Details of DPV and References
DPV NO: 360 September 1998
Family: Unallocated ssRNA+ viruses
Species: Raspberry bushy dwarf virus | Acronym: RBDV
This is a revised version of DPV 165
Raspberry bushy dwarf virus
A. Teifion Jones Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, Scotland, UK
M. A. Mayo Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, Scotland, UK
- Main Diseases
- Geographical Distribution
- Host Range and Symptomatology
- Transmission by Vectors
- Transmission through Seed
- Transmission by Grafting
- Transmission by Dodder
- Nucleic Acid Hybridization
- Stability in Sap
- Properties of Particles
- Particle Structure
- Particle Composition
- Properties of Infective Nucleic Acid
- Molecular Structure
- Genome Properties
- Relations with Cells and Tissues
- Ecology and Control
- Described by
Barnett & Murant (1970).
- Loganberry degeneration virus
Rev. appl. Mycol. 39: 603)
- Raspberry yellows virus (Cadman, 1952; Jones et al., 1982)
- Raspberry line pattern virus (Basak, 1971; Jones et al., 1982)
- Raspberry yellows virus (Cadman, 1952; Jones et al., 1982)
A virus with quasi-isometric particles about 33 nm in diameter, occurring in Rubus species world-wide. Virus particle preparations contain three ssRNA species of 5.4, 2.2 and 0.9 kb and a major protein of Mr 30,500. The virus is readily transmissible by inoculation of plant sap and infects a wide range of plants, often symptomlessly. It is seed- and pollen-borne in Rubus and spreads between plants in association with pollen. No vector is known.
The virus is involved with others in inducing the disease known as bushy dwarf (Cadman & Harris, 1951), or symptomless decline (Cadman, 1952) in Lloyd George red raspberry; affected plants are characterized by stunting and proliferation of canes and are prone to autumn fruiting. Despite its name, RBDV does not on its own cause bushy dwarf disease (Barnett & Murant, 1970); this disease results when RBDV infects plants already infected with the aphid-borne virus, black raspberry necrosis (Jones, 1979). However, in sensitive Rubus species and cultivars, RBDV is the causal agent of yellows disease (Jones et al., 1982) in which the leaves show pronounced chlorotic/bright yellow line-patterns or veinal yellowing; yellowed areas often coalesce to produce a partial or complete yellowing of affected leaves (Fig. 1; Daubeny et al., 1978; Jones et al., 1982). Circumstantial evidence suggests that premature defoliation, decreased vigour, leaf curling, necrosis, death of lateral shoots and increased winter kill are associated with infection by some isolates of the virus in some red raspberry cultivars (Wilson et al., 1983; Barbara et al., 1984). Following graft-inoculation, the virus induces chlorotic/bright yellow mottling and/or line-patterns in R. laciniatus, R. molaccanus, R. procerus, R. phoenicolasius, and also in Cydonia oblonga cv. C7/1 (Desvignes & Savino, 1975; Jones et al., 1982). The virus infects symptomlessly many Rubus species and cultivars, including blackberry, loganberry, red raspberry and black raspberry and, by graft-inoculation, Fragaria vesca and Prunus mahaleb (Barnett & Murant, 1970, 1971; Converse, 1973; Jones, 1986; Jones et al., 1982). Fruit on infected sensitive cultivars of raspberry may have a high proportion of aborted drupelets, a condition known as crumbly fruit (Fig. 2; Daubeny et al., 1970, 1978; Murant et al., 1974).
Probably occurs wherever Rubus is grown world-wide. It is reported from eastern and western Europe, Scandinavia, Russia, North and South America, Australasia and South Africa (summarized by Murant, 1987).
