Effects of root isoquinoline alkaloids from Hydrastis canadensis on Fusarium oxysporum isolated from Hydrastis root tissue


Tims, M.c.; Batista, C.

Journal of Chemical Ecology 33(7): 1449-1455

2007


Goldenseal (Hydrastis canadensis L.) is a popular medicinal plant distributed widely in North America. The rhizome, rootlets, and root hairs produce medicinally active alkaloids. Berberine, one of the Hydrastis alkaloids, has shown antifungal activity. The influence of a combination of the major Hydrastis alkaloids on the plant rhizosphere fungal ecology has not been investigated. A bioassay was developed to study the effect of goldenseal isoquinoline alkaloids on three Fusarium isolates, including the two species isolated from Hydrastis rhizosphere. The findings suggest that the Hydrastis root extract influences macroconidia germination, but that only the combined alkaloids--berberine, canadine, and hydrastine--appear to synergistically stimulate production of the mycotoxin zearalenone in the Fusarium oxysporum isolate. The Hydrastis root rhizosphere effect provided a selective advantage to the Fusarium isolates closely associated with the root tissue in comparison with the Fusarium isolate that had never been exposed to Hydrastis.

J
Chem
Ecol
(2007)
33:1449-1455
DOI
10.1007/s10886-007-9319-9
)MMUNICATION
Effects
of
Root
Isoquinoline
Alkaloids
from
Hydrastis
canadensis
on
Fusarium
oxysporum
Isolated
from
Hydrastis
Root
Tissue
Michael
C.
Tims
Charisma
Batista
Received:
21
March
2007
/Revised:
11
May
2007
/Accepted:
21
May
2007
/
Published
online:
5
June
2007
©
Springer
Science
+
Business
Media,
LLC
2007
Abstract
Goldenseal
(Hydrastis
canadensis
L.)
is
a
popular
medicinal
plant
distributed
widely
in
North
America.
The
rhizome,
rootlets,
and
mot
hairs
produce
medicinally
active
alkaloids.
Berberine,
one
of
the
Hydrastis
alkaloids,
has
shown
antifungal
activity.
The
influence
of
a
combination
of
the
major
Hydrastis
alkaloids
on
the
plant
rhizosphere
fungal
ecology
has
not
been
investigated.
A
bioassay
was
developed
to
study
the
effect
of
goldenseal
isoquinoline
alkaloids
on
three
Fusarium
isolates,
including
the
two
species
isolated
from
Hydrastis
rhizosphere.
The
findings
suggest
that
the
Hydrastis
root
extract
influences
macroconidia
germination,
but
that
only
the
combined
alkaloids—berberine,
canadine,
and
hydrastine—appear
to
synergistically
stimulate
production
of
the
mycotoxin
zearalenone
in
the
Fusarium
oxysporum
isolate.
The
Hydrastis
root
rhizosphere
effect
provided
a
selective
advantage
to
the
Fusarium
isolates
closely
associated
with
the
root
tissue
in
comparison
with
the
Fusarium
isolate
that
had
never
been
exposed
to
Hydrastis.
Keywords
Hydrastis
Goldenseal
Fusarium
F
oxysporum
F
solani
F
commune
Endophyte
Isoquinoline
alkaloid
Rhizosphere
Chemical
ecology
M.
C.
Tims
(M)
Department
of
Cell
Biology
and
Molecular
Genetics,
University
of
Maryland,
College
Park,
MD
20742,
USA
e-mail:
michael.tims@nist.gov
C.
Batista
F.
Hebert
School
of
Medicine,
Uniform
Services
University
of
Health
Sciences,
Bethesda,
MD
20814,
USA
Present
address:
M.
C.
Tims
Division
of
Analytical
Chemistry,
National
Institute
of
Standards
and
Technology,
100
Bureau
Drive,
Stop
8392,
Gaithersburg,
MD
20899-8392,
USA
4i
Springer
1450
J
Chem
Ecol
(2007)
33:1449-1455
Introduction
Goldenseal
(Hydrastis
canadensis
L.,
Ranunculaceae)
is
an
herbaceous
perennial
that
is
found
in
North
American
under
mesic
cove
forest
canopies
throughout
much
of
the
south
and
eastern
seaboard
and
north
into
Canada.
