Evaluating Growth, Biochemical, Physiological and Yield Responses in Maize with Activated Biochar under different moisture conditions: A Field Study

doi:10.21203/rs.3.rs-5021191/v1

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Evaluating Growth, Biochemical, Physiological and Yield Responses in Maize with Activated Biochar under different moisture conditions: A Field Study

https://doi.org/10.21203/rs.3.rs-5021191/v1

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published 23 Oct, 2024

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Amidst depleting water resources, rising crop water needs, changing climates, and soil fertility decline from inorganic modifications of soil, the need for sustainable agricultural solutions has been more pressing. The experimental work aimed to inspect the potential of organically activated biochar in improving soil physicochemical and nutrient status as well as improving biochemical and physiological processes, and optimizing yield-related attributes under optimal and deficit irrigation conditions. The field experiment with maize crop was conducted in Hardaas Pur (32°38.37'N, 74°9.00'E), Gujrat, Pakistan, from March to June in consecutive years, 2023 and 2024. The experiment involved the use of DK-9108, DK-6321, and Sarhaab maize hybrid seeds, with five moisture levels of evapotranspiration (100% ETC, 80% ETC, 70% ETC, 60% ETC, and 50% ETC) maintained throughout the crop seasons. Furthermore, activated biochar was applied at three levels: 0 tons/ha (no biochar), 5 tons per hectare, and 10 tons per hectare. The study's findings revealed significant improvements in soil organic matter, bulk density, nutrient profile and total porosity with biochar supplementation in soil. Maize plants grown under lower levels of ETC in biochar supplemented soil had enhanced membrane stability index (1.6 times higher) increased protein content (1.4 times higher), reduced malondialdehyde levels (0.7 times lower), improved antioxidant enzyme activity (1.3 times more SOD and POD activity, and 1.2 times more CAT activity), improved relative growth (1.05 times more) and enhanced yield parameters (grain yield 26% more) than control. Additionally, among the two biochar application levels tested, the 5 tons/ha dose demonstrated superior efficiency compared to the 10 tons/ha biochar dose.

Biological sciences/Plant sciences/Plant physiology

Earth and environmental sciences/Environmental sciences

Earth and environmental sciences/Environmental sciences/Environmental impact

Activated Biochar

Maize

Water Stress

Crop Evapotranspiration

Penman-Monteith Equation

Global environmental challenges are intensifying due to urbanization, altering climatic patterns, and exponential population growth; Pakistan is particularly vulnerable to the repercussions of these developments^1,2. Water scarcity is one of the consequences of these unusual weather patterns, mostly brought on by the increase in temperature. This has a direct impact on quality and quantity of agricultural productivity³. Drought conditions have become increasingly prevalent worldwide over the last century⁴. Numerous regions in Pakistan have been confronted with drought conditions ranging from moderate to severe⁵. The Pakistan Council of Research in Water Resources (PCRWR) predicts Pakistan will experience its worst water shortage by 2025⁶. Because of rising crop water demand and decreasing water supply, ensuring a sufficient food supply becomes an enormous challenge in the long run⁷. Elevated levels of CO₂ and temperature stimulate increased evapotranspiration; consequently, plants demand a greater quantity of water to meet their fundamental growth requirements⁸. Moreover, rising food demand can be met by overharvesting, but this can lead to soil deterioration and soil infertility if harvesting and crop sowing are not managed appropriately⁹. A variety of soil amendments, including manure, organic matter, lime, and gypsum, are utilised for the purpose of enhancing soil fertility¹⁰. However, integrating biochar into the soil is the most efficient and environmentally beneficial method for preserving soil fertility¹¹. The biochar is generated through the process of pyrolysis of organic waste underneath anaerobic conditions and the resultant solid product with a highly recalcitrant carbon proportion is called biochar¹². Biochar has many potential benefits as a soil amendment it improves soil structure, decreases bulk density of soil, and neutralizes acidic soil¹³. It also improves pore volume and soil porosity, enhances soil cation exchange capacity¹⁴, and facilitates a positive change in soil organic matter¹⁵, and increases the plant's available water level by improving the water retention capability of soil^16,17. The biochar stores carbon in the organic matter of soil, thus mediating in carbon sequestration¹⁸. It also helps to reduce the release of greenhouse gases in atmosphere¹⁹. These are the reasons that give a clear win-win indication about the use of biochar for yield improvement of crops under drought conditions²⁰.

To augment the efficacy of utilization of biochar in soil modification, the activation of biochar is an effective approach. The activation of biochar improves its surface area, pore size, and absorption ability²¹. This process of activation can be physical²², chemical²³, or biological²⁴. Different types of gases and steam are used in physical methods to activate biochar, but they can produce some hazardous chemicals in the environment²⁵. For the chemical activation of biochar, zinc chloride²⁶, hydrogen peroxide²⁷, and sodium hydroxide²⁸ are used. These chemicals may increase the porosity of biochar¹⁴, but the disadvantages are cost-effectiveness and leakage of chemicals during the process. The organic activation of biochar by extracellular enzymes secreted by microbes effectively increases the pore size and biochar surface area²⁹.

Maize is grown widely for food, medicine, and industrial purposes³⁰. As per the International Production Assessment Division (IPAD) of United States Department of Agriculture (USDA), the global production of maize was 1.16 billion MT in 2022 which is 8 percent lower than the previous year, while Pakistan comes at 15th number in the production ranking with annual maize yield of 9.2 million MT, which is less than one percent of global maize production. Pakistan’s maize yield has decreased by 9.4% (5.8 tons/ha) since the last year’s yield was 6.4 tons/ha. According to USDA, the decline in corn production in the year 2022 was due to delayed planting and a recent drought scenario. Therefore, to minimize the effects of drought on food security, using activated biochar could be a beneficial approach to handle the declining yield due to water stress. Due to increased agricultural activities and crop production to fulfill the needs of food for an increasing population, the soil is becoming less fertile, and its water retention capacity is getting poor³¹. Moreover, semi-arid and arid areas are becoming vulnerable to water stress due to changing climatic patterns³². Therefore, an easily accessible approach should be devised that can increase the yield and, at the same time, prevent soil from degradation. Thus, using organically activated biochar for maize under drought conditions could be helpful in problems addressed earlier. The fundamental goals of this research are to evaluate the potential of organically activated biochar implementation for improving soil quality and maize growth, biochemical, physiological, and yield-related attributes under optimum and deficit irrigation. And, to estimate the suitable level of activated biochar amendment for maize grown under water stress.

2.1. Biochar Production and Activation

Arabic tree (Acacia nilotica L.) timber chips were utilized to produce biochar. The reactor was heated at an average ramp of 20 ℃ until reaching 450 ℃, sustaining pyrolysis for three hours. The surface characteristics of biochar were further modified by the activation process, through a blend with vermicompost tea and perlite (1:1:1 ratio). For accelerated activation, a 2-liter liquid molasses solution was included, and the mixture was blended daily to clinch proper aeration. This process continued for two weeks until excess moisture disappeared, yielding a glossy black solid soil amendment.

2.2. Characterization of Research Site

The experiment was conducted from February to June, in 2023 and 2024, in the village Hardaas Pur, Gujrat, Pakistan (32°38.37’N, 74°9.00’E) to determine the optimal level of activated biochar under varying moisture conditions. The soil of the research site was a loam with little higher clay ratio (280 g/kg silt, 390 g/kg clay and 330 g/kg sand). Soil pH at the 0.00–0.15-meter layer was determined by ISO 10390 (2021) and soil EC was determined by the method of Rayment & Higginson³³. Elemental and structural analysis of soil samples (Fig. 1) was perfumed by SEM (scanning electron microscope) JSMS910 (JEOL, Japan). Soil saturation percentage, bulk density by core method, particle density, soil total porosity, soil organic matter, nitrogen, available potassium and phosphorus, oxidizable carbon and total organic carbon were determined by the Estefan method³⁴. The water content retention curve was fitted by using a non-linear Van Gunechten model³⁵ by adjusting the van Gunechtan parameters utilizing SWRC Excel solver function developed by Anlauf³⁶. The saturated hydraulic conductivity was determined by constant head method (Klute 1965). The unsaturated hydraulic conductivity was estimated by the Mualem-van Genuchten Model³⁵.

2.3. Experiment Design and Treatments

The successive field experiments were managed in the village Hardaas Pur, Gujrat, Pakistan (32°38.37’N, 74°9.00’E) to determine the optimal level of activated biochar under varying moisture conditions. Maize hybrids (DK-9108, DK-6321, and Sarhaab) seeds were sourced from Bayer, Corporate Lahore, Pakistan. Biochar was applied at three levels in the 20cm of soil top layer: no biochar (control, 0 tons per hectare), 5 tons per hectare, and 10 tons per hectare. The experiment utilized a split-plot design with 45 subplots (2 m by 2 m), each comprising three rows spaced 30 cm apart. Each maize hybrid was sown in a separate subplot, with seeds planted at a level of 3–5 cm and a frequency of 14 seeds per row. Experiment consists of 45 treatments with five moisture levels (100%, 80%, 70%, 60%, and 50% ETC) and three biochar application rates (0 t/ha, 5 t/ha, and 10 t/ha) as outlined in Table 1.

Table 1

Experiment Treatments Repeated in triplicates with three maize hybrid DK-9108, DK-6321 and Sarhaab
Sr. No	Abbreviation	Treatment
1	T1	ETC 100%, 0 tons biochar
2	T2	ETC 80%, 0 tons biochar
3	T3	ETC 70%, 0 tons biochar
4	T4	ETC 60%, 0 tons biochar
5	T5	ETC 50%, 0 tons biochar
6	T6	ETC 100%, 5 tons biochar
7	T7	ETC 80%, 5 tons biochar
8	T8	ETC 70%, 5 tons biochar
9	T9	ETC 60%, 5 tons biochar
10	T10	ETC 50%, 5 tons biochar
11	T11	ETC 100%, 10 tons biochar
12	T12	ETC 80%, 10 tons biochar
13	T13	ETC 70%, 10 tons biochar
14	T14	ETC 60%, 10 tons biochar
15	T15	ETC 50%, 10 tons biochar
ETC is indicating evapotranspiration

2.4. Crop Water Management and Irrigation Strategy

The crop water need was computed by the following equation projected by Food and Agriculture Organization for United States (FAO).

IN$\:\:=\text{E}\text{T}\text{C}-\text{P}\text{e}$

Where; IN is the net water requirement, ETc is crop evapotranspiration, and Pe represents effective rainfall. Moreover, the evapotranspiration was calculated by using this expression³⁷,

$$\:\text{E}\text{T}\text{c}\:={\text{E}\text{T}}_{\text{O}}\:\times\:\:{\text{K}}_{\text{C}}$$

ETo is reference evapotranspiration, and Kc is crop coefficient. Reference evapotranspiration was calculated by using Penman Monteith Eq. 3⁸,

$$\:\text{E}\text{T}\text{o}\:=\:\frac{0.41{\Delta\:}\left(\text{R}\text{N}-\text{G}\right)+{\gamma\:}\left[900/\left(\text{T}+273\right)\right]\:\text{U}\text{2\:}\left(\text{e}\text{s}-\text{e}\text{a}\right)}{{\Delta\:}+{\gamma\:}\left(1+0.3\:\text{U}\text{2}\right)}$$

Where; mean daily temperature at a height of 2 meters (°C), RN symbolizes net radiation, G represents the heat flux of soil in MJm²/day, Δ represents gradient of the vapor pressure-temperature curvature in k.Pa (°C) ⁻¹, γ is psychometric constant (kPa°C^− 1), U2 denotes daily wind rate at 2-meter elevation in meters per second, 'es' denotes the average saturation vapor pressure, while 'ea' signifies the real vapor pressure.

Calculations for ETo were performed in CROPWAT 8.0, entering geographic information (location, year, altitude (m), latitude (°), longitude (°), and weather variables including minimum temperature (°C), maximum temperature (°C), relative humidity (%), speed of wind (m/s), and sunlight hours. These variables for the experimental site were obtained from the Earth Observing System and Data Analytics (EOSDA, 2023), a satellite-based crop monitoring platform (Fig. 2). Crop stage-specific coefficients (Kc values) for maize, derived from³⁹, were employed in the experiment, varying from 0.35 to 1.20 based on the crop stage. Effective rainfall was estimated using the FAO/AGLW dependable rain formula in CROPWAT 8.0,

$$\:\text{P}\text{e}=(0.6\:\times\:\text{P})-3.33$$

If P ≤ 70mm

Pe is effective rainfall, while P is the total precipitation in mm.

