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Genetic transformation is an essential tool for the modern study of gene function and the genetic improvement of an organism. The genetic transformation of many fungal species is well established and can be carried out by utilizing different transformation methods including electroporation, Agrobacterium, biolistics, or polyethylene glycol (PEG)-mediated transformation. Due to its technical simplicity and common equipment requirements, PEG-mediated transformation is still the most commonly used method for genetic transformation in filamentous fungi. Here, we describe a PEG-based protocol developed for genetic transformation of
Stagonospora nodorum
, a fungal pathogen of wheat. This protocol is directly applicable to other fungi especially those in the Dothideomycete class of fungi.