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- Volume 66, Issue 12
- Article

oa Phytoplasma classification and phylogeny based on in silico and in vitro RFLP analysis of cpn60 universal target sequences
- Authors: Edel Pérez-López1 , Chrystel Y. Olivier2 , Mauricio Luna-Rodríguez3 , Tim J. Dumonceaux4,5
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1 1Instituto de Biotecnología y Ecología Aplicada (INBIOTECA), Universidad Veracruzana, Avenida de Las Culturas Veracruzanas Xalapa, Veracruz, México 2 2Agriculture and Agri-Food Canada, London Research and Development Centre, London, Ontario, Canada 3 3Laboratorio de Alta Tecnología de Xalapa - DGI, Universidad Veracruzana, Médicos 5, Unidad del Bosque Xalapa, Veracruz, México 4 4Agriculture and Agri-Food Canada, Saskatoon Research and Development Centre, Saskatoon, Saskatchewan, Canada 5 5Department of Veterinary Microbiology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
- Correspondence Tim J. Dumonceaux [email protected]
- First Published Online: 01 December 2016, International Journal of Systematic and Evolutionary Microbiology 66: 5600-5613, doi: 10.1099/ijsem.0.001501
- Subject: Evolution, Phylogeny and Biodiversity
- Received:
- Accepted:
- Cover date:
- This is an open access article published by the Microbiology Society under the Creative Commons Attribution License




Phytoplasma classification and phylogeny based on in silico and in vitro RFLP analysis of cpn60 universal target sequences, Page 1 of 1
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Phytoplasmas are unculturable, phytopathogenic bacteria that cause economic losses worldwide. As unculturable micro-organisms, phytoplasma taxonomy has been based on the use of the 16S rRNA-encoding gene to establish 16Sr groups and subgroups based on the restriction fragment length polymorphism (RFLP) pattern resulting from the digestion of amplicon (in vitro) or sequence (in silico) with seventeen restriction enzymes. Problems such as heterogeneity of the ribosomal operon and the inability to differentiate closely related phytoplasma strains has motivated the search for additional markers capable of providing finer differentiation of phytoplasma strains. In this study we developed and validated a scheme to classify phytoplasmas based on the use of cpn60 universal target (cpn60 UT) sequences. Ninety-six cpn60 UT sequences from strains belonging to 19 16Sr subgroups were subjected to in silico RFLP using pDRAW32 software, resulting in 25 distinctive RFLP profiles. Based on these results we delineated cpn60 UT groups and subgroups, and established a threshold similarity coefficient for groups and subgroups classifying all the strains analysed in this study. The nucleotide identity among the reference strains, the correspondence between in vitro and in silico RFLP, and the phylogenetic relationships of phytoplasma strains based on cpn60 UT sequences are also discussed.
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A supplementary figure and a supplementary table are available with the online Supplementary Material.
