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array:24 [ "pii" => "S0325754115001601" "issn" => "03257541" "doi" => "10.1016/j.ram.2015.10.006" "estado" => "S300" "fechaPublicacion" => "2016-01-01" "aid" => "75" "copyright" => "Asociación Argentina de Microbiología" "copyrightAnyo" => "2015" "documento" => "article" "crossmark" => 1 "licencia" => "http://creativecommons.org/licenses/by-nc-nd/4.0/" "subdocumento" => "fla" "cita" => "Rev Argent Microbiol. 2016;48:15-20" "abierto" => array:3 [ "ES" => true "ES2" => true "LATM" => true ] "gratuito" => true "lecturas" => array:2 [ "total" => 1599 "formatos" => array:3 [ "EPUB" => 43 "HTML" => 1065 "PDF" => 491 ] ] "itemSiguiente" => array:19 [ "pii" => "S0325754116000031" "issn" => "03257541" "doi" => "10.1016/j.ram.2016.01.001" "estado" => "S300" "fechaPublicacion" => "2016-01-01" "aid" => "84" "copyright" => "Asociación Argentina de Microbiología" "documento" => "article" "crossmark" => 1 "licencia" => "http://creativecommons.org/licenses/by-nc-nd/4.0/" "subdocumento" => "fla" "cita" => "Rev Argent Microbiol. 2016;48:21-6" "abierto" => array:3 [ "ES" => true "ES2" => true "LATM" => true ] "gratuito" => true "lecturas" => array:2 [ "total" => 1488 "formatos" => array:3 [ "EPUB" => 42 "HTML" => 984 "PDF" => 462 ] ] "en" => array:13 [ "idiomaDefecto" => true "cabecera" => "<span class="elsevierStyleTextfn">Original article</span>" "titulo" => "An acidic sphingomyelinase Type C activity from <span class="elsevierStyleItalic">Mycobacterium tuberculosis</span>" "tienePdf" => "en" "tieneTextoCompleto" => "en" "tieneResumen" => array:2 [ 0 => "en" 1 => "es" ] "paginas" => array:1 [ 0 => array:2 [ "paginaInicial" => "21" "paginaFinal" => "26" ] ] "titulosAlternativos" => array:1 [ "es" => array:1 [ "titulo" => "Actividad de una esfingomielinasa ácida tipo C producida por <span class="elsevierStyleItalic">Mycobacterium tuberculosis</span>" ] ] "contieneResumen" => array:2 [ "en" => true "es" => true ] "contieneTextoCompleto" => array:1 [ "en" => true ] "contienePdf" => array:1 [ "en" => true ] "resumenGrafico" => array:2 [ "original" => 0 "multimedia" => array:7 [ "identificador" => "fig0005" "etiqueta" => "Figure 1" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr1.jpeg" "Alto" => 1215 "Ancho" => 1229 "Tamanyo" => 73344 ] ] "descripcion" => array:1 [ "en" => "<p id="spar0015" class="elsevierStyleSimplePara elsevierViewall">Time-course of mycobacterial SMase activity. Mycobacterial cell fractions (2<span class="elsevierStyleHsp" style=""></span>μg of total protein) were tested for SMase activity at incubation time points between 0 and 60<span class="elsevierStyleHsp" style=""></span>min. Symbols represent the mean<span class="elsevierStyleHsp" style=""></span>±<span class="elsevierStyleHsp" style=""></span>SE of [<span class="elsevierStyleSup">14</span>C]-phosphorylcholine released by <span class="elsevierStyleItalic">M. tuberculosis</span> whole cell extracts of strains H37Rv (○) or CDC1551 (●) in three independent experiments performed in triplicate.</p>" ] ] ] "autores" => array:1 [ 0 => array:2 [ "autoresLista" => "Jorge Castro-Garza, Francisco González-Salazar, Frederick D. Quinn, Russell K. Karls, Laura Hermila De La Garza-Salinas, Francisco J. Guzmán-de la Garza, Javier Vargas-Villarreal" "autores" => array:7 [ 0 => array:2 [ "nombre" => "Jorge" "apellidos" => "Castro-Garza" ] 1 => array:2 [ "nombre" => "Francisco" "apellidos" => "González-Salazar" ] 2 => array:2 [ "nombre" => "Frederick D." "apellidos" => "Quinn" ] 3 => array:2 [ "nombre" => "Russell K." "apellidos" => "Karls" ] 4 => array:2 [ "nombre" => "Laura Hermila" "apellidos" => "De La Garza-Salinas" ] 5 => array:2 [ "nombre" => "Francisco J." "apellidos" => "Guzmán-de la Garza" ] 6 => array:2 [ "nombre" => "Javier" "apellidos" => "Vargas-Villarreal" ] ] ] ] ] "idiomaDefecto" => "en" "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S0325754116000031?idApp=UINPBA00004N" "url" => "/03257541/0000004800000001/v1_201603250025/S0325754116000031/v1_201603250025/en/main.assets" ] "itemAnterior" => array:19 [ "pii" => "S0325754115001509" "issn" => "03257541" "doi" => "10.1016/j.ram.2015.10.004" "estado" => "S300" "fechaPublicacion" => "2016-01-01" "aid" => "73" "copyright" => "Asociación Argentina de Microbiología" "documento" => "article" "crossmark" => 1 "licencia" => "http://creativecommons.org/licenses/by-nc-nd/4.0/" "subdocumento" => "fla" "cita" => "Rev Argent Microbiol. 2016;48:5-14" "abierto" => array:3 [ "ES" => true "ES2" => true "LATM" => true ] "gratuito" => true "lecturas" => array:2 [ "total" => 2171 "formatos" => array:3 [ "EPUB" => 35 "HTML" => 1603 "PDF" => 533 ] ] "en" => array:13 [ "idiomaDefecto" => true "cabecera" => "<span class="elsevierStyleTextfn">Original article</span>" "titulo" => "Expression and refolding of the protective antigen of <span class="elsevierStyleItalic">Bacillus anthracis</span>: A model for high-throughput screening of antigenic recombinant protein refolding" "tienePdf" => "en" "tieneTextoCompleto" => "en" "tieneResumen" => array:2 [ 0 => "en" 1 => "es" ] "paginas" => array:1 [ 0 => array:2 [ "paginaInicial" => "5" "paginaFinal" => "14" ] ] "titulosAlternativos" => array:1 [ "es" => array:1 [ "titulo" => "Expresión y renaturalización del antígeno protector de <span class="elsevierStyleItalic">Bacillus anthracis</span>: un modelo para evaluar el replegado de proteínas antigénicas recombinantes a gran escala" ] ] "contieneResumen" => array:2 [ "en" => true "es" => true ] "contieneTextoCompleto" => array:1 [ "en" => true ] "contienePdf" => array:1 [ "en" => true ] "resumenGrafico" => array:2 [ "original" => 0 "multimedia" => array:7 [ "identificador" => "fig0015" "etiqueta" => "Figure 3" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr3.jpeg" "Alto" => 1807 "Ancho" => 2667 "Tamanyo" => 570557 ] ] "descripcion" => array:1 [ "en" => "<p id="spar0030" class="elsevierStyleSimplePara elsevierViewall">Composite image of the dots, showing Coefficient <span class="elsevierStyleItalic">D</span> values above each dot while the number at the bottom indicates the order by decreasing coefficient <span class="elsevierStyleItalic">D</span> values. Letters and numbers on the top and left margins indicate original dot location, corresponding to the multiwell plate and the dot blot assay shown in <a class="elsevierStyleCrossRef" href="#fig0010">Fig. 2</a>.