Molecular characterization of Begomovirus infecting sweet...
Transcript of Molecular characterization of Begomovirus infecting sweet...
Indian Journal of Biotechnology
Vol 6, January 2007, pp 45-51
Molecular characterization of Begomovirus infecting sweet pepper in Oman
Akhtar J Khan*, Nadiya A Al-Saady, Madleen S Al-Mahruki, Muna Al-Oufi and Ali M Al-Subhi
Department of Crop Sciences, College of Agricultural and Marine Sciences, Sultan Qaboos University, P.O. Box-34, Al-
Khod 123, Sultanate of Oman
Received 18 October 2005; revised 16 January 2006; accepted 22 March 2006
Whitefly transmitted tomato yellow leaf curl is one of the most devastating viral disease of cultivated sweet pepper
(Capsicum frutescens grossum) and other vegetables in Oman. Infected sweet pepper plants showed typical begomovirus
symptoms as upward leaf curling, interveinal and leaf chlorosis, and growth stunting. Begomovirus infecting sweet pepper in
Oman was detected by polymerase chain reaction (PCR) using begomovirus specific degenerate primers
(PAL1v1978/PAR1c496 and AV494/AC1048). Core region (74-604 bp) of coat protein gene of the begomovirus was
amplified by PCR with tomato yellow leaf curl virus (TYLCV) specific degenerate primers (TycpV369/TycpC1023). Core
region of coat protein gene contains highly conserved regions and is used to identify the begomovirus infecting sweet
peppers. Virus identification was performed by percent sequence identity and parsimony analysis using core coat protein
gene sequences of sweet pepper virus with complete genome, core region of coat protein and coat protein gene sequences
from reference begomoviruses. The core region sequence identity of coat protein gene of sweet pepper virus from Oman
was 92.2, 96.5, 94.0, 93.8, and 96.5% with TomGV-Lebanon, TYLCV-Guadeloupe, TYLCV-Israel, TYLCV-Kuwait, and
TYLCV-Mexico, respectively. Phylogenetic trees and percent sequence identity with reference to begomoviruses permitted
the identification of sweet pepper virus as TYLCV based on tree position and extent of sequence identity. Phylogenetic
analysis revealed that sweet pepper tomato yellow leaf curl virus clustered with its closest relatives from Middle East
regions but formed a separate strain.
Keywords: sweet pepper, begomovirus, core region of coat protein gene
IPC Code: Int. Cl.8 C12N15/11, 15/34
Introduction Diseases of many vegetables, including sweet
pepper, caused by whitefly-transmitted geminivirus
are known worldwide1,2
. The typical symptoms of
sweet pepper disease caused by begomovirus are leaf
curl, leaf crumple, foliar mosaic, and mottle among
others. However, symptoms of a given virus-host
combination may vary depending on cultivars,
environmental conditions and virus strain. Infection of
a single host by two different viruses causing similar
symptoms is often noticed, which can complicate the
diagnosis of the disease and make the phenotype
symptoms an unreliable criteria for viral
identification. While DNA sequence comparison and
phylogenetic analysis with sequences of known
begomoviruses can evidently provide a reliable viral
identification3.
In Oman, exotic cultivars of sweet pepper are
cultivated each year during October-March in the
northern coastal region of Al-Batinah. The crop is
grown to meet the high domestic demand for fresh
produce. However, the major constraint in sweet
pepper cultivation is a disease, resulting in leaf
curling, mottling and growth stunting of plants,
caused by whitefly-transmitted geminivirus.
Geminiviruses are a group of plant DNA viruses
characterized by the geminate shape and particle size
of 18-25 nm4. Whitefly transmitted geminiviruses are
classified into the genus Begomovirus of the family
Geminiviridae, and are transmitted by Bemisia
tabaci5. The majority of the Begomoviruses are
known to have bipartite genome but single genome
(DNA-A) monopartite viruses are being reported, in
increasing numbers, mainly from the old world6.
