First Report of Clover yellow vein virus Infecting (Arsenic bush).

Clover yellow vein virus (ClYVV) is a member of the genus Potyvirus, family Potyviridae and was reported to infect many plant species, such as Ammi majus L., Phaseolus vulgaris L., Vicia faba L., Lens culinaris L., Borago officinalis L., Cicer arietinum L., Gladiolous gandavensis L., Glycine max L., Trifolium repens L., and Dendrobium sp. (Irey et al. 2006; Ortiz et al. 2009; Park et al. 2014; Yoon et al. 2022). Senna septemtrionalis (Viv.) H.S.Irwin & Barneby (arsenic bush), a species in the subfamily Caesalpinioideae, is widely distributed in tropical and subtropical regions (Datiles et al. 2022). In June 2021, virus-like symptoms of mosaic, chlorosis, and leaf-curling were observed in arsenic bush in Kunming, Yunnan province, China. Symptomatic leaves were collected from four arsenic bush (SS1-4), and asymptomatic leaves were collected from 3 additional arsenic bush plants (SS5-7) (eXtra S1). To identify the putative causal virus, sap of symptomatic leaves (SS1) was stained with 1 % phosphotungstic acid and observed under a transmission electron microscope (TEM). Potyvirus-like particles (about 750-800 nm  13 nm) were observed from the sample (eXtra S1). Total RNA was extracted from sample SS1 using TRIzol Reagent (Invitrogen, USA) and subjected to the Illumina NovaSeq platform for RNA-Seq. After trimming and quality control of raw data, 24,125,963 high-quality clean reads were assembled into 72,835 Unigenes using Trinity software. BLAST searches indicated that the nucleotide sequence of Unigene c29731 (10,893 nt) and its deduced amino acid sequence shared 82.62% to 96.45% and 92.60% to 99.19% identity with several ClYVV isolates, respectively. Unigene c29731 had the highest coverage ratio (88%) and the highest nucleotide sequence identity (96.45%) with ClYVV isolate IA-2016 (GenBank accession No. MK292120.1). The complete genome of ClYVV SS1 isolate (ClYVV-SS, GenBank accession No. OP868578) was determined using RT-PCR and 5' and 3' rapid amplification of cDNA ends (RACE) (Chen et al. 2001). A total of 224,936 out of 24,125,963 reads were mapped to the ClYVV-SS1 genome, yielding an average depth of coverage of 3,056.823 (min=1, max=7,859) at nucleotides from 1 to 9,324 of ClYVV genome (eXtra S2). BLASTN results indicated that the complete genome of ClYVV-SS1 shared 96.45% nucleotide sequence identity and 99% coverage ratio with ClYVV isolate IA-2016 genome. Phylogenetic analysis showed that ClYVV-SS1 and other ClYVV isolates clustered together (eXtra S2). RT-PCR was performed on samples (SS2-7) using a pair of primers of the coat protein gene (5'- TCCGACAAAGATAAGTTGAATGCTGGTG-3' and 5'-GAATCGTGCTCCAGCAATGTGA-3') designed from multiple sequences alignment (MSA). Using SS1 sample as positive control, amplicons of ~813 bp were obtained from three symptomatic samples (SS2-4) but not the asymptomatic ones (SS5-7). A total of 17 of 20 arsenic bushes developed symptoms of mosaic and leaf-curling approximately two weeks after mechanical inoculation with arsenic bush (SS1) sap, with 10 uninoculated plants used as control (eXtra S1). RT-PCR was performed for all tested plants. 17 symptomatic arsenic bushes tested positive for ClYVV, while all other samples tested negative. This confirmed that the symptomatic arsenic bushes were infected with ClYVV. To our knowledge, this is the first report of ClYVV infecting arsenic bush.