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Electron Microscopy of Viruses

and the pentameric penton base. By fitting the crystal structures into the EM density, a quasi-atomic model can be derived that describes the exact position of all capsomers and the interacting surfaces and protein loops between the capsomers. Figure 6 shows details of the fit of the crystal structure of the penton base into a penton base from the EM model. A number of different algorithms are available to perform these fits, some of which take into account the possibility of flexibility between protein domains. The fit in Figure 6 (arrow) shows density in the EM model that is not filled by density from the crystal structure. Some loops of the molecule are flexible and therefore invisible in the crystal structure, whereas the lower resolution of the EM model does allow imaging.

The Future In our opinion, the future of EM in virology lies in an extended use of the combination of structural data as shown above. Fitting crystal structures of intact proteins or domains into protein complexes (viral complexes or complexes between viral and host proteins) will show how proteins interact in the infected cell. Although the EM models have a resolution of only around 10 A˚, the information on the interface of the proteins in the complex is of near-atomic resolution. These techniques can be applied to regular viruses. For irregular viruses, such as influenza and paramyxoviruses, tomography will allow the construction of models with a resolution of around 40 A˚ that will show how the proteins in the virus particle interact. In particular, it will be interesting to see the packaging of the nucleocapsids in these viruses. At present we do not know how the DNA is packaged in adenovirus particles and it is possible that the icosahedral averaging techniques result in a loss of this information if the packing symmetry of the DNA is in fact not icosahedral. The same problem exists for capsid features that are not

icosahedral such as unique capsid vertices that may be used for the packaging of DNA in adenoviruses and herpesviruses. Such problems may be overcome by using reconstruction techniques that are not based on icosahedral symmetry. See also: Virus Particle Structure: Nonenveloped Viruses; Virus Particle Structure: Principles.

Further Reading Al-Amoudi A, Dubochet J, and Studer D (2002) Amorphous solid water produced by cryosectioning of crystalline ice at 113 K. Journal of Microscopy 207: 146–153. Al-Amoudi A, Chang JJ, Leforestier A, et al. (2004) Cryo-electron microscopy of vitreous sections. EMBO Journal 23: 3583–3588. Chiu W, Baker ML, Jiang W, Dougherty M, and Schmid MF (2005) Electron cryomicroscopy of biological machines at subnanometer resolution. Structure 13: 363–372. Dubochet J, Adrian M, Chang JJ, et al. (1988) Cryo-electron microscopy of vitrified specimens. Quarterly Reviews of Biophysics 21: 129–228. Fabry CM, Rosa-Calatrava M, Conway JF, et al. (2005) A quasi-atomic model of human adenovirus type 5 capsid. EMBO Journal 24: 1645–1654. Griffiths G, McDowall A, Back R, and Dubochet J (1984) On the preparation of cryosections for immunocytochemistry. Journal of Ultrastructure Research 89: 65–78. Grunewald K and Cyrklaff M (2006) Structure of complex viruses and virus-infected cells by electron cryo tomography. Current Opinion in Microbiology 9(4): 437–442. Harris JR (1997) Royal Microscopy Society Handbook 35: Negative Staining and Cryoelectron Microscopy. Oxford: Bios Scientific Publishers. Sartori N, Richter K, and Dubochet J (1993) Vitrification depth can be increased more than 10-fold by high pressure freezing. Journal of Microscopy 172: 55–61. Tokuyasu KT (1986) Application of cryoultramicrotomy to immunocytochemistry. Journal of Microscopy 143: 139–149. Tokuyasu KT and Singer SJ (1976) Improved procedures for immunoferritin labeling of ultrathin frozen sections. Journal of Cell Biology 71(3): 894–906. Wepf R, Amrein M, Burkli U, and Gross H (1991) Platinum/iridium/ carbon: A high-resolution shadowing material for TEM, STM and SEM of biological macromolecular structures. Journal of Microscopy 163: 51–64.

