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7:'
THE
~~
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S I S
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*
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MOEE OF ENTRANCE OF FUSARlm~
~"
INTO WHEAT SEEDLINGS
*
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1}
*
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FRANCIS O. HOU~ES
~~
7~
DEPARTMENT
OF BIOLOGY AND PUBLIO HEALTH
~$o
I
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TABLE
o F
UONTENTS
Introduction: The Disease
Page
(1)
Object of investigation
( 6)
Former Work
(9)
( 11)
Methods:
.Seedlliings
( 11)
Inoculating
( 15)
Killing and embedding
( 15)
Staining and mounting
( 18)
(20)
Experiments
Fusarium sp. Strain I
(21)
Fusarium sp. Strain II
(26)
Fusarium sp. Strain III
(33)
Alternaria sp.
(35)
Helminthosporium
sp.
(53)
Discussion of results
(56)
Summary of results
( 60)
Bibliography
( 61)
I N T ROD
U C T ION
FusariQ~-blight of the cereal crops is a fairly
wide-spread disease of wheat, barley, oats, ~ye and
many grasses of the United States.
It attacks both
the root systems, causing footrot, and the stalks
and leaves, causing wilt.
The causal organism is Gibberella saubinettii,
(Mont.) Sacc., a fungus closely related to the large
genus Fusarium.
This species has a number of other
names which will be found in the list of synonyms
on page
4.
The disease is prevalent throughout the eastern
states and in the central wheat producing area of
the United States.
In Europe it occurs in England,
France, Italy, Germany, Austria, Holland, Denmark,
Norway, Sweden and Russia.
It is also reported from
Siberia and Australia.
The loss of wneat from this disease in the states
reporting in 1917 was over ten million bushels according to the Plant Disease Survey (1).
( 1)
The total
lass
in all states was probably nearly twenty million
bushels.
The crops affected have a lower percentage of germination, an increased death rate among young seedlings,
wilting of full grown plants, and final blighting of
the heads containmng the seeds.
The kernels which are blighted may be very much
smaller than normal, light in weight, and unable to
germinate.
This condition obtains if the infection
is early in the development of the seed.
Later in-
fection causes but slight shrinking, slight decrease
in weight, with low percentage germination.
In-
fection just before ripening causes almost no change
in the appearance of the seed, except that light
pink spots may be observed.
Such seeds usually
germinate~ but wilt when still yow1g plants as a
result of infection from the seed.
The pink spots
on the infected seeds contain conidia characteristic
of the fungus, being found as well on other parts of
diseased plants at times.
(2)
The conidia are typically, sometimes all, 5-septate,
measuring 45p to 65p by 4.2 to 5.5p; 3-septate forms
measure 35 to 45p by 5 to 5.5p; seldom 4-septate;
,~
rarely 6-,
7-,
or more septate, 60-75p
by 4 to 5~. (1)
LIS
o
T
F
SYNONYMS
..
The proper name of the organism
blight
of wheat
is Gibberella
concerned
saubinettil
in the
(Mont.)
Sacc.
Synonyms
Gibberella
as given by Atanasoff
Gibbera
(D. and M.) S. 1879 in Michelia,
saubinettii
v.
(1)
1, p. 513.
1856,
saubinettii,
Syll. Gen. Spec. crypt.
p. 252.
Botryosphaeria
saubinettll
Ver handle
(Mont.) Niessl,
1872, in
Na turf. J,T;er.
",Brllnn. Bd.
10 ,
p. 195, pl. 4, fig. 29.
Fusarium
graminearum,
Schwabe,
1839, Fl. anhalt.,
v. 2, p. 285, pl. 6, fig. 7, Sacc. Syll.
v. 22, p. 1483-84,
Gibbera
pulicaris
(Fr.) f. zeae maydis,
Ascomyceten
roseum,
Rehm:
381. From New Jersey,
J. B. Ellis.
Fusarium
1913.
Autorum.
(4)
8, 1875,
Fusarium tropicalis, Rehm, 1898, in Hedwigia, Bd. 37,
p.
194.
Giberella tritici, P. Henn. 1902, in Hedwigia, Bd. 41,
p.
301.
Fusarium rostratum, App. and Wollenw., 1910, in
Arb. K. Biol. Anst. Land. un Forstw.,
Ed • 8~; p. 30 •
(5)
o B
J E C T
The object of this thesis investigation was to
discover the mode of entrance of the fungus causing
wheat blight into the wheat seedling.
Previous
workers have observed the death of the seedling after
inoculation, or leaf-spot after spores or mycelium have
been placed on the leaf.
1l0onid1a,
ascospores, and
mycelium of the organisms, when placed on normal
young plants, with or without wOlli1ding,cause 1nf1'ection.fI(1)
It has also been noted that lIthese
organisms can invade the tissues of the seed, straw,
and heads of the cereal crops after ripening and
1I
harvesting if the conditions are favorable.
