Rosmarinus officinalis essential oil as an effective antifungal and herbicidal agent

Abstract

In order to reduce the use of chemical pesticides, great interest has been focused on environment-friendly biological control agents and botanicals that preserve biodiversity. In this context, our study aimed to assess the antifungal and herbicidal activities of Rosmarinus officinalis essential oil (EO) to find an alternative to synthetic pesticides. The chemical composition of R. officinalis essential oil was determined by gaz chromatography-mass spectrometry analysis (GC-MS). Results showed that R. officinallis EO was rich in monoterpenes and the major constituents were 1,8-cineole (54.6%), camphor (12.27%) and α-pinene (7.09%). However, under laboratory condition, two tests were carried out. The first one consisted on the study of EO antifungal activity using ELISA microplates and the second one consisted on evaluating the effect of EO on seedling growth of weeds. It was confirmed that this EO significantly inhibits spore germination of Fusarium oxysporum, Fusarium culmorum, Penicillium italicum and at 6 mM, the percentage of inhibition reached 100% on Fusarium oxysporum. Indeed, EO slows down seedling growth of Trifolium incarnatum, Silybum marianum, and Phalaris minor. In fact, EO at 5 mM completely inhibits seed germination. On the other hand, another experiment was carried out to evaluate the herbicidal activity by spraying EO on weeds. This showed that a novel herbicide formulation was set up for the first time to improve the activity of R. officinalis EO on post-emergence. Overall, R. officinalis EO can be suggested as a potential eco-friendly pesticide and suitable source of natural compounds potentially usable as natural pesticides.

Full text

RESEARCH ARTICLE OPEN ACCESS
Rosmarinus ocinalis essential oil as an eective antifungal and
herbicidal agent
Soene Ben Kaab1,2,3, Iness B. Rebey2,4, Marwa Hana1, Chadi Berhal1, Marie L. Fauconnier4, Caroline De Clerck1,
Riadh Ksouri2 and Haissam Jijakli1
1University of Liege, Gembloux Agro-Bio Tech, Integrated and Urban Plant Pathology Laboratory, 2 Passage des Déportés 5030 Gembloux, Belgium.
2Biotechnology Center at the Technopole of Borj-Cedria (CBBC), Laboratory of Aromatic and Medicinal Plants, BP 901, 2050 Hammam-Lif, Tunisia.
3University of Tunis el Manar, Faculty of Mathematical, Physical and Natural Sciences of Tunis, University campus BP 2092-El Manar, Tunis,
Tunisia. 4University of Liège, Gembloux Agro-Bio Tech, Unit of General and Organic Chemistry 2 Passage des Déportés 5030 Gembloux, Belgium.
Spanish Journal of Agricultural Research
17 (2), e1006, 9 pages (2019)
eISSN: 2171-9292
https://doi.org/10.5424/sjar/2019172-14043
Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, O.A, M.P. (INIA)
Abstract
In order to reduce the use of chemical pesticides, great interest has been focused on environment-friendly biological control
agents and botanicals that preserve biodiversity. In this context, our study aimed to assess the antifungal and herbicidal activities
of Rosmarinus ocinalis essential oil (EO) to nd an alternative to synthetic pesticides. The chemical composition of R. ocinalis
essential oil was determined by gaz chromatography-mass spectrometry analysis (GC-MS). Results showed that R. ocinallis EO was
rich in monoterpenes and the major constituents were 1,8-cineole (54.6%), camphor (12.27%) and α-pinene (7.09%). However, under
laboratory conditions, two tests were carried out. The rst one consisted on the study of EO antifungal activity using ELISA microplates
and the second one consisted on evaluating the eect of EO on seedling growth of weeds. It was conrmed that this EO signicantly
inhibits spore germination of Fusarium oxysporum, Fusarium culmorum, Penicillium italicum and at 6 mM, the percentage of inhibition
reached 100% on Fusarium oxysporum. Indeed, EO slows down seedling growth of Trifolium incarnatum, Silybum marianum, and
Phalaris minor. In fact, EO at 5 mM completely inhibits seed germination. On the other hand, another experiment was carried out to
evaluate the herbicidal activity by spraying EO on weeds. This showed that a novel herbicide formulation was set up for the rst time
to improve the activity of R. ocinalis EO on post-emergence. Overall, R. ocinalis EO can be suggested as a potential eco-friendly
pesticide and suitable source of natural compounds potentially usable as natural pesticides.
Additional keywords: biological control; 1,8 cineole, fungicidal activity; bio-herbicidal activity; formulation.
Abbreviations used: EO (essential oil); PDA (potato dextrose agar); PDB (potato dextrose broth).
Authors’ contributions: SBK, MH, RK and HJ conceived and designed the research. SBK conducted the experiments. SBK and CB
analyzed the data. SBK, IBR, CDC and MLF wrote the manuscript. All authors commented, discussed, and approved the manuscript.
Citation: Ben Kaab, S.; Rebey, I. B.; Hana, M.; Berhal, C.; Fauconnier, M. L.; De Clerck, C.; Ksouri, R.; Jijakli, H. (2019).
Rosmarinus ocinalis essential oil as an eective antifungal and herbicidal agent. Spanish Journal of Agricultural Research, Volume
17, Issue 2, e1006. https://doi.org/10.5424/sjar/2019172-14043
Received: 06 Oct 2018. Accepted: 29 Apr 2019.
Copyright © 2019 INIA. This is an open access article distributed under the terms of the Creative Commons Attribution 4.0
International (CC-by 4.0) License.
Funding: University of Liège, Integrated and Urban Plant Pathology Laboratory, Gembloux Agro-Bio Tech; Laboratory of Aromatic
and Medicinal Plants, Biotechnology Center at the Technopole of Borj-Cedria (CBBC).
Competing interests: The authors declare no conict of interest.
