The Rhizoctonia complex represents a heterogeneous group of filamentous fungi that do not produce asexual spores and share general morphological characteristics, including the formation of mycelium and brown sclerotia (García, 2008). Rhizoctonia solani, the most significant species within the genus, affects various crops across families including Poaceae (e.g., corn, rice, wheat, barley, oats), Fabaceae (e.g., soybean, peanut, bean, alfalfa, chickpea, lentil, pea), Solanaceae (e.g., tobacco, potato), Amaranthaceae (e.g., sugar beet), Brassicaceae (e.g., canola), Rubiaceae (e.g., coffee), Malvaceae (e.g., cotton), Asteraceae (e.g., lettuce), Araceae (e.g., pothos), Moraceae (e.g., ficus), and Linaceae (e.g., flax) (Ajayi and Bradley, 2018; DGSV-CNRF, 2020). This pathogen can cause severe damage, resulting in losses of 80 to 100% of seedlings and final yield reductions up to 30%. According to the International Plant Protection Convention (IPPC) and the International Standard for Phytosanitary Measures (ISPM) No. 8, “Determination of Pest Status in an Area,” R. solani is present throughout areas planted with host crops in México, classifying it as a non-quarantine pest under ISPM No. 5, “Glossary of Phytosanitary Terms” (DGSV- CNRF, 2020).

Various control measures exist for managing pathogens, including certified clean seeds, land tillage, crop rotation to non-host species, seed treatments with fungicides, and, where available, the use of resistant crop varieties (Ajayi and Bradley, 2018). Chemical control through fungicides remains one of the most commonly employed strategies in modern agriculture due to its efficiency, speed, practicality, and economic viability (Jáquez-Matas et al., 2022). However, fungal populations targeted by these chemicals can develop resistance over time (Carmona and Sautua, 2017). Additionally, the ability of R. solani to overwinter as long-lived sclerotia in the soil and its capacity to infect a wide range of crops make crop rotation alone an insufficient management strategy, highlighting the necessity for alternative control options (Ajayi and Bradley, 2018; Akber et al., 2023).

The application of nanotechnology in agriculture is particularly interesting given the mass production of agricultural products required to meet global demand (Mridha, et al., 2025). Nanomaterials offer alternatives for improving, mitigating, and remedying collateral problems associated with modern agriculture (Rodríguez and Díaz, 2024).

Among these materials, oxide particles such as ZnO, TiO₂, CuO, Fe₂O₃ are commonly used in various fields (Lira et al., 2018; Zhu et al., 2019) TiO₂ nanoparticles (NPs) have applications in cosmetics, functional fibers, plastics, inks, paints, fine ceramics, and as food colouring, also serving as protective barriers in consumable packaging (Zhu et al., 2019). Ag NPs are valued for their antimicrobial activity and are incorporated into surfaces intended for food contact (Lira et al., 2018). CuO NPs are used in superconductors, thermal resistance applications, coating additives, antibacterial materials, and magnetic materials. ZnO NPs, apart from being toxic to human tumor cells, have demonstrated antimicrobial activity, either inhibiting or enhancing microbial growth (Zhu et al., 2019).

The antifungal activity of ZnO has been documented against several pathogens. For instance, ZnO particles synthesized via hydrothermal and co-precipitation methods demonstrated antifungal activity against Colletotrichum gloeosporioides, inhibiting spore germination and causing hyphal deformation (De la Rosa et al., 2018). Against Erythricium salmonicolor, the pathogen responsible for the pink disease in coffee, ZnO NPs ranging from 20 to 45 nm exhibited significant antifungal capacity (Arciniegas et al., 2017). Moreover, ZnO NPs have shown antifungal activity against Fusarium oxysporum by inhibiting mycelial growth and sporulation in vitro (González et al., 2021). Similarly, several studies have confirmed the antifungal activity of ZnO particles against Rhizoctonia solani (Al-Dhabaan et al., 2017; Khan and Siddiqui, 2021; Al-Zaidi et al., 2023).

However, Pariona et al. (2020) note that few studies have been conducted on the impact of ZnO particle morphology on their antifungal activity. Therefore, the present study aimed to synthesize ZnO microparticles (ZnO MPs) with three distinct morphologies to evaluate their in vitro antifungal activity against Rhizoctonia sp.

