Photosynthetic Parameters of Melons in Response to NO₃⁻ and NH₄⁺ as N Sources and Irrigation with Brackish Water High in Na⁺, Ca²⁺, and Cl⁻

Abstract

High levels of dissolved salts in irrigation water sources limit melon cultivation in northeastern Brazil. In this context, nitrogen fertilization has been employed as one strategy to alleviate the effects of salt stress on plants. This study aimed to evaluate the effect of different nitrogen sources on cantaloupe melon cultivation under fertigation and irrigation with water of the same salinity and different cationic concentrations (Na⁺ and Ca⁺). The research consisted of two experiments, each following a randomized complete block design in a 4 × 2 factorial arrangement with four replicates. The treatments included four levels of electrical conductivity of the nutrient solution (2.0; 3.0; 4.0; and 5.0 dS m⁻¹) and two nitrogen sources of different origins: NO₃⁻ [Ca(NO₃) and KNO₃] and NH₄⁺ [CH₄N₂O and NH₄H₂PO₄]. The following factors were chlorophyll pigments, chlorophyll a fluorescence, and fruit weight. Nitrogen fertilization with NH₄⁺ mitigated salt stress by increasing the synthesis of chlorophyll a and carotenoids in plants irrigated with NaCl-based saline water. Furthermore, there was no influence of nitrogen sources on chlorophyll a fluorescence. Finally, NO₃⁻ fertilization reduced the effects of salt stress on the leaf mass ratio, specific leaf area under Ca²⁺ fertigation, and relative growth rate of leaf area in melons under cationic prevalences of Na⁺ or Ca²⁺ (associated with Cl⁻).

1. Introduction

The melon (Cucumis melo L.) is a cucurbit with morphological characteristics of herbaceous plants, and its main product is the fruit, which is primarily consumed fresh [1]. In 2022, the global production reached 28.55 million tons of melon fruits [2] and Brazilian melon production, in the same year, amounted to 699.2 thousand tons across 27.4 thousand hectares. In Brazil, 97.5% of melon production comes from the Northeast Region, specifically the states of Rio Grande do Norte and Ceará contributing 77.5% of the national total. The state of Pernambuco produced 41.4 thousand tons, representing 5.92% of the national production [3].

Among the factors of melon production in this region of Brazil, it is worth highlighting nitrogen fertilization and irrigation. There are reports of the use of CH₄N₂O, Ca(NO₃) or (NH₄)₂SO₄ as N sources [4], with doses associated with the production function, since nitrogen is decisive in the cost of crop production. On the other hand, under saline conditions, lower Cl⁻ concentrations were found in plants exposed to high levels of NO₃⁻ and vice versa [4]. There are also reports for the cv. Gália (aeroponic system) that ammonium nutrition exerted a negative effect on plant growth and tended to increase the level of total N in plant tissue and resulted in higher Na and Cl and lower Ca, K, and NO₃ levels in plant tissue [4]. In fact, plants activate different pathways for assimilation depending on N source, either separately or in combination; plants that prefer NO₃⁻ may show symptoms of toxicity to NH₄⁺, while those that prefer NH₄⁺ may develop a less efficient NO₃⁻ uptake system [5].

This information is very important in the productive context of northeast Brazil, since brackish waters, predominantly in Na⁺, Ca²⁺, and Cl⁻, are used in the irrigation and fertigation of crops, including melon plants [6]. Melons plants are considered moderately salt-tolerant [7]; with reports of salinity limits of 3.3 dS m⁻¹ [8] and 2.5 dS m⁻¹ [9] for different growing situations, climatic conditions, and cultivars, the common point is that the reduction in the number of fruits per melon plant is the main cause of yield loss in the melon cultivars sensitive to salinity [10].

In terms of photosynthetic parameters, chlorophyll bio-synthesis and nitrogen metabolism in melon plants are found to be affected due to high salinity, i.e., leaf photosynthetic pigment and the fluorescence of chlorophyll a are important indicators of crop tolerance to salinity [11,12], as it is a consequence of the osmotic and water conditions’ effects on the photosynthetic efficiency of plants [13].

Moreover, the analysis of chlorophyll a fluorescence reveals the level of excitation energy that stimulates photosynthesis and helps distinguish the inhibition or loss in the electron transfer process of Photosystem II [14]. In this regard, it is also observed that the accumulation of salts in irrigation water hampers water absorption, which is essential for photosynthetic pigments, and thus, this excess of toxic ions in plant tissues causes an imbalance in metabolic and biochemical functions [15,16].

In light of this, several studies have been and continue to be conducted to evaluate the interaction between salinity and nitrogen [15,17,18], since nitrogen can be used to reduce deleterious effects of salinity, with its impact varying according to the form of nitrogen applied (nitrate or ammoniacal) and the plant species studied [19]. The present study is based on the hypothesis that nitrate-based fertilization mitigates the deleterious effects caused by fertigation with saline water inducing plant tolerance to salt stress by increasing the biosynthesis of photosynthetic pigments and photochemical efficiency, which reflects in higher fruit weight.

Therefore, we propose to compare (1) the effect of NO₃⁻ or NH₄⁺ fertilization as a mitigator of the negative effects of salinity using photosynthetic parameters and melon fruit weight as indicators and (2) fertigation ionic compositions on the photosynthetic parameters and fruit weight of melon plants irrigated with brackish water prevalent in Na⁺ or Ca²⁺ (associated with Cl⁻).

2. Materials and Methods

2.1. Location and Characterization of the Experimental Environment

Two experiments were conducted in a protected environment at the Fertigation and Salinity Experimental Station of the Department of Agricultural Engineering at the Federal University of Pernambuco (UFRPE/headquarters), located in the municipality of Recife, Pernambuco (8°01′05″ S latitude and 34°56′48″ W longitude, with an average altitude of 6.5 m).

The greenhouse used measures 7 × 21 m, with a ridge height of 3 m and a maximum arch height of 4.5 m, featuring nylon side screens and a 150-micron film roof. Temperature and relative humidity data, as well as global solar radiation, were collected daily using a digital thermohygrometer (Incotern) and a portable pyranometer (IF1307 Instrufiber), respectively, inside the greenhouse (Figure S1).

