scispace - formally typeset

Journal ArticleDOI

Molecular markers in Australian isolates of Rhizoctonia solani

01 Jun 1994-Fungal Biology (Elsevier)-Vol. 98, Iss: 6, pp 665-671

TL;DR: There was less variation in the RFLP patterns when random cloned fragments of DNA were used as probes and group specific patterns could be identified for all groups, but significant variation within some of the groups whereas other groups were more uniform.
Abstract: Isolates of Rhizctonia solani from different locations within Australia and Japan were analysed for restriction fragment length polymorphisms (RFLPs). These isolates belong to different anastomosis and pectic zymogram groups. Southern blots of restriction enzyme digested DNA were probed with a cloned 18S ribosomal RNA (rRNA) gene, or random cloned fragments of R. solani DNA. The patterns obtained with the rRNA probe revealed significant variation within some of the groups whereas other groups were more uniform. Group specific patterns could not be identified for all groups. There was less variation in the RFLP patterns when random cloned fragments of DNA were used as probes and group specific patterns could be identified for all groups.

Content maybe subject to copyright    Report

Mycol.
Res.
98
(6):
665-671
(1994)
Printed
in
Great
Britain
665
Molecular
markers
in
Australian
isolates
of
Rhizoctonia solani
P. A.
O'BRIEN
School
of
Biological and Environmental
Sciences.
Murdoch University. Murdoch.
WA6150,
Australia
Isolates of Rhizctonia solani from different locations within Australia
and
Japan were analysed for restriction fragment length
polymorphisms
(RFLPs).
These isolates belong to different anastomosis
and
pectic zymograrn groups. Southern blots of restriction
enzyme digested
DNA
were probed with a cloned 185 ribosomal
RNA
(rRNA) gene,
or
random cloned fragments
of
R.
solani DNA.
The patterns obtained
with
the rRNA probe revealed significant variation within some
of
the groups whereas
other
groups were
more uniform. Group specific patterns could
not
be identified for
all
groups. There was less variation in the
RFLP
patterns when
random cloned fragments
of
DNA
were used
as
probes and group specific patterns could be identified for all groups.
Isolates of many species
of
plant pathogenic fungi show
tremendous diversity in characteristics such
as
morphology
and pathogenicity. A useful concept has been to assign these
isolates
to
groups based
on
pathogenicity (pathotypes), or
anastomosis behaviour (anastomosis groups).
In
recent years the application of molecular biological
techniques such
as
restriction fragment length polymorphism
(RFLP)
have made great advances in clarifying the taxonomic
relationships of numerous species of fungi (Manicom
et
aI.,
1987; Braithwaite, Irwin & Manners, 1990; Levy
et
a/.,
1991).
These studies have revealed a hitherto unsuspected level of
variation
in
a number
of
species of phytopathogenic fungi,
and have shed light
on
the mechanisms of variation in those
species (Hulbert
& Michelmore, 1987).
Rhizocfonia
solani
Kuhn is an important pathogen of cereal
and legume crops in Australia. The incidence of disease due to
this pathogen has been increasing in recent years, and there
are no effective measures to control the spread
of
the
pathogen (Anderson, 1982; McNish, 1986). Isolates show
tremendous variation in morphological, and pathogenic
characteristics. These isolates are divided into anastomosis
groups (Ogoshi, 1987; Sneh, Burpee
& Ogoshi, 1991). To
some extent these
groups
correlate with pathogenicity, but
there
is
still considerable variation within groups, and new
groups are regularly identified (Sneh
et
al.,
1991). Due
to
the
lack of stable markers in
R.
solani
there
is
no information
of
the
mechanisms
by
which this variation
is
generated. Lack of
markers has also hindered studies
on
the population dynamics
of the pathogen in the soil, and
on
the epidemiology
of
the
pathogen.
In
this
study
we
have explored the possibility of
using
DNA
polymorphisms as genetic markers for
R.
solani.
The results show
that
markers can be detected using either
ribosomal RNA genes,
or
random cloned fragments
to
probe
Southern blots
of
restriction digested
R.
solani
DNA.
MATERIALS
AND
METHODS
Fungal strains
The names and characteristics of the isolates used in this study
are outlined
in
Table 1. All
of
the isolates are multinucleate,
and have
as
their teleomorph
Thanatephorus
cucumeris
(Frank)
Donk. The isolates are divided into pectic zymograrn groups
(ZG)
on
the basis
of
the pectic enzymes produced when
grown
on
pectin (Sweetingharn. Cruickshank & Wong, 1986;
Neate, Cruickshank
& Rovira, 1988; Cruickshank, 1990).
These pectic zymogram groups correspond to anastomosis
groups. The pathogenicity of the isolates is: ZGI, cereals and
legumes; ZG2, cereals and legumes; ZG3, legwnes; ZG4.
legumes; ZGS, crucifers; ZG6, legumes; ZG7,
potato;
ZG8,
soil saprotroph;
AGl,
cereals and legumes; AGs, soil;
AG6
and
7,
saprotroph (Sneh
et
al., 1991).
Plasmids
To
generate probes for
RFLP
analysis,
DNA
from the ZG3
isolate R16 was digested with
Hind
III
and the digest cloned
into the bacterial plasmid
pUCI8.
Plasmids were chosen at
random from this library and used as probes. The ribosomal
RNA
clone pTA2S0. 10 was supplied
by
R.
Appels, CSIRO
Division of Plant Industry, Canberra. This plasmid contains a
I kb sequence from the 18S rRNA
gene
of
wheat cloned into
pBR322 (Appels
& Dvorak, 1982). Plasmid
DNA
was prepared
from cultures of
E.
coli
strain JM83
by
the alkaline-SDS
method
(Sam
brook, Fritsch & Maniatis. 1990). and stored in
TE
buffer at -
20°C.
Growth
and
isolation
of
DNA
For analysis
of
RFLPs
mycelium was grown in Petri dishes