(1)
More particularly Atanasoff states lithecoleorhiza
and coleopt11e, which always die shortly after the
formation of the permanent roots and the appearance
of the first foliage leaf, seem to offer a ~ood
mediQ~ for the establishment of the various species
of Fusarium, which then penetrate into the tissues
of the permanent roots and the first foliage leaf,
( 6)
causing rotting and browning of the invaded portions. II
(1)
No histological evidence has been found for this
statement, and the work reported in this paper was an
attempt to obtain such a condition and to discover the
exact manner in which the fungus is able to penetrate
the tissues, whether into the cells, between the cells,
through stomata of the first leaf, or in some other
way.
It must be pointed out that there are possibilities
for mistakes in the macroscopic examination of infected seedlings.
Even after careful surface steril-
ization, seeds are almost sure to contain some fungus,
in many cases the organism of this disease; inoculation
ll
without apparent wounding may cause IIleaf-spot or
browning on account of osmotic conditions at the
point where the fungus is deposited.
So that an
investigator may place spores on the surface of a
leaf, observe browning of the point of supposed infection, wilt of the entire plant, browning of the
roots and all the symptoms of Fusarium seedling
(7)
blight
without
material.
any real action
of the infective
It is only by examining
sections
through
point of inoculation
that it can be shown whether
or not the organisms
used were effective.
(8)
the
FORMER WORK ALONG SIMILAR LINES
Histological investigations similar to the one
reported in this paper have been carried on for
many of the fungus diseases of plants.
In 1886 DeBary discovered that a species of
Botrytis entered the leaves of broad beans after
forming appresoria, under which the tissue softened and blackened.
Two years later, Ward described a lily disease
in which the hY~llie exuded a viscous fluid, attaching the tlITeads to the surface and softening the
cellulose walls to aid the entrance of the fungus.
Busgen showed in 1893 that Botrytis cinerea
formed appresoria and then il~ection threads, which
passed through the walls of the cells after these
had been softened by enzyme activity.
Dey in 1919 found that Colletotrichum lindemuthianum entered by means of a peg-like infection hypha
near which there was some enzyme action after pene-
tration.
A number of disease producing organisms also pass
through the stomata of the leaves, or send only
l~ustoria into the cells for nourishment.
( 10)
MET
(1)
HOD
S
SeOedlings:
Seedlings were grown from seed surface-
sterilized as follows:
50% alcohol .••..
30 seconds
Water .•....
30 minutes
2% HgCl2 in 50% alc.
2 minutes
Wash thoroughly in sterile water
The seeds were then grown on agar plates until
the young shoot was about- one half centimeter in
length.
( 11)
The sketch
seedling
on the next
at the proper
The appearance
prepared
The seeds
should
shows an enlarged
stage for inoculation.
of a number
carefully
page
agar
of the seedlings
plate
be arranged
is also
on a
shown.
with a sterile
needle
so that the first
leaf will develop
in a normal
position
easy and accurate
inoculation.
to allow
Not too many
single
plate
seedlings
since fungus
two of the seeds may spoil
seeds at some little
the medit~.
majority
and this fact
careful
should
( 12)
from
otherwise
on a
one or
sterile
on the surface
of
seeds will be in the
surface
be realized
seeds.
be grown
mycelia
distance
The infected
even with
should
sterilization,
in placing
the
( 13)
-~fI
'--~'~
+
~i~
( 14)
(2)
Infection:
Infection was in general accomplished
without woundi-ng, usually without the addition of
anything other than the suspension of spores or
mycelium in water.
(3) Killing and embedding:
At first the killing solution used was an
aqueous solution of chromic and acetic acids, made
up as follows:
Chromic acid
5%
acetic acid
Water
.25
gm.
2 cc.
100
cc.
This was found to be by no means so useful as
a killing solution of absolute alcohol, chloroform
and glacial acetic acid, as follows:
Absolute alcohol
6~-parts
Chloroform
3 parts
Glacial Acetic Acid
part.
The advantage of this killing solution is in the
rapidity with which the sections can then be brought
into paraffin, requiring only two days at the most,
whereas the first process should have at least a
week.
This solution has been reco~mended by other
workers for similar problems. (5,V)
The embedding process was as follows, using the
second killing fluid:
( 1)
Specimen killed at the end of a days work.
(2)
Killing fluid (until next morning)
(3)
95% alcohol
(4)
100% alcohol (over night)
16 hours.
1-6 hours.
16 hours.
(5) Mixtures of 1tto-a xylene and absolute
alcohol, 2Gto 1 xylene and alcohol, and pure
xylene, from a few minutes to 15 minutes apiece.
The pure xylene should be replaced once to ensure
removal of the alcohol.
