Penetration and infection of corn by Puccinia polysora Underw.


Santiago Oro, R.; Exconde, O.R.

Philippine Agriculturist 58(1/2): 50-60

1974


Puccinia polysora penetrated corn leaves through stomata directly by germ tube. Hyphal growth originating from just below the guard cells was strictly intercellular among the mesophyll cells, without invading the vascular elements. Haustoria were formed both in the mesophyll and epidermal cells. Uredia were produced from the pseudoparenchymatous funguous layer arising from the dense mycelial growth at the point of penetration. Ten days after inoculation, uredia erupted with the son developing at the point of inoculation. Fungal development pattern in the leaf sheath, midrib and husk was the same as in the leaf.

PENETRATION
AND
INFECTION
OF
CORN
BY
PUCCINIA
POLYSORA
UNDERWENT'
ROSALINDA
SANTIAGO-ORO
and
0.
R.
EXCONDE
2
Puccinia
polysora
penetrated
corn
leaves
through
stomata
directly
by
germ
tube.
Hyphal
growth
originating
from
just
below
the
guard
cells
was
strictly
intercellular
among
the
mesophyll
cells,
without
invading
the
vascular
elements.
Haustoria
were
formed
both
in
the
mesophyll
and
epidermal
cells.
Uredia
were
produced
from
the
pseudoparenchymatous
funguous
layer
arising
from
the
dense
mycelial
growth
at
the
point
of
penetration.
Ten
days
after
inoculation,
uredia
erupted
with
the
son
developing
at
the
point
of
inoculation.
Fungal
development
pattern
in
the
leaf
sheath,
midrib
and
husk
was
the
same
as
in
the
leaf.
INTRODUCTION
Tropical
corn
rust
caused
by
Puccinia
polysora
Underwent
is
one
of
the
major
disease
problems
of
corn
growers
in
the
Philippines.
Although
it
does
not
cause
as
much
damage
as
other
corn
diseases
like
the
downy
mildew,
corn
rust
is
considered
one
of
the
important
diseases
of
corn
in
the
country
today.
This
paper
reports
the
mode
of
penetration
and
histopathological
development
of
P.
polysora
on
corn
leaves,
leaf
sheaths,
midrib
and
husk.
MATERIALS
AND
METHODS
Inoculation.
Inoculation
of
1
-week
old
and
1
-month
old
plants
(Philippine
Hybrid
801)
was
done
late
in
the
afternoon
or
early
in
the
evening.
Two
methods
of
inoculation
were
used,
dry
and
liquid.
In
the
liquid
inoculation,
spores
were
washed
twice,
suspended
in
distilled
water
and
either
sprayed
with
an
atomizer,
dropped
in
the
whorl
or
infected
with
a
disposable
plastic
hypodermic
syringe.
Twenty
ppm
of
a
spreader
-
sticker
solution
(Tween
20)
were
added
to
100
ml
spore
suspension.
In
1
The
major
portion
of
this
thesis
was
submitted
by
the
senior
author
as
partial
fulfillment
of
the
requirements
for
the
degree
of
Master
of
Science
in
Plant
Pathology.
Supported
in
part
by
the
Upland
Crops
Production
Program.
CES
No.
3282.
UPCA
Journal
Paper
No.
73-44.
2
Respectively,
Instructor,
Central
Philippine
University,
Iloilo
City
and
Asso-
ciate
Professor,
Department
of
Plant
Pathology,
College
of
Agriculture,
U.P.
at
Los
Baiios
3720.
50
PENETRATION
AND
INFECTION
OF
CORN
51
the
dry
method
of
inoculation,
an
equal
amount
of
talc
was
mixed
with
uredospores
and
dusted
on
corn
leaves
previously
wiped
with
Tween
20
solution.
In
addition,
potted
plants
were
exposed
to
naturally
infected
field
for
3
days,
removed
and
then
placed
inside
nylon
cages.
Penetration
studies.
One
to
two-week
old
leaves
of
plants
were
detached
and
sprayed
with
the
uredospores
suspension
inside
a
petri
plate
lined
with
moist
tissue
paper.
These
inoculated
leaves
were
cut
and
examined
3
times
every
4
hrs.
and
every
12
hrs.
Clearing
and
staining
of
whole
mounts
were
made
with
the
use
of
a
saturated
aqueous
solution
of
chloral
hydrate
containing
0.1
to
0.2%
aniline
blue
and
heating
in
an
Arnold
Sterilizer
for
15
to
30
min.
