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Communication

The Inhibitory Activity of Salicylaldehyde Compounds on Aspergillus Species and Their Effects on Aflatoxin Production and Crop Seed Germination

by
Jong H. Kim
1,*,
Kathleen L. Chan
1,
William M. Hart-Cooper
2,
DeAngela Ford
1,
Kaydren Orcutt
2,
Siov Bouy L. Sarreal
1,
Jeffrey D. Palumbo
1 and
William J. Orts
2
1
Foodborne Toxin Detection and Prevention Research Unit, Western Regional Research Center, Agricultural Research Service, United States Department of Agriculture, 800 Buchanan St., Albany, CA 94710, USA
2
Bioproducts Research Unit, Western Regional Research Center, Agricultural Research Service, United States Department of Agriculture, 800 Buchanan St., Albany, CA 94710, USA
*
Author to whom correspondence should be addressed.
Appl. Sci. 2024, 14(21), 10000; https://doi.org/10.3390/app142110000
Submission received: 28 September 2024 / Revised: 28 October 2024 / Accepted: 30 October 2024 / Published: 1 November 2024
(This article belongs to the Section Food Science and Technology)
Browse Figures
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Abstract

:

Featured Application

Salicylaldehyde and 4-isopropyl-3-methylphenol can be used as potential antifungal and anti-mycotoxigenic agents for crops/foods to be processed.

Abstract

Crops such as tree nuts, corn and peanut are highly susceptible to infestation by the aspergilli Aspergillus flavus or Aspergillus parasiticus and subsequent aflatoxin (AF) contamination, a serious threat to public food safety. Conventional control of the aspergilli has been through the application of fungicides; however, certain fungicides at sub-optimal doses have been correlated with increased production of mycotoxins including AF. Natural products (NP) have been a potential source of antifungal agents. In this study, we performed risk assessment testing, for which thirteen NP/derivatives (generally recognized as safe) were examined at sub-inhibitory concentrations to determine the enhancement of AF production in aspergilli. We found that benzaldehyde derivatives or thymol (THY) enhanced AF production in aspergilli, while 4-isopropyl-3-methylphenol (4I3M), a synthetic analog of the NP THY and carvacrol, or salicylaldehyde (SLD) exerted a potent antifungal or mycotoxin-inhibitory effect. In seed testing (corn, pistachio kernels), SLD effectively prevented fungal growth as a fumigant, while 4I3M completely inhibited AF production at ≥1.0 mM. Therefore, we concluded that NP/derivatives that do not have any significant environmental impact can be a potent source of antifungal or anti-mycotoxigenic agents, either in their nascent form or as leads for more effective derivatives; however, NP should be applied at optimum concentrations to prevent the abnormal enhancement of mycotoxin production by fungi.

