401
September 2003
Family: Ophioviridae
Genus: Ophiovirus
Species: Citrus psorosis virus
Acronym: CPsV


Citrus psorosis virus

Robert G. Milne
Istituto di Virologia Vegetale, CNR, I-10135 Torino, Italy

M. Laura García
Instituto de Bioquímica y Biología Molecular, Universidad Nacional La Plata, 1900 La Plata, Argentina

Pedro Moreno
Instituto Valenciano de Investigaciones Agrarias, Moncada-46113, Valencia, Spain

Contents

Introduction
Main Diseases
Geographical Distribution
Host Range and Symptomatology
Strains
Transmission by Vectors
Transmission through Seed
Transmission by Grafting
Transmission by Dodder
Serology
Nucleic Acid Hybridization
Relationships
Stability in Sap
Purification
Properties of Particles
Particle Structure
Particle Composition
Properties of Infective Nucleic Acid
Molecular Structure
Genome Properties
Satellites
Relations with Cells and Tissues
Ecology and Control
Notes
References
Acknowledgements
Figures

Introduction

Citrus psorosis disease was first described by Swingle & Webber (1896) and shown to be graft-transmissible by Fawcett (1936). Associated virus particles were first described by Derrick et al. (1988). Strong evidence suggests that CPsV is the cause of psorosis but Koch's postulates have not been fulfilled due to lack of purified infectious preparations. The virus has three negative-sense ssRNAs each encapsidated in the same coat protein and forming fine filamentous particles, probably of three sizes, that appear as highly kinked closed circles.

Synonyms
Citrus ringspot (Wallace & Drake, 1968; Garnsey & Timmer, 1980).
Spirovirus (Derrick et al., 1993).

Main Diseases

The characteristic symptoms in adult citrus trees in the field are bark-scaling (Fig.1) and internal wood staining (Fig.2) in the trunk and limbs (Roistacher, 1991, 1993). In the common form of the disease (psorosis A), bark scaling first appears on trees 10-15 years old, restricted to the main trunk and limbs. Old leaves are usually symptomless but chlorotic flecks may occur in young leaves (Fig.3). There may be sparse foliage, dieback and reduced yield. In the more aggressive B form (Fawcett, 1932), extensive bark-scaling, also affecting thin branches, appears earlier, with chlorotic blotches (Fig.4) or pustules (Fig.5) on old leaves, and sometimes ringspots on fruits (Fawcett & Bitancourt, 1943; Roistacher, 1991; Whiteside et al., 1989; Timmer et al., 2000). Psorosis B usually causes severe decline and sometimes death.

Geographical Distribution

Psorosis-like symptoms occur in most citrus-growing regions worldwide. The distribution of the disease is well documented but reliable detection of CPsV itself is recent, and not yet widely applied in citrus-growing regions. Thus, in many countries CPsV has not been confirmed by biological, serological or molecular indexing. It has been confirmed in North and South America, South Africa and the Mediterranean basin.

Host Range and Symptomatology

CPsV has been detected serologically in different varieties of sweet orange (Citrus sinensis (L.) Osb.), sour orange (C. aurantium L.), lemon (C. limon (L.) Burn. f.), grapefruit (C. paradisi Macf.), clementine (C. clementina Hort. ex Tan.), satsuma (C. unshiu (Macf.) Marc.) and some mandarin hybrids like Fortune mandarin (C. clementina x C. tangerina Hort. ex Tan.) and Ortanique tangor (C. reticulata Blanco x C. sinensis), but it probably also infects other species.

Bark scaling is mainly a disease of sweet orange, mandarin and grapefruit, the most susceptible being sweet orange. Gum may accumulate below the scales and may impregnate the xylem producing wood staining and vessel occlusion. Chlorotic flecks in young leaves occur in many experimentally inoculated citrus species and hybrids, including sweet orange, mandarin (C. reticulata Blanco), clementine and satsuma, grapefruit, sour orange, lemon, rough lemon (C. jambhiri Lush), citron (C. medica L.), lime (C. aurantifolia (Christm.) Swing.), tangelo (C.reticulata x C. sinensis) and tangor. Some of these species, like sour orange, lemon or rough lemon, do not develop bark scaling though they may show intense young leaf symptoms. Poncirus trifoliata (L.) Raf. can harbour CPsV symptomlessly.

