Pectolytic and cellulolytic activity in Rhizopus-infected rin and nor tomato mutants Rhizopus stolonifer, Lycopersicon esculentum


Barkai-Golan, R.K.peliovitch, E.

Mycopathologia 82(2): 89-93

1983


Mycopathologia
82,89-93
(1983).
©
Dr
W.
Junk
Publishers,
The
Hague.
Printed
in
The
Netherlands.
Pectolytic
and
cellulolytic
activity
in
Rhizopus-infected
rin
and
nor
tomato
mutants
Rivka
Barkai-Golan'
&
E.
Kopeliovitch
2
Agricultural
Research
Organization,
The
Volcani
Center,
Bet
Dagan,
Israel
2
Faculty
of
Agriculture,
The
Hebrew
University
of
Jerusalem,
Rehovot,
Israel
Abstract
Polygalacturonase
(PG)
and
polymethylgalacturonase
(PMG)
activity
was
recorded
in
the
two
non-ripen-
ing
mutants
of
tomato
devoid
of
these
enzymes,
rin
and
nor
fruits,
following
Rhizopus
stolonifer
infection.
Activity increased
markedly
with
disease
development.
C
s
-cellulase
activity,
generally
found
in
healthy
rin
and
normal
fruit,
increased
with
maturity
as
well
as
with
disease
development.
The
effect
of
fungal
infection
on
C
s
-activity
was
particularly
pronounced
on
the
mature-green
nor
fruit,
in
which
only
traces
of
activity
were
recorded
prior
to
inoculation.
At
an
advanced
stage
of
disease
development,
infection
brought
pectolytic
and
cellulolytic
activity
in
the
standard
and
mutant
fruits
at
all
stages
of
maturity
to
a
similar
level,
regardless
of
the
differences
in
their
initial
enzyme
levels.
A
correlation
was
drawn
between
the
level
of
pectolytic
activity
and
the
rate
of
fruit
softening
in
Rhizopus-infected
mutant
fruits.
Tissue
softening
following
infection
was
attributed
to
the
activity
of
fungal
polygalacturonases.
The
relatively
high
levels
of
C
x
-cellulase
found
in
infected
mutants
during
the
early
stages
of
disease
development,
when
only
a
slight
or
no
softening
was
recorded,
suggested
a
lack
of
relationship
between
cellulolytic
activity
and
softening
of
tomato
tissues.
Introduction
Rin
and
nor
tomato
fruits
are
both
non-ripening
mutants
of
tomato,
which
lack
the
characteristic
climacteric
of
the
normal
fruit
and
fail
to
soften
(6,
8,
9,
11).
Comparing
the
enzymatic
activity
of
polygalacturonase
(PG)
and
C
x
-cellulase
of
rin
and
normal
tomatoes
during
maturation,
Buescher
&
Tigchelaar
(5)
found
that,
whereas
in
normal
fruit
both
PG
and
C
s
-activity
increased
during
ripening,
in
the
mutant
fruit
only
C
s
-activity
increased
and
PG
was
not
detected
at
all
during
fruit
develop-
ment.
Absence
of
PG,
or
the
presence
of
traces
only,
was
reported
also
for
the
nor-mutant
(8)
whereas
C
s
-activity
of
this
non-ripening
mutant
has
not
been
studied
before.
Both
these
mutants
were
reported
to
be
more
resistant
to
postharvest
pathogens
than
the
normal
fruit
(3,
4,
5,
9).
However,
following
inoculation
of
Rhizopus
stolonifer
through
wounds
into
the
peri-
carp
of
the
mutant
fruits,
a
typical
rot
of
the
normal
fruit,
which
is
characterized
by
softening
of
tissues
followed
by
a
total
fruit
collapse,
was
recorded
(4).
