An In Vitro Attempt for Controlling Severe Phytopathogens and Human Pathogens Using Essential Oils from Mediterranean Plants of Genus Schinus

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An In Vitro Attempt for Controlling Severe Phytopathogens and Human
Pathogens Using Essential Oils from Mediterranean Plants of Genus Schinus
Hazem Salaheldin Elshafie,1 Nadia Ghanney,2 Stefania Mirela Mang,1
Ali Ferchichi,2 and Ippolito Camele1
1School of Agricultural, Forestry, Food and Environmental Sciences, University of Basilicata, Potenza, Italy.
2Institut National Agronomique de Tunisie, 1002 Tunis and Arid Land Institute, Medenine, Tunisia.
ABSTRACT Growing concerns about food safety and environmental protection enhanced the need for new and safe plant
disease control strategies. The chemical composition of the three essential oils (EOs) extracted from leaves and fruits of
Schinus terebinthifolius and leaves of Schinus molle, growing in Tunisia, was studied by GC and GC-MS. In all, 12
compounds were identified. The oils were mainly composed of terpene compounds. a-Pinene, a-phellandrene, and d-limonene
were the major constituents. The aim of the current study was to evaluate the in vitro antimicrobial effectiveness of three EOs
derived from plants of genus Schinus and extracted from leaves and fruits of S. terebinthifolius and leaves of S. molle. Both
antifungal and antibacterial activities of the EOs were examined. The antifungal activity of the studied EOs was investigated
against Colletotrichum acutatum and Botrytis cinerea in comparison with the systemic fungicide azoxystrobin used at 0.8 lL
mL-1. The antibacterial activity was evaluated against three strains of Gram-positive (G+ve) bacteria (Bacillus megaterium,
Bacillus mojavensis and Clavibacter michiganensis) and four strains of Gram-negative (G-ve) bacteria (Escherichia coli,
Xanthomonas campestris, Pseudomonas savastanoi, and Pseudomonas syringae pv. phaseolicola) compared with the syn-
thetic antibiotic tetracycline at a concentration of 1600 lg mL-1. The minimum inhibitory concentration of the studied EOs
has been evaluated against the above microorganisms using the 96-well microplate method. Tested microorganisms exhibited
different levels of sensitivity to each tested EO. All investigated EOs reduced the fungal mycelial growth when used at low
concentrations from 250 to 1000 ppm and from 2000 to 8000 ppm against C. acutatum and B. cinerea, respectively. Higher
concentrations of the same EOs exhibited a fungicidal effect against both mitosporic fungi. The EO extracted from leaves of S.
terebinthifolius significantly inhibited the growth of tested bacterial strains. Nevertheless, E. coli showed a weak resistance
toward the same EO and a high resistance toward the other two tested EOs. Finally, P. savastanoi and P. syringae pv.
phaseolicola showed a high resistance toward all tested EOs.
KEY WORDS: � antimicrobial activity � chemical composition � GC-MS analysis � minimum inhibitory concentration
� plant essential oils
INTRODUCTION
The widespread use of traditional synthetic pesticideshas significant drawbacks, including air pollution, in-
creasing their costs, pesticide residues, and negative impact
on human health.1,2 For several years, a variety of synthetic
chemicals has been used as antimicrobial agents to inhibit
overall growth of plant pathogenic fungi.3 The growth of
public concern over the health and environmental hazards
associated with the increased levels of pesticide used in fruit
orchards and the lack of renewal of the use of licenses for
some of the most effective biocide molecules have led to
develop alternative, safe, and natural methods of postharvest
control.4,5 Development of natural antimicrobials and bio-
pesticides will surely help in decreasing the harmful impact
of synthetic pesticides and preventing their accumulation in
ecosystems.6,7
Plant essential oils (EOs) could be successfully exploited
as a source of alternative substances to synthetic pesticides,
usable for controlling various infectious plant pathogens.
