Rhodotorula subericola sp. nov., an anamorphic basidiomycetous yeast species isolated from bark of Quercus suber (cork oak)

Collection site and isolation of the strains

Cork was sampled in the province of Badajoz (Extremadura, Spain). Badajoz is in the west of Spain and occupies a surface area of 1500 km². The weather is continental with influence from the Atlantic Ocean, characterized by mild winters with minimum mean temperatures of 3.2 °C and maximum mean temperatures of 13.9 °C. Summers are warm with minimum mean temperatures of 17.0 °C and maximum mean temperatures of 34.3 °C. Badajoz has a rainy season from October to April with precipitation between 50 and 80 mm, while the rest of the year it is below 30 mm. Cork is produced by Quercus suber L. (Fam. Cupuliferae) and consists of the exterior layers of the bark beneath the epidermis, which develop extraordinarily in these species, becoming thick and of the peculiar spongy consistency which characterizes cork. Cork is extracted every 11 years from the trees. Then, cork planks are stored in the fields for some time, even years, before they are used to make cork stoppers.

Yeast strains Y-31^T and Y-20B were isolated in Spain from bark of cork oak at the initial stages of the manufacturing process of cork stoppers (Álvarez-Rodríguez et al., 2003).

Phenotypic characterization of yeast isolates

Morphology of the yeasts was studied using a light microscope (Nikon Eclipse E800). Physiological and morphological characterization of the strains was performed according to standard methods (Yarrow, 1998).

Sequencing and phylogenetic analysis

Yeast cells picked from 48-h-old colonies were directly used in PCR reactions. The D1 and D2 domains of the 26S rRNA gene were amplified using the external primers NL-1 and NL-4 (O'Donnell, 1993), and the internal transcribed spacers ITS and the 5.8S ribosomal gene were amplified using the external primers ITS-1 and ITS-4 (White et al., 1990). PCR reactions were performed in a PROGENE thermocycler (Techne) as follows: a first denaturation step at 95 °C for 5 min, followed by 40 cycles of 94 °C for 40 s, 55 °C for 40 s and 72 °C for 30 s, with a final extension of 10 min at 72 °C. The PCR products were cleaned with the Perfectprep Gel Cleanup (Eppendorf) and then directly sequenced using the BigDye terminator version 3.1 cycle sequencing kit (Applied Biosystems), following the manufacturer's instructions, in an Applied Biosystems automatic DNA sequencer, model 310. Primers NL-1 and NL-4 were used in the sequencing reactions to read both DNA strands of D1 and D2 domains of the 26S rRNA gene, and primers ITS-1 and ITS-4 were used in the sequencing reactions to read both DNA strands of the ITS1–5.8S–ITS2 rDNA region.

Sequences of the D1/D2 region of the 26S rRNA gene were edited and assembled using mega version 3.1 software (Kumar et al., 2004), and then subjected to a GenBank blastn search to retrieve sequences of closely related taxa.

The sequences of the D1/D2 region of the 26S rRNA gene were included in a multiple alignment generated using mega version 3.1. The Kimura two-parameter model was used for distance correction, and the neighbour-joining method (Saitou & Nei, 1987) was used for phylogenetic inference. Support for tree branches was evaluated by bootstrap analysis from 1000 heuristic searches (mega version 3.1). Sequence AF189901 representing the D1/D2 ribosomal region of Kondoa aeria CBS 8352^T was used as the tree outgroup.

Phylogenetic position of Y-31^T and Y-20B and phenotypic comparison with closely related species

Sequence variability within Y-31^T and Y-20B was absent in the D1/D2 region of the 26S rRNA gene and the ITS–5.8S rDNA region.

Phylogenetic analysis based on the D1/D2 region sequences indicated that the closest relative (75 % bootstrap) to R. subericola was R. cycloclastica (Fig. 1⇓). Alignment of the D1/D2 region sequences of the novel isolates and related taxa revealed 25 bp substitutions in 644 positions (96 % sequence identity) between the novel isolates and R. cycloclastica. These species cluster in a group (70 % bootstrap) with R. retinophila, R. terpenoidalis, Colacogloea peniophorae and R. philyla in the Microbotryum lineage. The Microbotryum lineage pertains to the subclass Microbotryomycetidae in the class Urediniomycetes of basidiomycetous yeasts (Scorzetti et al., 2002). The class Urediniomycetes is divided into four lineages: Microbotryum, Sporidiobolus, Erythrobasidium and Agaricostilbum. Scorzetti et al. (2002) divided the Microbotryum lineage into two weak clades, namely Colacogloea and Leucosporidium. Phylogenetic reconstructions including most of the members in the Microbotryum lineage (Alvaro Fonseca, 2004) produced a tree similar to the one reconstructed by Scorzetti et al. (2002). In the present study, the Microbotryum lineage appears to be divided into three clusters, Leucosporidium (76 % bootstrap), Colacogloea (70 % bootstrap) and a third cluster (87 % bootstrap) composed of the species Sporobolomyces falcatus, Rhodotorula foliorum, Rhodotorula sp. CBS 6370 and Rhodotorula diffluens. The lineage Microbotryum includes, in addition to members of the genera Rhodotorula, species from the genera Sporobolomyces, Leucosporidium and Colacogloea. Other members of the polyphyletic anamorphic genus Rhodotorula can also be found in the lineages Erythrobasidium and Sporidiobolus Scorzetti et al. (2002).

