High-Energy Emulsified Clove Essential Oil Nanoemulsion as a Natural Herbicidal Product: Germination Suppression and Seed Structure Alteration in Echinochloa crus-galli

TL;DR

Clove essential oil nanoemulsions can act as eco-friendly herbicides by suppressing weed seed germination and damaging seed structures.

Contribution

Development of high-energy emulsified clove essential oil nanoemulsions with herbicidal effects on Echinochloa crus-galli.

Findings

Clove EO nanoemulsions inhibited E. crus-galli germination and seedling growth.

Nanoemulsions damaged seed structures, particularly the endosperm, as observed via SEM-EDS.

Optimized nanoemulsions had stable droplet sizes and remained stable for at least four weeks.

Abstract

Clove (Syzygium aromaticum (L.) Merr. & L.M. Perry) essential oil (EO)-based nanoemulsions may have a promising future in eco-friendly herbicide development. Clove EO was found to have a high eugenol content of 87.27%. Organic-solvent-free nanoemulsions using clove EO as a bioactive ingredient were fabricated using ultrasonication and microfluidization emulsification methods. Fourier-transform infrared spectroscopy confirmed that both emulsification methods did not affect the EO components. The droplet size of optimized nanoemulsions was determined using dynamic light scattering. The smallest size of 66.9 nm was obtained by microfluidization at 20,000 psi and eight passes. Additionally, the smallest droplet size for a sonicated nanoemulsion was 103.9 nm, obtained by ultrasonication at 20% for 6 min. Transmission electron microscopy confirmed the droplet sizes of both optimized nanoemulsions. In a storage test, both optimized nanoemulsions were stored at 4 °C for at least four weeks. Finally, both nanoemulsions were evaluated on pre-emergence herbicidal activities against Echinochloa crus-galli. The results showed that both nanoemulsions inhibited E. crus-galli germination and seedling growth, and additionally, inhibited seed imbibition and α-amylase activity. Micro-morphological and ultrastructural analysis was observed using a scanning electron microscope and an energy dispersive X-ray spectrometer (SEM-EDS). SEM-EDS micrographs of the treated seeds showed that the seed structure was damaged, especially the endosperm, leading to the inhibition of seed germination.

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Full text

1. Introduction

Natural herbicides have potential utility in agriculture to reduce the current wide use of chemical weed control methods and support environmental and human health protection. Such herbicides can incorporate active components from organic acids, plant extracts, and essential oils (EOs), which are substances that are thought to be environmentally friendly, have minimal toxicity, and persist less [1]. Clove (Syzygium aromaticum (L.) Merr. & L.M. Perry) EO has been used as an active ingredient of natural herbicides [2,3,4]. The Asiatic region’s economic growth was enhanced by the commerce in cloves and the hunt for this valued spice. As one of the most precious spices, clove has been utilized for ages as a food preservative and for numerous therapeutic uses [5]. The main constituent of clove EO has been identified as eugenol [2,6]. The bioherbicidal efficacy of clove EO was first reported by Bainard et al. [2], who documented significant effects on the dry weight and growth of Chenopodium album, Sisymbrium irio, Melilotus indicus, and Raphanus raphanistrum seedlings. Moreover, application of the EO to weed seedlings as a natural bioherbicide resulted in membrane breakdown and a loss of tissue integrity, which led to electrolyte leakage. Despite the potential utility of such natural herbicides, they are still not widely used in the field because of their low water solubility, high volatility, unpredictable composition, and off-target effects, as well as their lesser environmental stability [7].

Nanotechnology has emerged as a likely avenue for addressing the issues of EO-based botanical herbicides. A wide variety of nanocarriers are available, including silica and carbon nanoparticles, microemulsions, and nanoemulsions, of which the last are especially remarkable prospects for agrochemical encapsulation and controlled release [8]. Numerous researchers have applied various emulsification preparation techniques to develop EO-based natural herbicides with smaller emulsion particles [9,10,11,12]. Nanoemulsions, which are colloidal dispersions with tiny droplets (r = 10~200 nm) of one liquid dispersed in another with which it is immiscible, such as oil droplets in water, are thermodynamically unstable mixtures [13,14]; however, the small droplet size allows the emulsion to persist over a long timescale [15].

