CULTURAL AND GENETICAL STUDIES OF
CERTAIN AGARICS

John B. Routien

A THESIS
Submitted to the Graduate School of Michigan State College
of Agriculture and Applied Science in partial ful­
filment of the requirements for the degree of
DOCTOR OF PHILO SOPHY

Department of Botany
1939

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ACKNOWLEDGMENTS
The writer wishes to express his appreciation
to Dr. E. A. Bessey for his suggestions and advice
throughout the progress of this investigation and for
his assistance in the preparation of this manuscript.
Acknowledgment is also made to Dr. H. J. Stafseth and
Professor C. D. Ball for their critical reading of the
the si s *

125811

TA3LE OF COITEEHTS

introduction

1

CULTURAL STUDIES
Review of literature

3

Materials and methods

7

Results

22

Discussion and conclusions

29

GEHETICAL STUDIES
Review of literature

32

Materials and methods

H1+

Results
Spore germination

1+9

Polarity

1+9

Geographic races

63

Interspecific pairings

63

Intergeneric pairings

61+

Discussion and conclusions

67

SUMMARY

72

LITERATURE CITED

75

PLATES

S3

1
INTRODUCTION

During the past two decades there has "been an increasing amount of
experimental study of the Agaricaceae.

Some of this has des.lt with the

development of the fruiting-body and the cytological phenomena that
accompany the growth and maturation of the sporophore.

Another part of

it deals with the more careful study of the fruiting-"body as an aid in
the preparation of a system of classification that would show the more
probable relationships of the mushrooms.

There have been a few papers

dealing with the culturing of Agarics for the production of frultingbodies, but most of this type of study has been incidental to other prob­
lems*

A very few workers have been engaged in the study of the physio­

logy of members of the Agaricaceae.
The subject of greatest interest to students of this group of
fungi in the past few years,

hov/ever, is what might be

called "sexuality."

It was known for a long time

that the mycelium of some

mushroomspossessed

clamp-connections. Kniep (^4-2, U3 ,
structures and the conjugate

was studying the

division of the nuclei in

formationof these
the cellswhen

Bensaude (5 ) reported in Coprinus fimetarius Fr* the existence of what
has been called "heterothallism."

Bensaude found that the mycelium

derived from the germination of a basidiospore was characterized by the
presence of only one nucleus in each cell and by the absence of clampconnections*

Hov/ever, when certain spores we re allowed to germinate and

the mycelia from these grown together, then the resultant mycelium was
dicaryon and had clamp-connections.

As a result of the work of numerous

investigators it has been found that there is a wide range of phenomena
involved in the Msexuality" (which includes heterothallism) of the Hymenomyceteae.

2
The present investigation was "begun with the hope that some con­
tribution could "be made to the study of the inheritance of characters
in the Agarics, a problem that has received little attention to date.
Before much progress could he made it was necessary to find suitable
media, artificial, if possible, for the development of fruiting-bodies
of the fungi studied and to determine the type of "sexuality" possessed
by each species.
This paper, therefore, consists of two main parts, which are as
follows:

a study of many kinds of culture media for the production of

fruiting-bodies and a study of the types of "sexuality" in the various
fungi.

Hie results of the genetical study were meager and are discus­

sed only briefly in this paper.

CULTURAL STUDIES
Review of literature
In most of the experiments dealing with the production of fruitinghodies of mushrooms in pure culture the investigators have used some
substance or substances upon which the particular fungi normally grow.
For example, sterile horse-dung has been used by Bohn (7)» Borriss
(8 , 9), Brunswik (15), Buller (17, IB). Hanna (33, 34), Lakon (46),
Oort (56 ) and Sass (63) for growing species of Coprinus.

Rabbit-dung

was used by G-ilmore (32) in her work with Psilocybe coprophila Fr.
Several workers, Hanna (35)» Humphrey (3B) and Mounce (50),have used
horse-dung mixed with sawdust or soil or horse-dung placed on or above
a layer of soil or used as a spawn to be placed in the soil.

In some

experiments horse-dung was used with some enriching material such as
malt extract.
Poole (57) found that a mixture of the dead roots and canes of the
dewberry plus prune juice was a good medium for the production of
fruiting-bodies of Collybia dryophila Fr.

It was discovered by Arnold

(2) that 0_. cirrata Fr. , C_. cirrata var. Cookei Bres. and C. tuberosa
Fr. formed typical sporophores when grown on the caps of Boletus luteus.
This was true when the caps were placed on the surface of wet soil and
the caps and soil sterilized by steam under pressure.

However, when

the soil was first sterilized by means of dry heat, the fungi would not
grow.

This same investigator found that Marasmius elongatipes Pk. would

fruit when it was grown on a medium consisting of powdered oak wood,
malt extract solution and peptone.
Thompson (6S) was able to secure fruiting-bodies of Nyctalis asterophora Fr. and H. parasitica (Bull.) Fr. on corn-meal and oat-meal agar.

h

Etter (25) secured good results with several species "by the use of
a medium consisting of corn-starch, corn-meal, sawdust from some wood
that the fungus normally grew upon and a strong solution of malt extract
that was added to the medium at the proper time,

Eaufert

(Ho)

obtained

fruiting-bodies of PIeurotus corticatus Fr. by the use of sawdust of
the basswood tree plus four per-cent malt extract solution.
Apparently very few attempts have been made to use vegetable mate­
rial itself for the production of fruiting-bodies*

Long and Harsch (hj)

found that the few species of Agarics that they tried as well as more
than thirty species of wood-rotting fungi developed fruiting-bodies on a
number of vegetables and fruits as well as on corn-meal and malt.

Of

the vegetables the parsnips and prunes were especially good; beans,
celery, beets, potatoes and alfalfa were useful to some extent.
Hot many workers have successfully used artificial media.

Gilmore

(32) used/a medium of glucose plus certain salts end peptone for Psilocybe
coprophila Fr.

Dickson (22, 23 , 2h) found that malt extract was suit­

able for Coprinus sphaerosporus (new species), and Sass (63 ) also found
this medium to be useful in his work with certain species of Coprinus.
The writer did not find in the literature any record of an attempt
to discover purely artificial media that would permit the production of
fruiting-bodies.

A medium that could be made up in exactly the same man­

ner every time and which would contain exactly the same materials would
be valuable for cultural and genetical studies.

With this in mind, the

writer tried to find such a medium for at least some fungi.

Lack of

notable success led him to try other types of media.
Careful study of the physical factors involved in the production of
sporophores has been almost entirely neglected.

Almost all of the

5

workers referred to in the preceeding paragraphs have stated that dif­
fuse sunlight is necessary for the formation of fruiting-bodies;
Dickson (22), however, found that normal sporophores of Coprinus
sphaerosporus would develop in the dark.

Long and Harsch (^7) say that

light is essential and that the character of the substrate is of secon­
dary importance.

Most of their work was done with such wood-rotting

fungi as Polyporus, Fomes, etc.
Borriss (8, 9), working with Coprinus lagopus Fr., reported that
certain rays of spectrum (UOO-^OO m u) are most effective in regard to
the production of fruiting-bodies and that rays longer than 6^0 m u are
of no value.

The greatest results were obtained by the use of rays

between UU0-U 50 m u.

Apparently of almost equal importance is mechani­

cal stimulation of the fruit-body primordium.

Temperature and humidity

were much less important.
Several workers, Etter (25), Hanna (35) an<3- Kaufert (*40), have
pointed out that the various colorings or hairs on the pileus or lines
on the stipe are apparently influenced by the relative humidity of the
atmosphere.

Freedom for the cap to expand outside of the container also

is necessary for the typical markings to develop.
Etter (25 ) and Kaufert (^0) have emphasized that the medium must
be porous and that there must be an abundant supply of readily-available
food materials.
In general, rather moderate temperatures (22°-27°C.) are best for
the growth of the mycelium and production of fruiting-bodies.
Another possible factor which has been hinted at in some reports,
Lakon (U6 ) and Gilmore (32), is indicated by the fact that certain fungi
have produced numerous typical fruiting-bodies during the summer months,

6
"but during the winter months these same fungi, under seemingly similar con­
ditions, form atypical sporophores or none at all.

The cause of this is

unknown.
The relation of the genetical constitution of the fungus to its
ability to form fruiting-bodies has been studied only within the last few
years.

At first it was thought that only the dicaryon mycelia would form

sporophores, and the appearance of the fruiting-bodies was suggested as a
criterion for judging whether or not a mycelium was monocaryon or dicaryon.
It was soon pointed out, however, that the monocaryon mycelia of certain
Agarics would produce fruiting-bodies.

A criticism that might have been

more pertinent, honrever, would be that, since so little was known concerning
the factors operative in the formation of fruiting-bodies, their absence
should not be interpreted as indicating that the hyphae were monocaryon.
Zattler (90) found in Schizophyllum commune Fr. that certain monosporous strains when grown together would produce typical fruiting-bodies,
while certain other strains when grown together would produce abortive
rudiments of sporophores.

This was interpreted as being due to a gene, g,

which is recessive and which operates toward the production of a rudimen­
tary fruit-body.
Gilmore (32) found evidence in Fsilocybe coprophlla that the produc­
tion of irregular fruiting-bodies by that fungus was controlled, in part,
by a gene that was neither dominant nor recessive.
Dickson (2U) found that abnormal fruiting-bodies were produced by
Coprinus macrorhizus Rhea.

His interpretation of results was that typical

fruiting-bodies would form only when the monosporous strains involved had
a full normal complement and that the abnormal sporophores were the result
of the presence in at least one of the mycelia of a number of recessive
genes and/or such chromosome aberrations as translocations and deletions.

7
Materials and methods
Tissue cultures of a number of Agarics were used in this study.
The cultures were secured by sceptically removing from the interior of
the stipe of a freshly-collected mushroom a small piece of tissue and
transferring it to acidified potato malt agar that had been poured into
a sterile Petri dish and allowed to harden*

If any mycelium developed,

it was transferred to slanted tubes of potato malt agar*

Prom these

tubes transfers were made to the various media that were to be tested.
Nearly all of the mushrooms that were studied were secured in
culture during the fall of 193& and during the growing seasons of 1937
and 193S.

With the exception of Coprinus radians Fr., which was cul­

tured from a sporophore that appeared on the campus of Northwestern
University, Evanston, Illinois, in 1935*

of the fungi were collected

either in the city of East Lansing, Michigan, or in the fields[or woods
nearby.

Since not all of the fungi were available from the start of the

study and because of difficulties in setting up large numbers of some
of the experiments, some of the species were grown on only a few of the
media while others were tested on all of them.
The species that were identified, cultured and tested on different
media are listed.

After each name is the approximate date upon which

the species was cultured.

They are as follows'*’:

Coprinus atramentarius

Fr. (October, 193^)» C. comatus Fr. (October, 193^),
1

fimetarius

The plants described by Peck and Longyear as well as Coprinus fimetaius xnacrorhizus Fr. are listed as they are given by Kauffman (Ul).
The author citations for the other species have been determined in
accordance with the rules of nomenclature aa formulated by the 1930
meeting of the International Botanical Congress.
In the determination
of the cita.tion of author the^works of Briquet (11), Fries (26, 27, 28,
29» 30). G-illet (31) &&& Quelet (5^) were consulted*

s

macrorhizus Fr. (June, 1937)» 0,. micaceus Fr. (September, 193&J August,
1937)* 0,» plicatllis Fr. (August, 1937)t

quadrifidus Peck (June, 1937)>

C. radians Fr. (June, 1935)* C.* radiatus Fr. (November, 1937)*

££mi-

lantanus Peck (August, 1936), G-alera crispa Longyear (July, 1937)*
✓
<
tenera (Fr.) Quelet (July, 1937)* Fanaeolus campanulatus (Fr.) Quelet
(September, 193^)» P* retirugis (Fr. ) Qjielet (September, 193&), P* soli/

dipes Peck (September, 193&), Psathyrella disseminata (Fr. ) Quelet
(June, 1937)* Psilocybe subviscida Pecic (June, 193*0, Stropharia semiglob at a (Fr.) Quelet (October, 193&)*
During the first eight months of testing of the media end fungi the
cultures were grown at room temperature (about 21°C.) in the laboratory.
Additional light was supplied at night by means of a 60 watt electric
light bulb that was suspended about three feet above the cultures.

During

the next four months the temperature varied from about 2U° to 35°C.» and
no additional light was supplied.

After that the temperature was again

near 21°C. for eight months; during this period, also, no supplementary
illumination was used.
In trying to find media, upon which the fungi would form fruitingbodies several types of media were tried.

The formulae and methods of

preparation of the media that were tested are listed below in several
groups.

Each group consists of media of the same general type.

The agar, malt extract and yeast extract were Difco products.

Unless

there is a statement to the contrary, it is to be understood that the
medium was tubed, sterilized at 15 pounds pressure for 30 minutes and
then slanted.
After the name of each medium there is a date to signify when that
medium was used in the tests.

Since all of this phase of the study was

9

simply a search, for a medium upon which typical sporophores would form,
the experiments were not duplicated.
A.

Natural media (with or without other materials added)
(1)

Sterile horse-dung (June, 1937)
Fresh horse-dung was placed in 250 ml. or 500 ml.
Erlenmeyer flasks, moistened with distilled water
and sterilized at 15 pounds for one and one-half hours.

(2)

Grass (June, 1937)
Grass from a lawn was dried and cut into small pieces.
Some of this was placed in Erlenmeyer flasks and enough
water added to saturate it. The material was sterilized
for one and one-half hours at 15 pounds pressure.

(3)

Decayed wood (June, 1937)
Wood from a much-decayed log of Fraxinus americana was
dried at 100°-1^0°C. and then powdered. 125 ml* of this
were placed in a 250 ml. or 500 ml. Erlenmeyer flask,
and 73 ml*
distilled water were added. After the wood
had lecome saturated the excess water was poured off and
the wood sterilized at 15 pounds pressure for one and onehalf hours.

(^•)

Sawdust (June, 1937)
125 ml. of fine sawdust from several deciduous trees were
soaked in distilled water for one hour. The excess water
was poured off and the contents of the flask were sterili­
zed at 15 pounds pressure for one and one-half hours.

(5)

Horse-dung straw (November, 1937)
A mixture of horse-dung and two-year old wheat straw was
planed in 250 ml. or 500 ml. Erlenmeyer fla.sks, moistened,
then sterilized at 15 pounds for one and one-half hours.

(6 )

Sand horse-dung decayed cottonwood (38) (Novernier, 1937)
Into 100 ml. and 250 ml. leakers was put enough sand to
make a layer three cm. deep. To the 100 ml. leakers were
added alout 100 ml. of a mixture of fresh horse-dung and
small pieces of partially decayed cottonwood.
In the
larger leakers only horse-dung was added. The materials
were saturated with distilled water and the excess poured
off. The leakers were covered with several pieces of
cheesecloth and were then sterilized at 15 pounds for one
and one-half hours.

(7)

Horse-dung agar (May, 1937)
Fresh horse-dung was extracted in distilled water for a
few hours, filtered through cheesecloth and cleared ly
means of an egg white. To this weie added 15 gm. agar.

10
(S)

Straw (June, 1937)
Two-year old wheat straw was cut into small pieces and
placed in 250 ml* or 500 ml. Erlenmeyer flasks. The straw
was saturated with distilled water, the excess water was
poured off and the contents of the flask sterilized at 15
pounds for one and one-half hours.

