Survival of Macrophomina phaseolina sclerotia in nitrogen amended soils


Filho, E.S.; Dhingra, O.D.

Phytopathologische Zeitschrift 97(2): 136-143

1980


The effect of N at 1, 3, 5 or 7 mg/g of soil, supplied as NaNO3 (NO3-N) or (NH4)2SO4 (NH4-N), on the survival of phytopathogenic M. phaseolina sclerotia was studied in 2 soils. The sandy clay loam and sandy loam soils were infested with 640 sclerotia/g of soil. The population of M. phaseolina declined gradually and significantly in both soils supplied with N from any source or at any concentration. In sandy clay loam soil the population dropped to 2-10 propagules if supplied with 3, 5 or 7 mg N/g of soil from either source. The major drop occurred 30-40 days after N amendment. In sandy loam soil the population dropped to 2 or 100 propagules if amended with 7 or 5 mg NH4-N/g of soil, respectively, but remained at 250 or 460 propagules/g of soil if N source was NO3. In both soils, NH3-N incited more rapid and drastic decline in population than NO3-N.

Phytopath.
Z.,
97,
136-143
(1980)
©
1980
Verlag
Paul
Parey,
Berlin
and
Hamburg
ISSN
0031-9481
/
ASTM-Coden:
PHYZA3
Departamento
de
Fitopatologia,
Universidade
Federal
de
Vicosa,
Brasil
Survival
of
Macrophomina
phaseolina
Sclerotia
in
Nitrogen
Amended
Soils
By
EDGAR
S.
FILHO
and
ONKAR
D.
DHINGRA
With
4
figures
Received
October
20,
1978
Macrophomina
phaseolina
(Tassi)
Goid.
[M.
phaseoli
(Maubl.)
Ashby,
Rhizoctonia
bataticola
(Taub.)
Butler]
is
a
pathogen
of
over
290
plant
species
(DHINGRA
and
SINCLAIR
1977).
It
causes
severe
disease
under
dry
and
warm
conditions,
with
soil
being
the
primary
source of
inoculum
(DHINGRA
and
SINCLAIR
1978).
Mycelial
growth
of
M.
phaseolina,
although
limited
in
natural
soils,
increases
with
increasing
temperature
upto
35
°
C
(NORTON
1953)
but
does
not
survive
in
soil
for
more
than
10
days
(GHAFFAR
1968,
GHAFFAR
and
AKHTAR
1968,
KovooR
1954,
MEYER
et
al.
1973).
The
fungus
can
colonize
organic
substrates
in
soil
(DHINGRA
et
al.
1976,
GHAFFAR
and
PARVEEN
1969,
RADHA
1956,
ZACHARIAH
1953).
Sclerotia
are
the
most
important
propagules
for
survival
of
M.
phaseolina
in
soil.
They
are
formed
in
the
host
during
parasitic
phase
and
released
into
the
soil
as
the
host
tissues
decay
(Coot(
et
al.
1973,
MEYER
et
al.
1974).
Sclerotia
are
generally
not
formed
in
free
soil
but
in
organic
substrate
colonized
by
the
fungus
(NORTON
1953).
Since
disease
potential
is
directly
proportional
to
inoculum
potential,
which
in
turn
is
directly
proportional
to
inoculum
density,
the
disease
potential
can
be
reduced
by
reducing
the
inoculum
density.
The
following
study
was
done
to
determine
if
nitrogen
amendment
of
soil
can
reduce
the
inoculum
density
of
M.
phaseolina.
Materials
and
Methods
The
soils
used
were:
Sandy
clay
loam
(coarse
sand
48
%,
fine
sand
21
%,
silt
7
%
and
clay
24
%),
total
nitrogen
(N)
0.08
%,
oxidizable
carbon
1.78
%,
moisture
holding
capacity
42
%,
and
pH
5.4,
obtained
from
a
Phaseolus
vulgaris
field
in
Ponte
Nova;
sandy
loam
(coarse
sand
33
%,
fine
sand
45
%,
silt
7
%
and
clay
15
%),
total
N
0.88
%,
oxidizable
U.S.
Copyright
Clearance
Center
Code
Statement:
0031-9481/80/9702-136302.50/0
800
700
I
I
o
1%
I
Time
I
N
Concentration
O
..,
600
5
;.
N
%
_
1
.
u
o
, \
0
5
0—
N
/;
/
N ,
al
Z
\
1 /
O
\
i•
-
•=
`5.,
400
0
i-
--
D
co
-
0.
0
a.
300
200
100
-
NO3
N
mg/g
Soil
Control
\
t
,
A
Survival
of
Macrophomina
phaseolina
Sclerotia
in
Nitrogen
Amended
Soils
137
carbon
1.01
%,
moisture
holding
capacity
32
%,
pH
5.4,
obtained
from
Glycine
max
field
in
Uberaba.
