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Article

Antifungal and Toxicological Evaluation of Natural Compounds Such as Chitosan, Citral, and Hexanal Against Colletotrichum asianum

by
Edson Rayón-Díaz
1,
Luis G. Hernández-Montiel
2,
Víctor Manuel Zamora-Gasga
1,
Jorge A. Sánchez-Burgos
1,
Surelys Ramos-Bell
1,
Rita María Velázquez-Estrada
1,
Juan Antonio Herrera-González
3 and
Porfirio Gutiérrez-Martínez
1,*
1
Laboratorio Integral de Investigaciones en Alimentos, Tecnológico Nacional de Mexico/Instituto Tecnológico de Tepic, Avenida Tecnológico #2595, Col. Lagos del Country, Tepic 63175, Nayarit, Mexico
2
Nanotechnology & Microbial Biocontrol Group, Centro de Investigaciones Biológias del Noroeste, La Paz 23096, Baja California Sur, Mexico
3
Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias, Campo Experimental Uruapan, Av. Latinoamericana 1101, Col. Revolución, Uruapan 60150, Michoacán, Mexico
*
Author to whom correspondence should be addressed.
Horticulturae 2025, 11(5), 474; https://doi.org/10.3390/horticulturae11050474
Submission received: 25 February 2025 / Revised: 25 April 2025 / Accepted: 25 April 2025 / Published: 28 April 2025
(This article belongs to the Special Issue Fungal Diseases in Horticultural Crops)
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Abstract

:
The Colletotrichum genus is one of the ten most relevant pathogenic fungi in the post-harvest sector owing to its high infection rate in tropical fruits; however, the search for alternatives to synthetic fungicides is crucial because of their adverse effects on health and the environment. This study evaluated the efficacy of chitosan (CH), citral (CT), and hexanal (HX) against Colletotrichum asianum, as well as the toxicological potential of these treatments. In in vitro tests, 1.0% CH, 0.03% CT, and 0.06% HX significantly inhibited fungal development in parameters of radial growth, sporulation, fungal biomass, and germination by 78–100% (p < 0.05). Furthermore, the toxicity index was low to moderate for most concentrations using cucumber and tomato seed germination as a study model. Toxicokinetic predictions suggest that CH, CT, and HX molecules do not pose a danger to human consumption, suggesting that they are promising alternatives to chemical fungicides for the control of phytopathogenic fungi.

1. Introduction

Mango (Mangifera indica L.) is a highly consumed fruit worldwide because of its outstanding nutritional value and contains carbohydrates, proteins, fats, minerals, and essential vitamins, such as vitamin A, vitamin B1, vitamin B2, and vitamin C [1,2]. Its climacteric characteristics accelerate post-harvest senescence, intensify ethylene production, and favor microbial proliferation, particularly in phytopathogenic fungi [3].
The development of a disease depends on environmental aspects, the pathogen itself, the characteristics of the host, and the actions of man. The genera of phytopathogenic fungi most frequently found in mango fruit are Lasiodiplodia, Cladospurium, and Colletotrichum [4]. Approximately 12 species of the genus Colletotrichum are associated with mango tissue infection worldwide, belonging to the C. gloeosporioides, C. boninense, C. cliviicola, and C. acutatum species complexes. Among these, Colletotrichum asianum is the second most prevalent species in Mexico, causing losses ranging from 20 to 46% during fruit storage and marketing [5,6]. The application of synthetic fungicides such as benomyl, carbendazim, mancozeb, and prochloraz to control Colletotrichum species requires strict regulations in exporting countries, as demonstrated by the Mexican Official Standard NOM-046-FITO-1995 in Mexico, which establishes phytosanitary standards for the movement of mangoes destined for export and the domestic market [7]. However, the use of synthetic fungicides has caused problems such as the accumulation of chemical residues in fruits, microbial resistance, and physiological alterations in trees [8,9]. These factors limit the effectiveness of controlling conventional phytopathogen methods and highlight the need for sustainable alternatives for the post-harvest management of fruits.
Advances in technologies related to eco-friendly products have transformed modern agriculture by promoting sustainable alternatives to conventional chemicals. Post-harvest treatments based on natural materials, such as chitosan and bioactive aldehydes, have shown high potential for controlling fungal diseases [10,11]. The antifungal effect of these treatments has been demonstrated at the postharvest stage, achieving a reduction in the severity of fungi such as Rhizopus (85%), Botrytis, and Penicillium in apples (48% and 62%, respectively); Colletotrichum in avocado and mango (100%) for applications starting at 1.0% reagent-grade chitosan [12,13,14,15]; and Penicillium in orange (100%), with applications starting at 150 mL·L−1 of citral [16]. These effects in fungi-treatment interaction cause alterations in the characteristics of the hyphae, permeability of the plasma membrane, leakage of intracellular compounds, and a reduction in the activity of succinate dehydrogenase (SDH) and malate dehydrogenase (MDH) enzymes related to the mitochondrial TCA cycle. The latter has only been reported for aldehyde activity [17,18,19,20,21]. It has been reported that applying treatments such as chitosan to fruits reduces respiration and ethylene production, maintains the sensory quality of fruits such as mangoes, and prevents oxidation and the development of pathogenic microorganisms [22,23,24]. The antifungal activity and improved shelf life of fruits owing to chitosan and bioactive aldehydes reinforce their potential as innovative tools for post-harvest management, minimizing environmental impacts and maximizing fruit protection and quality during national and international marketing.
To find alternatives for the control of Colletotrichum asianum in mangoes, in vitro treatments based on chitosan and bioactive aldehydes (citral (CT) and hexanal (HX)) were evaluated. The tests focused on analyzing the capacity of these compounds to inhibit fungal development. Additionally, the cytotoxic effects of the treatments were investigated using tomato and cucumber seeds as biological models to evaluate germination and root development parameters. In addition, in silico analysis was performed to characterize absorption, distribution, metabolism, elimination, and toxicology (ADMET) properties. This multidisciplinary approach will facilitate the development of safer and more effective treatments for controlling fungal diseases by integrating the analysis of their inhibitory activity and toxicological profile for future agri-food applications.

