Whitefly-mediated Inoculation Research Articles

Genetic potentiality of Pumpkin genotypes against Pumpkin yellow vein mosaic virus

Pumpkin (Cucurbita moschata Duch. ex Poir.), a nutritious vegetable, is susceptible to several diseases, with Pumpkin Yellow Vein Mosaic Virus (PYVMV) causing severe impact by affecting plants at all growth stages, leading to substantial yield losses, vegetable shortages, and malnutrition in Bangladesh. This study evaluated thirty pumpkin genotypes for PYVMV resistance, genetic potential, and yield characteristics, sourced from Advanced Chemical Industries (ACI) Agribusiness and various districts in Bangladesh. For agronomic and molecular assays, thirty pumpkin genotypes along with the standard check were planted following RCBD with three replications at BAU-GPB farm. Whitefly-mediated inoculation in a net house assay determined the Area Under Disease Progress Curve (AUDPC) under controlled conditions. Tissue Blot Immunoassay (TBIA), using rabbit antiserum specific to PYVMV, showed no cross-reactivity with non-target viruses, with consistent results across TBIA, AUDPC, and molecular assays. Infected younger leaves were collected for virus-specific molecular analyses. Three sets of SSR markers were used to identify genes responsible for PYVMV resistance. PYABFP & PYABRP and PYBHFP & PYBHRP primer sets were capable of producing expected fragment.ToLCV1100F & ToLCV1650R primer sets were used for final confirmation. Genotypes PK2, PK5, and PK17 showed superior performance with higher individual fruit weights, more fruits per vine, higher overall yields, and minimal viral infection compared to the check variety (Baromashi). In contrast, PK1, PK6, PK25, and PK27 exhibited poor yield and related traits. Marker analysis revealed the absence of resistance genes in PK1, PK3, PK4, PK6, PK7, PK8, PK9, PK10, PK11, PK12, PK13, PK14, PK15, PK16, PK18, PK19, PK20, PK21, PK22, PK23, PK24, PK25, PK26, PK27, PK28, PK29, and Baromashi (control), but confirmed the presence of resistance genes in PK2, PK5, and PK17. Consequently, PK2, PK5, and PK17 were recommended as PYVMV-resistant genotypes with high yielding genetic potentiality.

Evaluation of tomato hybrids for resistance to leaf curl virus disease and for high-yield production

The leaf curl disease of tomato (Solanum lycopersicum L.) caused by tomato leaf curl virus (ToLCV) poses a serious production constraint throughout the world. An evaluation for resistance to ToLCV and four horticultural traits was carried out using 10 parents and 45 F1 hybrids during the autumn of 2014–2015. Screening against ToLCV was done under both induced natural disease conditions and whitefly (Bemisia tabaci Genn.)-mediated inoculation, with symptoms ranging from symptomless to very severe infection. Under the natural disease conditions, seven parents and 20 F1 hybrids appeared symptomless until the fruit maturation stage. Using the artificial inoculation technique, six parents and 18 hybrids showed resistance at 60 days post inoculation. The correlation coefficients indicated that breeding for ToLCV-resistant hybrids enhanced total yield, fruit weight and total soluble solids content of fruits under natural disease conditions. Based on screening for resistance to ToLCV and four horticultural traits, we identified 4 tomato hybrids, namely CLN 138A × CLN 138B, CLN 138B × PVB-1, CLN 104 × CLN 154 and CLN 17 × PVB-1 to be favorable for commercial use. These hybrids showed resistance after undergoing the whitefly-mediated inoculation, produced 40% higher yield, and were either superior or at par for fruit weight, TSS and lycopene content when compared to the commercial control under natural disease conditions.

