Distribution and Research Advances of Citrus tristeza virus

Journal of Integrative Agriculture March 2012 2012, 11(3): 346-358 REVIEW Distribution and Research Advances of Citrus tristeza virus Sagheer Atta1, 2, ZHOU Chang-yong1, 3, ZHOU Yan1, 3, CAO Meng-ji1, 2 and WANG Xue-feng1, 3 1 National Citrus Engineering Research Center, Southwest University, Chongqing 400712, P.R.China 2 College of Plant Protection, Southwest University, Chongqing 400716, P.R.China 3 Key Laboratory of Horticultural Science for Southern Mountainous Regions, Ministry of Education, Chongqing 400715, P.R.China Abstract Citrus tristeza virus (CTV) is one of the most important causal agents of citrus diseases and exists as numerous strains. CTV is replicated in phloem cells of plants within the family Rutaceae and is transmitted by a few of aphid species. CTV epidemics have caused death of millions of citrus trees in many regions all over the world, where the sour orange (Citrus aurantium) was used as rootstock. Also the production of grapefruit (C. paradisi) and sweet orange (C. sinensis) has been affected by CTV strains. CTV gives uplift to three prominent syndromes, namely quick-decline (tristeza), stempitting and seedling-yellows. The disease is graft-transmissible in nature but not seed-transmitted. However, the tristeza disease in most citrus groves was a man-made problem created by the desire of horticulturists to introduce cultivars from other citrus growing areas. The utmost importance of the disease called for review articles in numbers of plant protection, epidemiology books, citriculture and proceedings. This review collects the information with respects to disease history, distribution host range, virus isolates association, identification and detection, transmission and management; especially on the current status of CTV prevailing and controlling in Pakistan. It provides valuable information for CTV disease and its controlling approaches. Key words: Citrus tristeza virus, epidemic, status in Pakistan, control INTRODUCTION AND DISEASE HISTORY The center of origin of most citrus cultivars is perhaps unknown but the ancient relatives of citrus are native to China, the Southeast Asia, the Malay Archipelago, New Caledonia, and Australia, and then co-evolved with the host. CTV dispersal to new regions mainly occurs through the movement and propagation of the infected plants or infected buds and then locally it is spread by a few of aphid species (Bar-Joseph et al. 1989; Timmer et al. 2000). However there is no proper evidence of seed transmission. As seed transmission does not occur we have to suppose an early adaptation of CTV by aphid transmission. At the start, fruits or seeds of citReceived 18 March, 2011 rus were brought from the site of origin to other regions of the world (Zaragoza 2007), which gives evidences that CTV was not dispersed at that time. At the end of the 19th century, with an increased botanical and commercial interest and value in citrus by the horticulturists, citrus plants were introduced from Asia to other regions and vast exotic citrus species were exchanged between collections (Roistacher 1981). In 1836, a foot rot epidemic caused by oomycetes of the genus Phytophthora sp. started in the Azores and later affected the Mediterranean countries, destroyed sweet orange trees [C. sinensis (L.) Osb.], and led to the adoption of Phytophthora tolerant rootstocks. That was to propagate citrus varieties on sour orange (C. aurantium L.), a foot-rot-resistant rootstock, highly adaptable to Accepted 1 June, 2011 Sagheer Atta, E-mail: [email protected]; Correspondenc ZHOU Chang-yong, Tel: +86-23-68349601, Fax: +86-23-68349592, E-mail: [email protected] © 2012, CAAS. All rights reserved. Published by Elsevier Ltd. Distribution and Research Advances of Citrus tristeza virus all types of soil and producing good bearing and fruits of excellent quality. Sour orange soon became almost the tremendous rootstock in the Mediterranean area and then in America. However, ultimately, the decision of using sour orange rootstock led to the dramatic effect that CTV has had on world citrus production. The new disease epidemic was dramatic for the citrus industry and caused the loss of almost 100 million trees propagated on sour orange and finally created the need for tristeza-tolerant rootstocks to rebuild a new citrus industry in the countries being affected. The most destructive epidemics of tristeza occurred in Argentina in 1930, then appeared in Brazil in 1937, was named tristeza. The epidemics occurred respectively in Ghana in 1938, California in 1939, Florida in 1951, Spain in 1957, Israel in 1970, and Venezuela in 1980, and the outbreaks have also been reported from other citrus groves, such as in Cyprus in 1989, Cuba in 1992, Mexico in 1995, the Dominican Republic in 1996, and Italy in 2002 (Bar-Joseph et al. 1989; Garnsey et al. 2000; Timmer et al. 2000; Gottwald et al. 2002; Davino et al. 2003). There are also indirect damages caused by CTV epidemics, such as the loss of sour orange rootstock which has the agronomic and horticultural qualities unmatched by any other rootstock, and the appearance of new problems related to the use of tristeza-tolerant rootstocks, such as the graft-transmissible diseases associated with the use of these rootstocks (Román et al. 2004). Today CTV is widespread in Israel, Morocco, India, China, Japan, Pakistan, Iran, Syria, Egypt, southern California and Florida of USA, Argentina, Brazil, South Africa, Tanzania, Australia, and southern Spain, and is moving into northern Spain (EPPO 2006) where is previously free of the disease. Although some citrus areas are free of the spread of CTV, but the threat continues for the areas having fewer species of aphids (Zhou 1997). HOST RANGE AND SYMPTOMS INDUCED 347 Aegle, Aeglopsis, Afraegle, Atalantia, Citropsis, Clausena, Eremocitrus, Hesperthusa, Merrillia, Microcitrus, Pamburus, Pleiospermium, and Swinglea (Timmer et al. 2000). The host in the non-citrus species Passiflora gracilis and P. coerulea using aphid vectors (Roistacher et al. 1988), and experimental infection in Nicotiana benthamiana (Gowda et al. 2005). Resistance to CTV isolates varies considerably and has been observed in Poncirus trifoliata (L.) Raf. (Rai 2006) and resistance to specific CTV strains has also been observed in Meiwa kumquat (F. crassifolia Swing), some pummelos and sour oranges (Asíns et al. 2004). CTV replication has been observed in protoplasts of trifoliate orange, pummelo or sour orange, suggesting that only CTV movement is impaired in these species (Albiach-Martí et al. 2004). CTV causes different disease syndromes on citrus plants depending on the virus strain, the variety of citrus and the scion rootstock combination (Moreno et al. 2008). Different CTV strains, generally referred to as seedling-yellows (CTV-SY), tristeza (CTV-T), stem-pitting (CTV-SP), and a mild type, have been widespread for many years. Any of these strains may exist in a citrus plant or they may occur together as a complex. Quick-decline or tristeza disease Sweet orange, mandarin (including Satsuma and Ponkan), Tankan, Iyo, Tangor, many varieties of tangelo and grapefruit are affected by this disease when grown on sour orange, pummelo or lemon rootstock (but not on rough lemon rootstock). The causal virus is either CTV-SY or CTV-T. When the adult tree is affected by such a combination, it turns yellowing and wilting rapidly and dies within a few years. If the tree is grafted onto resistant rootstock such as trifoliate orange or mandarin, it recovers immediately after grafting (Fraser 1952). Seedling-yellows disease BY CTV CTV affects almost all species, varieties, hybrids of genera Citrus and some species of Fortunella and also experimentally