Tomato yellow leaf curl virus (leaf curl)

Growing tomato in open fields is much more constraining than growing under cover. In all regions where TYLCV is endemic, seedlings need to be grown and kept in a glasshouse or a screenhouse in order to prevent early infection by whitefly feeding. In Thailand, farmers grow tomato plants in isolated paddy fields after rice harvesting. In Egypt the spread of TYLCV in open fields was successfully controlled by roguing infected plants and removing all overwintered tomato crops before the emergence of whitefly populations ( Mazyad et al., 1986 ). Similar measures were undertaken in Cyprus over 3 consecutive years resulting in a decrease in the incidence of the disease from 50 to 5% ( Ioannou, 1987 ). In the Dominican Republic, a mandatory 3 months host-free growing period which included bean, cucurbits, aubergine, melon, okra, pepper and tomato, significantly reduced the inoculum and lowered to a minimum the incidence of TYLCV ( Gilbertson et al., 2007 ). Early planting when the incidence of B. tabaci is low, effectively reduced TYLCV infection in Egypt, Israel and Cyprus. Interplanting with rows of TYLCV non-host trap plants such as squash and cucumber have been used to divert whiteflies from tomato in the Jordan Valley, delaying TYLCV infection by 2 months ( Al-Musa, 1982 ). Sticky yellow plastic polyethylene sheets have been used, with limited success, to decrease the pressure of the whitefly population ( Cohen et al., 1974 ). When economically feasible, growing tomatoes in greenhouses covered with insect-proof 50-mesh screens and double-doors constitutes the optimal solution for crop protection against the TYLCV whitefly vector ( Cohen and Berlinger, 1986 ; Ioannou, 1987 ). UV-absorbing plastic sheets or UV-absorbing 50-mesh screens, used as tunnels or as screenhouse covers, greatly reduced the infestation of tomato plants by B. tabaci and the incidence of TYLCV ( Antignus et al., 1998 ). Whitefly natural enemies ( Gerling, 1990 ) have not been used on a large scale in open fields.

Chemical Control

Applying systemic insecticides as soil drenches or regular spraying during the seedling stage can reduce the population of the B. tabaci vector and the incidence of TYLCV. Many insecticides such as organophosphates, carbamates and pyrethroids effectively reduce the whitefly population, whether sprayed one insecticide at a time, alternately, or together with oil and emulsifiers ( Makkouk and Laterrot, 1983 ). However, these insecticides provided only partial TYLCV control, even when sprayed daily ( Cohen and Antignus, 1994 ). Continued extensive use of insecticides is not only detrimental to the environment but it promotes the development of resistant whitefly populations ( Cahill et al., 1995 ). More potent insecticides with novel modes of action such as imidacloprid were introduced in the 1990s ( Elbert et al., 1990 ). Over the past few years imidacloprid has assumed major importance for control of B. tabaci in the field and in greenhouses. To be efficient, the insecticide has to induce the death of all whiteflies within 35-40 min before it succeeds in infecting the plant, thereby preventing inoculation of TYLCV, which is not always the case in the field ( Rubinstein et al., 1999 ). As for other commonly used insecticides, whitefly populations resistant to imidacloprid have been emerging in many places ( Castle et al., 2010 ).

Host-Plant Resistance

Breeding programmes aimed at producing tomato cultivars resistant to TYLCV started in the late 1960s and have expanded since. These programmes are based on the introgression into the domesticated tomato Solanum lycopersicum of resistance or tolerance found in some accessions of wild tomato species such as S. cheesmaniae, S . chilense, S. habrochaites, S. peruvianum and S. pimpinellifolium . Depending on the plant source, resistance was reported to be controlled by one to five loci, either recessive or dominant ( Zakay et al., 1991 ; Picó et al., 1996; Nakhla and Maxwell, 1998 ). The first commercial tolerant cultivar, TY20 (Hazera) carrying tolerance from S. peruvianum, showed delayed symptoms and the accumulation of viral DNA ( Pilowski and Cohen, 1990 ; Lapidot et al., 1997 ). More advanced cultivars derived from TY-20 include 8484 and 8472. Additional commercial hybrids tolerant to TYLCV include Anastasia (Brunsma), Silver, Beludo and Ulyses (Seminis), Fiona and Jackal (Sluis and Groot), TOP 21 (Clause), Saria and Gemstar (Petoseed), Hilario, Rex, Tycoon and Tyking, (Royal Sluis), Pitenza-TY and Pal-TY (Enza-Zaden). In heavily affected areas, these hybrids need to be protected with insecticides during the first weeks after planting to produce acceptable yields. Many experimental lines are being developed which show good levels of resistance against TYLCV and other begomoviruses affecting tomato worldwide. The breeding line TY172 originating from S. peruvianum is a symptomless carrier of TYLCV whether infected in the greenhouse or in the field; at least three loci may account for the resistance ( Friedmann et al., 1998 ). The breeding line 902 originating from S. habrochaites support little virus accumulation; another line derived from the same wild tomato progenitor, line 908, is tolerant to the virus. Tolerance is controlled by a dominant major locus, and resistance by two to three additive recessive loci (Vidavski and Czosnek, 1998). Nine hybrids based on these lines have been successfully tested in infested fields in Israel, Egypt, Morocco and Guatemala.

