Boxwood phyllosphere epiphytic and endophytic fungi in response to three common fungicides

Chlorothalonil and propiconazole are protectant and systemic fungicides commonly used in horticulture industries. However, their effects on non-target phyllosphere fungal communities in and on ornamental plants remain poorly understood. In this study, we used boxwood, an iconic landscape plant as a model system to examine the effects of these fungicides on the phyllosphere fungal communities. Shoot samples from boxwood cultivar 'Vardar Valley' (Buxus sempervirens) were collected before (0) and 1, 7, and 14 days after fungicide treatment in early summer and early fall. Total genomic DNA was extracted from the shoot washings and surface-disinfected plant tissue for identification of epiphytic and endophytic fungi using ITS amplicon and Nanopore MinION® sequencing. A total of 427 epiphytic and 152 endophytic fungal genera were identified. Cladosporium and Shiraia dominated the epiphytic and endophytic communities, respectively. Both protectant and systemic fungicides largely impacted epiphytic fungal community diversity and structure, while a stronger effect on the endophytic fungal community was observed in early fall. Fungicide application induced varied changes in fungal composition and trophic mode. However, the fungicide effect was not all suppressive but depended on fungicide chemistry. The co-occurrence networks of phyllosphere fungal communities were also impacted by fungicide application and several hub taxa were identified. These results revealed the boxwood phyllosphere fungal communities and demonstrated the potential impacts of protectant and systemic fungicides on epiphytic and endophytic fungi. They are fundamental for utilizing the full potential of microbiome while reducing horticulture's environmental footprint.