Synergistic effect of nano-chelated iron fertilizer and vermicompost tea on the stigma yield and phytochemicals of Crocus sativus L. in an alkaline soil

Abstract

Background

Saffron (Crocus sativus L.) is considered the most valuable spice in the world, and its cultivation requires specific soil conditions to maximize yield and quality. While saffron can grow in calcareous soils, the high pH levels typically associated with these soils can limit optimal production. Limited studies have demonstrated that applying nano-chelated iron fertilizer (NCIF) and solid vermicompost individually can enhance saffron growth and yield. However, the combined effects of NCIF and liquid vermicompost tea (VCT) in alkaline conditions remain unexplored in saffron or other plants. This study investigated the individual and combined impacts of NCIF soil drenching (0, 4, and 8 kg ha^− 1) and VCT foliar applications (0, 10, and 20%) on stigma yield and phytochemicals in various parts of saffron plants grown in an alkaline soil.

Results

VCT and NCIF, particularly in combination, significantly reduced electrolyte leakage, while enhancing total chlorophyll content, flower yield, and stigma biomass and length. The integrated application of 20% VCT and 8 kg ha^− 1 NCIF yielded the highest concentrations of safranal, crocin, picrocrocin, and total flavonoid content, alongside superior DPPH scavenging activity, and reducing power in stigma. Similar improvements in phenolic content and antioxidant properties, along with enhanced iron-chelating activity, were observed in the petals and leaves.

Conclusions

These novel findings underscore the synergistic effect of NCIF and VCT to improve saffron stigma yield, quality, and antioxidant properties under challenging soil conditions.

Introduction

Saffron (Crocus sativus L.), often referred to as the “golden spice” or “red gold”, is a geophyte that propagates exclusively through corms. This plant blooms between mid-October and late November in the Northern Hemisphere, producing violet petals with deep red stigmas and yellowish styles [1]. Its stigma is renowned for its unique bioactive compounds—safranal, crocin, and picrocrocin—that contribute to its distinctive aroma, vibrant color, and rich flavor [2]. Saffron has also garnered significant scientific interest for its potential therapeutic applications in combating cardiovascular disorders, Alzheimer’s disease, and cancer [3]. Soil fertility and optimal nutrient availability are critical factors for achieving both high yields and superior stigma quality [4, 5]. The application of bio- and organic fertilizers significantly enhances saffron production [6]. For instance, Jami et al. [7] reported that vermicompost, particularly when combined with mycorrhiza, boosted flower count, leaf chlorophyll content, leaf area, dry weight, and the concentration of crocin and picrocrocin in stigma. Saffron thrives in diverse climatic and soil conditions, with optimal growth observed in loose, well-drained, clay-calcareous soils enriched with organic matter, low bulk density, and a neutral to slightly alkaline pH [1]. However, high pH in soils reduces the availability of essential nutrients such as iron, phosphorus, and zinc [8], posing significant challenges to achieving sustainable saffron production. In Iran, saffron is often cultivated in alkaline and calcareous soils with low nutrient availability and insufficient organic matter. Therefore, implementing targeted soil management practices and developing innovative agricultural inputs are crucial for addressing these challenges [9]. A study demonstrated that the combined application of vermicompost and γ-aminobutyric acid effectively alleviates challenges associated with high soil pH and phosphorus deficiency in saffron cultivation [10]. Such findings suggest promising solutions to improve crop performance in alkaline soils.

Vermicompost, derived from the organic matter processed by earthworms, is a nutrient-rich material containing vitamins, amino acids, humic substances, enzymes, and phytohormones [11, 12]. This organic fertilizer promotes plant growth, boosts secondary metabolism in medicinal plants, and enhances crop yield under various stresses [12–15]. Vermicomposts are produced from different organic materials, and specific samples may exhibit high electrical conductivity (EC), which limits their application for plant fertilization in saline and alkaline soils [11, 16]. Over the past decade, innovative liquid derivatives such as vermicompost leachate, vermiwash, and vermicompost tea have emerged as practical alternatives to vermicompost [17, 18]. These liquid biostimulants are rich in soluble nutrients, bioactive compounds, beneficial microorganisms, and phytohormones, all of which play pivotal roles in enhancing plant growth and stress tolerance [17, 19–22].

Vermicompost tea (VCT) is produced by extracting nutrients and beneficial microorganisms from solid vermicompost through stirring and aeration [18]. The studies on VCT have predominantly focused on its potential for disease suppression and pest management [23–26] and its influence on microbial activity and improvement of nutrient availability in soils [27, 28]. For example, VCT soil amendment enhanced maize plant performance by reinforcing antioxidant defense mechanisms under salt stress [29]. Seed priming with VCT enhanced seed germination, suppressed Meloidogyne incognita, and improved growth parameters in tomato by increasing antioxidative enzyme activity and secondary metabolite production [30]. Despite these advancements, research on VCT foliar application and its impact on plant stress tolerance remain limited. A few studies have shown that VCT foliar application alleviated salinity effects on fennel [31] and saffron [32], as well as improved water-deficit tolerance in soybean [33] and Beta vulgaris [34]. However, the effect of VCT foliar application on flower yield and quality of saffron grown in alkaline soils remains unexplored and warrants further investigation.

