Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Ann. appl. Biol. (2002), 140:109-127 Printed in Great Britain 109 Breeding for resistance to whitefly-transmitted geminiviruses By MOSHE LAPIDOT1* and MICHAEL FRIEDMANN2 1 Department of Virology, Agricultural Research Organization, The Volcani Center, Bet Dagan 50250, Israel 2 Department of Plant Genetics, Agricultural Research Organization, The Volcani Center, Bet Dagan 50250, Israel (Accepted 11 March 2002; Received 27 December 2001) Summary Geminiviruses comprise a large and diverse family of viruses that infect a wide range of important monocotyledonous and dicotyledonous crop species and cause significant yield losses. The family Geminiviridae is divided into three genera, one of which is Begomovirus. Species of this genus are transmitted by the whitefly Bemisia tabaci in a persistent, circulative manner and infect dicotyledonous plants. Severe population outbreaks of B. tabaci are usually accompanied by a high incidence of begomoviruses. During the last two decades, there has been a worldwide spread of the B biotype of B. tabaci, accompanied by the emergence of whitefly-transmitted geminiviruses. Control measures in infected regions are based mainly on limitation of vector populations, using chemicals or physical barriers. However, under conditions of severe whitefly attack, none of these control measures has sufficed to prevent virus spread. Thus, the best way to reduce geminivirus damage is by breeding crops resistant or tolerant to the virus, either by classical breeding or by genetic engineering. A number of begomoviruses have been the subject of much investigation, due to their severe economic impact. This review considers the most severe viral diseases of four major crops (tomato, bean, cassava and cotton). The approaches taken to breed for resistance to these viral diseases should provide a perspective of the issues involved in breeding for begomovirus resistance in crop plants. Key words: Geminivirus, resistance, breeding, cassava, bean, tomato, cotton, Bemisia tabaci Introduction Geminiviruses are a large and diverse family of viruses that infect a wide range of important crop species, including both monocotyledonous and dicotyledonous plants, and cause significant yield loses. These viruses are characterised by the geminate morphology of the capsid and by circular single-stranded DNA (ssDNA) genomes. The family Geminiviridae is divided into three genera, based on genome structure, plant host and insect vector (Fauquet et al., 2000). Species of the genus Mastrevirus (Maize streak virus as type member) are transmitted by leafhoppers (Homoptera: Cicadellidae), have a monopartite genome, and generally infect monocotyledonous plants. Species of Curtovirus (Beet curly top virus, BCTV, as type member) are also transmitted by leafhoppers, have a monopartite genome, but with a different genetic organisation from that of the mastreviruses, and infect dicotyledonous plants. Species of Begomovirus (Bean golden mosaic virus, BGMV, as type member) are transmitted by the whitefly Bemisia tabaci (Gennadius) and infect dicotyledonous plants. Most begomoviruses of the Old World and all New World begomoviruses *Corresponding Author E-mail: [email protected] © 2002 Association of Applied Biologists have bipartite genomes (A and B components); however, some Old World begomoviruses, including Tomato yellow leaf curl virus (TYLCV) have a monopartite genome. All begomoviruses are transmitted by their whitefly vector in a persistent, circulative manner. That is, once the vector feeds on an infected host plant and acquires the virus, transmission of the virus can then occur within hours, and may continue for the life span of the vector (Cohen & Harpaz, 1964). However, as shown for TYLCV, transmission efficiency declines with time (Cohen & Harpaz, 1964). Control measures in infected regions have traditionally emphasised vector control (Cohen & Antignus, 1994; Polston & Anderson, 1997; Hilje et al., 2001; Palumbo et al., 2001), mainly by pesticides or physical barriers. Chemical control methods have been only partially effective, since whitefly populations can reach very high numbers, leading to intensive pesticide use (sometimes twice daily) in attempts to eliminate the vector before it transmits the virus. Furthermore, there are concerns that the vector may develop pesticide resistance and the intense application of pesticides may have deleterious effects on the environment (Pico et al., 1996; Palumbo et al., 2001). Physical barriers such 110 MOSHE LAPIDOT & MICHAEL FRIEDMANN as fine-mesh screens have been used in the Mediterranean Basin to protect crops (Cohen & Antignus, 1994). Recently, UV-absorbing plastic sheets and screens have been shown to inhibit penetration of whiteflies into greenhouses (Antignus et al., 1996; Antignus et al., 2001b). Furthermore, filtration of UV light was shown to hinder the whiteflies dispersal activity, and consequently reduce virus spread (Antignus et al., 2001a). However, adoption of physical barriers adds to production costs and these screens create problems of shading, overheating and poor ventilation. Therefore, the best way to reduce geminivirus damage is by breeding crops resistant or tolerant to the virus (Cohen & Antignus, 1994; Pico et al., 1996; Morales, 2001). Apart from virus transmission, whiteflies cause extensive damage to crops through excessive sap removal, excretion of honeydew that promotes growth of sooty mould fungi, and by inducing systemic disorders (Byrne & Bellows, 1991). For progress in the development of host plant resistance to whiteflies see Bellotti & Arias (2001). Breeding for resistance to begomoviruses, which is the subject of this review, presents particular challenges. This review will outline strategies that must be considered when attempting to breed for resistance. Since many of the viruses are not transmitted mechanically, in most cases protocols must be developed to utilise infective whiteflies to inoculate target plants with the virus. As a part of inoculation protocols, symptom severity scales need to be developed for each disease. Susceptible controls included in the screen should ideally become totally infected and show the highest symptom severity. Once efficient inoculation protocols have been developed, the crop can be screened to search for resistant genotypes. The starting material should consist of available cultivars and lines of the commercial species, and then progress to related species based on ease of interspecies crossability. In fact, in many cases, sources of resistance must be identified in wild species of the crops in question, and protocols must be defined to evaluate the levels of resistance observed after inoculation with the virus. The inheritance of resistance to begomoviruses has been studied for many years and several sources of resistance have been shown to be multigenic. Strategies to map the genes and develop molecular markers should facilitate the development of tools to sustain breeding programmes and allow development of resistance to multiple viruses. A prevalent problem in breeding for resistance to geminiviruses has been the lack (until recently) of consistency in viral nomenclature different viruses have the same name, names of viruses keep changing, acronyms that look like they imply strains actually signify very different viruses, etc. (Fauquet et al., 2000). The recent finding that recombination between species of geminiviruses happens relatively frequently adds to the already complex situation (Padidam et al., 1999; Fauquet et al., 2000). This has caused confusion for breeders and researchers, making communication difficult, slowing progress, and causing incorrect assumptions. Moreover, breeders may not understand the subtle but important distinctions in nomenclature, which may lead to mistakes. Recently, new guidelines for concise nomenclature of geminiviruses have been proposed, which upon implementation should clarify the confusion (Fauquet et al., 2000). It is also recommended, whenever embarking on a geminivirus resistance breeding project, to use molecular tools (such as cloning and sequencing) to accurately define the virus(es) in question. A number of begomoviruses have been the subject of extensive study due to their severe economic impact. This review considers the most severe geminiviral diseases affecting four major crops (tomato, bean, cassava and cotton). The approaches taken to breed for resistance to these viral diseases should indicate the issues involved in breeding for begomovirus resistance in crop plants. It should be noted, however, that in many regions there are commonly multiple infections of individual plants with several different viruses or even several different begomoviruses, so that breeding for resistance to a single virus may not provide a full solution to compound begomovirus infections. Definition of resistance A prevalent problem associated with breeding for viral resistance is the lack of standard terminology used by different researchers. Plant breeders do not always agree with plant pathologists in regards to the definition of resistance. Breeders are mainly interested in improving the performance of a plant variety under field conditions. Thus, yield and fruit quality are paramount. In contrast, plant pathologists place an emphasis on the fate of the virus in the plant. One of the more problematic terms is whether a plant in question is resistant to the pathogen itself, or to the effect of the pathogen symptoms of the disease. The following definitions of resistance are used in this review. A host plant is resistant if it can suppress the multiplication of a virus, and consequently suppress the development of disease symptoms. Regardless of the mechanism of resistance (the host may be resistant to establishment of infection, viral replication or viral spread within the plant), the final outcome is the same less virus accumulates in the resistant host. Resistance can range from very high (up to immunity in which case no virus accumulates in the plant rendering it, in fact, a nonhost), to moderate, or low. However, even for low resistance, the resistant plant will accumulate less Breeding for resistance to whitefly-transmitted geminiviruses virus than the susceptible host, and will express milder disease symptoms. Tolerance is a unique instance where in response to virus infection, the host expresses negligible or mild disease symptoms, but supports normal levels of virus multiplication. Thus, the plant rather than being resistant to the virus, tolerates the pathogen and despite its presence expresses mild symptoms and produces a good yield (Cooper & Jones, 1983; Walkey, 1985). Breeding for Begomovirus Resistance in Tomato (Lycopersicon esculentum L.) Several different begomoviruses, depending on the geographical areas of cultivation, affect tomatoes grown in tropical and subtropical regions. One of the most widespread and economically important viruses is Tomato yellow leaf curl virus (TYLCV), which in the Mediterranean Basin affects the crop mainly during the summer and autumn seasons. Yield losses due to the virus often reach 100% (Pico et al., 1996). A disease resembling that now known to be caused by TYLCV was first reported in 1939 in the Jordan valley, in what later became the state of Israel, and was associated with outbreaks of whiteflies (Avidov, 1946). Nearly 20 years later, farmers started large-scale cultivation of cotton in the Jordan and Bet Shean valleys, which probably supported the establishment of large whitefly populations, close to tomato fields. Indeed in 1959, concomitant with a heavy infestation of whiteflies, a disease of uncertain etiology destroyed the entire tomato crop in the area (Cohen & Harpaz, 1964). Diseased plants were sampled, and it was found that the disease was transmitted by whiteflies, hence the identification of the disease agent as a virus. At first the virus was identified (erroneously) as Tomato yellow top virus, which is transmitted by aphids. Later, it became apparent that a new virus, termed TYLCV, was the causal agent (Cohen et al., 1961; Cohen et al., 1963; Cohen & Harpaz, 1964). It took another 20 years before the geminate particle was observed by electron microscopy (Russo et al., 1980). Only in 1988 was TYLCV first isolated (Czosnek et al., 1988), and shortly after it was shown to be a monopartite geminivirus (Navot et al., 1991). Following comparisons of TYLCV isolates from distinct geographical regions, it became apparent that the name TYLCV has been given to a heterogeneous complex of begomoviruses (Moriones & NavasCastillo, 2000). Most of the isolates have a monopartite genome, as does the type member, although in one instance (Thailand isolate) a bipartite genome has been demonstrated (Rochester et al., 1994). During the last two decades, there has been a worldwide spread of the B biotype of B. tabaci accompanied by the emergence of whiteflytransmitted geminiviruses, including TYLCV 111 (Bedford et al., 1994; Polston & Anderson, 1997; Palmer & Rybicki, 1998; Moriones & NavasCastillo, 2000). TYLCV has been long known in the Middle East, North and Central Africa, Southeast Asia, and has later continued to spread to southern Europe where severe outbreaks have been reported (Czosnek & Laterrot, 1997; Nakhla & Maxwell, 1998). The virus has also been identified in the Caribbean (Polston & Anderson, 1997), Mexico (Ascencio-Ibanez et al., 1999), and in the Southeastern USA (Polston et al., 1999; Valverde et al., 2001). There have been strenuous, prolonged efforts to breed cultivars resistant to this damaging virus. An important step in the efforts to breed for TYLCVresistant cultivars has been the development of an appropriate virus inoculation and screening method to assess TYLCV resistance (Lapidot et al., 1997). In essence, 10-day-old seedlings are exposed to large numbers of viruliferous whiteflies, which upon feeding on the plants, transfer the virus practically with 100% efficiency (all susceptible controls become infected with TYLCV) (Lapidot et al., 1997). In fact, natural field exposure infection has been shown to be largely inefficient, as many plants escape infection, even under heavy inoculation pressure (Fig. 1). It was shown that only 50% of susceptible tomato plants were infected during the first month after planting them in an infected field. Moreover, despite high whitefly populations and available inoculum, 90 days after transplanting 10% of the susceptible plants still escaped infection (Vidavsky et al., 1998). Thus, it was concluded that selection of tomato plants based solely on the absence of symptoms in an infested field could be misleading (Vidavsky et al., 1998) (Fig. 1). Furthermore, natural field inoculation led to milder infection compared to artificial inoculation, probably due to late and unsynchronised infection (Pico et al., 1998). In order to search for new sources of resistance by inoculation of wild species of tomato, it was necessary to carry out individual inoculations in cages, since otherwise accessions of some wild species could escape infection as a consequence of non-preference by whiteflies (Pico et al., 1998). To analyse segregating populations for TYLCV resistance, a symptom severity rating system was developed, ranging from 0 for plants showing no visible symptoms, to 4 for plants showing very severe plant stunting, yellowing, pronounced cupping and curling of leaves, and cessation of plant growth (Fig. 2) (Friedmann et al., 1998). However, the most relevant evaluation of resistance is the effect of infection on total yield and yield components, as compared to equivalent non-infected plants (Lapidot et al., 1997). Usually, tests comparing different varieties are carried out under field inoculation, and no comparison is made to the full yield potential of 112 MOSHE LAPIDOT & MICHAEL FRIEDMANN Fig. 1. Field inoculation of susceptible tomato plants with Tomato yellow leaf curl virus (TYLCV). Tomato plants susceptible to TYLCV (cv. 5656) were transplanted to the field in September 2001, in an area abundant with whiteflies. The picture was taken 80 days following transplanting. I = Infected; H = Healthy. Fig. 2. Tomato yellow leaf curl virus (TYLCV) symptom severity rating in segregating F2 populations. Young (10day-old) tomato seedlings from an F2 population segregating for resistance were inoculated with TYLCV using viruliferous whiteflies. Symptom development was evaluated 30 days after inoculation according to the following scale: (0) no visible symptoms, inoculated plants show the same growth and development as non-inoculated plants; (1) very slight yellowing of leaflet margins on apical leaf; (2) some yellowing and minor curling of leaflet ends; (3) a wide range of leaf yellowing, curling and cupping, with some reduction in size, yet plants continue to develop; (4) very severe plant stunting and yellowing, and pronounced cupping and curling; plant growth stops. Breeding for resistance to whitefly-transmitted geminiviruses uninfected plants; information which is necessary for determining the effect of TYLCV infection on the yield. Nevertheless, even such imperfect tests can only be carried out on the most promising new varieties, as they are lengthy and costly. One of the major obstacles in the development of TYLCV-resistance is the assessment of the resistance level displayed by the plant. Plant age at the time of infection, inoculation pressure and growth conditions all have a major effect on the severity of symptoms induced by the virus (Pico et al., 1998; Lapidot et al., 2000). Thus, to facilitate the assessment of the level of resistance to TYLCV in resistant tomato plants, a panel of differential hosts has been developed to allow comparative scoring (Lapidot et al., 2001). The panel comprises seven homozygous tomato genotypes that exhibit different levels of TYLCV resistance, ranging from fully susceptible to highly resistant, based on symptom severity and virus content. It has been shown that when these plants are inoculated with TYLCV under different environmental conditions, although the score of each individual resistant genotype changes, its ranking on the scale does not. Thus, to evaluate disease resistance of a given (test) tomato genotype, the test genotype is inoculated alongside the panel of differential hosts, and within four weeks its level of resistance can be determined relative to its position on the resistance scale (Lapidot et al., 2001). Since all cultivars of tomato (Lycopersicon esculentum) are extremely susceptible to TYLCV, wild Lycopersicon species have been screened for their response to the virus (reviewed in Laterrot, 1992; Pico et al., 1996, 1999; Pilowsky & Cohen, 2000). Thus, breeding programmes have been based on the transfer of resistance genes from accessions of wild origin into the cultivated tomato. Progress in breeding for TYLCV resistance has been slow, primarily because of the complex genetics of the resistance, and the need to set up a reliable screen for resistance to the virus, which is dependent on the availability of viruliferous whiteflies (see above) (Lapidot et al., 1997; Vidavsky et al., 1998). The first commercial resistant cultivar, TY20, carrying resistance derived from L. peruvianum, showed delay in both symptoms and accumulation of viral DNA (Pilowsky & Cohen, 1990; Rom et al., 1993). Thereafter, advanced breeding lines with high levels of resistance from L. peruvianum (Lapidot et al., 1997; Friedmann et al., 1998), L. chilense (Zamir et al., 1994; Scott et al., 1996) L. pimpinellifolium and L. peruvianum (Vidavsky et al., 1998) and L. hirsutum (Vidavsky & Czosnek, 1998; Hanson et al., 2000) have been developed by different breeding teams and are being used extensively to breed high quality F1 hybrids. In addition, a number of resistant F 1 hybrids have been released for commercial production by several seed companies (Pico et al., 1998). The tomato line H24 has shown excellent resistance to strains of TYLCV from Taiwan and 113 south India (Kallo & Banerjee, 1990; Hanson et al., 2000). The genetics of resistance to TYLCV has been studied for a number of the resistant breeding lines mentioned above. In most cases, the sources of resistance to TYLCV appear to be controlled by multiple genes (reviewed in Pico et al., 1996, 1999). Line TY172, which exhibited the highest level of resistance in a field trial in which yield components were evaluated for various resistant cultivars and lines (Lapidot et al., 1997; Friedmann et al., 1998), is derived from L. peruvianum (Friedmann et al., 1998). It is a symptomless carrier of TYLCV, in which the virus titer is very low. Attempts to produce disease symptoms on TY172 plants by grafting with a susceptible infected donor were unsuccessful. Even when exposed continuously to very high levels of virus, line TY172 did not develop disease symptoms (Friedmann et al., 1998). The resistance in line TY172 is controlled by at least three interacting genes (Friedmann et al., 1998). The highly resistant line 902 is derived from a cross between two L. hirsutum accessions resistant to TYLCV, followed by crossing to L. esculentum and selfing resistant symptomless individuals (Vidavsky & Czosnek, 1998). In addition, an advanced breeding line (F 8 generation) derived from L. pimpenellifolium hirsute and a line derived from L. chilense that underwent four backcrosses to L. esculentum exhibit strong resistance as evidenced by symptomless scores (Vidavsky et al., 1998). The resistance in L. hirsutum appears to be controlled by two to three additive recessive genes (Vidavsky & Czosnek, 1998) and that of L. pimpinellifolium by one major gene (Vidavsky et al., 1998). The L. chilense LA1969 source is controlled by a major gene termed TY-1 and at least two more modifier genes (Zamir et al., 1994) and a number of commercial hybrids have been released carrying this resistance. The hirsutum source used in the AVRDC (Asian Vegetable Research and Development Center) breeding programme is controlled by two genes acting epistatically (Hanson et al., 2000). Wild tomato species have also been screened to identify sources of resistance to other begomoviruses affecting tomato worldwide. Several wild species accessions were shown to have varying degrees of resistance to a Venezuelan strain of Tomato yellow mosaic virus (ToYMV) which causes severe losses to tomato crops in Venezuela (Piven et al., 1995). This virus is transmitted both mechanically and by whiteflies. Interestingly, different accessions of wild species showed varying resistance to the virus, depending on the inoculation method utilised (Piven et al., 1995). The L. chilense accession LA1969, which is a good source of resistance to TYLCV (Zakay et al., 1991), as well as to the Taiwan strain of TYLCV (Chiang et al., 1984) was found to be highly resistant to ToYMV (Piven et al., 1995). However, it developed symptoms when inoculated 114 MOSHE LAPIDOT & MICHAEL FRIEDMANN mechanically, suggesting either avoidance of transmission, or inhibition of initial entry of the virus into plant cells as a mechanism of resistance (Piven et al., 1995). Breeding lines (F5 generation) derived from a cross with L. peruvianum were reported as having nonspecific immunity to several pathogens, including Tomato yellow top virus (TYTV) and Beet curly top virus (BCTV) (Thomas & Mink, 1998). Tomato mottle virus (ToMoV) is an important geminivirus affecting tomato in Florida (Polston et al., 1993). There is an ongoing programme at the University of Florida to introgress ToMoV resistance from accessions of L. chilense, a species difficult to use for gene transfer due to interspecific crossability barriers. In segregating populations from crosses to tomato, there was a high number of susceptible progeny, suggesting a multigenic control of resistance for all the L. chilense accessions studied (Scott et al., 1996). Several of the lines also had effective TYLCV resistance. Recently, a number of TYLCV-resistant tomato lines exhibited resistance to unrelated begomoviruses in a field trial in Guatemala (Mejia et al., 2001). Preliminary results showed the lines to have decreased symptoms and higher yields than a susceptible control. The resistant lines showed differential levels of infection with three begomoviruses known to be present in the area of the trial, Tomato severe leaf curl virus, Tomato golden mottle virus and Pepper golden mosaic virus (Mejia et al., 2001). Currently, inoculation protocols exist for TYLCV, which together with highly resistant breeding lines derived from different wild species, are already resulting in the commercial release of resistant cultivars suitable for production in regions where TYLCV is a major constraint. There is the additional issue of adaptability of a given resistant variety to the growing conditions of a particular region it is likely that local breeding programmes will need to incorporate the resistance from the cultivars being released into locally adapted cultivars. Such breeding programmes would benefit greatly from molecular markers linked to the resistance genes. In addition, molecular markers to the different sources of resistance will be required in order to allow the pyramiding of these genes. Further work is needed to ascertain whether the TYLCV-resistant cultivars will be resistant to other economically important begomoviruses. Breeding for Begomovirus Resistance in Common Bean (Phaseolus vulgaris) The common bean (Phaseolus