Direct penetration of Rhizopus stolonifer into stone fruits causing rhizopus rot


Baggio, J. S.; Goncalves, F. P.; Lourenco, S. A.; Tanaka, F. A. O.; Pascholati, S. F.; Amorim, L.

Plant Pathology 65(4): 633-642

2016


Rhizopus rot, caused by Rhizopus stolonifer, is a major postharvest disease of stone fruits. The disease is related to the occurrence of mechanical and physical damage; however, observations at a Brazilian wholesale market suggest that direct penetration can occur. Therefore, the penetration mechanisms of R.stolonifer in stone fruits were evaluated. To identify the production of enzymes that help with direct penetration by the pathogen, esterase activity, both in mycelial discs and in spore suspensions of the fungus in water and in modified Van Etten nutrient solution, was measured. Assays were also conducted to evaluate the growth of R.stolonifer on glucose or cutin as a sole carbon source. The pathogen grew on both media, and higher esterase activity was observed in the cutin medium. Wounded and unwounded peaches and nectarines were inoculated with R.stolonifer spore suspensions in water or in modified Van Etten nutrient solution. Wounded fruit inoculated with either of the R.stolonifer spore suspensions developed rhizopus rot, whereas unwounded fruit developed the rot only in the presence of spores in the modified Van Etten nutrient solution. Scanning electron and light microscopic examination showed the fungus can directly penetrate the nectarine cuticle. Diisopropyl fluorophosphate, a serine hydrolase inhibitor, prevented rot development in peaches. The results provide valuable evidence for the ability of R.stolonifer to directly penetrate unwounded stone fruits, probably due to the production of esterase enzymes.

Plant
Pathology
(2016)
65,
633-642
Doi:
10.1111/ppa.12434
Direct
penetration
of
Rhizopus
stolonifer
into
stone
fruits
causing
rhizopus
rot
J.
S.
Baggio,
F.
P.
Gongalves,
S.
A.
Lourengo,
F.
A.
0.
Tanaka,
S.
F.
Pascholati
and
L.
Amorim*
Departamento
de
Fitopatologia
e
Nematologia,
Escola
Superior
de
Agricultura
"Luiz
de
Queiroz",
Universidade
de
SS
-
0
Paulo,
Piracicaba,
SP
13418-900,
Brazil
Rhizopus
rot,
caused
by
Rhizopus
stolonifer,
is
a
major
postharvest
disease
of
stone
fruits.
The
disease
is
related
to
the
occurrence
of
mechanical
and
physical
damage;
however,
observations
at
a
Brazilian
wholesale
market
suggest
that
direct
penetration
can
occur.
Therefore,
the
penetration
mechanisms
of
R.
stolonifer
in
stone
fruits
were
evaluated.
To
identify
the
production
of
enzymes
that
help
with
direct
penetration
by
the
pathogen,
esterase
activity,
both
in
mycelial
discs
and
in
spore
suspensions
of
the
fungus
in
water
and
in
modified
Van
Etten
nutrient
solution,
was
measured.
Assays
were
also
conducted
to
evaluate
the
growth
of
R.
stolonifer
on
glucose
or
cutin
as
a
sole
carbon
source.
The
pathogen
grew
on
both
media,
and
higher
esterase
activity
was
observed
in
the
cutin
medium.
Wounded
and
unwounded
peaches
and
nec-
tarines
were
inoculated
with
R.
stolonifer
spore
suspensions
in
water
or
in
modified
Van
Etten
nutrient
solution.
Wounded
fruit
inoculated
with
either
of
the
R.
stolonifer
spore
suspensions
developed
rhizopus
rot,
whereas
unwounded
fruit
developed
the
rot
only
in
the
presence
of
spores
in
the
modified
Van
Etten
nutrient
solution.
Scanning
electron
and
light
microscopic
examination
showed
the
fungus
can
directly
penetrate
the
nectarine
cuticle.
Diisopropyl
fluorophos-
phate,
a
serine
hydrolase
inhibitor,
prevented
rot
development
in
peaches.
The
results
provide
valuable
evidence
for
the
ability
of
R.
stolonifer
to
directly
penetrate
unwounded
stone
fruits,
probably
due
to
the
production
of
esterase
enzymes.
Keywords:
esterases,
infection,
postharvest
diseases,
Prunus,
Rhizopus
stolonifer
Introduction
Considerable
postharvest
losses
are
reported
in
fruits
due
to
physical,
physiological
and
pathological
damage,
which
can
occur
during
harvesting,
grading,
packing
and
transportation
to
markets.
Physical
damage
to
fruit
is
important
for
pathogen
penetration,
especially
for
fungi
that
cause
postharvest
rots
(Agrios,
2005).
Postharvest
damage
during
marketing
can
cause
up
to
50%
loss
of
stone
fruits
(Martins
et
al.,
2006).
Studies
carried
out
in
a
wholesale
market
in
Brazil
showed
a
correlation
between
mechanical
damage
and
postharvest
diseases
in
stone
fruits
(Martins
et
al.,
2006;
Amorim
et
al.,
2008).
In
Brazil,
diseases
are
the
main
cause
of
price
reduction
among
all postharvest
damage
in
peaches.
For
example,
a
1%
increase
in
disease
incidence
results
in
a
0.91
and
1.24%
reduction
in
sale
price
in
wholesale
and
retail
markets,
respectively
(Lima
et
al.,
2009).
Rhizopus
rot,
caused
by
Rhizopus
stolonifer,
is
one
of
the
most
important
postharvest
diseases
of
stone
fruits
(Ogawa,
1995).
The
disease
occurs
mainly
on
ripe
fruit,
which
are
more
prone
to
wounds
and
have
higher
sugar
*E-mail:
Published
online
6
August
2015
©
2015
British
Society
for
Plant
Pathology
content.
After
2-3
days,
infected
fruits
become
soft
and
watery,
and
they
release
juices
with
a
fermented
or
acidic
odour.
Under
favourable
temperatures
and
humidity,
approximately
one
day
after
the
initial
symptoms
appear,
a
rapid
and
abundant
mycelial
growth
can
be
observed
on
the
surface
of
the
infected
fruit
and
the
pathogen
pro-
duces
long
mycelial
stolons
with
black
sporangia
and
spores
(Snowdon,
1990).
Rhizopus
spp.
are
cosmopolitan,
filamentous,
lower
fungi
found
in
soil,
decayed
fruits
and
vegetables,
and
commonly
observed
in
packing
houses
(Snowdon,
1990;
Massola
Junior
&
Krugner,
2011).
