This dissertation describes two different projects. The first is the development of an oral DNA vaccine delivery system for fish. A novel oral DNA vaccine delivery system was developed for Rainbow Trout by combining non-viral vectors (polycationic liposomes or polycationic polymer) to facilitate the DNA vaccine's uptake by cell membranes along with enteric-coated protection of the DNA embedded in microparticles to prevent DNA degradation in the gastrointestinal tract. Spray drying and spray coating bead techniques were employed in the preparation of the DNA vaccine microparticles. The spray drying technique allowed production of spherical shape enteric-coated microparticles with a particle size range of 0.18 to 20 ��m. Larger particle sizes of 40-50 mesh were obtained from the spray-coated bead technique. The resultant DNA vaccine microparticles were granulated with regular fish feed and given to fish to investigate the efficacy of the delivery system in providing protection against IHNV, and to demonstrate the ease of administration in fish. An in vivo fish trial experiment showed improvement in fish survival rate when fish were immunized with larger particle size DNA vaccine microparticles. Further research to find effective vector carriers for the DNA vaccine delivery system and to seek modifications of the delivery system that will still prevent the denaturation of plasmid DNA that will also facilitate membrane uptake of the DNA vaccine is needed in order to develop a safe, effective, and commercially viable vaccine to control the outbreak of IHNV. The second project of the dissertation is prediction of in vitro drug release profiles from a novel matrix tablet spray-coated with a barrier membrane using mathematical and statistical models. Tablets were prepared by direct compression followed by spray coating. The relationship of the amount of hydrophilic materials in the core tablets and barrier thickness on drug release mechanism was investigated using factorial design and regression analysis. Drug release characteristics were influenced and can be controlled by modifying the amount of hydrophilic materials in the core tablet and the barrier thickness. Mathematical equation generated from regression analysis of n-value, lag time, and percent drug release as a function of the amount of hydrophilic material and the amount of coating material applied can now be used as a tool for predicting and optimizing in vitro drug release from matrix tablets spray-coated with a barrier membrane. / Graduation date: 2003

The objective of this study was to characterize an unknown agent associated with decline in black raspberry (Rubus occidentalis) in Oregon. A virus was found consistently associated with decline symptoms of black raspberries and was named Black raspberry decline associated virus (BRDaV). Double stranded RNA extraction from BRDaV-infected black raspberry revealed the presence of two bands of approximately 8.5 and 7 kilobase pairs, which were cloned and sequenced. The complete nucleotide sequences of RNA 1 and RNA 2 are 7581 nt and 6364 nt, respectively, excluding the 3' poly(A) tails. The genome structure was identical to that of Strawberry mottle virus (SMoV), with the putative polyproteins being less than 50% identical to that of SMoV and other related sequenced viruses. The final 189 amino acids of the RNA-dependent- RNA-polymerase (RdRp) reveal an unusual indel with homology to AlkB-like protein domains, suggesting a role in repair of alkylation damage. This is the first report of a virus outside the Flexiviridae and ampeloviruses of the Closteroviridae to contain these domains. An RT-PCR test was designed for the detection of BRDaV from Rubus tissue. BRDaV is vectored non-persistently by the large raspberry aphid Amphorophora agathonica, the green peach aphid Myzus persicae, and likely nonspecifically by other aphid species. Phylogenetic analysis of conserved motifs of the RdRp, helicase, and protease regions indicate that BRDaV belongs to the Sadwavirus genus. To assess the rate of spread BRDaV, four newly planted fields of black raspberries (Rubus occidentalis) in Oregon were studied for three years. In an effort to characterize the suspected complexity of synergistic interactions between BRDaV and other Rubus-infecting viruses, the prevalence of ten additional Rubus viruses was also monitored in the study fields. The timing of BRDaV infection as it relates to aphid populations and flights was also determined. Testing of nearby vegetation identified several symptomless Rubus hosts of BRDaV, as well as detection in multiple cultivars of black raspberry and several non-Rubus weeds. It was determined that BRDaV spreads rapidly with a low aphid threshold and consistently is associated with decline of black raspberries in Oregon. / Graduation date: 2006

No description available.