- Keyword(s): taxonomy, Phytoplasma, chaperonin 60, differentiation, Diversity
© 2016 Her Majesty The Queen in Right of Canada as represented by the Minister of Agriculture | Published by the Microbiology Society
-
Andersen M. T., Liefting L. W., Havukkala I., Beever R. E..( 2013;). Comparison of the complete genome sequence of two closely related isolates of ‘Candidatus Phytoplasma australiense’ reveals genome plasticity. . BMC Genomics14:529. [CrossRef][PubMed]
-
Arnaud G., Malembic-Maher S., Salar P., Bonnet P., Maixner M., Marcone C., Boudon-Padieu E., Foissac X..( 2007;). Multilocus sequence typing confirms the close genetic interrelatedness of three distinct flavescence dorée phytoplasma strain clusters and group 16SrV phytoplasmas infecting grapevine and alder in Europe. . Appl Environ Microbiol73:4001––4010. [CrossRef][PubMed]
-
Bai X., Zhang J., Ewing A., Miller S. A., Jancso Radek A., Shevchenko D. V., Tsukerman K., Walunas T., Lapidus A. et al.( 2006;). Living with genome instability: the adaptation of phytoplasmas to diverse environments of their insect and plant hosts. . J Bacteriol188:3682––3696. [CrossRef][PubMed]
-
Botti S., Bertaccini A..( 2003;). Variability and functional role of chromosomal sequences in 16SrI-B subgroup phytoplasmas including aster yellows and related strains. . J Appl Microbiol94:103––110. [CrossRef][PubMed]
-
Chung W. C., Chen L. L., Lo W. S., Lin C. P., Kuo C. H..( 2013;). Comparative analysis of the peanut witches'-broom phytoplasma genome reveals horizontal transfer of potential mobile units and effectors. . PLoS One8:e62770. [CrossRef][PubMed]
-
Claisse O., Renouf V., Lonvaud-Funel A..( 2007;). Differentiation of wine lactic acid bacteria species based on RFLP analysis of a partial sequence of rpoB gene. . J Microbiol Methods69:387––390. [CrossRef][PubMed]
-
Clark G. W., Tillier E. R..( 2010;). Loss and gain of GroEL in the Mollicutes. . Biochem Cell Biol88:185––194. [CrossRef][PubMed]
-
Crooks G. E., Hon G., Chandonia J. M., Brenner S. E..( 2004;). WebLogo: a sequence logo generator. . Genome Res14:1188––1190. [CrossRef][PubMed]
-
Davis R. E., Zhao Y., Dally E. L., Lee I. M., Jomantiene R., Douglas S. M..( 2013;). ‘Candidatus Phytoplasma pruni’, a novel taxon associated with X-disease of stone fruits, Prunus spp.: multilocus characterization based on 16S rRNA, secY, and ribosomal protein genes. . Int J Syst Evol Microbiol63:766––776. [CrossRef][PubMed]
-
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. . Ann Phytopathol Japan33:259––266. [CrossRef]
-
Dumonceaux T. J., Green M., Hammond C., Perez E., Olivier C..( 2014;). Molecular diagnostic tools for detection and differentiation of phytoplasmas based on chaperonin-60 reveal differences in host plant infection patterns. . PLoS One9:e116039. [CrossRef][PubMed]
-
Gascuel O., Steel M..( 2006;). Neighbor-joining revealed. . Mol Biol Evol23:1997––2000. [CrossRef][PubMed]
-
Goh S. H., Potter S., Wood J. O., Hemmingsen S. M., Reynolds R. P., Chow A. W..( 1996;). HSP60 gene sequences as universal targets for microbial species identification: studies with coagulase-negative staphylococci. . J Clin Microbiol34:818––823.[PubMed]
-
Haakensen M., Pittet V., Ziola B..( 2011;). Reclassification of Paralactobacillus selangorensis Leisner et al. 2000 as Lactobacillus selangorensis comb. nov. . Int J Syst Evol Microbiol61:2979––2983. [CrossRef][PubMed]
-
