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. 2018 Oct 19;55(12):5123–5132. doi: 10.1007/s13197-018-3458-6

Inhibitory effects of acetophenone or phenylethyl alcohol as fumigant to protect soybean seeds against two aflatoxin-producing fungi

Sawai Boukaew 1,✉, Poonsuk Prasertsan 2
PMCID: PMC6233459  PMID: 30483009

Abstract

The antifungal activity of acetophenone and phenylethyl alcohol prevents seed contamination by Aspergillus flavus TISTR 3041 and A. parasiticus TISTR 3276. Their effects on seed germination were investigated. In vitro results showed that 100 µL L−1 acetophenone completely inhibited (by 100%) growth, conidial germination, and sporulation of the two aflatoxin-producing fungi, while phenylethyl alcohol showed only weak inhibitory activity even at 1000 µL L−1. Exposure to acetophenone at 100 µL L−1 for 6 h could completely kill (100% death) both fungal strains, while phenylethyl alcohol showed much lower efficacy (53.12%). In vivo results revealed that fumigation with 100 µL L−1 acetophenone for 24 h completely controlled (100%) A. flavus TISTR 3041 on soybean seeds during a 14-day test but exhibited weak efficacy on A. parasiticus TISTR 3276 (31.77%). Phenylethyl alcohol (1000 µL L−1) demonstrated weak inhibitory effect against both strains. The two volatile compounds had no adverse effects on seed germination. SEM confirmed that acetophenone could completely inhibit conidia germination, and abnormal growth of both fungal strains was observed. Thus, acetophenone has high potential to protect soybean seeds against aflatoxin-producing fungi.

Electronic supplementary material

The online version of this article (10.1007/s13197-018-3458-6) contains supplementary material, which is available to authorized users.

Keywords: Aflatoxigenic fungi, Acetophenone, Fumigant activity, Fungal ultrastructure, Phenylethyl alcohol

Introduction

Soybean (Glycine max (L.) Merrill) is one of the most important oil seed crops and protein sources in the world. Contamination of cereal and grains by fungi is a chronic problem in the warm and humid warehouses of tropical countries. Various microorganisms, especially Aspergillus flavus and A. parasiticus, which are the most common spoilage fungi in food and stored grains, are known to produce aflatoxins (Ng’ang’a et al. 2016). Protection against these is commonly pursued by fumigation. The fumigants can be acquired from microorganisms or from natural compounds (Svircev et al. 2007).

In recent years, synthetic pesticides have been replaced by other types of volatile compounds in the control of food spoilage or food-borne pathogens (Li et al. 2010; Wang et al. 2013; You et al. 2015; Zheng et al. 2013). Volatile compounds that are active against Penicillium italicum include dimethyl disulfide, dimethyl trisulfide, d-limonene, aromadendrene, isoledene, caryophyllene, phenylethyl alcohol, and acetophenone (Li et al. 2010). There are reports on the efficacy of isoamyl acetate, isoamyl alcohol, and phenethyl alcohol against A. brasiliensis (Ando et al. 2012), phenylethyl alcohol, and α-terpineol against A. flavus NKDW-7 (Prakash et al. 2015), heneicosane, 2-nonanone, 2-decanone, 2-methylpyrazine, heptadecane, diethyl phthalate, β-benzene ethanamine, BHT, gentisic acid, thymol, and n-hexadecanoic acid against Colletotrichum gloeosporioides (Zheng et al. 2013), and 2,4-di-tertbutylphenol against Phytophthora capsici (Sang and Kim 2012). The volatile compounds against F. oxysporum include acetophenone and benzyl alcohol (Raza et al. 2015), phenylethyl alcohol, 1H-indene, 2,3-dihydro-4,7-dimethyl, biphenyl, methyl N-(1H-benzimidazol-2-yl) carbamate (Yuelian and Qingfang 2013), and 1-phenylbut-3-ene-2-ol (Kavitha et al. 2010).

Several compounds derived from microorganisms or plant essential oils, including acetophenone and phenylethyl alcohol, have shown remarkable antimicrobial activities in previous studies (Li et al. 2010; Naznin et al. 2014; Prakash et al. 2015). They chemically belong to ketones or alcohols and are known as antimicrobial agents. They possess antifungal activity against anthracnose pathogen (Colletotrichum spp.) on chili (Boukaew et al. 2018).

