Biochar Improves Soil Fertility in Sandy Nutrient-Poor Soil, While Wood Distillate Modulates Nutrient Dynamics and Plant Physiological Responses in Basil (Ocimum basilicum L.) Plants

Abstract

The progressive decline in fertility in nutrient-poor sandy soils has increased interest in soil conditioners that improve nutrient availability. Rising costs and the need to restore biological fertility have shifted attention towards fertilizers that not only enhance productivity but also improve soil biological activity. This study aims to evaluate the effects of biochar (3% w/w; BC) and wood distillate (one irrigation intervention per week at 2% v/v; WD), applied individually or in combination (BC + WD), on a nutrient-poor soil, evaluating soil fertility and basil plant physiology and growth but also antioxidant responses in a pot experiment. Soil NPK content and enzymatic activity were assessed, while plant growth, macronutrient uptake, gas exchange, and antioxidant system responses were monitored after 28 and 56 days of treatment. BC treatment, followed by BC + WD treatment, increased soil P availability by 36% and 37%, respectively, after 56 days compared to untreated soil (CNT). A similar pattern was evidenced for the exchangeable K and pH of the soil. Although BC led to a reduction in soil enzymatic activity, the BC + WD treatment enhanced urease and acid phosphatase activity after 56 days by 26% and 7%, respectively, compared to CNT. Similarly, P uptake by plants was improved by BC + WD after 56 days, while potassium, K, uptake increased in both the BC and BC + WD treatments by 38% and 75% at the final sampling. BC or BC + WD resulted in improved photosynthesis and gas exchange, while WD influenced responses related to redox balance and antioxidant activity over time. Moreover, BC + WD slightly stimulated an increase in dehydroascorbate reductase (+52%), ascorbate peroxidase (+78%), and glutathione reductase (+41%) activity compared to CNT, enforcing the plant antioxidant system. Therefore, the positive antioxidant responses were primarily attributed to the use of BC rather than WD. Both BC and BC + WD proved to be effective and sustainable soil conditioners with beneficial effects on soil P and K availability, as well as certain enzymatic activities. For plants, the effects were more pronounced with BC treatment, showing antioxidant responses within the first 56 days. In general, BC improved soil fertility, and WD acted as a modulator of nutrient dynamics and plant physiological responses, especially when combined with BC.

1. Introduction

The sustainable management of soil aimed at enhancing crop quality and yield without affecting soil health has become one of the main challenges for agricultural practices. Progressive loss of organic matter, biodiversity, soil compaction and pollution, as well as biological desertification, are the main concerns in soil science [1]. Sandy soil, often used for high-income production, is the most exposed to nutrient deficiency because of its characteristics: high aeration, low soil organic matter (SOM), low cation exchange capacity (CEC), and use in agricultural practices involving frequent tillage and a high use of mineral fertilizers [2].

Biochar, a C-rich material obtained by the pyrolysis of biomass under anaerobic conditions, has increased in popularity and prevalence in recent years, due to the several positive changes in long-term soil fertility and significant environmental impact of C stocking [3]. Biochar’s effects on the physical properties of soil depend on the source material, the pyrolysis temperature peak and duration, as well as the characteristics of the soil, but usually involve an increase in water holding capacity and infiltration, an improvement in aggregate stability, and a reduction in nutrient loss [4]. Soil porosity increases with high doses in biochar application [5]. Therefore, bulk density decreases and soil aeration improves [6].

The application of this soil improver can also positively affect the chemical properties of the soil. For instance, Premalatha et al. [7], in their experiment with rising irrigation water salinity, noted that biochar produced an electrical conductivity (EC) decrease in soil irrigated with salted water, while EC increased in soil irrigated with unsalted water, demonstrating that biochar acts as an EC buffer. In the same experiment, the results showed that biochar has positive effects on total organic C and the availability of N, P and K and that this pattern is dose-dependent; also, the activities of phosphatase, urease and dehydrogenase are enhanced by biochar application. These results are also confirmed by Phares et al. [8], who showed a significant increase in total organic carbon (TOC), total N and available P. Soil enzymatic activity is also promoted by biochar. These experiments reported an interesting aspect of the effect of biochar on crop production, for instance, an increase in plant height, biomass, and flowering, and the biosynthesis of secondary metabolites, such as phenols, flavonoids, alkaloids and tannins.

Wood distillate, also known as wood vinegar or pyroligneous acid, is a liquid by-product of the pyrolysis process, the same involved in biochar production. In recent years, wood distillate has attracted farmers as a soil improver and as a plant protection product [9]. It is produced from the condensation of gases during biochar production [10] and it is mainly composed of organic acids, phenols, aldehydes, ketones and alcohols [11]. This product can enhance the availability of nutrients such as N, P, K and Ca, depending on many factors [12]. Wood distillate also affects soil enzymatic activity. For instance, Cantini et al. [11], in a study that aimed to develop a slow-release fertilizer, reported an inhibitory effect on urease activity due to wood distillate application. Koc et al. [13] found that wood distillate positively affects alkaline phosphatase and β-glucosidase activity. Regarding the effect on plants, Gu et al. [14] reported that the application of wood distillate at different water dilutions (between 1:100 and 1:400 v/v) can enhance the yield of Raphanus sativus L. in terms of root diameter and weight, supported by an increase in net photosynthetic rate. Carril et al. [15], in an experiment on three leguminous species, reported that wood distillate was effective for enhancing the plant yield of Lens culinaris L.

The interaction between biochar and wood distillate has recently gained increasing attention in the scientific community, especially to maximize the benefits induced by biochar and wood distillate and to compensate for the limitations of their individual applications. A few recent studies have been conducted on the interaction between these two materials [2,16,17,18]. However, the state of the art presents some gaps. For instance, their combination produces pH stabilization or a slight increase [2]. Becagli et al. [16] reported that TOC and dissolved organic carbon (DOC) were influenced by biochar but not affected by wood distillate and the combined treatment showed no differences compared to biochar alone. On the other hand, the combination of the two materials produced a significant increase in total N and available P and this improvement in nutrient availability was also found in plant biomass [16]. An increase in plant yield is reported by other studies after the application of both biochar and wood distillate [2,18].