Identification of Begomoviruses using serology is not
suitable, because high titre antisera are difficult to
prepare and lack sufficient specificity. Consequently,
DNA based molecular diagnostic techniques, such as
polymerase chain reaction (PCR) amplification, using
universal or specific primers and DNA sequencing
has supplemented serology for detection and
identification of begomoviruses. So far, classification
and phylogenetic relationships of begomoviruses have
been based upon complete monopartite viral genomes
___________
*Author for correspondence:
Tel: (+968) 24 141217; Fax: (+968) 24 413418
E-mail: [email protected]
INDIAN J BIOTECHNOL, JANUARY 2007
46
or the DNA-A component of the bipartite viruses,
coat protein (CP) and/or replication association
protein gene (Rep) trees7-9
. Although complete
genome sequences are desirable for identification and
classification of geminiviruses, this requires
considerable time and expense. Full-length CP gene
sequences are accepted by the International
Committee on the Taxonomy of Viruses (ICTV) for
the provisional classification of begomoviruses when
complete genome (monopartite virus) or DNA-A
component (bipartite virus) sequences are
unavailable10
. For sequences to be useful for
identification at both the species and strain/variant
levels, regions of the genome that contain both
conserved and variable elements should be targeted.
So far, CP gene has been proven useful for plant virus
detection and identification. PCR based studies have
established the diversity of tomato yellow leaf curl
virus in Sudan, showing that there are two distinct, yet
closely related, Begomovirus species associated with
tomato leaf curl disease11
. In this paper, authors
describe the molecular characterization of a tomato
yellow leaf curl virus (TLCV), a begomovirus
infecting sweet pepper in Oman.
Materials and Methods Sample Collection
To identify the causal agent(s), sweet pepper
samples showing typical leaf curl disease symptoms
were collected from different fields in Al-Batinah
region during 2004-2005. Samples were brought in
cool boxes from field and stored at -80oC until used
for DNA extraction.
Total DNA Extraction
Total DNA was extracted from symptomatic and
asymptomatic leaves of sweet pepper using
Dellaporta extraction buffer12
with some
modifications.
PCR
DNA samples extracted from symptomatic sweet
pepper plants were used as template for PCR. DNA
extracted from asymptomatic plants and sterile water
was used as negative control. Begomovirus specific
degenerate primer pairs PAL1v1978 (5’-
GCATCTGCAGGCCCACATYGTCTTYCCNGT-3)
and PAR1c496 (5’-AATACTGCAGGGCTTYCTR-
TACATRGG-3’)13
; and AV494 (5’-GCC(C/T)AT-
(G/A)TA(T/C)AG(A/G)AAGCC(A/C)AG-3’) and
AC1048 (5’-GG(A/G)TT(A/G/T)GA(G/A)GCATG-
(T/A/C)GTACATG-3’)14
were used to prime the
amplification of 1.1-1.4 kb and 500-600 bp product,
respectively. TYLCV core CP gene specific
degenerate primer pair TycpV369 (5’-
ACGCCCGYCTCGAAGGTTCG-3’) and TycpC1023
(5’-GTACAWGCCATATACAATAA-CAAGGC-3’)
was used to amplify 500-650 bp core CP region of the
virus genome15
. Fifty ng of each DNA preparation in
1 µL TE were added to ‘Ready-To-Go’ PCR beads
(Pharmacia Biotech, Sweden), followed by 1 µL of
each primer (10 pmoles) and 22 µL of sterile
deionized water for a final reaction mixture volume of
25 µL. The DNA was amplified by 35 cycles,
consisting of denaturation at 94oC for 30 sec (2 min
for cycle 1 and 45 sec for cycle 35), annealing at 55oC
for 1 min, and primer extension at 72oC for 2 min (5
min for cycle 35). After amplification, 4 µL aliquot
from each sample was electrophoresed in a 1%
agarose gel and visualized by staining with ethidium
bromide and UV illumination.
