Wettability of the Plant Growth Regulator 28-HB on Pepper Leaves at Different Developmental Stages

Abstract

Studying the wettability of plant growth regulators on crop leaf surfaces is essential for enhancing crop yield. In this study, the wetting behavior of the plant growth regulator 28-homo-brassinolide (28-HB), supplemented with different surfactants, was investigated on the adaxial and abaxial surfaces of pepper leaves at the seedling, early flowering, and fruiting stages. The microstructure of the leaf surface was characterized using an ultra-depth field microscope. The surface free energy (SFE) of the leaves was calculated using the Owens-Wendt-Rabel-Kaelble (OWRK) method. Additionally, the surface tension of the 28-HB solutions containing various surfactants, as well as the contact angles on pepper leaves at different growth stages, were measured. The experimental results indicate that the surface free energy (SFE) of pepper leaves significantly decreases with plant maturation. Specifically, the SFE of the adaxial leaf surface declined from 43.4 mJ/m² at the seedling stage to 26.6 mJ/m² at the fruiting stage, while the abaxial surface decreased from 27.5 mJ/m² to 22.5 mJ/m². At all growth stages, the relative polar component (R_P) of the adaxial surface was consistently higher than that of the abaxial surface and showed a gradual decline from 94.70% to 57.34% as development progressed. The contact angle measurement showed that the addition of surfactant decreased the contact angle of 28-HB on the leaf surface and increased the wetting area. Among the tested formulations, the addition of fatty alcohol ethoxylates (AEO-9) significantly reduced the contact angle to below 45°, and resulted in an adhesion tension below 30 mN/m and adhesion work lower than 105 mJ/m². These values indicate superior wetting performance compared to formulations containing sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide (CTAB). This study integrates the surface free energy characteristics of pepper leaves at different growth stages with the wetting performance of various surfactant systems, providing a quantitative basis for the selection and optimization of surfactants in agricultural spray formulations. The findings offer theoretical support for precise pesticide application strategies, enhancing pesticide adhesion and absorption on leaf surfaces, thereby improving pesticide utilization efficiency throughout the crop growth cycle.

1. Introduction

Plant growth regulators are naturally occurring or artificially synthesized compounds that can regulate plant metabolism and physiological functions, and they have been widely used in agriculture, forestry, and other related fields [1,2]. The integration of PGRs into agricultural practices in China holds significant importance for the advancement of the country’s agricultural development [3]. Currently, PGRs are mainly applied by preparing aqueous solutions of specific concentrations and spraying them onto crop leaves through nozzles [4,5]. The behavior of droplets impacting leaf surfaces is influenced by the physical characteristics of both the droplets and the leaf surfaces [6,7,8], as well as their relative motion states [9,10]. The regulatory effect of plant growth regulators (PGRs) depends not only on their active ingredients but also on whether these active compounds can be effectively transported to their target sites under suitable conditions. To enhance the penetration ability of PGRs, spray formulations typically include additives such as solvents, surfactants, and water conditioners, while also selecting appropriate treatment timings based on the plant’s growth stage. The adhesion and spreading behavior of PGR droplets on leaf surfaces directly affects the utilization efficiency of the active ingredients [11]. Optimizing the deposition and diffusion of PGRs on leaf surfaces is crucial for effective crop growth regulation, improving resource use efficiency, and reducing environmental pollution. Therefore, a deeper understanding of the interactions between leaf surface properties and PGR droplets to improve wettability has become a key research focus in plant protection [12].

Peppers are rich in vitamins and are among the most nutritionally valuable vegetables [13]. During the cultivation of peppers, it is essential not only to manage pests and diseases but also to apply plant growth regulators (PGRs) at different developmental stages to promote growth [14]. The interfacial structural properties of pepper leaves significantly influence the wetting behavior of pesticide droplets on plant foliage [15]. Key factors affecting droplet wetting include the chemical composition of cuticular waxes, surface topography, and structural appendages such as trichomes. Wang et al. [16] conducted a systematic analysis of the physical (e.g., roughness) and physicochemical properties (e.g., surface free energy, its components, and work of adhesion) of 60 plant species using scanning electron microscopy (SEM) and thermodynamic approaches. They found that surface roughness, free energy, and adhesion work significantly affect the wetting behavior of droplets. During plant growth, the wax composition in the leaf cuticular layer undergoes changes [17]. Ahmed [18] established a growth curve of pepper leaves based on measured leaf length and width and used SDS-PAGE to detect regulatory protein differences during leaf development. Maarseveen and Jetter [19] reported significant differences in the surface wax composition and wettability between the adaxial and abaxial leaf surfaces. Henningsen et al. [20] employed transmission electron microscopy (TEM) and scanning electron microscopy (SEM) to conduct a detailed characterization of the leaf surfaces of pepper plants measuring 50–70 cm in height. Their results indicated that pepper leaves are glabrous on the adaxial surface, with sparse glandular trichomes occasionally present on the abaxial surface. Both surfaces exhibited relatively high stomatal densities. Furthermore, the contact angle hysteresis values of water droplets on both leaf surfaces were comparable, suggesting similar wetting hysteresis behavior.

