Metabarcoding Profiling Reveals Microbiome Structure and Predicts Functional Shifts in Grapevines Challenged by Phyllosticta ampelicida

Black rot disease (BRD), caused by the still understudied Phyllosticta ampelicida, is spreading across several grape producing countries, posing a growing threat to the agroindustry. The role of the grapevine microbiome in defending against this pathogen, particularly in terms of microbiota structure and community homeostasis, remains unclear. In this study, we aimed to characterize the epiphytic phyllosphere microbiota of grapevines and identify shifts in microbial genetic structure associated with BRD symptoms. We sampled three vineyards of the cultivar "Touriga Nacional" in the Douro region (Portugal), collecting 20 leaves from (a) five healthy and (b) five BRD-symptomatic grapevines. The presence of P. ampelicida was confirmed in all symptomatic samples. Epiphytic bacterial DNA was extracted and sequenced using next-generation sequencing (NGS). Results indicate that although overall the diversity and richness indexes were not different in diseased plants compared to healthy ones, there was a reduction in OTU richness in black rot-affected grapevines. Diseased plants exhibited significant shifts in microbial network assemblages and showed an increased relative abundance of certain taxa, such as Acinetobacter, suggesting a possible recruitment of beneficial bacteria in response to biotic stress. Additionally, we observed a higher abundance of antibiotic resistance-related KEGG Orthologues (KOS) in symptomatic plants, raising potential concerns for human health. This study presents the first characterization of the grapevine phyllosphere epiphytic bacterial microbiota and its structural shifts in response to BRD.