Journal of Economic Entomology, 2017, 1–6

doi: 10.1093/jee/tox065
Research Article
Ecotoxicology

Separate and Combined Effects of Mentha piperata and

Mentha pulegium Essential Oils and a Pathogenic Fungus
Lecanicillium muscarium Against Aphis gossypii
(Hemiptera: Aphididae)
Asgar Ebadollahi,1,2 Mahdi Davari,3 Jabrael Razmjou,3 and Bahram Naseri3
1
Moghan College of Agriculture and Natural Resources, University of Mohaghegh Ardabili, Ardabil, Iran (ebadollahi@uma.ac.ir),
2
Corresponding author, e-mail: ebadollahi@uma.ac.ir, and 3Department of Plant Protection, College of Agricultural Sciences,
University of Mohaghegh Ardabili, Ardabil, Iran (mdavari@uma.ac.ir; ramjou@uma.ac.ir; b_naseri@uma.ac.ir)
Subject Editor: Murray Isman
Received 11 October 2016; Editorial decision 31 January 2017

Abstract
In the present study, the toxicity of essential oils of Mentha piperata L. and Mentha pulegium L. and pathogen-
icity of Lecanicillium muscarium (Zare & Gams) were studied in the melon aphid, Aphis gossypii Glover.
Analyses of the essential oils by GC–MS indicated limonene (27.28%), menthol (24.71%), menthone (14.01%),
and carvol (8.46%) in the M. piperata essential oil and pulegone (73.44%), piperitenone (5.49%), decane (4.99%),
and limonene (3.07%) in the essential oil of M. pulegium as the main components. Both essential oils and the
pathogenic fungus had useful toxicity against A. gossypii. Probit analysis indicated LC50 values (lethal concen-
trations to kill 50% of population; 95% confidence limits in parentheses) of M. piperata and M. pulegium essen-
tial oils as 15.25 (12.25–19.56) and 23.13 (19.27–28.42) ml/liter air, respectively. Susceptibility to the pathogenic
fungus increased with exposure time. Aphid mortality also increased when the essential oils were combined
with L. muscarium, although the phenomena was additive rather than synergistic. Mycelial growth inhibition of
L. muscarium exposed to the essential oils was also very low. Based on our results, M. piperata and M. pule-
gium essential oils and the pathogenic fungus L. muscarium have some potential for management of A.
gossypii.

Key words: Aphis gossypii, essential oil, chemical composition, Lecanicillium muscarium, toxicity

Aphis gossypii Glover (Hemiptera: Aphididae) is a globally import-
ant polyphagous pest on watermelon, cotton, and vegetables, ex-
plaining its common names as melon aphid or cotton aphid. It can
cause crop damage by direct feeding, or indirectly through honey-
dew production and transmission of >50 plant viruses (Gildow
et al. 2008). Although chemical insecticides are the main tool for
aphid management, the melon aphid has high potential to develop
resistance to a wide range of pesticides due to its high reproductive
capability (Wang et al. 2007, Herron and Wilson 2011, Matsuura
and Nakamura 2014). Further, other negative side effects of syn-
thetic insecticides such as effects on nontarget organisms and envir-
onmental and human health hazards have directed attention to the
consideration of least-toxic alternatives (Aktar et al. 2009, Watts
and Williamson 2015). These concerns along with consumer de-
mand for safe produce have increased consideration for eco-friendly
bio-agents for insect pest management.
Plant essential oils are biosynthesized in several organs such as
leaf, flower, stem, and root as secondary metabolites. They are
commonly extracted by hydro and steam-distillation methods and
consist of complex mixtures of volatile compounds (Bakkali et al.
2008). Essential oils are generally nontoxic to mammals and bio-
degradable in the environment (Isman 2006). Their applications in
the cosmetic, food, and pharmaceutical industries are well known
(Asbahani et al. 2015). Further, there is much evidence for their effi-
cacy in insect-pest management (Isman et al. 2011, Isman and
Grieneisen 2014).
Cosmopolitan entomopathogenic fungi with their eco-friendly
features are being used for integrated pest management. The genus
Lecanicillium is among the most important entomopathogenic fungi
(Gul et al. 2014). White-halo fungus, L. muscarium (Petch) Zare &
W. Gams., is a distinguished pathogen of arthropods that has been
isolated from several insect pests such as aphids, scales, thrips, and
whiteflies (Askary and Yarmand 2007, Anand et al. 2009, Shinde
et al. 2010). It has been commercialized as Mycotal and Verticillin,
and has displayed great potential for insect-pest management (De
Faria and Wraight 2007, Ali et al. 2013).