2.2. Characterization of Experimental Units

Thirty-two pots were used in Experiment I and another thirty-two pots in Experiment II. Each pot was placed on a masonry base measuring 0.20 × 0.20 × 0.20 m (height, length, and width), equidistant in all directions by 0.50 m. Additionally, 7.77 L pots were fitted with a drainage system consisting of fourteen holes, each 7 mm in diameter, at the lower end of the pot. A 45 cm diameter piece of Clarite mesh was placed at the bottom of the pot as a physical barrier to prevent soil loss. Finally, the pot was filled in layers, maintaining a density of 1.5 g cm⁻³, with a total of 9 kg of dry soil mass, corresponding to 10.170 kg of wet soil mass. (Figure 1).

Figure 1. Schematic drawing of the experimental units used in the research.

The soil used came from the municipality of Goiana, Pernambuco, with geographic coordinates of 7°33′38″ S and 35°00′09″ W, at an altitude of 13 m, and was collected from the 0 to 0.20 m layer. It was classified as Spodosol according to the American classification [20] and analyzed for its physical and chemical characteristics. Since the soil pH was within the range suitable for the crop and the soil did not contain exchangeable aluminum, liming was not necessary.

In the chemical characterization of the soil, the pH was 6.5. The phosphorus (P) concentration was 5.03 mg dm⁻³, while the levels of calcium (Ca²⁺), magnesium (Mg²⁺), sodium (Na⁺), and potassium (K⁺) were 1.75, 0.55, 0.08, and 0.07 cmolc dm⁻³, respectively. Aluminum (Al³⁺) showed a value of 0.00 cmolc dm⁻³, and the potential acidity (H⁺ + Al³⁺) was 0.69 cmolc dm⁻³. The sum of bases (SB) was 2.45 cmolc dm⁻³, the cation exchange capacity (CEC) was 3.14 cmolc dm⁻³, and the effective cation exchange capacity (ECe) was also 2.45 cmolc dm⁻³. The levels of copper (Cu), iron (Fe), manganese (Mn), and zinc (Zn) were 0.1, 4.0, 4.1, and 1.3 mg dm⁻³, respectively. Base saturation (V) was 78.03%, and the organic matter (OM) content was 5.53 g kg⁻¹.

In the physical characterization, the soil had 952 g kg⁻¹ of sand, 22 g kg⁻¹ of silt, and 26 g kg⁻¹ of clay, being texturally classified as sandy. The bulk density (Bd) was 1.50 g cm⁻³, the particle density (Pd) was 2.65 g cm⁻³, and the total porosity was 43.60%.

As for the chemical characterization of the irrigation water, the following values were observed: pH of 6.30 and electrical conductivity (EC) of 0.12 dS m⁻¹. The concentrations of potassium (K⁺), calcium (Ca²⁺), magnesium (Mg²⁺), sodium (Na⁺), ammonia, nitrate, alkalinity, and chlorides were 2.50, 0.90, 0.46, 5.40, 0.00, 0.02, 11.70, and 15.40 mg L⁻¹, respectively.

2.3. Experimental Design and Treatments: Experiments I and II

In both experiments, the experimental design was the same; however, in Experiment I, the levels of electrical conductivity of water (ECa) were obtained by dissolving NaCl in supply water (0.12 dS m⁻¹), while in Experiment II, the same levels were obtained by dissolving CaCl₂·2H₂O in the same supply water.

Thus, the experimental design used was a randomized block design in a 4 × 2 factorial scheme, with four blocks and four repetitions, totaling thirty-two experimental units for Experiment I and another thirty-two units for Experiment II.

The treatments consisted of four salinity levels of nutrient solution (2.0, 3.0, 4.0, and 5.0 dS m⁻¹) used in fertigation, with the fertilizer sources and nutrient quantities adopted based on the methodology proposed by Morais et al. [21]. To meet the nitrogen demand, two sources of different origins were used: for the nitrate source (NO₃⁻), calcium nitrate and potassium nitrate were used; and for the ammoniacal source (NH₄⁺), urea and MAP (monoammonium phosphate—composed of nitrogen and phosphorus) were used.

2.4. Irrigation Management

2.4.1. Determination of the Maximum Water Retention Capacity in the Soil, the Vase Capacity

The maximum water retention capacity of the soil (vase capacity) was determined according to the methodology of [17]. With due care to avoid the effect of precipitation, radiation, and evapotranspiration, in triplicate, three structures identical to the experimental units were exposed to saturation by capillarity until the ‘film’ was formed. They were then exposed to free drainage until they reached constant weight. Three 80 g samples of homogeneous masses of moist soil were then taken from the pots. The average water content (Equation (1) found in the three samples was taken as the maximum water retention capacity of the soil, 13%.

$$\mathrm{M}\mathrm{V}\mathrm{C}=\mathrm{ }{\displaystyle \frac{\mathrm{M}\mathrm{W}\mathrm{S}-\mathrm{M}\mathrm{D}\mathrm{S}}{\mathrm{M}\mathrm{D}\mathrm{S}}}\mathrm{ }\times \mathrm{ }100$$

(1)

in which

MVC = Moisture at vase capacity (g g⁻¹);

MDS = Mass of the dry sample (g);

MWS = Mass of the wet sample (g).

2.4.2. Determination of Current Soil Moisture and Irrigation Blade

(a)

Description of the moisture sensor and its calibration for Spodosol

For the daily monitoring actions of current soil moisture, Frequency Domain Reflectometry (FDR) was used, utilizing an EnviroSCAN communication port, produced by the Australian company Sentek, equipped with an RS 232 interface that records the electromagnetic signal. The soil moisture sensors are of the capacitive type ECH₂O, models EC-5, with dimensions of 8.9 cm in length, 1.8 cm in width, and 0.7 cm in thickness, and 5TE (METER GROUP, Inc., Pullman, WA, USA), with dimensions of 10 cm in length, 3.2 cm in width, and 0.7 cm in thickness. The calibration process, called ‘laboratory calibration’ of θ, was developed for the EC-5 sensor by plotting the probe readings versus θ derived from the gravimetric method for the type of soil used in the experiment. The equation was developed using Microsoft Excel^® regression, as shown in Equation (2) [22].

$$\mathsf{\theta }=0.953\mathrm{X}\mathrm{ }-\mathrm{ }0.0451$$

(2)

in which

ϴ = Soil moisture (m³ m⁻³);

Coefficients = 0.953 and −0.0451;

Sensor reading = X.

