Impact of Nitrogen Limitation, Irrigation Levels, and Nitrogen-Rich Biostimulant Application on Agronomical and Chemical Traits of Hydroponically Grown Cichorium spinosum L.

Abstract

This study investigates the effects of nitrogen fertilization, irrigation, and biostimulant application on the growth and nutrient composition of Cichorium spinosum L. The experimental design included two nitrogen rates (NR100 and NR30, 100% and 30% of plant requirements), two irrigation levels (WA100 and WA50, 100% and 50% of water availability,), and foliar application of a nitrogen-rich biostimulant (BS and NoBS, biostimulated or not biostimulated). In comparison to NR100, NR30 reduced agronomical parameters leaf number, leaf area, leaf fresh, and dry weight by 13.53%, 24.93%, 20.76%, and 15.00%, respectively, whereas dry matter content was increased by 7.64%. WA50 also resulted in reduction in the agronomical characteristics by 8.62%, 7.19%, 5.53%, and 5.26, respectively, whereas the dry matter content was not affected. BS positively affected the agronomical characteristics by 7.49%, 8.01%, 7.18%, and 5.56, respectively, whereas the dry matter content was not affected. The effects of nitrogen rates and water availability suggest the more pronounced impact of nitrogen compared to water stress on the agronomical characteristics. Biostimulant application partially mitigated the effects of NR30 but was ineffective against WA50. The nutrient content of the leaves was also affected. NR30 reduced leaf nitrate, calcium, and zinc content, but increased iron, manganese, and copper concentrations. WA50 altered magnesium and zinc levels: it increased the former and decreased the latter. The interaction between nitrogen and water stress notably affected the plants’ calcium content, which was higher under the NR100 x WA50 treatment. These findings provide significant insights into the perlite-based cultivation of C. spinosum L., and its resilience against drought stress. Moreover, the beneficial effects of sufficient nitrogen rates on leaf fresh weight of Cichorium spinosum L. outline the importance for improving nutrient solution management schemes. Biostimulant application demonstrated promising results and could, after further research, become a viable solution for maintaining optimal yields under nitrogen stress.

1. Introduction

The agricultural sector has long faced challenges in meeting the needs of populations. Today, these challenges have escalated and taken on a global scale. According to FAO (Food and Agriculture Organization), it is dire for the agricultural to intensify food production to meet the rising global population’s demands by 2050 [1]. Increasing demand for year-round production, driven by rising population and urbanization, has placed immense pressure on farmers and arable land [2]. At the same time, the impacts of climate change, including unpredictable weather patterns and shifting growing seasons [3], as well as soil degradation and desertification, coupled with the concentration of populations in cities and the need to produce more food with fewer resources, such as water and fertilizers, have significantly heightened the difficulties the agricultural sector must overcome [4,5]. The need for agricultural practices for maintaining high yields, while avoiding excessive fertilizer use and reducing nutrient leaching and environmental pollution, has become apparent the past decades [6]. In addition, consumer habits have been shifting towards more healthy lifestyles. Awareness surrounding the health benefits of functional foods [7,8], and especially plants rich in minerals and bioactive compounds [9,10], has resulted in a growing demand for such species, cultivars, and cultivation methods. While the regulations regarding health claims and functional foods has been a difficult task, for both businesses and regulatory frameworks [11], there is a growing body of evidence that supports beneficial effects of the bioactive compounds found in plant-based foods including wild leafy greens [12,13,14,15].

The Mediterranean basin is a highly vulnerable to climate change, further intensifying the challenges facing its agriculture [16]. Agriculture in the Mediterranean is predominantly small-scale, and local products are integral to the region’s diet. Several wild edible vegetables offer potential advantages, such as greater resilience to environmental stresses like drought, extreme temperatures, and pests, along with the ability to maintain high yields [17,18]. The cultivation of wild edible vegetables could contribute to food security, while stabilizing agricultural incomes, especially for local and small-scale growers. Cichorium spinosum L. is a wild leafy edible plant of the Asteraceae family. It grows naturally in rocky terrains at various altitudes in the Mediterranean region, from near the coast to up mountains. In the literature it can be also found with the names “spiny chicory” or “stamnagathi”, which is its native name in Greece. Its leaves grow in a rosette formation, have a bitter flavor, and are typically harvested at various growth stages to be boiled or even eaten raw in salads [19,20,21,22]. Stamnagathi, being a wild edible plant, has not been thoroughly examined despite of its promising chemical properties. There are few studies that have examined stamnagathi in agricultural settings, with some focusing on open field production while others concentrating on its cultivation in soilless agricultural systems [23,24,25,26,27]. Even though stamnagathi has an estimated demand of 2000 tons per year, there are few to no commercial growers that currently supply the mainstream markets. There have been few projects for large-scale open-field and perlite-based cultivation the past few decades but those companies are no longer actively present in the market. The demand is met by gathering stamnagathi in the wild or at the borders of growers’ field, which raises a lot of questions regarding quality and safety.

The excessive use of fertilizers, particularly nitrogen-based ones, is a major contributor to environmental pollution through nitrate leaching. As the global population grows, the demand for nitrogen fertilizers has surged, while concerns over their environmental impact have significantly increased in recent decades [28]. In plants, nitrogen is absorbed as nitrate or ammonium [29,30]. In the initial stage of nitrate assimilation, nitrate reductase [31] converts nitrate into nitrite, followed by the conversion of nitrite to ammonium by nitrite reductase [32,33]. Ammonium is then utilized in amino acids through reactions mediated by glutamine synthetase and glutamate synthase [34,35]. The cultivation of horticultural crops is especially prone to nitrate losses due to their high fertilizer requirements and shallow root zones. Therefore, improving nitrogen utilization efficiency is a difficult task affected by several factors, such as the hydraulic characteristics of the growing medium, the nitrogen and other nutrient levels in the irrigation water, and the timing of irrigation. Moreover, the needs for water and nutrients in plants are frequently determined by genetics and are specific to each species. Furthermore, root morphology and the interaction between canopy and the surrounding environment also affect the transpiration rate and therefore nutrient uptake. Precision fertigation, by delivering nutrients through irrigation, can improve the absorption of plant nutrients, such as nitrogen, while reducing nitrate runoff [36].

