Exploring the biodiversity of a wine region: Saccharomyces yeasts

associated with wineries and vineyards

L. Mercado1 and M. Combina

1,2

1EEAMendoza, Instituto Nacional de Tecnología Agropecuaria (INTA), 2Consejo Nacional de Investigaciones Científicas

y Técnicas (CONICET). San Martín 3853 (5507), Luján de Cuyo, Mendoza, Argentina

Saccharomyces yeasts have been used in the fermentation of food and drink products for thousands of years. Wine

production is a complex microbiological process involving different S. cerevisiae populations during fermentation.

Argentina is the fifth largest wine producer in the world and most of its grape and wine production is located in the west

side of the country on the eastern side of the Andes Mountains (between 22º and 42º S). In characterizing a wine region, it

is necessary to not only define its microclimate and soil characteristics, but also the yeast populations that are present in

the vineyards, grapes and wineries. The objective of the present study was to determine the native strains of

Saccharomyces cerevisiae present in the grapes, wineries and wines of the Zona Alta del Río Mendoza (ZARM) region.

This region is known worldwide for producing high quality Malbec wines. Two different, but related, aspects were

evaluated using a molecular approach for Saccharomyces strain differentiation. First, we tried to elucidate the origin of S.

cerevisiae yeasts involved in the spontaneous fermentation of grape musts by evaluating the distribution of these yeasts on

winery equipment and determining their contribution to the fermentation process. Their genetic relationships with

commercial yeasts were also evaluated. Secondly, we explored S. cerevisiae strain diversity in the ZARM vineyards. The

contribution of vineyard Saccharomyces strains to the population responsible for industrial spontaneous fermentations and

the level of genetic relationships between these populations were also evaluated. The results showed S. cerevisiae biota

resident in both wineries and vineyards. A wide diversity and dynamic behaviour were also found within and between

seasons, as was a variable contribution to the fermentation process as well as complex interactions with the commercial

yeasts used in the wineries. There was no evidence of a representative strain distributed throughout the viticultural region

evaluated. Complex genetic relationships at the molecular level between isolated yeasts which shared the same ecological

environment were found. Although a wide diversity was observed, these yeasts shared many characteristics, as evidenced

by molecular markers, which suggests that the strong selection pressure exerted by the fermentation process could have

generated variability at different levels. Knowledge about the biodiversity of native Saccharomyces strains is essential for

the preservation and exploitation of the oenological potential of wine grape growing regions. Although microorganism

biodiversity has hardly been considered before, it could be used alongside other tools to help face the effects of climate

change on viticulture and the winemaking process.

Key words: yeasts, Saccharomyces, wine, winery, vineyard

1. Introduction

The quality of wine is a direct consequence of the evolution of the microbiota of must during fermentation. Yeasts play

a central role in the fermentation process during winemaking. Saccharomyces cerevisiae, ‘the wine yeast’, is the most

important species involved in alcoholic fermentation [1]. In the past, wine fermentation was spontaneously carried out

by indigenous yeasts. This method is still applied by several wineries in Argentina to preserve the tipicity (or regional

character) of their wines. Yeasts naturally present in musts transform sugars into alcohol, carbon dioxide and other

important metabolites [2]. It is generally assumed that these yeasts are present on grapes and winery equipment,

although some controversy about this still exists [3]. Due to the extremely low occurrence and the difficulty in isolating

Saccharomyces from healthy undamaged grapes by direct plating, some authors have excluded a natural origin of these

yeasts, postulating instead only a ‘winery origin’ for them [4, 5, 6]. However, it has been shown that damaged grapes

are very rich depositories of microorganisms including Saccharomyces [7]. The contribution to the fermentation process

by flora present on winery equipment surfaces has been widely postulated [8, 9, 10, 11], but only confirmed recently by

direct sampling and the isolation of yeasts from these surfaces [12, 13 14].

Yeasts are part of the natural microbial communities of grapes [15]. It is generally thought that unique strains of

yeasts are associated with particular grape varieties in specific geographical locations and that significant diversity and

regional character, or ‘terroir’, are introduced into the winemaking process via this association [16, 17, 18, 19, 20].Thus,

the grapes of a region represent an important source of yeasts for starter culture development when trying to preserve

both yeast biodiversity and the regional influence on the characteristics of a wine [3].

Argentina represents an important wine producer in South America. Although it has an extensive history in oenology

and viticulture, very little is known about the ecology of the microorganisms involved in local fermentation. The

development of knowledge on the microbial ecology of local ecosystems is essential for understanding the winemaking

process and for generating products with local characteristics, allowing the development of modern winemaking

practices and the diversification of wine products.

In the present study, two different, but related, aspects were evaluated using a molecular approach for S.cerevisiae

strain differentiation. First, we tried to elucidate the origin of S. cerevisiae yeast involved in the spontaneous

_______________________________________________________________________________________

fermentation of grape musts by evaluating the distribution of these yeasts on winery equipment and determining their

contribution to the fermentation process. Their genetic relationships with commercial yeasts were also evaluated.

Secondly, we explored S.cerevisiae strain diversity in ZARM vineyards. The contribution of vineyard S.cerevisiae

strains to the population responsible for industrial spontaneous fermentations and the level of genetic relationships

between these populations were also evaluated.

