Species and varieties in the Rhizopus arrhizus-Rhizopus oryzae group as indicated by their DNA complementarity


Ellis, J.J.

Mycologia 77(2): 243-247

1985


Results of DNA renaturation experiments indicated that the 11 strs. tested can be accommodated in 3 taxa: R. arrhizus var. arrhizus, R. a. var. rouxii and R. a. var. delemar. Two isolates referred to R. oryzae (including the presumed type) showed > 95% relatedness with R. arrhizus var. arrhizus. R. arrhizus is retained as it has date precedence over R. oryzae.

Mycologia,
77(2),
1985,
pp.
243-247.
©
1985,
by
The
New
York
Botanical
Garden,
Bronx,
NY
10458
SPECIES
AND
VARIETIES
IN
THE
RHIZOPUS
ARRHIZUS
-
RHIZOPUS
OR
YZAE
GROUP
AS
INDICATED
BY
THEIR
DNA
COMPLEMENTARITY
J.
J.
ELLis
Northern
Regional
Research
Center,
Agricultural
Research
Service,
U.S.
Department
of
Agriculture,'
Peoria,
Illinois
61604
ABSTRACT
DNA
renaturation
experiments
gave
evidence
to
conclude
that
Rhizopus
arrhizus,
R.
oryzae,
R.
delemar,
Amylomyces
rouxii,
R.
delemar
var.
minimus,
R.
delemar
var.
multiplici-
sporus,
R.
arrhizus
var.
delemar,
R.
chungkuoensis
var.
isofermentarius,
and
R.
javanicus
var.
kawasakiensis
can
be
accommodated
in
three
taxa,
namely,
R.
arrhizus
var.
arrhizus,
R.
arrhizus
var.
rouxii,
and
R.
arrhizus
var.
delemar.
Key
Words:
Rhizopus,
DNA
renaturation,
taxonomy.
Numerous
attempts
have
been
made
to
clarify
the
species
concepts
of
Rhi-
zopus,
with
primary
emphasis
on
a
single
or
relatively
few
morphological
or
physiological
features
(2,
3,
10,
15).
Some
workers
attempted
to
rearrange
existing
data,
using
relatively
newer
techniques,
with
either
weighted
or
nonweighted
characteristics
to
place
strains
in
appropriate
species
and
to
show
relationships
(1).
Traditionally,
we
have
weighted
heavily
characteristics
of
the
sexual
stage
over
other
characteristics
exhibited
by
a
species.
Problems
arise
when,
as
is
the
case
for
many
species
of
Rhizopus,
the
sexual
stage
is
unknown
or
is
rarely
produced.
Thus,
we
have
depended
on
morphological
characteristics
of
the
spor-
angial
apparatus
and
attempted
to
support
our
viewpoint
with
physiological
data.
The
lack
of
definitive
information
on
the
numerous
described
species
of
Rhizopus
has
led
to
considerable
difficulty
in
determining
the
extent
of
variability
within
a
species
and
how
the
species
can
be
readily
distinguished.
In
particular,
it
has
been
most
difficult
to
attach
names
to
strains
belonging
in
the
Rhizopus
arrhizus—
R.
oryzae—R.
delemar
group
of
species
and
varieties.
To
delimit
the
taxa
and
the
morphological
characteristics
that
will
help
place
strains
in
those
taxa,
deoxyri-
bonucleic
acid
(DNA)
renaturation
studies
were
performed.
MATERIALS
AND
METHODS
Fungal
cultures.
The
fungal
strains
(TABLE
I)
used
in
this
study
are
maintained
in
the
Agricultural
Research
Service
Culture
Collection
(NRRL)
at
the
Northern
Regional
Research
Center.
Stock
cultures
were
grown
on
either
potato
dextrose
agar
(PDA)
or
yeast-malt
agar
(YM).
These
media
are
described
as
M-20
and
M-69,
respectively,
in
the
Mycological
Guidebook
(12).
For
DNA
extraction,
cultures
were
grown
in
YM
broth
(400
ml
per
1
liter
Erlenmeyer
flask)
for
30
h
on
a
reciprocal
shaker
at
28
C.
Cultures
were
harvested
by
filtering
with
suction
and
were
suspended
in
the
spermidine-spermine-sucrose
buffer
of
Morris
(as
cited
by
Timberlake,
14).
DNA
purification.
—DNA
was
extracted
and
purified
by
modifying
Marmur's
method
(8)
with
the
use
of
hydroxylapatite
(Bio-Gel
HTP,
Bio-Rad
Laboratories)
1
The
mention
of
firm
names
or
trade
products
does
not
imply
that
they
are
endorsed
or
rec-
ommended
by
the
U.S.
