Journal of Phytopathology and Pest Management 5(3): 55-66, 2018
pISSN:2356-8577 eISSN: 2356-6507
Journal homepage: http://ppmj.net/
∗
Corresponding author:
Amer F. Mahmoud,
E-mail: amermahmoud@aun.edu.eg
Telephone: +201024811706
55
Copyright © 2018
Potential resistance of certain sunflower cultivars
and inbred lines against charcoal rot disease
caused by
Macrophomina phaseolina
(Tassi) Goid
Marwa M. Taha
1
, Amer F. Mahmoud
1*
, Mohamed A. Hassan
1
, Adel M. Mahmoud
2
, Mohamed A. Sallam
1
1
Department of Plant Pathology, Faculty of Agriculture, Assiut University, 71526 Assiut, Egypt
2
Department of Agronomy, Faculty of Agriculture, Assiut University, 71526 Assiut, Egypt
Abstract
Keywords: Enzymes, charcoal rot, Macrophomina phaseolina, phenol contents, sunflower.
56
Introduction
Sunflower (
Helianthus annus
L.) is one
of the most important oil seed crops in
Egypt and many other countries.
Charcoal rot disease caused by the soil
borne pathogenic fungus
Macrophomina
phaseolina
is one of the serious diseases
of sunflower growers in Egypt and all
over the world (Jalil et al
.,
2013;
Aboshosha
et al.,
2007; Khan, 2001;
Mehdi et al
.
, 1988). This fungal pathogen
causes seedling blight, stem rot and
charcoal rot.
M. phaseolina
is reports as
the most fungal pathogens causing the
charcoal rot disease on more than 500
plant species (Purkayastha et al., 2006).
No chemical control currently exists for
management of charcoal rot disease, and
plant resistance for such disease has been
hard to identify. Due to soil borne nature
of the pathogen, control strategies other
than host resistance are not much
effective and economical. A yield loss
claimed by charcoal-rot in many courtiers
has been recorded up to 25% under
favorable conditions for the growth and
development of
M. phaseolina
(Jimenez
et al., 1983; Tikhonov et al., 1976;
Orellana, 1971). Planting resistant
sunflower mixed cultivars reducing the
effects of charcoal rot disease caused by
Macrophomina phaseolina,
compared to
the same cultivars planted individually
(Mahtab et al.,
2013; Muhammad et al.,
2011; Aboshosha et al.,
2008).
Development of resistant varieties is one
of the most important methods for
management of charcoal rot disease on
sunflower (Aboutalebi et al., 2014). It is
the cheapest source for management.
Therefore, this work was planned for
screening certain sunflower cultivars and
inbred lines against charcoal rot disease.
Materials and methods
Collection of sunflower cultivars and
inbreed lines:
Four cultivars and ten
inbreed lines were used in this study, two
Egyptian commercial sunflower cultivars
(Giza 102 and Sakha 53) were obtained
from (Agricultural Research and
Experiments Center, Giza, Egypt) and
two Russian sunflower varieties (Enosa
and Bozolok) obtain from Russia as
samples. Ten inbreed lines (S
5
) obtained
from Agronomy Department, Faculty of
Agriculture, Assiut University, Egypt.
Inbred lines were obtained from selfing
of open pollinated cultivar “Maiak”.
Causal pathogen:
Macrophomina
phaseolina
(Tassi) Goid
was isolated
from naturally diseased sunflower plants,
showing typical symptoms of charcoal
rot disease during 2013 growing season
from different fields in Assiut
Governorate. Identification of the
pathogen was carried out on 5-7 days old
culture using the morphological and
microscopic characteristics of mycelium
and spores according to Booth (1977),
Pitt (1979), Domsch
et al. (1980) and
Mahmoud and Budak (2011) and
confirmed by Mycological Center,
Faculty of Science, Assiut
University,
Egypt.
Disease assessment:
Pre-emergence
damping-off % was estimated by
counting the number of the non-
germinated seeds (2 weeks post-sowing).