Analysis of the resistance of Gossypium herbaceum to cotton leaf curl kokhran virus strain burewala and cotton leaf curl multan betasatellite

Gossypium herbaceum is resistant to cotton leaf curl disease but the nature of the resistance is poorly characterized. The reaction of G. herbaceum cv. Co Tiep Khac to infection by the Burewala strain of cotton leaf curl Kokhran virus (CLCuKoV-Bur) and cotton leaf curl Multan betasatellite (CLCuMuB) was characterized following whitefly-mediated inoculation and grafting. The CLCuKoV-Bur and CLCuMuB isolates used in this study had 97–100% nucleotide sequence identity with other isolates for which sequence information is accessible in databases. A high level of resistance was demonstrated in cv. Co Tiep Khac using 200 whiteflies per plant with no apparent disease symptoms even 90 days post-inoculation, and no detection of the virus-betasatellite complex, as shown by polymerase chain reaction (PCR) and rolling circle amplification (RCA)/PCR. Following graft-inoculation, mild foliar disease symptoms were observed in leaves proximal to the grafting sites of most of cv. Co Tiep Khac plants but not in apical leaves. The presence of CLCuKoV-Bur and CLCuMuB was detected by RCA/PCR but not by PCR and Southern hybridization in symptomless apical leaves of cv. Co Tiep Khac. In addition, less virus accumulated in cv. Co Tiep Khac compared to G. hirsutum cv. CIM-496. These results provided insights into the resistance of G. herbaceum cv. Co Tiep Khac to CLCuKoV-Bur and CLCuMuB.

Efficient Transmission and Propagation of Tomato Chlorosis Virus by Simple Single-Leaflet Grafting.

Tomato chlorosis virus (ToCV), a member of the genus Crinivirus, has caused an epidemic disease in tomato worldwide. ToCV is phloem-limited and transmitted by whiteflies in a semi-persistent manner, but not by mechanical inoculation. Experimental propagation of ToCV has been performed primarily by using whitefly-mediated inoculation. To develop a simple and convenient method for transmission of ToCV, we investigated grafting single-leaflets from tomato plants infected with ToCV to recipient tomato seedlings. Forty-one of 46 tomato seedlings tested were grafted successfully with single-leaflets infected with ToCV. Among them, 36 seedlings (87.8%) were systemically infected with ToCV and developed typical symptoms. Our results demonstrated that single-leaflet grafting could provide a sufficient amount of inoculum for the transmission of ToCV to the grafted seedlings.

Immunity to tomato yellow leaf curl virus in transgenic tomato is associated with accumulation of transgene small RNA.

Gene silencing is a natural defense response of plants against invading RNA and DNA viruses. The RNA post-transcriptional silencing system has been commonly utilized to generate transgenic crop plants that are "immune" to plant virus infection. Here, we applied this approach against the devastating DNA virus tomato yellow leaf curl virus (TYLCV) in its host tomato (Solanum lycopersicum L.). To generate broad resistance to a number of different TYLCV viruses, three conserved sequences (the intergenic region [NCR], V1-V2 and C1-C2 genes) from the genome of the severe virus (TYLCV) were synthesized as a single insert and cloned into a hairpin configuration in a binary vector, which was used to transform TYLCV-susceptible tomato plants. Eight of 28 independent transgenic tomato lines exhibited immunity to TYLCV-Is and to TYLCV-Mld, but not to tomato yellow leaf curl Sardinia virus, which shares relatively low sequence homology with the transgene. In addition, a marker-free (nptII-deleted) transgenic tomato line was generated for the first time by Agrobacterium-mediated transformation without antibiotic selection, followed by screening of 1180 regenerated shoots by whitefly-mediated TYLCV inoculation. Resistant lines showed a high level of transgene-siRNA (t-siRNA) accumulation (22% of total small RNA) with dominant sizes of 21 nt (73%) and 22 nt (22%). The t-siRNA displayed hot-spot distribution ("peaks") along the transgene, with different distribution patterns than the viral-siRNA peaks observed in TYLCV-infected tomato. A grafting experiment demonstrated the mobility of 0.04% of the t-siRNA from transgenic rootstock to non-transformed scion, even though scion resistance against TYLCV was not achieved.

Sweet pepper confirmed as a reservoir host for tomato yellow leaf curl virus by both agro-inoculation and whitefly-mediated inoculation.

