Crop loss due to fungal species also remains a serious problem1.
Emerging resistance of these species is seriously decreasing the number of effective
The crop production has tended to reduce the use of chemical in their products
due to increasing pressure of consumers or legal authorities or to adopt more
natural alternatives for crop protection. Plants and their essential oils are
potentially useful sources of antimicrobial compounds. Numerous studies have
been published on the antimicrobial activities of plant compounds against many
different types of microbes, including phyto-pathogenic fungi6.
The main constituents of essential oils were mono and sesquiterpenes including
carbohydrates, phenols, alcohols, ethers, acetates and ketones which are responsible
for their biological activity. Aromatic and medicinal plants produce a wide
variety of volatile terpenes and their oxygenated derivatives. Mixtures of these
substances, which are known as essential oils can be isolated from diverse parts
of plants by steam distillation. The antimicrobial properties of essential oils
are well recognized and their preparations are found applications as naturally
occurring antimicrobial agents in pharmacology, pharmaceutical botany, phyto-pathology,
medical and food preservation. Thus, the discovery of essential oil preparations
that possess antimicrobial activities has been the subject of many research
investigations of a wide variety of plant species6,7,8.
In an attempt to reduce the use of synthetic pesticides, extensive investigations
into the possible exploitation of plant compounds as natural commercial products,
that are safe for humans and the environment. Indeed, the search of natural
compounds and management methods alternatives to classical pesticides has become
an intense and productive research field 9,10.
The genus of Pistacia which belonged to the family of Anacardiaceae there
are 11 species which some of them used as ornamentals and some valued as fruit
tree. Pistacia trees are characteristic for the Mediterranean basin flora.
Five species of the genus grow naturally in the Mediterranean basin and Middle
East: P. lentiscus, P. atlantica, P. palaestina,
P. terebinthus and Pistacia vera originated in central Asia and is
cultivated throughout the Mediterranean region11.
The aims of this study were, in a first step to assay the main constituents
of the essential oil obtained from the leaves of P. lentiscus, P.
terebinthus and Pistacia vera growing in Tunisia. In a second step,
we assessed their antifungal potential against ten phyto-pathogenic fungi.
MATERIALS AND METHODS
Plant material: The leaves of P. lentiscus, P. terebinthus
and Pistacia vera were collected from the I.N.R.G.R.E.F. arboretums (National
Institute of Researches on Rural Engineering, Water and Forests). Five samples
collected from more than five different trees were harvested, mixed for homogenization
and used in three replicates for essential oil extractions. The specimen of
the plant was submitted to the herbarium division of the institute and identification
was confirmed in the Laboratory of Forest Ecology.
Isolation of the essential oils: The essential oils were extracted by hydrodistillation of fresh plant material (100 g of each sample in 500 mL of distilled water) using a Clevenger-type apparatus for 3 h according to the standard procedure described in the European Pharmacopoeia.
The oils were dried over using anhydrous sodium sulfate (a pinch 10 mL-1) and stored in sealed glass vials at 4°C before analysis. Yield was calculated based on dried weight of the sample (mean of three replications).
Gas chromatography-mass spectrometry: The composition of the oils was
investigated by GC and GC/MS. The analytical GC was carried out on an HP5890-series
II gas chromatograph (Agilent Technologies California USA) equipped with Flame
Ionization Detectors (FID) under the following conditions: the fused silica
capillary column, apolar HP-5 and polar HP Innowax (30 mx0.25 mm ID, film thickness
of 0.25 mm). The oven temperature was held at 50°C for 1 min then programmed
at rate of 5°C min-1 to 240°C and held isothermal for 4 min.
The carrier gas was nitrogen at a flow rate of 1.2 mL min-1; injector
temperature: 250°C, detector: 280°C; the volume injected: 0.1 mL of
1% solution (diluted in hexane). The percentages of the constituents were calculated
by electronic integration of FID peak areas without the use of response factor
correction. GC/MS was performed in a Hewlette Packard 5972 MSD System. An HP-5
MS capillary column (30 mx0.25 mm ID, film thickness of 0.25 mm) was directly
coupled to the mass spectrometry. The carrier gas was helium, with a flow rate
of 1.2 mL min-1. Oven temperature was programmed (50°C for 1
min, then 50-240°C at 5°C min-1) and subsequently held isothermal
for 4 min. Injector port: 250°C, detector: 280°C, split ratio: 1:50.