2. Materials and Methods

2.1. Reactivation of Colletotrichum Asianum Strain

Mycelial discs were obtained from the strain earlier isolated from mango fruits and identified as C. asianum (GenBank accession numbers ITS MN272369.1; GPDH MK376935.1) [25]. Mycelial discs 6 mm in diameter were placed on potato dextrose agar (PDA) medium (DIBICO S.A. de C.V., Cuautitlán Izcalli, Mexico). The plates were incubated at 25 ± 2 °C for 10 d (NOVATECH Incubator Mod. E160-ED, State of Mexico, Mexico). Once the strain was activated, it was used in experiments corresponding to the experimental stage.

2.2. Preparation and Application of Treatments

Commercial-grade chitosan (CH) from the Golden-Shell Pharmaceutical® brand was used (90% deacetylation degree, low molecular weight, high density, and 177 µm particle size). The proposed methodology followed that described by [14]. An acidified solution was prepared at 1.5% acidity in a volume of 1000 mL using commercial acetic acid (Members Mark®, 5% acidity). Subsequently, 40 g of chitosan was added to obtain a final concentration of 4% (w/v). The solution was mixed using a magnetic stirrer (IKA® C-MAG-HS7S1, Staufen, Germany) at 800 rpm for 24 h. Heat (80 °C) was applied for the first four hours to facilitate chitosan dissolution. Finally, dilutions were prepared from the stock solution, maintaining CH concentrations at 0.5%, 1.0%, and 1.5% (v/v), and mixed with PDA medium to a total volume of 25 mL.
Citral (CT) and hexanal (HX) were prepared following the methodology described by [26]. Certified reagent-grade compounds (Sigma-Aldrich®, St. Louis, MO, USA) were diluted in sterilized distilled water and 0.5% dimethyl sulfoxide (DMSO) in combination with 2% TWEEN 80 for citral and 2% TWEEN 20 for hexanal used as stabilizers (Sigma-Aldrich®, St. Louis, MO, USA). The mixture was stirred at 800 rpm for 2 h at ambient temperature using a magnetic stirrer (IKA® C-MAG-HS7S1, Staufen, Germany). Fixed volumes of 5 mL were added at concentrations of 0.03%, 0.05%, and 0.1% (v/v) for CT and 0.04%, 0.06%, and 0.08% (v/v) for HX, in combination with PDA medium, up to a total volume of 25 mL.
The “poisoned agar” method was used, which consisted of incorporating known concentrations of the treatments into the PDA medium before pouring it into Petri dishes. Distilled water (DW), 0.5% DMSO, and 1.5% commercial acetic acid (AAc) (pH 2.3) were used as controls to evaluate the possible inherent effects of these compounds on the development of C. asianum.

2.3. In Vitro Test

2.3.1. Mycelial Inhibition

Following the poisoned agar method, once the medium solidified with the treatment, C. asianum mycelial discs (6 mm in diameter) were placed in the center of the Petri dishes (n = 8). The plates were incubated at 25 ± 2 °C for 10 d. The radial growth of the mycelium was measured every 48 h until the end of the incubation period using a digital vernier with a precision of 0.01 mm. The final growth values were used to calculate the percentage of inhibition, with distilled water (DW) serving as the reference standard [25].

Sporulation

Once the mycelial inhibition test was completed, spore suspensions were prepared according to the method described by [25]. For this purpose, 10 mL of sterilized distilled water was added to the mycelia in each Petri dish, and the entire growth area was carefully scraped to release spores. The obtained suspension was manually homogenized, and 10 µL was collected for analysis. Spore counting was performed using a hemocytometer and Motic BA300 microscope (Xiamen, China) at 40× magnification. The final spore concentration per milliliter (spores/mL) was determined by averaging spore counts per treatment using a hemocytometer.

2.3.2. Fungal Biomass

The fungal biomass assay was performed according to the method proposed by [27] with some modifications. The previously described poisoned agar method was used, on which Whatman® #1 filter paper discs (8.8 cm diameter) were placed, covering the surface of the medium with treatment. Next, a disc of C. asianum mycelium (6 mm diameter) was placed in the center of the Petri dish and incubated at 25 ± 2 °C for 8 d (n = 8).
At the end of the incubation period, the filter paper containing the adhered mycelium was removed and placed on a watch glass to measure the initial weight by using an analytical balance (REDWAG Wagi Elektroniczne, AS 220/C/2, Radom, Poland). The samples were then placed in an oven (SERVIPLUS, Mod. JES70SE, Mexico City, Mexico) at intervals of 30 s at a temperature of approximately 60 °C until a constant weight was obtained. The dry weight of each sample was recorded in grams (g).

2.3.3. Percentage of Spore Germination

The C. asianum spore germination percentage was determined using the methodology proposed by [28] with some modifications. Thin 5 mm2 slices of PDA treatment were placed on a sterile slide. The slides were inoculated with 20 µL of spore suspension (8 × 106 spores/mL) and incubated at 25 ± 2 °C for 7 h. One hundred spores were counted under an optical microscope (Panthera, Motic BA300, Xiamen, China) with a 40× objective. Spores that were considered germinated were those with elongation of the germ tube double its initial size. The germination percentage was calculated using Equation (1), and the test was performed in triplicate for each treatment.
Germination% = (Number germinated spores/Number total spores) × (100)

2.4. Cytotoxicological Tests

For the cytotoxicological test of seeds, the methodology proposed by [29] was used, with some modifications. Tomato (Solanum lycopersicum D. var. cerasiforme) and cucumber (Cucumis sativus L. var. poinsett) seeds from Hortaflor® were used as biological models to evaluate the effects of the treatments.