Post-emergence damping-off% was
estimated by counting the number of
damping-off seedlings (4 weeks post-
sowing). Disease severity of charcoal rot
was assessed through visual observation
and symptoms on plants showing
symptoms of charcoal rot at the end of
the experiment (afusing numerical grades
ranging from (1 to 10) according to
57
Smith and Carvil (1997) and Mahmoud et
al. (2018) as follows:
(0)= No symptoms,
(1)= 0 ≤ 10 % stem infected area,
(2)= 10 ≤ 20 % stem infected area,
(3)= 20 ≤ 30 % stem infected area,
(4)= 30 ≤ 40 % stem infected area,
(5)= 40 ≤ 50 % stem infected area,
(6)= 50 ≤ 60 % stem infected area,
(7)= 60 ≤ 70 % stem infected area,
(8)= 70 ≤ 80 % stem infected area,
(9)= 80 ≤ 90 % stem infected area,
(10)= 90 ≤ 100 % stem infected area or
dead plant.
Where: (N) = number of plants in each
group of diseased plants; and (0, 1, 2
...10) = numerical grades of diseased
plants.
Greenhouse experiment:
Experiment
was carried out in 2015 growing season
at the greenhouse of Plant Pathology
Department, Faculty of Agriculture,
Assiut University, Egypt. Sterilized pots
(35 cm in diameter) were filled with
sterilized sandy
-
loam soil which mixed
thoroughly with the inoculums of
M.
phaseolina
at the ratio of 2% of soil
weight, then pots were irrigated. Soil
infestation was carried out 7 days before
seed sowing. Seed disinfestations were
carried out by dipping the seeds in
sodium hypochlorite solution (0.2%) for
2 minutes then rinsed several times with
sterilized water. Inoculums of the
pathogen was prepared by inoculating
sterilized 1000 ml conical flasks
containing barley medium (150g barley +
4g glucose + 200ml water) with
M.
phaseolina
and incubated at 28 ±2°C for
two weeks. Each pot was planted with 10
sunflower seeds for each cultivar. Four
pots were used as replicates. Pots
containing non infested soil mixed with
2% sterile barley medium were used as a
control. The plants were irrigated when
necessary and daily observed for
infection progress. Percentages of
survival plants were recorded after 2 and
4 weeks from sowing (as pre- and post-
emergence damping-off, respectively).
At the end of the experiment disease
severity was assessed.
Field experiment:
The experiment was
carried out in Assiut University Farm,
Assiut, Egypt during 2016 and 2017
growing seasons. The soil was divided
into plots (plot = 3× 2.1 m). Each plot
contained 4 rows, 60 cm a part. Each row
contained 6 hills spaced 20 cm. Every
hill was infested with the inoculum of
M.
phaseolina
by adding 50 g
/
hill then
covered with soil and irrigated at the
same time. The inoculum was prepared
as mentioned before in greenhouse
experiment. After 7 days, every hill was
sown with 5 sterilized seeds of each
cultivar and inbred and covered with soil.
After sowing all plots were irrigated.
Plots containing non infested sterile
barley medium (50g
/
hill) were used as
control. Randomize complete block
design with four replicates were used for
each treatment. Percentages of survival
plants were recorded after 2 and 4 weeks
from sowing. At harvest time (12 weeks
from planting) disease severity of
charcoal rot was assessed.
Determination of total phenol and
salicylic acid contents in sunflower
cultivars and inbreed lines, due to
58
infection by
M. phaseolina
:
Leaves of
sunflower cultivars and inbred line,
(infected and uninfected of two weeks
old plants), were immersed in liquid N2,
homogenized in 80% methanol (one
gram plant material in 10 mL) and stored
in the deep-freeze (-20°C) later, the
homogenate were centrifuged at 1000
rpm for 30 min at 4°C, the pellet was
discarded after addition of ascorbic acid
(0.1 gm. for 5 mL) and the homogenates
were evaporated in rotary evaporator at
65°C and repeated 3 times for 5 min. The
residues were dissolved in 5 mL of 80%
methanol. Four replicates were used each
treatment (Rapp & Zeigler, 1973).
Phenol
contents were determined using the
method described by Sahin et al. (2004)
as follows: The reaction mixture was
consisted of 0.02 methanol extract, 0.5
mL folin-ciocalteau reagent, 0.75 mL of
Na
2
CO
3
solution (20%) and 8 mL water.
The mixture incubated for one hr. at
37°C in water bath. Methanol was used
as blank. Total phenol contents were
assayed spectrophotometrically at 767
nm as mg/g plant fresh weight. Gallic
acid was used (0-5 mg) as a standard
curve. Total phenol = mg Gallic acid / g
plant material. Salicylic acid contents
were determined using the method
described by Dat et al
.