(9)

Straw malt-extract (July, 1938 )
This was prepared in the same manner as the straw medium
except that a solution of 30 gm*
malt-extract per liter
of distilled water was used in place of the water alone.

(10)

Straw potato mait-extract (July, 1937)
This was prepared in the same manner as the straw medium
except that a mixture of the extract of 200 gm. of potatoes
per liter of distilled water plus 15 gm. of malt-extract
per liter was used.

(11)

Potatoes (July, 1937)
Potatoes that had "been extracted in distilled wa,ter were
placed in Erlenmeyer flasks and sterilized at 15 pounds for
one-half hour.

(12)

Bread (August, 1937)
Small pieces of whole wheat and white "bread were placed in
Erlenmeyer flasks and saturated with distilled water. The
contents were then sterilized.

(13) Milk malt-extract agar (November, 1937)
Malt extract--------------------15 gm.
A g a r ------ ---------------------15 gm.
Pasteurized m i l k --------------- 1 liter
B.

Artificial media (including some natural nutrients)
(1*4-)

Xylose potato agar (April, 193S)
X y l o s e --------------------------12.5 gm.
A g a r ------*
— 15*0 gm.
Potato extract (200 gm. potatoes
in 1 liter distilled waiter----- 1 liter

(15)

Glucose aga.r (October, 193&; February, 1937)
Glucose------------------------- 10 gm.
A g a r ----------------------------15 gm.
Distilled w a t e r ---------------- 1 liter

(l6 ) Potato glucose agar (October, 193^5 February, 1937)
Glucose------------------------- 20 gm.
A g a r -------------------------- — 15 gm.
Potato extract (200 gm. potatoes in
1 liter distilled w a t e r ) ------- 1 liter

11

(17)

Stronger potato glucose agar (October, 193&'* February,
1937)
Glucose---------------------------- 10 gm.
Agar-------------------------------- 15 gm.
Potato extract (>+00 gm. potatoes
in 1 liter distilled w a t e r ) -------- 1liter

(IS)

Peptone potato glucose agar (October, 193^i February,
1937)
Glucose---------------------------- 20 gm.
Pep t o n e --------------------------- 2 gm.
Agar
15 gm.
Potato extract (200 gm. potatoes in
1 liter distilled water) --------1 liter

(19)

Modification of Piefer, Humphrey and Acree’s Medium
(6l) for Wood-destroying Fungi (May, 1937)
— ---- 1+0.00 gm.
Glucose — ------- — ---KHP0.
>+.00 gm.
Asparagine — —
— >+.00 gm.
(HHu )2HP0lj. —
2.00 gm.
MgSOh •7 H p Q -------------------------- 2.00 gm.
CaCOr?—
— 0.25 gm.
CaCLg
0.10 gm.
A g a r ------------------------------- 15.00 gm.
Distilled water —
1 liter

(20)

Modification of Leonian's (6l) Agar Medium (May, 1937)
Glucose
---------------- — ------ 6.0 gm.
Malt extract
•------- ------------6.0 gm.
EHpPOj,------------ ---------------- 1.2 gm.
MgS0}|.7Hg0
------------ -------- 0.6 gm.
Agar — ---------------------------- l6*0 gm.
Distilled w a t e r -----------—
1 liter

(21)

Glucose corn-meal agar (October, 193&; February, 1937)
Glucose------ -— ---------10.0gm.
Corn-meal
*------------------------ 20.0 gm.
Agar
---------------15*0gm.
Distilled w a t e r
1liter

(22)

Modification of Barnes’s (6l) Medium (June, 1937)
Glucose---------------------------->+.0 gm.
K P0U----------------------------- — 1.0 gm.
------------------------------ 1.0 gm.
KM).,----- — ------------------------ 1.0 gm.
A g a r ------------------------------- 25.0 gm.
Distilled w a t e r
1liter

(23 )

Potato mannose agar (June, 1937)
Mannose-------------------Agar
-------------------Potato extract (200 gm. potatoes in
1 liter of distilled water) —

1 .>+ gm.
l6.0gm.

1 liter

12
(2^)

Standard strength potato malt agar (th.roi3gh.out the
experiment)
Ma>lt extract------------- ----— -— 15*0 gm.
A g a r -----------— ----- 15*0 gm.
Potato extract (200 gm. potatoes in
1 liter of distilled v/ater) -------- 1liter

(25)

Twice standard potato malt agar (October, 193&J February,
1937)
Prepared as standard potato malt agar except that*4-00
gm. of potatoes were used in preparing the extract of
potatoes ra.ther than 200 gm.

(26 )

Half standard potato malt agar (July,
1937)
To 100 ml. of the twice standard potato malt extract
were added 300 ml. of distilled water.
To the resultant
*4-00 ml. were added 6.5 gm* of agar.

(27)

Soft potato malt agar (June, 1937)
Malt extract
15 gm.
5 gm.
A g a r -------- *
Potato extract (U00 gm. potatoes in
1 liter of distilled water)------- 1 liter

(28)

Potato malt agar - Czapek (6l) (May, 1937)
N a N O ? --------------------- --- ----- 1.00 gm,
KHgPOjj-— — ----------------- ---- 0.50 gm.
KC1 ------------------------------ 0.25 gm.
MgSOj^.7^20 --------------- --------- 0.25 gm.
FeSOij..7H2O ------------------------ trace
Malt extract---------------------- 5*00 gm.
A g a r -------------------------------5*00 gm.
Potato extract (^00 gm* potatoes in
1 liter of distilled ws-ter)------- 1 liter

(29)

Malt peptone agar (52) (June, 1937)
Malt extract------------ --------- 70*0 gm.
Peptone ---------------------------10.0 gm.
A g a r ---------------------------- — 20.0 gm.
Distilled w a t e r ------------------- 1 liter

(30)

Modification of mineral malt peptone agar (from
Naumov (52). (June, 1937)
( N H l ^ H P O ^ ------ ------------------ 2.0 gm.
K^HPOii----------------------------- 1*0 gm.
ZnSOij.
----------------------------U.O gm.
MgSOj^. 7H20 ------------------------ 0.5 gm.
Malt extract----------.-— 25-0 gm.
Peptone ---------------------------10.0 gm.
A g a r -------20 .0 gm.
Distilledw a t e r -----------------------1 liter

13
(31)

Mineral Potatomalt agar(November, 1937)
KEUPO^--------- -------------------- 1.000 gm.
C a C l g ------------------------------ 0.100 gm.
MgS0^..7H20---------------- --------- 0.600 gm.
NaNO^----------------------------- 1.000 gm.
FeCl?------------------------- ------ 0.020 gm.
E-zB0i------------------------- ---- 0 .0^0 gm.
BaCl 2------------- ------------ ----0.100 gm.
MnSOj,------------------------------- 0.100 gm.
CuSOj,— ------ ----------- ----------O.OOH gm.
ZnSOj,------------------------------- 0.0SO gm.
LiCl -7------------------------------- 0.00*4- gm.
ITapSTO-z---------------- ----------- 0.006 gm.
COINO-Jp---------------------------- 0.010 gm.
Ni(l\T0^)p--------------------------- 0.006 gm.
AICI3------------------------------- 0.010 gm.
These materials were added to 50 ml. distilled
water, dissolved and then added to 1 liter of
standard strength potato malt agar.

(32)

Dung extract malt agar (June, 1937)
Malt extract---------------------- 15*0 gm.
Agar — --------------------------- 15.0 gm.
Horse-dung extract (in distilled
w a t e r ) ---------------------------- 1 liter

(33)

Dung potato malt agar (May, 1937)
Fresh horse-dung was extracted 2*4 hours in J>00 ml. of
distilled water. The 200 ml. filtrate was diluted
to 1 liter. 500ml. of thismixture were
added to
500 ml. of anextract of *400 gm. potatoes in 1 liter
of water.
Tothis literof material were added the
following!
15*0 gm.
Malt extract------Agar — --15.0 gm.

(3^)

Soil extract potato malt agar (October, 193&J February,
1937)
Soil from the greenhouse was extracted 24 hours in 500
ml. distilled water. The 250 ml. filtrate from this was
added to 250 ml. of an extract of *400 gm. of potatoes
in 1 liter of distilled water. To this were added the
following:
Melt extract---------------------- 15*0 gm.
A g a r ------------------------------- 15*0 gm.

(35)

Soil dung potato maltagar (May, 1937)
Soil was extracted in distilled water and the filtra.te
diluted to 1 liter. To this liter was added horsedung which was extracted for 2*4 hours. 300 ml. of this
extract were added to 200 ml. of an extract of *400 gm.
of potatoes in 1 liter of distilled water. To this

Ik

mixture were added the following:
Malt extrant -------------------A g a r ----------------------------

7*5

7*5 gm.

(36 )

Gra,ss malt agar (May, 1937)
Grass from a lawn was cut into small pieces that
were soaked for IS hours in distilled water. The
filtra,te was diluted to 1 liter and to it were added
the following:
Malt extract---------------------15*0 gm*
Agar
l6.0 gm.

(37)

Leaf mold potato malt agar (October, 193&J February,

1937)
gm. of partly decayed leaves of beech and maple
trees were extracted 2U hours in J00 ml. of distilled
water. To the 500 ml. extract were added 200 gra.
potatoes. This was steamed for 1 hour. The filtrate
was restored to 5^0 ml. volume with distilled water
and to it were added the following:
Malt extract
------------------7*5 gm.
A g a r ------------------------------8.0 gm.
(3S)

Yeast potato malt agar (May, 1937)
Malt extract
15*0 gm*
Agar — -- ---- ---- ------- *
15*0 gm.
Fleischmann1s y e a s t --------------2 cakes
Twice standard potato extract
(see medium 2 h ) ----------------- — 1 liter

(39)

Yeast potato malt agar - Czapek (6l) (May, 1937)
To 1 liter of the medium prepared as in medium
number (27) were added U cakes of FIeischmann's
Yeast.

(*+0)

Soil dung yeast potato malt agar (May, 1937)
To 300 ml. of soil dung potato malt agar (medium
number 3*0 were added 2 cakes of Fleischmann*s
Yeast.

(*4-1) Yeast grass malt agar (May, 1937)
To 1 liter of the grass malt agar (medium number
35) were added k cakes of FIeischmann*s Yeast.
(*4*2)

Yeast extract potato malt agar (August, 1937)
Yeast extract---------------------- 10.0 gm.
Standard potato malt agar (medium 23 )— 1 liter

(U3 ) Live yeast potato malt agar (June, 1937)
To each slant of standard potato malt agar was added
a, suspension of vigorously growing cells of Sac char omyces cerevisiae. After the yeast cells had grown
for three days the various fungi were planted on the
slants.

15

(HU)

Mo&ification of Czapek* s (6l) Medium (October, 193&I
Feb ruary, 193 7)
HalTO-z

2.0 gm#

KHpFO^,------------------------------ 1.0 gm.
K O I -------------------------------- 0.9 gm.
MgS0],.7H20
0.9 gm.
FeS0^. 7H 20 ------------------------- 0.1 gm.
Sucrose
20.0 gm.
Agar
19*0 gm#
Distilled w a t e r -------------------1 liter
(U9)

Duggar's Medium (37)• (August, 1937)
N H ^ U O , ----------------------------- 1.0 gm.
ICHpPOj,
0*9 gm.
MgS0Ir7H20 ------------------------ 0.29 gm.
F e C l ? -----------------------------trace
Sucrose
9*0 gm*
Agar
9.0 gm.
Distilled w a t e r -------------------1 liter

(H6 )

Sucrose corn-starch, agar (January, 193^)
Sucrose
19*0 gm#
Soluble corn-starch
10.0 gm.
Agar
16.0 gm#
Distilled w a t e r ------------------1 liter

(U7)

Lactose milk agar (February, 193^)
Lactose
19*0 gm.
Agar
19*0 gm#
Pasteurized m i l k -----------------1 liter

(US)

Cellobiose potato agar (April, 193^)
Cellobiose------------------------- 35*0 gm#
A g a r ------------------------------- 19*0 gm#
Potato extract (200 gm. potatoes
in 1 liter of distilled water) --- 1 liter

(H9 )

Raffinose potato agar (June, 1937)
Raffinose--------------------------- 1.6 gm#
A g a r ------------------------------- l6.0 gm.
Potato extract (200 gm. potatoes
in 1 liter distilled water) ------ 1 liter

(90)

Mannitol potato agar (April, 193^)
Mannitol — ------------------------- 19*0 gm.
A g a r ------------------------------- 19*0 gm.
Potato extract (200 gm. potatoes in
1 liter of distilled water) ------ 1 liter

16
(51)

Modification of Claussen's (6l) Medium (May, 1937)
KH 2P0 4 --------------------------- 0.050 gm.
NHjiNO-7--------------------------- 0.050 gm.
MgSOu.7H 20
0.020 gm.
Ee^(?0j,
0.001 gm.
Inulin
2.000 gm.
Agar
15.000 gm.
Distilled w a t e r
1 liter

(52)

Czapek*s Glycerine Aga,r (6l) (July, 1938 )
Glycerine
30*0 gm.
HaNO-2---------------------------- 2.0 gm.
K2HP0i|--------------------------- 1.0 gm.
MgSOji.7 ^ 0 ----------------------- 0.5 gm.
K C 1 ------------------------------ 0.5 gm.
FeS0j|---------------------------- trace
Agar
15*0 gm.
Distilled w a t e r ------------------ 1 liter

(53)

Modification ofcorn-mealstarchsawdust medium
(June, 1937)
Corn m e a l
HS.O gm.
Corn starch
l6.0 gm.
Sawdust
8.0 gm.
Agar
20.0 gm.
Distilled w a t e r ------------- 1
liter

(5U)

Modification of Etter*s (25 ) Medium
(June, 193&)
Corn starch
16.0 gm.
Corn meal
50*0 gm.
Sawdus t
8 .0 gm.
The sawdust was a mixture of several woods. The above
quantities of the constituents were placed in 500 ml.
Erlenmeyer flasks and saturated with a solution of
25 gm. of malt-extract in 1 liter of distilled water.
Elasks were then sterilized. 'When fruiting-bodies
began to appear, more of the malt-extract solution was
added.

(55)

Prune agar (October, 193&; February, 1937)
120 gm. of pitted prunes were steamed in 1 liter of
distilled water for one-half hour. The filtrate was
made up to volume and to it were added IS gm. agar.

(56 )

Prune corn-meal agar (October, 193&J February, 1937)
An extract of prunes was prepared as in the prune agar
medium (number 55 )*
1 liter of the extract were added
the following:
Corn-meal------------------------ 20.0 gm.
A g a r -----------------15*0 gm.

(6l)

17

(57)

Oat agar (July, 193^)
100 gm* of rolled oats were steamed for one-half hour
in 1 liter of distilled water* To the filtrate were
added IS gm. agar.

(58)

Modification of Tubeuf1s (6l) Medium (June, 1937)
10.0 gm.
K^&POlj.--------------------------- 5*0 gm.
MgSO^.7H20 ----------------------- 1.0 gm.
S5 per-cent lactic a c i d
2.0 ml.
A g a r ------------------------------ 20.0 gm.
Distilled w a t e r ------------------ 1 liter

C. Addition of poisons or weakly antiseptic substances
(59)

Phenol potato malt agar (June, 1937)
The following solutions of phenol were made up:
100 per-cent (saturated water solution), 50 per-cent,
10 per-cent, 1 per-cent, 0.5 per-cent. One drop of
one of these solutions was added by means of a sterile
pipette to a test tube containing 10 ml. of sterile
potato malt agar of standard strength. The tube v/as
then slanted.

