Typescript, etc. / Digitized by Kansas State University Libraries

Thesis (M.S.)--University of Wisconsin--Madison, 1968. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1970. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

The uredial stage of Puccinia graminis Pers. f. sp. tritici Erikss. and Henn. is obligately parasitic on wheat in nature. The transition from the dormant uredospore to the vegetative state takes place on the plant by the sequential development of a germ tube and a series of specialized infection structures (appressoriurn, peg, vesicle, and infection hypha). Conditions favouring uredospore germination are different from those promoting differentiation. Infection structures will only develop in response to a delicately timed sequence of specific environmental stimuli. Thus, the precise timing of cellular events occurring during differentiation could be crucial for rust survival. The timing of nuclear development, DNA, RNA, and protein synthesis was investigated cytologica1ly in the differentiating uredospore apart from the host plant. For the in vitro production of infection structures, a liquid medium was developed, called MPG, which consisted of a Ca-K-PO₄ buffer, D-glucose, and 'Evans' peptone. There was significantly greater infection structure formation on this medium than on the Ca-K-PO₄ buffer alone. Nuclear behaviour in differentiated sporelings was found to differ from that in sporelings which were germinated without forming infection structures. The young germ tube was dikaryotic. Nuclei of germinating spores remained relatively unchanged during development. Divisions rarely occurred, and when they did in some older germ tubes, four was the maximum nuclear number. Septation soon followed to restore the binucleate condition. During differentiation, on the other hand, the nuclear number increased due to divisions both in the appressorium and in the vesicle. The mature appressorium normally had k nuclei, the mature vesicle 7 or 8. This number was subsequently reduced in the infection hypha to 1 or 2. In addition, two types of nuclear formations were regularly seen in the vesicle; bilateral nuclear clumps and specific migration patterns. Results of experiments using metabolic inhibitors indicated that the synthetic requirements for morphogenesis of the germ tube and the infection structures were also different. The germ tube did not appear to require either DNA, RNA, or protein synthesis, whereas the infection structures required all three. RNA synthesis, essential for the appressorium, was found to occur during the first 2 hours of germination even before the heat stimulus to induce infection structures was applied. The role of infection structures is still poorly understood. Therefore, studies were undertaken to determine the effect of differentiation both on infection of the host and on continued growth of the rust in axenic culture. Spores that were heat-shocked to induce differentiation gave a markedly higher infection count when placed on exposed host mesophyll than those which were only pre-germinated without heat shock. This suggested that the infection structures might be essential for plant infection, not merely for stomatal penetration. Attempts were made to produce vegetative colonies from single uredospores. Physically separate, thinly-seeded spores (l to 10 spores/mm² ) failed to initiate colonies on a defined AXENIC medium which normally supported growth if thickly-seeded (1000 to 2000 spores/mm² ). When thinly-seeded spores were first germinated or differentiated in MPG medium for periods of from 2 hours to k days, then transferred to the AXENIC medium, colonies were induced, each arising from a single uredospore. Colonies which had originated from differentiated sporelings sustained growth for a longer period than those from germinated ones, suggesting that infection structures are important for vegetative growth. When the above two-stage medium was used with single uredospores, each in a separate well of a plastic micnotest plate, no vegetative growth occurred. Germ tubes were shorter and differentiation rarely occurred in the isolated single spore condition as compared to physically separate spores in a common medium. This difference was independant of the volume of medium per spore. Attempts to use media, conditioned for varying time intervals with large numbers of differentiating spores, as a starting medium for single spores, proved unsuccessful. Attempts to isolate the differentiation stimulator to apply it to single spores also failed, although a germination inhibitor and stimulator were detected. Results of final experiments suggested that the use of glass vials as containers for single spores might yield more promising results. / Land and Food Systems, Faculty of / Graduate

Digitized by Kansas State University Libraries

A cytological study of sporogenesis, mature uredospores and hydrated uredospores of Puccinia graminis tritici was made to determine if a definitive nucleolus was present. Electron microscopy has shown that the nuclei in immature uredospores and associated fungal tissue of the uredosorus possess prominent nucleoli. Nucleoli whose average diameter was 1.7 microns were observed in 58 percent of the nuclear sections from immature uredospores. Presence of a nucleolus in ultrathin sections of mature uredospores is established. Nucleoli whose average diameter was 0.5 microns were observed in 6 percent of the nuclear sections from mature uredospores. Nucleoli were not observed in hydrated uredospores which had resumed active metabolism. The reduction in the size of the nucleoli in mature uredospores and absence of nucleoli in hydrated uredospores may indicate that ribosomal RNA synthesis is repressed as uredospores mature. / Land and Food Systems, Faculty of / Graduate

The development of our knowledge of the metabolism, nutrition., and physiology of the rust fungi has been restricted because of our inability to grow these fungi in axenic culture. Most of the physiological and biochemical investigations on rust fungi have been limited to studies of the germination and development of germ tubes. It is not known, whether failure of the rusts to grow axenically is associated with a metabolic block in essential biochemical pathways. The uredial stage of an Australian isolate (ANZ 126-6, 7) of wheat stem rust was grown on a relatively simple medium. Successful in vitro growth was obtained on a medium containing Czapek's minerals, glucose and an organic nitrogen source viz. yeast extract, peptone or BSA. Vegetative growth producing a discrete colony appeared to be dependent upon high density seeding, when conventional dry seeding techniques were followed. When a spore suspension in gelatin was used for inoculation consistent, reproducible vegetative growth at low density seeding was obtained. Water spore suspensions failed to support any growth on the same series of media, under identical conditions. Sodium citrate, host extract, and host protein fractions in general inhibited growth whereas pectin increased the lag phase. Spore-like bodies were obtained inside the colony, which were devoid of any pigmentation. It is suggested that the combination of physical and chemical factors surrounding the germinating spore play an important role in the in vitro development of rust fungi. / Land and Food Systems, Faculty of / Graduate

Call number: LD2668 .T4 1953 M6 / Master of Science

Call number: LD2668 .T4 1950 W6 / Master of Science

Call number: LD2668 .T4 1957 S38 / Master of Science