inoculated citrus relatives of the genera Seedlings of self-rooted trees of sour orange, Natsudaidai, lemon, Buntan, and grapefruit are affected by seedling-yellows disease after being infected with distinctive phenotype of some isolates of CTV (Albiach- © 2011, CAAS. All rights reserved. Published by Elsevier Ltd. 348 Marti et al. 2010). These trees become yellow, die back and stunt and sometimes a quite cessation of growth of sour orange, grapefruit or lemon seedlings takes place (Fraser 1952). The physiological mechanism associated with the induction of SY symptoms has not been understood yet. The SY reaction may sometimes be transient. If the affected trees are grafted onto a resistant rootstock, they could be recovered soon. Although it is not economically important but it can be assayed in the glass-house much easily as compared to stem-pitting and decline. Stem-pitting disease Most varieties of citrus are affected by stem-pitting disease, even if they are grafted onto a rootstock resistant to tristeza. Although few varieties of mandarin such as Satsuma and Ponkan are resistant to CTV but most of the citrus species such as grapefruit, sweet orange and Buntan and citrus relatives, tangelo, tangor, Iyo, Yuzu, and Natsudaidai are all susceptible. Stempitting results from abnormal vascular differentiation, and when the disease is severe, plants develop a large number of pits on both their trunks and their stems. Affected trees become dwarfed and show less vigor (Moreno et al. 2008) and occasionally die back. As a result, although there is profuse flowering, the trees bear only poor crops of small sized or irregularly shaped fruits. The severe strain of CTV-SP induced rind-oil spots, or brown spots with gumming on the fruits of some cultivars. TRANSMISSION AND EPIDEMIOLOGY Propagation of virus-infected buds is the cause of dispersal of CTV into new areas, while the aphid vector is responsible for local spread. The virus has also been experimentally transmitted to healthy plants by dodder (Cuscuta subinclusa) (Weathers and Hartung 1964) and by stem-slash inoculation with partially purified extracts (Garnsey et al. 1977) but these procedures are epidemiologically unimportant. CTV isolates have been described as differing in the symptoms induced in the field (da Graça et al. 1984) in the reaction induced on indicator plants and in aphid transmissibility (Ballester- Sagheer Atta et al. Olmos et al. 1988). These biological differences may affect the epidemiology of the disease and the damage produced by CTV in different citrus-growing areas. Four aphid species (Aphis gossypii, the cotton or melon aphid; A. spiraecola, the spirea aphid; Toxoptera aurantii, the black citrus aphid; and Toxoptera citricida, the brown citrus aphid) have been associated with the natural spread of CTV (Yokomi et al. 1994; Rocha-Peña et al. 1995). The most efficient vector of CTV worldwide is T. citricida (Rocha-Peña et al. 1995). The transmissibility of CTV isolates by T. citricida is 25 times higher than that of A. gossypii. T. citricida is found in Asia, Australia, Africa, Central and South America, and different Caribbean countries (Rocha-Peña et al. 1995; Halbert et al. 2004), which is also a serious pest of citrus while both feeding and breeding normally taking place on citrus (Roistacher 1991). CTV was first demonstrated as being aphid transmitted by using hundreds of aphids per plant to transmit the pathogen (Meneghini 1946). T. citricida transmission of CTV was reported to have no latent period, but with acquisition and inoculation periods being at least 30 min (Bar-Joseph et al. 1989). Also there have been some reports to record the acquisition and inoculation periods of CTV by T. citricida being in seconds (Retuerma and Price 1972). Some authors recognize the semi-persistent nature of CTV aphid transmission and additionally classify this transmission as bimodal (Chalfant and Chapman 1962). In bimodal transmission, virus acquisition can cluster around two periods, a short time period and a relatively long time period, and there is generally no change no matter the aphids are pre-acquisitionally fasted or not (Lim and Hagedorn 1977). Variable single aphid transmission rates for T. citricida have also been recorded, for instance, up to 25% (Yokomi et al. 1994), 0-55% (Broadbent et al. 1996), and 16.5-18.4% (Tsai et al. 2000). CTV isolates varied in their ability to be transmitted experimentally by the T. citricida (Yokomi et al. 1994). ELISA was used to detect CTV (EPPO 2004). A few reports demonstrated the detection of viruses in aphid vectors using the most sensitive PCR-based assays (Cambra et al. 2006) either by using RNA purification and RT-PCR (Mehta et al. 1997) or by RT-nested PCR (Olmos et al. 1999), a multiplex real-time PCR assay (Ananthakrishnan et al. 2010). A number of stud- © 2011, CAAS. All rights reserved. Published by Elsevier Ltd. Distribution and Research Advances of Citrus tristeza virus ies quantitatively estimated the number of viral targets in single aphids (Fabre et al. 2003). The spatial and temporal spread of CTV had also been studied in some citrus growing countries (Gottwald et al. 1999). These studies showed that the spread of CTV depended on the presence of T. citricida or A. gossypii as the predominant vector species. In areas where A. gossypii was predominant, CTV incidence increased from 5 to 95% in 8-15 years following a stair-step line and infected trees showed limited aggregation, and it was shown that new infections were not related with existing infected trees. Meanwhile, in areas where T. citricida was predominant, the same disease rate often occurred in only 2-4 years with a rapid and essentially consecutive increase, and aggregates of infected trees were common because the immediately spread to the trees adjacent to existing infections was frequent. The biology and feeding habits of these vector species perhaps were the causes for these distinct spread patterns (Gottwald et al. 1996). MOLECULAR CHARACTERISTICS OF CTV CTV, a member of the Closteroviridae (Bar-Joseph et al. 1972), genus Closterovirus, has a ~20 kb singlestranded, positive sense RNA genome (Karasev et al. 1995). This virus genome resides in a single RNA molecule, which produces an unusually large number of less-than-full-length viral RNAs during replication. There are ten genes in the 3´-portion of the genome that are expressed via 3´ coterminal sg mRNAs (Karasev et al. 1995). Each of the mRNAs produces two additional less-than-full-length RNAs: a negative-stranded RNA with sequence complementary to the sg mRNA and a 5´-terminal positive-stranded sgRNA produced by termination near the controller element upstream of the start of the mRNA, apparently during genomic RNA synthesis (Gowda et al. 2001). Hence, CTV produces 30 sgRNAs associated with its ten 3´-terminal genes. The most unusual sgRNAs are two small 5´-coterminal positive-stranded RNAs which have been referred to as ‘low molecular weight tristeza RNAs’ (LMT) (Che et al. 2001). LMT1 and LMT2 RNAs are ~750 and 650 nt respectively (Gowda et al. 2003). Both accumulate to high amounts at molar levels higher than that of the virion RNA. LMT1 RNA, which is produced 349 during replication by termination upstream of a previously unknown controller element that produces only minute amounts of a 3´-terminal mRNA-like sgRNA, but without known function (Ayllon et al. 2004). The mechanism of production of the smaller LMT2 has been unknown. The CTV genome consists of 12 open reading frames (ORFs) and potentially encoding at least 19 protein products (Karasev et al. 1995). The CTV virons are polarly coated with two separate coat proteins (CPs) p25 and p27, elaborated as major and minor (ca. 3%) CPs, respectively (Febres