Enhancing Soilless Production of Portulaca oleracea, Mesembryanthemum crystallinum and Valerianella locusta Through Nitrogen Form Ratio Optimization and Biostimulant Application

Abstract

Underutilized leafy greens are considered as functional plant species primarily due to their resilience to abiotic stress factors, low nutrient requirements, and high nutritional value. Over the past 30 years, many experiments have been conducted to identify nutrient-efficient species, cultivars, landraces, and ecotypes, but few have successfully entered mainstream agriculture. The integration of these species into advanced horticultural systems, such as hydroponics, has the potential to further strengthen their impact on sustainable agriculture by minimizing use of resources, enabling year-round cultivation, and improving the nutritional profile of the harvested produce. As leafy vegetables, a primary food safety concern is the accumulation of nitrates in the leaves. In hydroponics, this issue is usually addressed by balancing the NH₄-N/total-N ratio (N_r) in the nutrient solution. Provided that the plant responses to high ammonia supply are species-dependent, three wild leafy greens, iceplant, corn salad, and common purslane, were grown in a soilless culture, with perlite as the substrate, under low (0.04) and high (0.12) N_r on a molar basis. Additionally, the potential of protein hydrolysates (PH) and seaweed extracts (SW) to alleviate plant tolerance to excess ammonia supply was also investigated. In terms of yield, high N_r led to significant yield restrictions in iceplant that reached 28%, while on corn salad, it had a positive impact, with yield increasing by 18%. Both biostimulant applications enhanced iceplant productivity only under optimal N_r conditions (0.04). Apart from yield responses, biofertilizers had no substantial impact on the plant nutrient profile. In contrast, high N_r suppressed nitrate accumulation in fresh leaves, while enhancing micronutrient uptake in all three plant species. In conclusion, this study highlights the pivotal role of biostimulants as plant stress protectors and growth regulators and identifies the optimal N_r ratio for maximizing the yield and quality performance of corn salad, iceplant, and common purslane in soilless cultivation systems.

1. Introduction

According to the latest EU Agricultural Outlook, European farmers are encouraged to enhance plant diversity in response to consumer preferences, environmental sustainability, and ecosystem balance [1]. Integrating alternative crops, such as underutilized wild leafy vegetables, into soilless cultivation systems could increase biodiversity and improve the sustainability of the agriculture sector [2]. These species are often rich in essential minerals, vitamins, and antioxidants, indicating their high nutritional value and the potential to address “hidden hunger”, which affects more than two billion people globally [3,4]. Furthermore, wild leafy vegetables are typically resilient to abiotic stress and require low inputs [5,6,7,8,9,10], offering a sustainable option for cultivation and inclusion in the daily human diet. However, targeted research is needed to efficiently address their specific cultivation requirements and ensure consistent optimal product quality.

Purslane (Portulaca oleracea) is a nutrient-rich wild leafy vegetable containing significant amounts of essential minerals for the human diet, such as potassium, calcium, magnesium, and iron [11]. Its high concentration of bioactive compounds also makes it a valuable functional food with potential pharmacological benefits [12]. Purslane exhibits higher levels of linolenic acid, beta-carotene, and ascorbic acid concentrations than many conventionally cultivated leafy vegetables, further supporting its strong antioxidant properties [13,14]. Moreover, the presence of cholesterol-free omega-3 fatty acids constitutes it as a viable plant-based alternative to fish oils [15,16]. Physiologically, purslane employs C4 photosynthesis, but under stress conditions, it can switch to CAM, enhancing its adaptability to limited water availability and high temperatures [17]. Moreover, purslane demonstrates remarkable stress resilience, maintaining stable growth, net photosynthetic rate, and macronutrient concentrations (Na, K, Mg, and Ca) in leaf tissues under salinity stress up to 50 mM Na⁺ in the root zone [18]. Notably, low nitrogen supply enhances purslane’s phenolic and flavonoid content, thereby increasing its antioxidant capacity [19].

Iceplant (Mesembryanthemum crystallinum) is a halophytic plant species with notable physiological and biochemical adaptations that enhance its survival under saline conditions [20]. The epidermal bladder cells of this plant develop early and cover the leaves, stems, and peduncle. These cells expand rapidly in response to salt stress by accumulating water and sodium, reflecting substantial metabolic alterations in salt-treated tissues [21]. Under salinity stress conditions, up to 200 mM Na⁺ in the root zone, iceplant exhibits enhanced growth and increased accumulation of antioxidants, flavonoids, polyphenols, and D-pinitol. It is also rich in phenolic compounds, such as tannins and catechins, further contributing to its stress tolerance and nutritional value [22,23]. Given its resilience to salinity and high nutritional value, iceplant represents a promising alternative food source for arid regions facing challenges related to salinity and water scarcity [24]. Additionally, its mineral profile, primarily Na, K, and Mg content, gives it a natural salty taste, making it a promising candidate as a salt alternative in culinary applications [25].

Corn salad (Valerianella locusta) is widely used as a salad green [26] and is cultivated in greenhouses, both in soil and soilless systems [27]. The bioactive compound profile of corn salad enhances its nutritional value as a fresh vegetable in the human diet. The plant’s leaves contain high concentrations of carotenoids, phenolics, folic acid, sterols, fatty acids, and glucosinolates [28,29], indicating strong antioxidant capacity and potential antitumor effects [30]. In terms of fertigation practices, corn salad is a high-N-demanding plant. Under reduced N supply, plant growth is negatively affected, an effect that can be mitigated through the application of biostimulants [31]. Furthermore, eustress application in hydroponically grown V. locusta using nutrient solutions (NSs) with increased EC levels and different cationic compositions indicates that the species is sensitive to salinity, as yield is notably restricted when Na⁺ is used to increase EC instead of Ca²⁺ [10]. These findings highlight the importance of tailored fertigation strategies for optimizing the yield and nutritional quality of V. locusta.