</p>" ] ] ] "autores" => array:1 [ 0 => array:2 [ "autoresLista" => "María Elisa Pavan, Esteban Enrique Pavan, Fabián Martín Cairó, María Julia Pettinari" "autores" => array:4 [ 0 => array:2 [ "nombre" => "María Elisa" "apellidos" => "Pavan" ] 1 => array:2 [ "nombre" => "Esteban Enrique" "apellidos" => "Pavan" ] 2 => array:2 [ "nombre" => "Fabián Martín" "apellidos" => "Cairó" ] 3 => array:2 [ "nombre" => "María Julia" "apellidos" => "Pettinari" ] ] ] ] ] "idiomaDefecto" => "en" "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S0325754115001509?idApp=UINPBA00004N" "url" => "/03257541/0000004800000001/v1_201603250025/S0325754115001509/v1_201603250025/en/main.assets" ] "en" => array:20 [ "idiomaDefecto" => true "cabecera" => "<span class="elsevierStyleTextfn">Original article</span>" "titulo" => "PCR-based method for the rapid identification of astaxanthin-accumulating yeasts (<span class="elsevierStyleItalic">Phaffia</span> spp.)" "tieneTextoCompleto" => true "paginas" => array:1 [ 0 => array:2 [ "paginaInicial" => "15" "paginaFinal" => "20" ] ] "autores" => array:1 [ 0 => array:4 [ "autoresLista" => "Fernando Colabella, Diego Libkind" "autores" => array:2 [ 0 => array:2 [ "nombre" => "Fernando" "apellidos" => "Colabella" ] 1 => array:4 [ "nombre" => "Diego" "apellidos" => "Libkind" "email" => array:1 [ 0 => "libkindfd@comahue-conicet.gob.ar" ] "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">*</span>" "identificador" => "cor0005" ] ] ] ] "afiliaciones" => array:1 [ 0 => array:2 [ "entidad" => "Laboratorio de Microbiología Aplicada y Biotecnología, Instituto de Investigaciones en Biodiversidad y Medio Ambiente, INIBIOMA (CONICET – UNComahue), Quintral 1250, Bariloche 8400, Río Negro, Argentina" "identificador" => "aff0005" ] ] "correspondencia" => array:1 [ 0 => array:3 [ "identificador" => "cor0005" "etiqueta" => "⁎" "correspondencia" => "Corresponding author." ] ] ] ] "titulosAlternativos" => array:1 [ "es" => array:1 [ "titulo" => "Un método basado en la PCR para la identificación rápida de levaduras acumuladoras de astaxantina (<span class="elsevierStyleItalic">Phaffia</span> spp.)" ] ] "resumenGrafico" => array:2 [ "original" => 0 "multimedia" => array:7 [ "identificador" => "fig0005" "etiqueta" => "Figure 1" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr1.jpeg" "Alto" => 748 "Ancho" => 1314 "Tamanyo" => 56132 ] ] "descripcion" => array:1 [ "en" => "<p id="spar0015" class="elsevierStyleSimplePara elsevierViewall">Yeast strains tested with multiplex PhR, ITS3 and NL4. 1–7 <span class="elsevierStyleItalic">P. rhodozyma</span>: (1) CBS 5905 (Lineage C1/B); (2) CBS 7918<span class="elsevierStyleSup">T</span> (Lineage C2); (3) CRUB 1149 (Lineage A); (4) ATCC 24229 (Lineage D); (5) ZP 938 (Lineage E); (6) ZP 875 (Lineage F); (7) ZP 874 (Lineage B); (8) TSN-67; (9) <span class="elsevierStyleItalic">C. capitatum</span> CBS 7420; (10) <span class="elsevierStyleItalic">C. macerans</span> CBS 2206; N – negative control (no template).</p>" ] ] ] "textoCompleto" => "<span class="elsevierStyleSections"><span id="sec0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0025">Introduction</span><p id="par0005" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">Phaffia rhodozyma</span> (also known as <span class="elsevierStyleItalic">Xanthophyllomyces dendrorhous</span>) is a basidiomycetous yeast, forming orange-red colonies and known worldwide since, until now, it is the only yeast capable of producing the carotenoid pigment astaxanthin (3,3′-dihydroxy-β,β-carotene-4,4′-dione). This compound is of economic importance because it is the most expensive feed component for aquaculture and aviculture<a class="elsevierStyleCrossRef" href="#bib0205"><span class="elsevierStyleSup">8</span></a>. Moreover, <span class="elsevierStyleItalic">P. rhodozyma</span> is the only known carotenogenic yeast species that is able to vigorously ferment a number of sugars, including glucose, maltose, sucrose and raffinose<a class="elsevierStyleCrossRef" href="#bib0210"><span class="elsevierStyleSup">9</span></a>. Moreover, it has recently been discovered that this yeast has the ability to synthesize a UV-absorbing molecule called mycosporine-glutaminol-glucoside of applied interest<a class="elsevierStyleCrossRef" href="#bib0235"><span class="elsevierStyleSup">14</span></a>.</p><p id="par0010" class="elsevierStylePara elsevierViewall">The natural distribution and habitat of <span class="elsevierStyleItalic">P. rhodozyma</span> are much broader than previously thought. Initially, genetically similar isolates of this yeast were found in the Northern Hemisphere in association with slime exudates of trees, being obtained from Japan, Canada, Russia, Italy, Germany and USA<a class="elsevierStyleCrossRefs" href="#bib0180"><span class="elsevierStyleSup">3,22,27,30</span></a>. A novel and genetically distinct <span class="elsevierStyleItalic">Phaffia</span> population was later isolated in the Southern Hemisphere, from the sugary stromata of the <span class="elsevierStyleItalic">Cyttaria hariotti</span> fungus, a parasite of the <span class="elsevierStyleItalic">Nothofagus</span> trees in Argentina<a class="elsevierStyleCrossRef" href="#bib0240"><span class="elsevierStyleSup">15</span></a>. An even more genetically divergent strain was described in Chile, which might represent a novel species although a single strain is known<a class="elsevierStyleCrossRef" href="#bib0310"><span class="elsevierStyleSup">29</span></a>. More recently, David-Palma et al.<a class="elsevierStyleCrossRef" href="#bib0175"><span class="elsevierStyleSup">2</span></a> investigated the association of <span class="elsevierStyleItalic">Phaffia</span> with <span class="elsevierStyleItalic">Nothofagus-Cyttaria</span> in Australasia, the other region of the world where <span class="elsevierStyleItalic">Nothofagus</span> are endemic, and discovered an even higher <span class="elsevierStyleItalic">Phaffia</span> diversity, including two endemic and markedly divergent linages which represent putative new species, both with the capability to produce astaxanthin and MGG (unpublished results). In this work it was suggested that <span class="elsevierStyleItalic">Phaffia</span> adaptation to different tree hosts/niches has driven population structure, thus, it can be anticipated that novel lineages of this yeast might be yet undiscovered. Proof of this fact are the recent isolates found by Yurkov et al.<a class="elsevierStyleCrossRef" href="#bib0330"><span class="elsevierStyleSup">33</span></a>, and Contreras et al.<a class="elsevierStyleCrossRef" href="#bib0170"><span class="elsevierStyleSup">1</span></a> from soil and Antarctic-related environments, respectively. In this scenario and due to the biotechnological relevance of this genus, specific culture media and biochemical techniques that allow a more favorable isolation and simpler identification of <span class="elsevierStyleItalic">P. rhodozyma</span> were reported in previous works<a class="elsevierStyleCrossRefs" href="#bib0175"><span class="elsevierStyleSup">2,13,26</span></a>. This is particularly important considering that the environmental isolation of <span class="elsevierStyleItalic">P. rhodozyma</span> is not always easy due to its low abundance and because it is easily misidentified with other sympatric and carotenoid-accumulating yeasts of the genera <span class="elsevierStyleItalic">Rhodotorula</span>, <span class="elsevierStyleItalic">Cystofilobasidium</span> and <span class="elsevierStyleItalic">Dioszegia</span><a class="elsevierStyleCrossRef" href="#bib0245"><span class="elsevierStyleSup">16</span></a>. Not many yeast species are able to produce carotenoid pigments and these are distributed among a few lineages<a class="elsevierStyleCrossRef" href="#bib0255"><span class="elsevierStyleSup">18</span></a>. Those species accumulating mainly xanthophyll-like carotenoids that produce orange-type colonies (<span class="elsevierStyleItalic">Cystofilobasidium</span> and <span class="elsevierStyleItalic">Dioszegia</span>) have higher probabilities of misidentification with astaxanthin-producing yeasts like <span class="elsevierStyleItalic">Phaffia</span>. Other sympatric species such as many <span class="elsevierStyleItalic">Rhodotorula</span> spp. or few <span class="elsevierStyleItalic">Cryptococcus</span> spp. can produce salmon-pink or intense red colonies depending on the relative level of accumulation of less polar carotenoids like torulene, torularhodine and/or β-carotene<a class="elsevierStyleCrossRef" href="#bib0255"><span class="elsevierStyleSup">18</span></a>.