Several parameters, such as light and photoperiod, fertilization strategies, and genetics, affect nitrate assimilation and therefore its concentration in plants [37,38,39,40]. As a vital component of numerous biomolecules, nitrogen is indispensable to life, performing essential functions in the physiology and metabolism of living organisms [41,42,43]. The availability of nitrogen influences morpho-physiological plant traits, thereby impacting marketability and visual quality [44]. Since the 1970s, researchers have shown a keen interest in how nitrates build up in various vegetables [45,46]. Leafy vegetables are known to contain significant amounts of nitrates compared to other plant-based foods [47]. It is important to note that some wild leafy vegetables often have significant nitrate levels, even when gathered from non-agricultural and non-fertilized land. This should be considered when attempting to bring these species into intensive farming systems [48]. The potential risks associated with high nitrate content in consumed products, particularly the suspected link to various cancers, have led to precautionary measures to decrease nitrate levels in leafy vegetables [49,50,51]. To this end, a precise nitrogen supply can mitigate high nitrate content in leaves during harvest, while maintaining high yields [52,53]. Soilless culture methods are ideal for exerting high control over nutrient supply to the plants. Previous studies have demonstrated that some stamnagathi ecotypes can be cultivated under a total N concentration of 4 mM without a reduction in its biomass, compared to a total N concentration of 16 mM, demonstrating its genotypic resilience to low-N supply levels [54].

Drought poses a formidable challenge for agriculture, a concern that is increasingly relevant with climate change. The availability of water for agriculture is increasingly constrained worldwide, where water scarcity increasingly threatens global food accessibility, necessitating the adoption of effective irrigation management strategies [55,56]. As a result, the effective management of irrigation water becomes paramount in ensuring a consistent food supply [57]. Identifying the water stress thresholds of crops and utilizing reduced irrigation practices has emerged as a key strategy in water-scarce regions as a means to optimize water usage [58]. Within this context, soilless culture methods and deficit irrigation emerge as a viable approach. In soilless systems, the precise control of irrigation allows for the adaptation of water supply to plant needs, minimizing water wastage and maximizing resource utilization [59]. The impact of water deficiency itself on plants is multifaceted, shaped by the severity, length of exposure, and the plant’s growth stage. In order to avoid the disruption of plant–water relations, which can in turn decrease water use efficiency, impair photosynthesis, and compromise water and nutrient uptake, understanding each plant’s needs is crucial in soilless culture and deficit irrigation practices [60,61,62].

Biostimulants are considered a promising novel technology that can be applied via foliar spray or fertigation. These products could be utilized to reinforce nutrient uptake and improve crop yield and quality, as well as modulate plant metabolic processes [63,64,65,66,67,68,69]. As an upcoming technology, the global biostimulants market is experiencing rapid growth. Projections indicate that it could reach a value of USD 2.24 billion by 2018 [70] and is expected to increase to USD 5.1 billion by 2027 [71]. A type of biostimulant, protein hydrolysates are composed of free amino acids and oligo- or polypeptides formed through chemical techniques, including enzymatic hydrolysis. Foliar application via spraying appears to allow the biostimulant to enter through the cuticle, epidermis, and stomata of the leaf, eventually reaching the foliar mesophyll [72]. Biostimulant-induced plant growth is frequently attributed to their hormone-like activities [73,74,75,76]. Protein hydrolysates have been shown to reduce yield loss when nitrogen levels of the soil or nutrient solution are sub-optimal [77]. Moreover, protein hydrolysates have also demonstrated promising results under drought conditions for crops with high water demands, such as grapevines, especially in Mediterranean vineyards [78].

Taking all the abovementioned into consideration, here, a study was designed aiming to explore how a wild edible leafy vegetable could be utilized as a cultivated crop under limited nitrogen or limited irrigation conditions and whether the application of a novel biostimulant could counteract some of the negative effects of the induced stresses.

2. Materials and Methods

2.1. Experimental Setup and Design

On 30 December 2022, a greenhouse experiment was set up, running for a duration of 38 days. The experiment was carried out in an unheated greenhouse at the Laboratory of Vegetable Production, Agricultural University of Athens (AUA), located at coordinates 37°59′2″ N and 23°42′19″ E.

As plant material, stamnagathi (Cichorium spinosum L.) achenes of the mountainous ecotype were used after breaking down the achenes in a commercial blender and separating the seed from the debris using a Endecotts Fluid Bed Dryer, model FBD2000 (Endecotts Limited, Parsons Ln, Hope Valley, UK), and a sieve until the remaining mixture contained mostly clean seeds and little debris (Figures S1 and S2). Seeds where then sown in rockwool or peat depending on the type of experiment. Seeds were sown in AO 25/40 rockwool plugs (Rockwool B.V, Roermond, The Netherlands). During the seedling preparation stage, these rockwool sheets were fertigated manually every 2–3 days to maintain moisture with the control nutrient solution (NR100).

The seedlings were separated and transplanted 35 days after sowing (

Table 1. Timetable of the experiment.

Culturing Practices	Date	Days after Sowing
Sowing	30 December 2022	0
Transplanting	3 February 2023	35
Harvest	13 March 2023	73

). The time of transplanting depended on the seedling growth stage. Seedlings were considered ready for transplanting after the development of five true leaves. Seedling plugs were re-planted directly into perlite bags at a plant density of 20 plants m⁻². Prior to transplanting, the perlite bags were fertigated with the nutrient solution designated for each treatment.

