Mrakia fibulata sp. nov., a psychrotolerant yeast from temperate and cold habitats.

Tree fluxes are sugar-rich, sometimes ephemeral, substrates occurring on sites where tree sap (xylem or phloem) is leaking through damages of tree bark. Tree sap infested with microorganisms has been the source of isolation of many species, including the biotechnologically relevant carotenoid yeast Phaffia rhodozyma. Tree fluxes recently sampled in Germany yielded 19 species, including several psychrophilic yeasts of the genus Mrakia. Four strains from tree fluxes represented a potential novel Mrakia species previously known from two isolates from superficial glacial melting water of Calderone Glacier (Italy). The Italian isolates, originally identified as Mrakia aquatica, and two strains from Germany did not show any sexual structures. But another culture collected in Germany produced clamped hyphae with teliospores. A detailed examination of the five isolates (three from Germany and two from Italy) proved them to be a novel yeast species, which is described in this manuscript as Mrakia fibulata sp. nov. (MB 830398), holotype DSM 103931 and isotype DBVPG 8059. In contrast to other sexually reproducing Mrakia species, M. fibulata produces true hyphae with clamp connections. Also, this is the first psychrotolerant Mrakia species which grows above 20 °C. Spring tree fluxes are widespread and can be recognized and sampled by amateurs in a Citizen Science project. This substrate is a prominent source of yeasts, and may harbor unknown species, as demonstrated in the present work. The description of Mrakia fibulata is dedicated to our volunteer helpers and amateurs, like Anna Yurkova (9-years-old daughter of Andrey Yurkov), who collected the sample which yielded the type strain of this species.

Diversity and phylogeny of basidiomycetous yeasts from plant leaves and soil: Proposal of two new orders, three new families, eight new genera and one hundred and seven new species.

Nearly 500 basidiomycetous yeast species were accepted in the latest edition of The Yeasts: A Taxonomic Study published in 2011. However, this number presents only the tip of the iceberg of yeast species diversity in nature. Possibly more than 99 % of yeast species, as is true for many groups of fungi, are yet unknown and await discovery. Over the past two decades nearly 200 unidentified isolates were obtained during a series of environmental surveys of yeasts in phyllosphere and soils, mainly from China. Among these isolates, 107 new species were identified based on the phylogenetic analyses of nuclear ribosomal DNA (rDNA) [D1/D2 domains of the large subunit (LSU), the small subunit (SSU), and the internal transcribed spacer region including the 5.8S rDNA (ITS)] and protein-coding genes [both subunits of DNA polymerase II (RPB1 and RPB2), the translation elongation factor 1-α (TEF1) and the mitochondrial gene cytochrome b (CYTB)], and physiological comparisons. Forty-six of these belong to 16 genera in the Tremellomycetes (Agaricomycotina). The other 61 are distributed in 26 genera in the Pucciniomycotina. Here we circumscribe eight new genera, three new families and two new orders based on the multi-locus phylogenetic analyses combined with the clustering optimisation analysis and the predicted similarity thresholds for yeasts and filamentous fungal delimitation at genus and higher ranks. Additionally, as a result of these analyses, three new combinations are proposed and 66 taxa are validated.

A comparative study of the in vitro activity of iodopropynyl butylcarbamate and amphotericin B against Prototheca spp. isolates from European dairy herds.

The objective of this study was to assess the in vitro effect of iodopropynyl butylcarbamate (IPBC) and amphotericin B (AMB) on Prototheca zopfii genotype 2 and Prototheca blaschkeae isolates recovered from dairy herds of Belgium, France, Italy, Germany, and Poland. The combination of IPBC with AMB on Prototheca isolates and toxicity of IPBC to the bovine mammary epithelial cells were also evaluated. The in vitro activity of IPBC and AMB against 96 isolates of P. zopfii genotype 2 and 42 isolates of P. blaschkeae was performed. Minimum inhibitory concentrations (MIC) and minimum algicidal concentrations (MAC) of IPBC and AMB were determined. To determine any synergistic, additive, or antagonistic effect of the combination of IPBC and AMB, 2-dimensional checkerboard combination tests were also performed to calculate fractional inhibitory concentrations. Cytotoxicity analysis of IPBC to the bovine mammary epithelial cell line was performed using a 3-(4,5-dimethyl-2-thiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The MIC for 50 and 90% of isolates (MIC50 and MIC90, respectively) for IPBC were 4 and 8 mg/L versus 0.5 and 1 mg/L for AMB, respectively. The MIC profiles differed between P. zopfii genotype 2 and P. blaschkeae, with the latter species being more susceptible to both compounds. The MIC50 and MIC90 of IPBC were 4 and 8 mg/L for P. zopfii genotype 2 and 1 and 2 mg/L for P. blaschkeae, respectively. The MIC50 and MIC90 of AMB were both 1 mg/L for P. zopfii genotype 2 and 0.25 and 1 mg/L for P. blaschkeae, respectively. Both IPBC and AMB exhibited the ability to kill Prototheca spp. The MAC for 90% of isolates of IPBC was twice the MIC90, whereas an 8-fold increase of the MIC90 was algicidal in the case of AMB. Overall, the combined use of IPBC and AMB exhibited an increased algicidal effect, albeit the fractional inhibitory concentration index showed synergistic activity only against 3 P. zopfii genotype 2 isolates. For all the remaining isolates (87.5%), this combination produced only an additive effect. The MTT assay results showed both IPBC and AMB, at the concentrations employed in the study, to be nontoxic to the epithelial mammary gland cells (cell viability >90%). Notably, only IPBC at the highest concentration (i.e., 8 mg/L) exerted a slight cytotoxic effect on the cell line tested (mean cell viability: 88.54 ± 3.88 and 90.66 ± 3.0, after 2 and 4 h of MTT treatment, respectively). The anti-Prototheca activity of IPBC was here demonstrated for the first time. In addition, the combined use of IPBC with AMB enhanced each other's effect, creating an additive rather than synergistic interaction. Both agents, used at concentrations corresponding to MIC values against Prototheca spp., showed no toxic effect for the mammary epithelial cells. In conclusion, IPBC, used either alone or in combination with AMB, can be considered a promising option in the treatment armamentarium for protothecal mastitis in dairy cows.