The
rhizome,
rootlets,
and
root
hairs
produce
medicinally
active
alkaloids,
including
the
major
alkaloids
berberine,
canadine,
and
(3-
hydrastine.
Because
of
the
alkaloids,
Hydrastis
is
an
important
dietary
supplement,
which
currently
retails
at
a
price
of
over
$160
per
pound.
Indigenous
use
of
Hydrastis
by
native
Americans
was
widespread
for
a
variety
of
infectious
diseases
(Moerman
1986).
Today,
the
rhizome
and
rootlets
are
used
as
an
antibacterial
treatment,
for
skin
infections,
conjunctivitis,
otitis
media,
and
urinary
tract
infections.
However,
the
plant
has
been
aggressively
harvested
from
its
native
setting
(wildcrafted)
and
is
now
listed
in
Appendix
II
of
the
Convention
on
International
Trade
in
Endangered
Species
(CITES).
Because
of
harvesting
pressures
and
loss
of
habitat,
increasingly
more
of
the
raw
material
for
medicinal
plant
use
originates from
cultivated
sources
rather
than
from
wildcrafted
plants.
Our
understanding
of
reciprocal
rhizosphere
influence
on
formation
of
clinically
relevant
plant
secondary
metabolites
is
woefully
inadequate.
We
have
little
data
on
how
fungi
in
the
plant
root
rhizosphere
may
alter
the
ratios
and
makeup
of
medicinally
important
plant
metabolites
or
whether
those
same
plant
compounds
present
in
root
exudates have
a
selective
effect
on
the
composition
of
the
fungal
rhizosphere
community.
Although
individual
alkaloids,
berberine
and
hydrastine,
have
been
reported
to
have
antifungal
activity
(Cernakova
and
Kostalova
2002;
Goel
et
al.
2003),
neither
Hydrastis
alkaloids
in
combination
nor
the
Hydrastis
whole
root
extract
have
been
investigated
for
effect
on
fungi.
This
study
explores
whether
the
level
of
association
between
Fusarium
and
Hydrastis
can
be
distinguished
by
the
fungal
responses
to
alkaloid
treatments
and
if
the
treatment
effects
are
the
products
of
additive
or
synergistic
interactions
of
two
or
more
Hydrastis
alkaloids.
Materials
and
Methods
Fungal
Isolation
Three
media
were
prepared,
Czapeks
Dox
containing
penicillin
G
(10,000
Mimi)
and
streptomycin
(10,000
µgimp,
water
agar
containing
the
same
anti-
biotics,
and
carnation
leaf
agar
(Sigma,
St.
Louis,
MO,
USA).
Wildcrafted
and
commercial
H.
canadensis
seeds
(Horizon
Herbs,
Williams,
OR,
USA)
were
sterilized
in
10%
Clorox
and
plated
on
media.
After
5
d
at
room
temperature,
transfers
were
made
to
potato
dextrose
agar
(PDA),
and
after
7-10
d,
the
fungi
were
identified.
Hydrastis
canadensis
leaf,
stem,
rhizome,
and
rootlet
tissue
were
surface
sterilized
with
10%
Clorox
and
plated
on
water
agar.
Isolates
were
continuously
transferred
until
pure
cultures
were
growing.
Fusarium
cultures
were
identified
by
the
Fusarium
Research
Center,
Department
of
Plant
Pathology,
Pennsylvania
State
University
(PSU).
By
using
a
modified
staining
method
(Koske
and
Gemma
1989),
H.
canadensis
root
and
seed
tissue
was
examined
directly
for
arbuscular
mycorrhizae
(AM).
To
detect
filamentous,
non-AM
fungal
growth
in
situ,
root
tissue
was
dipped
in
95%
ethanol
and
flamed
briefly.
Root
tissue
was
cut
into
5-mm
sections,
and
seeds
were
stripped
of
their
pericarp.
Lactophenol
blue
was
added
to
each
tissue,
and
tissue
were
examined
microscopically
(x40).
