Tobacco necrotic dwarf virus
Central Research Institute,The Japan Tobacco & Salt Public Corporation, Midori-ku, Yokohama, Japan
Described by Kubo, Tamura & Fukuda (1976)
and Kubo &
A virus with RNA-containing isometric particles c. 25 nm in
diameter occurring in phloem cells. Transmitted by aphids in the
persistent manner, but not by inoculation with sap. It has a narrow
host range. Recorded only from Japan.
Causes stunting, and premature yellowing and death of the lower
and intermediate-aged leaves, of tobacco (Nicotiana tabacum
). Causes faint vein yellowing in spinach.
Recorded only from Japan.
Host Range and Symptomatology
Twenty species in five families are known to be susceptible;
solanaceous plants are the main hosts. Natural hosts are tobacco,
spinach and Capsella bursa-pastoris
. Spinach is the primary
overwintering host. Gomphrena globosa
can be infected experimentally. Severity of phloem
necrosis in tobacco plants varies between cultivars and is most
prominent in cv. MC 1 (Fig.1
- Diagnostic species
- Datura stramonium. Systemically infected leaves show
- Nicotiana sylvestris. Systemically infected leaves show
vein yellowing followed by interveinal chlorosis. Plants are
severely stunted. Leaves bearing veinal necrosis roll downward and
are malformed (Fig.3).
- Physalis floridana. Interveinal chlorosis and moderate
rolling of systemically infected leaves (Fig.4).
- Propagation species
- Physalis floridana is suitable for maintaining cultures
and is the best source of virus for purification.
- Assay species
- Nicotiana sylvestris is useful for insect transmission
tests. Symptoms appear about 10 days after infection (Fukuda, 1978).
Strains of the virus have not been studied in detail, but
several isolates are distinguishable from the type strain on the
basis of severity of reactions of Nicotiana
(S. Kubo, unpublished results).
Transmission by Vectors
The virus is transmitted by the aphid Myzus persicae
in the persistent manner (Kubo et al., 1976
also transmits the virus, but less
efficiently (M. Tamura, unpublished results). M. persicae
seems to be the only species that transmits the virus from the
overwintering host (spinach) to tobacco plants. Minimum acquisition
access period is 3 h and minimum inoculation access period is 30 min.
There is a latent period in the vector of 2 to 4 days, depending on
the dose of virus acquired. M. persicae
injected with purified
virus at high concentrations can transmit in 17 h. The aphid retains
ability to transmit the virus after moulting and for periods longer
than 30 days. In M. persicae
injected with purified virus
preparations, the virus particles are observed in the basal lamina
of salivary glands including the accessory ones and in abdominal
connective tissue cells, but there is no evidence for multiplication
of the virus in the aphids (Kuwata & Kubo, 1978
Transmission through Seed
Not seed-transmitted in N. tabacum
Strongly immunogenic in rabbits. Antiserum with a titre of
1/4096 (microprecipitin test) has been obtained by intramuscular
and intravenous injection (Kubo & Takanami, 1979
). The conventional
agar-gel double diffusion test is useful but, because of the low
concentration of the virus in plants, concentrated or partially
purified preparations must be used.
Serologically, the virus is closely related to potato leafroll
and distantly related to soybean dwarf
and carrot red leaf
viruses (Kubo & Takanami, 1978
; Roberts, Tamada & Harrison,
; Waterhouse & Murant, 1981
; S. Kubo, unpublished results).
Tobacco necrotic dwarf and potato leafroll viruses both have a
single-stranded RNA of M. Wt 2.0 x 106
and they share
many properties with other members of the luteovirus group
& Kubo, 1979b
Stability in Sap
Determined by inoculation of tobacco mesophyll protoplasts with
partially purified virus preparations. Thermal inactivation point
(10 min) is c.
80°C; infectivity is retained for more
than 6 months at 4°C (Kubo & Takanami, 1980
The following enzyme-assisted method is useful (Takanami &
). Powder frozen leaves of infected P. floridana
and disrupt in a mechanical blender in 0.1 M sodium citrate buffer
(2 ml/g leaf), pH 6.0, containing 1% Driselase (an enzyme preparation
which macerates plant tissue), and 0.1% thioglycollic acid. Shake the
suspension for 2 h at 25 to 28°C and then emulsify by adding 0.5
vol. of a 1:1 (v/v) mixture of chloroform and n
-butanol, and centrifuge
at low speed. Precipitate the virus from the aqueous phase by adding
polyethylene glycol (M. Wt 6000) to 8% (w/v) and NaCl to 0.4 M.