Marker-assisted breeding has allowed localization of TYLCV-resistance loci on the tomato genetic map and has been instrumental in developing new tomato varieties. Using polymorphic DNA markers, a TYLCV-tolerance gene originating from S. chilense LA1969, Ty-1, has been mapped to tomato chromosome 6, close to the nematode resistance Mi gene ( Zamir et al., 1994 ). Another locus associated with tolerance originating from S. pimpinellifolium has been mapped to chromosome 6 but to a locus different from Ty-1 (Chague et al., 1997). Polymorphic markers have also been used to map a loci coinded Ty-2 conferring resistance to ToLCV, originating from S. habrochaites to tomato chromosome 11 ( Hanson et al., 2000 ). A major partially dominant locus, termed Ty-3, was mapped to chromosome 6, near the Ty-1 locus ( Ji et al., 2007 ). Another TYLCV resistance locus, termed Ty-4, was mapped on the long arm of Chromosome 3 ( Ji et al., 2008 ). Another locus, termed Ty-5, was mapped to chromosome 4 using the breeding highly resistant line TY172, which originating from S. peruvianum ( Anbinder et al., 2009 ). The different sources of resistance were piled up to provide tomato plants with better resistance to TYLCV ( Vidavski et al., 2008 ). The genes conferring resistance have not yet been isolated. An RNAi-based genome-wide screening is under way to uncover the gene network sustaining TYLCV resistance in tomato lines issued from S. habrochaites ( Czosnek et al., 2011b ).

1. Pathogen-derived resistance

Virus-resistant transgenics have been developed in many crops by introducing either viral capsid protein or replicase gene encoding sequences. This concept has been called pathogen-derived resistance (PDR) ( Lomonossoff, 1995 ; Baulcombe, 1996 ). PDR has been the most common approach used to obtain resistance to TYLCV, including viral sequences that generate antisense RNA as well as the expression of full-length and truncated viral genes ( Polston and Hiebert, 2007 ). The CP gene was one of the first TYLCV genes evaluated for the ability to generate pathogen-derived resistance. Tomato plants expressing the TYLCV CP showed a delay in symptoms, a recovery from infection, and resistance upon repeated inoculations ( Kunik et al., 1994 ). Expression antisense RNA of the TYLCSV Rep gene provided a good level of resistance in Nicotiana benthamiana (Bendahmane and Gronenborn, 1997). The expression of sense and antisense constructs of truncated TYLCSV Rep in N. benthamiana also showed resistance ( Noris et al., 1996 ). When a truncated TYLCV-Mld Rep gene was expressed in tomato, the transgenic plants were resistant to TYLCV-Mld, but were susceptible to TYLCV-IL ( Antignus et al., 2004 ). A 2/5 TYLCV Rep gene construct conferred high levels of resistance and often immunity in both transformed N. tabacum and S. lycopersicum ( Yang et al., 2004 ).

2. Virus gene silencing

Viruses trigger plant antiviral mechanisms leading to the degradation of viral RNA via the formation of dsRNA derived from viral sequences, initiating post-transcriptional gene silencing (PTGS). This phenomenom was named RNA-mediated gene silencing. Some viruses establish themselves by expressing silencing supressor genes, which interfere with plant host silencing mechanisms ( Voinnet et al., 1999 ; Roth et al., 2004 ). RNA-mediated virus resistance proved to be more efficient than protein-mediated resistance but was highly-sequence dependent. Transgenic plants have been developed which exploited the mechanism of silencing via dsRNA ( Smith et al., 2000 ). Using a transformation cassette consisting of the 3’-end of the Rep (sense and antisense orientation) as the arms of the hairpin, and a castor bean catalase as the intron, the treated tomato plants showed immunity to TYLCV upon whitefly-mediated inoculation ( Fuentes et al., 2006 ). A similar approach was used with the 5’-end of the TYLCV CP gene and a maize ubiquitin intron, inducing resistance to TYLCV in tomato ( Zrachya et al., 2007 ). The intron-hairpin approach was used to express multiple TYLCV sequences in tobacco and tomato plants ( Abhary et al., 2006 ). The treated plants were symptomless and TYLCV DNA was not detected. In general, silencing was specific to the TYLCV strain; silencing using TYLCV sequences conferred resistance to TYLCV but not to other TYLCV species or strains, such as TYLCSV ( Noris et al., 2004 ).

3. GroEL

This novel approach is based on the observation that GroEL produced by whitefly endosymbiotic bacteria binds to the CP of TYLCV in the insect hemolymph, thereby protecting the virion from destruction and ensuring transmission ( Morin et al., 1999 ). TYLCV-GroEL binding has been exploited to generate TYLCV resistant tomato plants. Tomatoes transformed with GroEL under a phloem-specific promoter showed milder to no symptoms in plants in the R ₀ through R ₂ generation. GroEL/TYLCV complexes were readily detected in resistant plants. It was hypothesized that GroEL/TYLCV complexes formed in transformed plants and that these complexes were interfering in virus movement ( Akad et al., 2007 ).