Correspondence should be addressed to Soene Ben Kaab: Soene.benkaab@doct.Uliege.ac.be; s.kaab@yahoo.fr
Introduction
Agricultural production has always been threatened
by the presence of plant pathogens such as fungi,
bacteria,
and viruses (Kordali et al., 2016). Moreover,
weeds are another major issue. In fact, they com-
pete with crops for resources (water, nutrients,
light) and cause huge economic losses, up to 34%
in major crops (Araniti et al., 2015). Every year,
approximately 2.5 million tons of pesticides are used
on crops worldwide to ght plant diseases (Koul et
al., 2008) with consequences on human health, soils
and the environment (groundwater contamination
and development of weed resistance). This intensive
use has been recognized as one of the main drivers of
biodiversity losses (Schütte et al., 2017).
In the last few decades, there has been growing
interest in investigating eco-friendly alternatives, in
particular essential oil (EO)-based methods in order to
curtail pesticide use because pesticides cause extensi-
ve damage to agricultural and natural systems (Ben
Ghnaya et al., 2013). Moreover, the use of EOs obtai-
ned through a cheap production process can reduce
the frequent applications of certain synthetic pestici-
des that have deleterious eects on the environment
and human health (Pavela & Benelli, 2016). But EOs

Soene Ben Kaab, Iness B. Rebey, Marwa Hana, Chadi Berhal, Marie L. Fauconnier, et al.
Spanish Journal of Agricultural Research June 2019 • Volume 17 • Issue 2 • e1006
2
can also present a low health risk during application.
One of the great challenges for further research is to
design an ecient stabilization process so as to apply
EOs in elds. In the same vein, several studies have
pointed out that EOs may have not only antifungal
activity (Koul et al., 2008; Tian et al., 2012; Ahluwalia
et al., 2014; Hmiri et al., 2015; Boubaker et al., 2016)
but also the ability to inhibit weed seedling growth
(Uremis et al., 2009; Poonpaiboonpipat et al., 2013). In
the Mediterranean region and especially in Tunisia, the
most widespread botanical family is Lamiaceae, which
has antimicrobial properties (Pintore et al., 2002).
Among these aromatic plants, the most interesting
species is Rosmarinus ocinalis (R. ocinalis) which
is known for its antifungal activity (Angioni et al.,
2004; Giamperi et al., 2011; Hmiri et al., 2015) and
its richness in EOs characterized by the predominance
of monoterpenes – mostly 1,8 cineole –, camphor, and
α-pinene (Zaouali et al., 2010). Hence, the main aims
of this study were (1) to assess the antifungal activity
of R. ocinalis EO against three potential plant-
pathogenic fungi, (2) to evaluate its herbicidal activity
on three weed species for the rst time, and then (3)
to formulate a bioherbicide in order to enhance its
eciency and stability.
Material and methods
Plant material and essential oil extraction
In March 2014, R. ocinalis plants, which belong
to the Lamiacae family, were collected at the owering
stage in a naturally diversied mountain of the Selia-
na region in the northeast of Tunisia (36°06'47.9"N
9°35'30.0"E). The plants were identied by the botanist
of the Biotechnology Center of Borj-Cedria (CBBC).
All selected plants were shade-dried for 15 days at
30°C. One hundred grams of dried leaves and owers
were chopped and subjected to hydrodistillation using
a Clevenger-type apparatus for 2 h (Ben Jemia et al.,
2015). The essential oil was stored at 4°C in amber
vials.
GC–MS analysis
The EOs were analyzed by a gas chromatography-
mass spectrometry analyzer (Hewlett Packard HP5890
series II, USA) equipped with an HP-5 column coa -
ted with 5% phenyl methyl siloxane (30 m × 250 µm
× 0.25 µm). The carrier gas was helium, at a pressure
of 1 ml/min. The mass spectrometer (Agilent Tech-
nologies, USA) ionized the compounds at an electron
impact of 70 eV prior to identication. The program
was the following one: 40 °C for 1 min, then a 4 °C/
min increase up to 100 °C, 100 °C for 5 min, followed
by a 6 °C/min increase up to 200 °C, then 200 °C for
5 min, and nally a 15 °C/min increase up to 250 °C.
The total running time for each sample was 46 min.
The components were identied by comparison with
the W9N11.L library and calculated retention indexes
relatively to C8-C24 n-alkanes injected in the HP 5MS
column. The relative area percentages of the dierent
EO constituents were calculated from the peak areas of
the total ions.
Formulation
A formulation was used to mix the EOs in water
and facilitate the penetration of active molecules thro-
ugh the epicuticular waxes. It contained amphiphilic
substances to render interactions between polar
and non-polar parts possible. The compounds of
the formulation were chosen to allow better stabili-
ty, ecacy, and a small droplet size. The detailed
composition of the formulation is presented in Table 1.
Preparation of the culture media
Potato dextrose agar (PDA) was used to grow the
fungal pathogens in Petri dishes, while potato dextrose
broth (PDB) and tomato juice (V8) were used for
growth in ELISA microplates.
Fungal strains and preparation of the inoculum
The fungal species Fusarium oxysporum (MUCL
38936), Fusarium culmorum (MUCL28166) and
Penicillium italicum (MUCL 15608) were obtained
from the BCCM/MUCL Agro-food & Environmental
Fungal Collection (Louvain La Neuve, Belgium).
They were cultured on PDA and incubated at 20°C
under a 16h L: 8h D photoperiod.
A spore suspension was made by adding 10 mL of
sterile distilled water to 0.05% Tween 20 on the surface
of
a 14-day-old fungal colony. The surface was gently
Table 1. Composition of the formulated natural herbicide
based on the use of Rosmarinus ocinalis essential oil.
Composition % Content
Essential oil
Hazelnut vegetable oil
Tween 20
Span 80
Atplus UEP-100
Ethanol
Water
Total
3.4
3.4
0.7
0.3
0.25
0.5
91.45
100

Rosmarinus ocinalis essential oil as an eective antifungal and herbicidal agent
Spanish Journal of Agricultural Research June 2019 • Volume 17 • Issue 2 • e1006
3
et al., 2012; Ben Ghnaya et al., 2013). In order to know
if R. ocinalis EO had only slowed down ger mination
or completely inhibited it, a supplementary test was
carried out. It consisted in transferring the treated
seeds from lter paper moistened with EO at 5 mM to
agar solution, to check if germination might continue/
resume or not. But no seed had germinated after 5 days.
