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Journal of Wood Science

Official Journal of the Japan Wood Research Society

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Antifungal activity of essential oils isolated from Egyptian plants against wood decay fungi

Journal of Wood Science volume 59, pages 499–505 (2013)Cite this article

Abstract

The essential oils of eighteen Egyptian plants were extracted by hydrodistillation and their chemical compositions were analyzed by GC-MS. The antifungal activity of the isolated oils was evaluated against two wood decay fungi Hexagonia apiaria and Ganoderma lucidum in vitro. The essential oil of Artemisia monosperma showed the highest inhibitory effect against H. apiaria (EC50 = 31 mg L−1) and G. lucidum (EC50 = 53 mg L−1). The results of in vitro tests indicated that the essential oils of Cupressus sempervirens, Citrus limon, Thuja occidentalis, Schinus molle, A. monosperma and Pelargonium graveolens were the most potent inhibitors against both fungi. These six oils caused significant reduction of wood mass loss of Scots pine sapwood after 6 weeks of fungal exposure. The oil of C. limon revealed the highest reduction of wood mass loss caused by H. apiaria, while A. monosperma oil displayed the highest reduction of wood loss caused by G. lucidum. These results support the potential use of essential oils for wood protection against decay fungi.

Introduction

A wide range of organic and inorganic fungicides is currently in use for wood preservation industry [1]. However, because of the health concerns associated with the use of these chemical treatments, the recent trend has been to develop environmentally benign approaches for the protection of wood. In this context, the search for simple bioactive compounds derived from plants that can be used against wood decay fungi has been a research direction for ecologically safe products [2]. For instance, essential oils are known to contain a natural mixture of monoterpenes, sesquiterpenes, diterpenes, and hydrocarbons, with a variety of functional groups, giving them antibacterial, antifungal, antitermite, and insecticidal activities. To protect wood from fungal and other biological damage, natural preservatives such as essential oils could be an attractive alternative compared to the use of highly toxic traditional wood preservatives [3]. However, essential oils have a wide use in pharmaceutical and food industry as antimicrobial agents, but their use as wood preservatives has not been fully explored. In this study, the chemical composition of essential oils from eighteen Egyptian plants was described using gas chromatography-mass spectroscopy (GC-MS), and their antifungal activities against two common tropical wood decay fungi were investigated.

Materials and methods

Test fungi

Two tropical wood decay fungi (provided from Laboratoire d’Etudes et de Recherche sur le Matériau Bois, LERMAB, Nancy University, France) Ganoderma lucidum and Hexagonia apiaria were used in this study. These two fungi are common for causing wood decay and white rot and are used in European standard methods (EN 113) for evaluating wood decay. Fungal cultures were grown and maintained in 90-mm Petri dishes containing 20 mL of Malt Extract Agar medium (MEA); 4 % malt extract, 2 % agar at 27 °C and 75 % relative humidity (R.H).

The two tested fungi are among the many fungi that cause wood decay and white rot.

Plant materials

Various parts of eighteen plant species: Artemisia judaica L. (aerial parts), Artemisia monosperma Del. (leaves), Callistemon viminals (Sol. ex Gaertn.) G. Don (leaves), Citrus aurantifolia (Christm.) Swingle (fruit peels), Citrus limon (L.) Burm.f. (fruit peels), Citrus paradisi Macfad (fruit peels), Citrus sinensis (L.) Osbeck (fruit peels), Cupressus macrocarpa Hartw. ex Gordon (leaves), Cupressus sempervirens L. (leaves), Myrtus communis L. (leaves), Origanum vulgare L. (aerial parts), Pelargonium graveolens L’Her (leaves) Rosmarinus officinalis L. (leaves), Syzygium cumini L. Skeels (leaves), Schinus molle L. (leaves), Schinus terebinthifolius Raddi (leaves) Thuja occidentalis L. (leaves) and Vitex agnus-castus L. (leaves) were collected during the flowering stage from different locations of Alexandria, Behira and Matrouh Governorates, Egypt, in August 2010–April 2011. These plants are common ornamental trees and wild-grown herbs in Alexandria region north Egypt. The plant materials were identified by Prof. FathAllah Zaitoon of Plant Pathology Department, Faculty of Agriculture, Alexandria University. Voucher specimens have been deposited in Department of Pesticide Chemistry, Faculty of Agriculture, Alexandria University.

