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J. Plant Prot. and Path., Mansoura Univ., Vol.9 (11): 775 - 781, 2018
Antifungal Activity of Spearmint and Peppermint Essential Oils a
gainst
Macrophomina Root Rot of Cotton
Fathia S. El-Shoraky
1
and A. Y. Shala
2
1
Institute of Plant Pathology, Agricultural Research Center, Giza, Egypt
Email: felshoraki@yahoo.com
2
Medicinal and Aromatic Plants Research Department, Horticulture Research Institute.
Agricultural Research Center, Giza, Egypt. Email: awad.shala@yahoo.com
ABSTRACT
Essential oils as natural antifungal substances one of the alternative methods for plant disease control. The present study was
conducted during 2015 and 2016 to investigate the antifungal activity and oil constituents of volatile oils from spearmint (Mentha viridis
L.) and peppermint( Mentha piperita L.) against cotton root rot pathogen (Macrophomina phaseolina). Gas chromatographic analysis
revealed that spearmint volatile oil was constituted by carvone (60.16%) as a major component followed by 1,8 cineole (8.67%),
limonene (7.40 %), dihydro carvone (5.86 %), β- ocimene (4.29%) and pulegone (3.23%). While peppermint volatile oil was rich in
menthone (46.52%), menthol (25.88%), limonene (7.72%) menthyl acetate (3.90%), iso menthol (2.10%) and sabinene (2.03%). Both
essential oils with different concentrations were evaluated in vitro against three fungus isolates. The two tested oils exhibited 89.55
inhibition percent for the crude oils, against all the tested fungal isolates. Moreover, it was noticed that as oil concentrations decreased,
the inhibitory effect also decreased. At the same time, a highly significant effect of oils at all concentrations was observed during
sclerotial formation (number and size). The use of essential oils as seed treatment exhibited a highly significant reduction in disease
incidence of cotton which has been artificially infested with root rot pathogen, compared to fungicide and untreated control treatments
under the greenhouse conditions. This reduction was calculated to be between 4.56 and 100% compared with a 26.67% reduction with
the utilization of Topsin M treatment at the pre-emergence stage. At the post-emergence stage, all applied treatments were able to
decrease the percentage of root-rot incidence. Reduction ranged between 66.67 and 100% over the untreated control. Reduction in
disease incidence was reflected in a survival plants increase of 34.62–96.17% and 73.09–126.9% for spearmint and peppermint volatile
oils, receptively. Results in the current study demonstrated, that application of peppermint essential oils has an observer influence on the
plant growth (plant height), which differ significantly from this of spearmint oil.
Keywords: spearmint; peppermint; essential oil; cotton; Macrophomina phaseolina
INTRODUCTION
Macrophomina phaseolina (Tassi) Goid is one of
the most important soil borne pathogens, has a wide host
range and infected over 500 plant species in more than 100
plant families around the world(Khan, 2007). The fungus
responsible for causing a major disease of cotton
(Gossypium barbadense) and has been cause severe losses
from seedling to maturity of cotton. Despite its wide host
range, the genus Macrophomina includes only one species,
M. phaseolina. Variation in morphology and virulence
between isolates of M. phaseolina was described as
polyphagous and cosmopolitan fungus which attack
several species of cultivated plants, comprising sorghum,
soybean, cotton, and corn (Su et al., 2001).
Due to the development of resistance to the
synthetic fungicide and accumulation of residues, the
utilization of natural products is deemed one of the better
substitutes for fungal disease management (Gujar and
Talwankar, 2012). Many scholars are trying to find
effective natural products for controlling plant diseases
replacing synthetic pesticides (Kim et al., 2005). Various
plant extracts have been reported as a source of bio-
pesticide because it’s inhibited the growth of plant
pathogens and reduced the hazard impacts to human health
and the environment. Various medicinal plants which have
antifungal properties can also be employed as a source of
plant bio pesticides (Aslam et al., 2010). Plant bio-
pesticides are cheap, locally available, nontoxic, and easily
degradable (Hadizadeh et al., 2009). Furthermore, its
display structural diversity, complexity and rarely include
halogenated atoms. These can act completely as pesticides
(Neerman, 2003).
Lamiaceae family involves more than 4000 species
in 200 genera. Numerous species in lamiaceae family are
medicinal plants that apply in human disease remedy as
well as food in a raw and cooked form (Dhifi et al., 2011).
Mentha piperita is a perennial herbaceous plant belonging
to the family Lamiaceae. It is indigenous of the
Mediterranean region. Peppermint oil is the greatest
popular and extensively utilized essential oil in food,
pharmaceutical and cosmetic industries (Baser 1993). The
biological activity of peppermint essential oil against fungi
and bacteria have been formerly reported by Jirovetz et al.,
(2007) and Moghaddam et al., (2013) Furthermore, the
antifungal and antibacterial activities of the Mentha viridis
volatile oil components have been defined in the previous
literature (Singh, et al., 1994 and Mkaddem et al., 2009).