Emerging and Reemerging Virus Diseases of Plants G P Martelli and D Gallitelli, Universita` degli Studi and Istituto di Virologia Vegetale del CNR, Bari, Italy ã 2008 Elsevier Ltd. All rights reserved.

Introduction A number of RNA plant viruses constitute new threats to economically relevant vegetable crops, grapevine, and citrus. These are listed among new disease-causing agents,

and are therefore denoted ‘emerging or re-emerging’ being often new viruses or virus strains responsible for serious diseases. The emerging plant viruses and their known vectors are quickly expanding in new areas as a consequence of the increasing international trade and the

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large numbers of people traveling in many countries. Once introduced, the fitness of these viruses to new agroclimatic conditions increases, leading to the development of severe epiphytotics.

Vegetable Viruses Pepino Mosaic Virus Geographical distribution

Pepino mosaic virus (PepMV) was originally described in Peru on pepino (Solanum muricatum) and found to infect tomato and related wild species symptomlessly, in experimental trials. Since 1999, PepMV outbreaks have been reported almost simultaneously in many European countries (Austria, Bulgaria, Finland, France, Germany, Hungary, Italy, Netherlands, Norway, Poland, Slovakia, Spain, Sweden, Switzerland, Ukraine, and UK) where it is considered an emerging pathogen of tomato glasshouses. PepMV was also found in Canada, USA (Arizona, California, Colorado, Florida, Oklahoma, Texas), Ecuador, and Chile. After discovering the PepMV occurrence in tomato grown in Europe and North America, a survey carried out in central and southern Peru and Ecuador demonstrated that the virus was present in Peruvian tomato crops as well as in the following wild Lycopersicon species: L. chilense, L. chmilewskii, L. parviflorum, L. peruvianum, and in L. pimpinellifolium in Ecuador. Disease symptoms and yield losses

In pepino, the virus causes yellow mosaic in young leaves, whereas affected tomato plants show a wide array of symptoms. These include stunting of the whole plant, bubbling of the leaf surface, interveinal chlorosis, mosaic and green striations on stem and sepals. Foliar symptoms resemble hormonal herbicide damage, while lower leaves show necrotic lesions that resemble damage caused by water that dripped onto the plant. Fruits from early infected tomato plants may show blotchy ripening and gold marbling. Ripe fruits develop yellow speckles and spots that make them unmarketable. Observations by Dutch scientists indicate that symptoms are more readily seen during autumn and winter months, and are masked during warmer months. Yield losses caused by PepMV are probably below 5%, although surveys in glasshouse-grown tomatoes reported crop reductions up to 40%. The disease spreads very rapidly and crop losses may be significant if early action is not taken to eliminate infected sources. Although Peruvian virus strains seem to be little virulent, new strains of higher virulence could arise through recombination. Because of the adverse effects on quality and yield, PepMV is becoming one of the most important tomato pathogens in Europe.

