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. Bacterial and fungal pathogens cause diseases in humans and plants alike. Antibiotics and fungicides are used for disease control, but the microbes are able to adapt quickly to these drugs by mutation. Multiple drug resistance (MDR) is well investigated in human pathogens and causes increasing problems with antibiotic therapy. Driven by the continuous use of fungicides in commercial vineyards, three types of rapidly increasing multidrug resistant populations of the grey mould fungus Botrytis cinerea have appeared in French vineyards since the mid 1990s. Using a combination of physiological, molecular and genetic techniques, we demonstrate that these MDR phenotypes are correlated with increased drug efflux activity and overexpression of two efflux transporters. Just two types of mutations, one in a regulatory protein that controls drug efflux, and the other in the gene for an efflux transporter itself, are sufficient to explain the three MDR phenotypes. We also provide evidence that a subpopulation of the French MDR strains has migrated eastward into German wine-growing regions. We anticipate that by continuous selection of multi-resistant strains, chemical control of grey mould in the field will become increasingly difficult.

Shelf life is an important quality trait for many fruit, including tomatoes. We report that enrichment of anthocyanin, a natural pigment, in tomatoes can significantly extend shelf life. Processes late in ripening are suppressed by anthocyanin accumulation, and susceptibility to Botrytis cinerea, one of the most important postharvest pathogens, is reduced in purple tomato fruit. We show that reduced susceptibility to B. cinerea is dependent specifically on the accumulation of anthocyanins, which alter the spreading of the ROS burst during infection. The increased antioxidant capacity of purple fruit likely slows the processes of overripening. Enhancing the levels of natural antioxidants in tomato provides a novel strategy for extending shelf life by genetic engineering or conventional breeding.

The wheat intracellular immune receptor repertoire can be explored with the NLR-Annotator tool.

Summary: Arabidopsis thaliana is known to produce the phytoalexin camalexin in response to abiotic and biotic stress. Here we studied the mechanisms of tolerance to camalexin in the fungus Botrytis cinerea, a necrotrophic pathogen of A. thaliana. Exposure of B. cinerea to camalexin induces expression of BcatrB, an ABC transporter that functions in the efflux of fungitoxic compounds. B. cinerea inoculated on wild‐type A. thaliana plants yields smaller lesions than on camalexin‐deficient A. thaliana mutants. A B. cinerea strain lacking functional BcatrB is more sensitive to camalexin in vitro and less virulent on wild‐type plants, but is still fully virulent on camalexin‐deficient mutants. Pre‐treatment of A. thaliana with UV‐C leads to increased camalexin accumulation and substantial resistance to B. cinerea. UV‐C‐induced resistance was not seen in the camalexin‐deficient mutants cyp79B2/B3, cyp71A13, pad3 or pad2, and was strongly reduced in ups1. Here we demonstrate that an ABC transporter is a virulence factor that increases tolerance of the pathogen towards a phytoalexin, and the complete restoration of virulence on host plants lacking this phytoalexin.

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 ΔBcatrB 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.