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Pomme fruits are propagated vegetatively, thereby facilitating frequent viral transmission. The causative agent of apple rubbery wood disease, apple rubbery wood virus 2 (ARWV2), can infect apple and pear. The branches of ARWV2-infected, symptomatic trees are flexible due to the decreased lignification of the xylem. In this research, we reanalysed our small RNA (sRNA) HTS datasets to survey the presence of ARWV2 in Hungary. Validation of HTS using RT-PCR revealed infection in several cultivars. The following RT-PCR-based survey revealed the infection of 17 trees, including not only apple, but also pears, one quince, and a rootstock, without showing any rubbery wood symptoms. Analysis of the sRNA datasets allowed us to profile the sRNA pattern of ARWV2-infected and non-infected trees, and characterise the differential expression pattern of vsiRNAs, sRNAs, and miRNAs targeting the lignin biosynthetic pathway. The results confirmed that the gene-expression changes in the genes that regulate lignification cannot be directly correlated with the presence of the virus, which can explain its frequent latent presence. The variable titre and sequence of the virus, and mixed-infection status of the trees, make its reliable diagnostics challenging, which could be achieved as a result of further research.

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.