Resuspend the virus in 0.01 M borate buffer, pH 8.0, concentrate by
two cycles of differential centrifugation using 0.0l M phosphate buffer,
pH 7.6 to 8.0, as the resuspending medium. Further purification is
achieved by sucrose density gradient centrifugation. Virus yields
are 5 to 10 mg per kg leaf material, at least ten times more than
those obtained by methods that do not include the use of Driselase.
Properties of Particles
Purified virus preparations contain a single sedimenting component. The virus particles are disrupted in water and in the presence of low concentrations of sodium dodecyl sulphate or high concentrations of caesium salts (chloride, sulphate and formate).
The particles precipitate reversibly when kept at 4°C in 0.01M phosphate buffers with pH values below 7.6
(Takanami & Kubo, 1979a
; Kubo & Takanami, 1980
Sedimentation coefficient (s20,w): 115 S.
Isoelectric point: c. pH 5.3 (determined by electrofocusing).
Absorbance at 260 nm (1 mg/ml, 1 cm light path): 7.0 (not corrected
A260/A280: 1.80 (not corrected for
Particles are isometric, c.
25 nm in diameter in negatively-stained
Particle CompositionNucleic acid:
Single-stranded RNA of M. Wt c.
2.0 x 106
, estimated by electrophoresis in polyacrylamide
gels in non-denaturing conditions. Two minor RNA components of M. Wt
about 1 x 105
are also detected (Takanami & Kubo,
Protein: The coat protein has a M. Wt of c. 25,700
daltons, estimated by electrophoresis in polyacrylamide/SDS gels
(Kubo & Takanami, 1980). Each subunit of the coat protein contains
c. 219 amino acid residues: ala, 12; arg, 21; asx, 20; cys, 3; glx,
16; gly, 23; his, 4; ile, 10; leu, 23; lys, 10; met, 2; phe, 9; pro,
9; ser, 24; thr, 12; trp, 2; tyr, 6; val, 13 (S. Kubo, unpublished
Relations with Cells and Tissues
The virus particles are confined to phloem tissue, namely sieve
tubes, phloem parenchyma and companion cells (Kubo, Kuwata &
; Imaizumi & Kubo, 1980a
). They are found
in nuclei, cytoplasm and vacuoles of infected cells (Fig.5
Cytoplasm of the infected cells shows a fibrous appearance.
Crystalline arrays of virus particles are not found. Despite its
restriction to the phloem of intact plants, the virus can multiply
in tobacco mesophyll protoplasts inoculated in vitro
& Takanami, 1979
Epidermal cells of tobacco leaves have been infected with the
virus by rubbing inoculation; the virus is thought not to spread from
the primarily inoculated cells (Imaizumi & Kubo, 1980b).
The virus may be distinguished from potato leafroll
vein-distorting (Smith, 1946
) and tobacco yellow-net viruses
(Abeygunawardena, Karandawela & Bandaranayake, 1967
) by the
reactions of tobacco, P. floridana
, and D. stramonium
- Abeygunawardena, Karandawela & Bandaranayake, Trop. Agriculturist 123: 37, 1967.
- Fukuda, Ann. Phytopath. Soc. Japan 44: 398, 1978.
- Imaizumi & Kubo, Ann. Phytopath. Soc. Japan 46: 54, 1980a.
- Imaizumi & Kubo, Ann. Phytopath. Soc. Japan 46: 422, 1980b.
- Kubo & Takanami, Ann. Phytopath. Soc. Japan 43: 76, 1977.
- Kubo & Takanami, Ann. Phytopath. Soc. Japan 44: 398, 1978.
- Kubo & Takanami, J. gen. Virol. 42: 387, 1979.
- Kubo & Takanami, Ann. Phytopath. Soc. Japan 46: 423, 1980.
- Kubo, Tamura & Fukuda, Hatabako Kenkyu 73: 49, 1976.
- Kubo, Kuwata & Imaizumi, Ann. Phytopath. Soc. Japan 44: 60, 1978.
- Kuwata & Kubo, Ann. Phytopath. Soc. Japan 44: 399, 1978.
- Roberts, Tamada & Harrison, J. gen. Virol. 47: 209, 1980.
- Smith, Parasitology 37: 131, 1946.
- Takanami & Kubo, J. gen. Virol. 44: 153, 1979a.
- Takanami & Kubo, J. gen. Virol. 44: 853, 1979b.
- Waterhouse & Murant, Ann. appl. Biol. 97: 191, 1981.
Disease outbreak caused by tobacco necrotic dwarf virus in
tobacco cv. MC 1.
Severe phloem necrosis in MC 1 tobacco.
Stunting, interveinal chlorosis and malformation of leaves
in Nicotiana sylvestris.
Physalis floridana: (left) healthy, (right)
A group of virus particles in a section of a degenerate cell
of Physalis floridana. Bar represents 200 nm.
Virus particles from a purified preparation stained with 2%
phosphotungstate. Bar represents 100 nm.