Evaluation of the antifungal activity of the Iranian thyme essential oils on the postharvest pathogens of Strawberry fruits

Postharvest diseases cause considerable losses to harvested fruits and vegetables during transportation and storage. The aim of this study was to evaluate the antifungal potential of Thymus danensis and Thymus carmanicus against four postharvest pathogenic fungi (Rhizopus stolonifer, Penicillium digitatum, Aspergillus niger and Botrytis cinerea) which can reduce the shelf life of strawberry fruit. The chemical composition of plant oils was determined by capillary gas chromatography and mass spectrometry. Antifungal assays were carried out in vitro using PDA plates. Antifungal potential was found for 2 analysed essential oils. T. carmanicus oils have chemical compositions characterized by carvacrol (70%), p-cymene (12.4%) and γ-terpinene (2.5%) as the major components while the major constituents of the T. danensis were thymol (64.8%), α-terpinene (11.3%) and p-cymene (7.9%). Thymus sp. oils showed inhibitory effect even at low concentration (300 μl/L) against B. cinerea and R. stolonifer and showed inhibitory effect against A. niger and P. digitatum at 600 μl/L. Both essential oils tested in vivo at the preliminary concentration exhibited inhibitory activity against the four pathogens.


INTRODUCTION
Postharvest diseases cause heavy losses of fruits during storage. The species reported to damage strawberries during this period include Rhizopus stolonifer, Penicillium digitatum, Aspergillus niger, and Botrytis cinerea. There have been some studies on the effects of essential oils on postharvest pathogens (Bishop and Thornton, 1997). These essential oils are thought to play a role in plant defence mechanisms against phytopathogenic microorganisms (Mihaliak et al., 1991). Most of the essential oils have been reported to inhibit postharvest fungi in in vitro conditions (Bellerbeck et al., 2001;Hidalgo et al., 2002). However, the in vivo efficacy and practical activity of only a few of the essential oils have been studied. There are also some reports on essential oils in *Corresponding author. E-mail: nabigol@iau-abhar.ac.ir. Tel: +98 261 273 2498. Fax: +98 261 2248378. enhancing the storage life of fruit and vegetables by controlling their fungal rotting. Dubey and Kishore (1988) found that the essential oils from leaves of Melaleuca leucadendron, Ocimum canum and Citrus medica were able to protect several stored food commodities from biodeterioration caused by Aspergillus flavus and Aspergillus versicolor.
Thymol is an essential oil component from thyme and has been used as medicinal drug, food preservative, and beverage ingredient (Jain, 1985;Mansour et al., 1986). Fumigation of sweet cherries with thymol was effective in controlling postharvest grey mold rot caused by B. cinerea (Chu et al., 1999), and brown rot caused by M. fructicola (Chu et al., 2001). Fumigation with thymol at 30 mg/l reduced the incidence of grey mold rot from 35% in untreated fruit to 0.5%. Liu et al. (2002) also found that thymol was more effective for controlling brown rot symptoms on apricots, and fumigation of plums with relatively low concentrations such as 2 or 4 mg/l can greatly reduce postharvest decay without causing any phytotoxicity.
The main purpose of this study was to evaluate the antifungal potential of Thymus danensis and Thymus carmanicus oils against four important pathogenic fungi (R. stolonifer, P. digitatum, A. niger, and B. cinerea) which can reduce the shelf life of strawberry fruit.

Plant material
Plant materials of T. danensis and T. carmanicus were collected from Karaj in Tehran province (Iran) in August 2007. A voucher specimen was deposited at the herbarium of College of Agriculture and Natural Resource, University of Tehran, Iran.

