Biology Fungal Lab

1 Name: _______________________ THE WORLD OF FUNGI OBJECTIVES: 1. Critically observe and identify different species representing the major lineages of Fungi and the ways they live. 2. Describe common characteristics (reproductive structures, growth habits) of different fungal lineages. 3. Describe common symbioses in biology, including mycorrhizae, lichens, termite gut protists, and legume and cycad root nodules. TECHNOLOGY REQUIREMENT: This lab requires internet access on a computer. For videos and digital images referenced below, check the Digital Atlas available on D2L. Introduction to the Kingdom Fungi Fungi, like bacteria, are literally everywhere; thus far about 100,000 distinct species have been identified, and it is estimated that at least one million more species await discovery. Most fungi—the yeasts—are single celled. But the most familiar fungi are multicellular mycelia, composed of filamentous strands of cells named hyphae. Multicellularity—the repetition of individual cells, each with an efficient surface-to-volume ratio and nucleus—remains one of the major evolutionary innovations leading to the great diversification of animals and plants. The fungi, however, have achieved evolutionary success by using another solution to the problems faced by unicellular organisms. By flattening or spreading out the cells with the presence of multiple nuclei, fungi have fine-tuned the filamentous form of multicellularity. This coenocytic (multinucleate) habit is common in some fungal groups, like the zygomycetes. All fungi are heterotrophs, either saprobes (decomposers), parasites (feeding on living hosts), or mutualists that form equally beneficial symbioses with other organisms. Because of their filamentous form, each fungal cell is no more than a few micrometers from the soil or water, and is separated from it by only a thin, rigid cell wall made of chitin, a strong nitrogen-rich sugar. They obtain food by first secreting digestive enzymes onto a food source (such as decaying plant matter in the soil or skin cells between your toes), then absorbing dissolved inorganic and organic materials. The fungi, together with the bacteria, are the principal decomposers of organic matter. Fungi are particularly important in plant decomposition, especially the lignin that few other organisms can digest. It is estimated that the top 20 centimeters of fertile soil in a single hectare (2.5 acres) contain nearly five metric tons of fungi and bacteria! 2 The major fungal phyla are distinguished primarily by molecular phylogenetics. In mycelial groups that produce spores during sexual reproduction, identification can be made by the distinct sporangia (such as asci, basidia, and zygosporangia), and sometimes the larger reproductive fruiting bodies (termed mushrooms by the non-specialist). During sexual reproduction, individual mycelia will fuse their hyphal cells in a process of plasmogamy, which produces a multinucleate heterokaryotic (n + n) cell. These cells may continue to divide to produce the fruiting body and sporangia, where karyogamy (the fusion of the nuclei) occurs to produce the diploid zygote. At this point, the zygote will tend to undergo meiosis to produce the haploid spores, which are generally wind dispersed. Mycelial fungi can also produce spores in different sporangia during asexual reproduction. Yeasts are asexual and reproduce solely by budding, a form of mitotic division. Part I. Phylum Zygomycota (zygomycetes, black bread mold, fruit mold) The zygomycetes are the dominant mold- and mildew-producing fungi. Most are terrestrial fungi with multinucleate (coenocytic) hyphae, with the hyphae septate (divided into discrete cells with septa) only during the formation of fruiting bodies. Produce distinctive zygosporangia during sexual reproduction. 700 species described (but most still to be formally named). materials: Images of prepared slides of Rhizopus, the common black bread and fruit mold. 3 1. Observe the slides of Rhizopus and draw what you observe, labeling the hypae and zygosporangia. Pay special attention to look for unfused, about to fuse, and fused hyphae. 2. What is the adaptive significance of spores forming on ends of upright filaments? How do you think the spores are dispersed in this species? Rhizopus 3. Why do think these molds are so successful at contaminating food stuffs? 4. Why do you think molds and mildews are so difficult to treat when they occur in households? Part II. Phylum Ascomycota (ascomycetes, cup or sac fungi) Terrestrial and aquatic fungi; the hyphae are septate but the septae are perforated; complete septae are cut off only in fruiting bodies. Sexual reproduction involves the formation of the ascus sporangium, in which meiosis takes place and within which spores are formed. The hyphae in many ascomycetes are packed together into complex ascocarps, in which asci occur on the upperside of the “cups”. Yeasts are unicellular ascomycetes that reproduce asexually by budding. At least 25,000 species occur in lichens (see Part IV.). > 100,000 species. 