IJSEM Sign up for IJSEM eTOCs
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplementary Figures and Table
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via CrossRef
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cadez, N.
Right arrow Articles by Smith, M. Th.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cadez, N.
Right arrow Articles by Smith, M. Th.
Agricola
Right arrow Articles by Cadez, N.
Right arrow Articles by Smith, M. Th.
Int J Syst Evol Microbiol 53 (2003), 1671-1680; DOI  10.1099/ijs.0.02618-0
© 2003 International Union of Microbiological Societies

Hanseniaspora meyeri sp. nov., Hanseniaspora clermontiae sp. nov., Hanseniaspora lachancei sp. nov. and Hanseniaspora opuntiae sp. nov., novel apiculate yeast species

Neza Cadez1, Gé A. Poot2, Peter Raspor1 and Maudy Th. Smith2

1 University of Ljubljana, Biotechnical Faculty, Department of Food Science and Technology, Jamnikarjeva 101, 1000 Ljubljana, Slovenia
2 Centraalbureau voor Schimmelcultures, Yeast Division, PO Box 85167, 3508 AD Utrecht, The Netherlands

Correspondence
Maudy Th. Smith
smith{at}cbs.knaw.nl


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Fourteen apiculate yeast strains isolated from various sources in South Africa, North America and the Hawaiian islands were found to be genetically divergent from other Hanseniaspora–Kloeckera species by using randomly amplified polymorphic DNA (RAPD)-PCR. After cluster analysis of the RAPD-PCR fingerprints, five groups were recognized. DNA reassociation values among representatives of these groups and strains of Hanseniaspora–Kloeckera species revealed that the strains represent five novel species. Four are described here as novel species of Hanseniaspora: Hanseniaspora meyeri sp. nov. (type CBS 8734T), Hanseniaspora clermontiae sp. nov. (type CBS 8821T), Hanseniaspora lachancei sp. nov. (type CBS 8818T) and Hanseniaspora opuntiae sp. nov. (type CBS 8733T). The fifth novel species, which is represented by only a single strain, CBS 8772, is not introduced as a new taxon. Phylogenetic analyses of the D1/D2 region of the 26S rDNA and internal transcribed spacer (ITS) regions with 5·8S rDNA sequences placed H. meyeri, H. clermontiae, H. lachancei, H. opuntiae and strain CBS 8772 close to Hanseniaspora uvarum and Hanseniaspora guilliermondii. The key characteristics for standard physiological identification of H. clermontiae and H. lachancei were respectively maximal growth temperature and assimilation of 2-keto-D-gluconate. However, physiological characteristics did not allow the distinction of H. opuntiae and strain CBS 8772 from H. guilliermondii or H. meyeri from H. uvarum. These three novel taxa can be identified by either ITS sequencing or PCR-RFLP of ITS regions using restriction enzymes MboII and HinfI.

Abbreviations: ITS, internal transcribed spacer; RAPD, randomly amplified polymorphic DNA

The GenBank/EMBL/DDBJ accession numbers for the sequences determined in this study are AJ512451–AJ512461 (26S rDNA D1/D2 domain) and AJ512427–AJ512443 (ITS region and 5·8S rDNA).

Detailed DNA–DNA hybridization data for the novel strains, a D1/D2-based phylogenetic tree and results of PCR-RFLP of the ITS region are available as supplementary material in IJSEM Online.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
In a previous study (Cadez et al., 2002Go), the genetic diversity of apiculate yeasts belonging to the genera Hanseniaspora Zikes and Kloeckera Janke was determined using randomly amplified polymorphic DNA (RAPD)-PCR analysis. Cluster analysis of the combined fingerprints of 74 strains segregated the strains into six major clusters, each representing a currently recognized species in the genus Hanseniaspora (Smith, 1998aGo), and one separate cluster of five strains that were physiologically indistinguishable from Hanseniaspora uvarum (Cadez et al., 2002Go). Because the origins of these five strains were natural and man-made environments in North America and Africa only, nine additional strains from Hawaii and Mexico were included in rapid genomic fingerprinting. Eleven strains sharing five distinct RAPD-PCR banding patterns were subjected to further studies involving DNA–DNA reassociation determinations. In addition, the D1/D2 and internal transcribed spacer (ITS) regions (ITS1, ITS2) and the 5·8S rRNA genes were sequenced to determine phylogenetic relationships. Because standard physiological identification tests did not allow the separation of all Hanseniaspora and Kloeckera species, a combination of phenotypic and molecular tests is required for accurate identification of these novel Hanseniaspora species.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Yeast strains and their characterization.
The strains examined and their origin, location and DNA G+C contents are listed in Table 1Go. Physiological characteristics of the yeasts were determined according to the methods of Yarrow (1998)Go. Utilization of carbon compounds was tested in liquid media after incubation at 25 °C in tubes for 4 weeks with continuous shaking at 30 r.p.m. Assimilation of nitrogen compounds was examined by the auxonographic method after 1 week.


View this table:
[in this window]
[in a new window]
  		Table 1. Strains studied

Culture collections are abbreviated as follows: CBS, Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands; NCAIM, National Collection of Agricultural and Industrial Micro-organisms, Budapest, Hungary; NRRL, ARS Culture Collection, National Center for Agricultural Utilization Research, Peoria, IL, USA; UWO-PS, University of Western Ontario Plant Sciences Yeast Culture Collection, London, Ontario, Canada. Standard deviations of G+C content determinations were ±0·5 mol%. ND, Not determined; NK, not known.

