Antibacterial Activity of Sustainable Thymol Nanoemulsion Formulations Against the Bacterial Blight Disease on Cluster Bean Caused by Xanthomonas axonopodis

Abstract

The aim of the present study was nanoencapsulation of thymol to improve its poor water solubility and preservation of encapsulated thymol against environmental conditions. Another goal of the current investigation was to assess the antibacterial activity of thymol nanoemulsion as a sustainable biopesticide to control the bacterial blight of cluster bean. An oil-in-water (o/w) nanoemulsion containing thymol was prepared by a high-energy emulsification method using gum acacia and soya lecithin as natural emulsifiers/surfactants. The characterization of thymol nanoemulsion was carried out using dynamic light scattering (DLS), transmission electron microscope (TEM) and Fourier transform infrared spectroscopy (FTIR). A mean particle size of about 83.38 nm was recorded within 10 min of sonication. The stability analysis of optimized nanoemulsion showed kinetic stability up to two months of storage at room temperature. The thymol nanoemulsion was found to be spherical with a size ranging from 80–200 nm in diameter using transmission electron microscopy. Fourier transform infrared spectroscopy was used to study the molecular interaction between emulsifier/surfactant and thymol. The antibacterial studies of thymol nanoemulsion (0.01–0.06%, v/v) by growth inhibition analysis showed a potential antibacterial effect against Xanthomonas axonopodis pv. cyamopsidis (18–0.1 log CFU/ml). Further, in field experiments, foliar spray of the different concentration of thymol nanoemulsion (0.01–0.06%, v/v) significantly increased the percent efficiency of disease control (25.06–94.48%) and reduced the disease intensity (67.33–4.25%) of bacterial blight in cluster bean.

Introduction

Cluster bean [Cyamopsis tetragonoloba (L.) Taub] also known as “Guar” is an essential drought and temperature-tolerant legume crop grown in the arid and semi-arid region of the Indian subcontinent as well as to a lesser extent in South Africa, Australia, Texas in North America, Brazil, and Oklahoma. It is also grown in rain-fed regions in the states of Rajasthan, Haryana, Gujarat, Punjab, and Uttar Pradesh in India [1]. The cluster bean endosperm contains 19–43% galactomannan gum which has a wide range of industrial applications including textiles, paper, cosmetics, mining, petroleum, paints, ceramics, pharmaceuticals, food processing, oil, and well drilling [2]. Cluster bean suffers from several crop diseases caused by bacteria, viruses, and fungi [3]. Among all the diseases, bacterial blight is the most destructive disease caused due to Xanthomonas axonopodis pv. cyamopsidis (XAC). This bacterium limits cluster bean productivity in all growing regions and is responsible for up to 58% yield loss, especially in irrigated lands and dry upland environments where predisposing factors favour disease development to epidemic proportions [4].

The increased dependency on chemical pesticides has generated severe concerns about sustainability, health risks, and environmental effect [5]. All these negative impacts favour safe, variable, and eco-friendly biopesticides to replace hazardous chemical pesticides [6]. However, biopesticides had some serious drawbacks like poor shelf life, on-field stability, unpredictable performance under alternating climatic conditions, and most critically the high dose required for maximum coverage area. Modern farming is grasping nanotechnology as a novel method to fight overall difficulties of agricultural yield production, sustainability, food preservation, and environmental impact [7]. Nanopesticides have gained benefits due to their highly scientific approach, durability, enhanced efficacy of active ingredients, and dose reduction [8]. Various nanoformulation strategies have been proposed including nanoliposomes, polymeric micelle, nanoemulsions, nanocapsules, solid lipid nanoparticles, and the use of metal nanoparticles. By protecting pesticides in nanocapsules, nano-encapsulated pesticide formulations could help to decrease the phytotoxicity of the active ingredient, reduce the dose of pesticide, and thus enhance solubility and stability achieved [9]. The difficulties in the creation of encapsulated pesticides are ensuring great reproducibility, specificity, selectivity, and controlled release [10].

