Exercise 1. Introduction to Fungi
I.
Brief Survey of Fungi
This first exercise will serve as a brief introduction to the world of fungi, a large and
heterogeneous assemblage of microorganisms. There are about 70,000 named species of
fungi and this is believed to be about 5% of the total number of species that exist in nature.
We will not learn them all during this course.
Many of these organisms are detrimental, inciting a large number of plant diseases,
resulting in the loss of millions of dollars worth of economic crops each year, and an
increasing number of animal diseases, including a number of human maladies. On the other
hand, there is a long and rapidly growing list of useful fungi. They have been used in the
preparation of food and beverages for thousands of years. There are many fungi that are
themselves edible. Industry has used other fungi in the manufacture of many valuable organic
compounds, including organic acids, vitamins, antibiotics and hormones. They have been
used in the research laboratory to study metabolic pathways, mineral nutrition, genetics and a
variety of other problems. But perhaps their greatest contribution has been, and continues to
be, their role in recycling carbon and other essential elements in the ecosystem. Since all of
them are heterotrophic, they rely on organic material, either living or dead, as a source of
energy. Thus, many are excellent scavengers in nature, breaking down dead animal and
vegetable material into simpler compounds which become available to other members of the
ecosystem.
A number of fungal specimens and exhibits, some economically and ecologically
important, may be on display in the laboratory. Browse among these demonstrations and try
to get a perspective of the general nature of fungi, their diversity in growth form and
conspicuous underlying similarities among members within various taxonomic groups.
Undoubtedly, many of you already have observed some of these forms in their natural
habitats. You will be observing most of them in greater detail later in the semester.
Included in the display will be:a) fungal diseases of plants b) fungal diseases of animals,
including people c) fungi used in industry
d) fungi associated with wood decay e)
some mycological literature of historical interest
II.
Overview of Fungal Systematics and Taxonomy
• As with all taxonomy the names of various taxa each have a specific ending that refers to
their taxonomic level. The fungal kingdom has its own endings for many taxa. It would be of
considerable advantage for you to become familiar with these endings so that you know
immediately what taxonomic level is being referred to when you are confronted with a
particular name.
e.g. White button, commercial mushroom
Kingdom
Fungi
Phylum
-mycota
Basidiomycota
Subphylum
-mycotina
Basidiomycotina
Class
-mycetes
Hymenomycetes
Order
-ales
Agaricales
Family
-aceae
Agaricaceae
Genus
-Agaricus
Species
-Agaricus bisporus
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Naming the fungi
“What’s in a name? That which we call a Rose
by another name would smell as sweet.”
--William Shakespeare, Romeo and Juliet
Why do we use scientific names in naming fungi?
Another good reason for learning scientific names is that they show relationships between
species of a genus. If you learn one species in a genus, you are more likely to be able to
place other fungi into the same genus.
There is (theoretically) only one accepted scientific name for a particular species throughout
the world. This helps in the exchange of all sorts of information.
There are so many mushrooms and so relatively few people studying them that scientific
names are an essential requirement for learning about mushrooms. But you’re saying—
haven’t we managed to come up with common names for all the birds and all the mammals?
In North American there are fewer than 800 species of birds that have been seen and fewer
than 1500 species of mammals. In the whole world there are only about 9000 species of
birds and 4800 species of mammals. Worldwide, there are more than 70,000 species of
fungi that have been described to science. Several independent calculations have estimated
that this represents only about 5-10% of the total number of species that actually exist in
nature. This means that there are likely a total of 1-1.5 million fungal species. It’s no wonder
that they don’t all have common names! We don’t even know what most of them look like. If
you discover a new bird species, you are likely to make the front page of the New York Times.
If you discover a new fungal species, you won’t even make the newspaper in most cases. An
estimated 500 new species of fungi are described every year. The implication of this is that
many of the mushrooms you find will never show up in a field guide or even in the scientific
literature. Don’t get too frustrated with this. You may only be able to identify something to
genus or to a closely related species. You will find this to be more of a problem in “difficult”
genera, such as Cortinarius, Russula, and Amanita, for which species concepts are narrowly
and poorly defined, and for which there are many undescribed species.
Common names for the same species can be different in different geographical areas. (e.g.
hen of the woods vs. sheepshead, chicken of the woods vs. sulphur shelf, etc.). In recent
years many field guides have manufactured common names for many of the illustrated
mushrooms. This has led to a proliferation of common names for a single mushroom. Most
fungal species, despite attempts of some field guides to “make up” common names where no
previously existed, do not have common names. Common names can be used for more than
one taxon-- e.g. among plants, the common name “cedar” is used for at least five unrelated
genera of trees with aromatic wood.
Don’t be afraid of scientific names. You already know hundreds of scientific names for all
sorts of organisms. Every five-year-old kid knows the scientific name for Tyrannosaurus rex,
Brontosaurus, and all the other dinosaurs. Everyone knows the genus names for
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Hippopotamus and Rhinoceros. And what about Coleus? Or Rhododendron? Don’t be afraid
of Latin mushroom names. Moreover don’t be afraid to pronounce and use the names. We
don’t really know how the ancient Greeks and Romans pronounced these words (they left us
no tapes and hardly and CD’s…), so your way of pronunciation may be just as valid as
anyone else’s. There are definitely regional and international dialects for pronouncing the
names.
I have found that the easiest and best way to remember a scientific name is to learn what the
Greek and Latin roots mean. Most of the names can be directly translated to English and
convey some sort of distinguishing character of that fungus, often a color, shape, smell, taste,
relative size, microscopic characteristic, geographical location, or a name in honor of
someone.
The structure of scientific names is really quite easy to understand. A scientific name consists
of a binomial (two words), the genus name and the specific epithet, or specific term. The
scientific name is always italicized or underlined to denote that it is in a different language,
Latin. The genus name is always capitalized and the specific epithet is always lower case.
Note that referring to the second word in the scientific name as the “species” or the “species
name” is incorrect. The species name is the same as the scientific name and always consists
of two words. The genus name encompasses all the species within its defined limitations,
while the specific term signifies something more… well… specific. You can think of a scientific
name in terms of your own name. If you were to write your name as if it were a scientific
name, you would put your family name first followed by your given name. For example, my
name written as a scientific name would be Volk tom. Try it with your own name. Note that
this seeming “reversal” of first and last names is common is Asian cultures. For example,
Chen Lu, the Chinese figure skater, has the family name “Chen,” and “Lu” is what her mother
and her friends call her.
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Latin for Scientists
Why Latin? In the 1700’s and 1800’s Latin was the common language of scholars and
scientists. If you wanted your paper or scientific thoughts read by a wide audience and you
wanted it to appear to be scholarly, you wrote in Latin. This is much the same situation today
with English arguably being the common language of science, although you might get a
different point of view from the French and Russians.
It was during the late 1700’s into the 1800’s during the heyday of scientific Latin that the
binomial system of nomenclature was proposed and accepted. Carl Linnaeus (1707-1778)
was a Swedish Botanist/Mycologist who completed his medical degree in 1735, but was really
more interested in plants (and to some extent, fungi). Most medical doctors at that time also
knew their plants and fungi very well, because that’s where most of medicines came from
(and still do). In 1753, Linnaeus published his first book Systema Naturae (The Classification
of Nature), in which he attempted to classify all known animals and plants, the two kingdoms
recognized at the time. As his fame grew, other researchers began to send him specimens
from all over the world to fit into his classification schemes. As he became more acquainted
with these exotic organisms, he continued to modify his classification schemes. In 1753
Linnaeus published Species Plantarum (“Species of Plants”), in which he attempted to list and
describe (in Latin) all of the species that were known to exist at the time. Modern researchers
now agree that this work should be considered the starting point for nomenclature of plants
and fungi.
Linnaeus’ major practical contribution to science was arguably his binomial system of
nomenclature. Prior to his work, workers used a polynomial system of nomenclature,
sometimes a whole paragraph in Latin to describe an organism. These names were much to
long to be convenient. Moreover, someone could change the name just because he didn’t
like it. For example the briar rose was named by various scientists Rosa sylvestris inodora
seu canina and as Rosa sylvestris alba cum rubore, folio glabro. Linnaeus’s binomial system
grew out of a need for shorthand names for these polynomials. For example he called this
rose by the binomial Rosa canina, a much simpler name to use in conversation or writing.
Although Linnaeus did not invent the use of binomials, he was the first to use them
consistently and in such monumental works.
As with other scientific works of the time, Linnaeus published in Latin. It is interesting that
Carolus Linnaeus is the Latinized form of Carl von Linné, his real name. Latin is still used
today for scientific names and for original species descriptions for several reasons. First of
all, it is tradition; we already have a system in place, so why change it? Secondly, since Latin
is a dead language (no one speaks it any more despite assertions of a former US vicepresident to the contrary…), it is unchanging, and the words still have their same meanings
even hundreds of years later. Third, because of its tradition and ubiquity, people around the
world can still understand Latin, regardless of their native language. I can’t tell you the
number of times I have been able to read the Latin description of a fungus in a Japanese,
Chinese, French, or Russian journal without knowing much about those languages.
Since 1935, when botanists and mycologists were threatening to stop using Latin, the
International Code of Botanical Nomenclature (which also covers fungi) has required a Latin
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diagnosis for all new species descriptions. This ensures that people from all over the world
can get a basic understanding of the new species by reading the Latin description. For
example here’s the Latin description of Armillaria nabsnona Volk & Burdsall. Try reading it,
even if you don’t know any Latin.
Fungiformi, pileo 4-5 cm diam, aurantio-brunneo; lamellis adnatis vel decurrentibus; stipite 810 cm X 2-5 mm, brunneo; velo supero, affixo 2-3 cm ab apice; contextu 0.5-1 mm lato;
basidiis 25-35 x 5.5-6 μm, nodose-septatis; basidiosporis ovoideis vel subglobosis, (6-) 8-10 X
5.5-6.5 μm, inamyloideis; habitatio riparum, gregario ad lignum angiospermarum arborum.
If you’re already familiar with fungal terminology, you can probably get a pretty good
understanding of what the mushroom looks like, even though the Latin diagnosis does not
contain the huge amount of detail found in the rest of the original paper. You can find the
translation on the next page???
Mushroom shaped, pileus 4-5 cm diameter, orange-brown; gills adnate to decurrent; stipe 810 cm X 2-5 mm, brown; veil superior, affixed 2-3 cm from the apex; context 0.5-1 mm wide;
basidia 25-35 x 5.5-6 μm, nodose-septate (with clamps); basidiospores ovoid to subglobose,
(6-) 8-10 X 5.5-6.5 μm, inamyloid; habitat in riparian areas (streams), gregarious on the wood
of angiosperm trees.
A Short Latin Lesson
In Latin, each word has a particular ending based on its number, gender, case and
declension. Number means singular or plural. Gender in Latin has three choices, masculine,
feminine, or neuter. Unfortunately gender is not always based on reason or on physical
maleness or femaleness, and many inanimate objects or animals were give designations of
masculine or feminine by the Romans. There are a few rules you can learn, but these rules
are almost always violated. The case refers to the way the word is used in a sentence. There
are specific ending for the subject (nominative), possessive (genitive), direct object
(accusative), indirect object (dative) and object of a preposition (ablative). Of these, only the
nominative and genitive are used in scientific names. The genus name is always a singular
noun in the nominative case. The specific epithet is either an adjective that modifies it (and
thus matches it in gender, number, and case– but not necessarily declension) or another
noun in the genitive, not necessarily matching anything about the first word. Declension
refers to a group of words that share particular endings. In Latin there are five declensions,
cleverly numbered 1-5. Within each declension, the endings are the same in nouns and
adjectives, except for the third declension. The first, second and third declensions are the
most common, with the others being rather rare. This, along with the fact that you can put the
words into virtually any order you want in a sentence, explains why Latin is a dead language.
Latin is a dead language.
It’s plain for you to see.
It’s killed off all the Romans,
And now it’s killing me.
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Nouns
Case
Nominative
Genitive
Case
1st
F
2nd
M
2nd
N
3rd
M or F
Singular
Plural
Singular
Plural
Singular
Plural
Singular
Plural
Singular
Plural
-a
-ae
-ae
-arum
-us
-i
-i
-orum
-um
-I
-a
-orum
---is
-es
-um
---is
-a
-um
4th
M
Singular
Nominative -us
Genitive
-us
4th
N
5th
F
Plural
Singular
Plural
Singular
Plural
-us
-uum
-u
-us or –u
-ua
-uum
-es
-ei
-es
-erum
Adjectives—same as nouns except for 3rd declension
Case
1st
2nd
2nd
F
M
N
Singular
Nominative -a
Genitive
-ae
Case
3rd
N
Plural
Nominative -us
Genitive
-us
Plural
-ae
-us
-arum -i
4th
M
Singular
Singular
Singular
-i
-um
-orum -I
4th
N
3rd
M or F
Plural
Singular
-a
-is
-orum -is
3rd
N
Plural
Singular
Plural
-es
-ium
-e
-is
-ia
-ium
5th
F
Plural
Singular
Plural
-us
-uum
-u
-ua
-us or – -uum
u
Singular
Plural
-es
-ei
-es
-erum
Naming fungi in Latin and figuring out what Latin names mean
In a scientific name the first word is always a singular noun in the nominative case.
Conveniently, the ancient Romans have predetermined the gender and case. Unfortunately,
sometimes the gender makes no sense in terms of actual gender, especially with inanimate
objects. In English these are always neuter (it), but in Latin they can be masculine feminine
or neuter.
The second word (the specific epithet) may either be
 an adjective to modify it (and thus matches it in gender, number, and case– but not
necessarily declension)
 A noun that is unrelated in gender and case (rare)
 Or another noun in the genitive, indication possession or location, not necessarily
matching anything about the first word
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Let’s analyze some scientific names.
Agaricus bisporus Check the ending of the genus first. That’s what often determines the
ending of the epithet. Agaricus is a Latin word meaning “mushroom.” Remember that the
genus name is always nominative singular. In this case it ends in –us, which makes it 2nd
declension, masculine. The epithet bisporus means two-spored; thus it is an adjective
modifying the genus name and must match the genus name in number, case, and gender.
Let’s compare this to Agaricus campestris, the meadow mushroom. Agaricus is still a Latin
word meaning “mushroom.” However, you will notice that the campestris ending –is does not
match. “campester” in Latin means meadow and is a 3rd declension noun. Thus this epithet
has to be a noun. Checking the tables above, you see that –is is a genitive ending for nouns
in the 3rd declension genitive. Thus Agaricus campestris literally means “mushroom of the
meadow.”
There are some common endings in the 3rd declension that it will be helpful to know.
-loma means “fringe” and is always neuter 3rd declension
Hypholoma (hyphal fringe)
Tricholoma (hair fringe)
Hebeloma (blunt or dull fringe)
-cybe means “head” and is always feminine 3rd declension
Hygrocbye (moist head)
Inocybe (fiber head)
Gastrocybe (stomach head)
Agrocybe (field head)
Conocybe (cone head)
Dermocybe (skin head)
Clitocybe (close head)
Psilocybe (naked head)
-ceps means “head” and is always feminine 3rd declension
Claviceps (club head)
Cordyceps (heart head)
-myces means “fungus” and is always masculine 3rd declension
Hypomyces (below a fungus)
Tyromyces (cheese fungus)
Dacrymyces (teardrop fungus)
Zelleromyces (Zeller’s fungus)
-derma means skin and is always neuter 3rd declension
Scleroderma (hard skin)
Hyphoderma (hyphal skin)
Cystoderma (bladder skin- referring to the shape of the cells in the cuticle)
Ganoderma (lustrous skin)
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-opsis means “like” or “similar to” and is always feminine 3rd declension
Hygrophoropsis (like Hygrophorus)
Phyllotopsis (like gills)
Tricholomopsis (like Tricholoma)
Coprinopsis (like Coprinus)
Clavulinopsis (like Clavulina)
Ramariopsis (like Ramaria)
Fomitopis (like Fomes)
-ellus (-ella, ellum), ulus (-ula,-ulum), -ina, –idius are all diminutive—i.e. designating a smaller
size
Lentinus, Lentinellus, Lentinula
Marsmiellus (small Marasmius)
Xeromphalina (small, dry Omphalina)
Coprinellus (little Coprinus)
Galerina (little Galera – now a defunct genus)
Gomphidius (little Gomphus)
Cantharellus (little drinking cup)
Cantharellula (little Cantharellus)
Hydnellum (little Hydnum)
Crucibulum (little crucible)
Scutellinia (little shield)
-phyllum means gills and is always 2nd declension neuter
Schizophyllum (split gills)
Lyophyllum (loose gills)
Chlorophyllum (green gills)
Aphyllophorales (without bearing gills order)
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Authorities
The name or names you see after the Latin name of the fungus are known as the authorities,
the person who named or renamed the fungus. For example:
Agaricus magnivelaris Peck; Peck described this fungus
Tricholoma magnivelare (Peck) Redhead ---- In this case Peck is the person who described
the fungus first, as Agaricus magnivelaris Peck; Redhead is the name of the person who
moved the species to the genus Tricholoma. You cannot determine the original name from
the name of this species, but the person who moved the species to Tricholoma was required
to publish the original name before moving it to a new genus. Note that the second part of the
name, the epithet, remains the same. Sometimes the ending may change, because the
epithet must usually agree with the genus name in gender. There are specific Latin rules for
endings, of course.
A special case occurs with Elias Fries. All names published prior to 1821 are considered to
have been sanctioned by Fries if they are mentioned in his 1821 publication Systema
Mycologicum I. All names in this publication are automatically conserved, that is they
supersede any previously published epithets. This is designated by placing Fries' name after
a colon that follows the name of the pre-1821 person who originally described it. For example:
Armillaria mellea (Vahl:Fries) Kummer --- Vahl originally described this species [in 1755 as
Agaricus melleus Vahl ]. Fries sanctioned the name in 1821 [ as Agaricus melleus Vahl:Fries
]. Kummer moved this species to the genus Armillaria [ in 1871 ]. The information in the
brackets cannot be discerned from the name plus authorities, but can be found by consulting
the publications by the authors named. There is some controversy about whether Fries
should be included at all, since he merely sanctioned the name. A similar situation occurs
with rusts, smuts, and Gasteromycetes with Persoon's sanctioning 1801 publication Synopsis
Methodica Fungorum.
The International Code of Botanical Nomenclature (ICBN) governs the scientific names of all
plants and fungi, including lichens. This code is a medium sized book, with both English and
French versions; it can also be found online at http://www.bgbm.fuberlin.de/iapt/nomenclature/code/SaintLouis/0000St.Luistitle.htm . The ICBN provides rules
for valid and unambiguous publication of names. Although it is under constant revision,
problems can and do arise. A major problem still exists with fungi that were described before
the ICBN became widely accepted. Much of the code deals with rules for retroactively
accepting older names.
There are very specific rules for naming a new species. The taxon must be a previously
undescribed and it must have a Latin description or diagnosis (since 1935). In addition, there
must be designated a type specimen with the location where it will be kept (since 1953), the
description must be validly published in an accepted journal or other scientific location, and
the name cannot have been previously used. Rules for renaming a genus or species are also
very specific. If the species is validly published, the original epithet must be used; the
basionym (original name) and place of publication must be listed; and the newly recombined
name cannot have been previously used. These are oversimplifications, but you get the
general idea that name changes are not done frivolously. Complications may arise when
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referring to older literature, before the code was accepted. Much of the ICBN of over 100
pages deals with very specific rules with examples to assist taxonomists from making
mistakes.
The International Code of Botanical Nomenclature defines the type species:
 Art. 7.2. A nomenclatural type (typus) is that element to which the name of a taxon is
permanently attached, whether as a correct name or as a synonym. The nomenclatural
type is not necessarily the most typical or representative element of a taxon.
 Art. 10.1. The type of a name of a genus or of any subdivision of a genus is the type of a
name of a species (except as provided by Art. 10.4). For purposes of designation or
citation of a type, the species name alone suffices, i.e., it is considered as the full
equivalent of its type.
In actuality, there have been relatively few name changes due to nomenclatural reasons.
Most of the name changes you have endured are due to increase in knowledge about certain
species, and clarifying concepts about them. For example, what you formerly knew as
Armillaria ponderosa (matsutake) now is properly called Tricholoma magnivelare. Armillaria is
now restricted to wood decay fungi, with adnate to decurrent white gills, and black
rhizomorphs. Tricholoma magnivelare has none of these characteristics, being mycorrhizal,
with adnexed gills and no rhizomorphs, and therefore cannot remain in Armillaria. The epithet
ponderosum, on the other hand, was changed for a taxonomic reason—the oldest legal name
is Agaricus magnivelaris Peck. Thus the epithet magnivelaris must be used for the American
matsutake mushroom, Tricholoma magnivelare.
Another good example is Merulius tremellosus Schrad:Fr. For many decades it was kept out
of the genus Phlebia simply because of the folded to almost poroid hymenophore
configuration. However, Nakasone and Burdsall (1984) decided it really did have all the
characteristics of Phlebia and should be placed in that genus as Phlebia tremellosa
(Schrad:Fr.) Nakasone & Burdsall. Thus the type species of Merulius was gone, and the
genus Merulius is no more. Phlebia now includes fungi with hymenophores that may be
smooth, warted, folded, poroid or even toothed. Both of these are good examples of the use
of systematics in fungi.
Rules of the botanical code
There are several basic tenets to the code. For a name to be accepted, it must have been
validly published, after 1753, and be the earliest published name. The name must have been
published using the binomial system.
Mycological nomenclature is based on “types.” The type specimen is that dried collection of
fungi that are meant to forever represent the name the author has proposed. Since 1953 the
author must designate an unambiguous type specimen that matches the author’s original
description. This specimen must be deposited in a recognized herbarium. The type specimen
idea has proved very valuable, since now mycologists can extract DNA even from very old
specimens for experimentation.
The published description of the fungus must also include a Latin diagnosis. I can’t tell you
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the number of times I have been “saved” by being able to read a Latin diagnosis from a paper
otherwise written in Chinese, Russian, or Czech.
In 1994 in Tokyo, the code was changed to make the starting point for fungal nomenclature
Linnaeus (1753) instead of Fries (1821). The code also gave special “sanctioning” status for
names in Fries’ 1821 Systema Mycologicum, in which Fries attempted to list and describe
every fungus that had been named up to that point. This change in starting date led to name
changes for many fungi, once people started to read the old literature from 1753-1821. Such
name changes became so dreaded and perceived as so ludicrous that the code was changed
again to allow for the conservation of any name for any reason. Thus we are not forced to
change the name of a familiar fungus because of this procedural problem, provided a little
paperwork is done.
However the vast majority of name changes that have take place in the past 25 years have
been due to scientific advancement of our knowledge about a particular species. Many
species have been split up into a number of new species. For example we now know that
once we considered to be a single worldwide species, Armillaria mellea, the honey
mushroom, should now rightfully be considered at least 40 species worldwide, 11 in North
America. Laetiporus sulphureus, the chicken of the woods or sulfur shelf, has recently been
found to be at least five species in North America. These vary in their edibility and
pathogenicity, so this is not a purely academic exercise as some have complained. New
species are often discovered in previously understudied habitats and geographical areas. A
quick perusal of the scientific literature shows that “hotspots” for fungal species exist in places
like Michigan and North Carolina, not so surprisingly in the same places that have been
“hotspots” for mycologists.
So how should you decide which name to use for a fungal species?
You should determine the reasons for a name change and decide for yourself whether the
name change is valid. Take other people’s opinion into consideration. In the end, it’s all just
a matter of opinion
There are some mycologists, professional and amateur, who jump on the bandwagon for any
new name that is published. If you intend to use a new name for a fungus, you should
certainly understand the reason for the change from an old familiar name. Sometimes the
distinction is based on something that seems rather silly on the surface of it, but turns out to
be just an easy indicator of very different morphology, physiology, ecological niche,
geography, or even palatability. Sometimes that slight difference in spore size and shape or
on a DNA gel is just the easiest way to distinguish between species that no one has yet
bothered to look at very closely.
Mr. Spock wisely said, “A difference that makes no difference is no difference.” To cite an
example, if the spore size difference is the only difference between two populations, that
difference should make no difference in our view of the two populations as belong to the
same species. However, if that spore size difference is just the easiest way to distinguish
between species that really are quite different in their other traits, the spore size difference
makes a difference. Do you feel any different about taxonomy now?
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Introduction to the major groups of fungi and fungus-like organisms
Although much of fungal taxonomy is in a state of flux, the major groupings remain fairly
constant. You should become familiar with the following groups by careful observation of the
representative forms on demonstration.
Kingdoms Protista or
cell walls lacking or made of cellulose.
Stramenopila
Myxomycota
Acellular Slime Molds - Assimilative phase is a plasmodium:
cell walls lacking.
Dictyosteliomycota
Cellular slime molds. assimilative phase is amoebae that
Acrasiomycota
aggregate into a pseudoplasmodium and a reproductive
structure.
Plasmodiophoromycota
Endoparasitic slime molds. all are internal plant parasites,
single celled
Oomycota
water molds. mostly aquatic, some plant parasites.
reproductive cells with two flagella
Kingdom FungiAssimilative phase typically filamentous or unicellular, cell walls
TRUE FUNGI
present.
Chytridiomycota
Chytrids. - Unicellular or filamentous with aseptate vegetative
hyphae; tend to be aquatic. reproductive cells with one
flagellum, vegetative hyphae lack septa
Glomeromycota
Arbuscular mycorrhizal fungi, AMF, endomycorrhizae.
Reproduction via external mitotic spores.
Zygomycota
bread molds, etc. Sexual reproduction by formation of
zygospores from fused gametangia; vegetative hyphae lack
septa.
Ascomycota
sac fungi: cup fungi, yeasts, powdery mildews, morels - Sexual
spores borne internally in sacs, asci; septate hyphae
Basidiomycota
club fungi: gilled mushrooms, pore fungi, jelly fungi, puffballs,
rusts and smuts - Sexual spores borne externally on clubshaped basidia; septate hyphae.
"deuteromycetes"
deuteromycetes—imperfect fungi. No known sexual stages;
septate hyphae.
By the end of this lab you should be able to identify which of these major groups each of the
specimens on display represents. You should be able to do this by paying particular attention to the
essential forms of these major taxonomic groups. Refer to the list of fungi in "A Classification of Major
Groups of Fungi" in the preface to your lab manual. This initial treatment of taxonomy is necessarily
superficial. By the end of the course you will be able to identify most fungi to Order and many to
Species. Such detail is impossible at this time since you are unfamiliar with most of the different life
cycles, sexual and asexual reproductive stages, nutritional modes, vegetative growth forms,
physiology, genetics, morphology, and ecology that are necessary for complete taxonomy. It is
important that you quickly gain at least superficial understanding of the taxonomy of fungi, since in the
long run, much of your further work will be based on this understanding, and in the short term, you will
be encountering random representatives of most of these groups in your field work early in the
semester. In order to ensure this understanding, your instructor will provide a short quiz on this
material early in the semester. As you study each group during the semester, fill in the chart on the
following pages.
Biology/Microbiology 412/512 TJV
13
Vegetative
forms
Dictyosteliomycota
Myxomycota
Plasmodiophoromycota
Oomycota
Glomeromycota
Chytridiomycota
Zygomycota
Ascomycota
Hemiascomycetes
Plectomycetes
Pyrenomycetes
Discomycetes
Loculoascomycetes
Basidiomycota
Hymenomycetes
Gasteromycetes
Urediniomycetes
Ustilaginomycetes
“deuteromycetes”
Septa
Wall
constituents
Asexual
reproduction
Biology/Microbiology 412/512 TJV
14
Sexual Reproduction
Structures for
plasmogamy
Site of
karyogamy
Structures for
sexual spores
Life
Cycles
Major
representatives
Biology/Microbiology 412/512 TJV
15
Exercise 2. Vegetative Growth Forms and Introduction
to Reproduction
Vegetative Growth Forms
Since the fungi and near-fungi as a group represent a heterogeneous array of
microorganisms, you might expect a certain amount of variation in their vegetative growth
and modes of reproduction. In this exercise you will examine some of the variation to be
found among those forms that are usually studied by mycologists. It is questionable
whether certain of these examples can be considered fungi, but they have usually been
studied in the domain of mycology and ecologically "behave" like fungi; therefore they will
be included here. The vegetative growth form in a great majority of the fungi consists of a
system of thread-like, walled, more or less cylindrical, hyphae (singular = hypha) making up
what is called a mycelium (plural--mycelia). However, hyphae are not found in many of the
most simple forms nor in the slime molds, which do not have walls in the vegetative stage.
I. Unwalled form
The vegetative phase of the true slime molds is characterized by an unwalled,
multinucleate plasmodium that is capable of ingesting small particles of food (bacteria,
yeasts, etc.) and absorbing water soluble nutrients.
•
Remove the cover of the culture of Physarum (Myxomycetes) and examine different
parts of the plasmodium with various microscope objectives without a cover slip. First
use the dissecting microscope, then the compound microscope. Don't rack the
objectives into the agar. If you do, clean them immediately with lens paper and glass
cleaner. Does the plasmodium retain a consistent shape? Is any movement
detectable? Specifically where? Is there any periodicity to the movement, and if so,
what is the period? Are there any portions that do not exhibit movement and, if so,
where are they located in the plasmodium? Place the culture in the refrigerator for 5-10
minutes and quickly check the movement again. If it has changed, state specifically in
what way(s). Sever one of the plasmodial strands with a razor blade or scalpel (do not
cut the agar surface) and determine whether or not they can rejoin. Try altering the
environment in other ways to see the effects on the plasmodium. Your plasmodium may
be kept in the vegetative stage for several days if it is kept in a moist, dark environment
and is fed. Add 2-3 dry oat flakes to your plate culture. After placing the food in the
culture, make a reciprocal transplant of a portion of your plasmodium with a partner to
see whether or not segments of different plasmodia can fuse with one another. To do
Biology/Microbiology 412/512 TJV
16
this, gently scrape some of the plasmodium from the agar surface with a spatula and
transfer it to the alternate dish. Incubate the dishes in the laboratory drawer and
observe them the following period. What was the behavior in your culture? Add another
two oat flakes and incubate the cultures in the light on one of the laboratory shelves.
Note their behavior for the next few days and retain them for a later date.
• Observe large plasmodia on demonstration.
II. Walled forms
•
•
A. Non-filamentous forms
Mount a small (the size of a pinhead) fragment of yeast cake Saccharomyces cerevisiae
(Ascomycota) in a drop of I2KI reagent, place a clean cover slip over it, and examine the
cells microscopically. Do not confuse the yeast cells with the numerous, relatively large,
starch grains that will be darkly stained. Do the yeast cells have a definite shape or is
there considerable variation? Can you detect any subcellular structure? Compare your
observations with the electron micrograph.
Study stained sections of a potato tuber parasitized by a unicellular parasite
Synchytrium (Chytridiomycota). The parasite will be found just beneath the outer
surface of the tuber. Remember, potatoes have many starch grains, which will be
stained pink in your slides -- don't confuse the starch grains with the parasite. First,
scan the entire section with the lower power objective until you definitely locate the outer
surface of the tuber, not just the surface of the section. Then you should have no
difficulty in locating the parasite. If you do, ask the instructor to help you.
B. Filamentous forms
1. Hyphae with Septa
• Examine the young hyphae of Morchella (Ascomycota) or some other filamentous
fungus growing on agar by removing the petri plate cover and placing a clean cover slip
over the margin (edge) of the colony. Place the open culture on the microscope stage
and observe at all magnifications. Does the hypha have a regular geometric form or is it
irregular? How would you describe the general hyphal structure? What is the general
form of a hyphal tip (i.e. tapered, hemispherical, flat, etc.)? Starting at the tip and
moving back, can you distinguish any internal structure? Since hyphal nuclei are rarely
distinguishable in unstained, living material, what do you suppose the subcellular
particles are? Does the hypha appear to be segmented into cells; i.e. are septa (cross
walls; singular--septum) present?
•
After looking at the living mycelium, examine electron micrographs (Fig. 1-6) of
filamentous hyphae of Polythrincium trifolii (deuteromycetes). This is an intercellular
parasite (i.e. the hyphae will be growing between the cells of a host), so don't confuse
the fungus with the host. What cytoplasmic organelles are apparent that you did not
observe in the living material? Knowing the magnification of these electron
micrographs, you should be able to calculate the actual size of some of these structures.
Fill out the information requested on the supplementary sheet, using metric units,
and turn it in to the instructor for checking. Do the electron micrographs confirm your
conclusion about the presence or absence of septa? If they are present, is each cell of
Biology/Microbiology 412/512 TJV
17
the hypha isolated from adjacent cells? (Note: Remember that the hypha is a
3-dimensional object whereas these sections are 2-dimensional; look at several
micrographs before making a conclusion)
•
•
•
•
•
Examine the electron micrograph showing a complex septum found in some of the more
advanced fungi (Basidiomycota). Would you expect cytoplasmic continuity between
adjacent cells (the diagrammatic drawing will give you the best clues)?
2. Hyphae with no Septa (i.e. with aseptate hyphae)
Examine young hyphae of Saprolegnia (Oomycota) growing on nutrient agar. Remove
the culture cover and place culture dish on the microscope stage. Is the general shape
of a hypha and its tip like that of Morchella? Can you detect any subcellular structure or
septa in the hyphae?
3. Specialized Hyphal Structures
a. Rhizoids
Cultures of Rhizopus (Zygomycota), will be available for examination of the mycelium
(ignore the black reproductive structures at this time). Invert the entire plate (do not
remove the cover) and observe, with the lower and medium power objectives, the
mycelium that has grown onto the plastic surface. Do all of the hyphae resemble those
observed in Saprolegnia, or does there appear to be some hyphal specialization? If you
do detect specialization, what form does it have and what function do you think it
serves? Only a few fungi exhibit vegetative specializations such as this.
b. Rhizomorphs (demonstration)
The formation of hyphal cables, called rhizomorphs, is common among many of the
fleshy fungi such as Lycoperdon and Armillaria mellea (Basidiomycota). The cables are
usually large enough to be seen readily without magnification and may superficially
resemble small roots (the term means "root form"). These structures commonly occur
beneath the bark of decaying logs or under the litter on the forest floor. A large
rhizomorph contains hundreds of hyphae that are tightly packed together. The hyphae
on the surface of the rhizomorph may deposit heavy, thick, dark colored walls. Observe
habit specimens and any other demonstration material relating to these structures.
c. Sclerotia (demonstration)
Like certain slime molds, such as Physarum, that forms a resting stage called a
sclerotium (sing.) during dry or other adverse circumstances, some of the more
advanced fungi with cell walls may develop dense hyphal "knots" on the mycelium that
are also called sclerotia (pl.). They are characteristic of certain genera and species
such as Sclerotium (deuteromycetes) and Morchella (Ascomycota) and often attain
considerable size. The outer hyphae usually have thick resistant walls that are
commonly brown or black. Observe habit material and any other demonstrations that
are available.
Biology/Microbiology 412/512 TJV
18
Introduction to Asexual and Sexual Reproduction
Since fungi can be found in virtually every conceivable habitat where there is adequate
available organic material for survival, you might expect that a variety of reproductive
mechanisms have evolved to perpetuate the species. Although many of the fungi possess
the ability to reproduce both asexually and sexually, there is one large group
(deuteromycetes) for which only asexual mechanisms are known. In general, asexual
reproduction is more effective, producing greater numbers of dispersal units than sexual
reproduction. However, since it is an asexual process, the genetic variability among the
progeny is low. The asexual (imperfect) stage of a fungus is termed the anamorph, while
the sexual (perfect) stage is referred to as the teleomorph.
I. Asexual Reproduction
The most common method of asexual reproduction in fungi is by means of spores, a
general term for dissemination and resting structures in fungi. Fungal spores exhibit a
wide variability in coloration, surface sculpturing, size, shape, number of cells, cellular
arrangement, and the manner in which they are borne on the mycelium. All, or most, of
these features are used for identification of genera and species. Thus, for any given
taxon (genus, species), there are characteristic features of its spores that often
distinguish it from other taxa. Some fungi produce more than one type of spore.
Asexual spores may be delimited within a sac-like structure, a sporangium (plural -sporangia), in which case they are called sporangiospores, which may be motile or they