(60)

CuSO^ potato malt agar (June, 1937)
The following solutions of CuSOj, were made up:
2 U (l N was considered to be 159*63 gm. of CuSO^
per liter of solution), 1 IT, 0.5 K, 0.1 IT., 0.01 IT.
These solutions were sterilized at 15 pounds for 30
minutes. The rest of the procedure was the same as
for medium number 59*

(61)

HC1 potato malt agar (June, 1937)
The solutions were made up as follows: 1 IT, 0.5 IT*
0.1 IT, 0.01 IT. The rest of the procedure was the
same as for medium number 59*

(62)

KMnOj, potato malt agar (June, 1937)
The following solutions were made up: IN (IN was con­
sidered to be 31*6 gm. of KMNOij, per liter of solution
since the medium was acidic), 0.5 N, 0.1 N, 0.01 N.
The solutions were sterilized at 15 pounds for one-half
hour. The rest of the procedure was the same as for
medium number 59 •

(63 )

AlCl, potato malt agar (July, 1937)
The following solutions were made up: 5 per-cent, 1
1 per-cent, 0.1 per-cent, 0.01 per-cent. Petri dishes,
each containing one Van Tieghem ring, were sterilized.
Sach dish v/as then maneuvered in such a manner that the
ring came to lie at a distance from the edge of the
dish equal to about one-fourth of the dish's diameter.
In each ring were placed a few drops of one of the
solutions. Standard potato malt agar was slowly poured
into the dish without disturbing the ring. When the

18

agar was firm the fungus was planted about one-fourth
of the distance across the dish on the side of the dish
opposite to the ring. It was thought that as the
solution in the ring gradually diffused into the a^ar
it would come into contact with the mycelium and would
perhaps induce the formation of fruiting-bodies.
(6H)

ZnSO^ potato malt agar (July, 1937)
Solutions were made up as follows: 5 per-cent, 1 per­
cent, 0.1 per-cent, 0.01 per-cent. The rest of the
method was the same as for medium number 63.

(65 )

AglTO-z potato malt agar (July, 1937)
Solutions were made up as follows: 2 per-cent, 1 per­
cent, 0.1 per-cent, 0.01 per-cent. The rest of the
method was the same as for medium number 63*

(66)

Crystal violet potato malt agar (July, 1937)
The following solutions were made up: 1:2,000; 1:50,000;
1:100,000; 1:500,000. The solutions were not sterilized.
Otherwise, the remainder of the procedure was the same
as for medium number 63 *

(67 )

Brilliant green malt agar (July, 1937)
Solutions were made up as follows: 1:2,000; 1:50,000;
1:100,000; 1:500,000. The solutions were not sterilized.
Otherwise, the procedure was the same as for medium
number 63.

(68)

Urine potato malt agar (July, 1937)
Human urine was placed in the Van Tieghem rings. It was
not diluted or sterilized. Otherwise, the remainder of
the procedure was the same as for medium number 63*

(69 )

Boric acid potato malt agar (August, 1937)
----------— ----1*0 gm.
H-zBO?
Standard potato malt a g a r --------- 1 liter

B. Addition of "growth - stimulating" substances*
(70)

3-indo1epropIonic acid potato malt agar (June, 1937)
A stock solution was made by using 0.1 gm. of the a.cid
in 1 liter of distilled water to give a solution with a
strength of 500,000 A. E. per ml. Dilutions were made
to give solutions with strengths of 50»000 ; 12 ,500;
6,250; 1,500; 500 A. E. per ml. Each of these solutions
was filtered through a Berkefeld filter. One drop of a.
solution was added to 10 ml. of potato malt agar in test
tubes. This resulted in a final dilution within the
tubes to give solution of the following strengths: 25O,
62.5 , 31» 7*5 an(I 2*5 A. E. per ml. of medium. The test
tubes of medium were then slanted.

19

33.

(71)

3"inl°le^u 'tyrlc acid potato malt agar (February, 193&)
0.1 ml. of indole butyric acid ("auxiline” manufactured
by the Pennsylvania Chemical Corporation by the use of
3 .551U mg. of indole butyric acid per ml. of 40 per­
cent alcohol) was added aseptically to a tube containing
potato malt agar. This gave a solution of 0.0355 mg.
the acid per ml* of medium.

(72)

3-indolebutyric acid treatment (February, 193&)
1.0 ml. of indole butyric acid was added to 350 ml. of
sterile distilled water to give a solution containing
0.012U mg. of the acid per ml. of medium. This solution
was filtered through a Berkefeld filter and then tubed
aseptically. The cultures of different fungi were left
in different tubes of the solution for 22 hours and were
then transferred to slants of standard potato malt agar.

(73)

Vitamin B-^ potato malt agar (April, 193&)
The vitamin B^ had been prepared by the Williams and
Cline Method by Merck and Company, Rahway, New Jersey.
(a) Malt extract----- — --------- 3*5 gm*
Vitamin
--------— --- ----- 0.0001 gm.
A g a r -------------------------- 3.5 gm.
Standard potato extract
300*0 ml.
Tubed, sterilised and slanted.
(b) Prepared as part (a) except that 0.005 gm. of
vitamin B-^ was used.

(7*0

Theelin potato malt agar (April, 1938)
The theelin or esta-triene - ol - 3 ~ one “ 17 (67 ) was
secured from G. F. Cartland (the serial number was
(Lot no. I - 35 & V C 15) 12 - 11 - 36 ). The following
solutions were made up:
(1) 1 mouse unit per ml. of medium
(0.1^ ml. of the theelin solution was added
to 72 ml. of potato malt agar)
(2) 5 mouse units per ml. of medium
(0.72 ml. of the solution of theelin was
added to 72 ml. of the medium)
(3 ) 25 mouse units per ml* of medium
(3*5 ml*
the solution of theelin were
added to 72 ml. of potato malt agar)
All of these solutions were tubed and sterilized.

Modifications of Conditions of Growth
(75)

Additional light (September, 193& ~ June, 1937)
During the first part of the experiment the cultures
were kept by a window on the west side of the laboratory.
Supplementary light was afforded at night by means of a
60 watt electric light bulb that was suspended about 3
feet above the cultures.

20

(7b)

Change in H + ion concentration (October, 1937)
By the addition of HC1 or HaOH the H + ion concentra,tion of various lots of potato malt agar was adjusted
(with the aid of the La Motte colorimeter) so as to
give the following pH values for the media: ph 3*6*
pH 5*3 (the usual pH of standard potato malt aga.r) and
pH S.U.

(77)

Increase of food material (February, 1933)
(a) 20-25 ml* of standard potato malt agar were poured
into sterile Petri dishes and the fungi planted on
that medium*
(b) About 50“60 ml. of standard potato malt agar were
put into tubes 30 cm* long and 3*5 cm* wide and were
then sterilized*

(78) Transfer of cultures to a second medium (June, 1937)
Fungi were grown for three weeks in tubes of liquefied
potato malt agar at pH of U. Each fungous mat was then
washed aseptically in an individual Petri dish containing
sterile distilled water* Following this the cultures
were transferred to slants of standard potato malt agar*
(79) Water potato malt agar (June, 1937)
After the cultures had grown for IS days on slants of
potato malt agar 1 ml. of sterile distilled water was
added aseptically to each tube.
(50) Pottery potato malt agar (June, 1937)
To each ttmbe of standard potato malt agar were added a
few pieces of clay pottery. The tubes were then sterili­
zed. It was thought that the pottery might serve as a
foundation for the formation of fruiting-bodies*
(51) Repeated subculturing (May, 1937 “ Js
u l 1937)
As soon as the culture had covered the surface of the
slant of potato malt agar a transfer was made to a fresh
tube of the same kind of medium. This was repeated a
number of times. The final sub-culture was planted on
potato malt agar in a Petri dish and allowed to grow for
several weeks*
(52) Ultra violet (July, 1937)
This test was performed with only two species:
Coprinus comatus and _C. sp*
"(a) Cultures of the fungi were grown on potato malt
agar in Petri dishes for a week and were then
exposed (with the Petri dish cover removed) to
ultra violet rays from s. mercury-vapor lamp. The
exposures were made at a distance of about g cm.
for 5 minute, 15 minute and 30 minute intervals.
Immediately after the exposures the treated fungi
were subcultured on fresh potato malt agar in
Petri dishes.

21

(b)

Freshly poured plates of potato malt agar were
irradiated under the conditions outlined in part
(a). The fungi were then transferred to these
irradiated plates of medium from cultures that had
not been treated,

(S3)

Heat treatment (August, 1937)
Cultures of a number of the fungi were grown in repli­
cate for l4 days in tubes of slanted potato malt agar.
One tube of each fungus was heated for 10 minutes in
water held at the following temperatures: U0°C., 35°C.,
70°C., S0°C. At the end of the 10-minute period the
tubes were placed in cold water so that the temperature
of the tubes was quickly reduced to room temperature.
The cultures were then left at room temperature to
continue grovrth.

(SU)

Scarified potato malt agar (August, 1937)
Slants of potato malt agar were scarified thoroughly
by means of the transfer needle. The cultures were then
planted on the medium.

(S3)

Scarified cultures (August, 1937)
Cultures were grown on slants of potato malt agar for
20 days and were then lacerated by means of the transfer
needle either at one place or over the entire culture.

(86 )

Transfer of cultures to sterilized cultures (August, 1937)
Cultures that had been scarified were sterilized and the
medium again slanted. Onto these slants were planted
transfers of other species of mushrooms.

(87)

Revival of cultures (October, 1937 and June, 1939)
Cultures were started in December, 193&* of a number of
the fungi. They were kept in the laboratory until
October, 1937* At that time about 8 ml. of potato malt
agar were aseptically added to each dried culture in an
attempt to revive the fungi and perhaps cause fructi­
fication.
Another series was started in November, 1937i and
the medium was added to the tubes in June, 1939*

22
Hesuits
The chief interest in this part of the study was the value of
the various media for the production of typical fruiting-bodies.

Of

secondary interest were the variations in rate and manner of growth
of the various species on the different media.
Since most of the media were in test-tubes when tested, only the
relative amount of growth (good or poor as compared to the growth on
standard potato malt agar) and the presence or absence of fruitingbodies were usually recorded.

If sporophores did develop, careful

note was made of their appearance and growth.
The words "good" and "poor11 as applied to the growth of the
mycelium are very general terms to indicate the relative amount of
growth of most of the species on a medium*

In referring to the value

of a medium for supporting the growth of the fungi all of the species
that were tested were considered, although not all of them were identi­
fied.
The term tttypical,11 when applied to fruiting-bodies, means that
the sporophores looked very much like the wild specimens that the wri­
ter has seen.

This resemblance was in regard to the gross morphology

and color of the sporophore and the discharge of the spores*
It should be pointed out again that some of the media were tested
with only a few species.
ferent results.

A wider range of tests might have given dif­

The media upon which only a few (less than ten)

species were tried are as follows:

phenol potato malt agar, ultra

violet, straw potato malt extract and potatoes.

23

Growth of fungi on various media.

Growth of the fungi was good upon the following media:

straw,

straw malt-extract, straw potato malt, “bread, milk malt agar, stan­
dard potato malt agar, twice standard potato malt agar, potato malt
agar - Czapek, yeast extract potato malt agar, leaf mold potato malt
agar, soil extract potato malt agar, phenol potato malt agar (except
in the 100 and 50 an<i

some extent the 10 per-cent solutions),

CuSOij. potato malt agar, HOI potato malt agar, KMnO^ potato malt agar,

AlCl-^ potato malt agar, ZnS0j| potato malt agar (except in the stronger
concentrations), AgNO^ (except in the stronger concentrations),
crystal violet and brilliant green potato malt agars (except in the
stronger concentrations), boric acid potato malt agar, urine potato
malt agar, 3“in<iolePropionic acid potato malt agar, indole butyric
acid potato malt agar, 3”*i11dolebutyric acid treatment, Vitamin B 1
potato malt agar, theelin potato malt agar (the use of 25 mouse units
per ml. of medium gave an increase in growth), additional light,
alteration in pH from 3*6 to S.U, increase of food material, transfer
of cultures to a second medium, water potato malt agar, repeated subculturing, ultra-violet, heat treatment, revival of cultures (for
some species; others did not revive).
Some media supported good growth of about one-half of the species
and poor growth for the remainder of the species.
follows:

These media are as

glucose agar, potato glucose agar, stronger potato glucose

agar, glucose corn-meal agar, peptone potato glucose agar, yeast
potato malt agar, dung extract malt agar, soil dung yeast potato malt
agar, soil dung potato malt agar, yeast grass malt agar, live yeast

2U

potato malt agar, mineral malt peptone agar, modification of meal
starch sawdust medium, mineral potato malt agar, lactose milk

agar,

xylose potato agar, cellobiose potato agar, mannitol potato agar,
modification of Etter’s medium.
Growth was poor or entirely lacking on the following media:
decayed wood, sawdust, horse-dung, grass, horse-dung straw, sand
horse-dung decayed cottonwood, horse-dung agar, potatoes, malt peptone
agar, dung potato malt agar, modification of Claussen’s medium, modi­
fication of Leonian's agar medium, modification of Piefer, Humphrey
and Acree's medium, modification of Tubeuf 's medium, potato raffinose agar, modification of Barnes’s medium, potato mannose agar, half
standard potato malt agar, Euggar's medium, sucrose corn-starch agar,
Czapekrs glycerine agar, oatmeal agar, pottery potato malt agar,
transfer of cultures to sterilized lacerated cultures, scarified
potato malt agar.
Production of fruiting-bodies on various media.
Qfypical fruiting-bodies formed on the following media:

Straw

malt, straw potato malt, standard potato malt agar, twice standard
potato malt agar, pots.to glucose agar, strong potato glucose agar,
peptone potato glucose agar.
The modification of Etter’s medium was good for the production
of fruiting-hodies only of Coprinus quadrifidus.

Dung-extract malt

agar was good only for C_. radians, C_. semilantanus, Panaeolus campanulatus and Stropharia semiglobata.
Abnormal sporophores of Coprinus radians developed on potatoes.

25
These will be discussed later.
All of the media that have not been mentioned specifically in
regard to the production of fruiting-bodies were very poor for this
purpose.

Either no sporophores of any species formed or the fruiting-

bodies were quite small and abortive and did not develop.
The following species did not form frui ting-bodies on any of the
media upon which they were grown:

Coprinus atramentarius, £. comatus,

£. plicatilis, Gal era crispa, G. tenera, Psathyrella disseminata,, and
Psilocybe subviscida.
Stropharia semiglobata formed fruiting-bodies only on dung
extract malt agar.

Coprinus quadrifidus formed fruiting-bodies only

on the modified Etter's medium.

Coprinus fimetarius macrorhizus formed

fruiting-bodies only on bread, straw potato malt and potatoes.

Panaeolus

campanulatus, P. retirugis and P. solidipes formed typical fruitingbodies on only a few of the media (horse-dung, straw and straw potato
malt extrs,ct), but small sporophores were formed on several other media.
Coprinus radiatus formed typical sporophores on several media.
Coprinus semilantanus produced typical fruiting-bodies on standard
potato malt agar, twice standard pota,to malt agar, strong potato glu­
cose agar, and on horse-dung (plate 1, fig. l).