Host Range and Symptomatology
One isolate of the virus infected 55 species in 12 dicotyledonous families, most of
(Barnett & Murant, 1970,
Most isolates induce a
mild systemic mottle in some species of Chenopodiaceae and chlorotic or necrotic
local lesions in some species of Leguminosae. With the exception of isolates from
(Murant & Jones, 1976;
Jones et al., 1996),
isolates of the virus are readily transmitted between herbaceous plant hosts by
inoculation of sap. Transmission from Rubus hosts is best achieved in spring
or early summer by grinding source leaves in 2% nicotine solution or 1%
polyethylene glycol (M. Wt 6000).
- Chenopodium amaranticolor. Occasional transient chlorotic
local lesions in 4-6 days; systemic chlorotic rings and line-patterns after 7-10 days
- C. murale. Local necrotic lesions or rings in 5-8 days; not systemic.
- C. quinoa. Occasional transient chlorotic/necrotic local lesions in 4-6 days; systemic chlorotic rings and line-patterns after 7-10 days.
- Phaseolus vulgaris cv. The Prince. Under winter conditions, small necrotic local lesions after 3-5 days (Fig. 4); not systemic.
- Nicotiana clevelandii. Symptomless systemic infection.
- C. murale. Local necrotic lesions or rings in 5-8 days; not systemic.
- C. quinoa is suitable for maintaining cultures and for propagating
the virus for purification.
- P. vulgaris cv. The Prince is useful provided that assays are made under winter conditions (growth chamber conditions 20 °C, 5000 lux, 16 h photoperiod) but lesion development is inhibited by a component of C. quinoa sap that occurs in increased concentrations in plants grown in long days and high light intensity conditions (Barnett & Murant, 1970). Lesion numbers are increased by using phosphate buffer in the inoculum and by keeping bean plants in the dark for 1 day before inoculation. However, isolates maintained in culture by successive passage in C. quinoa sometimes lose their ability to induce lesions in P. vulgaris.
Several variants are known. Jones et al. (1996) placed isolates into one of three categories: S, isolates with serological characteristics, and restricted Rubus host range, similar to those of the Scottish type isolate D200; RB (resistance-breaking), isolates which are serologically indistinguishable from S isolates but are able to infect Rubus hybrids and cultivars resistant to S isolates; and B, isolates found in black raspberry (R. occidentalis) which are serologically distinguishable by spur formation (in gel double diffusion tests) from S and RB isolates and which appear to have a lower specific infectivity (Murant & Jones, 1976; Jones, 1986; Jones et al., 1996). Several laboratory and field variants of S and RB isolates are known which differ in symptom severity in herbaceous hosts and in at least one instance in serological properties (A. T. Jones unpublished data).
Transmission by Vectors
Transmission through Seed
The virus can be transmitted to seed of red and black raspberry through both
gametes; up to 77% of seedlings from infected red raspberry (R. idaeus,
Murant et al., 1974;
were infected. Up to 15% seed transmission was detected in R.
Only 1-2% seed transmission was detected in
F. vesca, and none in C. quinoa
(Murant et al., 1974).
In raspberry, the virus in pollen infects not only the seed resulting from fertilization but also the plant pollinated (Cadman, 1965; Murant et al., 1974). This appears to be the only means of spread in the field because, in plots containing infector plants, no spread was detected to healthy plants when these plants were prevented from flowering for 3 years, whereas most healthy plants allowed to flower became infected during the first two flowering seasons (Murant et al., 1974). Natural field spread can be rapid in infectible cultivars (Murant et al., 1974; Bulger et al., 1990).
Moderately immunogenic in rabbits; antisera with titres of 1/512 were produced (Barnett & Murant, 1970). Martin (1984) produced four mouse monoclonal antibodies to purified virus particles of an S isolate of RBDV. In immunodiffusion tests against polyclonal antiserum, the virus gives a single precipitin line with moderate dilutions of antiserum but additional lines may be present at a low dilution, suggesting that either smaller virus-related components exist or antibodies to plant proteins are present. Those virus isolates tested from black raspberry (B category isolates) are very similar to S and RB category isolates serologically, reacting almost to the titration end-point of antiserum to isolates in these categories, but they can be differentiated from them by the presence of spurs in gel double diffusion tests (Murant & Jones, 1976). All isolates react strongly in DAS-ELISA with either polyclonal or monoclonal antiserum (Jones et al., 1996). In Western blot analysis, polyclonal antiserum reacted strongly to isolates from all three categories, whereas the four monoclonal antibodies raised against an S category isolate reacted strongly with some isolates but only very weakly with others (A. T. Jones & W. J. McGavin, unpublished data).
