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FLAGELLAR STRUCTURES OF ZOOSPORES
OF SOME FUNGI AND ALGAE

THESIS FOR DEGREE OF MASTEh LF SCIENCE
MICHIGAN STATE COLLEGE

BERNARD ELLISON
I944

THESIS

 

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FIAGELLAR STRUC'IURES OF ZOOSPORES 01" SM FUNGI AND AIGAE

by
BERNARD 1I_*‘3“.I..I.ISOI\I

A 1531313
Sutmitted to the Graduate School of Michigan
State College of Agriculture and Applied

Science in partial fulfilment of the
requirements for the degree or

MASTER OF SCIENCE

Department of Botany and Plant Pathology

THESIS

 

 

ACKNCNLEDGSM’WT

I should like to eXpress my appreciation to Dr.
Ernst A. Bessey for suggesting this research problem and for
his valuable aid and advice. His interest and encouragement
have been a source of inspiration to me throughout the course
of the investigation and his suggestions and criticisms have
been most helpful.

I should also like to thank Mr. John M. Roberts for
many stimulating and valuable suggestions and for furnishing
me with certain material used in the investigation. Likewise
I should like to express my thanks to D. J. C. Walker of the
Agricultural College of the University of Wisconsin, and to
Dr. C. M. Haenseler of the New Jersey Experiment Station who
were good enough to furnish me with clubbed cabbage roots from
which I obtained the zoospores of Plasmodiophora brassicae. I
should like to thank Mr. Robert Hagelstein, Curator of Myxo-
mycetes, at the American Musieum of Natural History, New York,
for furnishing me with material and a number of helpful sug-
gestions.

In addition I should like to eXpress my appreciation
to all the members of the Botany and Plant Pathology Depart-
ment staff not only for aid given me from time to time on this
problem but also for the consideration and the simulating assoc-
iation I have had during my attendance at Michigan State College

of Agriculture and Applied Science.

/e(‘rzruesa
. v'. v. (z -4: 2.3 T4.» !. ‘J

STUDIES IN THE FLAGELLAR STRUCTURE OF SOME FUNGI AND ALGAE
INTRODUCTION

In those unicellular organisms posessing flagella it
has been shown that the latter may fall into two or possibly
three types (54, $5). These are: the ciliated flagellum or
flagellum pos§ssing tinsels, the whiplash type, and the type
having neither whiplash or tinsels. This last type of flagellum
is probably a modification of the whiplash type of flagellum.
The lash of the whiplash type is merely the extension of the
central core of the flagellum beyond the sheath. This exten-
sion or whiplash has been found to vary in length considerably
on individuals of the same species on the same slide and in
one case a living zoospore was seen by Couch (6) to suddenly ex-
tend the whiplash enormously. Under the circumstances it is
doubtful whether one should consider a flagellum in which the
whiplash is very short or not extended at all a distinct type
on a par with the other two distinctive types.

Certain botanists use the terms flagellum and cilium
interchangeably. It is the author's Opinion that this is a mis-
take in view of the fact that the term cilium when applied to a
swimming organelle of a unicellular organism is so well entrench-
ed in zoological literature as referring to those distinctive
structures (morphologically quite different from flagella) found
on.members of the subphylum Ciliophora, Phylum Protozoa. A fla-
gellum as contrasted to a cilium.is that relatively long, whip-
like, swimming organelle, characterized by having an outer sheath

surrounding an inner core; the inner core arising from a blephar-

cplast which is in turn connected to the nucleus by a rhizo-
plast. It is in this sense that the terms flagellum.and cilium
are used in this paper.

It is now considered that the position, number, type,
etemof the flagella are of phylogenetic importance and they are
being used more and more by mycologists as a means of classifi-
cation of certain groups. In most cases there is a consistent
correlation between the number and types of flagella and certain
physiological characteristics. Chief of these characteristics
is the cell wall composition and in some of the algae the nature
of the photosynthetic product is also correlated (5, 59).

It was:Dr. Bessey's (5) suggestion that probably the
primitive or ancestral flagellate of some of the algae and also
some of the lower fungi was a unicellular, biflagellate, green
alga having both the whiplash and the ciliated flagellum present,
as occurs in the algae of the group XanthOphyceae (or Heterokon-
tae). These two flagella come in the course of evolution, to
have a lateral position with one directed forward and the other
to the posterior. Thus we have, by the loss of one or the other
of the flagella a uniflagellate organism with the flagellum dir-
ected anteriad or posteriad. From such a generalized organism
have arisen other algae such as the heterocont green algae in
which the slimming state has been reduced and now represented
by zoospores. These algae may in turn produce a parasitic ser-
ies due to the loss of the chlorophyll and could reasonably be
regarded as the ancestral forms of the Chytridiales in the broad

use of the name. From this group others arose in turn through

the loss of one flagellum or other. These retaining both flagella
led to the Olpidiopsidaceae; those that lost the posterior, whip-

lash flagellum led to Rhizidiomyces and relatives, and by the loss
of the anterior, tinsel type flagellum arose the whole Olpidium

to Monoblepharis series (6). Likewise from the green flagellates

 

arose the Sarcodina in general and the Mycetozoa, Plasmodiophor-
ales, etc. series in particular through the loss of chlorophyll
and the progressive modification of the flagellum.into the pseudo-
podium.

