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From Vineyard Soil to Wine Fermentation: Microbiome Approximations to Explain the “terroir” Concept


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Wine originally emerged as a serendipitous mix of chemistry and biology, where microorganisms played a decisive role. From these ancient fermentations to the current monitored industrial processes, winegrowers and winemakers have been continuously changing their practices according to scientific knowledge and advances. A new enology direction is emerging and aiming to blend the complexity of spontaneous fermentations with industrial safety of monitored fermentations. In this context, wines with distinctive autochthonous peculiarities have a great acceptance among consumers, causing important economic returns. The concept of terroir, far from being a rural term, conceals a wide range of analytical parameters that are the basis of the knowledge-based enology trend. In this sense, the biological aspect of soils has been underestimated for years, when actually it contains a great microbial diversity. This soil-associated microbiota has been described as determinant, not only for the chemistry and nutritional properties of soils, but also for health, yield, and quality of the grapevine. Additionally, recent works describe the soil microbiome as the reservoir of the grapevine associated microbiota, and as a contributor to the final sensory properties of wines. To understand the crucial roles of microorganisms on the entire wine making process, we must understand their ecological niches, population dynamics, and relationships between ‘microbiome- vine health’ and ‘microbiome-wine metabolome’. These are critical steps for designing precision enology practices. For that purpose, current metagenomic techniques are expanding from laboratories, to the food industry. This review focuses on the current knowledge about vine and wine microbiomes, with emphasis on their biological roles and the technical basis of next-generation sequencing (NGS) pipelines. An overview of molecular and informatics tools is included and new directions are proposed, highlighting the importance of –omics technologies in wine research and industry.
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fmicb-08-00821 May 4, 2017 Time: 16:30 # 1
published: 08 May 2017
doi: 10.3389/fmicb.2017.00821
Edited by:
Sandra Torriani,
University of Verona, Italy
Reviewed by:
David Rodriguez-Lazaro,
University of Burgos, Spain
Braulio Esteve-Zarzoso,
Universitat Rovira i Virgili, Spain
Ignacio Belda
Alberto Acedo
Specialty section:
This article was submitted to
Food Microbiology,
a section of the journal
Frontiers in Microbiology
Received: 09 December 2016
Accepted: 21 April 2017
Published: 08 May 2017
Belda I, Zarraonaindia I, Perisin M,
Palacios A and Acedo A (2017) From
Vineyard Soil to Wine Fermentation:
Microbiome Approximations
to Explain the “terroir” Concept.
Front. Microbiol. 8:821.
doi: 10.3389/fmicb.2017.00821
From Vineyard Soil to Wine
Fermentation: Microbiome
Approximations to Explain the
terroir” Concept
Ignacio Belda1,2*, Iratxe Zarraonaindia3,4, Matthew Perisin1, Antonio Palacios1,5 and
Alberto Acedo1*
1Biome Makers Inc., San Francisco, CA, USA, 2Department of Microbiology, Biology Faculty, Complutense University of
Madrid, Madrid, Spain, 3Department of Genetics, Physical Anthropology and Animal Physiology, University of the Basque
Country, Leioa, Spain, 4IKERBASQUE – Basque Foundation for Science, Bilbao, Spain, 5Laboratorios Excell Iberica,
Logroño, Spain
Wine originally emerged as a serendipitous mix of chemistry and biology, where
microorganisms played a decisive role. From these ancient fermentations to the current
monitored industrial processes, winegrowers and winemakers have been continuously
changing their practices according to scientific knowledge and advances. A new
enology direction is emerging and aiming to blend the complexity of spontaneous
fermentations with industrial safety of monitored fermentations. In this context, wines
with distinctive autochthonous peculiarities have a great acceptance among consumers,
causing important economic returns. The concept of terroir, far from being a rural
term, conceals a wide range of analytical parameters that are the basis of the
knowledge-based enology trend. In this sense, the biological aspect of soils has
been underestimated for years, when actually it contains a great microbial diversity.
This soil-associated microbiota has been described as determinant, not only for the
chemistry and nutritional properties of soils, but also for health, yield, and quality of
the grapevine. Additionally, recent works describe the soil microbiome as the reservoir
of the grapevine associated microbiota, and as a contributor to the final sensory
properties of wines. To understand the crucial roles of microorganisms on the entire wine
making process, we must understand their ecological niches, population dynamics, and
relationships between ‘microbiome- vine health’ and ‘microbiome-wine metabolome.’
These are critical steps for designing precision enology practices. For that purpose,
current metagenomic techniques are expanding from laboratories, to the food industry.
This review focuses on the current knowledge about vine and wine microbiomes, with
emphasis on their biological roles and the technical basis of next-generation sequencing
pipelines. An overview of molecular and informatics tools is included and new directions
are proposed, highlighting the importance of –omics technologies in wine research and
Keywords: NGS, wine microbiome, vine health, soil microbiome, metagenomic analysis, bioinformatic tools and
databases, 16S rRNA gene sequencing
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Wine is a product with high sociocultural interest. In particular,
wines with distinctive autochthonous properties have a great
demand among consumers and collectors, causing important
economic consequences. It is well known that physical (climate)
and biological factors (soil, grape variety and fauna), as well as
viticulture and enological techniques work together to determine
the sensory-characteristics of a wine from a particular region,
establishing the concept of terroir. In this sense it should be
noted that, apart from these factors, recent studies highlight the
contribution of the native vine microbiota in the winemaking
process of wines from a particular region (Knight et al., 2015;
Bokulich et al., 2016). Additionally, results from Burns et al.
(2016),Grangeteau et al. (2017) correlate human-agronomical
practices in vineyards with the soil and grape microbiota and, also
with its later behavior at cellar, reinforcing the interdependence
between the anthropogenic and microbiological basis of terroir.
Microbes transform plant products into socio-economically
important products and fermented beverages, such as wine,
which is an extremely important sector for several countries. For
instance, the International Organization of Wine and Vine (OIV)
estimated in 2015 that the global wine-growing surface area was
7,534,000 hectares, with the biggest producer being Italy (18% of
the global total), followed by France (17.3%) and Spain (13.5%).
Outside the EU, the USA has the highest wine production
followed by Argentina, Chile and Australia (OIV, 2015).
Due to the economic importance of the grapevine, this
crop has received considerable interest among researchers;
although this attention mainly focuses on the plant genome
and transcriptome/metabolome to better understand how the
plant responds to the physical environment, abiotic stresses and
diseases (e.g., the International Grape Genome Program, IGGP).
However, plants cannot be considered a self-contained, isolated
organism, as plant fitness is a consequence of the plant per se and
its associated microbiota (Vandenkoornhuyse et al., 2015). Thus,
a more holistic conception should include plant-microorganisms
and microbe-microbe interactions.
Although the role of microorganisms at cellar stages has
been well investigated, the biological aspect of soils has not
received similar attention, when actually it contains a great
microbial diversity with important roles in plant nutrition and
health (Compant et al., 2010;Bhattacharyya and Jha, 2012).
Next-generation sequencing (NGS) approaches have uncovered
a higher than expected microbial diversity in both vine and
wine and discovering new microbial species, some with unknown
contributions to the organoleptic properties of wines (Bokulich
et al., 2016). Stable differences among microbial populations of
grape musts have been attributed to grape variety, geographical
area, climatic factors and vine and grape health, leading to the
concept of vine microbial terroir (Bokulich et al., 2014). This fact
has been reinforced at a phenotype-metabolome level by other
works such as Knight et al. (2015),Bokulich et al. (2016), and
Belda et al. (2016). The later observed distinctive and clustered
metabolic profiles (production of hydrolytic enzymes) for yeast
strains depending on their geographical origin. It has been also
observed that the origin of these microorganisms in musts is
the microbial consortia of grapes, with the original reservoir of
these microorganisms being vineyard soil (Zarraonaindia et al.,
2015). Thus, the microbiological aspects of wine production
are influenced by the vineyard and not just by the winery and
fermentative processes.
The maturation of grapes is a complex process that depends
on numerous factors (Kennedy, 2002). Traditionally, the most
common measured parameters include: sugar concentration,
acidity and aromatic and phenolic maturity. However, soil and
grape microbiological complexity throughout the cycle of the
vine and grape maturation is rarely taken into consideration.
Communities of microorganisms (fungi, yeast and bacteria)
associated with the vineyard play an important role in soil
productivity as well as disease resistance developed by the vine.
