Effects of film-forming Pichia and Candida yeasts on cider and wine as post-fermentation contaminants.

Film-forming yeasts are potential sources of defects in alcoholic beverages. The aim of this study is to assess the growth capacity of Pichia and Candida film-forming yeasts in cider and wine and the effects on their chemical composition. Cider, partially and fully fermented wine were inoculated with strains of C. californica, P. fermentans, P. kluyveri, P. kudriavzevii, P. manshurica, and P. membranifaciens to simulate a post-fermentative contamination. The former three species grew only in cider. Pichia manshurica and P. kudriavzevii displayed high viability in wine up to 13.18% (v v-1) ethanol. Significant changes in odour-active molecules from different chemical groups were observed in cider and wine in the inoculated samples, compared to the non-inoculated ones. Cider is more susceptible to contamination by all of the species tested, due to its low alcohol content, while P. membranifaciens, P. manshurica, and P. kudriavzevii are additionally potential spoilage agents of wine. This study highlights the risk of cider and wine contamination by film-forming yeasts. Their impact on aroma profiles depends on their ability to grow and their metabolism. This study contributes to an understanding of the possible physiological and metabolic mechanisms responsible for film formation and chemical changes in alcoholic beverages.

Fungicide sensitivity of grapevine bacteria with plant growth-promoting traits and antagonistic activity as non-target microorganisms.

This study evaluates the capacity of commercial formulations of synthetic fungicides to inhibit grapevine bacterial growth when sprayed on vineyards to control diseases, such as downy mildew, powdery mildew and secondary rots. Fungicide sensitivity plate assays were carried out on bacteria isolated from vineyards that were also identified and characterized for their plant growth-promoting (PGP) traits and antifungal activity. The high taxonomic variability of bacteria screened with different chemical classes of fungicides is one new finding of this study. Seven out of 11 fungicides were able to inhibit the growth of bacteria at a concentration corresponding to the maximum dose allowed by law in spray treatments of vineyards. Bacterial sensitivity to each fungicide varied greatly. Many sensitive isolates displayed PGP traits and/or antagonistic activity. This study shows the potential impact of fungicidal treatments on grapevine bacterial microbiota. The involvement of bacteria beneficial to the growth and health of plants underlines the importance of this investigation. Our data reveal that the control of a certain disease may be possible using fungicides that have no or low impact on natural non-target microbiota. Understanding the action mechanisms of the active ingredients in these products is a priority for the development of new eco-friendly pesticides.

Characterization of selected species of Pichia and Candida for their growth capacity in apple and grape must and their biofilm parameters.

Pichia and Candida species include biofilm-forming yeasts able to spoil foods and beverages. Strains belonging to 10 Pichia and Candida species isolated from apples, grape musts, and wines were analysed. They were subjected to molecular typing and characterized for their ability to grow and ferment must for cider and wine production, and for their biofilm properties. All strains grew similarly in apple and grape must. Glucose-fermenting strains displayed differentiated fermentation performances. Great variation in SO2 and ethanol sensitivity was observed among the strains. Pichia manshurica strains showed high tolerance to both molecules. Eleven and five surface-spreading biofilm (MAT) phenotypes were identified in solid and liquid media, respectively. Strains produced biofilms with variable thicknesses and widths in culture tubes. Cell adherence and aqueous-hydrocarbon biphasic hydrophobicity assays were carried out. Some Pichia manshurica and P. membranifaciens strains exhibited a high capacity to form a thick biofilm and had high cell adherence and hydrophobicity values. These strains could be more likely to colonize the internal surfaces of tanks. This study evidenced that some Pichia and Candida strains can proliferate during apple and grape must fermentation and may be detrimental the beverage quality, due to their specific biofilm properties.

Molecular and Physiological Properties of Indigenous Strains of Oenococcus oeni Selected from Nero di Troia Wine (Apulia, Italy).