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2 Journal of Economic Entomology, 2017, Vol. 0, No. 0
The main goal of present study was, therefore, to assess the sep-
arate and combined toxicity of essential oils isolated from two
reputed medicinal plants (Mentha piperata L. and Mentha pulegium
L.) with the entomopathogenic fungus L. muscarium against A. gos-
sypii under laboratory conditions.
Materials and Methods
Plant Materials and Essential Oils Extraction
The aerial part of M. piperata and M. pulegium was collected at the
flowering stage from Ardabil (38 220 45.000 N, 48 140 57.100 E),
Ardabil province, Iran. The collected species were identified by com-
paring with existing herbarium specimens at the University of
Mohaghegh Ardabili, Ardabil, Iran. Plant materials were washed
with distilled water to remove any dirt and debris and then air-dried
at 27 6 1  C. Fifty grams of the leaves of each plant with 500 ml dis-
tilled water were hydro-distilled using a Clevenger-type apparatus
(Goldis Company, Iran) for 3 h. Anhydrous sodium sulfate was used
to remove water from essential oils. Obtained essential oils were
stored in a refrigerator at 4  C prior to chemical analysis and
bioassays.
Chemical Composition of Essential Oils
Chemical analysis of M. piperata and M. pulegium essential oils was
performed by a GC (7890A Hewlett–Packard, Palo Alto, CA)
equipped with a split injector and a mass sensitive detector system
(5975C). Chromatographic separation was carried out on an HP-5
capillary column 30 m in length, 0.25 mm ID, and 0.25 mm in film
thickness. The mass was restrained in the EI mode at 70 eV. The in-
jector temperature was 250  C. Column temperature program com-
menced at 50  C for 3 min and increased by 10  C/min to 110  C
and by 10  C/min to 180  C. Helium (99.99%) was used as carrier
gas with a flow rate of 1 ml/min. Identification of spectra was car-
ried out by comparing retention times and mass spectra fragmenta-
tion with those in computer libraries (Wiley 7n.1 [Wiley, New York,
NY] and NIST [Standard Reference Data, Gaithersburg, MD]) and
with mass spectra from the literature (Adams 2001).
Insect Rearing
The melon aphid population was obtained from cucumber green-
houses in the Moghan region of Ardabil province. The aphid colony
was reared on cucumber seedlings for up to 10 generations in the
greenhouse of Faculty of Agriculture, University of Mohaghegh
Ardabili, Iran. To prepare the aphid colony, apterous aphids were
carried from infected plants to cucumber seedlings every week.
Insects were reared in a growth chamber kept at 25 6 2  C, 65 6 5%
RH, and a photoperiod of 14:10 (L:D) h.
Toxicity of Essential Oils
Based on primary experiments, estimated essential oils concentra-
tions (1.87–50 and 3.75–56.25 ml/liter air, respectively, for M. piper-
ata and M. pulegium essential oils) were applied accurately on 3-
cm-diameter pieces of filter paper (Whatman No. 1). These treated
filter papers were then appended to the lid of 9-cm-diameter Petri
dishes with 80 ml volume. Fresh cucumber leaf discs, 9 cm diameter,
were placed in the bottom of the Petri dishes above wet cotton pads
to maintain humidity. Twenty age-synchronized adult aphids were
placed in each Petri dish and the dishes sealed with Parafilm. All
procedures were identical for control groups except that essential
oils were not applied. Four replications were made for each concen-
tration and control groups and mortality was determined after 24 h.
