Nano ZnO and Bioinoculants Mitigate Effects of Deficit Irrigation on Nutritional Quality of Green Peppers

Abstract

Green peppers (Capsicum annuum L.) are a fruit vegetable with great culinary versatility and present important nutritional properties for human health. Water deficit negatively affects the nutritional quality of green peppers’ fruits. This study aimed to investigate the influence of zinc oxide nanoparticles (ZnONPs), associated with plant growth-promoting bacteria (PGPB), on the post-harvest nutritional quality of green peppers subjected to water deficit. In an open-field experiment, two irrigation levels (50 and 100% of crop evapotranspiration (Etc)), four treatments composed of a combination of ZnONPs, zinc sulfate (ZnSO₄), and PGPB (T1 = ZnSO₄ via leaves, T2 = ZnONPs via leaves, T3 = ZnONPs via leaves + PGPB via soil, T4 = ZnSO₄ via soil + PGPB via soil), and a control treatment (Control) were tested. Water deficit or water deficit mitigation treatments did not interfere with the physical–chemical parameters (except vitamin C content) and physical color parameters (except the lightness) of green peppers. On average, the water deficit reduced the levels of Ca (−13.2%), Mg (−8.5%), P (−8.5%), K (−8.6%), Mn (−10.5%), Fe (−12.2%), B (−12.0%), and Zn (−11.5%) in the fruits. Under the water deficit condition, ZnONPs or ZnSO₄ via foliar, associated or not with PGPB, increased the levels of Ca (+57% in the T2 and +69.0% in the T2), P, Mg, and Fe in the fruits. At 50% Etc, the foliar application of ZnONPs in association with PGPB increases vitamin C and mineral nutrients’ contents and nutritional quality index (+12.0%) of green peppers. Applying Zn via foliar as ZnONPs or ZnSO₄ mitigated the negative effects of water deficit on the quality of pepper fruits that were enhanced by the Bacillus subtilis and B. amyloliquefaciens inoculation. The ZnONPs source was more efficient than the ZnSO₄ source. The water deficit alleviating effect of both zinc sources was enhanced by the PGPB.

1. Introduction

Fruit vegetables are such important sources of vitamins and mineral nutrients in the human diet [1,2]. Due to their great versatility in cooking and their nutritional properties, green peppers in natura are one of the main vegetables consumed in Brazil [3]. In the semi-arid region of Brazil, green peppers are widely cultivated; however, water scarcity generally promotes a decrease in fruit productivity, which can also affect their nutritional quality [4].

Water deficit negatively affects the production and quality of vegetables such as tomatoes and peppers, especially during flowering, fruiting, and fruit maturation [5,6]. In some situations, water stress can increase the concentration of soluble solids, titratable acidity, and vitamin C but decreases other quality parameters, such as fruit size, firmness, and mineral elements’ content [6]. The water deficit decreases fruit yield, the translocation of photoassimilates, and the content of minerals such as calcium, potassium, magnesium, and iron in the fruits [5]. The decrease in mineral nutrients in fruits also occurs due to the decrease in the diffusion process and mass flow that contribute to the largest portion of the amount of nutrients that reach the roots and, subsequently, their redistribution to the fruits [7].

The foliar application of zinc, as zinc oxide nanoparticles (ZnONPs), in some plant species, including green peppers and other fruits and vegetables, can contribute to mitigating the negative effects of environmental stresses such as water deficit and saline stress on the plants and improving some aspects of the physical–chemical quality of fruits [8,9,10,11,12,13]. Due to their high specific surface, the main mode of action of ZnONPs would be through the stimulating effect on plant metabolism, through their interaction with biomolecules, promoting an increase in enzymatic activity and the synthesis of substances that act to mitigate oxidative stress in plants [14,15].

ZnONPs can also act as an efficient source of zinc (Zn) for plants, with slower release than conventional sources, such as zinc sulfate [8,16]. In plants, zinc is involved in the synthesis of several hormones, such as gibberellin, auxin, cytokinin, and abscisic acid [17]. In addition, Zn participates in the synthesis of chlorophyll, the development of chloroplasts, and the integrity of the cell membrane, providing protection, stability, and structure [17,18]. Furthermore, zinc plays a crucial role in fruiting, seed growth, and fruit maturation, increasing resistance to environmental stress and acting as a co-enzyme in the enzymatic processes related to protein synthesis, glucose metabolism, and DNA synthesis, being also essential for the production of auxins, which regulate root growth, cell expansion, and the beginning of flowering [19,20,21].

