Control of Puccinia triticina the causal agent of wheat leaf rust disease using safety resistance inducers correlated with endogenously antioxidant enzymes up-regulation

Abstract

The role of safe resistance inducers, hydrogen peroxide (H2O2), benzothiadiazole (BTH) and salicylic acid (SA), as environmentally safe compounds, compared to two commercial fungicides (Somi 8 and Tilt) was evaluated. Treatments against the wheat leaf rust, caused by Puccinia triticina, on the susceptible Egyptian wheat variety (Gemmiza 7) compared to the resistant variety (Misr-1) either with infection or without infection under field conditions for the 2 growing seasons (2014/15 and 2015/16) in Kafr-El-Sheikh, Governorate, Egypt were carried out. Hydrogen peroxide, BTH and SA treatments were effective to suppress the disease visible symptoms and disease severity percentage significantly. Somi 8 and tilt fungicides showed a highly significant reduction in disease severity (%) and disease visible symptoms, followed by the resistance inducers as compared to control susceptible variety infected or uninfected plants. Reactive oxygen species (ROS) such as: superoxide (O2.-) and H2O2 were increased earlier after the infection as a result of treatments, thereby, activities of antioxidant enzymes catalase (CAT), peroxidase (POX) and polyphenol oxidase (PPO) were increased compared to the control. Accordingly, the activities of antioxidant enzymes were increased significantly. Early accumulation of ROS levels could show a central role in killing or inhibiting the fungus and immunizing plants against disease symptoms by increasing the enzyme activities. The treatments were effective so that the chlorophyll a and b concentrations were increased and electrolyte leakage was decreased compared to control. Consequently, yields character values were increased significantly. Interestingly the results proved that the safe resistance inducers showed similar mechanisms to the resistant cultivar Misr-1, thus, it can be recommended to use these safe compounds as an alternative to the fungicides. © 2017, Egyptian Society for Biological Control of Pests. All rights reserved.