vulgaris) is a major crop that is severely affected by begomoviruses. Bean golden mosaic has become the most serious viral disease of common bean throughout Latin America and the Caribbean, and has caused severe damage to snap bean production in Florida (Blair et al., 1995; Bianchini, 1999; Singh et al., 2000b; Morales, 2001; Morales & Anderson, 2001). Disease symptoms include plant dwarfing and malformation, leaf chlorosis, abortion of flowers and pods, pod malformation, and reduction of seed yield and quality (Morales & Niessen, 1988; Bianchini, 1999). The causal agent of bean golden mosaic is Bean golden mosaic virus (BGMV). Studies of BGMV from different geographical regions revealed a number of strains of the virus, which form two groups, BGMV type I and II (Faria & Maxwell, 1999; Garrido-Ramirez et al., 2000; Morales & Anderson, 2001). Type I BGMV isolates are found in South America and are not sap-transmissible, whereas type II isolates are found in Central America, the Caribbean Basin and Florida, and are sap-transmissible. The nomenclature of BGMV was revised recently, and type II BGMVs were renamed Bean golden yellow mosaic virus (BGYMV) (Fauquet et al., 2000). Bean is also attacked by TYLCV, which has led to increased infection of both bean and tomato crops in Spain, in regions where they are inter-cropped (Navas-Castillo et al., 1999; Sanchez-Campos et al., 1999). Resistance to TYLCV has been observed in commercial varieties, but the inheritance has not yet been evaluated (Lapidot, 2002). A protocol for inoculation of bean with BGYMV by viruliferous whiteflies has been described, resulting in 100% infection of susceptible parental lines and uniform timing of symptom expression (Velez et al., 1998). Likewise, protocols for mechanical inoculation have been developed (Morales & Niessen, 1988). In analysing segregating material for resistance to BGYMV, several symptom severity ratings have been described (Morales & Singh, 1993; Bianchini, 1999). A strong effect of plant age on the incidence of infection was found when bean plants were inoculated mechanically with BGYMV. Infection rates dropped from 100% infection in 7-day-old plants, to 0% infection in 12day-old plants (Morales & Niessen, 1988). Similarly, the success rate of TYLCV infection by whiteflies was highly dependent on bean plant age. When 14-day-old susceptible bean plants were inoculated, nearly total infection was achieved but, when 12- or 26-day-old plants of the same variety were inoculated, the infection rates were only 40% and 34%, respectively (Lapidot, 2002). Thus the same phenomenon occurred in both studies a distinct dependence of infection success rates on bean plant age. There have been extensive programmes to search for sources of resistance and develop bean cultivars highly resistant to BGYMV. Several sources of resistance have been identified and utilised to breed Breeding for resistance to whitefly-transmitted geminiviruses resistant varieties. There are two major groups of common bean germplasm, the Middle American and the Andean groups, which apparently were domesticated separately, from different wild bean populations. Each group is divided into three races. For the Middle American group, Durango, Jalisco and Mesoamerica races are characterised by small and medium seeded black, small red, pink, white and pinto types. For the Andean group, the Chile, Nueva Granada and Peru races are characterised by medium and large seeded kidney bean, red mottled and snap bean types. Members of the group can be intercrossed, yet genetic barriers exist in some cases, necessitating the use of bridging lines (Morales & Singh, 1991). The Mesoamerica race includes the landraces Porrillo Sintetico and Turrialba 1 which are resistant to infection by BGYMV, showing delayed chlorosis. These have been used as the source of resistance for many of the cultivars that have been released (Bianchini, 1999; Singh et al., 2000a). However, under severe disease pressure, the resistance breaks down, leading to leaf chlorosis and yield losses (Singh et al., 2000b). The Durango race also contains landraces with resistance to BGYMV, such as Garrapato which possesses the gene bgm (or bgm1), which confers partial resistance, and differs from the resistance derived from Porrillo Sintetico (Urrea et al., 1996; Singh et al., 2000b). The combination of both sources of resistance led to the development of the highly resistant breeding line A429 (Velez et al., 1998; Singh et al., 2000c). This line expresses very attenuated symptoms upon infection, but lacks the desirable horticultural characteristics required for release commercially (Velez et al., 1998). The Nueva Granada race also possesses resistance to BGYMV, such as the line Royal Red, carrying the resistance gene bgm2. This line resists pod deformation caused by BGYMV, but is susceptible to leaf chlorosis (Morales & Niessen, 1988). These two recessive genes, bgm and bgm2 are non-allelic (Velez et al., 1998). The genetics of resistance to BGYMV was studied in a 8 ´ 8 complete diallel cross (Morales & Singh, 1991). The parents and hybrids were inoculated mechanically with BGYMV under greenhouse conditions, and different symptoms were scored on a scale of 1 (symptomless) to 9 (most severe symptoms). Resistances from the classic sources Porrillo Sintetico and Royal Red were shown to be largely additive (Morales & Singh, 1991). In another study using recombinant inbred lines derived from a cross between a genotype belonging to the Mesoamerica strain and a genotype of the Durango strain, several lines showed transgressive values for resistance or susceptibility as compared to the parent lines. This indicated that the genes controlling 115 BGYMV resistance in the parent lines were different and complementary (Morales & Singh, 1993). A recombinant inbred population derived from the cross of Dorado (resistant) and XAN176 (susceptible) was used together with random amplified polymorphic DNA (RAPD) markers to identify two quantitative trait loci (QTLs) linked to resistance to BGYMV (Miklas et al., 1996). The two QTLs had a cumulative additive effect that explained 60% of the phenotypic variation for resistance. As mentioned already, although resistant cultivars have been developed from single sources of resistance, they are not adequate under heavy inoculum pressure. Recent studies have shown that by pyramiding resistance genes derived from different races in P. vulgaris, highly resistant lines may be developed (Urrea et al., 1996; Velez et al., 1998; Singh et al., 2000a,b). For instance, the highly resistant line DOR 303 resulted from a combination of the Mesoamerica strain Porrillo Sintetico with the New Granada line Royal Red, (Velez et al., 1998). Other promising breeding lines were developed, such as line 9236-6, whose resistance was derived from the previously developed line A429, which showed resistance to chlorosis due to the single recessive gene bgm. Likewise, line 9245-94, whose resistance was derived from DOR303, was shown to carry the recessive gene controlling resistance to chlorosis, bgm2 (Velez et al., 1998). In addition, sources of resistance are found in the secondary gene pool of common bean (P. constaricensis, P. coccineus, P. polyanthus). Highly resistant lines have been developed from interspecific crosses between P. vulgaris and P. coccineus showing mild and delayed symptom expression and higher yields upon BGYMV infection than the susceptible controls, even under increased virus incidence (Bianchini, 1999; Singh et al., 2000b). The identification of different sources of resistance to BGYMV, together with the development of molecular markers to some of the sources, should facilitate the release of highly resistant cultivars based on the combination of resistance genes. Breeding for Resistance to Begomoviruses in Cassava (Manihot esculenta) Cassava (Manihot esculenta) is a staple food in many parts of the developing world, and especially in Sub-Saharan Africa where it is severely affected by begomoviruses. This crop presents a challenge for breeding for begomovirus resistance, due to its perennial habit and propagation by vegetative cuttings rather than by seeds. Consequently, the incidence of the disease increases during successive cycles of cultivation, and new crops are affected if diseased cuttings are used for propagation. 116 MOSHE LAPIDOT & MICHAEL FRIEDMANN Therefore, this has a direct bearing on the breeders evaluation of the level of resistance that is apparent in a given field. Cassava mosaic disease (CMD) is wide spread throughout Sub-Saharan Africa, South India and Sri Lanka and is the most important factor limiting cassava yields in many locations (Fauquet & Fargette, 1990; Fargette et al., 1996; Thresh et al., 1998b; Legg, 1999; Legg & Thresh, 2000). The disease is caused by a number of begomoviruses, occurring singly or together (Thresh et al., 1998c; Fondong et al., 2000b; Berry & Rey, 2001; Pita et al., 2001a). Currently, four distinct begomoviruses have been found associated with CMD in Africa: African cassava mosaic virus (ACMV), East African cassava mosaic virus (EACMV) (Swanson & Harrison, 1994), an ACMV-EACMV recombinant virus which has been referred to as either Ugandan variant virus (UgV) or as EACMV-Ug (Deng et al., 1997; Zhou et al., 1997) and South African cassava mosaic virus (SACMV) (Berrie et al., 1998). For a recent review on the diversity of African cassava begomoviruses see Pita et al. (2001a). Cassava yields in Africa are much lower than their full yield potential (5-10 tons ha-1 compared with 80 tons ha-1), mainly due to insect pests and diseases and the use of infertile soils (Fauquet & Fargette, 1990). In fact, CMD can cause yield losses of up to 95%, (Legg, 1999), and annual losses have been estimated at 30% (Fargette et al., 1996). The incidence of the disease coincides with the whitefly populations and with the rainfall cycle of the year (Legg & Thresh, 2000). Since cuttings used routinely to propagate the crop can also transmit CMD, sanitation and selection of healthy cuttings are an important component of control of the disease (Thresh & Otim-Nape, 1994; Gibson & Otim-Nape, 1997; Thresh et al., 1998a; Fondong et al., 2000a). The symptoms of CMD are affected by temperature, being suppressed above 35°C (Hahn et al., 1980; Fondong et al., 2000a). This has a direct bearing on screening for resistance. Consequently, field screening is carried out in areas with high inoculum pressure and where the average temperature is relatively low (below 30°C). A symptom severity rating ranging from 0 (no symptoms) to 5 (severe mosaic and distortion of the entire leaf), has been defined and used in several breeding programmes including those of IITA (International Institute of Tropical Agriculture, Ibadan, Nigeria), where there has been a longstanding programme to breed CMD resistant cassava cultivars (Hahn et al., 1980, 1989; Fargette et al., 1996). The evaluation of resistance to CMD is complicated by the fact that cuttings taken from infected plants may grow into symptomless plants, this being more prevalent in resistant cultivars (Gibson & Otim-Nape, 1997; Thresh et al., 1998b). This is a result of the virus not becoming fully systemic in highly resistant cultivars, and where the virus titer is also low. This leads to a slower spread of the virus, and thus, some cuttings, although taken from infected plants, may grow into healthy plants, a phenomenon termed reversion. Therefore, reversion is important in reducing the incidence of infection in the cassava stocks used for propagation (Gibson & Otim-Nape, 1997; Fondong et al., 2000a). Moreover, symptomless cuttings may arise as a result of exposure to high temperatures. Likewise, selection of cuttings from healthy rather than infected plants for propagation also affects the spread of the virus. Thus, in addition to the primary infection due to whitefly inoculation, there is also a secondary spread due to propagation from cuttings. This affects the incidence of the disease when monitored over a period of several years. Mathematical simulations to assess the interactions of the different parameters affecting CMD epidemics have been developed (Fargette & Vie, 1995), and have shown that host resistance, in combination with reversion and cutting selection, can potentially reduce the incidence of disease significantly. Resistant cultivars therefore not only suffer less yield loss than susceptible plants, but also are less likely to become heavily infected, even after many years of successive cropping, as long as their use is combined with selection of healthy cuttings and expression of reversion. No adequate level of resistance to CMD was found in the cultivated cassava species Manihot esculenta. Therefore, resistance