Begomoviruses cause great losses of tomato crops in Central America. Eight
begomoviruses were identified by sequencing PCR fragments. Four of these
were new viruses: Tomato severe leaf curl virus (ToSLCV, AF130415), Tomato
golden mottle virus (ToGMoV, AF132852), Tomato mild mottle virus (ToMiMoV,
AF131071) and Tomato yellow mottle virus (ToYMoV, AF112981) and four were
previously characterized: Pepper golden mosaic virus (PepGMV, AF136404)
Tomato mosaic Havana virus (ToMHV, AF139078), Tomato leaf curl Sinaloa
virus (ToLCSinV, AF131213) and Pepper huasteco yellow vein virus (PHYVV).
A general probe, consisting of the most conserved region of the CP gene
of Bean yellow golden mosaic virus (BGYMV, M91604), was used in non-radioactive
hybridization methods to detect these viruses. Specific probes, which
consisted primarily of the common region for each virus, were developed.
Specific primers for PCR identification were designed for each virus,
and the PCR fragments obtained from plant samples with these begomovirus-specific
primer sets were sequenced to confirm the primers’ specificity.
ToSLCV, ToMHV and ToMiMoV were present in tomatoes from Comayagua, Honduras.
ToYMoV and ToLCSinV were present only in samples from Costa Rica. ToSLCV,
ToGMoV, ToMiMoV, ToMHV, PepGMV, ToLCSinV and PHYVV were detected in tomatoes
in one valley in Guatemala. ToSLCV only existed in mixed infections with
another bipartite begomovirus, and no DNA-B was ever identified for this
virus. These detection methods were used to monitor begomoviruses in tomato-breeding
lines being developed in Guatemala for resistance to these viruses.
Tomato-infecting begomoviruses cause serious losses throughout
Central America (Morales and Anderson, 2001; Jones, 2003). In many locations,
incidence is 100% during the dry season and losses may exceed 60%. These
epidemics are associated with the changing agricultural practices, such
as continuous cropping of tomatoes and increased cultivation of hosts
for the natural vector, Bemisia tabaci (Polston and Anderson, 1997). Since
resistant cultivars are not available, the major control measures have
involved numerous applications of insecticides, e.g., applications every
third day. Losses have been so extensive in some areas of Guatemala and
Nicaragua that tomatoes are no longer grown. The begomoviruses associated
with tomatoes in Central America have not been molecularly characterized.
Thus, this study was undertaken to provide sequence data for the begomoviruses
associated with tomatoes collected in Central America, and these sequences
were used to develop specific molecular tools for detection of these begomoviruses
and to apply these tools in a tomato breeding program in Guatemala for
begomovirus resistance (Mejía et al., 2004).
Materials and Methods
Characterization of tomato-infecting begomoviruses from
Tomatoes exhibiting begomoviral symptoms (leaf curl, leaf crumple, yellow
mottle and dwarfing) were collected from Guatemala, Honduras, Costa Rica,
and Nicaragua. Tissues from young leaves for analysis were used for DNA
extraction. Tissues were stored either dried or frozen. Discs were cut
from fresh leaf tissue with a flame-sterilized, number 5 cork borer, placed
in sterilized 1.5-ml microfuge tubes and stored at -80°C. Dry samples
were stored as 0.5-mm-wide strips cut with a flame-sterilized blade, dried
at room temperature, placed in paper envelopes, and stored at room temperature.
DNA from plant tissue was extracted by a modified Dellaporta method (Rojas
et al., 1993) or by the Puregene DNA Isolation Kit, Cell and Tissue Kit
(Gentra Systems, Minneapolis, MN).