(6) Paraffin in a small lump added to the xylene
and allowed to dissolve slowly until saturation is
reached.
This step of the process can be carried
( 16)
out to advantage
containing
the specimens
incubator.
specimen
When
mens
poured
chilled
the mouth
during
in several
into a box made
quickly
in the present
of the tube
in a 370
and placing
convenient
can be heated
paraffin,
paper,
by closing
the day the
changes
of pure
of strips
and sectioned.
investigation
of
All speci-
were
cut
12p
i11:thickness.
If more
at hand
mould
than one piece
the portions
before
of infected
should
chilling
results
much
sections.
This
process
may be materially
to arrange.
to advantage
to make
in the
that one
the other
very favorable
in one way which
would
shortened,
day if necessary,
is not an excessive
easier
is
be
in the other.
even put into a single
days
sections,
By this means
may be secured
less noticeable
be arranged
in such position
will be cut into longitudinal
into cross
material
period
but two
and is usually
The two nights
are thus used
sure of the important
( 17)
and
killing
and dehydrating
(4)
processes.
Staining:
The stains
used were Delafield's
and a counterstain
haematoxylin,
the normal
to some extent;
portions
orange
always
on account
Since
the
takes both
red,
injured
injured
are
areas,
or of the osmotic
at the point
with
portions
in il~ected
of the fungus
of wounding
of inouulation,
the needle,
the
lIDportant of the two s~ains
the disease.
if a large
tissue
takes
only and show a brilliant
is by far the more
in locating
true
plant
in evidence
prevailing
or on account
The fungus
a purplish
red coloration.
conditions
eosin
giving
take the eosin
practically
either
of eosin.
Haematoxylin
This
is particularly
nQmber
of sections
must be examined
as is usually
the case
in paraffin
work;
characteristic
lesions
are once observed
pared under
all the powers
examination
to locate
portions
available,
similar
of the material
places
after
the
arid com-
additional
in other
may be cal-ried on with 'the
( 18)
lowest
power,
fatiguing
showing
movements
forty
millimeter
cases
in which
magnification
abnormal
temporary
sure tlmt this
a detectable
solutions,
water
used
eosin added
work
the many
with
it might
operations
different
trace
of
away
to this will
to greatly
facilitate
instead
through
and stains
all the alcohols
to a~out
This fact was tmfortunately
and it was evident
in the
enough
in
examination,
in gt~ damar.
of the round
one-fifteenth
to stain
A little
the tissues
to mOlmt
in pass-
strengths
the paraffin.
The use of this one bath
investigation
involved
alcohol
stain
and the specimen-~is ready
time required
be profitable
and to stain directly
to dissolve
one minute
strictly
in all
conditions.
through
in this
and avoiding
is to be recoBmended
ft~nish
ing slides
length.
field
For such work a
it is reasonably
to do away with
about
larger
of the eye.
lens
will
For rapid,
xylene
a much
trip
cuts dOID1 the
of its normal
not recognized
ill1tilall the work was complete
that
tIle haematoxylin
essential.
(19)
was not
EXPERIMENTS
During this investigation a few more than forty
experiments were carried out, using organisms of
three genera as follows:
Fusari~~ sp.
Strain
II
II
1\
II
II
II
I
II
III
Alternaria sp.
Helminthosporium sp.
Source of Organisms:
The first strain of Fusaril@ used was obtained
in the form of a portion of infected head of wheat
covered with the conQdia of Gibberella saubinettii.
The second strain was isolated from an infected seed
in the laboratory in the form of large masses of
conidia.
This was of questionable character, and was
probably a saprophytic species, but was used for comparison as the spores were so plentiful.
The third
strain was isolated from infected seed by Miss MacInnes,
(20)
who was working on this problem at the Institute at
the time;
the first strain was also obtained
I
through her kindness.
The Alternaria and Helminthospori~
were iso"lated
from infected seeds which they killed on plates
prepared for inoculation with the other fungi.
They
were used, not because they bore directly on the
question in hand, but because the Alternaria seemed
to be so persistent in attacking the wheat, and
because Helminthosporium had been recently (1920)
described as a cause of an important wheat disease.
(8).
It was hoped that work on these might point the
way to technique capable of demonstrating entrance
on the part of Fusari~~.
Fusari~~ sp. Strain I:
During the series of experiments involving the
strain of Fusari~~ from an infected head,. the
following points were brought out.
