Identification of Viral Diseases Affecting tobacco plant in Spring Young Dog, Republic of Korea

1. Identification of Viral Diseases Affecting tobacco plant in Spring Young Dog, Republic of
Korea
Belete, Mesele Tilahun1, 2
Gudeta, Workuitu Firmosa1,2
Lee SagnHee1,2
and Jae Son Moon 1, 2
1
Korea Research Institute of Bioscience and Biotechnology, 125 Gwahak-ro, Yuseong-gu,
Daejeon 34114, South Korea
2
University of Science and Technology (UST), Yuseong, Daejeon 34113, South Korea
Abstract: The RT-PCR detecting result of pooled sample showed that tobacco ringspot virus,
tobacco streak virus, tobacco necrosis virus, and tobacco mosaic viruses were infected tobacco
plant.
1. INTRODCUTION
Tobacco (Nicotiana tabacum) is a significant and vital crop, particularly in low-income agricultural
areas [1]. Tobacco is grown in over 125 countries, on over 4 million hectares of land. The global
tobacco crop is worth approximately US$20 billion, a small fraction of the total amount generated
from the sale of manufactured tobacco products. Tobacco crops are susceptible to a wide range of
diseases and viruses, which can infect them naturally or experimentally [1], [2].
Several viruses have been detected in tobacco plant all around world, even though only a few are
reported in Korea, some of these are: Tobacco ringspot virus (TRSV, family: Secoviridae, Genus:
nepovirus), Tobacco leaf curl virus (TLCV, family: Geminiviridae, Genus: Begomovirus), tobacco
mild green mosaic virus (TGMV, family: Virgaviridae Genus: Tombamovirus) and Tobacco Mosaic
Virus (TMV, family: Virgaviridae Genus: Tombamovirus) etc.
Other worldwide tobacco infected viruses are aphid-transmitted Potato Virus Y (PVY, Family: Potyviridae,
Genus: Potyvirus), Alfalfa mosaic virus (AMV, Family: Bromoviridae, Genus: Alfamovirus), thrips
transmitted Tomato spotted wilt virus (TSWV, Family: Bunyaviridae, Genus: Tospovirus), Cucumber
mosaic virus (CMV, Family: Bromoviridae, Genus: Cucumovirus) and Tobacco rattle virus (TRV, Genus:
Tobra- virus) are thought to be the most prevalent ones [1]–[5]. The study's main goals were to figure out
and determine what type of viruses affect tobacco plant in major growing areas in Korea, as well as their
occurrence and relevance.
Tobacco mosaic virus (TMV) was initially found out in tobacco, and could infect more than 350 various
plant species, consisting tomato, pepper, tobacco, cucumber, tomato, cucumber, etc. [4]. Tobacco necrosis
virus (TNV) is a tiny icosahedral fungal-transmitted virus with a wide host range that includes woody
plants, herbaceous, dicotyledonous and monocotyledon, however systemic infection is rare. Over 20
tobacco necrosis virus isolates and many associated satellite viruses (STNV) have been identified since it
was originally described in 1935 [6]. Tobacco ringspot virus (TRSV) has a wide host range, infecting both
woody and herbaceous plants and causing serious illnesses in tomato, blueberries, cucurbits, tobacco,
grapevine and soybean. Tobacco ringspot virus induced soybean bud blight, in particular, causes significant
yield reduction and low quality of seeds [3].