</p><p id="par0015" class="elsevierStylePara elsevierViewall">The objective of this work was to provide a rapid and accurate PCR-based detection method for astaxanthin-accumulating yeast isolates from environmental samples.</p></span><span id="sec0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0030">Materials and methods</span><span id="sec0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0035">Yeast strains</span><p id="par0020" class="elsevierStylePara elsevierViewall">Seven representative strains of the different <span class="elsevierStyleItalic">Phaffia</span> spp. clades described in David-Palma et al.<a class="elsevierStyleCrossRef" href="#bib0175"><span class="elsevierStyleSup">2</span></a> were used in this study (<a class="elsevierStyleCrossRef" href="#tbl0005">Table 1</a>). Strains ZP938 and ZP875 were kindly provided by Dr. J. Sampaio (UNL, Portugal). The <span class="elsevierStyleItalic">Phaffia</span>-related strain TSN-67 (astaxanthin and MGG non-producing, unpublished results) recently reported by Yurkov et al.<a class="elsevierStyleCrossRef" href="#bib0330"><span class="elsevierStyleSup">33</span></a> was kindly provided by the author (A. Yurkov) and was tested together with two species of the genus <span class="elsevierStyleItalic">Cystofilobasidium</span>. The latter species were included as controls due to their phylogenetic proximity to <span class="elsevierStyleItalic">P. rhodozyma</span> (also belonging to the order Cystofilobasidiales), habitat co-occurrence and similar appearance of their colonies.</p><elsevierMultimedia ident="tbl0005"></elsevierMultimedia></span><span id="sec0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0040">Primer design</span><p id="par0025" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">Phaffia</span> spp. specific primer was designed based on concatenated sequences of internal transcribed spacers (ITS) and D1D2 partial sequences of 20 <span class="elsevierStyleItalic">P. rhodozyma</span> strains (available at GenBank by May, 2011) and 5 <span class="elsevierStyleItalic">Cystofilobasidium</span> spp. Using the NCBI primer BLAST tool and then checked using Primer3. Both widely used ITS3 and NL4 and new <span class="elsevierStyleItalic">Phaffia</span> spp. specific primers are shown with full detail in <a class="elsevierStyleCrossRef" href="#tbl0010">Table 2</a>.</p><elsevierMultimedia ident="tbl0010"></elsevierMultimedia></span><span id="sec0025" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0045">Phylogenetic analysis</span><p id="par0030" class="elsevierStylePara elsevierViewall">rRNA gene sequences corresponding to the internal transcribed spacers 1 and 2 (ITS 1 and ITS 2), 5.8S rRNA of all the strains depicted in <a class="elsevierStyleCrossRef" href="#tbl0005">Table 1</a> were obtained from NCBI. DNA sequences alignment and analyses were performed using MEGA 5.0<a class="elsevierStyleCrossRef" href="#bib0285"><span class="elsevierStyleSup">24</span></a>. Alignments were carried out using CLUSTALW<a class="elsevierStyleCrossRef" href="#bib0290"><span class="elsevierStyleSup">25</span></a> and were edited manually when required. Phylogenetic analysis using Neighbor Joining were performed in MEGA 5.0, using the Kimura 2-parameter model as substitution model. The reliability of the NJ trees was assessed by bootstrap analysis including 1000 replications.</p></span><span id="sec0030" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0050">Cell and molecular methods</span><p id="par0035" class="elsevierStylePara elsevierViewall">Cell growth was performed on YMA agar plates (3<span class="elsevierStyleHsp" style=""></span>g/l yeast extract, 3<span class="elsevierStyleHsp" style=""></span>g/l malt extract, 5<span class="elsevierStyleHsp" style=""></span>g/l bactopeptone, 10<span class="elsevierStyleHsp" style=""></span>g/l glucose and 15<span class="elsevierStyleHsp" style=""></span>g/l agar), DNA was extracted using the protocol described previously in Libkind et al.<a class="elsevierStyleCrossRef" href="#bib0220"><span class="elsevierStyleSup">11</span></a> and purified using chloroform:isoamyl alcohol reagent (24:1, v/v). DNA was quantified using Shimadzu UV-1800 spectrophotometer and Multiplex PCR assays were performed in a Labnet MultiGene Gradient thermocycler, under the following conditions: denaturation at 95<span class="elsevierStyleHsp" style=""></span>°C for 5<span class="elsevierStyleHsp" style=""></span>min followed by 35 cycles of 95<span class="elsevierStyleHsp" style=""></span>°C 30<span class="elsevierStyleHsp" style=""></span>s, 55<span class="elsevierStyleHsp" style=""></span>°C 1<span class="elsevierStyleHsp" style=""></span>min, 72<span class="elsevierStyleHsp" style=""></span>°C 1<span class="elsevierStyleHsp" style=""></span>min, and a final extension at 72<span class="elsevierStyleHsp" style=""></span>°C for 7<span class="elsevierStyleHsp" style=""></span>min. PCR amplification was performed in a total volume of 25<span class="elsevierStyleHsp" style=""></span>μl, containing about 50<span class="elsevierStyleHsp" style=""></span>ng of total DNA, 1× Go<span class="elsevierStyleItalic">Taq</span> PCR buffer (Promega), 200<span class="elsevierStyleHsp" style=""></span>μM each dNTP (GE Healthcare), 1<span class="elsevierStyleHsp" style=""></span>U <span class="elsevierStyleItalic">Taq</span> polymerase (Go<span class="elsevierStyleItalic">Taq</span>, Promega) and 0.3<span class="elsevierStyleHsp" style=""></span>μM each primer. Gel electrophoresis was performed on 1<span class="elsevierStyleHsp" style=""></span>% agarose in 0.5× TBE, 90<span class="elsevierStyleHsp" style=""></span>V for 30<span class="elsevierStyleHsp" style=""></span>min.</p><p id="par0040" class="elsevierStylePara elsevierViewall">A 100<span class="elsevierStyleHsp" style=""></span>bp DNA size ladder (Highway) plus a lambda DNA digested with HindIII (Promega) were used as a molecular marker.</p></span></span><span id="sec0035" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0055">Results</span><p id="par0045" class="elsevierStylePara elsevierViewall">A single primer of 20 nucleotides called PhR (<a class="elsevierStyleCrossRef" href="#tbl0010">Table 2</a>) was designed to be used in combination with the universal primers ITS3<a class="elsevierStyleCrossRef" href="#bib0320"><span class="elsevierStyleSup">31</span></a> and NL4<a class="elsevierStyleCrossRef" href="#bib0215"><span class="elsevierStyleSup">10</span></a> to perform a multiplex amplification reaction that leads to the rapid detection of astaxanthin-producing yeasts of the genus <span class="elsevierStyleItalic">Phaffia</span>. The ability of this method to detect all proposed lineages of <span class="elsevierStyleItalic">Phaffia</span> sp. and at the same time retain the sufficient specificity to avoid false positives was evaluated. For this purpose, an up-to- date set of eight representative strains of all known <span class="elsevierStyleItalic">Phaffia</span> and <span class="elsevierStyleItalic">Phaffia</span>-related lineages were assayed, including the <span class="elsevierStyleItalic">Phaffia</span> sp. TSN-67 isolate reported by Yurkov et al.