Control of Aspergillus flavus in wheat grains using Cymbopogon flexuosus essential oil

Fungi are one of the main food spoilage agents. Numerous species when subjected to stress conditions produce secondary metabolites known as mycotoxins, which are mutagenic and carcinogenic substances. The fungus Aspergillus flavus is one of the main contaminants of grains and is known to produce Aflatoxin. Pesticides are used in agriculture to contain fungi and other pests, but they harm other species, the environment and the human health, in addition to the development of resistance to these substances in pest species. Natural alternatives have been sought to control these organisms. In this context, essential oils are a viable option against A. flavus. The aim of this study was to identify the main components and evaluate the effectiveness of lemongrass essential oil (Cymbopogon flexuosus) for controlling the fungus Aspergillus flavus. Initially, the effect of essential oil on mycelial growth of the fungus was assessed by in vitro tests at the doses: 0.05; 0.1; 0.2; 0.4; 0.6; 0.8; 1.6; 3.2; 6.4; and 12.8 μL mL-1. The minimum inhibitory concentration (MIC) was 0.8 μL mL-1. The in vivo test was performed at the following concentrations: 0.6; 0.8; and 1.6 μL mL-1. The results showed that the essential oil has fungicidal potential against A. flavus. The main component of the essential oil was citral.


Introduction
A. flavus is one of the main contaminants of grains (Liu, Li, Wu, Xiao, & Xing, 2021), causing large economic losses and potential health issues on humans and animals (Xu et al., 2021) because of the production of mutagenic and carcinogenic secondary metabolites, especially aflatoxin (Tian et al., 2021).
Pesticides are used in crops to fight pests, yet these substances do not only affect these organisms, they harm other species, the soil, the aquatic ecosystem and human health. Adding to the problem is the fact that field pests, including fungi, become more resistant to pesticides. Within this context, this study has investigated the possibility of replacing these substances by natural alternatives that do not cause environmental damage and harm to species. Essential oils have been presented as an alternative. Essential oils are extracted from aromatic plants. They are secondary metabolites known for their strong odor, volatility, medicinal, antiseptic, antibacterial, antiviral, and antifungal properties, being widely used in food, health, and pharmaceutical industries (Bakkali, Averbeck, Averbeck, & Idaomar, 2008). The Cymbopogon genus has been extensively studied, consisting of around 180 species, including different sub-species, varieties and sub-varieties. Extensive research has been conducted to prove the benefits of the Cymbopogon genus, including studies on the extract, essential oils, biological activity of the chemical compounds, and development. Pharmaceutical studies have tested the medical effects of Cymbopogon species such as anti-inflammatory, antimicrobial, antioxidant, and anticancer activities (Zahra, Hartati, & Fidrianny, 2020), as well as in the central nervous system (Rojek et al., 2021).
The C. flexuosus essential oil has the chemical compound citral as its main component, presenting anti-inflammatory effect on animal and human cells, as well as antimicrobial, antioxidant, and antifungal activities (Han, & Parker, 2017).
This study identified the main components and evaluated the efficacy of C. flexuosus essential oils and its major citral compound on the fungus A. flavus tested at different doses in vitro and in vivo experiments, and determined the minimal inhibitory concentration (CIM).

Materials and Methods
C. flexuosus essential oil was purchased from the commercial supplier Ferquima Company ® and was stored at 4º C. The tested doses were diluted in distilled water and dimethylsulfoxide 1% (DMSO) to obtain a homogeneous solution for the diffusion test with the substrate and microdilution.

Fungi and spores suspension
The fungus species A. flavus (IOC 4102 strain) from the Culture Collection of Filamentous Fungi was kindly provided by the Fundação Oswaldo Cruz. The fungal cultures were grown in potato, dextrose and agar medium (PDA), in petri plates at 30 ºC for seven days. To perform the spores collection, 15 mL of sterile distilled water were added to the Petri dishes with PDA medium with 7-day-old fungal colony growth. The fungal suspension was adjusted with sterile distilled water to the final concentration of 4,5 x 10 6 spores/mL -1 using a Neubauer chamber (Model: K5-0011, KAVIS, São José do Pinhais, Brasil). The suspension was stored at 4 ºC until use.

Microdilution at microplate
The minimum inhibitory concentration (MIC) of the essential oil (EO) on the fungus studied was determined by serial microdilution in 96-well microplates. The doses tested were defined from the results of the in vitro test, being evaluated the following essential oil doses: 1.8; 1.6; 1.4; 1.2; 1; 0.9; 0.8; 0.7; 0.6; 0.5; 0.45; 0.4; 0.35; 0.3; 0.25; 0.22; 0.2; 0.17; 0.15 and 0.12 μL mL -1 . For each dose tested four replications were performed in PD medium (potato and dextrose) with a solution containing essential oil, DMSO and spores suspension (10 7 ) and a control treatment without the essential oil. The plates were maintained in B.O.D (Biochemical Oxygen Demand) chambers (Model: SSBOD 120L, Benfer, São Paulo, Brazil), at 35 ºC for 72 hours. After this incubation period the results were analyzed visually. MIC was defined as the lowest essential oil concentration in which no fungal growth had occurred (Pandey, Rai, & Acharya, 2003;Dellavalle et al., 2011).

