High intragenomic, intergenomic, and phenotypic diversity in pulcherrimin-producing Metschnikowia yeasts indicates a special mode of genome evolution.

In molecular systematics, the delimitation of yeast species is based on the notion that the barcode differences are smaller within species than between them. The most widely used barcodes are segments of the chromosomal repeats coding for ribosomal RNAs that are homogenised in yeasts. The analysis of these segments of the type strains of ten species recently merged in Metschnikowia pulcherrima and 37 new isolates demonstrated that this is not the case in this species. The intragenomic diversity significantly exceeded the threshold gaps used to differentiate related yeast species. Large segments of the D1/D2 domains were not diverse within the genomes and could therefore be used to determine the taxonomic affiliation of the isolates. The genome structures of the isolates were compared by RAPD and the RFLP of the mitochondrial DNA. Both patterns were highly heterogeneous. The sequence analysis of the PUL4 gene (a member of the PUL gene cluster involved in pulcherrimin production) revealed very high intragenomic differences, suggesting that the genomes may be chimerised. Three phenotypic traits related to the antimicrobial antagonism characteristic of the species were also highly diverse and prone to reversible segregation resembling epigenetic processes (silencing and reactivation of regulators) rather than mutations and back-mutations. These features make M. pulcherrima unique among yeasts and indicate that it evolves in a non-standard way.

Exploring the interplay between the core microbiota, physicochemical factors, agrobiochemical cycles in the soil of the historic tokaj mád wine region.

A Hungarian survey of Tokaj-Mád vineyards was conducted. Shotgun metabarcoding was applied to decipher the microbial-terroir. The results of 60 soil samples showed that there were three dominant fungal phyla, Ascomycota 66.36% ± 15.26%, Basidiomycota 18.78% ± 14.90%, Mucoromycota 11.89% ± 8.99%, representing 97% of operational taxonomic units (OTUs). Mutual interactions between microbiota diversity and soil physicochemical parameters were revealed. Principal component analysis showed descriptive clustering patterns of microbial taxonomy and resistance gene profiles in the case of the four historic vineyards (Szent Tamás, Király, Betsek, Nyúlászó). Linear discriminant analysis effect size was performed, revealing pronounced shifts in community taxonomy based on soil physicochemical properties. Twelve clades exhibited the most significant shifts (LDA > 4.0), including the phyla Verrucomicrobia, Bacteroidetes, Chloroflexi, and Rokubacteria, the classes Acidobacteria, Deltaproteobacteria, Gemmatimonadetes, and Betaproteobacteria, the order Sphingomonadales, Hypomicrobiales, as well as the family Sphingomonadaceae and the genus Sphingomonas. Three out of the four historic vineyards exhibited the highest occurrences of the bacterial genus Bradyrhizobium, known for its positive influence on plant development and physiology through the secretion of steroid phytohormones. During ripening, the taxonomical composition of the soil fungal microbiota clustered into distinct groups depending on altitude, differences that were not reflected in bacteriomes. Network analyses were performed to unravel changes in fungal interactiomes when comparing postveraison and preharvest samples. In addition to the arbuscular mycorrhiza Glomeraceae, the families Mycosphaerellacae and Rhyzopodaceae and the class Agaricomycetes were found to have important roles in maintaining soil microbial community resilience. Functional metagenomics showed that the soil Na content stimulated several of the microbiota-related agrobiogeochemical cycles, such as nitrogen and sulphur metabolism; steroid, bisphenol, toluene, dioxin and atrazine degradation and the synthesis of folate.

Starmerella lactis-condensi, a yeast that has adapted to the conditions in the oenological environment.

The yeast Starmerella (Candida) lactis-condensi is considered a food contaminant microorganism. The aim of our research was to determine why St. lactis-condensi could become the dominant species of Essences, the top sweet wine speciality of Tokaj wine region in Hungary. We investigated the physiological properties of these yeasts based on parameters that may influence their ability to selectively proliferate and persist during maturation in wines with very high sugar content. These include glucose and fructose, alcohol, and sulphur tolerance. Our studies have shown that St. lactis-condensi is a fructophilic yeast that is able to adapt quickly to very high sugar concentrations (up to 500 g/L) in the Essences. The high glucose concentration inhibits its growth, as well as that of the St. bacillaris (Candida zemplinina) strains tested. The type and amount of sugars in the Essences, together with the sulphur and alcohol content, influence the composition of the dominant yeast biota. Analysis of (GTG)5 microsatellite in the nuclear genome and mtDNA-RFLP studies demonstrate that a diverse population of St. lactis-condensi occurs in the Tokaj wine region, in the Essences. This yeast species is characterised by both physiological and genetic biodiversity. GC-MS analysis of Essences colonised exclusively with these yeasts showed no deterioration in quality.