Basil (Ocimum basilicum L.), a member of the Lamiaceae family, is a versatile aromatic herb renowned for its culinary, medicinal, and nutraceutical applications, characterized by diverse chemotypes rich in essential oils like linalool, eugenol, and phenolic acids such as rosmarinic acid [19]. Studies on chemotypes revealed that cultivars and morphotypes of different basil varieties influence phenolic acid profiles and antioxidant capacities, making this plant species very versatile [19]. Moreover, under reduced solar radiation conditions, certain genotypes exhibited superior light use efficiency, yielding higher biomass and more branches [20]. Our previous research has demonstrated the utility of this plant species in assessing soil conditioners [16]. For instance, co-application of wood distillate and biochar enhanced soil organic carbon, microbial biomass, enzymatic activities (e.g., phosphatase and urease), and basil dry biomass by improving N and P availability [16]. Similarly, wood distillate mitigated bioplastic-induced stress in basil, increasing soluble proteins and ascorbic acid contents while reducing lipid peroxidation levels [21].

This study aimed to assess whether the biochar effects on soil, modulated by wood distillate application, could represent a key strategy for restoring nutrient-poor soils in terms of fertility. Indeed, considering the soil fertility loss expected in the coming years, the novelty of the work lies primarily in the use of marginal soils. Another important and scientifically novel aim of the present experiment was to evaluate the influence of the single and combined application of biochar and wood distillate on basil production and physiological mechanisms, with the hypothesis that biochar would have a greater impact on soil fertility than on plant responses, whereas wood distillate would be more effective in modulating physiological characteristics and nutrient uptake; the combination of these products is expected to integrate these positive effects. In addition, to the best of our knowledge, antioxidant responses were evaluated in plants grown in soil treated with biochar and/or wood distillate, thereby contributing to the advancement of scientific knowledge.

2. Materials and Methods

2.1. Experimental Set Up

The experiment was carried out in a greenhouse at the Department of Agriculture, Food and Environment of the University of Pisa, Italy (43°42′ N;10°25′ E) during the period from June to July 2025. The growing conditions were an average temperature of 31 °C, 65.8% relative humidity and a light intensity of 158.7 W m⁻². The sandy soil utilized for the experiment was collected close to Pisa at 5 m above sea level (43°40′ N; 10°19′ E), air dried and sieved to 2 mm. Soil characteristics are reported in

Table 1. Characterization of soil used for the experiment. EC: electrical conductivity; TOC: total organic carbon; WHC: water holding capacity.

	Unit	Value
Sand	%	94.5
Silt	%	5.0
Clay	%	0.5
pH		8.74
EC	μS cm−1	188.5
TOC	%	0.37
Total N	mg kg−1	390.12
C/N ratio		9.5
Available P	mg kg−1	12.15

.

Refrigerated basil seeds (O. basilicum cv. “Tigullio”) obtained during 2025 and purchased from the company Pagano Domenico e Figli (Scafati, Salerno, Italy) were sown in rock wool cubes, and seedlings were grown for 14 days in a commercial nutrient solution (Atami, Rome, Italy). At the transplanting phase, seedlings were chosen at the same phenological stage, characterized by the same number of leaves and plant height (i.e., four true leaves and an average plant height of 5 cm) and transplanted in 0.4 L pots.

The experiment consisted of three soil treatments: soil amended with 3% (w/w) biochar before transplanting (BC), the weekly fertigation per pot with 250 mL of wood distillate stock solution diluted to 2% (v/v; WD) and the combination of the two previous treatments (BC + WD). Untreated soil was used as the control (CNT), as reported in Figure 1.

The irrigation regime (three times per week with tap water to restore soil moisture to saturation capacity) was the same for all treatments (CNT, BC, WD and BC + WD) and, when the wood distillate was distributed for WD and WD + BC treatments, CNT and BC were fed with tap water. Forty basil plants (10 per treatment) were randomly distributed in the greenhouse. Soil and plant sampling were performed after 28 and 56 days. After 28 days basil plants were cut because they reached commercial maturity. Cutting may negatively affect the physiological and biochemical responses of regrowing plants by inducing oxidative stress. However, the same procedure was applied to all plants in the present experiment. Subsequently, basil plant re-growth was allowed, and the final sampling was carried out at 56 days.

2.2. Biochar and Wood Distillate

BC was applied at a rate of 3% (w/w) corresponding to 51 t ha⁻¹ and the source material was woodchips (30–50 g) from certified forest residues (Abies sp., Alnus sp., Castanea sativa, Fraxinus sp., Quercus sp., Robinia pseudoacacia) obtained by pyrogasification (BIODEA–RM Group, Arezzo, Italy). The average heating rate before reaching a peak of 1280 °C was 75–80 °C min⁻¹. The extreme temperature peak is explained by the maximization of wood distillate production at that temperature. This allows the same process to be used to efficiently produce both soil conditioners. The parameters used for BC characterization were analyzed through certified methods approved by Italian regulations (Legislative Decree 75/2015): pH 9.8, 400% water holding capacity, 8.7% organic carbon, <0.5% total N, and 0.034% total P. The TOC content was classified as Class 1 following the Guidelines for Certification of the International Biochar Initiative (IBI; http://www.european-biochar.org/en/ebc-ibi, accessed on 20 December 2025). The utilization of 1280 °C as the peak temperature of biochar production can initially affect the interaction with soil, making it slower but, at the same time, introducing a source of more stable carbon compared with biochar produced at lower temperatures [4].