Nitrogen (N) is a crucial macronutrient for plant physiology, serving multiple roles in plant physiology and metabolism. As a fundamental component of proteins, nucleic acids and chlorophyll, it directly influences plant growth, development, and metabolism [32]. It supports enzyme functions, cellular structure, and overall metabolic regulation as the key component of proteins. Plants primarily absorb N in two mineral forms, nitrate (NO₃⁻) and ammonium (NH₄⁺). In soilless crops, the addition of NH₄⁺ aims to effectively manage the pH in the root solution [33]. The reduction in root-zone pH was primarily determined by the NH₄⁺/total-N ratio, with the absolute NH₄⁺ concentration in the supplied nutrient solution having a lesser effect [7]. This NH₄⁺-induced pH decline is attributed to the preferential uptake of NH₄⁺ by plant roots. NH₄⁺ is preferentially absorbed by the plants when both NO₃⁻ and NH₄⁺ are available in the root zone, despite NO₃⁻ being the predominant form absorbed by most crop species [34]. This preference persists even though NO₃⁻ assimilation requires higher energetic expenditure. Following uptake, these inorganic N forms are assimilated into amino acids, mainly through the glutamine synthetase/glutamate synthase cycle pathway [35]. The assimilation of mineral N forms in organic compounds is strongly influenced by photosynthetically active radiation, affecting NO₃⁻ accumulation in leaves [32]. As a critical food safety parameter for human health, NO₃⁻ concentration in the edible parts of leafy vegetables is strictly regulated, and safety thresholds of 3000 to 5000 mg per kg of fresh weight have been established by the European Union (Regulation No. 2023/915).

Seaweed extracts (SW) and protein hydrolysates (PHs) can be used as effective biostimulants to enhance stress tolerance in vegetable crops. When applied to stressed plants, seaweed extracts boost the activity of antioxidant enzymes such as SOD, CAT, and APX, contributing to oxidative stress mitigation [36,37]. Additionally, these extracts can promote growth and nutrient uptake in crops like tomato and spinach [38,39,40,41]. Similarly, PHs function as potent biostimulants by enhancing nutrient use efficiency and activating both enzymatic and non-enzymatic antioxidant defense systems, supporting plant growth and physiological functions under abiotic stress conditions [42,43,44]. Moreover, PHs modulate phytohormonal balance, helping to maintain photosynthetic efficiency and reduce stress-induced damage [45].

The objective of this research is to develop cultivation protocols that can optimize open soilless (hydroponic) production of three underutilized leafy greens: iceplant (Mesembryanthemum crystallinum), corn salad (Valerianella locusta), and common purslane (Portulaca oleracea). To achieve this, the study examined a novel approach that combines different NH₄⁺/total-N ratios (N_r) in the nutrient solution with biostimulant applications (PH, and SW), evaluating their effects on plant growth, nutritional quality, and NO₃⁻ accumulation. Specifically, the study seeks to (a) determine the optimal NH₄⁺/total-N ratio that maximizes yield and minimizes NO₃⁻ accumulation in leaves while enhancing nutrient uptake, (b) evaluate the potential of biostimulants to alleviate stress caused by high ammonia supply and improve plant productivity, and (c) assess the impact of nitrogen form and biostimulants on the nutritional profile of these species to ensure food safety and quality.

2. Materials and Methods

2.1. Plant Material and Growing Conditions

Three independent experiments were conducted in a free-drainage soilless culture system, at the greenhouse facilities of the Laboratory of Vegetable Production, the Agricultural University of Athens (37°58′57.8″ N, 23°42′14.3″ E). The first experiment focused on purslane (Portulaca oleracea) with seeds (Fitotech Industrial and Commercial S.A., Athens, Greece) sown in rockwool trays (AO Plug, Grodan, Roermond, the Netherlands) on 30 March. At the stage of the first 2–3 true leaves, on 10 April, 5 seedlings were transplanted into 33 L perlite bags (Geoflor Hydro, Greece), with harvest occurring at commercial maturity on 27 April. The second experiment followed an identical protocol for corn salad (Valerianella locusta, ‘Elixir’ (HM. CLAUSE Sas, Portes-Les-Valence, France)), with sowing on 30 March and transplanting into the soilless system at the 2–3 true leaf stage on 10 April, while the experiment terminated on 6 May. The third experiment with iceplant (Mesembryanthemum crystallinum L.) also followed the same configuration, with sowing of the seeds (Fitotech Industrial and Commercial S.A., Athens, Greece) on 20 April, transplantation at the same developmental stage on 30 April, and final harvest on 18 May. In all three experiments, the perlite substrates were moistened with starter nutrient solution (NS) (

Table 1. Composition of the irrigation water, starter nutrient solution, and the two supplied nutrient solutions (high and low N_r).

Parameter	Unit	Water	Starter	High Nr	Low Nr
EC	dS m−1	0.30	3.00	2.40	2.30
pH		7.55	5.60	5.60	5.60
K+	mM	0.00	8.72	7.43	7.43
Ca2+	mM	1.00	5.25	4.10	4.10
Mg2+	mM	0.20	3.42	2.29	2.29
NH4+	mM	0.00	0.89	1.79	0.64
SO42−	mM	0.20	5.02	3.88	2.73
NO3−	mM	0.00	15.05	12.63	13.78
P	mM	0.00	1.65	1.40	1.40
Fe	μM	0.00	40.00	25.00	25.00
Mn	μM	0.00	5.00	8.00	8.00
Zn	μM	0.20	7.00	5.00	5.00
Cu	μM	0.00	0.80	0.75	0.75
B	μM	0.00	40.00	30.00	30.00
Cl−	mM	0.30	0.40	0.40	0.40
Na+	mM	0.60	0.60	0.60	0.60
HCO3−	mM	2.20	0.40	0.40	0.40
Nr	mol:mol		0.06	0.12	0.04

) prior to transplanting. The greenhouse maintained consistent environmental conditions throughout all three experiments (17–25 °C, 50–80% relative humidity) and the plants were grown under natural light (13 h day/11 h night), using the same soilless culture system configuration for comparability between species.

