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Microbiology and Biotechnology Letters

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Food Microbiology (FM)  |  Food Borne Pathogens and Food Safety

Microbiol. Biotechnol. Lett. 2022; 50(2): 202-210

https://doi.org/10.48022/mbl.2202.02004

Received: February 7, 2022; Revised: April 4, 2022; Accepted: April 25, 2022

Screening of Volatile Organic Compound-Producing Yeasts and Yeast-Like Fungi against Aflatoxigenic Aspergillus flavus

Rujikan Nasanit1*, Sopin Jaibangyang1, Tikamporn Onwibunsiri2, and Pannida Khunnamwong3,4*

1Department of Biotechnology, Faculty of Engineering and Industrial Technology, 2Department of Food Technology, Faculty of Engineering and Industrial Technology, Silpakorn University, Sanamchandra Palace Campus, Nakhon Pathom 73000, Thailand
3Department of Microbiology, Faculty of Science, Kasetsart University, Jatujak, Bangkok 10900, Thailand
4Biodiversity Center Kasetsart University (BDCKU), Bangkok 10900, Thailand

Correspondence to :
Rujikan Nasanit,      nasanit_r@su.ac.th
Pannida Khunnamwong,     fscipik@ku.ac.th

Abstract

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Aflatoxin contamination in rice has been documented in a number of studies, and has a high incidence in Asian countries, and as such, there has been a growing interest in alternative biocontrol strategies to address this issue. In this study, 147 strains of yeasts and yeast-like fungi were screened for their potential to produce volatile organic compounds (VOCs) active against Aspergillus flavus strains that produce aflatoxin B1 (AFB1). Five strains within four different genera showed greater than 50% growth inhibition of some strains of A. flavus. These were Anthracocystis sp. DMKU-PAL124, Aureobasidium sp. DMKU-PAL120, Aureobasidium sp. DMKU-PAL144, Rhodotorula sp. DMKU-PAL99, and Solicococcus keelungensis DMKU-PAL84. VOCs produced by these microorganisms ranged from 4 to 14 compounds and included alcohols, alkenes, aromatics, esters and furans. The major VOCs produced by the closely related Aureobasidium strains were found to bedistinct. Moreover, 2-phenylethanol was the most abundant compound generated by Aureobasidium sp. DMKU-PAL120, while methyl benzeneacetate was the major compound emitted from Aureobasidium sp. DMKU-PAL144. On the other hand, 2-methyl-1-butanol and 3-methyl-1-butanol were significant compounds produced by the other three genera. These antagonists apparently inhibited A. flavus sporulation and mycelial development. Additionally, the reduction of the AFB1 in the fungal-contaminated rice grains was observed after co-incubation with these VOC-producing strains and ranged from 37.7 ± 8.3% to 60.3 ± 3.4%. Our findings suggest that these same microorganisms are promising biological control agents for use against aflatoxin-producing fungi in rice and other agricultural products.

Keywords: Aflatoxin B1, Aspergillus flavus, biological control, volatile organic compounds

Graphical Abstract


Introduction

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Mycotoxins are toxic chemicals with low molecular weight that are produced by filamentous fungi as secondary metabolites. Aflatoxins are a family of mycotoxins produced by some strains of Aspergillus spp. such as A. flavus, A. parasiticus and A. nomius [1]. Aflatoxins are estimated to be responsible for the destruction of 25% or more of global food each year [2, 3]. Consumption of aflatoxin-contaminated food products can lead to disease and death in humans and other animals. Moreover, AFB1 is more hazardous than other aflatoxins and can cause cancer, with the majority of cases occurring in the liver [4]. Over 89 percent of the world's rice is produced in Asia, with China and India accounting for 55 percent of global output [5]. Rice is typically grown in subtropical climates with hot, humid temperatures that encourage the growth of fungi and the production of toxins. Aflatoxin contamination in rice has been documented in a number of studies, with a significant prevalence in Asian countries [6].