PCR products containing the common region and part of the Rep and CP genes
of DNA-A were obtained using degenerate geminiviral primer set (PRepv1978:
5’ GCCCACATYGTCTTYCCNGT 3’/PCPc715: 5’ TTDATRTTYTCRTCCATCCA
3’; Potter et al., 2003). These fragments were cloned in pCR2.1
vector (Original TA CloningR Kit, Invitrogen Corporation, Carlsbad, CA)
and sequenced using Big Dye Sequencing Kit™ (Biotechnology Center,
Madison, WI). Sequence analysis was accomplished by comparison with known
DNA sequences through the National Center for Biotechnology Information
BLAST program, the GCG program (Wisconsin Package Version 10.2, Genetics
Computer Group, Madison, WI) and by DNAMAN Version 5.2.9 software, Lynnon
Full-length PCR fragments were obtained for ToGMoV and ToSLCV. The PCR
overlapping primer set (PToGMoVAvf-NcoI: 5’ TAT CCC ATG GTT CTG
GAG CCT TTG CG 3’/PToGMoVAcf-NcoI: 5’TAT TCC ATG GGT TCC TCC
ATT TCC ACT CTC C 3’) was used to amplify the full-length fragment
of ToGMoV DNA-A, and the primer set PToGMoVBvf-ApaI: 5’ AAC AGG
GCC CAT AAA AAA TGA CCC GCG C 3’ / PToGMoVBcf-ApaI 5’ TTG
TGG GCC CGG GTA GGT AAA AAA TCG C 3’ was used to amplify the DNA-B.
Also, the overlapping primer set PToSLCVAvf-SacI: 5’ AAA AGA GCT
CTC TCT AAA ACT CTA TGT TGC TGG 3’ / PToSLCVAcf-SacI: 5’ AAA
AGA GCT CCC CCT GGT GTC CTG GC 3’ used to amplify DNA-A of ToSLCV.
A full-length clone of DNA-A of Pepper huasteco yellow vein virus (PHYVV,
AY044162) was provided by R. L. Gilbertson (University of California-Davis)
and was used as positive control for virus detection experiments. Bean
golden yellow mosaic virus full-length DNA-A clone pGAA1 (BGYMV, M91604)
(Gilbertson et al., 1991), was used as positive control target DNA for
the degenerate primer set PRepv1978/PCPc715. Tomato yellow leaf curl virus
(TYLCV) infectious clone, pTYEG14 (AY594174), was used as positive control
target DNA for this virus.
PCR protocols and begomovirus-specific primer design
A specific PCR primer pair was developed for each of eight tomato-infecting
begomoviruses (Table 1) using unique conserved
regions of the viral genome identified by sequence alignments for the
Rep gene and common region (data not shown). The specificity and robustness
of the specific PCR primer pairs were tested by PCR with standard viral
DNAs from sequenced clones.
PCR parameters for the eight sets of specific primers were optimized for
25-µl reactions containing: 2.5 µl of 2.5 mM deoxynucleotide
triphosphates (dNTPs), 2.5 µl of 10x buffer, 2.5 µl of 30
mM MgCl2, 0.2 µl of Taq DNA polymerase, 2.3 µl of Taq enzyme
diluent (Idaho Technology), 2.0 µl of each complementary and viral
sense primer at 10 µM, 5.0 µl of DNA extract, and H20. Taq
DNA polymerase, MgCl2, 10x buffer, and dNTPs were purchased from Promega
(Madison, WI) and used as indicated by the manufacturer. The target DNAs
from infected plants were diluted 10 times and the cloned viral DNAs were
used at 20 ng/µl. PCR cycle parameters for specific fragment amplification
with the specific primers were as follows: denaturation at 96°C for
30 sec, annealing temperature as in Table 1 for 30 sec, and extension
at 72°C for 35 sec for 30 cycles. The slope was set to 2.0. Annealing
temperature was dependent on the primer set (Table
1). All reactions were in thin-walled 0.2-ml Labsource® PCR tubes.
PCR-amplified fragments were separated by gel electrophoresis using 1.0%
to 1.5% GenePure LE agarose (IscBioExpress, Kaysville, UT). PCR reactions
were performed in a RapidCycler™ (Idaho Technology, Idaho Falls,
ID). To confirm the specificity of the PCR-based diagnostic tools, the
PCR fragments from reactions with the specific primer pairs were directly
sequenced using Big Dye Sequencing Kit™. Sequences of PCR fragments
from reactions with specific primer pairs that had a 91% or greater nucleotide
identity for 400 nt of the Rep gene and the common region to a known sequence
were considered to be the same begomovirus (Fauquet et al., 2003).