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Egyptian Journal of Biological Pest Control, 27(1), 2017, 101-110
Control of Puccinia triticina the Causal Agent of Wheat Leaf Rust Disease Using Safety
Resistance Inducers Correlated with Endogenously Antioxidant Enzymes Up-regulation
Hafez1, Y. M.; Kh. A. A. Abdelaal2; Naglaa A. Taha3; M. M. Badr4 and R. A. Esmaeil5
EPCRS Excellence Center and Plant Pathology & Biotechnology Lab., Dept. of Agricultural Botany,
hafezyasser@gmail.com
(1,4,5Plant Pathology Branch, 2Agricultural Botany Branch), Faculty of Agriculture, Kafrelsheikh University, 33516,
Kafr-Elsheikh, Egypt, 3 Plant Pathology Research Institute, Agri. Res. Center, Sakha Station, Egypt.
(Received: January 25, 2017 and Accepted: March 12, 2017)
ABSTRACT
The role of safe resistance inducers, hydrogen peroxide (H2O2), benzothiadiazole (BTH) and salicylic acid (SA), as
environmentally safe compounds, compared to two commercial fungicides (Somi 8 and Tilt) was evaluated. Treatments
against the wheat leaf rust, caused by Puccinia triticina, on the susceptible Egyptian wheat variety (Gemmiza 7) compared
to the resistant variety (Misr-1) either with infection or without infection under field conditions for the 2 growing seasons
(2014/15 and 2015/16) in Kafr-El-Sheikh, Governorate, Egypt were carried out. Hydrogen peroxide, BTH and SA
treatments were effective to suppress the disease visible symptoms and disease severity percentage significantly. Somi 8
and tilt fungicides showed a highly significant reduction in disease severity (%) and disease visible symptoms, followed
by the resistance inducers as compared to control susceptible variety infected or uninfected plants. Reactive oxygen
species (ROS) such as: superoxide (O2.-) and H2O2 were increased earlier after the infection as a result of treatments,
thereby, activities of antioxidant enzymes catalase (CAT), peroxidase (POX) and polyphenol oxidase (PPO) were
increased compared to the control. Accordingly, the activities of antioxidant enzymes were increased significantly. Early
accumulation of ROS levels could show a central role in killing or inhibiting the fungus and immunizing plants against
disease symptoms by increasing the enzyme activities. The treatments were effective so that the chlorophyll a and b
concentrations were increased and electrolyte leakage was decreased compared to control. Consequently, yields character
values were increased significantly. Interestingly the results proved that the safe resistance inducers showed similar
mechanisms to the resistant cultivar Misr-1, thus, it can be recommended to use these safe compounds as an alternative
to the fungicides.
Key words: Wheat leaf rust, Induced resistance, Puccinia triticina, benzothiadiazole, Salicylic acid,
Superoxide, H2O2.
INTRODUCTION
Wheat (Triticum aestivum L.) is one of the most
vital cereal crops in the world and considered the first
strategic food crop in Egypt. Wheat plants are
infected with several diseases such as: rusts, smuts
and other diseases. Wheat leaf rust caused by
Puccinia triticina, is one of the most common
diseases in Egypt. The fungus is an obligate parasite
in which suggested to be as an important disease of
wheat similar to stem rust (Leonard and Szabo, 2005)
and Fusarium head blight (Goswami and Kistler,
2004). Yield losses in wheat infected with P. triticina
are generally the result of the decreased number of
kernels per head and kernel weight (Kolmer, 2005).
Control of cereal diseases is usually carried out by
fungicide treatment, however, the fungicide
application is limited because of the development of
pathogenic strains with fungicide resistance, the
harmful effect on human health and the
environmental pollution (Hafez et al., 2016). The use
of alternative control treatments such as safe
resistance inducers namely, hydrogen peroxide
(H2O2) which used for plant immunization against
biotic and abiotic stresses as well as used for the
organic food production (Hafez et al., 2012),
benzothiadiazole (BTH) and salicylic acid (SA)
seems important (Bayoumi and Hafez, 2006; Hafez et
al., 2008; Hafez, 2013 and Hafez and El-Baghdady,
2013). BTH is a resistance inducer and a functional
analogue to SA. It induces a systemic acquired
resistance (SAR) during the activation of signal
transduction pathway and it has no anti-microbial
properties (Görlach et al., 1996). BTH and other
inducers protected several plant species against viral,
bacterial and fungal pathogens (Bayoumi and Hafez
2006 and Körösi et al., 2009). BTH suppressed the
gray mold caused by B. cinerea in strawberry (Terry
and Joyce, 2000), induced resistance against
Penicillium expansum in peach (Liu et al., 2005) and
pear fruits (Cao et al., 2005) during postharvest
storage. BTH protected white pepper fruits from B.
cinerea infection (Hafez, 2010). Salicylic acid (SA),
which exists in many plant organs, is an endogenous
signal molecule inducing plant defense response and
reducing populations of pathogens. Exogenous
application of SA in non-toxic concentrations was
effective in the regulation of biotic and abiotic
stresses (Xu and Tian, 2008).
As almost no studies were done in Egypt on the
control wheat leaf rust, using such resistance inducers
correlated with the exogenously ROS levels and
antioxidants, therefore, the aim of research was to

