Annals of Applied Biology ISSN 0003-4746

RESEARCH ARTICLE

Identification, characterisation and detection

of a new tospovirus on sweet pepper
Y.-H. Cheng1,† , Y.-X. Zheng2,† , C.-H. Tai2 , J.-H. Yen3 , Y.-K. Chen2 & F.-J. Jan2,4,5
1 Plant Pathology Division, Taiwan Agricultural Research Institute, Taichung, Taiwan
2 Department of Plant Pathology, National Chung Hsing University, Taichung, Taiwan
3 Agricultural Extension Center, National Chung Hsing University, Taichung, Taiwan
4 Agricultural Biotechnology Center, National Chung Hsing University, Taichung, Taiwan
5 NCHU-UCD Plant and Food Biotechnology Program, Biotechnology Center, National Chung Hsing University, Taichung, Taiwan

Keywords
Capsicum annuum; fruit deformation; mottle;
sweet pepper; Tospovirus.
Correspondence
Dr F.-J. Jan, Department of Plant Pathology,
National Chung Hsing University, Taichung
402, Taiwan.
Email: fjjan@nchu.edu.tw
†
These authors contributed equally to this
work.
Received: 29 May 2013; revised version
accepted: 15 September 2013; published
online: 31 October 2013.
doi:10.1111/aab.12084
Abstract
A putative virus-induced disorder showing mottling and deformation on
leaves and fruits of sweet pepper was observed in Taiwan in 2009. Crude
sap of symptomatic sweet pepper leaves reacted positively in enzyme-linked
immunosorbent assay (ELISA) with antisera to tospoviruses in the Watermelon
silver mottle virus (WSMoV) serogroup. A virus culture, TwPep3, isolated from
the symptomatic sweet pepper revealed isometric virions measuring about
80–100 nm in diameter via electron microscopy. Seedlings of sweet pepper
inoculated with TwPep3 showed similar symptoms as those observed in the
field. A 0.9-kb viral genome of isolate TwPep3 could be amplified using
degenerate primers for L RNA conserved region of tospoviruses but not using
those of tobamoviruses, potyviruses and Cucumber mosaic virus. The partial
L RNA sequence of isolate TwPep3 (KF383955) shared 75.1% and 82.4%
nucleotide identities with those of WSMoV and Tomato necrotic ringspot
virus (TNRV), respectively. To determine the taxonomic relationship of isolate
TwPep3, the full length of S RNA (KF383956) was sequenced. Sequence
analyses showed that the nucleocapsid (N) gene of TwPep3 shared 82–83%
and 87.9–89% nucleotide and amino acid sequence identities, respectively,
with those of five isolates of TNRV, the most closely related tospovirus.
Moreover, the NSs proteins and S RNAs of TwPep3 and TNRV shared only
78.7% and 77.6% amino acid and nucleotide sequence identities, respectively.
Based on our results, isolate TwPep3 should be considered as a new tospovirus
and we propose the name Pepper chlorotic spot virus (PCSV). A specific primer
pair designed in the N gene was successfully used in reverse transcription-
polymerase chain reaction for PCSV diagnostic work.

Introduction reported to infect peppers (Green & Kim, 1994; Pernezny

Pepper (Capsicum annuum L.) is native to the Central
and South America and is one of the most important
vegetables in the family Solanaceae. Viral diseases often
cause large losses of peppers worldwide. Potyviruses,
tobamoviruses, tospoviruses, begomoviruses and Cucum-
ber mosaic virus (CMV) are the most common viruses
infecting solanaceae crops (Green, 1991; Pernezny et al.,
2003). To date, at least 67 viruses of 25 genera have been
et al., 2003). In Taiwan, more than 13 viruses have been
reported to infect peppers. These viruses are Chilli veinal
mottle virus (ChiVMV) (Tsai et al., 2008), CMV (Lin &
Chen, 1976), Peanut stunt virus (PSV) (Huang et al., 1994),
Pepper mild mottle virus (PMMoV) (Green & Kim, 1991),
Pepper veinal mottle virus (PVMV) (Cheng et al., 2009), Pep-
per mottle virus (PepMoV) (Cheng et al., 2011), Potato virus
S (PVS) (Lu & Lu, 1981), Potato virus X (PVX) (Green &
Kim, 1991), Potato virus Y (PVY) (Green & Kim, 1991),