The hazardous influences of synthetic chemicals
fungicides result in a growing attention in exploiting
alternative and harmless treatments is a great challenge.
Previous reports elucidated the antifungal activity of
essential oils involving lemongrass, citronella, clove,
peppermint, thyme and oregano oils against various fungal
species (Viuda-Martos et al. 2007). Antifungal activities of
specific essential oils were noticed effective against
Rhizoctonia solani, Fusarium moniliforme and Sclerotinia
sclerotiorum (Muller et al., 1995), F. oxysporum (Bowers
and Locke, 2000), and F. solani, R. solani, Pythium
ultimum and Colletotrichum lindemuthianum (Zambonelli
et al., 1996). The mechanism of action of these compounds
against fungi may be correlated to their capability to
dissolve or disrupt the integrity of cell walls and
membranes (Isman and Machial, 2006). Also, essential oils
from eleven medicinal plants were effective in controlling
several soilborne cotton pathogenic fungi (Fusarium poae,
F. oxysporum, F. moniliforme, F. solani, Rhizoctonia
solani, Macrophomina phaseolina, and Sclerotium rolfsii)
under the greenhouse and field conditions (El-Shoraky,
2016). Moreover, essential oils of thyme and spearmint
Fathia S. El-Shoraky and A. Y. Shala
776
diminished in vitro growth of the pathogenic fungi, R.
Solani, Pythium ultimum var. ultimum, Fusarium solani
and Colletotrichum lindemuthianum (Zambonelli et al.,
1996). New biocides-based methods have advanced by
using formulations of essential oils. These formulations,
obtained from fennel, peppermint, caraway, oregano,
rosemary, and ginger, are emulsified with diverse fixed oils
(sesame, olive, cotton and soybean oils) to be operated as a
carrier (Bowers & Locke, 2000 and Mario et al., 2002).
Formulations derived from ginger has been utilized in
treating black rot caused by Alternaria alternata in tomato
fruits (Helal & Abdeldaiem, 2009).
In relation to M. phaseolina chemical control, there
are no fungicides registered for this pathogen. Thus, it is
necessary to evaluate natural alternatives as a fungicide and
their efficiency for controlling it. So, the present study
aimed to evaluate the antifungal potential of Mentha viridis
L. and Mentha piperita L. essential oils against three
isolates of M. phaseolina and to determine both essential
oil constituents. At the same time, to assess the essential
oils effectiveness as cotton seed treatments in controlling
Macrophomina root rot in cotton.
MATERIALS AND METHODS
Essential oils extraction and oil constituents
The tested plants were acquired from Medicinal
and Aromatic Plants Research Department, Horticulture
Research Institute, Agriculture Research Center (ARC),
Ministry of Agriculture, Egypt. Fresh herb of spearmint
(Mentha viridis) and peppermint (Mentha piperita L.)
Family Labiatae. These plants were chosen on the basis of
earlier knowledge on their antifungal activities (Singh, et
al., 1994 and Moghaddam et al., 2013). The essential oils
were extracted by steam distillation using Clevenger type
apparatus for 3 hours. The extracted oils were dried over
anhydrous sodium sulfate. Gas Chromatographic analysis
(GC) was determined for spearmint and peppermint
essential oils at Medicinal and Aromatic Plants Research
Department lab., Horticulture Research Institute,
Agriculture Research Center, Giza, Egypt which were
investigated by DsChrom 6200 Gas Chromatograph
prepared with a flame ionization detector for separation of
volatile oil constituents. The analysis circumstances were
as follows: The chromatograph apparatus was installed
with capillary column BPX-5, 5% phenyl (equiv.)
polysillphenylene-siloxane 30 m x 0.25mmID x 0.25 m
film. Temperature program ramp increase with a rate of 10
Cº / min from 70 to 200 Cº. Flow rates of gases were
nitrogen at 1 ml/min, hydrogen at 30 ml/min and 330
ml/min for air. Detector and injector temperatures were
300 Cº and 250 Cº, respectively. The obtained
chromatogram and report of GC analysis were analyzed to
calculate the percentage of essential oils main components.
The essential oils were then evaluated for antifungal
activity (El-Shoraky& Rashed, 2012).
