Bibliography: leaves 108-152 / viii, 152 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This study investigates the induction of resistance to the clover cyst nematode, Heterodera trifolii Goffart, an economic pest in white clover pastures that are a key to high milk yields in dairy cattle...it explores the potential of soil and rhizosphere bacteria to induce systemic resistance in white clover, Trifolium repens L. (summary) / Thesis (Ph.D.)--University of Adelaide, Dept. of Applied and Molecular Ecology, 2000

Bibliography: leaves 137-163. / vii, 163, [25] leaves, [13] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / his is a comprehensive study of induced homoeologous recombination along most of the complete genetic length of two homoeologous chromosomes in the Triticeae (7A of common wheat and 7Ai of Agropyron intermedium), using co-dominant DNA markers. Chromosome 7Ai was chosen as a model alien chromosome because is has been reported to carry agronomically important genes conferring resistance to stem rust and barley yellow dwarf virus on its short and long arms, respectively. / Thesis (Ph.D.)--University of Adelaide, Dept. of Plant Science, 1997

Bibliography: 136-146 / v, 146 leaves, [9] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--Dept. of Agronomy, University of Adelaide, 1987

Thesis (MSc)--University of Stellenbosch, 2000. / ENGLISH ABSTRACT: Grapevine leafroll is one of the most damaging viral diseases that affect many viticultural regions of the world. Numerous reports over the last few years have associated closterovirus-like particles with leafroll disease. To date, eight serologically distinct closteroviruses have been isolated from leafroll infected vines, of which grapevine leafroll associated closterovirus-3 (GLRaV-3) is the best characterized. Virus resistance in transgenic plants based on the expression of a virusderived gene is known as pathogen-derived resistance. The viral coat protein (CP) gene, which expresses a structural protein responsible for coating the virus particles, was used in the first demonstration of virus-derived resistance. Coat protein-mediated resistance is currently the most feasible and most widely used method to obtain virus resistance in crop plants. The CP gene of a South African isolate of GLRaV-3 infected grapevine was isolated, cloned and sequenced. Double stranded RNA (dsRNA) was extracted from GLRaV-3 infected material and a high molecular weight band, of -18 kb was identified from infected vines. The dsRNA was used as a template in a reverse transcription PCR together with GLRaV-3 CP gene specific primers for the amplification of the GLRaV-3 CP gene (975 bp). The GLRaV-3 CP gene was cloned into the pGem®-T Easy vector. Clones hosting the CP gene in the sense (pLR3CP+) and antisense (pLR3CP-) orientations respectively were obtained. The sequence obtained from these two clones showed 99.26 % similarity to the only other GLRaV-3 CP nucleotide sequence available. The GLRaV-3 CP gene was excised from pLR3CP+ and pLR3CP- and subcloned into a plant expression vector, pCAMBIA 3301 in the sense (pCamBLR3CP+) and antisense (pCamBLR3CP-) orientations respectively, therefore enabling sense and antisense gene expression in transgenic plants. The GLRaV-3 CP gene was also subcloned from pCamBLR3CP+ into another plant expression vector, pCAMBIA 2301 in the sense orientation and designated as pCVSLR3CP+. These three constructs were given to Dr. M. Vivier (Institute for Wine Biotechnology, Stellenbosch) for grapevine transformation experiments. Two of these constructs, pCamBLR3CP+ and pCamBLR3CP- as well as pCAMBIA 3301 were used to transform Nicotiana tabacum by Agrobacterium tumefaciens-mediated transformation. Plants were selected for their ability to withstand the herbicide, Basta. This resistance is due to the presence of a plant selectable marker gene on each of these constructs, known as the bar gene. PCR with GLRaV-3 CP gene specific primers showed no amplification of the GLRaV-3 CP gene in the plants transformed with pCamBLR3CP+ and pCamBLR3CP-. Southern blot analysis with the GLRaV-3 CP gene as hybridization probe showed no signal for these plants, thus confirming the PCR results. PCR with bar gene specific primers showed no amplification