First Report of Potato Virus S Andean Isolates Infecting Potato in Estonia

Abstract

Potato virus S (PVS, Carlavirus sigmasolani) is a regulated pathogen in EU seed potato (Solanum tuberosum L.) production programs. Phylogenetic studies have identified three lineages: ordinary (–O; spread worldwide), and two Andean phylogroups (–A, mainly found in Latin America, Middle East, and East Africa; and –RVC with a limited spread in the Andes region) (Topakaya et al. 2023). Recently, we reported the first complete genome of a PVS-O isolate in Estonia (PV764920; van der Sman et al. 2026). In September 2025, seed lots from 34 potato fields (100 tubers per lot) were analysed using high-throughput sequencing (HTS). Plants were grown out and pooled into ten subsamples for each lot. Total RNA was extracted using a Thermo Scientific KingFisher Flex system with the MagMAX™ Plant RNA Isolation Kit (Applied Biosystems). The subsamples from the same seed lot were subsequently pooled for library preparation using the TruSeq Stranded Total RNA with Ribo-Zero Plant kit (Illumina) and sequenced on an Illumina iSeq100 platform using an iSeq 100 i1 Reagent v2 (300-cycle) cartridge, producing 2 × 75 bp paired-end (PE) reads. Reads were demultiplexed according to the instrument workflow. All bioinformatic analyses were performed in Geneious Prime 2026.0.2. Raw reads were quality trimmed and filtered using BBDuk, then PE reads were merged and duplicate reads were removed using default settings. De novo assembly was carried out with SPAdes. Resulting contigs were screened by BLASTn against the NCBI virus database, revealing 71 contigs, ranging from 297 to 8547 nucleotides in length, with the highest nucleotide identity with PVS in 11 out of 34 libraries. Five complete or near-complete genomes were assembled. Two genomes (GenBank Acc. No. PZ397343-44) represented PVS-A isolates, showing the highest nucleotide identity with the Kazakhstan isolate MN095414 (99%), while three genomes (GenBank Acc. No. PZ397345-47) represented PVS-O isolates showing the highest nucleotide identity (98%) with European isolates PP856220, MF346599, and OL472247. PVS-A was once detected in the same library with PVS-O, and once with PVY and PLRV. In other libraries, PVS-O was detected alone (in seven cases), or together with PVY and PVM (once), or with PVY (once). No other viruses were found. Phylogenetic analysis of PVS genome sequences was performed in MEGA 12 using MUSCLE (1000 iterations), and maximum-likelihood phylogenetic tree reconstruction in RAxML 2.0.1 GTR+G+I model, 1000 bootstrap replicates tree visualized in iTOL, showing clustering of Estonian PVS genomes consistent with BLASTn results (Suppl. Fig. S1). In parallel, seed samples were tested for potato virus A, Y, X, M, S and potato leafroll virus, using RT-qPCR as described in van der Sman et al. (2025). RT-qPCR results confirmed the findings of HTS. PVS incidence ranged from 1% to more than 20%. Our two PVS-A findings originated from the neighbouring fields located in Viljandi county where the seed potato was grown from the seed tubers originated from Germany. Given that Andean PVS-A isolates have been mainly reported in association with material originating from Germany (Bragard et al. 2020), this finding may suggest a possible introduction pathway linked to imported seed material.