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A new tospovirus infecting sweet pepper
Tobacco mild green mosaic virus (TMGMV) (Li & Chang,
2005), Tobacco mosaic virus (TMV) (Green & Kim, 1991),
Tomato spotted wilt virus (TSWV) (Zheng et al., 2010) and
Tomato yellow leaf curl Thailand virus (TYLCTHV) (Shih
et al., 2010).
Pepper is cultured in the central and southeastern
Taiwan. In 2009, TSWV, the type species of Tospovirus,
was found on sweet pepper in central Taiwan (Zheng
et al., 2010). Tospoviral particles about 80–120 nm in
diameter are quasi-spherical lipoprotein-enveloped. The
genome of tospoviruses consists of three single-stranded
RNAs designated as S, M and L RNAs according to their
size. The S RNA encodes the nucleocapsid (N) and the
nonstructural protein (NSs) in the viral complementary
and viral strands, respectively. The N protein is the
structural protein and the NSs protein is a RNA silencing
suppressor. Similar to S RNA, M RNA encodes the
nonstructural protein (NSm) and glycoprotein precursor
(GnGc) in an ambisense organisation. The NSm protein
is the movement protein and GnGc is the precursor
of glycoproteins (Gn and Gc) that form the spikes
on the lipid envelope of viral particles. The L RNA
harbours one open reading frame (ORF) for L protein
in the viral complementary strand. The L protein is
the RNA-dependent RNA polymerase with the functions
of helicase, transcriptase, replicase and endonuclease.
Tospoviruses have a wide host range and are thrips-
transmitted (Jan et al., 2003).
A new disorder showing symptoms of mottle and
deformation on leaves and fruits was observed on net
house-cultured sweet peppers in Sinyi Township, Nantou
County, Taiwan in 2009 and 2010. In this study, the
causal agent was identified and characterised. It is a
new tospovirus for which the provisional name Pepper
chlorotic spot virus (PCSV) is given.
Materials and methods
Virus source and maintenance
Symptomatic plants of sweet pepper were collected
in Sinyi Township, Nantou County, Taiwan in 2010.
A virus culture (isolate TwPep3) was isolated from
symptomatic sweet peppers and maintained in the
systemic host, Nicotiana benthamiana, and a local lesion
host, Chenopodium quinoa, by mechanical inoculation
using 30-fold dilution of the inoculum prepared by
grinding infected leaf tissue in buffer 4 (0.033 M KH2 PO4 ,
0.067 M K2 HPO4 and 0.01 M Na2 SO3 , pH 7.0).
Electron microscopy
Ultrathin sections of the symptomatic leaves of N.
benthamiana plants infected with isolate TwPep3 were
108
Y.-H. Cheng et al.
prepared by the protocol described previously (Jan &
Yeh, 1995; Zheng et al., 2008).
Purification of nucleocapsid protein and production of
polyclonal antibody to TwPep3
Nucleocapsid protein (NP) of isolate TwPep3 was purified
by the procedures described by Zheng et al. (2008).
Polyclonal antibody (PAb) against the NP of isolate
TwPep3 was generated in a New Zealand white rabbit
as described previously (Jan & Yeh, 1995).
ELISA
The procedure of indirect enzyme-linked immunosorbent
assay [indirect enzyme-linked immunosorbent assay
(ELISA)] followed the method previously described
(Zheng et al., 2008). Rabbit antisera for TSWV, Capsicum
chlorosis virus (CaCV), Impatiens necrotic spot virus (INSV),
Peanut chlorotic fan-spot virus (PCFV), Watermelon silver
mottle virus (WSMoV), PVY, Bean common mosaic virus
(BCMV), Dasheen mosaic virus (DsMV), Turnip mosaic
virus (TuMV), Zantedeschia mild mosaic virus (ZaMMV),
Zantedeschia mosaic virus (ZaMV), PMMoV, TMV, Tomato
mosaic virus (ToMV), CMV, Carnation mottle virus (CarMV)
and TwPep3 were used for virus detection of the
symptomatic plants of sweet pepper and the test plants
inoculated with isolate TwPep3.
Host range and back inoculation
To characterise the biological properties of isolate TwPep3,
plants from 26 species representing six families were
used for a host-reaction test. Leaves of C. quinoa plants
inoculated with isolate TwPep3 were grinded in buffer 4
and the young leaves of the test plants were rubbed
with TwPep3 sap inocula and carborundum powder.
Inoculated plants were grown in a greenhouse for
at least 30 days. Virus presence in both symptomatic
and asymptomatic inoculated plants was confirmed by
indirect ELISA using the antiserum against isolate TwPep3
prepared in this study.
Immunoblotting
Immunoblotting was conducted as described previously
(Jan & Yeh, 1995; Zheng et al., 2008). Total protein extract
of mock-inoculated and virus-infected C. quinoa leaves
was separated in 0.8% gel by sodium dodecyl sulphate
(SDS)-polyacrylamide gel electrophoresis and then trans-
ferred to a polyvinylidene difluoride membrane (Perkin
Elmer, Waltham, MA, USA). After blocking in 10 mM
Tris, 0.9% NaCl, 0.25% gelatin, 0.1% Triton-X100 and
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Y.-H. Cheng et al. A new tospovirus infecting sweet pepper