It is important to understand the microbial consortia associated
with particular diseases, such as Esca,Eutypa,Botryosphaeria, and
Phomopsis diebacks, and also the dynamics of infection processes
in order to take preventive actions, especially at the most critical
moments (Figure 1). For instance, microbial insights are crucial
for defining strategies for the preparation of new plantings. At
this stage, it could be interesting to improve the microbiological
conditions of the soil by bioremediation and to avoid risk of cross
infection during pruning (Bertsch et al., 2013;Fontaine et al.,
The diversity and number of microorganisms that are
able to establish in an ecological niche in the soil and on
the vine will determine both the grapes’ health and the
variability of microorganisms that will be introduced in the
winery that further affect the fermentation processes and
wine maturation (Barata et al., 2012). Thus, with adequately
managed microbiome information, it could be possible to prevent
fermentation problems, volatile acidity increases, Brettanomyces
contamination and biogenic amines production. Knowing more
about the microbiological conditions of the vineyard allows the
winegrower to think about the reduction of chemical treatments
and performing them only when they are objectively necessary.
Additionally, this knowledge would help the winemaker to
use lower sulfur concentration at cellar stages and even
to decide the type of yeast and dose to be inoculated if
and when necessary (Figure 1). This is valued information
especially considering new enology trends, such as organic
Next-generation sequencing technologies enable the detection
and quantification of microorganisms present in vineyard soil,
grapes, as well as its transformation later in winery. The impact of
the microbiological component of terroir and how it contributes
not only to its quality but also in the organoleptic features
of the wine is considerable. This impact also contributes to
the sensory regional distinctiveness and the wine style of the
winery that currently plays an important role in differentiation
and competitiveness in the worldwide market. If something can
distinguish one vineyard from another, among other factors, it
certainly is its microbial community. In this context, the objective
of this review is to summarize the current knowledge about
the role of microbial communities in viniculture, highlighting
the contributions of NGS technologies and identifying new
scientific-industrial frontiers.
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FIGURE 1 | Current challenges on viticulture and enology assumable by NGS approaches; advisable technical improvements; necessities and
perspectives in data science. NFB means ‘Nitrogen Fixing Bacteria’ and PMB means ‘Phosphate Mobilizing Bacteria’.
Plants host a variety of microorganisms (fungi, yeast, and
bacteria) on and inside organs and their surrounding soil. Among
these inhabitants are both harmful and beneficial microbes
that are involved in crucial functions such as plant nutrition
and plant resistance to biotic and abiotic stresses, hence in
plant growth promotion, fruit yield, disease resistance and
survival (Lugtenberg and Kamilova, 2009;Compant et al., 2010;
Bhattacharyya and Jha, 2012).
Studies on microorganisms associated with grapevines have
been centered on the cultivable fungi (mainly yeast) or bacteria
that can have a negative economic impact, compromising
the yield and quality of the grapevine, as well as wine
production. Studies have focused on disease causing pathogens
(Agrobacterium vitis, Xylella fastidiosa, Erysiphe necator,
Phomopsis viticola, Fusarium spp., etc.) and microorganisms of
enological interest. The later species have been grouped into three
classes [reviewed in Barata et al. (2012)]: (1) easily controllable
or innocent species, without the ability to spoil wine when good
manufacturing practices are applied; (2) fermenting species
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responsible for sugar and malic acid conversion; and (3) spoilage
sensu stricto species responsible for wine alteration The most
widely known cultivable bacteria are acetic acid bacteria (AAB;
e.g., Acetobacter and Gluconacetobacter) and lactic acid bacteria
(LAB; e.g., Lactobacillus, Oenococcus, and Pediococcus). Among
yeasts, Saccharomyces members have attracted most of the
attention as they are the main fermentation agents commonly
used as inocula (e.g., Saccharomyces cerevisiae, S. bayanus, S.
pastorianus, and S. paradoxus among others), while other genera
are the most frequent wine spoilers (e.g., Brettanomyces/Dekkera,
Issatchenkia, Zygoascus, and Zygosaccharomyces).
While culture dependent methods have been useful to
detect and identify microbial organisms associated with
grapevine and grape products, and also to study in vitro
their metabolic properties (Belda et al., 2016), they have
led to a rather biased picture of the microbial community.
These methods neglect the larger, non-culturable fraction
that is believed to be as high as the 95–99% of the
microorganisms present (Amann et al., 1995;Curtis, 2002).
In wine environment, due to the stressful environment
associated to the addition of SO2, high ethanol concentration,
etc., a fraction of the bacteria and yeast enter in a Viable But
Non-Culturable state (VBNC) (Millet and Lonvaud-Funel, 2000;
Divol and Lonvaud-Funel, 2005). At this state cells do not
grow on culture media, however, they are still viable and
maintain a detectable metabolic activity (Yamamoto, 2000)
which may affect fermentation performance as well as flavor.
Examples of such microorganisms include Candida stellata,
Brettanomyces bruxellensis, S. cerevisiae, Zygosaccharomyces
bailii, etc. (Salma et al., 2013). Thus, in order to reach to these
VBNC microbiologists were driven to develop alternative
culture-independent techniques. Particularly, quantitative real
time PCR (qPCR) has been widely used to detect bacteria and
yeast considered to be wine spoilers and that have VBNC strains
responsible for the production of off-flavors or having a negative
impact on wine, e.g., Brettanomyces spp. (Tofalo et al., 2012).
Nowadays, qPCR is believed to be a rapid diagnostic tool to
detect the presence and quantify the abundance of particular
microorganisms of interest, however, when the objective is
not a targeted species, but rather a whole community analysis,
PCR-DGGE has been the classical method of choice. The later
technique is adequate to approximate the total community
profile and for comparative community structure analysis, but
it has several drawbacks mainly associated to biases related
with species richness estimates and its low sensitivity to detect
low abundance species (Neilson et al., 2013). For instance,
multiple bands could associate with single isolates. In addition,
multiple sequences might be associated with a single band
and preferential amplification biases between phylogenetically
diverse members of the community have been shown (Neilson
et al., 2013). Andorrà et al. (2010) compared the population
dynamics of microorganisms of grape must fermentation by
three culture independent techniques (DGGE, direct cloning of
amplified DNA, and qPCR) with plate counting, and evidenced
that the biodiversity observed in the must and at the beginning
of fermentation was much higher when DGGE or direct cloning
were used. However, the predominance of certain yeast such as
C. zemplinina and S. cerevisiae during fermentation limited the
detection of low abundant species. Thus, while DGGE is believed
to give a quick and non-expensive view of the community, it
skews microbial diversity estimates (David et al., 2014) and
it has a limited use to study diverse environmental samples
dominated by few species (Andorrà et al., 2010). When adding
NGS technique into the detectability comparition of culture
independent techniques to study yeast community in must
and ferments, the studies evidenced that larger numbers of
yeast species were detectable by NGS than by PCR-ITS-RFLP
or DGGE in grape samples. Moreover NGS detected species
in ferment samples that were undetectable with the two later
techniques (David et al., 2014). In addition, Wang et al. (2015)
analyzed Carignan and Granache grape must and fermentation
from three vineyards in Priorat (Spain) and found that NGS
detected all the species identified by the rest of methods (DGGE,
qPCR and culture dependent), whereas DGGE could just detect
the dominant species of non-Saccharomycetes class. Thus, NGS
showed to be more appropriate to understand must and wine
environment yeast communities (David et al., 2014;Wang et al.,
Next-generation sequencing technologies are providing a
powerful approach to achieve a more complete understanding of
the complexities of microbial communities and their impact on
plant growth, disease resistance/susceptibility, climate adaptation
and environmental remediation. This technology is enabling
researchers to simultaneously obtain information on thousands
of taxa as opposed to targeted approaches that detect only a
taxonomically predefined group. Thus, metagenomics coupled
with new bioinformatics tools, is allowing performance of more
complex multifactorial analyses and is becoming a powerful
strategy in diagnostics, monitoring, and traceability of products.
Its application in viticulture while recent is promising (Table 1),
as accumulating data suggest that there is a much higher
microbial diversity associated both with the plant (Leveau and
Tech, 2010;Pinto et al., 2014;Zarraonaindia et al., 2015) and
the fermentation process (Bokulich et al., 2012;Piao et al., 2015;
Pinto et al., 2015;Portillo and Mas, 2016;Stefanini et al., 2016)
compared to previous culture based studies. Most metagenomics
research in this field has focused on microbial monitoring during
fermentation to obtain a detailed description of the relevant
microbial populations associated with grape and must that might
lead to wine spoilage, an advance highly valuable for winemaking.
These NGS-enabled studies reflect a wider range of bacteria,
besides the commonly detected LAB and acetic acid species,
able to persist in fermenting musts of various grape varieties
(Bokulich et al., 2012;Piao et al., 2015;Portillo and Mas, 2016;
Stefanini et al., 2016). For instance, the first wine-related study
conducted in the wine environment with NGS was conducted by
Bokulich et al. (2012) during botrytized wine fermentation using
16S rRNA gene amplicon sequencing. These authors showed an
array of fluctuating low abundant taxa not traditionally associated
with wine, as well as atypical LAB communities during the
process. Similarly, results from Portillo and Mas (2016) suggested
that AAB are more abundant and dynamic than previously
thought during low or unsulfited wine fermentations, and seemed
to be independent of the grape variety. Interestingly, in this
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TABLE 1 | Research and industrial hallmarks of viticulture and enology led
by NGS approaches.