The effects of three applied factors, nitrogen rate (NR), water availability (WA), and biostimulant application (B), were investigated in this experiment. During the seedling preparation stage, the plants grew without the implementation of stresses. Irrigation and nitrogen deficit were applied after the transplanting phase. After transplanting, the cultivation was carried out under two isosmotic solutions with different nitrogen rates (NRs), namely the control nutrient solution (N100), with a total nitrogen at 13.64 mmol L⁻¹, and a limited nitrogen treatment with 30% (N30) that of the control, 4.55 mmol L⁻¹ (

Table 2. Chemical characteristics of the supplied nutrient solutions.

Element	Units	Control (NR100)	Limited Nitrogen (NR30)
NO3−	mmol L−1	12.00	4.00
NH4+	mmol L−1	1.64	0.55
NH4+/Total-N	-	0.12	0.12
K+	mmol L−1	6.71	6.98
Ca2+	mmol L−1	3.70	3.85
Mg2−	mmol L−1	2.07	2.16
SO42−	mmol L−1	3.11	4.41
H2PO4−	mmol L−1	1.46	1.46
Fe	μmol L−1	17.89	17.89
Mn2+	μmol L−1	9.36	9.36
Zn2+	μmol L−1	4.47	4.47
Cu2+	μmol L−1	0.73	0.73
B	μmol L−1	27.56	27.56
Mo	μmol L−1	0.52	0.52
Si	mmol L−1	0.00	0.00
Cl−	mmol L−1	0.40	6.00
Na+	mmol L−1	0.60	0.60
HCO3−	mmol L−1	0.40	0.40
Other properties
EC *	dS m−1	2.5	2.5
pH	-	5.6	5.6
Ψs **	MPa	−0.20	−0.20

* Electrical conductivity; ** osmotic potential.

). Water availability was applied at two levels: the control treatment, which supplied 100% of the plant’s needs (WA100), and the drought treatment, which was applied by reducing the irrigation supply to 50% (WA50). In the control group, plants were irrigated so that the target drainage fraction substituted by a suitable value of 30%, whereas drought treatments had a drainage of 0%. The drought treatment was initiated one week after the transplant. In addition, biostimulant application (B) with a hydrolyzed plant protein was carried out via foliar spray (BS), whereas the control plants received only water (NoBS). The biostimulant applied in this study was Tyson^® by Mugaver Fertilizers (Mugavero Teresa S.A.S, Contrada Canne Masche, Italy), a protein hydrolysate biostimulant rich in nitrogen (total nitrogen, 5.0%; organic nitrogen, 4.5%; organic carbon, 25%; and free amino acids, 13.4%), which was selected for its demonstrated beneficial effects in previous trials [67,68,69,76,79]. The solution was prepared as recommended (3 mL of Tyson^® per L of tap water), and 0.2 mL per plant was applied every 10 days (3 times throughout the experiment).

A split–split plot design was applied for the three main factors. The main plots contained two levels of nitrogen rates (NR100 and NR30). Subplots were divided into two levels of water availability (WA100 and WA50), and sub-subplots were dedicated to the biostimulant treatments (NoBS and BS). This design led to 8 unique treatment combinations (2 NR × 2 WA × 2 B), and each treatment was repeated in 6 perlite grow bags, with 6 plants per replicate, resulting in 288 plants (Figures S3 and S4).

2.2. Measurements and Statistical Analysis

In total, 18 plants per treatment were sampled from 6 grow bags. Each plant was collected to assess its individual agronomic traits. First, the number of leaves was counted by separating them manually by hand and placing them on a leaf area meter (model LI-3100C, LI-COR, Inc., Lincoln, NE, USA). A precision scale (model PE-3600, Mettler Toledo LLC, Columbus, OH, USA) was then used to record the fresh weight of each plant. The leaves were then dried in a ventilated oven (model STF-N 400, FALC Instruments S.L.R., Treviglio, Italy) at 65 °C until the recorded values stabilized for all samples, 7 days after placing them in the chamber. After the drying process, 3 plants per grow bag were combined and treated as a single sample, resulting in 6 replicates per treatment. The samples were then placed into a grinder at 6000–6500 rpm (MF 10 Microfine, IKA Werke, Staufen, Germany) and gathered into sealable plastic bags to prevent degradation before chemical analysis. Chemical analysis were carried out as previously presented in another publication by our group [80]. Leaf nitrate content was determined using the salicylic acid nitrification method [81]. Moreover, the dry ash method was used for the preparation of the aqueous tissue extract, which was then used for the determination of the leaf nutrient content. Flame atomic absorption spectroscopy (FAAS) was applied for the macro-nutrients calcium, magnesium, and iron and the micro-nutrients manganese and zinc (AA-7000, Shimadzu, Kyoto, Japan) [82]. Potassium and sodium were determined using flame photometry (Flame Photometer 410, Sherwood, Cambridge, UK) [83].

Statistical analysis was performed with the Statistica 10 software package by StatSoft Inc. for Windows (Tulsa, OK, USA). The data were analyzed using factorial ANOVA and Duncan’s multiple range test, with significance set at p ≤ 0.05 for all variables.

3. Results

3.1. Effects of Reduced Nitrogen Rates, Drought Stress, and Biostimulant Application on Agronomical Characteristics of Stamnagathi

Nitrogen rates (NRs) have significant effects on the agronomical characteristics of hydroponically cultivated stamnagathi (

Table 3. Main effects of nitrogen rate (NR), water availability (WA), and biostimulant application (B) on the leaf number (LN), leaf area (LA), leaf fresh weight (LFW), leaf dry weight (LDW), and dry matter content (DMC) of hydroponically grown stamnagathi.