<a class="elsevierStyleCrossRef" href="#bib0330"><span class="elsevierStyleSup">33</span></a> from soil and representative strains of the seven different lineages recently described by David-Palma et al<a class="elsevierStyleCrossRef" href="#bib0175"><span class="elsevierStyleSup">2</span></a>. Additionally, <span class="elsevierStyleItalic">Cystofilobasidium capitatum</span> and <span class="elsevierStyleItalic">Cystofilobasidium macerans</span> were included due to their phylogenetic proximity and, since they inhabit similar substrates and grow in morphologically similar colonies, they can be frequently confused with <span class="elsevierStyleItalic">Phaffia</span><a class="elsevierStyleCrossRefs" href="#bib0185"><span class="elsevierStyleSup">4,5,22,27,28</span></a>.</p><p id="par0050" class="elsevierStylePara elsevierViewall">The reverse PhR primer was designed to anneal in the conserved D1D2 domains of the 26S rRNA gene and to generate a 643<span class="elsevierStyleHsp" style=""></span>bp amplicon when it is used together with the universal forward primer ITS3. The universal reverse primer NL4 was included for amplification control since it will produce 1155<span class="elsevierStyleHsp" style=""></span>bp in any eukaryotic microorganism. When tested in our set of astaxanthin-producing strains we observed the specific <span class="elsevierStyleItalic">Phaffia</span> band (∼640<span class="elsevierStyleHsp" style=""></span>bp) for all <span class="elsevierStyleItalic">P. rhodozyma</span> lineages and even for strains of group E and F, which represent novel species of the genus <span class="elsevierStyleItalic">Phaffia</span> (<a class="elsevierStyleCrossRef" href="#fig0005">Fig. 1</a>). In most of these cases, the control band (∼1150<span class="elsevierStyleHsp" style=""></span>bp) was also present though displaying a weak signal.</p><elsevierMultimedia ident="fig0005"></elsevierMultimedia><p id="par0055" class="elsevierStylePara elsevierViewall">The <span class="elsevierStyleItalic">Phaffia</span> sp. strain TSN-67<a class="elsevierStyleCrossRef" href="#bib0330"><span class="elsevierStyleSup">33</span></a> was negative for the specific primer. Two nucleotide differences were detected in the 3′ end of the specific primer for this strain explaining the lack of amplification (<a class="elsevierStyleCrossRef" href="#tbl0015">Table 3</a>), which is in accordance with its relative distant phylogenetic relationship with the <span class="elsevierStyleItalic">Phaffia</span> clade (<a class="elsevierStyleCrossRef" href="#fig0010">Fig. 2</a>). Based on our phylogenetic analysis, the strain belongs to the order Cystofilobasidiales (Tremellomycetes, Agaricomycotina), occupying a basal position and having <span class="elsevierStyleItalic">P. rhodozyma</span> as the closest match, but showing more than fifty substitutions in the ITS region.</p><elsevierMultimedia ident="tbl0015"></elsevierMultimedia><elsevierMultimedia ident="fig0010"></elsevierMultimedia><p id="par0060" class="elsevierStylePara elsevierViewall">Carotenogenic species of the genus <span class="elsevierStyleItalic">Cystofilobasidium</span> were negative for the test since the specific band was absent due to the presence of 3 or more nucleotide substitutions mostly in the 3′ end. An environmental strain of <span class="elsevierStyleItalic">Dioszegia</span> spp., which was also tested, was negative (data not shown). <span class="elsevierStyleItalic">In silico</span> analysis of the remaining 5 <span class="elsevierStyleItalic">Cystofilobasidium</span> species, all <span class="elsevierStyleItalic">Dioszegia</span> known species and representative species of pigmented yeasts of the genus <span class="elsevierStyleItalic">Rhodotorula</span>, <span class="elsevierStyleItalic">Rhodosporidium</span> and <span class="elsevierStyleItalic">Cystobasidium</span> (ex-<span class="elsevierStyleItalic">Rhodotorula</span>) showed that at least 3 nucleotide differences were present in all cases (<a class="elsevierStyleCrossRef" href="#tbl0015">Table 3</a>). <span class="elsevierStyleItalic">Cystofilobasidium</span> species were those showing the lower number of substitutions while <span class="elsevierStyleItalic">Dioszegia</span> species differed in 4 or 5 substitutions. With the exception of <span class="elsevierStyleItalic">Cystobasidium minutum</span> (Cystobasidiales) that had only 3 substitutions, the red yeasts of <span class="elsevierStyleItalic">Rhodotorula</span> and <span class="elsevierStyleItalic">Rhodosporidium</span> had 6 or more substitutions.</p></span><span id="sec0040" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0060">Discussion</span><p id="par0065" class="elsevierStylePara elsevierViewall">Based on our results, the specific primer PhR, coupled with the universal primers ITS3 and NL4, has the required sensitivity and specificity for proper detection of relevant astaxanthin-accumulating yeasts of the genus <span class="elsevierStyleItalic">Phaffia</span>, here represented by all seven lineages recently described by David-Palma et al<a class="elsevierStyleCrossRef" href="#bib0175"><span class="elsevierStyleSup">2</span></a>. Despite the wide genetic diversity of this group of yeasts, the test detected all desired strains, differentiating them from other co-occurring, also carotenogenic and closely related yeast species, such as members of the genus <span class="elsevierStyleItalic">Cystofilobasidium</span> (<a class="elsevierStyleCrossRef" href="#fig0005">Fig. 1</a>).</p><p id="par0070" class="elsevierStylePara elsevierViewall">The specificity of the method for astaxanthin producing strains is based on the complete homology of the PhR primer to the 5′ portion of the D1D2 domains of the large ribosomal subunit (26S), and the presence of 2 or more nucleotide differences in the 3′ end for distantly related strains (TSN-67) and <span class="elsevierStyleItalic">Cystofilobasidium</span> spp.</p><p id="par0075" class="elsevierStylePara elsevierViewall">In a previous report, a new and innovative strategy for improving <span class="elsevierStyleItalic">P. rhodozyma</span> recovery rate in environmental samples was obtained<a class="elsevierStyleCrossRef" href="#bib0295"><span class="elsevierStyleSup">26</span></a>, which was successfully employed in the work of David-Palma et al<a class="elsevierStyleCrossRef" href="#bib0175"><span class="elsevierStyleSup">2</span></a>. A rapid identification method based on the simultaneous presence of astaxanthin and mycosporines was also described as a helpful tool for the screening of <span class="elsevierStyleItalic">P. rhodozyma</span> in a large set of new isolates. Although useful, this biochemical test is not as precise and reliable as DNA-based methods, hence we developed here a new molecular strategy (multiplex PCR reaction) for the accurate and rapid detection of astaxanthin-producing yeasts among environmental isolates or even for a rapid identity check of laboratory or production strains. In this regard, several successful developments with similar strategies for the molecular identification of <span class="elsevierStyleItalic">Saccharomyces</span> spp. and <span class="elsevierStyleItalic">Zygosaccharomyces</span> spp. have been reported<a class="elsevierStyleCrossRefs" href="#bib0200"><span class="elsevierStyleSup">7,19,21,23</span></a>.