Enological features addressed by
microbiome study
Interference of microorganisms in plant
Lugtenberg and Kamilova, 2009;
Compant et al., 2010;
Bhattacharyya and Jha, 2012;
Martins et al., 2013;
Vandenkoornhuyse et al., 2015
Microbial diversity in vineyard Leveau and Tech, 2010;
Pinto et al., 2014;
Zarraonaindia et al., 2015
Microbial diversity in wine fermentations Bokulich et al., 2012;
Piao et al., 2015;
Pinto et al., 2015;
Portillo and Mas, 2016;
Stefanini et al., 2016
Anthropogenic-agronomical practices
determining vineyard and wine
Burns et al., 2016;
Grangeteau et al., 2017
Microbial contribution to wine chemistry Verginer et al., 2010;
Bokulich et al., 2016
Terroir markers (microbial zoning) Bokulich et al., 2014, 2016;
Burns et al., 2015;
Knight et al., 2015
study yeast diversity and dynamics during wine fermentation
was assessed in addition to bacteria, evidencing that the genera
Hanseniaspora and Candida were dominant during the initial
and mid- spontaneous fermentation of Grenache grapes while
certain Candida and Saccharomyces species predominated at the
end of the fermentation. Other studies have demonstrated how
different fermentation techniques (spontaneous vs. inoculated)
affect the microbial community composition and its succession
during fermentation (Piao et al., 2015), and also how the previous
agronomical practices in the vineyard could play a critical
role in these population dynamics (Grangeteau et al., 2017).
These authors’ results indicated certain phyla are associated
with each particular technique. Interestingly, they observed that
Gluconobacter experienced a notable increase during organic
fermentation, which led the authors to conclude that this might
explain the increased susceptibility to wine spoilage in wines
produced using that technique.
These above-mentioned studies enhance our understanding
of microbial diversity during fermentation and allow the
identification microbial contamination sources. However, as
DNA sequencing approaches detects living as well as dead
microorganisms, it is still not clear to what extent these
microorganisms metabolically are active and capable of affecting
organoleptic properties of wine. The role of the microbiota
influencing the flavor, color and quality of wine, under a systems
biology perspective, remained elusive until recently.
While soil, weather, farming techniques and grape variety
contribute to the unique qualities of wine, adding distinctiveness
and thus market value, the contribution of the microbiota in
defining terroir is now in the spotlight of scientific research.
Regionally distinct wines are highly appreciated by consumers
and add value to the industry. In Spain alone there are
90 zones, which produce distinct so-called PDO wines, of
which 69 are Denomination of Origin (DO), 2 are Qualified
Denomination of Origin (DOCa), 7 are Quality Wine with a
Geographical Indication (Vino de Calidad) and 14 are Single
Estate Wine (Vino de Pago). While the chemosensory distinction
of wines from different growing regions has been previously
established [e.g., Loópez-Rituerto et al. (2012)], indigenous
microorganisms associated with grapes were shown to be able
to produce compounds responsible for the regional flavors
of the resulting wine, e.g., VOCs (Verginer et al., 2010). In
addition, Knight et al. (2015) experimentally demonstrated that
wine organoleptic characteristics are affected by the origin and
genetics of wild S. cerevisiae natural strains, providing objective
evidence for a microbial aspect to terroir.Bokulich et al.
(2014) showed that Cabernet Sauvignon must from different
growing regions in California could be distinguished based
on the abundance of several key fungal and bacterial taxa.
This differential must microbiota could potentially influence
wine properties and contribute to the regionalization of wine.
The later was further proved in Bokulich et al. (2016); these
authors demonstrated that both grape microbiota and wine
metabolite profiles were able to distinguish viticultural area
designations and individual vineyards within Napa and Sonoma
Counties in CA, USA. Interestingly, the vineyard microbiota
correlated with the chemical composition of the finished wines,
hinting at the possibility of predicting wine phenotypes prior to
fermentation. Nevertheless, wine aroma is defined by hundreds of
chemical compounds with different natures (i.e., higher alcohols,
esters, fatty acids, terpenes, thiols) causing a broad spectrum of
sensory thresholds, and also suffering synergies and antagonisms
(Belda et al., 2017). Thus, looking for microbial signatures
determining wine typicity, the sensorial characterization of wines
should consider not only chromatographic analysis (revealing
the diversity and concentration of aroma compounds), but also
developing serious sensorial or olfactometry analysis to reflect
the real perception of wine aroma or, at least, considering odor
activity values (OAVs) to correlate the real influence of microbial
species in wine aroma, as was addressed by Knight et al. (2015).
While grape and must have been more heavily researched,
Zarraonaindia et al. (2015) further hypothesized that the soil
and its associated microbiota influences wine characteristics.
First, per these authors’ studies, the aboveground bacterial
community was significantly influenced by soil edaphic factors
such as total carbon, moisture and soil temperature, which would
ultimately impact the quality of grapes due to changes in nutrient
availability for the plant. Second, soil bacterial communities
differed between the sampled vineyards in Long Island, New York
and those differences were reflected in the microbial composition
in vine roots. These root endophytes can shape the microbial
assemblages of aboveground organs by changing the endophytic
microbial loads in grapes. Third, a significant input of soil
microorganisms to grapes through epiphytic migration during
harvest was suggested. The later was also evidenced by Martins
et al. (2013), leading Zarraonaindia et al. (2015) to propose
that soil derived microorganisms could have a greater role than
previously anticipated in wine, as they will ultimately end up
in the fermentation tanks. The link between soil microbiota
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and terroir was further evidenced by Burns et al. (2015) who
identified distinctive microbial community profiles by American
Viticultural Areas (AVA).
The above summarized studies were conducted on grapevines
and wine and address microbial composition by means
of 16s rDNA PCR amplicon and ITS (Internal Transcribed
spacer) NGS sequencing for elucidating the bacterial and fungi
community, respectively. This marker gene amplification and
sequencing method, also called amplicon sequencing, has become
the method of choice to simultaneously detect multiple species
in must and wine environment since 2012 (see Bokulich et al.,
2012, 2016;Pinto et al., 2014, 2015;Knight et al., 2015, among
others). However, the particular experimental question of the
research to be conducted will determine the method mostly
suited to answer to the question. For instance, if the goal is to
track a particular microbial strain or genus from soil to must
to fermentation, then qPCR could be a more appropriate and
has the added benefit of absolute quantification (Neeley et al.,
2005). To detect a specific microbe, primers must be designed to
be highly specific for the microbe of interest. Often the primer
design can be completed by genome comparison of targeted and
non-targeted strains to find a unique gene or region. Another
strategy involves targeting a conserved gene (16S rRNA, gyrB,
rpoB) and making sure the primers mismatch off-target strains
particularly at the 30end. Single copy genes provide an added
bonus for absolute quantification. Microbe quantification by
qPCR, however, does not scale easily if the goal is to analyze
more than a few strains while amplicon sequencing is suited to
determine the community.
However, amplicon sequencing is not free of pitfalls,
and different biases have been described in multiple steps
of the process; First, DNA extraction method is one of the
key and limiting steps for metagenomic analysis by NGS.
Various approaches have been applied for environmental
DNA extraction, including freeze–thaw lysis (Herrick et al.,
1993), bead beating (Miller et al., 1999;Courtois et al., 2001;
Urakawa et al., 2010;Petric et al., 2011), liquid nitrogen grinding
(Ranjard et al., 1998), ultrasonication (Picard et al., 1992), hot
detergent treatment (Holben, 1994), use of strong chaotropic
agents like guanidinium salts (Porteous et al., 1997), and high
concentration of lysozyme treatment (Hilger and Myrold, 1991).
Furthermore, soil, grapes and wine are complex physicochemical
environmental samples that contain many interfering agents for
molecular analysis such as impurities, phenols, humic acid, fulvic
acid, metal ions and salts, and therefore additional purification
steps are necessary which can introduce bias by altering the
original community (e.g., a fraction of the community might
be lost through purification, etc.). There are several commercial
kits that could be used to fasten the process, however, the
selection of the best DNA extraction method and kit is not
straightforward as different DNA extraction methods can
produce different results (Keisam et al., 2016). Unfortunately,
there is no “gold standard” for DNA extraction method and one
should be selected on a case-by-case basis considering the aims,
specimens of the study and scalability (including simplicity,
cost effectiveness, and short handling time) and intended study
comparisons. An additional problem is the introduction of
contaminating microbial DNA during sample preparation.
