Loss of a major toxin gene cluster defines a metabolic schism and host-specific virulence in Botrytis pseudocinerea

Abstract Botrytis pseudocinerea is a cryptic fungal species, morphologically indistinguishable from the notorious plant pathogen Botrytis cinerea, yet their distinct ecological behaviours suggest fundamental biological differences. This study resolves the paradox of why B. pseudocinerea, despite intrinsic resistance to the fungicide fenhexamid, often fails to dominate agricultural ecosystems. Remarkably, we demonstrate that B. pseudocinerea VD165 exhibits superior vegetative growth and stress tolerance compared to B. cinerea B05.10, coupled with heightened virulence on solanaceous hosts like tomato and tobacco. A comprehensive bio-guided chemical investigation combined with targeted gene expression analysis reveals a fundamental schism in its secondary metabolism. The VD165 isolate of B. pseudocinerea produces a potent phytotoxic cocktail of botcinin polyketides, a strategy supported by the constitutive and infection-induced strong upregulation of the Bcboa6 and Bcboa9 biosynthetic genes. Critically, we establish that it has completely lost the botrydial sesquiterpene pathway, a primary virulence factor in B. cinerea. The significant accumulation of the upstream terpene precursor mevalonolactone provides definitive biochemical evidence for this truncated pathway. This metabolic switch—the evolutionary loss of one major toxin gene cluster and the compensatory upregulation of another—mirrors the co-regulatory mechanism previously demonstrated through genetic knockout of the botrydial pathway in B. cinerea and is a pivotal event that has shaped the unique pathogenic identity of B. pseudocinerea. This finding provides a model for how loss-of-function events in secondary metabolism can redefine host specificity and virulence in fungal pathogens—an evolutionary principle applicable across microbial taxa. Author Summary In our study, we investigated a biological puzzle. We focused on Botrytis pseudocinerea, a “cryptic” fungus that looks identical to the common grey mould pathogen, Botrytis cinerea. This ’hidden’ species is resistant to a major fungicide, yet paradoxically, it often fails to outcompete its fungicide-susceptible sibling in treated agricultural fields. We wanted to know why. We discovered that B. pseudocinerea has undergone a major evolutionary “trade-off”. During its evolution, it completely lost the gene cluster for botrydial, a primary toxin used by B. cinerea to infect plants like grapes. To compensate, its genome has permanently “super-charged” the production of a different chemical cocktail—a potent family of toxins called botcinins. This metabolic switch defines its identity: it is less effective on hosts like grapes, but has become a hyper-virulent specialist on other hosts, like tomato and tobacco. Our work provides a clear model of how “evolution by subtraction”—losing a key function—can be a powerful force in creating a new, specialized pathogen, solving an ecological mystery in the process. Competing Interest Statement The authors have declared no competing interest.