First Report of Paprika mild mottle virus Infecting pepper (Capsicum annuum L.) in China.

Paprika mild mottle virus (PaMMV), a tobamovirus from the Virgaviridae family, has been reported to infect pepper (Capsicum annuum L.) in the Netherlands (García-Luque et al., 1993), Japan (Hamada et al., 2003), Bulgaria (Ruíz del Pino et al., 2003), and Israel (Luria et al., 2018). Pepper has become the most widely planted vegetable and the most heavily consumed spicy condiment, with a large planting area (>21,000 km²) in China (Zou et al., 2021). However, the crop's productivity is limited by viral diseases. In August 2021, a survey of viral diseases was conducted across five pepper-growing regions in Linyi, Shandong Province, China, revealed that 5% to 10% of plants in most fields exhibited symptoms such as stunting, leaf narrowing, chlorosis, and crinkling of leaf tissue (Fig. S1-a). Subsequently, seven symptomatic samples (Fig. S1-b) were collected from a plot (118° 29' E, 34° 65' N) where thin-skinned peppers were grown. All samples tested positive in a Western blot assay using a polyclonal antibody for tobacco mosaic virus (TMV), indicating the presence of a tobamovirus (Fig. S1-c). To identify the virus, total RNAs were extracted from 10 different symptomatic pepper plant samples, using the RNeasy Plant Mini Kit (QIAGEN, Hilden, Germany) and combined into a single sample in equal amounts (100 ng/μL each). RNA-Seq was performed on this mixture using the Illumina NovaSeq 6000 (Illumina, San Diego, USA). Raw reads (6.92G) in fastq format were processed with in-house perl scripts to yield clean reads (6.68G), which were assembled into 780 contigs (mean length: 2036 bp) using Trinity software (v2.6.6). These contigs were then analysed using the NCBI BLASTX program (http://www.ncbi.nlm.nih.gov/blast) against the viral RefSeq database. The results demonstrated that the contigs exhibited an average genome coverage of 40.16%. Notably, one unique contig (6453 bp) was mapped to the PaMMV genome (reference sequence KX187305.1) with 98.34% identity. To verify the RNA-Seq result, a PaMMV-specific primer pair (forward: 5'-GAGTTCATAGAGGCAGTACC-3'; reverse: 5'-CTTCGATTTAAGTGGAGGGAT-3') was designed for amplifying an 800-nt fragment of the PaMMV coat protein (CP) gene by RT-PCR. The 800-nt fragment was successfully amplified from all the symptomatic samples (Fig. S1-d). Sequenced RT-PCR products (GenBank No. OR365081.1) showed 99.75% nucleotide identity with PaMMV isolates (OQ198318.1, China, goat) according to BLAST analysis. Mechanical transmission of PaMMV from three infected pepper plants collected in Linyi to healthy pepper plants confirmed pathogenicity. Three pepper cultivars (trade name: Qiemen, Tianyu, Haonong11) were tested and showed varying symptoms post-inoculation (Fig. S1-e). All cultivars were confirmed in the lab to be PMMoV-sensitive and exhibited significant viral disease symptoms after PMMoV inoculation (data not shown). Each variety was inoculated with at least 3 seedlings from different field-collected virulence sources. Qiemen (cultivar: sweet pepper) exhibited severe symptoms with necrotic spots, Tianyu (cultivar: Chaotian pepper) had mild leaf wrinkling, and Haonong11 (cultivar: Pickled pepper) showed yellow mottle and leaf shrinking. All tested seedlings were positive for PaMMV by RT-PCR (Fig. S1-f). To our knowledge, this is the first report of PaMMV infecting pepper in China, highlighting the need for vigilant monitoring to protect the chili industry and prevent the virus's spread to other crops.