The
two
soil
collection
sites
are
located
800
km
apart,
and
charcoal
rot
caused
by
M.
phaseolina
occurs
sporadically.
The
soils
were
air
dried
to
3
%
moisture
and
passed
through
a
2
mm
sieve.
One
kilogram
of
each
soil
was
infested
with
500
mg
of
dry
sclerotia
of
M.
phaseolina
prepared
as
described
by
DHINGRA
and
SINCLAIR
(1975)
and
mixed
in
twin
arm
motorized
mixer
for
24
hours.
One
hundred
gram
of
infested
soil
was
then
amended
with
either
sodium
nitrate
or
ammonium
sulphate
to
provide
1,
3,
5
or
7
mg
N/g
of
soil.
Both
salts
were
added
in
the
solution
form
ensuring
thorough
mixing.
The
soil
moisture
was
adjusted
to
45
%
of
the
moisture
holding
capacity.
Soil
not
supplied
with
N
served
as
control.
All
soils
were
placed
in
polyethylene
bags
of
300
g
capacity,
the
bags
with
soil
were
tied
and
incubated
at
30
°
C
in
a
humid
chamber
to
minimize
water
loss.
All
treatments
were
prepared
in
triplicate.
Samples
of
about
5
g
were
removed
every
10
days
from
each
treatment
for
50
days
to
determine
changes
in
M.
phaseolina
population.
The
samples
were
air
dried
for
24
hours
and
crushed
in
a
mortar
and
pestle.
The
selective
medium
for
M.
phaseolina
as
described
by
MEYER
et
al.
(1973)
for
population
estimation
in
soil
was
modified
by
increasing
the
concentrations
of
chloroneb,
mercuric
chloride
and
rose
bengal
to
312,
8.5
and
112
mg
per
liter
and
they
were
added
to
the
basal
medium
at
50-55
°C.
Adjustment
of
pH
was
elimi-
nated.
The
natural
pH
of
the
medium
varied
between
8.0
and
8.5.
The
population
of
M.
phaseolina
in
soil
samples
was
determined
by
sprinkling
50
mg
of
crushed
soil
on
modified
selective
medium
in
15
cm
petri
plates
in
three
replications.
All
the
data
were
analyzed
by
factorial
experimental
design
using
Tukey's
test
for
significant
differesice.
0
10
20
30
40
50
TIME
(in
days)
Fig.
1.
Changes
in
the
population
of
Macrophomina
phaseolina
in
sandy
clay
loam
soil
amended
with
different
concentrations
of
nitrogen
(N)
supplied
as
sodium
nitrate
(average
of
three
replications)
1
1%
I
Time
I
N
Concentration
600
0
0
u.
D
,
500
_
j
400
a.
300
1
.
'".."C
k\
A
1
'•••
.
;••
800
700
NH
4
N
mg/g
Soil
3
5
7
Control
200
100
138
FILHO
and
DHINGRA
Results
The
addition
of
N
either
as
NO
3
or
NH
4
at
any
dosage
incited
a
sig-
nificant
decline
in
M.
phaseolina
population
in
both
soils
(Figs.
1
to
4).
In
sandy
clay
loam
soil
the
population
declined
by
40
%
of
the
original
popula-
tion,
within
first
10
days
of
incubation
when
supplied
with
NO,-N,
without
significant
differences
at
different
levels
of
N
(Fig.
1).
In
NI-1
4
-N
supplied
soil,
the
population
declined
by
80
%
within
first
10
days
at
1
mg
N/g
soil
level,
whereas
in
soil
supplied
with
higher
levels
of
NH
4
-N,
the
decline
in
the
population
of
M.
phaseolina
was
significantly
less
(Fig.
2).
The
population
increased
significantly
between
10
to
20
day
incubation
period
in
all
treat-
ments,
dropping
drastically
thereafter,
reaching
2-10
propagules
in
soil
sup-
plied
with
3,
5
or
7
mg
N/g
of
soil.
The
major
drop
occurred
between
30
and
40
days
of
incubation,
showing
no
further
increasing
tendencies.
In
soil
applied
with
1
mg
N/g
of
soil,
although
population
declined
drastically
for
40
days
reaching
50
to
75
propagules,
showed
an
increasing
tendency
at
the
end
of
50
days
(Fig.
2).
Although
the
trend
of
population
decline
was
similar
whether
the
soil
was
supplied
with
NO,-N
or
NH
4
-N,
the
population
decline
of
0
10
20
30
40
50
TIME
(in
days)
Fig.
2.
Changes
in
the
population
of
Macrophomina
phaseolina
in
sandy
clay
loam
soil
amended
with
different
concentration
of
nitrogen
(N)
supplied
as
ammonium
sulphate
(average
of
three
replications)
700
-
J
600
0
cn
500
z
0
400
a.