The characterization of Oenococcus oeni strains isolated from Nero di Troia wine (Apulia, Italy) sampled in two distinct production areas was carried out. The two indigenous populations, consisting of 95 and 97 isolates, displayed high genetic diversity when analyzed by amplified fragments length polymorphisms (AFLP). Based on the UPGMA dendrogram obtained by AFLP analysis, the two populations displayed similar genotypes that grouped in the same clusters with a high level of similarity (>95%). One genotype was found in only one of the two areas. Representative strains of each cluster were analyzed for their enzymatic activities (esterase, ß-glucosidase, and protease), assayed in whole cells, and tested for their metabolic properties (consumption of L-malic acid, citric acid, acetaldehyde, and arginine) and growth parameters. Significant differences among strains, including the reference strain ATCC BAA-1163, were observed for all of these properties. Principal component analysis evidenced phenotypic differences among strains, and well separated some of them belonging to different genotypes. Strains exhibiting the best performances in most of these traits could be further investigated in order to select possible candidates as malolactic starters for Nero di Troia wine. This study provided insights on the population structure of O. oeni of a local winemaking area useful to the understanding of the regional diversity of this bacterium, an issue not yet completely resolved

In Vivo Endophytic, Rhizospheric and Epiphytic Colonization of Vitis vinifera by the Plant-Growth Promoting and Antifungal Strain Pseudomonas protegens MP12.

An evaluation was conducted of the colonization of Pseudomonas protegens MP12, a plant-growth promoting and antagonistic strain, inoculated in vine plants during a standard process of grapevine nursery propagation. Three in vivo inoculation protocols (endophytic, rhizospheric, and epiphytic) were implemented and monitored by means of both culture-dependent and independent techniques. Endophytic treatment resulted in the colonization of the bacterium inside the vine cuttings, which spread to young leaves during the forcing period. Microscopy analysis performed on transformed dsRed-tagged P. protegens MP12 cells confirmed the bacterium's ability to penetrate the inner part of the roots. However, endophytic MP12 strain was no longer detected once the plant materials had been placed in the vine nursery field. The bacterium also displayed an ability to colonize the rhizosphere and, when the plants were uprooted at the end of the vegetative season, its persistence was confirmed. Epiphytic inoculation, performed by foliar spraying of cell suspension, was effective in controlling artificially-induced Botrytis cinerea infection in detached leaves. The success of rhizospheric and leaf colonization in vine plants suggests potential for the future exploitation of P. protegens MP12 as biofertilizer and biopesticide. Further investigation is required into the stability of the bacterium's colonization of vine plants under real-world conditions in vineyards.

Epiphytic bacteria from withered grapes and their antagonistic effects on grape-rotting fungi.

The bacterial community in the surface of withered grapes, which are partially dehydrated in the post-harvest period to produce Italian passito wine, has been seldom investigated. Fifty epiphytic bacterial strains isolated from withered berries were identified and characterized. Genera such as Bacillus, Brevibacillus, Curtobacterium, Micrococcus, Pseudomonas and Staphylococcus have been identified by comparative sequence and phylogenetic analyses of 16S rRNA gene sequences. Bacillus was predominant and several taxa within this genus have been recognized. All isolates were characterized by PCR fingerprinting and assayed for osmotic tolerance, motility and antifungal activity. Several Bacillus strains displayed antagonistic effects on grape-rotting fungi such as Botrytis cinerea, Penicillium expansum and Aspergillus uvarum. The other strains were weakly or non-antagonistic on these fungi. Assay on antagonistic interactions among bacteria was also carried out. Bacillus strains, which exhibit swimming and swarming motility, have the potential to colonize the grape surface and to compete with their neighbours for space and resources. The occurrence of these isolates could reduce the contamination of fungal pathogens during grape withering. Epiphytic antagonistic bacteria could potentially be of interest for fungal biocontrol in the post-harvest processing of fruit and vegetables.

Correlating Noble Rot Infection of Garganega Withered Grapes with Key Molecules and Odorants of Botrytized Passito Wine.

Experimental passito wines with different percentages of naturally noble-rotten grapes of the Garganega variety were analyzed to evaluate key molecules and odorants related to the typical aroma and sensory profile of botrytized passito wine. Remarkable changes in the concentration of 1-octen-3-ol, 4-terpineol, benzaldehyde, N-(3-methylbutyl)acetamide, and sherry lactone 1 and 2 were observed between sound and noble-rotten wines. Wines were perceived to be different for floral, honey, figs, apricot, and caramel scents. By partial least square regression these descriptors were well correlated to samples. An important positive contribution of sherry lactones, N-(3-methylbutyl)acetamide, vanillin, benzaldehyde, and γ-butyrolactone to honey, apricot, and caramel was observed. It is conceivable that oxidative effects of Botrytis cinerea infection play an important role in the genesis of these chemical and sensory aroma markers. This study provides a predictive tool for winemakers that use natural grape withering to produce wines whose aroma profile is not standardized due to the seasonal variation of noble rot incidence.

