Revista Mexicana de Fitopatología ISSN: 0185-3309 [email protected] Sociedad Mexicana de Fitopatología, A.C. México Almeyda León, Isidro Humberto; Rocha Peña, Mario Alberto; Piña Razo, Jaime; Martínez Soriano, Juan Pablo The use of polymerase chain reaction and molecular hybridization for detection of phytoplasmas in different plant species in Mexico Revista Mexicana de Fitopatología, vol. 19, núm. 1, enero-junio, 2001, pp. 1- 9 Sociedad Mexicana de Fitopatología, A.C. Texcoco, México Available in: http://www.redalyc.org/articulo.oa?id=61219101 How to cite Complete issue Scientific Information System More information about this article Network of Scientific Journals from Latin America, the Caribbean, Spain and Portugal Journal's homepage in redalyc.org Non-profit academic project, developed under the open access initiative Revista Mexicana de FITOPATOLOGIA/ 1 The Use of Polymerase Chain Reaction and Molecular Hybridization for Detection of Phytoplasmas in Different Plant Species in Mexico Isidro Humberto Almeyda-León, Mario Alberto Rocha-Peña, INIFAP/Univ. Aut. De Nuevo León, Unidad de Investigación en Biología Celular y Molecular, Apdo. Postal 128- F, Cd. Universitaria, San Nicolás de los Garza, Nuevo León, México CP 66450; Jaime Piña-Razo, INIFAP-Centro de Investigación Regional del Sureste, Apdo. Postal 13 Suc. B, Mérida, Yucatán, México; and Juan Pablo Martínez-Soriano, CINVESTAV-Unidad Irapuato, Apdo. Postal 629, Irapuato, Guanajuato, México CP 36500. Correspondence to: [email protected] (Received: November 28, 2000 Accepted: March 8, 2001) Abstract. probes hybridized with DNA extracted from symptomless Almeyda-León, I.H., Rocha-Peña, M.A., Piña-Razo, J. and coconut palms from coconut lethal yellowing affected areas, Martínez-Soriano, J.P. 2001. The use of polymerase chain whereas, no hybridization was detected with DNA extracted reaction and molecular hybridization for detection of from healthy plants of all species tested. This is the first phytoplasmas in different plant species in Mexico. Revista report of molecular detection in Mexico of phytoplasmas Mexicana de Fitopatología 19:1-9. infecting cassava. The oligonucleotide primer pairs MMF/MMR, R16F2/R16R2, P1/Tint, P1/Bltvaint, P1/Ayint, P1/P7 and P3/P7 were used in Additional keywords: Primers, digoxigenin-DNA probes, polymerase chain reaction (PCR) for detection of coconut lethal yellowing, potato purple top, cassava, phytoplasmas in coconut palms (Cocos nucifera), marigold Caludovica palmata. (Tagetes erecta), potato (Solanum tuberosum), periwinkle (Catharanthus roseus), and cassava (Manihot esculenta). Resumen. Los pares de iniciadores MMF/MMR, R16F2/ There were differences in specificity in the amplification of R16R2, P1/Tint, P1/Bltvaint, P1/Ayint, P1/P7 and P3/P7, se DNA sequences depending of the primer pair set used and usaron en la reacción en cadena de la polimerasa (PCR), para plant species tested. Primer pair P1/Tint amplified single DNA la detección de fitoplasmas en cocotero (Cocos nucifera), fragments from all above plant species, including cempazúchitl (Tagetes erecta), papa (Solanum tuberosum), symptomless coconut palms collected from lethal yellowing teresita (Catharanthus roseus) y yuca (Manihot esculenta). (LY) affected areas. The primer set MMF/MMR yielded Se observaron diferencias de especificidad en las secuencias consistent results in the detection of phytoplasmas from de DNA´s amplificadas dependiendo del par de iniciadores coconut palms, marigold, and periwinkle phyllody. DNA utilizados y la especie de planta evaluada. El par de iniciadores fragments amplified with primer pairs R16F2/R16R2 and P1/ P1/Tint amplificó fragmentos de DNA de todas las especies Tint from the above plant species mentioned, were digested de plantas evaluadas, incluyendo plantas de cocotero with restriction endonucleases EcoR1 or AluI. Marigold and asintomáticas colectadas en áreas afectadas por el periwinkle showed an identical pattern and was similar in amarillamiento letal (AL). Los resultados fueron consistentes some extent to that of coconut palm. DNA fragments amplified con el par de iniciadores MMF/MMR en la detección de from coconut palm (LY269 bp) and periwinkle (PW1.2 bk), fitoplasmas en cocotero, cempazúchitl, y teresita con síntomas were cloned into vector pGEM-T and used to transform cells de filodia. Los fragmentos de DNA amplificados con los pares of Escherichia coli strain JM109; then, they were used as de iniciadores R16F2/R16R2 y P1/Tint, de todas las especies probes in DNA-DNA hybridization assays. These probes de plantas evaluadas fueron digeridos con las enzimas de are targeted to anneal to the operon of the ribosomal DNA of restricción EcoRI y AluI. El cempazúchitl y la teresita the 16S gen of phytoplasmas. Probe PW1.2 