2. No Virus Name Abrv. Country Accession no.
1 Satellite tobacco mosaic virus STNV USA V01468.1
2 Tobacco ringspot virus RNA 2 TRSV Korea KJ556850.1
Tobacco ringspot virus RNA 1 Korea KJ556849.1
3 Tobacco Mosaic Virus TMV China JF920727.1
4 Tobacco necrosis virus A TNV-A China AY546104.1
5 Tobacco necrosis satellite virus TNSV USA NC_001557.1
6 Tobacco virus 2 USA NC_034265.1
7 Tobacco vein distorting virus TVDV China EF529624.1
8 Tobacco bushy top virus TBTV China NC_004366.1
10 Tobacco vein clearing virus TVCV USA NC_003378.1
11 Tobacco mild green mosaic virus TMGMV USA NC_001556.1
12 Tobacco vein banding mosaic virus TVBMV China MH898883.1
13 Tobacco etch virus TEV USA KM282189.1
16 Tobacco curly shoot virus TbCSV India JX467693.1
17 Tobacco virus 1 TMV-1 China NC_027712.1
18 Tobacco yellow dwarf virus TYDV USA NC_003822.1
19 Tobacco rattle virus TRV Germany KX096038.1
20 Ethiopian tobacco bushy top virus ETBTV Zambia MT225089.1
23 Tobacco vein mottling virus TVMV USA NC_001768.1
24 Tobacco streak virus USA KM504248.1
25 Tobacco leaf curl Yunnan virus TbLCYNV China MN218671.1
26 Tobacco leaf curl virus(Tomato) Korea KT992047.1
30 Tobacco necrosis virus D (Glycine max) Canada MF158986.1
31 Tobacco yellow virus China MK751486.1
33 Tobacco Yellow crinkle virus Cuba NC_015627.1
34
Velvet tobacco mottle virus satellite RNA VTMoV USA NC_003906.1
35 Tobacco mosaic virus strain Ohio V TMV Ohio V USA FR878069.1
37 Tobacco leaf curl Zimbabwe virus Zimbabwe NC_002817.1
38 Tobacco leaf curl Cuba virus Cuba NC_035888.1
40 Tobacco curly shoot virus Tomato India JX467693.1
41 Velvet tobacco mottle virus Australia NC_014509.2
42 Tobacco mosqueado virus TMOSQV Brazil NC_030118.1
43 Tobacco leaf curl Thailand virus TLCV Germany NC_009553.1
Table 1. List of the major infecting Tobacco virus in worldwide and their location
2. Materials and methods
Sample collection and field tour took place between Jun 11- 12, 2021, when the plants have been before
their reproductive phase. We were collect tobacco plant having the virus-like symptoms of leaf
malformation, yellowing, necrosis and stunting. The sample collection field region are indicated in Fig.1
plants exhibiting viral symptoms have been chosen purposely at farming field.
A total of 5 tobacco plants virus-infected samples (n = 5) were collected from the province based observation of
viral disease symptoms estimation near a location Jinbo-Myeon, Korea. The samples were put in explicitly
labeled plastic bags and brought to Korean Research Institute of Bioscience and Biotechnology(KRIBB)
laboratory in building 6th
, 4th floor.

3. Fig1. Image of tobacco plants showing viral disease symptom on the farmers filed
Laboratory tests
The collected samples were individually ground to a fine powder and stored at −80 °C for further studying. The
samples (n = 5) were pooled for detecting. Total RNA was extracted from the pooled sample using the WizPrep™
Plant RNA Kit (Wizbiosolution, Seongam, Korea), and cDNA were synthesized with the 25-mer degenerate
primer N25 using Revert Aid Reverse Transcriptase (Thermo Scientific, Waltham, MA, USA), following
to the manufacturer’s instructions. The cDNAs were amplified by PCR with AccuPower ProFi Taq PCR
PreMix kit (Bioneer, Republic of Korea) using that of 7 oligo pair of primer tobacco virus according to the
thermal profile: 95 °C for 5 min followed by 36 cycles at 95°C for 20 seconds, annealing in temperature
appropriate to primers (table 1) for 30 seconds and extension step at 68°C for 1 minutes per 1 kb. The RT-
PCR amplified products were analyzed by 1% gel electrophoresis having EtBr staining and visualized under
UV light.
3. Results and Discussion
Fig 2. shows RT-PCR was used to analyze the 7 viruses, and a 1 % ethidium bromide-stained agarose gel
was visualized under UV light. The Laboratory RT-PCR detecting result showed that TRSV, TSV, TNV
and TMV virus were observed from pooled sample.
Virus name Abr. Expected Size result
1 Tobacco ring spot virus TRSV 222 +
2 Tobacco vein banding mosaic virus TVMV 515 -
3 Tobacco streak virus TSV 512 +
4 Tobacco necrosis virus TNV 781 +
5 Tobacco mosaic virus TMV 597 +
6 Tobacco mild green mosaic virus TMGMV 550 -
7 Tobacco etch virus TEV 326 -
Table 2: viral infectivity result of pooled tobacco sample: (+) infected sample and (-) shows non infected
virus).

4. The samples (n = 5) were pooled and the we were for detected seven viruses from pooled sample. The
result Fig demonstrated that the sample was infected with 4 viruses namely: tobacco ring spot virus
(TRSV), tobacco streak virus (TSV) tobacco necrosis virus (TNV), tobacco mosaic virus (TMV). Tobacco
ring spot virus: (family: Secoviridae Genus: Nepovirus ) bipartite ssRNA(+) genome composed of RNA-
1 ~ 7.5 kb and RNA-2 ~ 3.9 kb. Tobacco streak virus: (Family: Bromoviridae Genus: Ilarvirus) tripartite
linear ssRNA genome composed of RNA1, RNA2, RNA3. Tobacco necrosis virus (Family Tombusviridae
) monopartite (except Dianthovirus which is bipartite), ssRNA genome of 4-5.4 kb, which lacks a cap
structure and a poly(A)tail. Tobacco mosaic virus: (TMV, Family Virgaviridae Genus: tobamovirus)
monopartite, ssRNA genome of 6.3-6.5 kb. TMV is one of the most damaging viruses to crops, causing
significant production losses and reducing product quality.
4. Reference
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