Interactive effects of acacia biochar, maize hybrids, and irrigation levels on soil health and crop productivity

Biochar production and activation

Biochar was produced from Acacia nilotica wood through slow pyrolysis at 450 °C for 3 h in a temperature-controlled pyrolysis unit, as described by Jahan et al. (2022). For biochar activation vermicompost and perlite were used. Vermicompost was prepared using corn stover as the feedstock and red earthworm (Eisenia fetida) as the composting organisms. To activate the biochar, it was mixed with perlite and vermicompost in a 1:1:1 ratio by weight. For enhancing activation process, a 2-liter liquid molasses solution was added to the mixture, which was stirred daily for proper aeration. The activation process continued for three weeks until excess moisture had completely evaporated (Jahan et al., 2023).

The field experiment was carried out from February to June, 2023. The study site was located in a loamy soil region of Gujrat, (Pakistan) characterized by slightly basic pH. Prior to sowing, the soil was ploughed three times using a tractor and a rotavator to ensure proper soil tilth, followed by irrigation through field water channels. Three levels of activated biochar amendments were incorporated into the top 15 cm of soil; 0 tons ha⁻¹ (A0), 5 tons ha⁻¹ (A1), and 10 tons ha⁻¹ (A2). The primary objective of activated biochar application was to enhance soil water-holding capacity and organic matter content by improving soil structure and chemical interactions (Paetsch et al., 2018). Soil samples were collected to analyze their physico-chemical properties.

Soil analysis

The baseline edaphic characteristic of the field soil were as follows: 279 g kg⁻¹ silt, 389 g kg⁻¹ clay and 330 g kg⁻¹ sand, classifying it as loam. Structural and elemental analyses of both amended and non-amended soil were performed using Scanning Electron Microscopy coupled with Energy-Dispersive X-ray spectroscopy (SEM-EDX; JSM-5910, JEOL, Japan). Soil organic matter content was determined according to the method of Estefan, Sommer & Ryan (2013). Soil pH was measured in air-dried soil samples following the ISO 10390 standard. Electrical conductivity (EC) was assessed using the procedure of Rayment & Higginson (1992). Saturation percentage (Sp) was calculated using the protocol outlined by Estefan, Sommer & Ryan (2013). ${\displaystyle SP=\frac{{\text{Total weight of H}}_2\mathrm{O}}{\text{Weight of dried soil}}\times 100.}$

Field experiment and experimental design

The experiment was conducted using a split-split-plot design with three replications. The total experimental area was 4,640 m². Each replication was divided into main plots (72 m²) and sub-sub-plot of 4 × 4 m². A two m wide path separated each sub-plot and sub-sub plot and water channels were established along these paths to facilitate controlled irrigation and prevent horizontal flow of water and cross contamination between treatments. The maize hybrids DK-2088, DK-6317, and YH-5427 were sown manually at a rate of 25 kg ha⁻¹, by maintaining a plant-to-plant spacing of 12.5 cm. Sowing was carried out on February 18, 2023.

Irrigation requirements (IR) for crop

Three irrigation regimes were implemented in the field experiment; full irrigation (FI) at 100% of crop evapotranspiration (ETc), partially deficit irrigation (PDI) at 70% ETc, and severely deficit irrigation (SDI) at 50% ETc. Daily reference evapotranspiration (ET₀) was determined using the FAO Penman-Monteith equation (Allen et al., 1998). The reference evapotranspiration values (expressed in mm day⁻¹) along with crop coefficient (Kc) values specific to maize growth stages, were used to calculate the crop’s water requirement. The following standard equation, as given by the Food and Agriculture Organization (FAO), was used to estimate irrigation needs. ${\displaystyle IN=ETC-Pe}$ where Pe indicates the effective rainfall, ETc is crop evapotranspiration, IN is the net water requirement. The evapotranspiration was calculated further by using the following formula: ${\displaystyle ETC=KC\times E{T}_0}$ where; ET₀ is the reference evapotranspiration, and Kc is crop coefficient for maize. The reference evapotranspiration was measured by using the Monteith equation (Howell & Evett, 2004).

Assessment of plant physiological and biochemical stress biomarkers

Proline content in maize leaf was determined by the procedure of Bates, Waldren & Teare (1973). Using the Prochazkova et al. (2001) method, the lipid peroxidation of maize leaves was recorded. ${\displaystyle MDA=\frac{\left[\left(A532-A600\right)-\left[\left(A440-A600\right)\left(\frac{MAat532}{MAat440}\right)\right]\right]}{157000}\times 10^6.}$ MA represents the molar substance in sucrose at two wavelengths (532 nm and 600 nm, respectively).

