| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Department of Microbial, Biochemical and Food Biotechnology, University of the Free State, PO Box 339, Bloemfontein 9301, South Africa
2 Centre for Confocal and Electron Microscopy, University of the Free State, PO Box 339, Bloemfontein 9301, South Africa
Correspondence
Carolina Pohl
PohlCH.sci{at}mail.uovs.ac.za
 |
ABSTRACT |
|---|
 TOP ABSTRACT MAIN TEXTREFERENCES |
|---|
-D-glucoside, salicin, lactose, erythritol, ribitol, xylitol, citrate and ethanol. In addition, no extracellular starch production was observed and the isolate was able to grow in the absence of additional vitamins. On the basis of these results, we suggest that the new strain represents a novel species for which the name Cryptococcus anemochoreius sp. nov. is proposed [type strain CBS 10258T (=NRRL Y-27920T)].
An additional phylogenetic tree based on the sequences of the internal transcribed spacer region of strain CBS 10258T and related species is available as a supplementary figure in IJSEM Online.
|
MAIN TEXT |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
). Most species produce extracellular starch and those that do not, utilize inositol. It is well known that the genus Cryptococcus is polyphyletic, occurring in the Tremellales, Trichosporonales, Filobasidiales and Cystofilobasidiales (Fell et al., 2000; Scorzetti et al., 2002). In addition, members of this genus have been isolated from a diverse range of habitats worldwide and several reports exist for the isolation of Cryptococcus species, such as Cryptococcus aerius, Cryptococcus albidus, Cryptococcus flavus, Cryptococcus laurentii, Cryptococcus luteolus and Cryptococcus magnus, from the atmosphere (Fell & Statzell-Tallman, 1998). It is also known that the pathogenic Cryptococcus species, Cryptococcus neoformans, often occurs in the air as small, respirable particles (Ruiz & Bulmer, 1981; Sukroongreung et al., 1999). Due to the prevalence of HIV in the mining workforce of South Africa, a study of the diversity of culturable airborne fungi (which may pose health risks to immunocompromised workers) was conducted in an active gold mine. During this study, comparative samples of the atmosphere outside were taken in order to establish a possible source of the fungal species found indoors. Of the isolates obtained from the atmosphere outside the mine, 25.9 % were yeasts. The majority of these isolates belong to known species in the genera Candida, Rhodotorula, Filobasidium and Cryptococcus. However, a novel anamorphic basidiomycetous isolate was obtained and we propose the name Cryptococcus anemochoreius sp. nov. for this isolate on the basis of morphological, physiological and molecular characteristics.
Since this species was isolated from the atmosphere, it is difficult to speculate on the ecology of C. anemochoreius. However, trees belonging to the genera Pinus, Rhus, Schinus and Eucalyptus grow in the vicinity of the sampling site and since several reports exist regarding the association of Cryptococcus species with trees, especially Eucalyptus (Ellis & Pfeiffer, 1990; Sorrell & Ellis, 1997; Fortes et al., 2001; Randhawa et al., 2001; Granados & Castaneda, 2005), it is tempting to speculate that C. anemochoreius may be associated with one or more of these trees.
Yeast isolation and characterization
Air samples were taken in June 2004 in the Free State province in central South Africa using a SAS Super 90 single stage impactor. The sampling time was 30 seconds at 30 l min–1. In order to facilitate the isolation of yeasts, rose Bengal agar (5.0 g l–1 peptone, 10.0 g l–1 glucose, 1.0 g l–1 potassium phosphate, 0.5 g l–1 magnesium sulphate, 0.05 g l–1 rose Bengal, 0.1 g l–1 chloramphenicol, 15.0 g l–1 agar) was used. Plates were incubated at 25 °C and yeast colonies were purified on 2 % yeast–malt extract (YM) agar. Morphological and physiological tests were performed in duplicate according to standard techniques (Yarrow, 1998). In addition, scanning electron microscopy was performed on the isolate using a JEOL 6400 WIN-SEM instrument (Van Wyk & Wingfield, 1994).
rDNA sequence analyses
The 26S rDNA D1/D2 and internal transcribed spacer regions were PCR amplified and sequenced using primer pair NL1 (5'-GCATATCAATAAGCGGAGGAAAAG-3') and NL4 (5'-GGTCCGTGTTTCAAGACGG-3') (Kurtzman & Robnett, 1998) and primer pair ITS4 (5'-TCCTCCGCTTATTGATATGS-3') and ITS5 (5'-GGAAGTAAAAGTCGTAACAAGG-3') (White et al., 1990), respectively. The resulting sequence was compared with sequences obtained from GenBank. Sequences for phylogenetic analysis were aligned using CLUSTAL_X 1.83 (Thompson et al., 1997). Phylogenetic and molecular evolutionary analyses were conducted with MEGA version 3.1 (Kumar et al., 2004) using the neighbour-joining method with the Kimura two-parameter distance measure. Confidence values were estimated from bootstrap analysis of 1000 replicates. The three species of Filobasidium were used as outgroup.
