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1 Egypt. J. Phytopathol., Vol. 45, No. 1, pp. 1-15(2017)
Induction of Resistance in Pepper
Plants Against Potato Virus Y (PVY)NTN
by Two Medicinal and Aromatic Plant
Essential Oils and Their Major
Components
Radwa M. Shafie, A.A. Kheder and Amal A. Farghaly
Plant Pathol. Res. Inst., Agric. Res. Centre, Giza, Egypt.
he effects of essential oils of clove and fennel seeds and their
major components as foliar spray were screened for inducing
resistance against Potato Virus Y in vivo in both systemic and local
lesion hosts at different concentrations. Clove oil at conc. of 15% gave
the highest inhibitory effect against PVY infection than fennel oil. The
inhibition percentages were 85, 80 % in pepper as a systemic host and
up to 90, 87 %, respectively in Chenopodium amaranticolor as a local
lesion host when applied 48 hrs before virus inoculation. Whereas, the
inhibitory effect was less pronounced when essential oils were
sprayed on the tested plants 24 hrs before virus inoculation. However,
the severity of symptoms was assessed by visual inspection. ELISA
test was used to confirm the results in all cases. When the major
components of essential oils were applied individually, Eugenol gave
the highest inhibitory effect (90%) on PVY- systemically infected
pepper plants and 91.8% on Ch. Amaranticolor as local infected plants
when applied 48 hrs before virus inoculation followed by Anethol.
While, Limonene was less effective in reducing PVY infection.
Considerable increase in total protein content in pepper plants sprayed
with essential oils and their major components before virus
inoculation was recorded compared with healthy and infected control
plants. Moreover, SDS-PAGE revealed a new protein band 20 KDa
which was found only in pepper plants sprayed with clove oil at conc.
of 15%. Also new proteins 19, 20 KDa and 25 KDa were found only
in pepper plants sprayed with Eugenol. It has been suggested that, the
induced proteins may help to limit virus spread or multiplication.
Keywords: Clove seed oil, Fennel seed oil, Induced systemic
resistance, Major oil components, Potato Virus Y (PVY)
and SDS-PAGE.
Sweet pepper (Capsicum annum L.) is a member of the Solanaceous fruity
vegetables group. It is one of the most important, popular and favorite vegetable
crops cultivated in Egypt for local consumption and export (El-Bassiony et al.,
2010). Potato virus Y (PVY) is one of the most damaging viruses causing diseases in
pepper plant; it is widespread wherever pepper and potato are grown (Crosslin et al.,
2006). The virus isolate reacted with dark green mosaic, curl and deformation on
Capsicum annum L. cv. California Wonder (EL Banna et al., 2015). PVY is difficult
to control because of its extremely broad natural host range (Hafez, 1999), and the
ability to be transmitted by many aphid species in non-persistent manner (Mascia et
al., 2010).
T
RADWA M. SHAFIE et al.
Egypt. J. Phytopathol., Vol. 45, No. 1 (2017)
2
The phenomenon of systemic acquired resistance (SAR), refers to a distinct
signal transduction pathway which can make plants to be stimulated to defend
themselves against pathogens (Hunt and Ryals, 1996). Induced resistance is the
phenomenon that a plant; once appropriately stimulated, exhibits an enhanced
resistance up on ‘challenge’ inoculation with a pathogen (Ryals et al., 1994).
Induced resistance has been adopted as a general term and defined as ‘the process of
active resistance dependent on the host plant’s physical or chemical barriers,
activated by biotic or abiotic agents (inducing agents) (Kloepper et al., 1992). In the
few last years, there has been target interest in biologically active compounds
isolated from plant species for inhibiting different viruses on the plant as they are
safe substances for human and environment (Bezic et al., 2011). Systemic induced
resistance (SIR) can be applied as an alternative to the traditional methods of plant
protection. In addition, these compounds were easily biodegradable, non-phytotoxic,
more environment friendly and safe (Ebadollahi, 2011). Clove is one of the most
important aromatic plants in the world. Essential oils obtained from its flower have a
long history of use as natural antimicrobial agent and have recently been used in
several pharmaceutical, food and cosmetic products (Park et al., 2007). Fennel also
has a long history of herbal uses and widely cultivated for its edible strongly
flavored leaves and seeds which are used as culinary species (Roby et al., 2012).