Aphids were considered dead if no leg or antennal movements were
observed when gently prodded.
Virulence of Pathogenic Fungus
Lecanicillium muscarium isolate IRAN1768C was originally ob-
tained from the Institute of Iranian Plant Protection Researches,
Iran. The fungus was incubated on Potato Dextrose Agar (PDA) in
9-cm-diameter glass Petri dishes in darkness at 27  C for 30 d. The
single spore method was used for fungus purification. Conidia were
harvested by flooding with 0.02% aqueous Tween 20 and stirring
with a glass bar. Viability of conidia was verified before bioassay
with up to 95% germination after 24-h incubation on PDA medium.
Conidia were counted using a Neubauer hemocytometer (Witeg,
Germany) to standardize suspensions from 1  104 to 1  108 spore/
ml. Cucumber leaves were immersed in 10 ml conidial suspensions
for 20 s. The leaves were left to dry for 10 min in a laminar flow
hood and placed in 9-cm-diameter Petri dishes. Twenty adult aphids
were placed in Petri dishes containing 1% agar and 3-cm-diameter
cucumber leaves. In control groups, leaves were immersed in sterile
distilled water containing 0.02% Tween-80. All Petri dishes were
incubated at 22 6 2  C, 75 6 5% RH, with a photoperiod of 16:8
(L:D) h. Experiments were replicated four times and insect mortality
was recorded daily for 5 d. Cadavers were disinfected with 2% so-
dium hypochlorite, washed with sterile distilled water, and then
incubated on filter paper into a Petri dish. Aphids with fungal sporu-
lation were considered to have died from the fungus treatment.
Combined Effects of Essential Oils and Pathogenic
Fungus
To evaluate combined effects of essential oils and L. muscarium, calcu-
lated LC25 to LC45 of essential oils and a concentration of 104 spore/ml
of L. muscarium were used together against A. gossypii adults. The es-
sential oils were applied to filter paper pieces attached to the lid of Petri
dishes and the cucumber leaves were immersed in conidial suspensions.
Bioassays were as described above for toxicity of essential oils and viru-
lence of L. muscarium. Four replications were used for each treatment
and mortality was determined after 24 h.
Effects of Essential Oils on the Mycelial Growth of
Pathogenic Fungus
Three concentrations of essential oils (LC35, LC40, and LC45) were
emulsified separately in 20 ml 0.02% aqueous Tween-80 and then
each emulsion mixed thoroughly in 20 ml PDA at 45  C. The pre-
pared medium was poured into 9-cm-diameter Petri dishes. Five-
millimeter mycelium discs were taken from stock cultures with a
cork borer and placed in the center of each Petri dish. To avoid
evaporative loss of essential oils, Petri dishes were sealed with
Parafilm. Three replications were prepared for each treatment and a
control group and incubated at 22 6 2  C with a photoperiod of
16:8 (L:D) h. Mycelial growth was measured until discs in the con-
trol groups were full, 7 wk.
Data Analysis
Data were analyzed by ANOVA with SPSS 16 software (SPSS Institute
2007). Differences between means were determined using Tukey’s test
at P ¼ 0.05. Mortality percentages were corrected using Abbott’s for-
mula (Abbott 1925) when any mortality was observed in the control
groups (<10%). Lethal concentration (LC) values were calculated
using Probit analysis (Finney 1971). Mycelial growth inhibition by es-
sential oils and synergistic ratios were evaluated by the following for-
mulas: Inhibition Percentage (IP) ¼ C-T/C  100, where C and T are
Journal of Economic Entomology, 2017, Vol. 0, No. 0 3

