*Article* **Bioactive Compounds, Antioxidant Activity, and Mineral Content of Wild Rocket (***Diplotaxis tenuifolia* **L.) Leaves as Affected by Saline Stress and Biostimulant Application**

**Ida Di Mola 1,† , Spyridon A. Petropoulos 2,\* ,† , Lucia Ottaiano <sup>1</sup> , Eugenio Cozzolino <sup>3</sup> , Christophe El-Nakhel <sup>1</sup> , Youssef Rouphael <sup>1</sup> and Mauro Mori <sup>1</sup>**


**Featured Application: The results of the present work provide useful insights into the response of** *Diplotaxis tenuifolia* **L. plants to salinity stress and biostimulant application under successive crop cycles. Considering the complex effects of biostimulants on vegetable crops, providing new information regarding the positive effects of this agronomic practice under salinity stress and variable growing conditions will be useful for farmers and crop production stakeholders in their efforts to address the increasing soil salinization and degradation of the quality of irrigation water.**

**Abstract:** The availability of irrigation water of good quality is decreasing due to soil salinization and the deterioration of aquifers. Moreover, ongoing climate change severely affects crop production and necessitates the intensification of cropping systems in order to ensure food security at a global scale. For this purpose, the aim of the present study was to evaluate the mitigating effects of two natural biostimulants on *Diplotaxis tenuifolia* L. plants cultivated at different salinity levels (EC of 0 dS m−<sup>1</sup> , 2 dS m−<sup>1</sup> , 4 dS m−<sup>1</sup> , and 6 dS m−<sup>1</sup> ) and harvested at six consecutive cropping cycles. The tested factors showed a varied combinatorial effect on the tested parameters. These findings indicate the importance of considering growing conditions and cropping periods when applying biostimulants in *D. tenuifolia* plants under salinity stress. Antioxidant activity and bioactive compounds, such as total phenols, carotenoids, and total ascorbic acid, were variably affected by salinity, biostimulant application, and harvesting time, while mineral profile was also affected by the tested factors depending on the combination of factors. Finally, nitrate content showed decreasing trends with increasing salinity, while biostimulant application resulted in the higher accumulation of nitrates compared to the untreated plants. Although biostimulant application seems to alleviate the negative effects of salinity stress, the effect of growing conditions, as indicated by successive crop cycles, is also important for the response of *D. tenuifolia* plants to saline conditions and biostimulant application.

**Keywords:** antioxidant activity; chlorophylls; carotenoids; tropical plants extracts; protein hydrolysates; total phenols

#### **1. Introduction**

Modern crop production has to cope with increasing soil degradation and the low availability of irrigation water due to salinization, with major consequences on total and marketable crop yield as well as on the quality of the final products [1–3]. Moreover, approximately 20% of available agricultural land is affected by salinity [3], while it is expected that the current soil salinization trends will result in 50% of arable land being

**Citation:** Di Mola, I.; Petropoulos, S.A.; Ottaiano, L.; Cozzolino, E.; El-Nakhel, C.; Rouphael, Y.; Mori, M. Bioactive Compounds, Antioxidant Activity, and Mineral Content of Wild Rocket (*Diplotaxis tenuifolia* L.) Leaves as Affected by Saline Stress and Biostimulant Application. *Appl. Sci.* **2023**, *13*, 1569. https://doi.org/ 10.3390/app13031569

Academic Editor: Luca Mazzoni

Received: 31 December 2022 Revised: 21 January 2023 Accepted: 24 January 2023 Published: 26 January 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

salt-affected by 2050 [4], mainly due to poor agricultural practices and the impact of climate change [5]. In this context, vegetable crops are highly affected by the changing conditions, especially the leafy species that are considered to be more prone to salinity than other crops [4,5]. Therefore, cropping systems and agronomic practices have to be reconsidered in order to address the current scenario and ensure food security in the mid- and long-term, especially in the arid and semi-arid regions of the world that are most affected by salinization and irrigation water shortages [6–8]. Moreover, halophyte species, which include important bioactive compounds with health-beneficial properties, could be integrated into farming systems and contribute to food security as well as land reclamation [9–11]. Among the several cropping strategies for mitigating the adverse effects of salinity, biostimulant application is proposed as an innovative and ecofriendly tool that allows cultivation under unfavorable conditions due to abiotic and biotic stressors [12–15].

Several studies have reported the beneficial effects of biostimulants on various crops grown under saline conditions. For example, El-Nakhel et al. [16] recently suggested that the application of legume-derived protein hydrolysates significantly ameliorated the negative impacts of high salinity (EC levels up to 6 dS m−<sup>1</sup> ) in pot-grown spinach plants. Moreover, Lucini et al. [17] reported similar beneficial effects on the crop performance and metabolite profile of pot-grown lettuce plants subjected to high salinity. Rouphael et al. [18,19] also suggested that the biostimulants obtained from seaweeds or vegetal proteins may increase tolerance to high salinity in lettuce plants through alterations in metabolic processes that induce the biosynthesis of stress-related compounds. Van Oosten et al. [20] suggested that the key mechanisms of action that are related to the beneficial effects of protein hydrolysates and plant extracts rich in amino acids as well as peptides on crops are due to osmoprotection and the scavenging of radicals at the shoot level, in addition to metal chelation and improved nutrient availability at the rhizosphere level.

Irrigation with saline water may induce metabolic changes that affect the chemical composition of vegetable products, while it may also affect the visual appearance and marketability of leafy products [21]. Apart from negative effects, moderate salinity levels may improve the quality of vegetable products through the enhancement of nutraceutical compound content (e.g., phenolic compounds, carotenoids, tocopherols, vitamins, and other antioxidant compounds) or by contributing to the taste and aroma attributes [22]. Therefore, the combinatory effects of salinity and biostimulant application have to be extensively studied in order to identify those salinity thresholds and biostimulant products that allow the production of high-quality end-products without compromising marketable yield. Moreover, a thorough evaluation of germplasm variability has to be considered, since a differential response to salinity is expected depending on the genotype [23].

