Effect of the essential oils of Thymus vulgaris , Cinnamomum zeylanicum and Mentha piperita on fungal growth and morphology

This study aimed to evaluate the antifungal activity of essential oils of Thymus vulgaris L., Cinnamomum zeylanicum B. and Mentha piperita L. on some saprophytic fungi. Essential oils were extracted by hydro-distillation, and chemical composition was analysed by gas chromatography coupled with mass spectrometry (GC-MS). T. vulgaris had as major components, Thymol (35.12%), pcymene (25.36%) and γ–terpinene (12.48%). E-B-Caryophyllane (21.82%), E-Cinnamaldehyde (13.03%) and eugenol (12.15%) were primary in C. zeylanicum. Menthol (33.59%), menthone (18.47%) and αpinene (8.21%) were primary in M. piperita. Applying the micro-atmospheric method, essential oils were tested against Rhizopus oryzae Went & Prins, Rhizopus stolonifer Ehrenb, Aspergillus tamarii Taka, Aspergillus parasiticus Speare, Aspergillus flavus Link and Talaromyces purpureogenus purpureogenum. The minimum inhibitory concentrations were 3 to 8, 5 to 16 and 13 to 23 μL/75mL air space for T. vulgaris, C. zeylanicum and M. piperita, respectively. Means of percentage inhibition were compared through one-way ANOVA by the Tukey test. Scanning electron microscopy revealed fungal cell wall deformation after exposure to essential oil vapour. These essential oils can be exploited as alternatives to synthetic food preservatives.

been that of urgent necessity: lacking in spectacular appeal, it is, however, often neglected.Synthetic fungicidal powders often used include Mancozeb, Thirame, methyl Thiophanate, etc.However, the use of these chemicals as food preservatives poses several problems particularly, environmental pollution, toxicity to living organisms, persistence as residues in food products, resistance of pathogenic agents (Etter et al., 2003).
It is well established that certain plants and their metabolites possess antimicrobial properties (Nguefack et al., 2004).These properties are significantly due to their volatile fraction, that is, essential oils (Hulin et al., 1998) and the presence of other bioactive substances such as phenols.Essential oils have a very broad spectrum of action, since they inhibit the growth of microorganisms (Ambindei et al., 2014) as well as insects (Tatsadjieu et al. 2007).
Thymus, Mentha and Cinnamum species are essential oil producing plants, and are being exploited for different properties.In native medicine, flowering parts and leaves of Thymus species have been extensively used as herbal tea, tonic, carminative, antitussive and antiseptic, as well as for treating colds (Maksimovic et al., 2008;Rota et al., 2008).Several studies have revealed the anti-oxidant, viral, inflammatory and microbial potential of the essential oil of Thymus vulgaris (Nickavar et al., 2005;Sessou et al., 2012).
Cinnamon can serve as a blood and digestive tonic, as a natural food preservative, and also exhibits antibacterial as well as antifungal properties (Kalemba and Kunicka, 2003;Jazet et al., 2007).
The main objective of this study was to evaluate the antifungal activity of the essential oils of T. vulgaris, Cinnamomum zeylanicum and M. piperita against some food spoilage fungi.This is based on the hypothesis that essential oils of these plants can inhibit fungal growth and development.Specifically, the chemical composition of the essential oils, the percentage inhibition, the nature of inhibition, minimum inhibitory concentration (MIC), minimum fungicidal concentration (MFC) and effect of essential oils on fungal morphology were evaluated.

MATERIALS AND METHODS
Whole mature plant of T. vulgaris L., was harvested in Dschang (geographical coordinates: 5°27' North and 10°04' East); leaves of C. zeylanicum B. and whole plant of M.a piperita L. were harvested in Mbouda (5°38' North and 10°15' East) in the West Region of Cameroon.These plants were harvested in September 2013 and were identified in the National Herbarium in Yaounde -Cameroon where voucher specimens were kept.