Iron deficiency is a common issue in plants, particularly in alkaline media, where iron availability is severely limited owing to the formation of insoluble compounds [8, 35]. Iron is a vital micronutrient that plays a crucial role in chlorophyll synthesis and the function of certain enzymes. Its deficiency often manifests as chlorosis, reduced flowering, and compromised metabolic functions, ultimately impacting plant health and productivity [36]. To supply iron for plants, synthetic iron chelates such as Fe-EDTA and Fe-DTPA are commonly employed in agriculture. However, the stability of most of these iron chelates is often limited by soil conditions, particularly in high-pH soils, and more stable chelating agents such as o, o-EDDHA/Fe³⁺ are expensive [37]. Additionally, specific chelating agents, such as EDTA, exhibit low biodegradability in soils, and their prolonged persistence can raise significant environmental concerns. These chelates can modify the natural metal speciation in soils and aquifers, potentially increasing the bioavailability of toxic metals [36]. To address these issues, nano-chelated iron fertilizers (NCIF) have emerged as alternative and innovative inputs. Nano-chelated fertilizers are produced through the polymerization of organic acids and exhibit a robust absorption capacity across a broad pH spectrum (3 < pH < 11). Moreover, these fertilizers facilitate the gradual release of essential plant elements [38]. Therefore, applying nano-chelated iron fertilizers (NCIF) with such characteristics may effectively address the limitations of conventional iron chelates and provide a steady supply of iron to plants grown in alkaline soils. A study demonstrated that the application of a specific NCIF surpasses the effect of ethylenediamine di(2-hydroxy-5sulfophenylacetic) acid (EDDHSA) in improving dry stigma weight, fresh flower weight, flower count, leaf number, and leaf length of saffron [39]. However, the impact of NCIF on saffron quality and yield under alkaline conditions remains uncertain.

Saffron often experiences nutrient stress over time due to its multi-year lifecycle. Therefore, balanced nutrition—particularly maintaining adequate levels of N, P, K, and Fe in the soil and corms— is critical for achieving high production and profitability per unit area [40]. The high pH in soils reduces the availability of essential nutrients, especially iron, which in turn decreases saffron yield and quality [10]. VCT foliar application—which delivers nutrients directly through leaf stomata—can help address this challenge and reduce reliance on soil-based fertilizers in high- pH soils. On the other hand, enhanced solubility and stability of NCIFs in high pH can improve iron availability to plants in alkaline soils without issues associated to use of conventional iron chelates [38]. To the best of our knowledge, no prior study has examined the combined effects of NCIF soil drenching and VCT foliar application on saffron, particularly under alkaline conditions. Therefore, we hypothesized that the integrated application of NCIF and VCT could serve as an innovative and eco-friendly strategy to enhance saffron yield and quality under such conditions. The current study provides the first comprehensive evaluation of the synergistic effects of NCIF and VCT on saffron flowering and the phytochemical profiles of the stigma, petals, and leaves of plants grown in high-pH soils.

Materials and methods

Characteristics of nano-chelated iron fertilizer

In this study, we applied Khazra nano-chelated iron fertilizer, which is a product developed by Sodour Ahrar Shargh Company in Iran. This fertilizer has been produced using patented nanochelating technology, registered with the United States Patent and Trademark Office (USPTO), which employs a “self-assembly” method. This specific NCIF is characterized by a hydrocarbon base free of ethylene bonds and hormones with 9% iron. The analyses using transmission electron microscopy (TEM) and dynamic light scattering (DLS) have demonstrated a mean particle diameter of 80 ± 3.2 nm, a mean hydrodynamic diameter of 57 ± 2.0 nm, and a zeta potential of − 13.7 ± 2.5 mV [41]. This fully water-soluble fertilizer has stability across a broad pH from 3 to 11, making it effective in both acidic and alkaline soils. Several studies demonstrated that this nano-chelated fertilizer has superior absorption compared to conventional iron fertilizers, including chemical sulfates and with minimal application rate outperforming other iron chelates [38, 39, 41]. However, this study, for the first time, examined its efficiency for saffron cultivation in an alkaline soil.

Preparation of vermicompost tea

Vermicompost applied in this study was procured from a local market in Iran, and its chemical characteristics are detailed in Table 1. To produce aerated VCT, a handmade incubation system equipped with an air pump was employed as described by Ghaffari et al., [34]. The process involved mixing solid vermicompost and water with 1:10 (10%) and 1:5 (20%) ratios and incubating at room temperature for 5 days under continuous aeration. Then, the resulting compost tea was filtered through a two-layer system with a mesh size of 800 microns to ensure its refinement. This actively aerated VCT was immediately applied for saffron spraying in the field.

Table 1.

The characteristics of vermicompost applied for this study

	EC (ds/cm)	pH	O.C (%)	T.N.V %	Total P (%)	Total K (%)	Total N (%)	Fe mg kg− 1		Mn mg kg− 1	Zn mg kg− 1	Cu mg kg− 1
vermicompost	3.51	8.04	32.16	11	0.25	10.8	3.6	56.17	28.54		78.68	10.63

Soil and climatic characteristics of field

The current study was conducted in the second growth year of a saffron field located in Jarghoyeh, Isfahan, Iran. The study site is geographically positioned at 51˚72’ N and 32˚99’ E, with an elevation of 1624 m above sea level. Table 2 indicates a meteorological station report for monthly average temperature, precipitation, as well as maximum and minimum humidity during October 2011-October 2012 in the region where the research was performed in it.

Table 2.

Monthly average temperature, precipitation, and maximum and minimum humidity during October 2011-October 2012

	Oct.	Nov.	Dec.	Jan.	Feb.	Mar.	Apr.	May	Jun	Jul.	Aug.	Sep.	Oct.
Precipitation	0	28.1	17.7	0	18.9	2.3	26.1	13.5	4.7	0	0	0	3.6
Temperature	18.6	10.6	3.9	3.9	3.6	6.8	12.9	18.7	23.8	27.4	26.5	23.8	18.2
Maximum Humidity	47	73	95	83	73	57	70	63	41	36	34	42	53
Minimum Humidity	10	31	46	27	27	17	21	17	10	10	8	10	15

The soil of the experimental field exhibited alkaline properties with a pH of 8.5. Detailed physicochemical properties of the soil are outlined in Table 3.

Table 3.