The
leaf
sections
were
rinsed
in
plain
lactophenol
for
12
to
24
hrs
before
they
were
examined
under
the
microscope
(Hilu,
1965).
Clearing
was
done
in
several
changes
of
20
ml
sodium
hydroxide
to
which
was
added
4
drops
of
5%
sodium
hypochlorite
solution.
This
clearing
agent
was
changed
daily
until
the
materials
became
transparent,
rinsed
in
distilled
water
and
stained
with
cotton
blue.
Infection
studies.
Serial
sections
of
inoculated
leaves
were
made
by
fixing
the
materials
in
Farmer's
solution
consisting
of
3:1
absolute
ethyl
alcohol
and
acetic
acid
solution,
or
in
formalin
aceto-alcohol
com-
bination
(FAA),
and
dehydrating
the
materials
in
a
series
of
tertiary
butyl
alcohol.
Embedding
was
done
with
tissue
mat.
Paraffin
sections
were
cut
10-20
microns
thick
in
a
rotary
microtome
and
stained
with
safranin
and
fast
green.
In
the
later
part
of
the
study
of
older
leaves,
leaf
sheath,
husks
and
midribs,
sections
were
cut
with
the
freezing
microtome.
In
staining
fresh
sections
cut
by
means
of
the
freezing
microtome,
the
tissues
were
dehydrated
by
the
use
of
a
thin
nylon
cloth
which
served
as
a
container
and
support
in
the
process
of
changing
alcohols.
RESULTS
Inoculation.
In
either
dry
or
liquid
inoculation,
2
to
3
days
after,
tiny
yellowish
flecks
were
produced
on
the
newly
expanded
inoculated
leaf
blade.
Five
to
7
days
later,
the
uredium
was
formed
and
10
days
after,
inoculation
pustules
were
fully
erupted.
Potted
plants
exposed
for
3
days
in
the
field
near
heavily
infected
plants
developed
pustules
10
to
12
days
after
exposure.
Less
than
50%
of
the
total
number
of
plants
exposed
in
this
manner
developed
symptoms.
Erumpent
pustules
were
formed
sparingly
on
plants
inoculated
with
a
dry
mixture
of
uredos-
pore
and
talc
in
spite
of
wiping
the
leaf
blade
with
20
ppm
Tween
20
prior
to
inoculation.
Relatively
fewer
pustules
developed
in
either
cir-
cumstances
compared
to
whorl
inoculation.
On
leaves
injected
with
52
PHILIPPINE
AGRICULTURIST
uredospores
suspended
in
distilled
water,
pustules
developed
below
the
point
where
the
inoculum
was
introduced.
Seedlings
inoculated
just
after
the
first
2
leaves
have
emerged
from
the
ground
developed
symptoms
more
rapidly
than
when
they
were
inoculated
a
week
later.
On
one
-month
old
plants,
whorl
inocula-
tion
did
not
produce
the
same
symptoms
as
in
seedlings.
In
almost
all
cases,
no
symptoms
were
produced
at
all.
Whole
mount
sections
of
mature
leaves
inoculated
with
liquid
spore
suspension
showed
that
the
inoculum
tended
to
congregate
near
the
base
of
the
trichomes
made
up
of
hardened
epidermal
walls.
Penetration.
Actual
penetration
was
observed
at
least
4
hrs
after
inoculation.
As
the
germ
tube
came
out
of
the
uredospores,
the
proto-
plasm
moved
away
from
the
mother
spore
into
the
developing
tube,
thus
becoming
concentrated
on
the
newly
-formed
appressoria.
The
ap-
presoria
formed
from
enlarged
portion
of
the
tip
of
the
developing
germ
tubes
were
spherical
to
clavate
in
shape.
They
were
formed
even
without
the
stomata.
The
germ
tube
appeared
like
an
ordinary
hyphal
process
as
it
approached
the
stoma
but
upon
reaching
it,
the
tip
enlarged
to
form
the
appressorium
(Fig.
1).
In
the
majority
of
cases,
appressoria
were
formed
before
penetration
took
place.
One
or
more
appressoria
may
form
on
a
single
stoma.
In
a
few
instances,
germ
tubes
were
seen
to
grow
directly
above
a
closed
stoma
or
along
the
stomatal
Fig.
I.
Whole
mount
section
of
corn
leaf
showing
germ
tubes
of
P.
poiysora
ap-
p
roac
hi
ng
a
stoma
(arrow)
(520
X).
PENETRATION
AND
INFECTION
OF
CORN
53
slit.
