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Taggiasca table olives are typical of Liguria, a Northwestern Italian region, produced with a spontaneous fermentation carried out by placing the raw drupes directly into brine with a salt concentration of 8–12 % w/v. Such concentrations limit the development of unwanted microbes and favor the growth of yeasts. This process usually lasts up to 8 months. Yeasts are found throughout the entire fermentation process and they are mainly involved in the production of volatile organic compounds, which strongly impact the quality of the final product. The aim of this study was to evaluate the dynamics of autochthonous yeasts in brines and olives in a spontaneous process with no lye pre-treatment or addition of acids in the fermenting brine with 10 % NaCl (w/v) in two batches during 2021 harvest. Three hundred seventy-three yeast colonies were isolated, characterized by rep-PCR and identified by the D1/D2 region of the 26S rRNA gene sequencing. Mycobiota was also studied by 26S rRNA gene metataxonomics, while metabolome was assessed through GC–MS analysis. Traditional culture-dependent methods showed the dominance of Candida diddensiae , Wickerhamomyces anomalus , Pichia membranifaciens and Aureobasidium pullulans , with differences in species distribution between batches, sampling time and type of sample (olives/brines). Amplicon-based sequencing confirmed the dominance of W. anomalus in batch 1 throughout the entire fermentation, while Cyteromyces nyonsensis and Aureobasidium spp. were most abundant in the fermentation in batch 2. Volatilome results were analyzed and correlated to the mycobiota data, confirming differences between fermentation stages. Given the high appreciation for this traditional food, this study helps elucidate the mycobiota associated to Taggiasca cv. table olives and its relationship with the quality of the final product. • Taggiasca olives are spontaneously fermented in North West of Italy. • Yeasts as drivers of the Taggiasca olives fermentation • W. anomalus was detected as dominant specie with both molecular methods. • Fermentation stage was a key factor in the shift of the mycobiota and volatiles. [ABSTRACT FROM AUTHOR]

Alternaria alternata is a common fungal pathogen causing postharvest decay in table grapes. This study addressed the potential of autochthonous yeasts and bioactive compounds of natural sources to act as biocontrol agents (BCAs) against A. alternata in cold-stored table grapes. With this purpose, 19 yeast capable of growing at 0–1 °C were isolated from the surface of Red Globe table grapes. These isolates, along with the pre-isolated strain Metschnikowia pulcherrima RCM2, were evaluated as BCAs in wounded berries. From these results, six yeast isolates were pre-selected to be combined with bioactive compounds of natural sources, like phenolic compounds (PCs) of side streams of wine industry, including bunch stem extract (BSE) (5–25 %), and cane extract (CE) (5–25 %), and functional polysaccharides from shrimp waste such as chitosan (CH) (0.5 %). Then, the biocontrol efficacy of combined treatments beyond individual ones was compared. The results revealed that 4 yeast isolates, namely M. pulcherrima RCM2 and ULA146, and Aureobasidium pullulans FUL14 and FUL18, were the most effective. However, when combined with the natural bioactive compounds, their efficacy against A. alternata did not increase significantly. Notably, ULA146 and FUL18 demonstrated a biocontrol efficacy of 36–37 %, comparable to that of the treatment with commercial doses of SO 2 , which only showed a 27 % reduction in the lesion diameter. These findings highlight the potential of using psychrotrophic yeasts as BCAs against A. alternata in cold-stored table grapes. Combining these yeast strains with BSE, CE and CH did not increase BCAs efficacy against this pathogen at the concentrations tested. The development of effective biocontrol strategies for A. alternata could contribute to reducing reliance on chemically synthesized fungicides, promoting sustainable practices, aiming to improve the quality and safety of cold-stored table grapes. • Grape surface hosts biocontrol-capable psychrotrophic yeasts. • Effective yeasts were Metschnikowia pulcherrima and Aureobasidium pullulans. • Grapevine extracts slightly increased the yeast efficacy against A. alternata. • Joint application with chitosan affected yeasts performance. • Psychrotrophic yeasts are a promising alternative for A. alternata biocontrol. [ABSTRACT FROM AUTHOR]

Abstract 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. Highlights • Withered grapes harbored several epiphytic yeast-like fungi and yeasts. • Cell concentration significantly varied among berries, both sound and damaged. • High phenotypic diversity among Aureobasidium pullulans strains was found. • 35 yeast species were recognized and ascomycetes prevailed over basidiomycetes. • The sanitary state of withered grapes could affect the population structure. [ABSTRACT FROM AUTHOR]

