Factors influencing ingress of Xanthomonas pruni through peach leaf scars and subsequent development of spring cankers


Feliciano, A.; Daines, R.H.

Phytopathology 60: 1720-1726

1970


Leaf scars, obtained by the forcible removal of the entire leaf or by delaminating the petioles 7 days prior to their removal, were inoculated immediately and semimonthly from 15 July to 1 November with Xanthomonas pruni. The scars were located either at the tip or one third of the way down the twig. Summer cankers were produced following inoculations performed from 15 July to I October, whereas spring cankers resulted from inoculations made from 17 September to 1 November. Ingress of X. pruni occurred only at fresh scars resulting either from the forcible removal of leaves or from the removal of petioles that had been delaminated 1 and 3 days. Inoculated scars at the tip developed many more spring cankers than those one third of the way down the twigs. Histopathological investigations disclosed that X. pruni can enter the twig through the xylem vessel and intercellular spaces in the leaf base. Entrance through the xylem vessels appeared to be of no pathological importance, however, since the bacteria, once inside, could not move out to infect neighboring cells. Bacteria survived the winter in the intercellular spaces of the cortex, phloem, and xylem parenchyma and initiated cankers the following spring.

Factors
Influencing
Ingress
of
Xanthomonas
pruni
through
Peach
Leaf
Scars
and
Subsequent
Development
of
Spring
Cankers
Alberto
Feliciano
and
R.
H.
Daines
Postdoctoral
Fellow
and
Professor
—Research
Specialist,respectively,
Department
of
Plant
Pathology,
Rutgers
University,
The
State
University
of
New
Jersey,
College
of
Agriculture
and
Environmental
Science,
New
Brunswick
08903.
Portion
of
a
Ph.D.
thesis
submitted
by
the
senior
author
to
the
Graduate
School,
Rutgers
University,
The
State
University
of
New
Jersey.
The
authors
gratefully
acknowledge
the
assistance
of
Ascunia
3.
Feliciano
in
the
preparation
of
the
histological
materials.
Accepted
for
publication
15
June
1970.
ABSTRACT
Leaf
scars,
obtained
by
the
forcible
removal
of
the
entire
leaf
or
by
delaminating
the
petioles
7
days
prior
to
their
removal,
were
inoculated
immediately
and
semimonthly
from
15
July
to
1
November
with
Xanthomonas
pruni.
The
scars
were
located
either
at
the
tip
or
one
third
of
the
way
down
the
twig.
Sum-
mer
cankers
were
produced
following
inoculations
performed
from
15
July
to
I
October,
whereas
spring
cankers
resulted
from
inoculations
made
from
17
September
to
1
November.
Ingress
of
X.
pruni
oc-
curred
only
at
fresh
scars
resulting
either
from
the
forcible
removal
❑f
leaves
or
from
the
removal
of
petioles
that
had
been
delaminated
1
and
3
days.
Inoculated
scars
at
the
tip
developed
many
more
spring
cankers
than
those
one
third
of
the
way
down
the
twigs.
Histopathological
investigations
dis-
closed
that
X.
pruni
can
enter
the
twig
through
the
xylem
vessel
and
intercellular
spaces
in
the
leaf
base.
Entrance
through
the
xylem
vessels
appeared
to
be
of
no
pathological
importance,
however,
since
the
bacteria,
once inside,
could
not
move
out
to
infect
neighboring
cells.
Bacteria
survived
the
winter
in
the
intercellular
spaces
of
the
cortex,
phloem,
and
xylem
parenchyma
and
initiated
cankers
the
following
spring.
Phytopathology
60:1720-1726.
Additional
key
words:
leaf
abscission,
ligno-suberization.
In
bacterial
canker
of
peach
caused
by
Xanthomonas
pruni,
two
types
of
twig
cankers,
designated
by
Thorn
-
berry
&
Anderson
(13)
as
"spring"
and
"summer"
can-
kers,
are
recognized.
It
is
well
established
that
the
main
source
of
primary
inoculum
comes
from
spring
cankers
occurring
in
the
terminal
portions
of
twigs
produced
during
the
past
growing
season.
Goldsworthy
&
Wilson
(6)
demonstrated
that
fall
infections
of
twigs
resulted
in
the
development
of
cankers
the
following
spring;
how-
ever,
they
did
not
identify
the
site
of
bacterial
ingress
that
resulted
in
the
development
of
the
cankers.