0.02% SDS, pH 7.4, proteins were incubated with the Table 1 Comparison of the nucleotide (nt) and deduced amino acid (aa)
antiserum against NP of TwPep3, CaCV or WSMoV. Fol- sequence identities of S RNA (KF383956) and the encoded genes of Pepper
chlorotic spot virus (PCSV-TwPep3) with those of other tospoviruses
lowing the addition of alkaline phosphatase-conjugated
available in GenBank
IgG, nitro blue tetrazolium / (5-bromo-4-chloro-1H-
indol-3-yl) dihydrogen phosphate chromogenic sub- NSs N
strates (NBT/BCIP) were used to visualise hybridisation. Virusa Accession No.b S RNA nt (%) aa (%) nt (%) aa (%)

TNRV-TT1 FJ489600 77.6 78.5 78.7 82.9 88.3

Molecular cloning and sequence analysis TNRV-CR FN995637 – – – 82.0 89.0
TNRV-Q1510 HM113532 – – – 83.0 88.3
For identification of isolate TwPep3, partial fragments TNRV-T1 FJ946835 – – – 82.9 88.3
of the TwPep3 genome were amplified and cloned TNRV-PP1 FJ947153 – – – 82.9 87.9
GBNV U27809 60.8 58.3 47.7 62.6 60.0
following the procedure previously described (Zheng et al.,
CaCV KC953852 55.2 58.5 48.6 61.0 58.5
2008). Three primer pairs designed for the detection of WBNV EU249351 61.5 58.7 47.7 60.6 57.5
potyviruses (Hrp5 and Pot1) (Colinet & Kummert, 1993; CCSV AY867502 62.0 60.0 50.9 62.1 57.0
Chen et al., 2006), tospoviruses (gL3637 and gL4435c) WSMoV NC_003843 54.2 57.3 47.3 58.8 56.4
TZSV EF552433 60.6 59.4 51.5 58.9 54.7
(Chu et al., 2001a) and tobamoviruses (Tob-Uni1 and
MYSV AB038343 58.3 55.6 42.9 57.9 52.2
Tob-Uni2) (Letschert et al., 2002), and the specific primers TYRV AY686718 58.3 55.2 45.3 57.5 46.7
for the coat protein gene of CMV (Zheng et al., 2010) IYSV AF001387 58.0 55.9 45.1 57.6 45.4
were used for virus identification. The amplification of PolRSV EF445397 58.2 55.1 44.0 56.9 45.2
BeNMV NC_018071 50.9 47.2 21.8 52.6 34.2
TwPep3 N gene was performed using the primer pair UHP
TSWV AF020660 48.6 45.8 22.1 48.4 32.4
and AT (Hassani-Mehraban et al., 2007). For sequence SVNV HQ728387 49.4 45.7 22.5 49.6 31.5
investigation of TwPep3 S RNA, the primer, FJJ2011- MeSMV EU275149 51.0 47.9 18.5 50.8 30.8
179, designed from S RNAs of Tomato necrotic ringspot GRSV L12048 50.1 47.3 20.3 49.7 30.1
PNSV HE584762 48.2 46.6 19.8 47.7 29.7
virus (TNRV), Watermelon bud necrosis virus (WBNV),
ANSV GQ478668 – – – 50.5 29.3
Groundnut bud necrosis virus (GBNV) and CaCV was used to CSNV AB600873 49.1 44.8 20.4 50.8 29.1
amplify the NSs gene. The specific primers (FJJ2011-198, TCSV AF282982 – – – 50.2 28.9
FJJ2011-199, FJJ2011-201 and FJJ2011-202) designed ZLCV AF067069, – 45.5 21.9 49.7 27.5
from the identified sequences were used for amplification JN572104
INSV X66972 51.7 47.9 19.4 49.2 27.2
