Grafting Boosts Physiological Performance and Nutrient Acquisition of Cantaloupe Under Salt and Bicarbonate Stress in Soilless Culture

Abstract

Soil salinity and bicarbonate-induced alkalinity severely limit melon productivity by disrupting physiological and biochemical processes. This study evaluated the effectiveness of grafting an Iranian cantaloupe cultivar, ‘Til-e-Sabz’, onto Cucurbita maxima × C. moschata rootstock in mitigating salinity (10 mM NaCl; 2.7 dS m⁻¹) and alkalinity (10 mM NaHCO₃; 2.6 dS m⁻¹) stress in soilless culture. Compared to non-grafted plants, grafted plants exhibited 22–35% greater leaf area, 28–40% higher shoot and root fresh biomass, and 25–38% higher dry biomass under both stress conditions. Relative chlorophyll content (SPAD) and total chlorophyll were reduced by stress but remained 15–21% higher in grafted plants. Carotenoid content was also maintained at 10–14% higher levels in grafted plants compared to non-grafted controls. Proline and soluble protein accumulation increased significantly under stress, with grafted plants accumulating 18–25% more proline and 12–20% more protein, indicating enhanced osmotic adjustment. Sodium levels increased in the roots and shoots under stress. However, grafted plants maintained 30–45% lower Na accumulation relative to non-grafted plants. In contrast, grafted plants showed up to 27% higher phosphorus and 32% higher iron uptake, while maintaining greater potassium retention (18–24%) under both salinity and alkalinity. Overall, grafting significantly improved physiological resilience and ion homeostasis, leading to enhanced stress tolerance. These findings demonstrate that grafting is a promising agronomic strategy to sustain melon production in saline and alkaline environments associated with increasing soil and water degradation.

1. Introduction

Melon (Cucumis melo L.) is an annual species in the Cucurbitaceae family, valued for its agricultural and economic importance worldwide [1]. It encompasses diverse varieties known by names like cantaloupe, muskmelon, Galia, Charentais, rock melon, and snap melon, which contribute to classification challenges due to variations in rind texture (netted or smooth), shape, size, and color (green, yellow, white, with or without stripes) [2]. Iran ranks fifth globally in melon production, with approximately 0.9 million tons produced annually, and serves as a center for melon biodiversity and domestication, featuring five major indigenous types [3,4]. In 2019, global cucurbit production exceeded 238 million tons, with melons contributing nearly 27.5 million tons [3]. Archeological evidence suggests that melon domestication dates back thousands of years in regions such as China, Egypt, and Iran [5]. Among horticultural groups, Cucumis melo var. cantalupensis encompasses key commercial cultivars, including the large, netted Persian type; the smooth-rinded, aromatic Charentais; the firm, netted Harper; and the sweet, green-fleshed Galia, which boasts good shelf life [6,7]. Melons are moderately sensitive to salinity, with a threshold EC of 2.2–2.5 dS m⁻¹, beyond which yields drop by 10–15% per EC unit increase [8]. Salinity poses a significant productivity barrier in arid and semi-arid areas through osmotic stress and ion toxicity [9,10,11]. By 2050, combined salinization and drought may make half of the arable land unusable [12,13]. Salt stress impairs water uptake, causes ionic imbalances, reduces photosynthesis through stomatal closure, delays flowering, and activates defenses such as ion exclusion, osmolyte production, and membrane changes [14]. Root roles in tolerance are understudied [15]. It reduces plant height, leaf area, root length, and biomass in crops like melon, lettuce, and strawberry [16,17], while disrupting nutrient balances (high Na and Cl, low K, Ca, P, Mg) [14], leading to chlorosis, leaf drop, pigment shifts [18], and variable responses in cucurbits by cultivar [19,20]. Prolonged exposure causes pigment degradation, membrane damage, senescence [21], and lowered Fv/Fm ratios indicating photosynthetic stress [22], with PSII disruptions from chloride [23].

Bicarbonate-induced alkalinity, a key salinity factor, causes physiological changes like reduced shoot growth and photosynthesis [9]. It elevates pH, reducing micronutrient availability (Fe, Zn, Mn) [24], making iron insoluble and causing chlorosis in young leaves via impaired chlorophyll synthesis [25]. Alkaline conditions hinder root function, nutrient uptake, and development in crops like melon, watermelon, and tomato [26]. Bicarbonate alters apoplastic pH and enzyme activity for Fe reduction/uptake [27], with less impact on resistant peas [28]. It induces Fe-deficiency chlorosis in calcareous soils via precipitation and inactivation [29], possibly through hormonal signals [29], reduced root respiration [30], and altered nutrient dynamics [31].