In the Mediterranean basin, young leaves of CPsV-infected trees usually show chlorotic flecks and spots, particularly in the spring flush, but this symptom is also associated with concave gum, blind-pocket, impietratura and cristacortis diseases, which are not associated with CPsV and are of unknown but presumed viral origin (Klotz, 1973; Roistacher, 1991; Whiteside et al., 1989; Duran-Vila & Moreno, 2000; Timmer et al., 2000). Young leaf symptoms are not observed in field trees in California (Roistacher, 1993) or South Africa (Doidge, 1939). Trees infected by the psorosis B variant of CPsV show chlorotic blotches in old leaves with gummy pustules on the underside; irregular or ring-shaped spots, often depressed, are sometimes observed in the fruit rind. Trees propagated from psorosis B-infected buds are severely stunted and may die after 3 to 6 years.

Biological indexing for psorosis (Roistacher, 1991) is done by grafting tissue to young seedlings of sweet orange and keeping these in a cool greenhouse (18-24 °C). CPsV causes a shock reaction with leaf drop and shoot necrosis (Fig.6) in the first flush, and chlorotic leaf flecking and spotting in successive flushes. More specific diagnosis requires a cross-protection test using psorosis B as a challenge (Roistacher 1991, 1993). Sweet orange seedlings are graft-inoculated with tissue of the candidate tree, and later grafted with a source of psorosis B. Psorosis-free plants develop the severe psorosis B symptoms after 6 months, whereas in psorosis A-infected plants, such symptoms fail to develop. Psorosis B isolates of CPsV, less frequently found in the field, can be directly diagnosed on sweet orange, where they induce characteristic pustules and blisters.

Some isolates of CPsV can be mechanically transmitted to herbaceous test plants using young leaf extracts freshly prepared in cold Tris or phosphate buffer containing standard anti-oxidants and protectants (Garnsey & Timmer, 1980; Derrick et al., 1988; da Graça et al., 1991; Navas-Castillo et al., 1993; Navas-Castillo & Moreno, 1995; Alioto et al., 1999).

Diagnostic species

Sweet orange (cv. Pineapple or Mme. Vinous) seedlings. Shock and/or chlorotic flecks in young leaves.

Chenopodium quinoa. Chlorotic, then necrotic local lesions (Fig.7, Fig.8) in 4-10 days. Some virus isolates are not easily mechanically transmitted, and may have to be adapted to C. quinoa over several passages.

Gomphrena globosa. Necrotic local lesions with red halos in 10 days, followed by systemic necrosis (Fig.9).

Propagation species

Sweet orange seedlings grown at 18-26°C.

C. quinoa, and systemically infected G. globosa grown at high temperature (Alioto et al., 1999).

Strains

CPsV has many variants. Psorosis A and B appear to be different strains, and may occur in the same tree but in different tissues (Fawcett & Cochran, 1942; Wallace, 1957). However, serological or molecular differences between A and B have not been described.

Other isolates differing by symptom expression in different hosts or at different temperatures have been reported (Navas-Castillo & Moreno, 1993a, 1993b; Roistacher, 1993). The ability of some isolates to induce chlorotic blotches and ringspots in old leaves and fruits led for years to citrus ringspot being considered a separate disease (Wallace & Drake, 1968).

Some isolates differ in the size of their coat protein (García et al., 1994; Navas-Castillo & Moreno, 1995). Panels of monoclonal antibodies against the coat protein have differentiated many serotypes in samples from the Americas, Lebanon, Spain and southern Italy (Djelouah et al., 2000; Roistacher et al., 2000; Alioto et al., 2001; Martín et al., 2002). Sequence comparisons of the RNA3 of various CPsV isolates have shown that the carboxy-terminal half of the coat protein has fewer amino acid substitutions than the amino-terminal half (Alioto et al., 2003).

Isolates differ in the titre they reach in host plants and in their ease of mechanical transmission to test plants; the original isolate CRSV-4 (Garnsey & Timmer, 1980) appears to be one of the easiest to work with.

Transmission by Vectors

In most citrus areas psorosis seems to spread only through propagation of infected buds. There are reports of natural dispersal of psorosis-like bark scaling in Argentina (Pujol & Beñatena, 1965; Timmer & Beñatena, 1977), Uruguay (Campiglia et al., 1976) and Texas (Timmer, 1974); however, the presence of CPsV in these plants was not confirmed. No vector has been identified (but see NOTES).

Transmission through Seed

Seed transmision was reported in the citranges (C. sinensis x P. trifoliata) Carrizo (Bridges et al., 1965) and Troyer (Pujol, 1966) and in P. trifoliata (Campiglia et al., 1976) but adequate biological indexing was not done and other agents could have caused the observed symptoms. More recent tests of seed transmission have yielded negative results (Roistacher, 1993; D'Onghia et al., 2000, 2001a).