Isolation of essential oils

The aerial plant parts were partially dried at room temperature (26 ± 1 °C) for 5 days and the fruit peels were used fresh. Essential oils were extracted by hydrodistillation in a Clevenger-type apparatus for 3 h. The oils were dried over anhydrous sodium sulfate, and stored at 4 °C until used for GC-MS analysis and biological activity tests.

Analysis of essential oils

Essential oils were diluted in diethyl ether and 0.5 μl was injected into the gas chromatography (Hewlett Packard 5890)/mass spectrometry (Hewlett Packard 5989B) (GC-MS) apparatus. The GC column was a 30 m (0.25 mm i.d., film thickness 0.25 μm) HP-5MS (5 % diphenyl) dimethylpolysiloxane capillary column. The GC conditions were as follows: injector temperature, 240 °C; column temperature, isothermal at 70 °C for 2 min, then programmed to 280 °C at 6 °C/min and held at this temperature for 2 min; ion source temperature, 200 °C; detector temperature, 300 °C. Helium was used as the carrier gas at the rate of 1 mL/min. The effluent of the GC column was introduced directly into the ion source of the MS. Spectra were obtained in the EI mode with 70 eV ionization energy. The sector mass analyzer was set to scan from 40 to 400 amu for 5 s. The oil components were identified by comparison of their retention indices and mass spectra with the NIST Mass Spectral Library.

In vitro antifungal assay

Antifungal assessment of the isolated essential oils was conducted using a mycelial radial growth inhibition technique against G. lucidum and H. apiaria. The oils were diluted with acetone and added to sterilized MEA medium before pouring in 9-cm Petri dishes. The oils were tested at concentrations ranged from 10 to 1000 mg L−1. The discs of mycelial culture (0.5 cm diameter) of fungi, taken from 8-day-old cultures, were transferred aseptically to the center of the Petri dishes. The plates were incubated at 27 °C in the dark. Three replicates were setup for all concentrations and control. Colony growth diameter was measured after the fungal growth in the control treatments had completely covered the Petri dishes. Inhibition percentage of mycelial growth was calculated as follows:

$$ {\text{Mycelial growth inhibition (\% ) }} = \, \left[ {\frac{{\left( {{\text{DC}} - {\text{DT}}} \right)}}{\text{DC}}} \right] \, \times { 100} $$

where DC and DT are average diameters of fungal growth of control and treatment, respectively. Effective concentration that caused 50 % inhibition of mycelial growth (EC50) and its corresponding 95 % confidence limits were estimated by probit analysis [4].

In vivo antifungal assay

Based on the obtained data from the in vitro antifungal assays, six plant essential oils (C. sempervirens, C. limon, T. occidentalis, S. molle, A. monosperma and P. graveolens) were used for the in vivo antifungal assays. The assay was carried out as described by Mohareb et al. [5]. Mini-block specimens (5 × 10 × 50 mm, radial × tangential × longitudinal) from Scots pine sapwood (Pinus sylvestris) are used. Four replicates (12 blocks) for each treatment were oven dried at 103 °C, and weighed, then impregnated with the respective treating solutions of the different essential oils at three levels of concentrations, which varied based on the obtained EC50 values in the in vitro assessment. The tested concentrations were EC50 value that obtained from the in vitro assessment, two- and fourfold of EC50 [6]. The samples were then wiped with a paper tissue before the weight was recorded for control of uptake. The treated wood specimens were then conditioned by transferring them into air-tight containers for 1 week to allow any potential fixation to occur. Upon drying they were then packaged and sterilized by exposure to ethylene oxide gas, and exposed to an active growth of the test fungi (G. lucidum and H. apiaria) inoculated 2 weeks prior to the placement of the blocks in Petri dishes. Three specimens were placed in each test plate. A perforated plastic mat was placed between the fungi growing on malt agar and the test block to prevent contact of the blocks with the agar medium. The blocks were then exposed to the tested fungi at 27 °C and 75 % R.H. After 6 weeks of fungal exposure, the blocks were removed from the incubation, brushed carefully to remove any adhering mycelium and placed on racks to air-dry (for 3–4 days). Once air-dried, blocks were oven dried at 103 °C, after which all the blocks were weighed and percentages of mass losses were calculated.