WD applied at 2% v/v was produced by BIODEA–RM Group and was obtained from the same source material used to produce BC. The main WD characteristics were as follows: pH 2.8, density 1.04 kg L⁻¹, TOC 33.8 g L⁻¹, total N 0.43 g L⁻¹, organic acid 3.23%, phenolic compounds 13.0 g L⁻¹, and methanol 13.4 g L⁻¹.

2.3. Soil Analysis

Soil was characterized before the experimental trial (see Table 1) and after 28 and 56 days of treatments. Physical and chemical properties of the soil such as pH, texture and EC were determined by standard methods [22,23]. pH was measured in water at a 1:2.5 ratio; texture was determined, after 2 h agitation with 5% (w/v) sodium hexametaphosphate, using an Esenwein sedimentation tube, followed by weighing clay and silt + clay fractions and calculating the particle-size distribution percentages by difference, based on the different sedimentation timing [24]. EC was measured using a conductivity meter after aqueous extraction at 1:10 ratio and filtration. TOC was determined using the method by Walkley and Black [25]. N_tot content was determined by the Kjeldahl method [26]. Soil was digested with H₂SO₄ and catalyst, distilled with UDK 129 Kjeldahl distillation unit (VELP scientifica, Usmate, Italy) and titrated with HCl 0.1 N. Available P was determined spectrophotometrically (Perkin Elmer Lambda 25, Milan, Italy) by the modified molybdenum blue method applied to acid extracts [27]. All analyses were performed on three replicates, each consisting of soil collected from three pots.

Soil Enzymatic Activities

Acid phosphomonoesterase activity (phosphatase) was assessed following the method described by Eivazi and Tabatabai [28] based on the hydrolysis of p-nitrophenyl phosphate by soil enzymes at pH 6.5 and spectrophotometric measurement at 410 nm.

β-Glucosidase activity was measured using a 4-nitrophenyl-β-D-glucopyranoside substrate and, after an incubation period at 37 °C for 60 min, the produced p-nitrophenol was measured at 410 nm [29].

Urease activity was measured spectrophotometrically at 690 nm according to Kandeler and Gerber [30] by analyzing ammonia production after a 2 h incubation with a urea substrate at 37 °C.

Fluorescein diacetate (FDA) hydrolysis was measured with spectrophotometer at 490 nm according to Green et al. [31] by analyzing fluorescein production after 3 h of incubation with fluorescein diacetate substrate at 37 °C.

The soil alteration index three (SAI3) was used to evaluate the influence of the treatments on the quality. As reported in Puglisi et al. [32], SAI3 was determined by converting the enzyme activity data according to the following equation (1):

SAI3 = (7.87 × β-glucosidase) − (8.22 × phosphatase) − (0.49 × urease),

(1)

where enzyme activities were expressed as micromoles of p-nitrophenol per gram of soil per hour (for β-glucosidase and phosphatase), and as micrograms of urea per gram of soil per hour (urease). It is important to underline that the lower the value of this index, the greater the effect of treatments on soil key enzyme activities. Therefore, fertilization treatment is expected to result in a decrease in SAI3.

All analyses were performed on three replicates, each consisting of soil collected from three pots.

2.4. Plant Analysis

2.4.1. Plant Fresh and Dry Weight

At each sampling stage, plant biomass and the dry weight of five plants per treatment were measured. Dry weight was calculated after drying the plant material in a ventilated oven (Memmert GmbH Co., KG Universal Oven UN30, Schwabach, Germany) at 105 °C until constant weight was reached.

2.4.2. Leaf Relative Water Content (RWC)

According to Kachout et al. [33], with minor modifications, leaf samples from three different plants per treatment were immediately weighed to determine the fresh weight (FW) of each leaf for RWC determination. The leaf samples were then floated on distilled water for 24 h in darkness to obtain the fully turgid mass. After this period, fully hydrated leaves were dried with paper towels, and then weighed to determine the saturated weight (SW). Finally, the leaves were placed in a 105 °C oven for 48 h to measure the dry weight (DW). The RWC of each leaf was calculated as follows (2):

RWC = [((FW − DW))/((SW − DW))] × 100

(2)

2.4.3. NPK in Leaves

Leaf N content was determined using the same method applied to soil samples. P in leaves (n = 3) was measured spectrophotometrically following the molybdenum blue method according to Murphy and Riley [34], as modified by Benini et al. [35]. For leaf K (n = 3), about 0.2 g of dry powdered leaf samples were put in Teflon tubes with 8 mL of HNO₃ (70%, v/v) and incubated overnight at room temperature. Then, samples were mineralized at 200 °C for 40 min using a microwave digestion system (Start D, Milestone Srl, Sorisole, BG, Italy). Samples were then transferred to a final volume of 25 mL, adjusted by the addition of double-distilled water. After mineralization, cation concentrations were measured using Inductively Coupled Plasma Mass Spectrometry (ICPMS-2030, Shimadzu, Kyoto, Japan). All the analyses were performed on three biological replicates, each consisting of plants from three pots.

2.4.4. Gas Exchange and Photosynthetic Pigments

At 28 and 56 days, four fully expanded leaves (n = 20) were randomly selected from five plants per treatment. Gas exchange measurements were conducted between 11:00 a.m. and 1:00 p.m. using a portable infrared gas analyzer (LI-6400, Li-Cor, Lincoln, NE, USA). Measurements were performed under a photosynthetic photon flux density of 1500 μmol m⁻² s⁻¹ (according to ambient PAR conditions). The CO₂ concentration within the leaf chamber was maintained at 400 μmol mol⁻¹ using the CO₂ mixing system, with an airflow rate of 500 μmol s⁻¹. Once steady-state conditions were reached, the net photosynthetic rate (P_n), intercellular CO₂ concentration (C_i), and stomatal conductance (g_s) were recorded. Intrinsic water use efficiency (WUE_int) was calculated as the ratio of P_n to g_s.