Abstract Pectinolytic yeasts can be applied to winemaking with the purpose of improving sensory and technological properties of wine because of their enzymes secreted during vinification. In this work, the autochthonous yeast-like pectinolytic strain from D.O. San Rafael viticulture region, Aureobasidium pullulans GM-R-22, was used in co-culture with Saccharomyces cerevisiae IOC 18-2007 in microvinification trials with Malbec must applying pre-fermentative cold maceration (PCM). A. pullulans remained viable during PCM and S. cerevisiae growth and fermentative kinetics were not affected in mixed culture with respect to pure S. cerevisiae culture. High pectinolytic activity (9.13 U/mg) was detected in mixed A. pullulans vinification during PCM, in which conditions of low temperature (8 °C), low pH (3.8) and high sugar concentration (250.6 g/L) governed. Mixed A. pullulans wine showed enhanced colour compared with pure S. cerevisiae wine, characterised by higher colour index and percentage of red colour, lower tonality and percentage of yellow colour, and negative values of b * and h * indicating more bluish and purplish tonalities. Moreover, filtration time and turbidity diminished by a 40% in mixed A. pullulans wine. The presence of GM-R-22 strain improved the production of desirable volatile compounds, such as esters and norisoprenoids, which displayed the maximum odour activity values (OAVs), whereas this strain reduced the total content of higher alcohols when compared to pure S. cerevisiae fermentation. Sensory analysis indicated that A. pullulans impacted on wine highlighting the violet hue, plum jam aroma, body and equilibrium that are distinctive features of Malbec variety. A. pullulans GM-R-22 seems to be promising for applying to low-temperature red winemaking as an adjunct culture to S. cerevisiae to improve the wine quality and vinification process. Highlights • A. pullulans GM-R-22 did not modify the S. cerevisiae growth or fermentative kinetics. • Mixed fermentation showed high pectinase activity at 8 °C, pH 3.8, 250.6 g/L sugar. • Mixed A. pullulans wine showed enhanced colour, clarification and aroma properties. • Sensory analysis showed the A. pullulans impact on wine emphasising Malbec features. [ABSTRACT FROM AUTHOR]

Two Aureobasidium pullulans strains (L1 and L8), able to prevent postharvest fruit decay, were evaluated in order to elucidate how the competition for nutrients and space was involved in their activity against Monilinia laxa , the causal agent of peach brown rot. The competition for nutrients was studied by co-culturing pathogen conidia and antagonists in different conditions of nutrient availability and avoiding contact between them. Both antagonists prevented the germination of conidia of M . laxa in water, reducing germination rate by > 35%. However, L1 and L8 showed the lowest inhibition of conidial germination in peach juice at 5%, with a reduction of 12.6% and 13.9% respectively. HPLC amino acid analysis of peach juice revealed that the addition of the yeast suspension greatly modified their composition: asparagine was completely depleted soon after 12 h of incubation and was probably hydrolyzed to aspartic acid by the yeasts, as aspartic acid content markedly increased. Pure asparagine and aspartic acid were tested by in vitro trials at the concentrations found in peach juice: both influenced M . laxa growth, but in opposite ways. Asparagine stimulated pathogen growth; conversely, amended medium with aspartic acid significantly inhibited the conidia germination and mycelial development of M . laxa . Scanning Electron Microscopy revealed that both strains showed a great capability to compete with M . laxa for space (starting 8 h after treatment), colonizing the wound surface and inhibiting pathogen growth. This study clearly showed that A . pullulans L1 and L8 strains could compete with M . laxa for nutrients and space; this mode of action may play an important role in the antagonistic activity, especially in the first hours of tritrophic host-pathogen-antagonist interaction, although several other mechanisms can interact each other. [ABSTRACT FROM AUTHOR]

Modern agriculture relies on an integrated approach, where chemical treatment is reduced to a minimum and replaced by biological control that involves the use of active microorganisms. The effect of the antagonistic yeast-like fungus Aureobasidium pullulans on proteins and bioactive compounds (alkylresorcinols, sterols, tocols and carotenoids) in winter wheat grain and on the colonization of wheat kernels by fungal microbiota, mainly Fusarium spp. pathogens, was investigated. Biological treatment contributed to a slight increase contents of tocols, alkylresorcinols and sterols in grain. At the same time, the variation of wheat grain proteins was low and not significant. Application of A. pullulans enhanced the natural yeast colonization after six months of grain storage and inhibited growth of F. culmorum pathogens penetrating wheat kernel. This study demonstrated that an integrated approach of wheat grain protection with the use of the yeast-like fungus A. pullulans reduced kernel colonization by Fusarium spp. pathogens and increased the content of nutritionally beneficial phytochemicals in wheat grain without a loss of gluten proteins responsible for baking value. [ABSTRACT FROM AUTHOR]

