Yeast Isolation and Maintenance

The localities, substrates of isolation, MycoBank numbers and GenBank accession numbers are summarized in Table 1 (see Information S1 for details).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Table 1. List of strains and DNA sequences used in this study.

https://doi.org/10.1371/journal.pone.0046060.t001

Phenotypic Characterization

Morphological and biochemical/physiological characterization of the isolates was performed according to Kurtzman et al. [15] and Barnett et al. [16].

DNA Sequencing and MSP-PCR (Microsatellite-primed PCR) Fingerprinting

The D1/D2 domain of the large subunit (26S) rRNA gene was sequenced as described by Kurtzman and Robnett [17]. The ITS region (ITS1, 5.8S rRNA gene and ITS2) was amplified and sequenced as described by Péter et al. [18]. Alignments and phylogenetic trees were constructed with MEGA 5 [19], using the neighbor joining method with bootstrap analysis based on 10,000 random samplings. MSP-PCR fingerprinting followed the protocols described in Sampaio et al. [20], and used the primers (GTG)₅ and M13. MSP-PCR profiles with each primer were repeated twice for inference of reproducibility. Gel electrophoresis images were acquired with the GelDoc XR System software (Bio-Rad).

Nomenclature

The electronic version of this article in Portable Document Format (PDF) in a work with an ISSN or ISBN will represent a published work according to the International Code of Nomenclature for algae, fungi, and plants, and hence the new names contained in the electronic publication of a PLOS ONE article are effectively published under that Code from the electronic edition alone, so there is no longer any need to provide printed copies.

In addition, new names contained in this work have been submitted to MycoBank from where they will be made available to the Global Names Index. The unique MycoBank number can be resolved and the associated information viewed through any standard web browser by appending the MycoBank number contained in this publication to the prefix http://www.mycobank.org/MB. The online version of this work is archived and available from the following digital repositories: PubMed Central; LOCKSS.

Proposal of New Genus

Data analysis of partial rDNA sequences allowed the recognition of a new clade of basidiomycetous yeast species that originated from different geographic locations (Table 1, Fig. 1). Most of the species within this clade are capable of glucose fermentation, and some species can ferment other sugars as well (Table 2, Table S1). Phylogenetically, the clade is located in the Bulleromyces clade of Tremellales sensu Scorzetti et al. [14], and is surrounded by species belonging to Cryptococcus, a well-recognized polyphyletic genus of the class Tremellomycetes [21]. Although fermentative ability is an uncommon feature in basidiomycetous yeasts, it has been reported in species of Mrakia, Mrakiella and Xanthophyllomyces [22]–[24], and in Filobasidium capsuligenum [25]. The current diagnosis of the genus Cryptococcus accommodates the inclusion of fermentative isolates, but there are no currently recognized Cryptococcus species with this ability [21]. Cryptococcus aquaticus was once considered a fermentative Cryptococcus, but it was transferred to the genus Mrakiella, as M. aquatica [26]. As classification of yeasts in monophyletic taxa is highly desirable, the proposal of a new genus for this clade is justified rather than describing new yeast species of Cryptococcus, which would increase the polyphyletic nature of this genus. As the generic name Cryptococcus will be confined to the C. neoformans clade [27], we propose a new monophyletic genus, Bandoniozyma, to accommodate those species. Hitherto, the new genus contains only anamorphic yeasts, but it cannot be ruled out that sexual stages could be discovered in the future.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 1. ITS tree showing the phylogenetic relationship among the Tremellomycetes, with emphasis on the Bandoniozyma clade, obtained by neighbor-joining analysis using Mega 5.0.

Most species belong to the order Tremellales, with the exception of Cystofilobasidium capitatum (Cystofilobasidiales), Cryptococcus albidus (Filobasidiales), Trichosporum cutaneum (Trichosporonales), and the outgroup Farysizyma itapuensis (Ustilaginales). The numbers given on the branches are the frequencies with which a given branch appeared in 10,000 bootstrap replications. Bar, substitutions per nucleotide position.

https://doi.org/10.1371/journal.pone.0046060.g001

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Table 2. Selected physiological/biochemical test responses for differentiation of the newly proposed Bandoniozyma species. The full data can be seen in Table S1.

https://doi.org/10.1371/journal.pone.0046060.t002

The proposed new genus forms a sister clade to the Auriculibuller/C. laurentii/C. flavescens complex. According to Scorzetti et al. [14], the Bulleromyces clade is weakly supported in both D1/D2 and ITS trees, and requires a detailed study of its biology and molecular systematics. Several new yeast species have been described recently in the Bulleromyces clade [28]–[32]. Additionally, genetic diversity among the strains previously identified as C. laurentii has been recognized [33], [34]. Finally, the large number of new species in the presently discussed fermentative basidiomycetous yeast group emphasizes the need for a taxonomic revision of the Tremellales.

