Citrus leaf rugose virus
S. M. Garnsey
U.S. Horticultural Research Laboratory, ARS, USDA, Orlando, Florida 32803, USA
Institute of Food and Agricultural Sciences, University of Florida, Agricultural Research and Education Center, Lake Alfred, Florida 33850, USA
Described by Garnsey (1975).
- Citrus crinkly-leaf-type virus (Rev. Pl. Path. 49: 330c)
An RNA-containing virus with isometric particles 25 to 32 nm in diameter, which
sediment as four nucleoprotein components. Readily sap-transmissible to many citrus
and herbaceous hosts. Vector unknown, reported only from Florida.
Causes leaf flecking in Eureka lemon (Citrus limon
rugosity in Mexican lime (C. aurantifolia
), and severe stunting of young Duncan
grapefruit (C. paradisi
Reported only from one location in Florida
Garnsey & Purcifull, 1969
Host Range and Symptomatology
Restricted in nature to citrus but can be
transmitted mechanically to a wide range of herbaceous plants
- C. aurantifolia (Mexican lime). Variable rugosity of leaves
- C. limon (Eureka lemon). Pinpoint chlorotic flecks on expanding leaves,
persistent without leaf distortion
- C. paradisi (Duncan grapefruit). Severe stunting and chlorosis
- Phaseolus vulgaris (bean) cv. Red Kidney. Small, necrotic local lesions
- Etrog citron (C. medica) and most other citrus varieties are suitable for
maintaining cultures. Tender new growth is a good source for purification. Gomphrena
globosa, Nicotiana tabacum and Petunia hybrida can also be used.
- Phaseolus vulgaris cv. Red Kidney, Vigna unguiculata (Early Ramshorn
cowpea), and Crotalaria spectabilis can be used as local lesion hosts. Other
cvs. of bean and cowpea are also local lesion hosts
Two strains, which differ in their effect on Duncan grapefruit,
have been described
A culture has been submitted to the American type
culture collection as PV195.
Transmission by Vectors
Vectors unknown, but field spread is suspected
The virus has been experimentally transmitted as a contaminant on
Transmission through Seed
None reported. Nucellar seedlings from apparently
naturally infected seedling trees of Orlando tangelo (C. paradisi
) were free of the virus
Transmission by Dodder
The virus is moderately immunogenic. Antisera with titres of 1/256
to 1/512 in gel diffusion tests have been obtained. Gels containing 0.75% agar and
0.04% sodium azide are preferable to gels with NaCl (8.5%) or phosphate buffer
(0.05 M) added. A single precipitin band which curves to the antigen well
forms in gel-diffusion tests with purified virus preparations containing all four
components. Extracts from infected plants or purified virus mixed with sap from
healthy plants contain a single, faster-moving antigen that forms a straight precipitin
line when allowed to react in gel-diffusion plates with antisera prepared against
the virus. No spur forms at the junction of precipitin lines formed to fast and slow
). The virus can easily be detected serologically in extracts from
young citrus leaves
(Garnsey & Purcifull, 1969
Two strains distinguished by reaction in Duncan grapefruit
are serologically indistinguishable. The virus is serologically
citrus variegation virus and
citrus crinkly leaf virus;
formation in gel diffusion plates and a 4-fold or greater difference in homologous
and heterologous titres clearly show the viruses are not identical
Recently, a similar serological relationship to
Tulare apple mosaic virus
has been found (R. M. Lister, D. Gonsalves & S. M. Garnsey, unpublished data).
Tests for serological relationships to
viruses were all negative
Gonsalves & Garnsey, 1975c
Properties, structure and composition of the particles place citrus
leaf rugose virus in the
Cross protection between the two described strains of citrus leaf rugose virus
occurs in Duncan grapefruit plants
The virus affords at least
partial protection in Eureka lemon and Etrog citron plants against challenge
inoculation by citrus variegation virus
Citrus variegation virus
also protects Red Kidney bean plants against challenge inoculation with citrus
leaf rugose virus
(D. Gonsalves & S. M. Garnsey, unpublished data).
Stability in Sap
The thermal inactivation point in citrus leaf extracts
prepared in 0.05 M phosphate buffer or of purified preparations is between 60 and
65°C. Extracts of young citrus leaves prepared in phosphate buffer retain some
infectivity for 48 h at room temperature and remain highly infective for 48 h at
Virus can be purified from young, infected citrus or tobacco
leaves by the following method
Grind leaves in pH 7.4 buffer (1
g/3 ml) containing 0.02 M potassium phosphate, 0.01 M sodium diethydithiocarbamate,
and 0.02 M sodium thioglycollate. Squeeze extract through cheesecloth. Mix filtrate
thoroughly with 0.7 ml hydrated calcium phosphate gel/g tissue, and clarify by low
speed centrifugation. Subject supernatant fluid to two cycles of differential
centrifugation and resuspend final high-speed pellets in pH 7.2 buffer containing
0.005 M potassium phosphate and 0.005 M MgCl2
. The virus is further purified by
rate-zonal density-gradient centrifugation in sucrose gradients. Virus yields from
citrus leaves are about 3-5 mg/100 g tissue. Yields from tobacco leaves are about
1-2 mg/100 g tissue.
Properties of Particles
As shown in
purified preparations contain
four nucleoprotein components
These are named according to
increasing sedimentation velocities as: NP 4, NP 3, NP 2, and NP 1, respectively
(Gonsalves & Garnsey, 1975b
Purified NP 3 and NP 4 are not infective
alone or in combination
(Gonsalves & Garnsey, 1974
Preparations of NP 1 +NP 2
are moderately infective and become highly infective with the addition of NP 3. The
relative amount of the different components is similar, regardless of the host in
which the virus was propagated.
