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Fungal trunk diseases (FTDs) have been a significant threat to the global stone fruit industry. FTDs are caused by a consortium of wood-decaying fungi. These fungi colonize woody tissues, causing cankers, dieback, and other decline-related symptoms in host plants. In this study, a detailed screening of the fungal microbiota associated with the decline of stone fruit trees in the Czech Republic was performed. The wood fragments of plum and apricot trees showing symptoms of FTDs were subjected to fungal isolation. The partial internal transcribed spacer region, partial beta-tubulin, and translation elongation factor 1-α genes were amplified from genomic DNA extracted from fungal cultures. All isolates were classified, and the taxonomic placement of pathogenic strains was illustrated in phylogenetic trees. The most abundant pathogenic genus was Dactylonectria (31%), followed by Biscogniauxia (13%), Thelonectria (10%), Eutypa (9%), Dothiorella (7%), Diplodia (6%), and Diaporthe (6%). The most frequent endophytic genus was Aposphaeria (17%). The pathogenicity of six fungal species ( Cadophora daguensis , Collophorina africana , Cytospora sorbicola , Dothiorella sarmentorum , Eutypa lata , and E. petrakii var. petrakii ) to four Prunus spp. was evaluated, and Koch's postulates were fulfilled. All tested isolates caused lesions on at least one Prunus sp. The most aggressive species was E. lata , which caused the largest lesions on all four tested Prunus spp., followed by E. petrakii var. petrakii and D. sarmentorum . Japanese plum ( Prunus salicina ) and almond ( P. amygdalus ) were the most susceptible hosts, while apricot ( P. armeniaca ) was the least susceptible host in the pathogenicity trial., Competing Interests: The author(s) declare no conflict of interest.