Old tissues,
whether from leaf or sheatll,which become dry and
wilted, are almost impossible to section because
(21 )
of the air contained
toddrive
this out by any moderate
or heating,
apparatus
final
can liquids
Wheat
first
Dany
by forced
infected
leaf,
without
macroscopically,
later
of light
with no more
normal,
occurred.
fection
though
stabbing
was tried with
it easier
the hope
to germinate
different
but
perfectly
of the tissues
sometimes
due to the presence
carrying
that the spores
under
Since
of
in-
in any of the sections
the needle
(22)
con-
were made,
on the sheath.
with
the
were
lmd not been discovered
thus far,
~mder
The sections
deposited
coveril~
that no microscopic
and of temperatl~e
browning
these
this was repeated
showed
This was probably
the material
to allow
so far as could be dis-
Trials
success.
although
in enough
on the sheath
sections
had developed.
ditions
of soaking
woundill..g,by placing
was not harmed
times;
-lesions
process
of the paraffin.
in a drop of water
covered
It is impossible
and only by the use of an exhausting
penetration
spores
in the cells.
these
spores
would
conditions.
find
This
also failed, and suspicion began to be attached to
the spores.
While.these were being tested the
experiments with.varied temperature and light conditions
were repeated with the same result, and needle stabs
were made into these still healthy plants.' Results
in all cases were negative.
The spores were tested as follows:
(1) Spores were mixed under a cover slip in a drop of
water.
A p~ece of wheat epidermis from the sheath
was introduced,
and the whole allowed to remain for
a total of seven days, being examined frequently.
No germination was noted.
(2) As soon as this method seemed to be at fault, a
second test was made in which some air was admitted.
Negative results were obtained as before .. Germination
was not observed.
(3) More air was used in a deep cell with a .drop of
water on the cover glass and a supply of water in
the bottom of the cell.
Negative result.
(23)
(4) Spores
were
of a Petri
dish,
the spores,
placed
and various
as follows:
sugar broth,
Ordinary
tried without
pieces
was practically
not more
water
(5) A
comparative
acid,
of spores
of Strain
wheat
producing
Germination
be safe to say that
related
These
forms.
in from
Conclusion
twenty
from
sprghted
under
series
vestigation
hours.
that the spores
This was confirmed
the spores
(24)
were
of experiments:
concluded
was made;
but which
all the conditions
to some ill~avorable conditions
to germinate.
orig2nally
were not typical
blight,
to twenty-four
this
It was therefore
seed, which
showed
the large
II, isolated
wheat
dilute
were also
epidermis.
It would
of the organism
able
acetic
test was rlillwith
from an infected
exposed
added with
cultures
of wheat
on the top
than one per cent. of the spores
of germination.
tried
of water
reagents
dilute
absent.
signs
numbers
in drops
had been
and were
lill-
when an in-
had been kept
in
diffuse
light
indoors
throughout
a considerable
part
of the winter.
The observations
are therefore
on their
a confirmation
study of the effect
Fusarit~
(4).
in this article
diffuse
light,
to nearly
inability
to germinate
of a recently
of temperature
The substance
published
and light on
of the work reported
cs it affected' this work was that
especially
the same extent,
th2.n one percent.
indoors,
reduced
in the course
(25)
but also outdoors
germination
of a winter.
to less
Fusari~m
sp. Strain
Germination
place
II:
of these
in about
spores
was found
20 lll~S. in drops
to take
of water
supplied
with air.
About
twice
them
on successive
in droplets
fection
with
one hlli1dredyoung
days with these
of water
nas observed.
checks
not
No difference
seeds
This
experiment
to rule
the inoculated
could be detected.
were found
contaminating
fungi
as shoml by examining
the surro~mding
agar
the absence
surface
sterilized
seeds.
sterile
agar
and used
tests
tubes
(those made
with
These
region
of growths
were
on the
such as
of
isolated
in the next
III).
to be
and bacteria,
in the vicinity
Strain
(26)
seeds and
From a repetition
a few seedlings
cOlnillonly
are to be observed
No in-
out the infections
between
plate and noting
placing
was repeated,
of the seed.
free from
inoculated
spores,
in most
qf this experiment
relatively
were
on the sheath.
inoculated,
sure to be found
the check
seedlings
series
in
of
Since
seedling
best
this
to enter
foliage
leaf,
entering
growth
strain,
though
with
spot seemed
were
cut and killed
fluid;
thousands
but showed
no infection
To test the ability
hyphae
through
wheat,
other
sheath,
sections
somewhat
puzzling
in water
and
fluffy
on the leaf, and
with
SOIDe specimens
the concentrated
carried
through
were
paraffin,
to send
the death
cut from
plates
of these
for they
spores
after
time growth
Sections
of
and leaves
A light,
of these
on agar
at the end of which
suspected
in sections.
the tissues
and placed
noticeable.
been
to have developed.
rapidly
a
of the first
of spores
noticeable
these were
it was thought
of shoots
for two days.
leaf
killing
the stomata
A number
was especially
leaf of the
as well as the other~
it has never
in this way.
to remain
the first
wit4 no stomata,
through
inoculated
allowed
covering
is provided
to give
chance
were
the sheath
of the
leaf and
for three
days,
of the fungus
specimens
showed
was
were
complete
infection
without
lesions
explain
entrance.
