common after prolonged treatment with azoles. Factors that can explain this striking difference between agricultural and clinical situations are discussed. Attention is also paid to the risk of MDR development in plant pathogens in the future. Finally, the paper describes the impact of fungal drug transporters on drug discovery.

During pathogenesis, fungal pathogens are exposed to a variety of fungitoxic compounds. This may be particularly relevant to Botrytis cinerea, a plant pathogen that has a broad host range and, consequently, is subjected to exposure to many plant defense compounds. In practice, the pathogen is controlled with fungicides belonging to different chemical groups. ATP-binding cassette (ABC) transporters might provide protection against plant defense compounds and fungicides by ATP-driven efflux mechanisms. To test this hypothesis, we cloned BcatrB, an ABC transporter-encoding gene from B. cinerea. This gene encodes a 1,439 amino acid protein with nucleotide binding fold (NBF) and transmembrane (TM) domains in a [NBF-TM6]2 topology. The amino acid sequence has 31 to 67% identity with ABC transporters from various fungi. The expression of BcatrB is up regulated by treatment of B. cinerea germlings with the grapevine phytoalexin resveratrol and the fungicide fenpiclonil. BcatrB replacement mutants are not affected in saprophytic growth on different media but are more sensitive to resveratrol and fenpiclonil than the parental isolate. Furthermore, virulence of deltaBcatrB mutants on grapevine leaves was slightly reduced. These results indicate that BcatrB is a determinant in sensitivity of B. cinerea to plant defense compounds and fungicides.

Host resistance and fungicide treatments are cornerstones of plant-disease control. Here, we show that these treatments allow sex and modulate parenthood in the fungal wheat pathogen Zymoseptoria tritici. We demonstrate that the Z. tritici-wheat interaction complies with the gene-for-gene model by identifying the effector AvrStb6, which is recognized by the wheat resistance protein Stb6. Recognition triggers host resistance, thus implying removal of avirulent strains from pathogen populations. However, Z. tritici crosses on wheat show that sex occurs even with an avirulent parent, and avirulence alleles are thereby retained in subsequent populations. Crossing fungicide-sensitive and fungicide-resistant isolates under fungicide pressure results in a rapid increase in resistance-allele frequency. Isolates under selection always act as male donors, and thus disease control modulates parenthood. Modeling these observations for agricultural and natural environments reveals extended durability of host resistance and rapid emergence of fungicide resistance. Therefore, fungal sex has major implications for disease control.