Leaf sugar content was estimated by crushing 0.5 g of leaf material in a clean mortar with 10 mL of distilled water (DuBois et al., 1956). After crushing, the mixture was filtered. In a test tube, 0.1 mL of filtrate with one mL of phenol (5% v/v) was added. After incubation for one hour, at room temperature, five mL of concentrated H₂SO₄ was added in the test tube. The same procedure was repeated for the blank and filtrate from other treatments. The absorbance readings of the sample and blank was noted at 420 nm by using a spectrophotometer (UV-Vis; Shmadzu 35, RP China).

For antioxidant enzyme assay, maize (one g) leaves were ground in a mortar and mixed with five mL of phosphate buffer and the mixture was centrifuged at 13,000 g at 4 °C for 19 min, and the upper layer was used to test the enzyme activity. The peroxidase was assayed by the procedure of Naveh, Mizrahi & Kopelman (1981). ${\displaystyle POD=\frac{\Delta 485}{mgofProtein}.}$ For catalase, the reaction mixture (three mL) was prepared by mixing 0.2 mL of enzyme extract and 50 mM H₂O₂. After 5 min, titanium reagent was added to terminate the reaction (Taranishi et al., 1974). Absorbance readings were noted at 410 nm, and following formula was used to assess catalase: ${\displaystyle CAT=\frac{\Delta 410}{mgofProtein}.}$

Assessment of morphological parameters

The plant height and leaf area were determined by ImageJ software (v1.51j8, USA). The fresh and dry weights of the crop were measured at 15-day intervals. Leaf, shoot, and root fresh weights were determined by an analytical weighing balance. Later, the root, shoots, and leaves were dried inside an oven for 72 h at 60 °C, and their dry weights were determined.

Yield parameters

The yield parameters including hundred seed weight, cob length, yield kg hectare⁻¹ were determined by Parihar et al. (2018).

Economic analysis of yield

An economic analysis of the maize crop was conducted using standard procedures, and average values were calculated across replications. Total production costs were estimated by counting for all inputs, including seed cost, land rent, field preparation, fertilizers, plant protection measures, and labor wages, following the approach outlined by Gittenger (1982). The gross income was determined based on prevailing market price of maize grain at the time of harvest. Net profit or loss was then calculated by subtracting the total cost of production from gross income. To assess the economic feasibility of the treatments, the benefit-cost ratio (BCR) was determined by using the following formula: ${\displaystyle BCR=\frac{Grossincome}{Totalcost}.}$

FTIR analysis of maize grain

The Fourier-transform infrared (FTIR) spectra of dry maize seed powder were obtained using the KBr (potassium bromide) method, utilizing a Nicolet iS5 Thermo Scientific instrument (USA), following the procedure outlined by Celi, Schnitzer & Négre (1997). The infra-red spectra of the dry maize seed samples were obtained in the range of 4,000 to 500 cm⁻¹. Further processing and analysis of the spectra were conducted using Origin Pro 2023.

Statistical analysis

Data were statistically analyzed using a three-way analysis of variance (ANOVA) under the general linear model (GLM) framework in Minitab 17. Mean comparisons were performed using Tukey’s test at a 5% significance level. All reported values represent the mean values ± standard error of the mean (SEM), based on three replicates (n = 3). Fourier-transform infrared (FTIR) spectral data from soil and maize seed samples were plotted using Origin Pro 2019. To explore the relationship between soil amendments, soil properties and plant responses, heatmap analysis was conducted using R statistical software (version R-4.3.1) following the approach described by Barter & Yu (2018).

Physicochemical characteristics of soil

The saturation percentage (Table 1) was recorded to be considerably higher (10–13%) with A1 (5 tons ha⁻¹ activated biochar) and A2 (10 tons ha⁻¹ activated biochar) in soil than A0 (soil without activated biochar). Regarding soil pH, it was noted that activated biochar buffered the soil to attain neutral pH and it was improved by 11%–19% with A1 and A2 incorporation in soil in contrast to A0. Similarly, soil organic matter was enhanced (50%–109%) with A1 and A2 activated biochar amendments. The electrical conductance of soil was also improved (18%–30%) in A1 and A2 amended soil. The available phosphorus and potassium were considerably improved i.e., 50%–125% and 8%–21% higher, respectively, in A1 and A2 incorporated soil.