Thymol (2-isopropyl-5-methyl phenol), is a major phenolic constituent found in essential oil from plants belonging to the Lamiaceae family [11]. It is used widely in the agricultural sector due to its potent antimicrobial, antibacterial, and antioxidant nature [12]. Its hydrophobic nature limits its solubility profile and decreases its biological potential [13]. Furthermore, in the presence of temperature, oxygen, and light, thymol is physically and chemically unstable which reduces its efficiency [14]. Formulating the thymol into nanoemulsion droplets can solve these challenges of poor solubility profile and physical and chemical stability [15]. The nanoemulsion of thymol can be stabilized by using surfactants/emulsifiers of natural or synthetic origin. As a result, natural emulsifiers for the preparation of thymol nanoemulsions are urgently needed for their valuable and safe use in agriculture. Recently, thymol nanoemulsion using food biopolymers of botanical origin as emulsifiers & surfactants, including gum acacia and soya lecithin, has been prepared using a high-energy emulsification method [16].

The primary goal of this research is to prepare a stable thymol nanoemulsion using natural food biopolymers ( gum acacia and soya lecithin) as surfactants and emulsifiers for the assessment of its antibacterial potential against Xanthomonas axonopodis pv. cyamopsidis to control bacterial blight disease of cluster bean. The storage stability of nanoemulsion at room temperature for up to 60 days was also investigated. To date, very less studies on natural food biopolymers like lecithin and gum arabic in combination with the active ingredient (thymol) have been undertaken. As a result, the current study explores a new arena toward an eco-friendly and excellent solution using a compound of natural origin combined with a nanotechnology approach for crop protection and pest management in agriculture.

Materials and Methods

Materials

Thymol was procured from Sigma-Aldrich Corp. (St. Louis, MO). Lecithin soya (30%), Gum Arabic powder, and ethanol (99.9%) were obtained from Hi-Media Laboratories Limited (Mumbai, India). Guar seeds (HG-563) were purchased from CCSHAU, Hisar. The bacterial culture of Xanthomonas axonopodis was isolated manually from infected guar leaves.

Isolation and Purification of Bacterial Culture

Infected portions of plants such as leaves showing symptoms of leaf spots were used for isolation of Xanthomonas axonopodis pv. cyamopsidis. Infected plant parts were chopped and sterilized with 0.1% mercuric chloride solution for two minutes and rinsed systematically three times with distilled water for isolation. Leaves were crushed using a pestle motor and subjected to centrifugation for 3 min at 1300 rpm [17]. Collected the supernatant and then serial dilutions up to six-fold were made and 100 µl was spread on Yeast extract peptone glucose agar (YEPGA) Petri plates. The inoculated Petri plates were incubated for 48 h at 30 ± 2 °C. With the help of a sterilized inoculated loop, the suspected bacterial colonies were picked up and restreaked on the surface YEPGA medium and incubated for 48 to 72 h at 30 ± 2 °C to obtain bright yellow-coloured bacterial colonies. Preliminary characterization of Xanthomonas isolates was done based on microscopies studies, Gram staining, and various other biochemical tests as described in Bergey’s Manual of Systematic Bacteriology [2]. Bacterial isolate was found to be straight rods when observed under the microscope and the size ranged from 0.5 to 0.7 × 0.7 to 1.6 μm. They were found to be Gram-negative and produced yellow-colored colonies on the YEPGA medium. The purified bacterial colonies were maintained on YEPGA slants and kept refrigerated at 4 °C for further studies.

Preparation of Thymol Nanoemulsion

Nanoemulsions were obtained using a high-energy emulsification process as described previously with certain modifications [18]. Gum arabic and lecithin were used as natural surfactants, ethanol as a co-surfactant, thymol as an active ingredient (oil phase), and water as the aqueous phase. For the preparation of nanoemulsion, gum Arabic (5 mg/ml) and soy lecithin (10 mg/ml) were weighed in a ratio of 1:2 and mixed in 20 ml of distilled water and stirred at 600 rpm using a magnetic stirrer for 30 min at room temperature. Then, the oil phase [thymol (1 mg/ml) was mixed with ethanol (100 µl/ml] and added slowly in a dropwise manner into an aqueous phase and further stirred at 600 rpm for 15 min at room temperature. The prepared formulations were then sonicated for 10 min using a Probe sonicator (Bandelin-Sonoplus, Berlin). The energy was delivered through a sonicator probe, which generates disruptive forces and minimizes emulsion droplet diameter. The maximum power output of the probe sonication was 25 W at a frequency of around 20 kHz. It was observed that a 50 ml glass beaker kept in a 500 ml glass beaker with ice and pulse rate (10 s on/off) succeeded well enough to keep the temperature below 35 °C. As a stable formulation, the sample with no visible sign of phase separation, sedimentation, and flocculation was chosen by visual observation. Nanoemulsion sizes were monitored during sonication by the obtained sample at a definite time interval (0 min—10 min) and characterized by DLS and the droplet size was specified in nanometers (nm).