Post-emergence activity of the essential oil
Another experiment was performed to study the eect
of EO on 2-3-week-old T. incarnatum, S. marianum,
and P. minor plantlets under controlled conditions
(natural
photoperiod supplemented with articial light
if needed, with 20 ± 3°C according to the sunlight. The
relative humidity was 60 ± 3%). Only P. minor seeds were
sown in boxes, whereas T. incarnatum and S. marianum
seeds were sown in pots. The weed seeds were sown in
11-cm-diameter pots, and the plants were watered every
day. Once the weeds reached the 2-3-leaf stage, several
solu tions were sprayed. They consisted of 10 mL of
R. ocinalis EO at 7.5, 20, and 34 mM, formulated R.
ocinalis EO at 34 mM, adjuvants alone (as nega tive
controls), distilled water, and a commercial bio logical
herbicide containing 34 mM of pelargonic acid (as a
positive control). Three replications were conducted for
each treatment, in a completely ran domized manner. Seven
days after spraying, the trea ted weed plants were examined
to assess wilting, necrosis, and chlorosis. The percentage
of ecacy was calculated following the equation :
Percentage of ecacy (%) = *100
where N refers to the number of necrotic or withered
leaves, and T represents the total number of leaves.
Statistical analysis
Pre-emergence and post emergence tests were
conducted using a randomized block design with 3
replications. Statistical analyses were performed with
Minitab 17 Statistical Software (Minitab Inc., State Co-
llege, PA, USA). Results were examined statistically
using one-way analysis of variance (ANOVA) followed
by Tukey’s multiple range tests. The dierences between
individual means were considered signicant if p<0.05.
Results
Chemical composition of R. ocinalis essential oil
The EO obtained by hydrodistillation of dried R.
ocinalis owers and leaves had a clear green color
scratched to suspend the spores in the liquid. The spore
suspension was ltered through a sterilized double
layer of ne cloth to remove mycelial fragments. The
spore concentration was adjusted to 10
6
spores/mL with
a Bürker haemocytometer.
Evaluation of the antifungal activity
The antifungal activity of the EO was evaluated using
ELISA microplates with a randomized block design,
as
described by Kaddes et al. (2016). The growth
of each
pathogen was monitored in a volu me of 200
µL
containing diluted (3.10
-2
v/v) PDB medium for P. ita-
licum and F. oxysporum, and V8 medium for F. culmo-
rum, the inoculum, and the EO at 1, 3, and 6 mM.
The optical density of each well was measured at a
wa ve length of 630 nm every 24 h for 120 h, using a
spectrophotometer for ELISA plates. Eight replications
were conducted for each concentration, and tween 20 at
1% v/v was used as a negative control. The inhibition
percentages were then calculated using the following
equation:
where AV is the average value, ODX’(t=0) is the
optical density of the pathogen growth control just after
inoculation, ODX’(t=120) is the optical density of the
pathogen growth control after 120 h, ODHx (t=0) is the
optical density of the pathogen in association with the
EO just after inoculation, and ODHx (t=120h) is the
optical density of the pathogen in association with the
EO after 120 h.
Seed germination bioassay
Seeds of Phalaris minor were collected in Tunisia
from wheat elds. However, seeds of Trifolium incar-
natum and Silybum marianum were obtained from
ECOSEM industry in Belgium. They were sterilized
using 5% sodium hypochlorite for 2 min. Filter pa-
pers were placed in 11-cm-diameter Petri dishes and
moistened with 2 mL of Tween 1% solution (which
did not interfere with the dierent assays) for the
seedling control, or with EO solutions at 0.625, 1.25,
2.5, and 5 mM for the treated seedlings. Ten seeds of
T. incarnatum, S. marianum or P. minor were then
placed immediately in Petri dishes, and three replica-
tes were prepared for each EO concentration. All Petri
dishes were randomly placed in a growth chamber at
a temperature of 23±1°C, in the dark. The number of
germinated seedlings was counted, and their hypoco-
tyls and root lengths were measured after 7 days (Amri

Soene Ben Kaab, Iness B. Rebey, Marwa Hana, Chadi Berhal, Marie L. Fauconnier, et al.
Spanish Journal of Agricultural Research June 2019 • Volume 17 • Issue 2 • e1006
4
centration increased spore germination inhibition
of plant pathogens after 5 days of incubation. At the
lowest EO dose, P. italicum was less sensitive than F.
culmorum and F. oxysporum. In fact, that concentration
was the least eective one. Furthermore, at 6 mM, the
inhibition percentages of spore germination were very
high, i.e. 85.99%, 100%, and 95.40% for F. culmorum,
F. oxysporum, and P. italicum, respectively (Fig. 1).
Herbicidal activity of R. ocinalis essential oil
under laboratory conditions
The application of EO at 5 mM completely inhibited
seed germination of three weeds (T. incarnatum, S.
marianum and P. minor) (Table 3). Moreover, EO at 1.25
and 2.5 mM caused signicant delays in shoot and
root
growth of the same weeds after 7 days as compared to
the control. As far as germination is con cerned, T. incarna -
tum proved more resistant than S. marianum and P. mi nor
and exhibited no response at the lowest EO concentration.
By contrast, the EO had strong eects on the seedling
growth of these weeds, even at low concentrations.
Herbicidal activity of R. ocinalis essential oil
under greenhouse conditions
Studies in which EOs are applied in post-emergen -
ce conditions are scarce. For this reason, R. ocinalis
EO was sprayed on 2-3-week-old weed plants in another
set of experiments to determine its post-emergence
herbicidal activity. The treatment using 7.5 mM EO
showed weed resistance and no visual damage. At 20
mM, the EO caused a few symptoms of injuries on T.
incarnatum and P. minor (Table 4). However, at 34 mM,
the EO caused more visible injuries ranging from wilting
after 1 day and chlorosis after 3 days on T. incarnatum.
