Morphogenesis of the wheat stem rust uredospore
Creators: name: Wisdom, Carolyn Jean
Publisher: name: University of British Columbia
Year: 2010

description: 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.