Stability of Thymol Nanoemulsion

By storing the nanoemulsions at room temperature, the intrinsic stability of thymol nanoemulsions was investigated by monitoring particle size and zeta potential after every 7 days till 35 days. After 35 days, thymol nanoemulsion was visually examined for signs of instability (creaming or phase separation or flocculation). A particle size analyzer was used to assess the change in droplet size and zeta potential at different intervals of time to determine kinetic stability.

Characterization of Thymol Nanoemulsion

Particle Size, PDI, and Zeta Potential Measurements

Particle size, polydispersity index, and zeta potential of nanoemulsions were determined by dynamic light scattering technique with a 120 s equilibration time and 11 runs using the zeta sizer Nano ZS 90 (Malvern UK). DLS analysis was carried out for up to one month at a 7-day interval and then after 60 days of storage at 25 °C. DLS analysis also recorded data of particle size by number, as required for nanoemulsions interpretation.

Measurement of Turbidity

The turbidity of all formulated nanoemulsions was analyzed by measuring the absorbance at 600 nm from (0 min to 10 min) sonication after 2 months of undiluted nanoemulsion using UV–Vis spectrophotometer (SPECTRO star^Nano, BMG LABTECH, Germany). The results were calculated as mean ± SE.

FTIR and Transmission Electron Microscopy (TEM)

FTIR (Spectrum II, Perkin Elmer Spectrum, BX II, Germany) was employed to analyze the alternation in chemical bonds of oil molecules and active ingredients present in the emulsions. In the regions of 4000 to 500 cm⁻¹, the FTIR spectroscopy of thymol nanoemulsion and pure thymol was performed. A nanoemulsion drop was placed on the KBr pellet. A small drop of nanoemulsion (20 µl) was loaded on a carbon-coated copper grid for morphological assessment and stained with 2% phosphor tungstic acid and dried for 5 min at room temperature [19]. A transmission electron microscope with a 200 kV (TEM, TECNAI 200 kV, Fei, Electron optics) accelerating voltage was used to examine the prepared sample.

CFU Measurements and Growth Kinetics Analysis

Growth inhibition tests were used to investigate the antibacterial potential of the thymol nanoemulsion as reported earlier [20]. XAC colonies were inoculated into 50 ml YEPGA broth and cultured for 48 h in a rotary shaker incubator at 30 ± 2 °C. 50 μl of mother culture was added to 5.0 ml YEPGA broth supplemented with various doses of thymol nanoemulsions (0.01 to 0.06%, v/v) as well as water as negative control and Streptomycin sulphate (0.01%, w/v) as a positive control. Cultures were incubated at 30 ± 2 °C on a rotary shaker incubator at 200 rpm. A spectrophotometer was used to measure optical density at 600 nm to evaluate bacterial growth (SPECTRO star^Nano, BMG LABTECH, Germany). Colony-forming units were used to evaluate the number of viable cells.1 ml culture samples of various treatments from the stationary growth phase were obtained and diluted to 10⁶ folds for CFU measurements. To obtain, distinct colonies of diluted samples (100 μl each) were distributed using an L-shaped spreader on yeast extract peptone glucose agar medium Petri plates. The Petri plates were incubated for 48 h at 30 ± 2 °C. The number of viable cells after incubation was counted and compared to determine the antibacterial properties of the treatments.

