Sexual Reproduction in Fungi: A Lab on Zygomycetes | Study notes Cell Biology

Fungal Reproduction Lab 3/3/09 Page 1

FUN G A L R E P RODUCTION LAB

SE X U A L R EPRODUCTION IN A ZYGOM Y C E T E

In t r o d u c t i o n

The Fungal kingdom contains the simplest multicellular organisms. The

body of a fungus is characteristically made up of threadlike filaments that show

little specialization and are similar in many species. An individual fungal filament

is called a hypha; the entire mass of hyphae of a single fungus is called a

mycelium. The hyphae of a fungus may be septate, because they form cross

walls (septae) between nuclei, or they may be coenocytic and lack such cross

walls. Fungi exist predominantly in the haploid state and do not produce

multicellular diploid forms.

Most fungi reproduce asexually and sexually; both processes usually

involve the production of hyphae that form specialized structures. Sexually

reproducing fungi produce specialized hyphae that are of two different mating

strains. Because the two strains cannot be distinguished visually, they are

designated simply as either "+" or "-." Differences in the patterns and structures

involved in sexual reproduction are the main criteria used to separate the fungi

into four Divisions.

The genus

Phycomyces

is characteristic of the Division Zygomycota. It

reproduces asexually through the formation of sporangia born on top of

sporangiophores. Disintegration of a sporangium results in the release of spores,

which can then germinate and form new mycelia. The hyphae typically do not

form septae.

Phycomyces

reproduces sexually through the production of "+"

and "-" strains of hyphae. Septae are formed near the tips of the hyphae, and

the cells formed by these cross walls are called gametangia. When hyphae of

two different mating strains meet, their gametangia fuse; "+" and "-" nuclei then

fuse to form zygotes. The now single, fused cell containing numerous zygotes

enlarges and forms a hard, irregular wall around itself. This structure is called

the zygosporangium and it gives the division its name. Following a period of

dormancy, the zygotes in the germinating zygosporangium undergo meiosis.

The sporangium that germinates releases both "+" and "-" haploid spores into

the air. Each of these can then form new mycelia.

Ob j e c t i v e s

A. To observe sexual reproduction in a fungus.

B. To observe nonreproductive fungal hyphae.

C. To learn sterile techniques for culturing organisms in the laboratory.

Pr i n c i p l e s

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FUNGAL REPRODUCTION LAB

SEXUAL REPRODUCTION IN A ZYGOMYCETE

Intr od u ction The Fungal kingdom contains the simplest multicellular organisms. The body of a fungus is characteristically made up of threadlike filaments that show little specialization and are similar in many species. An individual fungal filament is called a hypha; the entire mass of hyphae of a single fungus is called a mycelium. The hyphae of a fungus may be septate, because they form cross walls (septae) between nuclei, or they may be coenocytic and lack such cross walls. Fungi exist predominantly in the haploid state and do not produce multicellular diploid forms. Most fungi reproduce asexually and sexually; both processes usually involve the production of hyphae that form specialized structures. Sexually reproducing fungi produce specialized hyphae that are of two different mating strains. Because the two strains cannot be distinguished visually, they are designated simply as either "+" or "-." Differences in the patterns and structures involved in sexual reproduction are the main criteria used to separate the fungi into four Divisions. The genus Phycomyces is characteristic of the Division Zygomycota. It reproduces asexually through the formation of sporangia born on top of sporangiophores. Disintegration of a sporangium results in the release of spores, which can then germinate and form new mycelia. The hyphae typically do not form septae. Phycomyces reproduces sexually through the production of "+" and "-" strains of hyphae. Septae are formed near the tips of the hyphae, and the cells formed by these cross walls are called gametangia. When hyphae of two different mating strains meet, their gametangia fuse; "+" and "-" nuclei then fuse to form zygotes. The now single, fused cell containing numerous zygotes enlarges and forms a hard, irregular wall around itself. This structure is called the zygosporangium and it gives the division its name. Following a period of dormancy, the zygotes in the germinating zygosporangium undergo meiosis. The sporangium that germinates releases both "+" and "-" haploid spores into the air. Each of these can then form new mycelia. Obje ct ive s A. To observe sexual reproduction in a fungus. B. To observe nonreproductive fungal hyphae. C. To learn sterile techniques for culturing organisms in the laboratory. Pr in cip les

Cultured "+" and "-" strains of Phycomyces blakesleeanus will be provided; these will have been grown on potato dextrose agar. You will place samples of both strains near each other in a fresh plate of the same kind of agar, using sterile technique. You will examine your plate one week later, at which time you will identify and examine zygosporangia as well as nonreproductive hyphae. Pr o ced ure s A. Be fo re yo u be gin t his ex er cise , ma ke ce rtain tha t you h av e all of the follow in g mate r ials re ad ily a va ila ble: Culture plates of "+" and "-" strains of Phycomyces blakesleeanus, grown on potato dextrose agar A culture plate containing potato dextrose agar Small forceps Sterile chamber, containing: Gas burner Striker Ethanol (70%), in a wash bottle Black Sharpie pen Dissecting microscope B. Pre par e a cu lt ure plate o f Ph yco m yces blak esleea nu s co nta in ing d iffe ren t m atin g st rains

Turn on the lights in the sterile chamber. Wash your hands thoroughly. Squirt some ethanol on the work surface of the sterile chamber; wipe with a paper towel. Discard the paper towel.
Obtain a fresh culture plate containing potato dextrose agar. Using the Sharpie pen, write your name, lab number and time, date, and " Phycomyces '+' and '-' on the lid of the plate. Place the culture plates of "+" and "-" strains of Phycomyces blakesleeanus and the fresh culture plate in the sterile chamber.
Flame sterilize the forceps. Remove the lid from the Phycomyces "+" plate and, keeping the lid in one hand, remove a piece of the hyphae. Replace the lid on the Phycomyces "+" plate.
Remove the lid from the fresh culture plate and keep the lid in one hand. Transfer the hyphae to the fresh culture plate. Replace the lid on the plate.
Repeat steps #3 and 4 above, removing a small piece of the hyphae from the Phycomyces "-" plate. The two pieced of hyphae should be about 2 cm apart.

Figure 1. Reproductive structures of the Zygomycete Rhizopus stolonifer

Asexual and sexual reproduction in bead mold, Rhizopus stolonifer,

illustrated in figure 1 is similar to that in Phycomyces. Most easily seen are the

branched hyphae that make up the mycelium. This consists of rhizoids that grow into the media and anchor the fungus and stolons that are runners across the media. Sporangiophores rise above the media and terminate in round structures called sporangia. When mature, the walls of the sporangium breaks down and releases haploid spores. The spores are dispersed in the air currents to new habitats. Spores that land on a suitable habitat that is warm and moist germinate into a new population of hyphae. When hyphae of two different strains meet, sexual reproduction may occur. Meeting hyphae come together and form gametangia. The gametangia fuse and develop into a diploid structure, zygosporangium that is resistant to adverse environmental conditions. The zygosporangium contains zygotes. Although most of the structures of Phycomyces resemble those of Rhizopus, the spiky zygosporangium of Phycomyces looks very different from that of Rhizopus. The zygosporangium remains dormant for a while and then the zygotes undergo meiosis, germinate a new haploid sporangium, and new haploid spores are released.

Sexual Reproduction in Fungi: A Lab on Zygomycetes, Study notes of Cell Biology

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