Persian Gulf Crop Protection

Persian Gulf Crop Protection, 3(1): 69-78 69
Persian Gulf Crop Protection
Available online on: www.cropprotection.ir
ISSN: 2251-9343 (online)
Volume 3 Issue 1, March 2014
Pages 69-78
Influence Of Antagonistic Fungaion The Root Infection Caused By
Macrophomina phaseolina On Okra.
Anam Mehwish Khanzada1
*, Dr. Abdul Mubeen Lodhi2
, Nadil Shah3
, Shagufta Rani Khanzada4
and Muhammad Siddique Khanzada5
.
Department of Plant Pathology, Sindh Agriculture University, Tandojam
(*Corresponding author e-mail: anam.mehwish.khanzada@gmail.com).
Abstract: Macrophomina phaseolina was isolated as root infecting fungi from okra plants showing
stunted growth, necrosis and severe plant mortality. The pathogenecity test, has confirmed that M.
phaseolina is an aggressive pathogen of okra plant. Bio-control agents including Trichoderma spp.,
Paecilomyces spp. and Gliocladium virens successfully checked the mycelial growth of the M.
phaseolina in dual assay test; however P. variotii followed by T. harzianum, T. pseudokoningii and T.
polysporum were the most effective bio-control agents in inhibiting the colony growth of test pathogen.
In pot experiment, application of bio-control agents greatly reduced the root infection in plants grown
in soil, artificially infested with M. phaseolina. Among six bio-control agents, T. harzianum and P.
variotii appeared as highly effective in reducing the root infection by test pathogen. The highest root
infection was found in plants treated with T. pseudokoningii followed by P. lilacinus. The application
of these bio-control agents also caused positive impact on plant growth. Moreover, these bio-control
agents also checked the pathogen infection and thus increased seed germination and decreased plant
mortality.
Key Words: Macrophomina phaseolina, Antagonistic fungi, Okra.

Introduction
Root rot caused by Macrophomina
phaseolina is considered as one of the most
destructive diseases of okra (Hafiz, 1986).
The fungus can also cause hallow stem,
root rot, pre-emergence and post-
emergence damping-off (Reuveni et al.,
1983). M. phaseolina is most often seen
during summer weather (Tosi and
Zazzerini, 1990 and Gulya et al., 2002).
About 5-100% yield losses due to this
disease have been reported (Vyas, 1981).
M. phaseolina does not survive more than
seven days in the mycelial form but it
sclerotia can survive over ten months in
soil (Ghaffar, 1968). It usually develops
when soil temperatures are 80-95o
F (27-
35o
C) for 2 to 3 weeks (Yang and Navi,
2003). Use of chemicals is common
practice for controlling the plant diseases.
During the past few decades various
approaches involving non-chemical means
of control were tested to bring down
inoculum densities to a level where
cultivation of high value crops has become
profitable. Therefore, in present study bio-
control agents will be evaluated against M.
phaseolina causing root rot of okra.
Materials and Methods
Isolation and Identification of
Macrophomina phaseolina: Isolation was
carried out from the infected okra plants
collected from Agriculture Research
Institute, Tandojam. The infected roots and
stems were washed with the tape water
thoroughly. Washed roots and stems were
cut into small pieces, these pieces were
then surface sterilized with 5% Sodium
hypochlorite (commercial bleach) for 1-1.5
minutes. The sterilized pieces dried onto
the tissue paper and then transferred on
petridishes containing PDA (Potato
Dextrose Agar) medium at five pieces per
petridish. These petridishes were incubated
at 30±1°C temperature for 7 days in
incubator. However, after every 24 hours
petridishes were observed for fungal
growth. The different fungal colonies
appeared were purified and identified on
the basis of colony characteristics as well
as morphological characteristics by using
keys of (Barnet and Hunter, 1972 and
1996).
Multiplication of M. phaseolina
Inoculum: In order to prepare large
quantity of M. phaseolina inoculum, its
sclerotia were obtained by growing the test
pathogen on sand+wheat meal substrate.
For this purpose 95 gm of sand and 5 gm
of wheat meal mixed together thoroughly
and then moistened with 10 ml sterilized
water. The substrate was transferred into
250 ml conical flask and sterilized in the
autoclave at 15Lbs for 20 minutes. Leaved
it 24 hours for cooling then added 5 mm
disc from actively growing M. phaseolina
pure culture in the conical flask and
incubated at room temperature for 4-6
weeks and shake the conical flask daily.
After 4-6 weeks the colour of the substrate
in the conical flask turned black due to the
sclerotial formation. The contents of the
conical flash were pored onto the 15µm
sieve and black tiny sclerotia present on
the surface of the sieve were collected in
the sterilized glass beaker for further use.
Pathogenicity Test of M. phaseolina: The
pathogenicity test of M. phaseolina, the
most frequent fungus was carried out on
local variety Sabz Pari.
Effect of Bio-Control Agents on Mycelial
Growth of M. Phaseolina: Different bio-
control agent’s viz., Trichoderma
harzianum, T. polysporidm and T.
pseudokoningii, Paecilomyces lilacinus,
Paecilomyces variotii and, Gliocladium
virens were evaluated against M.
phaseolina by dual assay method. For this
purpose, a 5 mm disc of M. phaseolina was
cut from freshly growing colony with the
help of cork borer and placed at one side of
PDA plate, on its opposite side placed
5mm disc of test bio-control agents and
tapped the petridish with tape. Then lined
the back side of petridish with the help of
marker and incubated it at 30±1ºC. The
growths of pathogen as well as bio-control
agents were measured daily, till the
colonies of both met. There were five
replications of each treatment.

