Introduction

Reclaimed semi-arid areas have low organic matter, poor soil structure, limited nutrient availability, and frequent exposure to salinity and drought1. These factors generally reduce nutrient uptake efficiency as well as the ability of plants to perform normal functions, resulting in reduced productivity and fruit quality2. Thus, the use of biostimulants with a positive environmental impact that can boost nutrient use efficiency and increase the tolerance of plants to stress will be critical to maintaining the production of olives in reclaimed land settings3.

The olive tree (Olea europaea L.) plays a pivotal role in the rural economy and lifestyle throughout the Mediterranean region4,5. It is highly prized for the olive oil it produces; a product celebrated for its extensive dietary and medicinal benefits. Olive oil is rich in monounsaturated fats particularly oleic acid6, antioxidants, vitamin E, tocopherols, and phenolic compounds, which collectively underpin its importance in the Mediterranean diet. A key feature of olive oil is its unique fatty acid profile: 70–85% of its unsaturated fats are monounsaturated (primarily oleic acid), supplemented by polyunsaturated fats (mainly linoleic acid), while saturated fatty acids (such as stearic and palmitic acids) constitute around 14% of its composition; the remaining minor compounds account for less than 2%7,8,9,10. Among the various cultivars, Arbosana, a cultivar originating from the Catalonia region of northeastern Spain, is widely recognized for producing high-quality extra virgin olive oil11. It is characterized by a balanced bitterness and pungency and has gained importance in several olive-growing regions, including Egypt12. The trees are compact and well adapted to high-density systems, with early bearing and high productivity, making the cultivar suitable for intensive orchards focused on both yield and oil quality13. These attributes make Arbosana an ideal selection for growers aiming to maximize productivity and achieve outstanding olive oil quality, especially within intensive farming systems14.

The period between late summer and early autumn is crucial for maximizing fruit development and bud preparation for the next season in olive trees. During the late summer through early autumn period, foliar application of chitosan nanoparticles (CHNP) and N-acetylthiazolidine-4-carboxylic acid (N-ATCA) can be done to enhance physiological and reproductive functions15. As natural elicitors, CHNP improves the hormone-regulated differentiation of flowers and improves the rate of fruit set, whereas N-ATCA increases the antioxidant capacity of the tree and aids with the metabolism of sulfur to support flower viability and fruit development16. Application of each of these products during this period will create the largest impact on fruit production for current season fruit production as well as future flowering, thereby enhancing yield, oil quality, and oxidative stability in semi-arid reclaimed soils.

Chitosan, a natural polymer derived from chitin, is a valuable substance obtained from the exoskeletons of crustaceans as shrimp, crab, and lobster17. Chitosan is produced by removing acetyl groups from chitin, the composition consists of repeating units of glucosamine linked by β-(1,4)-glycosidic bonds18. Chitosan possesses numerous advantageous properties, including biocompatibility, low toxicity, non-immunogenicity, and biodegradability, making it highly valuable in various applications19. Chitosan exhibits several beneficial effects on plants, making it a valuable tool in agriculture20. These effects include promoting plant growth, increasing tolerance to abiotic stress, improving nutrient uptake and efficiency21,22,23,24. Recent investigations have documented useful efficacy on growth, yield, fruit properties, and oil quality when applying chitosan at concentrations of 500, 1000, and 1500 ppm on Picual and Arbosana olive trees25,26. Nanotechnology, which manipulates materials at the nanoscale (1–100 nm), creates particles with unique physical and chemical properties that differ from their bulk forms. These properties such as a high surface-to-volume ratio, distinct shape, and enhanced thermal stability have driven its application in diverse sectors including medicine, engineering, and agriculture27,28. In farming, where conventional practices often lead to significant crop losses due to pests, poor soil, and nutrient deficiencies, nanotechnology offers innovative solutions29,30. Chitosan is a natural biopolymer that has attracted attention in agriculture as a potential ingredient for nanoparticle-based formulations. Chitosan as a nanoparticle primarily acts as a carrier for active ingredients; thus, enhancing their stability, controlling release and improving the bioavailability of active ingredients, than having a direct effect on the physiology of plants themselves31,32,33. Differentiating between the effects of nanotechnology as a delivery mechanism and the effects of the active compounds will provide a clearer picture of their individual contributions to plant productivity. Reported benefits associated with chitosan-based nanoparticles include improved seed germination, improved long-term and post-harvest quality of produce, improved fertigation, and increased photosynthesis, antioxidant activity, chlorophyll content and tolerance to stress This integration not only overcomes traditional agricultural challenges but also paves the way for more sustainable and efficient agrotechnological systems while maintaining food quality31,32,33. The technology and the active ingredient provide two different perspectives; one being that technology, when viewed as a delivery mechanism, allows for the development of a framework, where multiple methods of delivery via chitosan nanoparticles will help to resolve traditional agricultural issues and also support the production of crops in a more environmentally sound manner and with increased efficiency.

