© 2006 Plant Management Network.
Relative Distribution and Incidence of Viruses Associated with Disease Outbreaks in Tomato Fields in Trinidad
S. N. Rampersad, The University of the West Indies, Department of Life Sciences, St. Augustine, The Republic of Trinidad and Tobago
Rampersad, S. N. 2006. Relative distribution and incidence of viruses associated with disease outbreaks in tomato fields in Trinidad. Online. Plant Health Progress doi:10.1094/PHP-2006-0309-01-RS.
Tomato production in Trinidad has suffered considerable losses in yield and fruit quality due to infections of hitherto surmised etiology. In order to develop strategies for controlling viral diseases in tomato, the relative distribution and incidence of seven viruses that commonly infect tomato were determined. Of the 362 samples tested, Potato yellow mosaic Trinidad virus (PYMTV) was found in every farm except two and was present at relatively high incidence throughout the country. Tobacco mosaic virus (TMV) and Tobacco etch virus (TEV) were found in fewer farms and at lower incidences while the other viruses were absent. Single infections of either virus were more common than double infections and multiple infections were rare but present. The results indicated that PYMTV is the predominant and most important viral pathogen in tomato production systems in Trinidad; however, begomovirus disease management strategies will also have to accommodate control measures that target virus complexes in those areas affected by such multiple infections.
Tomato (Lycopersicon esculentum Mill.) is susceptible to many viruses, and considerable yield losses and diminished fruit quality can occur due to single or multiple viral infections (3). In Trinidad, tomato is an important commodity crop, and demands of the local as well as limited regional export markets are met by widespread and intensive cropping, especially during the dry season (January to June). Tomato production is more difficult during the rainy season and the incidence of bacterial and fungal diseases is higher.
The genus Begomovirus constitutes a group of geminiviruses (family Geminiviridae) many of which have a bipartite genome (19). Begomoviruses are vectored by the sweetpotato whitefly, Bemisia tabaci (Genn.) and infect a variety of dicotyledonous plant species. In Trinidad, the only tomato-infecting begomovirus reported to date is Potato yellow mosaic Trinidad virus (PYMTV) (24), although many other tomato-infecting begomoviruses have been reported in the wider Caribbean region (14). PYMTV is a double recombinant begomovirus and has since been classified as a species distinct from Potato yellow mosaic virus (PYMV), which was first identified in Venezuela (18). PYMTV was considered to be the causal agent of viral epidemics in tomato in Trinidad since the late 1980s (24), but no empirical studies have been carried out to determine the relative importance of begomoviruses versus other tomato-infecting viruses such as cucumoviruses, potyviruses, and tobamoviruses. A farmer survey conducted between July and August 2002 indicated that at least 40% of yield losses was due to virus infections.
The last survey of tomato-infecting viruses in the Caribbean region prior to the emergence of begomoviruses in tomato was carried out by Haque (1974) who identified Tobacco mosaic virus (TMV) and two other viruses, Potato leaf roll virus (PLRV) and Cucumber mosaic virus (CMV). This survey, however, was based only on symptomology.
Recent field inspections have revealed that symptoms observed in virus-infected tomato plants in Trinidad are, for the most part, caused by mosaic-inducing viruses. These symptoms include interveinal chlorosis, leaf malformations including leaf crinkling and curling, yellow mottling or mosaic, filiform leaf formation and, in some cases, stunted plant growth. Symptom severity varies depending on the time of infection and variety of tomato.
There is a need to conduct a survey of tomato-growing areas to determine the relative prevalence of tomato-infecting viruses to aid in the development of virus disease management strategies. Such a survey in Trinidad would reveal the identity of viruses associated with these outbreaks, and aid in determining the importance and contribution of begomovirus infection to the overall viral disease situation in tomato.
Therefore, the objectives of this study were (i) to determine the etiology of the mosaic disease affecting tomato, and (ii) to investigate the incidence and distribution of seven tomato-infecting viruses in tomato crops and selected nurseries throughout Trinidad.
