Aislamiento de fitoplasmas asociados a cuero de sapo en yuca

Contenido principal del artículo

Autores

Carlos betancourt https://orcid.org/0000-0001-6573-4230
Juan Manuel Pardo https://orcid.org/0000-0003-0496-3181
Jaime Muñoz https://orcid.org/0000-0002-8237-0499
Elizabeth Alvarez Cabrera

Resumen

En Colombia, el ‘‘cuero de sapo’’ es la enfermedad más limitante del cultivo de yuca, que ocasiona pérdidas en producción de raíces hasta del 90%. La presente investigación tuvo como objetivo, el aislamiento in vitro del fitoplasma asociado a cuero de sapo. Para ello, se emplearon medios de cultivo líquido y sólido, usando tejidos de raíces, peciolos, tallos, hojas y semillas de yuca, afectada por la enfermedad. Pruebas de PCR, qPCR, secuenciación, microscopia de luz y microscopia electrónica de transmisión fueron aplicadas, para verificar el crecimiento de fitoplasmas y descartar la presencia de otros microrganismos. Los resultados muestran que los medios permiten, consistentemente, el crecimiento de fitoplasmas, obteniendo colonias en medio sólido a partir de medio líquido. Las pruebas de PCR, qPCR y secuenciación confirmaron presencia de Cassava frogskin phytoplasma del grupo 16SrIII, en los dos medios de cultivo. Además, a partir de las colonias, se lograron fotografías de células con morfología y tamaño similares a las fitoplasmáticas. Es la primera vez, en el mundo, que se consolida información suficiente del aislamiento de fitoplasmas en medio artificial. Adicionalmente, se logró el aislamiento de Pigeon pea witches´ broom phytoplasma del grupo IX, a partir de tallos, peciolos y flores de vinca (Catharanthus roseus), con síntomas asociados a fitoplasmas. Este proceso permitió corroborar la efectividad del medio y la morfología de las células fitoplasmáticas, bajo microscopia electrónica.

Palabras clave:

Detalles del artículo

Licencia

Creative Commons License
Esta obra está bajo licencia internacional Creative Commons Reconocimiento-NoComercial 4.0.

 

Referencias

1. ALTSCHUL, S.; GISH, W.; MILLER, W.; MYERS, E.; LIPMAN, D. 1990. Basic local alignment search tool. J. Molecular Biology. 215:403-410. https://doi.org/10.1016/S0022-2836(05)80360-2

2. ÁLVAREZ, E.; MEJÍA, J.F.; LOKE, J.B.; HERNÁNDEZ, L.; LLANO, G.A. 2003. Detecting the phytoplasma-frogskin disease association in cassava (Manihot esculenta Crantz) in Colombia. Phytopathology. 93:S4. https://doi.org/10.1094/PDIS-93-11-1139

3. ÁLVAREZ, E.; MEJÍA, J.; LLANO, G.; LOKE, J.; CALARI, A.; DUDUK, B.; BERTACCINI, A. 2009. Characterization of a phytoplasma associated with frogskin disease in cassava. Plant Disease. 93:1139-1145. https://doi.or/10.1094/PDIS-93-11-1139

4. BERTACCINI A.; MARANI, F. 1980. Mycoplasma-like organisms in Gladiolus sp. plants with malformed and virescent flowers. Phytopath. Medit. 19:121-128.

5. BERTACCINI, A.; LEE, I.-M. 2018. Phytoplasmas: an update. In: Phytoplasmas: Plant Pathogenic Bacteria-I. En: Rao, G.P.; Bertaccini, A.; Fiore, N.; Liefting, L. (eds). Characterization and Epidemiology of Phytoplasma-Associated Diseases. Chapter 1. Springer, Singapure. p.1-29.

6. CALVERT, L.; CUERVO, M.: LOZANO, I.; VILLAREAL, N.; ARROYAVE, J. 2008. Identification of three strains of a virus associated with cassava plants affected by frogskin disease. J. Phytopathology. 156(11‐12):647-653. https.//doi.org/10.1111/j.1439-0434.2008.01412.x

7. CARVAJAL-YEPES, M.; OLAYA, C.; LOZANO, I.; CUERVO, M.; CASTAÑO, M.; CUELLAR, W. 2014. Unraveling complex viral infections in cassava (Manihot esculenta Crantz) from Colombia. Virus Research. 186:76-86. https://doi.org/10.1016/j.virusres.2013.12.011