Each sample was amplified with degenerative primer pair set for DNA-A
(PRepv1978/PCPc715) with the same PCR conditions used for the specific
PCR primer pairs except that the PCR cycle parameters for fragment amplification
were as follows: denaturation at 94°C for 1 min, annealing at 55°C
for 2 min, and extension at 72°C for 2 min for 30 cycles. These cycles
were followed by a final cycle of 94°C for 1 min, 61°C for 2 min,
and 72°C for 7 min, and then held at 18°C. Samples that tested
positive with the PRepv1978/PCPc715 primer pair were then tested with
the begomovirus-specific PCR primer pairs. The latter PCR cycle parameters
were used also with the TYLCV-specific PCR primer pair (PTYIRv21: 5’
TTG AAA TGA ATC GGT GTC CC 3’/PTYIRc287: 5’ TTG CAA GAC AAA
AAA CTT GGG ACC 3’) to monitor the existence of this virus in Central
America. These PCR reactions were performed in a MJ DNA Engine PT200 Thermocycler™
(MJ Research Inc., Waltham, MA).
Nucleic acid hybridization probes and hybridization protocols
Nucleic acid hybridization techniques were developed with non-radioactive
probes for dot blot detection of the eight tomato-infecting begomoviruses.
The specific probes for hybridization were designed using the common region
of the viral genomes. This region differs among begomovirus species but
is highly conserved within a given species. A general probe for hybridization
with begomoviruses was based on the highly conserved region of the CP
gene for Western Hemisphere begomoviruses. PCR primer pairs PBGGTv647
(5’ TAT GTG TAT ATC CGA TGT CAC ACG TGG 3’) and PBGGTc1048
(5’ CGA ATT TTC AAT GTC GCA TAT ACA GGG 3’) were developed
to produce the DNA for the general probe from DNA-A of clone pGAA1 of
BGYMV (Table 2). Virus-specific DNA for probe
labeling was produced by PCR with primer pairs listed (Table
2) and with cloned viral DNAs as targets. PCR conditions for DNA probe
amplification were the same as for the degenerate PCR primer pair.
The Alk Phos Direct Hybridization Kit™ (Amersham Pharmacia, Pisscatway,
NJ) was used for the non-radioactive labeling of probes according to the
manufacturer’s instructions. Hybridization techniques for the specific
probes used high stringency with positively-charged nylon membranes (Biodyne
A, Pall-Gelman, Ann Arbor, MI) at 65ºC according to the manufacturer’s
instructions. Low stringency was used with the CP-general probe at 55ºC
according to manufacturer’s instructions. The specificity of the
hybridization technique was confirmed by using full-length cloned viral
DNA of DNA-A from representative of different phylogenetic clusters (Faria
et al., 1994) of begomoviruses: Bean dwarf mosaic virus (pBDA1), Bean
golden mosaic virus (pBZA1), and Bean golden yellow mosaic virus (PGAA1)
were obtained as infectious clones (Gilbertson et al., 1991). Bean calico
mosaic virus was a full-length clone (pBCaMV-A2) and TYLCV was an infectious
clone (pTYEG14). These viral DNA targets were obtained by preparation
of the plasmids of the full-length clones and spotting 25 ng DNA on the
Also, viral DNA from PCR fragments prepared from the clones of the eight
tomato-infecting begomoviruses with the primer pair PRepv1978/PCPc715
served as positive control DNAs for evaluation of the hybridization methods.
Twenty-five ng of DNA of each of the eight targeted viruses were spotted
on each membrane as control targets. DNA from plant tissue was extracted
using the Dellaporta method and 5 µl of the DNA extract was spotted
on the membrane as the target for hybridization. The results of the hybridization
and PCR experiments were compared with those of the direct sequencing
of the PCR-amplified fragments to confirm the specificities of both techniques.
Utilizing the diagnostic tools in a tomato breeding program
for resistance to begomoviruses in Guatemala
Young tomato leaves were collected in March 2004 from different tomato
breeding lines and commercial lines expressing various disease severity
indexes (DSI, see Mejía et al., 2004). DNA was extracted from plant
tissue by Puregene DNA Isolation Kit, Cell and Tissue Kit (Gentra Systems,
Minneapolis, MN) and examined with both PCR and hybridization techniques.