Surveying Pathogenic Viruses in Tomato
Field sampling. Tomato fields were sampled in the dry season during the peak production period (April to June) in 2003. Collections were made from tomato-growing areas in five of the seven counties that comprise the island of Trinidad: St. George (East and West), St. Andrew, Victoria, St. Patrick, and Caroni (Fig. 1). Two counties (St. David and Mayaro) did not have tomato production at the time of sampling. Depending on the extent of tomato farming carried out at the time of the study, between two and five fields were sampled within each tomato-growing area. Plants in these fields were at the flowering or early fruit development stage. Tomato-growing areas were classified as clustered or isolated based on degree of geographical separation from other commercial fields. Field data on agronomic practices, cultivars grown, size of cultivated area, insecticide application, and general disease management practices were determined according to a formal farmer survey simultaneously conducted with sampling.
Sample collection. Out of approximately 300 to 350 plants observed along a diagonal transect (approximately 150 m in length), young leaves were collected from an average of 10 to 15 tomato plants showing symptoms of viral infection from each field visited (except for Sangre Grande where symptomless plants had to be sampled). The samples were placed in labeled polyethylene bags and transported to the laboratory for processing. Symptom severity was scored based on the following scheme: 0 = no symptoms; 1 = very mild symptoms (specks of pale green to yellow on parts of the leaf); 2 = moderate symptoms (yellow mosaic or mottling of leaf on some parts of leaf) with leaf distortion (leaf curling and crinkling); 3 = severe symptoms (yellow mosaic or mottling on entire leaf) with leaf distortion (leaf curling and crinkling); 4 = severe mosaic (interveinal chlorosis, yellow mosaic or mottling on entire leaf); and 5 = extremely severe leaf distortion and leaf size reduction. Symptom scores for the tomato-growing areas were based on the mean scores of symptomatic plants.
DAS-ELISA assays. DAS-ELISA using polyclonal antisera (4) was used to detect the presence of seven viruses. Polyclonal antisera for Tobacco mosaic virus (TMV), Cucumber mosaic virus (CMV), Potato virus Y (PVY), Tomato spotted wilt virus (TSWV), Potato leaf roll virus (PLRV), and Tobacco etch virus (TEV) was from Biorad (France), whereas that for detection of begomoviruses was from Bioreba AG-Switzerland and was raised against a recombinant coat protein of cabbage leaf curl virus (CaLCV).
One gram of infected leaf tissue was weighed and ground in 7 ml extraction buffer as 1:7 w/v (as recommended by the respective manufacturer for each antisera). The extracts were filtered through miracloth (Calbiochem-Novabiochem Corporation, La Jolla, California, USA), and 100 μl of the filtrate was used in the ELISA test. Two replicates were performed per sample. Antibodies were diluted as 1:1000 (for TMV, CMV, PVY, TSWV), 1:500 (for PLRV), and 1:100 (for TEV) in carbonate coating buffer (as recommended by the respective manufacturer for each antisera). An aliquot (100 μl) of each diluted antibody was used in the ELISA. Microtiter plates were incubated for 16 h at 4°C in a moist chamber. Each plate was aspirated and washed three times in an automated ELISA plate washer (Labsystems Oy, Helsinki, Finland) using ELISA wash buffer (as recommended by the respective manufacturer for each antisera). The infected leaf extract (100 μl) was added in duplicate to the plates and incubated for 16 h at 4°C in a moist chamber. After incubation, the plates were again aspirated and washed three times with ELISA wash buffer. Conjugated antibodies were diluted in conjugate buffer as recommended by the respective manufacturer. Diluted conjugated antibody (100 μl) was added to each well and incubated for 5 h at 30°C in a moist chamber. The plates were aspirated and washed three times with ELISA wash buffer and 100 μl of p-nitrophenyl phosphate (pNPP) substrate was added to each well. The plates were incubated at room temperature in the dark to allow color development. After approximately 120 min, the plates were read at a wavelength of 405 nm with an ELISA plate reader (Bio-Rad Laboratories, Inc., Hercules, California, USA). Positive and negative controls were provided for each virus by the respective manufacturer. A single column of wells each containing 100 μl of extraction buffer was also used a negative control. Samples were considered to be positive for a respective virus if the average absorbance readings of both replicates exceeded three times the mean of the negative control values (provided by the respective manufacturer) after 120 min of incubation (Table 1).
Table 1. Range of ELISA values obtained for negative and positive controls and for positive detection of Tobacco etch virus (TEV), Tobacco mosaic virus (TMV), and Begomovirus in tomato extracts.
