THE BIOGEOGRAPHY OF FERMENTATIVE YEAST POPULATIONS FROM THE VINEYARDS OF THE AZORES ARCHIPELAGO J. Drumonde-Neves a b, M. T. Lima a, D. Schuller b (a) Research Center for Agricultural Technology (CITAA) Department of Agricultural Sciences / University of Azores Campus de Angra do Heroísmo, 9700-042 Angra do Heroísmo, Portugal (b) Centre of Molecular and Environmental Biology (CBMA) / Department of Biology / University of Minho Campus de Gualtar, 4710-057 Braga, Portugal dschuller@bio.uminho.pt
AIMS Evaluate the biogeography of the fermentative yeast flora from the vineyards of the Azores Archipelago; To build a Saccharomyces cerevisiae strain collection as a resource for: - Preservation of yeast genetic diversity; - Selection and improvement of wine strains; - Equitable sharing of genetic data; - Evolutionary studies of this species in isolated island environments.
INTRODUCTION Azores Archipelago Corvo Flores Graciosa Terceira Faial Pico S. Jorge São Miguel 600 km Santa Maria
INTRODUCTION Non-abandoned vineyards Abandoned vineyards Azores Archipelago Corvo Flores Graciosa Terceira - Biscoitos Faial Traditional varieties Verdelho Arinto S. Jorge Pico Terrantez Hybrid varieties (American varieties) São Miguel 600 km Santa Maria
MATERIAL AND METHODS SAMPLING LOCATIONS Santa Maria São Miguel Terceira 3 Baía do Tagarete 2 Baía de S. Lourenço S. Vicente 10 Biscoitos 14 4 Sul 1 Baía da Maia 5 P. Delgada 6 Caloura 7 Vila Franca 8 Ponta Garça 9 Faial da Terra 13 Pesqueiro 12 Salga 11 Porto Martins Graciosa Pico 15 Vitória 16 Santa Cruz da Graciosa 17 Luz 18 Lagoa Outeiro Lagidos Candelária 19 23 22 21 Canada das Almas 24 S. Mateus 25 Terra do Pão Appellations of origin No appellations of origin Abandonned vineyards 20 Manhenha Faial S. Jorge Flores Norte Pequeno 27 28 29 Capelinhos 26 Varadouro Praia do Norte 35 Urzelina Fajã do Ouvidor 34 Fajã da Ribeira 32 da Areia Fajã dos Vimes 33 Fajã de S. João 31 30 Ponta do Topo 36 Santa Cruz das Flores
MATERIAL AND METHODS Traditional varieties SAMPLING Hybrid varieties Terrantez Arinto Verdelho Non-abandonned vineyards Abandonned vineyards Santa Maria São Miguel Terceira Graciosa 15 18 21 22 53 Pico 11 Faial 21 São Jorge Flores 1 6 27 24 65 41
MATERIAL AND METHODS Weight loss (g/l) Microvinification Isolation Species identification 0 g/ L g/ L ITS-RFLP 0-20 -40-60 -80-90 0 1 6 11 16 21 26 Day SEQUENCING S. cerevisiae strains identification Interdelta Sequences
RESULTS Number of yeast isolates Fermentation initial stage Fermentation final stage Total 2009 2640 1470 4110 2010 2250 1170 3420 Total 4890 2640 7530
RESULTS Yeast species occurring in initial fermentative stages Candida railenensis Candida diversa Issatchenkia hanoiensis Candida azymoides Hanseniaspora opuntiae Zygoascus meyerae Metschnikowia pulcherrima Pichia cecembensis Candida azyma Saccharomycopsis vini Hanseniaspora vineae Pichia membranifaciens Wickerhamomyces anomalus Williopsis californica Meyerozyma guilliermondii Candida fukuamaensis Candida fructus Candida inconspicua Candida quercitrusa 3 Unidentified species Pichia fermentans Hanseniaspora uvarum Candida zemplinina Issatchenkia terricola
RESULTS Yeast species occurring in initial fermentative stages Candida railenensis Candida diversa Issatchenkia hanoiensis Candida azymoides Hanseniaspora opuntiae Zygoascus meyerae Metschnikowia pulcherrima Pichia cecembensis Candida azyma Saccharomycopsis vini Hanseniaspora vineae Pichia membranifaciens Wickerhamomyces anomalus Williopsis californica Meyerozyma guilliermondii Candida fukuamaensis Candida fructus Candida inconspicua Candida quercitrusa 3 Unidentified species Pichia fermentans Hanseniaspora uvarum Candida zemplinina Issatchenkia terricola