Possible sources of DNA contamination include molecular
biology grade water, PCR reagents and DNA extraction kits
themselves. Contaminating sequences matching water-and
soil-associated bacterial genera including Acinetobacter,
Alcaligenes, Bacillus, Bradyrhizobium, Herbaspirillum, Legionella,
Leifsonia, Mesorhizobium, Methylobacterium, Microbacterium,
Novosphingobium, Pseudomonas, Ralstonia, Sphingomonas,
Stenotrophomonas, and Xanthomonas have been reported
previously. The presence of contaminating DNA is a particular
challenge for researchers working with samples containing a low
microbial biomass. In these cases, the low amount of starting
material may be effectively swamped by the contaminating DNA
and generate misleading results (Salter et al., 2014).
Second, DNA library preparation, based on fragment
amplification through PCR with barcoded primers, is another
step in which it is possible to introduce additional biases.
The choice of primers and targeted variable regions will bias
identification and quantification (Soergel et al., 2012;Bokulich
and Mills, 2013). Additionally, in any PCR- and primer-based
taxonomic investigation, members of a microbial community
may be omitted, distorted, and/or misrepresented, typically
due to primer mismatches or PCR biases (Acinas et al., 2005;
Hong et al., 2009;Lee et al., 2012;Pinto and Raskin, 2012;
Logares et al., 2014). On the contrary, primers might show
variability in their amplification efficiency by for example,
favoring certain species amplification (Baker et al., 2003;
Sipos et al., 2007;Klindworth et al., 2013). This preferential
amplification is thought to be derived from different sources
such as primer mismatches, the annealing temperature and PCR
cycle numbers (Sipos et al., 2007). For instance, Sipos et al.
(2007)’ studies evidenced that A. hydrophila and P. fluorescens
were preferentially amplified over both Bacillus strains when
the 63F primer was used (which contained three mismatches
against DNA isolated from the Bacillus strains), while the 27F
primer amplified all templates without bias. Interestingly, the bias
introduced by primer mismatches was reduced at lower annealing
Multiple primer pairs are available for marker genes, and
each pair is associated with its own taxon biases. Marker
gene databases are frequently updated, and the updated
information can include new microbial lineages with suboptimal
or poor binding to existing PCR primers; to maximize
taxonomic sensitivity in light of these new data, primers
may need to be periodically redesigned. A recent example in
the literature is the modification of the most common 16S
primers used 515f and 806r to remove know biases against
Crenarchaeota/Thaumarchaeota and the marine and freshwater
Alphaproteobacterial clade SAR11 (Apprill et al., 2015;Parada
et al., 2016).
Klindworth et al. (2013) evaluated the coverage and phylum
spectrum for bacteria and archaea of 175 primers and
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512 primer pairs in silico for three amplicon size classes
(100–400, 400–1000, >1000 bp), demonstrating the differences
in coverage and specificity among the studied primers. Besides,
this information represents a valuable guideline for selecting
primer pairs that could minimize the bias in PCR-based microbial
diversity studies. In the same way, probeBase1is an additional
online resource, providing the opportunity to evaluate the in
silico hybridization performance of oligonucleotides, as well as
finding suitable hierarchical probes that could target an organism
or taxon of interest at different taxonomic levels (Greuter et al.,
The ideal marker gene should have conserved regions that
flank variable regions. The conserved regions allow primer design
to amplify multiple taxons at ones. Ribosomal rRNA genes fit
this description and have been widely used for identification
of bacteria/archaea (16S) and fungi (ITS) (Gilbert et al., 2010).
However, ribosomal RNA genes show copy-number variation,
with very disparate number of copies per taxa (from one in
many species to up to 15 in some bacteria and to hundreds in
some microbial eukaryotes) biasing conclusions related to the
abundance of the organisms.
To evaluate the entire microbial community in the specific
case of the wine ecosystem, it is necessary to strike an
appropriate balance between amplifying all members of every
taxon (high coverage) and obtaining the highest taxonomic
resolution possible, e.g., to be able to discriminate among closely
related species (Figure 1). Each marker shows differences in its
discrimination power at intra-genera as well as at intra-species
level. Thus researchers must have that in mind when designing
their project, in order to choose the most appropriate molecular
marker to answer their particular question/s. For instance, primer
pair 515f/806r is the most widely used for targeting the V4
region of for bacteria/archaea (Parada et al., 2016), and this
combined with Illumina sequencing has been used to characterize
the microbiomes of numerous environments (Caporaso et al.,
2012), vine and wine environments among them. Data from
high diverse environments, as Sakinaw Lake, showed species
resolution level from 49.4% of the 16S V4 sequences classified
compare with 74.5% using full 16S. Although the relative
classification differences at the sequence level do not directly
translate to differences in community representation (Singer
et al., 2016). However, vine and wine samples have the added
difficulty in that mitochondrial and chloroplast DNA can be
amplified with these V4 region primers and thus grapevine
plastid sequences overwhelm the sequencing. Researcher have
two ways to avoid this problem: design primers that mismatch
mitochondrial/chloroplast sequences or add blocking reagents
that bind these sequences (Lundberg et al., 2013). Besides, the
V4 domain of the 16s rDNA gene is considered to be the most
suitable marker for capturing the bacterial community in wine,
as it is able to reliably discriminate LAB to genus-level (Bokulich
et al., 2012). However, in fermentative systems, some species of
LAB are considered wine spoilers while others exhibit malolactic
activity, thus it might be essential to reach to species level
(Bokulich and Mills, 2012) and/or strain level, in order to have
a more comprehensive view of the community. Unfortunately,
currently available amplicon sequencing markers are unable to
capture that level of resolution in all taxa. These limitations could
be overcome by combining several techniques such as genera
specific T-RFLP or qPCR and amplicon sequencing.
Third, important sources of artifacts are also derived from
the High-throughput sequencing technology chosen. While
pyrosequencing introduces homopolymer errors (indel error),
Illumina sequencing has average substitution errors at 0,0086
sequencing rate (Schirmer et al., 2015). Sequencing platforms
also show a disparity in sequencing depth (number of reads per
run) and read length. Illumina MiSeq is the most commonly
used sequencer for amplicon sequencing due to its high coverage
with a total nucleotide sequenced of 15GB allowing sequencing
the abundant and rare community giving a deep view of
the community composition. However, llumina sequencing is
characterized by a short variable region sequencing (2 ×300 bp
vs. 700 bp in 454). Currently, nearly full-length rRNA gene
sequencing is possible with PacBio and Nanopore technologies
(Benitez-Paez et al., 2016;Schloss et al., 2016).
Finally, one of the biggest limitation of amplicon sequencing
techniques relays on its inability to address a functional
characterization of the microbial communities. There are many
desired microbial functions in winemaking, mainly related to
alcoholic and malolactic fermentations, and diversity of genes
related to those functions may influence winemaking more than
just taxonomic diversity. In addition, closely related strains with
highly similar 16S rRNA gene or ITS sequences contain different
fermentation-related genes (Knight et al., 2015) and thus that
strain diversity remains hidden in current amplicon sequencing
studies. Single-cell genomics emerges as a potential strategy
that could help to obtain a deeper knowledge into species-
strain level diversity. This strategy is powerful when the targeted
organism is dominant or high abundant in a low species richness
ecosystem. However, in highly diverse ecosystems or when the
species to be targeted is low abundance, it may require a higher
sorting throughput, specific labeling with fluorescent probes or a
previous cultivation step, all of which could contribute to biases.
Alternatively, shotgun metagenomic sequencing would also
reveal functional genes in addition to rRNA genes, allowing a
more comprehensive genomic and functional representation
through whole-genome sequencing (WGS) of complete
communities, but the cost and the number of reads needed
to estimate the environmental population is high compared
to PCR-based approaches. Even more in wine samples, as a
very deep sequencing is required to detect microbes due to
an overabundance of plant DNA (Zarraonaindia et al., 2015),
making this method costly for a large number of samples.
Metatranscriptomics is emerging as a powerful technology
for the functional characterization of microbial communities
that can reveal both the taxonomic composition and active
biochemical functions of the detected organisms. These approach
is of especial interest in wine environment, as amplicon
sequencing is not able to discriminate among living or dead
organisms, nor the metabolically active or inactive organisms.
However, the high sequencing depth needed and the high cost
associated with the sequencing of each sample limits the number
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of samples that could be surveyed within a project currently.
In addition, challenges associated with this technique include
among others, the lack of established reference genomes to
annotate the short reads generated in the sequencing and the high
computational effort needed for the analyzes. Being a technique
still in its infancy, new analysis tools and standardized pipelines
are under development. In this context, the next section aims
to summarize critical concepts and sources of biases in NGS
Along with the relative ease with which thousands of organisms
can be detected in samples via 16S/ITS sequencing, a whole host
of bioinformatics approaches have been developed to extract
meaningful results from the large datasets that are generated.