Successive harvesting is a common practice in various leafy greens and, according to the literature, may significantly increase the overall yield when compared to single harvesting practices [24]. Moreover, aside from increased yields this technique is costas well as labor-efficient and may allow for more growth cycles throughout the growing period [25]. The number of harvests is dependent on the genotype and the growing conditions, since not all of the cultivars and growing periods are suitable for this technique due to susceptibility to inflorescence formation under specific temperature and photoperiod conditions [26]. Considering that successive harvesting may occur under variable growing conditions, this practice may also affect the chemical composition and quality of leafy vegetables, such as rocket [27].

Rocket (*Eruca sativa* Miller) and wild rocket (*Diplotaxis tenuifolia* L.) are two leafy vegetables of the Brassicaceae family with increasing commercial interest during recent years due to their special characteristics in terms of nutritional value, aroma, and taste [28]; however, in addition to the high nutritional value of its edible leaves there is great concern regarding the tendency of nitrate accumulation, which is considered to be an anti-nutritional factor and may pose significant threats to human health [29–31]. Therefore, special attention should be given to those agronomic practices that may contribute to the reduction in leaves' nitrate content, such as the cropping season and time of harvesting, nitrogen fertilization

rates, or nitrogen form, among others [29,32–34]. In this context, the use of biostimulants has been associated with an increased content of nitrate in leafy vegetables, although contrasting results have been reported in the literature [35–37]. These findings indicate that biostimulant application should be carefully considered in order to avoid any undesirable effects on the quality of the final product, since it seems that the impact on nitrate content depends on the species, the biostimulant product, and the cropping system [30].

In this work, a pot experiment was conducted in order to evaluate the effect of two biostimulatory products on the mineral contents and chemical profiles of *Diplotaxis tenuifolia* plants grown under saline conditions. Moreover, the agronomic practice of successive harvesting was implemented, aiming to determine any effects of growth stage on plants' responses to salinity and biostimulant application.

#### **2. Materials and Methods**

#### *2.1. Experimental Settings and Design, Crop Management, and Soil Sampling*

The experiment was carried out at the experimental field of the Department of Agricultural Science in Portici (Naples, 40◦ 48.8700 N; 14◦ 20.8210 E; 70 m a.s.l.) under a plastic greenhouse (2020–2021). The species chosen for the test was wild rocket (*Diplotaxis tenuifolia* L.) cv. "Reset", marketed by Maraldi Sementi Srl (Cesena—FC, Italy), with the following characteristics: green leaves with medium-sized lobes, high potential yield, and a noticeable flexibility in terms of growing conditions that makes it useful for cultivation in any season.

For the test, pots with a 0.38 m<sup>2</sup> surface and a 0.70 m height were used; they were filled with a sandy soil, whose physical and chemical properties are reported in Table 1.


**Table 1.** Physical and chemical properties of the test soil.

Three seedling groups per pot (about 15–20 seedlings each) were transplanted on 8 October 2020, and harvested 6 consecutive times from 25 November 2020 until 20 May 2021, hereafter referred to as I, II, III, IV, V, and VI. Regarding fertilization, only nitrogen in the form of ammonium nitrate (26%) was applied, provided at a rate corresponding to 18 kg ha−<sup>1</sup> per each cycle. For the first cycle, it was added 18 days after transplant (DAT), while in the successive cycles this was carried out at approximately 4 days after harvest (DAH) of the previous growing cycle, ranging between 2 and 9 DAH depending on the conditions. No pesticide treatments were carried out.

The experimental design was a split-plot design, with saline irrigation as the main experimental factor and biostimulant application as the subfactor. The saline irrigation treatments included the following: EC0: irrigation with tap water; EC2: irrigation with water that had an EC of 2.0 dS m−<sup>1</sup> ; EC4: irrigation with water that had an EC of 4.0 dS m−<sup>1</sup> ; and EC6: irrigation with water that had an EC of 6.0 dS m−<sup>1</sup> . There were three biostimulant application treatments: 1. untreated—control; 2. treated with Auxym®, a tropical plant extract hereafter referred to BA; and 3. treated with Trainer®, a protein hydrolysate derived from legumes, hereafter referred to BT. Both biostimulants are marketed by Hello-Nature Italia Srl (Rivoli Veronese, Italy), and they were applied as a foliar spray three times per each cycle (except for the last cycle, which was shorter; therefore, only two applications were performed) at doses of 2 mL L−<sup>1</sup> and 3 mL L−<sup>1</sup> for Auxym®and Trainer®, respectively. Starting with harvest II, the formation of new leaves was taken into account to decide when to perform the first biostimulant application. All of the treatments were replicated 3 times for a total of 36 pots.

The water losses were calculated by the Hargreaves method [38], and were fully restored by 26 irrigations over the six cycles. The desired EC for saline treatments was obtained by adding NaCl to tap water, as reported by Di Mola et al. [39]. A total of 28.5 L per pot was applied with 34.6, 69.1, and 103.7 g of NaCl per pot in the EC2, EC4, and EC6 treatments, respectively. At the first cycle, the first three irrigations were made with tap water for all of the treatments in order to promote the rooting and establishment of seedlings. To monitor the trend in the soil electrical conductivity, at each harvest the soil was sampled at a 0–20 cm depth and the soil water solution extraction method (1:5 dilution) was used to measure soil EC (EC1:5; Basic 30 CRISON electrical conductivity meter; Crison Hach, Barcelona, Spain).

The air temperature under the plastic greenhouse was monitored by a Vantage Pro2 (Davis Instruments, Hayward, CA, USA) weather station on an hourly basis. Data are reported as the daily minimum and maximum temperatures (Supplementary Figure S1).

#### *2.2. ABTS and Hydrophilic Antioxidant Activity, Total Phenols, and Total Ascorbic Acid Analysis*

At each harvest, fresh leaves for each treatment and replicate were collected and stored in a freezer at −80 ◦C, after which they were lyophilized for the determination of ABTS and hydrophilic antioxidant activity (ABTS AA and HAA, respectively), total phenols, and total ascorbic acid (TAA).

