First report of watermelon mosaic virus infecting faba bean ( L.) in China.

Faba bean (Vicia faba L.) is the fourth most cultivated temperate legume (Lyu et al., 2021). It is widely grown throughout the world as a nutritious legume crop. From March to April in 2023, field-grown faba bean plants with virus-like symptoms such as mosaic, discoloration, stunting, yellowing, chlorosis and leaf curling were observed at 13 different locations in Nanjing and Xinghua City in Jiangsu Province, China. A total of sixty-five leaf samples were collected from the symptomatic faba bean plants. The leaf morphology of these plants is deformed, and the growth and development are severely affected by the infected diseases. To identify the viruses that infected the faba bean plants, the samples were mixed, and the total RNA was extracted using the TRIzol reagent (Invitrogen, USA). A small RNA sequence library was constructed using the Trusses Small RNA Sample Prep Kit (Illumina, San Diego, USA) and analysed by high-throughput sequencing using an Illumina HiSeq 2500 platform (Lianchuan Biotechnology, Hangzhou, China). The obtained clean sequencing reads were mapped mainly to 11 viruses (with mapped read counts >20000). De novo assembly of the total reads was performed using ACGT101-miR and Velvet software (LCSciences, Houston, Texas, USA). The resulting assembled contigs were analysed by BLASTn and BLASTx at National Center for Biotechnology Information. Seventy-three contigs were matched to the genome sequence of watermelon mosaic virus (WMV) in the genus Potyvirus of the family Potyviridae. RT-PCR was used to detect WMV in the high-through sequencing sample using specific primers (WMV-F: 5'- TTAACCACATTGATGGTTGC-3', WMV-R: 5'- GACCCGAAATGCTAACTGTG-3') with an expected product of 924 bp. The sequencing results of PCR amplicon confirmed that the sample was infected with WMV (Fig. S1A). RT-PCR analysis of pooled samples from each location showed that samples from three locations were infected with WMV (Fig.S1B). To detect WMV in the individual sample collected from these three locations, a total of 18 samples were analysed by RT-PCR and four samples were positive for WMV (Fig.S1C). The symptoms of the four infected plants are shown in Fig.S1D. To determine the full-length sequence of the WMV isolate from faba bean, RT-PCR was performed using five overlapping primer pairs designed according to the WMV sequences obtained by high-throughput sequencing and the aligned WMV sequences. The amplified PCR products were then inserted into pMD19-T (Takara, Dalian, China) and sequenced. The full-length sequence of the faba bean WMV was assembled from the five overlapping sequences and deposited in GenBank as WMV-Fb isolate (accession number PP782053). The complete sequence of the WMV-Fb isolate has 10,044 nt and contains a 9,648 nt open reading frame encoding a polyprotein of 3,216 amino acids. It shares the highest nucleotide sequence identity (97.46%) with two WMV isolates (accession number OM948829.1 and OM948820.1) from Zucchini in Spain and had 97.40% nucleotide sequence identity with Sapindus mukorossi potyvirus strain pt164-pot-23 (accession number MN722418.1) from Sapindus mukorossi Gaertn. in Jiangsu, China at the complete genome level. To reveal the molecular evolutionary relationships between WMV-Fb and other isolates, a phylogenetic tree was constructed using the polyprotein nucleotide acid sequence of WMV-Fb and reported WMV isolates in the G1 (classical isolates), G2 and G3 (emerging isolates) groups (Bertin et al., 2020; Abdalla et al., 2021). Phylogenetic analysis showed that the WMV-Fb isolate was clustered in the G3 group (Fig.S1E). To the best of our knowledge, this is the first report of WMV infecting faba bean. WMV has a wide host range and causes severe losses in cucurbit crops, such as melon, watermelon, zucchini, and cucumber (Dong et al., 2017; Nematollahi et al., 2021). Our results provide a better understanding of the host range of WMV and suggest that WMV poses a potential threat to legume crops.