2. Saccharomyces winery populations and the origin of fermentative yeasts

It is generally assumed that spontaneous grape must fermentation occurs via the development of yeast ‘naturally’

present in the must, although the origin of these yeasts is a matter of discussion [4, 5, 6, 12, 13]. In order to elucidate the

participation of winery and grape Saccharomyces populations in spontaneous fermentations of Malbec musts from the

Zona Alta del Río Mendoza region (Argentina), Saccharomyces yeast associated with grapes and winery equipment

were analysed over two consecutive years [14]. The total winery yeast population was evaluated on all winery surfaces

in contact with grapes or must during processing, from harvest to the end of fermentation. Two sampling points were

considered: before vintage (BV) and during vintage (DV). At before vintage time, the processing equipment had been

cleaned, disinfected and not used since the previous year. The aim of this first sampling (BV) was to evaluate the ability

of the yeasts to survive between two consecutive seasons. At the second sampling point (DV) the winery surfaces were

sampled before the must was processed. This sampling (DV) was carried out to evaluate the winery micobiota present

during the must processing and its contribution to fermentation. The winery equipment was used to process other grapes

between both sampling points. Grape samples were directly obtained upon the arrival of grapes at the winery by random

sampling. The grapes were crushed and the tanks were filled. Samples of fresh must (M) were taken once the fresh must

was inside the fermentation tanks. During fermentation, two sampling points were considered: the beginning of

fermentation (BF), when the initial density of the must diminished in 0.01 g/mL and the end of fermentation (EF when

the density remained constant. The fermentations were conducted following standard winery practices without the

addition of commercial yeasts. Several commercial S.cerevisiae strains normally employed in ZARM viticulture region

wineries were also included in this study.

To find out the total yeast population, samples were spread onto two culture media (MEA+BR and WL) carried out

in triplicate using a serial dilution method. At the same time, an enrichment procedure (SelMed selective enrichment

medium according to Mortimer and Polsinelli [7]) was employed to allow multiplication of Saccharomyces from grapes,

where these yeasts could be present at numbers below the detection limit for direct isolation by plating. Saccharomyces

and non-Saccharomyces yeasts were rapidly discriminated according to their ability to grow in L-lysine medium

(Oxoid, Basingtoke, UK). This assignation was confirmed by a conventional yeast identification method following

some of the taxonomic criteria described by Kurtzman and Fell (1998) [21]. S.cerevisiae isolates were subsequently

differentiated at the strain level by using two molecular methods. Many techniques have been developed using the tools

offered by molecular biology and many of them are useful for identifying and characterizing yeasts at the molecular

level. The main molecular techniques proposed for the studies of S. cerevisiae strain diversity include: pulsed field

electrophoresis [22], mitochondrial DNA restriction analysis [23], interdelta element PCR amplification [24, 25] and the

amplification of polymorphic microsatellite loci [26-30]. The two molecular markers selected in this study still

represent the simplest and most widely used techniques for studying Saccharomyces biodiversity. We discussed about

their differences, usefulness and advantages in a previous work [31]. Firstly, we used interdelta PCR analysis, which

allows the amplification of DNA fragments between two delta elements. Delta elements are direct repeat elements

which flank the Ty1 retrotransposons that are dispersed on the S.cerevisiae nuclear genome at an amplifiable distance;

their number and position in the genome is strain specific and stable in about 50 generations [24]. Mitochondrial DNA

restriction fragment length polymorphism was also applied. This produces an unambiguous mitochondrial pattern

supported on specific restriction sites. The endonuclease Hinf I recognizes a high number of restriction sites in the

nuclear yeast DNA but only a few sites in the mitochondrial genome. Therefore, mitochondrial restriction fragments

can be easily separated by agarose gel electrophoresis [23]. Patterns of bands generated with each method were

combined to define the “pattern”, i.e., the strain.

2.1. Total yeast counts in different winery surfaces and grapes.

The total yeast counts on different winery surfaces showed an increasing number of total yeasts according to the

advancing vintage. The isolation frequency of Saccharomyces also increased. This confirms that the continuous passage

of must on winery equipment throughout the vintage season supports the development of yeasts present and may

introduce new ones. Furthermore, grape must exerts a positive selective pressure on Saccharomyces because of its high

sugar content, low pH and the presence of SO2 [15]. The yeast counts obtained from grapes and fermentation phases

were in agreement with counts previously reported [15, 32, 33]. As we expected, it was not possible to isolate

Saccharomyces by direct sampling from the grapes. Recovery by the enrichment method confirmed their low

population on this substrate. This fact has been reported before by several authors who questioned the presence of this

species in the vineyard ecosystem and postulated a ‘winery origin’ for it [5, 6, 12, 34]. Several studies demonstrated that

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Saccharomyces on grapes occurred at percentages below 0.1% of naturally occurring yeast biota, and that they were not

systematically widespread, since neither all plants nor all grape clusters harboured wine yeast [7, 35, 36].