Effect of Bio-Control Agents on M.
Phaseolina and Okra: A pot experiment
was conducted on local variety Sabz Pari at
Department of Plant Pathology, Sindh
Agriculture University Tandojam in the
month of December 2010, to evaluate the
effect of bio-control agents on plant
growth and disease development on okra.
The seeds of commonly growing okra
variety Sabz Pari were surface sterilized
with commercial bleach and sown into
thermopol glasses of 7 cm diameter filled
with the mixture of 190 gm sterilized
sandy soils in 1 cm depth. Prior to sowing,
the soil is artificially infested with the
pathogen inoculum at 40 sclerotia/gm of
soil. The took sterilized sandy loam soil
6700 scelrotia per 190 g soil and mixed
0.38g rice gain of Trichoderma spp. each
Trichoderma spp. mixed separately and
other bio-control agents, Paecilomyces
lilacinus, Paecilomyces variotii and,
Gliocladium virens inoculums of each
mixed two petridishes of pure culture per 3
thermopol glasses and without bio-control
agents glasses served as a control. The
uninoculated soil served as control. The
experiment was conducted complete
randomized block design with 3
replications. Seeds germinations were
recorded after 10 days of sowing, while
seedling mortality recorded after 30 days.
Plants were uprooted after one month of
sowing and data of plant growth as well as
root infection were recorded.
Results
Isolation and Identification:
Mcrophomina phaseolina was
predominantly found from the root of okra
plant collected from Tando jam. The
effected plant showed stunted growth, die-
back and pre-mature mortality. Due to high
disease severity large number of seedling
were died due to post-emergence damping-
off (Fig. 1). M. Phaseolina produced black
colour colony on PDA its produced tiny
black sclerotia under favorable
environments (Fig. 1). Microsclerotia are
black, spherical to oblong, and
occasionally irregular in shape.
Microsclerotia vary widely in size and
number, depending upon culture and host
material on which they are growing with
diameters of 60-200 µm common.
Effect of Bio-Control Agents on Mycelial
Growth of M. Phaseolina: All bio-control
agents significantly checked the mycelial
growth of the M. phaseolina is dual assay
test, as compared to control (Fig. 2).
Among six bio-control agents
Paecilomyces variotii was appeared as
most effective antagonist, in which
pathogen can produce only 32.0 mm
colony growth followed by T. harzianum
(43 mm), T. pseudokoningii, (45 mm) and
T. polysporum (45.5 mm). The maximum
colony growth of test pathogen was
recorded in plats of P. lilacinus (70.20
mm) followed by G. virens (48.0 mm)
(Fig.2).
Effect of Bio-Control Agents on M.
Phaseolina and Okra: Maximum plant
length was recorded in plants treated with
P. variotii (133.78 mm) and T. harzianum
(133.38 mm) followed by T. polysporum
(110.35 mm) and G. virens (99.67 mm).
Whereas, minimum plant length was
recorded in untreated plants (control)
(59.87 mm) followed by plants treated with
T. pseudokoningii (72.08mm) (Fig. 3).
Similar, trend was also observed in shoot
weight, where plants treated with T.
harzianum and P. variotii produce
maximum plant weight (53.890 mg and
51.540 mg). followed by P. lilacinus T.
polysporum and G. virens (30.410 mg,
28.767mg and 28.010 mg).The minimum
plant weight was recorded in untreated
plants followed by T. pseudokoningii
(13.112 mg) (Fig 4). All bio-control agents
increased seed germination as compared to
control. (Fig. 5). Maximum seed
germination were observed in T.
harzianum treated soil (97.56%) followed
by P. variotii (95.83%). Whereas,
minimum germination was observed in
untreated soil followed by soil treated with
either T. pseudokoningii or T. polysporum
(83.33%) (Fig. 5). Among six bio-control
agents minimum plant mortality occurred