N-ATCA is synthesized from the amino acid L-cysteine and serves as a precursor that is converted back into cysteine in plant34. This conversion triggers various metabolic processes, including the synthesis of thiol-rich proteins and the activation of the ascorbate–glutathione (AsA-GSH) cycle, which helps plants manage oxidative stress26. N-ATCA undergoes a series of biochemical reactions, resulted in formation of thioproline, formyl cysteine, and L-cysteine35. These compounds play fundamental roles in various biochemical reactions within plants, particularly in the synthesis of glutathione36. L-cysteine acts as a precursor for the synthesis of glutathione (GSH), a tripeptide molecule composed of L-cysteine, L-glutamate, and glycine37. Glutathione synthesis contributes to protein synthesis by facilitating the formation of disulfide bonds, which are crucial for stabilizing protein structures and maintaining their functionality38. Additionally, L-cysteine is involved in sulfur metabolism, redox regulation, and the biosynthesis of certain phytohormones, such as ethylene and brassinosteroids39;40. Recent studies have also shown its potential to induce disease resistance26,41. The application of N-ATCA on various crops, including New Castle apricot35, Starking Delicious apple, Bhagwa Kesar pomegranate42, and olive trees (Alshallash et al. 2023, has demonstrated improvements in vegetative growth, total chlorophyll content, and proline content. Furthermore, it has been associated with increased fruit set, colour, weight, size, and yield percentage43,44. The aim of this study is to evaluate the effects of foliar applications of chitosan nanoparticles (CHNPs), N-acetyl thiazolidine-4-carboxylic acid (N-ATCA), and their combinations on Arbosana olive trees under newly reclaimed lands conditions. Specifically, it investigates their impact on enhancing vegetative growth, flowering, fruiting, oil yield and quality of olive trees. The goal is to provide sustainable strategies for maximizing olive production, improving oil quality, and boosting crop yields.

Materials and methods

Plant material and experimental site

The study was conducted over two consecutive growing seasons (2021 and 2022) in a private orchard located in Wadi Al-Natrun, Egypt (30°24’01.7” N 30°02’20.6” E). The experiment utilized eight-year-old Arbosana olive trees (Olea europaea L.), planted at a density of 4 × 6 m under a drip irrigation system. The soil of the experimental site is typical of newly reclaimed sandy loam soils in the region. Standard horticultural practices surface drip irrigation techniques, utilizing 8 tree emitters (8 L/HR) per irrigation line via 2 irrigation lines. In addition, the distance from the trunk of the tree to the irrigation line was 50 cm on both sides of the tree. Moreover, irrigation scheduling was based on reference evapotranspiration (ETo) to ensure adequate water supply according to crop requirements under the prevailing climatic conditions. Pruning and pest management were uniformly applied to all trees. Arbosana olive trees were also treated with the prescribed fertilization program, applying 1000gN, 1500P2O5, and 500K2O per tree per year (the values refer to the amount applied). Fertilizer for the micronutrients was applied/taken at the rate of 300/150/100/50 and 50 mg from chelated Fe, Mn, Zn, Cu, and B as boric acid in March, May, and August. All trees received the recommended fertilization as per the guide of the Extension Service under the Ministry of Agriculture in Egypt. The experiment was conducted with the full knowledge and written permission of the private orchard owner, who agreed to the use of the orchard and plant material for research and publication purposes.

Methods

Preparation of chitosan nanoparticles (CHNPs)

Chitosan nanoparticles (CHNPs) were synthesized using the ionic gelation method described by Balusamy et al.45 with specific modifications as detailed below. Low molecular weight chitosan (1.0 g; Sigma-Aldrich; degree of deacetylation ≥ 75%) was dissolved in 100 mL of 1% (v/v) aqueous acetic acid under magnetic stirring (800 rpm) at room temperature (25 ± 2 °C) for approximately 4 h until complete dissolution. The pH of the solution was adjusted to 5.5 using 1 M NaOH. Sodium tripolyphosphate (TPP) was prepared as a 1% (w/v) aqueous solution and added dropwise (0.5 mL min⁻¹) to the chitosan solution under continuous stirring (1000 rpm). The chitosan: TPP volume ratio was maintained at 4:1. Nanoparticles formed immediately through electrostatic interaction between protonated amino groups of chitosan and negatively charged phosphate groups of TPP. The suspension was stirred for an additional 40 min to ensure complete cross-linking. The nanoparticles were collected by centrifugation at 12,000 rpm for 30 min, washed twice with distilled water to remove residual reagents, and re-dispersed in distilled water prior to characterization. The purified CHNPs were then characterized using Transmission Electron Microscopy (TEM; JEOL JEM-1230, Japan) operating at 120 kV to determine particle morphology and size distribution. The TEM image confirmed the formation of spherical nanoparticles with an average diameter of 20.3 nm (Fig. 1).