Abstract: The yeast-like fungus Aureobasidium pullulans PL5 is a microbial antagonist against postharvest pathogens of fruits. The strain is able to produce hydrolases, including glucanases, chitinases and proteases. The alkaline serine protease gene ALP5 from A. pullulans was cloned, inserted into the vector pPIC9 to construct pPIC9/ALP5, and then expressed in Pichia pastoris strain KM71. ALP5 had a molecular mass of 42.9kDa after 5days growth with 1% methanol induction at 28°C. The recombinant protease expressed in P. pastoris showed its highest activity under alkaline conditions (at pH10) and a temperature of 50°C. The antifungal activity of the recombinant protease was investigated against Penicillium expansum, Botrytis cinerea, Monilinia fructicola and Alternaria alternata in vitro and on apple. The recombinant protease reduced significantly the spore germination and the germ tube length of the tested pathogens in PDB medium. The highest level of protease efficacy was observed against M. fructicola and B. cinerea, whereas a lower efficacy was observed against P. expansum and A. alternata indicating a possible effect of the pathogen cell wall composition on the proteolytic activity of the recombinant protease. The presence of protease was able to cause the swelling of the hyphae of B. cinerea, under an optical microscope. The recombinant protease expressed in P. pastoris was more active against the pathogens in vitro than the same enzyme expressed in E. coli in previous studies. The efficacy of ALP5 was also evaluated against the pathogens in vivo on cv Golden Delicious apples. The protease was more efficient in controlling M. fructicola, B. cinerea and P. expansum than A. alternata. However, the extent of the activity was dependent on the enzyme concentration and the length of fruit storage. This study demonstrated the capacity of the alkaline serine protease to keep its enzymatic activity for some days in the unfavorable environment of the fruit wounds. The alkaline serine protease could be developed as a postharvest treatment with antimicrobial activity for fruit undergoing a short storage period. [Copyright &y& Elsevier]

Abstract: The influence of temperature and relative humidity (RH) on the activity of three biocontrol agents—the yeast Metschnikowia pulcherrima LS16 and two strains of the yeast-like fungus Aureobasidium pullulans LS30 and AU34-2—against infection by A. carbonarius and ochratoxin A (OTA) accumulation in wine grape berries was investigated in lab-scale experiments. The presence of wounds on grape skin dramatically favored infection of berries by A. carbonarius strain A1102, since unwounded berries showed very low levels of infection at all conditions of RH and temperature tested. Artificially wounded berries pre-treated with the biocontrol agents were inoculated with the ochratoxigenic A. carbonarius strain A1102 and were incubated for 5days at two levels of RH (60% and 100%) and three different temperatures (20, 25 and 30°C). The three biocontrol agents were able to prevent infections at 60% RH and 20°C. At 60% RH and 25°C only strain AU34-2 achieved some protection on day 5, whereas at 30°C a limited biocontrol efficacy was evident only up to day 2. At 100% RH, LS16, LS30 and AU34-2 showed effective protection of grape berries at 20°C until the 5th day of incubation. The three biocontrol agents achieved significant protection at higher temperatures only until the 2nd day after the beginning of the experiment: all three strains at 25°C, and only strain LS16 at 30°C. After 5days, the three biocontrol agents were able to significantly reduce the level of OTA in berries at all the conditions tested. This occurred even when protection from infection was not significant, except at 30°C and 100% of RH for all the three strains, and at 25°C and 100% of RH for strain LS16. The biocontrol agents displayed a higher rate of colonization on grape berries at 20 and 25°C than at 30°C. The higher value of RH (100%) appeared to increase the rate of colonization, in particular at 20 and 25°C. Taken together, our results emphasize the significant influence of environmental factors on the effectiveness of biocontrol against A. carbonarius as well as on OTA contamination in wine grape berries, and the need for biocontrol agents that can cope with the environmental conditions that are conducive to attack by A. carbonarius. [Copyright &y& Elsevier]

