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Botrytis cinerea is a necrotrophic fungus that causes considerable economic losses in commercial crops. Fungi of the genus Botrytis exhibit great morphological and genetic variability, ranging from non-sporogenic and non-infective isolates to highly virulent sporogenic ones. There is growing interest in the different isolates in terms of their methodological applications aimed at gaining a deeper understanding of the biology of these fungal species for more efficient control of the infections they cause. This article describes an improvement in the protoplast production protocol from non-sporogenic isolates, resulting in viable protoplasts with regenerating capacity. The method improvements consist of a two-day incubation period with mycelium plugs and orbital shaking. Special mention is made of our preference for the VinoTaste Pro enzyme in the KC buffer as a replacement for Glucanex, as it enhances the efficacy of protoplast isolation in B459 and B371 isolates. The methodology described here has proven to be very useful for biotechnological applications such as genetic transformations mediated by the CRISPR/Cas9 tool.

Botrytis cinerea, a necrotrophic fungus responsible for grey rot disease, causes substantial economic losses. However, recent studies have discovered distinct non-sporogenic and non-infective isolates of this species, such as the B459 field strain. Examination of these isolates is particularly intriguing in the context of the development of methodological applications that could be useful in the biocontrol of this phytopathogenic species. This investigation revealed that B459 exhibited a reduced growth rate yet displayed enhanced resilience to stressors like oxidative stress agents, SDS, ethanol, and PhITC. Notably, its ability to generate reactive oxygen species (ROS) and acidic compounds, crucial in plant–pathogen interactions, was impacted. Bio-targeted toxin identification assays and the metabolomic study of extracts obtained from fermentations at seven and fourteen days revealed that this strain does not biosynthesize botrydial and derivatives neither botcinin family toxins. Furthermore, its inability to infect tomato fruits, grape, and gerbera petals coincides with its lack of toxin production under culture conditions typically adapted for reference strain B05.10.

The genome sequencing of Botrytis cinerea supplies a general overview of the map of genes involved in secondary metabolite synthesis. B. cinerea genomic data reveals that this phytopathogenic fungus has seven sesquiterpene cyclase (Bcstc) genes that encode proteins involved in the farnesyl diphosphate cyclization. Three sesquiterpene cyclases (BcStc1, BcStc5 and BcStc7) are characterized, related to the biosynthesis of botrydial, abscisic acid and (+)-4-epi-eremophilenol, respectively. However, the role of the other four sesquiterpene cyclases (BcStc2, BcStc3, BcStc4 and BcStc6) remains unknown. BcStc3 is a well-conserved protein with homologues in many fungal species, and here, we undertake its functional characterization in the lifecycle of the fungus. A null mutant ΔBcstc3 and an overexpressed–Bcstc3 transformant (OvBcstc3) are generated, and both strains show the deregulation of those other sesquiterpene cyclase-encoding genes (Bcstc1, Bcstc5 and Bcstc7). These results suggest a co-regulation of the expression of the sesquiterpene cyclase gene family in B. cinerea. The phenotypic characterization of both transformants reveals that BcStc3 is involved in oxidative stress tolerance, the production of reactive oxygen species and virulence. The metabolomic analysis allows the isolation of characteristic polyketides and eremophilenols from the secondary metabolism of B. cinerea, although no sesquiterpenes different from those already described are identified.

Glucosinolates, the main secondary metabolites accumulated in cruciferous flora, have a major impact on fortifying plant immunity against diverse pathogens. Although Botrytis cinerea exhibits varying sensitivity to these compounds, current research has yet to fully understand the intricate mechanisms governing its response to glucosinolates. Different species of the genus Botrytis were exposed to glucosinolate-derived isothiocyanates, revealing that B. fabae, B. deweyae, and B. convolute, species with the mfsG transporter gene (Bcin06g00026) not detected with PCR, were more sensitive to isothiocyanates than Botrytis species containing that gene, such as B. cinerea, B. pseudocinerea, and B. byssoidea. This finding was further corroborated by the inability of species with the mfsG gene not detected with PCR to infect plants with a high concentration of glucosinolate-derived isothiocyanates. These results challenge established correlations, revealing varying aggressiveness on different plant substrates. An expression analysis highlighted the gene’s induction in the presence of isothiocyanate, and a bioinformatic investigation identified homologous genes in other Botrytis species. Our study underscored the importance of advanced biotechnology to help understand these proteins and thus offer innovative solutions for agriculture.