Moreover, the EO antimicrobial efficacy has extended their
use as a natural preservative for foodstuff conservation as
well as for prolonging their shelf life.7,8 The newly discov-
ered EO antimicrobial properties showed the possibility of
their use as natural biopesticides, which could be taken into
account by the pharmaceutical industry to develop innova-
tive and less risky therapies for some human diseases.2,9
The cashew family Anacardiaceae Lindl. includes more
than 700 species in 82 genera that are primarily distributed
pantropically. Some of its genera are also present in tem-
perate areas. Plants in this family are cultivated for their
Manuscript received 4 August 2015. Revision accepted 7 December 2015.
Address correspondence to: Prof. Ippolito Camele, School of Agricultural, Forestry,
Food and Environmental Sciences, University of Basilicata, Viale dell’Ateneo Lucano 10,
I-85100 Potenza, Italy, E-mail: ippolito.camele@unibas.it
JOURNAL OF MEDICINAL FOOD
J Med Food 00 (0) 2016, 1–8
# Mary Ann Liebert, Inc., and Korean Society of Food Science and Nutrition
DOI: 10.1089/jmf.2015.0093
1
edible fruits and seeds as well as are a source of their me-
dicinal compounds. From the chemical and economical
points of view, they produce a good quantity of resins and
tannins. Several species of the family belonging to genus
Schinus are cultivated also as ornamentals.10
The most common EOs of Anacardiaceae were extracted
from the Schinus molle L. and Schinus terebinthifolius
Raddi, native to Brazil and commonly known either as
Brazilian pepper tree or as Felfel Aareed. They are indige-
nous to South and Central America and also grow in tropical
and semitropical regions of the United States and Africa as
ornamental garden trees. The above-mentioned species are
characterized by production of an EO, which is responsible
for a peppery flavor. In some places, they have also been
used in the perfume industry.11 The leaves and reddish fruits
of S. terebinthifolius are rich in this EO that contains high
concentrations of monoterpene and sesquiterpene hydro-
carbons, as reported by El-Massry et al.12
S. terebinthifolius was traditionally used as an antibac-
terial, antiviral, diuretic, digestive stimulant, tonic, wound
healer, anti-inflammatory, and hemostatic as well as a me-
dicament to treat urinary and respiratory infections.13
El-Massry et al.12 have studied the antimicrobial activity
of different crude extracts, which have been obtained, using
dichloromethane or ethanol, from fresh leaves of S. ter-
ebinthifolius cultivated in Egypt. The crude extract obtained
using the first solvent exhibited higher antimicrobial activity
against some fungi and bacteria, such as Staphylococcus
aureus Rosenbach, Pseudomonas aeruginosa, Escherichia
coli, Aspergillus niger, Aspergillus parasiticus, and Candi-
da albicans, in comparison either to the crude extract ob-
tained by ethanol or to the EO from the same source.
Ghanney and Rhouma14 reported that crude leaf extracts
of S. terebinthifolius, prepared with methanol, ethanol, and
hot distilled water, had a clear antibiotic effect against
Agrobacterium tumefaciens Smith & Townsend, the causal
agent of crown gall disease in tomato plants. It is supposed
that some alkaloid and flavonoid compounds in them could
be responsible for the antibacterial activity.
dos Santos et al.10 have chemically characterized the
composition of two EOs extracted from the above two
Anacardiaceae by capillary GC and GC-MS. Twenty-seven
and 29 compounds were identified in EO obtained from
S. molle and S. terebinthifolius, respectively. The same re-
searchers showed that sesquiterpene and monoterpene hy-
drocarbons were present in high percentage in the leaf and
fruit EOs obtained from both Schinus species.
Affonso et al.15 analyzed the chemical composition of the
EO isolated from fruits of S. terebinthifolius using chro-
matographic analysis by GC-MS and quantified the relative
percentages of each component. In particular, 22 compo-
nents, including monoterpene and sesquiterpene, were
identified, with a yield of about 2.6% (w/w) of dry fruitweight. They also showed that its major constituents were
a-fenchene (20.75%), b-pinene (10.11%), b-myrcene (9.30%),
a-phellandrene (14.94%), limonene (20.81%), and iso-
sylvestrene (13.87%). In previous studies, Barbosa et al.16
reported that a-phellandrene and b-pinene represented, on
average, 7.0% and 1.5%, respectively, of the EO isolated
from fruits of S. terebinthifolius.