Statistical analysis

Statistical analysis was performed using the SPSS 16.0 software program (Statistical Package for Social Sciences, USA). The log concentration-response lines allowed determination of the EC50 values for the fungal bioassay according to the probit analysis [4]. The 95 % confidence limits for the range of EC50 values were determined by the least-square regression analysis of the relative growth rate (% control) against the logarithm of the compound concentration. The data of the in vivo experiments were analyzed by one-way analysis of variance (ANOVA). Mean separations were performed by Student–Newman–Keuls (SNK) test and differences at P < 0.05 were considered as significant (Cohort software Inc. 1985).

Results and discussion

Chemical composition of the isolated essential oils

The chemical composition of the eighteen essential oils obtained by hydrodistillation was analyzed using GC-MS. The major constituents of the essential oils and their percentages are given in Table 1. Some major components were prevalent in many of the tested plants, such as dl-limonene, α-pinene, β-pinene l-linalool, 1,8-cineole, sabinene, 4-terpineol and γ-terpinene but others were specific to the plant species. The major constituents of the essential oils mainly belonged to four chemical groups: oxygenated monoterpenes (i.e., α-thujone, β-thujone, chrysanthenone, 4-terpineol, l-linalool, d-pulegone, α-citral, β-citronellol camphor and linalool oxide), monoterpene hydrocarbons (i.e., dl-limonene, sabinene, γ-terpinene, β-pinene, β-carene, phellandrene and α-pinene), sesquiterpene hydrocarbons (i.e., bicyclogermacrene, α-elemene, β-elemene and trans-caryophyllene), and oxygenated sesquiterpenes (i.e., cedrol and elemol).

Table 1 Oil yield and major constituents of the essential oils isolated from Egyptian plants
Full size table

The chemical compositions of the isolated essential oils from C. aurantifolia, C. paradise, C. limon, C. sinensis, C. viminals, C. sempervirens, S. molle, C. macrocarpa, P. graveolens, R. officinals and M. communis are in accordance with those previously reported [7–13]. On the other hand, the major constituents of the essential oils isolated from A. monosperma, O. vulgare T. occidentalis and A. judaica were completely different with those previously reported on the chemistry of these oils [14–17]. Some of the major constituents of the essential oils of V. aguns, S. terebinthifolius and S. cumini were similar to those previously reported for the oils isolated from plants growing in Egypt and other countries around the world [18–20]. However, the percentages of constituents are differed. The differences in essential oil compositions could be due to several factors, such as geographical location, season, environmental conditions, nutritional status of the plants and other factors [21].

In vitro antifungal activity of essential oils

The inhibitory effects of 18 essential oils isolated from Egyptian plants were evaluated against two wood decay fungi, G. lucidum and H. apiaria. The essential oils revealed various mycelial growth inhibitions against the tested fungi depending on fungal species and tested oil. The essential oil of A. monosperma exhibited the highest inhibitory effect against H. apiaria with EC50 value of 31 mg L−1, while the oil of V. aguns was the less effective one (Table 2). The essential oils of A. monosperma, T. occidentalis, C. sempervirens, S. molle and C. limon showed promising antifungal activity against H. apiaria as their EC50 values were less than 100 mg L−1. On the other hand, the oils of C. paradisi, A. judaica, C. sinensis, S. cumini and P. graveolens revealed pronounced antifungal activity against this fungus as EC50 values were less than 200 mg L−1. The oil of A. monosperma caused the highest inhibitory effect against G. lucidum with EC50 value of 53 mg L−1, followed by the oils P. graveolens, S. molle, C. limon and C. sempervirens, while the oil of M. communis had the weakest antifungal effect (Table 3). In general, all the tested oils showed higher inhibitory effect against H. apiaria than G. lucidum.

Table 2 In vitro antifungal activity of essential oils against Hexagonia apiaria using mycelial growth inhibition method
Full size table
Table 3 In vitro antifungal activity of essential oils against Ganoderma lucidum using mycelial growth inhibition method
Full size table

To the best of our knowledge, there were no reported studies investigating the effectiveness of the tested essential oils against wood decay fungi, H. apiaria and G. lucidum. However, the antifungal activity of some of these oils against human pathogenic, food spoilage and plant pathogenic fungi was described. For example, the essential oils of C. limon, C. paradise, C. aurantium, M. communis and C. sinensis possessed antifungal activity against yeasts and food spoilage fungi [22, 23] and plant pathogenic fungi [24, 25]. In addition, the oil of O. vulgare showed effectiveness to inhibit the growth of food spoiling yeasts Candida albicans and C. krusei [26], and human pathogens Malassezia furfur, Trichophyton rubrum, and Trichosporon beigelii [25]. The antifungal activity of the oil S. terebinthifolius against Aspergillus flavus, Candida albicans, A. niger, Colletotrichum gloeosporioides, Penicillium digitatum, Trichoderma sp. and Helminthosporium oryzae was described [19, 27]. The oils of C. sempervirens and R. officinalis possessed antifungal against several fungi and yeasts species [25, 27, 28].

