Natural occurrence of broad bean wilt virus 2 on Mirabilis jalapa in China

Abstract

Mirabilis jalapa Libosch. is an annual ornamental herbaceous plant. Its leaves and roots are used as a traditional folk medicine that function in clearing heat and detoxifying, promoting blood circulation, regulating menstruation, and nourishing kidney (Annapoorani et al. 2014; Liu et al. 2020; Wang et al. 2018). Broad bean wilt virus 2 (BBWV-2), which belongs to the family Secoviridae, is transmitted by aphid in a non-persistent manner in the nature (Kondo et al. 2005) and mainly damages Vicia faba, pepper, yam and spinach (He et al. 2021). The leaves of M. jalapa on the campus showed shrinking (Supplementary Fig. 1A), yellowing (Supplementary Fig. 1B), mosaic (Supplementary Fig. 1D & 1E), and the whole plant had stunted and rough (Supplementary Fig. 1A & 1C) symptoms in the autumn of 2021. Eight plants (S21-S28) with these symptoms were harvested for total RNA extraction, siRNA mixture purification, and siRNA library made (NEBNext® Ultra™ II RNA Library Prep Kit for Illumina®, NEB, UK). The high-throughput siRNA sequencing with pair-end method was performed on Illumina Hiseq 2000 platform (Sangon, Shanghai, China). The raw sequencing data was treated with the Illumina’s CASAVA pipeline (version 1.8). The adaptor was removed and the reads were mostly distributed in 21-24 nt length area (Supplementary Fig. 2A). The contigs (∼12,500, Length > 350 bp) were obtained by de novo assembling with the Velvet Software 0.7.31 (k = 17), then the BLASTN was preformed against GenBank database. Surprisingly, 237 contigs showed significant nucleotide sequence similarities to the genome of BBWV-2. To determine the incidence of BBWV-2 to M. jalapa in campus garden, twenty-eight leaf samples were randomly collected from the garden. Leave extract and total RNA of the sample were tested for BBWV-2 by ELISA (Agdia, USA, SRA46202/0096) and RT-PCR assay, respectively. Twenty-two samples were infected compared with the positive control, and their readings of ELISA were above or parallel to the positive control (Supplementary Fig. 2B∼2D). The coding sequence (1,395 bp) of BBWV-2 movement protein (MP) was amplified by a specific pair of primers (Supplementary Table S1) according to the contigs, the results indicated that the 22 out of 28 samples (78.6%) tested positive for BBWV-2 by both ELISA and RT-PCR (Supplementary Fig. 2E). The MP fragment of BBWV-2 obtained from one of the sample was purified by TIANgel Midi Purification Kit (Tiangen, Beijing, China) and then cloned into pMD19-T (TaKaRa, Dalian, China) vector. Ten separate clones were selected and sequenced (Sangon, Shanghai, China) after PCR verification. The obtained sequences (GenBank accession No. OM416039) were analyzed by BLASTN and bioEdit software (version 7.2.3). According to the phylogenetic tree constructed by BBWV-2 MP sequences (Supplementary Fig. 3), the obtained MP sequences (OM416039, ON677747, and ON677748) were most related to the BBWV-2 MP sequences that from pepper (GenBank accession No. JX183228.1), they share the nucleotide identity of 84.87%. To determine the occurrence and distribution of BBWV-2 in other areas, another twenty-two samples were randomly collected for RT-PCR in different regions of Jiangsu Province, China (Supplementary Table S2). The BBWV-2 infection rate was 76.0% in the M. jalapa. In sum, this is the first report of BBWV-2 naturally infecting M. Jalapa in China.