2.2. Experimental Design

All three experiments employed identical two-factorial experimental designs to maintain comparability between the purslane, corn salad, and iceplant trials. The first factor investigated was the NH₄⁺/total-N ratio (N_r) in the NS with two treatment levels: a low N_r level of 0.04 and a high N_r level of 0.12 on a molar basis (Table 1). The second factor examined biostimulant application, comparing two products: a seaweed (SW: ‘Algastar’ liquid extract of Ascophyllum nodosum extract) (Table S1) and a protein hydrolysate (PH: Tyson) (Table S2), both supplied by Mugavero Fertilizers, Italy. Both biostimulants were applied via foliar spraying, according to the manufacturer’s recommendations. The spraying solution of SW contained Algastar at a concentration of 2 mL L⁻¹, while that of PH contained Tyson at a concentration of 3 mL L⁻¹. Biostimulant applications occurred twice during each crop cycle—on the 14th and the 21st of April for purslane and corn salad, and on the 3rd and 10th May for iceplant, with untreated plants serving as controls. The factorial combination of these two factors, N_r and biostimulant application, produced six distinct treatments: (1) high N_r—control; (2) high N_r—SW; (3) high N_r—PH; (4) low N_r—control; (5) low N_r—SW; and (6) low N_r—PH. Each treatment was replicated four times (n = 4), with each experimental unit consisting of one perlite bag containing five plants. This complete randomized design allowed for systematic evaluation of both individual and interactive effects of N_r and biostimulant application on plant performance.

2.3. Sampling and Laboratory Analysis

At harvest, the fresh weight (FW) of all the plants was recorded in order to calculate the yield (g plant⁻¹). One plant from each replication was collected, cleaned with distilled water, and dried to a constant weight at 65 °C to determine the mineral concentrations. The dried tissues were powdered using a blade mill and then subjected to the dry ashing procedure at 550 °C for 8 h. Subsequently, 10 mL of 0.25 M HCl solution was added to the ash of each sample to extract K⁺, Ca²⁺, Mg²⁺, Na⁺, Fe, Mn, and Zn [46]. The reduced N (N_red) content of the plant tissues was determined according to the Kjeldahl method, using a Labtec DT 220 digestion system coupled with a Tecator Kjeltec 8200 distillation unit (FOSS A/S, Hillerod, Denmark). To determine the mineral N (N_min) content, which corresponds to leaf nitrate (NO₃⁻) concentration, the dried tissue samples were subjected to hot-water extraction in a water bath at 80 °C for 1 h, while the nitrate concentration in plant extracts was quantified photometrically at 410 nm via the salicylic acid method [47]. The total-N concentration of the dried plant tissues was calculated as the sum of N_min and N_red values, while the leaf NO₃⁻ concentrations were also given as mg of NO₃⁻ per kg of fresh weight (FW) (ppm).

2.4. Partial Budget Analysis

The partial budget analysis was performed to appraise the net economic profits that the growers may accrue by employing the SW or PH biostimulants. The economic method described by Giordano et al. [48] was used for the evaluation. To calculate the added net return sustained by both SW and PH, the subsequent formula was employed: Added net return = added gross return − added variable costs.

2.5. Statistical Analyses

The experiment was conducted with a completely randomized design with four replications per treatment. Treatment effects were analyzed using two-way factorial ANOVA to evaluate: (1) the main effects of nitrogen ratio (N_r) and biostimulant application, and (2) their interaction effects. When ANOVA indicated significant differences (p < 0.05), post hoc comparisons were performed using Duncan’s multiple range test to identify specific treatment differences. The statistical analysis of the data, ANOVA and Duncan’s multiple range test were performed using STATISTICA 12.5 for Windows (StatSoft Inc., Tulsa, Oklahoma, USA). Graphical representation of data was performed using the GraphPad Prism 8 software (GraphPad Software, La Jolla, CA, USA).

3. Results

3.1. Evolution of EC and pH in the Drainage Solution

Figure 1 shows the EC and pH evolution in the drainage solution throughout the cultivation period, depending on different NH₄⁺/total-N ratio (N_r) treatments. In all three experiments, N_r had no significant impact on EC levels. For purslane and corn salad, EC ranged from 2.75 to 3.23 dS m⁻¹ and 2.69 to 3.22 dS m⁻¹, respectively. For iceplant, EC values were between 2.80 and 3.23 dS m⁻¹ for both N_r levels. Regarding pH, no significant differences were detected in the drainage solution for purslane and corn salad. During the cultivation period, pH decreased from 6.6 to 6.2, irrespective of the N_r treatment. In contrast, for iceplant, the pH values for the high-N_r treatment were consistently significantly lower, declining from 5.9 to approximately 5.1, compared to the low-N_r treatment, the pH values of which declined from 6.2 to approximately 5.5, indicating gradual acidification of the drainage solution in both cases.

3.2. Yield and Dry Matter Content

The plant fresh weight (FW) of iceplant and corn salad was significantly affected by both the N_r and the biostimulant application, while interaction between the two factors was detected in both species. Specifically, under low-N_r conditions, the application of SW and PH significantly enhanced iceplant FW by 20.4% and 23.0%, respectively, compared to the corresponding control no biostimulant application. In contrast, under high N_r the FW of iceplant was significantly lower compared to low N_r, irrespective of the biostimulant application (

Table 2. Impact of ammonium on N ratio (N_r) and biofertilizer application on fresh weight (FW) and dry matter content (DMC) (%) of purslane, iceplant and corn salad. SW: seaweed extract; PH: protein hydrolase.