8. CHAPMAN, G.; BUERKLE, E.; BARROWS, E.; DAVIS, R.E.; DALLY, E. 2001. A light and transmission electron microscope study of a black locust tree, Robinia pseudoacacia (Fabaceae), affected by witches´ broom and classification of the associated phytoplasma. J. Phytopathology. 149:589-597. https://doi.org/1046/j.1439-0434.2001.00673.x

9. CONTALDO, N.; BERTACCINI, A.; PALTRINIERI, S.; WINDSOR, H.; WINDSOR, G. 2012. Axenic culture of plant pathogenic phytoplasmas. Phytopathologia Mediterranea. 51(3):607-617. https://doi.org/1014601/Phytopathol_Mediterr-11773
10. CONTALDO, N.; SATTA, E.; ZAMBON, Y.; PALTRINIERI, S.; BERTACCINI, A. 2016. Development and evaluation of different complex media for phytoplasma isolation and growth. J. Microbiological Methods. 127:105-110. https://doi.org/10.1016/j.mimet.2016.05.031

11. DE SOUZA, A.; DA SILVA, F.; BEDENDO, I.; CARVALHO, C. 2014. A phytoplasma belonging to a 16SrIII-A subgroup and dsRNA virus associated with cassava frogskin disease in Brazil. Plant Disease. 98(6):771-779. https://doi.org/10.1094/PDIS-04-13-0440-RE

12. DENG, S.; HIRUKI, C. 1991. Amplification of 16S rRNA genes from culturable and nonculturable Mollicutes. J. Microbiology Methods. 14:53-61. http://dx.doi.org/10.1016/0167-7012(91)90007-D

13. DOI, Y.; TERANAKA, M.; YORA, K.; ASUYAMA, H. 1967. Mycoplasma or PLT group like microorganisms found in the phloem elements of plants infected with mulberry dwarf, potato witches’ broom, aster yellows or paulownia witches’ broom. Annal Phytopathology Society Japan. 33:259-266. https://doi.org/10.3186/jjphytopath.33.259

14. FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS - FAO. 2013. Save and Grow: Cassava. A Guide to Sustainable Production Intensification. Rome. Disponible desde Internet en: http://www.fao.org/docrep/018/i3278e/i3278e.pdf (con acceso 11/06/2016).

15. FUDL-ALLAH, A.; CALAVAN, E.; IGWEGBE, E. 1972. Culture of a mycoplasma-like organism associated with stubborn disease of citrus. Phytopathology. 62:729-731. https://doi.org/10.1094/Phyto-62-729

16. FUDL-ALLAH, A.; CALAVAN, E. 1973. Effect of temperature and pH on growth in vitro of a mycoplasma-like organism associated with stubborn disease of citrus. Phytopathology. 63:256-259. https://doi.org/10.1094/Phyto-63-256

17. GASPARICH, G.E.; WHITCOMB, R.F.; DODGE, D.; FRENCH, F.E.; GLASS, J.; WILLIAMSON, D.L. 2004. The genus Spiroplasma and its nonhelical descendants: phylogenetic classification, correlation with phenotype and roots of the Mycoplasma mycoides clade. Int. J. Syst. Evol. Microbiol. 54:893-918. https://doi.org/101099/ijs.002688-0

18. GHOSH, S.; RAYCHAUDHURI, S.; CHENULU, V.; VARAM, A. 1975. Isolation, cultivation and characterization of mycoplasma-like organisms from plants. Proc. Indian National Science Academy. 41(4):362-366.

19. GIBB, K.; PADOVAN, A.; MOGEN, B. 1995. Studies on sweet potato little-leaf phytoplasmas detected in sweet potato and other plant species growing in Northern Australia. Phytopathology. 85:169-174. https://doi.org/10.1094/Phyto-85-169

20. GIOVANNONI, S. 1991. The polymerase chain reaction. In: Stackebrandt, E.; Goodfellow, M. (eds). Nucleic acid techniques in bacterial systematics. John Wiley and Sons. New York, p.17. https://doi.org/10.1002/jobm.3620310616

21. GUNDERSEN, D.E.; LEE, I.-M.; SCHAFF, D.A.; HARRISON, N.A.; CHANG, C.J.; DAVIS, R.E.; KINGSBURY, D.T. 1996. Genomic diversity and differentiation among phytoplasma strains in 16S rRNA groups I (aster yellows) and III (X-disease). Internal J. Systematic Bacteriology. 46(1):64-75. https://doi.org/10.1099/00207713-46-1-64

22. HAMPTON, R.; STEVENS, J.; ALLEN, T. 1969. Mechanically transmissible mycoplasma from naturally infected peas. Plant Disease Report. 53:449-503.