Valorisation of Whey Permeate in Sequential Bioprocesses towards Value-Added Products-Optimisation of Biphasic and Classical Batch Cultures of Kluyveromyces marxianus.

Whey permeate is categorised as hazardous wastewater for aquatic environments, mainly due to its high lactose content. Therefore, it must be valorised before being released into the environment. One pathway for whey permeate management is its use in biotechnological processes. Herein, we present roads for whey permeate valorisation with the K. marxianus WUT240 strain. The established technology is based on two bioprocesses. During first, 2.5 g/L 2-phenylethanol and fermented plant oils enriched with different flavourings are obtained after 48 h biphasic cultures at 30 °C. The second process leads to a maximum of 75 g ethanol/L (YP/S = 0.53 g/g) after 96 h at 30 °C. Moreover, established whey permeate valorisation pathways reduced its biochemical oxygen demand and chemical oxygen demand values by 12- to 3-fold, respectively. Together, the present study reports a complete, effective, and environmentally friendly whey permeate management strategy while simultaneously enabling the acquisition of valuable compounds with substantial application potential.

Characterization of Zygosaccharomyces lentus Yeast in Hungarian Botrytized Wines.

Tokaj botrytized sweet wines are traditionally aged for several years in wood barrels or bottles. As they have significant residual sugar content, they are exposed to microbial contamination during ageing. Osmotolerant wine-spoilage yeasts are most commonly found in the Tokaj wine-growing region in the species Starmerella spp. and Zygosaccharomyces spp. For the first time, Z. lentus yeasts were isolated from post-fermented botrytized wines. Our physiological studies confirmed that these yeast strains are osmotolerant, with high sulphur tolerance and 8% v/v alcohol tolerance, and that they grow well at cellar temperature in acidic conditions. Low ß-glucosidase and sulphite reductase activities were observed, whereas protease, cellulase, and α-arabinofuranosidase extracellular enzyme activities were not detected. Molecular biology analyses carried out by RFLP analysis of mtDNA revealed no remarkable differences between strains, while microsatellite-primed-PCR fingerprinting of the (GTG)5 microsatellite and examination of chromosomal pattern revealed considerable diversity. The fermentative vigour of the tested Z. lentus strains was found to be significantly lower compared to the control Saccharomyces cerevisiae (Lalvin EC1118). It can be concluded that Z. lentus is a potential spoilage yeast in oenology which may be responsible for the initiation of secondary fermentation of wines during ageing.

Comparison and Intercorrelation of Various Bentonite Products for Oenological Properties, Elemental Compositions, Volatile Compounds and Organoleptic Attributes of White Wine.

Bentonite fining is one of the generally applied wine-making technological elements that may seriously affect wine components. The aim of this study was (i) to investigate the effect of 21 bentonite products on eight oenological parameters, 19 elements, 21 volatile organic compounds (VOCs) and 10 organoleptic properties of white wine; and (ii) to quantify intercorrelations among the parameters separately for each of the four quality attributes. Among oenological parameters, sugar, acidity, malic-, lactic-, citric acid and total phenol contents were significant among several bentonite products. The amounts of elements were the lowest in the control wine treatments (with exceptions of, e.g., Ni and Cu); and these values were significantly different from several bentonite products. The relative presence of the VOCs was above 100% for most VOCs, but it was below 100% for 1-propanol, 4-amino-1,5-pentandioic acid and butane-dioic acid, and diethyl ester in all treatments. For organoleptic parameters, the values of clearness, colour, flavour intensity and taste persistency was the lowest in the control wine treatment, while the values of flavour character, flavour quality, taste intensity, taste character, and overall harmony were the highest for the bentonite products of AP, EBE, M-SA, EBE, EBE, respectively. Results of correlation and factor analyses showed strong intercorrelative effects of bentonite fining on the four quality attributes. In conclusion, this study can help in the proper choice of a specific bentonite product in relation to complexity effects of bentonite fining.

