Molecular Detection and Partial Characterization of Tomato Yellow Leaf Curl
Virus in Sri Lanka
Tomato Yellow Leaf Curl Virus (TYLCV) is an important plant virus on one of the economically most important vegetable crops; tomato (Lycopersicon esculentum Mill.). This had not been molecularly detected before, in Sri Lanka. TYLCV-GN-SL was isolated from apparently infected tomato plants using modified Cetyltrimethyl Ammonium Bromide (CTAB) method in Gannoruwa. Associated Begomoviruses were detected using Deng 541/Deng 540 and AV 494/AC 1048 primer pairs. TYLCV was detected for the first time in tomato in Sri Lanka using P1V/P4C, TYLCV specific primer pair. Nucleotide sequence of coat protein of isolated TYLCV-GN-SL proved that the Indian strain of ToLC virus was closely related to Tomato Leaf Curl Sri Lanka Virus (TLCV-SL: 97%) and Tomato leaf curl Geminivirus (TLCGV: 93%) through direct sequencing data. TLCV-SL was confirmed as TYLCV isolate. TYLCV was molecularly detected from major tomato growing districts like Badulla, Nuwara-Eliya, Kandy and Matale in Sri Lanka.
October 04, 2012; Accepted: December 10, 2012;
Published: February 12, 2013
Tomato Yellow Leaf Curl Disease (TYLCD) caused by several viruses belonging
to different species, which all together are referred to as Tomato Yellow Leaf
Curl Virus (TYLCV) of the genus Begomovirus of the family Geminiviridae
(Czosnek, 2008) is one of the most devastating plant
disease in many tropical and subtropical regions in the world (Reddy,
2006). The virus is ranked third among Top 10 plant viruses
(Scholthof et al., 2011). In Sri Lanka, a high
incidence of TYLCD was reported during April to September (Ariyarathna
et al., 2004).
TYLCV is primarily transmitted by the sweet potato whitefly (Bemisia tabaci
Gannadius) in a persistent and circulative manner (Melzer
et al., 2009) but is not transmitted by the greenhouse whitefly (Trialeurodes
vaporariorum) (Sugano et al., 2011). It is
not mechanically or seed transmitted (Green and Kalloo, 1994;
Cerkauskas, 2005; Czosnek, 2008).
TYLCV genome is either monopartite or bipartite and has geminate (twinned)
particles size of 18-20x30 nm, consisting of two incomplete icosahedra joined
together in a structure with 22 pentameric capsomeres and 110 identical protein
subunits (Gafni, 2003; Czosnek,
2008). A functional coat protein with 260 amino acid length is essential
for host plant infection and insect transmission in TYLCV (Noris
et al., 1998). TYLCV was the first Begomovirus proven to possess
a single genomic DNA (ssDNA) and it has a 2787 nt (Czosnek,
2008). It encodes six partially overlapping Open Reading Frames (ORFs) that
are organized bi-directionally (Gafni, 2003) and a
long (313 nt) Intergenic Region (Czosnek, 2008).
Symptom development in tomato is in 2- 3 weeks after inoculation of TYLCV but
the viral DNA can be detected 7 days earlier (Ber et
al., 1990) and the highest viral concentration can be detected at 4
days before symptom appearance (Green and Kalloo, 1994).
Typical symptoms in tomato leaves are yellow (chlorotic) leaf edges, upward
leaf cupping, leaf mottling with dark green and yellow, leathery, reduced leaf
size and abnormally shaped foliage with evident vein clearing, short internodes,
erect terminal and axillary shoots, proliferation of axillary branch formation,
premature flower drop and loss of small fruits (Brown and
Nelson, 1988; Costa and Heuvelink, 2005; Reddy,
Symptomatological identification of TYLCV is unreliable (Momol
et al., 1999) and the Polymerase Chain Reaction (PCR) methods are
highly effective as a tool for rapid and large-scale diagnostics of TYLCV-infected
samples (Briddon and Markham, 1995). Apart from being
a tool useful for detection, the products of the diagnostic PCR reactions are
suitable for further characterization of the viruses (Briddon
and Markham, 1995).
Virus taxonomy must respond to the development of new technologies (Fauquet
and Stanley, 2003). The analysis of DNA sequences has become the tool of
choice, allowing to accurately identify the virus and to evaluate its relationship
with other TYLCV isolates (Czosnek, 2008). The phylogenetic
analysis is important in showing ancestral relationships among geminviruses
and is critical for virus taxonomy. Often, complete genomes or gene sequences
are used. However, where complete sequences are not available, partial nucleotide
sequences are useful in determining relationships among new and emerging geminiviruses
(Roye et al., 1999).