1. Introduction

Fungal infections/contaminations on food crops are serious problems since effective agents for treating fungicide-resistant fungi are often very limited [1,2]. The long-term application of fungicides, for example, strobilurins, phenylpyrrols, azoles, etc., to crop fields causes a selection pressure for the development of fungal resistance to conventional fungicides [3,4,5,6,7,8]. The administration of fungicides at “suboptimal” concentrations or time-points of fungal growth can also increase toxin production by fungi [9,10], thus negatively affecting the safe production of foods/crops as well as public food safety. For instance, the application of strobilurin fungicides at early growth stages has been identified as a risk factor for mycotoxin contamination in wheat [9]. Also, a chiral triazole fungicide prothioconazole could induce the synthesis of deoxynivalenol (DON) by the wheat pathogen Fusarium graminearum [10].
Aflatoxins (AFs) are a group of polyketide metabolites (difuranocoumarins) produced by the strains of Aspergillus section Flavi, for which Aspergillus flavus and Aspergillus parasiticus are the main species associated with AF contamination or outbreak [11]. AFs trigger acute toxicity in human/poultry, which is mainly due to the hepatotoxic, immune-modulatory, mutagenic/carcinogenic and teratogenic effects of AFs [12]. After consumption by human/poultry, AFs are metabolically activated by the hepatic cytochrome P450s (CYPs), leading to the formation of toxic, reactive AF-8,9-exo-epoxide; the resulting 8,9-dihydro-8-(N7-guanyl)-9-hydroxyAF adduct triggers mutations (GC to TA frameshift transversions) or other DNA damages such as chromosomal breaks, aberrations, etc. [13]. According to the International Agency for Research on Cancer (IARC), AFs (AFB1, AFB2, AFG1, AFG2) are characterized as Group 1 carcinogenic to humans [14]. Due to the risk of AF contamination in food/feed crops, United States Department of Agriculture (USDA) administers programs for AF testing of crops destined for export to foreign countries including those in the European Union (EU) [15].
AFs are commonly found in major food/feed crops including corn, peanuts, tree nuts (almonds, pistachios), dried fruits as well as milk [16,17]. Environmental factors, such as temperature, humidity/rain precipitation, drought, etc., affect the growth of AF-producing fungi on crops during cultivation, harvest and/or storage [17]. For example, temperature stress is the cause of fungal establishment on field crops, while high water activity and temperature trigger the growth of fungi in grains during storage [16].
Corn is highly susceptible to infection by AF-producing Aspergillus strains and subsequent AF contamination [18,19]. Aspergillus spores released from fungal mycelium/sclerotia present in soil are dispersed by wind, and then infect the developing inflorescences of corn [18,19]. Similarly, pistachios are also susceptible to AF contamination [20]. Of note, while the hull of pistachios remains intact during the natural split of shells prior to harvest, it is reported that around 1–4% of orchard pistachios exhibit an early split during growth (namely, the hulls split with the shell), which exposes the pistachio kernel to fungi as well as insect pests [21]. These early-split pistachios exhibit a higher level of AF contamination compared to the non-split nuts [21], thus requiring proper control measures for the prevention of AF contamination.
Innovative technologies have been developed to control AF contamination in foods/feeds, which include (but are not limited to) (1) gamma radiation (damaging microbial DNA) [22], (2) UV irradiation (destroying fungal cell wall and nucleic acids) [23], (3) ozone fumigation (detoxifying AFs) [24], (4) chemical agents (exerting AF reduction and degradation) [25], and (5) sorting techniques (differentiating AF-contaminated corn kernels) [26]. Of note, biological control has been developed as a sustainable AF control strategy for corn and pistachios, for which atoxigenic (namely, non-AF producing) A. flavus has been applied for long-term crop protection against Aspergillus strains [27,28,29].
Recently, A. parasiticus mutants resistant to anilinopyrimidine fungicides produced AFs at concentrations much higher than a wild-type strain [30]. Doukas et al. [31] also reported that A. parasiticus mutants resistant to the sterol-demethylation-inhibiting (DMIs) fungicides produced significantly higher levels of AF, where mutation at the cytochrome P450 gene cyp51A and overexpression of cyp51A gene and/or a multidrug resistance gene (ABC transporter protein) were responsible for DMI resistance in A. parasiticus [31].
Fludioxonil (FLU) is a commercial, phenylpyrrole fungicide, for which the antifungal effect is exerted through the intact signaling system responsible for oxidative/osmotic stress defense in fungi termed the “mitogen-activated protein kinase (MAPK)” pathway [32]. FLU disrupts fungal growth/survival by exerting excessive activation of the MAPK pathway, which triggers cellular energy drain [32]. This MAPK system is operated to protect fungal cells from environmental oxidative/osmotic stressors. However, fungi with mutations in the MAPK system develop FLU tolerance [32]; conversely, these fungal mutants are highly susceptible to oxidative stressors including redox-active compounds. Meanwhile, recent investigations have shown that the antifungal mechanism of action of certain drugs/compounds involves an oxidative stress response in fungi, which further contributes to fungal death. Therefore, these classes of drugs/compounds are defined as oxidative stress reagents [33,34,35,36].
Natural products (NP)/botanicals are potential sources of antifungal agents, food additives, etc. [37,38]. For example, natural phenolics serve as potent redox cycling agents, thus inhibit microbial growth via the destabilization of cellular redox homeostasis [39,40]. However, there are significant research gaps between NP utilization in food/feed products and their safety, thus requiring vigorous risk assessment studies [41]. We hypothesize that, as determined in conventional fungicides, if NPs or their structural derivatives are applied at “suboptimal” concentrations, certain NPs/derivatives can enhance mycotoxin production by the AF-producing aspergilli (A. flavus, A. parasiticus) due to the oxidative stress caused by the disruption of cellular redox homeostasis. It has been shown in A. parasiticus that AF production was triggered by increased oxidative stress [42]. A recent study also showed that botanicals, such as pomegranate peel extract, applied at suboptimal concentrations increased the production of AFB1 in A. flavus [43].
In this study, we investigated the effect of “sub-inhibitory” doses (namely, sub-optimal concentrations that still maintain fungal growth, but slightly less growth than “no treatment” controls; see Results and Discussion) of thirteen NP/derivatives, currently used as food/feed additives [44], against A. flavus and A. parasiticus by evaluating their enhancement of AF production (AFB1 and AFB2 for A. flavus; AFB1, AFB2, AFG1 and AFG2 for A. parasiticus) [45,46]. We reasoned that these NP/derivatives could serve as representatives for studying the safe use of various benzaldehydes, cinnamic acids and isopropyl methylphenol compounds enlisted as food/feed additives by the United States Food and Drug Administration (FDA) [44]. Currently, the safety of certain compounds selected (e.g., isopropyl methylphenols) is not conclusive, as published by the European Food Safety Authority [41]. Noteworthy, the FDA guideline indicates that each “food ingredient manufacturers/food producers should ensure marketed products are safe as well as compliant with all applicable regulatory requirements” [47]; hence, comprehensive evaluation of food/feed additives/substances is highly necessary. Compounds that did not enhance AF production were selected and tested further for their utility as potent antifungal agents in crop (corn) seeds.

2. Materials and Methods

2.1. Chemicals and Microorganisms

Chemicals and media, namely fludioxonil (FLU) and thirteen NP/derivatives (2,3-, 2,4-, 2,5-dihydroxybenzaldehyde; 3,5-dimethoxybenzaldehyde; 2-hydroxy-4-methoxy- and 2-hydroxy-5-methoxybenzaldehyde; 4-methyl- and 4-methoxycinnamic acid; salicylic acid; salicylaldehyde; thymol; carvacrol; and 4-isopropyl-3-methylphenol; Figure 1), were purchased from Sigma Aldrich Co. (St. Louis, MO, USA), except for dimethyl sulfoxide (DMSO; AMRESCO Co. (Solon, OH, USA)), ethanol (Thermo Fisher Scientific (Waltham, MA, USA)) and potato dextrose agar (PDA; BD Life Sciences (Franklin Lakes, NJ, USA)). Aspergilli (Aspergillus flavus NRRL 4212, Aspergillus parasiticus NRRL 2999) [48] and Penicillium expansum (W1, wild type; FR2, FLU resistant mutant) [49] were maintained at 28 °C on PDA.