Harrison N. A., Davis R. E., Oropeza C., Helmick E. E., Narváez M., Eden-Green S., Dollet M., Dickinson M..( 2014;). ‘Candidatus Phytoplasma palmicola’, associated with a lethal yellowing-type disease of coconut (Cocos nucifera L.) in Mozambique. . Int J Syst Evol Microbiol64:1890––1899. [CrossRef][PubMed]
-
Hedberg M. E., Israelsson A., Moore E. R., Svensson-Stadler L., Wai S. N., Pietz G., Sandström O., Hernell O., Hammarström M. L., Hammarström S..( 2013;). Prevotella jejuni sp. nov., isolated from the small intestine of a child with coeliac disease. . Int J Syst Evol Microbiol63:4218––4223. [CrossRef][PubMed]
-
Hill J. E., Penny S. L., Crowell K. G., Goh S. H., Hemmingsen S. M..( 2004;). cpnDB: a chaperonin sequence database. . Genome Res14:1669––1675. [CrossRef][PubMed]
-
Hodgetts J., Boonham N., Mumford R., Harrison N., Dickinson M..( 2008;). Phytoplasma phylogenetics based on analysis of secA and 23S rRNA gene sequences for improved resolution of candidate species of ‘Candidatus Phytoplasma’. . Int J Syst Evol Microbiol58:1826––1837. [CrossRef][PubMed]
-
Hogenhout S. A., Oshima K., Ammar el-D., Kakizawa S., Kingdom H. N., Namba S..( 2008;). Phytoplasmas: bacteria that manipulate plants and insects. . Mol Plant Pathol9:403––423. [CrossRef][PubMed]
-
IRPCM Phytoplasma/Spiroplasma Working Team-Phytoplasma Taxonomy Group( 2004;). ‘Candidatus Phytoplasma’, a taxon for the wall-less,non-helical prokaryotes that colonize plant phloem and insects. . Int J Syst Evol Microbiol54:1243––1255. [CrossRef][PubMed]
-
Kakizawa S., Oshima K., Jung H. Y., Suzuki S., Nishigawa H., Arashida R., Miyata S., Ugaki M., Kishino H., Namba S..( 2006;). Positive selection acting on a surface membrane protein of the plant-pathogenic phytoplasmas. . J Bacteriol188:3424––3428. [CrossRef][PubMed]
-
Kube M., Schneider B., Kuhl H., Dandekar T., Heitmann K., Migdoll A. M., Reinhardt R., Seemüller E..( 2008;). The linear chromosome of the plant-pathogenic mycoplasma ‘Candidatus Phytoplasma mali’. . BMC Genomics9:306. [CrossRef][PubMed]
-
Lee I.-M., Hammond R. W., Davis R. E., Gundersen D. E..( 1993;). Universal amplification and analysis of pathogen 16S rDNA for classification and identification of mycoplasmalike organisms. . Phytopathol83:834––842. [CrossRef]
-
Lee I. M., Gundersen-Rindal D. E., Davis R. E., Bartoszyk I. M..( 1998;). Revised classification scheme of phytoplasmas based on RFLP analyses of 16S rRNA and ribosomal protein gene sequences. . Int J Sys Bacteriol48:1153––1169. [CrossRef]
-
Lee I. M., Zhao Y., Bottner K. D..( 2006;). SecY gene sequence analysis for finer differentiation of diverse strains in the aster yellows phytoplasma group. . Mol Cell Probes20:87––91. [CrossRef][PubMed]
-
Links M. G., Dumonceaux T. J., Hemmingsen S. M., Hill J. E..( 2012;). The chaperonin-60 universal target is a barcode for bacteria that enables de novo assembly of metagenomic sequence data. . PLoS One7:e49755. [CrossRef][PubMed]
-
Marcone C., Lee I. M., Davis R. E., Ragozzino A., Seemüller E..( 2000;). Classification of aster yellows-group phytoplasmas based on combined analyses of rRNA and tuf gene sequences. . Int J Syst Evol Microbiol50:1703––1713. [CrossRef][PubMed]
-
Marqués A. M., Burgos-Díaz C., Aranda F. J., Teruel J. A., Manresa À., Ortiz A., Farfán M..( 2012;). Sphingobacterium detergens sp. nov., a surfactant-producing bacterium isolated from soil. . Int J Syst Evol Microbiol62:3036––3041. [CrossRef][PubMed]