The bioinformatics challenge comes in at least two parts
(I) preprocessing the datasets into a collection of representative
reads (or operational taxonomic unit – OTU) that can be
associated with databases of known species and (II) associating
the collection of species inferred in a sample (known and newly
detected) with properties of the sample in order to study the
relationships between the microbiome and the terroir.
In the first stage, the large amounts of raw sequencing reads
are processed (trimming adaptor sequences, merging forward
and reverse sequences, filtering on read quality) before finally
being dereplicated into a collection of unique sequences. There
is a lot of software available to perform these tasks and they
often are part of packages that offer an entire processing pipeline
(USEARCH, vsearch, FASTX-Toolkit) (Edgar, 2013;Rognes
et al., 2016). The unique sequences are then clustered according
to sequence similarity, choosing a relatively arbitrary cutoff at
97% identity (Seguritan and Rohwer, 2001), resulting in a set of
OTUs that are each assumed to be originating from a specific
organism. In other words, OTUs are proxies for microbial species
in the sample (Schloss et al., 2009;Caporaso et al., 2010).
Although conceptually simple, this step poses major
challenges both computationally and in terms of biases that
might potentially bleed into subsequent analysis. First of all, for
large sets of sequences, all against all pairwise alignments would
be prohibitive, e.g., 1 million of unique sequences (commonly
encountered), would require 1000 billion pairwise comparisons.
This has led to comprehensive bioinformatics pipelines for
OTU clustering, including the software pipelines mentioned
above (USEARCH, vsearch, swarm), which all rely on clever
heuristics (Edgar, 2013;Eren et al., 2013;Mahé et al., 2014, 2015;
Tikhonov et al., 2015;Rognes et al., 2016) in order to accelerate
this process at the expense of perfectly accurate clustering. The
second challenge is to avoid biases that can occur during OTU
clustering. The biases can be multifold; (a) different biological
species might have the same sequence and therefore be grouped
into one set, (b) sequencing errors or amplification errors
(including chimeric reads) or untrimmed sequences can group
sequences that have the same origin into separate groups. The
first issue will underestimate biological diversity whereas the
latter will overestimate it. Together these scenarios will corrupt
the accurate representation of the real biological makeup of the
terroir. This highlight again that it is important to quality trim
and filter the raw sequences to minimize the risk of including
artifacts in environmental data sets.
Finally, the curated OTUs are subjected to phylogenetic
assignment, which aims to identify what species or genus an
OTU most likely belongs to. This is achieved by comparing
them with taxonomically classified sequences at databases, such
as GreenGenes (for bacteria community characterization), SILVA
(bacteria and eucaryotes) and Unite (for Fungi) among others.
Again, a range of software is available (Qiime, UTAX, SINTAX,
stampa) (Caporaso et al., 2010;Edgar, 2013, 2016). This stage
is again a source of biases, partly because OTUs can represent
multiple species, there is ambiguous assignment, and because too
small differences that do exist could be ignored by these methods.
For instance, in the case of Oligotyping, a single base pair can
differentiate ecological strains (Eren et al., 2013). Furthermore,
and more generally, reference databases are themselves based
largely on predicted species rather than experimentally cultivated
species and can thus bias taxonomic assignment. Additionally,
different reference databases would yield different taxonomic
assignments as a function of completeness and quality of the
database (McDonald et al., 2012). Notably, if a given species
is not represented within the database, sequences derived from
that species would receive an incorrect assignment or remain
unclassified. This is aggravated for wine and soil associated
microbial sequences field, where reference databases lag behind
human-associated microbes. Increasing and curating robust
databases is a key goal for the scientific community (Figure 1).
There are also other methods allowing comparison of amplicons
derived from functional genes in which we might not know
percent identities that correspond to taxonomic levels, but
in some cases, are optimal to reflect geographical (and thus,
environmental) distance (Haggerty and Dinsdale, 2017). In
relation to wine related samples, cluster free methods show the
potential to define the microbial terroir at the strain or sub-OTU
level (Tikhonov et al., 2015;Eren et al., 2016).
Equipped with a dataset of biological entities in the terroir
(genus- or species-level), the second bioinformatics challenge
concerns associating the microbiome to the properties of the
terroir. Depending on the aim, this can be more or less
difficult. One goal is to use microbial community data to
classify soil samples into types and geographical regions and,
therefore, define the microbial terroir. Recently, Bokulich et al.
(2016) demonstrated the power of this approach for classifying
Californian regions and fermentation metabolites based on
microbial abundances in musts. However, if species or even
strain information is required to establish an association between
microbiome and specific wine making properties, then the
taxonomic assignment is essential and can make or break an
analysis depending on the resolution it achieves and the biases
it can prevent.
Apart from the nature of the question, generally, the structure
of OTU abundance data poses some challenges that need to
be carefully taken into account. Because the species can occur
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in very different abundances (often spanning several orders of
magnitude), the collection of species across samples can greatly
vary. This leads to a very sparse dataset, which is defined as
a dataset with many zero values. These zero values can be
problematic as they could entertain multiple hypotheses; for
instance, a zero count in a sample could be because a species
is not present, or because it just has not been detected. This
can lead to biased comparisons between samples. One way to
deal with this is to use distance metrics that do not consider
these situation (e.g., Bray-Curtis) or that specifically include
a phylogenetic tree that allows to relate species information
into meaningful groups. Preprocessing of OTU data from raw
counts to a value that makes samples comparable to each other
is the next step. This is also referred to as normalization and
there are number of analytical choices available (Segata et al.,
2011;Paulson et al., 2013) depending on whether low-abundance
species or high abundance species should have more of an
impact in the analysis inquestion. For instance, counts can be
converted to frequencies (divide the number of reads by the
total number of reads in the sample). The performance of
these techniques given OTU table peculiarities has been tested
elsewhere (McMurdie and Holmes, 2014;Weiss et al., 2016).
This is also a crucial step when applying machine learning
With a preprocessed dataset available, the probe community
level differences between samples, can be studied with supervised
and unsupervised machine learning techniques. Unsupervised
learning categorizes samples based on OTU abundances without
prior knowledge of the sample phenotypes. Principle component
analysis (PCA or more commonly PCoA) and clustering
algorithms can be used to gain a high level view over
differences in samples. These analyses are largely exploratory
and provide visual evidence of community differences. If
information regarding the terroir is available, or there are
some clearly defined groups that are to studied, supervised
learning techniques can be applied to for instance classify new
samples based on past community characterizations (Bokulich
et al., 2016). Distinct wine regions, types and tastes make
wine related samples well-suited for these classification methods
(Statnikov et al., 2013). Different software packages are available
to perform these methods and can be more or less adapted to
the study of metagenimics problems (vegan, phyloseq, Qiime,
Another method to extract knowledge from microbiome data
is to consider it as a network of interactions between individual
strains. Aside from the impact of single strains in plant health
(pathogens, symbionts) and wine characteristics, or spoilage
potential, these strains impact wine production not in isolation
but instead as members of complex microbial communities.
Much research now focuses on these community level effects that
can impact plant phenotypes such as flowering time (Wagner
et al., 2014).
These predictive technologies allow to make initial inferences
about whether these differentially abundant single OTUs cause
certain phenotypes. However, they will requires further testing,
likely with pure culture treatments. One excellent example
of going from correlation to causation is the use of pure
fungal and oomycete cultures in a common garden to confirm
single strain effects on the overall microbial community
structure associated with Arabidopsis thaliana (Agler et al.,
Defining the microbial terroir with bioinformatics is only
an early step to understand how microbes shape each step in
winemaking. Wine imparts its taste and smell via metabolites,
many derived from the grapes and many derived from or
modified by microbes. Identifying which microbes influence
these processes is key to defining how they affect the sensory
profile of wines. As we add genomic sequences to our
reference database we will be able to leverage annotated
sequences to predict metabolic capacity for each microbe.
Genome scale metabolic models (GSMM) combined with flux
balance analysis allows for analysis of metabolic outputs given
a set of inputs (Varma and Palsson, 1994). Furthermore,
GSMMs can expand to community-level models (Zomorrodi
and Maranas, 2012;Khandelwal et al., 2013;Louca and Doebeli,
2015) to uncover how microbes synergistically create complex
wine metabolite profiles. Going forward, it will be critical
not only to define which microbes created your favorite
wine but also how their metabolisms shaped the taste of
that wine. Thus, viticulture will benefit very much from the
generation of commercial platforms that enable studying vine
and wine microbiome and wine metabolome. Currently such
platforms are already in place, with WineSeqR
– (Biome
Makers, Inc.)2allowing wine microbiome characterization
through NGS and Wine ScreenerR
– (Bruker)3allowing
wine metabolome analysis by nuclear magnetic resonance.
