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APPLIED
AND
ENVIRONMENTAL
MICROBIOLOGY,
Feb.
1991,
p.
434-439
0099-2240/91/020434-06$02.00/0
Copyright
©
1991,
American
Society
for
Microbiology
Isolation
and
Identification
of
Vesicular-Arbuscular
Mycorrhiza-
Stimulatory
Compounds
from
Clover
(Trifolium
repens)
Roots
MURALEEDHARAN
G.
NAIR,'*
GENE
R.
SAFIR,2
AND
JOSE
0.
SIQUEIRA2t
Bioactive
Natural
Products
Laboratory,
Department
of
Horticulture
and
Pesticide
Research
Center,'
and
Department
of
Botany
and
Plant
Pathology,2
Michigan
State
University,
East
Lansing,
Michigan
48824
Received
6
September
1990/Accepted
26
November
1990
Two
isoflavonoids
isolated
from
clover
roots
grown
under
phosphate
stress
were
characterized
as
formonon-
etin
(7-hydroxy,4'-methoxy
isoflavone)
and
biochanin
A
(5,7-dihydroxy,4'-methoxy
isoflavone).
At
5
ppm,
these
compounds
stimulated
hyphal
growth
in
vitro
and
root
colonization
of
an
undescribed
vesicular-
arbuscular
mycorrhiza,
a
Glomus
sp.
(INVAM-112).
The
permethylated
products
of
the
two
compounds
were
inactive.
These
findings
suggest
that
the
isoflavonoids
studied
may
act
as
signal
molecules
in
vesicular-
arbuscular
mycorrhiza
symbiosis.
Vesicular-arbuscular
mycorrhizae
(VAM)
result
from
a
complex
sequence
of
interactions
between
fungal
hyphae
and
host
cells,
leading
to
a
functional
mutualistic
state
(6).
Plant
factors
stimulate
VAM
hyphal
growth
in
vitro
and
also
the
precolonization
phase
of
VAM
formation
(4,
11,
13,
25,
26,
29).
It
has
been
suggested
(14,
15)
that
the
quantity
rather
than
the
presence
of
specific
compounds
in
the
root
exudates
is
responsible
for
stimulation
of
fungal
growth
and
VAM
root
colonization
(36).
Other
studies,
however,
showed
no
rela-
tionship
between
root
exudation
and
VAM
infection
(2).
Viable
spores
of
most
VAM
fungal
species
readily
germinate
on
distilled
water
(39),
and
there
is
no
evidence
that
they
require
any
specific
host
factors.
However,
certain
compo-
nents
of
root
exudates
or
plant
cells
may
act
as
signal
molecules
capable
of
inducing
hyphal
growth,
branching,
differentiation,
and
host
penetration
(4,
5,
25).
An
earlier
report
from
this
laboratory
(11)
indicated
the
presence
of
a
transient
VAM-stimulating
factor
in
exudates
from
phosphorus-deprived
young
white
clover
seedlings.
This
study
also
indicated
that
the
quality
of
the
exudate
is
important
in
stimulating
VAM
hyphal
growth.
Similar
results
have
been
found
with
stressed
suspension-cultured
legume
cells
(29)
and
with
cultures
of
Ri
T-DNA-transformed
roots
(3-5).
The
studies
with
transformed
roots
also
indicated
that
root-inducing
factors
are
required
for
the
fungus
to
switch
from
preinfection
to
a
biotrophic
state.
However,
the
nature
of
these
highly
active
plant
factors
has
not
been
determined
to
date.
In
this
study,
we
report
the
isolation
and
identifica-
tion
of
VAM-stimulatory
compounds
from
clover
roots.
MATERIALS
AND
METHODS
General
analytical
methods.
'H
and
13C
nuclear
magnetic
resonance
(NMR)
spectra
were
recorded
on
a
Varian
Gemini
300
spectrometer
in
CD3OD
solutions
at
25°C.
Infrared
spectra
in
KBr
were
obtained
with
a
Perkin-Elmer
1600
series
FT-IR
spectrometer,
and
UV
spectra
were
recorded
on
a
Shimadzu
UV-265
spectrometer.
Melting
points
were
recorded
on
a
Thomas
model
40
micro
hot-stage
apparatus
and
are
uncorrected.
Mass
spectra
(MS)
were
obtained
on
a
JEOL
model
HX-110
mass
spectrometer.
