Development of a Protoplast‐Mediated Transformation System for <i>Monilinia fructicola</i> : A Tool for Functional Genomics

ABSTRACT Monilinia fructicola is a major causal agent of brown rot in stone fruit and is recognised as the most aggressive species within the genus Monilinia . Despite its economic impact, the molecular basis of its pathogenicity remains poorly understood, due to the lack of efficient genetic transformation tools. This study aimed to develop and optimise a robust protoplast‐mediated transformation protocol for M. fructicola , providing a foundational platform for functional genomics in this pathogen. To achieve this, different culture media and enzymatic combinations were tested to maximise protoplast yield and viability. Best results were obtained using hyphae grown in Murashige and Skoog (MS) medium supplemented with 0.1 M fructose, and enzymatic digestion with a combination of VinoTaste Pro and Yatalase, yielding up to 3 × 10 7 protoplasts g −1 of fresh mycelium. Regeneration rates reached up to 60% under optimal conditions. Competent protoplasts were transformed with a self‐propagating, unstable telomeric plasmid conferring fenhexamid resistance, achieving a transformation efficiency of 38.8%, within or above the range reported for other filamentous fungi. Upon subculturing transformants under nonselective conditions, the resistance marker was lost in fewer than 50% of cases, suggesting that plasmid persistence in M. fructicola is higher than expected. Although M. fructicola can be transformed efficiently using circular DNA, the limited rate of plasmid loss may require further optimisation to enable full utility in marker‐free genome editing strategies. This work provides an essential step forward in enabling genetic manipulation in M. fructicola , opening the door to advanced tools such as CRISPR/Cas9‐mediated genome editing.