was introgressed from other related Manihot species by interspecific crosses (Hahn et al., 1980), including the ceara rubber M. glaziovii (Fargette et al., 1996; Fregene et al., 2000). The generation of resistant germplasm dates back to the 1930s in Eastern Africa and Madagascar (Jennings, 1994; Cours et al., 1997; Fregene et al., 2000; Morales, 2001). The material generated by Storey in the 1930s (Storey, 1936) has been a major source of resistance for breeding programmes. An epidemic in Madagascar in the 1930s almost eliminated the cultivation of cassava and led to the generation and deployment of resistant varieties generated from inter-specific crosses (Cours et al., 1997). This, in combination with utilising CMDfree cuttings for propagation and roguing of diseased plants, has helped to maintain a low incidence of CMD. However, the varieties developed in Madagascar showed a lower level of resistance under high inoculum pressure in the Ivory Coast (Cours et al., 1997). Since cassava originated in Latin America, there are several related species there with potential for resistance to the virus (Fargette et al., 1996). Backcrosses to the cultivated cassava are needed, in order to increase the yield potential, both of the tuberous roots (major carbohydrate source) and of the leaves (used as a vegetable) (Jennings, Breeding for resistance to whitefly-transmitted geminiviruses 1994). In fact, a number of inter-specific hybrids have been developed which show good resistance and have been used in breeding programmes (Hahn et al., 1989; Fargette et al., 1996). Resistance from the interspecific cross in cassava has been reported to be recessive, polygenic and additive with a heritability of about 60% (Hahn et al., 1980, 1989). Lines with improved resistance have been developed both at IITA and at CIAT (Centro Internacional de Agricultura Tropical, Cali, Colombia), these retain the desirable horticultural traits and quality of the cassava products. Studies are beginning to emerge comparing the resistance of newly released CMDresistant cassava lines. The components of resistance to CMD were compared over a number of years for several cassava cultivars (Fargette et al., 1996). Significant correlations were found between symptom intensity, disease incidence and virus titre. Interestingly, a close relationship between disease incidence and yield reduction was not found when the yield was compared between infected and healthy plants of five selected cultivars (Fargette et al., 1996). This has a direct bearing on the efficacy of breeding programmes based on screening material according to disease symptoms and incidence. Therefore, the authors suggested that yield assessments should be included early in breeding programmes, in order to complement screens for symptom intensity, which are highly variable. However, it seems that a larger sample of cultivars should be assayed for yield loss under infection in order to test this lack of correlation. Nevertheless, the authors concluded that the use of highly resistant germplasm showing a high field resistance, combined with the selection of healthy cuttings for propagation, should limit the impact of CMD to a large extent (Fargette et al., 1996). A number of cultivars were assessed for resistance to CMD in multi-location trials in Uganda, where there is a severe ongoing epidemic (Otim-Nape et al., 1998). This is in part due to the intercropping of resistant and susceptible varieties, exposing the former to high inoculum pressures (Thresh et al., 1998b; Morales, 2001). There were considerable differences in the rate of spread of the disease among different sites in the country as well as large differences in the extent of field infection of susceptible genotypes. Nevertheless, a number of improved genotypes showed considerable resistance to infection and have been released for use in Uganda (Otim-Nape et al., 1998). Recently, several genotypes from the IITA germplasm collection were assessed for resistance to CMD and other major diseases affecting cassava in Africa, under natural infestation conditions (Fokunang et al., 2000). CMD incidence and severity reached a peak 3 months after infection, with the severity being high in 80% of the genotypes. In addition, the severity of the disease 117 was negatively correlated with tuberous root number and weight. Breeding for begomovirus resistance in cassava requires the standardisation of inoculation protocols that will address the phenomenon of reversion, so as to assess properly the resistance level of the material being screened. The development of molecular markers linked to resistance genes would be extremely valuable, as it would allow screening of symptomless propagation material to ascertain it comes from a resistant source. Breeding for Resistance to Begomoviruses in Cotton (Gossypum hirsutum L.) The two most prominent whitefly-transmitted diseases of cotton are cotton leaf crumple disease, and cotton leaf curl disease. Since high incidences of both diseases are associated with high whitefly populations, begomoviruses have been a major candidate as the causal agents of the disease. Indeed, Cotton leaf crumple virus (CLCrV), a bipartite begomovirus (Nadeem et al., 1997), was found to be associated with the crumple disease (Brown & Nelson, 1984; Briddon & Markham, 2000). Cotton crumple disease mainly affects cotton in the southern USA, and causes relatively mild symptoms since it usually appears late in the cotton growing season. This may explain why breeding cotton for resistance to CLCrV is progressing rather slowly. It has been shown that the resistance of Cedix, a highly resistant cotton cultivar to CLCrV from El Salvador, is controlled by two dominant and supplementary genes, which must occur together in order to confer full resistance (Wilson & Brown, 1991). Resistant lines, however, appear to have relatively low yields and this has complicated the development of elite, resistant cultivars. Recently, more efforts were made to find resistance in upland cotton to CLCrV (Natwick et al., 2000). In this study, the researchers developed a rating system for disease symptom severity, molecular tools for disease evaluation, and most importantly, confirmed that cotton leaf crumple disease is indeed induced by CLCrV. However, due to a lack of an alternative inoculation system, Natwick et al. (2000) have had to rely on field infection, which hinders progress. In the last decade cotton leaf curl disease (CLCuD) has become the major limiting factor of cotton production in Pakistan (Briddon & Markham, 2000). Recently, the disease spread to India (Briddon & Markham, 2000). Due to the severity of the disease, and the widespread presence of whitefly populations, the 1993 Pakistan cotton crop was declared a disaster (Brown, 1996). Since whiteflies transmit the disease, and the symptoms were indicative of geminivirus infection, a begomovirus was sought as the causal agent of the disease. Indeed, in 1993 a monopartite 118 MOSHE LAPIDOT & MICHAEL FRIEDMANN begomovirus was found in CLCuD-infected cotton plants, and named Cotton leaf curl virus (CLCuV) (Mansoor et al., 1993). However, full-length infectious clones of CLCuV were unable to induce the characteristic symptoms of CLCuD in cotton. Thus, other factors involved in inducing CLCuD were sought. In 1999, Mansoor and co-workers (Mansoor et al., 1999) found a smaller ssDNA molecule (termed DNA 1) that was associated with CLCuD. DNA 1 could self-replicate in plant cells but required CLCuV for spread in plants and for insect transmission. However, it was found that DNA 1 plays no part in inducing disease symptoms. Only recently, an additional small (c. 1350 nucleotides) ssDNA molecule (termed DNA b) was isolated from CLCuD-infected cotton plants (Briddon et al., 2001). DNA b requires CLCuV for replication and encapsidation, and when coinoculated with CLCuV to cotton plants, induces symptoms typical of CLCuD. Thus, the begomovirus (CLCuV) DNA b complex represents the infectious unit responsible for CLCuD (Briddon et al., 2001). Although resistant cotton cultivars are required, and despite considerable efforts, progress in breeding cotton for resistance to CLCuD has been slow, mainly since breeders have had to rely on field inoculation, and until very recently were unable to identify the disease-inducing agent. Nevertheless, resistant cotton cultivars have been developed in Pakistan, and some show high resistance and/or even seem to be immune. However, these cultivars have narrow adaptability to different growth conditions, and yield less than uninfected susceptible cultivars (Briddon et al., 2000; Rahman et al., 2001). The authors suggest that the highly resistant cultivars could be grown together with better-adapted tolerant cultivars, and thus diminish the spread of the disease while reaching acceptable yields. It was also found that the Pakistan native cotton (Desi, Gossypium arboreum) is resistant to the virus, but the majority of cotton cultivated in Pakistan is Gossypium hirsutum (Briddon et al., 2000; Rahman et al., 2001). This may indicate that the disease is not new to the Pakistan area, and that the wide use of susceptible hirsutum cultivars contributed to the current epidemics. Support of this suggestion comes from the finding that the most popular cotton varieties used in the last decade in Pakistan are highly susceptible to the disease (Briddon et al., 2000). In breeding for resistance, a strong environmental interaction with genotypes has been shown to confound the results. The level of resistance exhibited by different lines was affected by the year of the trials, perhaps as a result of differences in the incidence of the virus present in each year (Rahman et al., 2001). In order to select for resistant genotypes it would therefore be advisable to develop protocols of artificial inoculation using whiteflies carrying all the components of CLCuV. These studies should be done under strong inoculation pressure and consistent environmental conditions. Development of Molecular Markers Linked to Resistance to Begomoviruses It would greatly facilitate the development of resistant germplasm if molecular markers, based on polymorphism of DNA sequences, linked to resistance genes were identified to aid in the selection process during the breeding efforts. Without molecular markers, breeding for viral resistance is limited to a phenotypic screen of the resistant plants, i.e., selecting plants solely on the basis of the presence or absence of symptoms or on symptom severity. Since the begomoviruses are transmitted by whiteflies, the inoculations and screening are limited to the warmer seasons of the year. Moreover, the use of field inoculation, even in an area of very high activity of viruliferous whiteflies, is inefficient and can be misleading (Vidavsky et al., 1998) (Fig. 1). Thus, the use of molecular markers should enhance the efficiency and reliability of methods for screening for resistance, and should reduce the costs associated with classical breeding for resistance (Kelly, 1995; Chague et al., 1997). Identifying molecular markers linked to each source of resistance will enable the study and understanding of the genetic components that influence viral resistance. The mapping of resistance genes from different sources will indicate which genes may be common to different sources, and which may be distinct. This will be of great value in determining how to combine different sources of resistance in breeding programmes. Lander and Botstein (1989) suggested that by using molecular markers strategically positioned throughout the genome, QTLs could be identified, mapped, and analysed. Using L. chilense as the resistance donor, Zamir et al. (1994) mapped the TYLCV-resistance gene, TY-1, to chromosome 6. Two more resistance modifier genes were mapped to chromosomes 3 and 7 (Zamir et al., 1994). Another TYLCV-resistance gene, originating from L. pimpinellifolium and accounting for 27.7% of the resistance, has been mapped by using RAPD PCRbased markers to chromosome 6, but to a locus different from TY-1 (Chague et al., 1997). Recently, Tomato leaf curl Taiwan virus (ToLCTWV, termed by the authors as TYLCV Taiwan isolate) resistance originating from the wild tomato species L. hirsutum was mapped to chromosomes 8 and 11 (Hanson et al., 2000). By marker-assisted selection, the introgression on chromosome 11 was shown to confer resistance to both ToLCTWV and Tomato leaf Breeding for resistance to whitefly-transmitted geminiviruses curl Bangalore virus (ToLCBV; Hanson et al., 2000). It is likely that combining genes from different sources of resistance can lead to superior levels of resistance to begomoviruses. For instance, Pilowsky et al. (1997) developed the