3.1. General Biodiversity
There is a general assumption which indicates that the larger the environmental heterogeneity is the larger the diversity of complex organisms is, indicating that more heterogeneous habitats usually present higher species alpha diversity [42
]. However, despite the recognized central role of bacteria in the soils’ fertility, less knowledge has been reported concerning the link between the environmental heterogeneity and bacterial diversity [44
]. Several investigations have reported that locations highly different in their environmental and physicochemical parameters usually tend to be very different in their bacterial community composition too [46
], suggesting that soil heterogeneity increases bacterial beta diversity. Even though the relationship between soil environments and fungal diversity is less known [48
], some meta-analysis studies have indicated that, in addition to bacterial alpha diversity, fungal alpha diversity is higher in fields with crop rotations [49
] or in temperate deciduous forests [50
In this study, we analyzed the general microbial diversity (bacterial and fungal population), with the aim of determining the potential connections between soil and wine-related microbiota from different vineyards. The microbial diversity as alpha diversity of the vineyard soils was measured using the Shannon index (measure of the species richness and abundance), comparing the effect of two seasons (summer and winter), with very different conditions of temperature and humidity in the sampled region. The climate conditions in the center of Spain are characterized by cold and humid winters, while the summers are hot with very little and occasional rainfall. In addition, the impact of the textural characteristics of the soils (Figure A1
) were determined, studying their impact over microbial biodiversity [51
] (Figure 1
The seasonal effect on bacterial alpha diversity changes significantly between seasons. The complexity of bacterial communities has been generally described to be lower in winter than summer [52
], and our results are in agreement with this observation (Figure 1
a). As a result, we hypothesize about the possibility whether the soil bacterial community can be used as a new biological parameter to be considered in vineyard soils zoning strategies in viticulture soils or not. In contrast to bacterial diversity, fungal diversity did not change with the seasons (Figure 1
b) and showed a lower Shannon index and greater dispersion than the bacterial subset. The fungal community inhabiting soils was more homogeneous during seasons, maintaining the regional homogeneity of the studied soils. Bacterial populations showed a microscale effect due to their heterogeneity in summer and winter. Although fungi populations could become an indicator of regional character in vineyard blocks.
In this study, we analyzed bacterial diversity as a function of the textural characteristics: calcareous, clayey and sandy soils (Table A1
). A lower bacteria diversity was observed in the different types of soil (Figure 1
a). Although the differences in the Shannon index among clayey, calcareous and sandy soils using ANOVA test were slightly statistically significant (p
-value = 0.056), it was observed that the Shannon index is different between sandy and clayey soils. No differences were observed in soil types in the fungal subset analyses (Figure 1
Beta diversity was calculated as dissimilarity between soil samples, according to the ASVs extracted from the raw data curation process. In the non-parametric multi-dimensional scale ordering (NMDS), the ASVs of the group of bacteria (Figure 2
a) and the fungi subset (Figure 2
b) show the distances of each soil sample.
The bacterial population separates into two groups defined in the NMDS1 component (p
-value = 0.001). The bacterial subset present in soils in winter was observed for NMDS1 > 0, approximately. Furthermore, the samples whose bacterial population defined the summer season were located for NMDS1 < 0 (Figure 2
a). The textural characteristics of the soil were also statistically significant (p
-value = 0.001), having NMDS2 > −0.1 for sandy soils, approximately, and NMDS2 < −0.1 for the other two soils (calcareous and clayey).
Sorting based on stress index for the fungi group allowed separation in the NMDS2 component (p-value = 0.001). The population of fungi linked to winter were found in NMDS2 < 0. In samples collected in summer, the fungal population was observed in NMDS2 > 0. Statistical analyses of the textural characteristics were also slightly significant (p-value = 0.052) since this separation was not too clear to define as a function of the NMDS values.
Based on the genomic sequencing of the V4 16S rRNA gene region, it was possible to estimate the functional genes that the bacterial population could express in the soil. The estimated metabolic functions include enzymes involved, among others, in the biogeochemical cycles of carbon, nitrogen, phosphorus and sulfur (vectors at Figure 2
a). Based on that, the metabolic routes involved in organic carbon formation, organic nitrogen use and others (see Table A2
for a detailed list of the metabolic routes included) appeared more represented in winter samples. We can hypothesize that this could be because winter samples were collected in January, and a greater concentration of organic matter is accumulated in the soil (coming from fall autumn leaves). On the other hand, summer samples cluster matched the direction of the contribution of metabolic routes involved in sulfur metabolism (organic formation and use). This can be explained as summer samples were collected in early June and some routine sulfur-based treatments were applied in April and May for guaranteeing a healthy grape ripening.
