Grafting cultivars onto rootstocks is a widely used practice by the apple industry predominantly aimed at faster fruit bearing. Using high-throughput sequencing, we revealed the presence of recently described viral agents, namely apple hammerhead viroid (AHVd), apple luteovirus 1 (ALV-1), and citrus concave gum-associated virus (CCGaV), in germplasm collections and production orchards in the Czech Republic and Hungary. The HTS results were validated with RT-(q)PCR, and Northern blotting techniques. To obtain further insight about the presence of these agents, RT-PCR based surveys were carried out and showed their widespread presence alone or in mixed infections. The pathogens were present both in production areas and in feral samples. In addition, rootstock-to-scion transmission of ALV-1 and CCGaV was confirmed using commercial rootstock materials. Phylogenetic relationships based on partial sequences of distinct variants were also investigated. Furthermore, the rosy apple aphid was found to be ALV-1-positive, suggesting that it might be a potential vector of the virus.

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.