		Purslane		Iceplant		Corn Salad
Nr	Biostimulants	FW	DMC (%)	FW	DMC (%)	FW	DMC (%)
High		152.18	4.53	79.33 b	3.37 a	11.43 a	9.49 b
Low		147.29	4.37	110.72 a	2.76 b	9.34 b	9.98 a
	Control	154.58	4.43	89.15 c	3.16	10.23 b	9.72
	SW	143.38	4.43	99.05 a	3.05	10.11 b	9.81
	PH	151.13	4.47	97.56 ab	2.99	10.64 a	9.65
Interactions
High	Control	160.93	4.53	77.96 c	3.54	11.48 a	9.40
	SW	145.44	4.49	80.49 c	3.34	11.47 a	9.54
	PH	150.84	4.57	79.45 c	3.26	11.32 a	9.50
Low	Control	149.35	4.35	97.65 b	2.85	8.8 c	10.03
	SW	141.57	4.38	117.61 a	2.75	8.83 c	10.08
	PH	151.43	4.36	120.17 a	2.66	10.22 b	9.79
Statistical significance
Nr		NS	NS	***	***	***	***
Biostimulants		NS	NS	*	NS	*	NS
Nr X Biostimulants		NS	NS	*	NS	**	NS

In each column, means (n = 4) followed by different letters are significantly different according to Duncan’s multiple range test (p ≤ 0.05). Regarding the variance components, according to associated p values, *** (p ≤ 0.001), ** (p ≤ 0.01), and * (p ≤ 0.05) represent statistical significance, while NS signifies non-significance.

).

For corn salad, PH application under low N_r significantly increased FW by 16.1%, compared to the control (no biostimulant application). When supplied with high N_r, corn salad exhibited a significantly higher FW than under low-N_r treatment. No significant differences were observed in the fresh yield of purslane, regardless of the treatments applied (Table 2).

Regarding dry matter content (DMC), significant differences were detected only in iceplant and corn salad. High N_r increased iceplant DMC by 22.1% relative to low N_r, whereas corn salad DMC increased by 5.3% under low N_r. In all three species, biostimulant applications had no significant effect on DMC (Table 2).

3.3. Nitrogen and Leaf Nitrate Content

The N nutrition of all three plants was significantly affected by both N_r and biostimulant application. Particularly, in purslane N, the reduced forms (N_red), mainly organic N, increased significantly by 15.3% under high N_r while the mineral form of N (N_min) significantly reduced by 21.3% under high N_r and by 15.9% with PH application. Consequently, total-N increased by 11.7% whereas NO₃⁻ content reduced by 19.3% under high N_r. Furthermore, the application of PH biostimulant also reduced the NO₃⁻ content by 14.5%, compared to the control (

Table 3. Impact of ammonium to N ratio (N_r) and biofertilizer application on reduced N (N_red), mineral N (N_min), total-N (N_red + N_min) content as percentage of dry matter (%), percentage of N assimilation (Nred/total-N %) and leaf nitrate (NO₃⁻) content as mg of NO₃⁻ per Kg of fresh weight (FW) (ppm) of purslane, iceplant and corn salad. SW: seaweed extract; PH: protein hydrolase.

Main Effects		Purslane
Nr	Biostimulants	Nred (%)	Nmin (%)	Total-N (%)	Nass (%)	NO3− (ppm)
High		4.98 a	0.37 b	5.35 a	93.16 a	732 b
Low		4.32 b	0.47 a	4.79 b	90.15 b	907 a
	Control	4.64	0.44 a	5.07	91.35 b	855 a
	SW	4.60	0.45 a	5.04	91.01 b	873 a
	PH	4.75	0.37 b	5.12	92.56 a	731 b
Statistical significance
Nr		***	***	***	***	***
Biostimulants		NS	***	NS	**	***
Nr X Biostimulants		NS	NS	NS	NS	NS
Nr	Biostimulants	Iceplant
		Nred (%)	Nmin (%)	Total-N (%)	Nass (%)	NO3− (ppm)
High		4.85 b	0.33 b	5.18 b	93.52 a	496
Low		5.06 a	0.40 a	5.46 a	92.69 b	481
	Control	4.90 b	0.38	5.28 b	92.78 b	530 a
	SW	4.72 b	0.36	5.08 b	92.91 b	470 b
	PH	5.24 a	0.36	5.60 a	93.64 a	457 b
Statistical significance
Nr		*	***	**	***	NS
Biostimulants		***	NS	***	**	**
Nr X Biostimulants		NS	NS	NS	NS	NS
Nr	Biostimulants	Corn salad
		Nred (%)	Nmin (%)	Total-N (%)	Nass (%)	NO3− (ppm)
High		4.88	0.28 b	5.16	94.50 a	1191 b
Low		4.73	0.31 a	5.04	93.89 b	1358 a
	Control	4.83	0.30 a	5.13	94.15 ab	1295 a
	SW	4.77	0.31 a	5.08	93.92 b	1340 a
	PH	4.81	0.28 b	5.09	94.52 a	1194 b
Statistical significance
Nr		NS	***	NS	***	***
Biostimulants		NS	***	NS	**	***
Nr X Biostimulants		NS	NS	NS	NS	NS

In each column, means (n = 4) followed by different letters are significantly different according to Duncan’s multiple range test (p ≤ 0.05). Regarding the variance components, according to associated p values, *** (p ≤ 0.001), ** (p ≤ 0.01), and * (p ≤ 0.05) represent statistical significance, while NS signifies non-significance.

).

In iceplant, high N_r significantly decreased N_red by 4.2% and N_min by 17.5%, compared to low Nr, while PH increased N_red by 6.9% compared to the control (no biostimulant application). These changes led to a 5.1% decrease in total-N under high NH₄⁺ concentration in the root zone (high N_r) compared to low N_r. Regarding NO₃⁻ content in fresh leaf tissues, only biostimulant application (SW: Algastar; PH: Tyson) had a significant effect, reducing it by 11.3% and 13.8%, respectively (Table 3).

