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Molecular characterization of Tomato spotted wilt virus isolates from Montenegro

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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 sequences
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 viruses
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), efficiency
of thrips vectors in virus transmission and the difficulty
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 species
from at least 92 families, including both monocots
and dicots (Parrella et al., 2003; EFSA, 2012). It causes
stunting, necrosis, chlorosis, bronzing, ringspots and linepattern
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 RNAdependent
RNA polymerase, the medium (M) segment,
encoding the NSm protein and the Gn-Gc precursor glycoprotein
and the small (S) segment, encoding the NSs
and the N protein.
Data about virus presence and distribution in Montenegro
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, ornamentals,
tobacco, and weeds in Montenegro, with surveys
carried out in 2007-2009, and to determine the genetic relationship
of Montenegrin TSWV isolates, with a number
of isolates from Serbia, Italy and Brazil.

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