Herbicidal activity of medicinal plants essential oil using nanotechnology for saffron weed control saffron weed control using medicinal plants’ essential oil

Abstract

Weeds present a significant challenge to agricultural systems, particularly in the cultivation of high-value crops like saffron. In this study, allelopathic effects of ten medicinal and aromatic plants were screened on germination and growth of lettuce seedlings using the dishpack and cotton-swab methods. The results indicated that clove, ajwain, perovskia and cinnamon strongly inhibited the hypocotyl and radicle growth of lettuce. Then, essential oils (EOs) from the selected plants were formulated into nanoemulsions (in different HLB) and their properties (particle sizes and PDI) were evaluated. The nanoemulsion formulated herbicidal activity from Trachyspermum ammi (ajwain), Syzygium aromaticum (clove), Cinnamomum cassia (cinnamon), and Perovskia abrotanoides (Perovskia) EOs evaluated against common weeds (Agropyron repens, Bromus japonicus, Chenopodium album, Festuca spp). Nanoemulsion formulations with 1% essential oils and an HLB of 15 showed the smallest particle sizes, lower PDI, and greater stability at 4 °C. The results showed that nanoemulsion spraying induced morphological, physiological, and biochemical changes in the weeds. The application of clove, ajwain, perovskia and cinnamon EOs on Agropyron repens and Bromus sp. increased ion leakage. Cinnamon oil on Agropyron repens reduced chlorophyll index, SOD activity (40.7% ), and protein levels (79.83% ), while ajwain oil decreased catalase enzyme activity (39.56%). On Bromus, ajwain EOs caused the greatest reduction in chlorophyll index, while cinnamon and perovskia EOs reduced catalase activity (82.8% and 52.32%, respectively), and clove EO led to the most significant decrease in SOD activity (24%). Nanoemulsion spraying on Festuca caused increased ion leakage (93%) and reduced catalase enzyme activity with ajwain and cinnamon oils (74.87% and 63.07%, respectively), while clove and perovskia oils decreased SOD activity (45% and 41%, respectively). The application of 1% nanoemulsion solutions of natural essential oils caused visible leaf burn symptoms—such as yellowing, chlorosis, necrosis, and tip burn—in all tested weed species within the first five days, with cinnamon and ajwain oils leading to over 90% desiccation after seven days. The results suggest that essential oils, particularly cinnamon, ajwain and clove, could be effective natural herbicides with potential for use in sustainable weed management.

Introduction

Weeds and crops participate in intricate growth interactions that can impose significant economic burdens on agricultural systems. This relationship has played a vital role in the history of crop domestication, highlighting the need for effective strategies to manage weeds. In modern integrated weed management practices, the use of herbicides has gained widespread acceptance [35]. The last four to five decades have witnessed the introduction of more than 200 chemical compounds, signifying a major evolution in the techniques utilized for weed control. Weeds pose a significant challenge to the productivity of horticultural fields, adversely affecting both the quality and quantity of fresh and processed agricultural products. Consequently, the development of safe and effective weed management strategies is of paramount importance in the cultivation of horticultural crops [42]. The accumulation of toxins and chemical residues in fresh produce, including fruits and vegetables, represents a significant challenge in the cultivation of horticultural crops. The use of prevalent pesticide or herbicides poses a serious threat as they contribute to the pollution of these crops [48].

Integrated Weed Management (IWM) is recognized as an environmentally sustainable approach for managing weeds in agricultural system [35]. Four primary weed management strategies exist: physical methods (crop rotation, cover cropping, interrow hoeing, thermal techniques), chemical methods (bioherbicides), mechanical methods, and biological methods utilizing microorganisms [29]. The International Allelopathy Society (1999) defined allelopathy as the study of secondary metabolite production across organisms including plants, algae, bacteria, and fungi, and their growth impacts in biological and agricultural settings. Allelopathic compounds are chemical substances produced by specific plants, particularly those with medicinal and aromatic properties, which affect ecosystems through interactions with other compounds and microorganisms. Essential oils (EOs) represent a significant category of plant secondary metabolites increasingly applied as biopesticides, including herbicides, insecticides, fungicides, bactericides, and acaricides [29].

The genus Crocus, classified within the Iridaceae family, encompasses approximately 85 flowering species. The autumn-blooming C. sativus is extensively cultivated across Iran, India, Afghanistan, Greece, Morocco, Spain, and Italy, renowned as the world’s most costly spice with numerous health benefits attributed to three primary bioactive compounds: crocin, picrocrocin, and safranal [2]. Picrocrocin and safranal, derived from carotenoid oxidation, contribute to the spice’s characteristic bitter flavor and distinctive aroma, respectively [51].

Natural compounds derived from medicinal plants have the potential to diminish reliance on synthetic herbicides in weed control practices. This shift not only contributes to a reduction in environmental pollution but also supports the development of safer agricultural products. While essential oil herbicidal efficacy has been extensively studied in laboratory settings, field condition investigations remain scarce [50].

Nanotechnology has demonstrated its advantages across various fields, including drug delivery, detection, nanosensors [39], water purification, data management, and the development of nanoscale materials for healthcare and industrial uses. The integration of nanotechnology with natural products represents a rapidly advancing research area. Nanocarriers enable effective drug delivery for precise therapeutic agent transport to designated organ regions, specific tissues, or individual cells. The synergy between natural products and nanotechnology presents promising opportunities, as nanotechnology enables incorporation of multiple natural products. Nano-drug delivery systems significantly enhance bioavailability, targeting efficiency, and controlled release characteristics of natural products, addressing clinical limitations and amplifying pharmacological benefits [27, 28, 33, 34].

A significant challenge associated with the application of essential oils in agricultural practices is their inherent volatility. To address this issue, several strategies have been employed, such as the encapsulation of essential oils and their incorporation into nanoemulsions. Consequently, the secondary objective of the current study was to evaluate the inhibitory effects of essential oils on seed germination in field conditions by utilizing the most effective essential oil in conjunction with superabsorbent.

The utilization of natural compounds for the management of weeds and pests is of significant importance. This study examined the allelopathic properties of various specimens of medicinal and aromatic plants, employing the dish-pack, and cotton swab methods, with lettuce seeds serving as the experimental model. Subsequently, the most potent inhibitors were identified, and their extracts were applied as herbicides during post-germination phases of key weeds found in saffron cultivation.

Materials and methods

This research was conducted in three separate experiments.

Plant materials and target weed species selection

Plant materials

Plant samples were obtained from different sources, including plant science research centers (Agropyron repens, Bromus japonicus, Chenopodium album and Festuca spp.), local herbal shops (Syzygium aromaticum and Cinnamomum cassia) and Mashhad Botanical Garden (Trachyspermum ammi, Perovskia abrotanoides, Teucrium polium, Ocimum citriodorum, Ocimum gratissimum, Ocimum basilicum, Ferula gummosa and Dorema ammoniacum).

Target Weed Species Selection: The selection of target weed species for this study was based on their prevalence and economic impact in saffron cultivation systems. Four key weed species were chosen: Agropyron repens (L.) Beauv. (quackgrass), Chenopodium album L. (common lambsquarters), Bromus tectorum L. (cheatgrass), and Festuca pratensis Schreb. (meadow fescue). These species represent problematic weeds in saffron fields due to their competitive nature and interference with saffron development at critical growth stages.

