Tomato chlorosis virus in pepper: prevalence in commercial crops in southeastern Spain and symptomatology under experimental conditions

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1 Doi: /j x Tomato chlorosis virus in pepper: prevalence in commercial crops in southeastern Spain and symptomatology under experimental conditions I. M. Fortes, E. Moriones and J. Navas-Castillo* Instituto de Hortofruticultura Subtropical y Mediterránea La Mayora (IHSM-UMA-CSIC), Consejo Superior de Investigaciones Científicas, Estación Experimental La Mayora, Algarrobo-Costa, Málaga, Spain Tomato chlorosis virus (ToCV), a member of the genus Crinivirus (family Closteroviridae), has been present in Spain since at least 1997, causing annual epidemics of yellowing in protected tomato crops. In 1999, sweet pepper plants exhibiting stunting and symptoms of interveinal yellowing and mild upward curling in the leaves, were found to be infected with ToCV in a greenhouse heavily infested with the whitefly Bemisia tabaci in the province of Almería, southeastern Spain. This study investigated the prevalence of ToCV in tomato and pepper crops in the major growing areas of southeastern Spain (Murcia, Almería and Málaga provinces) over a 3-year period. In addition, an experimental system was developed for ToCV inoculation using B. tabaci as a vector, which allowed analysis of susceptibility of different pepper cultivars to the virus. The disease syndrome and yield losses induced by ToCV in pepper were also studied under experimental conditions, confirming severe yield reduction in infected plants. Keywords: Bemisia tabaci, Capsicum annuum, Closteroviridae, Crinivirus, symptomatology, whitefly transmission Introduction The occurrence of emerging viral diseases transmitted by whiteflies greatly limits sustainable production of economically important crops such as tomato (Solanum lycopersicum), one of the major vegetable crops worldwide (Navas-Castillo et al., 2011; Varma et al., 2011). Amongst others, Tomato chlorosis virus (ToCV) (genus Crinivirus, family Closteroviridae) is emerging as a problem worldwide. This virus causes a yellow leaf disorder syndrome in tomato plants, resulting in severe damage to production (Wisler et al., 1998a,b). ToCV is a typical member of the genus Crinivirus, limited to the phloem, transmitted in a semipersistent manner by three whitefly (Hemiptera: Aleyrodidae) species (Bemisia tabaci, Trialeurodes vaporariorum and T. abutiloneus), and with a bipartite genome of single-stranded RNA of positive polarity (Wisler et al., 1998b). Both RNA molecules are separately encapsidated in long and flexuous virions. RNA1 contains four open reading frames (ORFs), the two largest of which encode proteins associated with virus replication; RNA2 contains nine ORFs which encode proteins associated with functions including virus encapsidation, movement and whitefly transmission (Wintermantel et al., 2005; Lozano et al., 2006b, 2007). * jnavas@eelm.csic.es Published online 16 January 2012 Both genomic components encode proteins with RNA silencing suppression activity (Cañizares et al., 2008). Since the early 1990s, epidemics of ToCV have been continuously emerging in many countries, reaching an almost global distribution, including the Americas, Europe, Africa and nearby islands, the Middle East and Asia (Fiallo-Olivé et al., 2011; Navas-Castillo et al., 2011). In Spain, symptoms of tomato yellowing were first observed in Almería and Málaga provinces in 1997 (Navas- Castillo et al., 2000). In the following years, ToCV was reported from most of the main tomato-producing areas in southeastern continental Spain and the Canary Islands. Its prevalence in tomato crops in these regions has been reported to be high, frequently at levels of % (Lozano et al., 2006a; Velasco et al., 2008). Symptoms of ToCV infection in tomato include interveinal yellow chlorotic areas that initially develop on lower leaves, and then progress towards the upper leaves of the plant. Bronzing and red patches also often occur within the yellow areas, and the leaves become thickened and crispy with the margins slightly curled upward. Although no obvious symptoms are usually observed in tomato, fruit ripening is affected and flower abortion occurs, resulting in economic damage (our unpublished data). In addition to tomato plants, ToCV has a wide range of hosts, both natural and experimental, that comprises about 30 plant species from 13 different families (Font et al., 2004; Tsai et al., 2004; Morris et al., 2006; Wintermantel & Wisler, 2006; Trenado et al., 2007; Solórzano-Morales et al., 2011), including some important crops, ornamentals and weeds. 994 ª 2012 The Authors Plant Pathology ª 2012 BSPP