Generally, the major component of EO from leaves,
flowers, and/or fruits of S. terebinthifolius resulted to be a-
pinene (15.01–51.82%).17 Barbosa et al.16 reported the
following major components of EO isolated from unripe
fruits of S. terebinthifolius: a-cadinol (20.60%), d-cadinene
(15.48%), b-pinene (10.21%), and epi-a-muurolol (9.89%).
Finally, Richter et al.18 indicated a-pinene (16.9%), a-
phellandrene (21.1%), b-phellandrene (10.8%), and limo-
nene (23.7%) as the major constituents of EO from fruits of
the same Schinus species.
Regarding the biological characterization, S. ter-
ebinthifolius EOs, Siddiqui et al.19 have reported that they
have antifungal, healing, and antiallergic effects. In some
countries, S. terebinthifolius was used in folk medicine for
treatment of inflammatory and venereal diseases as well as
hemostatic, antirheumatic, pain relieving, antidiarrheic, and
a remedy for gingivitis and fever.15
Azoxystrobin is a fungicide acting, through the inhibition
of mitochondrial respiration, on spore production and ger-
mination as well as mycelial growth. The same active sub-
stance has been shown to explicate a phytotoxic effect on
some plants, such as certain apple and crab apple varieties.
Azoxystrobin is toxic to fish and aquatic organisms. It is
moderately persistent in soil, with a half-life of 1–4 weeks.
Azoxystrobin may present a leaching risk to groundwater.20
Due to the limited number of articles on the antimicrobial
activity of EOs extracted from plants of genus Schinus, the
current study has the goal to cast light on the composition
and antimicrobial activities of EOs obtained from leaves and
fruits of S. terebinthifolius and leaves of S. molle. The an-
tifungal activity of the studied EOs and their minimum
inhibitory concentrations (MIC) have been investigated
against Botrytis cinerea Pers. and Colletotrichum acutatum
J.H. Simmonds in comparison with the systemic fungicide
azoxystrobin. Moreover, their antibacterial activity has
been evaluated against three strains of Gram-positive (G+ve)
bacteria (Bacillus megaterium de Bary, Bacillus mojavensis
Roberts and Clavibacter michiganensis Smith) and four
strains of Gram-negative (G-ve) bacteria (E. coli Migula,
Xanthomonas campestris Pammel, Pseudomonas savastanoi
Janse, and Pseudomonas syringae pv. phaseolicola Van Hall)
compared to tetracycline antibiotic.
MATERIALS AND METHODS
Fungal isolates
The tested phytopathogenic fungi were monoconidic
isolates, stored at 4�C as pure cultures, and maintained in the
mycotheca of the School of Agricultural, Forestry, Food and
Environmental Sciences of the University of Basilicata,
Potenza, Italy. The fungal species were cultured on potato
dextrose agar (PDA) at 24�C – 2�C. The micromycetes used
were C. acutatum (isolate number 1778 from olive) and
B. cinerea (isolate number 1132 from strawberry). Identi-
fication of the two studied isolates was reached on the basis
2 ELSHAFIE ET AL.
of their microscopic morphological features and, succes-
sively, with molecular methods based on polymerase chain
reaction only for the isolate from olive.
