Yeasts volatile organic compounds (VOCs) as potential growth enhancers and molds biocontrol agents of mushrooms mycelia.

Volatile organic compounds (VOCs) produced by yeasts can positively affect crops, acting as antifungals or biostimulants. In this study, Aureobasidium pullulans and Metschnikowia pulcherrima were evaluated as potential antagonists of Trichoderma spp., common fungal pathogen in mushroom cultivation. To assess the biocontrol ability and biostimulant properties of the selected yeast species, in vitro co-culture and VOCs exposure assays were conducted. In both assays, VOCs produced by Aureobasidium spp. showed the stronger antifungal activity with a growth inhibition up to 30 %. This result was further confirmed by the higher volatilome alcohol content revealed by solid phase microextraction-gas chromatography mass spectrometry (SPME/GC-MS). Overall, Aureobasidium strains can be potentially used as biocontrol agent in Pleorotus ostreatus and Cyclocybe cylindracea mycelial growth, without affecting their development as demonstrated by VOCs exposure assay and Fourier-transform infrared spectroscopy (FT-IR). Conversely, M. pulcherrima was characterized by a lower or absent antifungal properties and by a volatilome composition rich in isobutyl acetate, an ester often recognized as plant growth promoter. As confirmed by FT-IR, Lentinula mycelia exposed to M. pulcherrima VOCs showed a higher content of proteins and lipids, suggesting an improvement of some biochemical properties. Our study emphasizes that VOCs produced by specific yeast strains are potentially powerful alternative to synthetic fungicide in the vegetative growth of mushroom-forming fungi and also able to modify their biochemical composition.

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.

Antagonistic Activities of Metschnikowia pulcherrima Isolates Against Penicillium expansum on Amasya Apples.

Postharvest fungal diseases cause serious fruit losses and food safety issues worldwide. The trend in preventing food loss and waste has shifted to environmentally friendly and sustainable methods, such as biological control. Penicillium expansum is a common postharvest contaminant fungus that causes blue mould disease and patulin formation on apples. This study aimed to provide biocontrol using Metschnikowia pulcherrima isolates against P. expansum, and to understand their antagonistic action mechanisms. In vitro, 38.77-51.69% of mycelial growth inhibition of P. expansum was achieved by M. pulcherrima isolates with the dual culture assay, while this rate was 69.45-84.89% in the disc diffusion assay. The disease symptoms of P. expansum on wounds were reduced by M. pulcherrima, on Amasya apples. The lesion diameter, 41.84 mm after 12 d of incubation in control, was measured as 24.14 mm when treated with the most effective M. pulcherrima DN-MP in vivo. Although the antagonistic mechanisms of M. pulcherrima isolates were similar, there was a difference between their activities. In general, DN-HS and DN-MP isolates were found to be more effective. In light of all these results, it can be said that M. pulcherrima isolates used in the study have an antagonistic effect against the growth of P. expansum both in vitro and in vivo in Amasya apples, therefore, when the appropriate formulation is provided, they can be used as an alternative biocontrol agent to chemical fungicides in the prevention of postharvest diseases.

Yeast sexes: mating types do not determine the sexes in Metschnikowia species.

Although filamentous Ascomycetes may produce structures that are interpreted as male and female gametangia, ascomycetous yeasts are generally not considered to possess male and female sexes. In haplontic yeasts of the genus Metschnikowia, the sexual cycle begins with the fusion of two morphologically identical cells of complementary mating types. Soon after conjugation, a protuberance emerges from one of the conjugants, eventually maturing into an ascus. The originating cell can be regarded as an ascus mother cell, hence as female. We tested the hypothesis that the sexes, female or male, are determined by the mating types. There were good reasons to hypothesize further that mating type α cells are male. In a conceptually simple experiment, we observed the early stages of the mating reaction of mating types differentially labeled with fluorescent concanavalin A conjugates. Three large-spored Metschnikowia species, M. amazonensis, M. continentalis, and M. matae, were examined. In all three, the sexes were found to be independent of mating type, cautioning that the two terms should not be used interchangeably.

RT-qPCR based quantitative analysis of ARO and ADH genes in Saccharomyces cerevisiae and Metschnikowia pulcherrima strains growth white grape juice.

