First report of four viruses infecting peony in Belgium: Cycas necrotic stunt virus, tobacco rattle virus, peony yellowing-associated virus 1 and lychnis mottle virus

Peonies (Paeonia spp.) are widely cultivated ornamentals and are typically vegetatively propagated to preserve desirable traits such as color, shape and scent. However, vegetative propagation can facilitate virus transmission through infected propagation material. As a result, viral infections have become a major concern for peony growers, with more than ten virus species reported in peonies worldwide (Lu et al., 2025). To investigate viruses present in peony propagation material from a Belgian nursery in 2023, the virome of five cultivars was analyzed using total RNA sequencing. The cultivars screened were ‘Mieke Bral’, ‘Old Faithful’, ‘Andreas Maschke’, ‘Pale Rozella’ and ‘Garden Treasure’ (https://americanpeonysociety.org/cultivars/peony-registry/). Total RNA was extracted from leaf samples of five cultivars (one plant per cultivar) using the Spectrum™ Plant Total RNA Kit (Sigma Aldrich). The five RNA extracts were pooled and sent to Admera Health (USA), which performed the following processes: ribosomal RNA removal (Qiaseq Fast Select Plant kit; Qiagen), library preparation (NEBNext® Ultra™ II Directional RNA Library Prep Kit; New England BioLabs) and Illumina sequencing (NovaSeq 6000, rRNA-depleted total RNA sequencing, 40M paired-end reads (2x150bp)). After quality filtering, reads were analyzed using an in-house bioinformatic pipeline. The following software was used: FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/), Cutadapt (Martin, 2011), Vsearch (Rognes et al., 2016), Ribodetector (Deng et al., 2022) and VirusDetect (Zheng et al., 2017). Cycas necrotic stunt virus (CNSV; Nepovirus cycas), tobacco rattle virus (TRV; Tobravirus tabaci), peony yellowing-associated virus 1 (PYaCV; unclassified Citrivirus species, Lu et al., 2025) and lychnis mottle virus (LycMoV; unclassified Secoviridae species, Jin et al., 2023) were identified. A total of 314 contigs were assigned to CNSV, 149 to TRV, 30 to PYaCV and 16 to LycMoV (Supplementary Table S1). Read mapping yielded genome coverages ranging from 8.65-100% and pairwise identities ranging from 96.4-99.6% relative to the closest GenBank sequences. The complete PYaCV genome and LycMoV RNA2 segment were reconstructed and deposited in GenBank (PZ617911, PZ617912). The remaining genomes or segments could not be reconstructed due to potential mixed viral variants in the pooled sample or insufficient coverage of LycMoV RNA1. RT-PCR was performed on the same individual (non-pooled) RNA extracts to confirm virus presence (Supplementary Table S2, Supplementary Figure S3). Primers targeting CNSV RNA2 were developed and confirmed the presence of the virus in ‘Old Faithful’ and ‘Garden Treasure’. ‘Old Faithful’ was co-infected with TRV (Robinson, 1992) and showed mosaic and light-green mottling on the leaves. TRV was also detected in ‘Andreas Maschke’. ‘Garden Treasure’ was co-infected with PYaCV (custom primers) and LycMoV (Shaffer et al., 2021). No clear symptoms were observed in the other infected cultivars. PCR products were Sanger sequenced (Macrogen) and submitted to GenBank (PZ280561-PZ280565). Cultivars ‘Mieke Bral’ and ‘Pale Rozella’ tested negative for all four viruses investigated in this study. To our knowledge, this is the first report of CNSV, TRV, PYaCV and LycMoV infecting peony in Belgium. These findings highlight the importance of virus screening in peony propagation material.