Maize (Zea mays L.) streak virus disease (MSD) is the most important virus disease in Africa but farmers are unaware of its status. A project was initiated to assess the current status of MSD and to breed for its resistance. Four populations comprised of two BC1F1 and two F2 progenies developed by backcrossing and selfing the F1 progenies of two crosses between a donor line (CMl 202) and two susceptible lines (CMl 321 and CMl 384) were developed. Conventional and molecular marker assisted selection (MAS) methods were used to screen for resistance to MSD in each of the four populations. To facilitate unbiased comparison, separate screening nurseries were established for MAS and conventional screening. The objectives of the study were five-fold; 1) to assess the status of MSD in Uganda and understand farmers' preferences and varietal selection criteria for maize using a participatory rural appraisal (PRA), 2) to screen for MSD resistance in early generations of segregating maize populations using conventional method, 3) to screen for resistance to MSD using SSR marker assisted selection , 4) to compare the effectiveness of marker assisted selection and conventional methods for selection for resistance to MSD, and 5) to compare costs associated with MAS and conventional selection methods. Results of PRA showed that unreliable rainfall and insect pests were the dominant constraints to maize productivity in Uganda. Diseases were ranked fifth among the production constraints . Maize streak virus disease was considered the most important disease constraint. Farmers showed common preference for high yielding and early maturing cultivars. However, farmers had other special preferences which were diverse and included large, white and high test density kernels for marketing, and sweet taste, particularly for home consumption. Farmers' research priorities included tolerance to drought, resistance to insect pests and diseases, sweetness, prolificacy, resistance to lodging, and drooping leaves because theyt cover the soil fast and prevent weed growth. Conventional screening for resistance to MSD showed that backcross and selfing populations segregated in 1:1 and 3:1 Mendelian ratios confirming the presence of one major gene with simple inheritance . Severity and incidence of disease were positively correlated suggesting a non-reference by the insects. In the selfing populations, the presence of complete esistance against MSD was suggested because frequency distribution patterns were highly skewed in favour of resistance. There was a decrease in disease severities with selection from BC1F1 to BC2F1 and from F2 to F3 generations indicating that high response to selection was achieved. On the other hand, one marker, umc1917, consistently polymorphic and eo-dominant was selected and used in MAS protocol. Results showed that the observed outcomes fitted the expected ratio of 1:2:1 for a F2 population and 1:1 for a BC1F1 population (X2 not significant). Evaluation of F3 and BC2F1 progeny selected using markers showed low disease severity suggesting that marker assisted selection was effective. However, the study showed that the presence of the O'Tl, was not consistent with symptom expression in the field. Evaluation of lines in three-way crosses identified ten potential lines that were high yielding, highly resistant to MSD and stable across three locations. Both MAS and conventional selection were equally effective in identifying high yielding lines although resistance was higher under MAS. Costs of MAS and conventional method varied depending on the units for comparison. The total costs of conventional method were higher than that of MAS in both first and second selection cycles. Comparing costs per row for conventional and costs per plant or data point for MAS showed that conventional selection was 2.4 times more expensive than costs per sample for MAS. However, costs per plant for MAS were 6.6 times higher than for conventional selection. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2006

Soybean rust (SBR) caused by Phakopsora pachyrhizi Syd. is an aggressive wind dispersed fungal disease which has spread around the world at an alarming rate in the last decade. The disease was first reported in South Africa (SA) in 2001. It has become well established in the province of KwaZulu-Natal. Reports are occasionally made from eastern Mpumalanga, late in the growing season, in years with good rainfall. Yield losses of 10 - 80% have been reported due to SBR infection. Literature was reviewed to better understand the pathogen in an attempt to find suitable disease management strategies. Many strategies involve delaying, rather than preventing, SBR infection. Of the two strategies to prevent infection, the use of fungicides was the only option for disease control in SA, as no resistant cultivars are available. Field trials were conducted to determine which fungicides are effective in controlling SBR. Further research was conducted to determine the timing, frequency