Effect of Nanoemulsion on Bacterial Blight Disease

Field experiments were performed at Guru Jambheswar University of Science &Technology, Hisar during the Kharif season (2021–2022) in a randomized block design with three replications under an average of 90% relative humidity. Each plot size was 4 rows × 1.5 m × 1.2 m with 0.4 m row-to-row spacing and the crop was sown with a spacing of 30 × 10 cm. Cluster bean seeds (HG-563) were collected from CCSHAU, Hisar, India. Xanthomonas. axonopodis pv. cyamopsidis were artificially inoculated after one month of germination under moist conditions maintained by water spraying during the daytime as per the previous method [21]. In short, the bacterial strain was grown on YEPGA medium plates for 48 h at 30 °C ± 2 °C to prepare the inoculum. The bacterial suspension was sprayed onto the leaf surface of cluster bean plants using a spray bottle. Foliar spray of thymol nanoemulsion (0.01–0.06%, v/v) along with water and Streptomycin sulphate as a negative and positive control was administrated after every 15 days of the first emergence of disease symptoms until run-off through a spray bottle. Leaves from each triplicate were chosen for observation to assess bacterial blight disease. Percent Disease intensity (PDI) was assessed early in the morning on a scale of 0 to 9 (leaves with no evident symptoms = 0; few individual lesions = 2; many individual lesions = 4; small patches of coalesced lesions = 6; medium-sized patches of coalesced lesions = 8; and large patches of consolidated lesions = 9) [22].

Furthermore, the disease intensity and PEDC (percent efficacy of disease control) was calculated by the formula given below [23, 24].

DI = Sum of all individual disease rating/total number of leaves examined–maximum rating × 100

PEDC = Disease index in control plots–disease index in treatment plots/disease index in control plots × 100

Statistical Analysis

The significance of the effect of Thymol nanoemulsion was evaluated by one-way analysis of variance (ANOVA) using Microsoft Excel. The significant effects were designated by p-value and all the experimental data were presented as mean ± standard deviation (SD).

Results

Droplet Size, PDI, and Zeta Potential Measurement

In a 100 ml glass beaker, sonication parameters were standardized for a 20 ml reaction volume containing gum arabic, thymol, soy lecithin, ethanol, and water. Initially, the coarse emulsion was made by mixing emulsifiers and surfactants and water followed by the addition of thymol as an oil phase using a magnetic stirrer at 600 rpm for 45 min. After that, a probe was placed in the center of the 100 ml glass beaker containing 20 ml reaction volume for sonication. A 100 ml glass beaker kept in a 500 ml glass beaker containing ice and then a 10-s pulse rate (on/ off) was found to be successful in keeping the temperature below 35 °C. As a result, droplet size decreased as sonication time increased from 0 min, 2 min, 4 min, 6 min, 8 min, and 10 min and the corresponding values obtained were 252.7 nm, 205.9 nm, 173.1 nm, 117.1 nm, 95.97 nm, 83.38 nm respectively (Fig. 1a). Similarly, when the sonication period increased (0 min to 10 min) polydispersity index values were reduced significantly (0.190, 0.227, 0.137, 0.144, 0.145, and 0.108) (Fig. 1b). The Zeta potential of nanoemulsion declined as sonication time increased (0 min to 10 min) and corresponding values obtained were −32.7, −34.9, −41.0, −47.8, −48.3, and −51.4 respectively (Fig. 1c).

Fig. 1.

DLS measurements of thymol nanoemulsions at 0 days a particle size (nm) of 0–10 min sonicated nanoemulsions b PDI of 0–10 min sonicated nanoemulsions c zeta potential (mV) of 0–10 min sonicated nanoemulsions

Stability of Thymol Nanoemulsion

Thymol nanoemulsions stability at room temperature (25 °C) was also examined methodically by monitoring particle size and zeta potential every 7 days to 35 days. After 35 days, phase separation and flocculation were also visually observed in nanoemulsions obtained by 0 min to 6 min of sonication whereas no phase separation was noticed in nanoemulsions obtained by 8 to 10 min sonication. The droplet size of nanoemulsion prepared by 0 min to 6 min of sonication increased up to 35 days, but it remained unchanged in samples obtained with 8 min to 10 min of sonication (Fig. 2a). However, zeta potential (mV) remained invariable in thymol nanoemulsions obtained by 8 min to 10 min of sonication in contrast to 0 min to 6 min of sonicated nanoemulsion (Fig. 2b). After 2 months of storage for 8 to 10 min, sonicated nanoemulsions showed stability in terms of particle size, PDI, and zeta potential with minor changes (Table 1). When compared to 0 – 6 min of sonicated nanoemulsion the size, polydispersity index, and zeta potential of the thymol nanoemulsions obtained by 8 to 10 min of sonication exhibited no significant changes after 2 months of storage. Zeta potential indicates colloidal stability due to electrostatic repulsion between droplets [25].

