The Impact of a Commercial Biostimulant on the Grape Mycobiota of Vitis vinifera cv. Barbera.

Reducing the use of fungicides, insecticides, and herbicides in order to limit environmental pollution and health risks for agricultural operators and consumers is one of the goals of European regulations. In fact, the European Commission developed a package of measures (the European Green Deal) to promote the sustainable use of natural resources and strengthen the resilience of European agri-food systems. As a consequence, new plant protection products, such as biostimulants, have been proposed as alternatives to agrochemicals. Their application in agroecosystems could potentially open new scenarios regarding the microbiota. In particular, the vineyard microbiota and the microbiota on the grape surface can be affected by biostimulants and lead to different wine features. The aim of this work was to assess the occurrence of a possible variation in the mycobiota due to the biostimulant application. Therefore, our attention has been focused on the yeast community of grape bunches from vines subjected to the phytostimulant BION®50WG treatment. This work was carried out in the CREA-VE experimental vineyard of Vitis vinifera cv. Barbera in Asti (Piedmont, Italy). The composition of fungal communities on grapes from three experimental conditions such as IPM (integrated pest management), IPM+BION®50WG, and IPM+water foliar nebulization was compared by a metabarcoding approach. Our results revealed the magnitude of alpha and beta diversity, and the microbial biodiversity index and specific fungal signatures were highlighted by comparing the abundance of yeast and filamentous fungi in IPM and BION®50WG treatments. No significant differences in the mycobiota of grapevines subjected to the three treatments were detected.

Genome Sequence of Oenococcus oeni OE37, an Autochthonous Strain Isolated from an Italian White Wine.

Oenococcus oeni OE37 is an autochthonous strain that was isolated from a Chardonnay wine from Piedmont (Italy) during spontaneous malolactic fermentation. Here, the OE37 genome sequence is presented, and a brief description of the main genes is reported.

Yeast distribution in Grignolino grapes growing in a new vineyard in Piedmont and the technological characterization of indigenous Saccharomyces spp. strains.

The aim of this study was to characterize the yeast consortium isolated from Grignolino grapes in a newly planted vineyard in Piedmont (Italy) via analysis of the intra-vineyard yeast distribution of grape samples from single rows. A two-phase approach allowed the identification of culturable yeasts present on grape skins and, through an enriching procedure via grape fermentation, the isolation of low frequency non-Saccharomyces and Saccharomyces spp. fermentative species, including S. paradoxus, which is highly unusual during grape fermentation, along with the intra-specific characterization of S. cerevisiae isolates. Culture-based molecular techniques revealed a grape yeast microbiota formed by (in order of abundance) Hanseniaspora uvarum, the yeast-like fungus Aerobasidium pullulans, Candida zemplinina, Pichia kluyveri, Candida californica, Curvibasidium cygneicollum, Meyerozima caribbica, Rhodotorula babjevae, Metschnikowia pulcherrima and Cryptococcus flavescens. Technological properties of isolated Saccharomyces spp. strains were analysed, identifying strains, including S. paradoxus, potentially suitable as an ecotypical starter for territorial wines.

Enhanced arginine biosynthesis and lower proteolytic profile as indicators of Saccharomyces cerevisiae stress in stationary phase during fermentation of high sugar grape must: A proteomic evidence.

A strain of Saccharomyces (S) cerevisiae (ISE19), which displayed an initial good adaptation to a high sugar medium with increased acetate and glycerol production but weak overall growth/fermentation performances, was selected during the alcoholic fermentation of Cortese grape must. To obtain insights into the metabolic changes that occur in the must during growth in particular conditions (high ethanol, high residual sugars and low nitrogen availability) leading to a sluggish fermentation or even fermentation arrest, comparative in-gel proteomic analyses were performed on cells grown in media containing 200g/L and 260g/L of glucose, respectively, while the YAN (Yeast Assimilable Nitrogen) concentration was maintained as it was. Two post-translationally different arginine synthases (pIs 5.6 and 5.8) were found in higher abundances in the high glucose-grown cells, together with an increased abundance of a glycosyltransferase involved in cell-wall mannans synthesis, and of two regulatory proteins (K7_Bmh1p and K7_Bmh2p) that control membrane transport. In parallel, a proteinase K-like proteolytic enzyme and three other protein fragments (Indolepyruvate decarboxylase 1, Fba1p and Eno1p) were present in lower abundances in the high glucose condition, where oxidative stress and cell cycle involved enzymes were also found to be less abundant. The overall results suggest that in stationary phase stress conditions, leading to stuck fermentation, S. cerevisiae ISE19 decreases cell replication, oxidative stress responses and proteolytic activity, while induces other metabolic modifications that are mainly based on cell-wall renewal, regulation of the solute transport across the cell membrane and de novo arginine synthesis.

