Antifungal activities of basil (Ocimum basilicum L.) extract on Fusarium species

The basil extract composition was determined by the GC-MS method and 38 different components were identified. The major components of the basil extract were estragol (86.72%), trans-α-bergamotene (2.91%), eucalyptol (2.67%), trans-ocimene (1.04%), linalool (0.72%), methyl-eugenol (0.71%), etc. The antifungal potential of the basil extract was tested against Fusarium oxysporum, F. proliferatum, F. subglutinans, and F. verticillioides isolated from cakes, using the agar plate method. Extract concentrations of 0.35 and 0.70% (v/v) significantly inhibited the growth of F. proliferatum (33.37 and 44.30%, respectively) and F. subglutinans (24.74 and 29.27%, respectively) whereas other investigated Fusarium species exhibited much lower sensitivity. The basil extract completely inhibited the growth of investigated Fusarium spp. at the concentration of 1.50% (v/v). Higher concentrations (0.35 and 0.70% (v/v)) reduced growth of aerial mycelium in all tested species. Strong medium pigmentation in the case of F. proliferatum and F. verticillioides was observed. The microscopic examination of the samples confirmed the presence of hyphae deformations with a frequent occurrence of fragmentations, thickenings and diminished sporulation. In addition to the basic, sensory, role the extract of basil has in the food product, it exerted significant antifungal properties, depending on its concentration.

In practice, the use of synthetic preservatives has been the main choice in attempts to prevent microbial spoilage of food commodities and, consequently, the biosynthesis of toxic metabolites.However, today, consumers demand a less use of synthetic preservatives and additives but still expect natural and, above all, safe food.Hence, comprehensive studies have been carried out recently on the use of essential oils, extracts and oleoresins extracted from spices and other aromatic herbs as alternative preservatives providing longer shelf-life and minimal changes in food quality (Benkeblia, 2004;Burt, 2004;Bakkali et al., 2008;Viuda-Martos et al., 2007, 2008;Fawzi et al., 2009;Tajkarimi et al., 2010).The main advantage of extracts is the absence of microbial contamination unlike natural spices which are frequently heavily loaded with microorganisms (McKee, 1995;Karan et al., 2005;Kocić-Tanackov et al., 2009).The antimicrobial activity of extracts has been attributed to many active phytochemicals, inclunding flavonoides, terpenoides, carotenoides, coumarins, and curcumines (Burt, 2004;Ceylan and Fung, 2004;Tepe et al., 2005;Viuda-Martos et al., 2007).
Basil is a popular culinary herb, and its essential oils have been used extensively for many years in the flavoring of confectionary and baked goods, condiments (example, ketchups, tomato pastes, chili sauces, pickles, and vinegars), sausages and meats, salad dressings, nonalcoholic beverages, ice cream, and ices (Suppakul et al., 2003).
The objective of this study was to evaluate the antifungal potential of the commercial basil (Ocimum basilicum L.) extract, intended for uses in food, against some Fusarium spp.isolated from cakes.

Basil extract
For the antifungal activity testing, a commercially available, food grade basil extract was provided from ETOL Tovarna arom in eteričnih olj d.d., Celje, Slovenia.

Determination of basil extract composition
The composition of extracts was determined by Gas Chromatography -Mass Spectrometry (GC-MS) analyses.The GC-MS analysis was carried out on a Varian T2100 GC-MS instrument equipped with data processor.A fused silica capillary column VF-5MS (30 m x 0.25 mm i.d., 0.25 m film thickness, Varian) was used for the separation of the sample components.The carrier gas ultra pure helium was passed through moisture and oxygen traps with constant flow rate of 0.62 cm 3 min.The following temperature program was used: injector temperature 230°C, initial temperature 40°C (held 5 min), temperature increase 5°C/min to 200°C and held at this temperature for 25 min.The mass spectrometer was operated in the electron ionization mode.The data acquisition was carried out in the scan mode (range 50 to 550 m/z).The injection volume was 1 μl.The compounds were identified by matching the mass spectra with NIST Mass Spectra Library stored in the GC-MS database.