 
RAPD-PCR analysis.
Genomic DNA was isolated as described by Möller et al. (1992)Go. Three microsatellite primers, (ATG)5, (GTG)5 and (GACA)4, were used in PCRs as described before (Cadez et al., 2002Go). A Perkin Elmer 2400 thermal cycler was programmed for 35 cycles of 1 min at 94 °C, 1 min at 48 °C [primer (ATG)5], 52 °C [(GTG)5] or 43 °C [(GACA)4] followed by 2 min at 72 °C. PCR products were separated on 2 % agarose gels in 1x TAE buffer. Ethidium bromide-stained gels were documented by GelDoc 2000 (Bio-Rad).

The RAPD-PCR profiles of each strain were combined in a composite fingerprint and a binary matrix was generated manually by scoring the absence (0) or presence (1) of each fragment. Further analyses were performed using NTSYS software package version 2.0 (Applied Biostatistics). Similarity values were calculated using the Dice coefficient and cluster analysis was performed using the UPGMA algorithm.

Genomic DNA analyses.
For DNA extraction, strains were grown for 2 days at 25 °C on a rotary shaker at 125 r.p.m. in 2 l yeast extract/malt extract broth (YM; Difco) using four 1 l flat-bottom flasks. Isolation and purification of DNA, determination of DNA base composition and DNA–DNA reassociations were performed according to Golubev et al. (1989)Go. Strains used in DNA–DNA reassociations are indicated in Table 1Go.

rDNA sequencing and phylogenetic analyses.
The D1/D2 domain of 26S rDNA was amplified using primers NL-1 and NL-4 (O'Donnell, 1993Go) and the ITS (ITS1, ITS2) and 5·8S rRNA gene regions were amplified using primers ITS1-F (Gardes & Bruns, 1993Go) and ITS4 (White et al., 1990Go). Amplification was performed under the following conditions: an initial denaturing step of 5 min at 94 °C, followed by 35 cycles of 40 s at 94 °C, 40 s at 55 °C and 30 s at 72 °C, with a final extension step of 7 min at 72 °C. The PCR products were purified using the Wizard PCR purification kit (Promega) according to the manufacturer's instructions and sequenced directly by using a Thermo Sequenase Cy5 Dye Terminator kit (Amersham Pharmacia Biotech). The primers used to sequence both strands of the D1/D2 domain and ITS1–5·8S rDNA–ITS2 regions were the same as for PCR amplification. The thermal cycling conditions were as follows: 25 cycles of 30 s at 94 °C, 30 s at 50 °C and 1 min at 72 °C. Purified sequencing reactions were loaded on ALFexpress II automatic DNA sequencer (Amersham Pharmacia Biotech).

The D1/D2 and ITS1–5·8S rDNA–ITS2 sequences were aligned automatically using the multiple sequence alignment program CLUSTAL X 1.8 (Thompson et al., 1997Go) and were adjusted manually. Positions where gaps existed in any of the aligned sequences were excluded. Further phylogenetic analyses employed the PHYLIP software package version 3.6 (Felsenstein, 2000Go). A distance matrix was obtained using DNADIST with Kimura's two-parameter method (Jin & Nei, 1990Go) and phylogenetic trees were constructed using the neighbour-joining method (Saitou & Nei, 1987Go). Stability of branches was assessed by bootstrap analysis (Felsenstein, 1985Go) in which 1000 replicates were used.

PCR-RFLP analysis of ITS.
The ITS1–5·8S rDNA–ITS2 regions were amplified using primers ITS1 and ITS4 as described previously (Cadez et al., 2002Go) and were digested directly with restriction enzymes DdeI (Promega), HinfI (Roche) and MboII (Promega) according to the instructions of the manufacturers. The digests were separated on 3 % agarose gels in 1x TAE buffer.

The molecular sizes of the ITS and 5·8S rDNA digests were determined by comparison to a DNA molecular marker (100 bp ladder; Gibco-BRL) using the Quantity One 4.0.3 (Bio-Rad) computer program.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Genetic diversity
Initially, the genetic diversity of 14 strains of the genus Hanseniaspora isolated from various sources in Africa, North America and Hawaii was analysed using RAPD-PCR analysis in comparison with the type and other representative strains of Hanseniaspora and Kloeckera species (Table 1Go). UPGMA clustering of the combined RAPD-PCR fingerprints obtained with microsatellite primers (ATG)5, (GTG)5 and (GACA)4 is shown in Fig. 1Go. The 14 strains segregated into five distinct groups, which clustered with the other Hanseniaspora and Kloeckera species at a similarity level below 40 %. On the basis of previous correlation between similarity values derived from RAPD-PCR analysis and DNA–DNA reassociations (Messner et al., 1994Go; Molnár et al., 1995Go), we predicted that the five groups were genetically divergent from currently accepted species of Hanseniaspora.



View larger version (30K):
[in this window]
[in a new window]
  		Fig. 1. UPGMA dendrogram of Hanseniaspora–Kloeckera strains based on combined RAPD-PCR fingerprints obtained with microsatellite primers (ATG)5, (GTG)5 and (GACA)4. The distance between strains was calculated using the Dice coefficient. The dotted line indicates a similarity level of 40 %.