For corn salad, neither N_r nor biostimulant application altered N_red and total-N. However, high N_r and PH application reduced N_min by 9.7% and 6.7%, respectively, compared to low N_r and no biostimulant application, which were also reflected in NO₃⁻ content, decreasing by 12.3% and 7.8%, respectively. Furthermore, N assimilation (N_ass), calculated as N_red to total-N, was significantly affected in all three species by both factors without any significant interactions between them. Specifically, N_ass was decreased under low N_r in both iceplant and corn salad, compared to low N, while the reverse was the case for purslane. As for the biostimulants, PH application consistently reduced NO₃⁻ and increased N_ass (Table 3).

3.4. Nutrient Concentrations in Plant Tissues

Regarding the K⁺, Ca²⁺ and Mg²⁺ concentrations in purslane, only Ca²⁺ was significantly affected by N_r. Specifically, plants supplied with high N_r exhibited a 30.2% reduction in Ca²⁺ concentration compared to those grown under low N_r (

Table 4. Impact of ammonium to N ratio (N_r) and biofertilizer application on K, Ca, Mg and Na content as mg per g of dry matter of purslane, iceplant and corn salad. SW: seaweed extract; PH: protein hydrolase.

Main Effects		Purslane
Nr	Biostimulants	K (mg g−1)	Ca (mg g−1)	Mg (mg g−1)	Na (mg g−1)
High		63.69	3.77 b	10.81	1.38
Low		67.00	5.40 a	10.74	1.32
	Control	64.89	4.91	10.94	1.29
	SW	64.29	4.64	10.91	1.47
	PH	66.44	4.13	10.50	1.32
Statistical significance
Nr		NS	***	NS	NS
Biostimulants		NS	NS	NS	NS
Nr X Biostimulants		NS	NS	NS	NS
Nr	Biostimulants	Iceplant
		K (mg g−1)	Ca (mg g−1)	Mg (mg g−1)	Na (mg g−1)
High		20.69	0.72	4.74	10.23
Low		20.15	0.74	4.65	8.42
	Control	20.00	0.68	4.52	9.17
	SW	19.75	0.70	4.77	9.50
	PH	21.44	0.80	4.81	9.33
Statistical significance
Nr		NS	NS	NS	NS
Biostimulants		NS	NS	NS	NS
Nr X Biostimulants		NS	NS	NS	NS
Nr	Biostimulants	Corn salad
		K (mg g−1)	Ca (mg g−1)	Mg (mg g−1)	Na (mg g−1)
High		57.80	0.58	4.07	0.44
Low		58.17	0.61	4.35	0.47
	Control	57.33	0.57	4.08	0.43
	SW	58.25	0.61	4.27	0.43
	PH	58.25	0.60	4.28	0.50
Statistical significance
Nr		NS	NS	NS	NS
Biostimulants		NS	NS	NS	NS
Nr X Biostimulants		NS	NS	NS	NS

In each column, means (n = 4) followed by different letters are significantly different according to Duncan’s multiple range test (p ≤ 0.05). Regarding the variance components, according to associated p values, *** (p ≤ 0.001) represent statistical significance, while NS signifies non-significance.

). In contrast, K⁺ content ranged from 63.69 to 67.00 mg g⁻¹ and Mg²⁺ from 10.50 to 10.94 mg g⁻¹ across all treatments, with no significant differences among either the N_r or the biostimulant application treatments (Table 4).

Similarly, in iceplant and corn salad, macronutrient concentrations (K⁺, Ca²⁺ and Mg²⁺) were not significantly affected by the two factors examined (N_r and biostimulant) (Table 4). In iceplant, K⁺ content ranged from 19.75 to 21.44 mg g⁻¹, Ca²⁺ from 0.68 to 0.80 mg g⁻¹ and Mg from 4.52 to 4.81 mg g⁻¹. For corn salad, K⁺ levels were 57.33–58.25 mg g⁻¹, Ca²⁺ 0.57–0.61 mg g⁻¹ and Mg 4.07- 4.35 mg g⁻¹. Additionally, neither N_r nor biostimulants significantly impacted Na content, which ranged from 1.29 to 1.47 mg g⁻¹ in purslane, 0.43 to 0.50 mg g⁻¹ in corn salad, and 8.42 to 10.23 mg g⁻¹ in iceplant.

In purslane leaf tissues, Fe (60.83–65.31 μg g⁻¹) and Mn (73.87 to 79.28 μg g⁻¹) concentrations remained stable and without any significant differences across treatments (

Table 5. Impact of ammonium to N ratio (N_r) and biofertilizer application on Fe, Mn, and Zn content as μg per g of dry matter of purslane, iceplant and corn salad. SW: seaweed extract; PH: protein hydrolase.

Main Effects		Purslane
Nr	Biostimulants	Fe (μg g−1)	Mn (μg g−1)	Zn (μg g−1)
High		64.30	75.61	49.60 a
Low		62.49	77.28	37.89 b
	Control	64.58	79.28	43.25
	SW	65.31	73.87	44.82
	PH	60.83	75.53	44.06
Statistical significance
Nr		NS	NS	***
Biostimulants		NS	NS	NS
Nr X Biostimulants		NS	NS	NS
Nr	Biostimulants	Iceplant
		Fe (μg g−1)	Mn (μg g−1)	Zn (μg g−1)
High		170.63 a	70.38	83.04 a
Low		81.70 b	70.29	59.34 b
	Control	125.12	68.80	69.82
	SW	133.74	68.04	73.16
	PH	120.48	73.92	70.81
Statistical significance
Nr		***	NS	***
Biostimulants		NS	NS	NS
Nr X Biostimulants		NS	NS	NS
Nr	Biostimulants	Corn salad
		Fe (μg g−1)	Mn (μg g−1)	Zn (μg g−1)
High		94.26 a	86.09 a	45.62 a
Low		87.54 b	78.52 b	38.59 b
	Control	91.73	84.62	44.30
	SW	87.70	79.8	38.75
	PH	92.64	82.12	42.94
Statistical significance
Nr		**	*	*
Biostimulants		NS	NS	NS
Nr X Biostimulants		NS	NS	NS

In each column, means (n = 4) followed by different letters are significantly different according to Duncan’s multiple range test (p ≤ 0.05). Regarding the variance components, according to associated p values, *** (p ≤ 0.001), ** (p ≤ 0.01), and * (p ≤ 0.05) represent statistical significance, while NS signifies non-significance.