Under water stress, several plant species can be favored by the presence of plant growth-promoting bacteria (PGPB) in the rhizosphere region [22,23,24,25,26]. Indirectly, PGPB can alleviate the effects of oxidative stress caused by drought, through the stimulation of enzymes from the antioxidant system such as catalase, superoxide dismutase, and peroxidase [27,28,29,30]. Plant inoculation with Bacillus amyloliquefaciens improved the survival of pepper plants under different stress conditions, resulting in a higher seedling growth rate and better physicochemical characteristics [31]. In the study conducted in [32], it was noted that the application of B. amyloliquefaciens to corn resulted in an increase in nutrient absorption and stimulated plant growth mechanisms, reflected in higher concentrations of amino acids, such as tryptophan, isoleucine, alanine, valine, tyrosine, and sugars, such as fructose and glucose. Similarly, for blueberry plants, inoculating the soil with different strains of B. amyloliquefaciens and B. subtilis increased the foliar chlorophyll content, nutrient absorption, and the levels of soluble solids, vitamin C, and anthocyanins in the fruits [33].

Although some studies already tested the influence of nano ZnO as a water stress alleviator [10,12,15], information is lacking on the effects of ZnO and PGPB, whether in combination or not, on the nutritional quality of fruiting vegetables such as green peppers exposed to water deficit, especially at low concentrations compared to the conventional zinc sulfate source.

In this present study, two main hypotheses were considered. The first hypothesis was that water deficit alters the physical–chemical quality and mineral composition of green pepper fruits. The second hypothesis is that zinc oxide nanoparticles (ZnONPs) applied via leaves, associated or not with PGPB, could mitigate the negative effects of water deficit on the physicochemical attributes of the quality and mineral composition of green pepper fruits and that ZnONPs would be more efficient than the conventional source of zinc (ZnSO₄*7H₂O).

The objective of this work was to evaluate how water deficit affects the nutritional quality of green peppers and whether the application of zinc sources could be capable of mitigating the possible negative effects of the lack of water. Therefore, the application of zinc oxide nanoparticles, associated or not with PGPB, on some physical–chemical parameters and mineral composition of green pepper fruits, from plants grown under two irrigation levels, was evaluated.

2. Materials and Methods

2.1. Characterization of the Experimental Area

The present research was carried out under field conditions, from December 2022 to April 2023, in the Experimental Farm area of the Center for Agro-food Science and Technology at the Federal University of Campina Grande (UFCG), Pombal-PB Campus. The Experimental Farm is located in the municipality of São Domingos-PB. According to the Köppen classification, the predominant climate is type Aw, hot and humid. Annual rainfall is around 800 mm, and the temperature range is always below 5 °C. The Experimental Farm is located at an altitude of 190 m, at the following coordinates: latitude 6°50′4″ S, longitude 37°53′9″ W. According to Gaussen’s classification, the Mediterranean-type bioclimate, also known as medium-dry northeastern, prevails, with a dry season that lasts between 4 and 6 months.

The soil tillage consisted of plowing and harrowing with a plow harrow. One day before planting soil fertilizer, soil sampling was carried out in the area, in the 0–20 cm layer for its chemical and physical characterization (

Table 1. Chemical and physical attributes of the soil used in the experiment.

Chemical	Value	Physical	Value
pH (CaCl2)	6.20	Sand (g kg−1)	444
P (mg kg−1)	291	Silt (g kg−1)	353
K+ (mg kg−1)	633	Clay (g kg−1)	203
Na+ (mg kg−1)	169	BD (g cm−3)	1.36
Ca2+ (mg kg−1)	1578	PD (g cm−3)	2.59
Mg2+ (mg kg−1)	562	TP (m3 m−3)	0.47
Zn2+ (mg kg−1)	1.69	FC (%)	12.87
H+ + Al3+ (cmolc dm−3)	2.30	PWP (%)	5.29
OM (g kg−1)	6.40	AWC (%)	7.58
V (%)	83.0
CEC (cmolc dm−3)	4.10	-	-

P, K⁺, Na^+, and Zn²⁺—Mehlich 1 extractant, H⁺ + Al³⁺—0.5 mol L⁻¹ calcium acetate extractant at pH 7, Ca²⁺, Mg²⁺—1 mol L⁻¹ KCl extractant, OM—organic matter, V—Base saturation, CEC—cationic exchange capacity, BD—bulk density, PD—soil particle density, TP—total porosity, FC—field capacity, PWP—permanent wilting point, AWC—available water content, pH (CaCl₂)—extractant CaCl₂ 2H₂O 0.01 mol L⁻¹.

) according to the methodology described by Embrapa [34].

During the experimental period, climatological data on temperature, relative humidity, and precipitation (Figure 1) were collected through the Agritempo website [35].