Preparation of treatments and experimental design

The experiment was laid out in a randomized complete block design (RCBD) with three replications per treatment. Each replicate consisted of three trees. 16 treatments were evaluated, including a control (sprayed with water) and various concentrations of N-acetyl-thiazolidine-4-carboxylic acid (N-ATCA) (purity ≥ 98%, Sigma-Aldrich (Merck): St. Louis, MO, USA.) N-ATCA and CHNPs, alone and in combination. Foliar applications were performed three times during each season: September 15th, October 1st, and October 15th. Each tree received 7 L of the respective spray solution. A handheld knapsack type of sprayer, which was calibrated to achieve standard sizes of drops and proper pressure, was used to apply the foliar sprays on the olive trees in a uniform manner throughout the canopy. The foliar applications were carried out during the late summer early autumn period, which represents a physiologically important stage in olive trees. During this stage, active oil accumulation occurs within developing fruits, while trees simultaneously undergo nutrient redistribution and bud differentiation for the following season. Foliar treatments applied at this stage may enhance metabolic activity, nutrient assimilation, and stress tolerance without inducing excessive vegetative growth. The application was completed so that the foliar sprays were applied evenly on each leaf until runoff from the leaf under calm weather conditions. A standard horticultural hollow cone nozzle manufactured by the sprayer manufacturer was used for the application; the nozzle produces a very fine to medium droplet size at a prescribed operating pressure and is suitable for general foliar applications. All solutions contained 0.1% Tween 20 as a surfactant, and the pH was adjusted to 5.5 using citric acid to optimize foliar absorption.

Pre-harvest foliar spray treatments were applied to olive trees across sixteen experimental groups. Treatment T1 served as the untreated water control. Treatments T2–T4 received chitosan nanoparticles (CHNPs) individually at concentrations of 500, 1000, and 1500 ppm, respectively. Treatments T5–T7 consisted of N-acetyl thiazolidine-4-carboxylic acid (N-ATCA) applied individually at 50, 100, and 150 ppm. The remaining treatments (T8–T16) represented all factorial combinations of the three CHNP concentrations (500, 1000, and 1500 ppm) with the three N-ATCA concentrations (50, 100, and 150 ppm), enabling evaluation of both individual and synergistic effects of the treatments on olive trees prior to harvest.

Analytical methods

Morphology of chitosan nanoparticles (CHNPs)

The CHNPs were characterized using a JEOL JEM-1230 High-Resolution Transmission Electron Microscope (HR-TEM) from Tokyo, Japan. The instrument operated at an accelerating voltage of 120 kV, providing high-resolution images for detailed analysis of the nanoparticles’ shape and size. For HR-TEM observation, a small drop of the CHNP suspension was placed onto a carbon-coated copper grid and allowed to dry at room temperature. Excess solution was removed using filter paper to obtain a thin, uniform film of nanoparticles on the grid surface. The samples were then examined directly without further staining under the JEOL JEM-1230 h-TEM operating at an accelerating voltage of 120 kV. This procedure followed standard protocols commonly used for nanoparticle visualization45.

Fourier transform infrared (FTIR) analysis

The chemical functional groups of the samples were identified using Fourier Transform Infrared Spectroscopy (FTIR). The spectra were recorded using an FTIR spectrophotometer in the range of 400–4000 cm⁻¹. Dried samples were finely ground and analyzed using the KBr pellet method. The obtained spectra were used to identify the main functional groups present on the sample surface.

Vegetative growth parameters

Vegetative growth was evaluated at the end of the growing season (December 1st) in both studied seasons. Fifteen mature, current-season shoots were randomly selected from the four original directions at mid-canopy height of each tree to ensure representative sampling. Shoot length (cm) was measured from the shoot base to the apical bud. The total number of fully expanded leaves per shoot was manually counted. The mean value per tree was calculated and used for statistical analysis.

Leaf mineral content

Leaf samples were collected in early December from the middle portion of current-season, non-fruiting shoots, following standard olive leaf sampling guidelines. Approximately 50–60 healthy, fully expanded leaves were collected per tree from all canopy orientations to minimize variability. Samples were washed with distilled water, oven-dried at 70 °C until constant weight, and ground to pass through a 0.5 mm stainless steel sieve.

Total nitrogen (N) was determined using the micro-Kjeldahl method as described by46. Briefly, 0.2 g of dried leaf powder was digested in concentrated sulfuric acid (H₂SO₄) in the presence of a catalyst mixture (K₂SO₄ and CuSO₄). After complete digestion and clarification, the ammonium formed was distilled with NaOH and titrated with standard HCl solution. Nitrogen concentration was calculated and expressed as a percentage of dry matter.

Phosphorus (P) content was determined colorimetrically according to47. An aliquot of the digested sample was reacted with ammonium molybdate and ascorbic acid reagent to form a phosphomolybdenum blue complex. Absorbance was measured at 882 nm using a spectrophotometer (Shimadzu UV-1800, Japan), and phosphorus concentration was calculated using a standard calibration curve.

Potassium (K) was measured using flame photometry following48. After wet digestion, potassium concentration in the extract was determined using a Jenway PFP7 flame photometer (Jenway, Bibby Scientific Ltd., UK). Calibration was performed using standard potassium solutions of known concentrations. Results were expressed as percentage of leaf dry matter. Results were expressed as a percentage of leaf dry matter.

