Ultrasonic Fabrication and Evaluation of Azoxystrobin Nanoemulsion against Plant Pathogenic Fungi

INTRODUCTION

Biological constraints, for example, fungal and bacterial pathogens, viruses, arthropods, and weeds, are responsible for major losses in quality and yield of crops and grasslands (Begna, 2020). Effective pest management is a major challenge in modern agriculture, with a need to consider control efficacy, cost affordability, environmental safety, toxicity towards non-target organisms, and sustainability of the production system (Khan et al., 2023). Despite progress in many technological fields, most management of these constraints is still based on the use of synthetic chemicals (Harun-Ur-Rashid and Imran, 2025). However, a large number of pesticides have already been withdrawn for regulatory reasons, because of their hazardous effects on the ecosystem or on the food chain, or because they have become ineffective as the result of increasing pesticide resistance (Lazarević-Pašti et al., 2025). These compounds are not being effectively replaced, causing serious difficulties for farmers in managing pests. Consequently, there is a renewed interest in the development of alternatives to synthetic pesticides.

The development of alternative fungus management techniques capable of reducing environmental issues while controlling infections that are both susceptible and fungicide-resistant plant protection by nanoemulsion has recently enabled the ability to use pesticides in an environmentally beneficial manner on a site-specific basis (Abd-Elsalam, 2024). Smart dispersion of nanopesticides is required to minimize pesticide dosage while increasing effectiveness. The efficacy, potential antifungal mechanisms, and synergy profiles with conventional fungicides are debated in relation to the potential use of metal NPs, biocontrol agents, and active ingredients of synthetic fungicides as nanofungicides, both as alternatives to conventional fungicides or/and as partners in the fight against fungicide resistance (Islam et al., 2024). Recent advances in nanotechnology have paved the way for the development of nanosized delivery systems. These systems enable effective fungicide delivery to target pathogens and enhance the bioavailability of fungicides while minimising environmental and human health risks (Elzein, 2024).

Azoxystrobin is a systemic methacrylate fungicide that is derived from the strobilurin family. As the first globally available germicide in the past decade, it attracted well-deserved attention on the development of its novel nanoformulations. Minimizing the size of its crystals to nanoscale is a popular research objective (Yao et al., 2018)

Azoxystrobin, a quinone outside inhibitor fungicide, reduced tobacco target spot caused by R. solani by 62%, but also affected the composition and diversity of other microbes on the surface and interior of treated tobacco leaves (Sun et al., 2023). High-throughput sequencing showed that the dominant bacteria prior to azoxystrobin treatment were Methylobacterium on healthy leaves and Pseudomonas on diseased leaves, and the dominant fungi were Thanatephorous (teleomorph of Rhizoctonia) and Symmetrospora on healthy leaves and Thanatephorous on diseased leaves. Both bacterial and fungal diversity significantly increased 1 to 18 days post treatment with azoxystrobin for healthy and diseased leaves.

The impact of azoxystrobin against pathogens on the soil microbiota and enzymes, as well as plant growth and development was assessed (Baćmaga et al., 2024). The laboratory experiment was conducted in three analytical terms (30, 60, and 90 days) on sandy clay (pH 7.0). Azoxystrobin was applied to soil in doses of 0.00, 0.110 and 32.92 mg/ kg d.m. of soil. Its 0.110 mg/ kg dose stimulated the proliferation of organotrophic bacteria and actinobacteria but inhibited that of fungi. It also contributed to an increase in the colony development index and a decrease in the ecophysiological diversity index of all analyzed groups of microorganisms.

 In order to control the release of fungicides in response to warm conditions, and enhance the efficacy, a series of thermo-responsive fungicide-loaded nanoparticles were developed (Baćmaga et al., 2024). The fungicide azoxystrobin, solvent, emulsifier Tween 80 and thermo-responsive component were combined to create thermal-response oil phases, conditions for emulsification were then optimized. The results indicated that the formula with 5 g azoxystrobin, 10 mL solvent, 6 mL Tween 80 and 2.5 g the proposed oil phase with the ability to transform from solid at 20 °C to soften at 31.5 °C. The optimal nanoparticles had a mean particle size of 162.1 nm, thermo-responsive morphological transformation between 20 °C and 30 °C, azoxystrobin crystal reforming after drying, the ability to attach to fungal spores and satisfied antifungal efficacy against P. nicotiana  and A. niger at 30 °C.