the mean colony diameters in control and treatments, respectively. Table 1. Chemical composition of essential oils isolated from
Synergistic Ratio (SR) ¼ A þ B/(A þ B), where A is mean mortality per- Iranian M. piperata and M. pulegium
centage of essential oil concentration, B is mortality of concentration
Components Retention Percentage
104 (spore/ml) of L. muscarium and A þ B and (A þ B) are expected time (min)
and observed mortality, respectively. SR < 0.7, indicates a synergistic M. M.
effect, whereas SR between 0.7–1.8 and SR > 1.8 indicate additive and piperata pulegium
antagonistic effects, respectively (Talebi Jahromi 2011). a-Pinene 6.04 0.63 0.68
5-Methylnonane 6.74 0.28 0.38
Results Sabinene 7.18 1.45 1.66
Ethylcyclohexane 7.55 – 0.60
GC–MS Analyses of Essential Oils (2-Methylpropyl)-cyclohexane 7.58 0.74 –
Limonene (27.28%), menthol (24.71%), menthone (14.01%), car- Decane 7.87 3.59 4.99
vol (8.46%), cis-q-menthan-3-one (5.40%), and trans-carene Limonene 8.81 27.28 3.07
(4.79%) were the major constituents in the essential oil of M. piper- c-Terpinene 9.62 0.38 –
ata. GC–MS analysis accounted for 99.94% of the total essential oil trans-Sabinene 9.86 0.32 –
components of M. piperata (Table 1). hydrate
Linalool 10.90 0.32 –
Pulegone (73.44%), piperitenone (5.49%), decane (4.99%), lim-
Menthone 12.47 14.01 0.99
onene (3.07%), and isopulegone (2.13%) were the main compo-
cis-q-Menthan-3-one 12.76 5.40 –
nents in the M. pulegium essential oil and 99.64% of this oil was Borneol 12.76 – 0.73
identified (Table 1). Isopulegone 13.07 – 2.13
Menthol 13.09 24.71 –
Toxicity of Plant Essential Oils Neoisomenthol 13.35 0.53 –
Results of bioassays indicated that the essential oils had considerable Bicyclo[3.2.2]non 13.51 0.45 –
-6-en-2-one
toxicity against adult females of A. gossypii. Lethal concentration
Dodecane 13.69 0.86 1.10
(LC) values of M. piperata and M. pulegium essential oils are shown Pulegone 14.88 0.36 73.44
in Table 2. Due to the overlap of their confidence limits, differences Carvol 15.05 8.46 –
between toxicity of M. piperata and M. pulegium essential oils were Piperitone 15.31 0.34 –
not significant (Table 2). Camphane 15.87 0.31 –
Likewise, there are no significant differences between the slopes 8-Hydroxy-c-4(5) 16.24 – 0.87
of the regression lines (1.26 and 1.51 for M. piperata and M. pule- -p-menthen-3-one
gium, respectively); their steep slopes indicate homogenous toxic re- trans-Carene 16.40 4.79 –
Piperitenone 17.69 – 5.49
sponse among aphids to these oils (Fig. 1).
Piperitenone oxide 18.33 – 0.79
b-Bourbonene 18.87 0.36 –
Virulence of L. muscarium Tetradecane 19.10 – 0.38
Lecanicillium muscarium showed significant insecticidal action Nonadecane 19.11 0.31 –
against A. gossypii with mortality positively correlated with concen- trans-Caryophyllene 19.76 1.88 1.12
tration and exposure time (Table 3). Probit analysis generated an b-Farnesene 20.61 0.31 –
b-Cubebene 21.30 1.39 –
LC50 value of 6.97  104 (4.74  103–3.08  105) spore/ml after 3 d.
Caryophyllene oxide 23.74 – 1.22
According to data in Table 3, LC50 values decreased with increasing
Viridiflorol 23.96 0.48 –
exposure time. LT50 values (time needed to kill 50% of the popula- Total 99.94 99.64
tion) are shown in Table 4. These values ranged from 78.18 h
(72.10–85.03) for the lowest concentration (104 spore/ml) to
31.50 h (24.40–37.93) for the highest concentration (108 spore/ml). similar result was obtained with M. piperata essential oil except
that observed mortalities were lower than expected at the con-
Toxicity of Essential Oils in Combination With L. centrations of 9.60 and 12.13 ml/liter air. However, for all com-
muscarium binations the Synergistic Ratio was between 0.7–1.8 and the
Mortality of A. gossypii was slightly increased when M. pule- observed phenomena therefore deemed to be only additive
gium essential oil was combined with the pathogenic fungus. A (Table 5).