Tomato yellow leaf curl virus (TYLCV), a member of the genus Begomovirus, has a single-stranded DNA genome. TYLCV can induce severe disease symptoms on tomato plants, but other hosts plants such as cucurbits and peppers are asymptomatic. A full-length DNA clone of a Korean TYLCV isolate was constructed by rolling-circle amplification from TYLCV-infected tomatoes in Korea. To assess relative susceptibility of sweet pepper varieties to TYLCV, 19 cultivars were inoculated with cloned TYLCV by agro-inoculation. All TYLCV-infected sweet peppers were asymptomatic, even though Southern hybridization and polymerase chain reaction analysis showed TYLCV genomic DNA accumulation in roots, stems, and newly produced shoots. Southern hybridization indicated that TYLCV replicated and moved systemically from agro-inoculated apical shoot tips to roots or newly produced shoots of sweet peppers. Whitefly-mediated inoculation experiments showed that TYLCV can be transmitted to tomatoes from TYLCV-infected sweet peppers. Taken together, these results indicate that sweet pepper can be a reservoir for TYLCV in nature.

A developmentally regulated lipocalin-like gene is overexpressed in Tomato yellow leaf curl virus-resistant tomato plants upon virus inoculation, and its silencing abolishes resistance

To discover genes involved in tomato resistance to Tomato yellow leaf curl virus (TYLCV), we previously compared cDNA libraries from susceptible (S) and resistant (R) tomato lines. Among the genes preferentially expressed in R plants and upregulated by TYLCV infection was a gene encoding a lipocalin-like protein. This gene was termed Solanum lycopersicum virus resistant/susceptible lipocalin (SlVRSLip). The SlVRSLip structural gene sequence of R and S plants was identical. SlVRSLip was expressed in leaves during a 15-day window starting about 40 days after sowing (20 days after planting). SlVRSLip was upregulated by Bemisia tabaci (the TYLCV vector) feeding on R plant leaves, and even more strongly upregulated following whitefly-mediated TYLCV inoculation. Silencing of SlVRSLip in R plants led to the collapse of resistance upon TYLCV inoculation and to a necrotic response along the stem and petioles accompanied by ROS production. Contrary to previously identified tomato lipocalin gene DQ222981, SlVRSLip was not regulated by cold, nor was it regulated by heat or salt. The expression of SlVRSLip was inhibited in R plants in which the hexose transporter gene LeHT1 was silenced. In contrast, the expression of LeHT1 was not inhibited in SlVRSLip-silenced R plants. Hence, in the hierarchy of the gene network conferring TYLCV resistance, SlVRSLip is downstream of LeHT1. Silencing of another gene involved in resistance, a Permease-I like protein, did not affect the expression of SlVRSLip and LeHT1; expression of the Permease was not affected by silencing SlVRSLip or LeHT1, suggesting that it does not belong to the same network. The triple co-silencing of SlVRSLip, LeHT1 and Permease provoked an immediate cessation of growth of R plants upon infection and the accumulation of large amounts of virus. SlVRSLip is the first lipocalin-like gene shown to be involved in resistance to a plant virus.

Expression of Stress-Response Proteins Upon Whitefly-Mediated Inoculation of Tomato yellow leaf curl virus in Susceptible and Resistant Tomato Plants

To better understand the nature of resistance of tomato to the whitefly (Bemisia tabaci, B biotype)-transmitted Tomato yellow leaf curl virus (TYLCV), whiteflies and TYLCV were considered as particular cases of biotic stresses and virus resistance as a particular case of successful response to these stresses. Two inbred tomato lines issued from the same breeding program that used Solanum habrochaites as a TYLCV resistance source, one susceptible and the other resistant, were used to compare the expression of key proteins involved at different stages of the plant response with stresses: mitogen-activated protein kinases (MAPKs), cellular heat shock proteins (HSPs, proteases), and pathogenesis-related (PR) proteins. The two biotic stresses-non-viruliferous whitefly feeding and virus infection with viruliferous insects--led to a slow decline in abundance of MAPKs, HSPs, and chloroplast protease FtsH (but not chloroplast protease ClpC), and induced the activities of the PR proteins, beta-1,3-glucanase, and peroxidase. This decline was less pronounced in virus-resistant than in virus-susceptible lines. Contrary to whitefly infestation and virus infection, inoculation with the fungus Sclerotinia sclerotiorum induced a rapid accumulation of the stress proteins studied, followed by a decline; the virus-susceptible and -resistant tomato lines behaved similarly in response to the fungus.