Transmission by Grafting

The virus is readily transmitted from citrus to citrus by grafting. It remains in callus grown from infected plants and can be transmitted by grafting these callus cells under a bark flap of the receptor plant (Duran-Vila et al., 1991; Navas-Castillo et al., 1995).

Transmission by Dodder

Psorosis A has been transmitted (Weathers & Harjung, 1964; Price, 1965).

Serology

Rabbits immunized with particle-enriched preparations have yielded low-titre antisera, usable in immunosorbent electron microscopy (ISEM) (Derrick et al., 1988) or in double antibody sandwich ELISA after absorption with healthy plant preparations (Garcia et al., 1997; D'Onghia et al., 1998; Roistacher et al., 2000). Another antiserum of similar quality was raised by injecting a rabbit with particle-enriched preparations, followed by a booster of recombinant coat protein raised in bacteria (P. Roggero and P. Moreno, unpublished results). Monoclonal antibodies (MAbs) have been raised and used together with rabbit antiserum in triple antibody sandwich ELISA for CPsV detection at all times of the year (Alioto et al., 1999, 2000, 2001; Martín et al., 2002). CPsV has also been detected by direct tissue-blot immunoassay using MAbs and selected plant tissues (Martín et al., 2002; D'Onghia et al., 2001b). One IgG MAb designated 13C5 recognises all CPsV isolates so far tested (Alioto et al., 1999; Martín et al., 2002). This MAb, though not the rabbit antiserum of Garcia et al. (1997), is able to trap the virions in ISEM (R.G. Milne and V. Masenga, unpublished data). Other MAbs to CPsV (Djelouah et al., 2000; Potere et al., 1999) have been used for diagnosis and strain differentiation.

Nucleic Acid Hybridization

CPsV can be detected by dot blot or tissue print hybridisation using 32P- or digoxigenin-labelled probes derived from RNA 3 (Barthe et al.,1998).

Relationships

CPsV is the type species of the genus Ophiovirus, the only genus in the family Ophioviridae. Lettuce ring necrosis virus (LRNV; Torok & Vetten, 2002), Mirafiori lettuce big-vein virus (MLBVV; formerly called Mirafiori lettuce virus; Roggero et al., 2000; Lot et al., 2002; van der Wilk et al., 2002), Ranunculus white mottle virus (RWMV; Vaira et al., 1997, 2003) and Tulip mild mottle mosaic virus (TMMMV; Morikawa et al., 1995) are other recognised ophiovirus species (van Regenmortel et al., 2000; see 8th Report of the ICTV, to be published 2004). A further ophiovirus not yet fully described is associated with freesia leaf necrosis disease (Vaira et al., 2003). All these viruses have similar particle morphologies, divided negative-sense ssRNA genomes of similar sizes, and coat proteins of similar sizes. CPsV is serologically unrelated to other ophioviruses, but RNA 3 and its product, the CP, share 46.5% and 30.9% nucleotide and amino acid sequence identities, respectively, with a Japanese isolate of MLBVV (Kawazu et al., 2003). The predicted protein product of RNA 1 of CPsV (280K) has some similarity in amino acid sequence to that of MLBVV and the partial sequence of RWMV (Naum-Ongania et al., 2003). It also has affinities with Lettuce big-vein-associated virus (formerly named Lettuce big-vein virus) (Sasaya et al., 2002; Vaira et al., 2003; van der Wilk et al., 2002).

Ophioviruses are not closely related to tenuiviruses or tospoviruses, but have affinities with viruses in the Mononegavirales, such as the Bornaviridae, Filoviridae, Paramyxoviridae and Rhabdoviridae (Naum-Ongania et al., 2003; Vaira et al., 2003; van der Wilk et al., 2002). However, the ophiovirus polymerases contain the SDD sequence which is a signature for segmented negative-strand RNA viruses (Orthomyxoviridae, Arenaviridae and Bunyaviridae) and not the GDNQ motif found in the Mononegavirales (Naum-Ongania et al., 2003).

Stability in Sap

The virus is labile in plant sap but if young citrus leaves with strong symptoms are homogenised in 0.05 M phosphate buffer, pH 7 or Tris buffer, pH 8, each containing 0.5% 2-mercaptoethanol, infectivity (local lesions in mechanically inoculated C. quinoa) can be retained for up to 2 h at 25°C or 24-48 h at 4°C (Garnsey & Timmer, 1980).

