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Int J Syst Evol Microbiol 56 (2006), 301-303; DOI  10.1099/ijs.0.63671-0
© 2006 International Union of Microbiological Societies

Geotrichum vulgare sp. nov., a novel asexual arthroconidial yeast

Michael Wuczkowski1, Chris Bond2 and Hansjörg Prillinger1

1 Austrian Center of Biological Resources and Applied Mycology (ACBR), Institute of Applied Microbiology (IAM), University of Natural Resources and Applied Life Sciences, Nußdorfer Lände 11, 1190 Wien, Austria
2 National Collection of Yeast Cultures (NCYC), Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, UK

Correspondence
Michael Wuczkowski
mwuczkow{at}edv1.boku.ac.at


   ABSTRACT
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 ABSTRACT
MAIN TEXT
 REFERENCES
 
Two strains of a novel yeast species were isolated from different habitats, from soil in an alluvial zone national park in Austria and from a drain in a Turkish soft drinks factory. Analysis of the nucleotide sequences of the D1/D2 region of their large-subunit rDNAs and PCR fingerprints show that the strains are members of the same species, described as Geotrichum vulgare sp. nov. Analysis of nucleotide sequences showed that this species is related to the ascogenous genus Galactomyces. The closest phylogenetic relative is Geotrichum silvicola, a recently described species. The type strain of Geotrichum vulgare is HA1379T (=CBS 10073T=NRRL Y-27915T).

Published online ahead of print on 26 August 2005 as DOI 10.1099/ijs.0.63671-0.

The GenBank/EMBL/DDBJ accession number for the large-subunit rRNA gene sequence of Geotrichum vulgare sp. nov. HA1379T is AJ511334.

A supplementary figure showing the appearance of arthroconidia of Geotrichum vulgare sp. nov. HA1379T is available in IJSEM Online.


   MAIN TEXT
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 ABSTRACT
 MAIN TEXT
REFERENCES
 
The anamorphic genus Geotrichum Link: Fries consists of 12 species having their teleomorphs in the genera Dipodascus Lagerheim and Galactomyces Redhead and Malloch (Barnett et al., 2000Go; de Hoog et al., 1986Go, 1998aGo, bGo; Kurtzman & Robnett, 1998Go; Pimenta et al., 2005Go). One species, Geotrichum silvicola Pimenta, Prasad, Lachance et Rosa, was described recently (Pimenta et al., 2005Go). Four strains of this species were isolated from flies of the genus Drosophila and two strains were found on larvae of the oak tasar silk worm, Antheraea proylei. The D1/D2 region of the large-subunit rDNA of Geotrichum silvicola differs by 15 substitutions from that of an undescribed species, designated by Kurtzman & Robnett (1998)Go as Galactomyces sp. NRRL Y-6418.

In this paper, a novel species of Geotrichum is described consisting of two strains from different habitats. One strain was isolated from soil in an alluvial zone forest in Austria and the other originated from a drain in a soft drinks factory in Turkey.

Yeast isolation and characterization
Strain HA1379T was collected in November 1999 from soil in an alluvial zone forest national park (‘Nationalpark Donauauen’) along the river Danube downstream from Vienna, Austria, near Mannswörth, as part of a biodiversity study on microfungi and yeasts (Wuczkowski et al., 2003aGo, bGo). The sampling and isolation procedures are described in Wuczkowski et al. (2003aGo, b)Go. Strain NCYC 3138 was isolated from a drain in a soft drinks factory in Turkey and deposited in the NCYC by Dr M. Stratford (Unilever Research, UK). Both strains were characterized by standard methods (Yarrow, 1998Go).

Sequencing and PCR-fingerprinting
Sequencing of the D1/D2 region of the 26S rDNA and PCR fingerprinting were performed as described in Wuczkowski et al. (2003aGo, b)Go.

Species delineation and identification
Strains HA1379T and NCYC 3138 show identical nucleotide sequences in the D1/D2 region of the 26S rDNA. The results of PCR fingerprinting (Fig. 1Go) show a high degree of similarity between the two strains and therefore it is highly likely that these strains belong to the same species. After growth on sporulation media (potato-dextrose agar, cornmeal agar, malt extract agar), asci were not found.