RESULTS Yeast species occurring in initial fermentative stages Candida railenensis Candida diversa Issatchenkia hanoiensis Candida azymoides Hanseniaspora opuntiae Zygoascus meyerae Metschnikowia pulcherrima Pichia cecembensis Candida azyma Saccharomycopsis vini Hanseniaspora vineae Pichia membranifaciens Wickerhamomyces anomalus Williopsis californica Meyerozyma guilliermondii Candida fukuamaensis Candida fructus Candida inconspicua Candida quercitrusa 3 Unidentified species Pichia fermentans Hanseniaspora uvarum Candida zemplinina Issatchenkia terricola
RESULTS Yeast species occurring in initial fermentative stages Candida railenensis Candida diversa Issatchenkia hanoiensis Candida azymoides Hanseniaspora opuntiae Zygoascus meyerae Metschnikowia pulcherrima Pichia cecembensis Candida azyma Saccharomycopsis vini Hanseniaspora vineae Pichia membranifaciens Wickerhamomyces anomalus Williopsis californica Meyerozyma guilliermondii Candida fukuamaensis Candida fructus Candida inconspicua Candida quercitrusa 3 Unidentified species Pichia fermentans Hanseniaspora uvarum Candida zemplinina Issatchenkia terricola
RESULTS S. Maria Yeast species occurring in initial fermentative stages 240 isolates 8 species S. Miguel 270 isolates 9 species Terceira 360 isolates 7 species Graciosa 420 isolates 9 species Pico 810 isolates 15 species S. Jorge 330 isolates 5 species Faial 180 isolates 5 species Flores 30 isolates 2 species
RESULTS Yeast species occurring in initial fermentative stages Corvo Flores Graciosa I. terricola Terceira Faial Pico S. Jorge M. pulcherrima São Miguel C. zemplinina Santa Maria
RESULTS Yeast species occurring in initial fermentative stages Corvo Flores 7,6 Graciosa 31.3% Faial 26.6% S. Jorge Pico 5,8 Terceira I. terricola 48.6% 0,8% São Miguel 0,4% Santa Maria
RESULTS Yeast species occurring in initial fermentative stages Corvo Flores 0,7 % Faial 8,5 % Graciosa 1,3 % Pico 8,9 % S. Jorge 1,9 % Terceira 43.1% São Miguel C. zemplinina 25.5% Santa Maria
RESULTS Yeast species occurring in initial fermentative stages Corvo Flores 8.5% Graciosa 0,% Terceira 0,% Faial 21.6% S. Jorge Pico M. pulcherrima 0,% 0,% São Miguel 0,% Santa Maria
RESULTS Yeast species occurring in initial fermentative stages Traditional varieties Hybrid varieties 47,25 % Hanseniaspora uvarum 53,41 % 19,21 % Metschnikowia pulcherrima 2,27% 14,60% Candida zemplinina 12,69 %
RESULTS Yeast species occurring in initial fermentative stages Non-abandoned vineyards Abandoned vineyards 45,22 % Hanseniaspora uvarum 65,64 % 11,57 % Metschnikowia pulcherrima 0,51% 0 % Candida azymoides 5,13%
RESULTS Number of yeast isolates Fermentation initial stage Fermentation final stage Total 2009 2640 1470 4110 2010 2250 1170 3420 Total 4890 2640 7530
g/l g/l g/l RESULTS Fermentations data Finished fermentations SAMPLES OF TRADITIONAL VARIETIES FROM NON-ABANDONED VINEYARDS 2009 2010 83,3 % 63,0 % SAMPLES OF HYBRID VARIETIES FROM NON-ABANDONED VINEYARDS 2009 2010 47,1 % 67,7 % SAMPLES OF HYBRID VARIETIES FROM ABANDONED VINEYARDS 2009 2010 33,3 % 47,1 % Samples from appellations of origin Samples from other winemaking regions
g/l g/l g/l RESULTS Fermentations data Fermentations finished by S. cerevisiae SAMPLES OF TRADITIONAL VARIETIES FROM NON-ABANDONED VINEYARDS 2009 2010 56,0 % 75,5 % SAMPLES OF HYBRID VARIETIES FROM NON-ABANDONED VINEYARDS 2009 2010 56,3 % 61,9 % SAMPLES OF HYBRID VARIETIES FROM ABANDONED VINEYARDS 2009 2010 75,0 % 50,0 % Samples from appellations of origin Fermentations completed by S. cerevisiae Samples from other winemaking regions