On a number of other

media this fungus formed only rudimentary fruiting-bodies.
Coprinus radians developed fruiting-bodies on a number of media
and on many more formed small but fairly typical sporophores.
^ ien

radians

was grown on potatoes the first frui ting-

bodies that developed were typical of the species except that only one
of them shed spores and deliquesced.

A little later, however, there

26

developed fruiting-bodies that were quite different from the normal
specimens*

They were as large as the more typical specimens but

differed in that the caps were distinctly yellow with a brownish
shade only at the top.

The scales on the cap were slightly paler

than in the normal specimens and seemed to be more pointed.
gills were crisped perpendicular to the long axis.

The

Within the next

few days more of these aberrant fruiting-bodies developed.

In these

the pilei were more brownish than yellow, but the gills were crisped.
Moreover, the gills were dry and remained united side by side.
edge of the cap was white and was inrolled.

The

The stipes of these as

well as the first specimens were coarsely striate.

Spores were not

shed from any of these sporophores, and there was no deliquescence
of the gills.
Tissue cultures from one of these unusual sporophores were
placed on standard potato malt agar, straw malt and horse-dung straw.
On all of these the fruiting-bodies that developed were nearly typical
as regards color of the pileus, but they were abnormal in that the
gills were crisped.

Moreover, there was considerable variation in

the formation of spores, their coloration, their discharge and the
deliquescence of the gills.

In no case was there a normal coloration

and discharge of spores with accompanying deliquescence.
About one-half of the cap of each sporophore that formed on the
potato malt agar* had colorless or very pale-brown gills.
due in some instances to failure of the spores to develop.

This was
In other

fruiting-bodies the spores developed but were only weakly pigmented*
Some of the fruiting-bodies discharged the spores that were most
deeply colored, whereas others did not.
normal.

The size of the spores was

27

The sporophores that formed on the straw malt medium were like
those on the potato malt agar except that the gills were more crisped,
there was no discharge of spores and the deliquescence resulted in
a somewhat dry and rather gummy mass,

The spores were normal in size

and shape but were either hyaline or pale brown*

Germination of the

spores was nearly 100 per-cent*
The mycelia of all of the transfers of these aberrant fungi were
very light brown or whitish as compared with the rich brown color of
the typical Coprinus radians*
Some of the cultures of Coprinus micaceus formed fruiting-bodies
on standard potato malt agar and twice standard potato malt agar*

General observations.
It was observed in most of the species that did form fruitingbocLies that the sporophores that were first formed after the fungus
was cultured were most typical.

After that the sporophores were

abnormal in some way, and this tendency to form rudimentary fruitingbodies continued to become more pronounced until finally only primordia
might be formed, or perhaps there would be no indication whatsoever of
the formation of any fruiting-bodies*
Coprinus micaceus
after it was cultured.

produced fruiting-bodies only immediately
The same was true of Stropharia semiglobata.

The three species of Panaeolus soon lost the ability to form fruitingbodies.

Coprinus semi1antanus gradually lost the ability of forming

sporophores, but the culture of that species that had been left for
over a year in the laboratory and was then revived immediately formed
typical fruiting-bodies.

Coprinus radians did not entirely lose the

28

ability to form fruiting-bodies, but the sporophores were not as large
as they were when the fungus was first cultured,

Coprinus quadrifidus

did not form any fruiting-bodies until it was grown upon Etter's medium.
An interesting observation is the fact that in all of the fungi,
with, perhaps, the exception of Coprinus radians, the production of
fruiting-bodies was greater and more typical during the months of April
to November.
On a number of the media the mycelia of at least some of the fungi
behaved in some unusual, manner, i.e. a change of color (from white to
pink, yellow, brown or black) or a change in the habit of growth.

29
Discussion and Conclusions
Of the species that did not fruit, Coprinus atramentarius (bj),
C. comatus (50 )

C. plicatilis (^-6) appear to "be the only ones that

have fruited for other workers*

However, the following species that

produced frui ting-bodies in pure culture seem not to have produced
fruiting-bodies in culture before;

Coprinus quadrifidus, 2* semi-

lantanus, Panaeolus retirugis, P. solidipes and Stropharia semiglobata*
One outstanding fact about the results is that so few media were
valuable for the production of fruiting-bodies.

The modification of

Etter's medium was good for only one species, and dung-extract agar
was of valxxe for only four species.
for a number of fungi*

Seven other media were suitable

This means that nearly SO of the 8J media,

tried were of no.value for the production of frui t-bodies.
An interesting feature is the fact that the media that were useful
were straw, the sawdust-corn meal-corn starch-malt extract medium of
Etter, dung-extract agar and an artificial medium consisting of potato
extrant and either malt extract or glucose.

Except for the fact that

the horse-dung was of so little value, these results are in agreement
with what has been found by most workers.
The reasons for the lack of fruiting-bodies on more of the natural
media, are unknown.
were being used.

It may have been due to the particular fungi that
It might be due to insufficient experimentation with

each medium to determine the best conditions for its use.

The pos­

sibility of the effect of light must not be overlooked, but the fungi
probably were exposed to as much illumination as was necessary.

It

is quite possible that the poor results were due to a lack of abun­
dant, readily-available food in a porous medium as emphasized by Etter
(25) and implied by Humphrey (38 ) and Mounce (50)•

30

The lack of fruiting-bodies on the artificial media, even on those
on which the mycelia grew abundantly, indicates that much more must be
learned concerning the chemical (sources of carbon and nitrogen, kind
and amount of mineral salts needed) and physical factors needed for
the production of fruiting-bodies.

A glance at the results will show

that the fungi grew well only on some of the natural media or on potato
malt agar alone or potato malt agar to which were added various sub­
stances*

Some of the artificial media allowed some of the fungi to

grow rapidly, but most of these media were poor for growth and of no
value for the formation of fruiting-bodies.
It has been mentioned before that a number of workers reported
that malt extract was very good for the growth of the Agarics that
were studied, and LaJPuze (*4-5) has shown that polysaccharides were "best
for the growth of certain wood-rotting fungi*

The reasons for the

value of malt extract for growth and fruit-body production are unknown*
It was thought that the use of poisonous or Mgrowth-promotingn
substances might induce the fungi to form sporophores.

Pailure to do

so must mean that the production of fruiting-bodies depends upon more
than the stimulus given by the substances as they were used in the
experiment.
The reasons for the gradual decrease or entire loss of the ability
to form fruiting-bodies can only be guessed at.

The reasons for the

decrease in fruit-body production during the winter months may be
related to the length of day.
In any work of this nature it should be kept in mind that the
fungi may be changing as the experiment proceeds.

It has been shown

that there may be changes in the fungi that are interpreted as dire to
changes in the genes.

If the production of fruiting-bodies depends

31
upon one gene (as G-ilmore (32) and Zattler (90) claim for certain
species) or upon several (as Dickson (2*0 claims), and if these genes
are capable of changing, then the problem is further complicated by
the fact that one may be working with a culture that would not produce
fruiting-bodies under any condition.

32
GENETI CAL STUDIES
Review of literature
Kniep (*42, *43» ^ 0

"been studying for several years the forma­

tion of clamp-connections in certain Agarics, hut Bensaude (5 ) was the
first to discover in the Basidiomycetes the phenomenon that has been
called "heterothallism."

She found in Coprinus fimetarius Er. that the

dicaryon condition of the hyphae and the presence of clamp-connections
could be brought about only when the mycelia derived from individual
spores of a sporophore were grown together in certain combinations.
Many individuals in various parts of the world, chiefly in Europe
and North America, have investigated the behavior of the mycelia
derived from the basidiospores of many species of Hymenomycetes.

Most

of this work has been done with members of the Agaricaceae, Polypora.ceae
and Thelephoraceae.
A species, or the spore from a fruit-body of that species, will
show one of two general types of behavior.
"heterothallic."

It may be "homothallie" or

Most writers today appear to be using the term

"heterothallic" in the sense that Nobles (55) used it.
this term means, " ... thatthe thalli which react

She says that

together to produce

a clamp-bearing mycelium contain nuclei of different genetic constitution
as regards the factors which determine for or against this reaction."
This definition, however, excludes those species that are not homothallic
and that do not form clamp-connections (the heterothallic species without
clamp-connec tions)•
If the four spores on the typical basidium differ in only one such
factor, there will be two groups of spores as regards their reactions
when paired with one another.

If, however, there are two such factors

located on different chromosome pairs, then there would be the possibility

33
of the spores' falling into either two or four reaction groups (or
"sexual phases" or "sexual groups"), depending upon the time of
disjunction of the pairs of chromosomes during meiosis in the
basidium.
The term "homothallic" now has come to mean that the mycelium
that develops from the spore rather soon becomes dicaryon without
being paired with any other mycelium; that is, pairing of two mycelia
is unnecessary.
The definitions that have just been given are not the same as
those originally applied to the terms.

When "homo thall ism" was first

used in the Phycomycetes it meant that all of the mycelia of a parti­
cular species were bisexual and any male and female nuclei could unite,
whether they were from the same mycelium or not.

"Heterothallism"

was the term applied to those species some mycelia of which produced
male organs and some female.

In such species the male cells had to

come from a mycelium other than the female.
When the Ascomycetes were studied It was found in some (as
Neurospora and Fleur age) that all of the mycelia from the uninucleate
spores had both male and female organs, as did the homothallic Phycoraycetes, but that the male cells would function only when they were
applied to the female organs of another mycelium.

There is a similar

situation in the Basidiomycetes except that there are no sex organs
(unless one wishes to consider the oidia as male gametes).
These phenomena could have been designated as examples of
"hermaphroditism with factors for self-sterility but mutual crossfertility" or as "heterothallism" with a change in the meaning of the
word "heterothallism."

Most of the workers have chosen to use the

3^
latter term.
As mentioned previously, the species that are "heterothallic"
may show tv/o groups of spores or mycelia.

The members of each group,

when paired among themselves on a medium, will not become dicaryon.
However, when any member of one group is paired with any member of
the other group the mycelium that develops will be dicaryon.
species is said to be bipolar.
a.

Such a

The two groups are designated as A and

These two letters are also used to indicate the two allelomorphic

genes that are assumed to exist in order to explain the results.
In other "heterothallic" species there are four groups of spores.
In normal cases the members of any one group will give rise to a
dicaryon mycelium only when they are paired with the members of one
of the other groups.

A species the spores of which behave in this

manner should be designated as quadripolar.

It is believed that the

quadripolar species have two different pairs of factors located on two
pairs of chromosomes.

The four groups of spores, and also the genes,

are labeled as AB, Ab, aB and ab.
Disjunction of both pairs of factors in the second division of
meiosis could result in either bipolar or quadripolar results.

Dis­

junction of one pair in the first division and of the other pair in
the second division would give only quadripolar results.

Disjunction

of both pairs in the first division would result in bipolarity.
Newton (53), Hanna (33)* Qp-intanilha (60), Bohn (7) and Brunswik (l6 )
(and Kniep, according to the latter) have studied the distribution
of the factors by isolation of the tetrads of spores of individual
basidia.

35
During the first years of study of these phenomena the investi­
gators emphasized the importance of the production of fruiting-hodies
in their pairing of monosporous nycelia.

If a sporophore developed

from a particular pairing, the two mycelia were considered to "be
“fertile«H

However, it

could not he considered

soon became apparent that this criterion
in at leastsome cases because monocaryon

mycelia of some species produce sporophores and because not all dicar­
yon nycelia would form fruiting-bodies.

At present, the emphasis is

placed upon the presence of clamp-connections as indicative of the
dicaryon state of the mycelium.

In almost all cases this is satis­

factory because the clamp-connections appear only when the cells have
two nuclei.

Buller (l£) and Newton (53) have also found that a branch

of a dicaryon hyp ha forms a much more acute angle
of a monocaryon hypha.

than does a branch

It has also been observed that the dicaryon

mycelia usually grow more rapidly than do the monocaryon, and sometimes
the dicaryon mycelium assumes an appearance quite distinct from that
of the monocaryon.

There are instances in which oidia are produced

by the monocaryon hyphae but not by the dicaryon (Buller (IS), Hanna
(33), Newton (53) and Vandendries (75))*

These characters have been

used to identify the diploidized (dicaryon) mycelium.

Some workers,

a.s Quintanilha (60 ) have attacked the problem by staining the nuclei.
The development of the clamp-connections and the conjugate divi­
sions have been worked out by Bensaude (5)f Buller (18), Kniep (U2, U3 ,
U^f), Noble (5^) and Quintanilha (60).

36
Discussions of the manner in which diploidization is accomplished
have "been given by Buller (IS), Noble (5U) and Qpintanilha (60).
In some homothallic species the hyphae develop clamp-connections*
In others no such structures are produced.

In the latter case most

workers have assumed that a species is homothallic if the mycelia do
not develop clamp-connections when grown in contact with a number of
different mycelia.

Some have stained the nuclei in order to he sure

that there are two nuclei in each cell of the hyphae.
Some of the workers in this field are the following:

Arnold (2),

Bohn (7), Brodie (12, 13 , lk), Brunswik (15, 16 ), Buller (17, IS, 19),
Chow (21 ), Dickson (22, 23, 2l+), Gilmore (32 ), Hanna (33 , 3I+. 35),
Heldmaier (36 ), Mounce (^+9, 50, 51)» Hewton (53)» Oort (56 ), Qpintanilha
(59, 60), Smith and Brodie (66 ), Vandendries (69 , 70, 71* 72, 73* 7^*
75, 76 , 77, 78 , 79, SO, SI, S2, S3, SU, 85, 86 , 87, 88 , 89) and Zattler
(90).
The exceptions to the scheme as outlined are numerous*

That an

apparently normally homothallic species may have a few spores that
are not homothallic and which are said to he heterothallic has heen
shown hy Sass (62 ) to he the case in a two-spored form of Coprinus
ephemerus.
There are a number of fungi that have heen recorded as homothallic
hy one worker and heterothallic hy another:
(73).

Mounce (^9* Vandendries

The sudden change of a monocaryon mycelium to the dicaryon state

has heen reported hy several workers:
(33 , 3U), Newton (53)

Chow (21), Dickson (22), Hanna

Vandendries (72, 7^).

Irregularities in the pairing reaction between spores of different
sexual phases have heen found hy various investigators for several
species.

Some of these workers are the following:

Brunswik (15),

37
Heldmaier (36 ), Oort (56 ) and Vandendries (7^)*
Some workers have reported that the monosporous mycelia of certain
species have remained the same ip their reaction capacities for many
yearsi

Hanna (3*0 and Mounce and Macrae (5l)*

Vandendries (8*0, how­

ever, has reported a number of species in which the mycelia changed
their reaction capacities, and any of the cases of the change of a
monocaryon mycelium to a dicaryon would come under this class of abnor­
malities.
In quadripolar species the only combinations that will result in
the diploidization of the mycelium and the formation of true clampconnections are as follows:

AB x ab and Ab x aB.

Buller (18), how­

ever, found that certain dicaryon mycelia would diploidize monocaryon
mycelia which, on theoretical grounds, they should not diploidize
(as 11(AB) x (Ab) -f (aB) or (Ab) x (AB) / (ab)” ).