A possible unique ecosystem in the endoglacial hypersaline brines in Antarctica.

Here, we present the results related to a new unique terrestrial ecosystem found in an englacial hypersaline brine found in Northern Victoria Land (Antarctica). Both the geochemistry and microbial (prokaryotic and fungal) diversity revealed an unicity with respect to all the other known Antarctic brines and suggested a probable ancient origin mainly due a progressive cryoconcentration of seawater. The prokaryotic community presented some peculiarities, such as the occurrence of sequences of Patescibacteria (which can thrive in nutrient-limited water environments) or few Spirochaeta, and the presence of archaeal sequences of Methanomicrobia closely related to Methanoculleus, a methanogen commonly detected in marine and estuarine environments. The high percentage (35%) of unassigned fungal taxa suggested the presence of a high degree of undiscovered diversity within a structured fungal community (including both yeast and filamentous life forms) and reinforce the hypothesis of a high degree of biological uniqueness of the habitat under study.

Molecular characterization of Prototheca strains isolated from Italian dairy herds.

One hundred sixty-one Prototheca spp. strains isolated from composite milk and barn-surrounding environmental samples (bedding, feces, drinking, or washing water, surface swabs) of 24 Italian dairy herds were characterized by genotype-specific PCR analysis. Overall, 97.2% of strains isolated from composite milk samples were characterized as Prototheca zopfii genotype 2, confirming its role as the main mastitis pathogen, whereas Prototheca blaschkeae was only sporadically isolated (2.8%). Regarding environmental sampling, 84.9% of isolates belonged to P. zopfii genotype 2, 13.2% to P. blaschkeae, and 1.9% to P. zopfii genotype 1. The data herein contradict previous hypotheses about the supposed exclusive role of P. zopfii genotype 2 as the causative agent of protothecal mastitis and, on the contrary, confirm the hypothesis that such pathology could be caused by P. blaschkeae in a few instances.

Early ecological succession patterns of bacterial, fungal and plant communities along a chronosequence in a recently deglaciated area of the Italian Alps.

In this study, the early ecological succession patterns of Forni Glacier (Ortles-Cevedale group, Italian Alps) forefield along an 18-year long chronosequence (with a temporal resolution of 1 year) has been reported. Bacterial and fungal community structures were inferred by high-throughput sequencing of 16S rRNA gene and ITS, respectively. In addition, the occurrence of both herbaceous and arboreous plants was also recorded at each plot. A significant decrease of alpha-diversity in more recently deglaciated areas was observed for both bacteria and plants. Time since deglaciation and pH affected the structure of both fungal and bacterial communities. Pioneer plants could be a major source of colonization for both bacterial and fungal communities. Consistently, some of the most abundant bacterial taxa and some of those significantly varying with pH along the chronosequence (Polaromonas, Granulicella, Thiobacillus, Acidiferrobacter) are known to be actively involved in rock-weathering processes due to their chemolithotrophic metabolism, thus suggesting that the early phase of the chronosequence could be mainly shaped by the biologically controlled bioavailability of metals and inorganic compounds. Fungal communities were dominated by ascomycetous filamentous fungi and basidiomycetous yeasts. Their role as cold-adapted organic matter decomposers, due to their heterotrophic metabolism, was suggested.

Short communication: isolation of Prototheca species strains from environmental sources in dairy herds.

Composite milk samples from 548 cows, and samples from feces, feed, bedding, water, liners (before and after milking), and the postdipping product were aseptically collected from 2 Italian dairy herds from February to November of 2006. Prototheca zopfii was isolated from 11.9% of milk samples, 15% of feces, and 33.3% of bedding samples. No viable cells of P. zopfii were observed in water before washing procedures, whereas 25 to 28.6% of samples from water used for washing both refrigeration tanks and milking equipment were contaminated with this yeast-like microalga. Analogously, the presence of P. zopfii was detected only on swabs collected from the liners after milking. Interestingly, in 1 of the 2 herds, water from the drinking trough was contaminated by viable cells of both P. zopfii and the related environmental species Prototheca stagnora. No viable cells were observed in cow feed. On the basis of the results presented herein, P. zopfii seemed to be widespread throughout the environments of dairy herds where outbreaks of bovine mastitis had occurred.

