TSWV isolates in GenBank from various countries. Characterization of Non-Structural Movement Protein (NSm) of. Tomato Spotted Wilt Virus From Pepper In ...
Characterization of Non-Structural Movement Protein (NSm) of Tomato Spotted Wilt Virus From Pepper In South of TURKEY
FM163371.1 spain 63
HM015518.1 spain
56
AY744490.1 USA: North Carolina
43 20
AY744491.1 USA: North Carolina AY870390.1 usa
36
AY744489.1 USA: North Carolina
35 28 41
AY870389.1 usa AY956380.1 USA: Florida
69
AY744488.1 USA: North Carolina
28
ABSTRACT Tomato spotted wilt virus (TSWV), belonging to Tospovirus genus in Bunyaviridae family has been known as only viral agent causing infections in insects and plants. TSWV consists of three genomic single-stranded RNA denoted as S, M, and L. The M RNA responsible for the expression of movement of the virus in host cells with the nonstructural protein translated by NSm region which is important for NSM gene-mediated resistance in durability. In this study, twenty-six pepper plants from Antalya and İçel provinces were found to be infected by TSWV in RT-PCR. Multiple alignment and phylogenetical comparison with homologous isolates retrieved from GenBank was carried out according to the NSm region of the local TSWV isolates. All turkish TSWV isolates showed high nucleotide homology, except one isolate (29) from Antalya. The isolate ’29’ was genetically different from all others and clustered with the variants of TSWV isolates in GenBank from various countries.
MATERIAL AND METHOD Isolates:
AY744487.1 usa AF208498.1 USA: Hawaii KT160281.1 USA: Pennsylvania
92
AY744482.1 usa
55
AY744484.1 USA: California
13
FM163373.1 spain
98
FM163372.1 Spain
5
AY744481.1 USA: California
40 236
AY744485.1 usa KJ575621.1 Italy KU308372.1 Poland
2
JN664253.1 china
21 2
AB921157.1 Japan AY744485.1 USA: California
30
KC261975.1 South Korea
86
HM581938.1 South Korea
49
TSWV isolates were collected in Antalya and İçel from March to June İn 2016. Double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was used to detect TSWV and positive samples were inoculated onto Nicotiana tabacum plants.
NSm genome sequencing: Total RNAs were purified from leaf and fruit tissues of TSWV positive samples with TRIZOL and used for RT-PCR with Moloney Murine Leukemia Virus (M-MuLV ) reverse transcriptase and Taq DNA polymerase (Thermo Scientific). TSWV-NSm forward «ACATTACACAAGCTCCTCTACC» and complementary «CCTTTAGGRATTATCAGCTTGC» primers were used described as Tentchev et al. (2013). Sequencing were performed by Medsantek(İstanbul-TURKEY) directly on RT-PCR products.
JF960236.1 China KU552117.1 China: Yunnan
100
KM657119.1 China
80
KM657118.1 China
48
KM657117.1 China
71
AB650469.1 South Korea Capsicum annuum KM365065.1 Australia
99
HM015516.1 Australia HM015512.1 Spain
100
87 KC261966.1 South Korea
31
32
KC261972.1 South Korea
14
AF208497.1 usa
24 8
AY744486.1 usa HQ830185.1 italy GQ373174.1 Serbia
42
GQ373175.1 Serbia
89
5
HQ246453.1 Serbia
33
GQ373176.1 Serbia
20 8
HM015515.1 Spain HM015521.1 Spain
33
KT717692.1 Australia
32
RESULTS AND DISCUSSION Tomato spotted wilt virus (TSWV), consists of S, M and L parts is the widespread causal agent around the World for large economic losses in horticultural and field crops. The nonstructural NSm protein of tomato spotted wilt virus (TSWV) is a putative viral movement protein responsible for cell-to-cell movement.
M
825 bp Fig. 1. Gel image of the NSm coding region of TSWV isolates. M: 100 bp marker
Mersin 71
35
Mersin 66 Mersin 70 Mersin91
77
Antalya 29 30 Mersin 63 90
9 19
5
Mersin 69
The results of phylogenetic analysis NSm ORF revealed that Antalya and Mersin isolates fell into two distinct clusters except Antalya 29 and Mersin isolates clustered two different groups. All Turkish isolates showed differentiation from TSWV isolates of other countries.
Mersin73
38
Mersin61
Fig. 3 Aminoacid substition of the nucleotide sequences of the partial NSm coding region of TSWV isolates.
Mersin 59 96 Mersin 64
Mersin 62
6
Mersin92 4 667 Mersin 94 3
Mersin 67 62 Mersin 57
Mersin 74 0
60 Antalya54
Antalya 52 77
Antalya 40 Antalya 36
58 11 12
Antalya 27 Antalya 46 Antalya 50
31 95
0.005
Nucleotide sequences corresponding to the NSm regions were aligned using CLUSTAL W and bootstrap analysis was applied using 500 bootstrap resampling of the phylogenetic trees with the neighbour-joining method implemented in MEGA (Tamura et al., 2007).
Mersin 65
50 17
Analysis of the genetic structure of TSWV isolates
Antalya 47
Fig. 2. Phylogenic analysis of the nucleotide sequences of the partial NSm coding region of TSWV isolates.
