Composition and in vitro antifungal activity of Bunium persicum , Carum copticum and Cinnamomum zeylanicum essential oils

Essential oils of Bunium persicum (Parsi Zira), Carum copticum (Ajwain) and Cinnamomum zeylanicum (cinnamon) were tested in vitro as growth inhibitors against six forma special of Fusarium oxysporum. Minimum inhibitory concentration (MIC) and median effective concentrations (EC50) values (μl/L) were also demonstrated. The antifungal activity of essential oils was assayed by poison food medium method (0, 100, 200, 300, 400, and 500 μl/L). Essential oils were extracted by means of hydro-distillation and afterwards, gas chromatography-mass spectrometry (GC-MS) analysis was performed to identify their components. The main constituents were cinnamaldehyd (77.51%), β-linalool (11.13%), αcubebene (1.18%) and 1S, CIS-calamenene (1.06%) for C. zeylanicum, thymol (52.95%), γ-terpinene (13.53%), p-cymene (11.28%) in C. copticum and γ-terpinene (24.02%), cuminaldehyde (20.1%), paracymene (13.09%), benzenemethanol alpha-propyl (13.01%), cyclopentane, 2-methyl-1-methylene-3-(1methylethenyl) (3.57%) in B. persicum. All three essential oils exhibited antifungal effect on the studied fungi species; but essential oil of C. zeylanicum was more effective. The growth of F. oxysporum f. sp. lycopersici, F. oxysporum f. sp. ciceri and F. oxysporum f. sp. melonis was completely inhibited by essential oil of C. zeylanicum at 200 ppm concentrations.


INTRODUCTION
Plant pathogens that include fungi, nematodes, bacteria and viruses can cause diseases or damages in plants (Montesinos, 2003).Among these, fungi are the main pathogen and cause many diseases in plants.Pathogenic fungi also cause yield losses in numerous economically important crops (Fletcher and Bender, 2006).The genus Fusarium is widely found in plant debris and crops (Marasas et al., 1984).Several species use of plant derived products as diseases control agents have been studied, since they tend to have low mammalian toxicity, less environmental effects and wide public acceptance (Lee et al., 2007).Particularly, essential oils were found to exert good antifungal activities both in vitro and in vivo conditions against a wide range of pathogens (Baruah et al., 1996;Caccioni et al., 1998;Reddy et al., 1998;Sharma and Verma, 2004).They may provide potential alternatives to control the agents that are currently used due to their bioactive chemicals content (Isman, 2000).
Parsi Zira (Bunium persicum) is a native plant of limited zones of the West Asia and it grows in northern areas of Khorasan, Kerman, and East of Zagros to Bandar Abbas and South of Alborz in Iran (Bonianpoor, 1995).Generally, Umbeliferae species including B. persicum have antimicrobial properties (Shetty et al., 1994).Sekine et al. (2007) studied antifungal activities of volatile compounds on 52 species against four phytopathogenic fungi and their results showed that B. persicum had the strongest antifungal activity.
The other studied species was Ajwain (Carum copticum) from Umbelliferae family.The mentioned plan is cultivated in black soils particularly along the riverbank throughout India, Iran, Egypt and Afghanistan.This plant grows in Sistan and Baluchestan, Azarbaijan, Esfahan, Khuzestan, Fars, Kerman and Khorasan provinces in Iran.Seeds of this plant are rich in thymol.
Cinnamon (Cinnamomum zeylanicum) is a plant of the Lauraceae family and native to Indonesia; but cultivated in Srilanka and India.The main chemical components of the essential oil, obtained from the leaves, are eugenol, eugenol acetate, cinnamic aldehyde and benzyl benzoate.
However, the aim of this study was to evaluate the efficacy of three natural essential oils in control of diseases caused by F. oxysporum.f. sp.lycopersici, F. oxysporum f. sp.ciceri and F. oxysporum f. sp.melonis at in-vitro condition.

Extraction of essential oils
Seeds of B. persicum and C. copticum, and stem bark of C. zeylanicum were powdered using a blender.The essential oil was extracted for 3 h by hydrodistillation, using Clevenger-type apparatus.Extracted oils were dried by anhydrous sodium sulfate, poured in opaque vials and were stored at 4°C till gas chromatography-mass-spectrometry (GC-MS) analysis.