Sporobolomyces agrorum sp. nov. and Sporobolomyces sucorum sp. nov., two novel basidiomycetous yeast species isolated from grape and apple must in Italy.

During a survey of yeast populations associated with grape and apple musts used for wine and cider fermentation, respectively, six pink-coloured ballistoconidia-forming yeasts belonging to the order Sporidiobolales (Basidiomycota) were isolated. Phylogenetic analysis inferred using sequences of the internal transcribed spacer (ITS), the D1/D2 domain of the large subunit rRNA gene, the small subunit (SSU) rRNA gene and DNA-directed RNA polymerase II subunit (RPB2) indicated that the six isolates were separated in two novel species. One of the new species, Sporobolomyces agrorum sp. nov., isolated from grape must, had Sporobolomyces roseus and Sporobolomyces metaroseus as its closest relatives, but showed four/two and 16 nucleotide substitutions in the D1/D2 and ITS regions, respectively, to these two species. The other novel species, Sporobolomyces sucorum sp. nov., was found in apple must and was closely related to Sporobolomyces pararoseus and Sporobolomyces patagonicus, but showed two/three and five substitutions in those two regions for its closest relatives. We detected additional representatives of this species, most of them isolated from grapes whose sequences were already available on public databases. A sexual stage could not be observed for the novel species.

Pseudomonas protegens MP12: A plant growth-promoting endophytic bacterium with broad-spectrum antifungal activity against grapevine phytopathogens.

Pseudomonas sp. MP12 was isolated from a soil sample collected in a typical warm-temperate deciduous forest near Brescia, Northern Italy. Phylogenetic analysis identified the species as Pseudomonas protegens. We evidenced in this strain the presence of the genes phlD, pltB and prnC responsible for the synthesis of the antifungal compounds 2,4-diacetylphloroglucinol (2,4-DAPG), pyoluteorin and pyrrolnitrin, respectively. P. protegens MP12 was also shown to produce siderophores and ammonia, yielded positive results with the indole-3-acetic acid test, and was capable of phosphate solubilization. Moreover, P. protegens MP12 exhibited inhibitory effects on in vitro mycelial growth of prominent grapevine (Vitis vinifera) phytopathogens such as Botrytis cinerea, Alternaria alternata, Aspergillus niger, Penicillium expansum and Neofusicoccum parvum. The strain showed activity even against Phaeomoniella chlamydospora and Phaeoacremonium aleophilum, which cause the devastating tracheomycosis/esca disease of grapevine trunks for which no efficacious control methods have been demonstrated so far. Furthermore, the MP12 strain manifested in vivo antifungal activity against B. cinerea on grapevine leaves. Culture-dependent and culture-independent analysis revealed the ability of P. protegens MP12 to efficiently and permanently colonize inner grapevine tissues. These results suggest that P. protegens MP12 could be worth of exploitation as an antifungal biocontrol agent for applications in viticulture.

Fungal Systematics and Evolution: FUSE 5.