Factor	Sources of Variation	LN (No. Plant−1)	LA (cm2 Plant−1)	LFW (g Plant−1)	LDW (g Plant−1)	DMC (%)
NR	NR100	16.78 ± 0.39 a	252.88 ± 4.96 a	17.87 ± 0.39 a	1.40 ± 0.03 a	7.85 ± 0.09 b
	NR30	14.51 ± 0.28 b	189.84 ± 5.65 b	14.16 ± 0.31 b	1.19 ± 0.02 b	8.45 ± 0.12 a
WA	WA100	16.35 ± 0.47 a	229.61 ± 9.37 a	16.47 ± 0.65 a	1.33 ± 0.04 a	8.23 ± 0.17 a
	WA50	14.93 ± 0.28 b	213.11 ± 7.01 b	15.56 ± 0.32 b	1.26 ± 0.03 b	8.07 ± 0.05 a
B	NoBS	15.08 ± 0.46 b	212.84 ± 9.59 b	15.46 ± 0.59 b	1.26 ± 0.04 b	8.25 ± 0.15 a
	BS	16.21 ± 0.32 a	229.88 ± 6.68 a	16.57 ± 0.40 a	1.33 ± 0.03 a	8.05 ± 0.08 a

The values presented in the table are mean values (n = 24) across treatments. In each column, mean within the same factor followed by different letters indicate significant differences according to Duncan’s multiple range test at p ≤ 0.05.

). Low nitrogen application (NR30) resulted in significantly inferior plant growth metrics compared to the control (NR100). Plants that underwent nitrogen stress exhibited lower leaf number, leaf area, fresh weight, and dry weight compared to those of NR100. Interestingly, the dry matter content was higher in NR30 compared to NR100. Water availability (WA) significantly influenced plant growth parameters in a similar trend to that of reduced nitrogen rates. Plants that underwent drought stress (WA50) demonstrated reduced leaf number, leaf area, fresh weight, and dry weight compared to plants that were irrigated sufficiently (WA100). Biostimulant application (B) also demonstrated a positive impact on plant growth in a similar manner to that of nitrogen rates. Plants sprayed with the nitrogen-rich biostimulant (BS) demonstrated higher leaf number, leaf area, fresh weight, and dry weight values, whereas dry matter content was not significantly affected.

The effects of nitrogen rates and water availability demonstrated significant differences in terms of the leaf fresh weight of the cultivated stamnagathi plants (Figure 1a). When the plants were cultivated under sufficient nitrogen conditions, drought stress (NR100-WA50) significantly reduced leaf fresh weight. When plants were cultivated under limited nitrogen conditions, the fresh weight was reduced but was not further diminished by drought stress (NR30-WA100 and NR30-WA50).

On the other hand, the biostimulant application appeared to have had a significantly positive effect on the leaf fresh weight of plants cultivated under limited nitrogen conditions (NR30-BS), even though that effect was not strong enough for the leaf fresh weight to reach values as high as those of plants cultivated with sufficient nitrogen (NR100-BS and NR100-NoBS), as illustrated in Figure 1b.

The interactions between nitrogen rate and water availability (NR × WA) revealed significant variations for leaf number and leaf area (

Table 4. The effects of nitrogen rate and water availability (NR × WA), nitrogen rate and biostimulants application (NR × B), and water availability and biostimulant application (WA × B) on leaf number (LN), leaf area (LA), and leaf dry weight (LDW) of hydroponically cultivated stamnagathi plants.

Factor Interaction	Sources of Variation		LN (No. Plant−1)	LA (cm2 Plant−1)	LDW (g Plant−1)
NR × WA	NR100	WA100	18.06 ± 0.48 a	267.99 ± 6.27 a	1.46 ± 0.04 a
		WA50	15.50 ± 0.31 b	237.77 ± 4.68 b	1.34 ± 0.02 a
	NR30	WA100	14.65 ± 0.4 bc	191.23 ± 7.74 c	1.21 ± 0.03 a
		WA50	14.36 ± 0.4 c	188.45 ± 8.55 c	1.18 ± 0.04 a
NR × B	NR100	NoBS	16.71 ± 0.58 a	252.55 ± 6.14 a	1.38 ± 0.04 a
		BS	16.85 ± 0.53 a	253.21 ± 8.07 a	1.41 ± 0.04 a
	NR30	NoBS	13.44 ± 0.21 c	173.13 ± 7.75 c	1.14 ± 0.03 a
		BS	15.57 ± 0.26 b	206.55 ± 4.74 b	1.25 ± 0.02 a
WA × B	WA100	NoBS	15.82 ± 0.77 a	221.53 ± 14.58 a	1.32 ± 0.05 a
		BS	16.89 ± 0.52 a	237.69 ± 11.95 a	1.35 ± 0.05 a
	WA50	NoBS	14.33 ± 0.41 a	204.15 ± 12.58 a	1.20 ± 0.04 a
		BS	15.53 ± 0.28 a	222.07 ± 5.72 a	1.31 ± 0.03 a

The values presented in the table are mean values (n = 12) across treatments. In each column, mean within the same factor interaction followed by different letters indicate significant differences according to Duncan’s multiple range test at p ≤ 0.05.

