Molecular plant-pathogen interactions with special reference to Eucalyptus Grandis polygalacturonase-inhibiting proteins and fungal polygalacturonases

Phytopathogenic fungi parasitise plants for survival. Knowledge of the interactions between plants and these fungi is of importance in designing control strategies. In this thesis the interactions of Eucalyptus polygalacturonase-inhibiting proteins (PGIPs) and fungal polygalacturonases (PGs) were characterised. About thirty years of research on the interaction between PGIPs and PGs was reviewed in Chapter 1. The gene sequences of the mature PGIP peptides from selected Eucalyptus species were determined in Chapter 2. The PGIP genes have very high similarities among themselves, signifying conservation of the function of PGIPs in this genus. Molecular characterisation of the endoPG gene of C. cubensis was presented in Chapter 3. This endoPG gene occurs more than two times in the genome of C. cubensis. The gene sequences of the other endoPGs remain to be determined. EndoPG production by C. cubensis, a Eucalyptus canker pathogen, was presented in Chapter 4. It was found that polygalactw:onases probably have a minor role in determining the levels of pathogenicity in different C. cubensis isolates. However, the hypovirus CHVl-173 has a role in reducing polygalacturonase production in infected hypovirulent C. cubensis isolates. Chapter 5 dealt with the interaction between Eucalyptus PGIPs and endoPGs from different fungal pathogens. Coniothyrium zuluense and Botryosphaeria dothidea produced more PGs than C. cubensis and Phytophthora cinnamomi in liquid culture. Disease tolerant Eucalyptus TAG5 clones produced PGIPs that are more effective in inhibiting endoPGs from C. zuluense than the susceptible Eucalyptus ZG14 clones. In Chapter 6 the gene sequence of the endoPG of Fusarium circinatum was detennined. This :fungus causes the . pitch canker disease in pine trees. This endoPG gene occurs as a single copy and is related to those of other Fusarium spp. The results presented in this thesis add to the current scientific knowledge pertaining to the role of PGIPs and PGs in pathogenic interactions, especially, between Eucalyptus PGIPs and fungal endoPGs. Interaction of Eucalyptus PGIPs and fungal endoPGs was demonstrated; therefore it is possible to genetically engineer Eucalyptus for more disease tolerant Eucalyptus plants using PGIP genes, especially, against Coniothyrium zuluense. This means that more research is needed to identity genes that will give a more global protection against many fungal pathogens.Thesis (PhD (Plant Biotechnology))--University of Pretoria, 2006.Geneticsunrestricte

Factors impacting on the microbiological quality and safety of processed hake

Problems with the safety and shelf life of export hake have been raised by the Namibian fishing industry. This prompted an investigation into the factors that may have an impact on the microbiological quality and safety of processed hake. Samples were collected along the processing line; the general microbiological quality (mesophylic and psychrotrophic aerobic plate counts), total Vibrio species and common fish spoilage bacterial counts were performed. The results constantly showed relatively high counts for the psychrotrophic and spoilage bacteria, indicating that most of these bacteria already formed part of the incoming fish. Hake is headed and gutted on board of fishing vessels and delivered to the factory only after 7 – 8 days for final processing. It is likely that this practise of heading and gutting the hake may have a negative effect on microbiological quality of the final product. A sharp increase in the mesophilic and sucrose fermenting Vibrio species counts were observed after filleting. It has been suggested that this contamination could be due to biofilms present in the distribution system for the treated sea-water used during processing. Although, sea-water could be an alternative source of water for marine fish processing plants, the  treatment and the quality of the water needs to be carefully managed.Key words: Hake fish, aerobic plate counts, Vibrio species, mesophiles, psychrophiles, spoilage bacteria

Development and use of microsatellites markers for genetic variantion analysis, in the Namibian germplasm, both within and between populations of marama bean (Tylosema esculentum)

Tylosema esculentum (marama) has long been identified as a candidate crop for arid and semi-arid environments due to its success in these environments and the high nutritional value of the seed. Molecular markers are essential for the assessment of the levels of genetic variation present within and between populations of marama as well for future marker-assisted breeding efforts. Microsatellites were isolated using a modified FIASCO enrichment technique. Eighty pairs of primers were designed to amplify across a selected set of perfect microsatellite repeats with greater than 5 repeat units. Of the 80 primer pairs screened, 76% were able to detect polymorphism and 21% gave monomorphic bands while the other 3% gave inconsistent results. Four of the polymorphic SSR’s were used for genetic variation analysis and have proved to be useful and informative markers for assessing intra-specific and interspecific variability of marama bean. Heterozygosity (H) within and between populations of marama bean in the Namibian germplasm ranged from 0.30 to 0.74. Some of the populations had low genetic variation while others had high genetic variation

Isolation and characterization of microsatellite repeats in Marama bean (Tylosema esculentum)

Marama bean [Tylosema esculentum (Burchell) Schreiber] occurs naturally in arid and dry parts of Southern Africa, including Botswana, Namibia and South Africa. Due to the high nutrient value of the seeds and tubers, rich in protein, oil and starch, it is a potential crop for arid areas where few conventional crops can survive. The objectives of this study were to design microsatellite primers based on microsatellite repeats identified in Marama bean and to determine their ability to detect polymorphisms for use in diversity characterization. DNA fragments of T. esculentum containing microsatellite loci were isolated by enrichment of genomic DNA by a modified FIASCO technique and sequenced. Nine Marama bean microsatellite libraries enriched for (AAG)7, (GTT)7, (AGG)7, (GAG)7, (CA)10, (CT)10, (TCC)7, (CA)15 and (CAC)7 were created and thirty microsatellite primers based on the microsatellite regions of marama bean were designed and screened for polymorphism. Polymorphic microsatellites that were identified can be used as markers in future breeding programs. The screening for the polymorphic SSRs is being carried out to reach a target of 50 or more polymorphic SSR markers. It appears that the marama bean germplasm has abundant polymorphic microsatellites as 77% of the 30 loci screened initially were polymorphic