of the intergenic region (IGR) and the 5 and 3 terminal GYSV AF013994 48.2 48.3 19.6 48.7 21.5
regions. The procedure to clone the 5 and 3 terminal PCFV AF080526 47.7 46.8 17.6 46.1 19.1
regions of S RNA was described previously (Zheng et al., a
Full names of tospoviruses are Tomato necrotic ringspot virus (TNRV),
2011). The Lasergene 7 software (DNASTAR, Madison,
Groundnut bud necrosis virus (GBNV), Capsicum chlorosis virus (CaCV),
WI, USA) was used to analyse the TwPep3 sequences. Watermelon bud necrosis virus (WBNV), Calla lily chlorotic spot virus
The cDNA sequence of TwPep3 was assembled using the (CCSV), Watermelon silver mottle virus (WSMoV), Tomato zonate spot
SeqMan program. The amino acid sequences of viral genes virus (TZSV), Melon yellow spot virus (MYSV), Tomato yellow ring virus
were deduced using the SeqBuilder program. The degree (TYRV), Iris yellow spot virus (IYSV), Polygonum ringspot virus (PolRSV),
of nucleic acid identity of the genomic RNAs and amino Bean necrotic mosaic virus (BeNMV), Tomato spotted wilt virus (TSWV),
Soybean vein necrosis virus (SVNV), Melon severe mosaic virus (MeSMV),
acid identity of the viral proteins among tospoviruses
Groundnut ringspot virus (GRSV), Pepper necrotic spot virus (PNSV),
was determined by the MegAlign program. The sequence
Alstroemeria necrotic streak virus (ANSV), Chrysanthemum stem necrosis
accession numbers of tospoviral L RNA used for sequence virus (CSNV), Tomato chlorotic spot virus (TCSV), Zucchini lethal chlorosis
analysis are GQ487713 for TNRV, KC953853 for CaCV, virus (ZLCV), Impatiens necrotic spot virus (INSV), Groundnut yellow spot
AF133128 for WSMoV, FJ822962 for Calla lily chlorotic virus (GYSV) and Peanut chlorotic fan-spot virus (PCFV).
b
spot virus (CCSV) and EF552435 for Tomato zonate spot Accession No.: FN995637, HM113532, FJ946835 and FJ947153 for N
virus (TZSV) in NCBI GenBank. The sequences obtained genes of TNRV isolates CR, Q1510, T1 and PP1, respectively; GQ478668
for ANSV N gene, AF282982 for TCSV N gene, AF067069 for ZLCV N gene
from GenBank for S RNA comparison are listed in Table 1.
and JN572104 for ZLCV NSs gene; the others for tospoviral S RNAs.
For calculating the distance matrices of the viral protein
sequences, MEGA4 software version 4.0 (Tamura et al.,
2007) was used. used in reverse transcription-polymerase chain reaction
(RT-PCR). To evaluate the primer specificity and to
identify tospoviruses in infected sweet pepper, plants
Virus detection
infected with CaCV and WSMoV, the two most closely
For disease monitoring and virus detection, a primer related tospoviruses to TwPep3, and primers in their
pair was designed in the N gene of TwPep3 S RNA and N genes were used as controls (Zheng et al., 2008).