2.5. Relative Growth and Yield Analysis

2.5.1. Relative increase in leaf area

The proportional growth in leaf area was deduced by the following expression by⁴⁰

L_1, Leaf area of initial harvesting, L_2, Leaf area of the next harvesting, T₁, Number of days of initial harvesting, and T_2, Number of days of the next harvesting

For leaf area (cm³), pictures of plant leaves were taken and processed in ImageJ software (NIH, v. 1.8.0_345 64-bit, Bethesda, MD, USA) using polygon selection in the software⁴¹.

$$\:\text{R}\text{e}\text{l}\text{a}\text{t}\text{i}\text{v}\text{e}\:\text{c}\text{h}\text{a}\text{n}\text{g}\text{e}\:\text{i}\text{n}\:\text{l}\text{e}\text{a}\text{f}\:\text{f}\text{r}\text{e}\text{s}\text{h}/\text{d}\text{r}\text{y}\:\text{w}\text{e}\text{i}\text{g}\text{h}\text{t}\:\left(\frac{\text{g}}{\text{d}\text{a}\text{y}}\right)=\frac{{\text{log}}_{\text{e}}{\text{w}}_{2\:-\:}{\text{log}}_{\text{e}}{\text{w}}_{1}}{{\text{T}}_{2}-{\text{T}}_{1}}$$

w_1, Leaf fresh weight of the initial harvesting, and w_2, Leaf fresh weight of the next harvesting

2.5.2. Relative increase in Shoot/Root Weight

The proportional expansion in root and shoot fresh and dry weight was calculated by using the following formula by⁴²;

$$\:\text{R}\text{e}\text{l}\text{a}\text{t}\text{i}\text{v}\text{e}\:\text{i}\text{n}\text{c}\text{r}\text{e}\text{a}\text{s}\text{e}\:\text{i}\text{n}\:\text{s}\text{h}\text{o}\text{o}\text{t}\:\text{o}\text{r}\:\text{r}\text{o}\text{o}\text{t}\:\text{f}\text{r}\text{e}\text{s}\text{h}/\text{d}\text{r}\text{y}\:\text{w}\text{e}\text{i}\text{g}\text{h}\text{t}\:\left(\frac{\text{g}}{\text{d}\text{a}\text{y}}\right)=\frac{{\text{log}}_{\text{e}}{\text{w}}_{2\:-\:}{\text{log}}_{\text{e}}{\text{w}}_{1}}{{\text{T}}_{2}-{\text{T}}_{1}}$$

While, the fresh and dry weight of root and shoot was recorded by electrical weighing balance.

2.5.3. Relative Growth Rate

The relative growth rate was deduced by the formula given below⁴³,

$$\:\text{R}\text{e}\text{l}\text{a}\text{t}\text{i}\text{v}\text{e}\:\text{g}\text{r}\text{o}\text{w}\text{t}\text{h}\:\text{r}\text{a}\text{t}\text{e}\:\left(\frac{\text{g}}{\text{d}\text{a}\text{y}}\right)=\frac{{\text{log}}_{\text{e}}{\text{W}}_{2\:-\:}{\text{log}}_{\text{e}}{\text{W}}_{1}}{{\text{T}}_{2}-{\text{T}}_{1}}$$

W_1, Initial dry matter of initial harvesting, and W_2, Initial dry matter of the next harvesting

2.5.4. Net Assimilation Rate

The net assimilation rate of the maize plants was calculated by⁴³.

$$\:\text{N}\text{e}\text{t}\:\text{a}\text{s}\text{s}\text{i}\text{m}\text{i}\text{l}\text{a}\text{t}\text{i}\text{o}\text{n}\:\text{r}\text{a}\text{t}\text{e}\:\text{o}\text{r}\:\text{E}\:\left(\frac{\text{m}\text{g}}{{\text{c}\text{m}}^{2}}\right)=\frac{1}{{\text{l}\text{o}\text{g}}_{\text{e}}{\text{L}}_{2}-\:{\text{l}\text{o}\text{g}}_{\text{e}}{\text{L}}_{1}}\times\:\:\frac{{\text{W}}_{2\:-\:}{\text{W}}_{1}}{{\text{T}}_{2}-\:{\text{T}}_{1}}$$

W_1, Primary dry matter of plant, W_2, Final dry matter of plant, L_1, Primary leaf area, L_2, Final leaf area, and T2 – T1, Time interval difference between harvestings

Plant sampling for relative growth analysis was taken three times with the interval of 15 days between each sampling, starting from the starting of vegetative stage.

2.6 Physiological and Biochemical Analysis

2.6.1. Cell membrane stability index assessment

The cell membrane stability index was calculated by the method of Premachandra et al.⁴⁴, and the modifications by Sairam⁴⁵. The Bradford’s method⁴⁶ was used to find the protein content. Lipid peroxidation or malondialdehyde content in the leaf was determined by following the method of Prochazkova et al.⁴⁷. The 2 ml of 0.1% trichloroacetic acid (TCA) was added to (100 mg) grounded leaf material. Then, the mixture was centrifuged. The top layer was separated and 4 ml of 0.5% TBA (thiobarbituric acid) and 1 ml of 20% TCA was mixed in it and warmed in a water bath for about 30 minutes at 95°C. The absorbance values were noted at 600 nm, 532 nm, and 440 nm, the malondialdehyde level was evaluated by using the following formula:

$$\:\text{M}\text{D}\text{A}\:=\:\frac{\left[\right({A}_{b}532\:-{A}_{b}600)-[({A}_{b}440-{A}_{b}600)\left(\frac{\text{M}\text{o}\text{l}.\:\text{A}\:\text{o}\text{f}\:\text{S}\text{u}\text{c}\text{r}\text{o}\text{s}\text{e}\:\text{a}\text{t}\:532\:\text{n}\text{m}}{\text{M}\text{o}\text{l}.\:\text{A}\:\text{o}\text{f}\:\text{S}\text{u}\text{c}\text{r}\text{o}\text{s}\text{e}\:\text{a}\text{t}\:440\:\text{n}\text{m}}\right)]}{15700}\:\times\:{10}^{6}$$

2.6.2. Antioxidant activity assessment

To assess antioxidant enzyme activity, leaf material was pulverized in a clean mortar using 5 ml phosphate buffer by keeping in an ice bath. Following this, the mixture was separated at 13,000 g for 20 minutes at 4°C, and the resulting top layer was analysed for antioxidant enzyme activity.

2.6.3. Superoxide dismutase activity

The photochemical reduction of nitro blue tetrazolium (NBT) inhibition was measured to assess the activity of SOD by using the Beauchamp & Fridovich⁴⁸ approach. Each reaction sample included 0.5 ml phosphate buffer, 0.2 ml of triton X, 0.1 ml of riboflavin (0.002 mM), 0.1 ml of enzyme extract, and 0.1 ml of methionine (13 mM). At 560 nm, the samples' absorbance was measured by UV spectrophotometer. To calculate SOD activity, the following formulas was used:

$$\:\text{I}\text{U}\:=\:\frac{\text{a}\text{b}\text{s}\text{o}\text{r}\text{b}\text{a}\text{n}\text{c}\text{e}}{50}\times\:100$$

IU is a global unit for enzyme activity

$$\:\text{S}\text{O}\text{D}\:=\:\frac{\text{I}\text{U}}{\text{m}\text{a}\text{s}\text{s}\:\text{o}\text{f}\:\text{p}\text{r}\text{o}\text{t}\text{e}\text{i}\text{n}}$$

2.6.4. Peroxidase activity

The process designated by Vetter et al.⁴⁹ was employed for the assessment of peroxidase activity, incorporating adjustments as suggested by Gorin & Heidema⁵⁰. The reaction combination consisted of 0.2 ml of enzyme extract, 1.8 ml of a 100 mM phosphate buffer (pH 7), 0.3 ml of 3 mM H₂O₂, and 0.1 ml of an aqueous solution containing 1% w/v p-phenylenediamine. The variations' absorbance was recorded for three minutes at 485 nm by UV spectrophotometer, and the peroxidase activity was evaluated using the formula:

$$\:\text{P}\text{O}\text{D}\:=\:\frac{\varDelta\:485}{\text{m}\text{g}\:\text{o}\text{f}\:\text{p}\text{r}\text{o}\text{t}\text{e}\text{i}\text{n}}$$

2.6.5. Catalase activity

To evaluate the catalase activity within the leaf sample, a reaction blend (3 ml) was organised, consisting of 0.2 ml enzyme extract, 2.6 ml potassium phosphate buffer with a pH of 7.2, and 0.2 ml of 15 mM H₂O₂.To stop the reaction, 2 ml of titanium reagent was added after 5 minutes. After centrifuging, the mixture for 10 minutes, values of absorbance at 410 nm were taken by UV spectrophotometer. This formula was used to determine the catalase activity:

$$\:\text{C}\text{A}\text{T}\:=\:\frac{\varDelta\:410}{\text{m}\text{g}\:\text{o}\text{f}\:\text{p}\text{r}\text{o}\text{t}\text{e}\text{i}\text{n}}$$

2.7. Yield Analysis

After harvesting of maize crop, following yield parameters were recorded such as cob length (cm), cob weight (g), and kernel number per cob, cob yield per plant, hundred grain weight, stover yield, and apparent water productivity.

The cob length (cm) was measured by processing the images of corn cobs in a java-based software, ImageJ (NIH version 1.8.0_345 64-bit, Bethesda, MD, USA)⁴¹. The cob weight (g) and thousand seed weight (g) were measured by electrical weighing balance, kernel number per cob were counted manually. Stover yield was calculated as the dry biomass after harvesting the cobs.

The grain yield of a plant was estimated by taking the weight of seeds per cob. Further, yield per hectare was estimated by multiplying the plant density according to layout by grain yield per plant.

$$\:\text{Y}\text{i}\text{e}\text{l}\text{d}\:\left(\frac{\text{k}\text{g}}{\text{h}\text{a}}\right)=\text{G}\text{r}\text{a}\text{i}\text{n}\:\text{y}\text{i}\text{e}\text{l}\text{d}\:\text{p}\text{e}\text{r}\:\text{p}\text{l}\text{a}\text{n}\text{t}\:\times\:\text{p}\text{l}\text{a}\text{n}\text{t}\text{i}\text{n}\text{g}\:\text{d}\text{e}\text{n}\text{s}\text{i}\text{t}\text{y}$$

$$\:\text{P}\text{l}\text{a}\text{n}\text{t}\text{i}\text{n}\text{g}\:\text{d}\text{e}\text{n}\text{s}\text{i}\text{t}\text{y}\:\text{p}\text{e}\text{r}\:\text{h}\text{e}\text{c}\text{t}\text{a}\text{r}\text{e}=\:\frac{\text{A}\text{r}\text{e}\text{a}\:\text{o}\text{f}\:\text{p}\text{l}\text{o}\text{t}\:\left({\text{m}}^{2}\right)}{\text{R}-\text{R}\:\text{S}\text{p}\text{a}\text{c}\text{i}\text{n}\text{g}\:\left({\text{m}}^{2}\right)}$$

The next formula was used to calculate the evident water productivity by following Shabbir et al.⁵¹

$$\:\text{A}\text{p}\text{p}\text{a}\text{r}\text{e}\text{n}\text{t}\:\text{w}\text{a}\text{t}\text{e}\text{r}\:\text{p}\text{r}\text{o}\text{d}\text{u}\text{c}\text{t}\text{i}\text{v}\text{i}\text{t}\text{y}\:\left(\text{k}\text{g}\:{/m}^{3}\right)=\text{A}\text{W}\text{P}=\frac{\text{G}\text{r}\text{a}\text{i}\text{n}\:\text{Y}\text{i}\text{e}\text{l}\text{d}\:\left(\text{k}\text{g}\:\right)}{\text{I}\text{r}\text{r}\text{i}\text{g}\text{a}\text{t}\text{i}\text{o}\text{n}\:\text{w}\text{a}\text{t}\text{e}\text{r}\:\text{s}\text{u}\text{p}\text{p}\text{l}\text{y}\:\left({m}^{3}\right)}$$

2.8. Statistical Analysis

The statistical analyses for the study were executed by using Minitab software (Ver. 21.2). Three-Way Analysis of Variance (ANOVA) in the framework of a General Linear Model (GLM) was employed to measure the influence of various growth, biochemical, physiological, and yield attributes. The main purpose of this analysis was to observe how the factors of amendments, evapotranspiration (ETC) levels, and maize hybrid influenced the growth, biochemical, and yield related parameters. To further explore significant differences among factor levels, Tukey Pairwise Comparison tests with a 95% Confidence Interval were conducted. This post-hoc analysis allowed a detailed examination of specific differences between factor levels while maintaining a high level of confidence. The covariance and correlation analysis of variables were performed in Visual Studio Code using Python libraries (kernel = Python 3.11.9) by constructing Principal Component Analysis (PCA) biplot and heatmap.

