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.

A link between host plant adaptation and pesticide resistance in the polyphagous spider mite Tetranychus urticae

“…MFS transporters are single-polypeptide carriers that work in symport/antiport (79,80), and studies in bacteria and fungi have identified roles in the transport of toxic substances (81)(82)(83). For example, overexpression of the mfsM2 gene in a sensitive strain of Botrytis cinerea, a fungal plant pathogen, led to drug-resistance levels similar to those of a fungicide-resistant B. cinerea strain (82). If spider mite MFS proteins function as efflux transporters, their up-regulation might result in a higher efflux of acaricides/ toxic allelochemicals or their metabolites out of spider mite cells.…”

Expansive Phenotypic Landscape of Botrytis cinerea Shows Differential Contribution of Genetic Diversity and Plasticity

“…Further, B. cinerea is known to have a large amount of standing genetic variation (Aguileta et al 2012;Alfonso et al 2000;Atwell et al 2015;Baraldi et al 2002;Fournier and Giraud 2008;Fournier et al 2002;Giraud et al 1997Giraud et al , 1999Ma and Michailides 2005;Munoz et al 2002;Rowe and Kliebenstein 2007;Staats and van Kan 2012). Several studies have shown that this genetic diversity within B. cinerea not only contributes to differential virulence in planta (Calpas et al 2006;Denby et al 2004;Rowe and Kliebenstein 2008;Schumacher et al 2012) but can also contribute to the development of fungicide resistant genotypes (Kretschmer et al 2009). In addition, recent work has identified natural allelic variants in B. cinerea that control the formation of sclerotia in response to UV light (Schumacher et al 2012).…”

Development of the novel fungicide fenpyrazamine

“…4 were approximately 8 and 3, respectively. 8) In this study, the resistance factors of MDR1 and MDR2 isolates to Ref. 4 were found to be 7.0 and 4.4, respectively.…”