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investigate the efficiency of some resistance inducers
compared to fungicides and resistant cultivar for
controlling the wheat leaf rust disease caused by P.
triticina as well as to clarify the resistance
mechanisms of treatments in relation with the ROS
levels and antioxidant activities.
MATERIALS AND METHODS
Plant materials
Field experiments were conducted during two
seasons (2014/15 and 2015/16) at the experimental
Farm of Kafr Elsheikh Governorate, Egypt.
Laboratory investigations were carried out at EPCRS
Excellence Center as well as Plant Pathology and
Biotechnology Lab. (Under accreditation of ISO
17025), Department of Agriculture, Kafr- El-Sheikh
University, Egypt. Evaluation of the efficiency of 3
resistance inducers and 2 fungicides against the wheat
leaf rust caused by Puccinia triticina on the
susceptible Egyptian wheat variety (Gemmiza 7)
compared to the resistant variety (Misr-1) either with
infection or without was carried out. Wheat grains
were sown in randomly plots (2×2.5m) at the rate of
40 g/plot. These experiments were laid out in
randomized complete block design, with 3
replications. All traditional cultural practices were
applied at the proper time, according to the Ministry
of Agriculture recommendations. The 2 used
fungicides and 3 inducers were applied as a foliar
spray 3 times before the infection. The first was in the
time of tillering stage (70 days after sowing), the
second was after 2 weeks from the first spray and the
3rd spraying was after one week from the second one.
The time of artificial inoculation was after one day of
the third spraying. Reactive oxygen species (ROS),
the antioxidant enzymes catalase (CAT) peroxidase
(POX) and polyphenol oxidase (PPO) enzymes were
determined 24, 48 and 72 hrs earlier after the
infection (ha) in the field and electrolyte leakage as
well. Yield characters such as 1000-grain weight (g)
was determined by the mean weight of random
samples of 1000 grains. Plant height was determined
by the mean height of random samples of 9 plants of
each treatment. Number of grains per spike was
counted as the average grains number of 9 random
spikes from the central rows of each treatment. Spike
length was counted as the average of the length of
spike of 9 random spikes from the central rows of
each treatment as described by Hafez et al. (2016).
Fungal inoculation
Under field conditions, mixed urediospores of P.
triticina were prepared by adding 1 mg of
urediospores to 20 gm of talc powder. The suspension
of spores was prepared by adding 1 gm of
urediospores to 20 ml of distilled water and trace of
mineral oil. After one day from the last spraying of
treatments, plants were uniformly inoculated with
freshly collected urediospores of P. triticina
(obtained from Wheat Disease Research Dept, Plant
Pathol. Res. Instit., Agric. Res. Center, Egypt)
according to the method approved by Tervet and
Cassel (1951) by spraying with previously prepared
spore suspensions as mentioned before and by dusting
with the mixed spores. Disease severity was assessed
every 7 days.
Treatment with fungicides and resistance inducers
Two fungicides as well as other compounds were
used in the present study. Tested fungicides were tilt
25% (0.5 gm/l) and somi 8 35% (1 gm/l) EC,
produced by the Shore Chemical Company and Kafr
El-Zayat chemical Company Limited, Cairo, Egypt,
respectively. Three resistance inducers were used in
the present study, benzo-(1,2,3)- thiadiazole-7-
carbothioic acid S-methyl ester (BTH), 4 Mm, H2O2
(5 ml /l) and salicylic acid (1 gm/l) were individually
tested for their effects on the infection type of wheat
leaf rust caused by artificial inoculation with P.
triticina urediospores under the field conditions
(Table 1).
Disease severity assessments
Artificially inoculated plants were carefully
examined to estimate the disease symptoms and
severity % of infected leaves by rust and infection
type depending on the modified scale The plant
reactions (Infection types) were expressed in 5 types
in the adult stage, where Immune = (0), Resistant =
(R), Moderately resistant = (MR), Moderately
susceptible = (MS) and Susceptible = (S) as described
Table (1): Tested treatments and concentration of wheat plants infected with P. triticina
No.
Treatments
Concentration per liter
1
2
3
4
5
6
7
8
9
Control susceptible variety
Control susceptible variety infected
Control resistance variety
Control resistance variety infected
Somi8 35%
Tilt 25%
H2O2
BTH
Salicylic acid
Sprayed with water only + natural infection
Artificially inoculation by leaf rust pathogen
Sprayed with water only + natural infection
Artificially inoculation by leaf rust pathogen
1 gm/l
0.5 gm/l
5 ml/ l
4 Mm/l
1 gm/l