may be borne at the tips or sides of hyphae, and are called conidia (sing. -- conidium).
In some fungi, the hyphae can break up into their component cells, which are then
called arthroconidia. Each cell serves as a dispersal unit and, if it is transported to a
suitable environment, will germinate to form a mycelium. If the protoplasts become
enveloped in a thick wall they are often called chlamydospores.
The simple splitting of a cell into two daughter cells (fission) is another means of
increasing the population and is characteristic of bacteria and some yeasts. Other
yeasts employ budding, in which a small outgrowth (bud or holoblastic conidium) is
formed on the parental cell. The nucleus of the parent cell divides and one daughter
nucleus migrates into the bud. The bud increases in size and eventually breaks off.
Biology/Microbiology 412/512 TJV
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II. Sexual Reproduction
Sexual reproduction involves the union of two compatible nuclei (not necessarily in
gametes) with a subsequent meiotic division yielding recombinant progeny. Compatible
nuclei are brought together by plasmogamy (fusion of protoplasm from different cells),
which may be temporally separated from karyogamy (fusion of nuclei) by several
minutes to several years depending on the species. A wide variety of sexual processes
have evolved among the fungi. You should become familiar with some terminology
associated with sexual reproduction in the fungi. The sex organs in fungi are called
gametangia (sing. gametangium), and may be differentiated into distinguishable male
and female organs that bear either differentiated sex cells (gametes) or one or more
"gamete nuclei." Male sex organs are called antheridia (sing. antheridium) while female
sex organs are known as oogonia (sing. oogonium). If a single mycelium is capable of
reproducing sexually it is homothallic whereas if two mycelia are required to reproduce
sexually, they are heterothallic. Heterothallic forms may have both male and female
gametangia on the same mycelium, but they are incompatible with one another. Sexual
structures and reproductive methods are key characters in the segregation of larger taxa
from one another (e.g., classes, orders, etc.). Hence, when examining the examples
provided in the laboratory, correlate them with specific taxonomic groups in your
classification outline.
III. You will be seeing many types of asexual and sexual reproduction as we study the
different groups of fungi. You should compare each group and note differences.
Biology 412/512
Exercise 2
Name_______________________________
Supplement Sheet 1
Calculate the actual size of the following structures in the electron micrographs,
giving all dimensions in micrometers (m = microns). You should know the following
metric equivalents: 1 meter (m) = l00 centimeters (cm) = 1000 millimeters (mm) =
1,000,000 micrometers (m).
Electron micrographs of a white clover leaflet infected with Polythrincium trifolii, a
deuteromycete.
Figure 1:
diameter of largest hypha _______________m
dimensions of host cell ____________ x ___________ m
Figure 2:
dimensions of largest nucleus ____________ x ____________ m
Figure 3:
dimensions of mitochondrion ____________ x ____________ m
dimensions of host cell chloroplast ____________ x _____________ m
thickness of septum ____________m
Figure 4:
dimension of largest microbody ____________ m
dimensions of largest crystal in the microbody ________ x _______ m
Figure 5:
thickness of hyphal wall ____________m
Biology/Microbiology 412/512
21
Biology 412/512
Exercise 2
Supplement Sheet 2
Measurement of Fungal Structures
Your compound microscope has four objective lenses: low, medium, high dry and
oil immersion. If you’ve never used an oil immersion lens before consult your instructor
for help. Note and record on the data sheet and on a label attached to your microscope,
the magnification of each objective lens. The magnification of each objective lens is
increased 10X by the ocular lenses. Calculate and record the total magnification of
objects when using each objective lens. Consult the instruction manual and/or ask the
lab instructor if you have any questions about the use of your microscopes.
The right eyepiece of your compound microscope contains an ocular micrometer.
A micrometer is a small, glass disc with a series of equally spaced, parallel lines (your
micrometers are 100 spaces long, each 10th line being numbered). Put a slide on the
stage and observe it with the low, medium, and high dry objectives. Note that the
relative distance between the lines on the ocular micrometer changes as you go from
one objective to another; i.e. the ocular scale represents a different length with each
objective. Thus, the scale is arbitrary. If it is to be used to measure objects, it must be
calibrated for each objective. This is accomplished with a stage micrometer, several of
which are available in the laboratory. Place a stage micrometer under the microscope.
The design of stage micrometers varies, depending upon the manufacturer, but all of
them have an accurately etched scale of parallel lines (consequently they are
expensive, so please be careful when handling them). On all stage micrometers,
regardless of the manufacturer, the distance between the most closely spaced lines is
10 m (= 1/100 of a millimeter). The micrometer (m), also known as the micron (), is
the basic unit for microscope measurements of fungal structures.
To calibrate the ocular micrometer, start with the low power objective and move the
stage micrometer so that the ocular scale is just above the stage scale and the two "0"
lines are matched. You may need to rotate the eyepiece so the two scales are parallel.
Starting at the opposite end of the scales (i.e. furthest from the "0" lines), look along
the scales until you find a line on the ocular scale that is matched exactly with a line on
the stage micrometer. In the right margin of the data sheets, record the number of
divisions between this line and the "0" line of the ocular micrometer; also record the
number of micrometers(m) that distance represents on the stage micrometer. Then
calculate the number of m/unit (small division) on the ocular micrometer and record it
on the data sheet and on a label attached to your microscope. Repeat the process for
the medium, high-dry and oil-immersion objectives. (You should be able to focus well
enough without oil to do this exercise; if you do use oil, be sure to clean the lens and
the slide.)
As you observe the structures listed on the next page in Exercise 2, measure and
record their dimensions. Have the instructor check your numbers to make sure you
understand how to use your micrometer. Size (in m) will be a useful character to note
and record as you observe various fungi and their structures this semester.
As an exercise in mental gymnastics, calculate the number of yeast cells in a 30 x
30 x 18 mm yeast cake, using the dimensions of the yeast cell you measured and
assuming the cell is a rectangular solid. Also calculate the number of miles this number
of cells would extend if they were placed end to end (1 kilometer = 0.6 miles). Record
these calculations on the back of the data sheet for checking.
Biology/Microbiology 412/512
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Exercise 2
Data Sheet
Microscope No. ______________
Calibration:
m/unit
objective
Total
magnification
(1 unit on ocular
micrometer is:)
_______X
____________X _____________m
_______X
____________X ____________m
_______X
____________X ____________m
_______X
____________X
____________m
Object Measured
Dimensions
Ascospore of Microsphaera
11 x 20 m
Human hair diameter
Diameter of hypha (Morchella)
Yeast cell
Synchytrium cell
Rhizopus spore
Biology/Microbiology 412/512
23
Exercise 3. Collection, Isolation and Cultivation of
Fungi
Fungi, and especially their spores, are present in astronomical numbers in almost all
natural environments: lakes, rivers, streams and oceans; rock debris, plant and animal remains,
as well as parasitic on almost all known types of organisms. Fungi that parasitize other fungi even
exist and are relatively common. In fact, wherever organic material exists fungi will be found
breaking it down into its elements and converting it to more fungal biomass. The biology of these
various kinds of fungi -- the eukaryotic, walled, heterotrophs -- forms the primary subject matter of
this course.
Objectives of the exercise:
(1) to analyze several environments for their fungal inhabitants, both macroscopic and
microscopic, and thereby to become acquainted with the major groups of fungi and related
organisms, where they can be found and in what relative proportions. A thorough, quantitative
analysis of all the fungi of even a single location would, of course, take much more time than we
have available and would be beyond the scope of this course, but you should be able to gain an
appreciation of the basic methodology and the conceptual problems involved.
(2) to become familiar with sterile techniques, the methods for obtaining pure cultures of
fungi and for keeping them that way. These techniques will be used at numerous points
throughout the semester and it is of the utmost importance that facility in them be acquired as
soon as possible. Read detailed instructions in the Appendix before the lab period.
(3) to stockpile in pure culture a representative variety of organisms for later identification
and study in subsequent laboratory exercises.
(4) to learn to recognize and distinguish between mycelial fungi, yeasts, actinomycetes
and bacteria as they appear in culture, macroscopically and microscopically.
Collecting fungi for the herbarium: Preparation of fungal specimens for long
term storage in the herbarium
Specimens from the field should be collected in brown-paper or wax-paper bags, each
collection in its own bag, to prevent mixing of the spores of different species. Plastic
bags are not an acceptable substitute, since they allow for accumulation of moisture
and subsequent rotting of the specimens, especially in hot, humid weather. It is better
to collect several specimens of the same fungus in various stages of development. And
it is almost always better to collect too many than too few – extras can always be
discarded later, but it’s not always easy to return to an area to collect additional
specimens.
Data about the collection should be written on a separate piece of paper to be inserted
in the bag or written on the bag itself. You should label the collection with your initials
and number your collections sequentially, either beginning with your first collection ever,
TJV-2, TJV 2, etc., or numbering beginning at "1" every year, e.g. TJV-2010-1, TJV2010-2, etc. Collection numbers, locations and tentative identification should be
entered in a collection book for easy reference.
Biology/Microbiology 412/512
24
Minimum data should include:
Your guess at the taxon represented, substrate (e.g. host), locality (as specific as
possible), city, state or province, date, and collector. Notes about the collection should
also be included, as well as things that were unusual about the collection.-- in short
include anything that might help in the identification and subsequent study of the fungus
or its culture.
It is especially important to try to determine the substrate as exactly as possible,
including species of tree where applicable, since many fungi have a limited host range
and knowing the species of substrate may aid greatly in the identification.
Identification of specimens.
Sometimes, especially with agarics, it is necessary to take extensive notes on the fresh
specimens with respect to color, smell, taste, etc. to aid in later identification.
Sometimes microscopic features must be examined using fresh specimens to note
location of oil droplets, pigments, etc. More often the [other] important characteristics
can be seen using rehydration of dried specimens, which is usually more convenient. If
cultures are to be made, they must obviously be made with fresh specimens. (see later
for various culture methods and media)
Drying specimens
Several commercial home dryers are available and are the most efficient option. Air
drying may be acceptable in areas and conditions of low humidity. A light bulb under a
drying rack in a box may be an acceptable inexpensive substitute.
Placing specimens in the herbarium
Dried specimens should be placed in an appropriate small box or packet, made out of
decay-resistant, acid-free paper (we use 100% cotton fiber paper). On the outside of
the box or packet there should be a label with all the collection information. If a label is
glued to the box it should be made out of the decay-resistant, acid-free paper and
appropriate glue should be used. Elmer'sTM glue, or its equivalent, works pretty well.
The original field label (either the separate sheet of paper or the cutout from the bag)
should be included in the package as well. Ideally the information should also be
entered into a computer database. There are databases available that will print labels
or packets directly from the database program (e.g. Paradox). Herbarium specimens
should be stored in a sealed, disinfected cabinet, if possible.
The boxes and packets can be stored in larger boxes in the herbarium, usually
arranged alphabetically by genus, although some herbaria store specimens according
to specific epithet, since the genus names can (and do! ) change.
Some herbaria use mothballs (naphthalene, para-dichlorobenzene, etc.) with the
specimens to prevent attack by insects, but there are probably human health problems
associated with long-term inhaling of these chemicals; at best, these chemicals are
merely unpleasant to work with. An alternative method is to freeze the specimens for
several days in a -40°C freezer at least once per year.. Specimens should be placed in
a plastic bag during freezing and left in the bag for a day or so after freezing to prevent
Biology/Microbiology 412/512
25
condensation of water as the specimens return to room temperature. This method
seems to work very well and eliminates the human exposure problems associated with
mothballs. The ideal herbarium should not have any specimens introduced into it that
have not undergone this freezing treatment. It is easier to prevent insect infestation
than to eliminate the problem once it occurs. Specimens that have been taken out to be
examined should be put through this procedure before returning them to herbarium
In tropical or other humid areas, the specimens will keep longer without contamination if
placed in ZiplocTM bags immediately upon removal from the drying apparatus.
Culturing fungi
I.
MEDIA
Five growth media will be available for your use. Ask instructors if you are uncertain about
which one(s) to use in a particular circumstance. The recipes below are “from scratch.”
Most of these media are also available as dehydrated powders.
•
Potato-Dextrose Agar (PDA)
(Satisfactory for growing many fungi, particularly phytopathogens & some bacteria)
glucose ............................................................................................................... 20 g
peeled potatoes, infusion from ......................................................................... 200 g
agar .................................................................................................................... 20 g
distilled water ................................................................................................. 1000 ml
pH 5.6
•
Czapek Solution Agar (CZ)
(A defined media useful for growing fungi, particularly soil fungi capable of utilizing
inorganic nitrogen. CZY has 0.5 gm of yeast extract added)
sucrose ............................................................................................................... 30 g
NaNO3 .................................................................................................................. 2 g
K2HPO4 ................................................................................................................. 1 g
MgSO4 ............................................................................................................... 0.5 g
KCl ..................................................................................................................... 0.5 g
FeSO4 .............................................................................................................. 0.01 g
agar .................................................................................................................... 15 g
distilled water ................................................................................................. 1000 ml
pH 7.3
•
Complete + Yeast (CYM)
(Useful for growing many saprophytic fungi, particularly wood degrading species)
Yeast extract......................................................................................................... 1 g
glucose ............................................................................................................... 10 g
peptone................................................................................................................. 2 g
KH2PO4 .............................................................................................................. 0.5 g
K2HPO4 ................................................................................................................. 1 g
MgSO4 ............................................................................................................... 0.5 g
Vitamin B1 ....................................................................................................... 120 ug
agar .................................................................................................................... 20 g
distilled water ................................................................................................. 1000 ml
pH 7.0
Biology/Microbiology 412/512
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•
Malt extract agar (MEA) 1.5%
(standard for wood decay fungi)
malt extract ......................................................................................................... 15 g
agar .................................................................................................................... 20 g
distilled water ................................................................................................. 1000 ml
•
Mycorrhizal Media (Palmer 1971)
KH2PO4 .............................................................................................................. 0.5 g
MgSO4.7h2O ...................................................................................................... 0.5 g
NH4Cl ................................................................................................................. 0.5 g
FeCl3 (1% soln.)............................................................................................10 drops
glucose .............................................................................................................. 5.0 g
malt extract ........................................................................................................ 5.0 g
agar ................................................................................................................. 15.0 g
distilled water ................................................................................................. 1000 ml
Additives to media: (when required, add to cooled media after autoclaving)
To ward against bacteria:
Streptomycin
0.1 g/l
To ward against ascomycetes and deuteromycetes:
Benomyl
0.4 ml stock soln./l
Benomyl stock solution:
add 0.5 g benomyl (trade name benlate) to 100 ml water, mix well, autoclave, cool to room
temperature, store in refrigerator, shake well before use. USE CAUTION — EXTREMELY
POISONOUS
II.
ISOLATION OF FUNGI FROM AIR, WATER OR SOIL
A.
Air
Although fungi do not truly inhabit the air, their spores are frequent passengers in air currents. A
qualitative sampling of fungal spores present in air can be done in the following simple way. The
results may also impress upon you the need for caution in exposing nutritive surfaces to air while
using sterile techniques.
1.
Expose plates of PDA agar to the air for periods of 30 sec, 1 min, 5 min, 10 min, and 30
min. Choose one or more indoor or outdoor locations to sample. Label plates with your name,
date, time, location and any other relevant information. Incubate plates in your drawer.
2.
Examine after several days.
Biology/Microbiology 412/512
27
B.
Water
Fungi that inhabit aquatic environments include some quite unusual types. They can be isolated
by means of a dilution technique in which a sample of pond or stream silt serves as the original
material, or the simple qualitative method outlined below can serve the purpose of demonstrating
their presence.
1.
Prepare about a dozen sterile split hemp seeds: Boil some hemp seeds in water for
several minutes, or use the pre-sterilized seeds provided. While holding a boiled seed against a
glass surface (e.g., the inside of a Petri dish) between the tips of a forceps, split the seed
transversely with a sterile scalpel or razor blade.
2.
Pour about 10 ml of a water sample in a sterile Petri plate and then place several half
seeds, cut end down, in the liquid as bait. Label plates appropriately and incubate at room
temperature for several days.
3.
Examine the hyphae emanating from the seeds. Compare these fungi with those that
appear on your agar plates.
C.
Soil
Three semi-quantitative methods are included below for your information. In most cases,
however, the following qualitative method is sufficient: Collect a small soil sample and label it with
your name, date, source location and any other relevant information. Add a small amount of your
soil sample to a sterile water "blank" and close lid securely. Agitate tube to disperse soil particles.
Use an inoculation loop to transfer a small amount of the water to appropriate media in a petri
plate and streak for isolated colonies. The instructor will demonstrate how to do this. Label plates
appropriately and incubate at room temperature.
Three semi-quantitative methods are included for your information. We won’t be doing any of
these in lab.
1. Soil Dilution and Plate Count Method
a. Weigh out 5 gm of a soil sample and suspend in 45 ml of sterile water in a 250 ml Erlenmeyer
flask on a mechanical shaker for 20-30 min. to disperse the soil particles well. This is a 1/10 or
10-1 dilution.
b. Withdraw with a sterile 10 ml pipette ten ml of the suspension and dilute 1/10 by transferring to
a 90 ml sterile water blank. For accuracy of dilution keep all suspensions as agitated as possible
immediately before withdrawing aliquots. The total dilution achieved so far is thus 1/100 or 10-2.
c.
Successive 1/10 dilutions can be made by withdrawing 1 ml aliquot from existing dilutions
and transferring them to 9 ml water blanks in test tubes. The test tubes can be shaken by
agitating them gently with a Vortex mechanical stirrer. Prepare in this way dilutions of 1/1000 (103
), 1/10,000 (10-4), 1/100,000 (10-5) and 1/1,000,000 (10-6).
d.
Distribute 1 ml aliquots of the 10-5 and 10-6 dilutions to the surface of solidified agar
nutrient media in Petri dishes. These dilutions should be sufficiently dilute so that the number of
spores and hyphal fragments per ml will not be excessive.
e.
Spread the 1 ml aliquot over the surface of the agar by means of a glass "hockey stick"
spreader that has been sterilized by alcohol-dipping and flaming. Incubate at 25°C.
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2.
Warcup Soil Plate Method
This method employs a slightly less violent method of dispersing the soil particles and hence
results in spore masses remaining more intact.
a.
Weigh out 0.01 gm of soil and transfer to a sterile, empty Petri dish. Crush the soil in the
plate gently with the tip of a flamed scalpel. Pour 10 ml of molten CZY medium that has been
kept in a 45°C water bath onto the plate and swirl gently with a rotary motion to disperse the soil
particles. Allow to solidify and incubate for several days at 25°C. Incubate the plates upside
down. Why?
b.
Examine the plates as in A5 above and compare the number and relative frequencies of
the colony types on a 1 gm of soil basis with the corresponding dilution and plate method.
3.
Waksman's Direct Inoculation Method
This method (a) uses a short enough incubation time so that spore germination cannot occur to
any great extent, and (b) allows for practically no dispersal of particles. It assays, in effect, for the
presence of actively growing hyphae in soil.
a.
Transfer with sterile forceps a block of soil (ca. 1 cm in diameter) to the center of a plate of
CZY agar. Incubate 24-48 hrs at 20°C.
b.
Examine the plates as in A5 and compare the results with both the Warcup method and
the dilution and plate count method.
D.
Regardless of the source of your inoculum, examine the plates after several days to one
week for (a) number of colonies, (b) relative frequencies of different types of colony: i.e., bacteria,
yeasts, actinomycetes and fungi, and (c) numbers of apparently different species of fungi (yeasts
and filamentous fungi). The four types of colony can be recognized with practice by examining
the underside of a Petri dish under the 10X objective of the compound microscope and carefully
focusing on the margins of individual colonies.
Filamentous fungi are easily recognized by the cottony appearance of the colonies and the
coarseness of the filaments. Actinomycetes usually have a chalky surface appearance, and
although filamentous like fungi, the filaments are exceedingly fine. Bacteria and yeasts present
similar surface appearances, but an examination of the margins, even at 100X magnification,
usually shows the considerable differences in the size of the cells: yeasts are several orders of
magnitude larger than bacterial cells. If in doubt, make a wet mount of the cells of the colony and
examine under the 43X objective.
Compare the relative frequencies of the different colony types from different sources and on the
different media used.
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III.
ISOLATION OF CULTURES DIRECTLY FROM FRUITING STRUCTURES
A.
Spore Drops
This is probably the easiest way to obtain cultures from most fruiting structures. The fruiting body
is suspended over agar, usually held to the top of the petri dish by Vaseline, stopcock grease or
tape. Care must be taken to orient the fruiting structure in its natural position, so that spores may
be forcibly ejected into the air rather than onto other surfaces of the fruiting body. Spores are
allowed to "rain" down on the agar surface for a few seconds, or sometimes overnight, depending
on the age and the spore productivity of the fungus. The spores are allowed to germinate, and
pure multispore cultures of the fungus can be obtained. If single spore isolates are desired for
mating studies or another reason, the spores must be spread out, allowed to germinate, and
picked singly under a dissecting microscope.
B.
Tissue Cultures
The advantage of this method is that the culture obtained is the same genetically as the mycelium
that formed the fruiting structure in the first place. This is particularly useful in mushroom
cultivation. To obtain this type of culture, a very small piece of the internal tissue of the fruiting
body is placed onto the appropriate medium. It helps to tear the fruiting body apart before
culturing it rather than cutting it all the way through. The latter methods tends to drag any surface
contaminants onto the internal tissues. It sometimes helps to have antibiotics in the medium,
streptomycin to ward against bacteria and benomyl to kill ascomycetes and deuteromycetes. Of
course, if these are what you wish to culture you should omit the antibiotics. Sometimes it helps
to keep out some competitors by pushing the piece of tissue down into the agar and covering it.
IV.
ISOLATING FUNGI IN PURE CULTURE
For most studies on the physiology, development and genetics of fungi it is imperative to have
pure cultures of the organisms. Pure culture isolation is simple in principle, occasionally
painstaking in practice. The basic goal is to isolate a small bit of inoculum of a fungus and to
grow it free of all other organisms. The smaller the bit of inoculum, the less likelihood that
contaminating organisms from the original isolate will still be present. Two types of fungal
structures are most often used for ensuring isolation in pure culture: (a) single spores, and (b)
hyphal tips. Individual fungi lend themselves better to one or another of these two methods. We
will attempt at this time two or the simplest types of purification.
A.
Streak Plating of Spores
1.
Choose a reasonably well isolated sporulating fungal colony from one of your plates. If it
is growing as a radial mycelium of uniform color and texture, chances are that it already consists
of only one fungal species. To confirm this, however, it will be necessary to streak some of its
spores onto a fresh plate of medium in such a way that new centers of growth are obtained, some
of which will have a high likelihood of having resulted from the growth of a single spore.
2.
Use an inoculating needle with a bent tip or a sharp tipped scalpel (#11). Sterilize the tip in
a flame and wet it with some sterile water. Touch the tip lightly to the surface of the colony to be
purified so that some spores adhere to it. Make a series of parallel streaks on a fresh plate of
PDA or other appropriate media, about 1" apart. Most of the spores will be deposited in the first
streak and progressively fewer in subsequent streaks. If streaking is done properly, the final
streak should deposit just one or two spores well separated from one another and the colonies
that result from them should be pure (i.e. derived from a single spore of one species).
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3.
Prepare two plates in this manner so that you can observe one plate microscopically while
keeping the other one sterile. Place one uncovered plate on the microscope stage and locate the
spores on the agar surface with the 10X objective. Measure and record the size, color, etc. of the
spores. Start at the initial inoculation point. If you have trouble focusing on the agar surface,
score the surface with your needle and focus on that first. Cover and incubate both plates in your
drawer for 24 hours and then observe the germinated spores on your non-sterile plate.
B.
Hyphal Tip Isolation
1.
Locate a hyphal tip emanating from a hemp seed or a non-sporulating agar colony. Under
the dissecting microscope use an sharp-pointed scalpel or chisel-pointed needle to slice the
terminal 2-3 mm of the hypha transversely. Attempt by means of the pressure of the slicing to
"cauterize" the wound and thereby to seal off the outward flow of protoplasm. If this is done the
hyphal tip should adhere to the needle.
2.
Withdraw the needle with adherent hyphal tip and deposit the tip on the surface of a fresh
plate of half strength cornmeal agar (½ CM). If possible, try to avoid breaking the surface of the
agar.
Half Strength Cornmeal Agar
(Corn meal agar is useful for the cultivation of phytopathological
and many other fungi. Dilute (½ CM) media promotes the growth of
widely separated hyphae.)
Cornmeal, infusion from .......................................................... 25 gm
Agar
............................................................. 15 gm
Water
........................................................... 1000 ml
3.
After several days mycelium should have developed, the surface of which may be
contaminated with bacteria. The mycelium that has penetrated the agar, however, should be free
of bacteria. This mycelium can be isolated free of bacteria in the following simple way: a large
square of agar (ca. 4 cm. square) is cut with a scalpel and inverted onto a sterile, empty Petri
dish. The underside of the agar can then be easily reached from the top and a small bit of agar
containing bacteria-free hyphae be excised and transferred to a fresh plate of nutrient medium (½
CM).
4.
If bacteria are still evident at the surface of the colony that results, repeat the inversionexcising process.
C.
Colonies that have been isolated in pure form and which have been checked
microscopically for the absence of bacteria and other kinds of fungi may then be routinely
transferred to fresh media in a culture tube by simply excising cubes of agar (containing
mycelium, of course) with a sterile arrowhead needle or scalpel. With some fungi a successful
transfer may be made from just the spores. Incubate the tube in your locker until you are confident
it is a pure culture. Label the tube with your name, show it to the instructor and display it with your
original plate culture. If the culture is not pure, repeat the process. Prepare a slide of the
sporulating apparatus in mounting fluid, draw and measure the size of the structures you see, and
have the instructor check it. Retain your slide. As you encounter representatives of the different
fungal groups this semester, keep the characteristics of your isolate in mind. Attempt to identify,
at least to genus level, before the end of the semester.
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FUNGI OF WISCONSIN
Trametes versicolor (Wulff:Fr.) Pilat
on Ulmus americana log
Hixon Forest, La Crosse County
October 17, 2005
coll: Tom Volk
Biology/Microbiology 412/512
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Exercise 4. Mating Type System Determination
The typical mating systems found in heterothallic fungi fall into one of three groups based on
the number of genes and alleles involved:
1.
One gene two alleles (unifactorial, bipolar heterothallism with two alleles): "A" and "a" as in
Neurospora crassa or "+" and "-" as in Rhizopus. In this system compatibility is based on
difference: A ´ a or + ´ - are compatible; A ´ A, a ´ a, "-" ´ "+" are all incompatible. Meiosis
yields two equal classes of spores.
2.
One gene with multiple alleles (unifactorial, bipolar heterothallism with multiple alleles): At the A
locus, there are many alleles; e.g., a1, a2, a3, ¼ an. Remember, there is only one allele in every
haploid strain and only two during diploid phases. Again, compatibility is the interaction of
different alleles during plasmogamy, e.g., a1 ´ a3. Matings among similar alleles, e.g., a3 ´ a3
lead to incompatible reactions. Again, meiosis of any diploid nucleus yields two equal classes
of spores.
3.
Two genes each with multiple alleles (bifactorial, tetrapolar system): In this system the mating
type loci each exhibit multiple alleles; e.g., the A and B locus, which are genetically unlinked,
may have 20-50 alleles at each locus, a1, a2, a3, ¼ an and b1, b2, b3, ¼ bn. However, once again
keep in mind that in any haploid spore only one of each allele is present; e.g., A1B1, and that in
a compatible mating A1B1 ´ A2B2, followed by nuclear fusion and meiosis, four types of spores
will result: A1B1, A2B2 (parental types) and A2B1 and A1B2 (recombinant types, by independent
assortment). So, although there are many allelic combinations possible, a single fruiting body
can generate only four types of spores.
To determine the nature of the mating system that is operative in any particular mushroom that you
might collect for study, the first step is to collect and isolate spores by the methods outlined in Figure 1.
Each group of students will isolate 15 germlings and transfer them to appropriate media in three petri
dishes, five per plate, well separated. Once the sporelings have grown into 1 cm colonies, 10
randomly selected colonies will be mated in all possible combinations to study mating reactions. Since
most basidiomycetes, including all those you will use, are heterothallic, it is not necessary to mate each
colony with itself. All of these crosses would be incompatible. The total number of colonies to be
analyzed per group may be derived by the formula
(n X n-1 ) /2
where n represents the total number of spores analyzed. Therefore, analysis of 10 spores would
translate into 45 matings for each group of students. Sterile technique is essential at all times.
Your identification of the mating type system operating in the mushroom of interest depends on
the nature of the mating reactions observed after placing the paired inocula to be tested in the middle
of a suitable agar medium. The hyphae that grow from the respective inocula will fuse, undergo
nuclear exchange, and the resulting mycelium will either be fertile (i.e., form clamp connections in the
case of most basidiomycetes, and/or fruiting bodies), or it will be sterile and remain vegetative. Your
results with spore by spore mating analysis should distinguish between mating systems dependent on
one gene and those dependent on two genes. You will not be able to distinguish between the two
kinds of single gene systems. Why not? You will, however, be able to distinguish single gene from
two gene systems. Random mating among spores from the single gene system will yield two kinds of
mating reactions: fertile and sterile. Random spore matings involving two gene systems will yield four
kinds of mating reactions: three sterile and one fertile. The fertile reactions can be detected
Biology/Microbiology 412/512
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cytologically in a few days by the appearance of a dikaryotic mycelium and subsequently the
development of the fruiting body. The nature of the incompatible reactions depends on the kind of the
heterokaryon formed from the mating reaction. The major characteristics of the four heterokaryons as
related to mating type gene interaction are listed in Table 1 below.
Table 1 Morphogenetic effects as related to the mating genes in mycelial interactions
leading to heterokaryosis in Schizophyllum commune. [= represents two incompatible
(because they are the same) alleles; ¹ represents two compatible alleles].
Clamp Connections:
Heterokaryon
Nuclear
Migration
Nuclear
Pairing
Conjugate
Division
Hook-cell
Formation
Hook-cell
Fusion
AB
Yes
Yes
Yes
Yes
Yes
A=B
Yes
No
Yes
No
No
AB=
No
Yes
Yes*
Yes
No
A=B=
No
No
No
No
No
* Only in the apical cell
•Data collecting from the mating reactions.
Score the mating reactions in Table 2 below using the abbreviations cited in Table 1 above.
AB
A=B
AB=
A=B=
1
1
2
3
4
5
6
7
8
9
10
Dikaryon
Common A heterokaryon
Common B heterokaryon
Common AB heterokaryon
2
3
4
5
6
7
8
9
10
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Exercise 5. Basidiomycota
Members of the Basidiomycota differ from all other fungi in that the meiotic spores,
basidiospores, are borne on (outside of) a specialized spore-producing structure called a
basidium. There is some variation in the form of the basidium and where it is borne in relation
to the mycelium (i.e. on or in a fruiting body (basidiocarp or basidioma) or in the absence of
one). The group encompasses a wide variety of forms, many of which you probably have
already encountered in nature. Included are four classes: the Hymenomycetes (familiar
mushrooms, boletes, jelly fungi and bracket fungi); the Gasteromycetes (puffballs, stinkhorns
and bird's-nest fungi); the Urediniomycetes, (the rusts) and the Ustilaginomycetes (the
smuts). The last two were previously grouped together as the Teliomycetes. Your textbook
(Alexopoulos Mims and Blackwell) is very hedgy about these upper classification levels. We
will use the classification scheme outlined here.
The mycelium of members of the Basidiomycota consists of well-developed, septate
hyphae. The septa of the Hymenomycetes and Gasteromycetes are complex and distinctive
and are called dolipore septa (see Ex. 2). The septa of the Teliomycetes are relatively simple
and similar to those of Ascomycetes. In some Hymenomycetes and Gasteromycetes the
hyphae may become aggregated into rhizomorphs (see Ex. 2). Basidiocarps arise from a
vegetative mycelium. Typically, the vegetative cells are dikaryotic (binucleate) although the
initial mycelium arising from a germinated basidiospore is monokaryotic (uninucleate). The
dikaryotic phase of many basidiomycetes is maintained by the production of clamp
connections, distinctive hyphal outgrowths which function to insure that two different nuclei are
allocated to each new cell following mitosis. Karyogamy and meiosis occur within the
basidium which then gives rise to haploid basidiospores. Therefore, the only diploid cell in
basidiomycetes is the basidium. Asexual reproduction in the group, where present, is by
means of budding, arthroconidia, chlamydospores, or oidia.
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I.
Hymenomycetes
All members in this class produce basidia in a palisade-like layer (a hymenium) which,
with few exceptions, are borne on basidiocarps that arise from a dikaryotic mycelium.
Furthermore, the hymenium is exposed prior to maturation of the fruiting body. The class is
divided into two subclasses (ending -mycetidae) based on whether or not the basidia are
septate.
A. Holobasidiomycetidae (basidia typically unicellular)
1. Agaricales (agarics)
This order includes the well-known mushrooms and toadstools. They are among the most
advanced of the orders, but we will study them first because of their familiarity. As evidenced
by the increasing number of field guides, the American public is becoming more interested in
this group. There has also been increased interest in their consumption, some for their
delicious palatable qualities, and some for their hallucinogenic properties. In contrast to the
relatively mycophobic Americans, Europeans (especially eastern Europeans) and Asians
(especially southeast Asians) are far more mycophilic, and mushroom hunting is part of their
cultural heritage. But beware! Some are deadly poisonous and every year many people die
from eating poisonous mushrooms.
The systematics of this order is in a state of flux. See classification scheme in preface.
Families—see supplement— Agaricales: Families and their characteristics
This order includes the well-known gilled mushroom-forming. (some authors include within this
order the boletes, which we will consider as a separate order.) Species in the Agaricales bear
their hymenia (basidia ± cystidia) on lamellae (gills). Most often the basidiocarp is fleshy,
being differentiated into a stipe (stalk) and pileus (cap). The young basidiocarp may be
covered by a sheath-like universal veil which is ruptured during maturation of the basidiocarp
and may remain as a more or less cup-shaped volva at the base of the stalk. The developing
gills may be covered by another veil (the partial veil) which ruptures during expansion of the
pileus. If it persists as a ring around the upper portion of the stalk, it is called an annulus.
Both the volva and annulus are used in identification.