The total nucleic acids were extracted from pure cultures
of isolate from olive with a commercial kit (QIAGEN,
DNeasy Plant Mini Kit), according to the manufacturer’s
instructions. The DNA was amplified using the universal
primer pair ITS4/ITS5.21 The amplicons obtained were di-
rectly sequenced, and the resulting sequences were com-
pared with those available in the GenBank nucleotide
archive using BLAST software.22
Bacterial isolates
The tested bacterial isolates were three strains of G+ve
bacteria (B. megaterium, B. mojavensis and C. michiga-
nensis) and four strains of G-ve bacteria (E. coli, X. cam-
pestris, P. savastanoi pv. savastanoi, and P. syringae pv.
phaseolicola). All tested bacterial strains were cultivated on
agar King B medium (KB), except for E. coli that was
cultivated on Luria-Bertani medium (LB). All isolates were
then incubated at 30�C – 2�C for 48 h. All tested bacterial
isolates have been previously identified and stored as pure
freeze-dried cultures at -20�C in the collection present at the
School of Agricultural, Forestry, Food and Environmental
Sciences of the University of Basilicata, Potenza, Italy.
Studied EOs
All studied EOs were obtained from leaves and fruits of S.
terebinthifolius and leaves of S. molle.
Plant material and extraction of EOs
For the extraction of studied EOs, the above plant organs
were collected from the Arid Land Institute of Medenine
(ALIM, southeast of Tunisia) and deposited in the Herbarium
of the Dryland Farming and Oasian Crop Department of the
same institute. Air-dried plant materials (200 g) were placed in
a 5-L round-bottom distillation flask and added 3 L of double-
distilled water. The EOs were obtained by steam distillation
for 3 h using a Clevenger-type apparatus. After removing
water traces with anhydrous sodium sulfate, the EOs were
stored at 4�C in a clean amber glass bottle until used.
Separation and analysis of oil components
The overall analysis was performed by GC/MS with a gas
chromatograph Shimadzu brand (GC 2010 Plus) coupled to a
QP 2010 Ultra mass spectrometer. The separation of EO
components was achieved by capillary column chromatogra-
phy on 0.25-lm-thick flash silica RTX-5MS (30 · 0.25 mm)
using helium as eluting gas, with a flow rate set of 1.2 mL min-1.
Samples (1 lL) were injected in split mode (leakage ratio,
1:50). The device was connected to a computer system
managing a mass spectrum library NIST 98 and driven by
software to monitor chromatographic analyses. Identification
of EO components was made through the comparison of their
retention indices with those of standard compounds of the
computerized database (NIST 98).
Antifungal activity test
The possible fungicidal inhibitory activity of EOs of S.
terebinthifolius and S. molle was determined as follows:
preparation of different concentrations of each EO, that is,
250, 500, and 1000 ppm in PDA and 0.2% Tween 20 in case
of C. acutatum and 2000, 4000, and 8000 ppm in case of B.
cinerea. Then, after that, 14 mL aliquots of each EO and
PDA were poured into Petri dishes. After the complete dry
off of the agar surface under laminar flow, 0.5 cm in diam-
eter fungal discs of the single above-mentioned mitosporic
fungi, cutoff from 96 h of fresh cultures, were singularly
inoculated in the center of each Petri dish prepared as above.
All plates were incubated at 24�C – 2�C for 4 days under
dark conditions. The diameter of fungal mycelial growth
was measured in mm. Petri dishes containing only PDA and
PDA + Tween 20 were also inoculated with fungal discs of
the same anamorphic fungi and used as a control. Each
treatment was carried out in triplicate. The fungitoxicity is
expressed as a percentage of growth inhibition (PGI) and
calculated according to the formula of Zygadlo et al.23
(Equation 1), herein reported, in comparison with that of the
synthetic fungicide azoxystrobin incorporated at 80 lL
100 mL-1 to PDA nutrient medium:
PGI (%)¼ 100 · (GC�GT)=GC, Equation (1)
where GC is the average diameter of fungal colony grown
on PDA alone (control), and GT is the average diameter of
fungal colony grown on PDA containing each EO.
Determination of MIC (96-well microplate method)
MIC was considered as the fungicidal effect of each EO
and is defined as the lowest concentration of each EO that
definitely inhibits the fungal growth. MIC was determined
on 96-well culture plates by a microdilution method using
microorganism suspension at a density of 108 spore mL-1.
