*3.3. Stomatal Conductance (gs), Photosynthesis (Pn), and Transpiration (Tr)*

Data in Table 2 show that irrigation quantity significantly (*p* < 0.001) affected the values of gs at all growth stages. At 83 DAS, PRD70 and PRD50 reduced the value of gs by 10% and 37% compared with FI, respectively. This is due to plant age, which reduces its activity. FI showed a higher Pn rate compared with the PRD treatments. At 63 DAS, Pn values under the FI plot were 10.685 μmol m−<sup>2</sup> s−1. This is a 7% and 16% increase compared with PRD70, and PRD50, respectively. Tr was significantly affected (*p* < 0.001) by irrigation quantity at all measured days. The lowest Tr values were observed under PRD50 treatments at 63 DAS.

However, FI treatments showed the highest Tr (4.046 mmol m−<sup>2</sup> s<sup>−</sup>1) at 35 DAS, which was not statistically different from PRD70. This finding indicates that the water deficit in PRD70 did not affect transpiration efficiency. At 83 DAS, PRD50 and PRD70 reduced the Tr values by 20% and 17.6%, respectively, compared with the FI treatments. In this study, the irrigation treatment significantly affected the gs, Pn, and Tr values, indicating that both Pn and Tr are controlled by gs, and they mutually affect each other [48,49]. Liu et al. [50] stated that under water-stressed conditions, gs decreases due to the closure of stomata to maintain leaf water status. However, there are opposing reports on the mechanism behind stomatal closure [51]. Although some studies suggest that chemical signals, such as abscisic acid (ABA) and pH are behind stomatal closure [50,52], others endorse that hydraulic signals, such as soil, root, and shoot resistances, are responsible for stomatal closure [53]. Many questions still arise related to the mechanism behind stomatal closure, even though many studies have been conducted [51]. Farooq et al. [54] stated that stomatal closure reduces the amount of carbon dioxide going into the parenchyma cells, which causes inhabitation of CO2 and light that ultimately affects plant photosynthesis efficiency.

**Figure 5.** Soil moisture for full irrigation (FI) and deficit partial rootzone drying under 70% and 50% of evapotranspiration (PRD70 and PRD50, respectively) at the left (L) and right (R) rootzone sides combined with: (**a**) Transparent mulch (WM) during the winter season (WS), (**b**) WM during the spring season (SS), (**c**) black mulch (BM) during the WS, (**d**) BM during the SS, (**e**) no mulch (NM) during the WS, and (**f**) NM during SS. FC, field capacity, and WP, wilting point.


**Table 2.** Analysis of variance of stomatal conductance (gs), photosynthesis (Pn), and transpiration (Tr) at the development stage (35 DAS), mid stage (63 DAS), and late stage (83 DAS) of squash growth during the winter (WS) and spring (SS) growing seasons.

WS: winter season; SS: spring season; WM: transparent mulch; BM: black mulch; NM: no mulch FI: full irrigation; PRD70 and PRD50: deficit partial rootzone drying under 70 and 50 of evapotranspiration, respectively; S, M and I: season, mulch and irrigation treatments, respectively; S × <sup>M</sup> × I: interaction between season, mulch and irrigation treatments; ns: not statistically significant; \*\*: significant at the 1% level (*p* < 0.01); \*: significant at the 5% level (*p* < 0.05); different letters indicate significant difference between treatments; bold letters and words indicate treatments names.

> Our results indicated that Pn decreased with a decrease in gs at the same stage of plant growth, but in different growth stages, a decrease in gs did not cause a decrease in Pn. For instance, at the mid stage (63 DAS), the Pn values increased despite gs reduction in both the mulch and irrigation treatments for the two growing seasons (Table 2). This could be explained by the squash leaves having reached their maximum area at this stage, when plants reach their peak values of most photosynthetic parameters [55]. The leaf gs, Pn, and Tr in the PRD treatments were significantly lower than that of FI at all measured days. In the PRD treatment, two sides of the root were alternately irrigated. The side of the root that undergoes a water deficit for a period induces ABA, which reduces gs, affecting both transpiration and photosynthesis efficiencies. However, the watered side of the root keeps the plant in a preferable situation [13]. In the current study, due to water stress under PRD treatment, plants induced ABA from the root to the leaves, resulting in the accumulation of ABA in the leaves causing stomatal closure [50,52]. Several studies showed that plants under PRD could enhance leaf Tr [56] and improve the Pn rate [57] compared to FI. These results are in agreement with other studies [5,58], which indicated that gs decreases with increasing water stress levels.