(1998) as follows:
Sample 500 µL homogenate were mixed
with 250 µL HCl (10N) and 1000 µL
methanol. Sample were incubated in a
water bath at 80°C for 2 h. neutralized
with 4-5 drops 1 M NaHCO
3
and 1000
µL methanol were added. The OD was
measured at 254 nm to calculate the
content of salicylic acid and expressed as:
Amount of total salicylic acid = µg/g
plant material.
Determination of oxidative enzymes:
For determination of oxidative enzymes
(peroxidase, polyphenenoloxidase and
catalase) leaves of sunflower cultivars
and inbred line, (infected and uninfected
of two weeks old plants), were treated
with Liquid N
2
and homogenized with
0.1 M Na-acetate buffer (pH 5.2) (for
one gram plant fresh weigh to10 mL
buffer), centrifuged at 1000 rpm for 30
min at 4°C and the oxidative enzymes
were determined in the supernatants.
Four replicates were used for each
treatment.
Determination of peroxidase activity
(PO):
Peroxidase activity was
determined using the method descried by
(Putter, 1974) as follows: Peroxidase
activity was determined
spectrophotometrically using quaiacol as
common substrate for peroxidases. The
reaction mixture was as follows: 0.2 mL
supernatant, 1 ml 0.1 M Na-acetate-
buffer PH 5.2, 0.2 mL 1% guaiacol and
0.2 mL 1% H
2
O
2
. The mixture was
incubated at 25°C for 5 min and then
measured at 436 nm. Extraction buffer
was used as blank. Enzyme activity was
calculated according to the change in
absorbency and was expressed as
enzyme in 1 mg protein.
Determination of polyphenoloxidase
activity (PPO):
Polyphenol oxidase
activity was determined using the
method described by Batra and Kuhn
(1975).
The reaction mixture was as
follows: 0.5 mL supernatant, 2 mL 50
mM sorensen phosphate buffer pH 6.5
(preparation of Sorensen phosphate
buffer, 6.8 gm. KH
2
PO
4
with 8.99 gm.
Na
2
HPO
4
2H
2
O solved in 1000 ml water
and 0.372 gm. EDTA was added the pH
adjusted to 6.5) and 0.5 mL substrate
59
Brenzcatechol (sigma Aldrich) at 37°C
for 2 hours and measured at 410 nm. PPO
activity = OD 410 nm/mg protein.
Determination of catalase activity:
Catalase activity was determined
spectrophotometrically (Aedi, 1984). The
homogenate of 0.5 mL for supernatant
were mixed with 3 mL from Sorensen
phosphate buffer pH 7.0 and 200 µL
H
2
O
2
30%. Sorensen phosphate buffer
was used as blank. Catalase activity was
determined spectrophotometrically using
spectrophotometer unicam UV calculated
at OD
240
nm. The enzyme activity was
expressed as changes in 240/mg
protein/min.
Statistical analysis:
The results were
analyzed using ANOVA test and the
means differences were regarded as
significant using LSD test at 5% level of
probability according to SAS
software
(SAS Institute, 1996).
Results
Reactions of certain sunflower
cultivars and inbred lines to infection
with charcoal rot disease caused by
Macrophomina phaseolina
under
greenhouse conditions:
The results of
this study are presented in Table (1). All
tested sunflower cultivars and inbred
lines were infected by
M. phaseolina
.
The susceptibility of the tested cultivars
to the disease is variable and varied
according to the tested cultivars and
inbred lines. There are a little difference
in the rate of susceptibility between the
tested sunflower cultivars and inbred
lines. In both tested season, sunflower
cultivars Giza 102 and Sakha 53,
produce the highest percentages of
survival plants (90% and 95%,
respectively) in pre- and post- emergence
damping-off as well as the lowest
percentages of charcoal rot disease
severity (10% for both cultivar).
Whereas, inbred line L
46
was the most
susceptible one and produced the lowest
percentages of survival plants (40%) in
pre- and post- emergence damping-off as
well as the highest percentages of
charcoal rot disease severity (70%).
Other tested inbred lines (L
49
, L
16
,
Bozolok, Enosa, L
7
, L
63
, L
60
, L
35
, L
36
and
L
26
) proved to be moderate resistant
lines. Whereas, inbred line (L
22
) showed
a moderate susceptible reaction.