In
the
present
work
a
host-pathogen
system,
in
which
the
host
is
a
non-softening,
enzymeless
rin
or
nor
tomato,
and
the
pathogen
-
the
pectolytic
fun-
gus
R.
stolonifer
(10)
-
was
used
to
study
the
effect
of
fungal
infection
on
the
pectolytic
and
cellulolytic
activities
in
the
mutant
fruit
and
their
relationship
to
the
softening
of
tomato
fruit.
Materials
and
methods
Tomato
variety
'Rutgers',
and
the
two
non-ripen-
ing
mutants
rin
and
nor,
which
were
both
partially
isogenic
to
'Rutgers'
(fifth
and
second
backcross,
respectively),
were
grown
during
spring
in
the
soil
100
80
60
20
UNINFECTED
INFECTED
Standard
0
rin
O
nor
Ath
/
/
/
/
/
/ /
/
/
/
.0
//
//
3
2
1
DIS
EA
SE
INDEX
(DI
)
1
2
3
DAYS
AFTER
INOCULATION
90
in
a
heated
glasshouse,
at
22
±
4
°
C.
The
fruits
were
harvested
at
three
stages
of
maturation,
'mature-
green',
'turning
point'
and
'mature'.
For
the
non-
ripening
mutant
fruits
the
'turning
point'
was
de-
fined
as
the
stage
of
changing
peel
color
from
green
into
yellow,
whereas
14
days
after
the
turning
point
was
referred
to
as
their
'mature'
stage.
The
harvest-
ed
fruit
was
inoculated
with
R.
stolonifer
(Ehrenb.
ex
Fr.)
Lind,
isolated
from
postharvest
rot
of
`Rutgers'
variety
tomato.
Inoculation
was
done
by
inserting
into
the
pericarp
through
a
2
mm
deep
wound,
a
uniform
sized
inocula
consisting
of
both
mycelium
and
spores
taken
from
the
periphery
of
3-day-old
single-spore
culture
on
potato-dextrose
agar
(PDA)
at
23
°
C.
Enzymatic
activities
of
inoculated
fruits
were
ex-
amined
24
h
and
3
days
after
inoculation
at
23
°
C.
Index
of
fungal
development
in
the
fruits,
defined
according
to
the
severity
of
symptoms
as
exhibited
by
rot
development
and
rate
of
softening
from
0
(apparently
healthy
tissues)
through
1
(slight
sof-
tening
at
the
site
of
inoculation)
to
5
(complete
fruit
rot
accompanied
by
tissue
collapse),
was
recorded
parallel
to
the
enzyme
tests.
Extracts
for
enzyme
tests
were
prepared
by
ho-
mogenizing
fruit
tissues
for
2
minutes
in
three
vol-
umes
(w
/
v)
of
1
M
NaC1
solution
in
an
osterizer
blender,
followed
by
filtration
of
the
liquid
fraction
as
described
previously
(2).
The
extracts
were
cen-
trifuged
at
15
000
X
g
for
10
min
at
4
°
C
to
obtain
a
supernatant
for
enzyme
activity
assay.
For
the
cel-
lulolytic
studies
the
homogenate
was
held
for
40
minutes
at
4-6
°
C
prior
to
centrifugation,
as
de-
scribed
by
Babbitt,
Powers
&
Patterson
(1).
For
PG
and
PMG
studies
the
supernatant
was
dialysed
against
water
for
24
h
at
4
°
C
prior
to
the
enzyme
determinations.
Preparations
from
non-inoculated
fruits
served
as
controls,
as
did
samples
from
inocu-
lated
tissues
boiled
for
60
min.
Relative
PG
and
PMG
activities
of
the
fruit
preparations
were
expressed
as
percentage
loss
in
viscosity
of
1%
(w/
v)
sodium
polypectate
(SPP,
Sigma
Chemical
Co.)
or
1%
pectin
(citrus
pectin,
Sigma
Chemical
Co.),
respectively,
after
60
min-
utes'
incubation,
whereas
viscometric
data
of
C
x
-
cellulase
were
recorded
after
5
h
incubation
with
1%
carboxymethylcellulose
(CMC,
City
Chemical
Corp.).