With the collection of relative frequency values (scaled frequency—SF) and the application of the calibration curve for the soil in question (Spodosol) developed by Almeida et al. (2018) [22], the current soil moisture was determined.

(b)

Determination of irrigation blade and irrigation interval

The irrigation interval adopted was, daily, with the irrigation blade, applied corresponding to the volume required to raise the soil moisture to the level equivalent to the pot capacity (13%). Once the irrigation blade was determined, its application was divided into three moments throughout the day, at 8:00 AM, 12:00 PM, and 3:00 PM. The irrigation blade was applied manually using a graduated beaker.

2.5. Fertigation Management

Preparation of the Nutrient Solution Used in Fertigation

In a water tank, 1000 L of supply water from UFRPE (ECw ≈ 0.12 dS m⁻¹) was placed. The quantity of fertilizers added was adapted from that proposed by Morais et al. [21], for both nitrogen sources: NO₃⁻ or NH₄⁺ (Table S1). Furthermore, it was ensured that the ECns value after adding the fertilizers remained the same, i.e., 2.0 dS m⁻¹, and after the preparation was completed, the nutrient solution was divided into smaller containers according to the treatment.

2.6. Salinization of Nutrient Solutions Used on Fertigation: Experiments I and II

After dissolving the nutrients and filling the smaller, properly identified reservoirs, the required amount of salts was added to achieve the desired electrical conductivity levels of the nutrient solution (ECsn). For the solubilization of NaCl and CaCl₂.2H₂O in Experiments I and II, respectively, the amounts of the respective salts (Table 1) were determined based on the equation proposed by [23].

Table 1. Ionic composition of nutrient solution used on fertigation.

Source of N: NO3−					Source of N: NH4+
Macronutrients (mEq L−1)
	Electrical conductivity of nutrient solutions—ECns (dS m−1)
Ions	2 dS m−1	3 dS m−1	4 dS m−1	5 dS m−1	2 dS m−1	3 dS m−1	4 dS m−1	5 dS m−1
NO3− (Exp 1 e 2)	2.75	2.75	2.75	2.75	0.00	0.00	0.00	0.00
NH4+ (Exp 1 e 2)	0.00	0.00	0.00	0.00	9.48	9.48	9.48	9.48
H2PO4− (Exp 1)	4.00	4.00	4.00	4.00	4.00	4.00	4.00	4.00
H2PO4− (Exp 2)	4.14	4.14	4.14	4.14	4.14	4.14	4.14	4.14
K+	11.87	11.87	11.87	11.87	11.87	11.87	11.87	11.87
Ca2+ (Exp 1)	1.62	1.62	1.62	1.62	1.62	1.62	1.62	1.62
Ca2+ (Exp 2)	1.62	3.04	3.54	5.47	1.62	3.04	3.54	5.47
Mg2+	1.11	1.11	1.11	1.11	1.11	1.11	1.11	1.11
SO42−	1.54	1.54	1.54	1.54	1.54	1.54	1.54	1.54
Micronutrients (ppm)
Ions	2 dS m−1	3 dS m−1	4 dS m−1	5 dS m−1	2 dS m−1	3 dS m−1	4 dS m−1	5 dS m−1
B	0.0225	0.0225	0.0225	0.0225	0.0225	0.0225	0.0225	0.0225
Cu	0.0225	0.0225	0.0225	0.0225	0.0225	0.0225	0.0225	0.0225
Fe	0.5625	0.5625	0.5625	0.5625	0.5625	0.5625	0.5625	0.5625
Mn	0.36	0.36	0.36	0.36	0.36	0.36	0.36	0.36
Mo	0.0036	0.0036	0.0036	0.0036	0.0036	0.0036	0.0036	0.0036
Zn	0.225	0.225	0.225	0.225	0.225	0.225	0.225	0.225
Salts (mEq L−1)
Ions	2 dS m−1	3 dS m−1	4 dS m−1	5 dS m−1	2 dS m−1	3 dS m−1	4 dS m−1	5 dS m−1
Na+ (Exp 1)	0.00	2.93	3.94	4.96	0.00	2.93	3.94	4.96
Na+ (Exp 2)	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
Cl− (Exp 1)	0.00	2.85	3.84	4.83	0.00	2.85	3.84	4.83
Cl− (Exp 2)	0.00	5.13	6.91	8.70	11.16	16.30	18.08	19.87

2.7. Description and Management of Culture

2.7.1. The Sowing, Transplanting, and Initiation of Treatments

The reference crop used was the F1 Caribbean Gold RZ hybrid melon of the cantaloupe Harper type. Sowing was performed manually in 55 cm³ tubes filled with Basaplant substrate, with one seed per tube.

After sowing, the seedlings were kept in the dark for the first few days after sowing—DAS. The seedlings were transplanted into pots (one seedling per pot) 15 DAS. During transplantation, the seedlings were positioned so that the collar region—the transition zone between the root system and the stem—was not buried and was level with the soil, avoiding excessive blade. At this stage, the seedlings had two definitive leaves and were selected based on uniform morphological characteristics (size, number of leaves, and vigor).

Initially, until 14 days after transplanting (DAT), irrigation for all treatments was conducted using a low electrical conductivity solution (ECns = 2.0 dS m⁻¹). From 15 DAT onward, the respective treatments were initiated, meaning the plants were irrigated with nutrient solutions containing salts as specified by the treatments. This occurred after the seedlings had acclimatized to the conditions of the greenhouse.

2.7.2. Plant Vertical Tutoring

After transplanting into the pots, the seedlings were tutored using strings to grow upright until reaching the height of the trellis (2.5 m) (Figure 2).

Figure 2. Arrangement of the plants after vertical tutoring.

2.7.3. Pruning Management

The plant was tutored to grow with a single stem, removing all shoots/stems up to the 11th node. At the 12th, 13th, and 14th nodes, hermaphroditic flowers (future fruits) were retained. For the remaining nodes, the procedure involved removing shoots and flowers to achieve optimal conditions, ensuring the fruit was as close as possible to the main stem. Shoots were removed from all internodes up to the 20th node. At the 23rd node, the apical bud was removed (topping), as per [21].