RFLPs
in Rhizoctonia
solani
Table
1.
Isolates
of
Rhizoctonia
solani
used in this
study
Geographical
ZG
AG
origin1/.
Source
R163
1.1
8
WA
1
R146
1.1
8
WA
1
JW92
1.1
8 SA
3
R1232
1.1
8
WA
1
1475
1.1
8 SA 2
H155
1.1
8 SA 2
H140
1.2
8 SA 2
1453
1.3
8 SA
2
1440
1.3
8 SA
2
1188
1.3
8 SA
2
R829
2
8
WA
1
R132 2
8
WA
1
R881 2
8
WA
1
R138 2 8
WA
1
1517
2
8 SA 2
R880 2 8
WA
1
R16
3
ND
WA
1
R103
3
ND
WA
1
RIO11
3
ND
WA
1
RI012
3
ND
WA
1
RI013
3
ND
WA
1
R917
3
ND
WA
1
R1026 3
ND
WA
1
R56
4
2.2
WA
1
R57 4
2.2
WA
1
R586 4
2.2
WA
1
R990
4
2.2
WA
1
R817 4
2.2
WA
1
R812 4
2.2
WA
1
BC108 4
2.2
SA 3
C96
4
2.2IIIB jap
3
RI-64
4 2-2IV
jap
3
R75 5
2.1
WA
1
R120
5
2.1
WA
1
PS4
5
2.1
Jap
3
T9-1
5
2.1
SA
3
1321
5
2.1
SA
2
1519
5
2.1
SA 2
R98 5
2.1
WA
1
TasD
6
ND
Tas
2
NZT7
6
ND
NZ
2
P40
7 3 SA
2
TasA
7
3 Tas 2
TasC
7 3 Tas
2
STG
7 3 SA
3
1323
8 4 SA
3
SCR117 8 4
SA 3
AH-1
4HG-1
Jap
3
RH-165
8
4HG-II
jap
3
WA,
Western
Australia; SA,
South
Australia; Tas, Tasmania; jap, Japan;
NZ,
New
Zealand.
1, Isolates
obtained
from
M.
Sweetingham, Dept.
of
Agriculture, Perth;
2,
isolates
obtained
from
R.
Cruickshank, University
of
Tasmania, Hobart,
Tasmania; 3, isolates obtained from
K.
Sivasitharnparam, University
of
Western Australia,
Perth;
ZG, pectic zymograrn
groups
(Sweetingharn
et
al.,
1986; Cruickshank, 1990); AG, anastomosis
groups
(Ogoshi, 1987); ND,
not
determined.
containing
20
ml
of
V8 medium for 6 d
at
25°
in the dark
without shaking.
The
mycelium was then harvested, washed
with ice cold
TE
buffer (10
mM
Tris/HC!
pH
8'0, I
mM
EDT
A),
lyophilized, and
DNA
isolated as described
by
Raeder
& Broad (1985).
666
Construction
of
an
R.
solani
DNA
library
R16 mycelium was inoculated into 500
ml
of GPY medium
(I
% glucose, I % peptone,
and
I % yeast extract) in a 2 I
Ehrlenmeyer flask. The culture was incubated for
6-7
d
at
25°
and 120 rpm. The mycelium
was
harvested, and extracted for
DNA
as described above.
The
DNA
was further purified
by
equilibrium centrifugation in a CsC! gradient (Sambrook
et
al.,
1990). The
DNA
was digested for 15
h)
with
Hind
III
(5
units
of
activity
I-lg-
1
DNA). Hind
III
digested pUC18 was treated
with calf intestinal phosphatase
(I
unit of activity
I-lg-
1
DNA)
at 37° for
30
min. Both
DNA
preparations were then
extracted
with
phenol-chloroform, with chloroform, and
ethanol precipitated.
The
pellet was washed with 70%
ethanoL dried and dissolved in
TE
buffer. The Hind
III
digested
R.
solani
DNA
was ligated to pUC18 overnight
at
15°, and the products
of
the
reaction transformed into
E.
coli
JM83
by
the
method
of
Hanahan (1983). Transformants were
selected
on
LB
agar containing 50
I-lg
ml-
l
ampicillin, and
X-gal (Sambrook
et
ai.,
1990).
Southern blotting
R.
solani
DNA
was digested
with
restriction enzyme
(5
units
of activity
I-lg-
1
DNA)
for 15 h under conditions specified
by
the supplier.
The
digest
was
fractionated
by
electrophoresis
through a
0'8%
agarose gel in TAE buffer at I
Vern-I.
The
DNA
was then
denaturated
and blotted
onto
Zeta Probe (Bio-
Rad) for 16 h using
I M ammonium acetate 20
mM
NaOH
as
the transfer buffer (Rigaud
et
al.,
1987). The membrane was
baked at 80° for 2
h,
and
prehybridized for I h
at
65°. Rapid
hybridization buffer (Amersham) was used at 0'1 ml
cm-
2
for
both
prehybridization
and
hybridization. After prehybrid-
ization, heat denatured
probe
was added
to
the bag and
hybridization carried
out
overnight
at
65°. The membrane was
rinsed briefly in 2
x
SSe,
O'
1%
SDS
at
room temperature and
at 65° for
30
min in
the
same solution. This was followed
by
two washes in
0'1
x
SSe,
0'1
% SDS for
30
min at 65°. The
membrane was
then
wrapped
in cellophane
film
and exposed
to
Kodak X-Omat
AR
X-ray film
at
- 80°.
Labelled hybridization probes were prepared
by
nick
translating
100
ng
plasmid
DNA
with 32p_dCTP. The nick
translation reagents were obtained from Amersham, and used
in accordance
with
their instructions. The reaction was
terminated
by
adding
EDT
A
to
20
mM.
The probe was used
directly after
heat
denaturation.
RESULTS
RFLPs in ribosomal
RNA
genes
A Southern blot prepared
with
fcoR I digested
R.
solani
DNA
was probed
with
a
wheat
rRNA
gene. A representative
sample of the data from this experiment
is
shown in Fig. 1, and
the results obtained
with
a larger number
of
isolates
is
given
in Table
2.
A prominent
4'8
kb band was common to all
of
the
groups, and
was
a consistent feature
of
the isolates within
these groups. The exceptions
to
this were ZG3, and ZG5. All
of
the ZG3 isolates contain a
2'2
kb band which
is
unique
to
this group. However,
only
one
isolate, R16, contains
the
4'8 kb band in addition
to
the
2'2 kb band.
Of
the six ZG5