Purification

Use young leaves with strong symptoms from greenhouse citrus, local lesions cut from C. quinoa leaves 5-8 days after inoculation, or systemically infected leaves of G. globosa grown at 30/25 °C (day/night) under strong lighting. Work rapidly in the cold. Homogenise with 10 volumes (w/v) of 0.05 M Tris-HCl, pH 8 containing 0.1% (w/v) ascorbic acid, 0.1% (w/v) cysteine and 0.5% (v/v) 2-mercaptoethanol (TACM), and squeeze through nylon stocking. Stir for 10 min with 8% (v/v) carbon tetrachloride or 10% (v/v) Freon-113, and centrifuge at low speed. Ultracentrifuge the aqueous phase (300,000 g for 60 min) and resuspend the pellet in TACM. As an alternative to this last step, add 10% (w/v) polyethylene glycol (Mol. Wt 20,000), 1% NaCl and 0.1% Nonidet P40 to the aqueous phase, stir for 45 min, centrifuge for 10 min at 15,000 g, and resuspend the pellet in TACM. Finally, centrifuge into a 10-40% linear sucrose gradient, prepared in TACM, for 2.5 h at 220,000 g (Derrick et al., 1988; Garcia et al., 1991; Alioto et al., 1999). No visible virus-containing bands are formed, but certain fractions ('top' and 'bottom' suitably recombined) are infective (Derrick et al., 1988; da Graça et al., 1991; García et al., 1991, 1994; Navas-Castillo et al., 1993). For further purification, dilute virus-containing fractions in TACM and centrifuge for 30 min at 500,000 g or for 60 min at 300,000 g. Resuspend the pellets in TACM, and centrifuge into a 10-40% caesium sulphate density gradient, prepared in TACM, for 70 min at 310,000 g., or for 19 h at 100,000 g at 4°C in 30% caesium sulphate. Again, no clearly visible virus-containing bands are formed, and virus particles are detected by electron microscopy (Alioto et al., 1999; Sánchez de la Torre et al., 1998).

Properties of Particles

The virus particles are physically stable for 24 h in cold TACM buffer. Ribonuclease treatment of infective extracts of C. quinoa abolishes infectivity (García et al., 1991). Particles survive in caesium sulphate but not in caesium chloride, and in 2% aqueous uranyl acetate but not in phosphotungstate (D. Alioto, E. Luisoni & R.G. Milne, unpublished data). They sediment in sucrose or caesium sulphate density gradients as two components (Derrick et al., 1988; Sánchez de la Torre et al., 1998).

Particle Structure

The particles are highly kinked, thread-like, circularized filaments about 3 nm in diameter, which occur in various configurations (see wire models, Fig.10), and are of two distinguishable contour lengths. The short particles are estimated to be 690-760 nm in length, the larger particles being about four times longer (Fig.11). The filaments show units repeating every 3 nm along the particle length (Fig.12); these may be images of the coat protein molecule, forming a doughnut through which the RNA strand passes. The open-circle particles easily collapse in vitro to form sinuous linear or branched duplex structures, first described by Derrick et al. (1988) and most easily seen after immunotrapping (Fig.13). These are about 9 nm in diameter, and about half the length of the circular contour (García et al., 1994; Milne et al., 1996; Milne, 2000; van Regenmortel et al., 2000).

Particle Composition

Three species of ssRNA have been detected in the particles, each encapsidated separately in the same protein. After deproteinization, dsRNA species are also found, corresponding to the three RNAs (Derrick et al., 1991; Naum et al., 2000; Sánchez de la Torre et al., 1998, 2002). In fact, ssRNAs of both polarities are separately encapsidated, although the negative strand is preponderant. With RNA3, for example, the negative strand is at least 50 times more abundant than the positive strand in particle preparations (Sánchez de la Torre et al., 1998).

Nucleic acid: The genomic ssRNAs are 8184 nt, 1644 nt and 1454 nt in length (Sánchez de la Torre et al., 1998, 2002; Naum et al., 2000; Naum-Ongania et al., 2003).

Protein: There is a single coat protein of 48-49 kDa in the CRSV-4 isolate (Derrick et al., 1988). Its size has been confirmed by sequencing the CP ORF (Barthe et al., 1998; Sánchez de la Torre et al., 1998). The CP size was estimated as 47 kDa in thirteen Spanish isolates, but 46 kDa in isolate P-126 (Navas-Castillo & Moreno, 1995). The Argentine isolate 90-1-1 had a CP estimated as 50 kDa (García et al., 1994).