Exactly 10 mL of liquid suspension was prepared from fresh
PDA fungalculture of 10 days old that was incubated at
24�C – 2�C. Spore formation was ascertained after 9 days of
incubation under a light microscope. The fungal suspension
was centrifuged at 1100 g for 5 min. Stock solution of each
EO was prepared in PDB liquid nutrient medium at 2000,
1800, 1600, and 1400 ppm in the case of C. acutatum and
10000, 9000, 8000, 7000, 6000, and 5000 ppm in the case of
B. cinerea. Each hole of the 96-well microplate was then
filled up with 200 lL of each single prepared EO suspension
and 100 lL of fungal suspension. After that, the 96-well
microplate was incubated for 12 days at 24�C – 2�C, and
then, the absorbance was read at k 630 nm using the ELISA
Microplate Reader instrument. All samples were tested in
triplicate. Azoxystrobin was used as the reference fungicide.
Technical procedures of Lehtinen et al.24 have been fol-
lowed with some minor changes.
To verify the fungicidal effect, fungal reculturing from
each well of the 96-well microplates has been performed on
PDA. The fungistatic effect of each tested EO was determined
by monitoring the lowest EO concentration that caused a
BIOCHEMICAL CHARACTERIZATION OF ESSENTIAL OILS OF SCHINUS SP. 3
significant reduction in fungal mycelial growth in comparison
with the positive control.
Antibacterial activity test
In these tests, the disc diffusion method of Bhunia John-
son25 has been used with some modifications as hereafter
explained: all tested bacterial strains, that is, B. megaterium,
C. michiganensis, X. campestris, B. mojavensis, P. savasta-
noi, and P. syringae pv. phaseolicola, were cultured on KB,
except for E. coli, which was cultured on LB selective sub-
strate. All bacterial cultures were incubated at 30�C – 2�C for
48 h. Bacterial suspension of each bacterial culture was
prepared in sterile distilled water at a concentration of 108
CFU mL-1 (OD = 0.2 nm). A mixture of 0.7% of soft agar and
bacterial suspension (9:1, v/v) was prepared, and 4 mL of this
suspension was poured into each 10 mL KB Petri dish 90 mm
in diameter. Blank discs (6 mm; Oxoid) were placed over KB
plate surfaces after complete solidification, and 20 lL ali-
quots from the EO suspension at the following concentra-
tions, original (TQ), 940, 470, and 235 lg lL-1 and
tetracycline at 1600 lg mL-1, were carefully applied over
discs. The antibacterial activity of the tested EOs was eval-
uated by measuring the inhibition zone diameter in mm.
Statistical analysis
Results obtained from the current research were statisti-
cally processed and subjected to analysis of variance, fol-
lowed by Tukey’s B and Duncan’s post hoc multiple
comparison tests, with a probability of P < .05 using SPSS
statistical software package version 13.0 (2004) to detect the
significance between the different concentrations of studied
EOs.
RESULTS AND DISCUSSION
Composition of Schinus EOs
The separation and analysis of EO components of the
ground fruits and leaves of the two species of Schinus in-
vestigated were performed by GC-MS, as described above.
The identification and quantification of EO components,
achieved using the NIST database, are reported in Table 1.
Identification of fungal isolates
The isolate from strawberry was identified as B. cinerea
based on the morphological features. The analysis of DNA
sequences obtained from the isolate from olive shows a high
similarity (99%—E value 0.0) with those present in the
GenBank for C. acutatum (accession numbers KM594093
and JN943492 confirming the morphological identification
by microscopic technique).
Antifungal activity test
All studied EOs have significantly reduced all tested
fungi, which exhibited a different sensitivity level to each
tested EO. Results of these trials showed that the studied
EOs were able to reduce the mycelial radial growth of
C. acutatum already at low concentrations (Fig. 1A). Higher
EO concentrations were needed to hold back the fungal
growth of B. cinerea (Fig. 1B).
The results of antifungal activity of S. terebinthifolius
EOs are in agreement with those of Gundidza et al.,26 who
found that these EOs exhibited antifungal activity against
Aspergillus flavus, C. albicans, and A. niger.