He called these

”illegitimate combinations” and considered that they were at least
quite similar to the ,,Durchbrechungskopulationen" that Brunswik (15 )
and Oort (56 ) had found earlier.
Qplntanilha (60), working with Coprinus fimetarius, was the first
to study these illegitimate combinations extensively and to use
cytological methods in his investigation.

He found that diploidization

occurred but that after the dicaryon condition was established it
persisted only in the apical cell.

This was due to the fact that the

elarap-connection never fused with the penultimate cell.

The result

was that one of the daughter nuclei produced by the conjugate division
of the two nuclei in the terminal cell was isolated from the bypha,
and the penultimate cell came to have only one nucleus.

The formation

of these false clamp-connections occurred only when the following

3S
mycelia were paired:

AB x a3 and Ab x ab.

The formation of false

clamp-connections seems to depend upon the possession by the two mycelia
of factors B or b in common at the same time that the factors A and a
are different.

Qpintanilha (60 ) has also reported a mutation in which

the monocaryon mycelium bears false clamp-connections and has two nuclei
in the apical cell.
In 1933 Vandendries and Brodie (87) reported a phenomenon that they
called "barrage sexuel” and which since then has usually been called
"barrage.”

They found that in certain combinations of the mycelia of

quadripolar species the aerial hyphae of the two mycelia would grow
towards each other until a space of only 3“5

separated them.

The

hyphae would then turn backward and would not occupy the space between
the two masses of mycelia.

The submerged mycelia would approach each

other until a space of about one mm. separated them.

The force causing

this mutual repulsion was not stopped by lead, paraffin, celluloid, mica,
glass and a number of other substances unless they were quite thick.
Vandendries (86) later reported that this barrage would not occur if the
pairing was done in a closed container.

Because strips of these materials

placed between the two mycelia merely decreased the intensity of the
reaction and because a water-tight partition of glass had no effect, the
authors suggested that the reaction might be due to some sort of radia­
tion between the two mycelia.
The barrage phenomenon was found to appear when the mycelia
ab x ab* or the mycelia a'b x a*bl were paired.

Brodie (lU) later found

that the barrage also appeared temporarily between the mycelia that
would diploidize each other (as AB x ab and Ab x aB).

Apparently bar­

rage appears in some species whenever recently isolated mycelia that

39
are genetically different for B and b are paired.

Many workers report

that they do not find this barrage.
Another interesting feature that has been found to exist in almost
every species that has been studied with this point in mind is the
existence of what have been called "geographic races."

Bauch (*0,

working with Ustilago longissima, has found that within a limited area
it is likely that various collections will be partially corape-tible.
This appears to be due to slight changes in one or both of the allelo­
morphic genes involved.

Bauch has found that there are a number of

multiple allelomorphs of each of the two allelomorphic genes that were
first found.

As the distance between the collections is increased the

collections are likely to be completely compatible.
On the basis of his work and that of others, Vandendries (Si)
formulated a law to the effect that all of the spores of a, fruiting-body
of a heterothallic species would form clamp-connections when grown as
mycelia in contact with the mycelia from spores of another fruit-body,
if the two sporophores were collected in the same general locality.

How­

ever, if the spores came from fruiting-bodies that were widely separated
(as America and Europe), then the mycelia would remain monocaryon.

This

reaction has been used by Buller and Newton (19) to identify a certain
Coprinus.
In spite of the fact that sometimes the mycelia from one sporophore
will not form clamp-connections when paired with mycelia from another
fruit-body that was collected in the immediate vicinity as has been
found by Vandendries (79, 89), and in spite of the fact that Mounce and
Macrae (5l) and Vandendries (77, 79. 89) found that mycelia from wi&efyseparated fruiting-bodies sometimes did form clamp-connections, this
rule is true for most of the species that have been studied*

ko
This "behavior of the n$rcelia "brings up the question of how to
explain all of the divergent results that have "been obtained.
the factors A, a, B and "b
According to Butler

At first,

werecalled sexual factors.
(20), Kniep later adopted theterm "copulation

factors" and assumed that the presence of different copulation factors
in the two mycelia was necessary for the diploidization process to occur.
Brunswik, according to Butler (20), attenpted to explain results
by assuming that homothallic and heterothallic species had the same
genotype except for the introduction or appearance in the heterothallic
species of one or two pairs of self-sterility factors.

Quintanilha

(59) has recently re-emphasized this theory.
Hartmann, according to Bose (10), thinks that every cell of the
mycelium is potentially sexual and that the normally dominant sex is
determined by some internal factor.

However, if this factor is weak,

then some external factor, such as the proximity of another gamete of
stronger sexual tendency,

will determine the behavior. This theory

would explain the resultsas being due to the relative

sexuality of the

two mycelia concerned.
In all of these theories it is assumed that mutations of the various
factors concerned would explain the abnormal (or unexpected) reactions
that sometimes occur, the changes in reaction-capacity of some mycelia
after a length of time, the change from the monocaryon to the dicaryon
state and the existence of geographic races.
Probably most workers have explained their results by the assump­
tion of the existence of multiple allelomorphs.

If the genetic consti­

tution of a mycelium is very similar to or very unlike that of another
mycelium (whether it be from the same fruiting-body or from one some
distance away), clamp-connections will not form.

However, if the

bl

mycelia are different "but not too dissimilar, then clamp— connections
will form when the mycelia are paired*
A suggestion made "by Vandendries (Si) on the "basis of his work
with Coprinus micaceus seems worthy of consideration in attempting to
explain the geographic races*

He assumes the existence of at least

two hereditary, genetically dominant factors within the members of a
species*

The spores must possess the same factor before it is possible

for them to form clamp-connections*

Then, and only then, will the

ordinary factors (A, a, B and b) come into play*

However, if the

mycelia possessed different dominant factors, it would be impossible
for the two to form clamps*
In order to explain the partially fertile (compatible) reactions
between certain geographic races of Coprinus micaceus (a suggestion
which would also be applicable to the results obtained by Mounce and
Macrae (51) with Fomes pinicola) Vandendries further assumed that the
two or more dominant factors (which may be called X and Y) could
mutate in such a manner that they would gradually grade into each
other*

In this group of specimens possessing a factor that is neither

X nor Y clamp-connections would form if the mycelia were of the proper
genetic constitution.
Chow (21) seemed to think that the idea of hermdT'phroditic self­
sterility elaborated by Ames (l) could be properly applied to the
Basidiomycetes.

According to this theory the cells of all mycelia

are alike in respect to their "sexual0 factors but possess factors
that make each mycelium self-sterile but cross-fertile (with another
particular mycelium).

42

It is customary to think of the "basidiospores in the Agarics
when the term "spore” is used, hut it has heen found that there are
other spores in a great many of the mushrooms.
as oidia, conidia and chlamydospores.

They are classified

'There are numerous references

in the literature to their occurrence and method of formation
(Bensaude (5). Brodie (12), Brunswik (15 ), Gilmore (32), Martens (48)
and Vandendries and Martens (88)).
It is quite evident, however, that there has heen confusion
in the literature because not all of the workers have used the term
"oidium” in the same sense.

This has heen pointed out in a recent

paper hy Brodie (13 ) who characterizes the oidia as ” ... short,
hyaline, rod-shaped cells with thin walls and dense protoplasmic
contents” which are ” ... formed hy the breaking up of mycelium into
segments” usually without the formation of transverse walls and which
are produced endogenously, not exogenously as the conidia are.
Brodie (13) believes that the oidia are most important as a means
of diploidization of monocaryon mycelium hut admits that in some cases
they may serve as a means of vegetatively propagating the fungus.
Prom a review of the literature Brodie (13) came to the conclu­
sion that monocaryon mycelia of most heterothallic species develop
oidia hut that, as far as is known, homothallic species do not produce
oidia.

He also decided that dicaryon mycelia rarely produce oidia

and then the oidia are often uninucleate.
The occurrence of uninucleate conidia on monocaryon mycelia and
usually dicaryon conidia on dicaryon mycelia have heen reported hy
Kaufert (40) for Pleurotus corticatus and hy Nobles (55)
allescheri.

Peniophora

Probably many of the references in the literature to

**■3
oidia should he considered as references to conidia.
Usually the conidia are h o m e on short sterigma-like branches,
hut Nobles (55) reported an oedocephaloid type of conidiophore in
Peniophoraallescheri, and Kaufert (40) reported coremia for Pleurotus
corticatus.
It has heen found hy many of the persons listed in the preceding
paragraphs that the asexual spores developed on either the monocaryon
or dicaryon mycelia behave in the same manner as did the parent hyphae
as regard the pairing reaction.
There have heen few attenpts to hybridize species or varieties of
Agarics.

Vandendries (84) paired monocaryon mycelia of Bypholoma

Candolleanum with H. velantinum; Pholiota aurivella with P. mutabilis
and Pleurotus Columbians with P. nidulans.
formed.

No clamp-connections were

However, crosses (70) between three unnamed species of

Panaeolus formed a dicaryon mycelium and also a rudimentary sporophore,
A cross hy the same worker (86 ) between the normal black form of
QmphaT ia maura and an albino form that he discovered developed clampconnections hut no sporophore*
Brunswik (15) crossed Panaeolus fimicola with P. campanulatus.
In one of the pairings he found clamp-connections*
Arnold (2) paired Collybia cirrata, £. cirrata var. cookei and
C. tuberosa in all combinations hut did not find clamp-connections.

44
Materials and methods
The species that were used in this phase of the study axe as fol­
lows'*' t

Anellaria separata (Pr.) Karst*, Coprinus fimetarius macror-

hizus Pr., C. micaceus Pr., CL plicatilis Pr*, CL radians Pr., CL semilantanus Peck, Haucoria semiorbicularis (Pr* } Quelet, Panaeolus papilionaceus (Pr*) Quelet, P. retirugis (Pr*) Quelet, Psathyrella disseminata
(Pr.) Quelet, Psilocybe foenisecii (Pr*) Quelet, P. subviscida Peck*
The culture of Coprinus radians was a tissue culture from a specimen
secured on the campus of northwestern University, Evanston, Illinois, in
June, 1935*

Monosporous cultures of Panaeolus papi 1 ionaceus and of P.

retirugis and some of Coprinus micaceus were secured from the Centraal"bureau voor Schimmelcultures, Baarn, Holland.

The two monosporous

strains of opposite sexual phase of Panaeolus papi 1 ionaceus were sent to
Baarn by R* Vandendries and had been obtained in Vienna in 1930*
two mono spore cultures of P. retirugis were sent to Baarn in 193^ from
Paris by A. Quintanilha.

The two mono spore cultures of Coprinus micaceus

were sent to Baarn in 1927 from Belgium by Vandendries.
The other species were collected either in the city of East Lansing,
Michigan, or within a mile of that place.
The mono spore cultures of Coprinus radians and CL semiiantanus
were secured through the use of fruiting-bodies that appeared in test
tubes or flasks of media in the laboratory.

Each of these had been in

culture for about two and one-half years when the mono spore cultures were
obtained.

Single-spore cultures of the other species were obtained from

1
The species named by
are listed as they are
citation of the others
Pries (26, 27, 28, 29,

Peck as well as Coprinus fimetarius macrorhizus
given by Kauffman (^S). For the correct author
the works of the following men were consulted:
30), Gillet (31), Karsten (39) and Quelet (52).

**5

the specimens that were collected.
The method of obtaining the spore cultures was essentially the
same as the method employed by Barnett (3)*

A small portion of the

pileus was fastened, gills down, to the inner surface of the lid of
a Petri dish by means of glue.

The lid was placed over the lower part

of the dish that contained hardened potato malt agar and was rotated.
There was thus an even distribution of the spores that were discharged
from the basidia.

It could be determined by examination of the surface

of the medium through the agar from the bottom of the dish by means
of a binocular microscope whether or not enough spores had fallen.

If

there were sufficient spores on the agar, the Petri dish was set aside
in a moist chamber until, by means of the microscope, one could see
that the spores had begun to germinate.
The removal of the germinating spores to tubes of the medium was
accomplished in the following manner.

The lid of the Petri dish was

removed and the spores located under high power magnification of the
dissecting microscope.

While observing the spores through the micro­

scope, a spore and a small amount of surrounding agar were removed on
the sharpened tip of an ordinary sewing needle that was held in the end
of a short holder for transfer needles.

The tip of the needle was then

gently stroked along the surface of an agar slant.
spore isolations were made for each species.

At least 30 mono­

The needle was never

flamed between separate spore isolation but was merely cleaned with a
piece of lens paper.

Sometimes even this was omitted.

Por work of such a nature that one does not need to know what
spores came from the same basidium this method is quick, easy and
efficient.

The amount of contamina-tion is practically zero.

There

kG
is only on© disadvantage that the writer encountered, and it may not
have "been due to the method itself.

Sometimes only a very small number

of the young hyphae that were transferred will continue to grow in the
tubes of medium.

If one does not remove a great many more spores than

he intends to use, he may not have as many cultures as would he desirable.
A different accession number was given to each sporophore from
which monospore isolations were made.

The various single-spore cultures

from any fruiting-body were designated as spores 1, 2, 3t etc. by
planing that number under a horizontal line above which was the acces­
sion number*
The mycelium of each mono spore culture was examined to determine
whether oidia were produced, whether the hyphae bore clanp-connections
and whether there were any features of the hyphae that would distinguish
them from hyphae of other species.

These observations are included in

the results for each species.
The medium used almost exclusively for spore germination was the
standard potato malt agar.

This was suitable for many of the species

whose spores did germinate, but the spores of some species would not
germinate on this medium.
were triedi

In those cases the following modifications

dung-extract agar, acidified (pH 4) potato malt agar, potato

malt agar to which a few drops of the ^uice of a lemon or a third of a
drop of 3-indol©butyric acid had been added, and the placing of a small
amount of the mycelium (tissue culture) of the fungus concerned on the
plates near the spores.
The mono spore cultures were maintained on potato malt agar, and
all of the pairings of these cultures were made on that medium.

Before

pairing of the cultures they were retransferred to different Petri

hi

dishes and allowed to grow for more than a week in order to ensure that
they would he growing vigorously,

The pairings were made in test-tubes

using transfers from the edges of the colonies on the Petri dishes*
The mated mycelia were allowed to grow for from two to four weeks
before they were examined for the presence of clamp-connections.

In

making the examination a small amount of mycelium was removed from the
edge of the growth in the tube at a position that would correspond to a
line drawn midway between the two paired cultures.

The mycelium was

studied under the high power or oil immersion lens of the microscope.
In most species the hyphae were so wide that the use of the high power
lens was sufficient*
In the case of one fungus (Ooprinus plicatilis) the mycelia that
appeared to have clamp-connections were studied further by growing the
hyphae on a nutrient medium in a manner suggested by Newcomer and
KenKnight^*.

The method as used by the writer was as follows:

a nutrient

medium was made up using the formula outlined below:
glucose----------------1*0 gm*
a g a r ----------— 1*2 gm*
gelatin----------2*0 gm*
distilled w a t e r
100 ml*
The ingredients were dissolved inthe water, and

the medium was then

sterilized at 15 pounds pressure for 30 minutes*
Into the lower half of a number of Petri dishes was placed one
watch glass and, resting on top of the glass, one microscope slide*

The

Petri dishes were then sterilized*
About two ml. of sterile distilled water were aseptically placed
in each watch glass under the microscope slide to furnish a humid

1

“

Their paper is in press.

Us
atmo sphere.

