Inoculum potential of Macrophomina phaseolina

Short, G.E.; Wyllie, T.D.

Phytopathology 68(5): 742-746

1977

Sodium hypochlorite was used to dissolve the melanin-like cementing agent that engulfs sclerotial cells of Macrophomina phaseolina. Then sclerotia were 'squashed' to enable enumeration of cells of the propagules. The number of cells per sclerotium was directly related to size of sclerotia; and sclerotium size appeared to depend on the available nutrients of the substrate on which the propagules were produced. Large sclerotia produced considerably more germ tubes than did small sclerotia when germinated on culture media. Sclerotia of M. phaseolina were sensitive to soil fungistasis; but in the spermosphere of soybean, sclerotia germinated within 2-3 mm of the seed surface and produced one to seven germ tubes per germinated sclerotium.

Ecology
and
Epidemiology
Inoculum
Potential
of
Macrophomina
phaseolina
G.
E.
Short
and
T.
D.
Wyllie
Former
Postdoctoral
Fellow
and
Professor,
respectively,
Department
of
Plant
Pathology,
University
of
Missouri-
Columbia,
Columbia,
MO
65201.
Published
with
the
approval
of
the
Director,
University
of
Missouri
Agricultural
Experiment
Station
as
Journal
Series
Paper
No.
7849.
Accepted
for
publication
14
October
1977.
ABSTRACT
SHORT,
G.
E.,
and
T.
D.
WYLLIE.
1978.
lnoculum
potential
of
Macrophomina
phaseolina.
Phytopathology
68:742-746.
Sodium
hypochlorite
was
used
to
dissolve
the
melanin-like
cementing
agent
that
engulfs
sclerotial
cells
of
Macrophomina
phaseolina.
Then
sclerotia
were
'squashed'
to
enable
enumeration
of
cells
of
the
propagules.
The
number
of
cells
per
sclerotium
was
directly
related
to
size
of
sclerotia;
and
sclerotium
size
appeared
to
depend
on
the
available
nutrients
of
the
substrate
on
which
the
propagules
were
Additional
key
words:
charcoal
rot,
decolorized
sclerotia.
produced.
Large
sclerotia
produced
considerably
more
germ
tubes
than
did
small
sclerotia
when
germinated
on
culture
media.
Sclerotia
of
M.
phaseolina
were
sensitive
to
soil
fungistasis;
but
in
the
spermosphere
of
soybean,
sclerotia
germinated
within
2-3
mm
of
the
seed
surface
and
produced
one
to
seven
germ
tubes
per
germinated
sclerotium.
Macrophomina
phaseolina
(Tassi)
Goid
causes
a
root
and
stem
rot
of
many
agronomic
crops,
including
soybean,
corn,
and
sorghum
(4,
13).
Multicellular
sclerotia
(26)
of
variable
size
(
I
I)
enable
the
fungus
to
survive
adverse
environmental
conditions
(4)
and
later
serve
as
sources
of
inoculum
for
infection
(3,
21).
Techniques
have
been
described
for
determination
of
population
densities
of
sclerotia
in
soil
(11,
12,
14,
25);
however,
the
number
of
cells
per
sclerotium
have
not
been
determined.
Such
information
may
more
precisely
elucidate
the
relationship
between
inoculum
density
and
disease
severity
(2,
5).
The
objectives
of
this
investigation
were:
(i)
to
examine
the
relation
between
substrate,
size
of
sclerotia,
and
number
of
cells
per
sclerotium;
and
(ii)
to
examine
germination
of
sclerotia
on
culture
media
and
in
spermosphere
(17,
23)
soil.
MATERIALS
AND
METHODS
Production
and
treatment
of
sclerotia.—Sclerotia
of
M.
phaseolina
strain
S
(27)
were
produced
in
flasks
containing
50
ml
of
potato-dextrose
broth
(PDB).
After
2-3
wk
of
incubation
at
33
C,
sclerotial
mats
were
rinsed
three
times
with
100
ml
of
sterile
water,
air-dried,
gently
ground
with
a
mortar
and
pestle,
and
placed
on
a
90-gm
(170-mesh)
sieve.
Sclerotia
were
collected
on
a
series
of
sieves
(170,
200,
270,
325,
and
500
mesh)
according
to
size
(>90
gm,
75-90
pm,
54-74µm,
45-53
Am,
and
25-44
Am
diameter,
respectively).
