In Vitro Sensitivity of Four Plant-Pathogenic Fungi Isolated from White Onion (<em>Allium fistulosum</em> L.) to Fungicides with Different Modes of Action

Jhon Andres Meneses-Mariño; Pablo Israel Alvarez-Romero; Eliana Granja-Guerra; Daniel Arturo Román-Robalino; Ana Francisca Tibúrcia Amorim Ferreira e Ferreira

doi:10.20944/preprints202609.0300.v1

Submitted:

03 September 2026

Posted:

03 September 2026

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Abstract

White onion (Allium fistulosum L.) is an economically important crop whose productivity and commercial quality can be affected by fungal diseases. This study evaluated the in vitro sensitivity of Fusarium sp., Penicillium sp., Alternaria sp., and Ulocladium sp. to six fungicides with different modes of action: the demethylation inhibitors (DMIs) tebuconazole and mefentrifluconazole, the quinone outside inhibitors (QoIs) pyraclostrobin and azoxystrobin, and the methyl benzimidazole carbamates (MBCs) thiophanate-methyl and thiabendazole. Fungicides were incorporated into potato dextrose agar (PDA) at concentrations of 0, 0.1, 1, 10, 100, and 500 ppm, and sensitivity was assessed based on mycelial growth rate (MGR) and median effective concentration (EC₅₀). Fungicide concentration significantly affected MGR (p < 0.05) in most fungus–fungicide combinations, although the magnitude of the response varied among fungal isolates and active ingredients. Fusarium sp. was highly sensitive to tebuconazole (EC₅₀ = 0.10 µg mL⁻¹), thiophanate-methyl (0.02 µg mL⁻¹), and thiabendazole (0.11 µg mL⁻¹), whereas Alternaria sp. showed high sensitivity to mefentrifluconazole (0.019 µg mL⁻¹), azoxystrobin (0.01 µg mL⁻¹), and pyraclostrobin (0.04 µg mL⁻¹). Penicillium sp. was highly sensitive to tebuconazole (0.07 µg mL⁻¹) and azoxystrobin (0.06 µg mL⁻¹), but showed markedly reduced sensitivity to mefentrifluconazole (749.53 µg mL⁻¹). Ulocladium sp. exhibited variable responses, ranging from high sensitivity to azoxystrobin (0.10 µg mL⁻¹) and thiabendazole (0.32 µg mL⁻¹) to moderate sensitivity to tebuconazole (7.46 µg mL⁻¹), pyraclostrobin (8.81 µg mL⁻¹), and thiophanate-methyl (6.90 µg mL⁻¹). Overall, the results demonstrated substantial fungus–fungicide-specific variation, including differences between active ingredients belonging to the same FRAC group. These findings provide baseline information for fungicide sensitivity monitoring and support the rational rotation of fungicides with different modes of action for sustainable disease management in white onion production.

Keywords:

Allium fistulosum

;

fungicide sensitivity

;

EC₅₀

;

mycelial growth

;

fungicide resistance

Subject:

Biology and Life Sciences - Plant Sciences

1. Introduction

White onion (Allium fistulosum L.), commonly known as Welsh onion, is a vegetable species native to the Far East [1] and is widely valued for its culinary and medicinal uses [2,3,4]. In 2024, the global cultivated area reached 250,443 ha, with a total production of 5,576,778.38 tons. The regional distribution of production was concentrated mainly in Africa (44.5%) and Asia (43.6%), followed by the Americas (4.9%), Oceania (4.8%), and Europe (2.2%). In Latin America, this crop is economically important because of its high demand for fresh consumption and its contribution to local agricultural systems. In Ecuador, approximately 11,111 ha were cultivated, with an estimated production of 98,846.97 tons [3]. At the local level, Santa Fe de Galán parish, in Chimborazo Province, has approximately 361.23 ha devoted to this crop [4].

Despite its economic importance, white onion production is constrained by diseases associated with phytopathogenic fungi that affect roots, foliage, and harvested tissues, potentially resulting in substantial yield and quality losses under favorable environmental conditions. Among the fungal genera associated with the crop are Fusarium, Alternaria, Penicillium, and Ulocladium, which may be involved in root deterioration, foliar lesions, and postharvest damage. In addition to reducing crop productivity and commercial quality, fungal diseases can increase production costs due to the need for repeated fungicide applications [5].

Chemical control remains an important component of fungal disease management. However, the recurrent use of fungicides with the same mode of action imposes selection pressure on fungal populations and may favor the development and establishment of resistant phenotypes. Fungicide resistance is therefore an important challenge for sustainable disease management because it can progressively reduce the efficacy of commonly used chemical groups, including demethylation inhibitors (DMIs), quinone outside inhibitors (QoIs), and methyl benzimidazole carbamates (MBCs) [6,7]. Consequently, understanding the sensitivity of fungal populations to fungicides with different modes of action is essential for designing effective resistance-management strategies.

In vitro sensitivity assays provide a useful approach for detecting differences in fungal responses to active ingredients before reduced efficacy becomes evident under field conditions. Fungal sensitivity can be quantified by evaluating parameters such as mycelial growth, spore germination, or biomass production across a range of fungicide concentrations. Dose–response data can then be used to estimate the median effective concentration (EC₅₀), defined as the concentration required to reduce fungal growth or another measured response by 50% relative to the untreated control [8,11]. EC₅₀ values provide a quantitative basis for comparing the relative sensitivity of fungal isolates and active ingredients and may contribute to the establishment of baseline sensitivity and the detection of shifts associated with fungicide resistance.

Sensitivity to fungicides can vary considerably among fungal species and isolates, even within the same population. Such variation may be influenced by genetic background, previous exposure to fungicides, selection pressure, application frequency, and resistance mechanisms such as alterations in target sites or increased activity of efflux systems [9]. Therefore, evaluating fungal isolates against fungicides belonging to different chemical groups and modes of action is important for identifying differences in sensitivity and supporting more rational fungicide use within integrated disease management programs [10].