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TwPep3, CaCV and WSMoV were maintained in C. quinoa

for the test of primer specificity. Sweet pepper plants A B
showing virus-like symptoms in Sinyi were collected for
TwPep3 detection by RT-PCR. The procedure of total RNA
extraction and RT-PCR was described previously (Zheng
et al., 2008).

Results
Symptoms and virus isolation
Symptomatic sweet peppers collected in Sinyi Township,
Nantou County showed chlorotic and necrotic spots
C D
on leaves, and bud necrosis. Fruits showed mottle and
deformation (Fig. 1A–1C). Leaf samples were tested by
ELISA with antisera produced against TSWV, CaCV, INSV,
PCFV, WSMoV, PVY, BCMV, DsMV, TuMV, ZaMMV,
ZaMV, PMMoV, TMV, ToMV, CMV or CarMV. Among
these antisera, only CaCV and WSMoV reacted positively
with the symptomatic leaves. A virus isolate, TwPep3, was
obtained from leaves of sweet peppers with symptoms of
chlorotic spots and fruit deformation via three consecutive
single-lesion isolation in C. quinoa and then established in
N. benthamiana.

Figure 1 Symptoms induced by Pepper chlorotic spot virus (PCSV-

Host range and back inoculation
From the 26 mechanically inoculated plant species, 19
were susceptible to TwPep3. Among the susceptible
species, C. annuum (sweet pepper and chili pepper),
Datura stramonium, Physalis minima, N. benthamiana,
Nicotiana glutinosa, Nicotiana occidentalis, Nicotiana rustica,
Nicotiana edwardsonii, Nicotiana tabacum var Xanthi,
Nicotiana tabacum var Samsun, Nicotiana tabacum var
Hicks, Nicotiana tabacum cv. White Burley and Vigna
unguiculata (cowpea) showed chlorotic and necrotic spots
on the inoculated and apical leaves. After inoculation
with TwPep3, seedlings of the sweet pepper cultivar
that was planted in Sinyi showed the chlorotic spots,
mottle, and deformation on leaves and the deformed
buds (Fig. 1D). TwPep3-infected sweet pepper fruits
showed the deformation symptom. Solanum lycopersicum
(tomato ‘Farmers 301’, ‘Reward’, ‘Feminine beauty’,
‘Double fortune’, ‘Sugar pearl’ and ‘Clever red’), C.
quinoa, Chenopodium amaranticolor, Gomphrena globosa,
Vigna radiata (mung bean) and Phalaenopsis hybrids ‘V3’
(Phal. Yukimai × Phal. Taisuco Kochdian) are local lesion
hosts. TwPep3-infected leaves of Phalaenopsis hybrids
‘V3’ revealed chlorotic spots at the outset. Then, these
spots developed to be necrotic. Based on the symptom
expression, ELISA results and infectivity assays, C.
quinoa, Citrullus lanatus (watermelon), Luffa acutangula
(luffa), Cucumis sativus (cucumber), Cucumis melo (melon),
Momordica charantia (bitter gourd), Vigna angularis (red
TwPep3) on sweet peppers. Field-observed leaf mottle (A), bud necrosis
(B), fruit mottle (C) and laboratory-inoculated leaf mottle and bud necrosis
(D).
bean), tomato ‘Hawlien-Yasu No. 5’ and ‘Tainan-Yasu
No. 6’ are nonhosts of TwPep3. To confirm these results,
all of the above-mentioned nonhosts were analysed at
least twice in independent experiments with three to five
replicates each.
Electron microscopy
The presence of roughly spherical enveloped virion
particles measuring 80–100 nm in diameter was observed
in ultrathin section of TwPep3-infected N. benthamiana by
electron microscopy (Fig. 2). The morphology of these
particles was typical of tospoviruses.
Serological analysis
The symptomatic peppers collected from the field, and
the N. benthamiana plant inoculated with TwPep3 reacted
with the antisera of CaCV and WSMoV in ELISA. To
distinguish TwPep3 from WSMoV and CaCV and to detect
this pepper-infecting virus, PAb against TwPep3 NP was
generated in a rabbit. In Western blot, a conspicuous
immunoreactive band with an approximate size of 31 kDa
was obtained in the crude sap of TwPep3-infected N.
benthamiana leaves with the TwPep3 antiserum (Fig. 3).

