Integrated Assays and Microscopy to Study the Botrytis cinerea–Strawberry Interaction Reveal Tissue-Specific Stomatal Penetration

Strawberry (Fragaria x ananassa) production has increased around the world, but crop quality and yield are threatened by fungal pathogens. Botrytis cinerea is a filamentous fungus that infects over 1400 species of crops, causing gray mold disease with devastating losses to horticulture worldwide, including strawberry. The heavy reliance on synthetic fungicides in the strawberry industry has led to the emergence of fungicide resistance in B. cinerea. Therefore, understanding the fundamental biology of B. cinerea is an important step in the search for novel antifungals. Although B. cinerea is one of the most serious pathogens of strawberry, this pathosystem is understudied compared to other plant hosts. Consequently, further evidence is needed on pathogen penetration and early disease development in strawberry tissues. Here, we adapted and advanced assays using detached strawberry leaves, fruits, and petals to study B. cinerea infection. These assays allow the comparison of the treatment effect on the same fruit, avoiding confounding from differential ripening, and facilitate the screening of fungicides or biocontrol agents. Through chlorophyll fluorescence analysis and scanning electron and confocal microscopy, we quantified lesions caused by B. cinerea in the early stages of infection in fruit and petals, and demonstrated that B. cinerea penetrates through the stomata of strawberry achenes, revealing a previously unrecognized infection route in this host. These data provide a deeper understanding of the B. cinerea–strawberry interaction and will serve as a foundation for future studies seeking novel antifungal treatments against B. cinerea.