Table 1: Reactions of certain sunflower cultivars and inbred lines to infection with charcoal rot
disease caused by Macrophomina phaseolina under greenhouse conditions.
Cultivars/ Inbred
lines
Survival plants relative to
control after 2 week (%)
Survival plants relative to
control after 4 week (%)
Charcoal rot disease
severity (%)
Sakha 53
Giza 102
Bozolok
Enosa
L
7
L
16
L
22
L
26
L
35
L
36
L
46
L
49
L
60
L
63
95
90
70
80
85
75
55
80
70
70
40
80
75
85
95
90
70
80
85
75
55
80
70
70
40
80
75
85
10
10
30
30
35
20
60
45
45
55
70
15
40
30
L.S.D.
0.05
17.75
17.75
22.35
60
Reactions of certain sunflower
cultivars to infection with charcoal rot
disease caused by
Macrophomina
phaseolina
in the field:
Data presented
in Table (2)
showed that tested sunflower
cultivars and inbred lines were varied in
their resistance to infection with
Macrophomina phaseolina.
In both tested
season sunflower cultivars Giza 102 and
Sakha 53, showed the highest
percentages of survival plants (90% for
both cultivar) in pre- and post-
emergence damping-off as well as the
lowest percentages of charcoal rot
disease severity (25% and 10%
respectively). Whereas, inbred lines L
46
and L
22
produce the lowest percentages
of survival plants (30% and 40%,
respectively) in pre- and post- emergence
damping-off as well as the highest
percentages of charcoal rot disease
severity (70% and 65% respectively).
Inbred lines (L
49
, L
16
, Bozolok, Enosa,
L
7
, L
63
and L
60
) proved to be moderate
resistant lines. While, inbred lines (L
26
,
L
35
andL
36
) showed an intermediate
effect.
Total phenol and salicylic acid
contentsin sunflower cultivars and
inbreed lines, due to infection by
M.
phaseolina
:
Data in Table (3) indicate
that each tested sunflower cultivars or
inbred lines showed higher amount of
total phenol contents in infected plants
with
M. phaseolina
than uninfected
plants. The highest amount of total
phenol contents was found in infected
plants of sunflower inbred line L
60
followed by Giza 102. While, L
46
inbred
line gave lower amount of total phenol
contents in infected plants than L
16
inbred line. Data in Table (4) showed
that infected plants in each tested
cultivars or inbred lines with
M.
phaseolina
produced higher salicylic acid
activity than uninfected plants (control).
Infected plants of L
49
inbred line showed
the highest amount of salicylic acid
followed by inbred line L
7
. The lowest
salicylic acid activity was found in
infected plants of inbred line L
46
.
Table 2: Reactions of certain sunflower cultivars to infection with charcoal rot disease caused by
Macrophomina phaseolina in the field during 2016 and 2017 growing.
Cultivars/
Genotypes
Survival plants relative to
control after 2 week (%)
Survival plants relative to
control after 2 week (%)
Charcoal rot disease
severity (%)
2016
2017
2016
2017
2016
2017
Sakha 53
Giza 102
Bozolok
Enosa
L
7
L
16
L
22
L
26
L
35
L
36
L
46
L
49
L
60
L
63
90
90
70
70
65
80
40
55
55
45
30
85
60
70
95
95
65
65
65
70
45
50
50
45
30
70
40
55
90
90
70
70
65
80
40
55
55
45
30
85
60
70
95
95
65
65
65
70
45
50
50
45
30
70
40
55
10
25
30
30
35
35
65
45
45
55
70
40
40
30
8.00
8.00
37.50
35.00
36.25
33.75
58.75
51.25
52.50
60.00
73.75
35.50
63.50
50.50
L.S.D. 0.05
19.35
17.06
19.35
17.06
31.83
17.69
61
Table 3: Determination of total phenol contents in healthy and
infected plants of sunflower cultivars and inbred lines growing
under greenhouse conditions.
