CYTOGENETIC CHARACTERIZATION AND IDENTIFICATION OF THE YEAST STRAINS ISOLATED FROM DOC DEALU MARE, VALEA CALUGAREASCA AREA

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1 CYTOGENETIC CHARACTERIZATION AND IDENTIFICATION OF THE YEAST STRAINS ISOLATED FROM DOC DEALU MARE, VALEA CALUGAREASCA AREA Elena Brinduse, Aurelia Tudorache, Laura Fotescu Research and Development Institute for Viticulture and Enology, Valea Calugareasca -iunie 2013-

2 INTRODUCTION The composition and the quality of wines are closely related to the yeast used for fermentation. In recent years, there has been growing the interest in using new autochthonous or local selected yeasts strains that are potentially better adapted to their local environments and can enhance the typical sensory properties of the wines produced in a given region. These yeasts would allow the production of wines with regional specificity, conferring traits that would distinguish these wines from the wines of other regions. In order to keep its place on the wine international market Romania should produce typical wines from local grape varieties by using like starter cultures autochtonous wine yeasts.

3 OBJECTIVES The isolation and purification of the yeast strains from Dealu Mare vineyard, Valea Calugareasca viticultural center; The characterization from cytogenetics and morpho-physiolocical point of view of the isolated autochtonous yeast strains; The taxonomic identification of the yeast strains; The built a collection of microorganisms proving high enological potential formed by the Saccharomyces and non-saccharomyces strains.

4 MATERIAL AND METHODS The origin of the yeasts has been Dealu Mare (Grand Hill in Romanian language) vineyard, Valea Calugareasca viticultural center. As biologic material have been used grape varieties for red wines recommended in Valea Calugareasca centre, respectively Cabernet Sauvignon (ecological and classical plantation), Merlot, Feteasca Neagra and Pinot Noir. The sampling has been performed during 3 harvest years, respectively 2009, 2010 and The samples have been prepared in two ways: 1) The berries have been harvested in aseptic conditions and introduced in an Erlenmeyer with sterile distillated water in order to collect the yeast from the grape surfaces. After 24 hours of shaking, from the suspension, plates with YEPD have been inoculated and cultivated during hours at 27 o C. 2) The berries have been crushed in aseptic conditions and the natural fermentation has occurred. During the fermentation, three moments for sampling have been chosen: beginning, middle and final phase of the fermentation.

5 MATERIAL AND METHODS Isolation of Yeast Strains 1 ml. probe sterile pipette 1 ml 1 ml 1 ml 1 ml 1 ml 9 ml 9 ml 9 ml 9 ml 9 ml 9 ml n x 0.1 ml x 0.1 ml 3 x 0.1 ml N = m x c x 10 where: N = the number of living microorganisms per gram of soil m = the average number of the colonies developed on the three Petri dishes c = inverse of the dilution used for inoculation 10 = the coefficient for reporting the results to 1 gram of sample.... Serial dilution (10-1 to 10-7 ) of the samples were prepared using peptone water as diluent and 0.1ml of each sample was inoculated on yeast extract peptone dextrose agar medium using spread plate method. Cultivation conditions: 27 C during hours SERIAL DILUTION TECHNIQUE

6 MATERIAL AND METHODS morphological and cultural characterization of yeast strains shape and cell dimension of the cell - on liquid media after 3 days cultivation on Wickerham media at 25 o C and on solid media YEPD; the pseudomycelia formation on potato-agar media - after 12 cultivation days; the sporulation on Gorodkova media-after 30 cultivation days; cultural characteristics on the synthetic media in test tubes.

7 MATERIAL AND METHODS Yeast physiological characterization RAPID BIOCHEMICAL TEST: testing the assimilation of 19 substrates and the analyzing of the data by the APILAB PLUS software. SUPPLEMENTARY CLASSICAL FERMENTATION AND ASSIMILATION TESTS for sugars and nitrates.

8 PHYSIOLOGICAL IDENTIFICATION KEYS SACCHAROMYCES SP. Physiological characteristic Inability to metabolize xylose Inability to metabolize trehalose Inability to grow using potassium nitrate, sodium nitrite and lysine Inability to use ethanol as the single carbon source Inability to arbutin split Genetic mechanism lack of xylose reductase and xylitol dehydrogenase genes lack of gene NTH1 and ATH1 lack of genes for nitrate reductase enzyme, LYC 1 gene for lysine N-6 acetyl transferase absence of gene for lactose fermentation lack of gene for β - glucosidases

9 PHYSIOLOGICAL IDENTIFICATION KEYS Non - SACCHAROMYCES SP. Carbon source Xylose Genetic mechanism Expression of xylose reductase and xylitol dehydrogenase genes Identification Candida spp.; Pichia spp.; Rodotorula mucilaginosa Starch Sta2 gene Candida spp., Pichia spp Methanol Hemicellulose Two alcohol oxidase genes genes, AOX1 and AOX2 Expression of xylose reductase, xylitol dehydrogenase, and xylulokinase genes Hansenula; Pichia, Candida Candida spp., Pichia spp