Accordingly, the objective of this study was to evaluate the in vitro sensitivity of four fungal genera, Fusarium sp., Penicillium sp., Alternaria sp., and Ulocladium sp., associated with white onion (Allium fistulosum L.) in Santa Fe de Galán parish, Chimborazo Province, Ecuador, to fungicides with different modes of action. Mycelial growth responses and EC₅₀ values were used to characterize differences in sensitivity among the fungal isolates and active ingredients, providing baseline information to support more efficient fungicide selection and sustainable disease management in white onion production.

2. Materials and Methods

2.1. Sample Collection

Samples were collected from an established white onion crop located in Santa Fe de Galán parish, Chimborazo Province, Ecuador (1°39′05″ S, 78°41′00″ W; 2,833 m a.s.l.). A total of 50 samples, comprising root and foliar tissues, were collected from plants exhibiting disease symptoms. The samples were individually placed in sterile plastic bags, transported under refrigerated conditions, and stored at 4–8 °C until subsequent fungal isolation and laboratory analysis.

2.2. Isolation of Fungi Associated with the Crop

Root and foliar tissues, with and without visible disease symptoms, were processed for fungal isolation. Tissue fragments (approximately 4 mm) were excised from the interface between healthy and symptomatic tissue using a sterile scalpel. The fragments were surface-disinfected by immersion in 70% ethanol for 30 s, followed by 2% sodium hypochlorite for 2 min, and subsequently rinsed with sterile distilled water for 2 min. The disinfected tissue fragments were placed on Petri dishes containing potato dextrose agar (PDA) and Rose Bengal medium supplemented with ampicillin and biconcilin. The plates were sealed with Parafilm and incubated at 25 ± 2 °C for 10 days.

After incubation, fungal colonies emerging from the tissue fragments were subcultured by transferring actively growing mycelial fragments to new Petri dishes containing PDA supplemented with 100 ppm chloramphenicol.. The cultures were incubated at 25 ± 2 °C for 10 days in the dark until pure fungal cultures were obtained for subsequent morphological identification [12,13]. From the fungal isolates recovered from the 50 root and foliar samples collected in Santa Fe de Galán, four genera were selected for the in vitro fungicide sensitivity assays: Fusarium sp., Penicillium sp., and Ulocladium sp. isolated from root tissues, and Alternaria sp. isolated from foliar tissue (Table 1).

Thefungicides evaluated in this study were classified according to the Fungicide Resistance Action Committee (FRAC) classification, based on their mode and site of action. Three FRAC groups were included: G1, corresponding to demethylation inhibitors (DMIs); C3, corresponding to quinone outside inhibitors (QoIs); and B1, corresponding to inhibitors of β-tubulin assembly during mitosis. The G1 group included the triazoles tebuconazole and mefentrifluconazole; the C3 group included the strobilurins pyraclostrobin and azoxystrobin; and the B1 group included the benzimidazoles thiophanate-methyl and thiabendazole (Table 2).

2.3. Plating of Fusarium sp., Penicillium sp., Alternaria sp., and Ulocladium sp.

Mycelial discs (approximately 5.13 mm in diameter) were obtained from actively growing cultures of Fusarium sp., Penicillium sp., Alternaria sp., and Ulocladium sp. using a sterile cork borer. Each mycelial disc was placed in a Petri dish containing PDA medium amended with the corresponding fungicide concentration. The plates were labeled, sealed with plastic film, and incubated in the dark at 25 ± 2 °C until the fungal colony in the untreated control (0 ppm) completely covered the Petri dish. The percentage of mycelial growth inhibition was determined relative to the untreated control. The resulting inhibition values were classified according to the scale established by [9] into three categories: low inhibition (0–25%), moderate inhibition (25–50%), and high inhibition (>50%) (Table 3).

2.8. Estimation of the Average Effective Concentration (EC₅₀)

The median effective concentration (EC₅₀) was estimated from the mycelial growth response of each fungal isolate to the different fungicide concentrations. Mycelial growth inhibition relative to the untreated control (0 ppm) was calculated for each fungicide concentration. The EC₅₀, defined as the fungicide concentration required to inhibit mycelial growth by 50% relative to the untreated control, was estimated from the relationship between the percentage of mycelial growth inhibition and the log₁₀-transformed fungicide concentration. EC₅₀ values were calculated in R using the ec50estimator package and were estimated separately for each fungal isolate and active ingredient.

2.9. Sensitivity Range

Fungicide sensitivity was classified according to the sensitivity ranges proposed by [15]. This classification was applied to the EC₅₀ values obtained for each fungal isolate and active ingredient, including tebuconazole, mefentrifluconazole, azoxystrobin, pyraclostrobin, thiophanate-methyl, and thiabendazole. Based on the corresponding EC₅₀ values, fungal isolates were assigned to the sensitivity categories shown in Table 4.

2.10. Classification of Fungicide Sensitivity

The sensitivity of the fungal isolates to the evaluated fungicides was classified according to the criteria proposed by [15]. The classification system establishes sensitivity categories based on predefined EC₅₀ ranges, allowing the response of each fungal isolate to be interpreted according to the concentration of active ingredient required to inhibit 50% of mycelial growth. Accordingly, lower EC₅₀ values indicate greater fungal sensitivity to the active ingredient, whereas higher values indicate a reduced sensitivity response.

This classification criterion was applied independently to each fungal isolate–fungicide combination. The evaluated active ingredients included tebuconazole, mefentrifluconazole, azoxystrobin, pyraclostrobin, thiophanate-methyl, and thiabendazole, representing different chemical groups and modes of action. Based on the EC₅₀ value obtained for each combination, the fungal isolates were assigned to the corresponding sensitivity category according to the ranges established by [15] and summarized in Table 4. This approach allowed comparison of sensitivity patterns among fungal isolates and active ingredients using a standardized classification criterion.