More
careful
in the peripheral
examination
showed
on the cells was progressive,
enclosed
many mycelial
as the infection
were barely
leaving
only
semblance
along
visible
and finally
the threads
of the cell structure
In a few places
but in all cases
peripheral
the fungus
on the inside
Within
faded
was much
entirely
by their
the sheath
seemed
ing almost
no trace
harm
penetrated
through
positions
penetrated,
to be going
wall.
(2$)
out
of branching
of mycelium
seemed
to have no
the cell walls,
of its attack,
to the surrolmding
away,
than that outside.
the mycelium
in passing
they
walls.
cells were
greater
thinner
to give a
that the concentration
difficulty
without
gradually
than in, as shown by the direction
and by the fact
which
in some sections
of mycelium
the lines of the former
rather
became
until
could
that the action
the cell walls
threads
proceeded
cells which
passing
portions
leav-
through
of the
Injuries to these heavily infected cells were not
shown by the e9sin stain, if they were present.
But
in some of the sections the cell walls were suspiciously
orange red and swollen at the points where normally
there are intercellular spaces.
Examination of these
areas under high powers showed that the hyphae of the
fungus, which stain deeply with haematoxylin, were
passing up through the longitudinal intercellular
spaces, enlarging these at times by crowding in,
three or more threads in a single space.
The walls
in the immediate vicinity were thicker than usual
and took the eosin as though injured.
A diagram of this condition is shown on the next
page, followed by a normal cell of similar appearance, stained only with haematoxylin, but showing
the normal intercellular spaces with the same
magnification.
Bothoof these diagrams are.camera
lucida drawings with an oil immersion lens.
The
total magnification is about fifteen hundred diameters as shown b~ the scales of microns attached.
(29)
Stalned
.eld'~haematoXYlin
with Dela
II
1",.1..,,1
\
)
,
«?
10/",-
I~
~-~~
~;;~-
( 1-
37 - 1
Dead shea th. )
~
~
Saprophytic organism passing through
(30)
intercellular
spaces.
Normal sheath cell,
spaces.
/
'"1.11111\
«r
Stain: Delafield's haema oxylin.
(31)
Ilf'
2~
No cases of entrance through the stomata were
observed, though in many sections fungus and
stomata were shown in good g~ose~oontact.
The
substomatal spaces were always clear and the
surroQ~dil~ cells in excellent condition.
Infection by this apparently saprophytic organism must have occurred by way of the cut ends of
the cells at the point where the fragment was
detached from the living plant.
In three days
plenty of opportunity was given for the fungus to
grow the length of the piece, a matter of a centimeter or less, and up tl~ough the cells from this
.wounded and unresistinB area.
Fusari~~
sp. Strain
Several
seedlings
when grown
surface
power
III:
on agar,
of tIle medium
spores
myceliwn
inoculate
of seedlings
than usual
from a number
further
negative
with a low
in tubes
of this strain.
some
resulted.
which
were much
less
with flli1gior bacteria
this
of
grown
strain.
One further
were
and used to
Sectioning
attempt
proved
also.
In order
to test the ability
stomata
tissues
of the first
of the wheat
experiment
similar
previously
described
leaves
the
containing
of plates
with
no infection.
by searching
the spores
No infectimns
selected
enter
with
as well.
plates
present
isolated
in an emulsion
contaminated
showed
were
were
Three
no organisms
in the vicinity
of the microscope,
These
showed
as determined
agar and inoculated
dead
which
and sheaths
sheath
strain
and leaf, an
carried
on with the
was performed.
inoculated
(33)
to
leaf, and to penetrate
to that
were
of this fungus
with
Living
emulsions
of the spores and killed after two days.
Other similar
portions were cut and allowed to remain three days on
agar plates before killing with the 80ncentrated killing
fluid.
Examination of a large nQmber of sections
showed no results from the inoculation.
Alternaria sp.:
A species of Alternaria was so frequently isolated
from the wheat seed after surface sterilization, and
was found to penetrate the epidermal cells so readily
that it was decided to inoculate a number of times
with this organism for the purpose of developing a
technique sufficient to demonstrate lesions in the
cells with ease and rapidity.
This result was
attained in the course of the work.
The spores were readily visible on the seedling under
low powers of the microscope, and so were more
readily watched than the Fusarium spores.
The latter
were found to be so transl~rent as to escape detection
even when present in considerable numbers.
Within two days after inoculation of wheat seedlings
with a number of these spores, the surface was found to
be punctured in many spots, and whole mounts of thin
pieces of the sheath were made.
These were stained
with safranin and mounted in glycerine.