in plants treated with T. harzianum
(13.0%) and P. variotii (16.6%) followed
by G. virens (51.0%) and T. polysporum
(53.430%) (Fig. 6). The maximum plant
mortality was found in untreated (control)
plants followed by plants treated with T.
pseudokoningii (83.0%) followed by P.
lilacinus (79.16%) (Fig. 6). Similarly,
application of bio-control agents greatly
reduce the root infection of okra plants
grown in soil artificially infested with M.
phaseolina. However, among bio-control
agents T. harzianum and P. variotii
appeared as highly effective in reducing
the root infection of test pathogen as only
14.0 and 20.0 % infection were recorded in
T. harzianum and P. variotii treated plant,
respectively. The highest root infection
was found in untreated (control) plants
followed by plants treated with T.
pseudokoningii (50%) and P. lilacinus
(44.0 %) (Fig. 7). Similarly, application of
bio-control agents greatly reduce the root
infection of okra plants grown in soil
artificially infested with M. phaseolina.
However, among bio-control agents T.
harzianum and P. variotii appeared as
highly effective in reducing the root
infection of test pathogen as only 14.0 and
20.0 % infection were recorded in T.
harzianum and P. variotii treated plant,
respectively. The highest root infection
was found in untreated (control) plants
followed by plants treated with T.
pseudokoningii (50%) and P. lilacinus
(44.0 %) (Fig.6g).
Discussion
The pathogenicity test, carried out during
present study on commonly growing okra
variety has confirmed that Macrophomina
phaseolina is an aggressive pathogen of
the okra. Its inoculation on test plants
significantly reduced seed germination and
plant growth, as well as increased the plant
mortality in okraIn present study, all
antagonistic fungi viz., Trichoderma
harzianum, T. polysporum and T.
pseudokoningii, Paecilomyces lilacinus, P.
variotii and Gliocladium virens
successfully checked the mycelial growth
of the M. phaseolina in dual essay test.
However, P. variotii followed by T.
harzianum T. pseudokoningii and T.
polysporum were the most effective bio-
control agents in inhibiting the colony
growth of the test pathogen. In pot
experiment, the application of these bio-
control agents also caused positive impact
on plant growth. Our result indicates that
plants treated with P. variotii and T.
harzianum followed by G. virens produced
more plant growth as compared to others.
Moreover, these bio-control agents also
checked the pathogen infection and thus
increased seed germination and decreased
plant mortality. The antagonistic effects of
different biological control agent against
M. phaseolina were well documented. Our
result were in confirmation to those
reported by (Sandoval, 2000) who
observed that T. harzianum showed
marked antagonistic and hyperparasitic
effect against M. phaseolina and other
pathogens, as well its application reduced
root infection and incidence of charcoal rot
in soybean. Similarly, (El-Mohommedy,
2004) reported that T. harzianum brought
86% reduction in the growth of M.
phaseolina which caused damping-off and
root rot disease in okra. Similarly (Gurjar
et al., 2004) and (Dawar et al., 2008)
tested many antagonistic fungi against M.
phaseolina and other pathogens and
observed that most of the antagonistic
fungi were effective against all pathogens.
Their application increased the seed
germination and plant growth as well as
reduced the disease severity in treated
plants. Malathi and (Doraisamy, 2004) also
observed that Trichoderma spp.
significantly inhabited the colony growth
of M. phaseolina. They also reported that
the application of T. harzianum brought
maximum reduction in pathogen infection
and enhanced the plant growth parameters.