The characteristics of the soil used for this experiment

	Silt %	Clay %		sand %	Avail. P (ppm)	Total N (%)	EC (1:5) (ms.cm-1)	pH	O.C (%)
soil	8	42	50		0.9	0.05	2.5	8.5	0.9

Agronomic practices and treatments

In the first year (i.e. 2010), the farm was prepared on July 5, using plowing, harrowing, and disking techniques. Saffron corms were purchased from Torbat-e-Heydarieh province, Razavi Khorasan, Iran (Coordinates: 35°25′N 59°09′E). Saffron corms averaging 8 g weight were dipped in a 1% fungicide water solution of copper oxychloride (Sumitomo Chemical Italia S.r.l., Milano, Italy) to minimize fungal diseases and hand-planted on July 10, at a depth of 20 cm during their dormancy period. To fertilization, 10 Mg ha⁻¹ cow manure at planting time, alongside 50 kg ha⁻¹ urea, 80 kg ha⁻¹ calcium superphosphate, and 50 kg ha⁻¹ potassium sulfate during the second irrigation were applied. An additional 50 kg ha⁻¹ of urea fertilizer was applied in mid-February. Weed control was carried out by hand in the autumn and spring in both years.

The present experiment was conducted in the second year of plant growth (i.e. October 2011-October 2012), as a factorial design in randomized complete blocks with three replications. The study included three levels of NCIF (0, 4, and 8 kg ha⁻¹) applied to irrigation water and three levels of foliar applications with VCT (0, 10, and 20%). Each plot was considered at a dimension of 2 × 2 m², which contained 8 rows, and the distance between rows was 25 cm. The distance between corms on rows was 25 cm and corms were planted at a depth of 15 cm.

After the last irrigation before flowering on October 22, stigma yield of the first year was harvested at November 2011. Then, the treatments of the current study were implemented from December 2011. NCIF soil drenching was performed through adding this fertilizer to water and irrigation on December 1 (i.e., after flower harvesting and at early vegetative regrowth). Subsequent irrigations were performed in the corm propagation stage in January and the corm filling stage in late April.

Foliar applications of VCT were performed three times at key phenological stages: post-harvest and early vegetative regrowth (December 1), mid-vegetative growth (January 1), and maximum leaf expansion (February 1). Leaf samples were collected on February 15 for biochemical analysis. Flowering occurred between October 25 and November 10; during this period, flowers were harvested daily to record flower count and fresh weight. Stigmas were carefully excised and shade-dried at room temperature to determine their length and dry weight. Finally, the samples were stored in airtight glass jars at approximately 10 °C with a moisture content of 8% for subsequent analysis.

Estimation of electrolyte leakage levels in leaves

To evaluate electrolyte leakage (EL) levels, fresh leaf discs were incubated in deionized water at 25 °C for 24 h, and the initial electrical conductivity (EC1) was measured. Subsequently, the discs were subjected to a high-temperature treatment at 90 °C for 2 h, followed by cooling to 25 °C to obtain the second electrical conductivity measurement (EC2). The electrolyte leakage was then calculated as follows [42]:

1

Quantification of chlorophyll content

The fresh leaves were macerated using acetone (80%, v/v), and after filtering, the absorbance of the extract was read at 663 and 645 nm. Then, total chlorophyll (TChl) content was calculated using the formula suggested by Arnon et al. [43].

Analysis of picrocrocin, safranal, and crocin in stigma

The apo-carotenoid contents in saffron stigma were analyzed following the methodology outlined in ISO 3632-1 standards [44]. The process involves preparing an aqueous saffron extract by dissolving 500 mg of powdered saffron (sieved to 0.5 mm) in 900 mL of distilled water, stirring for 1 h in the dark, and diluting to 1000 mL. Subsequently, a 1:10 dilution with deionized water is performed, followed by filtration using PTFE filters (15 mm diameter, 0.45 μm pore size). The extract is analyzed with a UV-Vis spectrophotometer to determine the concentrations of picrocrocin, safranal, and crocin by measuring absorbance at wavelengths of 257 nm, 330 nm, and 440 nm, respectively, using the following equation:

Where the absorbance values at 257, 330, and 440 nm, denoted as D, 10,000, represent the total extract dilution based on the quantity of saffron sample utilized, and the variable H accounts for the moisture.

Measurement of total flavonoid content in the stigma

The total flavonoid content (TFC) was determined by combining 1.5 ml of methanolic extract solution with glacial acetic acid and 0.1 mL of 10% AlCl₃ solution. After allowing the mixture to react for 40 min, the optical absorbance of the resulting flavonoid–aluminum complex was measured at 415 nm. TFC was expressed in terms of milligrams of Quercetin (QE) equivalents per gram of dry weight [45].

Quantification of total phenolic content in leaves and petals

The phenolic content was determined by extracting 0.3 g of powdered, dried leaves or petals in 80% methanol, followed by centrifugation at 5000 ×g for 5 min at 4 °C. Subsequently, 0.1 mL of the supernatant was combined with 2.8 mL of deionized water, 0.1 mL of 2% Folin-Ciocalteu reagent, and 2 mL of 2% sodium carbonate. The mixture was incubated for 30 min, and the optical density was measured at 750 nm. Total phenolic content (TPC) was quantified as milligrams of gallic acid equivalent per gram of dry weight [46].

Estimation of the antioxidant properties

The DPPH radical scavenging activity of extracts was assessed using the method outlined by Amooaghaie and Rajaei [47]. The procedure involved mixing 0.5 mL of the extract with 2.5 mL of a 0.004% DPPH solution, followed by incubation in darkness for one hour. Subsequently, absorbance decrease was measured at 517 nm. The scavenging activity was calculated using the formula:

The reducing power in extracts was assessed using a spectrophotometric method [47]. The procedure involved mixing 0.5 mL of the extract with 0.5 mL of phosphate sodium buffer (0.2 M, pH 6.6) and potassium ferricyanide [K₃Fe(CN)₆], followed by incubation at 50 °C for 20 min. After incubation, 0.5 mL of 10% trichloroacetic acid was added to the mixture, which was then centrifuged at 5000 rpm for 10 min. Subsequently, 2.5 mL of the supernatant was combined with 0.5 mL of distilled water and 0.1 mL of a 0.1% ferric chloride solution. The resulting blue-green coloration was measured at 700 nm using a spectrophotometer.

