Population study of the sclerotia of Macrophomina phaseolina in cotton fields


Sheikh, A.H.; Ghaffar, A.

Pakistan Journal of Botany 7(1): 13-17

1975


A technique was developed for the isolation and enumeration of Macrophomina phaseolina from soil. After wet sieving through 120 mesh screen the soil residue retained on 300 mesh screen was suspended in 0.5% Calcium hypochlorite solution and distributed on PDA Streptomycin, Demosan and rose bengal. M. phaseolina colonies were detected within 5 days giving containing Penicillin, 94% recovery. Naturally infested soils averaged 0-29 sclerotial propagules per gram of soil. More sclerotia were present in the surface soil than at 6" depths. A substantially higher sclerotial population detected in certain root rot infested patches as compared to healthy fields. In difference in the population of sclerotia between the healthy and root rot infested portions of cotton general no significant was fields was observed.

Pak.
J.
Bot.,
7
(1):
13-17,
1975.
POPULATION
STUDY
OF
THE
SCLEROTIA
OF
MACROPHOMINA
PHASEOLINA
IN
COTTON
FIELDS*
ABDUL
HAKEEM
SHEIKH
AND
ABDUL
GHAFFAR
Department
of
Botany,
The
University,
Karachi,
Pakistan.
Abstract
A
technique
was
developed
for
the
isolation
and
enumeration
of
Macrophomina
phaseolina
from
soil.
After
wet
sieving
through
120
mesh
screen
the
soil
residue
retained
on
300
mesh
screen
was
suspended
in
0.5%
Calcium
hypochlorite
solution
and
distributed
on
PDA
containing
Penicillin,
Streptomycin,
Demosan
and
rose
bengal.
M.
phaseolina
colonies
were
detected
within
5
days
giving
94%
recovery.
Naturally
infested
soils
averaged
0-29
sclerotial
propagules
per
gram
of
soil.
More
sclerotia
were
present
in
the
surface
soil
than
at
6"
depths.
A
substantially
higher
sclerotial
population
was
detected
in
certain
root
rot
infested
patches
as
compared
to
healthy
fields.
In
general
no
significant
difference
in
the
population
of
sclerotia
between
the
healthy
and
root
rot
infested
portions
of
cotton
fields
was
observed.
Introduction
Macrophomina
phaseolina
(Tassi)
Goid.,
is
known
to
produce
root
rot,
stem
rot
or
pod
rot
on
over
305
host
plants
in
different
parts
of
the
world
of
which
at-
least
40
economic
hosts
have
so
far
been
recorded
from
Pakistan
(Young,
1949;
Ghaffar
et
al,
1964).
The
fungus
is
believed
to
persist
in
soil
in
the
form
of
sclerotia
formed
on
infected
host
tissue
and
subsequently
released
in
soil
during
decaying
process
(Smith,
1969).
M.
phaseolina
is
widely
distributed
in
different
parts
of
the
world
indicating
its
persistence
in
diverse
environmental
conditions
with
variable
soil
types.
As
a
preliminary
to
the
biological
control
of
M.
phaseolina,
a
technique
for
the
direct
isolation
and
enumeration
of
sclerotial
propagules
from
soil
was
deve-
loped.
The
technique
was
subsequently
used
to
study
the
population
density
of
the
sclerotia
of
M.
phaseolina
in
cotton
fields
where
root
rot
was
found
to
be
severe.
Materials
and
Methods
M.
phoseolina,
isolate
KUMH
54,
previously
isolated
from
the
root
rot
specimen
of
cotton
was
used
in
this
study.
This
was
the
same
isolate
used
by
Ghaffar
et
al
(1969)
in
their
investigations.
The
fungus
was
grown
on
corn
meal
sand
medium
(5
%
w/w)
fo
r
tw
o
we
e
k
s
at
30°C
and
'the
sclerotia
separated
by
successive
floatation
in
distilled
water
and
decantation.
The
sclerotia
were
dried
at
room
temperature
and
mixed
in
soil
(0.005:
100g;
w/w,
sclerotia:
soil).
More
or
less
similar
technique
as
used
for
the
separation
of
sclerotia
of
Sclerotium
cepivorwn
(Papavizas,
1971)
and-M.
phaseo-
lina
(Papavizas
and
Klag,
1974)
was
used
in
this
experiment.
*This
research
has
been
financed
in
part by
a
grant
made
by
the
United
States
Department
of
Agri-
culture
under
PL-480.
14
A.
H.
SHEIKH
AND
A.
GHAFFAR
Twenty
g
of