Fig. 2.

a A comparative graphical representation of particle size and b zeta potential of thymol nanoemulsions from 0–35 days at an interval of 7 days prepared by different sonication time

Table 1.

Characterization of optimized thymol nanoemulsions at different sonication times after two months

Sonication time (min)	Particle size (nm)	PDI value	Zeta potential (mv)	Turbidity at 600 nm (O.D.)
0	540.2 ± 6.0	0.579 ± 0.3	−28.6 ± 0.7	3.513 ± 0.2
2	359.4 ± 5.4	0.526 ± 0.2	−30.7 ± 0.4	3.014 ± 0.2
4	231.3 ± 4.1	0.470 ± 0.1	−31.8 ± 0.6	2.170 ± 0.0
6	179.1 ± 2.8	0.437 ± 0.1	−37.1 ± 0.3	0.667 ± 0.1
8	114.0 ± 2.1	0.351 ± 0.1	−41.0 ± 0.5	0.485 ± 0.1
10	94.65 ± 1.3	0.288 ± 0.1	−46.3 ± 0.2	0.347 ± 0.0

Turbidity Measurement

The absorbance at 600 nm was used to measure the turbidity of the undiluted nanoemulsion. The turbidity of thymol nanoemulsions after two months is shown in Table 1. The turbid nature of the 0 min sonicated formulation (coarse emulsion) was due to the highest absorbance value of 3.513. With the lowest absorbance (600 nm) of 0.347, 2–6 min of sonicated nanoformulation were translucent and 8 to 10 min of sonicated nanoformulation were optically transparent. Thus, thymol nanoemulsion obtained after 10 min sonication was used for subsequent studies.

FTIR Spectroscopy

Figure 3 shows the FTIR spectrum of pure thymol and optimized thymol nanoemulsion to identify their molecular interactions. Thymol, as a phenolic compound with an amorphous structure, exhibited several distinct peaks [26], including 3639 cm⁻¹, 3599 cm⁻¹, 3572 cm⁻¹, and 3539 cm⁻¹ indicating OH stretching at the benzene group and 1660 cm⁻¹, 1639 cm⁻¹, 1613 cm⁻¹ indicating C = C stretching in the aromatic ring and the fingerprint region, where the stretching vibrations for C-O were located at 1243 cm⁻¹, 1157 cm⁻¹ and 1091 cm⁻¹ [27, 28] as well as 667 cm⁻¹ (alkyne C-H bend) in Fig. 3a. A smooth and broadened peak at 3423 cm⁻¹ was detected in the spectra of thymol nanoemulsion (Fig. 3b), indicating the existence of –OH stretching of carboxylic acids from gum acacia. The greater intensity of this peak was partly related to the hydrogen bonding between gum acacia and thymol [29]. The presence of a strong peak at 1631 cm⁻¹ demonstrated the appearance of C = C stretching in the aromatic ring of thymol, indicating the presence of thymol [30].

Fig. 3.

FTIR spectrum of a Pure thymol and b Thymol nanoemulsion

TEM

Figure 4(a) shows the TEM image and equivalent size distribution histogram of optimized thymol nanoemulsion (Fig. 4b). TEM micrograph displayed spherical droplets of 150 nm in 10 min sonicated thymol nanoemulsion (Fig. 4 a, b). Most of the particle size was below 150 nm according to the DLS analysis of the 10 min sonicated thymol nanoemulsion (Fig. 1 a). Thus, TEM results are also in agreement with the DLS analysis.

Fig. 4.