2.7.4. Pollination and Fruit Thinning

Cross-pollination was carried out manually, with daily frequency until all plants were pollinated. Starting from the beginning of flower opening, pollination occurred between 6:00 and 7:30 a.m., using the tips of the anthers from one plant (male organ of the flower) to transfer pollen to the stigmas (female organ of the flower) of another plant, typically using an average of three male flowers per female flower. After fruit set, ensuring no further fruit abortion occurred, thinning was performed as necessary to leave only one fruit per plant. The fruits were harvested when they reached maximum development and showed signs of tissue rupture at the abscission layer of the fruit peduncle.

2.7.5. Phytosanitary Management

Pest control (Bemisia tabaci, Aphis gossypii, and Diaphania nitidalis) and diseases (Sphaerotheca fuliginea and Pseudoperonospora cubensis) were managed through chemical intervention, with preventive applications of insecticides from the chemical groups Acetamiprid and Deltamethrin (soluble powder and emulsifiable concentrate, respectively), and fungicides from the chemical groups Chlorothalonil and Sulfur (concentrated suspension and soluble powder, respectively). For the control of invasive plants in the pots, manual weeding was carried out throughout the experiment period.

2.8. Analyzed Variables

2.8.1. Photosynthetic Pigments

The quantification of photosynthetic pigment levels (Chl a, Chl b, carotenoids, total Chl, Chl a/Chl b ratio, and total Chl/carotenoids ratio) was performed 43 days after transplanting, following the laboratory method developed by [24]. From the extracts, the concentrations of chlorophylls and carotenoids in the solutions were determined using a spectrophotometer at absorbance wavelengths (470, 646, and 663 nm), using Equations (3)–(8).

$\mathrm{C}\mathrm{h}\mathrm{l} a=12.21\mathrm{A}\mathrm{B}\mathrm{S}663-2.81 \mathrm{A}\mathrm{B}\mathrm{S}646$	Chl a: Chlorophyll a (mg g−1 FW *)	(3)
$\mathrm{C}\mathrm{h}\mathrm{l} b=20.13\mathrm{A}\mathrm{B}\mathrm{S}646-5.03 \mathrm{A}\mathrm{B}\mathrm{S}663$	Chl b: Chlorophyll b (mg g−1 FW *)	(4)
$\mathrm{C}\mathrm{a}\mathrm{r}=(1000\mathrm{A}\mathrm{B}\mathrm{S}470-1.82 \mathrm{C}\mathrm{h}\mathrm{l} a-85.02\mathrm{C}\mathrm{h}\mathrm{l} b)/198$	Car: Carotenoids (mg g−1 FW *)	(5)
$\mathrm{C}\mathrm{h}\mathrm{l}\mathrm{T}=17.3\mathrm{A}\mathrm{B}\mathrm{S}646+7.18\mathrm{A}\mathrm{B}\mathrm{S}663$	ChlT: Total Chlorophyll (mg g−1 FW *)	(6)
$\mathrm{R}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o} \mathrm{C}\mathrm{h}\mathrm{l} a/\mathrm{C}\mathrm{h}\mathrm{l} b=\mathrm{C}\mathrm{h}\mathrm{l} a/\mathrm{C}\mathrm{h}\mathrm{l} b$	dimensionless	(7)
$\mathrm{R}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o} \mathrm{C}\mathrm{h}\mathrm{l}\mathrm{T}/\mathrm{C}\mathrm{a}\mathrm{r}=\mathrm{C}\mathrm{h}\mathrm{l}\mathrm{T}/\mathrm{C}\mathrm{a}\mathrm{r}$	dimensionless	(8)
* FW: fresh weight of the leaf

2.8.2. Fluorescence the Chlorophyll a

Chlorophyll a fluorescence was measured 43 days after transplantation through initial fluorescence (F0—elétrons quantum⁻¹), maximum fluorescence (Fm—elétrons quantum⁻¹), variable fluorescence (Fv—elétrons quantum⁻¹), maximum quantum yield of Photosystem II (Fv/Fm—elétrons quantum⁻¹), photochemical efficiency in PSII (Fv/F0—elétrons quantum⁻¹), and basal quantum efficiency of the non-photochemical process (Fm/F0—elétrons quantum⁻¹) in leaves pre-adapted to darkness using leaf clamps for 30 min, between 8:00 and 10:00 AM, on the third fully expanded leaf counted from the apical bud, using a fluorometer (Brand: Fluorpen, Model: FP-100), acquired in Drasov, Czech Republic.

2.8.3. Biomass Allocation in the Photosynthetic Apparatus

For the Leaf Mass Ratio (LMR), the ratio between leaf mass and total dry mass of the plant (MDleaf/MDtotal) was calculated, expressing the fraction of non-exported dry matter, based on the equation from [25] according to Equation (9).

$$\mathrm{L}\mathrm{M}\mathrm{R}={\displaystyle \frac{\mathrm{M}\mathrm{D}\mathrm{l}\mathrm{e}\mathrm{a}\mathrm{f}}{\mathrm{M}\mathrm{D}\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}}}\mathrm{ }$$

(9)

in which

LMR = Leaf mass ratio (g g⁻¹);

MD_leaf = Leaf dry mass (g per plant);

MD_total = Total dry mass (g per plant).

The SLA values were obtained through the dry mass and area of the leaf discs, represented by the equation below and proposed by [26], according to Equation (10).

$$\mathrm{S}\mathrm{L}\mathrm{A}={\displaystyle \frac{\mathrm{M}\mathrm{S}\mathrm{f}\mathrm{o}\mathrm{l}\mathrm{h}\mathrm{a}\mathrm{s}}{\mathrm{L}\mathrm{A}}}$$

(10)

in which

SLA = Specific leaf area (g cm²);

MS = Leaf dry mass (g per plant);

LA = Leaf area (cm²).

Based on the leaf area data recorded at 15 and 57 days after harvest, the relative leaf area growth rate was determined according to the methodology [27], as described in Equation (11).

$$\mathrm{R}\mathrm{G}\mathrm{R}-\mathrm{L}\mathrm{F}={\displaystyle \frac{\mathrm{N}\mathrm{l} \mathrm{L}\mathrm{F}2-\mathrm{N}\mathrm{l} \mathrm{L}\mathrm{F}1}{(\mathrm{T}2-\mathrm{T}1)}}$$

(11)

in which

RGR-LF = Relative growth rate of leaf area (cm day⁻¹);

LF2 = Final leaf area measurement (cm²);

LF1 = Initial measurement of leaf area (cm²);

T2−T1 = Time interval (15–57 days);

Nl = Neperian logarithm.