The top views showed clearly that a large proportion
of the spores
had sent out hyphae,
had succeeded
in penetrating
epidermis.
ing tubes
Moreover
were
of the mycelium,
infection
length,
that the penetrat-
not of the same diameter
but were
threads.
from
the long cells of the
it was noted
of the nature
These
penetrating
departure
and that these
were usually
immediately
under
as the rest
of thin
of very
short
the point
of
the main hypha.
It was not easy to see in these whole mounts,
which
could be viewed
just how far these
how they
section
infection
penetrated.
surrounded
showed
the result
into the cell nearly
tl~ough
of the infection
of enzyme
thread
(36)
partition
tube was
strikingly
thread,
activity.
usually
to the opposite
a nearby
to
in profile.
area,
This area and the infection
nor
necessary
that the infection
by the proximity
inward,
proceeded,
It was therefore
by a cone-shaped
and suggesting
passing
threads
so as to see the process
The sections
changed
only from the surface
wall,
passed
sometimes
to another
cell.
In the whole
and their
observed
tached
have
mounts
surrounding
after
from
them.
This
observations
parasites
tl~t
affected
and spores
for about
for the process,
of Shear
of the genus
and Wood
germ-tubes
sue.
penetrated
These
conditions
surface
were able
than
the purpose
(6).
of the organism,
mediately
to withstand
washed
lightly
with the
They guessed
in such a contingency.
(37)
of the
from which
into the tis-
more
severe
attached
awaiting
and in a position
from
the surface
off by rain.
stage,
be ex-
of the organism
a short distance
of a resting
irrespective
as would
with appresoria,
the spore,
and easily
to
must have germinated
out hyphae
de-
in 1913 on weak
the spore as soon as this reached
sending
seemed
parallel
Glomerella
some of the species
plant,
had become
character
an interesting
threads
areas were
the same distance
the saprophytic
suggests
infection
All of the threads
of the time allowed
from
of these
cone-shaped
the hyphae
penetrated
pected
many
to the
They served
the death
to respond
im-
•
The writers mentioned did not see this happen,
but assumed it from the fact that pieces of tissue
showing no spores developed the fUl~US on incubation,
and from some observations on the surface of the
specimens in which bodies were made out to fit this
theory.
The difficulty of observation in this way
made the results uncertain.
Their microtome sections of specimen~ from which
the remnants developed the disease if incubated)
showed no such conditions.
They thought that this
was quite natural because of the minutemess of the
infection threads and because they would be hidden
in the structure of the cells.
Evidently they did not use a differential stain,
but only one which stained the whole section uniform,':"
lYe
Under these conditions it would not be surpris-
ing that they failed to see the tubes.
But by using
a stain such as eosin, with or without a background
stained with haematoxylin the points of infection
could hardly be missed.
They would stand out as
bright
orange
The words
red spots on a pale background.
of the authors
mentioned
were as follows:
( 6)
"Many :saces of Glomerella
appear
to be rather
majority
of cases
the dormant
weak
conditions
probable
IIIn the
explanation
of
which
have been
shown to be
instances
in leaves
and fruit
present
in so many
showing
no external
conidia
on ascospores
contact
with
ditions
of temperature
evidence
the plant
surface
are able
conditions
than the spores,
under
they come in
favorable
con-
and produce
to endure more unfavorable
and which
the epidermis.
penetra tes at first
is that the
whenever
and moisture,
which
a germ tube through
of disease
germinate
appresoria
ently
ordinary
parasites"
the most
infections
under
in turn send
This tube appar-
bllt:1.avV13;ry
shont distance
r
and does
little
a dormant
weakened
natural
harm
to the host
or inactive
or injured
death.
condition
or the organ
Bodies
resembling
(39)
cells,
until
remaining
in
the host becomes
infected
dies a
appresoria
have
been found
on the surface
upon which
the fungus
developed
and they are sometimes
surface
of lemons
difficult
found
and other
positively
leaf surface
apples
and leaves
in a moist
in abundance
citrou8
to identify
and trace
and the writers
of normal
chamber,
on the
fruits.
It is
these bodies 'on a
the germ tube in the tissue,
have as yet been lli1ableto devote
necessary
time
to this feature
to verify
the suggested
the
of the investigation
explanation
of the facts
observed.
Large
infected
series
of microtome
leaves,
the unused
of which
develop-
in a moist
chamber,
have
when
been
but the presence
been demonstrated
quite
with
is restricted
just penetrating
the surface
dormant
extent
infections
(40)
has nat
be
that the dormant
or germ
in-
tube
is correct.1I
would
the explanation
of apples
hyphae
This would
to a short hypha
The work done on Alternaria
to a remarkable
of fungus
certainty.
nat.ural if the supposition
fection
of presumably
portions
ed the fungus
studied,
placed
sections
seem to bear
offered
and other fruits.
out
for the
It is
probable
that
be examined
found
if the sections
with differential
that the appresoria
typical
of the latter
form,
stains
gave very decidedly
the effect
sections
showed
modified
area
in
thread;
whole mounts
of appresoria,
in the immediate
but
was due to the
vicinity
of the in-
tube.