A small drop of the liquefied medium was smeared aseptically

on the slide by means of a bent glass rod that had been dipped in alcohol
and then flamed.

When this had solidified a bit of the mycelium of a

culture was planted on this medium.

The dishes were kept in a moist

chamber for two to three days before examination.

In order to study

the cultures a drop of water was placed on the mycelium and the cover
slip placed on top of the water.
In recording the results of the search for clamp-connections in
any of the fungi a - sign means that none were found in that particular
mating.

A / sign means that clamp-connections were found.

In most of the / reactions the clamp-connections were so large
and so numerous that a brief survey of the preparation on the slide was
sufficient.

When the clamps were small or very infrequent it was neces­

sary to spend several minutes in study of the hyphae.

After all of

the matings for a species had been studied and the reactions recorded,
a table was prepared to show how many sexual phases there were and to
which one each of the mono spore cultures belonged.

If the reaction of

any particular pairing did not agree with the theoretical reaction,
the pairing was examined again very carefully.

In the case of Psilocybe

foenisecii and P. subviscida the writer failed to reexamine doubtful
results.
The term "conidium" has been used when definite conidiophores
could be found.

The term ^oidium*1 was used when the asexual spores

were small, rod-shaped, hyaline and with dense protoplasm, although in
no case did the author see just how they were formed.

U9
Results
Spore germination
The spores of the following species germinated in 12 to 1+8 hours
on standard potato malt agar:

Coprinus fimetarius macrorhizus, CL

micaceus, CL plicatilis, C^. quadrif idus (one collection), CL radians,
0,» semil ant anus, Haucoria semiorbiculari s, Psathyrella disseminata,
Psilocybe foenisecii, and P. subviscida (one collection).

The germina­

ted spores of CL quadrif idus did not survive the transfer to tubes of
the medium.

Spores of Anellaria separata germinated on dung-extract

agar.
Spores of the following species did not germinate on any of the
media that were used:

Ooprinus quadrif idus (several collections), CL

radiatus, Gal era crispa, G. tenera, Lepiota Friesii Lasch., Panaeolus
campanulatus, Psilocybe foeni secii (several collections) and Stropharia
semiglohata (several collections).

Polarity
Anellaria separata
In June, 193^»

mono spore cultures of this species (culture

number 12h) were secured.

The mycelium in all of the cultures was

appressed, and in some this was so extreme that the hyphae appeared to
be water-soaked.

Most of the mycelia were white, but a few were brownish.

Growth was rather vigorous.

The larger hyphae were h - 5

in diameter.

On some of the hyphae there were large, chlamydospore-like cells (plate I,
fig. 3).

Ho oidia or conidia were found.

The results of the pairing are shown in Table 1.
were large and numerous.

Clanp-connections

They were never seen on the enlarged cells or

on the thick hyphae that formed the chlamydo spore-like cells.

Ho

fruiting bodies developed, and oidia did not form on the dicaryon

50
cultures*

Attempts were made to demonstrate "barrage "by growing a few

of the proper mycelia in Petri dishes, but no aversion was observed*

Table 1 * Results of pairing of eighteen monosporous cultures of
Anellaria separata (culture 12*4*)* Paired March, 1939

Ah

AB
/

aB

/ Z 2 9 6 7 \ 6 /9 Z 7 Z Q

a Jb

I t 13 J 7 2 S 3Q

S /Q 14- 2^

44 4 4
4 44 4
4
A ~ 4 —h
4 44 4
4 44 4 4
44 444
44 —4 4
h
*
4 44 4
4 4 44 4

Ab

!-*

“

f
_4 4 4
44 _
4
-4 4 4 4 4
4 4 44 4
44 4 4 4 4
44 4 4 44
■+■

_

44
44 4
44 4
44 4

Coprinus fimetarius macrorhizus
Monospore cultures of this species (culture number 150) were
obtained in August, 1938*

AH. of the cultures grew very vigorously

with the production of a great amount of white, aerial mycelium*

In

all of the mycelia many, very small (50 - 100 ja. in width) sclerotia
were found.

The hyphae were ^ - 5ji in width.

Abundant oidia were

produced (plate I, fig. *0.
The results of the pairing appear in Table 2*

51

On about one-half of the dicaryon mycelia typical fruitingbodies developed*

Rudimentary sporophores formed on a number of

other dicaryon and on many monocaryon pairings*

Table 2 * Results of pairing of twenty-nine monosporous cultures of
Ooprinus fimetarius macrorhizus (culture 150). Paired in November, I93 S,

<Z

Z

3

S ’6 7 8 9 to f2 13 /4 /6 /& 192! 2Z23ZA-ZS'Z? zQ

/

4 44 4 4
4 4 4 44
4 4 4 44
44
4 4
4 4 4 44
4 44 4 4
+ 4 4 4 4
4 44 4 4
44 4
~ i~ 4 4
4 4 4 44 4
4 4 4 444
4 4 4 4 4 4
4 4 44 4 4
4 4 4 4 4 4
4 44 4 4 4
4 4 4 4 4 _f_
44 4 44
44 4 4 4 4
+ 4 4 4 44
4 44 4 4 4
4 4 4 4 44
4 4 4 44 4
4 4 4 44 4
4
4
4
4
4
4
4
4

Z

3
5

6
7

8
9
JO

JZ
A l

/3
/4
/6
18
19

2J
22

23

2.1
28
yj29
4 4- 4// - h 4
/5
4
11 4- 4
+ 4
4 4

4
4
4
4
4
4

4
4
4
4
4
4

4
4
4
4
4
4

4
4
4
4
4
4

4
4
4
4
4
4

44
1- 4
4 4
4 4
4 4
4 4

—

4
4
+
4
4
4

4
4
4
4
4
4

44 4 4
4 4 44
44 4 4
4 4 4 4
4 4 4 4
+ 4 44

4 4
4 4
4 4
4 4
44
4 4

4
4
4
4
4
4

4 4 4 4
4 4 4 4
44 4 4
4 44 4
4 4 44
4 4 4 4

4
4
4
4
4
4>

52
Coprinus mieaceus
Monospor© cultures of this species were secured from several
sources*

Cultures designated as l 6a were obtained from a fruiting-

body that developed near the laboratory in September, 193&*

Cultures

numbered 127 were obtained from a sporophore that developed about ^00
yards from where the sporophore for culture l 6a appeared*
of this sporophore were secured July, 1938*

Cultures

Culture S2 was a haploid

strain that was secured from Centraalbureau voor Schimmelcultures in
October, 1938*

Culture S3 was the opposite sexual strain and was

secured also from the Bureau*

Both cultures had been sent to the

Centraalbureau in 1927 from Belgium by Vandendries*
The mycelia of culture l 6a when first isolated grew vigorously
and produced a dense, \yhite mass of hyphae.

Oidia similar to those of

0 . fimetarius macrorhizus were produced*
Some of the cultures were paired soon after they were obtained*
Clamp-connections formed on some of the pairings, but no record was kept
of this experiment*

On one of the dicaryon mycelia typical sporophores

were formed (plate I, fig* 2).
When the cultures were used for pairing reactions in April, 1939.
they had changed in appearance and in action*

Most of the cultures

were producing a pink color along the edge of the slant and to some
extent in the n^celium that did not touch the sides of the container.
At this time no oidia were seen.

However, there were present in great

numbers some elliptical, somewhat pointed, hyaline spores (plate I,
fig* 7)*

These conidia developed on many of the hyphae at the tips of

usually club-shaped, sometimes constricted and often branching conidiophores (plate I, figs. 5 and 6 ).

These conidia were present on all of

53
the monocaryon mycelia*

Each culture also caused the formation of

some substance that had an odor very similar to that of the flesh of
ripe peaches.
The presence of the color, the presence of a new and quite dif­
ferent type of asexual spore, the production of a distinctive odor and
the pairing reactions would indicate that the cultures had become con­
taminated*

However, the fact that the color did not develop in all

of the culture^ that some of the cultures were powdery on the surface
and that some were not and the fact that contamination of all of the
nine cultures by the same fungus does not seem likely might indicate
that the differences

in the cultures may not

tion but to a change

in the fungus itself.

have been due to contamina­

Dilution plates were poured of all of the cultures in an attempt
to obtain cultures that had been derived from single spores*

Most of

these cultures that came (theoretically) from single spores also deve­
loped a pink color and all produced the spores and the llf^aity,, odor*
The nine mono spore cultures of l6a were paired in April, 1939* i&
all possible combinations.

In no pairing did clamp-connections develop*

Monosporous cultures of culture 127 grew vigorously with the pro­
duction of a dense mass of white, aerial hyphae.
very slender, brown,

In many of the cultures

rhizomorph-like strands of hyphaewere developed

where the mycelium came into contact with the sides of

the test-tube.

The rather wide hyphae developed oidia in abundance*
The results of the pairing of the mycelia are shown in Table 3*
In many cases the dicaryon hyphae were wider than the monocaryon, and
the clamps could be easily seen*

In others it was difficult to find

the clamp-connections because they were infrequent and were small*
fruiting-bodies formed*

Ho

5^

Table 3 . Results of pairing of monosporous cultures of C. micaceus
vculture 127). Paired April, 1939.
~ -------Ah

c,B
4 4_ 44
4
—
4
4 4 4
— 4 4 4
4
4
—
4 4
4
4
4

4

4
4~

4~

4

4 4
4 4- + -4+ — 4—
4~
-h
4
4 4~
4- 4 + 4
"

-

4
4

-

~

-

~

44~

4

-

4

4-

Culture 82 looked very much like cultures of 127 la growth and
color.

Culture 83* however, did not grow as rapidly, and it showed a

profusion of wide hyphae that had yellowish contents#

There was some

development of oidia on "both cultures#
Cultures 82 and 83 were paired in April, 1939*

clamp-connec­

tions were found on the mycelium that developed#
Coprinus plicatilis
Monospore cultures of this species (culture 109) were obtained in
June, 193S#

The mycelia were sppressed and white, and they grew rapidly#

Chlamydo spore-like cells were found on some of the hyphae.

On the

55
mycelia were found some structures that resembled oidia (plate II, fig.
2) and others that resembled conidia (plate II, fig. 2).
Sixteen cultures that were devoid of clamp-connections were cros­
sed in all combinations.

At first examination it was thought that the

species was bipolar, but a careful study of the structures that looked
like clamp-connections revealed that in some of the apparently dicaryoh
hyphae the short branch that turned backward from the tip cell in the
formation of a clamp did not fuse with the main hypha.

In order to

study this process better the nycelia that appeared to have clamp-con­
nections were grown on agar that had been placed on sterile glass slides
(as described in the discussion of methods).

By this method it could

clearly be seen what mycelia had the typical clasp-connections (plate I,
fig. 9) and what ones had the incomplete or false clamps (plate I, fig.
10) characteristic in some species of Illegitimate crosses.
The results of the pairing of the mycelia are given in Table
Since the illegitimate combinations could be identified by the presence
of the false clamp-connections, it was possible to assign each culture
to a particular sexual phase in the manner that Qpintanilha (6 0 ) has
done in C* fimetarius.

Ho fruiting-bodies developed.

56

sixteen monosporous cultures of £. plicaPaired in March, 1939* — L indicates an illegitimate

tidis (culture 109).
cross*

AB ^

ajb

X >S 14-Z& 7 /s'<T/6 2 J Z 4 Z 9 r
B lll^20Z6
X
X X X + X X X X
X X X X X X X XX X
L X X X —h X —
Xx X _
X X_
_
LX X X X —h X X
XX X X X X X X X X
X X X X X x_ X X X X
X X X X X X
X X _L X X X
X X X J_ ~h X
X LX X X X
X L
-X X X —I”
L
X X X X- X _
X
X
X X X X
X X -h X X X
X X X -h X -1X X X -h X X
—

c?£>

Coprinus radians
Monosporous cultures of this species (culture 10) were secured in
December, 1937* from a fruiting-body that developed on a tissue culture of
the fungus*

The spores began to germinate in seven hours*

Most of the

cultures were appressed with only a little aerial mycelium of powdery
appearance.

The cultures were whitish or very pale brown*

The powdery

appearance was due to the presence of many conidia (plate II, fig. 3)*
Ho fruiting-bodies developed on the mycelia.
A few of the cultures possessed much aerial mycelium that was dark
brown in color and that grew more rapidly than did the cultures men­
tioned above.

On some of these mycelia fruiting-bodies developed.

The

57
resemblance of these cultures to the tissue cultures used in the first
part of the experiments was so great that it seems probable that these
cultures just described were dicaryon.

The presence of clauqps could

not be considered in settling the question because the tissue cultures
did not show any clamp-connections*
Monosporous cultures were paired in all possible combinations*
The examination of the pairings did not reveal any clamp-connections*
Spores from the aberrant fruit-body (culture number 69a) of this
species that developed on the potatoes and which has been discussed
previously were germinated*

The appearance of the nycelia was quite

similar to that of the monosporous mycelia of culture 10 *
The monosporous mycelia of culture 69 a were also paired in all com­
binations, but no clamp-connections were found.
Coprinus semilantanus
Monospore cultures of this species (culture 21 Rev.) were obtained
in March, 1939* from a fruiting-body that developed in pure culture*
Two of the cultures formed a deeply wrinkled or convoluted, yellowish
colony that grew slowly.

The colonies could easily be disorganized by

disturbance with the transfer needle; the colonies crumbled apart.

The

mycelium above the surface of the agar consisted of chlamydospore-like
cells; the cells are illustrated by fig. H on plate II.
were filled with dense protoplasm.

The hyphae

The hyphae in the substratum were

pale but contained very large granules.
Two other cultures began to develop in the same manner as just
described but soon formed one or more small patches of typical, white,
aerial mycelium*
The remaining ten cultures began as did the others but very quickly
formed a large mass of white, aerial mycelium over the small, rugose

58

colony*

Purther growth was limited to this fluffy mycelium*

Oidia similar to those of C. fimetarius macrorhizus were produced
on the aerial mycelium*
The results of the pairing are given in Table 5*
tions were in all cases quite large and distinct*

The clamp—connec­

They were never

found on the wide hyphae that made up the rugose colonies that were used
in some of the pairings*

No oidia were produced on the dicaryon mycelium*

A few small but fairly typical fruiting-bodies developed on some of the
dicaryon mycelia, but fruiting-body primordia were formed on both the
dicaryon and monocaryon mycelia that were examined in the pairings*

Table 5* Results of pairing of fourteen monosporous cultures of C*
semilantanus (culture 21 Rev*) Paired May, 1939*
~"
A B

ab

Ab

4 —A
4

4 4

A B

A
A
4

A
AA~

4- 4 4

A b

—4 4
4 —
aB

4
_
|w
. |-

A
<7-

A

—

---- 4 4 4 4 4
4 —h 4 4 4 4
—4
4 4 -4 4 j
—

4

A

A
A
A
A
A
A

59
Haucoria semi orbicularis
Monosporous cultures of this species were secured from two fruitingbodies.

One of these (culture 106) sporophores was found on a lawn in

June, 193S.

The other (culture 112) was obtained in June, 193#* I*1 a

meadow about one and one—half miles from the place of collection of
culture number 1 06 .
The mycelia were appressed but formed a thin felt*
little aerial mycelium.