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Causal agent and classification PepMV (genus Potexvirus, family Flexiviridae) has helically constructed semirigid filamentous particles with a modal length of 508 nm and a diameter of 11 nm. Sequencing of a number of strains has shown that virions contain a single molecule of linear, positive-sense, single-stranded RNA (ssRNA) 6410–6425 nt in size. The genome organization is typical of the genus Potexvirus, with five open reading frames (ORFs): ORF1, encoding a putative replicase of 164 kDa; ORFs 2–4, coding for the triple gene block proteins 1–3 (TGBp ) of 26 kDa, 14 kDa, and 9 kDa, respectively; and ORF5, encoding the 25 kDa coat protein (CP). Phylogenetic analyses carried out on replicase, TGBp1, and CP amino acid sequences revealed that PepMV is closely related to narcissus mosaic virus (NMV), scallion virus X (SVX), cymbidium mosaic virus (CymMV), and potato aucuba mosaic virus (PAMV) (Figure 1). Tomato isolates of PepMV can be distinguished from the original isolate from pepino on the basis of symptomatology, host range, and sequence data. On the other hand, tomato isolates from Europe appear very similar to each other sharing nucleotide sequence identities higher than 99% and in the range of 95–96% with PepMV from L. peruvianum from Peru. Nucleotide sequence comparisons between US and the European tomato isolates show only 79–82% identity. Gene-for-gene comparison between sequenced isolates suggests that TGBp1 and TGBp3 are more suitable than either the replicase or CP gene products for discriminating virus isolates. Epidemiology PepMV is transmitted by contact and readily spread mechanically by contaminated hands, tools, shoes, clothing, and plant-to-plant contact. The virus is thought to remain viable in dry plant material for as long as 3 months. Seed transmission or surface seed contamination in tomato is suspected but not demonstrated. In the UK, the virus is frequently found in imported fruits. Infection of other solanaceous crops such as eggplant, tobacco, and potato has only been observed in experimental trials, while infection in pepper has not been demonstrated. Cucumber can be artificially infected but the virus does not appear to spread systemically in the plant. Alternative hosts that may serve as virus reservoirs were studied in Spain, where native plants with viruslike symptoms growing in or around tomato fields were collected and analyzed for the presence of PepMV. As many as 18 weed species were found to be infected. Because of the high similarity of tomato virus isolates, a common origin seems likely although where this origin is located is still unclear. The extent of PepMV distribution in Peru, together with the fact that many of the wild Peruvian Lycopersicon populations sampled were isolated and had not been manipulated by man, led to the conclusion that this virus has been present in the region for a

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Figure 1 (a) Strong symptoms of PepMV on tomato; (b) symptom of CYSDV on melon; (c) severe vein yellowing on cucumber infected by CVYV; (d) swelling at the graft union and death of the scion of a vine grafted on Kober 5BB, infected by Grapevine leafroll-associated virus 2; (e) a citrus grove affected by sudden death disease; and (f) a citrus plant killed by sudden death disease. (a) Courtesy of Dr. E. Moriones. (b) Courtesy of Dr. E. Moriones. (c) Courtesy of Dr. I. M. Cuadrado. (e, f) Courtesy of Dr. J.Bove`.

long time and that other factors might be involved in its spread. Myzus persicae does not transmit PepMV. In southern Spain a faster spread of PepMV was observed in bumble-bee pollinated greenhouses than in those with no pollinator insects. Control

Recommended control strategies for PepMV focus on sanitation. Use of certified seed lots, complete removal (including roots) of infected plants, limited access to affected rows, and sanitation of clothing and tools are all critical. The increasing concern caused by this new disease has led the European Commission to authorize member countries to take measures to prevent the spread of PepMV within the European Union. Criniviruses Criniviruses are whitefly-transmitted RNA viruses that cause plant diseases with increasingly important yield impact, consequent to the sudden explosion of whitefly populations in temperate regions in the last 10–20 years. All these viruses induce yellowing symptoms in their hosts, are generally phloem-limited, nonmechanically

transmissible, and have large ssRNA genomes. Cucurbit yellow stunting disorder virus (CYSDV), tomato infectious chlorosis virus (TICV), and tomato chlorosis virus (ToCV) are important emerging criniviruses. Cucurbit Yellow Stunting Disorder Virus Geographical distribution CYSDV was first observed in the southeastern coast of Spain, on melon and cucumber grown under plastic. The virus was also found in the Canary Islands, Egypt, France, Israel, Jordan, Lebanon, Mexico, Morocco, Portugal, Saudi Arabia, Syria, Texas, Turkey, and the United Arab Emirates. Disease symptoms and yield losses Protected crops of cucumber and melon show severe yellowing that starts as interveinal mottle of older leaves to develop into complete yellowing of the leaf lamina, except for the veins, followed by rolling, brittleness, and stunting. On the whole, symptoms are virtually indistinguishable from those caused by beet pseudo yellows virus (BPYV). Zucchini can also be infected but symptoms have not been described. Disease incidence in protected

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crops can easily reach 100%. In Spain and many other countries, CYSDV is considered the prevailing virus of protected cucurbit crops. Causal agent and classification