P.
A. O'Brien 667
ZGI
1234567RQ
ZG2 ZG5 ZG8
10
II
121314151617
181920212223
ZG3
24
252627
28
ZG4
29
30
31
32 33
34
-9.4
-6.6
-4.3
-9.4
-6.5
-4.3"~".
,~
....
-2.3
....
-2.0
:~j
-4.3
-2.3
-2.0
-2.3
-9.4
-6.5
-4.3
Fig.
1.
RFLPs in
the
ribosomal
RNA
genes
of
R.
salani.
EeaR
I
digested
DNA
was
electrophoresed
on
a
0'8%
agarose
gel,
and
blotted
onto
a
nylon
membrane.
The
membrane
was
probed
with
nick translated
pTA250.10.
Lanes:
(1)
RI63;
(2)
RI46;
(3)
R1232;
(4)
JW92;
(5)
1475;
(6)
HISS;
(7)
HI40;
(8)
1453;
(9)
1440;
(10)
R829;
(11)
R132;
(12)
R138;
(13)
R880;
(14)
R75;
(15)
R120;
(16)
PS4;
(17)
T9-1;
(18)
1321;
(19)
1519;
(20)
1323;
(21)
SCRIl7;
(22)
AH-l;
(23)
RHI-65;
(24)
RI6;
(25)
RI03;
(26)
RIOll;
(27)
RI013;
(28)
RI012;
(29)
R56;
(30)
R586;
(31)
R813;
(32)
BCl08;
(33)
C96;
(34)
RI-64.
Table
2.
Size
of
EeaR I
fragments
homologous
to
p
TA250.
10
AG
ZG
Lane
8
1
1 2 3
4
8
2
5
6
NO
3
7 8
4
9
10
2'1
5
II
12
13
14
IS
NO
6
16
3
7
17
18
4
8
19
20
14
9'7
8
5'6
5'1
5
4'5
0'6
0'6 0'6 0'6
No.
of
2 4 2 I
isolates
0'6 0'6
4 ]
2'2 2'2
0'6 0'6
I 5
0'6
0'6
9 I
0'6 0'6
0'6 0'6 0'6
I 2 I I 1
0'6
2
0'6
0'6
I 2
0'6
3
0'6
I
Isolates:
lane
(I)
R163,
1440;
(2)
RI46,
JW92,
HISS,
1232;
(3)
HI40,
1453;
(4)
]475;
(5)
R829,
R132,
R88I',
R138;
(6)
R880;
(7)
RI6;
(8)
R103,
41OII,
R1012,
R1013,
R1026';
(9) R56,
R813,
R57',
R586,
R990', R817',
R812',
BCI08,
C96;
(10)
RI-64;
(II)
R75;
(12) R120,
T9-I;
(13)
PS4;
(14)
1321;
(15)
1519;
(16)
TasO',
NZT7';
(17)
P40';
(18)
TasN,
TasC';
(19)
1323,
SCRII7,
RH-I65;
(20)
AH-1.
Oata
for
these
strains
are
not
shown
in
Fig. 1.
Fragment
sizes are
in
kbp.
isolates tested,
only
two contain the 4'8 kb band
(Fig.
I).
Other
bands observed in common among isolates from
different groups included a 9'7, and a
0'6
kb band. All isolates
tested contained the
0'6
kb band (Table
2).
In contrast the
9'7
kb band was
not
a consistent feature within
anyone
group. It was observed in some
of
the isolates from ZG1,
2,
4 and s
(fig.
I).
Bands
in
addition
to
the 4'8 kb band were observed
in
a
number of groups. These are unique
to
the groups they were
observed
in,
but
unlike the 4'8 kb band were
not
a consistent
feature of the isolates from those groups. The ZGI and ZG2
isolates contain a 2'8
kb
band. All
of
the ZG2 isolates tested
contain the 2'8 kb band,
but
its presence in the ZG1 isolates
is
variable. The
ZGs
(AG2-1) isolates were highly poly-
morphic. Five different patterns were obtained for six
isolates.
On
the basis of anastomosis behaviour this group
is
known to be highly polymorphic (Sneh
et
al.,
1991).
The variability
in
banding pattern corresponded more
closely with the classification
of
isolates into
AG
than with the
ZG
concept.
Of
the nine ZG4 isolates analysed (Table
2),
eight gave an identical pattern with a single
4'8
kb band. The
remaining isolate RI-64 gave a very different pattern from the
other
ZG4
isolates. This may be a reflection
of
the fact that
RI-64
belongs
to
a different anastomosis subgroup than C96.
From the
RFLP
patterns the Australian isolates belong
to
the
same subgroup as C96. Similarly, the ZG8 isolate AH-1 which
can be differentiated from the other ZG8 isolates
by
its
RFLP
pattern
(Fig.
I,
lane 22; Table
2),
belongs
to
a different
AG4
subgroup than the other
AG4
isolates (Table I). ZG1 and 2
isolates all belong
to
AG8, although these
two
ZG cannot be
clearly distinguished with the rRNA probe since the
characteristic 2'8, and 9'7 kb bands appear in
both
groups.
The results obtained with the rRNA probe show that this
probe would be very useful for detecting variation within the
group, and in clarifying relationships between clones from the
same group,
but
less useful for the identification of groups.