They
can
cause
postharvest
rots
in
many
vegetables
and
fruits
(Agrios,
2005),
including
potato,
aubergine,
watermelon,
melon,
cucumber
(Harter
8c
Weimer,
1922),
sweet
potato
(Walker,
1972),
grape
(Snowdon,
1990;
Tavares
8c
Silva,
2006),
strawberry
(Maas,
1998),
raspberry,
blackberry
(Davis,
1991)
and
stone
fruits
(Ogawa,
1995).
According
to
the
literature
(Harter
8c
Weimer,
1922;
Davis,
1991;
Ogawa
8c
English,
1991;
Maas,
1998;
Tavares
8c
Silva,
2006;
Bautista-Banos
et
al.,
2014),
Rhizopus
is
strictly
a
wound
parasite,
and
it
can
only
penetrate
host
tissues
through
fresh
wounds,
microwounds
and
bruises
made
by
harvesting,
handling
and/or
insects.
This
is
most
likely
due
to
its
inability
to
produce
specific
enzymes
that
help
in
the
penetration
process
(Nguyen-The
8c
Chamel,
1991).
Despite
these
claims,
the
presence
of
rhizopus
rot
was
observed
on
apparently
unwounded
stone
fruits
dur-
633
BSPP
0)
0
0
CL
CL
634
J.
S.
Baggio
et
al.
ing
surveys
carried
out
in
a
wholesale
market
in
Sao
Paulo,
Brazil
(authors'
unpublished
data),
suggesting
that
R.
stolonifer
has
additional
penetration
strategies.
It
is
known
that
R.
stolonifer
spores
require
external
nutrient
sources
to
germinate
(Medwid
8c
Grant,
1984;
Nguyen-
The
et
al.,
1989);
therefore,
it
is
possible
that
the
inabil-
ity
of
Rhizopus
to
directly
penetrate
its
hosts
is
due
to
its
incapability
to
germinate
on
an
intact
fruit
surface
where
external
nutrients
are
not
available.
If
the
fungus
cannot
germinate
it
is
unable
to
produce
specific
enzymes,
such
as
esterases,
which
can
help
to
penetrate
the
plant
cuticle
and
cell
wall.
The
production
of
esterases,
especially
cutinases
(EC
3.1.1.74),
is
essential
for
some
pathogens
to
directly
pen-
etrate
their
hosts;
for
example
Colletotrichum
gramini-
cola
in
corn
and
sorghum
(Pascholati
et
al.,
1993),
Uromyces
viciae-fabae
in
beans
(Deising
et
al.,
1992)
and
Pestalotia
malicula
in
plum
(Sugui
et
al.,
1998).
In
these
studies,
the
enzymes
were
found
in
spore
exudates
and/or
mucilage,
and
the
pathogens
were
capable
of
cutin
degradation.
To
confirm
the
presence
and
activity
of
these
enzymes,
diisopropyl
fluorophosphate
(DIPF),
an
inhibitor
of
serine
hydrolase
enzymes,
including
esterases
and
proteases
(Cohen
et
al.,
1967),
was
used.
DIPF
was
able
to
block
cutinase
activity,
and
prevent
disease
devel-
opment
on
corn
leaves
inoculated
with
C.
graminicola
(Pascholati
et
al.,
1993).
However,
reports
of
the
exis-
tence
of
enzymes
that
can
help
R.
stolonifer
penetrate
directly
into
fruits
are
rare;
most
of
them
refer
to
enzymes
of
the
parasitic
process
of
establishment
and
host
colonization
by
the
fungus
(Spalding,
1963;
Wells,
1968).
It
has
already
been
shown
that
the
pathogen
can
synthesize
enzymes
that
destroy
plant
tissues
(Sommer
et
al.,
1963);
however,
none
were
identified
as
degraders
of
fruit
cuticle
components
that
may
facilitate
penetra-
tion
by
the
fungus.
The
single
report
that
studied
the
enzymatic
degradation
of
nectarine
epidermis
by
R.
stolonifer
showed
that
the
pathogen
penetration
apparently
did
not
involve
cutinolytic
enzymes
(Nguyen-
The
8c
Chamel,
1991).
Despite
the
information
that
fruit
infection
by
R.
stolonifer
occurs
through
wounds
or
directly
by
myce-
lial
stolons,
producing
the
symptoms
known
as
nested
infection,
little is
known
about
the
mechanisms
of
fruit
penetration
by
spores
of
this
pathogen.
Therefore,
the
objective
of
this
study
was
to
evaluate
the
penetration
mechanisms
of
R.
stolonifer
in
stone
fruits.
Materials
and
methods
Pathogen
identification
One
isolate
of
R.
stolonifer
was
collected
from
diseased
peaches
obtained
from
a
wholesale
market
in
Brazil
and
identified
by
molecular
techniques.
DNA
was extracted,
and
the
ITS
region
of
the
gene
was
amplified
by
PCR
using
the
forward
primer
ITS1
(5'-
TTCCGTAGGTGAACCTGCGG-3'
and
the
reverse
primer
ITS4
(5'-TCCTCCGCTTATTGATATGC-3'
(White
et
al.,
1990).
PCR
products
amplified
by
these
primers
gave
a
fragment
of
900
bp.
The
purified
product
was
sequenced
and
compared
with
the
DNA
sequences
of
R.
stolonifer
deposited
at
GenBank
(accession
num-
bers
EU622265,
AF117935
and
AF117936).
The
fungus
was
grown
on
potato
dextrose
agar
(PDA)
(Oxoid
Ltd)
and
kept
at
25°C
for
3
days
under
constant
fluorescent
light
to
promote
mycelial
growth
and
sporulation.
Spore
germination
To
test
the
germination
of
R.
stolonifer
in
vitro,
spore
suspen-
sions
were
produced
by
adding
sterile
distilled
water
or
nutrient
solution
to
a
3-day-old
culture.
The
nutrient
solution
contained
20
g
glucose,
2
g
asparagine,
0.5
g
KH
2
PO
4
and
26
g
MgSO
4
in
1
L
water
(Van
Etten
et
al.,
1969).
Spore
concentration
was
determined
using
a
haemocytometer
and
adjusted
to
10
5
spores
mL
-1
.
Four
aliquots
(40
µ1..
each
one)
of
the
spore
sus-
pension
were
incubated
in
sterile
Petri
dishes.
The
Petri
dishes
were
kept
inside
a
plastic
container
with
water,
to
provide
high
humidity.
The
containers
were
kept
at
25°C
for
4,
6,
8,
12,
24
and
48
h.