The
appressoria
were
also
observed
not
only
on
top
of
a
stoma
but
they
attached
themselves
to
the
edge
of
the
stomatal
pores
where
the
2
guard
cells
meet.
Invasion
of
the
leaf
tissue.
A
thin
infection
hypha
was
produced
from
the
appressorium
and
grew
directly
through
the
guard
cells.
The
guard
cells
did
not
lyze
but
developed
more
affinity
to
the
stain
than
normal
cells.
As
soon
as
the
thin
infection
thread
reached
the
sub-
stomatal
chamber,
it
enlarged
to
the
normal
hyphal
size
and
produced
septa
just
below
the
guard
cells.
The
hyphae
were
at
first
thin
and
irregularly
septated.
Hyphal
growth
was
more
or
less
continuous
as
the
mycelium
developed
extensively
between
the
mesophyll
cells.
The
hyphae
grew
close
to
the
mesophyll
cells
deriving
nutrients
from
them
before
a
specialized
organ
for
absorption
was
formed.
Most
of
the
my-
celium
grew
laterally
among
the
sub
-epidermal
palisade
cells
invading
new
substomatal
chamber
and
forming
new
colonies
therein
(Fig.
2).
Haustoria
were
formed
within
the
mesophyll
and
the
epidermal
cells.
The
mycelium
continued
to
ramify
between
the
host
cells
but
it
did
not
invade
the
vascular
elements.
Hyphae
were
seen
growing
abun-
dantly
outside
the
bundle
sheath
of
the
vascular
tissues
but
no
haustoria
were
formed
inside
these
cells.
Fig.
2.
Cross
section
of
corn
leaf
showing
hyphal
tip
of
P.
polysora
(arrow)
from
adjacent
tissue
invading
a
neighboring
substomatal
chamber.
(520
X).
Dense
hyphal
colonies
were
first
formed
at
the
point
of
penetra-
tion
but
as
the
hyphae
continued
their
lateral
growth,
they
reached
new
substomatal
chambers.
In
these
substomatal
cavities
the
hyphae
54
PHILIPPINE
AGRICULTURIST
formed
well
-developed
mycelial
colonies
which
resembled
an
entangled
mass
of
wide
loose
threads.
This
mass
later
developed
into
a
pseudo
-
parenchymatous
layer
which
eventually
gave
rise
to
the
uredospores
(Fig.
3).
Fig.
3.
Portion
of
a
cross
section
of
a
corn
leaf
showing
substomatal
cavity
filled
with
hyphae
(a)
of
P.
poll/1mm
below
a
closed
stoma
(b)
(520
X).
The
protoplasm
of
the
fungus
moved
along
the
pseudoparenchy-
matous
region
into
the
tip
of
the
hyphae
causing
it
to
expand.
Because
of
this,
the
terminal
portion
became
enlarged
while
the
rest
of
the
mycelium
assumed
a
thin
-stalk
-like
structure
which
eventualy
became
the
pedicel
(Fig.
4).
As
more
protoplasm
was
concentrated
in
the
Fig.
4.
Longitudinal
section
of
corn
leaf
showing
intercellular
hyphae
(a)
the
pro-
toplasm
of
which
is
concentrated
in
the
hyphal
tips
(b),
and
eventually
becoming
the
uredospore
mother
cell
(520
X).
PENETRATION
AND
INFECTION
OF
CORN
55
developing
uredospore,
a
septum
was
laid
at
the
point
where
the
hyphal
tip
began
to
enlarge
so
that
the
structure,
now
spherical
in
shape,
be-
came
separated
from
the
stalk
by
means
of
a
septum.
The
uredospores
at
first
appeared
coarse
and
irregular
with
prominent
echinulations
but
as
they
matured,
they
took
on
a
smoother
appearance.
Several
stalk
-like
processes
resembling
sterile
paraphyses
were
formed
together
with
the
uredospores.
The
host
cells
were
not
visibly
affected
as
the
fungus
grew
and
developed
in
the
intercellular
spaces
and
as
it
produced
dense
hyphal
colonies
and
uredospores.
Except
for
a
reduction
in
the
size
and
amount
of
chloroplasts
in
infected
cells,
no
other
physical
change
was
evident
at
this
point.
As
more
uredospores
were
formed,
the
developing
fungus
pushed
back
the
mesophyll
cells
towards
the
inner
tissues
resulting
in
the
distortion
of
the
vascular
elements
located
around
the
area
(Fig.
5).
,c,
Fig.
5.
Cross
section
of
corn
leaf
showing
distorted
bundle
sheath
of
the
vascular
element
(arrow)
due
to
the
force
generated
by
the
developing
uredospores.