-
Mitrović J., Kakizawa S., Duduk B., Oshima K., Namba S., Bertaccini A..( 2011;). The groEL gene as an additional marker for finer differentiation of ‘Candidatus Phytoplasma asteris’-related strains. . Ann Appl Biol159:41––48. [CrossRef]
-
Mitrović J., Smiljković M., Seemüller E., Reinhardt R., Hüttel B., Büttner C., Bertaccini A., Kube M., Duduk B..( 2015;). Differentiation of ‘Candidatus Phytoplasma cynodontis’ based on 16S rRNA and groEL genes and identification of a new subgroup 16SrXIV-C. . Plant Dis99:1578––1583. [CrossRef]
-
Murray R. G., Stackebrandt E..( 1995;). Taxonomic note: implementation of the provisional status Candidatus for incompletely described procaryotes. . Int J Syst Bacteriol45:186––187. [CrossRef][PubMed]
-
Nei M., Li W. H..( 1979;). Mathematical model for studying genetic variation in terms of restriction endonucleases. . Proc Natl Acad Sci U S A76:5269––5273. [CrossRef][PubMed]
-
Nejat N., Vadamalai G., Davis R. E., Harrison N. A., Sijam K., Dickinson M., Abdullah S. N., Zhao Y..( 2013;). ‘Candidatus Phytoplasma malaysianum’, a novel taxon associated with virescence and phyllody of Madagascar periwinkle (Catharanthus roseus). . Int J Syst Evol Microbiol63:540––548. [CrossRef][PubMed]
-
Oshima K., Kakizawa S., Nishigawa H., Jung H. Y., Wei W., Suzuki S., Arashida R., Nakata D., Miyata S. et al.( 2004;). Reductive evolution suggested from the complete genome sequence of a plant-pathogenic phytoplasma. . Nat Genet36:27––29. [CrossRef][PubMed]
-
Oshima K., Maejima K., Namba S..( 2013;). Genomic and evolutionary aspects of phytoplasmas. . Front Microbiol4:230. [CrossRef][PubMed]
-
Park S.-H., Jung J.-H., Seo D.-H., Lee H.-L., Kim G.-W., Park S.-Y., Shin W.-C., Hong S., Park C.-S..( 2012;). Differentiation of lactic acid bacteria based on RFLP analysis of the tuf gene. . Food Science and Biotechnology21:911––915. [CrossRef]
-
Pérez-López E., Luna-Rodríguez M., Olivier C. Y., Dumonceaux T. J..( 2016a;). The underestimated diversity of phytoplasmas in Latin America. . Int J Syst Evol Microbiol66:492––513. [CrossRef][PubMed]
-
Pérez-López E., Olivier C. Y., Luna-Rodríguez M., Rodríguez Y., Iglesias L. G., Castro-Luna A., Adame-García J., Dumonceaux T. J..( 2016b;). Maize bushy stunt phytoplasma affects native corn at high elevations in Southeast Mexico. . Eur J Plant Pathol145:963––971. [CrossRef]
-
Quaglino F., Kube M., Jawhari M., Abou-Jawdah Y., Siewert C., Choueiri E., Sobh H., Casati P., Tedeschi R. et al.( 2015;). ‘Candidatus Phytoplasma phoenicium’ associated with almond witches'-broom disease: from draft genome to genetic diversity among strain populations. . BMC Microbiol15:148. [CrossRef][PubMed]
-
Ruiz A., Poblet M., Mas A., Guillamón J. M..( 2000;). Identification of acetic acid bacteria by RFLP of PCR-amplified 16S rDNA and 16S-23S rDNA intergenic spacer. . Int J Syst Evol Microbiol50:1981––1987. [CrossRef][PubMed]
-
Saccardo F., Martini M., Palmano S., Ermacora P., Scortichini M., Loi N., Firrao G..( 2012;). Genome drafts of four phytoplasma strains of the ribosomal group 16SrIII. . Microbiology158:2805––2814. [CrossRef][PubMed]
-
Shao J. Y., Jomantiene R., Dally E. L., Zhao Y., Lee I.-M., Nuss D. L., Davis R. E..( 2006;). Phylogeny and characterization of phytoplasmal NusA and use of the nusA gene in detection of group 16SrI strains. . J Plant Pathol88:193––201.