2.2. Corn and Pistachio Kernels

Corn seeds (Zea mays; Golden Bantam 12 Corn Seeds) were procured from True Leaf Market Co. (Salt Lake City, UT, USA). Unprocessed pistachio kernels (Pistacia vera) were obtained from Valley Orchard, LLC (Fresno, CA, USA). Seeds were maintained at room temperature (22 °C) until use.

2.3. Antifungal Assay

2.3.1. Petri Plate Assay on Segmented Dishes: Salicylaldehyde (SLD; Fumigant)

Determination of antifungal activity of SLD, a fumigant benzaldehyde derivative, on the surfaces of crop seeds was performed in segmented Petri plates (100 mm × 15 mm; VWR International Co. (Radnor, PA, USA)). These plates were divided into four sections, three of which were filled with corn or pistachio kernels (Figure S1). For the corn testing, corn seeds were soaked in conidial suspensions of A. flavus NRRL 4212 (1 × 104 CFU mL−1), overnight (16 h), and seeds were placed in segmented Petri plates (Total 10 seeds per plate; section 1: 3 seeds, section 2: 3 seeds, section 3: 4 seeds, section 4: SLD). SLD was dissolved in ethanol (60% v/v) and was applied onto a round Whatman™ Grade 1 qualitative paper (2.5 cm in diameter; GE Healthcare Bio-science Co. (Piscataway, NJ, USA)) (Note: Dimethyl sulfoxide can serve as an alternative solvent [50]). Total 120 μL of SLD or ethanol (control) was applied on Whatman™ paper (double layer) per each plate (SLD concentrations tested: 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 mM). Fungal spores on the surface of seeds were exposed to the volatilized SLD.
The plates containing SLD (or ethanol control) and seeds were then sealed with two layers of Parafilm® (American National Can Co. (Chicago, IL, USA)) to prevent escape of volatilized SLD. The plates were incubated at 28 °C (1, 2 or 3 days). The antifungal treatments, therefore, consisted of (1) control plates: no A. flavus NRRL 4212 without SLD vapor (ethanol only) for determining the level of “seedborne” fungal contaminants, (2) control plates: A. flavus without SLD vapor (ethanol only), and (3) treated plates: A. flavus with SLD vapor (0.1 to 1.0 mM on Whatman™ paper). After exposure to SLD, corn seeds were placed on PDA recovery plates and the level of fungal survival was monitored for 5–7 days at 28 °C. Throughout the experiments, corn seeds were not surface-sterilized before treatments.
A similar antifungal assay was performed using “unprocessed” raw pistachio samples (harvested from orchards), as described above, to determine seedborne fungal contaminants only (“w/o” A. flavus inoculation).

2.3.2. Raised Bed Assay (Large Scale Testing): SLD (Fumigant)

Determination of antifungal activity of SLD was performed further in a larger, raised-bed container (16 cm × 20 cm × 10 cm; KEHUI-Direct Co. (Shenzhen, Guangdong Province, China)). The application method of SLD was similar to that described above, where the volume (amount) of SLD applied was increased (proportional to the volume of sectioned Petri plates). Therefore, the antifungal treatments also consisted of (1) control plates: no A. flavus NRRL 4212 without SLD vapor (ethanol only), (2) control plates: A. flavus without SLD vapor (ethanol only) and (3) treated plates: A. flavus with SLD vapor (0.5, 1.0 mM) applied onto a Whatman™ filter paper (9 cm diameter). After SLD exposure, corn seeds were placed on PDA recovery plates and the level of fungal survival was monitored for 5–7 days at 28 °C.
To precisely evaluate the level of fungal contaminants (fungal burden) on the surface of corn seeds, the SLD-treated seeds (3 days) were rinsed (200 seeds/250 mL ddH2O), and the supernatants (200 μL/plate) were spread onto PDA recovery plates after 1:1, 1:10 and 1:100 dilutions. The level of fungal contamination (either “fungistatic” or “fungicidal” antifungal activity) on seed surfaces were monitored for 48 h at 28 °C. Results were based on five replicate plates.

2.3.3. Petri Plate Assay on PDA Plates: 4-Isopropyl-3-Methylphenol (4I3M; Non-Fumigant)

Determination of antifungal activity of 4I3M was performed on PDA. Corn seeds were soaked in water containing 0.1 to 1.0 mM of 4I3M (dissolved in ethanol, 60% v/v) and A. flavus NRRL 4212 (1 × 104 CFU mL−1), overnight (16 h); then, each corn seeds were transferred onto PDA recovery plates (10 seeds per plate), and fungal growth/survival was monitored at 28 °C for 5–7 days. Results were based on three replicate Petri plates (100 mm × 15 mm; Corning Inc. Life Sciences (Tewksbury, MA, USA)).

2.4. AF Analysis; HPLC

For AF analysis, A. flavus and A. parasiticus were treated with thirteen compounds (described above) at the respective “sub-inhibitory” concentrations (Table 1), 28 °C, for 7 days. Results were based on three replicate PDA plates (60 mm × 15 mm; Corning Inc. Life Sciences (Tewksbury, MA, USA)). The level of fungal growth at sub-inhibitory concentrations was initially determined based on fungal radial growth (% compared to untreated control) on PDA plates (average of triplicate) (See Section 3). AF analysis (AFB1, AFB2 for A. flavus; AFB1, AFB2, AFG1, AFG2 for A. parasiticus) was performed using an Agilent 1260 system (Palo Alto, CA, USA) with fluorescence detection at 365 nm excitation and 455 nm emission, as previously described [51]. Analysis was performed using OpenLAB CDS Chemstation Edition for LC & LC/MS Systems (Rev. C.01.08) (Agilent Technologies (Palo Alto, CA, USA)).