2.4.1. Seed Pre-Treatment

The seeds were subjected to a four-stage disinfection process: first, a rinse with distilled water for 30 s, followed by immersion in 10% sodium hypochlorite for 10 min, a bath in 70% alcohol solution for 60 s, and finally, two rinses with sterilized distilled water. The seeds were placed on sterile gauze and left to dry in a laminar flow hood.

2.4.2. Seed Treatment Application

After pretreatment, 20 seeds (10 tomatoes and 10 cucumbers) were randomly selected and placed in 90 mm diameter Petri dishes with Whatman® #1 filter paper discs. Then, 5 mL of the corresponding treatments at different concentrations was added to each dish. DW and 0.5% DMSO were used as the controls. The seeds were stored at 25 ± 2 °C for 8 d. The effects of the treatments were evaluated by measuring the radicle length and width and recording the data for each group. The test was performed in duplicate, and the results were analyzed using Equations (2)–(7).
REI is root elongation inhibition:
REI = 1 − (Treatment root length/Reference root length) × 100
RRE is relative root elongation:
RRE = (Treatment root length/Reference root length) × 100
RSG is relative seed germination:
RSG = (Treatment seeds germination/Reference seeds germination) × 100
GI is the Germination Index:
GI = (Relative seed germination × Relative root elongation)/100
NRGI is the Normalized Residual Germination Index:
NRGI = (Treatment seed germination − Reference seed germination)/Reference seed germination
NRREI is the Normalized Residual Root Elongation Index:
NRREI = (Treatment root elongation − Reference root elongation)/Reference root elongation
The phytotoxicity scale ranged from −1 (maximum toxicity) to positive values, indicating the stimulation of seed growth (hormesis). A 50% reduction in response variables, such as seed germination or root elongation, is considered potentially indicative of a chronic toxic effect, reflecting a significant decrease in the viability or growth of organisms in the long term. For this reason, the scale proposed by [30] indicates the following: (a) 0 to −0.25 low toxicity, (b) −0.25 to −0.50 moderate toxicity, (c) −0.5 to −0.75 high toxicity, and (d) −0.75 to −1 very high toxicity.
Histological sections were prepared according to the protocol published by [31] with some modifications. After 8 d of germination, the plant material was stored at 5 °C for 24 h. The roots were manually sectioned with a scalpel under a digital LED stereoscopic microscope (Motic® Model DM143, Xiamen, China), producing cross-sections approximately 0.5–1 mm thick. The tissues were mounted on a slide and stained with safranin and alcian blue to identify specific cell structures such as primary and secondary cell walls. Observations were made using a microscope (Panthera, Motic BA300, Xiamen, China) with a 40× objective.

2.4.3. In Silico Analysis of Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET)

The in silico analysis was performed according to the methodology proposed by [32]. The Swiss ADMET server was used to evaluate the physicochemical (size, polarity, solubility, saturation, flexibility, and lipophilicity) and pharmacokinetic (interaction with cytochrome P450 and lethal dose (LD50)) properties of the compounds used. In contrast, toxicological properties were analyzed using StopTox, pkCSM, Protox-II, and SwissADME tools. Simplified molecular input line entry system (SMILES) files of each compound (CH, CT, and HX) were introduced into the servers to evaluate their classification, prediction, and toxicological probability parameters.

2.5. Statistical Analysis

Data are expressed as the mean ± standard error for the response variables of mycelial growth, sporulation, and biomass, which were performed in duplicate, and as the mean ± standard error of three replicates in the variables of germination percentage and cytotoxicity in tomato and cucumber seed models. Results were analyzed by one-way analysis of variance (ANOVA) and Fisher’s LSD post-hoc tests to compare means, considering a statistical significance value of 0.05. The study was carried out using the Statistica v10 program (StatSoft, Data Analysis Software System, Tulsa, OK, USA).

3. Results

3.1. In Vitro Effect on the Development of Colletotrichum asianum

3.1.1. Inhibition of Radial Growth, Sporulation, and Fungal Biomass

CT, HX, and CH concentrations significantly reduced mycelial growth of C. asianum in vitro (p < 0.05). Low concentrations of HX and CH allowed mycelial growth on day 10 of evaluation (11.31 and 17.82 mm, respectively); however, it was significantly lower than that of the reference treatment (DW 90 mm) (Figure 1). The percentage of mycelial inhibition of C. asianum ranged from 78.9 to 100% in CH, CT, and HX treatments, showing a relationship with increasing applied concentration.
In addition, the capacity to generate reproductive cells and the total mycelial mass were affected (Table 1). Previous studies have reported that CH concentrations with different degrees of deacetylation and molecular weights can influence the morphology and growth kinetics of fungal strains such as A. niger, B. cinerea, F. oxysporum, R. stolonifer, and C. truncatum [33,34,35]. Moreover, this is the first report on the effects of CH, CT, and HX on fungal biomass against the development of phytopathogens.