This work bears
also a relation
to the investigations
the relation
species.
difference
between
parasite
an immune
and host
in its tt~n the fungus
to prevent
its entrance
Presumably
Alternaria
is unable
cells died
rapid
into other
to proceed
without
species
tIDsuited to the
suffered
sufficient
(41)
in immQ~e and
and a non-immupe
il~ected
of the wheat
(2) on
that the essential
was chemically
the immediately
into the cells
of some interest
out by Stakman
It was found
was that the former
parasite;
carried
between
non-immtme
ly, and
stained
that the appearance
be
at least
but only the thin infection
investigation
fection
it would
do not exist,
for in the present
were to
setting
quickchanges
cells.
further
up
destructive
wheat
changes
and reactions,
local
is concerned,
but disastrous
to the final
fection
after
as though
the death
the fup~us
of evolution
action
able
with
by the selection
It would
toward
tant variety
living
wheat
iwnediately.
perhaps
very variable
and it is
accustomed
to their
individuals
best
when a sufficiently
this resting
of selection
question,
point and await
seem to be on the road of evolution
parasitism
seem to be a parasite
process
of those
shall have been
to do away with
in the course
such an environment.
therefor~
complete
slowly
seem
it in extremely
the cell contents,
tllat it is being
to withstand
leaves
in-
It would
just the right
But this habit
contact
probable
had been adapted
to stop at
developments.
close
of the tissue.
so far as the
period
could
Thus Alternaria
(42)
time
content;
it
the
would
How soon such a
be completed
in a short
of time
to enable
and to enter
in the making.
in chemical
very long stretch
produced
resis-
is a doubtful
if the fungus
perhaps
only
if it is relatively
is
in a
stable.
Drawing. from whole mount
portion
of sheath,
showing
entrance
from germination
Stained
with
safranin,
(43)
of Alternaria
of spore
mounted
(1-25-1)
of
in glycerine.
\\
\\
\
\,
\
,\
\\
\
\
\
,
\
\
\
\
•••
~
(44)
I
I~
~
••
.3~
,
'tOr ..~
,
•
'~'For
,
"
..~ ,~
MIll/",
Detail of similar mount, ~mder oil immersion
showing infection tD~ead
and surrounding affected area
("1-29-1)
(45)
I
/
(/
I
)
/
" •••l •..•1
'?-
(46)
,~
Longitudinal
showing
germinating
Eosin
section
spores,
Haematoxylin
( 1-2 1- 1)
(47)
and infection
Stain
tube
\
\
\
(48)
Longitudinal
showing
infected
section
spot
~~ ~:. -:~ ~~
-!~
in perspective
.(.
"h
~~-
( 1-2 1-2 )
~}
i}
~'" ~-
-i(-
(49)
.;(-
-,~
"
~}
*
~~ ~-
I
J
\
\
(50)
Longitudinal
microtome
Thickness
Showing
Wall
section
twelve
characteristic
swollen
near
microns
zones with
one point
( 1- 32 -7)
(51 )
at surface
eosin
of infection.
l"..lnlll
9.-
1~
\
\
Helminthosporiu~
sp.:
Helminthosporilm
of seedlings
produced
A few sections
several
were
swelling
vicinity,
changing
of plwplisg
onto the sheaths
lesions
of th~se.
shown.
their
The fungus
chemical
staining
red with
eosin,
red of the normal
in four days.
In the sections
of the cell walls
cause a differential
a very bright
inoculated
microscopic
were made
lesions
remarkable
spores
caused
in the immediate
nature
reaction.
against
a
so as to
They
showed
the background
walls
stained
in part
examined
pointed
to entrance
with haematoxylin.
On the whole
between
spaces.
the cells
Diagrams
softening
showing
in outlining
a substance
capable
of the walls.
pages,
of the cell walls
the intercellular
from the point of attack,
the phenomena
on the following
color used
through
secrete
some distance
and of causing
istics
and passage
The flli1gusmust
of diffusing
found
the series
being
them.
(53)
observed
the staining
represented
will be
characterby the
LESION
CAUSED BY HELMINTHOSPORlillK.
Eosin Haematoxylin Stain
Oamera lucida drawing, under oil i~nersion
showing swe&len cell walls
(1-33-4)
(54)
...
' -.'
(f)
..~...
. ." -,,' ..' ..
-
"
•
'
'.
I
.
~:.\:.......
. ~":-.:< ..
(55)
D I S C U S S ION
o F
RES
U L T S
From the examination of several thousand paraffin
sections of the sheath and leaves after inoculation
with strains of Fusaril~ no evidence for the entrance
at this stage could be fOill1d.