0
300
Survival
of
Macrophomina
phaseolina
Sclerotia
in
Nitrogen
Amended
Soils
139
M.
phaseolina
was
significantly
higher
in
NH,-N
than
in
NO,-N
supplied
soil,
during
the
first
30
days
of
incubation
period
with
no
particle
difference
thereafter.
In
sandy
loam
soil
supplied
with
1
or
3
mg
N/g
soil
the
population
of
M.
phaseolina
entered
in
a
cycle
of
increase
and
decrease
(Figs.
3
and
4).
The
level
of
population
reached
at
each
decreasing
phase
was
significantly
lower
than
the
level
reached
during
earlier
declining
phase.
In
soil
supplied
with
5
or
7
mg
N/g
of
soil
the
population
declined
significantly,
and
gradually
over
the
test
period,
without
showing
an
increase
at
any
sampling
time.
The
population
reached
to
2
and
100
propagules/g
soil
in
soil
supplied
with
7
or
5
mg
NH
4
-N/g
soil,
respectively
(Fig.
3)
whereas
in
soil
supplied
with
same
quantity
of
NO,-N
the
population
of
M.
phaseolina
remained
at
250
and
460
propagules/g
soil
(Fig.
4).
Discussion
More
than
one
mechanism
appears
to
be
involved
in
the
ultimate
reduc-
tion
of
M.
phaseolina
population
in
nitrogen
supplied
soil.
Nitrogen
amend-
NH
4
—N
mg/g
Soil
800
5
41
\
--
\
\\
Control
fit
/
\\
A \
/
\\
./
./
A
.//
\.\
200
A
1%
I
Time
100
I
N
Concentration
a...
1
0
10
20
30
40
50
TIME
(in
days)
Fig.3.
Changes
in
the
population
of
Macro
phomina
phaseolina
in
sandy
loam
soil
amended
with
different
concentrations
of
nitrogen
(N)
supplied
as
ammonium
sulphate
(average
of
three
replications)
NO
3
-
N
1
3
5
7
Control
mg/g
son
z
_
-
0.
19_
.........
o.
-a
.o
800
A
1%
I
Time
100
N
Concentroticn
1
0
10
20
30
40
700
_
j
600
0
cr.
500
_
J
'I
400
0
300
200
140
FILHO
and
DHINGRA
TIME
(in
days)
Fig.
4.
Changes
in
the
populations
of
Macrophomina
phaseolina
in
sandy
loam
soil
amended
with
different
concentration
of
nitrogen
(N)
supplied
as
sodium
nitrate
(average
of
three
replications)
ment
of
soil
resulting
in
inoculum
density
decline
of
sclerotial
fungi
has
been
reported
(DHINGRA
and
SINCLAIR
1975,
GREEN
and
PAPAVIZAS
1973).
A
sig-
nificant
negative
correlation
between
population
of
M.
phaseolina
and
general
microbial
population
of
the
soil
was
found.
The
time
for
which
the
population
of
M.
phaseolina
decline
was
directly
correlated
to
the
amount
of
nitrogen
added
to
the
soil,
which
also
directly
correlated
to
the
time
for
which
general
microbial
population
of
soil
increased
(DHINGRA
and
SINCLAIR
1975).
The
viability
of
sclerotia
in
nitrogen
treated
soils
is
reduced
by
increased
microbial
activity
in
the
sclerosphere
forming
a
nutrient
gradient
resulting
in
the
leakage
of
nutrients
from
within
the
sclerotia.
Thus,
the
sclerotia
are
weakened
to
the
point
of
ungerminability.
Ko
and
LOCKWOOD
(1967),
and
GRIFFEN
(1972)
postulated
that
leakage
of
nutrients
from
sclerotia
is
accelerated
by
direct
stimulation
by
antagonists
or
by
the
osmotic
effects
of
rapid
removal
of
leachate
from
the
surface.
An
intense
microbial
activity
in
the
sclerosphere
of
M.
phaseolina
is
to
be
expected
in
soils
used
in
this
study,
since
the
carbon
content
of
soil
was
low.
The
viable
sclerotia
are
not
easily
digested
by
soil
organisms
because
of
their
melanin
rich
cell
walls
(GANGOPADHYAY
and
WYLLIE
1974,
HURST
and
WAGNER
1969).
Survival
of
Macrophomina
phaseolina
Sclerotia
in
Nitrogen
Amended
Soils
141
The
sclerotia
of
M.
phaseolina
germinate
normally
in
nutrient
amended
soils
and
abnormally
in
nonamended
soils
(AYANRU
and
GREEN
1974,
SMITH
1969).
