3.1. Determining the pH and Electrical Conductivity of the Crude Extracts
It was possible to establish a negative correlation between pH and electrical conductivity (EC) according to Pearson’s coefficient (R
2 = 0.9545); as one variable increased, the other decreased in a way that was close to linear (
Table 1).
Comparing the different extracts, the AA extract stands out both because its pH (3.48) is much lower than the others and because its EC (22.50) is much higher, while the other extracts show similar values for these parameters.
Based on the results, considering that pH can negatively affect plants when it is below 5.5 and that EC is an index of dissolved salts, which in high concentrations (above 2 dS/m) can cause extracts to be too conductive for plants, provoking cells into osmotic stress and thus having a negative impact on the development of the plants [
19]. It became clear that the AA extract, compared to the others, would have to be diluted with water in lower concentrations than the other raw extracts in order to achieve a neutral/slightly acidic pH and an EC close to 1 dS/m, which according to Hernández-Herrera et al. [
20] showed the best results for algae extracts made with
Sargassum liebmannii,
Padina gymnospora (
Phaeophyceae),
Caulerpa sertularioides and
Ulva lactuca (Chlorophyta), regarding germination and seedlings development of
Solanum lycopersicum L.; 0.2% seaweed concentrations showed the best results since plants are more sensitive to salinity during germination and seedling growth stages.
3.2. Determination of Crude Extract Solids
Table 2 shows the total solids (TS), volatile solids (VS) and fixed solids (FS) present in each extract. Given the previous information, namely the method of preparation and the raw material of the extracts, the AA extract, based on microalgae, was the statistically densest (
p < 0.05) and therefore had the highest TS values (118.499 ± 2.233); the CJ extract, prepared at the lowest concentration (0.035 g/mL), had the lowest TS value (10.335 ± 0.046). UA and GR extracts presented very similar values (23.595 ± 0.095 and 22.098 ± 0.113, respectively). These values were expected given that the method used to prepare them was similar, although the raw materials were partly different. Regarding VS, the AA extract showed the statistically (
p < 0.05) highest values (1015.250 ± 3.018), while GR showed the lowest (984.386 ± 2.936); only the AA extract showed statistically significant differences compared to other extracts. The AA extract had the highest amount of FS (15.662 ± 1.109), while CJ had the lowest (6.390 ± 2.273), with significant differences between these two (
p < 0.05).
It was not possible to compare these parameters with the literature as they depend on the concentration at which the extracts were prepared, the extraction methods and the material used, which is mostly unknown for UA, GR and AA, while for CJ, no information was found regarding these parameters.
3.4. Phenolic Compound and Antioxidant Content of Algae Extracts
Regarding phenolic compounds (
Figure 2), the CJ extract presented the lowest value of the four extracts, as expected, with 0.064 g GAE/L since the other extracts have
Ascophyllum nodosum (
Phaeophyceae), known for having relatively high amounts of phenolic compounds like phlorotannins that exert a powerful antioxidant activity [
22]. The GR extract had the highest phenolic content (0.489 g GAE/L), followed by the UA extract (0.449 g GAE/L) and AA (0.415 g GAE/L); these values are still lower than those reported by Ertani et al. [
23], with total phenolics ranging between 0.555 g/L and 1.933 g/L in five commercialized biostimulants from
Ascophyllum nodosum. Regarding antioxidants, the UA extract had the highest value (0.402 g AAE/L), followed by the GR extract (0.367 g AAE/L), with statistical differences with the CJ extract, which had relatively low values (0.020 g AAE/L). It was possible to establish a strong positive correlation for UA, CJ and GR extracts between PC and AO according to the Pearson coefficient (R = 0.9859), showing a close linear relationship between these two variables.
The AA extract, contrary to what was expected regarding the phenolic compounds present in this extract, showed no inhibition of the ABTS
+ radical or negative values, indicating a lack of antioxidant activity or a pro-oxidant effect, as described by Castro [
24], where in other algae extract with
Codium tomentosum, a green seaweed, it was not possible to obtain coherent and concordant results using this method. Possible future approaches to this parameter could include the evaluation of antioxidants using HPLC or other methods such as TEAC (Trolox Equivalent Antioxidant Capacity), ORAC (Oxygen Radical Absorbance Capacity) DPPH (2,2-Diphenyl-1-picrylhydrazyl) and FRAP (Ferric Reducing Antioxidant Power), which, like the ABTS method, evaluate antioxidant capacity through different oxidation/reduction mechanisms [
25].
3.5. Germination Tests
Figure 3 shows the germination percentage (GP) of turnip greens seeds seven, ten, and 14 days after incubation (DAI) and the influence that each extract had on their germination (different letters indicate significant differences between these groups (
p < 0.05)). The extracts CJ 0.05, AA 0.003 and AA 0.01 were the ones that had the greatest PG at the 14th DAI (96%), while the GR, for all its concentrations, showed the least promising results for PG 88%, 89.3% and 89.3%, respectively, for GR 0.05, GR 0.10 and GR 0.12.
