Most plant fungal pathogens that cause worldwide crop losses are understudied, due to various technical challenges. With the increasing availability of sequenced whole genomes of these non-model fungi, effective genetic analysis methods are highly desirable. Here, we describe a newly developed pipeline, which combines forward genetic screening with high-throughput next-generation sequencing to enable quick gene discovery. We applied this pipeline in the notorious soilborne phytopathogen Sclerotinia sclerotiorum and identified 32 mutants with various developmental and growth deficiencies. Detailed molecular studies of three melanization-deficient mutants provide a proof of concept for the effectiveness of our method. A master transcription factor was found to regulate melanization of sclerotia through the DHN (1,8-dihydroxynaphthalene) melanin biosynthesis pathway. In addition, these mutants revealed that sclerotial melanization is important for sclerotia survival under abiotic stresses, sclerotial surface structure, and sexual reproduction. Foreseeably, this pipeline can be applied to facilitate efficient in-depth studies of other non-model fungal species in the future.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.
Keywords: DHN melanin; Sclerotinia sclerotiorum; SsSMR1; forward genetic analysis; next-generation sequencing; sclerotial formation; soilborne phytopathogen.