3.1. Soil characterization

The results of soil physicochemical properties are presented in Table 2. Soil bulk density and particle density were considerably lower (13–23%; 9–23%) in 5 T/ha and 10 T/ha biochar supplemented soil respectively. However, the highest total porosity was recorded in soil amended with 10 tons/ha biochar amended soil that is 26% higher than the soil with no biochar supplementation. The EC (13%-45%), organic matter, nitrogen, total organic carbon and oxidizable carbon significantly enhanced with biochar supplementation in the soil by 87–130%. Similarly, available phosphorus (5%-9%) and potassium (26–34%) also improved considerably in biochar supplemented soil. Biochar supplementation in soil caused no significant effects on C/N ratio but caused a bit increase in pH from 7.6 to 7.9 in contrast to non-supplemented soil. The SEM-EDX analysis of soil mineral profile indicates a considerable improvement in O, C, Mg, Si, K, Ca, and Fe by 17.3–21%, 34–63%, 3-7.3%, 2–4%, 2–7%, 1–5%, and 7–8% in 5 tons/ha and 10 T/ha biochar supplemented soil, respectively, in contrast to soil without biochar supplantation. However, titanium content decreased in biochar supplemented soil. The SEM images of the soil samples also (Fig. 1) revealed an array of macro, meso and micropores in biochar supplemented soil. The water content (cm³/cm³) at five pressure heads was determined by pressure plates extractors and the fitted curve (from pF 0 to pF 4.5) is shown in Fig. 3a. The biochar supplemented treatments showed more water retention content as compared to non-biochar supplemented one. The mean saturated hydraulic conductivity is seemed to be decreased in biochar amendments as compared to control. 66.72 cm/day, 43.83 cm/day, and 51.30 cm/day saturated hydraulic conductivity has been noted in 0 tons/ha, 5 tons/ha and 10 tons/ha treatments respectively. The fitted curve for unsaturated hydraulic conductivity is represented in Fig. 3b.

Table 2

Physicochemical and nutrient status of soil
Parameter	0 Tons per hectare	5 Tons per hectare	10 Tons per hectare
Saturation percentage (%)	30 ± 2 (c)	35 ± 1.5 (b)	40 ± 1.16 (a)
Bulk density (Mg m^− 3)	1.75 ± 0.042 (a)	1.53 ± 0.036 (b)	1.38 ± 0.05 (c)
Particle density (Mg m^− 3)	2.84 ± 0.22 (a)	2.58 ± 0.16 (b)	2.18 ± 0.28 (c)
Total porosity (%)	62 ± 5.2 (c)	75 ± 4.7 (ab)	78 ± 1.3 (a)
EC (ds m^− 1)	0.827 ± 0.031 (c)	0.93 ± 0.03 (b)	1.2 ± 0.13 (a)
pH	7.63 ± 0.015 (b)	7.68 ± 0.015 (b)	7.97 ± 0.06 (a)
Organic matter (%)	0.363 ± 0.032 (c)	0.69 ± 0.01 (b)	0.85 ± 0.06 (a)
Available phosphorus (mg kg^− 1)	6.67 ± 0.15 (b)	7 ± 0.06 (a)	7.3 ± 0.1 (a)
Available potassium (mg kg^− 1)	62.3 ± 2.5 (b)	78 ± 1 (a)	83 ± 2.7 (a)
Total organic carbon (%)	0.21 ± 0.019 (c)	0.4 ± 0.0058 (b)	0.49 ± 0.037 (a)
Oxidizable carbon (%)	0.158 ± 0.014 (c)	0.3 ± 0.004 (b)	0.37 ± 0.028 (a)
Nitrogen (%)	0.024 ± 0.002 (c)	0.046 ± 0.0001 (b)	0.056 ± 0.004 (a)
Oxygen (%)	20.55 ± 1.21 (b)	24.1 ± 0.95 (a)	24.89 ± 1.32 (a)
Carbon (%)	4.8 ± 0.13 (c)	6.42 ± 0.21 (b)	7.82 ± 0.14 (a)
Magnesium (%)	1.51 ± 0.04 (c)	1.56 ± 0.062 (ab)	1.62 ± 0.01 (a)
Aluminum (%)	10.05 ± 0.02 (a)	9.31 ± 0.01 (b)	10.05 ± 0.031 (a)
Silicon (%)	38.17 ± 0.45 (b)	39.06 ± 0.54 (a)	39.57 ± 0.47 (a)
Potassium (%)	4.43 ± 0.035 (c)	4.51 ± 0.034 (bc)	4.75 ± 0.047 (a)
Calcium (%)	6.26 ± 0.023 (b)	6.29 ± 0.032 (b)	6.56 ± 0.04 (a)
Titanium (%)	1.083 ± 0.12 (a)	0.85 ± 0.028 (b)	0.93 ± 0.032 (b)
Ferric (%)	9.18 ± 0.15 (c)	9.82 ± 0.02 (ab)	9.895 ± 0.013 (a)
Carbon/nitrogen ratio	8.75 ± 0.09 (a)	8.7 ± 0.12 (ab)	8.75 ± 0.17 (a)
± value indicates standard error of three replicates while different letter in parenthesis
indicates significant differences at 95% confidence interval.

3.2. Crop water balance

The water balance of maize during the spring seasons is pictured in Fig. 4. The effective rainfall during the season was 102.95 mm in 2023 season, and only 5.78 mm in 2024. The total water deficit during the crop duration was 576.8 mm in 2023 season, and 714.73 mm in 2024 season. The water deficit met by manual irrigation by 576.8 mm, 453.2 mm, 391.3 mm, 329.5 mm, and 267.7 mm in 2023 season, and 714.73 mm, 573.97 mm, 503.59 mm, 433.21 mm, 362.83 mm in 2024 season, respectively in 100% ETC, 80% ETC, 70% ETC, 60% ETC, and 50% ETC sub-plots. The water deficit is dependent on the environmental conditions as well as crop coefficient (stage of crop growth). It has been seen that the highest water deficit from the crop was noticed at the dough stage in both seasons (12th week on charts) where the crop coefficient was highest (Kc = 1.20) with the counteract of other variables, like humidity, temperature, wind speed, and sunlight duration.

3.3. Relative growth analysis

The relative increase in fresh and dry weight of maize root and shoot is presented in Table 3. It is noted that water stress resulted in the reduction of the relative gain in weight of maize root and shoot, while biochar-supplemented soil have shown a better relative increase in the weight of maize root (42% more) and shoot (34% more). The highest relative increase in fresh and dry weight of maize plant was noted in DK-9108 maize grown in 5 tons/ha biochar amended soil at 80% ETC. The relative increase in fresh and dry weight of maize leaf, and leaf area (Table 4) showed substantial variations (P < 0.05). It is noted that water stress reduced the relative increase in the weight of maize leaf and leaf area. At the same time, plants from biochar-supplemented soil have shown a better relative increase in the weight of maize leaf (45% more) and leaf area (54% more). The highest relative rise in fresh and dry weight of maize leaf was noted in DK-9108 maize grown in 5 tons/ha biochar amended soil at 80% ETC, and the maximum relative increase in leaf area was observed in Sarhaab maize under 5 ton/ha biochar amendment at 80% ETC. The net assimilation and relative growth rate of maize hybrids is presented in Table 4. Water stress lowered the net assimilation rate and relative growth rate in maize, whereas biochar-supplied treatments improved the net assimilation rate (46% higher) and relative growth rate (35% higher). The highest net assimilation rate was found in DK-9108 maize grown in 5 tons/ha biochar amended soil at 80% ETC, and the highest relative growth rate was found in Sarhaab in 5 tons/ha biochar treated soil at 80% ETC.

