Botrytis cinerea loss and restoration of virulence during in vitro culture follows flux in global DNA methylation

Pathogenic fungi can lose virulence after protracted periods of culture but little is known of the underlying mechanisms. Here we present the first analysis of DNA methylation flux at a single-base resolution for the plant pathogen B. cinerea and iden-tify differentially methylated genes/genomic regions associated with virulence ero-sion during in vitro culture. Cultures were maintained for eight months with subcul-tures and virulence testing every month. Methylation-sensitive amplified polymor-phisms were performed at monthly intervals to characterise global changes to the pathogen's genome during culture and also on DNA from mycelium inoculated onto Arabidopsis thaliana after eight months in culture. Characterisation of culture-induced epialleles was assessed by whole-genome re-sequencing and whole-genome bisulfite sequencing. Virulence declined with time in culture and recovered after inoculation on A. thaliana. Variation detected by methylation-sensitive amplified polymorphisms fol-lowed virulence changes during culture. Whole-genome (bisulfite) sequencing showed marked changes on global and local methylation during culture but no sig-nificant genetic changes. We imply that virulence is a non-essential plastic character that is at least partly modified by changing levels of DNA methylation during culture. We hypothesise that changing DNA methylation during culture may be responsible for the high virulence/low virulence transition in B. cinerea and speculate that this may offer fresh opportunities to control pathogen virulence.