Journal of Plant Pathology

, Volume 101, Issue 1, pp 97–105 | Cite as

Biological and molecular characterization of Uruguayan citrus tristeza virus field isolates

  • Leticia RubioEmail author
  • Ana Bertalmío
  • Lester Hernández-Rodríguez
  • María José Benítez Galeano
  • Ana Arruabarrena
  • Fernando Rivas
  • Rodney Colina
  • Diego Maeso
Original Article


Citrus tristeza virus (CTV) is the causal agent of the most important viral disease of citrus. Symptoms that may affect the productive potential of citrus plants are observed in Uruguayan orchards even though resistant rootstocks are used. CTV is fully eliminated in propagative materials by the National Sanitation and Certification Program, but since the virus and its vector are widespread in the country, the risk of infection in the field persists. In this situation, using mild CTV strains in a cross-protection program would be a useful alternative to attempt to increase yield and quality of the local citrus industry. To this aim, this study assessed the biological and molecular characteristics of 32 local CTV isolates. Bioassays were conducted in a greenhouse with controlled conditions. Each isolate was graft-inoculated on Mexican lime, sweet orange, sour orange and Duncan grapefruit indicator plants. Symptoms and their intensity were evaluated. Molecular characterization was carried out by RT-PCR amplification, using primers for the p25, p20 and p23 genes. PCR products were sequenced, nucleotide sequences were aligned with international reference strains and phylogenetic trees were constructed. Results of the biological and molecular analysis showed the prevalence of severe CTV isolates with a high genetic variability. Two out of 32 characterized isolates were selected as mild CTV isolates to be tested as candidates for future cross-protection experiments. The survey showed a complex scenario for the management of CTV in Uruguay.


Closterovirus Closteroviridae CTV Biological indexing Phylogenetic analysis 



This research was funded by Instituto Nacional de Investigación Agropecuaria, Uruguay (project CT-06), through the National Program of Citrus, in collaboration with the Laboratorio de Virología Molecular, Centro Universitario Regional Litoral Norte de la Universidad de la República.


This study was funded by Instituto Nacional de Investigación Agropecuaria, Uruguay (project CT-06).

Compliance with ethical standards

Conflict of interest

The authors declare no conflicts of interest.

Ethical approval

The authors declare no animals were used in this research.


