Crimson Publishers Research Article

Wings to the Research

First Report of Tomato Spotted Wilt

Orthotospovirus Infecting Polygonum
Multiflorum in China
Qin Zhuo 1,2#, Xue Zheng2#, Shaozhi Zhang2, Xiaoman Ai3, Xiaofang Zhang3, Lihua
ISSN: 2637-7659
Zhao2* and Zhongkai Zhang2**
1
College of Agriculture, Yunnan University, Kunming 650504, Yunnan, China
2
Biotechnology and Germplasm Resources Institute, Yunnan Academy of Agricultural Sciences; Yunnan
Provincial Key Lab of Agricultural Biotechnology; Kunming 650205, Yunnan, China
3
Southwest Forestry University, Kunming 650224, Yunnan, China

Abstract
Tomato Spotted Wilt orthotospovirus (TSWV) is a member of the Orthotospovirus genus. It has
extensive host and causes serious disease in many crops. In May 2021, virus-like symptoms such as
chlorosis, mottling, leaf yellowing and necrosis were observed on Polygonum multiflorum (hereinafter
as P. multiflorum) leaves in Kunming, Yunnan Province, China. Spherical viral particles with a diameter
of 80-120nm were observed by TEM.RT-PCR and DNA sequencing analysis were performed, and the
*Corresponding author: Lihua Zhao, Bi- results showed that the sequence of N gene for this virus shared 99.20% identity with TSWV isolate
otechnology and Germplasm Resources from Shandong isolate (acc. no. MN861982.1).The obtained 777-bp consensus sequence named TSWN-N
Institute, Yunnan Academy of Agricultural was deposited in GenBank (acc. no. OP800910).Phylogenetic tree analyses suggested that TSWV obtained
Sciences; Yunnan Provincial Key Lab of Ag- from P. multiflorum plant samples clustered together into a large clade from Korea. Koch’s postulate was
ricultural Biotechnology; Kunming 650205, performed, and the virus was re-amplied from the inoculated plants. This is the first report of TSWV on P.
Yunnan, China
multiflorum in China and provides early warning of virus disease on medicinal materials.
Zhongkai Zhang, Biotechnology and Ger-
mplasm Resources Institute, Yunnan Keywords: Tomato spotted wilt orthotospovirus; Polygonum multiflorum; Electron microscopy; RT-PCR
Academy of Agricultural Sciences; Yunnan
Provincial Key Lab of Agricultural Biotech-
nology; Kunming 650205, Yunnan, China Introduction
Polygonum multiflorum (hereinafter as P. multiflorum) is a perennial herb that belongs
Submission: April 12, 2023
Published: May 05, 2023 to the genus Pleuropterus genus and Polygonaceae family [1]. It is a famous traditional Chi-
nese medicine and as a tuber has been used for treating disease such as blackening hair, an-
Volume 13 - Issue 1 ti-aging, anti-hyperlipidemia, antioxidant, anti-inflammatory, anticancer, hepatoprotection,
How to cite this article: Qin Zhuo, cardio-protection and improving age-related cognitive dysfunction [2]. Owing to its high me-
Xue Zheng, Shaozhi Zhang, Xiaoman Ai, dicinal value, P. multiflorum usually is distributed mainly in Sichuan, Yunnan, Guizhou and
Xiaofang Zhang, Lihua Zhao* and Zhongkai southern Shanxi and Gansu in China [1]. Diseases often occur in the planting process and cause
Zhang**, et al. First Report of Tomato serious economic losses. The most widespread disease on P. multiflorum is ceitocybe bescens,
Spotted Wilt Orthotospovirus Infecting
Polygonum Multiflorum in China. Mod rust disease, leaf spot disease and some fungal diseases or insect pests [3-5]; However, there
Concep Dev Agrono. 13(1). MCDA. 000801. are few reports about the virus that infects P. multiflorum. Polygonum ringspot virus (PolRSV),
2023. a recently described tospovirus [6], has been reported only on the weed species Polygonum
DOI: 10.31031/MCDA.2023.13.000801 dumetorum and Polygonum convolvolus in different geographic areas in Italy. So far, there have
Copyright@ Lihua Zhao and Zhongkai been no reports about the tomato spotted wilt virus infecting P. multiflorum [7,8]. In this pa-
Zhang. This article is distributed under per, symptomless P. multiflorum plants leaves were received from professionally cultivated
the terms of the Creative Commons fields for regular control of virus infection in May 2021. TEM and the molecular investigations
Attribution 4.0 International License, indicated that this virus was TSWV. This work was done to fulfill Koch’s postulate and verify
which permits unrestricted use and
redistribution provided that the original TSWV is able to infect P. multiflorum plants after artificial inoculation in fields.