Microorganisms that are sources of volatile compounds include Muscodor albus (Corcuff et al. 2011), Laetiporus sulphureus (Petrovic et al. 2013), Acremonium sp. (Yuelian and Qingfang 2013), Arabidopsis thaliana (Naznin et al. 2014), Aureobasidium pullulans (Francesco et al. 2015), Paenibacillus polymyxa WR-2 (Raza et al. 2015), Daldinia cf. concentric (Liarzi et al. 2016), Candida intermedia (Huang et al. 2011), Nocardia levis MK-VL_113 (Kavitha et al. 2010), Streptomyces philanthi RM-1-138 (Boukaew et al. 2013), S. platensis F-1 (Wan et al. 2008), and S. globisporus JK-1 (Li et al. 2010).

Essential oils containing acetophenone and phenylethyl alcohol can be obtained from several plants such as Angelica archangelica (Prakash et al. 2015) and Premna integrifolia Linn (Rahman et al. 2016). Volatile acetophenone and phenylethyl alcohol at 100 µL L−1 had an inhibitory effect on the disease incidence of P. italicum on Shatang Mandarin (Li et al. 2010). Phenylethyl alcohol at 2.0 µL mL−1 added to solid media could inhibit (by 100%) A. flavus NKDW-7 (Prakash et al. 2015).

To date, the antifungal activities of acetophenone and phenylethyl alcohol against food spoilage fungi have not been extensively investigated. The main purposes of this study were (1) to investigate the antifungal activities of acetophenone and phenylethyl alcohol on the suppression of mycelial growth, conidial germination, and sporulation of the two aflatoxin-producing fungi through fumigation, (2) to evaluate the efficacy of the two volatile compounds in controlling soybean seed contamination by aflatoxin-producing fungi and their effects on seed germination, and (3) to study the mode of action of acetophenone on the two aflatoxin-producing fungi.

Materials and methods

Microorganisms and inoculum production

Aflatoxin producing fungi Aspergillus parasiticus TISTR 3276 and A. flavus TISTR 3041 (Sangmanee and Hongpattarakere 2014) were obtained from the Thailand Institute of Scientific and Technological Research (TISTR), Phathumthani, Thailand. They were grown on potato dextrose agar (PDA; 39 g L−1; Difco Laboratory) at 4 °C. Spore inocula of pathogenic fungi were collected in 5 mL of water from 7-day-old cultures. The spore count was performed using a hemacytometer before dilution with distilled sterilized water to the required concentration.

Technical grade pure volatile compounds

Pure commercial compounds (technical grade) of acetophenone (Fluka, for GC, ≥ 99.5%) and phenylethyl alcohol (Sigma–Aldrich ≥ 99%, FCC, Kosher, FG) were used in this study.

Evaluation of antifungal activity of acetophenone and phenylethyl alcohol on mycelial growth and conidial germination of the two aflatoxin producing fungi

The two volatile compounds were tested for antifungal activity on mycelial growth and conidial germination of two aflatoxin-producing fungi, A. parasiticus TISTR 3276 and A. flavus TISTR 3041, on PDA using an antifungal bioassay (Li et al. 2012). Four small Petri dishes (50 mm diameter × 15 mm height) were placed inside a larger Petri dish (140 mm diameter × 20 mm height with 0.5 L inner volume). Three smaller dishes contained 5 mL of PDA inoculated with a 0.5 cm diameter fungal plug from the periphery of an actively grown culture of aflatoxin-producing fungi, while the fourth dish contained a piece of autoclaved filter paper (Whatman®), to which a volatile compound was added at the concentrations 1, 10, 100, and 1000 µL L−1 (droplet to filter paper in an enclosed Petri dish system). The Petri dishes were covered with a large lid, sealed with Parafilm M® and incubated at 28 ± 2 °C for 5 days. Equivalent amounts of sterile distilled water were used as a control. After the diameters of the colonies on the three plates were measured, the inhibition percentage was calculated using the following formula.