Nevertheless, contrary to what was observed at taxonomic (alpha and beta diversity) level, there is not a clear pattern clustering the soils samples coming from different soil types or collected at different seasons (Figure A2
). This can indicate that the taxonomic differences found between vineyard blocks are buffered at a functional level due to the high functional redundancy commonly found within soil microbial communities [8
3.2. Wine-Related Microbial Diversity
Since the soil has been reported as the main reservoir of microorganisms in the vineyard, and a notable co-occurrence of microorganisms exists among vineyard soils, grapes and musts [15
], it is of interest to study the presence, diversity and abundance of wine-related bacterial and fungal species in the studied soils. Soil microbiota has been described as important, not only for the chemical and nutritional properties of soils, but also for health, yield, and quality of the grapevine. Apart from being the origin of the fermentative microbiota that will reach the winery as part of the microbial consortia established in the grapes—which would be responsible for positive flavor compounds production or in the production of undesirable molecules (off-flavors, biogenic amines, etc.)—the soil microbiome has been directly co-related with some flavor characteristics of wines (via plant-microbiome interactions), such as the rotundone concentration found in Shiraz grapes from Australian Cool Climate areas [54
]. Thus, in response to the current trend of elaborating “single-vineyard” wines as a way to enhance the terroir
characteristics of each vineyard block, understanding the microbial signature of soils should be considered in future vineyard zoning works, when trying to define their fermentative potential. The raw data from the sequencing process were filtered, obtaining the abundances of the microorganisms previously described to be isolated from wine-related samples (Table A3
The WRB found in the meta-taxonomic studies of soils were filtered at the taxonomic level of family due to the limitations showed by the NGS-technique used in this work [55
]. The soil samples collected in winter and summer differ in the presence of the family Lactobacillaceae
, being of greater presence in summer and absent in winter, while Leuconostocaeae
appears in more plots in summer samples. Some examples of species from these families are Oenococcus
), mainly responsible for malolactic fermentation [56
]. In addition, various species of Lactobacillus
can cause spoilage of wine during bulk storage in the cellar and after bottling [57
]. No differences were observed by soil type, although in summer calcareous Samples 8 and 9 showed a similar abundance pattern. However, it is possible that in Plots 7–9 the absence of the Lactobacillaceae
family was due to an active limestone concentration of more than 5.1% (Table A1
). The pronounced prevalence of the Acetobacteraceae
family observed in winter stood out. The ability of acetic acid bacteria to convert ethanol in acetic acid is one of the main sources of wine spoilage. Both grapes and wine are subject to spoilage by this bacteria at different stages of the grape ripening and the winemaking processes [58
The wine-related fungi (WRF) present in the soils were the Cryptococcaceae
families (Figure 4
b). However, within some samples, no representatives of these families were found. In summer soils samples from Plots 2–4 and winter samples from Plots 5, 6, 8 and 9, no WRF families were detected. In the summer season, a clear prevalence of the family Saccharomycetaceae
was observed in Plots 1, 5, 6 and 9. Plot 8 did not present fungi of the family Cryptococcaceae
. Is important to highlight that the calcareous soils of Plots 8 and 9 showing the presence of the WRF family Cryptococcaceae
were the only ones that presented this family during summer. In winter, a high frequency of the family Debaryomycetaceae
was observed in Plots 1, 2, 4 and 7. The soil of Plot 3 showed the Pichiaceae
families, which were equally represented. Due to the succession of families Saccharomycetaceae
between summer and winter, WRF seems to be a better indicator for differentiating the seasonal fermentative potential among plots.
The beta diversity analyzed in the WRB families shows a clear distinction between winter and summer (Figure 4
). The component NMDS2 allowed good separation between the variations in the subset of bacteria. The winter samples were mainly arranged in NMDS2 < 0, while variations in summer samples qwew disposed in NMDS2 > 0.