> The gs, Pn, and Tr were significantly affected by mulching treatments. However, at 35 DAS, gs showed no significant difference (*p* > 0.05) between mulch and non-mulched treatments. At 63 DAS, compared with NM, WM and BM increased gs by 20% and 10%, respectively, indicating that plants under mulched treatments were healthier at the mid growth stage. At 83 DAS, the NM treatment reduced the Pn value by 11% and 12.5% compared with the BM and WM treatments, respectively (Table 2). At 35 DAS, the non-mulched treatments reduced the Tr by 19% and 22% compared with the BM and WM treatments, respectively. Our results agree with the findings described by Ibarra-Jiménez et al. [59] and Lira-Saldivar et al. [36], who found that plastic mulch significantly increased photosynthetic activity in zucchini plants compared with non-mulched treatments. This finding is due to the advantage of plastic mulch, which can control soil temperature, enhance soil moisture,

and elevate crop photosynthesis [60]. Yang et al. [61] and Zhang et al. [62] emphasized that soil hydrothermal state is an essential element in photosynthesis. The proper soil moisture and temperature situation under mulched treatments boost the movement of water from the deep soil to the surface soil by capillary and steam action, increasing the intercellular CO2 concentration in the ear-leaf [62]. These activities help increase carbon sources for leaf photosynthesis, thereby decreasing the limitations of stomatal factors [63] and leading to consistently higher Pn in mulched than non-mulched treatments.

Data in Table 2 indicate that gs, Pn, and Tr were significantly affected by growing season for all measured days. The highest gs was 0.3881 and 0.2912 mmol m−<sup>2</sup> s−<sup>1</sup> in the SS, and WS at 35 DAS, respectively. The Tr in SS and WS followed the same trend as gs; the highest Tr was observed at 35 DAS. At 63 DAS, the Pn value in the SS increased by 10%, compared with in the WS. Urban et al. [64] revealed that high temperatures affect all physiological processes in plants. Furthermore, Jones et al. [65] and Scherrer et al. [66] asserted that environmental factors, such as radiation, air temperature, and wind, affect the size of the stomata aperture. In this study, the physiological trend (gs, Pn, and Tr) could be explained by the environmental differences between the two growing seasons, where the SS had higher air temperature, radiation, and wind speed than the WS (Figure 1).

The effects of the growing season, mulch treatment, and irrigation quantities on gs, Pn, and Tr were significant at all squash stages (Table 2). This finding indicates that sowing squash during a suitable growing season and choosing a suitable combination of irrigation volume and plastic mulch could enhance squash physiological response, which would ultimately increase the yield and IWUE.

The gs was not significantly affected by interactions between growing season, mulch, and irrigation quantities, as shown in Table 2. However, at 83 DAS, the interaction between season and mulch showed a significance difference (*p* < 0.05). No interaction effect on Pn was observed, except for interaction between irrigation and mulch, which significantly affected (*p* < 0.05) Pn values at 35 DAS and 63 DAS. In 35 DAS, comparing with same irrigation strategies FI, BM increased Pn 3% and 20%, respectively compared with WM and NM. In PRD70, Pn values under BM and WM were not different, while BM and WM enhanced Pn values 37%, and 36%, respectively, compared with NM. in PRD50, BM increased Pn 21%, 39%, compared with WM and NM, respectively. Data in Table 2 indicate that there was no significant interaction between growing season, mulch, and irrigation on Tr, except after 83 DAS. This indicates that squash plants were not able to withstand environmental changes at a late stage of growth, and there was a water deficit due to the age of the plants. Tr values were reduced under PRD strategies compared with FI for both mulched and non-mulched treatments. At 63 DAS, WM increased Tr 6% and 14% compared with BM, and NM, respectively under FI strategy. Using PRD70 and PRD50 Tr values under WM was higher 18% and 38% compared with BM, and NM, respectively. At 83DAS, under FI, Tr under BM was higher 10%, and 17%, respectively, compared with WM and NM. In PRD 70, WM increased Tr 22%, and 40%, respectively, compared with BM and NM. in PRD50, Tr was reduced dramatically due to water stress. However, WM increased Tr by 9% while BM increased by 7%, compared NM.

#### *3.4. Chlorophyll Index (SPAD Value)*

The chlorophyll index (SPAD value) was statistically analyzed, as shown in Table 3. High chlorophyll content is a desired attribute, as it implies a low degree of photoinhibition of the photosynthetic apparatus [67]. Li et al. [42] suggested that SPAD values could perfectly trace the variations in chlorophyll content of plants. At 35 and 83 DAS, squash plants sown in the SS showed high chlorophyll content (SPAD value) compared with those sown in the WS. This could be due to the higher photosynthesis rate (Pn), observed in squash plants sown in the SS, compared with those sown in the WS (Table 2). Li et al. [68] and Peiguo and Mingqi [69] emphasized that the relative chlorophyll and photosynthetic rate interact positively with each other, as chlorophyll represents the primary chloroplast component of photosynthesis.