). However, Zn concentration was significantly influenced by N_r, with high N_r increasing Zn content by 30.9% compared to low N_r.

In iceplant, N_r significantly affected Fe and Zn concentrations, with Fe increasing by 109% and Zn by 40%, compared to low N_r (Table 5). No significant differences were observed in Mn concentration, which ranged from 68.04 to 73.92 μg g⁻¹ across all treatments.

In corn salad, high N_r led to significant increases in Fe, Mn and Zn concentrations of 7.7%, 9.6% and 18.2%, respectively. However, the application of biostimulants had no significant effect on Fe, Mn or Zn content in any of the tested plant species (purslane, iceplant, or corn salad).

3.5. Partial Budget Analysis

Table 6. Added net return for purslane, iceplant and corn salad obtained by the application of seaweed extract (SW) or protein hydrolysate (PH).

Species	Biostimulant	Yield Increase (kg ha−1)	Price (EUR kg−1)	Added Gross Return (EUR ha−1)	Added Variable Cost (EUR ha−1)			Added Net Return (EUR ha−1)
					Biostimulant Treatment	Application	Total
Purslane	SW	−2800.0	8.5	−23,800.0	81.0	200.0	281.0	−24,081.0
	PH	−862.5	8.5	−7331.3	51.0	200.0	251.0	−7582.3
Iceplant	SW	2475.0	41.0	101,475.0	81.0	200.0	281.0	101,194.0
	PH	2102.5	41.0	86,202.5	51.0	200.0	251.0	85,951.5
Corn salad	SW	−30.0	17.6	−528.0	81.0	200.0	281.0	−809.0
	PH	102.5	17.6	1804.0	51.0	200.0	251.0	1553.0

SW: 13.5 EUR/L; PH: 8.5 EUR/L.

shows the added net return obtained by the application of SW and PH on purslane, iceplant and corn salad.

The output underlined that biostimulants differently affected the added net return of the species. For purslane, negative values were found, independently of the biostimulant used. For iceplant, the application of both biostimulants gave positive outcomes in terms of added net return, with the peak in plants treated with SW (+101,194.0 EUR ha⁻¹). Lastly, corn salad showed different behavior depending on the biostimulant used with negative values when SW was applied and positive results when plants were treated with PH (+1553.0 EUR ha⁻¹).

4. Discussion

The integration of wild edible species and underutilized crops into highly intensive and productive agricultural systems, such as soilless culture, requires targeted research to understand and identify optimal agronomic practices for yield, quality and resource usage. To this end, effective and precise nutrition management is critical to optimize both yield production and product quality. For leafy greens, nutrient supply must be carefully regulated to minimize NO₃⁻ accumulation in edible tissues, ensuring compliance with the European Union’s maximum permissible nitrate limits (Regulation No. 2023/915) and safeguarding human health [8,9,10,49].

In soilless cultivation systems, both NH₄⁺ and NO₃⁻ forms are supplied to the plants. However, the ratio between the two nitrogen forms (N_r) is crucial for pH management in the root zone and N plant nutrition [50]. Plants preferentially take up NH₄⁺ up to certain levels due to its lower energy requirements for N assimilation into carbon skeletons compared to NO₃⁻ [32]. However, excessive NH₄⁺ can lead to ammonia toxicity, particularly under low-pH conditions in the root environment [51]. It has been observed that excessive consumption of sugars for ammonium assimilation caused by elevated NH₄⁺ levels can result in carbohydrate limitation and elevated ammonia within the cytoplast [52]. This can reduce the availability of carbohydrates for photosynthesis and respiration, alter cytoplasmic pH, and induce oxidative stress [53].

As demonstrated in the present study, the response of plants to N_r levels depends on species and cultivation system. These species-specific differences likely reflect distinct capacities for ionic balance and tolerance to ammonium stress. The results of the fresh yield for the three examined leafy greens, purslane, iceplant and corn salad, varied with N_r levels. In the case of purslane, there were no significant differences in FW between low (0.04) and high N_r (0.12) levels, whereas iceplant was negatively affected by high N_r and corn salad demonstrated a positive response. Similar variability exists among other leafy vegetables. For instance, lettuce FW increased with N_r up to 0.30 [54]. Furthermore, Wenceslau et al. [52] recommended an N_r level of 0.23 for iceberg lettuce in a floating hydroponic system, while toxicity symptoms were observed at N_r levels higher than 0.5. Furthermore, the findings of Bulgari et al. [55] suggested that rocket yield remained unaffected by different N_r values from 0 to 0.50, consistent with the findings for Cichorium spinosum L. (stamnagathi) by Chatzigianni et al. [7]. On the other hand, sowthistle (Sonchus oleraceus L.), a wild edible leafy green, exhibited better performance in hydroponic culture with nutrient solutions of an N_r of 0.05 or less rather than higher values [56]. Regarding the effect of N_r on golden thistle (Scolymus hispanicus L.), another wild edible species, Papadimitriou et al. [57] suggested that the FW of substrate-grown plants was higher under standard N supply when 10% of the nitrogen was supplied in the form of NH₄⁺ as compared to a supply of 20%.