Proposal of New Species

The species in the Bandoniozyma clade are mainly separated from each other based on the ITS sequences (Table 1, Figure 1), while the D1/D2 sequences of the 26S rDNA were less informative (showed 2 to 13 nucleotide substitutions). Pairwise comparisons between the species show that most of them have 14 to 29 ITS nucleotide substitutions with each other, except in the case of the comparisons between Bandoniozyma glucofermentans and Bandoniozyma aquatica, which will be discussed later. Intraspecific ITS variability is less than 2 nucleotide substitutions (viz., Bandoniozyma noutii CBS 8364^T, CBS 8365 and CBS 8368; Bandoniozyma tunnelae CBS 8024^T, CBS 6024 and CBS 6123; and B. glucofermentans CBS 10381^T and ATCC MYA-4761), unless in the case of B. complexa (CBS 11570^T, MA68d, CBS 12531, IMUFRJ 51948, CBS 12398 and UFMG-LD3.02), whose strains have 2 to 7 nucleotide substitutions in the pairwise comparisons. The biochemical/physiological profiles of all the species are given in Table 2 and Table S1. All the species assimilate D-glucose, D-xylose, L-rhamnose, sucrose, maltose, a-a-trehalose, cellobiose, D-glucitol, D-manitol, myo-inositol, D-gluconate, succinate, L-lysine, grow at 25°C and are DBB positive. None of the species ferment galactose, and assimilate methanol or nitrate.

The genus is comprised of two clearly separated groups of species (Figure 1). The first group is composed of Bandoniozyma tunnelae, Bandoniozyma noutii and Bandoniozyma visegradensis. The latter two species are represented by strains obtained mainly from plant exudates. It is significant to note that B. noutii is able to ferment sucrose and raffinose, which are two of the most common sugar components of plant exudates [35], [36]. While most species in the Bandoniozyma genus originated from tropical/subtropical areas and environmental samples, B. tunnelae and B. visegradensis are from Finland and Hungary, respectively, and B. tunnelae was obtained from a human-related substrate (Table 1).

Bandoniozyma glucofermentans was isolated from the gut of insects, while Bandoniozyma fermentans was isolated from an unidentified mushroom (Table 1). The type strain of B. glucofermentans was isolated from Amphix laevigatus (Coleoptera: Endomychidae), a mycophagous beetle [37]. Although an additional strain of B. glucofermentans was found from Canthon sp. (Coleoptera: Scarabaeidae), which is usually copro-necrophagous, some Canthon species have a generalist feeding behavior [38]. The hypothesis that B. glucofermentans and B. fermentans take part in the beetle/mushroom/yeast interaction model should be further investigated to evaluate if additional fermentative basidiomycetous yeasts occur in mushroom related sources. B. glucofermentans is closely related to Bandoniozyma aquatica, which differs from the former in the ITS sequence (5 substitutions), assimilation of D-arabinose, melibiose, raffinose, and growth in vitamin-free medium (Table 2). B. aquatica, like other Bandoniozyma strains discussed below, was isolated from lake water, suggesting that these yeasts can also be dispersed by water. The lake where this yeast was isolated is surrounded by Atlantic Rain Forest, and its occurrence in this aquatic environment could be considered the result of a run-off from plant or insects of this forest.

