The Metschnikowia pulcherrima Clade as a Model for Assessing Inhibition of Candida spp. and the Toxicity of Its Metabolite, Pulcherrimin.

Candidiasis is one of the most frequent infections worldwide. In this study, the antimicrobial properties of six strains belonging to the Metschnikowia pulcherrima clade were evaluated against twenty Candida and Candida-related Filobasidiella neoformans var. bacillispora (syn. Cryptococcus neoformans) of different origins, employing the agar cross method. The toxic effect of pulcherrimin, a red metabolite that is responsible for the antimicrobial activities of Metschnikowia spp., was evaluated in various experimental models. The results of agar tests showed that the selected M. pulcherrima strains inhibited the growth of the Candida and non-Candida strains. However, inhibition was dependent on the strain and the environment. The presence of peptone, sodium silicate, and a higher incubation temperature decreased the antifungal action of the M. pulcherrima strains. Pulcherrimin showed cytotoxic and antiproliferative activity, with oxidative stress in cells leading to apoptosis. More research is needed on the mechanism of action of pulcherrimin on somatic cells.

Moniliella zaluziensis sp. nov., a black yeast related to fruit trees of the Rosaceae family.

Our previous studies focused on the diversity of yeasts related to the aboveground parts of fruit trees, as well as the soil adjacent to these trees, located in the south-west of Slovakia. During these studies, we isolated two Moniliella strains: CCY 11-1-1T from the blossoms of a peach tree (Prunus persica) and CCY 11-1-2 from the soil adjacent to a pear tree (Pyrus communis), both found in the Malé Záluzie locality. The sequences of the D1/D2 domain of the large subunit (LSU) rRNA gene and the internal transcribed spacer (ITS) region were identical in both strains. They differed only by two nucleotide substitutions in the segment of the gene of translation elongation factor one alpha (TEF-1α). Phylogenetic analysis demonstrated that strains CCY 11-1-1T and CCY 11-1-2 formed a separate species in the clade of insect-associated members of the genus Moniliella. The strains differed from the closest species Moniliella oedocephalis by 23 nucleotide substitutions and 12 indels in the D1/D2 domain, more than 6 % in the ITS region (31 nt and 25 indels) and by 44 nt in the segment of TEF-1α. Therefore, these two strains are recognized as belonging to a novel species, for which we have proposed the name Moniliella zaluziensis sp. nov., derived from the locality of their origin, Malé Záluzie. The type strain of M. zaluziensis sp. nov. is CCY 11-1-1T.

Biological Activity of Pulcherrimin from the Meschnikowia pulcherrima Clade.

Pulcherrimin is a secondary metabolite of yeasts belonging to the Metschnikowia pulcherrima clade, and pulcherrimin formation is responsible for the antimicrobial action of its producers. Understanding the environmental function of this metabolite can provide insight into various microbial interactions and enables the efficient development of new effective bioproducts and methods. In this study, we evaluated the antimicrobial and antiadhesive action of yeast pulcherrimin, as well as its protective properties under selected stressful conditions. Classical microbiological plate methods, microscopy, and physico-chemical testing were used. The results show that pure pulcherrimin does not have antimicrobial properties, but its unique hydrophilic nature may hinder the adhesion of hydrophilic bacterial cells to abiotic surfaces. Pulcherrimin also proved to be a good cell protectant against UV-C radiation at both high and low temperatures.

Diversity of yeasts in the soil adjacent to fruit trees of the Rosaceae family.

Yeasts are common constituents of different types of soil. Their diversity depends on the season, the type and depth of the soil, the plant species, and the locality. In this study, diversity of yeasts isolated from the soil adjacent to five fruit trees (apple, appricot, peach, pear, and plum) in two localities (in Slovakia) in four sampling periods was examined. Our results demonstrated differences in the species richness and evenness among the yeast populations, which inhabited the soil beneath individual fruit tree species in both localities. Altogether, 32 ascomycetous and 27 basidiomycetous yeast species were discovered. The highest species richness was found in the soil adjacent to the apricot trees. Galactomyces candidum, Metschnikowia pulcherrima, Hanseniaspora uvarum, Schwanniomyces capriottii, and Tausonia pullulans, as well as the genus Apiotrichum, were present in soil samples in all samplings. Two species of the genus Holtermanniella (H. festucosa and H. takashimae) were exclusively isolated during Sampling IV in April. Cyberlindnera spp., Clavispora reshetovae, S. capriottii, and Trichosporon asahii were found only in one of two localities. Ascomycetous yeasts were present more frequently than their basidiomycetous counterparts in the three samplings (one in June and two in October); they formed from 65.6% to 70.8% of the total yeast population, whereas basidiomycetous yeasts prevailed in the April sampling (61.2%).