4 materials: Images of prepared slides of the cup fungus Peziza; mold-producing Aspergillus and Penicillium; and Saccharomyces cerevisiae, the baker’s and brewer’s yeast. Dried ascocarps of various ascomycetes. 5. Examine a prepared slide of Peziza showing a crosssection through the ascocarp and label hyphae, asci, and ascocarps. 6. How many spores are held within an ascus? What process produced these spores? 7. Observe several specimens of living, dried, or preserved ascocarps. Where are the asci produced on the ascocarp? Peziza 5 Asexual reproduction in ascomycetes: conidia Many ascomycetes reproduce asexually by sporangia called conidiophores; the spores are called conidia. A number of fungi are known to only reproduce in this way, as sexual stages remain unknown to science. These fungi often are recognized taxonomically as “Fungi Imperfecti” and form a polyphyletic group of ascomycetes. 8. Examine the prepared slides of Penicillium or Aspergillus, drawing (and labeling) the conidiophores with their conidia. Penicillium Aspergillus 9. What process produced conidia in these conidiophores? Penicillium is the ascomycete genus responsible for the flavor and blue veins in Roquefort, Gorgonzola, and other blue cheeses (P. roqueforti), and for the white moldy crust on Camembert and brie cheeses (P. camemberti). (Another species in this genus, P. chrysogenum, was used in the discovery of the first antibiotic, penicillin.) 10. Using a dissecting needle, remove a small thin piece of blue cheese containing part of a blue vein. View the wet mount and draw (and label) the mycelium, conidiophores, and conidia. Do the same with part of the white crust of a soft-rind cheese (such as Brie or Camembert), labeling the mycelium. Penicillium in blue cheese Mycelium from soft-rind cheese crust 11. Why does the presence of these ascomycete fungi in the cheese help preserve this food? 6 Single-celled yeasts Single-celled yeasts break down carbohydrates using fermentation into carbon dioxide and alcohol and several species have been domesticated for use in baking and production of beer and wine. Reproduction occurs by budding, in which a single cell divides into two unevenly sized cells, a larger mother cell and a much smaller daughter cell. 12. Examine a prepared slide of the yeast Saccharomyces cerevisiae, drawing (and labeling) cells dividing by budding. (You can observe living yeasts by adding a teaspoon of baking yeast to a half cup of warm [not hot!] water with a tablespoon of dissolved sugar, and then waiting a few minutes for a scum of carbon dioxide bubbles to be produced.) S. cerevisiae (prepared slide) 13. Given that yeasts are asexual, how do scientists know Saccharomyces is an ascomycete? Part IV. Phylum Basidiomycota (club fungi, mushrooms, smuts, rusts) Terrestrial fungi, including the mushrooms and toadstools, with the hyphae septate but the septa are perforated; complete septa cut off reproductive bodies. Sexual reproduction involves the formation of basidia, a club-like reproductive structure, where meiosis produces spores. Fruiting bodies are called basidiocarps. The basidiomycetes are the most diverse group of fungi, and include puffballs, shelf fungi, and two important groups of plant pathogens, rusts and smuts, and many mycorrhizae that occur as forest tree symbionts. > 50,000 species. 7 materials: Images of prepared slides of the shaggy mane fungus Coprinus; Fresh basidiocarps of Agaricus; dried specimens of basidiocarps, including gill spore prints and rhizomorphs. 14. Examine a prepared slide of Coprinus showing a cross-section through the basidiocarp gills, and label hyphae and basidia. 15. How many spores are held within a basidium? What process produced these spores? Coprinus 8 16. Basidiospores can be seen by placing a mushroom cap down on paper. As the cap dries, the spores are shed, leaving a spore print. Spore color is a valuable taxonomic character for identifying basidiomycetes. Draw a picture of a spore print. Spore print 17. Describe the color and pattern of the different spore prints, observing the relationship between the spores and the basidiocarp gills. Are the basidiocarp and the spores the same or different colors? 18. How could you estimate how many spores are present on the gill of a single mushroom? 