sign in sign up
<<
Home , Phytoplasma

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 in different plant 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 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. ( erecta), potato (Solanum tuberosum), periwinkle (), 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 . 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 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 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 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 -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. 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 (EM), first revealed the presence of phytoplasmas symptoms of phyllody and virescence (Navidad and in plants and (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, 1% (v/v) polyvinylpyrrolydone 40,000]. The (PCR) (Ahrens and Seemüller, 1992; Firrao et al., 1993; Harrison slurry was transferred to tubes, gently mixed and then et al., 1994a, 1994b; 1996) as well as molecular hybridization incubated for 30 min at 65°C with occasional mixing. The with labeled probes (Harrison et al., 1992; 1994a). Both resulting lysate was extracted with an equal volume of techniques have facilitated diverse studies to detect and chloroform-isoamyl alcohol (24:1) and centrifuged for 5 min identify phytoplasmas in plants (Harrison et al., 1992, 1994a, at 8,000 rpm at 4°C. The upper aqueous layer was transferred 1994b), in vectors (Rahardja et al., 1992), in ecological to a fresh tube and re-extracted with a 1/10 vol. warm at 65°C studies (Kirkpatrick et al., 1990), and in (Kuske CTAB/NaCl solution [10% (w/v) CTAB, 0.7 M NaCl], and an and Kirkpatrick, 1992). Some cloned probes, presumably equal volume of chloroform-isoamyl alcohol. The tubes were representing highly conserved chromosomal DNA once again centrifuged for 5 min at 8,000 rpm at 4°C. The sequences, detect a wide range of different phytoplasmas, aqueous phase was transferred to a fresh tube and 1 vol. whereas others appear to detect closely related phytoplasmas CTAB precipitation solution [1% (w/v) CTAB, 50 mM Tris- only (Harrison et al., 1993). In Mexico, there are several plant HCl pH 8.0, 10 mM EDTA pH 8.0] was added, and centrifuged diseases caused by phytoplasmas, two of which are of major for 5 min at 2,000 rpm at 4°C. The supernatant was removed importance: coconut lethal yellowing and potato purple top and the nucleic acid pellet was resuspended in 1 ml of high (Almeyda-León et al., 1998; Cadena-Hinojosa and Galindo- salt TE buffer (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA pH 8.0, Revista Mexicana de FITOPATOLOGIA/ 3