Percent weight of elements in the soil

The surface characteristics and elemental composition of soil samples is presented in Fig. 1A and Fig. S1. The carbon percentage in soil was increased by 54% to 83% in A1 and A2 soil amended with activated biochar as compared to A0. The oxygen content was also enhanced by 10% to 43% in soil with activated biochar in contrast to non-amended soil. Magnesium content in soil was enhanced (52% to 10%) in A1 and A2 soil than in A0. The aluminum content in soil increased by 41% to 27% in A1 and A2 than in A0. Silicon in soil was lowered by 12% to 20% in A1 and A2 in contrast to control A0. The potassium percentage was increased by 26% to 9% in activated biochar-amended soil. The calcium percentage decreased by 29% to 52% in A1 and A2 soil than to non-amended soil. The titanium percentage was declined by 8% to 16% in A1 and A2 soil as compared to A0. The iron percentage was improved by 45% to 18% in A1 and A2 soil amended with activated biochar then to A0.

Soil FTIR analysis

The FTIR spectra of the soil showed the intense bands in the activated biochar amended soil (Fig. 1B). The OH band at 3,620 cm⁻¹ is 32% and 6% higher in A1 and A2 then to A0 soil. While, C = C band at 1,456 cm⁻¹ is 18% to 13% higher in A1 and A2 then to A0. The Si-O band at 1,006 cm⁻¹ is 2% to 1.4% higher intensity in A2 and A1 soil then to A0 (Xu et al., 2020).

Weather conditions

The climatic data is presented in Fig. S2 for the year 2023, mean monthly temperatures ranged from 21.9 °C (February) to 36.7 °C (June), with a gradual increase over the months. Mean relative humidity varied between 30.9% (February) and 39.4% (March), while wind speeds fluctuated from 2.41 m/s (March) to 3.21 m/s (May). Average daily sun hours remained consistently high, ranging from 10.12 (February) to 11.60 (May). Total monthly rainfall was absent in February (0.00 mm), peaked in March (83 mm), and reached its highest in June (134.1 mm).

Estimation of biochemical stress indicators

The highest increase of sugar content was found in Dk-6317 (32% to 55%) as compared to Dk-2088 and Yh-5427. While lowest sugar content was found in Dk-2088 under all irrigation levels (Fig. 2A). The proline content was higher in Dk-2088 in contrast to Yh-5427, and DK-6317. While the lowest proline content under all irrigation levels was found in Dk-6317 (Fig. 2B). The protein content was higher in Dk-2088 (17% to 136%), as compared to Yh-5427 and Dk-6317. The lowest protein content was found in Yh-5427 (70% to 62%) under all irrigation (Fig. 3A). The lipid peroxidation in Dk-2088, Yh-5427, and DK-6317 were decreased by 33% to 91%, 34% to 61% and 38% to 70.4% in A1 and A2 soil as compared to A0 under full irrigation. The lowest lipid peroxidation was detected in Dk-6317 by 25% to 32% in A1 and A2 soil when compare to A0 under severely deficit irrigation conditions (Fig. 3B). Considerably enhanced leaf peroxidase (POD) activity was recorded in the plants of non-amended soil; however, comparatively low POD activity was recorded in the plants of activated biochar-amended soil. The POD activity reduced under water stress in Dk-2088, Yh-5427, and DK-6317 by 36% to 66%, 41% to 60%, and 57% to 64% in A1 and A2 then to A0 (Fig. 3C). The catalase activity enhanced considerably in non-amended soil. However, the reduction of catalase activity (116% to 84%) was found in Yh-5427 in contrast to Dk-2088 and Dk-6317 under full irrigation in A1 and A2 soil then to A0. The lowest catalase activity was found in Dk-6317 (−29% to −48%) (Fig. 3D).

Morphological parameters

The highest increase in shoot fresh weight (Table S1) was observed in Dk-6317 at the vegetative and maturity stages, with improvements ranging from 4% to 19% and 4% to 8%, while at the tasseling stage, shoot fresh weight was improved considerably in Yh-5427 by 4% to 8% respectively, under full irrigation in A1 and A2 soil. Under partially deficit irrigation conditions, improvement in shoot fresh weight was recorded in Dk-6317 at the vegetative, tasseling, and maturity stages, with enhancements ranging from 6% to 37%, 12% to 13%, 5.5% to 8% respectively, while the highest shoot fresh weight under severely deficit irrigation conditions was recorded in DK-6317 at the vegetative, tasseling and maturity stages i.e., 38% to 58%, 6% to 9%, and 4% to 11% respectively in treatments A1 and A2. Conversely, the greatest improvement in shoot dry weight was noted in Dk-6317 at the vegetative, tasseling, and maturity stages, with enhancements ranging from 99% to 161%, 5% to 21%, 36% to 55% respectively, under severely deficit irrigation conditions in treatments A1 and A2.