Sachs (40) suggested that the Saprolegniales origin-
ated from the Siphonales with Vaucheria close to the ancestral
form. There are several facts which support this hypothesis
one of the chief being that there is a strong serum reaction be-
tween Vaucheria and Saprolegnia. (26).

It was Dr. Bessey's opinion that investigation of the
flagellar structure of certain groups might yield additional in-
formation on the phylogeny of those groups. With this in mind
he suggested that flagellar studies be made on the Mycetozoa,
PlasmodiOphorales, Chytridiales, Saprolegniales and some of the
heterokont and coenocytic green algae. It was hoped that the
results would lend support to one of the theories of phylogeny
and relationship that had been based upon other data i.e. com-
parative morphology, serum reaction, etc.

MATERIALS AND METHODS

Zoospores were obtained from material collected by me

and also from.material obtained from other collectors. I shall

not go into the generalities of inducing germination in the var-

iOus organisms largely because there are a number of comprehen-
sive articles on this subject(lO, ll, 12, 14, 50, 57, 41).

In the author's experience there are no general methods which

are effective in inducing the germination in the different genera.
Each species, indeed each specimen, must be treated as an indi-
vidual and germination must be induced by a laborious process of
trial and error. Robert Hagelstein in a letter to the author
exPressed the opinion that the germination studies based on indi-
vidual specimens do not establish the germination characteristics
or requirements for a whole species by any means. The author is
in hearty accord with this Opinion.

Some of the various treatments suggested by different
authors as a means of inducing germination are as follows:
seeding the spores in distilled water; seeding the spores in
soil solutions of various concentrations; seeding the spores in
dilute nutrient solutions of various types; chilling; incuba-
tion at various temperatures; wetting and drying, etc. The author
has found most of these methods useful in individual cases but
useless as a general procedure for inducing either germination
or the formation of zoospores.

Germination was usually carried on in hanging drop
cultures in Van Tieghem cells or more effectively in culture
slides. The latter method was found to be the most satisfactory
in a number of cases for the reasons that when germination takes
a period of several days, condensation on the coverglass in a
hanging drOp culture will cause the drop to spread and some-
times be lost by running down the side of the cell. Furthermore371

cases where there seems to be a mass action effect in the ger-

-5...

mination the culture slide makes it possible to use a greater
number of spores than is possible in the hanging drOp. Temp~
erature was controlled when necessary by the use of incubators
but the majority of the zoospores were germinated at room temp-
erature. The specific methods of obtaining the germination

for each organism referred to in this paper will be discussed
farther on.

Zoospores to be stained for flagellar structures were
collected by means of a micro pipette and placed on a very clean
slide. These zoospores were killed by inverting the slide over
the fumes of a two percent solution of osmic acid. The slide
was allowed to dry for several hours and was then stained with
the b§ff1er flagellar stain technique (25). The relative time
of mordanting and staining was varied according to trial and
error to obtain the best results. In general it may be said
that treating the smear for a maximum time with the mordant and
a minimum time with the stain will produce a sharper staining
with less of the stain precipitating on the slide. After stain-
ing preparationéwere allowed to dry over night in a desiccator.
They were then mounted in Canadian balsam.

Cytological stains were made in cases where it was
necessary to determine whether biflagellate zoospores were ab-
normal or were in a stage of division. These preparations were
stained by an adaptation of the method deveIOped by Cotner (41
42). The strength of the stain was varied somewhat. It was
found that zoospores of the Mycetozoa stain more readily than

most other zoospores and that a weaker stain was more satis-

factory. Smears were prepared in the same way as those prepar- 2-_

ed for staining by the Lgffler stain. Rather than introducing
a drop of stain into the drop of water containing the dead zoo-
spores as Cotner did,it is satisfactory to apply the stain to
the dried smear. After staining, the slide is placed in a des-
iccator for twenty-four hours, cleared with clove oil and then
mounted in Canadian balsam.

The slides were studied under high dry and a clear-
ite oil immersion lens. Drawings were made with a camera lu-
cida and then photographed.

OBSERVATIONS
Stemonitis ferruginea Ehrenb. The specimen of g. fErruginea
used was obtained from John M. Roberts and was collected by him
in Indiamfla in July 1940. It was stored for approximately two
years with no particular attempt to keep the spores viable.
When germination of the spores was attempted in January, 1942,
they were found to germinate readily in distilled water at room
temperature. This germination was carried on in a hanging drop
culture using garden hose wdshers in place of the glass Van
Tieghem cell. The first germination was observed after forty-
eight hours and the spores had germinated up to eighty per-
cent in fifty-four hours. Zoospores were collected in a micro
pipette and tranferred to slides cleaned in hot soapy water and
then stored in ninety-five percent l100h01- The importance 0f
having the slides perfectly clean cannot be over emphasized be-
cause foreign material will cause the stain to precipitate on

the slide. It is likewise important to keep the culture reason-

’/

 

Fig. l. Stemonitis ferruginea

@ Flagellar structure of the swarm
’ .0113.