Coprinus and/or Psilocybe are representative of the "gill fungi." Look at habit material first,
to become familiar with the general growth form, and then look at prepared slides of
cross-sections of the cap and locate basidia and basidiospores. Also note the trama (inner
tissue of gills, composed here of tightly packed hyphal cells only) and compare with
Russula and Lactarius below.

Observe the spectacular cystidia of Pluteus cervinus exhibited on demonstration slides.

Observe fruiting bodies of representative genera and note the general form and structure.
Which genera were obviously enveloped by a universal veil when young? Prepare a slide
of a thin gill of one of the fresh basidiocarps on display. Note the particular form of the
Biology/Microbiology 412/512
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basidia and basidiospores of that species.

Examine other demonstration material related to this group. Included will be literature that
is used to identify specimens in this group as well as other macroscopic forms. See also
the supplement to this exercise. While perusing this material, note the range in form of the
agaric basidiocarps. What are some of the key characters used in the identification of the
gill fungi? The most deadly forms are in the genus Amanita. Assuming you are interested
in collecting gill fungi for consumption, what easily determined criteria would you use to
avoid collecting them? Using these criteria, would you eliminate edible species?
Characters used to separate the modern genera of gilled fungi
There has been an increased acceptance of an ever-multiplying number of genera in the past 50 years or so.
Gone are the days of Elias Magnus Fries (1794-1878), the father of modern mycology, when nearly any gilled
mushroom was placed in Agaricus. Modern genera are meant to convey to a mycologist some important features
about the fungus in question, including the fungus's critical role in the environment, not just superficial
morphology. When more characters of a fungus are studied, more cursory relationships can fall apart and new
relationships can be discovered. This leads to many familiar fungi being moved to different genera, as new
information is uncovered. Unfortunately most fungi do not have real common names, so we are stuck with some
inconvenient name changes. Some other reasons for name changes are discovery of an older name in the
scientific literature, splitting of a single species into more than one species, discovering a North American
species has been "masquerading" under a European name, and a few others.
The following are characters that are used to distinguish modern genera of the Agaricales.
Spore print color.
The first character asked for in most keys to the genera of Agarics is spore print color. Usually this can be
guessed at by looking at the color of the mature gills, but sometimes you can be easily fooled. Taking a spore
print is highly recommended. Some people take two spore prints, one on white paper and one on black paper, so
that the full nuances of color can be examined. If you have a Petri dish or other clear plastic or glass to use, the
spore print on these can be placed over any color background.
Melzer's solution-- reaction of the spores
Another major separating character is the reaction of the spores in Melzer's solution. The active ingredient is
Iodine, which stains starch a dark blue-black (an amyloid reaction) and stains certain other compounds reddish (a
dextrinoid reaction). Often it is only the ornamentation of the spores that reacts in Melzer's, and this must be
examined under the microscope. However even without a microscope the color change reaction can often be
seen by applying a drop of Melzer's reagent to the spore print. For this reaction, spore prints on paper should not
be used, because the paper itself has and amyloid reaction because of starch residues in it. Some genera have
spores that are ornamented, but not amyloid, although this is unusual.
Spore shape and ornamentation
Certain genera have very distinctive spores shape, such as the irregular and angular spore shape of Entoloma
species, the striate spores of Clitopilus, the cylindrical spore shape of Suillus, and the truncate spores of Pholiota.
Certain genera also have spores with distinctive ornamentation, such as some Russula species. Some have
spores that are ornamented but not amyloid, although this is unusual.
Cystidia—sterile microscopic appendages on the gills or pores among the basidia
Certain genera have very distinctive cystidia, such as the heavily branched cystidia of Mycena, the horned
cystidia of many Pluteus species, or the golden chrysocystidia of Stropharia.
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Nutritional mode
Modern genera of Agarics can be separated by their nutritional mode. Saprophytic fungi use dead organisms as
their source of organic material. Parasitic fungi use living organisms as their source of nutrition. Symbiotic
fungi live in association with another living organism in a mutually satisfying relationship. The mode of
nutrition is significant at the generic level.
Gill attachment
Some genera can be separated by the attachment of their gills to the stipe. Some examples are free, attached,
adnexed, adnate, and decurrent.
Unusual features
Some fungi have unusual features that characterize their genus. For example the presence of a volva in Amanita
or Volvariella, the cortina (cobwebby veil) in Cortinarius, latex in Lactarius, sphaerocysts (circular cells that
break apart easily) in Russula and Lactarius, serrate (toothed) gills in Lentinus and Lentinellus.
Presence or absence of a partial veil that becomes an annulus
This character, although losing some importance in many genera, is still a significant character for a number of
genera. E.g. Agaricus and Cystoderma nearly always have a partial veil and an annulus, while Mycena and
Russula almost never have a veil and annulus. However some genera now include annulate and non-annulate
species, such as Armillaria, Amanita, Agrocybe, Pleurotus, and Tricholoma.
Mode of development of the fruiting body
In some genera, fruiting bodies develop from an "egg" covered by a universal veil. In others the gills originate
deep inside the fruiting structure and are covered by a partial veil. In still other genera the gills develop on the
outside after the fruiting body is formed. This is not always an easy character to study.
Clamps vs. simple septa on the hyphae
Some fungi in some genera have small appendages at the septa of the hyphae called clamps. Some do not. This is
sometimes used to separate certain genera, although this character is not as important as in the polypores. For
example, this is a major distinction between Cantharellus (clamps) and Craterellus (simple septa).
Stipe attachment
Some genera have stipes that are mostly attached laterally and other have stipes that are mostly centrally
attached. This is often not a very reliable character due to inconsistencies in growth conditions. However in
some genera (e.g. Panellus, Phyllotopsis, Mycena, Tricholoma) it can be very reliable.
Peeling gills
Some fungi have gills that peel away from the flesh of the mushroom (e.g. Phylloporus, Paxillus, etc.). These
fungi are closely related to the boletes, which have pore layers that peel away easily from the flesh.
Structure of the gill trama
Some genera can be separated on the basis of the structure of the gill trama, the non reproductive, supportive
tissue of the gills. Some examples of gill trama structure are parallel, interwoven, convergent, divergent.
Structure of the pileipellis (cap cuticle)
The appearance of the cap surface (fuzzy, viscid, dry, etc) can be attributed to the microscopic structure of the
cap cuticle. These can be identified by microscopic examination or with practice can be observed with a hand
lens. See excellent articles on using a hand lens to study mushrooms by Walt Sundberg (McIlvainea 11 (1):15-22,
1993; McIlvainea 11(2): 48-60, 1994).
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2. Boletales (the boletes; some authors consider this a family, the Boletaceae, within the
Agaricales)
The fruiting bodies of this family are usually soft, fleshy, and mushroom-like, with a stalk and a
pileus, but instead of gills, these fungi have pores. The hymenium lines the pores that are
found on the underside of the cap. They are especially common in coniferous forests, many
of them forming mycorrhizae with the roots of pine and larch. They are also common on oak,
birch and aspen

3.
Observe habit specimens of this group, including Boletus, Suillus, Gyrodon, and/or
Strobilomyces, noting the pores on the under surface of the cap. What characteristics
could you use to distinguish a bolete from a polypore?
Russulales
Members of the Russulales are similar to the Agaricales except that the trama (inner tissue of
the gills) is composed of sphaerocysts or rounded cells in addition to typical elongated hyphal
cells and they have distinctive amyloid, ornamented spores. The presence of sphaerocysts
often renders the basidiocarps of Russulales somewhat brittle — a useful field character).

