Introduction
Tomato spotted wilt virus (TSWV; type
member of genus Tospovirus) is transmitted by thrips belonging to Dictyothripsbetae
Uzel, Thrips tabaci Lindeman, F. intonsatrybom and Frankliniella
occidentalis (most efficiently) infecting tomato around the globe (Ciuffo et al. 2008). The
quasi-spherical viral particles of TSWV range from 80 to 120 nm in diameter.
The capsid is surrounded by a lipid bilayer made of glycoprotein, which
consists of + ssRNA tripartite genome having small (S), medium (M) and large
(L) segments. Small segment contains 2900 nucleotides which encodes
non-structural protein silencing suppressor (NSs) and nucleocapsid protein
(Takeda et al. 2002). The medium segment having 4800 nucleotides encodes
precursor glycoprotein (Gn-GC) and movement protein (NSm). The large segment
comprises 8900 nucleotides which encodes RNA dependent RNA polymerase enzyme
(RdRp) involved in replication of the virus (Soellick et al. 2000).
It is reported that TSWV infects
more than 1300 plant species belonging to 92 families of mono and
dicotyledonous plants causing a loss of more than 1 billion USD annually
(Parrella et al. 2003). Typical symptoms caused by TSWV includes
stunting, necrosis, chlorosis, bronzing and ringspots (Adkins 2000). Along with other pathogens (fungi and bacteria), the
management of viral diseases is of supreme importance as the crop is infected
by at least 136 characterized viral species (Xu et
al. 2017). The amplified international trade and the global climatic
variations are causing more frequent emergence of new viruses infecting tomato
and other crops, while the previously reported viruses are becoming epidemics (Hanssen et al. 2010). It is critical to
understand the diversity and evolution of plant viral pathogens for their
management.
The molecular evolution study of
plant RNA viruses is very crucial to understand the parameters of virus
managements like geographical distribution, adaptation, and evolutionary
process (Lauring and Andino 2010). The information of Pakistani native plant
viruses regarding molecular characterization is not sufficient because of
unknown genetic variability. Due to extensive tomato cultivation over a large
area, a wide range of tomato infecting genetically diverse viral strains and
species may exist in Pakistan and their genetic exchange can cause the
evolution of new viral species. The current research work was conducted to
explore the molecular variability of Pakistani TSWV- isolates. The findings of
this study will be helpful in development of sustainable management strategies
for TSWV disease that will ultimately boost the quality of tomato production in
Pakistan.
Materials
and Methods
Samples collection
Symptomatic
tomato leaves were collected from plants exhibiting foliar ring spots,
stunting, necrosis, chlorosis, line pattern and bronzing associated with Tomato
spotted wilt virus. These samples were collected from farmer’s fields
during April to August 2018 survey from Bahawalpur, Faisalabad, Lahore and
Multan. Symptomatic and healthy leaf samples were tested for TSWV infection by
using TSWV-specific-Bioreba Agri Strip ELISA Assay.
RNA
extraction and Reverse transcriptase polymerase chain reaction (RT-PCR)
The
total RNA content of ELISA positive plant leaves was isolated by using TRIzol®
Reagent (Life Technologies, Carlsbad, U.S.A.) as per manufacturer’s protocol.
The isolated RNA was directly used in RT-PCR. TSWV-nucleocapsid protein
(N-gene) was amplified by using primers pair of CP-5Bam and CP3-Pst (Antignus et
al. 1997) in one-step RT-PCR. The reaction mixture was incubated at 42°C
for 60 min for the synthesis of genomic cDNA of TSWV. The initial denaturation
temperature was set at 95°C for 5 min and 35 cycles were set for template
amplification for 60 s at 94°C, 60 s at 58°C than extension for 60 s at 72°C
and final extension was done at 72°C for 10 min. The amplified PCR products
were separated in 1% agarose gel electrophoresis. The positive PCR products
were cleaned by using PCR purification kit (Quiagen) QIAquick® and sequenced in
opposite directions.
Sequence
analysis
The
Transeq program (EMBOSS) was used to translate the obtained nucleotide
sequences (Rice et al. 2000). The BLAST analysis of protein and
nucleotide sequences was done to identify the TSWV. Closely related sequences
were downloaded from NCBI and aligned by using CLUSTAL W program (Larkin et
al. 2007). Also, the percent identities of amino acid and nucleotide
sequences were recorded by BioEdit v. 7.2.6.1. The software MEGA 6 with default
parameters and 1000 bootstrap replications was used to construct the
phylogenetic tree and measure the evolutionary distance using neighbour joining
method (Tamura et al. 2013).