Essential oils and plant extracts can be applied to activate host defense mechanisms
as a potential management. Volatile constituents of Carum copticum and
Cymbopogon citrates were found to be the most potent in reducing the infectivity of
PVX and PVY on C. amaranticolor (Tripathi, 1985). The essential oils of fennel and
anise completely inhibited PVX, TMV and TRSV on C. amaranticolor (Shukla et
al., 1989). The efficacy of Lippa nodiflora, Datura metel and Thuja orientalis
extracts for inducing systemic resistance was tested against PVY on potato.
Spraying the foliage by the extracts exhibited a protection period to the plants
against PVY infection up to one month in the presence of virus source with the
vector Myzus persicae (Al-Ani et al., 2011). Essential oil of clove caused maximum
inhibition of Potato leaf roll virus replication (Iftikhar et al., 2013). The essential oil
of Artemisia and lemongrass and ginger resulted in more than 50% inhibition of
TMV on tobacco plants (Lu et al., 2013). Moreover, the constituents of these oils
like Limonene, Cineole, Zingiberene and Citronellal gave more than 40% inhibition
rate for TMV. Foliar application of eugenol induced systemic resistance against
Tomato yellow leaf curl virus (TYLCV) in tomato plants (Wang and Fan, 2014).
The objectives of this work are to investigate the efficacy of the foliar spraying
with two essential oils and their major components as natural resistance inducers
against PVY on pepper plants under greenhouse condition, and to study the
accumulation of PR-proteins which appear as a result of inducing resistance.
M a t e r i a l s a n d M e t h o d s
This work was carried out in the greenhouse belongs Virus and Phytoplasma Res.
Dep., Plant pathology Res. Institute, (ARC), Giza, Egypt. Seeds of pepper
(Capsicum annum L. cv. California Wonder) were obtained from Vegetable Disease
Res. Dept., and Chenopodium amaranticolor Cost & Reyn seeds were supplied from
INDUCTION OF RESISTANCE IN PEPPER PLANTS …......
Egypt. J. Phytopathol., Vol. 45, No. 1 (2017)
3
Virology greenhouse, Virus and Phytoplasms Res. Dept., Plant Pathology Research
Institute, ARC, Giza, Egypt.
Virus source:
An isolate of Potato virus Y (PVY)NTN isolated previously by Amer et al., 2004
was used in this investigation. The crude sap obtained from frozen leaves of pepper
(Capsicum annum L. cv. California Wonder) was inoculated into pepper plants
which exhibited mosaic and vein banding symptoms 14 days after inoculation.
Essential oil preparation:
The pure essential oil of Clove (Syzgium aromaticum L.), fennel (Foeniculum
vulgare L.) and their major components (active ingredient) (eugenol, caryophyllen
α- pinene) & (anethol, fenchone and limonene, respectively) were kindly supplied
from the National Organization for Drug Control and Research (NODCAR), Giza.
Dilutions of 5, 10, 12 and 15% of essential oils were prepared in distilled water
containing 0.1% Tween-20. Similarly, dilution of 1% of essential oil components
were prepared by mixing 1ml subsequently made up to 99 ml with sterilized distilled
water containing 0.1% Tween -20.
Induced systemic and localized resistance against PVY:
This experiment was conducted using Randomized Complete Block Design.
Essential oils were applied as foliar sprays. Twenty pepper seedlings were
transplanted in pots (ten plastic pots 20 cm. in diam. were used as a replicates in
each treatment by means of two plants per pot). Ten leaves of Ch. amaranticolor
were used as a replicates in each treatment. The effect of the four concentrations of
clove and fennel essential oils, i.e. 5, 10, 12 and 15% were estimated in two
experiments.
In the first experiment, pepper transplants (30 days old) were used as a systemic
host. Whereas, Ch. amaranticolor (30 days old) plants were used as a local lesion
host in the second experiment. Plants were sprayed with 100ml of each
concentration of 5 and 10%, 24 and 48 hrs before virus inoculation and with 12 and
15%, 48 hrs before virus inoculation. In check experiments two groups of plants
were used, the first were sprayed with buffer solution pH 7.4 (healthy control) and
the second were sprayed with distilled water containing 0.1% Tween-20, and then
plants were mechanically inoculated with PVY- infected sap 1ml/ plant (infected
control). Tested plants were observed daily for the appearance of systemic
symptoms on pepper plants or developing of local lesions on Ch. amaranticolor
leaves. Inhibitory effect of the tested essential oils was determined as described by
Devi et al. (2004) using the following equation:
Inhibition % = (A-B/ A) ×100, where A is the number of plants in check experiment
and B is the number of treated plants and after that inoculated by virus inoculums.