The Effect of Zn(II) Ions and Reactive Oxygen on the Uptake of Zinc and Production of Carotenoids by Selected Red Yeasts.

Three strains of red yeast Rhodosporidium kratochvilovae, Rhodotorula glutinis and Sporidiobolus salmonicolor were studied for their responses to the presence metal stress, oxidative stress and a combination of these stress factors. For all yeast strains, the production of ß-carotene increased in stress conditions. The combination of H2 O2 and Zn2+ significantly activated the pathways for the production of torularhodin in the strain R. glutinis (from 250 to 470 µg g-1 DCW) as well as ß-carotene (from 360 to 1100 µg g-1 DCW) and torulene (from 100 to 360 µg g-1 DCW) in Sp. salmonicolor. Strains of R. glutinis and Rh. kratochvilovae bound the majority of Zn(II) ions to the fibrillar part of the cell walls, whereas the strain Sp. salmonicolor bound them to both extracellular polymers and the fibrillar part of the cell walls. A decrease in the ability of yeasts to tolerate higher concentrations of Zn(II) in the presence of free radicals (hydrogen peroxide) was also found.

Characterization of a long-chain α-galactosidase from Papiliotrema flavescens.

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 ß-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.

Disentangling identity of species of the genus Taphrina parasitizing herbaceous Rosaceae, with proposal of Taphrina gei-montani sp. nov.

Five strains (CCY 058-007-001T, CCY 058-007-002, CCY 058-007-003, CCY 058-007-004 and CCY 058-007-005) of a novel parasitic yeast belonging to the genus Taphrina were isolated from leaf tissues of Geum montanum L. (Rosaceae), collected from the Vysoké Tatry Mountains, Slovakia. Genetic analyses revealed that these isolates differ by 15 unique substitutions in the ITS region and by six substitutions in the rns gene from all other species of the genus Taphrina analysed hitherto. The novel strains are also distinguished from all other species of the genus Taphrina by their morphology, biochemical properties and ecology. These strains represent a novel species, for which the name Taphrina gei-montani sp. nov. is proposed. The type strain is CCY 058-007-001T (=CBS 14159=BU001). The MycoBank number is MB815677. The present study also demonstrates that two distinct species of the genus Taphrina parasitize the herbaceous Rosaceae: Taphrina gei-montani sp. nov. on Geum montanum and Taphrina tormentillae on Potentilla species.

Yeasts associated with an abandoned mining area in Pernek and their tolerance to different chemical elements.

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).

A two-stage combined trickle bed reactor/biofilter for treatment of styrene/acetone vapor mixtures.

Performance of a two-stage biofiltration system was investigated for removal of styrene-acetone mixtures. High steady-state acetone loadings (above C(in)(Ac) = 0.5 g.m(-3) corresponding to the loadings > 34.5 g.m(-3).h(-1)) resulted in a significant inhibition of the system's performance in both acetone and styrene removal. This inhibition was shown to result from the acetone accumulation within the upstream trickle-bed bioreactor (TBR) circulating mineral medium, which was observed by direct chromatographic measurements. Placing a biofilter (BF) downstream to this TBR overcomes the inhibition as long as the biofilter has a sufficient bed height. A different kind of inhibition of styrene biodegradation was observed within the biofilter at very high acetone loadings (above C(in)(Ac) = 1.1 g.m(-3) or 76 g.m(-3).h(-1) loading). In addition to steady-state measurements, dynamic tests confirmed that the reactor overloading can be readily overcome, once the accumulated acetone in the TBR fluids is degraded. No sizable metabolite accumulation in the medium was observed for either TBR or BF. Analyses of the biodegradation activities of microbial isolates from the biofilm corroborated the trends observed for the two-stage biofiltration system, particularly the occurrence of an inhibition threshold by excess acetone.

The diversity of yeasts associated with grapes and musts of the Strekov winegrowing region, Slovakia.

Many different yeast species have been isolated from grapes and musts worldwide. The diversity and frequency of yeasts depend on a number of factors such as the grape variety, the physical damage of the grapes, the weather conditions and the chemical composition of must. A total of 366 isolates were associated with the three grape cultivars: Blue Frankish, Green Veltliner and Sauvignon blanc over four consecutive years. Yeast cultures were isolated from the grapes and from the fermenting musts after the first and seventh days. The ascomycetous yeasts of the genera Aureobasidium, Candida, Hanseniaspora, Metschnikowia, Pichia, Saccharomyces and Saccharomycopsis together with basidiomycetous yeasts of the genera Cryptococcus, Dioszegia, Filobasidium, Rhodotorula and Sporidiobolus were associated with the three grape varieties. Hanseniaspora uvarum, Metschnikowia pulcherrima, Pichia kluyveri, Pichia kudriavzevii and Sporidiobolus pararoseus were found on the berries in significant amounts. P. kluyveri and P. kudriavzevii were more associated with the damaged grapes, whereas Sp. pararoseus with intact ones. H. uvarum and M. pulcherrima were present on both types of grapes almost equally. The yeast composition and quantitative representation of yeast species varied over the grape varieties and the years examined. Although the basidiomycetous species formed a significant proportion of the yeast population in some individual grape variety/year combinations, the ascomycetous species were dominant.