Methods of producing nanoemulsions can mainly be classified into two types, low- and high-energy [16]. Low-energy emulsification methods employ only chemicals and ordinary stirring [13] and rely on either spontaneous or transitional phase inversion, which can be caused by raising the dispersed phase volume fraction or by changing the system’s hydrophilic–lipophilic balance (HLB). Despite their simplicity, these low-energy techniques have several restrictions. For example, they are usually impractical for large-scale applications due to requiring vast quantities of specific combinations of surfactants [17]. Accordingly, high-energy techniques like ultrasonication, microfluidization, and high-pressure homogenization are more often used for producing nanoemulsions; however, a machine is needed to generate strong forces [12]. Ultrasonication is a well-known high-energy technique that produces smaller-sized droplets. In this technique, the emulsion system experiences sinusoidal pressure change as a result of mechanical vibrations caused by ultrasonic waves (>20 kHz) [18]. Microfluidization is also notable for producing emulsions with homogeneous size distribution and smaller droplet sizes, especially in comparison to high-pressure homogenization. Furthermore, microfluidizers deliver stable nanoemulsions at low surfactant concentrations [12,13,19].

E. crus-galli, known as barnyardgrass, poses a continual danger to crop productivity and quality, particularly in the cultivation of rice and maize. This weed is quite problematic in agricultural fields due to its fast growth, tolerance to many environmental conditions, and prolific seed production [20]. This study aimed to determine the optimal conditions for fabricating a clove EO nanoemulsion-based natural herbicide using microfluidization and ultrasonication with a surfactant mixture comprising Tween 80 and Span 80 at HLB 12. Method parameters were optimized using droplet size as a criterion, and optimal nanoemulsions with the smallest droplet size for each emulsification method were evaluated for pre-emergence herbicidal activity against the weed E. crus-galli. To provide a mechanism understanding, the morphological and physiological effects on the germination process were also examined, demonstrating this nanoformulation’s potential as a sustainable natural herbicide made from industrial crops.

2. Materials and Methods

2.1. Materials and the Target Weed

Clove EO was purchased from Thai—China Flavours and Fragrances Industry Co., Ltd. (Nonthaburi, Thailand). The EO was extracted using the steam distillation process. The nonionic surfactants polyethylene sorbitan monooleate (Tween 80) and sorbitan monooleate (Span 80) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Mature seeds of barnyard grass (Echinochloa crus-galli (L.) Beauv.) were collected from paddy areas in Ladkrabang, Bangkok, Thailand. The collected seeds were incubated in a hot-air oven at a temperature of 40 °C for 48 h to break dormancy. Their germination capability was 95%.

2.2. Identification of Clove EO Constituents by Gas Chromatography/Mass Spectrometry (GC/MS)

Gas chromatography/mass spectrometry (GC/MS) using an Agilent series 6890N gas chromatograph (Agilent Technologies, Santa Clara, CA, USA) coupled to an Agilent 5973 mass detector (Agilent Technologies, Santa Clara, CA, USA) was applied to identify the constituents of clove EO. The analysis was carried out on a HP-5 silica capillary column (30 m; 0.25 mm ID; film thickness 0.25 µm). Operating conditions were as follows: oven temperature of 40 °C (3 min); temperature range of 10 °C to 100 °C (5 min); and flow rate of 1 mL/min of helium gas. The sample volume (0.2 µL) was injected into the capillary column in the split mode (1:50). The temperatures of the ion source and interface were 230 °C and 280 °C, respectively. Individual constituents were distinguished via comparison of their mass spectra (molecular mass and fragmentation pattern) with the internal reference mass spectra library (National Institute of Standards and Technology, NIST, 2014). Compound identification was deemed acceptable only when both the forward and reverse match scores were ≥800 in relation to the reference library. The relative amount of each individual component was expressed as the percentage of its peak area relative to the total peak area.

2.3. Nanoemulsion Preparation

An oil-in-water coarse emulsion of clove EO was produced by stirring with a nonionic surfactant mixture (Smix) at a hydrophilic–lipophilic balance (HLB) of 12 (Tween 80, HLB of 15.0; and Span 80, HLB of 4.3). The HLB value indicates a surfactant’s solubility, with lower values reflecting lipophilic characteristics and larger values indicating hydrophilic characteristics. HLB of Smix values were calculated using Griffin’s formula [21]:

$$[eqn]$$

where X_A_ is the mass fraction of surfactant A.