2.9. Data Analysis

The normality and homoscedasticity of the data was confirmed by the Shapiro–Wilk and Levene test (p ≤ 0.05), respectively. The data were subjected to analysis of variance using the F-test, and when significance was observed at the 0.01 and 0.05 probability levels, the quantitative factors (electrical conductivity levels) were compared using regression analysis, and the qualitative factors (nitrogen sources) were compared using the Scott-Knot mean test. The statistical analyses were performed using the SISVAR statistical software, version 5.7 [28].

3. Results

3.1. Photosynthetic Pigments

In Experiment I (waters salinized with NaCl), it was found that the interaction between treatments influenced (p ≤ 0.05) the levels of Chl a and carotenoids (Car). Individually, the electrical conductivity of the nutrient solution (ECns) affected (p ≤ 0.05) Chl b, Chl a/Chl b, ChlT/Car, and Chl T. In the first experiment, no influence (p > 0.05) of the N sources, individually, was observed (Table S2).

In Experiment II (waters salinized with CaCl₂), the electrical conductivity of the nutrient solution (ECns) significantly affected (p ≤ 0.05) Chl a, Chl b, Car, and Chl T, and it affected (p ≤ 0.01) Chl a/Chl b, ChlT/Car; still regarding the second experiment, individually, no significance was observed for the interaction between ECns and N sources (p > 0.05) (Table S3).

Under the prevalence of NaCl in the water, it was found that when NH₄⁺ was used as the N source, the Chl a content was reduced at a rate of 2.3696 mg g⁻¹ of FW for each unit increase in ECns; on the other hand, when NO₃⁻ was used as the N source, the Chl a content was minimum (9.17 mg g⁻¹ of FW) at an ECns estimated at 4.2 dS m⁻¹. In this sense, when comparing the N sources within each ECns, it was found that Chl a was 20.31% higher when NH₄⁺ was used as the N source, within the ECns of 3.0 dS m⁻¹ (Figure 3a).

Figure 3. (a,b) Chlorophyll a (Chl a); (c,d) Chlorophyll b (Chl b); (e,f) Carotenoids (Car); (g,h) Total Chl; (i,j) Chlorophyll a/Chlorophyll b ratio (Chl a/Chl b); and (k,l) Total Chl/Car ratio of melon plants under saline levels and nitrogen sources, 43 days after transplanting. ECns: electrical conductivity of the nutrient solution; LS: saline levels; SN: source nitrogen; CV: coefficient of variation; ** and *: significant at 0.01 and 0.05 probability levels, respectively; ns: not significant.

In the situation where the water was salinized with CaCl₂.2H₂O, that is, in Experiment II, a reduction (p ≤ 0.05) in the Chl a of 1.8107 mg g⁻¹ of FW was observed for each unit increase in ECns (Figure 3b).

The Chl b content was minimum (3.74 mg g⁻¹ FW) at ECns estimated at 4.2 dS m⁻¹, in plants under prevalence of Na⁺ in the brackish water. Additionally, an isolated effect was observed for nitrogen sources; the use of NH₄⁺ fertilization the Chl b content was 16.87% higher (Figure 3c). Under the prevalence of Ca²⁺ in the brackish water, the unit reduction was estimated at 8.89% for each dS m⁻¹ increment. (Figure 3d).

For carotenoid levels, under the prevalence of Na⁺, reductions of 0.9069 (NO₃⁻) and 0.599 (NH₄⁺) mg g⁻¹ FW were estimated for each unit increase in ECns, with losses calculated within the studied ECns range of 38.83% and 22.63%, respectively. When comparing the N sources within each ECns, it is noted that the carotenoid content is 15.43% higher (p ≤ 0.05) when NH₄⁺ was used as the N source (ECns of 3.0 dS m⁻¹) (Figure 3e). In Experiment II, prevalence of Ca²⁺, there was no effect of the interaction between treatments or of the nitrogen sources individually; however, a reduction in Car content of 0.64 mg g⁻¹ FW was observed for each dS m⁻¹ increment, with a total loss of 25.81% when comparing the content of plants exposed to 2.0 and 5.0 dS m⁻¹ (Figure 3f).

As total pigments levels, the unit losses were 4.5622 and 3.24 mg g⁻¹ FW for each dS m⁻¹ increase in ECns, under the prevalence of Na⁺ or Ca²⁺ in the brackish water, respectively, reaching total losses, within the ECns range studied, of 49.72% or 35.68%, respectively (Figure 3g,h). The Chl a/Chl b ratio was maximum (3.17 or 3.19 mg g⁻¹ FW) under prevalence of Na⁺ (ECns estimated in 3.05 dS m⁻¹) or Ca²⁺ (ECns estimated in 3.04 dS m⁻¹), respectively, with no influence of N sources being observed. (Figure 3i,j). For the Chl T/Car ratio, losses were observed of 0.53 (10.83%) and 0.17 (4.35%) mg g⁻¹ FW for each dS m⁻¹ increased to ECns and, comparing the Chl T/Car of plants under 2.0 and 5.0 dS m⁻¹, losses of the order of 41.48% and 14.28%, for the prevalence of Na⁺ or Ca²⁺ on the brackish water, respectively (Figure 3k,l).

3.2. Fluorescence the Chlorophyll a

The interaction between the treatments or the isolated effect of N sources did not influence (p > 0.05) the chlorophyll a fluorescence of melon plants in Experiments I and II. For the variables initial fluorescence (F₀), maximum fluorescence (Fm), and variable fluorescence (Fv), a significant effect (p ≤ 0.05) was observed, isolated for the ECns, and also for both experiments (Tables S4 and S5).

When comparing the results of plants under 2.0 and 5.0 dS m⁻¹, the Fo values drop from 792.16 to 585.27 electrons quantum⁻¹ (prevalence of Na⁺) and from 791.72 to 616.33 electrons quantum⁻¹ (prevalence of Ca²⁺), which imply a unitary loss of 68.96 and 58.46 electrons quantum⁻¹ per increased dS m⁻¹, respectively (Figure 4a,b).