Agropyron repens is a perennial rhizomatous grass (Poaceae, C3) with a noxious growth habit, identified among dominant perennial weeds in saffron (Crocus sativus L.) fields in Khorasan, Iran. Its vegetative spread and persistence make management challenging in these semi-arid systems where saffron’s weak and sparse canopy structure provides limited competition [22]. Chenopodium album, an annual C3 dicot, is also reported among the dominant weeds in these fields, characterized by its prolific seed production and persistence in the soil seed bank, enabling it to establish across varied environmental conditions[22]. Bromus tectorum, a C3 annual grass, has similarly been identified in saffron-producing areas of Iran, with its early-season growth and high seed production making it a problematic competitor during saffron establishment phases [22]. Festuca pratensis, another perennial C3 grass in the Poaceae family, forms part of the weed flora observed in Iranian saffron fields, contributing to the competitive pressure on saffron crops through its persistent growth and resource acquisition strategies [22]. These species highlight the need for integrated weed management strategies tailored to the unique ecological and agronomic conditions of saffron production systems in Iran.

Phenological Comparison and Interference Analysis:

Understanding the phenological relationship between saffron (Crocus sativus L.) and associated weed species is critical for designing effective weed management strategies in saffron cultivation systems. Saffron is a geophyte with an autumnal growth cycle, sprouting in autumn, flowering shortly thereafter, and entering dormancy during the summer, a pattern well adapted to Mediterranean and semi-arid climates [40]. In Iranian saffron fields, weeds display diverse phenological strategies—from annual species that complete their life cycle rapidly in early spring to persistent perennials—resulting in temporal overlaps with key growth stages of saffron that increase competition for resources [22]. Phenological analyses of both crop and weed species are therefore essential for optimizing the timing of control measures to reduce competitive pressure and support sustainable saffron production.

Based on the actual saffron cultivation calendar, saffron corms are planted from mid-July to late September, followed by fertilization in late September. Shoot emergence occurs when temperatures drop below 17 °C (late September to early October), triggering corm sprouting and initial growth. Flowering occurs from October to late November, which represents the most economically important period for saffron production. Post-harvest vegetative growth continues through winter and spring (December-June) with multiple irrigation and fertilization cycles supporting leaf development and corm maturation for subsequent seasons.

Table 1.

Phenological comparison of selected weeds with Crocus sativus

Species	Life Cycle	Germination Period	Peak Growth	Flowering Period	Senescence	Critical Interference Window
Crocus sativus	Perennial geophyte	Corm sprouting: Late Sept-Early Oct	Dec-June (vegetative growth)	Oct-Late Nov	July-August (dormancy)	-
Agropyron repens	Perennial grass	March-May, August-September	May-July, September-October	June-August	October-November	Pre-flowering establishment, winter-spring competition
Chenopodium album	Annual broadleaf	April-June	June-August	July-September	September-October	Summer dormancy period, early establishment interference
Bromus tectorum	Winter annual grass	September-November	March-May	April-June	June-July	Post-planting establishment, spring growth periods
Festuca pratensis	Perennial cool-season grass	March-May, August-October	April-June, September-November	May-July	December-February	Spring growth and autumn establishment periods

Weed-Saffron interference windows

The phenological analysis revealed that these weeds create competitive pressure during three critical windows in saffron cultivation.

1. Post-planting establishment period (September-October): Following saffron corm planting and initial sprouting, when the developing saffron plants are most vulnerable to competition for space, nutrients, and water resources.

2. Flowering period (late October to late November): The most economically critical phase when energy reserves are mobilized for flower and stigma production. Competition during this period can directly impact saffron yield and quality. Also, the presence of weeds on the farm makes the harvest process more difficult and costly.

3. Winter-spring vegetative growth period (December-June): During this extended period, multiple irrigation and fertilization cycles support leaf development and corm maturation for the next growing season. Weed competition during this phase affects the long-term productivity of saffron cultivation.

Experiment I: allelopathic activity of some medicinal plants using dish-pack method

To maintain the integrity of the volatile compounds, the drying process of the plants was tailored to the specific type of tissue, utilizing an oven set at temperatures ranging from 30 to 60 °C for a duration of one to three days.

Dish pack method

The assessment of allelopathic effects was conducted utilizing the dish pack method, as documented in prior (Fig. 1) [4, 43]. Lettuce seeds, specifically the Great Lakes 366 variety, were selected as the test organism due to their high germination reliability, responsiveness to inhibitory and stimulatory substances, and ease of procurement. The most potent allelopathic plants were chosen for the subsequent phase of the cotton swab experiment.

Fig. 1.

Dish pack method for screening allelopathic activity of volatile compounds of medicinal plants

Measurement of growth inhibition: Shoot and root inhibition percentages were assessed at two distances: near (41 mm apart, using two wells) and far (average of three distant wells). The inhibition percentage was calculated using the formula:

where:

• B = Mean shoot/root length in control wells.

• E = Mean shoot/root length in treatment wells.

Experiment II: allelopathic activity of medicinal plants essential oil using, cotton swab method

Essential oil extraction

In this experiment, the essential oils of ajwain (Trachyspermum ammi), clove (Syzygium aromaticum), cinnamon (Cinnamomum cassia), and Perovskia (Perovskia abrotanoides) were extracted. Essential oil extraction was performed utilizing the hydro distillation technique for four hours, employing a Clevenger-type apparatus with a distillation rate set at 3 ml/min, following the guidelines established by the European Pharmacopoeia [8, 17]. The extracted essential oils were subsequently gathered in dark, sealed, airtight glass vials, treated with anhydrous sodium sulfate to remove moisture, and then stored in a refrigerator maintained at a temperature of 4 °C.

Essential oil analysis by GC-MS: The EOs were analyzed by GC/MS as in the previously published paper. Briefly, the EOs analysis was conducted using a Varian 3400 GC-MS system, which featured a DB-5 fused silica column measuring 30 m in length and 0.25 mm in internal diameter, with a film thickness of 0.25 μm. The oven temperature was programmed to increase from 50 °C to 240 °C at a rate of 4 °C per minute, while the transfer line was maintained at 260 °C. Helium served as the carrier gas, operating at a linear velocity of 31.5 cm/s, with a split ratio set at 1:60. The ionization energy was fixed at 70 eV, with a scan time of 1 s and a mass range spanning from 40 to 300 atomic mass units. The identification of essential oil (EO) components was achieved by comparing their mass spectra against those stored in a computer library or with known authentic compounds, further validated through the comparison of their retention indices with those of authentic substances or published data [10]. Additionally, mass spectra from existing literature were referenced (Adams, 2007). Retention indices for all volatile constituents were calculated using a homologous series of n-alkanes [15]. The relative concentrations of individual components were calculated as percentages of the total peak area using area normalization without internal standard reference.