The iron-chelating activity of the extracts was evaluated using the method described by Carter [48]. Various concentrations of the extracts were incubated with 0.05 mL of FeCl₂·4 H₂O (2 mM), followed by the addition of 0.2 mL of ferrozine solution (5 mM) to initiate the reaction. Methanol was used to adjust the total volume to 0.8 mL, and the absorbance was measured at 562 nm after 10 min of incubation. A sample containing ethylenediamine tetraacetic acid (EDTA), which did not develop any color, was used as the positive control, while a sample without EDTA served as the negative control. The percentage of metal chelation was calculated using the formula:

where A₀ represents the absorbance of the control, and A₁ corresponds to the absorbance of the test samples.

Statistical analysis

The study was executed as a factorial arrangement in a completely randomized block design with three replications. All data were checked for normality through employing Kolmogorov-Smirnov and Shapiro-Wilk tests. Then, a two-way ANOVA was performed using Statistical Analysis System software (SAS Institute, Cary, NC, USA, Version 9.1) to evaluate the individual effects of VCT and NCIF, as well as their interaction (VCT × NCIF). Duncan’s multiple range tests were applied to compare means at P < 0.05. Pearson’s correlation analysis between variables was performed using PAST software (http://palaeoelectronica.org/2001_1/past/issue1_01.htm). The principal component analysis (PCA) and clustering analysis were conducted using the XLSTAT software (2016).

Results

Effect of NCIF and VCT on quantitative traits of the flower and stigma

The two-way ANOVA results revealed that the application of NCIF or VCT each had significant individual effects (p < 0.01) on all quantitative characteristics of the flower and stigma. However, the interaction effects of NCIF × VCT were not statistically significant for flower number, as well as stigma fresh weight and length (Table 4).

Table 4.

The effects of vermicompost tea (VCT) foliar spraying and nano-chelated iron fertilizer (NCIF) soil drenching on quantitative traits of flower and stigma of saffron plants grown in an alkaline soil

Treatments	Flower count (No. m− 2)	Fresh weight of flowers (g m− 2)	Fresh weight of stigma (g m− 2)	dry weight of stigma (g m− 2)	Stigma length (mm)
Individual effect of VCT
0	31.36c	28.22c	1.14c	0.39c	27.02c
10%	39.56b	37.55b	1.51b	0.51b	28.01b
20%	57a	46.78a	1.63a	0.60a	28.80a
Significance	**	**	**	**	**
Individual effect of NCIF
0	29.67c	28.78c	1.35c	0.47b	27.5c
4 kg ha⁻¹	41.67b	37b	1.52b	0.50b	27.94b
8 kg ha⁻¹	51.56a	46.78a	1.73a	0.57a	28.38a
Significance	**	**	**	**	**
Interaction effect of NCIF × VCT
Significance	ns	ns	ns	ns	ns

Significant differences (p<0.05) between means ± SE were identified by Duncan’s multiple-range tests and presented by different letters

**, and ns respectively refer to significance at p < 0.01 and no significant effect based on ANOVA analysis

As shown in Table 4, while the control plants grown in alkaline soil (i.e., 0 VCT or 0 NCIF) exhibited the lowest values for the flower and stigma quantitative characteristics, the single application of NCIF at both rates significantly increased these attributes. The soil drenching 8 kg ha⁻¹ NCIF was more effective and increased flower number, fresh weight of flowers, fresh and dry weight of stigma, and stigma length by an average of 73.77%, 62.54%, 69.29%, 21.27%, and 3.2%, respectively, compared to the control (0 NCIF in Table 4).

Similarly, the single VCT application at both concentrations positively affected all flower and stigma quantitative characteristics. The most pronounced effects were observed in the 20% VCT treatment, which increased flower number, fresh weight of flowers, fresh and dry weight of stigma, and stigma length by an average of 81.7%, 65.7%, 42.9%, 53.86%, and 6.58%, respectively, relative to the control (0 VCT in Table 4).

Effect of NCIF and VCT on total chlorophyll and electrolyte leakage

The two-way ANOVA results revealed that interaction effects of NCIF × VCT for TChl content and EL level in leaves were statistically significant (p < 0.01, data not shown).

As shown in Fig. 1, the application of 4 and 8 kg ha⁻¹ NCIF alone significantly enhanced TChl content, while decreasing EL level in leaves compared to the control. A similar trend was observed for foliar application of both VCT concentrations alone. All combinations of NCIF or VCT significantly increased TChl content and reduced EL level in leaves, indicating synergistic effects of two fertilizers on these variables. The most pronounced improvements, however, were achieved using the combined treatment of 20% VCT and 8 kg ha⁻¹ NCIF, which increased TChl content by 69% and decreased EL level by 44% compared to the control (Fig. 1).

Fig. 1.

The effect of vermicompost tea (VCT) foliar spraying and nano-chelated iron fertilizer (NCIF) soil drenching on total chlorophyll content (A) and electrolyte leakage level (B) in leaves of saffron plants grown in an alkaline soil. Significant differences (p < 0.05) between means ± SE were identified by Duncan’s multiple-range tests and presented by different letters

Effect of NCIF and VCT on bioactive compounds in stigma

The two-way ANOVA results revealed that interaction effects of NCIF × VCT for the concentrations of picrocrocin, crocin, safranal, and TFC in the stigma were statistically significant (p < 0.01, data not shown).

As shown in Fig. 2, the single application of 4 and 8 kg ha⁻¹ NCIF or 10% and 20% VCT significantly increased safranal, picrocrocin, and crocin levels as well as TFC compared to the control. All combinations of NCIF or VCT significantly increased the levels of these metabolites compared to the controls, indicating synergistic effects of two fertilizers on these attributes. The most pronounced improvements, however, were achieved through the combined treatment of 20% VCT and 8 kg ha⁻¹ NCIF, which led to substantial increases in picrocrocin, crocin, safranal, and TFC by 44.44%, 52.63%, 99.6%, and 52.15% compared to the controls (Fig. 2).

Fig. 2.