 
DNA relatedness
Subsequently, DNA–DNA reassociation analysis of strains of the five genetically divergent groups (I–V) was performed. Of group I, four selected isolates from South Africa, Hawaii and Georgia (USA) exhibited DNA relatedness values ranging from 91 to 100 %. Relatedness of 100 % was observed within groups II and III, the latter consisting of two strains, CBS 8818T and CBS 8819, isolated from a traditional tequila fermentation in Mexico and referred to by Lachance (1995)Go as atypical Hanseniaspora guilliermondii strains because of their lack of assimilation of 2-keto-D-gluconate. Among the Hawaii isolates, CBS 8733T and CBS 8820 of group IV, a relatedness value of 97 % was observed. Group V included only a single strain, CBS 8772, isolated from orange juice concentrate in Georgia (USA). Detailed DNA relatedness data for strains of groups I–V are available as supplementary material in IJSEM Online.

In Table 2Go, the mutual DNA relatedness values of the five groups and the seven currently accepted Hanseniaspora and Kloeckera species are shown. Mutual DNA relatedness values were usually low, ranging from 1 to 40 %. However, several intermediate values were found among groups; 62 % between groups I and II, 66 % between groups IV and V and 51 and 53 %, respectively, between group III and groups IV and V. From these values, it can be concluded that the groups represent five novel species, and, from the intermediate similarity values in particular, it can be inferred that groups I and II and groups III, IV and V represent two groups of diverging species complexes.


View this table:
[in this window]
[in a new window]
  		Table 2. DNA–DNA relatedness among taxa of Hanseniaspora and Kloeckera

Values are mean percentages of at least two determinations. The standard deviation for levels of reassociation within and among groups was <=5 %.

 
Sequence analysis and phylogenetic placement
The phylogenetic relationships of the four novel species and strain CBS 8772 to other Hanseniaspora and Kloeckera species were determined by sequence comparisons. The D1/D2 sequences of 26S rDNA of 11 selected strains were determined (GenBank accession numbers shown in Table 1Go) and aligned with those of the other Hanseniaspora and Kloeckera type strains. Pairwise comparisons revealed that sequence divergences among the six species and strain CBS 8772 belonging to the H. guilliermondii–H. uvarum cluster did not exceed 1 % (6 nucleotide substitutions), a value suggested by Kurtzman & Robnett (1998)Go to be the borderline of species separation. Nucleotide substitutions of more than 1 % are indicative of separate taxa, while differences of 0–3 nucleotides seem to be indicative of conspecificity or sister species. However, DNA–DNA reassociation may, as in the present case, detect genetic divergence that is not detectable by sequencing. The study of Boekhout et al. (1994)Go showed that H. uvarum and H. guilliermondii, species sharing low DNA relatedness [11–29 % (Meyer et al., 1978Go); 22 %, this study], had sequence divergence in the D1/D2 region of only 1 % (6 base substitutions). Consequently, the rates for D1/D2 substitutions between the recently diverged species complexes Hanseniaspora meyeri sp. nov.–Hanseniaspora clermontiae sp. nov. and Hanseniaspora lachancei sp. nov.–Hanseniaspora opuntiae sp. nov. were proportionally lower, as H. clermontiae differed by only two base substitutions (0·35 % sequence divergence) from H. meyeri and H. opuntiae differed by three base substitutions (0·5 % sequence divergence) from H. lachancei. Strain CBS 8772, a sister taxon to the species complex H. opuntiae–H. lachancei, differed by four (0·7 % sequence divergence) and five (0·9 % sequence divergence) base substitutions, respectively, from H. opuntiae and H. lachancei. The D1/D2-based phylogenetic tree is available as Supplementary Fig. A in IJSEM Online.

The placement of the four novel species and CBS 8772 within the H. uvarum–H. guilliermondii cluster by D1/D2 sequence analysis was found to have a low level of statistical support (bootstrap values <70 %) and, therefore, the non-coding ITS regions, with higher rates of sequence substitution, were applied to resolve the phylogenetic position of these very closely related yeasts (James et al., 1996Go; Oda et al., 1997Go).

The ITS1–5·8S rDNA–ITS2 sequences of the type and other strains of Hanseniaspora–Kloeckera species were determined and used to construct a phylogenetic tree by the neighbour-joining method (Fig. 2Go). Our tree topology is consistent with that reported previously (Esteve-Zarzoso et al., 2001Go). A high bootstrap value (100 %) supported the placement of H. meyeri and H. clermontiae in close relationship to H. uvarum and H. lachancei, H. opuntiae and strain CBS 8772 to H. guilliermondii, which is in agreement with phenotypic similarities and DNA reassociation results (Table 2Go). The short branches imply very close phylogenetic relationships between species of the H. uvarum–H. guilliermondii cluster. Detailed sequence analysis of the ITS1 and ITS2 regions of the genetically closer pairs revealed that H. meyeri differed from H. clermontiae (57–66 % DNA relatedness) by a single base substitution, H. lachancei differed from H. opuntiae (53–55 % DNA relatedness) by nine base substitutions and Hanseniaspora vineae differed from Hanseniaspora osmophila [38–60 % DNA relatedness (Meyer et al., 1978Go); 33 %, this study] by 17 base substitutions. This lack of correspondence between the degree of sequence divergence and DNA reassociation data for Hanseniaspora species supports the observation of Seifert et al. (1995)Go, that the rate of evolution in the ITS region varies from species to species.



