Soil geological description
In the area of Contrada Finocchio, where the first vineyard is located, a middle-upper Oligocene lithological succession outcrops; it consists of open shelf carbonates, mostly represented by thick bedded whitish marly limestones, alternating with white or greyish marls. The observed thickness of each level varies from few centimeters up to 80-100 centimeters. Sometimes are present intercalations of nummulitic and resedimented biocalcarenites. The above described lithological complex is ascribed to the lower portion of the so called Ragusa Formation, particularly to the Leonardo Member. The general characteristics of the succession are strongly influenced by the predominant presence of the compact limestones or of the more or less marly levels containing 20 to 60% clay in addition to other constituents. The permeability degree of the lithological complex is generally scarce due to the presence of very low permeable marly levels; however, the locally presence of limestone with high secondary permeability, due to fissuration induced by tectonic processes, allows a moderate drainage of the groundwater and the formation of small aquifers with local importance.
Eastward of Menfi town, where Contrada Bertolino and the second vineyard are located, largely outcrops a middle-upper Pliocene terrigenous succession locally known as Marnoso-arenacea del Belice formation. It consists, from bottom, of several teens of meters of fine to medium sandstones; the sandy particles are mainly rounded quartz grains, but levels of resedimented biocalcarenites also occurred. They upwards go to sandstones and coastal calcarenites and conglomerates. The substratum in the area is formed of up to 50 meters of thick package of hemipelagic shales and marls, and brownish siltstones with interbedded siltstones and calcarenite mudstones. The Marnoso-arenacea del Belice formation underlie the quaternary marine deposits mostly represented by lower Pleistocene yellowish partially cemented calcarenites and biocalcarenites alternating to thick beds of biocalcirudites, with thin intercalation of marls levels, conglomerates lens and calcareous sands and gravels. The total thickness of the quaternary complex varies from few meters, as in the interest area, up to few tens meters. The natural porosity characteristics of the above mentioned lithological complexes, specially of the calcarenites levels and of the small cemented sands, allows a good drainage and circulation of the groundwater partially confined by marly and loam/clay grained thin bedded intercalation with a low permeability.
Soil chemical analyses
The average values of physical and chemical characteristics of the two soils are reported in Supplementary Table 1. The pH resulted alkaline, but in an optimum pH range for plant nutrient uptake .
Root system distribution and development
Table 1 reports the roots density at two different distances from the vine trunk (0.40 and 1.00 m) dividing the roots in three diameter classes and the average number of roots/m2 found in the two soils along the dug trench. In those soil conditions, we found that the total roots number of the C soil was higher compared to ML one at 0.40 m distance from vine trunk, while the mean roots density did not vary significantly at 1.00 m distance from vine trunk. Considering the root size we found thinner roots (Ø < 1 mm) in ML soil at 1.00 m from the vine trunk, and more abundant thickest roots ( Ø 1 - 3 mm and Ø > 3 mm) for both distances (0.4 and 1 m) in C soil were the sandy texture allowed to a more roots diffusion (Table 1). We found some significant differences: an average ratio of 7.1 versus 3.8 at 0.4 m from vine trunk (p < 0.05) and 10.9 versus 4.3 at 1 m from vine trunk (p < 0.05) for ML and C, respectively (Supplementary Table 2).
For what concerns the vertical distribution, in C soil the major number of roots (62 roots / 0.04 m2) was located between 0.40 and 0.60 m of soil depth and at 0.40 m distance from vine trunk. Indeed, for ML soil, we observed the major number of roots at the same depth (0.40-0.60 m) but at 1.00 m distance from vine trunk (48 roots / 0.2 m2).
Bacterial community diversity and composition
The bacterial community was analyzed at both the family and the genus level: the number of retained sequences after chimera removal and taxonomical assignment ranged from 29,924 to 51,428. The diversity indices (Shannon and Simpson) indicate that in both soil types a significant difference between superficial and deep microbial composition occur (Tab. 2). Interestingly both the indices suggest that calcarenite superficial (CS) display the same diversity of deep marly limestone (MLD).
