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Background  The necrotrophic fungal pathogen  Pyrenophora tritici-repentis  ( Ptr ) causes tan (syn. yellow) spot of wheat and accounts for significant yield losses worldwide. Understanding the molecular mechanisms of this economically important crop disease is crucial to counteract the yield and quality losses of wheat globally. Substantial progress has been made to comprehend the race structure of this phytopathogen based on its production of necrotrophic effectors and genomic resources of  Ptr.  However, one limitation for studying  Ptr  in a laboratory environment is the difficulty to isolate high spore numbers from vegetative growth with mycelial contamination common. These limitations reduce the experimental tractability of  Ptr .    Results  Here, we optimized a multitude of parameters and report a sporulation method for  Ptr  that yields robust, high quality and pure spores. Our methodology encompasses simple and reproducible plugging and harvesting techniques, resulting in spore yields up to 1500 fold more than the current sporulation methods and was tested on multiple isolates and races of  Ptr  as well as an additional seven modern Australian  Ptr  isolates. Moreover, this method also increased purity and spore harvest numbers for two closely related fungal pathogens ( Pyrenophora teres  f.  maculata  and f.  teres ) that cause net blotch diseases in barley ( Hordeum vulgare ), highlighting the usability of this optimized sporulation protocol for the wider research community.    Conclusions  Large-scale spore infection and virulence assays are essential for the screening of wheat and barley cultivars and combined with the genetic mapping of these populations allows pinpointing and exploiting sources of host genetic resistance. We anticipate that improvements in spore numbers and purity will further advance research to increase our understanding of the pathogenicity mechanisms of these important fungal pathogens.    Supplementary Information  The online version contains supplementary material available at 10.1186/s13007-021-00751-4.

An optimized sporulation method for the wheat fungal pathogen Pyrenophora tritici-repentis