Optimization of ELISA for detection of begomoviruses. Detection of begomoviruses with ELISA required an optimization of the standard DAS-ELISA protocol. This involved testing the efficacy of two extraction buffers, different grinding ratios, and two antibody concentrations. Samples were ground in either a Tris/Sodium sulfite extraction buffer (0.05 M Tris [pH 8.5] to which 0.75 g Na2SO3 per 100 ml was added just before use) in a 1:5 and in a 1:10 w/v ratio or in the general extraction buffer (Bioreba AG, Reinach, Switzerland) in a 1:5 and in a 1:10 w/v ratio. Antibodies used were diluted 1:1000 and 1:500 in coating buffer and conjugate buffer, respectively.
Nursery survey for begomovirus-infected seedlings. Four nurseries located in the northern part of Trinidad were surveyed for begomovirus-infected tomato seedlings using DAS-ELISA during part of the dry season (May to June) in 2003. Preliminary results of a survey conducted in July to August 2002 in northern Trinidad had revealed that the highest incidence of infection was caused by tomato-infecting viruses. Hence, some nurseries that were the main suppliers of tomato seedlings for these farms were selected for this study. Of the four nurseries sampled, three were located in Aranguez/San Juan (St. George West) and were large commercial seedling suppliers to more than 50% of the farms surveyed. The last nursery was located in Tunapuna/Piarco (St. George East) and was a small-scale supplier. A total of 60 seedlings from all of these greenhouses (10 samples of ‘Kada,’ 30 samples of ‘Kada Hybrid 61,’ 10 samples of ‘Gempride,’ and 10 samples of ‘Akash’) were randomly sampled and tested for infection using ELISA. Kada Hybrid 61 appeared to be popular with home gardeners at the time of testing and explains why every nursery supplied this variety and propagated a relatively large number of seedlings. Data concerning the cultivar and age of seedlings tested in addition to general nursery practices were also recorded.
Analysis of data. ELISA data collected from individual tomato fields were pooled and results were summarized as infection percentages (single infections) according to tomato-growing area. Data was also analyzed to determine the percentage of dual or mixed infections: begomovirus with TMV, begomovirus with TEV, and begomovirus with both TMV, and TEV. Statistical significance was calculated based on chi-square analysis. A cut-off value for P being <0.001 was considered to be statistically significant; probability values greater than 0.001 were not considered to significantly contribute to virus incidence. The degree of contribution of each growing area to virus incidence was determined by removing those areas with very low infection percentages for begomovirus, TMV, and TEV from the data set. All analyses were performed using Minitab Statistical Software (Minitab Inc., State College, PA, USA).
Tomato cultivation practices. Fourteen tomato-growing fields within five counties were surveyed (Fig. 1). The major tomato-growing areas were Aranguez, Pasea/ Macoya, Trincity, Maloney, and Valencia, all situated in the north (St. George). Bonne Aventure was the only major growing area in the south (Victoria). These areas consisted of long-established, neighboring holdings that practiced mixed cultivations of a range of vegetable crops, including tomato. Only one farm (D’abadie) in the north (St. George) was a new agricultural development and the samples were collected from the very first tomato crop.
Although several cultivars were grown, the most commonly grown cultivars were "Akash" (55.6 %) followed by "Gempack" (29.6%) (Table 2). Plant density was 14,000 to 27,000 plants per ha with a between-plant spacing of 30 to 60 cm and a between-row spacing of 60 to 90 cm. All plants were staked irrespective of growth habit. Methods of irrigation included drip and furrow irrigation systems; one farm (Santa Cruz, north in St. George) utilized an overhead, semi-automated sprinkler system. Most farms routinely practiced some form of weed control, either manual weeding and/or herbicide use.
Table 2. Agronomic characteristics of the tomato-growing areas.
x Indicates those farms that used imidacloprid. Size of field expressed as the total size of all the fields tested per growing area (in hectares).
y Number of samples collected and tested per tomato-growing area.
Field applications of the insecticide imidacloprid (Admire, Bayer CropScience Inc., Research Triangle Park, NC) were used in five tomato-growing areas (Table 2). Large numbers of whiteflies in various stages of development were observed in tomato fields in the central (Caroni) and south in Princes Town (Victoria). However, there was no evidence of heavy whitefly infestation in fields in the north (indicated by an absence of eggs, nymphal or adult developmental stages on the leaf samples collected) regardless of insecticide use.