In vitro test
In the in vitro anti-fungal assay, the following C. flexuosus essential oil doses were tested: 0.05; 0.1; 0.2; 0.4; 0.6; 0.8; 1.6; 3.2; 6.4 and 12.8 µL/mL -1 . Two control treatments without essential oil were performed: one with fungus growing on PDA medium only; and the other with fungus growing in PDA medium added with DMSO to evaluate the influence of the surfactant on fungal growth.
Was added in the cast BDA substrate (45 °C), the essential oil solution was added to liquid PDA medium, diluted in 1% DMSO, to obtain the desired concentration. After homogenization, the substrate was poured to the Petri dishes and incubated with a 7mm mycelial disc of the PDA grown colony of the fungus in study. The control plates, without essential oil, were inoculated following the same process. Afterwards, all the plates were incubated at 30 ºC until the fungus covered the entire control plate, that is, between 7 and 10 days, which was considered the end of the incubation time. The experiment was carried out with five replications. The colony diameter was recorded daily with a digital caliper (Model: 150mm, MTX, São Paulo, Brazil). The colony size of each test sample was compared with the size of the control tests. The results were expressed as the diameter size of the microbial growth halo. The percentage of colony inhibition (PI) was calculated according to Billerbeck, Roques, Bessière, Fonvieille and Dargent (2001): PI= (C-T/C) x100 (Equation 1), where: C = average diameter of the mycelia in the control; T = average diameter of the mycelia in the treatment.
According to the in vitro assay results, the MIC test was performed with its major component, the Citral, purchased from the Sigma-Aldrich Company ® . The doses tested were: 0; 0.2; 0.4; 0.6; 0.8 and 1.6 µL mL -1 . PI was determined using Equation 1.

Analysis of the control of the fungus by essential oils in infected grains
Seeds of wheat were provided by Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA) (Wheat/Rio Grande do Sul). The analysis of efficacy of C. Flexuosus essential oil on the fungus growth was defined from the doses that showed the largest inhibition percentages (PI) of the mycelial growth in the in vitro assay, being 0.6; 0.8 and 1.6 µL mL -1 besides the control, which did not contain any treatment with essential oils. These doses where tested in different periods of time (10, 20 and 30 days) in a controlled environment (30°C).
In 1.4 L glass bottles, 200g of wheat grains were autoclaved at 121°C for 15 minutes. Afterwards, to each container, 2mL of the suspension at 10 7 spores/mL -1 of A. flavus was inoculated to the grains. The flasks were incubated in a controlled environment (30 °C) (B.O.D.) for 2 days to promote the fungus growth.
After 48h, filter paper sachets were soaked in different doses of essential oils. The sachets were placed in the bottles with the grains inoculated with the fungus. The control treatment received no dose of the essential oil and was analyzed at the different times that the treatment was applied. Each dose was tested with three replications. These procedures were carried out in a laminar flow cabinet (Model: SP-960/6-HI, SPLABOR, Presidente Prudente, Brazil). Each dose tested was added with the volume of essential oil equivalent to the filter paper sachet: 0.6 µL.mL -1 : 840 µL, 0.8 µL mL -1 : 1120 µL and 1.6 µL mL -1 : 2240 µL.
The efficacy of the essential oil was analyzed by collecting samples at each 10 days. Colony Forming Units (CFU) under the effect of each treatment were quantified. At the end of each time, 10g grains of wheat were collected and diluted in 90 mL in saline solution (NaCl 0.9%). Ten-fold serial dilutions (10 -1 to 10 -3 ) were prepared with this solution. A volume of 0.5 millimeter of each dilution were inoculated to Petri dishes with 20 mL of Sabouraud agar medium. The plates were incubated for 48 h at 30 ºC in B.O.D chambers. Plates containing 05 to 250 UFCs were counted and the percentages of inhibition (PI) of growth were calculated according to Tatsadjieu, Yaouba, Nukenine, Ngassoum, & Mbofung (2010): PI= C0-Ct/C0 x100 (Equation 02), where C0 = the number of colonies that receive no treatment; CT = the number of colonies that received the treatment with essential oils.

