3.1. Interactive Effects of Drought and Organic Fertilizers on Growth and Physiological Traits
Drought stress had a highly significant (
p ≤ 0.01) effect on growth (leaf area and height), shoot dry weight, and physiological (relative water content, chlorophyll content, and membrane stability) traits of
Brassica juncea plants at the vegetative stage (
Table 1). Significant interactions of soil moisture and fertilizer types (
p ≤ 0.01) were also recorded for all morpho-physiological traits except for root biomass (
Table 2; R
2 = 0.49,
Table S1). Concurrent with our results, water stress was shown to reduce plant height and leaf area in wild mustard and brassica species [
44,
45] and other plant species [
46,
47]. The impairment of mitosis and the elongation and expansion of cells as well as the down of turgor pressure caused by moisture stress result in reduced leaf area and growth [
9,
48,
49]. Additionally, a decline in leaf area, has been regarded as a morphological acclimatization mechanism to lessen water loss through transpiration. Optimal leaf area is critical for photosynthesis, which in turn, is the main driver of plant growth [
9,
50]. The effect of water stress significantly decreased the shoot dry weight and root dry weight by 76.2% and 74.9%, respectively (
Table 1). Other Brassica species have also shown reduction in dry matter production under water deficit condition largely due to its inhibitory effect on leaf development and consequently light interception and reduced carbon assimilation [
51].
Despite a general decline in plant height, leaf area, and shoot dry weight being observed along with soil moisture levels, the application of organic fertilizers improved these traits. Compared to the control, under severe drought stress, plant height and leaf area increased in the mixed fertilizer treatments, chitosan, and home-grown organic nutrient (CH + HO) by 33.14% and 24.2%, HO and ultra-green, UG (HO + UG) by 29.6% and 15.11%, and chitosan (CH) + UG + HO by 35.3% and 55.6%, respectively (
Table 1). Combined effects of drought and fertilizer types increased leaf area and height per plant with a greater magnitude than under drought stress alone, and this increase by combined drought stress and fertilizer types was synergistic too (
Table S1). The positive response of plant heights, leaf area, and shoot biomass in the mixed fertilizer treatments under severe water stress condition might partly be due to the augmented nutrient level. This could also partly be because of the better quality of organic matter by the addition of organic fertilizers, which conceivably increased the absorbing ability of the root systems for nutrients [
52]. In agreement with our results, application of chitosan stimulated plant growth and increased the availability and uptake of water and essential nutrients by alleviating drought stress [
28,
53]. In addition, it has been found that organic fertilizers modulate nutrients mobility and availability under varying soil water conditions thus ultimately enhancing plant growth [
54,
55]. In agreement with our results, it has been reported that application of chitosan reduced the negative impact of drought condition on dry matter [
11] and growth attributes [
56,
57]. However, application of fertilizers increased either shoot weight or dry weight at a different magnitude. In the mixed fertilizer and well-watered treatments, the highest shoot dry weight was acquired, and this signifies that mustard favors the regular application of water and fertilizers for optimum growth and development.
There was a significant general trend in decreasing of relative water content (RWC), membrane stability index (MSI), and chlorophyll content (SPAD) of mustard plants along with soil moisture stress intensity (
p < 0.05,
Table 1). However, overall, the application of fertilizers significantly increased the leaf RWC (28.41%), MSI (75.31%), and chlorophyll content (55.5%) under severe water stress condition as the case in the mixed fertilizers (CH + UG + HO) relative to the control suggesting a synergistic response. This synergetic effect was also supported by our generalized regression model, which revealed a significant interaction effect on MSI (Wald-χ2 = 236.71,
p ≤ 0.001) and RWC (Wald-χ2 = 29.92,
p ≤ 0.001) when comparing the combined (CH + UG + HO) effect over a single effect of UG (
Table S1). Looking at the effect of individual stress factors, soil moisture deficit induced significant decrease in RWC (35.43%), MSI (50.23%), and SPAD (45%) (
Table 1). Overall, the effects of combined fertilizers and drought stress on RWC, SPAD, and MSI were stronger than the effects of individual stress factors (
Table 1), but the level of the effect varied. Lower RWC by
B. juncea show its predisposition to drought stress that could be coupled with a discrepancy between water loss and water uptake and is a signal of the decline in turgor pressure of guard cells due to stomata closure [
58,
59]. In addition, the lower RWC reflects considerable reductions of leaf water potential, which ultimately lead to closure of stomata. This, in turn, favors stomatal resistance that could potentially reduce the rate of transpiration and an increase in leaf temperature [
60]. Consequently, higher temperature reduces membrane stability and permeability and, ultimately, different aspects of plant metabolism. Higher RWC in organic fertilizers treated plants might be due to improved water holding capacity [
61] and osmolytes as supported by the increase MSI. The decrease in MSI may be because under water deficit conditions the overproduction of reactive oxygen species (ROS) occurs, which disrupts the cell membrane by altering its phospholipid and fatty acid compositions [
62]. Hence, the ability of a plant to maintain MSI and integrity would explain its tolerance toward drought [
63].