Fungal strains
As test microorganisms, the following fungal strains from the genus Fusarium were used: F. oxysporum Schlecht., F. proliferatum (Matsushima) Nirenberg, F. subglutinans (W ollenw.& Reinking) Nelson, Toussoun and Marasas and F. verticillioides (Sacc.)Nirenberg (syn.F. moniliforme Sheld.).The fungal cultures were isolated from cakes and maintained on Potato Dextrose Agar (PDA) (Merck, Darmstadt) at 4°C as a part of the collection of the Laboratory for Food Microbiology at the Faculty of Technology, University of Novi Sad, Serbia.

Determination of the basil extract effect on the Fusarium spp. growth
The agar plate method was applied in the testing of the antifungal activity of basil extracts.The basic medium for the antifungal tests Kocić-Tanackov et al. 10189 was PDA.The medium was divided into equal volumes (150 mL), poured into Erlenmeyer (volume 250 mL) flasks and autoclaved at 121°C for 15 min after which it was cooled to 45°C.The extracts were added to the PDA to achieve the following concentrations: 0, 0.16, 0.35, 0.70, and 1.50 % (v/v).PDA containing different concentration of basil extract was poured in sterile Petri dishes ( 9 cm), 12 mL into each dish.Seven-day fungal cultures grown on PDA were used to prepare fungal spore suspension tests.Suspensions of fungi were prepared in a medium which contained 0.5% Tween 80 and 0.2% agar dissolved in distilled water and were adjusted to provide initial spore count of 10 6 spores mL by using a haemocytometer.For each extract dose and fungal species, including the controls, dishes were centrally inoculated by spreading 1 µL of a spore suspension (10 3 spores/ mL) using an inoculation needle.After inoculation, the Petri dishes were closed with a parafilm.
The effect of the basil extract on fungal growth was evaluated by a daily measurement of the diameter of the radial colony growth during 14 days of incubation at 25 2°C.The parafilms were removed from the Petri dishes in which no colony growth was observed after 14 days, and the dishes were further incubated for 16 days (30 days in total) at 25 2°C.In Petri dishes in which fungal growth was observed from 15 th to 30 th day, concentration of basil extract used was considered to be minimal inhibitory concentration (MIC).If there was no visible fungal growth after 30 days, fungal spores were transferred using a wet cotton baton to the PDA in which no basil extract was added and were incubated for 5 days at 25 2°C for the determination of fungicide effect (MFC).
The inhibitory effect of the basil extract on fungal growth after 14 days was calculated following the formula: Where, I is the inhibition (%); C is the colony diameter on the control dish (cm) and T is the colony diameter on the test dish (cm) (Pandey et al., 1982).
Changes in macroscopic and microscopic features of fungi were also observed and compared to the controls.The macroscopic features of spores were observed using a binocular, magnifying glass Technival 2, Carl Zeiss and the microscopic features using a microscope Aristoplan, Leitz.

