Natural Occurrence of cowpea mild mottle virus infecting sesame (Sesamum indicum L.) in Anhui Province, China.

Abstract

Sesame (Sesamum indicum L.) is one of the primary oilseed crops in China, and often intercropped with shorter crops like peanuts and soybeans. Cowpea mild mottle virus (CpMMV), a member of the Betaflexiviridae family, has been reported in numerous countries worldwide and can infect natural hosts including cowpeas, soybeans, common beans, peanuts, and tomatoes, causing symptoms such as leaf mottling, mosaic patterns, or spotted patterns on the infected leaves. CpMMV is transmitted by whiteflies in nature and by mechanical inoculation in laboratory settings (Iwaki et al., 1982). In September 2023, while surveying soybean virus diseases in Huang-Huai-Hai region of China, we observed sesame plants near a soybean field (longitude 115.76°E, latitude 32.89°N) showing stunted growth, leaf mottling, and mosaic patterns. These symptoms affected approximately one-third of the sesame plants in a 0.1-hectare field. To identify the virus associated with symptomatic leaves, two sesame samples were collected for small RNA deep sequencing. Total RNA was extracted using TRIZOL and sent to BGI for library construction and sequencing with the BGISEQ-500 sequencer. De novo assembly of sRNA reads was performed using Velvet software (version 1.2.10) as described (Su et al., 2016), followed by BLASTn and BLASTx searches against the nonredundant nucleotide and protein databases. CpMMV was identified from sesame plants, with twenty-three contigs ranging from 51 to 368 nucleotides showing similarity to CpMMV, covering 33.7% of the total CpMMV genome. The largest CpMMV contig, spanning 368 nucleotides (nt), exhibited 97% identity to CpMMV isolate Anhui_SZ_DN1383 (Genbank Accession No. MN908944.1) from soybean (Wei et al., 2020). To validate the presence of CpMMV in sesame, RNA from each sample was individually extracted, and CpMMV was detected by reverse-transcription polymerase chain reaction (RT-PCR) according to the manufacturer's instructions (Vazyme, Nanjing, China). Primers were designed based on two small RNA-assembled contigs spanning the CpMMV triple gene block protein 1 (TGBp1) and TGBp2 ORF (Forward: 5´-GGTACCAAAAGATAAGCTTGTTATCTTG-3´; Reverse: 5´-TTAGTACCGTCTCTGTAACAGCCA-3´). Both sesame samples tested RT-PCR positive for CpMMV. The PCR amplicon (597 nt) of these two sesame samples were purified and sequenced. Sequences shared 100% nucleotide identity between them. Nucleotide sequence comparisons confirmed the virus as CpMMV (Accession No. PP767740), exhibiting >99% identity to CpMMV isolate HN_SQ (MW354940.1). Phylogenetic analysis of the 597 nt amplicon, using MEGA7 with eighteen other CpMMV isolates, revealed that the CpMMV isolate from sesame was most closely related to soybean isolates HN_SQ (MW354940.1) and Anhui_SZ_DN1383 (MN908944.1). To fulfill Koch's postulates, healthy sesame leaves were rub-inoculated with crude extracts from CpMMV-infected field samples. RT-PCR confirmed systemic infection at 4 weeks post-inoculation, with symptoms of stunted height, leaf mottle, and mosaic mirroring those observed in the field. Previously, CpMMV has been experimentally documented to infect sesame (Thouvenel et al., 1982), but to our best knowledge, this is the first report of CpMMV infecting sesame under natural conditions. With widespread whiteflies in the Huang-Huai-Hai region of China, CpMMV poses a significant risk to sesame production and may serve as a reservoir, threatening nearby crops such as soybeans.