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.

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.

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.

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.

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.

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.

Microbial lipids production from wastes by Metschnikowia pulcherrima: a review / 生物工程学报

Biodiesel is an alternative fuel to addressing the energy shortage problem. Microbial lipids have attracted widespread attention as one of the potential feed-stocks for cost-effective and efficient biodiesel production. However, the large-scale production of microbial lipids is hampered by the complexity and the high cost of aseptic culturing approach. Metschnikowia pulcherrima is an oleaginous yeast with strong environmental adaptability. It is capable of utilizing a wide spectrum of substrates, and can be cultured under non-sterile conditions. Therefore, this yeast has great potential to replace the traditional oleaginous microorganisms, particularly in the area of recycling wastewater and solid waste for the production of biodiesel. Based on the analysis of lipid production and application conditions of M. pulcherrima, this review summarized the unique advantages of M. pulcherrima and the key factors affecting lipids production. We further discussed the feasibility of cultivating M. pulcherrima on various organic wastes under non-sterile conditions for lipids production. Moreover, we analyzed the challenges associated with M. pulcherrima's in the yield and mechanism for lipids production, and proposed perspectives for how to achieve efficient biodiesel production using this yeast.

Screening Wild Yeast Strains for Alcohol Fermentation from Various Fruits

Wild yeasts on the surface of various fruits including grapes were surveyed to obtain yeast strains suitable for fermenting a novel wine with higher alcohol content and supplemented with rice starch. We considered selected characteristics, such as tolerance to alcohol and osmotic pressure, capability of utilizing maltose, and starch hydrolysis. Among 637 putative yeast isolates, 115 strains exhibiting better growth in yeast-peptone-dextrose broth containing 30% dextrose, 7% alcohol, or 2% maltose were selected, as well as five alpha-amylase producers. Nucleotide sequence analysis of the 26S rDNA gene classified the strains into 13 species belonging to five genera; Pichia anomala was the most prevalent (41.7%), followed by Wickerhamomyces anomalus (19.2%), P. guilliermondii (15%), Candida spp. (5.8%), Kodamaea ohmeri (2.5%), and Metschnikowia spp. (2.5%). All of the alpha-amylase producers were Aureobasidium pullulans. Only one isolate (NK28) was identified as Saccharomyces cerevisiae. NK28 had all of the desired properties for the purpose of this study, except alpha-amylase production, and fermented alcohol better than commercial wine yeasts.