Fruit yield and quality

Fruit yield was determined at harvest by measuring the total fruit weight per tree. Oil percentage was subsequently determined from representative fruit samples collected from each treatment. Therefore, the evaluation of treatment effects in this study reflects their influence on the productivity and oil characteristics of the same growing season. To determine the total amount of extracted oil from the harvested fruit, an average sample weighing 100 g was obtained from each replicate. Each sample was extracted using a Soxhlet extractor with petroleum ether (boiling between 40 and 60°). The length of time for each extraction was 6 h. Any petroleum ether remaining in the oil/solvent mixture was removed using a rotary evaporator (Heidolph Instruments GmbH & Co. KG, Germany) set at 40°. The remaining oil was brought to a temperature of 105° using an oven and dried for 1 h to eliminate water and/or residual petroleum ether prior to recording the final weight of the oil collected from the extracted fruit49. Finally, an oil yield (kg/tree) was calculated for each tree based on the percentage of oil collected from the extracted sample divided by the tree’s total harvest weight. Fruit firmness (N/cm²) was measured using a digital penetrometer (Shimpo Instruments, Japan). Fruit color (Hue angle, saturation, brightness) was determined using a Minolta CR-400 colorimeter (Konica Minolta, Japan).

Oil quality analysis

Fatty acid methyl esters (FAMEs) were prepared according to ISO 12966-2:2017 and analyzed by gas chromatography (Agilent 6890, Agilent Technologies, USA).

Determination of Iodine Value (IV): The iodine value (IV) and oxidizability index (Cox) were calculated based on the fatty acid composition using established formulas41,43.The iodine values ​​for all oils were within the acceptable iodine value (IOC) range (75–94 g iodine/100 g).

Statistical analysis

Data were collected in a three-replicate, a randomized complete block design. The significance of differences among treatments was assessed using ANOVA (p < 0.05) in SPSS (v22.0). Duncan’s multiple range test was applied to distinguish significantly different means. Results are observed as the mean ± standard deviation (SD).

Results and discussion

Characteristics of the chitosan nanoparticles

TEM imaging revealed the morphological characteristics of the CHNPs, demonstrating their roughly spherical shape and smooth surface, with an average diameter of 20.3 nm calculated from measurements of 100 nanoparticles, as depicted in Fig. 1.

Fig. 1
Fig. 1The alternative text for this image may have been generated using AI.
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TEM of chitosan nanoparticles.

The chemical composition

Figure 2 shows that the primary infrared (IR) bands for olive fruit surfaces can be attributed to different types of vibrations. The vibration band at both 700 cm-1 and 758 cm-1 is due to mono-substituted aromatic ring vibrations and there are other ring-associated vibration frequencies around 841 cm-1 and 905 cm-150. There are other aromatic ring vibrations also associated with phenyl groups in very broad ranges of 1660 to 2000 cm-1 which have associated 1600 cm-1 (ring), 1450 cm-1 (Doublet, ring) related to aromatic ring C–H and CC vibrations dominate. In addition to the above, the examined bands associated with aliphatic and aromatic (symmetrical and asymmetrical) C–H stretch vibrations were identified. C–H aliphatic stretch (υC–H ali) occurs in the 2850 cm -1 to 3000 cm-1 range whereas aromatic C–H stretch (υC–H arom. ring) associated with phenol rings occurs in the 3000 cm-1 to 3100 cm-1 range51.

The primary IR (Infrared Spectroscopy) bands for the surface of olive fruit have been identified based on the data in Fig. 2. The bands at both 700 and 758 cm m-1 were identified as being associated with the mono-substituted aromatic ring vibrations, as indicated by previous studies (Fahmy and Friedrich, 2013). The mono-substituted aromatic rings also have other ring-associated vibrations, indicated by the bands found at 841 and 905 cm 152. Figure 4a also indicates the presence of other bands throughout the region of approximately 1660–2000 cm-1, corresponding to the various phenyl groups found in the aromatic structure of the surface of the olive fruit. The νC–H ring vibrations at 1600 cm-1 and 1450 and 1490 cm− 1 doublets correspond to CH and CC vibrations, with the predominance being noted in the greater number of bands between these two frequencies. The bands that were linked to aliphatic and aromatic component C–H stretching vibrations (symmetric and asymmetric) were also identified. Bands of aliphatic C–H stretching vibrations fall within a range of approximately 2850–3000 cm-1, while bands of aromatic C–H stretching vibrations associated with the phenyl ring can be found within a range of approximately 3000–3100 cm− 153.

Fig. 2
Fig. 2The alternative text for this image may have been generated using AI.
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FTIR spectra for the surface of olive fruit compared to that coated with N-ATCA and CHNPs layers.