Symptom description. Most samples showed virus-like symptoms including mosaic, mottling, chlorosis, crumpling and distortion of leaves and some degree of distorted and stunted growth. However, it should be noted that a small number of leaf samples showed symptoms of some disease, but were negative for all of the viruses tested. This may suggest some alternate injury (e. g., abnormal pH, herbicide injury, nutritional deficiencies, feeding damage by mites or insects) to these plants.
Optimized DAS-ELISA for detection of begomovirus. The Tris/Sodium sulfite (pH 8.5) extraction buffer (1:5 w/v) using an antibody concentration of 1:500 offered greater sensitivity, as indicated by a higher spectrophotometric reading, than the general extraction buffer (pH 7.4) using antibody concentrations of either 1:1000 or 1:500 (data not presented). Hence, the Tris/Sodium sulfite buffer system was used throughout the study. It should be noted that there were no problems associated with background in this ELISA test. The range of values for all negative and positive controls and those considered to be positive for virus detection is presented in Table 1.
Incidence and distribution of tomato-infecting viruses. Three viruses were detected in samples of tomato showing virus symptoms in Trinidad in 2003: TMV, TEV, and begomovirus. All samples tested negative for the presence of PVY, TSWV, CMV, or PLRV. Of a total of 362 samples tested, begomovirus, TEV, and TMV were detected in 56.4%, 11.6%, and 5.0% of samples respectively. Virus infection was not detected in any samples from two tomato-growing locations: Sangre Grande in the north (St. Andrew), and Bonne Aventure in the south (Victoria). However, while samples collected from Sangre Grande displayed no symptoms (but were still tested); those collected from Bonne Aventure had a mean symptom score of 3.4 (Table 2). These symptoms may have been due to other factors such as abnormal pH, herbicide or other chemical injury, nutritional deficiencies, or insect damage.
Distribution and incidence of begomovirus. Fields in the north in the St. George county (Santa Cruz, Aranguez, Pasea/Macoya, Maloney, Trincity/D’abadie, Valencia) and one field in the St. Andrew county (Vega Oropouche/Cumuto) (Table 3) had the highest frequency of begomovirus infection at 42.8% (frequency as the percentage of begomovirus infection all the locations).
Table 3. Percentage of infections of Tobacco mosaic virus (TMV), begomovirus, and Tobacco etch virus (TEV) according to tomato-growing area.
x Number of samples collected and tested per tomato-growing area.
Table 4. Percentage infection of dual and multiple infections of begomovirus with Tobacco mosaic virus (TMV) and with Tobacco etch virus (TEV) according to tomato-growing area.
x Number of samples collected and tested per tomato-growing area.
Overall, there were significant differences (P < 0.001) among tomato-growing areas with respect to incidence of begomovirus, even when Bonne Aventure and Sangre Grande were not factored into the analysis. However, when Penal (St. Patrick) and Debe (Victoria) were removed from the analysis, there were no significant differences in begomovirus infection percentages among tomato-growing areas (P = 0.093). This suggests that these two areas accounted for the significance obtained.
Distribution and incidence of TMV. The highest incidence of TMV was detected in three locations: Aranguez (north-St. George), Pasea/Macoya (north-St. George), and Penal (south-St. Patrick) (Table 3). Statistical analysis showed significant variation (P < 0.001) among single TMV infection percentages with respect to tomato-growing areas especially between those found in St. George East (27.5%) and St. George West (13.2%).
Distribution and incidence of TEV. TEV was detected in six tomato-growing areas: four in north Trinidad, St. George county (Aranguez, Pasea/ Macoya, Maloney, Santa Cruz); and two in the south (Penal - St. Patrick county and Grand Couva/Tortuga - Victoria county) (Table 3). Analysis of infection percentages of single infections of TEV among tomato-growing areas revealed significant differences (P < 0.001), due to lower infection percentages in Penal and Maloney.