In vitro antagonists against M. phaseolina:
Paper disc plate method (Loo et al., 1945) was
used. Circular disc (5 mm dia.) of Whatman filter (No. 1)
were cut and after dipping in different oils were located 1
cm inward from the periphery of Petri dishes at four
equidistance places, having in the center the inoculum of
the pathogen (M. phaseolina). Then, a 5 mm plug taken
from 7 days old culture of the test fungus was placed on
the center of PDA medium in a Petri dish and sealed with
parafilm, plates were incubated at 25+3 Cº for 5 days. At
the completion of the incubation period, the linear growth
of the mycelium was measured. Radial growth of M.
phaseolina was recorded and the inhibition percentage was
calculated using the following formula:
C = Radial growth of M. phaseolina in control (mm)
T= Radial growth of M. phaseolina in presence of treatment (mm)
Based on the preliminary screening results the
selected essential oils were prepared in paraffin oil and four
doses viz., 1:1, 1:2, 1:3 and 1:4 (crude oil: paraffin oil) and
applied on Whatman no.1 filter papers (5mm diameter),
control papers were treated with paraffin oil only, another
one was treated with Topsin M fungicide (Thiophenat
methyl 70%). The antifungal activity of essential oil was
evaluated on the mycelia growth of M. phaseolina
according to Boyraz and Ozcan (2006). Five-millimeter
mycelia discs taken from the borders of a 7 days old
culture were located in the middle of a (PDA) plate.
Circular disc (5 mm dia.) of Whatman filter (No. 1) were
cut and after dipping in different oils were placed 1 cm
inward from the periphery of Petri dishes at four
equidistance places, having in the center the inoculum of
the pathogen (M. phaseolina). The Petri plates were sealed
with parafilm and incubated at 24 Cº for 5 days (Arya, et
al., 2017).
Sclerotial formations:
The number and size of fungus sclerotia were
counted in fungal culture suspensions under the
microscope (at low power 10X). The fungal culture
suspension was prepared by vigorously shaking the 5 mm
mycelial disc of the fungus in 5 ml (FAA) (Parmar et al.,
2017).
In vivo experiments (pot experiments):
The present study was conducted at Cotton
Pathology Department, Sakha Agric. Res. St. Seeds of a
local cotton variety cv. Giza 96 were treated with different
concentrations of the tested oils. From the preliminary trial,
these concentrations hadn’t harmful impacts on seeds
germination. For comparison, some seeds were treated
with Topsin M fungicide at the recommended dose (3g/kg
seeds). Greenhouse experiments have been performed to
specify the efficiency of plant essential oils as a seed
treatment to manage cotton root rot (caused by
Macrophomina phaseolina). The potting mixture (soil)
infested with M. phaseolina was used. In this experiment
three agent’s viz., two essential oils (spearmint and
peppermint) and one fungicide (Topspin M) were used
individually for seed treatment. Essential oils were used at
1ml kg
-1
seed while fungicide was used at 3g/kg seed. In
case of control, seeds were sown in Macrophomina
inoculated soil without any agents.
Preparation of fungal inoculum and soil infestation:
Substrate for the growth of each isolate of M. phaseolina,
was prepared by used autoclaved sorghum medium (50 g
of sorghum grains and 40 ml of tap water) in 500ml glass
bottles. Every bottle was inoculated with five discs (0.5 cm
in diameter) of the 5-day-old culture of fungus. Bottles
were incubated at 25±1 Cº for 15 days. The inoculums
which used for soil infestation was a mixture of equal
amounts (w/w) of 3 isolates. 5% formalin solution was
used for soil and pots sterilizing for 15 min. Sterilized soil
J. Plant Prot. and Path., Mansoura Univ., Vol.9 (11), November, 2018
777
was covered with a polyethylene sheet for 7 days to
maintain the gas then left to dry for 2 weeks till all
formaldehyde traces were disappeared. Inoculum was
added at a rate of 3% (w/w) and mixed carefully with the
soil one week prior planting. The infested soil was
dispensed in diameter 25 cm plastic pots and these were
planted with treated seeds (10 seeds/pot) and kept in the
greenhouse. Pre- and post-emergence damping- off were
registered after 15 and 45 days from planting.
Statistical analyses:
Statistical analyses were performed using Assistat-
7.7 beta software for windows (Silva & Azevedo, 2009.).
A complete randomized design was used in these
experiments. The collected data was statistically analyzed
by Duncan´s Multiple Range Test for comparing means.
RESULTS
Gas chromatographic analysis for spearmint
essential oil (Chromatogram 1) revealed the presence of 15
components of which 11 components of them were
identified by the retention times obtained from pure
reference compounds. The identified components were α-
pinene ( 0.55 %), limonene (7.40 %), 1,8 cineole (8.67 %),
β- ocimene ( 4.29% ) , γ- terpineol (1.23%), dihydro
carvone (5.86 %), pulegone ( 3.23%), carvone (60.16 %),
dihydrocarveol acetate (0.93 %), β- caryophyllene (0.95 %)
, caryophyllene oxide (0.88 %) and the rest of numbers are
unknown these results were in accordance with Baser ,
(1993) and Mkaddem et al., (2009). While
chromatographic analysis for peppermint essential oil
revealed the presence of 15 components of which 9
components of them were identified (Chromatogram 2).