2.11. Statistical Analyses

Statistical analyses were performed separately for each fungal isolate–fungicide experiment. Mycelial growth rate (MGR) data were subjected to a one-way analysis of variance (ANOVA), considering fungicide concentration as the treatment factor. When significant differences were detected by ANOVA, treatment means were compared using Tukey’s honestly significant difference (HSD) test at the 5% significance level (p ≤ 0.05). All statistical analyses were performed in R (version 4.2.2) using the agricolae package. Comparisons among fungicide concentrations were conducted independently for Fusarium sp., Penicillium sp., Alternaria sp., and Ulocladium sp.; therefore, the statistical groupings represented by different letters correspond exclusively to comparisons among fungicide concentrations within each independent fungal isolate–fungicide experiment.

3. Results

3.1. Mycelial Growth Rate

The analysis of variance (ANOVA) showed that fungicide concentration had a significant effect (p < 0.05) on the mycelial growth rate (MGR) of the evaluated fungal isolates in most of the independent fungus–fungicide experiments. The magnitude of the response varied according to the fungal isolate and active ingredient. Tukey’s test (p ≤ 0.05) allowed the identification of statistically different groups among fungicide concentrations within each independent experiment (Figure 1, Figure 2, Figure 3, Figure 4, Figure 5 and Figure 6).

3.1.1. Effect of Tebuconazole on Mycelial Growth Rate

The ANOVA indicated a significant effect of tebuconazole concentration on MGR (p < 0.05). In Fusarium sp., the untreated control (0 ppm) showed the highest MGR (15.3 mm/day) and was assigned to group A. Increasing tebuconazole concentrations significantly reduced mycelial growth, with the lowest MGR (1.2 mm/day) recorded at 500 ppm, corresponding to group C. A significant reduction in MGR was also observed for Penicillium sp., Alternaria sp., and Ulocladium sp. In Ulocladium sp., the MGR decreased from approximately 8.3 mm/day in the untreated control (group A) to 1.6 mm/day at 500 ppm (group B). Overall, the highest concentrations were associated with the lowest MGR values for the four fungal isolates (Figure 1).

3.1.2. Effect of Mefentrifluconazole on Mycelial Growth Rate

The ANOVA showed a significant effect of mefentrifluconazole concentration on MGR (p < 0.05). In Fusarium sp., Tukey’s test separated the concentrations into five statistical groups. The untreated control showed the highest MGR (15.3 mm/day; group A), whereas the lowest MGR (6.7 mm/day; group E) was recorded at 500 ppm. In Penicillium sp., MGR decreased from approximately 7.5 mm/day at 0 ppm to 3.8 mm/day at 500 ppm. Significant reductions in MGR with increasing fungicide concentration were also observed for Alternaria sp. and Ulocladium sp. (Figure 2).

3.1.3. Effect of Pyraclostrobin on Mycelial Growth Rate

The ANOVA showed that pyraclostrobin concentration significantly affected MGR (p < 0.05) in Fusarium sp., Penicillium sp., and Alternaria sp. In Fusarium sp., the untreated control presented the highest MGR (17.1 mm/day; group A), while the lowest value was recorded at 500 ppm (9.4 mm/day; group C). In Penicillium sp., MGR decreased from 7.9 mm/day in the control to 5.2 mm/day at 500 ppm, with Tukey’s test separating the concentrations into groups A, AB, and B. Alternaria sp. also showed significant differences among concentrations. In contrast, no significant differences among pyraclostrobin concentrations were detected for Ulocladium sp. (p > 0.05), as indicated by the common statistical grouping across treatments (Figure 3).

3.1.4. Effect of Azoxystrobin on Mycelial Growth Rate

The ANOVA indicated a significant effect of azoxystrobin concentration on MGR (p < 0.05) for the evaluated fungal isolates. Tukey’s test showed differences among concentrations, with the untreated controls generally presenting the highest MGR values and fungicide-amended treatments showing lower growth. The response differed among Fusarium sp., Penicillium sp., Alternaria sp., and Ulocladium sp., but in all cases the lowest MGR values were associated with fungicide exposure compared with the corresponding untreated control (Figure 4).

3.1.5. Effect of Thiophanate-Methyl on Mycelial Growth Rate

The effect of thiophanate-methyl concentration on MGR differed among the evaluated fungal isolates. Significant differences among concentrations were detected for Fusarium sp., Alternaria sp., and Ulocladium sp. (p < 0.05), with reductions in MGR observed as fungicide concentration increased. In contrast, Penicillium sp. showed no significant differences among concentrations (p > 0.05), as indicated by the absence of separation among treatment means by Tukey’s test. Thus, the effect of thiophanate-methyl concentration was dependent on the fungal isolate evaluated (Figure 5).

3.1.6. Effect of Thiophanate-Methyl on Mycelial Growth Rate

The ANOVA showed a significant effect of thiabendazole concentration on MGR (p < 0.05) for the evaluated fungal isolates. Increasing concentrations were associated with significant reductions in mycelial growth. In Ulocladium sp., for example, the untreated control presented the highest MGR (7.1 mm/day; group A), whereas the lowest MGR was recorded at 500 ppm (3.9 mm/day; group C). Significant differences among concentrations were also observed for Fusarium sp., Penicillium sp., and Alternaria sp., with the higher concentrations generally belonging to statistical groups characterized by lower MGR values (Figure 6).

3.2. Estimation of the Median Effective Concentration (EC₅₀)

The EC₅₀ analysis revealed marked differences in the sensitivity of the evaluated fungal isolates to tebuconazole (Table 5). Fusarium sp. and Penicillium sp. exhibited the lowest EC₅₀ values, at 0.10 and 0.07 µg mL⁻¹, respectively. Both values were below 1.0 µg mL⁻¹ and, according to the established sensitivity ranges, these isolates were classified as highly sensitive to tebuconazole. In contrast, Ulocladium sp. showed an EC₅₀ of 7.46 µg mL⁻¹, within the 5–20 µg mL⁻¹ range, and was therefore classified as moderately sensitive. Alternaria sp. exhibited the highest EC₅₀ value (22.75 µg mL⁻¹), corresponding to the 20–100 µg mL⁻¹ range and resulting in its classification as highly resistant. Overall, the EC₅₀ values showed a clear gradient in response to tebuconazole, with Penicillium sp. and Fusarium sp. exhibiting the greatest sensitivity, followed by Ulocladium sp., whereas Alternaria sp. showed the lowest sensitivity among the evaluated fungal isolates.

