1 2 3 4 5 6 7 8 9 10 11 121314 15 16 17 18 19 20 21 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 International Journal of Food Microbiology xxx (2010) xxx–xxx FOOD-05174; No of Pages 8 Contents lists available at ScienceDirect International Journal of Food Microbiology j ourna l homepage: www.e lsev ie r.com/ locate / i j foodmicro Strain typing of Zygosaccharomyces yeast species using a single molecular method based on polymorphism of the intergenic spacer region (IGS) Petra Wrent, Eva-María Rivas, José M. Peinado, María-Isabel de Silóniz ⁎ Departamento de Microbiología. Facultad de Biología. Universidad Complutense de Madrid. C/José Antonio Novais, 2. 28040, Madrid, Spain ⁎ Corresponding author. Tel.: +34 91 3944965; fax: E-mail address: siloniz@bio.ucm.es (M.-I. de Silóniz) 0168-1605/$ – see front matter © 2010 Elsevier B.V. Al doi:10.1016/j.ijfoodmicro.2010.06.007 Please cite this article as: Wrent, P., et al. polymorphism of the intergenic spacer reg a b s t r a c t a r t i c l e i n f o 22 23 24 25 26 27 28 29 30 31 Article history: Received 17 December 2009 Received in revised form 27 May 2010 Accepted 12 June 2010 Available online xxxx Keywords: Yeast typing Zygosaccharomyces rouxii Z. mellis Z. bailii PCR-RFLP IGS rDNA Food spoilage yeast Unlike previously reported methods that need a combination of several typing techniques, we have developed a single method for strain typing of the Zygosaccharomyces bailii, Z. mellis and Z. rouxii spoilage species. Strains belonging to other species have also been included for comparison. We have demonstrated that the IGS-PCR RFLP method has a high discriminative power. Considering the three endonucleases used in this work, we have obtained a variability of 100% for Z. mellis and Z. rouxii strains and up to 70% for Z.bailii. We have also detected two misidentified Z. mellis strains (CBS 711 and CBS 7412) which have RFLP patterns with a set of bands characteristic of Z. rouxii strains. Sequencing of 26S rDNA D1/D2 domains and the 5.8-ITS rDNA region confirmed these strains as Z. rouxii. The method also groups three certified hybrid strains of Zygosaccharomyces in a separate cluster. +34 91 3944964. . l rights reserved. , Strain typing of Zygosaccharomyces yeast s ion (IGS), Int. J. Food Microbiol. (2010), doi:1 © 2010 Elsevier B.V. All rights reserved. 3233 59 60 61 62 63 64 65 66 67 Q1 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 1. Introduction It has been shown that yeasts are involved in the spoilage of an extensive range of foods according to their metabolic and physiolog- ical capabilities (Stratford, 2006). However, in order to establish contamination sources during food processing and thus avoid economic losses, it is essential not only to identify which species are present but also to discriminate them at strain level. In terms of spoilage ability some of the most dangerous yeasts are found in the Zygosaccharomyces genus. This genus includes osmoto- lerant, strongly fermentative yeasts that are able to resist weak-acid preservatives such as benzoic and sorbic acids (Casas et al., 2004). These physiological characteristics are responsible for their well- known ability to cause spoilage (Stratford, 2006). Among these yeasts are Z. mellis, isolated from honey, syrups and from low aw products in general (Stratford, 2006), and the Z. rouxii and Z. bailii species, commonly found in the food and drinks industries. Although the Z. rouxii species is involved in production of food such as miso and traditional balsamic vinegar (Solieri and Giudici, 2008), it is also frequently isolated from low aw spoiled foods like marzipan or nougat (Casas et al., 2004; Martorell et al., 2005). According to the zymological indicators defined by Sancho et al. (2000), they are considered to be some of the most dangerous yeasts for product stability in fruit pulps and concentrates. 83 84 85 86 87 In order for some of the Zygosaccharomyces species to be strain- typed, several molecular techniques have been studied. Törok et al. (1993) proposed an electrophoretic karyotyping, and Esteve-Zarzoso et al. (2003) the RFLP of mtDNA. Martorell et al. (2005) demonstrated that if the objective is to differentiate species belonging to the same genus, the best result is obtained by electrophoretic analysis. If, on the other hand, it is to characterize Z. bailli and Z. rouxii at strain level, they suggested the combination of RFLP and RAPD analysis. A combination of several typing techniqueswas therefore required (Maqueda et al., 2010). The intergenic region (IGS) of rDNA has the advantage that its locus is more variable than other existing loci investigated so far (Sugita et al., 2001). Several studies have exploited this region. Sequence analysis of the IGS region permitted the separation of clinical isolates of Cryptococcus neoformans into two varieties (Diaz et al., 2005; Diaz and Fell, 2000; Fan et al., 