Three
replications
were
performed.
Germination
of
sporangiospores
was
noted
when
the
germ
tube
length
was
equal
to
or
greater
than
the
diameter
of
the
spore.
The
percentage
germination
was
determined
by
counting
the
first
100
spores
observed
under
a
microscope
at
x400
mag-
nification.
The
experiment
was
carried
out
twice.
Qualitative
evaluation
of
esterase
activity
The
spore
suspensions,
obtained
as
described
above,
were
adjusted
to
two
inoculum
concentrations,
10
5
and
10
6
spores
mL
-1
.
The
suspensions
were
used
immediately
after
preparation
or
kept
at
25°C
on
a
100-rpm
shaker
for
4
or
8
h
to
promote
spore
germination.
Colletotrichum
graminicola,
a
corn
pathogen
known
to
produce
cutinase,
was
grown
on
oat-
meal
agar
and
kept
at
21°C
for
10-14
days
under
fluorescent
light
(Pascholati
et
al.,
1993).
A
spore
suspension,
used
as
a
pos-
itive
control,
was
obtained
by
adding
sterile
distilled
water
and
adjusting
the
concentration
to
10
5
spores
mL
-1
.
To
determine
esterase
activity,
an
assay
was
performed,
based
on
the
hydrolysis
of
indoxyl
acetate,
which
results
in
the
forma-
tion
of
insoluble
crystals
of
indigo
blue.
The
indoxyl
acetate
substrate
(35
mg)
was
dissolved
in
1
mL
acetone
and
added
to
49
mL
0.05
M
Tris-HC1
buffer
(pH
8.0)
to
give
a
final
concen-
tration
of
0.7
mg
mL
-1
indoxyl
acetate
(Pascholati
et
al.,
1993).
Subsequently,
spore
suspensions
were
prepared
in
water
or
nutrient
solution,
at
different
concentrations
(Table
1)
and
incubated
at
25°C
for
0,
4
or
8
h,
as
described
above.
Addition-
ally
mycelial
discs
of
the
fungus
(0.5
cm
in
diameter)
added
to
sterile
distilled
water
were
also
tested
for
esterase
activity.
Con-
trols
were
also
prepared
with
Tris-HC1
buffer
alone;
buffer
and
indoxyl
acetate
only;
and
C.
graminicola
spore
suspension
with
buffer
only
or
with
buffer
and
indoxyl
acetate.
After
adding
the
indoxyl
acetate
solution,
the
test
tubes
were
incubated
at
25°C
for
a
further
30
min
and
30
µ1..
aliquots
of
each
treatment
were
then
placed
on
a
polystyrene
Petri
dish
and
the
formation
of
insoluble
crystals
of
indigo
blue
observed
under
a
light
micro-
scope.
The
experiment
was
conducted
three
times.
Quantitative
evaluation
of
esterase
activity
Rhizopus
stolonifer
was
grown
on
a
liquid
medium
made
of
10
g
glucose,
2
g
asparagine,
1
g
KH
2
PO
4
,
0.5
g
MgSO
4
.7H
2
0,
0.2
mg
Fe
3
',
0.2
mg
Zn
2
',
0.1
mg
Mn
2
',
5µg
biotin
and
100
Plant
Pathology
(2016)
65,
633-642
Rhizopus
direct
penetration
into
peaches
635
Table
1
Qualitative
evaluation
of
esterase
activity
exhibited
by
Rhizopus
stolonifer
and
Colletotrichum
graminicola
Code
Treatment
Tris-HCI
(mL)
Indoxyl
C.
graminicola
acetate
(mL)
(mL)
a
R.
stolonifer
mycelium
T
i
4
T2
2 2
T3
3
T4
1
2
T5
4
5
disks
T6
2 2
5
disks
T7
3
T8
1
2
T3
1
2
Tio
1
2
Ti
1
2
R.
stolonifer
spore
suspension
Esterase
activity
b
mL
in
water,
10
5
spores
mL
-1
mL
in
water,
10
5
spores
mL
-1
mL
in
water,
10
6
spores
mL
-1
mL
in
nutrient
solution,
10
5
spores
mL
-1
mL
in
nutrient
solution,
10
6
spores
mL
-1
a
10
5
spores
mL
-1
.
b
(+)
presence
or
(-)
absence
of
indigo
blue
crystals
from
hydrolysis
of
indoxyl
acetate,
indicating
esterase
activity.
thiamine
in
1
L
water
(Lilly
&
Barnett,
1951),
and
the
same
medium
with
glucose
replaced
by
apple
cutin,
an
insoluble
polyester
of
plant
cuticle
(Kolattukudy,
1980).
Colletotrichum
graminicola
was
also
grown
in
both
media.
Fungal
mycelium
discs
(0.5
cm
in
diameter)
were
separately transferred
to
Erlenmeyer
flasks
containing
25
mL
of
the
medium.
Three
Erlenmeyer
flasks
were
used
for
each
treatment.
The
pathogens
were
not
added
to
the
control
treatments.
The
Erlenmeyer
flasks
were
kept
at
25°C
on
a
shaker
(100
rpm)
for
4
days
for
R.
stolonifer
and
10
days
for
C.
graminicola.
The
contents
of
the
Erlenmeyer
flasks
were
fil-
tered
(Whatman
no.
1),
and
the
resultant
filtrate
centrifuged
at
17
787
g
for
20
min
at
4°C.
The
supernatant
was
filtered
through
a
mixed
cellulose
esters
membrane
filter,
with
0.22
pm
pore
size
(MF-Millipore;
Merck)
and
the
filtrate
was
collected
inside
microtubes
and
assayed
in
a
spectrophotometer.
The
presence
of
esterase
activity
was
confirmed
by
detection
of
the
hydrolysis
of
p-nitrophenyl
butyrate
to
p-nitrophenol,
with
the
resultant
pro-
duct
measured
using
a
spectrophotometer
at
400
nm
(Pascholati
et
al.,
1993).
Fungal
growth
was
quantified
by
fresh
weight
of
the
biomass
after
removal
of
the
liquid
medium.
The
reaction
mixture
for
esterase
consisted
of
800
pi.
Tris-
HC1
buffer
(0.05
M
,
pH
8.0),
100
pL
enzyme
preparation
of
each
fungus
and
100
pL
stock
solution
of
p-nitrophenyl
buty-
rate.
Controls
for
the
enzyme
assays
were
performed
with
iden-
tical
reaction
mixtures
and
filtered
culture
medium,
which
had
not
been
exposed
to
the
fungus,
as
a
negative
control.