Sclerotia
also
were
produced
in
small
quantities
on
0.2%
water
agar,
and
size
determinations
were
made
microscopically
using
an
ocular
micrometer.
Sclerotia
from
soybean
stems
00032-949X/78/000125S03.00/0
Copyright
(c)
1978
The
American
Phytopathological
Society,
3340
Pilot
Knob
Road,
St.
Paul,
MN
55121.
All
rights
reserved.
[Glycine
max
(L.)
Merr.
`Amsoy
71']
were
collected
by
splitting
the
stem
longitudinally,
removing
the
pith
containing
the
sclerotia,
passing
the
pith
and
sclerotia
through
a
Wiley
mill
using
a
246-Am
(60-mesh)
sieve
(27),
and
then
sieving
as
with
the
PDB
culture-produced
sclerotia.
Sclerotia
were
decolorized
for
cell
enumeration
as
follows:
individual
sclerotia
were
placed
in
a
drop
of
6.0%
sodium
hypochlorite
on
a
microscope
slide
and
covered
with
a
coverslip.
After
10-30
min,
a
slight
amount
of
pressure
was
applied
to
the
coverslip
to
'squash'
the
sclerotium
for
cell
counting.
Germination
of
sclerotia.—Sclerotia
were
incubated
at
16,
24,
or
32
Con
2.0%
water
agar
(WA),
potato-dextrose
agar
(PDA),
or
chloroneb-mercuric
chloride-rose
bengal
agar
(CM
RA)
(12).
Sclerotia
were
examined
microscopically
every
24
hr
to
determine
percentage
germination
and
number
of
germ
tubes
per
germinated
sclerotium.
Sclerotia
were
observed
for
2
wk
when
incubated
at
16
C.
When
both
parameters
ceased
to
increase,
microscopic
observations
were
discontinued.
Sclerotium
germination
also
was
observed
in
the
spermosphere
of
soybean
using
a
modification
of
a
technique
previously
described
(17).
A
sandy
loam
soil
(58%
sand,
27%
silt,
and
15%
clay)
was
infested
with
sclerotia
with
diameters
of
45-54
gm
or
74-90
pm
collected
from
stem
tissue
and
from
PDB
culture,
respectively.
Amsoy
71
soybean
seeds
were
planted
in
soil
columns
(17)
I
cm
below
the
soil
surface;
soil
moisture
was
established
and
maintained
at
75%
of
moisture
holding
capacity.
Sclerotia
were
incubated
for
2
days
at
32
C
or
for
4
days
at
16
C
following
seed
placement.
Soil
was
embedded
in
agar,
and
then
blocks
of
agar-embedded
soil
containing
sclerotia
were
removed
at
defined
millimeter
increments
from
the
seed
(17)
and
placed
on
a
microscope
slide
in
a
drop
of
6.0%
sodium
hypochlorite
742
May
1978]
SHORT
AND
WYLLIE:
MACROPHOMINA
INOCULUM
POTENTIAL
743
B
•
•
•
D
_
O
t 1 I
0
a...31
..
—•
'0 0.
.
,
•
...
...
I
,. r/
..,,
4
ki
r
',
‘
‘":•
40
-
".
5
A
,
A
i
i
,
':
*
/ :
•
•
*'
P'
1•
••
•
0
4
'
..
i
N
•
i,
0
•
*
Fig.
I-(A
to
F).
Effect
of
sodium
hypochlorite
on
sclerotia
of
Macrophomina
phaseolina.
A)
Nontreated
sclerotium
(X1,100).
B)
Sodium
hypochlorite-treated
sclerotium
(X1,100).
C-D)
Scicrotia
decolored
in
sodium
hypochlorite
and
'squashed'
(X1,100).
E)
Decolored,
nongerminated
sclerotia
on
soil
smear(x1,100).
F)
Decolored,
germinated
sclerotium
on
soil
smear(x1,100).
Y
E
A
•
c•
4
si.-
-".
-,.
....
,,
•
..'
.N
.
t.
Q
v ,
.?
.
...7
.
0.
4,
Diameter
range
(
gy
m)
Proportion
(%)
Cells
per
sclerotium
(no.)
25-44
1
27
(14-44)
b
45-53
I
59
(28-98)
54-74
6
79
(53-102)
75-90
18
109
(76-195)
>90
74
214
(128-320)
25-44
27 27
(11-43)
45-53
22
47
(35-75)
54-74
51
69
(43-104)
25-44
I 1
24(14
,
43)
45-53
47
54-74
42
Soybean
tissue
Substrate
Potato
dextrose
broth
0.2%
water
agar
744
PHYTOPATHOLOGY
[Vol.