artificially
infested
soil
were
wet
sieved
through
120
mesh
(125
Ea.
pore
size)
and
300
mesh
(53
IA)
screen.
The
sclerotia
of
M.
phaseolina
upto
120
11
(Haigh's
'C'
strain
of
R.
bataticola)
are
retained
on
300
mesh
screen.
The
residue
obtained
on
300
mesh
screen
were
washed
in
running
tap
water
for
1
minute
and
transferred
into
a
beaker
containing
0.5
%
Ca0C1
which
was
made
up
to
100
ml
to
obtain
a
1:5
dilution.
The
sclerotial
suspension
was
continuously
agitated
by
magnetic
stirrer
and
one
ml
aliquot
were
distributed
on
the
strip
of
filter
paper
to
count
the
number
of
sclerotial
propagules.
One
ml
aliquot
were
also
removed
and
pippetted
on
to
the
surface
of
3
day
old
agar
plates
and
evenly
spread
The
plates
were
incubated
at
30°C.
Grayish
to
black
colonies
of
M.
phaseolina
were
detected
and
easily
identified
within
5
days.
Experimental
Results
I.
Isolation
and
enumeration
of
sclerotia
from
soil:
Of
the
different
media
tried,
PDA
containing
Penicillin,
Streptomye:n
(each
@
60mg/litre)
and
Demosan
(@,
300mg/litre)
with
or
without
rose
bengal
(@100mg/
litre)
gave
consistently
good
results,
(Fig.
1).
Use
of
Ca0C1
was
essential
since
it
prevented
the
fast
growing
mucorales
and
other
fungi.
It
may
be
mentioned
that
we
did
not
use
the
various
selective
media
as
suggested
by
Papavizas
&
Klag
(1974)
and
do
not
remove
the
Ca0C1
from
sclerotial
surface
before
its
transfer
on
the
agar
medium.
Approximately
6-10
M.
phaseolina
colonies
were
obtained
on
the
agar
plates.
Of
the
47-50
sclerotial
propagules/g
of
soil
as
recovered
on
the
strip
of
filter
paper,
44-47
M.
phaseolina
colonies
were
obtained
on
the
agar
plates
giving
approximately
94
%
recovery.
It
would
appear
that
non
removal
of
Ca0C1
did
not
result
in
any
appreciable
loss
of
germinability
of
sclerotia.
It
may
be
mentioned
that
sclerotia
of
M.
phaseolina
are
rindless
and
all
the
cells
of
the
sclerotia
have
thick
Hall
(Bryant
&
Wyllie
(1970).
A
B
4
41
2
4
—64
Fig.
1.
Receovery
of
M.
phaseolina
on
dilution
plates
oiontaining
PDA
with
different
antimicrobial
agents.
A—Soil
suspension.
B—Soil
suspension
in
Calcium
hypochlorite.
1.
PDA.
2.
PDA
+
Penicillin
l
Streptomycin.
3.
PDA
+
Penicillin
+
Streptomycin
+
Demosan.
4.
PDA
+
Penicillin
I
Streptomycin
±
Rose
bengal.
5.
PDA
Penicillin
'
Streptomycin
+
Demosan
+
Rose
bengal.
Macrophomina
SCLEROTIAL
POPULATION
IN
COTTON
FIELDS
15
II.
Population
of
M.
phaseolina
in
soils
of
cotton
field:
The
above
technique
was
used
for
isolating
sclerotia
of
M.
phaseolina
from
soil
and
estimating
its
inoculum
density.
Soil
samples
were
taken
at
random
from
upto
2"
and
6-9"
depth
of
cotton
fields
of
Rajawala
and
Risalewala
(Lyallpur),
Multan,
Setharja
and
Tandojam
where
root
rot
is
a
problem
and
the
plants
showed
wilting.
Sampling
was
carried
out
during
the
1974
cotton
growing
season.
For
comparison
soil
from
the
base
of
healthy
plants
were
also
obtained.
The
pH
of
-the
soil
samples
ranged
from
7.2-8.3.
Soil
was
kept
in
polythene
bags
and
were
air
dried
before
analysis.
Of
the
154
soil
samples
analysed,
the
population
of
sclerotia
of
M.
phaseolina
exhibited
a
substantial
degree
or
variability
since
a
population
of
0-29
sclerotial
propagules
per
g
of
soil
was
detected
(Table
1).
The
mean
population
counts
varied
significantly
with
the
localities
(pL
0.001).
Considerably
large
population
of
sclerotia
existed
in
Setharja,
Tandojam
and
Rajawala
and
relatively
small
population
in
Risalewala
and
Multan.
It
is
interesting
to
note
that
M.
phaseolina
sclerotia
were
detected
from
certain
healthy
cotton
fields
as
well.
In
general
no
signifiant
differ-
ence
in
the
population
of
sclerotia
between
the
healthy
and
root
rot
infested
portions
of
the
cotton
fields
was
detected.
In
Setharja
fields,
however,
a
substantially
higher
sclerotial
count
was
obtained
