3.1. Determination of Macro- and Micro-Elements of Hop Leaves
Macro elements data outlined a statistical difference at the ANOVA between the values of two fertilization times for each studied element (
Table 2).
In general N decreases from t1 (35.55 g/kg on average) to t2 (33.13 g/kg on average). This result is due to the N translocation from different plant organs to the cones [
2]. The lower N quantity was found in Treat 1 (in both times), which represents the treatment with the least N supplied. N shows the highest leaf concentration in Treat 2 (
Table 2), reflecting the N content in fertilization plans (
Table 1); Untreat is unexpectedly quite high, despite having received no N (
Table 2). Also P content in leaves shows differences between the two fertilization periods. P in t1 reaches the highest amount in Treat 4, even if P was not applied, while in t2, P presence was in accordance with the fertilization plan, with the highest amount in Treat 3 which corresponds to the highest amount of phosphorous (P
2O
5) applied (
Table 1). In the same period, Untreat leaves show the lowest P content. K shows differences only between the two fertilization periods, with a slight decrease between t1 and t2. Mg is involved in the enhancing of crop production [
33], and shows an increase in leaf content between the two fertilization periods, as a result of increased Mg amount. The Mg content shows the highest level in Treat 3 in t1, and Treat 4 in t2, but these results do not reflect the Mg contents of the fertilization plan. Calcium is not present in any treatment; nevertheless, Untreat in t2 has the highest Ca content. This difference is probably due to an altered response for the absorption of Ca due to the interaction with the other elements; it is well known that some elements can promote or inhibit the absorption of others, as in the case of an excess of P that inhibits the absorption of elements like Zn, Cu, Mn and Ca [
34,
35]. Sulphur shows significant differences only at t1 (
Table 2). In this case, the leaves of Treat 4 show the highest values, while the lowest values are found in Treat 1.
Also, the microelements leaf content shows differentiation between the two sampling periods (t1 and t2) (
Table 3). The elements that show the largest variations is Zn, followed by Mn and Fe. As they are involved in cone formation, the translocation of these microelements from leaves to cones is a likely hypothesis [
2]. The opposite trend may be observed for B, which might be due to a greater B intake in the second period, especially in Treat 4, that shows the highest leaf B concentration (
Table 3).
3.2. Anatomical Analysis of Leaves by Light Microscopy
The transverse section of Untreat leaves shows a large central vein and a thin leaf mesophyll (125.05 ± 2.62 μm) (
Figure 1A). The central vein consists of a large cribro-vascular bundle immersed in a parenchyma and surrounded, in the adaxial and abaxial part, by a collenchyma tissue. The foliar mesophyll appears to be made up of 2–3 layers of palisade tissue cells with an average thickness of 75.54 ± 6.21 μm (
Figure 1A). The spongy tissue is on average 23.28 ± 1.36 μm thick (
Figure 1B). The palisade tissue represents about 70%, while the spongy mesophyll is about 25% of the entire mesophyll/leaf blade. The upper epidermis is generally made up of large cells with a longitudinal diameter of 32.68 ± 1.41 μm and a transverse diameter of 15.48 ± 0.99 μm; these cells then appear elongated longitudinally. The thickness of the cuticle in the upper epidermis is 2.47 ± 0.61 μm (
Figure 1A). The cells of the lower epidermis are smaller than those of the upper epidermis: their average longitudinal diameter is 11.28 ± 0.98 μm, while the transverse one is 12.03 ± 1.02 μm. These cells then appear isodiametric (
Figure 1A). The thickness of the lower epidermis cuticle is 2.43 ± 0.61 μm.
To better understand the effect of foliar treatments in leaf anatomical parameters, data are summarized in
Table 4. The two sampling times (t1 and t2) did not show any differences from a structural point of view, and for this reason the data averages of the two times are shown in
Table 4.
The results show significant differences for some leaf anatomical features. The leaves of the Treat 2 showed a significant increase in cuticle, of both the upper and lower epidermis (
Figure 1B;
Table 4). In tomato, some authors [
36] indicate that the concentration of foliar N affects the thickness of the epidermal cuticle. It appears quite established that high concentrations of N lead to a decrease in the cuticle thickness; peach trees treated with different concentrations of N have produced fruits with different cuticle thickness [
5], and thinner if N concentration was high.
In our study organic nitrogen has positive effects on increasing the cuticle thickness. Furthermore, the percentage of palisade tissue was also found to be significantly lower in Treat 2. Treat 2 is characterized by a low input of K in exchange for a high input of organic N (
Table 1). Hu et al. [
37] indicated that, in
Brassica napus L., the major difference in leaf anatomical structure with reference to K treatments was that palisade tissue thickness was reduced with increased K supply, and spongy tissue cell size and tissue thickness were equally reduced under the K
120 vs. K
60 and K
0 treatments (
Table 4). In our study on hop, it results that a high input of N and low input of K lead to reduced growth of the palisade tissue.