1 M NaCl). Nucleic acids were precipitated by adding a 0.6 Maryland, U.S.A., or Perkin Elmer-Cetus Co., Norwalk, vol. of isopropanol, and pelletd by centrifugation for 15 min Connecticut, U.S.A.). To prevent evaporation, the reaction at 8,000 rpm at 4°C. The pellet was washed with 80% ethanol, was covered with a layer of 50 µl of mineral oil. The reactions dried and resuspended in 200 µl of TE. For washing the DNA, were performed in a DNA Thermal Cycler (Perkin Elmer) using 0.5 vol. of 7.5 M ammonium acetate were added and mixed the conditions reported by Harrison et al. (1994b) and Lee et thoroughly. Samples were kept on ice for 10 min, centrifuged al. (1993b). These PCR conditions were repeatedly evaluated for 10 min at 10,000 rpm and the aqueous supernatant was and finally standardized as follows: denaturation at 94°C for transferred to a fresh tube. A 0.6 vol. of isopropanol was 2 min, followed by 30 cycles at 94°C for 1 min, 55°C for 1 min, added, mixed and stored two hours at –20°C. DNA was 72°C for 1 min, and one additional extension run at 72°C for 10 recovered by centrifuging the samples for 10 min at 10,000 min. For samples extracted from symptomless coconut palms rpm. Pellets were rinsed twice with 1 ml of 70% ethanol, dried collected from lethal yellowing affected areas (LYAA), the and resuspended in 50 µl of TE buffer (10mM Tris-HCl pH 8.0 run was extended to 35 cycles with an polymerization time of and 1 mM Na2EDTA). DNA concentrations were determined 1 min 30 sec. Eight µl of the amplification reaction were taken spectophotometrically at 260 nm and samples then stored at and fractionated in a 1.5% agarose (or 6% polyacrylamide) 4°C until processed. gel containing 1X TBE (100 mM Tris-HCl, 90 mM boric acid, Oligonucleotide design. Two 20-mer oligonucleotides MMF/ 1mM Na2EDTA), stained with 0.5 mg/ml ethidium bromide MMR (Fig. 1), were designed in our lab (Martínez-Soriano et solution and visualized in a UV transilluminator (Sambrook et al., 1994) and targeted to the 197-219 and 454-472 positions al., 1989). on the 16S rRNA as reported in the Genebank 86.0 and EMBL RFLP analyses of PCR products. DNA from the five plant 41.0 database, (accession number AF222063) National Center species with symptoms suggesting phytoplasma, was for Biotechnology Information of the National Library of amplified by using the primer pair P1/Tint and R16F2/R16R2 Medicine (Bethesda, Maryland. USA). The DNA sequence and further purified with the GeneClean System (BIO 101 Inc.). of forward primer (MMF) was 5´- Between 5-8 µl of each PCR product (approx. 300-500 ng of AAGACCTAGCAATAGGTATG-3´, while that of the reverse DNA) was separately digested by the restriction enzymes primer (MMR) was 5´-TCAGCAATGATTTTTCC ATC-3´. EcoRI and AluI according to the manufacturer’s instructions. Some other sets of primers previously reported, such as R16F2/ The restriction products were then electrophoresed through R16R2 (Lee et al., 1993b); P1, P3, P7, Bltvaint, AYint, and Tint 6% polyacrylamide gels, stained with 0.5 mg/ml ethidium (Smart et al., 1996), and some combinations of them were also bromide and visualized by UV transillumination. included in the study. All sets of oligonucleotides were Cloning of DNA fragments and molecular hybridization. synthesized by Bio-Synthesis, Inc. (Lewisville, Texas, U.S.A.). DNA fragments of 269 bp and of 1.2 kb were amplified with PCR conditions. PCR was performed in a volume of 50 µl MMF/MMR and R16F2/R16R2, respectively, from DNA of using 50 ng of DNA, 25 pmoles of each primer, 1X PCR buffer coconut palms with LY, and from periwinkle with virescence (200 mM Tris-HCl pH 8.4, 500 mM KCl), 2mM MgCl2, 200 µM symptoms. Each fragment was cloned into vector pGEM-T in of each of the four deoxynucleotide triphosphates and 2.5 Escherichia coli JM109 cells according to suppliers units of Taq polymerase (GIBCO BRL, Inc, Gaithersburg, instructions (Promega, 1996). Transformant colonies were

Spacer Region

Gene 16S rRNA Gene 23S rRNA

P1 P3 P7 MMF/MMR 269 bp MMF MMR R16F2/MMR 330 bp P3/P7 350 bp R16F2 R16R2 R16F2/R16R2 1.2 kb lle tRNA P1/Bltvaint 1.45 kb P1/AYint 1.5 kb P1/Tint 1.6 kb P1/P7 1.8 kb Bltvaint AYint Tint

Fig. 1. Diagrammatic representation of a phytoplasma rRNA operon including the 16S, the 23S rRNA genes and the intergenic spacer region (SR). The positions of oligonucleotide primers used in PCR analysis are represented as arrows. The expected size of phytoplasma-specific PCR products generated from the SR, 16S and 23S rRNA primer pairs are indicated. The portion in gray represents tRNA Ile (Modified from Smart et al., 1996).