23. JAGOUEIX, S.; BOVÉ, J.; GARNIER, M. 1994. The phloem-limited bacterium of greening disease of citrus is a member of the alpha subdivision of the proteobacteria. Internal J. Systematic Bacteriology. 44:397-386. https://doi.org/10.1099/00207713-44-3-379

24. JAGOUEIX, S.; BOVÉ, J.; GARNIER, M. 1996. PCR detection of the two ‘Candidatus’ liberobacter species associated with greening disease of citrus. Molecular and Cellular Probes. 10:43-50. https://doi.org/10.1006/mcpr.1996.0006

25. JENSEN, J.; HANSEN, H.; LIND, K. 1996. Isolation of Mycoplasma genitalium strains fron the male urethra. J. Clinical Microbiology. 34:286-291.

26. KUBE, M.; MITROVIC, J.; DUDUK, B.; RABUS, R.; SEEMÜLLER, E. 2012. Current view on phytoplasma genomes and encoded metabolism. The Scientific World Journal. 2012:185942. https://doi.org/10.1100/2012/185942

27. LABRUNA, M.; WHITWORTH, T.; HORTA, M.; BOUYER, D.; MCBRIDE, J.; PINTER, A.; POPOV, V.; GENNARI, S.; WALKER, D. 2004. Rickettsia species infecting Amblyomma cooperi ticks from an area in State of São Paulo, Brazil, where Brazilian spotted fever is endemic. J Clin Microbiol. 42:90-98. https://doi.org/10.1128/JCM.42.1.90-98.2004

28. LEE, I.-M.; DAVIS R.E. 1986. Prospects for in vitro culture of plant pathogenic mycoplasma like organisms. Annual Reviews of Phytopathology. 24:339-354. https://doi.org/10.1146/annurev.pv.24.090186.002011

29. LEE, I.-M.; HAMMOND, R.H.; DAVIS, R.E.; GUNDERSEN, D.E. 1993. Universal amplification and analysis of pathogen 16S rDNA for classification and identification of mycoplasma-like organisms. Phytopathology. 83:834-842. https://doi.org/10.1094/Phyto-83-834

30. LEE, I.-M.; GUNDERSEN, D.E.; HAMMOND, R.H.; DAVIS, R.E. 1994. Use of mycoplasma like organism (MLO) group specific oligonucleotide primers for nested PCR assays to detect mixed MLO infections in a single host plant. Phytopathology. 84:559-566. https://doi.org/10.1094/Phyto-84-559

31. LEE, I.-M.; DAVIS, R.E.; GUNDERSEN, D.H. 2000. Phytoplasmas: phytopathogenic Mollicutes. Annual Review of Microbiology. 54:221-255. https://doi.org/10.1146/annurev.Micro.54.1.221

32. MARCONE, C. 2013. Movement of phytoplasmas and the development of disease in the plant. In: Weintraub, P.; Jones, P (eds). Phytoplasmas: Genomes, Plant Hosts and Vectors. CAB International. p.114-131. https://doi.org/10.1079/9781845935306.0114

33. MCCOY, R.E. 1979. Mycoplasmas and yellows diseases. In: Whitcomb, R.F.; Tully, J.G. (eds). The mycoplasmas. Academic Press. New York. 3:229-264.

34. MESEGUER, M.; BOGA, B.; ANDREU, L.; GRAU, G. 2012. Diagnóstico microbiológico de las infecciones por Mycoplasma spp. y Ureaplasma spp. Enfermedades Infecciosas y Microbiología Clínica. 30(8):500-504. https://doi.org/10.1016/j.eimc.2011.10.020

35. MUSETTI, R.; LOI, N.; CARRARO, L.; ERMACORA, P. 2002. Application of immunoelectron microscopy techniques in the diagnosis of phytoplasma diseases. Microscopy Res. Technique. 56:462-464. https://doi.org/10.1002/jemt.10061