Antiviral Properties of the Natural Polyphenols Delphinidin and Epigallocatechin Gallate against the Flaviviruses West Nile Virus, Zika Virus, and Dengue Virus.

The Flavivirus genus contains important pathogens, such as West Nile virus (WNV), Zika virus (ZIKV), and Dengue virus (DENV), which are enveloped plus-strand RNA viruses transmitted by mosquitoes and constitute a worrisome threat to global human and animal health. Currently no licensed drugs against them are available, being, thus, still necessary the search for effective antiviral molecules. In this line, a novel antiviral approach (economical, simple to use, and environmental friendly) is the use of natural compounds. Consequently, we have tested the antiviral potential of different polyphenols present in plants and natural products, such as wine and tea, against WNV, ZIKV, and DENV. So that, we assayed the effect of a panel of structurally related polyphenols [delphinidin (D), cyanidin (Cy), catechin (C), epicatechin (EC), epigallocatechin (EGC), and epigallocatechin gallate (EGCG)] on WNV infection, and found that D and EGCG inhibited more effectively the virus production. Further analysis with both compounds indicated that they mainly affected the attachment and entry steps of the virus life cycle. Moreover, D and EGCG showed a direct effect on WNV particles exerting a virucidal effect. We showed a similar inhibition of viral production of these compounds on WNV variants that differed on acidic pH requirements for viral fusion, indicating that their antiviral activity against WNV is produced by a virucidal effect rather than by an inhibition of pH-dependent viral fusion. Both polyphenols also reduced the infectivity of ZIKV and DENV. Therefore, D and EGCG impair the infectivity in cell culture of these three medically relevant flaviviruses.

Phage-host interactions analysis of newly characterized Oenococcus oeni bacteriophages: Implications for malolactic fermentation in wine.

Nowadays, only few phages infecting Oenococcus oeni, the principal lactic acid bacteria (LAB) species responsible for malolactic fermentation (MLF) in wine, have been characterized. In the present study, to better understanding the factors affecting the lytic activity of Oenococcus phages, fifteen O. oeni bacteriophages have been studied in detail, both with molecular and microbiological methods. No correlations were found between genome sizes, type of integrase genes, or morphology and the lytic activity of the 15 tested phages. Interestingly, though phage attack in a wine at the end of alcoholic fermentation seems not to be a problem, it can indeed represent a risk factor for MLF when the alcohol content is low, feature that may be a key point for choosing the appropriate time for malolactic starter inoculation. Additionally, it was observed that some phages genomes bear 2 or 3 types of integrase genes, which point to horizontal gene transfer between O. oeni bacteriophages.

An event-specific method for the detection and quantification of ML01, a genetically modified Saccharomyces cerevisiae wine strain, using quantitative PCR.

The availability of genetically modified (GM) yeasts for winemaking and, in particular, transgenic strains based on the integration of genetic constructs deriving from other organisms into the genome of Saccharomyces cerevisiae, has been a reality for several years. Despite this, their use is only authorized in a few countries and limited to two strains: ML01, able to convert malic acid into lactic acid during alcoholic fermentation, and ECMo01 suitable for reducing the risk of carbamate production. In this work we propose a quali-quantitative culture-independent method for the detection of GM yeast ML01 in commercial preparations of ADY (Active Dry Yeast) consisting of efficient extraction of DNA and qPCR (quantitative PCR) analysis based on event-specific assay targeting MLC (malolactic cassette), and a taxon-specific S. cerevisiae assay detecting the MRP2 gene. The ADY DNA extraction methodology has been shown to provide good purity DNA suitable for subsequent qPCR. The MLC and MRP2 qPCR assay showed characteristics of specificity, dynamic range, limit of quantification (LOQ) limit of detection (LOD), precision and trueness, which were fully compliant with international reference guidelines. The method has been shown to reliably detect 0.005% (mass/mass) of GM ML01 S. cerevisiae in commercial preparations of ADY.

Short-term response of different Saccharomyces cerevisiae strains to hyperosmotic stress caused by inoculation in grape must: RT-qPCR study and metabolite analysis.