View larger version (21K):
[in this window]
[in a new window]
  		Fig. 2. Phylogenetic tree showing the relationships of Hanseniaspora–Kloeckera species inferred from ITS1–5·8S rDNA–ITS2 sequences. The tree was constructed by using the neighbour-joining analysis. Bootstrap percentages above 50 % from 1000 bootstrap replicates are shown. The outgroup species was Saccharomyces cerevisiae. Bar, 2 % sequence divergence.

 
Identification
The diagnostic key for identification of Hanseniaspora and Kloeckera species using standard yeast identification procedures was supplemented from Smith (1998b)Go and Barnett et al. (2000)Go as shown in Table 3Go.


View this table:
[in this window]
[in a new window]
  		Table 3. Diagnostic key for identification of Hanseniaspora and Kloeckera species

 
H. lachancei can be distinguished from H. guilliermondii, H. opuntiae and strain CBS 8772 by its ability to assimilate 2-keto-D-gluconate as a sole carbon source and from the other Hanseniaspora and Kloeckera species by the presence of growth at 37 °C. H. clermontiae can be differentiated from its sister species H. meyeri and H. uvarum by the absence of growth at 30 °C and from Hanseniaspora valbyensis by its ability to assimilate 2-keto-D-gluconate. Conventional physiological criteria cannot distinguish H. meyeri from H. uvarum or H. opuntiae and strain CBS 8772 from H. guilliermondii. For accurate identification of these species, sequencing of the ITS regions is needed. As an alternative to sequencing, a rapid identification method, PCR-RFLP of ITS regions, can be applied. Restriction of the ITS regions with enzyme MboII separates H. meyeri from H. uvarum or H. opuntiae from H. guilliermondii and HinfI separates strain CBS 8772 from H. guilliermondii (Supplementary Fig. B in IJSEM Online).

Species descriptions
Of the five novel species, only four are described below. One taxon, represented by a single strain, will not be introduced until additional isolates are available. Nevertheless, it is included in the identification key.

Group I

Latin diagnosis of Hanseniaspora meyeri sp. nov.
In medio liquido post 48 horas 25 °C cellulae apiculatae, ovoideae vel elongatae, 2·5–12·5x1·5–6·0 µm, singulae vel binae; gemmatione bipolari repruducentes. Post unum mensem annulus tenuis et sedimentum formantur. In agaro farina Zeae maydis confecto pseudomycelium rudimentarium. In quoque asco 2–4 ascosporae petasiformes. Glucosum et cellobiosum fermentantur. Glucosum, cellobiosum, salicinum, arbutinum, glucono-{delta}-lactonum, 2-ketogluconatum, D-glucitolum, ethylaminum, lysinum et cadaverinum assimilantur. Non assimilantur galactosum, L-sorbosum, D-glucosaminum, D-ribosum, D-xylosum, L-arabinosum, D-arabinosum, L-rhamnosum, sucrosum, maltosum, trehalosum, methyl {alpha}-D-glucosidum, melibiosum, lactosum, raffinosum, melezitosum, amylum solubile, glycerolum, erythritolum, ribitolum, xylitolum, L-arabinitolum, D-glucitolum, D-mannitolum, galactitolum, inositolum, acidum D-glucuronicum, acidum D-galacturonicum, acidum DL-lacticum, acidum succinicum, acidum citricum, methanolum et ethanolum. Maxima temperatura crescentiae 30 °C. Crescit in medio addito 10 % NaCl, 50 % glucoso et 0·1 % cycloheximido. G+C acidi deoxyribonucleati 36·6–37·4 mol%. Typus: CBS 8734T (=NRRL Y-27513T) ex pomo Sapindi sp. in Hawaii, isolatus. In collectione zymotica Centraalbureau voor Schimmelcultures, Trajectum ad Rhenum lyophilus depositus.

Description of Hanseniaspora meyeri sp. nov.
Hanseniaspora meyeri (mey'er.i. N.L. gen. n. meyeri honouring Piet Meyer, a young, promising South African scientist interested in taxonomy, who died too young).

In YM liquid medium after 48 h at 25 °C, cells are apiculate, ovoid to elongate, 2·5–12·5x1·5–6·0 µm, single or in pairs. Budding is bipolar. A sediment is present. After 1 month, a very thin ring is formed. After 1 month at 25 °C, the streak culture on malt agar is cream-coloured, butyrous, smooth, glossy, flat to slightly raised at the centre, with an entire to slightly undulate margin. On cornmeal agar, a rudimentary pseudomycelium is formed. Asci containing two to four hat-shaped ascospores are observed after 2 weeks on 5 % Difco malt extract agar at 25 °C. Glucose and cellobiose are fermented. The following carbon compounds are assimilated: glucose, cellobiose, salicin, arbutin, glucono-{delta}-lactone, 2-ketogluconate and D-gluconate. The following are not assimilated: galactose, L-sorbose, glucosamine, D-ribose, D-xylose, L-arabinose, D-arabinose, L-rhamnose, sucrose, maltose, trehalose, methyl {alpha}-D-glucoside, melibiose, lactose, raffinose, melezitose, starch, glycerol, erythritol, ribitol, xylitol, L-arabinitol, D-glucitol, D-mannitol, galactitol, myo-inositol, D-glucuronate, D-galacturonate, DL-lactate, succinate, citrate, methanol and ethanol. Assimilation of nitrogen compounds: positive for ethylamine, lysine and cadaverine; negative for sodium nitrate and sodium nitrite. Growth at 30 °C is positive; no growth at 35 °C. Growth on YM agar with 10 % NaCl is positive. Growth on 50 % glucose (w/w)/yeast extract agar is weak. Growth in the presence of 0·1 % cycloheximide is positive. Diazonium blue B reaction is negative. G+C content of nDNA (Tm) is 36·6–37·4 mol%.