2.5. Overcoming FLU Tolerance of Mitogen-Activated Protein Kinase (MAPK) Mutants of P. expansum

The capability of 4I3M to overcome FLU tolerance of mitogen-activated protein kinase (MAPK) mutant of Penicillium expansum (FR2) was performed in PDA (triplicates) via the modified method described previously [34]. Six-well plates (Costar® TC-Treated Multiple Well Plates, Diameter: 34.8 mm; Corning Inc. Life Sciences (Tewksbury, MA, USA)) were prepared with PDA containing (1) ethanol only (control), (2) FLU (50 μM), (3) 4I3M (0.25, 0.5, 0.75, 1.0, 1.25 or 1.5 mM), and (4) FLU (50 μM) + 4I3M (0.25–1.5 mM); then, fungal spores (1 × 103 CFU) were spotted onto the center of PDA. The inoculated plates were incubated at 28 °C. Fungal growth (radial growth) was monitored for 3 to 5 days.

2.6. Statistical Analysis

Statistical analysis to determine Student’s t-test was performed using “Statistics to use” tool [52]. Paired data for (1) seed germination, aspergilli contamination and seedborne fungal contamination w/or w/o SLD treatment, and (2) AF production (viz., SLD vs. SA; 4I3M vs. THY or CARV) were analyzed, where a p value < 0.05 was considered significant.

3. Results and Discussion

3.1. Effects of Sub-Lethal Concentrations of NP/Derivatives on AF Production: A. flavus

To investigate the effects of sub-lethal concentrations (Supplementary Figure S2a,b) of NP or their structural derivatives on AF production by A. flavus, NPs/derivatives were applied to A. flavus at 0.1 to 1.0 mM, depending on types of compounds (Table 1). Most compounds marginally affected the growth of A. flavus at the test concentrations, except SLD, which prevented fungal growth at 1.0 mM. Then, the level of AF production (AFB1, AFB2) at each concentration point was compared to that of control (no treatment; defined as 100%). AF production with an SD value ± 4% was considered equivalent to the control in this study.
As shown in Figure 2a, 20 out of 52 concentration points (marked as an upper arrow (“^”)) exhibited enhanced AF (B1, B2) production compared to the untreated control (9 to 129%). As mentioned earlier, A. flavus produces AFB1 and AFB2 only, but not AFG1 and AFG2, whereas 2,4-D, 4I3M and 4-MOC did not enhance AF production at any concentrations examined. While CARV increased the AFB2 production at 0.2 mM (36% enhancement), both 4I3M and CARV at 0.4 mM showed the most potent anti-aflatoxigenic activity (81 to 97% inhibition), whereas the structural analog THY at 0.2 mM enhanced the AFB2 production at the highest level compared to other treatments (129% enhancement) (Order of AF inhibition (THY derivatives): 4I3M > CARV > THY; high to low).
Noteworthy is that, although SLD and salicylic acid (SA) are very close structural analogs (viz., only one additional oxygen in SA structure compared to SLD), SLD at 1.0 mM completely prevented the growth of A. flavus, while SA allowed fungal growth along with AFB1 enhancement at the same concentration.