During the winemaking process, glycerol synthesis represents the first adaption response of Saccharomyces cerevisiae to osmotic stress after inoculation in grape must. We have implemented an RT-qPCR (Reverse Transcription-quantitative PCR) methodology with a preventive evaluation of candidate reference genes, to study six target genes related to glycerol synthesis (GPD1, GPD2, GPP2 and GPP1) and flux (STL1 and FPS1), and three ALD genes coding for aldehyde dehydrogenase involved in redox equilibrium via acetate production. The mRNA level in three strains, characterized by different metabolite production, was monitored in the first 120 min from inoculation into natural grape must. Expression analysis shows a transient response of genes GPD1, GPD2, GPP2, GPP1 and STL1 with differences among strains in term of mRNA abundance, while FPS1 was expressed constitutively. The transient response and different expression intensity among strains, in relation to the intracellular glycerol accumulation pattern, prove the negative feedback control via the HOG (High Osmolarity Glycerol) signalling pathway in S. cerevisiae wine strains under winery conditions. Among the ALD genes, only ALD6 was moderately induced in the hyperosmotic environment but not in all strains tested, while ALD3 and ALD4 were drastically glucose repressed. The intensity of transcription of ALD6 and ALD3 seems to be related to different acetate production found among the strains.

Complementing DIGE proteomics and DNA subarray analyses to shed light on Oenococcus oeni adaptation to ethanol in wine-simulated conditions.

Direct addition of Oenococcus oeni starters into wine can cause viability problems. In the present study, the influence of ethanol in wine-simulated conditions on O. oeni has been evaluated by complementing microarray techniques and DIGE proteomics. Two different ethanol concentrations were studied. In 12% ethanol, pyrimidine anabolism was stimulated, but in 8% ethanol some energy-consuming biosynthetic pathways were limited. The most significant result was the stress response induced by alcohol that concerned both the cell-envelope and specific stress proteins. Interestingly, 8% and 12% ethanol triggered different stress responses: in mild ethanol stress (8%), chaperones with prevalent refolding activity (like HSP20) were over-expressed, whereas at higher alcohol concentration (12%), together with HSP20 and the refolding DNAJ/K, also chaperones having proteolytic activity (like ClpP) were induced. Furthermore the stress response repressor HrcA was downregulated only at 12% ethanol, suggesting that it controls stress pathways, which are different from those active at 8% alcohol. This result confirms that the HrcA system is operative in O. oeni where the CtrS system is prevalent. BIOLOGICAL SIGNIFICANCE: The use of malolactic starter cultures has become widespread to control the MLF process and to prevent off-flavors. There is significant interest in understanding the molecular mechanisms that O. oeni uses to adapt to harsh wine conditions. The overall results highlight that the alcohol-induced stress response involves not only biosynthesis of stress proteins but also envelope-linked mechanisms. From a practical point of view this research underlines the importance of starters acclimation to induce responses that would allow better adaptation to the wine. As a consequence, a well adapted starter can complete malolactic fermentation and improve the final wine quality.

Development of a new method for detection and identification of Oenococcus oeni bacteriophages based on endolysin gene sequence and randomly amplified polymorphic DNA.

Malolactic fermentation (MLF) is a biochemical transformation conducted by lactic acid bacteria (LAB) that occurs in wine at the end of alcoholic fermentation. Oenococcus oeni is the main species responsible for MLF in most wines. As in other fermented foods, where bacteriophages represent a potential risk for the fermentative process, O. oeni bacteriophages have been reported to be a possible cause of unsuccessful MLF in wine. Thus, preparation of commercial starters that take into account the different sensitivities of O. oeni strains to different phages would be advisable. However, currently, no methods have been described to identify phages infecting O. oeni. In this study, two factors are addressed: detection and typing of bacteriophages. First, a simple PCR method was devised targeting a conserved region of the endolysin (lys) gene to detect temperate O. oeni bacteriophages. For this purpose, 37 O. oeni strains isolated from Italian wines during different phases of the vinification process were analyzed by PCR for the presence of the lys gene, and 25 strains gave a band of the expected size (1,160 bp). This is the first method to be developed that allows identification of lysogenic O. oeni strains without the need for time-consuming phage bacterial-lysis induction methods. Moreover, a phylogenetic analysis was conducted to type bacteriophages. After the treatment of bacteria with UV light, lysis was obtained for 15 strains, and the 15 phage DNAs isolated were subjected to two randomly amplified polymorphic DNA (RAPD)-PCRs. By combining the RAPD profiles and lys sequences, 12 different O. oeni phages were clearly distinguished.

Putrescine production from different amino acid precursors by lactic acid bacteria from wine and cider.

