Introduction

Fungi rank second among pathogens that cause diseases in maize as the reason for the loss in maize crop (Mohammadi et al. 2011). In order to increase maize production, it is crucial to prevent maize diseases (Lanubile et al. 2015). When conditions are ideal, fungi are responsible for 50 to 80 percent of the damage to stored maize grains.

Aspergillus spp., Fusarium spp., Penicillium spp., Alternaria spp., Pythium spp., Rhizoctonia spp., and Rhizopus spp., which are soil- and seed-borne fungal pathogens, are responsible for many of the common seed diseases in maize (Ashiq 2015; Benkerroum 2020; Rózewicz et al. 2021).

These fungi are able to produce mycotoxins, which are toxicologically dangerous to both human and animals' health. Aflatoxin, ochratoxin, patulin, trichothecenes, and fumonisin are the primary mycotoxins (Ashiq 2015; Mahato et al. 2019; Jallow et al. 2021).

The use of pesticides to control fungi has expanded as their infestation has increased (Christensen et al. 2014). The excessive use of chemicals had a negative impact on the environment and human health (Al-Ansary et al. 2022).

Mycotoxins, according to Leslie et al. (2008), affect the quality of harvested crops and have an adverse effect on the health of individuals as well as animals. Mycotoxins have a wide range of structural variations, which causes a wide range of effects (Ismaiel and Papenbrock 2015).

A number of mycotoxins produced by Aspergillus species, including aflatoxins, were described by Hussain et al. (2013). Mycotoxins known as aflatoxins, which are hazardous to people and animals, are produced by A. flavus and A. parasiticus (Somda et al. 2008).

The prevention of mold contamination and growth and the detoxification of contaminated items are two types of strategies that can be used to manage aflatoxins (Lavkor and Var 2017; Benkerroum 2020). Similarly, pre- and post-harvest controls are two general types of techniques that can be used to prevent aflatoxins (AFS) contamination (Kabak et al. 2006; Lavkor and Var 2017). AF-resistant crop varieties, use of fungal and bacterial inhibitors, application of natural and chemical agents and appropriate field management techniques (such as crop rotation, irrigation, and soil cultivation) are the main control strategies used prior to harvest. Following harvest, control tactics focus on enhancing storage and drying conditions using biological, chemical, and natural irradiation (Varga et al. 2010; Agriopoulou et al. 2020).

The current study aimed to develop strategies for management of fungal seed-borne pathogens and reduce their aflatoxin production depending on the alternatives of conventional fungicides or fumigants as post-harvest treatments.

Materials and methods

Plant material

Twenty five samples (1 kg each) of white maize grains were collected randomly from different locations in Cairo, Giza, Qalyubia, Fayoum and Sharqia governorates, Egypt. Each sample was placed in dry sterile containers. Collected samples were labeled and kept separately in sealed paper bags and transported to the laboratory of biopesticides production, Plant Pathology Department, Research of Agricultural and Biological Institute, National Research Center, where they were stored at 4 °C refrigerator for further analysis.

Isolation of seed-borne fungi associated with maize grains

One hundred maize grains were taken from each of the 25 samples, surface sterilized by dipping in 1 % aqueous sodium hypochloride solution for 3 min, followed by three successive rinses in sterile distilled water Then grains were dried with sterilized filter papers in a laminar flow hood. From each sample, 100 grains were selected then plated on each of Potato Dextrose Agar (PDA) and Malt salted agar (MSA) media, 50 grains/medium in 5 replicates for 7 day at 25±2 °C (Dhingra and Sinclair 1985). The isolated fungi were single-spored and the cultures were transferred onto PDA slants at 4 °C for further studies (Cumagun 2012).

Frequency of fungal isolates associated with grains

The frequency of occurrence of each fungus species isolated from maize grains was calculated by the following formula (Tsedaley and Adugna (2016)):

Fungal isolate Frequency (%) = Number of occurrence of fungus species/total number of isolated fungi × 100.

Biological identification of more frequent maize associated seed-borne fungi

The biological identification of the fungal isolates had been done and published in previous work (Al-Ansary et al. 2022).

Molecular identification of more frequent maize associated seed-borne fungi

The molecular identification of the fungal isolates had been done by Sigma Scientific Services co.