2 Tomato chlorosis virus in pepper 995 In 1999, sweet pepper (Capsicum annuum) plants exhibiting symptoms of interveinal yellowing, mild upward leaf curling and stunting were observed in a greenhouse heavily infested with the whitefly B. tabaci in Almería province, southeastern Spain. Molecular hybridization and RT-PCR with specific primers showed that samples collected from plants with symptoms were infected with ToCV. This was the first report of ToCV infecting sweet pepper plants (Lozano et al., 2004), and two further papers have recently been published concerning reports from Brazil (Barbosa et al., 2010) and Costa Rica (Vargas et al., 2011). Attempts to transmit ToCV to pepper plants under controlled conditions, using B. tabaci and T. vaporariorum as vectors, have been reported to be unsuccessful (Morris et al., 2006; Wintermantel & Wisler, 2006), except for a recent report from Brazil in which a single pepper plant was infected by B. tabaci (biotype B; Barbosa et al., 2010). However, the precise relationship between ToCV infection and the disease syndrome and possible yield losses induced in pepper plants was unknown. The goal of this study was to determine the impact of ToCV on pepper cultivation. For this, the prevalence of ToCV in the major growing areas of pepper in southeastern Spain (Murcia, Almería and Málaga provinces) were first determined over a 3-year period. In addition, a system was developed for the inoculation of pepper plants with ToCV using B. tabaci as a vector, which allowed the experimental infection of different cultivars, and therefore characterization of the symptomatology caused by ToCV on this crop. Materials and methods Field sampling Field surveys of commercial pepper crops were conducted from 2005 to In 2005, leaf samples were collected from an open field pepper crop (Almayate, Málaga province, southern Spain), where symptoms indicative of ToCV infection were observed. Ten samples (young apical leaf) were collected from plants exhibiting mild chlorosis and abnormally elongated leaves; additionally, samples were also collected from 10 plants exhibiting a healthy appearance. During 2006 to 2008, systematic surveys were undertaken of pepper fields in the major growing areas in southeastern Spain (Málaga, Almería and Murcia provinces) using a W scheme (Campbell & Madden, 1990) and collecting about 30 samples (one young apical leaf per plant) per visited field. In each province and year, 10 fields were visited. An equivalent sampling scheme was carried out in commercial tomato crops from the same provinces, but in this case one middle height leaf was sampled per plant. Young apical and intermediate height leaves were selected for pepper and tomato, respectively, based on previous unpublished work in this laboratory that showed that these leaves contained the highest amount of viral RNA. In 2006, only tomato crops could be sampled in the Murcia province. Virus molecular detection Presence of ToCV RNA in leaf samples was determined either by tissue blot molecular hybridization or by reverse transcription-polymerase chain reaction (RT-PCR). For tissue blot hybridization, freshly cross-sectioned leaf petioles were squash-blotted on positively charged nylon membranes (Roche Diagnostics) and hybridized with a ToCV-specific probe obtained from the coat protein gene of isolate Pl-1-2 of a naturally infected tomato plant collected in Málaga province in 1997 and maintained in tomato cv. Moneymaker by periodic transmission with B. tabaci. Primers MA310 (+): 5 -ATGGAGAACAGT- GCCGTTGC-3 and MA311 ( ): 5 -TTAGCAACCAG- TTATCGATGC-3 were used to amplify, with RT-PCR, a 774 bp fragment from the ToCV coat protein gene. The obtained amplicon was cloned into pgem-t Easy (Promega) and, after linearization with SalI, was used for in vitro transcription using T7 RNA polymerase (Roche Diagnostics) and digoxigenin (DIG)-11-UTP to generate a negative-sense RNA probe. This probe proved to be useful for diagnosis of ToCV (Trenado et al., 2007; Gómez et al., 2010). RT-PCR was performed using the forward MA380 (5 -GTGAGACCCCGATGACAGAT-3 ) and reverse MA381 (5 - TACAGTTCCTTGCCCTCGTT-3 ) primers designed to amplify a 436 bp DNA fragment of the coat protein gene, based on the sequence of the Spanish AT80 99 ToCV isolate (Lozano et al., 2006b). Total RNA was extracted using TRIzol Reagent (Invitrogen) according to the manufacturer s instructions, and RT- PCR was performed using Superscript One-Step RT-PCR with Platinum Taq Kit (Invitrogen) under the following conditions: 50 C for 30 min, initial