The
stock
solution
of
13
mm
p-nitrophenyl
butyrate
was
diluted
in
the
same
reaction
buffer
to
give
a
final
concentration
of
1.3
mm.
The
enzyme
reaction
was
carried
out
for
3
min,
and
each
treat-
ment
was
repeated
six
times.
The
protein
concentration
of
the
samples
was
determined
by
a
Bradford
assay
(Bradford,
1976)
with
bovine
serum
albumin
as
the
standard.
Each
treatment
was
read
six
times.
Thus,
esterase
activity
was
expressed
as
the
absorbance
(A)
variation
per
minute
per
milligram
of
protein
(A
A
min
-1
mg
-1
of
protein).
Enzyme
activity
and
fungal
mass
data
were
subjected
to
anal-
ysis
of
variance
and
Tukey's
test
(P
<
0.05)
using
sTATIsTicA
soft-
ware
(Statsoft).
Inoculation
of
peaches
and
nectarines
with
IL
stolonifer
Each
experiment
consisted
of
four
treatments
with
30
replica-
tions.
Mature
fruits
(soluble
solids
from
8.35
to
8.85
Brix,
and
firmness
from
0.77
to
1.83
kgf)
were
surface-disinfected
with
0.5%
sodium
hypochlorite
solution
for
3
min.
The
fruits
were
placed
individually
on
plastic
nests
without
touching
each
other.
Treatments
evaluated
were:
(i)
wounded
fruit
inoculated
with
a
spore
suspension
in
water
(W-W);
(ii)
unwounded
fruit
inoculated
with
a
spore
suspension
in
water
(U-W);
(iii)
wounded
fruit
inoculated
with
a
spore
suspension
in
nutrient
solution
(W-N);
(iv)
unwounded
fruit
inoculated
with
a
spore
suspension
in
nutrient
solution
(U-N);
and
a
control
treatment
with
water
or
nutrient
solution
that
did
not
contain
pathogen
spores.
Fruits
in
treatments
(i)
and
(ii)
were
wounded
(1
mm
in
diam-
eter
by
3
mm
in
depth)
with
a
hypodermic
needle.
A
30-pL
ali-
quot
of
R.
stolonifer
spore
suspension
(10
5
spores
mL
-1
)
was
placed
over
the
equatorial
region
of
the
wounded
and
unwounded
fruits.
The
fruits
were
then
incubated
at
25°C
in
the
dark,
in
a
humid
chamber
for
24
h.
Rhizopus
rot
incidence
was
assessed
for
5
days
after
inoculation.
Two
cultivars
of
peaches
(Dourado
and
Chiripa,
two
experiments
each)
and
two
cultivars
of
nectarines
(Sunripe
and
Josefina,
one
experiment
each)
were
used.
The
final
incidence
of
disease
among
the
different
treatments
was
compared
by
nonparametric
comparison
tests
of
multiple
proportions
(Zar,
1999).
If
the
null
hypothesis
of
equal
propor-
tions
was
rejected,
a
Tukey-type
multiple
comparison
testing
among
the
proportions
was
carried
out
based
on
the
angular
transformation
(Zar,
1999).
Effect
of
DIPF
on
peaches
inoculated
with
R.
stolonifer
The
influence
of
DIPF
on
the
spore
germination
of
R.
stolonifer
and
the
infection
of
peach
by
the
pathogen
was
evaluated
on
polystyrene
Petri
dishes
and
inoculated
peaches,
respectively.
Spore
suspensions
were
prepared
in
nutrient
solution
as
described
above.
Aliquots
of
20
pi.
R.
stolonifer
spore
suspen-
sion
(10
5
spores
mL
-1
)
were
placed
on
a
Petri
dish
surface
and
20
pL
of
a
stock
solution
of
200
pm
DIPF
(Sigma-Aldrich)
in
Tris-HC1
buffer
(0.05
M,
pH
8.0)
added.
The
control
treatment
consisted
of
the
spore
suspension
alone.
The
Petri
dishes
were
kept
inside
a
germination
box
with
a
wet
filter
paper
to
produce
a
moist
chamber.
The
germination
boxes
were
kept
at
25°C
for
24
h
in
the
dark.
Four
aliquots
per
dish
and
three
dishes
per
Plant
Pathology
(2016)
65,
633-642
:
636
J.
S.
Baggio
et
al.
treatment
were
used.
Germination
assessment
was
based
on
the
observation
of
100
spores
under
the
light
microscope.
Mature
peaches
were
surface-disinfected
with
0.5%
sodium
hypochlorite
solution
for
3
min,
and
placed
individually
on
plas-
tic
nests
without
touching
each
other.
Treatments
evaluated
were:
(i)
40
µL
R.
stolonifer
spore
suspension;
(ii)
20
µL
spore
suspension
and
20
µL
of
a
stock
solution
of
DIPF
at
the
time
of
inoculation;
(iii)
20
µL
spore
suspension
and
20
µL
DIPF
1
h
after
inoculation;
(iv)
20
µL
spore
suspension
and
20
µL
DIPF
2
h
after
inoculation;
(v)
20
µL
spore
suspension
and
20
µL
DIPF
4
h
after
inoculation;
(vi)
20
µL
spore
suspension
and
20
µL
DIPF
6
h
after
inoculation;
(vii)
40
µL
nutrient
solution
without
R.
stolonifer
spores;
and
(viii)
20
µL
nutrient
solution
and
20
µL
DIPF
without
R.
stolonifer
spores.
Rhizopus
rot
inci-
dence
was
assessed
3
days
after
inoculation,
and
fruits
that
had
softened
in
the
region
of
pathogen
deposition
were
considered
diseased.
Five
peaches
per
treatment
were
used
and
the
experi-
ments
were
repeated
once.
The
disease
incidence
was
compared
among
the
different
treatments
by
nonparametric
comparison
tests
of
multiple
proportions
(Zar,
1999).
Nectarine
penetration
by
R.
stolonifer
observed
under
the
scanning
electron
microscope
(SEM)
Nectarines
were
surface-disinfected
with
0.5%
sodium
hypochlorite
solution
for
3
min
and
received
the
same
treat-
ments
previously
described
for
inoculation
of
peaches
and
nec-
tarines
with
R.
stolonifer.
The
fruits
were
kept
on
nests
inside
plastic
containers,
without
touching
each
other,
and
then
incu-
bated
in
a
humid
chamber
at
25°C
in
the
dark
for
10
h.
Six
fruits
per
treatment
were
used.