The type, CBS 8734T (=NRRL Y-27513T), was isolated from fruit of Sapindus sp., Hawaii. It has been deposited in the Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands.

Group II

Latin diagnosis of Hanseniaspora clermontiae sp. nov.
In medio liquido post 48 horas 25 °C cellulae apiculatae, ovoideae vel elongatae, 3·5–18·0x2·5–5·0 µm, singulae vel binae; gemmatione bipolari repruducentes. Post unum mensem annulus tenuis et sedimentum formantur. In agaro farina Zeae maydis confecto pseudomycelium rudimentarium. In quoque asco, 2–4 ascosporae petasiformes. Glucosum et cellobiosum fermentantur. Glucosum, cellobiosum, salicinum, arbutinum, glucono-{delta}-lactonum, 2-ketogluconatum, D-glucitolum, ethylaminum, lysinum et cadaverinum assimilantur. Non assimilantur galactosum, L-sorbosum, D-glucosaminum, D-ribosum, D-xylosum, L-arabinosum, D-arabinosum, L-rhamnosum, sucrosum, maltosum, trehalosum, methyl {alpha}-D-glucosidum, melibiosum, lactosum, raffinosum, melezitosum, amylum solubile, glycerolum, erythritolum, ribitolum, xylitolum, L-arabinitolum, D-glucitolum, D-mannitolum, galactitolum, inositolum, acidum D-glucuronicum, acidum D-galacturonicum, acidum DL-lacticum, acidum succinicum, acidum citricum, methanolum et ethanolum. Maxima temperatura crescentiae 25 °C. Crescit in medio addito 10 % NaCl, 50 % glucoso et 0·1 % cycloheximido. G+C acidi deoxyribonucleati 35·7–37·2 mol%. Typus: CBS 8821T (=NRRL Y-27515T) isolatus ex infecta Clermontia sp. in Hawaii. In collectione zymotica Centraalbureau voor Schimmelcultures, Trajectum ad Rhenum lyophilus depositus.

Description of Hanseniaspora clermontiae sp. nov.
Hanseniaspora clermontiae (cler.mon'ti.ae. N.L. gen. n. clermontiae of Clermontia, the genus name of the host plant).

In YM liquid medium after 48 h at 25 °C, cells are apiculate, ovoid to elongate, 3·5–18·0x2·5–5·0 µm, single or in pairs. Budding is bipolar. A sediment is present. After 1 month, a very thin ring is formed. After 1 month at 25 °C, the streak culture on malt agar is cream-coloured, butyrous, smooth, glossy, flat to slightly raised at the centre, with an entire to slightly undulate margin. On cornmeal agar, a rudimentary pseudomycelium is formed. Asci containing two to four hat-shaped ascospores are observed after 2 weeks on 5 % Difco malt extract agar at 25 °C. Glucose and cellobiose are fermented. The following carbon compounds are assimilated: glucose, cellobiose, salicin, arbutin, glucono-{delta}-lactone, 2-ketogluconate and D-gluconate. The following are not assimilated: galactose, L-sorbose, glucosamine, D-ribose, D-xylose, L-arabinose, D-arabinose, L-rhamnose, sucrose, maltose, trehalose, methyl {alpha}-D-glucoside, melibiose, lactose, raffinose, melezitose, starch, glycerol, erythritol, ribitol, xylitol, L-arabinitol, D-glucitol, D-mannitol, galactitol, myo-inositol, D-glucuronate, D-galacturonate, DL-lactate, succinate, citrate, methanol and ethanol. Assimilation of nitrogen compounds: positive for ethylamine, lysine and cadaverine; negative for sodium nitrate and sodium nitrite. Growth at 25 °C is positive; no growth at 30 °C. Growth on YM agar with 10 % NaCl is positive. Growth on 50 % glucose (w/w)/yeast extract agar is weak. Growth in the presence of 0·1 % cycloheximide is positive. Diazonium blue B reaction is negative. G+C content of nDNA (Tm) is 35·7–37·2 mol%.

The type, CBS 8821T (=NRRL Y-27515T), was isolated from Clermontia sp. rot, Hawaii. It has been deposited in the Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands.