Statistical analysis
Each antifungal test was carried out in 3 series and 2 replications.MS Excel was used to calculate means and standard deviations.Significant differences between values were determined by Duncan s multiple range test (p<0.05),following one-way ANOVA.
Effect of the basil extract on the Fusarium spp.growth The basil extract at the investigated concentrations exhibited the capacity to reduce or inhibit the growth of the Fusarium species.Tables 2 to 5 give the growth of the fungi F. verticillioides, F. oxysporum, F. proliferatum, and F. subglutinans, during the 14 days of the incubation.
Table 6 shows the inhibitory effect (%) of the basil extracts on the colony growth of Fusarium species on the 14 th day of incubation.
The growth of the investigated Fusarium spp. was observed already during the first day when the lowest concentration of the basil extract was applied (0.16% (v/v)).At this concentration, a statistically significant reduction in the colony growth compared to the control was determined on the 5 th day of incubation of F. verticillioides (Table 2), on the 1 st day of incubation of F. oxysporum (Table 3), on the 7 th day of incubation of F. sublutinans (Table 4), and on the 3 rd day of incubation of F. proliferatum (Table 5).Higher extract concentrations caused the growth delay of investigated Fusarium spp. with different inhibitory effects on the reduction of the growth rate.
The basil extract concentration of 0.35% (v/v) delayed the growth for a day only in F. proliferatum, while the growth of the other investigated Fusarium spp.occurred already on the first day of the incubation.At this concentration, statistically significant reduction in the colony growth compared to the control sample was determined in F. verticillioides on the 4 th day of incubation (Table 2), and for F. oxysporum, F. subglutinans, and F. proliferatum on the 1 st day (Tables 3 to 5).At the extract concentration of 0.70% (v/v), the growth of F. oxysporum, F. subglutinans, and F. proliferatum was observed on the 2 nd , 3 rd and 4 th day, respectively.The growth inhibition of all investigated Fusarium spp. at this concentration was statistically significant compared to the control sample.
The growth inhibition of Fusarium spp.after the 14 th day of incubation at the lowest concentration was insignificant, ranging from 8.08% (F.subglutinans) to 15.22% (F.proliferatum) (Table 6).Concentrations of 0.35 and 0.70% (v/v) significantly inhibited the growth of F. proliferatum (33.37 and 44.30%, respectively) and F. subglutinans (24.74 and 29.27%, respectively), while other investigated Fusarium spp.showed lower sensitivity.At these concentrations, the highest tolerance that is sensitivity was detected in F. veriticillioides, F. proliferatum, respectively.The complete inhibition of observed Fusarium species was obtained at the  Values followed by the same small letter (a-e) within the same row are not the significantly different (p>0.05) according to Duncan's multiple range test.Values followed by the same capital letter (A-N) within the same column are not the significantly different (p>0.05) according to Duncan's multiple range test.
concentration of 1.50% (v/v) after the 14 th day of incubation (Table 6).
A visible growth of the investigated Fusarium species was not observed after 30 days of incubation at the concentration of 1.50% (v/v).The transfer of inoculated spores free from extract did not result in spore germination; hence, this concentration is the minimum fungicide concentration (MFC) for the observed Fusarium species.The greatest reduction in the mycelial growth by increasing extract concentrations was observed in F.  Values followed by the same small letter (a-e) within the same row are not the significantly different (p>0.05) according to Duncan's multiple range test.Values followed by the same capital letter (A-N) within the same column are not the significantly different (p>0.05) according to Duncan's multiple range test.
proliferatum and F. subglutinans, which implies that their sensitivity is higher than sensitivity of other two species (Tables 2 to 5).
In general, data on the antifungal activity of basil essential oils and extracts against Fusarium species and other species such as Aspergillus species are rather scarce (Hitokoto et al., 1980;Baratta et al., 1998;Zollo et al., 1998;Basilico and Basilico, 1999;Özkan and Erkmen, 2001;Alpsoy, 2010).On the other hand, data on the effects of the essential oils and extracts obtained from other spice herbs against Fusarium species are more profuse than those of basil essential oils and basil extracts (Velluti et al., 2003).Furthermore, the antimicrobial activity of an essential oil is attributed mainly to its major compounds.The synergistic or antagonistic effect of one compound in minor percentage in the mixture has to be considered (Daferera et al., 2003;Velluti et al., 2003).This clearly suggests that the antifungal activity of essential oil or extract of each herb or even a genotype (Hasegawa et al., 1997) has to be investigated separately against a particular fungal species.Lis-Balchin et al. (1998) studied the antifungal activity of the methyl chavicol type of basil essential oil against F. culmorum (W.G.Smith) Sacc.and found that the oil exhibited 71% reduction in the mycelial growth of the tested species.Reuveni et al. (1984) studied the percentage of inhibition of principle constituents of basil against F. oxysporum.They found that eugenol demonstrated the highest percentage of inhibition (100%) against F. oxysporum, whereas for linalol and methyl chavicol, showed an inhibition of 26.4 and 30.3%, respectively.However, the results reported by Adigüzel et al. (2005) showed that none of the three tested basil extracts (ethanol, methanol, and hexane) exhibited antifungal activities against F. oxysporum.
Basil oil was found to significantly affect the growth of F. verticillioides after 7 days of incubation (Fandohan et al., 2004).It totally inhibited the growth of F. verticillioides at concentrations lower than 2.7 µL/mL.Basil oil remained effective over 21 days, with minimal inhibitory concentrations of 1.3 µL/mL.Soliman and Badeaa (2002) found that basil oil demonstrated fungistatic activity against F. verticillioides at concentration of 2000 and fungicidal activity at concentration of 3000 ppm.Essential oil from O. gratissimum L. originating from Kenya showed a strong inhibitory effect against fumonisin B 1 (FB 1 ) production by F. (Dambolena et al., 2009), which can be attributed to its high eugenol content.Jayashree and Subramanvam (1999) reported that the antiaflatoxigenic activity of eugenol was due to the inhibition of lipid peroxidation.
The investigated basil extract, in addition to the growth inhibitory effect, caused changes in the macro and micro morphology of fungi.At higher concentrations (0.35 and 0.70% (v/v)) reduced growth of aerial mycelium in all investigated Fusarium species was observed, accompanied with stronger medium pigmentation in the case of F. proliferatum, and F. verticillioides.At these concentrations, the microscopic examination confirmed the presence of hyphae deformations with a frequent occurrence of fragmentations, thickenings and diminished sporulation.In F. proliferatum, 0.70% (v/v) extract concentration caused granular appearance of hyphae and deviations as well as deformed phyalides.These deformations may be related to the changes at the cellular level (decrease in the oxygen uptake, reduction of the cellular growth, inhibition of the synthesis of lipids, proteins and nucleic acids, changes in the lipid profile of the cell membrane, and inhibition of the fungal cell wall synthesis (Adetumbi et al., 1986;Ghannoum, 1988;Gupta and Porter, 2001;Corzo-Martinez et al., 2007).For example, the investigations of cellular ultra structures of A. niger after exposure to etheric oils of thyme and ajoene extracted from garlic caused the absence of surface ornaments, thinning of cell walls, abruption of the cell membrane from the cell wall, surface hyphae deviations, and the destruction of cellular organelles (Yoshida et al.,1987;Rasooli et al., 2006).It can be assumed that active components of essential oils like thymol, eugenol, and carvacrol damage the biological membrane by bonding to essential enzymes integrated with the cellular membranes (Knobloch et al., 1989;Davidson, 2001).
Literature data suggest that essential oils can be practically applied in post-harvest disease control or mycotoxins inhibition on stored kernels (Soliman and Badeaa, 2002).Basil essential oil significantly diminishes the severity of grain infestation with F. verticillioides and inhibits the radial colony growth in comparison to the control (Kabore et al., 2006).Essential oil of O. gratissimum was found to be highly efficient against F. verticillioides producing high infection suppression figure of 95-100% (Nguefack et al., 2008).These findings showed that essential oils reduced the natural microbial load of kernels by 95 to 100%.The use of this oil at 200 ppm concentration significantly reduced (86%) the mycelial growth of F. verticillioides as well as spore germination at 500 ppm.The in vivo test showed that oil from O. basilicum reduced significantly the incidence of F. verticillioides in artificially inoculated corn, compared to the controls (Fandohan et al., 2004).A reduction of the toxin level up to 60%, however, was observed at concentrations of >4.8 µL/g and complete inhibition occurred at 6.4 µL/g.At the concentration of 4.8 µL/g, basil oil significantly adversely affected the germination of corn kernels.These findings suggest that at 4.8 µL/g concentration, basil oil can be recommended only for the treatment of kernels intended for human and animal consumption, but not for the treatment of kernels to be used as a seed material.