The effect of vermicompost tea (VCT) foliar spraying and nano-chelated iron fertilizer (NCIF) soil drenching on the content of picrocrocin (A), safranal (B), crocin (C), and total flavonoid content (D) in stigma of saffron plants grown in an alkaline soil. Significant differences (p < 0.05) between means ± SE were identified by Duncan’s multiple-range tests and presented by different letters

Effect of NCIF and VCT on the antioxidant activity of stigma

The two-way ANOVA results revealed that interaction effects of NCIF × VCT for DPPH scavenging activity and reducing power were statistically significant (p < 0.01, data not shown).

As shown in Fig. 3, fertilization with 4 kg ha⁻¹ NCIF alone did not enhance DPPH scavenging activity but increased reducing power. However, the application of 8 kg ha⁻¹ NCIF alone was more effective, significantly improving both DPPH scavenging activity and reducing power in the stigma. Additionally, a single application of 10% and 20% VCT significantly increased both attributes (Fig. 3A, B). All combinations of NCIF or VCT significantly influenced DPPH scavenging activity and reducing power in the stigma compared to the control, indicating synergistic effects of the two fertilizers on these variables. However, the most pronounced effects were observed with the combined application of 20% VCT and 8 kg ha⁻¹ NCIF, which increased DPPH scavenging activity by 68.77% compared to the control. Meanwhile, the highest reducing power was achieved using the combined application of 10% VCT and 8 kg ha⁻¹ NCIF, marking a 39.34% increase compared to the control (Fig. 3).

Fig. 3.

The effect of vermicompost tea (VCT) foliar spraying and nano-chelated iron fertilizer (NCIF) soil drenching, on DPPH scavenging activity (A), and reducing power (B) in the stigma of saffron plants grown in an alkaline soil. Significant differences (p < 0.05) between means ± SE were identified by Duncan’s multiple-range tests and presented by different letters

Effect of NCIF and VCT on TPC of petals and leaves

The two-way ANOVA results revealed that interaction effects of NCIF × VCT for total phenolic content (TPC) in petals and leaves were statistically significant (p < 0.01, data not shown).

As shown in Fig. 4, the single application of 4 and 8 kg ha⁻¹ NCIF or 10% and 20% VCT concentrations independently resulted in the significant increases in TPC of petal and leaves compared to the controls. Additionally, all combinations of NCIF or VCT significantly increased TPC in both petals and leaves compared to the control, indicating synergistic effects of two fertilizers on these variables. However, the most pronounced improvement in TPC was observed with the combined application of 20% VCT and 8 kg ha⁻¹ NCIF, which increased TPC levels in petals and leaves by 1.9, and 2.1 folds, compared to the controls. This synergistic effect underscores the potential benefits of integrating these treatments for optimizing phenolic content in plant tissues (Fig. 4).

Fig. 4.

The effect of vermicompost tea (VCT) foliar spraying and nano-chelated iron fertilizer (NCIF) soil drenching on total phenol content (TPC) in petal (A), and leaf (B) of saffron plants grown in an alkaline soil. Significant differences (p < 0.05) between means ± SE were identified by Duncan’s multiple-range tests and presented by different letters

Effect of NCIF and VCT on the antioxidant properties of petals and leaves

The two-way ANOVA results revealed that the application of NCIF or VCT each had significant individual effects (p < 0.01) on all antioxidant properties in petals and leaves. Additionally, significant interaction effects were demonstrated for DPPH scavenging activity in petals and leaves, as well as reducing power and chelating activity in leaves (p < 0.01 or p < 0.05). However, the interaction effects of NCIF × VCT for reducing power and chelating activity in petals were not statistically significant (Table 5).

Table 5.

The effects of vermicompost tea (VCT) foliar spraying and nano-chelated iron fertilizer (NCIF) soil drenching on quantitative traits of flower and stigma of saffron plants grown in an alkaline soil

Treatments	Petal DPPH scavenging activity (%)	Petal reducing power (∆OD700)	Petal iron-chelating activity (%)	Leaf DPPH scavenging activity (%)	Leaf reducing power ( ∆OD700)	Leaf iron-chelating activity (%)
Individual effect of VCT
0	63.62c	0.17b	48.9b	54.11c	0.124b	20.89b
10%	72.14b	0.22a	73.3a	78.00a	0.231a	27.89a
20%	77.51a	0.21a	67.8a	72.67b	0.238a	26.55a
Significance	**	**	**	**	**	**
Individual effect of NCIF
0	60.07c	0.17c	43.3c	60.55c	0.157c	21.67c
4 kg ha⁻¹	72.63b	0.20b	62.2b	69.22b	0.193b	25.11b
8 kg ha⁻¹	80.59a	0.23a	84.4a	75.00a	0.242a	28.55a
Significance	**	**	**	**	**	**
Interaction effect of NCIF × VCT
Significance	*	ns	ns	**	**	**

Significant differences (p<0.05) between means ± SE were identified by Duncan’s multiple-range tests and presented by different letters

*, **,  and ns  respectively refer to significance at p < 0.05; significance at p < 0.01 and no significant effect based on ANOVA analysis

As shown in Table 4, while the control plants grown in alkaline soil (i.e. 0 VCT or 0 NCIF) exhibited the lowest values for all antioxidant properties in both petals and leaves, the single application of NCIF or VCT at both rates significantly increased these attributes. Notably, the individual application of 8 kg ha⁻¹ NCIF or 20% VCT yielded optimal results in these antioxidant properties in both petals and leaves (Table 5).

As shown in Fig. 5, the combined application of 20% VCT and 8 kg ha⁻¹ NCIF led to the highest enhancement in DPPH scavenging activity in both petals and leaves, reducing power and chelating activity in leaves compared to the control. These findings highlight the synergistic potential of combining NCIF and VCT for maximizing antioxidant properties in various parts of the plant.

Fig. 5.