RESULTS and DISCUSSION
Molecular characterization of tomato-infecting geminiviruses
in Central America
PCR products containing the common region and part of the Rep and CP genes
of DNA-A were obtained (?1.4 kb) using degenerate geminiviral primer set
PRepv1978/PCPc715. These fragments were cloned and sequenced (Table
3). Seven different tomato-infecting begomoviruses were identified
in tomatoes growing in Central America. Four of these were new viruses:
Tomato severe leaf curl virus (ToSLCV), Tomato golden mottle virus (ToGMoV),
Tomato mild mottle virus (ToMiMoV) and Tomato yellow mottle virus (ToYMoV);
and three had been previously characterized: Pepper golden mosaic virus
(PepGMV) Tomato mosaic Havana virus (ToMHV) and Tomato leaf curl Sinaloa
virus (ToLCSinV). Based on the obtained sequences, specific primers were
designed and used to amplify full-length clones of DNA-A for ToGMoV and
ToSLCV (Table 3). Specific primers were also
used to amplify a full-length clone of the DNA-B of ToGMoV. No DNA-B was
ever identified for this for ToSLCV and it always exited in mixed infections
with another bipartite begomovirus (data not shown). DNA-A of ToSLCV was
also isolated in 1997 from cucumber growing near Sanarate, Guatemala (AF131735).
The primer set PPHYVVv/PPHYVVc specific for PHYVV was used to examine
tomato samples collected from Guatemala. Sequence analysis of the PCR-amplified
700-bp DNA fragment showed 97% nucleotide identity to PHYVV. This proved
the existence of PHYVV in Guatemala.
Sequences of the eight different tomato-infecting geminiviruses detected
in Central America in this study were analyzed together with twelve previously
identified, whitefly-transmitted geminiviruses. The relationships among
the 20 whitefly-transmitted geminiviruses were based on 805 nucleotides
of the N-terminus of the Rep gene. Sequence analysis showed that the eight
identified viruses are from different species. A phylogenetic tree (DNAMAN
software) indicated (Fig. 1) that ToYMoV and
ToGMoV are not related and they are not closely related to any of the
identified geminiviruses. ToLCSinV is in the same cluster as Potato yellow
mosaic Trinidad virus (PYMTriV), ToMiMoV grouped with Tomato rugose mosaic
virus (ToRMV). ToSLCV is most closely related to Squash leaf curl virus
(SLCV). Using the Phylogenetic tree (Fig. 1)
we can put the eight detected begomovirus in Central America in six different
groups. Group one has ToYMoV. Group two has PYMTriV, ToLCSinV, and ToMHV.
Group three has PHYVV. Group four has ToRMV, Potato yellow mosaic virus
(PYMV) and ToMiMoV. Group five has ToGMoV. Group six has ToSLCV, SLCV
Evaluation of PCR primer pairs designed for detection
of begomoviruses using cloned DNAs as the target DNAs
The begomovirus-specific PCR primer pairs developed for each of the seven
isolated viruses produced the expected fragments of ca. 400 bp. The specific
primers designed for PHYVV amplified the expected 700-bp fragment. All
primer pairs amplify only their respective viral DNAs. The specificity
of the primer pair for each virus was obtained by adjusting the annealing
temperature for each set of primers (Table 1).
Evaluation of hybridization probes designed for detection
of begomoviral DNAs with cloned DNAs and plant extracts
The PCR primer pairs (Table 2) designed to amplify
specific DNA probes from cloned viral DNAs gave the expected sizes, which
ranged from 120 to 700 bp. All probes were specific to their respective
viral targets and did not give hybridization signals with symptomless
tomatoes or from the other twelve different cloned viral DNAs (seven characterized
tomato viruses from this study plus the five standard viruses from different
phylogenetic clusters). The general probe gave strong signals with all
five different cloned, full-length viral DNAs from different clusters
of begomoviruses (BDMV, BGMV, BGYMV, BCaMV and TYLCV) and did not react
with the healthy tomato extract.