Vacuum
liquid
*
Corresponding
author.
t
Permanent
address:
Escola
Superior
de
Agricultura
de
Lavras,
MG-Brazil.
chromatography
and
preparative
thin-layer
chromatography
were
carried
out
on
silica
gel
adsorbants
by
using
CHCl3-
methanol
(MeOH)
solvent
systems.
Plant
material.
White
clover
(Trifolium
repens
L.
cv.
Ladino)
plants
(2
weeks
old)
were
used
for
all
exudate
and
extract
collections.
Plants
were
grown
in
sterile
square
glass
staining
dishes
containing
Hoagland
solution
with
and
with-
out
phosphorus
as
previously
described
(11).
At
2
weeks
of
age,
exudates
were
collected
from
the
roots
of
clover
seed-
lings
and
grown
with
or
without
phosphorus
as
follows.
Plants
were
taken
out
of
the
nutrient
solution
and
rinsed
with
sterile
distilled
water
several
times,
and
then
50
seedlings
were
placed
in
each
staining
jar
containing
sterile
distilled
water
(50
ml)
for
24
h.
The
distilled
water
in
which
seedling
roots
were
immersed
for
24 h
was
pooled
for
each
treatment
and
lyophilized
at
4°C.
Contamination
checks
were
done
after
7
and
14
days
during
plant
growth
by
plating
out
spent
nutrient
solution
on
water
agar
and
potato
dextrose
agar
at
the
time
of
nutrient
solution
replacement.
No
contamination
was
observed.
The
roots
collected
from
the
seedlings
of
the
same
experiment
(14
days
old),
as
well
as
root
exudates,
seeds,
and
plant
tops,
were
lyophilized
at
4°C
and
used
for
extraction.
Bioactive
compounds
from
the
lyophilized
roots.
Isolation
and
purification
of
the
active
compounds
for
VAM
hyphal
growth
were
carried
out
as
shown
in
Fig.
1.
The
extract
was
purified
by
vacuum
liquid
chromatography
as
follows.
Silica
gel
for
column
chromatography
(40
mesh)
(60
g)
was
made
into
a
slurry
in
CHCl3-MeOH
(4:1,
vol/vol)
and
poured
into
a
sintered
glass
filter
(fine)
fitted
with
a
Buchner
flask
connected
to
a
vacuum
line.
When
the
slurry
was
almost
dry
under
vacuum,
a
solution
of
the
extract
in
CHCl3-MeOH
(4:1)
was
applied
on
the
surface
of
the
silica
gel
and
eluted
with
the
same
solvent
system
(150
ml).
The
organic
extract
was
dried
in
vacuo
and
further
purified
by
preparative
thin-layer
chromatography.
Two
pure
fractions
thus
ob-
tained
were
used
for
bioassay
and
chemical
characterization,
and
they
were
called
clover
A
(Rf
=
0.68,
3.6
mg),
compound
1,
and
clover
B
(Rf
=
0.60,
2.8
mg),
compound
2.
Identical
fractions
were
obtained
from
the
root
exudates
as
well.
Since
the
compounds
were
in
larger
quantities
from
the
root
extracts,
further
purification
and
identification
of
the
active
components
were
carried
out
only
on
root
extracts.
Compound
1
(clover
A).
The
high-Rf
band,
colorless
nee-
dles
from
hot
MeOH;
mp,
255
to
256°C;
UV
(MeOH,
nm)
434
Vol.
57,
No.
2
VAM-STIMULATORY
COMPOUNDS
FROM
CLOVER
ROOTS
435
Lyophilized
clover
roots
(1g)
1.
Stirred
with
MeOH
(500
ml)
at
room
temperature
2.
Filtered
MeOH
extract
Residue,
discarded
1.
Evaporated
in
vacuo
2.
Vacuum
Uquid
Chromatography,
MeOH-CHCI3
MeOH-CHCI3
solution
(100
ml)
1.
Evaporated
in
vacuo
2.
Purified
by
TLC,
CHCI3-MeOH
(4:1,
v/v)
Active
Fractions
l
Clover
A
Formononetin
Inactive
Fractions(discarded)
Clover
B
Biochanin
A
FIG.
1.
Isolation
and
purification
scheme
for
clover
A
(for-
mononetin)
and
clover
B
(biochanin
A)
from
14-day-old
white
clover
roots.
TLC,
Thin-layer
chromatography.