  1. Akaike H (1974) A new look at the statistical model identification. IEEE Trans Autom Control 19:716–723CrossRefGoogle Scholar
  2. Albiach-Marti M, Mawassi M, Gowda S, Satyanarayana T, Hilf M, Shanker S, Almira E, Vives M, Lopez C, Guerri J, Flores R, Moreno P, Garnsey S, Dawson W (2000) Sequences of Citrus tristeza virus separated in time and space are essentially identical. J Virol 74:6856–6865CrossRefGoogle Scholar
  3. Anisimova M, Gascuel O (2006) Approximate likelihood-ratio test for branches: a fast, accurate, and powerful alternative. Syst Biol 55:539–552CrossRefGoogle Scholar
  4. Bar-Joseph M, Lee R (1989) The continuous challenge of Citrus tristeza virus control. Annu Rev Phytopathol 27:291–316CrossRefGoogle Scholar
  5. Benítez-Galeano M, Rubio L, Bertalmío A, Maeso D, Rivas F, Colina R (2015) Phylogenetic studies of the three RNA silencing suppressor genes of south American CTV isolates reveal the circulation of a novel genetic lineage. Viruses 7:4152–4168CrossRefGoogle Scholar
  6. Benítez-Galeano MJ, Castells M, Colina R (2017) The evolutionary history and spatiotemporal dynamics of the NC lineage of Citrus tristeza virus. Viruses 9:272. CrossRefGoogle Scholar
  7. Benítez-Galeano M, Vallet T, Carrau L, Hernández-Rodriguez L, Bertalmío A, Rivas F, Rubio L, Maeso D, Vignuzzi M, Moratorio G, Colina R (2018) Complete genome sequence of a novel recombinant Citrus tristeza virus: a resistance-breaking isolate from Uruguay. Genome Announc 6:e00442–e00418. CrossRefGoogle Scholar
  8. Bentancour C, Scatoni I, Morelli E (2009) Insectos del Uruguay. Universidad de la República, Facultad de Agronomía, Facultad de Ciencias, 658pGoogle Scholar
  9. Biswas K, Tarafdar A, Sharma S (2012) Complete genome sequence of mandarine decline Citrus tristeza virus of the northeastern Himalaya hill region if India: comparative analyses determine recombinant. Arch Virol 157:579–583CrossRefGoogle Scholar
  10. Broadbent P, Bevington KB, Coote BG (1991) Control of stem pitting of grapefruit in Australia by mild strain cross protection. In: Brlansky R, Lee R, Timmer L (eds) Proceedings of 11th conference of the International Organization of Citrus Virologists. IOCV, Riverside CA, pp 64–70Google Scholar
  11. Broadbent P, Brlansky R, Indsto J (1996) Biological characterization of Australian isolates of Citrus tristeza virus and separation of subisolates by single aphid transmissions. Plant Dis 80:329–333CrossRefGoogle Scholar
  12. Costa A, Nunes W, Corazza M, Zanutto C, Müller G (2010) Biological and molecular characterization of isolates of Citrus tristeza virus with potential for use in preimmunization programs. Summa Phytopathol 36:81–82CrossRefGoogle Scholar
  13. Da Graça J, van Vuuren S (2010) Managing Citrus tristeza virus losses using cross protection. In: Citrus tristeza virus complex and Tristeza diseases. APS Press, St. Paul MN, pp 247–260Google Scholar
  14. Dawson T, Money P (2000) Evidence for trifoliate resistence breaking isolates of Citrus tristeza virus in New Zealand. In: da Graca J, Lee R, Yokomi R (eds) Proceedings of the 14th conference of the International Organization of Citrus Virologists. IOCV, Riverside, CA, pp 69–76Google Scholar
  15. Dawson WO, Bar-Joseph M, Garnsey SM, Moreno P (2015) Citrus tristeza virus: making an ally from an enemy. Annu Rev Phytopathol 53:37–55CrossRefGoogle Scholar
  16. de Koch Brotos L, Boasso C (1955) Lista de las enfermedades de los vegetales en el Uruguay. Ministerio de Ganadería y Agricultura. Dirección de Agronomía. Laboratorio de Fisiología y Patología Vegetal. Publicación N° 106, 65pGoogle Scholar
  17. Folimonova S (2013) Developing an understanding of cross-protection by Citrus tristeza virus. Front Microbiol 4(75):1–9Google Scholar
  18. Francis M, Peyrou M, Perea B, Borde J, Fosali Y (1997) Caracterización de aislamientos del virus de la Tristeza de los cítricos (CTV) que causan pérdidas en pomelo injertado sobre Trifolia en Uruguay. In: IX Congreso Latinoamericano de Fitopatología.10/97. Montevideo, UruguayGoogle Scholar
  19. Garnsey S, Gumpf D, Roistacher C, Civerolo E, Lee R, Yokomi R, Bar-Joseph M (1987) Toward standarized evaluation of the biological properties of Citrus tristeza virus. Phytophylactica 19:151–157Google Scholar
  20. Garnsey M, Civerolo E, Gumpf D, Paul C, Hilf M, Lee R, Brlansky R, Yokomi R, Hartung J (2005) Biological characterization of an international collection of Citrus tristeza virus (CTV) isolates. In: Hilf M, Duran-Vila N, Rocha-Peña M (eds) Proceedings of the 16th conference of International Organization of Citrus Virologists. IOCV, Riverside, CA, pp 75–93Google Scholar
  21. Gonsalves D, Garnsey S (1989) Cross-protection techniques for control of plant virus diseases in the tropics. Plant Dis 73:592–597CrossRefGoogle Scholar
  22. Gottwald T, Garnsey S, Cambra M, Moreno P, Irey M, Borbón J (1996) Differential effects of Toxoptera citricida vs. Aphis gossypii on temporal increase and spatial patterns of spread of citrus tristeza. In: Moreno P, da Graca J, Timmer L (eds) Proceedings of the 13th Conference of International Organization of Citrus Virologists. IOCV, Riverside, pp 120–129Google Scholar