This study intends to fabricate an azoxystrobin nanoemulsion comprised of commercial and technical fungicide and evaluate antifungal potential of the nanoemulsion against phytopathogenic fungi A. alternata, F. oxysporum and R. stolonifer In vitro and In vivo. The research of this nanoformulation could lead to the discovery of a new fungicide that can be an alternative to harmful fungicides in crop protection.

MATERIAL AND METHODS

Fungicide and Chemicals

Azoxystrobin (Amistar 25% SC, Methyl (2E)-2-(2-{[6-(2-cyanophenoxy)pyrimidin-4-yl]oxy}phenyl)-3-methoxyprop-2-enoate) was supplied by El-Hoda Company (Wadi El Natrun area, El-Behira governorate, Egypt).Tween 80, catechol, 3,5-Dinitrosalicylic acid and dimethyl sulphoxide (DMSO) were procured from Sigma-Aldrich, St. Louis, MO, USA. Potato dextrose agar (PDA) was acquired from Oxoid Ltd., Basingstoke, Hampshire, UK. p-Aminobenzoic acid was obtained from Acros Organics, New Jersey, USA. Carboxymethyl cellulose was obtained from Kelong Chemical Agent Factory, Chengdu, China. Bromothymol blue was acquired from Merck, Germany.

Tested fungi

Phytopathogenic fungi, Alternaria alternata (Fries), Fusarium oxysporum (Schiech), and Rhizopus stolonifer(Ehren) were used in the present study. They obtained from the Microbiology Laboratory, Department of Plant Pathology, Faculty of Agriculture, Damanhour University, Damanhour, Egypt, and kept during the experiments on PDA medium at 27 ± 2°C.

Preparation of nanoemulsion containing fungicide

Nanoemulsion was prepared by the procedure reported by (Sugumar et al., 2014), with some modifications. The nanoemulsion was prepared in two phases. The coarse emulsion was prepared by stirring and then further emulsification using a high-energy ultrasonic process. Azoxystrobin nanoemulsion was prepared as follow: a.i (0.5 %), DMSO (44%), Tween 80 (15%), water (40.5%), sonication pulses (9 cycle/sec) at sonication power (75% of sonicator power (20 kHz)) for 15 min at 25^◦C. The formation is clear and transparent (Fig. 1) (Li & Chiang, 2012).

Fig. 1. Schematic illustration of the preparation of nanoemulsion containing azoxystrobin

SEM analysis was done by using a JEOL JSM-5410 (Japan) electron microscope with a W-source and operating at 25 kV. The sample was prepared on a glass slide (1×1 cm) after washing it with ethanol. A small drop of nanoemulsion has spread evenly over the glass slide and allowed air to dry. To make it conductive, gold coating with Jeol Quick Auto Coater was performed (JFC-1500). The slides were then subjected to SEM analysis under ambient conditions.

Particle size and poly dispersity index (PDI) assay

The mean droplet size and poly dispersity index (PDI) of nanoemulsion formulation was performed by a dynamic light scattering method using Zetasizer Nano ZS (Malvern Instruments, UK) at room temperature. Sample was diluted before measurements to 10% with deionized water to avoid multiple scattering effects. Emulsion droplet size was estimated by the average of three measurements and presented as mean diameter in nm. The higher the PDI value refers to the lower uniformity of globules size of nanoemulsion(Tyagi et al., 2012).