Chemicals. The reactives used were agar from the SIGMA ALDRICH brand, denatured ethyl alcohol 96% from the GOLDEN BELL brand, and distilled water. For the antifungal evaluation, PDA agar from the BD BIOXON brand was used, and as a positive control, the fungicide commercially named INTERTHIRAM 480 (ICF), with the active ingredient Thiram (Bis(dimethylthiocarbamoyl) disulphide), with a composition of Thiram 42% by weight, equivalent to 480 g of active ingredient per litre.

Synthesis of ZnO MPs. The hydrothermal synthesis method was carried out to obtain MPs with three distinct three-dimensional morphologies. These shapes were achieved by using zinc acetate as a precursor and varying the concentration of potassium hydroxide. The molar ratio between zinc acetate and potassium hydroxide was 1:9 for rod-shaped ZnO (ZnO-R), 1:3 for lamellar forms (ZnO-L), and 1:1 for hexagonal prisms (ZnO-P). For each synthesis, zinc acetate and potassium hydroxide were simultaneously dissolved in tri- distilled water (15 mL), followed by the dropwise addition of 96% ethanol (15 mL). The resulting solution was then transferred into an autoclave (46 mL) and placed in a muffle furnace at 160 °C for 18 h. After that, the precipitate particles were collected, transferred to 35 mL centrifuge tubes, and then washed twice with distilled water. This process involved centrifugation at 5000 rpm for 5 min, followed by decanting the supernatant each time. Finally, the plastic tubes containing the washed precipitate were placed in an oven at 80 °C for 12 h. After drying, the material was recovered, finely ground in an agate mortar, and weighed.

Characterization of MPs. The physicochemical characterization (size, shape) was conducted using scanning electron microscopy (Jeol JSM-7401F SEM). The structure was studied using X-ray diffraction (XRD) patterns, which were obtained with a Philips X-Pert diffractometer operating at 40 kV and 30 mA, utilizing Cu Kα radiation (λ = 0.15418 nm) in Bragg-Brentano geometry. The scanning angle 20 was between 20 and 80°, with a step size of 0.015°. The patterns were indexed for ZnO, and the JCPDS card # (00-036-1451) was used for reference (Sedefoglu, 2023).

Preparation of cultures poisoned with MPs. The poisoned culture media with ZnO MPs were prepared following the method described by Guerrero et al. (2007), with some modifications. Three types of MPs (ZnO-P, ZnO-L, and ZnO-R) were incorporated into the PDA culture medium.

The required amounts of MPs and inoculum concentration factor (ICF) were added to each Petri dish (90 mm diameter), followed by the necessary volume of PDA culture medium to achieve final concentrations of 200 (C), 500 (B), and 1000 (A) ppm. Plates containing only PDA medium were prepared as negative controls. Each concentration was prepared in triplicate. The culture media were previously held for 24 hours to evaluate their sterility.

Fungal Inoculation. The antifungal activity of ZnO MPs against Rhizoctonia sp. was evaluated using the method described by Derbalah et al. (2019) with some modifications. A strain of Rhizoctonia sp., isolated from a diseased potato acquired in Chihuahua Capital, was used for the assay. For this assay, a total of 39 Petri dishes were prepared: 27 for the ZnO MPs treatments, three independent replicates for the negative control, and nine for the positive control (ICF).

A 5 mm diameter agar disc containing a 7-day-old Rhizoctonia sp. culture was placed in each Petri dish, which was then incubated at 27 ± 1 °C for 7 days. Radial growth was measured every 24 h. using a calliper.

Microscopic analysis. After seven days of inoculation, wet mounts were prepared in duplicate. Small portions of the fungus were collected from the replicated Petri dishes and placed on slides with a drop of lactophenol cotton blue stain, covered with a coverslip, and observed under a ZEISS Primostar 1 microscope.

Statistical Analysis. A factorial design with three factors was carried out, whose general model is shown in equation (1):

yijkl = m + MPi + Cj + Dk + MPi*Cj + MPi*Dk + Cj*Dk + MPi*Cj*Dk + eijkl(1)

Where yijkl is the response variable (mycelial growth in mm), m is the overall mean, MPi represents the type of nanoparticle MP with levels ZnO-P, ZnO-R, ZnO-L, and ICF, Cj corresponds to the nanoparticle concentrations at 200, 500, and 1000 ppm, Dk denotes the days of growth monitoring (levels 1 to 7), l is the number of replicates, and e is the error term. The model also includes two-way interactions (MPi * Cj, MPi * Dk, and Cj * Dk) as well as a three-way interaction (MPi * Cj * Dk).