Table 2. Fumigant toxicity of essential oils of M. piperata and M. pulegium against adult female A. gossypii after 24 h

Essential oils na Slope 6 SE LC50 values with 95% confidence limits (ml/liter air) Sublethal concnb v2 (df ¼ 3)

LC25 LC30 LC35 LC40 LC45

M. piperata 120 1.26 6 0.13 15.25 (12.25–19.56) 4.45 5.85 7.55 9.60 12.13 4.36
M. pulegium 120 1.51 6 0.15 23.13 (19.27–28.42) 8.26 10.39 12.85 15.71 19.09 4.38

Lethal concentrations and 95% confidence limits (CL) were estimated using logistic regression (SPSS Institute 2007).
a
The number of aphids used in each replicate.
b
These concentrations were selected for combined bioassays with pathogenic fungus.
4 Journal of Economic Entomology, 2017, Vol. 0, No. 0

Effects of Essential Oils on L. muscarium Mycelial
Growth
Neither essential oil was particularly inhibitory to mycelial growth
of the fungus, with maximum inhibition after 7 wk for essential oils
of M. piperata and M. pulegium of 12.35% and 8.88%, respectively
(Table 6).
Discussion
Chemical compositions of essential oils of M. piperata and M. pulegium
have been recently reported. For example, pulegone (73.33%) and men-
thone (8.63%) were the main components of M. pulegium essential oil
in the study by Benayad et al. (2012). In the present study, pulegone was
also predominant (73.44%), but menthone was only a minor constituent
(0.99%) of M. pulegium. Conversely, piperitenone was a trace constitu-
ent in the M. pulegium essential oil studied by Benayad et al. (2012) but
a main constituent in the present work (5.94%). Karamaouna et al.
(2013) reported that menthol (34.6%), iso-menthone (14.6%), men-
thone (9.7%), and 1,8-cineole (8.3%) were the main components of the
essential oil of M. piperata. In the present study, menthol and menthone
were also major constituents of the essential oil of M. piperata but our
oil also had considerable proportions of limonene and carvol.
Differences in genetic makeup, seasonal variation, geography, and even
methods of plant drying and essential oil extraction may influence the
chemical composition of plant essential oils (Sefidkon et al. 2007,
Khanavi et al. 2013, Rocha et al. 2014, Riahi et al. 2015).
In the present study, toxicity of essential oils of M. piperata and
M. pulegium was evaluated against A. gossypii. Insecticidal effects
of these essential oils have been previously reported by Benayad
et al. (2012) and Karamaouna et al. (2013). Benayad et al. (2012)
investigated the fumigant toxicity of M. pulegium essential oil
against two major Coleopteran insect pests: Rhyzopertha dominica
7 increased with increasing exposure time.
M. piperata M. pulegium
Probit of mortality (%)
(F.) (Bostrichidae) and Sitophilus oryzae (L.) (Curculionidae). They
found that 3 ll of essential oil produced complete mortality after
24 h. In the study of Karamaouna et al. (2013), strong contact tox-
icity of M. piperata was shown against the vine mealybug,
Planococcus ficus (Signoret) (Pseudococcidae). However, the aphidi-
cidal effects of these essential oils have been evaluated in the present
study for first time, although susceptibility of A. gossypii to other
plant essential oils has been previously reported. For example, fumi-
gant toxicity of essential oil of Artemisia dracunculus L. against the
adults of A. gossypii was investigated by Mousavi and Valizadegan
(2014). They observed significant mortality of A. gossypii with an
LC50 value of 18.63 ll/liter. Good efficacy of essential oils of Sinapis
alba L., Cannabis sativa L., and Achillea millefolium L. has been
shown by spraying against A. gossypii in the study of Yankova et al.
(2014). Toxicity of Santalum austrocaledonicum Vieill against A.
gossypii in laboratory, greenhouse, and field bioassays has been re-
ported by Roh et al. (2015). Our results for insecticidal effects of es-
sential oils M. piperata and M. pulegium on A. gossypii are
consistent with those reported for other plant essential oils.
Pathogenicity of L. muscarium has been documented against sev-
eral insect pests (Askary et al. 1999, Marshall et al. 2003, Askary
and Yarmand 2007, Down et al. 2009). For example, Wang et al.
(2004) found strains V16063, V3450, and Vp28 of L. muscarium
had good virulence against Bemisia tabaci (Gennadius), with LC50
values, respectively, of 2.57  105, 6.03  105, and 6.05  105 spore/
ml after 8 d. Effects of L. muscarium isolates Lm1, Lm2, Lm3, Lm4,
Lm5, and Lm6 against nymphs of Ricania simulans (Walker)
(Ricaniidae) were studied by Guclu et al. (2010) at 1  107 spore/ml
after 7 d. Mortality percentage and LT50 values ranged from 50.95
to 74.76% and 2.34 to 3.90 days, respectively. In the present study,
strain IRAN1768C of L. muscarium indicated strong potential for
A. gossypii control. A concentration of 1  107 spore/ml produced
complete mortality after 4 d and susceptibility of A. gossypii