3.2. Effects of Sub-Lethal Concentrations of NP/Derivatives on AF Production: A. parasiticus

NPs/derivatives were also examined in A. parasiticus at the same concentrations applied to A. flavus (See above) (Table 1). As determined in A. flavus, most compounds moderately affected the growth of A. parasiticus at test concentrations, except SLD, which prevented fungal growth at as low as 0.5 mM, indicating A. parasiticus was more susceptible to SLD compared to A. flavus (growth prevention at 1.0 mM). Similarly, A. parasiticus was also slightly more susceptible to 4I3M (0.4 mM), THY (0.4 mM) and CARV (0.4 mM) compared to A. flavus (viz., less fungal growth; See Supplementary Figure S2), indicating “compound—fungal species specificity” exists with the NP/derivatives treatments.
As shown in Figure 2b, 16 out of 104 concentration points (marked as an upper arrow (“^”)) exhibited enhanced AF (B1, B2, G1, G2) production compared to the untreated control (5 to 110% enhancement), whereas 2,3-D, 2,5-D, 2H4M, 4I3M and 4-MEC did not enhance AF production at any test concentrations. As determined in A. flavus, while CARV increased the production of AFB2 and AFG2 at 0.2 mM (29% and 20% enhancement, respectively), both 4I3M and CARV at 0.4 mM exerted the most potent anti-aflatoxigenic activity (94% to 99% inhibition); conversely, the structural analog THY at 0.4 mM exerted the highest level of AF (B1, B2 G1, G2) enhancement compared to other treatments (62% to 110% enhancement). Therefore, analogous to A. flavus, the order of AF inhibition by THY derivatives was determined as: 4I3M > CARV > THY (high to low).
THY and CARV have been used as food/feed additives. For example, the feed additive product Biomin®DC-P (trade name) is a mixture of five compounds including THY, CARV, d-carvone, methyl salicylate and l-menthol, which are encapsulated with a hydrogenated vegetable oil [53]. The additive has been used in feed for chicken fattening, chickens reared for laying, etc. However, the safety of this additive for laying/breeding birds was not conclusive [41]. Since cereal grains are the main ingredients in animal feed, ensuring that feeds are free of risk factors, such as mycotoxins or mycotoxin triggering agents, is of high importance. As investigated in this study, THY or CARV could function as risk factors by exerting mycotoxin-enhancing capabilities; therefore, optimization of concentrations of ingredients such as THY or CARV for feed use is highly necessary.
Of note, 4I3M is the only compound that did not enhance AF production in both A. flavus and A. parasiticus. 4I3M is a synthetic derivative of THY and CARV, which was previously developed as an antimicrobial preservative or fungicide [54,55]; 4I3M has recently been examined as an ingredient of oral care products [56,57] that target either fungal or bacterial pathogens, including Candida species, Aggregatibacter actinomycetemcomitans, Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pneumoniae, Klebsiella pneumoniae, Porphyromonas gingivalis, etc. Compared to THY or CARV, 4I3M possesses color- or odor-neutral characteristics; hence, it is more appealing to consumers [58]. Of note, a previous study indicated that the structural analog THY affected fungal antioxidant defense system such as superoxide dismutases, glutathione reductase, etc. [59]. Therefore, we speculate that 4I3M might also affect a similar oxidative stress defense system in fungi. The determination of the precise mechanism of antifungal and/or anti-mycotoxigenic activity of 4I3M warrants future in-depth study.
Also interesting is that, except for THY and CARV, most AF types enhanced in A. parasiticus by NPs/derivatives were AFG1 or AFG2 (Table 1) but not AFB1 or AFB2 (A. flavus naturally does not produce AFG1 and AFG2) [45,46,60]. Yu et al. previously characterized the AF biosynthetic pathway gene cluster in aspergilli [61], in which at least 23 enzymatic reactions are involved. AFB1/AFG1 or AFB2/AFG2 are produced from the metabolic intermediates O-methylsterigmatocystin or dihydro-O-methylsterigmatocystin, respectively, by the action of oxidoreductase encoded by aflQ gene. At this moment, it is not clear why the effect of NP/derivatives in A. parasiticus is enhancing mostly AFG1/AFG2. Future investigation might elucidate the mechanism of action of differential AF enhancement by these compounds, and new control/intervention point(s) that can specifically inhibit AFG1 and/or AFG2 production.
As observed in A. flavus, while SLD at 0.5 mM prevented the growth of A. parasiticus, the structural analog SA still allowed the fungal growth at the same concentration, which coincided with AFG1 and AFG2 enhancement in A. parasiticus (structure–activity relationship). Therefore, we selected two compounds, viz., SLD (potent antifungal activity in both A. flavus and A. parasiticus) and 4I3M (highest anti-aflatoxigenic activity), and investigated further for their utility as potential antifungal and/or anti-aflatoxigenic agents using corn seeds.

3.3. Use of Salicylaldehyde (SLD) as an Antifungal Agent Targeting Corn Kernels: Petriplate Analysis (Small Scale)

SLD is a plant-derived volatile compound classified as Generally Recognized As Safe (GRAS) [62,63,64]. In this study, we investigated the antifungal efficacy of SLD (0.1 to 1.0 mM) as a fumigant by targeting A. flavus contaminated on corn kernels (seeds). The effect of SLD on seed germination frequency was also examined.
As shown in Table 2, SLD exhibited potent antifungal activity against A. flavus or seedborne fungal contaminants; the average number of A. flavus-contaminated corn kernels decreased from 6.8 (1-day exposure) to 3.9 (2-day exposure) and 2.9 (3-day exposure), respectively, as the exposure time increased from 1 to 3 days (see also Supplementary Figure S3). Likewise, the number of corn kernels having seedborne fungal contaminants also decreased from 3.6 (1-day exposure) to 0.6 (2-day exposure) and 0.3 (3-day exposure), respectively, as the incubation time increased.
A similar trend of SLD antifungal activity was also determined in the tree nut pistachio (Supplementary Figure S4). In this testing, no A. flavus was artificially inoculated onto the surface of pistachios, and therefore, antifungal testing was solely against farm-derived, seedborne fungal contaminants. We determined that the level of seedborne fungal contaminants on pistachios was decreased as the concentration of SLD (applied as a fumigant) increased (0.1 to 1.0 mM) (Figure S4). Complete inhibition of fungal growth was achieved at 0.6 mM of SLD (3-day exposure), thus indicating SLD can exert potent antifungal activity on different food crops. Tree nuts in California, USA, are mostly contaminated with pathogenic fungi, such as Alternaria sp., Aspergillus sp., Botrytis sp., Botryosphaeria sp., etc. [65].
Of note, coinciding with the SLD antifungal activity was the inhibition of corn seed germination at ≥0.6 mM (2- to 3-day exposure) (Table 2; Supplementary Figure S3). Therefore, results indicated that SLD could be utilized as a potent antifungal fumigant especially for the foods/crops “to be processed”, but not for the crop seeds for cultivation. A recent study also indicated that a benzaldehyde derivative, benzoic acid, which is a secondary metabolite released as a root exudate, is an inhibitor triggering plant autotoxicity, thus interfering with seed germination and root growth in Lactuca sativa L. [66]. As mentioned above, natural phenolics, such as benzaldehyde analogs, are potent redox cyclers that destabilize cellular redox homeostasis [39,40]. We surmised that, as determined in benzoic acid [66], SLD might also negatively affect redox homeostasis in corn seeds, thus resulting in the inhibition of seed germination. Other studies have described further the importance of redox homeostasis in seed germination/plant growth [67,68,69].
SLD has been used as a food flavoring agent and/or an intermediate for producing pharmaceuticals [64]. In the atmosphere, SLD exists as a vapor with vapor pressure value of 5.93 × 10−1 mmHg at 25 °C. Noteworthy is that SLD has been exempted from requiring a tolerance by the United States Environmental Protection Agency (EPA); hence, this compound can be applied as an inert ingredient up to 14% by weight of antifungal formulations [70]. Collectively, SLD could be applied as a fumigant during food/crop sanitation or storage. The identification of the precise mechanism of the structure–activity relationship, namely, differences in the antifungal/anti-aflatoxigenic efficacy between SLD and SA, warrants future in-depth investigation.