1995). Strain typing of Pichia anomala has also been achieved by analysing the sequence of the intergenic region 1(IGS1) (Bhardway et al., 2007). Other studies have focused on discriminating strains belonging to different yeast species, such as Phaffia rhodozyma and Xanthophyllomyces dendrorhous (Fell and Blatt, 1999). The Restriction Fragment Length Polymorphism (RFLP) of the IGS2 region of rDNA made it possible to differentiate between the physiologically similar dairy yeast species Kluyveromyces marxianus and K. lactis (Naumova et al., 2005). We were previously able to differentiate the Debaryomyces hansenii yeast species in foods through PCR-RFLP of the IGS region (rDNA) (Romero et al., 2005). This also allowed us to separate the Debaryomyces genus into species and varieties (Quirós et al., 2006). The aim of this investigation is to evaluate the usefulness of PCR- RFLP analysis of the IGS region of rDNA as a single typing method for pecies using a single molecular method based on 0.1016/j.ijfoodmicro.2010.06.007 http://dx.doi.org/10.1016/j.ijfoodmicro.2010.06.007 mailto:siloniz@bio.ucm.es http://dx.doi.org/10.1016/j.ijfoodmicro.2010.06.007 http://www.sciencedirect.com/science/journal/01681605 http://dx.doi.org/10.1016/j.ijfoodmicro.2010.06.007 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 Table 1t1:1 Strains studied, their origin, source of isolation, size of the amplified IGS region and code of the RFLP pattern obtained with HapII, HhaI and MboI endonucleases. t1:2 t1:3 Species and strains studied Source of isolation IGS (bp) Patterns t1:4 Zygosaccharomyces bailii (B) t1:5 CECT 1898T Apple juice 6300 BA1 BB1 BC1 t1:6 CYC 1226 Culture contaminant 6300 BA1 BB2 BC2 t1:7 CECT 1924 Unknown 6300 BA1 BB1 BC1 t1:8 CECT 11042 Grape 6300 BA2 BB3 BC5 t1:9 CECT 11043 Turbid wine 6300 BA3 BB4 BC3 t1:10 MAY(12) Mayonnaise 6300 BA4 BB5 BC4 t1:11 MAY(13) Mayonnaise 6300 BA4 BB5 BC4 t1:12 t1:13 Zygosaccharomyces bisporus (Bi) t1:14 CECT 11055T Beer 5500 BiA1 BiB1 BiC1 t1:15 CECT 11348 Beer 5500 BiA2 BiB2 BiC2 t1:16 t1:17 Zygosaccharomyces cidrii (C)a t1:18 CECT 10657T Cider 2900 CA1 CB1 CC1 t1:19 CECT 11349 Cider 2900 CA1 CB1 CC1 t1:20 t1:21 Zygosaccharomyces fermentati (F)a t1:22 CECT 11056T Sediment of pepermint 2900 FA1 FB1 FC1 t1:23 CECT 10382 Alpechin 2900 FA3 FB2 FC2 t1:24 CECT 10678 Drosophila sp. 2900 FA2 FB2 FC2 t1:25 t1:26 Zygosaccharomyces kombuchaensis (K) t1:27 CBS 8849T Kombucha tea 7000 KA1 KB1 KC1 t1:28 t1:29 Zygosaccharomyces lentus (L) t1:30 CECT 11040 Swiss wine yeast 6700 LA1 LB1 LC1 t1:31 CECT 11041 Wine 6700 LA2 LB1 LC1 t1:32 t1:33 Zygosaccharomyces mellis (M) t1:34 CECT 11057 T Honey 4200 MA4 MB3 MC3 t1:35 CBS 684 Honey 4200 MA1 MB1 MC1 t1:36 CBS 711b Strawberry juice 4200 RA19a RB12 RC24 t1:37 CBS 735 Fermenting honey 4200 MA5 MB5 MC1 t1:38 CBS 738 Fermenting honey 4200 MA1 MB4 MC1 t1:39 CBS 1091 Honey 4200 MA2 MB2 MC2 t1:40 CBS7277 Alpechin 4200 MA3 MB3 MC3 t1:41 CBS 7412b Honey 4200 RA20a RB13 RC25 t1:42 t1:43 Zygosaccharomyces microellipsiodes (Mi)a t1:44 CBS 427 T Apple juice 3000 MiA1 MiB1 MiC1 t1:45 t1:46 Zygosaccharomyces rouxii (R) t1:47 CECT 1232 T Grape juice 4200 RA1 RB1 RC1 t1:48 CECT 1231 Bombon 4200 RA9 RB1 RC8 t1:49 CECT 10132 Unknown 4200 RA4 RB7 RC16 t1:50 CECT 10137 Raisin 4200 RA2 RB6 RC14 t1:51 CECT 10312 Fig cake 4200 RA1 RB7 RC5 t1:52 CECT 10313 Fig cake 4200 RA1 RB1 RC5 t1:53 CECT 10350 Dried fig 4200 RA3 RB4 RC7 t1:54 CECT 10377 Phoenix dactilifera 4200 RA8 RB2 RC3 t1:55 CECT 10381 Molasses 4200 RA4 RB7 RC15 t1:56 CECT 10425 Honey 4200 RA7 RB10 RC22 t1:57 CECT 10427 Honey 4200 RA3 RB9 RC9 t1:58 CECT 10445 Plum jam 4200 RA10 RB8 RC6 t1:59 CECT 10633 Honey 4200 RA17 RB7 RC20 t1:60 CECT 11121 Grape juice 4200 RA2 RB7 RC14 t1:61 CECT 11136 Grapes 4200 RA6 RB3 RC10 t1:62 CECT 11189 White wine 4200 RA11 RB7 RC2 t1:63 CECT 11923 Soy sauce 4200 RA18 RB11 RC23 t1:64 CECT 11929 Orange and lemon juice 4200 RA14 RB8 RC17 t1:65 CECT 12003 Cherry 4200 RA4 RB7 RC21 t1:66 CECT 12004 Cherry 4200 RA12 RB7 RC11 t1:67 CYC 1484 Unknown 4200 RA3 RB5 RC4 t1:68 CYC 1486 Honey 4200 RA16 RB5 RC10 t1:69 CYC 1487 Nougat 4200 RA13 RB7 RC12 t1:70 CYC 1488 Honey 4200 RA4 RB7 RC9 t1:71 NCYC 1522 Salty bean 4200 RA5 RB6 RC15 t1:72 NCYC 1682 Miso 4200 RA21 RB12 RC24 t1:73 NCYC 3060 Soy sauce 4200 RA21 RB12 RC25 t1:74 NCYC 3061 Soy sauce 4200 RA22 RB12 RC26 t1:75 T2R Nougat fruit 4200 RA4 RB7 RC18 t1:76 Bch Chocolate bun 4200 RA5 RB6 RC18 t1:77Table 1 (continued) t1:78Species and strains studied Source of isolation IGS (bp) Patterns t1:77MAY(1) Liquid sugar 4200 RA15 RB3 RC13 t1:78MAY(15) Liquid sugar 4200 RA15 RB5 RC15 t1:79Es 14 Marzipan 4200 RA4 RB6 RC19 The first letter corresponds to the species: Zygosaccharomyces bailii (B), Z. bisporus (Bi), Z. cidri (C), Z. fermentati (F), Z. kombuchaensis (K), Z. lentus (L), Z. mellis (M), Z. microellipsoides (Mi), Z. rouxii (R), followed by the letter corresponding to the endonucleases: