Experiment 25A | Lab25 | Virtual Edge | Molb 2021 | College of Agriculture and Natural Sciences

Background & Introduction:

Fungi are a diverse group of eukaryotic organisms that usually grow best in dark, moist habitats.  They receive their energy from outside sources by secreting, into the environment, enzymes that digest organic matter.  Most fungi are  saprophytes; the organic matter from which they take their nutrients is dead.  Thus these organisms play an essential role in decomposition.  They are also economically important in food, beverage and antibiotic production.  Conversely, many fungal species are pathogenic, causing diseases that range from athletes foot to ergot.

Fungi include both the unicellular, non-filamentous yeasts and the multicellular, filamentous molds. Yeasts are typically spherical or oval in shape and are widely distributed in nature (found on fruits, leaves of trees, etc).  Yeasts reproduce asexually by budding, a process by which a new cell forms as a protuberance (bud) from the parent cell.  Molds come in many sizes, shapes, and forms, ranging from mushrooms, puffballs and large bracket fungi found on trees to the small colonies often found as contaminants on moldy fruits and cheeses. 

The body of a fungus is called the thallus.  It can range in size and can be either a single cell or multicellular.  The cells within the thallus are coated with a cell wall made of a strong polysaccharide called chitin.  Long, branched filaments called hyphae  further characterize a mold.  These hyphae form a tangled web that is referred to as mycelium.  Hyphae are composed of an outer cell wall and an inner lumen and are either septate (have cross walls) or coenocytic (protoplasm streams throughout).  

Most molds are capable of both sexual and asexual reproduction.  Asexual reproduction can occur either by central constriction of a parent cell to form two daughter cells or by spore formation.  There are several types of spore formation.  When hyphal fragementation occurs, the resulting spores are termed  arthrospores.  If the cells are surrounded by a thick wall before hyphal fragmentation, the spores are instead called chlamydospores. Spores that develop within a sac (sporangium) at a hyphal tip are referred to as sporangiospores.  If spores are not produced in a sac, but do develop at a hyphal tip or sidewall they are called conidiospores.  Finally, spores produced from a vegetative cell by budding are blastospores.  Sexual reproduction involves the union of two nuclei.  Sexual life cycles vary from one fungal species to another.  However, most have a diploid stage, which involves the formation of a spore that can survive in harsh external conditions.

Classification of Fungi:

Fungi have been classically characterized and classified by the appearance of their colony (color, size, etc), hyphal organization (septate or coenocytic), and the structure and organization of reproduction spores. More recently, however, ribosomal RNA sequences are being used to further categorize these organisms. Many disagreements exist in the literature regarding the placement of several genera into their respective phyla. The following classification scheme is based largely on Prescott et. al., Microbiology. Some useful information regarding hyphae type was retrieved from http://bugs.bio.usyd.edu.au/learning/resources/Mycology/contents.shtml and http://bugs.bio.usyd.edu.au/learning/resources/Mycology/StructureFunction/introduction.shtml. We will look at six fungal divisions. Two more (Urediniomycetes and Ustilaginomycetes) are often classified as Basidiomycota and as such are not listed as separate divisions here.

Table #1:

Group	Common Name	Hyphal Organization	Reproduction Characteristics	Example
Chytridiomycota	Chytrids	coenocytic hyphae  (if present)	Asexual: motile zoospores Sexual: sporangiospores	Allomyces
Zygomycota	Bread molds	coenocytic hyphae	Asexual: sporangiospores Sexual: zygospores	Rhizopus stolonifer
Ascomycota	Sac fungi	septate hyphae	Asexual: conidiospores Sexual: ascospores	Saccharomyces cerevisiae Aspergillus Penicillium
Basidiomycota	Club fungi	septate hyphae	Asexual: often absent Sexual: basidiospores	Mushrooms
Glomeromycota	Mycorrhizae	coenocytic hyphae	Only asexual reproduction known via spores or fragmentation	Acaulospora
Microsporidia	Often still referred to as protists	N/A	Asexual or sexual (complex life cycle)	Entero-cystozoon

Chytridiomycota (chytrids):
The simplest of the fungi, the chytrids are microscopic and found in freshwater, mud, soil and sometimes the rumen.

Zygomycota (bread molds):
Members of the subdivision Zygomycota have coenocytic hyphae. Asexual reproduction is via sporangiospores, which can be released from the sporangium and carried by air currents. When the spores reach an appropriate substrate, they germinate to produce new hyphae. Bread molds do not usually cause human disease. In fact, in some countries they are used in food production. Rhizopus, however, is an opportunistic human pathogen; it is especially dangerous to people with diabetes mellitus that is not well controlled.

Ascomycota (sac fungi): 
Members of the subdivision Ascomycota include molds that have septate hyphae and some yeasts. They are called sac fungi because their sexual spores, called ascospores, are produced in a sac or ascus. Asexual reproduction is via conidiospores. The Ascomycetes include fungi that cause chestnut blight and Dutch elm disease. Claviceps purpurea is a parasite on rye grass that causes ergot.

Basidiomycota (club fungi):
Basidiomycetes also possess septate hyphae. The sexual spores, called basidiospores, are produced by a club-shaped structure called a basidium. In mushrooms the basidia are found along the gills or pores on the underside of the cap. Some mushrooms produce toxins that are lethal to humans.

Glomeromycota (mycorrhizae)
Most are mycorrhizal fungi that form a mutualistic symbiosis with the roots of plants.

Microsporidia 
Obligate intracellular parasites of fish, humans and insects

Procedure:

Ascomycota

Make a wet mount slide of Saccharomyces cerevisiae using the following procedure:

Place 3-4 loopfuls of water on the slide.
Flame an inoculating loop and allow it to cool.  Pick up a single colony of Saccharomyces cerevisiae and mix it into the drop of water on the slide.
Place a cover slip over the mixture.  Be careful to avoid bubbles.

Observe the slide under high dry and oil immersion.  Try to identify the budding cells.  Make a sketch in the results section.

Observe the colonies of Aspergillus niger and Penicillium camembertii. Describe the morphology and draw an example of each colony in the results section. View the plates under the dissecting microscope. Make a sketch and try to identify the hyphae and buds. DO NOT REMOVE THE PLATES’ LIDS.

2. Basidiomycota

Observe the oyster, shitake and portabella mushrooms.  Note the gills on the underside of the cap.  Make a wet mount of a portabella gill using the following procedure:

Using a razor blade, cut out a portion of a single gill and place it onto a microscope slide. 
Place several drops of sterile water onto the gill and cover it with a cover slip.

Observe this slide under the low power and high dry objective lenses.  Make a sketch in the results section.  Be sure to label the basidia covering the gill.

3. Zygomycota

Observe the colonies of Rhizopus stolonifer.  Describe the morphology and draw an example of a colony in the results section.  View the plate under the dissecting microscope.  Make a sketch and try to identify the hyphae and sporangia filled with black spores.  DO NOT REMOVE THE PLATE’S LID.