Bandoniozyma complexa is represented by 10 strains isolated from different substrates and geographical regions (Table 1). These strains group in three different MSP-PCR fingerprinting profiles with primers M13 (Figure S1) and GTG₅ (data not shown). MSP-PCR fingerprinting is a methodology that displays whole genome profiles, and isolates that belong to the same species usually present identical or similar fingerprints [39]. B. complexa group I differs from group II by 7 nucleotide substitutions in the ITS sequences, assimilation of starch, glycerol, erythritol, L-arabinitol, galactitol, citrate and growth in the presence of 0.01% cycloheximide, while group III differs from group I by 4 ITS nucleotide substitutions, glucose fermentation and nitrite assimilation, and from group II by 3 substitutions, glucose fermentation, assimilation of starch, glycerol, erythritol, citrate, nitrite and growth in the presence of 0.01% cycloheximide (data not shown). In particular, glucose fermentation is negative for strains in group III and strongly positive in less than three days for groups I and II. The biochemical tests were confirmed in two different laboratories. Group III strain CBS 12398 does not form pseudohyphae nor true hyphae on Dalmau plate culture on corn meal agar, while strains belonging to groups I and II do. The different MSP-PCR profiles with two independent primers and phenotypic traits suggest these groups are separate species, but D1/D2 and ITS sequencing could not clearly differentiate them. It is possible that the strains in this complex have recently diverged, and the ITS sequences may not be the best molecular marker for differentiation of these putative species. Attempts were made to sequence other genes (EF1-alpha and cytochrome B), but sequencing problems prevented conclusive results (Information S2).

Bandoniozyma complexa group I strains were isolated from air from a timber factory in South Brazil, which dealt mostly with wood of Pinus spp., but also processed Ficus and Eucalyptus trees (Table 1), suggesting that it may have been air-dispersed from a plant-related origin. B. complexa group III strain CBS 12398 was isolated from pineapple in Taiwan, while three other strains from this group were isolated from lake water within an Amazon Forest ecosystem in Northern Brazil (Table 1, Figure S1), thus suggesting dispersion from a primary plant-related substrate. The fact that B. complexa group III isolates were found in two geographically distant tropical countries indicates that this group may have a wide distribution in tropical environments.

Bandoniozyma complexa group II strains CBS 12531, BD143 and BD149 were isolated from a biofilm associated with a corroded aluminum screw from an energy transmission tower in Southeast Brazil (Table 1), which contained several other species of filamentous fungi and yeasts [40]. Strain CBS 12531^T was shown to be conspecific with strain IMUFRJ 51948 by means of D1/D2 and ITS sequences, and both were isolated from geographically close locations in Brazil. Considering that strain IMUFRJ 51948 was obtained from a bromeliad, and that many Bandoniozyma strains were isolated from plant substrates and lake water, it could be suggested that association of B. complexa group II with the corrosion biofilm occurred after dispersion from a primary environmental source, although a role in biofilm formation cannot be excluded. Fungal influenced corrosion of a variety of materials, such as metals, minerals and concrete, is well known [41], [42], and there are reports of isolation of yeasts from substrates damaged by corrosion [43], [44].

Although a sexual stage could not be observed for any of the proposed Bandoniozyma species, B. visegradensis strain CBS 12505^T formed septate hyphae and presented conjugating cells and tubes (Figure 2). Studies concerning the reproductive behaviour of B. visegradensis and isolation of more strains of this species will probably improve the current circumscription of this group of yeasts.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 2. Bandoniozyma visegradensis CBS 12505^T a) Growth on 5% malt extract agar, 3 days, 25°C, b) Septate hypha in 5% malt extract broth, 19 days, 25°C, c) Curved buds connecting independent cells (c.1) or mother cell bud pairs (c.2), YM agar, 10 days, 25°C, d) Conjugating cells, YM agar, 10 days, 25°C, e) Conjugation tubes, 2% malt extract agar, 18 days, 6°C. Bar = 10 µm.

https://doi.org/10.1371/journal.pone.0046060.g002

We hypothesize that the fermentation ability of the Bandoniozyma cluster was acquired after its separation from the non-fermentative C. laurentii/C. flavescens clade. Secondarily, B. complexa group III and B. visegradensis may have lost the fermentative capacity that is typical of the whole group. As glucose fermentation in yeasts is a two-step reaction mediated by the enzymes pyruvate decarboxylase and alcohol dehydrogenase [45], the alternative hypothesis of multiple independent origins for the fermentative ability of the species in this group is highly improbable. The fermentative capabilities of some Bandoniozyma species (i.e. B. noutii, B. tunnelae, B. fermentans, B. glucofermentans, B. aquatica, B. complexa groups I and II) are unusual for basidiomycetous yeasts due to the strong glucose fermentation at 25–28°C, which is comparable to some ascomycetous species that vigorously ferment sugars.