Fungal species
Fungal species were isolated from contaminated stored grains from Ngaoundere-Cameroon, their DNA were extracted, amplified and sequenced.The corresponding DNA sequence was blasted in the NCBI gene bank (Ambindei et al., 2016).The six fungal strains used in this study were a strain each of Rhizopus oryzae, Aspergillus tamarii, Aspergillus parasiticus, Rhizopus stolonifer, Aspergillus flavus and Talaromyces purpureogenus.

Extraction and chemical composition analyses of essential oils
Essential oils from the different plants were extracted by hydrodistillation with the help of a Clevenger apparatus.Essential oils obtained were analysed by gas chromatography and gas chromatography coupled with mass spectrometry (GC/MS) (Jazet et al., 2010).
Gas chromatography was done in a Varian CP-3380 GC with flame ionization detector (FID) fitted with a fused silica capillary column (30 m x 0.25 mm coated with DB5, film thickness 0.25 μm).The operating conditions were: Injection temperature: 200°C; detection temperature: 200°C; temperature program 50 -200°C at 5°C/min; carrier gas nitrogen, with a flow rate of 1 mL/min.GC-MS analyses were performed using a Hewlett-Packard apparatus equipped with an HP1 fused silica column (30 m x 0.25 mm, film thickness 0.25 m) and interfaced with a quadrupole detector (GC quadrupole MS system, model 5970).Column temperature was programmed from 70 -200°C at 10°C/min; injector temperature was 200°C.Helium was used as carrier gas at a flow rate of 0.6 mL/min.The mass spectrometer was operated at 70 eV.The operating conditions were: temperature programming: 70 to 200°C at 10°C/min; Injection temperature: 200°C; Flow rate of vector gas (helium): 0.6 mL/min, injection volume: 0.1 µL of essential oil solution diluted at 10% in hexane.
Compounds were identified by comparing the calculated retention indices and the mass spectrum with those given in literature (Adams, 2007).

Antifungal activity
The micro-atmosphere method described by Sulaiman (2013) was used at varying amount of essential oils.The inhibiting action of the essential oil vapour is highlighted.The inoculum is placed at the center of a solidified medium in a Petri dish.A given quantity of essential oil is placed on the lid of the petri dish and the dish incubated in reverse position.The solution to be tested evaporates and the volatile phase carries on an inhibiting activity on the germ tested.The essential oil has no direct contact with the inoculum, only the essential oil vapour.

Preparation of culture medium
A potato dextrose agar w/chloramphenicol HIMEDIA brand culture medium was used.According to the manufacturer's instructions, 39.05 g of medium was suspended in 1000 mL distilled water.The suspension was heated to boil so as to enable complete dissolution of medium.It was then sterilised by autoclaving at 121°C at a pressure of 1 bar for 15 min.

Inoculation and application of essential oil
20 mL of culture medium was poured in a Petri dish of diameter 90 mm and height 10 mm.The resultant air space volume in the Petri dish was calculated to be 75 ml.After solidification, a 6 mm well was created at the centre of the medium and a mycelial disc from a two-day old pure culture of the respective fungus was deposited into the well.Pure essential oil at varying volumes of 5, 10, 15, 20 and 25 µL were placed on the lid of the Petri dish with the help of a micropipette, giving corresponding concentrations of 5, 10, 15, 20 and 25 μL/75 ml air space.Placed in a reverse position, the Petri dishes were sealed with parafilm so as to prevent cross contamination.Each concentration was repeated thrice.A negative control was carried out with no essential oil.No reference volatile antifungal substance was available, hence the absence of a positive control.The Petri plates were then incubated at 28±2°C.The incubation duration was dependent on the growth rate of the specific fungus.Mycelial growth diameter (in mm) was recorded after every 24 h until the control plates were completely covered.