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Table (2): Scale of disease severity assessments
Reaction
Description
Observation
R-Value
No Disease
No visible infection symptoms
0
0.0
Resistant
Visible chlorosis or necrosis, no uredia
R
0.2
Moderately resistant
Small uredia surrounded by chlorosis or necrosis areas
MR
0.4
Moderately Susceptible
Uredia medium size with no necrotic margins but possibly
some distinct chlorosis
MS
0.8
Susceptible
Large uredia with no necrosis and little or no chlorosis
S
1.0
by Roelf et al. (1992) in table (2). Disease severity
was estimated every week after inoculation in each
experiment.
Electrolyte leakage
Measurements were carried out as described by
Szalai et al. (1996) with some modification. Twenty
segments (1 cm2) of wheat leaves were individually
placed into flasks contained each 25 ml deionized
water (Milli-Q 50, Millipore, Bedford, Mass., USA).
Flasks were shaken for 20 hr at ambient temperature
to facilitate electrolyte leakage from injured tissues.
Initial electrical conductivity measurements were
recorded for each vial, using an Acromet AR20
electrical conductivity meter (Fisher Scientific,
Chicago, IL). Flasks were then immersed in a hot
water bath (Fisher Isotemp, Indiana, PA) at 80°C
(176°F) for 1 hr to induce cell rupture. The vials were
again placed on the Innova 2100 platform shaker for
20 hr at 21°C (70°F). Final conductivity was
measured for each flask. Electrolyte leakage
percentage for each bud was calculated as: initial
conductivity/final conductivity × 100.
Detection of O2·- and H2O2
O2·- and H2O2 were visualized as a purple color of
Nitro blue tetrazolium (NBT) and a brown color of
3,3-diaminobenzidine (DAB), respectively. Wheat
leaves (2 cm pieces) were vacuum infiltrated with 10
mM potassium salicylate buffer (pH 7.8) containing
0.1% w/v NBT or 0.1% w/v DAB. NBT- and DAB-
treated samples were incubated under daylight for 20
min and 2 hrs, respectively and subsequently cleared
in 0.15 w/v % trichloroacetic acid in ethanol:
chloroform 4:1 (v/v) for 1 day (Hückelhoven et al.,
1999). Cleared samples were washed with water and
placed in 50% glycerol prior, to be ready for
evaluation. Discoloration resulted by NBT or DAB
staining was quantified using a ChemiImager 4000
digital imaging system (Alpha Innotech Corp., San
Leandro, USA).
Biochemical assays of antioxidant enzymes
A weight of 0.5 g fresh treated wheat leaf material
was homogenized at 0-4˚C in 3 ml of 50 mM TRIS
buffer (pH 7.8), containing 1 mM EDTA-Na2 and
7.5% polyvinylpyrrolidone. The homogenates were
centrifuged (12,000 rpm, 20 min, 4˚C) and the total
soluble enzyme activities were measured
spectrophotometrically in the supernatant. All
measurements were carried out at 25˚C, using the
model UV-160A spectrophotometer (Shimadzu,
Japan). The activity of catalase (CAT) was
determined according to Aebi (1984). Polyphenol
oxidase (PPO) activity was determined according to
the method described by Malik and Singh (1980).
Peroxidase (POX) activity was measured of the crude
enzyme extract according to Hammerschmidt et al.
(1982).
Chlorophyll a and b concentrations
Chlorophyll a and b concentrations as mg/g fresh
weight of leaves were extracted. Leaf samples (0.5 g)
were homogenized with acetone (90% v/v), filtered
and make up to a final volume of 50 ml. Chlorophyll
concentrations were calculated
spectrophotometerically from the absorbance of
extract at 663 and 645 nm according to Lichtenthaler
(1987). It was determined 90 days after sowing.
Statistical analysis
The experiments were conducted in a completely
randomized design with 3 replicates for each
treatment. Data represent the mean ± SD. Student’s t-
test was used to determine whether significant
differences (P<0.05) existed between mean values
according to O'Mahony (1986).
RESULTS AND DISCUSSION
Effect of treatments on disease severity (%) and
disease symptoms
Safe resistance inducers significantly decreased
the disease severity as compared to the 2 fungicides
and showed similarity to the resistance cultivars
during the 2 growing seasons (Fig. 1). The treatments
were effective so that the disease symptoms
were significantly inhibited (Fig. 2). The best disease
control effect was achieved by Somi8 and Tilt
fungicide treatments, followed by other resistance
inducers, which showed a highly significant reduction
in disease severity (%) and also disease symptoms.
The obtained results are in agreement with
those obtained by El-Salamony (2002); Ata et
al. (2008); Mersha et al. (2012) and Hafez et al
(2014a, and b).

