Detection and quantification protocols for investigating the early stages of Botrytis cinerea interaction with strawberry reveal different infection strategies

Botrytis cinerea is a filamentous fungus that infects over 200 species of crops causing grey mold disease with devastating losses to agriculture worldwide. 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 the first step in the search for novel antifungals. Although B. cinerea is one of the most serious pathogens of strawberry ( Fragaria x ananassa ), few protocols have been specifically developed to study this pathosystem. Consequently, early development of pathogen penetration in strawberry is poorly understood. Here we developed assays using detached strawberry leaves, fruit and petals to study B. cinerea infection. These assays allow comparison of treatment effect on the same fruit, and facilitate the screening of fungicides or biocontrol agents. Through real-time PCR, chlorophyll fluorescence analysis, scanning electron and confocal microscopy, we quantified the lesion and fungal biomass of B. cinerea in the early stages of infection in fruit and petals, and demonstrated that B. cinerea penetrates through 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 .