Precision nano-fertilization as a strategy to enhance growth and sweetener accumulation in stevia under arid sandy soil conditions

2.1 Experimental site and soil properties

Field experiments were conducted during the summer seasons of 2023 and 2024 at the experimental farm, Agricultural Research Center (ARC), Giza, Egypt in Giza, Egypt (Latitude: 30.021° N, Longitude: 31.2103° E). The site is characterized by sandy-textured soils with inherently low fertility and limited nutrient retention capacity. A pre-sowing soil analysis (0-30 cm depth) revealed a pH of 7.6, electrical conductivity (EC) of 1.1 dS/m, and organic matter content of 0.43%. Available nitrogen, phosphorus, and potassium levels were 42 mg/kg, 5.8 mg/kg, and 110 mg/kg, respectively. Soil texture was determined using the hydrometer method and confirmed as sandy.

2.2 Plant material and cultivation practices

Stevia rebaudiana cv. Sugar High seedlings were sourced from a certified horticultural nursery in Egypt. Transplanting was carried out on March 15, 2023, for the first season, and March 17, 2024, for the second season, using 30-day-old seedlings. Each experimental plot measured 2 m × 2 m and was arranged in rows with 30 cm plant spacing and 60 cm row spacing, accommodating 20 plants per plot. Standard cultural practices were applied across all treatments as recommended. Harvesting occurred on August 10, 2023, and August 12, 2024.

2.3 Nano-fertilizer sources and treatment structure

All nano-fertilizers employed in this study were procured from the Central Laboratory for Agricultural Climate (CLAC), Giza, Egypt. Both transmission electron microscopy (TEM) imaging and energy dispersive X-ray spectroscopy (EDS) analysis were conducted specifically for the nano-fertilizers synthesized and applied in this study, to validate their morphology, elemental composition, and ensure consistency with nanoscale delivery criteria. Nanoparticle size distribution was analyzed from TEM images using ImageJ software. This method has been widely employed for accurate nanoparticle size determination and statistical analysis (Jabbar et al., 2022). The application of ImageJ for nanoscale structural analysis has also been demonstrated in materials science studies, confirming its suitability for particle size assessment (Hashim et al., 2018).

Representative micrographs (Fig. 1) depict the structural features of the nano-fertilizers:

• Nano-ZnO displayed irregularly clustered particles with diameters ranging between 23.06 and 27.6 nm.

• Nano-Fe₂O₃ particles were predominantly spherical with slight agglomeration, exhibiting a mean size range of approximately 30.4 to 34.2 nm.

• Nano-NPK particles appeared well-dispersed, primarily cubic or rounded in shape, with average dimensions of 30-32 nm; larger aggregates up to 1 µm were occasionally observed.

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Fig. 1.

Morphological and dimensional characterization of hybrid nanostructures via TEM; panels (a & b): Surface topography of nano-ZnO particles, panels (c & d): Nano-Fe₂O₃ particles, and panels (e & f): nano-NPK particles.

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All nano-formulations maintained a consistent nanometric size distribution below 100 nm, aligning with the physicochemical criteria for efficient nutrient uptake via foliar and root absorption. These structural properties make them well-suited for application in low-retention sandy soils. The elemental composition of each nano-fertilizer was validated through EDS, confirming the formulations as follows: Nano-ZnO (40 ppm Zn), Nano-Fe₂O₃ (50 ppm Fe), and Nano-NPK (20:20:20 NPK).

Experimental design and fertilizer treatments

The experiment followed a split-plot in Randomized Complete Block Design (RCBD) with six fertilization treatments and three replicates:

• T1 – Control: 100% recommended dose of conventional NPK (600 kg/ha of granular NPK 20:20:20)

• T2 – 100% nano-NPK (foliar application of 20:20:20 nano-formulation at 2 g/L)

• T3 – Nano-ZnO foliar application at a concentration of 40 ppm Zn (2 mL/L of ZnO nanoparticle suspension)

• T4 – Nano-Fe₂O₃ foliar application at 50 ppm Fe (2 mL/L of Fe₂O₃ nanoparticle suspension)

• T5 – Combined nano-fertilizer treatment: nano-NPK (2 g/L) + nano-ZnO (40 ppm) + nano-Fe₂O₃ (50 ppm)

• T6 – Integrated treatment: 50% conventional NPK (300 kg/ha of NPK 20:20:20) + 50% nano-NPK (1 g/L).