A report that the virus is related to apple chlorotic leaf spot closterovirus (Cadman, 1965, 1970) is disproved (Barnett & Murant, 1970). Although the particle morphology and mode of transmission through pollination suggests affinities with viruses in the genus Ilarvirus, it differs from them in sedimentation behaviour and in the number and sizes of its RNA species. It is recognized as the sole member of the genus Idaeovirus (Murant & Mayo, 1995).
Stability in Sap
In C. quinoa sap, a typical S category isolate lost infectivity after dilution 10-4, heating for 10 min at 65 °C or storage for 4 days at 22 °C; infectivity was stabilized by the presence of 0.02 M 2-mercaptoethanol but not by 0.02 M DIECA (Barnett & Murant, 1970). By contrast, a B category isolate in C. quinoa sap lost infectivity after dilution 10-2 or storage for 2-3 h at 20 °C (Murant & Jones, 1976).
Harvest inoculated and systemically infected leaves of C. quinoa about 10 days after inoculation and triturate in 4 volumes (w/v) of 0.05 M sodium phosphate buffer, pH 7 containing 0.01 M 2-mercaptoethanol. Adjust the pH to 4.8 with 0.5 M citric acid and centrifuge for 20 min at 3000 g. In some preparations following this treatment, the virus is in the pellet and in others in the supernatant fluid. If the virus is in the pellet, recover it by resuspension overnight in 0.05 M citrate buffer, pH 6.0, followed by low speed centrifugation to remove insoluble material. If the virus is in the supernatant fluid following the pH 4.8 step, precipitate it by adding PEG (M. Wt 6000) to 8% (w/v) and NaCl to 0.8% (w/v), resuspending the pellets in citrate buffer containing 0.01 M 2-mercaptoethanol. Concentrate the virus by differential centrifugation, and purify further by exclusion chromatography in columns of 2% agarose beads, and/or by centrifugation in sucrose density gradients (Murant, 1976).
Properties of Particles
The virus particles are unstable and readily disrupt in the presence of 0.01% sodium dodecyl sulphate, indicating that they are stabilized by protein-RNA linkages (Murant, 1976). The particles sediment as a rather broad band in sucrose density gradients, with so20,w of 115 S in 0.05 M citrate buffer, pH 6. Very rarely, preparations also contain particles c. 15 nm in diameter that sediment at c. 34 S and are serologically indistinguishable from the 33 nm diameter particles. When centrifuged to equilibrium at 20°C in solutions of CsCl or RbBr, formaldehyde-fixed virus particles formed a band at a density of c. 1.37 g cm-3 (Murant, 1976).
The virus particles are c. 33 nm in diameter, and appear quasi-isometric (Fig. 5) because they tend to collapse and deform on the electron microscope grid; they appear more spherical in shape when previously fixed in glutaraldehyde.
Nucleic acid: The virus particles contain three species of ssRNA which are
sometimes present in approximately equimolar amounts. However, in some
preparations and with some isolates, the smallest RNA species is present in much
greater molar concentration than the two larger species. Virus particle preparations
have A260/A280 of 1.62, suggesting
that particles contain about 24% RNA. The complete nucleotide sequences of the
three RNA species of a RB category isolate have been determined; they contain,
5449 nt (RNA-1; accession no. S51557;
Zeigler et al., 1992),
2231 nt (RNA-
2; accession no. S55890;
Natsuaki et al., 1991)
and 946 nt (RNA-3;
Mayo et al., 1991).
The way in which the RNA molecules are packaged in the
particles is unknown, but it is unlikely that all three RNA species are contained
within the same particle.