The aim of this work was to study the production of biogenic amines and particularly putrescine in lactic acid bacteria (LAB) related to wine and cider. We applied an analytical protocol that involves the use of PCR and TLC techniques to determine the production of putrescine from different precursors. Moreover, we also studied the ability of the Lactobacillus and Pediococcus tested to produce histamine and tyramine. The results showed that the majority of the Lactobacillus brevis analyzed harbour both AgDI and tdc genes and are tyramine and putrescine producers. Conversely, among the other LAB tested, only one Lactobacillus hilgardii and one Pediococcus pentosaceus produced putrescine. The AgDI gene was also detected in two other LAB (Lactobacillus mali and Pediococcus parvulus), but no putrescine production was observed. Finally, hdc gene and histamine production were found in strains (L. hilgardii 5211, isolated from wine, and Lactobacillus casei 18, isolated from cider) that were not putrescine producers.

Does Oenococcus oeni produce histamine?

The presence of histamine in wine and other fermented foods may pose a toxicological risk for consumers. Production of histamine by Oenococcus oeni, which is the main agent of malolactic fermentation in wine and thus very important for the wine industry, has been extensively analyzed with contradictory results. If histamine production by O. oeni strains is a widespread trait, enological practices will be affected and the use of non-producing commercial O. oeni starters should be strongly recommended to avoid histamine production during winemaking. However, a review of published data showed that most evidence strongly supports the view that O. oeni is not responsible for histamine production in wine. We therefore propose the adoption of common analytical methods and the introduction of publicly-available validated histamine-producing O. oeni reference strains as a common positive control in assays to resolve this important issue.

Degradation of ochratoxin a by Brevibacterium species.

The ability to degrade ochratoxin A was studied in different bacteria with a well-known capacity to transform aromatic compounds. Strains belonging to Rhodococcus, Pseudomonas, and Brevibacterium genera were grown in liquid synthetic culture medium containing ochratoxin A. Brevibacterium spp. strains showed 100% degradation of ochratoxin A. Ochratoxin α was detected and identified by high-performance liquid chromatography-mass spectrometry (HPLC-MS) as a degradation product in the cell-free supernatants. The degradation of ochratoxin A is of public concern for food and environmental safety, because it could contribute to the development of new biological ochratoxin A detoxification systems in foodstuffs. In this study, the degradation of ochratoxin A by bacteria belonging to the food chain was demonstrated for the first time.

Quantitative expression analysis of mleP gene and two genes involved in the ABC transport system in Oenococcus oeni during rehydration.

Oenococcus oeni is recognized as the principal microorganism responsible for malolactic fermentation, and the control of its activity is of primary importance in winemaking. The aim of this study was to quantify the levels of expression of the malate transporter gene (mleP) and of two genes putatively involved in the ATP-binding cassette transport system (oeoe_1651, oeoe_0550) to better understand the physiological response of bacteria during rehydration. These genes coding for transporters were studied in different rehydration media. Initially, three different statistical algorithms were used to identify suitable reference genes to be used for the normalization of expression data in O. oeni during rehydration, and to this purpose, the best genes found were ddl and gyrB. The results showed that the genes for transporters of malate and sugar (mleP, oeoe_1651) were activated immediately after a few minutes of rehydration, when specific medium compositions were used.

Identification of reference genes suitable for normalization of RT-qPCR expression data in Saccharomyces cerevisiae during alcoholic fermentation.

Expression data from RT-qPCR (reverse transcription quantitative PCR) needs to be normalized to account for experimental variability among samples caused by differential yields of the transcripts in RNA extraction or in the reverse transcription. The most common method is to normalize against one or more reference genes (RG). We have selected RGs suitable for normalization of RT-qPCR raw data in Saccharomyces cerevisiae during alcoholic fermentation. The RGs were evaluated by three different statistical methods. The suitability of the selected RG sets was compared with ACT1, a commonly used non-validated single RG, by normalizing the expression of two target genes. Expression profiles of the target genes revealed the risk of misleading interpretation of expression data due to an unreliable RG.

An RT-qPCR approach to study the expression of genes responsible for sugar assimilation during rehydration of active dry yeast.

A short reactivation period in aqueous media is required for active dry yeast (ADY) to be utilised in winemaking. Rehydration restores the active metabolic conditions necessary for good fermentative and competitive abilities. We used a reverse transcription-quantitative PCR (RT-qPCR) method with relative quantification to investigate the expression of seven hexose transporter genes (HXT1-7) and one invertase-encoding gene (SUC2) during ADY rehydration in water with or without sucrose. For this, seven candidate reference genes were evaluated, and the three most stably expressed genes were selected and used for mRNA normalisation. The results show that, during the rehydration in the presence of sucrose, yeast cells are able to immediately hydrolyse this sugar into glucose and fructose as soon as they are introduced in the medium. Subsequently, differential glucose/fructose uptake occurs, which is mediated by hexose transporters. At the transcriptomic level, there is a strong induction of the high-affinity transporters, HXT2 and HXT4, and the low-affinity transporters, HXT3 and HXT1, when ADY is rehydrated with sucrose, while HXT5 and HXT6/7 are expressed at high levels with a moderate tendency to decrease. In water, the HXT2 gene was the only one of the transporter genes studied that showed significant variations. These results suggest that during rehydration, expression is not simply regulated by the affinity to hexose but is also controlled by other mechanisms that allow the cell to bypass glucose control. Moreover, the expression of SUC2 showed little variation in media with sucrose, suggesting that other invertases and/or posttranscriptional controls exist.