HapII (A), HhaI (B), MboI (C) and finally a number corresponding to the pattern. CBS, Centraalbureau voor Schimmelcultures, The Netherlands; CECT, Colección Española de Cultivos Tipo, Spain. The remaining strains were isolated and identified in our laboratory. t1:80a Z. cidri and Z. fermentati are proposed as Lachancea species and Z.microellipsiodes as Torulaspora species (Kurtzman, 2003, FEMS Yeast 24,403–417). t1:81 b Strains identified in this study as belonging to Zygosaccharomyces rouxii species. t1:82 Zygosaccharomyces rouxii (R) 2 P. Wrent et al. / International Journal of Food Microbiology xxx (2010) xxx–xxx Please cite this article as: Wrent, P., et al., Strain typing of Zygosacch polymorphism of the intergenic spacer region (IGS), Int. J. Food Microb rapid discrimination at strain level for the Z. bailii, Z. mellis and Z. rouxii species. 2. Materials and methods 2.1. Strain and culture conditions Fifty-nine strains belonging to the Zygosaccharomyces genus and one strain of Saccharomyces cerevisiaewere used in this research. They were obtained from different Type Culture Collections or isolated in our laboratory from contaminated or spoiled products. The sources of isolation, obtained from information provided by collections or in our laboratory, are shown in Table 1. The strains were grown in Yeast Morphology Broth at 28 °C and routinely maintained on Yeast Morphology Agar (YMA) containing 0.5% (w/v) yeast extract (Difco Laboratories, Detroit, Mitch, USA), 0.3% (w/v) proteose-peptone No.3 (Difco), 0.3% (w/v) malt extract (Difco), 1% (w/v) glucose (Panreac Quimica S.A., Barcelona, Spain), and 2% (w/v) agar. 2.2. DNA isolation, amplification protocols and RFLP analysis DNA was isolated following the standard protocol of Querol et al. (1992), which includes a first enzymatic step to obtain spheroplasted cells, followed by the isolation and purification of DNA. Alternatively, the DNeasy plant minikit (Qiagen, Hilden, Germany) was used after the spheroplast step. The IGS region of the rDNAwas amplified by PCR in a Mastercycler gradient device (Eppendorf, Germany) using CNL12 (5′CTGAACGCCTCTAAGTCAG3′) and CNS1 (5′GAGACAAGCATATGAC- TACTG3´) forward and reverse primers respectively (Appel and Gordon, 1995). The region defined by these primers spans from base position 3046–3064 on the 26SrDNA (GenBank Accession no. AY048154) to base position 37-17 on the 18S rDNA (GenBank Accession no. J01353). A set of PCR reactions were carried out in microtubes containing a master mix with a final volume of: (i) 25 μl, containing target DNA (50 ng), 2 mMMg Cl2, 1 mM dNTPs (Ecogen, Madrid, Spain), 1 μM of each primer (Sygma–Genosys, Cambridge, UK), 1U Taq polymerase (Ecogen) and sterilized distilled water (MO Laboratories, Inc., USA) up to final volume. The thermal cycling parameters were as follows: an initial denaturation step at 94 °C for 85 s, followed by 35 cycles of 35 s at 95 °C (denaturation), 55 s at 58 °C (annealing) and a final extension at 72 °C for 10 min as previously developed in our laboratory (Romero et al., 2005; Quirós et al., 2006). In order to improve the results the following protocol was evaluated: (ii) 25 μl containing target DNA (100 ng), 12.5 μl 2xGC buffer I or II (TaKaRa bio IncShiga, Japan), 4 μl dNTP mixture (2.5 mM each) (TaKaRa), 1,25 μl of each primer (20 mM) (Sygma–Genosys, Cam- bridge, UK), 0.25 μl La TaqGC (TaKaRa) and sterilized distilled water (MO Laboratories, Inc) up to 25 μl. PCR conditions were an initial denaturation at 94 °C for 85 s and 35 cycles of denaturation at 95 °C aromyces yeast species using a single molecular method based on iol. (2010), doi:10.1016/j.ijfoodmicro.2010.06.007 http://dx.doi.org/10.1016/j.ijfoodmicro.2010.06.007 Original text: Inserted Text "´ " Original text: Inserted Text "´" Original text: Inserted Text "´ " 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150Q2 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 3P. Wrent et al. / International Journal of Food Microbiology xxx (2010) xxx–xxx for 35 s. For annealing, a gradient of 54.9 °C to 69.5 °C for 55 s was probed, followed by an extension for 10 min at 72 °C. PCR-amplified DNA fragments were separated in 1% (w/v) agarose gels (Bio-Rad), stained with 0.05% (v/v) ethidium bromide (Bio-Rad) and visualized under UV light. The 1 kb DNA ladder (MBI Fermentas) was used as a molecular size marker. PCR amplification products from the IGS region of DNA (20 μl) were digested without further purification using HapII, HhaI andMboI endonucleases (Amersham Pharmacia Biotech, Buckinghamshire, UK) (Romero et al., 2005; Quirós et al., 2006). Restriction fragments were separated on 2.5% (w/v) agarose gels, stained with 0.05% (v/v) ethidium bromide and visualized under UV light. The 100 bp DNA ladder (MBI Fermentas) was used as a molecular size marker. 