The identified components were α-pinene (0.80%),
sabinene (2.03%), limonene (7.72%), 1,8 cineole
(0.83%), menthone( 46.52 %), menthol ( 25.88 %), iso
menthol (2.10%), menthyl acetate (3.90%) and β-
caryophyllene (0.71 %), whereas the rest of numbers are
unknown these results were in harmony with the previous
studiesJirovetz et al., (2007) and Moghaddam et al., (2013)
.
Chromatogram 1. Spearmint essential oil components
Chromatogram 2. Peppermint essential oil components
Antifungal activity
The antifungal activity of two essential oils
(spearmint and peppermint) against mycelial growth of
three isolates of Macrophomina phaseolina is displayed in
(Table1). Statistical analysis (p = 0.05) showed that both
essential tested oils had antifungal activity against M.
phaseolina. The obtained results showed that all
concentrations of tested oils gave a reduction of fungus
growth which was found significantly superior over or
equal of the check fungicide. However, the two tested oils
showed more than 88 percent inhibition in crude oils
against all the tested fungal isolates. It was additionally
detected that when concentrations of oil reduced, the
inhibitory effect also reduced.
Sclerotial formation
ANOVA of the influence of two essential oils at
different concentrations and their interactions on the
sclerotial formation of three isolates of M. phaseolina is
shown in Table 2. Analysis of variance showed significant
influences of isolates and oils in addition to its
concentrations on sclerotial numbers. At the same time,
ANOVA revealed a non- significant effect of isolates and
oils on sclerotial size. A highly significant effect of oils
concentrations was observed on sclerotial formation
(number and size). Based on the significant effect of
interaction between oil concentrations and the other factors
(isolates and oils), (Fig. 1). Similarly; the tested two oils at
all concentrations decreased sclerotial formation. It was
also noticed that when concentrations of oil decreased, the
inhibitory effect also decreased.
Fathia S. El-Shoraky and A. Y. Shala
778
Table 1. The antifungal activity of two essential oils (spearmint and peppermint) against three Macrophomina
phaseolina isolates (cotton root rot pathogen)
Oils Isolates
Oil concentrations ( v:v )
Topsin M Control
C
rude
1:1
1:2
1:3
1:4
0
Spearmint
M1
Linear
growth
0.47
C
0.70
BC
0.70
BC
0.83
BC
1.00
C
4.50
A
1.10
B
4.50
A
In
%
A
89.5
6
84.44
84.44
81.5
6
77.78
0.0
75.56
--
M2
Linear
growth
0.53
E
0.97
DE
1.40
CD
1.80
C
1.97
C
3.63
B
0.60
E
4.50
A
In
%
88.22
78.44
68.89
60.00
56.22
19.33
86.67
--
M3
Linear
growth
0.47
F
1.20
DE
1.50
CDE
1.70
CD
1.93
C
3.73
B
0.93
EF
4.50
A
In
%
89.5
6
73.33
66.67
62.22
57.11
17.11
79.33
--
Peppermint
M1
Linear
growth
0.47
C
0.53
BC
0.53
BC
0.60
BC
0.63
BC
4.50
A
1.10
B
4.50
A
In
%
89.5
6
88.22
88.22
86.67
86.00
0.00
75.55
--
M2
Linear
growth
0.47
C
0.57
C
0.73
C
0.83
C
0.87
C
3.57
B
0.60
C
4.50
A
In
%
89.5
6
87.33
83.
78
81.
56
80.67
20.
67
86.67
--
M3
Linear
growth
.
0.47
D
1.50
BC
1.63
B
1.83
B
1.97
B
4.50
A
0.93
CD
4.50
A
In
%
89.5
6
66.67
63.
78
59.33
56.