3.4. Use of SLD as an Antifungal Agent on Corn Kernels: Raised Bed Analysis (Large Scale)

Antifungal efficacy of SLD was examined further in large containers (16 cm × 20 cm × 10 cm) for scaling up. As shown in Figure 3, application of SLD at 0.5 to 1.0 mM in the large containers completely inhibited the growth of A. flavus and/or seedborne fungal contaminants, while no treatment control (seedborne contaminants or A. flavus at 0.0 mM) allowed the growth of fungi. As determined in Petri plate assay (Section 3.3 above), SLD at 0.5 to 1.0 mM also inhibited seed germination (tested in the large containers).
We examined further the “fungal burden on the surfaces” of corn kernels after SLD treatment (See Figure 3). As shown in Figure 4, corn kernels treated with 0.5 to 1.0 mM of SLD prevented the survival of A. flavus or seedborne filamentous fungi (after 1:1, 1:10, 1:100 dilutions), while only some “yeast-like” contaminants (seedborne) were detected from the same seeds (1:1 (no dilution)); however, no indication of any microbial survival was determined with 1:10 and 1:100 dilution, respectively.
In comparison, high level of fungal contamination was detected in the control groups, viz., no SLD treatments (seedborne contaminants or A. flavus only). Therefore, results indicated that the effect of SLD antifungal activity (against A. flavus or seedborne filamentous fungi) seemed “fungicidal” where no filamentous fungi was survived after SLD fumigation. If necessary, standard methods such as modified protocols developed by the Clinical and Laboratory Standards Institute (CLSI) could be applied for the precise determination of fungistatic or fungicidal characteristic of SLD [71,72].
Future in-depth study is also necessary to confirm (1) the antifungal efficacy of SLD against broad spectrum of fungal pathogens/contaminants such as azole fungicide-resistant aspergilli recently identified in California tree nut farms [73], and (2) the effectiveness of SLD (as fumigant) for the sterilization of internal seeds when seeds are stored in piles.

3.5. Use of 4-Isopropyl-3-Methylphenol (4I3M) as an Antifungal and Anti-Aflatoxigenic Agent on Corn Kernels

Next, 4I3M was tested as an antifungal agent against A. flavus or seedborne fungi contaminated on corn kernels. As shown in Figure 5a, 4I3M exerted antifungal activity at or above 0.8 mM (tested up to 1.0 mM) without affecting seed germination, possibly being less redox-active than SLD. However, compared to SLD, 4I3M did not completely inhibit the growth of fungi on corn seeds at the highest concentrations tested (namely, 0.8–1.0 mM).
The effect of 4I3M on AF production by A. flavus and A. parasiticus was examined further at higher concentrations (0.5 to 1.5 mM). As shown in Figure 5b, there was an incremental increase in anti-aflatoxigenic activity of 4I3M as the concentration of the compound increased (from 0.5 to 1.5 mM); complete inhibition of AF production was achieved in both A. flavus and A. parasiticus at 1.0 to 1.5 mM of 4I3M.

3.6. Overcoming Fludioxonil Tolerance of Mitogen-Activated Protein Kinase (MAPK) Mutants of P. expansum by 4I3M

Penicillium expansum FR2 is a MAPK pathway mutant involved in oxidative stress signaling [49], thus showing tolerance to FLU, whereas it is more susceptible to oxidative stress reagents compared to the parental, wild-type strain. Using P. expansum (W1; wild type, parental strain) and FR2 (oxidative/osmotic stress MAPK mutant) as a “model” filamentous/mycotoxigenic (producing toxic patulins) fungal system, we examined the capability of 4I3M for overcoming FLU tolerance of P. expansum FR2. We tried to confirm the antifungal potential of 4I3M as a “redox-active” compound by targeting FR2. As shown in Figure 6, 4I3M at as low as 0.75 mM overcame FLU tolerance of P. expansum FR2. Of note, P. expansum FR2 exhibited higher sensitivity to 4I3M, as determined by the smaller colony size formed (viz., fungal growth: 58 ± 0% W1 vs. 22 ± 5% FR2 compared to the control). Therefore, results indicated that 4I3M is a redox-active compound, where the level of redox-activity (redox cycling) exerted by 4I3M was enough to induce the higher susceptibility of FR2 to 4I3M toxicity when compared to the parental strain W1.
Altogether, we speculated that 4I3M could also be applicable as a potent antifungal and/or anti-aflatoxigenic agent in foods/crops without affecting seed germination. There was a differential antifungal efficacy between 4I3M (non-fumigant) and SLD (fumigant), which might be due to the differences in the level of redox activity of the compounds. The order of antifungal efficacy was: SLD (complete inhibition of fungal growth) > 4I3M (complete inhibition of AF production) (high to low).
In summary, via the risk assessment testing using thirteen NP or their structural derivatives, we determined that several benzaldehyde/THY derivatives could enhance AF production if applied at sub-inhibitory concentrations in A. flavus and A. parasiticus. The results indicated that caution should be exercised in the use of NP/derivatives so as not to interfere with the safe production of foods/crops as well as public food safety. Therefore, the precise determination of optimum concentrations of NP/derivatives for usage is necessary to prevent abnormal enhancement of mycotoxins during industrial application.
Conversely, SLD and 4I3M appear to be an effective antifungal and/or anti-mycotoxigenic agents, where SLD prevented the growth of AF-producing A. flavus as a fumigant while 4I3M effectively disrupted AF production by A. flavus and A. parasiticus. The use of SLD as a fumigant could also warrant the minimal deposition of the compound on the surface of crops during application. Collectively, NPs or their structural derivatives, such as SLD or 4I3M, can be a potent source of antifungal or anti-mycotoxigenic agents, which can lower conventional pesticide burden as well as overcome fungicide resistance of fungal pathogens during food/crop production. The determination of the mechanism of antifungal and/or anti-mycotoxigenic activity of NP including SLD or 4I3M requires future in-depth investigation.
In conclusion, if technological advancements are further explored or achieved, the outcomes of this study will result in effective fungal and mycotoxin control, thus ensuring the safe production of foods/crops and new revenues for the food/crop industry. While not pursued in this study, in-depth determination of the “antifungal” efficacy of other NP/derivatives (in addition to SLD and 4I3M) can provide further insight as to the utility of various benzaldehydes, cinnamic acids or isopropyl methylphenol derivatives for safe food/crop production.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/app142110000/s1, Figure S1: Petri plate assay on segmented dishes; Figure S2: The level of fungal growth with the treatment of thirteen NP/derivatives at sub-lethal concentrations; Figure S3: Antifungal activity of salicylaldehyde tested against A. flavus and seedborne contaminants on corn seeds; Figure S4: Antifungal activity of salicylaldehyde tested against seedborne contaminants on pistachios.

