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Alfalfa Dwarf Disease, a Viral Complex Affecting Alfalfa Crop in Argentina *E

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Alfalfa Dwarf Disease, a Viral Complex Affecting Alfalfa Crop in Argentina *E Alfalfa dwarf disease, a viral complex affecting alfalfa crop in Argentina16 Trucco1, V.M.*, Bejerman1,2, N., de Breuil1,2, S., Cabrera Mederos1,2, D., Lenardon1,3, S., Giolitti1, F*. 1Instituto de Patología Vegetal - Centro de Investigaciones Agropecuarias - Instituto Nacional de Tecnología Agropecuaria (IPAVE-CIAP-INTA). 2Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina. 3 Cátedra de Fitopatología, Facultad de Agronomía y Veterinaria, Universidad Nacional de Río Cuarto, Córdoba, Argentina. *e-mail: [email protected], [email protected] KEYWORDS: Alfalfa dwarf disease, Medicago sativa, Viral complex, Field distribution, Aphis craccivora. Alfalfa (Medicago sativa L.) is considered the most important forage crop in Argentina and an essential component of bovine meat and milk production. It is well adapted to a large region in Argentina due to its high salinity and drought resistance as well as its efficient atmospheric nitrogen-fixing capacity (Basigalup et al., 2007). Alfalfa is the forage crop with the largest cultivated area in the country, over 3.2 million hectares, and it ranks fourth after soybean, wheat and maize (Basigalup pers.comm., 2018; «Bolsa de Cereales», 2018; «Bolsa de Comercio de Rosario», 2017). In 2010, a new viral disease, named alfalfa dwarf disease-ADD, was identified in Argentina. The symptoms include severe plant stunting and leaves with decreased size, distortion, chlorosis and the presence of vein enations and papillae on the abaxial surface (Figure 1). Initial field assays indicated that ADD presented incidences higher than 50%, yield decreases of dry mass of up to 30% and significantly reduced crop life (INTA-Informa, 2010). Plants infected with ADD were found to be co-infected with Alfalfa mosaic virus (AMV) and Alfalfa dwarf cytorhabdovirus (ADV). Subsequently, other three viruses were detected in the same plants: Bean leafroll virus (BLRV), Alfalfa enamovirus-1 (AEV-1) and Alfalfa leaf curl virus (ALCV). So far, these five viruses (collectively named as Argentine Alfalfa Viruses = AAV) were detected in alfalfa plants, with ADV and AEV-1 being virus species described for the first time worldwide. Methods for AAV detection were adjusted and the complete genome sequences of each virus were reported (Bejerman et al., 2011, 2015, 2016, 2017; Trucco et al., 2014, 2016). Taking into account that little is known about viral diseases affecting alfalfa and considering the importance of this forage crop for cattle production in Argentina, our objective was to generate data to elucidate the situation of the viral disease complex in this crop and the relative importance of each component of the disease complex; meeting this objective will be an important step toward adopting methods for their proper management. During 2010-2017, the geographical distribution and prevalence of ADD and AAV were evaluated in alfalfa- producing areas of Argentina, with 17 provinces being surveyed: Buenos Aires, Catamarca, Chaco, Córdoba, Entre Ríos, Jujuy, La Pampa, La Rioja, Mendoza, Neuquén, Río Negro, Salta, San Juan, San Luis, Santa Fe, Santiago del Estero and Tucumán. ADD was detected in all surveyed provinces, with a prevalence of 87.0% being recorded (147 alfalfa fields with at least one ADD-plant/169 total surveyed alfalfa fields). Notably, the 22 fields where ADD was undetected correspond to young alfalfa crops (less than one year old) (Figure 2). Except for BLRV in Chaco, all five viruses were detected in at least one of the ADD sampled fields from each surveyed province, with prevalence values ranging from 64 to 100%. Interestingly, AMV was the only one of the five viruses detected in all ADD-infected alfalfa samples, with a prevalence of 100% being recorded. Alfalfa is also cultivated in Argentina with the purpose of seed production, which is carried out in irrigated arid regions like San Juan province (Basigalup et al., 2007). A preliminary assay was conducted in Guanacache-San Juan; alfalfa plants showing symptoms of enations (characteristic symptom of ADD) were selected and the effect of AAV on seed production and symptom expression was analyzed. A decrease of up to 38% in seed production was observed in plants showing enations, with AMV being the virus with most negative impact, followed by ALCV. This result was confirmed via a principal component analysis, which showed that both *CITATION: Trucco, V.M., Bejerman, N., de Breuil, S., Cabrera Mederos, D., Lenardon, S., Giolitti, F. 2018. Alfalfa dwarf disease, a viral complex affecting alfalfa crop in Argentina. IN Proceedings. Second World Alfalfa Congress, Cordoba, Argentina. 