Chlorophylls and carotenoids were extracted from fresh leaf material from five plants per treatment according to Papadakis et al. [36]. Total chlorophyll (Chl) and carotenoid (Car) concentrations were determined according to Gisbert-Mullor et al. [37]. Data are expressed as mg Chl or Car per g of FW.

2.4.5. Oxidative Stress Marker

Leaf hydrogen peroxide (H₂O₂) content was determined following Velikova et al. [38]. Fresh tissue (0.1 g) was homogenized in 1% (w/v) TCA, centrifuged at 14,000× g for 15 min at 4 °C, and the supernatant was reacted with potassium iodide in sodium phosphate buffer (pH 7.0). H₂O₂ concentration was measured at 350 nm and expressed as µmol g⁻¹ FW, using a standard curve. All the analyses were performed on four leaves from four biological replicates, each consisting of plants from four pots.

2.4.6. Antioxidant Enzymes

Leaf tissue samples were ground in liquid nitrogen and extracted in 66 mM potassium phosphate buffer (KH₂PO₄/K₂HPO₄, pH 7.0) containing 1 mM EDTA. The homogenates were centrifuged at 14,000× g for 10 min at 4 °C, and the resulting supernatants were used for protein quantification and enzyme activity assays. Protein concentration was determined according to Bradford [39], using bovine serum albumin as the standard. All analyses were performed on five biological replicates, each consisting of plants from five pots.

Superoxide dismutase (SOD) activity was measured following Beauchamp and Fridovich [40] by monitoring the inhibition of nitro blue tetrazolium (NBT) photoreduction at 560 nm. One unit of SOD activity was defined as the amount of enzyme required to inhibit NBT reduction by 50%, and results were expressed as U μg⁻¹ protein min⁻¹.

Catalase (CAT) activity was assayed according to Lyons et al. [41] by recording the decrease in absorbance at 240 nm due to H₂O₂ decomposition. Activity was calculated using the molar extinction coefficient of H₂O₂ (39.4 mM⁻¹ cm⁻¹) and expressed as nmol H₂O₂ μg⁻¹ protein min⁻¹.

Ascorbate peroxidase (APX) activity [41] was determined by monitoring the oxidation of ascorbate at 290 nm, using an extinction coefficient of 2.8 mM⁻¹ cm⁻¹, and expressed as nmol AsA μg⁻¹ protein min⁻¹.

Dehydroascorbate reductase (DHAR) activity [41] was measured by following ascorbate formation at 265 nm (ε = 14 mM⁻¹ cm⁻¹), subtracting blank values, and expressed as nmol AsA μg⁻¹ protein min⁻¹.

Glutathione reductase (GR) activity [41] was assessed by monitoring NADPH oxidation at 340 nm (ε = 6.22 mM⁻¹ cm⁻¹) and expressed as nmol NADPH μg⁻¹ protein min⁻¹.

2.4.7. Antioxidant Molecules

Reduced (AsA) and oxidized (DHA) ascorbate forms were determined according to Kampfenkel et al. [42]. Fresh roots or leaves (0.1 g) were extracted in 6% w/v TCA and centrifuged at 14,000× g for 15 min at 4 °C. Total ascorbate was measured after reduction with dithiothreitol, whereas AsA was quantified without the reducing agent. Following incubation at room temperature, samples were reacted with N-ethylmaleimide, TCA, orthophosphoric acid, 2,2′-dipyridyl, and FeCl₃, and absorbance was recorded at 525 nm. DHA content was calculated as the difference between total ascorbate and AsA. Results were expressed as µmol g⁻¹ FW.

Reduced (GSH) and oxidized (GSSG) glutathione levels were determined following De Pinto et al. [43]. Total glutathione and GSSG were measured after derivatization with vinylpyridine utilizing the same extract used for ascorbate quantification. The enzymatic assay was performed by monitoring 5,5′-dithiobis(2-nitrobenzoic acid) reduction at 412 nm in the presence of glutathione reductase and NADPH. GSH content was calculated as the difference between total glutathione and GSSG. Results were expressed as µg g⁻¹ FW.

All the analyses were performed on three biological replicates, each consisting of plants from three pots.

2.5. Statistical Analysis

After checking the normality of distribution (Shapiro–Wilk test, 95% confidence interval) and the homoscedasticity by Bartlett test, a one-way ANOVA was carried out using the soil treatment (BC, WD and the combination) as the variability factor. Significant differences among treatments were determined by LSD Fisher post hoc test (p ≤ 0.05). GraphPad 9 software (GraphPad, La Jolla, CA, USA) was used for statistical analysis.

3. Results

3.1. Soil

The addition of BC to the soil significantly increased pH in both sampling times; WD had the opposite effect, but only at the first sampling, while no differences were observed after 56 days compared to CNT (

Table 2. Soil pH, electrical conductivity (EC), total N, available P and exchangeable K contents in biochar-enriched soil (B), in wood distillate-enriched soil (WD) and in soil enriched with the combination of biochar and wood distillate (BC + WD) after 28 and 56 days of treatment. Non-treated soil was utilized as control (CNT). Means were subjected to a one-way ANOVA with soil treatment as the variability factor, analyzing each time point separately. For each parameter, means followed by different letters indicate significant differences at p ≤ 0.05 using the post hoc LSD test. Lack of letters means no significant differences with the same statistical analysis.