highly TYLCV- resistant TY172 and TY197 lines by combining lines that expressed moderate levels of resistance to TYLCV. Thus, combining resistances derived from different wild tomato species, such as L. hirsutum, L. chilense, L. peruvianum and L. pimpinellifolium, could result in breeding lines expressing high and stable levels of resistance to TYLCV (Vidavsky et al., 1998). Molecular markers linked to each source of resistance would greatly simplify the process of combining different genes into breeding material. A codominant RAPD marker tightly linked to the bgm (or bgm1) gene from the common bean Garrapato resistant source was developed by applying bulked segregant analysis (Urrea et al., 1996). The marker R2 570/530 was then identified in lines likely to contain the bgm gene. The marker was also used to confirm that the resistant source DOR303 contains a resistance gene distinct from the bgm gene (Velez et al., 1998). Recently, highly resistant lines that combine different sources of resistance to BGYMV have been released and shown to contain both the R2 570/530 marker as well as a sequence characterised amplified region (SCAR) marker SW12 700 linked to a gene derived from the Porrillo Sintetico resistance (Singh et al., 2000b). Therefore, incorporation of molecular markers linked to different resistance genes allows for the pyramiding of resistances into breeding lines and cultivars, which greatly enhances the resistance to the virus. Markers enable breeders to identify which resistances have been bred into highly resistant lines, whose main source of resistance would otherwise remain unknown (Bianchini, 1999). A concerted effort is being made at CIAT and other institutes to establish a molecular genetics platform for cassava, which should facilitate and lead to the generation of molecular markers linked to CMD resistance (Fregene et al., 2001). This platform includes the generation of a molecular genetic map of cassava constructed with molecular markers such as restriction fragment length polymorphism (RFLP), RAPDs, microsatellites, and isozymes, initially estimated to cover approximately 60% of the cassava genome (Fregene et al., 1997). Since the generation of the map, markers have been continuously added to it, especially simple sequence repeat (SSRs) and expressed sequence tags (ESTs) (Fregene et al., 2001). Amplified fragment length polymorphism (AFLP) markers were used to screen CMD-resistant and susceptible cassava accessions and the resulting band patterns allowed the clustering of the accessions into groups based on geographical location and/or CMD resistance (Fregene et al., 119 2000). Although a particular AFLP marker linked to CMD resistance was not reported, the molecular genetic map being generated should lead to the development of molecular markers linked to CMD resistance from different accessions. Pathogen-Derived Resistance of Begomoviruses The breakthrough in recombinant DNA technology and the establishment of transformation and regeneration systems for plants has enabled the use of a genetic engineering approach for breeding. The concept of pathogen-derived resistance (PDR) was proposed by Sanford & Johnston (1985), who suggested engineering resistance by transforming a susceptible plant with gene sequences derived from the pathogen itself. It was suggested that the expression of certain key gene products of a pathogen by the plant either in an inappropriate form or amount, or at an inappropriate time during the infection cycle could disturb infection by the invading pathogen (Sanford & Johnson, 1985). The first manifestation of PDR was provided by the demonstration that transgenic tobacco plants expressing the tobacco mosaic virus (TMV) capsid protein (CP) were resistant to infection with TMV (Powell Able et al., 1986). Subsequently, there have been several successful attempts to generate virus resistance in transgenic plants based on this concept through the expression of virus-derived genes or genome fragments (Lomonossoff, 1995; Baulcombe, 1996). A number of PDR strategies have been investigated for the control of begomoviruses (Frischmuth & Stanley, 1993). These can be arranged into five categories: a) capsid protein-mediated resistance, b) movement protein-mediated resistance, c) defective interfering (DI) viral DNA, d) genes in antisense orientation, e) truncated or mutated replicase (Rep) (C1 or AC1) gene. The first two approaches involve expression of viral capsid or movement proteins in order to inhibit viral proliferation. The last three categories, despite differences in transgene construct used, all aim to inhibit viral replication by disrupting the activity of the Rep gene. This is not surprising, since the Rep gene encodes the only viral protein that is indispensable for geminiviral DNA replication (Brough et al., 1988; Elmer et al., 1988; Etessami et al., 1991). Capsid protein-mediated resistance Capsid protein-mediated resistance (CPMR) has been used extensively and successfully to induce resistance to many RNA viruses (Fitchen & Beachy, 1993). The first attempt to use CPMR for begomoviruses was by Kunik et al. (1994). Primary (R0) transgenic plants of an interspecific tomato 120 MOSHE LAPIDOT & MICHAEL FRIEDMANN hybrid transformed with the TYLCV CP gene (V1) showed a delay in symptom development and a recovery from viral infection with time. The resistance was manifested only by expression of the transgenic protein, since transgenic plants expressing the CP gene only at the RNA level showed no resistance to viral infection (Kunik et al., 1994). Sinisterra et al. (1999) transformed tobacco plants with a modified CP gene (30 nucleotides were deleted from the 5 end) of ToMoV. Inoculated plants varied in response from susceptibility to immunity. The transformed plants expressed the transgene transcript, but the transgene product could not be detected. However, there was a positive correlation between the presence of the transgene and resistance, although the correlation was not 100%. Moreover, in some cases the presence of the transgene gave rise to infected plants that expressed unusual symptoms. The authors concluded that the resistance could be due to a RNA-mediated mechanism (Sinisterra et al., 1999). The above reports are the only ones demonstrating successful resistance mediated by begomovirus CP. Azzam et al. (1996) have expressed the CP of BGYMV in transgenic beans. However, the transformed plants were susceptible to the virus and exhibited severe symptoms similar to those of nontransgenic plants. The transformed plants expressed the transgene transcript but not the viral CP, which may account for the lack of resistance. However, transgenic Nicotiana benthamiana plants that expressed the CP of ACMV also failed to express any level of resistance (Azzam et al., 1996; Frischmuth & Stanley, 1998). With TMV (an RNA tobamovirus), it has been demonstrated that CPMR affects early stages of viral penetration into cells, probably at the stage of viral disassembly (Fitchen & Beachy, 1993). While TMV is transmitted mechanically to leaf mesophyl cells, begomoviruses are transmitted by whiteflies into the phloem. Moreover, while TMV and other RNA viruses replicate in the cytoplasm of infected cells, begomoviruses replicate in the cell nucleus. These differences may explain why CPMR has not proven successful for begomoviruses. Movement protein-mediated resistance The expression of non-functional movement proteins (MP) in transgenic plants has been tested successfully as a PDR strategy for plant RNA viruses (Lapidot et al., 1993; Malyshenko et al., 1993; Beck et al., 1994). In general, the transgenic plants showed mediocre levels of resistance, which was expressed mainly as delayed and attenuated symptoms of infection, against the virus from which the transgene was derived. However, the resistance was mediated against a wide range of different viruses (i.e., widespectrum resistance), unlike CPMR (Cooper et al., 1995). Two movement proteins, BC1 and BV1, both encoded by DNA B of bipartite begomoviruses, are required for viral infectivity and systemic infection (Sanderfoot & Lazarowitz, 1996). The BC1 MP mediates viral cell-to-cell movement by increasing plasmodesmatal size exclusion limits, whereas the BV1 MP functions as a nuclear shuttle (Noueiry et al., 1994; Sanderfoot & Lazarowitz, 1996). The first suggestion that expression of a non-functional BC1 MP by transgenic plants may induce inhibition of begomovirus movement came from the work of von Arnim & Stanley (1992), who tested if BC1 MP from one virus (Tomato golden mosaic virus; TGMV) could support movement of another virus (ACMV). To do this, the ACMV AV1 gene (which encodes the viral CP) was replaced by TGMV BC1 gene. This recombinant construct was used to inoculate N. benthamiana plants, together with ACMV B DNA. The TGMV BC1 MP not only did not supplement ACMV movement, it actually inhibited it (von Arnim & Stanley, 1992). Indeed, transgenic tobacco plants expressing a mutated form of the BC1 gene of ToMoV showed resistance to this virus as well as to Cabbage leaf curl virus (CabLCV) (Duan et al., 1997b). The tobacco plants were transformed with the wild type form of BC1, which resulted in transgenic plants expressing viral disease-like phenotypes. One of the transgenic plant lines had an asymptomatic phenotype. In this line, the BC1 transgene underwent a spontaneous mutation, potentially resulting in the production of a protein from which the 119 C-terminal amino acids were deleted and 26 amino acids derived from an unidentified origin were incorporated (Duan et al., 1997a). Recently, transgenic tomato plants expressing the BV1 or BC1 MPs from Bean dwarf mosaic virus (BDMV) were generated (Hou et al., 2000). The transgenic plants expressing the BV1 MP appeared normal, whereas plants expressing the BC1 MP showed viral disease-like symptoms, although BDMV induces only a symptomless infection in tomato. One BC1 transgenic line did not show any disease-like symptoms. This line expressed a truncated BC1 protein, following a spontaneous mutation, which resulted in a deletion in the 3 region of the gene. Transgenic R0 plants, expressing either BV1 or BC1, showed a significant delay in the onset of infection following inoculation with ToMoV. R1 progeny plants also showed a delay in ToMoV infection, but to lesser extent than the R0 plants (Hou et al., 2000). These results demonstrate that begomovirus MPs, similar to RNA viruses MPs, can be used for development of PDR. However, to date, the level of resistance induced by begomovirus MPs is not sufficient for commercial use. Breeding for resistance to whitefly-transmitted geminiviruses Inhibition of viral replication Defective interfering (DI) resistance The first demonstration of PDR for geminiviruses was the expression of a defective interfering DNA B of ACMV in transgenic N. benthamiana plants (Stanley, 1990). Symptom severity and viral replication were reduced following inoculation with ACMV. No difference in disease severity or in viral replication was observed following inoculation with TGMV, or with BCTV (Stanley, 1990). Antisense resistance Antisense sequences of the Rep gene of TGMV strongly reduced symptom development and viral replication in transgenic tobacco plants (Day et al., 1991). When these plants were inoculated with other geminiviruses, a four-fold reduction in BCTV, but not ACMV DNA accumulation was found (Bejarano et al., 1994). Bendahmane & Gronenborn (1997) obtained almost complete suppression of TYLCV replication and disease symptoms in several lines of transgenic N. benthamiana plants expressing TYLCV Rep (C1) in the antisense orientation. Although a strict correlation between transgene transcript level and degree of resistance was not shown, plants with elevated levels of transcript consistently exhibited a higher degree of virus resistance. Incorporation of a hammerhead ribozyme to the Rep antisense construct did not improve the resistance level displayed by the antisense construct alone (Bendahmane & Gronenborn, 1997). Aragao et al. (1998) transformed beans with a construct containing antisense copies of the Rep and BC1 genes of BGMV (Brazilian isolate). Four lines of transgenic plants (R4 generation) were challenged with BGMV. Plants of two lines showed both delayed and attenuated viral symptoms. Plants of the other two lines developed typical BGMV symptoms. No correlation was found between transgene RNA level and resistance (Aragao et al., 1998). Truncated or mutated Rep gene N. benthamiana plants were transformed with the ACMV AC1 gene (Hong & Stanley, 1996). Although the AC1 RNA transcript was detected in the transgenic plants, the plants were unable to complement the infection of an ACMV AC1 mutant. Those plants