Thirteen new species are formally described: Cortinarius brunneocarpus from Pakistan, C. lilacinoarmillatus from India, Curvularia khuzestanica on Atriplex lentiformis from Iran, Gloeocantharellus neoechinosporus from China, Laboulbenia bernaliana on species of Apenes, Apristus, and Philophuga (Coleoptera, Carabidae) from Nicaragua and Panama, L. oioveliicola on Oiovelia machadoi (Hemiptera, Veliidae) from Brazil, L. termiticola on Macrotermes subhyalinus (Blattodea, Termitidae) from the DR Congo, Pluteus cutefractus from Slovenia, Rhizoglomus variabile from Peru, Russula phloginea from China, Stagonosporopsis flacciduvarum on Vitis vinifera from Italy, Strobilomyces huangshanensis from China, Uromyces klotzschianus on Rumex dentatus subsp. klotzschianus from Pakistan. The following new records are reported: Alternaria calendulae on Calendula officinalis from India; A. tenuissima on apple and quince fruits from Iran; Candelariella oleaginescens from Turkey; Didymella americana and D. calidophila on Vitis vinifera from Italy; Lasiodiplodia theobromae causing tip blight of Dianella tasmanica 'variegata' from India; Marasmiellus subpruinosus from Madeira, Portugal, new for Macaronesia and Africa; Mycena albidolilacea, M. tenuispinosa, and M. xantholeuca from Russia; Neonectria neomacrospora on Madhuca longifolia from India; Nothophoma quercina on Vitis vinifera from Italy; Plagiosphaera immersa on Urtica dioica from Austria; Rinodina sicula from Turkey; Sphaerosporium lignatile from Wisconsin, USA; and Verrucaria murina from Turkey. Multi-locus analysis of ITS, LSU, rpb1, tef1 sequences revealed that P. immersa, commonly classified within Gnomoniaceae (Diaporthales) or as Sordariomycetes incertae sedis, belongs to Magnaporthaceae (Magnaporthales). Analysis of a six-locus Ascomycota-wide dataset including SSU and LSU sequences of S. lignatile revealed that this species, currently in Ascomycota incertae sedis, belongs to Pyronemataceae (Pezizomycetes, Pezizales).

Yeast-like fungi and yeasts in withered grape carposphere: Characterization of Aureobasidium pullulans population and species diversity.

Yeast-like fungi and yeasts residing on carposphere of withered grapes for Italian passito wine production have been scarcely investigated. In the present study, isolates from single berries, both sound and damaged, of Nosiola, Corvina and Garganega varieties were analyzed at the end of the withering process. Great variation of cell concentration among single berries was observed. In sound berries, yeast-like fungi were significantly more frequent than yeasts. Species identification of isolates was carried out by BLAST comparative analysis on gene databases and phylogenetic approach. All yeast-like fungi isolates belonged to Aureobasidium pullulans. They displayed different culture and physiological characteristics and inhibitory capacity against phytopathogenic fungi. Moreover, PCR profile analysis revealed high genotypic similarity among these strains. A total of 35 species were recognized among yeast isolates. Ascomycetes prevailed over basidiomycetes. To the best of our knowledge, Naganishia onofrii and Rhodosporidiobolus odoratus were identified for the first time among yeasts isolated from grapes, must or wine. Hanseniaspora uvarum and Starmerella bacillaris were the most frequent species. Most species were found only in one grape variety (nine in Nosiola, 10 in Corvina and five in Garganega). The sanitary state of withered grapes could have an important impact on the structure of these epiphytic populations.

Changes in chemical and sensory properties of Amarone wine produced by Penicillium infected grapes.

There is very little information on effects of Penicillium on aroma of passito wine. This study analyzed chemical composition and sensory properties of Amarone wines produced from withered grapes artificially contaminated by P. expansum or P. crustosum. Changes in properties of the two wines were evident by comparing wines obtained from healthy and Botrytis cinerea infected grapes used as controls. Penicillium infection affected primary and volatile composition of Amarone wine. Sensory profiles of these wines, obtained by descriptive analysis, resulted in clear differences in the wines between themselves and the control wines. Partial least square regression analysis explained only partially the relationship between molecules and sensory descriptors, and showed the existence of complex interactions of compounds mainly involved in specific aroma attributes. GC-olfactive analysis showed a greater number of odour regions in P. crustosum wine compared to control wines. Useful insight was provided into understanding how Penicillium rotten grapes affect Amarone wine properties.

Yeast species diversity in apple juice for cider production evidenced by culture-based method.

Identification of yeasts isolated from apple juices of two cider houses (one located in a plain area and one in an alpine area) was carried out by culture-based method. Wallerstein Laboratory Nutrient Agar was used as medium for isolation and preliminary yeasts identification. A total of 20 species of yeasts belonging to ten different genera were identified using both BLAST algorithm for pairwise sequence comparison and phylogenetic approaches. A wide variety of non-Saccharomyces species was found. Interestingly, Candida railenensis, Candida cylindracea, Hanseniaspora meyeri, Hanseniaspora pseudoguilliermondii, and Metschnikowia sinensis were recovered for the first time in the yeast community of an apple environment. Phylogenetic analysis revealed a better resolution in identifying Metschnikowia and Moesziomyces isolates than comparative analysis using the GenBank or YeastIP gene databases. This study provides important data on yeast microbiota of apple juice and evidenced differences between two geographical cider production areas in terms of species composition.