Biolistic inoculation of plants with Tomato yellow leaf curl virus DNA

Tomato yellow leaf curl virus (TYLCV) full-length DNA was amplified by PCR and cloned into a bacterial plasmid. The cloned TYLCV DNA was excised from the plasmid, ligated and the resulting monomeric circular double-stranded TYLCV DNA was used to inoculate tomato ( Solanum lycopersicom) and datura ( Datura stramonium) plants by particle bombardment. The bombarded plants produced typical disease symptoms, similar to those produced following whitefly-mediated inoculation, albeit 5–7 days later than whitefly-inoculated plants. The success rate of inoculating tomato plants by particle bombardment averaged 37%, whereas with datura plants, it averaged 85%. With whitefly-mediated inoculation of TYLCV, the success rate of inoculation was also higher in datura plants than in tomato plants. Bombardment of datura plants with a linear form of TYLCV DNA also resulted in viral infection, with an inoculation success rate similar to that with the closed-circular TYLCV DNA. Bombarding datura plants with the bacterial plasmid containing the cloned TYLCV DNA did not result in viral infection, but bombardment with a bacterial plasmid containing a cloned dimer of TYLCV DNA yielded an infection rate of 50–100%. This is the first report of TYLCV inoculation of plants using particle bombardment of a cloned monomeric linear or closed-circular form of TYLCV double-stranded DNA.

Making a friend from a foe: expressing a GroEL gene from the whitefly Bemisia tabaci in the phloem of tomato plants confers resistance to tomato yellow leaf curl virus

Some (perhaps all) plant viruses transmitted in a circulative manner by their insect vectors avoid destruction in the haemolymph by interacting with GroEL homologues, ensuring transmission. We have previously shown that the phloem-limited begomovirus tomato yellow leaf curl virus (TYLCV) interacts in vivo and in vitro with GroEL produced by the whitefly vector Bemisia tabaci. In this study, we have exploited this phenomenon to generate transgenic tomato plants expressing the whitefly GroEL in their phloem. We postulated that following inoculation, TYLCV particles will be trapped by GroEL in the plant phloem, thereby inhibiting virus replication and movement, thereby rendering the plants resistant. A whitefly GroEL gene was cloned in an Agrobacterium vector under the control of an Arabidopsis phloem-specific promoter, which was used to transform two tomato genotypes. During three consecutive generations, plants expressing GroEL exhibited mild or no disease symptoms upon whitefly-mediated inoculation of TYLCV. In vitro assays indicated that the sap of resistant plants contained GroEL-TYLCV complexes. Infected resistant plants served as virus source for whitefly-mediated transmission as effectively as infected non-transgenic tomato. Non-transgenic susceptible tomato plants grafted on resistant GroEL-transgenic scions remained susceptible, although GroEL translocated into the grafted plant and GroEL-TYLCV complexes were detected in the grafted tissues.

Characterisation of a Quantitative Resistance to Vector Transmission of Tomato yellow leaf curl virus in Lycopersicon pimpinellifolium

Two wild genotypes from the same species Lycopersicon pimpinellifolium, WVA106 (susceptible) and INRA-Hirsute (so-called ‘resistant’), were compared with respect to their reaction to Tomato yellow leaf curl virus isolate Reunion (TYLCV-Mld[RE]), using both whitefly-mediated inoculation and graft inoculation. Disease incidence and symptom severity were scored. Presence and quantification of viral DNA were assessed by dot blot hybridisation. Upon insect inoculation, accession INRA-Hirsute showed a moderate resistance against TYLCV that was overcome by a high inoculation pressure obtained by increasing the cumulative number of inoculative whiteflies. Temporal analyses of the disease progress in relation to this criterion exhibited that the protection was quantitative, mainly reducing the TYLCV-Mld[RE] incidence by at maximum 50% at low inoculation pressure. When graft inoculated, the final TYLCV-Mld[RE] disease incidence was 100% in both susceptible and resistant genotypes with severe symptoms, suggesting a reduction of virus transmission by a vector resistance as a possible mechanism. Implications of using such type of resistance in breeding programmes are discussed.