Essential oil gas cromatography
The analysis of the essential oil constituencies were performed by gas chromatography with mass spectrometry (CG/EM) (Model: GCMS-QP2010, Shimadzu, Quito, Japan) according to the methodology of Lee, Garcia, Martinazzo and de Souza Teodoro (2020). The identification of the compounds were performed by comparing the mass spectrums obtained with the data of the equipment and by the Kovats Retention Index (IK) of each component (Lanças, 2009). The quantitative analysis of the main components of the essential oil, expressed as percentages, were performed by the peak area integration of normalization as described by Zhang, Chen, Wang e Yao, (2006).

Statistical analysis
The results were analyzed using the statistic program SISVAR ® , by analysis of variance and means compared by the Scott-Knott test at 5% significance level. The experiment was arranged in a completely randomized design (Ferreira, 2014).

Results and Discussion
The main component of C. flexuosus essential oil used in this study was Citral (72.0%), which is formed by neral isomers (40.0%) and geranial (32.0%; Table 1). The fungitoxic activities of the C. flexuosus essential oil on the A. flavus mycelial growth inhibition showed a significant difference of the mycelial growth of the fungus in study (Table 2).  (2007), except the value of the 13 peak, which is based on Choi (2003).  The incubation time of A. flavus was 10 days, and it showed growth between the 0.05 and 0.6 μL mL -1 doses, however the growth was inhibited from the 0.6 μL mL -1 dose. At doses below 0.4 μL mL -1 the diameter of the fungus inhibition halo ranged between 53 and 92 mm (total diameter of the Petri dish). At the 10 th day, a discreet growth was recorded (2 mm) at the 0.6 μL mL -1 dose. Chao, Young and Oberg (2000) evaluated the effects of C. flexuosus essential oil on microorganisms and found that neral (43.5%) and geranial (28.8%) were the major components in C. flexuosus, with fungitoxic activities against the Aspergillus genus.
Cymbopogon species have been reported as having antifungal activity against A. niger and A. fumigatus (Munda, Dutta, Pandey, Sarma and Lal, 2019) and A. flavus. Martinazzo, de Oliveira and de Souza Teodoro (2020) verified inhibition effects of C. citratus on A. flavus from the 0.6 µL mL -1 , achieving total control at 1.0 µL mL -1 . Achar et al. (2020) obtained total control at 2000 µL mL -1 , using the method of diffusion in medium.
Over the time of evaluation, the citral compound was efficient in the complete inhibition of the mycelial growth of A. flavus from the dose of 0.8 μL mL -1 (Table 3). At lower doses, the maximum growths recorded were: 74 mm (0.05 μL mL -1 ); 65 mm (0.2 μL mL -1 ); 21 mm (0.4 μL mL -1 ) and 05 mm (0. 6 μL mL -1 ). In the same way as with the other results, both for essential oils and citral, the doses at 0.6 μL mL -1 showed no significant differences between the control fungus and the doses that had 100% inhibition. Even though the inhibition of the fungus at the end of the incubation times may be a sign of microorganism development over time. Therefore, the dose at 0.8 μL mL -1 is recommended as the lowest dose for the control of the A. flavus fungus, both for the C. flexuosus essential oil and the citral compound.
The C. flexuosus essential oil evaluated in this study was also evaluated within its duration of antifungal efficacy over the days. The results of this study show that doses from 0.6 μL mL -1 of the C. flexuosus essential oil inhibited the fungal growth for at least 30 days, with efficacy of about 99.31% (Table 4).  Luo et al. (2004) investigated the effects of citral on A. flavus using broth dilution (MIC = 0.5 μL mL -1 ) and found that citral injured the wall and the membrane of A. flavus spore, decreasing its elasticity. After entering the cell, the citral modified the mitochondrial genetic expression, its reduplication and morphology, also in addition to altering the aggregation of protein-like macromolecules. This led cells, organelles and macromolecules to lose their normal structures and functions, which resulted in the loss of germination capacity of A. flavus spores, proving that citral has fungitoxic properties against fungi of the Aspergillus genus.
The antimicrobial activity of citral on A. flavus was also studied by López-Malo, Alzamora and Palou (2002), in which they found MIC of 1800 μL mL -1 (hydrogen potential of 3.5 and 4.5) and verified fungal growth inhibition for 60 days.

Conclusion
The essential oil of C. flexuosus showed in vitro fungicide effect against the fungus Aspergillus flavus, controlling fungal growth from the dose of 0.8 μL mL -1 . The MIC of the essential oil on A. flavus was 1.0 and 0.8 μL mL -1 .
The major component identified in the essential oil was the citral. The in vitro tests with citral showed fungicidal effect against the fungus from the dose 0.6 μL mL -1 .
The C. flexuous essential oil was proven to be efficient on stored contaminated wheat grains, maintaining the fungicidal capacity even after 30 days of incubation the best control of the fungal A. flavus in T. aestivum.