19. Observe the basidiocarps of Agaricus (Cremeni, portobello, and white button mushroom). Cut a thin slice of the stem or cap of the mushroom and make a wet mount. Do the same for the gills. Draw (and label) the mycelial hyphae, and basidia if visible. Agaricus stem/cap cross section Agaricus gill cross section 9 20. Basidiomycetes produce a variety of basidiocarps with variable morphologies. Look at some of the basidiocarps on display and list some features you would use to create a key that would distinguish between the different species (especially the forms called club fungi vs shelf fungi). Ecological roles of basidiomycetes: forest decomposers The mycelium of saprotrophic basiodiomycetes forms grows extensively throughout the leaf litter and dead wood as it decomposes plant matter. In wood, the mycelia penetrate through vascular cells, producing mycelia termed rhizomorphs (mycelial cords). Cellulose is decomposed quickly, but eventually lignin is also digested. The decomposition of wood is essential for cycling of nutrients and regeneration in forests and other terrestrial plant communities. The largest organism in the world, Armillaria, the honey mushroom, is a common brown rot basidiomycete whose soil and wood-dwelling mycelium covers more than 3 square miles in moist Oregon forests. 21. Observe available wood that displays rhizomorphs. Draw a representative sample, showing how the mycelium grows. Rhizomorphs in wood 10 Part IV. Symbioses in fungi (and other organisms) Although most people consider evolutionary adaptation from the perspective of ruthless competition (i.e., the common phrases “nature red in tooth and claw” or “survival of the fittest”), cooperation is often the better solution. Organisms that cooperate with those of other species are termed mutualists, and their mutually benefitting interactions can confer significant fitness benefits for both parties. When these interactions involve close physical contact, these interactions are sometimes termed a symbiosis. A key consideration in such co-evolutionary interactions is that each partner has increased fitness in the presence of the other partner; in other words, the benefits to each partner must outweigh the costs. In the remainder of today’s lab, we will focus on several famous mutualistic symbioses, several involving fungi, and consider the adaptive benefits each partner gains by cooperating with genetically unrelated organisms. Mycorrhizae Several families of basidiomycetes, some ascomycetes, and an unusual group of fungi in Phylum Glomeromycota (currently considered a monophyletic group sister to the asco- and basidiomycetes) form close associations with plant roots. These fungus/root associations are known as mycorrhizae and play an important role in plant water uptake. Ectomycorrhizae form sheaths around roots and penetrate between root cells. Arbuscular mycorrhizae (sometime termed endomycorrhizae) have hyphae that penetrate within the cell membranes of root cells. The benefit to plants is so immense, that it has been claimed that it is easier to list plant species that lack mycorrhizae (especially arbuscular ones) than to list those that do. materials: Images of prepared slides of pine (Pinus) seedlings with and without mycorrhizae. 22. Observe the prepared slides of pine seedlings with mycorrhizae and without mycorrhizae, drawing (and labeling) the plant and fungal cells. Pine seedling with mycorrhizae Pine seedling with mycorrhizae 23. Based on your observation, is the pine symbiosis an ectomycorrhizal or arbuscular mycorrhizal symbiosis? 11 24. What might the benefits of this association be to the fungal partner? To the plant partner? 25. How might such an association have evolved by natural selection? (Make sure to explain the environment where they occur and the adaptive benefits each partner gains.) 26. Recent evidence has also suggested that mycorrhizal fungi can transfer carbon reserves between different species of trees in a forest! How might this change the perspective of a forest ecologist studying competition between tree species? (Hint: might it be possible for a parent tree to assist its sapling offspring or other relatives living nearby in the forest?) 12 Ascomycetes as symbionts: Lichens Ascomycetes also have evolved to live symbiotically with photosynthetic organisms from two different kingdoms. The most familiar example is lichens, which are composed of a fungus (mycobiont) and either a cyanobacterial or green algal species (photobiont). What might be the benefits of this association to each of these very dissimilar organisms? materials: Images of prepared slides of lichen; living and dried specimens of various lichens. 27. Examine the lichens on display and note the different growth forms: crustose (encrusting), foliose (leafy), and fruticose (erect). Draw a representative example of each form. crustose foliose fructicose 28. Which habitats would these different morphologies be adaptive in, and why? 29. Looking at the sexually mature lichen sample, are the fungal partners in the lichen (mycobionts) ascomycetes, basidiomycetes, or zygomycetes? Why do you think this? 13 30. Observe the squash mount of a living lichen to observe its internal structure. Draw what you observe, identifying the fungal hyphae and algal/cyanobacterial cells. 