The EC₅₀ analysis revealed marked differences in the sensitivity of the evaluated fungal isolates to mefentrifluconazole (Table 6). Alternaria sp. exhibited the lowest EC₅₀ value (0.019 µg mL⁻¹), which was below 1.0 µg mL⁻¹ and resulted in its classification as highly sensitive. Ulocladium sp. showed an EC₅₀ of 1.17 µg mL⁻¹, within the 1–5 µg mL⁻¹ range, and was therefore classified as sensitive. In contrast, Fusarium sp. exhibited an EC₅₀ of 43.05 µg mL⁻¹, corresponding to the 20–100 µg mL⁻¹ range and resulting in its classification as highly resistant. Penicillium sp. showed the highest EC₅₀ value (749.53 µg mL⁻¹), exceeding 500 µg mL⁻¹, and was classified as very highly resistant. Overall, the EC₅₀ values demonstrated a pronounced gradient in response to mefentrifluconazole, with Alternaria sp. showing the greatest sensitivity, followed by Ulocladium sp., whereas Fusarium sp. and particularly Penicillium sp. exhibited substantially lower sensitivity to this fungicide.

The EC₅₀ analysis revealed differences in the sensitivity of the evaluated fungal isolates to azoxystrobin (Table 7). Alternaria sp., Penicillium sp., and Ulocladium sp. exhibited EC₅₀ values of 0.01, 0.06, and 0.10 µg mL⁻¹, respectively. All three values were below 1.0 µg mL⁻¹ and, according to the established sensitivity ranges, these isolates were classified as highly sensitive to azoxystrobin. Fusarium sp. showed a comparatively higher EC₅₀ value of 1.39 µg mL⁻¹, within the 1–5 µg mL⁻¹ range, and was therefore classified as sensitive. Overall, the EC₅₀ values indicated a high level of sensitivity to azoxystrobin among the four fungal isolates, with Alternaria sp. exhibiting the greatest sensitivity, followed by Penicillium sp. and Ulocladium sp., whereas Fusarium sp. showed the lowest relative sensitivity among the evaluated isolates.

The EC₅₀ analysis revealed differences in the sensitivity of the evaluated fungal isolates to pyraclostrobin (Table 8). Alternaria sp. and Fusarium sp. exhibited the lowest EC₅₀ values, at 0.04 and 0.35 µg mL⁻¹, respectively. Both values were below 1.0 µg mL⁻¹ and, according to the established sensitivity ranges, these isolates were classified as highly sensitive to pyraclostrobin. Penicillium sp. showed an EC₅₀ value of 1.17 µg mL⁻¹, within the 1–5 µg mL⁻¹ range, and was therefore classified as sensitive. In contrast, Ulocladium sp. exhibited the highest EC₅₀ value (8.81 µg mL⁻¹), corresponding to the 5–20 µg mL⁻¹ range and resulting in its classification as moderately sensitive. Overall, the EC₅₀ values indicated that Alternaria sp. exhibited the greatest sensitivity to pyraclostrobin, followed by Fusarium sp. and Penicillium sp., whereas Ulocladium sp. showed the lowest relative sensitivity among the evaluated fungal isolates.

The EC₅₀ analysis revealed differences in the sensitivity of the evaluated fungal isolates to thiophanate-methyl (Table 9). Fusarium sp. and Alternaria sp. exhibited the lowest EC₅₀ values, at 0.02 and 0.70 µg mL⁻¹, respectively. Both values were below 1.0 µg mL⁻¹ and, according to the established sensitivity ranges, these isolates were classified as highly sensitive to thiophanate-methyl. In contrast, Ulocladium sp. and Penicillium sp. showed higher EC₅₀ values of 6.90 and 9.27 µg mL⁻¹, respectively. These values were within the 5–20 µg mL⁻¹ range, resulting in the classification of both isolates as moderately sensitive. Overall, the EC₅₀ values indicated that Fusarium sp. exhibited the greatest sensitivity to thiophanate-methyl, followed by Alternaria sp., whereas Ulocladium sp. and Penicillium sp. showed comparatively lower sensitivity, with Penicillium sp. exhibiting the lowest relative sensitivity among the evaluated fungal isolates.

The EC₅₀ analysis revealed differences in the sensitivity of the evaluated fungal isolates to thiabendazole (Table 10). Alternaria sp., Fusarium sp., and Ulocladium sp. exhibited EC₅₀ values of 0.03, 0.11, and 0.32 µg mL⁻¹, respectively. All three values were below 1.0 µg mL⁻¹ and, according to the established sensitivity ranges, these isolates were classified as highly sensitive to thiabendazole. In contrast, Penicillium sp. showed a higher EC₅₀ value of 1.17 µg mL⁻¹, within the 1–5 µg mL⁻¹ range, and was therefore classified as sensitive. Overall, the EC₅₀ values indicated a high level of sensitivity to thiabendazole among the four fungal isolates, with Alternaria sp. exhibiting the greatest sensitivity, followed by Fusarium sp. and Ulocladium sp., whereas Penicillium sp. showed the lowest relative sensitivity among the evaluated isolates.

4. Discussion

The in vitro assays demonstrated substantial variation in the response of Fusarium sp., Penicillium sp., Alternaria sp., and Ulocladium sp. to the six fungicides evaluated. This variation was evident both in the reduction of mycelial growth across fungicide concentrations and in the EC₅₀ values obtained for each fungus–fungicide combination. Overall, the results indicate that fungicide sensitivity was determined not only by the chemical group and mode of action but also by the particular fungal isolate and active ingredient. This finding is relevant because compounds belonging to the same FRAC group did not necessarily produce equivalent responses, emphasizing the importance of evaluating individual active ingredients when designing fungicide-based disease management strategies.