Three
fruits
were
washed
with
Table
2
Rhizopus
stolonifer
spore
germination
(%)
in
water
or
nutrient
solution,
after
different
periods
of
incubation
Treatment
Water
Nutrient
solution
Incubation
(h)
Mean
a
Range
b
Mean
a
Range
b
4
1.6
1-3
7.3
6-8
6
5.3
2-8
83.6
82-88
8
7.6
6-8
90.5
85-95
12
7.8
4-13
96.2
95-98
24
7.9
5-11
100.0
48
9.6
4-15
100.0
a
Values
obtained
from
two
experiments
of
100
spores
each.
b
Minimum
and
maximum
values
of
spore
germination
from
the
two
experiments.
distilled
water
and
brushed
with
a
delicate
paintbrush
to
remove
the
pathogen
structures
from
the
nectarine
surface.
Fragments
(1
x
1
cm)
were
excised
from
the
inoculated
regions
of
fruit
from
all
treatments
for
analysis
by
scanning
elec-
tron
microscopy.
The
fragments
were
exposed
to
2%
(w/v)
osmium
tetroxide
(0s0
4
)
vapour
for
12
h
(Kim,
2008),
and
transferred
into
a
container
with
silica
gel
present
to
dry.
The
fragments
were
sputter-coated
(SCD
050
Bal
Tec)
and
examined
with
an
LEO
435
VP
scanning
electron
microscope
(Leo
Elek-
tronenmikroskopie
GmbH).
Nectarine
penetration
by
R.
stolonifer
observed
under
the
light
microscope
Unwounded
nectarines
were
surface-disinfected
and
inoculated
with
30
µL
of
R.
stolonifer
spore
suspension
(10
5
spores
mL
-1
)
in
nutrient
solution
or
with
30
µL
nutrient
solution
without
the
pathogen
(control).
The
fruits
were
kept
inside
plastic
containers
and
then
incubated
in
the
dark
in
a
humid
chamber
at
25°C
for
12
h.
Fragments
(1
x
1
cm)
were
excised
from
the
inoculated
regions
and
fixed
in
Karnovsky
solution
(Karnovsky,
1965).
The
samples
were
subsequently
dehydrated
through
a
graded
alcohol
series
and
embedded
in
methacrylate
(Historesin;
Leica
Instru-
ments).
Using
a rotating
microtome,
5-Am-thick
sections
were
cut
and
stained.
For
histological analysis,
samples
were
stained
with
toluidine
blue
(O'Brien
&
McCully,
1981)
and
permanent
slides
were
mounted
in
the
synthetic
resin
Entellan.
Slides
were
viewed
under
a
Zeiss
Axioskop
2
microscope
and
digital
images
were
captured
with
an
attached
camera
connected
to
a
computer.
Results
Spore
germination
The
spore
germination
of
R.
stolonifer
was
higher
in
the
presence
of
nutrient
solution
than
in
water
(Table
2;
Fig.
1).
Spore
germination
rates
in
water
ranged
from
1.6%
at
4
h,
to
9.6%
at
48
h,
whereas
in
nutrient
solution
the
germination
rates
were
higher
than
80%
after
6
h,
and
reached
100%
at
24
h.
Differences
in
the
morphology
of
the
spores
and
germ
tubes
of
R.
stolonifer
in
nutrient
solution
and
water
were
also
observed
(Fig.
1).
When
in
nutrient
solution,
the
structures
of
the
fungus
became
swollen,
thickened
and
granular
in
appearance.
The
germ
tubes
tended
to
clump
together
to
form
mycelial
pellets
after
extended
periods
of
incubation.
Figure
1
Germination
of
Rhizopus
stolonifer
spores
in
water
(a)
and
nutrient
solution
(b)
after
8
h
of
incubation.
In
(b)
thick
hyphae
can
be
seen.
Bars
represent
25
gm.
Plant
Pathology
(2016)
65,
633-642
(a)
100
80
60
-
,
to
-
20
Inc
ide
nce
(
%
o
f
disease
d
fru
it)
Wounded
98.3a
98
3a
96.78
Unwounded
0
Nuiden
solution
Water
Nutrien
solution
Ob
Water
Treatments
Rhizopus
direct
penetration
into
peaches
637
Qualitative
and
quantitative
evaluation
of
esterase
activity
For
qualitative
esterase
activity,
the
indigo
blue
colour
was
observed
30
min
after
addition
of
indoxyl
acetate
to
the
C.
graminicola
and
R.
stolonifer
suspensions
(Table
1).
In
the
case
of
the
R.
stolonifer
spore
suspension
in
nutrient
solution
(10
6
spores
mL
-1
,
treatment
T
11
)
with
shaking,
the
darkest
blue
colour
was
observed
after
8
h
Table
3
Fresh
mass
of
Rhizopus
stolonifer
and
Colletotrichum
graminicola
colonies
grown
on
culture
media
containing
glucose
or
cutin
as
a
sole
carbon
source
Fresh
mass
(g)
a
Carbon
source
R.
stolonifer
b
C.
graminicola
Glucose
5.97
Aa
4.99
Ab
Cutin
4.50
Ba
3.91
Bb
a
values
obtained
by
mean
of
three
repetitions.
Values
followed
by
the
same
upper
case
letters
in
the
columns
and
the
same
lower
case
let-
ters
in
the
rows
do
not
differ
among
them
according
to
Tukey's
test
(P
<
0.05).
b
Evaluations
carried
out
after
4
and
10
days
for
R.
stolonifer
and
C.
graminicola,
respectively.
(data
not
shown).
The
presence
of
indigo
blue
crystals
was
detected
inside
and
surrounding
the
spores
(not
shown).
Rhizopus
stolonifer
and
C.
graminicola
were
able
to
grow
on
culture
media
that
had
glucose
or
cutin
as
a
sole
carbon
source.
The
highest
fresh
mass
weight
values
were
obtained
when
the
fungi
were
grown
on
glucose
medium,
and
R.
stolonifer
grew
more
prolifically
than
C.
graminicola
(Table
3).
The
values
of
esterase
activity
were
0.198
and
0.140
AOD
min
-1
mg
-1
protein
for
C.
graminicola
and
0.011
and
0.044
A
A
min
-1
mg
-1
protein
for
R.
stolonifer,
in
glucose
and
cutin
culture
media,
respectively.
Rhizopus
rot
on
peaches
and
nectarines
In
all
experiments,
infection
of
peaches
and
nectarines
by
R.
stolonifer
did
not
occur
in
unwounded
fruits
when
the
spores
were
suspended
in
water
(U-W
treatment),
whereas
infection
occurred
in
unwounded
fruits
when
spores
were
suspended
in
nutrient
solution
(U-N
treat-
ment;
Figs
2
8c
3).