Group III

Latin diagnosis of Hanseniaspora lachancei sp. nov.
In medio liquido post 48 horas 25 °C cellulae apiculatae, ovoideae vel elongatae, 2·5–18·5x1·0–5·5 µm, singulae vel binae; gemmatione bipolari repruducentes. Post unum mensem annulus tenuis et sedimentum formantur. In agaro farina Zeae maydis confecto pseudomycelium rudimentarium. In quoque asco, 4 ascosporae petasiformes. Glucosum et cellobiosum fermentantur. Glucosum, cellobiosum, salicinum, arbutinum, glucono-{delta}-lactonum, acidum gluconicum, ethylaminum, lysinum et cadaverinum assimilantur. Non assimilantur galactosum, L-sorbosum, D-glucosaminum, D-ribosum, D-xylosum, L-arabinosum, D-arabinosum, L-rhamnosum, sucrosum, maltosum, trehalosum, methyl {alpha}-D-glucosidum, melibiosum, lactosum, raffinosum, melezitosum, amylum solubile, glycerolum, erythritolum, ribitolum, xylitolum, L-arabinitolum, D-glucitolum, D-mannitolum, galactitolum, inositolum, 2-ketogluconatum, acidum D-glucuronicum, acidum D-galacturonicum, acidum DL-lacticum, acidum succinicum, acidum citricum, methanolum et ethanolum. Maxima temperatura crescentiae 37 °C. Crescit in medio addito 10 % NaCl, 50 % glucoso et 0·1 % cycloheximido. G+C acidi deoxyribonucleati 34·8–35·6 mol%. Typus: CBS 8818T (=NRRL Y-27514T) isolatus e Agaves fermentatum in Mexico. In collectione zymotica Centraalbureau voor Schimmelcultures, Trajectum ad Rhenum lyophilus depositus.

Description of Hanseniaspora lachancei sp. nov.
Hanseniaspora lachancei (la.chan'ce.i. N.L. gen. n. lachancei honouring M.-A. Lachance, for his enormous contributions to yeast taxonomy and ecology).

In YM liquid medium after 48 h at 25 °C, cells are apiculate, ovoid to elongate, 2·5–18·5x1·0–5·5 µm, single or in pairs. Budding is bipolar. Sediment is present. After 1 month, a very thin ring and a sediment are formed. After 1 month at 25 °C, the streak culture on malt agar is cream-coloured, butyrous, smooth, glossy, flat to slightly raised at the centre, with an entire to slightly undulate margin. On cornmeal agar, a rudimentary pseudomycelium is formed. Asci containing four hat-shaped ascospores are observed on 5 % Difco malt extract agar at 25 °C. Glucose and cellobiose are fermented. The following carbon compounds are assimilated: glucose, cellobiose, salicin, arbutin, glucono-{delta}-lactone and D-gluconate. The following are not assimilated: galactose, L-sorbose, glucosamine, D-ribose, D-xylose, L-arabinose, D-arabinose, L-rhamnose, sucrose, maltose, trehalose, methyl {alpha}-D-glucoside, melibiose, lactose, raffinose, melezitose, starch, glycerol, erythritol, ribitol, xylitol, L-arabinitol, D-glucitol, D-mannitol, galactitol, myo-inositol, 2-ketogluconate, D-glucuronate, D-galacturonate, DL-lactate, succinate, citrate, methanol and ethanol. Assimilation of nitrogen compounds: positive for ethylamine, lysine and cadaverine; negative for sodium nitrate and sodium nitrite. Growth at 37 °C is positive, growth at 40 °C is negative. Growth on YM agar with 10 % NaCl is positive. Growth on 50 % glucose (w/w)/yeast extract agar is weak. Growth in the presence of 0·1 % cycloheximide is positive. Diazonium blue B reaction is negative. G+C content of nDNA (Tm) is 34·8–35·6 mol%.

The type, CBS 8818T (=NRRL Y-27514T), was isolated from fermenting agave juice, Mexico. It has been deposited in the Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands.

Group IV

Latin diagnosis of Hanseniaspora opuntiae sp. nov.
In medio liquido post 48 horas 25 °C cellulae apiculatae, ovoideae vel elongatae, 3·0–16·0x1·5–5·0 µm, singulae vel binae; gemmatione bipolari repruducentes. Post unum mensem annulus tenuis et sedimentum formantur. In agaro farina Zeae maydis confectopseudomycelium rudimentarium. In quoque asco, 4 ascosporae petasiformes. Glucosum et cellobiosum fermentantur. Glucosum, cellobiosum, salicinum, arbutinum, glucono-{delta}-lactonum, 2-ketogluconatum, acidum gluconicum, ethylaminum, lysinum et cadaverinum assimilantur. Non assimilantur galactosum, L-sorbosum, D-glucosaminum, D-ribosum, D-xylosum, L-arabinosum, D-arabinosum, L-rhamnosum, sucrosum, maltosum, trehalosum, methyl {alpha}-D-glucosidum, melibiosum, lactosum, raffinosum, melezitosum, amylum solubile, glycerolum, erythritolum, ribitolum, xylitolum, L-arabinitolum, D-glucitolum, D-mannitolum, galactitolum, inositolum, acidum D-glucuronicum, acidum D-galacturonicum, acidum DL-lacticum, acidum succinicum, acidum citricum, methanolum et ethanolum. Maxima temperatura crescentiae 37 °C. Crescit in medio addito 10 % NaCl, 50 % glucoso et 0·1 % cycloheximido. G+C acidi deoxyribonucleati 33·6–35·3 mol%. Typus: CBS 8733T (=NRRL Y-27512T) isolatus ex Opuntia megacantha in Hawaii. In collectione zymotica Centraalbureau voor Schimmelcultures, Trajectum ad Rhenum lyophilus depositus.