Treatment T5 (combined nano-NPK + nano-ZnO + nano-Fe₂O₃) was included to evaluate the maximum synergistic potential of a fully nano-based system. In contrast, Treatment T6 integrated nano-fertilizers with conventional NPK to test a partial substitution strategy, assessing whether a hybrid approach could achieve comparable agronomic gains while minimizing chemical inputs. This comparison supports practical scalability for growers transitioning to nano-based fertilization.

The application rates of nano-fertilizers used in this study were determined based on a combination of prior literature, preliminary greenhouse screening trials, and alignment with agronomically relevant nutrient thresholds. Specifically, the selected concentrations 40 ppm for Nano-ZnO, 50 ppm for Nano-Fe₂O₃, and the equivalent macro-nutrient content for Nano-NPK (20:20:20) were informed by published studies demonstrating efficacy in enhancing nutrient use efficiency and crop performance without inducing phytotoxicity (e.g., Raliya et al., 2015; Khan et al., 2020). These concentrations were validated through pilot trials under controlled conditions to ensure optimal physiological response in Stevia rebaudiana under sandy soil stress.

All foliar nano-fertilizer treatments were applied uniformly at 30, 60, and 90 days after transplanting using a calibrated handheld pressure sprayer to ensure consistent coverage across all treatments.

2.4 Data collection and measured parameters

The following traits were evaluated throughout the growing cycle and at final harvest:

• Plant Height (cm): Measured from the soil surface to the apex using a ruler

• Leaf Number: Total number of leaves per plant counted manually

• Leaf Area (cm²): Determined using a leaf area meter

• Fresh Biomass (g/plant): Total fresh weight of whole plants

• Dry Biomass (g/plant): Samples oven-dried at 70°C to constant weight

• Leaf Yield (g/plant): Total harvested leaf weight

• Leaf Dry Matter (%): determined by first recording the fresh weight of the harvested leaves using an electronic balance. The samples were then oven-dried at 70 °C until a constant weight was achieved. The dry matter content was calculated using the following formula:

  $\text{Leaf Dry Matter }\%=\text{Dry weight}/\text{Fresh weight}\times \text{1}00$

• Nutrient Uptake: Leaf tissues were digested with H₂SO₄-H₂O₂; nitrogen was determined by the Kjeldahl method, phosphorus by spectrophotometry, potassium by flame photometry, and Zn/Fe by atomic absorption spectrophotometry

• Stevioside and Rebaudioside A (mg/g): Quantified by HPLC (Agilent 1200 series) following methanolic extraction and filtration.

2.5 Statistical analysis

All experimental data from the 2023 and 2024 seasons were subjected to analysis of variance (ANOVA) using MSTAT-C software (Michigan State University, 1991) in an RCBD. Prior to conducting ANOVA, data were screened to ensure compliance with parametric assumptions, including normality and homogeneity of variances. Means were separated using the L.S.D. (Least Significant Difference) test at the 5% probability level (p ≤ 0.05). Standard errors (SE) were also calculated for each trait to quantify variability among replications and are presented alongside the mean values in the tables.

3.1 Vegetative growth attributes

Across both seasons, T5 (Combined nano-fertilizer treatment) emerged as the most effective treatment in promoting vegetative growth. It significantly outperformed all other treatments in plant height, number of leaves, and leaf area. In the first season (Table 1), T5 (combined nano-fertilizers) consistently produced the tallest plants, largest leaf number, and expanded leaf area, outperforming the conventional control (T1) across both seasons (Fig. 2). Nano-fertilizers play a pivotal role in regulating the physiological and biochemical processes of crops by enhancing nutrient availability and uptake efficiency. This improved nutrient dynamics stimulates key metabolic activities and promotes meristematic functions, leading to increased apical growth and expanded photosynthetic area. These effects are primarily mediated through enhanced cell division, chlorophyll biosynthesis, and leaf expansion, driven by hormone-regulated growth responses. (Schmidt et al., 2020; Acharya et al., 2024; Nazir et al. 2024; Ali et al., 2024; Muhammad et al., 2025).

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Fig. 2.

Effect of nano-fertilizer treatments on plant height (cm) of Stevia rebaudiana during the first and second seasons. Error bars represent standard error (SE), L.S.D values at 5% (2.73 and 3.83) for 1^st and 2^nd seasons, respectively.

