Details of DPV and References

DPV NO: 420 March 2008

Family: Tombusviridae

Genus: Carmovirus

Species: Carnation mottle virus | Acronym: CarMV

This is a revised version of DPV 7

Carnation mottle virus

Alan A. Brunt "Brayton", The Thatchway, Angmering, W. Sussex BN16 4HJ, UK

Giovanni P. Martelli Department of Plant Protection and Applied Microbiology, University of Bari and Institute of Plant Virology of the CNR, Bari section, Bari, Italy

Contents

Introduction

The origin of Carnation mottle virus (CarMV) is unknown. The virus was first detected in the UK, into which it was probably introduced in commercial stocks of carnations and/or pinks. It was described by Kassanis (1955) and Hollings & Stone (1970).

CarMV is an RNA-containing virus with isometric particles about 32-35 nm in diameter that mainly infects species in the Caryophyllaceae. It is transmitted mechanically but has no known vector. The virus is the type species of the genus Carmovirus which comprises 14 definitive and 8 tentative species and, together with seven additional genera (Tombusvirus, Dianthovirus, Aureusvirus, Avenavirus, Necrovirus, Panicovirus and Machlomovirus), is included in the family Tombusviridae (Lommel et al., 2005). The generic name is derived from the name of the type species, CARnation MOttle VIRUS.

Main Diseases

Depending on cultivar and/or environmental conditions, typical strains of the virus induce mild mottling in the young leaves (Fig. 1) and faint chlorosis in mature leaves of carnations (Dianthus caryophyllus) and pinks (Dianthus barbatus). In some carnation cultivars, the quality and quantity of cut flowers is reduced (Calderon & Arbelaez, 1999). In Begonia elatior and Begonia × cheimantha CarMV induces vein clearing and curling of leaves and flower breaking (Anonymous, 1984; Paludan & Begtrup, 1985). The leaves of Calla lily (Zantedeschia spp.) develop yellow mottling, yellow ringspot and a general mosaic (Chen et al., 2003b). The virus occurs symptomlessly in infected lettuce (Lactuca sativa) and Daphne × burkwoodii and Daphne odora (Tomlinson & Faithfull, 1976; Morris-Krsinich & Milne, 1977).

Geographical Distribution

The virus has a very wide geographical distribution which is probably co-incident with the commercial cultivation of carnations and pinks. This extensive distribution is probably due to inadvertent international exchange of infected plantlets and germplasm before the virus had been identified. Thus its introduction to many countries was accidental. However, dissemination now occurs less frequently and virus incidence is often lower due to the general availability of virus-tested plants and the use of certification schemes (Anonymous, 1991; 1993).

Host Range and Symptomatology

Although carnations (Dianthus caryophyllus) and pinks (D. barbatus) are its major natural hosts, the virus has also been reported to occur naturally in Zantedeschia spp. (Calla lily) in Taiwan (Chen et al., 2003b), Begonia elatior and Begonia × cheimantha in Denmark (Anonymous, 1984; Paludan & Begtrup, 1985), lettuce (Lactuca sativa) in the UK (Tomlinson & Faithfull, 1976), and Daphne × burkwoodii and Daphne odora in New Zealand (Morris-Krsinich & Milne, 1977).

The virus can be transmitted by mechanical inoculation of sap to a fairly wide range of experimental host species (43 of 98 tested in 15 dicotyledonous families; Kowalska, 1972), Transmission is easier if inhibitors of infection in carnation sap are removed by using partially purified virus preparations as inocula (Hollings & Stone, 1964).

Diagnostic species

Chenopodium amaranticolor. Chlorotic local lesions within a week (Fig. 2) turning necrotic in older leaves, followed by mild yellowish mottling of systemically infected leaves (Fig. 3) in short day conditions.

Chenopodium quinoa. Local chlorotic spots (Fig. 4) followed by systemic mottling and distortion of the leaves. It is essential that seedlings be grown under conditions that increase their susceptibility to systemic infection (Garcia-Castillo et al., 2001).

Gomphrena globosa. Local necrotic spots in 7-10 days (Fig. 5) not followed by systemic infection. This distinguishes CarMV from Carnation ringspot virus (genus Dianthovirus, family Tombusvidae).