Fig. 2. Cytological and neuro-

motor apparatus of _S_. ferruginea.

 

ably free of bacteria as they may if presant in large numbers
cause a precipitation of stain on the slide. The zoospores
were killed by exposure to fumes of osmic acid for thirty sec-
onds. The slides were allowed to dry in an inverted position.
This has the advantage of allowing the zoospores to become bet-
ter distributed over the slide because.it prevents them from
being attracted to the periphery of the drOp. It also helps
keep the slides dust free. A number of smears were taken for
staining. The time of mordanting and staining was varied frem
one half to one minute for the mordant solution and one to two
minutes for the stain. During the mordanting and staining the
slide was heated very gently over an alcohol burner with the
flame very low. The slides were never allowed to get so warm
as to be uncomfortable when placed on the back of the hand.
Slides selected as being most satisfactory had been treated with
the mordanting solution for three quarters of a minute and stain-
ed for one and one half minutes. Cytological stains were made
by treating a dried smear for five minutes with a .5 percent
aqueous solution of crystal violet. The stain was washed off
with distilled water and the slide allowed to dry for forty-
eight hours in a desiccator. It was then cleared with clove
oil and mounted in Canadian balsam,

Three types or modification of the flagella were dis-
cernable on the slides stained with the [peffler stain. They
were as follows: approximately 54 percent were of the ordinary
blunt ended type; 44 percent had slender whiplashes of variable

length; and 1.5 percent had the knobbed type of flagella. Bi-

flagellate 2003pores occurred in the ratio of fifteen biflagel-
late to seven hundred and sixty uniflagellate or 1.9 percent.
Ofthese biflagellate 2005pores the flagella were of various types
as follows: both flagella stubbed; one stubbed and one whip-'
lash; one stubbed and one knobbed. Combinations that were not
found were those having two whiplash flagella or a whiplash plus
a knobbed flagellum. No tinsel type flagella were found.

The cytological stain showed a large, dark staining
nucleus present in all cells. It was invariably located in the
anterior portion or flagellar end of the cell. A darker stain-
ing endosome is usually present and in some cases two are pres-
ent. The number of endosomes in the nucleus seems to have no
correlation with the number of flagella. In no case was there
more than one nucleus present in the cell regardless of the num-
ber of flagella present. Nor was there any indication that the
nuhleus was preparing to divide or was in any stage of division.

There is no apparent difference in the neuromotor ap-
paratus of either the uniflagellate or biflagellate cells. Even
in the uniflagellate zoospores the neuromotor apparatus is
clearly of a potentially dual nature. This supports the investi-
gations of the Japanese mycologists Sinoto and Iguasa who con-
tend that the zoospores of the Mycetozoa, whether uniflagellate
or biflagellate, have two bleparoplasts (29, 58).

Two blepharOplasts are present each with a rhizoplast
connecting it with the nucleus. Each blepharoplast gives rise
to a flagellum in the case of the biflagellate zoospores. There

is no apparent difference in the blepharOplasts whether they

produce a flagellum or not.

The zoospores were in various stages of becoming
amoeboid. Practically all of the zoospores showed the forma-
tion of pseudopodia while still in the actively swimming or fla-
gellated state. Other zoospores were seen that had completely
lost their flagella and were carrying on locomotion exclusively
by pseudOpodia. In these cases the entire neuromotor apparatus
had disappeared.

Stemonitis fusca (Roth) Rost.: The specimens of §. fusca used

 

in the flagellar studies were collected in the second college
woodlot during the month of April, 1945 and germination studies
were carried on immediately after collection in hanging drOp
cultures. It was found that the spores germinated readily in
either sterile river water or distilled water at twenty eight
degrees centigrade. Approximately seventy percent of the spores
germinated under these conditions within ninety-six hours.
Little or no germination was obtained at room temperature.

I As in the case of §. ferruginea the zoospores were
collected in a micro pipette and transferred to clean slides
which had been washed in soapy water and stored in ninety-five
percent alebhol. These zoospores were then killed by exposure
to the fumes of a two percent solution of osmic acid for thirty
seconds. The smears were then allowed to dry upside down in a
dust free chamber and were then stained both for flagellar struc-
ture and for cytological details. Again it was found that the
most satisfactory slide for studying flagellar structure was the

one that had been treated with the mordant solution for three

 

 

 

P—W‘
\
1

 

 

 

 

Fig. 3. Flagellar structure of

Stemonitis fusca.

Fig. 4. Neuromotor apparatus

Of go fimca.

-10-

quarters of a minute and with the stain solution for one and

one half minutes. Cytological stains were made by treating the
dried smear for five minutes with a .5 percent aqueous solution
of crystal violet. The slides were then cleared and destained
until satisfactory. The clearing and destainingMcarried on
while observing under the microscope and the process was stepped
by washing the oil of cloves off the slide with xylol. The slides
were then mounted in Canadian balsam.

