The members of the phylum Zygomycota are an interesting group of fungi in that they seem to bridge the gap between the so called "lower fungi" (slime molds and flagellate fungi) and the "higher fungi" (Ascomycota, Basidiomycota, and Deuteromycota). The Zygomycota typically reproduce asexually by means of non-motile sporangiospores. Sexual reproduction is by gametangial copulation and results in the formation of zygospores. Some species are heterothallic and have a bipolar mating type system which is biallelic but there are some which are homothallic. In most species, a well-developed coenocytic mycelium is formed, but in parasitic forms, the thallus is often much reduced. The cell wall is composed of chitin and chitosan. They are typically saprobic, but a number of species are parasites of man, other animals, fungi and plants. Some are useful in the production of pigments used for food coloring, production of modified steroids, alcohols and fermented oriental food such as tempeh. They are a diverse group occuring in a wide variety of habitats. For example, the class Trichomycetes is an assemblage of fungi which have a simple or branched thallus attached by an extracellular holdfast to either the cuticle lining the digestive tract or external surface of an arthropod. The association is thought to be a form of commensalism. This group will not be considered further in this exercise.
In today's exercise, we will examine some of the members of two orders of the class Zygomycetes - Mucorales and Entomophthorales. It is however worth mentioning an important order, the Endogonales. Members of this order form mycorrhizal associations with roots of higher plants. The association is thought to be symbiotic with the plants benefiting from the ability of the fungus to absorb scarce mineral from the soil. The fungus, on the other hand, obtain its supply of carbohydrates from the plant. It is now believed that these fungi are ubiquitous and that almost all plants have mycorrhizal associations for survival.
BIOLOGY OF ZYGOMYCETES
Sexual reproduction in the Zygomycetes is similar to the Oomycetes in which hormone signals are sent between mycelia of opposite mating types. These signals cause differentiation of the mycelium to form sexual structures. The signalling between the two mating types was first observed in 1924 by a mycologist named Bergeff using the species Mucor mucedo. The following is a summary of his initial experiments:
1. If both + and - strains were inoculated together in the same liquid culture and cell free extracts taken and used for inoculation of media for the two strains separately, the cell free extract could stimulate development of progametangia in both strains.
2. Cell free extracts of either + or - strains grown separately did not stimulate zygospore formation in either type.
3. If + spores are inoculated in cell free extract of - strain-grown culture extract, and following growth of the + strain, a cell free extract is taken, it could stimulate zygospore production in either strain.
4. Mixed culture filtrates from separate + and - cultures did not allow zygospore production in either strain.
The correct interpretation of these experiments indicate that there is a constitutive hormone produced by each strain, the + strain producing a hormone called + progamone and the - strain producing - progamone. These hormones induce each strain to produce another hormone, the + strain producing + gamone, and the - strain producing - gamone, which causes hyphae of each strain to develop structures called progametangia. When the progametangia from the different strains come into contact, it induces the formation of structures called gametangia. The zygosporangium is formed after the walls of the gametangia break down and a single zygospore is formed. The zygospore is the only diploid structure in the life cycle of zygomycetes.
SOME TERMS TO KNOW
When you try to key some of the fungi found in the class Zygomycetes, you will come across some terms which you will need to know in order to identify the genus.
apophysis: A swelling or a swollen filament at the end of a sporangiophore below the sporangium
circinate: twisted round, coiled
deliquescent: becoming liquid after maturing
sporangiola: a small sporangium without columella and having fewer than ten spores
sterigma (pl. sterigmata): an extension arising from the sporangiophore which gives rise to the sporangiospores
stolon: a runner going from one sporangium to the next
verticillate: whorled in appearance
merosporangium: a cylindrical sporangiolum
In this exercise, you will follow the given key to confirm the identity of the different species, draw and note their distinguishing characteristics and compare what you see under the dissecting and compound microscopes with the provided illustrations and electron micrographs.
I. ORDER MUCORALES
The Mucorales characteristically produce a profuse, well-developed mycelium that is fast growing. Most species are saprobic. Members of the Mucorales are among the most common of fungi and are readily isolated from a wide variety of habitats. Asexual reproduction is by means of sporangiospores in sporangia or sporangiola and sexual reproduction is by production of zygospores in zygosporangia.
A. FAMILY MUCORACEAE
In this family, all of the sporangia are borne singly with thin walls and multiple sporangiospores inside. The sporangia are always columellate.
1. Mucor - no rhizoids or stolons and the sporangium lacks an apophysis. Mucor spp. are commonly found in soil, dung, decaying vegetation, and stored grains. Some species are used commercially in the production of tofu, Chinese cheese and bean cake; others cause zygomycosis in humans.
a. Observe cultures growing on PDA and malt extract under the dissecting scope. Describe what you see.
b. Make a mount in water of sporangia of Mucor hiemalis and note the columella and sporangiospores. The sporangium wall is fragile and breaks away easily, leaving the columella exposed. The columella in intact sporangia can be best observed in young material lightly stained with cotton blue.
c. Some species of Mucor are known to exhibit dimorphism. Under normal conditions, they grow like normal mycelial fungi but when oxygen is limiting or when the carbohydrate content of media are higher than normal, they grow like yeast. We will try to see if M. hiemalis is dimorphic. We will inoculate malt extract broths with twice the amount of glucose called for by the recipe and shake in a 25°C incubator. Some PDA plates will also be inoculated and placed in a Brewer's jar.
d. Observe mated + and - strains and look for zygospores under the dissecting scope and prepare wet mounts.
