Disruption of two effector genes reveals their contribution to peach brown rot caused by <i>Monilinia fructicola</i>

Brown rot, caused by Monilinia spp., is a major disease of stone fruit, yet the molecular determinants of Monilinia fructicola pathogenicity, particularly effector-related mechanisms remain poorly defined. Here, two candidate effector genes were functionally analysed by targeted mutagenesis. Resistance cassettes (hygromycin or fenhexamid) flanked by ~1 kb homology flanks were used for protoplast-mediated transformation, and repeated single-spore isolation was applied to obtain pure transformants. PCR screening and qPCR expression analyses identified two homokaryotic knockouts of mfru030g00190 and several heterokaryotic knockdown mutants of mfru004g02710, both encoding small phytotoxic effector proteins. In vitro characterization revealed no major differences between mutants and the wild type in colony morphology, radial growth, or conidiation in different media (MEA and peach-based) and temperatures (15 °C, 25 °C), indicating that these genes are not required for vegetative development. By contrast, fruit inoculation assays on peach demonstrated that deletion of mfru030g00190 significantly reduced disease incidence, whereas mfru004g02710 knockdown mutants showed a more moderate decrease and no consistent reduction in lesion size. These outcomes support the view that necrotrophic fungi deploy redundant effector repertoires, where disruption of individual genes attenuates but does not abolish pathogenicity. This study establishes a practical workflow for generating and purifying M. fructicola mutants and provides direct evidence of the contribution of effectors to infection. The results lay a foundation for broader functional genomics in Monilinia and highlight the need to identify cognate host targets in Prunus, a key step toward leveraging effector biology for resistance breeding and durable disease-management strategies.