A negative statement such as this requires for
practical proof a far greater number of trials than
could well be carried on in the limited time allowed,
and should h?ve the benefit of virulent strains from
infected areas.
However, these ~xperiments seem
to point toward a resistance of the wheat in the
early seedling stage to infection through the sheath
covering the first leaf, and even throhgh8~he early
stages of the first leaf.
Former statements, claiming that the fungus is
able to enter at these points may well be questioned
somewhat carefully.
Most of the examinations re~~rt-
ed have been macroscopic in character, and might well
be ascribed to some other cause.
Some of the casual
remarks of other workers show that errors may have
occurred.
The three
Plants
surface
checks,
after
types
of possible
sterilized,
.with most
seeds,
spite of attempted
Plants
spores
inoculating
all the other
logical
the presence
Finally,
on account
inoculation,
It would
(1)
from
is made,
of spores
lesions
without
therefore
of the plant.
such treatment
of the sheath
and lesions
mean very
by histo~
correlated
with
in the vicinity.
simulating
of physical
injury
leaf-spot
may occur
to the tissues
by the
infection.
seem wise,
in the following
Strains
in
incidentally
if further
cal work were to be done on this general
to proceed
occur
have been inoculated
portions
examination
methods
or not,
OYer the sheath,
resulting
unless
symptoms
But this will
inoculated
by checks
by spraying
little
by
sterilization.
controlled
Infections
as developing
on the sheath.
whether
are as follows.
but not controlled
have been reported
inoculation
errors
problem,
manner:
of the organisms
(57)
histologi-
in as virulent
a
condition
(2)
as possible
Inoculation
coleorhiza,
first
should
should
foliage
be obtained.
be made
leaf,
and permanent
and into the seed, with a larger
than usual
really
to determine
whether
Fragments
of checks
the infection
of leaf, root,
be as small as possible,
in length,
lation
should
and sheath
not more
is
include
(4)
in duplicate
Specimens
killing
fluid
the point of inocu-
give
longitudinal
type of inoculation,
time limitations
with a
affected
areas
and cross
sections
with
for lesions
so
of each
eosin only if
should
some evidence
well,
show enough
(58)
sectioned
the use 'of haematoxylin.
are working
will
and should
in the concentrated
in this work,
and stained
16mm. lens until
that the stains
be killed
prohibit
(5) Examinations
portion,
at least.
should
mentioned
should
than half a centi-
and some of the unaffected
be obtained
as.to
number
roots,
due to the inoculation.
(3)
meter
on coleoptile,
be conducted
is obtained
and that the
to be detected
with
the 40mm. lens, when
of covering
more
this should
specimens
be used as a means
in a short
period
of time.
SUMMARY
(1)
The strains of Fusarium used'showed no ability
to enter the living tissues of the sheath or of
the first foliage leaf of young wheat seedlings.
(2)
Alternaria sp. was observed to be a frequent
weak parasite or saprophyte on the tissues of the
sheath.
It enters by a short a 6ho~~ infection
thread which proceeds but a short distance into
a single cell and then becomes dormant until the
infected organ dies.
(3) Helminthosporium was observed to produce lesions
involving swollen and modified cell walls.
It seems
to pass between the cells.
(4)
Rapid methods for embedding and for staining
paraffin sections Of small pieces of infected plant
tissues were used and described.
(60)
BIB
(1)
Fusarium
L I 0 G RAP
Blight
Dimitr
of mleat
Atanasoff.
Wisconsin
Published
Cereals.
Contribution
from
1:
XX:
in Cereal
Agr. Exp.
Experiment
of Plant
in Journal
Research
A Study
and Other
Agricultural
tion, and Bureau
(2)
H Y
Sta-
Industry.
of Agricultural
1-32.
Rusts.
Stakman.
Sta. Bull.
study of resistant
138.
Minn.
Histological
and susceptible
strains.
(3) The FusariuE
Problem.
Wollenweber.
1913:
Phytopathology.
Discussion
(4)
The Effect
of classification.
of Temperature
sp. causing
1920,
in the Physiology
Blackman
Annals
and Light
W11eat Scab.
Phytopathology
(5) Studies
111:24.
and Wellsfo~
of Botany
(61 )
on Fusarium
Jean MacInnes.
X: 52.
of parasitism.
and Dey.
30: 389-398,
1916.
II and V.
Methods
used are of value.
(6) Parasites
be16nging
Shear
and Wood.
Agricultuxe,
Bulletin
(7) Methods
Station,
u.
S. Dept.
of Plant
of
Industry.
#252.
Histology.
(8) A Helminthosporium
Minnesota,
1913.
Bureau
in Plant
Stalooan.
to the Genus Glomerella.
Disease
July
1920.
Agricultural
Bulletin
(62)
#191.
Chamberlain.
of Wheat
and Rye.
University
Experiment
of