Extracellular enzymatic activities and physiological profiles of yeasts colonizing fruit trees.

Yeasts form a significant and diverse part of the phyllosphere microbiota. Some yeasts that inhabit plants have been found to exhibit extracellular enzymatic activities. The aim of the present study was to investigate the ability of yeasts isolated from leaves, fruits, and blossoms of fruit trees cultivated in Southwest Slovakia to produce extracellular enzymes, and to discover whether the yeasts originating from these plant organs differ from each other in their physiological properties. In total, 92 strains belonging to 29 different species were tested for: extracellular protease, ß-glucosidase, lipase, and polygalacturonase activities; fermentation abilities; the assimilation of xylose, saccharose and alcohols (methanol, ethanol, glycerol); and for growth in a medium with 33% glucose. The black yeast Aureobasidium pullulans showed the largest spectrum of activities of all the species tested. Almost 70% of the strains tested demonstrated some enzymatic activity, and more than 90% utilized one of the carbon compounds tested. Intraspecies variations were found for the species of the genera Cryptococcus and Pseudozyma. Interspecies differences of strains exhibiting some enzymatic activities and utilizing alcohols were also noted. The largest proportion of the yeasts exhibited ß-glucosidase activity and assimilated alcohols independently of their origin. The highest number of strains positive for all activities tested was found among the yeasts associated with leaves. Yeasts isolated from blossoms assimilated saccharose and D-xylose the most frequently of all the yeasts tested. The majority of the fruit-inhabiting yeasts grew in the medium with higher osmotic pressure.

Yeasts and yeast-like organisms associated with fruits and blossoms of different fruit trees.

Yeasts are common inhabitants of the phyllosphere, but our knowledge of their diversity in various plant organs is still limited. This study focused on the diversity of yeasts and yeast-like organisms associated with matured fruits and fully open blossoms of apple, plum, and pear trees, during 2 consecutive years at 3 localities in southwest Slovakia. The occurrence of yeasts and yeast-like organisms in fruit samples was 2½ times higher and the yeast community more diverse than that in blossom samples. Only 2 species (Aureobasidium pullulans and Metschnikowia pulcherrima) occurred regularly in the blossom samples, whereas Galactomyces candidus, Hanseniaspora guilliermondii, Hanseniaspora uvarum, M. pulcherrima, Pichia kluyveri, Pichia kudriavzevii, and Saccharomyces cerevisiae were the most frequently isolated species from the fruit samples. The ratio of the number of samples where only individual species were present to the number of samples where 2 or more species were found (consortium) was counted. The occurrence of individual species in comparison with consortia was much higher in blossom samples than in fruit samples. In the latter, consortia predominated. Aureobasidium pullulans, M. pulcherrima, and S. cerevisiae, isolated from both the fruits and blossoms, can be considered as resident yeast species of various fruit tree species cultivated in southwest Slovakia localities.

Influence of pesticides on yeasts colonizing leaves.

The effect of nine different pesticides on the growth of yeasts isolated from the leaves of fruit and forest trees was investigated. Four insecticides (with the active ingredients: thiacloprid, deltamethrin, lambdacyhalothrin, and thiamethoxam) and five fungicides (with the effective substances: bitertanol, kresoxim-methyl, mancozeb, trifloxystrobin, and cupric oxychloride) were tested. The concentrations of chemicals were those recommended by the manufacturers for the spraying of trees. The yeast strains isolated from the leaves of fruit trees were not sensitive to any of the insecticides. The majority of yeast strains isolated from the leaves of forest trees were either not sensitive or only to a small extent. While Rhodotorula mucilaginosa and Pichia anomala were not affected by any insecticide, the strains of Cryptococcus laurentii and Rhodotorula glutinis showed the highest sensitivity. The effects of fungicides on the growth of isolated yeasts were more substantial. The fungicide Dithane DG (mancozeb) completely inhibited the growth of all yeasts. All strains isolated from fruit tree leaves were more resistant to the tested fungicides than those isolated from the leaves of forest trees. The most resistant strains from the leaves of fruit trees belonged to the species Metschnikowia pulcherrima, Pichia anomala, and Saccharomyces cerevisiae, whereas Cryptococcus albidus and C. laurentii, originating from the leaves of forest trees, showed the highest sensitivity to fungicides.