4.1. Response to Triazole Fungicides

The two triazole fungicides showed marked inhibitory activity, although their effects differed among the fungal isolates. At 1 ppm, tebuconazole produced 54.6% inhibition of Fusarium sp. and Penicillium sp., corresponding to high inhibition, whereas mefentrifluconazole resulted in 51.5% inhibition of Alternaria sp. and 35.3% inhibition of Ulocladium sp. These differences demonstrate that fungal responses to DMIs were dependent on both the active ingredient and the fungal isolate, rather than being uniform across the triazole group.

Triazole fungicides belong to the demethylation inhibitors (DMIs) and interfere with ergosterol biosynthesis, an essential process for maintaining fungal cell membrane integrity and function. Their activity is associated with inhibition of sterol 14α-demethylase, thereby disrupting normal sterol composition and restricting fungal growth [16]. Consequently, the reductions in mycelial growth observed in the present study are consistent with the biochemical activity expected for this fungicide group. Previous studies conducted in oregano [17], strawberry [18], and gladiolus [19] have similarly demonstrated the ability of triazole fungicides to suppress fungal development, supporting their effectiveness against several phytopathogenic fungi [20,21].

Nevertheless, the EC₅₀ results revealed substantial differences between tebuconazole and mefentrifluconazole. Fusarium sp. showed high sensitivity to tebuconazole, with an EC₅₀ of 0.10 µg mL⁻¹. This result agrees with previous evidence indicating that triazole-containing fungicides such as metconazole, prothioconazole, and tebuconazole are among the most effective chemical compounds against Fusarium spp. [31]. The strong response observed at relatively low concentrations suggests that ergosterol biosynthesis in the evaluated Fusarium isolate was effectively disrupted by tebuconazole.

Similarly, Penicillium sp. exhibited high sensitivity to tebuconazole, with an EC₅₀ of 0.07 µg mL⁻¹. The low concentration required to reduce mycelial growth by 50% is consistent with the high specificity of triazoles for sterol biosynthesis, a pathway essential for fungal membrane structure and cellular function [32]. However, this response contrasted sharply with that obtained for mefentrifluconazole, for which Penicillium sp. exhibited an EC₅₀ of 749.53 µg mL⁻¹. Such a pronounced difference between two fungicides belonging to the same FRAC group highlights the importance of evaluating active ingredients individually and suggests that sensitivity to one DMI cannot necessarily be extrapolated to another.

The response of Alternaria sp. further illustrates this active ingredient-specific pattern. This fungus exhibited an EC₅₀ of 0.019 µg mL⁻¹ for mefentrifluconazole, indicating very high sensitivity, whereas the EC₅₀ for tebuconazole was considerably higher (22.75 µg mL⁻¹). Previous research on A. alternata has reported a low risk of resistance to mefentrifluconazole, although overexpression-associated mechanisms can confer low levels of reduced sensitivity [33]. The strong contrast observed in the present study indicates that even fungicides targeting the same biochemical pathway can differ substantially in their biological activity against a particular fungal isolate.

For Ulocladium sp., the response was intermediate. Tebuconazole produced an EC₅₀ of 7.46 µg mL⁻¹, corresponding to moderate sensitivity, while mefentrifluconazole showed an EC₅₀ of 1.17 µg mL⁻¹, corresponding to sensitivity. These results are consistent with previous observations of susceptibility of dematiaceous fungi exposed to triazole fungicides [34]. Taken together, the results for DMIs demonstrate that classification based solely on fungicide group is insufficient to predict biological activity. Instead, the interaction between fungal isolate and specific active ingredient appears to be critical in determining the magnitude of the response.

4.2. Response to Strobilurin Fungicides

The QoI fungicides azoxystrobin and pyraclostrobin also reduced mycelial growth, but their inhibitory effects differed among the fungal isolates. At 1 ppm, pyraclostrobin produced 28.3% inhibition of Fusarium sp., while azoxystrobin resulted in 62.9% inhibition of Penicillium sp. In Alternaria sp., azoxystrobin and pyraclostrobin produced 31.8 and 35.9% inhibition, respectively, whereas azoxystrobin produced 48.6% inhibition of Ulocladium sp. Thus, although both fungicides belong to the same FRAC group, their relative activity varied according to the fungal isolate.

Strobilurins act primarily by binding to the quinol oxidation (Qo) site of cytochrome b in the mitochondrial cytochrome bc₁ complex. This interaction interrupts mitochondrial electron transport, reducing energy production and consequently restricting fungal growth and development [22]. The reductions in mycelial growth observed in the present study are therefore consistent with inhibition of mitochondrial respiration. Previous studies in strawberry [23,24], soursop [25], and walnut [26] have also reported effective suppression of fungal development by strobilurin fungicides.

The EC₅₀ values provided additional evidence of differences in sensitivity within this fungicide group. In Fusarium sp., azoxystrobin produced an EC₅₀ of 1.39 µg mL⁻¹, indicating sensitivity, whereas pyraclostrobin produced a lower EC₅₀ of 0.35 µg mL⁻¹, indicating high sensitivity. Previous studies have shown that azoxystrobin can reduce mycelial growth, conidial production, pathogenicity, and ATP production in Fusarium spp., supporting its potential for suppressing this fungal group [35]. However, the lower EC₅₀ obtained with pyraclostrobin in the present study suggests that, for the evaluated isolate, pyraclostrobin exerted stronger activity at lower concentrations.

Penicillium sp. also showed high sensitivity to the QoI fungicides, although the two active ingredients differed quantitatively. Azoxystrobin showed an EC₅₀ of 0.06 µg mL⁻¹, while the response to pyraclostrobin was comparatively lower. The high sensitivity to azoxystrobin is consistent with reports showing reductions in conidial germination and mycelial growth of Penicillium digitatum following azoxystrobin treatment [36]. These findings support the capacity of QoI fungicides to interfere effectively with fungal development, although sensitivity may differ among species and isolates.