With the L§ffler stain three modifications of the fla-
gella were observed. They were as follows: approximately
50.4'percent were of the plain type of flagellum; 14.8 percent
were of the whiplash variety, and 46 percent were of the knob-
bed variety. Biflagellate zoospores occurred as about 8 per-
cent of the total. In the biflagellate zoospores the flagella
occurred in the following combinations: plain plus plain; plain
plus whiplash; plain plus knobbed; whiplash plus knobbed; whip-
lash plus whiplash. None were seen in which both flagella were
of the knobbed variety. In no case was a flagellum present of
the tinsel type. The relative length of the flagella varied all
the way from markedly heterokont to isokont. The length of the
flagellum seemed to have no correlation with the type.

Zoospores of §.‘£ggg§ were the best studied as far
as the knobbed flagellum was concerned. These zoospores exhib-
ited the condition in a greater number of cases than zoospores
of any other genus or species. It is difficult to say what this
modification represents. Knobbed flagella have been observed

on members of the Chytridiales and interpreted variously as ab-

-11...

normal forms due to degeneration or due to immaturity (7, l, 15).
Neither of these eXplanations can be regarded as applicable in
this case. The percentage of zoospores having the knobbed fla-
gellum was seen to be as great in young, newly germinated cul-
tures as in old cultures. The percentage remained approximate-
ly the same in cultures germinated in both distilled water and
in river water. Also the temperature during germination did
not affect the percentage. Spores from other specimens were
used to make sure that the condition was not due to a peculiar-
ity of an individual specimen. The knobs on the flagella were
observed by Dr. Bessey and myself on living zoospores thus re—
moving the possibility that the feature was due to the treat-
ment the zoospores received during the killing or staining pro-
cesses.

'Without additional investigation it would be diffi-
cult to make any authoritative explanation of this type of fla-
gellum. Many authors regard the flagellum and the pseudopodium
as homologous. If this is true then the sheath of the flagel-
lum would represent the eetoplag. of the pseudOpodium and the
central cylinder the more fluid endoplasm. If the cylinder or
endOplasm.were for} some reason more fluid than is usually the
case a slight extension of the cylinder instead of remaining as
a whiplash would, due to surface tention, tend to collect into
a drop 0!:knob at the end of the flagellum. This idea is sup-
ported in part by the fact that those species having the knob-
bed flagellum on some of their zoospores also have whiplashes

on others of their zoospores. Thus the knobbed flagellum.may

-1 2...

represent a modification of the ef'the whiplash type and not a
third and distinct type of flagellum, and it must not be regard-
ed in all cases as being an abnormality due to age or environ-
ment.

It would be interesting to investigate this problem
further and determine if the knobbed condition is character-
istic of any particular groups, particularly the mycetozoa.

In'§.'§g§gg the dual nature of the neuromotor appar-1?.
atus is not as evident as it is in §. ferruginea. All zoospor- '
es remain potentially biflagellate in that they have the usual
two blepharoplasts. However, only one of the bleparoplasts has
a rhizoplast connecting it to the nucleus. In the case of the
uniflagellate zoospores it is always the blepharOplast which
does motjhave the nuclear cennection which produces the flagel—
lum. Exactly the same condition is found in the biflagellate
zoospores in regard to the neuromotor apparatus as is found in
the uniflagellate ones except that in the former case both bleph-
aroplasts of course produce a flagellum.

A large nucleus is invariably located in the anter-
ior portion of the cell just as in the case of g. ferruginea.
This nucleus has a darker staining endosome present and occas-
ionally two. There is no apparent correlation between the num-
ber of endosomes in the nucleus and the number of flagella on
the zoospore. In no case was there a zoospore seen with more
' than one nucleus or any condition that indicated that the nuc-

leus or the cell was preparing to divide or in any state of di-

vision.

fl

 

l
10“

 ‘

 

 

Fig. 5. Flagellar structure

of Fuligg septica.

Fig. 6. Neuromotor appar-

atus of E. septica.

-15-

Pseudopodia were present on all zoospores and the
zoospores were seen in all stages of becoming amoeboid. In
those in which the flagella had been completely absorbed the
neuromotor apparatus had likewise degenerated.

Fuligo septica (Linn.) Gmel.: Specimens used in the study were
collected shortly after the formation of the sporangium along
the railroad track by the "Stores“ building.~ These germinated
in four days in both distilled water and in sterile river water
at room temperature and also at 28 degrees centigrade. Time

of germination was cut down to 48 hours at room.temperature by
wetting and drying the spores. Germination was carried on in
culture dishes. This is the only onzof the many slime molds
which I germinated or attempted to germinate which was benefit-
ted by the wetting and drying method as recommended by Jahn
(14).

Three modifications of the flagellar structure were
seen in the following proportions: 52 percent the whiplash
type; 47 percent of the blunt ended type; and less than one
percent of the knobbed type. Since these are gii probably
all modifications of the whiplash type it would probably be
more accurate to say that one hundred percent of them were
of the whiplash type. None of the zoospores had flagella of
the tinsel type.

