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1 Russia Collection of Microorganisms, Institute for Biochemistry and Physiology of Micro-organisms, Russian Academy of Sciences, Pushchino 142290, Russia
2 Centro de Recursos Microbiologicos, Secção Autónoma de Biotecnologia, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal
3 Mendeleev Chemical–Technological University, Moscow 125820, Russia
Correspondence
Wladyslav I. Golubev
wig{at}ibpm.serpukhov.su
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The GenBank accession numbers for the 26S rDNA D1/D2 region sequences of C. perniciosus sp. nov. PYCC 5769T and C. nemorosus sp. nov. PYCC 5768T are AF472624 and AF472625.
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). On the basis of these observations, glucuronate medium was proposed for estimation of the density and species composition of hymenomycetous yeasts in natural communities (Golubev, 2000). With glucuronate agar, both higher counts and a wider diversity of such yeasts were revealed during the study of yeast communities from the phyllosphere and soil of the Prioksko-terrasny biosphere reserve (Moscow region, Russia) compared with the more commonly used malt extract or glucose/yeast extract/peptone agar.
Many of the isolates obtained with glucuronate agar were identified as Cryptococcus albidus (Saito) Skinner or Cryptococcus laurentii (Kufferath) Skinner according to taxonomic keys and criteria used for differentiation (Fell & Statzell-Tallman, 1998; Barnett et al., 2000). However, such identification results mainly from the large number of variable characteristics in the standard descriptions of these ubiquitous species. Yeast species that are easily identified usually exhibit specificity whereas, in many cases, yeasts that are considered ubiquitous are in fact heterogeneous assemblages of species that are difficult to identify by conventional methods. The taxonomic heterogeneity of Cryptococcus albidus and Cryptococcus laurentii has been recognized in molecular biochemical studies (Vancanneyt et al., 1994; Fonseca et al., 2000; Sugita et al., 2000). These two species are also heterogeneous with respect to sensitivity to mycocins (zymocins, killer toxins) secreted by tremelloid killer yeasts (W. I. Golubev, unpublished data). Mycocin typing showed that most of the isolates from the Prioksko-terrasny reserve differed greatly from the Cryptococcus albidus and Cryptococcus laurentii type strains. Subsequent closer inspection led us to the conclusion that the cultures represented undescribed species.
Strains
Strains VKM Y-2905T (isolate PTZ 59T) and VKM Y-2907 (isolate PTZ 441) were isolated, by plating on glucuronate agar, from turf and plants collected from a steppe plot with fescue/herbaceous vegetation in the Prioksko-terrasny biosphere reserve in September 1997 and 2000, respectively. Strain VKM Y-2906T (isolate PTZ 75T) was also isolated, on this medium, from herbaceous plants collected in an oak forest in the same reserve in September 1997. All other strains used (most of which were type strains) for the comparative studies or in tests for sensitivity to mycocins were from the Russia Collection of Microorganisms (VKM; http://www.vkm.ru/).
Morphological and physiological characterization
For morphological and physiological characterization, standard methods currently employed in yeast taxonomy were used (Yarrow, 1998). Micrographs were taken with an Olympus BX50 microscope, using phase-contrast optics, and an Olympus C-3030 Zoom digital camera. The ability to produce the brown and green colour effect was examined on Guizotia abyssinica creatinine agar (Staib, 1999).
Monosaccharide analysis
After hydrolysis of extracellular polysaccharides with 3 M trifluoroacetic acid (100 °C, 6 h), neutral sugar composition was detected with an LG-2000 carbohydrate analyser (Biotronic).
rDNA sequence analysis
For sequence analysis, total DNA was extracted according to the procedures described by Sampaio et al. (2001) and amplified using primers ITS5 and LR6. Cycle sequencing of the 600–650 bp region at the 5'-end of the 26S rDNA D1/D2 domain employed forward primer NL1 (5'-GCATATCAATAAGCGGAGGAAAAG-3') and reverse primer NL4 (5'-GGTCCGTGTTTCAAGACGG-3'). The internal transcribed spacer region was sequenced using the forward primer ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and the reverse primer ITS4 (5'-TCCTCCGCTTATTGATATGC-3'). Sequences were obtained with an Amersham Pharmacia ALF express II automated sequencer using standard protocols. Alignments were made with MegAlign (DNAStar) and corrected visually. PAUP* 4.0, version b8 (Swofford, 2000), was employed to perform phylogenetic analyses using the maximum-parsimony method. Bootstrap analyses were based on 1000 random resamplings (Felsenstein, 1985).