The complete bacterial community composition for each sample type at the family level is reported in Supplementary Tab. 3 and statistical results of pairwise comparisons are reported in Supplementary Tab. 4. Here, to simplify, we are describing results for the families which represent at least the 2% of the bacterial community (Fig. 1a). Regarding the calcarenite soil we can observe that the composition of superficial (CS) and deep (CD) samples is quite different as also reported by the statistical analysis in Supplementary Tab. 4 and in the PCoA analysis (Supplementary Fig. 1). It is worth nothing that the CD sample displays the higher number of taxa and the most different composition if compared with all the other samples. In detail, the superficial sample is richer in Nitrososphaeraceae and Bacillaceae whereas Sphingobacteriaceae and Comamonadaceae are more abundant in the 120 cm depth sample (Supplementary Tab. 4). Interestingly, pairwise comparison of superficial calcarenite and marly limestone samples revealed that at the family level only three showed significant differences in abundance. Among the three families, Bacillaceae and Nitrososphaeraceae are more abundant in CS sample than in MLS one. Regarding the ML soil we observed 21 families more abundant in superficial (MLS) sample and 28 families that are more represented in 120 cm depth (MLD) sample. Among the differentially abundant families Acidobacteriaceae, Sphingomonadaceae, Gemmatimonadaceae, Nitrospiraceae, Acidimicrobiaceae, Verrucomicrobia subdivision 3 are over-represented in MLS sample whereas Bacillaceae, Nitrososphaeraceae, Streptomycetaceae and Sphingobacteriaceae are over-represented in MLD sample. We then also compared the two deep soil types observing the wider number of differential abundant families. The families over-represented in MLD sample are 36, among them Nitrososphaeraceae, Bacillaceae, Hyphomicrobiaceae, Rhodospirillaceae, Chitinophagaceae and Sinobacteraceae which belong to the top selected families representing about the 60% of microbial composition. In parallel, 32 families are more abundant in CD: among them Sphingobacteriaceae, Comamonadaceae, Pseudomonadaceae, Nitrospiraceae, Acidobacteriaceae, Sphingomonadaceae and Cytophagaceae.
Regarding the bacterial composition at the genus level (Supplementary Tab. 5) we decided to retain only taxa representing at least the 2% of the overall community (Fig. 1b). Pairwise comparisons were done to underlay the significant differences among sample types (Supplementary Tab. 6). First, we started with CS and CD samples. There are 8 genera which are more present in the CS samples, whereas 21 genera are more abundant in CD samples. Among genera more present in CD soil we found Pedobacter, Flavobacterium, Variovorax and Pseudomonas. When comparing the CS and MLS soils we observed only few differences: 4 genera are more abundant in CS soil, among them the Flavobacterium genus, and only one is more present in MLS soil. Then, we compared MLS to MLD observing a high number of differentially abundant genera: 37 are more present in MLS whereas 50 are more present in MLD. Among genera more abundant in MLS we observed Acidobacterium, Sphingomonas, Nordella, Stenotrophobacter, Nitrospira and Vicinamibacter. In parallel, among the genera more abundant in MLD we found Flavobacterium, Pedobacter and Steroidobacter. Finally we also compared the genera of MLD and CD soils. As reported for the families, also for genera we found in this comparison the widest number of differentially abundant genera: 61 are more abundant in MLD samples, whereas 62 are more present in the CD soil. Among them Steroidobacter and Thiobacter are more present in MLD and Pedobacter, Flavobacterium, Variovorax, Acidobacterium, Nitrospira, Nordella, Vicinamibacter, Sphingomonas and Stenotrophobacter are more abundant in CD samples.
Fungal community diversity and composition
As for the bacterial also for the fungal community we analyzed at both the family and the genus level: the number of retained sequences after chimera removal and taxonomical assignment ranged from 40,294 to 73,785. The diversity indices (Shannon and Simpson) indicate that in both soil types a significant difference between superficial and deep microbial composition occur (Tab. 3). The difference is more marked in calcarenite than in marly limestone soils.