Grubbs and Kolmogorov-Smirnov tests were applied to detect outliers and assess the normality of the data, respectively. Subsequently, an analysis of variance (ANOVA) was performed to identify significant effects of the factors and their interactions. Finally, multiple comparison tests of means were conducted using Fisher’s least significant difference (LSD) test. All analyses were carried out using MINITAB 20.3 and OriginPro 2021, with a significance level of p ≤ 0.05.

Physicochemical characterization. The SEM micrographs for the three morphologies and the size distributions of the MPs are shown in Figure 1. The images for each morphology were included at low and high magnification. Most of them exhibit a hexagonal shape, with varying sizes for each type. The micrographs in Figures 1A and 1D show hexagonal prism particles (ZnO-P) with an average size of 8487 ± 2074 nm, and most of them exhibit marked dips on their surface (Figure 1D). In the case of the lamellar particles (ZnO-L), it can be observed that all of them are in different orientations (Figure 1B and Figure 1E). They have an average diameter of 508 ± 155 nm and show an approximate thickness of 60 nm, forming a smooth surface. In the case of rod-shaped particles (ZnO-R), the images show elongated hexagonal prism particles with a pointed shape (Figure 1C and Figure 1F), and they have

an average length of 2326 ± 1437 nm. These images confirm the three morphological types of particles.

Figure 1

The X-ray diffraction patterns show a similar profile for all obtained samples. Figure 2 shows that all patterns exhibit a polycrystalline structure. As we can see, the intensities do not change proportionally. However, the particles have distinct shapes, as seen in Figure 1. This patterns were indexed for ZnO using the JCPDS card No. 00-036-1451. These results confirm the crystallinity of the obtained particles for three morphologies.

Figure 2

Antifungal Activity of ZnO MPs Against Rhizoctonia sp.

Macroscopic appearance. Figure 3, shows the average growth measurements of Rhizoctonia sp. (in mm) over time. The values belong to the mean of three test antifungal replicates, and the error bars represent the standard deviation. On the first day, the negative control exhibited a growth of 13.5 ±0.42 mm. At the lowest concentration (200 ppm), ICF showed an increase of 11.9±0.57 mm. In contrast, for the ZnO-P sample, a growth of

12.6±2.11mm was measured, indicating that the nanoparticle treatments had minimal antifungal activity at this early stage. At the same concentration, the ZnO-L sample exhibited a diameter of 8.9±0.26 mm, and the ZnO-R sample showed an 8.3±0.36 mm diameter.

Figure. 3

After three days of incubation, the negative control reached 45.7±0.52 mm of growth, a value exceeded by both the minimum and medium concentrations of ICF, which recorded

47.2±2.5 mm and 46.4±1.25 mm, respectively. As well as by the minimum concentration of ZnO-P, which reached 47.7±5.05 mm. On the same day, the maximum concentration of ZnO-R displayed the highest antifungal activity, with a growth inhibition of 21±2.36 mm.

On the sixth day, the control exhibited a growth of 80.7±0.64 mm, which was exceeded by all three concentrations of ICF and by the minimum concentrations of ZnO-P and ZnO- R, which showed growths of 83±3.46 mm and 82.7±3.93 mm, respectively. These results highlight the lower antifungal effectiveness of these particular treatments under these conditions.

The inspection using images on the seventh day is presented in Figure 4, further illustrating the differences among treatments. In these images, the numbers (1, 2, and 3) represent the three times of repetition, and the letters A, B, and C denote the 1000, 500, and 200 ppm concentrations, respectively. At the lowest concentration (200 ppm, column C), the nanoparticle treatments did not show a notable difference compared to the negative control (Figure 4E). At the medium concentration (500 ppm, column B), sample ZnO-P (Figure 4A) was the treatment causing the least inhibition-apart from ICF-followed by ZnO-R (Figure 4C). In contrast, ZnO-L (Figure 4B) exhibited the strongest inhibition of Rhizoctonia sp. at this concentration. At the highest concentration (1000 ppm, column A), significant inhibition was observed in all treatments, with ZnO-R (Figure 4C) showing the highest antifungal activity, followed by ZnO-L (Figure 4B).

Another important observation was the presence of sclerotia-the resistant structures of Rhizoctonia sp.-which were detected in all treatments, including ICF, and a concentration of 200 ppm. At 500 ppm, only ZnO-P (Figure 4A) exhibited sclerotia, while at 1000 ppm, none of the treatments resulted in their formation.