Firstly, Tween 80 (72%) and Span 80 (28%) were mixed using a magnetic stirrer at 1500 rpm for 10 min to obtain the Smix solution. Then, clove EO was added to the Smix, and mixing continued for 10 min. Finally, DI water was added, and mixing continued for another 10 min to obtain the coarse emulsion. This coarse emulsion was fabricated into nanoemulsions by high-energy emulsification, namely, using microfluidization and ultrasonication.

2.3.1. Microfluidization Emulsification Method

The coarse emulsion was refined into nanoemulsion form using an M-110P microfluidizer processor (Microfluidics, Newton, MA, USA) at 25 °C, 10,000–25,000 psi, and 1–8 passes. The Z-shaped interaction chamber for the microfluidizer has a diameter of 87 µm. A total of 200 mL coarse emulsion was fed to the processor, which circulated the coarse emulsion in the direction of a microchannel-equipped interaction chamber. In the process, the high shear pressures caused the emulsion’s droplet size to decrease [22]. The obtained nanoemulsions were stored at 4 °C and evaluated in further experiments.

2.3.2. Ultrasonication Emulsification Method

The coarse emulsion was refined into nanoemulsion form using a 20 kHz ultrasonic processor (Model: CP750, Power: 750 Watts) from Cole-Parmer Instruments (Vernon Hills, IL, USA). An ultrasound probe with a tip of 13 mm diameter was used to generate disruptive forces at amplitudes of 20%, 40%, and 60% and intervals of 2, 4, 6, 8, and 10 min. To prevent overheating, the ultrasonic pulser operated 30 s ON and 30 s OFF. During processing, an ice water bath was used to reduce the heat generated. All experiments were performed in a 100 mL glass beaker. The obtained nanoemulsions were stored at 4 °C and evaluated in further experiments.

2.4. Nanoemulsion Characteristics

2.4.1. Droplet Size and Polydispersity Index (PI)

A dynamic light scattering (DLS) technique was used to determine each nanoemulsion’s average droplet diameter and polydispersity index (PI) using a Nanoplus 3 (MICROMERITICS, Kashiwa, Japan). Measurement parameters consisted of a temperature of 25 °C and a fixed scattering angle of 165°. In order to prevent the multi-scatter effect during droplet analysis, nanoemulsion samples were diluted with DI water at a ratio of 1:9. The program nanoPlus version 5.10/3.00 was used to compute each measurement, and five replications were performed. The nanoemulsion with the smallest droplet size was selected as the optimal formulation for further experiments.

2.4.2. Morphology

Droplet morphology of the optimized nanoemulsions was investigated using transmission electron microscopy (TEM) (HITACHI HT7700, Hitachi High-Tech Corp., Tokyo, Japan) operating at 80 kV to confirm droplet size. After allowing a drop of nanoemulsion to settle on a grid covered with carbon for ten minutes, a drop of 2 percent uranyl acetate was added, and the mixture was left to sit for thirty seconds.

2.4.3. Fourier-Transform Infrared (FT-IR) Spectroscopic Analysis

Fourier-transform infrared (FT-IR) spectra of pure EO, coarse emulsion, and optimal nanoemulsions were recorded from wave number 4000–400 cm^−1^ using a FT-IR spectrometer (Invenio-s, Bruker, Optics, Ettlingen, Germany) to identify the functional groups and covalent interactions, if present. Samples were formed as disks by grinding and pressing with potassium bromide (KBr). One mg of the dried material was combined with 100 mg of spectroscopic-grade KBr powder. Each spectrum was recorded as 32 scans at a resolution of 2 cm^−1^.

2.5. Herbicidal Activity Assay

Healthy and equal-sized seeds of E. crus-galli were selected for the evaluation of seed germination and seedling growth inhibition by clove EO-based nanoemulsions using the Petri dish assay. Optimized nanoemulsion treatments were prepared at concentrations of 75, 150, 300, and 600 ppm EO. For each assay, double germination papers were placed in a Petri dish, and 5 mL of treatment solution was added. Then, 20 seeds were placed onto the germination paper, and the cover was sealed. The dishes were kept for seven days in a growth chamber (LAC-1075-N, Longyue, Shanghai, China) (12/12 light/dark, at 25 ± 2 °C, humidity 80%). Distilled water and Smix solution served as controls. After completing the incubation, germinated seeds were counted, and the lengths of seedling roots and shoots (cm) were measured. A completely randomized design was used, with four replicate Petri dishes for each treatment. Percent inhibition of germination and seedling growth was determined using the following formula (1):