The CJ 0.05 extract showed the best results in terms of GP (7, 10 and 14 DAI), followed by the AA 0.003 extract, with significant differences when compared to the UA 0.12, GR 0.05, GR 0.10, and GR 0.12 extracts and control. The GR extract, in all its concentrations, showed the least results, even with lower values than the control. No treatment showed significant differences when compared to the control, although up to the 10th day, the control showed one of the highest values since seeds are sensitive to salinity during imbibition, despite the effect of salt being merely osmotic until a hydration threshold is surpassed [
26].
The control and extracts GR 0.05 and CJ 0.05 stimulated the radicle length (RL) the most (7.50; 9.86 and 8.40 cm, respectively), obtaining the highest results (
Figure 4). The lower the extract concentration, the greater was the radicle growth. This is because when the extract concentration is lower, there are less nutrients in the extract, so the radicles will be stimulated to grow, searching for nutrients. On the other hand, the treatments AA 0.01, AA 0.05 and AA 0.003, (2.27; 4.45 and 5.73 cm, respectively), were the ones that led to the lowest RL, which is in line with the same logic: knowing that this extract is the richest in nutrients, the roots were not stimulated to grow in search of them, resulting in lower radicle growth.
As for epicotyl length (EL), all the treatments, apart from the AA 0.003 (7.49 cm) and AA 0.005 (7.92 cm) extracts, showed significant differences when compared to the control (6.00 cm), which had the worst result in this parameter. This shows that all the extracts, except those mentioned, positively stimulated the growth and development of the seedlings. The UA 0.12 treatment showed the best EL values, followed by their lowest concentrations of UA 0.10 and UA 0.05 (11.100; 10.693 and 10.547 cm, respectively).
Considering all the parameters analysed in the germination tests and the economic factor, the extracts UA 0.12, CJ 0.05, GR 0.10 and AA 0.003 were chosen to be used in the test with the potted plants.
Among the selected concentrations, the UA 0.12 extract showed the highest PG% on the 14th day, as well as the highest EL of all the treatments, although there were no significant differences between the different concentrations.
The CJ 0.05 extract showed the best results in terms of GP and EL, in line with its higher concentrations, as well as a higher RL. Taking these factors into account, as well as the economic factor, this extract was selected at the lowest concentration tested for the pot test.
The GR 0.10 extract had a poor GP% compared to the other treatments, but it had the highest EL value and a modest RL value of its concentrations. GR 0.05 showed a very high RL, but a lower EL and GP%, than GR 0.10, leading to a disparity between EL and RL compared to the other treatments, which may be related to the lack of nutrients in the root and not to its biostimulant potential, so GR 0.10 was chosen.
Finally, the AA extract showed the lowest results in terms of RL, with only AA 0.003 showing a RL that was not significantly different when compared to the control and EL that was still higher than the control. We therefore opted for AA 0.003, which had the closest concentration to that suggested by the manufacturer.
3.6. Trial of Radishes in Pots with Extract Applications
The leaves on the control plants were generally more yellowish in colour while the plants treated with the other extracts were green (
Figure 5). With the Dualex Force-A measurements (
Table 4), we can see that the control plants, in general on day 47, showed lower flavonoid values in the leaves compared to the others, as well as large fluctuations in this parameter and, consequently, in the nitrogen balance index (NBI). Compared to the control plants, on average, plants treated with the extracts managed the heat, dehydration and radiation stresses better in increasing the production of flavonoids, proving the potential of extracts as stress-mitigating agents that protect and improve plants [
8,
27].
There were no statistically significant differences in the Chl and Anth parameters related to chlorophyll and anthocyanidins between the treatments (
Table 4). There were, however, significant differences in the flavonoid index (Flav) and, consequently, in the nitrogen balance index (NBI), even on the 34th day, when no differences were expected.
Comparing the values, where statistically significant differences were observed, it is worth highlighting the flavonoids in AA which, as well as being the only group to show an increase in flavonoids after the first foliar application, also showed the highest values for this parameter on the 44th day and on the 47th day after the second application. The other extract treatments, UA, CJ and GR, however, showed a drop in flavonoid values in the leaves between the 34th and 44th days and between the 44th and 47th days, although, on average, they are higher than the control.
In general, there were no significant statistical differences between treatments in terms of plant morphology, except for the vertical diameter of the root (
Table 5), where the UA and CJ extracts showed the highest values, with significant differences compared to the control related to higher growth. The control radishes, in all the parameters, showed greater mutual disparity (higher standard deviation) and less uniformity compared to the radishes subjected to treatments UA, CJ, GR and AA.