2.3. Sequence determination An analysis of sequences was carried out only on those strains which had a doubtful species classification. The 5.8-ITS rDNA region sequences were amplified by PCR using its1 (5′-TCCGTAGGT- GAACCTGCGG-3′) and its 4 (5′TCCTCCGCTTATTGATATGC3′) primers (White et al., 1990). The D1/D2 domains of the 26S rDNA were amplified using NL1 (5′-GCATATCAATAAGCGGAGGAAAAG-3′) and NL4 (5′-GGTCCGTGTTTCAAGACGG-3′) primers (Kurtzman and Rob- nett, 1998). PCR products were cleaned using the Ultraclean™ PCR clean-up Kit (MO-BIO, Larsband, USA) and 5 μM directly sequenced using the ABI PRISM 3730 DNA Sequencer (Applied Biosystems, Foster City, USA). Each set of sequences was aligned using the MegAlign program of DNASTAR software (Lasergene, Wisconsin, USA), with the most similar sequences obtained from the GenBank Nucleotide sequence Database by BLAST. Alignments were manually revised and edited in the insertion-deletion regions for which alignment was uncertain. 2.4. Data analysis Distance analysis was performed with TREECOM v1.3b software (Van de Peer and De Wachter, 1993). Distance estimations were calculated following the method described by Nei and Li (1979). The data on RFLP patterns were coded as binary tables, with 1 representing the presence of a fragment and 0 representing the absence of a fragment. The tree was inferred by the Neighbor-Joining algorithm. The robustness of the tree was estimated with bootstrap values on 1000 replicates. S. cerevisiae CECT 1942NT was the designated outgroup in the analysis. 3. Results The IGS region (rDNA) with a size lower than 4000 bp was amplified using the protocol (see method 2.2. (i) in Materials and methods). Those with a size higher than 4000 bp had to be amplified with La Taq GC polimerase for DNA fragments rich in G+C (GC buffer I). The annealing temperature was set at 55 °C for 55 s (see method 2.2. (ii) in Materials and methods). The PCR yielded a single fragment that ranged from 2900 bp for the Z. cidri (now Lachancea cidri, Kurtzman, 2003) species to 7000 bp for the Z. kombuchaensis species. As can be observed in Table 1, the size of the amplification products is identical when the strains belong to the same species. The digestion of the IGS amplification product reveals 37 different patterns forHapII, 32 forHhaI, and43 forMboI enzymes (Tables 1 and2). In general, high polymorphism was obtained for each species with the three enzymes assayed (Tables 1 and 2, Figs. 1 and 2). None of the endonuclease could be used independently to discriminate strains and species. The number of patterns varies depending on the endonuclease used. For example, for Z. bailii we obtained five patterns with the HhaI endonuclease and fourwith theMboI andHapII endonucleases (Tables 1 Please cite this article as: Wrent, P., et al., Strain typing of Zygosacch polymorphism of the intergenic spacer region (IGS), Int. J. Food Microb and 2, Fig. 2). However, in Z. rouxii the best discrimination between strainswas obtainedwith theMboI endonuclease (Tables 1 and 2, Fig. 1). The restriction patterns obtained with all three endonucleases, as illustrated in Table 1, enable us to differentiate all the Z. mellis and Z. rouxii strains examined in this study. For example, strains that show the RA1 patternwithHapII (Z.rouxii CECT 1232T, CECT 10312 and CECT 10313) can be differentiated if the other two enzymes are considered, namely, RA1/RB1/RC1, RA1/RB7/RC5 and RA1/RB1/RC5 respectively. When the strain patterns belonging to different species were analysed, together with the dendrogram (Fig. 3), some conflicting resultswere observed. Two strains, CBS 711 andCBS 7412, identified as Z. mellis, appeared inside the main cluster of Z. rouxii. The restriction profiles for the strains included in this cluster showed common bands with whichever endonucleases were assayed. However, the Z. rouxii CECT 11923 and CECT 10425 strains, together with Z. rouxii hybrids, appeared in a second cluster because of the lack of these common bands (Table 2). To solve this conflict, sequence analyses of ITS rDNA and the D1/D2 domains of 26S rDNA were performed (Table 3). The sequences studied showed that Z. mellis CBS 711 and CBS 7412 have 100% similaritywith strains belonging to Z. rouxii species (see Table 3). Two patterns were therefore added to Z. rouxii strains (Table 1). The CECT 11923 and CECT 10425 strains were also confirmed as Z. rouxii. 