10 RESULTS DALMAU diagram Strain Shape Size (μ) Cells Groups Budding type Liquid media Culture Solid media (colonies) Pseudmycelia Sporulation MI132 elliptical, oval 3,5 x 9,5 isolated, group of 2 polar no surface formation, fine deposit white-cream whole margins spherical MI133 elliptical, oval 4,5 x 8,5 isolated, group of 2 polar no surface formation, fine deposit white-cream whole margins - 2 oval MI326 MI6212 round oval oval 5,5 x 7,9 5,3 x 8,2 short chain, groups short chain polar polar no surface formation, sandy deposit Surface ring, fine deposit yellow-brown, striated on the surface grey-cream, shiny plane, flat, lobate margins oval 1-4 elliptical MI8216 elliptical, oval 4,2 x 9,8 isolated, group of 2 polar No surface formation, granular deposit brown-cream central wrinkled; whole margins oval

11 RESULTS Identification of strains Colony shape and colour White to creamy Creamy White to creamy White to creamy Creamy Pinkish red Pinkish Pinkish Creamy and spherical Colony surface appearance Smooth and flat Smooth and flat Smooth and flat Soft and smooth Smooth and flat Smooth and flat Smooth and flat Smooth and shiny Smooth, shiny; flat or raised Vegetative morphology Cell shape Arrangement Spherical cells Ellipsoidal cells Ellipsoidal cells Globose to oval cells Spherical cells Spherical cells Ellipsoidal cells Spherical cells Spherical or Elongated cells Singly budding Multipolar budding Candida colliculosa Candida utilis Singly budding Multipolar budding Multipolar budding Multipolar budding Singly budding Multipolar budding Ascospore formation Pellicle formation Pseudomycelium /true mycelium Singly budding Identity Candida pelliculosa Candida magnolia Cryptococcus albidus Rhodotorula mucilaginosa Rhodotorula mucilaginosa Rhodotorula glutinis Saccharomyces cerevisiae Creamy and spherical Smooth or moisten; flat or raised Spherical, elongated or oval cells Multipolar budding Saccharomyces cerevisiae

12 Aspects concerning the morphological and cultural characteristics of yeast strains

13 RESULTS - Carbon Assimilation Test using API 20 C AUX kit (BioMEIREUX) G G 2 A X A X G I S M N C L M S T M R L L K R Y D L A N O D A E A A A R L A U Y G A L O T L O R G G L C L C E Z F IDENTIFICATION / /- +/- +/- Saccharomyces cerevisiae INC / /- +/- - - INC + INC +/- +/ Rhodotorula mucilaginosa /- +/- INC /- +/- +/ /- INC + + +/ / /- Rhodotorula glutinis Candida utilis + +/ / /- Candida pelliculosa Candida magnolia Candida colliculosa + - +/ INC - INC /- + +/ / /- +/- +/- Saccharomyces cerevisiae Rhodotorula mucilaginosa INC / /- + +/- Cryptococcus albidus / /- +/- +/- Saccharomyces cerevisiae + +/ / INC INC + INC + +/ Candida magnolia / /- +/ /- +/- Saccharomyces cerevisiae /- +/ /- +/- +/ /- + + Rhodotorula mucilaginosa INC Legend: assimilate carbon source (+); do not assimilate carbon source (-); variable response (+/-); inconclusive (INC)

14 RESULTS Taxonomic identification test Assimilation capacity test results Yeast strain Glucose Sucrose Maltose Galactose Raffinose Mannitol Lactose Cellobiose Xylose Trehalose IDENTIFICATION MS / Candida famata MS2 + +/- +/- +/ /- +/- Candida colliculosa MS / / Cryptococcus albidus MS /- +/- + +/- - +/- + + Rhodotorula mucilaginosa MS /- + +/ /- Saccharomyces cerevisiae MS Candida guilliermondii MS / Candida lusitaniae MS Candida norvegensis Fermentative capacity test results MS1 +/- +/- +/ /- Candida famata MS2 + +/- +/- +/- - +/- Candida colliculosa MS Cryptococcus albidus MS Rhodotorula mucilaginosa MS5 + +/- +/- +/- - +/- Saccharomyces cerevisiae MS /- - - Candida guilliermondii MS /- +/- - + Candida lusitaniae MS Candida norvegensis Legend: assimilate/ferment carbon source (+); do not assimilate/ferment carbon source (-); variable response (+/-)

15 RESULTS Taxonomy of yeast trains (Barnett et all., 2000) Phylogenetic group ASCOMYCOTA Class HEMIASCOMYCETES Order SACCHAROMYCETALES Family CANDIDACEAE Family METSCHNIKOWIACEAE Family SACCHAROMYCETACEAE Phylogenetic group BASIDIOMYCOTA Class HYMENOMYCETES Order TREMELLALES Family SACCHAROMYCODACEAE Class UREDINIOMYCETES Order SPORIDIALES Family TREMELACEAE Family SPORIDIOBOLACEAE