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Y.-H. Cheng et al.
Figure 2 Electron micrographs of spherical enveloped viral particles in
ultrathin sections of Pepper chlorotic spot virus (PCSV)-infected Nicotiana
benthamiana tissues at magnifications of 7000× (upper panel) and
70 000× (lower panel).
Figure 3 Serological relationships of Pepper chlorotic spot virus (PCSV)-
TwPep3, Capsicum chlorosis virus (CaCV) and Watermelon silver mottle
virus (WSMoV) by Western blotting. Antisera against the nucleocapsid
proteins (NPs) of PCSV, CaCV and WSMoV were used to react with the
crude saps extracted from the Nicotiana benthamiana leaves infected
with PCSV (TP), CaCV (C) and WSMoV (W), and the healthy control (H).
Concurrently, TwPep3 reacted slightly with the CaCV and
WSMoV antisera.
Molecular cloning and sequence analysis
To identify the taxonomic relationships of TwPep3 and
other sweet pepper-infecting viruses, genome fragments
of TwPep3 were amplified by RT-PCR using degenerate
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A new tospovirus infecting sweet pepper
primers of potyviruses, tospoviruses and tobamoviruses,
and specific primers of CMV. One full-length cDNA
fragment around 900 base pair (bp) was amplified
from total RNA extracted from TwPep3-infected N.
benthamiana using primers gL3637 and gL4435c designed
in the conserved regions of tospovirus L RNAs (Fig.
4A). No fragment was amplified from TwPep3-infected
plants with primers of potyviruses, tobamoviruses and
CMV. After cloning and sequencing the 924-bp cDNA
fragment, 82.4%, 76.9%, 75.8%, 75.5% and 75.1%
nucleotide sequence identity was obtained with the
conserved L RNA region of TwPep3 and the corresponding
752-bp conserved region of TNRV, and the 927-bp
conserved region of CCSV, TRZSV, CaCV and WSMoV,
respectively. The deduced amino acid sequence of TwPep3
L RNA shared 96.4%, 83.1%, 82.8%, 82.5% and
83.1% identity with that of TNRV, CCSV, TZSV, CaCV
and WSMoV, respectively. These comparative sequence
analyses indicated that TwPep3 is a member of the genus
Tospovirus closely related to TNRV and tospoviruses in the
WSMoV serogroup (Zheng et al., 2011).
For more accurate determination of the TwPep3
taxonomic status, the sequence of N gene and S RNA
was determined. To sequence the S RNA of TwPep3,
five cDNA fragments were amplified by RT-PCR using
the primers designed by Hassani-Mehraban et al. (2007)
and those used in this study (Fig. 4B). After assembling
the sequences of these five cDNA fragments into contigs,
the S RNA of TwPep3 was found to be 2786 nucleotides
(nts) in length with an ambisense coding arrangement.
In the viral RNA strand, an ORF of 1359 nts between
nt 66 and 1424 encoded the nonstructural NSs protein
of 452 amino acids with a predicted molecular mass of
51.3 kDa. The NSs gene of TwPep3 shared 44.8–78.5%
nucleotide sequence identities and 17.6–78.7% amino
acid sequence identities with those of 22 tospoviruses
available in GenBank (Table 1). The N gene located
between nt 1874 and 2719 in the viral complementary
sense was 846 nts long and encoded the N protein of 281
amino acids. The calculated molecular mass of N protein
was 31.0 kDa. The sequence of TwPep3 N gene exhibited
46.1–83.0% nucleotide identities and 19.1–89.0% amino
acid sequence identities with those of 24 tospoviruses
available in GenBank (Table 1). The 5 and 3 untranslated
regions (UTRs) of the S RNA were 65 and 67 nts in length,
respectively. The IGR of S RNA was 449 nts long with
an A + U content of 77.3%. The S RNA, IGR, 5 and 3
UTR of isolate TwPep3 shared 77.6%, 52.7%, 81.5% and
86.2% nucleotide identities, respectively, with those of
TNRV.
Phylogenetic analysis of N and NSs proteins of TwPep3
and other tospoviruses revealed three clusters with
TwPep3 closely related to TNRV (Fig. 5).