of the bar gene in the plants infected with pCAMBIA 3301. The plants transformed with pCamBLR3CP+ and pCamBLR3CP- were also screened for the presence of the bar gene. Three of the eight plants tested showed amplification of the -560 bp bar gene. This result suggests that these plants were transformed with pCAMBIA 3301 (vector without the ligated GLRaV-3 CP gene) and not pCamBLR3CP+ or pCamBLR3CP- as had been expected. This project provides preliminary work for the subsequent transformation of grapevine with the GLRaV-3 CP gene, in an attempt to impart virus resistance. / AFRIKAANSE OPSOMMING: Wingerd rolblaar is een van die mees beskadigende virale siektes wat baie wingerd areas in die wêreld aantas. In Aantal verslae oor die afgelope jare het closterovirus partikels met wingerd rolblaar geassosieer. Tot hede, is agt serologiese onderskeibare closterovirusse geïsoleer vanuit geaffekteerde wingerde, waarvan wingerd rolblaar geassosieerde closterovirus-3 (GLRaV-3) die beste gekarakteriseerd is. Virus bestandheid in transgeniese plante gebaseer op die uitdrukking van gene afkomstig vanaf virusse, staan bekend as patogeen-afgeleide weerstand. Die virale kapsule protein (CP) geen vervaardig In strukturele protein wat verantwoordelik is vir die bedekking van die virus partikel. Dié geen was gebruik in die eerste demonstrasie van patogeen-afgeleide weerstand. Kapsuul protein-bemiddelde weerstand is tans die mees praktiese en algemene gebruikte metode om virus weerstand in plant gewasse te verkry. Die CP geen van In Suid Afrikaanse isolaat van GLRaV-3 geïnfekteerde wingerde is geïsoleer, gekloneer en die volgorde is bepaal. Dubbelstring RNA (dsRNA) was uit GLRaV-3 geïnfekteerde materiaal geëkstraheer en In hoë molekulêre gewig band van -18 kb is geïdentifiseer. Die dsRNA is gebruik as In templaat vir In omgekeerde transkripsie PKR saam met GLRaV-3 CP geen spesifieke inleiers vir die amplifikasie van die GLRaV-3 CP geen (975 bp). Die GLRaV-3 CP geen is gekloneer in die pGem®-T Easy vektor. Klone met die CP geen in die sin (pLR3CP+) en teensin (pLR3CP-) oriëntasies respektiewelik is verkry. Die volgorde wat verkry is vanuit hierdie twee klone dui op In 99.26 % ooreenstemming met die enigste ander GLRaV-3 CP geen volgorde wat beskikbaar is. Die GLRaV-3 CP geen is uit pLR3CP+ en pLR3CP- gesny en is gesubkloneer in In plant ekspressie vektor, pCAMBIA 3301 in die sin (pCamBLR3CP+) en teensin (pCamBLR3CP-) oriëntasies respektiewelik, wat die sin en teensin geen ekspressie in transgeniese plante in staat stel. Die GLRaV-3 CP geen was ook gesubkloneer vanaf pCamBLR3CP+ in In ander plant ekspressie vektor, pCAMBIA 2301 in die sin orientasie en is as pCVSLR3CP+ benoem. Hierdie drie konstruksies is aan Dr. M. Vivier (Instituut vir Wyn Biotegnologie, Stellenbosch) gegee vir wingerd transformasie eksperimente. Twee van hierdie konstruksies, pCamBLR3CP+ en pCamBLR3CP- asook pCAMBIA 3301 is gebruik om Nicotiana tabacum deur middel van Agrobacterium tumefaciens-bemiddelde transformasie te transformeer. Plante is geselekteer vir hul vermoë om die onkruiddoder, Basta, te weerstaan. Die teenwoordigheid van die plant selekteerbare merker geen, bar, op elke konstruksie lui tot dié weerstand. Die plante wat getransformeer is met pCamBLR3CP+ en pCamBLR3CP- is deur PKR saam met die GLRaV-3 CP geen spesifieke inleiers getoets, en geen amplifikasie van die GLRaV-3 CP geen is getoon nie. Southern blot analise met die GLRaV-3 CP geen as hibridisasie peiler het geen sein gewys vir hierdie plante nie, wat die PKR resultate bevestig. Die plante wat getransformeer is met pCAMBIA 3301 is deur PKR saam met die bar geen spesifieke inleiers getoets, en geen amplifikasie van die bar geen is getoon nie. Die plante wat getransformeer is met pCamBLR3CP+ en pCamBLR3CP- is ook getoets vir die teenwoordigheid vir die bar geen. Drie van die agt plante wat getoets is, het amplifikasie van die -560 bp bar geen getoon. Hierdie onverwagte resultate stel voor dat dié plante met pCAMBIA 3301 (vektor sonder die geligeerde GLRaV-3 CP geen) en nie met pCamBLR3CP+ en pCamBLR3CPgetransformeer is nie. Hierdie projek verskaf voorlopige werk vir die daaropvolgende transformasie van wingerd met die GLRaV-3 CP geen in 'n poging om virus bestandheid te verskaf.