CYSDV (genus Crinivirus, family Closteroviridae) has a narrow host range limited to the family Cucurbitaceae and is confined to phloem tissues. Virions are flexuous filaments with lengths between 750 and 800 nm. Genome consists of two molecules of ssRNA of plus-sense polarity designated RNA-1 and -2. RNA-1 is 9123–9126 nt long and contains five ORFs with papain-like protease, methyltransferase, RNA helicase, and RNA-dependent RNA polymerase domains in the first two overlapping ORFs, a small 5 kDa hydrophobic protein, and two further downstream ORFs potentially encoding proteins 25 and 22 kDa in size, respectively. RNA-2 is 7976 nt long and contains the hallmark ORFs of the family Closteroviridae, encoding, in the order, a heat shock protein 70 (HSP70) homolog, a 59 kDa protein, the major (CP) and the minor (CPm) CP. In the 30 -terminal region, RNA-1 contains an ORF potentially encoding a protein of 25 kDa which has no homologs in any databases, and RNA-2 has an unusually long 59 noncoding region. Subgenomic RNAs were detected in CYSDV-infected plants, suggesting that they serve for the expression of internal ORFs. CYSDV can be divided into two divergent groups of isolates. One group is composed of isolates from Spain, Lebanon, Jordan, Turkey, and North America, the other of isolates from Saudi Arabia. Nucleotide identity between isolates of the same group is greater than 99%, whereas identity between groups is about 90%. Epidemiology

Natural hosts of CYSDV are restricted to cucurbits: watermelon, melon, cucumber, and zucchini. Cucurbita maxima and Lactuca sativa are experimental host plants. The life cycle of CYSDV is dependent on its vector, the whitefly Bemisia tabaci, as viral outbreaks are associated with heavy infestations of whitefly biotypes A, B, and Q. Transmission of CYSDV by biotype B is more effcient than by biotype A, whereas biotype Q transmits as efficiently as biotype B. Trialeurodes vaporariorum was displaced by B. tabaci as the dominant whitefly along the southeastern coast of Spain when CYSDV took over BPYV as the agent of yellowing diseases of cucurbits. Acquisition periods of 18 h or more and inoculation periods of 24 h or more seem necessary for high transmission rates of CYSDV, which can persist for at least 9 days in the vector. The virus is not known to be seed-borne.

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and in reducing the incidence of the disease because the vector has a wide host range and quickly develops resistance to most of existing insecticides. Roguing infected plants and weeds that can act as hosts for the vector and removal of overwintering crops prior to the emergence of adult whiteflies may prove useful. This helps if applied over large areas and where there is no continuous cropping in glasshouses, which are the sites of whitefly survival and the source of virus spread throughout the year. Growing plants under physical barriers such as low-mesh tunnels may also have a positive effect. No resistant cultivars are currently available commercially but experimental evidence for delayed viral infection and decreased symptom severity in accessions of Cucumis sativus has recently been obtained. Tomato Infectious Chlorosis Virus Geographical distribution TICV infections have been reported from California, France, Greece, Indonesia, Italy, Japan, North Carolina, Spain, and Taiwan. This virus may also be present in the Czech Republic but the record has not been confirmed. Disease symptoms and yield losses Symptomatic tomato plants in open field and greenhouses exhibit interveinal yellowing in older leaves, followed by generalized yellowing. Symptoms can be confused with nutritional disorders (i.e., magnesium deficiency), pesticide toxicity, or natural senescence as older leaves may also turn red. Necrosis and occasional upward rolling of the leaves have also been reported. On the whole, infected plants are less vigorous and with fruits that may show delayed ripening. There is no information on the effects of TICV on artichoke and lettuce crops or ornamental species known to be natural hosts. Disease incidence may vary from one or few plants to severe outbreaks, depending on the abundance of whitefly populations. In California and Greece, disease incidence between 80% and 100% was reported. Causal agent and classification The natural hosts of TICV (genus Crinivirus, family Closterovidae) include members of the families Chenopodiaceae, Compositae, Ranuncolaceae, and Solanaceae where the virus is confined to phloem tissue. Virions are flexuos filaments with lengths between 750 and 800 nm. Genome consists of two molecules of ssRNA of positive polarity, denoted RNA-1 and RNA-2, whose nucleotide sequence has partially been determined. Both RNAs contain the hallmark ORFs of the family Closteroviridae.

