M.Sc. / As part of an investigation into differential gene expression in response to abiotic and chemical inducers of acquired resistance in tobacco, a PCR fragment of 660bp was repeatedly found in RNA preparations from treated cell suspensions by differential display analysis. The fragment (D1B) was isolated, purified, cloned and sequenced. The nucleotide sequence of the fragment was compared with sequences in the BLAST sequence database and was found to be homologous to the mitochondrial orfx genes from Arabidopsis thaliana, Beta vulgaris, Oenothera berteriana, Oryza sativa and Marchantia polymorpha. In order to obtain the full sequence of the gene specific primers were designed using the Arabidopsis sequence as template. The primers were designed to complete the 5’-end of the gene and were designed to overlap the D1B fragment previously found. A fragment (C3Y) of 460bp was isolated, purified, cloned and sequenced. The complete sequence (D1B and C3Y combined) was 851bp long and showed 96% homology with the Arabidopsis orfx gene on the nucleotide level and 87% homology on the translated amino acid level. The sequence was submitted to the Basic Local Alignment Search Tool (BLAST) database as accession gi: 24209907. In plant genomes, the orfx gene is closely linked to important structural genes such as the nad subunits of complex I (NADH: ubiquinone oxidoreductase). Orfx codes for a hypothetical protein that shows homology to the mttB (membrane targeting and translocation) gene found in E. coli. In bacteria the gene is essential because if deleted, the organism was no longer viable. Functional analysis of the bacterial gene revealed a novel pathway specific for membrane targeting and secretion of cofactor containing proteins, such as iron-sulphur (Fe-S) clusters, of which the mttB gene encodes one subunit. It is thought that a similar pathway might be responsible for the correct localisation and assembly of such Fe-S containing protein complexes in the inner mitochondrial membrane of higher plants. The differential display result may be indicative of a general up-regulation of mitochondrial gene expression in response to the triggering of plant defences or a possible specific effect on the expression of the orfx gene. A hypothesis was formulated that chemical inducers of plant defences affect the mitochondria of treated plant cells to result in increased production of reactive oxygen intermediates (ROI), similar to the oxidative microbursts proposed to be involved in systemic required resistance. Using a dichlorodihidrofluorescein (H2DCFDA) assay, it was found that salicylic acid (SA), benzo (1,2,3) thiadiazole-7-carbothioc acid S-methyl ester (BTH) and isonitrosoacetophenone (INAP) increased ROI production within cells in a dose dependant manner. The biochemical basis of this effect could possibly be related to the inhibition of the NADH:ubiquinone oxidoreductase activity of complex I of the mitochondrial electron transport chain by SA, BTH and INAP. / Prof. I.A. Dubery

No description available.

The relationship of geographic location, insecticide exposure, time of season and the genotype of red and green morphs of the tobacco aphid, Myzus nicotianae Blackman, on tobacco was investigated in Virginia in 1988 and 1989. Color morph and karyotype were found to be highly related. The translocated karyotype was associated with the red morphs and the normal karyotype was associated with the green morphs. The karyotype of red and green morphs did not change markedly from the beginning to the end of the tobacco growing season. Studies were also conducted to determine if the red and green morphs of the tobacco aphid were developing resistance to acephate (Orthene Tobacco Insect Spray), the most commonly used aphicide on flue-cured tobacco in Virginia. Slight resistance was present in both red and green aphid populations from several counties. Studies were also conducted to determine if males were present in tobacco aphid populations in Virginia. Cool temperatures and short photoperiods which initiate male production in Myzus persicae (Sulzer) with holocyclic life cycles did not cause the production of males in M. nicotianae native to Virginia. / Master of Science

A two year study was conducted to measure the impact of tobacco aphid, Myzus nicotianae Blackman, colonization and cumulative aphid-days on flue-cured tobacco, Nicotiana tabacum (L.). The objectives of the study included quantifying the response of tobacco production variables, cured leaf quality, and cured leaf chemical analysis to the level of cumulative aphid-days. Aphid populations and tobacco plant responses were recorded on an individual plant basis. A gradient in cumulative aphid-days was obtained through the use of temporally distributed aphid colonizations and selective insecticide use. Tobacco aphid populations resulted in yield reductions as great as 22 and 27% in 1988 and 1989, respectively, while gross economic returns were reduced 27 and 32% in the respective years. The responses of tobacco production variables were characterized by a decreasing negative slope; therefore, incremental losses were greatest at low levels of cumulative aphid-days. Regression models were developed to describe crop production responses as a function of cumulative aphiddays. The quality of the cured leaves (grade index) was also responsive to the level of cumulative aphid-days. Changes in both tobacco grade group and quality within a given group occurred with increasing cumulative aphid-days. The occurrence of nondescript tobacco was associated with large aphid populations. The chemical quality of the cured tobacco was also influenced by cumulative aphid-days. The total alkaloid content followed a linear function, while the level of reducing sugars was a nonlinear relationship with cumulative aphid-days. The study also reported on the disproportionate impact of aphid populations upon the within-plant responses of tobacco. A study was conducted to evaluate the impact of tobacco aphid management using eight different action thresholds. Comparison of the two most commonly recommended treatment thresholds (10 and 20% of plants with 50 or more aphids per leaf) revealed no significant differences in the number of remedial treatments required or the yield and gross economic returns. However, use of the latter threshold resulted in a delay of approximately one week for the first treatment and the retreatment interval. / Ph. D.