Fungicides are often used to control fungal infections in agricultural products. However, residues of these substances may persist in final products, posing a health risk to consumers. Consequently, interest in an alternative biocontrol technique to address these concerns has increased during the last decade. Numerous yeast strains have been shown to suppress aflatoxin-producing fungi in previous studies e.g. Candida parapsilosis, C. nivariensis, Kwoniella heveanensis, and Pichia anomala [7-10]. Most of these investigations focused on direct application of yeast cultures to agricultural products. However, when it comes to post-harvest treatment of commodities such as rice, maize, or wheat, it is necessary to consider the moisture content [9]. To circumvent this constraint, VOCs produced by microorganisms that may act against phytopathogenic fungi have been extensively investigated. Only a few studies on prospective strains of VOC-producing yeasts against aflatoxinproducing fungi have been reported [8-10]. Thus, the objectives of this study were to identify promising strains of yeast or yeast-like fungi that produce antagonistic VOCs against a variety of aflatoxin-producing A. flavus strains and to assess the efficacy of these strains in reducing AFB1 in fungal-contaminated rice grains.

Materials and Methods

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Microorganisms used in this study

A total of 147 strains of yeast and yeast-like fungi were obtained from the Khunnamwong laboratory, Department of Microbiology, Faculty of Science, Kasetsart University, Thailand. Aspergillus flavus A39 was provided by Dr. Amara Chinaphuti, Department of Agriculture (DOA), Thailand. This fungal strain was used as the main strain in screening and evaluating the ability of antifungal VOC-producing strains in this study due to its high production of AFB1. Five other strains of Aspergillus flavus that produce aflatoxins were obtained from the Kasetsart University Research and Development Institute (KURDI). These included A. flavus CH016, CH033, CH271, CH307, and CH464. Yeasts and filamentous fungi were grown in yeast malt extract (YM) and potato dextrose agar (PDA) (Titan Biotech LTD, India), respectively, at 28 ± 2℃.

Primary screening of VOC-producing strains against A. flavus A39

The microbial strains were first evaluated for their ability to produce anti-fungal VOCs by the dual culture method on a 2-partition Petri dish containing PDA. Briefly, yeasts and yeast-like fungi were grown in YM agar for 48 h. Each of these cultures was streaked on one side of a PDA partition-Petri dish and then incubated at 28 ± 2℃ for 48 h. A 5 mm diameter plug of 7 d-old A. flavus A39 mycelium was then inoculated onto the other side of the dish. As a control experiment, only fungal inoculation was performed. The dish was sealed and incubated in the dark at 28 ± 2℃ for 7 days. This experiment was performed in duplicate. The diameter of the fungal colony was measured, and the percentage of fungal growth reduction was calculated relative to the control experiment. The microbial strains that reduced mycelium growth by more than 10% in this step were selected for further study.

Selection of effective VOC-producing strains against A. flavus growth

The selected strains were further screened for their effectiveness against A. flavus using a face-to-face plate assay. These cultures were grown in yeast extract peptone dextrose (YPD, Y(1% yeast extract (Himedia, India), 2% bacteriological peptone (Himedia, India), and 2% dextrose (SRL, India)) broth for 24 h at 28 ± 2℃ in a shaking incubator at 150 rpm. Cell suspensions of 107 cells/ml were prepared in YPD broth. One hundred microliters of the cell suspension were spread onto PDA agar plates. The cultures were incubated at 28 ± 2℃ for 48 h. After incubation, the cover lid was replaced by a PDA agar plate that had previously been spotted with 20 μl of A. flavus spore suspension at 106 spores/ml. A control experiment was done without a yeast or yeast-like culture. The plates were sealed and incubated in the dark for 7 days at 28 ± 2℃. The experiment was performed in triplicate. The efficacy of antagonistic VOC-producing yeasts was revealed by the relative reduction of fungal growth compared to the control experiment.