Hydrastis
canadensis
soil
samples
were
collected
from
soil
adhering
to
the
rootlets
of
H.
canadensis
plants
and
from
a
site
devoid
of
H.
canadensis
or
any
other
herbaceous
plant.
Both
soils
were
screened
also
for
the
presence
of
AM
reproductive
spores
by
using
screens
of
increasingly
smaller
mesh.
In
addition,
soil
slurries
were
prepared
and
added
to
the
three
isolation
media.
After
3
d,
fungi
were
transferred
to
PDA
'4
Springer
J
Chem
Ecol
(2007)
33:1449-1455
1451
tubes
and
identified.
Corn
kernels
were
added
directly
to
both
soil
samples
to
isolate
pathogenic
fungi.
After
14
d,
kernels
were
plated
on
water
agar
containing
antibiotics.
Fungal
transfers
were
made.
Stock
Hydrastis
Extraction
and
Standards
Hydrastis
canadensis
tissue
was
air-dried
and
ground
(20
mesh)
with
a
Wiley
mill.
Root
and
rhizome
samples
(100
mg)
were
defatted
with
50
ml
n-heptane
for
2
h
on
a
Labline
Orbital
Shaker
at
100
rpm
in
dark
brown
bottles.
Samples
were
filtered
(Whatman
#1)
and
then
dried
for
2
h.
Fifty
ml
50%
methanol:
1%
acetic
acid
were
added,
and
samples
were
placed
on
a
shaker
(100
rpm),
25°C,
24
h.
The
sample
was
filtered
and
re-extracted
twice.
The
three
filtrate
samples
were
combined,
filter
sterilized
(Nalgene
0.45
µm),
evaporated,
and
stored
in
a
dark
bottle
at
5°C.
A
voucher
specimen
of
H.
canadensis
(#05001ARZ)
is
stored
in
the
Norton
Brown
Herbarium,
University
of
Maryland,
College
Park,
MD,
USA.
Alkaloid
standard
stock
solutions
(w/v)
of
0.12%
berberine
dihydrochloride
(Chromadex,
Santa
Ana,
CA,
USA),
0.02%
canadine
(Chromadex,
Santa
Ana,
CA,
USA),
0.08%
hydrastinine
hydrochloride
(Sigma-Aldrich,
St.
Louis,
MO,
USA)
were
prepared
and
filter
sterilized.
Spore
Counts
Seven
treatments
were
added
in
1-ml
increments
to
125-ml
Erlenmeyer
flasks
wrapped
in
aluminum
foil:
three
individual
alkaloid
standards,
all
three
alkaloids
combined
into
a
standard
mix,
a
0.2%
(dry
w/v)
Hydrastis
root
extract
(05001ARZ),
a
control
of
50%
methanol
(1%
acetic
acid),
and
water.
A
spore
suspension
of
three
Fusarium
isolates
5001AR
(Fusarium
oxysporum,
from
Hydrastis
rootlet
tissue),
PSU
0-1174
(Fusarium
commune,
from
PSU)
and
9001
ARC
(Fusarium
solani,
from
Hydrastis
rhizosphere
soil)
was
prepared.
Inoculated
carnation
leafs
were
added
to
distilled
water,
filtered
through
sterile
cheesecloth,
and
resuspended
in
0.1%
Tween
20.
One
milliliter
(10
6
conidia)
of
each
isolate
was
added
to
flasks
containing
treatments
and
incubated
in
25
ml
of
Czapeks
Dox
medium
for
0,1,2,3,
and
4
d.
Hyphae
were
filtered,
lyophilized,
and
dry
weight
was
determined.
Filtrate
was
also
collected,
lyophilized,
and
stored
at
5°C.
Chlamydospore
formation
and
macroconidia
germination
rates
from
lyophilized,
4-d-old
mycelia
were
determined
with
a
Bright
line
hemocytometer
and
light
microscopy
(x40).
Mycotoxin
Analysis
Freeze-dried
filtrates
were
analyzed
by
using
enzyme-linked
irnmuno-
sorbent
assay
(ELISA;
Veratox,
Neogen,
Lansing,
MI)
to
determine
if
Hydrastis alkaloids
altered
Fusarium
mycotoxin
production.