The antifungal activity of studied EOs is possibly due to
the contents of volatile constituents, especially monoterpene
and sesquiterpene fractions.12,27,28 For antifungal activity,
among those terpenes, the tri-terpenemoronic acid is con-
sidered the most important. It was isolated, for the first time,
from Schinus sp.28 Actually, moronic acid has been obtained
from other medicinal plants, such as Ozoroa mucronata and
Rhus javanica, and Brazilian propolis.28,29
The moronic acid exhibited various biological activities,
such as antitumor,30 anti-AIDS,31 antidiabetic,32 and anti-
microbial.12
The major components identified in EO of ripe fruits of S.
molle were a-phellandrene, b-pinene, b-phellandrene, lim-
onene, and cyclohexane methanol,4-ethyl-,alpha.,alpha.,
4-trimethyl-3-(1-methylethenyl)-,[1R-(1-a,3.a.,4.beta,)].33
Table 1. Major Components and Quantification of the Essential Oils Extracted
from Schinus terebinthifolius and Schinus molle
No. Compound Leaves of S. terebinthifolius Fruits of S. terebinthifolius Leaves of S. molle
1 a-Pinene 8.71 23.26 5.32
2 b-Pinene 0.66 0.82 4.65
3 a-Phellandrene 16.94 44.29 35.67
4 b-Phellandrene 0.48 0 0.34
5 Carene 0.17 0 0.19
6 d-Limonene 11.19 16.67 19.35
7 o-Cymene 4.22 1.97 1.56
8 b-Myrcene 1.72 2.32 2.34
9 Caryophyllene 1.37 1.56 3.84
10 c-Muurolene 0.38 3.33 0.23
11 Cyclohexanone,5-methyl-2(1-methylindene) 0 3.77 0
12 1–6-Cyclodecadiene,1-methyl-5-
methylene-8-(-1-methylethyl)-[S-(E-E)]-
18.69 0 19.20
4 ELSHAFIE ET AL.
The antifungal activity of moronic acid is due to the fungal
cell membrane damage, with an alteration of their perme-
ability and an inhibition of their respiration.26,34
Determination of MIC (96-well microplate method)
The effects of the studied EOs on the tested fungi resulted,
in this respect, variable. In particular, EO of S. ter-
ebinthifolius leaves strongly inhibited both tested fungi,
showing the lowest MIC values of 1400 and 6000 ppm
against C. acutatum (Fig. 2A) and B. cinerea (Fig. 3A), re-
spectively. EOs of S. terebinthifolius fruits and S. molle
leaves showed slightly minor effectiveness, exhibiting MIC
values of 1600 ppm against C. acutatum (Fig. 2B,C) and 5000
and 6000 ppm against B. cinerea (Fig. 3B,C), respectively.
The moderate mycelial growth inhibitory activity of
studied EOs against B. cinerea has recently also been re-
ported by El-Badawy and Abdelgaleil,35 who showed that
the EO of S. molle has a lower effect on B. cinerea than other
tested EOs extracted from Artemisia monosperma, Cu-
pressus macrocarpa, and Pelargonium graveolens. Ibrahim
and Al-Naser33 showed an analogous level of inhibitory
effect against the above anamorphic fungus for an n-hexane
EO extracted from the same Schinus species.
Antibacterial activity test
The EO extracted from leaves of S. terebinthifolius has
the most significant activity against the majority of tested
bacterial species, except for P. savastanoi and P. syringae
FIG. 1. Antifungal activity of the studied essential oils (EOs) against Colletotrichum acutatum (A) and Botrytis cinerea (B). Scale bars with
different letters indicate mean values significantly different at P < .05 according to Tukey’s test. Data are expressed as mean of three replicates –
SDs. Azoxy, azoxystrobin 0.8 lL mL-1.
FIG. 2. Minimum inhibitory concentration (MIC) of studied EOs (ppm) against C. acutatum (Microplate 96). (A) Schinus terebinthifolius
leaves, (B) S. terebinthifolius fruits, and (C) Schinus molle leaves; PDB+F: potato dextrose broth incorporated with tested fungi. Different letters
in each point are significant according to Tukey’s B test at P < .05. Data were obtained from three replicates.