22
0.00
79.33
--
Mean of
Linear
growth
0.48
f
0.91
e
1.08
de
1.27
cd
1.39
c
4.07
b
0.88
e
4.50
a
*mean values within rows followed by the same letter are not significantly different at p < 0.05
A
inhibition percent in fungal growth under different treatments used, calculated relative to fungal growth in untreated control
B
Concentrations of essential oils were calculated as (v: v) to the carrier oil
Table 2. Analysis of variance of interactions between isolates of Macrophomina phaseolina and essential oils at
different concentrations in vitro
Parameters and
Source of variation
DF
SS
MS
F
Sclerotial number
Isolates (a)
2
59.89575
29.94788
1175.0483
**
Error
-
a
6
0.15292
0.02549
Oils (b)
1
1.11446
1.11446
397.4597
**
Int. a x b
2
1.21798
0.60899
217.1879
**
Error
-
b
6
0.01682
0.00280
Concentrations (c)
6
313.1977
52.19962
22048.7848
**
Int. a x c
12
15.81619
1.31802
556.7214
**
Int. b x c
6
5.31679
0.88613
374.2960
**
Int. a x b x c
12
5.30286
0.44190
186.6577
**
Error
-
c
72
0.17046
0.00237
Sclrtotial size
Isolates (a)
2
0.12968
0.06484
3.8000
ns
Error
-
a
6
0.10238
0.01706
oils (b)
1
0.02032
0.02032
5.2245
ns
Int. a x b
2
0.69063
0.34532
88.7959
**
Error
-
b
6
0.02333
0.00389
Concentrations (c)
6
4.20762
0.70127
259.8824
**
Int. a x c
12
0.09810
0.00817
3.0294 **
Int. b x c
6
0.10190
0.01698
6.2941 **
Int. a x Tb x c
12
0.34381
0.02865
10.6176 **
Error
-
c
72
0.19429
0.00270
** Significant at a level of 1% of probability (p < .01)
* Significant at a level of 5% of probability (.01 =< p < .05)
ns Non-significant (p >= .05)
Fig .1. Effect of two essential oils {spearmint (o1) and peppermint (o2)} at different concentrations on sclerotial
formation (number and size m) of three Macrophomina phaseolina isolates (m1-m3), cotton root rot
pathogen
J. Plant Prot. and Path., Mansoura Univ., Vol.9 (11), November, 2018
779
Greenhouse experiments
The tested essential oils as seed treatment exhibited
a highly significant reduction in disease incidence of cotton
which has been artificially infested with root rot pathogens,
compared to fungicide and untreated control treatments
(Table 3). Seed treatment with spearmint essential oil gave
a significant reduction to emerged cotton seeds against the
invasion of pathogenic fungi at the pure oil and the first
two concentrations, at the pre-emergence stage.
Peppermint oil at all concentrations recorded from 100% to
50% protection compared with 27.27% protection when
Topsin M treatment was used, over the untreated control.
At the post-emergence stage, data also displayed
that all used treatments reduced the percentage of root-rot
incidence from 100% to 66.67% compared with the
untreated control. The oil treated seeds had a superior
impact on disease incidence by recorded plant survival
percentage over cent percent (126.9% for pure peppermint
oil). Results in the existing table demonstrated that all
concentrations of the tested oils caused an increase in the
survival plant percentage. The spearmint oil recorded
survival plant percentage ranged from 96.66 % to 34.62%
at the low concentration, while the peppermint oil recorded
survival percentage ranged from 126.9 to 73.09%. In this
regard, M. phaseolina appeared further sensitivity against
seed treatments through using both spearmint and
peppermint oil than fungicide.
Results in the current study revealed that applying
of peppermint essential oil have an observer effect on the
plant growth (plant height), which differ significantly from
spearmint oil.
Table 3. Incidence of cotton root rot caused by M. phaseolina and plant height in response to seed dressing with
two essential oils (spearmint and peppermint) under greenhouse conditions.
Oils &
parameters
Oil concentrations
Topsin
M control average
P
ure
1:1
1:2
1:3
1:4
0:1
Pre
emergence
%
Spearmint
D I
15.00
aB
18.33
aB
26.67 aAB
35.00 aA
38.33 aA
36.67 aA
26.67 aAB
36.67 aA
29.17
a
R%
59.1
50.01
27.27
4.5
5
-
4.53
0.0
27.27
Peppermint
D I
0.00
bD
6.67 bCD
13.33 bC
18.33 bBC
18.33 bBC
36.67 aA
26.67 aAB
36.67 aA
19.58
b
R%
100
81.81
63.65
50.01
50.01
0.0
27.27
average
7.50 d
12.50 cd
20.00 bc
26.67 b
28.33 ab
36.67 a
26.67 b
36.67
a
Post
emergence
%
Spearmint
D I
0.00
3.33
0.00
3.33
3.33
16.67
6.67
20.00
6.67
a
R%
100
83.35
100
83.35
83.35
16.65
66.67
Peppermint
D I
1.67
0.00
3.33
3.33
6.67
16.67
6.67
20.00
7.29
a
R%
91.65
100
83.35
83.35
66.67
16.65
66.67
average
0.8
4
b
1.67
b
1.67
b
3.33
b
5.00
b
16.67 a
6.6
7
b
20.00
a
Stand %
Spearmint
D I
85.00 bA
78.3
4
bAB
73.33 bABC
61.67 bCD
58.3
4
bDE
46.6
6
aEF
66.6
6
aBCD
43.33 aF
64.16
b
E%
96.17
80.78
69.24
42.33
34.62
7.69
53.87
Peppermint
D I
98.33 aA
93.33 aAB
83.33 aBC
78.33 aCD
75.00 aCD
46.67 aE
66.67 aD
43.33 aE
73.13
a
E%
126.93
115.39
92.31
80.78
73.09
7.69
53.87
average
91.66
a
85.83
ab
78.33 bc
70.00 cd
66.66 d
46.67 e
66.67 d
43.33 e
Plant height
Spearmint
D I
44.50 bC
56.77 bAB
59.00 bAB