Grafting enhances growth, physiological, and biochemical traits under alkalinity in melon [32], watermelon [26,33], cantaloupe landraces [34], and cucumber [9,35]. Originating in ancient East Asian practices for Solanaceous and Cucurbitaceae vegetables in the 15th–16th centuries [36], commercial grafting surged in Japan and Korea from the 1920s, using squash rootstocks for watermelon [37]. It expanded with plastic tunnels in the 1950s and reached Western agriculture by the 1990s via rootstock innovations and greenhouses [38,39]. Vigorous rootstocks improve water/nutrient absorption [40,41], boost vegetative growth, cytokinins, stress tolerance, and reduce chemical use [42], though excess vigor in tomatoes may need management like defoliation [43]. Grafting suits intensive systems with limited land for high yields [44]. It confers resistance to soilborne diseases like bacterial wilt, Fusarium, and nematodes, replacing fumigation and rotation amid environmental limits [45,46], and is proven effective in melon, tomato, cucumber, and watermelon [47]. Compatibility affects outcomes, sometimes lowering yields [48], but often boosts them under stress, plus fruit size and productivity [49,50]. Fruit quality varies by rootstock, environment, and species [51], with larger fruits and fewer disorders, but inconsistent sugar/flavor impacts [52]. Under salinity, grafting improves vegetative traits, yield, quality, mineral composition, and antioxidant activity in melons [25,32,34,53,54,55]. It enhances nutrient uptake (Ca, P, Mg) [56,57], supporting photosynthesis and resilience under salt/pH stress, with grafted cucumbers and melons showing superior biomass, chlorophyll, and nutrients versus non-grafted [50,58]. Grafting aids sustainable horticulture with resilience and environmental benefits [9]. The hypothesis posits that grafting melons onto vigorous pumpkin rootstocks improves tolerance to salinity and bicarbonate alkalinity via better water status, nutrient uptake, and physiology compared to non-grafted plants.

2. Materials and Methods

2.1. Experimental Design and Location

To evaluate the effects of grafting on melon tolerance to salinity and alkalinity stress, an Iranian melon accession ‘Til-e-Sabz’ was grafted onto the hybrid pumpkin rootstock Cucurbita maxima × C. moschata cultivar (ES113). ‘Til-e-Sabz’ is a local variety of Cantalupensis melons widely cultivated in Khorasan province, Birjand city, Iran. The experiment was conducted under soilless conditions in the greenhouse of the Faculty of Agriculture at Vali-e-Asr University of Rafsanjan (Rafsanjan, Iran) from March to July 2023. A factorial experiment was arranged in a completely randomized design (CRD) with four replicates. The factors included grafting (grafted and non-grafted) and stress treatment (10 mM sodium bicarbonate (≈2.6 dS m⁻¹), 10 mM sodium chloride (2.7 dS m⁻¹), and a non-stress control (2.3 dS m⁻¹)) and four sampling times (7, 17, 27, and 90 days post-transplanting).

2.2. Experimental Setup

Til-e-Sabz melon seeds with a purity of 99% and a germination of 93% were purchased with lot number 22319 from Dast Sabz Agriculture and Industry Company, Esfarayen, North Khorasan, Mashad city, Iran. The scion was sown 3 to 4 days earlier than the rootstock due to its relatively slower growth to ensure a stronger graft union. Seeds were germinated in a 1:1 perlite and coir pith mix. Hole grafting was performed at the cotyledon stage. Grafted plants were placed in a controlled environment (16–18 °C with relative humidity greater than 85%) to minimize water loss at the graft site. Initial acclimatization began after 6 days, with gradual increases in light intensity and a reduction in humidity. After twenty-three days’ acclimation, grafted and non-grafted plants were transferred to larger polystyrene pots for soilless culture. These pots were placed in hydroponic units, and nutrient solution application began the following day. From grafting until transplanting to the hydroponic system, all plants received the nutrient solution described in

Table 1. The nutrient solution and concentration used in this experiment [58].

Chemical Compounds	Chemical Formula	Final Concentration (mg L−1) (Seedling Stage to Fruit Set)	Final Concentration (mg L−1) (from Fruit Set to Harvest)
Macronutrients
Potassium Dihydrogen Phosphate	KH2PO4	270	270
Potassium nitrate	KNO3	200	200
Calcium nitrate	Ca(NO3)2	680	1357
Magnesium sulfate	(MgSO4, 7H2O)	500	500
Micronutrients
Iron chelates	Fe-EDDHA	25	25
Boric acid	H3BO3	7.5	7.5
Manganese sulfate	MnSO4, 7H2O	8.4	8.4
Copper sulfate	CuSO4, 5H2O	0.54	0.54
Molybdenum peroxide	MoO3	0.15	0.15
Zinc sulfate	ZnSO4, 7H2O	1.18	1.18

. Stress treatments began after 5 days, using 10 mM sodium bicarbonate and 10 mM sodium chloride. The soilless culture system consisted of three separate 300 L tanks, continuously aerated and filled with a nutrient solution. Each tank received one of the three treatments: salinity, alkalinity, or control. The tanks were covered with 4 cm-thick polystyrene sheets with holes for 4 L pots filled with perlite. Each tank contained two rows of pots: one for non-grafted and one for grafted plants. Each treatment consisted of four replicates, each containing four plants, for a total of sixteen plants per treatment. At each sampling point, one plant per replicate was harvested, resulting in a total evaluation of 192 plants across all treatments and sampling times, and one plant per pot was sampled every 10 days for growth parameter assessment. The final plant was harvested after fruit development. Nutrient solutions were prepared using water purified by a five-stage filtration system with an EC of 14 µS cm⁻¹. The nutrient formula used is shown in Table 1. Solutions were replaced weekly for the first two weeks and every five days thereafter. Plants were exposed to the stress treatments until the end of the experiment. Greenhouse conditions during the treatment period were maintained at 27 ± 3 °C (day) and 24 ± 3 °C (night) with 52.4 ± 5% relative humidity. Final harvests occurred after 3 months, and samples were transferred to the lab for analysis. Biometric parameters, including leaf area, plant height, and biomass, were measured on one representative plant per replicate at each growth stage, resulting in four plants per treatment for each sampling point. All biochemical analyses were performed in triplicate for each plant sample.