Local lesions were counted seven to ten days after inoculation. The percentage of
inhibition of local lesion formation by each treatment over the control was
calculated based on the number of local lesions produced using the formula
described by Madhusudhan et al. (2011) as:
I = C-T/C×100
Where I = inhibition percentage of local lesion formation over control, C = average
No. of local lesions in control leaves and T = average No. of local lesions in treated
RADWA M. SHAFIE et al.
Egypt. J. Phytopathol., Vol. 45, No. 1 (2017)
4
leaves with the essential oils (ten leaves of Ch. amaranticolor were used as a
replicates in each trial). The effect of major clove oil components (eugenol,
caryophyllen α- pinene) and fennel oil components (anethol, fenchone and limonene)
were also estimated at conc. of 1% against PVY on Pepper and C. amaranticolor
plants. Plants were sprayed with different oil components 24 and 48 hrs before virus
inoculation. In check experiments plants were sprayed with distilled water
containing 0.1% Tween-20, then plants were mechanically inoculated with PVY
infected sap. Tested plants were observed daily for the appearance of systemic
symptoms or local lesions and the percentage of inhibition for each treatment was
calculated as mentioned before.
Effect of PVY on leaf area of pepper plants:
Vegetative growth expressed as leaves area. Plant-1 of pepper plants were taken
to determine the effect of PVY on plants depending on date of infection and
compared with control (healthy) pepper plants. Leaf.Area.Plant-1 (cm2) was
measured using Image Analysis Software for Plant Disease Quantification (Assess
2.0 program).
Protein extraction:
Protein extraction was carried out according to Bollag and Edelstein (1993).
Using one gram fresh weight of leaves collected after five days of inoculation with
PVY from pre-treated pepper plants of each treatment and both healthy and infected
control plants. Total proteins were determined using bovine serum albumin as a
standard spectrophotometric method by Bradford (1976).
Sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS- PAGE):
Polyacrylamide gel electrophoresis (PAGE) was used to determine the
qualitative changes in the soluble proteins of pepper plants (healthy or infected with
PVY) as a result of spraying with essential oils or its major components. Twenty-
microliters of leaf samples (40 µg of protein) were subjected to electrophoresis in
15% polyacylamide prepared in 0.1% SDS (Bollag and Edelstein, 1993) and stained
with silver nitrate according to Sammons et al. (1981). Obtained protein gels were
scanned for band Rf using gel documentation system. Different molecular weights
(MW) of bands were determined against protein marker 66,25 and 18 kDa.
Statistical analysis:
Data were analyzed with the statistical analysis system SAS. All multiple
comparisons were first subjected to analysis of variance (ANOVA) comparisons
among means was carried out according to Duncan's multiple range test (Duncan,
1995).
R e s u l t s
Induced systemic resistance against PVY:
The results in Table 1 and Fig. 1 show that all four concentrations i.e., 5, 10, 12
and 15% of both essential oil of clove and fennel, in general gave encouraging
results of virus inhibition when compared with the control treatment. The efficiency
of inhibition was increased with the increasing the concentration and the time before
INDUCTION OF RESISTANCE IN PEPPER PLANTS …......
Egypt. J. Phytopathol., Vol. 45, No. 1 (2017)
5
virus inoculation. The conc. of 15% of the two essential oils tested was the most
effective one, as it significantly induced the highest systemic resistance against PVY.
Essential oil of clove gave the highest inhibition percentage (85 and 80%) at conc. of
15 and 12%, respectively when applied 48 hrs before virus inoculation. Meanwhile,
essential oil of fennel at the same conc. gave 80 and 70% inhibition, respectively.
The inhibitory effect was less pronounced when essential oils were sprayed on the
tested plants 24 hrs before virus inoculation. Least inhibitory percentage (10%) was
obtained using fennel oil at conc. of 5% when applied 24 hrs before virus
inoculation. ELISA test was used to confirm the results in all cases.