8 / Volumen 19, Número 1, 2001 differences in signal intensities were evident among DNA or P.L.T. group-like organism found in the elements samples (Fig. 7A and B). These differences can be attributed of plants infected with mulberry dwarf, potato witches to lower titers of pathogen DNA or the degree of sequence broom, aster yellows or paulownia witches broom. Annals similarity between probes and the DNA tested, or both as of the Phytopathological Society of Japan 33:259-266. suggested by Harrison et al. (1994a). The probe LY269 showed Doyle, J.J., Doyle, J.L. 1990. Isolation of plant DNA from fresh a high specificity with the samples from coconut and jipi tissue. Focus 12:13-15. palm and a weak reaction with DNA from periwinkle phyllody Firrao, G., Gobbi, E., Locci, R. 1993. Use of polymerase chain (Fig. 7A). The absence of hybridization between the probe reaction to produce oligonucleotide probes for LY269 and DNA from phytoplasmas from infected marigold, mycoplasmalike organisms. Phytopathology 83:602-607. cassava and potato, can be interpreted as a low degree of Harrison, N.A., Bourne, C.M., Cox, R.L., Tsai, J.H., Richardson, sequence similarity between the probe and the DNA tested. P.A. 1992. DNA probes for detection of mycoplasmalike The different DNA patterns obtained with the RFLP analyses organisms associated with lethal yellowing disease of (Figs. 6A and B), supports the above conclusion. The probe palms in Florida. Phytopathology 82:216-224. PW1.2 showed a positive reaction with all the samples tested Harrison, N.A., Richardson, P.A., Tsai, J.H. 1993. Detection (Fig. 7B), indicating that it has a lower annealing specificity and diagnosis of lethal yellowing: Conventional methods and can be used as a more universal probe with phytoplasmas and molecular techniques. Simposium Internacional sobre from diverse plant species. No hybridization was detected el Amarillamiento Letal del Cocotero. Mérida, Yucatán. between both probes and DNA from healthy plants, indicating CICY. 16 pp. the reliability of the test for the diagnostic purposes for Harrison, N.A., Richardson, P.A., Jones, P., Tymon, A.M., diseases caused by phytoplasmas. According to the overall Eden, S.J., Mpunami, A.A. 1994a. Comparative results, PCR proved to be more sensitive than molecular investigation of MLOs associated with Caribbean and hybridization for the detection when lower titers of pathogen African coconut lethal decline diseases by DNA exist. However, both approaches represent powerful tools hybridization and PCR assays. Plant Disease 78:507-511. for sensitive detection and diagnosis. To our knowledge, this Harrison, N.A., Richardson, P.A., Kramer, J.B., Tsai, J.H. 1994b. is the first report on the molecular detection on phytoplasmas Detection of the -like organism associated affecting cassava in Mexico. The use of both PCR and with lethal yellowing disease of palms in Florida by molecular hybridization with specific DNA probes are polymerase chain reaction. Plant 43:998-1008. currently in use in improvement programs and epidemiological Harrison, N.A., Richardson, P.A., Tsai, J.H., Ebbert, M.A., studies in coconut palm (Almeyda-León et al., 1998) and Kramer, J.B. 1996. PCR assay for detection of the potato. Phytoplasma associated with maize bushy stunt disease. Plant Disease 80:263-269. LITERATURE CITED ICSBSTM, 1993. Minutes of the interim meetings, 1 and 2. Ahrens, U., and Seemüller, E. 1992. Detection of DNA of plant International Committee on Systematic Bacteriology pathogenic mycoplasma-like organisms by a polymerase Subcommittee on the Taxonomy of . August chain reaction that amplifies a sequence of the 16S rRNA 1992, Ames, Iowa. International Journal of Systematic gene. Phytopathology 82:828-832. Bacteriology 43:394-397. Almeyda-León, I.H., Rocha-Peña, M.A. y Piña, R.J. 1998. Kirkpatrick, B.C., Fisher, G.A., Fraser, J.D., Purcell, A.H. 1990. Diagnóstico molecular del amarillamiento letal: Su Epidemiological and phylogenetic studies on western X- aplicación en estudios epidemiológicos. Memorias de la disease mycoplasma-like organisms. pp 288-302. In: Recent Primera Reunión Nacional de la Palma de Coco. Acapulco, advances in mycoplasmology. G. Stanek, G.H. Cassell, J.G. Guerero, México. 99-111 pp. Tully, R.F. Whitcomb (eds.). Gustav Fischer Verlag, New Cadena-Hinojosa, M. 1993. La punta morada de la papa en York. México. Incidencia y búsqueda de resistencia. Agrociencia Kuske, C.R., Kirkpatrick, B.C. 1992. Phylogenetic relationships 4:247-256. between the western aster yellows mycoplasmalike Cadena-Hinojosa, M., Galindo-Alonso, J. 1986. Reducción organism and other prokaryotes established by 16S rRNA de la incidencia de la“Punta Morada de la Papa” por medio gene sequences. International Journal of Systematic de fechas de siembra, genotipo de la planta y aplicaciones Bacteriology 42:226-233. de insecticidas. Revista Mexicana de Fitopatología 3:100- Lee, I-M., Davis, R.E., Sinclair, W.A., DeWitt, N.D., Conti, M. 104. 1993a. Genetic relatedness of mycoplasmalike organisms Chiykowski, L.N., Sinha, R.C. 1989. Differentiation of MLO detected in Ulmus spp. in the United States and Italy by diseases by means of symptomatology and vector means of DNA probes and polymerase chain reactions. transmission. Zentralb. Bakteriol. Hyg. (Suppl.) 20:280-287. Phytopathology 83:829-833. Davis, R.E., Sinclair, W.A. 1998. Phytoplasma identity and Lee, I-M., Hammond, R.W., Davis, R.E., Gundersen, D.E. 1993b. disease etiology. Phytopathology 88:1372-1376. Universal amplification and analysis of pathogen 16S Doi, Y., Teranaka, M., Yora, K., Asuyana, H. 1969. Mycoplasma rDNA for classification and identification of Revista Mexicana de FITOPATOLOGIA/ 9