We isolated several fermentative basidiomycetous strains from plant substrates (leaf surface, exudates and flowers) and mushroom, in addition to other possible secondary substrates, associated with the dispersion of these yeasts, such as insects, air, water, corroded aluminum screw, and human nail. These substrates were mostly sampled in tropical/subtropical areas (Brazil, Panama and Taiwan), but a temperate country (Finland) also yielded fermentative strains (B. tunnelae strains CBS 8024^T, CBS 6024 and CBS 6123). As the sampling areas are globally distributed, it seems that the main premise for the presence of these yeasts is the availability of simple sugars as substrates for fermentation. We foresee that more fermentative basidiomycetous yeast strains will be reported as new simple sugar-containing substrates are sampled.

Bandoniozyma Boekhout, Valente, Pagnocca, Rosa, Lee, Suh, Blackwell, Péter, & Fell gen. nov

Valente et al. 2012, gen.nov. [urn:lsid:imycobank.org:names: MB 563851.

Budding cells are globose, subglobose, ovoid or ellipsoidal. Asexual reproduction is by polar or multilateral budding. Colonies are white, cream- colored to yellowish, smooth and butyrous or mucoid. Hyphae or pseudohyphae may be present. Clamp connections may be present. Sexual reproduction was not observed. Ballistoconidia are not produced. Fermentation of glucose is generally present. Diazonium blue B and urease reactions are positive. Growth on myo-inositol and D-glucuronate are positive, but growth on nitrate is negative. Starch-like compounds are generally formed. Type species: Bandoniozyma noutii.

Etymology: The genus is named in honour of Robert (Bob) J. Bandoni, University of British Columbia, who dedicated his life to the study of the Tremellales.

The following species are accepted in the genus and can be differentiated by ITS sequencing and the biochemical/physiological tests included in Table 2.

1. Bandoniozyma noutii Boekhout, Fell, Scorzetti & Theelen sp. nov

Valente et al. 2012, sp. nov. [urn:lsid:imycobank.org:names: MB 563852.

Etymology: The specific epithet noutii refers to Robert (Rob) Nout, investigator of traditionally fermented foods from South America, Africa and Asia.

After growth for 7 days in 2% glucose medium at 25°C, a sediment and film are formed. Cells are ovoid, subglobose to globose, 4–6×4–5 µm, usually with polar budding but also with multilateral budding, with buds that may adhere into short chains (Figure 3a). Pseudohyphae are present and fall apart in filaments that measure 28–50(−70)×2–4 µm, and eventually become somewhat thick-walled, irregularly broadened or somewhat broadened on one side. After 5 days on 5% malt extract agar at 25°C, colonies are 10 mm in diameter, convex, grayish cream-beige, shiny, strongly mucoid, smooth, and with an entire margin that may form sectors. Cells measure 3.3–7.8×3–7 µm. Under a cover glass filaments and hyphae occur with cells measuring 8–40×2–2.5 µm. On Dalmau plate on yeast morphology agar, pseudohyphae occur with cells measuring 8–40×2–2.5 µm. Sexual reproduction is absent. Mixing the three available strains on potato dextrose agar, oat meal agar, corn meal agar, malt extract agar, glucose-yeast extract agar, and yeast extract malt extract agar did not show any indication of a mating reaction. Physiological/biochemical test responses can be seen in Table 2 and Table S1.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 3. Cell morphologies: a) Bandoniozyma noutii CBS 8364^T after 7 days in YM broth at 25°C. Bar = 10 µm, b) Bandoniozyma tunnelae CBS 8024^T after 7 days in YM broth at 25°C. Bar = 10 µm, c) Bandoniozyma fermentans CBS 12399^T grown in YMA for 3 day at 25

°C. Bar = 10 µm, d) Bandoniozyma glucofermentans CBS 10381^T after 7 days in YM broth at 25°C. Bar = 10 µm, e) Bandoniozyma aquatica CBS 12527^T grown in YEPD broth for 3 days at 25°C. Bar = 10 µm, and f) Bandoniozyma complexa CBS 11570^T grown in YEPD broth for 3 days at 25°C. Bar = 10 µm.

