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Botrytis cinerea Pers., the fungal plant pathogen and the causal agent of gray mould diseases in grapevine, is vastly responsible for substantial economic losses in table and wine grapes production worldwide by negatively affecting plant growth and causing the reduction of grape and wine quality. The conventional approach for pathogen control has been up to date based on synthetic fungicides with good effectiveness against pathogens but a negative impact on the environment. The growing level of harmful residues in the environment and some also detected in wines have led the European Union and many winemakers to limit the application of synthetic fungicides to earlier season. However, with a high risk of disease also late in the season, the need for other solutions is clear. Consequently, more and more research is focused on finding potential alternatives in the form of effective biological control agents. Although there are several reports of yeast’s biocontrol activity, they are up to date still poorly commercialized for such purposes. As the yeasts represent an important part of the grape microflora, competing with other microorganisms (including pathogens) for nutrients and space, we decided to examine the potential of autochthonous yeasts as "green" alternatives in fighting against phytopathogens such as B. cinerea in viticulture. With this aim, we tested the biocontrol activity of 119 different indigenous yeasts belonging to 30 different species of 17 genera against filamentous fungus B. cinerea, the causal agent of grey mould or botrytis bunch rot in grape. The yeasts were screened for putative multidimensional modes of action such as antifungal volatiles (VOC), in vitro inhibition of fungal mycelial growth, competition for nutrients, hydrolytic enzyme activities, and yeast tolerance to fungicides like copper, iprodione and cyprodinil/fludioxonil combination. With a qualitative detection of the hydrolysis activity by using screening methods based on solid medium with chitin or βD-glucosides as substrates, we found that many tested yeasts were capable of producing lytic enzymes with the ability to degrade the cell wall of phytopathogenic fungi and are potentially also able to produce VOCs via hydrolysis of grape glycosides as a result of β-glucosidase presence. Furthermore, we observed the capability of tested yeast to inhibit fungal mycelia growth on plate and assimilation of a wide variety of carbon sources however, no siderophore producers were detected. In general, the yeasts under observation were tolerant to the tested fungicides. Their fungicide resistance can indeed be regarded as a beneficial trait for potential biofungicide agent (PBA) candidates due to open possibilities of applications and combinations within low input pest management strategies in the vineyard. Finally, a field experiment in Pinot noir and Pinot gris vineyards was designed to study different combinations of optimized canopy microclimate manipulation (CMM) techniques and potential biocontrol agent (PBA) application. In experimental conditions, the ability of PBA’s to maintain appropriate population density for disease prevention was observed. In addition, the grape and wine quality parameters were analysed to observe the possible impact of implemented biocontrol yeast on final products. The biocontrol yeast Pichia guilliermondii ZIM 624 was selected and applied in experimental vineyards based on yeast testing results. We were able to detect and confirm PBA yeast’s suitable density on grapes until harvest. In the case of early defoliation for both varieties lower grape compactness was observed together with lower yield/ plant, regardless of PBA yeast/ no yeast application. Among grape basic quality parameters, the optimized techniques showed a positive effect on sugar content. Still, unexpectedly, in the treatments with biocontrol yeasts some trends toward higher acidity were noticed in Pinot gris.