Evaluation of breeding tomato lines partially resistant to Tomato yellow leaf curl Sardinia virus and Tomato yellow leaf curl virus derived from Lycopersicon chilense

Twelve tomato (Lycopersicon esculentum Mill.) advanced breeding lines derived from L. chilense and partially resistant to Tomato yellow leaf curl Sardinia virus (TYLCSV) were evaluated for their resistance to the species from Israel (Tomato yellow leaf curl virus, TYLCV). Two assays were carried out in two consecutive years, using agroinoculation and whitefly-mediated inoculation, respectively. Symptom severity, percentage of infection, and viral DNA accumulation (using molecular hybridization) were measured. In the first assay, the 12 breeding lines were agroinoculated with both virus species. Resistance to TYLCSV was confirmed for the 12 breeding lines, but only 6 of them showed resistance to TYLCV. During the second assay these six breeding lines were whitefly-inoculated with TYLCV. All lines showed high levels of partial resistance to TYLCV consisting in attenuation and delay in time of symptom development and reduction in virus accumulation when compared with the susceptible control. Three of these lines even accumulated significantly lower amounts of viral DNA than the resistant controls 'Anastasia' and 'Boludo' hybrids. These lines also display good horticultural traits, appropriate for the protected growing system and for the fresh market requirements. These advanced breeding lines are base material for developing commercial hybrids highly resistant to TYLCSV and TYLCV.

Resistance of tomato and sweet-pepper genotypes to Tomato leaf curl Bangalore virus and its vector Bemisia tabaci

The primary objective of this study was to identify sources of resistance to Tomato leaf curl Bangalore virus—Bangalore strain 4 (ToLCBV-[Ban4]) and its whitefly vector Bemisia tabaci (Gennadius) in tomato and sweet-pepper genotypes, because resistant cultivars did not exist. A total of 25 wild and cultivated tomato and nine sweet-pepper genotypes obtained from the Asian Vegetable Research and Development Centre, Taiwan and local cultivars were screened at the University of Agricultural Sciences, Bangalore, India, both by whitefly-mediated inoculation of virus in the glasshouse and natural infection in the field. Resistance to virus was assessed by disease incidence, symptom severity and DNA hybridisation tests. Resistance to B. tabaci was measured by counting the number of eggs, nymphs and pupal cases on the tomato genotypes. The wild species Lycopersicon peruvianum INRA sel. and L. chilense LA 1969 were resistant to ToLCBV-[Ban4] but highly susceptible to whiteflies, whereas L. hirsutum LA 1777 was resistant to both the virus and the vector. Among the L. esculentum genotypes, H-24, FL 744-6-9, FL 699 and FL 699 sp + were tolerant to ToLCBV-[Ban4], but were susceptible to whiteflies. Of the nine pepper genotypes inoculated with the virus, only five became infected, at low incidence levels and none were suitable for the development of B. tabaci. Use of tomato lines with resistance to both the virus and the vector offer the best way of controlling these related problems, and the prospects for tomato leaf curl virus disease management in South India are discussed.

Comparison of resistance to Tomato leaf curl virus (India) and Tomato yellow leaf curl virus (Israel) among Lycopersicon wild species, breeding lines and hybrids

The objective of this study was to screen wild and domesticated tomatoes for resistance to Tomato yellow leaf curl virus, Israel (TYLCV-Is) and Tomato leaf curl virus from Bangalore isolate 4, India (ToLCV-[Ban4]) to find sources of resistance to both viruses. A total of 34 tomato genotypes resistant/tolerant to TYLCV-Is were screened for resistance to ToLCV-[Ban4] under glasshouse and field conditions at the University of Agricultural Sciences, Bangalore, India. Resistance was assessed by criteria like disease incidence, symptom severity and squash-blot hybridization. All the tomato genotypes inoculated with ToLCV-[Ban4] by the whitefly vector Bemisia tabaci (Gennadius) produced disease symptoms. In some plants of the lines 902 and 910, however, the virus was not detected by hybridization. The tomato genotypes susceptible to ToLCV-[Ban4] by whitefly-mediated inoculation were also found susceptible to the virus under field conditions. However, there were substantial differences between genotypes in disease incidence, spread, symptom severity and crop yield. Despite early disease incidence, many genotypes produced substantially higher yields than the local hybrid, Avinash-2. Sixteen tomato genotypes from India resistant/tolerant to ToLCV-[Ban4] were also tested for TYLCV-Is resistance at the Hebrew University of Jerusalem, Rehovot, Israel. Accessions of wild species, Lycopersicon hirsutum LA 1777 and PI 390659 were the best sources of resistance to both viruses. Lines 902 and 910, which were, resistant to TYLCV-Is were only tolerant to ToLCV-[Ban4] and accession Lycopersicon peruvianum CMV Sel. INRA, resistant to ToLCV-[Ban4], was only tolerant to TYLCV-Is. Implications of using the resistant lines in breeding programme is discussed.