Its herbicidal activity reached up to 45%. S. marianum
was consistently more resistant than T. incarnatum and
P. minor at all concentrations. Pelargonic acid (used
as positive control at 3.4%) completely punctured T.
incarnatum and stopped P. minor and S. marianum
growth. We also used the same EO in a formulated
version to enhance the distribution, the coverage, and
the penetration of the active molecules. It presented a
high herbicidal activity, higher than the non-formulated
EO, which reached 71.33% against T. incarnatum. Six
hours after spraying the formulated EO, T. incarnatum
and P. minor leaves were already wilting.
Discussion
Our results show that R. ocinalis EO is an inte-
resting antifungal and herbicidal agent from which
and emitted a pungent smell. The extraction yield was
ca. 1.2% (w/v). The EO components identied by
gas chromatography/mass spectrometry (GC/MS) are
listed in Table 2. This process identied 98.71% of the
compounds present in the EO. The R. ocinalis EO was
characterized by the predominance of the monoterpene
class, among which 1,8 cineole, camphor, and α-pinene
were the most present. This class was followed by
ketones and alcohols, while esters and sesquiterpenes
were found in minor quantities.
Antifungal activity of R. ocinalis essential oil
In a dose-response bioassay, our results showed
that this EO had an interesting potential at dierent
concentrations (1, 3, and 6 mM). A rise in EO con-
Table 2. Chemical constituents of the essential oil
extracted from Rosmarinus ocinalis dried leaves and
owers.
Compounds RIaRIb(%)c
Monoterpene hydrocarbons 17.09
α-Thujene 928 910–935 0.31
α-Pinene 931 921–944 7.09
Camphene 950 936–965 3.09
β-Pinene 980 962–987 3.81
Myrcene 993 975–991 0.44
Phellandrene 1005 990–1009 0.10
γ-3 carene 1011 997–1027 0.27
p-Cymene 1026 1004–1029 1.39
γ-Terpinene 1062 1049–1069 0.41
α-Terpinene 1012 1154–1195 0.18
Oxygenated monoterpenes 80.19
Camphor 1143 1481–1537 12.27
1,8 cineole 1033 1021–1044 54.60
Borneol 1165 1653–1728 9.66
Terpinen 4 ol 1178 1165–1189 0.90
Terpineol 1189 1178–1203 2.76
Esters 0.72
Bornyl-acetate 1286 1264–1297 0.72
Sesquiterpenes 0.71
β-Caryophyllene 1421 1384–1430 0.62
α-Humulene 1455 1430–1466 0.04
γ Cadinene 1525 1498–1531 0.05
aCalculated retention indexes relatively to C8-C24 n-alkanes
injected in the HP 5MS column. bRetention indexes relatively
to C8-C24 n-alkanes injected in the HP 5MS column, based on
Babushok et al. (2011dimethyl silicone with 5% phenyl groups
(slightly polar). cRelative quantications were calculated by
dividing the peak area of each compound by the total area of
each chromatogram.

Rosmarinus ocinalis essential oil as an eective antifungal and herbicidal agent
Spanish Journal of Agricultural Research June 2019 • Volume 17 • Issue 2 • e1006
5
a more environment-friendly alternative to chemical
herbicides might be derived. The antifungal and her-
bicidal activities of EOs have been widely reported
in recent years (Pintore et al., 2002; Salamci et al.,
2007 Tian et al., 2012; Amri et al., 2012; Kaur et
al.,
2012; Ben Ghnaya et al., 2013; Ahluwalia et al.,
2014;
Bouabidi et al., 2015; Hmiri et al., 2015; Ali -
pour & Saharkhiz, 2016; Synowiec et al., 2017 ), but
to our knowledge, only a few studies have focused on
their eect on post emergence when sprayed on weeds
(Hazrati et al., 2017 ). R. ocinalis is largely used in
traditional medicine (Pintore et al., 2002; Ben Jemia
et al., 2015) and widely known for its antimicrobial
and antioxidant activities (Bozin et al., 2007; Celiktas
et al., 2007; Zaouali et al., 2010), but the present stu -
dy
unveils its herbicidal eect in pre-emergence and
post-emergence for the rst time. On the other hand,
GC-
MS
analysis of our R. ocinalis EO extracted
from dried leaves and owers identied 19 compounds
dominated by oxygenated monoterpenes including
1,8 cineole, camphor, and borneol. These results are in
agreement with Zaouali et al. (2010), who showed that
these three major components are also predominant
in the Tunisian R. ocinalis EO. However, their per-
Figure 1. Fungicidal activity of Rosmarinus ocinalis essential oil against three
plant pathogens (Fusarium oxysporum, Fusarium culmorum, and Penicillium
italicum) after 120 h. Dierent letters mean signicantly dierent results with the
same strain (p<0.05, Tukey’s statistical test).
Table 3. Inhibitory eects of Rosmarinus ocinalis essential oil extracted from leaves
and owers at the vegetative stage on the germination and seedling growth of Trifolium
incarnatum, Silybum marianum, and Phalaris minor after 7 days.
Weeds Dose (mM) Germination (%) Root length (cm) Shoot length (cm)
T. incarnatum Control
0.625
1.25
2.5
5
100.0±0.00 A
100.0±0.00 A
100.0±0.00 A
100.0±0.00 A
0.0±0.0 B
4.33±0.11 A
0.26±0.04 B
0.26±0.02 B
0.15±0.01 BC
0.0±0.0 C
3.95±0.16 A
0.85±0.01 B
0.63±0.01 B
0.31±0.02 C
0.0±0.0 D
S. marianum Control
0.625
1.25
2.5
5
86.67±8.89 A
80.00±13.33 A
70.00±6.66 AB
43.33±4.44 B
0.0±0.0 C
1.75±0.05 A
1.73±0.05 A
1.10±0.04 B
0.70±0.007 C
0.0±0.0 D
2.69±0.09 A
2.78±0.09 A
1.03±0.08 B
0.75±0.01 C
0.0±0.0 D
P. minor Control
0.625
1.25
2.5
5
86.66±4.44 A
56.66±4.44 B
36.66±4.44 C
16.66±4.44 D
0.0±0.0 E
2.5±0.17 A
2.24±0.04 A
1.79±0.03 B
0.9±0.07 C
0.0±0.0 D
5.11±0.11 A
4.72 ±0.10 A
3.82±0.12 B
2.22±0.19 C
0.0±0.0 D
Means followed by dierent capital letters in each column are signicantly dierent (p<0.05,
Tukey’s statistical test).