2.3. Light-Absorbing Compounds in Photosynthetic Apparatus

To quantify chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids, 0.25 g of fresh leaf tissue was finely ground in a porcelain mortar with 10 mL of 80% acetone until a homogeneous slurry was obtained. The resulting mixture was transferred into 20 mL Falcon tubes and centrifuged at 3500 rpm for 10 min. The absorbance of the supernatant was measured at 480, 510, 645, 652, and 663 nm using a T80 UV/VIS spectrophotometer (PG Instruments Ltd., Hereford, UK). The concentrations of pigments were calculated using the following equations as described by Arnon [59]:

$$\mathrm{Chlorophyll} \mathrm{a} (\mathrm{mg} {\mathrm{g}}^{-1}\mathrm{FM}) = [\left(12.7\times (\mathrm{A}663\right)- \left(2.69\times (\mathrm{A}645\right)]\times \left({\displaystyle \frac{\mathrm{V}}{1000}}\times \mathrm{W}\right)$$

$$\mathrm{Chlorophyll} \mathrm{b} (\mathrm{mg} {\mathrm{g}}^{-1}\mathrm{FM})=[\left(22.9\times (\mathrm{A}645\right)-\left(4.68\times (\mathrm{A}663\right)]\times \left({\displaystyle \frac{\mathrm{V}}{1000}}\times \mathrm{W}\right)$$

$$\mathrm{Total} \mathrm{Chlorophyll} (\mathrm{mg} {\mathrm{g}}^{-1}\mathrm{FM})=\left({\displaystyle \frac{\mathrm{A}652\times 1000}{34.5}}\right)\times \left({\displaystyle \frac{\mathrm{V}}{1000}}\times \mathrm{W}\right)$$

$$\mathrm{Carotenoids} (\mathrm{mg} {\mathrm{g}}^{-1}\mathrm{FM})=[\left(7.6\times (\mathrm{A}480\right)-\left(1.49\times \mathrm{A}510\right)]\times \left({\displaystyle \frac{\mathrm{V}}{1000}}\times \mathrm{W}\right)$$

where A = absorbance at the specified wavelength, V = volume of acetone used (10 mL), and W = fresh sample mass (0.25 g).

2.4. Relative Chlorophyll Content (SPAD)

To assess relative chlorophyll content (SPAD index), four leaves from the 3rd and 6th nodes of each plant were selected and measured using a SPAD-502 Chlorophyll Meter (Konica Minolta, Inc., 2-7-2, Marunouchi, Chiyoda-ku, Tokyo 100-7015, Japan).

2.5. Plant Water Relations

This section focused on leaf relative water content (RWC). Several 6 mm diameter leaf discs were punched from fresh leaves, and their fresh weight was measured immediately to calculate RWC. The discs were floated in distilled water in Petri dishes for 6 h to allow full turgidity. After soaking, the discs were blotted dry and weighed again to determine their turgid weight. Finally, the discs were oven-dried at 70 °C for 48 h to determine their dry weight. RWC was calculated using the following equation:

$$\mathrm{Relative} \mathrm{Water} \mathrm{Content} (\%) = 100 \times \left({\displaystyle \frac{\mathrm{F}\mathrm{r}\mathrm{e}\mathrm{s}\mathrm{h} \mathrm{M}\mathrm{a}\mathrm{s}\mathrm{s}-\mathrm{D}\mathrm{r}\mathrm{y} \mathrm{M}\mathrm{a}\mathrm{s}\mathrm{s}}{\mathrm{T}\mathrm{u}\mathrm{r}\mathrm{g}\mathrm{i}\mathrm{d} \mathrm{M}\mathrm{a}\mathrm{s}\mathrm{s}-\mathrm{D}\mathrm{r}\mathrm{y} \mathrm{M}\mathrm{a}\mathrm{s}\mathrm{s}}}\right)$$

2.6. Proline Concentration

For proline extraction, 0.5 g of fully expanded leaves was homogenized in a mortar with 5 mL of 95% ethanol. The extract was transferred to a Falcon tube and extracted twice more using 5 mL of 70% ethanol each time. The pooled extract was centrifuged at 3500 rpm for 10 min. The supernatant was used for proline quantification. To determine proline concentration, 1 mL of the alcoholic extract was diluted with 10 mL of distilled water, then mixed with 5 mL of ninhydrin reagent (prepared by dissolving 1.25 g ninhydrin in 30 mL glacial acetic acid and 20 mL of 6 M phosphoric acid). An additional 5 mL of glacial acetic acid was added, and the mixture was shaken briefly. The reaction mixture was incubated in a water bath at 100 °C for 45 min. After cooling, 10 mL of benzene was added, and the contents were thoroughly mixed using a mechanical shaker. The samples were left to stand for 30 min to allow phase separation. The absorbance of the benzene phase was read at 515 nm using a T80 UV/VIS spectrophotometer. A standard curve was prepared using L-proline at concentrations of 0, 31.25, 62.5, 125, 250, and 500 mgL⁻¹ [60].

2.7. Soluble Sugar Content

Soluble sugars were quantified using the same alcoholic extract prepared for proline measurement. A 0.1 mL aliquot of the extract was mixed with 3 mL of freshly prepared anthrone reagent (150 mg anthrone dissolved in 100 mL of 72% sulfuric acid). The reaction mixture was heated in a water bath for 10 min to develop color. After cooling, the absorbance was measured at 625 nm using a spectrophotometer. A glucose standard curve was constructed using concentrations of 0, 250, 500, 750, 1000, 1250, 1500, 1750, 2000, 2250, and 2500 mgL⁻¹ [61].

