Tomato yellow leaf curl virus (TYLCV), a Begomovirus in the family Geminiviridae, is the most devastating virus of the tomato plant in tropical and subtropical regions including Iran. The family Geminiviridae comprises plant viruses that have a circular, single-stranded DNA genome and geminate particles consisting of two incomplete icosahedra (Hull, 2002). Geminoviruses are classified into four genera based on the type of insect vector, host range, and genome organization (Rybicki et al., 2000). The genus Begomovirus includes species with monopartite or bipartite genomes such as TYLCV that are transmitted by whitefly, Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae) (Moriones and Navas-Castillo, 2000; Cohen and Nitzany., 1966). B. tabaci has developed resistance against insecticides in recent years (Dittrich et al., 1990) and therefore, a few viruliferous whiteflies may be enough for transmitting the virus to a large number of plants.
Chemical control methods as well as integrated pest management (IPM) strategies employed for controlling the vector have not been successful in decreasing the incidence of TYLCV on the tomato crop (Reynaud et al., 2003). Under these circumstances, breeding for resistance to TYLCV appears to be a promising and environmentally friendly approach for controlling the disease (Chague et al., 1997). Resistance to TYLCV has been reported in wild relatives of the cultivated tomato; S. peruvianum, S. hirsutum, S. pimpinellifolium and S. cheesmanii (Kasrawi et al., 1988; Geneif, 1984; Hassan et al., 1984). However, in some of these highly resistant wild accessions such as S.peruvianum LA385, TYLCV was detected by back indexing, hence such a resistance could also be viewed as tolerance (Kasrawi et al., 1988). Genetic analyses indicated that tolerance to TYLCV is controlled by five recessive genetic factors (Pilowsky and Cohen, 1990). Zamir et al. (1994) mapped a major TYLCV tolerance locus (TY-1) in the tomato wild relative S. chilence on chromosome 6. Chague et al. (1997) reported four Random Amplified Polymorphic DNA (RAPD) markers linked to a quantitative trait locus involved in the resistance to TYLCV. Resistance to TYLCV in S. pimpinellifolium IRNA-Hirsute is reported to be against the insect vector rather than the virus. In breeding programs for protection against begomoviruses, tolerant genotypes with low levels of infection but expressing reduced disease incidence may be discarded without full consideration of their epidemiological effects at the population level in the field (Delatte et al., 2006). It has also been shown that, acquisition of TYLCV from a tolerant or resistant plant, and its transmission by whiteflies are less efficient than those for a susceptible plant (Lapidot et al., 2001). Some sources of resistance to TYLCV may also show resistance to some other viruses such as Tomato curly stunt virus (ToCSV) (Pitersen and Smith, 2002).
Although in a breeding program, the evaluation of resistance level should correspond to the effect of infection on total yield and yield components (Lapidot et al., 1997), the severity of the infection and the level of viral accumulation can serve as indictors of resistance level (Pico et al., 1999). The tomato plant was introduced to Iran several centuries ago and has been subjected to many genetical changes resulting in an interesting diversity, reflecting the various climatic conditions in the country. In this study the Iranian tomato collection was screened for resistance to a newly emerged isolate of TYLCV from Southern Iran (TYLCV-Ir2). Resistance was evaluated based on the severity of the disease symptom and viral DNA amplification. The type of resistance was then analyzed by comparing graft-inoculated and whitefly inoculated S. peruvianum plants.
MATERIALS AND METHODS
Virus isolate: The TYLCV isolate used in this study inculation was a newly emerged isolate method known as TYLCV-Ir2 (accession EU085423 in NCBI gene bank) (Azizi, 2007) collected from the infected tomato fields of the Bandar Abbas region, South of Iran. The isolated virus was first identified based on the development of symptoms on tomato, bean (Phaseolus vulgaris), and wild tobacco species Nicotiana benthamiana and N. rustica plants. The TYLCV DNA fragment was then amplified by polymerase chain reaction (PCR), using a pair of TYLCV specific primers (Azizi, 2007). The virus was biologically purified by whitefly transmission and maintained in an insect-proof greenhouse and allowed to propagate in the tomato (S. lycopersicum cv. PS111) plants.