Morphological characterization of optimized thymol nanoemulsion a TEM micrograph (inset zoom image) b particle size distribution histogram of thymol nanoemulsion

In Vitro Antibacterial Activity

The prepared thymol nanoemulsion was subjected to investigation for antibacterial potential and disease control. Figure 5(a) shows the growth kinetics of Xanthomonas axonopodis pv. cyampsidis at various doses of nanoemulsion of thymol, distilled water as negative control, and 0.01% w/v Streptomycin sulphate as a positive control. The time-kill curve revealed a potential growth inhibitory impact through zero absorbance at 0.04 to 0.06% v/v concentration of nanoemulsion. The antibacterial potential was also assessed using log CFU/ml (Fig. 5 b). Thymol nanoemulsion at 0.04 to 0.06% (v/v) concentration suppressed bacterial growth, similar to the positive control. The results demonstrated that the prepared thymol nanoemulsion has considerable antibacterial activity against XAC.

Fig. 5.

Antibacterial activity of negative control (-ve CN, water), positive control (+ ve CN, streptomycin sulphate (0.01% v/v) and thymol nanoemulsions (NE 0.01–0.06%, v/v) a optical density (O.D.) at 600 nm and b Colony Forming Unit (CFU/ml)

Effect of Nanoemulsion on Bacterial Blight Disease

Due to its significant antibacterial activity against Xanthomonas axonopodis pv. cyamopsidis under in vitro conditions the field investigation was initiated to evaluate its efficacy against the incidence of bacterial blight in cluster bean. The soil temperature was 25–35 °C and the soil type during monsoon in the university was sierozem soil (Brownish-greyish surface on a lighter layer). Due to the rainy season, irrigation was required in the early stage of cropping only and weeds were removed from time to time manually by using a draw hoe and trowel. Our guar crop responds best even without fertilizer as they are growing in nutrient-rich soil. In the field experiments, bacterial blight disease symptoms were noticed after two weeks of artificial inoculation of Xanthomonas axonopodis pv. cyamopsidis.

After disease emergence, a foliar spray of water (negative control), antibiotics (positive control), and different concentrations of nanoemulsion of thymol (0.01–0.06%, v/v) were applied. After one week of application, data on the percent efficacy of disease control (PEDC) and disease intensity (DI) were collected (Fig. 6). Lesions expanded in negative control plants leading to disease severity of up to 78% and 2% disease severity was recorded in positive control plants. Thymol nanoemulsion-treated plants with different concentrations (0.01 to 0.06%) showed considerably lower disease severity up to 67% to 4% (Table 2). Excellent PEDC was observed in plants treated with 0.06%, v/v thymol nanoemulsion and 0.01%, v/v Streptomycin sulphate as a positive control.

Fig. 6.

Symptoms of bacterial blight disease on cluster bean plants in field experiments a Foliar spray of thymol nanoemulsion at 0.06%, v/v concentration b large precise necrotic lesions on leaves and blighting in negative control plants (water) c small yellow lesions at the tip of cluster bean leaves at 0.06%, v/v thymol nanoemulsion d little blighting in positive control cluster bean plants at 0.01%, v/v Streptomycin sulphate

Table 2.

Disease intensity and percent efficacy of disease control at various doses of thymol nanoemulsion, water, and streptomycin sulphate in a field experiment of cluster bean. The mean value in triplicate (each triplicate contains 3 plants sample)

Treatment (%), v/v	Disease intensity (DI) (%)	Percent efficacy of disease control (PEDC) (%)
Negative control (Water)	78.00 ± 2.1	0.00 ± 0.0
Positive control (0.01%, Streptomycin sulphate)	2.23 ± 2.9	96.44 ± 0.8
Thymol nanoemulsion
0.01	67.33 ± 4.6	25.06 ± 5.0
0.02	56.90 ± 3.4	37.14 ± 3.6
0.03	49.19 ± 2.3	58.96 ± 2.8
0.04	30.38 ± 1.4	66.34 ± 1.9
0.05	18.66 ± 0.9	78.35 ± 1.2
0.06	4.25 ± 0.8	94.48 ± 1.0

