Journal of Plant Pathology (2014),
96 (1), 201-205
Zindovic et al. 201
S
hort
C
ommuniCation
MOLECULAR CHARACTERIZATION OF
TOMATO SPOTTED WILT VIRUS
IN
MONTENEGRO
J. Zindovic
1
, M. Ciuffo
2
and M. Turina
2
1
Department of Plant Protection, Biotechnical Faculty, University of Montenegro, Podgorica, Montenegro
2
Istituto di Virologia Vegetale del CNR, Torino, Italy
SUMMARY
The presence and distribution of Tomato spotted wilt
virus (TSWV) on different vegetable, ornamental, tobacco
plants and weeds was investigated in the open field and
greenhouses in different locations of Montenegro. DAS
ELISA was used to identify the virus in 2072 samples
collected from 2007 to 2009. TSWV was detected in two
samples collected in 2007, none of the samples collected in
2008 and 50 samples collected in 2009. The sensitivity and
specificity of four different previously described primer
sets for TSWV detection were assessed by RT-PCR. The
N gene sequences were compared with those of 20 new
isolates from Montenegro, Serbia, Italy and Brazil and 27
others retrieved from the NCBI database. Nucleotide se-
quences comparison revealed high level of similarity (97-
99,9%) between Montenegrin and TSWV isolates from
other European countries. Phylogenetic analysis of the N
gene revealed seven distinct subgroups, showing that the
investigated isolates clustered with those from the same
geographic region, with the exception of one Montenegrin
(Is-103) and one Italian isolate (Gb-21).
Key words: Tomato spotted wilt virus, ELISA, RT-PCR,
diagnosis, phylogenetic analysis.
Tospoviruses are among the most damaging plant vi-
ruses worldwide (Peters et al., 1991; Pappu et al., 2009;
Turina et al., 2012). Tomato spotted wilt virus (TSWV), the
type member of the genus Tospovirus, family Bunyaviridae
(Plyusnin et al., 2011), is one of the ten most economically
important plant viruses (Scholthof et al., 2011) due to its
worldwide distribution, broad host range (Peters, 2003), ef-
ficiency of thrips vectors in virus transmission and the dif-
ficulty of controlling virus and vectors (Pappu et al., 2009).
Tospoviruses present in Europe include Tomato spotted
wilt virus (TSWV), Impatiens necrotic spot virus (INSV),
Iris yellow spot virus (IYSV) and Polygonum ringspot virus
(PolRSV) (Ciuffo et al., 2008; Pappu et al., 2009). These
viruses are transmitted by single or multiple thrips species,
among which Frankliniella occidentalis Pergande (Gardner
et al., 1935), F. intonsa Trybom (Wijkamp et al., 1995), Thrips
tabaci Lindeman (Pittman, 1927) and Dictyothrips betae
Uzel (Ciuffo et al., 2010) are widely distributed in Europe
(Turina et al., 2012); nevertheless, global spread of TSWV
epidemics can be strictly correlated to worldwide dispersal
of F. occidentalis, its most efficient vector (Jones, 2005).
TSWV has been reported to infect over 1300 plant spe-
cies from at least 92 families, including both monocots
and dicots (Parrella et al., 2003; EFSA, 2012). It causes
stunting, necrosis, chlorosis, bronzing, ringspots and line-
pattern symptoms (Adkins, 2000).
TSWV particles are quasi-spherical and enveloped, and
have a tripartite, ambisense, single-stranded RNA genome
composed of the large (L) segment, encoding an RNA-
dependent RNA polymerase, the medium (M) segment,
encoding the NSm protein and the Gn-Gc precursor gly-
coprotein and the small (S) segment, encoding the NSs
and the N protein.
Data about virus presence and distribution in Monte-
negro is scarce. Until recently (Zindovic et al., 2011), no
survey was done regarding TSWV in this region.
The aim of this study was to investigate the occurrence
and distribution of TSWV in different vegetables, orna-
mentals, tobacco, and weeds in Montenegro, with surveys
carried out in 2007-2009, and to determine the genetic re-
lationship of Montenegrin TSWV isolates, with a number
of isolates from Serbia, Italy and Brazil.
In 2007, 23 sites were inspected and 464 samples of
32 different species (23 families) were collected. In 2008,
1020 samples of 42 species (22 families) were taken from
28 locations. In 2009, 588 samples of 53 species (26 fami-
lies) were collected from 25 locations. Samples from open
fields and greenhouses were diverse in symptoms expres-
sion, with 55 samples showing tospovirus-like symptoms,
1995 samples showing general virus-like symptoms and 22
samples being symptomless.
All samples were analyzed with a commercial DAS-
ELISA diagnostic kit (Bioreba, Switzerland) according to
the manufacturer’s instructions, except that 100 μl reac-
tions were used. Positive and negative controls were in-
cluded in each test.
Edizioni ETS Pisa, 2014
Corresponding author:
M. Turina
Fax:+39.011.343809
E-mail:
[email protected]
202 TSWV in Montenegro
Journal of Plant Pathology (2014),
96 (1), 201-205
Of the 2072 samples tested, 52 were infected by TSWV
with an incidence of 0.4% (2/464) and 8.5% (50/588) in
2007 and in 2009, respectively. In 2008, none of the tested
samples (0/1020) was positive. The virus was detected in
two Dracaena sp. plants imported in Montenegro in 2007,
found in the flower market in Podgorica. In 2009, TSWV
was recorded in Capsicum annuum (33), Primula sp. (6),
Calceolaria sp. (4), Gerbera sp. (4), Aquilegia sp. (1), Petu-
nia sp. (1) and Chrysanthemum sp. (1) plants in three sites
in the Podgorica district and one site in the Herceg-Novi
district. All infected plants were from greenhouses and
symptomatic.