Cotton swab method. In this approach, the extracted essential oils from chosen medicinal and aromatic plants were subsequently employed to assess their effects on the germination of lettuce seeds, as outlined in our previously published research[32]. Briefly, the 20 mL glass vials underwent disinfection in an oven following thorough washing. A 0.75% (w/v) agar solution, sourced from Merck Inc. in Kenilworth, NJ, USA, was sterilized using an autoclave. Subsequently, seven lettuce seeds (Lactuca sativa, cultivar Great Lakes No. 366) were positioned on the agar surface. A double-tipped cotton swab was bisected and centered on the agar, ensuring that its cotton tip was suspended above the agar. Essential oil was then introduced into the cotton swab in precise volumes of either 1 or 3 µL, utilizing a Hamilton capillary syringe. Following this, the vials were sealed with a rubber cap and aluminum seal, and secured using a crimper. The vials were incubated in a germinator maintained at a temperature of 21 ± 2 °C in the absence of light. Control vials were prepared similarly, containing agar and seeds, with the swabs placed in the same manner; however, these swabs did not receive any essential oil injections, distinguishing them from the treated vials [32].

Measurement of growth Inhibition

Shoot and root growth inhibition percentages were calculated using the following formula [30].

where:

• T = Mean shoot/root length in treatment wells.

• C = Mean shoot/root length in control wells.

Experiment III. Herbicidal activity of selected essential oil as nanoemulsion

Nano-emulsion of essential oil

Selected essential oils with strong allelopathic activity were formulated as nanoemulsions using a combination of two surfactants Tween 80 (hydrophilic, HLB ~ 15) and Span 80 (lipophilic, HLB ~ 4.3) to generate mixtures with a range of hydrophilic-lipophilic balance (HLB) values. Specifically, HLB values of 4.3, 8, 9, 11, 12, and 15 were obtained by adjusting the ratios of these two surfactants. This broad HLB range was chosen to evaluate how the polarity of the surfactant system influences emulsion formation and potential allelopathic performance. For each formulation, 10 g of the surfactant mixture corresponding to a target HLB value were carefully weighed and mixed, followed by the addition of 1 milliliter of the selected essential oil.

Following the thorough vortexing and homogenization of the essential oil within the surfactant (with different HLB), the resultant mixture was adjusted to a total volume of 100 ml. During the incorporation of this mixture into distilled water, the solution was continuously agitated using a magnetic stirrer. Subsequently, sonication was conducted for a Duration of five minutes, employing a cycle of 20 s on followed by a 5-second off, at 60% of the device’s maximum power output (750 W) utilizing a Sonicate apparatus.

Nanoemulsion particle size evaluation: In this study, to evaluate the particle size and particle size distribution index (PDI) of nanoemulsion formulations, measurements were taken on days 1, 40, and 80. The 40-day interval was chosen because nanoemulsions typically maintain stable particle sizes During the initial weeks, with more significant changes occurring over longer periods. This interval also corresponds to key developmental stages of target weeds and crops, making it a relevant timeframe for assessing nanoemulsion stability During biologically active phases. Therefore, measuring every 40 days provided a balance between capturing meaningful changes and practical experimental design. The particle size was determined using a Malvern Zetasizer, model Vasco3, from Cordouan, France.

Herbicidal activity evaluation: Visual toxicity symptoms were evaluated in Agropyron repens, Chenopodium album, Bromus tectorum and Festuca pratensis. The seeds of various weed species were obtained from the research center affiliated with the Agricultural Jihad Organization and natural habitats in Isfahan province. Initially, the weed seeds were planted in seedling trays in the research greenhouses of Ferdowsi University of Mashhad, using a mixture of coir, agricultural soil, and perlite in a 1:2:1 ratio. After one month, the seedlings were transferred to one-kilogram plastic pots and maintained under greenhouse conditions until reaching the 7–8 leaf stage. The nanoemulsion solutions were applied via foliar spraying on all four weed species simultaneously between 9:00 and 11:00 AM, a time when the temperature was not at its peak. Four days after application, morphological and physiological traits were measured based on visible changes in the plants.

Ion leakage (IL) measurement

Ion leakage was measured four days post-spraying. For each replicate, five 1 cm discs were cut from the leaves and placed individually in test tubes. The leaves were washed with deionized water, and 10 mL of deionized water was added to each tube, which was then sealed with aluminum foil. The tubes were incubated for 24 h at room temperature. After mixing the contents with a shaker, the initial electrical conductivity (EC1) was measured using a conductivity meter (model CC-501). The tubes were then autoclaved at 120 °C for 20 min, cooled, and the secondary electrical conductivity (EC2) was measured. Ion leakage percentage (IL) was calculated using the formula [11].

IL= [EC1/EC2] ×100.

Chlorophyll index (SPAD)

The leaf chlorophyll content was measured using a handheld chlorophyll meter (SPAD-502 Plus, Konica Minolta, Japan) four days after post-emergence herbicide application. Measurements were taken on the leaves of Agropyron repens, Bromus japonicus, Chenopodium album and Festuca spp. The SPAD values were recorded, and the average chlorophyll index for each plant species was calculated.

Preparation of enzyme extracts

The extraction buffer was prepared using 50 mM sodium phosphate buffer (pH 7.0), 1 mM EDTA, 0.5% Triton X-100, 50 mM Tris, and 1% (w/v) polyvinylpyrrolidone (PVP). Leaf samples (0.1 g) were ground in liquid nitrogen using a pre-chilled mortar and pestle under cold conditions. The homogenized tissue was mixed with 1 mL of sodium phosphate buffer and centrifuged at 12,000 rpm for 20 min at 4 °C. The supernatant was collected and used for enzyme activity assay [53].

Catalase (CAT) specific activity assay

Catalase activity was measured by adding 50 µL of plant extract to a reaction mixture containing 3 mL of sodium phosphate buffer and 4.51 µL of hydrogen peroxide (H₂O₂). The decrease in absorbance at 240 nm was recorded over 90 s. Enzyme activity was expressed in micromoles of H₂O₂ decomposed per minute per gram of fresh weight using the following equation [7].

Which:

• ΔA: Change in absorbance at 240 nm over 90 s.

• TV: Total volume of the reaction buffer and extract (3 mL).

• EV: Volume of the extract (0.05 mL).

• D: Dilution factor.

• ε: Catalase extinction coefficient (39.4 mM⁻¹ cm⁻¹).

Superoxide dismutase (SOD) activity assay

The reaction mixture was prepared by combining 2 mL of sodium phosphate buffer, 200 µL of nitroblue tetrazolium (NBT), 400 µL of methionine, 200 µL of riboflavin, and 50 µL of plant extract. The samples were then exposed to a 15-watt fluorescent lamp for 15 min. After the light was turned off, the reaction was halted, and absorbance was measured. The reaction mixture without the enzyme extract, which exhibited the highest color intensity, was used as the control, while the reaction mixture without enzyme extract and light exposure was used to calibrate the spectrophotometer [25]. SOD activity was calculated using the following equation.

where U represents one unit of superoxide dismutase activity, defined as the amount of enzyme required to inhibit the photoreduction of NBT by 50%. ODc and ODs refer to the absorbance at 560 nm for the control and sample, respectively.

Protein content determination

The protein content was measured using the Bradford method [5]. The Bradford reagent was prepared by dissolving 0.05 g of Coomassie Brilliant Blue in 25 mL of 95% ethanol and 50 mL of 85% orthophosphoric acid, followed by dilution with distilled water. For protein quantification, 50 µL of the sample was mixed with 3 mL of the Bradford reagent, and absorbance was measured at 595 nm using a spectrophotometer.