In
similar
studies
using
forced
stream
inoculations,
Adam
et
al.
(I)
report
that
August
is
the
opt
time
in
Illinois
for
the
application
of
X.
pruni
to
peach
trees
for
the
development
of
high
incidence
of
spring
cankers.
Using
needle
inoculations,
these
workers
found
that
the
highest
incidence
of
spring
cankers
resulted
from
inoculation
of
twig
terminals,
and
that
spring
canker
development
was
reduced
as
the
distance
from
the
terminal
was
increased.
Lenticels
and
leaf
scars
are
suspects
as
sites
for
infec-
tion
resulting
in
spring
canker
development,
as
Smith
(12),
Rolfs
(11),
and
others
report
that
stoma
and
lenticel
infections
result
in
the
development
of
disease
symptoms
in
leaves
and
twigs.
In
addition,
leaf
scars
have
been
shown
to
be
the
site
of
bacterial
ingress
into
the
host
by
Horne
et
al.
(9),
Hewitt
(8),
Crosse
(4,
5),
and
others.
The
purpose
of
the
present
investigation
was
to
deter-
mine
whether
leaf
scar
infections
by
Xanthomonas
pruni
(E.F.S.)
Dows.
result
in
the
development
❑f
spring
cankers.
If
the
leaf
scar,
as
expected,
proved
to
be
an
important
site
for
bacterial
ingress
into
the
peach
twig,
the
intent
was
to
determine
(i)
the
effect
of
leaf
scar
age,
location
on
the
twig,
time
of
invasion,
and
development
❑f
twig
cankers
the
following
spring;
(ii)
the
path
taken
by
the
pathogen
during
invasion.
MATERIALS
AND
METTIODS.-Two-y
ea
r-oI
d
Sunhigh
and
Rio-Oso-Gem
peach
trees
used
in
these
experiments
were
grown
outdoors
in
3-gaI
porcelain
crocks
fi
lled
with
washed
sand.
They
were
watered
daily
with
nutrient
solution
to
insure
good
flushing.
The
solution
contained
nutrient
elements
in
the
following
ppm:
N(NO
a
),
45;
N(NH
4
),
22;
P,
15.5;
IC,
78;
S,
126;
Ca,
80;
Mg,
48;
Fe+
/.,
1;
B,
0.1;
Mn++,
0.25;
Zn++,
0.01;
Cu++,
0.01;
and
Mo++,
0.01.
In
addition,
Na
t
EDTA
was
used
to
prevent
precipitation
of
Fe+
+.
Inoculum
for
these
experiments
was
secured
from
leaves
showing
the
bacterial
spot
disease
previously
col-
lected
from
an
orchard
and
frozen
in
cubes
of
ice.
Forty
-eight
-hr
-old
broth
cultures
of
new
isolates
from
frozen
leaves
were
used
in
each
experiment.
The
iden-
tity
of
a
new
isolate
was
determined
by
its
sensitivity
to
X.
pruni
bacteriophage.
In
all
experiments,
four
trees
were
used
for
each
treat-
ment,
and
all
twigs
were
inoculated
accordingly.
Of
these,
one
tree
was
used
for
isolations
and
histological
investigations
and
three
trees
were
held
for
observations
on
canker
development.
Inoculation.
--Two
experiments
designed
to
evaluate
the
method
used
in
exposing
the
leaf
scar,
its
location
on
the
twig,
and
the
time
of
inoculation
on
the
incidence
of
spring
canker
development
were
conducted
in
1966
and
1967.
The
leaf
scars
inoculated
were
produced
at
the
tip
and
one-third
❑f
the
way
down
the
twig
accord
-
1720
December
19701
FELICIANO
AND
DAINES;
XANTHOMONAS
PRUNI
1721
ing
to
the
method
employed
by
Crosse
(5):
fresh
leaf
scars
were
exposed
(Method
A)
by
pulling
leaves
off
forcibly;
and
(Method
B)
by
removal
of
delaminated
petioles.
At
the
time
of
petiole
detachment
(7
days
after
delamination),
abscission
development
was
under-
way
and
the
exposed
leaf
scars
resembled
those
result-
ing
from
the
dropping
of
mature
leaves.