Biflagellate zoospores were common, amounting to a-
bout 26 percent of the total. These had the following combi-
nations of flagella: blunt ended plus blunt ended; blunt end-

ed plus whiplaSh; whiplash plus whiplash; and blunt ended plus

-14..

knobbed flagellum. The combination not observed was whiplash
plus knobbed. The relative length of the flagella varied all
the way from markedly heterokont to isokont.

Like the other members of the Mycetozoa studied two
blepharoplasts are always present in both the uniflagellate
and the biflagellate zoospores. In the case of the biflagel-
late condition each of the blepharoplasts produces a flagellum.
Only one of the blepharOplasts had a rhizoplast connecting
it to the nucleus in the case of the uniflagellate as well as
the biflagellate zoospores. In this regard the zoospores re-
semble those of Stemonitis fusca. In the uniflagellate zoospore
the single rhiz0p1ast connects the blepharoplast that does not
produce the flagellum.

A rather large but lightly staining nucleus is pres-
ent in the anterior part of the cell. No endosomes were ob-
served in the nuclei. In no case was there more than one nuc-
leus present in a cell. There was no indication that any of
the nuclei were in a stage of division or were preparing to
divide.

Plasmodigphora-brassicae_Woronin 1878: The flagellar struc-

 

ture of Plasmodiophora brassicae is of particular interest at
theepresent time due to a tendency on the part of some auth-

ors (17) to place the genus in the family Woroninaceae. This
is done largely on the basis of Ledinghamds research (19, 20,
21) which showed that some genera of the PlasmodiOphoraceae

(Plasmodiophora and Spongospora) had two flagella rather than

 

one as was stated in the original description. It has been

Fig. 7. Flagellar structure

Q

l

g of Plasmodiophora brassicae.

-15-

shown, howeVer, that the slime molds as a group are potential-
ly biflagellate (29, 58) in that they have two blepharoplasts
and so the posession of two flagella should not necessarily
make it impossible to consider the Plasmodiophorales as being
allied to the slime molds. As some other members of the Heron-
inaceae have been shown to have a tinsel type of flagellum and
the slime molds have only the whiplash or modified whiplash
type, the presence or absence of the tinsel flagellum on‘glgg-

modiophora should be a diagnostic character of some importance

 

and would tend to confirm its inclusibn in the Woroninaceae.

 

Peronospora line on the one hand or its alliance to the slime
mold group on the other.

The zoospores used in this study were obtained by
germinating spores from infected roots supplied by Dr. C. M.
Haenseler of the New Jersey Experiment Station and by Dr.
William.J. Hooker of the University of Wisconsin. A number
of different methoddas recommended in the literature for ob-
taining germination of the spores were tried with little success.
Chupp (4) recommended germinating the spores in a muck soil
filtrate (pH not specified) and incubating them at an optimum
temperature of 28 degrees Centigrade. He found that germination
dropped rapidly as the incubation temperature was lowered and
he was able to get little or no germination at room temperature.
He was unable to obtain infection of cabbage seedlings in the
greenhouse during the winter. He was also unable to obtain
the germination of the spores in distilled water. Wellman (57)

on the other hand found the optimum temperature for germination

-16...

to be not over twenty-five degrees centigrade with the percen-
tage of germination rapidly drOpping off as the incubation tem-
perature was raised. He reported germination as being good in
distilled water. Ledingham in a letter to the author reported
that he found germination to be satisfactory when the spores
were wet and dried a number of times and then incubated at room
temperature in tap water with a pH of 8. He also used distilled
water and obtained satisfactory germination. He did recommend,
however, that the spores be germinated in considerable quantity
because there seemed to be some mass effect. These ways were
tried as recommended including placing sterile, excised root

5 tips into the cultures in hope that the presence of the living
host tissue might have a stimulatory effect on the germination
of the spores. Rain water and melted snow water were tried.

The pH was manipulated by adding small amounts of lactic, acetic
and hydrochloric acid to the various media. Minute amounts of
hydrogen peroxide were added to the media in an attempt to sup-
ply oxygen to the spores. Oxygen and air were bubbled through
the cultures in an attempt to induce germination. Results were
either entirely negative or the germination was so very slight
that it was impossible to obtain the zoospores. A spore was
once observed germinating, by both Dr. Bessey and myself, which
had become attached to one of the root hairs of an excised root
placed in the culture. The spore case was seen to be split and
a small amount of protOplasm had oozed out. Attached to this
bit of protOplasm two actively beating flagella could be detect-

ed. Unfortunately this was lost in attempting to transfer the

A
r

-17-

rootlet from the culture dish to a slide for staining. Our ob-
servation confirms those of Hellman (57) on the germination of
the spore. He reports that flagella are produced almost immed-
iately after the first bit of cytoplasm oozes through the break
in the spore case and from that time on the partially germinated
spore swims about actively. As considerable time may elapsabe-
fore germination is complete it makes the last stages of germin-
ation exceedingly hard to observe. It is not altogether sur-
prising that spores of E. brassicae should vary in their require-
ments for germination because the existence of biological races
in E. brassicae has been shown by Walker (56).