68
to
decolor
the
sclerotia
(16).
The
slide
was
warmed
slightly
to
melt
the
agar,
and
a
soil
smear
was
made
for
microscopic
examination
of
sclerotium
germination.
RESULTS
Sclerotia
of
M.
phaseolina
were
thoroughly
decolored
by
6.0%
sodium
hypochlorite
in
less
than
0.5
hr
(Fig.
1-A,
B).
The
apparent
dissolution
of
the
black,
melanin-like
cementing
agent
(6)
also
permitted
the
sclerotia
to
be
'squashed'
to
such
an
extent
that
individual
cells
of
the
propagules
could
be
counted
easily
(Fig.
1-C,
D).
Decoloring
sclerotia
on
soil
smears
facilitated
a
positive
identification
of
nongerminated
(Fig.
l-E)
and
germinated
(Fig.
I-F)
sclerotia.
Cells
of
sclerotia
decolored
in
1.2%
to
6.0%
sodium
hypochlorite
were
no
longer
viable.
Effect
of
substrate
on
inoculum.—The
mean
diameter
and
standard
deviation
of
100
sclerotia
produced
on
PDB
(130
±
30
Am)
was
significantly
greater
than
when
sclerotia
were
produced
on
0.2%
WA
(63
±
8
Am)
or
in
stems
of
naturally-infected
field-grown
soybeans
(70±
18
Am).
Indeed,
74%
of
the
sclerotia
produced
on
PDB
had
diameters
greater
than
90µm;
whereas,
all
of
the
sclerotia
produced
on
0.2%
WA
or
naturally-infected
stems
of
soybeans
had
diameters
of
less
than
75
Am
(Table
1).
Finally,
the
number
of
cells
per
sclerotium
increased
directly
with
size
of
sclerotia
(Table
1).
Relation
of
inocula
characteristics
to
sclerotium
germination.—Large
sclerotia
(74-90
Am
diameter)
produced
twice
as
many
germ
tubes
when
germinated
on
2.0%
WA
as
did
smaller
sclerotia
(45-53
Am
diameter)
(Table
2).
Since
it
was
difficult
to
count
all
of
the
germ
tubes
produced
by
some
sclerotia,
a
maximum
of
10
germ
tubes
per
germinated
sclerotium
was
counted;
therefore,
the
total
number
of
germ
tubes
per
germinated
sclerotium
probably
was
underestimated,
particularly
among
the
large
(74-90
µm
diameter)
sclerotia,
and
among
sclerotia
germinating
on
PDA,
where
rapid
growth
obscured
later-
emerging
germ
tubes.
Temperature
affected
the
rapidity
of
sclerotium
germination,
but
the
final
number
of
germ
tubes
was
not
affected.
Particulate
matter
and
color
of
CMRA,
and
microbial
contamination
on
PDA,
resulted
in
selecting
WA
as
the
most
suitable
medium
to
assay
for
germinability.
Germination
of
sclerotia
(74-90
Am
diameter)
in
soil
at
32
C
was
confined
primarily
to
a
zone
within
2
mm
of
the
soybean
seed
(Table
3),
with
one
to
seven
germ
tubes
produced
by
each
germinated
sclerotium.
When
smaller
sclerotia
(45-53
µ
m
diameter),
obtained
from
stems
of
naturally-infected
soybeans,
were
incubated
in
soil
at
16
C,
sclerotium
germination
was
not
observed
beyond
2
mm
of
the
seed
surface.
DISCUSSION
Severity
of
diseases
caused
by
soilborne
pathogens
is
dependent
upon
many
factors,
including
the
inoculum
potential
of
the
infective
propagules
in
the
soil
(2,
5).
Inoculum
potential
measures
the
ability
of
the
pathogen
to
infect
its
host
(5).
Garrett
(8)
and
Baker
(2)
expressed
this
ability
in
terms
of
biological
energy
available
for
infection.
To
measure
inoculum
potential
in
fundamental
units
of
biological
energy
would
be
ideal,
but
in
this
instance,
energy
is
difficult
to
measure
in
an
absolute
sense
(2).
Consequently,
biological
energy
has
been
expressed
as
a
function
of
the
genetic
capacity
of
the
pathogen,
environmental
factors,
available
nutriment
(both
internal
and
external
to
the
propagule),
and
inoculum
density
(2).
