First report of zygocactus virus X ( <i>Potexvirus eczygocacti</i> ) and pitaya virus X ( <i>Potexvirus ecspitayae</i> ) infecting dragon fruit ( <i>Hylocereus</i> spp.) in Spain

In June 2024, mosaic, mottling and discoloration symptoms were observed on the cladodes of six dragon fruit varieties (Purple haze, Dark Star, Guatemalense, Roja, Amarilla and Isis golden), grown in a greenhouse located in the municipality of La Mojonera, Almeria, Spain. Samples from symptomatic plants were collected, and total RNA was extracted as previously described (Pallas et al. 1987), followed by small RNA purification using the RNA Clean and Concentrator Kit (Zymo Research, Irvine, CA, United States). A cDNA library was synthesized using NEB Next® Multiplex Small RNA Library Prep Set for Illumina® and analyzed by high throughput sequencing (HTS) using an NovaSeq X Plus and procedure SE50 at Sistemas Genomicos (Paterna, Spain). Adapter sequences were then removed with Cutadapt, followed by FastQC analysis to confirm successful trimming and to verify that only reads between 21 and 24 nt were retained. Plant small RNAs, including tRNAs, rRNAs, and other non-coding RNAs, were filtered out by aligning sequences with Bowtie2 against specialized databases such as Rfam and plant small RNA repositories (miRBase) (SRA record PRJNA1442788). The resulting clean, size-filtered reads, which ranged in number from 1,649,054 to 4,119,892 per sample, were then aligned to the complete genomes of five potexvirus species: cactus virus X (CVX; NC_002815.2), pitaya virus X (PiVX; NC_024458), zygocactus virus X (ZyVX; NC_006059.1), opuntia virus X (NC_006060.1), and schlumbergera virus X (NC_011659.1). Alignment percentages were used as an indicator of relative viral abundance, with the highest values observed for PiVX (27.28%), followed by ZyVX (18.59%) and CVX (14.66%). With respect to genomic coverage, the most complete representation was observed for PiVX (~99.9%), ZyVX (~99.0%), and CVX (~98.8%). Next, PiVX, ZyVX and CVX were confirmed by one-step RT-PCR using PiVX, ZyVX and CVX-specific primers designed from a region of the coat protein (CP) gene (Park et al. 2018). The expected amplicon sizes (approximately 678 and 690 nt) obtained for PiVX and ZyVX, respectively, were subsequently cloned and sequenced. The 678 nt CP sequences for the PiVX amplicons (GenBank PX655241) shared 98% identity with the CP gene of PiVX , Brazil (OR941748) and China isolates (OM802132) from Hylocereus sp. The cloned amplicon sequences for ZyVX (GenBank PX655240) shared 96 and 97% identity, with the CP gene of ZyVX dragon fruit isolates from Brazil (R941758) and Taiwan (KM288843), respectively. To confirm the presence of PiVX, ZyVX, and CVX, and the absence of OpVX and SchVX in the original greenhouse, 15 additional dragon fruit samples from plants expressing mosaic, mottling or discoloration symptoms, were collected and tested by RT-PCR (Park et al. 2018). showing that all samples were positive for these viruses, either as simple infections or as mixed infection. In addition, symptomatic dragon fruit greenhouse samples from Alicante (n=5) and Murcia (n=8), provinces were tested and presence of PiVX, ZyVX, and CVX infections were confirmed. PiVX has earlier been detected on plant species of the cactaceae in South Korea, USA, and Ecuador (Bae and Park, 2022; Singh et al., 2024; Espinoza-Lozano, et al., 2025), and ZyVX in dragon fruit plants in India, Brazil, China, Korea, and Ecuador (Parameswari et al., 2023; Espinoza-Lozano, et al., 2025). Previously, we reported CVX in Spain (Janssen et al., 2022). To the best of our knowledge, detection of PiVX and ZyVX by HTS followed by RT-PCR confirmed the first report of these two potexviruses in H. undatus in Spain. In a nutshell, to mitigate the dispersal of PiVX and ZyVX, diagnostic testing of these pathogens must be incorporated in the indexing process of dragon fruit propagating materials.