DNA Barcoding for Identification of ‘Candidatus Phytoplasmas’ Using a Fragment of the Elongation Factor Tu Gene
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{"title"=>"DNA Barcoding for Identification of 'Candidatus Phytoplasmas' Using a Fragment of the Elongation Factor Tu Gene", "type"=>"journal", "authors"=>[{"first_name"=>"Olga", "last_name"=>"Makarova", "scopus_author_id"=>"57197948259"}, {"first_name"=>"Nicoletta", "last_name"=>"Contaldo", "scopus_author_id"=>"35279369600"}, {"first_name"=>"Samanta", "last_name"=>"Paltrinieri", "scopus_author_id"=>"23568566200"}, {"first_name"=>"Geofrey", "last_name"=>"Kawube", "scopus_author_id"=>"55532230900"}, {"first_name"=>"Assunta", "last_name"=>"Bertaccini", "scopus_author_id"=>"7005650463"}, {"first_name"=>"Mogens", "last_name"=>"Nicolaisen", "scopus_author_id"=>"6603827466"}], "year"=>2012, "source"=>"PLoS ONE", "identifiers"=>{"pmid"=>"23272216", "doi"=>"10.1371/journal.pone.0052092", "sgr"=>"84871325028", "isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)", "scopus"=>"2-s2.0-84871325028", "issn"=>"19326203", "pui"=>"366299078"}, "id"=>"77a598a6-dd5d-39c6-aa45-94a5c335af16", "abstract"=>"BACKGROUND: Phytoplasmas are bacterial phytopathogens responsible for significant losses in agricultural production worldwide. Several molecular markers are available for identification of groups or strains of phytoplasmas. However, they often cannot be used for identification of phytoplasmas from different groups simultaneously or are too long for routine diagnostics. DNA barcoding recently emerged as a convenient tool for species identification. Here, the development of a universal DNA barcode based on the elongation factor Tu (tuf) gene for phytoplasma identification is reported.\\n\\nMETHODOLOGY/PRINCIPAL FINDINGS: We designed a new set of primers and amplified a 420-444 bp fragment of tuf from all 91 phytoplasmas strains tested (16S rRNA groups -I through -VII, -IX through -XII, -XV, and -XX). Comparison of NJ trees constructed from the tuf barcode and a 1.2 kbp fragment of the 16S ribosomal gene revealed that the tuf tree is highly congruent with the 16S rRNA tree and had higher inter- and intra- group sequence divergence. Mean K2P inter-/intra- group divergences of the tuf barcode did not overlap and had approximately one order of magnitude difference for most groups, suggesting the presence of a DNA barcoding gap. The use of the tuf barcode allowed separation of main ribosomal groups and most of their subgroups. Phytoplasma tuf barcodes were deposited in the NCBI GenBank and Q-bank databases.\\n\\nCONCLUSIONS/SIGNIFICANCE: This study demonstrates that DNA barcoding principles can be applied for identification of phytoplasmas. Our findings suggest that the tuf barcode performs as well or better than a 1.2 kbp fragment of the 16S rRNA gene and thus provides an easy procedure for phytoplasma identification. The obtained sequences were used to create a publicly available reference database that can be used by plant health services and researchers for online phytoplasma identification.", "link"=>"http://www.mendeley.com/research/dna-barcoding-identification-candidatus-phytoplasmas-using-fragment-elongation-factor-tu-gene", "reader_count"=>41, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>3, "Researcher"=>9, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>7, "Student > Postgraduate"=>1, "Student > Master"=>9, "Other"=>2, "Student > Bachelor"=>4, "Lecturer"=>1, "Lecturer > Senior Lecturer"=>3, "Professor"=>1}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>3, "Researcher"=>9, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>7, "Student > Postgraduate"=>1, "Student > Master"=>9, "Other"=>2, "Student > Bachelor"=>4, "Lecturer"=>1, "Lecturer > Senior Lecturer"=>3, "Professor"=>1}, "reader_count_by_subject_area"=>{"Unspecified"=>2, "Biochemistry, Genetics and Molecular Biology"=>5, "Agricultural and Biological Sciences"=>31, "Medicine and Dentistry"=>2, "Earth and Planetary Sciences"=>1}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>2}, "Earth