2.2. Distribution of S.cerevisiae strains in winery

Between 9 and 20 different Saccharomyces patterns were found on winery equipment at BV during 2001 and 2002

respectively, (Table 1 shows the results for 2002 as an example). Some surfaces exhibited a unique pattern, i.e. a single

yeast strain, while others harboured up to 10 different strains. The equipment surfaces evaluated previously for Malbec

grape processing (DV) exhibited an increasing number of different patterns, 22 patterns in 2001 and 35 in 2002. Despite

the diversity found in the winery, some strain patterns were present simultaneously and at both sampling times on more

than one equipment surface sampled (Table 1). All of the S.cerevisiae isolates found in the winery at BV were

considered as resident or ‘perennial’ strains. However, the S.cerevisiae patterns recovered from the winery for both

years, independent of the isolation sampling point, were also included as resident biota under the assumption that they

were present in low numbers at BV and were undetectable by the sampling method used.

It is important to point out the great diversity of S.cerevisiae strains found on the winery equipment. There are no

previous reports of such extensively recovered S.cerevisiae strains. The amount and diversity of wine yeasts present on

equipment depends on the standards of cleanliness of the winery and the nature of the surface. Irregular, unpolished

surfaces which are difficult to clean, for example pipes and crushers, may support dense populations of winery yeasts

[2].

Our results confirmed the presence of a particular Saccharomyces strain population resident in the winery. This

population was found to have a dynamic behaviour since it fluctuated from year to year and throughout the vintage

season, even though the weather conditions and cleaning protocols of the winery in both years were similar. Despite this

dynamism, it is important to note the existence of stable strains throughout the season and in consecutive years. Several

authors have observed the persistence of some yeast strains and their subsequent contribution to spontaneous

fermentations in wineries [4, 9, 13].

2.3. S.cerevisiae winery strains in grapes and fermentation

Despite the fact that the grapes were sampled from the same vineyard they exhibited different yeast patterns each year.

Although S.cerevisiae was found in low number on grapes, one of the predominant strains on the grapes was also

isolated from winery surfaces in 2001. This situation could be explained by contamination of the grapes with these

yeasts being present on the equipment used in harvest and transport. The presence of these yeasts on different

equipment and tools used in harvest operations has been previously demonstrated [15]. Our results showed a change in

yeast population composition on the grapes from year to year. The great diversity and heterogeneity in distribution of

the wine yeasts in vineyards have been previously demonstrated [35, 36, 37]; these differences can be attributed to the

fact that different bunches of grapes were sampled which may have had a distinct flora.

The S.cerevisiae population on fresh must consisted of three to five strains which showed different molecular

patterns from those previously described on grapes, although some of the yeast strains previously found in winery also

appeared in the fresh must samples. On the other hand, the whole strain population on fresh must could not be detected

later during fermentation. Interestingly, a strain population change was also observed between the grapes and fresh must

once it had been in contact with the winery equipment (Table 1).

During spontaneous fermentation, a succession of different S.cerevisiae strains was observed in both years. An

increased strain diversity was found during fermentation (Table 1) and a few strains were present at more than one

fermentation stage. Similar situations have been described in spontaneous fermentations where the dynamics of

different subpopulations throughout the fermentation stages were observed [11, 13, 37, 38, 39, 40]. In the present study,

a change in the yeast population during fermentation was observed from year to year. While in 2001 8 different strains

participated in must fermentation, in 2002 a total of 22 patterns were found during this process (Table 1). These results

agreed with those of other studies, where the micobiota of each year was characterized by the appearance of new strains

and by different isolation frequencies than the previously detected strains [10, 11, 37, 41]. Two different situations – a

large number of strains at low percentages and a smaller number of strains with one dominant strain – were found in

this study and have been previously reported [13, 36, 40, 41]. Curiously, and in agreement with Santamaría et al. (2005)

[13], in the fermentation where one strain was dominant, in the 2001 fermentation one strain represented 67 % and 29

% of strain population at beginning and end of fermentation respectively, it was coincident with a commercial yeast

which had not been previously inoculated in the must.

Some S.cerevisiae yeast strains found during fermentation were previously isolated from the winery equipment.

Around 30% and 60% of the yeast population at the end of fermentation in 2001 and 2002 corresponded to the winery

strains, respectively, most being ‘perennial’ yeast strains. This fact provides evidence of the contribution of winery

yeast to the industrial process through simple contact. It has been postulated that numerous different Saccharomyces

strains are present in the winery ecosystem and that the different conditions at each harvest (the chemical composition

of must, the winemaking process, the level of sulphitation and temperature, for example) could determine which

specific strains will develop during fermentation [13]. Similar results were previously observed by Ciani et al. (2004)

[12], who demonstrated that autochthonous S.cerevisiae strains in a winery predominate in natural fermentations and

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concluded that the contribution to the process by Saccharomyces resident on grapes was not significant or was absent.

Strains which had not previously been found on equipment surfaces, nor on the grapes or fresh must, were also found to

be involved in the fermentation process. Under our experimental sampling procedure for evaluating the winery surfaces,

some strains might not have been detected, resulting in a limited picture of the kinds of strains that actually occur in

winery. It could also be possible that these strains came from other equipment at the winery or other fermentation tanks.

Table 1 Distribution, as percentage, of Saccharomyces strains, defined by combination of amplification and restriction patterns,

isolated during 2002 season at ZARM region.