$$[eqn]$$

2.5.1. Scanning Electron Microscope and Energy Dispersive X-Ray Spectrometer (SEM-EDS) Analysis

Micro-morphological seed traits were examined using a scanning electron microscope and an energy dispersive X-ray spectrometer (SEM-EDS) at a model: JEOL JSM-IT500HR (JEOL Ltd., Tokyo, Japan). The seeds treated with the sample treatment were dehydrated using a sequence of increasing ethanol concentrations after being fixed in 70% ethanol. The seeds were then deposited on metallic stubs using double adhesive tape, coated with gold for six minutes in a sputtering chamber, processed at an accelerating voltage of 10 kV, and analyzed [23]. Depending on the size of the seeds, several image magnifications (from 37× to 2000×) were used.

2.5.2. Seed Imbibition

Seed imbibition and α-amylase activity were evaluated for both optimized nanoemulsions and the coarse emulsion. Treatment formulations consisted of the optimal nanoemulsion and coarse emulsion at concentrations of 150, 300, and 600 ppm, and the surfactant mixture solution at a concentration of 600 ppm. Water was used as a control. The process of seed imbibition was carried out according to Turk and Tawaha [24] with modifications. Briefly, 30 seeds of E. crus-galli were weighed (W_1_) and soaked in the treatment solution for 12, 24, and 36 h. After incubation, the seeds were washed and weighed again (W_2_). A completely randomized design was used, with four replicate Petri dishes for each treatment. Seed imbibition percentage was then determined as follows (2):

$$[eqn]$$

2.5.3. α-Amylase Activity

The dinitrosalicylic acid (DNS) method was used to assess α-amylase activity, as reported by Sadasivam and Manickam [25]. First, α-amylase was extracted from the seeds by grinding them with 4 mL of 0.1 M CaCl_2_ in an ice bath, followed by centrifugation at 10,000 rpm for 20 min at 4 °C. The supernatant was collected and stored at 4 °C for α-amylase activity measurement. The amylase reaction was initiated by mixing 1 mL of supernatant and 1 mL of 1% soluble starch in an acetate buffer solution (pH 5.5). Then, this mixture was incubated at 37 °C for 15 min. After incubation, 1 mL of DNS reagent was added to stop the reaction, and the mixture was boiled at 100 °C for 5 min, and then cooled in an ice bath. Finally, the absorption at 560 nm was measured using a UV/Vis spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and the α-amylase activity was calculated and reported in μmol maltose/min/g(FW). A completely randomized design was used, with four replicate Petri dishes for each treatment.

2.6. Statistics

A completely randomized design with four replications was used in all experiments. Data is expressed as the mean ± standard deviation (SD). Tukey’s multiple range test was used to compare the means (p < 0.05 considered significant).

3. Results and Discussion

3.1. Identification of Clove EO Constituents

The complex mixture, that is, clove EO, was interrogated using GC/MS. Table 1 presents the six identified chemical constituents of the EO from clove (S. aromaticum) bud, which constituted 99.30% of the total oil. Outstandingly, eugenol was far and away the dominant component (87.27% of total). Ahuja et al. [26] similarly reported eugenol to be the major constituent of S. aromaticum (clove) oil, making up 45–90% of the oil. Previously, eugenol was found to cause morphological and physiological changes in plant seedlings and to inhibit germination and growth, suggesting it can be used successfully to control grassy weeds. Eugenol additionally damages cellular membranes, which results in harm to plant tissue [27].