	28 Days	56 Days
pH
CNT	8.40 ± 0.04 c	8.57 ± 0.04 c
BC	8.58 ± 0.01 a	8.72 ± 0.01 a
WD	8.35 ± 0.01 d	8.56 ± 0.02 c
BC + WD	8.50 ± 0.02 b	8.62 ± 0.02 b
EC (μS cm−1)
CNT	228.00 ± 22.61	206.00 ± 6.24 b
BC	237.00 ± 12.29	248.33 ± 34.96 a
WD	235.67 ± 20.79	182.60 ± 10.39 b
BC + WD	266.67 ± 42.52	281.00 ± 20.66 a
Total N (mg kg−1)
CNT	501.67 ± 45.00 b	499.33 ± 10.69 b
BC	485.33 ± 35.92 b	529.67 ± 26.50 ab
WD	567.00 ± 37.04 a	480.67 ± 21.39 b
BC + WD	525.00 ± 7.00 ab	557.67 ± 42.19 a
Available P (mg kg−1)
CNT	15.62 ± 0.52 a	18.18 ± 1.64 b
BC	12.75 ± 1.47 b	17.59 ± 3.36 b
WD	13.81 ± 0.99 ab	24.73 ± 0.63 a
BC + WD	13.18 ± 0.86 b	24.86 ± 1.31 a
Exchangeable K (mg kg−1)
CNT	69.58 ± 1.28 b	35.53 ± 1.51 b
BC	239.62 ± 4.05 a	148.45 ± 7.60 a
WD	75.60 ± 3.01 b	32.47 ± 1.50 b
BC + WD	237.77 ± 5.02 a	166.97 ± 31.19 a

).

BC + WD induced a slight but significant pH increase compared to CNT on both samplings (Table 2). EC was positively affected by BC and BC + WD treatments after 56 days; no significant differences were observed at the first sampling time (Table 2).

Total N content increased in WD-treated soil after 28 days (by 11% compared to CNT, whilst, after 56 days, BC + WD treatment induced the highest N content in the soil (+10% compared to CNT). Differently, available P content was negatively affected by BC and BC + WD treatments after 28 days, while, in the final sampling, WD and BC + WD treatments enhanced P availability after 56 days, by 26 and 27%, respectively, compared to CNT. Exchangeable K was increased by 71% by BC and BC + WD treatments after 28 days, and by 77% on average after 56 days (Table 2).

Acid phosphatase activity was slightly suppressed by BC treatment after 56 days, while BC + WD had a significant positive influence on this enzymatic activity compared with CNT (Figure 2a).

β-glucosidase activity was reduced by BC (–4% after 28 days and 22% after 56 days) than CNT, while WD increased β-glucosidase enzymatic activity by 18 after 28 days and by 13% after 56 days compared with CNT (Figure 2b). Urease activity was increased by the BC + WD treatment compared with CNT after 28 and 56 days (+42% and +22%, respectively), whilst an increase in this enzyme was evidenced in BC + WD compared with BC and WD treatments only after 56 days (Figure 2c). FDA hydrolysis was positively affected by BC treatment at both sampling times, showing increases of 28 and 37% after 28 and 56 days, respectively, compared with CNT. At the final sampling, WD and BC + WD treatments had a positive effect compared with CNT, with increases of 40 and 43%, respectively (Figure 2d). SAI3 was reduced by the combined treatment at both sampling times (Figure 2e).

3.2. Plant

3.2.1. Plant Yield, Physiology and Elemental Results

Total plant fresh weight was significantly enhanced by BC exclusively after 56 days, whilst the plant dry weight reported higher values after 56 days of BC treatment when compared to BC + WD, even though no significant differences were found when compared to CNT (

Table 3. Total plant fresh weight and dry weight, leaf relative water content (RWC), leaf N, P and K contents in basil plants grown in biochar-enriched soil (B), in wood distillate-enriched soil (WD) and in soil enriched with the combination of biochar and wood distillate (BC + WD) after 28 and 56 days of treatment and a plant cut between the two time points. Plants grown in non-treated soil were utilized as control (CNT). Means were subjected to a one-way ANOVA with soil treatment as the variability factor, analyzing each time point separately. For each parameter, means followed by different letters indicate significant differences at p ≤ 0.05 using the post hoc LSD test. Lack of letters means no significant differences with the same statistical analysis.

	28 Days	56 Days
Total plant fresh weight (g plant−1)
CNT	5.29 ± 1.10	3.25 ± 0.26 b
BC	5.63 ± 0.86	4.36 ± 0.56 a
WD	5.47 ± 0.96	3.81 ± 0.28 ab
BC + WD	4.96 ± 0.74	3.70 ± 0.48 b
Total plant dry weight (g plant−1)
CNT	0.91 ± 0.21	0.65 ± 0.20 ab
BC	0.97 ± 0.12	0.77 ± 0.10 a
WD	0.91 ± 0.15	0.65 ± 0.04 ab
BC + WD	0.80 ± 0.15	0.60 ± 0.10 b
RWC
CNT	85.77 ± 1.67 a	81.83 ± 3.88 a
BC	85.70 ± 3.03 a	65.38 ± 10.32 b
WD	83.53 ± 2.43 b	67.14 ± 7.37 b
BC + WD	86.62 ± 1.44 a	74.39 ± 26.38 b
Leaf N (mg g−1 DW)
CNT	14.96 ± 0.15 a	12.37 ± 1.80
BC	12.24 ± 1.43 b	11.67 ± 1.41
WD	13.54 ± 1.16 ab	11.32 ± 1.13
BC + WD	12.30 ± 0.70 b	12.83 ± 1.41
Leaf P (mg g−1 DW)
CNT	3.47 ± 0.06	1.95 ± 0.07 a
BC	3.58 ± 0.26	1.59 ± 0.18 b
WD	3.65 ± 0.22	1.73 ± 0.12 ab
BC + WD	3.96 ± 0.13	1.65 ± 0.11 b
Leaf K (mg g−1 DW)
CNT	8.48 ± 0.95 c	14.03 ± 7.57 bc
BC	22.65 ± 3.96 b	19.39 ± 0.89 b
WD	9.25 ± 0.93 c	8.31 ± 1.36 c
BC + WD	29.33 ± 3.40 a	24.51 ± 5.03 a

).

The RWC was significantly reduced exclusively by WD treatment after 28 days and, in contrast, by all treatments after 56 days. Therefore, we can affirm that both total plant fresh weight and RWC were affected by an extra-effect induced by cut as well as by treatments, leading to highly variable results (Table 3).

