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1 Philipps-University Marburg, Department of Biology, Plant Ecology, Karl-von-Frisch-Straße, 35043 Marburg, Germany
2 Department of Biology, University of Western Ontario, London, Ontario, Canada N6A 5B7
Correspondence
Michael Brysch-Herzberg
Herzberg{at}students.uni-marburg.de
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AJ620185 and AJ620186.
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MAIN TEXT |
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), and later expanded to include four species from flowers and associated insects (Lachance et al., 1998, 2000). Together with a number of asexual species in the genus Candida, the genus Wickerhamiella forms a loose monophyletic clade of species that exhibit a relatively low value of DNA relatedness in the D1/D2 region (Lachance et al., 1998). Most species in the clade are associated with insects (Lachance et al., 2000). We now describe Candida bombiphila sp. nov.
The strains were isolated from the proboscis of a Bombus terrestris queen and from the honey pots in a nest of Bombus pascuorum bees. Isolation was performed by direct plating of the honey and by stroking the proboscis of a B. terrestris queen on YM agar (Yarrow, 1998) supplemented with chloramphenicol (100 mg l–1). The yeasts were purified and characterized using standard methods (Yarrow, 1998). Physiological tests were performed using replica plating (Lachance, 1987). Sexual cross-reactivity was evaluated by mixing actively growing cultures on YM agar, 10 % (w/w) malt-extract agar and on restricted growth medium (Yarrow, 1998). The mixtures were incubated at room temperature and observed periodically with an Axiophot microscope (Zeiss). Images were recorded digitally by the means of a Mega Fire electronic camera (Intas) and optimized for brightness and contrast with the PHOTOSHOP software package (Adobe).
The D1/D2 domain of the large-subunit rDNA was amplified from whole cells and then sequenced as previously described by Lachance et al. (1999). Known sequences for other species were retrieved from GenBank. The DNAMAN package (lynnon biosoft) was used to edit and align the sequences with the CLUSTAL W algorithm (Thompson et al., 1994), and to construct trees with the neighbour-joining algorithm.
Latin diagnosis of Candida bombiphila Brysch-Herzberg & Lachance sp. nov.
Cultura in extracto malti post dies 3 ad 25 °C cellulae globosae ad ellipsoideae (2·7–4·0x1·9–2·5 µm), singulae aut binae. Cultura in agaro malti post dies 10 ad 25 °C cremea et butyrosa. Pseudomycelium et mycelium verum formantur. Glucosum fermentatur. L-Sorbosum, D-ribosum, glycerolum, mannitolum, glucitolum, acidum succinicum, acidum citricum, acidum malicum (lente), acidum gluconicum, glucono-
-lactonum assimilantur, at non inulinum, sucrosum, raffinosum, melibiosum, galactosum, lactosum, trehalosum, maltosum, melizitosum, methyl 
-D-glucosidum, amylum, cellobiosum, salicinum, L-rhamnosum, D-xylosum, L-arabinosum, D-arabinosum, methanolum, ethanolum, erythritolum, ribitolum, xylititolum, galactitolum, inositolum, acidum lacticum, 2-keto-D-gluconatum, D-glucosaminum, N-acetyl-D-glucosaminum, acidum glucuronicum nec hexadecanum. Natrium nitricum, natrium nitrosum, ethylaminum, lysinum et cadaverinum assimilantur. Ad crescentiam vitaminae necessariae sunt. Augmentum ad 37 °C. Habitat Bombus spp. in Germania.
Typus in collectione zymotica Centraalbureau voor Schimmelcultures, Trajectum ad Rhenum, sub no. CBS 9712T deposita est.
Description of Candida bombiphila Brysch-Herzberg & Lachance sp. nov.
Candida bombiphila (bom.bi'phi.la. N.L. nom. fem. adj. bombiphila Bombus-loving, in reference to the source of isolation, bumblebees).
