The fungi lack photosynthetic pigments. As a group, they are heterotrophic and must obtain their food either from dead organic matter (saprophytically) or from living organisms (parasitically). Approximately 100,000 species have been described, and these are very widely distributed, particularly on land, although there are a number of common water-growing species. You will attempt to get some idea of the diversity of form that occurs among the fungi.
The fungi can be divided into six divisions: Myxomycophyta, Chytridomycota, and Oomycota (in kingdom Protista), Zygomycota, Ascomycota, and Basidiomycota (in kingdom Fungi). Classification of fungi is controversial and based primarily on their sexual reproduction. A seventh artificial division, Deuteromycota, includes all species for which no sexual stages are known at present. Deutero- means "duty"; thus mycologists consider it their duty to discover the sexual stages and classify these species into the natural divisions...easier said than done. Most are clearly members of Ascomycota and a few are clearly members of Basidiomycota.
The true fungi differ from the slime molds in producing a vegetative body which is composed of masses of fine branched filaments often having a cottony appearance. A single filament is called a hypha, while a mass of hyphae makes up the mycelium. Mycelia may form reproductive structures, usually both asexual and sexual. While the mycelia of different species are very much alike in gross appearance, the reproductive structures are distinctive.
You will look at a few rather typical representatives of the fungi. Make a record that will give some idea of the diversity of form that occurs among the fungi and will point out some of the differences among the kinds of fungi. You should use the life cycle diagrams at the end of this exercise to draw the structures at their respective positions.
The slime molds are characterized by a naked mass of protoplasm, a plasmodium, which moves in an amoeboid fashion and can engulf bacteria and other solid food materials. At the end of its growth phase, the plasmodium produces a spore-producing structure, the sporangium, from which spores are liberated to reproduce the organism.
Observe the cultures of Physarum. These have been grown on moist oatmeal on 2% agar; some are older and others were transferred yesterday onto plain agar. In these young cultures you can readily see the plasmodial stage under the dissection microscope or low power of the compound microscope. Observe streaming in the protoplasm. You may recall that the plasmodium is multinucleate and each nucleus is diploid.
Do all the streaming organelles flow in the same direction?   yes   no
Put an area of active flow under observation for several minutes.
Does the cytoplasm always flow at the same rate?   yes   no
Does the cytoplasm ever change its direction of flow?   yes   no
An older culture may have formed sporangia. The sporangium in Physarum is often a rather amorphous structure, but in other genera it has a very delicate, complex, and characteristic shape. Observe the sporangium of one of the other genera, such as Stemonitis or Arcyria.
These are considered by some to be the most primitive of the true fungi but are much more similar to some of the algae in appearance and reproduction than are any of the other fungi. Most species are aquatic, though several are terrestrial. Divisions grouped here include both saprophytes and parasites. You will look at a few representatives.
If available, observe the culture noting the bright orange color of some of the areas of the plate. Make a water mount of some scrapings in one of these areas, and observe under the microscope. You will notice that there are two structures which occur in pairs; one is nearly colorless (the female gametangium) the other is smaller and bright orange (the male gametangium). Originally these structures are single haploid cells which divide internally to form gametes.
The paired structure can, therefore, be considered part of a ____________-phyte generation.
The gametes released from both gametangia are motile, and you should be able to watch some of the gametangia in your wet mount release gametes after some time.
The female gamete is larger than the male gamete, so the type of fertilization is ________-gamy.
Later in the period, you should be able to observe larger gametes paired with smaller gametes in your wet mount (providing you have kept it moist). Zygotes appear in about 6 hours.
As a result of syngamy, these zygotes represent the diploid ____________-phyte generation.
Make a water mount of Saprolegnia if available by putting the slide in the water under one of the seeds or insects on which the fungus has been growing for about one day, and raising it so that the mycelium is spread out on the slide just as it is in the water. Observe with the microscope. If a living culture is not available, observe a prepared slide of Achlya.
The vegetative mycelium is coenocytic, so cross walls (septa) normally form only to cut off a reproductive cell. It may be possible to find evidence of both asexual (in the younger cultures) and sexual reproduction (in the older cultures). The asexual spores, produced in an elongated, slightly enlarged cell (zoosporangium) at the end of a hypha, are motile (zoospores). In a living culture you may find a zoosporangium about to discharge through an apical pore or what look like paramecia (really zoospores) swimming around. The prepared slide of Achlya may show these in a stained and fixed way (blah).
Its sexual reproduction is oogamous with several eggs produced in almost spherical oogonia and sperm nuclei in narrow antheridial hyphae growing around the oogonia. These should be observable in older cultures or prepared slides of Saprolegnia. Although you are not studying this life cycle in detail, you should be able to recall the basic ideas of the ____________-gamous life cycle. This life cycle will be investigated in more detail in the algae exercise. These fungi with cellulosic walls may represent algae which have lost their chloroplasts and thus are probably very remote relatives of true fungi. An interesting aspect of the Saprolegnia life cycle is that hyphae of only one strain are required for successful sexual reproduction; this condition is called homothallic.