In vivo antifungal activity of essential oils

The results of the in vivo inhibitory effects of the most potent essential oils against wood decay fungi, H. apiaria and G. lucidum are presented in Table 4. Six essential oils were evaluated as wood preservatives against the tested fungi in the presence of wood blocks of Scots pine sapwood. The results are shown as wood mass loss that caused by fungi after 6 weeks of the essential oils treatment at the three tested concentrations (EC50, two- and fourfold of the EC50) compared with the control (Table 4). In general, the reduction of wood mass loss was concentration dependent and all the tested oils significantly decreased the wood mass loss compared with the control. The oil of C. limon revealed the highest reduction of wood loss caused by H.apiaria (1.90, 1.11 and 0.99 % at concentrations of EC50, two- and fourfold of the EC50 compared with 7.87 % loss in the control). However, the oil of A. monosperma displayed the weakest reduction with wood loss percentages of 4.06, 3.68 and 1.97 % at the concentration of EC50, two- and fourfold of the EC50, respectively.

Table 4 Mass losses of pine blocks treated with different essential oils at EC50, two- and fourfold of the EC50 after 6 weeks exposure to Hexagonia apiaria and Ganoderma lucidum
Full size table

In the case of G. lucidum fungus, the oil of A. monosperma caused the highest reduction in wood mass loss at concentration of the EC50 value, while the oil of P. graveolens caused the lowest reduction. The oil of C. sempervirens was the most effective in the reduction of wood loss at concentration of fourfold of the EC50 (2.35 % compared with 8.94 % loss in the control). It can be notice that the wood mass loss is higher than that found in the case H. apiaria fungus. This result is in agreement with the present in vitro results in which the oils showed higher inhibitory effect against H. apiaria than G. lucidum. It has been reported that some of essential oils had a potential wood protection against wood decay fungi [29]. On the other hand, the inhibitory effect of essential oils against wood decay fungi was described [30–33].

The mechanism of antifungal action of essential oils is not completely understood. However, some authors gave several assumptions according to their observations. A majority of the studies on the mechanism of action of essential oils are accentuated on the interference of oxygenated monoterpenes with certain enzymatic reactions evolved in the cell wall synthesis. Further, the mode of antifungal action of the essential oils could be dependent on two different mechanisms. Some oil components may irreversibly disrupt the cell membrane structure by cross-linking reactions, causing a leakage of electrolytes and subsequent depletion of amino acids and sugars, while others may selectively be inserted into the lipid-rich portion of the cell membrane, thereby disturbing membrane function [34–36].

Conclusion

In summary, this study demonstrated the in vitro and in vivo antifungal activity of essential oils against wood decay fungi and potential use of essential oils, particularly the oils of C. sempervirens, C. limon, T. occidentalis, S. molle, A. monosperma and P. graveolens as preservatives for the control of wood decay caused by H. apiaria and G. lucidum. However, for the use of essential oils as alternatives of synthetic wood preservatives, further studies are required to evaluate toxicity and the effectiveness of treatment for long term on wood.

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Acknowledgments

This work was supported by the Alexandria University Research Fund (ALEX-REP, 2010–2011). Authors are grateful for Prof. Philippe Gérardin from LERMAB, Nancy University, France for providing the tested wood decay fungi.

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Authors and Affiliations

  1. Department of Forestry and Wood Technology, Faculty of Agriculture, Alexandria University, Elshatby, Alexandria, 21545, Egypt

    Ahmed S. O. Mohareb

  2. Department of Chemistry of Pesticides, Faculty of Agriculture, Alexandria University, Elshatby, Alexandria, 21545, Egypt

    Mohamed E. I. Badawy & Samir A. M. Abdelgaleil

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  1. Ahmed S. O. Mohareb
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  2. Mohamed E. I. Badawy
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Correspondence to Samir A. M. Abdelgaleil.

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Mohareb, A.S.O., Badawy, M.E.I. & Abdelgaleil, S.A.M. Antifungal activity of essential oils isolated from Egyptian plants against wood decay fungi. J Wood Sci 59, 499–505 (2013). https://doi.org/10.1007/s10086-013-1361-3

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