The ABTS AA was determined on 200 mg of freeze-dried sample extracted with methanol. The measurement of the absorbance was assessed spectrophotometrically (Hach DR 2000, Hach Co., Loveland, CO, USA) at 734 nm according to the methods of Re et al. [40]. The ABTS AA was expressed as mmol of Trolox per 100 g of dry weight (dw).

The HAA was assessed, after extraction with distilled water, by the N, N-dimethylp-phenylenediamine (DMPD) method [41]. It was measured spectrophotometrically at 505 nm, and the values were expressed as mmol of ascorbic acid per 100 g of dry weight (dw).

The TAA was also spectrophotometrically measured at 525 nm according to the procedure of Kampfenkel et al. [42].

The total phenolic content was determined spectrophotometrically at 765 nm according to the Singleton et al. method [43] and expressed as mg of gallic acid per 100 g−<sup>1</sup> of dw.

#### *2.3. Chlorophylls and Carotenoids Analysis*

Chlorophylls (chlorophylls a and b) and carotenoids were assessed spectrophotometrically on 1 g of fresh leaves, after extraction with ammoniacal acetone, according to the method described by Wellburn [44] at 662 and 647 nm for chlorophylls a and b, respectively, as well as at 470 nm for carotenoids. They were expressed as mg g−<sup>1</sup> fresh weight (fw).

#### *2.4. Mineral Content Analysis*

The determination of nitrate, Na, K, Ca, Mg, Cl, S, and P was carried out on 250 mg of dried and finely ground leaf tissues, suspended in 50 ml of ultrapure water (Milli-Q, Merck Millipore, Darmstadt, Germany), followed by a shaking water bath (ShakeTemp SW22, Julabo, Seelbach, Germany) at 80 ◦C for 10 min. The supernatant was filtered and processed on an ion chromatograph (ICS3000, Thermo Scientific™ Dionex™, Sunnyvale, CA, USA) as described previously by Rouphael et al. [45]. Mineral concentrations were expressed as mg g−<sup>1</sup> dw, while nitrate concentration was converted into mg kg−<sup>1</sup> fw.

#### *2.5. Statistical Analysis*

All of the results were subjected to a three-way analysis of variance (ANOVA), considering salinity levels (S), biostimulant application (B), and harvesting time (H) as factors. The analysis was performed with the SPSS software package (version 22, Chicago, IL, USA). When significant effects were detected, the means were separated using Tukey0 s honestly significant difference (HSD) test at *p* < 0.05.

#### **3. Results and Discussion**

#### *3.1. Soil Electrical Conductivity*

The soil electrical conductivity linearly increased in all of the saline treatments, but with different trends; at the last harvest the increase over the first harvest was more than double in EC2, and about three times in EC4 as well as EC6 (Figure 1). Moreover, the EC values were significantly higher in EC6 compared to the rest of the salinity levels, while the EC0 treatment presented the lowest overall values in all of the harvestings, being significantly lower than the rest of the treatments at the last four harvestings (II–IV). Finally, all of the salinity treatments differed among each other in the last three harvestings. Similarly to our findings, Mori et al. [46] also suggested an increase in soil EC values with the increasing salinity of the nutrient solution, which is attributed to the gradual buildup of Na and Cl in soil solutions due to inhibited water uptake from plants. Moreover, Schiattone et al. [47] also suggested a gradual increase in soil EC with increasing salinity and multiple harvestings in wild rocket plants, although the increase was less profound between different harvesting times of the same salinity level compared to the increase between the different salinity levels. According to Feng et al. [48], the application of saline irrigation water resulted in an increase in EC levels in soil solutions which evolved with the progression of the growing cycle, while similar findings were recorded for the use of brackish water in cotton plant irrigation [49]. *Appl. Sci.* **2023**, *13*, x FOR PEER REVIEW 6 of 18

**Figure 1.** Trends in soil electrical conductivity (dS m<sup>−</sup>1) over the six harvesting times (I–VI). Vertical bars above each mean indicate standard deviation (SD; n = 3). **Figure 1.** Trends in soil electrical conductivity (dS m−<sup>1</sup> ) over the six harvesting times (I–VI). Vertical bars above each mean indicate standard deviation (SD; n = 3).

#### *3.2. Antioxidant Activity and Compounds 3.2. Antioxidant Activity and Compounds*

The analysis of variance of the data about antioxidant activity (hydrophilic antioxidant activity (HAA) and ABTS assay), total phenol content, total ascorbic acid content (TAA), chlorophylls (a, b, and total chlorophyll), and total carotenoids is presented in the Supplementary Material (Table S1). The analysis did not show any significant interaction of the three tested factors, namely salinity (S), biostimulants (B), and harvesting time (H). On the other hand, significant interactions were recorded The analysis of variance of the data about antioxidant activity (hydrophilic antioxidant activity (HAA) and ABTS assay), total phenol content, total ascorbic acid content (TAA), chlorophylls (a, b, and total chlorophyll), and total carotenoids is presented in the Supplementary Material (Table S1). The analysis did not show any significant interaction of the three tested factors, namely salinity (S), biostimulants (B), and harvesting time (H). On the other hand, significant interactions were recorded between B × H, S × H, and S × B for

between B × H, S × H, and S × B for specific parameters, e.g., total phenols for the B × H interaction; HAA, ABTS, and TAA for the S × H interaction; and HAA, ABTS, total