https://doi.org/10.1371/journal.pone.0046060.g003

Strains investigated: CBS 8364^T ( = DBVPG 4489^T), CBS 8365 ( = DBVPG 4490), from exudate of Eriobotrya japonica (Rosaceae), Tijuca Forest, RJ, Brazil; and CBS 8368 ( = DBVPG 4499), from Flower of Pimenta dioica (Myrtaceae), Pau da Fome, Pedra Branca, RJ, Brazil, all isolated by G. Capriotti, DBVPG. The type strain has been deposited in Centraalbureau voor Schimmelcultures (CBS) and DBVPG Industrial Yeasts culture collections.

2. Bandoniozyma aquatica Brandão, Valente, Pimenta & Rosa sp. nov

Valente et al. 2012, sp. nov. [urn:lsid:imycobank.org:names: MB 563857.

Etymology: The specific epithet aquatica refers to the habitat (freshwater) from which this species was isolated.

After growth in yeast extract-malt extract-peptone-glucose broth at 25°C for 3 days, the cells are globose to subglobose, 3.1–4.7×2.8–4.9 µm, and occur singly or in pairs (Figure 3e). Asexual reproduction is by multilateral budding. Sediment is present. After 7 days at 25°C on yeast extract-malt extract-peptone-glucose agar, streak cultures are cream, butyrous, rugose, and glistening. On Dalmau plate cultures on corn meal agar after 10 days at 25°C, pseudohyphae are formed. Sexual reproduction was not observed. Ballistoconidia are not produced. Physiological/biochemical test responses can be seen in Table 2 and Table S1.

Strain investigated: UFMG-DH-4.20^T ( = CBS 12527^T, ATCC MYA-4876^T), from a freshwater sample, Ecological Reserve of Rio Doce, Brazil, isolated by L. Brandão. The type strain has been deposited in Universidade Federal de Minas Gerais (UFMG), American Type Culture Collection (ATCC) and Centraalbureau voor Schimmelcultures (CBS) culture collections.

3. Bandoniozyma complexa Landell, Pagnocca, Sette, Passarini, Garcia, Ribeiro, Lee, Brandão, Rosa & Valente sp. nov

Valente et al. 2012, sp. nov. [urn:lsid:imycobank.org:names: MB 801195.

Etymology: The specific epithet complexa refers to the phenotypic and genotypic variation observed in the strains presently maintained in the species.

In glucose-peptone-yeast extract broth or yeast extract-malt extract-peptone-glucose after 3 to 4 days at 25°C, the asexual cells are globose to subglobose and ovoid, and occur singly or in pairs, 1.8–4.6×2.0–4.3 µm, and occur singly or in pairs (Figure 3f). After 1 week on glucose-peptone-yeast extract agar or yeast extract-malt extract-peptone-glucose agar at 25°C, the streak culture is white, creamy, smooth, butyrous and glistening. After 3 weeks in Dalmau plate culture on cornmeal agar, pseudohyphae and true hyphae formation are variable. Sediment is formed. Asexual reproduction is by multilateral budding. Sexual reproduction was not observed. Mixing the available strains on corn meal agar did not show any indication of a mating reaction after 6 days at 25°C. On yeast extract-malt extract-peptone-glucose agar after 3 days at 25°C, colonies are smooth, mucoid to butyrous, glistening and cream-colored, and have an entire margin. Ballistoconidia are not produced. Physiological/biochemical test responses can be seen in Table 2 and Table S1.

Strains investigated: CBS 11570^T ( = ATCC MYA-4603^T, MA28a ^T) and MA68d, from air samples in Rio Grande do Sul, Brazil, isolated by J. Crestani; CBS 12531 ( = CBMAI 1003), from a corroded screw from an energy transmission tower in Suzano, SP, Brazil, isolated by M. Passarini; IMUFRJ 51948, from the bromeliad Neoregelia cruenta in Rio de Janeiro, Brazil, isolated by K. Garcia; CBS 12398 ( = BCRC 23285, PL04), from pineapple, Hsinchu, Taiwan, isolated by C-F Lee; UFMG-LR3.11, from freshwater of Lago Rico Lake, Parque Estadual do Cantão,TO, Brazil; UFMG-LD2.09 and UFMG-LD3.02, from freshwater of Lago de Dentro Lake, Parque Estadual do Cantão,TO, Brazil, all isolated by R. Pimenta & L.R. Brandão. The type strain has been deposited in American Type Culture Collection (ATCC) and Centraalbureau voor Schimmecultures (CBS) culture collections.