4.
Russula and Lactarius (all species exude a milky juice when fresh basidiocarps are broken)
are common Wisconsin genera. Fresh or preserved specimens will be available. Mount in
Melzer’s and observe the amyloid spores and the sphaerocysts.
Cantharellales (chanterelles)
Basidiocarps are typically funnel shaped with the hymenium smooth, wrinkled or folded to form
thick gill-like structures, usually forked. Species of Cantharellus and Craterellus are some of
the most delicious edible wild fungi. Preserved or fresh specimens will be on display.
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5.
Aphyllophorales
Recent years have brought many changes in the classification within this order, with no single
system being widely accepted. We will use a modification of the most widely accepted
scheme, that of Donk (1964. A conspectus of the families of Aphyllophorales. Persoonia
3:199-324). Note various ways that surface area is increased for spore dispersal.
a.
Thelephoraceae and Corticiaceae
The basidiocarps in this family vary from a thin weft of hyphae bearing basidia to
well-developed fructifications. The most common type is a tough, leathery or corky structure
which is crust-like, shelf-like, or upright with the hymenium covering a smooth or undulating
surface. Most of the members grow on wood, some of them being very common (e.g. species
of Stereum).

b.
Basidiocarps of several genera, including Thelephora, Xylobolus, Stereum,
Phanerochaete, and/or Phlebia, will be on display to illustrate the range in forms
encountered in these groups.
Clavariaceae
The members of this family often are referred to as the "coral fungi" because of the erect,
compact branching form exhibited by many species. However, some species have only
simple, club-shaped or columnar basidiocarps. Their texture is usually fleshy or waxy, the
hymenium extending over the entire surface except at the base.

c.
Basidiocarps of various representatives will be on display, including Clavaria, Ramaria,
and/or Clavariadelphus. The hymenium can be observed on prepared slides.
Hydnaceae
The common name "tooth fungi" is applied to these forms because there are often positively
geotropic spines on the basidiocarp. The hymenium covers the surface of these "teeth."
There is a considerable range in the form and texture of the basidiocarps, some of which can
be seen in the specimens on display, including Hydnum and Hericium.
d.
Polyporaceae
The "pore fungi," or "polypores" as these are commonly called, is a large, diverse group
commonly encountered on woody substrates, although not restricted to them. In many, the
hymenia line numerous pores or tubes on the underside of the basidiocarp, which may be
fleshy (Polyporus) leathery (Trametes), corky or woody (Fomes) in texture. They may be
bracket-like, stalked, or sessile in form and perennial or annual.

Examine basidiocarps of representative genera on display and note the range in general
growth form, texture, size, and shape of the pores, etc. Observe Ganoderma basidiocarps
that have been broken, and deduce whether it is most likely an annual or a perennial
structure. Also, examine sections through its pores to observe the hymenium.
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
Slides of Polyporus will be available for observation of basidia, sterigmata, and cystidia.
Don't confuse the basidia with the slightly longer, stouter, sterile cystidia that are intermixed
with the basidia.

Clamp Connections: Prepare a slide of a dikaryotic culture of Fomitopsis cajanderi (or
Schizophyllum commune) and observe the hyphae with clamp connections. These are a
characteristic microscopic feature of most basidiomycetes.

Some examples of wood rots and basidiocarps of the fungi that cause them will be on
display. Note differences in the manner in which the wood is affected. What causes the
different appearances of white rots and brown rots?
e.
Hymenochaetaceae
This family is a prime example of why hymenophore (spore bearing surface) configuration is
not the best character to use in family delimitations. The members of this family all have the
following microscopic characters in common: simple septa (no clamps), turn black in KOH (23%), may have thick walled setae or setal hyphae. Various hymenophore configurations are
included in this family: Observe specimens and make slides to see the hyphae and setae.
Hymenophore
configuration
Representative genera
Smooth
Toothed
poroid
Hymenochaete
Hydnochaete
Phellinus, Inonotus, Coltricia
6.
Schizophyllales
The basidiocarps in these forms are at first cupulate in form, later becoming discoid.
Schizophyllum commune is extremely common and has worldwide distribution. It has been
used to study genetics, physiology, and morphogenesis, and occasionally can infect humans.
Superficially it appears to be a gilled mushroom, but the hymenial layer consists of thick
lamellae that are split longitudinally with both edges folded back. They are not believed to be
homologous with the gills of the Agaricales. Observe both wild and cultured specimens.
7.
Dacrymycetales
This is a small group of saprophytic fungi whose fruiting bodies are mostly gelatinous to waxy
in texture and often brightly colored (yellow or orange). The basidia are non-septate; they are
typically slender and Y-shaped ("tuning fork" basidia), with one basidiospore being borne at
the tip of each arm. Superficially they may resemble members of the Tremellales (see below)
but the basidia are not septate. In addition they often are bright orange in color, making it
possible to distinguish them macroscopically from the Tremellales, with some experience.
Basidiocarps of Dacrymyces, Calocera, and/or Dacryopinax will be available for examination.
Prepared slides may be available.
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B.
Phragmobasidomycetidae aka Heterobasidiomycetidae
(septate basidia). The orders are based on the direction of the septation in the basidia,
whether transverse or cruciate
1.
Auriculariales
The distinguishing feature of this order is the transversely septate basidium (perpendicular to
the direction of growth), which resembles that found in the rusts. The basidiocarps tend to be
gelatinous, or somewhat leathery.

2.
Observe the habit specimens of Auricularia and/or Phleogena. The most common species
Auricularia auricula or A. polytricha are often used in oriental cooking and can be bought
dried under the name "cloud ear" or "tree ear." There may be prepared slides available.
Tremellales
Members of this order have cruciate-septate basidia—the septum is parallel to the direction of
growth and are usually found in the form of a cross. Most members of this order are wood
inhabiters and are commonly called "jelly fungi" because the basidiocarps are often
gelatinous. They shrink greatly and have a horny texture when dry, but quickly swell and
revive when it rains.

Examine habit specimens of Tremella, Pseudohydnum, Tremellodendron, and/or Exidia to
get an idea of the form and texture of basidiocarps in this group. There may be prepared
slides available. If fresh specimens are available look for cruciate-septate basidia.
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II. Gasteromycetes
In contrast to the preceding class, the fertile areas of members of this group develop within an
enclosed basidiocarp, at least until the spores are mature. The fertile portion of the
basidiocarp is called the gleba. Although treated as a separate class, the phylogenetic
relationships within the Gasteromycetes and other classes is poorly understood. Some of the
Gasteromycetes probably belong phylogenetically to the Agaricales, but others probably do
not. The sole feature that distinguishes the Gasteromycetes is that the spores mature within
an enclosed basidiocarp. Most of the several orders are mentioned here.
A.
Lycoperdales
This group includes the common puffballs, most species of which are edible, and some of the
earthstars. The peridium usually has two, sometimes four, layers. Inside, the basidia are
usually borne in a distinct hymenium that lines the small, scattered glebal chambers. At
maturity, the gleba breaks down into a dry, powdery mass of spores and capillitial threads, the
latter being thick-walled hyphae in this case.

Mature basidiocarps of representative genera will be on display (Lycoperdon, Calvatia,
Bovista, and/or Astraeus). For each, note whether there is a special structure for discharge
of the spores. In which genus (genera) is the double-layered peridium evident? Do the
two layers exhibit different physiological and/or morphological features?

Using the material specifically designated, make a water mount of the material inside of the
peridium, either by gently tapping the fruiting body to discharge the contents or by
removing a small portion with a forceps, whichever is most appropriate. Are both spores
and capillitial threads evident? Compare the contents if more than one species is
available.
B. Sclerodermatales.
These include the false puffballs, most of which are poisonous, as well as Pisolithus tinctorius
and some other puffball and earthstar shaped fungi. The key macroscopic character that
distinguishes the Sclerodermatales from the Lycoperdales is the organization of the gleba into
distinct locules, which can be observed in cross sections of immature sporocarps. Most are
dark colored from early development. Most of the members of this order are mycorrhizal,
although the ecological status of some members is unknown.

Observe specimens of Scleroderma Pisolithus, and/or Geaster, noting especially the thick
peridium and the locules.
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C. Phallales
These are the stinkhorns, so-called because of the offensive, sometimes nauseous, odor that
emanates from the exposed gleba. What function do you think this serves? The young
fruiting body is spherical to egg-shaped, often developing just below the ground surface at the
end of a stout rhizomorph.

Observe preserved specimens of these young basidiocarps. Is the rhizomorph apparent?
Looking at sectioned "eggs", you should be able to distinguish a white, leathery peridium
on the outside. In the center, you should see an elongate, white hollow core which will
eventually elongate into a stalk. A bell-shaped receptacle arises from the apex of the
stalk--this bears the dark-colored glebal mass. Phallus impudicus is sometimes called the
“Stinkmorchel” in Germany. Where do you suppose this name comes from?

Next examine mature fruiting bodies of Phallus. Are the structures you observed in the
"egg" apparent in the mature fructification? What happened to the peridium?

Compare the basidiocarp of Phallus with other genera in the order (Dictyophora, Lysurus,
Mutinus), noting similarities and differences, especially in relation to the receptacle.
D. Nidulariales (bird's-nest fungi)
Examine mature basidiocarps of Cyathus and Crucibulum. Inside the nest-shaped peridium,
you should see a cluster of "eggs"—these are peridioles, within which the basidia are borne.
Prior to maturation, the "nest" was covered by a thin, fragile layer of hyphae (called the
epiphragm), which flakes off at maturity. Is it apparent on any of the specimens on display?

E.

Study stained sections through a basidiocarp of Cyathus. Note that a highly coiled
funiculus is attached to each peridiolum. When wet, it is sticky and serves to adhere the
peridiolum to the substrate after it has been ejected. Can you see the hymenium and
basidiospores inside of the peridiolum? How do you suppose the basidiospores escape?
Sphaerobolales
Habit specimens may be available to show the fruiting bodies of Sphaerobolus (the spherethrower) before and after the discharge of the single peridiolum. Note that during the
violent discharge of the peridiolum the top of the fruiting body is broken off, exposing two
small cups. The inner cup is everted and the peridiolum is shot up to 2 m vertically and 4
m horizontally, an enormous distance considering the small size of the peridiolum (about
1-2 mm diameter).
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III. Urediniomycetes (Rusts)
Both the rusts and the smuts, (sometimes both are included in the class Teliomycetes) are
characterized by having a special thick-walled resting spore, the teliospore, in their life cycle.
The germinating teliospore functions as the basidium and gives rise to the basidiospores).
These are some of the most economically important basidiomycetes — parasites of
agricultural crops, commercially valuable timber, horticultural crops, etc.
This class includes several thousand species of parasitic fungi, commonly called "rusts," for
which ferns, gymnosperms and angiosperms serve as hosts. Up to the early 1950's they had
been considered obligate parasites, but since that time several have been cultured
successfully in the absence of host tissue. The intercellular mycelium is uninucleate in the first
stages but becomes binucleate in its later stages. Autoecious species complete their life cycle
on a single host species; but many rusts exhibit a peculiar life cycle in that they pass through a
part of their development on one host species and then must infect an entirely different host
species to complete their life cycle. These latter forms are heteroecious species.
Reproductively, the rusts can be relatively complex, some producing as many as five different
kinds of spores, but no fruiting body is involved. Unfortunately, there are several sets of
synonymous terms used to describe these various spore types and the associated
spore-bearing structures. The system used below seems to have widespread acceptance and
perhaps eventually will be universally accepted. Numbers are often used to designate the
different spore types, as illustrated below:
0.
Pycnia (spermagonia) bear haploid pycniospores (spermatia). They develop on
mycelia which arose from infection of the host by basidiospores. The pycniospores act
as sperm cells and effect fertilization of a receptive hypha.
I.
Aecia are more or less cup-shaped structures that produce chains of 1-celled, dikaryotic
aeciospores. They normally result from a fertilization process.
II.
Uredinia (uredia) bear urediniospores (urediospores) which are deciduous, 1-celled,
and act like conidia.
III.
Telia bear teliospores, both of which have variable structure among the many rust
species. Teliospores always produce basidia upon germination.
IV.
Basidia (promycelia) bear haploid basidiospores.
However, it should be emphasized that not all rust species produce all of these spore types.
Further elaboration on life histories will be taken up in the lecture.
Three families of rusts are traditionally recognized, Puccinaceae, Melampsoraceae and
Coleosporiaceae. You will see representatives of the first two families only.
1.
Pucciniaceae (teliospores stalked, arranged singly or in groups, but never in layers or
crusts)
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a.
Puccinia graminis will be used to illustrate the features of a rust life cycle, since it is one
of the most complex. This species has been the object of intensive study because of its
tremendous economic significance--the "black stem rust" of cereal crops. Refer to a life
cycle diagram such as the one on text p. 623.

Examine infected barberry leaves (Berberis vulgaris) macroscopically and under a
stereoscopic microscope, noting the clustered arrangement of the aecia (aecidioid aecia).

Observe prepared slides showing sections through infected barberry leaves. What effect,
if any, does the pathogen have on the host tissue? Can you detect any intercellular
mycelium? Scan the section to locate median sections through the flask-shaped pycnia
(stage 0). What is their relationship to the host tissue? Are they more prevalent on the
upper or lower leaf surface? Where are the pycniospores produced and how can they gain
access to the external environment for dispersal? The pycnium is the structure in which
the dikaryotic condition is established, arising by a fertilization process wherein a
pycniospore of one mating type fuses with a receptive hypha of a compatible mycelium.
The resulting dikaryotic hyphae grow to the bottom of the leaf where they form the aecial
primordia.

Scan the slide and locate a good section of an aecium (stage I). Are aecia more prevalent
on the upper or lower leaf surface? Can you suggest a selective advantage for this
distribution? Look closely at young aeciospores and the basal cells subtending the
aeciospore chains and note the nuclear situation.

The aeciospores are unable to infect barberry leaves; rather they must land on an
appropriate grass host to perpetuate the line. Soon after infection, the binucleate
mycelium begins to form large numbers of sporulating pustules--the uredinia.

Scrape a few urediniospores (stage II) from the wheat stems provided and mount them in
water or preferably cotton blue in lacto-phenol. How many cells comprise the spore? Can
you detect germ pores? What would you expect the nuclear situation to be in these
spores? What host(s) are these spores capable of infecting (you may want to refer to the
demonstration material)? A set of electron micrographs illustrate urediniospore
germination.

About the time the grain is ripening, the telial stage appears. The uredinia may be
converted to telia, or the mycelium may develop new telial pustules. Can you distinguish
telia from uredinia in habit material?

Prepare a mount of teliospores (stage III) and compare them with the urediniospores. In
what ways do they differ from one another?

Examine prepared slides showing uredinia and telia in section view. Be sure to note the
vegetative mycelium in these preparations. From these slides, and the material observed
above, characterize these two structures the best you can.
The teliospores are the overwintering stage, the two nuclei in each cell fusing with one
another to form diploid cells. Early in the spring, each cell germinates to form a cylindrical
basidium. The diploid nucleus migrates into this structure, undergoes meiosis, and forms

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four haploid nuclei. Septa are laid down, segregating the nuclei in separate cells. Each
cell of the basidium (stage IV) forms a sterigma on which a uninucleate, haploid
basidiospore is borne

See demonstration slide and illustration of P. graminis basidia (germinating teliospores).
Puccinia graminis stage IV is so ephemeral that habit specimens are unavailable, but you can
get some idea of what it looks like by observing the basidia of another rust, Gymnosporangium
juniperi-virginianae, the incitant of cedar-apple rust. In this species, the telial stage is
represented by the emergence of long, gelatinous, orange horns that emerge from galls, borne
on cedar or juniper hosts, in the spring.

Remove a small portion of one of these gelatinous telia with a forceps, mount it in a drop of
water and tease it apart with probes. Compare these teliospores with those of P. graminis.
Note the basidia emerging from the teliospores. Scan the slide and try to locate basidia
bearing sterigmata and basidiospores (there may be a few or none at all). The
basidiospores of P. graminis are only capable of infecting barberry; those of the
cedar-apple rust infect apple, crab apple, and hawthorn.
b.
Examine demonstration material of caeomoid and roestelioid aecia, and different types
of teliospores. Teliospore morphology is the basis for the taxonomy of the rusts.
Familiarize yourself with the taxonomic literature available.
2.
Melampsoraceae (teliospores not stalked, aggregated in layers, crusts, or columns)
Cronartium ribicola, the incitant of "white pine blister rust" will be used to demonstrate the
salient features of this family. This is also a heteroecious rust in which the pycnia and aecia
occur on white pine and the uredinia and telia on leaves of currant or gooseberry. Examine
the material in the same manner as you did with P. graminis.


There will be habit material of diseased pine branches for macroscopic examination--note
the large galls on which the aecia are produced (since they are very fragile, most or all of
them have been broken off, leaving only the gall).
Study prepared slides of the pycnial stage, which develops in the cortical region of young
branches. Note that they cover a large flat area compared to the minute, flask-shaped
pycnia of P. graminis. Other prepared slides will show sections through aecia.


If slides of uredinia are available, examine them and compare with those of P. graminis.
Finally, study prepared slides having sections through telia, which are elongated, horn-like
columns consisting of stalkless teliospores united laterally and terminally. These germinate
in place and produce basidia similar to those observed in Gymnosporangium.

Observe scanning electron micrographs of germinating teliospores and basidiospores.
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IV. Ustilaginomycetes (Smuts)
The Ustilaginomycetes are commonly known as the smut fungi, undoubtedly because of one
of the older meanings of the word (smutzen in German = “to stain”). The Ustilaginomycetes
are also characterized by having a special thick-walled resting spore, the teliospore, in their life
cycle the germinating teliospore functions as the basidium and gives rise to the basidiospores.
Along with the Urediniomycetes (rusts) these are some of the most economically important
basidiomycetes—parasites of agricultural crops, commercially valuable timber, horticultural
crops and many other.
This group is commonly referred to as the smuts because they form black, dusty spore
masses (the teliospores) which resemble soot or smut. In contrast to many rusts, which have
complex life cycles involving two unrelated hosts, smuts complete their life cycle on only one
host. They normally are parasitic on flowering plants, causing extensive losses in cereal
crops. No fruiting bodies are produced, nor is there any evidence of sex organs, although
successful sexual reproduction does involve the establishment of a dikaryotic mycelium having
compatible nuclei. Budding of basidiospores and conidia is a common method of asexual
reproduction.
When the mycelium reaches the sporulation stage, it usually forms masses of hyphae
comprised of numerous short cells. The protoplast rounds up and secretes a thick wall,
thereby becoming a teliospore (smut spore) which will give rise to a basidium. The hyphal
walls gelatinize, and the teliospores are released. Teliospore characteristics, along with host
range and disease symptomology, are the primary features used for the segregation of the
genera and species in this group. The spores exhibit various types of ornamentation, and they
may occur singly, in pairs, or in clumps called spore balls. These clumps may be comprised
exclusively of fertile cells, or they may include accessory sterile cells.
Selections from the following material may be available for your inspection. Whenever habit
material is available, note what effect the parasite has on the host and where the sporulation
occurs. You also will be able to characterize the teliospores for a number of species (check
your observations with the literature provided).
1.
Ustilaginaceae (transversely septate metabasidium with lateral and terminal
basidiospores)
Ustilago avenae: habit, prepare spore mount, Ustilago hordei: habit, prepare spore mount,
Ustilago maydis: habit, prepare spore mount, Ustilago neglecta: habit, prepare spore mount,
Ustilago utriculosa: habit, prepare spore mount, Sorosporium cenchri: habit, demonstration
slide
2.
Tilletiaceae (nonseptate metabasidium with only terminal basidiospores)
Tilletia brunkii: demonstration slide, Tilletia caries: demonstration slide
Tilletia foetida: habit, prepare spore mount, Tilletia pulcherrima: demo slide,
Urocystis agropyri: prepare spore mount
Urocystis anemones: habit, prepare spore mount
Urocystis colchici: habit, prepared slides
Urocystis occulta: demonstration slide
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Supplement 1: Agaricales: Families and their characteristics
Examine the material referred to below, as directed, depending on what species we find. Your lab notebook
should include sketches that emphasize the diagnostic characters of each family, and should also include
answers to the embedded questions. You will not be required to know taxa below the family level except
for as indicated by the lab questions. You should refer to supplemental information such as the field guides
to augment your understanding.
1. Amanitaceae
A white-spored mycorrhizal family of 2 genera. Sporocarps have free gills and a volva. They may
also have a partial veil, and/or “warts” on the cap.
Amanita rubescens - Revive a small piece of gill in 95% ethanol, followed by water, and then mount in a
drop of Melzer’s. Crush the gill slightly. Note the amyloid spores.
Amanita muscaria - Associated with conifers. Section the gill tissue to see the divergent gill trama. Mount
in water and Melzer’s to observe the reaction. Note the important macroscopic characteristics of the family
and observe the photograph of this species in a field guide such as Mushrooms of North America (MNA).
Amanita virosa - Associated with oaks. This species is deadly poisonous. Observe the specimen, note
important characters, and see a field guide.
2. Lepiotaceae
This is a saprophytic family which resembles the Amanitaceae macroscopically. Sporocarps have
free gills, appressed scales, and a “ring”, but no universal veil. Microscopically, spores of the Lepiotaceae
usually have an apical pore.
Chlorophyllum molybdites - Revive a piece of gill in ethanol and mount in water and Melzer’s. Crush
slightly and observe the thick walled spores with an apical pore. Chlorophyllum is exceptional in the
Lepiotaceae because it has a greenish spore print.
Lepiota americana - Observe the fresh specimen and compare to the description of the similar L. rhacodes
in a field guide.
What characters distinguish the Lepiotaceae from the Amanitaceae?
3. Hygrophoraceae
A predominantly (?) mycorrhizal family. The gills in this family look and fell waxy due to their
long, narrow basidia. All members of the Hygrophoraceae lack universal and partial veils.
Hygrophorus praetensis - Section and observe the divergent trama.
Hygrocybe conicus - Observe the specimen and note the illustration in MONENA. Crush in Melzer’s to
observe the smooth thin-walled spores and the long narrow basidia.
How could you distinguish the Hygrophoraceae from the Lepiotaceae and Amanitaceae?
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4. Tricholomataceae
This is a large and diverse white-spored family, of which we will observe some of the more common genera.
Laccaria ochropurpurea - Crush a piece of gill, and observe the spores in Melzer’s and KOH. This fungus
is an important ectomycorrhizal species. Compare spores to those of the Russulales (below).
Tricholoma flavovirens - Observe and check in MONENA. Why is this fungus not placed in the
Russulaceae or the Hygrophoraceae?
Armillaria mellea - Observe fresh material if available and check the illustrations in MONENA. Note the
partial veil. This fungus is an important root pathogen. The mycelium-laced wood it inhabits glows in the
dark (“foxfire”), and it can be identified in the field by its black rhizomorphs (shoestrings).
Pleurotus ostreatus - The Oyster mushroom. Note the laterally attached stipe. Crush a piece of gill tissue in
Melzer’s and observe the spores.
Marasmius oreades - Rehydrate a dried sporocarp and observe how this fungus “revives.” This is one of the
typical “fairy ring” mushroom found in lawns.
Gymnopus (=Collybia) subnudus - Prepare a slide of a dikaryotic culture of Collybia subnuda and observe
the hyphae with clamp connections. These are a characteristic microscopic feature of most basidiomycetes.
Lentinellus ursinus - Revive, section and mount in Melzer’s to observe the different hyphal types which
compose the pileus trama. Note the amyloid trama.
How could you distinguish mushrooms in the Tricholomataceae from the other white spored agaric
families?
5. Pluteaceae
A pink-spored family with free gills. The spores of members of this family are not angular as they
are in the Rhodophyllaceae..
Volvariella bombycina - Note the volva, free gills, and pink lamellae in this specimen and check with
MONENA.
Pluteus cervinus - Note the free lamellae and lack of volva. Make a section of the gills and mount in KOH.
Observe the spores, metuloids (thick walled cystidia), and convergent gill trama. Metuloids are also visible
in V. bombycina if you have trouble seeing them here.
6. Rhodophyllaceae or Entolomataceae - Salmon to Pink spores with attached gills.
Entoloma strictipes - Crush a piece of gill and mount in KOH. Observe the angular spore. This is an
ectomycorrhizal species.
Clitopilus prunulus - Mount a piece of gill in KOH. Observe the longitudinally grooved spores (look for
spores oriented on end).
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Entoloma abortivum - Note the aborted fruiting bodies involving both this species and Armillaria mellea.
Both species are common in this area. The precise nature of the relationship is a matter under study, but it
may be that the Entoloma is parasitizing the Armillaria (or vice versa . . .)
7. Coprinaceae - a saprophytic family. The spores have a pore and are purple-black in mass. Cap cuticle is
cellular (compare to the Strophariaceae, which has a filamentous cuticle).
Coprinus micaceous - section the pileipellis and mount in 3% KOH. Note the cellular cuticle
(polycystoderm) and the spores with an apical pore.
Coprinus atramentarius - crush, mount in 3% KOH. Note the spores with an apical pore and read about the
toxins in this fungus in Mushrooms Demystified.
Coprinus comatus - The “shaggy mane,” a prized edible species. Note the dried material and check the
photograph in a field guide.
Panaeolus foenisecii - Crush in 3% KOH. Observe the warted spores with an apical pore. Some reports
claim that this species contains a mild hallucinogen; other reports disagree. . .
Psathyrella hydrophila - Observe and check the life form in a field guide.
8. Bolbitiaceae - a saprophytic family closely related to the Coprinaceae. The spore color is yellow-orange,
to brown.
Bolbitius vitellinus - Observe its similarity to a Coprinus with brown spores. Crush a piece of gill in KOH
and observe the spores.
Agrocybe praecox - Section the cuticle in KOH and observe the pyriform cuticular cells (cystoderm) and
spores with an apical pore. How is the structure of the pileipellis related to the appearance of the mature
sporocarp?
9. Strophariaceae (Note: the intense purple/black color of the spore print fades in time).
Stropharia semiglobata - See the illustration in the field guide on display.
Hypholoma capnoides - Section in 3% KOH; observe the chrysocystidia and observe the filamentous
cuticle. Note the spores. Do they have an apical pore?
Psilocybe cubensis - Sorry, we have no material of this species at this time. This is the mushroom most
often cultivated (illegally) by hallucinogenic mushroom growers. Check its appearance in a field guide. The
macroscopic characters which define Psilocybe are - the spore print color, the viscid cap, the partial veil
which leaves an annulus, attached gills, and blue staining somewhere on the pileipellis, usually the stipe.
10. Agaricaceae - Free gills, annulus (usually), chocolate brown spore print, thick walled spores with a
small pore, or none. The Agaricaceae is very similar to the Lepiotaceae and differs only in the spore print
color.
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Agaricus bisporus - This mushroom is the common cultivated mushroom found in grocery stores; it does not
grow wild in the United States. Look at the basidia in KOH; how many spores per basidium? Look at the
spores carefully under oil immersion and try and see an apical pore; it is very small and you may or may not
be able to see it.
Agaricus sylvicola - Observe dried material and read description in a field guide.
11. Cortinariaceae
Galerina autumnalis - Deadly poisonous; see dried material and photograph and description in a field guide.
Note the viscid cap (when wet), the habitat on wood, and the fragile partial veil.
Cortinarius violaceus - Cortinarius is a very large genus with many species. Crush in 3% KOH, observe the
rugose spores.
Hebeloma crustuliniforme - All species in this genus are poisonous. Make a section and observe both the
cheilocystidia and the viscid cuticle.
Inocybe lanuginosa - Highly poisonous; all species in this genus are poisonous as well. Note the tuberculate
warted spores.
Crepidotus mollis - No stipe in this genus; always on wood. This genus is sometimes put in a family of its
own, the Crepidotaceae.
Pholiota squarrosa - This is a common brown-spored wood decomposer. Note the raised cap scales and
clustered habit on wood, both features typical of the genus.
12. Paxillaceae (only 4 N. American species)
Paxillus atrotomentosus - (Probably a wood decomposer). See dried material and read its description in a
field guide. What is the important feature here?
Paxillus involutus - (Mycorrhizal) Observe spores in Melzer’s.
13. Gomphidiaceae - mycorrhizal with conifers. This family is probably closely related to the Boletales.
The gills are decurrent, the spore print is smoky-brown, and there are distinctive cystidia in the
hymenium.
Chroogomphus rutilans - Section the pileus and mount in Melzer’s. Note the amyloid trama, spores and the
large cystidia.
Gomphidius glutinosus - Section and mount in Melzer’s. Note the non-amyloid trama, spores and the large
cystidia.
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Supplement 2 : Keys to Major classes and some orders of Macroscopic Basidiomycota, Plus families
of the Agaricales . Tom Volk 8/2005
Key to Major classes
1. Hymenium (fertile layer of cells) exposed to the air prior to maturation of the fruiting body……2
2. Fruiting body jelly-like. Basidia septate..……….…...Heterobasidiomycetes
2a. Fruiting body not jelly like. Basidia non-septate. ………….Hymenomycetes
1a. Hymenium not exposed to the air prior to maturation of the fruiting body…………..Gasteromycetes
Key to orders of Hymenomycetes (literally “mushrooms with a fertile layer called a hymenium”)
1.
Fruiting body not mushroom-like, or if mushroom like, with teeth ……..…………Aphyllophorales
1. Fruiting body mushroom-like, usually with a stem and a cap, underside with gills, blunt folds, pores or
smooth
……….. …………………………..………………………………………………..2
2. Mushroom with true gills…………………………………………………………….3
3. Mushrooms easily breaking up into small pieces, or with latex when cut, on soil—
mycorrhizal on roots of trees, white to buff spore print ………………………………..Russulales
3 Not as above, on various substrates including ground, wood, dung, grass, and others.
Spore print white, pink, or brown. Most mushrooms with gills key out here!!!…… Agaricales
2. Mushroom without true gills. May have blunt folds or pores………………………………….4
4. Mushroom smooth on the underside or with blunt ridges……...Cantharellales
4. Mushroom with pores………………………………………
5. Pores peeling from the cap, usually on the ground …………...Boletales
5. Pores not peeling from the cap, on wood or on the ground from buried
wood…Aphyllophorales (polypores)
Key to families of the Aphyllophorales (literally “not bearing gills”)
The key uses macroscopic characteristics to key out to Friesian family
1. Fruiting body turns black in 3% KOH (micr. with setae, no clamps )……….….
……Hymenochaetaceae
1. Fruiting body does not turn black in KOH (micr. characteristics otherwise)…….……..…..……………2
2. Hymenophore with pores ……………………………………………………… Polyporaceae s.l.
2. Hymenophore smooth or with teeth or upright branches
…………………………….…………………3
3. Hymenophore smooth………………………………………Corticiaceae and Thelephoraceae
3. Hymenophore with teeth or upright branches………………………………………………….4
4. Hymenophore with downward pointing teeth…………………….…………..Hydnaceae
4. Hymenophore with upright branches………………………………………..Clavariaceae
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Key to families of the Agaricales (literally “mushroom forming organisms”)
1. Spore print white to off-white……………………………………………………………………….2
2. Gills free from stipe ………………………………………………………………………...3
3. Mycorrhizal, often with a volva and/ or annulus………….………….Amanitaceae
3a. Saprophytic, never with volva, usually with annulus ..white spored Agaricaceae
(Lepiotaceae)
2. Gills attached to stipe, decurrent, or stipe absent ……………………………..…………….4
4. Fruiting body with a very brittle waxy consistency, always on the
ground…Hygrophoraceae
4. Fruiting body various consistency on various substrates …………..Tricholomataceae
1. Spore print some shade of brown or pink……………….……………………………..……………5
5. Spore print pink……………………………………………………………………………6
6. Gills attached, usually on the ground, (micr. spores angular) ………Rhodophyllaceae
6. Gills free, on wood (micr. Spores regular in shape, smooth) ….………...Pluteaceae
5. Spore print some shade of brown…………………………………………………………..7
7. Spore print orange brown to rusty brown ……………………………….…..……8
8. Spore print rusty brown to earth brown, gills free, on soil, small and relatively rare
…………………………………………………………………………...Bolbitiaceae
8. Spore print orange-rusty-yellow/brown, gills attached ..……Cortinariaceae
7. Spore print brown-black, chocolate brown, or violaceous brown ……………….……9
9. Spore print brown-black or black ……………………………………………….10
10. Spore print brown-black, gills attached, on wood or on ground
……………………………………………………..………….Coprinaceae
10. Spore print black, gills decurrent.…………..…………...Gomphidiaceae
9. Spore print some other shade of brown …………………………………11
11. Spore print violaceous brown to rusty brown; gills attached; usually
saprophytes on wood………………………………………..Strophariaceae
11. Spore print chocolate brown; free gills; on soil or debris ..Agaricaceae
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Key to orders of the Gasteromycetes (literally “stomach fungi”)
1. Fruiting body more or less spherical at maturity, with or without star-like appendages…………………2
2. Outer covering of fruiting body thick and hard…………………..………… Sclerodermatales
2. Outer covering of fruiting body papery thin ……….…………………………….Lycoperdales
1a. Fruiting body some other shape …………………………………………………………………………..3
3. Fruiting body larger, elongated, usually foul smelling, often phalloid …………………..Phallales
3. Fruiting body a small nest shaped structure, with “eggs” , no smell …………..……….…………4
4. Fruiting body larger than 2 mm with more than one “egg” at maturity…………………… Nidulariales
4. Fruiting body smaller than 2 mm with just one “egg” at maturity ………………Sphaerobolomycetales
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Exercise 6. Ascomycota
The Ascomycota includes organisms of great economic importance, both beneficial and
destructive. The mycelium of most members is comprised of septate hyphae, the walls of
which contain a large proportion of chitin. The septa are not complete; rather there is a septal
pore in the center, the cytoplasm between adjacent cells being continuous through this pore.
Asexual reproduction within the group may be accomplished by fission, budding,
fragmentation, arthroconidia, chlamydospores, or conidia, depending upon the species and
the environmental conditions. The asexual stage is often referred to as the anamorph or
imperfect stage. Many fungi with distinct affinities for the various ascomycete groups are
known only by their anamorph stage and are usually considered members of the
deuteromycetes. Because of their relationship to known ascomycetes, some of these species
will be considered in Ex. 6 instead of Ex. 7.
The one feature that distinguishes this group from all other fungi is the form of the
teleomorph or perfect stage. It invariably involves the development of an ascus, a sac-like
structure, which encloses ascospores, the meiotic products of reproduction. The asci of most
forms develop within a fruiting body known as an ascocarp or ascoma (pl. ascomata). The
manner in which the asci are borne is the main feature segregating the different classes of
ascomycetes. There is considerable controversy with regard to the classes of Ascomycota. It
would be a gross understatement to say the classification scheme is in a state of flux. We will
use a modification of the scheme outlined in your text. This taxonomic scheme is based on
the morphology of the ascocarp, which may or may not have evolutionary significance, as
much convergent evolution seems to have taken place. Your textbook does not even use class
names in the Ascomycota.
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I. Hemiascomycetes
Members in this class do not have ascocarps; their asci arise individually on the vegetative
thallus. They are believed to be either very primitive ascomycetes or degenerate forms. The
group includes the yeasts and yeast-like organisms (simple single-celled forms), a few
mycelial saprophytes growing on plant exudates, and the fungi that incite leaf-curl diseases on
various plant hosts.
A. Saccharomycetales
1. Asexual reproduction in this order may occur by disarticulation (Endomyces), or budding
(Saccharomyces, Endomycopsis, Candida). Some species produce a pseudomycelium,
which superficially resemble hyphae—however the cells act independently from one
another and not as a colony.
Cultures of 1 or more of the following genera will be available to observe the vegetative
structures and types of asexual reproduction.