212,
248,
300;
+KOH
206, 256,
336;
'H
NMR
(CD30D)
8
8.15
(1H,
s,
H-2),
8.05
(1H,
d,
J
=
9
Hz,
H-5),
7.45
(2H,
dd,
J
=
2,9
Hz,
H-2',
H-6'),
6.98
(2H,
dd,
J
=
2,9
Hz,
H-3',
H-5'),
6.85
(1H,
dd,
J
=
2,8
Hz,
H-6),
6.81
(1H,
d,
J
=
2
Hz,
H-8),
3.80
(3H,
s,
OMe);
13C
NMR
(CD30D)
8
152.22
(C-2),
124.43
(C-3),
175.61
(C-4),
127.71
(C-5),
119.26
(C-6),
154.22
(C-7),
102.30
(C-8),
156.45
(C-8a),
122.16
(C-4a),
123.87
(C-i'),
130.01
(C-2'),
113.61
(C-3').
159.20
(C-4'),
113.61
(C-5'),
130.01
(C-6'),
55.28
(OMe);
Cl
(+,
methane)-MS,
m/z
(%
int.)
269
(M+H,
100),
257
(12),
239
(5),
132
(8);
El
(+,
Dl)-MS,
m/z
(%
int.)
268
(M+,
100),
253
(M+
-
CH3,
40),
239
(20),
150
(35),
132
(100).
Compound
2
(clover
B).
The
low-Rf
band,
pale-brown
solid;
mp,
212
to
214°C;
UV
(MeOH,
nm)
210,
260;
+KOH
271,
326;
'H
NMR
(CD30D)
8
8.01
(1H,
s,
H-2),
7.40
(2H,
d,
J
=
8
Hz,
H-2',
H-6'),
6.90
(2H,
d,
J
=
8
Hz,
H-3',
H-5'),
6.35
(1H,
d,
J
=
1.8
Hz,
H-8),
6:25
(1H,
d,
J
=
1.8
Hz,
H-6),
3.75
(3H,
s,
OMe);
13C
NMR
(CD3OD)
8
155.78
(C-2),
117.20
(C-3),
178.30
(C-4),
160.30
(C-5),
116.92
(C-6),
160.80
(C-7),
103.50
(C-8),
160.10
(C-8a),
126.20
(C-4a),
55.80
(OMe);
El
(+,
Dl)-MS,
mlz
(%
int.)
284
(M+,
100),
269
(M+
-
CH3,
42),
204
(5),
180
(5),
156
(10),
132
(20).
Compound
3.
Compound
1
(50
mg)
was
methylated
(27)
by
dissolving
in
dry
acetone
(50
ml),
stirred
with
K2CO3
(1.2
g),
and
refluxed
(10
min).
Dimethyl
sulfate
was
added
to
this
mixture
(0.5
ml)
and
refluxed
until
no
more
starting
material
was
shown
by
thin-layer
chromatography
(6
h).
The
reaction
mixture
was
cooled,
the
solid
was
filtered
off,
and
the
filtrate
was
concentrated,
diluted
with
water,
and
extracted
with
ethyl
acetate
(EtOAc).
The
EtOAc
layer
was
washed
with
an
NaHCO3
solution
and
water
and
dried
over
anhydrous
MgSO4.
Removal
of
the
solvent
afforded
a
brown
solid
which
was
recrystallized
from
MeOH
to
yield
colorless
platelike
crystals,
compound
3
(45
mg);
mp,
155
to
156°C;
El
(+,
Dl)-MS,
m/z
(%
int.)
282
(M+,
100),
267
(M+
-
CH3,
35),
251
(3),
239
(15),
150
(35),
132
(100),
117
(20),
89
(20).
Compound
4.
Compound
2
(50
mg)
was
methylated
with
dimethyl
sulfate
in
acetone
with
K2CO3
as
in
the
case
of
compound
1,
and
the
work-up
of
the
reaction
mixture
afforded
pale
yellow
platelike
crystals
from
MeOH,
com-
pound
4
(40
mg);
mp,
158
to
160°C;
El
(+,
Dl)-MS,
m/z
(%
int.)
312
(M+,
100),
295
(10),
283
(15),
266
(20),
180
(10),
156
(15),
137
(8),
132
(30),
117
(5),
89
(5).
Spore
germination.