Control

Control of CYSDV consists in controlling B. tabaci, and on eliminating infection sources. Chemical control of B. tabaci has not been effective in preventing the spread

Epidemiology TICV is spread by the whitefly T. vaporariorum but not by B. tabaci and it is not mechanically transmitted. Besides

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tomato, natural infections have been detected in Callistephus chinensis, Chenopodium album C. murale, Cynara cardunculus, lettuce, Physalis ixocarpa (tomatillo), globe artichoke, Nicotiana glauca, Petunia hybrida, Picris echioides, Ranunculus sp., and Zinnia elegans.

limit populations of its broad range of vectors. The management strategies outlined for CYSDV apply also for ToCV.

Control

Geographical distribution Cucumber vein yellowing virus (CVYV) has been recorded form Greece, Israel, Jordan, Portugal, Spain, Sudan, and Turkey.

TICV has the potential to cause significant losses to tomato and other naturally susceptible crops if it becomes established. Control largely depends on the efficacy of treatments and strategies to limit populations of T. vaporariorum. Tomato seedlings for planting should come from disease-free stocks. The general management strategies outlined for CYSDV can also be adopted for TICV.

Tomato Chlorosis Virus Geographical distribution

ToCV infections have been reported from the US (Colorado, Connecticut, Florida, Louisiana), Europe (France, Greece, Italy, Portugal, Spain), Morocco, Israel, Puerto Rico, South Africa, and Taiwan. Disease symptoms and yield losses

Symptoms are very similar to those induced by TICV, that is, progressive yellowing of the whole plant. Apparently, there are no estimates of yield losses, although since its discovery, the virus represents a serious problem for tomato production in many parts of the world.

Cucumber Vein Yellowing Virus

Disease symptoms and yield losses In cucumber, CVYV causes pronounced vein clearing deformation of the leaves followed by a generalized chlorosis and necrosis of affected plants. Fruits show light to dark green mottling. Nonparthenocarpic cucumbers are symptomless carriers of CVYV while parthenocarpic cucumbers develop severe symptoms. Stunting was also observed in cucumber and melon and sudden death in protected melons in Portugal. In watermelon, symptoms are often light or not expressed, but splitting of the fruits has occasionally been observed. In zucchini, symptoms vary from chlorotic mottling to vein yellowing of the leaves, but symptomless infections have also been recorded. Symptoms severity may be increased by synergistic reactions with other viruses. Yield losses have not been quantified. CVYV could present a threat to cucurbits grown outdoors or under glasshouses, although its actual impact in the complex pathosystem affecting cucurbit crops in the Mediterranean and subtropical regions has not been determined.

Causal agent and classification

ToCV (genus Crinivirus, family Closteroviridae) virions are flexuous filaments encapsidating two molecules of positive-sense ssRNA denoted RNA-1 and RNA-2, whose complete nucleotide sequence has been determined. RNA-1 consists of 8595 nt organized into four ORFs and encodes replication-associated proteins. RNA-2 is 8244 nt long and putatively encodes nine ORFs comprising in the order, the HSP70 homolog, a 59 kDa protein, CP, and CPm, which may be involved in determining the unique, broad vector transmissibility of the virus. Phylogenetically, ToCV is closely related to sweet potato chlorotic stunt virus (SPCSV) and CYSDV. Epidemiology

ToCV is transmitted by T. vaporariorum, B. tabaci biotypes A and B, and T. abutilonea. Besides tomato, the natural host range includes pepper, Datura stramonium, Physalis wrightii, Solanum nigrum, and Z. elegans. Control

ToCV has the potential to cause significant losses to tomato if it becomes established. Control depends on the efficacy of treatments and strategies to