Plant material: Iranian collection of tomato germplasm consisting of 125 accessions of S. lycopersicum collected from across the country, and nine TYLCV tolerant S. lycopersicum accessions introduced by the Asian Vegetable Research and Development Center (AVRDC) and six accession of S. peruvianum were evaluated in this study. Eight plants of each accession were grown under greenhouse conditions (25 ± 2ºC, 12 h light and 70-80% relative humidity (RH)) and were tested against TYLCV infection. The experiment was replicated twice.
Whitefly maintenance and plant inoculation: Whiteflies (Bemisia tabaci, biotype B) were identified and collected from greenhouse grown potatoes, Karaj, Iran. Whitefly biotype B colonies were established on cotton plants and then transferred, reared and maintained on cabbage (Brassica oleracea) plants under a cage in the greenhouse at 25±2ºC. Whitefly mediated mass inoculation technique was used to inoculate plants (Pico et al., 1998). The insects were given a 24h acquisition access period to TYLCV- infected tomato source plants. Eight seedlings of each accession at the four-leaf stage were then inoculated by placing the pots of whitefly infected plants between the pots of tomato plants with the appropriate accessions to be examined. This was repeated four times. Plants were periodically shacked to obtain a uniform vector distribution and individually exposed to about 20 viruliferus whiteflies per plant for 10 days. After inoculation, plants were sprayed with the insecticide Imidacloprid (Confidor, Bayer, Germany) and kept in an insect-proof greenhouse for 5 weeks. The experiment was performed twice, during 2005 and 2006. To determine the type of TYLCV resistance, the resistant accessions were inoculated by grafting. After inoculation, plants were kept in an insect-proof greenhouse for 5 weeks. In each experiment, six plants from each resistant accession were tested. PCR detection assay was employed for detection of the virus in inoculated plants three and five weeks post-inoculation.
DNA extraction: Total DNA was extracted according to Dellaporta et al. (1983) with minor modifications. Two apex leaves (0.5 g) were collected from each of eight green house-grown tomato plants of each accession and used for DNA extraction. 0.1 g of fresh leaf tissue was grounded to a fine powder in liquid nitrogen. The homogenate was incubated in 600 ml of extraction buffer (100 mM Tris-HCl (pH 8), 50 mM EDTA, 500 mM NaCl, 10 mM 2-b-mercaptoethanol and 1% (w/v)SDS) at 65ºC for 10 min and mixed with a half volume of Chloroform: Isoamyl alcohol (24:1 v/v). The mixture was centrifuged at 11269 ´g for 15 min and the supernatant (500 ml) was transferred to a 1.5 ml microcentrifuge tube and the DNA was precipitated by adding 150 ml of sodium acetate (5 M, pH 5.2) and 600 ml of isopropanol. The pellet was washed with 70%(v/v) ethanol, air dried and dissolved in 100 ml of sterile double distilled water.
PCR amplification of viral DNA: TYLCV specific primers (TYLCV-F and TYLCV-R) amplifying a 670 bp fragment, were designed according to the conserved sequences of TYLCV-sar (EU143757), TYLCV-Is (DQ845787.1) (Pico et al., 1999) and TYLCV-Ir2 (EU085423)(Azizi 2007) available in the NCBI GenBank (Table 1). Conserved sequences of 18S rDNA of S. lycopersicum were used to design 18S rDNA-specific primers (18S F/18S R) acting as an internal control and amplifying a 406 bp fragment (Table 1). The optimized PCR procedure was carried out in a 25 ml reaction volume containing one unit of Taq DNA polymerase (Fermentas), 50 ng of plant DNA, 2.5 ml of 10X PCR buffer (500 mM KCl, Tris-HCl (pH 8.4)), 1.5 mM MgCl2, 0.5 ml of 10 mM dNTPs, 25 picomol of each primer and an appropriate volume of deionized H2O to make up to 25 ml. The optimal conditions for amplification were as follows: Initial denaturation at 94ºC for 4 min, followed by 25 cycles of denaturation at 94ºC for 1 min, annealing at 50ºC for 1 min, extension at 72ºC for 2 min and a final extension at 72ºC for 10 min. PCR was carried out in a thermocycler machine (Mastercycler ep Gradient) supplied by Eppendorff (Germany). PCR products were fractionated and assessed on 1% (w/v)TAE agarose gels.