Analysis of condensed and hydrolysable tannins from commercial plant extracts.

High performance liquid chromatography (HPLC)/DAD and MS qualitative and quantitative analyses of polyphenols, hydrolysable and condensed tannins from Pinus maritima L. and tannic acid (TA) extracts were performed using normal and reverse phase. Normal-phase HPLC was more suitable for pine bark (PBE) and tannic acid extracts analysis. The chromatographic profile revealed that P. maritima L. extract was mainly composed by polymeric flavanols (containing from two to seven units) and tannic acid (characterized by a mixture of glucose gallates containing from three to seven units of gallic acid). Concerning their antimycotic properties, P. maritima L. extract exhibited a broad activity towards yeast strains of the genera Candida, Cryptococcus, Filobasidiella, Issatchenkia, Saccharomyces: MICs from 200 to 4000 microg/ml (corresponding to 140-2800 microg/ml of active polyphenols) were determined. Conversely, no activity of tannic acid was observed over the same target microorganisms. Taken into consideration the above results of HPLC analysis and on the basis of the current literature, we may conclude that only 70.2% of polyphenols (recognized as condensed tannins) occurring in P. maritima L. extract can be apparently considered responsible for its antimycotic activity.

Fingerprinting of yeasts at the strain level by differential sensitivity responses to a panel of selected killer toxins.

We used differential sensitivities to a panel of twenty-five cell-free crude killer toxins to fingerprint forty-four Saccharomyces cerevisiae strains of different origin and all taxonomically certified by nDNA-nDNA reassociation. Cluster analysis of numerical data obtained by different growth inhibition areas observed in Petri dishes allowed the complete and reproducible discrimination of all S. cerevisiae strains.

In vitro antimycotic activity of some plant extracts towards yeast and yeast-like strains.

As part of screening aimed at the selection of novel antimycotic compounds of vegetable origin, leaf extracts of Camellia sinensis L., Cupressus sempervirens L. and Pistacia lentiscus L. and the seed extract of Glycine soja Sieb. et Zucc. were tested against yeast and yeast-like species implicated in human mycoses. Of the extracts only those of C. sinensis (obtained from a commercial preparation of green tea) exhibited broad activity towards Candida glabrata, Clavispora lusitatiae, Cryptococcus laurentii, Filobasidiella neoformans, Issatchenkia orientalis, Saccharomyces cerevisiae and Prototheca wickerhamii strains. MICs ranging from 300 to 4800 microg extract/mL (corresponding to 130-2010 microg/mL total polyphenols) were observed. Concentrations of the C. sinensis extract over 25 000 microg/mL caused a rapid decrease of viable cells of Fil. neoformans and its activity was dose-dependent. Tests carried out using the pure polyphenols present in C. sinensis extract composition, showed that only epicatechin-3-O-gallate (ECG) and epigallocatechin-3-O-gallate (EGCG) possess antimycotic activity.

Assessment of discriminatory power of three different fingerprinting methods based on killer toxin sensitivity for the differentiation of Saccharomyces cerevisiae strains.

AIMS: A panel composed of 44 taxonomically certified strains of Saccharomyces cerevisiae of different origin was used to evaluate the discriminatory power of three different fingerprinting methods based on sensitivity towards 24 killer toxins. METHODS AND RESULTS: Binary data matrix (BDM), triplet data matrix (TDM) and numerical data matrix (NDM) were used as fingerprinting methods. NDM possessed the highest discriminatory power, assessed through the Simpson's, and Hunter and Gaston's indices for the measurement of diversity. The upper limits of fingerprinting ability expressed by the three above methods have been also discussed. CONCLUSIONS: NDM determined a significant increase of discriminatory power than the use of BDM or TDM, in terms of an effective amplification of their fingerprinting efficacy. SIGNIFICANCE AND IMPACT OF THE STUDY: The NDM fingerprinting method could find application in control laboratories for the discrimination of yeast strains of industrial importance or covered by patent.

First large-scale isolation of Prototheca zopfii from milk produced by dairy herds in Italy.

A total of 1045 milk samples collected from infected and non infected quarters of 269 cows were investigated. This study showed that 4.7% of samples possessed cells of Prototheca spp. (10(6)cells/ml). The presence of other pathogenic microorganisms was also monitored. Prototheca spp. isolates were classified on the basis of current taxonomic guidelines and identified as P. zopfii. Susceptibility tests carried out in vitro by using 25 antibiotic compounds revealed that the strains of P. zopfii. were susceptible only to nystatin and amphotericin B (58 and 33% of total strains, respectively). The present study represents the first large-scale investigation carried out in Italy on the isolation of this achlorophyllous yeast-like microalga in milk samples produced by dairy herds.