First Report of Tomato Spotted Wilt Virus Isolated from Nasturtium (<i>Tropaeolum majus</i>) 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.

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