4. Bandoniozyma fermentans Lee sp. nov

Valente et al. 2012, sp. nov. [urn:lsid:imycobank.org:names: MB 563855.

Etymology: The specific epithet fermentans refers to the ability of the species to ferment glucose and other sugars.

After growth in yeast extract-malt extract-peptone-glucose broth at 25°C for 3 days, the cells are globose to subglobose, 1.9–4.4×2.3–4.6 µm, and occur singly or in pairs (Figure 3c). Asexual reproduction is by multilateral budding. Sediment is present. After 7 days at 25°C on yeast extract-malt extract-peptone-glucose agar, streak cultures are creamy, butyrous, smooth, and glistening. On Dalmau plate cultures on corn meal agar after 10 days at 25°C, neither pseudohyphae nor true hyphae are formed under the cover glass. Sexual reproduction was not observed. Ballistoconidia are not produced. Physiological/biochemical test responses can be seen in Table 2 and Table S1.

Strain investigated: CBS 12399^T ( = BCRC 23267^T, NU7M71^T), from fruiting body of an unidentified mushroom, Beinan, Taitung, Taiwan, isolated by C-F Lee. The type strain has been deposited in Bioresources Collection and Research Center (BCRC), Food Industry Research and Development Institute, Taiwan; and Centraalbureau voor Schimmelcultures (CBS) culture collections.

5. Bandoniozyma glucofermentans Suh & Blackwell sp. nov

Valente et al. 2012, sp. nov. [urn:lsid:imycobank.org:names: MB 563856.

Etymology: The specific epithet glucofermentans refers to the character of the species to ferment glucose.

In yeast extract-malt extract-peptone-glucose broth after 7 days at 25°C, cells are globose, subglobose, or ovoid, 2.5–5.0×2.5–6.5 µm, and occur singly or in pairs (Figure 3d). Pseudohyphae are present. On yeast extract-malt extract-peptone-glucose agar after 7 days at 25°C, colonies are cream colored, smooth, mucoid with a slightly filamentous edge. After 10 days of growth on Dalmau plate culture on cornmeal agar at 25°C, pseudohyphae and true hyphae are present. Aerobic growth is white to cream colored with a slightly fuzzy margin. Sexual reproduction was not observed. Sediment is formed. Ballistoconidia are not produced. Physiological/biochemical test responses can be seen in Table 2 and Table S1.

Strains investigated: CBS 10381^T ( = ATCC MYA-4760^T, NRRL Y-48076^T, BG 02-7-15-015A-1-1^T), from the gut of Amphix laevigatus (Coleoptera: Endomychidae), Barro Colorado Island, Panama; and ATCC MYA-4761 ( = NRRL Y-48077, BG 02-7-16-015A-1-1), from the gut of Canthon sp. (Coleoptera: Scarabaeidae), Barro Colorado Island, Panama, all isolated by S-O Suh and M. Blackwell. The type strain has been deposited in American Type Culture Collection (ATCC), Centraalbureau voor Schimmelcultures (CBS), and ARS culture collections (NRRL).

6. Bandoniozyma tunnelae Boekhout, Fell, Scorzetti & Theelen sp. nov

Valente et al. 2012, sp. nov. [urn:lsid:imycobank.org:names: MB 563853.

Etymology: The specific epithet tunnelae refers to Dr. E. Tunnela, Finland, who isolated the strains.

