First report of tomato spotted wilt virus (TSWV) isolated from nasturtium (Tropaeolum majus L.) with a serious leaf mosaic disease in China.

Tomato spotted wilt virus (TSWV, Genus Orthotospovirus, Family Tospoviridae) is a thrips-transmitted negative-stranded RNA virus with a large host range. Major economic losses caused by TSWV have been recorded in various crops such as tomato, pepper and lettuce (Pappu et al., 2009; Adams et al., 2017). Nasturtium (Tropaeolum majus L.), a common flowering plant species native to Peru, is generally cultivated as a garden and greenhouse ornamental plant in China. It is also known for its medical, edible and cosmetic values (Jakubczyk et al., 2018). In June 2019, a serious leaf mosaic disease (Figure S1) was observed in ~77% of T. majus plants in Beiling Park in Shenyang City, Liaoning, China. To reveal the possible viral agent associated with the disease, leaf tissue was collected from 9 symptomatic and 2 asymptomatic plants, followed by total RNA extraction from each of the samples using RNASimple Total RNA Kit (Tiangen, Beijing, China). The RNA from one representative symptomatic sample (Figure S1, d) was used to construct an rRNA-depleted library using the Ribo-Zero™ rRNA Removal Kit (Plant Leaf) (Illumina, CA, USA). The library was subjected to RNA-Seq using a BGISEQ platform (Shenzhen Huada Gene Science and Technology Service Co., Ltd., Shenzhen, China). A total of 109,279,540 quality-filtered reads were obtained using the CLC Genomics Workbench 9.5 software (Qiagen, Valencia, CA, USA). Clean reads were assembled into 88,091 contigs ranging in length from 200 to 19,695 bp using Velvet (Zerbino and Birney 2008). Among the contigs, three sized at 8801, 4617 and 2909 bp were found to share a sequence identity of 98.74-99.35% with the RNA segments (L, M and S) of TSWV. No sequences of other viruses, viroids included, were detected. Ten primer pairs were designed based on the TSWV contigs to obtain the full genome sequence of the virus (Table S1). Five, three, and two amplicons were obtained for the TSWV RNA segments L, M and S, respectively, from the same RNA sample used in the RNA-Seq process. These amplicons were then cloned into the pMD18T vector (TaKaRa, Dalian, China) and Sanger sequenced. The resulting sequences were assembled and analyzed using the DNAMAN version 8.0 (LynnonBiosoft, Quebec, Canada) and DNAStar version 6.0 software (DNAStar Inc, Madison, WI, USA) (Table S1). The RNA segments were determined to be 8,914 nt (L, accession no. MT241883), 4,791 nt (M, MT241884) and 2,922 nt (S, MT241885) in length. They shared a sequence identity of >99% with the "LL-N.05" (segment L, KP008128) and "SPAIN-1" (segment S, AY744479) isolates from tomatoes in Spain and the "Beijing" isolate (segment M, MH717046) from chrysanthemum in China. These results, together with phylogenetic analysis, suggest that the isolate (designated as isolate LN-HJL) was likely resulted from genome reassortment between other isolates. No reliable recombination events were detected in the RNA segments of LN-HJL by the RDP4 program (Martin et al., 2015). RT-PCR assay with primer pair S-1F/1R on all samples led to positive detection of TSWV only in the symptomatic samples (Figure S1, g), indicating that the virus was likely responsible for the disease symptoms observed in T. majus. To the best of our knowledge, this is the first report of the TSWV infection in T. majus in China, and the second in the world since the first report in the USA (Ie, 1964). TSWV-infected T. majus plants not only lose their aesthetic and economic values (Figure S1, f), but also may act as reservoirs for the spread of TSWV to other important crops. T. majus should therefore be monitored regularly for the virus and managed accordingly in China.

Full genome sequence of a novel potexvirus from Euonymus bungeanus Maxim based on RNA-Seq analysis.

In July 2018, a yellow mottle leaf disease was observed on the leaves of Euonymus bungeanus Maxim plants in Liaoning Province of China. Typical flexuous filaments (diameter, 13 nm; length, ca. 500 nm) were observed in extracts on the symptomatic leaves. Transcriptome sequencing indicated the presence of a potexvirus in the infected samples. The complete viral genome, determined to be 6,784 nucleotides in length, excluding the poly(A) tail, contains five open reading frames and is most closely related to that of euonymus yellow vein associated virus (EuYVAV, MF078061) (41.0%). Based on the coat protein gene, this virus shares the highest sequence similarity with cymbidium mosaic virus (CyMV, EF125179), with 43.9% nucleotide and 38.8% amino acid sequence identity, respectively. Phylogenetic analysis indicated that the virus clustered with potexviruses and is most closely related to EuYVaV. This virus is a distinct member of the genus Potexvirus, for which the name "euonymus yellow mottle associated virus" (EuYMaV) is proposed.

The complete nucleotide sequence and genome organization of a novel virus of the order Tymovirales isolated from Prunus davidiana (Carr.) Franch. in Liaoning, China.

In September 2017, a yellow spot leaf disease was noted on the leaves of Prunus davidiana (Carr.) Franch. plants in Liaoning, China, and spherical virions (approx. 30 nm in diameter) were later observed in preparations of symptomatic leaves. Subsequent deep sequencing of small RNA revealed the presence of a virus in these symptomatic leaves The complete genome of this viral isolate consists of 6,072 nucleotides, excluding the poly(A) tail. The virus showed the closest genetic relationship to grapevine-associated tymo-like virus, reported in Colmar, France (GaTLV, MH383239), which is the sole member of the newly proposed genus "Gratylivirus" within the order Tymovirales, which is currently unassigned to a particular family. The virus clustered closely with GaTLV in a phylogenetic tree constructed based on complete genomic sequences. On the basis of the nucleotide and amino acid sequences of the replicase and coat protein genes, this virus shares the highest (although still relatively low) sequence similarity with those of GaTLV (41.6%-60.8% identity), indicating that the virus is a distinct member of the order Tymovirales, for which the name "prunus yellow spot-associated virus" (PYSaV) is proposed. To our knowledge, this is the first report of a virus naturally infecting P. davidiana.

Complete genome sequence of a novel member of the family Potyviridae isolated from Phellodendron amurense Rupr. in Liaoning, China.

In September 2017, Phellodendron amurense Rupr. plants showing yellow ringspots on leaves were observed in Liaoning, China. Flexuous filamentous particles (~1000 × 13 nm) were observed in the sap prepared from symptomatic leaves. A virus was detected in the symptomatic leaves by sequencing small RNAs and assembling the genome sequence. The complete genomic RNA was found to be 10,457 nucleotides in length excluding the poly(A) tail and to have the closest phylogenetic relationship to rose yellow mosaic virus (RoYMV), the sole member of newly established genus Roymovirus in the family Potyviridae. The coat protein gene (CP) of this virus shares 49.2% nucleotide and 55.1% amino acid sequence identity with that of RoYMV. These results suggest that this virus, which was named "phellodendron yellow ringspot-associated virus" (PYRaV) is a new member of the genus Roymovirus.