Incidence
of
disease
caused
by
M.
phaseolina
has
been
directly
related
to
the
population
of
sclerotia
in
soil
(13,
24),
but
the
sclerotium
population
is
an
extremely
crude
measure
of
inocula
such
as
with
sclerotia
of
M.
phaseolina,
which
are
multicellular
(Fig.
1-A,
B,
C,
D)
and
capable
of
producing
multiple
germ
tubes
(Table
2,
Fig.
I-F)
and
capable
of
repeated
germination.
Our
method
of
decolorizing
and
'squashing'
sclerotia
has
made
it
possible
to
estimate
the
inoculum
potential
of
this
pathogen
on
a
numerical
cellular
basis.
Sodium
hypochlorite
previously
has
been
used
to
TABLE
I.
In
vivo
and
in
vitro
production
of
sclerotia
by
Macrophomina
phaseolina:
relation
of
propagule
size
to
number
of
cells
per
propagule
Characteristics
of
sclerotia
'Based
on
100
sclerotia
per
substrate.
'Mean
and
range
of
25
sclerotia
per
treatment.
May
1978]
SHORT
AND
WYLLIE:
MACROPHOMINA
INOCULUM
POTENTIAL
745
decolorize
microsclerotia
of
Verticillium
albo-atrum
(16),
which
then
were
stained
and
cleared
to
reveal
the
origin
of
germ
tubes.
However,
the
method
has
not
been
used
widely,
even
though
our
experience
with
M.
phaseolina
and
other
sclerotia-producing
fungi
suggests
a
wide
applicability.
For
example,
the
difficulty
in
differentiating
between
microsclerotia
and
certain
soil
particles
on
modified
soil
smears
(15)
was
resolved
easily
with
hypochlorite—each
sclerotium
was
seen
as
a
distinct
clump
of
cells
unlike
anything
else
in
the
soil
(Fig.
1-E,
F).
Unfortunately,
the
hypochlorite
treatment
was
lethal
to
the
cells;
thus,
the
ability
of
each
cell
to
produce
a
germ
tube
could
not
be
tested
directly.
However,
larger
sclerotia
produced
more
germ
tubes
than
smaller
sclerotia
(Table
2),
and
sclerotia
of
M.
phaseolina
are
capable
of
repeated
germination
(Short
and
Wyllie,
unpublished).
It
is
not
known
if
single
cells
are
capable
of
either
multiple
or
repeated
germination.
Regardless,
it
should
be
possible
to
determine
indirectly
the
relationship
between
cell
number
and
potential
number
of
germ
tubes
per
sclerotium.
Sclerotia
of
M.
phaseolina
buried
in
nona
mended
soil
failed
to
germinate
unless
suitable
nutrients
were
added
(3,
22).
Smith
(22)
found
the
amino
acid
fraction
of
root
exudate
from
sugar
pine
seedlings
to
be
particularly
stimulatory
to
germination,
and
M.
phaseolina
is
known
to
use
nearly
all
amino
acids
for
growth
(7).
Soybean
seed
exudates
contain
sucrose
and
fructose
(9)
compounds
also
known
to
stimulate
germination
of
sclerotia
of
M.
phaseolina
(1).
Sclerotia
of
M.
phaseolina
have
been
reported
to
germinate
in
the
rhizosphere
of
pine
seedlings
(21)
and
we
have
observed
sclerotium
germination
in
the
spermosphere
of
soybean
seeds
(Table
3).
The
maximum
distance
from
the
soybean
seed
at
which
sclerotia
germinated
(2-3
mm)
was
considerably
less
than
with
chlamydospore
germination
of
Fusarium
solani
(17,
23)
in
the
spermosphere
of
pea
(7-8
mm)
and
bean
(10-12
mm).
Sclerotia
of
M.
phaseolina
require
nearly
24
hr
to
germinate
under
optimum
conditions,
in
contrast
to
the
4-5
hr
required
for
chlamydospore
germination
of
F.
solani
f.
sp.
phaseoli
(23).
Competition
for
nutrients
(20)
at
the
onset
of
sclerotium
germination
(24
hr)
was
TABLE
2.
Effect
of
source
and
size
of
sclerotia,
substrate,
and
temperature
on
germination
of
sclerotia
of
Macrophomina
phaseolina
Characteristics
of
sclerotia
Germination
Temp
(C)
Germination
of
sclerotia
(%)
Germ
tubes
per
germinated
sclerotium
(apparent)
(no.)