Supplementary Table S3 presents the effect of biostimulant application and harvesting time on HAA, the ABTS assay, total phenols, and TAA content in *Diplotaxis tenuifolia* leaves, regardless of salinity level. As indicated in Supplementary Table S1, the only significant interaction was recorded in the case of total phenol content. The highest total phenol content (Figure 2) was recorded in *D. tenuifolia* leaves collected from plants treated with the Auxym® biostimulant and harvested at the fourth harvesting time, without being significantly different from the untreated plants harvested at the same harvesting time. On the other hand, the lowest overall value was recorded for the Auxym® biostimulant and the third harvesting time, without being significantly different from untreated plants of the first harvesting time. Moreover, for each biostimulant an increasing trend in total phenols was recorded until the fourth harvest, followed by a significant decline for the last two harvestings, especially in plants treated with biostimulants. In contrast to our study, Schiattone et al. [50] did not report a significant effect of biostimulants on total phenol content, while they suggested a significant effect of two biostimulants (azoxystrobin and extracts of yeast as well as brown algae) in wild rocket plants grown under nitrogen deprivation conditions. Moreover, Candido et al. [51] suggested a differential response of wild rocket plants to azoxystrobin and nitrogen deprivation depending on the cropping cycle, thus indicating that growing conditions are pivotal for plants' responses to abiotic stressors and/or biostimulant application. specific parameters, e.g., total phenols for the B × H interaction; HAA, ABTS, and TAA for the S × H interaction; and HAA, ABTS, total phenols, and TAA for the S × B interaction.

Supplementary Table S3 presents the effect of biostimulant application and harvesting time on HAA, the ABTS assay, total phenols, and TAA content in *Diplotaxis tenuifolia* leaves, regardless of salinity level. As indicated in Supplementary Table S1, the only significant interaction was recorded in the case of total phenol content. The highest total phenol content (Figure 2) was recorded in *D. tenuifolia* leaves collected from plants treated with the Auxym®biostimulant and harvested at the fourth harvesting time, without being significantly different from the untreated plants harvested at the same harvesting time. On the other hand, the lowest overall value was recorded for the Auxym®biostimulant and the third harvesting time, without being significantly different from untreated plants of the first harvesting time. Moreover, for each biostimulant an increasing trend in total phenols was recorded until the fourth harvest, followed by a significant decline for the last two harvestings, especially in plants treated with biostimulants. In contrast to our study, Schiattone et al. [50] did not report a significant effect of biostimulants on total phenol content, while they suggested a significant effect of two biostimulants (azoxystrobin and extracts of yeast as well as brown algae) in wild rocket plants grown under nitrogen deprivation conditions. Moreover, Candido et al. [51] suggested a differential response of wild rocket plants to azoxystrobin and nitrogen deprivation depending on the cropping cycle, thus indicating that growing conditions are pivotal for plants' responses to abiotic stressors and/or biostimulant application. Similarly, Giordano et al. [52] did not record a significant difference in two successive harvests in terms of the total phenol content of perennial wall rocket plants treated with two biostimulants, whereas TAA content significantly increased for both biostimulants over the control treatment. Moreover, Carillo et al. [53] reported a decrease in total phenols in lettuce plants where two successive harvests were implemented, while TAA content showed the opposite trend. On the other hand, Corrado et al. [54] did not observe any significant differences in the total phenol content of basil plants in two successive harvests, while Caruso et al. [35] indicated an increase in total phenol content in lettuce plants grown in two different seasons (winter and winter–spring) and subjected to two biostimulants (tropical plant extracts and legume-derived protein hydrolysates). The contrasting results in literature reports could be associated with differences in the genotypes tested, since, according to Ciriello et al. [55], a significant difference in the total phenol content of different basil genotypes was recorded in two successive harvestings.

Table 2 presents the effect of biostimulant application and salinity level on HAA, the ABTS assay, total phenols, and TAA content in *D. tenuifolia* leaves, regardless of the harvesting time. A varied response was recorded depending on the antioxidant activity assay. In particular, the non-salinized untreated plants (EC0 × BC treatment) recorded the highest and lowest values for HAA and ABTS AA assays, respectively, whereas an opposite trend was observed for plants that were grown under mid- to high-salinity levels and did not receive any biostimulant application (EC4 × BC). This result is in agreement with the findings of Di Mola et al. [56], who also suggested that non-salinized lettuce plants had the highest HAA, while El-Nakhel et al. [16] also reported a contrasting response of spinach plants to salinity stress and biostimulant application in regard to HAA and ABTS AA assays. Regarding total phenols and TAA content, no specific trends were recorded since no significant differences were recorded between most of the treatments, although the lowest overall content was recorded for the highest salinity level and the plants treated with the Trainer®biostimulant; however, El-Nakhel et al. [16] suggested a significant decrease in total phenols and TAA content for spinach plants treated with a legume-derived protein hydrolysate, whereas increasing salinity resulted in an increase in total phenol content and had no effect on TAA content. On the other hand, Rouphael et al. [36] noted a beneficial effect of biostimulant application on the total phenol content of spinach plants. According to Carillo et al. [53], no significant effects of salinity on the total phenol content, as well as the lipophilic and hydrophilic antioxidant activity, of lettuce plants grown under salinity stress were noted, while the same authors suggested fluctuating trends for TAA content.

**Figure 2.** Effect of biostimulant and harvesting time on total phenol content in *Diplotaxis tenuifolia* leaves, regardless of salinity level. Different letters indicate significant differences according to Tukey's honestly significant difference (HSD) test at *p* = 0.05; Roman numbers (I–VI) indicate the successive harvests; BC: no biostimulants added; BA: plants treated with Auxym®; and BT: plants treated with Trainer®. All data are expressed as mean ± SE (standard error), n = 3. **Figure 2.** Effect of biostimulant and harvesting time on total phenol content in *Diplotaxis tenuifolia* leaves, regardless of salinity level. Different letters indicate significant differences according to Tukey's honestly significant difference (HSD) test at *p* = 0.05; Roman numbers (I–VI) indicate the successive harvests; BC: no biostimulants added; BA: plants treated with Auxym®; and BT: plants treated with Trainer®. All data are expressed as mean ± SE (standard error), n = 3.

.