ToLCV affected tomato samples were tested with graft inoculation in Sri Lanka.
Infected samples were detected by DNA hybridization using 6i probes, which detected
the Indian monopartite tomato geminivirus by Dr. S.K. Green in Asian Vegetable
Research and Development Centre (De Zoysa, 1996). Tomato
leaf curl Sri Lanka virus-(Sri Lanka: Bandarawela: 1997)-ToLCSLV-(LK: Ban: 97)
species has been published (Fauquet et al., 2008).
This study was the first molecular detection and partial characterization of the TYLCV in Sri Lanka.
MATERIALS AND METHODS
The experiments were conducted at the Division of Plant Pathology, Horticultural Crops Research and Development Institute (HORDI), located in the Latitude of 7°16'N and the Longitude of 80°36'E from April to September 2012.
Sample collection and total DNA extraction: Samples from the tomato plants showing typical characteristic TYLCD symptoms and healthy tomato plants grown under insect proof conditions were collected with the avoidance of cross contaminations.
Total DNA was extracted from apparently infected and healthy samples using
CTAB extraction protocol previously described by Lodhi et
al. (1994) with slight modifications. Approximately 150 mg of tender
leaf tissues were homogenized using a mortar and pestle with 1.5 mL of pre-warmed
extraction buffer (2% (v/v) CTAB, 100 mM Tris-HCl (pH 8.0), 20 mM EDTA, 1.4
M NaCl and 0.2% (v/v) β-mercaptoethanol added just before use) with 50
mg of PVP. The homogenized sample was transferred into a 1.5 mL micro centrifuge
tube and incubated at 65°C for 30 min while mixing at 10 min intervals.
The tube was centrifuged at 10,000 rpm for 5 sec and 750 μL of supernatant
was transferred into a new tube and treated with 750 μL of chloroform:
isoamyl alcohol (24:1) followed by vortexing and centrifuging at 10,000 rpm
for 15 min. Aqueous layer was transferred into a new tube and 300 μL of
ice-cold isopropanol was added and mixed by inverting the tube slowly. The tube
was incubated overnight at -20°C. The DNA was pelleted down by centrifuging
at 10,000 rpm for 15 min. The resultant pellet was washed with 500 μL of
70% (v/v) ethanol by vortexing followed by centrifugation at 10,000 rpm for
5 min. The DNA pellet was dried until all the alcohol was evaporated and dissolved
in 50 μL of TE (Tris-EDTA) buffer (10 mM Tris-HCl (pH 8) and 1 mM EDTA
(pH8)), incubated at 37°C for 30 min and stored at -20°C until further
PCR amplification: PCR was carried out with oligonucleotide primers (Table 1).
A PCR was conducted in a reaction volume (25 μL) containing 14.8 μL of sterile water, 2.5 μL of 10X PCR buffer (500 mM KCl, 100 mM Tris-HCl (pH 9.1) and 0.1% Triton X-100), 2 μL of dNTP (2.5 mM), 2 μL of each Deng 541/Deng 540 (10 μM), 0.5 μL of MgCl2 (25 mM), 0.2 μL of Taq polymerase (5 IU μL-1) and 1 μL of template DNA (diluted 1:25 in water). Following thermal cycle programme was performed, 1 cycle (4 min at 94°C), 30 cycles (30 sec at 94°C, 30 sec at 58°C and 45 sec at 72°C) and 1 cycle (10 min at 72°C) in a thermocycler (Labnet Inc. U.S.A, Model: Labnet Gradient).
A PCR was conducted in a reaction volume (25 μL) containing 13.4 μL of sterile water, 2.5 μL of 10X PCR buffer (500 mM KCl, 100 mM Tris-HCl (pH 9.1) and 0.1% Triton X-100), 2 μL of dNTP (2.5 mM), 2 μL of each AV 494/AC 1048 (10 μM), 1.0 μL of MgCl2 (25 mM), 0.1 μL of Taq polymerase (5 IU μL-1) and 2 μL of template DNA (diluted 1:25 in water). Following thermal cycle programme was performed, 1 cycle (5 min at 94°C), 30 cycles (30 sec at 94°C, 30 sec at 50°C and 45 sec at 72°C) and 1 cycle (10 min at 72°C).