2.2. Treatments and Experimental Design

The experiment was carried out in a randomized block design, in a split-plot scheme, where the plots were two irrigation levels (50 and 100% of crop evapotranspiration (Etc)) and the subplots were four water deficit irrigation alleviate treatments (here named as treatments DHA) composed from nanoparticles of zinc oxide (ZnONPs), zinc sulfate (ZnSO₄), and plant growth-promoting bacteria (PGPB) (T1 = ZnSO₄ via leaves, T2 = ZnONPs via leaves, T3 = ZnONPs via leaves + PGPB via soil, T4 = ZnSO₄ via soil + PGPB via soil), and control treatment, with four blocks, totaling 40 experimental plots. These treatments were defined considering some premises. The supply of Zn via foliar application should be more efficient than that via soil application, and, therefore, the dose of Zn via foliar application should be many times lower than that applied to the soil. This was also considered by other authors [36,37,38]. Likewise, it was considered that Zn supplied as ZnONPs via foliar would be more efficient than Zn supplied by ZnSO₄ via soil or leaves. The biological component was to verify whether PGPB could increase the availability of Zn from the soil itself or increase the efficiency of Zn application via soil, in the form of ZnSO₄, since this form of Zn is susceptible to precipitation reactions, decreasing the availability of Zn for plants. In addition, the PGPB component could act as an attenuator of water deficit due to the production of beneficial substances in the root zone of green pepper.

The total area of the experiment corresponded to 22.8 m in length by 14.8 m in width (Appendix A). The plants were grown at a spacing of 1.2 m between rows and 0.5 m between plants in the row. Each subplot consisted of four 3.0 m long rows in which 20 plants were grown. A useful plot consisted of an area measuring 3.0 × 2.0 m in the central part of the plot, where 6 plants were evaluated. When installing the experiment, there was a space of 1.2 m between subplots and 2.0 m between blocks (Appendix A).

2.3. Seedlings’ Transplanting and Soil Fertilization

Pepper seedlings (Capsicum annuum L.) cv. ‘Kolima’ were produced in expanded polystyrene trays (Isopor^®), filled with coconut fiber as a substrate. The transplant was carried out when the seedlings had 4 to 6 true leaves. The seedlings were transplanted in ridges measuring 0.4 m wide and 0.30 m high. Based on the soil analysis (Table 1), planting and supplementary fertilization were carried out according to the recommendations of the Fertilization Recommendation Manual for the State of Pernambuco [39]. For planting fertilization, the formula of NPK 10-10-10 (10%N, 10% P₂O_5, and 10% K₂O) was applied at a dose of 1000 kg ha⁻¹ in a single dose and incorporated into the soil when lifting the planting rows. Further nitrogen and potassium fertilizations were applied via fertigation, according to the following schedule: 39.2 kg ha⁻¹ of urea (45% of N) and 15.8 kg ha⁻¹ of KCl (58% of K₂O) at 15 days after transplanting (DAT), 39.2 kg ha⁻¹ of urea and 15.8 kg ha⁻¹ of KCl at 30 days after transplantation, 39.2 kg ha⁻¹ of urea and 15.8 kg ha⁻¹ of KCl at 45 DAT, and 39.2 kg ha⁻¹ of urea and 15.8 kg ha⁻¹ of KCl at 60 DAT. All fertilizers used were obtained from Heringer^® Fertilizers, Rio Grande, Brazil.

2.4. Application of Treatments

As ZnONPs, the product nano zinc oxide (fr Sigma-Aldrich^®, Darmstadt, Germany) brand was used; it had 97% purity, a particle size smaller than 100 nm, and a specific surface area of 10.8 m²g⁻¹. ZnONPs’ suspensions and ZnSO₄*7H₂O (Vetec Fine chemistry Ltd., Duque de Caxias, Brazil)) solution (referred to in the text only as ZnSO₄) were prepared at concentrations of 0.125 g L⁻¹ (100 mg Zn L⁻¹) and 4.54 g L⁻¹ (1000 mg Zn L⁻¹), respectively. The treatments referring to bioinoculation (PGPB) consisted of the combination of two commercial products, one containing Bacillus subtilis BV-09 (Vittia Defense and Nutrition, Ltda., São Joaquim da Barra, Brazil) at a dosage of 1.0 × 10⁸ CFU mL⁻¹ and the other containing B. amyloliquefaciens (Vittia Defense and Nutrition, Ltd., São Joaquim da Barra, Brazil) at a dosage of 1.0 × 10⁹ CFU mL⁻¹. At the 15 DAT, 45 mL of each PGPB source were mixed with 10 L of water. From this mixture, 30 mL were applied to the soil, close to the stem of each plant. Each product was applied at a rate of 3 L ha⁻¹, totaling 6 L ha⁻¹.