Surfactants can significantly alter the deposition behavior of pesticide solutions on crop foliage [21,22,23]. Tredenick et al. [24] investigated the effects of surfactant concentration and surface inclination (0°, 45°, and 90°) on the behavior of water and surfactant droplets on wheat and pepper leaves. Parameters such as contact angle, droplet height, volume, and diameter were measured, and the influence of surfactant type and inclination angle on droplet evaporation characteristics—such as shape, contact angle, evaporation rate, and time—was experimentally demonstrated. Hagedorn et al. [25] explored how six surfactants with varying hydrophilic-lipophilic balance (HLB) values affected the structure of soybean leaf surface waxes and leaf wettability using scanning electron microscopy. Surfactants with HLB values below 10 reduced the size of wax structures and induced a transition from Cassie-Baxter wetting to an intermediate wetting state, thereby enhancing droplet adhesion. The physicochemical nature of surfactant solutions directly influences droplet adsorption and deposition behavior on leaf surfaces. Tamjidi et al. [26] examined isothermal, thermodynamic, and kinetic aspects of the adsorption process and found that the addition of anionic or cationic surfactants significantly altered the surface charge of the adsorbent, directly affecting its droplet adsorption capacity. Bao et al. [27] studied the impact dynamics of Triton X-series surfactants with varying hydrophilic ethylene oxide (EO) chain lengths on artificial superhydrophobic surfaces. Their results showed that changes in EO chain length affected molecular diffusion rates, receding contact angles, and adhesion properties, providing an effective strategy to regulate droplet deposition via surfactant design. Xu et al. [28] measured the contact angle and wetting area of droplets containing four different additives on waxy leaf surfaces and found that the presence of surfactants promoted the deposition of pure water droplets.

Most of the above-mentioned studies focus on how external additives modify surface tension to improve wettability, assuming the leaf surface as a static substrate. However, due to the dynamic nature of plant surfaces—which change significantly with developmental stage [29,30]—leaf wettability is not constant, but varies across different growth phases. Moreover, changes in the physicochemical properties of leaves during development may substantially affect the spreading behavior of 28-HB (28-homo-brassinolide) on the leaf surface.

In this study, an extended depth-of-field microscope was employed to systematically characterize the microstructures of the adaxial and abaxial leaf surfaces of pepper at the seedling, initial flowering, and fruiting stages, aiming to investigate the surface properties of leaves at different growth phases. The wetting and adhesion behaviors of 28-HB mixed with various types of surfactants on pepper leaves at different growth stages were evaluated by measuring contact angle, surface tension, and adhesion force. Furthermore, the regulatory mechanisms of surfactants on the wetting and deposition of 28-HB on leaf surfaces were elucidated. These findings provide theoretical guidance for the practical application of foliar pesticide sprays on pepper, potentially enabling the reduction in chemical pesticide usage while enhancing efficacy, thereby ensuring ecological safety and the quality of pepper products.

2. Materials and Methods

2.1. Experimental Materials

Peppers (Capsicum annuum L. Tianshuai 101) were obtained in April 2024 from Four Seasons Spring Nursery in Shouguang, China. They were subsequently transplanted into pots containing organic soil and grown under controlled laboratory conditions (relative humidity: 60–75%, temperature: 20–25 °C, photoperiod: 16 h light/8 h dark). Supplemental illumination was provided by a flat-panel LED lamp (WEN-2 Leafy Vegetable Spectrum, Shandong Weiga Optoelectronics Co., Ltd., Weifang, China). During the growth period, soil moisture was consistently maintained to ensure the healthy development of the pepper plants.

To investigate the leaf surface characteristics at different growth stages, healthy leaves were selected from three growth phases: seedling stage (approximately 25 days after growth), initial flowering stage (around 60 days), and fruiting stage (about 85 days). To ensure sampling consistency across different growth stages, three fully developed and fully expanded mature leaves were collected at each sampling time point. The sampling locations are illustrated in Figure 1. Leaves at the seedling stage were collected approximately 25 days after sowing, each plant having developed three true leaves from top to bottom, as shown in Figure 1A. Leaves at the early flowering stage were sampled around 60 days after sowing from the first fork of the stem, as shown in Figure 1B. At the fruiting stage, leaves were collected approximately 85 days after sowing from branches bearing fruit, as shown in Figure 1C. For each growth stage, experiments were conducted at three specific positions on the leaf, as illustrated in Figure 1D. All selected leaves were fresh and healthy at the time of sampling. To minimize errors caused by water loss and wilting, all measurements were conducted within one hour after detachment. The testing environment was maintained at a temperature of 20–25 °C and a relative humidity of 60–75%.

2.2. Leaf Surface Characterization

Healthy pepper plants at the seedling stage (25 days), early flowering stage (60 days), and fruiting stage (85 days) were selected, with five plants chosen from each stage. The leaf positions are shown in Figure 1. As illustrated in Figure 1D, the leaves were cut into leaf blocks measuring approximately 35 mm × 8 mm, avoiding the midrib during the cutting process to maintain flatness and measurement consistency. For each leaf, measurements were repeated three times on different regions of the same leaf block to ensure data reliability and reproducibility.