Statistical
Analysis
The
data
of
isoquinoline
alkaloid
treatment
effects
on
the
fungal
response,
including
macroconidial
germination
and
mycotoxin
production,
were
normally
distributed;
therefore,
they
were
analyzed
with
a
paired
t-test.
Results
and
Discussion
Results
from
screening
Hydrastis
tissue
and
surrounding
soil
provide
limited
evidence
of
a
selective
rhizosphere
effect.
From
native
Hydrastis
plant
populations,
F.
oxysporum
was
isolated
from
disease-free
rootlet
and
seed
tissue,
whereas
Pythium
spp.
was
isolated
from
seed.
The
F.
oxysporum
isolate
was
microscopically
detected
intracellularly
and
localized
near
rootlet
tips.
In
addition,
coenocytic
hyphae
were
also
detected
in
the
same
Hydrastis
root
pericycle
tissue
from
which
F.
oxysporum
(05001AR)
isolation
occurred,
although
the
4i
Springer
1452
J
Chem
Ecol
(2007)
33:1449-1455
unknown
fungus
was
never
isolated
in
culture.
Microscopic
examinations
of
stained
Hydrastis
root
tissue
provided
no
evidence
for
AM
fungi,
nor
were
AM
chlamydospores
found
in
any
of
the
soil
collections.
Mesic
cove
forests
where
Hydrastis
was
collected
are
not
particularly
conducive
to
high
populations
of
AM
fungi.
Fusarium
solani
and
Pythium
sp.
were
present
in
Hydrastis
rhizosphere
soil.
Species
isolated
from
non-rhizosphere
soil
include
Rhizoctonia
solani
and
Gliocladium
spp.
Isolates
from
commercial
Hydrastis
tissue
include
F
solani
from
rootlet
and
leaf
tissue,
and
F
oxysporum
isolated
from
seed.
No
fungi
were
isolated
from
rhizome
or
stem
tissue
of
any
Hydrastis
sample.
Although
the
Hydrastis
rhizosphere
soil
does
not
drastically
alter
the
fungal
community,
the
presence
of
F
oxysporum
within
Hydrastis
root
tissue
suggests
that
elements
of
Hydrastis
root
exudates
may
influence
the
level
of
association
of
Fusarium
species
with
root
tissue.
To
model
Hydrastis/Fusarium
interactions
at
the
host
plant
root
interface,
an
assay
was
designed
based
on
the
assumptions
that
berberine,
canadine,
and
hydrastine
are
present
in
Hydrastis
root
exudates,
as
evidenced
by
plant
cell
culture
experiments
(Maddox
et
al.
1999).
The
whole
root
extract
was
chosen
to
model
the
complex
of
compounds
present
in
Hydrastis
root
exudates.
Analysis
with
liquid
chromatography-mass
spectroscopy
(data
not
shown)
indicated
that
the
whole
root
extract
contained
berberine,
canadine,
and
hydrastine.
Based
on
previous
Hydrastis
extraction
studies
(Galeffi
et
al.
1997;
Gentry
et
al.
1998;
McNamara
et
al.
2004),
minor
alkaloids
and
non-alkaloid
components
should
also
be
present.
Three
Fusarium
species
exhibiting
different
levels
of
association
with
Hydrastis
root
tissue
and
rhizosphere
were
identified.
Treatment
effects
on
Fusarium
growth
and
spore
formation
were
not
significant
for
the
three
species
(data
not
reported).
However,
macroconidia
germination
rates
represented
a
significant
response
factor,
as
summarized
in
Fig.
1.
All
individual
alkaloid
treatments
significantly
inhibited
F
oxysporum
macroconidia
germination
(75-100%),
whereas
the
whole
root
extract
treatment
stimulated
germination
(25%).
Fusarium
solani
germination
was
significantly stimulated
by
the
whole
root
extract
(200%),
whereas
only
the
alkaloid
standard
treatment
inhibited
spore
germination
(75%).
