4.1. Soil Chemical Properties
The persistent and large increases in both soil organic C and total N pools observed in digestate-treated plots across the entire experimental period is consistent with what was reported, for instance, by Knoop, Dietrich, Heinrich, Dornack and Raab [
31], who found a 21-month lasting effect after the soil addition of digestate. Needless to say, most authors’ reports corroborate this finding [
37,
38,
39] However, it must be also noted that opposite results were shown by other authors [
20,
40]. For instance, Adani, et al. [
41] observed a 50% increase in labile carbon loss as a result of a 75% increase in the compost dose. Nevertheless, the potential storage of freshly added C pools through digestate amendment is strongly dependent on either the anaerobic biogas properties or the recipient soil characteristics [
42,
43,
44,
45]. We surmise that the pedogenetic features of the studied olive grove soil rich in fine-textured particles, poor in calcium carbonate, and with limited aeration produced soil fabric conditions suitable for physical protection and reduced mineralization of newly added organic pools, whatever their content of easily degradable organic substrates. Finally, total C and N readings from non-tilled soil confirm that the high clay content leads to the formation of stable microaggregates in which carbon is stabilized and sequestered in the long term, in accordance with what was reported by Six, et al. [
46] and Denef, et al. [
47].
It is also worth noting that soil EC, which is a highly variable soil chemical characteristic generally subjected to seasonal fluctuations due to continuously occurring soluble nutrient addition (mineralization and fertilization) and removal (leaching, plant uptake, and microbial immobilization) processes, remained significantly higher in DIG-treated plots. This finding suggests that soluble salt accumulation was the preferential process occurring in the olive grove soil as a consequence of a reduced internal drainage (Ksat), which can be estimated as low as 0.27 mm h−1 by considering the clay content. This physical feature protects groundwater from eutrophication and excessive N leaching, but rises the risk of secondary salinization under repeated and large applications of solid digestate. Finally, other than an immediate and ephemeral increase observed soon after the start of the trial, no significant variations in soil pH were appreciated over the entire experimental period in all treatments, thus confirming the strong buffering activity of the acid clay-rich olive grove soil.
4.2. Soil Exchangeable Micronutrients and Aluminum Content
Soil acidity is one of the main constraints to crop production because it exerts multiple critical influences on chemical and biological soil properties. In detail, it has a pronounced impact on the growth and activity of soil organisms, including plants, animals and microbes; it affects the mobility of many contaminants; and creates conditions of a nutrient imbalance and aluminum toxicity [
48]. Regarding this last point, it is needed to point out that aluminum toxicity is rarely a problem when the soil pH measured in CaCl
2 is above 4.8 (pHH
2O 5.2), because little aluminum exists in the solution or in exchangeable pools above this pH level. This is in full accordance with our findings from the acid olive grove soil, where aluminum was equally found at an estimated concentration around 1 µM in all three treatments: a value widely lower than the threshold value of 50 µM indicated by [
49] as the upper limit interfering with Al-sensitive plants, which represent almost one-third of currently cultivated olive germplasms. To sum up, in the present study, aluminum toxicity cannot be considered a factor influencing the olive tree physiology and productivity, either directly or indirectly, because it was found to be well below the critical threshold and not to have negatively altered the Ca/Al balance, which was always much larger than the critical value of one.
Note that in strongly acidic soils, as the one tested in the present research, the availability of macronutrients (Ca, Mg, K, P, N, and S), as well as two micronutrients, Mo and B, is strongly restricted. In contrast, the availability of micronutrient cations (Fe, Mn, and Cu, but also Zn and Ni) is increased, even to the extent of toxicity [
48]. Unbalanced and low Ca, Mg, and K contents are certainly a major pedogenetic-linked factor limiting olive tree yield and quality. Similarly, the parent material and pedogenetic processes can be invoked as main factors responsible for deficiencies of exchangeable Cu and Fe levels, irrespective of differing soil management treatments. Needless to say, iron toxicity can become a serious problem to plants at very low pH levels (usually less than 4.0) and under anaerobic conditions, which did not occur in the olive grove soil here.
Manganese is an essential plant nutrient whose toxicity, when taken up in excessive amounts, can represent a growth-limiting factor to olive tree plants, especially in acid soils derived from manganese-rich parent material [
50]. In fact, Mn becomes increasingly available as the pH drops, but unlike aluminum, Mn toxicity starts to appear at pH
H2O levels as high as 5.6. Given the chemical properties and nature of the parent material of the olive grove soil, not unexpectedly, the available Mn concentration in soil reached a value as high as 200 mg kg
−1 (above the 50 mg kg
−1 limit considered toxic to many crop species [
50,
51]), and it was further enlarged, rather than restricted, by the addition of the organic amendment, which increased its bioavailability (albeit not significantly), as observed in the present study. In fact, the Ca/Mn (moles to moles) ratio, calculated using the soil data, shows a clear declining trend across treatments, from 15.3 and 12.9 (in TILL and NT soil, respectively) to 10.2 (in DIG-treated soil), thus corroborating the role of the added organic matter in promoting Mn mobility in soil.