Figure 4. (a,b) Initial fluorescence (F₀); (c,d) maximum fluorescence (Fm), (e,f); variable fluorescence (Fv), 43 days after transplanting melon plants under saline levels and nitrogen sources. ECns: electrical conductivity of the nutrient solution; LS: saline levels; SN: source nitrogen; CV: coefficient of variation; **: significant at 0.01 probability levels; ns: not significant.

The increase in ECns also resulted in linear reductions in the Fm values for each unit increase in ECns. In Experiment I, the losses were at a rate of 6.60% per each increased dSm⁻¹; within the proposed ECns range, the calculated total reduction was 22.79%. In Experiment II, the Fm values were reduced by a 6.69% per unit increase in ECns and demonstrated a reduction of 23.18% when comparing the highest and lowest estimated values (Figure 4c,d).

Reductions of 7.63 and 7.51% on Fv was estimated for each dS m⁻¹ increased on ECns for Na⁺ or Ca²⁺ prevalence, using NO₃⁻ or NH₄⁺ as source of N. In fact, when comparing Fv of plants exposed to 2.0 and 5.0 dS m⁻¹, the losses reach 27.02 and 26.52% for Na⁺ or Ca²⁺ prevalence, respectively (Figure 4e,f).

3.3. Biomass Allocation in the Photosynthetic Apparatus

In Experiments I and II, it was found that the nitrogen sources influenced (p ≤ 0.01) the leaf mass ratio. In Experiment II, it was observed that the electrical conductivity of the nutrient solution (ECns) affected (p ≤ 0.05) the specific leaf area and the nitrogen sources (p ≤ 0.01). In Experiments I and II, it was found that the electrical conductivity of the nutrient solution (ECns) affected (p ≤ 0.01) the relative leaf area growth rate, as well as (p ≤ 0.01) the nitrogen sources.

For the prevalence of Na⁺ and Ca²⁺, the leaf mass ratio values were higher when NH₄⁺ was used as the N source, with values 4.20% and 5.17% higher, respectively, when comparing the maximum and minimum means obtained (Figure 5a).

Figure 5. (a) Leaf mass ratio, (b) specific leaf area, and (c–e) relative growth rate of leaf area 57 days after transplanting melon plants under saline levels and nitrogen sources. ECns: electrical conductivity of the nutrient solution; LS: saline levels; SN: source nitrogen; CV: coefficient of variation; ** and *: significant at 0.01 and 0.05 probability levels, respectively; ns: not significant. ^a,b For the leaf mass ratio, it was only significant for the nitrogen sources for both experiments, while for the specific leaf area, it was only significant for the experiment with CaCl₂.2H₂O.

In plants exposed to saline water with Ca²⁺ prevalence, the specific leaf area (SLA) was maximum (0.0456 g cm²) at the estimated ECns of 3.0 dS m⁻¹; it was also observed that the use of NO₃⁻ resulted in a 19.60% increase in specific leaf area (Figure 5b). The SLA of plants under sodium–saline water was not affected (p > 0.05) by the treatments.

For the prevalence of Na⁺ in the water, it was observed that when NO₃⁻ was used as the nitrogen source, the leaf area growth rate was reduced by 12.72% for each dS m⁻¹ increase in ECns; on the other hand, when NH₄⁺ was used as the nitrogen source, the decrease was 15.27% for each dS m⁻¹ increase in ECns (Figure 5c,d). When comparing the means for the prevalence of Na⁺ and Ca²⁺, the relative leaf area growth rate values were higher when NO₃⁻ was used as the nitrogen source, with values 10.48% and 8.61% higher, respectively, when comparing the means (Figure 5e).

4. Discussion

For each kg of N, Ca(NO₃) or KNO₃ is typically more expensive than CH₄N₂O; however, from the melon grower’s point of view, other aspects must also be considered. In addition to the supply of Ca²⁺ or K⁺ and the minimization of losses through volatilization, Ca(NO₃) does not imply soil acidification and is important for crops such as melons that demand Ca²⁺ throughout the cycle [29,30].

In this research, the same mass per liter of NO₃⁻ or NH₄⁺ was used; however, as expected, the increase in ECns implied a reduction in the supply of NO₃⁻ and NH₄⁺ (prevalence of Na⁺) and prevalence of Ca²⁺, throughout the crop cycle (Table S7). This reduction is a direct implication of the limitation that the osmotic issue imposes on the consumption of water and nutrients by plants; however, the results on the content of photosynthetic pigments or fruit weight, may be associated with the activation of different assimilation pathways depending on the N source, especially under high salinity conditions.

High salinity concentrations cause significant damage to plants, leading to declines in productivity and fruit quality in crops. Under salt stress, the physiological processes of plants are constantly affected in various ways, including high amounts of ions, such as Na⁺ or Ca²⁺, which cause the degradation of enzymatic structures and cellular organelles, thereby halting photosynthesis, respiration, and the synthesis of proteins and photosynthetic pigments in plants [31,32]. Furthermore, the accumulation of salts in the soil leads to physiological drought and nutrient imbalance in plants [33]; and the stress caused by the increase in salts makes plants generate reactive oxygen species (ROS) [31,34].

Além disso, pode-se sugerir que as altas concentrações elevadas de Na⁺ ou Ca²⁺, provenientes de águas salinizadas com NaCl ou CaCl₂, podem ter ocasionado redução no desenvolvimento do melão, comprometendo a absorção de nutrientes e resultando em um desequilíbrio nutricional nas proporções de Na⁺/K⁺, Na⁺/Ca²⁺ e Na⁺/Mg²⁺ [35].

In a study carried out by [36] in the Mediterranean area with melon crops (var. cantalupensis Naud. Alpes) with different types of irrigation and fertilization with nitrogen and potassium in a greenhouse, he was able to demonstrate that increased salinity is one of the main factors that reduce the yield of the most varied production parameters of the plant.

4.1. Photosynthetic Pigments

The use of NH₄⁺ as a N source, specifically for the ECns of 3.0 dS m⁻¹ (NaCl), increased the Chl a content by 20.31%; however, for the other salt concentration levels, the Chl a concentration does not change significantly. In fact, NH₄⁺ can increase chlorophyll content in plants, but the effect depends on the concentration and balance with other nutrients like K⁺. Anyway, while in similar situations mild ammonium stress induces chlorophyll accumulation, chlorosis seems to be an exclusive effect of severe ammonium toxicity [37], as observed for the other concentrations.