These tools are based on robust databases and allow both
producers and regulatory councils from appellations of origins
to establish ‘standard profiles’ for their wines, and better
understand the microbial and chemical bases of their distinctive
In this article, the impact of NGS technologies in vine
and wine microbiology has been reviewed. Regarding the
importance of microbiome in viticulture and enology, the role
of microorganisms in the chemical and nutritional properties
of vineyard soils, crop health and yield, and also in the
later fermentation performance and wine flavor are the main
challenges to explore using –omics tools. For that purpose,
certain technical aspects should be improved at laboratory
stages, such as universal DNA&RNA extraction protocols to
avoid biases, and improved sequencing approaches to increase
microbiome resolution and quantification. It is also important
to develop robust and curated databases to improve taxonomic
assignments (Figure 1). Finally, it is time to develop big data
works, using statistical data-mining and machine learning tools
to solve, in a holistic systems-biology view, the above-mentioned
challenges in wine industry.
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Belda et al. Microbiome in Enology and Viticulture
IB and AA conceived the work. IB, AP, and AA wrote the
“Introduction” section. IB and IZ wrote “The microbiome of
vine and wine: a review” section. AA, IZ, and MP wrote “NGS
microbial profiling: key steps, biases and limitations” section.
IZ and MP wrote “Bioinformatics and predictive methods to
uncover the microbial terroir” section. Finally, IB edited the final
version of the manuscript.
This study was funded by WineSeq Project, BiomeMakers Inc.
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Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.
Copyright © 2017 Belda, Zarraonaindia, Perisin, Palacios and Acedo. This is an
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Frontiers in Microbiology | 12 May 2017 | Volume 8 | Article 821
... Ainsi, de nombreuses évidences ont montré l'importance des microorganismes sur le produit fini (p.ex. Burns et al. 2015;Belda et al. 2017; ...
... Microbiota can then deeply affect the fitness of plants. In the case of grapevines, microorganism composition has been shown to not only influence plant growth and development but also the quality and the quantity of the resulting wine, for instance by shaping the volatile compound profiles in grapes Belda et al., 2017). Microorganisms are recruited from the soil microbial reservoir by the plant to form the rhizosphere from which a fraction of the microorganisms can colonise the inner part of the plant (i.e., the endosphere) . ...
Le concept d’holobionte considère l’unité fonctionnelle composée des plantes et de ces microorganismes. Il promeut une approche holistique à la gestion des cultures et, plus généralement, à la vision du vivant. Cependant, ce concept est sujet à débat en raison du manque de preuves expérimentales de son existence. De plus, la compréhension fine des mécanismes régissant l’assemblage des microorganismes associés aux plantes reste un des enjeux majeurs de l’écologie microbienne et de l’agriculture. Le but de cette thèse était de tester la validité du concept d’holobionte, d’étudier les facteurs qui impactent l’assemblage des communautés microbiennes de l’endosphère racinaire et d’analyser la dynamique intra- et inter-annuelle du microbiote de la vigne. Grâce à une expérimentation mise en place en association avec une pépinière viticole (greffage et plantes chimériques), nous avons pu démontrer l’existence d’un recrutement actif et déterminé de microorganismes par la plante, sous l’effet dominant du porte-greffe, ce qui nous a permis d’augmenter les preuves expérimentales à l’existence du concept d’holobionte. Dans le cadre d’une seconde partie, nous nous sommes intéressés aux dynamiques d’assemblages du microbiote de la vigne, grâce à un vaste plan d’échantillonnage mis en place au sein d’un domaine viticole. Nous avons mis en avant le rôle fondamental des facteurs environnementaux, de l’âge et du cépage dans l’assemblage du microbiote endosphérique racinaire de la vigne à petite échelle géographique et l'existence de patrons temporels intra-annuels marqués dans la structuration de ces communautés. Ces travaux fournissent un ensemble de connaissances nouvelles dans le domaine de l’écologie microbienne, soutiennent l’existence d’un terroir microbiologique et soulignent l’importance de la prise en compte de ce terroir microbien dans le cadre d’une gestion durable de la vigne.
... Enfin, un constat important est le manque de lien entre la qualité du sol et la qualité de la vigne et mê me la qualité du raisin et du vin. Bien que de ré centes publications é mettent des hypothè ses sur ces liens, aucune dé monstration n'a é té faite pour le moment [8] [21] [67]. Ce manque de transversalité peut s'expliquer par la complexité d'une approche multidisciplinaire mais aussi par la né cessité de mieux connaître chaque domaine (é cologie du sol, é cophysiologie et gé né tique de la vigne, oenologie…) avant de les croiser. ...
La filière viticole est associée à des enjeux économiques et à des problématiques environnementales qui nécessitent qu’elle s’engage dans la transition agroécologique de façon urgente. Dans cette transition, les sols et leur biodiversité constituent un levier écologique fondamental en renfort des leviers agronomiques classiques. A cette fin, bien connaître la qualité biologique des sols viticoles et évaluer l’impact des différentes pratiques et modes de production est indispensable. Cet article présente les résultats d’une synthèse et méta-analyse de la littérature scientifique faisant le bilan des connaissances et des lacunes sur la qualité biologique des sols viticoles.
... Además, cada vez hay más evidencia respecto de la activación microbiológica de los suelos debido a la actividad de las lombrices (de Menezes et al., 2018;Medina-Sauza et al., 2019), considerando que es justamente esa actividad microbiológica la principal responsable de los procesos tendientes al ciclo de elementos y secuestro de carbono en los suelos (Castellano et al., 2015;Bender et al., 2016). Especialmente en los sistemas vitícolas, en los cuales la microbiota ejerce diferentes efectos en la calidad del vino (Bokulic et al., 2016;Belda et al., 2017a), en el control de plagas y enfermedades (Pineda et al., 2017;Neher y Barbercheck, 2019) y en la diferenciación de origen o Terroir (Capozzi et al., 2015;Jara et al., 2016;Belda et al., 2017b; Gutiérrez-Gamboa y Moreno -Simunovic, 2019). Es evidente la relación de las variables utilizadas en este estudio, con los procesos o funciones ecológicas de interés para la viticultura, sin embargo, se precisa del continuo desarrollo y mejoramiento de herramientas prácticas de evaluación en el campo, que cuenten con la rigurosidad y coherencia científica. ...
... To overcome this trend, in recent decades, several winemakers have carried out spontaneous fermentations exploiting indigenous yeasts coming from the vineyard or vinery equipment, with the aim to improve the analytical and aroma complexity and to give peculiar and recognizable flavors to the final wine [5,9]. Indeed, recent studies demonstrated that specific grape varieties and the climate of a specific geographical area seem to influence the yeast community [10][11][12][13][14], indicating a variation of the microbial community of grapes in the function of regional distribution [15][16][17][18][19]. Recently, the correlation between the regional microbial community and the organoleptic characteristics of wine has attracted growing attention [11,20,21]. ...
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The relation between regional yeast biota and the organoleptic characteristics of wines has attracted growing attention among winemakers. In this work, the dynamics of a native Saccharomyces cerevisiae population was investigated in an organic winery. In this regard, the occurrence and the persistence of native S. cerevisiae were evaluated in the vineyard and winery and during spontaneous fermentation of two nonconsecutive vintages. From a total of 98 strains, nine different S. cerevisiae biotypes were identified that were distributed through the whole winemaking process, and five of them persisted in both vintages. The results of the oenological characterization of the dominant biotypes (I and II) show a fermentation behavior comparable to that exhibited by three common commercial starter strains, exhibiting specific aromatic profiles. Biotype I was characterized by some fruity aroma compounds, such as isoamyl acetate and ethyl octanoate, while biotype II was differentiated by ethyl hexanoate, nerol, and β-damascenone production also in relation to the fermentation temperature. These results indicate that the specificity of these resident strains should be used as starter cultures to obtain wines with distinctive aromatic profiles.
... Wine originally emerged as a serendipitous mix of chemistry and biology, where microorganisms played a decisive role. From these ancient fermentations to the current monitored industrial processes, winemakers have been continuously changing their practices according to scientific knowledge and advances (Belda et al. 2017). Grape fermentation can be performed by deliberated inoculation of yeast starter or allowing the microbes naturally associated with grapes to conduct the fermentation. ...
Full-text available
With the awakening of consumers' awareness of sustainable development issues and demand for terroir wines, natural wines provide opportunities for the future development of the wine industry. Microbiomes are integral to viticulture and winemaking, where various microorganisms can exert positive and negative effects on grape health and wine quality. Communities of microorganisms associated with the vineyard play an important role in soil productivity as well as disease resistance developed by the vine. Wine is a fermented natural product, and the vineyard serves as a key point of entry for quality-modulating microbiota, particularly in wine fermentations that are conducted without the addition of exogenous yeasts. Thus, the sources and persistence of wine-relevant microbiota in vineyards critically impact its quality. In this review, we first examined that mimicking natural ecological cultivation to improve microbial diversity can enhance vineyard ecological services and reduce external inputs; then we examined that grape berries naturally possess all the elements of winemaking, including the nutrients for microbial growth, driving forces for the microbiota succession, and the enzymatic system for biochemical reactions; finally, we examined food safety, stability, specific interventions, and sustainability of natural wine industry-scale practices.
