*3.3. Photosynthetic Pigments*

Intrinsic difference in photosynthetic pigments profile and content were observed between the genotypes (Table 3). Among non-salinized seedlings, chlorophyll (*a*, *b*, and total) and carotenoids content was higher in melon (CM11 and CM12), while the lowest concentration was observed in CM5. Compared to non-saline seedlings, chlorophyll *a* content significantly increased under salt stress in CMM-R1 and CMM-R2 (+47% and +61%), while it decreased in CMO 51–17 (−40%) and LC1 (−49%) and it did not vary in the other genotypes (Figure 3a). On the other hand, chlorophyll *b* content increased in CMM-R1 (+38%) and CMM-R2 (+64%) seedlings grown under saline conditions, while it decreased in LC1 (−51%), and was not affected by salinity in any of the other genotypes (Figure 3b). As compared to non-saline conditions, the total chlorophyll and carotenoid content increased under 150 mM NaCl in CMM-R1 (+44% and +52%, respectively), CMM-R2 (+62% and +54%), while they decreased in LC1 (−50% and −28%) and it did not vary for the other genotypes (Figure 3c,d).

**Figure 2.** Plant biomass accumulation, after one week of NaCl treatment, in term of shoot dry weight (**a**), root dry weight (**b**), shoot dry matter content (**c**), and root dry matter content (**d**) in seedlings of sixteen *Cucumis melo* L., six *Citrullus vulgaris* Schrad., two *C. maxima* × *C. moschata*, four *Lagenaria siceraria* (Molina) Standl. different genotypes, and *Cucurbita moschata* Duch. *cv* 51–17 and *Lu*ff*a cylindrica* Mill. Mean values ± standard errors; *n* = 3 followed by different letters within each parameter are significantly different based on Duncan post hoc (*p* < 0.05).

**Table 3.** Chlorophyll *a*, *b*, total carotenoids content, and electrolyte leakage in leaves of seedlings of sixteen *Cucumis melo* L., six *Citrullus vulgaris* Schrad, two *C. maxima* × *C. moschata*, four *Lagenaria siceraria* (Molina) Standl. different genotypes, and *Cucurbita moschata* Duch. *cv* 51–17 and *Lu*ff*a cylindrica* Mill. grown at 0 or 150 mM of NaCl. (1 week after the beginning of the treatment). Non significance or significance differences at *p* ≤ 0.05 or 0.01 are indicated as: ns and \*\* respectively.


**Figure 3.** Photosynthetic pigment content, after one week of NaCl treatment, in term of chlorophyll *a* (**a**), chlorophyll *b* (**b**), total chlorophyll (**c**), and carotenoids (*x* + *n*) (**d**) in seedlings of sixteen *Cucumis melo* L., six *Citrullus vulgaris* Schrad., two *C. maxima* × *C. moschata*, four *Lagenaria siceraria* (Molina) Standl. different genotypes, and *Cucurbita moschata* Duch. *cv* 51–17 and *Lu*ff*a cylindrica* Mill. Mean values ± standard errors; *n* = 3 followed by different letters within each parameter are significantly different based on Duncan post hoc (*p* < 0.05).

#### *3.4. Electrolyte Leakage*

Electrolyte leakage was significantly different between the genotypes (Table 3). Under 0 mM NaCl, CMM-R2, LS1, and CM2 showed the highest value of electrolyte leakage, while the lowest electrolyte leakage values were observed in CV4, LC1, LS4, and CM10 (Figure 4). Electrolyte leakage values were generally increased by salinity, although significant differences from control conditions were only observed in LS2 (+184%), CV13 (+243%), CM10 (+4662%), and CM13 (+509%) (Figure 4).

**Figure 4.** Electrolyte leakage (EL) in leaves, after one week of NaCl treatment, in seedlings of sixteen *Cucumis melo* L., six *Citrullus vulgaris* Schrad., two *C. maxima* × *C. moschata*, four *Lagenaria siceraria* (Molina) Standl. different genotypes, and *Cucurbita moschata* Duch. *cv* 51–17 and *Lu*ff*a cylindrica* Mill. Mean values ± standard errors; *n* = 3 followed by different letters within each parameter are significantly different based on Duncan post hoc (*p* < 0.05).

#### *3.5. Cluster Heat Map and Principal Component Analysis*

To obtain a detailed overview and to better distinguish the morpho-physiological changes induced by salinity on the tested genotypes, a cluster heat map and a principal component analysis (PCA) were conducted considering the percentage variation between 150 and 0 mM NaCl for all the aforementioned measured parameters.