Centrifugation and freeze thaw cycle test

Following centrifugation of the sample at 5000 rpm for 30 minutes, phase separation, creaming, and cracking were observed. These observations indicate a lack of the desired maximum stability for the nanoemulsion, which ideally should exhibit no phase separation (i.e., creaming or cracking). Successful formulations will subsequently undergo further thermodynamic stability testing. To assess the accelerated stability of the nanoemulsion formulation, we conducted a study involving temperature variations. The formulation was exposed to -21°C and 21°C, with each temperature condition maintained for at least 24 hours. All measurements were carried out in triplicate for reliability (Kadhim and Abbas, 2015).

Heating cooling cycle test

This test evaluated the impact of heating and cooling cycles on the stability of the nanoemulsion formulation. We stored the prepared nanoemulsion at 4°C and 40°C, with each temperature sustained for 48 hours. The formulation showed stability under these conditions and is therefore suitable for further investigation(Kadhim and Abbas, 2015).

Stability at 25°C

A 25 ml sample of freshly prepared nanoemulsion was transferred to a glass tube. Over a 4-week storage period, the nanoemulsion transitioned from a steady state to exhibiting creaming and coalescence (Badawy et al., 2017).

Viscosity and pH measurements

The dynamic viscosity of the nanoemulsion was measured without dilution using a Rotary Myr VR 3000 digital viscometer. An L1 and L1 spindle combination was employed, with measurements conducted at 100 rpm and 29.5°C. To ensure accuracy, each reading was taken following a two-minute sample equilibration period. All measurements were performed in triplicate, and the viscosity data are reported in mPa.s. pH value observations are integral to determining nanoemulsion stability, as shifts in pH can signal the occurrence of chemical reactions within the formulation. A digital pH meter was employed for all pH measurements  (Junyaprasert et al., 2007).

Fungicidal activity of azoxystrobin nanoemulsion on mycelial growth

The fungicidal activity was established through the mycelial radial growth technique, adapting the method by Badawy et al. (2014). Stock solution aliquots were added to PDA medium, which was subsequently poured into Petri dishes. A 5 mm mycelial plug of the target fungus was centrally inoculated onto each dish, followed by incubation in the dark at 27 ± 2°C. The percentage of fungal growth inhibition was determined by comparing radial growth to that of the control. Finally, the EC50 for each compound was calculated using probit analysis with SPSS 26.0 software (Finney, 1971).

Fungicidal activity of azoxystrobin nanoemulsion on spore germination

Spores of the tested fungi were obtained from two-week-old cultures maintained on PDA medium in 9-cm diameter Petri dishes. Cultures were grown under fluorescent illumination at 26°C. Spores were carefully dislodged from the agar surface using a sterile glass rod, and the resultant suspension was filtered through three layers of cheesecloth. The spore suspension was then diluted with sterile water to achieve an absorbance of 0.25 at 425 nm, which corresponds to a concentration of approximately 1.0×10⁶ conidia/mL. For the germination assay, 50 µL aliquots of the prepared spore suspension were dispensed into eppendorf tubes containing 500 µL of potato dextrose broth (PDB) medium, with azoxystrobin concentrations of either 500 or 750 mg/L. These tubes were incubated at 26°C for 24 hrs. Spore germination was subsequently observed microscopically. Samples were introduced into both counting chambers of a hemocytometer by carefully touching the coverslip edges with a pipette tip to facilitate capillary filling. Spore counting was performed using a Neubauer hemocytometer under a light microscope at 40x magnification. A spore was deemed germinated if the length of its germ tube was equal to or greater than the length of the spore itself. The counts of germinated and non-germinated conidia were recorded, and the inhibition of spore germination (%) was calculated based on these counts. All experiments were conducted in three replicates(Griffin, 1994).

