Agrobacterium tumefaciens-mediated transformation and T-DNA insertion characterisation of Didymella tanaceti and Stagonosporopsis tanaceti for infection visualisation in pyrethrum.

Agrobacterium tumefaciens-mediated transformation (ATMT) has been widely used in filamentous fungi for gene functional studies and for the introduction of fluorescent markers to investigate infection biology. In pyrethrum (Tanacetum cinerariifolium), the foliar pathogens Didymella tanaceti and Stagonosporopsis tanaceti cause significant yield losses. However, the infection biology of D. tanaceti remains poorly understood. In this study, ATMT was used to generate fluorescently labelled strains of D. tanaceti and S. tanaceti expressing mNeonGreen and tdTomato, respectively. Transformants were characterised for fluorescence stability, morphology, growth, and pathogenicity. Whole-genome sequencing revealed predominantly single-copy T-DNA insertions, with occasional partial insertions and small genomic rearrangements. Despite variation in fluorescence intensity among D. tanaceti transformants, stable expression of fluorescent proteins was confirmed in both species. Detached leaf assays demonstrated that transformed strains exhibited infection patterns comparable to their respective wild types. In D. tanaceti, germination occurred by 24 hours after inoculation (HAI), followed by hyphal growth and direct epidermal penetration by 72 HAI, leading to mesophyll colonisation. No major differences in infection timing or colonisation patterns were observed between wild type and transformed strains. In S. tanaceti, infection dynamics were consistent with previous descriptions, supporting the suitability of transformed strains for in planta studies. Fluorescently labelled strains of D. tanaceti and S. tanaceti are valuable tools for investigating infection biology and provide the first visual insights into early infection processes of D. tanaceti in pyrethrum. These findings lay the foundation for future studies incorporating quantitative approaches to better understand pathogen dynamics and interactions.