No description available.

Turnip mosaic virus (TuMV) infects a variety of crops, worldwide, including the economically relevant Brassicacea family. It was previously demonstrated that TuMV infection in tobacco protoplasts leads to an overall decrease of host protein. However, it remains unclear whether this phenomenon is due to the repression of plant gene transcription during the infection period or due to viral inhibition of host translation. In this study, quantification of various transcripts and protein products from infected tobacco was performed via real-time RT-PCR and ELISA. In comparison to the gamma-tubulin endogenous control, gene expression for the tobacco H3, HSP70 and granule-bound starch synthase was affected by TuMV infection with time. / Tobacco protein accumulation in whole leaf tissues was also significantly affected by increase of virus particles.

M.Sc. / Plant disease can be naturally suppressed by plant growth promoting rhizobacteria and endophytic / endorhizosphere bacteria. Apart from direct antagonism against pathogenic organisms, these plant growth promoting bacteria and endophytes can induce a form of systemic resistance (ISR) in plants. The main bacterial inducing component has been suggested to be the outer membrane lipopolysaccharides (LPS), found in the cell walls of Gramnegative bacteria. Burkholderia cepacia (Pseudomonas cepacia) is a bacterial endophyte that has potential as a biocontrol agent. Although a few studies have indicated that LPS from, certain Pseudorrionads has a protective effect in plants against disease, a controlled investigation has not been attempted previously with a purified preparation of LPS. LPS was isolated from the bacterial cell wall, prepared and characterized by denaturing electrophoresis. Characterization of the LPS also included the determination of 2-keto-3-deoxyoctonate, carbohydrate —, as well as the protein content. The purified LPS was found to possess activity as an elicitor of plant defence responses in tobacco where the induction of pathogenesisrelated (PR) proteins were investigated and electrophoretically analysed. An optimum LPS concentration range of 50-150 14/m1 was determined by studying cell death using the Evans blue procedure. Time and concentration ranges for LPS induced responses were established in cell suspensions, leaf discs, whole leaves and whole plants. It was determined that the PR-protein response could be optimally induced after four days following elicitation with 100 fag/ml LPS. Systemic induction of resistance was tested by treatment of the lower leaves and following the response in the upper leaves; as well as bacterial inoculation of the plant roots followed by PR-protein extraction of the leaves. Treatment of tobacco plants with LPS protected the plants against subsequent infection by the pathogen Phytophthora nicotianae, thereby suggesting a role for LPS as activators of systemic acquired resistance (SAR). It can be concluded from this study that the lipopolysaccharides from Burkholderia cepacia, that were used in this study, are effective local as well as systemic inducers of the defense PR-proteins in Nicotianae tabacum cv Samsun NN. The fact that protection is associated with PR-protein induction distinguishes it from the protection induced by rhizobacteria.

M.Sc. (Zoology) / Paratrichodorus meyeri De Waele & Killian, 1992 is extremely sensitive to surface sterilization; therefore in vitro culture could not be utilized for the mass culture of this nematode and another method had to be developed. Hermetically sealed glass containers were employed successfully for the reproduction of Paratrichodorus meyeri. With this method an average 12 times increase was achieved on tomatoes over a period of 60 days. Certain crops were evaluated in the greenhouse to establish their status as hosts for Paratrichodorus meyeri. The aim of this experiment was to determine which plants could be used as hosts for the reproduction of the nematodes in the greenhouse. It was found that tomatoes (cultivar Roma VF) and wheat (cultivar Gamtoos) were excellent hosts, tobacco (cultivar K51E) and onions (cultivar Bon accord) good hosts, tobacco (cultivar TL 38) and lettuce (cultivar Great Lakes) average hosts and sweetcorn (cultivar Golden Bantam), beetroot (cultivar Formanova) and cabbage (cultivar Sugar Loaf) unsatisfactory hosts for this nematode species. The aim was also to determine the effect of this nematode species on tobacco and cotton. Various inoculation levels of Paratrichodorus meyeri were tested on tobacco cultivars K51E, TL33 and RK and the cotton cultivar Acala 1517170. It was established that an inoculation level as low as 50 - 100ft! Qsoil had a negative effect on cotton (cult. Acala 1517170). A damage threshold value of 100ft! Q soil on the tobacco . cultivar K51E was established. It is also possible for tobacco cultivar RK to support this nematode species without any adverse effects it can even be beneficial for this particular cultlvar. It was also determined that a negative correlation exits between the initial number of nematodes inoculated and the final population.