We argue that, given the feeding material used for the anaerobic digestion process, the low micronutrient content, and the used application rate, the solid anaerobic digestate supplied in the present research did not contribute to significantly rise the metal load of the recipient soil. In fact, in accordance with several authors [
16,
19,
52], biogas digestates have generally very low amounts of heavy metals, which primarily depend on the quality of the input feedstock [
18]. Thus, direct enrichment of soil micronutrients cannot be considered here a relevant process linked to the soil addition of an agricultural solid digestate. From the other side, it cannot be excluded that reduced mineral N forms entering the soil with digestate addition could have promoted the nitrification process with the consequent release of additional acid cations capable of displacing Ca and Mg from the exchange complex, thus enabling their leaching to occur and a further decrease soil pH [
48]. If this is true, excess ammoniacal N sources entering the soil with a high clay content (45%) and low base saturation (<32%) would have increased the nutrient unbalance at the soil–water interface and further contributed to rise acidic conditions and soluble Mn excess. Finally, Mn forms complexes with organic ligands, but how these ligands are stable, and increases or decreases in Mn mobility in soil, and its availability to plants depend on several factors (soil pH, CSC, water and temperature regimes, and microbial activity), which make the question still matter of debate [
12,
53].
To conclude, whatever the role of the solid digestate addition on affecting Mn mobility in soil, it must be noted that the Ca/Mn values obtained for the field treatments were always below the critical threshold of 30, a finding representing a soil condition characterized by a constitutive excess of soluble Mn and Mn-related nutrient imbalance, which both severely limit plant growth and productivity.
4.3. Olive Tree Fruit Micronutrients and Aluminum Content
The metal content evaluation in drupes shows a similar Cu concentration in DIG and NT, suggesting a higher Cu uptake than TILL plants; this could be connected to higher CO
2 fixation and Fv’/Fm’ ratio, taking into account that Cu is a fundamental co-factor in plastocyanin function and, consequently, in photosynthetic activity [
54]. Furthermore, it is interesting to note how NPQ is composed by several factors, indicating not only a heat dissipation process but also the efficiency of other metabolic pathways related to photosystems II and I, like the quenching state transition (
qT) that indicates phosphorylated proteins transferring light energy from photosystem II to photosystem I [
55]. So, we can argue that the higher NPQ found in DIG and NT could be interpreted as a positive response, although it was not statistically different between DIG and TILL.
Noteworthy, the total Mn content in olive tree fruits was found within the expected range (4.9–8.7 mg kg
−1), whereas a slightly lower amount of Fe was observed (reference range: 18–25 mg kg
−1) [
56], suggesting that (i) olive plant tolerance to excess Mn involves an alteration of the distribution of the metal within the plant (i.e., favoring root accumulation while restricting shoot translocation, especially towards tree fruits) [
50]; and (ii) iron and manganese contents are negatively correlated, and excess Mn brings about inhibition of Fe uptake and metabolism [
51]. In fact, in addition to direct negative effects, excess Mn in the soil solution can interfere with the absorption, translocation, and metabolism of other essential nutrients, such as Ca, Mg, P, and Fe, thus magnifying their deficiencies. This becomes particularly relevant in acid soils where a nutrient imbalance is common.
From the other side, aluminum remained unchanged in olive tree fruits, thus mirroring the evenly distributed and tiny amount of soil exchangeable Al detected in all treatments.
4.4. Physiological Responses
Based on photosynthesis and stomatal conductance, it appears clear that soil tillage negatively affected the photosynthetic rate of olive leaves, thus providing values which are generally lower than those generally reported in the literature, as in [
50]. Then, it is not surprising that the chlorophyll fluorescence parameters—which allow us to estimate the photochemistry of the photosynthetic system II (PSII) from the fluorescence decline, termed “quenching”, and provide an early assessment of plant responses to most types of stresses [
57]—declined. In particular, the reduction of the Fv/Fm ratio (i.e., the maximum quantum yield of PSII), which was well below the threshold value of 0.7, indicates that olive tree plants were under stress conditions [
58]. To explain these findings, we argue that the mechanical disturbance due to the repeated tillage events could have altered the soil physical properties in a way that, given the fine-textured features of the olive grove soil and the rainfed cultivation regime, water loss through evaporation increased due to improved soil aeration. It is also worth considering that, within the context of global warming, soil water deficiency is being exacerbated by increasing temperatures and declining precipitation, as evidenced during the 2016–2017 experimental period (
Table S1), when compared to the time series data registered over the 2000–2015 period (average T 17.4 °C, annual rainfall 889.7 mm, [
26]. In brief, readings from physiological responses indicate that in conventionally tilled soil, factors other than Mn toxicity and a nutrient imbalance negatively affected the functionality of PSII in photosynthesis and hence olive tree productivity. We hypothesize that the tillage-induced water deficit, which was further exacerbated by the rainfed regime, would have resulted in a decline in the olive fruit number and oil content, but not in their biometric features. In a few words, water-stressed plants produced less olives of a similar size. This finding is in agreement with [
59,
60].
