Natural infection of Viola cornuta (Violaceae) with Cucumber
mosaic virus, subgroup I
JOEL ARNEODO*, SOLEDAD DE BREUIL*, SERGIO LENARDON** AND LUIS CONCI**
Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)
Instituto de Fitopatología y Fisiología Vegetal (IFFIVE) – Instituto Nacional de Tecnología Agropecuaria (INTA)
Keywords: Viola cornuta, Cucumber mosaic virus, electron microscopy, serology, AC-RT-PCR.
ABSTRACT: Plants of Viola cornuta displaying typical virus symptoms were observed during spring 2003
in a plant nursery in Córdoba, central Argentina. Electron microscopic examinations of symptomatic leaf
samples revealed the presence of isometric virus-like particles about 30 nm in diameter. Subsequent serologi-
cal analysis allowed the identification of the pathogen as a subgroup I strain of Cucumber mosaic virus
(CMV). These results were confirmed by antigen capture - reverse transcription - polymerase chain reaction
with specific CMV primers, and digestion with a restriction enzyme. This is the first report of CMV infecting
V. cornuta in Argentina.
2005, 29(2): 205-207
ISSN 0327 - 9545
PRINTED IN ARGENTINA
Commercial floriculture has the potential to become
an important industry in Argentina. Córdoba province,
with an area of ca. 650,000 m2 dedicated to this activity,
occupies the seventh position in the national ranking
(Informe INTA-JICA, 2003). Production can be affected
by several factors, including plant pathogens such as
fungi, viruses and, at a lesser extent, bacteria. There is,
however, a lack of information concerning the occurrence
and distribution of these infectious agents on the differ-
ent ornamental crops grown in central Argentina.
During spring 2003, plants of Viola cornuta L.
(Violaceae) showing flower color breaking, mild mo-
saic, leaf size reduction and general stunting were ob-
served in a plant nursery located near La Granja
(Córdoba province). The described symptomatology
suggested the presence of a plant virus, and studies were
conducted to elucidate the aetiology of the disease. This
work reports on the identification of the disease causal
agent, by means of electron microscope, serological and
Materials and Methods
Samples were collected from V. cornuta plants
showing the symptoms described above. Plant material
was stored at –20°C, except for electron microscope
observations (in this case, fresh leaf tissue was imme-
Address correspondence to: Dr. Luis R. Conci. IFFIVE-INTA,
Camino 60 Cuadras km 5˚ (X5020ICA), Córdoba, ARGENTINA.
FAX: (+54-351) 4974330. E-mail: email@example.com
Received on March 29, 2004. Accepted on December 30, 2004.
JOEL ARNEODO et al. 206
Leaf dip preparations were made from portions of
symptomatic leaves, cut several times with a razor blade
in a drop of PBS pH 7 + 0.01% (w/v) sodium sulphite
(Na2SO3). The leaf extracts were then transferred to car-
bon-coated Formvar grids for seven min. After wash-
ing with distillated water, these were negatively stained
with 2% uranyl acetate and examined with a JEOL 1220
transmission electron microscope.
On the basis of the results obtained by microscopic
analysis, symptomatic plants were tested for Cucumber
mosaic virus (CMV) using DAS-ELISA (Clark and
Adams, 1977). Leaf samples were ground in extraction
buffer (PBS pH 7 + 0.05% Tween 20 + 2% polyvinyl
pyrrolidone) at a 1:10 (w/v) dilution. Polyclonal and
monoclonal antisera (anti CMV, and anti-CMV sub-
groups I and II) (Agdia Inc., Elkhart, USA) were used.
Positive (supplied by Agdia) and negative (healthy V.
cornuta) controls were placed on each microtitre plate.
Absorbance at 405 nm was recorded after adding p-
nitrophenyl phospate as substrate; values higher than
three times the mean for healthy controls were consid-
Once the pathogen was serologically identified, an
antigen-capture (AC) (Rowhani et al., 1995; López
Lambertini et al., 1998) followed by reverse transcrip-
tion-polymerase chain reaction (RT-PCR) was per-
formed to confirm the presence of CMV in the analysed
plants. For AC, PCR tubes were coated with 50 µl of a
1:10 (w/v) dilution of tissue extract in carbonated coat-
ing buffer, and incubated overnight at 4°C. RT-PCR was
performed with two CMV-specific primers that flank
the CMV capsid protein gene (Rizos et al., 1992). The
following cycling parameters were used: 48°C for 45
min, 94°C for 4 min, (40 cycles of 94°C for 30 s, 40°C
for 30 s, 68°C for 1 min), and a final cycle of 68°C for
7 min. The amplified fragment was then digested with
restriction enzyme MspI (Promega Corporation, Madi-
son, USA) for subgroup identification (Rizos et al.,
1992). Digestions were carried out at 37ºC for 4 h. Both,
PCR and digestions products were run in agarose gels
(1.4% and 3% respectively) and stained with ethidium
Results and Discussion
Electron microscopic observations revealed the
presence of isometric particles, about 30 nm in diam-
eter, in leaves of symptomatic V . cornuta plants (Fig. 1).
The virus was identified as CMV by DAS-ELISA (data
not shown). When testing with subgroup-specific anti-
sera, the isolate under study was shown to belong to
subgroup I. CMV infection was confirmed by AC-RT-
PCR. As expected, a DNA fragment of approximately
870 bp was amplified from the analysed samples (Fig.
2). MspI digestion of PCR products yielded two frag-
FIGURE 1. Electron micrograph of a leaf dip
preparation stained in 2% uranyl acetate,
showing CMV particles. Bar: 100 nm.
FIGURE 2. RT-PCR amplification of CMV RNA.
M: 1 kb DNA ladder (Promega). Lane 1: positive
control (CMV-infected Trifolium repens). Lanes
2 and 3: different V. cornuta symptomatic plants.
Lane 4: negative control (healthy V. cornuta).
Cucumber mosaic virus on Viola cornuta
ments of approximately 340 and 540 bp (Fig. 3). This
pattern allowed the classification of the isolate within
subgroup I, in accordance with the serological results.
CMV , an ubiquitous plant virus, infects a wide range
of plant species around the world, among which many
ornamentals, and new hosts for this virus are described
greenhouse conditions. The accurate identification of
the pathogenic agent constitutes the first step towards
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FIGURE 3. Subgrouping of the CMV isolate by
restriction enzyme analysis. M: 100 bp DNA lad-
der (Promega). Lanes 1 and 2: MspI digestions
of RT-PCR products from different V. cornuta
every year. Subgroup I strains seem to be more frequent
than those of subgroup II (Crescenzi et al., 1993) and,
in general, induce a more severe disease (Boari et al.,
2000; Hord et al., 2001). In Argentina, the virus has
already been detected in various crops, including Apium
sp., Lactuca sativa, Calendula officinalis and several
Cucurbitaceae (compiled by Fernández Valiela, 1995).
More recently, it has been recorded infecting Capsicum
annuum (Atencio et al., 1997), Glycine max (Laguna et
al., 2002), Phaseolus vulgaris (Rodríguez Pardina et
al., 2002) and Arachis hypogaea (de Breuil et al., 2002).
This paper reports, for the first time, the occurrence of
CMV on V. cornuta.
CMV has a very broad range of natural reservoirs.
It can be easily transmitted by more than 80 aphid spe-
cies during brief probes, as well as by mechanical trans-
mission and by seed (Gallitelli, 2000). Therefore, the
disease can spread rapidly in plant nurseries or under
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