BIOCHEMICAL CHARACTERIZATION OF ESSENTIAL OILS OF SCHINUS SP. 5
pv. phaseolicola (Table 2), whereas EOs isolated from S.
terebinthifolius fruits and S. molle leaves showed a moder-
ate significantantibacterial effect against B. mojavensis and
a slightly significant activity against B. megaterium, C.
michiganensis, and X. campestris compared to that of S.
terebinthifolius leaves (Table 2). Generally, the tested G+ve
bacteria were more susceptible to the tested EOs than G-ve
ones.
The notable antibacterial activity of EO of S. ter-
ebinthifolius leaves has also been reported by Guerra et al.36
and Gundidza et al.26 against Yersinia enterocolitica, P.
aeruginosa, E. coli, Acinetobacter alcoaceticus, B. subtilis,
and Klebsiella pneumoniae.
According to the results of previous research, the anti-
bacterial activities of studied EOs are not only due to their
contents of monoterpene and sesquiterpene28 but also due to
the phenolic hydroxyl groups forming hydrogen bonds with
the active site of target enzymes.27 Therefore, the antimi-
crobial activity of the EO of S. terebinthifolius leaves could
be ascribed to the presence of biologically active com-
pounds, such as a-pinene, a-phellandrene, d-limonene, cy-
clodecadiene, and 1-methyl-5-methylene.
The low activity observed against E. coli, P. savastanoi,
and P. syringae pv. phaseolicola could be due to the pres-
ence of lipopolysaccharides in the outer membrane of the
G-ve bacteria, which make them inherently resistant to
external agents, such as hydrophilic dyes, antibiotics, and
detergents.37–39
The obtained antibacterial activity results are in agree-
ment with those of Cole et al.,39 who found that EO of S.
terebinthifolius fruits was active particularly against tested
G+ve bacteria, such as Corynebacterium sp., Bacillus sp.,
and Nocardia sp., while G-ve species, Enterobacter sp., E.
agglomerans, E. coli, and K. oxytoca, showed less sensi-
tivity to the tested EO.
Cole et al.39 have reported that the differences of antibac-
terial activity among G-ve and G+ve bacteria are due to the
structure of their cell walls. Whereas the G-ve bacteria have a
more complex cell wall composed of a thin peptidoglycan
layer and an outer membrane containing lipopolysaccharides,
FIG. 3. MIC of studied EOs (ppm) against B. cinerea (Microplate 96). (A) S. terebinthifolius leaves, (B) S. terebinthifolius fruits, and (C) S.
molle leaves; PDB+F: potato dextrose broth incorporated with tested fungi. Different letters in each point are significant according to Tukey’s B
test at P < .05. Data were obtained from three replicates.
