Background

Strawberries are valuable commercial fruit crops that provide critical income and food security for many farmers and consumers around the world. Strawberry plants can be affected by bacterial and fungal infections like gray mold, anthracnose, and powdery mildew, which can damage the plant, reduce fruit quality, and even lead to plant death (Yang et al., 2023a). Botrytis cinerea is the causing agent of the gray mold disease in almost all vegetable and fruit crops, including strawberry plants and post-harvest fruits (Dean et al., 2012; Yang et al., 2023b). This necrotrophic fungal phytopathogen causes an estimated $10–100 billion yearly loss (Fillinger and Elad, 2016). The airborne conidia are produced by B. cinerea in diseased plant tissues, posing a long-lasting threat as part of the secondary infection cycle. An outbreak of the gray mold disease will likely occur under low temperature and high humidity in greenhouse and field conditions. Importantly, B. cinerea is also widely accepted as the second most important fungal pathogen in the research field of molecular plant pathology (Dean et al., 2012), ranked after Magnaporthe oryzae (Chen et al., 2023) but before Fusarium graminearum (Yang et al., 2018). Although plant species such as Arabidopsis and tomato have been used as classic models to study plant response against B. cinerea (Sun et al., 2011), other economically valuable crop species, like strawberries, are not well studied (Petrasch et al., 2019), especially from the flower and fruit perspectives. This protocol provides a detailed method for assessing the response of strawberry plants to the infection by the gray mold fungus B. cinerea at different stages of plant development. It can be adapted for other research purposes (e.g., in vitro detached leaf/fruit inoculation) and other flowering plants with appropriate modifications. The main factors to consider are the inoculation method (fungal disc vs. fungal conidia depending on the plant tissues) and the inoculation dosage (different plant species have varying levels of resistance to the fungus). For example, leaves and fruits of strawberry, tomato, and tobacco can be detached from plants and kept in optimal in vitro conditions (e.g., low temperature and high humidity) for subsequent fungal disc/conidia inoculation. This protocol can also be combined with other methods in related research fields, such as plant–microbe interactions. For instance, some beneficial bacteria can induce plant defense mechanisms that make the plants more resilient to future attacks (Sahib et al., 2019; Zhao et al., 2021; Yang et al., 2022). These defense mechanisms are called induced systemic resistance (ISR) and systemic acquired resistance (SAR) (Chanda et al., 2011; Yang et al., 2023b). This protocol can be used to study ISR/SAR of flowering plants against the gray mold fungus and help develop strategies for plant disease management from the plant’s perspective. However, this protocol has not been tested yet in field conditions and should also be modified according to plant protection principles such as the plant disease triangle, the plant fitness tetrahedron, and the plant disease management hexagon (Yang et al., 2023a), which should take into account multiple factors rather than a single one.