First Report of broad bean wilt virus-2 in marigold (Tagetes erecta L.) in China

Broad bean wilt virus 2 (BBWV-2), a member of the genus Fabavirus in the family Secoviridae mainly transmitted by aphids, has been recognized as a severe pathogen affecting the production of horticultural and ornamental plants worldwide (Xia et al. 2020). The virus was reported to infect many plant species mostly belonging to the family Fabaceae in China (Wang et al. 2017). In August 2018, marigold plants with the symptom of mosaic were observed in the field of Huairou, Beijing (Figure S1). Total RNA was extracted from symptomatic leaf samples from a single plant with TRIzol reagent (Invitrogen, Carlsbad, CA) with a standard procedure following the manufacturer's instructions, and small RNAs were isolated for deep sequencing library construction with Illumina TruSeq Small RNA Sample Prep Kit (Illumina, San Diego, CA, USA). The high-throughput sequencing was carried out on an Illumina HiSeq2500 platform. After raw data process, 8,911,917 clean reads were gained and further de novo assembled into contigs with CLC Genomics Workbench software. BLASTN and BLASTX analysis against the GenBank database showed that 81 of the 9,495 assembled contigs shared high nucleotide (nt) sequence identity with the bipartite genome of BBWV-2 isolate Gyp (KX686589-KX686590 for RNA1 and RNA2, 89% of the genome coverage and 90% nt identity) and 34 with high nt sequence identity of cucumber mosaic virus(CMV) from Tagetes erecta (EU665000-EU665002 for RNA1-RNA3,89% of the genome coverage and 96% nt sequence identity) with sequence coverage ranging from 24-fold to 8,078-fold at different genome positions. To further confirm the presence of BBWV-2, a RNA2 specific primer pair targeting the coat protein region (F1423-1448,5-CTGACAGAGGAATACTATTTCCAAAG-3;R2692-2719,5-CCTGTAAAATTGATATCTCCGGACAAAC-3) was designed from the obtained HTS sequence and reverse transcription-polymerase chain reaction (RT-PCR) was conducted. The 1.3 kb amplicon was ligated to pMD19-T vector (TaKaRa, Dalian, China) and sequenced. Sequence analysis showed it (BBWV-2-marigold, MW322809) shared 99% nt sequence identity with the Gyp isolate infecting Gynura procumbens from South Korea (LC497425.1). Phylogenetic analysis constructed with MEGA6 with the CP nt sequence of other reported BBWV-2 isolates showed BBWV-2-marigold clustered closely with the isolates from South Korea infecting Gynura procumbens (Figure S2), in accordance with the sequence identity analysis. Further RT-PCR with primer pair targeting the RNA1 (F3025-3050 5-GACAGAGTGATATTCCTAATCGAGAT-3; R4035-4062CACTCAATGC AATAAAGGTCTGGCACCT) was conducted and specific bands with the expected size of 1.0 kb were obtained in the agarose gel (data not shown), which further confirmed the existence of BBWV-2.A total of 16 marigold leaf samples(7 from Huairou and 9 from Yanqing) with mosaic symptom were collected and tested by RT-PCR with the abovementioned primer pair, and 4 from Huairou were BBWV-2 positive. Sequence analysis showed that these 4 isolates shared 100% nt sequence identity with the former sequenced isolateBBWV-2-marigold. Furthermore, CMV specific primer pair targeting the CP (F: 5-ATGGACAAATCTGGATCTCCCAAT-3/R: 5-CTAAGTCGGG AGCATCCGTGAGAT-3) were designed to detect the existence of CMV in these samples and results showed that all these 16 samples were positive for CMV. To the best of our knowledge, this is the first report of BBWV-2 in marigold in China.These findings will assist investigations on the epidemiology of diseases caused by BBWV2 in China.