Prior to imaging, the leaf surfaces were gently cleaned. The prepared leaf blocks were first placed on a glass slide, which was then positioned on the stage of a 3D super-depth field microscope (VHX-900F, Keyence Corporation, Osaka, Japan). Both top-view and side-view images of the leaf surfaces were captured. During imaging, the universal zoom lens (model VH-Z20R, Keyence Corporation, Osaka, Japan) was adjusted to obtain images at 20× and 500× magnification, respectively, for the leaf surface morphology. The leaf veins were vertically cut using a double-edged blade, and the cross-sectional view of the leaf was captured under 500× magnification. By imaging the leaf at 500×, 3D images of the leaf structure were obtained, allowing for rotation of the 3D leaf to observe the overall structure. Roughness values were measured along the longitudinal direction parallel to the leaf veins, avoiding the main vein, with three evenly spaced measurements taken. Since the water content of the leaves tends to evaporate quickly after harvesting, all leaf surface observations were completed within two hours after sample preparation.

2.3. Preparation of Reagents and Test Solutions

28-homo-brassinolide (28-HB; CAS No. 110369-52-3), which has a significant yield-enhancing effect on peppers, was purchased from Yunnan Yunda Science and Technology Agrochemicals Co., Ltd. (Kunming, Yunnan, China). Sodium dodecyl sulfate (SDS) was obtained from Guangdong Wengjiang Chemical Reagent Co., Ltd. (Guangzhou, Guangdong, China). Cetyltrimethylammonium bromide (CTAB) was acquired from Shenzhen Jintenglong Industry Co., Ltd. (Shenzhen, Guangdong, China). Fatty alcohol polyoxymethylene ether (AEO9) was purchased from Shandong Yousuo Chemical Technology Co., Ltd. (Jinan, Shandong, China). Formamide and methanol, which were used as test liquids, were purchased from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China).

Specified amounts of CTAB, SDS, and AEO9 were individually weighed and dissolved in deionized water to prepare aqueous solutions with concentrations ranging from 1.00 × 10⁻⁶ mol/L to 5.00 × 10⁻² mol/L. Subsequently, each surfactant solution at a concentration of 1.00 × 10⁻² mol/L was separately mixed with the plant growth regulator 28-HB at 8.09 × 10⁻⁶ mol/L to form three individual mixed solutions. The pH of the solutions was neither adjusted nor measured during the preparation process.

2.4. Calculation of Leaf Surface Free Energy and Its Components

The Owens-Wendt-Rabel-Kaelble (OWRK) method is a technique used to calculate the surface free energy of a solid based on the contact angle of several liquids in contact with the solid [31]. When employing this method, the surface free energy is divided into a polar component (γ^p) and a dispersive component (γ^d). The interfacial free energy between a solid and a liquid is denoted as γ_sl. The relationship between γ_sl, γ^p, and γ^d is expressed by the OWRK method as follows [32]:

$${\gamma }_{sl}={\gamma }_l+{\gamma }_s-2{\left({\gamma }_l^d{\gamma }_s^d\right)}^{{\displaystyle \frac{1}{2}}}-2{\left({\gamma }_l^p{\gamma }_s^p\right)}^{{\displaystyle \frac{1}{2}}}$$

(1)

where γ_l and γ_s represent the surface free energy of the liquid and solid, respectively.

Young’s equation characterizes the wettability between a liquid and a solid. The relationship between the contact angle (θ) of the liquid on the solid surface and the free energy of the solid, liquid, and gas can be expressed as follows [33]:

$$\begin{array}{c}{\gamma }_l\cos \theta ={\gamma }_s-{\gamma }_{sl}\end{array}$$

(2)

By substituting Equation (1) into Equation (2), the following expression can be obtained:

$$\begin{array}{c}{\displaystyle \frac{\left(1+\cos \theta \right){\gamma }_l}{2{\left({\gamma }_l^d\right)}^{\frac{1}{2}}}}={\left({\gamma }_s^d\right)}^{{\displaystyle \frac{1}{2}}}+{\left({\gamma }_s^p\right)}^{{\displaystyle \frac{1}{2}}}{\left({\displaystyle \frac{{\gamma }_l^p}{{\gamma }_l^d}}\right)}^{{\displaystyle \frac{1}{2}}}\end{array}$$

(3)

As shown in Equation (3), treating it as a linear function, γ_s^d and γ_s^p are calculated from the slope and intercept of the fitted line, respectively. These values are then summed and solved to obtain γ_s. Formamide and methanol, with known surface tension, dispersive, and polar components, were used as test fluids in this experiment. The surface tension of formamide is 58.2 mN/m, with dispersive and polar components of 39.5 mN/m and 18.7 mN/m, respectively. For methanol, the surface tension is 23.7 mN/m, with dispersive and polar components of 16.7 mN/m and 7.0 mN/m, respectively. The contact angles of formamide and methanol on the adaxial surfaces of the pepper leaves at the seedling stage were measured by a contact angle meter, yielding values of 65.40° and 21.50°, respectively. At the seedling stage, the contact angles on the abaxial surface were 74.89° and 35.23°, respectively. At the early flowering stage, the contact angles on the adaxial and abaxial surfaces were 62.47° and 18.50°, and 75.00° and 35.40°, respectively. At the fruiting stage, the contact angles on the adaxial and abaxial surfaces were 48° and 24°, and 55° and 29°, respectively. The unknowns in Equation (3) are the dispersive and polar components of the leaf surface, which can be solved by solving the two equations.