Inhibition%=[(R1-R2)/R1]×100

where R1 = mycelial growth of the aflatoxin-producing fungi alone on PDA (control) and R2 = mycelial growth of the aflatoxin-producing fungi on PDA fumigated with acetophenone or phenylethyl alcohol.

For the conidial germination in this assay, four small Petri dishes (50 mm diameter × 15 mm height) were placed inside a larger Petri dish (140 mm diameter × 20 mm height with a 0.5 L inner volume). Fifty µL of spore inoculum (1 × 104 spore mL−1) of the aflatoxin-producing fungi were spread on three of the smaller dishes containing 5 mL of PDA and the fourth dish was filled with the gaseous components of each volatile compound that varied in concentrations of 1, 10, 100, and 1000 µL L−1 (droplet to filter paper in an enclosed Petri dish system). Equivalent amounts of sterile distilled water were used as a control. After incubation at 28 ± 2 °C for 24 h, the conidial germination on the smaller plates was observed and the inhibition of conidial germination was determined by the previously mentioned formula. There were three replicates for each treatment.

Evaluation of antifungal activity of acetophenone and phenylethyl alcohol on sporulation of the two aflatoxin-producing fungi on PDA plates and on soybean seeds

The effects of the two compounds on sporulation of the aflatoxin-producing fungi on PDA plates and on soybean seeds were investigated using an antifungal bioassay. In the PDA plate experiment, 50 µL of spore inoculum (1 × 104 spore mL−1) of the aflatoxin-producing fungi were spread on each of the three smaller dishes containing 5 mL of PDA and the fourth dish was filled with the gaseous components of each volatile compound matching liquid concentration 1, 10, 100, and 1000 µL L−1 (droplet to filter paper in an enclosed Petri dish system). Equivalent amounts of sterile distilled water were used as a control. After incubation at 28 ± 2 °C for 5 days, the total number of conidia per plate was assessed by mixing with sterile water in a serial dilution (1 × 103) after which the number of conidia was counted under a compound microscope.

Soybean seeds were prepared by soaking in 100 mL of distilled water for 5 h and then autoclaving at 121 °C for 15 min. Five soybean seeds were transferred onto each of the three smaller dishes (total of 15 seeds), and then a 50-µL spore inoculum of aflatoxin-producing fungi at 1 × 104 spore mL−1 was spread on each soybean seed while the fourth dish contained gaseous acetophenone or phenylethyl alcohol matching liquid concentration 1, 10, 100, and 1000 µL L−1 (droplet to filter paper in an enclosed Petri dish system). Equivalent amounts of sterile distilled water were used as a control. After incubation at 28 ± 2 °C for 5 days, the total number of conidia per soybean seed was assessed by mixing the infected soybean seeds with sterile water in a serial dilution (1 × 103) before counting the number of conidia under a compound microscope. There were three replicates for each treatment.

Fungicidal kinetics of acetophenone and phenylethyl alcohol against conidia of the two aflatoxin-producing fungi

To obtain the fungicidal kinetics of the two compounds, 50-µL spore inoculum (1 × 104 spore mL−1) of each of the two aflatoxin-producing fungi was spread on PDA. The cultures were fumigated with 100 µL L−1 of acetophenone or phenylethyl alcohol for 0, 1, 3, 6, 9, and 12 h. Equivalent amounts of sterile distilled water were used as a control. The cultures were removed after fumigation and incubated at 28 ± 2 °C for 48 h. The fungicidal kinetics were determined based on the total number of conidia alive on the PDA plate (Li et al. 2013). For each treatment, there were three replicates.

Effects of acetophenone and phenylethyl alcohol in protecting soybean seeds against contamination by the two aflatoxin-producing fungi and their effects on seed germination

The protective abilities of the two volatile compounds on soybeans against the two aflatoxin-producing fungi were evaluated in an antifungal bioassay. Fifteen soybean seeds were placed on three smaller dishes (five soybean seeds per Petri dish), then 50 µL of spore inoculum (1 × 104 spore mL−1) of the aflatoxin-producing fungi were spread on each soybean seed and on the fourth dish that contained the various concentrations of acetophenone or phenylethyl alcohol (1, 10, 100, and 1000 µL L−1, droplet on filter paper in an enclosed Petri dish system). Equivalent amounts of sterile distilled water were used as a control. The Petri dishes were covered with a large lid, sealed with Parafilm M®, and incubated at 28 ± 2 °C for 5 days. Then, the soybean seeds in the dishes were individually examined for infection under a stereomicroscope (Sumalan et al. 2013) and the protection of seeds was quantified based on infected seed counts as: Percentage of protection = [{(Control-treatment)/Control} × 100]. For each treatment, there were three replicates and the experiment was conducted three times.