and rate of fungicide applications for optimal control of SBR. Trials were evaluated for disease severity, seed yield and the effect of fungicides on seed quality. Fungicides from the triazole class of the sterol biosynthesis inhibiting group of fungicides were found to be the most effective in controlling SBR. A fungicide from the strobilurin group was found to be less effective than the triazoles at the suggested rate, but was found to be as effective when evaluated at higher dosage rates. Triazoles premixed with fungicides from the benzimidazole and strobilurin groups were also effective in controlling SBR. Timing of application was found to be critical for strobilurin fungicides, but not for triazole fungicides, which have a curative ability, unlike strobilurins. Strobilurin fungicides applied preventatively, before the appearance of disease symptoms were as effective as triazole fungicides applied after disease symptoms, but before infection levels had reached 10%. Across both wet and dry seasons two fungicide applications applied at 21d intervals at the R2 growth stage resulted in effective disease control. In wet seasons, a third fungicide application resulted in yields that were higher, albeit not statistically significant, than two fungicide applications. Assessments of individual fungicides for optimal dosage rate found that registered rates were already optimal for some fungicides, but for others it appeared as if alterations were necessary to the rate suggested for registration. This study was one of the first to extensively evaluate the efficacy of the new triazole and strobilurin fungicides on SBR control. The results have been shared globally, but particularly with newly affected countries in South and North America. Although this research has been groundbreaking, there are still many aspects of fungicide control which need to be studied in order to further optimise chemical control of SBR. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2005

Five potential resistance-inducing chemicals were applied to two sugarcane varieties (N12 and N27) in a pot trial with the aim of inducing resistance to nematodes in naturally-infested soil. BION® (acibenzolar-S-methyl), methyl jasmonate, cis-jasmone and 2,6-dichloroisonicotinic acid (INA) were applied as a foliar spray and suSCon® maxi (imidacloprid) applied to the soil. All chemicals were tested at two rates and plants were sprayed one week prior to being harvested at 7, 9 and 11 weeks of age. Meloidogyne and Pratylenchus infestation of sett and shoot roots was determined at each harvest. The activity of four pathogenesis-related proteins was examined at 7, 9 and 11 weeks using separate assays, these enzymes where chitinase, β-1,3-glucanase, peroxidase and polyphenol oxidase. Methyl jasmonate treatment produced significant increases in β-1,3-glucanase, chitinase and peroxidase activity. All other elicitor treatments showed little difference in enzyme activity from the Control. The effect of each treatment on plant growth was examined by recording the dried root and shoot biomass of each plant. No significant differences were seen (p<0.05; Holm-Sidak test). However, root and shoot dried biomass was highest in the N12 variety treated by suSCon® maxi. The infection of sugarcane with Ustilago scitaminea (sugarcane smut) is commonly identified visually by the presence of a smut whip. Identification of sugarcane smut infection can be determined prior to whip development by staining tissue sections with lactophenol cotton blue and examining plant tissues microscopically. This allows for a rapid determination of smut infection which can aid breeding programs. Smut infection is achieved in vitro by soaking sugarcane setts in smut spores collected from infected whips. Four methods of inoculation were examined. The method that most consistently caused infection involved allowing setts to germinate for 24 hours, before puncturing a bud with a toothpick, followed by submerging the sett in 1x10⁸ smut spores per mℓ. An elicitor of systemic acquired resistance called BION®, and an insecticide with resistance-inducing properties called Gaucho® (imidacloprid) were used as a sett soak treatments to induce resistance to sugarcane smut. The effect of each treatment at three concentrations on plant germination and growth was examined in the NCo376 variety. Smut spore germination on agar was examined in the presence of both treatments at three concentrations. Sugarcane setts were treated with a concentration that did not significantly reduce the germination of smut spores or sugarcane setts. Plants were infected with smut post treatment and allowed to grow for approximately one month until plants were between 8 and 10 cm in height. Smut infection was assessed by cutting longitudinal sections through the base of the shoot and staining each section with cotton blue lactophenol. Treatment with BION® and Gaucho® did not reduce smut infection. / M.Sc.Agric. University of KwaZulu-Natal, Pietermaritzburg 2013.