The effect of vermicompost tea (VCT) foliar spraying and nano-chelated iron fertilizer (NCIF) soil drenching on DPPH scavenging activity in petal (A), and leaf (B), reducing power (C) and iron-chelating activity (D) in the leaf of saffron plants grown in an alkaline soil. Significant differences (p < 0.05) between means ± SE were identified by Duncan’s multiple-range tests and presented by different letters

Multivariate analyses

The analysis of Pearson correlation (Fig. 6) reveals a predominantly positive relationship among the most of the evaluated traits. Only, the correlation between the fresh weight of stigma (S-FW) and its total flavonoid content (S-TFC) was found to be insignificant.

Fig. 6.

Pearson’s coefficient correlations between evaluated variables in saffron cultivated in an alkaline soil. Strong negative and positive correlations have been represented by large red and blue circles, respectively. The investigated variables include: Number and fresh weights of flowers (F-No., F-FW), fresh and dry weights and length of stigma (S-FW, S-DW, S-L), total chlorophyll (TChl), electrolyte leakage (EL), picrocrocin (Pic), Crocin (Croc), safranal (Saf), total flavonoid content in stigma (S-TFC), DPPH scavenging capacity and reducing power (S-DPPH, S-RP), total phenol content in leaves and petals (L-TPC, P-TPC), DPPH scavenging capacity, reducing power and iron-chelating activity in leaves and petals (L-DPPH, L-RP, L-chelate, P-DPPH, P-RP, P-chelate)

A strong positive correlation was observed between stigma dry weight and length (S-DW, S-L), and flower counts (F-No). Furthermore, the DPPH scavenging activity was strongly correlated with picrocrocin, crocin, and safranal levels, while the reducing power revealed a stronger correlation with TFC in the stigma. Additionally, significant positive correlations were observed between leaf and petal total phenolic content (L-TPC and P-TPC) and their respective DPPH scavenging activity, reducing power, and iron chelating activity. In contrast, EL level was negatively correlated with all attributes.

Principal Component Analysis (PCA) was conducted to elucidate the associations among different groups of variables and treatments. The PCA results revealed that the first principal component (PCA 1) accounted for 89.52% of the data variability, while the second principal component (PCA 2) explained 5.69%.

The findings of PCA indicate that the majority of variations in the evaluated traits (except EL level) were predominantly linked to the combined treatments, i.e. N4V10, N8V10, N4V20, and specifically N8V20. This analysis highlights the significant impact of combined treatments on the observed traits, providing valuable insights into the synergistic effects of fertilizers on these attributes. In contrast, the highest EL levels were obtained in N0V0 (control without treatment) and N4V0, N8V0, N0V10, and N0V20 (i.e., single treatments).

The clustering analysis also categorized the treatments, as illustrated in Fig. 8. The first cluster encompassed the combined treatments of N4V10, N4V20, N8V10, and N8V20, indicating that the simultaneous application of nano-chelated iron fertilizer and vermicompost tea had a significant impact on the evaluated traits. Among these, the treatment with the highest levels of both fertilizers (N8V20) led to the most pronounced effects on most attributes. The second cluster included N0V10, N0V20, N4V0, and N8V0 treatments, which were grouped with the control treatment in a cluster. This classification suggests that the individual application of these fertilizers, compared to their combination less affects the evaluated traits.

Fig. 8.

The clustering the different groups of treatments in saffron cultivated in an alkaline soil. The treatments were included: N0V0, N0V10, N0V20, N4V0, N8V0, N4V10, N4V20, N8V10, and N8V20, where N0, N4, and N8 represent nano-chelated iron fertilizer with concentrations of 0, 4, and 8 kg ha^− 1, and V0, V10, and V20 represent vermicompost tea at concentrations of 0, 10, and 20%

Discussion

Saffron is highly valued not only for the culinary and medicinal benefits of its stigma but also for the antioxidant properties found in its flowers and leaves [2]. The findings of this study provide the first evidence for the potential of combining NCIF and VCT as an innovative fertilization strategy for saffron cultivation in an alkaline soil.

VCT is a liquid fertilizer rich in essential nutrients [19]. Therefore, its foliar spraying enhanced saffron flower and stigma characteristics in an alkaline soil, with optimal results at a 20% concentration (Table 3). Similarly, VCT foliar application increased corm yield, as well as the number, fresh weight, length, and yield of saffron flowers and stigma under both saline and non-saline conditions [32]. It has also been demonstrated that partially substituting mineral fertilizers with VCT can enhance essential nutrient levels in plants. This treatment has resulted in higher onion bulb yield and quality [49] and enhanced seed yield and quality in wheat grains [50]. As illustrated in Table 1, vermicompost is rich in essential macro- and micro-nutrients, including N, P, K, Fe, Zn, Mn, and Cu. A substantial portion of these essential nutrients and bioactive compounds was likely preserved during the extraction process. Thus, VCT foliar application likely compensated mineral deficiencies induced by alkaline stress, promoting sustainable saffron flowering. Nevertheless, it is noticeable that VCT is a diluted extract, providing lower nutrient concentrations compared to solid vermicompost. However, the presence of beneficial microorganisms, humic substances, and plant growth-promoting hormones in VCT supports its role as an effective biostimulant for flower initiation and development. Arancon et al. [51] reported that humic acids extracted from vermicompost, comparable to commercial humic acids and indole-3-acetic acid (IAA), could enhance flower and fruit production in pepper plants. Another study demonstrated that in addition to the role of phytohormones present in vermicompost leachate, it primarily enhances salt tolerance in tomato plants by modifying endogenous levels of jasmonate and cytokinin in leaves [52]. Previous studies have shown that plant hormones [53] as well as humic acid and micronutrients [54] can effectively improve saffron yield. Therefore, these biostimulats frequently found in VCT, may be responsible for increasing flowering and stigma yield in saffron. Additionally, VCT can induce defensive responses in plants subjected to various abiotic stresses [17]. In the current study, VCT spraying also increased TChl content and reduced EL level in leaves (Fig. 1), which may further support saffron flowering in alkaline soils.