Volume injected: 0.1 mL of 1% solution (diluted in hexane); mass spectrometer:
HP5972 recording at 70 eV; scan time: 1.5 s; mass range: 40-300 amu. Software
adopted to handle mass spectra and chromatograms was ChemStation. The identification
of the compounds was based on mass spectra (compared with Wiley 275.L, 6th edition
mass spectral library). Further confirmation was done from Retention Index data
generated from a series of alkanes retention indices (relatives to C9-C28 on
the HP-5 column)12.
Antifungal activity assays: Eight plant pathogenic fungi were obtained
from the culture collection of the Tunisian National Institute of Agronomic
Research (INRAT). Cultures of each of the fungi were maintained on Potato Dextrose
Agar (PDA) and were stored at 4°C and in 1 mL of glycerol 25% at -20°C.
The fungal species used in this study were: Fusarium culmorum, F. avenaceum,
F. oxysporum, F. subglutinans, F. verticillioides,
F. nygamai, F. nygamai, Botrytis cinerea, Microdochium nivale
and Alternaria sp.. Antifungal activity was studied by using an in
vitro contact assay which produces hyphal growth inhibition13.
Essential oil was dissolved in 1 mL of Tween 20 (0.1% v/v) and then added into
20 mL PDA at 50°C to obtain a final concentration of 4 μL mL-1.
A mycelia disk of 5 mm in diameter, cut from the periphery of a 7 day-old culture,
was inoculated in the center of each PDA plate (90 mm diameter) and then incubated
at 24°C for 7 days. PDA plates treated with Tween 20 (0.1%) without essential
oil were used as control. Tests were repeated in triplicate. Growth inhibition
was calculated as the percentage of inhibition of radial growth relative to
the control using the following formula:
where, C is an average of three replicates of hyphal extension (mm) of controls and T is an average of three replicates of hyphal extension (mm) of plates treated with essential oil.
Statistical analysis: Data of germination, seedling growth and fungi
inhibition assays were subjected to one-way Analysis of Variance (ANOVA), using
the SPSS 17.0 software package. Differences between means were tested through
Student-Newman-Keuls (SNK) and values of p≤0.05 were considered significantly
different. To evaluate whether the essential oil constituents identified are
useful in reflecting the chemical relationships between species, 13 compounds,
detected in the oil samples with contents in the essential oils of 3.5% in at
last one species, were subjected to PCA and HCA using SPSS 17.0 software14.
RESULTS AND DISCUSSION
Yield and chemical composition of Pistacia species essential oils:
The mean yields of the three Pistacia leaf oils varied according to the
species from 0.14±0.026 for P. lentiscus to 0.24±0.03 and
0.28±0.02, respectively for P. terebinthus and P. vera.
||Figure 1: Average essential oils yield of three
Pistacia species. Values with different superscripts (a-b) are
significantly different at p = 0.05 (means of three replicates)
The Analysis of Variance (ANOVA) indicated that the oil yields were significantly
different between the species (p≤0.05). The average classification showed
the presence of two overlapping groups (Fig. 1) the first
group was constituted by P. terebinthus and P. vera which have
the high yield and the second group was only constituted by P. lentiscus.
Similar yield was obtained in Morocco for P. lentiscus (0.14%) and in
accordance with those obtained in Corsica by Castola et al.15.
Nevertheless, it is less than those reported by Duru et al.16
for the plants collected from Turkey (0.3%). On the other hand, P. vera
from Tunisia showed higher yield than that from Turkey (0.15%)16.
The chromatographic analysis (GC (RI) and GC/MS) of the essential oils allowed
the identification of 42 components (Table 1), representing
94.73-98.9% of the total oil content.
In the oil from P. lentiscus, the hydrocarbonated monoterpenes amounted
to 63.9%; on the other hand, the total sesquiterpenic fraction amounted to 31.3%
of the total oil. The main compounds are the monoterpenes α-pinene
(20.6%), limonene (15.3%) and β-pinene (9.6%), the oxygenated monoterpene
terpinen-4-ol (8.2%) and the sesquiterpene germacrene D (8.4%). Other compounds,
in a lesser amount, are α-phellandrene (3.85%) and α-terpineol (3.5%).