First Report of a Bean-Infecting Begomovirus from Macroptilium lathyroides in Puerto Rico That Is Distinct from Bean Golden Mosaic Virus.

Bean golden mosaic begomovirus (BGMV) was long suspected to cause bright yellow mosaic symptoms in Macroptilium lathyroides (L.), a weed common to Puerto Rico. M. lathyroides plants exhibiting bright yellow mosaic symptoms were collected from Puerto Rico during 1994 to 1999, and the biotic and molecular characteristics of the suspect begomovirus were determined. Symptoms in M. lathyroides, indistinguishable from those observed in field-infected plants, were reproducible by whitefly transmission (Bemisia tabaci type B) and biolistic inoculation of leaf extracts (1). In bean, Phaseolus vulgaris (L.) 'Topcrop,' the M. lathyroides virus caused green-yellow mosaic foliar symptoms and stunting, reminiscent of symptoms caused by BGMV from Puerto Rico (BGMV-PR). However, biolistic- or whitefly-mediated inoculation of M. lathyroides with BGMV-PR resulted in no discernible infection. Sequence analysis (2) of the coat protein (CP) gene (AF176092) and common region of the A (CR-A) (AF176093) and B (CR-B) (AF176094) components of the virus from M. lathyroides indicated that these sequences shared only 77.3 to 79.3% and 62.4 to 68.8% identity, respectively, with BGMV from the Dominican Republic, Honduras, Guatemala, Jamaica (JAM), and PR. Alignment of the M. lathyroides virus CP sequence with other well-studied begomoviruses indicate its closest relative is BGMV-PR (82%) and that it shares less than 73% identity with partial CP sequences of Macroptilium golden mosaic virus-JAM (AF089839, AF089840). The directly repeated CR sequences of the M. lathyroides virus, putatively involved in AC1 binding, are TGGTGACTGGTG and are distinct from TGGAGACTGGAG, the analogous direct repeat in BGMV-PR. We provisionally designate the new, previously undescribed begomovirus species from M. lathyroides, Macroptilium mosaic virus (MaMV). Results indicate MaMV-PR and BGMV are distinct, bean-infecting begomoviruses from the Caribbean and that MaMV-PR may pose a new threat to bean production, particularly where the type B vector is established.

Tomato Breeding Lines Resistant and Tolerant to Tomato Yellow Leaf Curl Virus Issued fromLycopersicon hirsutum

ABSTRACT Two tomato yellow leaf curl virus (TYLCV)-resistant plants from accessions LA1777 and LA386 of the wild tomato species Lycopersicon hirsutum have been crossed. The resulting resistant F1 plants were crossed with the domesticated tomato L. esculentum, and a series of selfing was performed. At each generation, individuals were selected for resistance (no symptoms and undetectable viral DNA) and tolerance (no symptoms but with detectable viral DNA) following controlled massive and repeated inoculations with viruliferous whiteflies. A stable BC1F4 line (denominated 902) that does not segregate for resistance was obtained. This line does not support virus accumulation, even upon extensive whitefly-mediated inoculation of young seedlings, and does not need protection with nets or insecticides. Another stable BC1F4 line (denominated 908) was tolerant to the virus. Both lines have good horticultural characteristics and bear 80- to 120-g red fruits. Analysis of segregation of susceptibility, tolerance, and resistance during the BC1F1 to BC1F4 crosses indicated that tolerance is controlled by a dominant major gene and resistance by two to three additive recessive genes. The resistant and tolerant lines do not need to be protected by insecticides or nets.