Yeasts colonizing the leaf surfaces.

The yeasts were isolated from the leaf surfaces of ten species of trees. The study site was a forest park (Zelezná Studnicka) of the Small Carpathians mountain range. One hundred and thirty seven yeast strains belonging to 13 genera were isolated from 320 samples of leaves and needles. Seventeen yeast species were isolated, but only seven occurred regularly: Aureobasidium pullulans, Cryptococcus laurentii, Pichia anomala, Metschnikowia pulcherrima, Saccharomyces sp., Lachancea thermotolerans, and Rhodotorula glutinis. The remaining species were isolated from the leaves and needles of three or less tree species. A. pullulans, Cr. laurentii, and P. anomala were the most frequently found species and they occurred on leaves and needles of all ten tree species. Saccharomyces sp. occurred in leaf samples collected from eight kinds of trees. M. pulcherrima and L. thermotolerans were found in samples collected from six species of trees. Both these species occurred almost always on the leaves of deciduous trees. Rh. glutinis was the most frequently isolated carotenoids producing species. We have found out that the ascomycetous and basidiomycetous species were present in the leaf samples in approximately equal frequency, contrary to the soil samples taken from this forest park, where the ascomycetous species were found rarely.

Killer activity of yeasts isolated from natural environments against some medically important Candida species.

Twenty-five yeast cultures, mainly of human origin, belonging to four pathogenic yeast species--Candida albicans, Candida krusei, Candida parapsilosis, and Candida tropicalis were tested for their sensitivity to ten basidiomycetous and eleven ascomycetous yeast species isolated from the water and soil environments and from tree leaves. The best killer activity among basidiomycetous species was exhibited by Rhodotorula glutinis, and R. mucilaginosa. The other carotenoid producing species Cystofilobasidium capitatum, Sporobolomyces salmonicolor, and S. roseus were active only against about 40% of the tested strains and exhibited weak activity. The broadest killer activity among ascomycetous yeasts was shown by the strains Pichia anomala and Metschnikowia pulcherrima. The species Debaryomyces castellii, Debaryomyces hansenii, Hanseniaspora guilliermondii, Pichia membranifaciens, and Williopsis californica did not show any killer activity. The best killer activity exhibited the strains isolated from leafy material. The lowest activity pattern was found among strains originating from soil environment.

Metal tolerance of yeasts isolated from water, soil and plant environments.

The tolerance of seventy yeast strains belonging to 15 species, isolated from water and soil environments as well as from tree leaves, to four heavy metals--copper, zinc, nickel and cadmium were studied. We have found that the interspecific and intraspecific variations in metal tolerance among studied strains were considerable. The highest interspecific variations were observed toward copper and cadmium. The strains of the species Sporobolomyces salmonicolor, Cryptococcus albidus, Cystofilobasidium capitatum, Saccharomyces cerevisiae, and Candida maltosa belonged to the most sensitive ones. In general ascomycetous yeasts were more tolerant to heavy metals than basidiomycetous ones. The differences among strains that came from various natural sources were also found. The most sensitive yeast population originated from untilled soil whereas the most tolerant population was isolated from tree leaves.

The diversity of yeasts in the agricultural soil.

One hundred and eleven yeast strains were isolated from 60 agricultural soil samples. The samples were taken from four various fields located in the southwest of Slovakia. Cryptococcus laurentii, Candida maltosa, Metschnikowia pulcherrima, and Sporobolomyces salmonicolor were the predominant species in the samples collected from all four types of fields. These species represented 78.4-86.6% of the total yeast counts.The results obtained enabled comparisons to be made between forest and agricultural soil yeast population. We have found out that the yeast population in tilled soils was significantly reduced. The number of yeasts in the tilled soils ranged from 40 to 6.8 x 10(3) CFU/g soil and the average number reached approximately 1.12 x 10(3). This number is more than ten times lower in comparison with the forest soils.

Effects of pesticides on yeasts isolated from agricultural soil.

The effect of six various pesticides on the growth of yeasts isolated from agricultural soil was investigated. Two herbicides (with the effective substances lactofen and metazachlor), two fungicides (with the effective substances fluquinconazole and prochloraz), and two insecticides (with the effective substances cypermethrin + chlorpyrifos and triazamate) were tested. It is evident that there are considerable differences in inhibition effects of studied pesticides. The fungicide with the effective substance prochloraz inhibited the growth of majority of yeast strains. The insecticide triazamate at concentration 0.6 mM restricted or inhibited growth of all tested strains. The strains of the genus Cryptococcus were the most sensitive to pesticides, while the strains of the species Cystofilobasidium capitatum, Debaryomyces occidentalis var. occidentalis, and Trichosporon cutaneum were the most resistant.