The foliar application of the suspensions’ ZnONPs or ZnSO₄ solution for each treatment (T1, T2, and T3) was carried out with the aid of a knapsack sprayer with a capacity of 20 L, with an average of 35 mL applied per plant. Neutral detergent was used as adjuvant in the concentration of 1 mL L⁻¹ in all foliar treatments. Regarding soil application, the treatments were applied close to the root zone of the plants using a beaker. For the T4 treatment, 50 mL of the ZnSO₄ solution was applied per plant, corresponding to a dose of 2.42 kg of ZnSO₄ per hectare. The mixture of bioinoculants was applied around 30 mL per plant. The treatments were applied 25 days after transplanting the seedlings, after homogenizing the plants in the field plots.

2.5. Irrigation Levels’ Application

After transplanting, the plants were irrigated by drip, with drippers spaced 0.20 m apart and with a nominal flow of 1.6 L h⁻¹. After emergence and standardization of the number and size of plants in the subplots, the plants were irrigated following different levels of water depth, which ran 32 days after transplanting.

The total irrigation required (TIR) was calculated using the following expression suggested by Bernardo et al. [40].

TIR = [(FC − WP) × Z × BD × f)]/10

where:

TIR corresponds to the initial total depth of water applied in mm;

FC is the soil moisture corresponding to field capacity, %;

WP is the soil moisture corresponding to wilting point, %;

Z is the effective depth of the pepper root system (30 cm);

BD is bulk density, g cm⁻³;

F is water availability factor (0.5).

The water irrigation uniformity test was carried out according to the Christiansen Uniformity Coefficient (CUC) evaluation methodology, proposed by Christiansen [41].

Control of the volume of water supplied to each irrigation level was carried out daily, at a standardized time, according to the ratio of the flow rate of the drippers to the time to reach the reference evapotranspiration proportions (Eto). As the time interval for each volume of the respective irrigation level was reached, the drip tapes corresponding to each irrigation level were successively disconnected. The irrigation depth corresponding to 100% of Etc was obtained by calculating Etc, according to the expression from Jessen [42].

Etc = Kc × Eto

where:

Etc is crop evapotranspiration, mm day ⁻¹;

Eto is reference evaporation, mm day ⁻¹;

Kc is crop coefficient (dimensionless).

The Kc values adopted for each crop depending on their phenological phases and the daily Eto values were obtained according to the FAO Penman–Monteith model [43]. During the experiment, climatological data were obtained from the automatic meteorological station in the municipality of São Gonçalo, Paraíba, as it is the closest to the experiment site, through the Agritempo platform [35].

The daily supply of irrigation depth was carried out through the irrigation time considering the characteristics of the cultivation system and the irrigation system according to the following expression suggested by Bernardo et al. [40].

$$\mathrm{I}\mathrm{t}={\displaystyle \frac{\mathrm{E}\mathrm{t}\mathrm{o}\times \mathrm{K}\mathrm{c}\times \mathrm{A}}{\mathrm{E}\mathrm{a}\times \mathrm{n}\times \mathrm{q}}}$$

where:

It is irrigation time, h;

Eto is reference evaporation, mm day⁻¹;

Kc is crop coefficient, dimensionless;

A is area occupied by one plant, m²;

Ea is application efficiency (0.90);

N is number of emitters per plant; and

Q is emitter flow rate, L h⁻¹.

2.6. Cultivation Practices and Phytosanitary Control

To avoid any interference from phytosanitary chemicals on the action of products containing PGPB, weed and pests control was carried out mechanically, manually, and/or with the use of hand hoes. During experimental period, no diseases were detected. In addition, horizontal staking was carried out using wooden stakes measuring an average of 1.60 m in length. The stakes were spread across the lines, being placed every 5 m. Ribbons were tied to the poles before the fruiting period to prevent the plants from tipping over due to the weight of the fruits.

2.7. Variables Evaluated

2.7.1. Physicochemical Analysis

At the time of the first fruit harvest, which occurred 70 days after transplanting, physical attributes of color, ascorbic acid (vitamin C), pH, soluble solids (SS), titratable acidity (TA), and SS/TA ratio were evaluated on the six commercial fruits from useful plots.

The physical attributes of fruit color were measured using a Konica Minolta CR-400™ digital colorimeter (Konica Minolta Co., Ltd., Tokyo, Japan), using the CIELAB system, which defines a three-dimensional color space with three axes in rectangular coordinates (L*a*b*). These coordinates represent lightness (L*), tones from red to green (*a positive to −a* negative), and tones from yellow to blue (*b positive to −b* negative). Furthermore, it defines cylindrical coordinates (L*, C*, H°), and the a* and b* values were converted into Hue angle (H°), representing the intensity of the color and Chroma (C*), indicating the purity of the color, according to [44,45]. The analysis was carried out outside, with one reading per fruit for each experimental plot.