Assessment of herbicide damage

Daily observations were conducted to evaluate visual damage in different weed species after applying the post-emergence herbicide. Symptoms such as chlorosis, necrosis, leaf desiccation, and scorch injury, were recorded. Additionally, regrowth and the emergence of new leaves were monitored. For a more detailed assessment, a binocular microscope was used to examine tissue damage at a finer scale.

Data analysis

Statistical analysis was performed using SAS software, while graphs were generated in Excel. Mean comparisons were conducted using the Least Significant Difference (LSD) test at a 5% significance level.

Results and discussion

Experiment I: Dish-pack method

Hypocotyl growth inhibition percentage

The allelopathic effects of different plants on lettuce seed hypocotyl growth showed that Syzygium aromaticum (clove) exhibited the highest inhibition at a close distance (41 mm), with 55.7% reduction. Ocimum citriodorum (basil) inhibited growth by 48.2% at a far distance and 47.8% at a close distance, while Ocimum gratissimum caused 46.2% and 45.7% inhibition at close and far distances, respectively. The inhibitory effects of Ocimum citriodorum and Ocimum gratissimum were not significantly different between two different distances, suggesting the presence of highly volatile compounds. However, Syzygium aromaticum showed significantly higher inhibition at a close distance, likely due to the concentration of active volatile compounds or the presence of less volatile compounds effective at short range. Teucrium polium, Ocimum citriodorum leaves, Ocimum gratissimum leaves, Ocimum basilicum flowers, and Cinnamomum cassia wood showed similar inhibitory effects on lettuce hypocotyl growth, with no significant differences among them, indicating potential antagonistic effects of their compounds. The lowest inhibitory effects on lettuce hypocotyl length in close distances were observed in Ferula gummosa (16.4%), Dorema ammoniacum (14.8%), and Proveskia abrotanoides (12.4%). However, the inhibitory effects of Ferula gummosa (20.4%) and Dorema ammoniacum (17%) were higher at greater distances, possibly due to antagonistic interactions among their compounds. Trachyspermum ammi was the only plant exhibiting a growth-promoting effect, significantly enhancing hypocotyl length in both close (9.6%) and distant (8.9%) treatments., showing a significant positive effect compared to other plants (Fig. 2).

Fig. 2.

Allelopathic effects of the plants used on lettuce hypocotyl length by the Dish pack method. “h” indicates the close well to the plant sample and “H indicates the distant well to the plant sample. L suggests the leaf organ and F denotes the flower organ

Radicle growth inhibition percentage

The strongest inhibitory effect on lettuce radicle length at close distances was observed in Syzygium aromaticum (67.5%), while it had a slight stimulatory effect (1.5%) at greater distances, likely due to low-volatility compounds. Ocimum gratissimum flowers inhibited radicle growth at both close (29%) and distant (35.6%) distances. Proveskia abrotanoides (22.7%), Ocimum citriodorum leaves (22.3%), and Ocimum basilicum leaves (21%) exhibited similar inhibitory effects at greater distances, with no significant differences. The weakest inhibition was observed in Ocimum citriodorum flowers (17.7%) and Ocimum gratissimum leaves (13%) at distant and close distances, respectively. Several plants exhibited radicle growth stimulation. The strongest effects at greater distances were recorded for Dorema ammoniacum (44.4%) and Trachyspermum ammi (40%), while at close distances, Trachyspermum ammi (34.7%) and Dorema ammoniacum (33.7%) had notable stimulatory effects. Cinnamomum cassia wood (37%) and Dorema ammoniacum (34.6%) also promoted radicle growth at close distances. Dorema ammoniacum maintained its stimulatory effect at greater distances (33.7%) without significant differences. Ocimum basilicum flowers promoted radicle growth (25%) at close distances (Fig. 3).

Fig. 3.

Allelopathic effects of the plants used on lettuce radicle by the Dish pack method. “h” indicates the close wells to the plant sample and “H indicates the distant well to the plant sample. L indicates the leaf organ and F indicates the flower sample

Experiment II: Cotton swab method

Inhibitory effects on hypocotyl growth

The essential oil treatments significantly affected lettuce hypocotyl growth. The average hypocotyl length in the control group was 18.21 mm, while after treatment, it reduced to 4.38 mm. On average, all treatments showed more than 75% inhibition of hypocotyl growth. Trachyspermum ammi oil caused complete inhibition, with no hypocotyls observed in germinated seeds. The oil of Ocimum gratissimum exhibited the highest inhibition (83.3% reduction), while Perovskia abrotanoides oil had the least effect, with a 73% reduction (Fig. 4).

Fig. 4.

Hypocotyle relative growth rate of lettuce in the Cotton Swab Method using different medicinal plant essential oils

Inhibitory effects on radicle growth

The essential oil treatments significantly inhibited radicle growth, with inhibition ranging from 77 to 92%. The average radicle length in the control group was 14.09 mm, which decreased to 2.41 mm after treatment. Trachyspermum ammi oil caused complete inhibition, with initial radicle growth followed by burn and stunted growth. The highest inhibition (92%) was observed with Ocimum gratissimum oil, followed by Syzygium aromaticum oil (87.7%). Oils of Ocimum basilicum, Ocimum citriodorum, and Perovskia abrotanoides caused similar inhibition, averaging 77.6% (Fig. 5).

Fig. 5.

Radicle relative growth rate of lettuce in the Cotton Swab Method

Gas chromatography-mass spectrometry (GC-MS)

The analysis of essential oils (EO) conducted through Gas Chromatography-Mass Spectrometry (GC-MS) revealed that the primary constituents of the essential oil derived from Provskia aboranoides included camphor at a concentration of 22.8%, eucalyptol at 18.7%, and alpha-pinene at 13.7%. In the case of basil (Ocimum basilicum), the predominant compounds were eucalyptol at 7.7% and Estragole at a significant 82.5%. For another basil variety, Ocimum gratissimum, the major components were identified as methyl cinnamate at 27.7% and linalool at 17.49%. Meanwhile, Ocimum citriodorum exhibited a different profile, with citral at 33.3%, neral at 24%, and geranial at 15.9% being the most abundant compounds. Cinnamaldehyde was found to be the dominant compound in cinnamon (Cinnamomum), comprising 86.93% of its essential oil. Lastly, the essential oil of the ajwain plant (Trachyspermum ammi) was characterized by thymol at 61.3%, gamma-terpinene at 20.2%, and o-Cymene at 15.6% (Table 2).

Table 2.