Similarly, Giordano et al. [52] did not record a significant difference in two successive harvests in terms of the total phenol content of perennial wall rocket plants treated with two biostimulants, whereas TAA content significantly increased for both biostimulants over the control treatment. Moreover, Carillo et al. [53] reported a decrease in total phenols in lettuce plants where two successive harvests were implemented, while TAA content showed the opposite trend. On the other hand, Corrado et al. [54] did not observe any significant differences in the total phenol content of basil plants in two successive harvests, while Caruso et al. [35] indicated an increase in total phenol content in lettuce plants grown in two different seasons (winter and winter–spring) and subjected to two biostimulants (tropical plant extracts and legume-derived protein hydrolysates). The contrasting results in literature reports could be associated with differences in the genotypes tested, since, according to Ciriello et al. [55], a significant difference in the total phenol content of different basil genotypes was recorded in two successive harvestings.

Table 2 presents the effect of biostimulant application and salinity level on HAA, the ABTS assay, total phenols, and TAA content in *D. tenuifolia* leaves, regardless of the harvesting time. A varied response was recorded depending on the antioxidant activity **Table 2.** Effect of salinity and biostimulant on hydrophylic antioxidant activity (HAA), ABTS antioxidant activity, total phenols, and total ascorbic acid (TAA) content in *Diplotaxis tenuifolia* leaves, regardless of harvesting time.


Different letters within each column indicate significant differences according to Tukey's honestly significant difference (HSD) test at *p* = 0.05. EC0: tap water (0 ds m−<sup>1</sup> ); EC2: 2.0 dS m−<sup>1</sup> ; EC4: 4.0 dS m−<sup>1</sup> ; EC6: 6.0 dS m−<sup>1</sup> ; BC: no biostimulants added; BA: plants treated with Auxym®; and BT: plants treated with Trainer®. All data are expressed as mean ± SE (standard error), n = 3.

Table 3 presents the effect of salinity level and harvesting time on HAA, the ABTS assay, and total ascorbic acid content in *D. tenuifolia* leaves, regardless of biostimulant application. HAA varied depending on the salinity level and the harvesting time, with the highest overall value being recorded for the non-salinized plants and the fourth harvesting time, whereas the highest salinity level and late harvesting (EC6 × VI) resulted in the lowest values for this particular assay. On the other hand, the EC6 × II and EC0 × VI treatments resulted in the highest and lowest values for the ABTS assay. Total phenol content was not affected by salinity levels and harvesting time, while for TAA content a decreasing trend with harvesting time was recorded, with the highest values being measured at the first harvesting time for all of the tested salinity levels. The lack of effect of harvesting time on the total phenol content (data not shown) of perennial wall rocket plants has been previously suggested by Giordano et al. [52], while Carillo et al. [53] did not record a significant effect of salinity on the antioxidant activity and total phenol content of lettuce plants.

**Table 3.** Effect of salinity and biostimulant on hydrophylic antioxidant activity (HAA), ABTS antioxidant activity, and total ascorbic acid (TAA) content in *Diplotaxis tenuifolia* leaves, regardless of biostimulant application.


Different letters within each column indicate significant differences according to Tukey's honestly significant difference (HSD) test at *p* = 0.05. EC0: tap water (0 ds m−<sup>1</sup> ); EC2: 2.0 dS m−<sup>1</sup> ; EC4: 4.0 dS m−<sup>1</sup> ; and EC6: 6.0 dS m−<sup>1</sup> . Roman numbers (I–VI) indicate the successive harvests. All data are expressed as mean ± SE (standard error), n = 3.

These contradictory results indicate that growing conditions are pivotal for the responses of *D. tenuifolia* plants to salinity stress and biostimulant application. This could be due to the fact that there is a combinatory effect on the plant protective mechanisms that induces the biosynthesis of antioxidant compounds as well as their activity. As reported in the literature and suggested by the results of the present study, when several factors are tested at the same time (e.g., biostimulant applications, harvesting time, salinity stress) the response of plants to studied parameters may vary depending on the experimental conditions. This response may also differ compared to the response to single factors. For example, in our study total phenol content was affected by salinity and harvesting time or salinity and biostimulant application, but no effect was recorded when salinity and harvesting time were considered. In the study by Bulgari et al. [57], a significant increase in total phenol content was observed for rocket plants grown in a floating system and subjected to EC levels of 3.5 dS m−<sup>1</sup> . Hamilton and Fonseca [58] reported a variable effect of salinity levels (up to 9.6 dS m−<sup>1</sup> ) on rocket plants (*Eruca sativa* and *Diplotaxis tenuifolia*) depending on the growing period (March to April and May to June). Similarly, El-Nakhel et al. [16]

recorded an increase in total phenols in spinach plants with increasing salinity. The same authors suggested a negative effect of a legume-derived protein hydrolysate compared to the control plants [16]. On the other hand, Corrado et al. [59] did not record a significant effect of salinity on total phenol content in two lettuce varieties, while they suggested a significant effect of harvesting time and genotype. According to Franzoni et al. [60], the benefits from biostimulant application on plants under stress could be associated with an increase in the expression of transcription factors involved in plant responses to stress, such as the induction of cuticular waxes, phospholipid and brassinosteroid biosynthesis, the regulation of sugar metabolism, and intracellular transport. Therefore, it seems that plant responses to external factors related to stress (e.g., salinity) or growth promotion (e.g., biostimulant) are highly affected by environmental and growing conditions. For this reason, further research is needed in order to define those conditions (e.g., the combinations of harvesting time, salinity level, and biostimulant product) that are favorable to the induction of secondary metabolism, allowing plants to better cope with salinity stress.