Abstract: An alkaline protease gene was amplified from genomic DNA and cDNA of the antagonistic yeast-like fungus Aureobasidium pullulans PL5, a biocontrol agent effective against Monilinia laxa on stone fruit and Botrytis cinerea and Penicillium expansum on pome fruits. An open reading frame of 1248bp encoding a 415-amino acid (aa) protein with a calculated molecular weight (M r) of 42.9kDa and an isoelectric point (pI) of 4.5 was characterized. The cDNAALP5 gene had an 18-amino acid signal peptide, one N-gylcosylation, one histidine active site, and one serine active site. The ALP5 gene with a M r of 1351bp contained two introns. One intron was of 54bp, while the other was of 50bp. Protein BLAST and phylogenetic tree analysis of the deduced amino sequences from the cDNAALP5 gene showed that the encoded protein had 100% homology to a protease enzyme (ALP2) of a sea strain of A. pullulans, suggesting that the protein ALP5 was an alkaline serine protease. Expression of ALP5 in Escherichia coli BL21 (DE3), followed by identification with Western-blotting, purification with Ni-NTA and analysis of enzymatic activity, yielded an homogeneous recombinant ALP5 which hydrolysed the substrate casein and inhibited the mycelial growth of the pathogens. At its optimal pH of 10.0 and reaction temperature of 50°C, the recombinant protease exhibited the highest activity towards the substrate casein, though the highest stability was at lower temperatures and pH between 7.0 and 9.0. This study provided the direct evidence that extracellular proteases secreted by the antagonist A. pullulans PL5 played a role in the biocontrol activities against some postharvest pathogens of apple and peach. [Copyright &y& Elsevier]

Abstract: The present study was undertaken with the purpose of selecting yeasts from wine grapes that are able to produce extracellular cold-active pectinases. After two consecutive selections yeast isolates were identified by pheno- and genotyping, and pectinolytic activity was preliminarily characterised at proximate winemaking conditions. Out of 1023 indigenous microorganisms isolated from grape skins of D.O. San Rafael (Mendoza, Argentina) viticulture region, 565 (55%) showed pectinolytic activity on plates and, among them, 96 (17%) were chosen in a primary selection. Ten isolates were finally selected for exhibiting the greatest activity at low temperature (12°C) and identified as Aureobasidium pullulans. GM-R-22 strain demonstrated the highest pectinolytic activity (0.751U/mL) at pH 3.5 and 12°C. Yeast pectinases were constitutively produced. This study is the first report about strains of A. pullulans producing pectinases which are able to show good activity at low temperature. These pectinolytic strains could be of interest in wine production. [Copyright &y& Elsevier]

A wide range of fresh fruits and vegetables is attacked by Penicillium species causing diseases during their postharvest handling. Many of these species are psychrotrophic and they are able to cause food spoilage at refrigeration temperature as happens with table grapes. After the harvest, grape bunches are stored inside boxes with SO 2 generator pads to reduce the contamination with fungal conidia. However, SO 2 residues are dangerous to people allergic to sulfites and they negatively affect the quality of fresh fruit. Biological control of phytopathogens with microbial antagonists naturally present on fruit surfaces could be helpful against postharvest diseases. The present study aimed to select native yeasts isolated from fermentation microenvironments and the surface of refrigerated grapes for their use in the biological control of P. expansum on table grapes stored in cold rooms. Non-pathogenic and pathogenic Penicillium species were isolated, and the four most aggressive pathogen isolates were identified as Penicillium expansum. Twenty yeast isolates identified as Aureobasidium pullulans , Cryptococcus magnus , Metschnikowia pulcherrima and Rhodotorula glutinis presented positive antagonistic activity against Penicillium expansum ; they controlled the development of at least one of the fungi, significantly reducing the disease incidence. The results showed that three antagonistic yeasts (M. pulcherrima 22, 36 and 43) reduced the disease incidence and severity of all 4 P. expansum isolates. It was also found that the fruit surface is not the only source for isolation of biological control agents. Microenvironments with different stress conditions could be a promising source to isolate antagonistic microorganisms. • Pathogenic and non-pathogenic Penicillium were found in table grapes in cold rooms. • The most aggressive pathogenic isolates were identified as Penicillium expansum. • Yeasts isolated from different sites were effective to control P. expansum. • Non-Saccharomyces yeasts reduced the pathogenicity of P. expansum isolates assayed. [ABSTRACT FROM AUTHOR]