SciELO - Scientific Electronic Library Online

vol.29 número2Changes in crossover distribution along a quadrivalent in a man carrier of a reciprocal translocation t(11;14)Reactive oxygen species in bovine embryo in vitro production índice de autoresíndice de materiabúsqueda de artículos
Home Pagelista alfabética de revistas  

Servicios Personalizados




  • No hay articulos citadosCitado por SciELO

Links relacionados



versión impresa ISSN 0327-9545

Biocell v.29 n.2 Mendoza abr./ago. 2005


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)

Address correspondence to: Dr. Luis R. Conci. IFFIVE-INTA, Camino 60 Cuadras km 5˚ (X5020ICA), Córdoba, ARGENTINA. FAX: (+54-351) 4974330. E-mail:

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 serological 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.

Keywords: Viola cornuta.Cucumber mosaic virus. Electron microscopy. Serology. AC-RT-PCR.


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 different ornamental crops grown in central Argentina.
During spring 2003, plants of Viola cornuta L. (Violaceae) showing flower color breaking, mild mosaic, leaf size reduction and general stunting were observed 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 molecular techniques.

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 immediately processed).
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 carbon- coated Formvar grids for seven min. After washing 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 subgroups 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 pnitrophenyl phospate as substrate; values higher than three times the mean for healthy controls were considered positive.
Once the pathogen was serologically identified, an antigen-capture (AC) (Rowhani et al., 1995; López Lambertini et al., 1998) followed by reverse transcription- polymerase chain reaction (RT-PCR) was performed 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 coating 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, Madison, 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 bromide.

Results and Discussion

Electron microscopic observations revealed the presence of isometric particles, about 30 nm in diameter, 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 antisera, the isolate under study was shown to belong to subgroup I. CMV infection was confirmed by AC-RTPCR. As expected, a DNA fragment of approximately 870 bp was amplified from the analysed samples (Fig. 2). MspI digestion of PCR products yielded two fragments 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.

. Electron micrograph of a leaf dip preparation stained in 2% uranyl acetate, showing CMV particles. Bar: 100 nm.

. 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).

. Subgrouping of the CMV isolate by restriction enzyme analysis. M: 100 bp DNA ladder (Promega). Lanes 1 and 2: MspI digestions of RT-PCR products from different V. cornuta symptomatic plants.

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 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 species during brief probes, as well as by mechanical transmission and by seed (Gallitelli, 2000). Therefore, the disease can spread rapidly in plant nurseries or under greenhouse conditions. The accurate identification of the pathogenic agent constitutes the first step towards its management.


1. Atencio FA, Gracia O, Zandomeni R, Grau O (1997). Detection of both subgroups I and II of Cucumber Mosaic Cucumovirus and their satellite RNAs on pepper in Argentina. Plant Disease 81: 695.         [ Links ]
2. Boari AJ, Maciel-Zambolim E, Carvalho MG, Zerbini FM (2000). Caracterização biológica e molecular de isolados do "Cucumber mosaic virus" provenientes de oito espécies vegetais. Fitopatologia Brasileira 25: 49-58.         [ Links ]
3. de Breuil S, Giolitti F, Nome C, Lenardon S (2002). Evidencias de Cucumber mosaic virus en cultivos de maní. XI Jornadas Fitosanitarias Argentinas. Río Cuarto, Córdoba. p. 73.         [ Links ]
4. Clark ME, Adams AN (1977). Characteristics of microplate methods of Enzyme Linked Immunosorbent Assay for detection of plant viruses. Journal of General Virology 34, 475-483.         [ Links ]
5. Crescenzi A, Barbarossa L, Gallitelli D, Martelli GP (1993). Cucumber mosaic cucumovirus populations in Italy under natural epidemic conditions and after a satellite- mediated protection test. Plant Disease 77: 28-33.         [ Links ]
6. Fernández Valiela MV (1995). Virus patógenos de las plantas y su control. Tomo I. Cuarta edición. Academia Nacional de Agronomía y Veterinaria, Buenos Aires. 701 pp.         [ Links ]
7. Gallitelli D (2000). The ecology of Cucumber mosaic virus and sustainable agriculture. Virus Research 71: 9-21.         [ Links ]
8. Hord MJ, García A, Villalobos H, Rivera C, Macaya G, Roossinck MJ (2001). Field survey of Cucumber mosaic virus subgroups I and II in crop plants in Costa Rica. Plant Disease 85: 952- 954.         [ Links ]
9. Informe INTA-JICA (2003). Estudio sobre la caracterización de la producción florícola en la República Argentina. Edited by INTEA, S. A., Buenos Aires. 293 pp.         [ Links ]
10. Laguna IG, Rodríguez Pardina P, Truol G, Ploper LD, Arneodo J, Galvez R, Herrera PS (2002). Evaluación de enfermedades virales de soja en Argentina bajo sistemas de labranza directa y convencional. XI Jornadas Fitosanitarias Argentinas. Río Cuarto, Córdoba. p. 63.         [ Links ]
11. López Lambertini P, Nome SF, Ducasse DA (1998). Detection of three Tospovirus RNA by multiplex genome amplification. III Encuentro Latinoamericano de Biotecnología Vegetal. 1 al 5 de junio, La Habana, Cuba.         [ Links ]
12. Rizos H, Gunn LV, Pares RD, Gillings MR (1992). Differentiation of Cucumber mosaic virus isolates using the polymerase chain reaction. Journal of General Virology 73: 2099-2103.         [ Links ]
13. Rodríguez Pardina P, Arneodo JD, Laguna IG (2002). Identificación y caracterización parcial del Cucumber mosaic virus presente en cultivos de poroto del NOA. XI Jornadas Fitosanitarias Argentinas. Río Cuarto, Córdoba. p. 79.         [ Links ]
14. Rowhani A, Maningas MA, Lile LS, Daubert SD, Golino DA (1995). Development of a detection system for viruses of woody plants based on PCR analysis of immobilized virions. Phytopathology 85: 347-352.         [ Links ]

Received on March 29, 2004.
Accepted on December 30, 2004.

Creative Commons License Todo el contenido de esta revista, excepto dónde está identificado, está bajo una Licencia Creative Commons