Location and research period

This study was conducted between July 2014 and September 2015 at the Plant Pathology Laboratory of the Universidad Autónoma de Chapingo, located in Texcoco, Chapingo, State of Mexico, and at the Organic Materials Laboratory of the Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, in Querétaro, State of Querétaro, Mexico.

Experimental design

A completely randomized design with a 3 × 3 × 5 factorial arrangement and three replications was used. The factors included the fungal species A. flavus, A. fumigatus, and F. verticillioides, which were provided by the Plant Pathology Laboratory of the Plant Protection Department of the Universidad Autónoma de Chapingo; the second factor was grain moisture content: 12, 15, and 18%; and the third, fungal treatments: L. tridentata extract (10 mL L⁻¹), azadirachtin (20 mL L⁻¹), aluminum phosphide (3 mg L⁻¹), acid mixture (ascorbic 1%, citric 0.25%, and lactic 0.25%), and a control without inhibitor application.

The experimental units consisted of 600 mL plastic bottles with 350 g of Criolla variety corn grains, previously disinfected using a broad-spectrum organic disinfectant (Virkon®), at a dose of 0.5 g L⁻¹.

Fungal inoculation and inhibitor incorporation

The inoculation of A. flavus, A. fumigatus, and F. verticillioides was performed by adding three milliliters of a 500,000 conidia mL⁻¹ suspension to each experimental unit. Twenty-four hours after this inoculation, three milliliters of each treatment were applied per experimental unit. Subsequently, the samples were maintained for twenty hours at 4°C to favor moisture homogenization.

The final moisture adjustment to 12, 15, and 18% was carried out using the formula described by ^{Pixton and Griffith (1971}), according to which the amount of water to add was determined based on grain mass and initial moisture content (percentage on wet basis). The amount of water necessary for the final moisture to be as desired was calculated, carefully adding the resulting volume and distributing it homogeneously through manual agitation, performing gentle circular movements for approximately two minutes. Finally, the experimental units were incubated at 27°C (± 2°C), sealed with polyethylene film to prevent moisture loss.

Variables evaluated

Fungal incidence in humid chamber was determined at 10, 20, and thirty days after inoculation (DAI). For this, twenty-five grains from each experimental unit were selected and placed in humid chamber with four replications per treatment. The humid chamber consisted of hermetic plastic containers of 15 × 10 × 5 cm, with a base of filter paper moistened with sterile distilled water and a plastic grid to avoid direct contact of grains with water. The chambers were maintained at 25 ± 2°C and relative humidity close to 95%, conditions that favor fungal development. After seven days of incubation, the percentage of fungal growth was quantified.

Additionally, fungal incidence in stored grains was determined at 30, 60, and ninety DAI, taking one hundred grains from each experimental unit. At ninety days of storage, aflatoxin concentration (B1, B2, G1, and G2) from A. flavus and A. fumigatus was evaluated. Twenty grams of grains were taken, which were ground and weighed, and then mixed with a methanol:water solvent (1:1, v/v) to extract aflatoxins. The suspension was mechanically agitated in an orbital shaker (IKA® KS 130 basic, Germany) at 150 rpm for five minutes and subsequently filtered to remove solid particles. The filtrate was passed through an immunoaffinity column with specific monoclonal antibodies for aflatoxins (Aflatest®, VICAM, United States). The column was washed with distilled water to remove impurities and aflatoxins were eluted with 1.0 mL of HPLC-grade methanol. The eluate was analyzed by high-performance liquid chromatography (HPLC) in an Agilent 1260 Infinity II equipment (Agilent Technologies, United States) equipped with fluorescence detector, using a C18 column (4.6 × 150 mm, 5 μm) and mobile phase water:methanol (60:40, v/v) at a flow of 1.0 mL min⁻¹.

Data analysis

An analysis of variance (ANOVA) was performed and, when significant differences between treatments were detected, Tukey's multiple mean comparison test (α = 0.05) was applied. In cases of significant interaction between factors, the interaction was decomposed to facilitate interpretation of results. Statistical analysis was performed using SAS® software version 9.3.

