Fungicide-Driven Evolution and Molecular Basis of Multidrug Resistance in Field Populations of the Grey Mould Fungus Botrytis cinerea

Abstract

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.

Author Summary

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.