Figure. 4

Microscopic appearance. Images acquired by the optical microscope are shown in Figure 5. The files A, B, and C belong to 1000, 500, and 200 ppm concentration, respectively. These images revealed long septate hyphae with consistent size in the negative control. The branching of these hyphae was perpendicular, and the tips of the terminal septa appeared slightly curved and narrow.

At the minimum concentration, treatments with ZnO-P and ZnO-L samples, as well as for the ZnO-P sample at the medium concentration and all three concentrations of ICF, exhibited the formation of hyphae resembling synema structures characterized by conidiophores grouped in tufts or brush-like formations (Ormeño, 2024). Additionally, a noticeable loss of plasma membrane integrity was observed, particularly in ZnO-R, where hyphal thickness varied, and in ZnO-L, where coiling of hyphae occurred at the medium concentration.

At the highest concentration (1000 ppm, file A), all treatments resulted in a complete loss of mycelial structure, with varying hyphal thickness, coiled hyphae, and condensation of material believed to be cytoplasm, as observed under the optical microscope (Figure 5).

Figure 5

Statistical Analysis. The results of the data analyses relating to the normality hypothesis are shown in Grubbs' test analysis, which shows the absence of outliers (p = 0.05). Similarly, the Kolmogorov-Smirnov test confirmed the normality hypothesis (p = 0.05).

The ANOVA summary is presented in Table 1. The F-value of the model was statistically significant (p < 0.05), indicating that the linear model was adequate for the analysis. The fitting parameter R^² value was 0.98, indicating that the model explains a significant amount of variance. Moreover, for all sources of variation, the calculated F- values were markedly higher than the corresponding FCritical values (F-value » FCritical), and in all cases the associated p-values were < 0.05. These results demonstrate that the effects observed are statistically significant and reinforce the reliability of the model.

Table 1

Source	DF	SS Adjusted	MS Adjusted	F-value	FCritic	p-value
MP	3	15052	5017.2	502.48	2.66	0.000
Concentration	2	12581	6290.4	630.00	3.05	0.000
Day	6	111770	18628.3	1865.68	2.13	0.000
MP*Concentration	6	4047	674.5	67.55	2.13	0.000
MP*Day	18	3246	180.3	18.06	1.65	0.000
Concentration*Day	12	3337	278.1	27.85	1.74	0.000
MP*Concentration*Day	36	2051	57.0	5.71	1.49	0.000
Error	168	1677	10.0

DF: degrees of freedom. SS: Sum square. MS: Medium Square. F: Fisher statistics, Calculated and Critical. p: probability.

The ANOVA revealed significant differences in the treatment, concentration, and day factors, as well as in the double and triple interactions regarding the response variable (growth). The results obtained through the ANOVA support the previous observations in Figures 3 and 4.

The comparison of means analysis using Fisher's LSD (Least Significant Difference) test is shown in the Figures 6A, 6B, and 6C, which correspond to the comparisons among treatments, concentrations, and days, respectively. As can be seen, the comparison of means at different levels for both treatment and concentration on the respective days reveals significant differences, as none of the intervals contains a value of 0.

Figure 6

Although XRD patterns confirm the same crystalline phase in all samples, SEM micrographs reveal significant morphological differences among particles. The observed variations in particle shape and size confirm that the synthesis conditions have a marked influence on morphology (Gulati et al., 2016; Moazzen et al., 2012; Phan and Nguyen, 2017; Shaba et al., 2021).

The macroscopic growth of fungi reveals concentration-dependent antifungal activity of ZnO nanoparticles against Rhizoctonia sp. Although minimal inhibition was observed at 200 ppm, particularly for ZnO-P and ICF, the ZnO-L and ZnO-R microparticles showed higher initial suppression of fungal growth at the same concentration. These qualities of the particles suggest that both morphology and surface properties influence their antifungal performance, as reported in previous studies too (Cruz et al., 2023; Mantecón, 2015).

The significant decrease in fungal growth observed for the ZnO-R sample at 1000 ppm indicates a particularly strong antifungal effect, which can be attributed to its elongated morphology, providing a larger active surface area and facilitating more effective interactions with fungal cells (Descanse et al., 2023). This morphology-dependent behaviour has been well-documented in the literature for ZnO nanostructures, where the shape and size are found to correlate with enhanced antimicrobial efficacy (Tsror, 2010; Mendes et al., 2024).