Source
Diameter
(Pm)
substrate'
Strain
S'
45-53
PDA
16
31
a'
3.0
a
PDA
32
33
a
3.2
a
Field'
45-53
WA
16
37
ab
6.4
b
WA
32
37
ab
4.8ab
Strain
S
45-53
CMRA
24
49
ab
6.3
b
WA
24
52
b
5.0
ab
Strain
S
45-53
WA
16
52
b
5.0
ab
WA
32
57
b
4.4
ab
Strain
S
75-90
WA
16
90
c
9.8
c
WA
32
91
c
8.7
c
"Potato-dextrose
agar
(PDA),
2.0%
water
agar
(WA),
or
chloroneb-mercuric
chloride-rose
bengal
agar
(CMRA).
'Sclerotia
produced
in
potato-dextrose
broth.
'Sclerotia
collected
from
stems
of
field-grown,
naturally-infected
soybeans.
'Means
in
each
column
followed
by
the
same
letter
do
not
differ
significantly
(P
=
0.05)
according
to
Tukey's
test.
TABLE
3.
Germination
of
Macrophomina
phaseolina
sclerotia'
at
32
C
in
the
spermosphere
of
Amsoy
71
soybean
Germ
tubes
per
Germination
germinated
sclerotium
Distance
from
seed
of
sclerotia
(apparent)
(mm)
(%)
(no.)
0-1
42
2.2
1-2
16
2.2
2-3
3
2.5
3-4
0
0.0
`Sclerotia
(74-90
pm
diameter)
were
obtained
from
PDB
cultures
of
M.
phaseolina
strain
S.
746
PH
YTOPAT
HOLOGY
[Vol.
68
probably
much
more
intense
than
at
the
onset
of
F.
solani
f.
sp.
phaseoli
chlamydospore
germination
(5
hr).
Other
factors
of
possible
importance
include
soil
moisture
and
temperature
(17,
23),
seed
quality
(18,
19),
and
fungistatic
level
of
the
propagule
(10).
LITERATURE
CITED
I.
AYANRU,
D.
K.
G.,
and
R.
J.
GREEN,
JR.
1974.
Alteration
of
germination
patterns
of
sclerotia
of
Macrophomina
phaseolina
on
soil
surfaces.
Phytopathology
64:595-601.
2.
BAKER,
R.
1965.
The
dynamics
of
inoculum.
Pages
395-403
in
K.
F.
Baker
and
W.
C.
Snyder,
eds.
Ecology
of
soil-
borne
plant
pathogens.
Univ.
Calif.
Press,
Berkeley.
571
3.
BHATTACHARYA,
M.,
and
K.
R.
SAMADDAR.
1976.
Epidemiological
studies
on
jute
diseases.
Survival
of
Macrophomina
phaseoli
(Maubl.)
Ashby
in
soil.
Plant
Soil
44:27-36.
4.
COOK,
G.
E.,
M.
G.
BOOSALIS.
L
D.
DUNKLE,
and
G.
N.
ODVODY.
1973.
Survival
of
Macrophomina
phaseoli
in
corn
and
sorghum
stalk
residue.
Plant
Dis.
Rep.
57:873-875.
5.
DIMOND,
A.
E.,
and
J.
G.
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1965.
The
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of
inoculum.
Pages
404-419
in
K.
F.
Baker
and
W.
C.
Snyder,
eds.
Ecology
of
soil-borne
plant
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Univ.
Calif.
Press,
Berkeley.
571
p.
6.
GANGOPADHYAY,
S.,
and
T.
D.
WYLLIE.
1974.
Melanin-like
compound
in
the
sclerotia
of
Macrophomina
phaseolina.
Indian
Phytopathol.
27:661-
663.
7.
GANGOPADHYAY,
S.,
and
T.
D.
WYLLIE.
1976.
Utilization
of
amino
acids
by
Macrophomina
phaseolina
(Tassi)
Goid.
J.
Biol.
Sci.
19:1-13.
8.
GARRETT,
S.
D.
1970.
Pathogenic
root-infecting
fungi.
Cambridge
University
Press,
London
and
New
York.
294
p.
9.
KEELING,
B.
L.
1974.
Soybean
seed
rot
and
the
relation
of
seed
exudate
to
host
susceptibility.