Dominant essential oil constituents in selected medicinal plants of the research

No	Plant Species	Main EO constituents	Retention time ((RT)
1
	Ocimum basilicum		19.31 12.97
2	Ocimum gratissimum		12.63 25.48
3	Ocimum citriodorum		21.67 20.64 20.08
4	Cinamommum		21.219
5 6	Provskia aboranoides Trachyspermum ammi	o-Cymene(15.6%)	16.9 12.23 13.7 29.44 17.7 15.96

Experiment III: Herbicidal activity of selected essential oil

Particle size analysis

The dimensions of nano-emulsions vary according to different hydrophilic-lipophilic balance (HLB) values. The nanoemulsion formulated using T. ammi essential oil at a concentration of 1% (V/V) demonstrated that an increase in the hydrophilic-lipophilic balance (HLB) resulted in a reduction in the size of the nanoemulsions. For the 1% essential oil nanoemulsions, the smallest particle sizes were observed at an HLB value of 15: 50 nm for Trachyspermum ammi (ajwain) (Fig. 6a), 12.84 nm for Syzygium aromaticum (clove) (Fig. 6b), and 95.96 nm for Perovskia abrotanoides (brazamble) (Fig. 6d). While, For the 1% Cinnamomum cassia (cinnamon) essential oil nanoemulsions, the solution with an HLB of 12 showed the smallest particle size of 77.78 nm (Fig. 6c). Other HLB values resulted in larger particle sizes, with the largest observed at HLB 4.3, where particle sizes exceeded 450 nm for ajwain (Fig. 6a) and brazamble (Fig. 6d), 250 nm for clove (Fig. 6b). While, cinnamon essential oil nanoemulsions, the solution with the HLB 15 solution showed particles greater than 188.9 nm (Fig. 6c). These results indicate that higher HLB values lead to smaller particle sizes in the nanoemulsions in all essential oil nanoemulsions except cinnamon (Fig. 6).

Fig. 6.

Particle Size (Z-Average) of Nanoemulsion Solutions (in Nanometers) Based on HLB Variation (4.3, 8, 9, 11, 12, and 15) for 1% Essential Oil Nanoemulsions of Different Plants (A: Trachyspermum ammi, B: Syzygium aromaticum, C: Cinnamomum cassia, D: Perovskia abrotanoides)

Polydispersity index (PDI)

In the 1% T. ammi essential oil nanoemulsions, the solution with an HLB value of 15 exhibited the lowest polydispersity index (PDI) of 0.15, indicating a more uniform particle size distribution. Conversely, the solution with an HLB of 4.3 had the highest PDI of 0.57, suggesting a broader distribution of particle sizes (Fig. 7a). For the 1% S. aromaticum essential oil nanoemulsion, the formulation with an HLB of 12 showed the lowest PDI (0.227), with a closely similar PDI of 0.245 observed in the solution with an HLB of 15. The remaining formulations had higher PDIs, indicating less uniformity in particle size distribution (Fig. 7b). In the 1% C. verum essential oil nanoemulsions, the solution with an HLB of 4.3 exhibited the lowest PDI (0.238), while the formulation with an HLB of 8 showed a slightly higher PDI of 0.268. Other solutions displayed even higher PDIs, particularly the one with HLB 11, where the PDI ranged from 0.50 to 0.60, reflecting a less consistent particle size distribution (Fig. 7c). For the 1% Provskia aboranoides essential oil nanoemulsion, the solution with an HLB of 4.3 had the lowest PDI (0.197), followed by the formulation with an HLB of 12 (0.232). Other formulations, especially at HLB 11, showed higher PDIs, with the solution at HLB 11 having a PDI of 0.30 (Fig. 7d). These results highlight that nanoemulsions with higher HLB values tend to exhibit lower PDIs, indicating more uniform particle size distributions. In contrast, lower HLB values result in higher PDIs, reflecting greater variability in particle sizes.

Fig. 7.

The variation in the polydispersity index (PDI) of nanoemulsion particles based on changes in HLB values (4.3, 8, 9, 11, 12, and 15) for 1% essential oil nanoemulsion solutions.of different plants (a: Trachyspermum ammi, b: Syzygium aromaticum, c: Cinnamomum cassia, d: Perovskia abrotanoides)

Stability evaluation of nanoemulsion particles over time (40 and 80 Days) based on Z-average

Over time, the particle size of 1% ajwain essential oil nanoemulsions increased across different HLB values, likely Due to nanoparticle aggregation. This growth led to sedimentation and reduced stability. Among the tested formulations, the nanoemulsion with HLB 15 exhibited the smallest particle size and the highest stability, maintaining a particle size of 167.1 nm on day 80, which was smaller than the initial size of the HLB 4.3 formulation (475.3 nm).

Similarly, the particle size of 1% clove essential oil nanoemulsions increased over time. The formulation with HLB 15 exhibited greater stability, with particles growing from 12.82 nm (day 1) to 147.89 nm (day 80), whereas the HLB 4.3 formulation showed a substantial increase from 252.3 nm to 754.83 nm over the same period.

For 1% cinnamon essential oil nanoemulsions, a gradual increase in particle size was observed, with the HLB 15 formulation reaching 272.9 nm by day 80. Although the HLB 12 formulation initially had smaller particles, it exhibited a greater size increase over time, demonstrating lower long-term stability. The clear appearance of the HLB 15 nanoemulsion further confirmed its superior stability. Interestingly, in 1% Perovskia abrotanoides essential oil nanoemulsions, particle size initially increased by day 40 but decreased by day 80. The HLB 15 formulation consistently exhibited the smallest particle size throughout the study, reaching 51.29 nm by day 80, indicating superior stability compared to other HLB values (Fig. 8).

Fig. 8.

Particle Stability (Based on Size) in Nanoemulsion Solutions with Different HLBs Over 40 and 80 Days After Preparation in Essential Oil Nanoemulsion Solutions.of different plants (a: Trachyspermum ammi, b: Syzygium aromaticum, c: Cinnamomum cassia, d: Perovskia abrotanoides)

Greenness index (SPAD)

Foliar application of the 1% cinnamon essential oil nanoemulsion significantly reduced the Greenness Index of Agropyron repens by 37.19% compared to the control, highlighting its strong inhibitory effect on chlorophyll content. However, no significant differences were observed among the tested essential oil treatments. In Bromus japonicus, all four essential oils significantly reduced Greenness Index values relative to the control, though their effects were statistically similar. Similarly, in Chenopodium album, nanoemulsions of Perovskia abrotanoides, Syzygium aromaticum, and Trachyspermum ammi resulted in a significant decline in Greenness Index compared to the control, but their inhibitory effects did not significantly differ from each other (Fig. 9).

Fig. 9.

Effects of Essential Oils Nanoemulsion on Greenness Index (SPAD) in Different Weed Species. Columns with shared letters indicate no significant difference at the 5% probability level based on the LSD test. The letters represent: (A = Agropyron repens) (B = Bromus japonicus) (CH = Chenopodium album) (C = Cinnamon essential oil) (D= Clove essential oil) (P = Perovskia abrotanoides essential oil) (T = Trachyspermum ammi essential oil)

These findings suggest that essential oil nanoemulsions can effectively suppress chlorophyll content in weeds, potentially impairing their photosynthetic capacity. The extent of inhibition, however, appears to be species-dependent, with A. repens exhibiting the highest sensitivity to cinnamon nanoemulsion. The overall reduction in Greenness Index across multiple weed species indicates that these nanoemulsions could serve as promising bioherbicidal agents, although further studies are needed to elucidate their long-term effectiveness and selectivity.

Ion leakage percentage

Applying the nanoemulsion solutions containing 1% essential oil significantly increased ion leakage across all tested species. In Bromus japonicus and Agropyron repens, all four essential oils significantly increased ion leakage compared to the control, although no significant differences were observed among the treatments. In Festuca spp., cinnamon and T. ammi essential oils caused a significant increase in ion leakage compared to the control, but their effects were statistically similar to those of the other treatments (Fig. 9). These results suggest that the application of essential oil-based nanoemulsions compromises membrane integrity in target weed species, potentially contributing to their inhibitory effects.