Distribution and incidence of dual and multiple infections. Dual infections of begomovirus with either TMV or TEV or multiple infections of all three viruses were detected (Table 3). Begomovirus infection occurred more frequently with TEV (29.6% of all locations tested and 8.0% of all samples assayed) than with TMV (11.1% of all locations tested and 2.4% of all samples assayed). Dual infections of begomovirus and TMV were found in two tomato-growing areas (Aranguez and Pasea/Macoya) located in north Trinidad (St. George). Dual infections of begomovirus and TEV were also found in a majority of fields in the north except for the tomato-growing area of Caroni, which is located in central Trinidad. Multiple infections involving all three viruses were rare (0.7% of all samples assayed) and found in only 7.4% of all locations tested. There were significant differences among infection percentages of dual and multiple infections of begomovirus with TMV, begomovirus with TEV, and begomovirus with both TMV and TEV (P < 0.001 for each case).
Survey of nursery seedlings for begomovirus infection. Seedlings were grown in open trays under an overhead screen. None of the seedlings, which were between five to eight-weeks-old, tested positive for begomovirus infection. There was also no evidence of whitefly infestation on the seedlings as indicated by an absence of eggs, nymphs, or adults on leaf undersides or elsewhere and there was no record of whitefly control on these seedlings at the time the survey was conducted. None of the seedlings displayed symptoms of infection. General nursery practices were similar in that seeds were not pre-treated with fungicides or insecticides; PROMIX potting mix (Premier Horticulture Inc., USA) was used by all nurseries in sowing tomato seeds and foliar application of NUTREX (Carbotech Iowa, Inc., USA) fertilizer was applied to seedlings three to five days post-emergence.
Conclusions and Recommendations
The study revealed that three viruses, TMV, begomovirus and TEV, were associated with viral disease outbreaks in tomato in Trinidad in 2003. The other viruses tested for (CMV, PVY, PLRV, and TSWV) were not detected, which indicates that that they were absent, occurred at too low a frequency or in titers too low to be detected by the sampling and/or testing method employed in this study. As this survey was conducted only in the dry season and in a single year (2003), a role for the other viruses in tomato viral disease in Trinidad cannot be ruled out.
The results indicated an overall high incidence of begomovirus infection. This suggests that begomovirus(es) may be the most important viral pathogen in tomato in Trinidad. As there no detected weed hosts of begomovirus (PYMTV) detected in Trinidad (16), it is likely that infected tomato plants are the primary inoculum source for begomovirus. This is also supported by a failure to detect virus infection in transplants. A similar finding was made for begomovirus Tomato yellow leaf curl virus (TYLCV) infecting tomato in the Dominican Republic (20).
The low incidence of begomovirus disease in ‘isolated’ locations (Sangre Grande and Bonne Aventure) compared to moderate to high incidence of disease in locations with "clustered" farms that practice overlapping cultivation provides indirect evidence that proximity to other fields affects disease incidence. Clustered fields may be exposed to cross-infections from one field to another as facilitated by short-distance migration of the whitefly vector (15). This assists in maintaining viruliferous whiteflies over consecutive generations (20). Aboul-Ata et al. (2000) reported a negative correlation between TYLCV incidence and distance from the primary field source of infection, which is consistent with the findings of this study. A high incidence of begomovirus infection was detected in the tomato production systems surveyed, despite chemical control of whiteflies and the use of "geminivirus-resistant" cultivars. These cultivars were bred for resistance against TYLCV, and they appear to be susceptible to begomovirus(es) in Trinidad based on having viral disease symptoms and systemic infections. Chemical control aimed at reducing vector numbers was inadequate at managing viral disease. The abaxial surface of every leaf sample collected from the Princes Town (Victoria) and Caroni areas (Caroni) was heavily infested with whitefly eggs, nymphs and/or adult insects despite imidacloprid use (Table 2), such that symptoms due to insect feeding damage and disease could not be easily distinguished.
Geminiviruses have been detected in multiple infections with other geminiviruses (8,11,12,21,23), but there have been limited reports of studies conducted to determine the frequency and incidence of geminiviruses in multiple infections with unrelated viruses in tomato. In Egypt, multiple infections of a begomovirus, Tomato yellow leaf curl virus (TYLCV), with PVY or CMV was detected at low incidences (1). Similarly in the Canary Islands, TYLCV was detected in mixed infection with Tomato chlorosis crinivirus (ToCV) in tomato, and in Mexico, begomoviruses were detected in multiple infections with a number of unrelated tomato viruses at low to moderate frequencies (6,9). Taken together with the results of this study, these findings demonstrate that multiple infections of two or more unrelated viruses (one of which is a begomovirus) in tomato is not uncommon. Although the observed extent of multiple infections of TEV and TMV with begomovirus was lower than might have been expected based on single infections, this study has illustrated that infection by one virus (TMV, TEV, or begomovirus) does not preclude infection by another invading virus species in tomato.