Phytopathology
64:1445-1447.
10.
LOCKWOOD,
J.
L.
1964.
Soil
fungistasis.
Annu.
Rev.
Phytopathol.
2:341-362.
1
1
.
MC
CAIN,
A.
H.,
and
R.
S.
SMITH,
JR.
1972.
Quantitative
assay
of
Macrophomina
phaseoli
from
soil.
Phytopathology
62:1098.
12.
MEYER,
W.
A.,
J.
B.
SINCLAIR,
and
M.
N.
KHARE.
1973.
Biology
of
Macrophomina
phaseoli
in
soil
studied
with
selective
media.
Phytopathology
63:613-620.
13.
MEYER.
W.
A.,
J.
B.
SINCLAIR,
and
M.
N.
KHARE.
1974.
Factors
affecting
charcoal
rot
of
soybean
seedlings.
Phytopathology
64:845-849.
14.
PAPAVIZAS,
G.
C.,
and
N.
G.
KLAG.
1975.
Isolation
and
quantitative
determination
of
Macrophomina
phaseolina
from
soil.
Phytopathology
65:182-187.
15.
PHIPPS,
P.
M.,
M.
K.
BEUTE,
and
K.
R.
BARKER.
1976.
An
elutriation
method
for
quantitative
isolation
of
Cylindrocladium
crotalaria
microsclerotia
from
peanut
field
soil.
Phytopathology
66:1255-1259.
16.
SCHREIBER,
L.
R.,
and
R.
J.
GREEN,
JR.
1963.
Effect
of
root
exudates
on
germination
of
conidia
and
microsclerotia
of
Verticillium
albo-atrum
inhibited
by
the
soil
fungistatic
principle.
Phytopathology
53:260-264.
17.
SHORT,
G.
E.,
and
M.
L.
LACY.
1974.
Germination
of
Fusarium
solani
f.
sp.
pisi
chlamydospores
in
the
spermosphere
of
pea.
Phytopathology
64:558-562.
18.
SHORT,
G.
E.,
and
M.
L.
LACY.
1976.
Carbohydrate
exudation
from
pea
seeds:
Effect
of
cultivar,
seed
age,
seed
color,
and
temperature.
Phytopathology
66:182-187.
19.
SHORT,
G.
E.,
and
M.
L.
LACY.
1976.
Factors
affecting
pea
seed
and
seedling
rot
in
soil.
Phytopathology
66:188-
192.
20.
SINGH,
R.
S.
1965.
Development
of
Pythium
ultimum
in
soil
in
relation
to
presence
and
germination
of
seeds
of
different
crops.
Mycopathol.
Mycol.
Appl.
27:155-160.
21.
SMITH,
R.
S.,
JR.
1963.
Epidemiology
and
host-parasite
relations
in
the
charcoal
root
disease
of
sugar
pine.
Ph.D.
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Univ.
Calif.
Berkeley.
98
p.
22.
SMITH,
W.
H.
1969.
Germination
of
Macrophomina
phaseoli
sclerotia
as
affected
by
Pinus
lambertiana
root
exudate.
Can.
J.
Microbiol.
15:1387-1391.
23.
STANGHELLINI,
M.
E.,
and
J.
G.
HANCOCK.
1971.
Radial
extent
of
the
bean
spermosphere
and
its
relation
to
the
behavior
of
Pythium
ultimum.
Phytopathology
61:165-168.
24.
WATANABE,
T.,
R.
S.
SMITH,
JR.,
and
W.
C.
SNYDER.
1967.
Populations
of
microsclerotia
of
the
soil-borne
pathogen,
Macrophomina
phaseoli,
in
relation
to
stem
blight
of
bean.
Phytopathology
57:1010
(Abstr).
25.
WATANABE,
T.,
R.
S.
SMITH,
JR.,
and
W.
C.
SNYDER.
1970.
Populations
of
Macrophomina
phaseoli
in
soil
as
affected
by
fumigation
and
cropping.
Phytopathology
60:1717-1719.
26.
WYLLIE,
T.
D.,
and
M.
F.
BROWN.
1970.
Ultrastructural
formation
of
sclerotia
of
Macrophomina
phaseoli.
Phytopathology
60:524-528.
27.
WYLLIE,
T.
D.,
and
G.
FRY.
1973.
Liquid
nitrogen
storage
of
Macrophomina
phaseolina.
Plant
Dis.
Rep.
57:478-
480.

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