Soene Ben Kaab, Iness B. Rebey, Marwa Hana, Chadi Berhal, Marie L. Fauconnier, et al.
Spanish Journal of Agricultural Research June 2019 • Volume 17 • Issue 2 • e1006
6
centages varied between 26.0-51.2%, 4.9-29.7% and
3.3-10%, respectively. These dierences in chemical
composition could be related to environmental fac -
tors
(the climate, the season, the soil), the genetic
diversity of the species, and the geographic conditi-
ons (Ben Ghnaya et al., 2013). Interestingly, the
monoterpenes identied as main constituents in our
EO have been described as powerful inhibitors of the
seed germination and growth of several plant species
(De Martino et al., 2010; Barton et al., 2014). These
compounds also showed antifungal activity (Ben
Ghnaya et al., 2013; Ahluwalia et al., 2014; Marei &
Abdegaleil, 2018).
In addition, EOs from plants of the Lamiaceae
family, and among them R. ocinalis, are known for
their antimicrobial activity (Hendel et al., 2016). In
our study, R. ocinalis signicantly inhibited the
spore germination of P. italicum, F. oxysporum, and F.
culmorum. F. oxysporum and F. culmorum have been
widely documented as the most important plant pests;
they cause substantial economic losses worldwide
(Hollingsworth & Motteberg, 2008). R. ocinalis
EO from Greece caused a dose-dependent inhibition
of the mycelial growth of ve fungi (Sclerotinia
sclerotiorum, Phytophthora nicotianae, Sclerotium
cepivorum, F. oxysporum, and Fusarium proliferatum)
(Pitarokili et al., 2002). In addition, Sardinian R.
ocinalis EO (450 and 900 µL/mL) showed a weak
activity against all tested fungi (Botrytis cinerea, F.
oxysporum lycopersici, Fusarium graminearum, F.
culmorum, and Rhizoctonia solani). On the other hand,
these EOs present multiple mechanisms of action due
to a large number of active compounds that reduces
the development of fungal resistance. For instance, a
recent study conrmed that 1,8 cineole alone had a low
antifungal power but showed an important synergistic
eect with α-pinene (Hmiri et al., 2015). These two
compounds were identied in our EO. In the same li-
ne, other reports suggested that 1,8 cineole combined
with terpinen-4-ol, also the major component of Me-
laleuca alternifolia EO, had a signicant synergistic
eect on the hyphal morphology of B. cinerea and its
ultrastructure as compared to the treatment using either
component alone. In fact, 1,8 cineole can penetrate the
cell and damage cellular organelles without aecting
membrane permeability. On the other hand, terpinen-
4-ol destroys membrane integrity and increases per-
meability, resulting in ion leakage and membrane dys-
functioning. Several studies reported that EOs could
cause structural and functional damage by disrupting
the membrane permeability and the osmotic balance
of the cell (Yu et al., 2015). Other studies have shown
that they can acidify the external medium and decrease
ATPase and dehydrogenase activities in Aspergillus
avus cells (Tian et al., 2012). Furthermore, EO from
seeds of Anethum graveolens showed fungicidal ac-
tivity against Sclerotinia sclerotiorum by inhibiting
mycelial growth and sclerotial germination. This eect
is the consequence of the inhibition of ergosterol
synthesis, malate dehydrogenase, and succinate dehy-
drogenase (Ma et al., 2015).
In parallel, to our knowledge, no study had yet
tackled the herbicidal activity of R. ocinalis EO.
In fact, our experiments highlighted the outstanding
inhibition of three dierent weeds after treatment
with our EO. This was seen on the percentage of ger-
mination, root growth, and hypocotyl length. In fact,
100% inhibition of germination and seedling growth
was observed with our EO at 5 mM. In this context,
Poonpaiboonpipat et al. (2013) showed that at 1 µL
and 2 µL/Petri dish of Cymbopogon citratus EO, there
was no signicant eect on shoot or root length, but
seedling length was shorter at 4 and 8 µL/Petri dish.
The strong phytotoxic activity was due to the pre-
sence of oxygenated monoterpenes, which is quite
similar to that of Tunisian Eucalyptus erthrocorys
Table 4. Herbicidal activity of Rosmarinus ocinalis essential oil (EO) extracted
from leaves and owers at the vegetative stage on weeds under greenhouse
conditions.
Treatment Dose
(%)
Trifolium
incarnatum
Silybum
marianum
Phalaris
minor
Negative control - 0.0±0.0 E0.0±0.0 C0.0±0.0 D
EO-free formulation - 0.0±0.0 E0.0±0.0 C0.0±0.0 D
EO 0.75 0.0±0.0 E0.0±0.0 C0.0±0.0 D
224±2.66 D0.0±0.0 C27.33±4.44 C
3.4 45±2.0 C0.0±0.0 C34.33±2.88 C
Formulated EO 3.4 71.33±2.44 B18±4.66 B46.33±2.22 B
Formulated pelargonic acid 3.4 100±0.0 A100±0.0 A100±0.0 A
Means followed by dierent letters in each column are signicantly dierent (p<0.05,
Tukey’s statistical test).