Pattern

Code

Winery before vintage Winery during vintage Grapes

Fresh

must

Fermentation Impact of the

pattern R CR PTR P Fa P Fb R CR TI TC BF EF

I02 20 Unique in winery II02 43 6.5 5 Winery and fermentation

III02 8.3 Unique in winery

IV02 8.3 6 Winery two surfacees

V02 20 20.5 5 Winery and fermentation

VI02 12.5 14 Winery repeated

VII02 20 Unique in winery

VIII02 5 Fermentation

IX02 6 20 Commercial

XLI 6 Unique in winery

XLII 12.5 6.5 6 10 Winery and fermentation

XLIIII 5 Fermentation

XLIV 6 20 Winery and fresh must

XLV 6 Unique in winery

XLVI 100 Unique in winery

XLVII 100 29 Winery repeated

XLVIII 20 Unique in winery

XLIX 6 Unique in winery

L 8.3 Unique in winery

LI 13.5 Unique in winery

LII 5 Fermentation

LIII 20 Unique in winery

LIV 14 Unique in winery

LV 33 Grape

LVI 6 7 Winery and fermentation

LVII 14 8.3 Repeated in winery

LVIII 6 Unique in winery

LIX 43 8.3 Repeated in winery

LX 6 Unique in winery

LXI 29 Unique in winery

LXII 14 Unique in winery

LXIII 8.3 Unique in winery

LXIV 13.5 12 15 5 Winery and fermentation

LXV 6 Unique in winery

LXVI 20 Fresh must

LXVII 8.3 Unique in winery

LXVIII 8.3 5 Winery and fermentation

LXIX 8.3 Unique in winery

LXX 6.5 7 Winery and fermentation

LXXI 6 Unique in winery

LXXII 6.5 Unique in winery

LXXIII 12.5 8.3 Unique in winery

LXXIV 8.3 Unique in winery

LXXV 6.5 21 5 Winery and fermentation

LXXVI 6 5 Winery and fermentation

LXXVII 7 Fermentation

LXXVIII 7 Fermentation

LXXIX 13.5 16 Repeated in winery

LXXX 67 Grape

LXXXI 20 Fresh must

LXXXII 20 Fresh must

LXXXIII Unique in winery

LXXXIV 12 7 10 Winery and fermentation

LXXXV 15 5 Commercial

LXXXVI 7 Fermentation

LXXXVII 7 Fermentation

LXXXVIII 5 Fermentation

LXXXIX 6.5 Unique in winery

XC 12.5 Unique in winery

XCI 6 Unique in winery

XCII 5 Fermentation

XCIII 8.3 6 10 Winery and fermentation

XCIV 10 Fermentation

XCV 20 Unique in winery

R: reception equipment; CR: crusher; PTR: pipe for must transport; PF: pipe for filling; TI: surface interior of tank; TC: exterior connections of the tank; BF: beginning of fermentation; FF: final of fermentation.

Our results also indicated commercial strain participation in fermentations conducted without yeast inoculation.

Similar findings were reported by other authors [9, 13]. Although the Malbec must under study was spontaneously

fermented in this winery, white wine fermentations are usually conducted by the inoculation of commercial yeasts.

These yeasts could remain on the equipment and may become predominant when spontaneous fermentations are

delayed, although their participation would decrease as the fermentation progressed, as was shown by our results.

Nevertheless, in this winery, no commercial strains were isolated on the equipment at the first sampling point,

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indicating that this yeast could not remain in the winery from year to year. Taking the contribution to the fermentative

population of the winery and commercial strains as a whole, they represented in average a 70 and 68% of strains found

in fermentation every season respectively.

In summary, the following findings can be highlighted: a) the low occurrence of Saccharomyces on grapes and its

limited participation in fermentation was confirmed; b) the population of S.cerevisiaes on fresh must showed a different

strain composition to the populations previously described on grapes, with only one exception. Moreover, 40% of the

strains in these samples were similar to those previously found in the winery equipment; c) a sequential substitution of

S.cerevisiae strains was observed during fermentation. Around 30% and 60% of the yeast population at the end of

fermentation had originated at the winery in the 2001 and 2002 vintages, respectively; d) the presence of

Saccharomyces on winery surfaces at the two different sampling points in both seasons was observed. A stable and

resident S.cerevisiae microbiota in the winery was confirmed, consisting of very diverse strains with most of them

showing a dynamic behaviour. Additionally, an important participation by the winery yeasts in fermentation was

demonstrated and their contribution was found to be dependent on the native yeast populations on must and the

oenological practices employed; e) commercial yeast strains were found during fermentation at different percentages

even though they were hardly found on the winery surfaces.

3. Vineyards and the noteworthy yeast biodiversity

One outcome of numerous reports is that the quality of a wine is a direct consequence of the yeast biota which

developed during fermentation [42]. In different wine regions around the world, there is currently a growing demand for

autochthonous strains of fermentative yeast with typical oenological characteristics representative of those particular

regions. These strains could be better adapted to the conditions of a particular region, including the soil and climate

conditions, the grape variety and the viticulture management and oenological techniques which are used there. Since the

importance of S. cerevisiae in wine production was already established, the use of foreign commercial yeast cultures has

been transformed into one of the most common practices for reducing the risk of spoilage of the wine. However, this

practice could prevent the production of some desirable or typical organoleptic characteristics of the wines and reduce

the diversity of the natural microbiota [43]. On the other hand, some studies have demonstrated a certain correlation

between S. cerevisiae strains isolated from grapes and wines and their geographical origin. The results of some studies

on yeast biodiversity in vineyards supported the theory that in spite of the great polymorphism observed, a population of

yeasts considered as particular to a viticulture region or ‘terroir’ might exist [44, 45, 46, 47]. Each viticulture region

constitutes an ecosystem where yeasts are included in the microbial diversity. The characterization of yeasts from a

viticulture region allows the determination of their population structure, their distribution in the vineyard and the

genetic relationships between them in relation to their geographical origins, and it also allows the evaluation of their

participation in fermentations. All of this information could be used in the selection of native yeasts and the

conservation of genetic resources.