In 5 % (w/w) malt extract after 3 days at 25 °C, the cells are ovoid, single or in parent-bud pairs and 2·7–4·0 µmx1·9–2·5 µm (Fig. 1a). On malt agar (10 %, w/w) after 10 days at 25 °C, colonies are cream-coloured, butyrous, convex to umbonate, with a smooth and glossy surface and an entire edge. Occasionally the colonies are convoluted with a lobate edge. Pseudomycelium is formed after 1 day on YM agar at 25 °C (Fig. 1b). True mycelium is formed after 2 weeks on malt agar (10 %, w/w) (Fig. 1c, d). Sexual activity has not been observed. Glucose is fermented weakly. Carbon compounds L-sorbose, D-ribose, glycerol, mannitol, glucitol, succinic acid, citric acid, malate (slow), D-gluconate and glucono--lactone are assimilated; no growth occurs on inulin, sucrose, raffinose, melibiose, galactose, lactose, trehalose, maltose, melezitose, methyl -D-glucoside, starch, cellobiose, salicin, L-rhamnose, D-xylose, L-arabinose, D-arabinose, methanol, ethanol, erythritol, ribitol, xylitol, galactitol, inositol, glucuronate, DL-lactic acid, 2-keto-D-gluconate, D-glucosamine, N-acetyl-D-glucosamine or hexadecane. Nitrate, nitrite, ethylamine, lysine and cadaverine are assimilated. Diazonium blue B reaction is negative. Growth on vitamin-free medium is negative. Growth on amino-acid-free medium is positive. Growth at 37 °C is positive. Acid production on chalk agar is slow. Gelatin liquefaction is negative. Casein hydrolysis is weak. Starch formation is negative. Growth on 60 % glucose/yeast extract agar is positive. Growth on YM agar with 5 % (w/w) NaCl is slow and growth on 10 % (w/w) NaCl is negative. No growth was observed in the presence of 0·01 % cycloheximide.
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Species delineation and identification
The D1/D2 sequence of the large-subunit rDNA of the type strain differs from the most closely related species, W. domercqiae, by 57 substitutions and three gaps (Fig. 2). Strain CBS 9713 (AJ620186) differs from the type strain by one substitution and one gap. Kurtzman & Robnett (1998) showed that in most cases distinct species differ by 1 % or more in these sequences; we conclude that our strains represent a novel, well-separated species. The origin and significance of strains with the designation UWOPS in Fig. 2 have been discussed by Lachance et al. (2001). They were included in the analysis for comparison and will be described as part of other studies. The sequence for strain NRRL Y-17858 was deposited in GenBank by Kurtzman & Robnett (1998).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Lachance, M. A. (1987). Approaches to yeast identification. In Yeast Biotechnology, pp. 33–51. Edited by D. R. Berry, I. Russell & G. G. Stewart. London: Allen & Unwin.
Lachance, M. A., Rosa, C. A., Starmer, W. T., Schlag-Edler, B., Barker, J. S. F. & Bowles, J. M. (1998). Wickerhamiella australiensis, Wickerhamiella cacticola, Wickerhamiella occidentalis, Candida drosophilae and Candida lipophila, five new related yeast species from flowers and associated insects. Int J Syst Bacteriol 48, 1431–1443.
Lachance, M. A., Bowles, J. M., Starmer, W. T. & Barker, J. S. F. (1999). Kodamaea kakaduensis and Candida tolerans, two new ascomycetous yeast species from Australian Hibiscus flowers. Can J Microbiol 45, 172–177.[CrossRef][Medline]
Lachance, M. A., Bowles, M. J., Mueller, C. & Starmer, W. T. (2000). On the biogeography of yeasts in the Wickerhamiella clade and description of Wickerhamiella lipophila sp. nov., the teleomorph of Candida lipophila. Can J Microbiol 46, 1145–1148.[CrossRef][Medline]
Lachance, M. A., Starmer, W. T., Rosa, C. A., Bowles, J. M., Barker, J. S. F. & Janzen, D. H. (2001). Biogeography of the yeasts of ephemeral flowers and their insects. FEMS Yeast Res 1, 1–8.[Medline]
Thompson, J. G., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673–4680.
van der Walt, J. P. & Liebenberg, N. V. D. W. (1973). The yeast genus Wickerhamiella gen. nov. (Ascomycetes). Antonie van Leeuwenhoek 39, 121–128.[Medline]
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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