In addition, the absence of sclerotia formation at higher concentrations is highly significant. Sclerotia serve as survival structures for Rhizoctonia sp., enabling it to persist under adverse conditions and constituting the primary inoculum for future infections (Tsror, 2010). The suppression of sclerotia at 1000 ppm in all treatments, particularly in ZnO-L and ZnO-R type MPs, suggests that these nanoparticles not only inhibit fungal growth but also interfere with the pathogen life cycle, offering a potential advantage over conventional treatments such as ICF (Mantecón, 2015).

The findings, as revealed by microscopic images, show that ZnO microparticles have a significant morphological effect on Rhizoctonia sp. hyphae, with the intensity of this effect increasing at higher concentrations. The presence of synema-like structures in ZnO-P and ZnO-L treatments, as well as in those with ICF, suggests that exposure to these agents may induce morphological adaptations or stress responses in the fungus (Ormeño, 2024).

The observed variability in the thickness and coiling of hyphae, especially with ZnO-R and ZnO-L sample treatments, indicates cell deterioration and could reflect alterations in cell wall growth and cytoskeleton stability, phenomena commonly reported in fungal cells exposed to nanomaterials. These morphology changes are consistent with previous studies describing nanomaterial-induced alterations in fungi growing (Desvani et al., 2018; Garrido, 2016).

At the highest concentration, the complete loss of mycelial morphology, along with cytoplasmic condensation, suggests severe cell damage. Such effects may be attributed to oxidative stress mechanisms, in which nanoparticles disrupt cellular organelles and increase the production of reactive oxygen species (ROS) (Fu et al., 2014; León et al., 2022). The role of ROS in nanoparticle-induced toxicity is widely documented, as they can cause lipid peroxidation, protein denaturation, and DNA damage, ultimately leading to cell death (Fu et al., 2014).

These results highlight the potential of ZnO microparticles, especially at high concentrations, to exert antifungal activity through physical alteration of cellular structures and biochemical mechanisms related to oxidative stress. This dual mode of action could position ZnO microparticles as a promising alternative or a complement to traditional antifungal agents in the management of pathogens such as Rhizoctonia sp.

Statistical analyses confirm the reliability of the experimental data and the suitability of employing parametric methods for further evaluation. The absence of outliers and verification of normality by the Kolmogorov-Smirnov test, along with residual plots, provide strong evidence supporting the integrity of the dataset. Such rigorous statistical validation is essential when evaluating biological phenomena, particularly in antifungal assays where variability can be high (Field, 2013).

Significant ANOVA results indicate that the factors of treatment type, concentration, and exposure time (in days) have a strong influence on fungal growth. The high correlation R² value (0.98) indicates an excellent fit of the model to the experimental data, suggesting that almost all of the variability in the growth measurements is explained by the

experimental variables and their interactions. These findings are consistent with the trends observed in Figures 3 and 4, which emphasize morphological and growth differences between treatments.

Fisher LSD test further clarifies the specific differences between treatment groups, concentrations, and days, revealing significant differences between all conditions evaluated. The fact that none of the confidence intervals include zero reinforces the conclusion that the treatments had measurable and statistically significant effects on fungal growth. Overall, the statistical results validate the experimental observations and confirm the efficacy of specific ZnO MPs treatments in inhibiting fungal growth, particularly at higher concentrations and with prolonged exposure times. These findings support the potential application of ZnO-R particles as antifungal agents, which justifies future research aimed at exploring their efficacy under different environmental conditions and against other phytopathogenic fungi.

Particles with different morphologies of ZnO exhibit a significant variation in antifungal activity. When comparing the activity of various morphologies of ZnO particles, it was concluded that none of the morphologies exhibited antifungal activity at the minimum concentration (200 ppm). However, at the medium concentration (500 ppm) and the maximum concentration (1000 ppm), all test nanoparticles exhibited an inhibitory effect, considering both growth and the presence of sclerotia. At the medium concentration, the laminar particles (ZnO-L) yielded the best results. In contrast, at the maximum concentration, the rod-type particles (ZnO-R) proved to be the most effective inhibitors. The effects of MP concentration, day, and their interactions were statistically significant for the growth of Rhizoctonia sp. Finding the “p” value less than 0.05.

This result confirms the potential for using particles with different morphologies in agriculture and represents a future possibility for using ZnO particles as fungicides. However, in vivo studies would be necessary to verify and visualize the effects of ZnO particles on plants.