denaturation at 94 C for 2 min, followed by 35 cycles of 94 C for 15 s, 50 C for 30 s and 72 C for 30 s, and a final extension at 72 C for 5 min. Virus transmission Whitefly-mediated transmission assays were conducted using adults of a B. tabaci biotype Q healthy population reared on melon (Cucumis melo cv. ANC 42, La Mayora- CSIC seed bank) in insect-proof screened cages. Transmission from naturally infected pepper to tomato plants was performed by using the shoots from 10 pepper plants collected in Málaga in 2005, shown to be infected by ToCV, as a virus source. Viruliferous whiteflies were obtained by providing a 48 h acquisition access period (AAP) to 50 healthy B. tabaci adults on each ToCVinfected pepper shoot within clip-on cages. Following AAP, each clip-on cage, containing the viruliferous whiteflies, was transferred to a tomato cv. Moneymaker (La Mayora-CSIC seed bank) test plant (three-leaf growth stage) for a 48 h inoculation access period (IAP). After IAP, whiteflies were eliminated from plants by insecticide spraying (Confidor 20 LS (20% imidacloprid) and Atominal 10 EC (10% pyriproxyfen)). Plants were maintained in a growth chamber (25 20 C day night, 70% relative humidity, with a 16 h photoperiod and

3 996 I. M. Fortes et al. photosynthetically active radiation at 250 lmol s )1 m )2 ) until analysed. The presence of ToCV in inoculated plants was analysed by molecular hybridization and RT- PCR at 15, 30 and 45 days post-inoculation (dpi). ToCVinfected plants were then maintained in an insect-proof greenhouse with temperature control (approximately 16 h day length, C day C night, and supplemental light when needed) by rooting of cuttings and periodical transmission using B. tabaci. The ToCV isolate present in one of these plants (isolate MM8) was used in subsequent transmission experiments. To determine the effect of the number of insects on the ToCV transmission efficiency from tomato to pepper plants, non-viruliferous B. tabaci adults were given a 48 h AAP on ToCV-infected tomato plants within insectproof cages. Then, groups of 10, 25 or 50 viruliferous whiteflies per test plant were released for a 48 h IAP into each of three wooden insect-proof cages containing 15 healthy pepper (cv. California Wonder, four-leaf growth stage) test plants. Fifteen pepper plants exposed to nonviruliferous whiteflies in equivalent conditions were used as a negative control. Following IAP, test plants were sprayed with insecticides and maintained in a greenhouse (see above) until analysed. The presence of ToCV in these plants was analysed by tissue blot molecular hybridization at 15 and 30 dpi. For ToCV transmission to different pepper cultivars, groups of 50 viruliferous B. tabaci whiteflies obtained from ToCV-infected tomato plants (see above) were deposited within clip-on cages on the third leaf from the apex of each pepper test plant (four-leaf growth stage) for a 48 h IAP. Experiments were carried out with five commercial pepper cultivars, representative of the three main types of pepper grown in southeastern Spain: Italian (Pescara RZ (Rijk Zwaan) and Spadi F1 (Vilmorin)); California (California Wonder (Ramiro Arnedo) and Yolo Wonder (Ramiro Arnedo)); and Lamuyo (Lamuyo F1 (Clause)). Thirty plants of each cultivar were used, 15 inoculated with viruliferous insects and 15 exposed to non-viruliferous whiteflies following the same procedure as for those which were used as mock-inoculated controls. After the IAP, test plants were sprayed with insecticides and maintained in a greenhouse (see above). At 15 dpi, test plants were transplanted into 25 L pots and maintained until 150 dpi in the same greenhouse. The presence of ToCV in these plants was determined by molecular hybridization at 15, 30 and 60 dpi. Evaluation of virus symptoms Symptoms were examined regularly in each ToCV-inoculated pepper plant and compared to the mock-inoculated control plants. In addition to observing foliar symptoms, plant height (cm) from the stem base to the apex at 60, 90, 120 and 150 dpi was recorded. Fruits were also collected periodically at the commercial green stage, and the total number of fruits and total weight per plant were determined. Data were analysed with a two-factor MANOVA, with yield, height and fruit number as dependent variables, and cultivar and presence absence of infection as independent variables. As the MANOVA revealed statistical differences (Wilks Lambda test for cultivar (F 12,365 =3Æ22, P <0Æ001), presence absence of infection (F 3,138 = 43Æ46, P < 0Æ001), and interaction between these two variables (F 12,365 =43Æ46, P =0Æ003)), each of the three dependent variables