#### *3.3. Chlorophyll and Carotenoid Content*

Table 4 presents the result of chlorophyll and carotenoid content in relation to salinity level, biostimulant application, and harvesting time. Considering that no significant interactions between the tested factors were detected, only the main effects of each factor are presented. Chlorophylls a and b as well as total chlorophyll content was significantly affected by biostimulant application, with Trainer®showing the highest content compared to the rest of the treatments. On the other hand, salinity level affected chlorophyll b and total chlorophyll content, with increasing salinity resulting in a significant decrease, especially at the highest salinity level tested. The effect of harvesting time on chlorophyll content did not show specific trends except for chlorophyll b, where a decrease was recorded with increasing salinity, whereas chlorophyll a and total chlorophyll content fluctuated over the growing period. Moreover, total carotenoid content showed a significant increase at low salinity (EC2), followed by a reduction with increasing salinity to levels similar to the control treatment. Finally, harvesting time seems to have a varied effect, with a slight increase at the second harvest followed by a decrease at subsequent harvesting, especially the late ones (harvests V and VI), where the lowest values were recorded.

The findings of our study are in agreement with reports in the literature, where it is suggested that salinity stress induces the disruption of the photosynthetic apparatus through the damage of chloroplasts and the decrease in chlorophyll content, especially in older leaves, which tend to accumulate more ions than younger ones [3]. Moreover, the application of biostimulants may mitigate the negative effects of abiotic stressors on chlorophyll content in leafy vegetables such as lettuce [61]. In harmony, El-Nakhel et al. [16] indicated a negative effect of increasing salinity on the chlorophyll content of spinach plants, while suggesting a positive effect from the application of a biostimulant that contained protein hydrolysates. The same authors recorded an increase in total carotenoids with increasing salinity, whereas biostimulant application had no significant effect on this parameter. Lucini et al. [17] noted a significant decrease in SPAD index values and chlorophyll fluorescence in lettuce plants grown under saline conditions, while biostimulant application (of plant-derived protein hydrolysates) only mitigated the negative effects on chlorophyll fluorescence and not those on the SPAD index. A similar finding with our study was reported by Caruso et al. [27], who indicated a positive effect of biostimulants on chlorophyll b content and no effects on carotenoid content, while they also mentioned that cropping season did not affect the abovementioned parameters. The single effect of biostimulant application (borage leaf or flower extracts) did not affect chlorophyll and carotenoid content in wild rocket plants, indicating the lack of effect in unstressed plants [62]. This argument was confirmed by Franzoni et al. [60], who suggested that borage extracts mitigated the negative effects of salinity on chlorophyll a fluorescence but had no effect on unstressed plants. It seems that growing conditions may affect the responses of plants to salinity and biostimulant application, since, according to Giordano et al. [52], a variable effect of protein

hydrolysates and plant extracts on the chlorophyll content of perennial wall rocket leaves was recorded, depending on harvesting time.


**Table 4.** Effect of salinity, biostimulant, and harvest on chlorophyll a, chlorophyll b, total chlorophylls, and carotenoid averages in *Diplotaxis tenuifolia*.

Different letters within each column and for the same factor indicate significant differences according to Tukey's honestly significant difference (HSD) test at *p* = 0.05. EC0: tap water (0 ds m−<sup>1</sup> ); EC2: 2.0 dS m−<sup>1</sup> ; EC4: 4.0 dS m−<sup>1</sup> ; EC6: 6.0 dS m−<sup>1</sup> ; BC: no biostimulants added; BA: plants treated with Auxym®; and BT: plants treated with Trainer®. The Roman numbers (I–VI) indicate the successive harvests. All data are expressed as mean ± SE (standard error), n = 3.

#### *3.4. Leaf Nutrient Composition*

The cation (Na, K, Ca, and Mg) concentrations were significantly affected by the second-degree interactions of salinity × biostimulant and salinity × harvest, except for K, which was also affected by the interaction of biostimulant × harvest (Supplementary Material, Table S2). In regard to the anions, Cl content was significantly affected by all three of the second-degree interactions; S content only by the S × H interaction and P content by the S × B interaction (Supplementary Material, Table S2). As for the nitrate content, it was affected by all of the second-degree interactions.

At lower salinity levels (EC0 and EC4) no significant differences were recorded between the biostimulant treatments, while at EC4 Auxym®application resulted in a significant decrease in Na content, which was not the case at the highest salinity level (EC6). At this salinity level, the highest overall Na content was recorded for this particular biostimulant without being significantly different from the other biostimulant treatment (Table 5). In regard to K concentration, it reached the highest value in plants that were not treated with biostimulants and which were irrigated with tap water, not being different from EC0 × BA, all of the treatments of EC2, and BA × EC4 (Table 5). For Mg concentration, only in EC2 was the treatment with Trainer®different from BC, while significant differences were also recorded from EC4 × BA and EC6 (both BC and BT treatments) (Table 5). In the plants irrigated with tap water or water with low salinity (EC2), BT elicited a higher value of Ca concentration in the leaves, but in EC2 it was not different from BA; in higher salinity levels no differences were recorded between plants treated and untreated with biostimulants (Table 5). Biostimulant application reduced Cl concentration in leaves up to moderate salinity levels (EC4 treatment), while at the highest salinity tested (EC6) no significant differences were recorded between BA, BT, and BC (Table 5). Sulfur content was not affected by the factors tested (data not shown). In regard to P concentration, at low (EC2) and moderate salinity stress (EC4) the plants treated with Trainer®showed higher values, contrary to what occurred in EC0 and EC6, where the treatment with BA elicited higher values, although no significant differences were recorded between the two biostimulant products (Table 5). In accordance with our study, Lucini et al. [17] reported that increasing salinity in a nutrient solution resulted in increased Na content in lettuce

leaves, whereas biostimulant application showed no significant effect. Similarly, K, P, Ca, and Mg content decreased with increasing salinity, while biostimulant application only affected P content [17]. Moreover, increased salinity has been associated with the disruption of nutrient and osmotic balance due to increased ratios of Na/K, Na/Ca, and Na/Mg [63].

**Table 5.** Effect of salinity and biostimulant application on minerals (Na, K, Ca, Mg, Cl, and P) and nitrate content in *Diplotaxis tenuifolia* leaves, regardless of harvesting time.