Extraction of fungal DNA

The fungal genomic DNA was extracted from single spored cultures of five fungal isolates recovered from maize grains collected from different governments in Egypt and grown on PD broth medium with the Quick-DNA Fungal Kit (Zymo Research, USA) according to the manufacturer's instructions.

Determination of the genomic DNA quantity and purity

The extracted DNA was estimated according to Sambrook et al. (1989) through reading the UV-absorbance at 260 and 280 nm using spectrophotometer to estimate the DNA quantity and purity.

PCR partial amplification and sequencing of internal transcribed spacer (ITS)

The fungal isolates were identified using molecular genetic analysis and internal transcribed spacer (ITS). A technique based on Boekhout et al. (1994) was used to get partial sequences of the isolate 18S rDNA. The gene’s divergent domain was amplified using two separate primers: the first primer (ITS1) sequence is 5' TCCGTAGGTGAACCTGCGG-3', while the second primer (ITS4) sequence is 5' TCCTCCGCTTATTGATATGC-3'. Utilized primer (12 ng) and 40 ng of the purified DNA sample were added to each polymerase chain reaction (PCR) bead. The amplified DNA products were electrophoresed on 1.0% agarose gel and 1X TBE (Tris-borate-EDTA) buffer at a constant 100 V for about 2 hours. The different band sizes were determined against Gene Ruler 100 bp DNA Ladder (Thermo SM0243) and the separated bands were stained with 0.5 µg/ml ethidium bromide and photographed using the Gel Documentation System with UV Transilluminator.

Fungal DNA purification

The PCR products were cleaned up using GeneJET™ PCR Purification Kit (Thermo K0701).

Isolate identification

The DNA sequencing of the purified PCR products were done with ABI 3730xl DNA sequencer (GATC Company, Germany) by using forward primer.

Evolutionary relationships of taxa

The DNA sequences of the fungal isolates were compared with the sequences available by the Basic Local Alignment Search Tool (BLAST) in the National Center for Biotechnology Information (NCBI), GenBank database (http://www.ncbi.nlm.nih.gov). The sequences were aligned together with those of reference taxa retrieved from public databases and the evolutionary distance was generated based on NCBI Neighbor Joining (Saitou and Nei 1987).

Source of standard Fungal isolate

An isolate of A. flavus isolates was kindly provided by Toxins and Food Pollutants Department, Food Industries and Nutrition Research Division, National Research Center. This isolate was known to induce aflatoxin and used as a standard isolate.

Essential oil

The ready-to-use Thyme essential oil was obtained from the Pressing and Extraction of Natural Oils Unit, National Research Center.

Direct contact assay

Antifungal activity was studied by using an in vitro direct contact assay. (Cakir et al. 2004; Gong et al. 2009).

Mycelial growth inhibition

Mycelial radial growth inhibition assay was conducted according to (Quiroga et al. 2001; Cakir et al. 2004; Song et al. 2004; Gong et al. 2009).

The percentage (%) of mycelial growth inhibition was determined as [(Mc – Mt)/Mc] ×100, where Mc average of five replicates of fungal mycelial growth measured on control agar medium, and Mt is an average of five replicates of fungal mycelial growth measured on treated agar medium with the treatments.

Inhibitory activity on spore germination

Spore germination inhibition assay was conducted according to (Liu et al. 2009; Wang et al. 2010).

The percentage (%) of the spore germination inhibition was determined as [(Gc – Gt)/Gc] ×100, where Gc is an average of five replicates of germinated spore in the control, and Gt is an average of five replicates of germinated spores in the treated ones.

Application methods

Two application methods i.e., Volatile and Carrier contact assays were tested with the acetic acid and thyme at different concentrations and evaluated for their capability to suppress aflatoxin production from A. flavus isolates; the one isolated throughout this study and the standard isolate, on maize grains during storage.

Volatile contact assay

The method of Paster et al. (1995), Abdallah (2005) and Bill et al., (2015) was modified as follow: The effect of volatile components on the treated maize grains with different concentrations of acetic acid and essential oils was tested in plastic containers. Maize grains treated without acetic acid or oils was used as the control. Three replicates were used for each treatment.

Carrier contact assay

Method by Wang et al. (2019) was modified as follow: Wheat bran as carrier was autoclaved at 120 °C for 60 min. Each of the tested acetic acid or oil added individually to the sterilized wheat bran with different concentrations, and then mixed thoroughly to ensure equal distribution of mixed carriers. The prepared mixture was added to sterilized maize grains. Three replicates were used for each treatment.