Field survey of tomato plants collected in Central America
The general and specific detection tools developed were applied to field-collected
tomato tissues from 1992-2004 in Guatemala, Honduras, Costa Rica, and
Nicaragua (Table 4). All 88 tomato plants
tested exhibited leaf curl, mottle, and/or golden mottle symptoms. DNA
was extracted by the Dellaporta method (Rojas et al., 1993) and tested
with PCR and hybridization methods. Seventy-three samples gave hybridization
signals with the CP-general probe. The degenerate DNA-A primer pair (PRepv1978/PCPc715)
detected begomoviruses in 82 samples. PCR primer pairs and hybridization
probes for the specific viruses detected the presence of tomato-infecting
begomoviruses in 82 of the 88 tomato samples collected from Central America
(Table 4). The PHYVV-specific probe and PHYVV-specific
primers were used only on samples collected from Guatemala in 2004, and
were useful in documenting the existence of this virus in Guatemala. Six
of the 88 tomato samples did not give PCR fragments with any of the primer
pairs or hybridization signals with any of the probes. Their symptoms
may have been due to viruses other than begomoviruses or environmental
factors. Our results are consistent with the many reports (Potter et al.,
2003) that have shown that PCR methods are more sensitive than hybridization
methods for detection of viruses. ToSLCV, ToGMoV, ToMHV, PepGMV, ToLCSinV,
ToMiMoV and PHYVV were detected in tomatoes in one valley near Sanarate
in Guatemala. ToSLCV, ToMHV and ToMiMoV were present in tomatoes from
Comayagua, Honduras. Only ToYMoV and ToLCSinV were present in samples
from Alajuela, Costa Rica. Only ToMiMoV and ToLCSinV were present in Nicaragua
in samples collected from Sebaco Valley in 1992. TYLCV was never identified
in any of the tomato samples examined in this study.
Confirmation of specificity of the PCR and hybridization
Positive and negative controls were included in each experiment to confirm
the specificity of the developed diagnostic tools. In each experiment,
the positive and negative controls reacted as expected. One hundred eleven
PCR-amplified fragments from thirty-nine tomato samples were directly
sequenced to confirm the specificity of the designed PCR-primer pairs.
One hundred six PCR-amplified fragments were obtained from tomato samples
collected in Guatemala in 2002, 2003 and 2004, and five PCR-amplified
fragments with primers PToYMoVC1v/PToYMoVC1c were from tomato samples
collected in Costa Rica in 1994 and 1996 (Table
4). All fragments obtained with virus-specific PCR primers had a 91%
or greater nucleotide identity with their respective viruses for 400 nt
or more for the Rep gene and the common region. Twenty-nine PCR fragments
obtained with the ToSLCV-specific primer pair were identified as ToSLCV,
twenty-eight as ToGMoV, thirteen as ToMiMoV, another thirteen as ToMHV,
eight as ToLCSinV, seven as PepGMV and eight more as PHYVV. These PCR
results agreed with the hybridization results.
The PCR and hybridization diagnostic tools developed were useful in monitoring
tomato-infecting begomoviruses in Central America. These diagnostic tools
are fast, sensitive, and accurate and can be used to detect these viruses
in many samples. With the non-radioactive hybridization method, the begomoviruses
can be identified and their concentration in the plants estimated. These
detection methods will have applications in epidemiological studies and
will be useful for monitoring begomoviruses in tomato germplasm in breeding
programs for resistance.
Application of PCR and hybridization detection methods
in a tomato breeding program in Guatemala
The PCR detection and hybridization protocols developed were utilized
in a program for breeding tomatoes resistant to begomoviruses in Guatemala
(see Mejía et al., 2004). The begomovirus resistant lines, Gh13
and Gc16, had begomovirus resistance genes from introgressions from Lycopersicon
hirsutum (Vidavsky and Czosnek, 1988) and Lycopersicon chilense (Scott
et al., 1995), respectively. Young tissues (thirty days after transplanting)
were collected from begomovirus susceptible line (M82) and F1 populations
(susceptible x resistant; resistant x resistant), and DNA was extracted
using the Gentra Extraction kit. Hybridization with CP-general probe (Fig.