Different letters within each column indicate significant differences according to Tukey's honestly significant difference (HSD) test at *p* = 0.05. EC0: tap water (0 ds m−<sup>1</sup> ); EC2: 2.0 dS m−<sup>1</sup> ; EC4: 4.0 dS m−<sup>1</sup> ; EC6: 6.0 dS m−<sup>1</sup> ; BC: no biostimulants added; BA: plants treated with Auxym®; and BT: plants treated with Trainer®. All data are expressed as mean ± SE (standard error), n = 3.

Nitrate content was significantly increased by biostimulant application for all of the tested salinity levels compared to the untreated plants (no biostimulants added), while a decreasing trend with increasing salinity was recorded for all of the biostimulant treatments (with or no biostimulants added) (Table 5). Similar results were recorded by El-Nakhel et al. [16], who also suggested an increase in nitrate content in spinach plants treated with a legume-derived protein hydrolysate, while increasing salinity also resulted in a significant decrease in nitrate. It seems that the high availability of Cl in the nutrient solution may impair nitrate uptake and decrease its content in plant tissue, while biostimulants may serve as nitrogen pools and contribute to the accumulation of nitrate in leaves without an increased uptake of exogenous nitrogen being observed [64,65]. In contrast, Bulgari et al. [62] reported a decrease in nitrate content in wild rocket plants treated with borage extracts, which indicates that biostimulant composition and the presence of nitrogenous compounds could be responsible for the increase in nitrate observed in other studies. Moreover, Bonasia et al. [57] recorded a variable effect of increasing salinity on nitrate content in wild rocket plants, depending on the cropping system and the genotype tested. Similarly, Giordano et al. [52] did not observe a significant effect of biostimulant application on the nitrate content of perennial wall rocket leaves despite the presence of nitrogen in one of the biostimulant products, which highlights the importance of harvesting time for the combined responses of plants.

As expected, the concentrations of Na and Cl increased when the salinity levels in the nutrient solution increased; in fact, the mean values of Na and Cl concentration were 4.89, 9.42, 13.29, and 15.62 g kg−<sup>1</sup> as well as 21.81, 31.87, 44.23, and 49.79 g kg−<sup>1</sup> for EC0, EC2, EC4, and EC6, respectively (Table 6). In addition, both elements also increased over the harvest periods, with values that were about double at harvest VI compared to I: 12.40 vs. 5.71 for Na and 43.44 vs. 22.40 for Cl, respectively. In contrast, Ca and Mg decreased with increasing salinity levels from EC0 (23.00 and 5.00 g kg−<sup>1</sup> , respectively) to EC6 (20.02 and 4.67 g kg−<sup>1</sup> , respectively), while both elements showed the highest value at harvest II (Table 6). In regard to the other two anions, S increased with the increase in salinity stress, but only EC2 was different from EC0; over the harvest periods, S concentration decreased until harvest IV and then increased, reaching, at harvest VI, a value that was not significantly different from that of harvest I (Table 6). Instead, P concentration decreased when the salt concentration incremented, but only EC6 was different from all of the other saline treatments; moreover, its concentration significantly decreased over the harvest periods (Table 6). Nitrate content recorded fluctuating trends, with a varied effect being observed at the various salinity levels and harvesting times, although a decreasing trend at the late harvesting times (harvests IV to VI) was noticed.


**Table 6.** Effect of salinity and harvesting time on minerals (Na, Ca, Mg, Cl, and S) and nitrate content in *Diplotaxis tenuifolia* leaves, regardless of biostimulant application.

Different letters within each column indicate significant differences according to Tukey's honestly significant difference (HSD) test at *p* = 0.05. Roman numbers (I–VI) indicate the successive harvests. EC0: tap water (0 ds m−<sup>1</sup> ); EC2: 2.0 dS m−<sup>1</sup> ; EC4: 4.0 dS m−<sup>1</sup> ; and EC6: 6.0 dS m−<sup>1</sup> . All data are expressed as mean ± SE (standard error), n = 3.

Similar results to our study have been reported by Malécange et al. [66], who studied the effect of a biostimulant product rich in free amino acids on lettuce crop performance and suggested an increase in nitrogen content in treated plants, regardless of the irrigation regime. On the other hand, considering that salinity stress is associated with the decreased water availability and increased osmotic potential of the nutrient solution, nitrate accumulation in plants subjected to salinity stress indicates its osmoregulatory activity [64]. This finding should be associated with protective mechanisms similar to those of halophytes, which tend to accumulate minerals under saline conditions as part of their defense against abiotic stressors [67].

On the other hand, K concentration significantly increased from harvest III to V in all of the biostimulant treatments, while a decrease was recorded for the last harvest (Figure 3A). Similar trends were suggested for Cl content, which gradually increased with harvesting time after harvest II (Figure 3B). Moreover, Cl content was higher in the leaves of plants untreated with biostimulants (41.01 vs. 34.89 g kg−<sup>1</sup> , mean value of BA and BT). Finally, contrasting trends were recorded in terms of nitrate content (Supplementary Table S4). In particular, the progress in harvesting time resulted in a decrease in nitrate content in plants that were not sprayed with biostimulants. In contrast, plants treated with either Auxym®or Trainer®recorded a steep decrease at the second harvest, followed by a significant increase at harvest III and fluctuating trends thereafter until harvest VI.

as mean ± SE (standard error), n = 3.

defense against abiotic stressors [67].

**Figure 3.** Effect of biostimulants and harvesting time on minerals K (**A**) and Cl (**B**) in *Diplotaxis tenuifolia* leaves, regardless of salinity level. Different letters indicate significant differences according to Tukey's honestly significant difference (HSD) test at *p* = 0.05. Roman numbers (I–VI) indicate the successive harvests. BC: no biostimulants added; BA: plants treated with Auxym®; and BT: plants treated with Trainer®. All data are expressed as mean ± SE (standard error), n = 3. **Figure 3.** Effect of biostimulants and harvesting time on minerals K (**A**) and Cl (**B**) in *Diplotaxis tenuifolia* leaves, regardless of salinity level. Different letters indicate significant differences according to Tukey's honestly significant difference (HSD) test at *p* = 0.05. Roman numbers (I–VI) indicate the successive harvests. BC: no biostimulants added; BA: plants treated with Auxym®; and BT: plants treated with Trainer®. All data are expressed as mean ± SE (standard error), n = 3.