Effectof azoxystrobin nanoemulsion on biochemical parameters

Effect of azoxystrobin nanoemulsionon poly-phenol oxidase (PPO) activity

The PPO activity was determined following the methodology described by Broesh (1954). PDA medium, supplemented with azoxystrobin nanoemulsion at concentrations corresponding to EC₅₀, 1/10 EC₅₀, 1/4 EC₅₀, and 1/2 EC₅₀, was prepared in 100 ml conical flasks. Fungal discs were inoculated onto the surface of this medium and incubated until complete hyphal growth was observed in the untreated control flasks. Subsequently, the culture medium was vacuum-filtered, and the filtrate was centrifuged for 15 minutes at 4000 rpm. The resulting supernatant, serving as the PPO source, was then used; 1 ml aliquots were combined with a reaction mixture consisting of 2.0 ml borate buffer (pH 9.0), 1.0 ml of 1% p-aminobenzoic acid, and 2.0 ml of 1% catechol. Enzyme activity was quantified spectrophotometrically by measuring absorbance at 575 nm after a one-hour incubation period in a 45°C water bath(Fuerst et al., 2011).The inhibition percentage of PPO activity was calculated from the equation:

I (%) = [(Ac - At)/Ac] * 100

where Ac is the absorbance in control and At is the absorbance in treatment.

Effect of azoxystrobin nanoemulsionon cellulase activity

 Fungal cultures were grown on PD medium amended with 3% carboxymethyl cellulose for 12 days at 27°C. The resulting culture medium was filtered through Whatman No. 1 paper to serve as the source of crude cellulase enzyme.One milliliter of the crude enzyme was combined with 2 ml of citrate buffer (pH 4.8), and this mixture was pre-warmed in a 50°C water bath for 30 minutes. Following this, 1 ml of azoxystrobin nanoemulsion, at specified concentrations (EC₅₀, 1/10 EC₅₀, 1/4 EC₅₀, and 1/2 EC₅₀), was introduced. The reaction mixture was then incubated at 28°C for 24 hours. To quantify the reaction, 3 ml of DNS reagent (prepared with 10 g 3,5-dinitrosalicylic acid, 10 g sodium hydroxide, 20 ml phenol, and 0.5 g sodium sulfate, brought to 1000 ml with distilled water) was added. Three replicates were prepared for each treatment, along with a positive control and an enzyme-free blank. After a final 15-minute incubation at 50°C in a water bath, the absorbance was read at 575 nm.(Helal et al., 2022). The inhibition percentage of cellulase activity was calculated from the equation:

I (%) = [(Ac – At)/Ac]*100

where Ac is the absorbance in control and At is the absorbance in treatment.

 Statistical analysis

Statistical analysis was done using the statistical package SPSS software version 26.0 (SPSS, Chicago, IL, USA). The log dose–response curves allowed determination of EC₅₀ for the bioassays according to probit analysis (Finney 1971). Statistical significance data was determined with one-way analysis of variance (ANOVA) by comparing means using SNK method at the probability of 0.05. The IC₅₀ value was estimated using probit analysis (LdP Line) (Steel and Torrie, 1980).

RESULTS AND DISCUSSIONS

Characterization of azoxystrobin nanoemulsion

Scanning electron microscope (SEM) estimation

SEM studied the morphology and shape of the nanoazoxystrobin and the data are presented in Fig. 2. The morphology of the nanoemulsion is extremely flexible, with spherical and sporadically triangular nanoemulsion detected in the micrograph.

The nanoemulsion exhibited a circular particle morphology, which aligns with the expected microscopic characteristics of microemulsions. Although the particles maintained their stability, their diameter was reduced upon 50-fold dilution (Leng et al., 2012).

(Elsharkawy et al., 2022) exposed that the Lambda-cyhalothrin nano-emulsions morphology is almost spherical and has size of 70.3 nm.

Fig. 2. Scanning electron micrograph of prepared azoxystrobin nanoemulsion. The SEM was performed on a JEOL JSM-1200EX II scanning electron microscope operating at an acceleration voltage of 25.0 kV.

Droplet size and PDI

The droplet's size was expressed as a mean diameter in nm (Mibielli et al., 2021; Silva et al., 2012). The droplet size of nanoemulsion was 116.41 nm. Nevertheless, the PDI value were 0.624 (Fig. 3).