The pH was measured using a digital benchtop pH meter, with direct readings taken from the homogenized pulp, according to [46]. The determination of vitamin C was carried out using the Tillman method. The results of the analyses were expressed in mg per 100 g of a sample of fresh fruit [46]. Titratable acidity was determined following the methodology recommended by [46]. The results were expressed in g of citric acid per 100 g of a sample of fresh fruit. Soluble solids’ content was determined directly in the homogenized pulp using a digital refractometer (model PR-100, Palette, Atago Co., Ltd., Tokyo, Japan). The results were expressed in °Brix [46]. The SS/TA ratio was calculated as the ratio between soluble solids and titratable acidity (SS/TA).

2.7.2. Mineral Nutrient Content

Six commercial fruits were separated from each subplot to take a sample consisting of 100 g of fresh fruit mass. These samples were dried in a forced air oven (65–70 °C) until constant weight, to later obtain the dry mass and water content. Subsequently, this material was crushed in a Wiley-type (Tecnal Scientific, Piracicaba, Brazil) mill and then sulfuric digestion, according to the methodology described in [47], was performed. Digestion was obtained in a Kjeldahl microdigester block (Tecnal Scientific, Piracicaba, Brazil), using samples of 0.2 g of dry plant material in 3 mL of concentrated sulfuric acid and 1 mL of hydrogen peroxide, with an initial temperature of 180 °C and a final temperature of 360 °C. In the sulfuric acid digestion stratum, the levels of the macronutrients calcium (Ca), potassium (K), phosphorus (P), and magnesium (Mg) and the micronutrients boron (B), manganese (Mn), iron (Fe), and zinc were determined by using the Inductively Coupled Plasma Optical Emission Spectrometry-ICP-OES (Thermo Scientific Co., Ltd., Bremen, Germany) technique. The element contents obtained in terms of dry mass content were converted to fresh mass contents, correcting the values by the water content of the fruits.

2.7.3. Overall Quality Index

A general fruit quality index (FQI), based on Funsueb [48], was generated with physical–chemical variables, physical color variables, and macro- and micronutrient content in the fruits. Initially, all values of the studied variables (X) were converted into percentages (Xi%) using the following expression:

$$\mathrm{q}\mathrm{i}={\displaystyle \frac{\mathrm{X}\mathrm{t}}{\mathrm{X}\mathrm{c}}}·100$$

where qi represents the value of a quality attribute X originating from treatment t and Xc represents the average value of attribute x obtained from control treatment.

FQI values were obtained, according to Funsueb [48], by using the geometric mean of the physical–chemical attributes and the mineral nutrient contents evaluated, according to the following expression:

$$\mathrm{F}\mathrm{Q}\mathrm{I}=(\prod _{\mathrm{i}=1}^{\mathrm{n}}\mathrm{q}\mathrm{i}{)}^{1/\mathrm{n}}$$

2.8. Statistical Analyses

The data were previously subjected to the normality test using the Shapiro–Wilk test [49]. The data obtained were subjected to analysis of variance, followed by a comparison of means using the Tukey test (p ≤ 0.05). These statistical analyses were performed using R statistical software 4.3.0 [50].

3. Results

3.1. Physicochemical and Physical Color Parameters

Irrigation depths and water deficit alleviate treatments (here named as treatments DHA) did not influence the fruit quality parameters of pH, SS, TA, SS/TA, C*, and Hue angle (

Table 2. Means ± standard error for pH values, soluble solids (SS), titratable acidity (TA), SS/TA ratio, chromaticity (C*), Hue angle (°Hue) of green peppers’ fruits as a function of irrigation levels and deficit irrigation treatments’ mitigation (DHA).

