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Nice abstraction from previous labs! The emphasis on sac fungus fruiting body variation is good.
Some questions would be nice.
Chytrids
Zygote Fungi
Sac Fungi
Introduction
Fungi are a unique group of organisms. Fungi can be defined as eukaryotic heterotrophs that have
absorptive nutrition and reproduce by spores. Spores are cells adapted for dispersal and/or dormant
survival. They can be either sexual (produced by meiosis) or asexual (produced by mitosis). Sometimes
spores are produced inside or on a special structure called a fruiting body.
The majority of fungi have a filamentous body (the individual strands are called hyphae). Hyphae are
microscopic tubes of protoplasm constrained by a cell wall. Hyphae are characterized by growth only at
the extreme tips, and typically branch extensively behind the tips. The resulting network is called a
mycelium. In the more primitive fungi the hyphae are continuous, without crosswalls (coenocytic or
aseptate). Hyphae separated at irregular intervals with cross-walls (septa) are considered more
advanced.
The more than 100,000 species of true fungi comprise a large and diverse group. Although all fungal
organisms were once classified into a single kingdom, current taxonomic schemes separate these true
fungi from organisms that resemble fungi in form or lifestyle, but are fundamentally different in several
ways. True fungi (placed in the Kingdom Fungi) have cell walls made of chitin and their nuclei are haploid
(although more than one nucleus can exist in each cell), The fungal-like organisms are placed in two or
three other kingdoms (depending on the taxonomic scheme--classification of fungi is under frequent
revision).
These organisms play a major role in the decomposition of organic matter in soil, increasing fertility in the
process. Fungi are used in industry to carry out fermentation reactions to produce organic acids, alcohols
and vitamins. They are the source of some antibiotics. Certain cheeses have their aroma and flavor
enhanced by fungal activity. On the other hand, fungi cause the destruction of food, fabrics, leather goods
and other commercial and industrial products. Fungi cause diseases in plants, animals and man. And
finally, there are the mushrooms. These common and sometimes colorful forms range from edible to
deadly poisonous, but the majority are really neither.
There are five phyla of true fungi: Chytridiomycota, Zygomycota, Ascomycota, Basidiomycota, and
Deuteromycota. This lab introduces you to The first three groups.
I.
Chytrids
The Chytridiomycota (often referred to as "chytrids") are an inconspicuous and little known group of fungi.
However, they have many important roles in nature. Many are saprotrophs in aquatic environments or wet
soils. Some species are parasites of algae and a few species are serious plant pathogens. A few
members parasitize small aquatic insects, and one genus is known to attack mosquito larvae. An
interesting group of chytrids are found in the rumen of herbivores and play a major role in the breakdown
of plant material. A few chytrids have also gained notoriety as newly discovered pathogens of amphibians.
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Chytrids as a group are good at degrading cellulose or chitin. Compared to most fungi, they are more
difficult to isolate and grow in the lab, but some can be obtained in culture by "baiting" them with suitable
substrates (such as boiled grass, hair, or insect exoskeletons).
The chytrids, believed to be ancestral to the other groups of true fungi, do have a cell wall composed of
chitin, but lack a true mycelium. They are typically unicellular but sometimes the body consists of
branched chains of cells. These fungi usually have tapering rhizoids that anchor them to their food
source, They are the only group of true fungi that produce a motile spore (called a zoospore). The
zoospores swim by means of a single posterior flagellum. There are several mechanisms for sexual
reproduction that have been identified, but details are unknown for most of the group. These fungi are
usually haploid, but some alternate between haploid and diploid phases.
A. Chytrid observation
Examine a demonstration plate of Phlyctochytrium acuminatum under the dissecting microscope. From
one of the other plates available, scrape off a small colony and prepare a wet mount on a slide. Gently tap
the coverslip to break up the clumps of cells. Observe under medium to high power. Note the single cells
with rhizoids. This cell will eventually be converted to a sporangium and release zoospores (zoospores
will not be present in our preparations). Make a sketch below of your observations.
II.
Zygote Fungi
Biologically, these fungi range from saprotrophs (live by decay) to weak plant parasites, to specialized
animal parasites, and finally to obligate parasites of other zygote fungi. They are common soil and air
inhabitants that grow rapidly so they can be troublesome as contaminants in laboratories and on food.
Some are used industrially to produce chemicals. One important group form an intimate mutuallybeneficial association with plant roots (called mycorrhizae) and facilitate phosphorus absorption from soil.