In vivo AFB1 reduction by VOC-producing strains

Rice grains were sterilized by autoclaving at 121℃, 15 psi for 15 min. The grains (10 g) were placed on one side of a 2-partition Petri dish. A fungal spore suspension (1 ml of 106 spores/ml) of A. flavus A39 was inoculated evenly onto the seeds. A cotton pad was put on the other side of the Petri dish, and 5 ml of sterile distilled water was pipetted onto the pad to produce moist condition for fungal growth. The lid was then replaced by a PDA plate containing yeast culture prepared the same way as described above. The plate was sealed and incubated at 28 ± 2℃ for 14 days in the dark. The control experiment was conducted without inoculation of a test culture. The experiment was performed in triplicate. AFB1 was extracted from 10 g of ground rice samples using 50 ml of 70% methanol. The mixture was shaken for 30 min and then filtered through Whatman No.4 filter paper. The AFB1 content in the sample was analysed using the ScreenEZ® Aflatoxin ELISA Test Kit (Siam Inter Quality, Thailand), according to the manufacturer’s instructions.

Scanning electron microscopy (SEM)

SEM was used to visualize the A. flavus structure on the rice grain surface after co-incubation with the VOCproducing strains. The grain samples were fixed with 2.5% glutaraldehyde and dehydrated with increasing ethanol concentration (20, 30, 50, 70, 90, and 100%). The samples were dried using a critical point dryer (CPD) and then coated with gold before being subjected to scanning electron microscopy (Hitachi SU8020, Hitachi High-Tech, Japan).

Identification of VOCs

Microbial strains (100 μl of 107 cells/ml cell suspension) were separately inoculated in a 10 ml vial that contained 3 ml of PDA. After incubation at 28 ± 2℃ in the dark for 48 h, the VOCs in the vial head space were analysed using solid-phase microextraction (SPME) (50/30 divinylbenzene/carboxen to polydimethylsiloxane) (Supelco, USA) and a gas chromatography/mass spectrometry (GC/MS) instrument (Agilent 7890A with 5975C inert MSD, Agilent Technologies, USA). The headspace samples were trapped with SPME at 30℃ for 45 min and subjected to the GC gas chromatography equipped with a DB-wax capillary column (30 m × 0.25 mm, 0.25 μm film thickness) (Supelco). Helium was used as a carrier gas. The column temperature was maintained at 40℃ for 2 min, increased to 200℃ at 5℃/ min, and then stabilized for 30 min for desorption. All mass spectra were identified based on the data system library [National Institute of Standards and Technology (NIST) 08]. The PDA vial without microbial culture underwent the same conditions and was used as a blank sample. The experiment was conducted in duplicate.

Data analysis

A one-way ANOVA with Tukey’s multiple comparisons test was performed using IBM SPSS Statistics for Windows, Version 20.0 to compare the efficacy of the VOC-producing strains on fungal growth and AFB1 reduction in rice grains. Significant differences were considered when the p-value was less than or equal to 0.05.

Results

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VOC-producing strains

The primary screening of the yeasts and yeast-like fungi that produced anti-fungal VOCs showed that 20 out of 147 strains isolated from pineapple leaves could reduce the mycelial growth of A. flavus A39 by more than 10%. These 20 strains were selected for further screening by the face-to-face method against the same A. flavus strain (Table 1). A. flavus growth was reduced by 27.3 to 63.0% by these 20 yeast and yeast-like fungi. The top 10 strains that inhibited A. flavus A39 growth by greater than 40% were then selected as promising VOCproducing strains against A. flavus. When tested against the other five strains of A. flavus, these antagonistic strains showed variable efficacy, from less than 5% to approximately 50%. Five strains, including Aureobasidium sp. DMKU-PAL120, Aureobasidium sp. DMKU-PAL144, Anthracocystis sp. DMKU-PAL124, Rhodotorula sp. DMKU-PAL99, and Solicoccozyma keelungensis DMKUPAL84, were selected for further study due to their ability to inhibit the growth of some A. flavus strains by more than 50%.

Table 1 . Reduction of fungal growth after incubation with the selected VOC-producing strains for 7 days using the face-toface method.