Recombination
analysis and selection pressure
The
software DnaSP (v. 5.10) was explored to detect the deletion and insertion of
nucleotides within studied sequences (Balasubramanian
and Selvarajan 2014). DnaSP version 5.0 was used to evaluate the
molecular diversity pattern at segregating sites by statistical analysis like
diversity of nucleotides at all sites, Fu, & Li’s F*, Fu, & Li’s D*and
Tajima’s D (Librado and Rozas 2009).
Results
Samples
collection
During field surveys, the TSWV infected samples were
showing necrosis, chlorosis and bronzing on foliar parts. These symptoms are
associated with TSWV infection.
Table 1:
Characteristics of nucleotide sequences of Pakistani TSWV isolates
|
Isolate |
Accession No |
(nt) |
(aa) |
A (%) |
C (%) |
G (%) |
U (%) |
|
TSWV-PK1 |
MN966565 |
777 |
259 |
34 |
20 |
24 |
22 |
|
TSWV-PK2 |
MN966566 |
777 |
259 |
33 |
20 |
24 |
23 |
|
TSWV-PK3 |
MN966567 |
777 |
259 |
34 |
20 |
23 |
23 |
|
TSWV-PK4 |
MN966568 |
777 |
259 |
33 |
20 |
24 |
23 |
|
TSWV-PK5 |
MN966569 |
777 |
259 |
33 |
20 |
24 |
23 |
* Number of
nucleotides (nt), encoded amino acids (aa) and percentages of
nucleotide bases i.e., A (Adenine), C
(Cytosine), G (Guanine) and U (Uracil)
Table 2: Genetic and molecular diversity of Pakistani TSWV isolates
|
Statistical test |
Values |
|
Evolutionary distance |
0.007-0.219 |
|
Tajima’s D |
2.10697 |
|
Fu, & Li’s D* |
-2.55465 |
|
Fu, & Li’s F* |
-2.78103 |
|
Ks* |
4.86231 |
|
Z |
34.27778 |
|
Snn |
0.55556 |
|
Fst |
0.05145 |
PCR amplification and analysis of sequences
The primer pair CP-Pst and CP5-Bam amplified the ~800
DNA fragments in each positive sample. The obtained amplified PCR fragment was
consisted of 777 nucleotides. The BLASTn analysis showed that nucleocapsid
protein gene (N-gene) is a part of small (S) fragment and located between 152nd
and 928th nucleotides of TSWV genome. Translate tool translated
the coding sequence into 259 amino acids. Few variations were observed by using
multiple sequence alignment of amino acid sequences of Pakistani TSWV isolates.
The central region of all isolates was observed to be conserved (Fig. 1). The
variations were in the 5’ and 3’ ends of N gene sequences.
The nucleotide sequences of five TSWV isolates (TSWV-PK1-5) were carefully
analysed and submitted into Genbank. Each sequence consisted of Uracil contents
33–34%, Guanine 18–19%, Cytosine 27–28% and Adenine 21–22% (Table 1). In BLAST
analysis the nucleotides and amino acid sequences of all Pakistani-TSWV
isolates showed 98.2–99.3% and 95.7–97.4% similarity with each other,
respectively. While they showed 97.2–99.3% and 92.7–97.8% nucleotide and amino
acid sequence similarity with previously reported TSWV isolates respectively.
Highest percentage identities were observed with TSWV-AJ295699 (Czech Republic)
and TSWV-AY744478 (USA) isolates.
Phylogenetic
reconstruction
The
phylogenetic tree was constructed among the sequences of nucleocapsid (N) gene
sequences of new TSWV Pakistani isolates (TSWV-PK1-5) and others previously
worldwide reported twenty-two TSWV-isolates through neighbour-joining. In
phylogenetic tree twenty-seven TSWV isolates making two major groups. Four
Pakistani TSWV isolates (TSWV1-2, 3 and 5) clustered with South Korean and
Turkish isolates in group-II. While one of the Pakistani TSWV isolate (TSWV-4)
clustered with Australian and South African isolates in Group-I (Fig. 2). Five
American isolates (KU179581, KU179561, KU179591, KU179577 and KU179513) in
group-II appeared to be rotted by Pakistani TSWV-isolates. The remaining
globally reported TSWV- isolates clustered in another subgroup separately.
Recombination
analysis and selection pressure
The
investigations of Single Nucleotide Polymorphism (SNP) and Insertions and
Deletions (INDEL) in nucleotide sequences were done to confirm the nature of
polymorphism among TSWV isolates. The INDELs were absent in studied sequences.
Pakistani isolates were holding 0.007–0.219 evolutionary distance among
themselves and 0.097–0.161 with isolates reported from other countries (Table
2). The highest evolutionary distance values were observed with isolates
AY879109 (0.026–0.029) and AJ296600 (0.024–0.039) and lowest with isolate
AJ296599 (0.009–0.013).
