Albugo tragopogonis is responsible for white rust of sunflower. It was first observed in 1929 in South Africa. Recently however, white rust has resulted in lodging exceeding 80% in some sunflower growing areas. Due to the obligate nature of the pathogen, studies of the biology, epidemiology and control of the disease has until now been limited to field trials and observations. Greenhouse trials are needed to understand the infection process, and to examine any resistance mechanisms used by the plant to defend itself against the pathogen. Presently, there is no practical artificial inoculation technique available and effective storage of the fungus is difficult. The purpose of these studies was to find new storage and inoculation techniques. Once the inoculation technique was optimized, the infection process of A. tragopogonis on susceptible and tolerant sunflower genotypes was examined. Infected leaves were collected from sunflower seedlings at the Grain-Crops Institute in Potchefstroom. Infected leaves were covered with plastic bags and freshly cut stems were placed in a cooler box filled with ice water. Some of the infected leaves were also placed in paper bags and allowed to dry for 24 h. Sporangia were collected using a vacuum device and stored in gelatin capsules at -20°C, -70°C or in liquid nitrogen directly after collection or following desiccation for 24 h. Sunflower seedlings at the four-leaf-stage were inoculated with freshly collected sporangia, or sporangia stored for 3, 5, 9, 12 and 15 mo. A zoospore suspension was prepared by allowing 105 sporangia/ml to germinate in distilled water for 3 h at 10°C. The zoospore suspension was then sprayed onto leaves until they were completely wet with a hand held garden spray bottle. Inoculated seedlings were covered with plastic bags to maintain high humidity and placed at 12°C for 16 h and incubated in a greenhouse until symptom development. Infection levels were assessed 10¬14 d after inoculation, using a scale of 1-5, with 1 indicating resistance and 5 indicating severe infection. Infection with fresh sporangia proved to be very consistent. Sporangia stored in capsules immediately after collection at -70°C after desiccation, produced the highest infection. Low levels of infection resulted from storage in liquid nitrogen or directly at -70°C. It is evident that successful storage may be obtained if the sporangia are dried before storage. These techniques to store and inoculate A. tragopogonis have proven to be reliable. Susceptible and tolerant genotypes were inoculated, using the spray bottle inoculation technique described above, to examine the difference in infection of A. tragopogonis. Leaves used for light microscopy were cut into 20 mm2 and those for scanning electron microscopy were cut into 5x5 mm pieces at 2, 4, 6, 8, 10, 12, 24, 36, 48, 72, 96, 120, 144 and 168 h time intervals after inoculation. The epidermis, palisade parenchyma and spongy parenchyma were chronologically stripped using the double-sided tape method. The material for the light microscope was prepared using the whole-leaf clearing and staining technique, the lactophenol-ethanol-analine blue technique and sectioning with freeze microtome. The material for SEM was prepared according to standard procedures and examined with a JEOL 840 SEM at 5 kV. Both the whole-leaf clearing and staining and the lactophenol-ethanol-aniline blue techniques proved to be unsuitable as most of the tissue was damaged by boiling. Sectioning with the freeze microtome was also unsuccessful. The SEM gave the most transparent results. This method gave us the ability to compare results with previous literature and to compare the infection process between of A. tragopogonis in the susceptible (RHA 358) and the tolerant (HYS 33) genotype.
Dissertation (MSc ( Plant Pathology))--University of Pretoria, 2005.