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A new tospovirus infecting sweet pepper
Figure 4 Reverse transcription-polymerase chain reaction (RT-PCR) of
Pepper chlorotic spot virus (PCSV)-TwPep3. (A) RT-PCR using degenerate
primers of potyviruses, tobamoviruses and tospoviruses, and specific
primers of Cucumber mosaic virus (CMV). (B) Sequencing strategy for
PCSV S RNA. Evaluation of primer specificity (C) and effectiveness in
field detection (D). Label of lanes: TP, PCSV-TwPep3; P, Potato virus Y;
PM, Pepper mild mottle virus; C, CaCV; CM, Cucumber mosaic virus; W,
WSMoV; H, healthy control; 1–10, 10 samples of sweet pepper collected
from fields in Sinyi.
Virus detection
To distinguish TwPep3 from other related tospoviruses,
a RT-PCR assay was developed with primers FJJ2012-67
and FJJ2012-68 designed in the TwPep3 N gene (Fig. 4B).
The expected size DNA product (0.9 kb) was amplified
from TwPep3-infected samples, whereas no amplification
occurred with CaCV- or WSMoV-infected samples (Fig.
4C). No DNA fragment was amplified from TwPep3-
infected samples with CaCV (FJJ2003-8/FJJ2003-10)
and WSMoV (FJJ2002-78/FJJ2002-79) primers (Zheng
112
Y.-H. Cheng et al.
et al., 2008). To evaluate the effectiveness of the primers
FJJ2012-67 and FJJ2012-68, 10 sweet pepper samples
with virus-like symptoms in Sinyi were collected and
tested by RT-PCR. Results showed the presence of TwPep3
in two samples (Fig. 4D) but not in the other eight
samples, which were all infected by TSWV (data not
shown).
Discussion
After investigation of virion morphology, serological
properties, genome sequence characteristics and back
inoculations, TwPep3 is a tospovirus and the causal agent
of leaf chlorosis, bud necrosis and fruit deformation on
sweet peppers in Sinyi. Sequence analyses indicated that
TwPep3 is closely related to TNRV (Chiemsombat et al.,
2010; Hassani-Mehraban et al., 2011; Seepiban et al.,
2011). The criteria for tospovirus special demarcation are
based on the sequence identity of the N protein, that
is, less than 90% amino acid identity (Fauquet et al.,
2005). Although TwPep3 is closely related to TNRV in
the phylogenetic analysis of tospoviral N proteins, the
N protein of TwPep3 shared only 87.9–89% amino acid
sequence identity with those of five TNRV isolates for
which information is available in GenBank (Table 1). The
N protein of the five TNRV isolates showed 97.5–100%
amino acid sequence identity to each other. Moreover,
the NSs proteins and S RNAs of TwPep3 and TNRV
shared only 78.7% and 77.6% amino acid and nucleotide
sequence identify, respectively. Together, our sequence
analyses indicated that TwPep3 is a new tospovirus for
which the name PCSV is proposed.
To date, the genus Tospovirus has 24 members (Table 1)
and three serogroups [TSWV, WSMoV and Iris yellow
spot virus (IYSV)] and some individual serotypes. In the
serological analyses, PCSV reacted positively with antisera
against WSMoV and CaCV N proteins, indicating that
PCSV belongs to the WSMoV serogroup, which includes
WSMoV, CaCV, CCSV, GBNV, TZSV and WBNV (Jan
et al., 2003; Zheng et al., 2011).
TNRV is a tospovirus infecting tomato and pepper in
Thailand (Chiemsombat et al., 2010; Hassani-Mehraban
et al., 2011) and is serologically related to WSMoV
serogroup (Seepiban et al., 2011). PCSV and TNRV have
similar host range and symptomatology. Both infected
Solanaceae plants but not cucurbits. A difference in host
reactions was observed on V. unguiculata and tomato.
Infection of V. unguiculata and tomato by TNRV is
localised and systemic, respectively (Hassani-Mehraban
et al., 2011), whereas infection of V. unguiculata by
PCSV is systemic. Six tomato cultivars showed localised
PCSV symptoms and two were symptomless. On N.
glutinosa, TNRV causes systemic mottling, vein necrosis,
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Y.-H. Cheng et al.