4. Discussion In food industries, strain typing can be considered from two perspectives: one for discriminating between different biotypes with specific properties, such as the production of volatile compounds for improving taste, ripening etc. (Andrade et al., 2009), as well as for monitoring the behaviour of a strain, for example, during the fermentation process (Suezawa et al., 2008), and the other for following one strain to determine the source of contamination, for example, to solve spoilage problems along the production chain in a specific industrial process (Martorell et al., 2005). Traceability is becoming a concept for providing safer food supplies and connecting the producer with the consumer (Regattiere et al., 2007). EC regulation 178/2002 (European Parliament, 2002) defines traceability as “the ability to trace and follow a food, feed, food-producing animals or ingredients through all stages of production and distribution”. In terms of food safety, it can tell us the history of the product. In the food spoilage context a method that attains a high grade of discrimination between strains allows us to follow the “traceable spoilage strain” along the production line. This provides the industrialist with a useful tool which may be required for legal demands. Our results show that the PCR-RFLP-IGS analysis presents a variability of 70% for Z. bailii with both HhaI and MboI endonucleases. Meanwhile, 100% variability was obtained for Z. mellis and Z. rouxii strains when the three enzymes were analysed together. This variability is higher than what has previously been reported for some of these species using other methods (Martorell et al., 2005; Suezawa et al., 2008). Moreover, if we analyse some of the strains with the same patterns some considerations should be made. For example, in Z. bailii the May (12) and May (13) strains were isolated in our laboratory from the same mayonnaise sample. It is therefore possible that they come from two colonies of the same strain. The CECT 1924 and CECT 1898 strains show the same RFLP pattern. Martorell et al. (2005) had previously studied them using RFLP mtDNA and RAPDs and found that they could not be distinguished by those methods. Although both come from Japan, the origin of only one of them is known (Table 1). Based on these results we propose that both isolates belong to the same strain. Other examples of identical RFLP pattern were found in the CECT 10657T and CECT 11349 strains of Z. cidri (now Lachancea cidri, Kurtzman, 2003). Both were deposited by the same author in the same year (1955) and were from the same origin: cider. Once again, we propose that both isolates belong to the same strain. aromyces yeast species using a single molecular method based on iol. (2010), doi:10.1016/j.ijfoodmicro.2010.06.007 http://dx.doi.org/10.1016/j.ijfoodmicro.2010.06.007 Original text: Inserted Text "then" Table 2t2:1 Restriction fragments (bp) obtained after the digestion of the IGS region of rDNA of species belonging to the Zygosaccharomyces genus with HapII, HhaI and MboI endonucleases. t2:2 t2:3 Species/No. (Code of Species) Restriction fragmentsa (bp) t2:4 Patterns Hap II (A) Patterns Hha I (B) Patterns Mbo I (C) t2:5 Zygosaccharomyces bailii/7. (B) t2:6 BB1 (1750+610+450+430+360+ 340+300+200+160) BC1 (2600+950+610+410+320+270) t2:7 BA1 (1000+900+700+530+290+ 270+200+170+140) BB2 (1900+1700+610+450+430+ 360+300+200+160) BC2 (2700+2600+950+610+410+ 320+270) t2:8 BA2 (1000+700+530+420+220+ 180+170+140) BB3 (2300+2000+610+450+430+ 350+330+300+200+160) BC3 (3000+950+610+410+320+270) t2:9 BA3 (1000+700+530+350+290+ 200+170+140) BB4 (2800+2500+610+450+430+ 360+300+200+160) BC4 (1300+1200+950+610+410+ 370+350+320+270) t2:10 BA4 (900+620+530+480+430+ 390+350+220+180+170+140) BB5 (2500+610+480+430+360+ 340+300+160) BC5 (2700+950+610+410+400+320+270) t2:11 t2:12 Zygosaccharomyces bisporus/2. (Bi) t2:13 BiA1 (1150+1100+630+510+430+ 370+300+220+170+120+90) BiB1 (1150+870+580+390+360+ 290+240+200+190+140+100) BiC1 (1500+1150+680+630+400+330+ 270+230+150) t2:14 BiA2 (1500+1200+630+600+510+ 430+370+300+170+120+90) BiB2 (1150+870+580+480+390+ 360+290+240+200+190+140+100) BiC2 (1500+1150+710+630+400+330+ 270+230+150) t2:15 t2:16 Zygosaccharomyces cidrii/2. (C) t2:17 CA1 (2000+320+270+210+150) CB1 (1180+760+730+300) CC1 (1100+460+330+320+250+210+ 170+110) t2:18 t2:19 Zygosaccharomyces fermentati/3. (F) t2:20 FA1 (1150+800+390+170) FB1 (1100+820+380+380+170) FC1 (750+690+390+320+250+210+110) t2:21 FA2 (1100+900+550+300+250) FB2 (1100+850+550+300+250) FC2 (1000+570+390+320+250+210+110) t2:22 FA3 (1500+600+320+270+210+150) t2:23 t2:24 Zygosaccharomyces kombuchaensis/1. (K) t2:25 KA1 (1600+1150+600+510+430+ 410+300+250+190+170+120) KB1 (1900+600+600+500+450+ 450+400+250+210+120) KC1 (1750+1250+1150+950+550+ 500+450) t2:26 t2:27 Zygosaccharomyces lentus/2. (L) LA1 (680+580+530+500+430+330+ 300+280+240+200+180+150) LB1 (1500+1300+900+600+580+ 500+420+300+270+200+150+90) LC1 (1100+850+610+590+550+450+ 320+310+270+250+150+110) t2:28 LA2 (730+580+530+500+430+330+ 300+280+240+200+180+150) t2:29 t2:30 Zygosaccharomyces mellis/10. (M) MA1 (1000+930+300+280+190+ 160+120+90) MB1 (1750+900+800+400+250) MC1 (1300+900+720+430+350+270+ 150+120+100) t2:31 MA2 (1000+930+480+300) MB2 (1750+850+500+400+390) MC2 (1300+900+750+450) t2:32 MA3 (1000+800+750+300+250) MB3 (1300+680+530+450+380+ 310+210) MC3 (1250+1000+450+410+260) t2:33 MA4 (1000+750+300+250 MB4 (1750+900+800+530+ 400+250) RC24 (760+670+520+500+410+350+ 