A
small
drop
of
X.
pruni
suspension
was
carefully
placed
on
both
types
of
leaf
scars
immediately
after
the
leaf
or
petiole
was
removed.
Control
plants
were
either
left
noninocu-
lated
or
a
drop
of
the
bacterial
suspension
was
placed
at
the
base
❑f
uninjured
petioles.
Both
types
of
leaf
scars
located
at
the
tips
and
one-
third
of
the
way
down
the
twigs
were
inoculated
on
15
July,
15
August,
17
September,
and
1
and
15
October.
On
1
November,
only
tip
inoculations
were
performed
since
all
but
the
terminal
leaves
had
normally
fallen.
In
a
second
experiment,
the
Sunhigh
cultivar
of
peach
was
used
to
determine
the
influence
of
leaf
scar
age
and
the
effect
of
varying
the
time
between
delamination
and
petiole
removal
on
infection.
Leaf
scars
produced
by
the
forcible
removal
of
undelaminated
petioles
(A)
were
inoculated
immediately
(0
hour),
1
day,
3
days,
and
7
days
after
exposure
of
the
scar.
The
effect
of
abscission
development
was
also
determined
by
detach-
ing
the
petiole
1
day,
3
days,
and
7
days
after
delamina-
tion
and
then
inoculating
the
scar.
These
treatments
were
also
made
at
two
locations:
the
tip
and
one
third
of
the
way
down
the
twig.
Isolation
and
identification.
—All
inoculated
twigs
were
observed
for
canker
development
and
isolations
made
semimonthly
to
determine
whether
infection
had
occurred
and
to
ascertain
the
longevity
of
X.
pruni
in
the
host
tissue
under
the
conditions
of
the
experiment.
All
yellow
bacteria
isolated
were
exposed
to
X.
pruni
bacteriophage
to
determine
whether
the
isolate
was
X.
pruni.
Two
cultures
of
X.
pruni
bacteriophage,
one
received
from
Harry
L.
Keil
(USDA)
and
the
other
isolated
and
purified
as
described
below,
were
used
in
these
studies.
A
culture
of
X.
pruni
bacteriophage
was
isolated
from
soils
taken
from
beneath
infected
peach
trees.
The
phage
was
purified
according
to
the
method
described
by
Anderson
(2),
and
the
agar
layer
method
(7)
was
used
in
testing
isolates
from
inoculated
twigs
for
plaque
formation.
TABLE
1.
Development
of
summer
and
spring
cankers
in
leaf
scars,
1967-1968
4
Histological
technique
--Histological
studies
of
inocu-
lated
twigs
were
made
to
determine
the
path
of
invasion
and
the
effect
on
the
host
tissue.
Sections
from
the
check
(noninoculated)
trees
were
likewise
taken
for
comparison.
Samples
were
taken
immediately,
1,
3,
5,
7,
9,
11, 15,
30,
and
60
days
after
inoculation.
Samples
were
cut
successively
into
5
-mm
segments
fi
xed
in
formalin-
acetic
acid
-alcohol.
The
fixed
materials
were
softened
in
a
solution
con-
taining
45
ml
of
52%
hydrofluoric
acid
and
7
mI
of
60%
ethyl
alcohol
for
9
days,
followed
by
washing
in
run-
ning
water
for
24
hr.
The
segments
were
dehydrated
in
a
tertiary
butyl
alcohol
series
and
embedded
in
Tissuemat
with
a
melt-
ing
point
of
56.5
C
(10).
The
tissues
were
cut
at
S
and
mounted
serially.
The
sections
were
stained
in
1%
solution
of
tbionin
in
5%
phenol
for
1
hr
and
counter
-
stained
in
orange
G
(saturated
solution
of
orange
G
in
cellosolve,
pure
cellosolve,
and
95%
alcohol,
1:1:I)
for
about
10
dips,
They
were
rinsed
quickly
in
a
solu-
tion
of
clove
oil,
cellosolve,
and
95%
alcohol,
1:1:1,
differentiated
in
a
solution
of
clove
oil,
absolute
alco-
hol,
and
xylol,
1:i:1
(10-12
dips),
cleared
in
xylol
(3
changes
of
xylol,
at
least
10
min
each
change),
and
mounted
in
permount
(3).