3.2. Effect of Microfluidization vs. Ultrasonication on Nanoemulsion Droplet Size

Nanoemulsions produced by microfluidization and ultrasonication methods were evaluated for droplet size and PI value. In microfluidization, the emulsification process involves the collision of two immiscible liquid flow streams that are passing through a narrow orifice in a microchannel. On the design level, this bears similarity to a high-pressure homogenizer, but the emulsion principle differs. First, an emulsion is divided into two streams as it flows through one channel; second, each stream is passed through its own fine channel; and third, the two streams collide in an interaction chamber. The collision of these fast-moving streams generates intense disruptive forces, leading to the formation of markedly smaller droplets [28]. High-energy microfluidization thus produces very stable and useful emulsions, although realizing the method’s full potential is dependent on the processing parameters and the properties of the material being treated [29]. In general, finer emulsions are produced by increasing the microfluidization pressure and cycle count [30]. The line graphs in Figure 1A,B depict the effect of microfluidization pressure (10,000–25,000 psi) and cycle count (1–8 passes) on the droplet size and PI of the nanoemulsion. Overall, droplet size decreased with increasing pressure and number of cycles. The initial coarse emulsion produced by stirring had a relatively large droplet size (260.5 nm and PI 0.309). After microfluidization, the smallest droplet size of 66.9 nm (PI = 0.156) was obtained under eight passes at a pressure of 20,000 (Figure 1A and Figure 2A). Dimak et al. [31] likewise fabricated peppermint EO nanoemulsions using microfluidization and reported that increasing the microfluidization pressure from 5000 to 20,000 psi can decrease droplet size from 130.2 to 69.8 nm.

Through the use of an acoustic field that generates interfacial waves, ultrasonic emulsification produces strong disruptive forces that cause the oil phase to explode into the water medium as droplets [32,33]. The irradiation period, power, and frequency all have a major impact on droplet size [34]. The line graphs in Figure 1C,D illustrate the effects of ultrasonication at amplitudes of 20–80% for 2–8 min. Overall, droplet size decreased with increasing sonication time, except for the nanoemulsion prepared at 80% amplitude, for which it increased with increasing time. Overprocessing from prolonged sonication can cause droplet coalescence [35]. The smallest droplet size (103.9 nm and PI 0.254) was obtained by applying the lowest amplitude (20%) for 6 min (Figure 1C and Figure 2B).

Comparing the results of the two high-energy emulsification methods, the nanoemulsion produced by microfluidization achieved both a smaller droplet size and a lower PI. Tang et al. [36] found that high-power ultrasonication and microfluidization techniques are able to produce nanoemulsions with minimal droplet diameters ranging from 150 to 170 nm, even given a pre-emulsion droplet size of 1 µm. Microfluidization provides superior physicochemical stability, but ultrasonic cavitation is comparatively adaptable, able to operate on larger quantities, and much more energy-efficient. However, ultrasonication also produces more heat and is not suitable for emulsion systems that contain materials sensitive to heat [34,37]. As clove EO is not very heat-sensitive, nanoemulsions based on it can be produced by either microfluidization or ultrasonication emulsification.

3.3. FT-IR Spectroscopic Analysis

Pure clove EO, the coarse emulsion, and the optimized ultrasonicated and microfluidized nanoemulsions were characterized using an FT-IR spectrometer to demonstrate the non-destructive nature of the preparation procedures (Figure 3). Since eugenol, a monoterpenoid, constitutes a significant portion of the oil, its characteristic peaks were observed in the 720–1250 cm^−1^ region [38]. Additional distinct peaks attributable to eugenol appeared at 1637, 1611, and 1511 cm^−1^, likely resulting from C=C stretching in the aromatic moiety. Minor terpenoids were responsible for the other peaks. All emulsions displayed similar patterns that included most of the peaks observed in pure clove oil. However, they also displayed a few additional bands, notably a very broad peak at 3332 cm^−1^ (O-H stretching, broad) and a strong peak at 1636 cm^−1^ (H-O-H bending), which could be attributed to water.

3.4. Morphological Characterization of Clove EO-Based Nanoemulsions

A transmission electron microscope was used to perform morphological analysis of the optimized nanoemulsions and confirm droplet size. Figure 4 presents TEM pictures of the nanoemulsions. The morphological analysis confirmed that the droplet sizes of both nanoemulsions correlated with their DLS results. The droplets were clearly visible, having a blank core representing the EO surrounded by the Smix, which was dispersed in water. As shown in Figure 4A,B, microfluidization produced droplets that were not entirely spherical but were homogeneously distributed. Meanwhile, ultrasonication produced spherical droplet shapes with spikes (Figure 4C,D). Other studies have reported clove nanoemulsion droplets to be spherical in shape [39,40,41]. The spike-like morphology observed in the TEM images of ultrasonicated nanoemulsion may be due to the temperature during preparation. The different shapes resulting from microfluidization and ultrasonication in this work may be attributable to the emulsification method affecting droplet shape and size. However, FT-IR results confirmed that both emulsification methods did not affect clove components.