2.5. Surface Tension and Contact Angle Measurement

Under controlled environmental conditions (temperature: 25 ± 0.5 °C; relative humidity: 35 ± 5%), the surface tension of surfactant and plant growth regulator (PGR) solutions at various concentrations was measured using the pendant drop method. All measurements were performed under the same temperature and humidity to ensure data comparability. Prior to measurements, the contact angle measurement system (OCA25, Dataphysics Instruments GmbH, Filderstadt, Baden-Württemberg, Germany) was calibrated using ultrapure water (surface tension of 72.8 mN/m at 25 °C) as a reference.

The test solution was used to lubricate the syringe chamber and then drawn into the syringe, which was mounted on the fixed support of the contact angle instrument. A droplet of 2 µL was dispensed using the instrument’s fully automated liquid dosing unit (injection accuracy: 0.005 µL). The droplet was recorded at a frame rate of 20 fps, and surface tension changes were monitored for 300 s. Each sample group was measured in five replicates, with the measurement error controlled within ±0.2 mN/m, and the average value was reported. Healthy, clean, and flat pepper leaves were cut into small sections (avoiding the midrib and any lesions), affixed to glass slides using double-sided adhesive tape, and placed on the sample stage of the OCA25 instrument. A 2 µL droplet of deionized water, surfactant solution, or PGR solution was deposited onto the leaf surface using the same dosing unit. The droplet profile was recorded at 20 fps, and the contact angle variation was tracked in real time from 0 to 300 s using the baseline-fitting method. All measurements were conducted at 25 ± 0.5 °C and 35 ± 3% relative humidity. Each type of leaf sample was measured ten times to reduce individual leaf variability [34].

2.6. Calculation of Adhesion Tension and Work of Adhesion

Wetting refers to the process by which one fluid displaces another at the solid-fluid interface. This process can be categorized into three modes: dipping, soaking, and spreading [35]. When a fluid encounters a solid surface, such as a leaf blade, the formation of adhesion typically involves the work of adhesion. The magnitude of the work of adhesion reflects the strength of interaction between the solid and the fluid: the greater the work of adhesion, the stronger the attachment between the fluid and the solid surface, and the more energy required to detach the fluid from the solid.

The adhesion tension and the work of adhesion of a droplet on a solid surface can be calculated using classical wetting theory. These parameters are determined by the contact angle of the liquid on the solid and can be expressed using Equations (4) and (5), respectively [36].

$$\begin{array}{c}\delta =\gamma \cos \theta \end{array}$$

(4)

$$\begin{array}{c}{W}_a=\gamma \left(\begin{array}{c}\cos \theta +1\end{array}\right)\end{array}$$

(5)

where δ is the adhesion tension of the droplet on the solid surface, W_a is the work of adhesion, γ is the surface tension of the solution, and θ is the contact angle of the droplet on the solid surface.

2.7. Data Analysis

All statistical analyses were performed using SPSS software (version 22, IBM Corp., Armonk, NY, USA). Prior to conducting one-way analysis of variance (ANOVA), the Shapiro–Wilk test was used to assess the normality of the data, and Levene’s test was applied to evaluate the homogeneity of variances, ensuring that the assumptions of ANOVA were met. If these assumptions were not satisfied, data were log-transformed before analysis. One-way ANOVA was employed to assess differences among treatments, followed by Tukey’s honestly significant difference (HSD) post hoc test for pairwise comparisons. Statistical significance was set at p ≤ 0.05. Each set of data consisted of three biological replicates with three measurements per biological replicate (n = 9). All results are presented as mean ± standard deviation (SD). These statistical procedures were used to assess the significance of differences in contact angle, surface roughness, and surface free energy at different growth stages and foliar conditions.

3. Results and Discussion

3.1. Surface Morphology of Pepper Leaves at Different Growth Stages

The surface morphology of crop leaves plays a crucial role in determining the spreading behavior of plant growth regulators [16]. As described in Section 2.2, five types of microscopic images of pepper leaves at different growth stages were acquired using a 3D ultra-depth-of-field microscope. Specifically, panels T1 and T2 represent 20× magnified images of the adaxial and abaxial leaf surfaces, highlighting surface textures and venation patterns. Panels T3 and T4 show 500× magnified images of the adaxial and abaxial surfaces, respectively, revealing microstructural features such as epidermal cells and trichomes. Panel T5 displays a cross-sectional micrograph of the leaf, illustrating its internal anatomical structure.

Figure 2 presents the surface micro-morphology of pepper leaves at different growth stages, including low-magnification (20×) and high-magnification (500×) images of both adaxial and abaxial surfaces, as well as cross-sectional views (T5) of the leaf structure. As shown in Figure 2A, at the seedling stage, the adaxial surface exhibited a darker green coloration, indicating higher chlorophyll content, and the main and lateral veins appeared relatively flat. In contrast, the veins on the abaxial surface were prominently raised. At higher magnification (T3 and T4), the adaxial surface displayed more compact and regularly arranged epidermal cells with a smoother texture and sparse trichomes. The abaxial surface; however, exhibited loosely arranged epidermal cells and a higher density of non-glandular trichomes, contributing to a rougher surface texture. The measured surface roughness values were 12.7 μm for the adaxial surface and 17.8 μm for the abaxial surface, confirming a smoother morphology on the adaxial side.