RFLPs
in
Rhizacfania
salani
668
Table
3.
Differentiation
of
groups
of
R.
solani
by
RFLP analysis with
random
cloned
DNA
fragments
as
probes
Probe
pRGL2-I
pRGL2-IO
pRGL2-12
pRGL2-AI
AG
ZG
7
1
8
2
3
2.2
2.1
4 5
8
1
8
2 3
2.2
2.1
4 5
8
1
8
2
3
2.2
2.1
4 5
8
1
8
2 3
2.2 2.1
3 4
4 5 7 8
>20
10
8'2 8'2
8
13
II
II
II
8'1
8'2
7'5
7
6
5'2
5'8
5'2
2-6
2'3
2'5
1'9
1'8 1'7
1'5
1'2 1'1
1'1
1'5
1'2
1'8 1'8
0'8
Southern
blots
of
fcoR I
digested
DNA
were
probed
with
nick
translated
random
cloned
fragments
from
isolate
RI6.
Isolates:
ZGI,
RI63;
ZG2,
R829;
ZG3,
RI6;
ZG4,
R56;
ZG5,
R75;
ZG7,
STG;
ZG8,
SCRII7.
Fragment
sizes are in kbp.
RFLPs
with
random
cloned fragments
To
generate additional probes for
RFLP
analysis,
R.
salani
DNA
was digested
to
completion with Hind
III,
and the digest
cloned into the plasmid
pUeI8
to
generate a library. Clones
Isolates:
lane
(1)
RI63;
(2)
RI46,
R1232,
1475',
HISS',
HI40';
(3)
1514',
1517',
R829, R132,
R88I,
R880;
(4)
RI6;
(5)
RI03,
RIO
II,
RI012,
RIO!;
(6) R56, R57, R586,
R990;
(7)
R75',
R120',
R98';
(8)
T9-I;
(9)
STG';
(10)
SCRII7.
,
Data
for
these
strains are
not
included in Fig.
2.
Fragment
sizes are in
kbp.
Table
4.
Size
of
fcoR I
fragments
homologous
to
pRGL2-IO
were selected from this library at random, and screened
to
determine the insert size. Clones with different sized inserts
were assumed
to
be different and were used as probes
in
hybridization reactions.
Initially a number of restriction enzymes were tested
to
determine which one would be most useful in generating
polymorphisms.
Of
the enzymes tested (feaR
I,
Hind
III,
BamH
I,
Pst
I, and feaR
V),
feaR I generated the most
polymorphisms (data
not
shown). Hind
III
and feaR V were
also very useful whilst
Pst
I, and BamH I generated very few
bands and thus were likely
to
be less useful in differentiating
groups.
A number
of
randomly cloned fragments were tested for
their ability
to
detect
RFLPs
in isolates from different groups.
Southern blots
of
feaR I digested
DNA
were probed with nick
translated random cloned fragments of
DNA
from ZG3
isolate RI6. Although the probes were derived from a ZG3
isolate, they hybridized
to
DNA
from isolates in other groups
(Table
3)
except for the following
two
probes (pRGL2-6, and
pRGL2-8) that were specific for
ZG3
isolates. All of the probes
were able
to
differentiate ZG3, ZG4, ZGs, and pRGL2-AI
was able to differentiate
ZGB from the other ZG (not tested
for pRGL2-IO). In each case the fragment pattern produced for
the ZG3 isolate
RI6
was more complex than the pattern
produced for the isolates from the other groups. This may be
a reflection
of
the fact that the probes originated from RI6.
Only
one
out
of
the four probes (pRGL2-I) was able
to
differentiate the
ZGI
and ZG2 isolates (Table 3). However,
the ZGI isolate used for this experiment was subsequently
found
to
be different from the other
ZGI
isolates (Table 3 &
4).
2
2'5
4
8
10
1'5
II
2'3
3
7
9
3 1
4
2.2
2.1
4 5
6 7 8
13
II
7
5'2
5'2
3
4 5
0'9
0'7 0'7
1 5
8
2
3
65
8
1
1 2
2'8
1'1 1'1
1'1
AG
ZG
Lane
No.
of
isolates