This research was an attempt to use a dicot transformation vector to transform a monocot. The initial purpose of this thesis was to transform orchids with the sense and antisense coat protein genes from the Odontoglossum ringspot virus (ORSV) in an effort to mitigate viral symptoms in transgenic plants using the transformation vector, Agrobacterium tumefaciens. However, it soon became apparent that much time would be needed to develop a transformation protocol. The transformation vectors used included the Agrobacterium tumefaciens disarmed strain LBA4404 with the binary plasmid pB1121, the disarmed strain At699 with the binary plasmid pCNL65, and the wild-type strain Chry5. The marker gene on the binary plasmids of both disarmed strains was p-glucuronidase (GUS).Several transformation protocols were used in an effort to determine if this transformation system would work on orchids. Transformation was not achieved even though a number of experimental conditions were varied. These included using two different types of orchid tissue, callus and protocorms; using two different species of orchids, Cattleya Chocolate Drop x Cattleytonia Kieth Roth and Cymbidium maudidum; varying the time the plant tissue was exposed to the bacteria from 1 hour to 96 hours; performing experiments with and without the wound signal molecule acetosyringone; and exposing the tissue to the virulent strains of A. tumefaciens mentioned previously.This research also developed GUS assay conditions necessary to decrease the number of false positives due to bacterial contamination. These conditions included chloramphenicol in the GUS assay buffer. / Department of Biology

Thesis (MSc (Genetics))--Stellenbosch University, 2008. / The vine mealybug, Planococcus ficus (Signoret), is a severe pest of grapevine in many grape and wine producing countries around the world. It is renowned not only for the considerable damage it infers to grapevine of its own accord, but in particular for its role in transmitting deleterious viral diseases such as grapevine leafroll disease, Kober stem grooving, Shiraz disease and corky bark. Incidentally, it is an exceptionally tenacious antagonist of grapevine, being resistant to both chemical and biological control mechanisms. As a result, finding an effective strategy for P. ficus control has become a main priority of viticultural industries worldwide. Possible implementation of biotechnological approaches to pest management has resulted in a need for P. ficus genetic data - of which there are currently very little available. The transcribed genes of an organism can be captured in a cDNA library, and the sequences of the various transcripts can then be characterized. In this study altogether five cDNA libraries were constructed from the transcribed sequences of Planococcus ficus (Signoret). Instrumental to their construction was the identification of an RNA extraction protocol that provided large quantities of high quality RNA from mealybugs. The five cDNA libraries were the result of a set of modifications to the Creator™ SMART™ cDNA Library Construction Kit (used for Primary Library construction), and differed mainly with regards to range of insert sizes they contain. Whereas an abundance of short fragments were found in the Primary Library (42% of screened inserts 60.5 kb, and 20% >1 kb), the Fractionated Libraries contained inserts of specific size ranges that were more-or-less equally represented. The broadest size range was found in Fractionated Library 4, for which a uniform distribution over the range 0.25 kb - 4 kb was observed. Average insert sizes of Fractionated Libraries 1 to 4 were estimated at 0.25 kb, 0.5 kb, 1 kb and 2 kb respectively. These results demonstrated the importance of using a protocol designed to circumvent the bias towards incorporation of shorter transcripts in cDNA libraries. Although the libraries were not exhaustively analyzed, the outcome of a pilot investigation indicated that 41% of the submitted sequences had matches in the non-redundant database of the National Center for Biotechnology Information (NCBI, E-value 6 10-5), and that approximately 82% of these were of insect origin. Moreover, two potential targets for an RNAi-mediated approach to P. ficus pest control were identified. With one exception, these sequences seemed to be unique to arthropods. Future research needs to investigate the efficiency by which these sequences are able to constrain P. ficus proliferation, and their suitability for grapevine transformation.