Both
treatments
with
wounded
fruits
showed
more
than
80%
of
rhizopus
rot
incidence
in
all
experiments
(Figs
2
8c
3).
Rhizopus
rot
occurred
in
96.7
and
93.3%
for
Dourado
and
Chiripa
peaches,
respectively,
in
the
U-N
treatment
Figure
2
Incidence
of
rhizopus
rot
in
Dourado
(a)
and
Chiripa
(b)
peach
cultivars
5
days
after
inoculation
with
Rhizopus
stolonifer.
The
data
represent
the
mean
of
two
experiments
of
30
fruits
each.
Bars
followed
by
the
same
letter
do
not
differ
significantly
at
the
5%
level
by
a
nonparametric
comparison
test
of
multiple
proportions
(Zar,
1999).
Figure
3
Incidence
of
rhizopus
rot
in
Sunripe
(a)
and
Josefina
(b)
nectarine
varieties
5
days
after
inoculation
with
Rhizopus
stolonifer.
Bars
followed
by
the
same
letter
do
not
differ
significantly
at
the
5%
level
by
a
nonparametric
comparison
test
of
multiple
proportions
(Zar,
1999).
(a)
Wounded
Unwounded
100
90a
83.3a
80
-
cu
60
0
'se
8
-
=
c
26.7b
20
-
0
Oc
Nutrien
Water
Nutrient
Water
solution
solution
Treatments
(b)
Wounded
Unwounded
100
100a
98.3a
93.3a
60
40
20
0
Ob
Nutrient
Water
Nutrien
Water
solution
solution
Treatments
(b)
Wounded
Unwounded
100
-
100a
100a
100a
80
-
60
40
20
0
Ob
Nutrient
Water
Nutrient
Water
solution
solution
Treatments
Plant
Pathology
(2016)
65,
633-642
638
J.
S.
Baggio
et
al.
Table
4
Disease
incidence
of
peaches
with
rhizopus
rot
symptoms
treated
with
diisopropyl
fluorophosphate
(DIPF)
at
different
periods
following
inoculation
with
Rhizopus
stolonifer
Time
of
DIPF
addition
to
the
fruit
after
inoculation
(h)
Incidence
(%)
a
Control
b
100
a
0
50
b
1
50
b
2
10
b
4
30
b
6
30
b
Values
followed
by
the
same
letters
in
the
column
do
not
differ
signifi-
cantly
at
the
5%
level
by
a
nonparametric
comparison
test
of
multiple
proportions
(Zar,
1999).
a
Values
obtained
by
mean
of
two
experiments
of
five
fruits
each.
b
Fruit
not
treated
with
DIPF.
(Fig.
2).
When
the
peaches
were
wounded,
the
disease
incidence
was
higher
than
95%
(Fig.
2).
rhizopus
rot
incidence
was
27
and
100%
for
Sunripe
and
Josefina
nectarines,
respectively,
in
the
U-N
treatment
(Fig.
3).
Effect
of
DIPF
on
peaches
inoculated
with
R.
stolonifer
Rhizopus
stolonifer
spores
treated
with
DIPF
showed
100%
germination
and
did
not
exhibit
abnormalities
in
their
development
(data
not
shown).
Some
inoculated
peaches
treated
with
DIPF
showed
rhizopus
rot
symp-
toms;
however,
the
disease
incidence
did
not
exceed
50%,
whereas
100%
of
the
non-treated
fruits
were
infected
(Table
4).
In
some
fruits,
DIPF
was
not
able
to
prevent
mycelial
growth
by
R.
stolonifer
on
peach
sur-
faces;
however,
rot
was
not
observed
and
the
mycelia
were
formed
only
as
the
result
of
spore
germination
in
the
nutrient
solution
(Fig.
4).
Nectarine
penetration
by
R.
stolonifer
observed
under
the
SEM
and
light
microscopes
There
was
neither
spore
germination
nor
penetration
on
unwounded
nectarines
inoculated
with
the
spore
suspen-
sion
of
R.
stolonifer
in
water
(Fig.
5a).
In
contrast,
fun-
gal
growth
around
the
wound
was
observed
on
wounded
fruit
inoculated
with
the
spore
suspension
in
water
(Fig.
5b).
Regardless
of
the
presence
of
wounding,
rhizopus
rot
developed
on
nectarine
surfaces
when
nutri-
ent
solution
was
used
(Fig.
5c,d).
A
swollen
hypha
at
the
end
of
the
germ
tube
was
observed
on
the
intact
surface
of
wounded
nectarines
inoculated
with
spore
suspension
in
water.
The
same
was
observed
on
wounded
and
unwounded
fruits
inoculated
with
spore
suspension
in
nutrient
solution
(Fig.
.5e).
Rhizopus
stolonifer
spores
in
water
or
nutrient
solution
placed
on
wounded
nectarines
germinated
and
penetrated
the
fruit
through
the
unwounded
surface,
showing
that
direct
penetration
can
occur.
Direct
penetration
of
the
intact
nectarine
surface
by
R.
stolonifer
was
observed
by
light
microscopy
after
inoculation
of
the
fruit
with
a
spore
suspension
in
nutri-
ent
solution
(Fig.
6a).
The
edge
of
the
hypha
became
swollen,
and
a
penetration
peg
breeched
the
intact
cuticle
of
the
nectarine
surface
(Fig.
6a).
The
same
process
was
also
observed
under
the
SEM,
with
a
swollen
penetration
structure
(appressoria-like)
at
the
end
of
the
germ
tube
(Fig.
6b,c).
When
R.
stolonifer
was
removed
from
the
fruit
surface,
appressoria-like
structures
remained
on
the
nectarine
surface
(Fig.
6d).
It
appeared
that
these
appres-
soria-like
structures
were
involved
in
penetration,
as
degradation
of
the
fruit
surface
was
observed
(Fig.
6b,c,
d).
Discussion
This
study
showed
that
R.
stolonifer
is
able
to
directly
penetrate
unwounded
stone
fruits
when
spores
germinate
in
an
external
nutrient
supply.
Esterases,
especially
cuti-
nases,
are
produced
by
the
fungal
mycelia
and
germ
tubes
after
spore
germination.
Penetration
of
healthy
fruits
by
R.
stolonifer
is
inhibited
when
an
esterase
inhi-
bitor
is
used.
This
study
confirmed
that
R.
stolonifer
was
unable
to
germinate
without
nutrients
in
the
spore
suspension,
in
accordance
with
the
results
of
Nguyen-The
et
al.