Protein: In polyacrylamide gel electrophoresis, particle preparations typically contain a single major protein of Mr c. 30,000 (Murant et al., 1986). However, some preparations, even highly purified ones, contain minor amounts of smaller proteins.
The genome comprises RNA-1 and RNA-2. RNA-3 is an exact copy of the 3'-most 946 nt of RNA-2 (Fig. 6). RNA-1 encodes a c. 190 kd polypeptide which contains three domains with sequence characteristics of methyltransferase, NTP-binding or RNA polymerase activities. RNA-2 encodes two polypeptides of which the 5'-most (39 kd) has some sequence similarities to movement proteins of other viruses. The second ORF encodes the coat protein (calculated M. Wt = 30,509) but this is synthesized in vitro by translation of RNA-3, not RNA-2 (Fig. 6) (Jones et al., 1996).
Relations with Cells and Tissues
In ultrastructural studies of infection in raspberry leaves showing yellows disease symptoms, Watkins et al. (1992) found extensive enlargement of chloroplasts, decreased cell vacuole volume and an increased production of vesicles. These features were not found in raspberry leaves showing pronounced yellowing induced by somatic mutation. No virus-like particles were detected in these studies. RBDV is not readily eliminated from infected Rubus by heat treatment alone. However, heat treatment followed by propagation from shoot tips or apical meristems eliminated the virus from some plants of R. idaeus (Murant et al., 1974; Lankes, 1995), R. strigosus (Mellor & Stace-Smith, 1976) and R. occidentalis (Converse, 1973). When using only tissue culture of meristem tips, it was necessary to subculture at least three times to eliminate the virus from raspberry (Theiler-Hedtrich & Baumann, 1989). Culturing axillary buds on a medium containing the antiviral compounds ribavirin and dodecyl- N-methylephedrinium bromide eliminated virus from some such cultures (Küdell & Buchenauer, 1989).
Ecology and Control
Because of its mode of transmission, the virus can be controlled in infectible cultivars only by planting virus-tested stock plants in isolation from sources of infection. This can be difficult because wild Rubus species may be infected with the virus (A. T. Jones, unpublished data; Credi et al., 1986). Where RB category isolates are known not to occur, effective control can be achieved by growing resistant cultivars (Jones et al., 1982; Jones, 1988).
The virus resembles
in its particle morphology and mode of transmission.
These features and some sequence similarities prompted the suggestion that RBDV
be classified in the family Bromoviridae
despite its having a bipartite genome
(Ziegler et al. 1993).
However, currently this suggestion is considered
Two ilarviruses, tobacco streak and its variants (Jones & Mayo, 1975; Stace-Smith, 1987) and apple mosaic (Baumann et al., 1987), are reported to infect Rubus naturally. However, unlike RBDV, most described isolates of these viruses induce systemic necrosis in C. quinoa and noticeable symptoms in several Nicotiana species. The virus is serologically unrelated to either of these viruses or to several other ilarviruses tested (Jones et al., 1996).
Progressive stages in the development of yellows disease symptoms in leaves of the red raspberry cv. Norfolk Giant.
Electron micrograph showing quasi-isometric virus particles in a purified particle preparation stained with uranyl formate. Bar = 100 nm.
Diagram showing the genome structure of RBDV based on the RNA sequences. Solid lines represent the RNA molecules. Boxes show the putative positions of the translation products of the ORFs. The positions of domains in the polyproteins are shown as: mtr, methyl transferase; NTP, NTP-binding; pol, RNA-dependent RNA polymerase; MP, residues shared among putative transport proteins. The dotted lines indicate that RNA-3 is derived from the 3'-terminal portion of RNA-2. The 12K translation product of RNA-1 has not been shown to be expressed and is therefore a putative product (Jones et al., 1996).
References list for DPV: Raspberry bushy dwarf virus (360)
- Barbara, Jones, Henderson, Wilson & Knight, Ann. appl. Biol. 105: 49, 1984.