The
tests
were
carried
out
in
0.1
M
citrate
buffer
at
pH
5
in
200
Fenske-Otswald
viscometers,
as
de-
scribed
previously
(2),
with
five
replicates
of
each
experiment.
Results
Uninfected
rin
and
nor
tomatoes
were
both
found
to
be
devoid
of
PG
and
PMG
at
all
stages
of
fruit
maturity,
except
for
traces
sometimes
re-
corded
in
the
mature
nor
mutant.
In
the
normal
fruit,
on
the
other
hand,
lack
of
pectolytic
activity
was
confined
to
the
mature-green
stage
only,
activi-
ty
increasing
with
fruit
maturity
(Figs.
1
and
2).
However,
24
h
after
Rhizopus
infection,
some
PG
_
O
SS
OF
VI
SC
OS
I
TY
OF
SO
D
IUM
POLY
PEC
TA
TE
(7.
)
MATURE-GREEN
TURNING
MATURE
Fig.
1.
Polygalacturonase
activity,
at
three
stages
of
fruit
maturity,
in
uninfected
standard
(0),
rin
(
)
and
nor
(A)
tomato
fruits,
and
Rhizopus
stolonifer-infected
fruits
(*,
♦,
respectively),
in
relation
to
disease
index
(-
-
-).
100
MATURE-GREEN
TURNING
MATURE
UNINFECTED
INFECTED
so
Standard
0
rin
0
nor
0
60
40
20
L
O
SS
O
F
VISC
OSITY
OF
P
ECTIN
PI
P
2
DAYS
AFTER
INOCULATION
5
/
/
/
/
1
3
2
DISEA
SE
IND
EX
(011
91
Fig.
2.
Polymethylgalacturonase
activity,
at
three
stages
of
fruit
maturity,
in
uninfected
standard
(0),
rin
(0)
and
nor
(L)
tomato
fruits,
and
Rhizopus
stolonifer-infected
fruits
(•,
•,
respectively),
in
relation
to
disease
index
(-
-
-).
I
I I
MATURE-GREEN
Standard
P
/
/
i
t
/
01
1
I
TURNING
t
I
/
/
I
t
I
1
MATURE
/ /
/
cf
/
I
t-
t
rin
/
p
/
Fe
/
/
/
Q
Fe
/
I
I
/
/
i
0
I
t
nor
/
f
-
/
/
))
TA'
, /
I
I
/
i
/
/
/
/
111
/
/
//
_.
I
t
0
1
I
I
1
2
3
1
2
3
1
2
3
DAYS
AFTER
INOCULATION
Fig.
3.
Cellulase
activity,
at
three
stages
of
fruit
maturity,
in
uninfected
(0-0)
and
Rhizopus
stolonifer-infected
(.
•)
fruits,
in
relation
to
disease
index
(0---0).
100
80
60
40
20
100
0
I
80
0
60
N
O
40
LL
20
0
100
80
60
40
20
5
4
3
2
1
5
4
3
2
1
5
4
3
2
DI
SEAS
E
INDE
X
ID
I)
92
and
PMG
activity
was
already
detected
in
both
mature-green
mutants,
as
well
as
in
the
mature-
green
normal
fruit,
all
of
which
were
devoid
of
activity
when
healthy.
Pectolytic
activity
increased
markedly
with
disease
index,
achieving
relatively
high
levels
3
days
after
incubation.
At
the
turning
point
stage,
fungal
infection
caused
an
increase
in
the
enzyme
levels
of
the
nor-
mal
fruit
and
similar
levels
were
recorded
in
the
two
enzymeless
mutants.
At
the
third
stage
of
maturity,
a
time
when
the
normal
healthy
fruit
exhibited
high
levels
of
PG
and
PMG,
fungal
effect
was
much
more
pro-
nounced
in
the
two
mutants,
than
in
the
normal
fruit.
Here,
again,
3
days
after
inoculation,
fungal
infection
brought
about
similar
high
enzyme
levels
in
the
mutants
and
in
the
normal
fruit,
regardless
of
the
marked
difference
in
the
initial
enzyme
level.