Biogenic amine production by contaminating bacteria found in starter preparations used in winemaking.

The aim of this work was to investigate if contaminating microorganisms, eventually present in bacteria and yeast preparations used as commercial starters in winemaking, have the ability to produce the biogenic amines histamine, putrescine and tyramine. Thirty commercial starters (14 yeasts Saccharomyces cerevisiae and 16 bacteria Oenococcus oeni) were cultured in synthetic broth and analyzed by TLC to detect amine production. Oenococcus oeni commercial preparations did not contain contaminants, but some yeast preparations resulted contaminated with amine-producing bacteria. Bacterial contaminants were isolated and analyzed for their ability to produce biogenic amines using HPLC and TLC. Decarboxylase genes were identified using PCR analysis followed by sequencing. Fermentations were performed in grape juice with two yeast commercial preparations containing bacterial contaminants to check if the potential biogenic amine production could happen also during winemaking. It was found that this production is possible; in particular, in the conditions used in this work, tyramine production was detected. Therefore, the results of this study have significance in relation to the risk of biogenic amines in wine. Moreover a novel species of Lactobacillus was found to be able to produce histamine.

Application of real-time RT-PCR to study gene expression in active dry yeast (ADY) during the rehydration phase.

During the industrial production of active dry yeast (ADY) and its subsequent use in winemaking, the yeast cell is subjected to drastic environmental changes that force it to undergo extensive metabolic modifications and changes in gene expression. In this study, we describe the use of real-time reverse transcription-polymerase chain reaction (RT-PCR) to monitor gene expression in ADY Saccharomyces cerevisiae during rehydration in different media. We used three statistical approaches to investigate the expression stability of eight potential reference genes during the rehydration process. The reference system thus obtained was used to normalize the expression values of three genes codifying for the ammonium transporters -- MEP1, MEP2, and MEP3 -- and two genes involved in the osmotic response-SIP18 and GPD1. The results suggested that for the target genes tested, the yeast reacted immediately to rehydration only when a fermentable carbon source was present in the medium. Furthermore, MEP2 expression was modulated by the ammonium concentration, indicating that nitrogen catabolite repression (NCR) is active during the rehydration phase.

Discrimination of Saccharomyces cerevisiae wine strains using microsatellite multiplex PCR and band pattern analysis.

We propose a rapid method for Saccharomyces cerevisiae strain identification based on multiplex PCR analysis of polymorphic microsatellite loci. Simple DNA extraction without the use of phenol, followed by a rapid PCR procedure optimised for multiplex amplification of loci SC8132X, YOR267C and SCPTSY7 and band pattern analysis of the fragments generated by agarose and polyacrylamide gel electrophoresis, has allowed us to distinguish among a panel of 30 tested commercial wine strains. This method was successfully performed in an ecological study where dominance between two strains was checked at two fermentation temperatures: 15 and 20 degrees C. The method should be useful for routine and low-budget discrimination of yeast strains, both in the wine and yeast production industries.

In vitro removal of ochratoxin A by wine lactic acid bacteria.

A study was carried out to determine the in vitro interaction between ochratoxin A (OTA) and wine lactic acid bacteria (LAB). Fifteen strains belonging to five relevant oenological LAB species were grown in liquid synthetic culture medium containing OTA. The portion of OTA removed during the bacterial growth was 8 to 28%. The OTA removed from the supernatants was partially recovered (31 to 57%) from the bacterial pellet. Cell-free extracts of three representative strains were produced by disrupting cells in a French pressure cell. The ability of crude cell-free extracts to degrade OTA was studied. OTA was not degraded by cell-free extracts of wine LAB strains, and no degradation products of OTA were detected in the high-performance liquid chromatograms of the methanol extract of the bacterial pellet. On the basis of these results, we conclude that OTA removal by wine LAB is a cell-binding phenomenon. The chemistry and the molecular basis of OTA binding to wine LAB remains unknown.