36. OLIVEIRA, S.A.S.; ABREU, E.F.M.; ARAÚJO, T.S.; OLIVEIRA, E.J.; ANDRADE, E.C.; GARCIA, J.M.P.; ÁLVAREZ, E. 2014. First Report of a 16SrIII-L phytoplasma associated with frogskin disease in Cassava (Manihot esculenta Crantz) in Brazil. Plant Disease. 98(1):153-154. https://doi-org/10.1094/PDIS-05-13-0499-PDN

37. OSHIMA, K.; KAKIZAWA, S.; NISHIGAWA, H.; JUNG, H.; WEI, W.; SUSUKI, S.; ARASHIDA, R.; NAKATA, D.; MIYATA, S.; UGAKI, M.; NAMBA, S. 2004. Reductive evolution suggested from the complete genome sequence of a plant pathogenic phytoplasma. Nature Genetics. 36:2729. https://doi.org/10.1038/ng1277

38. PINEDA, B.; LOZANO, J.C. 1981. Investigaciones sobre la enfermedad del "cuero de sapo" en yuca (Manihot esculenta Crantz). Cali, CIAT. 16p.

39. PITCHER, D.; WINDSOR, D.; WINDSOR, H.; BRABURY, J.; YAVARI, C.; JENSEN, J.; LING, C.; WEBSTER, D. 2005. Mycoplasma amphoriforme sp. nov., isolated from a patient with chronic bronchopneumonia. Internal J. Systematic and Evolutionary Microbiology. 55:2589-2594. https://doi.org/10.1099/ijs.0.63269-0

40. POGHOSYAN, A.; LEBSKY, V. 2004. Aislamiento y estudio ultraestructural de tres cepas de fitoplasmas causantes de enfermedades tipo “stolbur” en Solanaceae. Fitopatología Colombiana. 28(1):21-30.

41. PRIBYLOVA, J.; SPAK, J.; FRANOVÁ, J.; PETRZIK, K. 2001. Association of aster yellows subgroup 16SrI-B phytoplasmas with a disease of Rehmannia glutinosa var purpurea. Plant Pathology. 50:776-781. https://doi.org/10.1046/j.1365-3059.2001.00638x

42. SAMBROOK, J.; FRITSCH, E.; MANIATIS, T. 1989. Molecular Cloning. A Laboratory Manual. 2nd Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. 215p.

43. SIDDIQUE, A.; AGRAWAL, G.; ALAM, N.; REDDY, M. 2001. Electron microscopy and molecular characterization of phytoplasmas associated with little leaf disease of brinjal (Solanum melongena L.) and periwinkle (Catharanthus roseus L.) in Bangladesh. J. Phytopathology. 149:237-244. https://doi.org/10.1046/j.1439-0434.2001.00590.x

44. SMART, C.; SCHNEIDER, B.; BLOMQUIST, C.; GUERRA, L.; HARRISON, N.; AHRENS, U.; LORENZ, K.; SEEMULLER, E.; KIRKPATRICK, B. 1996. Phytoplasma-specific PCR primers based on sequences of the 16S-23S rRNA spacer region. Appl. Environm. Microbiol. 62:2988-2993.

45. SKRIPAL, I.G.; MALINOVSKAIA, L.P. 1984. Medium SMIMB-72 para aislamiento y cultivo de micoplasmas fitopatógenos. Microbiologicheskii Zhurnal. 46(2):71-75.

46. SUZUKI, M.; GIOVANNONI, S. 1996. Bias caused by template annealing in the amplification of mixtures of 16S rRNA genes by PCR. Appl. Environ. Microb. 62:625-630.

47. TULLY, J.G. 1993. Current status of the mollicute flora of humans. Clinical Infectious Disease. 17(Suppl.1):S2-S9.

48. TULLY, J.G. 1995. Culture medium formulation for primary isolation and maintenance of mollicutes. In: Razin, S.; Tully, J.(Eds). Molecular and Diagnostic Procedures in Mycoplasmology. Academic Press. San Diego. p.119-131. https://doi.org/10.1016/B978-012583805-4/50005-4

49. WATERS, H.; HUNT, P. 1980. The in vivo three dimensional form of a plant mycoplasma like organisms by the analysis of serial ultrathin sections. J. General Microbiology. 116:111-131. https://doi.org/10.1099/00221287-116-1-111

50. ZIV, A.; FUENTES, C. 2007. Improved purification and PCR amplification of DNA from elemental samples. FEMS Microbiology Letters. 272(2):269-275. https://doi.org/10.1111/j.1574-6968.2007.00764.x

Descargas

La descarga de datos todavía no está disponible.