</p><p id="par0080" class="elsevierStylePara elsevierViewall">The use of three primers in a multiplex reaction has the advantage of still obtaining an amplicon in a negative sample, therefore, the results are unambiguous and cannot be attributable to a failure in the amplification reaction. The cost, complexity and time required for the test is low enough to be performed in most microbiology labs in a routine manner and could be even expanded to perform a colony PCR assay. Our own experience shows rRNA gene amplification (such as the one implied here) of <span class="elsevierStyleItalic">Phaffia</span> strains and other basidiomycetous yeasts can be easily achieved using the direct colony PCR method described by Espinar et al.<a class="elsevierStyleCrossRef" href="#bib0180"><span class="elsevierStyleSup">3</span></a>.</p><p id="par0085" class="elsevierStylePara elsevierViewall">Ecological and biodiversity studies in new environments have led to the description of highly divergent astaxanthin-producing yeasts that were hitherto unknown. The biotechnological relevance of these new genetic lineages remains to be studied; however, the fact that other genetically different strains of <span class="elsevierStyleItalic">Phaffia</span> might be out there has been proved. Contreras et al.<a class="elsevierStyleCrossRef" href="#bib0170"><span class="elsevierStyleSup">1</span></a> recently obtained novel strains of <span class="elsevierStyleItalic">P. rhodozyma</span> from Antarctic environments, one of which showed improved astaxanthin production with respect to the average yields of wild strains. The methods described earlier<a class="elsevierStyleCrossRefs" href="#bib0230"><span class="elsevierStyleSup">13,26</span></a> in combination with the molecular method presented here should represent excellent tools for the successful hunting of more astaxanthin-accumulating yeasts in the wild.</p></span><span id="sec0045" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0065">Ethical disclosures</span><span id="sec0050" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0070">Protection of human and animal subjects</span><p id="par0090" class="elsevierStylePara elsevierViewall">The authors declare that no experiments were performed on humans or animals for this investigation.</p></span><span id="sec0055" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0075">Confidentiality of data</span><p id="par0095" class="elsevierStylePara elsevierViewall">The authors declare that no patient data appears in this article.</p></span><span id="sec0060" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0080">Right to privacy and informed consent</span><p id="par0100" class="elsevierStylePara elsevierViewall">The authors declare that no patient data appears in this article.</p></span></span><span id="sec0065" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0085">Conflict of interest</span><p id="par0105" class="elsevierStylePara elsevierViewall">The authors declare that they have no conflicts of interest.</p></span></span>" "textoCompletoSecciones" => array:1 [ "secciones" => array:12 [ 0 => array:3 [ "identificador" => "xres620785" "titulo" => "Abstract" "secciones" => array:1 [ 0 => array:1 [ "identificador" => "abst0005" ] ] ] 1 => array:2 [ "identificador" => "xpalclavsec635064" "titulo" => "Keywords" ] 2 => array:3 [ "identificador" => "xres620784" "titulo" => "Resumen" "secciones" => array:1 [ 0 => array:1 [ "identificador" => "abst0010" ] ] ] 3 => array:2 [ "identificador" => "xpalclavsec635065" "titulo" => "Palabras clave" ] 4 => array:2 [ "identificador" => "sec0005" "titulo" => "Introduction" ] 5 => array:3 [ "identificador" => "sec0010" "titulo" => "Materials and methods" "secciones" => array:4 [ 0 => array:2 [ "identificador" => "sec0015" "titulo" => "Yeast strains" ] 1 => array:2 [ "identificador" => "sec0020" "titulo" => "Primer design" ] 2 => array:2 [ "identificador" => "sec0025" "titulo" => "Phylogenetic analysis" ] 3 => array:2 [ "identificador" => "sec0030" "titulo" => "Cell and molecular methods" ] ] ] 6 => array:2 [ "identificador" => "sec0035" "titulo" => "Results" ] 7 => array:2 [ "identificador" => "sec0040" "titulo" => "Discussion" ] 8 => array:3 [ "identificador" => "sec0045" "titulo" => "Ethical disclosures" "secciones" => array:3 [ 0 => array:2 [ "identificador" => "sec0050" "titulo" => "Protection of human and animal subjects" ] 1 => array:2 [ "identificador" => "sec0055" "titulo" => "Confidentiality of data" ] 2 => array:2 [ "identificador" => "sec0060" "titulo" => "Right to privacy and informed consent" ] ] ] 9 => array:2 [ "identificador" => "sec0065" "titulo" => "Conflict of interest" ] 10 => array:2 [ "identificador" => "xack209299" "titulo" => "Acknowledgements" ] 11 => array:1 [ "titulo" => "References" ] ] ] "pdfFichero" => "main.pdf" "tienePdf" => true "fechaRecibido" => "2015-03-14" "fechaAceptado" => "2015-10-29" "PalabrasClave" => array:2 [ "en" => array:1 [ 0 => array:4 [ "clase" => "keyword" "titulo" => "Keywords" "identificador" => "xpalclavsec635064" "palabras" => array:4 [ 0 => "Astaxanthin" 1 => "Mycosporines" 2 => "<span class="elsevierStyleItalic">Xanthophyllomyces</span>" 3 => "Yeasts" ] ] ] "es" => array:1 [ 0 => array:4 [ "clase" => "keyword" "titulo" => "Palabras clave" "identificador" => "xpalclavsec635065" "palabras" => array:4 [ 0 => "Astaxantina" 1 => "Micosporina" 2 => "<span class="elsevierStyleItalic">Xanthophyllomyces</span>" 3 => "Levaduras" ] ] ] ] "tieneResumen" => true "resumen" => array:2 [ "en" => array:2 [ "titulo" => "Abstract" "resumen" => "<span id="abst0005" class="elsevierStyleSection elsevierViewall"><p id="spar0005" class="elsevierStyleSimplePara elsevierViewall">It has been recently found that the natural distribution, habitat, and genetic diversity of astaxanthin-producing yeasts (<span class="elsevierStyleItalic">i.e. Phaffia rhodozyma</span>, synonym <span class="elsevierStyleItalic">Xanthophyllomyces dendrorhous</span>) is much greater than previously thought. <span class="elsevierStyleItalic">P. rhodozyma</span> is biotechnologically exploited due to its ability to produce the carotenoid pigment astaxanthin and thus, it is used as a natural source of this pigment for aquaculture. <span class="elsevierStyleItalic">P. rhodozyma</span> was also capable of synthesizing the potent UVB sunscreen mycosporine-glutaminol-glucoside (MGG). Therefore, further environmental studies are needed to elucidate its ecological aspects and detect new potential strains for the production of astaxanthin and MGG. However, obtaining new isolates of <span class="elsevierStyleItalic">P. rhodozyma</span> and related species is not always easy due to its low abundance and the presence of other sympatric and pigmented yeasts. In this work we report a successful development of a species-specific primer which has the ability to quickly and accurately detecting isolates representing all known lineages of the genus <span class="elsevierStyleItalic">Phaffia</span> (including novel species of the genus) and excluding closely related taxa. For this purpose, a primer of 20 nucleotides (called PhR) was designed to be used in combination with universal primers ITS3 and NL4 in a multiplex amplification. The proposed method has the sensitivity and specificity required for the precise detection of new isolates, and therefore represents an important tool for the environmental search for novel astaxanthin-producing yeasts.