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3.2 Biomass accumulation and leaf yield

Fresh and dry biomass accumulation responded positively to nano-fertilizer applications, with T5 again exhibiting the highest performance. Nano-fertilizer applications enhanced fresh and dry biomass, with T5 recording the highest values (Figs. 3 and 4). The substantial increase in biomass and leaf yield under nano-Fe₂O₃ and nano-NPK treatments highlights the role of Fe in enzyme activation and enhanced metabolic flux, while nano-NPK improves nutrient partitioning and source–sink balance (Bhardwaj et al., 2022; Akhter et al., 2025).

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Fig. 3.

Influence of nano-fertilizer applications on fresh biomass (g/plant) of Stevia rebaudiana in both seasons. Error bars represent standard error (SE), L.S.D values at 5% (15.17 and 16.81) for 1^st and 2^nd seasons, respectively.

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Fig. 4.

Dry biomass (g/plant) response of Stevia rebaudiana to different nano-fertilizer treatments across two seasons. Error bars represent standard error (SE), L.S.D values at 5% (3.83 and 5.70) for 1^st and 2^nd seasons, respectively.

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3.3 Leaf dry matter percentage

The superior leaf dry matter percentage (LDM%) (Table 2) observed in T5 reflects enhanced photosynthate accumulation and improved water-use efficiency in sandy soils, likely facilitated by nano-enabled delivery of Fe and N, which improve carbon fixation and assimilate storage (Reddy et al., 2024; Singh et al. 2025; Muhammad et al., 2025).

3.4 Nutrient uptake efficiency

Nutrient uptake efficiency followed a similar trend. Nitrogen content in T5-treated plants reached 29.3 mg/g versus 20.4 mg/g in T1. Phosphorus and potassium uptake were also highest under T5, at 6.9 mg/g and 47.2 mg/g respectively. Macro- and micronutrient uptake was maximized under T5 (Figs. 5-9). The synergistic effect of nano-NPK with Fe and Zn enabled more efficient assimilation, owing to the higher mobility and foliar penetration of nanoparticles, which enhance root-to-shoot translocation and nutrient bioavailability (Ali et al., 2025; Dimkpa et al., 2017).

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Fig. 5.

Effect of nano-fertilizer treatments on nitrogen content in Stevia leaves during 2023 and 2024 growing seasons. Bars represent standard error (SE), L.S.D values at 5% (1.27 and 1.01) for 1^st and 2^nd seasons, respectively.

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Fig. 6.

Effect of nano-fertilizer treatments on potassium (K) uptake in Stevia rebaudiana leaves during 2023 and 2024 growing seasons. Bars represent standard error (SE), L.S.D values at 5% (2.46 and 2.38) for 1^st and 2^nd seasons, respectively.

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Fig. 7.

Effect of nano-fertilizer treatments on phosphorus (P) concentration in Stevia rebaudiana leaves during 2023 and 2024 growing seasons. Bars represent standard error (SE), L.S.D values at 5% (0.27 and 0.39) for 1^st and 2^nd seasons, respectively.

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Fig. 8.

Effect of Nano-ZnO and combined nano-fertilizer applications on foliar Zinc (Zn) content in Stevia rebaudiana leaves during 2023 and 2024 growing seasons. Bars represent standard error (SE), L.S.D values at 5% (0.08 and 0.08) for the 1^st and 2^nd seasons, respectively.

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Fig. 9.

Effect of varying nano-fertilizer treatments on iron (Fe) concentration in leaves of Stevia rebaudiana during 2023 and 2024 growing seasons. Bars represent standard error (SE), L.S.D values at 5% (0.19 and 0.20) for 1^st and 2^nd seasons, respectively.

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3.5 Sweetener content (Stevioside and Rebaudioside A)

Steviol glycosides (stevioside and rebaudioside A) were significantly enhanced under nano-fertilizer treatments, particularly T5 (Table 3). Mechanistically, Zn acts as a cofactor for glycosyltransferases that catalyze glycoside biosynthesis, while Fe enhances chlorophyll-driven carbon fixation, thereby increasing precursor availability. Nano-NPK contributes by improving carbohydrate partitioning, jointly stimulating secondary metabolite pathways (Puscaselu et al., 2019; Djéssica et al., 2022; Rafiq et al., 2024; Akhter et al., 2025).

