The phylum Ascomycota is possibly the largest group of fungi with an estimated 28,650 species representing 2,720 genera. This figure is on the rise as more and more of the teleomorphic stages of the Deuteromycota are being found and described as having ascomycete characteristics. Members of the Ascomycota are widespread in distribution and can be found in almost all types of habitats. Many are of considerable economic importance, as food, in industrial processes, and as causes of various diseases in plants and animals.

Characteristics of the ascomycetes:

1. sexual spores (meiospore n)-ascospores that develop in an ascus; forcibly discharged

2. ascus develops from a zygote, 2N vegetative cell, or ascogenous hyphae

3. ascogenous hyphae-dikaryotic either homokaryotic or heterokaryotic; not free-living

4. vegetative stage-one-celled or filamentous; hyphae with cells delimited by septa

5. septa perforate-permits nuclear migration and formation of heterokaryons

6. cell wall usually b,1-3 or 1-6 glucans or mannans, also chitin

7. all except Hemiascomycetes have ascocarps

8. many have asexual stage-pleomorphic

9. heterothallic or homothallic

10. heterothallic are bipolar and biallelic

11. sexual reproduction is by:
a. gametangial fusion: establishment of 2N or N+N mycelium occurs when there is a fusion of morphologically similar gametangia; this process is similar to Zygomycota.
b. gametangial contact: fusion of morphologically distinct gametangia; these are known as the antheridia and ascogonia.
c. spermatization: fusion of spermatia or conidia and ascogonia; spermatia are similar to single-celled asexual spores (conidia), but the spermatia do not germinate and cannot form a free- living state. On the other hand, the conidia can form a free-living state.
d. somatogamy: fusion of vegetative cells or hyphae.
e. conidiation: spores are able to function both as the fertilizing agent or conidia (Ex. Neurospora).

Once the 2N or N+N stage has been formed, the hyphae are now known as ascogenous hyphae and is not a free-living stage. The ascogenous hyphae go through unique process to form the ascus (refer to handout).

ANAMORPH + TELEOMORPH=HOLOMORPH

A simple but sometimes confusing nomenclature system describing the different stages of an ascomycete during its life cycle. This deals with whether asexual or sexual spores are being produced. If only the asexual spores are produced, the fungus is said to be in its anamorphic stage. If only the sexual spores are produced, the fungus is in its teleomorphic stage. Both the anamorphic and teleomorphic stages together form the holomorphic stage. The holomorph name of the fungus usually takes the name applied to the teleomorphic stage. For example, if Geotrichum candidum is only producing asexual spores or conidia, it is in its anamorph stage. When the same species is producing sexual spores it is given a teleomorphic name, Endomyces geotrichum. The holomorphic name of the fungus is also Endomyces geotrichum. Note that this is the same fungus given two different names depending on what kind of spores it is producing at the time. If the fungus produces two or more different types of asexual spores, it can have just as many different anamorphic names.

CLASSES OF ASCOMYCOTA

1. Hemiascomycetes-no ascocarp; asci come from diploid cells

2. Plectomycetes-ascocarp is a cleistothecium which does not have an opening through which ascospores escape; these ascomycetes rely on physical disruption of the ascocarp.

3. Pyrenomycetes-ascocarp is a perithecium which have an opening called an ostiole.

4. Discomycetes-ascocarp is an apothecium on which asci are exposed upon maturity; the apothecium is an open or cup-like structure upon which asci are formed.

5. Loculoascomycetes-ascocarp a pseudothecium which is similar to a perithecium; bitunicate asci.

6. Laboulbeniomycetes-ascocarp a perithecium, lack true mycelium, obligate parasites of insects.

I. Class Hemiascomycetes

The fungi in this class include the yeasts. They have asci that are borne free or naked on the surface of the host or substrate;that is they are not enclosed in any kind of fruiting body. The thallus is simple in structure and when the mycelium are formed, they are very scanty. These fungi do not form ascogenous hyphae and form diploid cells either by fusing with another cell of opposite mating type if they are heterothallic or with sibling if they are homothallic. Although this class is widely distributed, the members are often associated with ripening fruits and plant exudates. We will consider two orders in this class-the Endomycetales and Taphrinales.

A. Order Endomycetales-includes those Hemiascomycetes in which two cells fuse to form a zygote, which is then transformed into an ascus.

1. Family Dipodascaceae-members of this family have mycelial forms with multispored asci that do not proliferate.

Dipodascus-mycelium consists of irregular cells and bears long slender asci which taper toward the apex. The ascospores are small, one-celled, and hyaline.

Mount a small amount of material from a colony of Dipodascus sp. in a drop of water and look for asci at different stages. Note the large number of ascospores in each ascus.

2. Family Endomycetaceae

Species in this family differ from Dipodasceae in producing a small and generally definite number of ascospores (1-8) in each ascus. The mycelium is composed of well-developed hyphae. Asexual reproduction is through the production of arthrospores.

Geotrichum-filamentous fungus and a pathogen on fruits. Its teleomorphic stage, Endomycetales geotrichum, occurs when mycelium of opposite mating type come together and an ascus forms in between the two hyphae.

Make a mount of Geotrichum candidum growing in culture and look for arthrospores.

3. Family Saccharomycetaceae

This family represents the ascomycetous yeasts. Mycelium is scanty or lacking and the asci can contain 1-8 ascospores.

a. Saccharomyces-in this genus, the vegetative cells reproduce by budding and fermentation of sugars is vigorous. The ascospores are round and there are four present per ascus.

Make a mount from a culture of S. cerevisiae, the beer and wine yeast, and observe under the microscope. The vegetative cells are oval and vary considerably in size. Observe stages in budding and look for asci with ascospores which should be common in older cultures.

b. Schizosaccharomyces-the vegetative reproduction in this genus is mainly by fission.

