Comprehensive Virome Profiling of Apple Mosaic Disease-Affected Trees in Iran Using RT-PCR and Next-Generation Sequencing

Apples (<i>Malus domestica</i>), one of Iran's oldest cultivated fruit crops, hold considerable economic importance. In this study, 170 apple leaf samples representing various commercial cultivars were collected across the country. RT-PCR screening targeted five common apple-infecting viruses and two viroids: apple chlorotic leaf spot virus (ACLSV), apple stem pitting virus (ASPV), apple stem grooving virus (ASGV), apple green crinkle-associated virus (AGCaV), apple mosaic virus (ApMV), apple scar skin viroid (ASSVd), and hop stunt viroid (HSVd). To identify additional or novel agents, 40 RT-PCR-negative samples were pooled into two composite groups and analyzed using next-generation sequencing (NGS). NGS was also performed on individual samples with mixed infections to retrieve full genomes. RT-PCR confirmed the presence of ACLSV, ASPV, ASGV, AGCaV, ApMV, and HSVd. NGS further revealed three additional pathogens: citrus concave gum-associated virus (CCGaV), apple hammerhead viroid (AHVd), and apricot vein clearing-associated virus (AVCaV), which were subsequently detected across the collection by RT-PCR. AGCaV was most prevalent (47.6%), followed by ACLSV (45.8%), HSVd (27.6%), AVCaV (20.5%), ASGV (17%), AHVd (15.2%), ASPV (14.1%), CCGaV (4.7%), and ApMV (3.5%). Mixed infections occurred in 67% of samples. Phylogenetic analysis based on CP genes (ACLSV, ASGV, AGCaV) and full genomes (AVCaV, AHVd) clustered Iranian isolates together, suggesting a common origin. This is the first report in Iran of AGCaV, CCGaV, ApMV, and AVCaV in apple, and notably, the first global report of AVCaV in a non-Prunus host. The findings provide the first comprehensive assessment of the sanitary status of apple trees in Iran.