An
undescribed
VAM,
a
Glomus
spe-
cies
(INVAM-112)
(24a),
subsequently
referred
to
as
Glomus
112,
was
grown
in
sorghum
(Sorghum
vulgare)
pot
cultures
in
the
greenhouse
for
4
months
and
stored
at
4°C
before
use.
This
VAM-infested
soil
was
wet
sieved
(12),
and
spores
were
suspended
by
centrifugation
in
a
Ficoll
gradient
(10,
35,
45,
and
60%).
Organic
debris
was
carefully
removed
from
the
spore
suspension
by
hand
with
a
Pasteur
pipette
under
a
dissecting
microscope.
Fungal
chlamydospores
were
then
surface
sterilized
with
a
1:1:1
solution
of
2%
(wt/vol)
chlo-
ramine-T,
0.02%
(wt/vol)
streptomycin
sulfate,
and
sodium
lauryl
sulfate.
After
incubation
under
vacuum
for
30
min,
chlamydospores
were
washed
with
sterile
distilled
water.
Surface-sterilized
chlamydospores
were
stored
at
4°C
until
used.
A
medium
designed
for
root
organ
culture
was
modified
to
contain
no
phosphorus
(11).
Fractions
obtained
as
in
Fig.
1
were
incorporated
into
the
agar
medium
at
5
ppm.
Only
uncontaminated
chlamydospores
were
used
in
our
experi-
ments.
Glomus
112
spores
(30
per
plate)
were
transferred
to
five
replicate
plates
for
each
root
exudate
fraction
to
be
tested
(11).
All
plates
were
incubated
at
25
to
27°C
in
the
dark.
Hyphal
elongation
was
monitored
at
5-day
intervals.
Hyphal
elongation
data
include
the
mean
hyphal
lengths
per
spore
and
consider
only
those
spores
that
germinated.
This
experiment
was
repeated
twice.
VAM
colonization
and
plant
growth.
The
effects
of
syn-
thetic
formononetin
(prepared
by
American
Cyanamid
Co.)
and
biochanin
A
(Sigma
Chemical,
St.
Louis,
Mo.)
on
VAM
formation
and
subsequent
plant
growth
were
examined
in
growth-chamber
experiments.
In
all
experiments,
water-
soaked
clover
seeds
were
pregerminated
on
filter
paper,
inoculated
with
Rhizobium
leguminosarum
biovar
trifolii
(peat-commercial
inoculum-Nitragin),
and
transplanted
at
a
rate
of
one
plant
per
cell
into
plastic
inserts
with
individual
cells
(4
by
5.5
cm),
each
containing
80
g
of
a
sand-soil
mix.
The
bulked
sand-soil
mix
was
steam
sterilized
for
2
h,
air
dried,
and
stored
until
use.
Chemical
analysis
of
the
soil
indicated
a
neutral
to
slightly
alkaline
pH,
low
to
moderate
fertility,
and
a
low exchange
capacity.
The
soil
was
inocu-
lated
with
Glomus
fasciculatum
obtained
from
pot
culture.
Dried
inoculum
was
incorporated
thoroughly
into
the
soil
mix
at
rates
sufficient
to
achieve
a
final
spore
density
ranging
from
2
to
4
spores
per
g
of
soil.
Prior
to
transplanting,
10
ml
of
5-ppm
solutions
of
each
test
compound
were
delivered
into
each
cell
by
a
pipette
dispenser,
unless
otherwise
indicated.
The
isoflavonoid
compounds
were
first
dissolved
in
a
small
volume
of
methanol
and
then
transferred
to
water.
Solvent
controls
were
also
prepared
by
using
similar
amounts
of
methanol
(0.02%
of
the
final
volume)
in
water.
Plastic
inserts
were
placed
in
plastic
trays
and
transferred
to
a
full-light
growth
chamber
(400
,umol/m2
per
s)
with
a
14-h
day
and
30
and
25°C
day
and
night
temperatures,
respec-
tively.
For
each
four-cell
unit,
a
plastic
petri
dish
bottom
(90
VOL
.
57
,
1991
APPL.
ENVIRON.
MICROBIOL.
15
10
5
1
5
10
5
0
Control
A
B
Fractions
2.
Hyphal
growth-stimulatory
effects
of
clover
A
and
clo-
by
15
mm)
was
used
as
a
base
to
avoid
chemical
contami-
nation
and
to
facilitate
watering.