Chlorophyll content has been used as a proxy to determine the tolerance of plants to water stress [
64]. In the present study, chlorophyll content progressively decreased by 45% (from 46.82 to 25.75 when the mustard plants were exposed to drought stress) (
Table 1). This reduction in SPAD could be associated with oxidative damage of chlorophyll pigment complexes and light-harvesting proteins modulating chlorophyll synthesis [
65,
66]. Aligned with our results, drought-induced reductions in chlorophyll contents are quite common in Brassica species and cultivars [
51]. In contrast, some studies have shown increased chlorophyll content under moderate and intensive drought stress [
67,
68]. However, application of fertilizer either singly or in mix improved the total chlorophyll content under severe drought stress compared to the control (
Table 1). Under severe water deficit condition, the maximum SPAD recorded was about 46.68 when treated with mix of CH and HGON. Our results showed a significant coupling effect of water stress and organic fertilizers on the chlorophyll content, based on SPAD measurements (
Table 1). Application of organic fertilizers further promoted accumulation of chlorophyll content in plants [
69]. Other studies have also shown that increasing chlorophyll content by organic fertilizer treatments under drought stress conditions may be due to increased activity of drought responsive enzymes [
68]. The highest chlorophyll content of
B. juncea plants under drought stress was observed in combined fertilizer treatment that could be due to positive effects of fertilizers that enhance N supply. It has been indicated that the formation of photosynthetic pigments mainly relies on the available nitrogen content in plants [
70]. The higher amounts of chlorophyll could be correlated with the content of nitrogen (r = 0.91), which stimulates the chlorophyll biosynthesis process [
71].
3.2. Interactive Effects of Drought and Organic Fertilizer on Nutrients Content
Analysis of variance revealed a significant effect of soil moisture level, fertilizer type, and their interaction on content of macronutrients (Ca, K, P, N, C, S, Na, Mg) and micronutrients (Fe, Zn) in mustard leaves (
p < 0.001, Tables 4, 5 and
Table S1). However, there was not a significant effect detected for Cu across all factors (
Table 1) as well the interaction effect on Mn. Drought stress and associated reduction in soil moisture can reduce plant nutrient uptake by reducing nutrient supply through mineralization [
72], but also by reducing nutrient diffusion and mass flow in the soil [
73]. The decline in the diffusion rate of nutrients towards the roots and their transport to the shoots could be due to the concurrent declines in membrane stability recorded. Although water stress without application of fertilizers decreased the content of all the macronutrients studied contrastingly, the K content was increased by 62.56% (
Table 3). Etienne et al. [
21] also reported that drought stress during the vegetative stage induces an increase in the concentration of K in leaves. Under water deficit conditions, maintaining sufficient K status may facilitate osmotic adjustment that enhances higher turgor pressure and RWC [
74]. Nevertheless, the decline in RWC in the present study did not support this presumption. Thus, alternatively, the increase in leaf K content could be associated with its critical role in overcoming drought-induced oxidative damage via inhibition of reactive oxygen species (ROS) production [
75]. In addition, drought stress caused a significant (
p ≤ 0.05) decrease in the Na concentration in the leaves despite fertilizer application. However, our results also revealed a contrasting pattern between K and Na content in response to severe water stress (r = −0.86,
p ≤ 0.01). It has been thought that Na content may up- or downregulate the function and accumulation of other compounds or nutrients such as K involved in osmotic adaptation [
21,
76]. The ionomic content of vegetative tissue can be influenced in a very unclear way by mineral uptake and availability as well as by drought [
21], which supports our results.
On the other hand, effects of combined fertilizers and water stress on macro- and micronutrients were stronger than the effects of individual stress factors, but the magnitude of effects varied for the specific nutrients (
Table 3,
Table 4 and
Table 5). Though there was an improvement in Mg concentrations following application of organic fertilizers under severe soil moisture, a general decline was still observed along with soil moisture gradients. This pattern mirrors with the reduction in chlorophyll content (
Table 3), as Mg plays a paramount role in chlorophyll synthesis [
77]. In agreement with Chatelain et al.’s [
78] report, in the present study, Mg concentrations were lower in the leaves of plants treated with chitosan. Ca decreased significantly in the leaves of mustard plants treated under severe water stress irrespective of fertilizers applied. This might be linked with restricted phloem mobility that negatively impacts the usual unidirectional transport of Ca during vegetative stage of the plant under water deficit conditions [
79]. An increase in Ca contents in mixed fertilizers (CH + HO, HO + UG, CH + UG and CH + UG + HO) when compared with a single fertilizer (CH, HO, and UG) suggests a synergetic effect (
Table 3). There was a general pattern in the decline of N and P contents along water stress levels with or without application of fertilizers (
Table 3). Waraich et al. [
80] have also indicated that plants decrease N and P uptake with a decrease in soil moisture. Importantly, a combination of fertilizers had a synergetic effect on phosphorus content when compared with the single effect of each fertilizer under severe water stress compared to the control (
Table 3 and
Table S1). The combined application of fertilizers (CH + UG + HO) showed twofold (68.53%) increase in P compared to the control under severe water stress. Application of organic fertilizers enhances uptake of macronutrients such as N and P in plants under different water stress levels [
9,
81].
