This laboratory exercise will mark the start of our detailed study of the different groups of fungi and specific fungal species representative of each group. As has been suggested, you are advised to draw each fungal specimen shown in the lab with great accuracy and write a detailed description to help you in remembering their appearance and names.

CLASSIFICATION OF FUNGI

Classification is a way to describe the relatedness of organisms. The classification of fungi has traditionally been based on gross morphology, development, and physiology of the organism. Although sequences of DNA and protein provide better information about the relatedness of two species, the more traditional approach to classification will serve as our system to indicate the relationships among fungi we observe in lab. The 6-Kingdom classification by Alexopolus, Mims, and Blackwell(Introductory Mycology, 4th edition) will be used and we will consider the following phyla of fungi which contain organisms traditionally classified as fungi.

Kingdom Protoctista

Phylum Myxomycota
Phylum Plasmodiophoromycota
Phylum Dictyosteliomycota
Phylum Acrasiomycota

Kingdom Stramenopila

Phylum Labyrinthulomycota
Phylum Hyphochytridiomycota
Phylum Oomycota

Kingdom Fungi

Phylum Chytridiomycota
Phylum Zygomycota
Phylum Ascomycota
Phylum Basidiomycota
Phylum Deuteromycota

Slime molds are intriguing organisms and are useful as tools to investigate fundamental biological processes such as cell differentiation, cell movement, and the mechanics of nuclear division. They are different from other organisms traditionally classified as fungi because they

1. lack cell wall in the vegetative stage which is a plasmodium
2. have ingestive metabolism (instead of absorptive)

The Slime Molds are spread in two kingdoms and five phyla.

A. Kingdom Protoctista

1. Phylum Myxomycota (Free-living true slime molds)

Members of this phylum are called true slime molds or plasmodial slime molds. They have flagellated cells called swarm cells which fuse to form a plasmodium. Sporophores arising from the plasmodium are the site of meiosis. The life cycle of a typical myxomycete is shown here. Some species from the class Myxomycetes have been investigated by molecular and cellular biologists because of its unique properties. When in the plasmodial stage, which is composed of several hundred nuclei, nuclear (mitotic) division occurs synchronously. Other phenomena include formation of the plasmodium from two cells of opposite mating type and differentiation of the plasmodium into fruiting bodies. These phenomena of nuclear synchronization and cellular differentiation are studies directly connected to cancer research.

A. Identification of the myxomycetes

Many of the morphological characteristics of the myxomycetes can be recognized using the naked eye or a dissecting microscope. Be careful with the specimens because they have very delicate structures which can be damaged by even gentle poking and prodding. In order to key these fungi out, you will need to know some definitions:
Peridium: the outside covering or wall of a fructification

Sporangia: sac-like structure, containing protoplasm which is converted tospores

Plasmodiocarp: a curved or branched, vein-like fruiting structure of some of the myxomycetes

Capillitium: sterile, thread-like structures present among spores in the fruiting bodies

Columella: sterile structure within a sporangium or other fructification; extension of a stalk

Aethalium: large, sometimes massive cushion-shaped fructification; group of sporangia which have not separated into individual units

Plasmodium: naked, multinucleate mass of protoplasm moving in amoeboid fashion

Your goal for this lab is to learn how to use a dichotomous key to identify the specimens presented. Please draw what you see; describe the characteristics of the specimen; and then write down the order, family, genus and species of the specimen.

B. Observing protoplasmic streaming Protoplasmic streaming is a characteristic of many cells but the streaming observed in myxomycete plasmodia is on an unusually massive scale. Obtain a growing plasmodium of Physarum polycephalum. Using a microscope and without opening the plate, focus on one big vein, and observe the movement of protoplasm. Some questions to help you in your observations:
Is the streaming unidirectional?
Is the rate in which streaming occur constant or variable?
Does it ever stop?
Does streaming occur in all of the veins at the same time, rate and direction?
Does streaming continue even if smaller veins join together?

C. 'Pet' Physarum polycephalum

Transfer a small agar block containing a growing plasmodium onto a new oat flake agar plate. This will be your 'pet' Physarum . Using your creativity, you can do whatever you like with it to answer your own question. A 'class pet' will serve as a control. This Physarum will be grown under optimal conditions, ie. fed with oatflakes as needed, kept in the dark at optimum temperature of 25 °C. Next week, we will compile all your observations to help us better understand the growth and life cycle of this myxomycete.

2. Phylum Plasmodiophoromycota (Endoparasitic true slime molds)

Members of this phylum are obligate endoparasites of vascular plants, algae, and other fungi. Its somatic phase consists of a plasmodium that develops within the host cells. Important species in this phylum include: Plasmodiophora brassicae, (the causal organism of clubroot of cabbage), Spongospora subterranea, (causal agent of potato powdery scab), and Polymyxa graminis (which is parasitic on wheat and other cereals and also known to be the vector of some important plant viruses). Using the prepared slides, examine host cells containing sporangia or sori and resting spores of these plant pathogens under the microscope.

3. Phylum Dictyosteliomycota (Dictyostelid Cellular Slime molds)

The cellular slime molds differ from the true slime molds in that their stalks consist of walled cells. Their unit of structure is a uninucleate, naked, haploid myxamoeba that feeds by engulfing bacteria. These myxamoebae aggregate to form a pseudoplasmodium, also known as a slug or grex. In this pseudoplasmodium, the component myxamoebae do not fuse; they cooperate as members of a well-organized community until sporulation occurs.

4. Phylum Acrasiomycota (Acrasid Cellular Slime Molds) In the Acrasiomycota, the aggregating myxamoebae do not form streams in developing pseudoplasmodia, their sorocarps do not have well-defined sori and sorophores, and their myxamoebae have lobose pseudopodia.

B. Kingdom Stramenopila

1. Phylum Labyrinthulomycota (Net Slime Molds)-have thallus consisting of branched tubes within which amoeboid cells crawl. They have heterokont (whiplash and tinsel flagella) spores. One important species is Labyrinthula macrocystis, a pathogen of eel grass.

Things to do:

1. identify to genus level two myxomycete specimens using the dichotomous key.
2. observe protoplasmic streaming in Physarum polycephalum
3. set-up "pet" P. polycephalum project
4. follow the life cycle of Dictyostelium discoideum by observing plates grown at different days. Try to see a moving slug, a pseudoplasmodium, culmination, etc.
5. examine prepared slides of Plasmodiophora brassicae, Spongospora subterranea, and Polymyxa graminis.