Irrigation Depth (%)	Treatments DHA	pH	SS (°Brix)	TA (g per 100 g)
	Control	5.443 ± 0.464 aA	4.858 ± 0.175 aA	0.109 ± 0.018 aA
	T1 (ZnSO4 via leaves)	5.432 ± 0.265 aA	4.975 ± 0.325 aA	0.109 ± 0.001 aA
50% Etc	T2 (ZnONPs via leaves)	5.370 ± 0.204 aA	4.767 ± 0.639 aA	0.130 ± 0.018 aA
	T3 (ZnONPs via leaves) + PGPB via soil	5.443 ± 0.252 aA	4.792 ± 0.382 aA	0.119 ± 0.022 aA
	T4 (ZnSO4 via soil) + PGPB via soil	5.437 ± 0.388 aA	5.289 ± 0.615 aA	0.103 ± 0.011 aA
	Mean	5.425 A	4.936 A	0.114 A
	Control	5.392 ± 0.292 aA	5.233 ± 0.183 aA	0.119 ± 0.013 aA
	T1 (ZnSO4 via leaves)	5.438 ± 0.263 aA	4.992 ± 0.185 aA	0.114 ± 0.011 aA
100% Etc	T2 (ZnONPs via leaves)	5.350 ± 0.479 aA	5.054 ± 0.042 aA	0.109 ± 0.001 aA
	T3 (ZnONPs via leaves) + PGPB via soil	5.282 ± 0.342 aA	5.183 ± 0.745 aA	0.114 ± 0.021 aA
	T4 (ZnSO4 via soil) + PGPB via soil	5.246 ± 0.138 aA	5.042 ± 0.203 aA	0.125 ± 0.021 aA
	Mean	5.341 A	5.101 A	0.116 A
		SS/TA	C *	°Hue
	Control	42.792 ± 4.477 aA	20.581 ± 0.910 aA	50.673 ± 4.500 aA
	T1 (ZnSO4 via leaves)	45.820 ± 2.991 aA	20.809 ± 1.900 aA	53.451 ± 5.700 aA
50% Etc	T2 (ZnONPs via leaves)	37.420 ± 4.065 aA	21.514 ± 0.842 aA	50.670 ± 3.060 aA
	T3 (ZnONPs via leaves) + PGPB via soil	42.878 ± 2.473 aA	21.276 ± 2.196 aA	50.859 ± 6.110 aA
	T4 (ZnSO4 via soil) + PGPB via soil	46.971 ± 5.969 aA	21.161 ± 1.872 aA	50.692 ± 4.560 aA
	Mean	43.176 A	21.068 A	51.269 A
	Control	44.246 ± 5.600 aA	20.607 ± 2.472 aA	50.311 ± 1.222 aA
	T1 (ZnSO4 via leaves)	44.029 ± 3.802 aA	21.164 ± 1.079 aA	50.755 ± 5.330 aA
100% Etc	T2 (ZnONPs via leaves)	46.549 ± 3.840 aA	21.911 ± 2.091 aA	50.959 ± 4.060 aA
	T3 (ZnONPs via leaves) + PGPB via soil	53.822 ± 2.628 aA	22.170 ± 0.629 aA	49.863 ± 1.485 aA
	T4 (ZnSO4 via soil) + PGPB via soil	41.123 ± 6.286 aA	23.012 ± 2.622 aA	50.230 ± 2.891 aA
	Mean	45.954 A	21.773 A	50.424 A

Within each variable, means followed by the same lowercase letters do not differ for irrigation depth, and means followed by the same uppercase letters do not differ for DHA treatments by Tukey’s test (p ≤ 0.05).

). In most of the variables evaluated, the average values obtained on the 50% Etc irrigation level were quite similar to those obtained on the 100% Etc, with variations of less than 5%. Among the DHA treatments, the T4 treatment provided an increase of 10.45% in the C* value and the T3 treatment increased the SS/TA ratio by 17.79%, both under the 100% Etc.

The interaction of irrigation depth and DHA treatments influenced the ascorbic acid content (Figure 2a) and color lightness (L*) (Figure 2b). The 50% Etc irrigation level increased the ascorbic acid content by approximately 35.1% in the control treatment and 51.0% in the T4 treatment compared to the 100% Etc irrigation level. Under the 50% Etc irrigation level, the T3 treatment and T4 treatment promoted an 18.6% increase in the ascorbic acid content, in relation to the control, while the T1 and T2 treatments had similar performances to the control. Under the 100% Etc irrigation level, the T3 treatment increased the ascorbic acid content by 72.7% in relation to the control. At this irrigation level (100% Etc), treatments T1, T2, and T4 were inferior to treatment T3 but provided 30.5% more ascorbic acid than the control treatment. The 50% Etc irrigation level increased the L* value in treatment T1 and decreased it in treatments T3 and T4 compared to the irrigation level of 100% Etc. Under water restriction, DHA treatments did not change the L* value, while under 100% Etc, the T1 treatment decreased the value of this parameter.

3.2. Mineral Nutrient Content

The effect of DHA treatments on mineral nutrient content in green pepper fruits was dependent on irrigation depths (p < 0.05). The water deficit (50% Etc) reduced the levels of Ca (Figure 3a), K (Figure 3b), and P (Figure 3d) in the DHA control and T1 treatments but did not change the levels of these nutrients in the other treatments compared to the 100% Etc irrigation level. The 50% Etc irrigation level reduced the Mg (Figure 3c) and Fe (Figure 3f) contents in the treatments’ control, T1, and T4 and the B contents (Figure 3e) in the T4 treatment. The water deficit reduced the levels of Mn (Figure 3g) and Zn (Figure 3h) in treatments T1 and T4. On overage, the water deficit reduced the levels of Ca by 13.2%, Mg by 8.5%, P by 8.5%, K by 8.6%, Mn by 10.5%, Fe by 12.2%, B by 12.0%, and Zn by 11.5% in the fruits.