In
contrast,
the
germination
rate
of
the
non-Hydrastis
isolate,
F.
commune,
was
inhibited
by
all
treatments;
especially
by
the
whole
root
extract
treatments
(40%).
The
components
responsible
for
the
inhibition
of
macroconidia
germination
of
the
Hydrastis
isolates
appear
to
be
berberine
and
canadine.
None
of
the
major
alkaloid
compound(s)
found
in
the
whole
root
extract
stimulated
any
isolate,
whereas
only
the
Hydrastis
isolates
were
stimulated
by
the
whole
root
extract.
The
results
suggest
that
non-alkaloid
components
and/or
minor
alkaloids
were
stimulatory
singly
or
synergistically,
although
additional
experiments
are
required
to
be
certain
of
the
active
component.
Because
disease
symptoms
were
absent
in
the
Hydrastis
root
tissue
from
which
the
F.
oxysporum
was
isolated,
this
suggests
that
the
isolate
is
nonpathogenic
and
may
be
endophytic.
Gunawardena
et
al.
(2005)
noted
that
Pisum
sativa
(pea)
root
exudates
stimulated
macroconidia
germination
in
F
solani
species
complex
(Nectria
haematococca)
in
a
host-specific
pattern
and
influenced
the
establishment
of
the
endophytic
relationship.
Although
the
F
commune
isolate
exhibited
the
highest
macroconidia
germination
rates,
only
the
Hydrastis
isolates
responded
positively
to
the
whole
root
extract
treatment
by
increasing
germination
rates.
Growth
within
Hydrastis
root
tissue
occurred
only
with
the
F
oxysporum
isolate,
suggesting
that
signal
compounds
present
in
the
extract
may
be
influencing
the
endophytic
status.
Changes
to
the
cultural
media
pH
can
influence
fungal
response.
Both
F
commune
and
F
solani
exhibited
a
similar
pattern
of
pH
change
with
treatments,
except
that
the
changes
in
the
pH
values
for
F
commune
whole
root
extract
and
methanol
treatments
lagged
slightly
(data
not
shown).
The
change
in
pH
values
for
F
oxysporum
whole
root
extract
and
methanol
treatments
were
similar
to
the
F
commune
isolate.
However,
the
initial
pH
values
'4
Springer
F.
oxysporum
MI
F.
commune
F.
solani
70.0%
60.0%
50.0%
40.0%
30.0%
20.0%
-
10.0%
-
0.0%
whole
plant
he
rberine
ran
ad
i
ne
hyd
rasiine
alkaloids
methanol
J
Chem
Ecol
(2007)
33:1449-1455
1453
%
maeroett
n
idia
l
g
erm
in
a
t
ion
Fig.
1
Isoquinoline
alkaloid
effect
on
germination
of
macroconidia
by
three
Fusarium
spp.
grown
in
Czapeks
Dox
liquid
media.
Treatments—berberine
0.12
mg
ml-1,
canadine
0.02
mg
ml-1,
hydrastine
0.08
mg
ml-1,
and
whole
root
extract
2.00
mg
ml—1—were
dissolved
in
1
ml
of
50%
methanol/1%
acetic
acid
solvent
and
added
to
25
ml
of
culture
medium
containing
1.0
ml
fungal
inoculum
(approx.
106
conidia
m1-1).
Samples
(N=12)
expressed
per
mg
of
dry
weight
mycelia
on
d
4
of
the
assay
(error
bars
the
standard
deviation
of
the
error,
*P<0.05,
**P<0.01,
paired
t-test)
of
individual
alkaloid
treatments
were
higher
than
the
other
isolates
(pH
6.5
vs.
pH
4.75),
and
remained
fairly
constant
during
the
study.
It
is
unclear
which
element
was
buffering
the
media—the
basicity
of
alkaloid
treatments
or
the
response
of
the
endophytic
isolate.
In
either
case,
treatment
replicates
were
randomized
with
respect
to
both
fungus
and
treatment,
which
suggests
that
the
pH
pattern
exhibited
is
unique
to
the
endophytic
isolate
and
is
not
because
of
differences
in
pH
between
alkaloid
medium
and
the
control.
Production
and
excretion
of
mycotoxins
might
also
play
a
role
during
interactions
at
the
rhizoplane.