Tetragonia expansa. Faint local chlorotic or necrotic lesions in about a week, followed by systemic infection.

Saponaria vaccaria (Pink Beauty). Vein chlorosis 8-10 days after inoculation followed by systemic mottling and malformation of the leaves and stunting (Hakkaart, 1974).

Dianthus barbatus (Sweet William). Some clones react with local yellowish spots in about a week followed by systemic chlorotic mottling. Other clones are immune.

Propagation species

Carnation, C. quinoa and S. vaccaria are suitable hosts for virus propagation and purification.

Assay species

C. amaranticolor and C. quinoa are useful local lesion hosts (Bansal & Singh, 1980) and will detect attenuated forms of the virus.

Transmission of virus to experimental hosts can detect and indicate the presence of CarMV but other methods are required for its specific identification.

Strains

Biological variants

The PSR strain (Hollings & Stone, 1964) induces necrotic local lesions in some hosts which develop chlorotic spots when inoculated with the serologically indistinguishable type strain.

Naturally-occurring attenuated (mild) strains of the virus that have little obvious effect on infected plants have been reported (Poupet et al., 1972; Tomlinson & Faithfull, 1976). An attenuated strain eliciting large chlorotic lesions in C. amaranticolor about three weeks after inoculation (Fig. 5) was isolated from carnation following heat treatment or meristem tip culture (Hollings & Stone, 1962). On serial passage, this strain reverted to the typical rapidly multiplying form with which it was serologically identical.

Serological variants

Variants differing serologically from the type strain (serological differentiation index = 1-2) have been reported from Canada (Kemp & Fazekas, 1966) and Italy (Rana & Castellano, 1984).

Molecular variants

The virus is genetically stable, as shown by the comparative study of the sequence of 21 geographically distinct isolates. However, coat protein (CP) analysis identified two distinct groups of strains, denoted PK and AN, based on the co-variation between amino acids at position 164, located in the S domain (see later) and at position 331, located in the P domain (Canizares et al., 2001). An Indian virus isolate belongs to a putative third group (PN) (Singh et al., 2005).

Transmission by Vectors

No transmission by Myzus persicae and Macrosiphum euphorbiae (Hollings & Stone, 1964). Unconfirmed persistent transmission by Aphis gossypii reported from India (Singh & Singh, 1989).

Transmission through Seed

None found (Hollings & Stone, 1964).

Transmission by Dodder

None found (Hollings & Stone, 1964).

Serology

The virus is an efficient immunogen. Polyclonal antisera with titres up to 16,384 raised in rabbits immunized with purified virus preparations yield a single precipitin band in gel-diffusion tests (Hollings & Stone, 1964; 1970). Monoclonal antibodies (MAbs) have been produced (Jordan & Guaranga, 2002; Wu et al., 2005). Early detection and identification methods were agglutination tests, i.e. virobacterial (Chirkov et al., 1984) or high density latex (Polak, 1980; 1983; Kawano & Takahashi, 1997), and micro-precipitin tests (Zhang, 1994). However, since all strains of the virus, except the attenuated forms, can be detected in crude sap by gel-diffusion tests, these have been widely used (Hollings & Stone, 1964; Oertel, 1977; Odinets et al., 1983). Although the reliability of gel-diffusion tests can be hampered by the seasonal fluctuations of virus titre (Kemp, 1967), because of their simplicity and specificity, they are still very useful despite the availability of more advanced serological procedures. ELISA has been used in the double antibody sandwich direct (DAS-ELISA) or indirect (DASI-ELISA) formats (Devergne et al., 1982; Eskenazy et al., 1983; Lommel et al., 1982; 1983a; 1983b; Polak, 1983; Moran et al., 1985; Severin & Gonzalez, 1996; Jordan & Guaranga, 2002; Chen et al., 2003a; 2003b; Singh et al., 2005), especially for testing large numbers of specimens (Rodoni et al., 1994). Tissue-Blotting ELISA is reported to be as sensitive as ELISA and Dot-ELISA (Zhang, 1999). The specificity and sensitivity of ELISA has been improved by the use of monoclonal antibodies; using MAbs Wu et al. (2005) detected virus in plant sap diluted 800-fold and as little as 0.1 ng in virus preparations.