Evaluation of whitefly-mediated inoculation techniques to screen Lycopersicon esculentum and wild relatives for resistance to Tomato yellow leaf curl virus

For two consecutive years nine hybrids and three varieties of tomato, four Lycopersicon peruvianum and four Lycopersicon chilense accessions were screened for Tomato yellow leaf curl virus (TYLCV) resistance. Three inoculation techniques using Bemisia tabaci, the vector of TYLCV, were compared: (1) artificial mass inoculation-simultaneous infection of cultivated and wild material in greenhouses; (2) artificial cage inoculation-individual infection in insect-proof cages; (3) natural field infection. Artificial inoculations led to higher levels of infection, but different patterns of response to each inoculation technique were found depending on the resistance level. Tomato varieties showed an important fruit set reduction after both artificial and natural inoculations. In contrast, field infection was milder in tomato hybrids, in which yield was barely affected. These hybrids showed a wide range of reactions with the two artificial inoculation techniques, but infection was always more severe after mass inoculation. Extreme severity of mass infection made it difficult to differentiate among variable degrees of resistance that were more reliably detected with cage inoculation. The hybrids F3524, F3522, Fiona, and Tyking showed the highest level of resistance. F3524 and F3522 had an acceptable yield in field and cage assays, but their resistance collapsed under massive conditions of infection. Tyking and Fiona exhibited the best response in all conditions, although their yield was moderately reduced in mass assays. Mass inoculation was not adequate for the screening of wild Lycopersicon. Some susceptible plants escaped infection, probably as a consequence of non-preference mechanisms and loss of vector infectivity. Individual inoculation in cages prevented the risk of non-infection, ensuring 100% disease incidence. This technique allowed the selection of highly resistant wild sources. L. chilense LA 1969 and LA 1963 had the highest level of resistance with the three inoculation techniques. L. peruvianum PI-126944 and L. chilense LA 1932, which were only tested in mass and field conditions, also exhibited a promising response. The results proved that the inoculation technique influences the response of tomato and wild Lycopersicon spp to TYLCV. It is concluded that artificial cage inoculation, although more time-consuming, is the most efficient, adequate, and reliable technique to screen both cultivated and wild Lycopersicon species for resistance to TYLCV.

Replication of tomato yellow leaf curl virus (TYLCV) DNA in agroinoculated leaf discs from selected tomato genotypes.

The leaf disc agroinoculation system was applied to study tomato yellow leaf curl virus (TYLCV) replication in explants from susceptible and resistant tomato genotypes. This system was also evaluated as a potential selection tool in breeding programmes for TYLCV resistance. Leaf discs were incubated with a head-to-tail dimer of the TYLCV genome cloned into the Ti plasmid of Agrobacterium tumefaciens. In leaf discs from susceptible cultivars (Lycopersicon esculentum) TYLCV single-stranded genomic DNA and its double-stranded DNA forms appeared within 2-5 days after inoculation. Whiteflies (Bemisia tabaci) efficiently transmitted the TYLCV disease to tomato test plants following acquisition feeding on agroinoculated tomato leaf discs. This indicates that infective viral particles have been produced and have reached the phloem cells of the explant where they can be acquired by the insects. Plants regenerated from agroinfected leaf discs of sensitive tomato cultivars exhibited disease symptoms and contained TYLCV DNA concentrations similar to those present in field-infected tomato plants, indicating that TYLCV can move out from the leaf disc into the regenerating plant. Leaf discs from accessions of the wild tomato species immune to whitefly-mediated inoculation, L. chilense LA1969 and L. hirsutum LA1777, did not support TYLCV DNA replication. Leaf discs from plants tolerant to TYLCV issued from breeding programmes behaved like leaf discs from susceptible cultivars.

Infection of tomato by the tomato yellow leaf curl virus: susceptibility to infection, symptom development, and accumulation of viral DNA

Symptom development in tomato plants following whitefly-mediated inoculation with tomato yellow leaf curl virus (TYLCV) was related to the occurrence of viral DNA using a specific DNA probe. Although disease symptoms were first observed 15 days post-inoculation, viral DNA could be detected 7 days earlier. TYLCV-DNA concentrations reached an optimum 4 days before symptoms appeared. The highest concentrations of TYLCV-DNA were found in rapidly growing tissues (shoot apex, young leaves, roots) and in the stems; the lowest concentrations were found in the older leaves and cotyledons. Plants were also inoculated on specific sites. Young leaves and apices were the best targets for virus inoculation. In these tissues, the viral DNA replicated at the site of inoculation and was transported first to the roots, then to the shoot apex and to the neighboring leaves and the flowers. Inoculation through the oldest leaves was inefficient.