104
Fig. (1): Effect of treatments on the disease severity
% of wheat leaves infected with Puccinia triticina
the causal agent of leaf rust disease. Cont. S.W.:
control susceptible wheat variety (Gemmiza 7)
sprayed with water only. Cont. R.W.: control
resistance variety (Misr-1) sprayed with water
only. Cont. S..Inf.: control susceptible variety
infected by P. triticina. Cont. R. Inf.: control
resistance variety infected by P. triticina. Somi 8
and tilt fungicides and other treatments:
susceptible wheat leaves infected by P. triticina
treated with somi 8, tilt, hydrogen peroxide
(H2O2), Benzothiadiazole (BTH) and salicylic
acid (SA).
Levels of reactive oxygen species (ROS) and
activity of antioxidant enzymes
All treatments were able to increase early after
infection the levels of endogenous ROS mainly
superoxide (O2•-) and hydrogen peroxide (H2O2) in
infected wheat by leaf rust fungus. The brown and
purple discoloration and spots in all treatments are the
indicators of H2O2 intensity and high levels of O2•- as
compared to the control treatments. Discoloration of
leaves resulted by NBT or DAB staining was
visualized using naked eye (Fig. 3) and quantitatively
as well (Fig. 4). Results also showed that the activities
of catalase (CAT), peroxidase (POX) and polyphenol
oxidase (PPO) were significantly increased in
infected wheat leaves compared to control treatments
early particularly 48 and 72 hrs after infection (hai)
(Fig. 5). This may be due to the inhibiting or killing
action of ROS to the fungal pathogen early after
infection, therefore, there was no chance for the
pathogen to grow or propagate (Hafez, 2010, Hafez et
al., 2014 a and b, Abdelaal et al., 2014 and Omara et
al., 2015). The results are supported by previous
researches (Hafez, 2009 and 2010, and Hafez and El-
Baghday, 2013). Data of the present study indicated
that high levels of ROS after infection stimulated, as
a result, increased antioxidant enzyme activities
therefore, immunized plants against disease infection.
Several studies indicated that SA and BTH can cause
ROS accumulation through the mitochondrial
electron transport inhibition (Norman et al., 2004) or
antioxidant enzymes (Bayoumi and Hafez, 2006). It
was suggested that H2O2 induces SA accumulation
(Van Camp et al., 1998). The up-regulation of CAT,
POX and PPO plays a pivotal role against viral,
bacterial and fungal infections (Hafez et al., 2012 and
2014 a and b).
Electrolyte leakage
Electrolytes leakage (EL) percentage is an
indicator of cell membrane permeability. Susceptible
wheat plants infected with P. triticina and treated
with safe, resistant inducers and fungicides (Tilt and
Somi 8) compared to the infected resistant variety
showed the highest significant reduction in
electrolyte leakage on both seasons (2014 and 2015)
compared to control of the infected susceptible
variety which showed a significant increase of the
membrane permeability on both seasons (Fig. 6). The
highest significant reduction in electrolyte leakage
was obtained later, after appearance of natural
infection in the field 24, 48 and 72 hrs. Likewise, non-
traditional treatments could alter resistance or
susceptibility of plants to infection through their
effects on cell membrane permeability (Hafez et al.,
2014a and b). High temperature stress induced
susceptibility in maize by increasing electrolyte
leakage (Garraway et al., 1989). This might result in
loss of host cell constituents which may be used by
invading pathogen as a source of nutrients. The
present results indicated that the treatments protected
cell membranes during the pathogen attack, while the
cell membrane of the untreated wheat plants was
affected by the pathogen infection and lost its
constituents. Results of the present study are in
agreement with those obtained by (Garraway et al.,
1989; Houimli et al., 2010 and Hafez, 2014 a and b).
Chlorophyll a, b and total concentrations
Chlorophyll a, b and total chlorophyll
concentrations were increased in all treated infected
wheat plants compared to the control of susceptible
variety in both seasons (Fig. 7). The increase of
chlorophyll a, b and total concentrations may be due
to the pivotal role of these treatments in improvement
physiological and biochemical aspects such as
photosynthetic capacity, antioxidant activity and
increase leaves longevity, chlorophyll concentrations
as well (Abdelaal et al., 2014; Hafez et al., 2014 a
and b as well as Abdelaal, 2015).
Effect of treatments on yield characters
All studied yield characters were affected by
treatments in both seasons, particularly fungicides
(Somi 8 and Tilt) compared to the control susceptible