The Zygomycota are typically mycelial. The mycelium is coenocytic (non-septate) except at the point
where reproductive structures are formed. The distinctive morphological feature of this group (nicknamed
the "zygote fungi") is the production of a sexual resting spore called a zygospore. Asexual reproduction
is usually by spores borne on sporangia in remarkably large numbers.
A. The Genus Rhizopus
Rhizopus is a ubiquitous diverse terrestrial genus of saprophytes and plant root pathogens. The species
featured in lab is commonly called black bread mold for reasons you can probably guess.
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(1) Examine a demonstration
plate of Rhizopus stolonifera
under the dissecting
microscope. The mycelium is
growing extensively over the
plate. The asexual sporangia
are very numerous and appear
as tiny dark dots on the
mycelium. Each sporangium
contains thousands of
sporangiospores. The stalk
supporting a sporangium is a
sporangiophore.
(2) Prepare a wet mount from
a culture plate. The sporangia
tend to break and you may see
the sporangial wall folded over.
Note the numerous dark
spores. Look for the swollen
tip of the sporangiophore
(inside the sporangial wall).
This is the columella. The
hyphae that connect the
sporangiophores are called
stolons.. Rhizoids (root-like
hyphal branches) develop from
the base of the
sporangiophores. Label the
structures in Figure 18-1.
(3) Examine a prepared slide
of Rhizopus showing sexual
Figure 18-1. Rhizopus, Vegetative Hyphae and Asexual Spores.
reproduction. Rhizopus is an
example of a fungus that is heterothallic. This means that two different strains of mycelia (designated as
+ and - strains) are needed in order for sexual reproduction to occur. Organisms that have no such
restriction are referred to as homothallic. Strands of the + and - strains send out lateral projections
(progametangia) toward one other. After contact is established, the progametangia become walled off
and are then termed gametangia. The walls between the two then dissolve and fertilization occurs. A
thick-walled zygospore is formed. The zygospore is a resting stage that typically persists after the hyphae
have died. Label the structures in Figure 18-2.
Figure 18-2. Sequence of Events in Rhizopus Sexual Reproduction.
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B. The Genus Pilobolus
Pilobolus is one of the most unusual members of the Zygomycota. It is commonly found on dung of
grazing animals. It has a remarkable mechanism for spore dispersal by explosively discharging its jetblack sporangia toward a light source. The mycelium forms a basal swelling called a trophocyst. A
sporangiophore grows from the trophocyst, elongates and forms a subsporangial swelling and a dark
sporangium. The subsporangial wall ruptures at maturity and the sporangium is shot off (propelled by the
hydraulic pressure in the sporangiophore). The sticky sporangium adheres to whatever it hits.
Cultures can be started by placing fresh horse manure in an open glass container. Sporangiophores
usually develop in 4 to 7 days. Observe the dish with the dissecting microscope.
III. Sac Fungi
The Ascomycota (nicknamed "sac fungi") are a very large group of fungi that occupy a broad range of
habitats, including terrestrial and even marine environments, where fungi are remarkably scarce. This
group is of great importance to human affairs. Sac fungi are important in food spoilage and destruction of
paper, fabrics, and wood. Dutch elm disease, apple scab and chestnut blight are examples of destructive
plant diseases caused by some members of this group. A few genera include human pathogens. The
brewing and baking industries utilize a group of sac fungi known as yeasts. Some of our antibiotics also
result from the biochemical activities of certain sac fungi (like Penicillium).
Although there are some single celled forms (called yeasts), most sac fungi form a mycelium. However,
unlike the zygote fungi, the hyphae are septate. The asexual spores (conidia) are non-motile, and can be
produced in a variety of ways but are never inside a sporangium. Sexual reproduction involves the
production of one or more sporangia (asci, singular ascus) with spores. In may sac fungi the asci are
found in layers inside or on a fruiting body termed an ascocarp. Primitive sac fungi may lack ascocarps.
A. Cleistothecium-producing fungi
1. Eurotium: Blue Mold.
Blue mold gets its common name from the color of its conidia (which have blue walls). Obtain a culture
plate and observe it with a dissecting microscope. The patches of blue-gray contain the conidia, which
are produced at the end of a specialized hypha (the conidiophore). The conidiophores of this fungus
have a swollen tip. Intact conidiophores with their conidia resemble toilet bowl brushes.