Author Contributions

Conceptualization, J.H.K.; methodology, J.H.K.; validation, J.H.K., K.L.C., D.F. and S.B.L.S.; formal analysis, J.H.K., K.L.C., D.F. and S.B.L.S.; investigation, J.H.K. and W.M.H.-C.; resources, J.D.P., W.M.H.-C. and K.O.; data curation, K.L.C. and D.F.; writing—original draft preparation, J.H.K.; writing—review and editing, W.M.H.-C., K.O., K.L.C., D.F., S.B.L.S., J.D.P. and W.J.O.; visualization, J.H.K., K.L.C. and D.F.; supervision, W.J.O.; project administration, W.J.O. All authors have read and agreed to the published version of the manuscript.

Funding

This research was conducted under the USDA-ARS CRIS project 2030-42000-054-000-D.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

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

Acknowledgments

The authors thank Keith Meeks, Ipura Food Safety Inc., Madera, California, United States for the constructive discussions.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Natural products or structural derivatives tested in this study. (1) 2,3-Dihydroxybenzaldehyde (2,3-D), (2) 2,4-Dihydroxybenzaldehyde (2,4-D), (3) 2,5-Dihydroxybenzaldehyde (2,5-D), (4) 3,5-Dimethoxybenzaldehyde (3,5-D), (5) 2-Hydroxy-4-methoxybenzaldehyde (2H4M), (6) 2-Hydroxy-5-methoxybenzaldehyde (2H5M), (7) Salicylaldehyde (SLD), (8) Salicylic acid (SA), (9) 4-Isopropyl-3-methylphenol (4I3M), (10) Thymol (THY), (11) Carvacrol (CARV), (12) 4-Methylcinnamic acid (4-MEC), (13) 4-Methoxycinnamic acid (4-MOC).
Figure 1. Natural products or structural derivatives tested in this study. (1) 2,3-Dihydroxybenzaldehyde (2,3-D), (2) 2,4-Dihydroxybenzaldehyde (2,4-D), (3) 2,5-Dihydroxybenzaldehyde (2,5-D), (4) 3,5-Dimethoxybenzaldehyde (3,5-D), (5) 2-Hydroxy-4-methoxybenzaldehyde (2H4M), (6) 2-Hydroxy-5-methoxybenzaldehyde (2H5M), (7) Salicylaldehyde (SLD), (8) Salicylic acid (SA), (9) 4-Isopropyl-3-methylphenol (4I3M), (10) Thymol (THY), (11) Carvacrol (CARV), (12) 4-Methylcinnamic acid (4-MEC), (13) 4-Methoxycinnamic acid (4-MOC).
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Figure 2. Types of AF enhanced by NPs or structural derivatives applied at sub-inhibitory concentrations in (a) A. flavus or (b) A. parasiticus. Upper arrow (^), Enhanced AF production compared to the “no treatment” control. Asterisk (*), p < 0.05 compared to the structural analogs (viz., SLD vs. SA; 4I3M vs. THY or CARV). PSA, p value compared to SA. PT, p value compared to THY. PC, p value compared to CARV.
Figure 2. Types of AF enhanced by NPs or structural derivatives applied at sub-inhibitory concentrations in (a) A. flavus or (b) A. parasiticus. Upper arrow (^), Enhanced AF production compared to the “no treatment” control. Asterisk (*), p < 0.05 compared to the structural analogs (viz., SLD vs. SA; 4I3M vs. THY or CARV). PSA, p value compared to SA. PT, p value compared to THY. PC, p value compared to CARV.
Applsci 14 10000 g002aApplsci 14 10000 g002b
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Figure 3. Antifungal activity of SLD tested at large scale.
Figure 3. Antifungal activity of SLD tested at large scale.
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Figure 4. Fungal burden on seed surface after SLD treatment.
Figure 4. Fungal burden on seed surface after SLD treatment.
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Figure 5. Antifungal and anti-aflatoxigenic activity of 4I3M: (a) antifungal activity of 4I3M; (b) inhibition of AF production by 4I3M.
Figure 5. Antifungal and anti-aflatoxigenic activity of 4I3M: (a) antifungal activity of 4I3M; (b) inhibition of AF production by 4I3M.
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Figure 6. Overcoming tolerance of mitogen-activated protein kinase (MAPK) signaling mutant of P. expansum (FR2) to FLU by 4I3M.