was subsequently analysed with a two-factor ANOVA. Significant differences between infected plants and mock-inoculated plants within cultivars were tested using the Tukey HSD post hoc test. Both tests were performed using the program STATISTICA (StatSoft Inc.) Results ToCV infections in commercial pepper crops Molecular hybridization (Fig. 1a) and RT-PCR amplification showed that the 10 pepper plants collected in a commercial crop in the province of Málaga in 2005 and exhibiting mild chlorosis were infected by ToCV, whereas all symptomless plants from the same plot tested negative. It should be highlighted that plants with (a) (b) (c) Figure 1 Detection of Tomato chlorosis virus (ToCV) by tissue blot molecular hybridization (two leaf petiole squash blots per sample) with a digoxigenin-labelled RNA probe in (a) 10 pepper plants showing mild chlorosis and elongated leaves (upper row) and 10 symptomless plants (lower row) collected from a commercial crop in 2005 in Málaga province; and (b) eight out of 10 tomato plants inoculated with B. tabaci using pepper plants as a virus source at 15 days post-inoculation. (c) Detection of ToCV in the same plants as in (b) by RT-PCR using primers for the coat protein gene. C, ToCV-infected (+) and healthy ()) controls; M, 1 kb ladder molecular weight marker; X, no sample.

4 Tomato chlorosis virus in pepper 997 Table 1 Prevalence (percentage of infected plants) of Tomato chlorosis virus (ToCV) in pepper and tomato commercial crops in southeastern Spain (Málaga, Almería and Murcia provinces) from 2006 to Systematic surveys following a W shape sampling pattern were conducted in 10 plots per crop species, province and year, with 30 samples analysed per plot (a) Year Crop Province Pepper Málaga 8Æ2 8Æ3 10Æ9 Almería 0Æ3 0 0 Murcia 0 0 Tomato Málaga 62Æ9 27Æ3 47Æ5 Almería 18Æ0 26Æ6 21Æ0 Murcia 32Æ0 38Æ3 15Æ6 (b), data not available. symptoms exhibited a characteristic elongation of leaves not observed in symptomless plants and not previously associated with ToCV infection in pepper (Lozano et al., 2004). The prevalence of ToCV infection was then studied in commercial pepper and tomato crops in southeastern Spain (Málaga, Almería and Murcia provinces) from 2006 to 2008 based on a systematic sampling. A total of 300 samples per crop species, province and year were analysed by tissue blot hybridization, and the results are shown in Table 1. Clear differences were observed among sampled regions both for pepper and tomato crops. Apart for one single plant in Almería in 2006, ToCV infection in pepper was only detected in fields in Málaga province, with a prevalence of about 10%. In contrast, ToCV infections were frequent in tomato in the three provinces, with Málaga province usually having a higher prevalence (27 63%), followed by Murcia (16 38%), and Almería (18 27%). ToCV transmission ToCV was readily transmitted by B. tabaci biotype Q from field-infected pepper plants collected in 2005 to tomato cv. Moneymaker for eight out of the 10 tomato test plants infected, as deduced by molecular hybridization and RT-PCR (Fig. 1b,c) respectively. One of these isolates derived from field-infected pepper plants (isolate MM8) was maintained in tomato plants by periodical B. tabaci transmission and rooting of cuttings. ToCV was readily transmitted by B. tabaci Q biotype from MM8-infected tomato source plants to pepper (cv. California Wonder), although a clearly heightened transmission efficiency was observed when the number of viruliferous insects per plant was increased (n = 10, 25, 50) (Fig. 2a). At 30 dpi, 73% of the pepper test plants were infected using 50 whiteflies per plant, whereas <20% infection was observed when using 10 whiteflies per plant, with intermediate results with 25 whiteflies per plant. Therefore, subsequent experiments used 50 viruliferous whiteflies per plant. Differences in susceptibility were observed between pepper cultivars. Although all five cultivars assayed could Figure 2 Evolution of Tomato chlorosis virus (ToCV) infection in (a) pepper cv. California Wonder plants, inoculated using different number of B. tabaci (biotype Q) adult viruliferous whiteflies per plant; and in (b) pepper plants of different cultivars inoculated using 50 B. tabaci (biotype Q) viruliferous adults per plant. At each time post-inoculation, plants were tested by tissue blot molecular hybridization with a digoxigenin-labelled RNA probe specific to ToCV. be infected, Pescara emerged as the most susceptible cultivar, with 100% infected plants at 30 dpi, whereas Lamuyo was the least susceptible cultivar, with 53% infection; California Wonder, Yolo Wonder and Spadi