Induced local resistance against PVY :
Data obtained from Table 2 showed that all tested concentrations of the two
essential oils significantly reduced the number of local lesions produced by PVY on
Chenopodium amaranticolor and increased the percentages of inhibition against
PVY (Fig. 1). This effect was the highest with the concentration of 15% and
decreased gradually by reducing the concentration from 12 to 5%. Essential oil of
clove at conc. of 10, 12 and 15% gave percentages of inhibition 77, 88 and 90%
respectively, when applied 48 hrs before virus inoculation. Essential oil of fennel
was less effective in reducing the local lesions produced by PVY on Ch.
amaranticolor than essential oil of clove (75, 80 and 87, respectively).
Fig. 1. Pepper and Ch. amaranticolor plants showing the effect of different
concentrations of clove oil on systemic and local infections produced by PVY. A: Control
(infected pepper); B: Healthy non-treated; D, E, G and H treated pepper plants with 15,
12, 10 and 5% conc. respectively; C: Ch. amaranticolor infected control; F and I: Ch.
amaranticolor treated plants with 15 and 5% conc., respectively.
RADWA M. SHAFIE et al.
Egypt. J. Phytopathol., Vol. 45, No. 1 (2017)
6
Table 1. Effect of different concentrations of clove and fennel essential oils on
inhibition percentages of Potato virus Y (PVYNTN) systemically infected pepper
plants
N=Mean number of infected plants I (%) = Inhibition
Table 2. Effect of different concentrations of clove and fennel essential oils on
inhibition percentages expressed as local lesions number produced by PVY on
Ch. amaranticolor
N=Mean number of local lesions I (%) = Inhibition
24 hrs before inoculation
Concentration (%)
Treatment
10
5
ELISA
I (%)
N.
ELISA
I (%)
N.
0.262
35
13
0.296
20
16
Clove
0.283
25
15
0.341
10
18
Fennel
0.977
0
20
0.977
0
20
Control
1.5
0.9
1.6
1.9
LSD at 0.05
48 hrs before inoculation
Concentration (%)
Treatment
15
12
10
5
ELISA
I
(%)
N.
ELISA
I
(%)
N.
ELISA
I
(%)
N.
ELISA
I (%)
N.
0.164
85
3
0.188
80
4
0.198
75
5
0.211
50
10
Clove
0.170
80
4
0.193
70
6
0.207
60
8
0.226
40
12
Fennel
0.977
0
20
0.977
0
20
0.977
0
20
0.977
0
20
Control
1.6
2.0
1.6
1.4
1.3
1.7
1.3
1.4
LSD at
0.05
24 hrs before inoculation
Concentration (%)
Treatment
10
5
ELISA
I (%)
N.
ELISA
I (%)
N.
0.249
66.7
4.0
0.270
50
6.0
Clove
0.368
58.3
5.0
0.293
41.7
7.0
Fennel
0.989
0
12
0.989
0
12
Control
1.3
1.2
1.4
0.6
LSD at 0.05
48 hrs before inoculation
Concentration (%)
Treatment
15
12
10
5
ELISA
I
(%)
N.
ELISA
I
(%)
N.
ELISA
I
(%)
N.
ELISA
I (%)
N.
0.143
90
1.0
0.162
88
1.2
0.173
77
2.3
0.199
70
3.0
Clove
0.158
87
1.3
0.179
80
2.0
0.185
75
2.5
0.206
65
3.5
Fennel
0.989
0
10
0.989
0
10
0.989
0
10
0.989
0
10
Control
1.2
1.4
1.6
1.2
1.3
0.5
1.5
1.2
LSD at
0.05
INDUCTION OF RESISTANCE IN PEPPER PLANTS …......
Egypt. J. Phytopathol., Vol. 45, No. 1 (2017)
7
Effect of PVY on leaf area of pepper plants:
Results showed that leaves area of infected plants are significantly smaller (2.50
cm2) than Healthy plants (6.54 cm2). Leaf area of treated plants with 12%, 15%
clove, 12% and 15% fennel and control (PVY-infected) were 4.80, 5.29, 2.62, 2.81
and 2.50 cm2, respectively. These results showed that the virus has a great effect on
leaf area of infected plants compared to healthy control plants (6.54 cm2).