Moreover, the most notable enhancement in root fresh weight was observed in Dk-6317, i.e., ranging from 107% to 153% at the vegetative stage (Table S2). At the tasseling and maturity stages, the most significant improvements in root fresh weight were observed in Dk-6317 i.e., 54% to 150%, 42% to 155% under severely deficit conditions in treatments A1 and A2 compared to treatment A0. Under severely deficit irrigation, the highest increase in root dry weight at the vegetative stage, tasseling, maturity stages 79% to 208% and 7% to 80% in A1 and A2 then to A0.

In terms of leaf fresh weight (Table S3), the highest improvement at the vegetative stage was found in Yh-5427 ranging from 15% to 89%, while at tasseling and maturity stages, improvement was detected in Dk-6317, i.e., ranging from and 21% to 87% and 15% to 37% respectively, under severely deficit irrigation in treatments A1 and A2 compared to treatment A0. Under severely deficit irrigation, the highest increase in leaf dry weight at the vegetative stage was found in Dk-6317 i.e., 128% to 220%. Regarding plant height, the highest improvement at the vegetative stage was observed in DK-2088 by 44% to 47%. At the tasseling and maturity stages, the highest improvements were found in YH-5427 by 6% to 21% and 30% to 35% in treatments A1 and A2 compared to A0 under partially deficit irrigation.

Regarding plant height (Table S4) at the vegetative stage, the highest improvement was observed in DK-2088 by 43% to 47%. At the tasseling and maturity stages, the highest improvements were found in YH-5427 by 5% to 21% and 30% to 35% in treatments A1 and A2 compared to A0 under partially deficit irrigation. Across all growth stages, the most substantial increase in leaf area under partially deficit irrigation was observed in YH-5427 by 19% to 42% in treatments A1 and A2 compared to A0 (Table S4). At the vegetative and maturity stages, the highest increase in leaf area was found in Dk-6317 by 28% to 37% and 8% to 11%, respectively, while at the tasseling stage, highest improvement was observed in YH-5427 by 19% to 44% under severely deficit irrigation in treatments A1 and A2 compared to A0. Regarding grain fresh weight, the most substantial increase was observed in Dk-2088 (45% to 130%) under full irrigation in treatments A1 and A2 compared to treatment A0 (Table S4).

Yield attributes

After experiencing water stress, plants grown in non-amended soil exhibited significantly lower yields (Fig. 4). However, activated biochar-amended soil demonstrated notable enhancements in the hundred seed weight of Dk-2088, Yh-5427, and Dk-6317, ranging from 5% to 49%, 9 to 55%, and 51% to 65% respectively, in A1 and A2 respectively, compared to A0 under partially deficit irrigation. Similarly, the hundred seed weight also improved considerably in activated biochar amended soil under severely deficit irrigation, with Dk-2088 exhibiting an increase of 29% to 52%, Yh-5427 showing an increase of 33% to 67%, and Dk-6317 displaying an increase of 31% to 45% in treatments A1 and A2 compared to A0 (Fig. 4A). The yield per hectare of maize was significantly influenced by various doses of activated biochar (Fig. 4B). Application of activated biochar enhanced yield per hectare in Dk-2088 by 5% to 15%, in Yh-5427 by 13% to 27%, and in Dk-6317 by 28% to 33% in A1 and A2 compared to A0 under full irrigation. Furthermore, under partially deficit irrigation, the yield per hectare of Dk-2088 increased by 9% to 24%, in Yh-5427 by 18.4% to 27%, and in Dk-6317 by 26% to 34% when soil treated with A1 and A2 compared to A0. Similarly, yield per hectare under severely deficit irrigation increased in Dk-2088 by 11% to 29%, in Yh-5427 by 17% to 14% and in Dk-6317 by 11% to 24% in soil amended with activated biochar (A1 and A2) compared to non-amended soil. The highest increase in cob length (Fig. 4C) was observed in Dk-2088, ranging from 8% to 27%, compared to Yh-5427 and Dk-6317. Regarding stover yield, notable enhancements were observed in Dk-2088, Yh-5427, and Dk-6317, ranging from 15% to 34%, 29% to 36% and 8% to 59%, respectively, under full irrigation in A1 and A2 compared to A0. Conversely, under partially deficit irrigation, stover yield (Fig. 4D) increased by 7% to 38% in Dk-2088, by 22% to 28% in Yh-5427, and by 22% to 40% in Dk-6317 when comparing treatments A1 and A2 to A0. Under severely deficit irrigation conditions, stover yield increased by 30% to 58% in Dk-2088, by 18% to 33% in Yh-5427, and by 47% to 65% in Dk-6317 in soil treated with activated biochar (treatments A1 and A2).