GC-MS analysis of essential oil
GC-MS analysis was done at 250°C on an Agilent 6890 gas chromatograph at 70 eV.The GC column was as follows: HP-5MS; the size of fused silica capillary was 0.25 × 3000 µm with film thickness of 0.25 µl.In addition, the GC-MS was operated under the following condition: the initial temperature was 50°C and was heated for 5 min, then it was heated up to 240°C at the rate of 3°C per min.Meanwhile, carrier gas (helium) flow was 0.8 ml/min.Identification of compounds was done based on the following explanation: the retention indices were calculated for all volatile constituents using a homologous series of n-alkanes C6 to C24.Moreover, the method of mass spectra was used for the identification of individual compounds with those of similar compounds from a database (Wiely/NBS library) or with authentic compounds and it was confirmed by the comparison of their retention indices with authentic compounds or with those reported in the literature (Ozcan et al., 2006).For quantification purpose, relative area percentages obtained by flame ionization detector (FID), were used without using correction factors.

Bioassay of pathogens
Samples of infected chickpea, tomato and cantaloupe (Melon) with Fusarium wilt symptoms were collected from Kurdistan, Fars and Markazi provinces of Iran.Pathogens were cultured on potato dextrose agar (PDA) at 26 ± 2°C and were stored at 5 to 8°C.Isolates of F. oxysporum recovered from infected plants were tested for their pathogenicity on chickpea, tomato and cantaloupe plants.Seeds sown in sterilized soil were infested with the pathogens in 10 cm diameter pots at a rate of 10 6 cfu/g.Control pots were without any inoculation of pathogen.Pots watered daily and after five weeks treatments were indexed and wilted plants were recorded over time for disease occurrence and pathogen re-isolated from diseases plants.

In vitro antifungal efficacy measurements
The fungal toxicity of the essential oils was evaluated against pathogens by using poisonous media technique (Mishra and Dudey, 1994).Five concentrations (100, 200, 300, 400, and 500 ppm) were mixed with PDA media in 40°C.Requisite amounts of the essential oils were dissolved separately in 0.5 ml of 0.1% Tween-80 and then were mixed with PDA medium for even distribution of the oil in PDA media.For control sets, the requisite amount of sterilized water in place of the oil was added to the medium.
The PDA with added oils was then poured into 9 cm Petri dishes.For inoculation, mycelium was taken from the periphery of 7-day old stock cultures.Plugs of mycelium were removed with a 6 mm diameter cork borer, inverted, and where placed in the center of each Petri dish.Plates were sealed with parafilm to prevent realization of volatile compounds.Four replicate plates were sited up for each concentration, and were plates incubated in the dark at 26 ± 2°C.Fungi toxicity was expressed in terms of percentage of mycelia growth inhibition and was calculated (Pandey et al., 1982).The percentage of growth inhibition of mycelia was calculated by formula I = [(dc-dt)/dc × 100], where I = percentage of growth inhibition, dc = radial growth of pathogen in control, and dt = radial growth of pathogen in treatment.For detection of fungistatic or fungicide effect in which oil was inhibited, fungal disc was reinoculated onto the fresh PDA media and revival of fungal growth was recorded at 26 ± 2°C after 10 days.Then, median effective inhibitory concentration (EC50) of essential oils was measured.