The average droplet size of the nanoemulsions typically falls within the range of 20-500 nm (Badawy et al., 2017). The small size of the droplets in nanoemulsions gives them some advantages over conventional emulsions. These advantages include higher optical clarity, higher stability to droplet aggregation and gravitational separation, and higher bioactivity of encapsulated components (McClements, 2012). The nanoemulsions have emerged as alternative drug carriers because they increase the dissolution rates and bioavailability of several poorly soluble drugs in water (Feng et al., 2018).

Zeta potential

The nanoemulsion exposed negative value of zeta potential equal -0.0914 mV (Fig. 3). The zeta potential is a better manner to improve sample stability and save time inspired in shelf-life tests.  It is considered a powerful indicator of nanoemulsion stability, resist flocculation and aggregation for longer times, and is associated with to surface potential of the droplets (Benita & Levy, 1993). The negative values are necessary for droplet-droplet repulsion and thus enhanced nanoemulsion stability (Bruxel et al., 2012). The high stability of formulations with zeta potential values is associated with repulsive forces that exceed attracting Van der Waals forces, resulting in dispersed particles and a deflocculated system (Mahdi et al., 2011).

Nano-azoxystrobin showed a Z-average size of 418.8 nm and conductivity of 0.036 mS/cm. Additionally, the zeta potential of nano-azoxystrobin was slightly highly negative (−14.1 ± 3.15), and the PDI value was somewhat higher (0.466), with a low viscosity of 0.8872 mpa.s, which the substance’s low oil content may have caused (Elshaer et al., 2025).

Fig. 3: A typical particle size distribution by a dynamic light scattering of the formulated azoxystrobin nanoemulsion

Stability tests of nanoemulsion

The nanoemulsion was stable at centrifugation of 5000 rpm, heating cycle, and freeze-thaw cycle for 4 weeks. This formulation was not observed in any phase separation. Nanoemulsions are inherently thermodynamically stable systems, characterized by their specific compositions of oil, surfactant, and water, which prevent separation, creaming, cracking, or coalescence. Centrifugation serves as an accelerated test for stability, as it can induce sedimentation or creaming, thereby demonstrating the influence of gravitational force on emulsion degradation(Tadros et al., 2004). These tests were performed to confirm from the stability, low surfactant formulation with a nanoemulsion size droplet and stable physicochemical properties.

Viscosity and pH

It has been measured the viscosity to ensure the better delivery of the formulation, so it has been recorded 37 mpa.s. Viscosity highly influenced by several factors such as disperse phase, volume fraction, colloidal interactions, droplet size, archeology of component phases, and droplet charge.The pH of azoxystrobin nanoemulsion was 7.45 (McClements, 2015).

Antifungal activity of azoxystrobin nanoemulsion against A. alternata, F. oxysporum and R. stolonifer

Table 1 presents a comprehensive analysis of the antifungal activity of three formulations of azoxystrobin: Technical (T), suspension concentrate (SC), and nanoemulsion (NE) against three fungal pathogens: A. alternata, F. oxysporum, and R. stolonifer. The EC₅₀ values, which indicate the concentration required to inhibit 50% of fungal growth, vary significantly among the different formulations and pathogens. For A. alternata, the nanoemulsion (NE) shows the highest antifungal potency with an EC₅₀ of 244.77 mg/L, suggesting that this formulation is more effective than the SC and Technical formulations, which have EC₅₀ values of 1533.34 and 3746.09 mg/L, respectively. This trend indicates that the nanoemulsion may enhance the bioavailability and efficacy of azoxystrobin against this pathogen.

Similarly, for F. oxysporum, the NE formulation again demonstrates superior antifungal activity with an EC₅₀ of 333.75 mg/L, compared to 1408.11 mg/L for SC formulation and 1330.83 mg/L for T formulation. This consistent performance of the nanoemulsion across different pathogens highlights its potential as a preferred formulation in agricultural applications. In the case of R. stolonifer, the NE formulation has an EC₅₀ of 517.51 mg/L, while the SC formulation shows a slightly higher EC₅₀ of 613.99 mg/L, indicating that both formulations are effective, but the nanoemulsion still holds an advantage. The slope values and confidence limits further support the reliability of these results, with lower confidence limits indicating a more precise estimate of the EC₅₀ values. Overall, the data suggests that the nanoemulsion of azoxystrobin is the most effective formulation against the tested fungal pathogens, which could lead to improved disease management strategies in agricultural practices.