The
presence
of
suberization
or
lignification
was
de-
termined
by
using
standard
solution
of
Sudan
IV
in
70%
alcohol
or
0.1
g
phloroglucin
in
10
cc
of
95%
alcohol
(10),
respectively.
REsur-rs.—Type
and
location
of
leaf
scars
and
time
of
inoculation
on
incidence
of
spring
cankers.
—Observa-
tions
for
canker
development
were
started
about
1
week
after
inoculations
were
made.
Summer
and
spring
can-
kers
were
distinguished
according
to
the
criteria
of
Thornberry
&
Anderson
(13);
results
are
given
in
Table
1.
Only
fresh
leaf
scars
obtained
by
the
forcible
removal
of
leaves
provided
suitable
infection
courts
for
the
inoculation
methods
employed.
Fresh
leaf
scars
pro-
tected
by
an
abscission
layer
or
the
base
of
the
petiole
of
intact
leaves
were
unsuitable
for
infection.
Spring
cankers
resulted
from
inoculations
made
between
17
September
and
I
November
(17
September,
1
October,
P
=
<
.05;
15
October
and
I
November,
P
= <
.01).
high
incidence
of
summer
cankers
resulted
from
inocu-
Rio-Oso-Gem
peach
cultivar
following
inoculations
of
fresh
Location
Date
of
inoculations
15
Aug.
1
Sept.
17
Sept,
1
Oct.
15
Oct.
1
Nov.
Summer
canker
incidence
Tip
83
85
83
68
One-third
of
the
way
down
64
65
69
39
0
Spring
canker
incidence
Tip
0 0
55
56
83
85
One-third
of
the
way
down
0
5
4
a
Data
represent
average
percent
of
twigs
observed
which
showed
cankers.
1722
PHYTOPATHOLOGY
TABLE
2.
Effect
of
leaf
scars
age
at
time
of
inoculation
on
development
of
spring
cankers
in
Sunhigh
peach
cultivar.
15
Oct.
1967n
Period
of
time
between
leaf
removal
or
petiole
delamination
and
inoculation
of
leaf
scar
0
hr
I
day
3
days
7
days
Location
BI
BI
B2
BI
B2
BI
B2b
Tip
61
0
44
0
38
0
0
One-third
of
the
way
down
the
twig
23
.30
0
0
0 0
a
Data
represent
average
percentage
of
twigs
observed
which
showed
cankers,
b
Treatment
RI:
fresh
Leaf
scars
obtained
by
forcibly
re-
moving
Ieaf.
Treatment
B2:
leaf
scars
obtained
by
removal
of
petioles
1,
.3,
and
7
days
after
delamination.
lations
made
from
15
July
to
1
October.
Tip
inocula-
tions
resulted
in
the
development
of
more
cankers
than
did
inoculations
one-third
of
the
way
down
the
twig.
Table
2
shows
the
effect
of
leaf
scar
age
on
the
inci-
dence
of
spring
cankers
on
Sunhigh
peach
trees.
Since
the
15
October
1966
inoculations
gave
a
high
percentage
of
spring
cankers,
and
since
normal
defoliation
at
nodes
one-third
of
the
way
down
the
twig
had
not
occurred
at
that
time,
15
October
was
picked
as
the
date
for
inocu-
lations
of
leaf
scars
of
various
ages,
types,
and
locations
on
twigs
in
the
1967-1968
experiments.
Leaf
scars
lo-
cated
at
the
tips
of
twigs
inoculated
immediately
after
the
forcible
removal
of
intact
leaves
showed
the
highest
incidence
of
spring
cankers
(Fig.
1).
Similarly
produced
leaf
scars
inoculated
24
hr
or
more
after
leaf
removal
did
not
result
in
the
development
of
spring
cankers
at
either
location
(tip
or
one-third
of
the
way
down
the
twig).
Results
obtained
with
leaf
scars
produced
by
the
removal
of
the
previously
delaminated
petiole
(B2)
clearly
demonstrate
the
protective
effect
of
stimulating
abscission
prior
to
inoculation.
A
decrease
in
disease
incidence
occurred
as
time
between
delamination
and
removal
of
the
petiole
increased.
Scars
located
one-third
of
the
way
down
the
twig
became
immune
to
infection
earlier
than
those
located
at
the
tip.