Table 2. Antibacterial Activity of Different Concentrations for Studied Essential Oils (lg ll-1)
Essential oils Concentration
Diameter of inhibition zone (mm)
Gram positive Gram negative
B. meg C. mich B. moj E. col X. cam P. sav P. syr
S. terebinthifolius (leaves) TQ 35.33 – 2.5a 37.33 – 0.6a 23.67 – 1.5b 8.67 – 1.5b 16.00 – 1.0b 0.00b 0.00b
940 lg lL-1 24.00 – 1.0b 27.00 – 2.0b 15.33 – 0.6c 4.00 – 1.0b 12.67 – 1.2c 0.00b 0.00b
470 lg lL-1 13.67 – 1.5cd 11.33 – 1.2e 3.67 – 1.2e 0.00c 3.33 – 0.6e 0.00b 0.00b
235 lg lL-1 0.00f 3.67 – 0.6f 0.00f 0.00c 0.00f 0.00b 0.00b
S. terebinthifolius (fruits) TQ 15.33 – 1.5c 25.33 – 1.5b 30.33 – 1.6a 0.00c 9.67 – 1.2d 0.00b 0.00b
940 lg lL-1 10.67 – 0.6de 21.00 – 1.0c 18.33 – 1.6c 0.00c 5.33 – 0.6e 0.00b 0.00b
470 lg lL-1 3.67 – 1.2f 20.33 – 1.5c 9.33 – 0.6d 0.00c 3.33 – 0.6e 0.00b 0.00b
235 lg lL-1 0.00f 15.33 – 0.6d 0.00f 0.00c 0.67 – 0.6f 0.00b 0.00b
S. molle (leaves) TQ 33.67 – 1.5a 0.00g 32.67 – 2.5a 0.00c 23.33 – 1.5b 0.00b 0.00b
940 lg lL-1 22.00 – 2.0b 0.00g 25.67 – 2.1b 0.00c 16.00 – 1.0b 0.00b 0.00b
470 lg lL-1 13.00 – 1.0cd 0.00g 15.00 – 1.0c 0.00c 10.00 – 1.0d 0.00b 0.00b
235 lg lL-1 8.67 – 1.2e 0.00g 10.00 – 1.0d 0.00c 4.67 – 0.6e 0.00b 0.00b
Tetracycline 1600 lg mL-1 45.00a 45.00a 45.00a 45.00a 45.00a 45.00a 45.00a
Values are recorded as the mean diameter of inhibition zone (mm) – standard deviation. Values followed by the different letters in each vertical column are
significantly different according to Tukey’s B test at P < .05. Data were obtained from three replicates.
TQ, original concentration; E. col, Escherichia coli; B. meg, Bacillus megaterium; C. mich, Clavibacter michiganensis; X. cam, Xanthomonas campestris; B. moj,
Bacillus mojavensis; P. sav, Pseudomonas savastanoi, and P. syr, Pseudomonas syringae pv. phaseolicola.
6 ELSHAFIE ET AL.
the G+ve ones are protected by a wall predominantly com-
posed of one type of macromolecule (peptidoglycan).
CONCLUSION
The implementation of EO treatments especially for
controlling some fungal and bacterial human or plant dis-
eases is very promising nowadays compared to synthetic
fungicides and antibiotics. Results from the current study
showed that the three tested EOs can be potentially used for
controlling the two studied phytopathogenic fungi C. acu-
tatum and B. cinerea in a dose-dependent manner. In par-
ticular, all tested EOs were able to control C. acutatum at
low concentrations ranging from 250 to 1000 ppm rather
than B. cinerea, which has been controlled at higher con-
centrations ranging from 2000 to 8000 ppm.
In addition, the studied EOs were effective in controlling
most of the investigated bacterial species: P. savastanoi and
P. syringae pv. phaseolicola showing a complete resistance
toward all of them. E. coli exhibited a moderate resistance only
toward EO extracted from leaves of S. terebinthifolius and a
complete resistance toward the other two tested EOs extracted
from fruits of S. terebinthifolius and leaves of S. molle.
In conclusion, the current study validates, in an organized
way, that most of the studied plant EOs possess substantial
antimicrobial properties against several plant or human
pathogenic microorganisms. This can explain their use in folk
medicine and pharmaceutical industry for curing several se-
rious diseases. Other trials are recommended in the near future
to study the in vivo antimicrobial efficacy of crude EOs
against several phyto- and human diseases. Moreover, the
isolation and chemical identification of most of their bioactive
substance(s) and the evaluation of its effectiveness compared
to synthetic fungicides and/or antibiotics would also be ad-
visable. Further studies are needed to determine which of the
compounds characterizing our studied EOs are responsible
for the observed antimicrobial activity, as well as to under-
stand the exact mechanism of action of the compound(s).
ACKNOWLEDGMENTS
We are grateful to Prof. Gian Luigi Rana for the critical
revision of the article. We also appreciate the assistance of
Prof. Michèle Parisien in revising the English editing.
Thanks for the technical assistance from C.T. Michele
Palumbo.
AUTHOR DISCLOSURE STATEMENT
No competing financial interests exist.
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