Discussion

In the present study, thymol nanoemulsion was successfully obtained using natural emulsifiers (gum arabic and lecithin) as a sustainable biopesticide against the bacterial blight of cluster bean. The nanoemulsion was developed by a high-energy emulsification method with a nanometric dimension and confirmed stability for two months at room temperature. A nanoemulsion's droplet size is determined by the components' interactions, the processing parameters used, and the surfactants' adsorption into the oil phase [31]. The method of ultrasonication was selected for the production of nanoemulsions because it encourages the development of small particles since the sonicator generates a variety of disruptive forces that promote turbulence and cavitation in the formulation [32]. This method has lower manufacturing costs, less surfactant concentration, and easier cleaning and operation. Thymol nanoemulsion that had been sonicated for 10 min produced the most stable nanoemulsion, with a z-average of 83.38 ± 4.2 nm, a PDI of 0.108 ± 0.02, and zeta potential of −51.4 ± 2.6 mV. In comparison to 10 min of sonication, nanoemulsion created by 0 to 8 min of sonication had larger droplet diameters and a higher PDI value. Increasing the sonication time from 0 to 10 min significantly decreased particle size from 252.7 to 83.38 nm and PDI value from 0.190 to 0.108 demonstrating that sonication time affects the size, polydispersity index, and ultimately the stability of nanoemulsion [33]. The majority of commercial applications require that nanoemulsions maintain their physical stability, or little to no change in particle size, during the course of their shelf life. Long-term stability is achieved by reducing particle size and narrowing the size distribution, which inhibits coalescence, flocculation, and Ostwald ripening [34]. The higher zeta potential of nanoemulsion is an important characteristic of stability which contributed to higher electrostatic repulsion among droplets [25]. A similar result was reported previously for thymol nanoemulsion obtained by ultrasonication method using thymol, saponin, and water indicating strong antibacterial potential, growth-promoting effects, and disease control [14]. Thus, thymol nanoemulsions prepared by 0 to 8 min of sonication showed higher zeta potential as compared to 10 min sonicated thymol nanoemulsion (Fig. 2b). The phosphate group of lecithin was primarily responsible for the negative zeta potential (−51.4 mV) of the thymol nanoemulsion. Lecithin has a stronger hydrophobic character and would adhere to the oil/aqueous edge which were surrounded by gum Arabic with higher hydrophilicity [29]. Chemical deformation of the oil phase may be brought on by intense high pressure and cavitations during the sonication process of emulsification [14, 35]. Practically, a high surfactant/emulsifier concentration is maintained to produce nanoemulsions with smaller particle sizes and greater stability [13]. However, in this study, we managed to produce a substantially smaller particle size and an incredibly stable nanoemulsion by keeping the gum acacia/lecithin concentration very low. Higher surfactant/emulsifier concentrations generally add toxicity and reduce the biological activity of the primary nanoemulsion component [36]. As a result, the thymol nanoemulsion produced in the current work with low concentrations of gum acacia and lecithin can be used in crops and foods without harming the environment. After 25–35 days of storage, a nanoemulsion created with less than 6 min of sonication showed evidence of phase separation by creaming and flocculation. However, the intensity of creaming was lesser in 8 min sonicated thymol nanoemulsions as compared to 0 to 6 min. The results strengthen the fact that higher sonication time gives more kinetic energy to nanoemulsions [37].

The size of the prepared nanoemulsions determines their turbidity also. The reduced turbidity of sonicated nanoemulsion after 8–10 min can be attributed to a minimum particle size that results in low scattering and makes the obtained nanoemulsion systematically transparent [38]. Keeping this in view, FTIR spectra of pure thymol and thymol nanoemulsion were studied for any chemical change in thymol and its interaction with gum acacia and lecithin. Along with a broad peak at 3423 cm⁻¹ which showed H-OH interaction and a sharp peak at 1631 cm⁻¹ which showed C=C stretching in thymol nanoemulsion, specifying that thymol was chemically stable and simply entrapped within gum acacia and lecithin [30]. TEM micrograph showed spherical-shaped droplets of 150 nm in 10 min sonicated thymol nanoemulsion (Fig. 4 a, b). Most of the particle size was below 150 nm according to the DLS analysis of the 10 min sonicated thymol nanoemulsion (Fig. 1 a). Thus, TEM results are in agreement with the DLS analysis. Previous research confirmed that the majority of essential oil nanoemulsions have a spherical appearance [39]. 10 min sonicated thymol nanoemulsion showed strong antibacterial potential against Xanthomonas axonopodis pv. cyamopsidis. At the concentration of 0.01–0.06% (v/v), it significantly inhibited bacterial growth (Fig. 5a, b), and specifically, no bacterial colony was observed in 0.04 to 0.06% (v/v) of thymol nanoemulsions which was similar to positive control. Traditionally, thymol is known as a reliable antibacterial agent [14, 40].