et al. 1996). The minor CP is associated with small amount of two other proteins p65, a homolog of cellular heat shock protein of the 70 kDa family (HSP7O), and a large protein, p61. The 12 ORFs of CTV are expressed through different number of ways including, proteolytic processing of polyprotein, translational frame shifting and up to 32 different 5´- and 3´subgenomic RNAs (Che et al. 2003). The two mechanisms are used to show the protein encoded by the 5´half of the genome which encodes ORFs 1a and 1b. A large, ~400 kDa polyprotein encoded by ORF1a is proteoltically processed by virus encoded proteases (Karasev et al. 1995). ORF 1b is translated by a +1 frame-shift. The second mechanism is used to express the 3´-coterminal ORFs 2 to 11. From the early genomic characterization of CTV, it is obvious that defective RNAs (dRNAs) are with almost all known CTV isolates. Most of the CTV dRNAs consist of the two genomic termini with extensive internal deletion. CTV isolates have multiple defective RNAs with various large sizes (Zhou et al. 1997; Ayllón et al. 1999b; Che et al. 2003). Recently CTV dRNAs were categorized in six classes (Batuma et al. 2004). Different factors contribute to the biological diversity of CTV isolates, such as genetic variation following super-infection with multiple isolates, homologous RNA recombination between sequence variants, the presence of defective RNAs and top working to new varieties (Ayllón et al. 1999a, 2006; Roy and Brlansky 2004; Vives et al. 2005). The 5´ half of the genome consists of two ORFs encoding protein associated with viral replication. ORF 1a encodes a large, ~400 kDa polyprotein, that includes two papain-like protease domain, a methyltransferaselike domain and a helicase-like domain. The ORF 1b encodes an RNA dependant RNA polymerase-like do- © 2011, CAAS. All rights reserved. Published by Elsevier Ltd. Sagheer Atta et al. 350 main (Satyanarayana et al. 1999). The 3´ half of the genome encodes ten genes that are not required for replication in protoplasts (Ayllón et al. 2003) with five gene-blocks. The five gene-block is unique to closteroviruses, and encodes a small, 6 kDa hydrophobic protein (ORF3), a 65 kDa cellular heat-shock protein homolog (HSP70h, ORF4), a 61 kDa protein (ORF5), and a tendem pair of structural proteins, a 27 kDa capsid protein (CPm. ORF6) duplicate followed by the 25 kDa (CP ORF7) (Pappu et al. 1994; Karasev et al. 1995). The small p6 is single-span transmembrane protein not required for virus replication or assembly, which exists in ER and functions in Beet yellow virus (BYV), another member of Closterovirus, for cell to cell movement (Peremysolve et al. 2004). p65 is the homologous of HSP70 heat-shock proteins which together with p61 and two capsid proteins are required for virion assembly (Satyanarayana et al. 2004). Protein p20 accumulates in amorphous inclusion bodies of CTV infected cells (Gowda et al. 2000). The product of 3´-most ORF (ORF11), p23, is a multifunctional protein with no homologue in other members of Closterovirus, that: (i) binds RNA molecule in non-sequence specific manner (Lopez et al. 2000); (ii) contains a zinc finger domain that regulates the synthesis of plus- and minus-strand molecules and controls the accumulation of plus strand RNA during replication (Satyanarayana et al. 2002); (iii) is an inducer of CTV like symptoms in transgenic C. aurantifolia plant (Ghorbel et al. 2001); and (iv) is a potent suppressor of intracellular RNA silencing in Nicotiana tabacum and N. banthamiana (Lu et al. 2004). The p33, p18 and p13 genes are involved in infection and movement in some hosts (Tatineni et al. 2008). The complete nucleotide sequence of CTV has been determined in at least nine distinct isolates (Karasev et al. 1995; Albiach-Martí et al. 2000; Suastika et al. 2001; Vives et al. 2005; Ruiz-Ruiz et al. 2006). Phylogenetic analysis of the complete sequences reported for nine CTV isolates revealed three main clusters that included (i) the severe SP isolates, T318A from Spain (Ruiz-Ruiz et al. 2006), SY568R from California (Vives et al. 2005), NuagA from Japan (AB046398) (Suastika et al. 2001), and VT from Israel (Mawassi et al. 1996); (ii) the mild isolates, T30 from Florida (Albiach-Martí et al. 2000) and T385 from Spain; and (iii) T36 from Florida (Karasev et al. 1995), Qaha from Egypt (AY340974) and a Mexican isolate (DQ272579). Within-group nucleotide identities were over 97.5%, whereas the lowest identity (75.6%) was between VT and Qaha. DIAGNOSIS OF CTV CTV isolates vary in their pathogenicity and also contain various genomic virus variants (Zhou et al. 2007) that can be detected by aphids or graft transmission to different citrus host species. The sub-isolates segregated in this way can be differentiated by pathogenicity tests in different hosts, by dsRNA patterns (Moreno et al. 1993) or by serologically using specific monoclonal antibodies (Permar et al. 1990; Cambra et al. 1993). It has been demonstrated that the haplotype distribution of two CTV genes can be altered after host change or aphid transmission (Ayllón et al. 1999). Molecular hybridizations and single-strand conformation polymorphisms analysis of the coat protein gene (Rubio et al. 1996) have been used to differentiate the Mediterranean CTV isolates. The best diagnosis method for CTV is to graft-inoculate indicator seedlings of Mexican lime and observe them for vein-clearing, leaf cupping, and stem-pitting (Roistacher 1991). Electron and light microscopy can be used to identify CTV particles and inclusions, but DAS-ELISA (Cambra et al. 1979) revolutionised the diagnosis for testing a large number of samples during surveys of large citrus areas for CTV control in nurseries and for epidemiological studies. The production of monoclonal antibodies specific to CTV (Permar et al. 1990) and others reported by (Nikolaeva et al. 1996) solved the problems of specificity and increased sensitivity of ELISA tests. A mixture of two monoclonal antibodies (3DF1 and 3CA5) or their recombinant versions (Terrada et al. 2000) can recognise all CTV isolates tested from different international collections. A detailed description and characterization of these monoclonal antibodies has been summarised (Cambra et al. 2000a). Tissue print-ELISA (Cambra et al. 2000b) for CTV detection allowed the sensitive indexing of thousands of samples simply and without the need to prepare extracts. A number of di- © 2011, CAAS. All rights reserved. Published by Elsevier Ltd. Distribution and Research Advances of Citrus tristeza virus agnostic procedures based on specific detection of viral RNA were developed, including molecular hybridization with cDNA or cRNA probes (Barbarossa and Savino 2006). PCR-based assays have been developed and modified (Zhou et al. 2001) based on immunocapture (Nolasco et al. 1993) or print or squash capture (Cambra et al. 2000c). A simple procedure has been described to perform nested-PCR in a single closed tube (Olmos et al. 1999) which allowed CTV detection in single aphids and in plant tissues. A co-operational PCR system (Co-PCR) (Olmos et al. 2002) has been described, which supplying similar sensitivity to nested PCR. Real-time RT-PCR protocol is more sensitive and allows the detection and quantification of genomic RNA copies in infected citrus tissues or in viruliferous aphids (Saponari et al. 2008). qPCR also is becoming more and more useful as a method for gene expression analysis (Vaudano et al. 2009). Through BD-PCR (bi-directional reverse transcription-polymerase chain reaction) analysis, a 392-bp fragment specific for the mild strains was amplified and a 320-bp fragment specific for the severe strains was produced (Jiang et al. 2008). The RFLP (restriction fragment length polymorphism) analysis for RT-PCR products of the CP gene with restriction enzyme Hinf I identified seven groups (Zhou et al. 2007; Jiang et al. 2008). RTPCR amplification patterns with primer set specific for several CTV genotypes (Hilf et al. 2005) for 5´UTR sequence types I, II and III (Ruiz-Ruiz et al. 2006) or for three groups of isolates differing by their p23 sequence (Sambade et al. 2003). Single-strand conformation polymorphism (SSCP) analysis of different gRNA regions (Sambade et al. 2007) has been used to characterize the population structure of CTV isolates and select specific variants for sequencing, thus allowing estimates of the genetic diversity within and between isolates (Ayllón et al. 2006). The phylogenetic analysis of p23 showed a high intra-isolate sequence variability suggesting that re-infections could contribute to the observed variability and that the host can play an important role in the selection of the sequence variants present in these isolates (Iglesias et al. 2008). Polymorphism analyses of p23, p25 and p27 genes showed that most isolates contained high intra-isolate variability (Iglesias et al. 2008). 