Percentage inhibition
The percentage inhibition of fungal growth was calculated as compared to the control without essential oil by the formula: Where Dc (mm) = diameter of fungal growth in the control dish; Dt (mm) = diameter of fungal growth in the test dish

Nature of inhibition
The fungistatic or fungicidal nature of the active essential oil was determined by transferring the inoculated discs from the Petri dishes which had 100% inhibition to a new plate with fresh PDA/chloramphenicol agar medium without essential oil.The absence of growth signifies death of inoculum hence the essential oil was fungicidal; the presence of growth signifies that essential oil was fungistatic.
For each fungal strain, a 3 x 6 factorial design (three essential oils, six concentrations) was used for data analyses.Means of percentage inhibition were compared by the Tukey test through one-way ANOVA using OriginPro 8.0 software.Graphs were plotted using Microsoft Excel 2016.

Effect of essential oil on fungal morphology
The effect of essential oil vapour on fungal morphology was studied by observation of exposed and non-exposed fungi species with a scanning electron microscope (Hafedh et al., 2010).At the end of incubation, fungal mycelia bearing distinct features were harvested with the help of sterile forceps and air-dried.This was to eliminate water from fungal hyphae and to preserve surface structure and prevent collapse of the cells when exposed to the SEM's high vacuum.Before viewing, dried samples were mounted in a JEOL JSC 1200 model coater and coated with a thin layer of gold to prevent the build-up of an electrical charge on the surface and to give a better image.The SEM used was a JEOL scanning electron microscope, JSM 5600 LV model.

Extraction yield of essential oils
M. piperita had the highest extraction yield (4.20%), followed by T. vulgaris (2.93%) and C. zeylanicum (1.44%).These differences in yield might be due to the different metabolic rates and specific intrinsic properties of the plants.M. piperita and T. vulgaris from Pančevo, Serbia, (Soković et al., 2007) had yields of 3.2 and 3%, respectively.C. zeylanicum from Cocotomey-Atlantique, Southern Benin had a yield of 1.1% from dried leaves (Yehouenou et al., 2012).Differences among same plants might be as a result of differences in harvest time, the agro-ecological zones, postharvest treatments and processing conditions.

Chemical composition of essential oils
The compounds and their respective percentages present in the different essential oils as analyzed by GC/MS are shown in Table 1.All three essential oils were each composed of more than thirty compounds, most of which were monoterpenes, especially oxygenated monoterpenes.