Nucleic Acid Hybridization

Molecular assays using non-radioactive probes were developed for detecting virus in infected carnation leaves (Sanchez-Navarro et al., 1996; Pallas et al., 1999). Dot blot hybridization was 125 times more sensitive than DAS-ELISA (Sanchez-Navarro et al., 1999). RT-PCR and Immuno-capture PCR are also very effective detection methods (Kong et al., 2002; 2003; Singh et al., 2005). More recently, Multiplex PCR has been used (Raikhy et al., 2006). Analysis of dsRNA has been used occasionally for virus detection (Yamashita et al., 1996) but is far less specific than other molecular procedures.

Relationships

In phylogenetic trees, CarMV clusters with all the other species of the genus Carmovirus, the closest being Saguaro cactus virus (Canizares et al., 2001). No serological relationships have been reported with any of other members of the same or seven other genera of the family Tombusviridae. Type and PSR strains protect against one another in cross-protection tests (Hollings & Stone, 1964).

Stability in Sap

The virus is very stable. In carnation and C. quinoa sap its thermal inactivation point (10 min) is about 90°C, dilution end point is 10^-5 (carnation) to 10^-7 (C. quinoa), and the infectivity survives from 70 (carnation) to 90 days (C. quinoa) at room temperature (18-20°C) (Hollings & Stone, 1964; Waterworth & Kaper, 1972). At the same temperature, the attenuated form of the virus was recovered after 13 months and virus lyophilized in sap of D. barbatus stored under vacuum retained infectivity for over 10 years (Hollings & Stone, 1970). Most of the virus infectivity was retained (92-94%) when frozen for 1 h at -20°C or -70°C and thawed. However, it decreased markedly following freeze-drying at pH 5.5-8.5, except when 0.5% lysine was added prior to the treatment (Fukumoto & Tochihara, 1986; 1990).

Purification

Method 1. (Hollings & Stone, 1964). Carnation or pink leaves minced with 0.05 M phosphate buffer, pH 7.6, containing 0.1% thioglycollic acid (w/v = 1/1.25) are squeezed through cheesecloth and the slurry treated with n-butanol to a final volume of 8.5%. The mixture is stored overnight at 2°C and the virus is separated from the aqueous phase by differential centrifugation. Final pellets are resuspended in phosphate buffer and subjected to sucrose density gradient centrifugation for 2 h at 25,000 rpm. Preparations made with borate buffer are cleaner but have about half the infectivity and serological activity than those extracted with phosphate buffer. Precipitation with ammonium sulphate (40% saturation) or ethanol (50%) or by acidification to pH 4.8 also gives acceptable virus preparations. Similar results are obtained by precipitation with polyethylene glycol (Sivers et al., 1976; Zhang, 1993).

Method 2. (Waterworth & Kaper, 1972). Stems and leaves of systemically infected C. quinoa plants are blended in 2-3 vol of 0.025 M phosphate buffer pH 7.0, squeezed through cheesecloth and the juice centrifuged at 3,500g for 10 min. The supernatant is treated with powedered activated charcoal at 5 g/100 ml juice and subjected to low-speed centrifugation (3,500g for 10 min). The supernatant is then mixed with 1 to 2 ml of Mg-treated bentonite/100 ml juice, incubated for 10 min and centrifuged again at low-speed. Clarified sap is centrifuged at 105,000g for 1.3 h, pellets are resuspended in the extraction buffer and subjected to sucrose density gradient centrifugation at 98,000g for 1.5 h. Rate-zonal centrifugation produces a single opalescent band where most of the infectivity is concentrated. Virus yield ranges from 230 to 350 mg/kg of infected tissue. Yields of up to 500 mg/kg of infected tissue were obtained with preparations purified from D. barbatus (Tremaine, 1970).

Properties of Particles

Sedimentation coefficient: CarMV sediments as a single component with a sedimentation coefficient (s_20,w) of 122-126 S (Tremaine, 1970; Waterworth & Kaper, 1972).

Molecular weight: 7.6-7.7 × 10⁶ (Tremaine, 1970; Waterworth & Kaper, 1972).