In the literature, Amhamdi et al.17
in Morocco reported β-myrcene (39.2%) limonene (10.3%), β-gurjunene
(7.8), germacrene (4.3%), α-pinene (2.9%), as the most abundant compound
in P. lentiscus essential oils. In a previous study, the chemical composition
of essential oils of Pistacia lentiscus L. from Tunisian populations
have been reported by Douissa et al.18,
data revealed from this study showed the abundance of α-pinene 17%, δ-terpinene
(9%) and terpen-4-ol (12%).
||Table 1: Chemical composition of the essential
oils extracted from the leaves of three Pistacia species
In the oil from P. terebinthus, the hydrocarbons monoterpenes amounted
to 83.12%, with a total sesquiterpenes amount of 9.4% (7.1% sesquiterpene hydrocarbons
and 2.3% of oxygenated sesquiterpenes) of the total oil. α-terpinene (41.34%),
α-pinene (19.24%), δ-terpinene (6.99%) and α-terpinolene (8.02%)
were the most abundant among the hydrocarbonated monoterpenes.
||Figure 2: Principal component analysis
of 13 compounds for the leaf essential oils of three Pistacia species
The global chromatographic analysis of P. vera oil showed a complex
mixture consisting mainly of monoterpene hydrocarbons and small amounts of oxygenated
mono and sesquiterpenes. It was dominated by monoterpene hydrocarbons (79.08%)
and oxygenated monoterpenes (8.32%), while oxygenated and sesquiterpenes hydrocarbons
were only present in small percentage (respectively, 4.45 and 2.88%). The major
components detected in the oil were α-pinene (16.07%) and α-terpinene
(32.44%). The chemical composition of P. vera and P. terebinthus
was previously reported in Turkey by Duru et al.16
and it have shown that essential oils of these two species were rich in oxygenated
monoterpenes like terpinen-4-ol and α-terpineol. These differences found
between the main constituents of oils obtained from Pistacia species
growing in Tunisia and these from the same species but growing in Turkey and
other countries could be related particularly to climate, soils and the genetic
background of tree.
Principal Components Analysis (PCA) and Hierarchical Cluster Analysis (HCA),
to evaluate whether the identified essential oil components may be useful in
reflecting the chemotaxonomic relationships in the three Pistacia species,
13 compounds with contents in the essential oils of minimum 3.5% in at last
one species (Table 2), were selected for the PCA and the HCA.
The PCA horizontal axis explained 57.95% of the total variance and the vertical
axis a further 42% (Fig. 2). The HCA based on the Euclidean
distances between groups indicated two groups of species (A and B), identified
by their essential oil chemotypes with a dissimilarity >20 (Fig.
3 and 4). Group B was further divided into two Subgroups
(P. vera and P. terebinthus) with a dissimilarity <1. P.
lentiscus stand out in both HCA and PCA analyses, forming separate group
||Table 2: Content in the essential oils extracted
from the leaves of three Pistacia species of the 13 compounds selected
for the principal component and the hierarchical cluster analysis
Since the essential oil components within the same group were significantly
correlated and tend to vary in the same way, we considered each group as a chemotype
(Fig. 3 and 4). In fact group A was reduced
to P. lentiscus which was highly positively correlated with the vertical
axis with an essential oil distinguished by high contents of α-pinene,
limonene, β-pinene and terpinen-4-ol. P. terebinthus, forming the
subgroup B1, which was highly positively correlated with the horizontal axis,
shared with P. vera forming the subgroup B 2 a high content of α-terpinene
(32.44-41.34%), however, it was specified by a considerable percentage of limonene
Antifungal activity of essential oils of three Pistacia species:
Essential oils isolated from leaves of P. lentiscus, P. terebinthus
and Pistacia vera were tested for their antifungal activity against ten
plant pathogenic fungal species.
||Figure 3: Principal component analysis of three
Pistacia species based on their chemical composition
||Figure 4: Dendrogram obtained by cluster analysis
based on the Euclidean distances between groups of the leaf essential
oils of three Tunisian Pistacia species
According to obtained results in Table 3, all essential oils
showed significant inhibition of fungal growth, this study also indicated that
the antifungal activity is variable depending on the fungal strain and tested
oils. Essential oils of P. terebinthus were most effective against F.
avenaceum and F. verticillioides when compared with P. lentiscus
and P. vera. Our results are in agreement with the literature, in
fact, the antimicrobial activity of essential oils and extracts from species
belonging Anacardiaceae family were reported. Essential oils of Pistacia
species collected from Turkey have been reported to have antifungal activity
against Fusarium sambucinum, Rhizoctonia solani and Pythium
ultimum16; essential oils of Pistacia
lentiscus grown in Tunisia have been studied by Douissa et al.18
and data reveled from this study show an important antimicrobial activity. In
general, there was a correlation between the antifungal activity and percentage
of some major components. Table 1 indicated that tested oils
contain similar major components like α-pinene, limonene and α-terpinene
which are known by their antimicrobial activity.
||Table 3: Antifungal activity of essential oil
extracted from leaves of three Pistacia species
Indeed, the antimicrobial activity of monoterpenes suggests that they diffuse
into pathogens and damage cell membrane structures19.