The cluster heatmap shows that the main clustering factor was the genotypes regardless of the species, highlighting common response traits to the increase of NaCl between the different genotypes (Figure 5). The genotypes formed two main clusters. The first was occupied by the watermelon genotype CV2 that clustered alone, mainly as a consequence of an increase in leaf number, root length, shoot diameter, and shoot and root dry weight, together with a decrease in electrolyte leakage and photosynthetic pigment content (Figure 5). The second main cluster is split into two sub-groups. In order of distance, the first sub-group arranges bottle gourd LS2, LS3, and LS4 with melon CM8 and the interspecific hybrids CMM-R1 and CMM-R2, together into several further sub-sets characterized by similar positive variations in photosynthetic pigment content and negative variation in electrolyte leakage and root dry matter content (Figure 5). The second sub-group contains most of the genotypes and is organized into two further subsets, one of which has two divisions. The first subset is determined by similar variation in shoot and root dry matter content and electrolyte leakage grouping melon (CM1, CM6, and CM7, CM15 and CM16) and watermelon CV3 and bottle gourd LS1 genotypes together (Figure 5). Furthermore, the CM1 genotype showed the highest positive variation in shoot diameter, while CM6 and CM7 showed the highest increase in dry root weight (Figure 5). The decrease observed under saline conditions in photosynthetic pigments, root length, and dry weight, together with shoot length and diameter and leaf number accounted for the formation of the second subset. This is further subdivided into two divisions, the first of which contains watermelon (CV1, CV4, and CV5), melon (CM4, CM5, CM11, CM12, and CM13), and luffa (LC1) genotypes. Among these genotypes, LC1, CM4, and CV5 showed the highest decrease in photosynthetic pigment contents, shoot diameter and root length, and dry weight. A decrease in root dry weight was also observed in CM11, CM13, and watermelon CV1 (Figure 5). The second and last subdivision is explained mainly by a decrease in shoot and root dry matter and places, together melon (CM2, CM3, CM9, and CM10), watermelon CV6, and *C. moscata* CMO 51–17. Among the genotypes, melon (CM2, CM3, and CM10) showed the highest decrease in shoot dry matter, while melon CM3, the highest increase in electrolyte leakage and CV6 also showed the highest increase in shoot length (Figure 5).

**Figure 5.** Cluster heat map analysis of the percentage variation to the increase of NaCl level of all the analyzed parameters in seedlings of sixteen *Cucumis melo* L, six *Citrullus vulgaris* Schrad., two *C. maxima* × *C. moschata*, four *Lagenaria siceraria* (Molina) Standl. different genotypes, and *Cucurbita moschata* Duch. *cv* 51–17 and *Lu*ff*a cylindrica* Mill. grown in a floating system in a nursery greenhouse. It was generated using the ClustVis online software [18] with Euclidean distance as the similarity measure and hierarchical clustering with complete linkage.

The PCA of the relative percentage variation of all the analyzed parameters in seedlings grown at 150 mM NaCl as compared with the control (0 mM NaCl) highlighted that the first three principal components (PCs) were related with eigenvalues higher than 1 and explained 64.5% of the total variance, with PC1, PC2, and PC3 accounting for 32.6%, 20.1%, and 11.9% respectively (data not shown). Both the species and the genotype contributed to the separation of PC1 and PC2, as highlighted in the PCA output, revealing common trait variations among genotypes regardless of the species (Figure 6). The melon genotypes CM3, CM4, CM5, CM11, CM13, and CM14, together with the watermelon genotypes CV1, CV3, and CV4, were concentrated in the upper right quadrant of the PCA output and positively correlated to an increase in electrolyte leakage (Figure 6). The watermelon genotypes CV2 and CV5, together with CMO 51–17, luffa LC1, bottle gourd genotype LS1, and melon genotypes CM2, CM10, and CM15 were concentrated in the negative lower right quadrant of PCA output and were correlated to an increase under saline conditions of shoot length and root dry matter content (Figure 6). The interspecific hybrid CMM-R1, together with melon CM8, CM9, CM12, and bottle gourd LS2 and LS3 were concentrated in the upper left quadrant of the PCA output (Figure 6) and together with the interspecific hybrid CMM-R2 (located in the lower left quadrant), were characterized with an increase in photosynthetic pigment content under saline conditions. In the left lower quadrant depicted genotypes CM1, CM6, CM7, and CM16, together with bottle gourd LS4 and watermelon CV6 and were characterized with increase in leaf number, shoot diameter, shoot dry weight, and dry biomass accumulation and root length under saline conditions (Figure 6).