Drought during the vegetative stage in
B. juncea induces a decrease in the amount of almost all micronutrients such as Zn, Fe, Mn, and Cu (
Table 3). The observed decrease in the accumulation of microelements in terms of both stress factors might indicate the inhibition of the transport of these ions to the leaves of mustard. In stress conditions, a decrease in Mn, Fe, Cu, and Zn levels may suggest a reduction in the synthesis of enzymes, especially of superoxide dismutase, which possesses these ions [
82]. However, the magnitude of these micronutrients was slightly improved with further treatment of organic fertilizers under severe water stress (
Table 3). There was also a significant interaction between fertilizer types and soil moisture levels for Fe in the leaves. Maximum amount of Fe (7.19 mg/mL) in
B. juncea leaves was recorded in HO-treated plants under well-watered conditions.
Plants differ widely in nutrient accumulation and demand across developmental stages and species-specific survival strategies in response to biotic and abiotic factors imposed [
83,
84]. Here, specifically, we further focus to unravel the effect of severe water stress on macronutrient concentrations of
B. juncea leaves within vegetative stage across different time and fertilizers treatment. Compared to the macronutrients content before the fertilizer application (0 DAT) there was a significant increase in K, Ca, P, Na, and Mg across time (
Figure 1 and
Figure 2) under severe drought stress. While Na content followed the opposite pattern of K content (
Figure 2), other micronutrients such as Fe, Mn, Cu, and Zn revealed a non-significant increase in magnitude (data not shown). These dynamics in content of macronutrients within mustard leaves could be ascribed to difference in nutrient mobility and the physiological demand during vegetative growth. Most of these nutrients are relatively immobile in plant tissues which may affect their transport than accumulation, particularly calcium, iron, and zinc [
85]. Contrasting with reproductive stage, the concentrations of the mobile nutrients such as P and K increased in during shoot elongation at early growth stage [
83]. Further, nitrogen regulates the development of plant organs and exerts stronger control on vegetative growth than reproductive growth [
86]. The K content was significantly increased at early vegetative growth stage than late vegetative growth stages under severe drought stress while fertilizers were applied. Similarly, Fernandez-Escobar et al. [
87] have also shown that K concentration was higher at early vegetative growth stage than late vegetative growth stages. The poorly mobile nutrient Ca accumulated gradually in the leaves across time.
3.3. Relationships between Traits
Overall, total chlorophyll content (SPAD) was positively associated with RWC (r = 0.841), height (r = 0.79), LA (r = 0.826), MSI (r = 0.703), shoot biomass (r = 0.855), Mg (r = 0.828), Ca (r = 0.816), N (r = 0.908), C (r = 0.76), P (r = 0.81), and Mn (r = 0.774) significantly during the vegetative stage of mustard (
p ≤ 0.001,
Table 6). The strong association of SPAD with morphological traits (e.g., height and LA) might be linked to directly or indirectly to the effect of chlorophyll content on photosynthesis which could also ultimately impact shoot biomass and carbon content [
88]. The positive association between SPAD and Mg mirror with its central location in chlorophyll structure and its integral role in modulating several physiological and biochemical processes containing the activation of plant growth enzymes [
89,
90]. Leaf area was positively correlated with leaf N concentrations (r = 0.81) in our dataset. There were negative correlations between leaf K and Ca (r = −0.01), K and Na (r = −0.049), and K and Mg (r = −0.384) contents. The reduction in leaf K contents as a function of the increase in Mg and Na content might be due to the antagonistic effect among these nutrients for competing for the same site of the carriers in the membrane [
91,
92]. However, these correlations between leaf nutrient contents may not necessarily be maintained as these rely on soil moisture availability and nutrient uptake [
19]. In sum, these findings imply that drought and fertilizer application may also modify cross talk between nutrients contents and hence modify the composition in the leaf. Micronutrients such as Cu, Mn, Zn, and Fe were positively correlated with the majority of morpho-physiological traits and macronutrients except their negative association with RSR, Na (Mn and Zn), root biomass (Cu and Fe), and K (Mn) (
Table 6).