Opravili smo dve hitri molekularni tehniki za identifikacijo enoloških kvasovk, ki so bile izolirane iz vinogradov z ekološko in intergrirano pridelavo grozdja. Najprej smo določili 15 Saccharomyces kvasovk s pomočjo analize multipleks PCR med zbranimi 131 izolati, nato smo s pomočjo ribotipizacije ITS določili približno 98 kvasovk do nivoja vrste, ki so bile potrjene tudi s sekvenciranjem regije ITS. Določili smo 16 vrst vinskih kvasovk: Hanseniaspora uvarum, H. valbyensis, H. opuntiae / H. uvarum, Lachancea thermotolerans, Candida tropicalis, Candida sp. / Starmerella sp., Metschnikowia pulcherrima, Pichia kudriavzevii, P. terricola, P. kluyveri, Wickerhamomyces anomalus, Kodamaea ohmeri, S. bacillaris, Saccharomyces cerevisiae, Saturnispora diversa in Torulaspora delbrueckii. Ugotovili smo, da so najdene kvasovke običajne naseljevalke grozdne mikroflore ter da je način pridelave grozdja vplival na pojavljanje določenih vrst v obravnavanih vinogradih. Nadalje smo testirali kvasovke na tvorbo sideroforov, tvorbo protiglivnih hlapnih organskih spojin (HOS), hidrolitično aktivnost in toleranco kvasovk na fungicide, kot so baker, iprodion, fluazinam ter kombinacijo ciprodinil / fludioksonil. Za kvalitativno detekcijo hidrolitične aktivnosti smo uporabili presejalne teste na osnovi trdnega gojišča z dodanim hitinom ali β-D-glukozidi kot substratom in ugotovili, da je veliko testiranih kvasovk sposobnih proizvajati litične encime, ki bi lahko bili odgovorni za razgradnjo celičnih sten fitopatogenih gliv in potencialno sposobni proizvajati HOS s hidrolizo grozdnih glikozidov zaradi prisotnosti β-glukozidaz. Prav tako smo opazili, da so te encimske aktivnosti običajno odvisne od seva. Na splošno so kvasovke tudi tolerantne na testirane fungicide. Ugotovili smo tudi, da je odpornost biofungicidnih kvasovk na fungicide koristna lastnost za razvoj biofungicidov v prihodnosti pri aplikacijah v strategijah z zmanjšanim vnosom pesticidov. We performed two rapid molecular techniques for the identification of enological yeasts that were collected from vineyards with organic and integreated pest management. First we distinguised 15 Saccharomyces yeasts with multiplex PCR analysis from collected 131 isolates, further on we identified approx. 98 isolates to species level based on ITS ribotyping that were confirmed also with ITS sequencing. Overall we distinguished 16 wine yeast species: Hanseniaspora uvarum, H. valbyensis, H. opuntiae / H. uvarum, Lachancea thermotolerans, Candida tropicalis, Candida sp. / Starmerella sp., Metschnikowia pulcherrima, Pichia kudriavzevii, P. terricola, P. kluyveri, Wickerhamomyces anomalus, Kodamaea ohmeri, S. bacillaris, Saccharomyces cerevisiae, Saturnispora diversa and Torulaspora delbrueckii. We observed that obtained yeasts are common inhabitants of grape microflora, and that applied pest managament in studied vineyards had an influence on occurence of certain species. Thereafter, we tested yeasts for siderophore production, antifungal VOCs production, hydrolysis activity and yeast tolerance to fungicides like copper, iprodione, fluazinam and cyprodinil / fludioxonil combination. With a qualitative detection of the hydrolysis activity by using screening methods based on soild medium with chitin or β-D-glucosides as substrates, we recognized that many tested yeasts were able to produce lytic enzymes that could be responsible for cell wall degradation of phytopathogenic fungi, and potentially able to produce VOCs from the hydrolysis of grape glycosides due to the β-glucosidase presence. We also observed that these enzymatic activities are usually strain-dependent. In general yeasts were also tolerant to tested fungicides, and we concluded that fungicide resistance of biofungicide yeasts is beneficial trait for future biofungicide development for possible applications in low input pest management strategies.

Kemijske in senzorične lastnosti vina so poleg genetskih danosti ter geo-klimatskih okoliščin odvisne tudi od strategije tehnologije pridelave, ki jo izbere vinar. V to strategijo spada tudi izbira seva kvasovke. Vinski trg postaja vse bolj zahteven in željan nečesa novega, zato vse več vinarjev poskuša pridobiti drugačne lastnosti vin z uporabo drugačnih sevov kvasovk, oziroma z uporabo kombinacij različnih vrst kvasovk. V diplomski nalogi smo preučevali vpliv različnih kombinacij kvasovk na potek fermentacije ter na osnovne kemijske in senzorične lastnosti pridelanega jabolčnega vina. Poskus je bil izveden na laboratorijski skali. Rezultati poskusa so pokazali, da različne vrste kvasovk različno vplivajo na senzorične lastnosti vina, nekoliko manj pa na kemijske lastnosti. Rezultati nakazujejo, da različne kvasovke različno hitro porabljajo sladkorje, kar vpliva tudi na hitrost poteka fermentacije. Ob opazovanju osnovnih kemijskih lastnostih smo opazili razlike med kvasovkami oz. kombinacijami kvasovk pri parametrih kot so hlapne kisline in reducirajoči sladkorji, medtem ko v vsebnosti alkohola in pH vrednostih končnih jabolčnih vin med obravnavanimi fermentacijami nismo opazili razlik. Senzorična analiza vonja in barve jabolčnih vin je potrdila vpliv kvasovk na barvo, intenzivnost, trajnost in vrsto vonja. In addition to genetic predisposition and geo-climatic growing conditions, the chemical and sensory properties of wine depend also on the production strategies chosen by the winemaker. These strategies include the decisions on yeast strains used for the fermentation. The wine market is becoming increasingly demanding and often desires something new, therefore more and more winemakers are trying to acquire different properties of wines by using different strains of yeasts or combinations of different types of yeasts. In the diploma thesis we studied the influence of various yeast combinations on the course of fermentation and the basic chemical and sensory properties of the produced apple wine. The work was done on laboratory scale. The results indicated different types of yeasts to have stronger influence on sensory properties than on the observed chemical characteristics of the wine. We have found that various yeasts use sugars in a different manner and therefore the rate of fermentation is different. Within chemical properties, the biggest differences were found in the content of volatile acidity and reducing sugars, whereas the differences in pH and alcohol level were only minor. Sensory analysis confirmed the yeasts influence theon color, intensity, persistence and type of flavour.

The aim of the study was to investigate and select indigenous yeasts Saccharomyces cerevisiae and non-conventional yeasts that were isolated during this and previous studies, and than evaluate them for wine production by performing micro-scale vinification experiment Namen študije je bil preučevanje in selekcija avtohtonih kvasovk Saccharomyces cerevisiae in ne-konvencionalnih kvasovk, ki so bile izolirane tekom te in prejšnje študije, ter jih s pomočjo izvedbe vinifikacijskega poskusa v manjšem obsegu ovrednotiti ali so primerne za proizvodnjo vina.