In vitro inhibitory effect of essential oil vapour on fungal growth
Not all essential oil concentrations showed total inhibition;  inhibition at 5 μL for T. vulgaris and C. zeylanicum, and at 20 μL for M. piperita.According to the post-hoc Tukey test, there was no significant statistical difference (p>0.05) per concentration of T. vulgaris and C. zeylanicum, whereas M. piperita showed significant statistical difference (p<0.05) in inhibition rate at volumes up to 15 μL.Specifically, the percentage inhibition of M. piperita varied from 44.71 ± 3.21 (for 5 μL) to 100% (20 μL).As illustrated in Figure 2, the higher the amount of essential oil, the greater the percentage inhibition, with 100% inhibition being attained with 10 μL essential oil of T. vulgaris and C. zeylanicum, and 25 μL for M. piperita.For M. piperita, there was a positive correlation between percentage inhibition and amount of essential oil.After nine incubation days, the Petri plates with 5 μL essential oil had the least percentage inhibition (57.38±4.32% for M. piperita).Differences in percentage inhibitions of T. vulgaris and C. zeylanicum were not significant statistically (p>0.05) at all amount of essential oils within the incubation period.
Figure 3 is an illustration of the inhibition of growth of A. parasiticus by different volumes of essential oils after eight days of incubation.As in the other cases, the least percentage inhibition was obtained at 5 μL with values of 49.61±4.50,84.65±3.12 and 87.01±2.42%for M. piperita, C. zeylanicum and T. vulgaris, respectively.Differences in percentage inhibitions at all amounts of essential oils were not significant statistically for T. vulgaris and C. zeylanicum, meanwhile M. piperita showed statistically different (p<0.05)results per amount of essential oil up to 15 μL.The result of the percentage inhibition of essential oils on R. stolonifer after five incubation days is as illustrated in Figure 4.
T. vulgaris and C. zeylanicum exhibited total inhibition at 10 μL, while M. piperita showed total inhibition at 15    was recorded by M. piperita with a volume of 5 μL.There was no significant difference (p>0.05) in the activity of T. vulgaris and C. zeylanicum as both showed total inhibition at all volumes applied.However, at amounts of 15 μL and lesser, M. piperita showed statistically significant (p<0.05)results from the other essential oils, and at higher volumes, all three essential oils had similar outcomes.After noting the volume of essential oil for which total inhibition was exhibited from the preliminary tests, the minimum amount of each essential oil required for total inhibition was determined and is shown in Table 2. Except for A. tamarii and A. flavus, there was no significant statistical difference between the MIC values for T. vulgaris and C. zeylanicum.Meanwhile, MIC values for M. piperita were statistically different from the other essential oils for all fungal species except for A. flavus were results with C. zeylanicum were statistically the   same (p>0.05).On a general note, T. vulgaris was the most active, followed by C. zeylanicum then M. piperita.
The antifungal activity of these essential oils is a function of their individual chemical compositions.Essential oil components either exhibit synergism, additive, antagonistic or can portray individual properties (Burt, 2004).The presence of phenolic compounds in the different essential oils renders them good antifungal agents.Thymol, linalool, carvacrol and eugenol are indications of an outstanding antifungal potential (Hyldgaard et al., 2012) as in the case of T. vulgaris essential oil.The relative high percentage of cinnamaldehyde and its derivatives in C. zeylanicum essential could be responsible for its antifungal activity (Carmo et al., 2008).In addition, the presence of other components such as caryophyllane (21.82%), α-pinene (6.31%), eugenol acetate (4.3%) could also confer antifungal activity.
The carvacrol precursor p-cymene, on its own, is not an excellent antifungal agent (Aligiannis et al., 2001;Bagamboula et al., 2004), but will boost the activities of components with functional side groups (Ultee et al., 2000;Rattanachaikunsopon and Phumkhachorn, 2010).The presence of p-cymene therefore in the essential oils, especially in T. vulgaris and C. zeylanicum is a strong indication of increased antifungal activity.Menthol and menthone components of essential oils are very good antifungal compounds (Sulaiman, 2013).These two components, in addition to α-pinene, limonene may be responsible for the antifungal activity of M. piperita.The presence of phenolic compounds like carvacrol, thymol, eugenol and menthol increase the antimicrobial activity of essential oils.This is attributed to the presence of an aromatic nucleus and a phenolic -OH group known to be reactive and to form hydrogen bonds with active sites of target enzymes (Velluti et al., 2003).

Nature of inhibition
Upon re-inoculation of mycelial discs of Petri dishes that showed total inhibition in freshly prepared culture medium, the three essential oils showed different nature of inhibitions, depending on their concentration.Even at the maximum tested concentration of 25 µl/75 ml air space, the action of M. piperita essential oil was fungistatic on all tested fungi.C. zeylanicum was fungicidal at 25 µl/75 ml air space to R. oryzae and T. purpureogenus, and fungistatic to the other tested fungi species.T. vulgaris was fungicidal to T. purpureogenus at