Thesis (MScAgr)--Stellenbosch University, 1962. / ENGLISH ABSTRACT: no abstract available / AFRIKAANSE OPSOMMING: geen opsomming

Wheat (Triticum aestivum and T. Durum) is an extremely important agronomic crop produced worldwide. Wheat consumption has doubled in the last 30 years with approximately 600 million tons consumed per annum. According to the International Maize and Wheat Improvement Center, worldwide wheat demand will increase over 40 percent by 2020, while land as well as resources available for the production will decrease significantly if the current trend prevails. The wheat industry is challenged with abiotic and biotic stressors that lead to reduction in crop yields. Increase knowledge of wheat’s biochemical constitution and functional biology is of paramount importance to improve wheat so as to meet with this demand. Pesticides and fungicides are being used to control biotic stress imposed by insect pest and fungi pathogens but these chemicals pose a risk to the environment and human health. To this effect, there is re-evaluation of pesticides currently in use by the Environmental Protection Agency, via mandates of the 1996 Food Quality Protection Act and those with higher perceived risks are banned. Genetic resistance is now a more environmental friendly and effective method of controlling insect pest and rust diseases of wheat than the costly spraying with pesticides and fungicides. Although, resistant cultivars effectively prevent current prevailing pathotypes of leaf rust and biotypes of Russian wheat aphid from attacking wheat, new pathotypes and biotypes of the pathogen/pest may develop and infect resistant cultivars. Therefore, breeders are continually searching for new sources of resistance. Proteomic approaches can be utilised to ascertain target enzymes and proteins from resistant lines that could be utilised to augment the natural tolerance of agronomically favourable varieties of wheat. With this ultimate goal in mind, the aim of this study was to elucidate the mechanism and synchronicity of wheat resistance to leaf rust (Puccinia triticina) and Russian wheat aphid (Duiraphis noxia) SA1. To determine the resistance mechanism of the wheat cultivars to leaf rust infection and Russian wheat aphid infestation, a proteomics approach using two-dimensional gel electrophoresis was used in order to determine the effect of RWA SA1 on the wheat cultivars proteome. Differentially expressed proteins that were up or down regulated (appearing or disappearing) were identified using PDQuestTM Basic 2-DE Gel analysis software. Proteins bands of interest were in-gel trypsin digested as per the protocol described in Schevchenko et al. (2007) and analysed using a Dionex Ultimate 3000 RSLC system coupled to an AB Sciex 6600 TripleTOF mass spectrometer. Protein pilot v5 using Paragon search engine (AB Sciex) was used for comparison of the obtained MS/MS spectra with a custom database containing sequences of Puccinia triticina (Uniprot Swissprot), Triticum aestivum (Uniprot TrEMBL) and Russian wheat aphid (Uniprot TrEMBL) as well as a list of sequences from common contaminating proteins. Proteins with a threshold of ≥99.9 percent confidence were reported. A total of 72 proteins were putatively identified from the 37 protein spots excised originating from either leaf rust or Russian wheat aphid experiments. Sixty-three of these proteins were associated with wheat response to stress imposed by RWA SA1 feeding while 39 were associated with infection by Puccinia triticina. Several enzymes involved in the Calvin cycle, electron transport and ATP synthesis were observed to be differentially regulated suggesting greater metabolic requirements in the wheat plants following aphid infestation and leaf rust infection. Proteins directly associated with photosynthesis were also differentially regulated following RWA SA1 infestation and P.