Assay for sensitivity to mycocins
The assay medium used to determine sensitivity to mycocins contained 0·5 % glucose, 0·25 % peptone, 0·2 % yeast extract, 5 % glycerol and 2 % agar with 0·05 M citrate/phosphate buffer (pH 4·5). A 0·05 ml aliquot of an aqueous suspension (105 cells ml-1) of each strain being examined for sensitivity was put on the surface of the agar medium and thoroughly dispersed with a spreader, and a loopful of strain VKM Y-2905T, VKM Y-2906T or VKM Y-2907 was then streaked on the plate. The plates were incubated at 18 °C until growth of the lawn strains appeared.
Phenotypic affiliation of isolates
The morphological and physiological properties of isolates PTZ-59T (=VKM Y-2905T), PTZ-75T (=VKM Y-2906T) and PTZ-441 (=VKM Y-2907) placed them in the genus Cryptococcus Vuillemin, based on the following characteristics: asexual reproduction, no fermentation, positive urease test, assimilation of i-inositol and D-glucuronate and production of starch-like compounds and xylose-containing extracellular polysaccharides. Strains VKM Y-2905T and VKM Y-2907 were identical in cultural, morphological and physiological characteristics. Strain VKM Y-2906T was similar, but it differed from the other two by the ability to grow at 30 °C and in the rate of utilization of some carbon sources (Table 1). The properties of all three cultures fit the standard description of Cryptococcus laurentii (Kufferath) Skinner (Fell & Statzell-Tallman, 1998; Barnett et al., 2000). However, the novel isolates were distinct from the type strain of this species in the assimilation of sorbose, trehalose, inulin, erythritol, glycine, creatine and creatinine and in the ability to produce the green colour effect (Table 2). No mating reactions were observed between the type strain of Cryptococcus laurentii and the novel isolates or among strains VKM Y-2905T, VKM Y-2906T and VKM Y-2907.
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). Taxonomic specificity of sensitivity to mycocins has already been shown (Golubev & Boekhout, 1995; Golubev et al., 1996; Boekhout & Scorzetti, 1997; Golubev, 1998; Buzzini & Martini, 2000) and the dissimilarities revealed suggest that the strains under study cannot be assigned to Cryptococcus laurentii but, instead, represent separate species.
Killing patterns
Initially, the novel isolates engaged our attention because their killer activity was expressed within the pH range 3·5–6·0 (citrate/phosphate buffer). Weak growth inhibition of sensitive yeasts was observed at pH 3·5 and 6·0, and there was no activity at pH 7·0. The broadest growth-inhibition zones developed at pH 4·5. The mycocins produced by these strains were active mainly against teleomorphs and anamorphs of the Filobasidiales and the Tremellales (Tables 3 and 4). Only a few of the anamorphs among the Cystofilobasidiales (Itersonilia perplexans, Tausonia pamirica, Udeniomyces pyricola, Udeniomyces puniceus) and the Trichosporonales (Trichosporon coremiiforme, Trichosporon cutaneum, Trichosporon dulcitum, Trichosporon moniliiforme, Trichosporon rugosum) were sensitive. All uredinomycetous and ustilaginomycetous yeasts as well as ascomycetous ones (62 species of 54 genera) were insensitive to the mycocins. The complete list of killing patterns is available at http://www.crem.fct.unl.pt/dimorphic_basidiomycetes/. The action spectrum of the mycocins secreted by strains VKM Y-2905T and VKM Y-2907 was significantly broader than the host range of the mycocin produced by strain VKM Y-2906T (Table 3). The type strain of Cryptococcus laurentii also produces a mycocin that shows fungicidal activity directed against filobasidiaceous and tremellaceous yeasts (Golubev & Kuznetsova, 1989). However, there were numerous differences in the killing patterns of the mycocins secreted by the novel isolates and Cryptococcus laurentii VKM Y-1665T (Table 4).
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were compared to confirm that the novel isolates were distinct from Cryptococcus laurentii and represent undescribed species. The sequences of the D1/D2 domain of the 26S rDNA were used to infer the phylogenetic position of strains VKM Y-2905T and VKM Y-2906T. The strains were found to be closely related to each other (four nucleotide differences) and to belong to the Cryptococcus laurentii (type strain) clade of the Tremellales. Fifteen to sixteen differences were observed in the D1/D2 region between Cryptococcus laurentii CBS 139T (=VKM Y-1665T) and the novel species. Strains VKM Y-2905T and VKM Y-2906T appear to have as their closest teleomorph (9–13 nucleotide differences) the recently described tremellaceous dimorphic fungus Papiliotrema bandonii (Sampaio et al., 2002). Mating reactions have not been observed between culture VKM Y-2917 of the latter and the novel isolates.