Strains1Reduction of fungal growth (%)2
A. flavus A39A. flavus CH016A. flavus CH033A. flavus CH271A. flavus CH307A. flavus CH464
Anthracocystis sp. DMKU-PAL12463.0 ± 1.3a31.2 ± 0.5c39.0 ± 3.2bc44.8 ± 0.8a36.0 ± 1.3abc35.3 ± 4.9a
Rhodotorula sp. DMKU-PAL9959.2 ± 4.6ab33.1 ± 1.7c41.7 ± 2.7b38.2 ± 4.5ab34.7 ± 10.8abc4.8 ± 3.7b
Aureobasidium sp. DMKU-PAL14454.7 ± 2.5abc31.2 ± 5.6c19.2 ± 1.4d13.3 ± 1.7de17.8 ± 4.2d6.0 ± 5.3b
Aureobasidium sp. DMKU-PAL12052.2 ± 5.8abcd38.1 ± 3.3bc39.0 ± 1.2bc24.6 ± 2.1cd28.9 ± 3.1bc13.3 ± 10.4ab
Papiliotrema sp. DMKU-PAL8347.7 ± 8.3abcde48.5 ± 2.8ab27.8 ± 2.0cd19.9 ± 1.9cd26.2 ± 2.7bc3.5 ± 2.4b
Tremella sp. DMKU-PAL5243.3 ± 9.9bcdef37.6 ± 2.3bc27.8 ± 3.1cd31.6 ± 3.7bc25.3 ± 8.2bc2.3 ± 1.2b
Solicoccozyma keelungensis DMKU-PAL8442.0 ± 5.2bcdef52.0 ± 6.9a52.9 ± 0.8a44.4 ± 3.3a49.8 ± 3.6a34.1 ± 22.0a
Papiliotrema sp. DMKU-PAL9841.4 ± 4.5cdef34.6 ± 2.6c42.6 ± 3.2b25.0 ± 2.2cd41.3 ± 3.1ab14.5 ± 4.4ab
Meyerozyma caribbica DMKU-PAL2541.4 ± 11.8 cdef37.6 ± 5.4bc29.1 ± 5.8cd6.7 ± 0.8ef20.9 ± 7.8d2.3 ± 8.6b
Hannaella sp. DMKU-PAL5940.7 ± 5.8 cdef34.6 ± 3.9c24.2 ± 7.9d26.5 ± 9.8c23.6 ± 5.8bc15.8 ± 4.2ab
Rhodotorula sp. DMKU-PAL10939.5 ± 5.7 cdef-----
Papiliotrema sp. DMKU-PAL6938.8 ± 4.4 cdef-----
Saitozyma sp. DMKU-PAL838.2 ± 4.5 cdef-----
Goffeauzyma sp. DMKU-PAL3937.5 ± 2.8 cdef-----
Jaminaea angkorensis DMKU-PAL4436.3 ± 3.2def-----
Pseudozyma sp. DMKU-PAL6035.0 ± 3.8 def-----
Saitozyma sp. DMKU-PAL7333.7 ± 1.7 ef-----
Hannaella sp. DMKU-PAL5830.5 ± 3.5 ef-----
Hannaella sp. DMKU-PAL6229.3 ± 1.1f-----
Solicoccozyma keelungensis DMKU-PAL9027.3 ± 5.8 f-----

1Microbial strains are ordered according to their reduction of A. flavus A39 growth.

2Each value is the mean ± SE (n = 3). Different letters indicate significant difference (p < 0.05) of fungal growth reduction in the same column.

Effects of emitted VOCs on AFB1 reduction and fungal growth on contaminated rice grains

As seen in Fig. 1, the VOCs produced by these strains exhibited antagonistic activity against A. flavus, resulting in a decrease in the AFB1 content of fungal contaminated rice grains ranging between 37.7 ± 8.3% and 60.3 ± 3.4%. Although, the AFB1 reduction was not significantly different between them (F4,10 = 2.006, p = 0.170), both Aureobasidium strains (DMKU-PAL120 and DMKU-PAL144) seemed to have a lesser impact on AFB1 reduction. The SEM micrographs (Fig. 2) demonstrated that co-incubation with these VOCs-producing strains resulted in a lower density of the fungal mycelium and spores on rice surfaces compared to the control treatment. Additionally, the released VOCs disrupted the structure of the fungal mycelium and affected sporulation. Such results may have led to the reduction of aflatoxin production when co-incubated with these antagonistic strains.