At
all
stages
of
maturity,
pectolytic
activity
was
correlated
to
the
rate
of
fruit
softening
following
Rhizopus
infection,
as
was
indicated
by
the
disease
index
(Figs.
1
and
2).
Both
uninfected
mature-green
mutants,
similar
to
the
uninfected
normal
fruit,
exhibited
C
x
-cel-
lulase
activity,
although
considerable
levels
were
found
only
in
the
normal
and
rin
fruits.
Cellulolytic
activity
in
all
fruits
increased
with
disease
severity.
However,
relatively
high
levels
were
recorded
24
h
after
inoculation
when
only
a
slight
softening
at
the
site
of
inoculation,
if
at
all,
was
recorded
(Fig.
3).
Fungal
infection
brought
cellulolytic
activity
to
similar
levels
in
all
three
fruits,
regardless
of
the
differences
in
the
enzymatic
levels
of
their
healthy
tissues,
and
almost
blurred
the
differences
existing
initially
at
the
various
stages
of
maturity
(Fig.
3).
Discussion
The
effect
of
Rhizopus
infection
on
the
enzymat-
ic
activity
of
rin
and
nor
mutants
was
expressed
by
both
increasing
the
cellulolytic
activity
normally
found
in
uninfected
fruits
and
by
causing
detectable
levels
of
PG
and
PMG
activity,
normally
absent
in
healthy
tissues.
The
enzymatic
activity
in
the
two
mutant
fruits
following
inoculation
suggests
no
re-
lationship
between
the
resistance
of
these
mutants
to
fungal
infection
(3,
4,
5,
9)
and
the
enzyme
potential
of
the
fungus
within
their
tissues.
Much
similarity
has
been
found
between
PG
and
PMG
activities
underlying
Rhizopus
infection
of
toma-
toes.
However,
PMG
activity
is
not
necessarily
due
to
a
single
enzyme.
Being
composed
of
several
components,
of
which
PG
is
an
important
one,
the
reduction
of
pectin
viscosity
could
be
due
to
a
combination
of
enzymes.
The
general
similarity
between
Figures
1
and
2
tends
to
support
this.
In
all
cases,
pectolytic
activity
of
the
infected
mutant
fruit
was
directly
related
to
the
rate
of
induced
fruit
softening.
The
fact
that
softening
of
infected
fruit
pro-
gresses
gradually
from
the
site
of
infection,
whereas
softening
of
the
normal
uninfected
fruit
occurs
even-
ly
over
the
whole
fruit,
may
suggest
that
the
induced
softening
is
the
result
of
activity
of
fungal
enzymes
rather
than
of
the
ability
of
the
fungus
to
trigger
the
mechanism
of
fruit
softening.
However,
in
order
to
establish
whether
pectolytic
activity
in
infected
to-
mato
fruits
is
of
host
or
of
pathogen
origin,
differ-
ential
identification
of
fungal
and
fruit
polygalac-
turonases
should
be
followed.
The
relatively
high
levels
of
C
x
-cellulase
recorded
in
infected
mutant
fruits
at
early
stages
of
disease
development,
when
only
slight
or
no
softening
has
yet
been
induced,
suggest
a
lack
of
relationship
between
softening
and
the
level
of
cellulase
activity
in
tomato
fruits,
supporting
the
assumption
of
Hobson
(7)
that
the
contribution
of
cellulase
to
softening
is
of
minor
importance
in
tomatoes
and
that
fruit
softening
is
associated
with
the
activity
of
pectolytic
enzymes.
Acknowledgements
We
thank
Mrs
Rachel
Karadavid
for
technical
assistance.
Rin
and
nor
mutant
tomatoes
were
kindly
supplied
by
Dr
E.
C.
Tigchelaar,
Purdue
University,
Lafayette,
Indiana.
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new
ripening
mutant,
non-ripening
(nor).
Tomato
Genet.
Coop.
23:
33-34.