61.67 bA
57.00 bAB
49.33 aBC
50.10 aABC
50.67 aABC
53.63
b
R%
-
12.18
12.0
4
16.44
21.71
12.49
-
2.65
-
1.13
Peppermint
D I
84.67 aA
78.33 aA
80.60 aA
89.00 aA
81.67 aA
49.33 aB
50.10 aB
50.67 aB
70.55
a
R%
67.10
54.59
59.07
75.65
61.18
-
2.65
-
1.13
average
64.58 b
67.55 ab
69.80 ab
75.334 a
69.34 ab
49.33 c
50.10 c
50.67 c
The Tukey Test at a level of 5% of probability was applied
Lower case letters for columns upper case letters for rows
The averages followed by the same letter do not differ statistically between themselves
DISCUSSION
Success in controlling plant diseases has been
documented from the use of artificial fungicides. It has side
effects, such as possible toxicity to plants, animals, and
man as well as adverse environmental impact. Fungicides
can also be costly, and indiscriminate application could
result in resistance, which can counteract their
effectiveness. Depending on the previously mentioned
constraints, alternative approaches such as using natural
products applications (plant extracts and essential oils) may
have a potential role in crop protection. Plant essential oils
are commercially produced from numerous botanical
sources. In our study, the growth inhibition and sclerotial
formation of M. phaseolina causing root rot in cotton have
been tested at various concentration of two essential oils in
vitro. The obtained results showed that the mycelial growth
of M. phaseolina isolates has been influenced differently
by the two tested essential oils. The highest efficiency
against M. phaseolina was registered for peppermint oil at
all tested concentrations, followed by spearmint oil, with
the same efficiency.
Effect of tested oils at various concentrations on
sclerotial formation was noticed negatively associated with
the inhibition of growth. The essential oils have the
capability to penetrate and disrupt the fungal cell wall and
cytoplasmic membranes, permeablise them and finally
damage mitochondrial membranes. Then changes in
electron flow through the electron transport system in the
mitochondria damage the lipids, proteins and nucleic acid
contents of the fungal cells (Arnal-Schnebelen et al.,
2004). Essential oils could also reduce the membrane
potential resulted into the leakage of radicals, cytochrome
C, calcium ions and proteins. Thus, permeabilization of
outer and inner mitochondrial membranes leads to cell
death by apoptosis and necrosis (Yoon et al., 2000).
Sharma and Tripathi (2008) stated that essential oils
perform on the hyphae of the mycelium, provoking the exit
of constituents from the cytoplasm, the loss of rigidity of
the hyphae cell wall and resulting in its collapse. The
activity of essential oils not only in their capability to cross
the cell wall, but also their capacity to damage cellular
enzyme system, including that relating to energy
Fathia S. El-Shoraky and A. Y. Shala
780
production (Conner and Beuchat 1984). Thus, there has
been grown interest in research on, natural antifungal
substances utilization, which might substitute synthetic
fungicides or participate to the development of new disease
control agents.
In the current study, it is well confirmed the control
impact of essential oils from peppermint and spearmint at
different concentrations against important root rot
pathogen of cotton (M. phaseolina). Inhibition of the
fungal growth might be due to the main components such
as menthone, menthol, and limonene in peppermint
essential oil in addition to carvone, 1,8 cineole, limonene,
dihydro carvone, and β- ocimene in spearmint essential oil.
Furthermore, it is possible that the other minor components
may act together synergistically in each oil as has
previously been recommended (Moghaddam et al., 2013).
The obtained data demonstrated that use of both
essential oils as seed treatment for cotton with the same or
supreme anticipated output which obtained with Topsin M.
Therefore, it is concluded that the tested –essential oils
have potential bio-fungicides against the major root rot
pathogen of cotton and as such contributed to characterize
these essential oils as “broad spectrum” bio-fungicidal
chemicals. Nguefack et al., (2008), concluded that essential
oils from Cymbopogon citratus, Ocimum gratissimum
and Thymus vulgaris, has potential bio-fungicides against
the major seed-borne pathogens of rice. Helal, (2017)
revealed that soaking squash seeds in biocides formulating
peppermint oil leads to a significant decline of damping off
and wilt diseases and caused a 10-fold increase in the
plants survival which grown in non-infested soil, compared
with the non-treated plants. In our study, the obtained
results showed that peppermint and spearmint essential oils
have an inhibiting impact on the pathogens growth of
cotton root rot in vitro, and their efficacy is prolonged to
protect seed germination or plant invasion in vivo test.