(250
X).
56
PHILIPPINE
AGRICULTURIST
The
epidermal
cells
were
separated
from
the
adjacent
palisade
tissues
as
the
fungus
continued
to
develop,
and
formed
a
wedge
between
them.
The
epidermal
cells
became
expanded,
forming
a
thin
elevated
cover
over
the
uredium
and
in
the
process,
tearing
the
inner
walls
(Fig.
6).
The
stoma
remained
intact
for
a
while
over
the
uredium
and
when
the
walls
could
no
longer
contain
the
force
generated
by
the
growing
fungus,
they
broke
and
exposed
the
uredium
containing
both
young
and
old
uredospores
(Figs.
7A
and
B).
In
all
instances
where
infection
was
Fig.
6.
Cross
section
of
corn
leaf
showing
a
developing
uredium.
Note
the
destruction
of
the
inner
epidermal
wall
(a),
spiny
young
uredospore
(b),
and
a
smoother
mature
one
(c)
(520
X).
A
a
7.
Cross
section
of
corn
leaf
showing
(A)
the
epidermis
(arrow)
broken
by
the
developing
uredium
and
(131
exposed
uredium
bearing
both
young
(a)
and
old
(b)
uredospores
alike.
Stalk
-like
processes
(c)
forming
to-
gether
with
the
uredospores
are
presumed
to
be
sterile.
(130
X).
PENETRATION
AND
INFECTION
OF
CORN
57
obtained,
the
plants
developed
sori
and
pustules
erupted
at
the
upper
leaf
surface
in
the
site
of
infection.
Growth
of
the
hyphae
and
the
development
of
the
pustules
were
limited
between
the
main
veins.
Necrotic
yellow
margins
surrounded
the
pustules.
Invasion
of
the
midribs,
leaf
sheaths
and
husks.
Naturally
infected
midribs
and
leaf
sheaths
developed
pustules
on
both
surfaces.
Abun-
dant
mycelial
growth
was
observed
from
the
point
of
penetration
on
the
upper
epidermal
surface
of
the
leaf
sheath
and
the
midrib.
The
hyphae
of
the
fungus
invaded
the
intercellular
spaces
laterally
and
ver-
tically
among
the
loosely
-arranged
mesophyll
cells
towards
the
lower
epidermis.
Fungous
hyphae
grew
extensively
over
a
large
area
form-
ing
colonies
between
the
sub
-epidermal
cells
directly
below
the
vascular
elements
or
in
adjacent
tissues.
Unlike
in
the
leaves
the
vascular
element
did
not
limit
fungal
growth
towards
the
lower
epidermis.
Haustoria
were
formed
at
the
point
of
penetration
and
in
the
adjacent
area
where
the
pseudoparenchymatoi.s
and
sporogenous
layer
of
fungus
were
produced.
The
haustoria
were
spherical
to
clavate
in
shape
later
becoming
cylindrical.
They
formed
several
lobes
and
appeared
as
gnarled
fist
-like
structures
(Fig.
8).
The
invaded
cells
at
this
point
became
devoid
of
chloroplasts.
Some
necrotic
cells
appeared
to
be
filled
with
several
hyphal
processes
consisting
of
enlarged
and
knob
-like
hyphal
tips.
-4
di
Fig.
8.
Longitudinal
section
of
corn
leaf
sheath
showing
gnarled,
fist
-like
haus-
torium
(arrow).
Note
the
dense
hyphal
growth
outside
the
invaded
cell.
(520
X).
58
PHILIPPINE
AGRICULTURIST
On
the
husk,
the
fungus
penetrated
through
the
stomata
of
the
outer
cover
and
grew
downward
to
form
colonies.
Active
fungal
growth
and
development
of
uredospores
generally
occur
among
the
lower
sub
-
epidermal
spaces.
The
thick-walled
outer
epidermis
inhibits
spore
pro-
duction
and
where
the
epidermal
cells
were
especially
thick
and
failed
to
expand,
the
uredospores
were
few
and
assumed
an
irregular
to
angular
appearance
(Fig.
9).
The
surrounding
mesophyll
cells
which
were
af-
11-
Fig.
9.
Cross
section
of
a
corn
husk
showing
irregular
shape
of
uredospores
(a)
due
to
the
non
-expansion
of
the
thick
upper
epidermal
wall.
Note
the
extensive
hyphal
growth
(b)
and
fewer
uredospores
formed.
(130
X).
fected
became
abnormal
in
shape.