The incidence of TYLCV infection and the type of observed symptoms in tested tomato accessions are summarized in Table 2. Cultivated tomato accessions (S. lycopersicum) exhibited a range of TYLCV symptoms including yellowing (Fig 1a), purple vein (Fig 1b), leaf curling (Fig 1c), stunting (Fig 1d) and reduced leaflet size (Fig 1a and 1b). In contrast, all six accessions of S. peruvianum remained symptomless and had normal growth, such as that of healthy plants (Tables 2 and 3). Based on phenotypic evaluation, most of the S. lycopersicum accessions lacked resistance and developed severe symptoms. Only nine accessions showed a very weak symptom and were thus considered as tolerant genotypes (Table 3).
Moreover, a DNA fragment of expected size (670 bp) and a 406 bp 18S rDNA fragment were amplified using TYLCV specific primers and specific primers 18SF/18SR, respectively for the accessions of S. lycopersicum. However, no viral DNA was detected in any of the six accessions of S. peruvianum. (Fig 2).
Three and five weeks following the graft-inoculation of the six S. peruvianum accessions with an infected S. lycopersicum accession, plants were assessed for the development of disease symptoms and amplification of viral DNA by PCR (Fig 2 and Table 4). Development of the disease symptoms was delayed, and no viral DNA was amplified three weeks post graft-inoculation (Table 4). However, five weeks after graft-inoculation, the expected fragment of 670 bp related to viral DNA along with very weak disease symptoms including small leaflets and curling were detected in all the tested plants of S. peruvianum (Fig and Table 4).
In order to identify potential sources of natural resistance to TYLCV, the Iranian collection of cultivated tomato, S. lycopersicum and its wild relative, S. peruvianum, were evaluated based on symptom development and amplification of viral DNA following whitefly inoculation. Accessions of S. lycopersicum exhibited a varying range of disease symptoms and lacked resistance. Most of them were found to be highly susceptible to TYLCV-Ir2 and only nine accessions showed a very mild symptom and considered as tolerant (Table 3). On the contrary the six accessions of S. peruvianum tested in this study showed no disease symptom and were assessed as resistant to TYLCV-Ir2 when inoculated with whiteflies. These wild relatives of cultivated tomato have previously shown to be the possible source of resistance against the disease (Morals and Anderson, 2001; Pico et al., 1996).
Accessions CLN2114DC1F1-180-31-9-11-12 and CLN2114DC1F1-2-29-20-23-14-0 that have in earlier studies been reported TYLCV tolerant (AVRDC, 2001), were found susceptible in this study. In addition, accession CLN2114DC1F1-2-29-20-23-14-0 showed a much faster development of disease symptoms than other tested accessions. This difference in reaction could be due to the virus strain, vector genotype or altered feeding conditions of the vector (Delatte et al., 2006; Navas-Castillo et al., 1999). Viruses transmitted by B. tabaci are deposited within the phloem through salivation. Therefore, altered feeding behavior could result in a significant decrease in the incidence of several begomoviruses that is usually interpreted as resistance to insect vector. This has been reported in studies with the Rice ragged stunt virus transmitted by planthopper Nilaparvata lugens (Stål) (Hemiptera: Delphacidae) to rice (Parejarearn et al., 1984) and Maize mosaic virus (MMV) transmitted by the planthopper Peregrinus maidis (Ashmead) (Hemiptera: Delphacidae) to maize (Dintinger et al., 2005).