The best germination obtained was obtained under the
following conditions: infected roots which had been frozen and
thawed several times over a two month period were maicerated in
a mortar. Distilled water was added and the mixture stirred up
to place the liberated spores in suspension. The coarser mater-
ial was allowed to settle to the bottom and the supernatant liq-
uid with the spores in suspension was decanted off and strained
through cheese cloth. It was then placed in the centrifuge and
rotated slowly to throw the coarser plant material to the bot-
tom. The spore suspension was then poured into another centri—
fuge tube and centrifuged again, this time more rapidly so that
the spores were thrown down. The water containing most of the
bacteria was then poured off. Sterile distilled water was added
and the spores stirred up into a suspension again. They were
centrifuged once again. This washing was repeated a number of

times depending on how numerous the bacteria were originally.

—18-

The spores were eventually left to germinate in a Syracuse watch
glass containing sterile distilled water made just acid by add-
ing acetic acid with litmus paper as the indicator. The spores
were incubated at twentyfive degrees centigrade. On the third
day a fair degree of germination had taken place.

2003pores were collected by means of a micro pipette
and placed on clean slides. The zoospores were then killed
and fixed by a forty-five second eXposure to the fumes of a two
percent solution of omnic acid. They were than stained by means
of the L§ffler technique. Those slides selected as most satis-
factory were those that were mordanted for forty—five seconds
and stained for one minute.

The zoospores were all of the biflagellate type and
in.most cases markedly heterokont. In some of them the shorter
flagellum was so reduced as to be almost unnotiégble. In one
zoospore found the two flagella were very nearly isokont. The
one flagellum was only slightly shorter than the other. The
flagella were in all cases of the blunt ended type. No tinsels
or whiplashes were to be found. In regard to the absence of
whiplashes this agrees with the work done by Ledingham. ~In his
work he used the Cotner stain. This type of stain is good for
showing the whiplash. In none of the zoospores in his photomi-
crographs is there a whiplash type of flagellum to be seen. No .

zoospore had a tinsel type of flagellum. No cytological stains

were made.

Nowakowskiella'gfi. Schroter (1892:82): The zoospores used were

obtained from Mr. John M. Roberts from a culture that heyuas

n-nd—.m____—._e_afl .

 

 

Fig. 8. Flagellar structure of

the swarm cell of Nowakowski-

 

’ 6119. 220

Fig. 9. Flagellar structure
of the swarm cells of Synchy-

trium decipiens.

-19-

using for his research on the general cytology of the fUngus.
Liberation of the zoospores was induced by mounting the zoo-
sporangia in a hanging drop of distilled water and chilling
slightly. This caused almost instant release of the zoospores.
Zoospores were collected with a micro pipette and transferred

to clean slides where they were killed and fixed by a thirty
second eXposure to the fumes of a two percent osmic acid solu-
tion. The smears were allowed to dry in a dust free chamber

and stained with a L§ffler stain. These preparations were mount—
ed in Canadian balsam. The best results were obtained by treat-
ing the slides with mordant for three quarters of a minute and
with the stain for half a minute. Cytologically stained mounts
were obtained from Mr. Roberts. They had been stained by the
Cotner method.

Two types of flagella were observed on the slides
stained by the LBffler method. These were the definite whip-
lash type and the blunt ended flagellum. The whiplash varied
in length from very short to some having the whiplash as long
as the flagellum itself. It was found that in the cases where
the whiplash was extremely long there was a corresponding reduc-
tion in the length of the flagellum proper. Less than one per-
cent of the 2008pores had two flagella. These flagella were
both of the whiplash type. Mr. Roberts interprets these bi-
flagellate but uninucleate zoospores as abnormal fsnms.

The cytological stains show that the flagellum arises
from a basal granule or blepharoplast located just under the

cell membrane. A rhizoplast connects the blepharoplast with

Fig. 10. Flagellar structure

of the zoospore of Eythium.gp,

 

‘3 9

’ Fig. 8. Flagellar structure of

the swarm cell of Clamydomonas

81').
i

, «‘A,__ _,

-20-

the nuclear cap. The rhiz0plast seems to pass near a large re-
fractile vacuole in most cases. The significance of this is not
clear. The abnormal biflagellate zoospores have not yet been
investigated satisfactoryily.
Synchytrium decipiens Farlow: The specimen used was collected
by Dr. Bessey on a leaf of Amphicarpa dioica. The zoosporangia
were scraped from the sori on the leaf and transferred to a drop
of distilled water in a hanging drop culture. They germinated
in three hours at room temperature. The zoospores were collect-
ed by a micro pipette and transferred to clean slides where they
were killed and fixed by exposing them to the fUmes of a two
percent solution of osmic acid for thirty seconds, then dried in
a dust free chamber for twenty-four hours. These smears were
stained with a L§ffler stain.