351 STATUS OF CTV IN PAKISTAN Pakistan is generally considered among the top 10 leading citrus-growing countries of the world both in production and quality. The growing area of citrus in Pakistan is about 193 211 ha with an annual production of 2 459 500 tons (Anonymous 2008). It contributes 2% of citrus fruit to the world’s production and earns a major source of foreign exchange for the country. Citrus is grown in all four provinces of Pakistan, but Punjab contributes almost 97% of the production of the country. Generally the growing area and production for different kinds of fruits and particularly for citrus have been increasing since the 1960’s due to the increasing demand in the domestic and foreign markets (Khan 1992). In Pakistan fungal and bacterial diseases of citrus have been documented since 1920 through different sources, however virus and virus-like diseases infecting different citrus species could not receive due attention for a long time because of the lack of proper facilities for the detection and characterization (Mughal 2004). Tristeza is a devastating disease in the citrus groves of Pakistan. In Pakistan only limited numbers of surveys have been made to test the presence of the disease in citrus orchards. Survey was made for citrus virus and virus-like diseases in the N.W.F.P and Punjab provinces. Along with other diseases tristeza was detected only in a few trees and confirmed by ELISA and electron microscopy (EM) (Catara et al. 1988). Investigation by EM, threadlike particles of CTV were found in phloem tissues of the columella. CTV was also confirmed by ELISA tests (Grimaldi and Catara 1989). Again a survey was carried out and more than fifty orchards and ten nurseries were sampled in different areas of the Punjab. ELISA tests and EM observations showed that CTV was present in the varieties in different districts. Mosambi was the most affected variety (7 positive out of 35) among the Mosambi, Bloodred and Pineapple sweet orange (Catara et al. 1991). Anwar and Mirza (1992) conducted a survey in 14 localities and in five districts, viz., Sahiwal, Sargodha, Faisalabad, Lahore, and Sheikhupora, and confirmed the prevalence of CTV by ELISA test with the highest infection (18.8%) in Sahiwal, followed by Sargodha (13.20%) and Faisalabad (13.13%), while no infection was found in Lahore district. In NWFP, symptoms of © 2011, CAAS. All rights reserved. Published by Elsevier Ltd. 352 vein-clearing and chlorosis were observed in young leaves of C. aurantium, C. limon cv. Eureka and C. sinensis by grafting and mechanical inoculation (Arif et al. 2005). Extensive surveys of the major citrus groves in Punjab and N.W.F.P and ELISA tests showed that the CTV incidence in Bhalwal and the Punjab were 44.61 and 48.46%, and in Mardan and N.W.F.P were 37.39 and 40.86%, respectively in 2006-2007 (Iftikhar et al. 2009). CONTROL STRATEGIES FOR CTV According to the incidence of CTV, strategies to control CTV vary depending on the virus strains and citrus varieties in each particular region (Garnsey et al. 1998). Quarantine and bud wood certification programs are useful measures to prevent introduction of CTV into countries where CTV does not exist yet. In citrus groves where CTV incidence is low, the disease can be suppressed by eradication or suppression programs (Gottwald et al. 2002). And in the CTV endemic regions CTV tolerant rootstocks, mild strain cross protection and genetically engineered resistance combined with certification programs are the potential ways to deal with the problems (Lee and Rocha-Pena 1992). Mild strain cross protection is the only best available management method that can be applied to control SP with mild CTV strain where disease is impossible to control by eradication or suppression. This technique has been widely used to control CTV on large scale in commercial citrus plantations, especially with Pera sweet orange in Brazil (Costa and Müller 1980), grapefruit in Australia (Broadbent et al. 1991), South Africa, Japan, and limes in India (Lee and Rocha-Pena 1992). These isolates were collected from old trees of the same cultivar that have been grown for years showing only mild or no symptoms. Cross protection is now being implemented in the CTV endemic regions such as Florida (Lee and Brlansky 1990). But unfortunately, the cross protection strategy had less success in other areas or with other varieties (Broadbent et al. 1991), which showing that cross protection perhaps has to depend on the varieties, CTV strains and environmental conditions prevalent in each region. Citrus sp. such as C. reticulata, C. volkameriana and C. jambhiri (Rangpur lime) are used as root stocks and somehow are tolerant Sagheer Atta et al. to QD-inducing CTV isolates and some hybrid rootstocks including citranges (C. sinensis×P. trifoliata) and citrumelos (C. paradisi×P. trifoliata) are also being used as CTV tolerant rootstocks to control CTV in some citrus growing areas. The presence of other economically important diseases such as citrus blight, viroids, and undesirable horticultural practices limit the usefulness of these rootstocks (Garnsey et al. 1987). Moreover, some CTV isolates induce SP symptoms in the scions regardless of the tolerance of their rootstocks (Bar-Joseph et al. 1989). Hence, these rootstocks do not give control against CTV-SP isolates where these isolates are widespread. Genetic engineering gives the specific trait of transgenic plants by incorporating a specific gene into the plants genome without changing the other desirable characteristics. Recent advances in molecular biology and breeding to incorporate resistance genes in commercial varieties have given best results to tackle the problem of crop losses due to pathogens. Plant transformation techniques have opened new vistas and possibilities for the development of sources of virus resistance compared with conventional breeding methods. However, different and complex genetic characteristics of reproductive citrus biology along with their larger plant size have put a great check on genetic improvement through conventional breeding. The first transgenic strategy concept in which a complete or partial gene is introduced into plant to obtain a specific resistance was proposed by (Sanford and Johnson 1985) as pathogen-derived resistance (PDR). PDR for a plant virus was first performed in 1986 (Powell-Abel et al. 1986) by introducing the CP gene of Tobacco mosaic virus (TMV) into transgenic tobacco plant which ultimately showed resistance to TMV infection and PDR to CTV was for the first time confirmed by the incorporation of the CP gene of CTV strain in