Table 3. Effect of PVY on leaf area of pepper plants
Effect of the major components of clove and fennel essential oils on inhibition
percentages of PVY on pepper plants:
The activity of the major components in clove oil (Eugenol, caryophyllene and
α-pinene) and in fennel oil (anethol, fenchone and limonene) were individually
evaluated against PVY (Table 4). Data revealed that all tested oil components gave
significant inhibitory effects on PVY- systemically infected pepper plants. The
inhibitory effect of eugenol was stronger in the reduction of PVY infection with the
rate of 90% when applied 48 hrs before virus inoculation. Also, the percentage of
inhibition of PVY with anethol was 85% followed by caryophyllene (80%). Lower
inhibitory effect was obtained when these components were applied 24 hrs before
virus inoculation. Least effect was obtained with limonene and fenchone (45% and
50%), respectively, when applied 24 hrs before virus inoculation.
Effect of major oil components of clove and fennel on inhibitory percentages of local
lesion number produced on Ch. amaranticolor:
Data presented in Table (5) revealed that all tested oil components gave
significant inhibitory effect on number of local lesion produced on Ch.
amaranticolor leaves inoculated with PVY. The highest inhibitory effect (91.8%)
was recorded with eugenol when applied 48 hrs before virus inoculation followed by
anethol (86.4 %) and caryophyllene (82.7%) while limonene gave the lowest record
(72.7%). Lower inhibitory effect was obtained when these components were applied
24 hrs before virus inoculation.
Leaf number
Healthy
control
Infected
control
Clove
12 %
Clove
15 %
Fennel
12 %
Fennel
15 %
Area
Area
Area
Area
Area
Area
1
5.41
2.68
4.56
4.40
3.17
2.16
2
6.85
2.24
4.82
5.94
1.97
2.93
3
6.72
2.78
4.41
5.60
3.01
3.28
4
5.45
2.13
4.45
4.24
3.04
2.30
5
6.84
2.24
5.08
5.92
1.41
2.85
6
6.57
2.90
4.29
5.50
2.94
2.64
7
5.49
2.23
4.63
3.85
3.20
2.26
8
6.62
2.29
4.90
5.78
2.08
2.94
9
7.19
2.77
4.92
5.47
3.05
3.26
10
8.22
2.75
5.95
6.16
2.30
3.49
Average
6.54
2.50
4.80
5.29
2.62
2.81
RADWA M. SHAFIE et al.
Egypt. J. Phytopathol., Vol. 45, No. 1 (2017)
8
Table 4. Effect of major components of clove and fennel essential oils on
inhibition percentages of Potato Virus Y (PVY)NTN systemically infected pepper
plants
Treatment
Time of application
24 hrs before inoculation
48 hrs before inoculation
N.
Inhibition (%)
ELISA
N.
Inhibition (%)
ELISA
Eugenol
6
70.0
0.281
2
90.0
0.121
Caryophyllene
8
60.0
0.253
4
80.0
0.183
α-pinene
9
55.0
0.301
6
70.0
0.212
Anethol
7
65.0
0.237
3
85.0
0.143
Fenchone
10
50.0
0.268
5
75.0
0.199
Limonene
11
45.0
0.352
7
65.0
0.240
Control
20
0
0.999
20
0
0.999
LSD at 0.05
0.4
0.7
0.3
0.5
N=Mean number of infected plants
Table 5. Effect of major components of clove and fennel essential oils on
inhibition percentages expressed as local lesions number produced by PVY on
Ch. amaranticolor
N=Mean number of local lesions
Total protein:
Protein content was determined in pepper plants sprayed with different
concentrations of clove and fennel essential oils and their major components. Data in
Table (6), indicated that protein content were significantly increased in treated
pepper plants compared with healthy and infected control plants. The highest
increased was observed in pepper plants sprayed with eugenol, anethol and clove oil
at conc. of 15% before 48 hrs of virus inoculation. Higher protein levels (1.77 mg/g
FW) was observed in plants sprayed with eugenol followed by anethol (1.73 mg/g
FW) compared with healthy and infected control plants (1.07, 1.21 mg/g FW,
respectively). While pepper plants sprayed with fennel and clove essential oil at 5%
was the lowest protein levels (1.32 mg/g FW, 1.40 mg/g FW, respectively).