Catalase activity

The foliar application of 1% nanoemulsion solutions containing cinnamon and T. ammi essential oils on Festuca spp. significantly reduced catalase activity by 74.87% and 63.07%, respectively, leading to weed suppression. Similarly, in Bromus japonicus, cinnamon and P. abrotanoides essential oils caused a substantial decline in catalase activity by 82.8% and 52.32%, respectively. In A. repens, T. ammi essential oil reduced catalase activity by 39.56%, significantly weakening the weed (Fig. 10).

Fig. 10.

Effects of Essential Oil Nanoemulsion on Ion Leakage in Different Weed Species. Columns with shared letters do not show significant differences at the 5% probability level based on the LSD test. The abbreviations used represent: (A = Agropyron repens), (B =Bromus japonicus), (CH = Chenopodium album), (F = Festuca spp.), (C = Cinnamon essential oil), (D = Syzygium aromaticum essential oil), (P = Perovskia abrotanoides essential oil), (T = Trachyspermum ammi essential oil)

Superoxide dismutase (SOD) activity

Foliar application of 1% nanoemulsion of cinnamon essential oil significantly reduced SOD activity by 40.7% in (A) repens, leading to weed suppression. Similarly, a 1% clove oil nanoemulsion reduced SOD activity by 24% in (B) japonicus, effectively controlling the weed. In Festuca spp., 1% nanoemulsions of clove and P. abrotanoides essential oils significantly reduced SOD activity by 45% and 41%, respectively (Fig. 11).

Fig. 11.

Effects of Nanoemulsion Essential Oil Inhibitory Intensity on Catalase Activity in Different Weed Species. Columns with shared letters do not show significant differences at the 5% probability level based on the LSD test. The abbreviations used represent: (A = Agropyron repens), (B = Bromus japonicus), (CH = Chenopodium album), (F = Festuca spp.), (C = Cinnamon essential oil), (D = Syzygium aromaticum essential oil), (P = Perovskia abrotanoides essential oil), (T = Trachyspermum ammi essential oil)

Protein content

Foliar application of 1% cinnamon and clove essential oil nanoemulsions resulted in a substantial reduction in protein content in Agropyron repens weed by 79.83% and 87.7%, respectively, ultimately leading to its suppression. Likewise, treatment with a 1% clove essential oil nanoemulsion significantly decreased protein content in Chenopodium weed by 53.7%, effectively inhibiting its growth. No significant variations were observed among the other treatments (Fig. 12).

Fig. 12.

Effect of Nanoemulsion Essential Oils on SOD Activity in Different Weed Species. Columns sharing the same letters are not significantly different at the 5% probability level based on the LSD test. The abbreviations represent: (A = Agropyron repens), (B = Bromus japonicus), (CH = Chenopodium album), (F= Festuca spp.), (C = Cinnamon essential oil), (D = Syzygium aromaticum essential oil), (P = Perovskia abrotanoides essential oil), (T = Trachyspermum ammi essential oil)

Qualitative observation of leaf burn intensity

The results showed that the application of 1% nanoemulsion solutions from all essential oils caused yellowing, chlorosis, necrosis, and tip burn on the leaves of Bromus within the first five days. However, the application of cinnamon and clove essential oils led to more than 90% desiccation of Bromus weeds after seven days (Fig. 13).

Fig. 13.

Effect of Essential Oils Nanoemulsion on Protein Content in Different Weed Species. Columns sharing the same letters are not significantly different at the 5% probability level based on the LSD test. The abbreviations represent: (A = Agropyron repens), (B = Bromus japonicus), (CH = Chenopodium album), (F = Festuca spp.), (C = Cinnamon essential oil), (D = Syzygium aromaticum essential oil), (P = Perovskia abrotanoides essential oil), (T = Trachyspermum ammi essential oil)

The utilization of a 1% nanoemulsion solution across all essential oils caused noticeable yellowing, chlorosis, necrosis, tip burn, and green burn on the leaves of the Agropyron repens plant within the initial five days. In contrast, the application of cinnamon and ajwain essential oils resulted in over 90% desiccation of this weed after seven days (Fig. 14).

Fig. 14.

Bromus weeds after treatment with essential oil nanoemulsion solutions.of different plants (a: Trachyspermum ammi, b: Syzygium aromaticum, c: Cinnamomum cassia, d: Perovskia abrotanoides), Compared to Distilled Water and Control Samples

The application of 1% nanoemulsion solution in all essential oils resulted in yellowing, chlorosis, necrosis, and tip burning of the leaves in Festuca plant within the first five days. However, the use of cinnamon and ajwain essential oils led to more than 90% desiccation of the Festuca weed after 7 days (Fig. 15). These findings indicate that while the nanoemulsion solution adversely affects the foliage of the Festuca plant, certain essential oils, particularly cinnamon and ajwain, can effectively desiccate the weed, demonstrating their potential as effective herbicidal agents.

Fig. 15.

Agropyron repens after treatment with Solutions of essential oil nanoemulsion solutions.of different plants (A: Trachyspermum ammi, B: Syzygium aromaticum, C: Cinnamomum cassia, D: Perovskia abrotanoides), Compared to Distilled Water and Control Samples

Similarly, the application of the 1% nanoemulsion solution across all essential oils produced yellowing, green burn, tip burn, along with slight chlorosis and necrosis in the leaves of Chenopodium album within the first five days. This indicates a consistent pattern of phytotoxicity associated with the treatment. Once again, the use of cinnamon and ajwain essential oils proved effective, leading to more than 90% desiccation of the Chenopodium album weed after a period of seven days. These findings highlight the significant impact of specific essential oils on the health and viability of these plant species (Fig. 16).

Fig. 16.

Festuca weed after treatment with Solutions of essential oil nanoemulsion solutions.of different plants (A: Trachyspermum ammi, B: Syzygium aromaticum, C: Cinnamomum cassia, D: Perovskia abrotanoides), Compared to Distilled Water and Control Samples

Fig. 17.

Chenbopidium album weed after treatment with essential oil nanoemulsion of different plants (A: Trachyspermum ammi, B: Syzygium aromaticum, C: Cinnamomum cassia, D: Perovskia abrotanoides), Compared to distilled water and control samples

Discussion

Allelopathic effects of essential oils on weed growth

The ability of medicinal and aromatic plants to transfer volatile chemical compounds and impact surrounding organisms is increasingly recognized. The interactions between plants must be considered in modern agricultural practices, especially as global efforts aim to reduce the use of harmful chemicals by introducing biological and ecological alternatives [29]. One such alternative is harnessing the chemical interactions between plants. In an ecosystem, plant-to-plant interactions can result in side effects that may influence neighboring plants through competition and allelopathy. Competition involves the active acquisition of limited resources by one organism, leading to the depletion of those resources and inhibiting the growth of other organisms [35]. Allelopathy, on the other hand, occurs when the growth of one plant is hindered by chemicals released by another species. Researchers have demonstrated the effectiveness of allelopathic properties in certain plant species for controlling the growth of other plants, including weeds. For example, essential oils from Trachyspermum ammi, Thymus vulgaris, and Pelargonium have shown significant allelopathic effects on seed dormancy and seedling growth, especially in weeds such as lettuce (Lactuca sativa) [32]. [32]. Also, the aqueous extract of saffron corm (SC) remnants significantly affected the germination percentage, mean germination time, and hypocotyl-to-radicle length ratio in lettuce seedlings [26].