Prepare a wet mount by gently scraping off a few conidiophores from the plate. Observe the conidia and
how they are produced.
Also note the tiny yellow bodies elsewhere on the plate. These are examples of a type of ascocarp called
a cleistothecium. With a dissecting needle, remove a few ascocarps from the culture and prepare a wet
mount. Observe under medium to high power. Gently press on the coverslip to rupture the cleistothecia
and look for asci with ascospores.
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2. Powdery mildews
These fungi are obligate parasites of plants and cause a group of plant
diseases known as powdery mildews. The name comes from the white,
powdery appearance of the asexual stage of the organism. They produce
a different kind of cleistothecium than Eurotium. Examine various leaves
infected with the organism. Look for the dark, round cleistothecia that
occur on the upper surface of the host. These cleistothecia have hyphal
appendages that differ in ways that can be used to identify the particular
genus.
B. Perithecium-producing fungi
Figure 18-3. Ascocarp of
A perithecium is another type of ascocarp produced by some sac fungi.
Powdery Mildew.
Unlike cleistothecia, the perithecia have an opening such as a pore or slit,
which allows the spores to escape. Although some perithecia are
globular in shape, many are flask-shaped with a neck terminating in a pore. At the base of the cavity
inside are the asci.
The fungus Sordaria fimicola produces perithecia. Mutant strains of this fungus are available that differ in
the color of their ascospores. The normal (wild type) color is dark green to brown. Observe plates on
which the wild type has been crossed with a mutant that produces tan ascospores. Mature perithecia will
appear black. Look for them along the line of overlap between the two strains.
Select 4 or 5 mature perithecia from the line of fusion using a dissecting needle and transfer them to a
slide. Add a small drop of water a coverslip. Examine the mount under the medium power. Gently press
down on the coverslip with your needle until the perithecia rupture
and discharge the asci. Manipulation of the coverslip will help fan
out the asci for easier examination.
Note that each ascus contains eight ascospores. During ascus
development a single nucleus first undergoes meiosis to produced
four cells, but then each cell divides by mitosisto make a total of
eight. Non-hybrid perithecia have ascospores of only a single color.
However, hybrid perithecia will contain four dark-green and four tan
spores. If the color of ascospores alternates in groups of two, a
genetic event called crossover has occurred, which involves an
actual exchange of genes between homologous chromosomes.
Otherwise, the hybrid asci will have spores segregated in two
groups of four by color. Careful analysis of spore color grouping
allows the estimation of crossover frequency.
Figure 18-4. Sordaria hybrid
perithecium with asci and
ascosopes. Second and third
asci from bottom had no
crossover during development.
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C. Apothecium-producing fungi
Cup or disc-shaped fruiting bodies are called apothecia. They are characterized by having the layer of
asci (called the hymenium) exposed at maturity. Apothecia can vary in size from barely visible to the
naked eye to several centimeters in diameter. Sometimes the cup may be supported on a stalk. The
apothecia are generally soft and fleshy and may be brightly colored. They are produced on the ground, on
wood or leaves and on animal dung in the cool early spring or fall weather.
1. (1) Peziza: a cup fungus.
a.
Examine the fruiting body of Peziza or other cup fungi
on demonstration. The exposed surface of the
apothecium bears the asci and ascospores. Label the
habit sketch of Peziza shown in Figure 18-5a.
Observe the the hymenium (fertile layer) bearing the
asci and ascospores in greater detail. Examine a
prepared slide of Peziza . A portion of a hymenium is
illustrated in figure 18-5b.
b.
2) Morels. The morels are one of the most highly prized
edible fungi. The apothecia are not cup-shaped but
have a spongelike cap with many ridges and pits. The
asci and ascospores line the pits of the cap. Because
they have a stalk and cap, they appear to be
mushrooms, but unlike true mushrooms, they are hollow
inside. Morchella is a readily recognized genus that
fruits in the spring. Observe demonstration material.
Figure 18-6. Morel Ascocarp.
Figure 18-5. Peziza. a. Ascocarps. b. Detail
of hymenium showing asci and ascospores.
D.
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D. Non-Ascocarp Producing Sac Fungi—The Yeasts
Yeasts are simple single cells that do not produce a mycelium or ascocarps. Most yeasts reproduce
asexually by a process called budding in which new daughter cells arise as an outgrowth from a mother
cell. A few yeasts multiply by fission (one cell splits symmetrically into two). Sexual reproduction involves
the formation of an ascus containing ascospores.