A
significantly
higher
incidence
of
canker
development
was
obtained
from
inoculations
of
Ieaf
scars
at
the
tip
than
of
those
located
one-third
of
the
way
down
the
twig.
Leaf
abscission
in
relation
to
infection.
—Leaf
scars
resulting
from
the
forcible
removal
of
leaves
located
at
the
tips
of
twigs
resulted
in
mechanically
damaged
fi
bers,
vessels,
sieve
tubes,
and
epidermal
and
paren-
chyma
cells.
Except
for
four
to
fi
ve
layers
of
collapsed
and
dried
cells
on
the
exposed
surface
a
day
after
leaf
removal,
no
barrier
was
observed
within
the
tissues
of
the
scars
that
could
provide
mechanical
protection
against
invasion
by
the
bacteria.
In
a
few
cases
in
the
1
November
inoculations,
a
trace
of
ligno-suberization
was
observed
in
the
area
of
the
protective
layer
even
before
the
removal
of
the
leaf.
Generally,
ligno-suberiza-
tion
of
the
protective
layer
in
such
scars
is
completed
in
5-7
days
after
leaf
removal.
By
the
9th
day,
the
[Vol.
60
protective
layer
has,
by
division,
become
several
cells
thick.
In
abscission
zones
of
leaves
located
one-third
of
the
way
down
the
twig,
Iigno-suberization
started
as
early
as
15
October
and
was
completed
just
after
leaf
fall.
Fresh
leaf
scars
resulting
from
the
removal
of
petioles
I
and
3
days
after
delamination
exhibited
very
little
or
no
ligno-suberization
(15
October
1967
inoculation).
They
apparently
did
not
differ
anatomically
from
scars
produced
by
the
forcible
removal
of
the
leaf,
except
that
tearing
was
less
when
the
petiole
had
been
delami-
nated.
Ligno-suberization
was
found
to
be
almost
com-
plete
in
leaf
scars
located
one-third
of
the
way
down
the
twig
when
they
were
produced
by
the
removal
of
petioles
that
had
been
delaminated
for
7
days.
Pathological
anatomy.
--Bacteria
were
not
observed
in
any
of
the
sections
prepared
following
inoculation
of
leaf
scars
resulting
from
the
removal
of
petioles
that
had
been
delaminated
for
7
days.
Leaf
scars
resulting
from
the
removal
of
petioles
that
had
been
delaminated
for
f
and
3
days
and
those
that
were
formed
by
forcible
removal
of
leaves
allowed
bacterial
invasion.
Once
the
bacteria
entered
the
host,
no
apparent
dif-
ference
in
the
course
taken
in
invading
the
tissues
was
observed
among
the
different
treatments.
One
day
after
inoculation,
bacteria
were
observed
at
the
surface
of
scars
and
some
had
penetrated
the
xylem
vessels
of
the
three
vascular
bundles
of
the
leaf
trace.
On
the
3rd
day,
the
bacteria
had
penetrated
the
intercellular
spaces
2-3
layers
of
parenchyma
cells
of
the
cortex
and
also
of
the
xylem
and
phloem
parenchyma.
By
the
5th
day,
invasion
of
the
cortex
had
extended
0.5
to
1.0mm
below
the
surface
of
the
leaf
scar.
By
the
9th
day,
some
cortical
cells
had
been
colonized
and
the
depth
of
inter-
cellular
invasion
had
increased
(Fig.
2).
By
the
11th
day,
invaded
cortical
cells
were
completely
disorganized
and
contained
masses
of
bacteria.
During
the
months
of
July,
August,
and
September,
periderm
was
forming
across
the
leaf
scar
4-5
days
after
forcible
defoliation,
and
was
well
developed
by
the
9th
day.
Obvious
formation
of
periderm
subsequent
to
bac-
terial
invasion
was
observed
by
the
30th
day
after
inocu-
lation,
with
a
phellogen
and
2-3
layers
of
phellem
sur-
rounding
and
isolating
the
invaded
area.
By
this
time,
the
vascular
bundles
of
the
leaf
trace
were
completely
invaded.
Lysigenous
cavities
formed
by
the
collapse
of
parenchyma
cells
and
filled
with
bacteria
surrounded
the
xylem
vessels.