Mexican lime (Domínguez et al. 2002). This concept of PDR has been confirmed in several plant-virus systems (Dasgupta et al. 2003). This strategy used in the transformation of other citrus hosts could not give clear or best results (Febres et al. 2003). Viral sequences other than CP genes have been explored to engineer PDR to plant viruses. Non-coding sequences from the 5´ and 3´ UTR changed viral genomes of plants as well as satellite RNAs and D-RNAs to produce transgenic plants © 2011, CAAS. All rights reserved. Published by Elsevier Ltd. Distribution and Research Advances of Citrus tristeza virus resistant to viruses (Nelson et al. 1993; Zaccomer et al. 1993). Even though the results had some variation with different plant-virus systems, the transformation of non-structural genes in transgenic plants is a promising strategy for developing virus resistance, especially for movement protein and replication associated proteins such as RdRp (Pappu et al. 1995). Constructs derived from the CTV 3´-UTR were used to transform sweet orange protoplasts and grapefruit plants but conclusive results on protection at the whole plant level were not reported (Febres et al. 2003). Transgenic limes showing the p23 gene from a severe or from a mild CTV isolate displayed leaf symptoms of similar intensity, which was associated with the accumulation level of the p23 protein (Fagoaga et al. 2005). It is clear evidence indicating that disease induction in the host may not be a side-effect of silencing suppression but a consequence of disruption of the miRNA metabolism (Lewsey et al. 2007). In short, these results indicate that p23 is an important CTV pathogenicity determinant that interferes with plant development specifically in Citrus species and relatives (Fagoaga et al. 2005). Transgenic p23UI-N. benthamiana were resistant to infection with a viral vector made of Grapevine virus A (GVA)+p23U (GVA-p23U), as indicated by the absence of the chimeric virus from inoculated plants. Inoculation of transgenic p23UI Alemow plants with CTV resulted in delayed appearance of symptoms in 9 out of the 70 transgenic plants. However, none of the plants showed durable resistance, as indicated by the obtaining of similar Northern hybridization signals from both transgenic and non-transgenic citrus plants (Batuman et al. 2006). Superinfetion exclusion or homologous intereference a phenomenon in which a primary viral infection prevents a secondary infection with same or closely related trait (Svetlana et al. 2010) showed that superinfection exclusion of CTV occurred only between the isolates same strain and with different strain. CONCLUSION AND FUTURE CHALLE NGES Tristeza epidemic is still going on in many citrus regions including Pakistan although 70 years have passed away since its first epidemic. Tristeza decline may cause more losses in future and even destroy the citrus 353 industry of some countries. Dispersal of tristeza induced diseases in Pakistan is increasing very rapidly, which may be able to destroy the citrus industry of Pakistan as well as that of the neighboring countries. While some management strategies may eventually tackle the problem and restore the citrus production, such as replacement of declining trees with new trees on tristeza tolerant rootstocks, resistance genes, cross protection, identification of pathogenicity determinants for different disease syndromes. Citrus production can be increased through nurseries running on a scientific and professional basis. Certified citrus nurseries are needed to solve the problem caused by CTV. Better understanding of the relationship among CTV isolates, host plants and vectors should be strengthened, which has been absolutely limited in Pakistan. The combination of using certified budwood programs plus MSCP strategy is certainly the best way to control the losses induced by CTV. Acknowledgements This study was supported by the National Natural Science Foundation of China (30471205), the Special Fund for Agroscientific Research in the Public Interest, China (20090300406), the Program for Changjiang Scholars and Innovative Research Team in University, China (PCSIRT, IRT0976), and the Chinese Scholarship Council, China (CSC). Special thanks go to Prof. Li Zhongan, Southwest University, China, for his kind help. References Albiach-Martí M R, Grosser J W, Gowda S, Mawassi M, Satyanarayana T, Garnsey S M. Dawson W O. 2004. Citrus tristeza virus replicates and forms infectious virions in protoplast of resistant citrus relatives. Molecular Breeding, 14, 117-128. Albiach-Martí M R, Mawassi M, Gowda S, Satyanayanana T, Hilf M E, Shanker S, Almira E C, Vives M C, López C, Guerri J, Flores R, Moreno P, Garnsey S M, Dawson W O. 2000. Sequences of Citrus tristeza virus separated in time and space are essentially identical. Journal of Virology, 74, 6856-6865. Albiach-Marti M R, Robertson C, Gowda S, Tatineni S, Belliure B, Garnsey S M, Folimonova S Y, Moreno P, Dawson W O. 2010. The pathogenicity determinant of Citrus tristeza virus causing the seedling-yellows syndrome maps at the 3´-terminal region of the viral genome. Molecular Plant Pathology, 11, 55-67. Ananthakrishna G, Venkataprasanna T, Roy A, Brlansky R H. 2010. Characterization of the mixture of genotypes of a Citrus tristeza virus isolate by reverse transcription- © 2011, CAAS. All rights reserved. Published by Elsevier Ltd. 354 quantitative real-time PCR. Journal of Virological Methods, 164, 75-82. Anonymous. 2008. Pakistan Statistical Year Book. Federal Bureau of Statistics, Govt of Pakistan. Anwar M S, Mirza M S. 1992. Survey of tristeza virus in Punjab (Pakistan). In: Proceedings of the 1st International Seminar on Citriculture in Pakistan. University of Agriculture Faisalabad. pp. 413-416. Arif M, Ahmed A, Ibrahim M, Hassan S. 2005. Occurrence and distribution of virus and virus-like diseases of citrus in north-west frontier province of Pakistan. Pakistan Journal of Botany, 37, 407-421. Asíns M J, Bernet G P, Ruiz C, Cambra M, Guerri J, Carbonell E. 2004. QTL analysis of Citrus tristeza virus-citradia interaction. Theory of Applied Genetics, 97, 1145-1154. Ayllón M A, Gowda S, Satyanarayana T, Dawson W O. 2004. Cis-elements at the opposite ends of the Citrus tristeza virus genome differ in initiation and termination of subgenomic RNAs. Virology, 322, 41-50. Ayllón M A, Gowda S, Satyanayanana T, Karasev A V, Adkins S, Mawassi M, Guerri J, Moreno P, Dawson W O. 2003. Effects of modification of the transcription initiation site context on Citrus tristeza virus subgenomic RNA synthesis. Journal of Virology, 77, 9232-9243. Ayllón M A, López C, Navas-Castillo J, Mawassi M, Dawson W O. 1999a. New defective RNAs from Citrus tristeza virus: evidence for a replicase driven template switching mechanism in their generation. Journal of General Virology, 80, 817-821. Ayllón M A, Rubio L, Moya A, Guerri J, Moreno P. 1999b. The haplotype distribution of two genes of Citrus tristeza virus is altered after host change or aphid transmission. Virology 255, 32-39. Ayllón M A, Rubio L, Sentandreu V, Moya A, Guerri J, Moreno P. 2006. Variations in two gene sequences of Citrus tristeza virus after host passage. Virus Genes, 32, 119-128. Ballester-Olmos J F, Pina J A, Navarro L. 1988. Detection of a tristeza-seedling yellows strain in Spain. In: Timmer L W, Garnsey S M, Navarro L, eds., Proceedings of the 10th Conference of the International Organization of Citrus Virologists (IOCV). IOCV, University of California, Riverside, CA. pp. 28-32. Barbarossa L, Savino V. 2006. Sensitive and specific digoxigeninlabelled RNA probes for routine detection of Citrus tristeza virus by dot blot hybridization. Journal of Phytopathology, 154, 329-335. Bar-Joseph M, Garnsey S M, Gonsalves D. 1979. The