Syphilis is one of the most exciting diseases explored in paleopathology and, therefore, tracing back its origin and development has provided a prolific debate. The combination of paleopathological data with historical sources, iconography, and archaeological contexts were the primary sources used to reconstruct its historical path. However, there are some limitations to paleopathological diagnosis due to the nature of bone reaction to stimuli. In addition, historical sources are subjected to a bias of social and cultural nature and the knowledge of those who wrote them. Hence, ancient DNA analysis offers the possibility of acquiring proof of cause by identifying pathogens in an organism. We undertook a metagenomic study of a skeleton exhumed from the Royal Hospital of All Saints (Portugal), renowned for treating syphilis from the 16th century onwards. The skeleton had previously been diagnosed with syphilis according to paleopathological analysis. However, the metagenomics analysis showed no presence of the pathogen associated with syphilis (i.e., Treponema pallidum) but revealed pathogenic microorganisms related to respiratory diseases (pneumonia), nonspecific bone infections (osteomyelitis), and oral bacterial pathologies as well as Hansen’s disease (also known as leprosy). The results are exciting and demand a reappraisal of the observed bone changes, recontextualizing their characterization as syphilis related. They prove that past reconstruction of health and disease diagnoses based on assessing human osteological remains of known context (such as a syphilitic hospital) may bias interpretations and, therefore, caution is recommended, not forgetting that the absence of evidence is not evidence of absence (in this case of syphilis) in life.
Wine presents the most distinct geographic signatures among all agricultural products, and these geographic characteristics of the wine are enhanced by the actions of indigenous microorganisms. China is one of the largest wine-producing and consuming nations in the world. The wine-related microbial resources are abundant in China, although their geographic distribution patterns and their contribution to the aroma of wine remain to be elucidated. In the present study, Cabernet Sauvignon samples from four wine-producing regions in China were subjected to high-throughput sequencing and HS-SPME-GC-MS techniques to study the diversity and dynamics of the microorganisms that were present during the spontaneous fermentation process and to provide a preliminary understanding of the contribution of these microorganisms to the volatile components of the wine. The results revealed significant differences in the microbial diversity in the grape musts among different vineyards, which led to significant differences in the composition of the volatile metabolites of the produced wine. Moreover, while the fermentation process was observed to have reshaped the structure of the microbial community, specific characteristics of the vineyard were retained at the completion of the fermentation process. The associations between microbiota diversity and wine chemicals suggested that the dominant species during the fermentation process largely determined the volatile components of the wine. The present study enhances the understanding of Chinese wine terroir and provides a scientific basis for maintaining the regional microbial biodiversity to sustain viticulture and winemaking.
Understanding the diversity and evolution of microorganisms during wine fermentation is essential for controlling its production. However, the flavors profiles associated with microbiota changes during the spontaneous fermentation have not yet been described in detail. In this study, we explored the correlations between microbial community with physicochemical properties and flavor components during the spontaneous fermentation of Cabernet Sauvignon wine. Microbial community diversity at different fermentation stages was identified using high-throughput sequencing, and physicochemical properties and volatile compounds were identified through fermentation features testing and headspace solid phase microextraction gas chromatography mass spectrometry. First, the diversity of the fungi community decreased with the fermentation process, whereas the bacteria did not change significantly until the end of the fermentation. Second, the changes of the fermentation environment had reshaped the diversity and composition of the microbial community. Finally, Aureobasidium, Cladosporium, Filobasidium, Hanseniaspora, Hannaella, Saccharomyces, Alternaria, Wickerhamomyce, Starmerella, Candida, Papiliotrema, Bradyrhizobium, Gluconobacter, Leuconostoclia, Comamonas, Acetobacter, and Massilia, were significantly correlated with changes of physicochemical properties and volatile compounds. Overall, our research results provide important insights for understanding the metabolically active microbiota, which is conducive to the expression of wine "terroir".
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In this study, the evolution of the yeast microflora present on the berry surface, during the ripening of Barbera grapes, was monitored. Sampling was performed in three vineyards located in the “Nizza” Barbera d’Asti DOC zone and different methodologies have been employed. A culture-dependent method based on the identification of strains grown on solid media by ARDRA (Amplified Ribosomal DNA Restriction Analysis) and the D1-D2 domain of ribosomal 26S DNA capillary sequencing was coupled to NGS (Next Generation Sequencing) targeting ITS (Internal Transcribed Sequence) amplicons with the Illumina MiSeq platform. By using culture-dependent techniques, the most frequently detected species was the yeast-like fungus Aureobasidium pullulans, which was dominant in the culturable fraction. Among yeasts, the presence of oligotrophic basidiomycetes such as Cryptococcus spp., Rhodotorula graminis and Sporidiobolus pararoseus was observed at the beginning of ripening. Afterward, upon approaching the harvest, a succession of oxidative or weakly fermentative copiotrophic species occurs, such as Saturnispora diversa, Issatchenkia terricola, Hanseniaspora opuntiae, Starmerella bacillaris and Hanseniaspora uvarum. The massive sequencing revealed a larger number of species, respect to the culture-dependent data. Comparing the two different approaches used in this work, it is possible to highlight some similarities since Aureobasidium, Rhodotorula and Sporobolomyces were detected by both methods. On the contrary, genera Hanseniaspora, Issatchenkia and Saturnispora were revealed by culture-dependent methods, but not by NGS, while Saccharomyces spp. were identified, with low frequency, only by NGS. The integrated application of NGS sequencing and culture-dependent techniques provides a comprehensive view of mycodiversity in the wine-growing environment, especially for yeasts with low abundance.
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Towards a more sustainable wine industry, distinct green strategies can be adopted by wineries, including solar power, recycled building materials, living soil roofs, natural ventilation, geothermal heating and cooling systems, and drought-tolerant landscaping. Sustainable winery architecture is also a global trend that needs to be developed. Principal aspects to consider for a wine sustainable production are developed with technological innovations in the fields of CO2 reuse, water management and saving, renewable energy, good practices in oenology and winemaking processes, functional biodiversity management, valorisation of winemaking by-products and climate change adaptation. This chapter offers to the readers a broad perspective on these aspects, serving as introductory overview for the topics developed in the following sections.
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Wine is a complex matrix that includes components with different chemical natures, the volatile compounds being responsible for wine aroma quality. The microbial ecosystem of grapes and wine, including Saccharomyces and non-Saccharomyces yeasts, as well as lactic acid bacteria, is considered by winemakers and oenologists as a decisive factor influencing wine aroma and consumer’s preferences. The challenges and opportunities emanating from the contribution of wine microbiome to the production of high quality wines are astounding. This review focuses on the current knowledge about the impact of microorganisms in wine aroma and flavour, and the biochemical reactions and pathways in which they participate, therefore contributing to both the quality and acceptability of wine. In this context, an overview of genetic and transcriptional studies to explain and interpret these effects is included, and new directions are proposed. It also considers the contribution of human oral microbiota to wine aroma conversion and perception during wine consumption. The potential use of wine yeasts and lactic acid bacteria as biological tools to enhance wine quality and the advent of promising advice allowed by pioneering -omics technologies on wine research are also discussed.
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Background VSEARCH is an open source and free of charge multithreaded 64-bit tool for processing and preparing metagenomics, genomics and population genomics nucleotide sequence data. It is designed as an alternative to the widely used USEARCH tool (Edgar, 2010) for which the source code is not publicly available, algorithm details are only rudimentarily described, and only a memory-confined 32-bit version is freely available for academic use. Methods When searching nucleotide sequences, VSEARCH uses a fast heuristic based on words shared by the query and target sequences in order to quickly identify similar sequences, a similar strategy is probably used in USEARCH. VSEARCH then performs optimal global sequence alignment of the query against potential target sequences, using full dynamic programming instead of the seed-and-extend heuristic used by USEARCH. Pairwise alignments are computed in parallel using vectorisation and multiple threads. Results VSEARCH includes most commands for analysing nucleotide sequences available in USEARCH version 7 and several of those available in USEARCH version 8, including searching (exact or based on global alignment), clustering by similarity (using length pre-sorting, abundance pre-sorting or a user-defined order), chimera detection (reference-based or de novo), dereplication (full length or prefix), pairwise alignment, reverse complementation, sorting, and subsampling. VSEARCH also includes commands for FASTQ file processing, i.e., format detection, filtering, read quality statistics, and merging of paired reads. Furthermore, VSEARCH extends functionality with several new commands and improvements, including shuffling, rereplication, masking of low-complexity sequences with the well-known DUST algorithm, a choice among different similarity definitions, and FASTQ file format conversion. VSEARCH is here shown to be more accurate than USEARCH when performing searching, clustering, chimera detection and subsampling, while on a par with USEARCH for paired-ends read merging. VSEARCH is slower than USEARCH when performing clustering and chimera detection, but significantly faster when performing paired-end reads merging and dereplication. VSEARCH is available at under either the BSD 2-clause license or the GNU General Public License version 3.0. Discussion VSEARCH has been shown to be a fast, accurate and full-fledged alternative to USEARCH. A free and open-source versatile tool for sequence analysis is now available to the metagenomics community.