Pathogenicity bioassay
Six isolates of pathogens were obtained from diseased potato, chickpea, and cantaloupe.These isolates belong to F. oxysporum f. sp.lycopersici (F27 and F37), F. oxysporum f. sp.ciceris (F3 and F6) and F. oxysporum f. sp.melonis (F12 and F22).In-vitro antifungal activities of oils showed that all studied essential oils had inhibitory effect on tested isolates.The essential oil obtained from the bark of C. zeylanicum showed the highest antifungal activity against all fungi tested.Mycelial growth of F. oxysporum f. sp.cicer (F3 and F6) and F. oxysporum f. sp.lycopersici (F27) were completely inhibited by essential oil of C. zeylanicum at a concentration of 100 ppm (Table 4), whereas meclial growth was inhibited completely in F. oxysporum f. sp.melonis (F12 and F22) and F. oxysporum f. sp.lycopersici (F37) at a concentration of 200 ppm.
The results of antifungal activity assays showed that C. copticum oil had inhibitory effects on fungi growth at a broad spectrum.All tested fungi species growth were reduced completely by 500 ppm, and in some cases, the growth of some fungal form special was completely inhibited by 100 ppm (Table 5).Parsi Zira essential oil had less antifungal activity than other oils.Its inhibitory effects on F. oxysporum f. sp.ciceri, F. oxysporum f. sp.melonis and F. oxysporum f. sp.lycopersici isolates were 42.45 to 84/95%, 71.65 to 100% and 48.55 to 100%, respectively (Table 6).
It is evident from Table 4 that cinnamon oil showed fungicidal activity against all the pathogens at 100 ppm concentration except F12, F22 and F37 isolates.In the latter three isolates, the oil exhibited fungicidal activity at 200 ppm.Ajwain and essential oils showed fungistatic activity against all pathogens at studied concentrations (Tables 5 and 6).

Determination of EC 50
The EC 50 values for different essential oils against pathogens were calculated according to the liner relation between inhibitory probit and logarithm of concentration.Results of EC 50 for different essentials oils against different pathogens are presented in Table 7.The cinnamon essential oil showed remarkable effects

DISCUSSION
Several studies are going on to explore the potential of essential oils as antifungal agents (Rasul et al., 2011;Sami et al., 2010;Delespaul et al., 2000;Giamperi et al., 2002;Lee et al., 2007;Mares et al., 2004;Mishra and Dudey, 1994;Reuveni et al., 1984).In this study, three essential oils have been studied against three forma special of F. oxysporum.This fungus is plant pathogenic and is responsible for disease at field conditions.So, considering the importance of essential oils as ecofriendly agents, they were studied against this fungus.The inhibition extent of fungal growth was dependent on the concentration and source of used essential oils.It is interesting to note that cinnamon bark oil exhibited high antifungal activity against all isolates of different forma special of F. oxysporum (Table 4) followed by Ajwain oils (Table 5).The literature is also silent on the mode of action of the essential oils when used as postharvest fungitoxicants (Ttipathi and Dubey, 2004).A substance may inhibit the growth of fungi either temporarily (fungistatic) or permanently (fungicidal).Cinnamon bark essential oil was proved to have fungistatic action at 100 ppm concentration for some isolates and fungicidal action at 200 ppm concentration for all studied isolates.Moreover, Ajwain and Parsi Zira had fungistatic mode of action in all studied concentrations (Tables 5 and 6).Conclusively, examination of various concentrations in this study showed promising prospects for the utilization of cinnamon bark oil as a sustainable eco-friendly botanical fungicide.
letter(s) in the same column are significantly different using Duncan's multiple range tests (p < 0.05).Letter in parenthesis indicates fungistatic or fungicidal nature, S = fungistatic and C = fungicidal nature.
letter(s) in the same column are significantly different using Duncan's multiple range test (p < 0.05).Letter in parenthesis indicates fungistatic or fungicidal nature, S= fungistatic and C= fungicidal nature.
letter(s) in the same column are significantly different using Duncan's multiple range tests (p< 0.05).Letter in parenthesis indicates fungistatic or fungicidal nature, S = fungistatic and C = fungicidal nature.

Table 1 .
Components of C. zeylanicum essential oil identified by GC-MS analysis.

Table 2 .
Components of C. copticum essential oil identified by GC-MS analysis.

Table 3 .
Components of B. persicum essential oil identified by GC-MS analysis.

Table 4 .
Antifungal activities of essential oils of Cinnamomum zeylanicum against different form specials of F. oxysporum.

Table 5 .
Antifungal activities of essential oils of C. copticum against different form specials of F. oxysporum.

Table 6 .
Antifungal activities of essential oils of B. persicum different form specials of F. oxysporum.

Table 7 .
Antifungal activity of essential oils against different fungal isolates.Data are expressed as LC50 values in µl per l, representing the concentration to effect 50% of the fungal challenged.