Table 3

Impact of Activated Acacia Wood Biochar Amendment on Relative Increase in Root and Shoot Growth of Three Maize Hybrids Under Varied Moisture Conditions
Treatment	Relative Increase in Root Fresh Weight (g/day)	Relative Increase in Root Dry Weight (g/day)	Relative Increase in Shoot Fresh Weight (g/day)	Relative Increase in Shoot Dry Weight (g/day)
V1, E 100%, 0T	0.088 ± 0.002 (k-m)	0.086 ± 0.002 (k-m)	0.099 ± 0.002 (k)	0.098 ± 0.002 (k)
V1, E 80%, 0 T	0.069 ± 0.003 (q-s)	0.067 ± 0.003 (q-s)	0.069 ± 0.002 (o-q)	0.067 ± 0.002 (o-q)
V1, E 70%, 0 T	0.05 ± 0.003 (u-w)	0.049 ± 0.003 (v-x)	0.06 ± 0.003 (q-r)	0.058 ± 0.003 (q-r)
V1, E 60%, 0 T	0.046 ± 0.003 (v-w)	0.045 ± 0.003 (w-x)	0.049 ± 0.002 (u-v)	0.047 ± 0.002 (u-v)
V1, E 50%, 0 T	0.033 ± 0.002 (x)	0.032 ± 0.001 (y)	0.037 ± 0.003 (x)	0.036 ± 0.003 (x)
V1, E 100%, 5 T	0.102 ± 0.002 (g-j)	0.101 ± 0.002 (g-j)	0.106 ± 0.003 (h-k)	0.105 ± 0.003 (h-k)
V1, E 80%, 5 T	0.119 ± 0.003 (a-c)	0.117 ± 0.003 (a-c)	0.122 ± 0.003 (b-e)	0.121 ± 0.002 (b-e)
V1, E 70%, 5 T	0.108 ± 0.002 (d-h)	0.106 ± 0.001 (d-h)	0.115 ± 0.003 (e-h)	0.114 ± 0.001 (e-h)
V1, E 60%, 5 T	0.081 ± 0.003 (m-o)	0.08 ± 0.003 (m-o)	0.074 ± 0.003 (m-o)	0.073 ± 0.001 (m-o)
V1, E 50%, 5 T	0.066 ± 0.003 (r-s)	0.065 ± 0.002 (r-s)	0.05 ± 0.002 (t-v)	0.049 ± 0.002 (t-v)
V1, E 100%, 10 T	0.095 ± 0.003 (j-k)	0.094 ± 0.003 (j-k)	0.101 ± 0.002 (j-k)	0.1 ± 0.003 (j-k)
V1, E 80%, 10 T	0.111 ± 0.003 (c-g)	0.109 ± 0.003 (c-g)	0.117 ± 0.003 (d-g)	0.115 ± 0.003 (d-g)
V1, E 70%, 10 T	0.103 ± 0.003 (f-j)	0.102 ± 0.003 (f-j)	0.108 ± 0.003 (g-j)	0.107 ± 0.002 (g-j)
V1, E 60%, 10 T	0.074 ± 0.003 (o-r)	0.073 ± 0.003 (o-r)	0.063 ± 0.003 (p-r)	0.062 ± 0.002 (p-r)
V1, E 50%, 10 T	0.055 ± 0.002 (t-v)	0.053 ± 0.002 (t-w)	0.041 ± 0.003 (v-x)	0.04 ± 0.002 (v-x)
V2, E 100%, 0 T	0.097 ± 0.003 (i-k)	0.095 ± 0.004 (i-k)	0.108 ± 0.003 (h-k)	0.106 ± 0.004 (h-k)
V2, E 80%, 0 T	0.078 ± 0.003 (n-q)	0.076 ± 0.003 (n-q)	0.082 ± 0.003 (l-m)	0.08 ± 0.002 (l-m)
V2, E 70%, 0 T	0.062 ± 0.002 (s-t)	0.061 ± 0.002 (s-t)	0.072 ± 0.003 (n-p)	0.071 ± 0.003 (n-p)
V2, E 60%, 0 T	0.057 ± 0.002 (t-u)	0.055 ± 0.002 (t-v)	0.064 ± 0.003 (p-r)	0.063 ± 0.004 (p-r)
V2, E 50%, 0 T	0.049 ± 0.003 (u-w)	0.048 ± 0.003 (v-x)	0.045 ± 0.002 (u-x)	0.043 ± 0.002 (u-x)
V2, E 100%, 5 T	0.112 ± 0.003 (c-f)	0.11 ± 0.002 (c-f)	0.119 ± 0.003 (d-f)	0.118 ± 0.003 (d-f)
V2, E 80%, 5 T	0.128 ± 0.003 (a)	0.126 ± 0.003 (a)	0.136 ± 0.003 (a)	0.134 ± 0.004 (a)
V2, E 70%, 5 T	0.116 ± 0.002 (b-d)	0.115 ± 0.002 (b-d)	0.129 ± 0.003 (a-c)	0.128 ± 0.003 (a-c)
V2, E 60%, 5 T	0.09 ± 0.002 (k-l)	0.089 ± 0.002 (k-l)	0.087 ± 0.003 (l)	0.085 ± 0.004 (l)
V2, E 50%, 5 T	0.075 ± 0.003 (o-q)	0.074 ± 0.003 (o-q)	0.071 ± 0.002 (o-p)	0.069 ± 0.002 (o-p)
V2, E 100%, 10 T	0.105 ± 0.002 (e-i)	0.103 ± 0.003 (e-i)	0.114 ± 0.003 (e-h)	0.112 ± 0.003 (e-h)
V2, E 80%, 10 T	0.116 ± 0.003 (b-d)	0.115 ± 0.003 (b-d)	0.125 ± 0.003 (b-d)	0.123 ± 0.003 (b-d)
V2, E 70%, 10 T	0.11 ± 0.003 (c-g)	0.109 ± 0.003 (c-h)	0.119 ± 0.003 (d-f)	0.118 ± 0.004 (d-f)
V2, E 60%, 10 T	0.082 ± 0.002 (l-o)	0.08 ± 0.002 (l-o)	0.072 ± 0.003 (n-p)	0.07 ± 0.003 (n-p)
V2, E 50%, 10 T	0.069 ± 0.002 (p-s)	0.068 ± 0.002 (p-s)	0.059 ± 0.001 (r-t)	0.058 ± 0.001 (r-t)
V3, E 100%, 0 T	0.091 ± 0.002 (k-l)	0.089 ± 0.003 (k-l)	0.104 ± 0.003 (i-k)	0.102 ± 0.004 (i-k)
V3, E 80%, 0 T	0.061 ± 0.003 (s-t)	0.059 ± 0.003 (s-u)	0.075 ± 0.003 (m-o)	0.074 ± 0.003 (l-n)
V3, E 70%, 0 T	0.055 ± 0.003 (t-v)	0.054 ± 0.002 (t-w)	0.066 ± 0.003 (o-r)	0.065 ± 0.002 (o-r)
V3, E 60%, 0 T	0.052 ± 0.003 (u-v)	0.05 ± 0.002 (u-w)	0.051 ± 0.003 (s-t)	0.049 ± 0.003 (s-u)
V3, E 50%, 0 T	0.042 ± 0.003 (w)	0.041 ± 0.003 (x)	0.04 ± 0.003 (w-x)	0.038 ± 0.003 (w-x)
V3, E 100%, 5 T	0.104 ± 0.003 (e-j)	0.102 ± 0.003 (e-j)	0.113 ± 0.002 (e-h)	0.112 ± 0.001 (e-h)
V3, E 80%, 5 T	0.122 ± 0.003 (a-b)	0.12 ± 0.003 (a-b)	0.13 ± 0.002 (a-b)	0.128 ± 0.002 (a-b)
V3, E 70%, 5 T	0.113 ± 0.003 (b-e)	0.111 ± 0.003 (b-e)	0.124 ± 0.003 (b-d)	0.123 ± 0.003 (b-d)
V3, E 60%, 5 T	0.085 ± 0.003 (l-n)	0.083 ± 0.003 (l-n)	0.08 ± 0.003 (l-n)	0.079 ± 0.003 (l-m)
V3, E 50%, 5 T	0.071 ± 0.004 (p-r)	0.07 ± 0.004 (p-r)	0.059 ± 0.002 (r-s)	0.058 ± 0.003 (r-s)
V3, E 100%, 10 T	0.101 ± 0.003 (h-j)	0.1 ± 0.003 (h-j)	0.107 ± 0.003 (h-k)	0.106 ± 0.003 (h-k)
V3, E 80%, 10 T	0.112 ± 0.003 (c-e)	0.111 ± 0.003 (c-e)	0.12 ± 0.003 (c-f)	0.119 ± 0.003 (c-f)
V3, E 70%, 10 T	0.106 ± 0.003 (e-i)	0.104 ± 0.002 (e-i)	0.112 ± 0.003 (f-i)	0.111 ± 0.003 (f-i)
V3, E 60%, 10 T	0.078 ± 0.003 (n-p)	0.077 ± 0.004 (n-p)	0.068 ± 0.002 (o-r)	0.066 ± 0.003 (o-r)
V3, E 50%, 10 T	0.061 ± 0.003 (s-t)	0.06 ± 0.003 (s-t)	0.049 ± 0.003 (u-w)	0.047 ± 0.003 (u-w)
Note: The different letters in parenthesis after standard deviation represent significant variations among the treatments, as determined by the Tukey test and 95% confidence interval.
Abbreviations: V1; DK-9108, V2; DK-6321, V3; Sarhaab, E; Evapotranspiration, T; tons biochar per hectare

Table 4

Impact of Activated Acacia Wood Biochar Amendment on Relative Increase in Leaf Growth, and Leaf Area of Three Maize Hybrids Under Varied Moisture Conditions
Treatment	Relative Increase in Leaf Fresh Weight (g/day)	Relative Increase in Leaf Dry Weight (g/day)	Relative Increase in Leaf Area (cm²/day)	NAR (g cm^− 2 day^− 1)	RGR (g/day)
V1, E 100%, 0 T	0.043 ± 0.002 (j-k)	0.042 ± 0.002 (l-m)	0.032 ± 0.001 (n-p)	4.61 ± 0.215 (m-o)	0.071 ± 0.004 (j-q)