Figure 6. Overcoming tolerance of mitogen-activated protein kinase (MAPK) signaling mutant of P. expansum (FR2) to FLU by 4I3M.
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Table 1. Types of AF enhanced by NP or structural derivatives applied at sub-inhibitory concentrations to A. flavus or A. parasiticus.
Table 1. Types of AF enhanced by NP or structural derivatives applied at sub-inhibitory concentrations to A. flavus or A. parasiticus.
NPs/DerivativesTest Concentrations (mM)A. flavusA. parasiticus
2,3-Dihydroxybenzaldehyde (2,3-D)0.1, 0.2B1, B2- 1
2,4-Dihydroxybenzaldehyde (2,4-D)0.5, 1.0- 1G2
2,5-Dihydroxybenzaldehyde (2,5-D)0.5, 1.0B1, B2- 1
3,5-Dimethoxybenzaldehyde (3,5-D)0.5, 0.75B1, B2G2
2-Hydroxy-4-methoxybenzaldehyde (2H4M)0.1, 0.2B2- 1
2-Hydroxy-5-methoxybenzaldehyde (2H5M)0.1, 0.2B1, B2G1
Salicylaldehyde (SLD)0.5, 1.0B2- 2
Salicylic acid (SA)0.5, 1.0B1G1, G2
4-Isopropyl-3-methylphenol (4I3M)0.2, 0.4- 1- 1
Thymol (THY)0.2, 0.4B2B1, B2, G1, G2
Carvacrol (CARV)0.2, 0.4B2B2, G2
4-Methylcinnamic acid (4-MEC)0.5, 1.0B2- 1
4-Methoxycinnamic acid (4-MOC)0.5, 1.0- 1G1, G2
1 No enhancement. 2 No growth.
Table 2. Antifungal activity of salicylaldehyde tested against A. flavus and seedborne contaminants.
Table 2. Antifungal activity of salicylaldehyde tested against A. flavus and seedborne contaminants.
1 Day 2 Day 3 Day
SLD (mM)# Seed Germinated# Af
Contaminated		# Seedborne Contaminated# Seed Germinated# Af
Contaminated		# Seedborne Contaminated# Seed
Germinated		# Af
Contaminated		# Seedborne Contaminated
0.0 (Seed only) 1100101001010010
0.0 (Seed only) 210010901010010
0.0 1910091006100
0.0 27100101007100
0.1 1610310100190
0.1 2610081000101
0.2 13645100172
0.2 23645100051
0.3 17656823100
0.3 2546232130
0.4 12010030001
0.4 2446271000
0.5 1863113001
0.5 2573111000
0.6 1464000000
0.6 2336121000
0.7 1493000000
0.7 2583000000
0.8 1573001000
0.8 2483101000
0.9 1573000000
0.9 2473001000
1.0 1574000000
1.0 2483001000
Average4.96.83.62.83.90.60.92.90.3
SD1.72.52.23.64.40.82.04.20.6
p-value---0.0160.010.00010.00010.010.0001
1 First replicate (ten seeds were exposed to SLD in three independent sections of segmented Petri plate). 2 Second replicate (ten seeds were exposed to SLD in three independent sections of segmented Petri plate).
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Kim, J.H.; Chan, K.L.; Hart-Cooper, W.M.; Ford, D.; Orcutt, K.; Sarreal, S.B.L.; Palumbo, J.D.; Orts, W.J. The Inhibitory Activity of Salicylaldehyde Compounds on Aspergillus Species and Their Effects on Aflatoxin Production and Crop Seed Germination. Appl. Sci. 2024, 14, 10000. https://doi.org/10.3390/app142110000

AMA Style

Kim JH, Chan KL, Hart-Cooper WM, Ford D, Orcutt K, Sarreal SBL, Palumbo JD, Orts WJ. The Inhibitory Activity of Salicylaldehyde Compounds on Aspergillus Species and Their Effects on Aflatoxin Production and Crop Seed Germination. Applied Sciences. 2024; 14(21):10000. https://doi.org/10.3390/app142110000

Chicago/Turabian Style

Kim, Jong H., Kathleen L. Chan, William M. Hart-Cooper, DeAngela Ford, Kaydren Orcutt, Siov Bouy L. Sarreal, Jeffrey D. Palumbo, and William J. Orts. 2024. "The Inhibitory Activity of Salicylaldehyde Compounds on Aspergillus Species and Their Effects on Aflatoxin Production and Crop Seed Germination" Applied Sciences 14, no. 21: 10000. https://doi.org/10.3390/app142110000

APA Style

Kim, J. H., Chan, K. L., Hart-Cooper, W. M., Ford, D., Orcutt, K., Sarreal, S. B. L., Palumbo, J. D., & Orts, W. J. (2024). The Inhibitory Activity of Salicylaldehyde Compounds on Aspergillus Species and Their Effects on Aflatoxin Production and Crop Seed Germination. Applied Sciences, 14(21), 10000. https://doi.org/10.3390/app142110000

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