Lisansky (1989) indicated that insecticidal activity of Beauveria

R² = 0.9701 R² = 0.9616
6 bassiana (Balsamo) Vuillemin can be enhanced by addition of coco-
nut oil. He concluded that a greater number of fungal conidia pene-
5 trate the mouth parts of insects because of the lipophilic properties
of the oil. In the study of Sabbour and Abd-El-Aziz (2010), patho-
4 genicity of three microbial agents including Isaria fumosorosea
Wize, Metarhizium rileyi (Farl.) Kepler, S.A. Rehner & Humber,
3 and Lecanicillium lecanii (Zimmerm.) Zare & W. Gams. was im-
proved in combination with essential oils of Eugenia caryophyllata
2 L., Cuminum cyminum L., and Brassica nigra L. against the broad
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 bean beetle, Bruchidius incarnatus (Boh.). They suggest that struc-
tural degradation of the insects’ integument might facilitate penetra-
Log of concentration
tion by the fungus germ tube. The pathogenicity of L. muscarium
Fig. 1. Concentration–mortality response lines for different concentrations of may be increased by essential oils of M. piperata and M. pulegium in
M. piperata and M. pulegium essential oils against adult female A. gossypii. the present study by a similar mechanism.

Table 3. Virulence of L. muscarium against adult female A. gossypii

Time (day) na Slope 6 SE v2 LC50 value with 95% confidence limits (spore/ml)

1 120 0.33 6 0.06 0.46 5.20  109 (5.58  108–5.83  1011)

2 120 0.38 6 0.05 1.84 2.26  106 (1.21  106–5.46  106)
3 120 0.53 6 0.06 6.73 6.97  104 (4.74  103–3.08  105)
4 120 0.63 6 0.09 3.37 3.72  103 (8.31  102–9.37  103)
5 120 0.90 6 0.25 0.39 8.58  102 (17.27–3.50  103)

Lethal concentrations and 95% confidence limits (CL) were estimated using logistic regression (SPSS Institute 2007).
a
The number of aphids used in each replicate.
Journal of Economic Entomology, 2017, Vol. 0, No. 0 5

Table 4. LT50 values of L. muscarium against adult female A. gossypii

Concn (spore/ml) na Slope 6 SE v2 LT50 values with 95% confidence limits (h)
4
10 400 4.10 6 0.41 3.05 78.18 (72.10–85.03)
105 400 4.32 6 0.38 10.59 60.99 (44.37–78.65)
106 400 4.59 6 0.38 7.56 51.03 (37.07–62.41)
107 400 4.72 6 0.40 8.94 40.45 (28.82–50.97)
108 400 5.49 6 0.54 5.46 31.50 (24.40–37.93)

Lethal times and 95% confidence limits (CL) were estimated using logistic regression (SPSS Institute 2007).
a
The total number of aphids used for each concentration within 5 d.