2.8. Protein Quantification

The Bradford method [62] determined the soluble protein content. Fresh leaf tissue (0.5 g) was weighed and thoroughly ground in a porcelain mortar with 6.25 mL of Bradford extraction buffer (Tris-HCl). The homogenate was transferred to a Falcon tube and stored at refrigeration temperature (approximately 4 °C) for 24 h without agitation. After incubation, the sample was centrifuged at 16,000 rpm for 30–40 min at 2–4 °C. A 0.1 mL aliquot of the resulting supernatant was transferred into a 15 mL Falcon tube and mixed immediately with 5 mL of Bradford reagent. The mixture was vortexed quickly and left to stand at room temperature for 25 min. The absorbance of the developed blue color was measured at 595 nm using a spectrophotometer (T80 UV/VIS Spectrometer, PG Instruments Ltd., Unit 4, The Stables Eagle Road, Hereford, UK).

2.9. Vegetative Parameters

The vegetative characteristics assessed in this study included leaf number, leaf area, stem height, stem diameter, and fresh and dry weights of the root, stem, and leaves. Stem height was measured using a ruler, while stem diameter was determined 5 cm above the grafting point using a digital caliper. Leaf area was recorded using a CI-202 Leaf Area Meter (CID Bio-Science, Inc., Camas, WA, USA). To choose fresh weight, plants were separated at the crown into three parts (leaves, stem, and roots) and each part was weighed individually using a precision balance. Samples were dried in an oven at 70 °C for 48 h for dry weight measurements and then weighed again.

2.10. Nutrient Element Analysis

The elements assessed in this study included potassium, iron, and phosphorus, measured in both roots and shoots. For sample preparation, 0.5 g of oven-dried and finely ground plant material (shoot or root) was placed in a crucible and first ashed at 250 °C for 30 min, followed by combustion at 550 °C for 3 h in a muffle furnace until white ash was obtained. The resulting ash was dissolved in 5 mL of 2N hydrochloric acid (HCl), and the final volume was adjusted to 50 mL with distilled water. This solution was directly used to analyze K, Na, and Fe. Potassium and Na were determined using a flame photometer (Model of JENWAY Bibby Scientific Ltd., Stone, Staffordshire, UK)), while Fe concentration was measured using atomic absorption spectrophotometry (Model GBC AVANTA, GBC Scientific Equipment, Melbourne, Australia). Phosphorus content was determined using the yellow ammonium molybdate–vanadate colorimetric method [63]. In this procedure, 5 mL of the digested extract was mixed with 10 mL of ammonium molybdate–vanadate reagent, and the final volume was adjusted to 50 mL with distilled water. The absorbance was then measured at 470 nm using the exact spectrophotometer.

2.11. Statistical Analysis

The experiment was conducted using a completely randomized design (CRD) with a three-factor arrangement and four replications per treatment. Data were analyzed using SAS software version 9.4 (SAS Institute Inc., Cary, NC, USA). A multi-factor ANOVA was performed to examine the main effects and interaction effects of grafting, salinity, and alkalinity stress across different sampling times. This allows for a more detailed comparison of the severity of salinity and alkalinity stress, as well as the effectiveness of grafting in mitigating each stress condition. Post hoc comparisons of means were conducted using Duncan’s Multiple Range Test (DMRT), with statistical significance determined at p ≤ 0.05.

3. Results

3.1. Photosynthetic Pigments

According to the analysis of variance, only the main effects of stress, grafting, and harvest time, along with their two-way interactions, had significant impacts on the chlorophyll a level. The mean comparison indicated an apparent reduction in chlorophyll a content in grafted and non-grafted plants under stress conditions. The highest chlorophyll a concentration was found in grafted plants under control conditions, whereas the lowest was observed in non-grafted plants exposed to alkalinity (Figure 1A). Further analysis showed that both grafting and developmental stages also affected chlorophyll content. Across all four sampling times, grafted plants consistently had higher chlorophyll concentrations than non-grafted ones (Figure 2B). The results also indicated that as the plants progressed through their growth stages, the level of chlorophyll in their leaves declined under stress, particularly in alkaline conditions. The highest chlorophyll a content was observed in the first harvest under control conditions, while the lowest was recorded at the fourth harvest under alkalinity stress. Notably, no significant differences were found among the sampling times under alkalinity stress (Figure 3C).

Chlorophyll b content was significantly influenced (at the 1% probability level) by the individual effects of stress, grafting, and time, as well as the interactions between grafting × stress, grafting × time, and stress × time. The comparison of treatment means indicated that grafted and non-grafted plants exhibited reduced chlorophyll b levels under stress conditions compared to the control. However, this reduction was not statistically significant in grafted plants (Figure 2A). Further analysis revealed a consistent and significant difference in chlorophyll b content between grafted and non-grafted plants across all developmental stages, with grafted plants maintaining, on average, 41% higher levels (Figure 2B). Moreover, under both salinity and alkalinity stresses, chlorophyll b content progressively declined over time, with alkalinity exerting a more pronounced adverse effect than salinity (Figure 2C).