The strongest overall response to the strobilurins was observed in Alternaria sp. Azoxystrobin and pyraclostrobin showed EC₅₀ values of 0.01 and 0.04 µg mL⁻¹, respectively, placing both within the highly sensitive category. Similar inhibitory effects against Alternaria spp. have been reported in citrus [37], and studies conducted in mandarin have provided additional evidence supporting the activity of these fungicides [38]. The consistently low EC₅₀ values obtained for both QoIs suggest that mitochondrial respiration represents a highly sensitive target in the Alternaria isolate evaluated in this study.

For Ulocladium sp., azoxystrobin showed an EC₅₀ of 0.10 µg mL⁻¹, indicating high sensitivity, whereas pyraclostrobin produced a considerably higher EC₅₀ of 8.81 µg mL⁻¹, corresponding to moderate sensitivity. Previous assays in garlic support the effectiveness of fungicides from this group against associated fungal pathogens [39]. Nevertheless, the marked difference between the two QoI fungicides observed here again demonstrates that a shared mode of action does not necessarily imply equivalent sensitivity.

This distinction has practical implications for resistance management. Although differences in EC₅₀ may help identify the most active compound against a particular isolate, azoxystrobin and pyraclostrobin share the same primary target site. Therefore, differences in their in vitro efficacy should not be interpreted as evidence that alternating these two active ingredients alone constitutes rotation between independent modes of action. Rather, their use should be integrated with fungicides belonging to different FRAC groups to reduce continuous selection pressure on the same biochemical target.

4.3. Response to Benzimidazole Fungicides

The benzimidazole fungicides thiophanate-methyl and thiabendazole also exhibited isolate-dependent activity. At 1 ppm, thiophanate-methyl produced 33.2% inhibition of Fusarium sp., whereas thiabendazole produced 29.4% inhibition of Penicillium sp. and 48.4% inhibition of Alternaria sp. In Ulocladium sp., inhibition at the same concentration was lower, reaching 24.2% with thiophanate-methyl and 14.9% with thiabendazole. These results demonstrate that the magnitude of inhibition at a single concentration alone does not fully describe fungicide sensitivity and should therefore be interpreted together with the complete concentration-response relationship and EC₅₀ estimates.

Benzimidazole fungicides act primarily by interfering with β-tubulin polymerization and microtubule assembly. Because microtubules are essential for chromosome segregation and other cellular processes during mitosis, disruption of their formation can inhibit cell division, hyphal development, and spore germination [27,28]. The reductions in mycelial growth observed in the present study are consistent with this mechanism of action. Previous studies in crops such as soursop [29] and bean [30] have also reported inhibition of fungal development following treatments with fungicides acting on these cellular processes.

Among the evaluated fungi, Fusarium sp. showed particularly high sensitivity to both MBC fungicides. Thiophanate-methyl and thiabendazole exhibited EC₅₀ values of 0.02 and 0.11 µg mL⁻¹, respectively. These low values indicate that relatively small concentrations were sufficient to reduce mycelial growth by 50%. Previous trials conducted in sugarcane reported 92.1% inhibition of fungal mycelial growth following treatment with thiabendazole [40], supporting the strong antifungal activity of this active ingredient.

In contrast, Penicillium sp. exhibited a markedly different response to the two benzimidazoles. Thiophanate-methyl showed an EC₅₀ of 9.27 µg mL⁻¹, corresponding to moderate sensitivity, whereas thiabendazole exhibited an EC₅₀ of 1.17 µg mL⁻¹ and was classified as sensitive. This approximately eight-fold difference between the two EC₅₀ values indicates substantially lower sensitivity to thiophanate-methyl than to thiabendazole under the experimental conditions. Previous studies have documented reduced sensitivity and resistance to benzimidazoles in Penicillium populations [41], providing a relevant context for the comparatively high EC₅₀ observed in the present study.

However, the elevated EC₅₀ observed for thiophanate-methyl should be interpreted cautiously. Because the present study evaluated in vitro sensitivity of selected isolates rather than the sensitivity distribution of a representative pathogen population, the results indicate reduced relative sensitivity but do not by themselves demonstrate field resistance. Confirmation of resistance would require a broader collection of isolates, appropriate baseline sensitivity data, and ideally complementary phenotypic or molecular evidence.

Alternaria sp. was highly sensitive to both MBC fungicides, with EC₅₀ values of 0.70 µg mL⁻¹ for thiophanate-methyl and 0.03 µg mL⁻¹ for thiabendazole. Previous research in tobacco reported a relatively low frequency (6.0%) of resistance in A. alternata populations [42]. The low EC₅₀ values obtained in the present study are consistent with a sensitive phenotype, particularly for thiabendazole. Nevertheless, continued exposure to fungicides with a single target site may increase selection pressure, making sensitivity monitoring important even when initial isolates show high susceptibility.

A similarly contrasting pattern was observed for Ulocladium sp. Thiophanate-methyl exhibited an EC₅₀ of 6.90 µg mL⁻¹, corresponding to moderate sensitivity, whereas thiabendazole showed a substantially lower EC₅₀ of 0.32 µg mL⁻¹ and was classified as highly sensitive. The effectiveness of MBC fungicides against fungal diseases has also been documented in mango [43]. The large difference between the two active ingredients in the present study suggests that their biological activity against Ulocladium sp. was not equivalent despite their shared mode of action.

4.4. Overall Implications for Fungicide Sensitivity and Disease Management

When the three fungicide groups are considered together, a central finding of this study is the heterogeneity of sensitivity among fungus–fungicide combinations. No single active ingredient exhibited the same relative activity against all four fungal isolates. Furthermore, substantial differences were observed between active ingredients belonging to the same FRAC group. This was particularly evident for mefentrifluconazole and tebuconazole in Alternaria sp. and Penicillium sp., and for thiophanate-methyl and thiabendazole in Penicillium sp. and Ulocladium sp.