Treatment
Time of application
24 hrs before inoculation
48 hrs before inoculation
N.
Inhibition
(%)
ELISA
N.
Inhibition
(%)
ELISA
Eugenol
3.5
65.0
0.288
0.9
91.8
0.113
Caryophyllene
5.0
50.0
0.262
1.9
82.7
0.172
α-pinene
6
40.0
0.293
2.5
77.3
0.203
Anethol
4.0
60.0
0.250
1.5
86.4
0.136
Fenchone
5.5
45.0
0.278
2.2
80.0
0.180
Limonene
6.5
35.0
0.368
3.0
72.7
0.220
Control
10
0
0.979
11
0
0.979
LSD at 0.05
0.3
0.9
1.0
0.4
INDUCTION OF RESISTANCE IN PEPPER PLANTS …......
Egypt. J. Phytopathol., Vol. 45, No. 1 (2017)
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Table 6. Effect of different concentration of clove and fennel essential oils and
their major components on total protein content (mg/g FW) in pepper plants
Protein pattern profile:
Pepper plants sprayed with essential oil of clove and fennel at 5, 10, 12 and 15%
applied 48 hrs before virus inoculation showed variation in number of protein
patterns, molecular weight and density of protein bands compared with untreated
infected control or healthy one, new pattern of protein was observed (Fig. 2A). A
new protein 20 kDa was found only in pepper plants sprayed with clove oil at conc.
of 15%. Extra bands between 16 to 45 kDa were released with high density in the
treated pepper plants but not identical to those in infected control and healthy plant.
Fig. (2B) show some changes in the protein patterns of pepper leaves pre-treated
with major oil constituents at conc. of 1% relative to control. De-novosynthesized
proteins were detected in treated leaves which were undetectable in control leaves.
Treatment with eugenol resulted in the detection of two similar induced proteins
(with molecular weights of 19, 20) in approximately similar amounts and 25 KDa.
Treatment with anethol and caryophyllene gave similar proteins, with molecular
weight of 23 and 24 kDa in different amounts. Treatment with anethol,
caryophyllene and α-pinene gave protein band with molecular weight of 24 kDa.
Treatments with eugenol and caryophyllene gave protein band with molecular
weight of 38 kDa.
Treatment
Protein content(mg/g FW)
48 hrs before inoculation
Clove 5%
1.40
Clove 10%
1.56
Clove 12%
1.63
Clove 15%
1.71
Healthy control
1.07
Infected control
1.21
L.S.D.at 0.05
0.14
Fennel 5%
1.32
Fennel 10%
1.41
Fennel 12%
1.51
Fennel 15%
1.60
Healthy control
1.07
Infected control
1.21
L.S.D.at 0.05
0.07
Eugenol
1.77
Caryophyllene
1.68
α-pinene
1.53
Anethol
1.73
Fenchone
1.62
Limonene
1.46
Healhy control
1.07
Infected control
1.21
L.S.D.at 0.05
0.08
RADWA M. SHAFIE et al.
Egypt. J. Phytopathol., Vol. 45, No. 1 (2017)
10
Fig. 2. SDS-PAGE analysis of: (A): Total protein extracted from pepper leaves pre-
treated with essential oil of clove: Lane M molecular mass markers, Lane 1: infected
unsprayed pepper leaves, Lane 2: healthy pepper leaves, Lane 3-6 pepper plants sprayed
with clove oil at conc. of 5, 10, 12 and 15%, respectively and (B): Total protein extracted
from pepper leaves pre-treated with the major oil components. Whereas: Lane M:
markers, Lane 7: infected unsprayed pepper leaves, Lane 2: healthy pepper leaves, Lane
1,8 are pepper plants sprayed with α-pinene and caryophyllene. Lane 3-6 are pepper
plants sprayed with fenchone, limonene, eugenol and anethole, respectively.