Mount a small portion of a colony of S. octosporus and observe under the microscope. The vegetative cells are hyaline and tend to be elongate. Observe dividing cells. Also look for various stages in cell conjugation and ascus formation. Mature asci are elongate and usually contain eight one-celled ascospores.

c. Hansenula-the shape of the ascospores of this genus is different from the more common circular shapes of the two genera above.

Mount a colony of H. saturnus and look for asci containing ascospores and discover for yourself why it was given its name.

NOTE:We will also project on the TV screen some live cells of the above species and hopefully be able to watch the yeats in action, budding and release of ascospores.

B. ORDER TAPHRINALES

The Taphrinales includes species in which the ascus arises from binucleate cells that form directly from the mycelium. The asci are erumpent through the host epidermis and are borne free on the host. All are parasitic on higher plants and some cause economically important plant diseases. Usually, eight one-celled ascospores are formed, but the number varies from four to many in different species. There is a single family,the Taphrinaceae, and one genus.

Taphrina-Examine an infected leaf showing the effects of the infection by Taphrina deformans. Observe prepared slides of sections through peach leaves infected with this fungus and look for asci and ascospores.

II. CLASS PLECTOMYCETES

In members of this class, the asci are small, evanescent, and are produced at different levels within the ascocarp. Most species have a completely enclosed ascocarp called a cleistothecium, but in some it is ostiolate. Tehy are widespread in occurrence, but are often associated with seeds, soils, and some are animal parasites. An important order in this class are the plant pathogens commonly referred to as powdery mildews.

A. ORDER EUROTIALES

The Eurotiales includes those plectomycetes which form their asci in completely enclosed ascocarps. The ascocarps are small, spherical, and are formed on a well-developed mycelium. The asci are globose to sub-globose, usually eight-spored, and are evanescent, freeing the ascospores inside the ascocarp. The anamorph is usually phialidic.

Talaromyces-The ascocarps of this genus are usually white or yellow. The asci are formed in short chains and the ascospores are hyaline. The conidial state is in the genus Penicillium.

Examine the bright yellow ascocarp of Talaromyces vermiculatus under the dissecting scope. Mount several ascocarps in water and crush gently to examine the asci and ascospores.

B. OREDER ONYGENALES

In this order, the ascocarp frequently consists of loose arrangment of hyphae, and a distinct peridium is lacking. The anamorph is arthroscopic or aleuriosporic. Many species are keratinophilic and are common dermatophytes.

1. FAMILY GYMNOASCACEAE

These are plectomycetes with the clestothecial wall consisting of loose network of hyphae.

Nannizzia-The peridial hyphae of this genus lack joints at the septa and the appendages are not well-defined. The free ends of the hyphae are spine-like, with lateral branches. The ascospores are hyaline.

Examine cultures of Nannizzia gypsea under the dissecting scope without the opening the plates. Examine demonstration slides of intact and crushed ascocarps in water. Observe the peridial hyphae, asci, and ascospores.

C. ORDER MICROASCALES

This order contains several srious plant pathogens.

1. FAMILY OPHIOSTOMATACEAE

These species have very small ascocarps with very long ostiolar necks, usually immersed in the substrate.

Ceratocystis-the ascocarps of this genus are dark, very small, with very long ostiolar necks. The ascospores are one-celled and hyaline. Examine photographs of Ceratocystis ulmi, the causal organism of Dutch elm disease.

D. ORDER ERYSIPHALES

Members of this order are also known as the powdery mildews. They have completely enclosed ascocarps that contain one or several asci. All are obligate parasites and cannot be cultivated in vitro. They form superficial hyaline mycelium that produces haustoria. There are a number of ways used to identify the genera of powdery mildews. One is by characterizing the appendages present on the cleistothecium. There are three different appendage morphologies used to identify the powdery mildews:circinoid, dichotomously branched, and bulbous based appendages. These are illustrated in attached figures. In addition, the species are also identified based on the number of asci in the cleistothecium and the conidial stages present on the host plant. There is a single family, Erysiphaceae.

We will examine the following species:

1. Erysiphe-this genus has ascocarps containing many asci and mycelioid appendages. Examine prepared slides of cleistothecia. Mount several ascocarps from dried material in a drop of water. Observe the appendages, then crush gently and look for asci and ascospores. Examine other slides of the conidial state, Oidium, and note how the conidia form in chains on conidiophores with swollen bases.

2. Uncinula-the ascocarps contain many asci and the appendages are rigid with a circinate or re-curved tip. Examine the leaves infected with this fungus under the dissecting scope. Mount several ascocarps in a drop of water and observe the appendages. Crush the ascocarps gently and look for asci and ascospores.

3. Phyllactinia-In this genus, there are many asci and the appendages are stiff, with a bulbous base. The conidial state belongs in the genus Ovulariopsis. Mount several ascocarps ina drop of water and observe the appendages.

NOTE:On the basis of the conidial state alone (anamorph) it is often not possible to distinguish the perfect genera.

PREPARATION FOR NEXT LABORATORY

Isolating ascomycetes from animal dung

We hope to isolate several species of ascomycetes from these dung to be used in our next lab. In addition, we can also follow the succession of fungi in different animal dung and know which comes first and which comes after. There are five kinds of dung available. They are all dry and odorless so there should be no zone of repugnance around them. They are rabbit dung, deer dung, cow dung, and an older and fresher version of elk dung. We will set this up as a class with each group being responsible for the dung of their choice.

Obtain small clean jars and dessicator jars (for the more massive dungs). Wet several layers of paper towels with distilled water and let the excess water drip off. Line the jars with wet paper towels. Carefully place the dung pellets/pieces/blobs on the wet paper towels inside the jars. Cover and incubate at room temperature.