Bark and Grape Microbiome of Vitis vinifera: Influence of Geographic Patterns and Agronomic Management on Bacterial Diversity.

In recent years, the concept of "microbial terroir" has been introduced in the frame of the more renowned notion of "vitivinicultural terroir,' since several studies demonstrated that wine characteristics are related to regional microbial community compositions. Most of the existing research focused on grape berries microbiota, since it can directly impact wine quality. In this work we studied, for the first time through next-generation sequencing, the epiphytic bacterial community of vine bark and its relationships with grape microbiota. The study was carried out in two Italian wine appellations (situated in different regions) to explore the impact of biogeography, and the influence of two agronomical practices (biodynamic and conventional) was evaluated as well. Overall, our results show that grapevine bark harbors a rich epiphytic microbiota and displays a higher microbial biodiversity than grape berry. Moreover, this study suggests that geographic and anthropogenic factors impact both bark and grape bacteriomes, but to a different extent. The evidence of a "microbial terroir" seems to be even more marked in bark than in berries, possibly due to its permanence over time and to its physical proximity with soil. The importance of vine trunk bark, as potential source of inoculum for grapes and as interesting bacterial diversity habitat, is evidenced. This opens new fields of investigation, not only for researchers that aim at describing this little-known habitat within the vineyard, but also for stakeholders from the wine industry that want to understand the roles of microorganisms on the entire winemaking process, from vineyard to cellar.

Botrytis euroamericana, a new species from peony and grape in North America and Europe.

A novel species of Botrytis isolated from peony in Alaska, USA, and grape in Trento District, Italy, was identified based on morphology, pathogenicity, and sequence data. The grape and peony isolates share sequence homology in the glyceraldehyde-3-phosphate dehydrogenase (G3PDH), heat shock protein 60 (HSP60), DNA-dependent RNA polymerase subunit II (RPB2), and necrosis- and ethylene-inducing protein 1 and 2 (NEP1 and NEP2) genes that place them in a distinct group closely related to B. aclada, a globally distributed pathogen of onions. Genetic results were corroborated with morphological and pathogenicity trials that included two isolates of B. cinerea and two isolates of B. paeoniae from peony in Alaska and one isolate of B. aclada. The authors observed differences in colony and conidia morphology and ability to cause lesions on different host tissues that suggest that the grape and peony isolates represent a distinct species. Most notably, the grape and peony isolates did not colonize onion bulbs, whereas B. aclada readily produced lesions and prolific sporulation on onion tissue. The new species Botrytis euroamericana is described herein.

Linking wine lactic acid bacteria diversity with wine aroma and flavour.

In the last two decades knowledge on lactic acid bacteria (LAB) associated with wine has increased considerably. Investigations on genetic and biochemistry of species involved in malolactic fermentation, such as Oenococcus oeni and of Lactobacillus have enabled a better understand of their role in aroma modification and microbial stability of wine. In particular, the use of molecular techniques has provided evidence on the high diversity at species and strain level, thus improving the knowledge on wine LAB taxonomy and ecology. These tools demonstrated to also be useful to detect strains with potential desirable or undesirable traits for winemaking purposes. At the same time, advances on the enzymatic properties of wine LAB responsible for the development of wine aroma molecules have been undertaken. Interestingly, it has highlighted the high intraspecific variability of enzymatic activities such as glucosidase, esterase, proteases and those related to citrate metabolism within the wine LAB species. This genetic and biochemistry diversity that characterizes wine LAB populations can generate a wide spectrum of wine sensory outcomes. This review examines some of these interesting aspects as a way to elucidate the link between LAB diversity with wine aroma and flavour. In particular, the correlation between inter- and intra-species diversity and bacterial metabolic traits that affect the organoleptic properties of wines is highlighted with emphasis on the importance of enzymatic potential of bacteria for the selection of starter cultures to control MLF and to enhance wine aroma.

Post-harvest proteomics of grapes infected by Penicillium during withering to produce Amarone wine.