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  		Fig. 1. PCR fingerprints of the two isolates of Geotrichum vulgarewith three different primers. M, Length marker.

 
The nearest relative to the novel species is Geotrichum silvicola, a recently described species which belongs to a clade of several Galactomyces species (Pimenta et al., 2005Go). Geotrichum silvicola shows 13 substitutions within the D1/D2 region of the 26S rDNA when compared with the strain HA1379T.

The sequence was compared with already published sequences retrieved from GenBank via the CLUSTAL_X program (Thompson et al., 1997Go). A neighbour-joining tree was constructed with the TREECON software package (Van de Peer & De Wachter, 1994Go), using the Kimura model (Kimura, 1980Go). Bootstrap values were calculated by 500 replications (Fig. 2Go). On the basis of the results of this study, it is evident that the new strains represent a novel species for which the name Geotrichum vulgare is proposed with strain HA1379T as the type strain.



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  		Fig. 2. Neighbour-joining tree based on nucleotide sequences of the D1/D2 region of the 26S rDNA of Geotrichum vulgare and its relatives. Percentage bootstrap values (shown at branch nodes) were obtained from 500 replications. Bar, 5 % sequence divergence.

 
Typical characteristics for the members of this clade and also for Geotrichum vulgare are the assimilation of D-glucose, D-galactose, L-sorbose, glycerol, DL-lactate, succinate and ethanol as the carbon source. Some differences were observed when the novel species was compared with Galactomyces geotrichum. The novel species does not use ribitol, D-mannitol, D-glucono-1,5-lactone or citrate. In order to separate Geotrichum vulgare from Geotrichum silvicola, its closest relative, assimilation of D-mannitol and citrate can be used, as Geotrichum vulgare does not assimilate these substrates. In addition, Geotrichum vulgare does not grow at 37 °C.

Latin diagnosis of Geotrichum vulgare Wuczkowski, Bond et Prillinger sp. nov.
Cultura in agaro malti post dies 7 (24 °C) plana, sicca, capillata et candida. In agaro Solanum tuberosum et glucosum post dies 7 mycelium verum et arthroconidia formantur. Asci nec ascosporae non formantur. Glucosum non fermentatur. D-Glucosum, D-galactosum, L-sorbosum, D-xylosum, sucrosum (var.), glycerolum, D-glucitolum, 2-keto-D-gluconatum (lente), DL-lactatum (lente), succinatum, ethanolum, propan-1,2-diolum, butan-2,3-diolum et assimilantur, at non D-glucosaminum, D-ribosum, L-arabinosum, D-arabinosum, L-rhamnosum, maltosum, {alpha},{alpha}-trehalosum, methyl {alpha}-D-glucosidum, cellobiosum, salicinum, arbutinum, melibiosum, lactosum, raffinosum, melezitosum, inulinum, amylum solubile, erythritolum, ribitolum, xylitolum, L-arabinitolum, D-mannitolum, galactitolum, myo-inositolum, D-glucono-1,5-lactonum, 5-keto-D-gluconatum, D-gluconatum, D-glucuronatum, citratum et methanolum. Ethylaminum et cadaverinum assimilantur et non natrium nitricum, natrium nitrosum et lysinum. Ad crescentiam vitaminae externae non necessariae sunt. Augmentum in 30 °C, at non 37 °C. Typus HA1379T, collectione zymotica Austrian Center of Biological Resources and Applied Mycology (ACBR) (=CBS 10073T=NRRL Y-27915T).

