A Practical Guide to Two-Stage Sporulation of Pyricularia oryzae: Method Comparison Using Strains from Diverse Grass Hosts

A Practical Guide to Two-Stage Sporulation of Pyricularia oryzae: Method Comparison Using Strains from Diverse Grass Hosts | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A Practical Guide to Two-Stage Sporulation of Pyricularia oryzae: Method Comparison Using Strains from Diverse Grass Hosts Jie-Hao Ou, Kazuyuki Okazaki, Akito Kubota, Guan-Ying Huang, Yi-Nian Chen, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7110509/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Nov, 2025 Read the published version in Plant Methods → Version 1 posted 9 You are reading this latest preprint version Abstract Background Pyricularia oryzae is a major fungal pathogen responsible for significant yield losses in rice. In recent years, diverse pathotypes have emerged as threats to other economically important grasses, including ryegrass, oats, wheat and foxtail millet. Research on host–pathogen interactions involving this species requires reliable spore production for inoculation. However, as a hemibiotrophic pathogen, P. oryzae often sporulates poorly on artificial media and typically requires specialized two-stage protocols for consistent spore production. Although several such methods have been developed, all were optimized for rice-derived strains and have not been systematically evaluated across strains from other hosts. Moreover, published protocols often lack detailed procedural information and troubleshooting guidance, posing challenges for researchers working in newly affected regions. Results We evaluated five two-stage sporulation protocols, including a novel method developed in this study. We then assessed their efficacy using 23 Pyricularia spp. strains collected from 13 Poaceae hosts, focusing on spore yield, technical feasibility and cost. The newly developed filter paper method consistently supported spore production and was compatible with cryopreservation at -40 °C, overcoming limitations associated with the short shelf life of fresh spores. Comparative analysis revealed strain-specific differences in sporulation levels across methods, with no apparent correlation with phylogenetic lineage. Step-by-step protocols with accompanying illustrations are also provided, along with practical recommendations for selecting suitable methods based on laboratory conditions, available resources, and research objectives. Conclusions This study provides a comparative evaluation of sporulation methods for P. oryzae strains from a range of grass hosts. The results serve as a practical reference for researchers conducting virulence profiling, resistance breeding, and disease management studies. These findings are particularly relevant for laboratories in regions that are currently affected by, or at risk of, outbreaks caused by this important plant pathogen. Pyricularia oryzae conidia production sporulation methods comparative protocol Poaceae virulence assay resistance screening grey leaf spot rice blast Full Text Additional Declarations No competing interests reported. Supplementary Files SuppFile1Filterpapermethod.pdf SuppFile2IRRImethod.pdf SuppFile3Mycelialmatmethod.pdf SuppFile4Corngrainmethod.pdf SuppFile5TARImethod.pdf SuppFile6SetupofSporulationEnvironment.pdf SuppFile7Strainlist.docx Cite Share Download PDF Status: Published Journal Publication published 18 Nov, 2025 Read the published version in Plant Methods → Version 1 posted Editorial decision: Revision requested 02 Oct, 2025 Reviews received at journal 02 Oct, 2025 Reviewers agreed at journal 02 Oct, 2025 Reviews received at journal 01 Sep, 2025 Reviewers agreed at journal 12 Aug, 2025 Reviewers invited by journal 15 Jul, 2025 Editor assigned by journal 14 Jul, 2025 Submission checks completed at journal 14 Jul, 2025 First submitted to journal 12 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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In recent years, diverse pathotypes have emerged as threats to other economically important grasses, including ryegrass, oats, wheat and foxtail millet. Research on host–pathogen interactions involving this species requires reliable spore production for inoculation. However, as a hemibiotrophic pathogen, \u003cem\u003eP. oryzae\u003c/em\u003e often sporulates poorly on artificial media and typically requires specialized two-stage protocols for consistent spore production. Although several such methods have been developed, all were optimized for rice-derived strains and have not been systematically evaluated across strains from other hosts. Moreover, published protocols often lack detailed procedural information and troubleshooting guidance, posing challenges for researchers working in newly affected regions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e We evaluated five two-stage sporulation protocols, including a novel method developed in this study. 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The results serve as a practical reference for researchers conducting virulence profiling, resistance breeding, and disease management studies. 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