and Planetary Sciences"=>{"Earth and Planetary Sciences"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>31}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>5}, "Unspecified"=>{"Unspecified"=>2}}, "reader_count_by_country"=>{"Brazil"=>1}, "group_count"=>1}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/523152"], "description"=>"<p>Two pairs of primer cocktails were used for universal amplification of the <i>tuf</i> barcode from all phytoplasma strains employed in this study in a nested PCR assay. Tuf 340/Tuf890 and Tuf400/Tuf835 primer cocktails were used in direct and nested PCR respectively. Each primer cocktail contained slightly different variants of the same primer mixed in equimolar amounts. The nucleotide sequences of the general sequencing primers M13F and T7 are underlined with a single and a double line, respectively. Primer positions correspond to the positions in the <i>tuf</i> gene of ‘<i>Ca.</i> P. asteris’ strain AY-WB (Genbank accession number CP000061).</p>", "links"=>[], "tags"=>["amplification", "dna"], "article_id"=>193645, "categories"=>["Microbiology", "Genetics", "Plant Biology", "Evolutionary Biology"], "users"=>["Olga Makarova", "Nicoletta Contaldo", "Samanta Paltrinieri", "Geofrey Kawube", "Assunta Bertaccini", "Mogens Nicolaisen"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0052092.t001", "stats"=>{"downloads"=>0, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Primers_used_for_amplification_of_the_tuf_DNA_barcode_/193645", "title"=>"Primers used for amplification of the <i>tuf</i> DNA barcode.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2012-12-18 01:00:45"}
  • {"files"=>["https://ndownloader.figshare.com/files/523120"], "description"=>"<p>K2P genetic distances were calculated within (intra) and between (inter) phytoplasma groups. Minimum and average mean K2P inter-group divergences were calculated for all phytoplasma groups. Intra-group divergences could only be calculated for groups with more than one representative. K2P inter-group distance values greater than K2P intra-group distance values (or average inter−/intra-group divergence ratios >1) indicate that inter- and intra- group divergences do not overlap and suggest the presence of a barcoding gap. d, sequence divergence distance; n/a, not available; n/c, not calculated.</p>", "links"=>[], "tags"=>["barcode", "k2p", "intra-", "inter-group"], "article_id"=>193607, "categories"=>["Microbiology", "Genetics", "Plant Biology", "Evolutionary Biology"], "users"=>["Olga Makarova", "Nicoletta Contaldo", "Samanta Paltrinieri", "Geofrey Kawube", "Assunta Bertaccini", "Mogens Nicolaisen"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0052092.t002", "stats"=>{"downloads"=>1, "page_views"=>10, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Comparison_of_the_tuf_barcode_mean_K2P_intra_and_inter_group_divergences_/193607", "title"=>"Comparison of the <i>tuf</i> barcode mean K2P intra- and inter-group divergences.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2012-12-18 01:00:07"}
  • {"files"=>["https://ndownloader.figshare.com/files/522937"], "description"=>"<p>Pairwise Kimura-2-parameter average distances between groups were determined for 91 and 66 phytoplasma strains from 19 groups for <i>tuf</i> and 16Sr, respectively. Note that inter-group sequence divergence in the 420–444 bp <i>tuf</i> barcode is much higher than divergence in the 1,2 kbp 16Sr gene fragment.</p>", "links"=>[], "tags"=>["pairwise", "inter-group", "k2p"], "article_id"=>193429, "categories"=>["Microbiology", "Genetics", "Plant Biology", "Evolutionary Biology"], "users"=>["Olga Makarova", "Nicoletta Contaldo", "Samanta Paltrinieri", "Geofrey Kawube", "Assunta Bertaccini", "Mogens Nicolaisen"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0052092.g003", "stats"=>{"downloads"=>0, "page_views"=>8, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Distribution_of_the_pairwise_inter_group_mean_K2P_sequence_divergence_/193429", "title"=>"Distribution of the pairwise inter-group mean K2P sequence divergence.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-12-18 00:57:09"}