M.Sc. / in the current study the effect of the addition of methyl Jasmonate (MeJA), chitosan, a cell wall elicitor prepared from Phytophthora nicotlanae to tobacco cells and the subsequent defense responses elicited in these cells were Investigated. The defense responses investigated can be divided into three categories according to the time scale whereby resistance responses in plant cells are induced: early events which included the analysis of lipid peroxidation, the induction of lipoxygenase (L0)0 enzyme activity as well as the changes in phosphoprotein profiles; intermediate to later responses which included investigations of peroxidase (POD) activity, lignin content, phytoalexin content and phenolic content and also late responses which included studies of pathogenesis-related proteins (PR) and 13-1,3-giucanase activity. An approach also followed in this study was the addition of MeJA to tobacco cells for 24 h followed by the addition of either the cell wall elicitor or chitosan as a secondary elicitors, to investigate possible preconditioning or sensitisation by MeJA. Results obtained in this study revealed the time and concentration dependent accumulation of phytoalexins (secondary metabolites) when MeJA was added to tobacco cells and an optimal concentration of MeJA to use in further studies was determined as 1 mM. MeJA was the most effective inducer of lipid peroxidation (22 fold induction), a response observable after 2 h of exposure to MeJA. Conditioning with MeJA, followed by both chitosan (19 fold induction) and elicitor (25 fold induction) led to an earlier accumulation as well as significant increases in the levels of malondialdehyde, the product of lipid peroxidation. LOX enzyme activity was significantly increased by the addition of MeJA (6 fold Induction), chitosan (4 fold induction) and elicitor (3.8 fold induction). Conditioning with .MeJA, followed by both chitosan (3.3 fold induction) and elicitor (3.9 fold Induction) also led to noteworthy increases in enzyme activity. Analysis of the phosphoprotein profiles do not reveal the accumulation of phosphorylated proteins when MeJA was added to cells and very little accumulation of such proteins when chitosan was added. Phosphorylated proteins could be observed in cells treated with elicitor and In the cases where conditioning with MeJA, followed by secondary elicitation with either chitosan or elicitor, was studied, the differential induction of phosphorylated cellular proteins could also be observed. No significant induction of POD activity could be observed under any of the conditions, except for a possible slight increase in POD activity starting at 16 - 24 h after the elicitor had been added and a more definite increase after 24 h which was sustained up to 48 h after the addition of MeJA. PAGE of peroxidase, followed by activity staining revealed the presence of a slow migrating anionic peroxidase as well as a fast migrating peroxidase. Conditioning with MeJA, followed by secondary elicitation with both chitosan and elicitor revealed enhanced POD activity as well increased induction of a fast migrating anionic peroxidase on PAGE gels. MeJA was a more effective inducer of elevated levels of lignin content than the elicitor or chitosan and the addition of MeJA to tobacco cells led to a 2.2 fold increase in the lignin content, a response observed after 24 h and sustained up to 48 h. Chitosan as secondary elicitor did not lead to any increase in lignin content, but the cell wall elicitor as secondary agent significantly increased the lignin content after 40 - 48 h. Analysis of phenolic content did not show any significant increases In the total soluble phenolics when the agents were used on their own and only the phenolic content of the MeJA-conditioned cells, followed by the addition of chitosan showed a slight increase. In this case, the HPLC analysis of the phenolics also revealed a shift In the profiles for phenolics. SDS-PAGE of PR proteins revealed the induction of constitutive as well as new proteins when MeJA and elicitor, but not chitosan were used as elicitation agents. However, In the MeJA-pretreated cells addition of both chitosan and elicitor led to increased accumulation of PR proteins with molecular masses ranging from 6 - 70 kDa. Results from the i3-1,3-glucanase activity assay indicate a strong induction (4-5 fold) when MeJA and elicitor (4 fold), but not when chitosan was added to cells. Conditioning effects were revealed when both chitosan (3 fold induction) and elicitor (2.5 fold induction) were used as secondary elicitors. The increases in intensities of bands with molecular masses ranging from 31- 35 kDa observed on SOS-PAGE gels where chitosan and elicitor were added as secondary agents corresponded in a time dependent manner with the increased levels obtained in thep-1,3-glucanase activity assay.

