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Cryptococcus flavescens and C. terrestris are phenotypically indistinguishable sister species that belong to the order Tremellales (Tremellomycetes, Basidiomycota) and which may be mistaken for C. laurentii based on phenotype. Phylogenetic separation between C. flavescens and C. terrestris was based on rDNA sequence analyses, but very little is known on their intraspecific genetic variability or propensity for sexual reproduction. We studied 59 strains from different substrates and geographic locations, and used a multilocus sequencing (MLS) approach complemented with the sequencing of mating type (MAT) genes to assess genetic variation and reexamine the boundaries of the two species, as well as their sexual status. The following five loci were chosen for MLS: the rDNA ITS-LSU region, the rDNA IGS1 spacer, and fragments of the genes encoding the largest subunit of RNA polymerase II (RPB1), the translation elongation factor 1 alpha (TEF1) and the p21-activated protein kinase (STE20). Phylogenetic network analyses confirmed the genetic separation of the two species and revealed two additional cryptic species, for which the names Cryptococcus baii and C. ruineniae are proposed. Further analyses of the data revealed a high degree of genetic heterogeneity within C. flavescens as well as evidence for recombination between lineages detected for this species. Strains of C. terrestris displayed higher levels of similarity in all analysed genes and appear to make up a single recombining group. The two MAT genes (STE3 and SXI1/SXI2) sequenced for C. flavescens strains confirmed the potential for sexual reproduction and suggest the presence of a tetrapolar mating system with a biallelic pheromone/receptor locus and a multiallelic HD locus. In C. terrestris we could only sequence STE3, which revealed a biallelic P/R locus. In spite of the strong evidence for sexual recombination in the two species, attempts at mating compatible strains of both species on culture media were unsuccessful.

Although Cryptococcus laurentii has been considered saprophytic and its taxonomy is still being described, several cases of human infections have already reported. This study aimed to evaluate molecular aspects of C. laurentii isolates from Brazil, Botswana, Canada, and the United States. In this study, 100 phenotypically identified C. laurentii isolates were evaluated by sequencing the 18S nuclear ribosomal small subunit rRNA gene (18S-SSU), D1/D2 region of 28S nuclear ribosomal large subunit rRNA gene (28S-LSU), and the internal transcribed spacer (ITS) of the ribosomal region. BLAST searches using 550-bp, 650-bp, and 550-bp sequenced amplicons obtained from the 18S-SSU, 28S-LSU, and the ITS region led to the identification of 75 C. laurentii strains that shared 99-100% identity with C. laurentii CBS 139. A total of nine isolates shared 99% identity with both Bullera sp. VY-68 and C. laurentii RY1. One isolate shared 99% identity with Cryptococcus rajasthanensis CBS 10406, and eight isolates shared 100% identity with Cryptococcus sp. APSS 862 according to the 28S-LSU and ITS regions and designated as Cryptococcus aspenensis sp. nov. (CBS 13867). While 16 isolates shared 99% identity with Cryptococcus flavescens CBS 942 according to the 18S-SSU sequence, only six were confirmed using the 28S-LSU and ITS region sequences. The remaining 10 shared 99% identity with Cryptococcus terrestris CBS 10810, which was recently described in Brazil. Through concatenated sequence analyses, seven sequence types in C. laurentii, three in C. flavescens, one in C. terrestris, and one in the C. aspenensis sp. nov. were identified. Sequencing permitted the characterization of 75% of the environmental C. laurentii isolates from different geographical areas and the identification of seven haplotypes of this species. Among sequenced regions, the increased variability of the ITS region in comparison to the 18S-SSU and 28S-LSU regions reinforces its applicability as a DNA barcode.