In the present study, purslane FW and DMC remained unaffected by the two N_r treatments applied. Spyrou et al. [49] observed a 19.3% yield reduction in purslane grown in a floating hydroponic system when N_r increased from 0.07 to 0.14, while Chrysargyris et al. [58] found that purslane FW was reduced in an NFT hydroponic system when an N_r over 5% was supplied. The contradictory findings can mainly be attributed to the differences in the cultivation systems. These findings indicate that optimal N_r values are not universal but depend on the species and the cultivation system. In both studies, the plants were cultivated in water culture systems, i.e., hydroponics, whereas in the present study, a substrate-based system comprising perlite was employed. A key distinction is that in soilless substrate systems, the volume of the root solution is substantially less than in hydroponic (i.e., water culture) systems [59]. The higher solution volume per plant in hydroponic systems can sustain elevated NH₄⁺ concentrations over extended periods. This phenomenon occurs despite the preferential uptake of NH₄⁺ by the plants, as the total amount of N absorbed is considerably lower than the available NH₄⁺ concentration of the root solution. Conversely, the limited root zone solution volume in substrate systems increases the likelihood of NH₄⁺ depletion. Notably, the application of biostimulants had no effect on plant growth parameters, suggesting minimal stress impact under both N_r levels. These findings highlight the need to tailor fertigation strategies not only to plant species but also to the cultivation system.

Corn salad demonstrated a positive response to elevated N_r levels with increased NH₄⁺ supply leading to enhanced plant FW. The slight increase in DMC under low N_r suggests that those plants can more efficiently utilize NH₄⁺-N to cover their needs due to its lower energy demand for assimilation into organic compounds [53,60]. Both biostimulants further promoted plant growth under low NH₄⁺ supply by increasing the FW. These findings align with Di Mola et al. [31], who suggested that plant-based protein hydrolysate biostimulant can effectively mitigate stress under reduced N availability in V. locusta. All the above highlight PH biostimulants’ capacity to improve N nutritional status and stimulate growth under N-limited conditions in corn salad. Consequently, the results suggest that corn salad tolerated elevated NH₄⁺ levels and benefited from biostimulant application, leading to enhanced growth.

In contrast, a clear negative effect of high NH₄⁺ application has been observed in the case of iceplant. FW significantly decreased under high N_r while DMC increased, indicating that the plants were under stress. The pH levels in both N_r treatments showed a downward trend, a finding that is in accordance with the observations of Rincón-Cervera et al. [61] in soilless iceplant crops, with significantly lower values in high-N_r treatment due to high NH₄⁺ supply [33]. However, pH was maintained at a level of over 5 for the entire growing cycle, which is the lower safety level for the plants [50]. It is thus evident that the observed growth restriction is predominantly ascribed to ammonia toxicity in the roots of iceplant [53]. However, both biostimulants enhanced plant growth, but only under optimal NH₄⁺ supply. Choi et al. [62] recently reported that vegetal-derived PH biostimulants, such as ‘Tyson’ in the present study, can effectively protect hydroponically grown lettuce plants from yield loss caused by low NO₃:NH₄, supporting the role of biostimulants in counteracting ammonia toxicity due to high NH₄⁺ supply.

Regarding N metabolism, the findings of the present study indicate that high N_r in the nutrient solution resulted in a promotion of N assimilation across all three plants. This phenomenon can be attributed to the N assimilation cycle, whereby NH₄⁺-N is directly incorporated into glutamate via the glutamine synthetase pathway, whereas NO₃⁻ requires energy-intensive reduction to NH₄⁺ via nitrate and nitrite reductases [63]. Hence, NH₄⁺-N assimilation is a less energy-consuming process [32]. Therefore, under high N_r, NO₃⁻ concentration in the leaves of purslane and corn salad was restricted, primarily because higher NH₄⁺ availability suppresses NO₃⁻ uptake and assimilation. Indeed, an increased supply of NH₄⁺ is an effective practice for reducing the accumulation of NO₃⁻ in wild edible leafy greens, which are tolerant to elevated NH₄⁺ concentrations in the root zone, such as C. spinosum [7] and S. oleraceus [56]. In the case of M. crystallinum plants, despite NH₄⁺ stress, no differences in the leaf NO₃⁻ concentration were detected. This finding suggests that the assimilation of N was not affected while the differences in total-N were ascribed to reduced NO₃⁻ uptake. In addition, the cultivation of purslane plants in a water culture system, in conjunction with the imposition of elevated levels of NH₄⁺ in the root zone, resulted in a decline in their FW, while the leaf NO₃⁻ concentration remained unaffected [49], mirroring iceplant responses in this study.

Elevated NO₃⁻ concentrations in food are associated with an increased risk of cancer [64,65]. As such, NO₃⁻ concentration in the edible parts of leafy vegetables is recognized as a pivotal quality index for ensuring human safety. The European Union has established regulatory measures for the maximum permissible limits of nitrate content for certain leafy greens, including lettuces, spinach and rucola, ranging from 2000 to 5000 mg kg⁻¹ (Regulation No. 2023/915). Although there are no defined limits for the species examined in this study, the respective values for the other leafy greens serve as indicators. In this study, NO₃⁻ concentrations in corn salad remained below 2000 mg kg⁻¹, consistent with findings by Voutsinos-Frantzis et al. [10]. Manzocco et al. [66] reported that hydroponically grown crops with 18 mM N (NO₃⁻ + NH₄⁺) in the nutrient solution can accumulate up to 4700 mg kg⁻¹ of NO₃⁻. Meanwhile, purslane and iceplant exhibited even lower NO₃⁻ levels (<1000 mg kg⁻¹), reflecting high product quality. Furthermore, Spyrou et al. [49] observed that hydroponic purslane grown in a 15 mM N solution can accumulate up to 2900 mg kg⁻¹ of NO₃⁻, suggesting that the present study’s lower values may result from cultivation conditions. The low leaf NO₃⁻ concentration in all plant species may also be attributed to high solar radiation, given that the cultivation took place in spring under Mediterranean conditions [67]. An alternative approach to N nutrition management for soilless-grown leafy greens is to reduce N supply during the final days before harvest, which can limit NO₃⁻ accumulation while maintaining yield and enhancing the plants’ antioxidant compound content [68,69,70].