The influence of fumigation of acetophenone and phenylethyl alcohol on soybean seed germination was investigated. Peanut and maize seeds were also investigated for comparison. One hundred seeds were fumigated with 100 µL L−1 of each volatile compound for 24 h. They were placed on moist paper and kept in a plastic box (120 mm width × 170 mm length × 68 mm height with 1 L inner volume), which was incubated at 28 ± 2 °C for 10 days. Distilled water was used as the control treatment. The toxicity to seed germination is reported as percentage of seed germination, along with stem and root lengths after the incubation.

Effects of the fumigation period with acetophenone to control the two aflatoxin-producing fungi on soybean seeds

The efficacy by the fumigation period with acetophenone to control the two aflatoxin-producing fungi on soybean seeds was investigated using an antifungal bioassay. Five soybean seeds were placed on each of three small dishes, then a 50-µL spore inoculum (1 × 104 spore mL−1) of the two aflatoxin-producing fungi was spread on each soybean seed while the fourth dish contained acetophenone at 100 µL L−1 (the minimum dose that completely controlled soybean seed infection). Equivalent amounts of sterile distilled water were used as a control. The four small dishes were placed in a large Petri dish, which was covered with a lid, sealed with Parafilm M®, and fumigated for 0, 6, 12, or 24 h. After 14 days of incubation at 28 ± 2 °C, the fungal colony growth on the soybean seeds was examined under a stereomicroscope. The seed contamination index (SCI) was determined using the formula (Doolotkeldieva 2010): % SCI = [(Number of contaminated seeds/Total number of seeds) × 100]. For each treatment there were three replicates (five soybean seeds each) and the experiment was conducted three times.

Mode of action of acetophenone against the two aflatoxin-producing fungi by scanning electron microscopy (SEM)

The biomass of A. flavus TISTR 3041 and A. parasiticus TISTR 3276 obtained from the 5-day-old cultures grown on PDA with and without acetophenone treatment were subjected to scanning electron microscopy (SEM) to determine the possible mode of action of acetophenone. The fungal biomass preparation and the SEM observation followed the procedure described by Boukaew and Prasertsan (2014). The mode of action of acetophenone was assessed from the ultrastructure alterations of the two fumigated fungal strains.

Statistical analysis

Duncan’s Multiple Range Test (DMRT) was used at 95% statistical significance (P < 0.05). The Statistical Package for the Social Sciences (SPSS) software version 15 for Windows was used to analyze the data.

Results

Evaluation of the antifungal activity of acetophenone and phenylethyl alcohol on mycelial growth and conidial germination of the two aflatoxin-producing fungi

The antifungal activities of acetophenone on mycelial growth and conidial germination of A. flavus TISTR 3041 and A. parasiticus TISTR 3276 were stronger than those of phenylethyl alcohol (Fig. 1). Significant reductions in fungal growth and conidial germination were observed in a dose-dependent manner (P < 0.05). At 100 µL L−1 fumigation, only acetophenone completely inhibited (100%) the fungal growth (Fig. 1a) and conidial germination (Fig. 1c) of the two aflatoxin-producing fungi. Phenylethyl alcohol showed only weak inhibitory activities on the fungal growth (Fig. 1b) and the conidial germination (Fig. 1d) even at 1000 µL L−1.

Fig. 1.