hybridized with mostraron un patrón idéntico y algo similar al de cocotero. DNA samples from all plant species tested, including jipi palm Los fragmentos de DNA amplificados de cocotero (LY269 (Caludovica palmata). Probe LY269 hybridized only with pb) y teresita filodia (PW1.2 kb), se clonaron en el vector DNA extracted from coconut and jipi palms, and periwinkle, pGEM-T y se utilizaron para transformar células de la cepa but not with DNA of marigold, potato and cassava. Both JM109 de Escherichia coli, y posteriormente se usaron como 2 / Volumen 19, Número 1, 2001 sondas para pruebas de hibridación DNA-DNA. Estas Alonso, 1986). Resistance to both is a part of continuous sondas son dirigidas para aparearse con el operon del DNA breeding improvement programs (Cadena-Hinojosa and ribosomal 16S de fitoplasmas. La sonda PW1.2 hibrida con el Galindo-Alonso, 1986; Cadena-Hinojosa, 1993; Piña-Razo, DNA de todas las plantas evaluadas, incluyendo la palma jipi 1995). The objective of this study was to optimize the PCR (Caludovica palmata). La sonda LY269 hibridiza solamente and molecular hybridization techniques, for the easy and con el DNA extraído de cocotero, palma jipi y teresita filodia, rapid detection of phytoplasmas in coconut palms and potato, pero no con el DNA extraído de cempazúchitl, papa y yuca. and some other plant species, in order to incorporate them Ambas sondas hibridan con DNA extraído de cocotero into cultivar improvement programs and epidemiological asintomático colectada en áreas afectadas por el AL, pero no studies. hibridizaron con el DNA extraído de plantas sanas de todas las especies evaluadas. Aquí se reporta por primera vez la MATERIALS AND METHODS detección molecular de fitoplasmas infectando a la yuca en Plant material. Tissue samples from several plant species México. were collected from different regions of the country. These included coconut palms (Cocos nucifera L.), both Palabras clave adicionales: Iniciadores, sondas DNA- symptomless and with symptoms of lethal yellowing disease digoxigenina, amarillamiento letal del cocotero, punta morada (LY), from the municipalities of Champotón, Escárcega and de la papa, yuca, Caludovica palmata. Carmen, in Campeche state; and Progreso in Yucatán state, where coconut lethal yellowing disease is endemic. Healthy Phytoplasmas (ICSBSTM, 1993) (formerly known as coconut palm samples were collected from Tampico in mycoplasmalike organisms), are wall-less prokaryotes that Tamaulipas state, a region in northeastern Mexico, still free affect many plant species throughout the world (McCoy et of LY. Other plant species samples with phytoplasma-like al., 1989). Historically, the detection of phytoplasmas has symptoms included: jipi palm (Caludovica palmata R.) with been hampered by the inability to culture these symptoms of lethal decline (Calkiní, Campeche); marigold microorganisms in vitro. Pathogen detection for disease (Tagetes erecta L.) with symptoms of phyllody (Texcoco, diagnosis has primarily relied on comparative studies of their Mexico); potato (Solanum tuberosum L.) with symptoms of biological properties (Chiykowski and Sinha, 1989). Electron purple top; periwinkle [Catharanthus roseus (L.) G. Don] with microscopy (EM), first revealed the presence of phytoplasmas symptoms of phyllody and virescence (Navidad and in plants and insects (Doi et al., 1969). However, this Monterrey, Nuevo León, respectively); and cassava (Manihot technique is time consuming, expensive, requires skilled esculenta Krantz) with symptoms of supergrowth (Cárdenas, personnel and is not suited for analysis of large numbers of Tabasco). In all cases, plant tissue from healthy looking samples. The presence of phytoplasmas in plants has also plants collected from distant fields was included as controls. been demonstrated by treating plant tissues with 4´- DNA extraction. DNA from plant tissue was extracted 6´diamidino-2-phenylindole (DAPI), a reagent that binds to following a protocol developed in our laboratory, by DNA and fluoresces under UV radiation (Russel et al., 1975). combining steps of the methods reported by Doyle and Doyle Both EM and DAPI have low sensitivity and can fail when (1990) and Rogers and Bendisch (1985, 1988). One gram of pathogen concentration in the tissues analyzed is low, and fresh tissue was ground in liquid nitrogen and suspended in neither are feasible for large scale studies. In the last 10 years, 2 ml of warm 2-ME/CTAB extraction solution [2% (w/v) several molecular techniques have been developed to study cetyldimethylethylammoniunbromide (CTAB), 100 mM Tris- phytoplasmas in different organisms. These techniques have HCl pH 8.0, 20 mM EDTA, pH 8.0, 1.4 m NaCl, 0.2% (v/v) 2- relied mostly on the use of the polymerase chain reaction mercaptoethanol,