D i s c u s s i o n
The objectives of this study were induction of systemic resistance in pepper
plants against virus infection. Two plant essential oils and their major components
were screened for inducing resistance against PVY. The results suggested that the
infection either systemically or locally was reduced in tested plants when compared
to untreated ones. Foliar treatment with essential oil of clove and fennel showed
high activity in reducing the number of PVY- infected pepper plants. Similar results
were noticed by Iftikhar et al. (2013), who mentioned that foliar treatment with
clove and fennel essential oil significantly reduced Potato leaf roll virus infection in
potato plants. Lippia nodiflora extracts were highly effective in inducing systemic
resistance in potato plants against PVY infection (Al-Ani et al., 2011). Lavender
essential oil had the ability to inhibit Tomato spotted wilt virus multiplication and
spread of virus infection in systemically infected tomato plants (Kobeasy et al.,
2013).
Essential oil of clove at 15% conc. was the most effective in reducing local
lesion number produced by PVY on Ch. amaranticolor followed by the same conc.
of fennel oil. These findings are in accordance with the previous work as mentioned
by Mohamed (2010) on garlic oil which reduced local lesion number produced by
Potato virus Y on Ch. amaranticolor. Essential oil of fennel totally inhibited the
INDUCTION OF RESISTANCE IN PEPPER PLANTS …......
Egypt. J. Phytopathol., Vol. 45, No. 1 (2017)
11
formation of local lesions produced by Potato Virus X on C. amaranticolor (Shukla
et al., 1989). Essential oil of Melaleuca alternifolia was effective as it significantly
decreased lesion numbers produced by Tobacco mosaic virus on Nicotiana glutinosa
(Bishop, 1995). Essential oil of Satureja montana inhibited local lesions number
produced by Cucumber mosaic virus on Ch. amaranticolor (Dunkic et al., 2010).
The average leaf area of treated plant with clove and fennel oil prior to PVY
infection showed a great effect on leaf area of infected plants compared to control
plants (healthy). Results demonstrated that there are significant differences between
leaf area of different clove and fennel treated plants compared with the control. The
result was in agreement with Faragette et al. (1988). The obtained results showed
that pepper plants pre-treated with eugenol and anethol exhibited significant reduced
infection percentages of PVY. These results are in agreement with (ChunMei, 2013)
who mentioned that eugenol significantly reduced the severity of Tomato yellow leaf
curl virus infection (TYLCV) when applied as a foliar spray. Eugenol also induced
(H2o2), peroxidase and polyphenol oxidase in tomato plants. Significant reduction of
local lesion number produced by CMV or TMV was detected when the thymol and
carvecrol was applied on Ch. amaranticolor (Dunkic et al., 2010). Sprays of the
limonoids, nimbin and nimbidin, compounds that occur in the leaves and seeds of
neem oil reduced local lesion formation on C. amaranticolor after mechanical
inoculation with Potato Virus X (Verma, 1974).
Pepper plants infected with Potato virus Y showing high content of total protein
compared to healthy plants. However, there was a progressive increase in protein
content in plants treated with clove and fennel essential oils and their major
components. This result agreed with that obtained by Haque et al., (2005), who
showed that Zucchini yellow mosaic virus infection increased the protein content of
pumpkin leaves compared to healthy ones. The increased protein content in virus
infected plants was due to increased activity of RNA synthetase or RNA polymerase
(Rao et al., 1989). The treated plants also show high protein content compared to
infected control . This may be due to the formation of new antiviral protein. This
agrees with that obtained by Abdel-Shafi (2005). Electrophoretic studies using
sodium dodecyl sulphate polyacrylamide gel electrophoresis indicated that foliar
treatment with two essential oils and their major components induced resistance
against PVY, thus resulted in inducing new proteins, which were not found in the
healthy or infected untreated plants. It has been suggested that, the induced proteins
may help to limit virus infection or multiplication (Chen et al., 2006). Furthermore,
low molecular proteins are responsible of the process of virus inhibition. These
induced proteins have been defined as pathogenesis related proteins, they are
implicated in plant defense because of their anti- pathogenic activities. The
continuous accumulation of newly induced proteins may help in the localization of
viral infection (Van-Loon et al., 1997). Based on current knowledge, it can be
concluded that induce systemic resistance resulting from the expression of several
parameters, including De-novo synthesis of pathogenesis related proteins (PR)
(Walter et al., 2007). This work may be a step; where more investigations are
needed to formulate these compounds to make them more easy to use.
RADWA M. SHAFIE et al.
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12
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(Received 15/01/2017;
in revised form 28/02/2017)
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