α-pinene, which was found in appreciable amounts in the oils of this study,
has been reported to be the cause of the antifungal activity of oil from Pistacia
lentiscus20. In another report it was
demonstrated that the antimicrobial activity of essential oil was associated
with phytochemical components such as monoterpenes21.
Sokovic kand Griensven22, described antifungal
activity of limonene and α-pinene against Verticillium fungicola
and Trichoderma harzianum. Thus, the antifungal activity of the oil in
this study is not attributed only to the high proportions of hydrocarbonated
monoterpenes, however, other major or trace components in the oil could give
rise to its antifungal activity. Indeed, there are synergistic and antagonistic
interactions between oil components. The mode of action of essential oils was
investigated by many authors who suggested that the antimicrobial activity is
produced by interactions provoked by terpenes in the enzymatic systems related
with energy production and in the synthesis of structural components of the
microbial cells23. Other reports suggested
that the components of the essential oils cross the cell membrane, interacting
with the enzymes and proteins of the membrane such as the H+/ATPase
pumping membrane, so producing a flux of protons toward the cell, exterior which
induces changes in the cells and ultimately their death. Besides, several authors24,25,26
reported that the antimicrobial activity is related to ability of terpenes to
affect not only permeability but also other functions of cell membranes, these
compounds might cross the cell membranes, thus penetrating into the interior
of the cell and interacting with critical intracellular sites. In addition,
Daferera et al. 27 reported that the
fungitoxic activity of essential oils may have been due to formation of hydrogen
bonds between the hydroxyl group of oil components and active sites of target
New trends in crop protection lead to a reduction in the levels of pesticides
or/and to the use of naturally-derived pesticide from plants, animal
or microbial origin. Among natural substances, essential oils and extracts from
several types of plants used as flavouring agents are known to possess many
biological activities and seem to be suitable for different types of products
as bio-herbicide. Our study could give the solution, which in its first part
had focused on the correlation between the chemical composition and the effectiveness
as antifungal agents of essential oils extracted from Tunisian Pistacia
- Pimentel, D., S. McNair, J. Janecka, J. Wightman and C. Simmonds et al.,
2001. Economic and environmental threats of alien plants,
animals and microbial invasions. Agric.
Ecosyst. Environ., 84: 1-20
- Ghasemi, Y., A. Khalaj, A. Mohagheghzadeh, A.R. Khosravi and M.H. Morowvat,
2007. Composition and antimicrobial activity of the
essential oil and extract of Hypericum elongatum. J.
Appl. Sci., 7: 2671-2675
- Koudou, J., P. Edou, L.C. Obame, I.H. Bassole and G. Figueredo et al.,
2008. Volatile components, antioxidant and antimicrobial
properties of the essential oil of Dacryodes edulis G. don from gabon.
Applied Sci., 8: 3532-3535
- Shonouda, M.L., S. Osman, O. Salama and A. Ayoub, 2008. Insecticidal
Effect of Chrysanthemum coronarium L. flowers on the pest Spodoptera
littoralis boisd and its parasitoid Microplitis rufiventris Kok.
with identifying the chemical composition. J.
Applied Sci., 8: 1859-1866
- Nour, A.H., S.A. Elhussein, N.A. Osman, N.E. Ahmed, A.A. Abduelrahman, M.M.
Yusoff and A.H. Nour, 2009. Antibacterial activity
of the essential oils of sudanese accessions of Basil (Ocimum basilicum
Applied Sci., 9: 4161-4167
- Amri, I., H. Lamia, S. Gargouri, M. Hanana and M. Mahfoudhi et al.,
2011. Chemical composition and biological activities
of essential oils of Pinus patula. Nat.
Prod. Commun., 6: 1531-1536
- Amri, I., S. Gargouri, L. Hamrouni, M. Hanana, T. Fezzani and B. Jamoussi,
2012. Chemical composition, phytotoxic and antifungal
activities of Pinus pinea essential oil. J.
Pest Sci., 85: 199-207
- Amri, I., E. Mancini, L. De Martino, A. Marandino and L. Hamrouni et
al., 2012. Chemical composition and biological
activities of the essential oils from three Melaleuca species grown
in Tunisia. Int.