|| Oligonucleotide primers used to detect TYLCV Begomovirus
associated with tomato
|K: G or T, R: A or G, S: C or G, W: A or T, Y: C or T, B:
C, G or T, V: A, C or G
PCR was conducted in a reaction volume (25 μL) containing 14.3 μL of sterile water, 2.5 μL of 10X PCR buffer (500 mM KCl, 100 mM Tris-HCl (pH 9.1) and 0.1% Triton X-100), 2 μL of dNTP (2.5 mM), 2 μL of P1V/ P4C (10 μM), 1.0 μL of MgCl2 (25 mM), 0.2 μL of Taq polymerase (5 IU μL-1) and 1 μL of template DNA (diluted 1:25 in water). Following thermal cycle programme was performed, 1 cycle (4 min at 94°C), 30 cycles (1 min at 94°C, 1 min at 55°C and 2 min at 72°C) and 1 cycle (10 min at 72°C). PCR products were stored at-20°C.
PCR products (5 μL of each) were subjected to 1% (w/v) agarose gel electrophoresis with 2 μL of loading dye at 80 volts for 1 hour in TBE buffer and stained with ethidium bromide (0.5 μg mL-1) and visualized under UV transilluminator and photographed by a digital camera. The results were verified against DNA marker (Vivantis).
Molecular characterization: Twenty micro liters of PCR product of ~520
bp consistently amplified using Deng 541/Deng 540 from TYLCV confirmed sample
was sent to the Gene Tech Pvt. Ltd, 54, Kitulwatte Road, Colombo 08, Sri Lanka
for sequencing. Direct sequenced data of CP was subjected to FASTA analysis
based on close sequence identity and the length of the sequences. Followed by,
Begomovirus of highly similar sequences were downloaded from GenBank
with the accession numbers provided by the FASTA output. Phylogenetic tree and
molecular evolutionary relationships were determined through MEGA 4.0 software
using neighbor-joining method (Tamura et al., 2007).
Detection of the presence of TYLCV in major tomato growing districts in
Sri Lanka: Naturally infected tomato plants showing typical characteristic
symptoms of TYLCD along with healthy tomato plants were collected from Badulla,
Nuwara-Eliya, Kandy and Matale Districts. Characteristic features of the infected
plants and varieties were recorded and photographed. Tender leaf samples were
used for DNA extraction using modified CTAB method. Extracted DNA samples were
amplified with degenerate universal primers (Deng et
al., 1994) for Begomovirus and TYLCV specific primers (Navot
et al., 1992). All the conditions as above relevant with primers.
PCR products were subjected to gel electrophoresis with DNA marker (Vivantis).
RESULTS AND DISCUSSION
Molecular detection of TYLCV: Apparently infected tomato plants with
yellow colour margins, dark green mid veinal area, shrinking and curling of
the leaflets and the stunting of the plant (Fig. 2) were subjected
to DNA extraction.
Diluted DNA sample (1:25) amplified with the degenerate primers (Deng
et al., 1994) yielded the 520 bp size of band (Fig.
1a) suggesting the presence of a Begomovirus in suspected sample.
For the identification and confirmation of all the TYLC viruses and ToMoV, it
had been recommended the PCR with Dengs degenerate primers (Anonymous,
2004). PCR amplification resulting Begomovirus specific band showed
the presence of Begomovirus in sample showing typical TYLCD.
||Detection of Begomovirus presence of the suspected
tomato sample (a) Detection of Begomovirus in infected tomato sample
using degenerate primers (Deng et al., 1994),
Lanes-M: 100 bp marker (Vivantis), 1: Infected tomato sample, 2:
Healthy tomato sample, 3: Water control and (b) Detection of Begomovirus
using Wyatt and Browns and Dengs degenerate primers, Lanes-M:
100 bp marker (Vivantis), 1: Infected bean sample, 2: Infected tomato
sample (Wyatt and Browns degenerate primers), 3: Infected cassava
sample (Dengs degenerate primers), 4: Water control
|| TYLCV infected tomato plant
The same set of degenerate primers had been used for the detection of Begomoviruses
in infected tomato-Banglore and naturally infected weed hosts (Reddy,
PCR primers that anneal to two highly conserved sequences within the most highly
conserved gene of the whitefly transmitted geminivirus subgroup would be useful
for broad-spectrum PCR based virus detection. Highly conserved DNA motifs within
the capsid gene of subgroup III geminiviruses are potentially suitable as degenerate
priming sites (Wyatt and Brown, 1996). The AV 494/AC
1048 primer pair is ideal because of the anticipated conservation and hence
their apparently universal nature. DNA sequence of the 550 bp core capsid gene
fragment was amplified by another set of degenerate primers (Wyatt
and Brown, 1996). The selected sample that is shown typical TYLCD symptoms,
amplified with the above degenerate primers, resulting 550 bp PCR products (Fig.