Plants
were
watered
daily
with
distilled
water,
from
both
the
bottom
and
the
top,
and
allowed
to
grow
for
up
to
4
weeks.
At
the
end
of
the
growth
period,
plants
were
harvested
and
assessed
for
growth.
Roots
were
washed
free
of
soil
and
cleared,
stained
(31),
and
assessed
for
VAM
colonization
by
a
line
intersect
method
(18).
Experiments
were
conducted
by
using
a
completely
randomized
design
with
at
least
12
plants
per
treatment
and
were
repeated
at
least
twice.
Experimental
data
were
sub-
jected
to
statistical
analysis
by
using
an
MSTAT
statistical
package
(MSTAT-C,
a
microcomputer
program
for
the
de-
sign,
management,
and
analysis
of
agronomic
research
ex-
periments,
1988,
Michigan
State
University,
East
Lansing,
Mich.).
RESULTS
The
purified
fractions
from
lyophilized
clover
root
exu-
dates
and
extracts
showed
hyphal
growth
stimulation
in
vitro
(Fig.
2).
Growth
stimulation
was
observed
1
week
after
spores
were
plated,
and
further
increases
were
evident
after
2
weeks.
Root
extract
fractions
were
structurally
identical
to
the
exudate
fractions,
evidencing
the
presence
of
both
formononetin
(clover
A)
and
biochanin
A
(clover
B)
in
the
exudates
and
in
the
extract
as
well
(Fig.
3).
An
identical
extraction
procedure
was
carried
out
on
clover
tops
and
seeds
used
to
produce
roots
for
extraction.
Analyses
of
the
extracts
from
the
seeds
and
plant
tops
did
not
indicate
the
presence
of
either
clover
A
or
clover
B.
These
extracts
were
not
active
on
VAM
hyphal
growth
and
hence
were
not
investigated
further.
A
similar
lack
of
activity
was
found
in
extracts
of
roots
obtained
from
clover
plants
grown
in
1
Formononetin
R'
=
OH,
R"
=
H
2
Biochanin
A
R'
=
R"
=
OH
3
R'=
OMe,
R"
=
H
4
R'=
R"
=
OMe
FIG.
3.
Structures
for
clover
A
(formononetin),
clover
B
(bio-
chanin
A),
and
their
methylated
products.
phosphate-supplemented
Hoagland
solution.
It
was
interest-
ing
to
observe
that
90-day-old
clover
roots
obtained
under
phosphate
stress
had
lower
concentrations
of
clover
A
and
clover
B
(data
not
presented)
and
also
showed
the
presence
of
another
related
isoflavonoid,
which
was
not
characterized
because
of
its
reduced
activity
on
VAM
hyphal
elongation
in
vitro.
The
effects
of
synthetic
isoflavonoids
on
VAM
root
colonization
and
growth
of
clover
plants
are
given
in
Table
1.
Both
formononetin
and
biochanin
A
enhanced
clover
root
colonization
by
G.
fasciculatum
and
plant
growth,
whereas
methylated
compounds
were
inactive
at
the
concentrations
tested.
DISCUSSION
The
stimulatory
effects
of
growth
roots
on
VAM
are
well
documented
(4,
5,
25,
26).
In
this
study,
for
the
first
time
we
believe,
compounds
capable
of
stimulating
hyphal
growth
and
VAM
colonization
were
isolated
and
identified.
These
compounds
were
identified
as
the
isoflavonoids
formonone-
tin
and
biochanin
A.
Both
are
constitutively
found
in
le-
gumes,
are
derived
from
the
phenylpropanoid
pathway,
and
have
also
been
regarded
as
phytoalexins
(42).
Spectral
analyses
of
both
clover
A
and
clover
B
confirmed
the
identity
of
these
compounds
as
formononetin
and
bio-
chanin
A,
respectively.
1H
NMR
spectra
of
clover
A
and
synthetic
formononetin
are
identical
(Fig.
4).
The
cross
peaks
observed
in
the
long-range
COSY
experiment
of
formononetin
in
dimethyl
sulfoxide-d6
are
assigned
in
Fig.
5.
This
experiment
unambiguously
proved
that
clover
A
is
7-hydroxy,4'-methoxy
isoflavone,
compound
1,
and
cannot
TABLE
1.