Heatmap analysis of interaction of soil properties and plant growth characteristics

A heatmap analysis (Fig. 5) was conducted to examine the interactions among different levels of activated biochar applications i.e., 0 tons ha⁻¹(A0), 5 tons ha⁻¹ (A1), 10 tons ha⁻¹ (A2), across three maize hybrids, DK-2088 (V1), Yh-5427 (V2), DK-6317 (V3) under three moisture level full irrigation (FI), partially deficit irrigation (PDI), and severely deficit irrigation (SDI). The results revealed that the A2 treatment under FI significantly improved soil organic matter and mineral content, and plant physiological and biochemical parameters across all three hybrids (A2+FI+V1, A2+FI+V2, and A2+FI+V3). In contrast, A0 under SDI showed marked reductions in soil health and plant performance (A0+SDI+V1, A0+SDI+V2, A0+SDI+V3). Notably, both activated biochar levels (A1 and A2) improved soil quality and plant responses under stress conditions, with the A2 offering the most substantial benefits even under SDI (A2+SDI+V1, A2+SDI+V2, A2+SDI+V3).

Economic analysis of maize

The average net returns from maize cultivation varied significantly among the three hybrids and irrigation regimes, depending on the level of activated biochar application (Table S5). For Dk-2088, net returns ranged from 1,303 to 1,526 USD under full irrigation, 796 to 1,087 USD under partially deficit irrigation, and 463 to 637 USD under severely deficit irrigation. For Yh-5427, returns ranged from 1,323 to 1,628 USD under FI, from 908 to 1,065 USD under PDI and from 402 to 603 USD under SDI. Meanwhile, Dk-6317 exhibited the highest economic performance, with returns ranging from 1,552 to 1,771 USD under FI, 1,080 to 1,259 USD under PDI and 215 to 645 USD under SDI. These gains were recorded in plots amended with activated biochar at A1 and A2 levels, while the lowest returns were consistently observed in non-amended soil across all moisture regimes.

FTIR analysis of maize grains

FTIR analysis revealed significant changes in peak intensities of key functional groups across maize hybrids (Fig. 6). The hydroxyl group (3,281 cm⁻¹) showed the highest intensity increases in hybrid Dk-2088 under partially and severely deficit irrigation (PDI and SDI), with 3.25–3.89% and 1.44–3.62% increases in A1 and A2 compared to A0. Conversely, a decline in this peak was observed in Dk-6317 under similar conditions. The methyl/methylene group (2,926 cm⁻¹) exhibited marked enhancement in Yh-5427 under full and PDI conditions, particularly under A2 (up to 29%). Dk-6317 showed notable increases in fatty acid-associated peaks (1,747 cm⁻¹) across all irrigation regimes, peaking at 7% under SDI in A2. Amide I (1,624 cm⁻¹) and Amide II (1,536 cm⁻¹) intensities increased substantially, especially in Yh-5427 under PDI (up to 9.44%) and full irrigation (11%) respectively, while Dk-2088 showed a moderate increase under SDI. The phospholipid peak (1,152 cm⁻¹) increased significantly in Yh-5427 under PDI and SDI, with peak enhancements of 6.92% and 3.96% in A2, respectively. Phytic acid (1,050 cm⁻¹) intensity increased only in Dk-6317 under PDI and SDI, with the highest increment reaching 12.2% in A2. Meanwhile, Fe–O (584 cm⁻¹) intensity markedly increased in Dk-6317, reaching 24.9% under FI and A2. The MgO (786 cm⁻¹) and ZnO (710 cm⁻¹) peaks showed maximal intensities in Yh-5427 under SDI, particularly under A2 (18.2% and 25.5%, respectively). Lastly, lipid (1,755 cm⁻¹) and protein (1,614 cm⁻¹) peaks were highest in Dk-6317 and Yh-5427 respectively under PDI in A2, both exhibiting 5.7% increase compared to A0. Overall, the A2 amendment consistently led to enhanced biochemical signatures, especially under water-deficit conditions.