Vegetative Growth

The results in (Fig. 3A and B) demonstrate that foliar application of chitosan nanoparticles (CHNPs) and N-acetylthiazolidine-4-carboxylic acid (N-ATCA) had significant and multifaceted effects on the growth of Albosana olive trees. (Fig. 3A and B) clearly demonstrates that all treatments positively affected plant growth compared to the control group (T1), and this effect was even more pronounced in the second season, indicating a potential cumulative benefit. The most significant improvement was observed in the combined treatments. Specifically, treatment T16 (1500 ppm CHNPs + 150 ppm N-ATCA) consistently achieved the highest values ​​for shoot length and number of leaves per shoot. T16 increased shoot length by 2.34 ± 0.18 cm and leaf number by 5.6 ± 0.4 compared to the control group.

Fig. 3
Fig. 3The alternative text for this image may have been generated using AI.
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Effect of foliar application of chitosan nanoparticles (CHNPs) and N-acetyl thiazolidine-4-carboxylic acid (N-ATCA) on vegetative growth of Olea europaea cv. Arbosana during two growing seasons. (A) Shoot length and (B) number of leaves per shoot under different treatments (T1–T16). Data are presented as mean ± SE.

Leaf nutrients

The results in (Fig. 4A-C) demonstrate that the foliar application of chitosan nanoparticles (CHNPs) and N-acetylthiazolidine-4-carboxylic acid (N-ATCA) had significant and multifaceted effects on the leaf nutrients of Arbosana olive trees. The improved nutritional status shown in Fig. 3 directly supports enhanced growth. Treatment T16 also resulted in the highest accumulation of the main macronutrients nitrogen (N), phosphorus (P), and potassium (K) in leaf tissue (Fig. 4A, B and C respectively). Treatment T16 significantly increased leaf nutrient content compared to the control, with nitrogen (N) at 2.15 ± 0.12%, phosphorus (P) at 0.43 ± 0.03%, and potassium (K) at 1.62 ± 0.08%.

Fig. 4
Fig. 4The alternative text for this image may have been generated using AI.Fig. 4The alternative text for this image may have been generated using AI.
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Influence of foliar application of CHNPs and N-ATCA on leaf macronutrient content of Olea europaea cv. Arbosana. (A) Nitrogen (N), (B) phosphorus (P), and (C) potassium (K) concentrations under different treatments (T1–T16). Values represent mean ± SE.

Fruit characteristics

Fruit and oil yield (kg/tree)

As shown in Fig. 5A and B, foliar spraying of CHNPs and N-ATCA significantly improved several fruit parameters in Arbosana olive trees. The data in (Fig. 5A and B) demonstrate that these positive effects directly translate into economic benefits. All treatments increased fruit and oil production per plant, with the combination with the highest CHNP concentration (1500 ppm) showing the greatest effect. Notably, T15 (1500 ppm CHNPs + 100 ppm N-ATCA) achieved the highest yield, even surpassing T16. This suggests a subtle interaction: the highest N-ATCA concentration (150 ppm) was optimal for vegetative growth (T16), while the slightly lower concentration (100 ppm) favored resource allocation to both fruit and oil production. Treatment T15 significantly increased total fruit production to 18.4 ± 1.2 kg per tree, with oil production also enhanced to 4.6 ± 0.3 kg per tree, resulting in a marked improvement in both fruit and oil yield compared to the control.

Fig. 5
Fig. 5The alternative text for this image may have been generated using AI.
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Effect of CHNPs and N-ATCA foliar treatments on productivity of Olea europaea cv. Arbosana. (A) Total fruit yield per tree and (B) oil yield per tree under different treatments (T1–T16). Data are expressed as mean ± SE.

Fruit firmness and colour

The data of fruit firmness and colour highlight the significant, multifactorial effects of foliar sprays of CHNPs and N-ATCA on the firmness (Figs. 6) and color (Figs. 7A-C) of Arbosana olive trees. The two treatments differentially modified fruit ripening and quality. Figure 5 shows that fruits treated with CHNPs alone maintained greater firmness, likely due to the film-forming properties of chitosan, which reduces respiration and delays senescence. In contrast, N-ATCA treatment resulted in softer fruits, indicating a faster ripening process. The color analysis confirms this effect on fruit ripening. The combined treatments, particularly T15 and T16, produced the darkest fruits, characterized by the lowest hue angle (indicating a shift from green to purple/black; Fig. 7A), which is associated with the accumulation of anthocyanin’s and other phenolic compounds, and the highest color saturation (indicating increased color intensity; Fig. 7B). This advanced color characteristic is a key visual indicator of optimal oil accumulation and quality.

Fig. 6
Fig. 6The alternative text for this image may have been generated using AI.
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Effect of foliar application of CHNPs and N-ATCA on fruit firmness of Olea europaea cv. Arbosana under different treatments (T1–T16). Values represent mean ± SE.

Fig. 7
Fig. 7The alternative text for this image may have been generated using AI.
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Effect of CHNPs and N-ATCA treatments on fruit color parameters of Olea europaea cv. Arbosana. (A) Hue angle, (B) color saturation (chroma), and (C) color index under different treatments (T1–T16). Data are presented as mean ± SE.