BACKGROUND: Yeast biosynthesizes fusel alcohols in fermentation through amino acid catabolism via the Ehrlich pathway. ARO8 and ARO9 genes are involved in the first step of the Ehrlich pathway, while ADH2 and ADH5 genes are involved in the last step. In this study, we describe RT-qPCR methods to determine the gene expression level of genes (ARO8, ARO9, ADH2, ADH5) found in Saccharomyces cerevisiae (Sc) and Metschnikowia pulcherrima (Mp) strains growth pasteurized white grape juice. METHODS AND RESULTS: We used RNA extraction and cDNA synthesis protocols. The RT-qPCR efficiency of primer pairs was evaluated by generating a standard curve through serial dilution of yeast-derived cDNA. Method performance criteria were determined for each RT-qPCR assay. Then, we evaluated the gene expression levels of the four genes in all samples. RNA extraction and cDNA synthesis from yeast samples demonstrated the method's capability to generate high-yield, high-purity nucleic acids, supporting further RT-qPCR analysis. The highest normalized gene expression levels of ARO8 and ARO9 were observed in SC1, SC4, and SC5 samples. No significant difference in ADH2 gene expression among Mp strains was observed during the examination of ADH2 and ADH5 genes (p < 0.05). We observed no expression of the ADH5 gene in Mp strains except MP6 strain. The expression of ADH2 and ADH5 genes was higher in Sc strains compared to Mp strains. CONCLUSIONS: The results suggest that the proposed RT-qPCR methods can measure gene expression of ARO8, ARO9, ADH2, and ADH5 in Sc and Mp strains growing in pasteurized white grape juice.

Enhancing the flavour quality of Laiyang pear wine by screening sorbitol-utilizing yeasts and co-fermentation strategies.

This study took a novel approach to address the dual challenges of enhancing the ethanol content and aroma complexity in Laiyang pear wine. It focused on sorbitol as a pivotal element in the strategic selection of yeasts with specific sorbitol-utilization capabilities and their application in co-fermentation strategies. We selected two Saccharomyces cerevisiae strains (coded as Sc1, Sc2), two Metschnikowia pulcherrima (coded as Mp1, Mp2), and one Pichia terricola (coded as Tp) due to their efficacy as starter cultures. Notably, the Sc2 strain, alone or with Mp2, significantly increased the ethanol content (30% and 16%). Mixed Saccharomyces cerevisiae and Pichia terricola fermentation improved the ester profiles and beta-damascenone levels (maximum of 150%), while Metschnikowia pulcherrima addition enriched the phenethyl alcohol content (maximum of 330%), diversifying the aroma. This study investigated the efficacy of strategic yeast selection based on sorbitol utilization and co-fermentation methods in enhancing Laiyang pear wine quality and aroma.

Isolation of local strains of the yeast Metschnikowia for biocontrol and lipid production purposes.

The bioprospection of indigenous microorganism strains with biotechnological potential represents a prominent trend. Metschnikowia yeasts exhibit diverse capabilities, such as ethanol reduction in winemaking, biocontrol potential, and lipid production. In this work, local Metschnikowia strains were isolated from different fruits by their ability to produce pulcherrimic acid, a molecule that has been linked to biocontrol activity and that binds iron giving colored colonies. Five strains were selected, each from one of five distinct sources. All of them were identified as M. pulcherrima. All five were able inhibit other yeasts and one M. pulcherrima, called M7, inhibited the growth of Aspergillus nidulans. The selected strains accumulated lipid bodies in stationary phase. Certain non-conventional yeasts like Hanseniaspora vineae are very sensitive to biomass drying, but cell extracts from M. pulcherrima added to the growth media as a source of antioxidant lipids increased their tolerance to drying. All strains isolated showed good stress tolerance (particularly to heat) and have nutrient requirements similar to a commercial M. pulcherrima strain. In addition, the M7 strain had a good growth in sugarcane and beet molasses and behaved like Saccharomyces cerevisiae in a growth medium derived from agricultural waste, a persimmon hydrolysate. Therefore, the isolation of local strains of Metschnikowia able to grow in a variety of substrates is a good source of biocontrol agents.

Heterologous pulcherrimin production in Saccharomyces cerevisiae confers inhibitory activity on Botrytis conidiation.