Description of Hanseniaspora opuntiae sp. nov.
Hanseniaspora opuntiae (op.un'ti.ae. N.L. gen. n. opuntiae of Opuntia, the genus name of the host plant).

In YM liquid medium after 48 h at 25 °C, cells are apiculate, ovoid to elongate, 3·0–16·0x1·5–5·0 µm, single or in pairs. Budding is bipolar. Sediment is present. After 1 month, a very thin ring and a sediment are formed. After 1 month at 25 °C, the streak culture on malt agar is cream-coloured, butyrous, smooth, glossy, flat to slightly raised at the centre, with an entire to slightly undulate margin. On cornmeal agar, a rudimentary pseudomycelium is formed. Asci containing four hat-shaped ascospores are observed on 5 % Difco malt extract agar at 25 °C. Glucose and cellobiose are fermented. The following carbon compounds are assimilated: glucose, cellobiose, salicin, arbutin, glucono-{delta}-lactone, 2-ketogluconate and D-gluconate. The following are not assimilated: galactose, L-sorbose, glucosamine, D-ribose, D-xylose, L-arabinose, D-arabinose, L-rhamnose, sucrose, maltose, trehalose, methyl {alpha}-D-glucoside, melibiose, lactose, raffinose, melezitose, starch, glycerol, erythritol, ribitol, xylitol, L-arabinitol, D-glucitol, D-mannitol, galactitol, myo-inositol, D-glucuronate, D-galacturonate, DL-lactate, succinate, citrate, methanol and ethanol. Assimilation of nitrogen compounds: positive for ethylamine, lysine and cadaverine; negative for sodium nitrate and sodium nitrite. Growth at 37 °C is positive, growth at 40 °C is negative. Growth on YM agar with 10 % NaCl is positive. Growth on 50 % glucose (w/w)/yeast extract agar is weak. Growth in the presence of 0·1 % cycloheximide is positive. Diazonium blue B reaction is negative. G+C content of nDNA (Tm) is 33·6–35·3 mol%.

The type, CBS 8733T (=NRRL Y-27512T), was isolated from Opuntia megacantha in Hawaii. It has been deposited in the Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands.


   ACKNOWLEDGEMENTS
 
We are indebted to Dr Marc-André Lachance (University of Western Ontario, Canada) for kindly supplying many isolates and DNA sequences and to Dr Dénes Dlauchy and Professor Tibor Deak (National Collection of Agricultural and Industrial Micro-organisms, Hungary) for providing cultures. Jernej Jakse is gratefully acknowledged for helpful advice pertaining to nucleotide sequencing.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Barnett, A. A., Payne, R. W. & Yarrow, D. (2000). Yeasts, Characteristics and Identification, 3rd edn. Cambridge: Cambridge University Press.

Boekhout, T., Kurtzman, C. P., O'Donnell, K. & Smith, M. T. (1994). Phylogeny of the yeast genera Hanseniaspora (anamorph Kloeckera), Dekkera (anamorph Brettanomyces), and Eeniella as inferred from partial 26S ribosomal DNA nucleotide sequences. Int J Syst Bacteriol 44, 781–786.[Abstract/Free Full Text]

Cadez, N., Raspor, P., De Cock, A. W. A. M., Boekhout, T. & Smith, M. Th. (2002). Molecular identification and genetic diversity within species of the genera Hanseniaspora and Kloeckera. FEMS Yeast Res 1, 279–289.[Medline]

Esteve-Zarzoso, B., Peris-Torán, M. J., Ramón, D. & Querol, A. (2001). Molecular characterisation of Hanseniaspora species. Antonie van Leeuwenhoek 80, 85–92.[CrossRef][Medline]

Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791.[CrossRef]

Felsenstein, J. (2000). PHYLIP – phylogenetic inference package, version 3.6 (alpha). University of Washington, Seattle, USA.

Gardes, M. & Bruns, T. D. (1993). ITS primers with enhanced specificity for basidiomycetes – application to the identification of mycorrhizae and rusts. Mol Ecol 2, 113–118.[Medline]

Golubev, W. I., Smith, M. T., Poot, G. A. & Kock, J. L. F. (1989). Species delineation in the genus Nadsonia Sydow. Antonie van Leeuwenhoek 55, 369–382.[CrossRef][Medline]

James, S. A., Collins, M. D. & Roberts, I. N. (1996). Use of an rRNA internal transcribed spacer region to distinguish phylogenetically closely related species of the genera Zygosaccharomyces and Torulaspora. Int J Syst Bacteriol 46, 189–194.[Abstract/Free Full Text]

Jin, L. & Nei, M. (1990). Limitations of the evolutionary parsimony method of phylogenetic analysis. Mol Biol Evol 7, 82–102.[Abstract]

Kurtzman, C. P. & Robnett, C. J. (1998). Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit (26S) ribosomal DNA partial sequences. Antonie van Leeuwenhoek 73, 331–371.[CrossRef][Medline]

Lachance, M. A. (1995). Yeast communities in natural tequila fermentation. Antonie van Leeuwenhoek 68, 151–160.[CrossRef][Medline]