3.5. Storage Stability of the Clove EO-Based Nanoemulsions

The stability of a nanoemulsion is essential to its use in a variety of agricultural products. Droplet size and PI are both important indicators of emulsion stability [42]. In this study, the optimized nanoemulsions were stored at 4 °C for five weeks, with droplet size and PI evaluated at weekly intervals. According to Somala et al. [4], the mean droplet size of a nanoemulsion kept at 4 °C is expected to remain good over time, whereas emulsions kept at 45 °C develop larger droplets. Such increases in droplet size at high temperature may be due to the Brownian motion of dispersed droplets resulting in coalescence or flocculation [43]. The line graphs in Figure 5A,B present the effects of storage at 4 °C on the droplet size and PI of clove EO-based nanoemulsions. For both nanoemulsions, droplet size grew as time increased from 0 to 5 weeks. After the first week of storage, the droplet size of both nanoemulsions increased substantially; in later weeks, it continued to increase more slightly, finally reaching above 200 nm by the end of the storage period. Meanwhile, the PI of both nanoemulsions decreased after the first week of storage and remained less than 0.25 at all time points (Figure 5B). According to Pongsumpun et al. [42], nanoemulsions are regarded as having a narrow distribution when their PI value is less than 0.3. Taken together, these findings indicate strong stability of the clove EO-based nanoemulsions during the evaluated storage period.

3.6. Effect of Clove EO-Based Nanoemulsion on Germination and Growth of E. crus-galli

The inhibitory effect of the clove EO nanoemulsion-based natural herbicide on E. crus-galli seed was evaluated using the Petri dish bioassay. Table 2 and Figure 6 present the effects on seed germination and seedling growth. Both nanoemulsions and the coarse emulsion showed a dose-dependent inhibitory effect that increased with increasing concentration of the treatment solution. Complete inhibition of germination was achieved in both nanoemulsions at a concentration of 600 ppm. Overall, both nanoemulsions exhibited the same effect in terms of germination inhibition. The nanoemulsion at a concentration of 600 ppm could completely inhibit seed germination because the seed structure, especially the endosperm, was damaged, preventing hydrolysis or mobilization of the embryo. This mechanism is further detailed in Section 3.7 and Section 3.8. In a clove EO-based nanoemulsion, it is the chemical components that provide the phytotoxic impact, as supported by research showing that isolated chemicals like eugenol impede the growth and germination of some weeds [2,26,44]. Specifically, seedlings of redroot pigweed and common lambsquarters were either killed or badly harmed by clove oil, with growth decreased by 99% and 91% respectively [2]. Additionally, both nanoemulsions showed a higher potential than the coarse emulsion, which could be explained by the small particle size that makes it easier for the seeds’ membranes to be penetrated [22]. Apart from particle size, evaporation and oxidation are two further characteristics that limit the inhibitory potential of oil components [9,10,45]. In this regard, after applying active compounds to weed seeds, it is critical to ensure their effective release and rapid contact with plant cells.

In the present evaluation, there was a dose-dependent trend of decreased shoot and root length. All treatment solutions (except those with a concentration of 75 ppm) inhibited root development more than shoots because the roots were in direct contact with the treatment solution and lacked cuticle cover, which permits monoterpenes to freely penetrate. Thus, the concentration of monoterpenes in the roots is higher than in the shoots, explaining the stronger inhibitory effect [26]. Our findings showed that, when compared to conventional coarse emulsion, clove EO nanoemulsion-based natural herbicide with high-energy emulsification demonstrated greater weed control efficiency at lower dosages.