3.1.2. Germination Percentage

Spore germination varied significantly depending on the treatment (p < 0.05). DMSO (0.5%) and DW showed the highest germination rates (100%), with no significant differences. CH treatment resulted in the lowest germination rate, regardless of the concentration. HX and CT showed variability depending on concentration, with 0.08% HX presenting the lowest germination within its group and 0.05% CT the highest. AAc (1.5%) exhibited intermediate germination rates (Figure 2). Germination is the first stage in the development of phytopathogenic fungi and is linked to the development of infections in various plant tissues [36]. The application of CH at concentrations starting from 0.5% and bioactive aldehydes at low concentrations (<0.1%) decreases the development of the germ tubes of pathogens belonging to genera such as Aspergillus, Alternaria, Botrytis, and Colletotrichum from 70 to 100% [13,14,28,36,37].
The germination results are shown in Figure 3. The average concentrations of each treatment had different effects on spore morphology. Complete inhibition of germ tube development was observed only with the application of CH and AAc. CT and HX partially inhibited growth. The DMSO and DW treatments resulted in normal spore germination development, consistent with the effects on CH–spore and aldehyde–spore interactions mentioned in the previous subsection.

3.2. Cytotoxicological Effect on Seed Germination

3.2.1. Radicle Development in Tomato and Cucumber Seeds

The results of this study revealed significant differences (p < 0.05) in the development of tomato and cucumber roots when exposed to CH, CT, and HX treatments. Notably, cucumber seeds treated with CH exhibited a significant decrease in germination, whereas this effect was less pronounced in the tomato seed model. However, in the cucumber model, seeds exposed to CT and HX demonstrated improved germination compared with the tomato model. These results indicate that the sensitivity of each plant model to the evaluated treatments may differ. Furthermore, seeds treated with DMSO exhibited a stimulating effect on root length, suggesting a potential role in promoting root growth, whereas AAc treatment completely inhibited root germination (Figure 4).
Significant differences were observed in the germination indices of the tomato and cucumber seeds (p < 0.05). The reference treatment (DW) resulted in normal germination values, whereas the DMSO treatment stimulated germination in the cucumber seed model. The calculated values of the normalized residual germination percentage index (NRGI) indicated low toxicity levels in the DW and DMSO treatments, as well as in CH at 0.5% and in HX at all concentrations across both study models. The CT treatment exhibited low toxicity in the cucumber seed model but moderate to high toxicity in the tomato seed model. The remaining concentrations of CH (1% and 1.5%) presented moderate to high germination toxicity indices (Table 2).

3.2.2. Effect on Root Tissue

Histological sections of the root tissue from seeds treated with CH, CT, and HX showed alterations in their development (Table 3). Safranin, which stains lignified components and the secondary cell wall, and alcian blue, which stains acidic polysaccharides and the primary cell wall, confirmed the proper formation of these crucial structures in cell development. CH treatment disrupted the radicle formation cycle, as evidenced by the irregular staining and structural disorganization observed in the seed tissue histological sections, which is related to the phytotoxic effect mentioned in the previous section. In addition, acidified solutions such as AAc treatment can alter cellular processes essential for germination, potentially by affecting internal pH regulation [38,39]. In the CT and HX treatments, slight alterations were observed in the formation of primary and secondary walls. However, it did not interrupt seed germination or radicle development.

3.2.3. Toxicokinetic Predictions of Treatments

The characteristics and processes related to the administration of a compound in the human body are evaluated on the basis of its toxicokinetic properties, including absorption, distribution, metabolism, elimination, and toxicity (ADMET). These properties are essential for determining their possible toxicological and pharmacological effects. In this study, the chemical molecules of CH, CT, and HX treatments were analyzed using in silico tools such as “SwissADME”, “pkCSM”, “Protox-II”, and “StopTox”, which provided a comprehensive view of the ADMET profile and the possible risk associated with the toxicity of these treatments. These analyses range from rapid assessments of basic pharmacokinetic properties (SwissADME and pkCSM) to more comprehensive evaluations covering a broad spectrum and identifying potential risks at target sites and acute toxicity (Protox-II and StopTox), which are crucial for regulatory considerations.
The chemical properties of CH, CT, and HX molecules were different for molecular weight, topological polar surface area (TPSA), hydrogen acceptors and donors, and their prediction in the toxicity class, which is related to the median lethal dose (LD50) established in mg/kg of body weight values and categorized on a scale of six different classes, according to the Protox-II platform (Table 4). The CH molecule obtained the highest values in TPSA as well as in its H+ donors and acceptors, attributes that confer its high reactivity and compatibility with other organic and inorganic molecules. CT and HX molecules presented similar characteristics in their TPSA, indicative of their bioavailability and rapid intestinal absorption (95.317 and 95.788%, respectively) according to the pkCSM platform.
The models used for the in silico prediction of the molecules were the ranges in “organic toxicity” with specific models for hepatotoxicity and “toxicity points” with models for carcinogenicity, immunotoxicity, mutagenicity, and cytotoxicity, in addition to some “toxicological routes” based on data from the Tox21 platform and “toxicity targets” to predict possible interactions with nuclear receptors, enzymes, and proteins involved in toxic responses. Toxicological analysis of CH, CT, and HX molecules resulted in “inactive” predictions with a high probability for most targets. Based on the parameters of the models analyzed, there is a low probability that these compounds cause specific damage, demonstrating their safe application according to the in silico test (Table 5 and Figure 5).