Pulcherrimin is an iron (III) chelate of pulcherriminic acid that plays a role in antagonistic microbial interactions, iron metabolism, and stress responses. Some bacteria and yeasts produce pulcherriminic acid, but so far, pulcherrimin could not be produced in Saccharomyces cerevisiae. Here, multiple integrations of the Metschnikowia pulcherrima PUL1 and PUL2 genes in the S. cerevisiae genome resulted in red colonies, which indicated pulcherrimin formation. The coloration correlated positively and significantly with the number of PUL1 and PUL2 genes. The presence of pulcherriminic acid was confirmed by mass spectrometry. In vitro competition assays with the plant pathogenic fungus Botrytis caroliana revealed inhibitory activity on conidiation by an engineered, strong pulcherrimin-producing S. cerevisiae strain. We demonstrate that the PUL1 and PUL2 genes from M. pulcherrima, in multiple copies, are sufficient to transfer pulcherrimin production to S. cerevisiae and represent the starting point for engineering and optimizing this biosynthetic pathway in the future.

Genome-scale metabolic models reveal determinants of phenotypic differences in non-Saccharomyces yeasts.

BACKGROUND: Use of alternative non-Saccharomyces yeasts in wine and beer brewing has gained more attention the recent years. This is both due to the desire to obtain a wider variety of flavours in the product and to reduce the final alcohol content. Given the metabolic differences between the yeast species, we wanted to account for some of the differences by using in silico models. RESULTS: We created and studied genome-scale metabolic models of five different non-Saccharomyces species using an automated processes. These were: Metschnikowia pulcherrima, Lachancea thermotolerans, Hanseniaspora osmophila, Torulaspora delbrueckii and Kluyveromyces lactis. Using the models, we predicted that M. pulcherrima, when compared to the other species, conducts more respiration and thus produces less fermentation products, a finding which agrees with experimental data. Complex I of the electron transport chain was to be present in M. pulcherrima, but absent in the others. The predicted importance of Complex I was diminished when we incorporated constraints on the amount of enzymatic protein, as this shifts the metabolism towards fermentation. CONCLUSIONS: Our results suggest that Complex I in the electron transport chain is a key differentiator between Metschnikowia pulcherrima and the other yeasts considered. Yet, more annotations and experimental data have the potential to improve model quality in order to increase fidelity and confidence in these results. Further experiments should be conducted to confirm the in vivo effect of Complex I in M. pulcherrima and its respiratory metabolism.

Candida massiliensis sp. nov. Isolated from a Clinical Sample.

The majority of Candida species are known as non-pathogenic yeasts and rarely involved in human diseases. However, recently case reports of human infections caused by non-albicans Candida species have increased, mostly in immunocompromised hosts. Our study aimed to describe and characterize as thoroughly as possible, a new species of the Metschnikowia clade, named here Candida massiliensis (PMML0037), isolated from a clinical sample of human sputum. We targeted four discriminant genetic regions: "Internal Transcribed Spacers" of rRNA, D1/D2 domains (28S large subunit rRNA) and part of the genes encoding Translation Elongation Factor 1-α and ß-tubulin2. The genetic data were compared to morphological characters, from scanning electron microscopy (TM 4000 Plus, SU5000), physiological, including the results of oxidation and assimilation tests of different carbon sources by the Biolog system, and chemical mapping by Energy-Dispersive X-ray Spectroscopy. Lastly, the in vitro antifungal susceptibility profile was performed using the E-test™ exponential gradient method. The multilocus analysis supported the genetic position of Candida massiliensis (PMML0037) as a new species of the Metschnikowia clade, and the phenotypic analysis highlighted its unique morphological and chemical profile when compared to the other Candida/Metschnikowia species included in the study.

Novel Metschnikowia yeasts from the gut of green lacewing in Japan.