α-Galactosidases are assigned to the class of hydrolases and the subclass of glycoside hydrolases (GHs). They belong to six GH families and include the only characterized α-galactosidases from yeasts (GH 27, Saccharomyces cerevisiae). The present study focuses on an investigation of the lactose-inducible α-galactosidase produced by Papiliotrema flavescens. The enzyme was present on the surface of cells and in the cytosol. Its temperature optimum was about 60 °C and the pH optimum was 4.8; the pH stability ranged from 3.2 to 6.6. This α-galactosidase also exhibited transglycosylation activity. The cytosol α-galactosidase with a molecular weight about 110 kDa, was purified using a combination of liquid chromatography techniques. Three intramolecular peptides were determined by the partial structural analysis of the sequences of the protein isolated, using MALDI-TOF/TOF mass spectrometry. The data obtained recognized the first yeast α-galactosidase, which belongs to the GH 36 family. The bioinformatics analysis and homology modeling of a 210 amino acids long C-terminal sequence (derived from cDNA) confirmed the correctness of these findings. The study was also supplemented by the screening of capsular cryptococcal yeasts, which produce the surface lactose-inducible α- and β-galactosidases. The production of the lactose-inducible α-galactosidases was not found to be a general feature within the yeast strains examined and, therefore, the existing hypothesis on the general function of this enzyme in cryptococcal capsule rearrangement cannot be confirmed.Graphical Abstract

During a study of microorganisms associated with maize (Zea mays) cultivation, yeasts were isolated from overwintered stalks, cobs and surrounding soil, which were collected from an agricultural field in south-central Illinois, USA. Predominant among isolates were two species of Cryptococcus (Cr. flavescens, Cr. magnus) and a red yeast that D1/D2 LSU rRNA gene sequences revealed to be a new species of the basidiomycete yeast genus Occultifur. The species, which was not detected in the same field during the growing season, is described here as Occultifur kilbournensis (MycoBank number MB 811259; type strain NRRL Y-63695, CBS 13982, GenBank numbers, D1/D2 LSU rRNA gene, KP413160, ITS, KP413162; allotype strain NRRL Y-63699, CBS 13983). Mixture of the type and allotype strains resulted in formation of hyphae with clamp connections and a small number of apparent basidia following incubation on 5% malt extract agar at 15 °C for 2 months. In view of the uncertainty of the life cycle, the new species is being designated as forma asexualis. From analysis of D1/D2 and ITS nucleotide sequences, the new species is most closely related to Occultifur externus.

Botrytis cinerea, the causal agent of grey mould, is a predominant agent causing extensive postharvest and quality losses of apples in Tunisia and worldwide. Efforts to manage this disease have met with limited success. For this reason, the use of microorganism preparations to control fungal diseases as an alternative to fungicides became an urgent need. From a total of 60 epiphytic yeasts, 10 were assessed in vitro against B. cinerea and selected isolates showing antagonism were evaluated for their ability to suppress the grey mould in vivo. On Petri plates, the most promising strains (three strains of Aureobasidium pullulans, one Cryptococcus flavescens, and one Citeromyces matritensis) showed a zone of inhibition against the pathogen fungus not exceeding 10 mm. In vivo, these isolates showed a remarkable antifungal activity since they significantly reduced disease severity on apples from 63% to 95% compared to the control. In conclusion, the work has demonstrated that the three strains, L7 of Aureobasidium pullulans, L2 of Citeromyces matritensis, and L10 of Cryptococcus flavescens, were highly effective and can be used as potential biocontrol agents in controlling the post-harvest decay of apples caused by B. cinerea.

Four plants, Cirsium arvense (creeping thistle), Equisetum arvense (field horsetail), Oxalis acetosella (wood sorrel) and Phragmites australis (common reed), which grew in an abandoned Sb-mining area in Pernek (Malé Karpaty Mts., Slovakia), were investigated for the yeast species. Yeasts were isolated from both the leaves of the plants and the soil adjacent to the plants. In total, 65 yeast cultures, belonging to 11 ascomycetous and 5 basidiomycetous yeast species, were isolated. The species most frequently isolated from both the soil and leaf samples were Trichosporon porosum , Galactomyces candidus and Candida solani , whereas Aureobasidium pullulans , Candida tsuchiyae and Sporidiobolus metaroseus were isolated exclusively from the plant leaves. All the yeast species isolated were tested for their tolerance to two heavy metals (Cd, Zn) and three metalloids (As, Sb and Si). The yeasts isolated from both the leaves and soils exhibited a high tolerance level to both As and Sb, present in elevated concentrations at the locality. Among the yeast species tested, Cryptococcus musci , a close relative to Cryptococcus humicola , was the species most tolerant to all the chemical elements tested, with the exception of Si. It grew in the presence of 200 mmol/L Zn, 200 mmol/L Cd, 60 mmol/L As and 50 mmol/L Sb, and therefore, it can be considered as a multi-tolerant species. Some of the yeast species were tolerant to the individual chemical elements. The yeast-like species Trichosporon laibachii exhibited the highest tolerance to Si of all yeasts tested, and Cryptococcus flavescens and Lindnera saturnus showed the same tolerance as Cryptococcus musci to Zn and As, respectively. The majority of the yeasts showed a notably low tolerance to Cd (not exceeded 0.5 mmol/L), which was present in small amounts in the soil. However, Candida solani , isolated from the soil, exhibited a higher tolerance to Cd (20 mmol/L) than to As (2 mmol/L).