Cloning of PCR Amplified DNA
The PCR amplified DNA fragments using CP gene
specific primer pairs TycpV369/TycpC1023 were
purified using the Wizard PCR DNA purification
system (Promega Corp., Wisconsin, USA). The
purified DNA was cloned using Promega pGEM-T
Easy Vector System II cloning kit according the
manufacturer’s instructions (Promega Corp.,
Wisconsin, USA). Recombinant plasmids were
screened by blue/white color screening on indicator
plates and PCR amplification using
TycpV369/TycpC1023 primers.
Sequencing and Phylogenetic Analysis
A cloned TycpV369/TycpC1023 fragment from
infected pepper virus was sequenced to verify
phylogenetic relationships; the sequence was
deposited in NCBI Gene Bank under the Accession
Number DQ132859.
Automatic sequencing was performed on an ABI
Prism Model 377 (PE Applied Biosystem, CA, USA)
with TycpV369/TycpC1023 primers using Big Dye
terminator chemistry16
at DNA sequencing facility of
Sultan Qaboos University.
DNA sequence of sweet pepper virus CP gene was
analyzed in BLAST on NCBI nucleotide database to
obtain information on sequence similarity. CP and
complete genome sequences of 31 geminiviruses
(Table 1) were aligned by the CLUSTAL W method
with MegAlign suite of LaserGene 6.0 software
KHAN et al: IDENTIFICATION OF BEGOMOVIRUS FROM SWEET PEPPER IN OMAN
47
(DNASTAR, Inc. Madison, WI, USA). The sequences
were visually inspected for logical placement of gaps
and manually adjusted where necessary. Fifteen CP
sequences belonging to geminivirus were separately
aligned including Oman strain to analyze CP
sequence identity using MegAlign suit of Lasergene
5.01 (Table 2). Cladistic analyses were performed
with PAUP (Phylogenetic Analysis Using Parsimony,
version 4.0b10)17
. Uninformative characters were
excluded from analyses. A phylogenetic tree was
constructed by a heuristic search via random stepwise
addition, implementing the tree bisection and
reconnection algorithm to find the optimal
phylogenetic tree(s). Beet curly top virus (Curtovirus),
a geminivirus was designated as outgroup to root the
tree. The analysis was replicated 1000 times.
Bootstrap analysis was performed to estimate stability
and support for the inferred clades18
.
Results Survey of Disease
Ten sweet pepper grown agricultural farms,
averaging 10-15 acres in size, were surveyed in Al-
Batinah, northern region of the Sultanate of Oman.
Mainly green cultivars of Sweet peppers were grown
on farms either under shade house or open field
during September to February. All the farms surveyed
were 5-10 km apart from each other and had varying
degree of leaf curl disease incidence. The disease
incidence ranged from 5 to 45% in older crops (data
not shown). Further, the incidence on sweet peppers
was high on field grown plants as compared to those
grown under shade house.
Symptoms in Diseased Plants
Typical leaf curl symptoms were observed on sweet
peppers infected with TYLCV. Peppers exhibited
upward curling of leaf margins with interveinal and
marginal chlorosis, and thickening of veins (Fig. 1).
In severe infection, yellow of the leaves was evident.
Infected leaves were often found thicker than the
normal ones. Detection of TYLCV by PCR Assays
A total of sixty samples (6 sample from each
farm) collected from infected sweet pepper yielded
Table 1—List of viruses compared in the present study, their origin and NCBI gene accession numbers
Virus strains Acronyms Origin Gene Accession No.