Effects
of
synthetic
isoflavonoids
on
growth
and
VAM
root
colonization
of
white
clover
plants
after
4
weeks
of
growth
in
the
presence
of
Glomus
112
Compounds
Clover
top
fresh
wt
(mg)a
VAM
colonization
(%)a
Control
38
c
33
bc
Formononetin
50
a
65
a
Biochanin
A
49
ab
63
a
Compound
3
38
bc
36
b
Compound
4
34
c
23
c
a
Means
followed
by
the
same
letter
did
not
differ
by
the
least
significant
difference
test
at
P
-
0.05.
o
S._
0
a)
FIG.
ver
B.
436
NAIR
ET
AL.
VAM-STIMULATORY
COMPOUNDS
FROM
CLOVVR
ROOTS
437
I'
5
3'
2I',6'
5'
8
B
8.4
8.2
8.0
7.8 7.6
7.4
7.2
7.0
6.8
6.6
PPM
FIG.
4.
'H
NMR
spectra
(300
MHz)
for
clover
A
(A)
al
synthetic
formononetin
(B)
in
CD30D.
The
4'-methoxy
region
in
t:
spectrum
is
not
shown.
I
II
be
the
isomer
7-methoxy,4'-hydroxy
isoflavone.
Long-range
COSY
experiments
showed
a
strong,
clear
correlation
of
the
methoxy
group
to
the
H-3'
and
H-5'
protons
and
weaker
correlation
to
the
H-2'
and
H-6'
protons
(Fig.
5).
The
7-OH
group
(B
10.80)
gave
cross
peaks
with
the
H-5
proton
as
well.
The
7-methoxy,4'-hydroxy
isomer
of
formononetin
had
been
synthesized
earlier
(21),
and
its
spectral
characteristics
were
different
from
that
of
clover
A.
Our
13C
NMR
data
on
formononetin
(clover
A)
and
biochanin
A
(clover
B)
differ
slightly
from
the
published
data
(30)
since
we
used
only
CD30D
as
the
solvent
instead
of
the
CD30D-dimethyl
_
sulfoxide-d6
mixture.
Clover
B
was
identical
to
biochanin
A
in
every
respect.
Methylation
of
both
formononetin
and
biochanin
A
resulted
in
good
yields
of
the
permethylated
products,
i.e.,
compounds
3
and
4,
respectively.
It
was
important
to
prepare
the
methylated
products
of
these
com-
pounds
to
ascertain
the
structural
activity
relationship
to
VAM
stimulation.
Synthetic
formononetin
and
biochanin
A,
but
not
the
methylated
forms,
caused
a
significant
increase
in
VAM
root
colonization
and
growth
of
clover
plants.
The
reasons
why
methylation
eliminated
the
VAM-stimulatory
effects
of
these
isoflavonoids
cannot
be
determined
until
the
Lnd
biological
bases
of
the
effects
that
these
compounds
have
on
the
VAM
are
elucidated.
Formononetin
has
been
found
as
a
stress
metabolite
in
soybeans
(28)
and
in
the
roots
and
forage
of
several
legumes
(8,
16,
18,
41).
In
forage,
formononetin
concentrations
range
from
14
ppm
in
alfalfa
to
1,700
ppm
in
red
clover
(16).
This
compound
was
also
found
in
greater
quantities
in
clover
root
I
-2.0
-3.0
-4.0
-5.0
-6.0
E
a.
-7.0
a,
-8.0
-9.0
-10.0
-11.0
I
I
T
I
VI
11.0
10.0
9.0
8.0
7.0
6.0
5.0
4.0
3.0
2.0
PPM
FIG.
5.
Nuclear
Overhauser
effect
spectrum
for
clover
A
obtained
from
a
long-range
COSY
experiment
in
100%
dimethyl
sulfoxide-d6.
I
l
VOL.
57,
1991
APPL.
ENVIRON.
MICROBIOL.
extracts
than
was
any
other
nod
gene-inducing
flavone
(10).
Its
concentration
in
clover
plants
is
reduced
by
seedling
age,
light
intensity,
fertilization
(34,
35),
and
plant
pathogens.
VAM
have
been
shown
to
increase
accumulation
of
gly-
ceollin
and
other
isoflavonoids
but
not
biochanin
A
and
formononetin
(23,
24).
The
activity
of
these
two
isoflavonoid
compounds
on
fungal
growth
or
VAM
formation
has
never
been
studied,
but
they
are
known
to
be
active
on
other
fungi
(43).