Genetic
differentiation and gene flow analysis
In
gene flow analysis, the gene differentiation coefficient value (Fst) was
observed as 0.05145, which is less than standard value 0.33 (Table 2). This
recorded value between world and Pakistani TSWV isolates show a frequent gene
flow among them.
The
genetic differentiation analysis was observed on the basis of three permutation
statistical test as Snn; 0.55556, Z;34.27778and Ks*; 4.86231; (Table 2) (Balasubramanian and Selvarajan 2014).
Haplotype
and nucleotide diversity
Haplotype
and nucleotide diversity from all sites were performed by using statistical
test like Fu, & Li’s F*, Fu, & Li’s D ⃰ and Tajima’s D. These
statistical tests were performed to determine molecular diversity pattern at
segregation sites. The values recorded in these statistical tests were found
negative i.e., Fu, & Li’s D ⃰ (-2.55465), Fu, & Li’s F
⃰ (-2.78103) and one positive result from Tajima’s D (2.10697) (Table 2).
These negative results indicate low polymorphism frequency in Pakistani
Tomato spotted wilt virus (TSWV) population (Tsompana
et al. 2005).
Discussion
In the present study, the N gene of TSWV was selected for the
characterization, because the nucleocapsid gene is considered as vital in
classification of plant viruses as it encodes for the synthesis of capsid
protein. Moreover, the interaction of plant viruses with their hosts and their
life cycle are also dependent on capsid protein gene (Moury and Simon 2011). The reports of International Committee of
Taxonomy of Viruses have suggested that, the nucleocpasid genes of plant viruses
belonging to same specie hold < 80% nucleotide and amino acid sequences
similarities (Ismaeil et al. 2015).
In current study the amino acid and nucleotide sequences of all Tomato
spotted wilt virus Pakistani isolates were 95.7–97.4% and 98.2–99.3%
similar to each other, which mean that all of the isolates were in close association
with each other as compared to world isolates. Also, the Pakistani isolates
clustered in same group in phylogenetic reconstruction. These findings were
also supported by the detection of frequent gene flow among Pakistani isolates.
Moreover, the TSWV population was observed to be under purifying or negative
selection as neutrality test like Fu, & Li’s F*, Fu,
& Li’s D ⃰ and Tajima’s D, showed negative values. Evolutionary distance describes the divergence of homologous sequences
from their common ancestors (Rosenberg 2005).
The lower genetic distance was observed among Pakistani isolates, which also
strengthens the close association of Pakistani TSWV isolates.
Conclusion
The present study concludes that there exists a close association among
Pakistani isolates and comprehensive surveys for detection and molecular
characterization based on complete genomic sequence of the studied virus from
different crops is recommended. This will be helpful to develop sustainable and
comprehensive strategies for the management of TSWV in future.
Author Contributions
The author AA, MNA and WA, contributed to designing and
executing lab work and basic writeup.
Conflict
of Interest
The authors declare that they have no conflict of interest.
Data Availability
The data will be made avaialble on acceptable requests to the
corresponding author.
%201061-1065%20SC_files/image002.png)
Fig. 1: Amino acid sequence alignment of five Pakistani Tomato
spotted wilt virus isolates
%201061-1065%20SC_files/image004.png)
Fig. 2: A 1000 bootstrap consensus tree based on nucleotide
sequences of small (S) fragment; partial nucleocapsid (N-gene) of five Tomato
spotted wilt virus isolates from Pakistan (dotted) and 22 database
sequences
Ethics
Approval
Not applicable.
References
Lauring AS, R Andino (2010). Quasispecies theory and the behavior of
RNA viruses. PLoS Pathog 6; Article e1001005
Rice P, I Longdenand, A Bleasby (2000). EMBOSS: The European molecular
biology open software suite. Trends Genet 16:276‒277
Rosenberg MS (2005). Evolutionary distance estimation
and fidelity of pair wise sequence alignment. BMC Bioinform 6:102-110
Soellick TR, JF Uhrig, GL Bucher, JW Kellmann, PH Schreier (2000). The
movement protein NSm of Tomato spotted wilt tospovirus (TSWV): RNA
binding, interaction with the TSWV N protein, and identification of interacting
plant proteins. Proc Natl Acad Sci USA 97:2373‒2378
Takeda A, K Sugiyama, H Nagano, M Mori, M Kaido, K Mise, S Tsuda, T
Okuno (2002). Identification of a novel RNA silencing suppressor, NSs protein
of Tomato spotted wilt virus. FEBS Lett 532:75‒79
Xu C, X Sun, A Taylor, C Jiao, Y
Xu, X Cai, X Wang, C Ge, G Pan, Q Wang (2017). Diversity, distribution and
evolution of tomato viruses in China uncovered by small RNA sequencing. J Virol 91; Article
e00173-17