Department
of
Agriculture
over
other
firms
or
similar
products
not
mentioned.
243
244
MYCOLOGIA
TABLE
I
SOURCE
OF
STRAINS
NRRL
1469
Rhizopus
arrhizus
Fischer.
Received
from
Blakeslee
collection
as
C
606
in
June,
1940.
Blakeslee's
notes
say
"Mucor
arrhizus
(=Rhiz.
arrhizus)
evidently
fr.
Cen-
trastelle."
(sic)
NRRL
1472
R.
delemar
(Boid.)
Wehmer
&
Hanz.
Received
from
Blakeslee
collection
as
C
608
in
June,
1940.
Blakeslee's
notes
say
"prob.
fr
.
Centralstelle."
NRRL
2871
R.
javanicus
var.
kawasakiensis
Takeda
&
Takamatsu.
Received
from
Institute
for
Fermentation,
Osaka,
as
IFO
4801
Type,
in
June,
1960.
NRRL
2872
Rhizopus
delemar
var.
minimus
Takeda.
Received
from
Institute
for
Fermentation,
Osaka,
as
Takeda
7301,
in
June,
1960.
Isolated
from
Indonesian
ragi.
NRRL
2873
R.
chungkuoensis
var.
isofermentarius
Takeda.
Received
from
Institute
for
Fermen-
tation,
Osaka,
as
IFO
4726
Type,
in
June,
1960.
NRRL
2915
R.
microsporus
v.
Tiegh.
Received
from
H.
Schoch,
University
of
Basel,
in
December,
1960.
Originally
from
Centraalbureau
voor
Schimmelcultures.
NRRL
3133
R.
oryzae
Went
&
Geerligs.
Received
from
Centraalbureau
voor
Schimmelcultures
in
May,
1964,
as
Went
strain
and
presumed
type
strain.
NRRL
3142
R.
oryzae
Went
&
Geerligs.
Isolated
from
Chinese
yeast
obtained
from
the
Taiwan
Sugar
Co.
in
April,
1964.
NRRL
3258
R.
delemar
var.
multiplicisporus
Inui,
Takeda
&
Iizuka.
Received
from
H.
Iizuka,
University
of
Tokyo,
as
IAM
6258,
in
January,
1967.
NRRL
5129
R.
arrhizus
var.
delemar
Mauvernay,
Laboureur
&
Labrousse.
Received
from
Mu-
seum
of
Nat.
Hist.
Paris
as
No.
1916,
in
September,
1970.
No
Latin
diagnosis
in
U.S.
Patent
No.
3,513,073
of
May
19,
1970.
NRRL
5866
Amylomyces
rouxii
Calmette.
Neotype
strain
isolated
January,
1974,
from
Look
Pang
(sweet
fermented
glutinous
rice)
made
in
Thailand.
chromatography
(7).
Purity
of
DNA
preparations
was
checked
by
their
absorbancy
ratios
at
230/260
nm
and
260/280
nm
as
well
as
by
their
thermal
denaturation
profiles.
DNA
renaturation.
—The
extent
of
DNA
relatedness
was
measured
spectropho-
tometrically
as
described
by
Seidler
and
Mandel
(11)
and
Kurtzman
et
al.
(4).
These
workers
have
shown
that
results
from
this
method
are
comparable
to
relatedness
values
obtained
using
radioisotope
techniques.
The
main
steps
in
the
spectrophotometric
techniques
are
the
following.
Purified
DNA
was
sheared
by
two
passages
through
a
French
press
cell
at
10,000
psi.
Following
filtration
through
a
micropore
filter
(0.45
Am,
from
Millipore®
Corp.),
the
sheared,
double-stranded
DNA
was
dialyzed
(Spectrapore
membrane
tubing
with
12,000
mol
wt
cut-off)
at
3-5
C
for
3
da
against
0.001x
SSC
(SSC
=
standard
saline
citrate,
0.15
M
NaC1,
0.015
M
trisodium
citrate,
pH
7.0)
with
0.001
M
EDTA.
The
DNA
solution
was
freeze-dried
and
redissolved
in
deionized-distilled
water.
DNA
renaturations
were
monitored
in
a
Gilford
260
recording
spectrophotometer
equipped
with
an
elec-
tronically-heated,
four-place
cuvette
holder.