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Latin diagnosis of Cryptococcus nemorosus W. Golubev, Gadanho, Sampaio et N. Golubev sp. nov.
In aqua glucosum et extractum fermenti et peptonum continente, post dies 3 cellulae subglobosae et ovoidae (3·4–6·0x5·1–7·7 µm), incapsulatae, singulae vel binae. Post unum mensem sedimentum et annulus formantur. In agaro morphologico (Difco) post unum mensem cultura in striis griseoalbida, glabra, nitida, mucosa; margo integra. Ballistoconidia nullae. Nec pseudomycelium nec mycelium formatur. Non fermentat. Glucosum, galactosum, L-sorbosum (exigue), N-acetylglucosaminum, glucosaminum (lente), ribosum (lente), xylosum, L-arabinosum, D-arabinosum (lente), rhamnosum, sucrosum, maltosum, trehalosum, 
-methylglucosidum (lente), cellobiosum, salicinum, arbutinum, melibiosum, lactosum, raffinosum, melezitosum, inulinum (exigue), amylum (lente), xylanum (lente), glycerolum (lente), ribitolum, xylitolum, arabinitolum (exigue), glucitolum, mannitolum, galactitolum, inositolum, glucono-1,5-lactonum, 2-ketogluconatum, 5-ketogluconatum, glucuronatum, lactatum (exigue), succinatum, citratum, glucaratum et ethanolum assimilantur neque erythritolum, acidum quinicum et glycinum. Kalium nitricum, kalium nitrosum, creatinum et creatininum non assimilatur. Vitamina externa at crescentiam necessaria sunt. In medio cum 50 % glucoso crescit (exigue). Crescit in 30 °C, non crescit in 35 °C. Urea finditur. Materia amyloidea iodophila formantur. Mannosum, xylosum, galactosum et glucosum in polysaccharides externis demonstrable est. Typus VKM Y-2906T (=PYCC 5768T) conservatur in collectione microorganismorum Russian Academy of Sciences, Pushchino, Rossia et in collectione zymotica lusitanica.
Description of Cryptococcus nemorosus W. Golubev, Gadanho, Sampaio et N. Golubev sp. nov.
Cryptococcus nemorosus (ne.mo.ro'sus. L. adj. nemorosus pertaining to woods or groves, referring to the origin of the isolate).
After 3 days in glucose/yeast extract/peptone broth, cells are subglobose and oval [width/length ratio 1·1–1·6 (mean 1·3)], 3·4–6·0x5·1–7·7 µm (mean 4·5x6·0 µm) with capsules and single or in pairs (Fig. 2a). After 1 month, there is a sediment and a ring. After 1 month on yeast morphology agar (Difco), the streak culture is greyish white, smooth, glistening and slimy (running to the bottom of the slant); the border is entire. No ballistoconidia are observed. After 10 days in slide cultures on corn-meal agar, neither pseudomycelium nor true mycelium is produced. Fermentation is absent. Assimilation of carbon and nitrogen compounds is indicated in Table 1. Growth in vitamin-free medium is negative. Grows at 30 but not at 35 °C on malt extract agar. Grows on 50 % (w/w) glucose/yeast extract agar but not on 60 % (w/w) glucose/yeast extract agar. Growth in the presence of 10 % NaCl is positive (weak); no growth in the presence of 15 % NaCl. Hydrolysis of urea and production of starch-like compounds are positive. Monosaccharide composition of extracellular polysaccharides: mannose, xylose, galactose and glucose (molar ratio 1 : 0·5 : 0·3 : 0·2). Production of brown or green colour effects (Staib agar) is negative. Sensitive to mycocins produced by Bullera sinensis var. lactis VKM Y-2826, Cryptococcus laurentii VKM Y-1627, VKM Y-1628, VKM Y-1665, Cryptococcus podzolicus VKM Y-2247 and Cystofilobasidium bisporidii VKM Y-2700.
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Latin diagnosis of Cryptococcus perniciosus W. Golubev, Gadanho, Sampaio et N. Golubev sp. nov.