Grafting, sampling time, and their two-way interactions (stress × time, grafting × stress, and grafting × time) all had significant effects (p < 0.01) on total chlorophyll content. Grafted melon plants retained higher total chlorophyll concentrations than non-grafted ones under stress conditions. Furthermore, alkalinity stress resulted in a greater reduction in chlorophyll content than salinity stress (Figure 3A). The data also demonstrated that the total chlorophyll content was highest at the first sampling time in both grafted and non-grafted plants. However, grafted plants consistently outperformed their non-grafted counterparts (Figure 3B). The highest value (3.62 mg/g fresh weight) was recorded in the first harvest under non-stress conditions, while the lowest (2.83 mg/g fresh weight) was observed in plants exposed to alkalinity stress during the fourth sampling (Figure 3C).

Analysis of variance for carotenoid content indicated that the main effects of stress, time, and their interactions (stress × time and stress × grafting) were insignificant at the 5% level. However, grafting alone and its interaction with time significantly affected carotenoid levels (p < 0.01). Grafted plants showed approximately 10% higher carotenoid concentrations than non-grafted ones (Figure 4A). Although stress exposure led to a reduction in carotenoids, the change was not statistically significant. Mean comparisons for the grafting × time interaction revealed that the highest carotenoid levels occurred in grafted plants at the first harvest. In contrast, the lowest were observed in non-grafted plants at the second harvest (Figure 4B).

3.2. SPAD Index

The SPAD readings, which indirectly reflect chlorophyll concentration, were significantly influenced by the individual and combined effects of grafting, stress, and sampling time. Stress treatments decreased the SPAD index in both grafted and non-grafted plants, with alkalinity stress having a more detrimental impact than salinity stress. Specifically, under salinity and alkalinity, the SPAD index dropped by approximately 14% and 21%, respectively, in grafted plants, and by 11% and 14%, respectively, in non-grafted ones compared to the control (Figure 5A). Grafted plants exhibited consistently higher SPAD values than non-grafted ones across all four sampling times (Figure 5B). The highest greenness index was recorded in the control treatment at the first sampling, while the lowest was seen under alkalinity stress at the final sampling time (Figure 5C).

3.3. Relative Water Content

Analysis of variance revealed that stress treatment, grafting, sampling time, the interaction of stress × time, the interaction of grafting × time, and the three-way interaction among these factors significantly influenced the relative water content (RWC) of the leaves. However, the interaction between grafting and stress alone did not significantly affect RWC at the 5% level. According to the results, plants exposed to stress exhibited lower RWC values than those under non-stress conditions. Moreover, grafted plants consistently maintained higher relative water content than non-grafted ones. The comparison of treatment means revealed that the highest RWC (82%) was recorded in grafted plants during the first harvest under non-saline and non-alkaline conditions. Interestingly, no statistically significant differences in RWC were observed among different harvest times in grafted plants. In contrast, the lowest RWC (68.5%) occurred in non-grafted plants under alkalinity stress. This treatment showed no significant difference from non-grafted plants exposed to salinity at the fourth harvest (Figure 6).

3.4. Proline Content

The variance analysis demonstrated that stress, time, grafting treatments, and the grafting × stress interaction had significant effects (p < 0.01) on proline accumulation in melon leaves. However, the grafting × time, stress × time, and three-way interactions were insignificant at the 5% level. Grafted plants accumulated about 18% more proline than non-grafted ones (Figure 7A). Proline content increased significantly under both stress treatments, with salinity and alkalinity raising levels by approximately 10% and 13%, respectively, compared to control conditions (Figure 7B). Moreover, early growth stages had lower proline concentrations than the third sampling time (Figure 7C). The grafting × stress interaction revealed that the highest proline accumulation occurred in grafted plants under alkalinity stress (Figure 7D).

3.5. Soluble Sugars

According to ANOVA, stress, time, and the interactions between grafting × time and stress × time significantly affected soluble sugar content. The stress × time interaction indicated that the highest sugar content was observed in stressed plants at the fourth time point, whereas the lowest occurred in control plants at the first sampling (Figure 8A). Similarly, the grafting × time interaction showed that grafted and non-grafted plants had higher soluble sugar levels at the fourth sampling time (Figure 8B).

3.6. Protein Content

The analysis of variance showed that stress, time, and grafting each had a significant effect (p < 0.01) on leaf soluble protein content. At the same time, their interactions were not statistically significant at the 5% level. Grafted plants exhibited around 8% higher soluble protein levels than non-grafted ones (Figure 9A). Stress treatments caused a marked increase in soluble protein content, with salinity and alkalinity leading to approximately 13% and 24% increases, respectively, compared to control plants (Figure 9B). Additionally, plants sampled at earlier growth stages had higher protein levels than those at the third and fourth stages (Figure 9C).

3.7. Plant Growth

According to the analysis of variance, the effects of stress, grafting, harvest time, and their interactions were all statistically significant at the 1% probability level. Mean comparisons revealed that grafted plants produced a higher number of leaves compared to non-grafted ones. Furthermore, the results showed that salinity and alkalinity stresses reduced leaf number in grafted plants by 13% and 36%, respectively, compared with the control. In non-grafted plants, the reductions were more severe at 39% under salinity and 48% under alkalinity stress, indicating a greater sensitivity to alkalinity. As illustrated in Figure 10, the highest leaf count was recorded in grafted plants at the fourth harvest under non-stress conditions, whereas the lowest count occurred at the first harvest (Figure 10).