The aim of this study was to explore the diversity of fermentative yeast species in a non-vineyard and vineyard environment, and exploit their oenological potential. For this purpose we collected plant and soil material in the vineyard (located in Črniče in Vipava Valley) planted with Vitis vinifera L. and in the forest (forest in Vipava Old Castle), and by using a selective medium with high ethanol and sugar content we managed to obtain 35 isolates. By sequencing the D1/D2 domain of the 26S rDNA we identified nine different yeast species. The oenological potential of identified yeasts was further investigated by employing a series of tests, such as growth at low pH values, tolerance to ethanol and sulfur, and H2S production ability. Based on the results of these tests we selected four different yeast species for the microvinification of Vitis vinifera L. cv. ‘Pinot Noir’ grape juice. A commercial strain Saccharomyces cerevisiae Lalvin D47 was also included as a control in the fermentation experiment. Fermentation monitoring was done by measuring daily weight loss and microbiological analysis in the middle of fermentation. For the final wines, sugars were determined and sensorial evaluation performed. Among four tested yeasts, S. paradoxus IVV 32 and Torulaspora delbrueckii IVV 36 appeared to be the most promising for winemaking, but further studies should be carried out. Namen študije je bil preučiti pestrost fermentativnih kvasovk v ne-vinogradniškem in vinogradniškem okolju ter raziskati njihov enološki potencial. V ta namen smo vzorčili različen rastlinski in talni material v vinogradu (nahajajočem se v Črničah v Vipavski dolini) in gozdu (Stari grad nad Vipavo), iz katerega smo s pomočjo uporabe selektivnega gojišča z visoko vsebnostjo sladkorja in etanola uspeli izolirati 35 izolatov. S sekvenciranjem domene D1/D2 na 26S rDNA smo določili devet različnih vrst kvasovk. Z izvedbo serije testov, kot je rast pri nizkih vrednostih pH-ja, tolerance na etanol in žveplo ter sposobnosti tvorbe H2S, smo nadalje raziskali enološki potencial določenih kvasovk. Na podlagi rezultatov teh testov smo za izvedbo mikrovinifikacije grozdnega soka sorte Vitis vinifera L. cv. ‘Modri pinot’ izbrali štiri različne vrste kvasovk, kot kontrolo smo vključili tudi komercialni sev Lalvin D47 vrste Saccharomyces cerevisiae. S spremljanjem dnevne izgube teže in mikrobiološko analizo sredi fermentacije smo izvedli monitoring fermentacije. Končnim vinom pa smo določili vsebnost sladkorjev in izvedli senzorično oceno. Med štirimi testiranimi kvasovkami, sta se seva IVV 32 vrste S. paradoxus in IVV 36 vrste Torulaspora delbrueckii pokazala kot najbolj obetajoča za proizvodnjo vina, vendar pa so potrebne nadaljnje raziskave.

Human exploitation of yeast fermentation dates back to the Neolithic. S. cerevisiae has been the most important yeast used for numerous fermentations of biotechnological interest, including grape fermentation for wine production. Despite its abundant use, the molecular mechanisms controlling alcoholic fermentation are rather unclear and the choice of S. cerevisiae as an inoculum is often the consequence of a mere habit, rather than the result of rational analyses. In this work we focused on the role of different yeasts in the winemaking process. I was interested in understanding how the specific strain used for inoculum could influence the wine aroma formation. Furthermore, I wished to investigate the yeast genetics and ecology by characterizing their population and strain diversity in one of the wine regions of Slovenia. To evaluate the effect of the yeast species on the fermentation outcome, we performed successive fermentations with five different species in combination with the industrial strain S. cerevisiae Lalvin T73. The experiment showed that at least two more yeast species, Kazachstania gamospora and Zygosaccharomyces kombuchaensis have good potential to be applied in the winemaking. , The main conclusions of this study are the possibility of: i) expanding the palette of alternative starters to widen the aromatic components ii) co–fermenting using two different yeast species. Mixed yeast culture fermentations are present in the natural context, but this practice has been neglected in biotechnological processes. Positive results of wine fermentations with non – conventional yeast urged us to explore the diversity of Slovenian wine region natural isolates. The resulting yeast collection counts more than 1200 strains for which phenotype and genotype have been defined. Numerous isolates, including non – Saccharomyces species, showed promising oenological and biotechnological traits because of their capacity of rapid utilization of various carbon sources, growth at low pH and at presence of copper sulfite and potassium metabisulfite. Sampling also revealed sharp discrimination between the ecological niches of S. cerevisiae and S. paradoxus, which is a striking feature of European vineyards. While S. cerevisiae habitats are strongly associated with human activity, S. paradoxus was mainly isolated from the forest sources. Profound analysis of the collected data could give some explanations to the driving forces of S. cerevisiae domestication and S. paradoxus geographic isolation population structure.