The combined interpretation of MGR and EC₅₀ is therefore important. MGR measurements demonstrated how fungal growth changed at individual fungicide concentrations, whereas EC₅₀ provided an integrated quantitative measure of the concentration required to reduce growth by 50%. Consequently, isolates showing apparently moderate inhibition at a particular concentration could nevertheless exhibit low EC₅₀ values when the entire concentration-response relationship was considered. The two parameters should thus be viewed as complementary rather than interchangeable indicators of fungicide response.

From a phytosanitary perspective, the results provide baseline information on the in vitro sensitivity of fungi associated with white onion cultivation in Santa Fe de Galán. Active ingredients such as tebuconazole against Fusarium sp. and Penicillium sp.; mefentrifluconazole against Alternaria sp.; azoxystrobin and pyraclostrobin against Alternaria sp.; and thiophanate-methyl and thiabendazole against Fusarium sp. exhibited particularly low EC₅₀ values. Conversely, the high EC₅₀ observed for mefentrifluconazole in Penicillium sp. and the comparatively elevated values obtained for some MBC–fungus combinations identify responses that warrant further investigation.

Nevertheless, these findings represent in vitro sensitivity under controlled conditions and should not be directly extrapolated to field efficacy. Fungicide performance under field conditions may be influenced by application timing, dose, coverage, environmental conditions, pathogen population structure, host phenology, fungicide persistence, and previous exposure of fungal populations to particular modes of action. Similarly, an elevated EC₅₀ in an individual isolate should not automatically be considered evidence of established resistance in the local pathogen population.

Future studies should therefore expand the number of isolates collected from different white onion production areas and growing seasons to establish population-level baseline sensitivity distributions. Such studies would allow discrimination between natural variability in sensitivity and true shifts associated with selection pressure. Integrating phenotypic sensitivity assays with molecular characterization of resistance-associated mechanisms would further strengthen resistance monitoring. Ultimately, these data could support rational rotation of fungicides with different modes of action and reduce repeated selection pressure on the same fungal target, contributing to more sustainable disease management in white onion production.

5. Conclusions

The in vitro sensitivity assays demonstrated that the response of the fungal isolates associated with white onion varied according to both the active ingredient and fungicide concentration. Tebuconazole showed strong inhibitory activity at low concentrations, particularly against Fusarium sp. and Penicillium sp., whereas azoxystrobin also produced high inhibition of Penicillium sp. at low concentrations. These results confirm that the magnitude of mycelial growth inhibition was dependent on the specific fungus–fungicide combination.

The EC₅₀ analysis further demonstrated marked differences in sensitivity among the evaluated fungal isolates. Fusarium sp. was highly sensitive to tebuconazole, pyraclostrobin, thiophanate-methyl, and thiabendazole, and sensitive to azoxystrobin. Alternaria sp. showed high sensitivity to mefentrifluconazole, azoxystrobin, pyraclostrobin, thiophanate-methyl, and thiabendazole, while exhibiting substantially lower sensitivity to tebuconazole. Ulocladium sp. showed high sensitivity to azoxystrobin and thiabendazole, sensitivity to mefentrifluconazole, and moderate sensitivity to tebuconazole, pyraclostrobin, and thiophanate-methyl. In Penicillium sp., the response was particularly dependent on the active ingredient, ranging from high sensitivity to tebuconazole and azoxystrobin to moderate sensitivity to thiophanate-methyl and markedly reduced sensitivity to mefentrifluconazole.

Overall, the results demonstrate that fungicides belonging to the same FRAC group did not necessarily produce equivalent sensitivity responses, emphasizing the importance of selecting active ingredients according to the fungal target rather than solely according to their chemical group. The MGR and EC₅₀ values generated in this study provide initial baseline information on the in vitro sensitivity of fungal isolates associated with white onion production in Santa Fe de Galán, Ecuador, and may contribute to the rational selection and rotation of fungicides with different modes of action. However, because these findings were obtained under controlled in vitro conditions and from selected fungal isolates, further studies involving larger pathogen populations and field evaluations are required before extrapolating these results to resistance status or field efficacy.

Funding

This study was supported by the FIASA Project No. FIASA-CA-2024-029, “Microbioma como estrategia para mitigar futuras pandemias vegetales, buscando un mecanismo de resiliencia en papa y maíz al cambio climático en la provincia de Chimborazo”, funded by the Fondo de Investigación para la Agrobiodiversidad, Semillas y Agricultura Sustentable (FIASA), Ecuador.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are available upon request to the corresponding author.

Acknowledgments

The authors thank the Fondo de Investigación para la Agrobiodiversidad, Semillas y Agricultura Sustentable (FIASA), Ecuador, for supporting this study through Project No. FIASA-CA-2024-029, “Microbioma como estrategia para mitigar futuras pandemias vegetales, buscando un mecanismo de resiliencia en papa y maíz al cambio climático en la provincia de Chimborazo”.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Mycelial growth rate (MGR) of (A) Fusarium sp., (B) Penicillium sp., (C) Alternaria sp., and (D) Ulocladium sp. in PDA medium supplemented with different concentrations of tebuconazole. Different letters indicate significant differences among concentrations according to Tukey’s test at the 5% significance level (p ≤ 0.05).

Figure 2. Mycelial growth rate (MGR) of (A) Fusarium sp., (B) Penicillium sp., (C) Alternaria sp., and (D) Ulocladium sp. in PDA medium supplemented with different concentrations of mefentrifluconazole. Different letters indicate significant differences among concentrations according to Tukey’s test at the 5% significance level (p ≤ 0.05).

Figure 3. Mycelial growth rate (MGR) of (A) Fusarium sp., (B) Penicillium sp., (C) Alternaria sp., and (D) Ulocladium sp. in PDA medium supplemented with different concentrations of pyraclostrobin. Different letters indicate significant differences among concentrations according to Tukey’s test at the 5% significance level (p ≤ 0.05).

Figure 4. Mycelial growth rate (MGR) of (A) Fusarium sp., (B) Penicillium sp., (C) Alternaria sp., and (D) Ulocladium sp. in PDA medium supplemented with different concentrations of azoxystrobin. Different letters indicate significant differences among concentrations according to Tukey’s test at the 5% significance level (p ≤ 0.05).