Causal agent and classification CVYV (genus Ipomovirus, family Potyviridae) has filamentous particles and is transmitted by B. tabaci but, unlike other whitefly-transmitted viruses, it can also be transmitted by mechanical inoculation. CVYV genome consists of a single molecule of plus-sense ssRNA of 9751 nt (ALM 23 isolate) containing most hallmarks of the genome of members of the family Potyviridae. The absence of a coding region for the helper component-proteinase seems to be a distinctive trait of CVYV. Two CVYV strains have been recognized from Israel and Jordan, that is, CVYV-Is and CVYV-Jor. The two strains induce similar vein-clearing symptoms in cucumber and melon, but CVYV-Jor infections in cucumber are more severe. CVYV is more closely related to Sweet potato mild mottle virus (SPMMV) than any other species in the family Potyviridae. Epidemiology CVYV naturally infects cucumber, melon, watermelon squash, and zucchini and several weed species are also natural hosts of the virus (e.g., Ecballium elaterium, Convolvulus arvensis, Malva parviflora, Sonchus oleraceus, Sonchus asper, and Sonchus tenerrimus). All experimental hosts belong to the

Emerging and Reemerging Virus Diseases of Plants

family Cucurbitaceae and include Cucurbita moschata, Cucurbita foetidissima, and Citrullus colocynthis. CVYV is semipersistent in its white-fly vector (B. tabaci) which retains the virus for less than 6 h. Therefore, individuals moving to nonhost plants may not remain viruliferous long enough to transmit the virus. Aphis gossypii and M. persicae are not vectors. Whether CVYV is seedborne has not been determined. Control

CVYV management is mainly based on the use of virusfree stock plants as well as of B. tabaci populations as already outlined for CYSDV. Several detection methods based on molecular hybridization and polymerase chain reaction (PCR) have been developed and used for screening Cucurbitaceae germplasm that can show some degree of resistance to CVYV.

Graft Incompatibility in Grapevines Geographical Distribution Cases of graft union disorders have been documented from Europe (‘Kober 5BB incompatibility’, ‘Syrah decline’), California, New Zealand, Italy, Australia, and Chile (‘Young vine decline’), and again California (‘Roostock stem lesions’, ‘Necrotic union’, and ‘Stem necrosis’), but are likely to occur also in other viticultural countries.

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Causal Agents and Classification An ordinary strain of grapevine leafroll-associated virus 2 (GLRaV-2) is consistently associated with Kober 5BB incompatibility in Europe, appears to be involved in California’s young vine decline, and was detected in diseased Chilean and Argentinian grapes. GLRaV-2, a definitive member of the genus Closterovirus (family Closteroviridae), has flexuous filamentous particles c. 1600 nm long an RNA genome 15 528 nt in size made up of nine ORFs. A virus originally detected in cv. Redglobe in California called grapevine rootstock stem lesion-associated virus (GRSLaV) proved to be a molecular and biological variant of GLRaV-2 (GLRaV-2 RG). Other molecular variants of GLRaV-2 were reported from New Zealand, Chile, and Australia in association with young vine decline conditions. Based on the differential responses of a panel of 18 rootstocks, up to five different graft-transmissible agents inducing incompatibility were detected in California. Of these, only GLRaV-2 RG, the putative agent of roostock stem lesion, was identified. The agents of necrotic union and stem necrosis are unknown. Equally unkown is the agent of Syrah decline, although there is circumstantial evidence that grapevine rupestris stem pitting-associated virus (GRSPaV) may have a bearing on its aetiology. GRSPaV, a definitive member of the genus Foveavirus (family Flexiviridae), has filamentous particles c. 730 nm in length, a genome 8726 nt in size comprising five or six ORFs, and occurs in nature as a family of molecular variants.