There was very

All of the mycelia grew vigorously*

No oidia

were seen, but chlamydospore-like cells were present*
The re stilts of the pairings are given in Tables 6 and 7*
Table 6 * Results of pairing of seventeen monosporous mycelia of
semiorbicularis (culture 106). Paired in January, 1939*

A
4-/2L IS/6 Zo 23 24-26z £ r5~ // / 4 /7/6ZZ Z5z?'
f
-4 44 444
4 "

4 4 444 4 44
4 44 4 4 4 44
44 4 44 44 4
4 4 4 4 4 444
44 4 4 444 4
44 4 444 44
44 4 4 444
4 ±i±l±44 44
l

4 4 4 4 4 4 44 4
h4 4 4 4 444 4
44 44 4 4 4 4 4
44 4 4 + 44 44
f
-4 44 444 4
—
h4 4 4 4 44 4 4 4
”
44 4 4
44 44 4
44 4444
h4 4 "

60

{Cable 7* Results of pairing eight monosporous cultures of IT. semiorbicularis (culture 112)* Paired January, 1939*
”

/ 3 4- <5T 7

6 &
4
4
4
4
4
4

A
a

4
4"
4
4
4
4-

+ 4 4- -4 4 -f
-h 4 4- 4- -4 4

Pse/fchyrella disseminata
The mono spore cultures of this species (culture 13S) were obtained
in July, 193^» a "fc ‘k*ie base of a stump in a wooded area*

All of the

mycelia were white, and all of the mycelia grew very vigorously*

$hen

the cultures were grown on agar in Petri dishes there was considerable
variation in the appearance of the various mycelia*
the mycelia formed an aerial mass*

About one-half of

In some this consisted of loosely

interwoven hyphae, whereas in others the aerial portion was very dense
and compact.
The remainder of the mycelia were much more appressed with a
scant amount of aerial hyphae#

In some of these cultures the hyphae

were so appressed that the colony appeared water-soaked.

In several

cultures there were tufts of hyphae that protruded from the surface of
the colony at a U-5 degree angle*
appearance.

Several colonies were of a powdery

In one of the cultures with appressed hyphae the hyphae

grew to the left; in another culture the hyphae grew to the right*
The hyphae were either quite wide or quite narrow.

The wide hyphae

6l
were often compacted into small rhizomorph-like strands.
Seventeen monosporous mycelia were paired in all combinations in
December, 193&*
The pairings of three of the mycelia with the other mycelia were
carefully examined.

Failure to find clamp-connections in any of these

combinations seemed to indicate that no clamp-connections would be
found.

Therefore, the pairings were not studied further.

Ho fruiting-

bodies developed.
Psilocybe foenisecii
Monospore cultures of this species (culture 1 3 6 ) were obtained
in July, 1938*

Eh© cultures were mostly appressed with only a small

amount of white, aerial mycelium.

In each culture there were many small,

irregularly-shaped, cream-colored knots of hyphae.
grew slowly.

All of the cultures

Chlamydospore-like cells were rather abundant#

Ho oidia

were found.
The results of the pairing are given in Table S.

Failure to find

clamp-connections wherever spore number 5 was involved may have been
due to the fact that the clamps were on very narrow hyphae and were
infrequent.

Ho fruiting-bodies developed.

Table S>. Results of pairing of nine monosporous mycelia of Psilocybe
foenisecii (culture 1 3 6 ). Paired August, 193^.
A3

A 8

#3

Psilocybe subviscida
Monospore cultures of this species (culture 116 ) were obtained
in June, 193&*

The cultures were very similar in appearance and growth

to those of Naucoria semiorbicularis.

There were no asexual spores.

The results of the pairings are given in Table 9.

In almost all

of these pairings the hyphae had to be examined by means of oil immer­
sion lens because they were so narrow.

Another difficulty in this

species was the fact that the damp-connectlons were often infrequent
in various pairings.

In some of the pairings marked as having clamps

only two or three such structures were seen in the entire preparation.

Table 9* He suits of pairing of seventeen monosporous mycelia of P.
subviscida (culture 116). Paired in July, 1938).

63

Greographic races
Coprinus mieaceus
Five mycelia of culture l6a were paired in all combinations with
ten mycelia of culture 127 that represented all sexual phases.

No

clamp-connections were found in any of the pairings.
Cultures 82 and S3 (two compatible mycelia) were paired in all com­
binations with nine mycelia of culture l 6a.

No clamp-connections were

found.
Cultures 82 and S3 were paired in all combinations with ten mycelia
of culture 127*

No clamp-connections were found.
Naueoria semiorbicularis

Eight mycelia of culture 112 were paired in all combinations with
eight mycelia of culture 106.

The results are given in Table 10.

Table 10. Results of pairing of mycelia of N. semiorbicularis (culture
1 1 2 ) with mycelia of culture 106 of the same species.
/o 6

r4- S // /S

r/ 4 4 4 4 4 474Z O4Z 7
/

3 44
~ 4 4 4 4+ 4
4- 4 4 4 4 4 4 44
//Z J. 5 4 4 4 4 4 4 4 4
6 4 4 4 4 4 4 4f
—
J
4
4
4
—
4
7
4
8 4 4 4 44 4 + 4
JO
-+ 4 4 4 4 4 4
K. 4

Interspecific pairings
Coprinus fimetarius macrorhizus x C. semilantanus
Four mycelia of C* semilantanus (culture 21 Rev.), representing
all sexual phases, were paired in all combinations with eight mycelia

6k
°

.5.* fimetarius macrorhizus (culture 1 5 0 ) that represented all sexual

phases.

No clamp-connections were formed.

aversion between the mycelia.

There was no sign of

In most of the pairings £. semilantanus

was overgrown by the other fungus, but in a number of such cases it
still formed primordia of fruiting-bodies.

C. fimetarius macrorhizus x C* micaceus
Four nycelia of £. micaceus (culture l 6a) were paired with eight
mycelia of (3. fimetarius macrorhizus (culture 1^0) in all combinations.
No clamp-connections developed.

There was no sign of aversion.

Four mycelia of C3. micaceus (culture 127) were paired in all combi­
nations with eight mycelia of (3. fimetarius macrorhizus (culture 150)*
No clamp-connections were formed.

There was no sign of aversion.

C. micaceus x O.plicatilis
Four mycelia of £. micaceus (culture 127), representing all sexual
phases, were paired with eight mycelia of
No clamp-connections were found.

plicatilis (culture 109 ).

There was no sign of aversion.

Fanaeolus papilionaceus x P. retirugis
Cultures 85

^6 (two compatible mycelia of P. papilionaceus)

were paired with cultures 88 and 89 (two compatible mycelia of P.
retirugis).

No clamp-connections were found.

There was no sign of

aversion.
Intergeneric pairings
Anellaria separata x Naucoria semiorbicularis
Three mycelia of A. separata (culture 1214-) were paired with eight
mycelia of N. semiorbicularis in all combinations.

In all of the

65

pairings the cultures of Raucoria grew the more rapidly*
often completely covered the mycelium of Anellaria*

This fungus

In gj^ ^preparations

the number of clamp-connections varied from one to three*

Since they

were so infrequent the pairings were studied a second time.

That time

no claurp-connections were found in any of the pairings that at first
were recorded as positive*

One of the clamp-connections that was most

clear is illustrated by plate II, fig* 5*

results that are given

in Table 11 are the results obtained the first time that the pairings
were observed*

There was no sign of aversion.

Table 11* Results of pairing of three monosporous mycelia of Anellaria
separata with eight mycelia of Raucoria semiorbicularis*
//2

/c
r/
r/ 3 4- 5 ^ 66 7 8 ~7o>
— + -h
/z+\ //
—
—
—
+| —1 + |-|-1 -J -I
\ / 7 M +
r

—

—

—

—

-

—

-

—

Anellaria separata x Panaeolus retirugis
Cultures 88 and 89 (compatible mycelia of P* retirugis) were paired
with eight mycelia of A. separata in all combinations.

There was seen

only one structure that resembled a clamp—connection.

It is illustrated

by plate II, fig* 6 .

There was no sign of aversion.

Anellaria separata x Panaeolus papilionaceus
Cultures 85 and 86 (compatible mycelia of P. papilionaceus) were
paired in all combinations with eight mycelia of A* separata.
connections were found.

Ro claiip-

There was no sign of aversion.

Raucoria semiorbicularis x Panaeolus papilionaceus
Cultures 85 and 86 (compatible mycelia of P. papilionaceus) were
paired with six mycelia of R. semiorbicularis (culture 106).

Ro clamp-

66
connections were found.

There was no sign of aversion*

Naucoria semiorbicularis x Panaeolus retirugis
Cultures 88 and 89 (compatible mycelia of P. retirugis) were
paired in all combinations with six mycelia of IT. semiorbicularis
(culture 106).

Ho clamp-connections were found.

There was no sign

of aversion.

Coprinus plicatilis x Psathyrella disseminata
Pour mycelia of Psathyrella disseminata (culture 138) were paired
with eight mycelia of Coprinus plicatilis (culture 109)*
connections were found.

There was no sign of aversion*

Ho clamp-

67
Discussion and conclusions
The presence of oidia or conidia and the variation between mono­
sporous mycelia of some species is in agreement with what other
workers have found.
Coprinus radians was found to be either homothallic or heterothailic.

On the basis of clamp-connections alone it could not be

decided into which class it fell.

Vandendries (71)# working with a

fungus that he called (3. radians Desm., found that clamp-connections
were produced and that the species was bipolar.

According to

Vandendries (7^0* Brunswik never found clanrp-connections in this
species but proved it to be bipolar by observing what pairings pro­
duced fruiting-bodies.

This method might have yielded definite

results if it had been used for this species by the author.

Chow (21),

using six mycelia, also reported that the species is bipolar.
Psathyrella disseminata, also, since it did not produce clampconnections or fruiting-bodles, may be either homothallic or heterothallic.

Vandendries (80, 82), on the basis of the distribution of

clamp-connections, has stated that this species is bipolar.

He also

found that there were various irregularities in the pairing reactions.
Coprinus fimetarius macrorhizus ((3. macrorhizus that Buller (18)
speaks of) was found to be bipolar but with so many mycelia in one of
the groups that one might suspect that there really are four sexual
phases.

The absence of barrage, however, would indicate that the

species is not quadripolar.

Buller (18) reports that Uewton found this

species to be heterothallic.
Since Coprinus fimetarius (or C. lagopus, according to Buller (18)
and Quintanilha (5 9 )) is quadripolar, it is interesting that this
variety (or species) is bipolar.

There immediately arises the question

6s
whether the variety has only one allelomorphic pair of genes affecting
the polarity or whether there are two pairs#

If there are two pairs,

then the disjunction of these pairs must he of such a nature that the
spores are usually bipolar#
Naucoria semiorbicularis was found to be bipolar#

Sass (63 ) sug­

gested on the basis of cytological study that H# semiorbicularis form
bispora was probably homothallic#

He found that some of the spores

were apparently uninucleate and that others were binucleate#

Gene tic al

study of this variety would probably help explain the relation of the
distribution of nuclei to the spores and the polarity of the fungus#
Coprinus plicatilis was found to be quadripolar and to form false
clamp-connections in certain Hillegitimate” crosses,
Coprinus semilantanus was found to be quadripolar.
Anellaria separata was found to be quadripolar#
Psilocybe foenisecii, on the basis of study of nine mycelia, was
found to be irregularly quadripolar.
When Coprinus micaceus (culture l 6a) was first cultured it was
found that it was heterothallic, but the type of polarity was not deter­
mined#

After the change in the cultures appeared, it was impossible to

work out the polarity because no clamp-connections were produced.
Coprinus micaceus (culture 127) might be interpreted by some as
being bipolar with many irregularities#

Others might consider it to

be quadripolar with numerous irregularities#

Since the species has

been found by Vandendries ( 7 0 to tie quadripolar and to show numerous
abnormalities in some cases (Si), one is prejudiced in favor of the
thought that the specimen that was studied was quadripolar#
mycelia should have been used in the pairings#

More

69
Psilocybe subviscida has been shown in the results (Table 9) to
be bipolar but with many irregularities.

It is possible, however, to

arrange the results in such a manner that the species would be thought
to be quadripolar*

It can not be stated definitely which type of

polarity the species possesses*
No irregularities were observed in the pairing of mycelia of the
following species:

Anellaria, separata, Coprinus fimetarius macrorhizus,

Coprinus plicatilis and Naucoria semiorbicularis*
There were a few irregularities in Coprinus semil ant anus and
Psilocybe foenisecii and numerous abnormalities (or perhaps failure In
some cases to find the clamps?) In C_* micaceus (culture 127)
Psilocybe subviscida*
Throughout the paper the terms 11homothallie” and HheterothallicH
have been used in the sense that other workers appear to be using them,
although the author thinks that perhaps other terms would be better to
indicate that the phenomena appear to be due to the existence of one
or two pairs of self-sterility factors*

That is, the two terms as used

here do not mean the same as they do when used to explain conditions in
the Phycomycetes*
In no species was there any evidence of barrage such as found by
Vandendries and Brodie (S7) 3n some fungi.
Palse clamp-connections were found only in Coprinus plicatilis*
In regard to geographic races it was found in Naucoria semior­
bicularis that spores from two fruiting-bodies about one and one-half
miles apart were almost completely compatible In all combinations*
In Coprinus micaceus the cultures l 6a and 127* separated by about
300 yards, did not produce clamps in any of the pairings*

This was

70
on^ r fco

expected, however, since none of the mycelia of l6a would

form clamp-connections when paired among themselves in all combinations*
Mycelia of l 6a also failed to form clamp-connections when paired
with two njycelia (cultures 82 and S3) from Belgium.
Mycelia of culture 127 of

micaceus from East Lansing failed to

form clamp-connections when paired with the two mycelia (S2 and S3)
from Belgium.
These results are in agreement with those of other workers in that
the spores from sporophores that are not distant one from another are
usually nearly entirely compatible (as in Naucoria semiorbicularis),
whereas spores from fruiting-bodies that are far apart (Coprinus
micaceus) are usually incompatible in all combinations.

It should be

pointed out here, and there appears to be no reference to this in the
literature, that confusion may arise from the fact that workers do not
explain what they mean by such terms as HnearH and Mfar,f in relation
to these geographic races.
For the present, at least, the theory of multiple allelomorphs
of the self-sterility factors appears to explain the results of study
of geographic races.

The author is inclined to favor the suggestion

that Vandendries (Si) made in regard to the existence of dominant,
hereditary factors that determine primarily whether or not two mycelia
from different fruiting-bodies could form clamp-connections.
Of the five interspecific crosses that were attempted, clamp-con­
nections were not found in any of the pairings.

This is in general

agreement with the results of other workers who have found clamps to
be very few or lacking in such crosses.
In the six intergeneric crosses that were attempted, trhrcro were

71
|
Tl'li^Wp I

there were no clanp—connections found in the pairings of

Anellaria separat a and Panaeolus papilionaceus, Naucoria semi or­
bicular is and P. papilionaceus, II. semiorbicularis and P. retirugis
^°Prlnus plicatilis and Psathyrella disseminata.
In the pairings of Anellaria separata and Naucoria semiorbicularis
a very few clamp-connections were found in four of the twenty-four
matings.

In the pairings of A. separata and Panaeolus retirugis only

one structure that resembled a clamp-connection was found.
It should be emphasized that most of the previous work deeding
with interspecific crosses in the Agarics involved only a few spores.
The writer also did not use many spores, but the number was greater
than most workers have employed.

It seems to the author that many

interspecific and intergeneric crosses could be obtained if large num­
bers of mycelia were used.