V1, E 80%, 0 T	0.035 ± 0.003 (l-o)	0.033 ± 0.002 (n-q)	0.025 ± 0.001 (s-u)	3.95 ± 0.207 (i-k)	0.061 ± 0.004 (p-t)
V1, E 70%, 0 T	0.031 ± 0.002 (n-r)	0.029 ± 0.002 (p-t)	0.019 ± 0.001 (v-x)	3.74 ± 0.225 (l-n)	0.057 ± 0.005 (r-u)
V1, E 60%, 0 T	0.026 ± 0.002 (p-s)	0.025 ± 0.002 (s-u)	0.015 ± 0.001 (y-aa)	2.94 ± 0.132 (x)	0.051 ± 0.005 (t-v)
V1, E 50%, 0 T	0.02 ± 0.002 (s)	0.019 ± 0.002 (u)	0.01 ± 0.001 (ab)	2.35 ± 0.246 (u-x)	0.035 ± 0.003 (w)
V1, E 100%, 5 T	0.056 ± 0.003 (g-i)	0.053 ± 0.002 (i-k)	0.039 ± 0.001 (j-m)	6.37 ± 0.291 (w-x)	0.082 ± 0.003 (h-j)
V1, E 80%, 5 T	0.072 ± 0.002 (c-d)	0.071 ± 0.002 (c-e)	0.054 ± 0.001 (c-e)	8.14 ± 0.123 (u-x)	0.111 ± 0.003 (a-c)
V1, E 70%, 5 T	0.06 ± 0.002 (f-h)	0.058 ± 0.002 (g-i)	0.042 ± 0.001 (i-k)	7.62 ± 0.16 (r-v)	0.097 ± 0.005 (d-f)
V1, E 60%, 5 T	0.038 ± 0.001 (k-m)	0.037 ± 0.001 (m-o)	0.029 ± 0.001 (p-r)	4.25 ± 0.228 (t-w)	0.077 ± 0.004 (h-n)
V1, E 50%, 5 T	0.031 ± 0.002 (m-r)	0.029 ± 0.002 (p-t)	0.018 ± 0.001 (w-z)	3.17 ± 0.176 (p-t)	0.066 ± 0.005 (l-r)
V1, E 100%, 10 T	0.052 ± 0.003 (i)	0.051 ± 0.002 (j-k)	0.036 ± 0.001 (m-n)	5.33 ± 0.262 (o-r)	0.075 ± 0.004 (i-o)
V1, E 80%, 10 T	0.063 ± 0.003 (e-g)	0.061 ± 0.002 (f-h)	0.048 ± 0.001 (f-g)	7.45 ± 0.264 (p-t)	0.095 ± 0.004 (d-g)
V1, E 70%, 10 T	0.054 ± 0.002 (h-i)	0.052 ± 0.002 (i-k)	0.038 ± 0.001 (k-m)	5.78 ± 0.204 (o-r)	0.089 ± 0.003 (e-h)
V1, E 60%, 10 T	0.032 ± 0.002 (m-p)	0.031 ± 0.002 (o-s)	0.024 ± 0.001 (s-u)	3.72 ± 0.14 (n-p)	0.071 ± 0.003 (j-q)
V1, E 50%, 10 T	0.025 ± 0.002 (q-s)	0.023 ± 0.001 (t-u)	0.013 ± 0.001 (aa-ab)	2.74 ± 0.28 (o-r)	0.046 ± 0.005 (u-w)
V2, E 100%, 0 T	0.051 ± 0.002 (i)	0.049 ± 0.002 (j-k)	0.043 ± 0.001 (h-i)	5.9 ± 0.262 (k-m)	0.076 ± 0.004 (h-o)
V2, E 80%, 0 T	0.043 ± 0.002 (j-k)	0.042 ± 0.002 (l-m)	0.031 ± 0.002 (o-q)	4.35 ± 0.242 (i)	0.067 ± 0.005 (k-r)
V2, E 70%, 0 T	0.038 ± 0.003 (k-m)	0.036 ± 0.002 (m-o)	0.027 ± 0.001 (q-t)	3.92 ± 0.256 (i-k)	0.064 ± 0.003 (o-s)
V2, E 60%, 0 T	0.031 ± 0.002 (m-q)	0.029 ± 0.002 (p-t)	0.021 ± 0.001 (u-w)	3.36 ± 0.22 (v-x)	0.059 ± 0.005 (q-u)
V2, E 50%, 0 T	0.027 ± 0.001 (p-s)	0.026 ± 0.001 (r-t)	0.016 ± 0.001 (x-aa)	2.85 0.302 (t-w)	0.05 ± 0.002 (t-v)
V2, E 100%, 5 T	0.066 ± 0.002 (d-f)	0.065 ± 0.002 (e-g)	0.055 ± 0.001 (c-d)	7.7 ± 0.209 (u-x)	0.097 ± 0.004 (d-f)
V2, E 80%, 5 T	0.089 ± 0.003 (a)	0.087 ± 0.002 (a)	0.063 ± 0.001 (a)	9.15 ± 0.143 (p-t)	0.118 ± 0.003 (a)
V2, E 70%, 5 T	0.075 ± 0.001 (b-c)	0.074 ± 0.002 (b-d)	0.058 ± 0.002 (b-c)	8.45 ± 0.256 (j-l)	0.106 ± 0.004 (a-d)
V2, E 60%, 5 T	0.053 ± 0.003 (h-i)	0.052 ± 0.002 (i-k)	0.04 ± 0.002 (i-l)	6.45 ± 0.249 (n-p)	0.085 ± 0.005 (f-i)
V2, E 50%, 5 T	0.035 ± 0.002 (l-n)	0.034 ± 0.002 (n-p)	0.031 ± 0.002 (o-q)	3.91 ± 0.277 (i-k)	0.078 ± 0.005 (h-m)
V2, E 100%, 10 T	0.062 ± 0.003 (e-g)	0.061 ± 0.002 (f-h)	0.051 ± 0.002 (e-g)	6.35 ± 0.126 (f-g)	0.084 ± 0.005 (g-i)
V2, E 80%, 10 T	0.077 ± 0.002 (b-c)	0.076 ± 0.001 (b-c)	0.058 ± 0.002 (b-c)	8.32 ± 0.202 (i-j)	0.107 ± 0.004 (a-d)
V2, E 70%, 10 T	0.066 ± 0.003 (d-f)	0.065 ± 0.002 (e-g)	0.047 ± 0.001 (g-h)	7.37 ± 0.169 (e-g)	0.11 ± 0.003 (a-c)
V2, E 60%, 10 T	0.043 ± 0.002 (j-k)	0.042 ± 0.002 (l-m)	0.036 ± 0.002 (l-m)	5.36 ± 0.282 (b-d)	0.079 ± 0.003 (h-k)
V2, E 50%, 10 T	0.033 ± 0.002 (m-p)	0.031 ± 0.002 (o-r)	0.024 ± 0.002 (s-u)	3.13 ± 0.205 (c-g)	0.066 ± 0.005 (m-r)
V3, E 100%, 0 T	0.049 ± 0.002 (i-j)	0.047 ± 0.002 (k-l)	0.036 ± 0.002 (l-m)	4.91 ± 0.25 (i)	0.074 ± 0.004 (i-p)
V3, E 80%, 0 T	0.04 ± 0.001 (k-l)	0.039 ± 0.001 (m-n)	0.027 ± 0.001 (q-t)	4 ± 0.197 (d-g)	0.064 ± 0.004 (o-s)
V3, E 70%, 0 T	0.035 ± 0.002 (l-n)	0.034 ± 0.002 (n-p)	0.023 ± 0.002 (t-v)	3.8 ± 0.175 (g-h)	0.061 ± 0.003 (q-t)
V3, E 60%, 0 T	0.029 ± 0.002 (n-r)	0.027 ± 0.002 (q-t)	0.019 ± 0.002 (w-y)	3.16 ± 0.252 (s-w)	0.065 ± 0.003 (n-s)
V3, E 50%, 0 T	0.024 ± 0.003 (r-s)	0.023 ± 0.002 (t-u)	0.014 ± 0.002 (z-aa)	2.5 ± 0.149 (o-s)	0.039 ± 0.003 (v-w)
V3, E 100%, 5 T	0.064 ± 0.002 (e-f)	0.062 ± 0.002 (f-g)	0.044 ± 0.002 (h-i)	7.12 ± 0.253 (q-u)	0.078 ± 0.002 (h-m)
V3, E 80%, 5 T	0.08 ± 0.003 (b)	0.078 ± 0.002 (b)	0.06 ± 0.002 (a-b)	8.55 ± 0.215 (n-q)	0.113 ± 0.004 (a-b)
V3, E 70%, 5 T	0.068 ± 0.003 (d-e)	0.066 ± 0.002 (e-f)	0.051 ± 0.002 (e-g)	7.97 ± 0.252 (h-i)	0.099 ± 0.004 (c-e)
V3, E 60%, 5 T	0.049 ± 0.003 (i-j)	0.048 ± 0.002 (k-l)	0.035 ± 0.001 (m-o)	5.73 ± 0.143 (i-k)	0.08 ± 0.004 (h-j)
V3, E 50%, 5 T	0.033 ± 0.002 (m-p)	0.031 ± 0.002 (o-r)	0.025 ± 0.001 (r-u)	3.57 ± 0.211 (d-g)	0.071 ± 0.003 (j-q)
V3, E 100%, 10 T	0.056 ± 0.002 (g-i)	0.055 ± 0.002 (h-j)	0.041 ± 0.002 (i-k)	5.93 ± 0.249 (a-c)	0.079 ± 0.004 (h-l)
V3, E 80%, 10 T	0.071 ± 0.002 (c-d)	0.069 ± 0.002 (d-e)	0.052 ± 0.001 (d-f)	7.76 ± 0.164 (b-f)	0.101 ± 0.003 (b-e)
V3, E 70%, 10 T	0.06 ± 0.002 (f-h)	0.058 ± 0.002 (g-i)	0.043 ± 0.001 (h-j)	6.09 ± 0.27 (b-e)	0.095 ± 0.003 (d-g)
V3, E 60%, 10 T	0.035 ± 0.003 (l-o)	0.033 ± 0.002 (n-q)	0.028 ± 0.001 (q-s)	4.37 ± 0.177 (a)	0.074 ± 0.005 (i-o)
V3, E 50%, 10 T	0.028 ± 0.002 (o-r)	0.026 ± 0.002 (r-t)	0.018 ± 0.001 (w-z)	2.93 ± 0.262 (a-b)	0.053 ± 0.003 (s-u)
Note: The different letters in parenthesis after standard deviation represent significant variations among the treatments, as determined by the Tukey test and 95% confidence interval.
Abbreviations: V1; DK-9108, V2; DK-6321, V3; Sarhaab, E; Evapotranspiration, T; tons biochar per hectare