Endomyces and Endomycopsis (place coverslip on culture; examine hyphae at edge of
colony)
Saccharomyces (mount in phloxine)
2. Sexual reproduction occurs through cell fusion followed by nuclear fusion and subsequent
meiosis producing ascospores within an ascus.
• A photograph illustrating the asci and ascospores of Saccharomyces cerevisiae, the
bakers' and brewers' yeast, will be on demonstration. What serves as the ascus in this
species? Since ascospores are haploid meiotic products, what would you conclude to be
the ploidy of the common vegetative cell?
• Observe the following stages of sexual reproduction using cultures and/or prepared slides
of Schizosaccharomyces: cell fusion, asci, ascospores.
Archiascomycetes
A. Schizosaccharomycetales
Fission yeasts. Asexual reproduction in this order may occur by simple fission

observe cultures of Schizosaccharomyces octosporus (mount in phloxine)
B. Taphrinales
All of the members in this order are parasitic on vascular plants, often inducing bizarre
malformations of diseased tissues and organs. In their natural habitats, they have a true
mycelium comprised of binucleate cells. However, on artificial media, they have a yeast-like
growth, multiplying by budding. Asci arise from binucleate cells (proasci) that proliferate on
the surface of the host and resemble chlamydospores. Eight ascospores are delimited; but in
some species these spores reproduce by budding within the ascus, resulting in a large number
of secondary ascospores.
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•
Examine habit material of several hosts infected by different species of Taphrina. Note the
organs invaded and the type and extent of malformation(s) induced in the host.
•
Taphrina deformans is the most important species economically, inciting the destructive
"leaf curl" of peach trees. Study prepared slides showing sections through diseased
leaves. Note the increased thickness of the leaf wherever the parasite is present.
Compare healthy and diseased portions of the leaf to determine what pathological changes
have occurred in response to the parasite. Is there any evidence of hypertrophy
(uncontrolled cellular enlargement)? Of hyperplasia (uncontrolled cellular division)? The
mycelium may be difficult to observe in the mesophyll.
Looking along the leaf surface, identify the layer of proasci from which asci will develop,
and mature asci with ascospores. How many ascospores are delimited? Is there any
evidence of ascospore budding?
•
Compare the asci of T. deformans with those of T. johansonii and T. caerulescens, which
are depicted in illustrations on demonstration.
•
Also study demonstrations that will illustrate cytological details not observable on the
slides.
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II. Plectomycetes
This class undoubtedly is not a natural assemblage of fungi, but more research is required
before a better system can be proposed. As presently conceived, the features distinguishing
this group from all other fungi are the development of more or less globose asci scattered in
the ascocarp (Eurotiales) or broadly clavate asci that form a single basal layer (Erysiphales).
A. Eurotiales (Plectascales, Aspergillales)
Gymnoascaceae (incompletely closed ascocarps)
Eurotiaceae (closed ascocarps or cleistothecia)
Arthrodermataceae
The ascocarp of most forms in this order is a cleistothecium (pl. cleistothecia), which is a
completely closed structure. In most cases, the fructification has a firm, sharply defined
peridium (wall), although in some forms (the Gymnoascaceae) there is only a loose weft of
hyphae surrounding the clustered asci (in one genus there is no sterile envelope). Many
members have conspicuous conidial stages.
1. Gymnoascaceae
•
Examine young cultures of Byssochlamys nivea by removing the cover and placing the
culture on the microscope stage. Note the long chains of conidia. Place a cover glass on
the culture and determine how they are borne on the mycelium; i.e., determine whether or
not there are specialized conidiophores. Also look for thick-walled, pear-shaped
chlamydospores, which are somewhat larger than the conidia and are borne singly at the
tips of short side branches. This illustrates that some fungi produce more than one type of
asexual spore.
The larger hyphae of this species have a distinctive form and are called racquet
hyphae--one end of the cell is somewhat swollen. There are some members of the
deuteromycetes having similar hyphae which, along with other features, leads some
mycologists to believe these species may be related to one another. Many of the fungi in
this group are dermatophytes, that is, they get their nutrition from the hide, hair, nails,
horns, or skin of animals, including humans. The presence of racquet hyphae is often
used as a preliminary taxonomic character to identify the fungus as a prelude to treatment
of the disease.
•
Demonstration material illustrating several human diseases caused by these
dermatophytes (Epidermophyton, Trichophyton and Microsporum) may be on display.
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2. Eurotiaceae (Aspergillaceae)
Members of this family are among the most widely distributed in nature. Most of them have
well-formed cleistothecia with well-defined, often dense, peridia. Consequently, the asci can
not be seen unless the ascocarp is broken open. The cleistothecia in these species are
always minute structures, usually less than 1 mm in diameter. A large number of species in
the group, belonging to the genera Eurotium, Sartorya and Emericella, have an Aspergillus
anamorph (conidial) stage whereas another large group, belonging to the genera Eupenicillium
(Carpenteles) and Talaromyces, have a Penicillium anamorph.
Many species in this group are of considerable economic importance, especially members of
Penicillium and Aspergillus. The antibiotic penicillin is produced by Penicillium notatum and P.
chrysogenum. Blue cheese is ripened primarily through the efforts of P. roqueforti, while P.
camemberti plays a similar role in camembert and brie cheeses. Several Aspergillus species
are used in industry, most notably A. niger which is used to produce citric acid. Potent
mycotoxins, including aflatoxin, are produced by Aspergillus flavus.
1. See the demonstration material on the development of penicillin. Some of this work
occurred at the University of Wisconsin during the 1940's.
2. Sexual reproduction in Eurotiaceae
•
Examine a plate culture of Eurotium chevalieri under a dissecting microscope or with the
lower powers of a compound microscope. You should see masses of small, yellow
spheres--these are the cleistothecia. Scrape off some of the most mature ones (where
would they occur in the streak?) and mount them in a drop of water on a slide. While
looking at them under the microscope, crush them by applying pressure to the cover glass
with a pencil eraser or your fingernail until you see the asci released. Observe them with
high magnification and find mature asci containing ascospores.
•
Prepared slides showing vertical sections through a similar culture (Eurotium sp.) will also
be available for examination. Note the distribution of the asci in the ascocarp and compare
the asci with those you observed from living material.
•
Cultures of Emericella nidulans, with huge black cleistothecia, may also be available.
3. The asexual spores of members of this group (as in almost all Ascomycota) are known as
conidia. They are usually borne at the tips of hyphae and therefore are exposed to the
environment. The above slides of Eurotium will also illustrate the Aspergillus anamorph
(imperfect stage), which imparted the greenish coloration in the cultures you looked at
above. The conidiophores, on which the conidia are borne, have a very distinctive
structure—compare it with the Penicillium anamorph.
•
Two morphological types of Penicillium are generally recognized, with monoverticillate and
asymmetric conidiophores, as shown on the two sets of slides provided. How can you
distinguish between these two types?
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•
Tube cultures of a number of species of Aspergillus and Penicillium will be on display to
give you some idea of the range of cultural characteristics displayed by each genus. The
collection will include some species that produce ascocarps and others for which no
teleomorph is known.
3. Arthrodermataceae
The ascocarp of most forms in this order is a gymnothecium (pl. gymnothecia). It is similar in
shape to a cleistothecium, which is a completely closed structure. In most cases, the
cleistothecium has a firm, sharply defined peridium, but in a gymnothecium there is only a
loose weft of hyphae surrounding the clustered asci. Most members have conspicuous
conidial stages.
They include the Arthroderma, the teleomorphs of most of the dermatophyte fungi
(anamorphs Epidermophyton, Microsporum, and Trichophyton) and Ajellomyces, the
teleomorph of some systemic human pathogens (anamorphs Histoplasma capsulatum and
Blastomyces dermatitidis.) We cover these groups more extensively in Medical Mycology.