Morphology and physiology of a strain of Bullera alba was studied. The fungus could be distinguished from Cryptococcus laurentii var. flavescens only on the base of ballistospore formation. The isolate studied formed a true septate mycelium.

Cryptococcus flavescens (previously reported as C. nodaensis), a biological control agent of Fusarium head blight, has been previously shown to have improved desiccation tolerance after cold adaptation. The goal of the current study was to determine the effect of cold adaptation on the physicochemical properties of C. flavescens that may be responsible for its improved desiccation tolerance. The results show that cold adaptation improves liquid hyperosmotic shock tolerance and alters the temperature dependence of osmotic shock tolerance. Fluorescence anisotropy was used to characterize differences in the membrane fluidity of C. flavescens with and without cold adaptation. Force curves from atomic force microscopy showed a significant increase in the cell wall spring constant after cold adaptation. Cold adaptation of C. flavescens during culturing was shown to produce smaller cells and produced a trend towards higher CFU yields. These results suggest that cold adaptation significantly alters the membrane properties of C. flavescens and may be an effective method of improving the desiccation tolerance of microorganisms. In addition, we provide information on the correct naming of the isolate as C. flavescens.

This report describes the first documented case of subcutaneous infection due to Cryptococcus flavescens in a dog. The chief symptoms of the patient dog were abscessed lesions on the dorsal muzzle, right eyelid, and lower jaw. Biopsy specimens from the lesions on the dorsal muzzle and lower jaw showed pyogranulomatous inflammation with numerous yeast cells. The patient dog was diagnosed with a subcutaneous fungal infection and orally received 5 mg/kg itraconazole once a day for 2 months, the abscesses disappeared. After 1 month at the end of treatment, the skin lesions did not redevelop. Isolates from the biopsy specimens were identified as C. flavescens by molecular analysis as well as morphologic and biochemical examination, indicating that C. flavescens is a potential canine pathogen.

The ability of yeasts to attach to hyphae or conidia of phytopathogenic fungi has been speculated to contribute to biocontrol activity on plant surfaces. Attachment of phylloplane yeasts to Botrytis cinerea, Rhizoctonia solani, and Sclerotinia homoeocarpa was determined using in vitro attachment assays. Yeasts were incubated for 2 d on potato dextrose agar (PDA) prior to experimentation. A total of 292 yeasts cultured on PDA were screened for their ability to attach to conidia of B. cinerea; 260 isolates (89.1%) attached to conidia forming large aggregates of cells, and 22 isolates (7.5%) weakly attached to conidia with 1 or 2 yeast cells attached to a few conidia. Ten yeasts (3.4%), including 8 isolates of Cryptococcus laurentii, 1 isolate of Cryptococcus flavescens, and an unidentified species of Cryptococcus, failed to attach to conidia. All non-attaching yeasts produced copious extracellular polysaccharide (EPS) on PDA. Seventeen yeast isolates did not attach to hyphal fragments of B. cinerea, R. solani, and S. homoeocarpa after a 1 h incubation, but attachment was observed after 24 h. Culture medium, but not culture age, significantly affected the attachment of yeast cells to conidia of B. cinerea. The 10 yeast isolates that did not attach to conidia when grown on agar did attach to conidia (20%-57% of conidia with attached yeast cells) when cultured in liquid medium. Attachment of the biocontrol yeast Rhodotorula glutinis PM4 to conidia of B. cinerea was significantly greater at 1 × 10 7 yeast cells·mL -1 than at lower concentrations of yeast cells. The ability of yeast cells to attach to fungal conidia or hyphae appears to be a common phenotype among phylloplane yeasts.Key words: adhesion, biological control, Cryptococcus laurentii, Rhodotorula glutinis.