from
the
root
rot
infested
patches
as
compared
to
the
healthy
parts
of
the
fields
at
both
the
depths
examined.
TABLE
1.
Scleratial
population
of
Macrophornina
phaseolina
in
soil.
Locality
Patches
No.
of
Soil
depth
Samples
(inches)
pH
Population/g
of
soil
range
Range
Average
1.
Rajawala
Healthy
15
2
7.4-7.6
9-29
16.2
15
6
7.2-7.6
2-12
5.8
Root
rot
10
2
7.4-7.6
5-25
15.1
10
6
7.4-7.6
1-13
6.2
2.
Risalewala
Healthy
4
2
7A-8.0
0-1
0.5
4
6
7.3-8.0
0-11
3.0
Root
rot
8
2
7.6-7.9
0--1
0.1
8
6
7.6-8.3
0-0
0.0
3.
Multan
Healthy
3
2
7.8-7.9
1-8
3.7
3
6
7.7-7.9
1-4
2.3
Root
rot
5
2
7.8-7.9
0-23
5.6
5
6
7.8-7.9
0-1
0.6
4..Setharja
Healthy
11
-
2
7.5-8.0
1-13
4.9
11
6
7.5-7.9
0-14
3.8
Root
rot
13
2
7.5-7.9
2-21
6.7
13
6
7.6-7.9
0-15
,
5.2
5.
Tandojam
Healthy
3
2
7.7-7.8
10-25
15.7
3
.•
6
7.7-7.8
6-11
8.5
Root
rot
5
2
7.7-7.8
6-15
10.8
5
6
7.6-7.8
3-19
,
9.8
154
Total:
16
A.
H.
SHEIKH
AND
A.
GHAFFAR
Analysis
of
variance
(Based
on
3
randomly
chosen
replicates)
Source
of
variation
SS
df.
M.S.
Main
effects
Localities
(L)
948.5
4
237.1
9.34***
Healthy
Vs
Disease
(H)
43.0
1
43.0
1.69
n.s.
Observations
at
different
depths
(D)
191.0
1
191.0
7.53**
First
order
interactions:
LxH
75.5
4
18.87
0.74
n.s.
DxH
0.5
1
0.5
0.020
n.s.
DxL
210.3
4
52.5
2.071
n.s.
Second
order
interaction:
LxDxH
197.2
4
49.3
1.945
n.s.
Treatments:
1666.0
19
87.68
3.45**
Error:
1014.0
40
25.35
Total:
2680.0.
59
Levels
of
significane:
"p.
0.01
•••p.
0.001
n.s.
Non-significant.
The
population
of
sclerotia
of
M.
phaseolina
varied
significantly
with
the
depth
(p
0.01).
Population
counts
in
most
localities
were
considerably
higher
in
the
surface
soil
(2'
depth)
in
comparison
to
soil
collected
from
6-9'
depth.
Conclusion
The
occurence
of
relatively
greater
number
of
sclerotial
propagules
of
M.
-
phaseolina
in
the
surface
soil
would
indicate
that
the
chances
of
colonization
of
plant
roots
soon
after
emergence
would
be
more.
This
observation
is
further
sub-
stantiated
by
Hussain
&
Ghaffar
(1975)
who
found
greater
degree
of
colonization
of
cotton
roots
by
M.
phaseolina
in
the
proximal
portion
as
compared
to
the
distal
part.
It
would
suggest
that
deep
ploughing
and
turning
over'
ót
the
soil
may
aid
considerably
in
reducing
the
sclerotial
population
of
M.
phaseolina
in
the
surface
soil.
The
numerical
threshold
for
infection
by
M.
phaseolina
on
cotton
is
not
known
although
the
sclerotial
densities
in
soil
have
been
correlated
with
increased
disease
incidence
in
bean
(Watanabe
et
al,
1967).
The
presence
of
0-29
sclerotial
pro-
pagules
in
the
healthy
portions
of
the
cotton
fields
as
compared.to
0-25
sclerotial
propagules
in
the
diseased
portions
would
indicate
that
infection
on
roots
may
be
present
without
obvious
above
ground
symptoms
and
presumably
certain
edaphic
factors
interact
with
fungal
propagules
to
produce
wilting.
Water
stress
has
been
attributed
to
be
a
more
important
predisposing
factor
in
Macrophomina
infection
of
sorghum
(Hsi,
1961;
Edmunds,
1964)
and
cotton
(Ghaffar
&
Erwin,
1969).
On
the
other
hand
symptoms
of
charcoal
rot
(M.
phaseolina)
development
in
soybean
(Glycine
max)
have
been
ascribed
to
enzyme
release
and
toxin
production
(Bryant
&Wyllie,
1970;
Dhingra
&
Sinclair,
1974).
Macrophomina
SCLEROTIAL
POPULATION
IN
COTTON
FIELDS
17
References
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W.E.
and
T.B.
Wyllie,
1970.
Pectolytic
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O.D.
and
J.B.
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1974.
Isolation
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by
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L.K.
1964.
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W.H.
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the
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