Figure 5. Mycelial growth rate (MGR) of (A) Fusarium sp., (B) Penicillium sp., (C) Alternaria sp., and (D) Ulocladium sp. in PDA medium supplemented with different concentrations of thiophanate-methyl. Different letters indicate significant differences among concentrations according to Tukey’s test at the 5% significance level (p ≤ 0.05).

Figure 6. Mycelial growth rate (MGR) of (A) Fusarium sp., (B) Penicillium sp., (C) Alternaria sp., and (D) Ulocladium sp. in PDA medium supplemented with different concentrations of thiabendazole. Different letters indicate significant differences among concentrations according to Tukey’s test at the 5% significance level (p ≤ 0.05).

Table 1. Fungal isolates obtained and used in the experimental trials.

Pathogen	Site	Crop	Sample type
(A) Fusarium sp.	Santa Fe de Galán	White onion	Root
(B) Penicillium sp.	Santa Fe de Galán	White onion	Root
(C) Alternaria sp.	Santa Fe de Galán	White onion	Foliar
(D) Ulocladium sp.	Santa Fe de Galán	White onion	Root

Table 2. Concentration of the active ingredient for preparing the stock solution.

Mode of action	Chemical group	Active ingredient	Trade name	Active ingredient concentration
G1	Triazoles	Tebuconazole	Stratego® SC	250 g.L^-1
G1	Triazoles	Mefentrifluconazole	Belanty®	400 g.L^-1
C3	Strobirulins	Pyraclostrobin	Comet® 25 CE	250 g.L^-1
C3	Strobirulins	Azoxystrobin	Amistar 50 WG	500 g.kg^-1
B1	Benzimidazoles	Thiophanate-methyl	Novak® 500 SC	500 g.L^-1
B1	Benzimidazoles	Thiabendazole	Mertect 500 SC	500 g.L^-1

Table 3. Percentage Inhibition Scale [14].

Sensitivity	Range
Low inhibition	0% - 25%
Moderate inhibition	25% - 50%
High inhibition	> 50%

Table 4. Sensitivity scale proposed by [15].

Sensitivity	Range
Very sensitive	<1,0 μg/mL
Sensitive	1-5 μg/mL
Moderately resistant	5-20 μg/mL
High resistance	20-100 μg/mL
Very high resistance	>500 μg/mL

Table 5. Effective concentration (EC₅₀) of tebuconazole for Fusarium sp., Penicillium sp., Alternaria sp., and Ulocladium sp.

	Code	DL₅₀	Phenotype	Sensitivity
Tebuconazole	Fusarium sp.	0,10	<1,0 µg.mL^-1	Very sensitive
	Penicillium sp.	0,07	<1,0 µg.mL^-1	Very sensitive
	Alternaria sp.	22,75	20-100 µg.mL^-1	High resistance
	Ulocladium sp.	7,46	5-20 µg.mL^-1	Moderately sensitive

Table 6. Effective concentration (EC₅₀) of mefentrifluconazole for Fusarium sp., Penicillium sp., Alternaria sp., and Ulocladium sp.

	Code	DL₅₀	Phenotype	Sensitivity
Mefentrifluconazole	Fusarium sp.	43,05	20-100 µg.mL^-1	High resistance
	Penicillium sp.	749,53	> 500 µg.mL^-1	Very high resistance
	Alternaria sp.	0,019	<1,0 µg.mL^-1	Very sensitive
	Ulocladium sp.	1,17	1-5 µg.mL^-1	Sensitive

Table 7. Effective concentration (EC₅₀) of azoxystrobin for Fusarium sp., Penicillium sp., Alternaria sp., and Ulocladium sp.

	Code	DL₅₀	Phenotype	Sensitivity
Azoxystrobin	Fusarium sp.	1,39	1-5 µg/ml^-1	Sensitive
	Penicillium sp.	0,06	<1,0 µg/ml^-1	Very sensitive
	Alternaria sp.	0,01	<1,0 µg/ml^-1	Very sensitive
	Ulocladium sp.	0,10	<1,0 µg/ml^-1	Very sensitive

Table 8. Effective concentration (EC₅₀) of pyraclostrobin for Fusarium sp., Penicillium sp., Alternaria sp., and Ulocladium sp.

	Code	DL₅₀	Phenotype	Sensitivity
Pyraclostrobin	Fusarium sp.	0,35	<1,0 µg/ml^-1	Very sensitive
	Penicillium sp.	1,17	1-5 µg/ml^-1	Sensitive
	Alternaria sp.	0,04	<1,0 µg/ml^-1	Very sensitive
	Ulocladium sp.	8,81	5-20 µg/ml^-1	Moderately sensitive

Table 9. Effective concentration (EC₅₀) of methyl thiophanate for Fusarium sp., Penicillium sp., Alternaria sp., and Ulocladium sp.

	Code	DL₅₀	Phenotype	Sensitivity
Methyl thiophanate	Fusarium sp.	0,02	<1,0 µg/ml^-1	Very sensitive
	Penicillium sp.	9,27	5-20 µg/ml^-1	Moderately sensitive
	Alternaria sp.	0,70	<1,0 µg/ml^-1	Very sensitive
	Ulocladium sp.	6,90	5-20 µg/ml^-1	Moderately sensitive

Table 10. Effective concentration (EC₅₀) of thiabendazole for Fusarium sp., Penicillium sp., Alternaria sp., and Ulocladium sp.

	Code	DL₅₀	Phenotype	Sensitivity
Tiabendazole	Fusarium sp.	0,11	<1,0 µg/ml^-1	Very sensitive
	Penicillium sp.	1,17	1-5 µg/ml^-1	Sensitive
	Alternaria sp.	0,03	<1,0 µg/ml^-1	Very sensitive
	Ulocladium sp.	0,32	<1,0 µg/ml^-1	Very sensitive

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In Vitro Sensitivity of Four Plant-Pathogenic Fungi Isolated from White Onion (Allium fistulosum L.) to Fungicides with Different Modes of Action