The
latter
were
frequently
fi
lled
with
bacteria
but
had
intact
walls
(Fig.
3).
No
histological
evidence
was
found
to
indicate
that
bacteria
can
successfully
penetrate
through
the
lateral
walls
of
invaded
xylem
vessels
to
infect
neighboring
parenchyma
cells,
nor
can
bacteria
from
infected
cells
move
into
the
xylem
vessels
(Fig.
4).
Likewise,
migra-
tion
was
never
observed
to
occur
between
vessels
of
a
singIe
vascular
bundle.
Considerable
attention
was
given
to
the
area
where
the
infected
leaf
trace
joins
the
healthy
vascular
cylinder
of
the
main
stem.
There
was
no
indication
that
the
bacteria
moved
from
infected
to
healthy
vessels
even
in
cases
where
two
vessels
lay
side
by
side.
In
some
instances,
tyloses
were
found
inside
December
19701
FELICIANO
AND
DAINES:
XANTROMONAS
PRUNI
1723
the
infected
xylem
vessels;
however,
they
probably
did
not
significantly
obstruct
movement
of
the
bacteria
since
bacteria
were
observed
above
and
below
the
tyloses.
Infection
of
the
phloem
occurs
fi
rst
through
the
me-
chanical
wounding
created
by
Leaf
removal
and
later
by
the
invasion
of
bacteria
from
the
cortical
parenchyma.
Bacteria
are
commonly
observed
in
the
intercellular
4
spaces
of
the
phloem
parenchyma.
As
bacterial
pockets
increase
in
size,
adjacent
cells
are
crushed,
including
the
sieve
elements
and
parenchyma
cells
(Fig.
5).
Bac-
teria
from
the
phloem
area
readily
invade
the
cambium.
In
some
cases,
however,
invasion
extends
only
from
cortical
parenchyma
to
the
phloem
fi
bers,
and
is
checked
by
periderm
formation
before
it
invades
the
other
phloem
elements
(Fig.
6).
4
Tf
4
3
k
y
,
V
4
t
?
e
'
9
Fig.
1-3.
1)
Diseased
and
healthy
peach
twigs
at
the
shuck
split
stage.
Left:
Diseased
twig
shows
a
spring
canker
which
resulted
from
the
inoculation
of
a
Ieaf
scar
located
at
the
tip
of
the
twig
on
1
November.
2)
Longitudinal
section
of
inoculated
twig
showing
intercellular
invasion
(arrows)
of
the
cortical
parenchyma
(x900).
3)
Longitudinal
section
through
the
vascular
bundle
of
the
leaf
trace
showing
lumina
of
the
xylem
vessels
filled
with
bacteria
(bac)
(a)
in
comparison
with
the
healthy
xylem
vessels
(b)
(X430).
"
4
'
'
-
‘11
4
4
,
r
41
0
1
70
h
0
.1#
1
c
p
e
'T
r
.
1
IV
.
4
0
wF
a
a
.
Ci
4,
.
4
4
*
•'
i f
46)1
i
I
der-.
ee
fr
.4
lhF
P
f
I
0.
t
1
724
FELICIANO
AND
DAINES:
XANTHOMONAS
PIWNI
1725
Apc.
dr_
ir
Ifr
'40
December
1970
-
do
410
,
ip
f
a;
4
.7T
:P
A
,e•
1.
74.*
t.
AL
a
A
5.
061Ww
b
4.1P
-
J
li
e
.
'tt
"or
#e•
.
_
Fig.
6.7.
6)
Transverse
section
through
the
stem
show-
ing
infected
phloem
fibers
(pf)
effectively
walled
off
by
periderm
(pd).
Note
also
the
periderm
separating
the
in-
vaded
(ic)
from
the
uninvaded
cortical
(uc)
tissues
(X200).
7)
Longitudinal
section
of
a
diseased
twig
terminal
showing
a
leaf
scar
through
which
infection
had
occurred,
Such
in-
fection
results
in
the
development
of
spring
cankers,
a
point
of
inoculation;
b
=
path
of
invasion;
c
normal
leaf
scar
(x
to).
Periderm
formed
around
invaded
areas
during
the
summer
and
early
fall
months
(July,
August,
September,
and
1
October
inoculations)
but not
during
the
late
fall
(15
October
and
1
November
inoculations)
and
winter
months.