The study of withered grape infection by Penicillium, a potentially toxigenic fungus, is relevant to preserve grape quality during the post-harvest dehydration process. This report describes the first proteomic analysis of Amarone wine grapes, infected by two strains of Penicillium expansum (Pe1) and Penicillium crustosum (Pc4). Protein identification by MS analysis allowed a better understanding of physiological mechanisms underlying the pathogen attack. The Pe1 strain had a major impact on Vitis vinifera protein expression inducing pathogenesis-related proteins and other protein species involved in energy metabolism. A greater expression of new Penicillium proteins involved in energy metabolism and some protein species related to redox homeostasis has been observed on grapes infected by Pc4 strain. Moreover, the new induced proteins in infected grapes could represent potential markers in withered grapes, thus creating the chance to develop case-sensitive prevention strategies to inhibit fungal growth.

Diversity of bacterial endophytes in 3 and 15 year-old grapevines of Vitis vinifera cv. Corvina and their potential for plant growth promotion and phytopathogen control.

This study represents the first investigation on ecology of endophytic bacteria isolated from 3 and 15 year-old vine stems of Vitis vinifera cv. Corvina. The analysis was performed by means of culture-dependent techniques. The obtained results showed that new grapevine endophytic genera are being discovered. Moreover, Bacilli and Actinobacteria are frequently isolated from 3 year-old plants, whereas Alpha- and Gamma- Proteobacteria classes are more prevalent in the 15 year-old plants. Shannon-Wiener (H) index and analysis of rarefaction curves revealed greater genus richness in young grapevine plants. Furthermore, results evidenced an increase of genotypic group number within specific genera (e.g., Rhizobium and Pantoea). Among isolated strains from 3 and 15 year-old stems, respectively, 34 and 39% produce siderophores; 22 and 15% secrete ammonia; 22 and 21% produce indole-3-acetic acid; 8.7 and 41% solubilize phosphate. Besides, two strains isolated from 15 year-old grapevines showed 1-aminocyclopropane-1-carboxylate deaminase activity. Antifungal activity analysis evidenced that two Bacillus strains possess growth antagonistic effect toward all the tested fungal strains. Therefore, the present study extends our knowledge of the diversity of the endophytic bacteria by providing new insights into the complexity of the grapevine microbiome.

Description of a taxonomically undefined Sclerotiniaceae strain from withered rotten-grapes.

A necrotrophic member of the Sclerotiniaceae family (herewith named strain C10) isolated from withered rotten-grapes, is described. Interestingly, the fungus has no defined taxonomic position since it has been impossible to attribute it to an existing genus. Phylogenetic analysis of partial sequences of glyceraldehyde 3-phosphate dehydrogenase (G3PDH), heat shock protein 60 (HSP60) and DNA-directed RNA polymerase II subunit (RPB2), revealed that strain C10 is distantly related to Amphobotrys and Botrytis. This evidence clearly distinguishes this new Sclerotiniaceae member from other taxa of the family. Moreover, its morphological characteristics did not match those of Amphobotrys and Botrytis. Infectivity assays demonstrated that strain C10 could be a potential postharvest pathogen of withered grapes. This study revealed the taxonomic importance of this strain suggesting the existence of a possible new genus, a theory that requires further investigation.

Occurrence and infection of Cladosporium, Fusarium, Epicoccum and Aureobasidium in withered rotten grapes during post-harvest dehydration.

Fungi like Cladosporium, Fusarium, Epicoccum and Aureobasidium can occur on withered grapes causing spoilage of passito wine. There is little or no information on the pathogenic role of these fungi. This study describes the isolation, incidence and identification of several isolates from different withered rotten grapes. Representative isolates grouped in several phenotypes were identified by phylogenetic analysis of internal transcribed spacer, actin or elongation factor gene sequences. Isolates of Cladosporium and Fusarium were ascribed to different species, of these C. ramotenellum, C. halotolerans and F. graminearum were isolated from Vitis vinifera for the first time. All Epicoccum and Aureobasidium isolates belonged to E. nigrum and A. pullulans, respectively. Random amplified DNA polymorphism analysis showed high level of heterogenicity among Epicoccum and Fusarium isolates. Infection assays were carried out to evaluate infectivity in some strains under different withering conditions. Fusarium spp. strains had similar infectivity, while significant variability was observed among Cladosporium spp. and E. nigrum strains. A. pullulans resulted particularly infective. This study provided insights into the occurrence and infection of these fungi in fruit-drying rooms with important implications towards control management during the withering.