Some demonstration materials will be on display.
B. Elaphomycetales
This order has only one genus, Elaphomyces, the hart's or deer truffle. Ascocarps are formed
underground by these ectomycorrhizal fungi. They are very common but rarely seen.
C. Erysiphales
As considered here, all forms are assigned to a single family, the Erysiphaceae. Your
textbook is unsure of where to place them taxonomically, considering them in Chapter 15—
Other filamentous ascomycetes. We will consider them in the Plectomycetes.
The Erysiphales have large, globose or broadly clavate, persistent, asci that form a single
basal layer in the cleistothecium. Since none of them have been successfully cultured on
artificial media they are considered obligate parasites, inciting diseases known as powdery
mildews. Some species are able to parasitize a wide range of hosts whereas others are
restricted to a single host species. The disease may be extremely destructive (e.g. on grape,
roses, apples, cucurbits) or may have no apparent effect on the host (e.g. lilac).
1. Examine habit material of various hosts parasitized by a variety of powdery mildews, noting
the superficial mycelium, the small, dark cleistothecia, and any deformation, necrosis
and/or chlorosis of the host.
2. Erysiphe graminis
•
Examine prepared slides showing cross-sections of grass leaves infected with Erysiphe
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graminis. Where are the vegetative hyphae most profuse? Locate haustoria in the
epidermal cells. What is their general form? Examine diagrams. The asexual
reproductive phase (Oidium anamorph) will also be apparent in these slides. Do there
appear to be specialized conidiophores? Note how the conidia are borne on the
conidiophore and ascertain how they are produced. Would you say that all of the conidia
produced by a conidiophore are most likely the same genotype? Why?
A typical example of ascus development occurs in Erysiphe graminis. Sexual reproduction
is initiated when a nucleus passes from an antheridium (the male gametangium) into the
female gametangium called an ascogonium, but nuclear fusion is delayed. One to several
"fertile" hyphae (ascogenous hyphae) grow out from the ascogonium, each cell being
binucleate (one nucleus derived from the antheridium, the other from the ascogonium).
Some of these cells enlarge and form the asci. While the asci are developing, sterile
hyphae proliferate around the cluster of ascogenous hyphae, forming a "shell" (peridium)
around the fruiting body. The ascocarp is spherical without any natural opening to the
external environment--this is known as a cleistothecium and is typically found in the
Plectomycetes.
•
Looking at asci sectioned longitudinally on the prepared slides, ascertain exactly what
happens during ascospore development, paying particular attention to the nuclear behavior
and the manner in which the ascospores are delimited. The protoplasm that is not
included within the ascospores is called the epiplasm.
•
From the dried specimens provided, scrape off a few ascocarps (they are brown or black in
color) with a moist half-spear and mount them in water on a slide. While looking at them in
the scope, gently press on the coverslip to crack the peridium and expose the asci. Are the
number of asci similar to the number you could see on the prepared slides? Are
ascospores visible?
3. The number of asci within an ascocarp, as well as the form of characteristic and
conspicuous external appendages arising from the ascocarp wall, are used for generic
segregation in the group. A number of unnamed specimens will be provided in the
laboratory. Using the key appended to this exercise, identify the six genera and
demonstrate to the instructor your evidence for these identifications. Place a specimen
under a stereoscopic microscope, scrape off a few well developed (dark colored)
ascocarps, and mount them in a drop of mounting fluid. Return the habit material to the
appropriate container. To determine the number of asci, break open the ascocarps by
pressing on the cover glass with a probe while looking through the microscope with low
magnification.
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A KEY TO THE COMMON GENERA OF THE ERYSIPHACEAE*
A. Appendages unbranched, typically hyaline, coiled or hooked at the tip…UNCINULA
B. Appendages stiff-looking, sharp-pointed, with a bulbous swelling at the base
…………………………………………………………………………….. PHYLLACTINIA
C. Appendages hyaline or dark, dichotomously branched at the tip
1. One ascus per cleistothecium .....................................................PODOSPHAERA
2. Several asci per cleistothecium ................................................. MICROSPHAERA
D. Appendages more or less flexuous, often dark, simple or irregularly branched, without tip
peculiarities or basal swelling
1. One ascus per cleistothecium ................................................... SPHAEROTHECA
2. Several asci per cleistothecium ............................................................ ERYSIPHE
*A total of about 20 genera of powdery mildew fungi have been described. Only six of these
are known to occur in the United States.
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III. Pyrenomycetes
This is the largest class in the Ascomycota and is characterized by development of ostiolate
perithecia containing unitunicate (single walled) asci that form a basal hymenium. There is
considerable confusion and controversy over the classification of this group, some authorities
recognizing a single order with several families, while others recognize several orders. Since
the taxonomy is in a great state of flux, we will use a simpler system based on the color of the
ascocarps, the form of the ascospores, and the position of the asci in the ascocarp.
The taxonomic scheme your book uses is
The scheme we will use is:
Hypocreales
Hypocreaceae
Nectriaceae
Clavicipitaceae
Sphaeriales
Microascales and Ophiostomatales
Hypocreales
Melanosporales
Clavicipitales
Microascales
Microascaceae
Ceratocystidaceae
Phyllachorales
Ophiostomatales
Diaporthales
Xylariales
Sordariales
Sordariaceae
Chaetomiaceae
Coniochaetaceae
Lasiosphaeriaceae
Meliolaes
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A. Sphaeriales
The fungi placed in this order usually have dark colored (brown or black) perithecia whose
walls are brittle or leathery in texture. Paraphyses (sterile hyphae that arise from the tissue at
the base of the asci) may be interspersed among the asci while periphyses (also sterile
hyphae) may be found in the ostiolar canal. The perithecia may be scattered over the
mycelium, or may be confined in or on a stroma (a dense mat or pad of sterile hyphae).
1. Neurospora is one of the best known genera, certain species having been used extensively
in cytological, physiological, and genetic investigations. While some species of
Neurospora are heterothallic (self-incompatible), both species we will examine are
homothallic.
•
Under a stereoscope or the low power objective of your scope, examine plate cultures of
Neurospora tetrasperma. (Do not open the plates if the pink-colored conidia forming
anamorph Monilia is present. It is a terrible lab contaminant.) What is the general form of
the ascocarp (you may want to roll some around to get a better perspective)? With higher
powers, examine the substrate surface. Can you observe anything other than mycelium or
ascocarps? Examine the underside of the petri dish lid. Is anything detectable? Remove
a few ascocarps, crush them in water on a slide, and observe the asci. How many
ascospores in each ascus? Is there any variability?
•
Prepared slides of N. tetrasperma having vertical sections through the ascocarps will be
available for supplementary study. Be sure to locate a perfectly median section. In what
way(s) does this ascocarp differ from those of Erysiphe? Fruiting bodies of this type are
called perithecia and are typically formed by members of the Pyrenomycetes. Observe the
ostiolar canal, paraphyses and periphyses. What do you think might be the functions of
the paraphyses and periphyses?
•
Also examine demonstration material related to this genus.
•
Examine cultures of Neurospora terricola as you did with Neurospora tetrasperma. The
number of ascospores per ascus here is more typical of the ascomycetes. How many are
there?
2. Chaetomium has perithecia that are covered with long hairs. Examine cultures and make
slide mounts to ascertain their form. Crush the perithecia on the slide to find asci. In this
genus the asci disappear shortly after the ascospores mature. Note the characteristic
lemon shape of the ascospores.
3. Some ascomycetes are homothallic while others are heterothallic. You will be supplied a
plate culture of a species of Sordaria, a pyrenomycete, for which you are to determine
whether it is homothallic or heterothallic by mating it with known tester strains. The latter
will be supplied in labeled culture dishes along with sterile nutrient agar plates. Before
starting, design an inoculation pattern that will tell you without doubt what form your culture
is. Remember, negative results are of little use--positive results (i.e., perithecia) must be
obtained to make a definite conclusion. For inoculating the plates, use small agar blocks
containing hyphal fragments. Be sure to label the position of each inoculum on the bottom
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of the plate (why not the top?), and use sterile technique. Keep your unknown culture until
your results (culture plate) and conclusion are accepted by the instructor. Incubate the
cultures in your locker drawer and check them periodically. Use the following questions to
guide your observations when asci are produced:
a) Is Sordaria fimicola homothallic or heterothallic?
b) Why are there usually eight ascospores per asci in Ascomycetes?
c) Why do some asci contain ascospores all the same color and others have spores of
two colors?
d) In the asci with two colors of ascospores, what arrangements of the two spore types do
you find?
e) What is the relationship of crossing over in meiosis to the arrangement of ascospores in
an ascus?
4. Cryphonectria parasitica (=Endothia parasitica) incites the destructive chestnut blight
disease. The long-necked perithecia develop in an orange-colored stroma that breaks
through the bark of the tree. Examine the habit material and slide on demonstration. The
native American chestnut has been virtually wiped out by this pathogen, although there is a
large grove of 160 acres of about three-thousand chestnut trees near West Salem,
Wisconsin that has been only recently infected.
5. Groups of perithecia, or large numbers of individual asci may be borne on (or embedded
in) large, macroscopic fruiting structures, usually a carbonaceous stroma. Preserved
and/or dried habit specimens will be provided in the laboratory as well as prepared slides.
(your textbook places these in a separate order, the Xylariales)
•
Examine habit specimens of Xylaria and Daldinia and note the form of the stroma. These
are very common genera in Wisconsin.
•
Study prepared slides of at least one genus, noting where the perithecia are borne and in
which plane the fruiting bodies were sectioned. Locate the asci on the slide and determine
where they would be borne on the habit specimens. Do the concentric zones in the
Daldinia stroma represent annual layers? What is the basis for your conclusions?
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B. Microascales and Ophiostomatales
Members in these orders produce their asci in "beaked" ascocarps (perithecia) which have
definite ostioles (openings) through which the ascospores are discharged. Classification of
this group is in question because some mycologists consider the presence of a perithecium
(characteristic of Pyrenomycetes) to be more important than the occurrence of scattered asci
within the ascocarp (characteristic of Plectomycetes). Current thinking places them in the
Pyrenomycetes.
These orders includes a number of economically important forms; e.g. the incitants of oak wilt
and Dutch elm diseases, species responsible for "blue stains" of lumber, and others.
The two orders are separated on slim basis (mainly the form of the conidiophore), so we will
not consider them separately. The two genera we will study were in fact considered to be in
the same genus until recently.
•
In the laboratory we will have habit material showing an undetermined species of
Ceratocystis growing and fruiting on rough-cut lumber to illustrate the type of ascocarps
found in this group. Note the extremely long necks by which the ascospores are
discharged through the ostiole at the apex. Note also the bluish coloration in the wood,
which greatly reduces its commercial value, mainly because it indicates decreased strength
of the wood.
Two well-known and widespread tree diseases in North America are incited by members of
this group: Dutch elm disease by Ophiostoma ulmi (formerly Ceratocystis ulmi) and oak wilt by
Ceratocystis fagacearum. It is known that O. ulmi was introduced on the east coast about
1930 on a shipment of elm logs from Europe via Canada. Since that time it has spread
westward causing extremely heavy losses. The disease is spread by two species of bark
beetles that develop under the bark of elms and subsequently feed on the young twigs.
•
Sections of elm branches with larval galleries will be on display. The fungus grows and
sporulates in these galleries. The beetle larvae pupate in the tunnels and eventually
transform into adult beetles. As the adults crawl through the tunnels to emerge to feed on
young elm twigs, their bodies are brushed by spores of the fungus. When the beetles
chew through the bark of the young twigs, some of the spores are deposited in the wound.
After germination, the mycelium grows into the xylem, inhibiting translocation. The tree
eventually wilts and dies.
•
If cultures of the anamorph stage of O. ulmi (Graphium) are available, examine them with a
dissecting microscope. What type of sporulating structures do you find? Do the spore
masses appear to be adapted for dispersal by beetles? In what way? Preserved
specimens of adult bark beetles will also be on display.
•
Observe any other demonstration exhibits concerning Ophiostoma or Ceratocystis.
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C. Hypocreales
Fungi that have bright-colored (orange, yellow, blue, etc.), ostiolate perithecia with soft or waxy
walls are assigned to this order. Apical paraphyses, sterile hyphae which arise from the roof
of the ascocarp and grow downward between the asci, instead of paraphyses, may be
present. Other than the color and texture of the ascocarp, they have many features in
common with the Sphaeriales. The perithecia may be scattered over the mycelium or be
borne in, or on, a stroma.
1. Neocosmospora exhibits features typical of this order. Examine plate cultures, mount a
few ascocarps in a drop of water, and crack them open to determine the character of the
asci and ascospores.
2. Nectria is a stromatic form commonly found on living and dead branches of woody plants.
Reddish-orange perithecia are clustered on small, cushion-like stromata that break through
the bark during maturation. Examine habit specimens and prepared slides showing vertical
sections through ascocarps and stroma. Can you detect the apical paraphyses among the
asci? What is the character of the ascospores? Conidiophores and conidia were borne on
these stromata prior to the differentiation of the perithecia.
3. Examine habit specimens of a basidiomycete mushroom fruiting body parasitized by
Hypomyces, which produces a bright orange stroma on the surface of the host. What
abnormalities can you detect in the host? Examine a specimen under a stereoscope. Is
there any evidence of perithecia in the stroma? After looking at prepared slides of crushed
mounts of the stroma, and a demonstration slide showing a section through the stroma,
determine the location of the hymenium.
4. Examine demonstration material relating to Gibberella, (anamorph= Fusarium) species of
which cause "foolish seedling disease" of rice, red ear rot of corn and other grain diseases.
D. Clavicipitales
Members of this order produce their perithecia within a well-developed stroma. Each of the
elongate asci has a thick cap perforated by a pore through which the ascospores escape. The
ascospores are thread-like and in many species become multicellular at maturity.
1. Claviceps purpurea parasitizes a variety of grasses, including cultivated forms such as
wheat, barley and rye, inciting the disease known as ergot. The fungus invades the
ovaries, developing dark, purple-brown, elongated sclerotia that project out like spurs from
the host plant (the French word for spur is ergot). Prior to the development of this large
sclerotium, the mycelium forms a minute stroma that bears conidia (Sphacelia). The
sclerotium develops directly beneath the stroma and pushes it out of the host fruiting head.
Upon maturation, the sclerotium falls to the ground and overwinters. In spring, stalked
stromata (the teleomorph stage) emerge from the sclerotium. The stalks are light purple
and the fertile stromatal heads are pinkish in color.
•
Examine infected heads of rye, quack grass, orchard grass, and any other habit material,
noting the general form of the sclerotia and their general location in the head. Look for the
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small conidial stroma that may persist at their apices (they are easily broken off, so all
sclerotia may not have them).
•
Study prepared slides having sections of the stroma. What is its general configuration?
Where are the conidiophores borne and what is their shape (i.e. simple, branched, long,
short, etc.)?
•
Next, examine habit material of "germinated" sclerotia, noting the general form.
•
Then study prepared slides having sections through the fertile heads. How would you
characterize the structure (be sure to search for median sections) and where would you
place the fungus in the taxonomic scheme? Examine any other demonstration material
that may be available.
The sclerotia contain a number of poisonous alkaloids, which, if ingested, cause a
condition known as ergotism or "St. Anthony's fire," which can be deadly in animals,
including humans. With modern milling methods, ergotism in humans has been greatly
reduced, but it is more common in domestic animals that graze on infected grasses or in
fields in which the sclerotia are lying. The sclerotia are used for the preparation of a
powerful abortifacient, which is also used to control hemorrhaging during childbirth. A drug
used in the treatment of migraine headaches is also produced. The sclerotia were also
one of the original sources of LSD.
2. Cordyceps also illustrates the salient features of the group. Species of this genus are
parasitic on insects, spiders, and fruiting bodies of certain fungi.
•
Examine habit material of Cordyceps parasitizing arthropods. In nature these are usually
inconspicuous, but can be very common in certain areas.
•
A specimen of Cordyceps parasitizing a hypogeous Elaphomyces ascocarp will be on
demonstration. The large perithecial stromata are differentiated into a stout, sterile stalk
and a conical, fertile head.
•
In a prepared slide, locate a median section of a perithecium and note its characteristic
features, especially the thread-like ascospores.
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IV. Discomycetes
The ascocarps in this class are apothecia, which typically have the asci exposed at maturity,
the exception being the Tuberales.
Taxonomic scheme in textbook
Medeolariales
Rhytismales
Ostropales
Cyttariales
Helotiales
Leotiaceae
Sclerotiniaceae
Dermateaceae
Geoglossaceae
Orbiliaceae
Gyalectales
Lecanorales
Caliciales
Pezizales
Pezizaceae
Tuberaceae
Terfeziaceae
Elaphomycetaceae
Glaziellaceae
Otidiaceae
Sarcoscyphaceae
Sarcosomataceae
Thelobolaceae
Ascobolaceae
Pyronemataceae
Ascodesmidiaceae
Morchellaceae
Helvellaceae
Taxonomic scheme we will use:
Helotiales
inoperculate
epigeous
rounded ascospores
Phacidiales
inoperculate
epigeous
threadlike ascospores
Pezizales
operculate
epigeous
various
Tuberales
inoperculate
hypogeous
usually spiny ascospores
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A. Helotiales
This is the "core" group of the inoperculate Discomycetes which develop cup-, club-, or
disc-shaped ascocarps above ground. The asci release their spores through an apical,
circular pore.
1. Habit material of the apothecia of Geoglossum, Spathularia, Mitrula and Leotia will be
available for your inspection. Study sections of one or more of these forms and determine
where the hymenium is located and the character of the ascospores.
2. Chlorociboria (Chlorosplenium) is also a distinctive member of the order. C. aeruginosum
grows as a saprophyte on dead trunks and branches on the forest floor. The hyphae
contain a green pigment (not chlorophyll!) that imparts a greenish coloration to the
substrate. The apothecia are stalked, often asymmetrical, cup-shaped to funnel-form at
the apex, where the asci are borne. Examine displays on demonstration. Please do not
touch the apothecia--they are very fragile when dried.
B. Phacidiales
This is a somewhat heterogeneous group of uncertain affinities.
1. Examine habit specimens of Hypodermella ampla on jack pine needles and Lophodermium
on pine needles. These two genera incite "needle cast" diseases of conifers.
2. The general structure in this group is exemplified by Rhytisma, which incites "tar spot"
diseases of various angiosperms. The small, dark fruiting bodies are stromata within which
apothecia develop.
•
•
Examine the conspicuous black stromata of Rhytisma acerinum as they appear on living
leaves of maple and also as they appear on overwintered leaves. In the latter, note the
radiating ridges which mark the location of elongated apothecia developing beneath. Habit
material of other species may also be on demonstration.
Study prepared slides having sections through mature ascocarps of Rhytisma andromedae
growing on leaves of bog rosemary. The lower portion of the stroma is stained green while
the upper portion covering the apothecia is dark. What is the character of the ascospores?
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C. Pezizales
This is a large group of operculate Discomycetes; i.e. the asci have a hinged lid-like structure,
an operculum, at the apex (less frequently a longitudinal slit) which permits escape of the
ascospores. Undoubtedly all of you have seen representatives of this order in nature.
•
To formulate some idea of the variation in the character of the apothecia, examine the
habit material on display. The collection will include some of the following common
genera:
Gyromitra
Helvella
Morchella
Peziza
Scutellinia
Urnula
Verpa
Sarcoscypha
•
Study prepared slides having apothecial sections of Scutellinia (= Lachnea) and/or Peziza
to determine where the hymenium is located. Note the paraphyses intermixed with the
asci.
•
Slides showing sections through the pileus (cap) of Morchella will also be available for
examination. Examine the direction the ascus tips are pointing. “Why” would they do this?
•
Examine water mounts of hymenial fragments from preserved specimens of Morchella or
use a fresh specimen from the order if available. The fruiting bodies of the genus (known
as the morels) are highly prized by those who like to eat mushrooms (mycophagists).
While the vegetative mycelium of the morel can be grown easily, it is quite difficult to get
them to fruit in culture.
D. Tuberales
This is a relatively small order whose ascocarps are usually subterranean. The European
forms include the well known "truffles," which are highly prized by gourmets.They range in size
from a few millimeters to several centimeters in diameter. They are more or less globose,
often fleshy structures with a smooth to irregular surface. The asci, which are elongate to
globose in form, may line one or more chambers that open to the outside. However, in many
forms, the asci appear to be scattered and embedded within a closed ascocarp, which most
mycologists interpret as a highly modified apothecium. Most of the species have been found
in Europe or the Pacific coast states.
•
Ascocarps of Tuber will be on display. Also examine sections on prepared slides, noting
the distribution and form of the asci. Do they have “typical” 8-spored asci?
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V. Loculoascomycetes
Fungi that have double-walled (bitunicate) asci borne in locules (cavities) in a stroma are
placed in this group. The ascocarp, then, can be called an ascostroma. Several orders are
recognized, but you will have an opportunity to see a representative of only one of them.
A. Pleosporales
In this order, the fructifications usually look remarkably like true perithecia, and often are called
pseudothecia. The asci are usually clavate and intermixed with pseudoparaphyses. At
maturity, each locule in the ascostroma usually has a well-defined ostiole through which the
ascospores escape.
A common Wisconsin species is Apiosporina morbosa (= Dibotryon morbosum) which incites
the "black knot" disease of plum and cherry. The mycelium is intercellular in the twigs of the
host, inducing remarkable aberrations in the differentiation of host tissues. About a year after
infection takes place, an extensive stroma breaks through the outer bark (about mid-May in
southern Wisconsin); and this is soon covered with a greenish brown velvety coating of
conidiophores and conidia. During the summer the stroma surface becomes papillate, each
papilla being a young pseudothecium. The asci develop slowly in the locules, and do not
mature until the following spring. Typically, the roof of the locule breaks off permitting escape
of the ascospores.
•
Examine habit specimens of black knot stromata, macroscopically and with a hand lens or
stereoscopic microscope. What would you say were the general characteristics of the
galls? Be sure to note the closely crowded papillae on the surface.
•
Before looking at diseased tissue microscopically, examine prepared slides of sections of a
normal two-year-old twig. You should be able to distinguish pith, secondary xylem,
secondary phloem, remains of the cortex and the beginning of an outer corky layer.
•
After seeing the normal pattern of tissue differentiation, examine sections through young
galls showing the asexual reproductive phase. What effects has the fungus had on the
pattern of host differentiation? Locate the stroma (it will have a greenish stain) on the
surface of the twig. Can you detect any intercellular hyphae below it? Are the stromatic
hyphae different than the vegetative hyphae? Note the form of the conidiophores and
conidia on the stromatal surface.
•
Next study sections through an older stroma that has pseudothecia. How would you
describe the general structure of a pseudothecium, its asci and ascospores? Are
pseudoparaphyses apparent?
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Exercise 7. The "deuteromycetes"
There are a large number of fungi for which there is no known "sexual" (teleomorph)
stage; i.e. only the "asexual" (anamorph) stage is known. Since most of them have conidial
stages similar to those found among the ascomycetes, it is believed that a majority have
ascomycetous affinities. A great many are probably ascomycetes whose perfect stages have
remained undetected. Indeed, every year mycologists report the discovery of the teleomorph
stages of some of these forms, in which case they are removed from the deuteromycetes,
renamed, and reclassified on the basis of the newly acquired information. Many of these
continue to be known by their anamorph names as well as their teleomorph names. Consult
the Dictionary of Fungi for synonyms. It seems likely that a large number of species never do
develop a teleomorph stage, perhaps having lost the ability in the course of evolution,
reproduction by conidia or other means having proved adequate for survival. There is some
controversey about whether "the deuteromycetes" are needed in classification schemes, since
the affinities of almost every fungus can be determined by morphological or molecular biology
methods. The short answer is that, philosophically, we do not need "the deuteromycetes," but
the classification scheme does serve many practical and historical purposes. The names are
also a key to the vast literature on these fungi.
The manner in which the deuteromycetes are classified, based on morphology of the
sporulating structures and morphology and color of the conidia, is admittedly an artificial one.
But there is need for some system to bring order into this vast assemblage of this second
largest group of fungi. We will not go into a great amount of detail about distinguishing the
species. Classification of deuteromycetes is extremely detailed, but it can be done with some
practice. We spend much more time on this in Medical Mycology, particularly in the lab.
Be sure to consult the demonstration materials and literature available in lab.
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I. Coelomycetes
This class includes all forms that produce conidia in pycnidia or acervuli, the former
mimicking perithecia and the latter being a more or less dense mat of hyphae bearing
conidiophores.
A. Sphaeropsidales
These are the pycnidial forms. Note the difference between a pycnidium (the asexual structure
discussed here) and a pycnium (the sexual structure found in many rusts, also known as a
spermagonium).
Septoria and Phoma are large genera of common parasites on angiosperms.

Examine habit material of both genera and note effects on the host tissue.

Examine pycnidia of Septoria under a stereoscopic microscope.

Observe prepared slides of Septoria or another member of this group to get some idea of
the general structure of pycnidia. What is their general form? Are the spores similar to
one another? How do the spores escape from the interior of the structure?

Cultures of Phoma may also be available for examination. Make a water mount of a few
pycnidia and crush to see the spores.
B. Melanconiales
This order encompasses those forms that produce conidiophores on acervuli.


Examine habit material of representative forms (e.g. Gloeosporium, Cylindrosporium,
Colletotrichum, Marssonina) under a stereoscopic microscope.
Prepared slides of Colletotrichum and/or Gloeosporium showing sections through acervuli
may be available for microscopic observations.

Cultures of Cryptomela and/or Pestalotia may be available for examination.
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II. Hyphomycetes
Like the previous class, these are mycelial forms, but the conidiophores and conidia, when
produced, are not associated with pycnidia or acervuli. (The class Blastomycetes, which we
will not examine, accommodates the yeast-like forms.) The way in which the conidiophores are
borne is the primary basis for segregation of the other three orders. The conidiophores are
aggregated as columnate synnemata (coremia) in the Stilbellales, on pad-shaped sporodochia
in the Tuberculariales, and scattered, mostly single and separate, in the Hyphomycetales.
Selected representatives will be provided for examination, as cultures, prepared slides
and/or demonstration material. The cultures should be observed with a stereoscopic
microscope and water mounts prepared for observation at higher magnifications. Note the
manner in which the conidiophores and conidia are borne.
A. Hyphomycetales (conidiophores scattered, mostly single)
•
Moniliaceae (hyaline or brightly colored hyphae and/or spores):
Aspergillus (slides)
Thielaviopsis (slides)
Fusarium (cult. demo. slide)
Trichothecium (cult.)
Penicillium (slides)
photos of Epidermophyton,
Trichophyton, and Microsporon
•
Dematiaceae (dark colored hyphae and/or spores):
Alternaria (cult.)
Cladosporium (habit)
Bipolaris (habit)
Polytrincium (slides, habit)
B. Stilbellales (synnemata or coremia)
Graphium (cult.)
Pennicillium claviforme (cult.)
C. Tuberculariales (sporodochia)
Fusarium (cult., demo. slide)
Tuberculina (habit)
Tubercularia (demo. slide, habit)
III. Mycelia Sterilia: Members of the Mycelia Sterilia do not produce any spores (except for
chlamydospores in some genera).
•
Observe the demonstration culture of Sclerotium.
IV. Many of the fungi you isolated in pure culture (see Ex. 3) are probably members of the
deuteromycetes. Compare your cultures and slides of sporulating structures to the
demonstration cultures. Identify at least one of them to species for your culture
collection.
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Exercise 8. Zygomycota
The Zygomyocota, the Chytridiomycota (Ex. 9), and the Oomycota are sometimes
known as the "lower" fungi because they are considered more primitive than the
Basidiomycota and Ascomycota. Their reproductive structures are generally smaller and less
complex (i.e. fewer types of cells are differentiated) and their vegetative hyphae do not have
septa, making their mycelia coenocytic. (Note that most mycologists no longer include the
Oomycota in the kingdom Fungi.)
The chief characteristic of members of the Zygomycota is the development of sexual
structures called zygospores, which arise from the fusion of two gametangia. A number of
species for which there is no known sexual stage are also placed in the Zygomycota--this is
because they exhibit other characteristics so typical of the group that they can be assigned
here with complete confidence. Included among these secondary features are the
development of characteristic sporangia or sporangiola and the complete absence of motile
cells. A number of them have evolved highly specialized mechanisms for spore dispersal,
which will be pointed out later.
Nutritionally, the group ranges all the way from saprophytes through weak facultative
parasites to specialized parasites of animals to obligate parasites of members of their own
group. One order of Zygomycetes, the Glomales (Now considered by many to be placed in
their own phylum, the Glomeromycota) is of considerable interest because it contains
mycorrhizal associates of a wide range of herbaceous and woody hosts, including many
important crop plants; see Ex. 11. Associations of Fungi with other Organisms. Two classes
are generally recognized, but there will be representatives of only the largest one, the
Zygomycetes, in the laboratory.
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I. Zygomycetes
A. Mucorales
The members of this order are chiefly saprophytic, living on such things as dung and decaying
plants and animals. However, there are a few facultative and obligate parasites in the group.
Most mucorales have a well-developed, coenocytic mycelium although solid septa may be laid
down at the bases of the reproductive structures and only infrequently in the vegetative
mycelium.
1. Asexual Reproduction
The "mucors" usually develop sporangia and sporangiospores when reproducing asexually.
The sporangia, borne on aerial sporangiophores, may be relatively large, containing many
spores; they may be small, containing only a few spores (in which case they are called a
sporangiolum instead of a sporangium); or they may have only a single spore, in which case
they are often referred to as a conidium (pl. conidia), even though they are not true conidia.
Cylindrical sporangia, called merosporangia, are produced by other genera.
The probable evolutionary sequence of spore development in this group can be studied. The
evolutionary trend in this group is from a sporangium containing many spores that are
dispersed as a unit (e.g. Pilobolus) to a sporangium containing many spores that are released
in small groups in mucous masses (Rhizopus, Zygorhynchus, Circinella) to a terminal
sporangium with mucous-encased spores and sporangiola containing a few spores
(Thamnidium) to no sporangia and many sporangiola containing a few spores
(Cunninghamella) to no sporangia and many single-spored sporangiola, each of which
behaves like a conidium (Mycotypha).

Cultures of representative genera will be available for you to observe this probable
evolutionary progression. Note also the coenocytic nature of the mycelium.

Observe the electron micrographs in Kerry O’Donnell’s book on demonstration.

Additional materials may be available for Pilobolus, a small, very common dung fungus
with amazing phototrophic and propulsionary abilities.
2. Sexual Reproduction
Sexual reproduction in this group is characterized by the fusion of the entire contents of two
multinucleate gametangia. The gametangia are more or less spherical multinucleate cells
borne as terminal swellings on the tips of short side branches called suspensors. When the
gametangia are formed in pairs, the walls between them dissolve and the contents mix,
forming a zygospore by the deposition of a thick wall.
It was in the Mucorales, of which Rhizopus is a member, that the phenomenon of sexual
incompatibility in fungi was discovered. Species that could produce zygospores on a single
mycelium were called homothallic; species that required two compatible thalli to form
zygospores were called heterothallic. Since the two compatible strains could not be
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distinguished morphologically, they were labeled "+" and "-". Both homothallic and
heterothallic species may occur in the same genus.
a. Examine prepared slides of Rhizopus to become familiar with the structures involved in this
type of sexual reproduction. The slides have both the asexual and sexual stages on them, so
don't confuse the two. The zygospores will be large, black, almost spherical structures, with a
warty surface, between two greenish stained suspensors, one of which may be quite inflated.
b. Heterothallic or Homothallic: Observe the control and experimental plates of several
species (Absidia, Mucro bacilliformis, Mucor hiemalis, Phycomyces, and/or Rhizopus) with
your dissecting and compound microscopes and look for evidence of sexual reproduction—the
production of zygospores. You may need to prepare slides for some of the species. Do not
confuse the zygospores with the asexual sporangiospores which will also be present on most
plates. You may open any of the plates which are not parafilmed.
Control Plates were inoculated with a spore suspension from only one kind of culture.
Experimental Plates:

If a homothallic species, plates were inoculated with spore suspensions from the same
culture (i.e. 1 & 2 are the same).

If a heterothallic species, plates were inoculated with spore suspensions from two
compatible mating types (i.e. 1 = + and 2 = - ).
1) Identify which species are homothallic and which are heterothallic.
2) Note the characteristic form of the zygospores for each species.
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B. Glomales (=Glomeromycota)
This order includes all of the known endomycorrhizal fungi -- those symbiotic fungi that
actually penetrate the root cells of many vascular plant species. They are known as the
vesicular-arbuscular (VA) mycorrhizae since they form conspicuous vesicles and/or
arbuscules, haustoria-like growths, within the root cells they inhabit. In addition, coarse
angular hyphae project into the soil surrounding the infected root, thereby increasing the
absorptive surface of the fungus-plant association. Endogone and Glomus are common
genera.

Observe the prepared slides showing root cross-sections with abundant arbuscules.

A second set of slides, whole stained mounts of roots, show the vescicles, large bulbous
hyphal swellings.

Compare demonstration material on endo- and ectomycorrhizae. We will observe these in
greater detail in a later lab.
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C. Entomophthorales
This order encompasses a much smaller number of species than the mucors. A majority of
them parasitize insects, although they have also been found as parasites on lower plants and
humans, and several colonize dung. The mycelium of these forms usually is not nearly as
extensive as that found in the mucors, and exhibits a strong tendency to form septa and then
fragment into hyphal bodies (arthroconidia), which may multiply by fission or budding and
eventually produce a conidiophore bearing a conidium at its tip. In some species, they may
serve as gametangia and fuse with one another to form zygospores. However,
chlamydospores (vegetative cells that have laid down a thick wall) are often the principal
resting structures.

Plate cultures of Basidiobolus ranarum may be available for microscopic study. Remove
the cover of the plate and place a cover glass near the margin of the colony. Observe
hyphae, gametangia in various stages of development (the youngest will be near the
margin), and mature zygospores. Note the diagnostic "beaks" associated with the
gametangia and zygospores. Also search for discharged conidia on the agar surface.

A demonstration slide showing an aphid parasitized by Entomophthora fumosa will be on
display. Note the abundance of hyphal bodies. Does this suggest a possible use for these
fungi in biocontrol of insects? What is their nuclear situation? You will also see conidia and
conidiophores. What is their nuclear condition?

Another demonstration slide will show aphids whose body cavities are completely filled with
chlamydospores of an unknown species of Entomophthora.

If available, examine cultures of Entomophthora coronata that have been in one-sided
illumination and note the pattern of spore dispersal. Remove the cover, invert it, and
observe discharged spores microscopically.
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Exercise 9: “Fungi” with motile cells
The Chytridiomycota, along with the Oomycota and Plasmodiophoromycota are placed by
some authors together in the division Mastigomycota because of the presence of motile cells
in the life cycles of these fungi. We will consider each of the three classes as separate
unrelated phyla -- in at least two kingdoms! All have a unicellular, non-septate filamentous or
plasmodial thallus and reproduce asexually by means of motile spores. Various methods of
sexual reproduction are employed.
I. Chytridiomycota
The one characteristic that distinguishes members of this class from all other fungi is the
production of motile cells (zoospores and/or gametes) which have a single, posterior, whiplash
flagellum. The most simple forms are unicellular while the most complex forms have a true
mycelium. All of them are coenocytic, i.e., they do not form regularly spaced cross-walls.
A. Chytridiales
Members of this order are commonly referred to as "chytrids" and they represent some of the
simplest true fungi. A majority of them are found in aquatic habitats, although certain
members of the group are common in terrestrial habitats. A considerable number of forms are
known to live as saprophytes on submerged organic debris; others parasitize algae,
microscopic animals, aquatic filamentous fungi, and pollen grains that have fallen into the
water. An appreciable number attack terrestrial angiosperms; among these are incitants of
some important diseases of cultivated plants.
Reproductive structures in chytrids are either holocarpic or eucarpic. In holocarpic species,
the entire thallus is used up in forming reproductive structures. In the more complex members
of the group, the thallus is comprised of a number of nucleated centers connected by a system
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of generally anucleate rhizomycelial threads. The nucleated areas are transformed into, or
serve as initiation sites for the development of, reproductive structures. Thus, the entire
thallus is not used up during the reproductive process and is referred to as eucarpic.
1. Holocarpic chytrids
a. Synchytrium endobioticum, incites an important disease of Irish potato known as "black
wart." However, it is much more destructive in Europe than in the USA.

Habit material will be available for examination.

Various stages in its life history, which differs from that of S. decipiens, are depicted in the
figures on demonstration. Note that it has a haploid phase and a diploid phase, the latter
giving rise to dormant, resistant, thick-walled resting sporangia.