Essential oils extraction and GC and GC-MS analysis
About 200 g dried material (plant material was dried at ambient temperature and shade condition) of each species were cut into pieces, ground and then submitted to hydrodistillation for 3 h using a Clevenger type apparatus. The obtained essential oils were dried with anhydrous sodium sulphate and stored at 4°C before analysis and use for antifungal study.
The composition of the oils was analysed by GC and GC-MS. The GC apparatus (Shimadzu model GC-7A, Tokyo, Japan) was combined with a Flame Ionization Detectors (FID, Shimadzu, Tokyo, Japan) equipped with DB-1 column (60 m x 0.25 mm ID, film thickness 0.25 µm). The oven temperature was programmed to increase from 60 to 250°C at a rate of 3°C/min and finally held isothermal for 4 min. The carrier gas was helium at a flow rate of 1.1 ml/min. Detector temperature was adjusted to 280°C and injector temperature to 250°C (Adams, 2001).
After optimization of GC conditions, each sample was injected to the GC-MS (Shimadzu GC-MS model QP-1100 EX, Tokyo, Japan). GC-MS was equipped with the column of DB-1 (30 m x 0.25 mm ID, film thickness of 0.25 mm). The GC temperature program was also used for GC-MS, for ionizing a voltage of 70 eV, which was used in MS analysis. The components were identified by comparison of their mass spectra with those of NIST98 library data of the GC-MS system and Adams libraries spectra (Adams, 2001), as well as by comparison with the compounds' elution order with their retention indices reported in the literature (Adams, 2001). Retention indices of the components were determined relative to the retention times of a series of n-alkanes with linear interpolation. Major compounds of both oils were obtained from Merck and Sigma-Aldrich companies.

Fungal strains
B. cinerea UTBC113, A. niger UTAN118, P. digitatum UTPD111 and R. stolonifer UTRH110 were obtained in mycological collection of department of plant protection, University of Tehran.

In vitro antifungal assay
The antifungal tests were carried out in vitro according to the method described by Pitarokili et al. (2003) using Petri dishes 8 cm in diameter containing potato dextrose agar (PDA). The essential Nabigol and Morshedi 9865 oils were dispersed as an emulsion in water using Tween 20 (0.05%) and added to PDA immediately before it was filled into the Petri dishes at a temperature of 45-50˚C. The concentrations tested were 150, 300, 600 and 1200 µl/L. The controls included the same quantity of Tween 20 mixed with PDA. The phytopathogenic fungi were inoculated immediately after preparation of the Petri dishes by placing in the centre of each plate a 6 mm diameter disk of the test species, cut with a sterile cork borer from the periphery of actively growing cultures on PDA plates. The Petri dishes were incubated in the dark at a temperature of 24˚C. Mean growth rates were calculated from five replicates of each fungal species every 24 h until fungi in the control filled the Petri dishes completely. The measurement of the fourth day was used to determine the minimum inhibitory concentration (MIC) and the EC50 values (concentration causing 50% inhibition of mycelial growth on control media). In addition, the antifungal activity of major compounds of both oil were determined. Fungi toxicity was expressed as the percentage of mycelial growth inhibition. EC50 values were calculated from the data subjected to Probit analysis (statistical software SPSS 10.0 Inc., Chicago, IL). To check the fungicidal or fungistatic properties, parts of the media from plates without mycotic growth were transferred into new PDA plates; no fungal growth after an incubation of 10 days was indicative of fungicidal activity.

In vivo assay
Strawberries (Fragaria ananassa Duch., cv Selva) were obtained from a commercial greenhouse, washed in 1% sodium hypochlorite and distilled water twice and after drying were dipped in each fungi conidial suspension (105 conidia per ml) for 1 min. Essential oils were prepared as emulsion in water using Tween 20 (0.05%) and then fruit treated with these emulsions and same quantity of Tween 20 as control. Spraying, dipping and fumigation were the methods of treatment. Fruits were placed in plastic trays and sealed with PVC stored in 25˚C until fungi in control covered their surface completely. Each fruit divided to 8 pieces and observation of disease symptoms in each pieces estimated equal 12.5% of disease incidence. The data were collected from 4 replicates each of 8 fruits.

Chemical composition of the essential oils
By hydrodistillation, the dried materials of T. danensis and T. carmanicus yielded 5.1 and 6.3% (v/w) of essential oils respectively. The chromatographic analyses resulted in the identification of 30 components, representing 98.1-99.6% of the oils. Quantitative and quali-tative analytical results by GC-MS are shown in Table 1. About 30 compounds of the oils were determined. T. carmanicus oil has chemical compositions characterized by carvacrol (70%), p-cymene (12.4%) and γ-terpinene (2.5%) as the major components while the major constituents of the T. danensis were thymol (64.8%), α-terpinene (11.3%) and p-cymene (7.9%).
There are considerable differences between the oils compositions obtained in this study and those previously reported (Nickavar al., 2005). This may be attributable that the amounts of the essential oil differ by geographic region, collection time, altitude and climate.

