We assess the role of DNA methylation in regulating virulence in culture using whole-methylome analysis of virulent and reduced virulence-derived cultures of Botrytis cinerea. DNA methylation plays a role on in vitro culture-induced loss of virulence in Botrytis cinerea

Pathogenic fungi can lose virulence after protracted periods of culture but little is known of the mechanisms governing its regulation. Here we assess the role of DNA methylation in regulating virulence in culture using whole-methylome analysis of virulent and reduced virulence-derived cultures of Botrytis cinerea. Virulence of B. cinerea declined during the eight month culture period and recovered to original levels after one fungal generation on A. thaliana leaf material. A time-course of culture samples were analysed with Methylation-sensitive amplified polymorphisms (MSAPs) showing that epi/genetic variation followed virulence changes during culture. Whole-genome resequencing showed no significant genetic changes during culture, however, bisulfite sequencing showed significant changes both on global and local methylation patterns with 1,577 differentially methylated regions overlapping with functionally known genes. We suggest that virulence is a non-essential plastic character regulated by DNA methylation during protracted in vitro culture. We propose DNA methylation as a regulator of the high virulence/low virulence transition in B. cinerea and as a potential mechanism to control pathogenicity.