). Plants under the combination treatment of high nitrogen and full irrigation (NR100-WA100) exhibited the highest leaf number and leaf area compared to their water-starved counterparts. Plants that underwent the low nitrogen treatment and were fully irrigated (NR30-WA100) scored values that were as low as the ones of reduced irrigation (NR30-WA50) for leaf dry weight and leaf area. The leaf number of the NR30-WA100 did not statistically differ from that of NR30-WA50 and NR100-WA50. The effects of nitrogen rate and biostimulant application (NR × B) demonstrated some significant differences (Table 2). The highest leaf area and leaf number values were observed in the treatment where the supplied nitrogen was at a sufficient level (NR100), regardless of the biostimulant application. On the other hand, biostimulant application resulted in a significant increase in the leaf number and leaf area of plants that were cultivated under limited nitrogen conditions (NR30-BS) compared to the untreated plants (NR30-NoBS). Nevertheless, the biostimulant application partially mitigated the nitrogen stress since the observed values were lower compared to the plants cultivated under the NR100 treatments. The leaf dry weight, on the other hand, appeared unaffected in these interactions. Additionally, the interaction between drought stress and biostimulant application (WA × B) did not have a significant impact on growth parameters (Table S1).

Dry matter content was affected significantly in all three interactions of the factors. As illustrated in Figure 2a, the effects of nitrogen rates and water availability demonstrated that plants under the combination of high nitrogen and full irrigation treatments (NR100-WA100) exhibited the lowest dry matter content, whereas the combination of nitrogen stress and sufficient irrigation (NR30-WA100) resulted in the highest dry matter content.

The interaction between nitrogen rate and biostimulant application (NR × B) significantly altered the dry matter content (Figure 2b). Under sufficient nitrogen, dry matter content values remained low for both treated (NR100-BS) and untreated plants (NR100-NoBS) and did not significantly differ from each other. On the other hand, under low nitrogen rates, non-sprayed plants (NR30-NoBS) demonstrated the highest values, whereas biostimulant application (NR30-NoBS) reduced the dry matter content to an intermediate level.

The effects of water availability and biostimulant application (WA × B, Figure 2c) revealed that under sufficient irrigation conditions, the biostimulant (WA100-BS) reduced the dry matter content to levels statistically similar to those of plants cultivated under drought conditions regardless of the biostimulant application (WA30-BS, WA30-NoBS), while the highest dry matter content was observed on plants that were sufficiently irrigated and where no biostimulant application took place (WA100-BS).

The effect of all three factors, namely nitrogen rate, water availability, and biostimulant application (NR × WA × B), affected significantly the dry matter content (Figure 3). The highest dry matter content was recorded in the NR30-WA100-NoBS treatment. NR30-WA100-BS was significantly lower compared to the forementioned treatment but higher compared to the statistically similar NR100-WA100-NoBS and NR100-WA100-BS. The dry matter content of the rest of the treatments lingered between NR30-WA100-BS and NR100-WA100-NoBS/BS. On the other hand, the differences in leaf number, leaf area, leaf fresh weight, and leaf dry weight could not be attributed to the interaction of the factors, according to Duncan’s multiple range test (

Table 5. The effects of nitrogen rate, water availability, and biostimulant application (NR × WA × B) on leaf number (LN), leaf area (LA), leaf fresh weight (LFW), and leaf dry weight (LDW) of hydroponically cultivated stamnagathi plants.

NR	WA	B	LN (No. Plant−1)	LA (cm2 Plant−1)	LFW (g Plant−1)	LDW (g Plant−1)
NR100	WA100	NoBS	18.08 ± 0.71	264.08 ± 8.22	19.22 ± 0.74	1.46 ± 0.05
		BS	18.03 ± 0.72	271.91 ± 9.98	19.10 ± 0.73	1.46 ± 0.07
	WA50	NoBS	15.33 ± 0.49	241.02 ± 6.71	16.31 ± 0.43	1.31 ± 0.04
		BS	15.67 ± 0.42	234.51 ± 6.87	16.85 ± 0.34	1.36 ± 0.02
NR30	WA100	NoBS	13.56 ± 0.28	178.99 ± 12.00	12.70 ± 0.61	1.18 ± 0.05
		BS	15.75 ± 0.36	203.46 ± 7.74	14.84 ± 0.20	1.24 ± 0.02
	WA50	NoBS	13.33 ± 0.34	167.27 ± 10.33	13.61 ± 0.42	1.10 ± 0.04
		BS	15.39 ± 0.41	209.63 ± 5.93	15.49 ± 0.44	1.25 ± 0.04

The values presented in the table are mean values (n = 6) across treatments. No significant differences were found according to Duncan’s multiple range test at p ≤ 0.05.

).

3.2. Effects on Leaf Chemical Characteristics

The nitrogen rates (NRs) of the supplied nutrient solution significantly affected leaf nitrate, calcium, and iron content (Figure 4a–c) as well as manganese, zinc, and copper content (Figure 4d,e). Plants that were cultivated under limited nitrogen conditions (NR30) had significantly reduced nitrate (Figure 4a) and calcium (Figure 4b) content compared to the control (NR100). On the contrary, iron content (Figure 4c) increased in nitrogen-deprived plants. In addition, the manganese and copper content of nitrogen-stressed plants demonstrated a statistically significant increase compared to plants cultivated under sufficient nitrogen conditions. On the contrary, leaf zinc content was lower in nitrogen-stressed plants.

Drought level also affected the leaf nitrate content, magnesium, and zinc content (Figure 5a–c). Plants that were cultivated under water stress (WA30) conditions exhibited reduced nitrate and zinc levels, but magnesium levels in the leaves were increased compared to sufficiently irrigated plants (WA100).

The effects of nitrogen rates and water availability (NR × WA) had significant effects on the leaf calcium content (Figure 6, Table S2). Under sufficient nitrogen (NR100), drought stress (WA50) lead to an increase in the calcium content of the leaves, whereas under limited nitrogen (NR30) drought did not significantly affect the calcium content. Hence, the highest calcium content was observed in plants that were sufficiently fertilized but were inadequately irrigated (NR100 and WA50) compared to the other factor combinations.

The differences in the leaf nutrient content due to the effects of all three factors nitrogen rate, water availability, and biostimulant application (NR × WA × B) could not be attributed to the interactions of the factors according to Duncan’s test (

Table 6. The effect of nitrogen rate, water availability, and biostimulant application (NR × WA × B) on leaf nitrate, potassium, magnesium, and calcium contents of hydroponically cultivated stamnagathi.