Disease Symptoms and Yield Losses The increased use of grapevine clonal material is revealing unprecedented and widespread conditions of generalized decline that develop dramatically in certain scion–rootstock combinations. Newly planted vines grow weakly, shoots are short, leaves are small sized, with margins more or less extensively rolled downwards, and the vegetation is stunted. The canopy shows autumn colors off season so that leaves turn reddish in red-berried varieties or yellow in white-berried varieties much earlier than normal. A prominent swelling forms at the scion/rooststock junction (‘Kober 5BB imcompatibility’, ‘Young vine decline’) sometimes accompanied by necrosis at the graft union (‘Necrotic union’), and variously extended necrotic lesions develop on the roostock stem (‘Roostock stem lesion’, ‘Stem necrosis’). Severely affected vines decline and may die within 1 or 2 years. Syrah decline is a severe disease characterized by early reddening of the leaves and swellings with grooves and deep cracks at the graft union. Appearance of graft union disorders depends more on the rootstock than on the scion. For instance, European grape varieties grafted on tolerant rootstocks (e.g., Freedom, Harmony, Salt creek, 03916, 101-14) exhibit a green canopy and perform well, whereas varieties grafted on susceptible roostocks (e.g., Kober 5BB, 5C, 1103P, 3309) develop a discolored canopy, decline, and may die.

Transmission GLRaV-2 and GRSPaV have no known vectors, but GRSPaV is pollen- and seed-borne. Infected propagative material is the major means for dissemination of both viruses. Control Use of certified virus-free scionwood and rootstocks is recommended. Currently known graft incompatibility agents can be eliminated with reasonable efficiency by heat therapy, meristem tip culture, or a combination of the two. If scionwood is infected, the use of sensitive rootstocks is to be avoided.

Sudden Death of Citrus Geographical Distribution Citrus sudden death (CSD) has only been reported from the State of Sao˜ Paulo in Brazil, where it has already killed about 1 million trees. It has the potentiality for spreading to other Brazilian States and neighboring countries.

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Disease Symptoms and Yield Losses

Control

CSD is a destructive disease first observed in 1999 in Brazil on sweet orange and mandarin trees grafted on Rangpur lime (Citrus limonia) or Volkamer lemon (Citrus volkameriana). Outward symptoms of infected trees and modifications of the bark anatomy at the bud union resemble very much those elicited by citrus tristeza, the major difference being that CSD develops on trees grafted on tristeza-resistant rootstocks. Symptoms of CSD are characterized by a generalized discoloration of the leaves which are pale green initially, then turn yellowish and abscise. With time, defoliation becomes more intense, the trees do not push new vegetation, and the root system decays. A rapid decline and death of the plant ensues, due also to phloem degeneration in the graft union region. The phloem of the susceptible rootstocks Rangpur lime and Volkamer lemon shows a characteristic yellow strain. Until their sudden collapse, infected trees bear a normal crop.

Production and distribution of healthy scion material to be grafted onto tolerant rootstocks (Cleopatra mandarin, Swingle citrumelo) can help restraining the spread of CSD in newly established groves. Grafting the affected trees above the graft union with seedlings of tolerant roostocks allows their recovery.

Causal Agent and Classification All trees affected by CSD host a strain of CTV and, with 99.7% association, a spherical virus denoted citrus sudden death-associated virus (CSDaV). This virus is a tentative member of the genus Marafivirus (family Tymoviridae), has isometric particles about 30 mn in diameter, a ssRNA genome 6805 nt in size with a high cytosine content (37.4%), encompassing two ORFs. ORF1 codes for a large polyprotein with a predicted Mr of 240 kDa which is processed to yield the replication-associated proteins (methyltransferase, helicase, and RNA-dependent RNA polymerase), a papain-like protease, and two CP subunits 21 and 22 kDa in size, respectively. One of the CP subunits is produced from the cleavage of the C-terminus of the polyprotein, the other is translated from a subgenomic RNA. ORF2 codes for a putative protein 16 kDa in size, with some relationship with a movement protein of viruses belonging to the sister genus Maculavirus (family Tymoviridae). It has not been ultimately established whether CTV or CSDaV are the causal agents of CSD, but their consistent association with the disease suggests that both may have a bearing on its etiology. Epidemiology CSD is transmitted by grafting and can be disseminated with propagation material. Natural spreading is by aphids (Toxoptera citricida) with a temporal and spatial pattern similar to that of citrus tristeza. CSDaV was detected in T. citricida that had fed on infected trees and was transmitted experimentally to healthy plants. Since marafiviruses are not aphid-borne, it was hypothesized that CSDaV may use CTV as a helper virus.

