Antifungal effect of the tea tree essential oil (Melaleuca alternifolia) against Penicillium griseofulvum and Penicillium verrucosum

2.1 Samples collection

Samples were taken at random from Martil city, in northern Morocco, on batches of products offered for sale to the public. Eight samples of lentil and nine of dry grapes were analyzed in our laboratory.

2.2 Isolation of Penicillium strains

The isolation of the Penicillium strains from the samples was carried out at the Environmental and Food Biotechnology Research Team (EFBRT) at the Normal High School of Tetouan, Morocco. To do, external face of food particles were disinfected and they were placed directly on Malt Extract Agar (MEA) (Samson and Gams, 1984). The incubation was at 25 °C for 7 days.

2.3 The spore suspension

In this study, all cultured strains were prepared from a spore suspension. For each Penicillium isolate, the spores were aseptically recovered and placed in a sterile solution of 0.1% Tween 80 distilled water for dispersion. A mother solution of spores was obtained, and its concentration was adjusted to 10⁷ spores/ml by enumeration in the Thomas cell.

2.4 Identification of isolated Penicillium

The isolation step allows separating the strains suspected to be Penicillium (Asehraou et al., 1992). For identification and confirmation steps, cultures were grown on three standard media: (MEA), Czapeck Yeast extract agar (CYA) (Samson, 1981), and 25% Glycerol Nitrate agar (G25N) (Pitt and Hocking, 2009). An inoculum, taken from the suspension of spores using a sterile loop, was cultured in petri dishes of CYA, MEA, and G25N. These were then incubated at 25 °C for 7 days (Khaddor et al., 2007).

The identification of isolates was based on the determination key of Pitt and Hocking (2009) and Visagie et al. (2014). This identification was mainly based on the morphological features and the biochemical characteristics of the isolates. The macroscopic and microscopic parameters were examined. Then, a possible production of mycotoxins by the identified strains was highlighted.

2.5 Research for mycotoxins

Two ml of 10⁷ spore/ml suspension of each identified strain was inoculated into three culture media, namely CYA medium, Base medium (MB) and liquid YES (Khaddor et al., 2007). The media were incubated at 25 °C for 10 days (Pitt, 1988). After incubation, the culture in YES liquid medium was subjected to Wattman paper filtration to remove the mycelium and then another filtration through a Millipore filter (0.45 µm) to be sterilized. Then, 25 ml of chloroform was added to the solid media (CYA and MB), and to the YES liquid medium and stirred for 2 min. The chloroform phase was recovered and filtered through a layer of anhydrous sodium sulfate. The filtrate was concentrated in a rotavapor and then evaporated to dryness under a stream of nitrogen. The mycotoxins were sought in the concentrated filtrate, and highlighted by Thin Layer Chromatography (TLC). The TLC plates used are 60 Kieselguhr F254. This method was performed as reported by Bouhoudan et al. (2018).

2.6 Chemical characterization of EO

Essential oil of M. alternifolia L. was supplied, in May 2015, by the Ain Lahjer women’s cooperative - Nouara - Dar Ben Karrich - Tetouan. The EO was obtained by hydrodistillation of dried aerial parts for 90 min in a device of Clevenger type. The resulting oil was dehydrated with anhydrous sodium sulfate and stored at 4 °C in the dark (Benayad et al., 2012). The chemical composition analysis of this EO was carried out by Gas Chromatography coupled with Mass Spectrometry (GC–MS). This method was done as described by Louhibi et al. (2015) and Derwich et al. (2009). The chemical characterization of EO M. alternifolia L. was performed on a gas chromatograph Trace GC Ultra-ITQ900 thermoscientific, equipped with a polar capillary column TG-SQC thermoscientific (60 m × 0.25 mm, 0.5 μm film thickness). Oven temperature program was from 50 °C to 240 °C at 5 °C/min. Injector temperature, 250 °C. Carrier gas Helium, flow rate 1 ml/min. The volume of injected sample was 1 μl (20 μl of EO diluted in 1000 μl of hexane). These analyses were conducted at the National Institute of Agricultural Research (INRA) of Tangier.

2.7 Antifungal effect of M. alternifolia EO on P. griseofulvum and P. verrucosum

In this study, two toxigenic isolates of Penicillium such as P. verrucosum and P. griseofulvum, isolated respectively from dry grapes and lentil, were subjected to the effect of different concentrations (Table 1) of M. alternifolia EO. The different concentrations of EO were prepared from a stock solution. The stock solution was obtained by dilution (1/10: v/v) of EO in distilled water containing 0.2% agar (Khaddor et al., 2007, 2006). Volumes corresponding to the concentrations (Table 1) were placed, each, in a 100 ml erlenmeyer flask. A volume of culture medium (MB, CYA and, liquid and solid YES) was added to each flask until a final volume of 25 ml. Then, each medium was inoculated with 0.5 ml of the spore solution. The control consists of 25 ml of culture medium added with 0.5 ml of distilled water at 0.2% agar. All these cultures were incubated at 25 °C for 10 days. Experiments were conducted in triplicate.

The media then was filtered by Buchner vacuum filter and Whatman N°1 filter to separate the mycelia from the liquid medium. The mycelia were then rinsed with hot distilled water to remove any trace of the culture medium. The dry weight was measured after placing the mycelia in an oven set at 80 °C for 24 h. The effect of the EO on the Penicillium growth was assessed by comparing the dry weight of the mycelium and the control.