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The effects of different anthropic activities (vineyard: phytosanitary protection; winery: pressing and sulfit-ing) on the fungal populations of grape berries were studied. The global diversity of fungal populations (moulds and yeasts) was performed by pyrosequenc-ing. The anthropic activities studied modified fungal diversity. Thus, a decrease in biodiversity was measured for three successive vintages for the grapes of the plot cultivated with Organic protection compared to plots treated with Conventional and Ecophyto pro-tections. The fungal populations were then considerably modified by the pressing-clarification step. The addition of sulfur dioxide also modified population dynamics and favoured the domination of the species Saccharomyces cerevisiae during fermentation. The non-targeted chemical analysis of musts and wines by FT-ICR-MS showed that the wines could be discriminated at the end of alcoholic fermentation as a function of adding SO 2 or not, but also and above all as a function of phytosanitary protection, regardless of whether these fermentations took place in the presence of SO 2 or not. Thus, the existence of signatures in wines of chemical diversity and microbiology linked to vineyard protection has been highlighted.
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While paradigms of macroecology are challenged by the high rates of reproduction, dispersal and horizontal gene exchange of bacterial communities, environmental DNA sequencing makes community profiles accessible. We test fundamental hypotheses of macroecological theories, showing that both taxonomic and functional classifications have distinct biogeographical variation across distance and environments depending on trophic composition. Studies spanning the global oceans. Taxonomic and functional profiles were obtained from metagenomes and were compared across oceanographic regions and tested for patterns of co-occurrence. The influences of sampling method (filter size), environmental variables and geographical distribution were compared with distance-based linear models to test predictors of taxonomic and functional composition. Macroecological drivers were compared with bacterial community structure to test four biogeographical hypotheses: (1) no biogeographical patterns, (2) community structure reflects environmental dissimilarity, (3) community structure reflects distance, (4) community structure reflects environment and distance. Bacterial families were clustered into four trophic groups – phototrophic, oligotrophic, eutrophic and copiotrophic – by changes in abundance across oceanographic regions and co-occurrence with core functions. Changes in community composition were best modelled by longitude for free-living communities and dissolved oxygen for mixed communities of free-living and particle-associated bacteria. Both microhabitat and community assignment had an impact on biogeographical patterns, with taxonomic compositions following our hypotheses 2 and 4 and functional gene compositions following hypotheses 3 and 4. We described four trophic groups adding to the current dichotomy of the classification of marine bacteria as oligotrophic or copiotrophic. Taxonomic composition of mixed communities reflected environmental differences but not geographical distance, whereas functional gene composition in free-living communities was independent of environmental dissimilarity and reflected geographical distance. Patterns of biogeography in bacterial communities differed depending on the description of taxa or function. Therefore, we developed a new paradigm for bacterial ecology which shows that some aspects of bacterial evolution depend on trophic complexity, history and current environmental conditions.
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Characterizing the community structure of naturally occurring microbes through marker gene amplicons has gained widespread acceptance for profiling microbial populations. The 16S ribosomal RNA (rRNA) gene provides a suitable target for most studies since (1) it meets the criteria for robust markers of evolution, e.g., both conserved and rapidly evolving regions that do not undergo horizontal gene transfer, (2) microbial ecologists have identified widely adopted primers and protocols for generating amplicons for sequencing, (3) analyses of both cultivars and environmental DNA have generated well-curated databases for taxonomic profiling, and (4) bioinformaticians and computational biologists have published comprehensive software tools for interpreting the data and generating publication-ready figures. Since the initial descriptions of high-throughput sequencing of 16S rRNA gene amplicons to survey microbial diversity, we have witnessed an explosion of association-based inferences of interactions between microbes and their environment.
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Unlabelled: Regionally distinct wine characteristics (terroir) are an important aspect of wine production and consumer appreciation. Microbial activity is an integral part of wine production, and grape and wine microbiota present regionally defined patterns associated with vineyard and climatic conditions, but the degree to which these microbial patterns associate with the chemical composition of wine is unclear. Through a longitudinal survey of over 200 commercial wine fermentations, we demonstrate that both grape microbiota and wine metabolite profiles distinguish viticultural area designations and individual vineyards within Napa and Sonoma Counties, California. Associations among wine microbiota and fermentation characteristics suggest new links between microbiota, fermentation performance, and wine properties. The bacterial and fungal consortia of wine fermentations, composed from vineyard and winery sources, correlate with the chemical composition of the finished wines and predict metabolite abundances in finished wines using machine learning models. The use of postharvest microbiota as an early predictor of wine chemical composition is unprecedented and potentially poses a new paradigm for quality control of agricultural products. These findings add further evidence that microbial activity is associated with wine terroir Importance: Wine production is a multi-billion-dollar global industry for which microbial control and wine chemical composition are crucial aspects of quality. Terroir is an important feature of consumer appreciation and wine culture, but the many factors that contribute to terroir are nebulous. We show that grape and wine microbiota exhibit regional patterns that correlate with wine chemical composition, suggesting that the grape microbiome may influence terroir In addition to enriching our understanding of how growing region and wine properties interact, this may provide further economic incentive for agricultural and enological practices that maintain regional microbial biodiversity.
Metagenomics experiments often characterize microbial communities by sequencing the ribosomal 16S and ITS regions. Taxonomy prediction is a fundamental step in such studies. The SINTAX algorithm predicts taxonomy by using k -mer similarity to identify the top hit in a reference database and provides bootstrap confidence for all ranks in the prediction. SINTAX achieves comparable or better accuracy to the RDP Naive Bayesian Classifier with a simpler algorithm that does not require training. Most tested methods are shown to have high rates of over-classification errors where novel taxa are incorrectly predicted to have known names.
Little is known about the hierarchical effects of management practices, soil attributes and location factors on structure of vineyard soil microbiota. A hierarchical effect occurs when the specific influence of an experimental factor (e.g. cover crop type, compost application) on soil-borne bacterial communities is greater within a subset composing the larger set but not across the entire set (e.g. bacterial communities only respond to a management practice within a subset of soil types but not across the entire set composed of all soil types). To address this concept, we measured differences in soil bacterial and archaeal diversity in wine-grape vineyard soils throughout Napa Valley, California. We describe how vineyard management practices influence soil resources, which in turn determine shifts in soil-borne bacterial communities. Soil bacterial communities were structured with respect to management practices, specifically cover crop presence and cover crop mix, tillage, and agricultural system designation, i.e. conventional, organic and biodynamic production systems. Distinctions with respect to management were associated with differences in pH and soil resource pools: total carbon and total nitrogen of the <53 and 53–250 μm particulate organic matter fractions, and potentially mineralizable nitrogen. Findings in this study suggest management practices in vineyard production systems directly influence soil microbial community structure, as mediated by shifts in soil resource pools. However, hierarchical effects occur, in which β-diversity is more strongly affected by specific management practices only within certain soil types, tillage or no-till soils or winegrowing region. This work allows for subsequent assessments of interrelationships of vineyard management, microbial biodiversity and their combined influence on soil quality, vine health, and berry quality.
Cultivation-independent investigation of microbial ecology is biased by the DNA extraction methods used. We aimed to quantify those biases by comparative analysis of the metagenome mined from four diverse naturally fermented foods (bamboo shoot, milk, fish, soybean) using eight different DNA extraction methods with different cell lysis principles. Our findings revealed that the enzymatic lysis yielded higher eubacterial and yeast metagenomic DNA from the food matrices compared to the widely used chemical and mechanical lysis principles. Further analysis of the bacterial community structure by Illumina MiSeq amplicon sequencing revealed a high recovery of lactic acid bacteria by the enzymatic lysis in all food types. However, Bacillaceae, Acetobacteraceae, Clostridiaceae and Proteobacteria were more abundantly recovered when mechanical and chemical lysis principles were applied. The biases generated due to the differential recovery of operational taxonomic units (OTUs) by different DNA extraction methods including DNA and PCR amplicons mix from different methods have been quantitatively demonstrated here. The different methods shared only 29.9-52.0% of the total OTUs recovered. Although similar comparative research has been performed on other ecological niches, this is the first in-depth investigation of quantifying the biases in metagenome mining from naturally fermented foods.