Beet curly top virus BCTV USA M24597
Tomato begomovirus ToBV-MT Mayotte AJ620914
Tomato begomovirus ToBV-FR France AJ620916
Tomato geminivirus ToGV-DR Dominique Republic AF068640
Tomato geminivirus ToGV-LB Lebanon AF065823
Tomato geminivirus ToGV-SD Sudan AF058030
Tomato yellow leaf curl virus TYLCV-FR France AJ842306
Tomato yellow leaf curl virus TYLCV-GD Guadeloupe AY319646
Tomato yellow leaf curl virus TYLCV-IS Israel AF058020
Tomato yellow leaf curl virus TYLCV-KU Kuwait AF065822
Tomato yellow leaf curl virus TYLCV-MX Mexico U65089
Tomato yellow leaf curl virus TYLCV-OM Oman DQ132859
Tomato yellow leaf curl virus TYLCV-RU Reunion AJ842307
Tomato yellow leaf curl virus TYLCV-RU Reunion AJ842308
Tomato yellow leaf curl virus TYLCV-EG Egypt AY594174
Tomato yellow leaf curl virus TYLCV-PG Portugal AF105975
Tomato yellow leaf curl virus TYLCV-IS Israel AB110218
Tomato yellow leaf curl virus TYLCV-JO Jordan AY594175
Tomato yellow leaf curl virus TYLCV-SP Spain AF071228
Tomato yellow leaf curl virus TYLCV-JP Japan AB014346
Tomato yellow leaf curl virus TYLCV-AL Almeria AJ489258
Tomato yellow leaf curl virus TYLCV-DR Dominique Republic AF024715
Tomato yellow leaf curl virus TYLCV-CU Cuba AJ223505
Tomato yellow leaf curl virus TYLCV-PR Puerto Rico AY134494
Tomato yellow leaf curl virus TYLCV-IR Iran AJ132711
Tomato yellow leaf curl virus TYLCV-ML Mali AY502934
Cassava begomovirus CBM-MZ Mozambique AF329243
Cassava begomovirus CBM-SZ Swaziland AF329235
South African Cassava mosaic virus SACMV-SA South Africa AF155806
Tomato begomovirus ToBV-MYDB Mayotte:Dembeni AJ865341
INDIAN J BIOTECHNOL, JANUARY 2007
Table 2--Comparison of nucleotide sequence of coat protein gene of begomovirus infecting sweet pepper from Oman with other begomoviruses
Coat protein gene sequence identity (%) Gemini virus strains* 1 2 3 4 5 6 7 8 9 10 11 12 13 14
SACMV-SA TomBV-MT TornBV-FR TomGV-DR TomGV-LB TomGV-SD TYLCV-FR TYLCV-GD TYLCV-IS TYLCV-KU TY LCV-MX TYLCV-OM TYLCV-RU TYLCV-RUSG
*Abbreviations of virus strains and descriptions are given in Table 1 I
I were aligned using Bio Edit program and analyzed in BLAST. NCBI BLAST results revealed that sequence - from symptomatic sweet pepper showed 98% identity with TYLCV reported from Egypt and Kuwait; 97%
I identity to TYLCV from Lebanon, Israel, and Jordan; 95% identity to TYLCV strains from Puerto Rico and Iran; 92% identity to TYLCV strain from Mali; 94% identity to Sudan strains; 85% identity to Cassava mosaic virus from South Africa; and 87% identity to tomato begomovirus from many countries (Table 2). Comparison of CP gene sequences of many begomoviruses from different parts of the world showed that TYLCV sequence from Oman is 96.5% identical to those reported from Guadeloupe and Mexico; 94% identical to strains from Israel; 93.8% identical to Kuwait; and more than 90% identical to
Fig. I-Sweet pepper plants infected with begomovirus tomato begomovirus strains from Dorninique Republic, Lebanon, and Sudan (Table 2).
begomovirus specific 1.2 kb products when amplified by PCR using the primer pair PALlv1978/PARlc496 Phylogenetic Analyses and 650 b~ with primer pair AV494 and AC1048 Results of phylogenetic analyses on core CP gene (Figs 2A & B). A 602 bp product was amplified by of T ~ C V from- sweet pepper in oman pig. 3) PCR using the TyLCV core CP gene specific primer revealed that sweet pepper virus clusters with TYLCV pairs T ~ c ~ V 3 6 9 and T ~ c ~ C 1 0 2 3 pig. 2C). PCR sbains from Kuwait, and Israel and TomGV from products were amp1ified plant Lebanon but formed a separate clade indicating a new samples unless otherwise mentioned. strain. The closest relative of TYLCV from Oman CP Gene Sequence Analysis was tomato begomovirus from Lebanon and Sudan.
