Metagenomic Analysis of Marigold: Mixed Infection Including Two New Viruses.

Marigold plants with symptoms of mosaic, crinkle, leaf curl and necrosis were observed and small RNA and ribo-depleted total RNA deep sequencing were conducted to identify the associated viruses. Broad bean wilt virus 2, cucumber mosaic virus, turnip mosaic virus, a new potyvirus tentatively named marigold mosaic virus (MMV) and a new partitivirus named as marigold cryptic virus (MCV) were finally identified. Complete genome sequence analysis showed MMV was 9811 nt in length, encoding a large polyprotein with highest aa sequence identity (57%) with the putative potyvirus polygonatumkingianum virus 1. Phylogenetic analysis with the definite potyviruses based on the polyprotein sequence showed MMV clustered closest to plum pox virus. The complete genome of MCV comprised of dsRNA1 (1583 bp) and dsRNA2 (1459 bp), encoding the RNA-dependent RNA polymerase (RdRp), and coat protein (CP), respectively. MCV RdRp shared the highest (75.7%) aa sequence identity with the unclassified partitivirus ambrosia cryptic virus 2, and 59.0%, 57.1%, 56.1%, 54.5% and 33.7% with the corresponding region of the definite delta-partitiviruses, pepper cryptic virus 2, beet cryptic virus 3, beet cryptic virus 2, pepper cryptic virus 1 and fig cryptic virus, respectively. Phylogenetic analysis based on the RdRp aa sequence showed MCV clustered into the delta-partitivirus group. These findings enriched our knowledge of viruses infecting marigold, but the association of the observed symptom and the identified viruses and the biological characterization of the new viruses should be further investigated.

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.