The findings of various studies emphasize the potential of essential oils to manage weed growth in agricultural systems. A study evaluated the inhibitory effects of 112 essential oils on lettuce seed germination, revealing that Ajwain (Trachyspermum ammi), oregano (Origanum vulgare), and geranium (Pelargonium graveolens) oils had the strongest effects on seed dormancy. Similarly, in a 2020 study on lettuce seeds, essential oils at concentrations as low as 1 µL/mL showed significant effects on germination and seedling growth in the vapor phase Moreover, Perovskia abrotanoides and Trachyspermum copticum have demonstrated strong inhibitory effects on the growth of lettuce roots and shoots, with a 90% inhibition rate observed in some tests [4]. Furthermore, lavender (Lavandula angustifolia) essential oil, at a concentration of 25 µL/mL, exhibited complete inhibition of root growth in lettuce seedlings, confirming the potency of certain essential oils in regulating plant growth [19]. Other studies, such as those by (Jaime et al., [21], also highlighted the significant inhibitory effects of eucalyptus oil (Eucalyptus globulus) on various weed species, particularly Portulaca oleracea, Echinochloa crus-galli, and Lolium multiflorum. The primary compound in eucalyptus oil, 1,8-cineole, played a crucial role in these effects, reinforcing the utility of volatile oils in weed control. Additionally, rosemary (Rosmarinus officinalis) essential oil, rich in monoterpenes such as α-pinene and camphor, has proven effective in suppressing weed seedling growth and could contribute to the development of biocidal essential oil-based herbicides [16]. Other previous studies demonstrated that essential oil components such as linalool, carvacrol, thymol, and eugenol exhibit significant phytotoxic effects, including reduced germination and seedling growth in both weeds and crops, thereby supporting their potential use as natural herbicides [49].

The results from these studies align with present research, further affirming the potential of plant essential oils in agricultural management. Variations in the allelopathic effects observed across studies can be attributed to differences in plant material used for oil extraction, harvest season, and chemotypes present in different plant populations. These factors may influence the chemical composition of the oils and consequently their efficacy in inhibiting plant growth. In conclusion, essential oils with allelopathic properties hold great promise for integrated weed management, offering a sustainable alternative to chemical herbicides. Continued research into the chemical composition and optimal application methods of these oils can enhance their effectiveness and contribute to the development of eco-friendly agricultural practices.

Nanoemulsion stability and droplet size control

The results of this study demonstrated that nanoemulsion formulations with an HLB value of 15 produced the clearest solutions across all tested essential oils, with droplet sizes consistently below 100 nm, confirming their nanoscale characteristics. The stability of these nanoemulsions aligns with previous findings, as smaller droplet sizes enhance physical stability and prevent phase separation over time. It has been established that Ostwald ripening is a key factor influencing droplet growth, particularly at higher temperatures [44]. The slower growth rate at lower temperatures is attributed to the Lifshitz-Slezov-Wagner (LSW) theory, emphasizing the role of absolute temperature in nanoemulsion stability. Furthermore, the surfactant-to-oil ratio plays a crucial role in droplet size reduction, as reported by [36], while optimal surfactant concentration is essential to balance hydrophilic-lipophilic properties [54].

The effectiveness of nanoemulsion-based herbicidal formulations largely depends on droplet size and physical stability, which directly influence the bioavailability and absorption of essential oils into weed tissues. Smaller droplet sizes (< 100 nm) ensure a higher surface-area-to-volume ratio, facilitating deeper penetration through the cuticular layer and enhancing phytotoxic effects on target weeds [46]. Studies have shown that nanoemulsions loaded with essential oils such as Cymbopogon nardus [46] or Satureja hortensis [20] demonstrate strong inhibitory effects on seed germination and weed biomass, particularly when formulated with optimized surfactant ratios and droplet size control.

Consistent with previous research, our findings indicate that formulations with an appropriate HLB value not only enhance transparency but also maintain stable particle distribution [6]. The strong electrostatic repulsion between charged droplets, influenced by zeta potential, further contributes to stability by preventing particle aggregation [52]. Additionally, mixed surfactant systems have been shown to optimize HLB balance and improve stability through synergistic interactions [14].

From an agricultural perspective, maintaining a stable nanoemulsion ensures efficient foliar application, as increased surface contact and prolonged wetting time enhance absorption [6, 36]. Also, the application of nano techniques has been shown to significantly enhance the stability and controlled release of plant essential oils, thereby preserving their bioactivity and improving their functional efficacy in various biological systems [37]. However, excessive surfactant concentrations may pose phytotoxicity risks, highlighting the need for precise formulation strategies. Overall, selecting an appropriate surfactant composition and HLB value is critical to achieving a stable, uniform nanoemulsion with enhanced bioavailability and efficacy for agricultural applications.

Herbicidal properties of plant-derived essential oils

The interaction between weeds and crops leads to significant economic costs in agricultural systems. Herbicides play a crucial role in weed management but pose environmental and public health risks, including pollution, food insecurity, and human health hazards. Moreover, herbicide-resistant weed species continue to emerge, with 36 new cases reported in 2017 [32]. Natural herbicides, derived from biological sources, are considered environmentally friendly alternatives, though their short half-life and potential toxicity to non-target organisms necessitate careful evaluation. Their mechanism of action resembles plant-pathogen interactions and allelopathy, disrupting weed defense systems and inducing enzyme production that degrades cell walls, ultimately leading to plant death [9].

Essential oils are lipophilic natural compounds derived from plants, containing secondary metabolites such as monoterpenic alcohols or oxygenated monoterpenes. They have been identified as potential natural herbicides, with their herbicidal activity attributed to allelopathic compounds. These natural substances represent a promising alternative for biological weed control. The herbicidal effects of essential oils primarily involve oxidative damage, ion leakage, reduced cellular respiration, and disruption of the waxy cuticle. Plant-derived herbicides are effective against a broad spectrum of weeds and serve as natural alternatives to synthetic selective herbicides [46].

The findings from our study align with existing literature on the chemical composition and biological activities of essential oils (EOs) from various plant species. For instance, the EO of Deverra tortuosa is rich in monoterpene hydrocarbons, with α-pinene (24.47–28.56%) and sabinene (16.2–18.6%) being predominant components [24]. Similarly, our analysis of Perovskia abrotanoides EO identified camphor (22.8%), eucalyptol (18.7%), and α-pinene (13.7%) as major constituents, indicating a comparable monoterpene profile. The antioxidant properties observed in D. tortuosa EO, demonstrated significant free radical scavenging activity. This activity is attributed to its high monoterpene content, particularly α-pinene and sabinene [24]. Our results with P. abrotanoides EO, which also contains substantial amounts of α-pinene, suggest a similar mechanism underlying its antioxidant potential.