Fungal incidence in humid chamber

The ANOVA indicated that the interaction between the three evaluated factors was not significant for fungal incidence in grains subjected to humid chamber. However, the double interactions inhibitor × fungus (Table 1) and humidity × fungus (Table 2) were significant, so the interactions were decomposed for better interpretation of results.

The humid chamber evaluation showed that, during the first ten days after inoculation (DAI), the applied inhibitors managed to partially reduce fungal incidence, particularly in A. flavus, while A. fumigatus and F. verticillioides showed high colonization capacity even under antifungal treatments. This agrees with reports by ^{Magan and Aldred (2007}) and ^{Bryła et al. (2022}), who indicate that the germination and growth rate of Fusarium and Aspergillus spp. depends on both water availability and the intrinsic tolerance of each species to inhibitory compounds.

In this study, the organic acid mixture and aluminum phosphide showed the lowest incidence values in A. flavus at ten DAI, but without significant differences compared to the control at twenty and thirty DAI. This loss of efficacy is associated with the low residual activity of the products, which according to ^{Hassan et al. (2012}) and ^{Rivera-Escareño et al. (2025}), can be less than ten days under favorable conditions for fungal development. The rapid increase in incidence in subsequent evaluations reflects that the humid chamber favors an environment close to humidity saturation (near 95% RH), an optimal condition for sporulation of these fungi (^{Milani, 2013}; ^{Suleiman, Rosentrater & Bern, 2013}).

Likewise, A. flavus consistently presented lower incidence values than the other species, which coincides with previous observations by ^{Mureithi (2010}), who documented that this species presents slower growth in whole grain than A. fumigatus or F. verticillioides under high humidity. However, its capacity to produce aflatoxins even at low colonization rates highlights the importance of its early control (^{Gong et al., 2016}).

In the moisture × fungus interaction (Table 2), it was observed that the effect of grain moisture content on F. verticillioides incidence was significant, where the incidence value was reduced by 7.2% when moisture decreased from 18% to 15%, and by 21.7% when moisture decreased from 15% to 12% at ten DAI. The incidence of A. flavus and A. fumigatus was not significantly reduced with the reduction of grain moisture.

At twenty DAI, the ANOVA detected that only the moisture × fungus interaction was significant, so no significant differences were observed between products; significant differences were still observed between the inhibitor × fungus interaction, but this was due to a greater influence of the fungus factor. This can be clearly observed in the groups formed in the multiple mean comparison (Table 1), where, for all inhibitors, A. flavus presented the lowest incidence values compared to the other species.

At thirty DAI, similar results were evident, but with less clear trends, because all recorded incidence values were equal to or close to 100%. It is important to emphasize that the residual activity of these products decreases over time. When the moisture × fungus interaction was analyzed, the trend was similar to what occurred with the inhibitor × fungus interaction. The statistical differences observed were mainly due to the inoculated fungi, with very low incidence values in A. flavus, while between A. fumigatus and F. verticillioides no significant differences were present.

In the first ten DAI there was a greater influence of the applied inhibitors, which was not evident in subsequent evaluations. This is because the products limit fungal growth but do not eliminate the inoculum, so when placed in humid chamber, mycelial development continues and can be detected rapidly. The differences detected between fungi are due to the fact that, during storage, A. flavus presented much slower growth than A. fumigatus and F. verticillioides, both in culture medium and during development in inoculated grains.

These results confirm that, although the evaluated inhibitors can delay the onset of growth, effective control under high humidity conditions requires complementary strategies, such as rapid grain drying, use of hermetic containers, and application of inhibitors with greater persistence, in line with recommendations by ^{Magan and Olsen (2004}) and ^{EFSA Panel on Plant Health et al. (2022)}.

