Molecular Characterization of a Begomovirus Affecting
Tomato in Colombia and Identification of
Resistance Sources
Martínez A.K1., Morales, F.J1. and Vallejo, F.A2.
1Virology Research Unit, International Center for Tropical Agriculture (CIAT), 2 Universidad Nacional de Colombia, Palmira
Screening for resistance to ToYMV
Ten viruliferous whiteflies were used in the screening of genotypes based upon the results obtained
in the preliminary experiment. High disease incidence scores were observed for most of the
The whitefly, Bemisia tabaci Genn., is an important pest of many industrial and food crops in selected tomato genotypes inoculated with the Colombian isolate of ToYMV, particularly 14 days
tropical and sub-tropical agricultural regions of the world, including Colombia. This whitefly species after inoculation (DPI). Disease severity scores differed among the genotypes tested. FLA 653-3-1-
is considered a complex of different biotypes capable of inducing severe direct and indirect damage 0 had an average score of 1.0 and symptoms were observed only after careful visual inspection,
to many crops as pests and vectors of plant viruses. In the past, B. tabaci was restricted to whereas ‘UNAPAL Maravilla’ and ‘Kyndio’ had scores of 3.4 and 3.5, respectively, showing moderate
agricultural regions under 1000 m of altitude, but following the introduction of a new B. tabaci, this to severe symptoms, including mosaic, yellowing, and foliar/plant malformation (Table 1).
pest expanded its geographic and host range. In the Cauca Valley of Colombia, tomato and snap bean
plantings were severely affected by the biotype B of B. tabaci and previously unreported Table 1. Evaluation of selected tomato genotypes to a Colombian isolate of Tomato yellow mosaic virus.
begomoviruses. In the absence of virus-resistant cultivars, farmers have been trying to control
whiteflies using a myriad of toxic insecticides with disappointing results. Investigations conducted 14 DPI 21 DPI 28 DPI
at CIAT have clearly shown that the local B. tabaci populations have developed resistance to all of
Genotype
the traditional insecticides whiteflies. It is, thus, necessary to implement an integrated whitefly Dis. Dis. Dis. Dis. Dis. Dis.
DBH DBH DBH
management strategy based primarily on the development of virus-resistant cultivars complemented inc. Sev. inc. Sev. Inc. Sev.
by other whitefly control strategies, such as cultural practices, use of new and more selective
insecticides, and use of biological control agents. Kyndio 100 70 2.8 A 100 80 3.0 A 100 80 3.5 A
The objectives of this study were to further characterize the begomovirus that affects U. Maravilla 100 60 2.9 A 100 60 3.1 A 100 60 3.4 A
tomato crops in the Cauca Valley and identify tomato genotypes possessing resistance to this new
CLN 2026D 50 40 1.3 B 90 70 1.7 B 100 70 2.5 B
begomovirus that might be selected as parental materials to incorporate resistance to this pathogen
in the susceptible local tomato cultivar ‘UNAPAL Maravilla’. FLA 456-4 70 10 0.9 BC 70 50 0.8 C 70 100 0.7 C
FLA 496 66 0 0.8 BC 66 16 0.8 C 66 33 1.0 C
FLA 478 30 0 0.3 C 30 0 0.3 C 30 0 0.3 C
FLA 653 20 0 0.2 C 40 20 0.4 C 100 20 1.0 C
The virus isolate was obtained from an affected tomato planting in Rozo, Cauca Valley,
Colombia. Viral DNA was extracted from young leaves of infected tomato plants and then amplified Regarding viral DNA detection, the DBH was slightly more reliable (91%) to detect viral DNA in
using specific degenerate primers for geminiviruses. The PCR products were cloned to obtain the symptomatic test plants than PCR (80%). In the case of FLA 653-3-1-0 and FLA 496-11-6-1-0, virus
complete DNA-A sequence. Phylogenetic analyses were done using MEGA version 4. Bootstrap detection started at 21 DPI, despite a low disease incidence. For FLA 478-6-3-1-11, which had the
analysis was performed for 500 trials using the same program. lowest disease incidence and severity scores, viral DNA was not detected. On the contrary, viral
In order to determine the number of viruliferous whiteflies needed to reach a disease DNA levels in FLA 456-4 were 100%. Susceptible genotypes ‘UNAPAL Maravilla’ and ‘Kyndio’ showed
incidence of 70-100%, using individual insect proof cages, a preliminary experiment was conducted disease incidence levels of 100% 14 DPI but lower viral DNA accumulation in infected test plants
under controlled greenhouse conditions using a completely randomized design with 10 replicates of (60-80%) (Figure 3).
individual tomato plants. Data were analyzed by ANOVA and Duncan Multiple Range test (P<0.05)
A B C
using SAS version 7 for Windows (SAS Institute Inc., Cary, NC, USA).