**Figure 6.** Principal component (PC) loading plot and scores of principal component analysis of the percentage of variation between 150 mM and 0 mM of NaCl of all the parameter analyzed in seedlings of sixteen *Cucumis melo* L. (CM), six *Citrullus vulgaris* Schrad (CV), two *C. maxima* × *C. moschata* (CMM-R), four *Lagenaria siceraria* (Molina) Standl. (LS) different genotypes, and *Cucurbita moschata* Duch. *cv* 51–17 and *Lu*ff*a cylindrica* Mill. grown in a floating system in a nursery greenhouse.

#### **4. Discussion**

It is well known that plants' response to different NaCl concentrations differs between species and even within genotypes of the same species. In this study, high variability in various morphological and physiological traits between the tested genotypes even from the same species were observed. Furthermore, different responses to NaCl were identified, confirming the high variability within the assessed germplasm collection.

According to Munns [19], a saline environment in the root zone triggers the activation of various mechanisms in plant physiology, morphology, and metabolism. This takes place in two main phases. Firstly, the presence of NaCl in the soil creates osmotic stress in the roots due to different solute concentration, causing the activation of multiple metabolic pathways that in the short term lead to stomata closure to reduce evapotranspiration and preserve water, and net photosynthesis reduction [1,20,21]. Morphological change primarily includes the growth inhibition of aerial organs like shoots and a reduction in the number of leaves. In the current experiment, 150 mM of NaCl significantly reduced the number of leaves only in melon (CM5), though a decreasing trend was observed in most genotypes. Shoot growth was significantly inhibited by salinity in most genotypes with the exception of watermelon (CV3, CV5, CV6) and luffa (LC1), which turned out to be more salt tolerant for this trait. The root system serves as an interphase between plant and soil, and its anatomy determines root performance [21]. Generally, root growth in a saline environment is inhibited less than the aerial organs [22]. In our study, root growth was not significantly affected by salt stress as the main factor, however, our data highlight a genotypic-specific response to salt concentration since root growth was promoted in salt-treated seedlings of CMM-R1, CMM-R2, and watermelon (CV3) and decreased in watermelon (CV6) and melon (CM14).

Salinity stress leads to a decrease in plant biomass, mainly in the epigean organs and this is ascribed to a decrease in CO2 fixation by photosynthesis due a reduction in stomatal conductance, together with nutrient disorders caused by the toxic action of Na<sup>+</sup> and Cl<sup>−</sup> ions inhibiting Ca2<sup>+</sup> and K<sup>+</sup> uptake [1,21–23].Under conditions of 150 mM NaCl, shoot dry weight was negatively affected only in melon (CM2, CM5, CM7, CM8, CM12, and CM13), *C. moscata* (CMO 51–17), watermelon (CV1 and CV2), and in the interspecific hybrid CMM-R1, the latter was the most salt sensitive compared with the other genotypes. In particular, watermelon (CV3), the interspecific hybrid CMM-R2, and bottle gourd (LS1) significantly increased shoot dry weight in response to salinity. On the other hand, shoot dry matter accumulation increased in saline conditions in bottle gourd (LS1, LS3, LS4) and in watermelon (CV2, CV3, and CV5) and melon (CM1) and both the interspecific hybrids CMM-R1 and CMM-R2. Many salinity tolerance traits in grafted plants are associated with the root system. A salt tolerant rootstock genotype alleviates the deleterious shoot growth inhibition, allocating more biomass to the root system, increasing the root surface able to uptake water and nutrients, resulting in a higher growth rate and biomass accumulation [24,25]. Accordingly, with 150 mM NaCl, root dry weight did not decrease in any of the genotypes except for watermelon (CV5), and root dry matter accumulation was significantly enhanced in melon (CM1, CM4, CM5, CM6, CM7, CM8, CM15, and CM16) as well as watermelon (CV1, CV2, CV3, CV4, and CV5), while it decreased in melon (CM3, CM10, and CM11) and CMM-R1 and CMM-R2. Several studies have reported the growth depressing effect of salt on melon [26–28], cucumber [3,29], mini-watermelon [30], tomato [31], and lettuce [32] grown hydroponically under greenhouse conditions. The second phase of the salt stress starts with the accumulation of sodium and chloride ions in the leaves and their degrading action on cell membranes and chloroplast membranes. This degrades the tonoplast together with chlorophyll molecules because of chlorophyllase enzyme [33] as well as the interference of salt ions with pigment-protein complexes which accelerate leaf senescence and abscission [34]. Contradictory information is available in literature on salt stress related to chlorophyll contents among the same species and to others. In fact, studies on melon *cv.* Parnon and *cv.* "Tempo F1", under saline conditions, reported a decrease in chlorophyll content [35,36] in line with studies on pumpkin [37], cucumber [38], tomato [31], canola and wheat [39,40]. Conversely, other

authors observed an increase of photosynthetic pigments in different melon genotypes [41–43] in line with studies on other species like hot pepper [44], sunflower [45], and sesame [46].