PCR amplified DNA fragments of 602 b using These results support the conclusion that sweet pepper I P 9 . TYLCV CP gene specific primer pairs , were T K C V from Oman is closely related to other
sequenced. One PCR positive symptomatic sample TYLCV strains from Middle East region, but forms a. fiom each farm, i.e. a total of 10 samples, was separate strain and can be named as TYLCV-Oman sequenced for phylogenetic analysis. The sequences strain.
KHAN et al: IDENTIFICATION OF BEGOMOVIRUS FROM SWEET PEPPER IN OMAN
49
Discussion Geminiviruses constitute an important group of
pathogen characterized by a circular single stranded
DNA genome and unique geminate particle
morphology19
. The TYLCV, a member of whitefly-
transmitted Geminiviruses, is a worldwide threat for
cultivated tomatoes and other agricultural crops3,20-22
.
Al-Batinah region is located on northern part of the
Sultanate of Oman along the coastal line and
comprises more than 80% of the total arable land.
During the survey in Al-Batinah region, 5-10%
incidence of leaf curl disease was found on sweet
peppers grown under shade house, while 35-45% on
those grown under open field. The low disease
incidence on plants under shade house could be due to
restricted movement of whiteflies, whereas in field
population of whiteflies was found in great
abundance. In addition, most of the field grown sweet
pepper crops were located adjacent to tomato field,
which showed more than 80% incidence of leaf curl
disease.
TYLCV was first recorded in Israel as a whitefly-
transmitted virus in tomato (Lycopersicon
esculentum) crops23-25
. It has been identified on the
basis of molecular data and placed in the Genus
Begomovirus, Family Geminiviridae. TYLCV occurs
in most eastern Mediterranean countries26
and parts of
sub-Saharan Africa, Asia, Australia, and the
Caribbean27
. In Europe two species of TYLCV are
present21
, comprising tomato yellow leaf curl virus
(TYLCV) syn TYLCV-IL, and tomato yellow leaf
curl Sardinia virus (TYLCSV) (using the most recent
taxonomic nomenclature described3).
All fragments obtained with TYLCV coat protein
gene specific PCR primers had a 90% or greater
nucleotide identity with their respective viruses. The
begomovirus specific primers yielded expected
product confirming the presence of virus in infected
sweet pepper samples14,28-31
. The CP gene is the most
highly conserved gene in the genus Begomovirus14
.
The CP gene sequence is used to predict strains,
species and taxonomic lineages of begomoviruses32
.
International Committee on Taxonomy of Viruses
(ICTV) accepts the classification of begomoviruses
based on CP sequence when full length genomic
sequences are not available9,33
.
Alignment of core CP sequences of different
begomoviruses with CP sequence of the present
Oman isolate and phylogenetic analysis of aligned
sequences clearly indicates that virus strain isolated
Fig. 2 (A-C)—PCR amplification of begomovirus isolated from
field grown sweet pepper in Oman: A. A product of 1.2 kb
amplified by using PAL1v1978 and PAR1c496; B. 620 bp
product amplified by primers AV494 and AC1048; & C.620 bp
product amplified by primers TycpV369 and TycpC1023 specific
to core CP region of TYLCV.
INDIAN J BIOTECHNOL, JANUARY 2007
50
from sweet pepper in Oman is a TYLCV strain. CP
sequence analysis and phylogenetic tree
reconstruction permits a predition of relatedness
among begomoviruses that is highly similar to the
outcome from alignment of begomovirus CP gene
sequences8,10
. The core CP region has highly
conserved amino acid sequences interrelated with
variable bases in all members of Begomovirus. These
features provide useful molecular tool to perform
preliminary identification of begomoviruses. The core
CP sequence contains sufficient conserved and
variable regions that virtually mimic the overall
composition of the complete CP sequence32
. Padidam
et al8 have used highly variable 5’ end of the CP gene
sequence ( ~ 200 nt) as a molecular marker to reveal
differences between closely related strains of
begomoviruses. However, complete reliance on
highly variable region may complicate the
begomovirus identification that otherwise can be done
by the conserved region. Present results from core CP
gene sequence alignment and construction of
phyologenetic tree using CP sequences and complete
genome sequences have clearly established that the
virus infecting sweet pepper from Oman is a discrete
strain of TYLCV belonging to the genus Begomovirus
and the name tomato yellow leaf curl virus-Oman
(TYLCV-OM) is proposed for the same.