NR	WA	B	NO3 (mg Kg−1)	K (mg g−1)	Mg (mg g−1)	Ca (mg g−1)
NR100	WA100	NoBS	2186.71 ± 181.75	70.00 ± 1.15	4.20 ± 0.10	10.00 ± 0.24
		BS	2301.36 ± 247.95	58.00 ± 4.16	3.96 ± 0.38	8.95 ± 0.35
	WA50	NoBS	1990.53 ± 158.94	69.33 ± 4.67	5.04 ± 0.26	11.06 ± 0.18
		BS	2084.01 ± 240.42	67.33 ± 3.53	4.83 ± 0.61	10.88 ± 0.39
NR30	WA100	NoBS	1464.52 ± 33.14	66.67 ± 1.33	4.41 ± 0.48	9.39 ± 0.03
		BS	1305.19 ± 43.17	61.33 ± 0.67	4.56 ± 0.23	10.37 ± 0.23
	WA50	NoBS	1132.46 ± 79.41	63.33 ± 5.21	5.01 ± 0.22	9.38 ± 0.50
		BS	1092.81 ± 108.86	64.00 ± 1.15	5.48 ± 0.73	9.30 ± 0.64

The values presented in the table are mean values (n = 6) across treatments. No significant differences were found according to Duncan’s multiple range test at p ≤ 0.05.

,

Table 7. The effect of nitrogen rate, water availability, and biostimulant application (NR × WA × B) on leaf iron, sodium, manganese, zinc, and copper content of hydroponically cultivated stamnagathi.

NR	WA	B	Fe (μg g−1)	Na (mg g−1)	Mn (μg g−1)	Zn (μg g−1)	Cu (μg g−1)
NR100	WA100	NoBS	31.89 ± 8.18	2.64 ± 0.22	26.83 ± 6.42	23.61 ± 2.64	23.95 ± 0.31
		BS	31.97 ± 3.36	2.56 ± 0.36	26.73 ± 1.47	27.74 ± 3.14	23.77 ± 0.25
	WA50	NoBS	40.61 ± 10.69	2.93 ± 0.37	28.46 ± 4.27	20.05 ± 0.54	23.05 ± 0.08
		BS	24.65 ± 10.65	3.12 ± 0.60	24.16 ± 4.70	17.86 ± 2.49	23.73 ± 0.27
NR30	WA100	NoBS	72.58 ± 4.02	2.48 ± 0.52	39.31 ± 2.31	21.64 ± 2.13	25.16 ± 0.48
		BS	84.66 ± 0.36	2.08 ± 0.08	44.95 ± 2.11	21.25 ± 2.04	25.46 ± 0.44
	WA50	NoBS	76.77 ± 3.70	2.40 ± 0.24	37.77 ± 0.53	18.14 ± 0.34	25.11 ± 0.05
		BS	76.52 ± 7.94	2.45 ± 0.14	34.83 ± 1.92	13.67 ± 1.48	24.98 ± 0.65

The values presented in the table are mean values (n = 6) across treatments. No significant differences were found according to Duncan’s multiple range test at p ≤ 0.05.

and Table S3).

4. Discussion

4.1. Dry Matter Content as a Stress Indicator

In order to maintain turgor in low water conditions, plants invest in thicker and stiffer cell walls which is often expressed as increased dry matter content. This has higher carbon costs per unit leaf area and results in slower growth. Thicker leaves increase the plant’s resilience to environmental factors and decrease the possibility of the plant being consumed by a herbivore in the wild [84]. Thus, dry matter content can be used as an indicator of the degree of stress. Stamnagathi plants cultivated under nitrogen-deficient, fully irrigated and devoid of biostimulants (NR30-WA100-NoBS), had the highest dry matter content (Figure 3), which suggests that this treatment group underwent the most stress, regardless of being sufficiently irrigated. Additionally, drought stress applied in perlite grow bags led to the buildup of nutrients and salts, including the buildup of nitrogen, which could also have affected cultivation in a positive way, given that stamnagathi is known to be salt- and drought-tolerant. Based on current research, the growth of stamnagathi is primarily affected by nitrogen rates. Even though in our experiment the most profound effect on leaf dry matter continent was attributed mainly to the supplied nitrogen, the effect of the nitrogen-rich biostimulant was shown to also have an effect. Foliar application on plants cultivated under nitrogen-deficit and fully irrigated conditions (NR30-WA100-BS) had a significant effect on reducing the dry matter content by 9.90% compared to non-sprayed plants (NR30-WA100-NoBS). Nevertheless, the lowest dry matter content was recorded in plants that were cultivated under sufficient nitrogen and water conditions, regardless of biostimulant application (NR100-WA100-NoBS and NR100-WA100-BS).

Notably, it was observed that foliar application with a protein hydrolysate biostimulant strongly decreased leaf dry matter content independently of the supplied nitrogen rates, suggesting a partial alleviation of the stress [85]. The increases in dry matter content also observed in other experiments were related mainly to nitrogen regimes rather than biostimulant application. For example, El-Nakhel et al. [77] reported that dry matter content was in fact increased under nitrogen limitation conditions but was not significantly affected by the biostimulant application. In another experiment, Schiattone et al. [86] reported that dry matter content was increased by water deficit and by nitrogen limitation but also reported that the seaweed derived biostimulant used in their experiment did not have a significant effect on the dry matter content, whereas the azoxystrobin biostimulant caused a reduction. According to Consentino et al. [76], the low water availability was believed to cause a low plant nitrogen uptake, similarly to our experiment, which consequently affected the dry matter content by reducing it, favoring the effect of nitrogen availability over water availability. In their report, even though the biostimulant was able to enhance some of the agronomical characteristics, it did not appear to significantly affect the dry matter content.