3.7. SEM-EDS-Based Structural Characterization

The SEM-EDS technique was used to study the micro-morphological and ultrastructural analysis of the treated seeds. After 36 h of soaking, E. crus-galli seeds treated with clove EO-based nanoemulsion at the highest concentration were analyzed for seed structure. Overall, based on SEM-EDS observations, the nanoemulsion had no negative effect on the outer structure of the treated seed. However, the nanoemulsion can disrupt the inner structure of the seed, resulting in reduced embryo development. Figure 7 shows the fertile lemma of the treated seeds with clove EO-based nanoemulsions produced using microfluidization at 20,000 psi for eight passes and ultrasonication with an amplitude of 20% for 6 min. The fertile lemma shape of the treated seed with clove EO-based nanoemulsion was no different from that of the control treatment (water). Similarly, the fertile palea shape of the treated seeds with clove EO-based nanoemulsion was also no different from that of the control treatment (Figure 8). However, when studying a cross-section of the treated seed, the effect of the nanoemulsion on the treated seed structure showed that the embryo and endosperm in the seed were not uniform (Figure 9). Especially, starch granules in the endosperm of the seed treated were damaged by the nanoemulsion. The application of the nanoemulsion induced a deformed starch granule in both treated seeds. In the control treatment, amyloplasts containing polyhedral starch granules were observed in the endosperm (Figure 9G). When the tested seed was soaked in the nanoemulsion, polyhedral starch granules were altered compared to the control treatment (Figure 9H,I). Toyosawa et al. [46] described that the polyhedral, sharp-edged shape of starch granules results from the granules expanding until they are tightly packed within the septum-like structure, which functions as a mold, and the amyloplast envelope; therefore, intact granules must maintain this polyhedral morphology. Starch granules of mature seed become a deformed structure. This is caused by abnormalities in starch synthesis or a weak amyloplast structure, which means that it cannot maintain its polyhedral shape. Therefore, the nanoemulsion may affect the stability of the starch granule structure or cause erosion, causing the previously sharp-edged shape to disappear. In a comparison between emulsification methods, the starch granules of the seed treated with nanoemulsions produced by ultrasonication (amplitude of 20%, 6 min) showed slightly more deformation than those of microfluidization treatment (20,000 psi, 8 passes). Additionally, the aleurone layer functions primarily to synthesize hydrolytic enzymes (α-amylase, dextrinase, α-glucosidase, and proteases) and secrete them into the underlying starchy endosperm to degrade stored food reserves [47]. The live aleurone layer surrounds the endosperm, the starch-rich part of the seed that perishes during desiccation and seed development. Despite being seen as dead, the endosperm continues to engage in a number of metabolic processes, including redox processes, which are essential for germination [48]. In Figure 9A, the aleurone layer appeared clearly normal, leading to embryo development. Cuboid cell shape is closely packed in the aleurone layer (peripheral endosperm), which is the part of the endosperm that remains active throughout seed maturity [48]. In contrast, Figure 9B shows that the aleurone layer of the seed treated with the nanoemulsion was not clearly visible because it may be deformed or damaged. In Figure 9C, the aleurone layer showed slight deformation. Therefore, the impaired function of the aleurone layer led to a decreased synthesis of α-amylase. Consequently, starch reserves were not hydrolyzed or mobilized for utilization, resulting in the failure of embryo development. As a result, the physiological consequence was impaired starch degradation and mobilization, according to the results of α-amylase activities of the treated seed. Consequently, the development of the embryo was inhibited by both nanoemulsions (Figure 9K,L). Normally, the aleurone layer exhibits programmed cell death during the final stage of seed germination. It has oil bodies and aleurone proteins. It is known that gibberellin produced in the germinating embryo is released through the scutellum to the aleurone layer, where it uses its accumulated reserves to cause the release of hydrolytic enzymes, such as α-amylase, which are then released into the endosperm [48]. In Figure 9A, the scutellum section presented expansion and development. The nanoemulsion also affected scutellum development (Figure 9B,C). In SEM-EDS results, Figure 9A showed evidence of embryo development by observing the radicle and plumule of the seed. On the other hand, the seeds treated with the nanoemulsion showed no clearly observable radicle and plumule (Figure 9B,C). Additionally, embryo meristematic tissue was observed in Figure 9M–O. The embryo of the treated seeds presented that its meristematic tissue was deformed (Figure 9N,O). Therefore, the nanoemulsion inhibited seed germination by destroying seed structures involved in the germination process.

3.8. Effect of Clove EO-Based Nanoemulsion on Seed Imbibition and α-Amylase Activity

Seed imbibition is the process of water adsorption by dry seeds, and the first stage of the seed germination process. When the seed begins imbibition, the embryo regains its biological activity and starts synthesizing gibberellin (gibberellic acid; GA), particularly in the scutellum, the single layer of tissue separating the embryo and endosperm. This GA is transported through the scutellum to the aleurone layer, the outer, living layer of the endosperm. Then, the aleurone layer receives a signal from GA, and it is stimulated to synthesize several hydrolytic enzymes, especially α-amylase [48].