Leaf N content was initially reduced by BC and BC + WD treatments when compared with CNT, whilst no significant differences were found between treatment at the end of the experiment. Leaf P content was reduced by BC and BC + WD by 19 and 15%, respectively, when compared to CNT, exclusively after 56 days of treatment, with a result in contrast with soil P availability. Leaf K content was enhanced by BC (+62%) and BC + WD (+71%) when compared with CNT after 28 days, whilst, after 56 days, only BC + WD treatment induced the increase in leaf K content (Table 3).

Gas exchange results are reported in Figure 3.

P_n was significantly higher in all treatments with respect to CNT after 28 days, whilst, after 56 days, BC and WD alone enhanced this parameter (Figure 3a). After 56 days no differences between CNT and the combination of the treatments were observed (Figure 3a). The g_s was increased by BC treatment after 28 days and by all treatments after 56 days when compared with CNT (Figure 3b). The C_i was reduced by WD and BC + WD after 28 days, whilst it was enhanced by all treatments after 56 days with the highest values after WD treatment (Figure 3c). WUE_int was increased by WD and BC + WD when compared to CNT after 28 days (Figure 3d).

Total chlorophyll content was increased by BC treatment with respect to CNT at both samplings, whilst the total carotenoid content reported the highest values after WD treatment at 28 days with significant differences exclusively with BC + WD treatment (

Table 4. Total chlorophyll and carotenoid content in basil leaves of plants grown in biochar-enriched soil (B), in wood distillate-enriched soil (WD) and in soil enriched with the combination of biochar and wood distillate (BC + WD) after 28 and 56 days of treatment and a plant cut between the two time points. Plants grown in non-treated soil were utilized as control (CNT). Means were subjected to a one-way ANOVA with soil treatment as the variability factor, analyzing each time point separately. For each parameter, means followed by different letters indicate significant differences at p ≤ 0.05 using the post hoc LSD test.

	28 Days	56 Days
Total chlorophyll content (μg g−1FW)
CNT	265.31 ± 51.60 b	226.72 ± 30.91 b
BC	418.62 ± 91.67 a	295.99 ± 35.29 a
WD	301.59 ± 34.63 b	234.88 ± 56.61 b
BC + WD	335.60 ± 35.76 b	218.52 ± 26.04 b
Total carotenoid content (μg g−1FW)
CNT	58.24 ± 6.39 ab	46.15 ± 16.25 b
BC	52.03 ± 13.00 ab	69.43 ± 12.93 a
WD	62.70 ± 9.44 a	47.78 ± 16.49 b
BC + WD	49.97 ± 4.37 b	55.03 ± 12.64 ab

). After 56 days, BC treatment induced an increase in carotenoid content when compared with CNT (Table 4).

3.2.2. Reactive Oxygen Species (ROS) and Antioxidant Responses

The leaf level of H₂O₂ was affected by treatments exclusively after 28 days, whilst no significant differences were reported after 56 days (Figure 4a). The H₂O₂ content was reduced by BC and WD treatments Figure 4a).

Antioxidant enzyme responses mainly involve BC and BC + WD treatments. The SOD and CAT activities were enhanced by BC and BC + WD treatments after 28 days (Figure 4b,c). After 56 days, CAT was reduced by BC + WD treatment when compared to controls (Figure 3c). No significant differences were found in APX activity between treatments and CNT after 28 days. Differently, at 56 days, an increase was evidenced in plants subjected to WD and BC + WD treatments and a reduction in those subjected to BC treatment, when compared with CNT leaves (Figure 4d). DHAR activity was reduced by WD treatment at 28 days. Differently, at 56 days, BC and BC + WD treatments increased the activity of this enzyme (Figure 4e). GR activity was enhanced by BC and BC + WD treatments after 28 and 56 days (Figure 4f).

The AsA content was reduced by the BC + WD treatment after 28 days with respect to CNT and no significant differences were observed after 56 days (Figure 5a).

The DHA content was increased by WD treatment after 28 days and it was reduced by all treatments after 56 days when compared with CNT leaves (Figure 5b). The GSH content was higher in BC-treated plants and lower in BC + WD-treated plants than CNT plants at 28 days. Conversely, at 56 days, the BC + WD treatment induced the highest GSH content, whilst the BC treatment induced the lowest (Figure 5c). The GSSG content had the highest values in leaves subjected to WD treatment, followed by BC treatment at 28 days. After 56 days, WD and BC + WD induced lower GSSG than CNT (Figure 5d).

4. Discussion

The present work is an advancement in understanding the effects of using C-storage products, such as biochar and wood distillate, in nutrient-poor soil. Moreover, investigating the interaction between plants and fertilized soil provides holistic insights useful for understanding the timeline of soil improver effects, identifying plant species adaptable to the chemical properties of these improvers, addressing specific nutrient deficiencies, determining appropriate improver doses, etc. The combination of the improvers may be an attempt to reduce their required dosages in nutrient-poor soil and to exploit the same fertilizer production process to obtain two useful products, thereby reducing production costs and application costs in agricultural scenarios.

4.1. The Combination of BC and WD Induces an Increase in Available P and Exchangeable K in a Nutrient-Poor Soil After 56 Days of Treatment, Even Though the Effect of BC Is More Remarkable than That of WD

BC and WD have opposite chemical properties and thus this aspect can affect the availability of nutrients in soil. BC is an alkaline improver, whilst WD has an acid pH due to the richness in organic acids. Becagli et al. [44] reported that WD treatment on a loamy–sandy soil had no effect on soil pH; in our case, pH modification was significant after a few weeks, likely due to soil characteristics such as high sand percentage and low TOC. In contrast, Fang et al. [45], in their experiment on BC and WD interaction, showed a slight pH increase with BC treatment, while BC + WD produced a more consistent pH increase. On the other hand, Sheng and Zhou [46] demonstrated a more drastic increase in pH in acidic soil than in alkaline soil because of the higher buffer capacity of alkaline soil whose initial TOC was high. Therefore, in the present experiment, the utilization of a sub-alkaline soil (pH 8.74) poor in TOC and the slight acidization induced by WD application modified soil pH, resulting in a slight increase in the combination and a more drastic increase with the BC application. However, BC demonstrated a more efficient alkaline effect than WD, especially after 56 days of treatment.