3.3. Anatomical Analysis of Leaves by Electronic Microscopy (SEM)
Leaf stomata were observed in the different treated samples (
Figure 2a,b).At the first period of treatments (t1) the stomatal density of Untreat and Treat 1 exhibited no statistical difference with a respective 671.64 and 689.55 stomata per mm
2 (
Figure 2a), while Treat 3 showed the greatest value (794.03 stomata per mm
2;
Figure 2b) (
Table 5). At the second period (t2), again Untreat and Treat 1 showed no statistically significant difference (674.6 and 710.45 stomata per mm
2), Treat 2 displayed an increase of stomata (728.36 stomata per mm
2), while Treat 3 and Treat 4 recorded lower values (662.69 and 567.16 stomata per mm
2, respectively) in comparison with Untreat (
Table 5). Comparing the treatments at t1 and t2, it can be observed that while Untreat remains substantially unchanged, Treat 1 shows a slight increase in stomatal density, while all the other treatments (Treat 2, Treat 3 and Treat 4) show a decrease in the number of stomata per unit area (
Table 5). Regarding stomata diameters, higher values were measured in Untreat (19.4 μm), Treat 4 (19.9 μm) and Treat 3 (19.7 μm) at t1, and in Treat 2 (18.5 μm) and Treat 4 (18.7 μm) at t2 (
Table 5). At t1, Treat 3 showed higher PD/ED (1.97), while at t2 all treatments recorded a lower ratio compared to Untreat (
Table 5).
Two types of glandular trichomes were identified on hop leaves [
38]: large peltate-type known as lupulin glands, found on the abaxial side (
Figure 2c), and stalked capitate glands, called also bulbous trichomes, which are much smaller, fewer in number, and consist of a stalk and four head cells, occurring on both adaxial and abaxial surfaces, located mostly along the vessels, but rarely present on the lamina (
Figure 2d black arrow). Since the latter trichomes are present in small numbers, only lupulin glands were considered in this study. At t1 all treatments showed no statistical differences with Untreat (
Table 5). At t2, while Untreat remained unchanged, all treatments underwent a decrease in the density of glandular trichomes (
Table 5). Two types of non-glandular hair are present on hop leaf: the predominantly cystolith-containing hairs are generally restricted to the adaxial leaf surfaces and contain calcium carbonate crystals (
Figure 2e,f), as already described by Dayanandan and Kaufman [
39]; the second is the longer and rarer type of slender trichomes present only on the midribs and major veins of the abaxial leaf surfaces (
Figure 2d black triangle). Since the cystoliths are more abundant and uniformly distributed, only this type was considered in this investigation. At t1, Treat 3 had the same density of Untreat, in practice (12.85 and 12.7 hairs per mm
2, respectively), while Treat 1, Treat 2 and Treat 4 outlined a small decrease (
Table 4). At t2, Treat 2 and Treat 3 showed no statistical differences with Untreat, while Treat 1 and Treat 4 recorded the respective highest (16.84 hairs per mm
2) and lowest density (7.68 hairs per mm
2).
The polar and equatorial diameter ratio (PD/ED) is associated with guard cell shape and is an important stomata feature. A higher ratio is correlated with an elliptic shape, characteristic of more functional stomata. Conversely, a rounded shape (smaller PD/ED) is associated with lower stomata functionality [
40]. Thus, in this study, in the first period, Treat 3 recorded the best results in terms of increased number of stomata and their functionality (PD/ED ratio), while in the second period all treatments showed a worse stomata functionality, compared to Untreat.
The non-glandular trichomes analysed are hairs containing cystoliths. The cystoliths (
Figure 2d,e) are intracellular mineralized inclusions present in the leaf of some Angiosperm families (Urticaceae, Moraceae, Ulmaceae, Cucurbitaceae and Acanthaceae). They consist mostly of calcium carbonate but a small amount of magnesium is also present [
41], which probably helps to stabilize the amorphous calcium carbonate [
42]. The high density of hairs containing cystoliths in Treat 1 at time t2 could be a consequence of the higher dose of Mg provided in this treatment; analogously, the lower Mg value in Treat 4 might be explained by the absence of this element.
3.8. DPPH Free Radical Scavenging Activity Test
In t1, Treat 3 e Treat 4 showed the highest amount of radical scavenging activities with 162.54 and 166.51 Trolox equivalent respectively (
Figure 7); their difference with the other treatments was the higher presence of K, Fe and Zn in these two treatments (
Table 1); thus, probably the increase of radical scavenging activity was promoted by one or all the microelements, especially Fe, supplied to the plants. In the second part of the study, at t2, the radical scavenging activity decreased (except for Treat 1) with no significant differences among the treatments (
Figure 7), despite a higher activity registered in Treat 1, which in t2 was differentiated from the other treatments for the a higher Mg supply. In t2, only Treat 2 contained Zn and Fe (in lower quantity compared to Treat 3 and Treat 4 in t1), but no K, and the results indicate either a lower impact of this elements on antioxidant activity in t2, or that the quantities involved are not sufficient to increase this activity. In the literature, the studies on the effects of fertilization on free radical scavenging activity are more frequently related to the nitrogen fertilization: Coria-Cayupán et al. [
46], in a study on lettuce, observed the low antioxidant activities in leaves in the presence of urea and mineral nitrogen fertilization, in comparison with other fertilization treatments. In another study on marigold under different organic foliar fertilizations, free radical scavenging activities were higher in the treatments that implied less nitrogen quantity, and lower in the treatments richer in nitrogen [
50]. Nonetheless, these studies do not help to elucidate the results that we obtained, because in the first period the high radical scavenging activity was observed in the treatments with high N content.