EC0: tap water (0 ds m<sup>−</sup>1); EC2: 2.0 dS m−1; EC4: 4.0 dS m−1; and EC6: 6.0 dS m−1. All data are expressed

Similar results to our study have been reported by Malécange et al. [66], who studied the effect of a biostimulant product rich in free amino acids on lettuce crop performance and suggested an increase in nitrogen content in treated plants, regardless of the irrigation regime. On the other hand, considering that salinity stress is associated with the decreased water availability and increased osmotic potential of the nutrient solution, nitrate accumulation in plants subjected to salinity stress indicates its osmoregulatory activity [64]. This finding should be associated with protective mechanisms similar to those of halophytes, which tend to accumulate minerals under saline conditions as part of their

On the other hand, K concentration significantly increased from harvest III to V in all of the biostimulant treatments, while a decrease was recorded for the last harvest (Figure 3A). Similar trends were suggested for Cl content, which gradually increased with harvesting time after harvest II (Figure 3B). Moreover, Cl content was higher in the leaves of plants untreated with biostimulants (41.01 vs. 34.89 g kg−1, mean value of BA and BT). Finally, contrasting trends were recorded in terms of nitrate content (Supplementary Table S4). In particular, the progress in harvesting time resulted in a decrease in nitrate content in plants that were not sprayed with biostimulants. In contrast, plants treated with either Auxym® or Trainer® recorded a steep decrease at the second harvest, followed by a significant increase at harvest III and fluctuating trends thereafter until harvest VI.

The increase in nitrate content with biostimulant application is already reported in the literature [16,30]; however, our findings indicate the importance of growing conditions in plant responses to nitrate accumulation, including soil properties, light intensity, and nitrogen form, among others [29,68]. Moreover, Bantis et al. [69] also suggested a higher nitrate content in the first harvest of rocket plants in an experiment where two successive harvests were implemented, a finding which is in agreement with our results for the plants that received no biostimulants. Regarding the nutrients profile, Caruso et al. [27] suggested a variable effect of growing conditions (winter and winter–spring cropping seasons) on wall rocket plants, while biostimulant application resulted in increased content for most of the nutrients (except for S, where no effects were recorded). According The increase in nitrate content with biostimulant application is already reported in the literature [16,30]; however, our findings indicate the importance of growing conditions in plant responses to nitrate accumulation, including soil properties, light intensity, and nitrogen form, among others [29,68]. Moreover, Bantis et al. [69] also suggested a higher nitrate content in the first harvest of rocket plants in an experiment where two successive harvests were implemented, a finding which is in agreement with our results for the plants that received no biostimulants. Regarding the nutrients profile, Caruso et al. [27] suggested a variable effect of growing conditions (winter and winter–spring cropping seasons) on wall rocket plants, while biostimulant application resulted in increased content for most of the nutrients (except for S, where no effects were recorded). According to the same authors, biostimulant application is associated with changes in root architecture that facilitate nutrient uptake, translocation, and assimilation, with the activity of signaling molecules or the expression of genes involved in macronutrient transportation through cell membranes [35]. Moreover, Giordano et al. [52], who tested the same biostimulant products in perennial wall rocket plants, reported that Trainer®and Auxym®contain bioactive compounds and peptides that may promote root growth as well as nutrient uptake, and therefore affect the mineral profile of leaves.

#### **4. Conclusions**

The results of the present work highlight the importance of biostimulant application in alleviating the negative effects of salinity stress on the chemical composition and mineral profile of *Diplotaxis tenuifolia* plants; however, a varied response in relation to harvesting time was recorded for most of the studied parameters, which indicates the pivotal effect of growing conditions in addition to the complexity of plants' responses to biostimulants and salinity stress. In conclusion, further research is needed in order to suggest those conditions that allow the alleviation of the negative effects of salinity stress through biostimulant application and prescheduled harvesting time. Moreover, special consideration is needed regarding the nitrate content of leaves, which tend to increase with biostimulant application, while moderate salinity and proper harvesting time seem to reduce the health risks associated with nitrate accumulation in *D. tenuifolia* leaves.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/app13031569/s1, Table S1: Analysis of variance of hydrophylic antioxidant activity (HAA), ABTS antioxidant activity, total phenols, total ascorbic acid (TAA), chlorophylls (a, b, and total chlorophyll), and total carotenoids; Table S2: Analysis of variance of minerals content; Table S3: Effect of biostimulant and harvesting time on hydrophylic antioxidant activity (HAA), ABTS antioxidant activity, total phenols, and total ascorbic acid (TAA) content in *Diplotaxis tenuifolia* leaves, regardless of salinity level; Table S4: Effect of biostimulants and harvest on minerals (Na, K, Ca, Mg, Cl, S, and P) and nitrate content in *Diplotaxis tenuifolia* leaves, regardless of salinity level.

**Author Contributions:** Conceptualization, I.D.M., E.C. and M.M.; methodology, S.A.P. and Y.R.; software, L.O. and C.E.-N.; validation, I.D.M., L.O. and C.E.-N.; formal analysis, S.A.P.; investigation, Y.R. and M.M.; resources, E.C.; data curation, L.O.; writing—original draft preparation, I.D.M. and S.A.P.; writing—review and editing, C.E.-N. and Y.R.; visualization, I.D.M. and S.A.P.; supervision, E.C. and M.M.; project administration, E.C.; funding acquisition, Y.R. and M.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The datasets generated for this study are available on request to the corresponding author.

**Acknowledgments:** The authors would like to thank Sabrina Nocerino, Maria Eleonora Pelosi, and Luca Scognamiglio for their support in the laboratory work.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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