Aflatoxin production

Detection and analysis of aflatoxin was performed using liquid chromatography–electrospray ionization–tandem mass spectrometry (LC-ESI-MS/MS) with an ExionLC AC system for separation and SCIEX Triple Quad 5500+ MS/MS system equipped with an electrospray ionization (ESI) for detection. The instrument data were collected and processed using the SCIEX OS 1.6.10.40973 software (Hu et al. 2017).

To determine the in vitro antifungal efficacy of three different concentrations (1/2 EC90, EC90 and 2EC90) of acetic acid and thyme essential oil, maize grains were divided into two methods, i.e.,, Volatile assay and Carrier contact assay, 1.0 Kg for each concentration of each treatment in both methods. Maize grains were primarily sterilized in Erlenmeyer flask for 20 min at 121 °C using autoclave.

Maize grains in both volatile and carrier contact methods were divided into two groups, each group inoculated with 20 mL/kg (106 spores/ml) of conidial suspension of one of the two A. flavus fungal isolates, then treatments with different concentrations were added and then maize was stored for 30 days. Untreated and inoculated maize grains were used as control treatments. Samples were taken from each method after 15 and 30 days of storage and AFB1 was extracted from maize grains.

Statistical analysis

The median effective concentrations (EC50 and EC90) against tested fungi were calculated using coefficient equation between probit of means of inhibition (%) and logarithm of concentrations used according to (Ramadan et al. 2007).

Results

Isolation of seed-borne fungi associated with maize grains

From the twenty five samples of maize grain collected from different governorates, the results showed that the most dominant genera isolated on PDA were Aspergillus (three species) followed by Penicillium sp. and Fusarium spp with average frequency of 62.5, 22.5 and 13.3%, respectively, while on MSA average frequency was 58.9, 29.4 and 8.3%, respectively. The other fungal genera such as Alternaria sp. (1.1–0.0 %) and other (0.9 & 0.5%) were also isolated with low frequency of occurrence (Fig.1).

Fig. 1
figure 1

Mean frequency of isolated fungi of Maize seeds from different Governorates in Egypt on: a PDA and b MSA

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A. flavus was the most frequent species among the Aspergilli group -in both PDA and MSA media- with frequency of occurrence 30.8 and 35.6 %, respectively, followed by A. niger with frequency of occurrence 26.0 and 19.9 %, respectively, while A. terrus was the least frequent isolate among the Aspergilli with frequency of occurrence 5.7 and 3.5 %, respectively.

Molecular Identification of more frequent maize associated seed-borne fungi

Following DNA extraction from pure fungal strains and concentration measurement by spectrophotometer, the ITS1 and ITS4 primers were used to amplify the area of the rDNA repeat unit that includes the ITS from the fungal strain's genomic DNA. (Fig. 2) demonstrate that after amplification, roughly 632 bp were produced.

Fig. 2
figure 2

Agarose gel (1.2%) stained with ethiudium bromide shows the PCR products of ITS regions of the five fungal isolates obtained from maize grains using ITS1 and ITS4 primers. M: 100 bp DNA Ladder

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After the DNA sequencing of the purified PCR products with ABI 3730xl DNA sequencer (GATC Company, Germany), the obtained DNA sequence with the identified fungal strains from NA01 to NA05 (Table 1) were conserved in the GenBank.

Table 1 Accession number, closest phylogenetic relative, and identity percent of five fungal isolates (NA01 to NA05) obtained from Maize grains
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Evolutionary relationships of the identified strains

See Fig. 3

Fig. 3
figure 3

Phylogenetic trees of the taxonomic position of the isolated fungal strain: a (NA01), b (NA02), c (NA03), d (NA04) and e (NA05) isolated from maize grains was created using the Neighbor-Joining method, based on the ITS sequences and other closely related species in database

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Determination of antimycotoxigenic activity

The antifungal effect of thyme and acetic acid was examined in vitro by the food poison (bi culture) technique. The EC50 and EC90 values of the thyme and acetic acid against mycelial growth and spore germination of isolated A. flavus and reference isolate of A. flavus are shown in Table 2.