In alkaline conditions, the availability of iron is often reduced, which can hinder the normal growth and flowering of plants [8, 35]. A previous study has demonstrated the critical role of iron supplementation in optimizing saffron flowering and stigma yield [55]. In the present study, NCIF soil drenching enhanced flower and stigma yields (Table 3), showcasing the efficiency of this fertilizer for addressing iron deficiency in saffron grown in an alkaline soil. Iron is an essential micronutrient critical for chloroplast function, chlorophyll synthesis, enzyme activation, and photosynthetic efficiency—all of which contribute to the energy needed for flowering [36]. Additionally, iron plays a direct role in pollen development and fertility [56] and regulates the flowering time [57]. Wang et al. [58] also found that iron deficiency stress in Chinese cabbage reduced flowering by inducing ethylene production. The nanoscale size of particles in NCIF allows for better penetration into plant tissues and increases surface area for interaction with root systems. Additionally, high stability of NCIF under alkaline conditions improves iron adsorption in plants. Consequently, effective iron delivery enhances Chl biosynthesis and photosynthetic process which in turn supports flowering and stigma yield.

In this study, PCA results demonstrated that the combined treatment of NCIF and VCT, rather than their individual applications, enhanced TChl content, supporting flowering and stigma yield (Fig. 7). Similarly, synergistic effects of Fe nanocomplex and nitrophenolate-based biostimulant increased essential nutrient availability and Chl content, and consequently enhanced growth and physiological performance of tomato seedlings [59]. Koocheki and Seyyedi [9] also reported that soil amendment with cattle manure and vermicompost, in combination with foliar applications of Fe-EDTA and Zn-EDTA, enhanced nitrogen and phosphorus uptake and increased saffron corm yield in a calcareous soil.

Fig. 7.

Visualizing the association between various groups of variables and treatments by employing the principal compound analysis (PCA)-biplot graph in saffron cultivated in an alkaline soil. Please see the abbreviation of variables in the caption of Fig. 6. The treatments were included: N0V0, N0V10, N0V20, N4V0, N8V0, N4V10, N4V20, N8V10, and N8V20, where N0, N4, and N8 represent nano-chelated iron fertilizer with concentrations of 0, 4, and 8 kg ha^− 1, and V0, V10, and V20 represent vermicompost tea at concentrations of 0, 10, and 20%

Integrating VCT or NCIF into saffron cultivation practices not only increased flower and stigma yields (Table 3) but also boosted the concentrations of crocin, picrocrocin, safranal, and TFC in saffron stigma (Fig. 2). Likely, humic substances, bioactives, beneficial microorganisms, and plant growth hormones present in VCT could evoke gene expression or the activity of enzymes involved in the biosynthesis pathways of these metabolites. Supporting this idea, VCT treatment significantly upregulated CVOMT and EOMT transcripts in the peltate glandular trichomes and increased the accumulation of chavicol and methyleugenol in the essential oil of basil [60]. It is also known that co-factors such as Fe²⁺, ascorbate, and 2-oxoglutarate are instrumental in facilitating oxidation reactions catalyzed by key enzymes involved in the biosynthesis of diverse flavonoids [61]. Additionally, iron delivery by NCIF probably optimized chlorophyll biosynthesis and photosynthesis, resulting in better availability of primary precursors for the production of these metabolites in saffron stigma. Maleki Farahani et al. [39] also found that the application of a specific NCIF increased chlorophyll b, total chlorophyll, and leaf area, which were closely linked to higher levels of picrocrocin, safranal, and crocin in saffron stigma. In another study, iron chelate (EDDHA-Fe) application increased corm and stigma yields and safranal concentrations [62]. These finding further highlight the critical role of iron in enhancing saffron stigma quality. Importantly, the combined application of VCT and NCIF compared to their individual usage resulted in higher levels of picrocrocin, safranal, crocin, and TFC in saffron stigma (Fig. 2). This underscores the synergistic effect of VCT and NCIF for optimizing saffron quality. Likely, improved iron availability, along with VCT biostimulatory effects, creates a balanced nutritional status, critical hormonal levels, and better photosynthetic efficiency for the biosynthesis of these valuable metabolites. Similarly, synergistic effects of humic acid and micronutrients (Fe, Zn, Mn, Cu) enhanced saffron flower yield, and stigma safranal, crocin, and picrocrocin contents [54]. Further molecular research is necessary to clarify the specific mechanisms by which VCT and NCIF interact to influence gene expression or enzyme activity related to the production of these metabolites. Crocin, picrocrocin, and safranal are crucial in defining the marketing characteristics of saffron, including the color, taste, and aromatic fragrance of the stigmas. These apo-carotenoids have significant value in culinary, medicinal, and industrial applications [2]. For example, studies have shown the antioxidant activity of crocin [63] and its potent anti-depressant properties [64]. Flavonoids are antioxidant compounds that play a crucial role in plant defense mechanisms and human health [65]. In the present study, a positive correlation was recorded between apo-carotenoid and flavonoid contents with DPPH scavenging activity and reducing power in the stigma (Fig. 6). This outcome underscores the significant role of these metabolites in enhancing the antioxidant properties of the stigma. Therefore, the synergistic effects of VCT and NCIF not only improve the quality and market value of saffron stigma but also enhance the health benefits for human consumption.

Soil alkalinity leads to oxidative stress in plants, as evidenced by the highest EL levels in control saffron plants (Fig. 1B). To overcome stressful conditions, plants activate various adaptive mechanisms, including the increased production of phenolic compounds [41, 44–46, 66, 67]. Phenolic compounds due to their critical molecular structure can scavenge free radicals and play a significant role in mitigating oxidative stress in plants [65]. In saffron also a negative correlation between TPC and EL level in leaves confirmed this concept (Fig. 6). The applications of VCT and NCIF significantly increased TPC (Fig. 4) while reducing EL level in leaves (Fig. 1B). Likely, the positive effect of VCT and NCIF on chlorophyll content and photosynthesis supports the supply of precursors for phenol biosynthesis, resulting in the mitigation of membrane oxidative damage in leaves. Similarly, the application of iron oxide nanoparticles significantly reduced oxidative stress induced by alkaline stress in bell peppers via activating antioxidant enzymes and boosting the production of phenolic compounds and flavonoids [68].