Vinification without Saccharomyces: Interacting Osmotolerant and "Spoilage" Yeast Communities in Fermenting and Ageing Botrytised High-Sugar Wines (Tokaj Essence).

The conversion of grape juice to wine starts with complex yeast communities consisting of strains that have colonised the harvested grape and/or reside in the winery environment. As the conditions in the fermenting juice gradually become inhibitory for most species, they are rapidly overgrown by the more adaptable Saccharomyces strains, which then complete the fermentation. However, there are environmental factors that even Saccharomyces cannot cope with. We show that when the sugar content is extremely high, osmotolerant yeasts, usually considered as "spoilage yeasts", ferment the must. The examination of the yeast biota of 22 botrytised Tokaj Essence wines of sugar concentrations ranging from 365 to 752 g∙L-1 identified the osmotolerant Zygosaccharomyces rouxii, Candida (Starmerella) lactis-condensi and Candida zemplinina (Starmerella bacillaris) as the dominating species. Ten additional species, mostly known as osmotolerant spoilage yeasts or biofilm-producing yeasts, were detected as minor components of the populations. The high phenotypical and molecular (karyotype, mtDNA restriction fragment length polymorphism (RFLP) and microsatellite-primed PCR (MSP-PCR)) diversity of the conspecific strains indicated that diverse clones of the species coexisted in the wines. Genetic segregation of certain clones and interactions (antagonism and crossfeeding) of the species also appeared to shape the fermenting yeast biota.

Volatile Organic Compounds (VOCs) of Endophytic Fungi Growing on Extracts of the Host, Horseradish (Armoracia rusticana).

The interaction between plant defensive metabolites and different plant-associated fungal species is of high interest to many disciplines. Volatile organic compounds (VOCs) are natural products that are easily evaporated under ambient conditions. They play a very important role in inter-species communication of microbes and their hosts. In this study, the VOCs produced by 43 different fungal isolates of endophytic and soil fungi during growth on horseradish root (Armoracia rusticana) extract or malt extract agar were examined, by using headspace-gas chromatography-mass spectrometry (headspace-GC-MS) and a high relative surface agar film as a medium. The proposed technique enabled sensitive detection of several typical VOCs (acetone, methyl acetate, methyl formate, ethyl acetate, methyl butanol isomers, styrene, beta-phellandrene), along with glucosinolate decomposition products, including allyl cyanide and allyl isothiocyanate and other sulfur-containing compounds-carbon disulfide, dimethyl sulfide. The VOC patterns of fungi belonging to Setophoma, Paraphoma, Plectosphaerella, Pyrenochaeta, Volutella, Cadophora, Notophoma, and Curvularia genera were described for the first time. The VOC pattern was significantly different among the isolates. The pattern was indicative of putative myrosinase activity for many tested isolates. On the other hand, endophytes and soil fungi as groups could not be separated by VOC pattern or intensity.

Diversity of Candida zemplinina isolates inferred from RAPD, micro/minisatellite and physiological analysis.

Among non-Saccharomyces wine yeasts, Candida zemlpinina is one of the frequently isolated and oenologically important species. It is mostly known from European winemaking areas and it has become one of the key species of non-Saccharomyces wine yeasts to study. Investigating the diversity of C. zemplinina isolates is important for a deeper understanding of the non-Saccharomyces wine yeasts and for the yeast starter industry, as numerous researches have pointed to the potential use of this species in winemaking. For assessing the biodiversity of a larger number of strains, RAPD and micro/minisatellite PCR is often the method of choice, however, this technique is often unstandardized. Whereas some laboratories use these methods for species identifications, others apply RAPD primers for determining intraspecies diversity. In this study, we have tested 5 different RAPD and micro/minisatellite primers on strains of C. zemplinina isolated from different locations. We show that after a rigorous PCR-optimization aimed at reproducibility and comparability of band patterns with these PCR-reactions, diversity of different strains from a wide range of geographic locations is relatively low. The analysis of several oenologically important physiological traits of the strains showed a relatively low level of diversity as well. We also demonstrate that the intraspecific diversity of C. zemplinina observable with different techniques (RAPD, micro/minisatellite or physiological analysis) may be fairly different and not necessarily comparable.