P.
A. O'Brien
ZGI
2
3 4
ZG2
5 6
7
-23
-9.4
-6.5
-4.3
-2.3
8
9
ZG3
10
II
12
ZG4
13
14
ZG3 ZG4
15
16
17
18
669
-23
-6.5
-4.3
-2.3
Fig_
2.
Southern blot of feaR I digested
R.
solani
DNA probed with
nick
translated
pRGL2-IO.
Lanes:
(1)
RI63,
(2)
RI46,
(3)
1232;
(4)
R829,
(5)
R132,
(6)
R88I,
(7)
R880;
(8)
RI6,
(9)
RI03,
(10)
RI011,
(11)
RlO12,
(12)
RI013, (13)
R56,
(14)
R57,
(15)
R9I7,
(16)
R8I7,
(17)
R8I7,
(18)
R8I2.
Size
of A
Hind
III
fragments
are
given
in
kbp.
To
assess the level
of
variation
between
isolates
of
the same
group, a
number
of
isolates from each group were analysed
with the
probes
pRGLl-IO
and
pRGLl-AI.
The
results
of
these experiments are
given
in Tables 3, and 4, and a
representative sample
of
the
data
is given in Fig.
Z.
For
both
of
the probes
the
level
of
intragroup variation was less than
observed with
the
rRNA
probe.
Of
twelve
ZG
1 +Z (AG8) isolates tested with pRGLl-IO,
al!
were identical
except
for
RI63
which
gave
a different
pattern
from the
other
isolates
with
both
probes (and also
with
the
rRNA
probe). Identical patterns were also obtained
for these isolates
with
the
pRGLl-AI
probe.
No
variation was
observed
between
the
ZG4
isolates with either probe. The
ZG3
isolates
could
be
distinguished
by
the presence
of
a 0-7
and
a
5-Z
kb
bands
in
the
pRGLl-IO
patterns. The
exception
to
this was the isolate
RI6
which in addition
to
these
two
bands, contained a 0-9 kb band.
Probe
pRGLZ-AI
gave
a
more
complex
pattern
with
the
ZG3
isolates than
with
isolates from
other
ZG.
On
the
basis
of
the
patterns
obtained
with
this
probe, the
ZG3
isolates can
be
divided into three subgroups.
One
of
these consists
of
RI6.
This probe also
appeared
to
differentiate
between
groups
4,
5,7
and
8 although
the
sample
size for
groups
5, 7
and
8
was
very
small.
There was also less variability in
the
ZG5 isolates_ Identical
patterns were
obtained
for three
of
the
four isolates
tested
with pRGLZ-IO, Table 4).
The
number
of
ZGs
isolates
tested
with
pRGLl-AI
was
too
small
to
draw
any
conclusions
regarding intragroup variation, as was
the
number
of
ZG7
and