PCA and HCA demonstrated that the combined application of VCT and NCIF had a superior effect on TPC in both leaves and petals (Figs. 7 and 8). This outcome highlights their synergistic effect to mitigate oxidative stress in saffron grown in an alkaline soil. In line with our findings, the combined application of iron nanoparticles with humic acid (Fe-NPs-HA) outperformed the effect of traditional iron sources or iron nanoparticles alone in promoting the growth of rose-scented geraniums in alkaline soil conditions. The superior effect of Fe-NPs-HA on photosynthetic pigments and nutrient uptake markedly boosted the levels of phenolic compounds, anthocyanins, and flavonoids, and improved the essential oil composition of this plant [69]. In addition to abiotic stresses, various elicitors, including nanomaterials [70], hormones [66, 71], and beneficial microorganisms [72] can also stimulate secondary metabolism and phenolic accumulation in plants. For instance, VCT treatment [73] and drip irrigation with nano- or chelated iron [74] enhanced phenolic contents in tomato plants under non-stress conditions. Likely, foliar application of VCT boosted TPC in saffron leaves and petals either via its beneficial microorganisms or by delivering humic acids and hormones. Similarly, the application of organic fertilizers not only elevated safranal and picrocrocin levels in stigma but also increased the total flavonoid and phenolic contents in saffron petals under non-stress conditions [6]. Iron can also act as a heavy metal and induce ROS generation, stimulating secondary metabolism in plants. It has been demonstrated that low levels of ROS can act as signaling molecules that promote growth and secondary metabolism, while excessive ROS levels can lead to oxidative stress and cellular damage [46, 75].

Pearson’s correlation coefficients and PCA indicated that higher TPC induced by VCT and NCIF was positively correlated with increased levels of DPPH scavenging ability, reducing power, and iron-chelating activity in leaves and petals (Figs. 6 and 7). Similarly, VCT fertilization increased TFC, TPC, and DPPH scavenging activity in Ocimum basilicum L [76]. Tavallali et al. [77] also found that nano-chelated Fe(III)-aminolevulinic acid, as an innovative Fe source, more effectively than Fe-EDDHA, enhanced phenolic contents and DPPH scavenging activity in Portulaca oleracea L. It is noticeable that saffron petals are rich in anthocyanins, flavonoids, and other phenolic compounds with significant antioxidant properties. These characteristics make them highly valuable for use in the pharmaceutical and cosmetic industries. Additionally, saffron petals can be utilized as natural colorants or preservatives within the food industry, offering a natural alternative to synthetic options [2, 78, 79]. Saffron leaves also contain antioxidant phenolic compounds that can be utilized for medicinal purposes or as natural feed additives in livestock farming [2, 80]. Therefore, the synergistic effect of VCT and NCIF on TPC increases the value of saffron petals and leaves as antioxidant resources for human consumption and various applications in the food and cosmetic industries.

Conclusion

This study provides the first evidence for the synergistic effects of VCT foliar application and NCIF soil drenching on enhancing both the quantity and quality of saffron in alkaline soils. These fertilizers significantly improved floral characteristics, stigma yield, bioactive compounds, and antioxidant activities. The higher concentrations of crocin, picrocrocin, safranal, and flavonoids present in the stigma contribute to better quality and offer potential health benefits for consumers. Additionally, these fertilizers increased phenolics and antioxidant activities in the petals and leaves, opening up new possibilities for their use in various industries. However, the current study was performed only in a single location and one season and did not include microbial analysis of VCT. Future studies should focus on identifying bioactive compounds and microbial populations present in VCT to provide deeper insights into the action mechanism of this bio-stimulant. Further research is essential for optimizing the application rates and timing of these innovative inputs across various soil types, diverse climates, and agricultural systems. Both VCT and NCIF are economic and eco-friendly alternatives to conventional fertilizers, reducing the risk of increasing soil pH and EC as well as chemical runoff. Despite these advantages, their adoption is not without challenges. Scaling up VCT production requires investment in infrastructure and training for farmers. Additionally, finding a solution for the consistency of VCT quality is essential to achieve desired outcomes. Similarly, NCIF may be cost-prohibitive for small-scale farmers in developing regions. Furthermore, the precise application of NCIF requires specialized knowledge to avoid overuse, which may lead to soil or water contamination. The increased application of nano-chelated fertilizers may raise significant environmental issue and human food safety concerns that require careful evaluation. Long-term field trials are essential for assessing the persistence and biodegradability of these inputs. Furthermore, these trials should evaluate their impacts on soil chemistry, microbial communities, nutrient cycling processes, and food web dynamics. Such studies are crucial to ensure that the benefits of nanofertilizers do not compromise environmental sustainability or public health.

Abbreviations

ACC

Aminocyclopropane carboxylic acid

ANOVA

Analysis of variance

DPPH

2,2-diphenyl-1-picrylhydrazyl

EC

Electrical conductivity

EL

Electrolyte leakage

EDTA

Ethylenediamine tetraacetic acid

EDDHSA

Ethylenediamine di(2-hydroxy-5sulfophenylacetic) acid

HCA

Hierarchical clustering analysis

NCIF

Nano-chelated iron fertilizer

PCA

Principal component analysis

QE

Quercetin

ROS

Reactive oxygen species

TChl

Total chlorophyll

TFC

Total flavonoid content

TPC

Total phenolic content

VCT

Vermicompost tea

Authors’ contributions

R.A. supervised and organized the study, while M.A advised it and contributed to VCT preparation. Z.H conducted the experiments. R.A., M.Gh. and Z.H. analyzed the data, and wrote MS. All authors reviewed and approved it for publication.

Funding

The study was conducted without any specific financial support from commercial, public, or non-profit funding organizations. The authors would like to thank the Plant Science Department of Shahrekord University, Iran, for the financial support of this research.

Data availability

The data that support the findings of this study are available from the second author (Z.H) upon reasonable request.

Declarations

Ethics approval and consent to participate

Not applicable. This manuscript does not involve researching about humans or animals.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.