The complete fungal community composition for each sample type at the family level is reported in Supplementary Tab. 5 and statistical results of pairwise comparisons are reported in Supplementary Tab. 6. Here, to simplify, we are describing results for the families which represent at least the 2% of the bacterial community (Fig. 2a). As a first general observation we can note that at least the family level, the composition of both CD and MLD soils is almost the same. There is only one family (Phaffomycetaceae, which is not in the representative group above the 2%) that shows a differential abundance as reported in Supplementary Tab. 6. When comparing the CS and CD soils we observed 4 families more representative of CS samples and 6 more abundant in CD samples. Among families more present in CD samples, we observed Botryosphaeriaceae, Togniniaceae and Chaetomiaceae which are in the group above the 2% threshold. Then we compared the CS and MLS samples: 8 families were more abundant in CS samples (among them the Clodosporiaceae) and 13 were more abundant in MLS soil. Among the latter we found Dermataceae and Togniniaceae. Finally, we also compared MLS and MLD samples: 13 are the families more abundant in MLS (among them the Dermataceae family) and 10 families are more present in MLD ones. Among the last group Botryosphaeriaceae and Chaetomiaceae are above the 2% threshold.
The complete fungal community composition for each sample type at the genus level is reported in Supplementary Tab. 9 and statistical results of pairwise comparisons are reported in Supplementary Tab. 10. Here, to simplify, we are describing results for the families which represent at least the 2% of the bacterial community (Fig. 2b). Similarly to what observed for the family level, also for the genus level minimal differences occurred between the CD and MLD soils. In specific we observed only one genus more abundant in CD (Stemphylium) and one more abundant in MLD (Cyberlindnera), both accounting for less than the selected threshold. When comparing CS and CD samples we observed 18 genera more abundant in CS samples (among them only Dactylonectria is above the 2% threshold) and 6 genera more abundant in CD samples. Among the latter we found Neofusicoccum, Camarosporium, Phaeoacremonium and Humicola. Comparing then the composition of two different superficial soils (CS vs MLS) we found the wider number of differentially abundant genera (54 in total), 25 more present in CS and 29 more present in MLS. Looking at the genera above the threshold, Chaetomium is more abundant in CS whereas Laetinaevia and Phaeoacremonium are more present in the MLS soil. The last pairwise comparison regards the MLS and MLD samples. The number of genera more present in MLS samples is 28 (Laetinaevia and Dactylonectria are above the 2% threshold) whereas 19 are more abundant in MLD (Camarosporium and Neofusicoccum are above the threshold).
Since specific fungal genera can have an important impact on plant development we decided also to focus on pathogenic (Fig. 3) and mycorrhizal (Fig. 4) fungi. Looking at the pathogenic ones, we can observe that in both soils Neofusicoccum species are more abundant in the 120 cm depth samples than in superficial ones. On the contrary Ilyonectria is more abundant in both soil types in the superficial layer. In addition, in calcarenite soil we observed a significant accumulation of Phaeoacremonium genus in the CD samples. Moreover, the genus Cadophora is more abundant in superficial samples of marly limestone (MLS) soil than in deep (MLD) ones. Regarding the mycorrhiza, in both soils the Glomus genus is more abundant in the 120 cm depth samples. In the case of Rhizophagus genus, it shows an opposite pattern, being more abundant in CD than CS and MLS than MLD samples respectively. Finally, the genus Funneliformis was detected only in the MLS sample.
Community structure is always represented by two independent factors: the diversity and the complexity of taxa present in each sample. Diversity indices (Taxa, Shannon, Simpson and Evenness), representing species richness and evenness, were calculated for both bacterial and fungi in CS, CD, MLS and MLD as reported in Tab. 2 and Tab. 3 respectively. In addition, after the bioinformatics classification of amplicons for both the 16S, ITS and the two communities analyzed together, we reduced the dataset of each biological replicate to a bi-dimensional scaling using a Bray-Curtis distance matrix and plotting the results in corresponding non-metric multidimensional scaling (NMDS), as reported in Supplementary Fig. 2. A cluster heatmap of 16S and ITS communities considered together is reported in Supplementary Fig. 3. Moreover, co-occurrence analyses of 16S, ITS and the two communities analyzed together are reported in Supplementary Fig. 4, 5 and 6. Statistical analyses of co-occurrence relationship are reported in Supplementary Tab. 11-12-13.
Wine sensory analyses
Results of tasting are reported in Fig. 5. The results show a good differentiation between the two wines. Regarding the olfactory scents, the wine obtained from the C soil resulted richer and more interesting in general, with higher scores for orange blossom and elegance but also in terms of pleasantness and floral retro olfactory sensations. The wine obtained from the ML soil was richer in ripe fruit notes such as melon, pear and citrus. In mouth no differences has been detected as for sapidity, body and acidity.