closteroviruses: a distinct group of elongated plant viruses. Advances in Virus Research, 25, 93-168. Bar-Joseph M, Loebenstein G, Cohen J. 1972. Further purification and characterization of threadlike particles associated with the citrus tristeza disease. Virology, 50, 821-828. Bar-Joseph M, Marcus R, Lee R F. 1989. The continuous Sagheer Atta et al. challenge of Citrus tristeza virus control. Annual Review of Physiology, 27, 291-316. Batuman O, Che Y, Moskowits O, Cohen M, Mawassi M, Bar-Joseph M. 2004. Variation in composition and biology of Citrus tristeza virus defective RNAs. APS Annual Meeting, Anaheim, California. Batuman O, Mawassi M, Bar-Joseph M. 2006. Transgenes consisting of a dsRNA of an RNAi suppressor plus the 3´ UTR provide resistance to Citrus tristeza virus sequences in Nicotiana benthamiana but not in citrus. Virus Genes, 33, 319-327. Broadbent P, Bevington K B, Coote B G. 1991. Control of stem-pitting of grapefruit in Australia by mild strain cross protection. In: Proceedings of the 11th Conference of the International Organization of Citrus Virologists (IOCV). IOCV, University of California, Riverside, CA. pp. 64-70. Broadbent P, Brlansky R H, Indsto J. 1996. Biological characterization of Australian isolates of Citrus tristeza virus and separation of sub-isolates by single aphid transmission. Plant Disease, 80, 329-333. Cambra M, Bertolini E, Olmos A, Capote N. 2006. Molecular methods for detection and quantitation of virus in aphids. In: Cooper I, Kuhne T, Polischuk V, eds., Virus diseases and Crop Biosecurity. Springer, Dordrecht, The Netherlands. pp. 81-88. Cambra M, Camarasa E, Gorris M T, Garnsey S M, Gumpf D J, Tsai M C. 1993. Epitope diversity of isolates of Citrus tristeza virus in Spain. In: Proceedings of the 12th Conference of the International Organization of Citrus Virologists (IOCV). IOCV, University of California, Riverside, CA. pp. 33-38. Cambra M, Gorris M T, Marroquín C, Román M P, Olmos A, Martinez M C, Hermoso de Mendoza A, López A, Navarro L. 2000a. Incidence and epidemiology of Citrus tristeza virus in the Valencian Community of Spain. Virus Research, 71, 85-95. Cambra M, Gorris M T, Román M P, Terrada E, Garnsey S M, Camarasa E, Olmos A, Colomer M. 2000b. Routine detection of Citrus tristeza virus by direct Immunoprinting-ELISA method using specific monoclonal and recombinant antibodies. In: Proceedings of the 14th Conference of the International Organization of Citrus Virologists (IOCV). IOCV, University of California, Riverside, CA. pp. 34-41. Cambra M, Moreno P, Navarro L. 1979. Detección rápida del virus de la tristeza de los cítricos (CTV) mediante la técnica inmunoenzimática ELISA-sandwich. Ann. INIA, Ser. Prot. Veg. 12, 115-125. (in Spanish) Cambra M, Olmos A, Gorris M T, Marroquín C, Esteban O, Garnsey S M, Llauger R, Batista L, Peña I, Hermoso de Mendoza A. 2000c. Detection of Citrus tristeza virus by print capture and squash capture-PCR in plant tissues and single aphids. In: Proceedings of the 14th Conference of the International Organization of Citrus © 2011, CAAS. All rights reserved. Published by Elsevier Ltd. Distribution and Research Advances of Citrus tristeza virus Virologists (IOCV). IOCV, University of California, Riverside, CA. pp. 42-49. Catara A, Azzaro A, Davino M, Grimaldi V, Hussain M, Salim A, Mirza M S. 1991. A survey for tristeza and greening in Punjab (Pakistan). In: Proceedings of the 11th Conference of the International Organization of Citrus Virologists (IOCV). IOCV, University of California, Riverside, CA. pp. 166-170. Catara A, Azzaro A, Mughal S M, Khan D A. 1988. Virus, viroids and prokaryotic diseases of citrus in Pakistan. In: Proceedings of the 6th Conference of the International Organization of Citrus Virologists (IOCV). IOCV, University of California, Riverside, CA. pp. 957-962. Chalfant A S, Chapman R K. 1962. Transmission of cabbage viruses A and B by the cabbage aphid and the green peach aphid. Journal of Economical Entomology, 55, 584-590. Che X, Dawson W O, Bar-Joseph M. 2003. Defective RNAs of Citrus tristeza virus analogous to Crinivirus genomic RNAs. Virology, 310, 298-309. Che X, Piestun D, Mawassi M, Satyanarayana T, Gowda S, Dawson W O, Bar-Joseph M. 2001. 5´-Coterminal subgenomic RNAs in Citrus tristeza virus infected cells. Virology, 283, 374-381. Costa A S, Müller G W. 1980. Tristeza control by cross protection: a US-Brazil cooperative success. Plant Disease, 64, 538-541. Dasgupta I, Malathi V J, Mukherjee S K. 2003. Genetic engineering for virus resistance. Current Science, 84, 341-354. Davino S, Davino M, Sambade A, Guardo M, Caruso A. 2003. The first Citrus tristeza virus outbreak found in a relevant citrus producing area of Sicily, Italy. Plant Disease, 87, 314. Domínguez A, Hermoso de Mendoza A, Guerri J, Cambra M, Navarro L, Moreno P, Peña L. 2002. Pathogenderived resistance to Citrus tristeza virus in transgenic Mexican lime (Citrus aurantifolia (Christm.) Swing.) plants expressing its p25 coat protein gene. Molecular Breeding, 10, 1-10. EPPO. 2006. Citrus tristeza closterovirus. Distribution maps of quarantine pests for Europe. Data sheet on quarantine pesrs of Europe. Fabre F, Kervarrec C, Mieuzet L, Riault G, Vialatte A, Jacquot E. 2003. Improvement of Barley yellow dwarf virusPAV detection in single aphids using a fluorescent real time RT-PCR. Journal of Virology Methods, 110, 51-60. Fagoaga C, López C, Moreno P, Navarro L, Flores R, Peña L. 2005. Viral-like symptoms induced by the ectopic expresión of the p23 of Citrus tristeza virus are citrus specific and do not correlate with the pathogenicity of the virus strain. Molecular Plant-Microbe Interactions, 18, 435-445. Febres V J, Ashoulin L, Mawassi M, Frank A, Bar-Joseph M, Manjunath K L, Lee R F, Niblett C L. 1996. The p27 355 protein is present at one end of Citrus tristeza virus particles. Phytopathology, 86, 1331-1335. Febres V J, Niblett C L, Lee R F, Moore G A. 2003. Characterization of grapefruit plants (Citrus paradisi Macf.) transformed with citrus tristeza closterovirus genes. Plant Cell Reports, 21, 421-428. Fraser L. 1952. Seedling-yellows, an unreported virus disease of citrus. Agricultural Gazette of New South Wales, 63, 125-131. Garnsey S M, Barrett H C, Hutchinson D J. 1987. Identification of Citrus tristeza virus resistance in citrus relatives and its potential applications. Phytophylactica, 19, 187-191. Garnsey S M, Gonsalves D, Purcifull D E. 1977. Mechanical transmission of Citrus tristeza virus. Phytopathology, 67, 965-968. Garnsey S M, Gottwald T R, Yokomi R K. 1998. Control strategies for Citrus tristeza virus. In: Hadidi A, Khetarpal R, Koganezawa H, eds., Plant Virus Disease Control. APS Press, St. Paul, MN. pp. 639-658. Garnsey S M, Gottwald T R, Hilf M E, Matos L, Borbón J. 2000. Emergence and spread of severe strains of Citrus tristeza virus isolates in the Dominican Republic. In: da Graça J V, Lee R F, Yokomi R K, eds., Proceedings of the 14th Conference of the International Organization of Citrus Virologists (IOCV). IOCV, Riverside, CA. pp. 57-68. Ghorbel R, López C, Moreno P, Navarro L, Flores R, Peña L. 2001. Transgenic citrus plants expressing the Citrus tristeza virus p23 protein exhibit viral-like symptoms. Molecular Plant Pathology, 2, 27-36. da Graça J V, Marais L J, von Broembsen L A. 1984. Severe tristeza stem pitting decline of young grapefruit in South Africa. In: Proceedings of the 9th Conference of the International Organization of Citrus Virologists (IOCV). IOCV, University of California, Riverside, CA. pp. 62-65. Gottwald T, Polek M, Riley K. 2002. History, present incidence, and spatial distribution of Citrus tristeza virus in the California Central Valley. In: Duran-Vila N, Milne R G, da Graça J V, eds., Proceedings of the 15th Conference of the