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Food Microbiology (FM) | Food Borne Pathogens and Food Safety
Microbiol. Biotechnol. Lett. 2022; 50(2): 202-210
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, firstname.lastname@example.org
Pannida Khunnamwong, email@example.com
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
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
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.
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.
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
The selected strains were further screened for their effectiveness against
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
SEM was used to visualize the
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.
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
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
As seen in Fig. 1, the VOCs produced by these strains exhibited antagonistic activity against
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
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
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,
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
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 . 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
In conclusion, our findings revealed that certain yeast and yeast-like fungi strains can produce anti-fungal volatiles against
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.
The authors have no financial conflicts of interest to declare.
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