Citations
More filters

Journal ArticleDOI
Lilyann Novak Frazer1, David R. Moore1Institutions (1)
TL;DR: This listing covers the period May 1, 1997 through to June 30, 1997, which roughly corresponds with the British Mycological Society's Special Interest Committees.
Abstract: This listing covers the period May 1, 1997 through to June 30, 1997. Citations are arranged in groups which roughly correspond with the British Mycological Society's Special Interest Committees. All correspondence about this item should be addressed to the Executive Editor. Reprints of this feature will not be available.

211 citations


Journal ArticleDOI
TL;DR: Results suggest that sequence analysis of ITS rDNA regions of R. solani may be a valuable tool for identifying AG subgroups of biological significance.
Abstract: Sequence analysis of the rDNA region containing the internal transcribed spacer (ITS) regions and the 5.8s rDNA coding sequence was used to evaluate the genetic diversity of 45 isolates within and between anastomosis groups (AGs) in Rhizoctonia solani. The 5.8s rDNA sequence was completely conserved across all the AGs examined, whereas the ITS rDNA sequence was found to be highly variable among isolates. The sequence homology in the ITS regions was above 96% for isolates of the same subgroup, 66-100% for isolates of different subgroups within an AG, and 55-96% for isolates of different AGs. In neighbor-joining trees based on distances derived from ITS-5.8s rDNA sequences, subgroups IA, IB and IC within AG-1 and subgroups HG-I and HG-II within AG-4 were placed on statistically significant branches as assessed by bootstrap analysis. These results suggest that sequence analysis of ITS rDNA regions of R. solani may be a valuable tool for identifying AG subgroups of biological significance.