- Barnett & Murant, Ann. appl. Biol. 65: 435, 1970.
- Barnett & Murant, Annals Phytopath. Numéro hors série: 129, 1971.
- Basak, Bull. Acad. polon. Sci., Ser. Sci. Biol. 19: 681, 1971.
- Baumann, Casper & Converse, Virus Diseases of Small Fruits, USDA Agriculture Handbook No. 631, p. 229, 1987.
- Bulger, Stace-Smith & Martin, Pl. Dis. 74: 514, 1990.
- Cadman, Ann. appl. Biol. 39: 495, 1952.
- Cadman, Pl. Dis. Reptr 49: 230, 1965.
- Cadman, Virus Diseases of Small Fruits and Grapevines, University of California Press, Berkeley, p. 149, 1970.
- Cadman & Harris, Rep. E. Malling Res. Stn for 1950: 127, 1951.
- Converse, Phytopathology 63: 780, 1973.
- Credi, Shier & Stace-Smith, Acta Hort. 186: 17, 1986.
- Daubeny, Freeman & Stace-Smith, J. Am. Soc. hort. Res. 95: 730, 1970.
- Daubeny, Stace-Smith & Freeman, J. Am. Soc. hort. Res. 103: 519, 1978.
- Desvignes & Savino, Acta Hort. 44: 139, 1975.
- Jones, Ann. appl. Biol. 86: 199, 1977.
- Jones, J. Hort. Sci. 54: 267, 1979.
- Jones, Crop Res. 26: 127, 1986.
- Jones, Aspects appl. Biol. 17: 179, 1988.
- Jones & Mayo, Ann. appl. Biol. 79: 297, 1975.
- Jones, Murant, Jennings & Wood, Ann. appl. Biol. 100: 135, 1982.
- Jones, Mayo & Murant, The Plant Viruses, Vol. 5: Polyhedral Virions and Bipartite RNA Genomes, Plenum Press, NY, p. 283, 1996.
- Küdell & Buchenauer, J. Phytopath. 124: 332, 1989.
- Lankes, Acta Hort. 385: 70, 1995.
- Martin, Can. J. Pl. Path. 6: 264, 1984.
- Mayo, Jolly, Murant & Raschke, J. gen. Virol. 72: 469, 1991.
- Mellor & Stace-Smith, Acta Hort. 66: 63, 1976.
- Murant, C.M.I./A.A.B. Descriptions of Plant Viruses 165, 4 pp., 1976.
- Murant, Virus Diseases of Small Fruits, USDA Agriculture Handbook No. 631, p. 229, 1987.
- Murant & Jones, Acta Hort. 66: 47, 1976.
- Murant & Mayo, Virus Taxonomy - Classification and Nomenclature of Viruses: 6th Rept Int. Committee on Taxonomy of Viruses, Springer-Verlag, Vienna. (Arch. Virol. Suppl. 10), p. 458, 1995.
- Murant, Chambers & Jones, Ann. appl. Biol. 77: 271, 1974.
- Murant, Mayo & Raschke, Acta Hort. 186: 23, 1986.
- Natsuaki, Mayo, Jolly & Murant, J. gen. Virol. 72: 2183, 1991.
- Ormerod, Rep. E. Malling Res. Stn for 1969: 165, 1970.
- Ormerod, Rep. E. Malling Res. Stn for 1971: 127, 1972.
- Stace-Smith, Virus Diseases of Small Fruits, USDA Agriculture Handbook No. 631, p. 235, 1987.
- Theiler-Hedtrich & Baumann, J. Phytopath. 127: 193, 1989.
- Watkins, Roberts & Jones, Ann. appl. Biol. 121: 151, 1992.
- Wilson, Knight & Barbara, Pl. Path. 32: 357, 1983.
- Ziegler, Mayo & Murant, Arch. Virol. 131: 483, 1993.
- Ziegler, Natsuaki, Mayo, Jolly & Murant, J. gen. Virol. 73: 3213, 1992.