Figure 1. Scerotia of Macrophomina phaseolina.
(a)
(b)

(c)
Figure 1. (i). Effect of different bio-control agents (a) P. variotii (b) P. lilacinus and (c) T. harzianum on
mycelial growth of M. Phaseolina.
(d)
(e)
(f)
Figure 2. (ii) Effect of different bio-control agents (d) T. pseudokoningii (e) T. polysporum and (f) G. virens on
mycilial growth of M. Phaseolina.

Table 1. Effect of different bio-control agents on mycelial growth of M. phaseolina.
S. NO Bio-control agents
Incubation
days
Diameter of pathogen
in interaction
(mm)
Diameter of bio-
control agent
(mm)
1 Paecilomyces variotii 3
32.0 d 58
2 Paecilomyces lilacinus 4 70.2 a 19.8
3 Gliocladium virens 4 48.0 b 42.0
4 Trichoderma pseudokoningii 5
43.0 c 47.0
5 Trichoderma polysporum 4
45.42 c 44.6
6 Trichoderma harzianum 4
45.0 c 45.0
7
Macrophomina phaseolina
(Control)
3 90.0
d
b
c
a a
c
e
0
20
40
60
80
100
120
140
T.pseudokoningii
T.polysporum
P.lilacinus
P.variotii
T.harzanium
G
.virens
control
PlantLength(mm)
T. pseudokoningii T. polysporum P. lilacinus P. variotii T. harzanium G. virens control
Figure 3. Effect of different bio-control agents on Plant length and of okra plants inoculated with M. phaseolina.

d
b
aa
bb
c
0
20
40
60
T.pseudokoningii
T.polysporum
P.lilacinus
P.variotii
T.harzanium
G
.virens
control
PlantWeight(mg)
Figure 4. Effect of different bio-control agents on Plant weight and of okra plants inoculated with M. phaseolina.
d
d c b a
c
e
0
20
40
60
80
100
T. pseudokoningii
T. polysporum
P. lilacinus
P. variotii
T. harzanium
G
. virens
control
Plantgermination(%)
Figure 5. Effect of different bio-control agents on Plant germination and of okra plants inoculated with M.
phaseolina.
b
c
b
e
f
d
a
0
20
40
60
80
100
T.pseudokoningii
T.polysporum
P.lilacinus
P.variotii
T.harzanium
G
.virens
control
PlantMortality(%)
Figure 6. Effect of different bio-control agents on Plant mortality and of okra plants inoculated with M.
phaseolina.

b
d
c
e
f
d
a
0
20
40
60
80
T.pseudokoningii
T.polysporum
P.lilacinus
P.variotii
T.harzanium
G
.virens
control
PlantInfection(%)
Figure 7. Effect of different bio-control agents on Plant root infection and of okra plants inoculated with M.
phaseolina.
d
d
b
e
d
c
a
0
20
40
60
80
100
T. pseudokoningii
T. polysporum
P. lilacinus
P. variotii
T. harzanium
G
. virens
control
Inhabition(%)
Figure 8. Effect of different bio-control agents on inhabitation percentage of M. phaseolina on PDA plates.
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