Table 1: Antifungal activity of the technical, suspension concentrate, and nanoemulsion of azoxystrobin against A. alternata, F. oxysporum, and R. stolonifer

^a The concentration causing 50% mycelialgrowth inhibition.

^b Slope of the concentration-inhibition regression line ± standard error.

^c   Intercept of the regression line ± standard error.

^d Chi square value.

T: Technical, NE: Nanoemulsion and SC: Suspension Concentrate.

Antifungal activity of azoxystrobin and their formulations on spore germination of A. alternata, F. oxysporumand R. stolonifer

 Table 2 presents the effects of different treatments on the spore germination of three fungal species: A. alternata, F. oxysporum, and R. stolonifer. For A. alternata, the control group showed minimal spore germination inhibition at 2.27%, The NE treatment at 750 mg/L resulted in a strong effect of 68.18%. While the SC treatment also demonstrated a significant inhibition, with 59.09% inhibition at the same concentration. The T treatment showed less effectiveness, with a maximum inhibition of 48.86% at 750 mg/L. In the case of F. oxysporum, the control group again had low inhibition at 2.17%. The NE treatment at 750 mg/L was even more effective, reaching 66.30%, while the SC treatment at 750 mg/L achieved 52.17% inhibition,  The T treatment had a maximum inhibition of 35.87% at 750 mg/L, indicating that the other treatments were more effective in controlling spore germination. For R. stolonifer, the control group showed no inhibition of spore germination. The NE treatment at 750 mg/L resulted in 51.02% inhibition, similar to the SC treatment at the same concentration. The T treatment was less effective, with only 24.49% inhibition at 750 mg/L. Overall, the data suggest that both NE and SC treatments are effective in inhibiting spore germination across all three fungal species, with NE showing particularly strong results in A. alternata and F. oxysporum .

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Table 2. Effect of the technical, suspension concentrate, and nanoemulsion of azoxystrobin on spore germination of A. alternata, F. oxysporum and R. stolonifer

Different letters in the same column indicate significant differences according to the Student-Newman-Keuls (SNK) test (P ≤ 0.05). T: Technical, NE: Nanoemulsion and SC: Suspension Concentrate.

The effects of azoxystrobin and difenoconazole on mycelial growth, spore germination, and control of brown spot were reported (Wang et al., 2016). Both mycelial growth and spore germination bioassay results exhibited that sensitivity of A. alternata to difenoconazole was lower than azoxystrobin. Azoxystrobin and the compound of azoxystrobin plus difenoconazole delivered excellent control efficiency on tobacco brown spot in field. Disease control efficacies for three sprays of azoxystrobin at doses of 0.094, 0.19 and 0.28 Kg a.i./ha, of azoxystrobin plus difenoconazole at 0.15, 0.22 and 0.29 Kg a.i./ha, and of difenoconazole at 0.12 Kg a.i./ha were between 86.00% and 89.67%, between 86.14% and 89.23%, and between 55.14 and 58.41%, respectively.

Effect of azoxystrobin and its formulations on poly-phenol oxidase activity of A. alternata, F. oxysporum and R. stolonifer