4. Discussion

The application of treatments such as CH, CT, and HX against phytopathogenic fungi in tropical fruits shows differential sensitivity, meaning that the inhibitory effects vary depending on the treatment. In particular, inhibition depended on the concentration of each treatment. For example, increasing the concentration of chitosan inhibited different stages of fungal development [40].
Our results on the in vitro effects of applying bioactive aldehydes align with those reported by [41], who observed a 100% inhibition of C. gloeosporioides and L. theobromae development. Additionally, there was 75.5% and 60% inhibition, respectively, at 24 h. Likewise, individual treatments with CH and Mentha piperita essential oil at 10 mg/mL achieved total inhibition of C. gloeosporioides and C. brevisporum development [42]. However, regarding the in vitro control of C. asianum using biological treatments, an inhibitory effect of 28.19% was reported according to [43], and the in vitro application of GRAS compounds showed inhibition levels ranging from 28.6% to 38.2% [25]. Therefore, our results are promising for controlling the development of this same species of phytopathogenic fungus.
The antimicrobial activity of chitosan is influenced by several factors, including molecular weight, degree of deacetylation, solvent pH, temperature at which the treatment is applied, and type of pathogen. The latter is related to the sensitivity of the plasma membrane, mainly those that present greater fluidity and are rich in polyunsaturated fatty acids [44,45]. Also, it has been mentioned that chitosan is most effective when the pH is between the ranges of 4.4 and 6.1. This is related to the availability of positive charges in the functional groups with greater reactivity (NH2 and primary OH) and their interaction with the negatively charged phospholipids found in the fungal cell wall through electrostatic interactions [46,47,48].
Recently, it has been suggested that the polycationic nature of chitosan could explain its impact on adhesion to cell membrane components, such as proteins and phospholipids, generating a positive impact on the inhibition of strain development and the release of intracellular substances, fragmenting the formation of cellular structures and restricting the transport of solutes into the cell [18]. This can result in low availability of phosphate ions and metals such as Ca2+ and Mg2+, which are essential for activities related to the formation of the plasma membrane, as well as limitations in the metabolic processes of the spore, such as the excretion of metabolites and changes in intracellular pH, blocking melanin formation, and the inhibition of appressorium formation [46,49,50,51].
In the case of antifungal activity by the application of bioactive aldehydes, such as CT and HX, most studies consider the use of low concentrations (<0.1%), centralizing their fungitoxic activity as the main inhibitory mechanism [52]. In particular, morphological changes are linked to disruption of the fungal cell membrane [53]. In addition, the application of CT and HX has been related to the blocking of the synthesis of ergosterol and sorbitol present in the cell wall and membrane, as well as the reduction in the activities of mitochondrial dehydrogenases, generating an increase and accumulation of reactive oxygen species in the spore, and the inhibition of the activity of enzymes involved in the synthesis of the cell wall and energy production, such as glucan synthases and chitinases, affecting the development of the fungus [54,55,56]. Regarding the inhibition of fungal development, effects have been reported on the activity of chitin synthase, an enzyme related to specific pathways for the formation of chitin in some phytopathogenic fungi, specifically those linked to the pathways of amino acid and nucleotide sugar metabolism [19], which would help explain the antifungal activity of the compounds evaluated. However, a more complete understanding of this phenomenon requires further research.
The results of the cytotoxicity assay were in agreement with those reported in previous studies. Ref. [57] mentioned that CH concentrations greater than 0.5% can affect the germination capacity of arugula seeds, so the applied concentration factor plays an important role in decreasing root development. These findings suggest that the test conditions (exposure time and pH of the medium), as well as the high concentrations of the treatments, influence the magnitude of phytotoxicity; therefore, the decrease in the viability and vigor of seeds exposed to these agents can be attributed to various mechanisms, such as oxidative stress, alterations in nutrient absorption, and deregulation of physiological processes. Future studies should concentrate on refining methods to detect negative impacts on crops early and on continuously enhancing strategies that encourage the sustainability of agri-food systems [29,58,59]. Additionally, the histological test results contrast with those reported by [60,61], who described a stimulating effect on seed development at concentrations of less than 0.4% chitosan. However, concentrations of less than 1% CH showed conservation effects on zucchini seeds, preserving them for up to three months without affecting radicle development [39]. This could be related to the formation of a film on the surface of the seed, which limits its water absorption and respiration phases [59,62]. Concerning CT, a decrease in tissue conformation was observed compared with the reference treatment. An herbicidal effect has been previously reported, generating alterations in root development, auxin content, and the absence of root hairs in the short term, which could be related to the low elongation and formation of cell walls at the same time as the control [63,64,65]. Additionally, no effects on seed germination have been reported by applying HX, which opens up the possibility of generating more evidence of its stimulating or antagonistic effects in similar study models.
Finally, in silico analysis of CH, CT, and HX molecules demonstrated that the presence of these compounds is safe and has a high probability of being predicted to be “inactive” if it interacts with some targets related to organic toxicity. These results are consistent with those reported for the predictive toxicological analysis of organic molecules and are similar to those reported in previous studies [66,67,68]. However, our approach is distinguished by the integration of advanced prediction platforms, which, in combination with complementary validation models, strengthens the evaluation of the antifungal potential of these organic molecules. This comprehensive methodology allows for a more precise and reliable characterization of the safety and efficacy profile of treatments.

5. Conclusions

CH, CT, and HX treatments demonstrated favorable effects in inhibiting the development of C. asianum in in vitro tests, observed starting at concentrations of 1% in CH, 0.03% in CT, and 0.06% in HX. In cytotoxicological tests on seeds, low to moderate toxicity was evident in the evaluated treatments, whereas in silico analysis indicated the absence of negative effects as long as the doses within the LD50 parameters were respected. These results highlight the potential of these compounds as safe and effective alternatives for the control of phytopathogens, providing new opportunities to evaluate their impact in other study models. In particular, future omics analyses could provide more detailed information on the molecular mechanisms involved in the interaction of these treatments with fungal cells and their impact on other biological systems.