Physiological differences between the members of the cluster where Y-31^T and Y-20B were included ranged from four to eight properties (Table 1⇓). Y-31^T and Y-20B were the only members of the group able to use galactose as sole carbon source and nitrate as sole nitrogen source. Y-31^T and Y-20B were investigated for basidiospore formation on cornmeal agar, 5 % malt extract agar and sucrose-yeast extract agar, but after 6 weeks of growth and observation no basidiospores were found. A loopful of cells of each isolate was mixed on a plate of cornmeal agar to obtain a dikaryotic hyphal phase, but after several days of observation no hyphal growth was detected, therefore we concluded that mating did not occur between the two isolates.

Latin diagnosis of Rhodotorula subericola Belloch, Villa-Carvajal, Álvarez-Rodríguez et Coque sp. nov.

In medio liquido dextrosum et peptonum et extractum levidinis continenti post 3 dies ad 25 °C cellulae sunt singulae aut binae, ovoidae aut cylindricae, 1.5–3×3.5–7 μm. Post unum mensem sedimentum adest. In agaro malti cultura aquosa mucosescens vel glutinosescens, partim cremea, impellucida, glabra, nitida. Pseudomycelium non formantur. Fermentatio nulla. d-Glucosum, sucrosum, α-trehalosum, d-glucitolum, d-mannitolum, ribitolum, ethanolum, d-gluconatum, d-glucosaminum, N-acetyl-d-glucosaminum, d-glucuronatum, citratum, succinatum, xylitolum assimilantur, at non d-galactosum, l-sorbosum, cellobiosum, lactosum, maltosum, melibiosum, melezitosum, raffinosum, inulinum, amylum solubile, d-arabinosum, l-arabinosum, d-ribosum, d-xylosum, l-rhamnosum, glycerolum, erythritolum, l-arabinitolum, galactitolum, meso-inositolum, methanolum, dl-lactatum, methyl α-d-glucosidum, salicinum, hexadecanum nec saccharatum. Glucosminum, l-lysinum, natrium nitricum et natrium nitrosum assimilantur neque ethylaminum et imidazolum. Sine vitaminis externis supplementis crescens. Maxima temperatura crescentiae: 30 °C. Gelatinum non liquescit et arbutinum non finditur. Acidum non formantur. Non crescit in 50 % glucoso. Materia amyloidea non formantur. Ureum hydrolysatur.

Typus: Y-31^T (=CECT 11976^T=CBS 10442^T), ex suber isolata. Holotypus lyophilus conservatur in collectione culturarum Colección Española de Cultivos Tipo, Hispania.

Description of Rhodotorula subericola Belloch, Villa-Carvajal, Álvarez-Rodríguez & Coque sp. nov.

Rhodotorula subericola [su.be.ri′co.la. L. n. suber -eris the cork-oak, cork-tree, cork; L. suff. -cola (from L. n. incola) inhabitant, dweller; N.L. n. subericola inhabitant of cork tree, cork].

In GPY medium after 3 days at 25 °C, cells are ovoid to cylindrical (1.5–3×3.5–7 μm) and occur singly or in parent–bud pairs (Fig. 2⇓). After 1 month at 25 °C, sediment is present. Streak culture on malt agar is viscous to mucoid, partly hyaline, partly creamish-opaque, smooth and glistening. No true hyphae or pseudohyphae develop in Dalmau plate cultures. Fermentation is negative. It assimilates d-glucose, sucrose, α-trehalose, d-arabinose, d-xylose, d-glucitol, d-mannitol, ribitol, ethanol, citrate (weak), succinate, d-gluconate, d-glucosamine, N-acetyl-d-glucosamine, d-glucuronate and xylitol. Does not assimilate d-galactose, l-sorbose, cellobiose, lactose, maltose, melibiose, melezitose, raffinose, inulin, soluble starch, l-arabinose, d-ribose, l-rhamnose, glycerol, erythritol, l-arabinitol, galactitol, inositol, methanol, dl-lactate, methyl α-d-glucoside, salicin, hexadecane or saccharate. Assimilation of nitrogen compounds: positive for nitrate, nitrite, l-lysine and glucosamine, negative for ethylamine and imidazole. Growth in vitamin-free medium is positive. Growth at 25 and 30 °C is positive. Growth at 37 °C is negative. Gelatin liquefaction and arbutin hydrolysis are negative. Acid formation on chalk agar is negative. Growth on 50 % glucose/yeast extract is negative. No starch-like substance is produced. Urease activity is positive.

The type strain, Y-31^T (=CECT 11976^T=CBS 10442^T), was isolated from a cork sample in Spain.