First report of Lily virus X infecting Paris polyphylla var. yunnanensis in Yunnnan, China.

Lily virus X (LVX) is a positive-sense ssRNA virus belonging to the genus Potexvirus in the family Alphaflexiviridae. LVX is known to infect plants of the genera Lilium and Tricyrtis in the family Liliacea. LVX was first reported in an asymptomatic lily (Lilium formosanum) from England (Stone, 1980), but has been shown to infect plants in the Netherlands (Chen et al. 2005), the United States (Jordan et al. 2008) and Japan (Nijo et al. 2018). To date, the complete genomes of two LVX isolates from the Netherlands and Japan have been reported. Paris polyphylla var. yunnanensis, known as Dianchonglou in China, is a perennial plant of the family Melanthiaceae (formerly belonging to the family Trillium). In China, its rhizome is commonly used as an antispasmodic agent for stroke and cancer treatment (Chang et al. 2017). From 2019 to 2022, leaf mottle and shrinkage which are typical symptoms of viral infections were observed on the leaves of P. polyphylla var. yunnanensis plants in Dianchonglou fields in Qujing, Yunnan. Disease incidence ranged from 19% to 45% across 5 fields (90 plants per field) in Qujing. To identify the possible viral pathogen(s) associated with the disease, the mirVanaTM miRNA isolation Kit was used to extract total RNA was from a mixed sample pool of 5 symptomatic leaf samples collected from the 5 fields. RNA sequencing library was constructed using TruSeqTM RNA sample preparation kit. Sequencing on the Illumina HiSeqTM 2500 platform (Illumina, USA) with 125-bp paired-end reads yielded 23,077,786 raw reads. 22,534,100 clean reads were obtained by removing reads of low quality and poly-N using Trimmomatic software (Bolger et al. 2014). By utilizing the paired-end splicing method in Trinity software (Grabherr et al. 2011) the the raw reads were De novo assembled into 184,596 contigs, of which 303 were related to viruses, including Paris mosaic necrosis virus (PMNV), Pear alphapartitivirus (PAPV), Dahlia mosaic virus (DMV), and Lily virus X (LVX). BLASTn analysis revealed that 12 contigs (lengths ranging from 344 nt to 5,981 nt, query cover 6% to 99%) were most similar (57.32% to 91.67% nt identities) to the genome sequences of LVX, suggesting a possible infection of LVX in the plants. To confirm the result, a full-length genomic sequence of LVX was obtained by reverse transcription polymerase chain reaction (RT-PCR) using specific primers designed based on the sequence of the assembled contigs. The PCR products were cloned into pGEM-T vector (Promega Corporation, USA) and sequenced using the Sanger method (Sangon Biotech, Shanghai, China). The obtained full-length genomic sequence of the LVX isolate (LVX-PP, accession number OM100017) was 5,981 nt in length. BLASTp analysis demonstrated that the putative Rep and CP of LVX-PP shared 76.27% to 81.05% and 80.81% to 81.82% aa sequence similarities with that of other LVX isolates, respectively. Maximum-likelihood phylogenetic trees inferred from the Rep and CP aa sequences showed that LVX-PP clustered closely with LVX isolates. The leaf samples were further analyzed using a lily virus X (LVX) ELISA kit (DEIAPV181, Creative Diagnostics, U.S.A.). Healthy P. polyphylla var. yunnanensis leaves were taken as a negative control and buffer solution as a blank control. The results showed a positive reaction for all five symptomatic plants (OD = 1.259 ± 0.007) relative to the negative (OD = 0.099) and blank (OD = 0.073) controls. These results indicate that LVX can infect P. polyphylla var. yunnanensis. To our knowledge, this is the first report that LVX has been detected in P. polyphylla var. yunnannensis. This study will serve as an important reference for the study of the host range of LVX. Further studies will be required to determine how LVX spreads between P. polyphylla var. yunnannensis and other host plants.