Cultivars/
inbred lines
Total phenol contents (mg Gallic acid / g Fresh weight)
Infected
Non infected
Mean
Sakha 53
Giza 102
Bozolok
Enosa
L
7
L
16
L
22
L
26
L
35
L
36
L
46
L
49
L
60
L
63
5.280
5.959
5.242
5.179
5.140
2.610
5.080
5.180
5.154
5.082
2.480
5.820
6.340
5.482
5.240
5.598
5.200
5.141
5.079
2.515
5.070
5.178
5.143
2.688
2.420
5.058
5.144
5.260
3.760
5.779
5.221
5.160
5.109
2.563
5.075
5.179
5.149
3.885
2.450
5.439
5.742
5.371
L.S.D. at 5%:
Cultivars (A) = 0.014, Infection (B) = 0.005,
Interaction (A×B) = 0.019.
Table 4: Determination of salicylic acid in healthy and infected
plants of sunflower cultivars and inbred lines growing under
greenhouse conditions.
Cultivars/
inbred lines
Salicylic contents (µg / g plant material)
Infected
Non infected
Mean
Sakha 53
Giza 102
Bozolok
Enosa
L
7
L
16
L
22
L
26
L
35
L
36
L
46
L
49
L
60
L
63
1.217
0.949
0.541
0.525
1.912
0.541
0.769
0.411
1.380
0.618
0.161
2.117
0.181
1.249
0.655
0.212
0.459
0.483
0.533
0.386
0.533
0.147
0.586
0.390
0.106
0.219
0.121
0.521
0.936
0.581
0.500
0.504
1.223
0.464
0.651
0.279
0.983
0.504
0.134
1.168
0.151
0.885
L.S.D. at 5%: Cultivars (A) = N.S., Infection (B) = N.S.,
Interaction (A×B) = N.S.
Peroxidase activity (PO):
Data in Table
(5) indicated that each tested sunflower
cultivars or inbred lines showed higher
amount of peroxidase activity in infected
plants with the
M. phaseolina
than
uninfected plants. The highest amount of
peroxidase activity was found in infected
plants of sunflower inbred line L
49
followed by Giza 102. Inbred line L
46
gave lower amount of peroxidase activity
in infected plants than L
60
inbred line.
Polyphenoloxidase activity (PPO):
Data in Table (6) showed that, infected
plants in each tested cultivars or inbred
lines with
M. phaseolina
produced higher
polyphenoloxidase activity compared
with uninfected plants. Infected plants of
Enosa cultivar showed the highest
amount of polyphenoloxidase followed
by Sakha 53. The lowest
polyphenoloxidase activity was found in
infected plants of inbred line L
22
.
62
Table 5: Determination of peroxidase activity in healthy and
infected plants of sunflower cultivars and inbred lines growing
under greenhouse conditions.
Cultivars/
inbred lines
Peroxidase activity (unit / mg protein)
Infected
Non infected
Mean
Sakha 53
Giza 102
Bozolok
Enosa
L
7
L
16
L
22
L
26
L
35
L
36
L
46
L
49
L
60
L
63
2.935
6.465
1.430
4.328
3.674
3.57
2.414
2.282
0.914
1.264
0.147
8.554
0.183
0.888
2.164
4.106
0.607
1.922
1.058
1.524
0.921
1.448
0.427
0.601
0.020
1.892
0.024
1.892
2.888
3.803
1.019
2.798
1.776
2.547
1.749
1.865
1.205
0.933
0.084
4.481
0.104
1.832
L.S.D. at 5%:
Cultivars (A) = 2.586, Infection (B) = 0.977,
Interaction (A×B) = N.S.
Table 6: Determination of polyphenoloxidase activity in healthy
and infected plants of sunflower cultivars and inbred lines growing
under greenhouse conditions.
Cultivars/
inbred lines
Polyphenoloxidase activity (unit / mg protein)
Infected
Non infected
Mean
Sakha 53
Giza 102
Bozolok
Enosa
L
7
L
16
L
22
L
26
L
35
L
36
L
46
L
49
L
60
L
63
3.933
2.664
1.680
4.228
3.821
3.428
0.562
1.461
0.902
1.298
0.604
2.553
1.465
0.730
0.686
2.577
0.675
2.367
3.316
0.831
1.143
0.866
0.583
0.679
0.436
0.279
0.271
0.666
2.310
2.621
1.178
3.298
3.569
2.130
0.853
1.164
0.743
0.989
0.520
1.416
1.736
0.698
L.S.D. at 5%: Cultivars (A) = 1.211, Infection (B) = 0.458,
Interaction (A×B) = N.S.
