Cellular, physiological and molecular approaches to investigate the antifungal and anti-aflatoxigenic effects of thyme essential oil on Aspergillus flavus
Introduction
Aspergillus flavus, a widespread fungal species belonging to Aspergillus section Flavi, is one of the most important producers of highly toxigenic secondary metabolites, including aflatoxin B1 (AFB1) (Perrone et al., 2014). This toxin is a potent carcinogenic, mutagenic and hepatotoxic aflatoxin with worldwide occurrence in important food and feed crops (Lewis et al., 2005). Apart from the AFB1 carcinogenic potential, the frequent consumption of food contaminated with AFB1 can also result in immune suppression, growth faltering in children and reduced life expectancy (Shephard, 2008). Thus, the presence of these fungi and subsequent aflatoxin contamination in food and feed is of great concern in terms of food safety management worldwide.
One of the most effective strategies to reduce mycotoxin contamination in food and feed is to prevent the development of toxigenic fungi on susceptible substrates (Kabak, Dobson, & Var, 2006). These chemical fungicides are still being widely applied for controlling these microorganisms, even with increasing public concern regarding chemical residues and the proliferation of resistance in pathogen populations (Tripathi & Dubey, 2004). For these reasons, there has been significant interest in the development of alternative fungal control methods that are environmentally sound and possess biodegradable properties.
In this context, essential oils (EOs) have been reported as promising antifungal natural products that constitute effective alternatives or complements to replace synthetic fungicides (Bakkali et al., 2008, Lang and Buchbauer, 2012). They are a mix of volatile compounds characterized by a strong odor and are formed by the secondary metabolism of aromatic plants (Bakkali et al., 2008). In addition to their well-documented antimicrobial properties (Burt, 2004), most of the EOs are considered ‘generally recognized as safe’ (GRAS) as food additives by the Food and Drug Administration (FDA), which makes them a potential bioresource of eco-friendly antifungal agents (Tisserand & Young, 2013, chap. 15).
Among the biological activities of EOs, the antifungal and anti-aflatoxigenic properties of thyme EO have been reported against Aspergillus spp. (Bluma and Etcheverry, 2008, Kohiyama et al., 2015, Omidbeygi et al., 2007). Most of the studies focus on describing the inhibitory effect of thyme EO on mycelium growth and aflatoxin production under several growth conditions and at different concentrations; however, to our knowledge, the antifungal mechanisms of this EO at the cellular and molecular levels are still not well understood.
Therefore, the aim of this study was to employ different approaches that involve cellular, physiological and molecular analyses to better understand the antifungal mechanism of thyme EO effects on A. flavus. Based on a growth rate inhibition assay, cell apoptosis, that is programmed cell death or a cell suicide response to events (Saikumar et al., 1999.), such as nuclear condensation and damage to the plasma membrane was investigated in the present study. Moreover, to give new insight into the anti-aflatoxigenic property of this EO at molecular level in A. flavus, we also evaluated AFB1 production and especially the transcriptional profile of genes related to the secondary metabolism and mechanism of virulence of this species in contact with thyme EO.
Section snippets
Fungal strain
The A. flavus strain MC119 used in this study was provided by the Laboratory of Mycotoxins (C-119) located in the Institute of Biology at the National Autonomous University of Mexico, in Mexico City [Instituto de Biología, Universidad Nacional Autónoma de México]. The strain was previously identified by morphological features as A. flavus, and its aflatoxigenic potential was identified by a thin-layer chromatography (TLC) method (Davis, Diener, & Eldridge, 1966). However, for accurate
Molecular identification of A. flavus isolate
The identity of the fungal isolate was confirmed to be A. flavus based on the β-tubulin sequence analysis. Results were considered significant at a query coverage of 99% and a percent identity of 100% to the A. flavus strain CBS 108.24. The nucleotide sequence is available in the GenBank database under the accession number MK580962.
Chemical characterization of thyme EO
The results of the GC–MS analysis of the thyme essential oil are demonstrated in the Table 2 and Fig. S2. A total of 28 compounds were identified, with thymol
Discussion
In recent years, considerable attention has been given to the potential of plant-based products to be used as alternatives to synthetic chemicals by the food and agriculture industries. In this context, EO and its bioactive compounds have been extensively studied by scientists worldwide, especially because they possess high efficiency as antifungal agents and eco-friendly characteristics. In the present study, we used different approaches to investigate the antifungal and anti-aflatoxigenic
Conclusion
In the present study, the fungal growth inhibition effect of thyme EO was related to its ability to disrupt the integrity of the plasma membrane and induce apoptosis in the hyphae of A. flavus. Moreover, thyme EO significantly reduced the AFB1 production of A. flavus in vitro. This anti-aflatoxigenic property was also attributed to the down-regulation of secondary metabolism gene (laeA) and to the modulation of hydrolase gene expression involved in fungal colonization and establishment. These
CRediT authorship contribution statement
Rodrigo C. Oliveira: Conceptualization, Methodology, Investigation, Writing - original draft. Magda Carvajal-Moreno: Methodology, Project administration, Supervision, Investigation, Investigation, Methodology. Benedito Correa: . Francisco Rojo-Callejas: Supervision, Investigation, Methodology.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
The authors thank the Instituto de Biología, Universidad Nacional Autónoma de México (IBUNAM) and University of Sao Paulo, Sao Paulo, Brazil for the data analysis. The authors also thank IBUNAM’s personnel: Pedro Mercado from the Technical Secretary, Laura Márquez, Joel Villavicencio, Jorge López, Alfredo Wong, Celina Bernal, Diana Martínez and Julio César Montero who provided valuable assistance with imaging, computer analysis and design. Additionally, we thank Georgina Ortega Leite and
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