2. Rhizopus - Colonies of Rhizopus produce rhizoids and stolons and the spherical sporangia have an apophysis.
a. Mount sporangiospores of Rhizopus nigricans in water and observe sporangia, spores, columella, stolons and rhizoids. The columella is best seen in young sporangia.
b. Observe the mated + and - strains under the dissecting scopes and observe stages in the formation of zygosporangium. Try and see if you can follow the stages in the attached life cycle of Rhizopus.
3. Zygorynchus - This genus resembles Mucor, but the species are all homothallic, with unequal suspensors. They are commonly isolated from soil.
a. Make a mount of sporangia and zygosporangia of Z. heterogamus and observe under the microscope.
4.Absidia - this genus is characterized by pyriform sporangia which are borne terminally on sporangiophores arising along the stolons. The zygosporangia are borne between opposed suspensors which often bear a whorl of circinate appendages on one or both suspensors.
a. Observe cultures of A. spinosa under the dissecting scopes and mount some sporangia and observe under the microscope. Record all your observations.
5.Phycomyces - the sporangia are borne on very long, unbranched sporangiophores over 8 cm tall, and which have a metallic luster.
a. Observe Phycomyces bleekesleanus growing on big jars. Note the rate of growth by measuring sporangiophore heights in cultures growing in the two jars inoculated at two different times.
b. Phototrophism and Geotrophism in Phycomyces - Phycomyces is known to exhibit positive phototrophism and a negative geotrophic response. To demonstrare these responses, we will cover one jar with foil and just expose a small area to a light source and rest the jar horizontally. The other jar will be covered with foil entirely. Both jars will be kept at 25 C. During the next lab period, we will observe the responses of these cultures.
c. Examine plates of mated + and - strains under the dissecting scopes and observe the tong-shaped gametangia and stages of zygosporangiun formation. Note the suspensors which bear branched, finger-like projections. Compare this zygosporangium with that of Rhizopus.
6.Circinella - This genus is characterized by globose columellate sporangia with spherical sporangiospores. The sporangiophores are branched and mostly circinate.
a. Observe cultures of C. muscae under the dissecting scope and mount some sporangia. Note their location - borne terminally on circinate branches.
B. FAMILY PILOBOLACEAE
Pilobolus, Pilaira, and Utharomyces are genera belonging to the Pilobolaceae. The first two genera are commonly found growing on the dung of herbivores where they produce distinctive asexual reproductive structures. Their sporangia are columellate, with a thick, cutinized wall and are forcibly discharged.
1. Pilobolus (the hat or discus thrower) - In Pilobolus, the sporangium is borne on a swollen subsporangial vesicle.
a. Examine fresh material growing on rabbit dung under the dissecting scope. Locate the sporangia and subsporangial vesicle and note the numerous water droplets present in these structures. Observe sporangial discharge.
b. Light responses of Pilobolus - Like Phycomyces, Pilobolus is also affected by light. The sporangium is shot towards a light source. We will try to examine these responses by doing some simple experiments.
1. What happens to the sporangium if there is no light? We will remove the cover of one culture and replace it with a paper cup which we will cover with foil entirely to simulate total darkness.
2. How accurate is the shooting of the sporangium towards a light source? We will do the same thing as above, only that we will put a hole through the foil on top of the inverted cup and replace this hole with a clear cellophane tape or plastic. We will then point a light directly towards this hole.
3. Does the intensity of light affect the sporangium discharge? Same as above except that we will cover the hole with an opaque plastic.
4. Does wavelength have an effect? Same as above but this time cover one hole with a piece of red cellophane while the other culture will have a hole covered with green cellophane.
5. We will expose one culture under diffuse ligth, that is not to be covered with the inverted paper cup and see the effect if light is coming from many directions.
C. FAMILY THAMNIDIACEAE
The sporangia of this family when present is columellate. The sporangiophore bearing the sporangia is branched and bears secondary sporangiophores with sporangiola.
1. Thamnidium - In Thamnidium, the branches bearing the sporangiola are dichotomously branched.
a. Observe cultures of T. elegans under the dissecting scope. Make a mount of in water and observe the sporangiola under the microscope.
D. FAMILY CUNNINGHAMELLACEAE
In this family, there are no sporangia formed and there are only one celled sporangiola present. Some mycologists refer to these asexual propagules as conidia.
1. Cunninghamella - In this genus, the sporangiophores are branched and indefinite in length, with the branches ending in fertile vesicles. The sporangioles can be smooth or rough mostly ovoid or round.
a. Observe cultures of Cunninghamella sp. under the dissecting scope. Make a mount in water and observe under the microscope.
b. Observe plates of mated + and - strains and look for zygospores and prepare a wet mount. Record all observations.
E. FAMILY MORTIERELLACEAE
Members of this family have sporangia without a columella. Their zygosporangia are surrounded by a large number of short hyphae that form a cottony envelope around them.
1. Mortierella - Found commonly in soil with sporangia formed on awl-shaped erect sporangiophore which may or may not be branched.
a. Observe cultures of M. isabellina under the dissecting scope. Mount some sporangia and sporangiophores and note their characteristics as stated above and in the key.
F. FAMILY SYNCEPHALASTRACEAE
This family contains the single species, Syncephalastrum recemosum, a heterothallic inhabitant of soil and dung and of worldwide distribution.
1. S. racemosum - The merosporangia of this genus are borne on swollen tips of sporangiospores. Observe cultures under the dissecting scope and make a mount of the sporangiospores and observe merosporangia under the microscope.
II. ORDER ENTOMOPHTHORALES
This is a small order of mycelial fungi that parasitize animals such as insects, or they occur saprobically in soil or on dung. Asexual reproduction is often with spores that are forcibly discharged. There is one family, the Entomophthoraceae.
In Entomophthora, the spores are smooth, produced outside the host and forcibly discharged. Examine prepared slides of sections through an infected fly and observe the sporogenous cells and spores and their relation to the host.