Figure 5 Phylogenetic relationships of NSs (A) and N (B) proteins of Pepper chlorotic spot virus (PCSV) and other tospoviruses. Sequence sources are
listed in Table 1. Phylogenetic analyses were conducted using MEGA4 software. The neighbour-joining algorithm with 1000 bootstrap replicates was
used to produce the dendrogram.
top necrosis (Hassani-Mehraban et al., 2011), necrotic
rings and ringspots (Seepiban et al., 2011), whereas PCSV
causes chlorotic and necrotic spots. Therefore, N. glutinosa,
V. unguiculata and tomato might serve as indicator plants
to distinguish PCSV and TNRV.
WSMoV (Yeh et al., 1992), PCFV (Chu et al., 2001b),
CCSV (Lin et al., 2005), CaCV (Zheng et al., 2008), Melon
yellow spot virus (MYSV) (Chen et al., 2010), TSWV
(Zheng et al., 2010) and PCSV are the seven tospoviruses
reported in vegetable crops and ornamentals in Taiwan.
Antibodies and antisera have been developed for the
diagnosis of these tospoviruses. In this study, the antisera
against the N protein of PCSV, CaCV and WSMoV cross-
reacted. Hence, a RT-PCR assay was developed for the
detection of PCSV.
Primers gL3637 and gL4435c designed in the conserved
region of tospoviral L RNAs (Chu et al., 2001a) can be used
to discover new tospoviruses with an expected 0.82-kbp
cDNA size production. A 924-bp cDNA was amplified by
RT-PCR with primers gL3637 and gL4435c from PCSV-
infected plants. A comparative analysis of PCSV cDNA
sequence with the L RNA sequence of CCSV, CaCV,
TZSV and WSMoV showed no extra nucleotide on the
conserved region of PCSV L RNA. The size of the amplified
PCSV cDNA was larger owing to misannealing of primer
gL3637 and primer gL4435c not only hybridising to the
expected site but also to an unexpected site located
Ann Appl Biol 164 (2014) 107–115
© 2013 Association of Applied Biologists
A new tospovirus infecting sweet pepper
upstream. Therefore, primers FJJ2012-67 and FJJ2012-
68 designed in the PCSV N gene were used to detect
PCSV specifically (Fig. 4B and 4C) and to diagnose the
disease caused by PCSV in the field (Fig. 4D). In field
survey of PCSV with RT-PCR, 2 of 10 symptomatic pepper
samples were PCSV-positive, whereas the other 8 were
PCSV-negative, but were infected by TSWV (data not
shown).
Pepper and tomato are two important vegetable
crops for which tospoviruses are economically significant
pathogens. In general, the pepper-infecting tospoviruses
also infect tomato, and vice versa. CaCV infects tomato
and capsicum in Australia (McMichael et al., 2002),
and Phalaenopsis orchid (Zheng et al., 2008) and tomato
(Huang et al., 2010) in Taiwan. CaCV, TSWV (Zheng et al.,
2010) and PCSV are the three tospoviruses reported in
solanaceae plants in Taiwan. So far, each of these three
viruses infects one solanaceae plant (tomato or pepper)
in the field although, as reported in this study, PCSV
also infects Phalaenopsis orchid. Because tospoviruses
are transmitted by thrips, CaCV, TSWV and PCSV are
potentially able to infect tomato, pepper, tobacco, potato,
eggplant and even other solanaceae plants in Taiwan.
There are more works on PCSV that warrant further
investigations, such as its distribution, its epidemiological
features, that is, seed transmission and thrips dispersal,
and the development of management strategies.
113
A new tospovirus infecting sweet pepper
Acknowledgements
We are grateful to Dr Chung-Jan Chang, Professor
Emeritus of the University of Georgia, for his critical
review of the manuscript. This work was partially sup-
ported by grant from National Chung Hsing University
and Council of Agriculture, Agricultural Research Insti-
tute (No. NCHU-TARI 10101), Taiwan, grants from the
National Science Council (Nos. NSC-101-2911-I-005-301
and NSC-102-2911-I-005-301) and the Ministry of Edu-
cation, Taiwan, under the ATU plan.
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