LAB 3
III. BAITING
ISOLATION OF FUNGI BY BAITING
Many fungi may be isolated from soil and water through the use of preferential substrates, or baits. The bait should ideally allow only the desired organism to develop. Human hair and nails, cow horn, cast snake skin, shrimp exoskeleton, dead insects, boiled seeds, and pollen are a few examples of common baits.
Isolation of aquatic species
Baiting from solid matter: Place a small amount of the solid substrate, which can be either plant tissue or soil, in the bottom of a petri dish. Add sterile ditilled water to a depth of about 5 mm. Gently place the bait in the dish. Cover the dish. A sterile beaker can be used to bait from larger amounts of solid matter.
Baiting from liquid: Pour the liquid of choice; which can be pond, lake, tap, or aquarium water; into the bottom of a petri dish to a depth of about 5 mm. Gently place the bait in the dish. Cover the dish.
Petri dishes used for baiting should be set aside in a spot where they can remain undisturbed for the duration of the baiting period. This period can last from 2 days to one week.
QUESTIONS
1. Draw and describe the different fungi you baited. Include in your description where your soil and water samples came from and what kind of baits you used.
2. What will be the most logical bait for the following types of fungi? Explain your answers. (a) plant pathogens (b) fungi with ligninase (c) fungi with cutinase (d) fungi with amylase (e) animal pathogens
3. What other methods can you use to isolate fungi from the soil?
LAB 4
GENETIC SYSTEMS AND MANIPULATION
The fungi we will be working with this semester have many diverse ways of propagating themselves, interesting and bizarre sexual structures, and easily scorable phenotypes of progeny which will become useful when we wish to follow traits of the two parents after a sexual cross has been completed.
Genetic manipulation of fungi has been perhaps one of the most useful and powerful tools in deciphering biochemical pathways, developing new pharmaceutical drugs, determining the roles of certain fungal genes in human and plant parasitism, and increasing the vigor and productivity of fungi involved in industrial processes. Fungi are haploid which makes them ideal tools for genetic analysis. They can be readily grown in the laboratory and complete their life cycles in a relatively short period of time (1-4 weeks). They have asexual and sexual reproduction and some have their meiotic products arranged in an orderly fashion that reflects the precise order of meiotic events, and their biochemical pathways and cellular organization are virtually identical to those of higher eukaryotes. In addition, they have alternative genetic systems called heterokaryosis and parasexuality. As an example of the importance of fungi in regard to the study of human physiology, the first gene identified as being involved in causing cancer in humans (RAS) was first discovered and characterized in Saccharomyces cerevisiae. Many human mascular diseases can also be studied indirectly because of the fact that fungi possess a similar organization of their cytoskeleton as do human cells.
Fungi reproduce both sexually and asexually. Asexual reproduction can be accomplished by: (1) fragmentation of hyphae, (2)budding of somatic cells, (3)fission of somatic cells, (4)production of mitotic spores. In general, asexual reproduction is more important for the propagation of the species because it results in the production of numerous individuals. Sexual reproduction, on the other hand, occurs less frequently than asexual reproduction. It is a means of generating genetic variability that can help the fungus survive by adapting to new environments. The generalized life cycles of the different groups of fungi are shown in the handout. Study the cycling from asexual to the sexual phases in some of the groups. (What similarities and differences can you detect?)
Sexual Reproduction
The usual chain of events during a sexual cross of a fungal species is as follows:
1. In the case of a heterothallic species, two fungal isolates of compatible mating types must be present. In order for them to be compatible, each must possess a different mating type allele at their mating type locus. For the Ascomycota, these alleles are usually designated A and a. These two isolates should not be thought of as a male and female because frequently each strain can produce both male and female structures.
2. Anastomosis occurs between two genetically different hyphae and the nuclei can etihter immediately fuse to form diploids (2N) or remain as dikaryotic mycelium (N+N).
3. After the nutrient source has been depleted by the fungus or if other conditions are conducive to mating (temperature, moisture, etc.), the nuclei fuse, if they have not already, and meiosis will proceed.
4. The meiotic spores, now haploid in most cases (the Oomycota are an exception), germinate and the life cycle repeats itself.
Mendelian Segregation
The inheritance of speciike any other organisms, follows the simple rules Mendel developed in the 1800s. The principle of segregation, or Mendel’s first law states that allelesof a gene will be distributed equally among the gametes produced by an organism. Mendel’s second law or the principle of independent assortment states that alleles of different genes will assort independently of eanh other. This has been modified to refer to alleles of genes or different chromosomes. This second principle will become evident when we discuss the tetrapolar mating system of the Basidiomycota.
Things to Do:
Sordaria mating
This will be done in pairs. Obtain a solidified plate of Sordaria crossing medium and inoculate with two agar blocks containing mycelia of wild-type and two agar blocks of tan mutant of Sordaria fimicola as shown in the student’s guide. In this exercise, we will learn about gene mapping and the concepts of linkage in Sordaria, in addition to being able to follow its life cycle and understand meiotic events in its development.