Table 2 Heatmap showing EC50 and EC90 (ppm) of thyme and acetic acid on mycelial growth and spore germination of isolated and reference A. flavus isolates
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Both thyme and acetic acid had an obvious significant inhibitory activity against isolated A. flavus mycelial growth, where there EC50 is 0.8704, 0.0016 mg/mL, respectively and EC90 is 1.6074, 0.0075 mg/mL, respectively with regression equations y = 3.7538x−6.0351 and y =1.4905x + 4.6916, respectively and coefficient of determination R2 = 0.872, R2 = 0.9701, respectively according to Table 2 and Fig. 4a. While, mycelial growth of reference isolate of A. flavus was significantly affected by thyme and acetic acid, as their EC50 is 0.8054, 0.0018 mg/mL, respectively and EC90 is 2.0808, 0.0088 mg/mL, respectively with regression equations y = 2.425x−2.0467 and y =1.4522x + 4.6249, respectively and coefficient of determination R2 = 0.9288, R2 = 0.9954, respectively according to Table 2 Fig. 4b.

Fig. 4
figure 4

Correlation coefficient between the logarithm of the treatments concentrations and the probit of means of a isolated A. flavus mycelial growth b reference A. flavus mycelial growth c isolated A. flavus spore germination inhibition (d reference A. flavus spore germination inhibition

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Both thyme and acetic acid had an obvious significant inhibitory activity of spore germination against isolated A. flavus, where there EC50 is 0.7309, 0.0036 mg/mL, respectively and EC90 is 1.2616, 0.0129 mg/mL, respectively with regression equations y = 4.2188x−7.0821 and y = 1.8219x + 3.9773, respectively and coefficient of determination R2 = 0.75, R2 = 0.9757, respectively according to Table 2 Fig. 4c. While spore germination of reference isolate of A. flavus was significantly affected by thyme and acetic acid, as there EC50 is 1.1303, 0.0042 mg/mL, respectively and EC90 is 1.8781, 0.0215 mg/mL, respectively with regression equations y = 4.5344x−8.8444 and y =1.4129x + 4.116, respectively and coefficient of determination R2 = 0.9547, R2 = 0.9394, respectively according to Table 2 Fig. 4d.

Detection and determination of aflatoxin production

The effect of thyme essential oil and acetic acid on two types of aflatoxins, i.e., AFB1 and AFB2 production by two A. flavus isolates (isolated and reference) in sterilized maize grains, at two different assays (volatile and carrier) and two incubation periods was studied after 15 and 30 days using LC-ESI-MS/MS. Both of them had a significant impact on AFB1 and AFB2 accumulation.

In the untreated controls, the major levels of only AFB1 and AFB2 were observed after 15 days of storage in both volatile and carrier contact assays then they began to decrease till it reached the lowest level after 30 days of storage.

After 15 days of storage

Determination of aflatoxin B1

Volatile contact assay indicated that thyme and acetic acid treatments after 15 days of incubation were able to reveal important reduction of AFB1 accumulation produced from isolated A. flavus, where there EC50 is 1.0304, 0.1008 mg/mL, respectively and EC90 is 1.8415, 1.6853 mg/mL, respectively with regression equations Y = 5.0763x−10.295 and Y =1.0464x + 4.9036, respectively and coefficient of determination R2 = 0.8977, R2 = 0.9067, respectively (Table 3 and Fig. 5a). In carrier contact assay, the thyme and acetic acid treatments after 15 days of storage also had an obvious significant inhibitory activity against AFB1, where there EC50 is 5.9729, 0.1588 mg/mL, respectively and EC90 is 16.6064, 0.2724 mg/mL, respectively with regression equations Y = 2.8823x−5.8841 and Y = 5.4646x−7.0274, respectively and coefficient of determination R2 = 0.7905, R2 = 0.9454, respectively according to Table 3 Fig. 5b.

