Neofabraea alba, an agent of apple Bull’s Eye Rot – multi-approach insights from Polish orchards

Bull’s eye rot, caused by fungi of the genus Neofabraea (syn. Pezicula , Phlyctema, Gloeosporium ), is an important postharvest disease of apples worldwide. This study aimed to identify and characterize Neofabraea species associated with Bull’s Eye Rot in Polish orchards. Apples with disease symptoms were collected from 53 locations across five voivodships. The developed protocol for the selective isolation of fungal strains of the genus Neofabraea from symptomatic apples allowed the collection of 155 strains. This protocol involved apple tissue surface sterilization with 70% ethanol and incubation at 18°C, effectively reducing contamination by non-target fungi. Molecular identification using Sanger sequencing, followed by a Whole-Genome Sequencing approach, confirmed that the isolates belonged to Neofabraea alba . The isolates were assessed for chemical sensitivity using Biolog™ PM21D plates, revealing growth inhibition by several substrates, including sodium selenite, magnesium chloride, trifluoperazine, and nystatin, with substrate-dependent variability within functional groups. The fungicide-sensitivity test showed that three of ten commercial fungicides exhibited the highest inhibitory effects, while four fungicides showed no activity, indicating complete resistance. The Biolog™ ECO Plate analysis showed differences in the stress response of microbial communities inhabiting the BER symptomatic tissue of apples, depending on variety. Additionally, metataxonomic analyses revealed slight variety-dependent differences, with fungi belonging to Dermateceae predominating at infection sites. These findings improve understanding of Neofabraea sp. pathogens in Poland and support the development of sustainable disease management strategies aligned with the European Green Deal.