The
reactions
were
carried
out
in
5x
SSC-20%
DMSO
with
75
µg
DNA
per
ml
for
a
single
strain
or
a
mixture
of
37.5
µ
g
DNA
for
each
of
the
paired
strains,
i.e.,
four
fused
quartz
cuvettes
were
used:
one
contained
only
water,
SSC,
and
DMSO
(blank);
two
contained
DNA
of
one
strain
each;
and
the
fourth
contained
the
DNA
mixture
of
the
two
strains.
Pre-
ceding
renaturation,
the
DNA
was
denatured
by
raising
the
temperature
to
90
C
and
holding
the
reaction
mixtures
at
that
temperature
for
10
min.
Then
the
temperature
was
lowered
3
C/min
until
it
reached
the
incubation
temperature
of
55
C.
This
temperature
corresponds
to
T
n
.,
25
C
which
was
determined
experi-
ELLIS:
RHIZOPUS
DNA
245
TABLE
II
REASSOCIATION
OF
NUCLEAR
DNA
IN
SPECIES
OF
Rhizopus
NRRL
No.
Name
2915b
R.
microsporus
5866
Amylomyces
rouxii
1469
R.
arrhizus
var.
arrhizus
3133
R.
oryzae
3142
R.
oryzae
1472'
3142
3133
1469
5866
2915b
18'
26
24
19
25
100
65
96
96
95
65
97
97
60
97
65
a
R.
delemar
var.
delemar.
b
Two
different
fermentations
and
extractions
of
DNA
for
this
strain.
Average
per
cent
of
2-3
reassociations.
mentally
in
the
reaction
buffer.
The
A
260
readings
for
the
four
cuvettes
were
recorded
successively
at
10
sec
intervals
for
the
4-9
h
necessary
to
reach
C0t
.
5.
Two
to
three
determinations
were
made
for
each
pairing.
Per
cent
renaturation
was
calculated
by
Seidler
and
Mandel's
method
(11)
using
the
C0t
.
5
values.
Determination
of
base
composition.
The
guanine
plus
cytosine
(G+
C)
content
of
the
nuclear
DNA
preparations
was
calculated
from
2-3
buoyant
density
de-
terminations
in
cesium
chloride
gradient
(20
h,
44,000
rpm)
(9,
13).
Measurements
were
made
in
a
Spinco
model
E
analytical
ultracentrifuge
equipped
with
an
elec-
tronic
scanner.
RESULTS
AND
DISCUSSION
A
summary
of
the
results
of
DNA
renaturation
experiments
of
strains
selected
as
most
representative
of
Rhizopus
microsporus,
R.
arrhizus,
the
presumed
type
of
R.
oryzae,
a
second
strain
of
R.
oryzae,
and
the
neotype
strain
of
Amylomyces
rouxii
are
presented
in
TABLE
II.
Rhizopus
microsporus
was
chosen
to
be
"the
obvious
distinct
species."
The
extent
of
DNA
relatedness
of
this
species
with
other
strains
listed
in
TABLE
II
is
quite
low,
and
no
pair
shows
more
than
26%
complementarity.
When
DNA
from
the
strains
of
A.
rouxii,
R.
arrhizus
var.
arrhizus,
and
R.
oryzae
were
interacted,
all
showed
more
than
95%
relatedness.
The
first
clue
that
strain
NRRL
1472
R.
delemar
was
somewhat
different
in
its
DNA
make-up
is
shown
by
its
60-65%
complementarity
with
the
aforementioned
strains
that
gave
high
relatedness
with
one
another.
Further
indication
is
seen
in
TABLE
III,
where
results
are
given
of
renaturations
between
strains
of
R.
delemar
var.
delemar
and
R.
arrhizus
var.
'arrhizus
with
strains
of
varieties
thought
to
De
synonyms
of
R.
delemar
var.
delemar
on
a
morphological
basis.
Indeed,
these
TABLE
III
REASSOCIATION
OF
NUCLEAR
DNA
FOR
STRAINS
OF
Rhizopus
delemar
NRRL
No.
Name
NRRL
1472'
NRRL
1469b
85'
71
89
67
100
70
96
73
95
73
2872
R.
delemar
var.
minimus
3258
R.
delemar
var.
multi
plicisporus
5129
R.
arrhizus
var.
delemar
2873
R.
chungkuoensis
var.
isofermentarius
2871
R.
javanicus
var.
kawasakiensis
a
R.
delemar
var.
delemar.
b
R.
arrhizus
var.
arrhizus.
Average
per
cent
of
2-3
reassociations.