Under Ca²⁺ prevalence, in no concentration tested, the use of NH₄⁺ or NO₃⁻ implied gains in melon Chl a. Actually, in both cases, the reduction in chlorophyll a content can be attributed to problems of membrane stability [27] and to increased degradation and/or inhibition of the synthesis of this pigment by the activity of enzymes such as chlorophyllase, hydroxylase, and dioxygenase [38]. Similar results were found by [39], who mentioned a reduction in the levels of Chl a in mini watermelon plants due to increased water salinity.

Like Chl a, under Na⁺ prevalence, the use of NH₄⁺ also implied gains of Chl b of 21.7%, in isolated analysis, without significative interaction with ECns. On the other hand, there is no difference in Chl b content when using NO₃⁻ or NH₄⁺ as N source, in plants exposed to brackish waters with a prevalence of Ca²⁺. These results reinforce the comments of [5] that in some situations compared to NO₃⁻, NH₄⁺ assimilation in plants requires less energy.

The Na⁺ prevalence also increase the Car content by 15.43% (3.0 dS m⁻¹) with NH₄⁺ as source of N. Although the source of N did not benefit the Car content at other concentration levels, it is noted that the use of NH₄⁺ (reduction of 22.63%) mitigated the losses in relation to the use of NO₃⁻ (reduction of 38.83%), when comparing the Car content of plants exposed to 2.0 and 6.0 dS m⁻¹. The reduction in Car content in melon can be attributed to the degradation of ß-carotene and the reduction in zeaxanthin formation; these results were observed in the present work. With different intensities, the production of carotenoids was affected [40].

Under salinity conditions (NaCl), the content of Chl a, Chl b, and Car was higher when NH₄⁺ was used as the N source. On the other hand, for the same salinity levels tested (CaCl₂), the use of NH₄⁺ or NO₃⁻ as a N source does not affect photosynthetic pigments. In summary, under the same ECns range, the content of Chl a, Chl b, and Car will be more or less affected by the use of NO₃⁻ or NH₄⁺ depending on the ionic prevalence of the brackish water, Na⁺ or Ca²⁺.

Testing NH₄⁺/NO₃⁻ ratios under non-salinity conditions, Ref. [41] found that the use of NH₄⁺ alone in yellow passion fruit plants, implied restrictions on the accumulation of dry biomass and growth; however, the isolated use of NO₃⁻ benefited the growth of plants, which presented higher levels of chlorophyll a, b, total, and carotenoids than plants with the other proportions of N. On the other hand, Ref. [37] added that excessive NH₄⁺ accumulation in tissues is toxic for plants and exclusively NH₄⁺-based nutrition enhances this effect. However, they consider that moderate NH₄⁺ stress induces chlorophyll accumulation and that chlorosis is a result of severe NH₄⁺ toxicity, as verified by these authors for Arabidopsis thaliana accessions. Finally, Ref. [5] add that physiological effects of NH₄⁺ and NO₃⁻ have confirmed that tea plants prefer NH₄⁺ as the dominant nitrogen (N) source.

The use of NO₃⁻ or NH₄⁺ did not affect Chl T, but the prevalence of Ca₂⁺ implied lower accumulated losses than the prevalence of Na⁺, within the same ECns range. On the other hand, from 3.17 and 3.19 dSm⁻¹, the Chl a/Chl b ratio decreased, considering the prevalence of Na⁺ or Ca²⁺, respectively, evidencing the effect of the increased salt concentration in the nutrient solution. The reduction in total chlorophyll levels measured in plants under stress can be associated with the presence of ions at toxic levels that dehydrate mesophyll cells, inhibiting enzymes involved in carbohydrate metabolism and some stages of chlorophyll synthesis [42]. Since chlorophyll is the most important pigment for capturing light energy, its reduction leads to a decrease in photosynthesis. A decrease in total chlorophyll content in melons stressed by salinity was also observed by [43].

The pigment ratios of Chl a/Chl b and total Chl/carotenoids in the leaves showed a tendency for reduction with the increase in salinity, which can be related to the plant response to environmental changes with increasing stress. With the prolongation of ECns levels, chlorophyll synthesis gradually decreased, which occurs due to the reduction in the light energy absorption capacity of chloroplasts under high salt stress, leading to a decrease in chlorophyll content [44]. Ref. [39] also found similar results, as all chlorophyll variables in melons showed a trend of reduction with the increase in water salinity levels.

The higher efficiency of the NH₄⁺ source in the presence of Na⁺ can be attributed to the reduced energy consumption during its assimilation and the activation of metabolic pathways adaptive to osmotic stress, as reported by [5,37]. In contrast, in waters rich in Ca²⁺, the presence of this cation contributes to membrane stability and ionic homeostasis, [45]. This reduces pigment degradation and promotes a more balanced metabolism, regardless of the N source used.

4.2. Fluorescence the Chlorophyll a

The inhibition of the accumulation of Na⁺ and Cl⁻ ions due to the antagonistic effects of NH₄⁺ and NO₃⁻ [46,47], which is used as a tolerance inducer, depends on several aspects. There are mentions in the literature of the economic advantages of using NH₄⁺ (moderate stress) compared to NO₃⁻ [48]; however, there are also reports of the enhancement of the effects of salinity when there are greater accumulations of NH₄⁺ in plant tissues. For [49], interactions between N nutrition and salinity vary depending on plant age, salt stress level, duration, and application method.

In this research, the effect caused by the use of NH₄⁺ as a source of N on photosynthetic pigments is not perceived when analyzing the fluorescence of chlorophyll a. In fact, the basic fluorescence signal has characteristic levels that reflect the plant’s ‘status’ at that moment, in relation to its own metabolism and its interaction with the environment in which it is found [50]. In other words, the positive correlation between nitrogen concentrations and leaf chlorophyll content [51] can be affected by other factors, such as salinity.

From this point of view, the use of NO₃⁻ or NH₄⁺ in salinity conditions does not affect the photochemical efficiency of plants; however, for the same salinity, the results can be analyzed differently depending on the ionic prevalence of the water Na⁺ or Ca²⁺. Although, in the present work, it is clear that the losses are associated with the increase in the concentration of salts.