In the present study, M. phaseolina revealed more
sensitivity against seed treatments by applying peppermint
oil than treatments by spearmint oil and Topsin M. The
application of peppermint oil leads to a significant
reduction of damping- off diseases and induced an increase
in the survival of the plants compared with the non-treated
plants. The present observation displayed that increasing
essential oils concentration led to a significant decline in
disease incidence.
CONCLUSION
The results of this study have shown that volatile oil
from M. piperita has strong remarkable antifungal activity
against M. phaseolina than M. viridis. Gas
Chromatographic analysis of volatile oil from M. viridis
was determined, and its main component was carvone.
While the main component of mentha piperita was
menthone. The study results can be used in the
management of plant pathogenic fungi.
REFERENCES
Arnal-Schnebelen, B.; F. Hadji-Minaglou; J. F. Peroteau; F
Ribeyre and V.G. de Billerbeck (2004). Essential
oils in infectious gynaecological disease: a
statistical study of 658 cases. Int. J. Aromatherapy.
14(4): 192-197.
Arya, P.; Godara, S.L.; Bimla and A. Jat.(2017). Efficacy
of antagonists against Macrophomina phaseolina
inciting dry root rot disease of groundnut. J.
Pharmaco.& Phytochem. 6(6): 1171-1173.
Aslam, A.; Naz, F.; Arshad, M.; Qureshi, R. and C.
A.Rauf, (2010). In Vitro antifungal activity of
selected medicinal plant diffusates against
Alternaria solani, Rhizoctonia solani and
Macrophomina phaseolina. Pak. J. Bot., 42: 2911-
2919.
Baser, K., (1993).Volatile oils of Anatolian Labiatae: a
profile. Acta Hort., 333:217–238.
Bowers, J.H. and L.C. Locke, (2000) Effect of botanical
extracts on the population density of Fusarium
oxisporum in soil and control of Fusarium wilt in
the green house. Plant Dis., 84: 300–305.
Boyraz , N. and M. Ozcan ( 2006). Inhibition of
phytopathogenic fungi by essential oil, hydrosol,
ground material and extract of summer savory
(Satureja hortensis L.) growing wild in Turkey.
Intern J. Food Micro., 107:238-242.
Conner, D.E. and L. M. Beuchat, (1984). Effect of
essential oils from plants on growth of food
spoilage. yeasts. J. Food Sci., 49: 429-434.
Dhifi, W.; Litaiem, M.; Jelali, N.; Hamdi, N. and W. Mnif
(2011). Identification of a new chemotype of the
plant Mentha aquatica grown in Tunisia: Tunisian
folkloric medicine. World J . Microbiol .&
Biotechnol., 25 (12) :2227–2238.
El-Shoraky, F. S.(2016). Efficacy of some essential oils
applied as seed treatment for control seedling
diseases of cotton. Egy. J. Plant Por. Res., 4(4):21-
43.
El-Shoraky, F. S. and N. M. M. Rashed (2012).
Antimicrobial and pesticidal potentiality of
essential oils of some medicinal plants against
deterioration of cumin seeds. J. Plant Prot. & Path.,
Mans. Univ., 3 (12): 1253 – 1268.
Gujar, J. and D. Talwankar, (2012). Antifungal potential of
crude plant extract on some pathogenic fungi.
World J. Sci.& Techno., 2: 58-62.
Hadizadeh, I.; Peivastegan, B. and M. Kolahi, (2009).
Antifungal activity of nettle (Urticad ioica L.),
colocynth (Citrullus colocynthis L. Schrad),
oleander (Nerium oleander L.) and konar (Ziziphus
spina-christi L.) extracts on plants pathogenic
fungi. Pak. J. Bio. Sci., 12: 58-63.
Helal, I. M. (2017). Control of damping-off disease in
some plants using environmentally safe biocides.
Pak. J. Bot., 49(1): 361-370.
Helal, I.M.M. and M.H. Abdeldaiem. (2009). Inhibition of
green and blue rots in orange fruits by using clove
and thyme essential oils treated by gamma
radiation. Arab J. Nucl. Sci. & App., 42: 257-268.
Isman, M.B. and C.M. Machial (2006) Pesticides based on
plant essential oils: from traditional practice to
commercialization. In M. Rai and M.C. Carpinella
(eds.), Naturally Occurring Bioactive Compounds,
Elsevier, BV, pp 29–44.