In order to microbiologically characterize the “Zona Alta del Río Mendoza” viticulture region, eight different

vineyards spread all over this region were selected. The grape sampling design included ten different sampling points

distributed throughout the sub-area of the vineyard evaluated. The grapes obtained from each sample point were

individually processed to determine the distribution of Saccharomyces within the vineyard. Afterwards, the vineyards

were harvested and the grapes were transported to six different wineries located in the same viticulture region, avoiding

mixing with other grapes, for spontaneous fermentation.

3.1. Saccharomyces populations from vineyards

We previously reported the low populations of Saccharomyces on mature healthy grapes and the difficulty in isolating

these yeasts from grapes using direct isolation methods [14, 48]. Therefore, an enrichment procedure was applied in

order to recover the S.cerevisiae strains from the grapes. The samples were aseptically crushed and the musts obtained

were allowed to ferment. The yeasts were later isolated. Using this procedure means that the results obtained reflect the

S.cerevisiae strains able to develop in the conditions imposed. It must be taken into account that conditions in a

vineyard are quite different from those in fermentation, and that the populations which are isolated and characterized

only reflect those yeasts with particular competitive traits allowing them to survive the fermentation process, i.e. yeasts

with some oenological interest. This methodology could give a ‘distorted’ picture of the Saccharomyces populations

present in vineyards, but it did not contradict the aims of this work and it is a commonly used strategy for the study of

vineyard yeast populations [36, 49, 50, 51, 52].

A combination of molecular patterns obtained by interdelta PCR and RFLP mtDNA allowed 1020 S.cervisiae

patterns to be differentiated. The selection of these molecular markers for the characterization of Saccharomyces

populations was found to be very useful in a study of closely related S.cerevisiae strains, as was as their simplicity and

low cost, as previously discussed [31]. The vineyards of ZARM exhibited a variable number of S.cerevisiae strains: 9 to

36 different molecular patterns in total were observed per vineyard (Table 2). A non-homogeneous distribution of these

yeasts was verified; the different sampling sites in the vineyards harboured between 1 to 12 different patterns. In some

cases, one or two sites showed great diversity whereas in others S.cerevisiae strains could not be isolated (Figure 1).

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Surprisingly, regardless of belonging to the same well-defined homogeneous ecosystem (ZARM region), each vineyardexhibited different Saccharomyces populations, i.e. each vineyard constituted a defined area with a particular yeastpopulation. Only two vineyards (NL and CZ), closely located to one another, had three patterns in common (Table 2).

The vineyards were classified as low polymorphic or highly polymorphic according to the level of Saccharomycesdiversity found.

High polymorphism vineyards: this group comprised six vineyards where more than 20 molecular patterns werefound, identified as CR, NL, NP, ID, CZ and T. Some patterns were found in different sites of the same vineyard, butthey only represented a small fraction of the total number of patterns, and 78% to 93% of these were unique patterns(Table 2). In general the sampled sites exhibited a combination of different patterns, only a few sites in these vineyardspresented only one pattern on their grapes. In general terms, the Saccharomyces populations of these vineyards werehighly diverse and randomly distributed, with each vineyard site showing different subpopulations.

It has been proposed the use of the ratio between the number of isolates and the number of patterns as anapproximate estimate of biodiversity [37]. According to this criterion, the highly polymorphic vineyards of ZARMshowed values of 1.9 to 3.6, corresponding to greater diversities than those detected in the other viticulture regions.Similar studies were carried out in two South African regions located in the Western Cape [36, 46], where the authorsfound different populations in the costal and continental areas, with the former being more diverse than the latter. Theseven coastal vineyards showed high variability with diversity indices from 2 to 15, while the continental vineyardswere the least variable with diversity values of 4 to 30. Another survey conducted in vineyards in the Vinho Verderegion in Portugal [37], other than finding important strain diversities and variable populations among vineyards andseasons, found a diversity factor of 5.45, indicating a more minor diversity than reported in the present work. Theseauthors [37] mentioned that their results produced similar diversity values to previously published surveys on thegenetic diversity of autochthonous oenological S.cerevisiae strains in other regions with a viticulture tradition, such asBordeaux [8]; Champagne and the Loire Valley )[16] in France; Tarragona [9], Priorato [11, 39] and La Rioja [53] inSpain; Toscana, Sicilia [54] and Collio [55] in Italy; Amyndeon and Santorini [40] in Greece, and Switzerland [56]. Thisfact indicates that the diversity found in the present study of ZARM vineyards was, in general, superior to thoseobserved in different regions of the viticultural world. A survey undertaken in another important Argentinean wineregion, Comahue, in the Neuquén province [57], showed a similar diversity as that observed in the present study.According to these comments, the ‘Old world’ vineyards (European) could exhibit a lower diversity than ‘New world’vineyards (Argentinean and South African), which can be understand considering the differences between both groupsof viticultural regions, including the length of time that the viticultural tradition took to develop, and also differences inthe length of the vineyards. The European vineyards are small and have had a lot of practice in vineyard managementand winemaking, which has been carefully maintained in order to obtain a product with an expression of the ‘terroir’[58]. In the ‘New world’, viticulture and winemaking are recent developments, and generally vineyards represent largeextensions of land located in wide ‘wine regions’. This latter fact could be the reason why no ‘regional’ strains werefound in this study. However, in the present study, a small group of closely located strains were found in two vineyards,which could be correlated with the concept of ‘terroir’ since the area including both vineyards approximatelycorresponded to the European ‘terroir’ [59].