Figure 1.Reduction of AFB1 in rice grains contaminated with A. flavus A39 after co-incubation with the antagonistic strains at 28 ± 2℃ for 14 days. Data are means of triplicates (n = 3) with a bar indicating the standard deviation (SE). The same letter indicates no significant difference (p > 0.05).

Figure 2.Scanning electron micrographs of fungal structure on rice surfaces contaminated with A. flavus A39 after co-incubation with the VOC-producing strains at 28 ± 2℃ for 14 days. (A) A. flavus, (B) A. flavus + DMKU-PAL84, (C) A. flavus + DMKUPAL99, (D) A. flavus + DMKU-PAL120, (E) A. flavus + DMKU-PAL124, (F) A. flavus + DMKU-PAL144. co, conidia; cp, conidiophores; my, mycelia

Identification of VOCs

Various classes of VOCs such as alcohols, alkenes, aromatics, esters and furans were produced by these microorganisms, ranging between 4 to 14 compounds under the studied condition (Table 2 and Fig. 3). Three alcohols, including 2-methyl-1-butanol, 3-methyl-1- butanol, and 2-phenylethanol, were detected in common among these strains. These compounds were produced in a high proportion by Anthracocystis sp. DMKU-PAL124, Rhodotorula sp. DMKU-PAL99, and Solicoccozyma keelungensis DMKU-PAL84. Two Aureobasidium strains generated different volatilomes. Additionally, the most abundant compounds produced by both strains were also distinct. Aureobasidium sp. DMKU-PAL120 emitted the highest level of 2-phenylethanol (50.16%), whereas Aureobasidium sp. DMKU-PAL144 released the greatest proportion of methyl bezeneacetate (52.74%). The prominent peaks on the chromatograms at about 6.80, 15.37, 10.38, 20.47, and 25.11 min are not VOCs generated by the microbial strains, but are the identical substances observed in the head space of the PDA control.

Table 2 . Volatile organic compounds produced by the selected strains identified by SPME-GC/MS.

Retention time (min)Possible compoundMW (g mol-1)Relative peak area ± SE1 (%)
Aureobasidium sp. DMKU-PAL120Aureobasidium sp. DMKU-PAL144Anthracocystis sp. DMKU-PAL124Rhodotorula sp. DMKU-PAL99So. keelungensis DMKU-PAL84
1.222-Fluoropropene60.070.87 ± 0.010.15 ± 0.00ndndnd
1.52Ethyl acetate88.111.35 ± 0.060.72 ± 0.10ndndnd
1.612-Methyl-1-propanol74.122.37 ± 0.151.17 ± 0.07nd1.88 ± 0.101.93 ± 0.19
1.94Benzene78.11nd1.44 ± 1.05ndndnd
2.512,5-Dimethylfuran96.13nd0.36 ± 0.034.93 ± 1.073.00 ± 0.151.88 ± 0.09
2.58Ethyl propanoate102.131.56 ± 0.120.87 ± 0.06ndndnd
2.953-Methyl-1-butanol88.1525.63 ± 1.478.85 ± 0.3257.91 ± 2.1249.01 ± 10.8933.66 ± 1.90
3.022-Methyl-1-butanol88.1513.32 ± 1.157.14 ± 0.1228.96 ± 0.0031.97 ± 9.3144.07 ± 1.61
3.371,4-Pentadiene68.12ndndndnd5.58 ± 0.19
4.38Ethyl butanoate116.161.07 ± 0.001.27 ± 0.03ndndnd
5.493-Methylbutanoic acid102.13ndndnd5.89 ± 0.003.67 ± 1.37
6.453-Methyl-1-butyl acetate130.181.73 ± 0.230.52 ± 0.14nd2.74 ± 0.00nd
13.942-Phenylethanol122.1650.16 ± 4.3714.41 ± 1.828.20 ± 0.005.52 ± 1.649.21 ± 0.76
16.00Methyl benzeneacetate150.171.95 ± 0.2252.74 ± 8.37ndndnd
16.13Naphthalene128.17nd7.11 ± 0.32ndndnd
18.03Ethyl benzeneacetate164.20nd3.30 ± 0.60ndndnd

1Mean value of the percentage of the peak area over the total area of the peaks in the chromatogram of the strains grown on PDA.

nd = not detected.