See also: Pepino Mosaic Virus; Plant Virus Diseases: Economic Aspects; Plant Virus Diseases: Fruit Trees and Grapevine; Virus Databases.

Further Reading Boubals D (2000) Le de´pe´rissement de la Syrah. Compte-rendu de la re´union du Groupe de Travail National. Progre´s Agricole et Viticole 117: 137–141. Ce´lix A, Lo´pez-Sese´ A, Almarza N, Go´mez-Guillamo´n ML, and Rodrı´guez-Cerezo E (1996) Characterization of cucurbit yellow stunting disorder virus, a Bemisia tabaci-transmitted closterovirus. Phytopathology 86: 1370–1376. Duffus JE, Liu HY, and Wisler GC (1996) Tomato infectious chlorosis virus – A new clostero-like virus transmitted by Trialeurodes vaporariorum. European Journal of Plant Pathology 102: 219–226. Greif C, Garau R, Boscia D, et al. (1995) The relationship of grapevine leafroll-associated virus 2 with a graft incompatibility condition of grapevines. Phytopathologia Mediterranea 34: 167–173. Lecoq H, Desbiez C, Dele´colle B, Cohen S, and Mansour A (2000) Cytological and molecular evidence that the whitefly-transmitted cucumber vein yellowing virus is a tentative member of the family Potyviridae. Journal of General Virology 81: 2289–2293. Legin R and Walter B (1986) Etude de phe´nome`nes d’incompatibilite´ au greffage chez la vigne. Progre´s Agricole et Viticole 103: 279–283. Maccheroni W, Allegria MC, Greggio CC, et al. (2005) Identification and genomic characterization of a new virus (Tymoviridae family) associated with citrus sudden death disease. Journal of Virology 79: 3028–3037. Meng B and Gonsalves D (2003) Rupestris stem pitting associated virus of grapevines: Genome structure, genetic diversity, detection, and phylogenetic relationship to other plant viruses. Current Topics in Virology 3: 125–135. Roman MP, Cambra M, Juares J, et al. (2004) Sudden death of citrus in Brazil: A graft-transmissible bud union disease. Plant Disease 88: 453–467. Rubio L, Soong J, Kao J, and Falk BW (1999) Geographic distribution and molecular variation of isolates of three whitefly-borne closteroviruses of cucurbits: Lettuce infectious virus, cucurbit yellow stunting disorder virus, and beet pseudo-yellows virus. Phytopathology 89: 707–711. Soler S, Prohens J, Dı´ez MJ, and Nuez F (2002) Natural occurrence of Pepino mosaic virus in Lycopersicon species in Central and Southern Peru. Journal of Phytopathology 150: 49–53. Uyemoto JK, Rowhani A, Luvisi D, and Krag R (2001) New closterovirus in ‘Redglobe’ grape causes decline of grafted plants. California Agriculture 55(4): 28–31. Van der Vlugt RAA, Cuperus C, Vink J, Stijger ICMM, Lesemann DE, Verhoeven JTJ, and Roenhorst JW (2002) Identification and characterization of Pepino mosaic potexvirus in tomato. Bulletin OEPP/EPPO Bulletin 32: 503–508. Verhoeven JTJ, van der Vlugt RAA, and Roenhorst JW (2003) High similarity between tomato isolates of Pepino mosaic virus suggests a common origin. European Journal of Plant Pathology 109: 419–425. Wisler GC, Duffus JE, Liu H-Y, and Li RH (1998) Ecology and epidemiology of whitefly-transmitted closteroviruses. Plant Disease 82: 270–279.