g/l g/l g/l RESULTS S. cerevisiae microflora composition SAMPLES OF TRADITIONAL VARIETIES FROM NON-ABANDONED VINEYARDS Total number of S. cerevisiae strains Number of S. cerevisiae strains per fermentation 2009 2010 80 41 4 3 1 7 1 2 2 23 19 2 5 7 3 1 1 5 6 8 4 1 5 2 2 2 3 2 SAMPLES OF HYBRID VARIETIES FROM NON-ABANDONED VINEYARDS 2009 2010 52 275 Strains 37 14 1 1 3 9 21 1 1 1 2 10 2 2 2 3 3 3 2 1 3 2 2 SAMPLES OF HYBRID VARIETIES FROM ABANDONED VINEYARDS 6 4 11 3 9 3 7 16 4 2 2009 2010 36 29 Samples from appellations of origin Fermentations completed by S. cerevisiae Samples from other winemaking regions
g/l g/l g/l RESULTS S. cerevisiae microflora composition per island SAMPLES OF TRADITIONAL VARIETIES FROM NON-ABANDONED VINEYARDS 4 3 1 7 1 2 2 23 19 2 5 7 3 1 1 5 6 8 4 1 5 2 2 2 3 2 SAMPLES OF HYBRID VARIETIES FROM NON-ABANDONED VINEYARDS 14 1 1 3 9 21 1 1 1 2 10 2 2 2 3 3 3 2 1 3 2 2 SAMPLES OF HYBRID VARIETIES FROM ABANDONED VINEYARDS St.ª Maria S. Miguel Terceira Graciosa Pico Faial S. Jorge Flores 6 4 11 3 9 3 7 16 4 2
g/l g/l g/l RESULTS S. cerevisiae microflora composition per island SAMPLES OF TRADITIONAL VARIETIES FROM NON-ABANDONED VINEYARDS 15 12 3 12 46 8 4 3 1 7 1 2 2 23 19 2 5 7 3 1 1 5 6 8 4 1 5 2 2 2 3 2 SAMPLES OF HYBRID VARIETIES FROM NON-ABANDONED VINEYARDS 16 2 3 12 9 4 22 11 14 1 1 3 9 21 1 1 1 2 10 2 2 2 3 3 3 2 1 3 1 4 SAMPLES OF HYBRID VARIETIES FROM ABANDONED VINEYARDS 16 7 1 4 0 2 1 4 2009 2010 St.ª Maria S. Miguel Terceira Graciosa Pico Faial S. Jorge Flores 6 7 4 16 23 0 3 4 0 2 6 7 4 11 3 9 3 16 4 2
CONCLUSIONS Within this study we showed that the vineyards of the Azores islands harbor a high yeast biodiversity and a high genetic diversity of S. cerevisiae strains. The comparison of the microflora from the different ecosystems of the islands showed correlations between the predominating species, geographic location of the islands, type of vineyard and grape variety. The species H. uvarum was predominant in seven islands of the Azorean Archipelago, independent of the grape varieties and the type of vineyard. However, this species was present in abandoned vineyards in considerably higher (20% more) proportions than in non-abandoned vineyards.
CONCLUSIONS The high percentage of C. zemplinina in Santa Maria and São Miguel differentiates the yeast microflora of this group of islands, pointing towards a correlation between geographic origin of the grapes and the yeast microflora composition. The species M. pulcherrima, appears clearly associated with the traditional grape varieties, being only found in Graciosa and Pico, the islands with higher proportion of such grape varieties. However, these data will be completed with results obtained from the samples collected in 2010.
CONCLUSIONS The genetic diversity of S. cerevisiae was more dependent on the sampling year than on the geographic origin, grape variety or type of vineyard, showing that ecologically meaningful affirmations require repeated sampling campaigns. This points to additional biotic and abiotic factors that affect the distribution and abundance of yeast communities in vineyards of the Azorean islands.
ACKNOWLEDGMENT J. Drumonde-Neves is recipient of a fellowship from the DRCT (M3.1.2/F/006/2008). This work was financially supported by the European Community s Seventh Framework Program (FP7/2007-2013) under grant agreement nº 232454 and the Portuguese Science Foundation (Fundação para a Ciência e Tecnologia, FCT), within the project AGR-ALI/103392/2008. We appreciate the kind assistance of the Serviços de Desenvolvimento Agrário do Faial, Graciosa, Pico, Santa Maria, S. Jorge e S. Miguel, and numerous wine producers. UNIÃO EUROPEIA FUNDOS ESTRUTURAIS