Family Chrysopidae is known to harbor specific gut yeasts. However, no studies have been conducted outside of a limited number of these green lacewing species, and the diversity of yeasts in the family as a whole is not known. Therefore, we collected 58 Chrysopidae adults (9 species, 6 genera, 2 subfamilies) in Japan and isolated yeasts from all individuals. The results showed for the first time that not only subfamily Chrysopinae but also subfamily Apochrysinae have gut yeasts. We obtained 58 yeast isolates (one from each host individual), all of which were of the genus Metschnikowia. 28S rDNA- and ITS-based phylogenetic analysis showed that the isolates were divided into three clades, designated clade I, II, and III. Clade I contains two previously described Chrysopidae gut yeasts (M. picachoensis and M. pimensis) as well as a one of our new species named M. shishimaru. Clade II is a new clade, with at least two new species named M. kenjo and M. seizan. Clade III contains the previously described species M. noctiluminum, a Chrysopidae gut yeast, and one of our isolate (We have not described it as new species). However, the phylogenetic relationship between our isolate and M. noctiluminum was unclear. These results indicate that the Japanese Chrysopidae gut yeasts consist mainly of three undescribed species and that they are more unique than those found in previous surveys. The results of this study indicate that Chrysopidae gut yeasts are more diverse than previously thought and should be investigated in various geographical regions in the future.

Metschnikowia ahupensis f.a., sp. nov., a new yeast species isolated from the gut of the wood-feeding termite Nasutitermes sp.

The gut of xylophagous insects such as termites harbours various symbiotic micro-organisms, including many yeast species. In a taxonomic study of gut-associated yeasts, two strains (ATS2.16 and ATS2.18) were isolated from the gut of the wood-feeding termite Nasutitermes sp. in Maharashtra, India. Morphological and physiological characteristics and sequence analyses of the ITS and D1/D2 region of the large subunit rRNA gene revealed that these two strains represent a novel asexual ascomycetous yeast species in the genus Metschnikowia. The species differs from some of its close affiliates in the genus in its inability to utilize ethanol and succinate as the sole carbon source and growth in high sugar concentrations (up to 50 % glucose). In contrast to most members of Metschnikowia, the formation of ascospores was not observed on various sporulation media. Moreover, whole-genome sequencing was used to further confirm the novelty of this species. When compared with other large-spored Metschnikowia species, average nucleotide identity values of 79-80 % and digital DNA-DNA hybridization values of 16-17 % were obtained. The name Metschnikowia ahupensis f.a., sp. nov. is proposed to accommodate this novel yeast species, with ATS2.16 as the holotype and strains NFCCI 4949, MTCC 13085 and PYCC 9152 as isotypes. The MycoBank number is MB 844210.

Bees just wanna have fungi: a review of bee associations with nonpathogenic fungi.

Bee-fungus associations are common, and while most studies focus on entomopathogens, emerging evidence suggests that bees associate with a variety of symbiotic fungi that can influence bee behavior and health. Here, we review nonpathogenic fungal taxa associated with different bee species and bee-related habitats. We synthesize results of studies examining fungal effects on bee behavior, development, survival, and fitness. We find that fungal communities differ across habitats, with some groups restricted mostly to flowers (Metschnikowia), while others are present almost exclusively in stored provisions (Zygosaccharomyces). Starmerella yeasts are found in multiple habitats in association with many bee species. Bee species differ widely in the abundance and identity of fungi hosted. Functional studies suggest that yeasts affect bee foraging, development, and pathogen interactions, though few bee and fungal taxa have been examined in this context. Rarely, fungi are obligately beneficial symbionts of bees, whereas most are facultative bee associates with unknown or ecologically contextual effects. Fungicides can reduce fungal abundance and alter fungal communities associated with bees, potentially disrupting bee-fungi associations. We recommend that future study focus on fungi associated with non-honeybee species and examine multiple bee life stages to document fungal composition, abundance, and mechanistic effects on bees.

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.

Fate of carbon in synthetic media fermentations containing Metschnikowia pulcherrima or Meyerozyma guilliermondii in the presence and absence of Saccharomyces cerevisiae.