J. Plant Prot. and Path., Mansoura Univ., Vol.9 (11), November, 2018
781
Jirovetz, L.; Wlcek, K.; Buchbauer, G.; Gochev, V.;
Girova, T.; Dobreva, A.; Stoyanova, A. and E.
Schmidt (2007). Chemical composition and
antifungal activity of essential oils from various
Bulgarian Mentha × piperita L. cultivars against
clinical isolates of Candida albicans. J. Essent. Oil
Bearing Plants., 10(5): 412-420.
Khan, S. N. (2007). Macrophomina phaseolina as causal
agent for charcoal rot of sunflower. Mycopath.,
5(2): 111-118.
Kim, D. I.; Park, J. D.; Kim, S. G; Kuk, H.; Jang, M. J. and
S. S. Kim (2005). Screening of some crude plant
extracts for their acaricidal and insecticidal
efficacies. J. Asia-Pacific Entom. 8: 93–100.
Loo, Y.H., Shell, P.S. and H.H. Thornberry (1945). Assay
of streptomycin by the paper disc plate method. J.
Bacteriol. 50(6):701-709.
Mario, D.; Giovanni, S.; Stefania, D. and B. Emanuela
(2002). Essential oil formulations useful as a new
tool for insect pest control. Pharm. Sci. Tech. 3: 13.
Mkaddem, M.; Bouajila, J.; Ennajar, M.; Lebrihi, A.;
Mathieu, F. and M. Romdhane (2009).Chemical
composition and antimicrobial and antioxidant
activities of Mentha (longifolia L. and viridis)
essential oils. J. Food Sci., 74 (7):358–363.
Moghaddam, M.; Pourbaige, M.; Tabar, H.K.; Farhadi, N.
and S. M. A. Hosseini (2013).Composition and
antifungal activity of peppermint (Mentha piperita)
essential oil from Iran. J. Essent. Oil-Bearing
Plants. 16(4):506–512.
Muller, R.F.; Berger, B. and O. Yegen (1995). Chemical
composition and fungi toxic properties to phyto
pathogenic fungi of essential oils of selected
aromatic plants growing wild in Turkey. J. Agric.
Food Chem., 43: 2262–2266.
Neerman M. F. (2003). Sesquiterpenes lactones a diverse
class of compounds found in essential oils
possessing antibacterial and antifungal properties.
Int. J. Aromatic., 13:114-120.
Nguefack, J.; Leth, V.; Lekagne Dongmo, J.B.; Torp, J.;
Zollo, P. H. A. and S. Nyasse (2008). Use of three
essential oils as seed treatments against seed-borne
fungi of rice (Oryza sativa L.). American-Eurasian
J. Agric. & Environ. Sci., 4 (5): 554-560.
Parmar, H.V.; Kapadia, H.J. and C. M. Bhaliya (2017).
Efficacy of different fungicides against
Macrophomina phaseolina (Tassi) Goid causing
castor root rot. Int. J. Chemical Studies. 5(5): 1807-
1809
Sharma, N. and A. Tripathi (2008). Effects of Citrus
sinensis (L.) Osbeck epicarp essential oil on growth
and morphogenesis of Aspergillus niger Van
Tieghem. Microb. Res., 163 (3): 337 – 344.
Silva, F. de A. S. e. & Azevedo, C. A. V. de.( 2009)
Principal Components Analysis in the Software
Assistat-Statistical Attendance. In: world congress
on computers in agriculture, 7, Reno-NV-USA:
American Society of Agricultural and Biological
Engineers.
Singh, J.; Dubeyd, A.K. and N. N. Tripathi (1994).
Antifungal Activity of Mentha spicata. Int. J.
Pharmacogn., 32 (4):314–319.
Su, G.; Suh, S. O.; Schneider, R. W. and J. S. Russin
(2001). Host specialization in the charcoal rot
fungus, Macrophomina phaseolina.
Phytopathology. 91:120-126.
Viuda-Martos M.; Ruiz-Navajas Y.; Fernandez-Lopez J.
and J.A. Perez- -Alvarez (2007). Antifungal
activities of thyme, clove and oregano essential oils.
J. Food Safety, 27 (1): 91–101.
Yoon, H.S.; Moon, S.C.; Kim, N.D.; Park, B.S.; Jeong,
M.H. and Y.H, Yoo ( 2000). Genistein induces
apoptosis of RPE-J cells by opening mitochondrial
PTP. Biochem. Biophys. Res. Commun. 276(1):
151-156.
Zambonelli, A.; Aulerjo, A.Z.D.; Bianchi, A. and A.
Albasini (1996). Effects of essential oils on
phytopathogenic fungi in vitro. J. Phytopathology.
94: 491-495.
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