31. Are the photobionts in this specimen green algae or bacterial cyanobacteria? Why do you think this? Lichen (squash mount) 32. What might the benefits of this association be to the fungal partner? To the plant partner? Root nodules in cycads and legumes Fertilizers (especially in the form of nitrogen needed for production of amino acids and chlorophyll, but also phosphorous for ATP and potassium for DNA and ATP) helps plants grow faster. This is especially critical in nutrient-poor soils, such as are found in tropical rain forests and other habitats. Organic nitrogen originates from nitrogen-fixing bacteria, and two types of plants—the cycads and dicot legumes (peas, beans, soy beans, clover, and peanuts in Family Fabaceae)—regularly culture such bacteria in specialized root nodules. The bacterial partner in cycads is typically the cyanobacterium Nostoc and that in legumes is typically the alphaproteobacterium Rhizobium. materials: Images of prepared slides of cycad rot nodules; clover plants with root nodules. 33. What might the benefits of this association be to the plant partner? To the bacterial partner? 14 34. Observe the prepared slide of the cycad root nodule in a compound microscope. The cyanobacteria tend to occur within a circular zone in the middle of the root nodule, and is sometimes greenish in color. Draw what you Cycad root nodule (cross-section) observe, identifying the plant and cyanobacterial cells. 35. Observe the fresh clover specimen in the dissecting microscope to identify the swollen root nodules containing nitrogen-fixing bacteria. Nodules with the most bacteria are red because of the production of leghemoglobin that sequesters oxygen to make an anoxic environment in the nodule. Draw the roots, and describe where they occur, and how many there are. Clover roots with nodules 36. Farmers regularly practice crop rotation, planting corn and wheat with soy beans (or other legumes) during alternate years. Why is crop rotation a useful practice for farmers to do? 15 37. Make a squash mount of a soybean root nodule to observe the legume root nodule.) Draw what you observe, identifying the plant and bacterial cells. Soybean root nodule Protist mutualists for wood digestion in termites Termites are famous for their damage to wood! But it turns out that these insects lack the enzymes to digest the main components in wood (lignin and cellulose). So how do they do it? Their hindgut is filled with cultures of bacteria and flagellated metamonad protists (themselves an interesting protistan group because they are all anaerobic and lack mitochondria!) that digest the cellulose in the wood ingested by the termites, producing simple sugars that are absorbed and used as a nutrient by the termite. The most common protist genus is Trichonympha. None of these organisms can digest the lignin, so termites defecate lignin-rich feces. materials: Images of prepared slide of termite flagellates; living termite with living protistan mutualists. 38. Observe the prepared slide of a termite hindgut in a compound microscope. You may have to search to find the flagellates as many bits of Flagellates in termite hindgut wood and partially digested material from the gut of the termite are also present on the slide and can easily be confused with the outline of a flagellate. The flagellates look like little “hairy” cells or microscopic lint balls. Draw what you observe, identifying the flagellates. 16 39. If available, you can instead observe living endosymbionts by dissecting out the hindgut of living termites. You can do this yourself, or ask your professor to do it for you.1 a. Obtain a microscope slide, coverslip, and two needles or sharp probes. b. Method I (“forceps dissection method”): i. Use the forceps to obtain a termite and hold it on the microscope slide. ii. Using the two needles, place one on the front end of the termite and one on the abdomen. When these needles are pulled in opposite directions, the alimentary canal will open and reveal the milky-colored gut fluid, which contains a wealth of microbes within. iii. Use the pipette to place a drop of distilled water on the slide near the alimentary canal. This will allow the protozoa to swim from the alimentary canal into the water and allow for easier viewing. c. Method II (“milking method”): i. Use the pipette to place a drop of water on the slide. ii. Use the forceps to grasp the front end of the termite and use a second pair of forceps to gently milk the hindgut, allowing the hindgut contents to drop on to the slide into the drop of water. d. Quickly cover the drop of water/gut fluid with a coverslip to preserve an anoxic environment for the anaerobic microbes living in the gut. 1 Modified from Patterson, R. 2016. I can’t live without you!: A close-up examination of microorganisms involved in mutually beneficial symbiotic relationships. American Society for Microbiology resource guide. 17 40. Observe the video of a living hindgut community in the compound microscope, drawing and identifying what you observe (including plant debris and flagellated protists). The protists will be most vivid if Living termite hind gut commu …
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