</p></span>" ] "es" => array:2 [ "titulo" => "Resumen" "resumen" => "<span id="abst0010" class="elsevierStyleSection elsevierViewall"><p id="spar0010" class="elsevierStyleSimplePara elsevierViewall">Recientemente, se ha encontrado que la distribución natural, el hábitat y la diversidad genética de levaduras productoras de astaxantina (p. ej., <span class="elsevierStyleItalic">Phaffia rhodozyma</span>, sinónimo <span class="elsevierStyleItalic">Xanthophyllomyces dendrorhous</span>) son mucho mayores de lo que se pensaba. <span class="elsevierStyleItalic">P. rhodozyma</span> se explota biotecnológicamente debido a su capacidad para producir el pigmento carotenoide astaxantina y, por lo tanto, se utiliza como una fuente natural de este pigmento para la acuicultura. También se encontró que esta levadura es capaz de sintetizar el potente protector solar UVB micosporina-glutaminol-glucósido (MGG). Por lo tanto, más estudios ambientales para dilucidar sus aspectos ecológicos y detectar nuevas cepas potenciales productoras de astaxantina y MGG son necesarios. Sin embargo, la obtención de nuevos aislamientos de <span class="elsevierStyleItalic">P. rhodozyma</span> y especies relacionadas no siempre es fácil debido a su baja abundancia y a la presencia de otras levaduras simpátricas y pigmentadas. En este trabajo se describe el desarrollo exitoso de un cebador especie-específico que tiene la capacidad de detectar rápidamente y con precisión cepas representativas de todos los linajes del género <span class="elsevierStyleItalic">Phaffia</span> previamente reportados (incluyendo nuevas especies del género) y excluir especies estrechamente relacionadas. Para ello, se diseñó un cebador de 20 nucleótidos (denominado PhR) para ser utilizado en combinación con los cebadores universales ITS3 y NL4 en una amplificación multiplex. El método propuesto tiene la sensibilidad y la especificidad requerida para la detección precisa de nuevos aislamientos y, por lo tanto, representa una importante herramienta para la búsqueda ambiental de nuevas levaduras productoras de astaxantina.</p></span>" ] ] "multimedia" => array:5 [ 0 => array:7 [ "identificador" => "fig0005" "etiqueta" => "Figure 1" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr1.jpeg" "Alto" => 748 "Ancho" => 1314 "Tamanyo" => 56132 ] ] "descripcion" => array:1 [ "en" => "<p id="spar0015" class="elsevierStyleSimplePara elsevierViewall">Yeast strains tested with multiplex PhR, ITS3 and NL4. 1–7 <span class="elsevierStyleItalic">P. rhodozyma</span>: (1) CBS 5905 (Lineage C1/B); (2) CBS 7918<span class="elsevierStyleSup">T</span> (Lineage C2); (3) CRUB 1149 (Lineage A); (4) ATCC 24229 (Lineage D); (5) ZP 938 (Lineage E); (6) ZP 875 (Lineage F); (7) ZP 874 (Lineage B); (8) TSN-67; (9) <span class="elsevierStyleItalic">C. capitatum</span> CBS 7420; (10) <span class="elsevierStyleItalic">C. macerans</span> CBS 2206; N – negative control (no template).</p>" ] ] 1 => array:7 [ "identificador" => "fig0010" "etiqueta" => "Figure 2" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr2.jpeg" "Alto" => 1652 "Ancho" => 1603 "Tamanyo" => 135687 ] ] "descripcion" => array:1 [ "en" => "<p id="spar0020" class="elsevierStyleSimplePara elsevierViewall">Neighbor joining phylogenetic tree of <span class="elsevierStyleItalic">Phaffia</span>, based on internal transcribed spacer sequences. Bootstrap values (1000 replicates) and lineages are indicated. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The percentage of trees in which the associated taxa clustered together is shown next to the branches. The outgroup is constituted by <span class="elsevierStyleItalic">Cystofilobasidium capitatum</span> (CBS 7420) and <span class="elsevierStyleItalic">Cystofilobasidium macerans</span> (CBS 2206).</p>" ] ] 2 => array:7 [ "identificador" => "tbl0005" "etiqueta" => "Table 1" "tipo" => "MULTIMEDIATABLA" "mostrarFloat" => true "mostrarDisplay" => false "tabla" => array:3 [ "leyenda" => "<p id="spar0030" class="elsevierStyleSimplePara elsevierViewall">C., <span class="elsevierStyleItalic">Cyttaria</span>; N., <span class="elsevierStyleItalic">Nothofagus</span>; n.a., Not applicable.</p>" "tablatextoimagen" => array:1 [ 0 => array:2 [ "tabla" => array:1 [ 0 => """ <table border="0" frame="\n \t\t\t\t\tvoid\n \t\t\t\t" class=""><thead title="thead"><tr title="table-row"><th class="td" title="table-head " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">Species \t\t\t\t\t\t\n \t\t\t\t</th><th class="td" title="table-head " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">Strain \t\t\t\t\t\t\n \t\t\t\t</th><th class="td" title="table-head " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">Lineage<a class="elsevierStyleCrossRef" href="#tblfn0005"><span class="elsevierStyleSup">a</span></a> \t\t\t\t\t\t\n \t\t\t\t</th><th class="td" title="table-head " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">Origin \t\t\t\t\t\t\n \t\t\t\t</th><th class="td" title="table-head " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">Substrate \t\t\t\t\t\t\n \t\t\t\t</th><th class="td" title="table-head " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">Reference \t\t\t\t\t\t\n \t\t\t\t</th></tr></thead><tbody title="tbody"><tr title="table-row"><td class="td" title="table-entry " rowspan="5" align="left" valign="middle"><span class="elsevierStyleItalic">Phaffia rhodozyma</span><br><span class="elsevierStyleItalic">(Xanthophyllomyces dendrorhous)</span></td><td class="td" title="table-entry " align="left" valign="top">CRUB 1149 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">A \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">Bariloche, Argentina \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">Water sample near <span class="elsevierStyleItalic">C. hariotii</span> on <span class="elsevierStyleItalic">N. pumilio</span> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top"><a class="elsevierStyleCrossRef" href="#bib0240">15</a> \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">ZP 874 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">B \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">Tasmania, Australia \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top"><span class="elsevierStyleItalic">C. gunnii</span> on <span class="elsevierStyleItalic">N. cunninghamii</span> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top"><a class="elsevierStyleCrossRef" href="#bib0175">2</a> \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">CBS 5905 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">C1 and B (hybrid strain) \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">Kyoto, Japan \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">Exudate of <span class="elsevierStyleItalic">Fagus crenata</span> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">Type strain of <span class="elsevierStyleItalic">P. rhodozyma</span><a class="elsevierStyleCrossRef" href="#bib0250"><span class="elsevierStyleSup">17</span></a> \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">CBS 7918<span class="elsevierStyleSup">T</span> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">C2 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">Moscow, Russia \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">Exudate of <span class="elsevierStyleItalic">Betula verrucosa</span> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">Type strain of <span class="elsevierStyleItalic">X. dendrorhous</span><a class="elsevierStyleCrossRef" href="#bib0195"><span class="elsevierStyleSup">6</span></a> \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">ATCC 24229 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">D \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">Hiroshima, Japan \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">Exudate of <span class="elsevierStyleItalic">Cornus brachypoda</span> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top"><a class="elsevierStyleCrossRef" href="#bib0250">17</a> \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top"><span class="elsevierStyleItalic">Phaffia</span> sp. I \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">ZP 938 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">E \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">Queensland, Australia \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">Leaves of <span class="elsevierStyleItalic">N. mooreii</span> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top"><a class="elsevierStyleCrossRef" href="#bib0175">2</a> \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top"><span class="elsevierStyleItalic">Phaffia</span> sp. II \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">ZP 875 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">F \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">Tasmania, Australia \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top"><span class="elsevierStyleItalic">C. gunnii</span> on <span class="elsevierStyleItalic">N. cunninghamii</span> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top"><a class="elsevierStyleCrossRef" href="#bib0175">2</a> \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top"><span class="elsevierStyleItalic">Phaffia</span> sp. \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">TSN-67 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">n.a. \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">Baden-Württemberg, Germany \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">Soil adjacent to <span class="elsevierStyleItalic">Picea abies</span> and <span class="elsevierStyleItalic">Fagus sylvatica</span> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top"><a class="elsevierStyleCrossRef" href="#bib0330">33</a> \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top"><span class="elsevierStyleItalic">Cystofilobasidium capitatum</span> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">CBS 7420 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">n.a. \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">France \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top"><span class="elsevierStyleItalic">Larus marinus</span> (great black-backed gull) \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top"><a class="elsevierStyleCrossRef" href="#bib0265">20</a> \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top"><span class="elsevierStyleItalic">Cystofilobasidium macerans</span> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">CBS 2206 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">n.a. \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">Unknown \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">Dew-retted flax straw \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top"><a class="elsevierStyleCrossRef" href="#bib0225">12</a> \t\t\t\t\t\t\n \t\t\t\t</td></tr></tbody></table> """ ] "imagenFichero" => array:1 [ 0 => "xTab1017854.png" ] ] ] "notaPie" => array:1 [ 0 => array:3 [ "identificador" => "tblfn0005" "etiqueta" => "a" "nota" => "<p class="elsevierStyleNotepara" id="npar0005">Following David-Palma et al.<a class="elsevierStyleCrossRef" href="#bib0175"><span class="elsevierStyleSup">2</span></a>.</p>" ] ] ] "descripcion" => array:1 [ "en" => "<p id="spar0025" class="elsevierStyleSimplePara elsevierViewall">List of yeast strains tested with <span class="elsevierStyleItalic">P. rhodozyma</span> specific primers</p>" ] ] 3 => array:7 [ "identificador" => "tbl0010" "etiqueta" => "Table 2" "tipo" => "MULTIMEDIATABLA" "mostrarFloat" => true "mostrarDisplay" => false "tabla" => array:1 [ "tablatextoimagen" => array:1 [ 0 => array:2 [ "tabla" => array:1 [ 0 => """ <table border="0" frame="\n \t\t\t\t\tvoid\n \t\t\t\t" class=""><thead title="thead"><tr title="table-row"><th class="td" title="table-head " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">Primer \t\t\t\t\t\t\n \t\t\t\t</th><th class="td" title="table-head " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">Sequence \t\t\t\t\t\t\n \t\t\t\t</th><th class="td" title="table-head " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">Reference \t\t\t\t\t\t\n \t\t\t\t</th></tr></thead><tbody title="tbody"><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">ITS3 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">5′-GCA TCG ATG AAG AAC GCA GC-3′ \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">White et al.<a class="elsevierStyleCrossRef" href="#bib0320"><span class="elsevierStyleSup">31</span></a> \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">PhR \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">5′-GAC TTG TAC ACA GGC CGG CA-3′ \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">This work \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">NL4 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">5′-GGT CCG TGT TTC AAG ACG G-3′ \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">Kurtzman and Robnett<a class="elsevierStyleCrossRef" href="#bib0215"><span class="elsevierStyleSup">10</span></a> \t\t\t\t\t\t\n \t\t\t\t</td></tr></tbody></table> """ ] "imagenFichero" => array:1 [ 0 => "xTab1017853.png" ] ] ] ] "descripcion" => array:1 [ "en" => "<p id="spar0035" class="elsevierStyleSimplePara elsevierViewall">Set of primers used in this work</p>" ] ] 4 => array:7 [ "identificador" => "tbl0015" "etiqueta" => "Table 3" "tipo" => "MULTIMEDIATABLA" "mostrarFloat" => true "mostrarDisplay" => false "tabla" => array:2 [ "tablatextoimagen" => array:1 [ 0 => array:2 [ "tabla" => array:1 [ 0 => """ <table border="0" frame="\n \t\t\t\t\tvoid\n \t\t\t\t" class=""><thead title="thead"><tr title="table-row"><th class="td" title="table-head " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">Specie \t\t\t\t\t\t\n \t\t\t\t</th><th class="td" title="table-head " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">Sequence \t\t\t\t\t\t\n \t\t\t\t</th><th class="td" title="table-head " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">Number of mismatches \t\t\t\t\t\t\n \t\t\t\t</th></tr></thead><tbody title="tbody"><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top"><span class="elsevierStyleItalic">P. rhodozyma</span> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">5′-GAC TTG TAC ACA GGC CGG CA-3′ \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">0 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">TSN-67 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">5′-GAC TTG TAC ACA GGC CG<span class="elsevierStyleBold"><span class="elsevierStyleUnderline">A</span></span> C<span class="elsevierStyleBold"><span class="elsevierStyleUnderline">G</span></span>-3′ \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">2 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top"><span class="elsevierStyleItalic">Cystofilobasidium</span> spp.