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UBC Theses and Dissertations

Host : parasite interactions of rusts and axenic culture of Melampsora species Lane, William D.

Abstract

The thesis is composed of two sections. The first section comprising Chapters I and II is designed to examine possible interactions between isolated flax protoplasts and germinating rust urediospores, or axenically grown rust mycelium in an attempt to obtain evidence for or against the theory that toxins play a role in resistance and susceptibility in the flax:flax rust system. A number of previously published observations form the basis of the toxin theory of resistance. These can be summarized as follows: necrotic cells are found surrounding the flax rust infections; the cytoplasm of the rust and the host never come into direct contact; plant cells some distance removed from the site of infection become necrotic; the necrotic area around an infection can be. extended away from the site of infection when an electric current is passed through the leaf, the necrosis extending in the direction of the positive pole; and the characteristics of the necrotic area vary depending on the virulence and resistance genes involved in the interaction. Chapter I describes the isolation of flax protoplasts, and their growth and. development in vitro. The medium used and other cultural conditions were, found to influence both the type of development and the longevity of cultured protoplasts. When cultural conditions were optimal, some protoplasts divided mitotically, and many remained alive for a month or longer. Information obtained from these experiments formed the basis of the experimental procedures employed in the work described in.Chapter II. In Chapter II the toxin theory of resistance and susceptibility was examined using a protoplast bursting bioassay to detect any fungal toxins, if they were present in the test solution. Protoplasts are suitable for a bioassay of this kind because they are fragile and sensitive and burst when exposed to toxic substances. The bursting response is easy to detect and to quantify. Because axenic cultures of flax rust are now available a search for evidence of extracellular products with toxic properties is therefore possible. Spore germination medium is also a possible source of fungal toxins. If the protoplasts isolated from either resistant or susceptible varieties, or both, were burst when incubated with exudate from axenic cultures or spore germination medium this would constitute evidence in support of the toxin theory. The search for toxic substances in both axenic culture exudate and spore germination medium yielded negative results. These, together with other previously published results, argue against the involvement of toxins in the flax: flax rust system. An alternative explanation, consistent with the observations which originally suggested the toxin theory, is that, when the fungus invades the intact leaf, toxins are produced by the host plant. This proposal is discussed. The second section of the thesis, comprising Chapters III and IV, describes techniques for growing rusts axenically. In Chapter III a technique is described for the isolation of colonies of flax rust from infected cotyledons. The technique depends on the digestion of host cell walls with hydrolytic enzymes and washing the liberated colonies free from adhering flax protoplasts. Using this method it is possible to collect large numbers of flax rust colonies with only a few host cells adhering to them. The isolated colonies can be used as a source of uncontaminated fungal tissue. Axenic cultures were established using colonies isolated in this way as inoculum. The results in Chapter IV are an extension and modification of those in Chapter III and describe the axenic culture of poplar rust for the first time. Surface sterilized leaf pieces of black cottonwood leaves centered on uredial infections of poplar rust (Melampsora occidentalis) were placed pustule side up on a completely defined agar base medium. All the pustules grew for the first two weeks and after 4 months about 30% of them became established as vegetative axenic culture colonies on the agar medium. The leaf tissue did not grow. The axenic cultures were successfully subcultured to fresh medium. Some produced spores in culture. The colonies were capable of reinfecting excised leaves of the host in vitro. The new axenic culture techniques described in Chapter III and IV are simpler than previous methods and have several other advantages.

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