  • {"files"=>["https://ndownloader.figshare.com/files/282917", "https://ndownloader.figshare.com/files/282975", "https://ndownloader.figshare.com/files/283035", "https://ndownloader.figshare.com/files/283052", "https://ndownloader.figshare.com/files/283074"], "description"=>"<div><h3>Background</h3><p>Phytoplasmas are bacterial phytopathogens responsible for significant losses in agricultural production worldwide. Several molecular markers are available for identification of groups or strains of phytoplasmas. However, they often cannot be used for identification of phytoplasmas from different groups simultaneously or are too long for routine diagnostics. DNA barcoding recently emerged as a convenient tool for species identification. Here, the development of a universal DNA barcode based on the elongation factor Tu (<em>tuf</em>) gene for phytoplasma identification is reported.</p> <h3>Methodology/Principal Findings</h3><p>We designed a new set of primers and amplified a 420–444 bp fragment of <em>tuf</em> from all 91 phytoplasmas strains tested (16S rRNA groups -I through -VII, -IX through -XII, -XV, and -XX). Comparison of NJ trees constructed from the <em>tuf</em> barcode and a 1.2 kbp fragment of the 16S ribosomal gene revealed that the <em>tuf</em> tree is highly congruent with the 16S rRNA tree and had higher inter- and intra- group sequence divergence. Mean K2P inter−/intra- group divergences of the <em>tuf</em> barcode did not overlap and had approximately one order of magnitude difference for most groups, suggesting the presence of a DNA barcoding gap. The use of the <em>tuf</em> barcode allowed separation of main ribosomal groups and most of their subgroups. Phytoplasma <em>tuf</em> barcodes were deposited in the NCBI GenBank and Q-bank databases.</p> <h3>Conclusions/Significance</h3><p>This study demonstrates that DNA barcoding principles can be applied for identification of phytoplasmas. Our findings suggest that the <em>tuf</em> barcode performs as well or better than a 1.2 kbp fragment of the 16S rRNA gene and thus provides an easy procedure for phytoplasma identification. The obtained sequences were used to create a publicly available reference database that can be used by plant health services and researchers for online phytoplasma identification.</p> </div>", "links"=>[], "tags"=>["dna", "barcoding", "elongation", "tu", "gene"], "article_id"=>115790, "categories"=>["Microbiology", "Genetics", "Cell Biology", "Evolutionary Biology"], "users"=>["Olga Makarova", "Nicoletta Contaldo", "Samanta Paltrinieri", "Geofrey Kawube", "Assunta Bertaccini", "Mogens Nicolaisen"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0052092.s001", "https://dx.doi.org/10.1371/journal.pone.0052092.s002", "https://dx.doi.org/10.1371/journal.pone.0052092.s003", "https://dx.doi.org/10.1371/journal.pone.0052092.s004", "https://dx.doi.org/10.1371/journal.pone.0052092.s005"], "stats"=>{"downloads"=>23, "page_views"=>12, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/DNA_Barcoding_for_Identification_of_Candidatus_Phytoplasmas_Using_a_Fragment_of_the_Elongation_Factor_Tu_Gene__/115790", "title"=>"DNA Barcoding for Identification of ‘<em>Candidatus</em> Phytoplasmas’ Using a Fragment of the Elongation Factor Tu Gene", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2012-12-18 01:36:30"}