260+170) t2:34 MA5 (1100+1000+930+300+280+ 190+160+120+90) MB5 (1750+1050+900+400+250) RC25 (760+650+500+500+410+ 350+310+260+170) t2:35 RA19 (970+700+500+400+380+ 300+290+160) RB12 (960+700+630+450+420+ 210+190) t2:36 RA20 (970+700+480+400+310+ 300+290+160) RB13 (960+700+680+630+450+ 420+210+190) t2:37 t2:38 Zygosaccharomyces microellipsiodes/1. (Mi) MiA1 (1100+650+370+180+160) MiB1 (800+670+610+320+350) MiC1 (800+550+370+270) t2:39 t2:40 Zygosaccharomyces rouxii/33. ® RA1 (970+700+500+400+300+ 290+160) RB1 (960+700+630+450+420) RC1 (760+670+520+500+410+ 350+260+200+170) t2:41 RA2 (970+700+480+400+300+ 270+250+160) RB2 (960+700+600+450+420) RC2 (760+520+410+350+260+170) t2:42 RA3 (970+700+480+400+300+ 270+160) RB3 (960+700+630+450) RC3 (760+520+410+350+300+260+170) t2:43 RA4 (970+700+480+400+300+ 290+160) RB4 (960+720+700+600+ 450+420) RC4 (760+520+410+350+310+260+170) t2:44 RA5 (970+700+480+400+300+ 290+270+160) RB5 (960+720+700+630+450) RC5 (760+520+500+410+350+260+170) t2:45 RA6 (970+700+480+400+330+ 300+270+160) RB6 (960+720+700+630+450+ 420+210) RC6 (760+650+500+410+350+260+170) t2:46 RA7 (1200+700+410+380+320+ 210+180+90) RB7 (960+740+700+630+450+ 420+210) RC7 (760+650+500+410+350+290+ 260+170) t2:47 RA8 (970+700+480+400+350+ 300+270+160) RB8 (960+790+700+600+450+420) RC8 (760+650+520+410+350+260+ 210+170) t2:48 RA9 (970+700+480+400+350+ 300+290+160) RB9 (960+700+630+450+420+180) RC9 (760+650+520+410+350+260+170) t2:49 RA10 (970+700+480+420+400+ 300+270+160) RB10 (1500+800+700+400+350+ 310+190+120+80) RC10 (760+650+520+410+350+310+ 260+170) 4 P. Wrent et al. / International Journal of Food Microbiology xxx (2010) xxx–xxx Please cite this article as: Wrent, P., et al., Strain typing of Zygosaccharomyces yeast species using a single molecular method based on polymorphism of the intergenic spacer region (IGS), Int. J. Food Microbiol. (2010), doi:10.1016/j.ijfoodmicro.2010.06.007 http://dx.doi.org/10.1016/j.ijfoodmicro.2010.06.007 Maribel Tachado Maribel Texto de reemplazo (R) 255 256 257 258 259 260 t2:51 Table 2 (continued) t2:52 Species/No. (Code of Species) Restriction fragmentsa (bp) t2:53 Patterns Hap II (A) Patterns Hha I (B) Patterns Mbo I (C) t2:50 RA11 (970+700+500+400+300+ 270+160) RB11 (1500+850+700+400+350+ 290+120+80) RC11 (760+650+520+500+410+350+ 310+260+170) t2:51 RA12 (970+700+500+400+300+ 290+200+160) RB12 (1500+850+680+400+360+ 350+190) RC12 (760+670+520+500+410+350+ 310+260+170) t2:52 RA13 (970+700+500+400+350+ 300+290+270+200+160) RC13 (760+700+500+410+350+260+170) t2:53 RA14 (970+700+500+450+400+ 300+290+160) RC14 (760+700+520+410+350+290+ 260+170) t2:54 RA15 (970+700+500+480+400+ 300+290+160) RC15 (760+700+520+500+410+350+ 260+170) t2:55 RA16 (970+700+500+480+400+ 370+300+290+270+160) RC16 (760+700+520+500+410+350+ 300+260+170) t2:56 RA17 (970+700+480+400+300+ 290+200+160) RC17 (760+700+650+520+500+410+ 350+260+170) t2:57 RA18 (1300+800+700+410+ 380+320+210+180) RC18 (760+700+670+520+500+410+ 350+310+260+170) t2:58 RA21 (1300+730+700+410+380+ 350+320+210+180+160+90) RC19 (760+700+690+650+520+500+ 410+350+310+260+170) t2:59 RA22 (1300+730+700+410+380+ 320+210+180+160+90) RC20 (760+650+520+500+410+350+ 260+170) t2:60 RC21 (760+700+650+520+500+410+ 350+310+290+260+170) t2:61 RC22 (1700+700+630+500+400+ 250+170) t2:62 RC23 (1700+800+700+500+400+250) t2:63 RC24 (1700+800+750+700+650+ 500+400+270+170+160+130) t2:64 RC25 (1700+800+650+500+400+ 270+170+160+130) t2:65 RC26 (1700+800+650+500+400+ 270+160+130) Z.cidri and Z. fermentati are proposed as Lachancea species and Z.microellipsiodes as Torulaspora species (Kurtzman, 2003, FEMS Yeast 24,403–417). t2:66 a Some fragments could be duplicated.t2:67 Zygosaccharomyces rouxii/33. ® 5P. Wrent et al. / International Journal of Food Microbiology xxx (2010) xxx–xxx One interesting result, although it was not part of our objective, was that the method enabled us to detect two misidentified strains of Z. mellis (CBS 711 and CBS 7412). These strains presented a restriction profile of the IGS region of rDNA which included the common bands described above for the greater part of Z. rouxii examined (Table 2). The D1/D2 domain 26S rDNA and ITS sequence analysis showed that Fig. 1. Zygosaccharomyces rouxii strain typing. Some PCR-RFPLs patterns of the IGS region of rDNA with MboI endonuclease. Lanes 1 and 9 correspond to the 100 bp DNA ladder (MBI Fermentas). Fig. 2. Zygosaccharomyces bailii strain typing. Some PCR-RFPLs patterns of the IGS region of rDNA with HapII endonuclease. Lanes 1 and 9 correspond to the 100 bp DNA ladder (MBI Fermentas). Please cite this article as: Wrent, P., et al., Strain typing of Zygosaccharomyces yeast species using a single molecular method based on polymorphism of the intergenic spacer region (IGS), Int. J. Food Microbiol. (2010), doi:10.1016/j.ijfoodmicro.2010.06.007 http://dx.doi.org/10.1016/j.ijfoodmicro.2010.06.007 Maribel Tachado Maribel Texto de reemplazo (R) Maribel Tachado Maribel Texto de reemplazo 7 261 262 263 264 265 266 267 268 269 270 Fig. 3. Dendrogram based on the IGS-PCR restriction analysis of Zygosaccharomyces species isolated in our laboratory or from Type Culture Collections. Distance estimations were calculated following the method described by Nei and Li (1979). Robustness of the tree was estimated with bootstrap values on 1000 replicates indicated as a percentage. Saccharomyces cerevisiae CECT194nNT was used as the outgroup species. *Z. cidri and fermentati are proposed as Lachancea species and Z. microellipsiodes as Torulaspora species (Kurtzman, 2003, FEMS yeast 24, 403–417). ** Strains identified in this study as Z.rouxii are shown in Table 3. 