This
provides
an
explanation
for
the
high
per-
centage
of
spring
cankers
developing
from
the
late
-fail
inoculations.
In
the
spring,
overwintering
bacteria
usually
occur
in
the
innermost
layer
of
the
cortex,
a
zone
of
parenchyma
cells
rich
in
starch
and
abutting
the
phloem
fi
bers.
From
here
they
migrate
through
intercellular
spaces
into
the
rest
of
the
cortex,
which
is
eventually
completely
de-
stroyed,
and
into
the
phloem
via
groups
of
parenchyma
cells
among
the
fi
ber
strands.
DiscussroN.—The
present
investigation
serves
to
in-
crease
our
understanding
of
the
time
and
site
of
ingress
of
X.
Pruni
into
the
tissue
of
peach
twigs,
and
some
of
the
factors
influencing
penetration
into
the
host
tissues.
Xantkomonas
pruni
invades
peach
twigs
in
the
au-
tumn
through
fresh
leaf
scars.
In
nature,
this
is
accom-
plished
most
effectively
by
wind
-driven
rain
causing
the
forcible
removal
of
leaves
and
at
the
same
time
deposit-
ing
inoculum
on
the
peach
leaf
scars.
The
incidence
of
spring
cankers,
therefore,
can
vary
from
year
to
year
depending
on
the
weather
conditions
during
the
period
when
effective
infection
can
occur,
In
New
Jersey,
this
period
occurs
from
September
to
early
November.
This
study
emphasizes
the
importance
of
fresh
wounds
produced
by
tearing
for
the
ingress
of
bacteria
through
the
leaf
scars
(Fig.
7).
Histological
studies
revealed
that
suherized
protective
layers
are
formed
prior
to
normal
leaf
fall
(Fig.
8),
thus
providing
de-
fenses
against
bacterial
ingress.
Formation
of
the
pro-
tective
layer
also
explains
the
experimentally
observed
decrease
in
disease
incidence
as
the
interval
increased
between
delamination
and
detachment
of
petiole
and
inoculation.
That
the
tip
of
the
twig
is
the
part
most
susceptible
to
infection
and
provides
the
most
favorable
conditions
for
the
overwintering
of
the
bacteria
was
earlier
demon-
strated
by
Adam
et
al.
(1),
and
has
been
confirmed
by
this
study.
Under
the
conditions
of
the
present
experi-
ments,
formation
of
the
protective
layer
and
its
subse-
quent
ligno-suberization
starts
in
the
month
of
October
Fig.
4-5.
4n)
Transverse
section
through
the
stem
showing
masses
of
bacteria
Onto
in
the
xylem
parenchyma.
Note
the
healthy
vessels
(hv)
besides
the
compressed
parenchyma
cells
(coc)
(
X430).
413)
Transverse
section
through
the
healthy
stem
showing
the
normal
appearance
of
the
xylem
parenchyma
cells
(arrow)
(X430).
5a)
Transverse
section
through
the
stem
showing
pockets
of
bacteria
(ph)
in
the
intercellular
spaces
of
the
phloem
elements
(
x200).
5b)
Transverse
section
of
the
stem
showing
healthy
phloem
elements:
phloem
fi
bers
(pf),
sieve
tubes
(st),
and
phloem
parenchyma
(pp)
(X430).
1726
.)•
.
.
1
J
!
-
_
ifs
P4
PHYTOPATHOLoGY
Fig.
8.
Free
-hand
longitudinal
section
(approximately
I00
p.
thick)
of
the
leaf
scar
just
after
normal
leaf
faIl
showing
a
well-suberized
protective
layer
(spI)
already
formed
(x100).
in
petioles
located
one-third
❑f
the
way
down
the
twig,
and
in
late
October
and
early
November
in
leaves
lo-
cated
at
the
tip
of
the
twigs.
Bacteria
were
never
ob-
served
to
invade
the
protective
layer
in
leaf
scars
of
the
following
types:
those
located
one-third
of
the
way
down
the
twig
and
resulting
from
the
removal
of
petioles
that
had
been
delaminated
3
and
7
days
before
inoculation,
or
those
formed
by
forcible
removal
of
leaves
at
either
the
tip
or
one-third
of
the
way
down
the
twigs
24
hr
before
inoculation
(15
October
1467).