Only uniflagellate zoospores were found to occur.
These flagella were all of the whipash or blunt ended type. The
whips were relatively short, varying from none at all to about
one micron in length. Approximately fifteen percent had no
whiplash at all. No cytological studies were made because no
biflagellate zoospores were found.
Pythium.gp, Pringsheim (1858): The specimen used was collected
by placing a hemp seed in a petri dish with water from the Red
Cedar River. A pure culture was obtained as far as fungi were
concerned by transferring a number of 2008porangia into a petri
dish containing some hemp seeds in sterile river water. Zoo-
sporangia were induced to germinate by placing them in distil-

led water in a hanging drop culture and chilling slightly. Zoo-

-21-

spores were produced within an hour. These zoospores were col-
lected by means of a micro pipette and because of their great
numbers placed in drops of distilled water on clean slides.
These were then eXposed to the fumes of a two percent solution
of osmic acid for thirty seconds. The smears were dried for
twenty-four hours and then stained by the L§ffler method.

The flagellar stains showed a very consistant type
of flagellation throughout. There was a "flimmer' or tinsel
type flagellum densely covered with cytoplasmic filaments and a
whiplash type of flagellum. The whiplash flagellum was remark-
ably constant in having whiplashes of approximately the same
length, the length of the whiplash being about .8 of a micron.
In no other form studied was the flagellation so constant. No
cytological stain was made because the flagellation was consis-
tent in all cases with that described for the Species.

Clamydomonas‘gp. Ehrenberg, 1855: The specimens of Clamydomon-

 

g£_used were collected from a sand culture growing in the green-
house. These were placed on clean slides and killed by expos-
ure to the fumes of two percent osmic acid for a minute and a
half. It was found that in general algae required a longer ex-
posure to ggmic acid fumes to preperly kill and fix than did

the zoospores of fungi. The smears were left to dry in a dust
free chamber for twenty-four hours and then stained with the
L§ffler stain. 'The treatment which seemed to give the best re-
sults was to treat with the mordant for two minutes and with

the stain for half a minute. These were then dried for twenty-

four hours and mounted in Canadian balsam.

-22-

The flagellar stain showed the two flagella to be of
the whiplash type. The length of the whiplash variied from
none at all to about half a micron in length. Approximately
twenty-five percent showed no whiplash at all on either flagel-
lum, thirty percent with a whiplash on one of the flagella, and
forty-five percent with whiplashes on both flagella.

No cytological stain was made because the flagella-
tion was consistant with that described for the genus and also
because Smith in his text (Cryptogamic Botany Vol. I) gives a
very good description and drawing of the neuromotor apparatus
for Clamydomonas.

Cladophora glomerata Kutzing, 1845: The Cladgphora used in
these studies was collected in late June from rocks in the

Red Cedar River. A number of the terminal cells were seen to

be swollen and rounded and it was supposed that they were about
to produce zoospores. To initiate zoospore production in algae
it is suggested that one variously treat the alga by removing it
from.swiftly running water to still; placing in the dark; place-
ing in bright light; placing in distilled water, etc. None of
these ways seemed to have the desired effect. When the swol-
len cells were placed in distilled water plasmoptosis took place
and the contents of the cells were discharged in an amorphous
mass. Finally the cells were left in a hanging drop culture

of sttrile river water. After three days what were thought to
be zoospores were observed to be swimming around in the drOp

and the enlarged cells were seen to have been emptied through

a round hole usually on the side of the cell. The swarmers )9cre

 

of the gametes of Cladophora

glomerata.

(2:;5 f Fig. 12. Flagellar structure
' 3

Fig. 15. Flagellar structure of
the swarm cells of Botgydium

granulatum.

 

 

-25-

collected in the usual manner with a micro pipette and trans-
ferred to clean slides.‘ They were exposed to the fumes of two
percent osmic acid for a minute and allowed to dry for several
hours and then stained with the L§ffler stain. It was found
best to apply the mordant for one and three-fourths minutes and
the stain for half a minute. Upon examination it'was seen that
they were gametes rather than zoospores as they had only two,
flagella. The gametes were markedly heterokont with the longer
flagellum about three times longer than the shorter flagellum.
One zoospore was seen to have a well develOped whiplash on the
longer flagellum. In all other cases the two flagella were all
of the blunt ended variety. No cytological stains were made.
é‘number of gametes appeared to have only one flagellum, but
this may be explained by the fact that the shorter flagellum
could very easily be hidden from.view by the cell body. None
of the gametes had tinsel type flagella.

Botgydigm granulatum (L.) Grev: The specimens used in this
study were collected by me both in the spring and fall in Field
19. According to the description of the genus and species they
are supposed to produce zoospores when flooded with water. I
was unable to stimulate zoospore production in this way. Instead
aplanospores were produced which probably represented encysted
zoospores. It was found that these aplanospores could be sown
on Culture media made by adding agar to a soil filtrate solution.
On this medium the spores germinated to form a germ tube. This
eventually grew to form an irregularly shaped body which in a-

bout a weeks time produced 2003pores. These zoospores escaped

-24-

through a hole dissolved in the cell wall at almost any point.
These zoospores were collected with a micro pipette, killed

and fixed by exposure to the fumes of two percent osmic acid

for a minute and a half and then allowed to dry in a dust free
chamber for twenty-four hours. The smearSwere then stained with
a Lgffler stain. It was found most satisfactory to mordant for
a minute and a half and stain for half a minute. The stained
specimens were dried for twenty-four hours and mounted in Can-
adian balsam.

