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

Abstract

15 Little is known about the mechanisms causing loss of virulence in pathogenic fungi as a 16 result of protracted culture. We studied the extent to which patterns of DNA methylation 17 varied between virulent and reduced virulence derivative cultures of Botrytis cinerea, 18 and identify the genes/genomic regions affected by these epigenetic modifications. 19  B. cinerea was cultured in vitro for eight months involving subculture every four weeks. 20 Fungal conidia were harvested at every four-week subculturing stage and inoculated onto 21 Arabidopsis thaliana Col-0 plants for virulence testing. Global epi/genetic changes in B. 22 cinerea during culture were assessed using methylation-sensitive amplified 23 polymorphisms (MSAPs) on mycelium from eight different sub-culture time points and 24 from mycelium recovered after eight months in culture and then inoculated onto A. 25 thaliana. Culture induced epi/allele characterisation was carried out by whole genome 26 sequencing and bisulfite sequencing of gDNA from samples after two and eight months 27 in culture and after 8 months in culture and following inoculation onto an A. thaliana 28 plant. 29 Virulence declined with time in culture and recovered after one fungal generation on A. 30 thaliana. MSAP data show that epi/genetic variation followed virulence changes during 31 culture. Whole genome sequencing showed no significant genetic changes during 32 culture. Conversely, bisulfite sequencing showed significant changes both on global and 33 local methylation patterns. 34 We suggest that virulence is a non-essential plastic character regulated by DNA 35 methylation during protracted in vitro culture. We propose DNA methylation as a 36 regulator of the high virulence/low virulence transition in B. cinerea and as a potential 37 mechanism to control pathogenicity. 38 39 40Botrytis cinerea is an ascomycete responsible for grey mould on hundreds of dicot plants that 41 is able to feed on different plant tissues (1) and causes annually up to $100 billion in losses 42 worldwide (2). The wide variety of symptoms on different organs and plant species may 43 suggest that B. cinerea has a large 'arsenal of weapons' to attack its host plants. B. cinerea is 44 well documented as a capable saprotroph and necrotroph with genetic types showing a trade-45 off between saprotrophic and necrotrophic capabilities (3). As other pathogens, B. cinerea 46 undergoes transcriptional and developmental regulation to govern the outcome of 47 pathogen/host interactions. Population dynamics between the two types have been linked to 48 resource availability. Interestingly, chances in virulence levels have also been observed 49 during protracted in vitro culture of B. cinerea (4). In fact, pathogenic fungi are notorious for 50 losing virulence when successively subcultured in vitro. Degenerate cultures have been 51 reported in a wide range of pathogenic fungi (5) but very little is known about why the 52 cultures degenerate. Different factors have been described as poss...

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Successive passaging through an apple host of six low-virulent <i>Neonectria ditissima </i>isolates increased virulence in one of them

“…Reversible epigenetic modifications modulating virulence have been observed following protracted in vitro culturing of Botrytis cinerea. In that study, loss of virulence, a common trait in many fungi during repeated sub-culturing, was accompanied by changes in global and local DNA methylation (Breen et al 2016). Since isolate ICMP14098 has been in culture for many years, it is possible that the original virulence of this isolates was lost in a similar way.…”

“…Since isolate ICMP14098 has been in culture for many years, it is possible that the original virulence of this isolates was lost in a similar way. In B. cinerea, virulence could be reinstated by inoculating the plant host Arabidopsis thaliana, reversing the epigenetic modifications (Breen et al 2016). A similar mechanism may be occurring in ICMP14098.…”

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“…Moreover, the pathogens with the greatest economic impact are fungi that cause serious damage or death (Fedele et al, 2020 andNotte et al, 2021). Among the fungal pathogens, we mention Botrytis cinerea which represents the major pathogen of strawberries worldwide (Breen et al, 2022).…”