Conclusions
The obtained results suggest that the investigated basil extract, besides improving a sensory appearance of food, could be used as preserving agent against Fusarium species (F. oxysporum, F. proliferatum, F. subglutinans, and F. verticillioides) which are known as frequent contaminants of food commodities.Further investigations should deal with broadening the practical application of the basil extract especially with regards to its role in protection of cereals and cereal products against Fusarium species and their metabolites.

Table 1 .
Chemical composition of basil extract.
a Relative area percentage.

Table 2 .
Effect of the basil extract on the colony growth of F. verticilioides.
Values followed by the same small letter (a-e) within the same row are not the significantly different (p>0.05) according to Duncan's multiple range test.Values followed by the same capital letter (A-N) within the same column are not the significantly different (p>0.05) according to Duncan's multiple range test.

Table 3 .
Effect of the basil extract on the colony growth of F. oxysporum.

Table 4 .
Effect of the basil extract on the colony growth of F. proliferatum.
Values followed by the same small letter (a-e) within the same row are not the significantly different (p>0.05) according to Duncan's multiple range test.Values followed by the same capital letter (A-N) within the same column are not the significantly different (p>0.05) according to Duncan's multiple range test.

Table 5 .
Effect of the basil extract on the colony growth of F. subglutinans.

Table 6 .
Inhibitory effect (%) of basil extract on colony growth of Fusarium species after 14 days of incubation.