6 P. Wrent et al. / International Journal of Food Microbiology xxx (2010) xxx–xxx the phenotypic identificationwas not correct.We therefore concluded that these two strains belong to the Z. rouxii species (Fig. 3, Table 3). Two Z. rouxii strains (CECT 11923 and CECT 10425) were confirmed as Z. rouxii in this study by sequencing. Although the undoubted value of using simple gene sequences (e.g. 26 S rDNA D1/ Please cite this article as: Wrent, P., et al., Strain typing of Zygosacch polymorphism of the intergenic spacer region (IGS), Int. J. Food Microb D2) to identify yeasts is recognized, this method has some limitations for the identification of hybrids (James et al., 2005). Moreover, the fact that both strains appear together with the hybrids (NCYC1682, NCYC 3060 and NCYC 3061) identified by James et al. (2005) in a separate cluster (Fig. 3) may indicate that they could also be hybrids. These aromyces yeast species using a single molecular method based on iol. (2010), doi:10.1016/j.ijfoodmicro.2010.06.007 http://dx.doi.org/10.1016/j.ijfoodmicro.2010.06.007 Table 3t3:1 GenBank accession numbers of the strains included in the study, and strains that presents a 100% similarity with them. t3:2 t3:3 Accession number t3:4 Strain 5.8S-ITS (strain) D1/D2 26S rRNA gene (strain) t3:5 Z. rouxii CECT 10425 FN431888 FN431893 t3:6 AB363048.1 (ZR510) AB363047.1 (ZR14) AB363045.1 (TSY-5) t3:7 AB363044.1 (TO) AB363043.1 (TA10101) AB363040.1 (No.3) t3:8 AB363038.1 (KS02) AB363035.1 (H14-8-1) AB363033.1 (H14-5-1) t3:9 AB363032.1 (H14-4-1) AB363031.1 (H14-1-2) AB363030.1 (-601) t3:10 AB302811.1 (IFO 1877) AB302810.1 (IFO 1876) AB302807.1 (IFO 1812) t3:11 AB302805.1 (IFO 0845) AB302801.1 (IFO 0596) AB302800.1 (IFO 0525) t3:12 AB302799.1 (IFO 0523) AB302798.1 (IFO 0521) AB302794.1 (IFO 0510) t3:13 AB302793.1 (IFO 0506) AB302792.1 (IFO 0505) AM947682.1 (ABT301) t3:14 AM947680.1 (ATCC 42981) AM943657.1 (ATCC 42981) AJ966342.2 (ABT301) t3:15 AY524006.1 AY524006.1 (NRRL Y-2547) AJ555406.1 (NCYC3042) t3:16 Z. rouxii CECT 11923 FN431886 FN431887 t3:17 AB363062.1 (ZR510) AB363061.1 (ZR14) AB363060.1 (ZR-1) CU928181.1 (CBS 732) AB363046.1 (ZR-1) AB363042.1 (SR-4) t3:18 AB363057.1 (SR-4) AB363056.1 (SR-2) AB363055.1 (KS03) AB363041.1 (SR-2) AB363039.1 (KS03) AB363037.1 (KS01) t3:19 AB363054.1 (KS01) AB363053.1 (KF-4) AB363051.1 (H14-7-1) AB363036.1 (KF-4) AB363034.1 (H14-7-1) AB302813.1 (IFO 1960) t3:20 AB302834.1 (IFO 1960) AB302827.1 (IFO 1130) AB302826.1 (IFO 0845) AB302812.1 (IFO 1914) AB302809.1 (IFO 1814) AB302808.1 (IFO 1813) t3:21 AB302824.1 (IFO 0596) AB302820.1 (IFO 0513) AB302819.1 (IFO 0512) AB302806.1 (IFO 1130) AB302804.1 (IFO 0740) AB302803.1 (IFO 0686) t3:22 AB302818.1 (IFO 0511) AM943656.1 (ATCC 42981) AM943655.1 (CBS 732) AB302797.1 (IFO 0513) AB302796.1 (IFO 0512) AB302795.1 (IFO 0511) t3:23 AY046189.1 (Unknown) AB302791.1 (IFO 0494) AM943656.1 (ATCC 42981) AM943655.1 (CBS 732) t3:24 AM911009.1 (YSF21) AM911008.1 (YSF34) AJ783434.1 (ESAB21) t3:25 AJ716118.1 (CBS 9714) U72163.1 (Unknown) t3:26 Z. mellis CBS 711 FN431889 FN431892 t3:27 AB363062.1 (ZR510) AB363061.1 (ZR14) AB363060.1 (ZR-1) CU928181.1 (CBS 732) AB363046.1 (ZR-1) AB363042.1 (SR-4) t3:28 AB363057.1 (SR-4) AB363056.1 (SR-2) AB363055.1 (KS03) AB363041.1 (SR-2) AB303639.1 (KSO3) AB393037.1 (KSO1) t3:29 AB363054.1 (KS01) AB363053.1 (KF-4) AB363051.1 (H14-7-1) AB363036.1 (KF-4) AB363034.1 (H-14-7-1) AB302813.1 (IFO1960) t3:30 AB302834.1 (IFO 1960) AB302827.1 (IFO 1130) AB302826.1 (IFO 0845) AB302812.1 (IFO1914) AB302809.1 (IFO1814) AB302808.1 (IFO1813) t3:31 AB302825.1 (IFO 0740) AB302824.1 (IFO 0596) AB302820.1 (IFO 0513) AB302806.1 (IFO1130) AB302804.1 (IFO0740) AB302803.1 (IFO0686) t3:32 AB302819.1 (IFO 0512) AB302818.1 (IFO 0511) AM943656.1 (ATCC 42981) AB302797.1 (IFO0513) AB302796.1 (IFO0512) AB302795.1 (IFO0511) t3:33 AM943655.1 (CBS 732) AM279465.1 (ABT301) AB302791.1 (IFO0494) AM943665.1 (ATCC42981) AM943655.1 (CBS 732) t3:34 AM911009.1 (YSF21) AM911008.1 (YSF34) AY524005.1 (PYCC3693) t3:35 AJ783434.1 (ESAB21) AJ716118.1 (CBS9714) U72163.1 (Unknown) t3:36 Z. mellis CBS 7412 FN431890 FN431891 t3:37 AB363062.1 (ZR510) AB363061.1 (ZR14) AB363060.1 (ZR-1) AB363046.1 (ZR-1) AB363042.1 (SR-4) AB363041.1 (SR-2) t3:38 AB363057.1 (SR-4) AB363056.1 (SR-2) AB363055.1 (KS03) AB363039.1 (KS03) AB363037.1 (KS01) AB363036.1 (KF-4) t3:39 AB363054.1 (KS01) AB363053.1 (KF-4) AB363051.1 (H14-7-1) AB363034.1 (H14-7-1) AB302813.1 (IFO 1960) AB302812.1 IFO 1914 t3:40 AB302834.1 (IFO 1960) AB302827.1 (IFO 1130) AB302826.1 (IFO 0845) AB302809.1 (IFO 1814) AB302808.1 (IFO 1813) AB302806.1 (IFO 1130) t3:41 AB302825.1 (IFO 0740) AB302824.1 (IFO 0596) AB302820.1 (IFO 0513) AB302804.1 (IFO 0740) AB302803.1 (IFO 0686) AB302797.1 IFO0513 t3:42 AB302819.1 (IFO 0512) AB302818.1 (IFO 0511) AM943656.1 (ATCC 42981) AB302796.1 IFO0512 AB302795.1 IFO0511 AB302791.1 (IFO 0494) t3:43 AM943655.1 (CBS 732) AM279465.1 (ABT301) AM943656.1 (ATCC 42981) AM943655.1 (CBS 732) AM911009.1 (YSF21) t3:44 AM911008.1 (YSF34) AY524005.1 (PYCC 3693) AJ783434.1 (ESAB21) Sequences retrieved from GenBank. t3:45 Accession number printed in bold corresponds to sequences obtained in the current study.t3:46 7 P.W rent et al./ International Journal of Food M icrobiology xxx (2010) xxx–xxx Please cite this article as: W rent, P., et al., Strain typing of Zygosaccharom yces yeast species using a single m olecular m ethod based on polym orphism of the intergenic spacer region (IG S),Int.J.Food M icrobiol.(2010),doi:10.1016/j.ijfoodm icro.2010.06.007 http://dx.doi.org/10.1016/j.ijfoodmicro.2010.06.007 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306Q3 307 308 309 310 311 312 313 314Q4 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 Q5 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 8 P. Wrent et al. / International Journal of Food Microbiology xxx (2010) xxx–xxx authors suggest that yeast hybrids may be more abundant than previously thought; in fact, Solieri et al. (2007) recently reported new Z. rouxii hybrids. For this reason, further studies are under way in our laboratory to clarify the nature of these strains. Note that this method also permits all the hybrid strains analysed so far to be distinguished, as shown in Tables 2 and 3. On the other hand, during this study, we have developed a modification of the method previously described by Romero et al. (2005) and Quirós et al. (2006) (Materials and methods). After the spheroplasted treatment, the DNA extraction was done in half the time using DNeasy (see Materials and methods 2.3). Changing the DNA polymerase and the PCR conditions (see Material and methods 2.3 (ii)) made it possible to amplify IGS regions with a size as high as 7000, 6700 or 6000 bp. The amplicon size of the IGS is different in seven out of the nine species of Zygosaccharomyces tested (Table 1). For example, a size of 6300 bp corresponds to Z. bailii and 5500 bp to Z. bisporus. Although this technique is unreliable as a method of identification, in practice relatively few yeast species are responsible for the majority of food spoilage by yeasts (Stratford, 2006) and some specific associations are frequent and often predictable (Fleet, 2006). This generally narrows the candidates for yeast spoilage in a specific food. Typingmethodologies are usually applied after the identification process. For example, Z. mellis and Z. rouxii may easily be identified from colonies by the size of the amplicon of the 5.8S-ITS region and the fragments obtained after digestion with three restriction endonucleases (Barata et al., 2008; Esteve-Zarzoso et al., 1999, 2003, yeast-id.com (CECT)). In conclusion, the PCR-RFLP analysis of the IGS region of rDNA is a fast and single molecular typing method producing clear and reproducible restriction RFLP patterns. It constitutes a typing method for the Z. bailii, Z. mellis and Z. rouxii species as well as for other species belonging to the Zygosaccharomyces genus. The method also dis- criminates hybrid strains of Zygosaccharomyces. It does not require sequencing technologies and as a consequence is easier to implant in the routine of an industry laboratory. 5. Uncited reference Kurtzman, 1990 Acknowledgements This study was supported in part by projects of the Spanish Ministry of Education and Science (AGL2005-02630) and the Complutense University of Madrid (UCM-BSCH GR58/08). The sequence analyses were performed at the Genomics Unit of the Madrid Science Park, Moncloa Campus. References Andrade, M.J., Rodríguez, M., Casado, E.M., Bermúdez, E., Córdoba, J.J., 2009. 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Maribel Tachado Maribel Texto insertado 2006 Maribel Tachado Maribel Texto de reemplazo 2.2 Maribel Tachado Maribel Texto de reemplazo 2.2 Strain typing of Zygosaccharomyces yeast species using a single molecular method based on polymorphism of the intergenic sp... Introduction Materials and methods Strain and culture conditions DNA isolation, amplification protocols and RFLP analysis Sequence determination Data analysis Results Discussion Uncited reference Acknowledgements section11