  • {"files"=>["https://ndownloader.figshare.com/files/522806"], "description"=>"<p>The <i>tuf</i> barcode tree largely follows the branching pattern of the 16S rRNA tree. Numbers at the nodes indicate bootstrap values; bars, substitutions per nucleotide position; asterisk, strains, whose 16S rRNA gene was sequenced in this study; 16S rRNA GenBank sequence accession number is indicated following the strain acronym; 16Sr group and subgroup are in parentheses; <i>A. laidlawii</i> (accession number NC010163) was used as an outgroup.</p>", "links"=>[], "tags"=>["trees", "barcode", "16s", "ribosomal", "rna"], "article_id"=>193298, "categories"=>["Microbiology", "Genetics", "Plant Biology", "Evolutionary Biology"], "users"=>["Olga Makarova", "Nicoletta Contaldo", "Samanta Paltrinieri", "Geofrey Kawube", "Assunta Bertaccini", "Mogens Nicolaisen"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0052092.g002", "stats"=>{"downloads"=>1, "page_views"=>33, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_NJ_trees_of_the_tuf_barcode_a_and_the_R16F2n_R16R2_fragment_of_the_16S_ribosomal_RNA_gene_b_/193298", "title"=>"NJ trees of the <i>tuf</i> barcode (a) and the R16F2n/R16R2 fragment of the 16S ribosomal RNA gene (b).", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-12-18 00:54:58"}
  • {"files"=>["https://ndownloader.figshare.com/files/522718"], "description"=>"<p> <b>bp </b><b><i>tuf</i></b><b> barcode from phytoplasma-infected plants and absence of amplification from healthy plants.</b> Lanes 1–10: healthy plants; lanes 11–22: phytoplasma-infected plants. Lanes: 1–apple, 2–aster, 3– grapevine, 4–lettuce, 5–maize, 6–tobacco, 7–periwinkle, 8–plum, 9–potato, 10–oat, 11–CA, 12–CoP, 13–FD-AS, 14–JR-1, 15–LUM, 16–NJ-AY, 17–PrB, 18–RuS, 19–AP-15, 20–ASHY4, 21–ASLO, 22–BF, lanes M–1 kb DNA ladder.</p>", "links"=>[], "tags"=>["amplification"], "article_id"=>193206, "categories"=>["Microbiology", "Genetics", "Plant Biology", "Evolutionary Biology"], "users"=>["Olga Makarova", "Nicoletta Contaldo", "Samanta Paltrinieri", "Geofrey Kawube", "Assunta Bertaccini", "Mogens Nicolaisen"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0052092.g001", "stats"=>{"downloads"=>1, "page_views"=>7, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_PCR_amplification_of_the_420_8211_440_/193206", "title"=>"PCR amplification of the 420–440", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-12-18 00:53:26"}
  • {"files"=>["https://ndownloader.figshare.com/files/522996"], "description"=>"<p>‘<i>Ca.</i> P. oryzae’ and ‘<i>Ca.</i> P. aurantifolia’ showed considerably higher intra-group divergences, suggesting the presence of subgroups in these groups, not recognized by the 16Sr-based phylogeny. Average Kimura-2-parameter distances were calculated for the 16S rRNA and <i>tuf</i> sequences of the same strains, n – number of phytoplasma strains within a group. Strains used in the analysis: ‘<i>Ca.</i> P. asteris’ group 16SrI – A-YA, CA, KVE, CHRYM, HYDP, NJ-AY, GD-1, AY-1, AYBG, RV, AY-J24126, AY-2192, AVUT; ‘<i>Ca.</i> P. aurantifolia’ group 16SrII – PEP, SPLL, SEPT, TBB-KG, WBDL, CoP, PrB, FAP, VCP; ‘<i>Ca.</i> P. oryzae’ group 16SrXI – NGS, NGS-BS, BVK; ‘<i>Ca</i>. P. trifolii’ group 16SrVI – CP-1, PWB, LUM, BLL, CPS; ‘<i>Ca</i>. P. pruni’ group 16SrIII – MW1, VAC, GR1, LNI, CR, SP1, CX, BF, GVX.</p>", "links"=>[], "tags"=>["k2p", "within-group"], "article_id"=>193490, "categories"=>["Microbiology", "Genetics", "Plant Biology", "Evolutionary Biology"], "users"=>["Olga Makarova", "Nicoletta Contaldo", "Samanta Paltrinieri", "Geofrey Kawube", "Assunta Bertaccini", "Mogens Nicolaisen"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0052092.g004", "stats"=>{"downloads"=>2, "page_views"=>8, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Average_K2P_within_group_sequence_divergence_/193490", "title"=>"Average K2P within-group sequence divergence.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-12-18 00:58:10"}

PMC Usage Stats | Further Information

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  • {"unique-ip"=>"7", "full-text"=>"7", "pdf"=>"5", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2016", "month"=>"4"}
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  • {"unique-ip"=>"13", "full-text"=>"15", "pdf"=>"3", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2016", "month"=>"6"}
  • {"unique-ip"=>"9", "full-text"=>"8", "pdf"=>"1", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"3", "cited-by"=>"0", "year"=>"2016", "month"=>"7"}
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Relative Metric

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