Figure 3.SPME-GC/MS chromatograms of VOCs produced by five strains of the selected yeast and yeast-like fungi on PDA after incubation at 28℃ for 2 days.

Discussion

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Microorganisms can produce VOCs through their metabolic pathways. These compounds belong to different classes, including acids, alcohols, aldehydes, esters, ketones, benzenoids, pyrazines, sulfides, terpenes, etc. [11, 12]. Some VOCs are antagonistic to other microorganisms. Studies of such compounds produced by various microorganisms for use as biological control agents against pathogenic fungi that cause damage to agricultural crops have increased in recent years [13-15]. In the present study, strains of yeast and yeast-like fungi isolated from pineapple leaves produced volatile compounds that inhibited the development of A. flavus. Some of them showed potential effects on the development of mycelium and sporulation, which led to an AFB1 reduction of up to 60% in the rice grains contaminated with a high number of A. flavus spores (105 spores/g).

In the current study, various classes of VOCs were produced by the selected strains. 2-methyl-1-butanol, 3- methyl-1-butanol, 2-phenylethanol, and methyl bezeneacetate were the main antagonistic agents produced. Various strains of epiphytic and endophytic yeasts have been reported for their ability to produce VOCs that have potential effects on phytopathogenic fungi. For example, Kwoniella heveanensis DMKU-CE82, an endophyte isolated from corn leaf tissues, produced VOCs closely matched to 2-methyl-1-butanol, 3-methyl- 1-butanol, hydrazine-1-1-dimethyl, and butanoic acid-3- methyl that were active against A. flavus [9, 16]. Candida intermedia C410 isolated from a healthy strawberry leaf produced 1,3,5,7-cyclooctatetraene and 3-methyl-1-butanol as the most abundant compounds that had an effect against Botrytis fruit rot [17]. Pichia kudriavzevii KKP 3005, P. occidentalis KKP 3004, and Meyerozyma quilliermondii, Meyerozyma caribbica KKP 3003 that were isolated from grapes and rye grains produced various types of VOCs such as ethyl esters of medium-chain fatty acids, phenylethyl alcohol, and acetate ester. These compounds had an antagonistic activity against common plant pathogens such as Botrytis cinerea, Mucor spp., Penicillium chrysogenum, P. expansum, A. flavus, Fusarium cereals, and F. poae [18]. Reports demonstrated that different strains of a microbial species may produce different VOCs. For example, the main compounds produced by two strains of Aureobasidium pullulans (L1 and L8) were 2-phenylethanol, 1-butanol-3-methyl, 1-butanol-2-methyl, and 1-propanol-2-methyl [13]. While another A. pullulans strain (PI1) emitted alcohols (ethyl alcohol, 1-butanol-3- methyl, and 2-phenylethanol) and esters (ethyl acetate and isoamyl acetate [19]. In our study, two strains of Aureobasidium sp. (DMKU-PAL120 and DMKU-PAL144) produced distinct VOC profiles, with different major compounds (Table 2). Both strains are closely related to A. thailandense. However, they are 98.3% identical, indicating that they should be different strains of the same species (P. Khunnamwong, personal communication). These results suggest that the different profiles of VOCs may be produced by closely related strains under the same growing condition. We also discovered that the VOCs produced by the different strains of So. keelunggensis had varying effects on mycelium growth (Table 1). Moreover, this is the first report showing the capability of So. keelungensis to produce antifungal volatiles.