1b). In Turkey, 15 symptomatic tomato samples were tested for Begomovirus
infection by using PCR with Wyatt and Browns degenerate Begomovirus
primers (Koklu et al., 2006). Begomovirus
infected bean samples were amplified with Wyatt and Browns degenerate
primers resulting 550 bp of bands. Infected cassava sample amplified with Dengs
degenerate primers showed the 520 bp band as a reference. The results showed
further, the association of Begomovirus in infected tomato plant sample
showing typical TYLCD.
DNA sample, which was positive for the presence of Begomovirus using
Dengs and Wyatt and Browns degenerate primers, amplified with the
TYLCV specific primer (P1V and P4C) resulting 1650 bp PCR product (Fig.
3). P1V and P4C primers anneal with the 6-80 and 2054-2071 positions of
the viral genome, respectively.
||Detection of TYLCV in infected tomato sample using TYLCV specific
primers, Lanes-M: 100 bp marker (Vivantis), 1: Infected tomato sample,
2: Healthy tomato sample, 3: Water control
Navot et al. (1992) reported similar results
using total DNA extracted from the TYLCV infected tomato samples in Israel.
TYLCV specific primers P1V (corresponds to the viron positive strand) and P4C
(corresponding to complementary viron strand) were used for the detection of
TYLCV and Immuno-captured (IC) PCR was employed in Palestine (Sawalha,
Based on the PCR with TYLCV specific primer, it clearly showed the presence of TYLCV in the tomato sample, with typical TYLCD symptoms. However, the healthy sample was not found positive with both Begomovirus specific primes and TYLCV specific primers. This is the first attempt made for molecular detecting of TYLCV in Sri Lanka.
The nucleic acid hybridization with Sri Lankan probe SL 14 confirmed the presence
of TYLCV in samples of T-245 and Thilina varieties sent to Taiwan in 2000 (Ariyarathna
et al., 2004). Tomato plant samples showing leaf mottling, curling,
yellowing, purpling, discoloration, distortion and stunting, spotted on nitrocellulose
membranes were sent to Asian Vegetable Research and Development Centre (AVRDC)
for DNA hybridization tests using Indian tomato geminivirus probe and found
negative results (De Zoysa, 1996).
Molecular characterization of TYLCV in tomato: FASTA format of the sequence,
denoted as TYLCV-GN-SL (Tomato yellow leaf curl virus-Gannoruwa-Sri Lanka) was
used for analysis. Other background nucleotide fragments were removed from the
direct sequence data that compared with several other TYLCV sequences.
|| FASTA format of sequenced data compared with 29 isolates
|Presence of TYLCV in major tomato growing districts in Sri
It has resulted 491 nt fragment as a partial sequence of TYLCV-GN-SL. This
type of contaminations is common in direct sequencing process. However, sequencing
after cloning with a suitable vector could be much more reliable. FASTA format
of sequenced data was compared with 29 isolates of other Begomoviruses
(Table 2) of highly similar sequences and a phylogenetic tree
was made using MEGA 4.0 software. The CP region of the virus displayed different
similarity percentages with other related viruses (Fig. 4).
TYLCV-GN-SL isolate was grouped together with Tomato Leaf Curl Sri Lanka Virus (TLCV-SL: AF274349.1) of 97%, tomato leaf curl geminivirus (TLCGV: AF321930.1) of 93% as a group in a small cluster, they were found to be very much genetically similar. Further, many of the related Begomoviruses denoted as ToLCV from India have been form a separate cluster but related to a small cluster, where TYLCV-GN-SL isolate included.
TYLCV-GN-SL isolate has been proved to be TYLCV using specific primers suggested
by Navot et al. (1992). Therefore, information
generated through this study seem sufficient for confirmation of Tomato leaf
curl Sri Lanka virus-(Sri Lanka: Bandarawela: 1997) (ToLCSLV- LK: Ban: 97; AF274349)
shown high similarity with TYLCV-GN-SL proved as a TYLCV, the nomenclature of
the ToLCSLV may be changed as TYLCV.