Because plants lack a circulating adaptive immune system, they have evolved multicomponent defense mechanisms to protect themselves against pathogen attack. These defense mechanisms/responses are either constitutively active in the plant, or they are inducible by pathogens. Understanding of the plant response to pathogen attack has advanced rapidly in recent years. Bacterial and fungal pathogenicity factors have been isolated, and mechanisms that are utilized by the plant to recognize the pathogen and initiate a plethora of defense mechanisms have been identified. In contrast to the well-documented effects of LPS on mammalian cells, the effects of LPS on plant cells have been far less studied. The present study focused on the involvement of lipopolysaccharides (LPS) isolated from the outer cell wall of the Gram-negative bacteria, Burkholderia cepacia (strain ASP B 2D), and yeast elicitor (YE, a cell wall preparation from Saccharomyces cerevisiae) on the molecular mechanisms and components involved in signal transduction and defense-related responses in suspension cultured cells derived from tobacco plants (Nicotiana tabacum cv. Samsun). LPS was extracted, analyzed by denaturing electrophoresis and characterized with regard to 2-keto-3-deoxyoctonate (KDO) content, carbohydrate content, and protein content. The purified LPS and YE were found to trigger defense- and resistance-related responses in the tobacco cells. These responses included a rapid influx of Ca2+ into the cytoplasm of transgenic aequorin-transformed tobacco cells, the production of reactive oxygen species (ROS) during the oxidative burst, alkalinization of the extracellular culture medium of the cells, and changes in the protein phosphorylation patterns of the cells. Time- and concentration-dependent studies for the induction of perception and signal transduction-related responses by YE and LPS indicated that 100 µg.ml-1 of either elicitor was sufficient to induce significant responses in the cells. YE and LPS both induced a rapid transient increase in cytosolic Ca2+ levels, returning to basal levels after seconds, followed by a second, larger and long-term increase in cytosolic Ca2+. The YE-induced cytosolic Ca2+ influx was 7.5 fold higher than that of LPS. Luminol-dependent chemiluminescence measurements of hydrogen peroxide (H2O2) produced during the YE- and LPS-induced oxidative burst reactions indicated 3.5 fold higher levels of H2O2 induced by YE than that induced by LPS. Total inhibition of H2O2 production by YE- and LPS-induced cells was observed upon treatment of the cells with the H2O2-degrading enzyme, catalase. ROS production was also analyzed by the H2DCF-DA-derived fluorescence assay. The degree of ROS production by YE-treated cells was larger than that of cells treated with LPS, suggesting that YE is a more potent inducer of plant defense responses than LPS. Categorization of the origins of the oxidative bursts, induced by YE and LPS, by the addition of a ROS scavenger (NAC), inhibitors of ROS production (DPI and DDC) and a nitric oxide scavenger (PTIO) indicated that YE and LPS induced different quantities of the same ROS species. The induced ROS included O2-·, H2O2 and perhaps other ROS species as well. In addition, both YE and LPS induced a remarkable burst of nitric oxide (NO), as determined by the 97% and 95% respective inhibitions of the H2DCF-DA-derived fluorescence by the nitric oxide scavenger PTIO. Alkalinization of the extracellular culture medium of the tobacco cells was observed after treatment of the cells with YE and LPS. Both of these elicitors induced a significant increase in extracellular pH from resting pH values of 5.7 to pH 6.3 by YE, and 6.0 by LPS. Notably, the YE-induced response returned to near basal pH levels after 50 min, while the LPS-induced response showed no signs of declining and fluctuated around pH 5.9 for the duration of the experiment. YE and LPS both induced the hyperphosphorylation of two distinct proteins with approximate molecular masses of 28 kDa and 2 kDa. Changes in the pattern of the [32P]-radiolabeled proteins pp28 became visible after 20 min of YE-elicitation and 30 min of LPS-elicitation and changes in pp2 phosphorylation became visible after 20 min treatment of the cells with both elicitors. Addition of the protein kinase inhibitor, staurosporine, to the cells followed by subsequent elicitation by YE or LPS, resulted in inhibition or abolishment of the elicitor-induced responses during the oxidative burst, extracellular alkalinization, and protein phosphorylation. In contrast, the addition of the protein phosphatase inhibitor, calyculin A, was found to mimic elicitor action in