Biochanin
A
has
been
shown
to
reduce
growth
of
a
Rhizoctonia
sp.
(44)
and
to
stimulate
growth
of
Penicillium
digitatium
(19).
Formononetin
effects
on
non-VAM
fungi
ranged
from
low
antifungal
activity
to
growth
stimulation
(9,
19).
Other
flavonoids
have
been
shown
to
enhance
growth
of
litter-decomposing
fungi
when
applied
at
a
concentration
of
5
ppm
(20).
More
recently,
the
flavonoids
hesperetin
and
apigenin
were
shown
to
stimulate
hyphalVrowth
of
the
VAM
fungus
Gigaspora
margarita,
but
their
enct
on
root
coloni-
zation
was
not
reported
(29).
Hesperetin
and
other
fla-
vonoids
are
also
considered
"xenognosins"
in
root-infecting
parasitic
weeds
(22);
this
association
resembles
a
mycor-
rhizal
association.
Since
both
formononetin
and
biochanin
A
were
found
in
clover
roots,
increased
hyphal
growth
in
vitro
by
six-
to
ninefold
over
controls,
and
increased
root
colonization,
these
compounds
may
function
as
signal
molecules
in
VAM
symbiosis.
The
stimulatory
effects
of
these
isoflavonoids
on
VAM
root
colonization
and
plant
growth
have
been
con-
firmed
in
other
studies
with
clover
and
other
nonlegumes
(37,
38)
and
are
VAM
mediated.
When
these
compounds
are
applied
in
soil
free
of
VAM
propagules,
they
show
no
stimulatory
effect
on
either
plant
growth
or
nodulation
(37).
In
general,
flavonoids
function
as
signal
molecules
in
Rhizo-
bium-legume
symbiosis
by
inducing
transcription
of
bacte-
rial
nodulation
genes
(33).
Nevertheless,
neither
formonon-
etin
nor
biochanin
A
is
a
good
inducer
of
nod
gene
activity
on
Rhizobium
leguminosarum
biovar
trifolii.
On
the
con-
trary,
they
may
act
as
anti-inducers
(10,
32).
Since
biochanin
A
and
formononetin
stimulate
growth
of
non-nodulating
plant
species
in
the
presence
of
VAM
(37,
38),
it
is
likely
that
they
promote
plant
growth by
stimulating
VAM
formation.
Mechanisms
by
which
these
isoflavonoids
stimulate
VAM
formation
cannot
be
determined
from
our
work.
Isofla-
vonoids
have been
suggested
to
alter
membrane
permeabil-
ity,
enzymatic
activity,
or
DNA
replication
(1)
in
other
fungi.
In
VAM,
the
isoflavonoids
may
act
as
(i)
the
plant
signal
that
stimulates
the
free-living
fungus
to
use
its
endogenous
spore
reserves
(4),
(ii)
inducers
for
genes
controlling
symbiosis
and
saprophytic
growth
repressed
as
the
result
of
coevolution
(7,
40),
and
(iii)
a
plant
signal
for
hyphal
differentiation
into
appressorium
and/or
arbuscule
(17)
as
in
the
case
of
parasitic
weeds
(22).
Nevertheless,
the
increase
in
root
colonization
may
simply
result
from
the
enhanced
hyphal
growth
which
may
increase
the
fungus-root
contact
that
is
essential
during
the
preinfection
phase
of
this
symbiosis.
These
results
along
with
those
presented
elsewhere
(37,
38)
constitute
evidence
that
the
isoflavonoids
formononetin
and
biochanin
A
are
involved
in
the
stimulatory
effects
of
clover
roots
towards
the
VAM.
However,
the
definitive
proof
for
the
involvement
of
these
compounds
as
signal
molecules
in
VAM
symbiosis
requires
further
biological
investigations.
ACKNOWLEDGMENTS
We
are
grateful
to
Sharon
Walton
and
Robert
Keller
for
technical
assistance.
We
also
thank
Marinos
Los
and
his
research
group
for
the
formononetin
sample
and
the
two-dimensional
COSY
NMR
spectrum
of
clover
B.
This
work
was
supported
in
part
by
a
grant
from
the
American
Cyanamid
Company
and
Michigan
State
University
Agricultural
Experiment
Station.
J.O.S.
is
a
recipient
of
a
scholarship
from
CNPq-Brazil.
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