Morphological change of fungi exposed to essential oil vapour
Morphological change due to exposure to essential oil vapour was studied in order to determine the site of action.Because of their relatively rigid vegetative and distinct features (Sporangiophores (specialised aerial hyphae), sporangia and spores), R. oryzae and R. stolonifer were used to elaborate the change in fungal morphology after exposure to essential oil vapour.Figure 7 shows images of R. oryzae unexposed and exposed to C. zeylanicum vapour as taken by an optical microscope.R. oryzae not exposed to essential oil vapour displayed characteristic rigid hyphae bearing sporangia and spores.This rigid vegetative body was evident by the lactophenol cotton blue that stains the chitin portion of the fungal cell wall, giving it a good contrast.Upon exposure to essential oil vapour, the vegetative bodies of the fungi lost their rigidity, leading to poor or no development of sporangia.Since these spore forming bodies were absent, it justified the complete absence of fungal spores.At the level of the hyphae, treated samples showed poor colouration when stained with lactophenol cotton blue dye, because a higher microscopic magnification (40×) was used.The major component of the fungal cell wall, chitin, was therefore not well developed.
The 3-dimensional views of R. oryzae and R. stolonifer unexposed and exposed to C. zeylanicum essential oil vapour as viewed with a scanning electron microscope (SEM) are as shown in Figures 8 to 11.In the absence of essential oil (Figures 8 and 10), R. oryzae and R. stolonifer showed distinct fungal structures: rigid hyphae, bearing sporangia with spores.The presence of essential oil vapour led to abnormal fungal growth.As a result, hyphae were not well developed, lost fungal rigidity, had no sporangium formation and hence, complete absence of spores.The lost in rigidity implies poorly developed cell wall, leading to a change in morphology.These findings are complementary with that of other researchers who put to evidence the poor development of A. niger by C. zeylanicum essential oil (Carmo et al., 2008).This disruption of the cell wall will definitely lead to leakage of cytoplasmic content of cell.These modifications in the cytological structure may be related to the interference of the essential oil with enzymes responsible for cell wall synthesis (Shukla et al., 2000).
The antifungal property of essential oils could involve inhibition of extracellular enzymes synthesis and the disruption of the cell wall structure resulting in lack of cytoplasm, damage of integrity and ultimately mycelial death.Cytoplasm granulation, cytoplasm membrane rupturing, cytoplasm hyperacidity and break down of the electron transport chain are some structural and metabolic events possibly related to the antifungal property of essential oils (Lopez-Diaz et al., 2002;Hyldgaard et al., 2012).It is also reported that essential oils are able to interfere with the mitochondrial membrane system by a membrane-disruptive activity closely associated with the enzymatic reactions, such as respiratory electron transport, protein transport and coupled phosphorylation (Atanda et al., 2006;Rasooli et al., 2006).

Conclusion
The antifungal activity of the vapour phase of the essential oils of T. vulgaris, C. zeylanicum and M. piperita could be attributed to major components present in the respective essential oils.The high concentration of   oxygenated monoterpene contributed to the increased antifungal activity of the essential oil against tested fungi.Improper development of fungi in the presence of essential oils portrays the fungicidal potential of these essential oils.The fact that essential oils of T. vulgaris, C. zeylanicum and M. piperita are generally recognised as safe gives more virtue for these natural products to be applied conveniently, especially in the vapour phase, as a food preservative instead of synthetic fungicides with multiple site effects.

Figure 1 .
Figure 1.Percentage inhibition of essential oils on R. oryzae after 5 incubation days.

Figure 2 .
Figure 2. Percentage inhibition of essential oils on A. tamarii after 9 incubation days.

Figure 3 .
Figure 3. Percentage inhibition of essential oils on A. parasiticus after 8 incubation days.

Figure 4 .
Figure 4. Percentage inhibition of essential oils on R. stolonifer after 5 incubation days.

Figure 5 .
Figure 5. Percentage inhibition of essential oils on A. flavus after 8 incubation days.

Figure 6 .
Figure 6.Percentage inhibition of essential oils on T. purpureogenus after 9 days.
letter on the same column are not statistically different (p<0.05) according to the Tukey test.

Figure 7 .
Figure 7. Unexposed (A) and exposed (B) R. oryzae to C. zeylanicum essential oil vapour as viewed with an optical microscope at 20 and 40x magnification, respectively after staining with lactophenol cotton blue.

Figure 8 .
Figure 8. R. oryzae unexposed to essential oil vapour as viewed with a SEM.

Figure 9 .
Figure 9. R. oryzae exposed to C. zeylanicum essential oil vapour as viewed with a SEM.

Figure 10 .
Figure 10.R. stolonifer non-exposed to essential oil vapour as viewed with a SEM.

Table 1 .
Chemical composition of essential oils of the studied plants.