Within each irrigation level, the most prominent DHA treatments were T2 and T3. Under the 50% Etc irrigation level, the T2 treatment increased the contents of K, Mg, Fe, and Mn, the T3 treatment increased the contents of Ca, K, Mg, P, B, and Fe, and the T4 treatment increased the Ca contents in green peppers. Under a 100% Etc water supply, the T2 treatment increased the Ca and Zn contents and the T4 treatment increased the Ca and Mn contents. Compared to the control treatment, the most notable increases occurred for Ca (69%) with the T4 treatment, under the level of 50% Etc, and for zinc (57%), with the T2 treatment, under 100% Etc.

There was a significant interaction between irrigation depths and DHA treatments for the general fruit quality index (FQI) (Figure 4). The water deficit decreased the FQI in the control, T1, and T4 treatments, while in the other DHA treatments, there was no difference between the irrigation levels for this variable. At the irrigation level of 50% Etc, treatments T2, T3, and T4 increased the FQI value by 8.5%, 12.3%, and 10.1%, respectively, in relation to the control treatment; however, these treatments were similar to each other. At the irrigation level of 100% Etc, treatments T1, T2, T3, and T4 increased the FQI in relation to the control treatment.

4. Discussion

4.1. Physicochemical Attributes and Color Parameters

Among the evaluated physical–chemical attributes (ascorbic acid content, pH, titratable acidity (TA), soluble solids (SS), and SS/TA ratio) and physical color parameters (L*, C*, and Hue angle), only the ascorbic acid and the L* parameter were affected by water deficit and DHA treatments. Water deficit increased the ascorbic acid in the treatments’ control and T4. Water stress also increased the ascorbic acid content in mango [13], maize [51], and tomato [52]. Ascorbic acid plays crucial roles in plant metabolism, such as protecting cells against oxidative damage and acting as a cofactor for enzymes involved in the production of flavonoids and phytohormones [51,53]. The increase in ascorbic acid content under water stress, therefore, is a plant response to adjust to the stress condition, playing an important role in detoxifying reactive forms of oxygen (ROS) and thus avoiding damage to the plant’s general metabolism [54,55,56].

Contrary to the results obtained in this present study, an increase in the levels of SS, TA, and the SS/TA ratio and the changes in the color parameters C* and Hue angle, in fruits from plants that were subjected to water deficit, was observed in other works [56]. The increase in SS content, TA (titratable acidity), and the color parameters in fruits under water deficit is generally attributed to the increase in the synthesis of metabolites and the increased activity of hexokinase enzymes due to the senescence process [57] and the loss of water by the fruit [58]. However, in eggplant, water restriction (50% Etc) decreased ascorbic acid levels, the C* parameters, and Hue angle [59]. According to these authors, this decrease was probably due to the decrease in the photosynthetic rate and, consequently, in photoassimilates and pigments. In this present study, under water restriction, the triggering processes to increase the levels of SS, TA, and the SS/TA ratio and the changes in the C* parameters and Hue angle were probably counterbalanced by the decrease in the production of photoassimilates, with the decrease in the photosynthetic rate. In this sense, an increase or decrease observed in the levels of metabolites (SS, organic acids, ascorbic acid, and carotenoids) in tomato fruits grown under water deficit (60% Etc) was dependent on the stage of fruit maturation (green or ripe) and the metabolites considered [60].

The DHA treatments affected only the ascorbic acid levels and, slightly, the fruit luminosity (L*). As already highlighted, ascorbic acid levels were the attribute most sensitive to water deficit. For this attribute, the similarity of the values obtained between the irrigation regimes (50% Etc and 100% Etc) in treatments T1 (foliar ZnSO₄), T2 (foliar ZnONPs), and T3 (foliar ZnONPs + PGPB) indicates that Zn applied as ZnSO₄ or ZnONPs possibly attenuates the negative effects of water deficit on ascorbic acid biosynthesis, which was enhanced by the inoculation of plants with BPCP. ZnONPs at a dose of 500 mg L⁻¹ increased the antioxidant activity in peppers in the germination phase [8] and ascorbic acid content in bell pepper fruits applied at a dose of 30 mgL⁻¹ via foliar [13]. The application of Zn as ZnSO₄ also increased SS contents in red pepper [29]. In plants, Zn performs important functions such as activating enzymes that protect against oxidative stress (such as superoxide dismutase), synthesizing tryptophan, which is a precursor of IAA (indole acetic acid), and acting on cell division and the integrity of the cell membrane [7,18,21]. In turn, the synergistic effect of PGPB with Zn may be due to the increase in the biological activity of the soil and/or production of plant growth-stimulating substances in the rhizosphere, such as organic acids, hormones, and enzymes, which can favor the photosynthetic rate and photoassimilates [25,26,61].