<a class="elsevierStyleCrossRef" href="#tblfn0010"><span class="elsevierStyleSup">a</span></a> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">5′-GAC TTG TAC ACA GGC C<span class="elsevierStyleBold"><span class="elsevierStyleUnderline">AA</span></span> C<span class="elsevierStyleBold"><span class="elsevierStyleUnderline">G</span></span>-3′ \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">3 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top"><span class="elsevierStyleItalic">Cystobasidium minutum</span><a class="elsevierStyleCrossRef" href="#tblfn0015"><span class="elsevierStyleSup">b</span></a> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">5′-GAC T<span class="elsevierStyleBold"><span class="elsevierStyleUnderline">CA</span></span> TAC AC<span class="elsevierStyleBold"><span class="elsevierStyleUnderline">G</span></span> GGC CGG CA-3′ \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">3 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top"><span class="elsevierStyleItalic">Dioszegia</span> spp.<a class="elsevierStyleCrossRef" href="#tblfn0020"><span class="elsevierStyleSup">c</span></a> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">5′-GAC TT<span class="elsevierStyleBold"><span class="elsevierStyleUnderline">A</span><span class="elsevierStyleUnderline">G</span></span>AC AC<span class="elsevierStyleBold"><span class="elsevierStyleUnderline">G</span></span> G<span class="elsevierStyleBold"><span class="elsevierStyleUnderline">T</span></span>C CGG CA-3′ \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">4 \t\t\t\t\t\t\n \t\t\t\t</td></tr></tbody></table> """ ] "imagenFichero" => array:1 [ 0 => "xTab1017852.png" ] ] ] "notaPie" => array:3 [ 0 => array:3 [ "identificador" => "tblfn0010" "etiqueta" => "a" "nota" => "<p class="elsevierStyleNotepara" id="npar0010">All species of <span class="elsevierStyleItalic">Cystofilobasidium</span> have at least 3 substitutions.</p>" ] 1 => array:3 [ "identificador" => "tblfn0015" "etiqueta" => "b" "nota" => "<p class="elsevierStyleNotepara" id="npar0015">ex <span class="elsevierStyleItalic">Rhodotorula minuta</span><a class="elsevierStyleCrossRef" href="#bib0325"><span class="elsevierStyleSup">32</span></a>.</p>" ] 2 => array:3 [ "identificador" => "tblfn0020" "etiqueta" => "c" "nota" => "<p class="elsevierStyleNotepara" id="npar0020">All known species of <span class="elsevierStyleItalic">Dioszegia</span> have at least 4 substitutions (tested <span class="elsevierStyleItalic">in silico</span>).</p>" ] ] ] "descripcion" => array:1 [ "en" => "<p id="spar0040" class="elsevierStyleSimplePara elsevierViewall">PhR primer specificity based on the nucleotide sequence of the annealing region</p>" ] ] ] "bibliografia" => array:2 [ "titulo" => "References" "seccion" => array:1 [ 0 => array:2 [ "identificador" => "bibs0005" "bibliografiaReferencia" => array:33 [ 0 => array:3 [ "identificador" => "bib0170" "etiqueta" => "1" "referencia" => array:1 [ 0 => array:2 [ "contribucion" => array:1 [ 0 => array:2 [ "titulo" => "Identification and analysis of metabolite production with biotechnological potential in <span class="elsevierStyleItalic">Xanthophyllomyces dendrorhous</span> isolates" "autores" => array:1 [ 0 => array:2 [ "etal" => false "autores" => array:6 [ 0 => "G. 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Sampaio for providing valuable strains and information for this study.</p>" "vista" => "all" ] ] ] "idiomaDefecto" => "en" "url" => "/03257541/0000004800000001/v1_201603250025/S0325754115001601/v1_201603250025/en/main.assets" "Apartado" => array:4 [ "identificador" => "37863" "tipo" => "SECCION" "en" => array:2 [ "titulo" => "Microbiología básica" "idiomaDefecto" => true ] "idiomaDefecto" => "en" ] "PDF" => "https://static.elsevier.es/multimedia/03257541/0000004800000001/v1_201603250025/S0325754115001601/v1_201603250025/en/main.pdf?idApp=UINPBA00004N&text.app=https://www.elsevier.es/" "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S0325754115001601?idApp=UINPBA00004N" ]
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2024 June | 14 | 3 | 17 |
2024 May | 20 | 2 | 22 |
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2022 October | 12 | 10 | 22 |
2022 September | 24 | 20 | 44 |
2022 August | 18 | 20 | 38 |
2022 July | 21 | 8 | 29 |
2022 June | 33 | 15 | 48 |
2022 May | 42 | 8 | 50 |
2022 April | 57 | 11 | 68 |
2022 March | 99 | 17 | 116 |
2022 February | 105 | 16 | 121 |
2022 January | 79 | 18 | 97 |
2021 December | 64 | 16 | 80 |
2021 November | 52 | 14 | 66 |
2021 October | 15 | 12 | 27 |
2021 September | 41 | 21 | 62 |
2021 August | 25 | 6 | 31 |
2021 July | 26 | 9 | 35 |
2021 June | 18 | 9 | 27 |
2021 May | 22 | 13 | 35 |
2021 April | 45 | 24 | 69 |
2021 March | 16 | 6 | 22 |
2021 February | 13 | 11 | 24 |
2021 January | 17 | 12 | 29 |
2020 December | 13 | 16 | 29 |
2020 November | 11 | 12 | 23 |
2020 October | 9 | 8 | 17 |
2020 September | 14 | 12 | 26 |
2020 August | 16 | 9 | 25 |
2020 July | 24 | 12 | 36 |
2020 June | 25 | 3 | 28 |
2020 May | 26 | 14 | 40 |
2020 April | 18 | 4 | 22 |
2020 March | 24 | 14 | 38 |
2020 February | 16 | 9 | 25 |
2020 January | 11 | 6 | 17 |
2019 December | 16 | 17 | 33 |
2019 November | 11 | 7 | 18 |
2019 October | 10 | 2 | 12 |
2019 September | 13 | 12 | 25 |
2019 August | 8 | 11 | 19 |
2019 July | 19 | 27 | 46 |
2019 June | 48 | 28 | 76 |
2019 May | 87 | 20 | 107 |
2019 April | 67 | 8 | 75 |
2019 March | 9 | 5 | 14 |
2019 February | 13 | 7 | 20 |
2019 January | 18 | 7 | 25 |
2018 December | 20 | 16 | 36 |
2018 November | 28 | 5 | 33 |
2018 October | 9 | 8 | 17 |
2018 September | 45 | 7 | 52 |
2018 August | 28 | 8 | 36 |
2018 July | 6 | 7 | 13 |
2018 June | 6 | 2 | 8 |
2018 May | 5 | 6 | 11 |
2018 April | 7 | 0 | 7 |
2018 March | 3 | 0 | 3 |
2018 February | 8 | 1 | 9 |
2018 January | 6 | 3 | 9 |
2017 December | 14 | 2 | 16 |
2017 November | 10 | 6 | 16 |
2017 October | 11 | 11 | 22 |
2017 September | 10 | 7 | 17 |
2017 August | 14 | 2 | 16 |
2017 July | 8 | 8 | 16 |
2017 June | 38 | 12 | 50 |
2017 May | 16 | 25 | 41 |
2017 April | 9 | 36 | 45 |
2017 March | 17 | 14 | 31 |
2017 February | 18 | 5 | 23 |
2017 January | 11 | 10 | 21 |
2016 December | 24 | 9 | 33 |
2016 November | 30 | 8 | 38 |
2016 October | 44 | 12 | 56 |
2016 September | 45 | 13 | 58 |
2016 August | 45 | 3 | 48 |
2016 July | 41 | 4 | 45 |
2016 June | 65 | 39 | 104 |
2016 May | 43 | 26 | 69 |
2016 April | 35 | 14 | 49 |
2016 March | 6 | 1 | 7 |