Table 3 Heatmap showing EC50 and EC90 (ppm) of thyme and acetic acid on aflatoxin B1 produced by isolated and reference A. flavus isolates after 15 days of storage
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Fig. 5
figure 5

Correlation coefficient between the logarithm of the thyme and acetic acid concentrations and the probit of means of AFB1 content produced by a isolated A. flavus and b reference A. flavus under volatile contact assay and AFB1 content produced by c isolated A. flavus and d reference A. flavus under carrier contact assay

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Also, volatile contact assay showed that thyme and acetic acid treatments after 15 days of incubation had significant inhibitory effect on AFB1 levels produced from reference A. flavus, where there EC50 is 0.0045, 0.0013 mg/mL, respectively and EC90 is 0.0744, 0.0134 mg/mL, respectively with regression equations Y = 1.0542x + 4.3072 and Y =1.2623x + 4.8554, respectively and coefficient of determination R2 = 0.9976, R2 = 0.9918, respectively (Table 3 Fig. 5c). In carrier contact assay, the thyme and acetic acid treatments after 15 days of storage also were effective in the inhibition of AFB1 accumulation, where there EC50 is 4.3877, 0.002 mg/mL, respectively and EC90 is 45.7908, 4.3841 mg/mL, respectively with regression equations Y = 1.2567x + 0.4228 and Y = 0.382x + 4.8888, respectively and coefficient of determination R2 = 0.9142, R2 = 0.9845, respectively (Table 3 Fig. 5d).

Determination of aflatoxin B2

In volatile contact assay, acetic acid had the ability to completely inhibit the accumulation of AFB2 produced by isolated A. flavus after 15 days of storage in compare to the control. However thyme was able to reduce AFB2 accumulation produced from isolated A. flavus, where there EC50 is 0.8482 mg/mL and EC90 is 1.4336 mg/mL with regression equation Y = 55.6165x−11.448 and coefficient of determination R2 = 0.7527 (Table 4 and Fig. 6a). In carrier contact assay, the thyme and acetic acid treatments after 15 days of storage also had an obvious significant inhibitory activity against AFB2, where there EC50 is 7.007, 0.0894 mg/mL, respectively and EC90 is 11.5019, 0.1431 mg/mL, respectively with regression equations Y = 5.9469x−17.869 and Y = 6.2618x−7.2177, respectively and coefficient of determination R2 = 0.7872, R2 = 0.75, respectively (Table 4 and Fig. 6b).

Table 4 Heatmap showing EC50 and EC90 (ppm) of thyme and acetic acid on aflatoxin B2 produced by isolated and reference A. flavus isolates after 15 days of storage
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Fig. 6
figure 6

Correlation coefficient between the logarithm of the thyme concentrations and the probit of means a Effect of different concentrations of thyme on AFB2 content produced by isolated A. flavus under volatile contact assay. Correlation coefficient between the logarithm of the thyme and acetic acid concentrations and the probit of means of AFB2 content produced by b isolated A. flavus and c reference A. flavus under carrier contact assay

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In volatile contact assay, both thyme and acetic acid had the ability to completely inhibit AFB2 accumulation produced by reference A. flavus after 15 days of storage in compare to the control. On the other hand, in carrier contact assay, the thyme and acetic acid treatments after 15 days of storage also were effective in the inhibition of AFB2 accumulation, where there EC50 is 1.6428, 0.0024 mg/mL, respectively and EC90 is 4.6534, 0.0166 mg/mL, respectively with regression equations Y = 2.8306x−4.102 and Y = 1.4949x + 4.4433, respectively and coefficient of determination R2 = 00.9627, R2 = 0.75, respectively (Table 4 and Fig. 5c).

After 30 days of storage

After 30 days of incubation, thyme and acetic acid treatments had the ability to reduce AFB1 content produced by isolated A. flavus, where there EC50 is 0.5163, 0.0096 mg/mL, respectively and EC90 is 20.2622, 1.1595 mg/mL, respectively with regression equations Y = 0.8031x + 2.8213 and Y =0.6146x + 4.3967, respectively and coefficient of determination R2 = 0.9902, R2 = 0.9882, respectively under the condition of volatile contact assay (Table 5 and Fig. 7a). On the other hand and under carrier contact assay, thyme and acetic acid treatments also had an obvious significant inhibitory activity against AFB1, where there EC50 is 10.0956 0.0759 mg/mL, respectively and EC90 is 44.5858, 7.1775 mg/mL, respectively with regression equations Y = 1.9843x−2.9454 and Y = 0.6478x + 3.7821 (Table 5 and Fig. 7b).