Low polymorphism vineyards: two vineyards, identified as CB and S, had a notably low diversity (Table 2), whereonly ten and nine S.cerevisiae strains were isolated, respectively. According to the sampling plan, ten sites wereevaluated in the area of each vineyard, but Saccharomyces could not be isolated from three sites of vineyard S and twosites of vineyard CB (Figure 1). Some patterns were highly distributed, for example, pattern 3S was found in five sitesout of seven where Saccharomyces was recovered in this particular vineyard, and pattern 2CB was isolated from foursites of vineyard CB. Moreover, both of these strains showed molecular patterns corresponding to commercial strains.

Fig. 1 Different patterns found inten sampling points of eightvineyards of ZARM region.

Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology A. Méndez-Vilas (Ed.)

©FORMATEX 2010 1047

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Additionally, pattern 1CB, found in two sites of vineyard CB, also coincided with another commercial strain. In

addition to the important presence and spread of commercial yeasts, the low diversity vineyards showed a drastic

decrease in S.cerevisiae diversity, as evidenced by the small number of strains isolated here. Moreover, high

biodiversity values (ratio of number isolates/number of strains) of 4.9 and 6.2 were observed for vineyards S and CB,

respectively. Coincidentally, both vineyards are located in a very close proximity to the winery, indicating that the

currently used oenological practices, such as the intensive use of commercial inoculates, could affect the biodiversity of

the vineyard ecosystem, decreasing polymorphism and replacing the native biota with foreign organisms [59].

Table 2 Diversity of S.cerevisiae strains found in vineyards from ZARM wine region of Mendoza, Argentina

Vineyard code

Total yeast

populations Number of distinct

patterns

Number of

patterns present in

unique

vineyard site

Number of

patterns present in more than

one vineyard

site

Common patterns

between vineyards

Number of

vineyard patterns found

in

fermentations

Commercial patterns in

vineyard (Log cfu/mL

±sd) Pattern Site Incidence

(%)

Hig

h p

oly

morp

his

m

T 4.92 ± 0.31 36 30 6 - 2 - - -

NL 3.31 ± 1.12 25 21 4 2 = 12 CZ

3 = 5 CZ 6 = 4 CZ

2 - - -

CR 3.21 ± 0.76 31 27 4 - 3 - - -

CZ 4.97 ±0.61 35 32 3 4 = 6 NL 5 = 3 NL

12 = 2 NL

2 - - -

ID 4.05 ±0.71 26 22 4 - 2 1

1 3

5

10

45.5 25

75

33.3 25 3 25 NP 3.58 ±1.13 23 18 5 - 1 6 4 12. 5

Low

poly

morp

his

m CB 2.81 ±1.17 9 7 2 - 2

1 1 100

5 80

2

2 55.6

4 100

7 100 9 100

S 2.31 ±0.71 10 5 5 - 3

3

1 16.7

3 100 4 61.5

6 85.7

8 60

9 7 33

8 20

3.2. Saccharomyces populations in industrial spontaneous fermentations

With the aim of examining the dynamics of Saccharomyces during spontaneous fermentations at an industrial scale in

different wineries from the ZARM region, the previously sampled vineyards were harvested and the grapes were

processed in six different wineries also located in the ZARM region. The spontaneous fermentations were conducted

according to the standard protocols of the different companies. Samples were taken at specific stages of the

fermentation process, defined in terms of must density, for comparison. The fermentations evaluated under industrial

conditions showed different levels of S.cerevisiae present at the beginning of the processes (BF), even though mature

healthy Malbec grapes were always used and the wineries have a similar level of technology, knowledge and prestige in

the production of quality wines. On the other hand, the intermediate stage (MF) of fermentation showed yeast counts

more homogeneous than those initially recorded, with a predominance of S.cerevisiae in all cases, which became the

exclusive species by the end of fermentation.

The fermentations evaluated showed a highly polymorphic nature and a variable behaviour of Saccharomyces

populations in each case. Moreover, the Saccharomyces populations were different in different fermentations, with 5-21

patterns observed in total. In the different fermentation stages, different numbers of patterns were observed, with a

maximum of 13. The dynamics of S.cerevisiae patterns during the fermentations were different and two main

tendencies were observed: fermentations with a high polymorphism at the start with the number of patterns involved

decreasing during the process; and the other fermentations which showed a low polymorphism in the beginning with

increasing populations co-existing until the end of the process. The low polymorphic vineyards (S, CB) showed only a

few strains at the beginning of fermentation and the presence of commercial strains, previously found in the vineyards,

participated in both processes.