TMV was detected in 21.4% of the locations tested. TMV infection was confined to farming areas with a history of tomato production. It may have been initially introduced through plant injury caused by crop workers, contaminated equipment and/or the smoking practices of the farmers. The virus can survive for many years in dead, dried plant material in addition to living, which makes it difficult to inactivate (13,22,25). It is suspected that failure to detect and subsequently eradicate TMV from these farms has led to its persistence in these agricultural areas.
TEV was found in 42.8% of all locations surveyed. TEV is transmitted in a non-persistent manner by aphids, and its occurrence in tomato is usually associated with other TEV-infected solanaceous crops (especially pepper) and weed species which act as virus reservoirs (2,10,25). Pepper was commonly found to be co-cultivated with tomato in many farms sampled including those in which TEV was detected. It is likely that viruliferous aphids moved the virus from infected pepper and weeds into tomato and this can happen in a short period of time (5). This probably accounted for the high incidence of TEV-infection in individual farms (Santa Cruz, Aranguez and Pasea/Macoya – St. George) surveyed in this study. Overall, however, among all farms tested, incidence of TEV infection was considered to be low to moderate.
The control of TMV in tomato may be achieved through (i) production of seeds from virus-free crops, (ii) seed treatment with 10% trisodium phosphate solution for 15 to 20 min (10), and (iii) minimum handling of seedlings. TEV disease control may involve (i) isolation of tomato crop from other solanaceous crops, cucurbits and weeds that may act as virus/vector hosts; (ii) minimizing multi-cropping with many common hosts to TEV; and (iii) treatment with aphicides to reduce aphid populations. Roguing and destruction of diseased plants during early stages of growth would assist control of both TMV and TEV. These methods should be used in conjunction with those outlined for begomovirus control (17) as part of an integrated approach to virus disease management in tomato in Trinidad.
The cropping practices revealed by this study demonstrate that crop patterns and proximity of fields are contributors to virus epidemics. In Trinidad tomato production is concentrated, overlapping, and without seasonal restrictions. New crops and old abandoned fields are often adjacent to one another with no separation in time or space. The practice of poly-culture also ensures that a constant resource of hosts for both vector and virus is present throughout the year. This results in perpetuation of vector and virus populations with a concomitant and continuous source of infection and infestation of the new crop from the reservoirs in the old crop. A similar situation contributed to epidemics of TYLCV in the Dominican Republic, and effective management was obtained by institution of a host-free period (20).
The author wishes to thank Christine Caruth and P. Umaharan for their assistance in the collection of samples and field data analysis.
1. Aboul-Ata, A. E., Awad, M. A. E., Abdel-Aziz, S., Peters, D., Megahed, H., and Sabik, A. 2000. Epidemiology of tomato yellow leaf curl begomovirus in the Fayium area, Egypt. OEPP/EPPO Bull. 30:297-300.
2. Averre, C. W., and Gooding, G. V. 2000. Virus diseases of greenhouse tomato and their management vegetable disease information. Note 15 (VDIN-0015), Coll. of Agric. and Life Sci., Plant Path. Ext., N. Car. State Univ.
3. Brown, J. K., and Bird, J. 1992. Whitefly-transmitted geminiviruses and associated disorders in the Americas and the Caribbean Basin. Plant Dis. 76:220-225.
4. Clark, M. F. 1981. Immunosorbent assays in plant pathology. Ann. Rev. Phytopathol. 19:83-106.
5. Fondufe, G. Y., Irwin, M. E., Bottenberg, H., and Kampmeier, G. E. 1995. Aphids and disease spread in crops. INHS Rep., November-December 1995.
6. Garzon-Tiznado, J., and Martinez-Carrillo, J. 2001. Past and present status of viruses infecting Chili pepper in Mexico. In: Proc. of the 16th Int. Pepper Conf. Tampico, Tamaulipas, Mexico. November 10-12, 2002.
7. Haque, S. Q. 1974. Status of virus diseases of horticultural crops in the commonwealth Caribbean. Univ. of the West Indies, Faculty of Agric. Ann. Rep. for 1974.