Author Contributions

Conceptualization, methodology, formal analysis, writing—original draft preparation, E.R.-D.; Visualization, cytotoxicological assay, histological assay E.R.-D. and P.G.-M.; ADMET analysis, E.R.-D., J.A.S.-B. and P.G.-M.; Data curation, E.R.-D., V.M.Z.-G. and. L.G.H.-M.; Writing—review and editing, L.G.H.-M., V.M.Z.-G., J.A.S.-B., S.R.-B., R.M.V.-E., J.A.H.-G. and P.G.-M. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Acknowledgments

The Secretariat of Science, Humanities, Technology, and Innovation (SECIHTI) supported this work through a scholarship (826679) awarded to Edson Rayón Díaz.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Radial growth of the fungus Colletotrichum asianum for 10 d in vitro with CH, CT, and HX application. Different letters in the graph indicate significant differences between treatments by day (mean ± SE) (n = 8). DW: distilled water; DMSO: dimethyl sulfoxide 0.5%; AAc: acetic acid; CH: chitosan; CT: citral; HX: hexanal.
Figure 1. Radial growth of the fungus Colletotrichum asianum for 10 d in vitro with CH, CT, and HX application. Different letters in the graph indicate significant differences between treatments by day (mean ± SE) (n = 8). DW: distilled water; DMSO: dimethyl sulfoxide 0.5%; AAc: acetic acid; CH: chitosan; CT: citral; HX: hexanal.
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Figure 2. Effects of CH, CT, and HX on the germination of Colletotrichum asianum. Different letters in the graph indicate significant differences between treatments (mean ± SE) according to Fisher’s LSD test (p < 0.05) (n = 3). DW, distilled water; DMSO, is dimethyl sulfoxide at 0.5%; AAc, acetic acid at 1.5%; CH, chitosan; CT, citral; HX, hexanal.
Figure 2. Effects of CH, CT, and HX on the germination of Colletotrichum asianum. Different letters in the graph indicate significant differences between treatments (mean ± SE) according to Fisher’s LSD test (p < 0.05) (n = 3). DW, distilled water; DMSO, is dimethyl sulfoxide at 0.5%; AAc, acetic acid at 1.5%; CH, chitosan; CT, citral; HX, hexanal.
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Figure 3. Spores exposed to CH, CT, and HX treatments and their effects on germ tube development were seen at 40× magnification after 7 h of germination. DW, distilled water; DMSO, dimethyl sulfoxide; AAc, acetic acid; CH, chitosan; CT, citral; HX, hexanal.
Figure 3. Spores exposed to CH, CT, and HX treatments and their effects on germ tube development were seen at 40× magnification after 7 h of germination. DW, distilled water; DMSO, dimethyl sulfoxide; AAc, acetic acid; CH, chitosan; CT, citral; HX, hexanal.
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Figure 4. Effect of CH, CT, and HX treatments at different concentrations on radicle development in tomato and cucumber seeds at 25 °C: (A) RRE, relative radicle elongation; and (B) RSG, relative seed germination. Values are expressed as the mean ± standard error (n = 20). Different letters in the graph indicate significant differences between treatments (mean ± SE) according to Fisher’s LSD test (p < 0.05). DW, distilled water; DMSO, dimethyl sulfoxide; AAc, acetic acid; CH, chitosan; CT, citral; HX, hexanal.
Figure 4. Effect of CH, CT, and HX treatments at different concentrations on radicle development in tomato and cucumber seeds at 25 °C: (A) RRE, relative radicle elongation; and (B) RSG, relative seed germination. Values are expressed as the mean ± standard error (n = 20). Different letters in the graph indicate significant differences between treatments (mean ± SE) according to Fisher’s LSD test (p < 0.05). DW, distilled water; DMSO, dimethyl sulfoxide; AAc, acetic acid; CH, chitosan; CT, citral; HX, hexanal.
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Figure 5. The organ-specific toxicity analysis of (A) CH, (B) CT, and (C) HX; predicted fragment contribution of acute oral and dermal toxicity, respectively: (D,E) CH; (F,G) CT; and (H,I) HX, obtained from ProTox-II and STopTox 1.0 analysis. The green color is non-toxic and the red color is toxic according to LD50. CH, chitosan; CT, citral; HX, hexanal.
Figure 5. The organ-specific toxicity analysis of (A) CH, (B) CT, and (C) HX; predicted fragment contribution of acute oral and dermal toxicity, respectively: (D,E) CH; (F,G) CT; and (H,I) HX, obtained from ProTox-II and STopTox 1.0 analysis. The green color is non-toxic and the red color is toxic according to LD50. CH, chitosan; CT, citral; HX, hexanal.
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Table 1. Effects of different CH, CT, and HX concentrations on in vitro development of Colletotrichum asianum.
Table 1. Effects of different CH, CT, and HX concentrations on in vitro development of Colletotrichum asianum.
TreatmentsConcentration
(%)		Sporulation
(106 Spores/mL)		Biomass Dry Weight (g)
DW-8.28 ± 2.84 c0.612 ± 0.006 c
DMSO0.54.52 ± 2.79 abc0.630 ± 0.011 c
AAc1.50.00 ± 0.00 a0.000 ± 0.000 a
CH0.56.40 ± 2.17 b0.604 ± 0.021 c
1.00.21 ± 0.21 a0.000 ± 0.000 a
1.50.00 ± 0.00 a0.000 ± 0.000 a
CT0.030.00 ± 0.00 a0.000 ± 0.000 a
0.050.00 ± 0.00 a0.000 ± 0.000 a
0.10.00 ± 0.00 a0.000 ± 0.000 a
HX0.048.41 ± 5.81 c0.252 ± 0.123 b
0.060.00 ± 0.00 a0.325 ± 0.123 b
0.080.00 ± 0.00 a0.318 ± 0.121 b
DW: distilled water; DMSO: dimethyl sulfoxide 0.5%; AAc: acetic acid; CH: chitosan; CT: citral; HX: hexanal. Values are expressed as the mean ± standard deviation (n = 8). Different letters in the table indicate significant differences between treatments (mean ± SD). Fisher’s LSD test (p < 0.05).
Table 2. The effects on seed germination and radicle elongation in S. lycopersicum var. Cerasiforme and C. sativus L. var. Poinsett seeds were exposed to CH, CT, or HX for 8 days at 25 °C.
Table 2. The effects on seed germination and radicle elongation in S. lycopersicum var. Cerasiforme and C. sativus L. var. Poinsett seeds were exposed to CH, CT, or HX for 8 days at 25 °C.
TreatmentGI %TsREI %TsNREI TsNRGI Ts
DW100 ± 0.00 a0.00 ± 0.00 d0.00 ± 0.00 a0.00 ± 0.00 a
DMSO 0.5%39.35 ± 14.30 b53.88 ± 11.26 c−0.53 ± 0.11 b−0.19 ± 0.10 abc