exhibited intermediate susceptibilities of 80, 73 and 73% at 30 dpi, respectively (Fig. 2b). Although a significant number of plants already exhibited infection at 15 dpi, it was only at 30 dpi that all the infected plants could be detected, and therefore 30 dpi was chosen for analysis of plants in subsequent experiments. ToCV symptoms and yield losses on pepper Pepper plants experimentally infected with ToCV MM8 (15 plants of each of the five cultivars tested) exhibited a significant growth reduction at 120 dpi (from 21% for Spadi to 34% for Pescara) compared to mock-inoculated plants (P <0Æ001) (Figs 3a and 4a), although differences were already observed from 60 dpi (not shown). At approximately 60 dpi, all ToCV-infected pepper plants also exhibited symptoms of mild interveinal yellowing on leaves (Fig. 4b). Yellowing symptoms first developed on lower leaves and then advanced upwards towards the middle part of the plant. In later stages, ToCV-infected plants exhibited a characteristic upward rolling and thickening of lower leaves (Fig. 4c), and some leaves located at the middle height of the infected plants showed a sharp end, giving a characteristically elongated

5 998 I. M. Fortes et al. (a) (a) (b) (c) (b) (c) (d) Figure 3 Effect of Tomato chlorosis virus (ToCV) infection on (a) plant growth, (b) fruit yield and (c) fruit number of infected (white bars) or mock-inoculated (black bars) pepper plants of different cultivars. Plant height was taken at 120 days post-inoculation. Yield was measured as total weight of commercial green stage fruit per plant. Mock-inoculated plants include both plants exposed to healthy B. tabaci whiteflies and plants exposed to viruliferous insects that finally escaped from infection, as deduced by molecular hybridization. Asterisks indicate significant differences (P < 0Æ05) between ToCV-infected plants and mock-inoculated plants within each cultivar. morphology (Fig. 4d). None of the previously indicated symptoms were observed in any mock-inoculated plant, nor in any of the inoculated plants that escaped infection. All five pepper cultivars showed a statistically significant yield reduction (measured as total weight of commercial green stage fruit per plant) as a result of ToCV infection, compared to mock-inoculated plants (P-values of all post hoc tests <0Æ005). The estimated yield losses ranged from 45% for Spadi to 75% for Pescara (Fig. 3b). This yield reduction was due both to reduction in fruit size (Fig. 4e) and fruit number. The number of fruits was significantly higher in mock-inoculated plants than in infected plants for all cultivars (P-values of post hoc tests <0Æ05), except Yolo Wonder (P = 0Æ45) (Fig. 3c). Discussion (e) Figure 4 Effect of Tomato chlorosis virus infection on pepper (cv. Pescara) plants inoculated using viruliferous B. tabaci (50 whiteflies per test plant) and maintained under controlled conditions. (a) Plant growth reduction in an infected plant (right) in comparison to a mock-inoculated plant (left); symptoms of (b) interveinal yellowing, (c) upward curling, and (d) abnormal sharp end, observed in leaves of infected plants; and (e) reduction in size of fruits from infected plants (right) in comparison to those from mock-inoculated plants (left). In the last three decades, whitefly populations, mainly from B. tabaci, have increased in number and distribution throughout the world, particularly in tropical, subtropical and temperate areas. This has been accompanied by the emergence of viral diseases transmitted by this insect. These diseases affect many economically important crops, resulting in yield reductions and significant economic losses, caused mainly by members of the genera Begomovirus (family Geminiviridae) and Crinivirus (family Closteroviridae) (Wisler et al., 1998a; Jones, 2003; Morales, 2007). Tomato-yellowing caused by the crinivirus ToCV is a good example of a whitefly-transmitted disease emerging in many countries worldwide (Navas-Castillo et al., 2011). Due to the increasing

