Grapevine (Vitis vinifera L.) cultivation is an important agricultural sector worldwide. Its expansion into new areas, like Kazakhstan, brings significant phytosanitary risks. Viroids, such as grapevine yellow speckle viroid 1 (GYSVd-1) and hop stunt viroid (HSVd), are RNA pathogens that threaten vineyard productivity. They can cause a progressive decline through latent infections. Traditional diagnostic methods are usually targeted and therefore not suitable for thorough surveillance. In contrast, modern high-throughput sequencing (HTS) methods often face challenges due to their high costs and complicated sample preparation, such as ribosomal RNA (rRNA) depletion. This study introduces a simplified diagnostic workflow that overcomes these barriers. We utilized the latest Oxford Nanopore V14 cDNA chemistry, which is designed to prevent internal priming, by substituting a targeted oligo(dT)VN priming strategy to facilitate the sequencing of non-polyadenylated viroids from total RNA extracts, completely bypassing the rRNA depletion step and use of random oligonucleotides for c DNA synthesis. This method effectively detects and identifies both GYSVd-1 and HSVd. This workflow significantly reduces the time, cost, and complexity of HTS-based diagnostics. It provides a powerful and scalable tool for establishing strong genomic surveillance and phytosanitary certification programs, which are essential for supporting the growing viticulture industry in Kazakhstan.

Grapevine viruses reduce harvests and degrade fruit quality, but their genetic diversity in Kazakhstan has remained unexplored. We collected symptomatic leaves from local vineyards and recovered eleven fragments of the coat-protein gene: one from grapevine fanleaf virus, five from grapevine virus A, and five from grapevine leafroll-associated virus 3. After Sanger sequencing, we compared these fragments with more than one thousand international counterparts to place the Kazakh strains on the global family tree. The results reveal a clear spectrum of genetic diversity that mirrors each virus’s route of spread. Grapevine virus A, which is moved both mechanically and by insects, proved the most variable; grapevine fanleaf virus, carried by dagger nematodes and pruning sap, had intermediate variability; and grapevine leafroll-associated virus 3, moved only by mealybugs and scales, was highly conserved. All Kazakh sequences fell inside established foreign lineages, showing that the viruses were imported multiple times rather than evolving locally. Grapevine virus A will require broad-coverage or multiplex PCR primers to avoid false negatives, whereas the stable leafroll virus can be monitored with a single high-sensitivity assay. Combined with vector management—mealybug control for leafroll, and nematode testing for fanleaf—these data lay the groundwork for a national clean-plant program and more resilient vineyards across Central Asia.