**Table 3.** Analysis of variance of the chlorophyll index (SPAD value) at the development stage (35 DAS), mid stage (63 DAS), and late stage (83 DAS) of squash growth during winter (WS) and spring (SS) growing seasons.

ns: not statistically significant, \*\*: significant at the 1% level (*p* < 0.01), \*: significant at the 5% level (*p* < 0.05); different letters indicate significant difference between treatments; bold letters and words indicate treatments names.

Mulched treatments significantly affected (*p* < 0.001) chlorophyll index values (Table 3). Our study showed that the SPAD value of mulch treatments was significantly higher than non-mulched treatments. The primary reason for the high SPAD value with mulch treatment could be that the film mulch changed the soil water content (Figure 5) and the heat environment in the root area of the squash, causing a change in the physical and chemical properties of the soil, which accelerated root system growth. Kante et al. [70] showed that a reduction in the chlorophyll content of plant leaves was directly associated with root growth. This result follows the same trend as the findings of Hugar et al. [71], Nasrullah et al. [72], and Iqbal et al. [73], who found that soil mulch enhances chlorophyll content compared with non-mulched treatments.

Drought stress reduced the chlorophyll index at all growth stages. PRD70 and PRD50 reduced the chlorophyll content. Under conditions of water stress, chlorophyll content declines as a result of damage to chloroplast membranes and structure and photo-oxidation of chlorophyll [74–76]. The reduction of leaf chlorophyll values due to a water deficit has been reported for squash [23], cabbage [58], cotton [73], and wheat [67] crops.

Chlorophyll index values were not significantly affected by the interactions between S×M×I. However, the interaction between S × M was significant (*p* < 0.05) at all measured days. At 63 DAS, the interaction effect between mulch and irrigation treatments on SPAD value was significance. In FI treatments, BM increased SPAD value 6% and 23% compared with NM. In PRD70, the SPAD values under BM and WM were not different. BM and WM both increased SPAD values 17% compared with NM. Under PRD50, WM increased SPAD values 3% and 24 %, respectively compared with BM and NM. Overall, FI and BM improved Pn, Tr and SPAD value.

### *3.5. Fruit Quality*

Table 4 shows the statistical analysis of squash fruit quality, total soluble solids (TSS), total acidity (TA), and vitamin C (VC) under mulch and irrigation treatments for the WS and SS. The fruit qualities of the FI treatment were significantly different (*p* < 0.001) to

those of the PRD treatments. Squash plants under the PRD50 treatments reduced TSS, TA, and VC by 17%, 25%, and 19%, respectively, compared with the FI treatment. The severe water stress treatment (PRD50) negatively affected the squash fruit quality. This finding could be explained by the water deficit causing a reduction in fruit water potential [25]. These results are in agreement with the findings of Al-Ghobari and Dewidar [24], Abd El-Mageed et al. [34], Kuslu et al. [77] and Zhang et al. [25], who found that water-stressed treatments reduced fruit qualities compared with non-stressed water. Fruit quality under PRD can be affected by many factors, including plant type, developmental stage, soil type, and environmental conditions [62].


**Table 4.** Analysis of variance of squash fruit quality, total soluble solids (TSS), total acidity (TA), and vitamin C (VC) for winter and spring growing seasons.

ns: not statistically significant, \*\*: significant at the 1% level (*p* < 0.01), \*: significant at the 5% level (*p* < 0.05); different letters indicate significant difference between treatments; bold letters and words indicate treatments names.

Mulching significantly affected *(p* < 0.0001) all fruit quality attributes. Mulch treatments increased the TSS, TA, and VC by 16%, 16%, and 13%, respectively, compared with non-mulched treatments. This result is consistent with those of Lira-Saldivar et al. [36] and Li et al. [78], who found that soil mulching enhances fruit quality, compared with non-mulching. Abd El-Mageed et al. [34] indicated that mulch could reduce the influence of water stress on squash fruit quality, as mulch reduces soil evaporation, while preserving soil moisture content near the root zone.

Growing seasons did not significantly (*p* > 0.05) affect fruit quality, except for TSS. The interaction effect between S×I on TSS and TA was significant (*p* < 0.001). However, there was no significant (*p* > 0.05) difference in the value of VC. Squash fruit qualities were not significantly affected by the interactions of S × M × I. In contrast, the effect of the interaction of S × M showed a significant difference (*p* < 0.05) between all fruit qualities.