Species delineation and identification
The D1/D2 sequence was compared with related sequences and phylogenetic analyses (Fig. 1) indicated that Cryptococcus anemochoreius represents a novel species in the Cryptococcus laurentii phylogenetic group 1 as described by Sugita et al. (2000) and Takashima et al. (2003). Within the C. laurentii phylogenetic group 1, C. anemochoreius occupies a relatively isolated position with Papiliotrema bandonii (20 nucleotide differences), Cryptococcus perniciosus (25 nucleotide differences), Cryptococcus nemorosus (21 nucleotide differences) and Cryptococcus sp. CBS 8363 (17 nucleotide differences) forming a subclade of nearest relatives. The relatively low bootstrap value between C. anemochoreius and the P. bandonii subclade (of 68 %) may increase with the isolation of more strains related to C. anemochoreius. Sequence data and phylogenetic analysis of the internal transcribed spacer region confirmed the positioning of C. anemochoreius in the C. laurentii phylogenetic group 1 and supported the recognition of C. anemochoreius as a novel isolate (a phylogenetic tree based on the internal transcribed spacer sequences is available as Supplementary Fig. S1 in IJSEM Online). Physiologically Cryptococcus anemochoreius can be distinguished from these related taxa as indicated in Table 1. Based on these differences, we conclude that the new isolate represents a novel species, Cryptococcus anemochoreius sp. nov.
|
|
-methyl-D-glucosidum, salicinum, lactosum, glycerolum, erythritolum, ribitolum, xylitolum, L-arabinitolum, citratum, propan-1,2-diolum, butan-2,3-diolum, acidum malatum et acidum butyratum non assimilantur. Non crescit in methanolo vel ethanolo. L-Lysinum, cadaverinum et ethylaminum assimilantur neque nitratum, nitritum, creatininum, creatinum, glucosaminum et imidazolum. Ureum finditur. Materia amyloidea non formatur. Vitaminum externum crescentiae non necessarium. In medio 0.01 % cycloheximidum vel 1 % acidum aceticum vel 50 % glucosum continente non crescit. Non crescit 37 °C. Typus praeservatus in collectione zymotica Centraalbureau voor Schimmelcultures, Utrecht, Neerlandia (holotypus lyophilizatus CBS 10258T) et in collectione zymotica Agricultural Research Services United States Department of Agriculture, Peoris, Illinois (isotypus NRRL Y-27920T).
Description of Cryptococcus anemochoreius Pohl, J. L. F. Kock, P. W. J. van Wyk & Albertyn sp. nov.
Cryptococcus anemochoreius (a.ne.mo'cho.rei.us. Gr. n. anemos wind; Gr. adj. choreios of, or belonging to, a dance; N.L. masc. adj. anemochoreius referring to the windblown nature of the isolate).
In yeast-malt extract (YM) broth after 3 days of growth at 25 °C, cells are globose to elongate (2.8–6.4 µmx2.3–3.7 µm). Polar budding is observed and cells occur as parent bud pairs (Fig. 2). On YM agar after 7 days at 25 °C, colonies are smooth with an entire margin and salmon coloured. In Dalmau plate cultures on YM agar, no pseudohyphae or true hyphae can be observed. No fermentation is observed. Able to assimilate D-glucose, D-galactose, L-sorbose, D-glucosamine, D-ribose (weakly), D-xylose, L-arabinose, D-arabinose, sucrose, maltose, trehalose, cellobiose, arbutin, melibiose, raffinose, melezitose, inulin, starch, D-glucitol, D-mannitol, galactitol, myo-inositol, D-gluconate, D-glucuronate, D-galacturonate, DL-lactate (weakly) and succinate (weakly), but not L-rhamnose, methyl -D-glucoside, salicin, lactose, glycerol, erythritol, ribitol, xylitol, L-arabinitol, citrate, propane-1,2-diol, butane-2,3-diol, malic acid or butyric acid. No growth is observed on methanol and ethanol as carbon sources. L-Lysine, cadaverine and ethylamine are utilized as nitrogen sources, but not nitrate, nitrite, creatinine, creatine, glucosamine or imidazole. Urea is hydrolysed, but no extracellular starch is produced. Vitamins are not necessary for growth, however, no growth is observed in the presence of cycloheximide (0.01 %), 1 % acetic acid or 50 % glucose. No growth is observed at 37 °C.
|
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Fell, J. W. & Statzell-Tallman, A. (1998). Cryptococcus Vuillemin. In The Yeasts, a Taxonomic Study, 4th edn, pp. 742–1767. Edited by C. P. Kurtzman & J. W. Fell. Amsterdam: Elsevier.