Fig. 1

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Effects of different concentrations of acetophenone (a, c) and phenylethyl alcohol (b, d) on the mycelial growth and conidial germination inhibition of A. flavus TISTR 3041 and A. parasiticus TISTR 3276 on PDA plates. Different letters above bars indicated significant differences (ANOVA, P < 0.05; Duncan’s multiple range test)

Evaluation of antifungal activity of acetophenone and phenylethyl alcohol on sporulation of the two aflatoxin-producing fungi on PDA plates and on soybean seeds

Figure 2 indicates that sporulation inhibition depended on the concentration of acetophenone or phenylethyl alcohol. The two compounds only delayed sporulation at low concentrations and complete inhibition was achieved at higher concentrations. At 100 µL L−1 fumigation dose, only acetophenone completely inhibited (100%) sporulation of both A. flavus TISTR 3041 and A. parasiticus TISTR 3276 on PDA plates (Fig. 2a) and on soybean seeds (Fig. 2c). Phenylethyl alcohol completely inhibited (100%) sporulation only of A. flavus TISTR 3041 at the concentration of 1000 µL L−1 on the PDA plate (Fig. 2b) and at 100 µL L−1 on soybean seeds (Fig. 2d).

Fig. 2.

Fig. 2

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Effects of different concentrations of acetophenone and phenylethyl alcohol on sporulation inhibition of A. flavus TISTR 3041 and A. parasiticus TISTR 3276 on PDA plates (a, b) and on soybean seeds (c, d). Different letters above bars indicate significant differences (ANOVA, P < 0.05; Duncan’s multiple range test)

Fungicidal kinetics of acetophenone and phenylethyl alcohol against conidia of the two aflatoxin-producing fungi

Fungicidal kinetics of acetophenone and phenylethyl alcohol revealed that more than 50% death of the two aflatoxin-producing fungi occurred at 3 h and 6 h fumigation, respectively (Fig. 3). A significant reduction of conidia germination appeared as exposure time increased (P < 0.05). The conidia of the two aflatoxin-producing fungi were completely killed (100% death) at 6 h of fumigation with acetophenone (Fig. 3a). However, phenylethyl alcohol showed only weak antifungal activity (51.53% and 53.12% death of A. flavus TISTR 3041 and A. parasiticus TISTR 3276, respectively) (Fig. 3b).

Fig. 3.

Fig. 3

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Growth inhibition kinetics of the conidia germination of the tested A. flavus TISTR 3041 and A. parasiticus TISTR 3276 by acetophenone (a) and phenylethyl alcohol (b) fumigation and incubated at 28 ± 2 °C. Different letters above bars indicate significant differences (ANOVA, P < 0.05; Duncan’s multiple range test)

Effects of acetophenone and phenylethyl alcohol on protection of soybean seeds against contamination by the two aflatoxin-producing fungi and their effects on seed germination

The effects of acetophenone and phenylethyl alcohol fumigation in protecting soybean seeds from contamination by A. flavus TISTR 3041 and A. parasiticus TISTR 3276 are shown in Fig. 4. Obviously, the protection of soybean seeds against contamination depended on the concentration of either volatile compound. At 100 µL L−1 acetophenone fumigation dose, total protection of soybean seeds was achieved (100%) (P < 0.05) (Fig. 4a). Therefore, the minimum effective dose of acetophenone on soybean seeds was 100 µL L−1. In contrast, phenylethyl alcohol provided no inhibition of either fungal strain on soybean seeds at any concentration tested (Fig. 4b). Thus, further studies on the effects of the fumigation period on the two aflatoxin-producing fungi and the mode of action were performed with acetophenone only.

Fig. 4.

Fig. 4

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Effects of different concentrations of acetophenone (A) and phenylethyl alcohol (B) fumigation on the protection of soybean seeds inoculated with A. flavus TISTR 3041 and A. parasiticus TISTR 3276 after incubation for 5 days at 28 ± 2 °C. Different letters above bars indicated significant differences (ANOVA, P < 0.05; Duncan’s multiple range test)

The effects of acetophenone and phenylethyl alcohol on seed germination were tested on soybean, peanut, and maize seeds. The results revealed that the two volatile compounds had no significant adverse effects on seed germination or on stem and root length of the seeds compared to the control (P < 0.05) (Table S1; Supplementary data). This indicates that acetophenone and phenylethyl alcohol fumigation can be applied to control spoilage microorganisms during seed storage.