  23. Guindon S, Dufayard J, Lefort V, Anisimova M, Hordijk W, Gascuel O (2010) New algorithms and methods to estimate maximum-likelihood phylogenies: 3.0. Syst Biol 59:307–321CrossRefGoogle Scholar
  24. Harper S (2013) Citrus tristeza virus: evolution of complex and varied genotypic groups. Front Microbiol 4(93):1–18Google Scholar
  25. Harper S, Dawson T, Pearson M (2010) Isolates of Citrus tristeza virus that overcome Poncirus trifoliata resistance comprise a novel strain. Arch Virol 155:471–480CrossRefGoogle Scholar
  26. Hernández-Rodríguez L, Bertalmío A, Arruabarrena A, Rubio L, Rivas F, Benítez-Galeano MJ, Colina R, Maeso D (2017) First report of the Citrus tristeza virus trifoliate resistance-breaking (RB) genotype in 'Newhall' sweet Orange in South America. Plant Dis 101:1063CrossRefGoogle Scholar
  27. Hilf M, Karasev A, Albiach-Marti M, Dawson W, Garnsey S (1999) Two paths of sequence divergence in the Citrus tristeza virus complex. Phytopathology 89:336–342CrossRefGoogle Scholar
  28. Iglesias NG, Gago-Zachert SP, Robledo G, Costa N, Plata MI, Vera O, Grau O, Semorile LC (2008) Population structure of Citrus tristeza virus from field Argentinean isolates. Virus Genes 36:199–207CrossRefGoogle Scholar
  29. Koizumi M (1991) Citrus tristeza virus field isolates from declined of dwarfed citrus trees in Japan. In: Brlansky R, Lee R, Timmer L (eds) Proceedings of the 11th conference of the International Organization of Citrus Virologists. IOCV, Riverside, pp 25–30Google Scholar
  30. Moreno P, Ambros S, Albiach-Marti M, Guerri J, Peña L (2008) Plant diseases that changed the world Citrus tristeza virus: a pathogen that changed the course of the citrus industry. Mol Plant Pathol 9:251–268CrossRefGoogle Scholar
  31. Müller IA and Campiglia HG (1981) Nivel de "stem pitting" (SP) en naranja 'Valencia' (Citrus sinensis (L) Obs.) y tangor 'Ellendale' (C. reticulata blanco x C. sinensis (L) Obs.) injertados en diferentes portainjertos. In Müller IA, ed. Investigaciones agronómicas Año 2 (1), pp. 79–82. UruguayGoogle Scholar
  32. Müller G, Costa A, Castro J, Guirado N (1988) Results from preimmunization tests to control the Capao Bonito strain of tristeza. In: Timmer L, Garnsey S, Navarro L (eds) Proceeding of the 10th conference of the International Organization of Citrus Virologists. IOCV, Riverside, CA, pp 82–85Google Scholar
  33. Posada D (2008) jModelTest: phylogenetic model averaging. Mol Biol Evol 25:1253–1256CrossRefGoogle Scholar
  34. Rocha-Peña M, Lee R, Lastra R, Niblett C, Ochoa-Corona F, Garnsey S, Yokomi R (1995) Citrus tristeza virus and its aphid vector Toxoptera citricida: threats to citrus production in the Caribbean and central and North America. Plant Dis 79:437–443CrossRefGoogle Scholar
  35. Roistacher C (1988) Observations on the decline of sweet orange trees in coastal Peru caused by stem-pitting tristeza. Plant protection bulletin India 36:19–26Google Scholar
  36. Roistacher C, Bar-Joseph M (1987a) Transmission of Citrus tristeza virus (CTV) by Aphis gossypii and by graft inoculation to and from Passiflora spp. Phytophylactica 19:179–182Google Scholar
  37. Roistacher C, Bar-Joseph M (1987b) Aphid transmission of tristeza virus: a review. Phytophylactica 19:163–167Google Scholar
  38. Roistacher C, da Graça J, Müller G (2010) Cross protection against Citrus Tristeza Virus a review. In: Brlansky R, Lee R, Timmer L (eds) Proceedings of the 17th conference of the International Organization of Citrus Virologists. IOCV, Riverside, CA, pp 1–27Google Scholar
  39. Rubio L, Ayllon M, Kong P, Fernandez A, Polek ML, Guerri J, Moreno P, Falk B (2001) Genetic variation of Citrus tristeza virus isolates from California and Spain: evidence for mixed infections and recombination. J Virol 75:8054–8062CrossRefGoogle Scholar
  40. Sambade A, Lopez 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. Arch Virol 148:2325–2340CrossRefGoogle Scholar
  41. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729CrossRefGoogle Scholar
  42. Weng Z, Barthelson R, Gowda S, Hilf ME, Dawson WO, Galbraith DW et al (2007) Persistent infection and promiscuous recombination of multiple genotypes of an RNA virus within a single host generate extensive diversity. PLoS One 2(9):e917CrossRefGoogle Scholar
  43. Yokomi R, Selvaraj V, Maheshwari Y, Saponari M, Giampetruzzi A, Chiumenti M, Hajeri S (2017) Identification and characterization of Citrus tristeza virus isolates breaking resistance in trifoliate orange in California. Phytopathology 107:901–908CrossRefGoogle Scholar

Copyright information

© Società Italiana di Patologia Vegetale (S.I.Pa.V.) 2018

Authors and Affiliations

  1. 1.Instituto Nacional de Investigación Agropecuaria (INIA), Estación Experimental INIA Salto GrandeSaltoUruguay
  2. 2.Laboratorio de Virología Molecular, Centro Universitario Regional Litoral Norte (CENUR- Litoral Norte)Universidad de la RepúblicaSaltoUruguay

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