Figure 10A illustrates the effect of the clove EO-based nanoemulsions on seed imbibition of E. crus-galli. Over the course of the imbibition period, the percentage of imbibition increased. After 24–36 h, seeds soaked in the Smix solution (600 ppm) had the highest adsorption, achieving the greatest imbibition percentage. Meanwhile, after 12 h, seeds soaked in water showed less imbibition than those soaked in nearly all nanoemulsion treatments (the exception being the microfluidized nanoemulsion at 150 ppm). Water has a high surface tension, which causes it to slowly penetrate into seeds. At the end of the assay (36 h), ultrasonicated nanoemulsions showed a lower imbibition rate compared to the same concentration of microfluidized nanoemulsion. Additionally, the highest concentration of ultrasonicated nanoemulsion (600 ppm) showed the overall lowest imbibition rate. Additionally, the imbibition results correlated with the SEM-EDS micrograph showing that the seed of the control treatment was higher than the treated seed with nanoemulsion. As in previous results, the PI value of the microfluidized nanoemulsion was smaller than that of the ultrasonicated nanoemulsion. A system with a variety of particle sizes is a characteristic feature of ultrasonic preparation [49]. The inhibition of seed imbibition was observed in ultrasonicated nanoemulsion because of the higher packing density of the dispersed phase on the seed coat. Sohn and Moreland [50] described that a wide particle size distribution significantly increases packing density compared to monodisperse systems, because smaller particles fill the voids between larger particles. However, the higher seed imbibition with the microfluidized nanoemulsion is attributable to its smaller droplet size.

Figure 10B illustrates the effect of the clove EO-based nanoemulsions on seed germination, as indicated by α-amylase activity. Following imbibition, seed germination is initiated by catabolic processes [11]. α-Amylase is the primary enzyme involved in starch mobilization, and its activity is crucial for seed germination [51]. During the germination process, α-amylase breaks starch down into smaller organic molecules, thereby providing necessary nutrients and energy [51]. After 12 h of treatment, α-amylase activity was not significantly different in any treatment solution. However, at 24 h, the control (water) exhibited the highest α-amylase activity, while all nanoemulsions and the Smix solution were not significantly different; this reflects that the Smix solution and the nanoemulsions potentially slow the enzyme activity. Finally, after 36 h, α-amylase activity exhibited a dose-dependent trend, decreasing with increasing nanoemulsion concentration, and the ultrasonicated nanoemulsion at 600 ppm resulted in the lowest activity. Overall, ultrasonicated nanoemulsions produced lower α-amylase activity than did microfluidized nanoemulsions, consistent with the seed imbibition result (Figure 10A). In Figure 9I, starch granules of the seed with sonicated nanoemulsion were covered with the packing density of the dispersed phase. The coating on starch granules may prevent enzymes from binding to the substrate [52]. Additionally, clove EO was rich in eugenol, which can reduce the expression of alpha-amylase, especially RAmy3B and RAmy3E, because both of these genes play a crucial role in the breakdown of starch in the aleurone layer to release nutrients for germination [48,53]. Taken together, these results support that clove EO-based nanoemulsions prepared using high-energy emulsification methods can inhibit seed germination of E. crus-galli by disrupting seed imbibition and α-amylase activity.

4. Conclusions

This study demonstrates the feasibility of utilizing clove essential oil as an effective natural herbicide produced through nanoemulsion technology. Our results showed that the major compound in clove EO was eugenol. Clove EO nanoemulsion-based natural herbicides were prepared using a nonionic surfactant mixture and two high-energy emulsification methods, namely microfluidization at 20,000 psi with eight passes (66.9 nm) and ultrasonication at 20% amplitude for 6 min (103.9 nm). FT-IR confirmed that these preparation methods did not degrade the components of the EO. The nanoemulsions were stable in refrigerated storage for at least four weeks. In bioassays, they showed strong inhibitory effects on the germination and seedling growth of E. crus-galli, including inhibiting both seed imbibition and α-amylase activity. SEM-EDS micrograph of the cross-section of E. crus-galli seed is the key to clarifying the mechanism of germination inhibition. The endosperm of the seeds was damaged by the nanoemulsion, leading to failed embryo development. These findings encourage the use of a clove EO-based nanoemulsion as a natural herbicidal product for sustainable weed management. The authors expect that this work will encourage research into natural herbicides as a sustainable solution to environmental and health pollution issues. Further, this study also suggests that this potential natural herbicide product could develop as a promising candidate for weed management in field-level applications. Future product development and large-scale field applications are made possible by this dose-reduction capability and scalable production technique, which also improves its cost-effectiveness and commercial viability.