About
70
%
of
the
sclerotia
germinated
within
4
days
in
soils
used
in
this
study,
whether
amended
or
nonamended
with
nitrogen
(unpublished
data).
The
germtube
and
subsequent
hypha
is
readily
attacked
by
soil
bacteria
and
actinomycetes
whose
population
increases
tremendously
in
nitrogen
supplied
soils
(KovooR
1954,
RADHA
1956,
NORTON
1953,
DHINGRA
and
SINCLAIR
1975).
Thus
the
formation
of
new
persistent
propagules
in
reduced.
This
aspect
is
found
in
Figure
4
where
the
population
in
soil
supplied
with
1
or
3
mg
N/g
of
soil
showed
a
cycle
of
increase
and
decrease.
Each
decrease
was
significantly
higher
than
the
earlier
decrease.
This
suggests
that
sclerotia
germinated
inter-
mittently
and
hyphae
could
not
form
new
persistent
propagules.
Another
factor
which
affects
viability
of
sclerotia
and
formation
of
new
propagules
is
the
availability
of
carbon.
Since
the
carbon
was
very
low
in
relation
to
nitrogen,
the
microbial
population
which
increased
tremendously
in
response
to
nitrogen
immobilized
most
of
the
available
carbon.
Thus
the
developing
hyphae
from
germinating
sclerotia
are
starved
for
lack
of
energy.
A
certain
amount
of
vegetative
growth
is
necessary
before
sclerotia
formation
occurs
(LIvINGsToN
1945).
Carbon
starvation
may
also
make
M.
phaseolina
hyphae
more
susceptible
to
antagonistic
organisms.
A
significantly
smaller
population
decline
in
NO
3
-N
supplied
sandy
loam
soil
than
in
sandy
clay
loam
soil
could
not
be
explained
from available
data.
The
population
declined
more
rapidly
and
reached
lower
levels
in
NH
4
-N
supplied
than
in
NO
3
-N
supplied
soils.
This
may
be
because
NH
4
-N
is
more
readily
utilized
by
soil
organisms
than
NO
3
-N
(GRAY
and
WILLIAMS
1971).
Although
the
degree
of
population
decline
in
two
soils
supplied
with
nitrogen
varied
significantly,
the
tendency
in
all
cases
was
for
a
significant
decline.
This
suggests
that
a
practical
biological
control
of
M.
phaseolina
by
nitrogen
fertilization,
which
can
be
fit
into
the
cultural
practices,
can
be
developed.
Summary
The
effect
of
nitrogen
(N)
at
1,
3,
5
or
7
mg/g
of
soil,
supplied
as
sodium
nitrate
(NO
3
-N)
or
ammonium
sulphate
(NH
4
-N),
on
the
survival
of
Macro-
phomina
phaseolina
sclerotia
was
studied
in
two
soils.
The
sandy
clay
loam
and
sandy
loam
soils
were
infested
with
640
sclerotia
per
gram
of
soil.
The
population
of
M.
phaseolina
declined
gradually
and
significantly
in
both
soils
supplied
with
N
from
any
source
or
at
any
concentration.
In
sandy
clay
loam
soil
the
population
dropped
to
2
to
10
propagules
if
supplied
with
3,
5
or
7
mg
N
per
gram
of
soil
from
either
source.
The
major
drop
ocurred
between
30
to
40
days
after
N
amendment.
However
in
sandy
loam
soil
the
population
dropped
to
2
or
100
propagules
if
amended
with
7
or
5
mg
NH
4
-N
per
gram
of
soil
respectively,
but
remainded
at
250
or
460
propagules
per
gram
of
soil
if
nitrogen
source
was
NO
3
.
In
both
soils,
NH
3
-N
incited
more
rapid
and
drastic
decline
in
population
than
NO3-N.
142
FILHO
and
DHINGRA
Zusammenfassung
Reduktion
der
Sklerotienzahl
von
Macrophomina
phaseolina
im
Boden
durch
Stickstoffdiingung
In
der
vorliegenden
Arbeit
wurde
der
Effekt
von
1,
3,
5
und
7
mg
N/g
Boden,
verabreicht
als
Nitrat-
oder
Ammoniumsulfat-N,
auf
den
Abbau
von
Sklerotien
von
M.
phaseolina
untersucht.
Ein
sandiger
Lehm
und
ein
sandiger
Ton
wurden
mit
je
640
Sklerotienig
Boden
infiziert.
Die
M.
phaseolina-Popu-
lation
sank
in
beiden
Boden
und
bei
alien
N-Verfahren
drastisch
ab,
wobei
die
Reduktion
zwischen
30
und
40
Tagen
nach
der
N-Verabreichung
am
griiiken
war.
Literature
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Authors'
address:
Departamento
de
Fitopatologia,
Universidade
Federal
de
Vicosa,
36.570
Vicosa,
MG
(Brazil).