Table 5 Heatmap showing EC50 and EC90 (ppm) of thyme and acetic acid on aflatoxin B1 produced by isolated and reference A. flavus isolates after 30 days of storage
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Fig. 7
figure 7

Correlation coefficient between the logarithm of the thyme and acetic acid concentrations and the probit of means of AFB1 content produced by a isolated A. flavus under volatile contact assay and b reference A. flavus under carrier contact assay c isolated A. flavus under volatile contact assay and d reference A. flavus under carrier contact assay

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While in volatile contact assay, both treatments of thyme and acetic acid possessed significant inhibitory effect on AFB1 levels produced from reference A. flavus, where there EC50 is 0.0159, 0.0209 mg/mL, respectively and EC90 is 0.1617, 0.0551 mg/mL, respectively with regression equations Y = 1.2707x + 3.4733 and Y =1.2623x + 4.8554, respectively and coefficient of determination R2 = 0. 9137, R2 = 0.9239, respectively (Table 5 and Fig. 7c). Although, treatments with thyme and acetic acid also had great effect in AFB1 accumulation inhibition under carrier contact assay, where there EC50 is 0.2608, 0.002 mg/mL respectively and EC90 is 5.992, 4.3841 mg/mL, respectively with regression equations Y = 0.3808x + 4.0799 and Y = 0.382x + 4.8888, respectively and coefficient of determination R2 = 0. 9051, R2 = 0.9845, respectively (Table 5 and Fig. 7d).

Determination of aflatoxin B2

Both thyme and acetic acid treatments using volatile and carrier contact assay had the ability to completely inhibit AFB2 accumulation produced by both isolated and reference A. flavus isolates after 30 days of storage in compare to the control.

Discussion

Grains are considered as one of the most economic important crops worldwide as well as in Egypt (Al-Ansary 2023). It’s found to be suitable for infection with a number of fungi, i.e.,, Aspergillus spp., Penicillium spp. and Fusarium spp. (Mansfield et al. 2008; Hussain et al. 2013).

In this study, corn grain samples were collected from different governorates in Egypt for detection of the presence of fungi-producing aflatoxin.

The experimental results showed that three genera of fungi (Aspergillus spp., Penicillium sp. and Fusarium spp.) were noted to be the most frequently fungi isolated from the corn grains. Out of them, three species of Aspergillus (A. flavus, A. niger and A. terreus) were isolated. The findings of this study could be suppoted by the work of Amadi and Adeniyi (2009); Goko et al. (2010); Krinjaja et al. (2013); Baka et al. (2014); Madbouly et al. (2014); Tsedaley and Adugna (2016); Shabana et al. (2022); Oldenburg et al. (2017) and Gromadzka et al. (2019) who also found that Aspergelli, Fusaria, and Penicilli were the three most prevalent post-harvest pathogenic fungi.

Results of isolation frequency in this study mentioned that Aspergillus spp. had the frequent presence and high level genera. These findings agree with the literature as Goko et al. (2010). On the other hand, Elwakil et al. (2020) stated that the most prevalent species were F. verticillioides (100%), Penicillium spp. (96.7%), A. flavus (80%), and A. niger (83.3%).

The five fungal isolates were biologically and molecularly identified using ITS region among using two primers flanking a PCR product of 632 nts. Results of molecular identification confirmed the biological identification as they were documented in GenBank under the accession numbers of OQ135182.1, OQ135183.1, OQ135184.1, OQ135185.1 and OQ135186.1 representing the strains of A. flavus, A. niger, A. terreus, F. verticillioides and T. verruculosus respectively. Results of molecular identification agreed with some investigations (Galletti et al. 2019; Gaige et al. 2020; Rahm et al. 2020; Tran et al. 2021) that revealed that maize grains were accompanied by several fungal pathogens, including A. flavus, A. niger, Penicillium spp., F. verticillioides and others, which causes various diseases in the majority of the world maize-growing areas.

At the level of inducing aflatoxin, the strain of A. flavus (OQ135182.1) was tested for aflatoxin production in fungal-infected grains compared to the standard strain.

Management of aflatoxigenic fungi was conducted using the selected strain as well as the standard. This was done by using two materials (thyme essential oil and acetic acid).