Study prepared slides having sections through diseased tubers. Again, don't confuse the
large, numerous starch grains with the parasite. You should be able to locate the parasite
easily after looking at the habit material. The thick-walled resting sporangia (diploid thalli)
are most conspicuous in these slides although you may find large thin-walled haploid thalli
at the surface of tuber section.

Slides illustrating stages in the development of sporangia (haploid) may also be on
demonstration.
b. Synchytrium decipiens is a common chytrid in Wisconsin that parasitizes the leaves, stems
and fruits of hog peanut, a weedy vine.

Examine diseased habit material (leaves) with a stereoscopic microscope and the lesions
that result from invasion by this parasite. Some of the blister-like pustules may have
broken open, leaving minute craters on the leaf surface. You may be able to see
numerous, small golden-colored granules (zoosporangia) within the craters.

After looking at the habit material, study prepared slides showing various stages in the
development of the organism. Note especially the nuclear situation in the vegetative
thallus (prosorus) and the zoosporangia, which serve as dispersal units of the parasite. Is
the parasite intercellular or intracellular? How many dispersal units are produced by a
single vegetative thallus (i.e., 1, 2, a few, or many)? What is left of the vegetative thallus
after it has produced zoosporangia?
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2. Eucarpic Chytrids
Members of the genus Physoderma parasitize higher plants and possess a relatively complex
vegetative thallus.
a. Physoderma alfalfae (formerly known as Urophlyctis alfalfae) causes "crown wart" disease.

Examine preserved specimens of alfalfa infected with P. alfalfae. Is the disease name
appropriate; i.e., does it describe a manifestation of the disease?

Next examine prepared slides with sections of a diseased host. First scan the section with
the low power objective until you find the reproductive structures of the parasite
(=thick-walled resting sporangia). Do they appear to be scattered more or less randomly?
After studying the illustrations in the reprint provided, which depicts most of the
developmental stages, can you provide an explanation for the pattern of their distribution?
Look for remnants of the rhizomycelial threads in the stained section (they will appear
green in these sections). How does their abundance compare with that in the illustrations?
How can you account for the difference?
b. Examine demonstration displays of corn leaves that have been invaded by Physoderma
zeae-maydis, which incites "brown-spot" disease. What is the effect of the pathogen on
the host? How does it compare with that of alfalfa invaded by P. alfalfae? Mount resting
sporangia of P. zeae-maydis for microscopic examination.
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B. Blastocladiales
Members of this order are chiefly water or soil inhabitants. They possess a true mycelium and
are characterized by the development of thick-walled, resistant sporangia that usually have
pitted walls. Another feature common to members of this group is the nuclear cap appressed
to the nucleus in motile cells (zoospores and/or gametes). Allomyces is one genus that has
been studied intensively. Two species will be used to illustrate two parts of the life cycle.
Allomyces is a form that reproduces sexually with haploid thalli developing male and female
gametangia that bear motile gametes that are released from the gametangia upon maturation.
These haploid gametes will fuse in pairs and eventually proliferate into a diploid mycelium that
will produce the diploid sporangia. Meiosis occurs within the thick-walled resistant sporangia
to produce haploid zoospores. Zoospores will develop into haploid thalli that bear the
gametangia.
1. Asexual Reproduction

Examine diploid thalli of Allomyces arbusculus and note the sporangia. How are the
sporangia borne on the mycelium? Are all of them alike (this is most easily determined by
mounting a bit of the thallus in mounting fluid instead of water)? Are zoospores present in
the culture dish?

Examine electron micrographs of the zoospores of two genera in this order. Note the
posterior whiplash flagellum characteristic of the motile cells of all members of the
Chytridiomycota.
2. Sexual Reproduction

Examine cultures of Allomyces macrogynous with a dissecting microscope or low power
objective. The gametangia usually occur in pairs, the smaller, orange, male gametangia
are borne above the larger, non-pigmented female gametangia. Do not confuse the
gametangia on the haploid thalli with the larger sporangia on the diploid thalli. Dissect out
a portion of the mycelium bearing gametangia, mount in a drop of water, add a coverslip
and observe at high magnification. Are any gametes apparent either within or outside of
the gametangia? Is it possible to distinguish between male and female gametes?
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II. Oomycota
This phylum, closely related to the algae and probably most accurately placed with the algae
in the Kingdom Protista, includes organisms that reproduce asexually by means of biflagellate
zoospores, each bearing one tinsel flagellum and one whiplash flagellum, which are borne in
sporangia. The vegetative thallus ranges from a unicell to a profusely branched mycelium.
The majority are eucarpic, the formation of oospores being characteristic of all but the most
simple forms. Usually, four orders are recognized, but you will see representatives of only two
of them.
A. Saprolegniales
This is the group that are familiarly known as "water molds," for most of them occur
abundantly in clear waters, although some species are soil-inhabiting. Most members of the
Saprolegniaceae, the largest family in the order, have a relatively coarse, rapidly-growing,
coenocytic mycelium (see Ex. 2), which, at least in aquatic habitats, commonly consists of two
types of filaments: rhizoid-like hyphae that penetrate the substrate and coarser hyphae that
radiate into the water as a conspicuous cottony mass. Reproductive structures typically are
found on these latter hyphae. Although they are mostly saprophytes, a few parasitize fish and
fish eggs and others cause serious diseases of sugar beets, peas and other crops. One or
more macroscopically examples of these parasitic forms may be on display
1. Asexual Reproduction

Observe cultures of Achlya or Saprolegnia to study the development of sporangia at the
tips of hyphae. How do these differ from Allomyces sporangia? Can you observe
zoospores emerging from the sporangia? How are new sporangia formed after the old one
is emptied in the two different genera?
2. Sexual Reproduction
In most fungi, no gametes are differentiated; rather, a nucleus or nuclei serve the same
function. In these situations, gametes are never released from the gametangia. Instead, the
nucleus (or nuclei) are transferred directly from one gametangium to another. In some, the
"male" nuclei pass into the female gametangium through a pore dissolved in the gametangial
walls at the point of contact. In other species like Saprolegnia & Achlya, a special structure,
the fertilization tube, serves as the conducting element for the migrating nucleus.
Examine corn meal agar (CM) or water cultures of Saprolegnia and/or Achlya growing on
hemp seed and look for the large, globose female gametangia, oogonia (sing. oogonium),
which are typically delimited at the ends of short side branches. Look for various stages of
development (the most mature stages will be near the substrate while the youngest stages will
be nearer the tips of the hyphae). Young oogonia will be completely filled with dense
protoplasm that will appear dark by transmitted light. In somewhat older oogonia, the contents
will have cleaved (divided) into oospheres (eggs). After fertilization, the oospores (fertilized
eggs) develop a thick, dark wall and are very conspicuous. (In some species,
parthenogenesis is common and aboospores, which look exactly like oospores, develop
without fertilization taking place.) How many oospheres are delimited within an oogonium?
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Look for antheridia (male gametangia) appressed to the outside of the oogonial wall. These
may be more easily distinguished if a small portion of the mycelium is mounted in a drop of
water on a glass slide. For best results, tease the hyphae apart with probes before applying
the coverslip. A fertilization tube grows from the multinucleate antheridium, through the
oogonial wall, and to each of the oospheres. Nuclei pass from the antheridium into the
oospheres where fertilization occurs.
B. Peronosporales
The members of this order represent the highest development in the Oomycota. Many
species are destructive parasites of economic plants. Although most members are terrestrial,
some are soil inhabitants and others are found in aquatic habitats. Sporangia are formed
during asexual reproduction, but they differ in form from those in the Saprolegniales. Sexual
reproduction involves differentiation of oogonia and antheridia, a single oosphere, surrounded
by a layer of periplasm, being delimited in each oogonium. The classification of the
Peronosporales is based primarily on the features of the sporangia and sporangiophores
(sporangium-bearing hyphae). (You will recall that most other fungal classification is based on
the sexual state.)
1. Pythiaceae
Members of this family are considered to be the most primitive in the order. They include both
aquatic and terrestrial species, many of the latter inciting serious diseases of economic crops.
For example, species of Pythium are important "damping off" fungi, causing disintegration of
the lower parts of various kinds of seedlings.
Pythium and Phytophthora are the two principal genera in the family, which is distinguished
from the Peronosporaceae and Albuginaceae by the fact that the sporangiophores are not
different from the somatic (vegetative) hyphae. Sporangiophores in these genera are never
"determinant" structures; i.e., never fixed in size, shape and growth. New sporangiophores
may arise from old ones, or from old sporangia, as long as conditions are favorable for
continued asexual reproduction.
a. The genus Pythium comprises over 80 species of aquatic and terrestrial fungi. Whenever
zoospores are produced (many species do not form them), they are always formed outside of
the sporangium. After sporangia are formed in water, the cytoplasm flows out of a long,
delicate tube to form an external vesicle, where it is cleaved into biflagellate zoospores. These
begin to swim inside the vesicle and are liberated when the vesicle membrane ruptures.
Sporangia may be filamentous (indistinguishable from vegetative hyphae), lobulate, or
spherical. In all species, sporangia may germinate directly (by forming hyphae); in some
cases, they may also germinate indirectly (by forming zoospores).
Sexual stages of Pythium paroecandrum and P. mammillatum
 Cut out and examine under low power and oil immersion the surfaces of 1 cm square
sections of agar from cultures of the above fungi on cornmeal agar. Examine P.
paroecandrum first. Note the spherical sporangia and oospores. Surrounding the latter will
be the old oogonial walls. You should also be able to find some younger oogonia, some of
which will have antheridia attached. This species provides good examples of typical sexual
stages in Pythium.
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
Next, examine inverted agar blocks from plates of P. mammillatum. Try to locate several
sexual units (oogonia and antheridia) formed at the very bottom surface of the agar and
observe these under oil immersion.
b. Asexual and Sexual stages of Phytophthora cactorum
Phytophthora is the other most important genus in the group. It differs from Pythium in that
zoospores are formed directly inside of the sporangia, never in an external vesicle. Sporangia
of Phytophthora spp. are characteristically lemon- or pear-shaped. Oospores appear similar
to those of many Pythium spp., but are usually larger and have thicker walls.

Asexual Reproduction: Obtain a culture (V8 agar) of Phytophthora cactorum that has been
flooded with distilled water to promote zoosporangial development. Observe the surface of
the colony, especially at the edge, with the medium power to observe sporangia. Place the
culture in a refrigerator for 15-30 minutes. Remove the culture and observe periodically for
zoospore discharge. What is the geometric form of the zoosporangium? How are the
zoospores released (i.e., does the sporangial wall crack open, is there an exit papilla,
etc.)?

Sexual Reproduction: Observe CM agar cultures of P. cactorum with your compound
microscope. Excise a 1 cm2 section of a colony and invert it on a glass slide (i.e., so the
bottom side is now uppermost). Place a cover slip on the agar block and observe the
sexual structures on the surface (do not confuse with zoosporangia that may also be
present). Note oogonia containing thick-walled oospores. Club-shaped antheridia also may
be present.
c. Phytophthora infestans is the incitant of the destructive "late blight" disease of Irish potato.

Examine potato leaves infected by this organism with a hand lens or stereoscopic
microscope. Note the whitish mass of sporangiophores in the necrotic areas which have
grown out from the interior of the leaf. (Do not confuse the epidermal hairs of the leaf,
which are much coarser, with the sporangiophores.)

Mount a few sporangiophores in a drop of water (use a stereoscopic microscope to locate
them) and examine them microscopically. Note the nodal swellings along the
sporangiophore--a sporangium developed at each of these nodes. Would you conclude
the sporangiophore exhibits indeterminate or determinate growth? Is the sporangiophore
branched or unbranched? Can the sporangia serve as dispersal structures?
In P. infestans, sexual reproduction is initiated when two compatible mating types come
together. Apparently this is a rare occurrence in North America and Europe, but does occur in
Central and South America (where the potato is native) with moderate frequency.

Demonstration slides, prepared from cultures in which the two mating types were grown
together, will be available to examine antheridia, oogonia and oospores. How many
oospores do you find in an oogonium?
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2. Albuginaceae
Members of this family are commonly called "white rusts," although none of them are true
rusts. All of them are obligate parasites of vascular plants, and belong to a single genus
(Albugo).
a. Asexual Reproduction
 Examine habit material of diseased host plants (shepherd's purse, radish, horseradish,
Amaranthus, etc.). What kinds of abnormalities are induced by the parasites? Note the
white blister-like lesions on the hosts.

Study prepared slides showing sections through lesions of this type. Specifically, what
causes the lesion (i.e., what do you observe inside of the pustule)? What is the
arrangement and structure of the sporangiophores? Do they have indeterminate or
determinate growth? How do the sporangia arise? Are they detachable? Are they
uninucleate or multinucleate? When conditions are cool and moist, the protoplast of a
sporangium will cleave into several zoospores (indirect germination) whereas under warm,
dry conditions, the sporangium will form a germ tube (direct germination). Examine the
host tissue beneath a pustule and locate the vegetative mycelium. Is it intercellular or
intracellular?
b. Sexual Reproduction
In Albugo the gametangia (oogonia, antheridia) are borne terminally on the vegetative hyphae.
Would they be intercellular or intracellular? Are the young oogonia uninucleate or
multinucleate? Is the oosphere uninucleate or multinucleate? What is the nuclear situation of
the periplasm which surrounds the oosphere? Answer these questions by observing the
prepared slides.


Two sets of slides will be available to study various stages of sexual development, one is
better for the earlier stages and the other is better for the more mature stages. In this
fungus, large, globose, multinucleate oogonia develop in the intercellular spaces. There
follows an internal cleavage wherein a single oosphere is cut out, which is surrounded by a
peripheral band of multinucleate protoplasm called the periplasm. A small, grayish,
centrally located spot (the coenocentrum) in the oosphere should not be confused with the
nucleus. A more or less triangular-shaped antheridium becomes appressed to the
oogonial wall and a fertilization tube passes through the periplasm and to the egg. One or
more nuclei pass from the antheridium into the oosphere where nuclear fusion occurs,
converting the oosphere to an oospore. The oospore wall has three layers, the outer layer
being smooth or variously sculptured or ornamented. The mature oospore often has a
crescent-shaped oil droplet centrally located and several, well-defined nuclei. The
instructor may ask you to demonstrate various stages of sexual development.
Observe the demonstration slide of oospores in host tissue.
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3. Peronosporaceae
This is the most highly advanced of the three families in the order. All of the members are
obligate parasites of vascular plants inciting diseases known as downy mildews. Since a
number of their hosts are cultivated commercially, they are important economically.
a. Asexual Reproduction

Observe demonstrations of various hosts infected by members of this group. The display
will include Plasmopara viticola parasitizing leaves, stems, tendrils and fruits of grape,
species of Peronospora on various hosts, and perhaps others. What portion of the fungus
do the white, fuzzy patches represent?

To determine this, remove a small portion of the mass (with a half-spear or fine-tipped
forceps) from the material provided and mount it in a very small drop of water; add a drop
of alcohol, and a drop of mounting fluid; tease the material apart with probes and apply a
cover glass. What are the structures? Are they branched, and if so, what is the pattern?
Are any dispersal structures apparent? If there are, what do they look like, how are they
borne, etc.?

Compare these structures with comparable ones of other genera that are on
demonstration. Would you say they would provide a good basis for generic identifications?
What appear to be the most important features for making your conclusion?

Study a prepared slide of a longitudinal section of a grape stem infected with Plasmopara
viticola and locate the vegetative hyphae of the pathogen; note their form and nuclear
condition. What host tissue has the greatest abundance of hyphae? While examining the
slide, look for small, club-shaped or bulbous haustoria (sing. haustorium) that penetrate the
host cell wall and function in nutrient absorption (oil immersion may be helpful). An
electron micrograph of a haustorium will be on demonstration to illustrate additional details.
Does the haustorium penetrate the plasmalemma? Knowing the magnification of the
micrograph, what are the actual dimensions, in mm, of the haustorium?

Study sections through crucifer stems infected with Peronospora parasitica and locate the
large, irregularly shaped haustoria in the cortex or pith cells. What is their nuclear
situation? Can you find remnants of the intercellular mycelium?
b. Sexual Reproduction

Examine demonstration exhibits of the sexual structures of Peronospora parasitica, which
resemble those of Plasmopara. How do they compare with those found in Albugo?
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III. Plasmodiophoromycota (Endoparasitic Slime Molds)
The Plasmodiophoromycota are organisms of uncertain affinities. We are considering them
here because they do produce flagellated zoospores. They also resemble the Myxomycetes
(see Ex. 10) in a number of ways: (a) they produce naked, coenocytic plasmodia, (b) they
reproduce by means of spores which germinate to form biflagellate myxamoebae, and (c)
sexual reproduction occurs via isogametic copulation of the biflagellated myxamoebae.
They differ from the Myxomycetes, however, in that they are obligately endoparasitic on higher
plants, algae and aquatic fungi. Secondly, an alternation of haploid and diploid plasmodial
generations appears to occur. Thirdly, no organized fruiting bodies are formed during the
process of sporulation, the spores being produced as loose clusters -- sori -- within the host
cells. Finally, certain cytological features are peculiar: the akaryotic stage of the plasmodium,
and cruciform nuclear division. To a considerable extent, the genera are distinguished on the
basis of whether the spores are free or are associated with one another in a particular and
characteristic fashion forming spore balls or cystosori (sing. cystosorus).
A. Plasmodiophora brassicae
1. Examine the preserved material of cabbage root infected with the club-root organism,
Plasmodiophora brassicae. Note the hypertrophied areas of the root; these contain the
parasite in greatest abundance. With a sharp razor blade, prepare thin sections of a
rootlet and mount in water. Note the small size of the individual spores of the
intracellular sori, and the lack of enclosing structures around the spore mass.
2. Study prepared slides of infected tissues and compare the infected and normal cells for
size and contents. To what is the hypertrophy due: increase in cell size, increase in
cell number, or both? Examine cells which contain the parasite in the plasmodial stage
as well as cells in which sori are in various stages of formation.
3. If material is available, prepare hanging drop slides of fresh infected cabbage root, and
observe periodically for the germination of the spores. How do the myxamoebae
compare with those of the Myxomycetes?
B. Spongospora subterranea
1. Examine potato tubers infected with the powdery scab organism, Spongospora
subterranea. Study stained sections of tuber lesions. Note the clustered arrangement
of the spores into cystosori (spore balls) and the presence of several cystosori per host
cell. Here too note the hypertrophy caused by the parasite. What is the distribution of
infected cells as compared with club-root disease?
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Exercise 10. Slime molds-- Dictyosteliomycota and
Myxomycota
The slime molds are organisms having questionable affinities, and it is unlikely whether
these phyla are more than remotely related to the true fungi, or even to one another.
However, it has been the mycologists who have contributed most of our knowledge about
them, so we will include them here. They all share the property of forming an amoeboid,
naked vegetative stage -- a plasmodium or pseudoplasmodium capable of ingesting
particulate food material -- and principally for this reason we will consider them together.
I. Dictyosteliomycota (cellular slime molds)
The cellular slime molds represent what might be considered the most primitive
microorganisms of fungal affinities, although they are really very much like protozoa in many
respects. We study them here primarily because mycologists have been responsible for
discovering them and elucidating many features of their biology. Not only are the cellular
slime molds interesting in themselves, but they are widely used in experimental studies of
morphogenesis and differentiation since the life cycle is short and simple, and the number of
different types of cells comprising the fruiting body is small. Dr. Kenneth Raper (U.W.Madison Departments of Botany and Bacteriology), eminent mycologist famous also for his
work on developing penicillin in the 1940's, published the authoritative treatise, The
Dictyostelids, shortly before his death in 1987.
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In our study of these organisms we will attempt to become familiar with the course of events in
their life history from spore to spore. Cultures of Dictyostelium discoideium and/or D.
purpureum will be available for examination in the laboratory. The medium was inoculated
with a streak of Escherichia coli (a bacterium), on which the myxamoebae feed. The slime
mold was inoculated at one end of this streak. Thus, the myxamoebae feed and multiply
progressively along this streak. Eventually, the myxamoebae will form aggregates of cells
(pseudoplasmodia) which will differentiate into small fruiting bodies (sorocarps). By looking
along the line of bacterial inoculum, you should be able to observe progressive stages in
sorocarp development.


Examine the cultures by placing the petri plate on the microscope stage.
Scan the culture with the low power objective to become familiar with various
developmental stages and where they are located. What is the general pattern of sorocarp
development? How can the sorocarp be characterized (e.g. does it have a stalk or is it
sessile, what is the general form of the fruiting body)?

Remove one or two sorocarps from the designated cultures with a forceps, and mount
them in water. (The sorocarps are very delicate, and collapse rapidly when exposed to
air.) Put on a cover slip and examine them at high magnification. What is the cellular
constitution of the sorocarp? Are all of the cells alike or do they differ from one another? If
they differ, in what way(s), and what function(s) do you suppose they perform?

Examine intermediate stages of development and determine whether or not the
myxamoebae retain their individuality (you may want to try adding a stain if available).

Finally, place a clean cover slip over the frontier of the colony (where the bacteria have
been ingested) to observe the vegetative myxamoebae. Examine them with the "high-dry"
or oil immersion objectives. Decreasing the light intensity may aid your observations. Do
the cells have a consistent, regular shape? Are any subcellular structures discernable,
such as nuclei, food vacuoles or contractile vacuoles?

Compare your observations with any light micrographs (pictures) or electron micrographs
that are available. What details do they show that you could not observe?
There is another phylum of cellular slime molds, the Acrasiomycota, which we will not consider
here.
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II. Myxomycota (true slime molds)
A. The vegetative state
The principal vegetative stage of Myxomycota (commonly called myxomycetes) is a naked,
coenocytic mass of protoplasm called the plasmodium. This plasmodial stage is capable of
existing saprophytically in the manner of many mycelial fungi, but in addition nutrition is often
holozoic, by engulfment of large particles and intact microorganisms. The following study of
the plasmodium of Physarum polycephalum will illustrate some of the chief vegetative
properties of the true slime molds.
1. Using water agar (WA) cultures, first observe the overall form, color and texture of the
plasmodium. Note the absence of enclosing wall structures, the amoeboid movement of the
plasmodial front, and the repeated branching and anastomosing of the protoplasmic mass.
Follow the course of its spreading, indeterminate growth for a period of several hours by
outlining the plasmodial front at intervals with a wax pencil. How would you distinguish
between growth and movement in a form such as this and how might growth be measured
quantitatively?
2. Under the microscope observe the streaming of the protoplasm in the individual veins. Is
there a difference in streaming between the center and periphery of a vein? With a fine
needle attempt to break the membrane of the plasmodium. What changes in flow occur?

Examine the streaming in a number of veins for at least five minutes each and note the
periodic reversals in the direction of flow. Record your observations with respect to time
and direction of flow in different portions of the plasmodium. Is there an inherent rhythm
for the plasmodium as a whole?