Genome Properties

CPsV is a negative stranded RNA virus, as shown by the composition of purified virions and by the characteristics of the RNA-dependent RNA polymerase (see RELATIONSHIPS). The gene arrangement is shown in Fig.14. ORF 1 of RNA1, at the 5' end of the negative strand, (accession numbers AY224663 and AF149101-AF149107) encodes a 280 kDa protein containing characteristic RNA-dependent RNA polymerase domains; ORF 2 of RNA1, separated from ORF 1 by 109 nt, encodes a 24 kDa protein of unknown function and with no homologues in the database (Legarreta et al., 1999; Naum et al., 2000; Naum-Ongania et al., 2003). RNA2 (accession number AF218572) contains a single ORF encoding a putative protein of 54 kDa without significant similarity to any reported protein but including a motif resembling a nuclear localization signal. In the 3'-terminal untranslated region of RNA 2 there is a putative polyadenylation signal (Sánchez de la Torre et al ., 2002). RNA3 (accession numbers AF036338, AF036926, AF060855) contains one ORF encoding the coat protein (Barthe et al., 1998; Sánchez de la Torre et al ., 1998).

The 3'-terminal regions of the three RNAs are very similar. In contrast, their 5'-terminal regions differ considerably in sequence (Naum-Ongania et al., 2003). The circular morphology of the virions suggests a panhandle structure formed by base-pairing between the terminal regions of each RNA, and in line with this view, the first 75 nt of the RNA 1 termini show 41% complementarity. However, Naum-Ongania et al. (2003) observed that secondary-structure predictions (MFOLD program, Walter et al., 1994) do not support the existence of a panhandle, or of a "corkscrew" structure as proposed for Influenza A virus (Flick & Hobom, 1999) and Mirafiori lettuce big-vein virus (van der Wilk et al., 2002).

Relations with Cells and Tissues

CPsV is not restricted to the phloem. Tissue immunoprints show that it also invades parenchyma cells (D'Onghia et al., 2001b; Martín et al., 2002). No virus particles or any specific inclusion or cytopathology have been detected in thin sections of infected tissues (R.G. Milne & V. Masenga, unpublished results).

Ecology and Control

CPsV has not been found naturally infecting hosts other than citrus. Spread mainly occurs through grafting but the virus may be spread naturally in the New World by an unknown vector (see TRANSMISSION BY VECTORS and NOTES). The virus can be controlled through certification programmes (Navarro, 1993).

Notes

A virus from India with flexuous filamentous particles, unrelated to CPsV but formerly called 'citrus ringspot virus', is now named Indian citrus ringspot virus (Rustici et al., 2000, 2002).

Apart from CPsV, other diseases and disorders can cause bark scaling resembling psorosis; these are Bahia bark scaling (Santos Filho et al., 2001a, 2001b), leprosis, foot rot caused by Phytophthora, Rio Grande gummosis, and genetic disorders such as lemon shell bark and sunscald (Whiteside et al., 1989; Timmer et al., 2000). Recently, psorosis-like bark scaling has been noted in some trees apparently free of CPsV and of these other agents (Martín et al., 2002).

No natural vector is known for CPsV but transmission through soil by zoospores of the fungal vector Olpidium brassicae has been demonstrated for two ophioviruses, Mirafiori lettuce big-vein virus (Lot et al., 2002) and Lettuce ring necrosis virus (Bos & Huijberts, 1996; Torok & Vetten, 2002) and indicated for a third, Tulip mild mottle mosaic virus (T. Morikawa & T. Natsuaki, personal communication).

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Figure 1

Psorosis bark scaling in the trunk of a sweet orange tree.

Figure 2

Staining caused by CPsV in the wood of a sweet orange tree.

Figure 3

Chlorotic flecks and spots induced by CPsV in a young leaf from the second flush after graft-inoculation.

Figure 4

Symptoms of psorosis B in an old leaf of sweet orange, showing chlorotic spots on the upper side.

Figure 5

Symptoms of psorosis B in an old leaf of sweet orange, showing gummy pustules on the underside.

Figure 6

Shock symptom in the first flush of a sweet orange seedling after graft-inoculation with a CPsV-infected bud.

Figure 7

Necrotic local lesions in Chenopodium quinoa following mechanical inoculation with a CPsV preparation.

Figure 8

Necrotic local lesions in Chenopodium quinoa following mechanical inoculation with a CPsV preparation, a further example.

Figure 9

Necrotic lesions in Gomphrena globosa caused by systemic infection with CPsV.

Figure 10

Wire models of the smallest CPsV particle in various configurations.

Figure 11

Partially purified preparation of CPsV showing particles of two sizes (large and small arrows). Uranyl acetate negative stain. Bar = 100 nm.

Figure 12

Negatively stained particle of CPsV showing the beaded structure of the nucleocapsid (arrows) (with permission of Journal of General Virology). Bar = 100 nm.

Figure 13

Crude sap preparation of CPsV-infected greenhouse-grown sweet orange leaf, after trapping with monoclonal antibody 13C5. Note particles in the duplex form. Bar = 100 nm.

Figure 14

Scheme of the CPsV genome.