Effects of the fumigation period with acetophenone on control of the two aflatoxin-producing fungi on soybean seeds

The efficacy of acetophenone was tested during a 24-h fumigation period for control of A. flavus TISTR 3041 and A. parasiticus TISTR 3276 on soybean seeds (Fig. 5). Fumigation using acetophenone for 24 h significantly reduced the soybean seed contamination by the two aflatoxin-producing fungi (P < 0.05), whereas the fumigation periods of 6 h and 12 h had no preventive effect (100% SCI) compared to the control. Interestingly, the 24-h fumigation treatment exhibited significant reduction and completely controlled A. flavus TISTR 3041 infection on soybean seeds during the 14-day test (P < 0.05). A. parasiticus TISTR 3276 appeared to be more resistant to acetophenone fumigation than A. flavus TISTR 3041.

Fig. 5.

Fig. 5

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Duration of fumigation with acetophenone of soybean inoculated with A. flavus TISTR 3041 and A. parasiticus TISTR 3276 after incubation for 14 days at 28 ± 2 °C. Different letters above bars indicate significant differences (ANOVA, P < 0.05; Duncan’s multiple range test). Seed contamination index (SCI) was calculated as SCI % = [(Number of contaminated seeds/Total number of seeds) × 100]

Mode of action of acetophenone on the two fumigated aflatoxin-producing fungi assessed by scanning electron microscopy (SEM)

Ultrastructure alterations of the two aflatoxin-producing fungi 5 days after treatment with acetophenone are seen in the SEM images of Fig. 6. In the control, there were no morphological changes in either A. flavus TISTR 3041 (Fig. 6a) or A. parasiticus TISTR 3276 (Fig. 6d, e). The conidiophores appeared globular and formed spherical heads containing swollen conidia with normal appearing smooth cell walls. The morphological changes were evident after exposure to acetophenone in both fungal strains. Exposure to acetophenone at 100 µL L−1 completely inhibited conidia germination of A. flavus TISTR 3041 (Fig. 6b, c), whereas damage and morphological alterations of conidia were exhibited along with abnormal growth of A. parasiticus TISTR 3276 (Fig. 6f). Markedly shriveled and crinkled cell walls and flattened hyphae of the two aflatoxin-producing fungi were also evident in the images (Fig. 6b, c, and f).

Fig. 6.

Fig. 6

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Scanning electron micrographs (SEM) of normal hypha, conidiophore, and spores of A. flavus TISTR 3041 (a) and A. parasiticus TISTR 3276 (d, e) without exposure to acetophenone (the control) after incubation at 28 ± 2 °C for 5 days. Mycelia treated with acetophenone at 100 µL mL−1 of A. flavus TISTR 3041(b, c) and A. parasiticus TISTR 3276 (f) after incubation at 28 ± 2 °C for 5 days. Red circle indicates the size of the vesicle of A. parasiticus TISTR 3276 exposed to acetophenone (e) and the damage can be compared to control without exposure to acetophenone (f)

Discussion

The efficiency of acetophenone and phenylethyl alcohol as volatile antimicrobial agents has been reported in various studies (Huang et al. 2011; Li et al. 2010; Naznin et al. 2014; Prakash et al. 2015; Yuelian and Qingfang 2013). However, the antifungal activities of the two volatile compounds against aflatoxin producing A. flavus and A. parasiticus have not been extensively investigated. In the present investigation, only fumigation with acetophenone (100 µL L−1) completely inhibited (100%) mycelial growth and sporulation as well as conidial germination of the two aflatoxin-producing fungi on PDA and on soybean seeds. This effective concentration of acetophenone (100 µL L−1) is identical to that inhibiting P. italicum in an in vitro test (Li et al. 2010). In addition, acetophenone-derived Mannich bases have the potential for developing novel antifungal agents against dermatophytes (Gul et al. 2001, 2002). Phenylethyl alcohol showed only weak antifungal activity in the in vitro test, and no efficacy against aflatoxin-producing fungi on soybean seeds even at 1000 µL L−1 in this study, or at 100,000 µL L−1 in the prior study by Li et al. (2010). However, the mycelial growth of A. flavus NKDW-7 was completely inhibited (100%) using phenylethyl alcohol at 2.0 µL mL−1 (2000 µL L−1) (Prakash et al. 2015). The effective dose of phenylethyl alcohol varies by microorganism, being 50 µg mL−1 (50,000 µL L−1) for F. oxysporum f. sp. cubense (Yuelian and Qingfang 2013).