2) and with the eight begomovirus-specific probes gave positive signals
for seven begomoviruses (Table 5). Only ToYMoV
was not detected in these samples; this results agree with the previous
survey data, since this virus has not been detected in Guatemala (Table
4). Each line and F1 germplasm was infected with at least two different
begomoviruses except the resistant F1 germplasm, XA175 (Gh13 x Gc16),
which did not have a positive hybridization signal with any of the probes.
Hybrid XA175 had no viral symptoms. However, when the degenerate PCR primers
were used with samples form XA175, PCR fragments were detected, and both
ToGMoV- and ToMiMoV-specific primer sets gave PCR fragments. Since no
hybridization signals were detected from these plants, the viral titer
is very low; and this might explain the lack of symptoms. Strong hybridization
signals were noted with each of the other two hybrid lines, which had
one resistant parental line crossed with M82 (Table
5). The hybrid line XA176 (G16c x M82) has resistance genes from the
Lycopersicon chilense; and it had a moderate symptoms (DSI = 2.5) and
positive hybridization signals with the CP-general probe and with five
different begomovirus-specific probes (Table 5).
The hybridization signal was strong with the CP-general probe, the ToSLCV-specific
probe and the ToMHV-specific probe (Fig. 2)
but the signals were weak with the ToGMoV-, ToMiMoV- and PHYVV-specific
probes. Also, another hybrid line XA188 (G13h x M82), which has resistance
genes from Lycopersicon hirsutum, had very slight symptoms (DSI = 1) and
had positive hybridization signals with the CP-general probe and five
different begomovirus-specific probes (Table 5).
The CP-general probe and the begomovirus-specific probes for ToSLCV and
PepGMV gave medium hybridization signals with this hybrid, whereas the
hybridization signals of the specific probes for ToGMoV, ToMiMoV and PHYVV
were weak. PCR-amplified viral DNA fragments of the expected sizes were
detected in all plants that had positive hybridization signals (Fig.
3). In a few cases, viral DNA fragments of the expected size were
successfully PCR-amplified from samples with no positive hybridization
signals (Table 5). The susceptible line M82 was
infected with at least six begomoviruses in one plant (ToSLCV, ToGMoV,
ToMiMoV, ToMHV, ToLCSinV, and PHYVV). In another M82 plant, five begomoviruses
(ToSLCV, ToGMoV, ToMHV, ToLCSinV, and PHYVV) were detected. In a third
plant, only two begomoviruses (ToSLCV and ToMHV) were identified. Commercial
hybrids, Elios, Marina, Silverado, and Tango, were infected with ToSLCV
and ToGMoV. The specific diagnostic tools detected ToSLCV in all lines
and hybrids except the highly resistant hybrid XA175, ToGMoV in all lines
and hybrids, PepGMV only in the resistant line XA188 and Elios, ToMiMoV
in the line M82 and four hybrids (XA175, XA176, XA188, and Silverado),
but not in Tango, Elios and Marina. ToMHV was detected in M82 and all
hybrids except the resistant hybrid XA175 and Silverado. ToLCSinV was
found only in the susceptible line M82. PHYVV was detected in M82 and
XA176, Elios, and Silverado (Table 5). For all
the samples that gave positive hybridization signals with the CP-general
probe and/or fragments with the degenerate PCR primers set (PRepv1978/PCPc715),
there was at least one begomovirus detected with a virus-specific diagnostic
These data indicate that tomato hybrids being developed for Guatemala
must have resistance to multiple begomoviruses. Also, since it is expected
that TYLCV will eventually be found it Central America, all begomovirus-resistant
tomato hybrids for this region should have resistance to both bipartite
and monopartite begomoviruses.
This research was supported partially by a grant from Bean-Cowpea CRSP/USAID
grant, a CDR/USAID grant no. TA-MOU-01-C21-008, Universidad de San Carlos
de Guatemala, and the College of Agricultural and Life Sciences, University
of Wisconsin-Madison. The authors thank Lane Milde for assistance in designing
and testing the specific primers for PHYVV.
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