In the higher plants the number of pollina­

tions that are made in order to secure crosses, even between species,
is large.

Grosses have been obtained between the following genera in

the higher plants: Zea x Tripsacum, Zea x Euchlaena, Triticum x Secale,
Triticum x Agropyron, Tri ticum x Aegilops; In the Rosaceae successful
crosses have been made between Sorbus x Aronia, Pyrus x Sorbus and
Oydonia x Pyrus, and in the Orchidaceae the crosses of Laelia x Cattleya are numerous.

It is quite possible that crosses between many

genera of the Agaricaceae could also be obtained, if one were to work
with numbers as great as were used in securing the crosses mentioned
above•

72
SUMMARY
1*

Seventeen species of the Agaricaceae were grown on eighty-

seven media, chiefly in test-tubes, in an affort to find a medium
that would allow the production of fruiting-bodies.
media nearly all of the seventeen species were used*

On most of the
On many of the

media a number of other unidentified species were also used.
2.

The following species did not form fruiting-bodies on any of

the media that were tried:

Coprinus atramentarius, £. comatus, £.

plicatilis, G-alera crispa, 0. tenera, Psathyrella disseminata and
Psilocybe subviscida*
3*

Coprinus radians, C. radiatus and £. semil ant anus formed

fruiting-bodies on a number of media*

C. fimetarius macrorhizus,

C* micaceus, Panaeolus campanulatus, P* retirugis and P. solidipes
formed sporophores on only a few media.

C. quadrifidus formed fruiting-

bodies only on a modification of Btter*s medium.

Stropharia semi-

globata formed fruiting-bodies only on dung-extract malt agar.
U.

The media, that were most valuable for sporophore production

were straw with water or malt extract, modification of Btter*s medium,
dung-extract agar, potato malt agar and potato glucose agar.
5.

Oidia, conidia or chlamydospore-like cells were found on the

monocaryon mycelia of most of the species, but none were found on
mycelia that were known to be dicaryon.

If the tissue cultures of

Coprinus radians are dicaryon, then that species produces conidia on
dicaryon nycelium.
6*

Coprinus fimentarius macro rhi zus and Naucoria semi orbicularis

were found to be bipolar.
7*

Ooprimis plicatilis, £. semilantanus, Anellaria separata and

73
Psilocybe foenisecii were found to be quadripolar*
S.

There were so many irregularities in the pairing reactions

of Coprinus micaceus (one fruiting-body) and of Psilocybe subviscida
that it can not be stated definitely whether they were bipolar or
quadripolar.
9*

Probably the former is quadripolar and the latter bipolar,

Since Coprinus radians and Psathyrella disseminata did not

produce clamp-connections in any of the pairings, it can not be decided
whether they are homothallic or heterothallic.

The same applies to a

culture of Coprinus micaceus which at first was heterothallic.

After

about two and one-half years in culture the fungus became so changed
(or contaminated?) that no clamp-connections would form when the mycelia
were paired.
10.

The existence of two geographic races was demonstrated in

Naucoria semiorbicularis.

The two fruiting-bodies were collected about

one and one—half mile s apart •
Ho conpatibility was found between mycelia of two fruiting-bodies
of Coprinus micaceus that were collected about 300 yards apart.

One

of these cultures was the one that had changed during its period of
culture on artificial media.

No compatibility was found between

mycelia of £• micaceus secured in East Lansing, Michigan, and mycelia
secured in Belgium.
11.

Barrage was not found in any of the species.

12.

Palse clamp-connections were found in certain combinations

of the mycelia of Coprinus plicatilis.
13.

Of the five interspecific crosses that were attempted, clamp-

connections were not found in any of the pairings*
14.

Of the six intergeneric crosses that were attempted, a few

7*

clamp-connections were found in certain of the pairings of Anellaria
separata and Naucoria semiorhiculari s, and a structure that might he
considered to he a clamp was found in one pairing of A. separata and
Panaeolus retirugis.

75
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76
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77
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23* ____

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2*4-*

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25 .

Utter, Bessie E.

wood-rot fungi.

Ibid. 50 * 219 ~ 2*4-6.

Hew media for developing sporophores of

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26.

Fries, Elias.

27*

__

28.

193&*

1929*

Systema Mycologicum 1. 1821.
* Flenchus Fun go run.
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1828.

Epicrisis Systematis geologic!.

I836 - 1638

29._________________ • Monographia Hymenomycetum Sueciae.
30._________________ . Hymenonycetes Europaei.
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32.

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187*4-.

187*4-*

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3U.

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35 #
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Les Hymenomycbtes.

1857*

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11
f|
Heldmaier, Clara. Uber die Beeinflussbarkeit der Sexualitat

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36 .

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Hirt, Ray.

The biology of Polyporus gilvus (Schw.) Fries.

Bulletin of the Hew York State College of Forestry at Syracuse University.
Volume 1 (la) February, 1928.

IS
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Humphrey, S. S*

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39*

Note on the production of fruiting—"bodies

Karsten, P. A.

Rysslands, Finalnds, och den Skandinaviska

Halfons Hattsvampar. Bidrag till Kannedom af Finlands N&tur och Folk 32!
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Kaufert, Frank.

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*11.

The production of asexual

spores by Pleurotus

Mycologia 27 ! 333 “ 3^1* 1935*

Kauffman, C, H.

The Agaricaceae of Michigan.

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*42.

Kniep, Hans.

Beitrage zur Kenntnis der Hymenomyceten. III.

ti
tTber die konjugierten Teilungen und die phylogenetische Bedeutung der
Schnallenbildungen.
*
m3•

Zeitschrift fur Botanik J: 36S - 398. 1915*

_____ __________ .

«
Beitrage zur Kenntnis der Hymenomyceten.

V. fiber die Entstehung der Paarkemigkeit der Zellen des Schnallenmycels.
Ibid. 9l 81 - 118. 1917*
*4*4.

■

Die l,multipolaretl Sexualitat der Hymeno­

myceten und deren D©utung durch M. Hartmann.
*45.

La Fuze, H. H.

Ibid. 22! 266 - 273*

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Coprinus.
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Lakon, Georg B.

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Die Bedingungen der Fruchtkorperbildung bei

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fungi on artificial media.

19^7*

Pure cultures of wood-rotting

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191S.
148.

Martens, P.

Alternance de phases et sexualite" dans un cycle

conidien chez Pholiota aurivella.

Comptes Rendus Hebdomadaires des

79
Seances de l*Acad^mie des Sciences (Paris) 195: 821 - 823.
*49*

Mounce, Irene.

1932.

Homothallism and the production of fruit-

bodies by monosporous mycelia in the genus Coprinus*

Transactions

of the British Mycologies! Society 7: 198 - 2l6. 1921.
50*

.

The production of fruit-bodies of Coprinus

comatus in laboratory cultures.
51 •

Ibid. 8 : 221 - 226. 1921.

_______________ _ snd R. Macrae.

Pomes pinicola.

Interfertility phenomena in

Canadian Journal of Research Series C 16: 35** ” 377•

1938.
52.

Naumov, N. A.

Methods of geological and Phytopathological

Investigations (Russian).
Leningrad.
53*

Imperial Printing Office.

Moscow and

1937*

Newton, Dorothy E.

of Coprinus Rostrupianus.
5*4. Noble, Mary.

The bisexuality of individual strains

Annals of Botany *40: 105 - 128.

1926.

The morphology and cytology of T^rphula

Trifolii Rostr. Ibid. (new series) 1: 67 - 98* 1937*
55*

Nobles, Mildred K.

Conidial formation, mutation and hybridi­

zation in Peniophora allescheri.

l^cologia. 28: 286 — 3^1 •

1935*

56 . Oort, A. J. P. Die Sexualitat von Coprinus fimetarius.
Recueil des Travaux Botaniques Neerlandais 27: 85 - 1*49*
57.
culture.
58.

Poole, R. P.

The fruiting of Collybia dryophila in pure

Mycologia 20: 31 “ 35*
Qpslet, M. L.

1930*

1928.

Les Champignons du Jura et des Vosges.

59 . Qpintanilha, A.

1872.

Le probleme de la sexuality chez les

Basidiomycetes. (Recherches sur le genre Coprinus). Boletim da Sociedade
Broteriana Ser. 2: 3 ~ 99*
go.

1933*
.

Cytologie et g^netique de la sexualite

so
chez les I^rmenomycetes.

61.

Ibid* 10: 289 - 332.

Rawlins, T. E.

methods.

Phytopathological and botanical research

John Wiley and Sons.

62.

Sass, John E.

Coprinus.

New York.

1928.

63 *__________________ •

cytological basis for homothallism

and heterothallism in the Agaricaceae.

61+.
mycete.

American Journal of Botany

1929.

________________ *

The cytology of a diploid sterile Hymeno-

Iftrcologia 2*+: 229 - 232.

65.

1933•

Aberrant heterothallism in a homothallic

Science 6S; 5^8.

16 : 663 - 702.

1935.

Smith, Alexander E.

1932.

Investigations oftwo-spored forms

tfy-cologia 26: 305 “ 331 *

the genus %-cena.

in

193^*

66 . ________________ . and Harold J. Brodie.

Cultural characters

and pairing reactions of monosporous nycelia and development of the
fruit-body of Pholiota (Flammula,) polychroa.

533 - 5W .
67.

1935.
Sobotka, Harry.

Wilkins Co. Baltimore.

68.
culture.

69.

Eye tali s parasitica and N. asterophora in

Vandendries, Rene.

193&*

Recherches sur le determinisms sexuel des

M^moire Couronne de I 1 Academie Royale de Belgique
1923 (cited in the next paper).

___________

Basidiomycetes.

Williams and

193&*

Mycologia 28: 222 - 227*

2me Serie 5: 1 - 98.

. Houvelles recherches sur la sexuality des

Bulletin de la Soci^td" Royale Botanique de Belgique.

2me Serie 5&J 73 “ 97*
■ji,

The Chemistry of the Sterids.

Thompson, G. E.

Basidiomycetes.

70.

Botanical Gazette $6:

1923*
.

Recherches exp Crimen tales sur la bipolarite

SI

sexuelle des Basidiomycetes.
72*

________

Ibid. (2 Ser. T 7) 57: 75 - 7S.

1921+.

. Contribution nouvelle a l'^tude de la

sexuality des Basidiomycetes.
73* _______

La Cellule 35: 129 - 155.

1925*

. L'hetero-homothallism dans le genre Coprinus*

Bulletin de la Societe Royal Botanique de Belgique (2 Ser. T 7) 57:
139 - lH6.

1925.

7^* ______________ . Les mutations sexuelles des Basidiomycetes.
Ibid. (2Ser. S - 9 ) 5S: 2S - 37 .
75* _______ —

•

1925.

Recherches experimentales prouvant la fixite

du sexe dans Coprinus

radians

Desra. Bulletin Trimestriel de

Mycologique de France

*4-1: 35^

- 37^-*1925*

la Societe

76* ______________ . La tetrapolarisexuelle des Coprins. Bul­
letin de la Societe Royale Botanique de Belgique (2 Ser. S - 9 ) 5^:

iso - is6.

1925.

77* ____________ Le comportement sexuel du Coprin mieace dans
ses rapports avec la dispersion de l'espece.
78.

Ibid. 60: 62 - 65.

1927*

. Les mutations sexuelles, 1 ,hetero-homothal-

lisme et la sterilite

entre races geographiques de Coprinus micaceus.

Memoir© de la Academie Royale de Belgique Cl. Sci.
79*

*

8°9s 3 “ 50. 1927*

Comment resou&re le probleme sexuel du

Coprin micace.

Bulletin de la Societe Royale Botanique de Belgique

6l: 123 - 135*

1928.

SO.

•La bipolarit^ sexuelle chez "Coprinus dis-

seminatus" Pers. Ibid. 62: 133 - 136 *
gl#

.

1929*

Les relations entre souches etrangeres

expliquees par les aptitudes sexuelles des individus parthenogeniques
chez Coprinus micaceus.

Bulletin Trimestriel de la Societe Mycologique

de France ^ 5 : 216 - 2^8.
82. _____

1929.
.

Conduite sexuelle de "Psa/bhyrella dis­

seminata" et essais de determination des valeurs relatives des
realisateurs selon Hartmann.

Bulletin de la, Academie Royale de

Belgique Cl. Sci. 16 : 1235 - 12^9.

1930 .

S3* __________________ • Les polarites sexuelles de Coprinus
tergiversans.

Bulletin Trimestriel de la Society Mycologique de

France *4-7• 36 - ^3*

1931*

8*4-.

.

Ibid. 49s 130 - 165*

sexuel des Hym^nomycetes.
85*

.

chez Pleurotus pinsitus.

86 .

Uouvelles investigations dans le domaine
1933*

La polarit^ sexuelle et le regime conidien

Ibid. 50: 203 ~ 212.

193^*

_________ ____ • Les modalites sexuelles des Basidiomycetes

Bulletin de la Soci^t^ Royale Botanique de Belgique
Ser. 70: 66 - 86*

1937*

87 .

• ©t H. J. Brodie.

Houvelles investigations

dans le domaine de la sexualite des Basidiomycetes.

165 - 210.

2me

La Cellule U2:

1933*

88 .

. et P. Martens.

et diploide, chez Phollota aurivella.

89.

11
Le cycle conidien, haploide

Ibid. 4l: 337 “ 3&6.

. et Gerard Robyn.

1932*

Houvelles recherches

experimentales sur le comport ement sexuel de Coprinus micaceus.
Memoire de la Academie Royale de Belgique Cl. Sci. U°9: 3 ~ 117*
90.

Zattler, Fritz.

II.
1929

Vererbungstudien an Hutpilzen (Basidio-

myzeten). Zeitschrift fur Botanik l 6 : U33 “ ^99*

192^.

S3

Plate I

Pig. 1 . Sporophores of Coprinus semilantanus on horse dung
Pig. 2. Sporophores of CL micaceus on potato malt agar; these
are the product of the pairing of two monosporous
mycelia of culture l6a soon after the mycelia were
secured.
Pig. 3* Chlaraydospore-like cells of Anellaria separata.
Pig. fy-. Oidia of Coprinus fimetarius macrorhizus.
Pig. 5* Conidia, and conidiophores of CL micaceus (culture l 6a).
These are typical of conidia, that developed on a film
of agar on a slide.
Pig. 6 . Conidia and conidiophores of CL micaceus (culture iGa)
that were mounted in water for examination.
Pig. 7* Conidia of (3. micaceus (culture l6&) in a water-mount.
Pig. S. Stages in germination of conidia of CL micaceus
(culture l 6a) growing on a film of agar on a slide.
Pig. 9. True clamp-connections of CL plicatilis.
Pig, 10.Pal.se clamp-connections of CL plicatilis.

PLATE I

Sk

Plate II

Fig.

* Coprinus plicatilis.

A portion of a mycelium

"bearing false clamp-connections*
Fig.

. Conidia and oidia-like cells of £. plicatilis*

Fig.

• Conidia and conidiophores of £. radians*

Fig.

* Chlamydospore-1 ike cells from a rugose colony
of £. semilantanus *

Fig.

• Clamp-connection found in one of the pairings
of Anellaria separata with Naucoria semiorhicularis.

Fig. 6 * Clamp-like structure found in one pairing of
Anellaria separata with Panaeolus retirugis.

PLATE II

2

m