AAc 1.5%0.00 ± 0.00 d100.00 ± 0.00 a−1.00 ± 0.00 d−1.00 ± 0.00 d
CH 0.5%3.87 ± 1.65 cd95.77 ± 1.44 ab−0.95 ± 0.01 cd−0.13 ± 0.13 ab
CH 1%6.00 ± 5.41 cd98.26 ± 0.43 ab−0.97 ± 0.00 cd−0.47 ± 0.07 abcd
CH 1.5%0.42 ± 0.22 d99.02 ± 0.40 a−0.98 ± 0.00 cd−0.66 ± 0.11 bcd
CT 0.03%2.77 ± 2.59 cd96.91 ± 2.43 ab−0.93 ± 0.02 cd−0.48 ± 0.24 abcd
CT 0.05%1.11 ± 0.64 d98.05 ± 0.48 ab−0.97 ± 0.00 cd−0.48 ± 0.19 abcd
CT 0.1%3.56 ± 3.21 cd94.51 ± 0.40 ab−0.94 ± 0.04 cd−0.61 ± 0.20 abcd
HX 0.04%24.63 ± 5.46 bcd70.32 ± 5.32 c0.69 ± 0.05 b0.17 ± 0.06 abc
HX 0.06%15.39 ± 1.89 bcd75.88 ± 5.56 bc−0.75 ± 0.05 bc−0.30 ± 0.11 abc
HX 0.08%27.78 ± 6.25 cd69.72 ± 7.41 c−0.69 ± 0.07 b−0.07 ± 0.03 ab
TreatmentGI %CsREI %CsNREI CsNRGI Cs
DW100 ± 0.00 ab0.00 ± 0.00 b0.00 ± 0.00 ab0.00 ± 0.00 c
DMSO 0.5%278.73 ± 108.15 b−112.26 ± 85.62 b1.12 ± 0.85 b0.30 ± 0.18 c
AAc 1.5%0.00 ± 0.00 a100.00 ± 0.00 a−1.00 ± 0.00 a−1.00 ± 0.00 a
CH 0.5%5.26 ± 3.20 a94.26 ± 2.19 a−0.94 ± 0.02 a−0.21 ± 0.19 bc
CH 1%0.02 ± 0.02 a99.90 ± 0.10 a−1.00 ± 0.00 a−0.91 ± 0.08 ab
CH 1.5%0.04 ± 0.04 a99.73 ± 0.76 a−0.99 ± 0.00 a−0.94 ± 0.05 ab
CT 0.03%115.55 ± 97.40 ab15.43 ± 58.49 ab−0.13 ± 0.60 ab−0.02 ± 0.27 c
CT 0.05%86.91 ± 28.98 ab19.63 ± 23.41 ab−0.19 ± 0.23 ab0.05 ± 0.05 c
CT 0.1%82.68 ± 70.15 ab20.90 ± 57.06 ab−0.20 ± 0.57 ab−0.25 ± 0.21 abc
HX 0.04%71.18 ± 21.58 ab23.83 ± 26.05 ab−0.24 ± 0.26 ab−0.04 ± 0.14 c
HX 0.06%124.89 ± 88.82 ab−4.03 ± 63.45 ab0.04 ± 0.63 ab−0.05 ± 0.20 c
HX 0.08%108.01 ± 45.08 ab−19.33 ± 39.55 ab0.02 ± 0.47 ab0.08 ± 0.08 c
GI, germination index%; REI, root elongation inhibition%; NREI, normalized residual elongation index; NRGI, normalized residual germination index. Normalized ranges from low toxicity (0 to −0.25), moderate toxicity (−0.25 to −0.50), high toxicity (−0.50 to −0.75), and very high toxicity (−0.75 to −1). Ts Tomato seeds; Cs Cucumber seeds. DW, distilled water; DMSO, dimethyl sulfoxide; AAc, acetic acid; CH, chitosan; CT, citral; HX, hexanal. Different letters in the table indicate significant differences between the treatments. Data are presented as the mean ± standard error (n = 20).
Table 3. Cross-section of root tissue and radicle development in cucumber seeds exposed to different concentrations of CH, bioactive aldehydes, and control treatments.
Table 3. Cross-section of root tissue and radicle development in cucumber seeds exposed to different concentrations of CH, bioactive aldehydes, and control treatments.
Cross-section of root tissueHorticulturae 11 00474 i001Horticulturae 11 00474 i002Horticulturae 11 00474 i003
Radicle
development		Horticulturae 11 00474 i004Horticulturae 11 00474 i005Horticulturae 11 00474 i006
TreatmentDWDMSO 0.5%AAc 1.5%
Cross-section of root tissueHorticulturae 11 00474 i007Horticulturae 11 00474 i008Horticulturae 11 00474 i009
Radicle
development		Horticulturae 11 00474 i010Horticulturae 11 00474 i011Horticulturae 11 00474 i012
TreatmentCH 0.5%CH 1.0%CH 1.5%
Cross-section of root tissueHorticulturae 11 00474 i013Horticulturae 11 00474 i014Horticulturae 11 00474 i015
Radicle
development		Horticulturae 11 00474 i016Horticulturae 11 00474 i017Horticulturae 11 00474 i018
TreatmentCT 0.03%CT 0.05%CT 0.1%
Cross-section of root tissueHorticulturae 11 00474 i019Horticulturae 11 00474 i020Horticulturae 11 00474 i021
Radicle
development		Horticulturae 11 00474 i022Horticulturae 11 00474 i023Horticulturae 11 00474 i024
TreatmentHX 0.04%HX 0.06%HX 0.08%
DW, distilled water; DMSO, dimethyl sulfoxide at 0.5%; AAc, acetic acid; CH, chitosan; CT, citral; HX, hexanal (n = 3).
Table 4. Analysis of chemical properties of CH, CT, and HX molecules.
Table 4. Analysis of chemical properties of CH, CT, and HX molecules.
TreatmentMolecular Weight (g/mol)Topological Polar Surface Area (TPSA)Hydrogen Bond Donors CountHydrogen Bond Acceptors CountPredicted Toxicity ClassLD50 (mg/kg)
CH1526.45808 Å22948250
CT152.2317.07 Å2014500
HX100.1617.07 Å20153240
CH, chitosan; CT, citral; HX, hexanal.
Table 5. Predictions of organ-specific toxicity of CH, CT, and HX molecules obtained from SwissADME, pkCSM, ProTox-II.
Table 5. Predictions of organ-specific toxicity of CH, CT, and HX molecules obtained from SwissADME, pkCSM, ProTox-II.
ClassificationTargetCHCTHX
PredictionProbabilityPredictionProbabilityPredictionProbability
Organ toxicityHepatotoxicityInactive0.74Inactive0.69Inactive0.73
Toxicity endpointsCarcinogenicityInactive0.69Inactive0.88Inactive0.59
ImmunotoxicityInactive0.83Inactive0.99Inactive0.97
MutagenicityInactive0.62Inactive0.98Inactive0.97
Tox21-Nuclear receptor signaling pathwaysCytotoxicityInactive0.65Inactive0.82Inactive0.75
Aryl hydrocarbon receptor (AhR)Inactive0.97Inactive1.0Inactive1.0
Androgen receptor (AR)Inactive0.92Inactive1.0Inactive1.0
Androgen receptor ligand binding domain (AR-LBD)Inactive0.96Inactive0.95Inactive1.0
AromataseInactive0.98Inactive1.0Inactive1.0
Estrogen receptor alpha (ER)Inactive0.69Inactive0.94Inactive0.78
Estrogen receptor ligand binding domain (ER-LBD)Inactive0.94Inactive0.96Inactive1.0
Peroxisome proliferator-activated receptor gamma (PPAR-Gamma)Inactive0.99Inactive1.0Inactive0.99
Tox21-Stress response pathwaysNuclear factor (erythroid-derived 2)-like 2/antioxidant responsive element (nrf2/ARE)Inactive0.98Inactive0.99Inactive1.0
Heat shock factor response element (HSE)Inactive0.98Inactive0.99Inactive1.0
Mitochondrial membrane potential (MMP)Inactive0.95Inactive0.98Inactive0.95
Phosphoprotein (tumor suppressor) p53Inactive0.95Inactive1.0Inactive1.0
ATPase family AAA domain-containing protein 5 (ATAD5)Inactive0.98Inactive1.0Inactive1.0
CH, chitosan; CT, citral; HX, hexanal.
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Rayón-Díaz, E.; Hernández-Montiel, L.G.; Zamora-Gasga, V.M.; Sánchez-Burgos, J.A.; Ramos-Bell, S.; Velázquez-Estrada, R.M.; Herrera-González, J.A.; Gutiérrez-Martínez, P. Antifungal and Toxicological Evaluation of Natural Compounds Such as Chitosan, Citral, and Hexanal Against Colletotrichum asianum. Horticulturae 2025, 11, 474. https://doi.org/10.3390/horticulturae11050474