Several studies have reported the impact of synthetic volatile organic compounds on phytopathogenic fungi. Some of them are the same compounds majorly emitted by the microorganisms in the present study. For example, a study demonstrated that the half-maximal inhibitory concentration (IC50) of synthetic 3-methyl-1-butanol for suppression of mycelial growth and of conidial germination of Botrytis cinerea were 70.2 ± 1.6 μl/l and 90.8 ± 0.4 μl/l, respectively. While phenylethanol was more efficient than 3-methyl-1-butanol in inhibiting mycelial growth (IC50 = 29.9 ± 5.0 μl/l), it was less effective in suppressing conidial germination (IC50 > 500.0 μl/l) [17]. A study showed that 2-phenylethanol influences fungal development by causing membrane disruption and inhibiting DNA, amino acid, and protein synthesis [20]. It also reduced the expression of all aflatoxin genes in A. flavus [21]. Rezende reported that fungi exposed to 2-methyl-1-butanol and 3-methyl-1-butanol experienced oxidative stress and changes in plasma membrane permeability [22]. Yalage Don et al. demonstrated that exposure to a mixture of VOCs, including ethanol, 2-methyl-1-propanol, 3-methyl-1-butanol, and 2-phenylethanol, resulted in ROS accumulation and electrolyte leakage in B. cinerea and Alternaria alternata [23]. Among the selected strains in this study, Aureobasidium sp. DMKU-PAL144 was the only strain that produced methyl benzeneacetate as the dominant compound that could affect A. flavus. This compound has recently been reported as a major compound produced by a biocontrol bacterium, Bacillus mycoides BM02 [24]. Additionally, it was observed that the synthetic methyl benzeneacetate inhibited spore germination of Fusarium oxysporum f. sp. lycopersici, a phytopathogenic fungus that causes tomato wilt, by up to 70–80% when it was used in a fumigant at a concentration of 10 mg/ml. This information supports our findings that the antagonistic microorganisms that produce these compounds have the potential to combat fungi, which in our study are the aflatoxigenic A. flavus. Such results may have led to the reduction of aflatoxin B1 content in the fungal- contaminated rice grains when co-incubated with these antagonists. Another study reported that 2-phenylethanol was most effective against a variety of phytopathogenic fungi, including B. cinerea, Penicillium expansum, P. digitatum, and P. italicum. However, when tested against Colletotrichum acutatum, it was less active than 2-methyl-1-butanol and 3-methyl-1-butanol [13]. This phenomenon was also observed in our study, where the same VOC-producing strain had varying effects on different strains of A. flavus under the same treatment condition (Table 1). As a result, a combination of the antagonists or active compounds may be required to effectively control different strains of pathogens.

For the last decade, antagonistic microorganisms have become a viable alternative to synthetic fungicides for controlling phytopathogenic fungi in agricultural products. These microbial agents are saprophytic and not pathogenic to plants, animals, and humans [25]. Therefore, application of these agents satisfies consumer demand for fungicide/pesticide-free agriculture. In our study, all microbial strains were isolated from pineapple leaves (P. Khunnamwong, personal communication). They are saprophytes and hence have the potential to be used safely as biocontrol agents against A. flavus in agricultural commodities. On the other hand, VOCs generally have a low molecular weight and readily enter the gas phase by vaporizing at 0.01 kPa at a temperature of approximately 20℃ [26]. Moreover, these compounds are active at low concentrations. Therefore, they can diffuse throughout the atmosphere and exert their inhibitory effect against the target pathogens. Furthermore, these compounds do not contaminate food commodities. Having these characteristics, they pose no significant threat to human health and the environment [27].

In conclusion, our findings revealed that certain yeast and yeast-like fungi strains can produce anti-fungal volatiles against A. flavus strains that produce AFB1. Additionally, this is the first report showing the ability of Solicoccozyma keelungensis to emit antifungal VOCs. Furthermore, 2-methyl-1butanol, 3-methyl-1butanol, 2- phenylethanol, and methyl benzeneacetate were the main active VOCs that inhibited mycelial growth and sporulation in A. flavus strains. Our findings suggest that these VOC-producing microorganisms could be promising biological control agents in agricultural products to combat phytopathogenic and mycotoxigenic fungi. However, more research into the practical use of these beneficial VOCs or of the microbe itself for fungal control in agricultural products is required.

Acknowledgments

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The authors are grateful to Associate Professor Dr. Nantana Srisuk of Kasetsart University, the director of the research program.

This work was supported by Kasetsart University Research and Development Institute, KURDI under Grant no. FF(KU)18.64.

Conflict of Interest

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The authors have no financial conflicts of interest to declare.

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