Fungal incidence in storage

The ANOVA detected significant statistical differences between factors, in the triple interaction inhibitor × fungus × moisture, and in the double interaction inhibitor × fungus. Considering the inhibitor × fungus interaction, the greatest influence was due to the fungus factor. The slowest growth was recorded in A. flavus in storage, consistent with what was observed under humid chamber conditions (Table 3). The applied inhibitors had no effect on the development of inoculated fungi; in fact, their effect showed no differences from the control without inhibitor application. Among the inoculated fungi, the lowest incidence was observed in A. flavus, followed by A. fumigatus and F. verticillioides, since these last two presented statistically similar incidence averages (Figure 1a).

Additionally, no significant differences were observed in mycelial growth at thirty or sixty DAI. However, at ninety DAI significant differences were detected in growth and sporulation under all treatments (Table 3). Recent studies support these latency times under similar conditions. For example, ^{Shi et al. (2023}) reported changes in fungal community and mycotoxin production in stored corn in temperature ranges of 20-30°C, confirming the late development of A. flavus during prolonged storage. Likewise, ^{Muga, Marenya and Workneh (2019}) demonstrated that aflatoxin contamination increases considerably when grain moisture exceeds 18%, especially if storage is prolonged, consistent with observations of higher incidence and sporulation at ninety DAI.

Although A. flavus presented slower growth, this fungus can develop from 13% moisture, like other Aspergillus species. However, the optimal moisture content for its development is 18% (^{Mureithi, 2010}), while Fusarium species require higher moisture levels. According to ^{Birzele and Prange (2003}), the development of Fusarium species in grains with 15% moisture content depends on storage time, but develops rapidly from 18% moisture content.

Statistical differences were highly significant between the different grain moisture percentages studied in storage (Figure 1b). Incidence averages decreased to lower values with lower grain moisture content, confirming that this is one of the most important factors for fungal development in storage (^{Milani, 2013}; ^{Suleiman et al., 2013}).

As recorded in the inhibitor × fungus interaction, the effect of inhibitors does not present a defined trend, but is strongly influenced by other factors, such as fungal species, moisture, or storage time. In evaluations performed in humid chamber, from twenty DAI onward, inhibitors already presented unstable behavior.

Figure 1 Average incidence (%) of mycotoxigenic fungi in stored corn grains, according to a) moisture contents (%), and b) fungal species. Texcoco de Mora, State of Mexico, Mexico, 2015. 

To achieve effective inhibition of fungi in stored grains, recent studies have demonstrated that acetic acid is effective at lower concentrations than previously reported. For example, ^{Haggag et al. (2024}) showed that both acetic acid and thyme have strong bioactive effect in vapor phase and can notably inhibit the accumulation of aflatoxins B1 and B2 produced by Aspergillus flavus, with EC₅₀ values for acetic acid of only 0.0013 mg mL⁻¹ in volatile contact (approximately 1.3 mg kg⁻¹). Additionally, ^{Sorathiya, Melo, Hogg and Pintado (2025}) reviewed sustainable applications of organic acids in food preservation and highlighted that acetic acid can contribute to prolonging shelf life and preventing fungal deterioration in grains without leaving toxic residues.

Figure 2 Average incidence (%) of mycotoxigenic fungi in stored corn grains, according to treatment with fungal inhibitors. Texcoco de Mora, State of Mexico, Mexico, 2015. 

Regarding the evaluated treatments, Larrea tridentata extract showed relatively low incidences in A. flavus and F. verticillioides at 12% moisture (Figure 2), which coincides with reports by ^{Rivera-Escareño et al. (2025}), who document that its phenolic compounds possess antifungal action through cell membrane alteration and inhibition of key enzymes in fungal metabolism. However, its efficacy decreased with increased moisture and storage time, suggesting the need for repeated applications or controlled-release formulations.