To further characterize TSWV isolates from pepper,
primula, calceolaria, gerbera, aquilegia, petunia and dra-
caena, the virus was mechanically transferred from ELISA-
positive samples onto Nicotiana benthamiana as described
by Roggero et al. (2002). Six TSWV isolates from Italy,
three from Serbia and two from Brazil were also included
in these bioassays (Table 1). Brazilian and Italian isolates
were present in the collection of virus isolates of the In-
stitute of Plant Virology in Turin (Italy). Serbian isolates
were kindly provided by Prof. Branka Krstic from the
Faculty of Agriculture, Belgrade (Serbia). All Montenegrin
and Serbian TSWV isolates, as well some Italian isolates,
produced chlorotic local lesions on inoculated leaves of
N. benthamiana followed by systemic mottling. The Italian
isolates P-202/1, I-186 and Miz-3, as well as Brazilian iso-
lates VE-223 and VE-225 caused local necrotic lesions on
inoculated N. benthamiana leaves followed by systemic ne-
crosis and leaf distortion. Two types of symptoms caused
by different TSWV isolates on N. benthamiana had also
been reported by Chatzivassiliou et al. (2000).
Only the isolates from two symptomatic dracaena plants
could not be mechanically transmitted, which may be due
to one or more of the factors limiting mechanical transmis-
sion of TSWV (Stobbs et al., 1992).
To characterize the genetic variability of TSWV iso-
lates, RT-PCR was run with total RNAs extracted from
infected leaves of N. benthamiana and N. tabacum cv. Sam-
sun using the RNeasy Plant Mini Kit (Qiagen, Germany)
or TRIZOL reagent (Gibco BTL, Life Technologies, USA).
The suitability of four primer sets targeting three regions
(N, NSs and NSm) of the virus genome was evaluated. The
first-strand cDNA was synthesized by reverse transcription
of total RNA using random hexamer primers and the M-
MLV enzyme (Invitrogen, USA). PCR amplifications were
Table 1. Tomato spotted wilt virus isolates analyzed in this study.
Isolate
Country
Region/Locality
Year
Original host
Accession no. (N gene)
Is-367
Serbia
Kragujevac
2007
Dahlia sp.
GU369728
Is-603
Serbia
Novi Knezevac
2007
Solanum lycopersicum
GU369723
Is-739
Serbia
Mladenovac
2007
Nicotiana tabacum
GU369724
Gb-21
Italy
Campania
1997
Gerbera sp.
GU369718
T-1003
Italy
Apulia
2005
S. lycopersicum
GU369725
P-202/1
Italy
Sicily
1999
Capsicum annuum
GU369721
P-240
Italy
Sicily
2001
C. annuum
GU369722
I-186
Italy
Latium
2000
Latuca sativa
GU369719
Miz-3
Italy
Campania
1997
Solanum melongena
GU369720
VE-223
Brazil
Sao Paulo
1994
S. lycopersicum
GU369726
VE-225
Brazil
Sao Paulo
1994
C. annuum
GU369727
Is-103
Montenegro
Dusici, Podgorica
2009
C. annuum
GU339505
Is-141
Montenegro
Tolosi, Podgorica
2009
Calceolaria sp.
GU339506
Is-228
Montenegro
Tolosi, Podgorica
2009
Petunia sp.
GU339507
Is-246
Montenegro
Tolosi, Podgorica
2009
Primula sp.
GU339508
Is-278
Montenegro
Tolosi, Podgorica
2009
Primula sp.
GU355940
Is-279
Montenegro
Tolosi, Podgorica
2009
Primula sp.
GU369715
Is-288
Montenegro
Tolosi, Podgorica
2009
Aquilegia sp.
GU355939
Is-334
Montenegro
Mojdez, Herceg-Novi
2009
Gerbera sp.
GU369716
Is-344
Montenegro
Mataguzi, Podgorica
2009
C. annuum
GU369717
Table 2. Primer sets used for detection of Tomato spotted wilt virus.
Primer
Region Sequence 5’ to 3’
Annealing t(°C) /time
Size of amplicon
Reference
S70-for
S890-rev
NSs
CACAGTACCAATAACC
CATCTCCTGCAACCTTGAAC
55°C/1 min
820 nt
Qiu et al. (1998)
S574-for
S1433-rev
NSs
GTCTTGTGTCAAAGAGCATACCTATAA
TGATCCCGCTTAAATCAAGCT
55°C/1 min
860 nt
Tsompana et al. (2005)
S1983-for
S2767-rev
N
CCCTCGAGGCTTTCAAGCAAGTTCTGCG
GCTCTAGAGCCATCATGTCTAAGGTTAAGCTCAC
50°C/1 min
780 nt
Tsompana et al. (2005)
M284-for
M962-rev
NSm
GAAGGAACATCTTCCTTTGG
CCTCTTCCTCTTCAACTGATCTCTC
55°C/1 min
680 nt
Qiu et al. (1998)