J. Mol. Sci., 13: 16580-16591
- Zanic, K., S. Goreta, S. Perica and J. Sutic, 2008. Effects
of alternative pesticides on greenhouse white fly in protected cultivation.
Sci., 81: 161-166
- Dudai, N., A. Poljakoff-Mayber, A.M. Mayer, E. Putievsky and H.R. Lerner,
1999. Essential oils as allelochemicals and their potential
use as bioherbicides. J.
Chem. Ecol., 25: 1079-1089
- Zrira, S., A. Elamrani and B. Benjilali, 2003. Chemical
composition of the essential oil of Pistacia lentiscus L. from Morocco:
A seasonal variation. Flavour
Frag. J., 18: 475-480
- Adams, R.P., 2001. Identification of Essential Oil
Components by Gas Chromatographye Quadrupole Mass Spectrometry. Allured Publisher,
Carol Stream, IL., USA.
- Cakir, A., S. Kordali, H. Zengin, S. Izumi and T. Hirata, 2004. Composition
and antifungal activity of Hypericum hyssopifolium and Hypericum
Frag. J., 19: 62-68
- Sokal, R.R. and J.F. Rohlf, 1995. Biometry the Principles
and Practice of Statistics in Biological Research. 3rd Edn., WH Freeman and
Co., New York, USA., pp: 887.
- Castola, V., A. Bighelli and J. Casanova, 2000. Intraspecic
chemical variability of the essential oil of Pistacia lentiscusL. from
Syst. Ecol., 28: 79-88
- Duru, M.E., A. Cakir, S. Kordali, H. Zengin, M. Harmandar, S. Izumi and
T. Hirata, 2003. Chemical composition and antifungal
properties of essential oils of three Pistacia species. Fitoterapia,
- Amhamdi, H., F. Aouinti, J.P. Wathelet and A. Elbachiri, 2009. Chemical
composition of the essential oil of Pistacia lentiscus L. from Eastern
Nat. Prod., 3: 90-95
- Douissa, F.B., N. Hayder, L.C. Ghedira, M. Hammami, K. Ghedira, A.M. Mariotte
and M.G.D. Franca, 2005. New study of the essential
oil from leaves of Pistacia lentiscus L. (Anacardiaceae) from Tunisia.
J., 20: 410-414
- Sikkema, J., J.A. de Bont and B. Poolman, 1995. Mechanisms
of membrane toxicity of hydrocarbons. Microbiol.
Mol. Biol. Rev., 59: 201-222
- Magiatis, P., E. Melliou, A.L. Skaltsounis, I.B. Chinou and S. Mitaku, 1999.
Composition and antimicrobial activity of the essential
oils of Pistacia lentiscus var chia. Planta
Med., 65: 749-752
- Matasyoh, J.C., J.J. Kiplimo, N.M. Karubiu and T.P. Hailstorks, 2007. Chemical
composition and antimicrobial activity of essential oil of Tarchonanthus
Chem., 101: 1183-1187
- Sokovic, M. and L.J.L.D. van Griensven, 2006. Antimicrobial
activity of essential oils and their components against the three major pathogens
of the cultivated button mushroom, Agaricus bisporus. Eur.
J. Plant Pathol., 116: 211-224
- Omidbeygi, M., M. Barzegar, Z. Hamidi and H. Nafhdibadi, 2007. Antifungal
activity of thyme, summer savory and clove essential oils against Aspergillus
flavus in liquid medium and tomato paste. Food
Control, 18: 1518-1523
- Cristani, M., M. DArrigo, G. Mandalari, F. Castelli and M.G. Sarpietro
et al., 2007. Interaction of four monoterpenes
contained in essential oils with model membranes: Implications for their antibacterial
Agric. Food Chem., 55: 6300-6308
- Lucini, E.I., M.P. Zunino, M.L. Lopez and J.A. Zygadlo, 2006. Effect
of monoterpenes on lipid composition and sclerotial development of Sclerotium
cepivorum Berk. J.
Phytopathol., 154: 441-446
- Tatsadjieu, N.L., J.P.M. Dingamo, M.B. Ngassoum, F.X. Etoa and C.M.F. Mbofung,
2009. Investigations on the essential oil of Lippia
rugosa from Cameroon for its potential use as antifungal agent against
Aspergillus flavus Link ex. Fries. J.
Food control., 20: 161-166
- Daferera, D.J., B.N. Ziogas and M.G. Polissiou, 2000. GC-MS
analysis of essential oils from some Greek aromatic plants and their fungitoxicity
on Penicillium digitatum. J.
Agric. Food Chem., 48: 2576-2581