Figure 2B shows the leaf surface characteristics at the early flowering stage. The veins on the abaxial side became more prominent and convex, while the adaxial surface still retained a higher chlorophyll content, as indicated by its darker appearance. High-magnification images revealed the emergence of undulating epidermal cell patterns on the adaxial surface, whereas the abaxial surface exhibited more complex microstructures and increased surface deposits. The surface roughness increased to 21.7 μm on the adaxial side and 38.6 μm on the abaxial side. At the fruiting stage (Figure 2C), both surfaces showed visibly raised veins, with the abaxial side presenting more pronounced surface patterns. The cuticle layer became more distinct, and the adaxial surface continued to display higher chlorophyll accumulation. Surface roughness further increased to 40.8 μm on the adaxial surface and 50.5 μm on the abaxial surface. The cross-sectional image (T5) revealed a thicker epidermis and well-developed palisade tissue on the adaxial side, whereas the abaxial surface had a thinner cuticle and a loosely distributed spongy mesophyll structure.

In summary, the leaf surface morphology—including epidermal cell architecture, trichome density, and cuticle thickness—underwent progressive changes during plant development. The abaxial surface consistently exhibited higher surface roughness and more complex microstructures, which are likely to influence droplet behavior and wettability on the leaf surface.

3.2. Surface Free Energy and Its Components in Pepper Leaves

Surface free energy (SFE) is a fundamental thermodynamic property of solid surfaces and plays a critical role in determining whether a solid can be wetted by a liquid [37]. In this study, the SFE of pepper leaves at the seedling, early flowering, and fruiting stages was determined using the widely adopted Owens-Wendt-Rabel-Kaelble (OWRK) method. Figure 3A–C show the SFE and its components on the leaf surfaces at each stage. At the seedling stage, the adaxial and abaxial surfaces had SFEs of 43.4 mJ/m² and 27.5 mJ/m², respectively. At the primordial stage, the values were 34.2 mJ/m² (adaxial) and 26.2 mJ/m² (abaxial), while at the fruiting stage, the SFEs decreased further to 26.6 mJ/m² (adaxial) and 22.5 mJ/m² (abaxial). At all stages, the SFE of the adaxial surface was consistently higher than that of the abaxial surface, and both decreased gradually as the leaves matured. According to Nairn and Forster et al. [38], hydrophobic surfaces exhibit low surface free energy, whereas hydrophilic surfaces show high surface free energy. Therefore, it can be inferred that the adaxial surface of pepper leaves is more hydrophilic than the abaxial surface at all growth stages and that the overall hydrophilicity of both surfaces declines as the leaf matures.

The surface free energy (SFE) of 22.5 mJ/m² obtained for the abaxial surface of pepper leaves at the fruiting stage is comparable to the SFE of 20 mJ/m² reported for the non-polar solid polytetrafluoroethylene (PTFE) [37]. PTFE is known for its extreme hydrophobicity, exhibiting a water contact angle of 115°, suggesting that the abaxial surface of pepper leaves at the fruiting stage is highly hydrophobic. In contrast, the adaxial surface at the seedling stage exhibited an SFE of 43.2 mJ/m², which is close to the value of 40 mJ/m² reported by Gizer et al. [39] for poly (methyl methacrylate) (PMMA). The polar component (γ^P) and the dispersive component (γd) of the surface free energy are indicators of hydrophilicity and hydrophobicity, respectively [40].

To better describe the relative contributions of the polar and dispersive components to the total surface free energy, we define two dimensionless parameters: R_p (the ratio of the polar component to the total SFE, R_d = γ^d/γ^t) and R_d (the ratio of the dispersive component, R_d = γ^d/γ^t), where γ^t is the total surface free energy. By definition, R_p + R_d = 1. Figure 3D presents the R_p values for the adaxial and abaxial surfaces of pepper leaves at different growth stages. At the seedling stage, the adaxial and abaxial R_p values were 94.70% and 74.37%, respectively; at the primordial stage, 81.67% and 42.37%; and at the fruiting stage, 57.34% and 16.93%, respectively.

These results indicate that at all stages, the adaxial surfaces had higher R_p values than the abaxial surfaces and that R_p decreased progressively as the plant developed. According to Zheng et al. [41], a higher R_p correlates with stronger hydrophilicity, while a higher R_d reflects stronger hydrophobicity, reducing the leaf’s wettability. Therefore, it can be inferred that the adaxial surface of pepper leaves is more hydrophilic than the abaxial surface at all growth stages, and the overall hydrophilicity of both surfaces decreases as the leaf matures. This conclusion aligns with the trends observed in the total surface free energy values.

The surface of pepper leaves at the seedling, flowering, and fruiting stages is covered by a waxy layer. Based on the previously obtained data on leaf surface roughness, it was observed that the roughness of the adaxial surface was consistently lower than that of the abaxial surface, regardless of the growth stage (seedling, early flowering, or fruiting). Furthermore, the surface roughness of the pepper leaves gradually increased with leaf maturation. For surfaces with a waxy coating, greater roughness typically correlates with higher hydrophobicity [42]. This supports the conclusion that the hydrophilicity of the adaxial surface is stronger than that of the abaxial surface, with the hydrophilicity ranking as follows: seedling stage > first-flowering stage > fruiting stage. These findings are consistent with our previous conclusions drawn from both surface-free energy and its components.