If the Saprolegnia culture you are using was made with dead insects, the hyphae that you observed are therefore [   saprobic   parasitic   ] on the flies.
Saprolegnia can also be found growing on live fish, making the hyphae [   saprobic   parasitic   ] on the fish.
Prepared slides will show you how downy mildew of grapes infect plant leaves. The species shown is probably Plasmopara viticola growing on grape leaves. You should be able to find this organism growing inside the cells of the grape leaves. Make a sketch of what you find and label it completely.
DO NOT OPEN ANY RHIZOPUS CULTURE if available! Examine the lid of the Rhizopus cultures with the dissecting microscope, if available.
These cultures will show only asexual reproduction; notice the obvious, dark sporangia borne at the ends of upright branches of the coenocytic mycelium. These contain a great many, small, black spores which are easily wind distributed (DO NOT OPEN THE CULTURE)! These spores may contaminate the building and cause allergic reactions in some persons. The upright sporangiophores are connected to each other by stolons and to the medium by rhizoids. Rhizopus and Phycomyces are heterothallic, so two strains are required for successful sexual reproduction.
|Why do you think that Rhizopus cultures show no sexual reproduction?|
If available, examine the plate cultures of Phycomyces under the dissecting microscope. In this case, two strains were inoculated at opposite sides of the Petri dish. Where the two strains have grown together, you should be able to observe a dark line composed of dark structures called zygospores. In the zygomycetes, hyphae of the two strains contact each other and the hyphal tips each divide off to form a gamete cell. The two gamete cells, which are in contact, fuse to form the zygote. The zygote develops a thick, dark wall, and enters a resting period as a zygospore. Meiosis occurs inside this resting spore or inside a sporangium that grows out of it.
This is the largest group of fungi and includes many economically-important species: some are pathogens of higher plants, others produce valuable substances such as alcohol or penicillin. All are characterized by a sac-like structure, called an ascus, which contains ascospores. The ascus is produced after sexual fusion, normal within an ascocarp, a structure of characteristic size and shape formed by an aggregation of hyphae. The asci occur in large numbers, forming a continuous layer, the hymenium, in the ascocarp; the ascospores are produced by meiosis. Asexual reproduction is usually by means of conidiospores cut off mitotically at the ends of certain special hyphae.
Observe slide cultures of Penicillium, the Fungi Imperfecti stage of an ascomycete, which is a saprophyte commonly occurring on rotting fruit, etc. Observe under the microscope. If these are not available, a prepared slide showing three fungi: Penicillium, Aspergillus, and Rhizopus together will provide a source for sketches. These imperfect stages are haploid in most cases.
Is the mycelium septate or coenocytic?   septate   coenocytic
Do you find any hyphae producing conidia?   yes   no
Yeast (Saccharomyces) is also an ascomycete, although it does not produce a typical mycelium. Obtain a drop from the yeast culture and observe under the microscope. If the culture is actively growing, the cells will be budding, a form of asexual reproduction by cutting off small cells at the surface. Yeast breaks down sugars to alcohol and carbon dioxide (fermentation) and is used commercially to produce both products; for example, in wine and bread making. A plate culture may contain cells that have fused to form a zygote that becomes and ascus and ultimately contains ascospores. Make a wet mount of a tiny bit of the culture to see if you can find asci containing ascospores.
How many ascospores are there in each ascus?   1   2   4   8
A plate of Sordaria should be available. Use a dissection needle or two to pluck one of the tiny black structures from the agar. This structure is a perithecium. This ascocarp is flask-shaped and has an apical opening pointing up toward the cover of the dish. Put two or three perithecia in a drop of water on a microslide. Add a cover slip and observe under low power. If water tension was sufficient, the perithecium may have ejected a bundle of asci. If not, you should remove the slide from your microscope and provide slight pressure on the cover slip with your thumb. Not too much pressure...just enough to crack open the ascocarp. Observe.
Are the vegetative hyphae septate or coenocytic?   septate   coenocytic
If septate, is the septum complete or incomplete?   complete   incomplete
How many ascospores do you find in each ascus?   1   2   4   8
Sordaria is homothallic and produces a perithecium (ascocarp) even when only one strain is present. When two genetically different strains are present, they hybridize where the mycelia come into contact. Please notice that syngamy is a two-step process (plasmogamy and karyogamy). In most other organisms the two steps occur more-or-less simultaneously. In higher fungi (Ascomycetes and Basidiomycetes) these steps are separated in time and a protracted dikaryotic (heterokaryotic) phase results. The fusion cells (the results of plasmogamy) produce the dikaryotic perithecium which is lined by the hymenium consisting of many asci. In each ascus, the two nuclei united (karyogamy occurred) and the diploid cell divided meiotically (four products) and then mitotically to produce eight haploid ascospores. The ascus contains these spores and, by virtue of small diameter, holds them in order of their production by meiosis. The combinations of black and tan spores held in a hybrid ascus of Neurospora (a related ascomycete) prompted the discovery of meiosis!