A₂₆₀/A₂₈₀ ratio: 1.48-1.66 (Waterworth & Kaper, 1972).

Isoelectric point: pH 5.2 (Tremaine, 1970).

Extinction coefficient: E₂₆₀ = 49.05 (Waterworth & Kaper, 1972).

Electrophoretic mobility: -8 × 10^-5 cm² sec^-1 volt^-1 at pH 7.0 in 0.02 ionic strength buffer (Tremaine, 1970).

Inactivation by ultraviolet irradiation and by chemicals: Exposure to U.V. rays to 30 min and to 2.0% formaldeyde or tri-sodium orthophospate destroyed most to the infectivity (Hollings & Stone, 1964).

Particle Structure

Particles are T=3 icosahedra 32-35 nm in diameter, with a rounded contour and a granular surface appearance (Fig. 6). The protein coat (CP) is composed of 180 identical subunits each of which is folded into the following three distinct domains: R, the N-terminal internal domain interacting with RNA; S, the shell domain, constituting the capsid backbone; and P, the C-terminal domain clustering in pairs that protrude from the particle surface to form 90 projections (Morganova et al., 1994). The particle structure of CarMV particles is thus similar to that of Turnip crinkle virus (TCV), another member of the Carmovirus genus (Hogle et al., 1986).

Particle Composition

Nucleic acid: Monopartite, single-stranded, linear, positive sense RNA with a molecular weight of 1.4 x10⁶, and sedimentation coefficient of 23 S. RNA accounts for about 18% of the particle weight and has the following molar percentages of nucleotides: G27, A30, C19, U24 (Tremaine, 1970); G26, A28, C22, U24 (Waterworth & Kaper, 1972).

Protein: Protein content in the particles is about 82%. CP subunits each consist of 348 amino acids and have a molecular weight of 38 kDa (Guilley et al., 1985; Canizares et al., 2001).

No lipids or other components have been detected in the particles.

Properties of Infective Nucleic Acid

RNA extracted from purified virions is infectious at concentrations as low as 4.7x10⁵ µg/ml (Waterworth & Kaper, 1972).

Genome Properties

Reviewed by Morris & Carrington (1988) and Russo et al. (1994). Complete genome sequences are available of CarMV isolates from the USA (Guilley et al., 1985; X02986), Spain (Vilar et al., 2001; AJ304989), China (Zhang et al., 2002; AF192772 = NC_001265) and India (Raikhy et al., 2006; AJ811998). A large (3,850 bp) cDNA fragment of the viral genome was cloned by Carrington & Morris (1984) and, more recently, a full-length infectious cDNA clone of the Chinese isolate has been made (Zhang et al., 2002). Viral genomes are 4,003-4,005 nucleotides in size, have sequences that are 95 to 97% identical (Canizares et al., 2001) and have the same structural organization. Regardless of the isolate, the genome contains four open reading frames (ORFs) which, in the order from the 5' to the 3' terminus, code for replication-associated proteins (ORF1 and ORF1-RT), movement proteins (ORF2 and ORF3), and the coat protein (ORF4). The genome is very compact, having non-coding regions of limited size (Fig. 7). Genomic RNA acts as a messenger for the translation of a 27 kDa protein from ORF1. By translational readthrough of the amber termination codon of ORF1, a protein of 86 kDa is synthesized which contains, in the readthrough portion, the GDD motif of RNA-dependent RNA polymerases (RdRp). Genes downstream of ORF1-RT are expressed via the synthesis of two subgenomic RNAs, 1.7 nt and 1.45 nt in size (Fig. 7). The centrally located ORFs 2 and 3 encode proteins of 7 and 9 kDa, respectively, both of which are involved in cell-to-cell transport of virus. In particular, the 7 kDa protein (p7) is an RNA-binding soluble protein (Vilar et al., 2001), whereas the 9 kDa protein (p9) has two transmembrane domains that allow its anchoring to the endoplasmic reticulum, with the N and C termini facing the cytoplasm (Vilar et al., 2002). The N and C segments of p9 interact with the soluble RNA-bound p7 to accomplish the cell-to-cell movment of the virus. ORF 4 is the coat protein gene encoding a 38 kDa protein.