16 RESULTS Taxonomy of yeast strains at the genus and species level CANDIDA colliculosa CLAVISPORA lusitaniae famata lusitaniae HANSENIASPORA uvarum magnoliae pelliculosa KODAMAEA ohmeri sphaerica stellata PICHIA anomala utilis jadinii GEOTRICHUM capitatum DEBARYOMYCES hansenii penicillatum KLOECKERA apiculata CRYPTOCOCCUS albidus neoformans RHODOTORULA glutinis minuta mucilaginosa SACCHAROMYCES TORULASPORA cerevisiae delbruecki

17 RESULTS Taxonomy of yeast strains isolated from grapes surface C. famata C. lusitaniae 20,0% 2,5% 7,5% 12,5% 2,5% 2,5% 20,0% 5,0% 5,0% 5,0% 2,5% C. magnoliae Candida utilis G. capitatum Kl. apiculata D. hansenii S. cerevisiae H. uvarum 7,5% 10,0% 15,0% 2,5% Cryptococcus albidus Cryptococcus neoformans Rhodotorula glutinis Rhodotorula minuta Rhodotorula mucilaginosa Without fermentative power With fermentative power Non-sporogene Sporogene Genus No. % Genus No. % No. % No. % Cryptococcus 3 7,5 Candida Geotrichum 1 2,5 Rhodotorula 5 12,5 Kloeckera 8 20 Debaryomyces 5 12,5 Saccharomyces 1 2,5 Hanseniaspora 1 2,5 TOTAL

18 RESULTS Taxonomy of yeast strains isolated from must GENUS Beginning of AF* Middle phase of AF End phase of AF No. % No. % No. % Candida 19 47, ,5 Geotrichum 1 0,9 Kloeckera 2 5 Clavispora 2 3 Kodamaea 1 0,9 Pichia 3 4,5 Debaryomyces 9 22,5 5 4,3 Saccharomyces 3 7, , Torulaspora 5,2 5 7,5 Rhodotorula 7 17, ,1 2 3 * AF Alcoholic Fermentation

19 RESULTS Germplasm collection GENUS SPECIES NO. OF STRAINS CANDIDA colliculosa 5 famata 33 lusitaniae 4 magnoliae 2 sphaerica 2 stellata 1 utilis 10 GEOTRICHUM capitatum 2 penicillatum 1 KLOECKERA apiculata 2 CRYPTOCOCCUS albidus 1 neoformans 2 RHODOTORULA glutinis 5 minuta 7 mucilaginosa 11 CLAVISPORA lusitaniae 2 HANSENIASPORA uvarum 1 KODAMAEA ohmeri 1 PICHIA anomala 1 jadinii 2 DEBARYOMYCES hansenii 14 SACCHAROMYCES cerevisiae 105 TORULASPORA delbruecki 11 TOTAL 225

20 RESULTS Strain data sheet CANDIDA LUSITANIAE - strain 27 ICDVV Valea Calugareasca Name of institution: Registration number: 0,001 Data of entry in the collection: 2010 Origin: PN B-1 Maintenance medium: Conservation form: Morphological and ultrasrtuctural diversity Culture characteristics on liquid medium disposed in inclined tube YEPD in paraffin oil Colony white to pink, moderate groth, deposit presents a fine appearance, with complete disintegration gas bubblets formation at the medium surface Moderate growth of "H" form Sowing route thin, wet-looking, diffuse, opaque culture disposed in Petri dishes Cell shape: Grouping mode: Cell size (µ) Pseudomiceliu, spors: Physiological characteristics fermentation test assimilation test Colonies large, round, with entire edge, convex profile, smooth type "S", opaque, glossy surface and viscous ellipsoidal or spherical cell singly budding (8,4-4,8)x(7,2-3,6) pseudohiphae, no sporulation Pozitive: Glucose; Sucrose; Trehalose. Variabile: Galactose; Maltose. Negative: Lactose. Pozitive: Glucose; Maltose; Sucrose; Trehalose; D-Xylose; Glycerol; Cellobiose; L-Rhamnose; D-Ribose; D-Mannitol; Ribitol; D-Glucitol; Salicin; DL-Lactic acid; Succinic acid. Variabile: Galactose; Melezitose; D-Arabinose; L-Arabinose; L- Sorbose; Citric acid. Negative: Potassium nitrate; Lactose; Raffinose; Melibiose; Galactitol; Erythritol; Inositol; Soluble Starch. Oenological importance Date of subcultivation February, 2013 Extracellular enzymatic activity

21 CONCLUSION During there have been isolated, purified and identified 252 yeast strains from Dealu Mare vineyard- Valea Calugareasca centre. These strains belong both to Ascomycota and Basidiomycota and we have identified 24 species belonging to 12 genera, from which 7 are sporogene. The prevalence rate of different yeast strains isolated in the research shows that yeast varies in their morphological and physiological characteristics. A collection of microorganisms with 225 yeast strains, proving high enological potential formed by the Saccharomyces and non-saccharomyces strains was built.