There
seems
to
be
no
anatomical
explanation
for
the
resistance
to
infection
shown
by
the
24
-hr
-old
leaf
scar
produced
by
the
forcible
removal
of
the
leaf.
With
the
inoculation
method
employed,
however,
a
drying
out
of
the
surface
cells
seems
to
have
been
sufficient
to
preclude
infection
from
occurring.
In
cases
where
ligno-
suberization
of
the
protective
layer
is
not
complete
at
the
time
of
inoculation,
whether
or
not
infection
occurs
depends
on
whether
the
bacteria
can
penetrate
into
the
leaf
base
before
completion
of
the
periderm
layer.
Since
development
of
the
periderm
occurs
more
rapidly
in
early
fall
than
during
October
and
early
November,
in-
fections
❑ccurring
during
the
latter
period
have
a
much
[Vol.
60
greater
chance
of
becoming
established
than
do
earlier
infections,
Invasion
of
xylem
vessels
by
X.
pruni
appears
to
be
of
little
pathological
importance,
as
bacterial
cells,
once
inside,
apparently
do
not
move
out
through
the
lateral
walls.
However,
X.
pruni
can
successfully
invade
inter-
cellular
spaces
and
parenchymatous
cells.
Numerous
ob-
servations
made
during
this
investigation
suggest
that
invasion
of
host
tissues
by
X.
pruni
receives
little
or
no
assistance
from
the
dissolving
action
of
pectic
or
cellulolytic
enzymes.
LITERATURE
CITED
1.
AnAm,
A.
V.,
D.
POwELL,
&
H,
W.
ANDERSON.
1955.
Time
of
peach
infection
by
Xanthomonas
pruni
in
relation
to
spring
canker
incidence.
Phytopathology
45:285-287.
2.
ANDERSON,
H.
W.
1928.
Bacteriophage
of
Bacterium
pruni.
Phytopathology
18:144
(Abstr.).
3.
CREACER,
D.
B.,
&
E.
P.
MATHERLY.
1962.
Bacterial
blight
of
poinsettia.
Histopathological
studies,
Phyto-
pathology
52:103-110,
4.
CROSSE,
J.
E.
1951.
The
leaf
scar
as
an
avenue
of
infection
for
cherry
bacterial
canker
organism
Pseu-
domonas
morsprunorum
Worrnald,
Nature
168:560.
5.
CROSSE,
J.
E.
1956.
Bacterial
canker
of
stone
fruits.
II.
Leaf
scar
infection
of
cherry.
J.
Hort.
Sd.
31
(3):212-224.
6.
GOLDSWORTHY,
M.
C,,
&
R.
A.
'Mum.
1952.
Terminal
bud
and
shoot
infections
and
the
overwintering
of
Xanthomonas
pruni
in
peach
trees.
Plant
Dis,
Reptr.
36(11):408-409.
7.
GRATIA,
A.
1936.
Des
relation
numeriques
entre
bac-
teries
lysogenes
et
particules
de
bacteriophage.
Ann.
Inst.
Pasteur
57:652-676,
8.
IlEwirr,
W.
B.
1938.
Leaf
scar
infection
in
relation
to
olive
-knot
disease.
Hilgardia
12:41-71.
9.
Hoame,
W.
T.,
W.
B.
PARKER,
&
L.
L.
DAWES.
1912.
The
method
of
spreading
of
the
olive
knot
disease.
Phytopathology
2
:
101-105.
10.
JoHANsEN,
D.
A.
1940.
Plant
Microtechnique.
McGraw-
Hill
Book
Co.,
N.
Y.
523
p.
11.
Ror.rs,
F.
M.
1915.
A
bacterial
disease
of
stone
fruits.
N.
Y.
Cornell
Agr.
Exp.
Sta.
Mem.
8:375-436.
12,
SMITH,
E.
F.
1903.
Observations
on
a
hitherto
unre-
ported
bacterial
disease,
the
cause
of
which
enters
the
plant
through
ordinary
stomata.
Science
17:456-
457.
13.
THORNBERRY,
H.
H.,
&
H.
W.
ANDERSON.
1933.
Over
-
wintering
of
Phytomonas
pruni
on
peach.
Phyto-
pathology
23:787-801.