11-14 November, 2018. Instituto Nacional de Tecnología Agropecuaria (INTA), http://www.worldalfalfacongress.org/ Instituto Nacional de Tecnología Agropecuaria 84 viruses were positively correlated with symptom expression and negatively correlated with seed production; no important correlation was observed with the remaining viruses. On the other hand, this result is supported by the finding that plants naturally co-infected with AMV and ALCV showed symptoms that resemble those of alfalfa dwarf disease, suggesting that their synergism could cause the development of ADD symptoms. Nevertheless, additional assays need to be conducted to confirm the contribution of this co-infection to symptom development (e.g. simple and mixed infection with AMV and ALCV on the same alfalfa genetic base and evaluation both under greenhouse and field conditions). The viruses can spread to an agricultural crop mainly due to the use of infected seeds, the behavior of vectors and the presence of alternative hosts. Seed transmission of AAV and ADD was evaluated. Seed transmission was recorded only for AMV (3.75%); thus, no seed transmission of ADD was observed. Vector transmission assays were carried out with aphids (Aphis craccivora) collected from ADD-infected alfalfa plants in the field. Symptoms of chlorosis and vein enations were recorded in alfalfa plants tested with five and 10 aphids/healthy plant; transmission of ADD by A. craccivora was confirmed at a rate of 2 and 10%, respectively. Moreover, all five viruses were detected in aphids collected in alfalfa fields, indicating that aphids are natural carriers of AAV. It is known that AMV, ALCV and BLRV can be transmitted by Aphis craccivora; however, the potential of this aphid as vector of ADV and AEV-1 needs to be confirmed. Moreover, the other aphid species that colonize alfalfa crop in Argentina will be evaluated as potential vectors. Several weed species (Trifolium repens, T. pratense, Dichondra repens, Melilotus spp., Sonchus oleraceus and Talinum paniculatum) growing adjacent to ADD-infected alfalfa plants and showing virus-like symptoms were collected and evaluated as possible natural reservoirs of AAV. Except for T. paniculatum, AMV was detected in all weed species, and both ADV and AEV-1 were detected in T. repens. All the other weeds tested negative for the remaining viruses. This is the first record of AMV-infected T. pratense in Argentina, and of Dichondra repens as natural host of AMV, and T. repens of both ADV and AEV-1 worldwide. Additional field sampling including more weed species will be carried out. The knowledge gained in this work will be useful to design management strategies for this disease and to generate tools that help minimize the negative effect of ADD on alfalfa crops. Figure 1. Symptomatology of ADD. Stunting and chlorosis in affected plants (A and B), decreased size and leaflet deformations (C-G), chlorosis on margins of the leaflet (G), and vein enation and papillae on the abaxial surface of leaves (C-F). Instituto Nacional de Tecnología Agropecuaria 85 Figure 2. Map of Argentina showing the sampling sites of ADD during 2010-2017. Black and white circles indicate the positive and negative detection of ADD, respectively. ACKNOWLEDGMENTS This work was partially supported by PNPV-1135022 (INTA). Instituto Nacional de Tecnología Agropecuaria 86 BIBLIOGRAPHY Basigalup, D. H., Rossanigo, R., & Ballario, M. V. (2007). Panorama actual de la alfalfa en la Argentina. In D. H. Basigalup (Ed.), El cultivo de la alfalfa en la Argentina (pp. 13-25). Eds. INTA. Bejerman, N., Giolitti, F., De Breuil, S., Trucco, V., Nome, C., Lenardon, S., & Dietzgen, R. G. (2015). Complete genome sequence and integrated protein localization and interaction map for alfalfa dwarf virus, which combines properties of both cytoplasmic and nuclear plant rhabdoviruses. Virology, 483, 275-283. doi:10.1016/j.virol.2015.05.001 Bejerman, N., Giolitti, F., Trucco, V., de Breuil, S., Dietzgen, R. G., & Lenardon, S. (2016). Complete genome sequence of a new enamovirus from Argentina infecting alfalfa plants showing dwarfism symptoms. Archives of Virology, 161(7), 2029-2032. doi:10.1007/s00705-016-2854-3 Bejerman, N., Nome, C., Giolitti, F., Kitajima, E., de Breuil, S., Pérez Fernández, J., et al. (2011). First Report of a Rhabdovirus Infecting Alfalfa in Argentina. Plant Disease, 95(6), 771-771. doi:10.1094/PDIS-10-10-0764 Bejerman, N., Trucco, V., de Breuil, S., Pardina, P. R., Lenardon, S., & Giolitti, F. (2017). Genome characterization of an Argentinean isolate of alfalfa leaf curl virus. Archives of Virology, 1-5. doi:10.1007/s00705-017-3673-x Bolsa de Cereales. (2018). http://www.bolsadecereales.com/. Accessed August 15, 2018. Bolsa de Comercio de Rosario. (2017). Estimaciones de producción. https://www.bcr.com.ar/Pages/gea/estimaProd.aspx. Accessed July 19, 2017. INTA-Informa. (2010). Descubren una nueva enfermedad en alfalfa. http://intainforma.inta.gov.ar/?p=2693. Accessed October 29, 2015. Trucco, V., de Breuil, S., Bejerman, N., Lenardon, S., & Giolitti, F. (2014). Complete nucleotide sequence of Alfalfa mosaic virus isolated from alfalfa (Medicago sativa L.) in Argentina. Virus Genes, 48(3), 562-565. doi:10.1007/s11262-014-1045-0 Trucco, V., de Breuil, S., Bejerman, N., Lenardon, S., & Giolitti, F. (2016). Bean leafroll virus (BLRV) in Argentina: molecular characterization and detection in alfalfa fields. European Journal of Plant Pathology, 146(1). doi:10.1007/s10658-016-0899-5 Instituto Nacional de Tecnología Agropecuaria 87 .
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