All
Fusarium
isolates
were
screened
for
mycotoxins
at
the
end
of
the
growth
phase
on
d
4
(Table
1).
Ueno
et
al.
(1977)
reported
that
the
F.
commune
isolate
was
capable
of
producing
the
mycotoxin
zearalenone
(ZON)
in
culture.
The
results
indicate
that
the
mycotoxin
was
present
in
the
water
treatment
during
our
assay.
All
other
treatments,
particularly
berberine,
canadine,
and
whole
root
extract,
inhibited
the
production
of
ZON
in
the
F.
commune
isolate,
which
did
not
produce
any
other
mycotoxin.
The
F.
solani
isolate
did
not
produce
detectable
mycotoxin
levels
in
any
treatment.
All
alkaloid
treatments
were
inhibitory
to
fumonisin
production,
especially
berberine.
Canadine
significantly
stimulated
T-2
mycotoxin
production
by
the
F.
oxysporum
endophyte,
whereas
hydrastine
had
less
of
a
stimulatory
effect.
The
alkaloid
standard
treatment
significantly
stimulated
production
of
ZON
by
F.
oxysporum.
The
whole
root
extract,
canadine,
and
methanol
treatments
had
no
effect,
whereas
berberine
and
hydrastine
treatments
had
an
inhibitory
effect
on
ZON
production.
Because
the
ratios
of
major
alkaloids
were
approximately
equal
between
the
Springer
1454
J
Chem
Ecol
(2007)
33:1449-1455
Table
l
Mycotoxin
production
by
Fusarium
in
media
containing
isoquinoline
alkaloids
Fungus
Treatment
Mycotoxin
T-2
fumoni
sin
ZON
...
.
.
-
r
°
..
0
7
^.
V
.
.
,
F.
oxysporum
whole
root
extract
0.05
0.00*
1.95
berberine
0.06
0.00* 0.00*
canadine
0.24*
1.05
1.87
hydrastine
0.11
3.98
0.00*
alkaloid
mix
0.04
3.17
7.61*
methanol
0.00
5.77
3.03
water
0.00
3.19
0.00
F.
commune
whole
root
extract
0.00
0.00
0.00*
berberine
0.00 0.00
0.00*
canadine
0.00 0.00
0.00*
hydrastine
0.00
0.00
1.07
alkaloid
mix
0.00 0.00
2.32
methanol
0.00 0.00
1.30
water
0.00
0.00
6.57
Correlation
coefficient
of
calibration
curves
(P
<
0.01)
0.9863
0.9984
0.9996
The
data
for
this
graph
was
collected
from
the
same
experiment
as
Fig.
1.
Sample
measurements
(N=3)
were
taken
of
mycelia
filtered
from
culture
on
d
4
using
Neogen
ELISA
fluorescence
plate
reader.
Results
expressed
as
ng
metabolite/mg
dry
weight
mycelia.
*P<0.05,
paired
t-test
whole
root
extract
and
alkaloid
standard
treatments,
the
results
suggest
a
synergistic
effect
among
the
three
alkaloids
that
was
able
to
overcome
the
inhibitory
effects
of
berberine
and
hydrastine
in
the
alkaloid
standard
treatment
but
not
in
the
whole
root
extract
treatment.
It
is
possible
that
minor
alkaloids
or
non-alkaloid
components
of
the
whole
root
extract
prevented
the
synergistic
interaction.
Overall,
mycotoxin
mass
values
were
near
the
limits
of
detection
for
the
Neogen
ELISA
kit
used
in
this
study.
However,
preliminary
screening
indicated
that
the
F.
oxysporum
endophyte
is
metabolically
more
active
in
producing
mycotoxins
and,
thus,
may
be
more
competitive
with
other
rhizosphere
fungi
at
the
Hydrastis
rhizoplane.
Springer
J
Chem
Ecol
(2007)
33:1449-1455
1455
As
cultivated
Hydrastis
roots
are
used
more
frequently
for
the
manufacture
of
commercial
herbal
medicine
rather
than
wildcrafted
sources,
future
work
is
required
to
understand
if
the
endophytic
status
of
F.
oxysporum
alters
the
alkaloid
content
and
ratios
of
Hydrastis
root
tissue.