In terms of the average total weight of the radish plants, the ones treated with UA were the heaviest (60.08 ± 15.29), and the control plants were the lightest (42.02 ± 28.69). The CJ, GR and AA treatments showed similar average values, with AA being slightly heavier.
When evaluating the weight of the aerial part, treatment AA had the highest values (19.52 ± 5.99), followed by treatment UA (19.15 ± 3.87), C (18.62 ± 17.02) and treatments CJ (17.02 ± 4.46) and GR (16.30 ± 5.31), which were lower than C. While the control had the highest number of leaves per plant (6.8 ± 1.19), the AA treatment had the lowest (5.7 ± 0.67), contrasting with the weight of the aerial part through a more robust aerial part and a more vigorous and denser stem (
Figure 5). On average, regarding leaf evaluation, all the treatments had larger leaves (M) than the control (S) (
Figure 1).
Regarding the weight of the root, an edible and valuable part of the radish, the results for weight as a function of diameter are in agreement with those obtained by Godlewska et al. [
27]; all the treatments resulted in a higher weight compared to the control, with the UA extract showing the best results, with an average of 80% more weight compared to the control, while the CJ, GR and AA treatments showed an average of 41.72%, 42.72% and 48.26% greater weight, respectively, when compared to the control. In general, the foliar application of seaweed extracts led to a mitigation of the abiotic stress on the plants and better vegetative growth and, consequently, better and higher radish yields; this is in accordance with the work of Mahmoud et al. [
28].
As for the nutritional properties of the radishes treated with different algae, they are shown in
Table 6.
As for moisture (%), the results were similar to those obtained by Ruzzi et al. [
29]. Radishes treated with the GR extract and control stood out from the others (
Table 6) and were significantly wetter than radishes from the AA treatment. Radishes treated with CJ (90.62 ± 0.26) and UA (90.56 ± 0.38) showed no significant differences with the others. The GR extract induced a higher % of protein in the radishes, and similarly to the water content of the radishes, only the GR treatment and the control produced significantly more protein than CJ. These quantities are in accordance with those obtained by Yousaf et al. [
30] between 1.62% and 3.02% protein by dry matter in a study with application of nitrogen and magnesium fertilizer in radishes. There were no statistically significant differences in phosphorus (
p < 0.05) production in radishes. The AA treatment had the highest phosphorus value (706.10 ± 63.83) compared to the other radishes subjected to the different treatments, while the control had the lowest values (363.39 ± 9.78), even lower than the 441 mg by 100 g dry matter mentioned by Godlewska et al. [
27].
The extracts that led to a decrease in phenolic compounds (PC) in the radish leaves (
Table 3) were the ones that showed the highest content of these compounds in the edible roots (
Table 6), similar to what was reported by Godlewska et al. [
27], where the application of some biological plant extracts to the leaves led to a decrease in total phenolic compounds in the leaves, but subsequently led to an increase in the radish roots. Plants treated with AA showed similar values to the control at root level regarding PC and AO. The GR treatment showed the highest values of PC (0.12121) and AO (0.0754 ± 0.0000), with all treatments except AA (0.0554 ± 0.0009) showing significant differences compared to the control. PC, C and AA yielded lower values than the ones described by Gamba et al. [
31] (ranging between 0.061 to 0.146 mg GAE/g fm).
Based on the results obtained, all the extract treatments resulted in a notable stimulation of plant growth/yield, as well as improved nutritional properties. The UA extract stood out as the treatment that caused the greatest increase in plant weight. The GR extract, on the other hand, stood out in terms of nutritional quality, having promoted a higher % of crude protein as well as more phenolic compounds and antioxidants. The AA extract led to healthier aerial parts, with greater weight and more flavonoid content and an increase in phosphorus levels in the roots, although it showed similar values to the control in terms of phenolic compounds and antioxidants in the roots. As for the CJ extract, it showed promising results, and possible future approaches could involve merging it with another extract like UA and GR. We can conclude that the treatments contributed to mitigating abiotic stress (particularly water stress) results that are in line with the work of Rasul et al. [
32], in which the application of an extract based on
Ascophyllum nodosum led to the triggering of cellular mechanisms in
Arabidopsis thaliana that contributed to its growth and tolerance to water stress, as well as reducing the production of reactive oxygen species (ROS). We can also conclude that the extracts promoted the absorption and assimilation of nutrients, in agreement with the study by Chami and Galli [
33] where the application of seaweed-based extracts led to a reduction in total fertilization units by 13%, improving the harvest and increasing agronomic efficiency five- to nine-fold compared to the conventional nutrition program, thus playing a crucial role in achieving sustainable agriculture and increased resilience to climate change.