Our isolate being genetically similar with most of the geminiviruses from tomatoes
in India, it can be suggested that Sri Lanka TYLCV is a strain of ToLCV in Southern
India. As a whole, the Indian ToLCV isolates are different from the tomato geminiviruses
found in other regions of East and Southeast Asia and Australia (Muniyappa
et al., 2000). Point mutation in one or several nucleotides can lead
to the genetic diversity of viruses. Such genetical events in viral genome can
result in many isolated strains that are genetically very much similar. Genomic
recombination in geminiviruses, not only between the variants of the same virus
but also between species and even between genera, has resulted in rapid diversification.
Due to the rapidly increasing number of geminiviruses that are being isolated,
there has been an urgent need for an improved system of classification and nomenclature
(Fauquet and Stanley, 2003; Mehrotra
and Aggarwal, 2003; Varma and Malathi, 2003).
The <89% of nucleotide identity threshold between full-length of DNA-A component
nucleotide sequences for Begomovirus species as a species demarcation
criteria is very important.
||Phylogenetic tree showing the genetic relationship of TYLCV-
GN- SL isolated from Sri Lanka to other related and begomoviruses. The numbers
appearing at each node indicate the percentage of supporting bootstrap samples
Proposed 89% threshold is not an absolute criterion and it is possible for
viruses to share more than 89% identity yet be classified as distinct species
on the strength of their biological properties and vice versa (Fauquet
and Stanley, 2003).
Geographical location of Sri Lanka being very close to India can be a risk of trans-boundary-migration of virus infected plants, viruliferous whitefly vectors etc. Genetic similarity of TYLCV-GN-SL with related Indian isolates implies this type of trans boundary migration.
It is important to sequence of the full genome of the TYLCV in Sri Lanka for
the obtaining of more information for further classification. In several cases,
the CP of TYLCV was more homologous to the CP of other whitefly-transmitted
geminiviruses occurring in the same region than to TYLCV isolates from other
regions, a fact that may point to adaptation of the geminivirus to its vector
(Czosnek and Laterrot, 1997).
Tomato samples collected from Badulla, Nuwara-Eliya, Kandy and Matale districts
showed characteristic symptoms of TYLCV identified (Table 3).
||Visual diagnosis and molecular detection of TYLCV from different
|YM: Yellow color leaf margin area, Y: Yellowing of leaflets,
DG: Dark green veinal area, C: Curling, S: Shrinking of leaflets, R: Leaf
size and growth reduction, +: Positive results with degenerate primers (Deng
et al., 1994)
The PCR studies using universal primers showed that all samples were infected
with Begomovirus and resulted 520 bp bands (Fig. 5).
It suggested that TYLCD is one of the major constraints in major tomato growing
||Molecular detection of TYLCV from major tomato growing districts,
Lanes-M: 100 bp marker (Vivantis), 1, 2: Infected, 3: Healthy tomato
samples (Badulla), 4, 5: Infected, 6: Healthy tomato samples (Nuwara: Eliya),
7, 8: Infected, 9: Healthy tomato samples (Kandy), 10, 11: Infected, 12:
Healthy tomato samples (Matale)
TYLCD is the most important and common virus disease of tomato in Sri Lanka
and it is reported in all tomato-growing areas. In 1998, 40% TYLCV infection
of variety T- 146 was reported in Katugastota while 90% of TYLCV infection in
variety Caribo was reported at Marassana (Ariyarathna et
al., 2004). According to the survey conducted, TYLCD incidence was high
in Kandy, Matale and Nuwara- Eliya districts. It affected over 25% of the tomato
cultivation and disease severity in some areas has gone up to 75% (Anonymous,
2004). Yield losses in tomato due to TYLCV usually ranged from 50-75% and
even up to 100% but may be as high as 100% making tomato production unprofitable
(Green and Kalloo, 1994). It is important to take the
control measures in the beginning of tomato cultivation. In addition, concerning
of the epidemiology of TYLCV is important within major tomato growing areas
due to the presence of TYLCV. The TYLCV management practices should be concerned
with destroying of even single tomato plant infected with TYLCV to control spreading
We thank all the staff members of the Horticultural Crops Research and Development Institute (HORDI), Gannoruwa, Peradeniya and Department of Plant Sciences, Faculty of Agriculture, Rajarata University of Sri Lanka.
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