Acknowledgement
Authors are thankful to the Sultan Qaboos
University for financial support (AJK) to carry out
this work. We thank Dr David Dennison of SQU for
his help in DNA sequencing.
References 1 Brown J K, Molecular markers for the identification and
global tracking of whitefly vector-begomovirus complexes,
Virus Res, 71 (2000) 233-260.
2 Brown J K, The molecular epidemiology of begomoviruses,
in Trends in plant virology, edited by J A Khan & J Dykstra
(The Haworth Press, New York) 2001, 279-316.
3 Fauquet C M, Mayo M A, Maniloff J, Desselberger U & Ball
L A, Virus taxonomy, VIIIth Report of the ICTV
(Elsevier/Academic Press, London) 2005
4 Fauquet C M, Bisaro D M, Briddon R W, Brown J K,
Harrison B D et al, Revision of taxonomic criteria for species
demarcation in the family Geminiviridae, and an updated list
of begomovirus species, Arch Virol, 148 (2003) 405-421.
5 Harrison B D & Robinson D J, Natural genomic and
antigenic variation in whitefly-transmitted geminiviruses
(begomoviruses), Ann Rev Phytopathol, 37 (1999) 369-398.
6 Sharma P, Rishi N & Malathi V G, Molecular cloning of coat
protein gene of an Indian cotton leaf curl virus (CLCuV-
HS2) isolate and its phylogenetic with others members of
Geminiviridae, Virus Genes, 30 (2005) 85-91.
7 Brown J K, Ostrow K M, Idris AM & Stenger D C, Chino
del tomate virus: Relationships to other begomoviruses and
the identification of A component variants that affect
symptom expression, Phytopathology, 90 (2000) 546-552.
8 Padidam M, Beachy R N & Fauquet C F, Classification and
identification of geminiviruses using sequence comparisons,
J Gen Virol, 76 (1995) 249-263.
9 Rybicki E P, A phylogenetic and evolutionary justification
for three genera of Geminiviridae, Arch Virol, 139 (1994)
49-77.
10 Mayo M A & Pringle C R, Virus taxonomy—1997, J Gen
Virol, 79 (1998) 649-657.
11 Idris A M & Brown J K, Evidence for interspecific
recombination for three monopartite begomoviral genomes
associated with the tomato leaf curl disease from central
Sudan, Arch Virol, 150 (2005) 1003-1012.
12 Dellaporta S L, Wood J & Hicks J B, A plant DNA mini
preparation: Version II, Plant Mol Biol Rep, 1 (1983) 19-21.
Fig. 3—single most parsimonious tree reconstructed by PAUP
(ver 4b10) of sweet pepper virus CP gene sequences with other
reference begomovirus CP sequences fron GeneBank database.
BCTV-US (Curtovirus) was included as outgroup to root the tree.
Bootstrap values are placed on major nodes in accordance with
50% confidence statistics. (Abbreviations of virus strains and
descriptions are given in Table 1)
KHAN et al: IDENTIFICATION OF BEGOMOVIRUS FROM SWEET PEPPER IN OMAN
51
13 Rojas M R, Gilbertson R L, Russell D R & Maxwell D P,
Use of degenerate primers in the polymerase chain reaction
to detect whitefly-transmitted Geminiviruses, Plant Dis, 77
(1993) 340-347.
14 Wyatt S D & Brown J K, Detection of subgroup III
geminivirus isolates in leaf extracts by degenerate primers
and polymerase chain reaction, Phytopathology, 86 (1996)
1288-1293.