Table 5. Toxicity of M. piperata and M. pulegium essential oils in combination with 104 (spore/ml) of L. muscarium against adult female A.
gossypii after 24 h

Essential oils Concn of essential oils (ml/liter air) A AþB (A þ B) SR

M. piperata 4.45 26.25 6 0.25 28.75 31.25 0.92

5.85 31.25 6 0.48 33.75 35.00 0.96
7.55 37.50 6 0.29 40.00 40.00 1.00
9.60 42.50 6 0.64 45.00 43.75 1.03
12.13 48.75 6 0.48 51.25 50.00 1.03
M. pulegium 8.26 26.25 6 0.25 28.75 33.75 0.85
10.29 31.25 6 0.25 33.75 38.75 0.87
12.85 36.25 6 0.48 38.75 43.75 0.89
15.71 43.75 6 0.25 46.25 48.75 0.95
19.09 47.50 6 0.29 50.00 52.50 0.95

A and B are mean mortality percentage of adult females of A. gossypii exposed to essential oils and 104 spores/ml of L. muscarium, respectively. The value of B
is 2.50 6 0.29. A þ B and (A þ B) are expected and observed mortality percentage, respectively. SR is Synergistic Ratio ¼ Expected mortality/Observed mortality.
Since SR values were between 0.7–1.8, observed phenomena are considered additive for all essential oils concentrations.

Table 6. Inhibition by M. piperata and M. pulegium essential oils (at concentrations equivalent to LC35, LC40, and LC45) on mycelial growth
of the pathogenic fungus L. muscarium

Essential oil Concn (ml/liter air) Time (wk)

1 2 3 4 5 6 7

M. piperata 7.88 3.51i 5.38h 5.67h 6.47g 6.60g 7.02fg 8.11ef

9.60 5.26h 6.45g 7.09fg 7.65f 8.12ef 8.33e 8.88e
12.13 8.77e 9.68d 10.64c 11.17bc 11.67b 11.84b 12.35a
M. pulegium 12.85 1.75g 2.15g 2.84 fg 4.12ef 4.57e 5.56d 4.40d
15.71 3.51f 3.23f 4.25e 5.29d 6.09c 7.46b 8.11a
19.09 5.26d 5.38d 6.38d 6.47c 7.61b 8.33a 8.88a

Inhibition percentage of treatments for each essential oil compared with Tukey’s test at P ¼ 0.05; means followed by the same letters are not significantly
different.

Susceptibility of L. muscarium itself to the plant essential oils essential oils and L. muscarium in the management of A. gossypii.
has not been previously evaluated. According to our results, inhib- However, future studies need to evaluate effects of these agents in
ition of the mycelial growth of L. muscarium was very low at the the field applications against A. gossypii and other pests, separately
concentrations of M. piperata and M. pulegium essential oils used. and in combination with other bio-pesticides.
Therefore, essential oils in the present study may be used in combin-
ation with L. muscarium.
The utilization of entomopathogenic fungi and plant essential Acknowledgments
oils as natural biocontrol agents should lead to fewer negative side
This research project was supported by the University of Mohaghegh
effects compared to synthetic chemical insecticides. In the present
Ardabili, Ardabil, Iran.
study, L. muscarium showed great potential in the control of A.
gossypii, and the essential oils of M. piperata and M. pulegium also
showed considerable insecticidal effects. Furthermore, combined ap-
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