3.4. Biochemical and Physiological Parameters

The analysis of variance of biochemical and physiological parameters at the vegetative and reproductive stages revealed significant outcomes regarding physiological and biochemical attributes (Table 5 and Table 6).

Table 5

Impact of Activated Acacia Wood Biochar Amendment on Membrane Stability Index, Malondialdehyde, and Protein Content, of Three Maize Hybrids Under Varied Moisture Conditions
Treatment	Membrane Stability Index (%)		Malondialdehyde (nmol/ml)		Protein content (mg/g)
	MSI Veg	MSI Rep	MDA Veg	MDA Rep	PC Veg	PC Rep
V1, 100% E, 0T	48.08 ± 2.52 (j-m)	38.08 ± 2.52 (lm)	4.34 ± 0.11 (c-e)	5.74 ± 0.22 (d-f)	4.35 ± 0.21 (n-q)	4.71 ± 0.21 (n-q)
V1, 80% E, 0T	33.07 ± 2.3 (op)	27.57 ± 2.3 (n-q)	4.87 ± 0.11 (c)	6.26 ± 0.22 (b-e)	3.83 ± 0.22 (p-w)	4.19 ± 0.25 (p-u)
V1, 70% E, 0T	27.31 ± 2.77 (p-r)	23.81 ± 1.77 (o-q)	5.61 ± 0.21 (b)	7.01 ± 0.21 (a-b)	3.23 ± 0.23 (u-y)	3.59 ± 0.23 (t-w)
V1, 60% E, 0T	23.05 ± 2.19 (qr)	20.05 ± 1.22 (q)	6.13 ± 0.12 (a-b)	7.53 ± 0.25 (a)	3.48 ± 0.28 (s-x)	3.93 ± 0.23 (r-v)
V1, 50% E, 0T	11.11 ± 2.14 (s)	11.87 ± 1.22 (r)	6.23 ± 0.13 (a)	7.63 ± 0.24 (a)	3.56 ± 0.24 (r-w)	3.84 ± 0.28 (s-v)
V1, 100% E, 5T	54.84 ± 1.24 (e-k)	40.12 ± 1.96 (j-m)	4.07 ± 0.31 (e)	5.47 ± 0.23 (e-f)	6.05 ± 0.24 (g-i)	6.43 ± 0.21 (g-i)
V1, 80% E, 5T	58.32 ± 1.77 (d-g)	50.65 ± 2.39 (d-g)	4.19 ± 0.13 (d-e)	5.59 ± 0.26 (d-f)	7.85 ± 0.21 (c-d)	8.23 ± 0.19 (c-e)
V1, 70% E, 5T	54.9 ± 1.4 (e-k)	47.5 ± 2.16 (e-j)	4.28 ± 0.11 (d-e)	5.68 ± 0.21 (d-f)	7.39 ± 0.24 (d-e)	7.74 ± 0.25 (de)
V1, 60% E, 5T	47.94 ± 1.77 (k-m)	41.74 ± 2.25 (i-m)	4.32 ± 0.21 (c-e)	6.03 ± 0.22 (c-f)	4.86 ± 0.2 (l-o)	5.21 ± 0.21 (l-o)
V1, 50% E, 5T	33.85 ± 2.28 (op)	28.55 ± 2.12 (no)	4.73 ± 0.21 (c-d)	6.37 ± 0.24 (b-d)	3.09 ± 0.22 (v-y)	3.44 ± 0.24 (u-w)
V1, 100% E, 10T	50.22 ± 2.75 (h-l)	39.22 ± 1.91 (lm)	4.05 ± 0.17 (e)	5.45 ± 0.24 (f)	5.45 ± 0.23 (i-l)	5.81 ± 0.23 (i-l)
V1, 80% E, 10T	57.6 ± 2.27 (e-h)	50.8 ± 2.27 (d-g)	4.35 ± 0.29 (c-e)	5.79 ± 0.25 (d-f)	6.13 ± 0.22 (g-i)	6.52 ± 0.19 (g-i)
V1, 70% E, 10T	53.92 ± 1.76 (e-k)	48.22 ± 1.76 (e-i)	4.44 ± 0.28 (c-e)	5.84 ± 0.25 (c-f)	6.13 ± 0.24 (g-i)	6.49 ± 0.23 (g-i)
V1, 60% E, 10T	41.97 ± 1.84 (mn)	37.77 ± 1.84 (lm)	4.58 ± 0.16 (c-e)	6.16 ± 0.23 (c-f)	4.01 ± 0.25 (p-t)	4.38 ± 0.25 (p-s)
V1, 50% E, 10T	26.98 ± 1.49 (p-r)	23.28 ± 2.34 (o-q)	4.84 ± 0.14 (c)	6.63 ± 0.21 (b-c)	3.09 ± 0.22 (w-y)	3.46 ± 0.24 (u-w)
V2, 100% E, 0T	55.35 ± 2.1 (e-k)	42.35 ± 2.9 (h-l)	1.68 ± 0.12 (i-l)	2.75 ± 0.22 (j-l)	5.39 ± 0.23 (i-l)	5.83 ± 0.25 (i-l)
V2, 80% E, 0T	43.5 ± 2.52 (lm)	39 ± 2.52 (lm)	1.89 ± 0.1 (i-l)	2.97 ± 0.24 (j-l)	5.13 ± 0.23 (k-m)	5.59 ± 0.22 (j-m)
V2, 70% E, 0T	30.82 ± 2.1 (op)	27.32 ± 2.1 (n-q)	2.06 ± 0.11 (i-j)	3.14 ± 0.23 (i-l)	5.05 ± 0.25 (l-n)	5.47 ± 0.24 (k-m)
V2, 60% E, 0T	31.18 ± 2.6 (op)	28.18 ± 2.6 (no)	2.16 ± 0.13 (h-i)	3.44 ± 0.26 (h-k)	2.71 ± 0.24 (x-y)	4.32 ± 0.24 (p-t)
V2, 50% E, 0T	28.14 ± 2.23 (o-r)	27.6 ± 2.23 (n-p)	3.37 ± 0.11 (f)	4.47 ± 0.25 (g)	3.87 ± 0.24 (p-v)	3.16 ± 0.25 (v-w)
V2, 100% E, 5T	61.23 ± 2.82 (c-e)	47.23 ± 1.82 (e-k)	0.48 ± 0.14 (r-s)	1.58 ± 0.26 (o)	7.05 ± 0.24 (e-f)	7.53 ± 0.25 (ef)
V2, 80% E, 5T	76.24 ± 2.19 (a)	69.57 ± 2.26 (a)	0.64 ± 0.18 (q-s)	1.74 ± 0.28 (o)	9.86 ± 0.22 (a)	10.36 ± 0.21 (a)
V2, 70% E, 5T	72.22 ± 1.61 (a-b)	64.82 ± 2.23 (a-b)	0.7 ± 0.11 (q-s)	1.82 ± 0.22 (n-o)	7.84 ± 0.22 (c-d)	8.32 ± 0.23 (cd)
V2, 60% E, 5T	55.54 ± 2.52 (e-j)	49.34 ± 2.52 (e-h)	0.74 ± 0.25 (q-s)	1.89 ± 0.3 (m-o)	5.89 ± 0.24 (g-k)	6.35 ± 0.24 (g-j)
V2, 50% E, 5T	52.61 ± 2.69 (f-k)	47.31 ± 2.69 (e-k)	0.97 ± 0.11 (o-r)	2.85 ± 0.24 (j-l)	3.36 ± 0.23 (t-x)	3.79 ± 0.22 (s-v)
V2, 100% E, 10T	57.71 ± 2.19 (e-g)	44.71 ± 2.19 (f-l)	0.41 ± 0.32 (s)	2.33 ± 0.23 (l-o)	5.92 ± 0.23 (g-j)	6.33 ± 0.22 (g-j)
V2, 80% E, 10T	65.78 ± 2.48 (b-d)	59.31 ± 2.56 (bc)	0.74 ± 0.1 (q-s)	2.59 ± 0.22 (l-n)	8.68 ± 0.23 (b)	9.11 ± 0.23 (b)
V2, 70% E, 10T	59.76 ± 2.51 (c-f)	54.06 ± 2.51 (c-e)	0.86 ± 0.08 (p-s)	2.78 ± 0.24 (j-l)	6.49 ± 0.22 (f-g)	6.95 ± 0.22 (fg)
V2, 60% E, 10T	53.03 ± 1.83 (f-k)	48.83 ± 2.83 (e-i)	1.06 ± 0.17 (n-q)	2.79 ± 0.24 (j-l)	4.37 ± 0.24 (m-p)	4.84 ± 0.24 (m-p)
V2, 50% E, 10T	43.76 ± 2.34 (lm)	40.06 ± 2.34 (j-m)	1.12 ± 0.1 (m-q)	3.11 ± 0.26 (i-l)	3.92 ± 0.22 (p-u)	4.41 ± 0.22 (p-s)
V3, 100% E, 0T	51.63 ± 2.18 (g-k)	39.63 ± 2.18 (lm)	2.65 ± 0.14 (g-h)	3.88 ± 0.35 (g-i)	5.17 ± 0.24 (j-l)	5.58 ± 0.24 (j-m)
V3, 80% E, 0T	35.03 ± 2.78 (no)	34.53 ± 2.78 (mn)	2.7 ± 0.15 (g-h)	3.92 ± 0.23 (g-i)	4.89 ± 0.23 (l-o)	5.31 ± 0.23 (k-o)
V3, 70% E, 0T	30.22 ± 1.7 (o-q)	26.72 ± 2.69 (o-q)	2.99 ± 0.14 (f-g)	4.2 ± 0.22 (g-h)	4.25 ± 0.24 (o-s)	4.64 ± 0.25 (o-r)
V3, 60% E, 0T	27.45 ± 1.98 (p-r)	24.45 ± 1.5 (o-q)	3.11 ± 0.12 (f-g)	4.4 ± 0.24 (g)	3.69 ± 0.24 (p-w)	4.09 ± 0.23 (p-u)
V3, 50% E, 0T	21.08 ± 2.33 (r)	20.54 ± 1.4 (pq)	4.39 ± 0.1 (c-e)	5.6 ± 0.21 (d-f)	2.56 ± 0.22 (y)	2.99 ± 0.23 (w)
V3, 100% E, 5T	57.58 ± 2.35 (e-h)	43.58 ± 2.35 (g-l)	1.42 ± 0.11 (k-o)	2.64 ± 0.4 (k-m)	6.33 ± 0.25 (f-h)	6.75 ± 0.25 (g-h)
V3, 80% E, 5T	66.79 ± 2.11 (bc)	57.89 ± 2.11 (b-d)	1.56 ± 0.17 (j-n)	2.78 ± 0.29 (j-l)	8.25 ± 0.23 (b-c)	8.66 ± 0.23 (bc)
V3, 70% E, 5T	60.05 ± 2.71 (c-f)	52.65 ± 2.71 (c-e)	1.63 ± 0.13 (i-m)	2.86 ± 0.2 (j-l)	7.53 ± 0.21 (c-e)	7.92 ± 0.22 (c-e)
V3, 60% E, 5T	49.89 ± 1.27 (i-l)	43.69 ± 1.27 (g-l)	1.67 ± 0.19 (i-m)	3.11 ± 0.22 (i-l)	4.91 ± 0.22 (l-o)	5.31 ± 0.22 (k-o)
V3, 50% E, 5T	43.01 ± 2.48 (lm)	37.71 ± 2.48 (lm)	2.05 ± 0.19 (i-j)	3.53 ± 0.24 (h-j)	3.21 ± 0.24 (u-y)	3.64 ± 0.24 (s-w)
V3, 100% E, 10T	53.77 ± 2.83 (e-k)	42.77 ± 2.83 (h-l)	1.34 ± 0.11 (l-p)	2.56 ± 0.23 (l-n)	5.63 ± 0.24 (h-l)	6.02 ± 0.24 (h-k)
V3, 80% E, 10T	60.56 ± 2.64 (c-e)	53.76 ± 2.64 (c-e)	1.69 ± 0.11 (i-l)	2.91 ± 0.24 (j-l)	7.45 ± 0.24 (d-e)	7.87 ± 0.24 (de)
V3, 70% E, 10T	57.02 ± 2.29 (e-i)	51.32 ± 2.29 (d-f)	1.79 ± 0.19 (i-l)	3.02 ± 0.22 (j-l)	6.28 ± 0.24 (f-h)	6.71 ± 0.24 (g-h)
V3, 60% E, 10T	44.04 ± 2.28 (lm)	39.84 ± 2.28 (k-m)	1.97 ± 0.18 (i-k)	3.42 ± 0.21 (h-k)	4.31 ± 0.25 (n-r)	4.73 ± 0.25 (n-q)
V3, 50% E, 10T	28.17 ± 1.98 (o-r)	24.47 ± 1.98 (o-q)	2.15 ± 0.13 (h-i)	4.17 ± 0.22 (g-h)	3.59 ± 0.23 (q-w)	3.98 ± 0.24 (q-u)
Note: The different letters in parenthesis after standard deviation represent significant variations among the treatments, as determined by the Tukey test and 95% confidence interval.
Abbreviations: V1; DK-9108, V2; DK-6321, V3; Sarhaab, E; Evapotranspiration, T; tons biochar per hectare