6 Tomato chlorosis virus in pepper 999 prevalence of ToCV throughout the world, a better knowledge of its host range and syndromes caused on cultivated hosts, virus vector relationships, and the potential impact on production of affected crops, is crucial to implement more effective and durable crop management practices. Here, the prevalence and potential impact of ToCV infections in pepper is analysed, and for the first time the symptoms and yield losses induced in plants are described. Systematic sampling of commercial pepper crops of the major growing areas in southeastern Spain from 2006 to 2008 indicated that infected plants were detected almost solely in the Málaga province, and at moderate prevalence in comparison to tomato crops. There are some observations that could explain the restriction of ToCV in pepper to Málaga province. First, although not measured in this study, B. tabaci populations were usually larger in pepper crops of the Málaga province, whereas very low population numbers were observed during samplings in Almería and Murcia. This is probably related to the widespread use of effective biological control management of B. tabaci in pepper in the two latter provinces, especially in Almería, where it is in use over nearly all the pepper growing area (van der Blom, 2009). It is also worth noting that T. vaporariorum, also a vector of ToCV, is virtually absent in tomato and pepper crops in the sampled areas. Therefore, the role of this whitefly in the epidemiology of this virus in southern Spain can be considered negligible. The differences observed for pepper were not so evident for tomato crops, which exhibited 16 38% prevalence of ToCV among sampled plants per province and year, probably related to a much less effective control of B. tabaci populations in this host species (van der Blom, 2007). Nevertheless, it should be noted that during , low ToCV prevalence was observed in tomato crops in comparison to that reported in previous years (up to % of plants infected) (Lozano et al., 2006a; Velasco et al., 2008). Furthermore, based on the present studies, some pepper varieties are more difficult to infect than tomato, and this could also influence the observed differences in prevalence in the field. In Murcia province, differences in ToCV prevalence between tomato and pepper crops might also be associated with the geographical separation between the growing areas of both host species, with almost no overlap. In contrast, both pepper and tomato crops overlap spatially and temporally in Almería and Málaga. Another possibly important factor is that in Málaga there is a more complex agriculture system, with a continuous overlap of greenhouse and open field crops in the same area; this probably contributes to the maintenance of a higher level of ToCV inoculum in either tomato or pepper crops. In addition, weeds which can serve as hosts of ToCV are also more abundantly associated with these crops in Málaga. In order to obtain more insight into the potential impact of ToCV infection in pepper, a better understanding of the symptoms caused by this virus in pepper plants was needed. An experimental system that facilitated the infection of pepper plants using B. tabaci as a vector was established, which comprised the use of 50 biotype Q viruliferous whiteflies per test plant, with plants at the fourleaf growth stage. Previous attempts to transmit ToCV experimentally to pepper plants using B. tabaci or T. vaporariorum (Morris et al., 2006; Wintermantel & Wisler, 2006) had been unsuccessful, with the exception of a single infected pepper plant obtained in Brazil using naturally infected pepper source plants and B. tabaci biotype B (Barbosa et al., 2010). Wintermantel & Wisler (2006) suggested that their inability to infect pepper could be due to a difference in susceptibility between varieties. Their results could be partly explained by the results here, as great differences in susceptibility were observed among the five pepper varieties tested. However, numerous attempts previously carried out in this laboratory to infect California Wonder pepper plants (which exhibited a moderate susceptibility) with ToCV had also been unsuccessful. The fact that in the present work a ToCV isolate obtained from pepper was used, in contrast to a tomato isolate being used in previous attempts, and that genetic variability was observed in Spanish ToCV isolates (Lozano et al., 2009), apparently associated with tomato or pepper host plant (unpublished results), might have contributed to the differences in transmissibility observed. Therefore, the study of host adaptation to ToCV isolates is an aspect which needs further study. It also remains to be determined whether the specific vector used in the aforementioned works influenced the obtained results, because differences in ToCV transmission efficiency to tomato plants, and virus persistence in the vector, have been found to depend on the different whitefly species or biotypes (Wintermantel & Wisler, 2006). Previous host range studies for ToCV were performed using T. vaporariorum, T. abutiloneus or B. tabaci biotypes A and B (Morris et al., 2006; Wintermantel & Wisler, 2006), whereas this work used B. tabaci biotype Q, the most widespread in southern