In addition to the variability in the number of patterns found in different stages of the process, a different permanence

of these patterns during fermentation was also observed. In general, a substitution of patterns was verified, with

multiple strains conducting the fermentation with no predominance. This result suggests that strains found in the

different stages could exhibit different physiological characteristics, with some of them more adapted to tolerate the

high osmotic pressure at the initial stage, with others having faster growth or more tolerance to ethanol [60]. This

_______________________________________________________________________________________

alteration in the strains is characteristic of spontaneous fermentation and constitutes a strength whereby the risk of

sluggish and stuck fermentations is reduced, and the strains could contribute metabolites which provide a greater

complexity to the wine [59].

The participation of vineyard strains in spontaneous fermentation was low or even nonexistent. Although in most

fermentations the patterns originated from vineyard (one to three in each case), these patterns did not display a

significant prevalence in the different fermentations (Table 3). Curiously, in just one case (the low polymorphic

vineyard S), the fermentative population mainly came from the vineyard. The population involved in this process had a

very limited diversity and was homogenous throughout the process with three patterns present at all stages; moreover,

two of these persistent patterns corresponded to a commercial strain. The different yeast patterns found in the

fermentations did not coincide with the vineyard patterns but they could have corresponded to isolates originally present

in the vineyard samples but which were not recovered under the conditions used for isolation, or they could have

represented winery strains incorporated by ‘contamination’ during the processing of musts in the winery.

Table 3 Diversity of S.cerevisiae strains found in spontaneous fermentations from ZARM wine region of Mendoza, Argentina

Vineyard

name

Fermentation

stage

Total number of

different patterns

Number of

patterns

present in

unique stage

Number of patterns

presents in more than

one stage

(pattern code)

Number of

patterns from

vineyard

(pattern code)

Number of

commercial

pattern

(pattern code)

T B 10 7 3 (36-37-40) 1 (36) 1 (40)

M 7 4 3 (36-37-40) 1(36) 1(40)

F 7 4 2 (36-40) 2 (16-36) 1 (40)

NL B 3 3 - 1(20) -

M 6 5 2 (29-33) - -

F 13 11 2 (29-33) 1(13) -

CR B 1 1 0 0 1(33)

M 10 8 4(3-32-36-44) 2(3-31 -

F 9 6 4(3-32-36-44) 1(3) -

CZ B 6 5 6(4-28-36-37-38-39) 2(4-28) -

M 7 5 7(4-28-36-37-38-39-40) 1(4) 1(40)

F 2 1 1(40) - 2(40-46)

ID B - - - - -

M 6 3 3(1-25-28) 2(1-25) 2(1-25)

F 7 4 3(1-25-28) 2(1-25) 2(1-25)

NP B 9 7 2(23-28) 1 (23) -

M 7 3 4(23-28-33-36) 1 (23) -

F 8 5 3(23-33-36) 1 (23) -

CB B 2 1 1(10) - -

M 10 8 3(2-10-18) 2(1-2) 2(1-2)

F 8 6 2(2-18) 1(2) 1(2)

S B 3 0 3(3-9-10) 2(3-9) 3(3-9-10)

M 3 0 3(3-9-10) 2(3-9) 3(3-9-10)

F 5 2 3(3-9-10) 3(3-9-11) 3(3-9-10)

-: none; ns: not sampled; B:beginning of fermentation; M: middle of fermentation; F: final of fermentation

3.3. The impact of commercial yeasts in vineyards and fermentations

The molecular patterns of S. cerevisiae strains isolated from the vineyards and fermentations were compared with a

set of 30 commercial strains which included those widely used in the wineries of the ZARM region. S. cerevisiae

isolates from four vineyards coincided with commercial strains (Table 1). Moreover, within this group, the low

polymorphic vineyards CB and S showed a wide distribution and high incidence of such strains. As commented above,

both vineyards are located next to the respective cellar, suggesting a transfer of yeasts from the winery to the vineyard.

Two other vineyards which belonged to the ‘highly polymorphic’ group of vineyards (NP and ID) also presented some

patterns coincident with commercial strains. The NP vineyard is located approximately 200 m from the winery where

the grapes were processed; this distance could explain why the commercial strains were not spread throughout this

vineyard and why the global diversity of S.cerevisiae populations found there was not affected, as it was the case of

vineyards S and CB. Moreover, another difference found was that the commercial strains present in vineyard NP were

not found later in the corresponding fermentation. On the other hand, vineyard ID is also located near a winery (300 m)

and this could be the cause of the presence of commercial strains on the grapes from this vineyard. These results agreed

with those of previous works, which suggested that the dispersal of commercial strains is mainly mediated by water

runoff, macerated grape skin at dumping sites and different vectors, such as insects or others, which would be

responsible for their presence at distances greater than 1000 m from the cellar [51].