201 citations



Journal ArticleDOI
TL;DR: Methods based on internal transcribed spacers (ITS) ribosomal DNA (rDNA) polymorphism and pectic zymograms (ZG) were compared for their use in routine identification of Rhizoctonia solani isolates occurring in flower bulb fields and it is proposed to assign AG 2 isolates pathogenic to crucifers and tulip to ZG5-1.
Abstract: Methods based on internal transcribed spacers (ITS) ribosomal DNA (rDNA) polymorphism and pectic zymograms (ZG) were compared for their use in routine identification of Rhizoctonia solani isolates occurring in flower bulb fields. Thirty three AG 2-t isolates, pathogenic to tulips, could be distinguished from AG 1-IC, AG 2-2IIIB and AG 2-2IV, AG 3 and AG 5 by means of ITS rDNA fragment length and after digestion with EcoR I from AG 4 and AG 5. AG 2-t isolates and two Japanese isolates, pathogenic to crucifers and tulips, had an estimated fragment size of 710 bp, whereas Dutch AG 2-1 isolates, non-pathogenic to tulips, showed an estimated fragment size of 705 bp on agarose gel. Digestion of AG 2-t and AG 2-1 isolates with EcoR I, Sau3A I, Hae III and Hinc II revealed four and five distinct ITS rDNA digestion patterns, respectively. In AG 2 isolates 2tR114, 21R14 and 21R61 a double digestion pattern, indicating different ITS sequences within an isolate, was found. The observed ITS fragment length polymorphism between isolates pathogenic and non-pathogenic to tulips were considered too small to be used in routine screening of field isolates. Sequencing of AG 2 isolates 21R01, 21R06, 2tR002 and 2tR144 showed a total ITS rDNA fragment length of 715, 713, 714, and 728 bp. As an alternative to ITS rDNA fragment length polymorphism, pectic enzyme patterns were studied using a commercially available vertical gel-electrophoresis system and non-denaturing polyacrylamide gels amended with pectin. Anastomosis tester isolates AG 1 to AG 11 revealed different ZG. Fifty AG 2-t isolates and five AG 2-1 isolates belonged to a homogeneous pectic zymogram group. We propose to assign AG 2 isolates pathogenic to crucifers and tulip to ZG5-1. AG 2-1 isolates, non-pathogenic to tulip, formed a heterogeneous group with 4 distinct ZG. Pectic zymography provides an easy, quick and unambiguous method for routine identification of large numbers of field isolates. Such a technique is needed for research on the dynamics of Rhizoctonia populations to develop environmentally friendly control measures of rhizoctonia disease in field-grown flower bulbs.

48 citations


Cites background from "Molecular markers in Australian iso..."

  • ...…of isolates of R. solani include genomic restriction fragment length polymorphism (Vilgalys and Gonzalez, 1990; Jabaji-Hare et al., 1990; O’Brien, 1994), random amplified polymorphic DNA (Duncan et al.,1993; Yang et al., 1995), polymorphism of the internal transcribed spacer (ITS) of…...

    [...]


Journal ArticleDOI
TL;DR: The UP-PCR technique revealed a greater degree of heterogeneity between the groups studied, and PCR-ribotyping with AluI and MspI is sufficient for analysis of distance between populations.
Abstract: Forty seven strains of the black yeasts, Aureobasidium pullulans and Hormonema dematioides, and the type strain of Hormonema macrosporum were examined using PCR-ribotyping and universally primed PCR with subsequent hybridization. Four groups (populations) were distinguished within A. pullulans with PCR-ribotyping, which largely coincided with UP-PCR/hybridization groups. The UP-PCR technique revealed a greater degree of heterogeneity between the groups studied. Five strains identified as Hormonena dematioides on the basis of physiological and morphological data formed a group recognizable with PCR-ribotyping and UP-PCR/hybridization, which also included H. macrosporum. Aureobasidium pullulans is characterized by the absence of RsaI restriction sites in rDNA amplified with primers 5.8S-R and LR7, while Hormonema species possessed several bands after RsaI digestion. For analysis of distance between populations, PCR-ribotyping with AluI and MspI is sufficient. Strains of A. pullulans produce exopolysaccharides in liquid media with different nitrogen sources, while the strains of Hormonema synthesize minor amounts of polysaccharides in media with peptone. Populations of A. pullulans differ slightly from each other in their optimal, medium-dependent production of polysaccharides.