Factors impacting on the microbiological quality and safety of processed hake

Problems with the safety and shelf life of export hake have been raised by the Namibian fishing industry. This prompted an investigation into the factors that may have an impact on the microbiological quality and safety of processed hake. Samples were collected along the processing line; the general microbiological quality (mesophylic and psychrotrophic aerobic plate counts), total Vibrio species and common fish spoilage bacterial counts were performed. The results constantly showed relatively high counts for the psychrotrophic and spoilage bacteria, indicating that most of these bacteria already formed part of the incoming fish. Hake is headed and gutted on board of fishing vessels and delivered to the factory only after 7 – 8 days for final processing. It is likely that this practise of heading and gutting the hake may have a negative effect on microbiological quality of the final product. A sharp increase in the mesophilic and sucrose fermenting Vibrio species counts were observed after filleting. It has been suggested that this contamination could be due to biofilms present in the distribution system for the treated sea-water used during processing. Although, sea-water could be an alternative source of water for marine fish processing plants, the treatment and the quality of the water needs to be carefully managed.We would like to thank the Ministry of Education of Namibia for funding this research, the Department of Microbiology and Plant, Pathology University of Pretoria for making the research facilities available and the Fish Processing Plant in Walvis Bay, Namibia for permitting us to use their facility. We would also like to thank Ms B. Kachigunda for data analysis

A review of geographical distribution of marama bean [Tylosema esculentum (Bruchell) Schreiber] and genetic diversity in the Namibian germplasm

Marama bean [Tylosema esculentum (Burchell) Schreiber] occurs naturally in the drier areas of Southern Africa, including Botswana and Namibia. The implementation of molecular breeding is important to advance the process of securing the world’s food supply. The development of molecular markers is vital for mapping important traits that can then be followed in subsequent breeding programs. This study assessed the distribution of marama bean in Namibia and isolated microsatellite regions for microsatellite primer design for the purpose of determining genetic diversity and construction of molecular genetic maps for marama. This will greatly enhance the process of domesticating marama bean, currently a wild plant that is still picked wild and unsustainably. The geographical distribution was geo-referenced using Geographic Positioning System (GPS) points and microsatellites were isolated from the germplasm using a modified FIASCO technique. The study revealed widespread, but patchy distribution of marama bean in Namibia. Five Marama bean microsatellite enriched libraries were created. The initial results provided vital information for the ongoing marama bean conservation function and improvement program

DEVELOPMENT AND USE OF MICROSATELLITE MARKERS IN MARAMA BEAN

Marama bean [ Tylosema esculentum (Burchell) Schreiber] occurs naturally in arid parts of southern Africa. Due to the high nutrient value of the seeds and tubers; richness in protein, oil and starch; it is a potential crop for arid areas where few conventional crops can survive. Microsatellites are becoming the molecular marker system of choice because they are multiallelic and generally more informative. Recently, the development of SSR enrichment techniques has increased the efficiency of SSR characterisation in new species. The aim of the study was to develop SSR’s for detection of polymorphisms in Marama bean. The microsatellite regions of the genome were the main focus for potential to be used in Marama bean genetic diversity studies. Microsatellite loci were isolated from the Marama bean germplasm using a modified FIASCO enrichment technique. Nine Marama bean microsatellite libraries, enriched for (AAG)7, (GTT)7, (AGG)7, (GAG)7, (CA)10, (CT)10, (TCC)7, (CA)15 and (CAC)7, were created. Of the 80 primers designed, 76% were able to detect polymorphism. Four of the SSR’s were used for a genetic variation analysis and have proved to be useful and informative for genetic diversity studies.Le haricot Marama [ Tylosema esculentum (Burchell) Schreiber] est produit naturellement en milieux arides de l’Afrique du Sud. Etant donné la valeur nutritionnelle des ses graines, carottes, sa richesse en protein, en huiles et amidon, le haricot Marama est considéré comme une culture potentielle pour des milieux arides où peu de cultures conventionnelles peuvent survivre. Dans ces conditions, les microsatellites deviennent de plus en plus un système moléculaire de choix parce que elles sont multialléliques et généralement plus informatives. Récemment, le développement des techniques d’enrichissement SSR a augmenté l’efficacité de la caractérisation de SSR dans de nouvelles espèces. L’objet de cette étude était de développer SSR pour la détection du polymorphisme dans le haricot Marama. Les régions microsatellitaires du génome étaient le principal point d’intérêt pour des études de la diversité génétique du haricot Marama. Les microsatellites loci étaient isolés du germoplasme du haricot Marama en en utilisant une technique d’enrichissement FIASCO modifié. Neuf librairies microsatellitaires du haricot Marama, enrichies pour (AAG)7, (GTT)7, (AGG)7, (GAG)7, (CA)10, (CT)10, (TCC)7, (CA)15 et (CAC)7, étaient créées. Parmi les 80 désignées, 76% étaient capables de détecter le polymorphisme. Quatre des SSR étaient utilisées pour une analyse de la variation génétique et ont prouvé être plus utiles et informatives pour des études de la diversité génétique