After growth for 7 days in 2% glucose medium at 25°C, a sediment is formed, cells are ellipsoidal, ovoid, subglobose to globose, 5–9×2.5–7 µm (Figure 3b), usually with polar budding but also with multilateral budding, with clavate budding cells that may adhere into short chains; with pseudohyphae that fall apart in filaments that measure 28–50(−70)×2–4 µm, eventually becoming somewhat thick-walled, irregularly broadened or somewhat broadened on one side. After 5 days on 5% malt extract agar at 25°C colonies are 25–35 mm in diameter, flat to somewhat raised, cream-colored, shiny, mucoid, smooth, and with an entire to somewhat eroded margin. Cells are ovoid, subglobose to globose, 3.8–5.5×3.3–6 µm, with polar to multipolar budding. Under a cover glass filaments and hyphae occur with cells that measure 20–70×2–5 µm. On Dalmau plate on yeast morphology agar extensive hyphae and loosely branched pseudohyphae occur that laterally form blastoconidia near the septa, with cells measuring 15–30×2–3×4–7 µm; usually broadened at one end and remain catenulate; the broadened part may give rise to globose to ellipsoidal thick-walled chlamydospore-like cells that may release through endosporulation. Sexual reproduction is absent. Mixing the three available strains on potato dextrose agar, oat meal agar, corn meal agar, malt extract agar, glucose-yeast extract agar, and yeast extract malt extract agar did not show any indication of a mating reaction. The whole cell hydrolyzates of CBS 6024 contain glucose (main), galactose, mannose, xylose, arabitol, mannitol and glucuronic acid. Physiological/biochemical test responses can be seen in Table 2 and Table S1.

Strains investigated: CBS 8024^T ( = DBVPG 7000^T), from human nail in Finland; CBS 6024 ( = DBVPG 6992, PYCC 4857), from unknown source; and CBS 6123 ( = DBVPG 6993), from unknown source, all isolated by E. Tunnela. The type strain has been deposited in Centraalbureau voor Schimmelcultures (CBS) and DBVPG Industrial Yeasts culture collections.

7. Bandoniozyma visegradensis Péter & Dlauchy sp. nov

Valente et al. 2012, sp. nov. [urn:lsid:imycobank.org:names: MB 563854.

Etymology: The specific epithet visegradensis refers to Visegrád, Hungary, the place where the type strain was isolated.

After 3 days on 5% malt extract agar at 25°C, the streak culture is mucoid, cream-colored to yellowish, smooth, slightly raised and glistening. The margin is entire. Cells are formed by multilateral budding, subspheroid, ovoid or ellipsoid, 3–9×4–13 µm, and occur singly and in pairs (Figure 2a). In 5% malt extract after 3 days at 25°C, a compact sediment is present, but a pellicle is absent. Following 1–3 weeks of incubation short pseudohyphal and septate hyphal fragments are present. The septate hyphae may bear clamps (or pseudoclamps) (Figure 2b) and the cells of the pseudohyphae may be connected by curved buds reminiscent of clamps of dikaryotic hyphae. After 3 weeks an incomplete climbing pellicle is present. On Dalmau plate culture on corn meal agar after 7 days at 25°C, pseudohyphae and true hyphae are absent, and conjugation tubes are present. Following prolonged incubation on several agar media, including 2% malt extract, corn meal, potato dextrose and yeast extract-malt extract-peptone-glucose agars, characteristic curved buds are interconnecting some cells. The connected cells may be independent (Figure 2c.1) or mother cell-bud pairs (Figure 2c.2). In addition, conjugating tubes and conjugations were also observed. The conjugation tubes may be simple (Figure 2d) or twisting and occasionally are ramified (Figure 2e). Formation of basidia and basidiospores was not observed even after 10 weeks incubation. The additional media acetate agar, glucose-peptone-yeast extract agar and vegetable juice agar (V8) were also applied to try to induce sexual state following incubation at 25°C for 10 weeks. The formation of conjugation tubes was abundant upon isolation of the strain, but has decreased during maintenance after subsequent subculturing. The formation of the conjugation tubes could be stimulated by incubating the cultures at lower temperatures (6°C or 15°C) and could be fully restored by freezing and thawing the strain in liquid nitrogen (i.e. freezing in 10% glycerol, direct immersion in nitrogen without stepwise cooling, thawing after 1 day in 37°C water bath). Ballistoconidia are not produced. Physiological/biochemical test responses can be seen in Table 2 and Table S1.

Strain investigated: CBS 12505^T ( = NRRL Y-48783^T, NCAIM Y.01952^T), from exudate of oak (Quercus cerris) in the Pilis Mountains near Visegrád, Hungary, isolated by G. Péter. The type strain has been deposited in the National Collection of Agricultural and Industrial Microorganisms in Budapest (Hungary), Centraalbureau voor Schimmelcultures (CBS) and ARS culture collections (NRRL).