First Report of Cowpea Polerovirus 2 Naturally Infecting <i>Macroptilium atropurpureum</i> in China

Macroptilium atropurpureum (DC.) Urb., commonly known as siratro, is a perennial weed belonging to the Fabaceae family. It is used as a cover crop with many crops such as maize, passion-fruit and rice (Xu et al. 2024). In July 2022, siratro plants exhibiting symptoms of leaf shrinking (Figure S1) were observed growing near cowpea (Vigna unguiculata L. Walp), fields in Sanya, located in the south of Hainan Province, China. To investigate the possible causal agents of the symptoms, 14 leaf samples were collected that includes two asymptomatic (M1 and M11) and 12 symptomatic (M2 - M10 and M12 - M14). Total RNA was individually extracted using the TRIzol Reagent (Life, USA). Subsequently, the 14 RNA extracts were then pooled into two composite samples (M1-M7, and M8-M14), from which two cDNA libraries were prepared using the VAHTS Universal V8 RNA-seq Library Prep Kit for MGI (Vazyme, China). High-throughput sequencing was performed on the DNBSEQ-T7 (Beijing Genomics institution, China) generating paired-end reads of 150 bp. De novo assembly using SPAdes v3.15.5 (with default parameters) yielded, 135,011 and 130,549 contigs from the two pooled samples respectively. BLASTN analysis of assembled contigs identified 170 virus-related sequences: 140 contigs mapped to Cowpea mild mottle virus (CPMMV) (NC_014730), with nucleotide (nt) identities ranging from 71.11% to 83.45%, 29 contigs matched to Bean common mosaic virus (BCMV) (NC_003397), showing 71.99% to 97.72% identities, and one contig, 4,095 nt in length, aligned with Cowpea polerovirus 2 (CPPV2) (NC_034247) sharing 93.64% identity. The CPPV2 contig contained an open reading frame (ORF) located at positions 201-2165 nt, encoding 654 amino acids (aa) protein, that exhibited a similarity of 90.06% to the P1 of BE179 (YP_009352253.1). To validate the presence of the three viruses, the 14 siratro samples were tested by reverse transcription - polymerase chain reaction (RT-PCR) using virus-specific primers pairs encompassing the viral coat protein (CP) genes (Table S1). Out of the 14 samples, three (M1, M7 and M8) were singly infected with CPMMV, while four exhibited mixed infections including two with CPMMV and BCMV (M2 and M3) and two with CPMMV and CPPV2 (M9 and M14) (Figure S2). Consequently, no clear association could be established between the observed symptoms and any single virus. Gel-purified PCR products were cloned into the pMD19-T Vector (Takara, China), and transformed into Escherichia coli DH5α cells (Sangon, Shanghai), and two clones per sample were selected for Sanger sequencing. The CPMMV sequences (PP790736 - PP790742) shared 96.90% - 100.00% nt identity with each other and 97.64% nt identity with sequence of CPMMV-JS (MT366555), CPPV2 (PP790745 and PP790746) shared 98.16% nt identity with each other and 97.66% identity with isolate CPPV2-GD (MZ042662) whereas BCMV (PP790747 and PP790748) shared 99.08% nt identity with each other and 97.85% nt identity with BCMV (KC832501). All three viruses identified in this study are previously known to infect leguminous plants (Gambley et al. 2022; Kwak et al. 2022; Palanga et al. 2017; Saqib et al. 2010). However, this is the first report of CPPV2 infecting siratro, thereby expanding our knowledge its known host range. Additional surveys and biological studies are currently being conducted to assess the significance of M. atropurpureum as a potential virus reservoir for viruses that could impact crop plants.