which showed some level of resistance to ACMV were either asymptomatic or produced delayed and attenuated symptoms, and accumulated less viral DNA. None of the lines showed resistance to the related geminiviruses TGMV and BCTV, demonstrating the virus-specific nature of the resistance (Hong & Stanley, 1996). Resistance to Tomato yellow leaf curl Sardinia virus (TYLCSV), accompanied by a substantial 121 reduction in viral replication, was demonstrated in transgenic N. benthamiana plants expressing the TYLCSV Rep gene which had a truncated 3 end. The Rep protein was predicted to comprise only the first 210 amino acids (Noris et al., 1996). However, resistance was eventually overcome, and the inheritance was not Mendelian. Brunetti et al. (1997) transformed tomato plants with the same construct and found that 1) accumulation of high levels of the truncated Rep protein was required for resistance, 2) high accumulation of the transgene product resulted in plants with a curled phenotype, and 3) the resistance did not extend to an unrelated geminivirus. Recently, it was shown that the resistant plants were susceptible to inoculation with a related strain of TYLCSV (TYLCSV-ES) (Brunetti et al., 1997). It was shown that the truncated Rep acts as a trans-dominant-negative mutant, inhibiting (but not abolishing) transcription of the Rep gene of the invading virus, and consequently, inhibiting viral replication. Moreover, the C4 ORF, which is located within the truncated Rep gene coding sequence, does not participate in resistance induction (Brunetti et al., 2001). An analysis of mutations in the putative NTPbinding site of the Rep gene of BGYMV demonstrated that the NTP-binding domain is required for replication (Hanson et al., 1995). Thus, the authors proposed that mutations in this motif might serve as dominant negative mutations for PDR and that they would interfere with viral replication. Sangare et al. (1999) mutated the Rep of ACMV altering the putative NTP-binding site, and transformed transgenic N. benthamiana plants with this mutant. When transgenic plants expressing the mutated Rep were inoculated with ACMV, a delay in symptom appearance occurred and symptom attenuation was seen. The resistant plants also accumulated less viral DNA than control plants. It was found that a high level of expression of the mutated Rep gene was essential for development of resistance (Sangare et al., 1999), as noted for transgenic tomato plants expressing a truncated Rep of TYLCSV (Brunetti et al., 1997). Resistance to ToMoV in both tobacco and inbred tomato lines has been generated using a full length Rep gene of ToMoV (Polston et al., 1998). The Rep gene provided stable resistance, was not associated with an altered phenotype, and was successfully passed through five generations of tomato. Following inoculation, no evidence of viral replication could be found in transformed plants, suggesting this transgene can confer very high levels of resistance to ToMoV. Resistance and horticultural qualities of T4 generation plants were evaluated in the field for three production seasons. Yields of transformed plants were found to be equivalent to the untransformed parents in the absence of ToMoV 122 MOSHE LAPIDOT & MICHAEL FRIEDMANN and greatly superior in the presence of ToMoV. Polston & Hiebert (2001) transformed inbred tomato genotypes with a truncated Rep gene of TYLCV (containing approximately 40% of the gene). The truncated Rep conferred a very high level of resistance to TYLCV (Florida isolate) T 1 generation plants were symptomless following inoculation and virus DNA was undetectable. T2 generation plants were inoculated in the greenhouse and later transplanted to the field. The plants remained symptomless under field conditions, and the phenotype of the transformed plants was normal (Polston & Hiebert, 2001; Polston et al., 2001). Transgenic tobacco plants transformed with the same construct were immune to TYLCV, but were susceptible to CabLCV and to ToMoV. The authors concluded that the truncated Rep appears to confer immunity to TYLCV in tobacco and tomato, but the resistance appears to be narrow spectrum (Polston et al., 2001). Transient assay for resistance One of the major drawbacks of using transgenic plants for testing viral constructs for PDR is the amount of time and effort required for plant transformation and regeneration. Thus, transient expression assays have been developed, by cotransformation of suspension cells or protoplasts with intact virus and the viral gene product in question. This transient expression assay allows the rapid screening of many potential dominant mutants, by studying the effect of the different viral products and/or mutants on viral replication. Indeed, the transient expression of intact or truncated ACMV Rep gene product caused a marked reduction in the ACMV DNA replication in tobacco protoplasts (Hong & Stanley, 1996). Hanson & Maxwell (1999) used a transient expression assay in tobacco suspension cells to demonstrate that several different BGMV and BGYMV Rep gene mutants, with mutations in the NTP-binding or DNA-nicking domains, were potent trans-inhibitors of geminivirus replication. Moreover, these mutants inhibited the replication of different BGMV and BGYMV isolates as well as replication of BDMV (Hanson & Maxwell, 1999). Recently, transient expression of the Tomato leaf curl New Delhi virus (ToLCNDV) truncated Rep (capable of producing the N-terminal 160 amino acids) was found to inhibit viral DNA accumulation in tobacco protoplasts and in N. benthamiana plants (Chatterji et al., 2001). In vivo assays, conducted by co-bombarding N. benthamiana plants with infectious clones of ACMV, Pepper huasteco yellow vein virus and Potato yellow mosaic virus, indicated inhibition of replication of heterologous viral DNA by the truncated Rep of ToLCNDV. The authors postulated the potential of using Rep proteins with mutations in the oligomerisation and DNA binding domain (both mapped to the first 160 amino acids) to interfere with viral DNA replication (Chatterji et al., 2001). It seems that the most promising PDR approach is the transformation of susceptible plants with mutated (especially truncated) forms of the viral Rep gene. However in some instances, the transgenically expressed Rep induced undesirable phenotypes. Progress is being made in the assessment of this approach as commercial cultivars (as opposed to test plants such N. benthamiana) have recently been tested in field experiments (Polston & Hiebert, 2001). Concluding Remarks Similar approaches to the breeding for resistance to begomoviruses have been taken for a number of major crops. In general, protocols have been defined for inoculation with the identified casual agent of the disease, ideally under controlled conditions. In most cases resistance has been transferred from related species and efforts are being made to develop molecular markers for the sources of resistance. The release of resistant cultivars is already ongoing, with variable levels of success. However, new viruses and strains of the begomovirus group keep on emerging. This is mainly a result of changes in cultivation habits, the continuing world-wide spread of different biotypes of whiteflies, and the relatively high frequency of recombination among geminiviruses (Padidam et al., 1999; Pita et al., 2001b). Another major challenge is that often two or more viruses co-infect a crop. These new challenges necessitate the development of plants that express multi-viral resistance. One approach will be the identification and/or development of resistance sources with a widespectrum of resistance, i.e., resistance against multiple viruses. Another approach to achieve multivirus resistance will be by combining genes conferring resistances to different viruses, that is, pyramiding resistance genes. This poses to the breeder two major problems: how to distinguish between the different resistance genes to be combined, and a need to continuously develop novel sources of resistance to the new emerging viruses. Molecular markers linked to resistance genes can be instrumental in achieving the pyramiding of resistance genes, as it will allow identification of resistance genes. It will also circumvent the problem of phenotypic selection of plants infected with a number of viruses, which could mask the effects of certain resistance genes. The combination of classical breeding with PDR can create an unlimited pool of resistance genes; since the genome of the new viruses that are able to Breeding for resistance to whitefly-transmitted geminiviruses infect the crop will serve as a source of resistance genes. In addition, resistance genes introgressed from wild species may complement those transgenic plants with PDR that provides only partial resistance. Moreover, viral sequences shared by many begomoviruses may be utilised for the development of PDR with a wide-spectrum of resistance. Acknowledgments Contribution from the Agricultural Research Organization, the Volcani Center, Bet Dagan, Israel. Number 534/01. We wish to thank our colleagues for their advice and by making data (published and unpublished) available: Claude Fauquet, Douglas Maxwell, Francisco Morales, Meir Pilowsky and Jane Polston. We are grateful to Shlomo Cohen and Victor Gaba (Dept. of Virology, Volcani Center) for critically reviewing this manuscript. References Antignus Y, Lapidot M, Cohen S. 2001a. Interference with UV vision of insects: an IPM tool to impede epidemics of insect pests and insect associated virus diseases. In Virusinsect-plant interactions, pp. 331-350. Eds K F Harris, O P Smith and J E Duffus. New York: Academic Press. Antignus Y, Nestel D, Cohen S, Lapidot M. 2001b. Ultravioletdeficient greenhouse environment affects whitefly attraction and flight behavior. Environmental Entomology 30:394-399. Antignus Y, Mor N, Joseph B, Lapidot M, Cohen S. 1996. Ultraviolet-absorbing plastic sheets protect crops from insect pests and from virus diseases vectored by insects. Environmental Entomology 25:919-924. Aragao F J L, Ribeiro S G, Barros L M G, Brasileiro A C M, Maxwell D P, Rech E L, Faria J C. 1998. Transgenic beans (Phaseolus vulgaris L.) engineered to express viral antisense RNAs show delayed and attenuated symptoms to bean golden mosaic geminivirus. Molecular Breeding 4:491-499. Ascencio-Ibanez J T, Diaz-Plaza R, Mendez-Lozano J, Monsalve-Fonnegra Z I, Arguello-Astorga G R, RiveraBustamante R F. 1999. First report of tomato yellow leaf curl geminivirus in Yucatan, Mexico. Plant Disease 83:1178. Avidov H Z. 1946. Tobacco whitefly in Israel. Tel Aviv: Hassadeh Publishing House (in Hebrew). Azzam O, Diaz O, Beaver J S, Gilbertson R L, Russell D R, Maxwell D P. 1996. Transgenic beans with the bean golden mosaic geminivirus coat protein gene are susceptible to virus infection. Annual Report of the Bean Improvement Cooperative 39:276-277. Baulcombe D C. 1996. Mechanisms of pathogen-derived resistance to viruses in transgenic plants. Plant Cell 8:18331844. Beck D L, van Dolleweerd C J, Lough T J, Balmori E, Voot D M, Andersen M T, OBrien I E W, Forster R L S. 1994. Disruption of virus movement confers broad-spectrum resistance against systemic infection by plant viruses with a triple gene block. Proceedings of the National Academy of Sciences, USA 91:10310-10314. Bedford I D, Briddon R W, Brown J K, Rosell R C, Markham P G. 1994. Geminivirus transmission and biological characterisation of Bemisia tabaci (Gennadius) biotypes from different geographic regions. Annals of Applied Biology 125:311-325. 