15 Sedegui M, Nakhla M K, Evans T A, Maxwell D P & Arifi
A, Detection of tomato yellow leaf curl virus and tomato
yellow leaf curl Sardinia virus with a general probe and
species specific probes in tomato samples from Mexico,
Phytopathology, 92 (2002) S74.
16 Heiner C R, Hunkapiller K L, Chen S M, Glass J I & Chen E
Y, Sequencing multimegabase-template DNA with BigDye
terminator chemistry, Genome Res, 8 (1998) 557-561.
17 Swofford D L, PAUP: Phylogenetic analysis using
parsimony (and other methods) Version 4 (Sinauer
Associates, Sunderland, Massachusetts, USA) 2002.
18 Felsenstein J, Confidence limits on phylogenies: an approach
using the bootstrap, Evolution, 39 (1987) 783-791.
19 Goodman R M, Single stranded-DNA genome in a whitefly-
transmitted plant virus, Virology 83 (1977) 171-179.
20 Czosnek H & Laterrot H, A world-wide survey of tomato
yellow leaf curl viruses, Arch Virol, 142 (1997) 1391-1406.
21 Moriones E & Navas-Castillo J, Tomato yellow leaf curl
virus, an emerging virus complex causing epidemics
worldwide, Virus Res, 71 (2000) 123-134.
22 Crescenzi A, Comes S, Napoli C, Fanigliulo A, Pacella R et
al, Severe outbreaks of tomato yellow leaf curl Sardinia virus
in Calabria, Sothern Italy, Comm Agric Appl Biol Sci, 69
(2004) 575-580.
23 Cohen S & Nitzany F E, Transmission and host range of the
tomato yellow leaf curl virus, Phytopathology, 56 (1966)
1127-1131.
24 Cohen S & Antigus Y, Tomato yellow leaf curl virus
(TYLCV), a whitefly-borne geminivirus of tomatoes, Adv
Dis Vector Res, 10 (1994) 259-288.
25 Czosnek H, Ber R, Navot N, Zamir D, Antignus Y et al,
Detection of yellow leaf curl virus in lysates of plants and
insects by hybridization with a viral DNA probe, Plant Dis,
72 (1988) 949-951.
26 Fauquet C M, Sawyer S, Idris, A M & Brown J K, Sequence
analysis and classification of apparent recombinant
begomoviruses infecting tomato in the Nile and
Mediterranean basins, Phytopathology, 95 (2005) 549-555.
27 Paximadis M, Idris A M, Torres-Jerez I, Villarreal A, Rey M
E C et al, Symptom phenotype, host range and molecular
characterization of geminiviruses with a common tobacco
host in the old and new world, Arch Virol, 144 (1998) 703-
717.
28 Deng D, McGrath P F, Robinson D J & Harrison B D,
Detection and differentiation of whitefly-transmitted
geminiviruses in plants and vector insects by the polymerase
chain reaction with degenerate primers, Ann Appl Biol, 125
(1994) 327-336.
29 Saiki R K, Gelfand D H, Stoffel S, Scharf S J, Higuchi R et
al, Primer directed enzymatic amplification of DNA with a
thermostable DNA polymerase, Science, 301 (1988) 260-
262.
30 Shih L, Tsai W S, Green S K & Hanson P M, Molecular
characterization of a new begomovirus from India, Plant Dis,
87 (2003) 598.
31 Swangjit S, Chatchawankanphanich O, Chiemsombat P,
Attathom T, Dale J et al, Molecular characterization of
tomato infecting begomoviruses in Thailand, Virus Res, 109
(2005) 1-8.
32 Brown J K, Idris A M, Torress-Jerez I, Banks G K & Wyatt
S D, The core region of the coat protein gene is highly useful
to establishing the provisional identification and
classification of begomoviruses, Arch Virol, 146 (2001)
1581-1598.
33 Rybicki E P, A proposal for naming geminiviruses: A reply
by the Geminiviridae Study Group Chair, Arch Virol, 143
(1998) 421-424.