As the causative agent of black shank, Phytophthora nicotianae is a serious threat to tobacco cultivation in South Africa. Research presented in this dissertation describes pathogenicity studies and control measures for P. nicotianae on tobacco. Special attention is given to the population structure of P. nicotianae in South Africa. The implications of these genetic studies in breeding and selection programs against P. nicotianae were also evaluated. The first chapter of this dissertation represents a literature review on black shank and available control measures for P. nicotianae on tobacco. The mechanisms of pathogenicity and the life cycle of P. nicotianae are also treated in detail. Special reference is made to the maintenance of genetic diversity in Phytophthora species and particularly P. nicotianae. This literature review also highlights the fact that very few studies have been conducted to determine the genetic structure of P. nicotianae populations. The success of South African breeding programs for tobacco cultivars with P. nicotianae resistance is to some degree dependent on the selection of isolates with high levels of aggressiveness. The research presented in chapter two provides information on cultivar resistance and selection of P. nicotianae isolates for future breeding programs. Significant differences in levels of aggressiveness were found between P. nicotianae isolates. Furthermore, race 0 and 1 of P. nicotianae occurred in most of the tobacco growing regions in South Africa. Selected Race 0 and 1 isolates were thus used to evaluate black shank resistance of 11 commercially planted tobacco cultivars. Commercially planted cultivars differed significantly in their resistance to race 0 and 1. Cultivars LK33/60 and OD1 were highly resistant to race 0 but susceptible to race 1 while cultivars Vuma/3/46 and LK3/46 were highly resistant to both race 0 and 1. Chapter three reports on the use of metalaxyl treatments combined with resistance in tobacco cultivars for control of P. nicotianae. One hundred and thirty two isolates of P. nicotianae were screened for sensitivity to metalaxyl. P. nicotianae isolates from most tobacco farms were metalaxyl sensitive. The results further indicated that the use of metalaxyl in combination with moderately resistant cultivars effectively reduced black shank in the field. The outcome of this study provided useful information for the implementation of an economically viable combination of disease resistance and metalaxyl as the basis for a P. nicotianae management program in South Africa. Chapter four of this dissertation deals with the development of a rapid seedling-' based screening technique to assay tobacco for resistance to P. nicotianae. This technique was validated by comparing it to a stem inoculation technique commonly used on adult plants. A strong positive correlation was found between results of the seedling assay and adult plant trials for all isolates and cultivars tested. P. nicotianae isolates could also be characterized as race 0 or I using both stem inoculation and the rapid seedling assay. The ability to screen large numbers of tobacco plants rapidly at the seedling stage allows for the testing of large germplasm resources in a systematic manner and under standard conditions. This may help in the timely development and release of more black shank resistant cultivars. In chapter five, a population study on P. nicotianae in South Africa is presented. One hundred and five P. nicotianae isolates were collected from the Northern Highveld and Lowveld regions, as well as from both citrus and tobacco hosts in South Africa. Levels of phenotypic diversity were determined in populations of P. nicotianae using RAPD markers. Among the 105 P. nicotianae isolates analysed 79 different RAPD phenotypes were found, where 35 of the isolates were found to be clonal. The high number of RAPD phenotypes (79) in relation to the sample size (105), the presence of both the Al and A2 mating type and high levels of phenotypic diversity in the P. nicotianae population indicate a sexually outcrossing P. nicotianae population in South Africa. This sexual outcrossing may mean that P. nicotianae is likely to remain a constant threat to tobacco and citrus cultivation, since new genotypes with the potential to overcome resistance genes in commercial cultivars are likely to emerge. All chapters of this dissertation deal with some aspects of black shank control and breeding for resistance to P. nicotianae. This dissertation provides new knowledge on variation in levels of aggressiveness, race distribution and the development of metalaxyl resistance in the South African P. nicotianae populations. This also represents the first study on the genetic diversity of P. nicotianae populations in South Africa. The results presented here not only show the possible occurrence of sexual reproduction, but also indicate the presence of clones and discreet phenotypic groups of P. nicotianae. This information will be applied in future tobacco breeding programs to select breeding lines with resistance against a number of specific P. nicotianae races and phenotypic groups. / Thesis (DPhil)--University of Pretoria, 2006. / Microbiology and Plant Pathology / Unrestricted