4.2. Nitrogen Rate, Water Availability, and Biostimulant Application: The Effects on Agronomical and Chemical Characteristics of C. spinosum L.

Looking at the main effects, it was clearly demonstrated that reducing the available nitrogen of the fertigation solution led to a reduction in all agronomical values, except for the dry matter content (Table 1). Compared to the control nitrogen rate treatment (NR100), limiting nitrogen rate to 30% (NR30) had a significantly negative on important agronomical factors. Leaf number, leaf area, leaf fresh weight, and leaf dry weight were reduced by 13.53%, 24.93%, 20.76%, and 15.00%, respectively. Nevertheless, there was a 7.64% increase in the dry matter content. The importance of nitrogen for photosynthesis, and therefore growth, has been well documented for staple crops and mainstream vegetables [87,88]. There is a strong asymptomatic relationship between biomass and nitrogen supply which is expressed through a linear relationship until a certain plateau is reached where the crops grow at the genetically determined potential rate and further nitrogen supplementation would only result in wastage or even have negative consequences on the cultivation, through chemical imbalances or salinity increases [30,42,89]. Even though the yield reduction in stamnagathi was observed due to nitrogen limitation, the overall product was up to commercial standards in terms of size and yield. To a less severe extent, reducing irrigation to 50% (WA50) led to a reduction in the number of leaves, leaf area, fresh leaf weight, and dry leaf weight by 8.62%, 7.19%, 5.53%, and 5.26%, respectively. Dry matter content was not affected by reduced irrigation. Additionally, drought has also been known to hinder growth rates, shifting plant metabolism towards certain defense mechanisms that consume valuable resources [90,91]. Finally, biostimulant application demonstrated a significant role in stress alleviation by increasing all agronomical values and reducing the dry matter content. In more detail, the use of the nitrogen-rich biostimulants did have a positive effect on agronomical traits, slightly boosting leaf number, leaf area, fresh leaf weight, and dry leaf weight by 7.49%, 8.01%, 7.18%, and 5.56%, respectively, with no notable impact on dry matter content. In the context that the main limiting factor was the nitrogen supply and that the biostimulant used in this experiment was a nitrogen rich protein hydrolysate, the stress alleviation through foliar application is a rather promising result. Other researchers have also observed the beneficial effects of nitrogen-rich biostimulants when applied to nitrogen stress crops [92].

Even though stamnagathi is a wild edible plant, and a relative resilient species to various types of stresses [27,93,94], its yield was found to be substantially affected by nitrogen, and water limitations in this experiment that took place in a semitransparent greenhouse cultivation in a soilless culture system consisted of perlite bags. Furthermore, the main findings demonstrated that nitrogen limitations (Figure 4a–f) also led to a reduction in nitrate, calcium, and zinc in the leaf tissues by 41.66%, 5.97%, and 16.27%, whereas iron, manganese, and copper levels were significantly increased by 140.49%, 47.69%, and 6.56%. On the other hand, water limitation (Figure 5a–c) led to a decrease in nitrates and zinc by 13.20%, and 26.02% but an increase in magnesium by 18.93%. Under drought conditions, magnesium, which is vital for energy conservation and protein synthesis, uptake and availability decreases, hindered normal growth [95]. The increase in magnesium in this case could possibly play a role in the explanation of the resilience of stamnagathi towards drought stress. In both cases, nitrate levels were measured as a quality indicator in regard to the EU regulations [96,97] and not as an indicator of the total nitrogen in the leaf tissues. Differences between nitrate levels among the treatments are related to the metabolism rate and the results are clearly justified by the amount of nitrate supplied through the irrigation integrals and the time period needed to sufficiently conduct nitrate assimilation [98,99,100].

4.3. Nitrogen Limitations Overshadow the Effects of Water Stress in the Cultivation of C. spinosum L.

The results derived from the two-way ANOVA demonstrated that the interactions between nitrogen and water availability as well as biostimulant application and nitrogen limitations were important. Stamnagathi is usually grown in areas where water scarcity is the main limiting factor [101]. Hence, its resilience towards water scarcity was expected. Moreover, its small size compared to the size of the perlite bags, and thus their water storage, could also suggest that to apply severe drought stress conditions for stamnagathi the irrigation amount and frequency or the size of the perlite bags could be further reduced. On the one hand, it would be important to conduct measurements of the substrate moisture before initiating irrigation rather than implementing a pre-defined irrigation regime, as it has been suggested by Nemali et al. [102]. Nevertheless, it is the premise of the open-loop cultivation systems that adequate drainage of the nutrient solution is required in order to keep salt accumulation under control. In other words, irrigating perlite-based cultivation is not only based on substrate moisture but also EC levels, since salts tend to accumulate, and plants might experience drought stress, i.e., osmotic stress, symptoms due to the increased salinity if the substrate is not sufficiently irrigated. This has resulted in a leaching fraction that should range between 20 and 40% of the supplied volume [103,104]. In this experiment, the leaching content of the drought stress was 0%.