No
marked
injury
to
the
host
cells
towards
the
inner
surface
with
thin
epidermal
cells
was
observed.
In
general,
the
pattern
of
growth
and
development
of
the
fungus
in
the
husk
were
similar
to
those
in
the
leaf
sheath
and
midrib.
DISCUSSION
Hooker
(1954)
reported
that
hypodermic
inoculation
of
P.
sorghi
was
superior
to
other
liquid
suspension
methods.
In
this
study,
whorl
inoculation
gave
better
results
than
hypodermic
injections
of
the
liquid
PENETRATION
AND
INFECTION
OF
CORN
59
inoculum.
Hypodermic
inoculation
of
older
leaves
of
the
same
plant
resulted
in
the
formation
of
fewer
pustules
than
when
the
inoculum
was
deposited
in
the
whorl.
Penetration,
which
is
almost
always
accompanied
by
appressorial
formation,
was
effected
through
the
stomata.
Direct
penetration
also
occurred.
This
is
similar
to
the
findings
of
Weber
(1922)
and
Hilu
(1965)
on
corn
infected
by
P.
sorghi.
The
pattern
of
development
of
the
fungus
in
this
study
conforms
with
the
observation
of
Wellensiek
(1927).
He
noted
that
the
forma-
tion
of
sub-stomatal
vesicles
seemed
to
be
more
characteristic
of
an
incompatible
reaction,
the
structure
serving
as
a
resting
organ
during
the
first
few
days
after
penetration.
He
also
noted
that
in
a
susceptible
leaf,
vesicle
may
or
may
not
be
formed
and
fungal
growth
was
more
or
less
continuous.
No
fungal
structure
was
found
to
invade
the
vascular
elements
confirming
the
earlier
reports
of
Hilu
(1965),
Van
Dyke
and
Hooker
(1969).
Rice
(1927)
reported
that
obligate
parasites
establish
successful
relationship
with
their
hosts
by
forming
haustoria
in
the
host
cells.
In
this
study,
haustoria
were
abundantly
produced
in
the
mesophyll
cells
and
occasionally
in
the
epidermal
cells.
The
haustoria
varied
in
shape
from
spherical
to
elongate
or
cylindrical,
later
becoming
multilobed
as
described
by
Hilu
(1965).
Formation
of
the
uredia
occurred
mainly
below
the
point
of
pene-
tration.
The
mycelium
continued
to
invade
new
intercellular
spaces
and
substomatal
chambers,
forming
intercellular
haustoria
as
they
moved
along.
In
the
leaf,
leaf
sheath
and
midrib,
most
uredia
formed
on
the
upper
surface
because
the
epidermal
cells
did
not
offer
great
resistance
to
fungal
growth.
The
production
of
uredospores
in
substomatal
chambers
exerted
pressure
in
two
ways:
pushing
the
epidermal
cells
upwards
causing
it
to
expand,
and
pushing
the
epidermal
tissues
downward
resulting
in
distorted
cells
of
abnormal
shapes.
The
thin
-walled
bundle
sheaths
of
the
vascular
elements
were
also
deformed
although
they
actually
ex-
hibited
some
kind
of
resistance
to
the
pathogen
itself.
These
observa-
tions
were
also
recorded
by
Hilu
(1965).
60
PHILIPPINE
AGRICULTURIST
LITERATURE
CITED
HILU,
H.
M.
1965.
Host
-pathogen
relationships
of
P.
sorghi
in
nearly
isogenic
resistant
and
susceptible
seedling
corn.
Phytopathology.
55:
563-569.
HOOKER,
A.
L.
1954.
Relative
efficiency
of
various
methods
of
inducing
field
infections
with
Helminthosporium
turcicum
and
Puccinia
sorghi.
Pl.
Dis.
Reptr.
38:
173-177.
RICE,
M.
A.
1927.
The
haustoria
of
certain
rusts
and
the
relation
between
host
and
pathogen.
Torrey
Bot.
Club.
Bull.
54:
63-153.
VAN
DYKE,
G.
C.
and
A.
L.
HOOKER.
1969.
Ultrastructure
of
host
and
para-
site
interactions
of
Zea
mays
with
Puccinia
sorghi.
Phytopathology.
59:
1934-
1946.
WEBER,
G.
F.
1922.
Studies
on
corn
rust.
Phytopathology.
12:
89-97.
WELLENSIEK,
J.
L.
1927.
The
nature
and
resistance
in
Zea
mays
L.
to
Puccinia
sorghi
Schw.
Phytopathology.
17:
815-825.