Messner, R., Prillinger, H., Altmann, F., Lopandic, K., Wimmer, K., Molnár, O. & Weigang, F. (1994). Molecular characterization and application of random amplified polymorphic DNA analysis of Mrakia and Sterigmatomyces species. Int J Syst Bacteriol 44, 694–703.[Abstract/Free Full Text]

Meyer, S. A., Smith, M. Th. & Simione, F. P., Jr (1978). Systematics of Hanseniaspora Zikes and Kloeckera Janke. Antonie van Leeuwenhoek 44, 79–96.[CrossRef][Medline]

Möller, E. M., Bahnweg, G., Sandermann, H. & Geiger, H. H. (1992). A simple and efficient protocol for isolation of high molecular weight DNA from filamentous fungi, fruit bodies, and infected plant tissues. Nucleic Acids Res 20, 6115–6116.[Free Full Text]

Molnár, O., Messner, R., Prillinger, H., Stahl, U. & Slavikova, E. (1995). Genotypic identification of Saccharomyces species using random amplified polymorphic DNA analysis. Syst Appl Microbiol 18, 136–145.

Oda, Y., Yabuki, M., Tonomura, K. & Fukunaga, M. (1997). A phylogenetic analysis of Saccharomyces species by the sequence of 18S-28S rRNA spacer regions. Yeast 13, 1243–1250.[CrossRef][Medline]

O'Donnell, K. (1993). Fusarium and its near relatives. In The Fungal Holomorph: Mitotic, Meiotic and Pleomorphic Speciation in Fungal Systematics, pp. 225–233. Edited by D. R. Reynolds & J. W. Taylor. Wallingford, UK: CAB International.

Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.[Abstract]

Seifert, K. A., Wingfield, B. D. & Wingfield, M. J. (1995). A critique of DNA sequence analysis in the taxonomy of filamentous ascomycetes and ascomycetous anamorphs. Can J Bot 73, 760–767.

Smith, M. Th. (1998a). Hanseniaspora Zikes. In The Yeasts, a Taxonomic Study, 4th edn, pp. 214–220. Edited by C. P. Kurtzman & J. W. Fell. Amsterdam: Elsevier.

Smith, M. Th. (1998b). Kloeckera Janke. In The Yeasts, a Taxonomic Study, 4th edn, pp. 580–581. Edited by C. P. Kurtzman & J. W. Fell. Amsterdam: Elsevier.

Smith, M. T. & Poot, G. A. (1985). Conspecificity of Hanseniaspora nodinigri and Hanseniaspora vineae: comparison by DNA reassociation. Antonie van Leeuwenhoek 51, 151–153.[CrossRef][Medline]

Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–4882.[Abstract/Free Full Text]

White, T. J., Bruns, T., Lee, S. & Taylor, J. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR Protocols: a Guide to Methods and Applications, pp. 315–322. Edited by N. Innis, D. Gelfand, J. Sninsky & T. White. London: Academic Press.

Yarrow, D. (1998). Methods for the isolation, maintenance and identification of yeasts. In The Yeasts, a Taxonomic Study, 4th edn, pp. 77–100. Edited by C. P. Kurtzman & J. W. Fell. Amsterdam: Elsevier.




This article has been cited by other articles:


Home page
 Appl. Environ. Microbiol.Home page
A. A. Nisiotou, A. E. Spiropoulos, and G.-J. E. Nychas
Yeast Community Structures and Dynamics in Healthy and Botrytis-Affected Grape Must Fermentations
Appl. Envir. Microbiol., November 1, 2007; 73(21): 6705 - 6713.
[Abstract] [Full Text] [PDF]

Home page
 Appl. Environ. Microbiol.Home page
A. A. Nisiotou and G.-J. E. Nychas
Yeast Populations Residing on Healthy or Botrytis-Infected Grapes from a Vineyard in Attica, Greece
Appl. Envir. Microbiol., April 15, 2007; 73(8): 2765 - 2768.
[Abstract] [Full Text] [PDF]

Home page
 Int. J. Syst. Evol. Microbiol.Home page
N. Cadez, P. Raspor, and M. Th. Smith
Phylogenetic placement of Hanseniaspora-Kloeckera species using multigene sequence analysis with taxonomic implications: descriptions of Hanseniaspora pseudoguilliermondii sp. nov. and Hanseniaspora occidentalis var. citrica var. nov.
Int J Syst Evol Microbiol, May 1, 2006; 56(Pt 5): 1157 - 1165.
[Abstract] [Full Text] [PDF]

Home page
 Int. J. Syst. Evol. Microbiol.Home page
M.-A. Lachance, C. P. Ewing, J. M. Bowles, and W. T. Starmer
Metschnikowia hamakuensis sp. nov., Metschnikowia kamakouana sp. nov. and Metschnikowia mauinuiana sp. nov., three endemic yeasts from Hawaiian nitidulid beetles
Int J Syst Evol Microbiol, May 1, 2005; 55(3): 1369 - 1377.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplementary Figures and Table
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via CrossRef
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cadez, N.
Right arrow Articles by Smith, M. Th.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cadez, N.
Right arrow Articles by Smith, M. Th.
Agricola
Right arrow Articles by Cadez, N.
Right arrow Articles by Smith, M. Th.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
INT J SYST EVOL MICROBIOL MICROBIOLOGY J GEN VIROL
J MED MICROBIOL ALL SGM JOURNALS