several aspects, including extracellular alkalinization, the oxidative burst and protein phosphorylation, even in the absence of elicitors or any other stimulus. Thus, a fine balance between the actions of certain protein kinases and protein phosphatases is an essential component of signal transduction during YE and LPS elicitation of tobacco cells but the identification and characterization of the staurosporine-sensitive protein kinases and their substrates are necessary to gain a better understanding of the chemosensory perception and signal transduction of the YE and LPS elicitor signals in plant cells. Moreover, the question of whether these perception and transduction mechanisms are connected with a reduced activity of a protein phosphatase, or with the increased activity of a protein kinase, or even a combination of both, remains to be elucidated. / Prof. I.A. Dubery

M.Sc. (Biochemistry) / Our comprehension of pathogen perception and defense response mechanisms that play key roles in the resistance of plants against pathogen attack have progressed substantially within the recent years. Recognizing the molecular mechanisms involved in pathogen perception is the basis of understanding the signalling networks that are involved, including the transcriptional regulation of plant defense genes. This has proven to be a great challenge in plant pathology and, as such, has attracted much attention. The receptor-like kinases (RLKs) constitute one of the largest classes of plant defense genes in Arabidopsis thaliana, and contains, inter alia, the well-known leucine-rich repeats-RLKs (LRR-RLK), as well as the S-domain receptor-like kinases (SD-RLKs) that have been shown to be involved in pathogen perception and not only self-incompatibility (SI) as originally discovered. Some members of these RLKs are able to detect pattern-associated molecular patterns (PAMPs), which are conserved pathogen-derived molecules, and trigger a battery of basal defense responses. The transcriptional activation and expression levels of RLKs are dependent on the variation in promoter architecture as a result of the number, location, order and class of cis-elements found in a promoter sequence. It is hypothesized that candidate RLK genes involved in PAMP surveillance are triggered and transcriptionally regulated in response to perception of PAMPs, and that the intensity of response is relative to the promoter architecture. The primary objective was to identify SD-RLKs and LRR-RLKs which demonstrated up-regulation in response to PAMPs. The SD-RLKs (At1g11330, At1g61430 and At1g61610) and LRR-RLKs (At1g51850, At2g19190 and At5g45840) were selected on the basis of microarray data (Nürnberger - TAIR accession set 100808727) and the Genevestigator database, and characterized utilizing bioinformatics tools. Here, molecular techniques were used to show that the selected RLK genes were responsive to PAMP inductions. Furthermore, this study explored which cis-elements and their corresponding transcription factors (TFs) are found in the promoter of plant defense genes and that may be involved in transcriptional regulation thereof...

M.Sc. / Chemically-induced plant defense responses were investigated in tobacco cell cultures. The inducing conditions were as follows: chitosan (C), an elicitor (E) prepared from Phytophthora nicotianae, isonicotinic acid (INA), isonicotinamide (IND) and isonitrosoacetophenone (INAP) as well as the addition of INA, IND and INAP as conditioning agents (primary elicitors) followed by secondary elicitation with either chitosan or elicitor. The defense responses investigated included determinations of phenylalanine ammonia-lyase (PAL) activity, total soluble phenolic content, specific phenolic profiles, phytoalexin content, (3- 1,3-glucanase activity and electrophoretic analyses of pathogenesis-related proteins (PR). The compounds, 4-(3-methyl-2-butenoXy)isonitrosoacetophenone (0-INAP) and 2-isonitrosoacetophenone (INAP) were successfully synthesized from the starting materials p-hydroxyacetophenone and acetophenone respectively. The organic synthesis of 0-INAP involved the formation of a prenyl ether.of p-hydroxyacetophenone, followed by a nitrosation reaction using butyl nitrite as the source of the nitroso group, on the a-carbon atom adjacent to the carbonyl group. The synthesis of INAP only required a nitrosation reaction on the a-carbon atom adjacent to the carbonyl group. The yields of 0-INAP and INAP were 12 - 15 % and 80 %, respectively. An evaluation of the properties of 0-INAP indicated that the compound, dissolved in methanol, has a molar extinction coefficient of 16 5001.mor.cm - ' at A. 302 nm. The compound possesses