mycoplasmalike organisms. Phytopathology 83:834-842. Cocotero. Centro de Investigación Regional del Sureste. Lee, I-M., Gundersen, D.E., Hammond, R.W., Davis, R.E. 1994. INIFAP. México (mimeo). Use of mycoplasmalike organism (MLO) group-specific Rahardja, U., Whalon, M.E., García, S.C., Yan, Y.T. 1992. Field oligonucleotide primers for nested-PCR assays to detect detection of X-disease mycoplasmalike organism in mixed-MLO infections in a single host plant. Paraphlepsius irroratus (Say) (Homoptera: Cicadellidae) Phytopathology 84:559-566. using a DNA probe. Environmental Entomology 21:81-88. Lee, I-M., Gundersen, D.E., Bertaccini, A. 1998. Phytoplasma: Rogers, S.O., Bendich, A.J. 1985. Extraction of DNA from Ecology and genomic diversity. Phytopathology 88:1359- milligram amounts of fresh, herbarium and mummified plant 1366. tissues. Plant Molecular Biology 5:69-76. Martínez-Soriano, J.P., Almeyda-León, I.H., Piña-Razo, J., Rogers, S.O., Bendich, A.J. 1988. Extraction of DNA from Rocha-Peña, M.A., Byerly-Murphy, K.F. 1994. Detection plant tissues. Plant Molecular Biology Manual A6:1-10. of Phytoplasma sp. associated with lethal yellowing of Russel, W.C., Newman, C., Williamson, D.H. 1975. A simple coconut palms using polymerase chain reaction. Revista cytochemical technique for demonstration of DNA in cells Mexicana de Fitopatología 12:75-80. infected with and viruses. Nature 253:461- McCoy, R.E., Cauldwell, A., Chang, C.J., Chen, T.A., 462. Chiykowski, L.N., Cousin, M.T., Dale, J.L., de Leeuw, G.T.N., Sambrook, J., Fritsch, E.F., Maniatis, T. 1989. Molecular Golino, D.A., Hackett, K.J., Kirkpatrick, B.C., Marwitz, R., Cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Petzold, H., Sinha, R.C., Sugiura, M., Whitcomb, R.F., Yang, Laboratory Press, Cold Spring Harbor, N.Y. I.L., Zhu, B.M., Seemüller, E. 1989. Plant diseases associated Smart, C.D., Schneider, B., Blomquist, C.L., Guerra, L.J., with mycoplasma-like organisms. Pages 545-640. In: The Harrison, N.A., Ahrens, U., Lorenz, K.H., Seemüller, E., Mycoplasmas Vol. 5. , Acholeplasmas and Kirkpatrick, B.C. 1996. Phytoplasma-specific PCR primers Mycoplasms of Plants and Arthropods. R.F. Whitcomb, based on sequences of the 16S-23S rRNA spacer-region. J.G. Tully (eds). Academic Press, Inc., San Diego, CA. Applied and Environmental Microbiology 62:2988-2993. Piña-Razo, J. 1995. Plan Nacional de Investigación en