Description of Geotrichum vulgare Wuczkowski, Bond & Prillinger sp. nov.
After 7 days on malt extract-yeast extract agar at 24 °C, colonies are white, flat, dry and powdery to finely hairy. Arthroconidia are abundantly present (see Fig. 3Go and Supplementary Fig. S1. in IJSEM Online). Hyphae are 3–7 µm wide with early disarticulation into cubic arthroconidia. Arthroconidia are 3–5 µm wide and 5–70 µm long. On slide cultures with potato-dextrose agar after 1 week, abundant true mycelia and arthroconidia are formed. Glucose is not fermented. The following carbon compounds are assimilated: D-glucose, D-galactose, L-sorbose, D-xylose, sucrose (variable), glycerol, D-glucitol, 2-keto-D-gluconate (weak), DL-lactate (weak), succinate, ethanol, propane-1,2-diol and butane-2,3-diol. No growth occurs on D-glucosamine, D-ribose, L-arabinose, D-arabinose, L-rhamnose, maltose, {alpha},{alpha}-trehalose, methyl {alpha}-D-glucoside, cellobiose, salicin, arbutin, melibiose, lactose, raffinose, melezitose, inulin, starch, erythritol, ribitol, xylitol, L-arabinitol, D-mannitol, galactitol, myo-inositol, D-glucono-1,5-lactone, 5-keto-D-gluconate, D-gluconate, D-glucuronate, citrate or methanol. The following nitrogen compounds are assimilated: ethylamine and cadaverine but not nitrate, nitrite or lysine. Growth in vitamin-free medium is positive. Growth at 30 °C and weak growth at 35 °C; no growth at 37 °C. No growth in 10 % sodium chloride or in 50 % glucose. Starch is not produced. Growth occurs at 0·01 % cycloheximide, growth at 0·1 % is variable. Urease activity is negative, Diazonium blue B test is negative. No production of extracellular amyloid compounds. Strain habitats include soil from an alluvial zone forest (Austria) and a drain in a Turkish soft drinks factory.



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  		Fig. 3. Differential interference contrast micrograph of Geotrichum vulgare HA1379T. Vegetative cells and hyphae after 1 week growth on potato-dextrose agar at 25 °C. Bar, 20 µm.

 
The type strain of Geotrichum vulgare, HA1379T (=CBS 10073T=NRRL Y-27915T), was isolated from soil. The epithet vulgare (common) refers to the diverse habitats from where the strains have been isolated.


   ACKNOWLEDGEMENTS
 
We thank Steve James and Ian Roberts at the NCYC for determining the 26S rDNA sequence of NCYC 3138 and for critical appraisal of the manuscript.


   REFERENCES
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 ABSTRACT
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Barnett, J. A., Payne, R. W. & Yarrow, D. (2000). Yeasts: Characteristics and Identification, 3rd edn, pp. 388–394. Cambridge: Cambridge University Press.

De Hoog, G. S., Smith, M. T. & Guého, E. (1986). A revision of the genus Geotrichum and its teleomorphs. Stud Mycol 29, 1–131.

De Hoog, G. S., Smith, M. T. & Guého, E. (1998a). Galactomyces Redhead & Malloch. In The Yeasts, a Taxonomic study, 4th edn, pp. 209–213. Edited by C. P. Kurtzman & J. W. Fell. Amsterdam: Elsevier.

De Hoog, G. S., Smith, M. T. & Guého, E. (1998b). Geotrichum Link: Fries. In The Yeasts, a Taxonomic study, 4th edn, pp. 574–579. Edited by C. P. Kurtzman & J. W. Fell. Amsterdam: Elsevier.

Kimura, M. (1980). A simple model for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16, 111–120.[CrossRef][Medline]

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]

Pimenta, R. S., Alves, P. D. D., Corrêa, A., Jr, Lachance, M.-A., Prasad, G. S., Rajaram, Sinha, B. R. R. P. & Rosa, C. A. (2005). Geotrichum silvicola sp. nov., a novel asexual arthroconidial yeast species related to the genus Galactomyces. Int J Syst Evol Microbiol 55, 497–501.[Abstract/Free Full Text]

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

Van de Peer, Y. & De Wachter, R. (1994). TREECON for Windows: a software package for the construction and drawing of evolutionary trees for the Microsoft Windows environment. Comput Appl Biosci 10, 569–570.[Free Full Text]

Wuczkowski, M., Druzhinina, I., Gherbawy, Y., Klug, B., Prillinger, H. & Kubicek, C. P. (2003a). Species pattern and genetic diversity of Trichoderma in a mid-European, primeval floodplain-forest. Microbiol Res 158, 125–133.[Medline]

Wuczkowski, M., Sterflinger, K., Kraus, G. F., Klug, B. & Prillinger, H. (2003b). Diversity of microfungi and yeasts in soils of the alluvial zone national park along the river Danube downstream of Vienna, Austria (‘Nationalpark Donauauen’). Austrian J Agricult Res 54, 109–117.

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.





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