Upon examination it was seen that the zoospores were
markedly heterocont. The longer flagellum was invariably of
the tinsel type while the shorter flagellum was without tin-
sels and was of the blunt ended variety. In no case was a def-

inite whiplash seen. No cytological stain was made.

Ulothrix 223 Kutzing, 1855: The Ulothrix used in these studies

was found growing in the pool in the botany greenhouse. It was
placed in culture dishes filled with distilled water and zoo-
spores formed in many of the cells. Various ways were tried to
induce the liberation of the zoospores from the cells but with
no success. Finally it was found that by manipulating the fil-
aments vigorously with a teasing needle a number of the gravid
cells could be broken and the zoospores liberated.

The zoospores were collected and placed on slides in
the usual manner. They were killed by a‘QO second exposure to
the fumes of a two percent osmic acid solution and dried for
twenty-four hours, then stained with a L§ffler stain. It was

found most satisfactory to mordant for one minute and stain for

 

Fig. 14. Flagellar structure

of the swarm spores of Ulothrix

E2.

-25-

one minute. The slides were dried and mounted in Canadian bal-
sam in preparation for examination.

It was found that all four flagella were consistent-
ly of the same length and all of the blunt ended variety. None
were seen with tinsel type flagella. No cytological stain was
made.

SUIfliARY MID CONCLUSIONS

Mycetozoa and PlasmodiOphora: The work done on this research
problem has confirmed the contention of Sinoto and Yuasa (29,
58) that the swarm cells of the Mycetozoa have two blepharo-
plasts. It shows that the Mycetozoa may be regarded as poten-
tially biflagellate and thadas Gilbert (10) and the Japanese
authors mentioned above pointed out, actually, two flagella

are frequently found. The writer definitely demonstrated their

presence in Stemonitis ferruginea, S. fuses and Fuliao septica.

 

 

It establishes the type of flagellum for these fonns and by in-
ference the entire Mycetosoa as being the whiplash type or a
modification of that type, i.e. blunt ended or knobbed.

A germinating spore of Plasmodiophora brassicae was

 

seen to have two actively boating flagella thus confirming Led-
ingham's investigation that indicated that the zoospores of E.
brassicae were biflagellate. The investigations of the fla-
gella types of this swarm cell lend support to the view that

the PlasmodiOphorales are closely allied to the Mycetozoa in that
their flagellation is of the Mycetozoa type. Certain authors
wish to place the members of the Plasmodiophorales in the fan-

ily Woroninaceae. Some members of this fmnily however have

--25_-

been shown to have the tinsel type for one flagellum and the
whiplash type for the other. Therefore 2. brassicae, the type
genus and species of the family and order Plasmodiophoraceae
and the PlasmodiOphorales, must of necessity be excluded from
relationship to the Woroninaceae due to its type of flagel-
lation.

Pythiumv223: The work done on Pythium g2, confirms the work
of Vlk and Couch (6, 54, 55) and helps to establish the flagella-
tion of the Peronosporales as being the tinsel plus the whip-
lahs type.

§ynchytrium decipiens and Nowakowskiella.gp.: This investi-
gation establishes the flagellar type of these two genera to

be of the whiplash type. This would strongly suggest that the
flagellar type for the entire Chytridiales (in the narrow sense
of the term) should be of the whiplash or modified whiplash
type.

Chlamydomonas s .: Vlk's investigation showing the flagella

of Chlamydomonas to be of the whiplash type are confirmed by
my results which show them to be of the whiplash or modified
whiplash type.

Botrydium_granulosum: These investigations show the flagella

 

of this form to be of the tinsel plus whiplash type and helps,
with the investigations by Vlk, to establish this as the type
of flagellation for the Heterokont algae.
Cladophora_glpmerata: The zoospores of this genus still re-
main uninvestigated. The gametes, however, are now seen to

be markedly heterokont and to have blunt ended flagella, nei-

-27-

ther of the tinsel type.

Ulothrix 22,: The flagella of this genus are seen to be of
the blunt ended type by these investigations. This confirms
Vlk's findings and helps to establish this as the type for the
group as a whole.

Some interesting deductions and observations should
be mentioned as follows: Evidence has been presented in this
paper that the ordinary blunt ended flagellum as well as the
knobbed flagellum are only modifications of the whiplash type.
The knobbed flagellum, as described in the literature prior
to this paper is described as an abnormal and degenerate con-
dition due to age etc. This is clearly not the case in the
organisms reported in this paper. Instead the indication is
very strong that the knobbed flagellum is a modification of the
whiplash type flagellum and not degenerative in its nature.

It is interesting to note that in all the organisms investi-
gated so far in which there are the two types of flagella,
tinsel plus whiplash, and which are of different length (het-
erokont) it is always the longer of the two flagella which

is of the tinsel type.

l.

2.

5.

4.

5.

6.

7.

8.

9.

10.

ll.

12.

15.

ll».

15.

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