BC influence on EC is probably correlated with the release of available cations in soil circulating solution; BC continuously releases ions which contribute to enhance soil EC. Joseph et al. [47] reported the quick ion dissolutions that lead to a rapid increase in EC. Our results show that after 56 days this effect becomes clear and as already seen for pH, BC is more effective than WD in the combination. Indeed, the EC increase is also evident in BC + WD treatment.

The total N content did not change with BC treatment in the present study, and this result is confirmed by Feng and Zhu [48], who analyzed the effect of different biochar carbon/fertilizer-nitrogen ratios on a silt loam soil. This pattern could be due to the short time of the experimental trial but also to the combination of two phenomena: (i) the immobilization of NH₄⁺ once adsorbed by BC; (ii) in nutrient-poor soil, the high C/N ratio increases microbial biomass, which affects nitrification and denitrification processes [49,50]. Moreover, root exudates in the present experiment further increased this ratio. This behavior is supported by results of other soil parameters. The FDA hydrolysis parameter increased following BC application after 28 days, and this result was confirmed by other authors [51]. Moreover, SAI3 is a data reduction process that involves the activities of three key enzymes, which are converted into scores reflecting positive or negative changes in the soil conditions (alteration). Meyer et al. [52] reported that SAI3 was correlated with SOM content and plant yield performance. Analyzing a set of amended and unamended soils, Puglisi et al. [32] observed that soils with more negative SAI3 values had higher TOC. Some studies [53,54] confirmed the tendency of SAI3 scores to become increasingly negative with increasing soil TOC and soil quality. In this study, in all treatments, the SAI3 values became more negative from 28 to 56 days, thus suggesting an overall amelioration of soil quality and soil TOC during basil cultivation and confirming the increase in the C/N ratio in both samples with BC application. After 28 days, the values were slightly more negative in the enriched soils and especially in those fertilized with BC + WD. At 56 days, only BC + WD showed a significantly lower value. This highlights that the combination of BC with WD can maintain high soil biomass activity [16]. These patterns demonstrate that the application of appropriate amounts of B and WD may be helpful for the microorganisms of soil, leading to a higher biological quality of soil in the root environment.

Therefore, when the soil C/N ratio increases, the N demand of microbes increases above N availability and N becomes the limiting factor relative to C [48]. In the present experiment, the SAI3 indicates the increase in microbial activities, especially under the combined BC + WD treatment, suggesting also an increase in organic carbon mineralization and a release of mineral N useful for the microbial community. This can explain the maintenance or little decrease in N content in BC treatment after 28 days. In contrast, the total N content increase after 56 days of BC + WD treatment is correlated with the increase in the urease activity. Becagli et al. [16] reported a similar result for the activity of this enzyme during the application of BC + WD, confirming a promoting effect of BC treatment more than that of WD utilization. Therefore, it is not completely clear whether the increase in microbial activity is related to the higher N mineral source induced by the urease activity even though it is evident, as BC can increase soil CEC absorbing NH₄⁺.

However, the WD treatment, after 28 days, also induced an increase in N content. Sharma et al. [55] noted that organic acids in WD could facilitate N release and increase N availability in the soil. Additionally, Seok and Park [56] observed that NO₃⁻-N leaching did not increase in the WD treatment, indicating that WD could prevent nitrification and reduce nitrate leaching, confirming the total N content increase in after 28 days of WD treatment in the present experiment.

The availability of soil P was negatively affected after 28 days by BC and BC + WD treatments; this effect is likely due to BC adsorption of HPO₄²⁻ that reduces P availability under a short-term experiment [57]. At the same time, the presence of plants in the system induced a higher uptake of P in leaves, reducing this nutrient in the soil. After 56 days, WD and BC + WD treatments significantly improve this macronutrient in soil. This effect may be due to the pH decrease, which induces the release of a higher number of phosphate ions from the soil. This pattern was confirmed by the increase in phosphatase activity in the WD + BC treatment after 56 days.

Biochar obtained from wood is rich in K and its introduction in soil rapidly releases this macronutrient [58]. However, a reduction between the first and second samplings can be noted, and this pattern could be due to the presence of the basil plants. Indeed, basil plants are interested in K luxury consumption and are able to deplete soil potassium [59]. Luxury K consumption can produce several problems in soil, due to an excess of K being taken away from the soil and not replenished. The K luxury consumption, in the present experiment, was likely stimulated by nutrient deficiencies of the soil under investigation. At the same time, the K luxury consumption by plants induced higher g_s values in BC treatments after 28 and 56 days, since this macro-element is directly connected with the stomatal opening [60].