246
MYCOLOGIA
TABLE
IV
GUANINE
PLUS
CYTOSINE
CONTENT
OF
NUCLEAR
DNA
FROM
Rhizopus
STRAINS
NRRL
No.
Name
G
+
C
(mol%)
1469
R.
arrhizus
36.6
(±0.22)a
3133
R.
oryzae
36.7
(±0.21)
1472
R.
delemar
36.1
(±0.07)
5866
Amylomyces
rouxii
35.5
(±0.12)
2915
R.
microsporus
43.9
(±0.35)
a
Mean
of
2-3
determinations
standard
deviation).
so-called
varieties
gave
at
least
85%
complementarity
with
strain
NRRL
1472
R.
delemar
var.
delemar,
whereas
they
gave
at
least
10%
less
complementarity
with
strain
NRRL
1469
R.
arrhizus
var.
arrhizus.
The
results
of
a
determination
of
G+C
content
of
the
DNA
from
pertinent
strains
are
shown
in
TABLE
IV.
The
only
obvious
distinction
noted
is
that
for
R.
microsporus.
All
other
G+C
ratios
fall
within
a
1.2
mol%
range,
consistent
with
their
demonstrated
conspecificity.
With
the
exception
of
R.
microsporus,
it
is
evident
from
the
data
presented
that
one
is
examining
the
DNA
of
a
single
species,
if
one
follows
the
lead
of
workers
investigating
yeasts.
Strains
of
some
heterothallic
species
of
yeasts
have
indicated
genetic
exchange
when
complementarity
has
been
as
low
as
25%
(5).
This
viewpoint
is
acceptable
on
a
morphological
basis
in
the
current
investigation
because
R.
arrhizus
has
quite
a
wide
morphological
variation
in
a
number
of
its
characteristics,
namely,
columellae,
rhizoidal,
sporangial,
sporangiophore,
and
sporangiospore
size
and
shape.
Because
of
the
wide
range
of
variation
it
seems
logical
to
retain
the name
R.
arrhizus
Fischer,
even
though
Fischer
(2)
stressed
the
applanate
nature
of
the
columella
in
this
species
(R.
arrhizus,
described
in
1892,
has
date
precedence
over
R.
oryzae
Went
&
Geerl.,
described
in
1895).
Many
strains
seen
by
the
author
in
culture
produced
at
least
a
few
columellae
that
were
more
broad
than
high,
al-
though
not
consistently
so.
Also,
most
recent
workers
have
accepted
R.
nodosus
as
a
synonym
of
R.
oryzae,
yet
it
has
been
described
as
having
applanate
columellae
(see
Lendner,
6,
fig.
45).
Although
the
neotype
strain
of
A.
rouxii
gave
a
very
high
percentage
of
reas-
sociation
with
strains
of
R.
arrhizus
and
should
probably
be
a
part
of
the
type
variety
on
this
basis,
the
author
proposes
to
retain
the
taxon
as
a
variety
for
convenience
because
(1)
it
is
propagated
primarily
by
hand
in
association
only
with making
fermented
ragi
materials,
(2)
it
has
not
been
isolated
from
nature
insofar
as
the
author
can
determine,
and
(3)
strains
can
be
readily
identified
on
a
morphological
basis.
The
probable
type
strain
of
R.
delemar,
namely
NRRL
1472,
as
well
as
the
strains
listed
in
TABLE
III,
have
sporangiospores
distinctly
larger
than
strains
of
R.
arrhizus
var.
arrhizus.
They
can,
therefore,
be
relegated
to
varietal
status
on
a
morphological
basis.
The
amount
of
complementarity
of
these
strains
with
strain
NRRL
1472
and
with
NRRL
1469
R.
arrhizus
var.
arrhizus
indicates
that
the
strains
listed
in
TABLE
III
are
synonymous
with
strain
NRRL
1472.
They
have
DNA
like
R.
arrhizus
enough
to
be
the
same
species,
yet
have
lower
(ca.
65%)
DNA
complementarity
with
representative
strains
as
well
as
morphological
dif-
ferences
to
classify
them
as
variety.
In
conclusion,
the
author
proposes:
(1)
that
the
name
R.
arrhizus
var.
arrhizus
Fischer
be
retained
with
NRRL
1469
designated
as
the
neotype
strain;
(2)
that
R.
oryzae
Went
&
Geerl.
be
reduced
to
synonymy
under
R.
arrhizus;
(3)
that
A.
ELLIS:
RHIZOPUS
DNA
247
rouxii
be
reduced
to
R.
arrhizus
var.
rouxii
(Calmette)
Ellis
with
the
basionym
A.
rouxii
Calmette.