123 Bejarano E R, Lichtenstein C P. 1994. Expression of TGMV antisense RNA in transgenic tobacco inhibits replication of BCTV but not ACMV geminiviruses. Plant Molecular Biology 24:241-248. Bellotti A C, Arias B. 2001. Host plant resistance to whiteflies with emphasis on cassava as a case study. Crop Protection 20:813-824. Bendahmane M, Gronenborn B. 1997. Engineering resistance against tomato yellow leaf curl virus (TYLCV) using antisense RNA. Plant Molecular Biology 33:351-357. Berrie L C, Palmer K E, Rybicki E P, Rey M E C. 1998. Molecular characterisation of a distinct South African cassava infecting geminivirus. Archives of Virology 143:2253-2260. Berry S, Rey M E C. 2001. Molecular evidence for diverse populations of cassava-infecting begomoviruses in Southern Africa. Archives of Virology 146:1795-1802. Bianchini A. 1999. Resistance to bean golden mosaic virus in bean genotypes. Plant Disease 83:615-620. Blair M W, Bassett M J, Abouzid A M, Hiebert E, Polston J E, McMillan R T Jr, Graves W, Lamberts M. 1995. Occurrence of bean golden mosaic virus in Florida. Plant Disease 79:529-533. Briddon R W, Markham P G. 2000. Cotton leaf curl virus disease. Virus Research 71:151-159. Briddon R W, Mansoor S, Bedford I D, Pinner M S, Markham P G. 2000. Clones of cotton leaf curl geminivirus induce symptoms atypical of cotton leaf curl disease. Virus Genes 20:19-26. Briddon R W, Mansoor S, Bedford I D, Pinner M S, Saunders K, Stanley J, Zafar Y, Malik K A, Markham P G. 2001. Identification of DNA components required for induction of cotton leaf curl disease. Virology 285:234-243. Brough C L, Hayes R L, Morgan A J, Coutts R H A, Buck K W. 1988. Effects of mutagenesis in vitro on the ability of cloned tomato golden mosaic virus DNA to infect Nicotiana benthamiana plants. Journal of General Virology 69:503-514. Brown J K. 1996. Distribution and genetic variability of whitefly-transmitted geminiviruses of cotton,. In Proceedings Beltwide Cotton Conferences, pp. 275-276. Nashville, TN, USA: National Cotton Council. Brown J K, Nelson M R. 1984. Geminate particles associated with cotton leaf crumple disease in Arizona. Phytopathology 74:987-990. Brunetti A, Tavazza R, Noris E, Lucioli A, Accotto G P, Tavazza M. 2001. Transgenically expressed T-Rep of tomato yellow leaf curl Sardinia virus acts as a trans-dominantnegative mutant, inhibiting viral transcription and replication. Journal of Virology 75:10573-10581. Brunetti A, Tavazza M, Noris E, Tavazza R, Caciagli P, Ancora G, Crespi S, Accotto G P. 1997. High expression of truncated viral rep protein confers resistance to tomato yellow leaf curl virus in transgenic tomato plants. Molecular PlantMicrobe Interactions 10:571-579. Byrne D N, Bellows T S. 1991. Whitefly biology. Annual Review of Entomology 36:431-457. Chague V, Mercier J C, Guenard M, Courcel A de, Vedel F. 1997. Identification of RAPD markers linked to a locus involved in quantitative resistance to TYLCV in tomato by bulked segregant analysis. Theoretical and Applied Genetics 95:671-677. Chatterji A, Beachy R N, Fauquet C M. 2001. Expression of the oligomerization domain of the replication-associated protein (Rep) of Tomato leaf curl New Delhi virus interferes with DNA accumulation of heterologous geminiviruses. Journal of Biological Chemistry 276:25631-25638. Chiang B T, Maxwell D P, Green S. 1984. Leaf curl virus in Taiwan. Tomato Leaf Curl Newsletter 5:3. Cohen S, Antignus Y. 1994. Tomato yellow leaf curl virus, a whitefly-borne geminivirus of tomatoes. Advances in Disease and Vector Research 10:259-288. 124 MOSHE LAPIDOT & MICHAEL FRIEDMANN Cohen S, Harpaz I. 1964. Periodic, rather than continual acquisition of a new tomato virus by its vector, the tobacco whitefly (Bemisia tabaci Gennadius). Entomologia experimentalis et Applicata 7:155-166. Cohen S, Nitzany F E, Harpaz I. 1963. Experiments in the control of yellowing top virus in tomatoes. Tel Aviv: Hassadeh Publishing House (in Hebrew) 43:576-578. Cohen S, Nitzany F E, Vilde T. 1961. The tomato yellow top virus in Israel. Hassadeh Tel Aviv (in Hebrew). 42:139-140. Cooper J I, Jones A T. 1983. Responses of plants to viruses: proposals for the use of terms. Phytopathology 73:127-128. Cooper B, Lapidot M, Heick Dodds J A, Beachy R N. 1995. A defective movement protein of TMV in transgenic plants confers resistance to multiple viruses whereas the functional analog increases susceptibility. Virology 206:307-313. Cours G, Fargette D, Otim-Nape G W, Thresh J M. 1997. The epidemic of cassava mosaic virus disease in Madagascar in the 1930s-1940s: lessons for the current situation in Uganda. Tropical Science 37:238-248. Czosnek H, Laterrot H. 1997. A worldwide survey of tomato yellow leaf curl viruses. Archives of Virology 142:1391-1406. Czosnek H, Ber R, Navot N, Zamir D, Antignus Y, Cohen S. 1988. Detection of tomato yellow leaf curl virus in lysates of plants and insects by hybridization with a viral DNA probe. Plant Disease 72:949-951. Day A G, Bejarano E R, Buck K W, Burrell M, Lichtenstein C P. 1991. Expression of an antisense viral gene in transgenic tobacco confers resistance to the DNA virus tomato golden mosaic virus. Proceedings of the National Academy of Sciences, USA 88:6721-6725. Deng D, Otim-Nape G W, Sangare A, Ogwal S, Beachy R N, Fauquet C. 1997. Presence of a new virus closely associated with cassava mosaic outbreak in Uganda. African Journal of Root Tuber Crops 2:23-28. Duan Y P, Powell C A, Purcifull D E, Broglio P, Hiebert E. 1997a. Phenotypic variation in transgenic tobacco expressing mutated geminivirus movement/pathogenicity (BC1) proteins. Molecular Plant-Microbe Interactions 10:10651074. Duan Y P, Powell C A, Webb S E, Purcifull D E, Hiebert E. 1997b. Geminivirus resistance in transgenic tobacco expressing mutated BC1 protein. Molecular Plant-Microbe Interactions 10:617-623. Elmer J S, Brand L, Sunter G, Gardiner W E, Bisaro D M, Rogers S G. 1988. Genetic analysis of tomato golden mosaic virus II. The product of the AL1 coding sequence is required for replication. Nucleic Acids Research 16:7043-7060. Etessami P, Saunders K, Watts J, Stanley J. 1991. Mutational analysis of complementary-sense genes of African cassava mosaic virus DNA A. Journal of General Virology 72:10051012. Fargette D, Vie K. 1995. Simulation of the effects of host resistance, reversion, and cutting selection on incidence of African cassava mosaic virus and yield losses in cassava. Phytopathology 85:370-375. Fargette D, Colon L T, Bouveau R, Fauquet C. 1996. Components of resistance of cassava to African cassava mosaic virus. European Journal of Plant Pathology 102:645654. Faria J C, Maxwell D P. 1999. Variability in geminivirus isolates associated with Phaseolus spp. in Brazil. Phytopathology 89:262-268. Fauquet C, Fargette D. 1990. African cassava mosaic virus: etiology, epidemiology, and control. Plant Disease 74:404411. Fauquet C M, Maxwell D P, Gronenborn B, Stanley J. 2000. Revised proposal for naming geminiviruses. Archives of Virology 145:1743-1761. Fitchen J H, Beachy R N. 1993. Genetically engineered protection against viruses in transgenic plants. Annual Review of Microbiology 47:739-763. Fokunang C N, Akem C M, Dixon A G O, Ikotun T. 2000. Evaluation of a cassava germplasm collection for reaction to three major diseases and the effect on yield. Genetic Resources and Crop Evolution 47:63-71. Fondong V N, Thresh J M, Fauquet C. 2000a. Field experiments in Cameroon on cassava mosaic virus disease and the reversion phenomenon in susceptible and resistant cassava cultivars. International Journal of Pest Management 46:211-217. Fondong V N, Pita J S, Rey M E C, Kochko A de, Beachy R N, Fauquet C M. 2000b. Evidence of synergism between African cassava mosaic virus and a new double-recombinant geminivirus infecting cassava in Cameroon. Journal of General Virology 81:287-297. Fregene M, Bernal A, Duque M, Dixon A, Tohme J. 2000. AFLP analysis of African cassava (Manihot esculenta Crantz) germplasm resistant to the cassava mosaic disease (CMD). Theoretical and Applied Genetics 100:678-685. Fregene M, Angel F, Gomez R, Rodriguez F, Chavarriaga P, Roca W, Tohme J, Bonierbale M. 1997. A molecular genetic map of cassava (Manihot esculenta Crantz). Theoretical and Applied Genetics 95:431-441. Fregene M, Okogbenin E, Mba C, Angel F, Suarez M C, Janneth G, Chavarriaga P, Roca W, Bonierbale M, Tohme J. 2001. Genome mapping in cassava improvement: challenges, achievements and opportunities. Euphytica 120:159-165. Friedmann M, Lapidot M, Cohen S, Pilowsky M. 1998. A novel source of resistance to tomato yellow leaf curl virus exhibiting a symptomless reaction to viral infection. Journal of the American Society for Horticultural Science 123:10041007. Frischmuth T, Stanley J. 1993. Strategies for the control of geminivirus diseases. Seminars in Virology 4:329-337. Frischmuth T, Stanley J. 1998. Recombination between viral DNA and the transgenic coat protein gene of African cassava mosaic geminivirus. Journal of General Virology 79:12651271. Garrido-Ramirez E R, Sudarshana M R, Gilbertson R L. 2000. Bean golden yellow mosaic virus from Chiapas, Mexico: characterization, pseudorecombination with other bean-infecting geminiviruses and germ plasm screening. Phytopathology 90:1224-1232. Gibson R W, Otim-Nape G W. 1997. Factors determining recovery and reversion in mosaic-affected African cassava mosaic virus resistant cassava. Annals of Applied Biology 131:259-271. Hahn S K, Terry E R, Leuschner K. 1980. Breeding cassava for resistance to cassava mosaic disease. Euphytica 29:673683. Hahn S K, John C, Isoba G, Ikoutun T. 1989. Resistance breeding in root and tuber crops at the International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria. Crop Protection 8:147-168. Hanson S F, Maxwell D P. 1999. Trans-dominant inhibition of geminiviral DNA replication by bean golden mosaic geminivirus rep gene mutants. Phytopathology 89:480-486. Hanson S F, Hoogstraten R A, Ahlquist P, Gilbertson R L, Russell D R, Maxwell D P. 1995. Mutational analysis of a putative NTP-binding domain in the replication-associated protein (AC1) of bean golden mosaic geminivirus. Virology 211:1-9. Hanson P M, Bernacchi D, Green S, Tanksley S D, Venkataramappa M, Padmaja A S, Chen H, Kuo G, Fang D, Chen J. 2000. Mapping a wild tomato introgression associated with tomato yellow leaf curl virus resistance in a cultivated tomato line. Journal of the American Society for Horticultural Science 125:15-20. Hilje L, Costa H S, Stansly P A. 2001. Cultural practices for Breeding for resistance to whitefly-transmitted geminiviruses managing Bemisia tabaci and associated viral diseases. Crop Protection 20:801-812. Hong Y, Stanley J. 1996. Virus resistance in Nicotiana benthamiana conferred by African cassava mosaic virus replication-associated protein (AC1) transgene. Molecular Plant-Microbe Interactions 9:219-225. Hou Y, Sanders R, Ursin V M, Gilbertson R L. 2000. Transgenic plants expressing geminivirus movement proteins: abnormal phenotypes and delayed infection by Tomato mottle virus in transgenic tomatoes expressing the Bean dwarf mosaic virus BV1 or BC1 proteins. Molecular Plant-Microbe Interactions 13:297-308. Jennings D L. 1994. Breeding for resistance to African cassava mosaic geminivirus in East Africa. Tropical Science 34:110122. Kallo G, Banerjee M K. 1990. Transfer of tomato leaf curl virus resistance from Lycopersicon hirsutum f. glabratum to L. esculentum. Plant Breeding 105:156-159. Kelly J D. 1995. Use of random amplified polymorphic DNA markers in breeding for major gene resistance to plant pathogens. HortScience 30:461-465. Kunik T, Salomon R, Zamir D, Navot N, Zeidan M, Michelson I, Gafni Y, Czosnek H. 1994. Transgenic tomato plants expressing the tomato yellow leaf curl virus capsid protein are resistant to the virus. Bio-Technology 12:500-504. Lander E S, Botstein D. 1989. Mapping Mendelian factors underlying quantitative traits using RFLP linkage maps. Genetics 121:185-199. Lapidot M. 2002. Screening common beans (Phaseolus vulgaris) for resistance to tomato yellow leaf curl virus. Plant Disease 86:429-432. Lapidot M, Friedmann M, Pilowsky M, Cohen S. 2001. Development of a universal scale for evaluation of TYLCVresistance level in tomato plants. Tomato Breeders Round Table, Antigua, Guatemala. Lapidot M, Gafny R, Ding B, Wolf S, Lucas W J, Beachy R N. 1993. A dysfunctional movement protein of tobacco mosaic virus that partially modifies the plasmodesmata and limits virus spread in transgenic plants. Plant Journal 4:959970. Lapidot M, Friedmann M, Lachman O, Yehezkel A, Nahon S, Cohen S, Pilowsky M. 1997. Comparison of resistance level to tomato yellow leaf curl virus among commercial cultivars and breeding lines. Plant Disease 81:1425-1428. Lapidot M, Goldray O, Ben-Joseph R, Cohen S, Friedmann M, Shlomo A, Nahon S, Chen L, Pilowsky M. 2000. Breeding tomatoes for resistance to tomato yellow leaf curl begomovirus. EPPO Bulletin 30:317-321. Laterrot H. 1992. Resistance genitors to tomato yellow leaf curl virus (TYLCV). Tomato Leaf Curl Newsletter 1:2-4. Legg J P. 1999. Emergence, spread and strategies for controlling the pandemic of cassava mosaic virus disease in east and central Africa. Crop Protection 18:627-637. Legg J P, Thresh J M. 2000. Cassava mosaic virus disease in East Africa: a dynamic disease in a changing environment. Virus Research 71:135-149. Lomonossoff G P. 1995. Pathogen-derived resistance to plant viruses. Annual Review of Phytopathology 33:323-343. Malyshenko S I, Kondakova O A, Navarova J K, Kaplan I B, Taliansky M E, Atabekov J G. 1993. Reduction of tobacco mosaic virus accumulation in transgenic plants producing non-functional viral transport proteins. Journal of General Virology 74:1149-1156. Mansoor S, Bedford I D, Pinner M S, Stanley J, Markham P G. 1993. A whitefly-transmitted geminivirus associated with cotton leaf curl disease in Pakistan. Pakistan Journal of Botany 25:105-107. Mansoor S, Khan S H, Bashir A, Saeed M, Zafar Y, Malik