4.2. BC Induces More Positive Effects on Physiological and Biochemical Mechanisms than WD in Basil Plants

As seen for the most soil parameters, BC also affected the plant production more than WD in the short treatment utilized in the present experiment. Indeed, the total plant weight was enhanced by BC treatment after 56 days. Although plant biomass after the cut had not yet reached, at 56 days of treatments, the levels observed after 28 days (a slowdown clearly caused by the cut), BC was the only treatment that increased fresh weight after 56 days, suggesting that this treatment specifically promoted re-growth after the cut by accelerating the production of plant biomass. At the same time, RWC decreased under all treatments, and plant dry weight was similar between the BC treatment and the control. It is well known that BC induces soil retention of water and an increase in water uptake in plants, increasing their fresh biomass (as reviewed by Lentini et al., [61]). Given the similarities in terms of dry weight, in the present experiment, the increase in plant biomass cannot be related to water uptake, but rather to the increase in nutrient uptake, such as K (Table 2 and Table 3), as already reported by other authors in Medicago ciliaris and soyabean [62,63]. However, leaf N and P contents were not involved in the increase in plant productivity induced by BC treatment, in contrast to the findings of Becagli et al. [16]. The observed dissimilarities may be induced due to differences between basil genotypes, as evidenced by Bajomo et al. [19]. After an initial (at 28 days) decrease in leaf N induced by BC and BC + WD, no significant changes were observed after 56 days in leaf N. Indeed, the pattern of soil N content was reversed by BC and the combination of BC + WD. Probably the length of the experiment was too short to highlight the role of N in plant response to these fertilizers. Regarding P availability, the pattern of leaf P after 56 days did not match soil P availability, but rather reflected soil P availability after 28 days. This behavior could be due to the initial increase in uptake of available soil P in WD- and BC + WD-treated plants, whilst a slowdown of the P uptake appeared to occur after 56 days, inducing a maintenance of a higher content of soil available P in WD and BC + WD treatments. This slowdown may be attributed to biochemical limitations and oxidative stress induced by WD treatment, likely due to the cut carried out on basil plants (deducible from the lack of AsA and glutathione turnover and marked accumulation of DHA and GSSG; Figure 4b,d) [64]. Indeed, the effect of the cut can be attributed to oxidative stress in plants due to two main reasons: (i) the direct contact of oxygen with cellular components and (ii) enzymatic oxidative stress induced by the activity of browning enzymes such as peroxidases and polyphenol oxidases inducing the conversions of phenols in quinones and, thus, in melanoidins [65].

The g_s values were also increased by BC treatment after both samplings (28 and 56 days). This result aligns with findings of other authors that utilized the BC treatments in stress conditions such as salt stress [66] or drought stress [67]. The stomatal opening is directly correlated to positive hydraulic signals and ion balance (especially by the K osmoregulatory action [60]) and to the reduction in abscisic acid content induced by BC [68] and negatively impacted by environmental stresses such as nutrient deficiency in the present experiment. Similarly to g_s values, chlorophyll and carotenoid contents also increased with BC treatment after 28 and 56 days and after only 28 days, respectively. This increment is already documented in wheat plants grown in salt-stressed soil treated with 1% w/w BC because of the enhanced nutrient availability and hydraulic exchanges induced by BC in stressed environmental conditions [69]. Antioxidant responses to BC application were evident after both 28 and 56 days. After 28 days, BC enhanced SOD and CAT activities, promoting ROS detoxification, particularly of H₂O₂. The increase in SOD activity may reflect the nutrient-poor soil conditions, as this enzyme catalyzes the dismutation of superoxide radicals into H₂O₂. Consistently, the lowest H₂O₂ level observed under BC treatment, together with the increased CAT and APX activities, responsible for converting H₂O₂ to O₂ and water, indicate enhanced protection against oxidative cell damage [70]. Different patterns were noted by Gharred et al. [63] in drought-stressed M. ciliaris plants after 42 days of BC treatment, the only report investigating plant antioxidant response to BC. These authors associated the decrease in SOD and APX activities with the buffering effect of BC on drought stress by regulating the activity of protective enzymes. However, in our study, a slight reduction in APX activity was found after 56 days of BC treatment, partially confirming findings by Gharred et al. [63] and suggesting that the enzymatic ROS detoxification induced by BC required more time in basil compared to other species such as M. ciliaris.

Similarly, the delayed activation of antioxidant defenses was also evident in WD-treated plants, in which an unbalanced redox state in the Halliwell–Asada–Foyer cycle after 28 days was recorded. Low DHAR and GR activities impaired ascorbate and glutathione turn-over, leading to DHA and GSSG accumulation (Figure 4). However, after 56 days, the pattern was completely reversed, indicating recovery of redox homeostasis.

Finally, in the combined BC + WD treatment, BC prevailed over WD, as antioxidant responses closely resembled those observed under BC alone, with high SOD, APX, DHAR and GR activities and increased GSH content.

Given the little information available in the literature, some aspects need further elucidation, including differences among plant species, experimental duration, and fertilizer concentration.

5. Conclusions

Biochar and wood distillate, applied alone or in combination, proved effective in improving the fertility of a nutrient-poor sandy soil and in modulating basil growth and physiological responses. Biochar was the main driver of changes in soil chemical properties, increasing pH, electrical conductivity, and especially exchangeable K, while also enhancing overall microbial activity and soil quality indicators. These improvements translated into positive plant responses, including higher fresh biomass, improved photosynthetic performance, increased stomatal conductance, and greater accumulation of photosynthetic pigments. The combined application of biochar and wood distillate showed clear positive effects over time, particularly by increasing soil P availability (+27% after 56 days), and the activity of key soil enzymes involved in nutrient cycling, such as urease (+37%) and acid phosphatase (+7%) compared with control soils. The reduction in the SAI3 index (–31%) under the combined treatment further indicated an improvement in soil biological quality and organic matter turnover. At the plant level, biochar strongly stimulated antioxidant defenses, promoting the coordinated activation of enzymatic systems. Wood distillate alone had more limited short-term effects but contributed to modulating plant redox balance. Therefore, it can be concluded that biochar increased soil fertility, whereas wood distillate improved nutrient dynamics and influenced the plant physiological responses. Indeed, biochar acts as a structural and nutritional soil amendment with gradual but persistent effects, while wood distillate functions as a biological enhancer that can intensify and anticipate biochar benefits, allowing a reduction in biochar application rates and, consequently, lower overall treatment costs. Despite the clear obtained results in soil, plants and their interactions, the limitations associated with the short duration of the experiment, the use of a single plant species as well as the greenhouse-controlled conditions compared with open field experiments, may have led to different results. Further long-term and field-scale studies utilizing different plants species are needed to assess the durability of these effects and their implications for crop yield and quality.