Sanoubar and collaborators in 2016 [47] indicated that chlorophyll concentration in stressed tissues can be construed as an index of tissue tolerance to NaCl. Accordingly, our results indicate a good tolerance in most of the genotypes, in particular in the interspecific hybrids CMM-R1 and CMM-R2, where photosynthetic pigments significantly increased under saline condition. Contrarily, in luffa, the decreasing chlorophyll content in response to salinity could be associated with greater sensitivity to salt stress [21]. Adaptation mechanisms induced by salt stress generally influence the light harvesting complex due to a faster decrease of chlorophyll *a* content as compared to chlorophyll *b*, which leads to an increase in the chlorophyll *a*/*b* ratio [34]. In our experiment, the chlorophyll *a*/*b* ratio was modified under saline condition only in CMO 51–17 and watermelon (CV1) where chlorophyll *a* decreased and chlorophyll *b* did not vary. Different hypotheses have been formulated to explain the increase in photosynthetic pigments in response to salinity. The first considers the morphological modifications associated with the decrease of leaf area due the smaller cell size, resulting in an enhancement of photosynthetic pigments concentration [45,46]. A second hypothesis, proposed by Garcia-Valenzuela and collaborators [48], associated the increase of photosynthetic pigments to the faster response that is generally observed in the biosynthesis of pigments as compared to the cell growth rate [48]. To date, however, most the credited hypothesis relates the increase observed in photosynthetic pigments to a short term acclimatization response to salinity and that a prolonged exposure would in any case be detrimental [49]. The ability to isolate or exclude sodium and chloride is a key factor to consider as a salt tolerance trait [27,28]. The longer the salt stress is prolonged, the higher the accumulation of sodium and chloride inside the plant is. To cope with this osmotic disorder, plants exclude uptake of these compounds primarily through root exclusion, and subsequently through compartmentation, by isolating salt in vacuoles. However, the higher the ability to isolate (salt) in the vacuole is, the less the cell membrane is damaged. Moreover, if the concentration increases inside the cytosol, plants start to synthesize different organic solutes to maintain the osmotic turgor and to reduce the deleterious effect of salt due to cell membrane degradation and the increase of reactive oxygen species (ROS) [19,21,50]. In our experiment, electrolyte leakage increased dramatically only in LS2, CV4, CM10, and CM13, revealing a high index of internal damage due to the presence of sodium and chloride inside the leaf cells of these genotypes which must be considered a sensitive response to salt stress.

#### **5. Conclusions**

Salinity stress leads to a decrease in plant growth with more pronounced deleterious effect in the shoot rather than the roots. Similar physiological and morphological salt adaptation traits were observed in different genotypes from different species. In light of the above consideration, we identified *C. maxima* × *C. moscata* interspecific hybrid CMM-R2, melon genotypes (CM6, CM7, CM10, and CM16), together with watermelon (CV2 and CV6) and bottle gourd (LS4) as salt tolerant genotypes and possible candidates as salt resistant rootstock to be introduced in grafting programs. Contrarily, luffa and melon (CM1, CM2, CM3, CM4, CM5 CM8, CM9, CM11, CM12, CM13, CM14, and CM15), watermelon (CV1, CV3, CV4, and CV5), together with CMO 51–17 and bottle gourd LS1 proved salt sensitive. In conclusion, this study provides information to growers, scientists, extension specialists, and breeders on the behavior of the tested genotypes. Further research on these genotypes is needed to clarify their performance in saline environments in the long term and their compatibility with commercial grafted varieties.

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

**Funding:** This work was carried out during an internship mobility agreement between the University of Bologna and the Ege University financed by the LLP Erasmus Placement Program (2014) and supported by Ege University Scientific Research Projects Coordination Unit with project number 2013-ZRF-001.

**Acknowledgments:** The authors are thankful to Sofia Siepi, Elyar Saeedi and Gamze Çılgın for their help collecting data and technical assistance during the experiment.

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