Table 6

Impact of Activated Acacia Wood Biochar Amendment on Antioxidant Enzymes (SOD, POD, and CAT) Assay of Three Maize Hybrids Under Varied Moisture Conditions
Treatment	Superoxidase dismutase (U/mg)		Peroxidase (U/mg)		Catalase (U/mg)
	SOD Veg	SOD Rep	POD Veg	POD Rep	CAT Veg	CAT Rep
V1, 100% E, 0T	3.08 ± 0.49 (s)	30.67 ± 2.95 (z)	3.08 ± 0.49 (v)	4.72 ± 1.09 (s)	16.59 ± 1.19 (t)	36.52 ± 1.99 (w)
V1, 80% E, 0T	5.74 ± 0.81 (p-r)	35.92 ± 2.15 (w-z)	5.74 ± 0.81 (s-u)	7.33 ± 0.82 (p-s)	19.49 ± 1.07 (q-t)	42.28 ± 2.27 (v-w)
V1, 70% E, 0T	9.07 ± 0.86 (j-o)	50.65 ± 2.83 (r-u)	8.01 ± 0.91 (q-t)	8.91 ± 0.35 (m-q)	23.43 ± 1.52 (m-r)	54.35 ± 2.89 (p-u)
V1, 60% E, 0T	10.06 ± 0.83 (h-k)	63.2 ± 2.68 (n-q)	11.06 ± 1.1 (n-p)	18.92 ± 0.72 (e-g)	23.22 ± 1.2 (m-r)	67.12 ± 2.1 (k-m)
V1, 50% E, 0T	12.5 ± 0.95 (f-h)	90.09 ± 2.44 (h-j)	14.89 ± 0.89 (k-l)	22.1 ± 0.79 (d)	38.71 ± 1.08 (e-f)	85.32 ± 3.54 (g-i)
V1, 100% E, 5T	5.19 ± 0.34 (r-s)	33.57 ± 3.1 (x-z)	5.19 ± 0.67 (u-v)	6.54 ± 0.73 (q-s)	21.03 ± 1.12 (p-t)	55.3 ± 3.33 (p-u)
V1, 80% E, 5T	6.86 ± 0.84 (o-r)	42.39 ± 2.17 (u-x)	6.87 ± 0.49 (r-u)	10.73 ± 0.47 (j-n)	26.41 ± 1.3 (k-n)	62.93 ± 3.51 (l-o)
V1, 70% E, 5T	7.44 ± 0.64 (l-r)	59.26 ± 2.41 (o-r)	7.44 ± 0.58 (q-u)	12.3 ± 0.86 (h-k)	29.48 ± 1.76 (i-l)	68.01 ± 2.87 (k-l)
V1, 60% E, 5T	16.19 ± 0.96 (e)	90.45 ± 3.19 (g-k)	18.05 ± 0.64 (j)	20.81 ± 0.3 (d-e)	37.81 ± 1.33 (e-f)	81.59 ± 3.1 (h-j)
V1, 50% E, 5T	28.4 ± 1.11 (c)	101.34 ± 3.09 (d-f)	26.34 ± 0.73 (e-f)	26.05 ± 0.97 (c)	54.46 ± 1.51 (b)	115.73 ± 3.28 (c)
V1, 100% E, 10T	5.11 ± 0.88 (r-s)	31.19 ± 3.2 (z)	5.11 ± 0.53 (u-v)	5.08 ± 0.65 (s)	19.22 ± 1.8 (r-t)	48.74 ± 2.43 (u-v)
V1, 80% E, 10T	5.91 ± 0.59 (p-r)	40.5 ± 3.07 (v-y)	5.91 ± 0.78 (s-u)	6.47 ± 0.59 (q-s)	21.61 ± 1.41 (o-s)	52.5 ± 2.48 (r-u)
V1, 70% E, 10T	8.01 ± 1.01 (k-p)	54.34 ± 2.78 (q-s)	9.07 ± 0.92 (p-r)	6.83 ± 0.77 (q-s)	26.09 ± 1.00 (l-o)	59.05 ± 2.89 (m-s)
V1, 60% E, 10T	13.32 ± 0.7 (f-g)	79.75 ± 2.38 (k-l)	14.37 ± 0.83(k-m)	11.38 ± 0.91 (j-m)	32.55 ± 1.19 (g-j)	77.2 ± 3.14 (i-j)
V1, 50% E, 10T	22.68 ± 0.94 (d)	93.79 ± 2.79 (f-j)	22.45 ± 0.54 (g-h)	17.99 ± 0.94 (f-g)	48.84 ± 1.72 (c-d)	111.32 ± 3.08 (c-d)
V2, 100% E, 0T	6.2 ± 0.57 (p-r)	36.67 ± 3.25 (w-z)	6.18 ± 0.49 (s-u)	9.54 ± 0.8 (l-p)	21.05 ± 1.62 (p-t)	50.79 ± 2.78 (s-v)
V2, 80% E, 0T	7.2 ± 0.84 (m-r)	44.23 ± 2.93 (t-w)	7.2 ± 1.14 (q-u)	10.25 ± 0.29 (j-o)	29.08 ± 1.11 (i-l)	51.05 ± 2.64 (r-u)
V2, 70% E, 0T	9.35 ± 0.62 (j-n)	63.61 ± 3.24 (n-p)	11.51 ± 0.91 (n-p)	12.14 ± 0.49 (h-l)	33.12 ± 1.14 (g-i)	62.87 ± 3.08 (l-p)
V2, 60% E, 0T	14.69 ± 0.49 (e-f)	94.14 ± 3.63 (f-i)	24.31 ± 0.47 (f-g)	19.51 ± 0.72 (d-g)	35.92 ± 1.41 (e-g)	83.88 ± 2.74 (h-i)
V2, 50% E, 0T	20.59 ± 0.7 (d)	118.59 ± 2.46 (b-c)	24.69 ± 0.58 (f-g)	30.57 ± 0.63 (b)	55.32 ± 1.18 (b)	103.79 ± 2.7 (d-e)
V2, 100% E, 5T	6.9 ± 0.6 (n-r)	47.62 ± 2.51 (s-v)	6.9 ± 0.32 (r-u)	11.02 ± 0.55 (j-n)	29.12 ± 1.23 (i-l)	61.32 ± 3.17 (l-q)
V2, 80% E, 5T	7.92 ± 0.78 (k-q)	54.75 ± 2.27 (p-s)	7.92 ± 0.55 (q-t)	13.38 ± 0.96 (h-j)	36.4 ± 1.67 (e-g)	67.99 ± 3.11 (k-l)
V2, 70% E, 5T	11.51 ± 0.76 (g-j)	77.25 ± 3.17 (k-m)	9.35 ± 0.62 (p-r)	18.56 ± 0.59 (e-g)	36.35 ± 1.45 (e-g)	79.71 ± 3.13 (i-j)
V2, 60% E, 5T	26.92 ± 0.68 (c)	103.57 ± 3.28 (d-e)	28.59 ± 0.94 (d-e)	26.39 ± 0.65 (c)	47.3 ± 1.21 (d)	95.75 ± 2.9 (e-f)
V2, 50% E, 5T	38.58 ± 0.7 (a)	134.67 ± 2.86 (a)	34.8 ± 0.58 (a)	36.62 ± 1.07 (a)	63.13 ± 1.41 (a)	128.84 ± 3.11 (a)
V2, 100% E, 10T	7.36 ± 0.4 (l-r)	41.26 ± 3.00 (v-y)	7.36 ± 0.86 (q-u)	10.63 ± 0.7 (j-o)	22.04 ± 1.22 (n-r)	53.25 ± 2.95 (q-u)
V2, 80% E, 10T	7.45 ± 1.02 (l-r)	47.35 ± 1.98 (s-v)	7.45 ± 0.79 (q-u)	9.55 ± 1.09 (l-p)	27.58 ± 1.04 (k-m)	57.56 ± 2.6 (o-t)
V2, 70% E, 10T	12.08 ± 0.45 (g-i)	70.47 ± 2.29 (m-n)	12.09 ± 0.99(m-o)	11.58 ± 0.85 (i-l)	34.45 ± 0.94 (f-h)	66.31 ± 3.25 (k-n)
V2, 60% E, 10T	21.31 ± 0.62 (d)	99.37 ± 3.24 (d-g)	19.16 ± 0.94 (i-j)	17.28 ± 0.84 (g)	45.03 ± 1.3 (d)	90.14 ± 3.22 (f-h)
V2, 50% E, 10T	32.46 ± 0.67 (b)	126.74 ± 2.84 (a-b)	31.46 ± 0.55 (b-c)	25.13 ± 1.08 (c)	54.14 ± 1.59 (b)	118.1 ± 2.56 (b-c)
V3, 100% E, 0T	5.51 ± 0.53 (q-s)	44.91 ± 2.14 (t-w)	5.47 ± 0.93 (t-v)	6.25 ± 0.85 (r-s)	17.49 ± 1.02 (s-t)	48.5 ± 3.02 (u-v)
V3, 80% E, 0T	7.76 ± 0.82 (k-q)	51.75 ± 1.7 (r-t)	6.83 ± 0.82 (r-u)	8.09 ± 0.28 (o-r)	21.75 ± 1.85 (o-s)	50.85 ± 2.19 (s-v)
V3, 70% E, 0T	7.41 ± 0.34 (l-r)	68.69 ± 2.62 (m-n)	7.41 ± 0.76 (q-u)	10.85 ± 0.36 (j-n)	25.18 ± 1.11 (l-p)	55.68 ± 2.97 (p-u)
V3, 60% E, 0T	12.6 ± 0.59 (f-g)	95.64 ± 2.14 (e-i)	12.6 ± 0.59 (l-n)	20.3 ± 0.87 (d-f)	29.25 ± 1.82 (i-l)	73.12 ± 2.94 (j-k)
V3, 50% E, 0T	16.52 ± 0.64 (e)	117.29 ± 2.18 (c)	23.05 ± 0.93 (g-h)	26.95 ± 1.02 (c)	47.34 ± 1.73 (d)	97.29 ± 2.03 (e-f)
V3, 100% E, 5T	6.97 ± 0.68 (n-r)	33.1 ± 2.83 (y-z)	6.97 ± 1.09 (r-u)	8.87 ± 0.92 (m-r)	25.12 ± 1.11 (l-p)	59.48 ± 2.73 (l-r)
V3, 80% E, 5T	8.11 ± 0.66 (k-p)	42.74 ± 2.11 (t-w)	8.11 ± 1.05 (q-s)	11.21 ± 1.06 (j-n)	30.64 ± 0.95 (h-k)	64.36 ± 2.36 (l-o)
V3, 70% E, 5T	9.78 ± 0.85 (i-l)	57.26 ± 2.54 (o-r)	9.78 ± 0.85 (o-q)	14.3 ± 0.57 (h)	34.2 ± 1.63 (f-h)	73.28 ± 3.12 (j-k)
V3, 60% E, 5T	21.61 ± 0.64 (d)	84.99 ± 2.92 (j-l)	20.94 ± 1.04 (h-i)	21.61 ± 0.84 (d)	39.81 ± 1.16 (e)	93.58 ± 2.32 (f-g)
V3, 50% E, 5T	33.2 ± 1.06 (b)	98.85 ± 2.62 (d-h)	32.1 ± 0.62 (b)	31.83 ± 0.65 (b)	62.86 ± 1.28 (a)	124.94 ± 3.17 (a-b)
V3, 100% E, 10T	6.35 ± 0.37 (p-r)	36 ± 1.95 (w-z)	6.35 ± 0.65 (s-u)	4.93 ± 0.93 (s)	20.43 ± 1.26 (q-t)	50.34 ± 1.73 (t-v)
V3, 80% E, 10T	6.87 ± 0.58 (o-r)	46.46 ± 2.93 (s-v)	7.97 ± 0.48 (q-t)	6.6 ± 1.06 (q-s)	23.81 ± 1.35 (m-q)	54.07 ± 3.11 (q-u)
V3, 70% E, 10T	9.54 ± 0.5 (j-m)	63.86 ± 3.21 (n-o)	9.33 ± 0.79 (p-r)	8.64 ± 0.74 (n-r)	28.35 ± 0.82 (j-l)	58.45 ± 2.33 (n-t)
V3, 60% E, 10T	16.8 ± 0.71 (e)	88.53 ± 2.44 (i-k)	16.94 ± 0.81 (j-k)	14.07 ± 1.05 (h-i)	36.55 ± 1.44 (e-g)	80.63 ± 2.64 (i-j)
V3, 50% E, 10T	26.52 ± 1.03 (c)	106.86 ± 2.28 (d)	29.21 ± 1.09 (c-d)	20.63 ± 0.42 (d-e)	52.72 ± 1.55 (b-c)	114.68 ± 3.25 (c)
Note: The different letters in parenthesis after standard deviation represent significant variations among the treatments, as determined by the Tukey test and 95% confidence interval.
Abbreviations: V1; DK-9108, V2; DK-6321, V3; Sarhaab, E; Evapotranspiration, T; tons biochar per hectare

Primarily at the vegetative stage, soil supplemented with biochar exhibited a significantly higher membrane stability index, protein content, and antioxidant enzyme activity compared to non-amended soil. The biochar-amended maize showed 1.62 times higher MSI, 1.43 times improved protein content, 1.47 times increased SOD activity, 1.24 times increased POD activity, and 1.21 times more CAT activity than non-amended maize. While the malondialdehyde concentration was more noted in non-amended maize than in biochar-amended maize. Non-biochar-amended maize exhibited 0.63 times more MDA content than biochar-amended maize.

At the reproductive stage, soil amended with biochar exhibited a significantly higher membrane stability index, protein content, and antioxidant enzyme activity compared to non-amended soil. The biochar-amended maize showed 1.61 times more MSI, 1.39 times more protein content, 1.13 times more SOD activity, 1.38 times more POD activity, and 1.22 times more CAT activity than non-amended maize. While the malondialdehyde concentration was noted to be higher in non-amended maize than in biochar-amended maize. Non-biochar-amended maize exhibited 77% more MDA content than biochar-amended maize.

Moreover, at both stages, the 5 tons/ha biochar amendment exhibited better responses in terms of biochemical and physiological parameters, compared to the 10 tons/ha as well as 0 tons/ha biochar amendments. Among the ETC levels, 80% ETC was superior compared to 100%, 70%, 60% and 50% ETC. Whereas, among maize varieties, the effective order was DK-6321, Sarhaab, and DK-9108.

3.5. Yield Analysis

The three-way ANOVA of maize hybrids, biochar supplementation levels and moisture regimes indicated that thousand seed weight and total seed weight (g) has revealed significant outcomes (Fig. 5). Reduced water levels resulted in a 1000-seed weight and total seed weight reduction of maize hybrids. Whereas, the application of biochar enhanced these weights of maize hybrids. Soil amended with 5 tons/ha of biochar significantly enhanced the thousand seed weight compared to non-amended soil by 16%, and total seed weight by 29%. Deficit irrigation resulted in grain yield and stover yield reduction of maize hybrids. Whereas, the integration of biochar enhanced the grain and stover yield of maize hybrids (Fig. 6). Soil amended with 5 tons/ha of biochar significantly elevated the grain yield and stover yield compared to non-amended soil by 26%. The apparent water productivity under deficit irrigation declined considerably (Fig. 7). Whereas, the application of biochar enhanced the apparent water productivity of maize hybrids. Soil amended with 5 tons/ha of biochar significantly enhanced the apparent water productivity compared to non-amended soil by 33%.

3.6. Principal Component Analysis (PCA) and Heatmap

The PCA biplot (Fig. 8) shows the first two principal components (PC1 and PC2), explaining 70.28% and 20.56% of the variance, respectively. The arrows indicate each variable's contribution to the principal components. Variables pointing in the same direction (e.g., membrane stability index, growth rate, weights, net assimilation rate, yield parameters) are positively correlated and mainly contribute to PC1. Variables in opposite directions (e.g., malondialdehyde content, stress indicators) are negatively correlated. The arrow length shows the variable's contribution strength. Ellipses represent 95% confidence intervals for treatment groups, highlighting variability. The red ellipse (0 t/ha Biochar) is distinct from the blue (10 t/ha Biochar) and green (5 t/ha Biochar) ellipses, indicating treatment differences. Higher Biochar treatments (5 t/ha and 10 t/ha) cluster together, separate from 0 t/ha, showing significant treatment effects. Different point shapes (triangles, squares, circles) denote the impact of ETC and variety on principal components, with clustering within ellipses suggesting intertwined effects with biochar treatments.

The heatmap (Fig. 9) shows the relationships between treatments and various biochemical, physiological, and yield parameters for maize. Darker colors (black/purple) indicate lower values, while lighter colors (yellow) indicate higher values. The combination of 5 t/ha biochar and 80% ETC yields the highest values across most parameters, suggesting it is the most favorable treatment. Water stress (50% ETC) significantly reduces growth and yield but increases stress-related biochemical responses. Different maize varieties respond uniquely to moisture and biochar levels, with DK-6321 showing the best tolerance to water stress, followed by SARHAAB and DK-9108.

4.1. Soil physicochemical properties

The study yielded significant findings, particularly concerning bulk density, particle density, total porosity, electrical conductivity, pH levels, organic carbon, organic matter, oxidizable carbon, phosphorus levels, and potassium levels. The most notable improvements were evident in soils modified with activated biochar compared to non-amended soil. However, it is noteworthy that a slight increase in electrical conductivity and pH in biochar-treated soil could be attributed to the presence of salts from the biochar's ash content, as noted by Liang et al.⁵² and Saeed et al.⁵³ Biochar has been noticed to stimulate microbial activity, promoting the decomposition of organic residues and thereby influencing the turnover of soil organic matter⁵⁴. The introduction of biochar in soil alters its physical properties, fostering improved water infiltration and reducing the risk of water runoff, consequently increasing the saturation percentage¹⁰. The observed increase in organic carbon and oxidizable carbon content signifies a positive impact on soil health and fertility, as organic carbon plays a primary role in nutrient retention, water-holding capacity, improved microbial activity, and overall soil structure. This finding aligns with the notion that biochar acts as a stable organic carbon pool and C/N ratios in the soil, thereby influencing overall carbon dynamics^55,56. The biochar enhances the nutrient level, including nitrogen, available phosphorus, and potassium, in the soil by increasing the cation exchange capacity of soil particles⁵⁷.