The data presented in Table 3 highlights the fungicidal activity of different formulations of azoxystrobin on the polyphenol oxidase enzyme of three fungal species: A. alternata, F. oxysporum, and R. stolonifer. The results indicate varying levels of effectiveness among the formulations, with the nanoemulsion (NE) showing the lowest IC₅₀ values for both A. alternata and F. oxysporum, suggesting a higher potency compared to the (SC) formulation and technical grade (T). For A. alternata, the IC₅₀ value for the nanoemulsion is 0.07 mg/ml, significantly lower than the 0.46 mg/ml for SC and 2.38 mg/ml for T grade. Similarly, for F. oxysporum, the NE formulation has an IC₅₀ of 0.26 mg/ml, while SC and T show higher values of 1.19 mg/ml and 1.01 mg/ml, respectively. Furthermore, R. stolonifer exhibits a different response, with the NE formulation showing an IC₅₀ of 0.38 mg/ml, which is lower than the SC 0.68 mg/ml, but both are more effective than the technical grade at 1.81 mg/ml. These findings suggest that the nanoemulsion formulation may enhance the bioavailability and efficacy of azoxystrobin against certain fungal pathogens, particularly in the case of A. alternata and F. oxysporum, while the response varies for R. stolonifer.

Table 3. Effect of the technical, suspension concentrate, and nanoemulsion of azoxystrobin on poly-phenol oxidase activity of A. alternata, F. oxysporum and R. stolonifer

^a The concentration causing 50% enzyme inhibition.

^b Slope of the concentration-inhibition regression line ± standard error.

^c Chi square value.

T: Technical, NE: Nanoemulsion and SC: Suspension Concentrate.

(Sundravadana et al., 2007) showed to analyse the induction of lignification-related enzymes and phenolic content in rice to blast disease caused by Pyricularia grisea using azoxystrobin. The severity of rice blast was reduced through treatment by azoxystrobin. Increased production of secondary metabolite – phenolic and lignification – related enzymes, namely, POD, PPO and PAL were detected in rice plants treated with azoxystrobin.

Azoxystrobin at three different concentrations, namely, 31.25, 62.50 and 125 g a.i. ha⁻¹ mancozeb (1 kg ha⁻¹) and Pseudomonas fluorescens (10 kg ha⁻¹) were evaluated for their efficacy in inducing defense enzymes in tomato against A. solani and Septoria lycopersici. The activity of PO, PPO, PAL, β-1, 3 glucanase, chitinase, catalase and defense-inducing chemicals (total phenols) was found to be increased in azoxystrobin and P. fluorescens-treated tomato plants. The activity of these enzymes and chemicals was higher in azoxystrobin (125 g a.i. ha⁻¹) and P. fluorescens-treated tomato plants challenge inoculated with the pathogens compared to other treatments (Anand et al., 2007).

Effect of azoxystrobin and its formulations on cellulase activity of A. alternata, F. oxysporumand R. stolonifer

Table 4 presents the fungicidal activity of different formulations of azoxystrobin against the cellulase enzyme produced by various fungal species, including A. alternata, F. oxysporum, and R. stolonifer. The data indicates that the nanoemulsion (NE) formulation exhibits the lowest IC₅₀ values across all tested fungi, suggesting a higher efficacy in inhibiting the cellulase enzyme compared to the technical (T) and suspension concentrate (SC) formulations. For A. alternata, the NE formulation shows an IC₅₀ of 0.15 mg/ml, significantly lower than the SC and T formulations, which have IC₅₀ values of 0.84 mg/ml and 1.59 mg/ml, respectively. Similarly, for F. oxysporum, the NE formulation has an IC₅₀ of 0.22 mg/ml, while the SC and T formulations are less effective with IC₅₀ values of 1.14 mg/ml and 1.22 mg/ml. In the case of R. stolonifer, the NE again demonstrates superior performance with an IC₅₀ of 0.46 mg/ml compared to 0.50 mg/ml for SC and 1.03 mg/ml for T formulation. These results highlight the potential of nanoemulsion formulations in enhancing the antifungal activity against cellulase-producing fungi, which could be beneficial for agricultural applications where cellulase activity is detrimental to crop health.

Table 4. Effect of the technical, suspension concentrate, and nanoemulsion of azoxystrobin on cellulase activity of A. alternata, F. oxysporum and R. stolonifer

^a The concentration causing 50% enzyme inhibition.

^b Slope of the concentration-inhibition regression line ± standard error.

^c Chi square value.

T: Technical, NE: Nanoemulsion and SC: Suspension Concentrate.