8. Holt, J., Colvin, J., and Muniyappa, V. 1999. Identifying control strategies for tomato leaf curl disease using an epidemiological model. J. App. Ecol. 36:625-633.
9. Idris, A. M., Lee, S. H., and Brown, J. K. 1999. First report of chino del tomate and pepper huasteco geminiviruses in greenhouse-grown tomato in Sonora, Mexico. Plant Dis. 83:396.
10. Jones, J. B., Jones, J. P., Stall, R. E., and Zitter, T. A. 1991. Compendium of tomato diseases. American Phytopathological Society, St. Paul, MN.
11. Melgar, J. C., Rivera, J. M., Krigsvold, D., Doyle, M. M., Aquilar, E., Rueda, A., Brown, J., and Martyn, R. 2002. Identification, distribution and epidemiology of plant virus pathogens that threaten pepper/tomato and cucurbit production in Honduras (Phase 1). 5th Seminar of Integ. Pest Man. in Non-trad. Export Crops. June, 2002, Guatemala.
12. Paplomatas, E. J., Patel, V. P., Hou, Y. M., Noueiry, A. O., and Gilbertson, R. L. 1994. Molecular characterization of a new sap-transmissible bipartite genome geminivirus infecting tomatoes in Mexico. Phytopathology 84:1215-1224.
13. Pfleger, F. L., and Zeyen, R. J. 1991. Tomato-tobacco mosaic virus disease. Pub. FS-01168, Plant Pathol. Comm. and Ed. Technol. Serv., Univ. of Minn. Ext. Serv.
14. Polston, J. E., and Anderson, P. K. 1997. The emergence of whitefly-transmitted geminiviruses in tomato in the Western Hemisphere. Plant Dis. 81:1358-1369.
15. Ramoppa, H. K., Muniyappa, V., and Colvin, J. 1998. The contribution of tomato and alternative host plants to tomato leaf curl virus inoculum pressure in different areas of South India. Ann. App. Biol. 133-187-198.
16. Rampersad, S. N. 2005. A study of dicotyledonous weed species as hosts of Potato yellow mosaic Trinidad virus (PYMTV). Plant Pathol. J. 4:157-160.
18. Roberts, E. J. F., Buck, K. W., and Coutts, R. H. A. 1988. Characterisation of potato yellow mosaic virus as a geminivirus with a bipartite genome. Intervirology 29:162-169.
19. Rojas, M. R., Hagen, C., Lucas, W. J., and Gilbertson, R. L. 2005. Exploiting Chinks In The Plant’s Armor: Evolution And Emergence Of Geminiviruses. Ann. Rev. Phytopathol. 43:361–94.
20. Salati, R., Nahkla, M. K., Rojas, M. R., Guzman, P., Jaquez, J., Maxwell, D. P., and Gilbertson, R. L. 2002. Tomato yellow leaf curl virus in the Dominican Republic: Characterization of an infectious clone, virus monitoring in whiteflies and identification of reservoir hosts. Phytopathology 92:487-496.
21. Sanz, A. I., Fraile, A., Garcia-Arenal, F., Zhou, X., Robinson, D. J., Khalid, S., Butt, T., and Harrison, B. D. 2000. Multiple infection, recombination and genome relationships among begomovirus isolates found in cotton and other plants in Pakistan. J. Gen. Virol. 81:1839-1849.
22. Sikora, E. J. 1994. Tobacco mosaic virus. Plant Dis. Notes., Alabama Coop. Ext. Syst. ANR-867.
23. Torres-Pacheco, I., Garzon-Tiznado, A., Brown, J. K., Becerra-Flora, A., and Rivera-Bustamante, R.F. 1996. Detection and distribution of geminiviruses in Mexico and the Southern United States. Phytopathology 86:1186-1192.
24. Umaharan, P., Padidam, M., Phelps, R. H., Beachy, R. N., and Fauquet, C. M. 1998. Distribution and diversity of geminiviruses in Trinidad and Tobago. Phytopathology 88:1262-1268.
25. Zitter, T. A., and Providenti, R. 1984. Vegetable crops: Virus diseases and disorders of tomato. Coop. Ext. Fact Sheet., Cornell Univ., Ithaca, New York.