The commercial strains were also found in some spontaneous fermentation evaluated in the present study, even

though commercial strains were not found in the corresponding grapes (Table 1 and 2). In these cases, it could be

_______________________________________________________________________________________

inferred that they were incorporated during winemaking in the cellar. Previous studies demonstrated the horizontal

transfer of yeasts in wineries by ‘cross contamination’, mediated by winery equipment employed daily in the processing

of musts, which transferred microorganisms from tank to tank [9, 61]. The present work emphasizes the real situation in

Argentinean oenology, where all the wineries evaluated corresponded to commercial industrial companies where

commercial yeast cultures are commonly used for fermentation. This fact could explain the presence of commercial

yeasts in the spontaneous fermentations evaluated.

It is noteworthy that only four different commercial strains were detected in the grapes and fermentations. Moreover,

one of them was repetitively found in four vineyards and five fermentations. This commercial strain is intensively and

widely used in the viticulture region evaluated. This fact could explain its generalized presence, in addition to its

characteristics which allow it to survive in the vineyard and in the winery, permitting it to compete with the other strains

present in these ecosystems.

The Saccharomcyes biodiversity results arising from this ecological area, the ZARM wine region, provide evidence

of the importance of evaluating different aspects of microbial diversity in order to assess the complete picture of yeasts

involved in the winemaking environment. Moreover, knowledge of the biological patrimony of yeasts is essential to

their maintenance, and is the source of the genetic background needed to obtain starter strains which are able to fully

develop the typical flavours and aromas of wines originating from different grapevine cultivars [2] and to ensure the

conservation of gene pools of primary importance for the preservation of productive activities based on yeast mediated

processes [62].

4. Molecular relationships among S.cerevisiae strains

These results show a ‘picture’ of Saccharomyces populations present in the ZARM wine region and illustrate the

populations of S. cerevisiae present in grapes, winery equipment and spontaneous fermentations. The analysis of

coincidences in the molecular patterns clearly visualized a huge polymorphism at this level. The identity among two

isolates was assigned based on total coincidence in the corresponding patterns of bands. But this criterion had some

limitations, for example: were the two patterns which were considered different by only one band totally different? Or

did some relationship exist among them that could be detected? In order to answer these questions a cluster approach

was proposed. Pattern of bands were compared after the estimation of size band and similarities based on the Dice

coefficient and dendrograms were constructed using the UPGMA method. A subset of S. cerevisiae strains representing

winery-fermentation isolates and commercial strains was selected and the corresponding patterns were compared. A

third molecular marker was used in order to gain information about the molecular relationships between the groups of

strains. The microsatellite analysis using six different loci were applied according to Jubani et al. (2007) [30]..

As shown in the dendrograms, different similitude coefficients were observed according the molecular marker used

(Figure 2). These results provided evidence of polymorphism at different genetic levels, with the nuclear and

mitochondrial markers allowing different groupings of strains. Three clusters of strains were repetitively conserved

independently of the molecular pattern utilized in the construction of the dendrogram; they mostly included isolates

from winery equipment. Moreover, the repetitive cluster of these isolates, inferring a monophyletic origin, and the slight

relationship with the commercial strains suggested an American origin accompanied by microevolutionary events in

recent times.

On the other hand, the rest of the isolates from the winery and the fermentations showed a random clustering

according to the molecular marker applied; this result suggests some kind of change at the nuclear level, which could

occur at a different rate in the nucleus with respect to the mitochondria, during the lifecycle of the yeast. Recent studies

demonstrated a low stability of the genome in wine yeast [2], which may be due to the high reorganization capacity of

its genome by Ty-promoted translocation, mitotic recombination and gene conversion [19, 63, 64]. Alternatively, it has

also been proposed that ethanol and acetaldehyde introduce breaks in the DNA, with a much higher mutation rate on the

mitochondrial genome. This may be due to a higher efficiency of the yeast nuclear DNA repair system compared with

the mitochondrial system that lacks a proofreading activity [19, 65]. The set of commercial strains included in this

analysis showed different relationships with those isolated from the winery equipment and fermentation strains. Some

of them clustered separately in the different analysis but others did not. Only one was always clustered with an isolate

from spontaneous fermentation. According to these results, the European strains were not restricted to a sole cluster, as

was previously found [19]. The existence of some genetic relationships between a few ‘winemaking-related’ strains

with some of the commercial strains would support the hypothesis that some native American strains proceeded from

European strains, as was recently suggested [30]. Conversely, it was also suggested that the existence of ‘genomic

resemblance’ among the native Saccharomyces and commercial strains, caused by centuries of positive selection during

wine production could lead to the same characteristics being explored during the process of selection for commercial

cultures [66]. It was suggested that resemblance in phenotype is reflected in genotypic characteristics [66].

The results of the studies presented here showed the complex relationships found at the molecular level among the

yeast isolates that share the same ecological environment. Moreover, this study revealed that, despite an abundant

diversity, these yeasts share many genetic characteristics.

_______________________________________________________________________________________

Figure 2 Dendrograms showing molecular relationships based on PCR interdelta (A), RFLP mtDNA (B) y SSR

(C) for 28 S.cerevisiae isolates and 7 commercial selected strains. Cophenetic correlation: (A),r = 0,88019; (B), r =

0,85498; (C), r = 0,88078. Clusters repetitively grouped were indicated with roman numbers I, II and III.

A

B

C

I

III

II

III

I

II

III

I

II

_______________________________________________________________________________________

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