The individual effects of grafting, stress, and harvest time as well as the interactions between stress and time, stress and grafting, grafting and time, and the combined effect of all three factors were significant at the 1% level. Mean comparisons showed that the largest leaf area was observed in grafted plants at the fourth harvest under non-stress conditions. The imposition of stress led to a marked decline in leaf area, with alkalinity stress causing a more pronounced reduction than salinity stress in grafted and non-grafted plants. Interestingly, the leaf area of grafted plants under stress at the fourth harvest was comparable to that of non-grafted plants grown under non-stress conditions (Figure 11).

The variance analysis results for root fresh weight indicated that grafting, stress treatments, sampling time, and all interactions among these factors had a statistically significant effect at the 1% level. A comparison of treatment means that salinity and alkalinity stress significantly reduced root fresh weight in grafted and non-grafted plants. However, grafted plants consistently exhibited greater root fresh mass than their non-grafted counterparts. Additionally, the data showed that as the plants matured, their responsiveness to stress intensified, with more apparent differences between stressed and non-stressed conditions. According to Figure 12A, the maximum root fresh weight (110.63 g) was recorded in grafted plants at the fourth harvest under non-stress conditions. The minimum value (2.5 g) occurred in non-grafted plants during the second harvest under salinity stress. It was also observed that the ‘Til-e-Sabz’ cultivar displayed a higher sensitivity to alkalinity stress than to salinity regarding root fresh weight. Analysis of variance for root dry weight, confirmed that the main effects of grafting, stress, and time, along with their interactions, including the three-way interaction, were all significant at the 1% level. Overall, non-grafted plants exhibited lower root dry weights compared to grafted ones. The application of stress resulted in a notable reduction in root dry weight for both plant types, particularly during the third and fourth harvests. The highest dry root mass was recorded in grafted plants at the fourth sampling time under non-stress conditions, while the lowest was observed in non-grafted plants at the first harvest under alkalinity stress. Interestingly, during the first and second harvests, differences in root dry weight between treatments were not statistically significant (Figure 12B).

Based on the results of the variance analysis for shoot fresh weight, the three-way interaction of grafting, stress, and harvest time was significant at the 1% level. Grafted plants produced greater fresh shoot biomass than non-grafted ones across treatments. Stress application reduced shoot fresh weight in both grafted and non-grafted plants, with the reduction being especially noticeable at the third and fourth harvests. The highest value was recorded in grafted plants at the fourth harvest under non-stress conditions, while the lowest was found in non-grafted plants at the first harvest subjected to alkalinity stress. No statistically significant differences were observed between treatments during the first and second sampling times (Figure 13A). Similarly, grafting, stress, harvest time, and interactions significantly affected shoot dry weight. Stress treatments substantially reduced dry biomass in the shoot.

Grafted plants, when grown without stress and harvested at the fourth time, exhibited the highest shoot dry weight. Conversely, the lowest dry weight was found in non-grafted plants subjected to alkalinity at the second harvest. No meaningful differences were detected between treatments during the earlier sampling times (Figure 13B).

3.8. Nutrient Elements

Grafting, stress, and the interaction between these two factors significantly affected phosphorus content in both roots and shoots. Both salinity and alkalinity significantly decreased phosphorus concentrations in melon plants. Root phosphorus declined by 13% under salinity and 24% under alkalinity, while shoot phosphorus decreased by 13% and 29%, respectively, compared to the control. Grafted plants showed a clear advantage, retaining more phosphorus in roots and shoots under stress conditions. The highest root (64%) and shoot (60%) phosphorus levels were recorded in grafted plants under non-stress conditions. In comparison, the lowest values—43% in roots and 35% in shoots—were observed in non-grafted plants exposed to alkalinity stress (Figure 14A,B).

Analysis of variance revealed that grafting, stress treatments, and their interaction had a significant effect on potassium content in melon roots and shoots. Treatment means comparisons showed that the highest root potassium level was recorded in non-grafted plants under non-stress conditions. At the same time, the lowest value occurred in non-grafted plants exposed to salinity (Figure 14C,D). Both salinity and alkalinity stress reduced potassium concentrations in roots and shoots, with salinity exerting a more severe impact than alkalinity (Figure 14C,D).

Based on the ANOVA results, root and shoot iron content were significantly influenced (p < 0.01) by grafting, stress, and their interaction. Mean comparisons showed that iron levels declined under salinity and alkalinity in grafted and non-grafted plants. Specifically, the root iron content decreased by approximately 35% and 49% under salinity and alkalinity, respectively, compared to the control, while the shoot iron content dropped by around 35% and 50% under the same treatments. As shown in Figure 15A,B, the highest iron concentrations in both roots and shoots were observed in grafted plants under control conditions. In contrast, the lowest values were recorded in non-grafted plants subjected to alkalinity stress.

Grafting, stress treatment, and their interaction had a statistically significant effect on sodium accumulation in both roots and shoots. The mean comparison revealed that salinity and alkalinity stress significantly increased sodium concentrations in melon plants. Specifically, sodium content in the roots increased by 140% and 94% under salinity and alkalinity stress, respectively, compared to the control. Sodium levels rose by 86% and 106% in the shoots under the same respective stress conditions. Furthermore, grafted plants accumulated less sodium in roots and shoots under stress conditions than non-grafted ones. The lowest sodium levels were observed in grafted plants under control conditions, while the highest levels occurred in non-grafted plants exposed to salinity stress (Figure 15C,D).