While sequentially inoculating non-Saccharomyces yeasts with Saccharomyces cerevisiae can lower the alcohol contents of wine, the abilities of these yeasts to utilize/produce ethanol or generate other byproducts remained unclear. Metschnikowia pulcherrima or Meyerozyma guilliermondii were inoculated into media with or without S. cerevisiae to assess byproduct formation. Both species metabolized ethanol in a yeast-nitrogen-base medium but produced the alcohol in a synthetic grape juice medium. In fact, Mt. pulcherrima and My. guilliermondii generated less ethanol per gram of metabolized sugar (0.372 and 0.301 g/g, respectively) compared to S. cerevisiae (0.422 g/g). Sequentially inoculating each non-Saccharomyces species with S. cerevisiae into grape juice media achieved up to 3.0% v/v alcohol reduction compared to S. cerevisiae alone while producing variable glycerol, succinic acid, and acetic acid concentrations. However, neither non-Saccharomyces yeasts released appreciable CO2 under fermentative conditions regardless of incubation temperature. Despite equivalent peak populations, S. cerevisiae produced more biomass (2.98 g/L) than the non-Saccharomyces yeasts while sequential inoculations yielded higher biomass with Mt. pulcherrima (3.97 g/L) but not My. guilliermondii (3.03 g/L). To reduce ethanol concentrations, these non-Saccharomyces species may metabolize ethanol and/or produce less from metabolized sugars compared to S. cerevisiae but also divert carbon towards glycerol, succinic acid, and/or biomass.

Survey of the yeast ecology of dehydrated grapes and strain selection for wine fermentation.

In this study we investigated the yeast population present on partially dehydrated Nebbiolo grapes destined for 'Sforzato di Valtellina', with the aim to select indigenous starters suitable for the production of this wine. Yeasts were enumerated, isolated, and identified by molecular methods (5.8S-ITS-RFLP and D1/D2 domain sequencing). A genetic, physiological (ethanol and sulphur dioxide tolerance, potentially useful enzymatic activities, hydrogen sulphide production, adhesive properties, and killer activity) and oenological (laboratory pure micro-fermentations) characterization was also carried out. Based on relevant physiological features, seven non-Saccharomyces strains were chosen for laboratory-scale fermentations, either in pure or in mixed-culture (simultaneous and sequential inoculum) with a commercial Saccharomyces cerevisiae strain. Finally, the best couples and inoculation strategy were further tested in mixed fermentations in winery. In both laboratory and winery, microbiological and chemical analyses were conducted during fermentation. The most abundant species on grapes were Hanseniaspora uvarum (27.4 % of the isolates), followed by Metschnikowia spp. (21.0 %) and Starmerella bacillaris (12.9 %). Technological characterization highlighted several inter- and intra-species differences. The best oenological aptitude was highlighted for species Starm. bacillaris, Metschnikowia spp., Pichia kluyveri and Zygosaccharomyces bailli. The best fermentation performances in laboratory-scale fermentations were found for Starm. bacillaris and P. kluyveri, due to their ability to reduce ethanol (-0.34 % v/v) and enhance glycerol production (+0.46 g/L). This behavior was further confirmed in winery. Results of this study contribute to the knowledge of yeast communities associated with a specific environment, like those of Valtellina wine region.

Biocontrol Potential of Antagonistic Yeasts on In Vitro and In Vivo Aspergillus Growth and Its AFB1 Production.

Aspergillus flavus is a major aflatoxin B1, posing significant health concerns to humans, crops, and producer fungi. Due to the undesirable consequences of the usage of synthetic fungicides, biological control using yeasts has gained more attention. In this study, eight isolates of epiphytic yeasts belonging to Moesziomyces sp., Meyerozyma sp. and Metschnikowia sp., which have been identified as antagonists, were isolated from different plants, including grapes, blueberries, hawthorns, hoskiran, beans and grape leaf. While volatile organic compounds (VOCs) produced by Moesziomyces bullatus DN-FY, Metschnikowia aff. pulcherrima DN-MP and Metschnikowia aff. pulcherrima 32-AMM reduced in vitro A. flavus mycelial growth and sporulation, only VOCs produced by Metschnikowia aff. fructicola 1-UDM were found to be effective at reducing in vitro AFB1 production. All yeasts reduced the mycelial growth of A. flavus by 76-91%, while AFB1 production reduced to 1.26-10.15 ng/g and the control plates' growth was 1773 ng/g. The most effective yeast, Metschnikowia aff. Pulcherrima DN-HS, reduced Aspergillus flavus growth and aflatoxin B1 production on hazelnuts. The AFB1 content on hazelnuts reduced to 333.01 ng/g from 536.74 ng/g. To our knowledge, this is the first report of yeasts isolated from plants being tested as potential biological control agents to reduce AFB1 production on hazelnuts.

Finding a correct species assignment for a Metschnikowia strain: insights from the genome sequencing of strain DBT012.