Rosmarinus ocinalis essential oil as an eective antifungal and herbicidal agent
Spanish Journal of Agricultural Research June 2019 • Volume 17 • Issue 2 • e1006
7
EO, renowned for its overwhelming phytotoxic eect
(Ben Ghnaya et al., 2013). In this context, among 12
EOs tested on weeds, caraway, thyme, peppermint, and
sage oils were classied as the most phytotoxic ones
owing to the existence of oxygenated monoterpenes
in a 64.1–93.3% range (Synowiec et al., 2017). In line
with this, among six monoterpenes tested by Gouda et
al. (2016), 1,8 cineole and (S)-limonene were showed
to inhibit Echinochloa crus-galli shoot growth. The
major components of EOs are very important for their
biological activity, but even the minor ones could
have signicant synergistic eects (Synowiec et al.,
2017). Many other individual compounds identied
in R. ocinalis, such as α-terpineol, citronellal,
citronellol, and α-pinene, have been conrmed to have
phytotoxic activity (Zhang et al., 2014). In contrast,
among 25 EOs, only those containing volatile pheno-
lic compounds such as thymol, carvacrol, eugenol,
alcohols or ketones, showed strong phytotoxic eect
on dierent weed seeds, even though the mode of
action of all these compounds has not yet been detailed
and a number of eects and hypotheses have been
reported by many authors. Several authors assume
that EOs act by causing biochemical and physiologi-
cal changes in seedling growth (De Martino et al.,
2010). For instance, Cymbopogon citrates EOs notably
slowed down α-amylase activity in E. crus-galli seeds
(Poonpaiboonpipat et al., 2013). Another clear exam ple
is Artemisia sp. EO: it induced reactive oxygen species
production, which in turn caused damage re sulting in
lipid peroxidation, decreased membrane u idity, and
nally increased membrane leakiness and inactivated
receptors, enzymes and ion channels (Kaur et al.,
2012). Moreover, 1,8 cineole inhibited root growth
and stopped DNA synthesis through several steps
(Koitabashi et al., 1997).
We applied R. ocinalis EO not only in pre-emer-
gence tests but also for the rst time in post-emergence
tests, by spraying it on weeds under greenhouse
conditions. Based on the visual damage induced by
this EO on weeds three days after spraying, herbicidal
properties were noticed. Necrosis and wilting leaves
were observed at a concentration of R. ocinalis EO
starting from 20 mM. Similar results showed that the
spraying of Cymbopogon citratus EO from 1.25 mM
to 10 mM on E. crus-galli leaves caused wilting,
and the leaves exhibited desiccation symptoms. In
addition, this EO decreased the chlorophyll a, b and
carotenoid contents, and caused electrolyte leakage,
indicating membrane disruption and loss of integrity
(Poonpaiboonpipat et al., 2013). Monoterpenes, which
are present at 80.19% in our R. ocinalis EO, may
aect plant photosynthesis, energy metabolism, and the
biosynthesis of secondary metabolites such as phenolic
compounds (Gouda et al., 2016). In addition, it has been
conrmed that the penetration of monoterpenes through
the cell wall and cell membrane can cause cellular
potassium leakage that inhibits glucose-dependent
respiration. A recent study showed that the spraying
of a nano-emulsion of Satureja hortensis EO reduced
the weed chlorophyll content, and increased electrolyte
leakage and cell membrane disruption (Hazrati et al.,
2017).
We investigated a formulation of R. ocinalis
EO as a bioherbicide for the rst time, based on
the following observations: (1) as R. ocinalis EO
is lipophilic, it does not dissolve well in water; (2)
in the same line, the reported herbicidal eect of
Satureja hortensis EO in the absence of tween 20
was lower on control weeds; and (3) EOs contain
terpenoids that are volatile, thermolabile, and may be
easily oxidized and hydrolyzed (Pavela et al., 2016).
For these reasons, we used an emulsier providing
better stability, e cacy and persistence for the for-
mulation. An ionic surfactant reduced the eective
concentration of eucalypt oil for a high herbicidal
activity against P. minor (Batish et al., 2007). Based
on that, a recent study showed that a formulation
containing palm oil, tween 20 and span 80 improved
the herbicidal activity of metabolites from Phoma sp.
(Todero et al., 2018).
To our knowledge, this is the rst report that links
the chemical composition of Tunisian R. ocinalis
EO to its fungicidal and bio-herbicidal eects on plant
pathogens and weeds, respectively. Moreover, the
formulation of the bio-herbicide based on Tunisian R.
ocinalis EO was attempted in this work for the rst
time. Hence, this work opens new perspectives on the
application of Tunisian R. ocinalis EOs as a novel
biocontrol strategy against harmful plant pathogens
and weeds. It also paves the way for new strategies
and pathways for the biopesticide industry to create
alternative chemical pesticides designed to be less
harmful to the environment and human health than
current ones. For agronomic applications, we found
that R. ocinalis EO could be used as a biofungicide
at low concentrations between 1 mM and 6 mM with-
out any phytotoxic eect in post-emergence tests. At
concentrations higher than 20 mM, this EO can be
used as a post-emergence bioherbicide. According to
our preliminary results, the use of EOs in the formu-
lation of bioherbicides can oer new prospects for
the sustainable production and practical use of EOs.
To go further in the experiments, it could be really
interesting to determine the modes of action of R.
ocinalis EO on weeds and fungi and try to improve
the eectiveness and stability of the bioherbicidal R.
ocinalis EO formulation.

Soene Ben Kaab, Iness B. Rebey, Marwa Hana, Chadi Berhal, Marie L. Fauconnier, et al.