Azadirachtin, a limonoid extracted from neem (Azadirachta indica), presented variable behavior. Although in some cases it reduced A. fumigatus incidence at 15% moisture, its effect was limited against higher moistures. This agrees with findings by ^{Kilani-Morakchi et al. (2021}), who highlight that azadirachtin is photo- and thermosensitive, with rapid degradation under environmental conditions, which shortens its residual activity in storage.

The acid mixture (ascorbic, citric, and lactic) showed moderate initial control, attributable to the reduction of grain surface pH and disruption of fungal homeostasis (^{Hassan et al., 2012}). However, its effectiveness declined as moisture and exposure time increased, which agrees with reports by ^{Alcalde (2025}) about the need to maintain low water activity conditions to enhance organic acid action.

Regarding aluminum phosphide, while it is widely used in postharvest management for its efficacy against pests, its role in fungal control is limited and its use is declining due to its high toxicity, acute poisoning risks, and regulatory restrictions in several countries (^{Yadav et al., 2021}; ^{Saldivia-Tejeda, 2018}). Additionally, certain fungi such as Aspergillus spp. and Penicillium spp. can tolerate phosphine or even solubilize phosphide, reducing its fungicidal effectiveness (^{Mendes et al., 2013}; ^{Rinu, Malviya, Sati, Tiwari & Pandey, 2013}). The transition toward safer alternatives, such as essential oils, plant extracts, and organic acids, responds to both food safety demands and the need to reduce environmental impact.

Collectively, these results suggest that none of the evaluated options provides prolonged control alone under elevated moisture conditions and extended storage (Figure 2). This reinforces the recommendation to integrate inhibitors with other practices, such as rapid drying and use of hermetic systems, to maximize protection against mycotoxigenic fungi and minimize risks to human health and the environment.

Aflatoxin concentration

Table 4 presents the results corresponding to aflatoxin accumulation in corn grains inoculated with A. flavus and A. fumigatus under different grain moisture contents, treated with fungal inhibitors and stored for ninety DAI.

In the evaluation performed at ninety DAI, there were no significant differences in fungal incidence according to applied inhibitors and in the inhibitor × fungus and moisture × fungus interactions. Only significant differences were observed between grain moisture percentages during storage. However, ^{Ghorbanian, Razzaghi-Abyaneh, Allameh, Shams-Ghahfarokhi and Qorbani (2008}) observed that, although mycelial growth reduction from azadirachtin 85% treatment is limited, it does reduce aflatoxin production by Aspergillus parasiticus.

It can be observed that after ninety DAI, aflatoxin production occurred only in grains with 18% moisture, with only traces accumulated at 15% moisture, and no mycotoxins were found when storage moisture was 12%. The detected mycelial growth was low at ninety DAI, evidencing that until sixty DAI, fungal development was scarce, and therefore, mycotoxin production was also low.

The observation of significant aflatoxin production only in grains with 18% moisture after ninety DAI is consistent with recent studies that indicate the critical influence of moisture and temperature on the synthesis of these toxins. ^{Muga et al. (2019}) reported that, although grain moisture content alone was not significant, the combination of high temperature (30°C) and high relative humidity (90%) produced elevated aflatoxin levels in corn, reaching up to 11,179 mg kg⁻¹ (≈ 11 mg kg⁻¹) at 18% moisture. This supports the findings obtained in this work in prolonged storage contexts. Likewise, ^{Villers (2014}) highlights that, during postharvest storage in warm and humid environments, aflatoxin production can increase exponentially, being practically nonexistent when moisture is maintained below 12%. The low levels or absence of mycotoxins at 12% conditions precisely reflect this critical barrier.

The probable causes of this dynamic include higher water activity that facilitates A. flavus germination and promotes its secondary metabolism (toxin synthesis), in addition to possible late release of spores or fungal metabolites under persistent moisture conditions. In contrast, lower moistures significantly delayed mycelial development and toxin production. Additionally, storage time is one of the factors that also strongly influences interaction with other factors, such as moisture and temperature (^{Suleiman et al., 2013}; ^{Cassini et al., 2005}), which can be reflected in the results obtained in the present study.