Apple viruses pose significant threat to global apple production. In this study, HTS technology was used to investigate the apple virome in the Czech Republic. Previously reported viruses, including ACLSV, ASPV, ASGV, ApMV, AGCaV, and CCGaV, were confirmed, and near-complete genomes were assembled. Additionally, two novel viruses, ARWV1 and ARWV2 were identified for the first time in the Czech Republic. Phylogenetic analyses showed low genetic variability among ARWV2 isolates, suggesting a possible recent introduction or limited diversification. In contrast, ARWV1 isolates displayed distinct clustering in the coat protein coding region, separating symptomatic and asymptomatic samples, indicating a potential involvement of genetic determinants in symptom expression. Mixed infections were prevalent, with multiple molecular variants of ACLSV, ASPV, and AGCaV detected within individual samples, along with co-infections involving viruses from different families. Recombination analysis identified frequent recombination events in ACLSV and ASPV, often involving non-apple parental sequences, suggesting their potential for cross-host infections. Additionally, an interspecific recombination event was detected in an almond ApMV isolate, with PNRSV as a minor parent. These findings highlight the impact of agricultural practices on viral evolution and host adaptation. This study demonstrates the utility of HTS as a powerful tool for uncovering viral diversity, recombination events, and evolutionary dynamics.

Plum pox virus (PPV) is one of the most destructive pathogens affecting stone fruit trees. It causes sharka disease and severe yield losses. The genetically modified plum cultivar ‘HoneySweet’ was developed to provide long-lasting resistance to PPV via RNA interference. Long-term field trials of ‘HoneySweet’ have been conducted in the Czech Republic since 2001, involving the artificial inoculation of the cultivar with PPV alone, and with apple chlorotic leaf spot virus (ACLSV) and prune dwarf virus (PDV) in combination. This study evaluates the impact of viral infection on tree growth after 24 years in the field. Growth parameters—trunk cross-sectional area (TCSA) and canopy volume—were measured and analysed using ANOVA and Tukey’s test. The results show that infected trees exhibit significantly reduced growth compared to non-infected controls, with the strongest inhibition observed in trees inoculated with PPV + PDV + ACLSV. The presence of ACLSV had the most pronounced negative effect on growth, while PDV did not significantly influence tree vigour. These findings emphasise the importance of using virus-free rootstocks and certified planting material to prevent growth suppression in HoneySweet orchards.