The findings of the current study confirm the herbicidal potential of plant-derived essential oils, in line with previous reports that D. tortuosa EO exhibited significant inhibition of germination and seedling growth in various weed species [24]. In the present study, the results demonstrate the significant impact of essential oils (EOs) from various plant species, including cinnamon (Cinnamomum cassia), ajwain (Trachyspermum ammi), and others, on weed suppression through nanoemulsion formulations. These findings highlight that these EOs, particularly cinnamon and ajwain, caused significant damage to weed species such as Bromus japonicus, Agropyron repens, Festuca spp., and Chenopodium album. This damage was indicated by increased ion leakage, decreased catalase and superoxide dismutase (SOD) activity, reduced protein content, and severe leaf desiccation. The increase in ion leakage following the application of EO nanoemulsions is consistent with previous studies that found similar disruptions in plant cell membranes, suggesting that these oils compromise membrane integrity. According to the results of the present study, essential oil emulsions (EOEs) of aromatic plants such as Mentha piperita, Pelargonium graveolens, Matricaria chamomilla, Chrysopogon zizanioides, Pogostemon patchouli, Mentha arvensis, and aqueous extracts of Andrographis paniculata showed that the application of essential oils led to an increase in electrolyte leakage in treated weeds, with significant increases observed compared to the control group [31]. Similar studies have reported that essential oils, such as cinnamon oil, also cause a significant rise in electrolyte leakage in plants. Specifically, cinnamon oil treatments resulted in higher electrolyte leakage in T. incarnatum and L. perenne, aligning with the findings of the current research, which highlights the detrimental effects of essential oils on membrane integrity. The observed increase in electrolyte leakage in response to the application of essential oils in the present study can be explained by the uncontrolled production and accumulation of reactive oxygen species (ROS), as noted in previous research [41].

The significant reduction in antioxidant enzyme activities (catalase and superoxide dismutase) following nanoemulsion application can be attributed to the disruption of cellular homeostasis and the overwhelming of plant defense mechanisms. Under normal conditions, antioxidant enzymes function as the primary defense system against oxidative stress by scavenging reactive oxygen species (ROS) [45]. However, the essential oil nanoemulsions induced excessive ROS production that exceeded the cellular antioxidant capacity, leading to enzyme system collapse. Enhanced level of ROS causes oxidative damage to lipid, protein, and DNA leading to altered intrinsic membrane properties like fluidity, ion transport, loss of enzyme activity, protein crosslinking, inhibition of protein synthesis, DNA damage, ultimately resulting in cell death [45]. The observed increase in electrolyte leakage and membrane permeability indicates that the nanoemulsions compromised cellular integrity, disrupting the subcellular compartmentalization necessary for proper enzyme function. The balance between production and elimination of ROS at the intracellular level must be tightly regulated and/or efficiently metabolized. This is necessary to avoid potential damage caused by ROS to cellular components as well as to maintain growth, metabolism, development, and overall productivity of plants. When cellular membranes are damaged, the spatial organization of antioxidant enzymes is disrupted, and their cofactors and substrates become unavailable, resulting in decreased enzymatic activity [18]. Furthermore, the severe oxidative stress, as evidenced by elevated proline accumulation, triggers a cellular emergency response that diverts metabolic resources away from enzyme synthesis and maintenance toward immediate survival mechanisms. The modulation and enhancement of the expression of genes that encode ROS detoxifying enzymes are commonly used to increase the tolerance against abiotic stresses, but under extreme conditions, this system becomes overwhelmed. This metabolic shift, combined with the direct oxidative damage to enzyme proteins themselves, explains why catalase and SOD activities declined progressively until complete cellular death occurred. The irreversible nature of this process demonstrates that once the antioxidant defense system is overwhelmed, the cascade of oxidative damage becomes self-perpetuating, ultimately leading to the observed herbicidal effects.

ROS accumulation can lead to oxidative stress [38], causing lipid peroxidation and damage to membrane structures, similar to the effects observed by [47], who reported that nano-encapsulated Satureja hortensis essential oil induced membrane leakage in Amaranthus retroflexus. In conclusion, the findings from this study, in combination with previous research, suggest that EO-based nanoemulsions can effectively disrupt plant cell membranes, destroy cellular components, and impair plant growth. This mechanism may underlie the observed phytotoxicity in the present study, supporting the potential use of EO nanoemulsions in sustainable weed management practices.

The root exudates of Chenopodium murale enhanced antioxidant enzyme activity in both Arabidopsis thaliana and Triticum aestivum, particularly catalase and peroxidase activities [13]. In a study on the herbicidal effects of essential oils from Nepeta species, the oils reduced germination, root, and shoot growth of wild mustard and wild oat. The oils also caused chlorophyll reduction and electrolyte leakage, indicating cell membrane damage, with N. menthoides showing stronger herbicidal activity [12]. Similar effects were observed with other essential oils, including Mentha piperita [23], Calotropis procera [3], and Persicaria lapathifolia [1], which inhibited germination and reduced growth in weeds. Our study aligns with these findings, confirming that plant-derived essential oils exhibit significant herbicidal properties by affecting seed germination, growth, and oxidative stress in target weeds, supporting their potential use as biological herbicides.

The present study demonstrated that foliar application of essential oils from various plants on the weed led to visible damage within seven days, including chlorosis, electrolyte leakage, and signs of cellular membrane disruption. Additionally, an increase in proline levels indicated oxidative stress induced by the oils. The results highlighted that the essential oils exhibited herbicidal potential and could serve as a viable biological herbicide for selective control of these weeds in crops. The observed damage ranged from chlorosis to complete necrosis, with the severity increasing over time. Notably, after the first application, weeds showed no recovery, and the damage was irreversible after seven days, leading to 100% damage [12]. These findings align with the results of the current study, supporting the potential use of plant-derived essential oils for effective weed control.

Study limitations

A recognized limitation of the present study is the absence of phytotoxicity assessment on Crocus sativus. However, the weed species selected for evaluation, including Agropyron repens, Bromus japonicus, Chenopodium album, and Festuca spp., are among the most widespread and competitive species commonly associated with saffron cultivation systems, particularly in arid and semi-arid regions of Iran. The observed herbicidal efficacy of essential oil nanoemulsions, particularly those derived from Cinnamomum cassia and Trachyspermum ammi, suggests their potential utility in the biological management of these problematic weeds. The findings of this study provide an initial scientific basis for the application of such formulations in saffron agroecosystems. To ensure the selectivity and safety of these nanoemulsions for saffron, future investigations should focus on comprehensive phytotoxicity evaluations involving germination performance, seedling development, and key physiological indicators of Crocus sativus under both controlled and field conditions. These assessments are essential for determining appropriate application rates and ensuring compatibility with saffron cultivation practices.

Abbreviations

CAT

Catalase

EO

Essential Oils

ROS

Reactive Oxygen species

SOD

Superoxide Dismutase

PVP

polyvinylpyrrolidone

Author contributions

Majid Azizi and Hoda Sajedimehra: Performing experimental works, Data curation, Formal analysis, Investigation, Methodology, Supervision, Visualization, Writing - original draft. Hamed Kaveh, Mansoureh Nazari: Conceptualization, performing experimental works, Investigation, Project administration, Validation, Visualization, Writing - review & editing. Seyedeh Faezeh Taghizadeh: Data analysis, Validation, Visualization, Writing - review & editing. All authors approved the final version.

Funding

This research has been financially supported by the Saffron Institute, University of Torbat Heydarieh, under the Grant number 177287.

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Availability of data and materials:

Competing interests

The authors declare no competing interests.