(1989).
(a)
S
Figure
4
Peaches,
3
days
after
inoculation
with
a
drop
of
Rhizopus
stolonifer
spore
suspension
in
nutrient
solution,
treated
(a,
b)
or
not
treated
(c)
with
diisopropyl
fluorophosphate
(DIPF)
2
h
after
inoculation.
Mycelia!
growth
was
observed
around
the
site
of
deposition
of
the
spore
suspension
in
(a).
(c)
Plant
Pathology
(2016)
65,
633-642
(a)
(b)
..r
(c)
;
(e)
1
„le-
4
.-,
C
)
:
.
.
Rhizopus
direct
penetration
into
peaches
639
Figure
5
Scanning
electron
microscopy
of
Rhizopus
stolonifer
on
nectarines.
(a)
Unwounded
nectarine
with
nongerminated
R.
stolonifer
spores
(arrows)
in
water,
bar
=
50
gm;
(b)
germinated
R.
stolonifer
spores
(arrow)
in
water
around
a
nectarine
wound
(*),
bar
=
200
gm;
(c)
unwounded
nectarine
surface
with
germinated
R.
stolonifer
spores
in
nutrient
solution,
bar
=
300
gm;
(d)
germinated
R.
stolonifer
spores
in
nutrient
solution
around
a
nectarine
wound
(*),
bar
=
1
mm;
(e)
appressoria-like
structures
(arrows)
at
the
end
of
germ
tubes
(GT)
of
germinated
spores
(S)
in
nutrient
solution
and
penetration
of
the
intact
surface
by
the
pathogen,
bar
=
20
gm.
Thus,
spore
germination
requires
exogenous
sources
of
carbon
and
nitrogen
as
the
spores
contain
insufficient
endogenous
carbon
to
germinate
in
water
(Medwid
Grant,
1984).
The
fungus
requires
from
3
to
5
h
in
nutrient
solution
to
germinate
(Harter
8c
Weimer,
1922)
and
produce
infective
hyphae
(Srivastava
8c
Walker,
1959).
When
in
nutrient
solution,
germinated
spores
become
swollen
and
form
thickened
mycelia,
as
previ-
ously
observed
for
Rhizopus
arrhizus
and
R.
stolonifer
(Buckley
et
al.,
1968).
Rhizopus
stolonifer
spores
treated
with
indoxyl
acetate
showed
a
blue
colour,
as
a
consequence
of
the
formation
of
insoluble
indigo
blue
crystals
due
to
esterase
activity.
These
crystals
were
observed
in
the
C.
graminicola
spore
matrix
(Pascholati
et
al.,
1993)
and
U.
viciae-fabae
(Deising
et
al.,
1992).
These
fungi
produce
esterases,
especially
of
the
cutinase
type,
which
facilitate
spore
adhesion
to
the
host
cuticle
and
direct
penetration.
In
the
present
study
it
was
noted
that
germinated
R.
stolonifer
spores
produced
greater
amounts
of
esterases
than
nongerminated
spores.
This
observation
suggests
that
R.
stolonifer
spores
have
constitutive
ester-
ase
enzymes.
Therefore,
any
incapacity
of
R.
stolonifer
to
penetrate
directly
is
most
probably
due
to
the
absence
of
spore
germination
and
esterase
production.
Rhizopus
spores
release
several
kinds
of
amino
acids,
enzymes
and
other
proteins
during
their
germination
(Van
Etten
et
al.,
1969).
The
fungus
invades
the
host
using
pectinases,
such
as
polygalacturonases
and
pectin
methyl
esterases,
which
macerate
the
host
tissue
during
infection
and
colonization
(Srivastava
et
al.,
1959;
Spald-
ing,
1963;
Wells,
1968).
In
strawberries,
the
fungus
infects
wounded
fruits
and
after
its
establishment
secretes
a
pectolytic
enzyme
responsible
for
degrading
the
middle
lamella,
causing
subsequent
cellular
breakdown
and
fruit
softening
(Maas,
1998).
Germinating
Rhizopus
spores
can
release
these
enzymes,
and
pectolytic
enzymes
are
produced
even
when
the
germination
process
is
inhibited
and
new
fungal
colonies
are
not
formed
(Sommer
et
al.,
1963).
According
to
those
authors,
only
these
enzymes
were
produced
by
R.
stolonifer
and
were
responsible
for
colonization.
However,
the
present
study
demonstrates
that
the
fungal
spores
can
also
produce
esterases,
which
may
be
important
for
the
infection
process.
The
indoxyl
acetate
solution
added
to
the
mycelial
discs
of
R.
stolonifer
showed
a
darker
blue
colour
than
Plant
Pathology
(2016)
65,
633-642
640
J.
S.
Baggio
et
al.
Figure
6
Light
and
scanning
electron
microscopy
of
Rhizopus
stolonifer
on
nectarines.
(a)
Swollen
hypha
(SH)
of
R.
stolonifer
and
a
penetration
peg
(arrow)
invading
the
intact
nectarine
surface,
12
h
after
pathogen
inoculation,
bar
=
20
gm;
(b)
appressoria-like
(AL)
structures
at
the
end
of
R.
stolonifer
germ
tube
and
direct
penetration
into
unwounded
nectarine,
bar
=
10
gm;
(c)
R.
stolonifer
spores
(S),
germ
tubes
(GT)
and
appressoria-like
structures
(arrows)
in
nutrient
solution
on
unwounded
nectarine,
bar
=
20
gm;
(d)
appressoria-like
structures
(arrows)
of
R.
stolonifer
after
removal
of
the
pathogen
mycelium
with
water
and
paint
brush,
bar
=
20
gm.
(b)
'..14%.
4.
AL
(C)
(d)
I
the
spore
suspension,
suggesting
that
the
vegetative
struc-
tures
produce
higher
amounts
of
esterases,
as
has
been
observed
in
tomatoes
inoculated
with
R.
stolonifer
(Velazquez-Del
Valle
et
al.,
2005).
Both
inoculation
methods
produced
100%
infection,
but
the
mycelium
was
more
invasive
than
the
spores
due
to
the
higher
pro-
duction
of
pectolytic
enzymes
responsible
for
cellular
breakdown
(Velazquez-Del
Valle
et
al.,
2005).
Rhizopus
stolonifer,
grown
on
a
medium
with
cutin
as
the
sole
carbon
source,
grew
and
produced
esterase
enzymes.