Table 7: Determination of catalase activity in healthy and infected
plants of sunflower cultivars and inbred lines growing under
greenhouse conditions.
Cultivars/
inbred lines
Catalase activity (unit / mg protein/ min)
Infected
Non infected
Mean
Sakha 53
Giza 102
Bozolok
Enosa
L
7
L
16
L
22
L
26
L
35
L
36
L
46
L
49
L
60
L
63
3.144
3.232
3.049
3.043
3.091
3.040
3.146
3.876
3.147
3.150
2.925
3.147
3.144
3.243
2.851
3.143
3.044
2.949
3.017
2.080
3.145
2.785
3.145
3.045
2.076
3.043
3.037
3.155
2.998
3.188
3.047
2.996
3.054
2.560
3.146
3.331
3.146
3.098
2.501
3.095
3.091
3.199
L.S.D. at 5%:
Cultivars (A) = 0.011, Infection (B) = 0.004,
Interaction (A×B) = 4.774.
63
Catalase activity (CAT):
Data in Table
(7) indicated that, each tested sunflower
cultivars or inbred lines showed higher
amount of catalase activity in infected
plants with the
M. phaseolina
than
uninfected plants. The highest amount of
catalase activity was found on infected
plants of inbred line L
26
followed by L
63
.
While, L
46
inbred line gave lower amount
of catalase activity in infected plants than
L
16
inbred line.
Discussion
In the present study, we evaluated
fourteen sunflower cultivars and inbred
lines for infection by
M. phaseolina
.
Most of the tested sunflower cultivars or
inbred lines (Giza 102, Sakha 53, Enosa,
Bozoloke, L
7
, L
16
, L
22
, L
26
, L
35
, L
36
, L
46
,
L
49
, L
60
and L
63
) were varied in their
susceptibility to charcoal rot disease
caused by
M. phaseolina.
Obtained
results showed that sunflower cultivars
Giza 102 and Sakha 53 produced the
highest percentages of survival plants in
pre- and post- emergence damping-off as
well as the lowest percentages of
charcoal rot disease severity and they
proved to be resistant cultivars. Whereas,
L
46
was the most susceptible inbred line.
The resistance in sunflower cultivars can
be attributed to increase the content of
certain enzymes after infection (Papaiah
& Narasimha, 2014; Roldan Serrano et
al., 2007; Ramanathan et al.,
2001;
Kirstensen et al., 1999; Flott et al.,
1989). In this study, salicylic acid (SA)
concentration, phenolic compounds and
the enzymatic activates or peroxidase
(PO), polyphenoloxidas (PPO) and
catalas (CAT) were determined in both
infected and uninfected of sunflower
cultivars and inbred lines. The results
showed that the highest amount of total
phenol contents were detected in the less
susceptible sunflower cultivars (Giza
102 and Sakha 53). While, the lowest
amount of total phenol contents was
detected in inbred line L
46.
Phenolics
might play an important role in plant
defense, phenols are essential for the
biosynthesis of lignin, which consider an
important structural component of plant
cell walls and most notably
phytoalexins. The oxidative enzymes
played an important role in plant
diseases resistance. Infected plants of
sunflower cultivars Giza 102 and Sakha
53 cultivar showed a highest level in all
determined enzymes activity (Catales,
peroxidase and polyphenoloxidase).
However, the lowest activity of enzymes
found in susceptible sunflower L
46
line.
The oxidative enzymes played an
important role in plant diseases
resistance
(Saraswathi & Reddy, 2012;
Cherif et al., 2007; Yang et al.,
2002;
Avdiushko et al., 1993).
Such results are
in line of that reported by Sulman et al.
(2001), Mydlarz and Harvell (2007),
Aboshosha et al.
(2008) and Papaiah and
Narasimha (2014). They mentioned that
many plants enzymes are involved in
defense reaction against plant pathogen.
These include oxidative enzymes such as
peroxidase, polyphenoloxidase, catales
in the formation of lignin and other
oxidative phenols that contribute to
formation of defense barrier for
reinforcing the cell structure
.
The less
susceptible cultivars identified in the
present study are expected to possess
diverse resistance genes and could be
64
efficiently used as parents to improve
resistance to charcoal-rot disease
therefore, plant breeders should exert
more efforts to improve and produce
these cultivars to be used widely to
overcome damping-off and charcoal-rot
diseases (Mahmoud et al., 2015).