K A, Briddon R, Stanley J, Markham P G. 1999. Identification of a novel circular single-stranded DNA 125 associated with cotton leaf curl disease in Pakistan. Virology 259:190-199. Mejia L, Czosnek H, Vidavsky V, Lapidot M, Friedmann M, Pilowsky M. 2001. Evaluation of tomato germplasm resistant to TYLCV for resistance to bipartite whitefly transmitted geminiviruses in Guatemala. APS Caribbean Division Meeting, La Habana, Cuba. Miklas P N, Johnson E, Stone V, Beaver J S, Montoya C, Zapata M. 1996. Selective mapping of QTL conditioning disease resistance in common bean. Crop Science 36:13441351. Morales F J. 2001. Conventional breeding for resistance to Bemisia tabaci-transmitted geminiviruses. Crop Protection 20:825-834. Morales F J, Anderson P K. 2001. The emergence and dissemination of whitefly-transmitted geminiviruses in Latin America. Archives of Virology 146:415-441. Morales F J, Niessen A I. 1988. Comparative responses of selected Phaseolus vulgaris germ plasm inoculated artificially and naturally with bean golden mosaic virus. Plant Disease 72:1020-1023. Morales F J, Singh S P. 1991. Genetics of resistance to bean golden mosaic virus in Phaseolus vulgaris L. Euphytica 52:113-117. Morales F J, Singh S P. 1993. Breeding for resistance to bean golden mosaic virus in an interracial population of Phaseolus vulgaris L. Euphytica 67:59-63. Moriones E, Navas-Castillo J. 2000. Tomato yellow leaf curl virus, an emerging virus complex causing epidemics worldwide. Virus Research 71:123-34. Nadeem A, Weng Z, Nelson M R. 1997. Cotton leaf crumple virus and cotton leaf curl virus are two distantly related geminiviruses. Molecular Plant Pathology On-Line http:// www.bspp.org.uk/mppol/1997/0612nadeem/. Nakhla M K, Maxwell D P. 1998. Epidemiology and management of tomato yellow leaf curl virus. In Plant Virus Disease Control, pp. 565-583. Eds A Hadidi, R K Khetarpal and H Koganezawa. St. Paul: APS Press. Natwick E T, Cook C, Gilbertson R, Seo Y, Turini T. 2000. Resistance in upland cotton to the silverleaf whitefly transmitted cotton leaf crumple disease. Proceedings Beltwide Cotton Conferences, National Cotton Council, San Antonio, USA, pp. 164-167. Navas-Castillo J, Sanchez-Campos S, Diaz J A. 1999. Tomato yellow leaf virus-Is causes a novel disease of common bean and severe epidemics in tomato in Spain. Plant Disease 83:2932. Navot N, Pichersky E, Zeidan M, Zamir D, Czosnek H. 1991. Tomato yellow leaf curl virus: a whitefly-transmitted geminivirus with a single genomic component. Virology 185:131-161. Noris E, Accotto G P, Tavazza R, Brunetti A, Crespi S, Tavazza M. 1996. Resistance to tomato yellow leaf curl geminivirus in Nicotiana benthamiana plants transformed with a truncated viral C1 gene. Virology 224:130-138. Noueiry A O, Lucas W J, Gilbertson R L. 1994. Two proteins of a plant DNA virus coordinate nuclear and plasmodesmal transport. Cell 76:925-932. Otim-Nape G W, Thresh J M, Bua A, Baguma Y, Shaw M W. 1998. Temporal spread of cassava mosaic virus disease in a range of cassava cultivars in different agro-ecological regions of Uganda. Annals of Applied Biology 133:415-430. Padidam M, Sawyer S, Fauquet C M. 1999. Possible emergence of new geminiviruses by frequent recombination. Virology 265:218-225. Palmer K E, Rybicki E P. 1998. The molecular biology of mastreviruses. Advances in Virus Research 50:183-234. Palumbo J C, Horowitz A R, Prabhaker N. 2001. Insecticidal control and resistance management for Bemisia tabaci. Crop Protection 20:739-766. 126 MOSHE LAPIDOT & MICHAEL FRIEDMANN Pico B, Diez M J, Nuez F. 1996. Viral diseases causing the greatest economic losses to the tomato crop. II. The tomato yellow leaf curl virus a review. Scientia Horticulturae 67:151-196. Pico B, Diez M J, Nuez F. 1998. Evaluation of whiteflymediated inoculation techniques to screen Lycopersicon esculentum and wild relatives for resistance to tomato yellow leaf curl virus. Euphytica 101:259-271. Pico B, Ferriol M, Diez M J, Nuez F. 1999. Developing tomato breeding lines resistant to tomato yellow leaf curl virus. Plant Breeding 118:537-542. Pilowsky M, Cohen S. 1990. Tolerance to tomato yellow leaf curl virus derived from Lycopersicon peruvianum. Plant Disease 74:248-250. Pilowsky M, Cohen S. 2000. Screening additional wild tomatoes for resistance to the whitefly-borne tomato yellow leaf curl virus. Acta Physiologia Plantarum 22:351-353. Pilowsky M, Friedmann M, Lapidot M, Nahon S, Shlomo H, Cohen S. 1997. TY-172 - a new source of resistance to tomato yellow leaf curl virus. Eucarpia Tomato, Jerusalem, Israel, p. 50. Pita J S, Fondong A, Sangare A, Kokora R N N, Fauquet C M. 2001a. Genomic and biological diversity of the African cassava geminiviruses. Euphytica 120:115-125. Pita J S, Fondong V N, Sangare A, Otim-Nape G W, Ogwal S, Fauquet C M. 2001b. Recombination, pseudorecombination and synergism of geminiviruses are determinant keys to the epidemic of severe cassava mosaic disease in Uganda. Journal of General Virology 82:655-665. Piven N M, Uzcategui R C de, Infante H D. 1995. Resistance to tomato yellow mosaic virus in species of Lycopersicon. Plant Disease 79:590-594. Polston J E, Anderson P K. 1997. The emergence of whiteflytransmitted geminiviruses in tomato in the western hemisphere. Plant Disease 81:1358-1369. Polston J E, Hiebert E. 2001. Engineered resistance to tomato geminiviruses. Proceedings of the Florida Tomato Institute. University of Florida, Naples, FL, pp. 19-22. Polston J E, McGovern R J, Brown L G. 1999. Introduction of tomato yellow leaf curl virus in Florida and implications for the spread of this and other geminiviruses of tomato. Plant Disease 83:984-988. Polston J E, Hunter W B, Abouzid A M, Hiebert E. 1998. Field performance of tomatoes transformed with Tomato mottle virus Rep gene. Second International Workshop on Bemisia and Geminiviruses, San Juan, Porto Rico, p. 61. Polston J E, Hiebert E, McGovern R J, Stansly P A, Schuster D J. 1993. Host range of tomato mottle virus, a new geminivirus infecting tomato in Florida. Plant Disease 77:1181-1184. Polston J E, Yang Y, Sherwood T, Bucher C, Frietas-Astua J, Hiebert E. 2001. Effective resistance to tomato yellow leaf curl virus (TYLCV) in tomato and tobacco mediated by a truncated TYLCV Rep gene. 3rd International Geminivirus Symposium, Norwich, UK, p. 82. Powell Abel P, Nelson R S, Hoffmann B De N, Rogers S G, Fraley R T, Beachy R N. 1986. Delay of disease development in transgenic plants that express the tobacco mosaic virus coat protein gene. Science 232:738-743. Rahman H, Khan W S, Khan M, Sha M K N. 2001. Stability of cotton cultivars under leaf curl virus epidemic in Pakistan. Field Crops Research 69:251-257. Rochester D E, DePaulo J J, Fauquet C M, Beachy R N. 1994. Complete nucleotide sequence of the geminivirus tomato yellow leaf curl virus, Thailand isolate. Journal of General Virology 75:477-485. Rom M, Antignus Y, Gidoni D, Pilowsky M, Cohen S. 1993. Accumulation of tomato yellow leaf curl virus DNA in tolerant and susceptible tomato lines. Plant Disease 77:253257. Russo M, Cohen S, Martelli G P. 1980. Virus-like particles in tomato plants affected by the yellow leaf curl disease. Journal of General Virology 49:209-213. Sanchez-Campos S, Navas-Castillo J, Camero R, Soria C, Diaz J A, Moriones E. 1999. Displacement of tomato yellow leaf curl virus (TYLCV)-Sr by TYLCV-Is in tomato epidemics in Spain. Phytopathology 89:1038-1043. Sanderfoot A A, Lazarowitz S G. 1996. Getting it together in plant virus movement: Cooperative interactions between bipartite geminivirus movement proteins. Trends in Cell Biology 6:353-358. Sanford J C, Johnson S A. 1985. The concept of parasitederived resistance: deriving resistance genes from the parasite own genome. Journal of Theoretical Biology 115:395-405. Sangare A, Deng D, Fauquet C M, Beachy R N. 1999. Resistance to African cassava mosaic virus conferred by a mutant of the putative NTP-binding domain of the Rep gene (AC1) in Nicotiana benthamiana. Molecular Biology Reports 5:95-102. Scott J W, Stevens M R, Barten J H M, Thome C R, Polston J E, Schuster D J, Serra C A. 1996. Introgression of resistance to whitefly-transmitted geminiviruses from Lycopersicon chilense to tomato. In Taxonomy, Biology, Damage, Control and Management Bemisia : 1995, pp. 357367. Ed D Gerling. Andover, Hants, UK: Intercept. Singh S P, Morales F J, Teran H. 2000a. Registration of bean golden mosaic resistant dry bean germplasm GMR 1 and GMR 5. Crop Science 40:1836. Singh S P, Morales F J, Miklas P N, Teran H. 2000b. Selection for bean golden mosaic resistance in intra- and interracial bean populations. Crop Science 40:1565-1572. Singh S P, Teran H, Munoz C G, Takegami J C. 2000c. Registration of multiple-disease resistant carioca dry bean A 801 and A 804 germplasm. Crop Science 40:1836-1837. Sinisterra X H, Polston J E, Abouzid A M, Hiebert E. 1999. Tobacco plants transformed with a modified coat protein of tomato mottle begomovirus show resistance to virus infection. Phytopathology 89:701-706. Stanley J. 1990. Characterisation and exploitation of plant virus DI DNA. The Exploitation of Micro-organisms in Applied Biology, Aspects of Applied Biology, No. 24. pp. 87-95. Storey H H. 1936. Virus diseases of East African plants: VI. A progress report on studies of the diseases of cassava. East Africa Journal of Agricultural Sciences 2:34-39. Swanson M M, Harrison B D. 1994. Properties, relationships and distribution of cassava mosaic geminiviruses. Tropical Science 34:15-25. Thomas P E, Mink G I. 1998. Tomato hybrids with nonspecific immunity to viral and mycoplasma pathogens of potato and tomato. HortScience 33:764-765. Thresh J M, Otim-Nape G W. 1994. Strategies for controlling African cassava mosaic geminivirus. Advances in Disease and Vector Research 10:215-236. Thresh J M, Otim-Nape G W, Fargette D. 1998a. The control of African cassava mosaic virus disease: phytosanitation and/ or resistance? In Plant Virus Disease Control, pp. 670-677. Eds A Hadidi, R K Khetarpal and H Koganezawa. St. Paul: APS Press. Thresh J M, Otim-Nape G W, Fargette D. 1998b. The components and deployment of resistance to cassava mosaic virus disease. Integrated Pest Management Reviews 3:209224. Thresh J M, Otim-Nape G W, Thankappan M, Muniyappa V. 1998c. The mosaic diseases of cassava in Africa and India caused by whitefly-borne geminiviruses. Review of Plant Pathology 77:935-945. Urrea C A, Miklas P N, Beaver J S, Riley H. 1996. A codominant randomly amplified polymorphic DNA (RAPD) marker useful for indirect selection of bean golden mosaic virus resistance in common bean. Journal of the American Breeding for resistance to whitefly-transmitted geminiviruses Society for Horticultural Science 121:1035-1039. Valverde R A, Lotrakul P, Landry A D. 2001. First report of Tomato yellow leaf curl virus in Louisiana. Plant Disease 85:230. Velez J J, Bassett M J, Beaver J S, Molina A. 1998. Inheritance of resistance to bean golden mosaic virus in common bean. Journal of the American Society for Horticultural Science 123:628-631. Vidavsky F, Czosnek H. 1998. Tomato breeding lines resistant and tolerant to tomato yellow leaf curl virus issued from Lycopersicon hirsutum. Phytopathology 88:910-914. Vidavsky F, Leviatov S, Milo J, Rabinowitch H D, Kedar N, Czosnek H. 1998. Response of tolerant breeding lines of tomato, Lycopersicon esculentum, originating from three different sources (L. peruvianum, L. pimpinellifolium and L. chilense) to early controlled inoculation by tomato yellow leaf curl virus (TYLCV). Plant Breeding 117:165-169. von Arnim A, Stanley J. 1992. Inhibition of African cassava mosaic virus systemic infection by a movement protein from the related geminvirus tomato golden mosaic virus. Virology 187:555-564. 127 Walkey D G A. 1985. Applied Plant Virology. New York: WileyInterscience. pp. 236-242. Wilson F D, Brown J K. 1991. Inheritance of response to cotton leaf crumple virus infection in cotton. Journal of Heredity 82:508-509. Zakay Y, Navot N, Zeidan M, Kedar N, Rabinowitch H, Czosnek H, Zamir D. 1991. Screening Lycopersicon accessions for resistance to tomato yellow leaf curl virus: presence of viral DNA and symptom development. Plant Disease 75:279-281. Zamir D, Ekstein-Michelson I, Zakay Y, Navot N, Zeidan M, Sarfatti M, Eshed Y, Harel E, Pleban T, van Oss H, Kedar N, Rabinowitch H D, Czosnek H. 1994. Mapping and introgression of a tomato yellow leaf curl virus tolerance gene, TY-1. Theoretical and Applied Genetics 88:141-146. Zhou X, Liu Y, Calvert L, Munoz C, Otim-Nape G W, Robinson D J, Harrison B D. 1997. Evidence that DNA-A of a geminivirus associated with severe cassava mosaic disease in Uganda has arisen by interspecific recombination. Journal of General Virology 78:2101-2111. 128 MOSHE LAPIDOT & MICHAEL FRIEDMANN