Ann.
Inst.
Pasteur
6:
611,
1892,
and
(4)
that
R.
delemar
be
reduced
to
R.
arrhizus
var.
delemar
(Wehmer
&
Hanz.)
Ellis
with
the
basionym
R.
delemar
Wehmer
&
Hanz.,
in
Hanzawa-Mykol.
Zentbl.
1:
77,
1912.
ACKNOWLEDGMENTS
I
thank
C.
P.
Kurtzman
for
helpful
guidance
and
suggestions
with
many
aspects
of
the
DNA
experiments
and
M.
Mahoney
and
S.
Janson
for
technical
assistance.
LITERATURE
CITED
1.
Dabinett,
P.
E.,
and
A.
M.
Wellman.
1973.
Numerical
taxonomy
of
the
genus
Rhizopus.
Canad.
J.
Bot.
51:
2053-2064.
2.
Fischer,
A.
1892.
Phycomycetes.
In:
Rabenhorst's
Kryptogamenflora,
Die
Pilze,
IV.
505
p.
3.
Inui,
T.,
Y.
Takeda,
and
H.
Iizuka.
1965.
Taxonomical
studies
on
genus
Rhizopus.
J.
Gen.
Appl.
Microbiol.,
Tokyo
(Suppl.)
Vol.
11.121
p.
4.
Kurtzman,
C.
P.,
M.
J.
Smiley,
C.
J.
Johnson,
L.
J.
Wickerham,
and
G.
B.
Fuson.
1980.
Two
new
and
closely
related
heterothallic
species,
Pichia
ainylophila
and
Pichia
mississippiensis:
characterization
by
hybridization
and
deoxyribonucleic
acid
reassociation.
Int.
J.
Syst.
Bac-
teriol.
30:
208-216.
5.
,
H.
J.
Phaff,
and
S.
A.
Meyer.
1983.
Nucleic
acid
relatedness
among
yeasts.
Pp.
139-
166.
In:
Yeast
genetics.
Fundamental
and
applied
aspects.
Eds.,
J.
F.
T.
Spencer,
D.
M.
Spencer,
and
A.
R.
W.
Smith.
Springer-Verlag.
6.
Lendner,
A.
1908.
Les
Mucorinees
de
la
Suisse.
K.-J.
Wyss,
Berne.
180
p.
7.
Markov,
G.
G.,
and
I.
G.
Ivanov.
1974.
Hydroxyapatite
column
chromatography
in
procedures
for
isolation
of
purified
DNA.
Anal.
Biochem.
59:
555-563.
8.
Marmur,
J.
1961.
A
procedure
for
the
isolation
of
deoxyribonucleic
acid
from
microorganisms.
J.
Mol.
Biol.
3:
208-218.
9.
Schildkraut,
C.
L.,
J.
Marmur,
and
P.
Doty.
1962.
Determination
of
the
base
composition
of
deoxyribonucleic
acid
from
its
buoyant
density
in
CsCl.
J.
Mol.
Biol.
4:
430-433.
10.
Scholer,
H.
J.,
E.
Muller,
and
M.
A.
A.
Schipper.
1983.
Mucorales.
Pp.
9-59.
In:
Fungi
pathogenic
for
humans
and
animals.
Part
A,
Biology.
Ed.,
D. H.
Howard.
Mycol.
Ser.
3.
Marcel
Dekker,
Inc.,
New
York.
11.
Seidler,
R.
J.,
and
M.
Mandel.
1971.
Quantitative
aspects
of
deoxyribonucleic
acid
renaturation:
base
composition,
state
of
chromosome
replication
and
polynucleotide
homologies.
J.
Bacteriol.
106:
608-614.
12.
Stevens,
R.
B.
(Ed.)
1974.
Mycological
guidebook.
Univ.
Washington
Press,
Seattle.
703
p.
13.
Szybalski,
W.
1968.
Use
of
cesium
sulfate
for
equilibrium
density
gradient
centrifugation.
Methods
Enzymol.
12B:
330-360.
14.
Timberlake,
W.
E.
1978.
Low
repetitive
DNA
content
in
Aspergillus
nidulans.
Science
202:
973-975.
15.
Zycha,
H.,
R.
Siepmann,
and
G.
Linnemann.
1969.
Mucorales.
Eine
Beschreibung
aller
Gat-
tungen
and
Arten
dieser
Pilzgruppe.
J.
Cramer,
Lehre.
355
p.
Accepted
for
publication
September
8,1984