Now shut off your microscope lamp for several seconds and then turn it on again. Repeat
this procedure, varying the period of light-dark switching and see if you can alter the rate of
flow reversal by means of this simple sort of photoinduction.
3. The plasmodium of Physarum is a phaneroplasmodium, and is just one of the three basic
types found among Myxomycetes. The other types are (a) the aphanoplasmodium (e.g.,
Stemonitis), an inconspicuous network of fine, transparent strands in which the protoplasm is
not visibly differentiated into ecto- and endoplasm, and (b) the protoplasmodium (e.g.,
Echinostelium), a microscopic plasmodium not differentiated into veins.
4. Plasmodium may be cultivated as follows: transfer a bit of plasmodium to the center of a
moistened filter paper in a Petri dish. Sprinkle the plasmodium daily with sterile, finely
pulverized oat flakes. The plasmodium will continue to grow as long as it is fed and kept
moist.
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B. Sclerotization and Sporulation
The further development of the plasmodium occurs in accordance with the conditions of
nutrition, moisture, light, Ph, etc. The two principal avenues of development are (a)
sclerotization, the conversion to a hard, dry resistant state of the protoplasm, and (b)
sporulation, the formation of elaborate fruiting structures associated with the production and
dispersal of spores.
1. Drying of the plasmodium ultimately leads to the production of sclerotia. Take welldeveloped filter paper cultures of Physarum and remove the covers from the plates so that
they will dry slowly. Observe drying plasmodia from time to time in order to observe the
production of macrocysts, spherical resistant bodies of which sclerotia are composed.
2. The reversibility of sclerotization can be confirmed by placing the sclerotium on fresh,
moistened filter paper and allowing it to germinate.
3. Sporulation generally occurs in association with depletion of the medium and desiccation
and is stimulated by light. Take two well developed filter paper plasmodia. Place one in
constant illumination and one in the dark. Keep both moistened, but only feed the one in the
dark. Examine periodically for the production of fruiting bodies, and observe the stages which
lead to the differentiation of a typical sporangium.
C. The Reproductive Stage
The range of variation in the structure of myxomycete fruit bodies can best be appreciated by
examining a representative series of specimens. In examining them the following points
should be observed especially:
(a) the size and shape of the fruit body, i.e., whether it is sporangiate, aethalioid or
plasmodiocarpic; its attachment to the substrate, i.e., the presence or absence of a
hypothallus and stalk.
(b) the color, shape, branching and sculpturing of the network of hairs that constitutes the
capillitium and the presence or absence of lime in the capillitium.
(c) the nature (if present) of the outer covering layer, the peridium, and the presence or
absence of lime in the peridium.
(d) the color of the spore mass and the shape, size and sculpturing of the individual spores.
Representative examples of most of the following orders of Myxomycota will be available,
although we not be putting much emphasis on these orders:
I. Ceratiomycales Ceratiomyxa
II. Liceales
Lycogala
III. Trichiales
Trichia, Hemitrichia, Arcyria
IV. Stemonitales
Stemonitis
V. Physarales
Physarum, Diderma, Diachea, Fuligo, Badhamia
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The following sequence of observations will be useful in studying myxomycetes:
1. examination under the dissecting microscope for a study of the general morphology and
habit.
2. examination of a wet mount in water or lactophenol cotton blue for a study of the capillitium
and spores. The high dry objective of the compound microscope can be used, but oil
immersion will be necessary for careful examination of surface sculpturing.
3. application of dilute (0.1N) HCl to the side of a wet mount to determine whether lime is
present in the capillitium and/or peridium. If the lime is present, material should be seen to
dissolve with the production of bubbles of CO2.
We are lucky to have a set of slime molds identified by G.W. Martin, the world expert on slime
molds, who was a professor at the University of Iowa. Be sure to look at the slime molds from
his collection, acquired by Dr. Allen Nelson during his early tenure in this department.
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Exercise 11. Associations of Fungi with other Organisms
Although many fungi undoubtedly are saprophytic in nature, a large number are capable of
becoming intimately associated with living forms, including other fungi. In many cases, this
association is a parasitic one and many of our plant diseases can be attributed to the fungi,
some of which you already have seen. In other situations, the fungus contributes to the
welfare and survival of the host, supplying it with one or more essential growth factors. Thus,
a mutualistic relationship may become established. Refer to the "Classification of Major
Groups of Fungi" in the preface and review the characteristics of all of the groups you have
observed this semester as you consider the examples of fungi that associate with other
organisms.
I. Parasites
The fungi as a group are capable of parasitizing a wide spectrum of organisms, but vascular
plants serve as hosts for the majority. Some parasites are limited to a single host species, or
even a particular portion of the host, while others are not as exacting in their substrate
requirements. A number of host-parasite associations will be available for examination;
wherever possible, determine the physical relationship of the parasite with the host tissues as
precisely as possible and note any morphological and/or anatomical abnormalities induced in
the host. Also determine where the parasite belongs in the classification scheme.
A. Algae, Fungi, and Plants as hosts (See lab copies of Greene's Fungi Parasitic on Plants in
Wisconsin and Farr et al’s Fungi on Plants and Plant Products in the United States.)
•
Study preserved material of Stephanodiscus parasitized by Podochytrium. What kind of
plant is Stephanodiscus? What is the structure of a mature thallus of the parasite (you will
probably not be able to detect the very fine rhizoidal threads that penetrate the interior of
the host)? Knowing that zoospores with a single, posterior, whiplash flagellum are
produced in the sporangia, where would you place it in the classification scheme?
•
Observe demonstration slides showing filamentous algal hosts (Spirogyra) parasitized by
Myzocytium, a member of the Oomycetes. When young, the vegetative thallus is a
multinucleate, unbranched hypha. Is it an intracellular or intercellular parasite? The hypha
eventually become segmented, each segment developing into a multinucleate sporangium.
After zoospores are cleaved, the sporangium develops an "exit tube" that provides a
means for the zoospores to escape the confines of the host cell wall. Exit tubes will be
apparent on one of the demonstrations.
•
Examine demonstration slides of Syncephalis (a mucor) parasitizing Rhizopus. Note the
thick sporangiophore terminated by a bulbous vesicle that bears finger-like mesosporangia
within which asexual spores are cleaved. How is the parasite attached to the host? How
do you imagine the parasite obtains nutrients?
•
A number of fungi incite destructive diseases of cultivated and native fruit trees.
Blumeriella jaapii (=Higginsia hiemalis) is the causal organism of "leaf spot" disease of sour
cherries, a destructive disease in Wisconsin. Preserved habit specimens of cherry leaves
exhibit typical lesions associated with the Cylindrosporium anamorph stage of the fungus,
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which develop while the leaves are on the trees. If the infection is heavy, the leaves
usually turn yellow and fall off. Mount some conidia from the material provided for
microscopic examination and examine prepared slides having sections through infected
leaves. How would you characterize the Cylindrosporium stage? The teleomorph stage
develops in overwintered leaves. What can you say about the nutritional status of this
organism during its life history? Study prepared slides having sections of overwintered
leaves. How would you characterize the teleomorph stage and where would you place it in
the classification scheme? Examine any other material relevant to this organism.
•
"Brown rot" is a destructive disease of plums, peaches and cherries incited by species of
Monilinia (=Sclerotinia). The attacked fruits rot rapidly on the tree, conspicuous conidial
masses developing on their surfaces (Monilia stage). The rotting fruit soon drops to the
ground and is transformed into a mummified pseudosclerotium, a dry, hard mass
comprised of fungal sclerotial tissue intermingled with remains of the host tissues. Fruiting
structures appear the following spring, arising from buried, or partially buried, mummies on
the ground. Examine preserved specimens of infected fruits. Pseudosclerotia will also be
on display--note their texture and the small, brownish patches of conidia. After looking at
preserved specimens of fruiting bodies borne on pseudosclerotia, and prepared slides
showing sections through the fruiting bodies, determine as best you can where it should be
assigned in the classification scheme.
•
Venturia inaequalis incites the widely-occurring and destructive disease of apples known
as "apple scab." The parasitic mycelium develops in a subcuticular position on the leaves
and fruits. Observe "scab" lesions on host material. Then examine prepared slides having
sections through a lesion and characterize the conidial (Spilocaea) stage; i.e. what is the
form of the conidiophore and conidia? When the infected leaves and fruits fall from the
tree in autumn, the mycelium continues growth saprophytically and penetrates into the
interior of the host, where it gives rise to the teleomorph stage that does not mature until
the following spring. Study stained sections through dead, overwintered apple leaves and
search for fruiting structures. Note the very distinctive spores that are produced.
•
As mentioned in a previous exercise, the rusts constitute a large group of parasitic fungi,
many of which are extremely destructive to commercially valuable species and horticultural
plantings. They are a wide-spread group commonly encountered in nature. You already
have looked at one of the most economically destructive rusts, Puccinia graminis, in
exercise 5. Habit specimens of a number of representatives will be on display. Note what
macroscopic effect, if any, the pathogen has on the development of the host. Also note the
variation in aecial (I) structure.
Although the lesions induced in the hosts by many of the rusts may be indistinguishable
from one another macroscopically, they are different microscopically. For example,
compare the structure of the teliospores of Puccinia graminis (see exercise 5) with those
produced by Puccinia podophylli and Phragmidium, using the dried specimens provided for
this purpose to make microscopic mounts. Prepared slides showing telia of Cronartium
ribicola and Melampsora bigelowii will also be available for examination. There may also
be differences in aecial structure. Study any demonstration material that is available.
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B. Animal hosts
Although a majority of fungal parasites use plants as hosts, there are also a number of
pathogens that parasitize animals, including humans. Medical mycology is an expanding field
of research and treatment, especially in tropical and subtropical regions.

Species of Entomophthora are parasitic on insects. Once infection is accomplished, an
insect rarely, if ever, survives. Study prepared slides of longitudinal sections through a
house fly that was killed by Entomophthora muscae. First become familiar with the various
parts of the insect (head, thorax, abdomen) using the lower powers of the microscope.
Then determine the distribution of the parasite in various regions of the host. What host
tissues appear to be most resistant to attack? What is the nuclear situation of the
vegetative hyphae? Are hyphal bodies apparent? Locate conidiophores and conidia,
noting especially where the former emerge from the insect's body. What is the nuclear
situation of each? Compare this parasite with the species of Entomophthora you studied in
exercise 8. There will be one or more demonstrations illustrating the forcible discharge of
conidia from flies killed by E. muscae--note the white haze of spores around the body.

Massospora is another member of the Entomophthorales that parasitizes locusts
(Magicicada). Examine habit specimens of parasitized hosts. Note how the terminal
abdominal segments have fallen away, thereby exposing the internal abdominal fungal
mass. The spores of this species are not forcibly ejected; rather, they are dispersed
through movement of the insect.

Septobasidium, a member of the Hymenomycetes, is one of the most interesting insect
parasites known among the fungi, forming some remarkable host-parasites interactions
with scale insects. Examine habit material of Septobasidium basidiocarps, which are
crust-like structures that grow over scale insect colonies. They hyphae penetrate some of
the insects to extract nourishment, which in turn is obtained from the plant host. The
parasitized insects are not killed rapidly, so in effect they are serving as a nutrient pump for
the fungus. These parasitized insects are sterile, but other members of the colony not
penetrated by the fungus are fertile, neatly protected from predators by the overlying fungal
mat. Examine demonstration slides having sections through a basidiocarp. The scale
insects are hemispherical and tightly appressed to the woody twig. Note the mycelial mat
that covers them.

The final insect parasites that we will consider belong to the genus Cordyceps. Flies, ants,
beetles, caterpillars, mealy bugs, etc. are attacked, killed, and mummified. Coremia are
usually found in the conidial stage. Erect, clavate or stalked perithecial stromata also grow
out from the mummified insect. Observe habit specimens on larvae of the June beetle and
other insects. What variation in form do you find? Prepared slides showing stromata in
section view will be available for study.

Arthobotrys (deuteromycetes) is a predatory nematophagous fungus. Note the specialized
hypha that trap the nematodes.

Examine demonstration slides having sections of a wild goose lung that is parasitized by a
fungus. After locating sporulating structures in the air sacs (alveoli), you shouldn't have
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trouble assigning it to the proper genus since you have seen representatives in a previous
exercise. Can you detect mycelium in the lung tissue?

Peruse demonstration exhibits illustrating various fungal diseases of other warm-blooded
animals, including humans. Do any particular taxonomic groups of fungi appear to be
prominent?
II. Mutualists
A. Mycorrhizae
The symbiotic association between fungal hyphae and the roots of higher plants is known
as a mycorrhiza (pl. mycorrhizae). This is a widespread phenomenon involving members of
most major groups of land plants, although its significance has become recognized and
appreciated by biologists only relatively recently. Mycorrhizal fungi enhance nutrient uptake
from soil and are of great antiquity. The earliest known vascular plants, Rhynie fossils from
400 million years ago, show evidence of mycorrhizae. Although several different general
types of mycorrhizae have been described in the literature, only two types, the
ectomycorrhizae and the endomycorrhizae, will be represented in the laboratory.
Ectomycorrhizae are characterized by having an external mantle of mycelium surrounding
the terminal portion of the root. It inhibits root hair formation and greatly reduces the
elongation of the short lateral roots, often inducing branching in the latter. Examine habit
specimens on demonstration under a stereoscope. Photographs will also be on display. The
mycelia of ectomycorrhizae usually penetrate into the intercellular spaces of the root cortex,
forming what is called a Hartig net. Examine material illustrating this interrelationship. Most
ectomycorrhizae are basidiomycetes (Amanita, Russula, Suillus, Boletus, etc.) but
representatives of other groups (truffles-Ascomycotina) also form these associations. Live
material may be available for making your own slides.
Endomycorrhizae slides will also be available. One set of slides shows root cross sections in
which arbuscules, haustoria-like growths, are abundant. What function might these have? The
other set of slides (whole stained mounts of roots) show the vesicles, relatively large, bulbous
swellings of the hyphae. What function might these have? Because of these structures, the
endomycorrhizae are also known as vesicular-arbuscular (VA) mycorrhizae. All the known
endomycorrhizae (Endogone and Glomus) are members of the order Endogonales
(Zygomycotina). What kind of sexual reproductive structure would they produce?
Preparing slides for observation of mycorrhizae. Roots were collected from nature washed
in distilled water to remove the soil, and boiled for one hour in xx?% KOH to bleach out
pigments. They were rinsed again several times in distilled water.
Mount the smallest parts of the roots you can find and look for evidence of fungi. Do you find
ectomycorrhizae or endomycorrhizae?
Non-photosynthetic plants parasitize fungi. Observe the demonstration materials for
heterotrophic plants. About 200 species of plants parasitize fungi that have a mycorrhizal
association with photosynthetic plants. These include indian pipe (Monotropa uniflora and
related spp.), beech drops, pine drops, squaw root, and broom rape.
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B. Lichens
A lichen thallus is a composite plant body made up of a fungus (mycobiont) and an alga or
cyanobacterium (photobiont or phycobiont) that form a symbiotic relationship. The photobiont
is usually a green alga (Chlorophyta), but Cyanobacterium (formerly called blue-green algae)
photobionts are also common. The mycobiont is almost always an ascomycete; a few are
basidiomycetes or deuteromycetes. It is generally acknowledged that the photobionts may
exist in the free-living, nonlichenized state but that the mycobionts grow and reproduce only in
association with an alga. Furthermore, it is the mycobiont that is responsible for the physical
form of the lichen thallus. Consequently, the binomial that is applied to the lichen thallus
refers only to the fungus and not the algal symbiont.
The symbionts can be isolated and cultured, but when separated do not exhibit the
characteristic form of the intact lichen. The lichen thallus is not only a discrete physical entity
but it is also a distinct physiological system. Many of the metabolites found in the thallus are
not produced by the isolated symbionts nor by other free-living fungi and algae.
1. Lichenized deuteromycetes
Obtain a fragment of bark with the lichen Lepraria on it. The Lepraria will appear as a thin,
greenish growth covering the bark surface. In Wisconsin this genus is often found in humid,
shaded habitats growing on rocks, on bark at the base of trees, or perhaps over mosses and
other lichens.
Lepraria exhibits the simplest type of lichen thallus. With a dissecting microscope observe
that the thallus appears as a scurfy, powdery crust. With the tip of a half-spear, scrape a
small portion of the thallus into a drop of alcohol and then add a drop of water on a
microscope slide, apply a cover slip and examine with a compound microscope. The basic
structural unit of Lepraria consists of a small number of green algal cells held together in
discrete clusters by strands of hyaline fungal hyphae. The phycobiont is a member of the
Chlorophyta and is considered by some to belong to the genus Chlorella. The cells tend to be
more irregular in shape and of larger size than they would be if they were free-living. The
fungal hyphae are septate, thick-walled and colorless; these are characteristic of nearly all
mycobionts. The balls of fungal and algal cells may be referred to as soredia and serve as
vegetative propagules. Although many other genera of lichens produce soredia, only Lepraria
has a thallus consisting solely of a layer of diffuse soredia.
2. Lichenized Ascomycota
Of the 33,000 known species of ascomycetes, more than half (18,000) are obligate
lichen-formers. In this exercise the lichenized ascomycetes will be studied in groupings based
on the growth forms of the lichen thallus rather than taxonomic affinities of the mycobionts.
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NOTE ON TECHNIQUE
To observe the composite structure of lichen thalli it is necessary to make thin freehand
sections. This will require a bit of practice. Place a piece of thallus on a wooden block and
moisten it with a drop of water. With the aid of a dissecting microscope and a sharp razor
blade, cut a series of thin sections. As proficiency is acquired, sections of 0.5 mm may be
routinely cut. Gently transfer the best sections to a microscope slide and mount in water or
mounting fluid. Sections of ascocarps may be cut in a similar manner; however, it is often
easier to section them when the ascocarps are moistened and cut in place on the thallus.
Foliose Lichens
Characteristically the foliose thallus is flat, leaf-like, and usually lobed; spreading over the
substrate, it is attached at intermittent points.
•
Observe the foliose thallus of Physcia with a dissecting microscope. The elongate lobes
are rather thick and are affixed to the substrate by pale or dark rhizines (slender root-like
processes). The upper surfaces of the thallus (examine young lobes) are densely spotted
with whitish marks, structures that are assumed to facilitate gas exchange. Abundant
ascocarps are present on the upper surface of the thallus. The apothecia are cup-shaped;
a white powder often obscures the black color of the hymenial surface. Note the persistent
rim of white thalloidal tissue that surrounds the edge of the apothecium.
•
Select one of the prepared slides of Physcia to study the cellular structure of the thallus;
focus on a portion of the section that was cut perpendicular to the cortices. Note that the
mycobiont and phycobiont are organized into specialized tissue layers. The uppermost
layer of tissue is the upper cortex, that is composed of several layers of closely packed
isodiametric fungal cells.
Directly beneath the upper cortex is another zone, the algal layer, in which the green algae
(stained red) and the mycobiont grow intermingled. Between the algal layer and the lower
cortex is the thick, cottony medulla. The medulla consists only of loosely interwoven
hyphae (stained blue-green). The lower cortex is thinner than the upper. Rhizines also
may be visible in the section.
Observe a section of a mature apothecium. It is in the formation of fruiting bodies that the
close relationship of the lichenized ascomycetes to free-living forms can be most clearly
seen. The ascocarps of lichens are unique in two ways: (1) algae are an integral part of
the mature fruiting body, and (2) the fruiting bodies are long lived, producing spores
repeatedly over the years. Many of the tissues and structures of Physcia apothecia are
identical to other ascomycetes. The hymenium lines the interior of the cup-shaped disc
and contains asci and paraphyses. The epithecium, a thin coating of cellular or gelatinous
material, which normally covers the hymenial surface and imparts the color to the disc,
probably will not be visible in the prepared material. Below the hymenium is the
sub-hymenium (hypothecium) from which the hymenium arises. Underneath the
hypothecium is the algal layer (often discontinuous) and finally the medulla.
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To better study the ascospores and asci, fresh material should be sectioned; you may find
the structures more clearly visible if they are stained with iodine (Melzer's Reagent). Each
ascus contains eight spores arranged in two rows. The ascospores have brown walls and one
transverse septum.
•
Examine a piece of Lobaria quercizans, an eastern species found in Wisconsin. The
thallus was attached to the bark substrate by dark rhizines, some of which may still be
visible. In addition, the younger portions of the under surface tend to be covered by a mat
of hairs that may also aid in attachment. On the under surface slight swellings that are
called cephalodia, might be detectable. The much wrinkled upper surface of the thallus
supports a large number of apothecia.
Prepared slides of L. quercizans are available to show x-sectional views of the pycnidia,
cephalodia, and the thallus structure. It will be necessary to examine more than one slide.
The pycnidia develop below the algal layer and are stained pink. They produce
uninucleate pycnidiospores that are thought to function as male gametes. Pycnidial
production is extremely common in lichens. When viewed in near-median section, pycnidia
are seen as globose or flask-shaped organs having a cortical-like peripheral wall. The
interior of the pycnidium contains many branched and anastomosing pycnidiophores, which
bear minute pycnidiospores. The spores fill the interior of the pycnidium and are eventually
released through the ostiole onto the surface of the thallus.
In addition to the green alga (Myrmecia) contained in the algal layer, there are blue-green
algae in the cephalodia. In the prepared slides, cephalodia will be seen scattered
throughout the medulla, composed of closely packed clusters of Nostoc intermixed with
hyphae. A layer of medullary hyphae surrounds the blue-green algal colony. The
symbiotic Nostoc shows a markedly different morphology from that of free-living varieties.
The Lobaria thallus is thus a complex consisting of a mycobiont and two different
phycobionts. It has been shown that the cephalodial Nostoc supplies the mycobiont with
nitrogenous compounds. It has also been demonstrated that the mycobiont receives
photosynthetic products from the phycobionts. The green algal symbiont supplies
carbohydrates in the form of ribitol (a 5-carbon alcohol), and the cyanobacterium
phycobiont provides glucose to the mycobiont.
•
The genus Parmelia sensu lato has the widest distribution and largest number of species
of all foliose lichen genera. In recent years, the genus Parmelia has been segregated into
many smaller genera. Three representatives (Xanthoparmelia conspersa, Punctelia
bolliana, and Flavoparmelia caperata) are available for study; all are common to North
America. Locally, the first two species are found in abundance covering boulders on the
talus slopes surrounding Devil's Lake. Flavoparmelia caperata is frequently collected on
tree trunks throughout Wisconsin. Compare the colors and physical characteristics of the
upper and lower surfaces of the three species. By what methods do you think these
species most often reproduce?
•
Easily recognized by its orange color, Xanthoria fallax appears to be among those lichens
least susceptible to pollution. It is probably the most common lichen in the city of Madison,
the bark of elms being a common substrate. Note its superficial resemblance to Physcia.
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Umbilicate Lichens
The umbilicate thallus is, as the name implies, attached by a single, more or less central,
strand of tissue that distinguishes these lichens from the true foliose lichens. It may be of
interest to note that the success of the American Revolution was influenced by a member of
this group. It is reported that George Washington's troops ate Umbilicaria ("Rock Tripe")
boiled as a soup during the hard winter at Valley Forge. Two species of Umbilicaria are
provided for study, both of which grow in abundance on the shaded rock outcrops in Devil's
Lake State Park.
Umbilicaria mammulata is one of the largest lichens; specimens 56 cm in diameter have been
collected in the Smoky Mountains. This rather impressive lichen is very brittle when dry and
should be moistened before handling. The upper surface is smooth and lacks distinctive
characters. Examine the ventral side and note the umbilicus at the point of attachment to the
substrate and the blackish lower surface.
Fruticose Lichens
The fruticose thallus is hair-like, shrubby, or strap-shaped and consists of a main branch
with or without numerous lateral branches; it is attached to the substrate only at basal points.
•
Usnea is often used as the classic example of a fruticose lichen. It grows on the trunks of
trees, and occasionally attains a length of 1 m. Because of its frequently pendulous habit it
is often mistaken for Spanish Moss (Tillandsia usneoides), which is not a moss but is a
member of the pineapple family.
Examine a moist specimen and note the cylindrical shape of the tapering branches. Grasp
a main branch and pull until it breaks; a construction similar to electrical wire will be
revealed. A tough white core, or medullary cord, forms the central portion of the branch
and is surrounded by the more fragile "insulation" of the algal layer and cortex.
•
Evernia prunastri, known as "Mousse des Chenes," is one of the ingredients in some of the
quality French perfumes; it is cited in Gerard's Herball as a "moss that partakes of the
(Quercus) bark of which it is engendered. It is to be used in compositions that serve for
sweet perfumes and that take away wearisomeness." This may be the first report of a
lichen "high."
•
Letharia vulpina is perhaps the most striking of all fruticose lichens; it grows in the high
Sierras on juniper and in the Cascades on ponderosa pine. The yellow color of the thallus
is imparted by the pigment vulpinic acid. L. vulpina has been used as a wolf poison.
"Dead carcasses are stuffed with a mixture of lichen and powdered glass and exposed in
the haunts of wolves in time of frost." Henneguy, who insists on the non-poisonous nature
of vulpinic acid, says that the wounds caused by the glass render the internal organs
extremely sensitive to the action of the lichen.
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•
A few thalli of a small Wisconsin Ramalina are available for macroscopic observation.
Notice the stiff, erect, and shrub-like thallus. Plate-like apothecia are abundant on most of
the specimens, either along the margins or on the tips of the branches. This genus, like all
the other fruticose ones on display, has a Chlorophyta phycobiont in the algal layer.
•
In contrast to the habit of the preceding Ramalina, R. reticulata is long and pendulous; it
occurs on high branches in fog communities along the west coast. The unusual growth
patterns of the thallus is readily apparent. Each large branch, or frond, is a delicate open
network from tip to base.
•
Cladonia is an intriguing fruticose genus that has a foliose growth phase in its life cycle.
Examine the bark samples on display and note the tiny foliose thalli (squamules) on them.
The green squamules represent the young thalli of a Cladonia. Eventually the primary
squamules produce upright branches that are characteristic of the genus.
C. cristatella has the common name of British Soldier or Match Stick Lichen. It is endemic
to N. America, occurring on old wood and tree stumps. Some of the branches (podetia)
also bear, at their apices, scarlet apothecia that contain hyaline aseptate spores.
•
Cladina (the reindeer lichen genus). Cladina mitis is a soil species that grows in large
clumps in open habitats. Lateral branches tend to be produced in whorls of 3 or 4; it is
believed that one whorl is produced each year. The axils of the branches are perforated
and reveal a hollow center. The most apical branches are short, recurved, and pointed.
Cladina rangiferina is another mat-forming Cladina. Compare the color and branching
pattern of C. rangiferina to C. mitis. Both species have a northern distribution and are
found in great abundance in the Arctic tundra. Research has demonstrated that both of
them serve as the most important food source for reindeer (Caribou). In some districts of
the arctic USSR, 80 to 95% of the winter food of reindeer consists of lichens.
Crustose Lichens
Crustose thalli form a flat crust on, or in, the substratum and adhere firmly by their entire
lower surface; consequently, they cannot be easily removed. The thallus usually lacks a lower
cortex. Unlike most of the foliose and fruticose lichens, the crustose genera are distinguished
according to ascocarp characters.
•
With a hand lens, examine the large colonies of Porpidia albocaerulescens growing on
quartzite boulders. The large orbicular, grey-green thallus forms a thin continuous crust
over the rock. At the circumference of the thallus is a prominent pale border called the
hypothallus, which for the most part is composed of fungal hyphae without any definite
tissue organization or algal layer. Scattered over the surface are black apothecia.
•
Look at the apothecia of Tephromela atra; the white thalline margin forms a definite rim
around the black hymenial surface. Tephromela and Porpidia both produce hyaline,
non-septate, ellipsoidal spores.
•
The genus Graphis ("script lichens") forms lirella-form ascocarps. The fruiting bodies are
black (but not carbonaceous), elongate structures in which the "disc" is a mere slit.
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3. Lichenized Basidiomycota
A small number of basidiomycetes are lichenized; most of them are tropical and appear to
have affinities with the non-lichenized family Thelephoraceae.
The most common lichenized basidiomycete, Cora pavonia, is found in damp forests growing
profusely in diffuse light on branches in much the same manner as a shelf fungus. When wet
the thallus is dark blue-green, the color being imparted by the blue-green algae residing in the
algal layer. Note the concentric growth zones on the upper and lower surfaces of the thallus.
The flesh-colored hymenial tissue is located on the underside of the thallus and is composed
of typical basidia and paraphyses. Between the fertile areas the undersurface is covered with
a fine tomentum. Only Cora and two or three other genera of basidiomycetes form what can
clearly be described as a lichen association.
References:
Ahmadjian, V. 1967. The lichen symbiosis. Blaisdell Publishing Co., Waltham, Mass.
Ahmadjian, V. and M.E. Hale. 1973. The Lichens. Academic Press, New York. 176 p.
Thompson, John. See lab materials.
Lichen Supplement to Fungal Classification
(modified from Hale and Culberson, 1970)
Phylum: Ascomycota
Class: Pyrenomycetes
Order: Sphaeriales
Genera:
Class: Discomycetes
Order: Lecanorales
Genera:
Dermatocarpon
Acarospora
Alectoria
Caloplaca
Cetraria
Cladina
Cladonia
Collema
Evernia
Hypogymnia
Lasallia
Lecanora
Lecidea
Letharia
Lobaria
Order: Ostropales
Genera:
Graphis
Order: Caliciales
Genera:
Coniocybe
Phylum: Basidiomycota
Class: Hymenomycetes
Order: Agaricales
Genera:
"deuteromycetes"
Genera:
Cora
Lepraria
Parmelia
Peltigera
Pertusaria
Physcia
Porpidia
Ramalina
Rinodina
Stereocaulon
Sticta
Tephromela
Thamnolia
Umbilicaria
Usnea
Xanthoria
Mycocalacium
Omphalina
Cyphelium