4.2. Mineral Nutrient Content

Among the minerals evaluated, green pepper exhibited higher levels of K, P, Mg, and Ca, in that order, followed by Fe, B, Zn, and Mn. Depending on the ADH treatments, the levels of these minerals were considerably reduced by the water deficit. Water deficit (80% Etc) reduced the contents of K and Ca in pepper [62] and decreased the contents of Mg, K, Cu, and Zn in tomato under 50% Etc [59] and Ca and B in strawberry under 70% Etc [63]. The process of nutrient accumulation in fruits depends on factors such as the genetic potential of the species, supply via soil or leaves, and nutrient mobility in the phloem [7]. Nutrient supply via soil depends on many factors, including soil moisture [64]. Mass flow and diffusion are the two main mechanisms that deliver mineral nutrients to plant roots [7]. The decrease in mineral nutrient content in pepper fruits was possibly due to the lower amount of these nutrients in contact with the plant’s roots, since Ca, Mg, K, Fe, Mn, B, and Zn reach the roots, mainly by mass flow, while K and P reach the roots by diffusion [7,64].

Under water deficit, treatments T2 (ZnONPs via leaf + PGPB) and T3 (ZnSO₄ via soil+PGPB) had a greater influence on the mineral composition of pepper fruits, since they increased the levels of K, Ca, Mg, P, Fe, B, and Mn. On the other hand, when there was no water restriction, these treatments (except for Ca and Zn) did not have the same performance in relation to the control, that is, only the T4 treatment (ZnSO₄ via leaf) increased the Mn and B contents. The treatments T2, T3, and T4 clearly increased the Zn concentration to the irrigation level of 100% Etc, but this did not occur under water deficit. Foliar application of ZnONPs increased the contents of K, Ca, P, Mg, and Zn in pepper fruits [13]. The role of Zn in cell division and the growth of fine roots contributes to a greater absorption of water and nutrients by the pepper plants and their subsequent translocation to the fruits [65,66,67]. The presence of PGPB in treatments T2 and T3 enhanced the beneficial effect of zinc on the accumulation of nutrients in pepper fruits. The role of beneficial microorganisms such as Bacillus subtilis and B. amyloliquefaciens in mitigating water stress in various crops appears to be due to their direct effects on plants and indirectly on the soil [24,25,26]. These bacteria possibly contributed to increasing the absorption of water and nutrients by peppers, due to the increase in the photosynthetic rate with a better adaptation of metabolism and osmotic adjustment under water deficit [68,69] and/or due to the increase in the solubility of poorly soluble forms of nutrients in the soil as well as by increasing the biological activity of the soil [26].

The FQI index, as a measure of the general fruit quality, revealed, in a summarized way, the negative effect of water deficit on the quality of pepper fruits and the role of Zn as an attenuator, both in the form of ZnONPs and in the form of ZnSO₄, as well as PGPB. This effect was more pronounced in treatments T2 and T3, in which no significant differences were observed between the irrigation levels tested. On the other hand, the superiority of treatments T2, T3, and T4 for the FQI index revealed the stimulating effect of Zn and BPCP, even under adequate irrigation (100% Etc).

Finally, it is worth highlighting that the first hypothesis considered in this work was confirmed for the ascorbic acid content and the mineral element content. The second hypothesis can be considered confirmed based on the mineral nutrient content and the general quality index (FQI) and considering that the dose of Zn supplied via ZnONPs was 10 times lower than the dose of Zn via ZnSO_4*7H₂O, thus proving the highest efficiency of ZnONPs. Although water deficit increased the ascorbic acid content in fruits, the lack of water decreased the overall quality of fruits, especially in terms of mineral nutrients, especially when considering the role of these nutrients in human nutrition [69].

5. Conclusions

The principal hypothesis of this work was that water deficit would alter the nutritional quality of green pepper fruits and that the supply of zinc, associated or not with beneficial bacteria, would attenuate the possible negative effects of water scarcity on fruit quality. The study reveals significant findings regarding the effects of water deficit and zinc treatments on the nutritional quality of peppers. Water deficit increased ascorbic acid levels in green pepper fruits, without altering soluble solids’ levels, color parameters (C* and Hue angle), and titratable acidity but decreased mineral nutrient levels. The application of Zn via foliar, in the form of zinc oxide nanoparticles (ZnONPs) or zinc sulfate (ZnSO₄), mitigated the negative effects of water deficit on the quality of pepper fruits. The ZnONPs’ source was more efficient than the ZnSO₄ source. The water deficit alleviating effect of both zinc sources was enhanced when the plants were inoculated with Bacillus subtilis and B. amyloliquefaciens.

The findings of this present work, together with other research, are important in terms of new technological perspectives for adapting plants to water deficit, whether in a semi-arid climate or in climates that are already suffering from the effects of climate changes.