105
Fig. (2): Effect of treatments on disease symptoms of wheat leaves infected with P. triticina 2 weeks after
infection.
A
B
C
A
B
C
Fig. (3): Effect of treatments on brown discoloration of hydrogen peroxide (upper rows) and purple
discoloration of superoxide (lower rows) of wheat leaves infected with Puccinia triticina 24 (A), 48 (B)
and 72 (C) hrs after infection.

106
Fig. (4): Effect of treatments on levels of hydrogen peroxide and superoxide of wheat leaves infected with
Puccinia triticina 24, 48 and 72 hrs after infection (hai).
Fig. (5): Effect of treatments on activity of antioxidant enzymes catalase (CAT), peroxidase (POX) and
polyphenol oxidase (PPO) of wheat plants 24 ,48 and 72 hrs after the appearance of natural infection (hai)
with P. triticina during the two seasons (2014 and 2015). Means of 3 measurements in each of two
independent experiments ± SD are shown.

107
Fig. (6): Effect of treatments on electrolyte leakage % of wheat plants 24, 48 and 72 hrs after the appearance
of natural infection (ahi) with P. triticina during the two seasons, 2014 and 2015.
Fig. (7): Effect of treatments on chlorophyll a, b and total chlorophyll of wheat plants infected with P. triticina
during the two seasons, 2014 and 2015.

108
Fig. (8): Effect of treatments on yield characters of infected wheat plants with leaf rust caused by P. triticina
during the two growing seasons 2014 and 2015.

109
variety (Fig. 8). The highest values of plant height,
Spike length, 1000- grain weight (g) were obtained
by fungicides (Somi 8 and Tilt), followed by safe
resistance inducers treatments compared to control
susceptible variety. The highest value of the number
of grains per spike was obtained by fungicides,
control resistant variety and compared to the
control susceptible variety infected in both seasons.
These results may be due to the positive effects of
treatment compounds in improving growth and yield
of infected wheat plants. Similar results were
conducted in barley plants (Hafez et al., 2014a and
Hafez et al., 2016).
In conclusion, the application of safe resistance
inducers (H2O2, BTH and SA) to clear up the effect
and mechanisms of these inducers against wheat leaf
rust disease caused by P. triticina fungus correlated
with the levels of ROS (O2·- and H2O2) as well as the
antioxidant activities compared to the fungicides.
Here, it was possible to induce ROS early after
infection on susceptible wheat variety, which
inhibiting or killing the fungus, consequently, up-
regulation of the antioxidants occurred, accordingly,
the fungus was suppressed. Interestingly, the effect
of the safe resistance inducers against P. triticina was
similar to the effect of resistant wheat variety. One
can recommend applying, such as these safe inducers
which could be used in the practical field and safety
use in the organic food production to control and
protect plants against fungal infections and perhaps
other microorganism, in addition to that improving
the growth and yield.
ACKNOWLEDGMENT
The authors thank the staff of the EPCRS
Excellence Centre as well as Plant Pathology and
Biotechnology Lab., Dept. of Agric. Botany, Fac. of
Agric., Kafr- Elsheikh University, Kafr-Elsheikh,
Egypt.
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