Fell, J. W., Boekhout, T., Fonseca, A., Scorzetti, G. & Statzell-Tallman, A. (2000). Biodiversity and systematics of basidiomycetous yeasts as determined by large-subunit rDNA D1/D2 domain sequence analysis. Int J Syst Evol Microbiol 50, 1351–1371.[Abstract]
Fortes, S. T., Lazera, M. S., Nishikawa, M. M., Macedo, R. C. & Wanke, B. (2001). First isolation of Cryptococcus neoformans var. gattii from a native jungle tree in the Brazilian Amazon rainforest. Mycoses 44, 137–140.[CrossRef][Medline]
Golubev, W. I., Gadanho, M., Sampaio, J. P. & Golubev, N. W. (2003). Cryptococcus nemorosus sp. nov. and Cryptococcus perniciosus sp. nov., related to Papiliotrema Sampaio et al. (Tremellales). Int J Syst Evol Microbiol 53, 905–911.
Granados, D. P. & Castaneda, E. (2005). Isolation and characterization of Cryptococcus neoformans varieties recovered from natural sources in Bogota, Colombia, and study of ecological conditions in the area. Microb Ecol 49, 282–290.[CrossRef][Medline]
Kumar, S., Tamura, K. & Nei, M. (2004). MEGA3: Integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5, 150–163.
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]
Randhawa, H. S., Mussa, A. Y. & Khan, Z. U. (2001). Decaying wood in tree trunk hollows as a natural substrate for Cryptococcus neoformans and other yeast-like fungi of clinical interest. Mycopathologia 151, 63–69.[CrossRef][Medline]
Ruiz, A. & Bulmer, G. S. (1981). Particle size of airborne Cryptococcus neoformans in a tower. Appl Environ Microbiol 41, 1225–1229.
Sampaio, J. P., Weiss, M., Gananho, M. & Bauer, R. (2002). New taxa in the Tremellales: Bulleribasidium oberjochense gen. et sp. nov., Papiliotrema bandonii gen. et sp. nov. and Fibulobasidium murrhardtense sp. nov. Mycologia 94, 873–887.
Scorzetti, G., Fell, J. W., Fonseca, A. & Statzell-Tallman, A. (2002). Systematics of basidiomycetous yeasts: a comparison of large subunit D1/D2 and internal transcribed spacer rDNA regions. FEMS Yeast Res 1497, 1–23.
Sorrell, T. C. & Ellis, D. H. (1997). Ecology of Cryptococcus neoformans. Rev Iberoam Micol 14, 42–43.[Medline]
Sugita, T., Takashima, M., Ikeda, R., Nakase, T. & Shinoda, T. (2000). Intraspecies diversity of Cryptococcus laurentii as revealed by sequences of internal transcribed spacer regions and 28S rRNA gene and taxonomic position of C. laurentii clinical isolates. J Clin Microbiol 38, 1468–1471.
Sukroongreung, S., Eampokalap, B., Tansuphaswadikul, S., Nilakul, C. & Tantimavanich, S. (1999). Recovery of Cryptococcus neoformans from the nasopharynx of AIDS patients. Mycopathologia 143, 131–134.[CrossRef]
Takashima, M., Sugita, T., Shinoda, T. & Nakase, T. (2003). Three new combinations from the Cryptococcus laurentii complex: Cryptococcus aureus, Cryptococcus carnescens and Cryptococcus peneaus. Int J Syst Evol Microbiol 53, 1187–1194.
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.
Van Wyk, P. W. J. & Wingfield, M. J. (1994). Ultrastructure of ascus arrangement and ascospore development in Ophiostoma seticolle. Mycologia 86, 607–614.
White, T. J., Bruns, T., Lee, S. & Taylor, J. W. (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 M. A. Innis, D. H. Gelfand, J. J. Sninsky & T. J. White. San Diego, CA: 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.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