Oil quality parameters

Fatty acids composition

The results in Table 1 presented the fatty acid profiles of olive oil in Arbosana olive trees processed under the various treatments (T1-T16) used in this study, with Oleic acid, the most desirable monounsaturated fatty acid in olive oil, remained the predominant component, ranging from 58.2 ± 1.8% to 60.7 ± 1.5% across treatments. Treatment T1 had the greatest quantity of oleic acid, while Treatment T5 contained the least amount. This indicated that the treatments had minimal impact on the oleic acid concentration and only affected the absolute amount produced. Palmitic acid was the predominant saturated fatty acid across all treatments, with values ranging from 19.36 ± 0.42% to 21.35 ± 0.51%, showing no statistically significant differences among treatments. The palmitic acid level was highest in Treatment T5 and lowest in Treatment T1. Linoleic acid was the predominant polyunsaturated fatty acid, with values ranging from 9.29 ± 0.37% to 12.46 ± 0.44% across treatments, with statistically significant differences observed among some treatments, with higher levels generally occurring in Treatments T5-T7and T10, indicating that the treatments have had a moderate effect on increasing the level of polyunsaturated fatty acids.

Stearic acid content differed among treatments, with the highest value recorded in T15 (4.59 ± 0.21%) and the lowest in T16 (2.84 ± 0.18%). Palmitoleic acid also showed treatment-dependent variation, with values ranging from 0.72 ± 0.05% to 1.14 ± 0.07%, but the maximum value (3.24%) was found in sample T5. All other fatty acids, such as myristic, pentadecanoic, margaric, arachidic, behenic, tricosanoic and lignoceric acid, only have low percentages (< 1%) in all treatments. Oleic acid is the largest component of the fatty acid profile, followed by palmitic acid and linoleic acid which can be associated with high-quality olive oil. The presence or absence of the individual fatty acids in treatments caused minimal changes in the proportion of fatty acids and, therefore, did not negatively impact the quality characteristics of the essential oils.

Table 1 Physicochemical characteristics of the experimental soil at the olive orchard before treatment application.
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Iodine value and calculated oxidizability value

Table 2 shows that the foliar application of chitosan nanoparticles (CHNPs) and N-acetylthiazolidine-4-carboxylic acid (N-ATCA) had significant and complex effects on the oxidative stability of Arbosana olive oil. The oxidative stability of oil, a key factor affecting its shelf life and market value, is assessed using the iodine (IV) value and the index of oxidation (Cox), as detailed in Table 3. While some individual N-ATCA treatments (T5 and T7) recorded high Cox values ​​(above 2.05), indicating reduced stability, the combined treatment was highly effective in mitigating this issue. For example, the COX value of the oil produced by T16 (1500 ppm CHNPs + 150 ppm N-ATCA) was 1.811, which is lower than that of the control group (1.768) and significantly lower than the high COX values ​​in the individual treatments. This suggests that CHNPs may exert a stabilizing effect by enhancing the antioxidant capacity of the fruit, thus compensating for the instability that may be caused by the premature ripening caused by N-ATCA.

Table 2 Effect of foliar application of chitosan nanoparticles (CHNPs) and N-acetyl thiazolidine-4-carboxylic acid (N-ATCA) on vegetative growth parameters of Olea europaea cv. Arbosana during the two experimental seasons.
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Principal Component analysis

PCA clearly differentiated the effects of pre-harvest foliar treatments on olive trees during the 2021 and 2022 seasons. Most of the variability among the 11 variables under study was explained by the first two components, as PC1 and PC2 components explained 82.5% and 9.4% of the overall variation in one season and 79.2% and 9.7% in the other season, respectively. PC1 was predominantly linked to yield components number of leaves and shoot length, N%, P%, K%, oil%, and attributes related to fatty acids, while PC2 was mostly impacted by fruit physical characteristics like firmness, hue angle, and brightness value. The combination treatments (CHNPs + N-ATCA; T8–T16), single applications (T2–T7), and the control (T1) were clearly distinguished from one another in the score plots. Higher combined concentrations (1000–1500 ppm CHNPs with 100–150 ppm N-ATCA) were specifically found on the positive side of PC1, suggesting better oil qualities, greater nutrient accumulation, and increased vegetative development. Comparatively weaker physiological responses were reflected in the closer clustering of the control and lower-dose single treatments. These results point to a synergistic relationship between CHNPs and N-ATCA that probably improves metabolic activity and nutrient uptake efficiency, which eventually leads to increased productivity and oil quality Fig. 8.

Fig. 8
Fig. 8The alternative text for this image may have been generated using AI.
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Principal component analysis (PCA) biplot illustrating the multivariate response of olive trees to 16 foliar treatments consisting of CHNPs (0, 500, 1000, and 1500 ppm), N-ATCA (0, 50, 100, and 150 ppm), and their combinations. The measured 11 parameters included shoot number, number of leaves, leaf N%, P%, and K%, yield, oil percentage, firmness, hue angle, saturation value and brightness value. PC1 and PC2 explained 82.5% and 9.4% of the total variance in 2021 and 79.2% and 9.7% in 2022, respectively.