Sedimentation coefficients (s20, w) (svedbergs): 79 (NP 4), 89
(NP 3), 98 (NP 2), and about 105 (NP 1). Calculations not made for infinite dilution.
Isoelectric point: between pH 4.4 and 5.0.
Absorbance at 260 nm (1 mg/ml, 1 cm light path): 5.3 for preparations containing
all four components.
Ratios of 1.40 to 1.46 were found for purified preparations containing
all four components. Similar ratios were found for NP 3 and for a mixture of NP 1 and
Particles are essentially isometric, with some heterogeneity
Particle diameter correlates positively with the sedimentation rate
of the components:
. 24.8 nm (NP 4), c
. 26.3 nm (NP 3), c
. 31.3 nm (NP 2), and
. 32.2 nm (NP 1)
(Gonsalves & Garnsey, 1976
Particles are partially
disrupted in phosphotungstic acid unless fixed in formaldehyde.
Particle CompositionNucleic acid:
RNA, single-stranded. The amount of RNA in the particles has
not been accurately determined, but based on
ratios is estimated to be
similar to that of
Tulare apple mosaic
tobacco streak viruses
(12-14%). The RNAs
in NPs 1, 2, 3 and 4 are designated as RNAs 1, 2, 3 and 4, respectively
(Gonsalves & Garnsey, 1974
Their M. Wts are: c.
1.1 x 106
0.7 x 106
, and c.
0.3 x 106
respectively. Sedimentation data indicate that two molecules of RNA 4 are probably
enclosed in one NP 4 particle. Very small amounts of a fifth RNA (RNA 4a) are usually
observed in gels. The RNAs are not infective singly. However, non-infective mixture
of RNAs 1+2+3 became highly infective with the addition of either RNA 4 or coat
(Gonsalves & Garney, 1975a
Coat protein and RNA-4 of
citrus variegation virus or
alfalfa mosaic virus
and coat protein from
tobacco streak virus
are also effective
(Gonsalves & Garnsey, 1975c
RNA 4 or coat
protein from citrus leaf rugose virus will activate non-infective mixtures of RNAs
1+2+3 from alfalfa mosaic virus, and from citrus variegation virus.
Protein: A single polypeptide species with M. Wt of c. 26,000
based on SDS-polyacrylamide gel-electrophoresis.
Relations with Cells and Tissues
The virus is present in most plant tissues.
Cellular associations, and occurrence of inclusions have not been investigated.
The virus has many properties similar to those of citrus variegation
Tulare apple mosaic
viruses. However, although these viruses are related, they
are easily distinguished by biological properties and serological differences. Citrus
variegation virus does not produce rugose symptoms in Mexican lime, and citrus leaf
rugose virus does not cause severe distortion and variegation patterns in Etrog
citron. Citus leaf rugose virus causes only local necrotic symptoms in the bean and
cowpea varieties tested, whereas citrus variegation virus commonly causes systemic
symptoms. Sedimentation profiles for the nucleoprotein components of the two viruses
are different, with citrus leaf rugose virus showing more NP 4 and poorer separation
of NP 1 and NP 2
(Gonsalves & Garnsey, 1975b
in the nucleic acid species of these two viruses also exist
(Gonsalves & Garnsey, 1975b
Similarly, citrus leaf rugose virus differs in biological and physical
properties from Tulare apple mosaic virus. The latter causes chlorotic local lesions
in cucumber (Cucumis sativus
), vein banding in Gomphrena globosa
necrotic local and shock symptoms in tobacco, none of which is caused by citrus leaf
rugose virus. Tulare apple mosaic virus is less stable, and the sedimentation profile
of its nucleoprotein components is different.
- Garnsey, Proc. Fla St. hort. Soc. 81: 79, 1968.
- Garnsey, Proc. 6th Conf. int. Organisation of Citrus Virologists 1974: 169, 1974.
- Garnsey, Phytopathology 65: 50, 1975.
- Garnsey & Purcifull, Proc. Fla St. hort. Soc. 81: 56, 1969.
- Gonsalves & Garnsey, Virology 61: 343, 1974.
- Gonsalves & Garnsey, Virology 64: 23, 1975a.
- Gonsalves & Garnsey, Virology 67: 311, 1975b.
- Gonsalves & Garnsey, Virology 67: 319, 1975c.
- Gonsalves & Garnsey, Proc. 7th Conf. int. Organisation of Citrus Virologists, 1976: 109, 1976.
Rugose symptoms in leaf of Mexican lime (Citrus aurantifolia).
Chlorotic flecks in leaf of Eureka lemon (C. limon).
Necrotic local lesions in primary leaf of Red Kidney bean (Phaseolus
(Top) Ultraviolet absorbance profile of virus nucleoprotein
centrifuged 14 h in 20-50% sucrose gradient at 113,000 g. (Bottom)
RNA preparation electrophoresed in 2.5% polyacrylamide gel. Arrows indicate direction
of sedimentation or migration.
Purified virus (natural mixture of all four components) stained with 1%
uranyl acetate. Bar represents 100 nm.
Reaction of antiserum to citrus leaf rugose virus (A) to different antigen
sources in gel-diffusion plate. Well 1
contains purified citrus leaf rugose virus; well 2, purified virus plus sap from
healthy Citrus excelsa; well 3, sap from infected C. excelsa; and well
4, sap from healthy C. excelsa.