3.3. Changes in Contact Angle of 28-HB on Leaves of Peppers at Different Growth Stages

The contact angle is a convenient and rapid method to characterize wettability [43], which is influenced by both the surface morphology and chemistry of the solid, as well as the properties of the liquid [44]. In this study, the surface morphology and roughness of pepper leaves were analyzed using 3D microscopy, and surface free energy and its components were calculated. The results indicate that both surface roughness and surface free energy change during plant development, thereby influencing leaf wettability.

To assess the wettability of pepper leaf surfaces, the contact angles on the adaxial and abaxial sides of the leaves at three different growth stages were measured over time (e.g., Figure 4). The contact angles of solutions on the leaves varied with time, and the rate of change in contact angles on both the adaxial and abaxial surfaces at the fruiting stage was lower compared to the seedling and early flowering stages. During the initial 30 s of measurement, the droplet quickly spread over the leaf surface due to the strong interfacial tension between the solid and liquid phases. Liquid molecules near the solid–liquid interface experienced strong adhesive forces, accelerating the movement of the contact line. As a result, the contact angle decreased rapidly upon contact with the leaf surface. This phase corresponds to the kinetic wetting regime, where inertial and capillary forces dominate. As time progressed, viscous resistance within the moving droplet began to counteract the spreading motion, gradually slowing the contact line velocity until it reached a critical point [45]. Between 30 and 60 s, the rate of change in the contact angle decreased, indicating a transition to a more quasi-static wetting regime. However, dynamic adjustments were still observed, as the contact line approached a stable configuration. In this stage, the system attempted to satisfy the Young’s equation, which defines the equilibrium contact angle based on interfacial tensions. If the contact angle deviates from the equilibrium state, the droplet may retract or spread further until mechanical equilibrium is reached. Notably, for droplets on both adaxial and abaxial leaf surfaces at the seedling stage, a sharp decline in contact angle was observed near 300 s. This may be attributed to evaporation-induced changes in droplet volume and shape, which enhance capillary forces and lead to further spreading. Wang et al. [46] also reported that the contact angle of sweet pepper leaves stabilized within 30 to 36 s. Therefore, in subsequent experiments, the stabilized contact angle at 30 s after the droplet made contact with the leaf surface was chosen for measurement.

Table 1. Stabilized contact angles of surfactants and their 28-HB mixtures on the foliage of peppers at the seedling, early flowering, and fruiting stages.

Growth Stage	Placement	Deionized Water (°)	28-HB (°)	28-HB + CTAB (°)	28-HB + SDS (°)	28-HB + AEO9 (°)
Seedling	Adaxial	65.49 ± 1.25	62.76 ± 0.75	53.66 ± 0.95	55.58 ± 2.20	40.24 ± 1.17
	Abaxial	68.49 ± 1.20	78.76 ± 2.25	44.46 ± 0.92	55.58 ± 2.23	28.24 ± 1.08
Early flowering	Adaxial	82.49 ± 1.35	72.76 ± 0.72	63.66 ± 0.96	45.58 ± 1.23	46.24 ± 2.57
	Abaxial	98.43 ± 1.80	78.78 ± 0.66	62.93 ± 1.57	42.71 ± 1.51	45.75 ± 0.74
Fruiting	Adaxial	83.77 ± 0.61	73.35 ± 1.59	63.11 ± 0.85	42.85 ± 0.84	29.05 ± 0.54
	Abaxial	92.71 ± 0.30	89.19 ± 0.24	72.53 ± 0.43	60.03 ± 0.78	42.49 ± 0.50

Note: Values are mean ± standard deviation.

shows the stabilized contact angles of 28-HB and its mixtures with surfactants on the adaxial and abaxial leaf surfaces of pepper plants at the seedling, early flowering, and fruiting stages. The contact angles of deionized water and 28-HB (8.09 × 10⁻⁶ mol/L) on the adaxial surfaces were consistently smaller than those on the abaxial surfaces across all growth stages. This observation is consistent with the previously calculated higher surface free energy of the adaxial surface, indicating that the adaxial surface is more hydrophilic than the abaxial surface. To enhance the adhesion and spreading of 28-HB on the leaf surface, the addition of appropriate surfactants is necessary to improve its wettability. As can be seen from Table 1, the contact angle of 28-HB on the adaxial and abaxial surfaces of the leaves of peppers at the seedling stage, the early flowering stage, and the fruiting stage decreased, regardless of the addition of the anionic surfactant SDS, the cationic surfactant CTAB, or the nonionic surfactant AEO9. Overall, the lowest contact angles of droplets were observed on both the adaxial and abaxial surfaces of pepper leaves at the seedling and fruiting stages after the addition of AEO9 to 28-HB, compared to the addition of SDS and CTAB. At the seedling stage, the lowest contact angles were observed with the addition of AEO9 and SDS to 28-HB on the adaxial and abaxial surfaces, respectively. These results suggest that the nonionic surfactant AEO9 is most effective at improving wettability at the seedling and fruiting stages, whereas a combination of AEO9 and SDS may be selectively employed at the early flowering stage. It should be noted that these recommendations are specific to pepper and may not be directly generalizable to other species without further verification.

