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. / Microbiology and Plant Pathology / unrestricted

The Sugarcane Industry contributes approximately 400 000 jobs and ZAR 8 billion annually to South Africa’s economy. Due to climate change and the subsequent threat posed by disease, these figures have been on the decline. Brown rust, a contributor to this decline is caused by the basidiomycete Puccinia melanocephala Syd. and P. Syd., which previously resulted in 50% yield losses in susceptible varieties. This highlighted the need for improved screening and breeding techniques which will result in the replacement of susceptible varieties. The objectives of this study were to: a) Adopt and optimise a glasshouse whorl inoculation screening technique applicable for mass screening of large populations. b) Develop a rapid and cost effective rust resistance screening technique using detached leaves. c)Utilise two flanking marker sets (R12H16 and 9O20-F4-PCR primers) for the rust resistance Bru1 gene in a diagnostic polymerase chain reaction (PCR) to identify rust resistant genotypes lacking Bru1 and possessing either quantitative resistance or an alternative major qualitative resistance gene. d) Correlate rust phenotypic data to AFLP marker data for the Linkage Disequilibrium (LD2) mapping population. e) Utilise suppression subtractive hybridization (SSH) profiling on rust challenged genotypes to discover differentially expressed genes between susceptible and resistant (susceptible Bru1 negatives taken away from resistant Bru1 negatives); and resistant genotypes (resistant Bru1 positives taken away from resistant Bru1 negatives). 4 Results from the glasshouse whorl inoculation trials showed the technique could be reliably used to screen large populations, as two independently conducted pot trials showed highly correlated rust ratings. A visually assessed detached leaf assay (DLA) was developed using selected genotypes. Chlorophyll fluorescence and SPAD readings were used in the DLA to determine the leaf photochemical efficiency (PIABS) with relation to chlorophyll content, resulting in reduced assessment time of at least two days. PCR diagnostics revealed 31% of LD2 did not possess either flanking marker, 8% had one or the other marker, and 61% had both markers. The overall rust phenotypic ratings (rating scale of 0-10) and Bru1 status of the genotypes was used to group the population, with the Bru1 negative genotypes containing all three rating categories (resistant 0-3.5; intermediate 3.51-6.5; susceptible 6.51-10); while the Bru1 positive genotypes were all resistant. The phenotypic data was correlated to AFLP data using the Pearson product-moment correlation coefficient and stepwise multiple linear regression employed to build marker based models to use for predicting non-Bru1 mediated resistance. SSH analysis was then subsequently conducted on genotypes selected on the basis of Bru1 status and AFLP correlation data. Two subtraction cDNA libraries were constructed and the cDNA inserted into electro-competent Escherichia coli cells. PCR on transformed cells revealed cDNA inserts ranging from 200- 1300bp. BLAST analysis of the cDNA sequences indicated the presence of high proportions of disease and drought stress related sequences in the libraries. Analysis of the sequences in both libraries showed that the resistant Bru1 negative genotypes contained oxidative stress related sequences which were however absent in the Bru1 positive resistant genotypes. The library comparing the Bru1 negative resistant genotypes against the Bru1 negative intermediate and susceptible genotypes showed a higher proportion of differentially expressed sequences coding for putative disease resistance proteins, highlighting their presence in the resistant genotypes. Both subtraction libraries also contained high proportions of a leucine rich repeat protein coding cDNA which contained a conserved domain homologous to that of a disease resistance protein conferring resistance to Pseudomonas syringae in Arabidopsis thaliana. The outcomes of this study will subsequently enable an improved understanding of sugarcane-rust resistance mechanisms and improved breeding and screening techniques for sugarcane by identifying SSH and AFLP markers linked to rust resistance QTLs or alternative R genes. / Thesis (M.Sc.Agric)-University of KwaZulu-Natal, Pietermaritzburg, 2012.

The first serious outbreak of brown rust of sugarcane caused by Puccinia melanocephala Syd. & P. Syd. was reported in India in 1907. It was first reported in South Africa (SA) in 1941 on the variety Co301 and is now present in almost all the sugarcane growing areas of the world. In SA, it is now described as an important disease of sugarcane, causing yield losses of up to 26% in susceptible varieties. Within the SA sugar industry, rust is controlled through the use of resistant varieties as it is the most economical method of control. However, most of the newer varieties that are being released have an intermediate resistance rating for rust. An integrated management approach for the control of rust is therefore being investigated. Aspects investigated in this study included environmental conditions required for development of the disease i.e. epidemiology, the use of silicon (Si) as a cultural control method against brown rust and identification of gene sequences expressed in response to brown rust infection. For the epidemiology study, inoculated plants were incubated in a dew chamber at different temperatures and leaf wetness periods. The choice of leaf wetness duration and temperature was based on urediniospore germination studies. The optimum temperature for urediniospore germination and disease development at > 98% relative humidity was found to be between 20 and 25°C with nine hours of leaf wetness. Silicon has been shown to reduce the incidence of diseases and pests in a number of crops. The ability of sugarcane to accumulate Si and the location of Si deposition was established using two uptake and deposition trials. Different concentrations of Si were applied to the plant and accumulation in the roots, stalks, old leaves and young leaves was determined using inductively coupled plasma optical emission spectrometry, with accumulation found to be roots > old leaves > stalks > young leaves. Silicon deposition in the leaves was determined using energy dispersive X-ray mapping on freeze dried specimens and significant differences were found between the upper epidermis, lower epidermis and mesophyll with the most Si being deposited in the lower epidermis. For disease severity, plants were naturally infected with rust and rated weekly. A significant decrease in disease severity and area under disease progress curve was noted when the Si concentration increased, indicating that Si has potential in reducing rust incidence. Currently, the most reliable and economical method of managing brown rust is with the use of resistant varieties. Identification of resistance within breeding lines is therefore important. For this part of the study, suppression subtractive hybridization was used as a tool to identify differentially expressed genes between a susceptible and resistant variety and a susceptible and intermediate variety, in response to brown rust infection. Two efficient subtracted cDNA libraries were generated and differentially expressed sequences were identified within each library. The results of this study show potential for the development of molecular markers which could be used for the early identification of brown rust resistance during the breeding process. This study forms a firm basis on which an integrated management strategy, for the management of brown rust in the SA sugar industry, could be designed. The cDNA sequences identified could be further investigated and used to develop molecular markers to select for rust resistant varieties, the epidemiology results together with further field data could be used to develop a disease prediction model and Si has potential in the field to reduce brown rust severity. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2009.