The evolution of citrus tristeza virus (CTV) from outbreaks occurred in Calabria, Italy, was compared with that of CTV outbreaks reported previously in another two proximal Italian regions, Sicily and Apulia. Examination of four genomic regions (genes p20, p25 and p23, and one fragment of open reading frame 1) showed two recombination events, and phylogenetic analysis disclosed two divergent CTV groups in Calabria: one formed by severe and the other by mild isolates. This analysis, together with others involving population genetic parameters, revealed a low migration rate of CTV between the three Italian regions, as well as significant differences in selective pressures, epidemiology and demography, all affecting the genetic structure of CTV populations. This research was carried out in the frame of the national project "Armonizzazione della diagnosi e valutazione del rischio di patogeni da quarantena e nocivi ai vegetali e ai prodotti vegetali (ARNADIA-ARON)", supported by the Italian Ministry of Agriculture. A. Fontana has been the recipient of a postdoctoral fellowship from "Progetto cofinanziato POR Calabria FSE 2007/2013-Obiettivo Operativo M2", and D. E. Debreczeni the recipient of a FPU predoctoral fellowship from the Spanish Ministry of Education and Science. Fontana, A.; Debreczeni, DE.; Albanese, G.; Davino, S.; Flores Pedauye, R.; Rubio, L. (2014). Evolutionary analysis of Citrus tristeza virus outbreaks in Calabria, Italy: two rapidly spreading and independent introductions of mild and severe isolates. European Journal of Plant Pathology. 140(3):607-613. doi:10.1007/s10658-014-0489-3 Abou Kubaa, R., D’Onghia, A. M., Djelouah, K., Savino, V., & Saponari, M. (2012). Characterization of Citrus tristeza virus isolates recovered in Syria and Apulia (southern Italy) using different molecular tools. Phytopathologia Mediterranea, 51, 496–504. Acosta-Leal, R., Duffy, S., Xiong, Z., Hammond, R., & Elena, S. F. (2011). Advances in plant virus evolution: translating evolutionary insights into better disease management. Phytopathology, 101, 1136–1148. Albanese, G., Schimio, R., Fontana, A., Ferretti, L., Palmeri, V., Campolo, O., et al. (2010). Assessment of Citrus tristeza virus (CTV) incidence in Calabria, southern Italy: results of a three-year survey. Phytopathologia Mediterranea, 49, 27–34. Albiach-Martí, M. R., Mawassi, M., Gowda, S., Satyanarayana, T., Hilf, M. E., Shanker, S., et al. (2000). Sequences of Citrus tristeza virus separated in time and space are essentially identical. Journal of Virology, 74, 6856–6865. Bar-Joseph, M., Marcus, R., & Lee, R. F. (1989). The continuous challenge of Citrus tristeza virus control. Annual Review of Phytopathology, 27, 291–316. Davino, S., Davino, M., Sambade, A., Guardo, M., & Caruso, A. (2003). The first Citrus tristeza virus outbreak found in a relevant citrus producing area of Sicily, Italy. Plant Disease, 87, 314–314. Davino, S., Rubio, L., & Davino, M. (2005). Molecular analysis suggests that recent Citrus tristeza virus outbreaks in Italy were originated by at least two independent introductions. European Journal of Plant Pathology, 111, 289–293. Davino, S., Willemsen, A., Panno, S., Davino, M., Catara, A., Elena, S. F., et al. (2013). Emergence and phylodynamics of Citrus tristeza virus in Sicily, Italy. PLoS ONE, 8, e66700. Domingo, E., & Holland, J. (1997). RNA virus mutations and fitness for survival. Annual Review of Microbiology, 51, 151–178. Efron, B., Halloran, E., & Holmes, S. (1996). Bootstrap confidence levels for phylogenetic trees. Proceedings of the National Academy of Sciences of the United States of America, 93, 7085–7090. Febres, V., Ashoulin, L., Mawassi, M., Frank, A., Bar-Joseph, M., Manjunath, K., et al. (1996). The p27 protein is present at one end of Citrus tristeza virus particles. Phytopathology, 86, 1331–1335. Ferretti, L., Fontana, A., Sciarroni, R., Schimio, R., Loconsole, G., Albanese, G., et al. (2014). Molecular and biological evidence for a severe seedling yellows strain of Citrus tristeza virus spreading in Calabria (Southern Italy). Phytopathologia Mediterranea, 53, 3–13. Fu, Y. X., & Li, W. H. (1993). Maximum likelihood estimation of population parameters. Genetics, 134, 261–1270. Harper, S. J. (2013). Citrus tristeza virus: evolution of complex and varied genotypic groups. Frontiers in Microbiology, 4, 93. Hilf, M., & Garnsey, S. (2000). Characterization and classification of Citrus tristeza virus isolates by amplification of multiple molecular markers. Proceedings of the Fourteenth Conference of the International Organization of Citrus Virologists, 14, 18–27. Hudson, R. R. (2000). A new statistic for detecting genetic differentiation. Genetics, 155, 2011–2014. Hudson, R. R., Boos, D. D., & Kaplan, N. L. (1992). A statistical test for detecting geographic subdivision. Molecular Biology and Evolution, 9, 138–151. Karasev, A., Boyko, V., Gowda, S., Nikolaeva, O., Hilf, M., Koonin, E., et al. (1995). Complete sequence of the Citrus tristeza virus RNA genome. Virology, 208, 511–520. Kimura, M. (1985). The neutral theory of molecular evolution. Cambridge: Cambridge University Press. Kosakovsky Pond, S. L., Posada, D., Gravenor, M. B., Woelk, C. H., & Frost, S. D. (2006). GARD: a genetic algorithm for recombination detection. Bioinformatics, 22, 3096–3098. Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan, P. A., McWilliam, H., et al. (2007). Clustal W and Clustal X version 2.0. Bioinformatics, 23, 2947–2948. Librado, P., & Rozas, J. (2009). DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics, 25, 1451–1452. Lu, R., Folimonov, A., Shintaku, M., Li, W., Falk, B. W., Dawson, W. O., et al. (2004). Three distinct suppressors of RNA silencing encoded by a 20-kb viral RNA genome. Proceedings of the National Academy of Sciences of the United States of America, 101, 15742–15747. Martín, S., Sambade, A., Rubio, L., Vives, M. C., Moya, P., Guerri, J., et al. (2009). Contribution of recombination and selection to molecular evolution of Citrus tristeza virus. Journal of General Virology, 90, 1527–1538. Martín, D. P., Lemey, P., Lott, M., Moulton, V., Posada, D., & Lefeuvre, P. (2010). RDP3: a flexible and fast computer program for analyzing recombination. Bioinformatics, 26, 2462–2463. Moreno, P., Ambrós, S., Albiach-Martí, M. R., Guerri, J., & Peña, L. (2008). Citrus tristeza virus: a pathogen that changed the course of the citrus industry. Molecular Plant Pathology, 9, 251–268. Moya, A., Holmes, E. C., & González-Candelas, F. (2004). The population genetics and evolutionary epidemiology of RNA viruses. Nature Reviews Microbiology, 2, 279–288. Nagy, P. D. (2008). Recombination in plant RNA viruses. In M. J. Roossinck (Ed.), Plant virus evolution (pp. 133–164). Berlin: Springer. Nei, M., & Kumar, S. (2000). Molecular evolution and phylogenetics. USA: Oxford University Press. Niblett, C. L., Genc, H., Cevik, B., Halbert, S., Brown, L., Nolasco, G., et al. (2000). Progress on strain differentiation and its application to the epidemiology of Citrus Tristeza Virus. Virus Research, 71, 97–106. Pappu, H. R., Pappu, S. S., Manjunath, K. L., Lee, R. F., & Niblett, C. L. (1993). Molecular characterization of a structural epitope that is largely conserved among severe isolates of a plant virus. Proceedings of the National Academy of Sciences of the United States of America, 90, 3641–3644. Rubio, L., Ayllón, M. A., Kong, P., Fernández, A., Polek, M. L., Guerri, J., et al. (2001). Genetic variation of Citrus tristeza virus isolates from California and Spain: evidence for mixed infections and recombination. Journal of Virology, 75, 8054–8062. Rubio, L., Guerri, J., & Moreno, P. (2013). Genetic variability and evolutionary dynamics of viruses of the family Closteroviridae. Frontiers in Microbiology, 4, 151. Ruiz-Ruiz, S., Soler, N., Sánchez-Navarro, J., Fagoaga, C., López, C., Navarro, L., et al. (2013). Citrus tristeza virus p23: determinants for nucleolar localization and their influence on suppression of RNA silencing and pathogenesis. Molecular Plant-Microbe Interactions, 26, 306–318. Sambade, A., López, C., Rubio, L., Flores, R., Guerri, J., & Moreno, P. (2003). Polymorphism of a specific region in gene p23 of Citrus tristeza virus allows discrimination between mild and severe isolates. Archives of Virology, 148, 2325–2340. Silva, G., Marques, N., & Nolasco, G. (2012). The evolutionary rate of Citrus tristeza virus ranks among the rates of the slowest RNA viruses. Journal of General Virology, 93, 419–429. Tajima, F. (1989). Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics, 123, 585–595. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., & Kumar, S. (2011). MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution, 28, 2731–2739. Vives, M. C., Rubio, L., López, C., Navas-Castillo, J., Albiach-Martí, M. R., Dawson, W. O., et al. (1999). The complete genome sequence of the major component of a mild Citrus tristeza virus isolate. Journal of General Virology, 80, 811–816. Vives, M. C., Rubio, L., Sambade, A., Mirkov, T. E., Moreno, P., & Guerri, J. (2005). Evidence of multiple recombination events between two RNA sequence variants within a Citrus tristeza virus isolate. Virology, 331, 232–237. Weir, B. S., & Cockerham, C. C. (1984). Estimating F-statistics for the analysis of population structure. Evolution, 38, 1358–1370.

Evolutionary analysis of Citrus tristeza virus outbreaks in Calabria, Italy: two rapidly spreading and independent introductions of mild and severe isolates

Abstract