1
Four experimental tomato lines originally developed in Florida, USA, from S. chilense X S. 1 1
lycopersicum namely FLA 496-11-6-1-0, FLA 478-6-3-1-11, FLA 456-4, and FLA 653-3-1-0 were 2 2 2
tested for begomovirus resistance. The tomato genotypes ‘Kyndio’, ‘UNAPAL Maravilla’, and line 3 3 3
CLN2026D were selected as susceptible controls (Figure 1) 4 4 4
5 5 5
6 6
6
7
7 + - 7 + - + -
Figure 3. Dot blot hybridization of tomato genotypes inoculated with a Colombian strain of Tomato yellow mosaic virus.
Horizontal numbered lines correspond to 10 individual plants assayed for each test genotype, except for FLA 496-11-6-
1-0 and FLA 653-3-1-0 that included only six and five test plants, respectively. A: 14 days post inoculation (DPI), B: 21
DPI, C: 28 DPI. Each spot corresponds to an individual plant. Dark spots indicate presence of viral DNA. (+): infected
susceptible plant (positive control), (-): healthy plant (negative control). Horizontal lanes correspond to: 1) FLA 478-6-3-
1-11, 2) FLA 496-11-6-1-0, 3) CLN 2026D, 4) Kyndio, 5) FLA 456-4, 6) UNAPAL-Maravilla, and 7) FLA 653-3-1-0.
A B
Figure 1. Whitefly-mediated inoculation of ToYMV using Individual insect proof cages
Disease incidence (number of plants infected over total inoculated) and severity were evaluated
two weeks after inoculation., whereas symptom severity was rated according to the 0-4 scale used
by Piven et al. (1995), where 0= no symptoms and 4= severe symptoms. Begomovirus infection was
confirmed by PCR and dot blot hybridization (DBH).
The selected test genotypes were also evaluated under field conditions for their agronomic
characteristics. Plants were evaluated in a randomized complete block design using three replicates
and five plants per replicate. Fruit production parameters evaluated were yield per plant, average
fruit width, length, and weight. Data were analyzed by ANOVA followed by Duncan Multiple Range
test (P<0.05) using SAS version 7 for Windows (SAS Institute Inc., Cary, NC, USA).
A B C D
Figure 4. Symptoms observed at 28 days post inoculation. A- Unapal-Maravilla. (susceptible), B- FLA 478-6-3-1-0
(Resistant)
Begomovirus Characterization Agronomic Characterization
The complete (2596 nt) sequence of the DNA-A component of the Colombian tomato begomovirus With respect to the FLA lines and in the absence of the virus, FLA 478-6-3-1-11 had the lowest
(accession number EU518935) showed a nucleotide identity of 91% when compared to the DNA-A yield (1.53 kg/plant) but the highest fruit weight (182.8 g), whereas FLA 456-4 had the smallest
component of Tomato yellow mosaic virus (ToYMV)–[Guadaloupe] (AY120882). Aminoacid identities E F G
fruits (55.4 g) but a high yield (1.80 kg/plant). The ‘Kyndio’ hybrid showed the highest yield (2.26
for the capsid protein were 90-98% when compared to ToYMV isolates from Panama, Guadaloupe kg/plant). According to the tomato descriptors published by the International Plant Genetic
and Venezuela. Resources Institute (1996), fruit shape for lines FLA 478-6-3-1-11, FLA 496-11-6-1-0 and FLA 653-
Not surprisingly, the Colombian tomato begomovirus can be observed in the ToYMV cluster of a 3-1-0 was ‘slightly flattened’ and for line FLA 456-4 it was ‘rounded’ with orange fruits (Figure 5).
phylogenetic tree generated with the DNA-A sequences of selected begomoviruses (Figure 2). This
cluster includes the different ToYMV strains characterized in different geographic locations of the
Caribbean region.
A B C D
E F G
Figure 5. Fruits of Solanum spp. materials tested for begomovirus resistance. A- cv. Unapal Maravilla, B- hyb. Kyndio, C-
FLA 478-6-3-1-11, D- FLA 653-3-1-0, E- FLA 496-11-6-1-0, F- FLA 456-4 y G- CLN 206 D.
The Tomato yellow mosaic virus is the begomovirus present in tomato crops of the Cauca Valley.
Based on our investigation, it is evident that lines FLA 653-3-1-0, FLA 496-11-6-1-0 and FLA 478-6-
3-1-11 could be used as sources of resistance to ToYMV.
The need to genetically improve tomato for their resistance to begomoviruses known to attack
tomato in Latin America is quite evident and the development of begomovirus-resistant cultivars
should be the foundation of any integrated pest management project.
IPGRI. 1996. Descriptores para el tomate (Lycopersicon spp.). Instituto Internacional de Recursos
Fitogenéticos, Roma, Italia.
Piven, N.M., de Uzcátegui, R.C. & Infante, H.D. 1995. Resistance to tomato yellow mosaic virus in
Figure 2. Phylogenetic tree based on nucleotide sequences available for the A component of selected begomoviruses species of Lycopersicon. Plant Dis. 79: 590-594.
using MEGA 4.0. Bootstrap indices are shown at each node.