Spain and in the western Mediterranean basin (Simón et al., 2001). Therefore, vector efficiency of the different whiteflies for transmission to pepper is an aspect that should also be further studied. Before this study, only circumstantial observations linked ToCV infections in pepper with yellowing and stunting symptoms (Lozano et al., 2004). However, knowledge of the precise symptomatology caused by ToCV in pepper plants is crucial to determine the actual importance of ToCV infections in this crop. Under controlled conditions, it was shown unequivocally that ToCV infections cause a reduced growth in infected plants, in addition to interveinal yellowing, upward curling and thickening in leaves (at the middle bottom part of the plant) and significant commercial yield loss. Similar effects were observed in five commercial pepper cultivars comprising the three basic types of peppers grown in southeastern Spain (Italian, California and Lamuyo), although differences in susceptibility appeared to exist between them. The observed symptoms strongly resembled those reported for the first ToCV-infected pepper

7 1000 I. M. Fortes et al. plants observed in Almería in 1999 (Lozano et al., 2004). In addition, middle leaves of infected plants had a distinctive more-elongated shape with a sharp end. This latter symptom was also observed under field conditions in the pepper plants collected in Málaga in 2005, these being the original source of the ToCV isolate used in this work. Therefore, it can be concluded that stunting growth of the plant, accompanied by symptoms of curling, interveinal yellowing and abnormal elongation in leaves, are characteristic of ToCV infections in pepper. More important economically is that commercial fruit yield of infected pepper plants was shown here to be significantly decreased, because of both fruit number and fruit size reduction. Although a similar effect of ToCV infection has been observed on tomato crops grown in Florida and Spain (Wisler et al., 1998a; Navas-Castillo et al., 2000), to the authors knowledge no quantitative data on yield reduction are available. It should be highlighted that the pepper cultivars studied in this work showed a gradation in terms of growth and yield reduction when infected by ToCV: this is highly correlated with their susceptibility to ToCV infection in terms of prevalence of infected plants. Thus, the cultivar Pescara, with 100% of the plants becoming infected under the controlled conditions tested here, exhibited the highest growth and yield reduction, whilst cultivars Spadi and Lamuyo, that exhibited the lowest susceptibility to infection, also presented the lowest growth and yield reduction. Efforts have been undertaken for tomato to locate virus resistance in plants and, although no commercial resistance is available, two Solanum accessions have recently been identified as potential sources of ToCV resistance to be incorporated in tomato (García-Cano et al., 2010). However, to the authors knowledge, no information on sources of resistance to ToCV has been described in pepper. Inoculation methodologies described in this work can help to implement programmes looking for sources of resistance in this host species. In summary, this study demonstrated the presence of ToCV causing infections in commercial pepper crops in Spain and defined the symptomatology of the disease syndrome caused in this host species. The resulting significant yield losses could severely limit pepper production in areas where increased prevalence occurs, which in turn may be determined by changes in vector populations. This can then pose a new threat to pepper, a crop of great economic importance worldwide. A more complex epidemiological situation for ToCV is also predicted in those areas favourable to virus presence, mainly if both whitefly vectors, B. tabaci and T. vaporariorum, are present and tomato and pepper crops overlap. Acknowledgements This work was partially supported by grants P08- AGR from Consejería de Economía, Innovación y Ciencia, Junta de Andalucía, Spain (CEIC) and AGL C02-01 AGR from Ministerio de Ciencia e Innovación, Spain (MICINN), both cofinanced by FEDER-FSE. E.M. and J.N.C. are members of the Research Group AGR-214, partially funded by CEIC. I.M.F. was recipient of a fellowship from MIC- INN. The authors wish to thank M. Montserrat for her guidance in statistical analysis and everyone who helped us on field surveys, particularly J.M. Aguilar, M. Berenguer, J.F. Campos, R. Gómez, S. García- Andrés, A. Lacasa, F. Monci, A. Monserrat, P. Navas, A.F. Orílio, R. Tovar, H.P. 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