The contact angle on the adaxial and abaxial surfaces of pepper leaves at the early flowering stage after the addition of SDS to 28-HB was similar to that of AEO9 added to 28-HB, whereas the contact angle of CTAB added to 28-HB was larger. This indicates the importance of considering the compatibility between the properties of the pepper leaf surface and the liquid. Since the solid surface free energy and liquid surface tension result from the combined effects of polar and dispersive forces, we analyzed the solid and liquid from the perspectives of R_d and R_p. The surface free energies of the adaxial and abaxial leaf surfaces at the first-flowering stage of peppers were 34.2 mJ/m² and 26.2 mJ/m², respectively (see Section 3.2). At this stage, the R_P of the adaxial surface was 81.67%, and that of the abaxial surface was 42.37%. The polar component of the SDS solution was 29.12%, the AEO9 solution was 26.46%, and the CTAB solution had a polar fraction of 41%. The R_d of the solutions determined the contact angle on the pepper leaves: the larger the R_d, the smaller the contact angle of the solution on the leaves. After the addition of CTAB, SDS, and AEO9 surfactants, the contact angle of 28-HB on pepper leaves was reduced to varying degrees (Figure 5).

3.4. Influence of Droplet Surface Tension and Plant Growth Stage on the State of Droplets on Pepper Leaves

The spreading behavior of droplets is not only influenced by the surface free energy of the solid but is also constrained by the surfactant used [47]. The surface tension of 8.09 × 10⁻⁶ mol/L 28-HB was 57.41 mN/m. When 28-HB (8.09 × 10⁻⁶ mol/L) was mixed with CTAB, SDS, and AEO9 at concentrations of 1.00 × 10⁻² mol/L, the surface tensions of their mixtures were 39.36 mN/m, 34.08 mN/m, and 32.47 mN/m, respectively.

To investigate the effect of surfactants on the contact angle of droplets on pepper leaves and to examine whether the contact angle differed at various growth stages, the two factors—surface tension and growth stage—were analyzed using ANOVA with SPSS software. The results of the experiment are shown in

Table 2. Effect of foliar properties and solution surface tension on contact angle of droplets on pepper leaves.

Scheme	SS	df	MS	F	p
Growth stage	1748.133	5	349.627	209.785	<0.001
Surface tension	13,413.949	3	4471.316	2682.904	<0.001
Growth stage × Surface tension	2637.957	15	175.864	105.523	<0.001

Note: SS, sum of squares; df, degrees of freedom; MS, mean square; F, test statistic; p, probability value. Growth stage (adaxial and abaxial surfaces of pepper leaves at the seedling, primordial, and fruiting stages); surface tension (28-HB, 28-HB + CTAB, 28-HB + SDS, 28-HB + AEO9). A p-value < 0.05 indicates statistical significance.

. It was found that the changes in contact angle of droplets on pepper leaves were significant (p < 0.05) after the addition of CTAB, SDS, and AEO9 to 28-HB. There were also significant differences in the contact angles of the same solutions on the adaxial and abaxial surfaces of pepper leaves at the seedling, early flowering, and fruiting stages. As shown in Table 1, the contact angle of the mixture of 28-HB and CTAB on the abaxial surface of pepper leaves at the seedling stage was 44.46°, whereas the contact angle on the abaxial surface at the early flowering stage was 62.93°, and at the fruiting stage, it was 72.53°. Although there are no obvious trichome on that surface of pepper leaves, changes in surface roughness at different growth stages also affected the contact angle. Therefore, the growth stage significantly affected the contact angle of droplets on pepper leaves (p < 0.05). Based on these results, it can be inferred that different growth stages of peppers require different surfactants to be matched with pesticides.

3.5. Adhesion of 28-HB to Pepper Leaves

During the process of droplet adhesion and spreading on a plant leaf, a force is generated that opposes the surface tension of the droplet and contributes to the stable adhesion of the droplet to the leaf surface, known as the adhesion tension. In other words, the droplets, when near the leaf blade in preparation for the wetting process, generate a maximum separation force required by the leaf blade. When the adhesion tension exceeds the surface tension of the droplet, the droplet is more likely to be deposited on the blade, promoting droplet spreading [48]. Based on the contact angles of droplets on pepper leaves at different growth stages, the corresponding adhesion tension and work of adhesion were calculated using Equations (4) and (5), and the results are shown in

Table 3. Contact angle, adhesion tension, and adhesion function on pepper leaves at seedling, first-flowering, and fruiting stage.