Spanish Journal of Agricultural Research June 2019 • Volume 17 • Issue 2 • e1006
8
References
Ahluwalia V, Sisodia R, Walia S, Sati OP, Kumar J, Kundu A,
2014. Chemical analysis of essential oils of Eupatorium
adenophorum and their antimicrobial, antioxidant and
phytotoxic properties. J Pest Sci 87: 341-349. https://doi.
org/10.1007/s10340-013-0542-6
Alipour M, Saharkhiz MJ, 2016. Phytotoxic activity and
variation in essential oil content and composition of rosemary
(Rosmarinus ocinalis L.) during dierent phenological
growth stages. Biocatal Agric Biotechnol 7: 271-278. https://
doi.org/10.1016/j.bcab.2016.07.003
Amri I, Gargouri S, Hamrouni L, Hanana M, Fezzani T,
Jamoussi B, 2012. Chemical composition, phytotoxic and
antifungal activities of Pinus pinea essential oil. J Pest Sci
85: 199-207. https://doi.org/10.1007/s10340-012-0419-0
Angioni ALA, Arra ANB, Ereti ELC, Arile DAB, Oroneo
VAC, Abras PAC, Sanita D, Porcell V, 2004. Chemical
composition, plant genetic dierences, antimicrobial and
antifungal activity investigation of the essential oil of
Rosmarinus ocinalis L. J Agric Food Chem 52: 3530-
3535. https://doi.org/10.1021/jf049913t
Araniti F, Mancuso R, Lupini A, Giofrè S, Sunseri F, Gabriele
B, Abenavoli M, 2015. Phytotoxic potential and biological
activity of three synthetic coumarin derivatives as new
natural-like herbicides. Molecules 20: 17883-17902. https://
doi.org/10.3390/molecules201017883
Babushok VI, Linstrom PJ, Zenkevich IG, 2011. Retention
indices for frequently reported compounds of plant essential
oils. J Phys Chem Ref Data 40 (4): 043101-1–043101-47.
https://doi.org/10.1063/1.3653552
Barton AFM, Clarke BR, Dell B, Knight AR, 2014. Post-
emergent herbicidal activity of cineole derivatives. J Pest Sci
87 (3): 531-541. https://doi.org/10.1007/s10340-014-0566-6
Batish DR, Singh HP, Setia N, Kohli RK, Kaur S, Yadav S,
2007. Alternative control of littleseed canary grass using
eucalypt oil. Agron Sustain Dev 27: 171-177. https://doi.
org/10.1051/agro:2007008
Ben Ghnaya A, Hanana M, Amri I, Balti H, Gargouri S, Jamoussi
B, Hamrouni L, 2013. Chemical composition of Eucalyptus
erythrocorys essential oils and evaluation of their herbicidal
and antifungal activities. J Pest Sci 86: 571-577. https://doi.
org/10.1007/s10340-013-0501-2
Ben Jemia M, Tundis R, Pugliese A, Menichini F, Senatore
F, Bruno M, Kchouk ME, Loizzo MR, 2015. Eect of
bioclimatic area on the composition and bioactivity of
Tunisian Rosmarinus ocinalis essential oils. Nat Prod Res
29: 213-222. https://doi.org/10.1080/14786419.2014.94230
5
Bouabidi W, Hanana M, Gargouri S, Amri I, Fezzani T, Ksontini
M, Jamoussi B, Hamrouni L, 2015. Chemical composition,
phytotoxic and antifungal properties of Ruta chalepensis L.
essential oils. Nat Prod Res 29: 864-868. https://doi.org/10.1
080/14786419.2014.980246
Boubaker H, Karim H, Hamdaoui AE, Msanda F, Leach D,
Bombarda I, Vanloot P, Abbad A, Boudyach EH, Ben AA,
2016. Chemical characterization and antifungal activities
of four Thymus species essential oils against postharvest
fungal pathogens of citrus. Ind Crop Prod 86: 95-101.
https://doi.org/10.1016/j.indcrop.2016.03.036
Bozin B, Mimica-Dukic N, Samojlik I, Jovin E, 2007.
Antimicrobial and antioxidant properties of rosemary and
sage (Rosmarinus ocinalis L. and Salvia ocinalis L.,
Lamiaceae) essential oils. J Agric Food Chem 55 (19):
7879-7885. https://doi.org/10.1021/jf0715323
Celiktas OY, Kocabas EEH, Bedir E, Ozek T, Baser KHC,
2007. Antimicrobial activities of methanol extracts and
essential oils of Rosmarinus ocinalis, depending on
location and seasonal variations. Food Chem 100: 553-
559. https://doi.org/10.1016/j.foodchem.2005.10.011
De Martino L, Mancini E, De Almeida LFR, De Feo V,
2010. The antigerminative activity of twenty-seven
monoterpenes. Molecules 15: 6630-6637. https://doi.
org/10.3390/molecules15096630
Giamperi L, Fraternale D, Ricci D, 2011. The in vitro action
of essential oils on dierent organisms. J Essent Oil Res
14: 312-318. https://doi.org/10.1080/10412905.2002.969
9865
Gouda NAA, Saad MMG, Abdelgaleil SAM, 2016. PRE
and POST herbicidal activity of monoterpenes against
barnyard grass (Echinochloa crus-galli). Weed Sci 64:
191-200. https://doi.org/10.1614/WS-D-15-00045.1
Hazrati H, Saharkhiz MJ, Niakousari M, Moein M, 2017.
Natural herbicide activity of Satureja hortensis L.
essential oil nanoemulsion on the seed germination and
morphophysiological features of two important weed
species. Ecotoxicol Environ Saf 142: 423-430. https://doi.
org/10.1016/j.ecoenv.2017.04.041
Hendel N, Larous L, Belbey, L, 2016. Antioxidant activity
of rosemary (Rosmarinus ocinalis L.) and its in vitro
inhibitory eect on Penicillium digitatum. Int Food Res
J 23: 1725-1732.
Hmiri S, Harhar H, Rahouti M, 2015. Antifungal activity
of essential oils of two plants containing 1, 8-cineole as
major component : Myrtus communis and Rosmarinus
ocinalis. J Mater Environ Sci 6: 2967-2974.
Hollingsworth CR, Motteberg CD, 2008. Agronomic and
economic responses of spring wheat to management of
fusarium head blight. Plant Dis 92 (9): 1339-1348. https://
doi.org/10.1094/PDIS-92-9-1339
Kaddes A, Parisi O, Berhal C, Ben Kaab S, Fauconnier MF,
Nasraoui B, Jijakli MH, Massart S, De Clerck C, 2016.