The spores are important to survival and spread of aflatoxigenic fungi (Rabea et al. 2003). Both acetophenone and phenylethyl alcohol had strong antifungal activity against A. flavus TISTR 3041 and A. parasiticus TISTR 3276. More than 50% of conidia were killed in all groups at an exposure time of 3 h for acetophenone and at 6 h for phenylethyl alcohol. In addition, the antifungal activity gradually strengthened with exposure time. Exposure for 6 h of the conidia to acetophenone could completely kill 100% of the conidia of the two aflatoxigenic fungi, while phenylethyl alcohol failed to kill the conidia completely under the same conditions.

Although the in vitro tests of acetophenone and phenylethyl alcohol are important first steps to check their antifungal potential, in vivo tests are still needed to confirm the results. Acetophenone fumigation on soybean seeds could decrease the growth of A. flavus TISTR 3041 and A. parasiticus TISTR 3276 considerably with 100% inhibition at 100 µL L−1. Therefore, at this effective concentration of acetophenone, soybean seeds could be significantly protected from infection by the two aflatoxin-producing fungi (P < 0.05), while the effective dose of the phenylethyl alcohol was 1000 µL L−1. Therefore, the minimum effective dose of acetophenone at 100 µL L−1 was 10 times lower than the dose of 1000 µL L−1 used to control P. italicum in the in vivo test reported by Li et al. (2010). This may be due to the different treatment methods and different types of organism used.

In general, some compounds produced by microorganisms (Boukaew et al. 2011) or plant essential oils (Kordali et al. 2009; Qiming et al. 2006; Young and Bush 2009) have been reported to inhibit seed germination. In this study, the fungicides acetophenone and phenylethyl alcohol were found to be effective for controlling the two aflatoxin-producing fungi in colonizing soybean seeds during 14 days of storage. In addition, the two volatile compounds at a concentration of 100 µL L−1 had no adverse effects on seed germination, or early stem length or root length of the soybean, compared to the control.

According to the in vivo assay, increasing the fumigation period of acetophenone to 24 h resulted in a significant reduction in soybean contamination, by potent fungicidal effects (P < 0.05). Therefore, fumigation with 100 µL L−1 of acetophenone for 24 h provided complete (100%) protection of the soybean seeds from infection by A. flavus TISTR 3041 during the 14 days of incubation. A. parasiticus TISTR 3276 appeared to have more resistance to acetophenone fumigation than A. flavus TISTR 3041 at the concentrations tested. The minimum fumigation period of acetophenone for protecting soybean seeds was 24 h. Exposure of the seeds to acetophenone and phenylethyl alcohol exhibited powerful antifungal activity against the two aflatoxin-producing fungi. Similar results have been obtained by several researchers with soybeans and other crops (Chavan and Padule 2005; Nasir 2003).

Results from in vitro and in vivo testing showed that acetophenone completely inhibited (100%) growth, conidial germination, and sporulation of A. flavus TISTR 3041 and A. parasiticus TISTR 3276 at a concentration of 100 µL L−1. The data and SEM results confirmed that acetophenone could damage the hyphae, conidiophores, and spore germination of the two aflatoxin-producing fungi. Some chemical compounds including natural cinnamaldehyde, citral, and eugenol have been reported to damage the hyphae, conidiophores, and spores of Aspergillus spp. (Hua et al. 2014).

In conclusion, the present study demonstrated that acetophenone exhibits higher efficacy than phenylethyl alcohol against A. flavus TISTR 3041 and A. parasiticus TISTR 3276 in both in vitro tests and on soybean seeds. There were no adverse effects on soybean seed germination, so acetophenone in particular could potentially be an effective fumigant to prevent fungal spoilage of seeds used as food.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 16 kb) (16.2KB, docx)

Acknowledgements

This research work was financially supported by the Agricultural Research Development Agency (Public Organization) (PRP5905021490) and Thailand Research Fund (RTA6080010).

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