antifungal activity against Cladosporium cucumerinum, Penicillium expansum and Aspergillus niger as well as the ability to scavenge superoxide radicals which was indicated by a decrease in the chemiluminescence signal produced in a reaction mbdure of hydrogen peroxide, horseradish peroxidase, the chemiluminescence probe, MCLA, and increasing concentrations of 0-INAP. The addition of INA to tobacco cells at a - final concentration of 12.5 iimol.g -1 cells or 2.5 mM did not lead to significant changes in PAL activity, but conditioning with INA, followed by chitosan as well as elicitor led to a 2.5-fold and a 4.3-fold induction respectively. INA as well as INA + C and INA + E led to significant increases in the total soluble phenolic content, and the HPLC analyses of these phenolics indicated the significant induction of a phenolic-like compound with a peak at Rt = 1.7 min. which possibly indicates isonicotinic acid, for INA + C and INA + E. A whole range of phytoalexins were detectable after the addition of INA to tobacco cells and conditioning with INA followed by chitosan induced the phytoalexin, lubimin, several hundred-fold. PR proteins were also induced by INA and a prominent band of 11- 13 kDa was induced after conditioning with INA, followed by secondary elicitation with the elicitor and especially with chitosan. (3-1,3-glucanase activity was also induced by INA; INA + E and particularly INA + C led to increases of 2.5-fold and 4.5-fold in 13-1,3-glucanase activity respectively. The addition of IND to tobacco cells at a final concentration of 12.5 pmol.g -1 cells or 2.5 mM led to a 2.6-fold induction in PAL activity after only 6 h, but conditioning with IND, followed by secondary elicitation did not lead to any significant changes. IND at the earlier time interval (24 h vs. 48 h) as well as IND + C and IND + E led to increases in the total soluble phenolic content, - and the HPLC analyses of these phenolics indicated the significant induction of a phenolic-like compound with ,a peak at Rt = 1.7 min. which possibly indicates isonicotinic acid, for IND + C and IND + E. A whole range of phytoalexins were detectable after the addition of IND to tobacco cells and conditioning with IND followed by chitosan induced the phytoalexin, solavetinone, several hundred-fold. PR proteins were also induced by IND and prominent bands of 34 kDa and 39 - 40 kDa were induced for IND + ELIC. (3-1,3-glucanase activity .was also induced by IND; however, secondary elicitation with chitosan did not lead to increases in enzyme activity, although a twofold increase was detectable for IND + ELIC, compared to IND 72. The addition of INAP to tobacco cells at a final concentration of 6.3 pmol.e cells or 1.25 mM led to a 1.7-fold induction in PAL activity after only 6 h, a response that was still detectable after 30 h; however, conditioning with INAP, followed by secondary elicitation did not lead to any noteworthy changes. INAP 24 as well as INAP 48 did not lead to significant changesin the total soluble phenolic content, but INAP + C and INAP+ E led to increases of 3.3-fold and 3.5-fold, respectively. HPLC analyses of the induced phenolics showed the significant induCtion of a phenolic compound with a peak at Rt = 14.5 min. which possibly indicate p-coumaric acid, for INAP + C and INAP + E. A whole range.of phytoalexins were detectable after the addition of INAP to tobacco cells, but the addition of a secondary elicitor led to a decrease in phytoalexin accumulation. PR proteins were also induced by INAP and conditioning with INAP, followed by especially the elicitor, led to the induction of a whole range of PR proteins with molecular masses ranging from 11 - 68 kDa. (3-1,3-glucanase activity was significantly induced (60-fold compared to control) by INAP 48; however, secondary elicitation led to a decrease in (3-1,3-glucanase)

M.Sc. (Botany) / Cereals such as oats, rye, rice, barley, maize and wheat are a major source of food worldwide. Wheat (Triticum aestivum L.) is the largest winter cereal crop produced in South Africa (Crop Estimates Committee, 2011; South African Department of Agriculture, Forestry and Fisheries, www.daff.gov.za/crop estimates). Wheat production in South Africa includes both the summer and winter rainfall areas. Like other crops wheat is cultivated under monoculture conditions to increase yield per hectare. This increases the risk to pathogen exposure, as monocultures are genetically very similar or even identical, which makes them particularly vulnerable to both abiotic and biotic stresses (Haile, 2001). Both of these stressors negatively influence crop yield (Peterson and Higley, 2001; Wang et al., 2003).