Tzortzakis and Economakis (2007); Martínez (2012) and Daniel et al. (2015) mentioned that essential oils can comprise more than 60 individual components in which up to 85% of the EO can be mainly composed of major components, while the remaining 15% is simply a trace amount (Senatore 1996). Several active compounds, including: aldehydes, phenols, and alcohols show significant bioactivities against fungi development (Jerković et al. 2019). Also acetic acid and other weak organic treatments can be successfully replace chemical treatments as they have an important role such as: preservatives in the food sector (Kang et al. 2003), reduce the development of fungi (Hassan et al. 2015), and prevent them from producing mycotoxin (Guimaries et al., 2018). These qualities inspired scientists to employ it for seed treatment as well as plant protection (Szopińska 2013; El-Saidy and El-Hai 2016; Rioux et al. 2016; Dorna et al. 2018; Escamilla et al. 2019). It was shown that acetic acid vapors at different concentrations inhibited the growth of Alternaria sp., Aspergillus spp., F. moniliforne, and Penicillium spp. as well as their ability to produce spores (Abdalla 2005; Morsy et al. 2000a, b; Luz et al. 2021).

The ability of thyme essential oil and acetic acid to inhibit the aflatoxin was detected within detection of their effect on mycelial growth, spore germination and aflatoxin production among two methods (volatile and carrier contact assay).

Acetic acid and EOs application techniques range from fumigation to combining seeds, to soaking seeds in the treatment solutions, to loading treatments on carrier in order to suppress seed-borne diseases. EOs and acetic acid should be applied practically (volatile and carrier contact applications) as inhibitors of mold growth because they are generally regarded as safe. Acetic acid and EOs have strong bioactive effect in the vapour phase, which makes them attractive as fumigants for stored product protection (Čvek et al. 2010).

Results revealed that the two materials were significantly effective on mycelial growth and spore germination inhibition. The findings of thyme essential oil effect on the toxgenic fungi could be supported by the work of Sinha et al. (1993) and Thanaboripat et al. (2004) who demonstrated that A. flavus growth in maize grain was inhibited with citronella EO. Simillarly, EOs were reported to affect the growth of F. culmorum (Sahab et al. 2014; Perczak et al. 2019) and Aspergillus flavus, A. parasiticus, A. ochraceus, and F. verticilloides (Soliman and Badeaa 2002). On the other hand, Nesci et al. (2011) observed that thyme oil couldn’t reduce the counts of Aspergillus section Flavi in stored peanut. Results presented by El-Aziz et al. (2015) indicated that the tested toxigenic fungi were sensitive to the essential oils particularly to thyme and cinnamon. These results were confirmed by many researchers, as the mycelium treated with thyme oil showed alteration in the morphology of the hyphae, which appeared collapsed and caused a reduction in mycelial growth. (Rad et al. 2011; Eweis et al. 2012). While the experimental result of acetic acid treatment was in harmony with earlier results which showed that acetic acid as post-harvest treatments directly inhibit pathogen growth, spore germination and aflatoxin production by affecting the active sites of enzymes and cellular metabolism (Arrebola et al. 2010; Bozik et al. 2017). Organic acids according to Kang et al. (2003) and Wang et al. (2021), lower the pH of the fungal cell, which requires a lot of energy to maintain intracellular pH equilibrium. Fungal growth will be constrained as a result of the significant energy consumption required for this task. Kang et al. (2003) found that when applying acetic acid, fungal respiration was inhibited and suggested that the action of acetic acid in its dissociated form would be more potentiated and exerted within the cell.

Results of aflatoxin reduction with thyme EO and acetic acid proved their significant inhibitory activity against AFB1 and AFB2 produced by both isolated A. flavus and standard A. flavus strains under volatile and carrier assays after 15 and 30 days of storage. Similarly, Abd El-Aziz et al. (2015) proved that A. flavus and A. parasiticus produced less aflatoxins (B) when exposed to studied essential oils compared to the control especially when thyme and cinnamon applied. Numerous researchers have backed up this finding (Rad et al. 2011; Eweis et al. 2012). Additionally, it has been demonstrated that the thyme oil can effectively stop fungi growth and the production of its toxins (Rasooli and Owlia 2005; Kumar et al. 2008; Ismaiel and Papenbrock 2015).

Chang et al. (2022) stated that who stated that volatile contact assay had stronger antifungal activity as a post-harvest treatment than carrier contact assay and this work agrees with our proved results.

As a conclusion, the natural products as thyme and acetic acid can be utilized to suppress fungal seed-borne pathogens and their aflatoxin production in stored maize grains.