Materials and Methods

Samples

Surveys were conducted in May 2021 in Kunming, Yunnan Province, China. Symptoms

Modern Concepts & Developments in Agronomy 1235

 MCDA.000801. 13(1).2023 1236

were observed on individual P. multiflorum of the same variety dis- TSWV-R primer annealing temperature 58 °C), 72 °C extension for
playing virus-like symptoms including foliar viral symptoms con- 45s, 35 cycles, 72 °C further extension for 10 min.
sisting of chlorosis, mottling, leaf yellowing and necrosis. The five
Target gene clones and sequencing analysis
sample leaves of P. multiflorum were collected from diseased parts
of plants and stored in a refrigerator at -80 °C on the same day for The target fragment DNA was recovered and purified. The re-
RT-PCR. covered products were connected to D18-T vector using the fast
DNA ligation kit. The ligation products were transferred into E.coli
Transmission electron microscopy DH5αcompetent cells. The positive clones were selected and sent
The virion morphology and size of the virus in P. multiflorum to Beijing Qingke Biotechnology Co., Ltd. for sequencing, and the
were confirmed by Transmission Electron Microscopy (TEM). Fol- sequencing results were analyzed by BLAST alignment analysis.
lowing the methods of introduction, negative staining was per- Phylogenetic analyses
formed for infected and healthy leaves and was observed under a
Phylogenetic analyses were conducted using MEGA version 6.0;
transmission electron microscope with an accelerating voltage of
The maximum likelihood that the (ML) tree was constructed with
80kV [9].
1000 replicates as the guide value to evaluate the reliability of the
RT-PCR amplification resulting tree [10].
Total RNA was extracted from symptomatic and symptomless Result
leaves using Trizol reagent (Nanjing, Norvezan Biotechnology) ac-
cording to the manufacturer’s protocol. The total RNA extracted TEM detection
above was reverse transcribed to complete the first-strand cDNA In May 2021, virus-like symptoms on leaves consisting of chlo-
synthesis according to the HiScriptIII 1st Strand cDNA Synthesis Kit rosis, mottling, leaf yellowing and necrotic were observed and ap-
(Nanjing, Norvezan Biotechnology). The primers were designed by peared on approximately 80% of P. multiflorum in the fields in Kun-
primer 5 and Table 1. PCR reaction system of 25μL: 10×PCR reaction ming, Yunnan (Figure 1A). The five samples with symptoms and
buffer (including Mg2+) 2.5μL, dNTP Mix 2μL, 10μmol/L upstream symptomless were taken for Transmission Electron Microscopy
and downstream primers each of 0.5μL, rTaq DNA polymerase (TEM) detection using negative staining [9], respectively. The re-
0.25μL, cDNA 1.25μL, ddH2O 18μL. Amplification conditions: 94 °C sults revealed spherical particles of 80-120 nm in diameter, similar
pre-denaturation 30s, 98 °C denaturation 10s, 53 °C annealing 30s to Orthotospovirus (Figure 1B).
(TospS-3 W/Tosp-3 primer annealing temperature 53 °C, TSWV-F /

Figure 1: Electron micrograph of virus particles found in P. multiflorum leaves.

A. Pictures of the leaf symptoms.
B. Viruses under electron microscopy.

PCR detection and sequence and cloned into the pMD18-T vector for Sanger sequencing (Takara,
Dalian, China). BLASTn-analysis revealed that the 5 amplicons were
In order to identify the exact virus, five samples of P. multiflorum
identical and shared 99.20% nucleotide sequence identity with To-
with symptoms and symptomless were tested by RT-PCR. Total RNA
mato spotted wilt orthotospovirus isolate Qingdao from Tobacco
was extracted using the RNA-easy Isolation Reagent (Vazyme, Nan-
(acc. no. MN861982.1). One sequence was deposited in the Gen-
jing, China), followed by reverse transcription (RT)-PCR with pairs
Bank under the accession number OP800910. Koch’s experiments
of universal primers for N gene of Orthotospovirusviruses (Table
also were performed and the virus from the infected plants was
1). A 777-bp amplicon was obtained from each sample (Figure 2)

Mod Concep Dev Agrono Copyright © Lihua Zhao and Zhongkai Zhang
 MCDA.000801. 13(1).2023 1237

successfully transmitted onto healthy P. multiflorum plants (n=5) distortion symptoms, but also tested positive for TSWV by RT-PCR
upon mechanical inoculation as the plants not only developed foliar with the N-specific primers (Table 1).

Figure 2: Presence of sequence in P. multiflorum plant samples by RT-PCR amplification.

M: DL2000 DNA Maker; A. Tomato spotted wilt virus genus virus amplification;
B. TSWV N gene fragment amplification.
Table 1: Primers used to amplify gene sequence.

Primers Sequence (5′-3′)

TospS-3 W GC (a/t) GTTCCAGGGTT (a/g)
Tosp-3 AGAGCAATCGAGGCGCTA ATAA
TSWV-F 5′-ATGTCTAAGGTTAAGCTCA-3′
TSWV-R 5′-AGCAAGTTCTGCAAGTTTTG-3′

Phylogenetic analysis The results are showed that the isolate of P. multiflorum grouped
with several TSWV isolates (e.g., LC505372.1, MK372883.1 and
The phylogenetic tree was prepared using the available amino
KP684518.1) from Korea and South China (Figure 3).
acid sequence of N protein of P. multiflorum and the N gene cor-
responding amino acid sequence of TSWV from different regions.

Figure 3: A phylogenetic tree constructed based on the tomato spotted wilt virus N gene sequence.

Mod Concep Dev Agrono Copyright © Lihua Zhao and Zhongkai Zhang
 MCDA.000801. 13(1).2023 1238

Discussion pleuropterus multiflorus. Journal of Tropical and Subtropical

Botany 29(4): 439-450.
Tomato spotted wilt orthotospovirus (TSWV), belonging to the
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family, is an important thrips-borne RNA virus [11]. It is the plant
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plants in 84 families, and it has caused serious economic losses to
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Acknowledgement
15. Pappu SS, Papp, HR, Gitaitis RD, Gay JD (1998) First report of
The authors acknowledge financial support from Important tomato spotted wilt tospovirus infection of watermelon in
plan of research and development (202103AF140003); Yunnan Georgia. Plant Dis 82(3): 351.
Fundamental Research Projects (202101AT070269).
16. Al-Saleh MA, Al-Shahwan I M, Amer MA, Shakeel MT, Ahmad MH,
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