It also appears that when cultivated commercially in well fertilized lands and soilless culture systems, stamnagathi and other wild edible greens benefit from the available nitrogen, as it enhances their yields [105]. Based on our results, we consider nitrogen availability to outweigh the water stress. Under nitrogen limitation conditions, the limitation of water availability did not demonstrate any significant reduction in fresh weight (Figure 1a) and agronomical characteristics (Table 4), with the exception of dry matter content being affected (Figure 2a). The fresh weights of NR100-WA50, NR30-WA100, and NR30-WA50 were decreased by 13.47%, 28.13%, and 24.06%, respectively, compared to the control treatment (NR100-WA100). Leaf number was reduced by 14.17%, 18.88%, and 20.49% for NR100-WA50, NR30-WA100, and NR30-WA50 compared to the control. Leaf area was also reduced by 11.28%, 28.13%, and 24.06% for NR100-WA50, NR30-WA100, and NR30-WA50, respectively, compared to the control. Moreover, the leaf dry weight of NR100-WA50, NR30-WA100, and NR30-WA50 was reduced by 8.22%, 17.12%, and 19.18%, respectively. On the other hand, the dry matter content of the aforementioned treatments was increased by 5.64%, 15.73%, and 5.90% compared to the control. Interestingly, the dry matter content of nitrogen- and irrigation-stressed plants (NR30-WA50) was reduced by 8.49% compared to plants that were sufficiently irrigated (NR30-WA100). In our experiment, stamnagathi dry matter content in this occasion can be misleading, since the rest of the agronomical characteristics of NR30-WA50 and NR30-WA100 did not demonstrate significant differences between the two treatments but were clearly reduced compared to those of plants cultivated under sufficient nitrogen conditions. To that end, water limitations only played an important role in reducing the values of agronomical characteristics when nitrogen was sufficiently available. In addition, this could a result of the difference in the nitrogen amount between NR100-WA100 and NR100-WA50, which was around 0.6 mmol plant⁻¹ per irrigation, whereas the nitrogen difference between NR30-WA100 and NR30-WA50 was only 0.2 mmol plant⁻¹ per irrigation.

For domesticated cultivars that have lost their resilience to various stresses, it is often observed that higher nitrogen rates could lead to better growth attributes, such as the number of leaves, leaf area, fresh biomass, and dry biomass [106]. Moreover, water deficit is known to hinder photosynthesis, nutrient absorption, and plant growth processes [107]. Nevertheless, Chen Ru et al. [108] observed that plants cultivated under insufficient nitrogen supply are likely to cope with one or several abiotic stresses better than plants that are abundantly supplied with nitrogen, though the exact reasons and mechanism behind this notion are not clearly documented. Li and Yaosheng Wang [109], in a proteomics focus study on combined stresses in barley, observed that compared to either drought or nitrogen stress alone, their combined effects led to the development of extensive signaling pathways related either to energy, carbohydrate, or amino acid metabolisms. Jiaxin Hu et al. [110] studied the short-term and long-term water and nitrogen use efficiencies of temperate grasslands under nitrogen and drought stress conditions. Their results demonstrated that in the long-term, drought presented a trade-off between nitrogen and water use efficiency, whereas nitrogen supply enabled a positive correlation between the two. Perhaps this positive correlation also played a part in our experiment when it came to the comparison of sufficiently fertilized plants.

4.4. Promising Results and Limitations of Nitrogen Stress Alleviation by Foliar Nitrogen-Rich Biostimulant Application

As far as biostimulant application is concerned, it was found to partially alleviate nitrogen stress (Table 4) when the biostimulant was applied on nitrogen-deprived plants (NR30-BS) by increasing leaf number by 15.85%, leaf area by 19.30%, and leaf fresh weight by 15.36% compared to non-sprayed plants (NR30-NoBS). The effect of the biostimulant was insignificant in terms of providing any level of resilience to water-stressed plants (Table 4). Hence, a nitrogen-rich biostimulant such as the one used in this experiment could be an eco-friendly solution to counter the negative effects of nitrogen limitations, even though it was not capable of completely alleviating the negative effects. Our results are in agreement with those of El-Nakhel et al. [77], whose research stated that biostimulants should be used as complementary additions to fertilizers and not as replacements. Nevertheless, research on dosage, frequency of application, and method of application should be further investigated.

4.5. Combined Nitrogen and Water Stress Affects Leaf Calcium Content of C. spinosum

As can be seen from chemical analysis, only calcium demonstrated a significant effect that can be attributed to the interactions between nitrogen and water availability (Figure 6). To that end, calcium was found to be higher by 15.72% in plants that were sufficiently fertilized but were irrigation starved (NR100-WA50) compared to the control. This could perhaps be attributed to the use of calcium in osmorythmisis since it is essential for plant recovery from dehydration through the activation plasma membrane ATPase, which pumps nutrients back after stress-induced membrane damage [111].

4.6. Optimizing the Fertigation Deficit Cultivation of C. spinosum

Optimizing crop production while implementing fertigation deficit can be a complicated task. Our experiment highlights nitrogen’s dominant influence on stamnagathi’s growth and resilience, overshadowing water stress effects. Future experiments could further fine-tune the cultivation of this wild edible vegetable by integrating environmental factors such as temperature, humidity, light intensity, and water availability of the substrate to optimize water and nutrient use reduction. Moreover, the applied biostimulant partially reduced nitrogen stress. Further investigation into dosage and application frequency could further define the usability of the studied product. Nevertheless, a plethora of biostimulants has surged onto the market and further investigation into different biostimulants could benefit cultivation practices for small-scale growers and local producers. Nevertheless, precise nitrogen management remains crucial for optimal cultivation outcomes. These findings provide a foundation for improving stamnagathi farming practices in soilless systems.

5. Conclusions

This study evaluated the individual and combined effects of nitrogen levels, drought stress, and biostimulant application on stamnagathi. The findings highlighted that even for a wild edible plant like stamnagathi, nitrogen availability is a key determinant for a high yield cultivation. While water stress impacted the plants, nitrogen limitations had a more profound effect, leading to reduced growth and altered nutrient composition. The application of a nitrogen-rich biostimulant partially mitigated the negative effects of nitrogen deficiency but was less effective under water stress conditions. These findings suggest that for optimal stamnagathi cultivation, especially in soilless systems, the careful management of nitrogen levels is essential. Future studies should explore more nuanced approaches to irrigation and biostimulant use to enhance plant resilience and productivity.