Antifungal activity
Incorporation of different essential oil concentrations (150, 300, 600 and 1200 µl/L) in PDA showed a considerable reduction on the growth of four common pathogens of strawberry fruits (Figure 1). The inhibition of the fungi depends on the concentration of the essential oils. The essential oils presented noticeable antifungal activity. Complete minimum inhibitory concentration (inhibition 100%; MIC) of both oils against B. cinerea, and R. stolonifer were 300 µl/L. The MIC of T. danensis and T. carmanicus against P. digitatum and A. niger was 600 µl/L (Table 3). The minimum fungicidal concentration (MFC) of the oils of T. Danensis and T. carmanicus against B. cinerea, and R. stolonifer was 1200 µl/L whereas a fungicidal effect was not observed on P. digitatum and A.niger even at high concentration ( Table  2).The major compounds of both oils (p-cymene, αterpinene, Thymol and carvacrol) also exhibit antifungal activity (Figure 2 and Table 3). The MIC of p-cymene and α-terpinene was 600 µL/L on B. cinerea and R. stolonifer and 1200 µL/L on A. niger, whereas the MFC of the both compounds on all pathogens was more than 1200 µL/L. The MIC of Thymol and carvacrol against R. stolonifer was 300 and 150 µL/L respectively. In addition, the MFC of thymol and carvacrol was 600 µL/L against B. Cinerea and 1200 and 600 µL/L against R. stolonifer respectively, whereas it was more than 1200 µL/L on A. niger (Table 3). However, thymol and carvacrol exhibited fungicidal activity at the highest concentration (1200 µL/L) whereas both oils didn't show fungicidal activity at this concentration.
Both essential oils tested in vivo at the preliminary concentration exhibited inhibitory activity against four pathogens ( Figure 3). T. carmanicus oil had better result and significantly decrease the disease incidence in strawberry fruits. These essential oils were more effective against A. niger on strawberry fruits. In the case of A. niger, dipping of fruits in T. carmanicus and T. danensis and essential oils causes less disease incidence with 18 and 30%, respectively. In all cases, dipping and spraying the essential oils were better than fumigation. Biological activity of essential oils depends on their chemical composition which is determined by the genotype and influenced by environmental and agronomic conditions (Marotti et al., 1993). The antifungal and antibacterial activity exhibited by Thymus genus essential oil has been demonstrated by several researchers (Rasooli and Mirmostafa, 2003). Antifungal and antibacterial activity of carvacrol and thymol as main constituents of T. danensis were reported in different studies (Consentino et al., 1999). Therefore, considerable antifungal activity of these oils observed in this study can be attributed to the presence of these compounds in the oils and their synergistic effects. Sensitivity of fungal species to plant essences is different and depends on essence type and dose of application. Difference in antifungal activity of essences depends on the ingredients. It is possible that one compound results in antifungal activity alone or in synergism with other compounds (Plotto et al., 2003). In the study, B. cinerea, and R. stolonifer were the most sensitive fungus that were completely in-hibited at a concentration of 300 µl/L whereas P. digitatum and A. niger were the most resistant fungus which cannot be inhibited completely even at 1200 µl/L.
Recently, the exploitation of natural products to control decay and prolong storage life of perishable commodity    has received more attention. Biologically, active natural products have the potential to replace synthetic fungicides (Tripathi and Dubey, 2004). The essential oil is one of the plant extracts applicable for the management of fungal rotting of fruit and vegetables, thereby prolonging shelf life (Meepagala et al., 2002). Most of the essential oils have been reported to inhibit postharvest fungi under in vitro conditions (Bellerbeck et al., 2001). However, the in vivo efficacy and practical activity of only some essential oils have been studied.
In conclusion, examination with various concentrations of these oils exhibited promising prospects for the utilization of essential oils against a wide spectrum of microorganisms without leaving detectable residues. So essential oils can be used as a source of sustainable eco-friendly fungicides.