The effects of grafting, stress, and their interaction were all significant at the 1% probability level. Mean comparisons revealed that chloride accumulation increased in both grafted and non-grafted plants under stress from salinity and alkalinity. Root chloride content increased by approximately 10% under salinity and 25% under alkalinity, relative to the control. Shoot chloride levels showed more pronounced increases, with about 94% under salinity and 53% under alkalinity stress. Figure 15E,F illustrate that the lowest chloride concentrations in roots and shoots were observed in grafted plants under non-stress conditions. At the same time, the highest levels were recorded in non-grafted plants subjected to salinity stress.

4. Discussion

Photosynthesis is a vital biochemical process for plant food production and growth [64]. Chlorophyll content indicates plant health [65], while carotenoids, isoprenoid molecules in photosynthetic and non-photosynthetic tissues, act as accessory pigments for light harvesting, energy transfer to chlorophyll, and antioxidant protection by scavenging oxygen radicals and dissipating excess energy via the xanthophyll cycle [66,67,68]. Classified as hydrocarbons like lycopene and beta-carotene or xanthophylls, carotenoids protect chlorophyll from photo-oxidation and transfer short-wavelength energy [67,68]. Under salt stress, melon exhibited reduced chlorophyll a and b, total chlorophyll, and carotenoids compared to controls due to chloroplast and thylakoid degradation [69,70], decreased pigment synthesis enzymes [71], increased chlorophyllase activity [72], inhibited chlorophyll biosynthesis from proline competition (sharing glutamate precursor) [73], magnesium and potassium deficiencies with a lowered K/Na ratio [74], and oxidative stress causing lipid peroxidation and chlorophyll breakdown [65]. Similar declines occurred under sodium bicarbonate in melon [25,34,54,55], watermelon [26,33], cantaloupe landrace [34], cucumber [9,35], tomato [75], Hyssopus officinalis [76], grapevine [77], tomato and pepper seedlings [76,78], gerbera [79], and strawberry [23]. Reduced chlorophyll weakens growth and stress resistance [80]. Bicarbonate-induced chlorophyll loss stems from chlorophyllase degradation [81], magnesium precipitation at high pH [82], sodium imbalance [83], and iron deficiency [84]. Carotenoid reduction under stress involves beta-carotene degradation and zeaxanthin formation [85,86], with increased protoporphyrinogen IX deaminase synthesis aiding tetrapyrrole precursors for chlorophyll and porphyrin [87,88].

Leaf relative water content (RWC) reliably measures plant tissue water status and is superior to cellular water potential due to its link to cell volume and balance between water content and transpiration [89,90]. Water use efficiency represents dry matter per water unit consumed [91]. Salt stress in melon decreased RWC and efficiency (Figure 10), likely from reduced root growth, stomatal closure, impaired root water absorption/transport, and Na/Cl accumulation [92,93]. Bicarbonate stress causes nutritional disturbances [77,94], root growth reduction, and hindered development from decreased water uptake [95]. In hydroponics, high sodium bicarbonate induces iron deficiency chlorosis despite ample water [96]. Yang et al. [97] noted greater water content decline under alkaline than salt stress. RWC decreases were reported in melon under salinity and alkalinity [13,22,26,34,54,55]. Minimal RWC reduction in resistant genotypes indicates better osmotic adjustment and stress resistance [79].

Plants resist stresses by accumulating osmolytes like proline and soluble carbohydrates to lower osmotic potential for water uptake under deficits [98,99]. These non-disruptive metabolites include sugars (sucrose, fructose), alcohol sugars (glycerol, methylated inositol), complex sugars (trehalose, raffinose, fructans), ions (potassium), charged metabolites (glycine betaine, dimethyl sulfonium propionate), and amino acids (proline) [62,100]. Osmolytes stabilize enzymes against ions, water stress, and denaturants [101], with proline mitigating ion effects, enhancing thermal stability [102], acting as a compatible solute for osmotic adjustment [103,104,105], storing carbon/nitrogen, and protecting from radicals [106]. Salt stress (Na+) prompts proline accumulation in cytosol for toxicity mitigation, transport to vacuoles, and balance [104]; glutamine synthetase activation boosts proline [107,108]. Alkaline stress inhibits root growth, lowers leaf water potential, and increases proline [108,109]. High alkalinity reduces soluble sugars from ion toxicity, damaging metabolism [82]. Sugars, photosynthetic products, and fuel physiological processes [110], influenced by elements like K, Fe, Zn, Cu, Mn in carbohydrate metabolism [15], are disrupted by bicarbonate uptake interference [33]. Grafted cantaloupe landrace showed higher proline as a stress barrier [34].