The application of PH biostimulants, composed of peptides and amino acids, enhanced N assimilation in all three plant species and effectively reduced NO₃⁻ accumulation in their edible parts. It is evident that multiple physiological mechanisms are collectively involved in this plant response to PH biostimulants. Firstly, it is important to note that key enzymes involved in nitrogen metabolism, including nitrate reductase, glutamine synthetase, and glutamate synthase, have been shown to lead to increased conversion of NO₃⁻ into organic nitrogen compounds. This process is stimulated by PHs through enzymatic and molecular mechanisms, including the upregulation of related gene expression [71,72,73], both accelerating NO₃⁻ assimilation, thereby reducing its accumulation in leaves.

Additionally, PH biostimulants provide readily available organic nitrogen sources (amino acids and short peptides), which are absorbed by roots and leaves and directly used in protein synthesis, supplement N, and reduce the plant’s dependence on the energy-demanding nitrate reduction pathway [74]. Furthermore, they contain bioactive peptides that act as signaling molecules, activating changes in gene expression related to N assimilation pathways, including transcriptional upregulation [75]. Amino acids also improve the efficiency of N uptake by regulating the activity of membrane transporters [76,77]. Collectively, these mechanisms enhance nitrogen use efficiency (NUE) and reduce NO₃⁻ levels in edible plant tissues, offering a promising strategy to improve both crop growth and product quality.

In purslane, Ca²⁺ concentration in the leaves significantly reduced under high N_r, while the Ca:Mg ratio also decreased. This suggests that the well-known competitive uptake mechanism between cations [53] was in favor of Mg²⁺ (NH₄⁺-Mg²⁺) rather than Ca²⁺ (NH₄⁺-Ca²⁺) [7]. The increased N_r in the nutrient solution also enhanced the uptake and accumulation of micronutrients such as Fe, Mn, and Zn in plant tissues of all three species. As demonstrated by White and Broadley [60], partial hydrolysis of metallic micronutrients, such as Fe, Mn, and Zn, has been observed in aqueous solutions, thereby rendering them unavailable to plants. Τhis response is primarily attributed to the rhizosphere acidification induced by NH₄⁺ uptake [78]. The higher availability of H⁺ near the root cells, caused by their export from the cells in favor of NH₄⁺ uptake, can increase the availability of these micronutrients. Higher leaf concentrations of Mn have also been reported in C. spinosum when the N_r increased from 0.05 to 0.25 [7]. Similarly, an increase in NH₄⁺ supply in lettuce crops has elevated the concentration of Fe [54] and Zn [62].

Based on the results of this study, growers aiming to cultivate purslane, iceplant, and corn salad in substrate-based soilless systems should adjust N_r in the supplied solution and follow a biostimulant application strategy depending on each species’ requirements. For purslane, the N_r can be maintained within the tested range (0.04–0.12) without compromising yield. To enhance product quality by reducing nitrate (NO₃⁻) accumulation in edible tissues, growers may either use a high N_r (0.12) or apply protein hydrolysate (PH) biostimulants weekly. Corn salad benefits from a higher N_r (0.12), which increases FW while reducing NO₃⁻ concentrations. Supplementing with PH biostimulants further reduces leaf NO₃⁻ levels, enhancing food safety. Iceplant should be cultivated under low N_r (0.04) to prevent ammonium stress and yield losses, while applying either seaweed extracts or PH biostimulants can boost yield and further decrease leaf NO₃⁻ content.

The economic evaluation revealed that the added net return obtained by the application of biostimulants highly depends on the species and biostimulant used. Specifically, for purslane, the added net return was always negative, suggesting a non-economic convenience for both treatments. Contrariwise, for iceplant, both biostimulants gave positive added net return, with a peak obtained by SW treatments. Moreover, corn salad’s added net return in response to biostimulant application depends on the biostimulant employed. Indeed, the application of SW determined negative added net return values, whereas the use of PH gave positive results. In general, the added variable costs for all species are very low; thus, the differences recorded can be attributable to the differences in yield performance and in price per kg, which in turn influence the added gross return. In fact, the negative values recorded for purslane are linked to the non-significant effect of biostimulants on yield and to the low price per kg, compared to the other two species. Furthermore, the peak recorded in iceplant can be attributed to the high increase in yield and to the very high price per kg (EUR 41.0).

5. Conclusions

This study demonstrates that optimizing the NH₄⁺/total-N ratio (N_r) and applying biostimulants significantly influence yield performance, nitrate accumulation, and nutrient composition in underutilized leafy greens cultivated in soilless systems. The responses were highly species-dependent: corn salad exhibited improved fresh weight under high N_r (0.12), whereas iceplant suffered yield losses due to NH₄⁺ toxicity, classifying it as an ammonium-sensitive species. In contrast, purslane maintained stable yields across N_r levels (0.04–0.12), reflecting its moderate NH₄⁺ tolerance. Notably, protein hydrolysate (PH) biostimulants enhanced nitrogen assimilation efficiency and reduced nitrate accumulation in edible tissues, underscoring their potential to improve both crop productivity and food safety. Furthermore, elevated NH₄⁺ supply enhanced the uptake of micronutrients (Fe, Mn, Zn), likely due to rhizosphere acidification favoring their solubility. These findings emphasize the need for species-specific nitrogen management strategies in soilless cultivation, integrating tailored N_r adjustments and biostimulant applications to optimize yield, nutritional quality, and resource-use efficiency. Moreover, the economic evaluation suggested that the application of both biostimulants is not convenient for purslane, but it is highly recommended for iceplant, and that—for corn salad—PH is the recommended biostimulant to increase the added net return. This study is preliminary and serves as a starting point for evaluating soilless cultivation and exploring the impact on the biochemical profiles and nutritional value of these three species. Additionally, future research should explore the molecular mechanisms underlying PH biostimulant efficacy and refine NH₄⁺ thresholds for additional underutilized species to expand their sustainable production in controlled environments.