1. BuddingSaccharomyces
Saccharomyces cerevisiae is the yeast used in the
majority of commercial fermentations. Make a wet
mount from a sugar solution to which a moist yeast cake
has been added. Examine the mount under high power.
Look for budding cells.The yeast cells are reproducing by
forming lateral protrusions called buds. Look for the
large vacuoles in mature cells.of Schizosaccharomyces
Figure 18.7. Saccharomyces cerevisiae.
2. Schizosaccharomyces
Schizosaccharomyces octosporus is a fission yeast. The cells divide by symmetrical fission rather than by
budding. Make a wet mount from the material provided. Observe the pattern of division in the cells.
Schizosaccharomyces also forms an ascus containing eight ascospores. Prepare a wet mount from the
provided culture and add a drop of iodine. The iodine reacts with starch in the cell walls of the ascospores
to make them more visible. Label 18-8.
a.
b.
Figure 18-8. Schizosaccharomyces octosporus. a. Asexual Reproduction by fission. b. Ascus with
ascospores.
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E. Plant Diseases Caused by Sac Fungi.
Observe the demonstration material provided on several well known plant diseases.
1. Black Knot of Plum and Cherry
The fungus causes conspicuous black-knotty swelling on twigs and
branches of Prunus species.. Infected trees become worthless after a
few years as stunting and limb death occur. Conidia and ascospores
both produce hyphae that invade both healthy and injured wood. The
disease can be controlled with pruning to remove infected parts or by
spraying with protectant fungicides.
Figure 18-9. Black Knot on a
Twig.
2. Ergot of Rye
The disease is most prevalent on rye but also occurs on other grasses. When
the grass heads out, the fungus invades the heads. The result is a sticky,
sugary mass of conidia that attracts insects. Splashing rain and the insects
disseminate the conidia. By the time the seeds mature, the fungus has
developed dark, resistant structures called sclerotia or ergots. These sclerotia
eventually develop perithecia with asci and ascospores, typically in the next
spring. The ascospores are capable of infecting new rye plants.
Figure 18-10.
Sclerotia Formed
by Ergot on Rye
Heads.
The economic losses from the disease are usually minimal but the sclerotia are
poisonous to humans or animals that eat the contaminated grain or even bread
made from it. The toxic substance is a strong alkaloid, specifically a lysergic
acid derivative (LSD). Although the sclerotia can be removed with modern
cleaning machinery, livestock poisoning still remains a problem.
Examine a mount of an infected rye plant. Note the elongated sclerotia
developing on the grain head.
3. Dutch Elm Disease
This may be the ultimate example of a plant disease imported from another continent with catastrophic
results. Introduced into the United States from Europe, it is the among the most destructive shade tree
diseases in this country. All elm species are affected but the fungus is most severe on American elm.
The disease may kill branches and entire trees in anywhere from a few weeks to a few years from the
time of infection. The fungus is a vascular parasite that plugs the water conducting system (xylem) of the
tree, resulting quickly in defoliation.
Bark beetles carry the fungal spores from dying or dead trees and logs to healthy trees. The beetles
establish brood galleries in diseased or dead trees, in which they lay their eggs. When larvae develop into
mature insects, dissemination of the fungus continues. The fungus can also be spread from infected
trees to healthy ones via root grafts too.
The asexual (conidial) stage plays the major role in the disease cycle. Sexual reproduction is apparently
rare in nature.
Systemic fungicides offer some control of Dutch elm disease, but are not very dependable. Disease
resistant varieties have been developed but they have neither the desirable shade nor size traits of
American elm. Observe demonstration material of the disease.
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KEY WORDS
thallus (pl. thalli)
vegetative
hypha (pl. hyphae)
coenocytic (aseptate)
septum (pl. septa)
mycelium (pl. mycelia)
fruiting body
zoospore
rhizoid
sporangium (pl. sporangia)
zygospore
sporangiophore
stolon
heterothallic
homothallic
conidium (pl. conidia)
conidiophore
ascus (pl. asci)
hymenium (pl. hymenia)
ascocarp
ascospore
cleistothecium (pl. cleistothecia)
perithecium (pl. perithecia)
apothecium (pl. apothecia)
budding
fission
wild type
sclerotium (pl. sclerotia)
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Answer Sheet, Laboratory 8