Evaluation of the eect of two volatile organic compounds
on barley pathogens. Molecules 21: 1-10. https://doi.
org/10.3390/molecules21091124
Kaur S, Singh HP, Batish DR, Kohli RK, 2012. Artemisia
scoparia essential oil inhibited root growth involves
reactive oxygen species (ROS)-mediated disruption

Rosmarinus ocinalis essential oil as an eective antifungal and herbicidal agent
Spanish Journal of Agricultural Research June 2019 • Volume 17 • Issue 2 • e1006
9
of oxidative metabolism: In vivo ROS detection and
alterations in antioxidant enzymes. Biochem Syst Ecol 44:
390-399. https://doi.org/10.1016/j.bse.2012.06.015
Koitabashi R, Suzuki T, Kawazu T, Sakai A, 1997. 1,8-Cineole
inhibits root growth and DNA synthesis in the root apical
meristem of Brassica campestris. J Plant Res 110: 1-6.
https://doi.org/10.1007/BF02506836
Kordali S, Usanmaz A, Cakir A, Komaki A, Ercisli S, 2016.
Antifungal and herbicidal eects of fruit essential oils of
four Myrtus communis genotypes. Chem Biodivers 13 (1):
77-84. https://doi.org/10.1002/cbdv.201500018
Koul O, Walia S, Dhaliwal GS, 2008. Essential oils as green
pesticides: Potential and constraints. Biopestic Int 4: 63-
84.
Ma B, Ban X, Huang B, He J, Tian J, Zeng H, Chen Y, Wang
Y, 2015. Interference and mechanism of dill seed essential
oil and contribution of carvone and limonene in preventing
sclerotinia rot of rapeseed. PLoS One 10: 1-15. https://doi.
org/10.1371/journal.pone.0131733
Marei GIK, Abdelgaleil SAM, 2018. Antifungal potential and
biochemical eects of monoterpenes and phenylpropenes
on plant. Plant Protect Sci 54: 9-16. https://doi.
org/10.17221/9/2017-PPS
Pavela R, Benelli G, 2016. Essential oils as ecofriendly
biopesticides? Challenges and constraints. Trends
Plant Sci 21: 1000-1007. https://doi.org/10.1016/j.
tplants.2016.10.005
Pintore G, Usai M, Bradesi P, Juliano C, Boatto G, Tomi
F, Chessa M, Cerri R, Casanova J, 2002. Chemical
composition and antimicrobial activity of Rosmarinus
ocinalis L. oils from Sardinia and Corsica. Flav Fragr J
17: 15-19. https://doi.org/10.1002/j.1022
Pitarokili D, Couladis M, Petsikos-Panayotarou N, Tzakou
O, 2002. Composition and antifungal activity on soil-
borne pathogenes of the essential oil of salvia sclarea
from Greece. J Agric Food Chem 50: 6688-6691. https://
doi.org/10.1021/jf020422n
Poonpaiboonpipat T, Pangnakorn U, Suvunnamek U, Teerarak
M, Charoenying P, Laosinwattana C, 2013. Phytotoxic
eects of essential oil from Cymbopogon citratus and its
physiological mechanisms on barnyardgrass (Echinochloa
crus-galli). Ind Crops Prod 41: 403-407. https://doi.
org/10.1016/j.indcrop.2012.04.057
Salamci E, Kordali S, Kotan R, Cakir A, Kaya Y, 2007.
Chemical compositions, antimicrobial and herbicidal
eects of essential oils isolated from Turkish Tanacetum
aucheranum and Tanacetum chiliophyllum var.
chiliophyllum. Biochem System Ecol 35: 569-581. https://
doi.org/10.1016/j.bse.2007.03.012
Schütte G, Eckerstorfer M, Rastelli V, Reichenbecher W,
Restrepo-Vassalli S, Ruohonen-Lehto M, Saucy AGW,
Mertens M, 2017. Herbicide resistance and biodiversity:
agronomic and environmental aspects of genetically
modied herbicide-resistant plants. Environ Sci Eur 29
(1): 5. https://doi.org/10.1186/s12302-016-0100-y
Synowiec A, Kalemba D, Drozdek E, Bocianowski J, 2017.
Phytotoxic potential of essential oils from temperate
climate plants against the germination of selected
weeds and crops. J Pest Sci 90 (1): 407-419. https://doi.
org/10.1007/s10340-016-0759-2
Tian J, Ban X, Zeng H, He J, Chen Y, Wang Y, 2012. The
mechanism of antifungal action of essential oil from dill
(Anethum graveolens l.) on Aspergillus avus. PLoS
One 7 (1): e30147. https://doi.org/10.1371/journal.
pone.0030147
Todero I, Confortin TC, Luft L, Brun T, Ugalde GA, Almeida
TCD, Arnemann JA, Zabot GL, Mazutti MA, 2018.
Formulation of bioherbicide with metablites from Phoma
sp. Sci Hortic 241: 285-292. https://doi.org/10.1016/j.
scienta.2018.07.009
Uremis I, Arslan M, Sangun MK, 2009. Herbicidal activity of
essential oils on the germination of some problem weeds.
As J Chem 21: 3199-3210.
Yu D, Wang J, Shao X, Xu F, Wang H, 2015. Antifungal
modes of action of tea tree oil and its two characteristic
compenents against Botrytis cinerea. J Appl Microbiol
119 (5): 1253-1262. https://doi.org/10.1111/jam.12939
Zaouali Y, Bouzaine T, Boussaid M, 2010. Essential oils
composition in two Rosmarinus ocinalis L. varieties
and incidence for antimicrobial and antioxidant activities.
Food Chem Tox 48: 3144-3152. https://doi.org/10.1016/j.
fct.2010.08.010
Zhang J, An M, Wu H, Liu DL, Stanton R, 2014. Phytotoxic
activity and chemical composition of aqueous volatile
fractions from Eucalyptus species. PLoS One 9 (3):
e93189. https://doi.org/10.1371/journal.pone.0093189