21 citations


References
More filters

Book
15 Jan 2001
Abstract: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years. No other manual has been so popular, or so influential. Molecular Cloning, Fourth Edition, by the celebrated founding author Joe Sambrook and new co-author, the distinguished HHMI investigator Michael Green, preserves the highly praised detail and clarity of previous editions and includes specific chapters and protocols commissioned for the book from expert practitioners at Yale, U Mass, Rockefeller University, Texas Tech, Cold Spring Harbor Laboratory, Washington University, and other leading institutions. The theoretical and historical underpinnings of techniques are prominent features of the presentation throughout, information that does much to help trouble-shoot experimental problems. For the fourth edition of this classic work, the content has been entirely recast to include nucleic-acid based methods selected as the most widely used and valuable in molecular and cellular biology laboratories. Core chapters from the third edition have been revised to feature current strategies and approaches to the preparation and cloning of nucleic acids, gene transfer, and expression analysis. They are augmented by 12 new chapters which show how DNA, RNA, and proteins should be prepared, evaluated, and manipulated, and how data generation and analysis can be handled. The new content includes methods for studying interactions between cellular components, such as microarrays, next-generation sequencing technologies, RNA interference, and epigenetic analysis using DNA methylation techniques and chromatin immunoprecipitation. To make sense of the wealth of data produced by these techniques, a bioinformatics chapter describes the use of analytical tools for comparing sequences of genes and proteins and identifying common expression patterns among sets of genes. Building on thirty years of trust, reliability, and authority, the fourth edition of Mol

215,117 citations


Journal ArticleDOI
Douglas Hanahan1, Douglas Hanahan2Institutions (2)
TL;DR: Competition with both transforming and non-transforming plasmids indicates that each cell is capable of taking up many DNA molecules, and that the establishment of a transformation event is neither helped nor hindered significantly by the presence of multiple plasmid molecules.
Abstract: Factors that affect the probability of genetic transformation of Escherichia coli by plasmids have been evaluated. A set of conditions is described under which about one in every 400 plasmid molecules produces a transformed cell. These conditions include cell growth in medium containing elevated levels of Mg2+, and incubation of the cells at 0 degrees C in a solution of Mn2+, Ca2+, Rb+ or K+, dimethyl sulfoxide, dithiothreitol, and hexamine cobalt (III). Transformation efficiency declines linearly with increasing plasmid size. Relaxed and supercoiled plasmids transform with similar probabilities. Non-transforming DNAs compete consistent with mass. No significant variation is observed between competing DNAs of different source, complexity, length or form. Competition with both transforming and non-transforming plasmids indicates that each cell is capable of taking up many DNA molecules, and that the establishment of a transformation event is neither helped nor hindered significantly by the presence of multiple plasmids.

10,867 citations


Journal ArticleDOI
U. Raeder1, P. Broda1Institutions (1)
TL;DR: A general, simple and inexpensive method for the isolation of DNA from filamentous fungi, starting from freeze‐dried mycelium 01–015% by weight, which allows the processing of many samples in parallel.
Abstract: We describe a general, simple and inexpensive method for the isolation of DNA from filamentous fungi. Starting from freeze-dried mycelium 01–015% by weight can be isolated as high molecular weight DNA suitable for restriction and ligation in 2 h. The preparation can be done in Eppendorf tubes and allows the processing of many samples in parallel. We have used the method with the basidiomycetes Phanerochaete chrysosporium, Coprinus cinereus and the ascomycete Aspergillus nidulans and others have used it with Trichoderma reesei, Aspergillus niger and for the isolation of DNA from tomato plants.

1,635 citations


Journal ArticleDOI
TL;DR: The role of environmental exposures in the development of phylogeny is studied in the context of infectious disease and infectious disease.
Abstract: INTRODUCTION . . . . . . . . . . . . . . . .. ..... . . . . . . . . .. . . . . . .. . . . . . . . . . . . . . . . . . . . . .. . ...... .. . . . . . . .... . . . . . 338 MOLECULAR PHYLOGENY AND MICROBES . . . . . . . . . . . .. . . . . . . . . .... . . ... . . . . .... .. . . . . . 338 Ribosomal RNAs as Indicators of Phylogeny.. . . . ..... . . . . .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338 Analysis of Population

1,061 citations


"Molecular markers in Australian iso..." refers background in this paper

  • ...Ribosomal RNA genes are present in hundreds of copies per genome in fungi, and they contain sequences that are highly variable (Olsen et al., 1986)....

    [...]

  • ...This may be a consequence of the fact that the rRNA genes can undergo significant variation without affecting biological function (Olsen et al., 1986)....

    [...]



Performance
Metrics
No. of citations received by the Paper in previous years
YearCitations
20151
20141
20102
20091
20051
20031