This
study
provides
the
first
evidence
that
medicinally
active
Hydrastis
alkaloids
influence
Fusarium
isolates
in
the
rhizosphere.
Acknowledgements
I
acknowledge
Paul
Strauss,
for
permission
to
wildcraft
goldenseal
on
United
Plant
Savers
land
and
Alex
Johnson
for
contributions
to
the
ELISA
detection.
This
study
is
part
of
the
Ph.D.
dissertation
research
undertaken
by
Michael
C.
Tims.
References
CERNAKOVA,
M.,
and
KOSTALOVA,
D.
2002.
Antimicrobial
activity
of
berberine—a
constituent
of
Mahonia
aquifolium.
Folia
Microbiol.
(Praha)
47:375-378.
GALEFFI,
C.,
COMETA,
M.
F.,
TOMASSINI,
L.,
and
NicoLETE,
M.
1997.
Canadinic
acid:
an
alkaloid
from
Hydrastis
canadensis.
Planta
Med.
63:194.
GENTRY,
E.
J.,
HANUMAN,
J.
B.,
KESHAVARZ-SHOKRI,
A.,
MORTON,
M.
D.,
VELDE,
D.
V.,
TELIKEPALLI,
H.,
and
MrrscHER,
L.
A.
1998
Antitubercular
natural
products:
berberine
from
the
mots
of
commercial
Hydrastis
canadensis
powder.
Isolation
of
inactive
8-oxotetrahydrothalifendine,
canadine,
13-hydrastine,
and
two
new
quinic
acid
esters,
hycandinic
acid
esters-1
and
-2.
J.
Nat.
Prod.
61:1187-1193.
GOEL,
M.,
SINGH,
U.
P.,
JHA,
R.
N.,
PANDEY,
V.
B.,
and
PANDEY,
M.
B.
2003.
Individual
and
combined
effect
of
(+/—)-alpha-hydrastine
and
(+/—)-beta-hydrastine
on
spore
germination
of
some
fungi.
Folia
Microbiol.
(Praha)
48:363-368.
GUNAWARDENA,
U.,
RODRIGUEZ,
M.,
STRANEY,
D.,
ROMEO,
J.
T.,
VANETTEN,
H.
D.,
and
HAWES,
M.
C.
2005.
Tissue-specific
localization
of
pea
root
infection
by
Nectria
haematococca.
Mechanisms
and
consequences.
Plant
Physiol.
137:1363-1374.
KOSICE,
R.
E.,
and
GEMMA,
J.
N.
1989.
A
modified
procedure
for
staining
mots
to
detect
VA-mycorrhizas.
Mycol.
Res.
92:486-505.
MADDOX,
R.
M.,
ZELDIN,
E.
L.,
and
MCCOWN,
B.
H.
1999.
Use
of
nodule
cultures
grown
in
bioreactors
for
mass
propagation
of
goldenseal.
In
Vitro
Cell
Dev.
Biol.
35:51-52.
MCNAMARA,
C.,
PERRY,
N.
B.,
Fou,Err,
J.
M.,
PARMENTER,
G.
A.,
and
DOUGLAS,
J.
A.
2004.
A
new
glucosyl
feruloyl
quinic
acid
as
a
potential
marker
for
mots
and
rhizomes
of
goldenseal,
Hydrastis
canadensis.
I
Nat.
Prod.
67:1818-1822.
MOERMAN,
D.
E.
1986.
Medicinal
plants
of
native
America,
vol.
1.
Reg.
Univ.
Mich.
Museum
Anthropol.,
Ann
Arbor,
pp.
910.
UENO,
Y.,
ISwI,
K.,
SAWANO,
M.,
OHTSUBO,
K.,
and
MATSUDA,
Y.
1977.
Toxicological
approaches
to
the
metabolites
of
fusaria.
XI
trichothecenes
and
zearalenone
from
Fusarium
species
isolated
from
river
sediment.
Jpn.
J.
Exp.
Med.
47:177-184.
Springer