Discussion

The expansion of worldwide olive production requires a focus on developing ways to enhance olive oil production in the most difficult conditions such as on reclaimed semi-arid land. In a two-year study, we have demonstrated that foliar (leaf) application of Chitosan Nanospheres (CHNP) and Nitrogen Acetylthiazolidine-4-carboxylic acid (N-ATCA) can promote both the agronomic performance and oil quality of Arbosana olive tree production. The strongest finding from our research was the synergistic effect or interaction between CHNP and N-ATCA. There was a consistent advantage of combining CHNP and N-ATCA over each alone for all tested growth and production parameters including vegetative growth, yield, fruit maturation, and oil stability. Based on our findings, we propose a working model that synthesizes these variables into a single, unified, biological system of CHNP/N-ATCA interaction. Foliar or seed applications (50–100 ppm) increase photosynthesis, chlorophyll content, and hormone balance, leading to taller plants and more fruit-bearing nodes54.

The synergetic use of chitosan nanoparticles (CHNPs) and N-acetylthiazolidine-4-carboxylic acid (N-ATCA) contributed positively to yield and quality through the combination of supportive flowering and fruiting55. CHNPs, which are used as natural elicitors in plants, stimulate phytohormone signalling pathways that encourage flower bud differentiation, which increases fruit set56. In addition, N-ATCA is a cysteine analog, increases antioxidant activity, and participates in sulphur metabolism, treatment enhancing pollen viability and flower development57. The combination of CHNPs and N-ATCA possibly enhances stress tolerance, nutrient translocation, and hormone balance through synergistic effects, leading to increased fruit retention, uniform fruit growth, and optimal oil biosynthesis. Overall, both treatments contribute to vegetative vigour and reproductive performance by achieving increased fruit yield and improved oil quality in this study.

The synergistic effects on vegetative plant growth and nutrient accumulation noted in our studies, especially from the combination of T16 (1,500 ppm-charged hydrogel nanoparticles or CHNPs and 150 ppm of nitrate-amine type carboxylic acid or N-ATCA) appear to have different, yet complementary modes of action that maximize the plants’ ability to conserve their resources for further use. Related chitosan trials on ‘Barhi’ date palm show dose-dependent increases (0.2% chitosan boosted new leaves, length, and area via hormone stimulation)58. On an individual basis, we believe the primary function of CHNPs is to assist in the plant’s acquisition and transportation of nutrients. Their small size and positive charge make them good candidates for foliar absorption and cellular entry. Additionally, they may enhance the permeability of cellular membranes and improve the ability of the plant to assimilate and store key macronutrients (nitrogen, phosphorus, and potassium)59. Similarly, it appears that N-ATCA is not directly involved in the plant’s acquisition and transportation of nutrients; rather, it prepares the plant for optimum use of those nutrients by priming the internal metabolic environment of the plant. N-ATCA, as a precursor to the amino acids cysteine and glutathione, likely enhances the responsiveness of the plant’s antioxidant system and prevents energy from being diverted away from growth in semi-arid regions due to oxidative stress60. The priming of the internal metabolic environment may also create an environment more conducive for the production of hormones involved in the regulation of cell division and elongation (Algarni et al., 2022). Therefore, the synergy observed with CHNPs increasing the “aspects” (or supply) of growth-promoting resources and N-ATCA improving the plant’s “capabilities” (or ability to utilize these resources) contributes to the vigour with which vegetative plants have developed in this study over the two growing seasons.

The finding that optimal growth (T16) and highest yield of fruit/oil (T15: 1500 ppm CHNPs + 100 ppm N-ATCA) occurred with different ratios signifies a refined mechanism by which combinations of nutrients interact to affect source-sink relationship or partitioning. The nutrient distribution/manipulation via biostimulants creates a mechanism to adjust the plant’s strategy of resource allocation. Spray Nano-chitosan at 100 ppm for highest yield, and fruit quality in Ewais mango. These nanomaterials enhance nutrient uptake, stress tolerance, and enzyme activity without toxicity risks61. The increased concentration of N-ATCA in T16 likely enhances the metabolic activity and increases the sink strength in the vegetative tissues. In contrast, lower, but still advantageous N-ATCA concentration in T15 and CHNPs appear to support and shift the sink strength towards the reproductive tissues. This is in agreement with previous reports regarding the ability of biostimulants to modulate the relationship between sources and sinks, as shown by62. Improvements in photosynthetic efficiency and nutrient transport via the use of CHNPs63 may provide the essential photoassimilates and nutrients required to increase the yield, thus eliminating the potential of a trade-off occurring between vegetative and reproductive growth (T15, T16).