Salinity and alkalinity reduced melon growth parameters (stem length, leaf number, area) in grafted and non-grafted plants, but pumpkin rootstock grafting improved them under stress. Salinity’s high osmotic potential hinders root water absorption [111], resulting in reduced uptake and growth [112], temporary leaf water loss [113], decreased cell division/elongation for smaller leaves [114], altered photosynthate transport, reduced aerial growth, stomatal closure, photosynthesis inhibition, ion imbalances [115], shortened stem/root [116], and lowered weight/dry matter [117]. Salinity challenges arise from osmotic reduction and Na toxicity [118]. Alkalinity from carbonate/bicarbonate raises pH, insolubilizing micronutrients (Fe, Zn) [115] and limiting growth via nutritional disorders and bicarbonate toxicity [119,120]; effects stem from pH-induced element insolubility [83]. Bicarbonate reduces shoot growth, leaf number, weights, and stem length [120], linked to chlorosis-lowered photosynthesis [76] from degraded chlorophyll synthesis via reduced Fe transport/solubility. Li et al. [121] noted reduced Coix lacryma-jobi growth (stem weight, leaf area, number, biomass). Stress limits leaf area by preventing maximum cell growth [85], decreasing photosynthesis [122] and photosynthate availability [123]. Bicarbonate-reduced leaf area was reported in cucumber [35], strawberry [23], melon and kiwifruit [12,18], maize, and Coix lacryma-jobi [121]. Bicarbonate harms plasma membranes, reducing root growth [124]. Sodium bicarbonate lowered lettuce leaf area, number, weights, length/width, photosynthesis, and conductance [18]; controls had higher aerial nutrients, and alkalinity increased root accumulation, severely affecting aerial growth [125]. Growth declines under stress in melon [49], cucumber [9,35], and tomato [75]. Resistant rootstocks enhance growth under stress [69], as in grafted cucumber under cadmium [126], copper, and high calcium nitrate; Citrullus lanatus on Cucurbita/Lagenaria [127]; watermelon on pumpkin [128]; cucumber on pumpkin under salinity [129]; melon on pumpkin under stress [130]; and cucumber on pumpkin with higher weights [131], due to strong roots controlling Na [130]. Grafted cucumber on pumpkin showed better alkaline growth from improved element transport via a higher root-to-shoot ratio/surface [33,132].

Salinity–nutrient relations in crops are complex [127,133], inducing disorders and reducing yields [57,134] via availability, absorption competition, transport, and distribution [111]. High leaf Na causes osmotic/metabolic issues, toxicity, and dry matter reduction [135]. Na interferes with K [136], similarly to hydrated radii complicating distinction [137]. Na maintains turgor but cannot replace K for photosynthesis, protein synthesis, and enzymes [138]. Alkalinity inhibits root activity, bicarbonate metabolism, and nutrient uptake [139]. Na is non-essential but aids osmosis/water uptake and is toxic at high levels [138,140]. Salinity increased melon Na in aerial parts/roots and grafting affected levels; high environmental Na replaces K [141] and competes with reducing essential ions [142].

Bicarbonate increased maize Na in roots/shoots and was higher in roots [143]; no leaf Na effect was observed in roses [144]. Lower grafted Na resulted from exclusion [24]; grafted cucumber had lower Na under salinity [33]; grafted melon/Cucurbita had lower Na/Cl [130]; and Luffa rootstock reduced cucumber Na transport [145]. K maintains root osmotic potential for water/mineral transport under turgor [138,146]. Salinity reduced root K; high Na inhibited uptake due to similarity [147], competition on transporters, or membrane instability leakage [148]. Higher aerial K indicates selectivity for tolerance [147].

Grafted cantaloupe improved nutrient uptake under stress [34]. Cl’s roles beyond photosynthesis light reactions are unclear; it is absorbed via H+/Cl− symporters/channels [113], accumulates under salinity, and is root stable [149]. Salinity increased melon root/shoot Cl; pistachio sensitive cultivars had higher Na/Cl [150]. Beans increased Cl under salinity, and silicon reduced it by minimizing NO₃/Cl competition [151]. K aids N metabolism [2], counters NO₃, and stores in vacuoles; supplements promote NO₃ and reduce Cl under salinity [138,152].

Bicarbonate responses vary [153]; raised vascular pH precipitates/inactivates P, Fe, Zn and disrupts growth/yield via interactions [109]. Bicarbonate reduced melon P in leaves/roots [101]; optimal P was observed at pH 5.5–7 [154], and higher pH forms low-solubility compounds with Ca/Mg [155]. Hydroponics favor acidic pH to prevent precipitation of Fe²⁺, Mn²⁺, PO₄³⁻, Ca²⁺, and Mg²⁺ above pH 7 [58].

Salinity reduces root growth/P absorption; H₂PO₄/Cl compete, and high Cl lowers P [156]. Grafted watermelon on squash reduced alkalinity effects, increasing P/Fe solubility [33]; and grafted cucumber had higher P under salinity [43]. Fe in oxidation–reduction [157] binds heme/non-heme proteins, preventing damage [158]; cytochromes (porphyrin-Fe) in chloroplast/mitochondrial chains and nitrate reductase [159,160] are essential for growth [17,35]; soil Fe is sufficient, but bioavailability is limited by factors/genotypes [161].

Bicarbonate reduced leaf Fe [162]; 10 mM decreased leaf Fe Vs. 0 mM and reduced pepper tissue Fe [162]. No root Fe effect was observed in some tomatoes/tobacco, and it decreased in greenhouse tomatoes [163]. Grafted/non-grafted watermelon Fe reduced with bicarbonate, more so in non-grafted; roots were higher than shoots [33].

5. Conclusions

Salinity and alkalinity significantly reduced key growth, physiological, and biochemical traits in cantaloupe, including leaf area, plant biomass, relative water content, and photosynthetic pigments. Grafting onto Cucurbita rootstock substantially improved stress tolerance, leading to higher growth, greater osmolyte accumulation, and enhanced water status under both salt and bicarbonate stress. Grafted plants exhibited more substantial nutrient uptake, with enhanced acquisition of potassium, phosphorus, and iron, and lower sodium accumulation, compared to non-grafted controls. Superior physiological performance in grafted plants resulted in improved yield components, including increased fruit number and size, even in degraded environments. These results highlight grafting as a practical and sustainable strategy to enhance cantaloupe resilience, productivity, and fruit quality when exposed to saline and alkaline stress in soilless cultivation systems.