References
Aboshosha SS, Attaalla SI, El-Korany AE,
El-Argawy E, 2008. Protein analysis and
peroxidase Isozymes as Molecular
Markers for resistance and susceptibility
of sunflower to Macrophomina
phaseolina. International Journal of
Agriculture and Biology 1: 28–34.
Aboshosha SS, Attaalla SI, El-Korany AE,
El-Argawy E, 2007. Characterization of
Macrophomina phaseolina isolates
affecting sunflower growth in El-Behera
governorate, Egypt. International Journal
of Agriculture and Biology 9(6): 807–
815.
Aboutalebi R, Dalili A, Rayatpanah S,
Andarkhor A, 2014. Evaluation of
sunflower genotypes against charcoal
rots disease in vitro and in vivo
condition. World Applied Sciences
Journal 31(4): 649–653.
Aedi H, 1984. Catalase in vitro In: method of
enzymology. Academic press, New
York, USA, 105: 121–126 pp.
Avdiushko SA, Ye XS, Kuc J, 1993.
Detection of several enzymatic activities
in leaf prints of cucumber plants.
Physiology and Molecular of Plant
Pathology 42: 441–454.
Batra GK, Kuhn, CW, 1975. Polyphenol
oxidase and peroxidase activities
associated with acquired resistance and
its inhibition by 2-thiouracil in virus
infected soybean. Physiological Plant
Pathology, 5: 239–248.
Booth C, 1977. Fusarium laboratory guide to
the identification of the major species.
Commonwealth Mycological Institute
Kew, Surrey, England, 1–158 pp.
Cherif M, Arfaoui A, Rhaiem A, 2007.
Phenolic compounds and their role in
bio – control and resistance of chickpea
to fungal pathogenic attacks. Tunisian
Journal of Plant Protection 2(1): 7–21.
Dat JF, Foyer CH, Scott IM, 1998. Changes
in salicylic acid and antioxidants during
induced thermotolerance in mustard
seedlings. Plant Physiology 118: 1455–
1461.
Domsch KH, Gams W, Anderson TH, 1980.
Compendium of soil fungi. Academic
press. A subbsidiary of Harcout Brace
Jovanovich, Publishers, London,
England, 1: 859 pp.
Flott B, Moerschbacher B, Reisener H, 1989.
Peroxidas isozyme patterns of resistant
and susceptible wheat leaves following
stem and rust infection. New Phytologist
111: 413–21.
Gomez KA, Gomez AA, 1984. Statistical
procedures for agricultural research. 2
nd
ed., John Willey, USA, 680 pp.
Jalil S, Sadaqat HA, Tahir HN, Shafaullah
Hayat A, Ali N, 2013. Response of
sunflower under charcoal rot
Macrophomina phaseolina stress
conditions. International Journal of
Scientific Engineering and Research 1:
89–92.
Jimenez DRM, Blance LMA, Sackston WE,
1983. Incidence and distribution of
charcoal rot of sunflower caused by
65
Macrophomina phaseolina in Spain.
Plant Disease 67: 1033–1036.
Khan A, 2001. Yield performance,
heritability and interrelation ship in some
quantitive traits in sunflower. Helia
24(34): 35–40.
Kirstensen BH, Bloch H, Rasmussen SK,
1999. Barley coleoptile peroxidases,
purification, molecular cloning and
induction by pathogens. Plant
Physiology 120: 501–512.
Mahmoud A, Budak H, 2011. First report of
charcoal rot caused by Macrophomina
phaseolina in sunflower in Turkey. Plant
Disease 95(2): 223.
Mahmoud AF, Abou-Elwafa SF, Shehzad T,
2018. Identification of charcoal rot
resistance QTLs in sorghum using
association and in silico analyses.
Journal of Applied Genetics 59(3): 243–
251.
Mahmoud AF, Hassan MI, Amein KA, 2015.
Resistance potential of bread wheat
genotypes against yellow rust disease
under Egyptian climate. The Plant
Pathology Journal 31(4): 402–413.
Mahtab R, Alireza DSR, Abasali A, Masoud
SNA, 2013. Study of reaction of
sunflower lines and hybrids to
Macrophomina phaseolina (Tassi) Goid.
causal agent of charcoal rot disease.
World Applied Sciences Journal 21:
129–133.