AMA Style

Rayón-Díaz E, Hernández-Montiel LG, Zamora-Gasga VM, Sánchez-Burgos JA, Ramos-Bell S, Velázquez-Estrada RM, Herrera-González JA, Gutiérrez-Martínez P. Antifungal and Toxicological Evaluation of Natural Compounds Such as Chitosan, Citral, and Hexanal Against Colletotrichum asianum. Horticulturae. 2025; 11(5):474. https://doi.org/10.3390/horticulturae11050474

Chicago/Turabian Style

Rayón-Díaz, Edson, Luis G. Hernández-Montiel, Víctor Manuel Zamora-Gasga, Jorge A. Sánchez-Burgos, Surelys Ramos-Bell, Rita María Velázquez-Estrada, Juan Antonio Herrera-González, and Porfirio Gutiérrez-Martínez. 2025. "Antifungal and Toxicological Evaluation of Natural Compounds Such as Chitosan, Citral, and Hexanal Against Colletotrichum asianum" Horticulturae 11, no. 5: 474. https://doi.org/10.3390/horticulturae11050474

APA Style

Rayón-Díaz, E., Hernández-Montiel, L. G., Zamora-Gasga, V. M., Sánchez-Burgos, J. A., Ramos-Bell, S., Velázquez-Estrada, R. M., Herrera-González, J. A., & Gutiérrez-Martínez, P. (2025). Antifungal and Toxicological Evaluation of Natural Compounds Such as Chitosan, Citral, and Hexanal Against Colletotrichum asianum. Horticulturae, 11(5), 474. https://doi.org/10.3390/horticulturae11050474

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