3.6 Integrated assessment

The combined nano-fertilizer treatment (T5) consistently delivered superior outcomes across vegetative growth, biomass accumulation, nutrient uptake, and steviol glycoside synthesis, underscoring the central role of synergistic nutrient interactions at the nanoscale. This performance is attributable to the unique properties of nanoparticles, including high surface area, enhanced solubility, and controlled release, which facilitate greater foliar absorption and more efficient translocation compared to bulk fertilizers (Dimkpa et al., 2017; Raliya et al., 2015).

The observed improvements in growth attributes are linked to nutrient-mediated physiological processes. Zinc nanoparticles act as cofactors for enzymes such as glycosyltransferases and dehydrogenases, promoting metabolic activity and secondary metabolite biosynthesis (Puscaselu et al., 2019; Rafiq et al., 2024). Iron contributes to chlorophyll development and photosynthetic efficiency, thereby increasing precursor availability for both biomass and glycoside accumulation (Schmidt et al., 2020; Reindinger et al., 2012). In parallel, nano-NPK enhances nitrogen assimilation and carbohydrate partitioning, stimulating source-sink relationships and assimilate allocation (Bhardwaj et al., 2022; Nazir et al., 2024). Together, these mechanisms explain the consistent superiority of combined nano-treatments.

A notable finding was the significant enhancement of steviol glycoside concentration. This is consistent with previous reports that Zn and Fe nanoparticles stimulate enzymatic pathways of secondary metabolism and upregulate glycosylation reactions involved in stevioside and rebaudioside A biosynthesis (Djéssica et al., 2022; Khan et al., 2020). The agronomic implication is that nano-fertilizers not only increase yield but also improve crop quality and economic value, particularly for high-value crops such as Stevia rebaudiana. In Stevia rebaudiana, steviol glycoside biosynthesis depends on both photosynthetic carbon fixation and glycosyltransferase-mediated glycosylation reactions (Puscaselu et al., 2019). Nano-fertilizers facilitate nutrient entry through stomata, cuticular pores, and root apoplastic/symplastic pathways (Dimkpa & Bindraban, 2017), thereby enhancing chlorophyll biosynthesis (via Fe), carbohydrate metabolism (via NPK), and enzyme activation (via Zn). This physiological synergy explains the higher stevioside and rebaudioside A accumulation observed under T5.

The environmental sustainability dimension is also crucial. Sandy soils are characterized by poor nutrient retention and high leaching losses. The nano-formulations applied here reduced nutrient losses through gradual release and improved uptake efficiency, which aligns with the broader goals of sustainable agriculture and reduced agrochemical pollution (Dimkpa & Bindraban, 2018; Liu & Lal, 2015). These outcomes suggest that nano-enabled fertilization can simultaneously address productivity and environmental challenges in marginal soils. The superior performance of nano-fertilizers can be attributed to their high solubility, gradual release, and enhanced bioavailability (Liu & Lal, 2015). These properties ensured a steady nutrient supply during critical growth phases, preventing stress-induced fluctuations common in sandy soils. In Stevia, this stability supported continuous photosynthesis and substrate flow into glycosyltransferase pathways, thereby stimulating stevioside and rebaudioside A accumulation (Djéssica et al., 2022).

While nano-fertilizers demonstrated improved nutrient use efficiency and reduced leaching in sandy soils, potential long-term ecological impacts should be considered. Reports indicate that nanoparticles may accumulate in soil and alter microbial communities, with possible transfer into food chains (Wang et al., 2024; Mubeen et al., 2023). Multi-season and multi-site studies are therefore required to confirm their environmental safety.

Finally, the inclusion of T6 (50% nano + 50% conventional NPK) highlights the potential for transitional adoption strategies. Although less effective than T5, T6 outperformed the conventional control, indicating that even partial substitution with nano-formulations enhances productivity and sustainability. This approach may be particularly attractive for smallholders in resource-limited settings, where gradual adoption reduces costs while maintaining yield improvements (Ali et al., 2024; Akhter et al., 2025; Muhammad et al., 2025).

Collectively, these findings provide strong evidence that integrated nano-macro and nano-micronutrient strategies enhance plant growth, yield, secondary metabolism, and sustainability under sandy soil conditions. By combining agronomic, biochemical, and environmental benefits, nano-fertilization represents a climate-smart solution for Stevia production in arid regions.