International Organization of Citrus Virologists. IOCV, Riverside, CA. pp. 83-94. Gottwald T R, Garnsey S M, Cambra M, Moreno P, Irey M, Borbón J. 1996. Differential effects of Toxoptera citricida vs. Aphis gossypii on temporal increase and spatial patterns of spread of citrus tristeza. In: Proceedings of the 13th Conference of the International Organization of Citrus Virologists (IOCV). IOCV, University of California, Riverside, CA. pp. 120-129. Gottwald T R, Gibson G J, Garnsey S M, Irey M. 1999. Examination of the effect of aphid vector population composition on the spatial dynamics of citrus tristeza virus spread by stochastic modeling. Phytopathology, 89, 603-608. © 2011, CAAS. All rights reserved. Published by Elsevier Ltd. 356 Gowda S, Stayanarayana T, Ayllón M A, Albiach-Marti M R, Mawass M, Rabindran S, Garnsey S M, Dawson W O. 2001. Characterization of the cis-acting elements controlling subgenomic mRNAs of Citrus tristeza virus: production of positive- and negative-stranded 3´terminal and positive-stranded 5´-terminalRNAs. Virology, 286, 151-154. Gowda S, Stayanarayana T, Ayllón M A, Moreno P, Flores R, Dawson W O. 2003. The conserved structures of the 5´ nontranslated region of Citrus tristeza virus are involved in replication and virion assembly. Virology, 317, 50-64. Gowda S, Satyanayanana T, Davis C L, Navas-Castillo J, Albiach-Martí M R, Mawassi, M, Valkov N, Bar-Joseph M, Moreno P, Dawson W O. 2000. The p20 gene product of Citrus tristeza virus accumulates in the amorphous inclusion bodies. Virology, 274, 246-254. Gowda S, Satyanarayana T, Robertson C J, Garnsey S M, Dawson W O. 2005. Infection of citrus plants with virions generated in Nicotiana benthamiana plants agroinfiltrated with binary vector based Citrus tristeza virus. In: Proceedings of the 16th Conference of the International Organization of Citrus Virologists (IOCV). IOCV, University of California, Riverside, CA. pp. 23-33. Grimaldi V, Catara A. 1989. Detection of citrus tristeza and greening in Pakistan through electron microscopy. Journal of Phytopathology, 126, 17-21. Halbert S E, Genc H, Çevik B, Brown L G, Rosales I M, Manjunath K L, Pomerinke M, Davison D A, Lee R F, Niblett C L. 2004. Distribution and characterization of Citrus tristeza virus in South Florida following establishment of Toxoptera citricida. Plant Disease, 88, 935-941. Hilf M, Mavrodieva V A, Garnsey S M. 2005. Genetic marker analysis of a global collection of isolates of Citrus tristeza virus: Characterization and distribution of CTV genotypes and association with symptoms. Phytopathology, 95, 909-917. Iftikhar Y, Khan M A, Rashid A, Mughal S M, Iqbal Z, Batool A, Abbas M. 2009. Occurrence and distribution of citrus tristeza Closterovirus in the Punjab and NWFP, Pakistan. Pakistan Journal of Botany, 41, 373-380. Iglesias N G, Gago-Zachert S P, Robledo G, Costa N, Plata M I, Vera O. 2008. Population structure of Citrus tristeza virus from field Argentinean isolates. Virus Genes, 36, 199-207. Jiang B, Hong N, Wang G P. 2008. Characterization of Citrus tristeza virus strains from southern China based on analysis of restriction patterns and sequences of their coat protein genes. Virus Genes, 37, 185-92. Karasev A V, Boyko V P, Gowda S, Nikolaeva O V, Hilf M E, Koonin E V, Niblett C L, Cline K, Gumpf D J, Lee R F, Garnsey S M, Lewandowski D J, Dawson W O. 1995. Complete sequence of the Citrus tristeza virus RNA genome. Virology, 208, 511-520. Sagheer Atta et al. Khan I A. 1992. Virus and virus like diseases of citrus. In: Proceedings of 1st International Seminar on Citriculture. Pakistan. 2-5 Dec., 1992. University of Agriculture, Faisalabad, Pakistan. pp. 343-352. Lee R F, Brlansky R H. 1990. Variation in the severity of citrus tristeza virus isolates from groves with quick decline. Proceedings of the Florida State Horticulture Society, 102, 1-3. Lee R F, Rocha-Pena M A. 1992. Citrus tristeza virus. In: Kumar J, Chaube H S, Singh U S, Mukhopadhyay A N, eds., Plant Disease of International Importance. Disease of Fruit Crops. Prentice Hall, Englewood Cliffs, NJ. vol. III. 456. pp. 226-249. Lewsey M, Robertson F C, Canto T, Palukaitis P, Carr J P. 2007. Selective targeting of miRNA-regulated plant development by a viral counter-silencing protein. The Plant Journal, 50, 240-252. Lim W L, Hagedorn D J. 1977. Bimodal transmission of plant viruses. In: Harris K F, Maramorosch K, eds., Aphids as Virus Vectors. Academic Press, New York, USA. pp. 237-251. Lopez C, Navas-Castillo J, Gowda S, Moreno P, Flores R. 2000. The 23-kDa protein coded by the 3´-terminal gene of Citrus tristeza virus is an RNA-binding protein. Virology, 269, 462-470. Lu R, Folimonov A, Shintaku M, Li W X, Falk B W, Dawson W O, Ding S W. 2004. Three distinct suppressors of RNA silencing encoded by a 20-kb viral RNA genome. Proceedings of the National Academy of Sciences of the United States of America, 101, 15742-15747. Mawassi M, Mietkiewska E, Gofman R, Yang G, Bar-Joseph M. 1996. Unusual sequence relationships between two isolates of Citrus tristeza virus. Journal of General Virology, 77, 2359-2364. Mehta P, Brlansky R H, Gowda S, Yokomi R K. 1997. Reverse transcription polymerase chain reaction detection of Citrus tristeza virus in aphids. Plant Disease, 81, 10661069. Meneghini M. 1946. Sôbre a natureza e transmissibilidade da doença “tristeza” dos citrus. Biológico, 12, 285287. (in Portuguse) Moreno P, Ambros S, Albiach-Marti M R, Guerri J, Pena L. 2008. Citrus tristeza virus: a pathogen that changed the course of the citrus industry. Molecular Plant Pathology, 9, 251-268. Moreno P, Guerri J, Ballester-Olmos J F, Albiach R, Martínez M E. 1993. Separation and interference of strains from a citrus tristeza virus isolate evidenced by biological activity and double-stranded RNA (dsRNA) analysis. Plant Pathology, 42, 35-41. Mughal S M. 2004. Symptomatology, detection, distribution and management of virus and virus-like diseases of citrus in Pakistan. In: Proceedings of the 1st International Conference on Citriculture. University of Agriculture, Faisalabad. pp. 97-105. Nelson A, Roth A D, Johnson J D. 1993. Tobacco mosaic © 2011, CAAS. All rights reserved. Published by Elsevier Ltd. Distribution and Research Advances of Citrus tristeza virus virus infection of transgenic Nicotiana tabacum plants is inhibitted by antisense constructs directed at the 5´ region of viral RNA. Gene, 127, 227-232. Nikolaeva O V, Karasev A V, Powell C A, Gumpf D J, Garnsey S M, Lee R F. 1996. Mapping of epitopes for Citrus tristeza virus-specific monoclonal antibodies using bacterially expressed coat protein fragments. Phytopathology, 86, 974-979. Nolasco G, de Blas C, Torres V, Ponz F. 1993. A method combining immunocapture and PCR amplification in a microtiter plate for the routine diagnosis of plant viruses and subviral pathogens. Journal of Virological Methods, 45, 201-218. Olmos A, Bertolini E, Cambra M. 2002. Simultaneous and co-operational amplification (Co-PCR): a new concept for detection of plant viruses. Journal of Virological Methods, 106, 51-59. Olmos A, Cambr M, Esteban O, Gorris M T, Terrada E. 1999. New device and method for capture, reverse transcription and nested PCR in a single closed tube. Nucleic Acids Research, 27, 1564-1565. Pappu H, Karasev A V, Anderson A J, Pappu S, Hilf M E, Febres V, Eckloff R M G, McCaffery M, Boyko V, Gowda S, Dolja V V, Koonin E V, Gumpf D J, Cline K, Garnsey S M, Dawson WO, Lee, R F, Niblett C L. 1995. Citrus tristeza virus represents a new virus group encoding a HSP70 protein. Virology, 199, 35-46. Peremyslov V V, Pan Y W, Dolja V V. 2004. Movement protein of a closterovirus is a type III integral transmembrane protein localized to the endoplasmic reticulum. Journal of Virology, 78, 3704-3709. Permar T A, Garnsey S