The type of resistance expressed by S. peruvianum accessions was studied using graft-inoculation of TYLCV and was compared to whitefly inoculations. TYLCV resistance observed in whitefly-inoculated S. peruvianum accessions was overcome five weeks after plants were graft-inoculated (Table 4). These results revealed that TYLCV resistance in these accessions can be overcome when plants are infected by graft-inoculation which is not a common method of natural infection in the field. The resistance against vector inoculation of TYLCV in S. peruvianum could be in accordance with its acyl sugar content, known to be a whitefly repellent (Liedl et al., 1995). Five genomic regions were detected as being associated with acyl sugar production in S. peruvianum (Mutschler et al., 1996). However, to our knowledge there is no report on the success of transferring these factors into a cultivated S. lycopersicum for development of resistance to TYLCV, probably due to unfavorable linkages. Although S. peruvianum accessions were not resistant when graft-inoculated, they are still considered major sources of resistance to TYLCV as whitefly transmission is the normal transmission mechanism in the field. TYLCV resistant accessions of S. peruvianum have also been reported to be suitable sources of resistance to Tomato leaf curl virus (ToLCV) and may show resistance to other tomato begomoviruses as well (Pitersen and Smith, 2002).
Virus titer in plant tissue is an indicator of resistance or susceptibility of plants to the virus. Low levels of virus titer and decreasing virus accumulation rate in plant tissue indicate the presence of a resistance mechanism in the plant (Pico et al., 2001; Lapidot et al., 1997; Rom et al., 1993; Pilowsky and Cohen, 1974). Thus TYLCV accumulation has been used as an indicator for resistance, but not as the sole indicator (Lapidot et al., 1997). In the present study despite the high levels of similarities in symptom development, there were considerable differences in TYLCV concentration among accessions (Table 5). There were also tolerant accessions showing no clear symptoms, but accumulating a high virus titer. Therefore, an assessment of virus titer in the plant along with the phenotypic evaluation of the disease severity is required for evaluation of TYLCV resistance. In general, based on both phenotypic and molecular evaluations, five categories of accession were identified (Table 5): accession with severe symptoms and high concentrations of viral DNA, accessions with severe symptoms but relatively low concentrations of TYLCV, accessions with mild or weak symptoms but high levels of TYLCV concentration and accessions with weak symptoms and low TYLCV concentrations. These two latter groups were considered as tolerant and finally a group of S. peruvianum which showed no symptoms and no TYLCV DNA amplification upon whitefly inoculation was considered as resistant. Tolerance and resistance are relative terms, largely related to the rate of virus replication (Pilowsky and Cohen, 1990). Results obtained here, also confirmed this point (Table 5). The accessions reported as tolerant by AVRDC (CLN2114DC1F1-180-31-9-11-12 and CLN2114DC1F1-2-29-20-23-14-0) did have a relatively low virus concentration but three weeks post-inoculation; TYLCV concentration increased in these accessions like in any other susceptible accession. In addition, the accession CLN2114DC1F1-2-29-20-23-14-0 was the fastest among all accessions in showing clear disease symptoms such as yellowing and leaf curling. These differences in reaction can be due to differences in virus strain or vector genotypes which can lead to the susceptibility of a resistant accession (Navas-Castillo et al., 1999).
The resistance mechanism in these wild species has been reported to be associated with the presence of exudates from trichom glands on the leaf surface, in which whiteflies become entrapped (Channaryappa and Shivashankar, 1992). A quantitative resistance to vector transmission of TYLCV was also reported in S. pimpinellifolium (Delatte et al., 2006). The type of resistance in S. peruvianum accessions observed in this study may be related to the insect vector as they become infected by graft-inoculation. The activity of B. tabaci on the tomato leaf surface was also studied. Trichomes on the leaf surface of S. peruvianum accessions were shorter and much denser compared to the trichomes of S. lycopersicum. Whitefly populations were also observed on these resistance accessions in different life stages including egg, nymph and adult whiteflies. Therefore, resistance may not be due to the lack of feeding by the vector, but the change in the feeding behavior. This may affect the virus transmission efficiency via the vector (Delatte et al., 2006). Nevertheless, further studies are required to characterize the impact of insect feeding behavior on plant resistance.
This study was conducted under the Project 25-8311 of Seed and Plant Improvement Institute and financially supported by Agricultural Research, Education and Extension Organization of Iran.