Previous
investigations
of
Pestalotia
malicola
also
revealed
intense
esterase
activity
when
the
fungus
was
grown
on
cutin
medium
(Sugui
et
al.,
1998).
In
the
present
study,
the
esterase
activity
of
R.
stolonifer
was
fourfold
higher
on
cutin
medium
than
glucose
medium.
This
observation
suggests
that
R.
stolonifer
must
pro-
duce
cutinase-type
enzymes
in
order
to
use
this
carbon
source.
This
is
supported
by
an
investigation
of
Fusarium
solani
f.
sp.
pisi,
a
pea
pathogen,
which
could
grow
on
cutin
medium
and
exhibited
esterase
activity,
99.7%
of
which
was
due
to
cutinase
(Stahl
8c
Schafer,
1992);
mutants
lacking
the
cutinase
gene
did
not
produce
esterases
and
their
growth
was
inhibited
on
the
cutin
media.
Cutinase
is
important
for
fungal
pathogenicity,
and
essential
for
fungal
penetration
through
the
host
cuticle
layer
(Ettinger
et
al.,
1987).
For
example,
cuti-
nase-deficient
mutants
of
Colletotrichum
gloeosporioides
lost
their
pathogenicity
and
ability
to
cause
anthracnose
on
papaya
(Dickman
8c
Patil,
1986).
The
insertion
of
cutinase
genes
in
Mycosphaerella
sp.,
a
pathogen
that
penetrates
papaya
exclusively
through
wounds,
enabled
it
to
penetrate
unwounded
fruits
directly
(Dickman
et
al.,
1989).
Diisopropyl
fluorophosphate
is
an
inhibitor
of
serine
hydrolase
enzymes,
including
esterases
and
proteases
(Cohen
et
al.,
1967),
and
it
is
able
to
block
cutinase
activity
(Pascholati
et
al.,
1993).
Diisopropyl
fluorophos-
phate
can
prevent
disease
development
on
corn
leaves
inoculated
with
C.
graminicola (Pascholati
et
al.,
1993).
In
the
present
study,
DIPF
decreased
infection
of
unwounded
peaches
by
R.
stolonifer,
most
probably
due
to
the
inhibition
of
cutinase,
but
it
did
not
inhibit
myce-
lium
formation
on
the
peach
surface.
Although
some
treated
peaches
became
infected,
this
was
probably
a
result
of
the
product's
volatility
or
the
pathogen's
fast
development
on
the
fruit.
Rhizopus
stolonifer
infection
has
already
been
reported
for
several
hosts.
The
infection
on
grapes
can
occur
due
to
the
release
of
cellular
substances
caused
by
injuries
to
berry
pedicels
or
incomplete
connections
between
berry
and
pedicel
(Lisker
et
al.,
1996;
Tavares
8c
Silva,
2006).
Furthermore,
it
was
found
that
the
removal
of
the
rasp-
berry
receptacle
during
harvest
resulted
in
a
cavity,
which
released
substances
that
promoted
a
favourable
humidity
and
nutrient
supply
for
Rhizopus
development
(Davis,
1991).
However,
none
of
the
previous
reports
showed
direct
penetration
of
Rhizopus
into
the
hosts.
The
addition
of
peach
juice
to
R.
stolonifer
spore
sus-
pension
was
found
to
promote
pathogen
growth
and
nectarine
infection
(Nguyen-The
et
al.,
1989).
The
Plant
Pathology
(2016)
65,
633-642
Rhizopus
direct
penetration
into
peaches
641
authors
reported
that
cuticular
microcracks
became
lar-
ger
and
the
pathogen
could
develop
and
produce
pecti-
nolytic
enzymes
inside
these
microcracks,
which
hydrolysed
the
fruit
epidermis
cell
wall.
In
the
present
study,
no
microcracks
were
observed
using
the
scanning
electron
microscope,
showing
that,
in
their
absence,
R.
stolonifer
penetrated
unwounded
nectarine
surfaces.
Therefore,
it
seems
that
wounds
are
more
important
for
juice
release
than
for
pathogen
penetration.
The
released
substances
from
wounds
enabled
R.
stolonifer
germina-
tion
and
the
subsequent
production
of
enzymes
capable
of
breaking
the
fruit
surface.
As
observed
on
grape
sur-
faces
inoculated
with
R.
stolonifer
(Lisker
et
al.,
1996),
the
present
investigation
also
showed
that
spores
germi-
nated
on
nectarines
near
the
wounds
and
then
developed
around
and
inside
the
wounds.
Similarly,
pathogen
pene-
tration
was
not
observed
through
disruptions
or
natural
openings
such
as
stomata
(Lisker
et
al.,
1996).
An
SEM
study
of
P.
malicola
by
Sugui
et
al.
(1998)
showed
that
germ
tubes
of
this
pathogen
penetrated
the
host
cuticle
of
quince
fruit
and
plums
directly
and
the
pathogen
developed
over
and
under
the
cuticle
surface,
as
observed
for
R.
stolonifer
in
nectarines
in
the
present
investiga-
tion.
This
study
showed
that
R.
stolonifer
was
able
to
infect
and
cause
fruit
rot
in
unwounded
peaches
and
nectarines
due
to
spore
germination
in
nutrient
solution
placed
on
the
fruit
surface.
In
contrast,
pathogen
spores
suspended
in
water
were
not
able
to
penetrate
unwounded
stone
fruit
tissues.
The
disease
incidence
in
unwounded
fruits
inoculated
with
R.
stolonifer
in
nutrient
solution
was
as
high
as
in
wounded
inoculated
fruits,
showing
that
even
in
the
absence
of
wounds
the
pathogen
is
able
to
pene-
trate
its
host
if
there
is
an
external
source
of
nutrients
provided.
When
the
spores
were
suspended
in
water
the
fruit
surface
most
probably
had
insufficient
nutrients
to
support
spore
germination
and,
consequently,
pathogen
penetration.
These
results
may
have
practical
implica-
tions
for
stone
fruit
transportation
and
storage
because
one
damaged
fruit
can
release
nutrients
essential
for
pathogen
spore
germination
and
subsequent
penetration
of
nearby
healthy
and
unwounded
fruits.
Acknowledgements
This
work
was
supported
by
contract
no.
2011/03034-8
from
Fundacao
de
Amparo
a
Pesquisa
do
Estado de
Sao
Paulo
(FAPESP).
The
authors
also
thank
Dr
Ricardo
Harakava,
scientific
researcher
at
Instituto
Biologic°,
Sao
Paulo,
for
technical
assistance
on
the
molecular
identifi-
cation
of
R.
stolonifer.
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