M, Gumpf D J, Lee R. 1990. A monoclonal antibody that discriminates strains of Citrus tristeza virus. Phytopathology, 80, 224-228. Powell-Abel P A, Nelson R S, De B, Hoffman 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. Rai M. 2006. Refinement of the Citrus tristeza virus resistance gene (Ctv) positional map in Poncirus trifoliata and generation of transgenic grapefruit (Citrus paradisi) plant lines with candidate resistance genes in this region. Plant Molecular Biology, 61, 399-414. Retuerma M L, Price W C. 1972. Evidence that tristeza virus is stylet-borne. Food and Agriculture Organization Plant Protection Bulletin, 20, 111-114. Rocha-Peña M A, Lee R F, Lastra R, Nibblet C L, OchoaCorona F M, Garnsey S M, Yokomi R K. 1995. Citrus tristeza virus and its aphid vector Toxoptera citricida. Plant Disease, 79, 437-445. Roistacher C N. 1981. A blueprint for disaster. I. The history of seedling yellows disease. Citrograph, 67, 4-24. Roistacher C N. 1991. Graft-transmissible diseases of citrus. In: Handbook for Detection and Diagnosis. FAO, Rome. 357 Roistacher C N, Dodds J A, Bash J A. 1988. Cross protection against citrus tristeza seedling-yellows and stem-pitting viruses by protective isolates developed in green-house plants. In: Proceedings of the 10th Conference of the International Organization of Citrus Virologists (IOCV). IOCV, University of California, Riverside, CA. pp. 91-100. Román M P, Cambra M, Juárez J, Moreno P, Duran-Vila N, Tanaka F A O, Alves E, Kitajima E W, Yamamoto P T, Bassanezi R B, Teixeira D C, Junior W C J, Ayres A J, Gimenes-Fernandes N, Rabenstein F, Girotto L F, Bové J M. 2004. Sudden death of citrus in Brazil: a grafttransmissible bud union disease. Plant Disease, 88, 453-467. Roy A, Brlansky R H. 2004. Genotype classification and molecular evidence for the presence of mixed infections in Indian citrus tristeza virus isolates. Archives of Virology, 149, 1911-1929. Rubio L, Ayllón M A, Guerri J, Pappu H, Niblett C L, Moreno P. 1996. Differentiation of citrus tristeza closterovirus (CTV) isolates by single-strands conformation polymorphism analysis of the coat protein gene. Annal of Applied Biology, 129, 479-489. Ruiz-Ruiz S, Moreno P, Guerri J, Ambrós S. 2006. The complete nucleotide sequence of a severe stem pitting isolate of Citrus tristeza virus from Spain: comparison with isolates from different origins. Archive of Virology, 151, 387-398. Sambade A, Ambrós S, López C, Ruiz-Ruiz S, Hermoso de Mendoza A, Flores R, Guerri J, Moreno P. 2007. Preferential accumulation of severe variants of Citrus tristeza virus in plants coinoculated with mild and severe variants. Archive of Virology, 152, 1115-1126. Sambade A, López C, Rubio L, Flores R, Guerri J, Moreno P. 2003. Polymorphism of a specific region in the gene p23 of Citrus tristeza virus allows differentiation between mild and severe isolates. Archive of Virology, 148, 22812291. Sanford J C, Johnston S A. 1985. The concept of parasitederived resistance genes from the parasite’s own genome. Journal of Theoretical Biology, 113, 395-405. Saponari M, Manjunath K, Yokomi R K. 2008. Quantitative detection of Citrus tristeza virus in citrus and aphids by real-time reverse transcription-PCR (TaqMan). Journal of Virological Methods, 147, 43-53. Satyanarayana T, Gowda S, Ayllón M A, Albiach-Martí M R, Rabindram R, Dawson W O. 2002. The p23 protein of Citrus tristeza virus controls asymmetrical RNA accumulation. Journal of Virology, 76, 473-483. Satyanayanana T, Gowda S, Ayllón M A, Dawson W O. 2004. Closterovirus bipolar virion: evidence for initiation of assembly by minor region. coat protein and its restriction to the genomic RNA 5´. Proceedings of the National Academy of Sciences of the United States of America, 101, 799-804. Satyanayanana T, Gowda S, Boyko V P, Albiach-Martí M © 2011, CAAS. All rights reserved. Published by Elsevier Ltd. 358 R, Mawassi M, Navas-Castillo J, Karasev A V, Dolja V, Hilf M E, Lewandowsky D J, Moreno P, Bar-Joseph M, Garnsey S M, Dawson W O. 1999. An engineered closterovirus RNA replicon and analysis of heterologous terminal sequences for replication. Proceedings of the National Academy of Sciences of the United States of America, 96, 7433-7438. Schwarz R E. 1965. Aphid-borne virus diseases of citrus and their vectors in South Africa. B. Flight activity of citrus aphids. South African Journal Agricultural Science, 8, 931-940. Suastika G, Natsuaki T, Terui H, Kano T, Ieki H, Okuda S. 2001. Nucleotide sequence of Citrus tristeza virus seedling yellows isolate. Journal of General Plant Pathology, 67, 73-77. Folimonova S Y, Robertson C J, Shilts T, Folimonov A S, Hilf M E, Garnsey S M, Dawson W O. 2010. Infection with strains of Citrus tristeza virus does not exclude superinfection by other strains of the virus. Journal of Virology, 84, 1314-1325. Tatineni S, Robertson C, Garnsey S M, Bar-Joseph M, Gowda S, Dawson W O. 2008. Three genes of Citrus tristeza virus are dispensable for infection and movement throughout some varieties of citrus. Virology, 376, 297-307. Terrada E, Kerschbaumer R J, Giunta G, Galeffi P, Himmler G, Cambra M. 2000. Fully “recombinant enzyme-linked immunosorbent assays” using genetically engineered single-chain antibody fusion proteins for detection of Citrus tristeza virus. Phytopathology, 90, 1337-1344. Timmer L W, Garnsey S M, Graham J H. 2000. Compendium of Citrus Diseases. APS Press, St Paul, MN. Tsai J H, Liu Y H, Wang J J, Lee R F. 2000. Recovery of orange stem-pitting strains of Citrus tristeza virus following single aphid transmissions with Toxoptera citricida from a Florida decline isolate of CTV. Proceedings of the Florida State Horticultural Society, 113, 75-78. Vaudano E, Costantini A, Cersosimo M, Prete V D, GarciaMoruno E. 2009. Application of real-time RT-PCR to Sagheer Atta et al. study gene expression in active dry yeast (ADY) during the rehydration phase. International Journal of Food Microbiology, 129, 30-36. Vives M C, Rubio L, Sambade A, Mirkov, Moreno P, Guerri J. 2005. Evidence of multiple recombination events between two RNA sequence variants within a Citrus tristeza virus isolate. Virology, 331, 232-237. Weathers L G, Hartung M K. 1964. Transmission of citrus viruses by dodder, Cuscuta subinclusa. Plant Disease Report, 48, 102-103. Yokomi R K, Lastra R, Stoetzel M B, Damsteegt D, Lee R F, Garnsey S M, Gottwald T R, Rocha-Peña M A, Niblett C L. 1994. Establishment of the brown citrus aphid in Central America and the Caribbean Basin and transmission of the Citrus tristeza virus. Journal of Economical Entomology, 87, 1078-1085. Zaccomer B, Cellier F, Boyer J C, Haenni A L, Tepfer M. 1993. Transgenic plants that express genes including the 3´ untranslated region of the Turnip yellow mosaic virus (TYMV) genome are partially protected against TYMV infection. Gene, 136, 87-94. Zaragoza S. 2007. Aproximación a la historia de los cítricos. PhD thesis, Origen, dispersión y evolución de su uso y cultivo, Universidad Politécnica de Valencia. (in Spanish) Zhou C Y, Deborah H, Rachael C, Patricia B, John B. 2001. A method for micro and rapid extraction of Citrus tristeza virus (CTV) nucleic acid applied to RT-PCR amplification. Journal of Fujian Agricultural University, 30, 200. (in Chinese) Zhou C Y. 1997. Occurrence guidelines and outlook of Citrus tristeza virus in China. In: Proceedings of 1st Chinese Symposium on Plant Virus and Viral Diseases Control Researches. China Agricultural Scientech Press, Beijing. pp. 182-187. (in Chinese) Zhou Y, Zhou C Y, Song Z, Liu K H, Yang F Y. 2007. Characterization of Citrus tristeza virus isolates by indicators and molecular biology methods. Agricultural Sciences in China, 6, 573-579. (Managing editor ZHANG Juan) © 2011, CAAS. All rights reserved. Published by Elsevier Ltd.