Previous research has demonstrated that thymol nanoemulsions have a more significant effect on the inhibition of bacteria growth as compared to poorly water-soluble bulk thymol [41]. This could be understood by the fact that uniformly dispersed nano-droplets of thymol nanoemulsion can easily penetrate and disrupt the microbial membrane [42]. Due to its potential antibacterial effects against Xanthomonas axonopodis pv. cyamopsidis in vitro experiments, we carried out field experiments to study its further protective efficacy against bacterial blight incidence in cluster bean. Negative Control (water) showed significantly higher disease intensity (78 ± 2.16), whereas incredibly lower disease intensity (30.38–4.25%) was found in 0.04 to 0.06% of thymol nanoemulsion (Table 2). The pattern of lesion formation and its further incorporation was visually distinguishable in control (water) and thymol nanoemulsion (Fig. 6).). To determine the effectiveness of a Thymol nanoemulsion, one-way ANOVA was used as a statistical method. Data on disease severity were used in the statistical analysis, and the three treatments were compared. The results were significant, with a p-value of less than 0.05, according to the analysis of variance (Table 2). As can be seen from the data in Table 2, foliar treatments with thymol nanoemulsion (0.06%) significantly showed lower disease severity as compared to water-based negative control which was nearly similar to positive control ( streptomycin sulphate) spray for cluster bean plants. The results confirmed that thymol nanoemulsion (0.06%) was more successful and showed the highest percent efficiency of disease control which was nearly similar to positive control. A significantly lower disease intensity and higher PEDC of thymol nanoemulsion in field experiments could be explained by the antibacterial activity of thymol nanoemulsion [43]. The observed results confirmed the potential antibacterial effects of thymol nanoemulsion which was nearly the same as the positive control (Streptomycin sulphate). For thymol to be used in the future as a plant growth promoter, more understanding of the mechanisms governing its interactions with plants would be advantageous. Additionally, the field application of the produced nanoemulsion for disease control and yield is being studied.

Conclusions

In the present study, thymol nanoemulsion was successfully obtained using natural emulsifiers (gum acacia and soya lecithin) as a sustainable biopesticide against the bacterial blight of cluster bean. The nanoemulsion was prepared using a high-energy emulsification method with a nanometric dimension and confirmed stability for two months at room temperature. In field experiments, the prepared thymol nanoemulsion demonstrated strong antibacterial potential against Xanthomonas axonopodis pv. cyamopsidis. This eco-friendly nanotechnology-based approach can efficiently overcome the pathogenic episodes faced by guar gum farmers. Nanotechnology-based agrochemical solutions lead to efficient crop production and more prosperous farmers. The application of natural nano compounds in pest management can also overcome key issues like resistance and toxicity resulting in enhanced crop production.

Author Contribution

Pooja Choudhary: Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review & editing; Gaurav Bhanjana: Review and Editing, Data Analysis; Neeraj Dilbaghi: Conceptualization, Data curation, Investigation, Methodology, Supervision, Writing – review & editing; Sandeep Kumar: Resources, Writing – review & editing.

Funding

Pooja Choudhary is grateful to the Council of Scientific and Industrial Research (CSIR), New Delhi, [Ref. No. 09/752(0083)/2018/EMR-1] for providing financial support to her in the form of a Junior Research Fellowship (JRF). Dr. Gaurav is also thankful to CSIR, Govt. of India for providing CSIR-SRA (No. B-12998 dated 31 March 2023).

Data Availability

All data supporting the results and discussions of this article are provided as tables and figures.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical Approval and Consent to Participate

Not applicable.

Consent for publication

Not applicable.