It is worth noting, however, that the prevalence of Ca²⁺ resulted in lower accumulated losses when comparing Fo and Fv of plants under 2.0 and 5.0 dS m⁻¹ and greater losses of Fm in the same ECns range. Ca²⁺ deficiency in plants implies a reduction in Fm (verified in tomato plants), in addition to damaged cell membranes (in peach plants) and impaired biosynthesis of chlorophyll and chlorophyll precursors [52]. On the other hand, excess induces the closure of leaf stomata, thus reducing photosynthesis; it affects the absorption and utilization of magnesium, and the efficiency of light energy utilization in leaves is reduced [53].

The reduction in initial fluorescence signals occurs in response to the decreased ability to transfer excitation energy from the light-harvesting system to the reaction center, likely due to the decrease in leaf water potential [54,55]. Furthermore, this reduction indicates that the activity of chlorophyll a was diminished, leading to less energy generation, which affects the proper functioning of Rubisco and, consequently, the CO₂ carboxylation process [56]. According to [57], the absence of differences in Fo for the salinity levels, as well as the reduction, could indicate plant tolerance to salinity. Like us, [35] also found a reduction in Fo in mini watermelon cultivation, as the electrical conductivity of the water increased.

Variable fluorescence and maximum fluorescence were reduced with the increase in ECns, without influence from nitrogen sources. Variable fluorescence refers to the plant’s ability to transfer energy from electrons ejected from pigment molecules to the formation of NADPH, ATP, and reduced ferredoxin. Thus, its reduction can indicate that the photosynthetic apparatus was damaged by salt stress, impairing the function of Photosystem II, with negative effects on the photosynthetic process [56]. This decrease may be related to the intensity and duration of exposure to saline conditions, caused by the accumulation of various toxic ions that impaired the plants’ photosynthetic apparatus. As a result, the functioning of the reaction centers of Photosystem I and II is compromised throughout the exposure period, in relation to the stress factor [55].

Furthermore, salinity reduces energy capture at the reaction centers, directly affecting maximum fluorescence. This likely happens because the excessive accumulation of specific ions causes an imbalance in the plant’s metabolic activity, leading to the formation of reactive oxygen species, which limits the energy activity of photosynthetic pigments [58]. A study by [59] found similar results when evaluating Japanese cucumber under different electrical conductivity levels of the nutrient solution, where they observed decreases in both variable fluorescence and maximum fluorescence as the electrical conductivity of the nutrient solution increased.

4.3. Biomass Allocation in the Photosynthetic Apparatus

In this study, the leaf mass ratio was better for fertilization with NO₃⁻ in both Experiment I and II. This may be related to research showing the existence of competition in absorption between nitrate and chloride, so that increasing the nitrate concentration in the root zone can reduce chloride uptake by the plant [60].

Another point to consider is that with the increase in salinity, the specific leaf area was negatively affected, which may be related to the adjustment of the crop’s water potential and the reduction in the soil water potential, which may have allowed the maintenance of water uptake and cell turgor, consequently leading to a reduction in the plant’s specific leaf area [61]. Thus, these results agree with a study conducted by [62]. When evaluating some melon genotypes grown in substrate under saline stress, they reported a decrease in specific leaf area with the increase in ECsn.

The decrease in the relative growth rate may occur due to photosynthetic limitations resulting from stomatal closure, which leads to lower CO₂ assimilation [63,64]. Thus, salt accumulation negatively affects water absorption by plants, which is a determining factor in the reduction in photosynthetic and metabolic processes, impacting plant growth as ECa increases [65]. The decrease in the relative growth rate of melon is mainly due to the reduction in leaf area and number of leaves, which can be corroborated by the study of [66], where it was demonstrated that high levels of electrical conductivity reduce stomatal conductance, transpiration, and photosynthesis, leading to a reduction in leaf area in melon plants of the Sancho and Caribbean Gold cultivars. According to [67], increasing electrical conductivity to 5.0 dS m⁻¹ also resulted in a reduction in both the number and area of leaves. Furthermore, nitrate fertilization mitigated stress on the relative growth rate in both the Na⁺ and Ca²⁺ experiments, which may be associated with the fact that when NH₄⁺ is used as the nitrogen source, there is a reduction in photosynthesis that coincides with increased energy suppression, indicating low energy efficiency, as evidenced by the study of [68] on zucchini (cv. Caserta), where nitrate and ammonium sources were used, and the best performance of photosynthesis-related variables under salt stress occurred with NO₃⁻.

The association between NO₃⁻ and waters with a predominance of Ca²⁺ suggests a synergy that promotes greater nitrate transport and higher efficiency in carbon metabolism. This interaction may have contributed to the maintenance of leaf mass and a more stable osmotic balance, mitigating the negative effects of salinity on vegetative growth.

5. Conclusions

Under salinity conditions, the content of Chl a, Chl b, and Car of melon will be more or less affected by the use of NO₃⁻ or NH₄⁺ depending on the ionic prevalence of brackish water, Na⁺ or Ca²⁺. The use of NH₄⁺ as a N source (Na⁺ prevalence) in melon fertigation provides an increase of 20.31% in Chl a content (3.0 dS m⁻¹); 21.7% in Chl b content; and 15.43% (3.0 dS m⁻¹) in carotenoids content, in relation to when NO₃⁻ is used. Plants respond to the increase in salt concentration by altering the relationship between Chl a and other protective pigments, as seen in the Chla/Chlb and ChlT/Car ratios. The use of nitrogen sources, whether NO₃⁻ or NH₄⁺, does not affect the photochemical efficiency of hybrid F1 Caribbean Gold RZ cantaloupe-type melon plants fertigated with nutrient solution up to 5.0 dS m⁻¹, regardless of the prevalence of Na⁺ or Ca²⁺. Melon plants respond to the stress caused by the increase in the electrical conductivity of the nutrient solution in terms of specific leaf area (this only for water with Ca²⁺) and the relative leaf area growth rate. Additionally, both are mitigated by the use of NO₃⁻ as the nitrogen source, and the same occurs for the leaf mass ratio, in which stress was reduced with NO₃⁻ fertilization.