After observing the wild-type asci from the ascocarp demonstration plate, you might want to record some of the combinations you see in the asci inside perithecia taken from the Sordaria crossing demonstration plate. The hybrid perithecia should be found at the borders between the zones of wild-type (black) and mutant (tan) mycelia. In study time, try to demonstrate what chromosomal arrangements in meiosis would give rise to the order of spores you have observed.
You have seen a sequence of ascocarps of increasing enclosure. What might be the adaptive advantage of enclosing the asci?
These are only the beginning of the complexity of forms in ascomycetes. Before you finish your observations of this group, you should look at prepared slides of some larger ascocarps:
Daldinia, an ascomycete in which the perithecia are embedded in a larger body called a stroma. Find the asci in a perithecium and determine the number of ascospores in the asci.
Helvella, the saddle fungus with an exposed and folded humenium. The gross structure of this ascocarp looks very much like a mushroom with a “cap” and a “stalk.” Your slide may only show the folded cap. Try to find the asci and count ascospores in each.
Morchella, the morel mushroom. This one is considered a delicacy to eat. The hymenium is highly convoluted and in the form of a cone on the end of a stalk. Examine the hymenium closely for asci. Count the ascospores in several asci.
Tuber, the truffle. This is also a large edible fungus. The ascocarp is hypogeous and is a cleistothecium. Dogs and pigs are used to locate them by scent. Huge prices (higher than gold) are paid per ounce for gourmet varieties of truffles.
While this division is not as large as Ascomycota, it is probably better known to you. It includes the common mushrooms, toadstools, bracket fungi, puffballs, and the rusts and smuts. The latter two groups are parasitic on many of our important crop plants. We are rarely aware of the vegetative mycelium of the first four groups; it grows in the organic matter of soils, in rotting logs, etc. The mushroom, etc., with which we are familiar, is the reproductive structure or basidiocarp formed by an intricate mass of interwoven dikaryotic hyphae. Basidiocarps vary greatly in appearance in different genera, but all bear basidia which are usually arranged in a layer of hymenium. The basidium, like the ascus, is the site of karyogamy and meiosis. The basidium is a club-like cell at the end of which are four short stalks, each of which bears one of the four basidiospores.
Pull apart parts of the rotting wood if available in the laboratory. The network of fine threads is vegetative mycelium; it may give a root-like appearance which is called a rhizomorph. You might also see these in a culture of wood-rot fungi: Armillariella...the mycelia are bioluminescent in this case. Give your eyes a good five minutes to adjust to darkness and observe the plates.
Observe the gross aspects of a common edible mushroom (Agaricus). Look at the under-surface of the cap of the mushroom; note the gills radiating from the center to the periphery. The surface of the gills is a hymenium made up of closely packed basidia bearing basidiospores.
Examine prepared slides of Coprinus, a mushroom, which shows sections of gills lined by basidia with basidiospores. Most of the latter will have become detached from the basidia during the preparation of the slide. Note the intertwined hyphae which make up the tissue of the gills.
Are these hyphae septate or coenocytic?   septate   coenocytic
Closely examine basidia along the surface of the lamellae (gills) to find one showing all four basidiospores on the tips of four sterigmata (tips). If you find a good one, call the instructor over so the rest of the class can see this.
Schizophyllum is a bracket fungus usually found on tree bark. The white basidiocarp is fan-shaped and usually grows out over the surface of the bark with gills facing the bark. This species has been studied widely in fungal physiology, genetics, and morphogenesis. The instructor will help you establish three cultures of this fungus (using "sterile" technique) which you can orient horizontally, vertically, and upside-down to observe the effect of gravity on basidiocarp formation. As basidiocarps form (may take days to weeks time) be sure to observe their shape and orientation with respect to gravity. Make sketches of their orientation and shape on the back of this page.
Some mushrooms you pick up on a hike in the woods will not have gills under their caps. They may have a sponge-like hymenium composed of may pores. These are the so-called polypore mushrooms. Examine a slide of some of this hymenium in Polyporus. Locate basidia and basidiospores.
There will likely not be time in class to examine the slides of Puccinia graminis, the barberry-wheat rust. Too bad because this is a wonderfully interesting life cycle involving two hosts and three separate kinds of spores. As you might guess, our survey of the fungi has been very cursory and superficial. We have looked at a kingdom-and-a-half in a very short time. At many universities there might be a whole department studying fungi!
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