Relations with Cells and Tissues

CarMV replicates very actively in cytoplasm of the host cells, resulting in the synthesis of numerous virus particles in the cells of different tissue types. Thus, the virus is found in stems, leaves, flowers, roots and apical meristems. No inclusion bodies of diagnostic value are produced, as in the case of other members of the same genus (Martelli & Russo, 1984). Virus particles occur in the cytoplasm of parenchyma cells and vascular tissues, are frequently associated with membranes or fill bleb-like extrusions of the tonoplast in the vacuoles, and, occasionally, are present in the nuclei (Robleda, 1973; Morris-Krsinich & Milne, 1977). Long distance movement of the virus in phloem tissues of C. quinoa is influenced by environmental factors (e.g., temperature and light intensity) (Garcia-Castillo et al., 2001).

Ecology and Control

CarMV is considered to be invasive because, as it is stable in vitro and highly infectious, it is readily transmitted mechanically from infected to healthy plants. Because the virus does not always induce obvious symptoms in young carnation and pinks plantlets, there is a constant risk that it could be accidentally disseminated internationally in infected stocks, which may lead to an infection incidence as high as 100% in glasshouse crops (Lommel et al., 1982; Hollings et al., 1977; Makkouk & Shehab, 1980). However, the risk is now less as virus-tested stocks are generally available and certification schemes administered by the European and Mediterranean Plant Protection Organization (Anonymous, 1991) and by the Council of the European Communities (Anonymous, 1993) are being implemented in some countries. CarMV is transmitted by contact between infected and healthy plants but, more usually, with virus-contaminated hands and equipment during horticultural practices (Hollings & Stone, 1964). The virus also occurs in fresh river and sea waters (Koenig & Lesemann, 1985; Kontzog et al., 1988) and susceptible plants can also be infected if grown in moist soil or Perlite containing infective virus (Goethals et al., 1973; Keldysh et al., 2005).

The virus is best controlled by the production, propagation and distribution of virus-tested stock plants, within the framework of certification schemes. Virus was reported to be eliminated from plants kept at 38°C for 2 months (Brierley, 1964) but this was not confirmed by Hollings & Stone (1964). Virus-tested plants, however, have been produced in many countries by meristem-tip culture (Quak, 1957; Paludan, 1965; Stone, 1968; Hollings & Stone, 1964; 1970; Kowalska, 1974; Rybalko & Kharuta, 1978; Pena-Iglesias et al., 1979; Poupet et al., 1981; Devergne, 1984; Fawzy et al., 1992). Meristem-tip culture was shown to be more effective if plants from which meristems are taken were first subjected to heat therapy (38°C for 3-5 weeks) (Goethals & van Hoof, 1971; Kowalska, 1974). Virus-tested stocks should be grown under carefully controlled conditions that reduce the possibility of becoming re-infected. Thus, virus spread can be greatly reduced by the application of elementary sanitary precautions such as handling plants individually and as infrequently as possible, using sterile cutting knifes or blades to take cuttings from each plant and growing virus-tested plants in sterile media (Goethals et al., 1973). Resistance to CarMV has been obtained experimentally by transferring the virus coat protein gene to plants, but such genetically modified carnations have not yet been used commercially (Bae & Yu, 2002; Yu & Bae, 2002).

Notes

Several other viruses with isometric particles 30-35 nm in diameter infect carnations. Three of these, Carnation ringspot virus (CRSV), Carnation Italian ringspot virus (CIRV) and Carnation yellow stripe virus (CYSV) are members of the family Tombusviridae, but are included in other genera, i.e. Tombusvirus (CIRV), Dianthovirus (CRSV), and Necrovirus (CYSV), respectively (Lommel et al., 2005). All these viruses differ from CarMV in host range, molecular properties and cytopathological features, and are serologically unrelated with and phylogenetically distant from it (Lommel et al., 2005). Even more diverse are Carnation cryptic virus 1 (CCV-1) and Carnation cryptic virus 2 (CCV-2), which are a definitive (CCV-1) and a tentative species (CCV-2) in the genus Alphacryptovirus, family Partitiviridae (Ghabrial et al., 2005).

Acknowledgements

The authors thank Dr. M.G. Bellardi, University of Bologna, Italy, for providing Fig. 1.

Figures

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