Metschnikowia pulcherrima is an important yeast species that is attracting increased interest thanks to its biotechnological potential, especially in agri-food applications. Phylogenetically related species of the so-called 'pulcherrima clade' were first described and then reclassified in one single species, which makes the identification an intriguing issue. Starting from the whole-genome sequencing of the protechnological strain Metschnikowia sp. DBT012, this study applied comparative genomics to calculate similarity with the M. pulcherrima clade publicly available genomes with the aim to verify if novel single-copy putative phylogenetic markers could be selected, in comparison with the commonly used primary and secondary barcodes. The genome-based bioinformatic analysis allowed the identification of 85 consensus single-copy orthologs, which were reduced to three after split decomposition analysis. However, wet-lab amplification of these three genes in nonsequenced type strains revealed the presence of multiple copies, which made them unsuitable as phylogenetic markers. Finally, average nucleotide identity (ANI) was calculated between strain DBT012 and available genome sequences of the M. pulcherrima clade, although the genome dataset is still rather limited. Presence of multiple copies of phylogenetic markers as well as ANI values were compatible with the recent reclassification of the clade, allowing the identification of strain DBT012 as M. pulcherrima.

Croatian white grape variety Marastina: First taste of its indigenous mycobiota.

The indigenous vineyard mycobiota contribute both to wine quality and vineyard sanitary status. Wines made from same grape variety but from different geographical locations are appreciated for their diversity. Because no information on indigenous mycobiota of Croatian grapevines is available, the aim of the present study was to start filling this knowledge gap by characterizing the indigenous mycobiota of Marastina variety. The use of metataxonomic approach has enabled the identification of 25 different fungal genera present on Marastina grape berries collected from 11 vineyards located within the Croatian coastal winegrowing region of Dalmatia (Northern Dalmatia, Dalmatian hinterland, Central and Southern Dalmatia). The substantial regional and local scale differences in their distribution were observed. Overall, Aureobasidium was the dominant genus followed by Cladosporium and Metschnikowia. Botrytis and Plenodomus were associated with the vineyards located in Central and Southern Dalmatia, whereas Pichia was associated with Northern Dalmatia vineyards. The largest abundance of Buckleyzyma, Cladosporium, Eremothecium, Fusarium, Papiliotrema, and Rhodotorula was observed in Dalmatian hinterland. Moreover, data suggested that climate conditions and soil type partially influenced the distribution of fungal communities. The local-scale differences emerged also for the physicochemical characteristics of fresh musts. The high malic acid content supported the development of Metschnikowia, and inhibited Fusarium growth, whereas a positive correlation between Erysiphe and pH values was observed. Sporobolomyces and Cystobasidium were negatively associated with high glucose concentration. The revealing of Marastina indigenous mycobiota provided information on the members of fungal community negatively influencing the grapevine sanitary status as well as those which could be employed in disease biocontrol.

Development of a TaqMan real-time quantitative PCR assay to detect Metschnikowia bicuspidata in Chinese mitten crab Eriocheir sinensis.

Milky disease of Chinese mitten crab Eriocheir sinensis caused by Metschnikowia bicuspidata is a novel disease with high mortality. No effective treatment is currently available, but a rapid, accurate detection method is required for the prevention and control of the disease. In this study, the genome-sequencing results of M. bicuspidata and similar species were used for comparative genomic analysis for genes specific to M. bicuspidata. A quantitative PCR (qPCR) detection method for M. bicuspidata was then established using the specific primers and probes designed according to the sequence of a hypothetical protein gene specific to M. bicuspidata. The assay was found to have a high degree of repeatability and reproducibility, with a linear dynamic range (R2 = 0.998) extending over 9 log10 dilutions and a high efficiency (100.7%). Furthermore, the method showed high sensitivity, being able to detect at least 11.3 copies µl-1 of recombinant plasmid, and strong specificity, without any cross-reaction with any of the 9 species of yeast that are closely related to M. bicuspidata or any of 16 species of pathogenic bacteria commonly observed in aquatic animals. The established method was used to examine 138 apparently healthy crabs collected from 22 farms, with 21 samples (15.2%) found to be M. bicuspidata-positive. Thus, the developed qPCR assay is a specific, sensitive, stable, and rapid diagnostic method for the detection and quantification of M. bicuspidata DNA from E. sinensis tissues.