-
Streten C., Gibb K. S..( 2005;). Genetic variation in Candidatus Phytoplasma australiense. . Plant Pathology54:8––14. [CrossRef]
-
Tamura K., Stecher G., Peterson D., Filipski A., Kumar S..( 2013;). mega6: molecular evolutionary genetics analysis version 6.0. . Mol Biol Evol30:2725––2729. [CrossRef][PubMed]
-
Tani A., Sahin N., Kimbara K..( 2012;). Methylobacterium gnaphalii sp. nov., isolated from leaves of Gnaphalium spicatum. . Int J Syst Evol Microbiol62:2602––2607. [CrossRef][PubMed]
-
Thompson J. D., Gibson T. J., Plewniak F., Jeanmougin F., Higgins D. G..( 1997;). The clustal_x windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. . Nucleic Acids Res25:4876––4882. [CrossRef][PubMed]
-
Town J., Annand H., Pratt D., Dumonceaux T., Fonstad T..( 2014;). Microbial community composition is consistent across anaerobic digesters processing wheat-based fuel ethanol waste streams. . Bioresour Technol157:127––133. [CrossRef][PubMed]
-
Tran-Nguyen L. T., Kube M., Schneider B., Reinhardt R., Gibb K. S..( 2008;). Comparative genome analysis of ‘Candidatus Phytoplasma australiense’ (subgroup tuf-Australia I; rp-A) and ‘Ca. Phytoplasma asteris’ strains OY-M and AY-WB. . J Bacteriol190:3979––3991. [CrossRef][PubMed]
-
Valiunas D., Jomantiene R., Davis R. E..( 2013;). Evaluation of the DNA-dependent RNA polymerase β-subunit gene (rpoB) for phytoplasma classification and phylogeny. . Int J Syst Evol Microbiol63:3904––3914. [CrossRef][PubMed]
-
Wei W., Davis R. E., Lee I. M., Zhao Y..( 2007;). Computer-simulated RFLP analysis of 16S rRNA genes: identification of ten new phytoplasma groups. . Int J Syst Evol Microbiol57:1855––1867. [CrossRef][PubMed]
-
Wei W., Lee I. M., Davis R. E., Suo X., Zhao Y..( 2008;). Automated RFLP pattern comparison and similarity coefficient calculation for rapid delineation of new and distinct phytoplasma 16Sr subgroup lineages. . Int J Syst Evol Microbiol58:2368––2377. [CrossRef][PubMed]
-
Yang Z..( 2007;). PAML 4: phylogenetic analysis by maximum likelihood. . Mol Biol Evol24:1586––1591. [CrossRef][PubMed]
-
Zahariev M., Dahl V., Chen W., Lévesque C. A..( 2009;). Efficient algorithms for the discovery of DNA oligonucleotide barcodes from sequence databases. . Mol Ecol Resour9:58––64. [CrossRef][PubMed]
-
Zeigler D. R..( 2003;). Gene sequences useful for predicting relatedness of whole genomes in bacteria. . Int J Syst Evol Microbiol53:1893––1900. [CrossRef][PubMed]
-
Zhao Y., Wei W., Lee I. M., Shao J., Suo X., Davis R. E..( 2009;). Construction of an interactive online phytoplasma classification tool, iPhyClassifier, and its application in analysis of the peach X-disease phytoplasma group (16SrIII). . Int J Syst Evol Microbiol59:2582––2593. [CrossRef][PubMed]
-
Zhao Y., Wei W., Davis R. E., Lee I.-M..( 2010;). Recent advances in 16S rRNA gene-based phytoplasma differentiation, classification and taxonomy. . In Phytoplasmas: Genomes, Plant Hosts and Vector, pp. 64––92. Edited by Weintraub P., Jones P.. Wallingford, UK:: CABI Publishing;.
-
Zhao Y., Davis R. E., Wei W., Shao J., Jomantiene R..( 2014;). Phytoplasma genomes: evolution through mutually complementary mechanisms, gene loss and horizontal acquisition.. . In Genomics of Plant-Associated Bacteria, pp. 235––271. Edited by Gross D. C.. Berlin Heidelberg:: Springer-Verlag;.
-
Zhao Y., Davis R. E., Wei W., Lee I. M..( 2015;). Should ‘Candidatus Phytoplasma’ be retained within the order Acholeplasmatales?. Int J Syst Evol Microbiol65:1075––1082. [CrossRef][PubMed]

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