In aqua glucosum et extractum fermenti et peptonum continente, post dies 3 cellulae subglobosae et ovoidae (3·4–5·1x5·1–6·8 µm), incapsulatae, singulae vel binae. Post unum mensem sedimentum et annulus formantur. In agaro morphologico (Difco) post unum mensem cultura in striis albida aut cremea, glabra, nitida, mucosa; margo integra. Ballistoconidia nullae. Nec pseudomycelium nec mycelium formatur. Non fermentat. Glucosum, galactosum, L-sorbosum (lente), N-acetylglucosaminum, glucosaminum (exigue), ribosum (lente), xylosum, L-arabinosum, D-arabinosum (lente), rhamnosum (lente), sucrosum, maltosum, trehalosum, -methylglucosidum (lente), cellobiosum, salicinum, arbutinum, melibiosum, lactosum, raffinosum, melezitosum, amylum (lente), xylanum (lente), glycerolum (lente), ribitolum (lente), xylitolum, arabinitolum (exigue), glucitolum, mannitolum, galactitolum (lente), inositolum, glucono-1,5-lactonum, 2-ketogluconatum, 5-ketogluconatum, glucuronatum, lactatum (lente), succinatum, citratum (lente), glucaratum et ethanolum (exigue) assimilantur neque inulinum, erythritolum, acidum quinicum et glycinum. Kalium nitricum, kalium nitrosum, creatinum et creatininum non assimilatur. Vitamina externa at crescentiam necessaria sunt. In medio cum 50 % glucoso non crescit (exigue). Non crescit in 30 °C. Urea finditur. Materia amyloidea iodophila formantur. Mannosum, xylosum, galactosum et glucosum in polysaccharides externis demonstrable est. Typus VKM Y-2905T (=PYCC 5769T) conservatur in collectione microorganismorum Russian Academy of Sciences, Pushchino, Rossia et in collectione zymotica lusitanica.
Description of Cryptococcus perniciosus W. Golubev, Gadanho, Sampaio et N. Golubev sp. nov.
Cryptococcus perniciosus (per.ni.ci.o'sus. L. adj. perniciosus pernicious, referring to mycocin production by the isolates studied).
After 3 days in glucose/yeast extract/peptone broth, cells are subglobose and oval [width/length ratio 1·2–1·8 (mean 1·4)], 3·4–6·0x5·1–8·5 µm (mean 4·3x6·0 µm) with capsules, single or in pairs (Fig. 2b). After a month, there is a sediment and a ring. After 1 month on yeast morphology agar (Difco), streak cultures are whitish to cream-coloured, smooth, glistening and slimy (growth runs to the bottom of the slant); the border is entire. No ballistoconidia are observed. After 10 days in slide cultures on corn-meal agar, neither pseudomycelium nor true mycelium is produced. Fermentation is absent. Assimilation of carbon and nitrogen compounds is indicated in Table 1. Growth in vitamin-free medium is negative. Grows at 25 but not at 30 °C on malt extract agar. Grows on 50 % (w/w) glucose/yeast extract agar but not on 60 % (w/w) glucose/yeast extract agar. Growth in the presence of 10 % NaCl is positive (weak); no growth in the presence of 15 % NaCl. Hydrolysis of urea and production of starch-like compounds are positive. Monosaccharide composition of extracellular polysaccharides: mannose, xylose, galactose and glucose (molar ratio 1 : 0·6 : 0·4 : 0·1). Production of brown or green colour effects (Staib agar) is negative. Sensitive to mycocins produced by Bullera sinensis var. lactis VKM Y-2826, Cryptococcus laurentii VKM Y-1627, VKM Y-1628 and VKM Y-1665 and Cryptococcus podzolicus VKM Y-2247.
The type strain, PTZ 59T (=VKM Y-2905T=PYCC 5769T), was isolated from turf of a steppe plot in Prioksko-terrasny biosphere reserve (Moscow region, Russia).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Sampaio, J. P., Weiss, M., Gadanho, 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.
Staib, F. (1999). The green colour effect (GCE) of the killer strain Cryptococcus laurentii CBS 139 on Staib agar. Mycoses 42, 103–106.[Medline]
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Swofford, D. L. (2000). PAUP*. Phylogenetic Analysis Using Parsimony (*and Other Methods). Sunderland, MA: Sinauer.
Vancanneyt, M., Coopman, R., Tytgat, R., Hennebert, G. L. & Kersters, K. (1994). Whole-cell protein patterns, DNA base composition and coenzyme Q types in the yeast genus Cryptococcus Kutzing and related taxa. Syst Appl Microbiol 17, 65–75.
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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