Abstract
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 identify differentially methylated genes/genomic regions associated with virulence erosion during in vitro culture. Cultures were maintained for eight months, with subcultures and virulence testing every month. Methylation-sensitive amplified polymorphisms 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 followed virulence changes during culture. Whole-genome (bisulfite) sequencing showed marked changes in global and local methylation during culture but no significant genetic changes. We imply that virulence is a non-essential plastic character that is at least partly modified by the 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.
Highlights
Whole-genome bisulfite sequencing (WGBS) in Ascomycetes has revealed that this group of fungi present repeated loci silenced by methylation and active genes methylated within exonic regions
In accordance with previous reports [6–9], the virulence of B. cinerea cultures progressively decreased with culture age but recovered after one passage of in vivo infection on
Concurrent with these changes, methylationsensitive amplified polymorphisms (MSAPs) profiles showed a similar progressive increase in deviation from the starter culture profiles consistent with previous reports of accumulative genetic/methylome change for other species when exposed to prolonged periods of culture [28]
Summary
We present the first analysis of DNA methylation flux at a single-base resolution for the plant pathogen B. cinerea and identify differentially methylated genes/genomic regions associated with virulence erosion during in vitro culture. Cultured fungal strains lose virulence irrespective of whether the parent culture originated from a single or multiple-spore colony [5], discounting the additional possibility of culture-induced selection favouring non-virulent strains The reversibility of this aspect of the observed phenotype comes in response to changes in the growing environment and so should be viewed as a plastic feature of the pathogen [12]. We explore possible links between the erosion of pathogenicity from B. cinerea during protracted in vitro culture and dynamics of DNA methylation across their genomes.
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