2.8 Effect of M. alternifolia EO on mycotoxins production by P. griseofulvum and P. verrucosum

Culture media prepared at different concentrations of M. alternifolia EO inoculated with both Penicillium species were also used to study the effect of EO on production of mycotoxins. They were incubated at 25 °C for 10 days. The extraction of any mycotoxins was carried out by adding 25 ml of chloroform to each erlenmeyer flask followed by stirring for 10 min. The chloroform phase was recovered and filtered through a layer of anhydrous sodium sulfate. The chloroform recovered was then evaporated under vacuum in the rotary evaporator. The resulted extracts were supplemented with 0.5 ml of the chloroform-methanol extraction solvent (2/1: V/V) to be analyzed by TLC.

Toxigenesis study was made according to the method reported by Khaddor et al. (2007) and Bouhoudan et al. (2018). Mycotoxins standards used by the reference of migration forehead (Rf) were patulin (P), citrinin (C), ochratoxin A (OTA), penicillic acid (PA), and griseofulvin (Gi) (Cunniff and Association of Official Analytical Chemists, 1995). Fifty µl of each extract and standard solutions (1 mg/ml) were spotted on TLC plates. Elution systems used were: toluene-ethyl acetate - formic acid (5/4/1, v/v/v) and chloroform - acetone - 2-propanol (85/15/20, v/v/v). The plates were examined in daylight and by ultraviolet 365 and 254 nm after spraying the spots by ANIS (p-anisaldehyde solution) and 8 min heating to 120 °C.

2.9 Statistical analysis

The Student’s t-test was used to determine the statistical significance of experiments performed in triplicates. Statistical values including means and standard deviation from the obtained data were calculated using Statistical Tools for High-Throughput Data Analysis; STHDA (http://www.sthda.com/french/rsthda/rsthda.php). Differences were considered significant if p < 0.05.

3.1 Identification of Penicillium strains and their mycotoxins

Color conidia (shades between blue, green and/or gray), roughness or smoothness of stipes on CYA and MEA, spherical mature conidia or ellipsoidal, colony diameter on CYA and MEA, structure and number of branches of penicil on MEA, were the characters analyzed and compared to the identification keys of Pitt and Hocking (2009). In this study, we isolated 14 strains of Penicillium. These strains belong to seven species (Table 2).

Our results showed that food samples of lentil were contaminated with eight strains belonging to five species of Penicillium. While, six strains belonging to three species of Penicillium were isolated from the samples of dry grapes. The isolated toxigenic species were P. verrucosum and P. griseofulvum.

Our findings showed that mycotoxins were produced only in YES medium. The metabolic profile showed that the Terrestric acid was produced by both Penicillium species. The Ochratoxin A production was revealed in the profile of P. griseofulvum (Table 3).

As P. verrucosum and P. griseofulvum were, in this work, mycotoxin-producing species, they were subject to the study of the effect of M. alternifolia EO.

3.2 Antifungal effect of M. alternifolia EO on P. verrucosum and P. griseofulvum

The CG-SM essays showed that the major component of the M. alternifolia EO was the terpinene-4-ol with a percentage of 23.06%. The identification of this EO component was done by comparing the mass spectrum of each peak separated by GC with those reported as references such in specialized bibliographies of mass spectrometry (Brophy et al., 1989; Hammer et al., 2006).

3.2.1

3.2.1 Effect of M. alternifolia on mycelial growth of P. verrucosum and P. griseofulvum

A significant decrease in dry weight (p < 0.05) relative to the control was noted for the mycelia of both species: P. verrucosum and P. griseofulvum. The graphic representation of the dry weight of P. verrucosum in relation to the dry weight of control (Fig. 1) showed that the lowest concentration (0.08%) significantly reduced the dry weight of mycelium to 60% of control dry weight. The decrease in dry weight was proportional to the increase in the concentration of EO. At 2.75%, the EO completely inhibits growth of P. verrucosum in the liquid YES.

Close

Fig. 1

Effect of M. alternifolia EO on the growth of P. verrucosum and P. griseofulvum.

Export to PPT

As for P. griseofulvum, inhibition of mycelial growth was significant from the 0.16% concentration (Fig. 1). Inhibition of mycelial growth was greater at the highest concentration of EO. Total inhibition of mycelial growth resulted in 2.75% of M. alternifolia EO for both species Penicillium in liquid YES.

3.2.2

3.2.2 Effect of M. alternifolia EO on mycotoxins production of P. verrucosum and P. griseofulvum

As we mentioned before, both species of Penicillium produce their mycotoxins only in YES medium. TLC performed to demonstrate the effect of EO on mycotoxin production by both fungal species also revealed that these mycotoxins were not produced in CYA and MB media. The production of Terrestric acid by both Penicillium species, and Ochratoxin A by P. Griseofulvum was inhibited from the lowest concentration of M. alternifolia EO.

The TLC plate (Fig. 2) showed that the production of the terrestric acid by P. verrucosum was inhibited at increasing concentrations of the EO of M. alternifolia represented respectively by B, C, D, E, F, and G. The yellow spot, in position A, represents the terrestric acid produced in the control culture in liquid YES medium.

Close

Fig. 2

Picture of a TLC plate showing the disappearance of the production of terrestric acid by P. verrucosum at increasing concentrations of M. alternifolia EO.

Export to PPT