Mehdi SS, Mehdi SA, 1988. Charcoal rot
resistance in sunflower cultivars at
various plant densities. Pakistan Journal
of Scientific and Industrial Research 31:
786–787.
Muhammad A, Javed MZ, Shahnaz D, 2011.
Screening of sunflower (Helianthus
annus L.) varieties against charcoal rot
fungus, Macrophomina phaseolina
(Tassi) Goid. FUUAST Journal of
Biology 1: 43–46.
Mydlarz LD, Harvell CD, 2007. Peroxidase
activity and inducibility in the see fan
coral exposed to a fungal pathogen.
Comparative Biochemistry Physiology
146: 54–62.
Orellana RG, 1971. The response of
sunflower genotype to natural infection
of Macrophomina phaseolina. Plant
Disease Reporter 54:157.
Papaiah S, Narasimha G, 2014. Peroxidase
and polyphenol oxidase activities in
healthy and viral infected sunflower
(Helianthus annuus L.) leaves. Bio
Technology 9(1): 01–05.
Pitt JI, 1979. The genus Penicillium and its
teleomophic states Eupenicillium and
Talaromyces. Mycobank Literature,
Academic Press London, England, 1:
634 pp.
Purkayastha S, Kaur B, Dilbaghi N,
Chaudhury A, 2005. Characterization of
Macrophomina phaseolina, the charcoal
rot pathogen of cluster bean, using
conventional techniques and PCRbased
molecular markers. Plant Pathology
55(1): 106–116.
Putter J, 1974. Peroxidase, In: Bergm eyer,
H.U. (ed), Methoden der enzymatischen
Analyses, Verlag Chemie, Weinheim,
Germany, 725–731 pp.
Ramanathan A, Vidhiyasekaran P,
Samiyappan R, 2001. Two pathogenesis-
related peroxidases in greengram (Vigna
radiate (L.) Wilczek) leaves and
cultured cells induced by Macrophomina
phaseolina (Tassi) Goid and its elicitors.
Micropiology Research 156: 139–144.
66
Rapp A, Ziegler A, 1973. Bestimmung der
phenolcarbonsäuren in rebblättern,
weintrauben and wein mittels polamyid –
Dünnschichtchromatographie. Vitis 12:
226–236.
Roldan serrano A, Luna del Castillo J, Jorrin
Novo J, Ferdan Ocana A, Gomez
Rodriguez MV, 2007. Chitinase and
peroxidase activities in sunflower
hypocotyls: effects of BTH and
inoculation with Plasmopara halstedii.
Biologia Plantarum 51(1): 149–152.
Sahin F, Guluce M, Daferera D, Sokmen A,
Sokmen M, Polissiou M, Agar G, Ozer
H, 2004. Biological activities of the
essential oils and methanol extract of
origanum vulgare ssp. Vulgare in the
Eastern Anatolia region of Turkey. Food
Control 15: 549–557.
Saraswathi M, Reddy MN, 2012. Phenolic
acids associated with Sclerotium rolfsii
in groundnut (Arachis hypogaea L.)
during pathogenesis. International
Journal of Plant Pathology 3 (2): 82–88.
SAS Institute, 1996. SAS User’s Guide:
Statistics. Version 6.03 Edition. SAS
Institute Inc., Cary, NC, USA.
Smith GS, Carvil ON, 1997. Field Screening
of Commercial and Experimental
Soybean Cultivars for Their Reaction to
Macrophomina phaseolina. Plant
Disease 81 (4): 363–368.
Sulman M, Fox G, Osman A, Inkerman A,
Williams P, Michalowitz M, 2001.
Relationship between total peroxidase
activity and susceptibility to black point
in mature grain of some barley cultivars.
Proceeding of the 10
th
Australian Barley
Technical Symposium.
Tikhonov OI, Nedelko OK, Persestova TA,
1976. Infection period and spreading
pattern of Sclerotium bataticola in
sunflower. VII: International Sunflower
Conference. Abstracts of papers,
Krasnodar, Russia.
Yang KH, Tsaus WL, Shieh GJ, Ho CL, Tsai
JN, Lin CY, Thseng FS, 2002. Screening
the peanut (Arachis hypogaea L.)
germplasm for resistance to pod rot
disease. Journal of Agricultural
Research of China, 51(3): 12–19.