The soil's SEM-EDX examination reveals that carbon, oxygen, magnesium, silicon, potassium, calcium, aluminium, and iron are the elements with the largest concentrations, followed by trace amounts of sodium and titanium. About 98% of the earth's crust is made up of oxygen, silicon, iron, aluminium, calcium, potassium, and sodium. The other 2% is made up of other elements. Additionally, feldspar and quartz dominate the continental crust⁵⁸. Silica particles (also known as SiO₂) contain a high Si and O concentration, indicating quartz material (almost 50% by weight). Also, most of the pure silica particles come from natural sources⁵⁹. Furthermore, potassium and sodium belong to the feldspar group (sometimes known as alkali feldspars). The constituents of the soil under present study mostly include iron-bearing particles, silicified quartz, and feldspar. However, all of the samples (from 0 T/ha, 5 T/ha, 10 T/ha biochar supplemented soil) showed carbon content because of the peat formation⁶⁰, but highest carbon content detected in 10 tons ha^–1 supplemented soil might be due to addition of recalcitrant carbon from the biochar¹⁰. The biochar application in soil improves its structure which helps to enhance its water retention capability⁶¹. The presence of fine particles in biochar may lead to the decrease of saturated hydraulic conductivity⁶².

4.2. Influence of biochar supplementation on maize growth

Insufficient water availability led to reduced root and shoot biomass, and comparative growth rate in plants. This decline may be due to various factors, such as, limited water uptake due to limited soil moisture accessibility, root and shoot cell damage by oxidative stress, resource distribution by prioritizing functions over root and shoot development and loss of nutrient absorption⁶³. Conversely, when biochar was applied into the soil, it significantly improved root and shoot, dry and fresh weight under water stress conditions. This improvement maybe due its advantageous effects, such as its ability to hold moisture in the soil, increasing nutrient availability through improved cation exchange capacity (CEC), promoting healthier root growth through a supportive soil structure, reducing soil compaction, mitigating oxidative stress, and fostering beneficial soil microbial communities⁶⁴. These mutual benefits create a more helpful atmosphere for roots to access water and nutrients and shoots to continue photosynthesis and growth, eventually resulting in increased root and shoot mass even when facing water stress challenges.

Water stress led to decrease in leaf live and dehydrated mass weight, as well as a decline in leaf area in which are the consequences of limited water availability, which hinders the plant's ability to maintain turgor pressure, cell expansion, and overall leaf growth. Moreover, water stress can induce oxidative stress in leaves, damaging their growth and functioning^65,66. On the flip side, when the biochar is applied in soil, it triggered an intensification in leaf live and dehydrated mass weight, as well as an expansion in leaf area under water stress conditions⁶⁷. This improvement is accredited to several valuable effects of biochar, including enhanced soil moisture retention, improved nutrient availability, and reduced oxidative stress⁷. Biochar's capacity to reduce water stress ultimately results in healthier and more vigorous leaves, with increased biomass and leaf area even in the face of water restrictions.

There has been decline in net assimilation rate (NAR) of plants due to water stress which is primarily associated to the plant's fight to preserve optimal photosynthesis rates due to limited water availability. As a result, reduced photosynthetic activity hampers carbon assimilation, leading to decreased biomass production and, consequently, lower NAR⁶⁸. Although, biochar application to soil can significantly boost the net assimilation rate (NAR) of plants under water stress. This enhancement is driven by biochar’s capacity to enhance soil water retention, promote nutrient availability, and make conditions favorable for leaf development, eventually leading to increased carbon assimilation and subsequently higher NAR, even under hydric stress⁶⁹.

4.3. Influence on Physiological, biochemical and yield attributes

Water stress typically leads to increased oxidative stress in plant cells, which can result in membrane damage and instability. Dehydrated plants exhibited a reduction in MSI due to membrane lipid peroxidation triggered by the accretion of reactive oxygen species (ROS)⁷⁰. This is common effect of all abiotic stresses mainly heavy metals, salinity, drought and heat and reported well in the literature^66,71,72. In contrast, biochar-mixed soil provided a more favorable environment for plants under water stress. Biochar's ability to enhance water retention and nutrient availability helps maintain cell turgor and reduces oxidative stress⁷³. Consequently, plants grown in biochar-amended soil revealed higher MSI values in contrast to those in non-amended soil under similar water stress situations. This observation highlighted the latent of biochar as a soil amendment to recover the membrane stability of plants and their capability to cope with water scarcity. Also, the membrane stability index (MSI) in maize is lower during the reproductive stage as compared with the vegetative stage, primarily as a consequence of water stress experienced during the reproductive phase⁷⁴.

The water stress stimulated oxidative stress in plants, resultant in amplified malondialdehyde content as a marker of lipid peroxidation and cellular damage⁷⁵. However, the application of biochar in soil can significantly alleviate the effects of water stress on MDA levels due to its water retention properties that help maintain soil moisture, reducing the severity of water stress and subsequently lowering oxidative stress. Additionally, biochar's ability to enhance nutrient availability and foster beneficial microbial communities in the rhizosphere can further bolster the plant's resilience to water stress, limiting the accumulation of MDA⁷⁶. This double act of biochar in extenuating water stress and reducing MDA content underscores its potential as a sustainable soil amendment for enlightening plant health and tolerance to environmental challenges. The levels of malondialdehyde content were higher during the reproductive stage in comparison to the vegetative stage, linked to more severe condition⁷⁷.

Protein content in plants was seen to be decreased under limited moisture level as the reduced water availability restricts various metabolic processes, including protein synthesis. The hydration-deficiency-related decline in protein content can result in a cooperated capability for plants to carry out essential functions and bear environmental contests⁷⁸. Though, the biochar application in the soil exhibits a mitigating effect on decrease in protein content. As a result, protein synthesis and metabolic procedures can continue more efficiently to increase protein content under drought situations⁷⁹. The protein content was higher during the reproductive stage in comparison to the vegetative stage⁸⁰, possibly due to decrease in photosynthesis at initial stages of crop while the protein content was seen higher at reproductive stage which is possibly due to new stress proteins expression⁸¹.

The hydric stress sources oxidative damage in plants, leading to the overrun of reactive oxygen species (ROS), which can damage cellular components⁸². Subsequently, the superoxidase dismutase, peroxidase, and catalase activity increased as a resistance mechanism to counteract reactive oxygen species and ease cellular damage⁸³. This is an innate behavior of plants to uplift antioxidant enzymes activities as first line of defense against abiotic stresses and is documented well in previous literature^66,84,85. Though, the introduction of biochar into the soil moderated the effects of water stress on these antioxidant enzymes. The ability of activated biochar enhances the retention of soil water and lightens the strictness of drought, thus, reducing the need for excessive ROS scavenging by antioxidant enzymes, i.e. SOD, POD, and CAT resulting enzymes in more concentration⁸⁶. The SOD, POD, and CAT activity has been seemed to be increased in water stress conditions, and application of biochar in soil resulted in growing of these enzymes activity and this increase in activity due to link with better plant and soil relations⁸⁷. Moreover, biochar's influence on soil nutrient accessibility and microbial communities indirectly influences enzyme activity⁸⁸. The activity of peroxidase and catalase at reproductive stage was noted to be more than vegetative stage⁸⁹, where a decrease in the activity of superoxidase dismutase activity has been noted at reproductive stage in parallel to vegetative stage⁹⁰. The dual action of biochar, in moderating water stress and controlling enzyme activity, highlights its potential as a sustainable soil supplement for enhancing plant flexibility to environmental stressors.

The water stress exerted a negative impact on crop yield parameters such as cob length, cob weight, kernel number, total seeds per cob, grain yield per hectare, stover yield, and apparent water productivity, as reduced soil moisture hinders various physiological processes in plants⁹¹. However, the mixture of biochar into soil lessened the negative impacts of water stress on different yield limiting factors. The water retention properties of activated biochar help maintain consistent soil moisture levels, thus mitigating the severity of water stress and supporting essential physiological functions⁹². Also, biochar improves nutrient availability, soil structure, and endorses beneficial microbial communities, all of which contribute to better crop resilience and higher yield parameters, even in the condition of water stress.

An interesting finding in this study relates to the dosage of activated biochar. It was obvious that the 5 tons/ha biochar amendment yielded more promising outcomes in terms of physiological, biochemical, and yield parameters as compared to the 10 tons/ha biochar amendment. This observation elevates the likelihood of diminishing returns associated with higher biochar doses, potentially attributed to a limitation in nitrogen (N) uptake at the higher dosage level⁹³. In essence, the optimal biochar dosage seems to strike a delicate balance between enhancing plant responses and avoiding nutrient oversaturation, highlighting the importance of fine-tuning biochar application rates for maximal benefits in agricultural practices.

In conclusion, this study planned to investigate the use of activated biochar to enhance maize relative growth, and yield-related features under optimal and deficit irrigation conditions. The main findings of research demonstrate the organically activated biochar have potential for improvement in maize growth, biochemical and physiological attributes, and yield, particularly under deficit irrigation, as can be witnessed by enhancement in apparent water productivity and thousand seed weight. More specifically, it was observed that 5 tons per hectare amendment of activated biochar was more effective than a 10 tons per hectare amendment in achieving these positive outcomes. The significance of our study lies in the convenience of activation method used for biochar, which can potentially benefit a broader range of researchers. Also, the limited availability of field trials in this research area points towards the newness of our findings. It is evident that the utilization of activated biochar holds considerable potential for enhancing soil health and agricultural sustainability. However, it is essential to emphasize that, conducting ongoing field experiments is requisite for a thorough assessment of its long-term advantages. Such continuing studies will not only deepen our understanding of biochar's enduring impact but also contribute valuable insights towards the adoption of green farming practices in the years to come. Our findings contributed to the rising frame of knowledge on this topic and opened tracks for further exploration in the field of agricultural sustainability and water resources planning.

Availability of data and materials

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethical approval and consent to participate

We declare that the manuscript reporting studies do not involve any human participants, human data or human tissues. So, it is not applicable.

Our experiment follows with the relevant institutional, national, and international guidelines and legislation.

Consent for publication

Not applicable

Competing interests

The authors have no relevant financial or non-financial interests to disclose.

Funding

Researchers Supporting Project Number (RSP2024R182) King Saud University, Riyadh, Saudi Arabia

Authors Contribution:

MBN; Experimentation and Methodology, SJ; Supervision and Validation, AR; writing-original draft preparation and Statistical analysis, AAS; Conceptualization, Data curation and Formal analysis, VR & MAE; Conceptualization and Investigation. All authors read and approved the final manuscript.

Acknowledgments: The authors would like to extend their sincere appreciation to the Researchers Supporting Project Number (RSP2024R182) King Saud University, Riyadh, Saudi Arabia.

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No competing interests reported.

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Evaluating Growth, Biochemical, Physiological and Yield Responses in Maize with Activated Biochar under different moisture conditions: A Field Study

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Abstract

Figures

1 Introduction

2 Material and methods

2.1. Biochar Production and Activation

2.2. Characterization of Research Site

2.3. Experiment Design and Treatments

2.4. Crop Water Management and Irrigation Strategy

2.5. Relative Growth and Yield Analysis

2.5.1. Relative increase in leaf area

2.5.2. Relative increase in Shoot/Root Weight

2.5.3. Relative Growth Rate

2.5.4. Net Assimilation Rate

2.6 Physiological and Biochemical Analysis

2.6.1. Cell membrane stability index assessment

2.6.2. Antioxidant activity assessment

2.6.3. Superoxide dismutase activity

2.6.4. Peroxidase activity

2.6.5. Catalase activity

2.7. Yield Analysis

2.8. Statistical Analysis

3 Results

3.1. Soil characterization

3.2. Crop water balance

3.3. Relative growth analysis

3.4. Biochemical and Physiological Parameters

3.5. Yield Analysis

3.6. Principal Component Analysis (PCA) and Heatmap

4 Discussion

4.1. Soil physicochemical properties

4.2. Influence of biochar supplementation on maize growth

4.3. Influence on Physiological, biochemical and yield attributes

5 Conclusion

Declarations

References

Additional Declarations

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