The biostimulants had different effects on fruit ripening and had complementary effects on fruit ripeness, which is very important in determining when to harvest and what the oil quality will be. The biostimulant materials CHNPs only delayed fruit ripening by delaying senescence, resulting in fruit that remained firmer longer than un-treated fruits. This increased fruit firmness may have been due to the formation of a semi-permeable film that inhibits respiration and reduces the loss of moisture from the fruit64. Chitosan maintained higher pulp firmness by strengthening cell walls and reducing softening enzymes mango cv. ‘Zibda65. Similarly, Nano-chitosan outperforms standard forms, maintaining aril texture via better silica/calcium uptake synergy in Wonderful Pomegranate Fruits66 Conversely, N-ATCA stimulated the fruit to ripen sooner by stimulating the accumulation of pigments and producing fruit that became softer sooner probably by influencing the metabolic rates of pig-ment formation and ethylene production21. Therefore, the combination treatments (T15, T16) have created a fruit that has optimal skin coloration (a primary indicator of oil accumulation) and does not exhibit excessive softening prior to harvest. Preharvest foliar sprays of chitosan, nano-chitosan, and calcium chloride on ‘Barhi’ date palms indirectly improved physical properties like fruit firmness and weight loss by reinforcing cell walls and slowing metabolic degradation67. The overall improved balance of these reciprocally interacting effects is an important part of managing the best harvest windows for producing oil of the highest quality from this fruit type. The overall response of the fruit to the combined treatment is representative of the final product of oil. The oils produced from all of the treatments met the EVOO standards for oil production. However, the oils produced with the combined treatments produced higher-quality oils than those produced from the individual treatments. There was a trend of producing consistently improved oil fatty acid profiles including slightly higher levels of oleic acid than oils produced from either of the two single treatment types. It is especially significant to note that in most of the other treatments, the minimum levels of oleic acid were close to being reached for this cultivar, while the oils from the biostimulant combination treatments were still being produced at levels that will ensure very good oil quality for this cultivar. It is likely that the combined treatments of biostimulants enhanced the production of oleic acid by minimizing environmental stresses on lipid synthesis68. Similarly, at the tree and fruit level, exposure to lower growing temperatures tends to increase the proportion of oleic acid and reduce more highly unsaturated fatty acids (especially linoleic and linolenic) in the final olive oil, within the normal agronomic range of Mediterranean climates69. In addition to the aforementioned findings, the combination treatment (T16) has been shown to demonstrate oxidation values that are not significantly different from that of controls, even though there is an increase in unsaturation with increased quality improvement. Therefore, it appears that the stabilization mechanisms have been activated to a greater extent than merely by changes in the fatty acid profile.

The prolonged oil stability observed under the combined treatments may have resulted from two contributing factors: (1) a slight increase in oleic acid content, which enhances oxidative resistance, and (2) a potential improvement in antioxidant capacity, possibly associated with higher levels of phenolic compounds and tocopherols. However, as these antioxidant parameters were not directly quantified in the present study, this interpretation remains speculative. Although secondary antioxidants (polyphenols and tocopherols) were not measured in our study, they play an important role in promoting oxidative stability70. In addition, both CHNPs and N-ATCA induce the activation of plant defense and antioxidant pathways71. Therefore, the combination of these two treatments may result not only in the improvement of the underlying composition of oil but could provide it also with extra protection due to the potential increase in protective antioxidants, thus producing a more durable product as observed in other studies72. Therefore, this holds promise as a future area of research to further quantify the antioxidant compounds present in oil.

Environmental impact, N-acetylthiazolidine-4-carboxylic acid (N-ATCA) is a cysteine derivative that functions as a plant bioregulator and stress-mitigating compound and is typically applied at very low concentrations73. Similarly, chitosan nanospheres are biodegradable materials derived from natural sources and are widely considered environmentally safe biostimulants74. Therefore, the combined use of CHNPs and N-ATCA represents a sustainable approach for improving olive productivity and oil quality while reducing reliance on conventional agrochemicals, particularly under semi-arid reclaimed soil conditions.

Conclusion

This two-year study conclusively demonstrated that foliar spraying of chitosan nanoparticles (CHNPs) and N-acetylthiazolidine-4-carboxylic acid (N-ATCA) is a highly effective strategy for stimulating plant growth in olive orchards cultivated on newly cultivated sandy loam soils in Arbosana olive trees. The combined application of the two compounds was most effective, highlighting their strong synergistic effect. While the formulation with the highest concentration (T16: 1500 ppm chitosan nanoparticles + 150 ppm N-ATCA) was most beneficial in promoting plant growth and nutrient uptake, the formulation with a slightly lower N-ATCA concentration (T15: 1500 ppm chitosan nanoparticles + 100 ppm N-ATCA) proved most effective in maximizing fruit and oil yield. These treatments positively impacted fruit ripening and quality. Importantly, the pursuit of higher yields did not compromise the essential quality of the olive oil, which consistently met the standards of extra virgin olive oil. This combination also helped mitigate the potential decline in oxidative stability associated with the use of N-ATCA alone. Therefore, the combined use of CHNPs and N-ATCA offers a promising, sustainable agricultural practice that can help increase overall yields, improve oil quality, and enhance the olive cultivation in semi-arid and newly reclaimed areas.