First Report of Tomato spotted wilt virus infecting

Strawflower (Helichrysum bracteatum, Asteraceae) , an annual or biennial herb, is one of the most popular flowers in the world because of the colorful flowers and the long flowering period. However, the ornamental plants belonging to Asteraceae are susceptible to numerous viruses such as cucumber mosaic virus (CMV) (Cucumovirus, Bromoviridae) , potato virus Y (Potyvirus, Potyviridae), tomato mosaic virus (ToMV) (Tobamovirus, Virgaviridae), tobacco mosaic virus (TMV) (Tobamovirus, Virgaviridae), chrysanthemum virus B (CVB) (Carlavirus, Betaflexiviridae), tomato aspermy virus (TAV) (Cucumovirus, Bromoviridae), tomato spotted wilt virus (TSWV) (Orthotospovirus tomatomaculae, Tospoviridae), and impatiens necrotic spot virus (INSV) (Orthotospovirus impatiensnecromaculae, Orthotospovirus) resulting in severe yield loss (Verma et al. 2003; Raj et al. 2007; Kondo et al. 2011; Liu et al. 2014; Marys et al. 2014; Min et al. 2020; Gautam et al. 2021; Read et al. 2022; Supakitthanakorn et al. 2022). Among these viruses, the TSWV, a thrips-transmitted negative-stranded RNA virus, is well known to cause viral disease in several plant species while is less reported in Helichrysum, especially in China. In April 2021, viral attack symptoms were detected on the leaves of H. bracteatum during our routine checks in the greenhouse located at Shunyi District, Beijing, China, such as wilting, shrinking, chlorotic blotches, chlorotic ring spots. To investigate the virus infecting H. bracteatum, in total of 25 symptomatic and 5 asymptomatic leaves were sampled and tested by the effective double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) using antisera against CMV, PVY/PVX, ToMV, TMV, CVB, TAV, TSWV, INSV, separately (Agdia, USA). Only the TSWV showed positive in symptomatic samples, and asymptomatic samples were all negative, which implied TSWV infection. To further confirm the virus type of TSWV isolated from H. bracteatum samples, the genomic RNA of the virus was isolated using reverse transcription and polymerase chain reaction (RT-PCR), and then was cloned, sequenced and analyzed. Total RNA of five symptomatic leaves (ELISA-positive) were extracted using the FastPure Plant Total RNA Isolation Kit (Vazyme, China), and then were reverse transcribed by HiScript II Reverse Transcriptase (Vazyme, China). Each genome segments were amplified using Phanta Max Super-Fidelity DNA Polymerase (Vazyme, China) with TSWV-specific primers newly designed and listed in Table S1. The PCR setup was as follow: 95°C for 30 s, followed by 35 cycles at 95°C for 30 s, 55°C for 30 s, and 72°C for 1.5 min, with a final extension at 72°C for 10 min. All PCR products were cloned into the TA/Blunt-Zero vector (Vazyme, China) and sequenced (GENEWIZ, Inc.). We assembled and then analyzed the evolutionary relationship of three genomic fragments, that is, TSWV-BJFC-Hb S (2923 bp), M (4785 bp) and L (8971 bp) using the BLAST tools. The results showed high similarity with TSWV-henan isolated from pepper in China (99.6% to TSWV-S (MT799179.1), 99.8 % to TSWV-M (MT799178.1) and 99.8 % TSWV-L (MT799177.1)). These sequences have been submitted to the GenBank (OM982910, OM982911 and OM937131). Taking all of these evidences together, the viral disease observed in H. bracteatum was closely associated with TSWV. TSWV is currently widespread in China, infecting Nasturtium, Chrysanthemum and cowpea (Xiao et al. 2015; Hu et al. 2018; Yu et al. 2021). Epidemics of TSWV have also been reported in several other countries such as Korea, North Carolina, Turkey and India (Renukadevi et al. 2015; Koehler et al. 2016; Kwak et al. 2021; Erilmez, S. 2022). This is the first report of TSWV infection on H. bracteatum in China. Due to the fast spread and serious economic losses of TSWV, the rapid detection may be the essential way to prevent this viral disease among crops (Macharia et al. 2014).