Pepper Leaves	Solution	Contact Angle (°)	Adhesion Tension (mN/m)	Work of Adhesion (mJ/m2)
Adaxial surface of pepper leaves at seedling stage	28-HB	62.76	26.28	83.69
	28-HB + CTAB	53.66	23.32	62.68
	28-HB + SDS	55.58	19.26	53.34
	28-HB + AEO9	40.24	24.79	57.26
Abaxial surface of pepper leaves at seedling stage	28-HB	78.76	11.19	68.60
	28-HB + CTAB	44.46	28.09	67.45
	28-HB + SDS	55.58	19.26	53.34
	28-HB + AEO9	28.24	28.61	61.08
Adaxial surface of pepper leaves at early flowering stage	28-HB	72.76	17.01	74.42
	28 HB + CTAB	63.66	17.46	56.82
	28-HB + SDS	45.58	23.85	57.93
	28 HB + AEO9	46.24	22.46	54.93
Abaxial surface of pepper leaves at early flowering stage	28-HB	78.78	11.17	68.58
	28 HB + CTAB	62.93	17.91	57.27
	28-HB + SDS	42.71	25.04	59.12
	28 HB + AEO9	45.75	22.66	55.13
Adaxial surface of pepper leaves at the fruiting stage	28-HB	73.35	16.45	73.86
	28 HB + CTAB	63.11	17.80	57.16
	28-HB + SDS	42.85	24.99	59.07
	28 HB + AEO9	29.05	28.39	60.86
Abaxial surface of pepper leaves at the fruiting stage	28-HB	89.19	0.812	58.22
	28 HB + CTAB	72.53	11.82	51.18
	28-HB + SDS	60.03	17.02	51.10
	28 HB + AEO9	42.49	23.94	56.41

Note: The contact angle error range in the table has been shown in Section 3.4. The error of adhesion tension is within ±2 mN/m; the error of adhesion work is within ±3 mJ/m².

. From the data in Table 3, it can be observed that there were no negative values for the adhesion tension and work of adhesion of 28-HB on pepper leaves. The contact angle of 28-HB on the adaxial surface of pepper leaves at the young stage was smaller compared to other stages, and as a result, the corresponding adhesion tension and work of adhesion were lower at this stage. Conversely, at the other stages, the adhesion tension and work of adhesion were greater. Upon adding surfactants, although the surfactant reduced the contact angle of droplets on the leaves at the seedling stage, there was no corresponding increase in adhesion tension and work of adhesion. Furthermore, the work of adhesion of 28-HB on pepper leaves at the seedling, first-flowering, and fruiting stages was greater than that observed after surfactant addition at the same growth stages. Therefore, the reduction in contact angle after adding surfactant did not necessarily result in a corresponding increase in the work of adhesion, and surface tension played a certain role, leading to a less pronounced trend in adhesion tension and work of adhesion on the pepper leaves.

The surface tension of the solution used in this experiment ranged from 30 mN/m to 80 mN/m. The adhesion tension of the solution fluctuated in response to changes in the surface tension and contact angle. Specifically, the fluctuation range of adhesion tension for droplets on the abaxial surface of pepper leaves at the early flowering stage ranged from −15 mN/m to 25 mN/m, while the fluctuation range on the leaves at other stages was larger. The fluctuation of the work of adhesion ranged between 50 mJ/m² and 70 mJ/m². When the droplets were on the adaxial leaves of pepper seedlings, the work of adhesion ranged from 50 mJ/m² to 105 mJ/m², showing a greater range of fluctuation. This suggests that the addition of surfactant prompted 28-HB to release more energy required for the leaf-wetting process during the seedling stage of peppers.

4. Conclusions

This study investigated the wetting behavior of 28-homobrassinolide (28-HB), in combination with different surfactants (CTAB, SDS, and AEO-9), on the adaxial and abaxial surfaces of pepper (Capsicum annuum) leaves at three developmental stages (seedling, early flowering, and fruiting). The major findings are as follows:

(1)

With increasing plant maturity, the leaf surface roughness increased markedly from 12.7 μm at the seedling stage to over 50 μm at the fruiting stage, indicating progressively more complex surface structures. At all growth stages, the adaxial surface exhibited higher surface free energy (SFE) than the abaxial surface. However, both the SFE and the ratio of polar components (R_P) decreased over time. Specifically, the adaxial SFE declined from 43.4 mJ/m² at the seedling stage to 26.6 mJ/m² at the fruiting stage, while the abaxial SFE decreased from 27.5 mJ/m² to 22.5 mJ/m². The R_P on the adaxial surface dropped significantly from 94.70% to 57.34%. The addition of AEO9 to 28-HB resulted in a significant reduction in the contact angle on pepper leaves, with values approaching 45° or below. In comparison to 28-HB mixed with SDS and CTAB, 28-HB with AEO9 showed better wettability performance on both the adaxial and abaxial surfaces of the leaves across all growth stages.

(2)

The addition of surfactants significantly enhanced the wettability of 28-HB on pepper leaf surfaces (p < 0.05). Compared with SDS and CTAB, the mixture of 28-HB with AEO-9 resulted in the greatest improvement, reducing the contact angle to below 45°, the adhesion tension to below 30 mN/m, and the work of adhesion to below 105 mJ/m². This enhanced wetting effect was observed consistently on both leaf surfaces across all developmental stages.

These findings suggest that selecting appropriate surfactants based on leaf surface characteristics and developmental stage can markedly improve leaf wettability, potentially increasing the efficacy of foliar-applied agrochemicals. However, the experiments were conducted under controlled laboratory conditions and focused solely on pepper, without accounting for varietal differences or environmental factors. Only three growth stages were examined. While biological replicates were included to control variability, broader applicability should be validated through further studies across different species and under field conditions.