On the other hand, solid digestate addition, which brought about a marked and persistent increase in total soil C and N pools, seemed to alleviate the adverse physiological effects found in olive plants from tilled plots in terms of increased CO2 fixation, stomatal conductance, and transpiration, even though they never reached response values as high as observed in NT-treated olive plants. Three possible interacting factors could be invoked to explain this result.
First is the reduced soil water loss due to an improved soil structure and the greater water-holding capacity of digestate-derived organic matter. Needless to say, solid anaerobic digestate exerts beneficial effects on soil physical properties, in full accordance with what was reported by several authors [
19,
53]. Moreover, it has been shown that digestate-released organic matter can contrast salt addition effects, thus enhancing soil water availability [
61]. We also observed that under rainfed conditions and in a fine-textured soil, the no-tillage practice was even more effective in maintaining the soil water-holding capacity and alleviating any water stress, as shown by the photosynthesis-related parameters that were always higher in NT than in both tilled plots (either amended or not). Both these findings are consistent with Gimenez, et al.’s [
62], who reported photosynthetic rates ranging between 7 and 18 µmol CO
2 m
−2 s
−1 when olive tree plants were grown under non-limiting water availability. On the other hand, the photosynthetic rate < 5 µmol CO
2 m
−2 s
−1 observed in conventionally tilled plots is certainly a clue of a stress response due to a water deficit. To sum up, adverse plant responses due to unfavorable physical conditions created in the clay soil by the tillage event were partially mitigated by the soil incorporation of solid digestate.
Second is a bio-stimulating effect on plant growth due to hormones and hormone-like molecules released from the solid digestate into the soil. Although poorly investigated, this topic was recently addressed by [
63,
64,
65], who went on to show the presence of auxin-like properties located in a fraction of the dissolved organic matter extracted from the digestate from full-scale digester plants. Within this context, the potential for the application of digestate-derived bioactive compounds used as biofertilizer has been proposed by Du, et al. [
66]. Needless to say, anaerobic digestion treatment has increased enormously during the last years and has become a very important sector for waste treatment and renewable energy production in Europe [
67]. As a by-product of the anaerobic digestion process, digestate is of great potential interest not only as a soil amendment [
15] but also as a source of biostimulant molecules [
68], which are concentrated in the most biologically reactive DOM fraction and are directly involved in plant nutrition processes [
69,
70]. Further inside, due to the photosynthetic performances observed in DIG treatment that were higher or equal to TILL, a lower oil yield and mesocarp oil percentage cannot be explained with a reduced C fixation rate, but with a hormone-like activity due to digestate-released humic substances. That is also consistent with the acceleration of the drupe ripening process in plants from DIG-treated soil, which could in turn justify the lower force needed to detach them from branch, in accordance with [
71,
72]. Moreover, less decreased photosynthetic performances observed in DIG than in TILL justify the difference in plant productivity (in terms of fruits per tree and fruit yield) found between these two treatments. Then, it becomes clear that digestate addition exerted a mitigating action on plant physiology and productivity when compared to unamended tillage. To sum up, it cannot be excluded that a pool of bioactive organic molecules entering the soil after amendment with digestate directly and indirectly affected the olive tree’s physiology, and hence its productivity, following a dose-response relationship (hormetic effect) [
73].
Third are changes in soil microbial activity and compositional shifts in prokaryotic community composition, which in turn altered the soil nutrient release and the nutrient use efficiency, as well as rhizosphere interactions at the soil–plant interface. Needless to say, it is known that soil incorporation of anaerobic digestate affects the soil microbial activity and brings about changes in the phylogenetic composition of prokaryotic communities [
16,
17,
18,
20,
74,
75,
76,
77]. In two parallel papers focused on olive grove soil, it was found that major changes involved the phylogenetic shift in the r/K-strategist balance, being copiotrophic bacterial taxa favored (such as Proteobacteria) and oligotrophic taxa (i.e., Acidobacteria) constrained [
25], accompanied by higher microbial C-use efficiency and a long-lasting release of soluble C and N forms [
78]. This would lead to the conclusion that over the long term, digestate addition helped create soil ecological conditions that were less unfavorable (or less constraining) to olive tree growth than the conventional tillage did.