Made available in DSpace on 2015-03-26T13:30:49Z (GMT). No. of bitstreams: 1 texto completo.pdf: 307446 bytes, checksum: 91b73feb2376192f69ab47a6f21c9d48 (MD5) Previous issue date: 2014-02-17 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Insecticide use is still the main method of arthropod pest management. Due to the social concern about environmental sustainability, new compounds have been used in an attempt to reduce the environmental impact of insecticides mainly regarding the risks to mammals, environmental persistence, pollution and development of insecticide resistance. Within this scenario new compounds and particularly bioinsecticides have been increasingly used. Several studies have been carried out to measure the efficacy of pest control agents and its effects on non-target organisms. However, beyond the investigation of the lethal effects of these compounds, the study of sublethal effects on natural enemies is also necessary and circumscribed to few species. Pirate bug predators (Heteroptera: Anthocoridae) play an important role as biological control of many insect pests, some which of great economic importance like the tomato leafminer Tuta absoluta. Blaptostethus pallescens Poppius (Anthocoridae) is a little-known species, but frequently found in tomato fields exposed to insecticides used for pest control, but little is known about those effects in this predator. This study assessed the lethal effects of three insecticides - chlorpyrifos, deltamethrin and azadirachtin - to B. pallescens using their field label rates. Azadirachtin was subsequently selected for assessment of its sublethal effects on behavior and reproduction due to tis lower acute mortality (< 25%) to predator adults after 48 hours of exposure. The insecticides chlorpyrifos and deltamethrin caused 100 % mortality within 4 hours, whereas the survival of insects exposed to azadirachtin was similar to the untreated control. Thus, further studies were conducted with azadirachtin to investigate its sublethal effects. Aiming to evaluate the effects of this insecticide in behavior, adult predators were placed in arenas treated with and without azadirachtin residues for the measurement of parameters such as number of stops, resting time, distance walked and walking velocity. There were no significant differences between treatments. Irritability and repellence to azadirachtin were assessed by the period and the proportion of individuals respectively remaining in the area containing insecticide residue on partially treated arenas. The results indicated that B. pallecens do not exhibit irritability nor repellence to azadirachtin. Subsequently predator couples were subjected to four treatments with azadirachtin untreated couple, treated female (and untreated male), treated male (and untreated female), and treated couple - where female longevity data, daily fecundity, fertility and F1 sex ratio were recorded. These data were used in a projection matrix that generated data of net reproductive rate, population growth rate and generation time for each treatment. The results indicated that there was difference between treatments. Thus, azadirachtin seems to be safer for the predator B. pallescens and therefore a good candidate for the use in pest management programs against T. absoluta. / O uso de inseticidas ainda é a principal forma para o controle de pragas. Em decorrência da preocupação social quanto à sustentabilidade, novos compostos vêm sendo usados na tentativa de reduzir o impacto ambiental de inseticidas, os chamados bioinseticidas. Estudos têm sido feitos para mensurar a eficiência de inseticidas para o controle de pragas e seus efeitos a organismos não-alvo. No entanto, além da investigação dos efeitos letais destes compostos, é necessário o estudo dos efeitos subletais em inimigos naturais, estes estudos vêm aumentando, mas ainda se concentram em poucas espécies. Os percevejos predadores da família Anthocoridae desempenham importante papel no controle biológico de muitas pragas, como a traça-do-tomateiro Tuta absoluta (Meyirick) (Lepidoptera: Gelechiidae), de grande importância econômica. Uma espécie pouco conhecida, mas frequentemente encontrada em campos de tomate, é Blaptostethus pallescens Poppius (Hemiptera: Anthocoridae) que, portanto vem sendo exposta aos inseticidas usados para o controle da praga, mas pouco se sabe sobre estes efeitos neste predador. Neste trabalho foram avaliados os efeitos letais de três inseticidas - clorpirifós, deltametrina e azadiractina ao B. pallescens usando suas respectivas doses de campo. Posteriormente, azadiractina foi selecionada para avaliação de efeitos subletais no comportamento e na reprodução. Os inseticidas clorpirifós e deltametrina causaram 100% de mortalidade em até 4 horas, enquanto que os insetos expostos a azadiractina tiveram sobrevivência semelhante ao controle. Com o objetivo de avaliar os efeitos deste inseticida no comportamento de locomoção, insetos foram colocados em arenas tratadas com resíduos de azadiractina e controle para a mensuração de parâmetros do caminhamento como número de paradas, tempo parado, distância percorrida e velocidade. Não houve diferença significativa entre os tratamentos. Para avaliação da irritabilidade e repelência foi mensurado o tempo e a proporção de indivíduos, respectivamente, que permaneceram na área contendo resíduos de inseticidas em arenas parcialmente tratadas. Os resultados indicaram que B. pallecens não exibiu irritabilidade e repelência mediante a exposição aos resíduos de azadiractina. Posteriormente casais foram submetidos a 4 tratamentos com azadiractina casal sem tratamento inseticida (i.e., controle), apenas fêmea tratada, apenas o macho tratado e o casal tratado onde foram coletados dados de longevidade das fêmeas, fecundidade diária, fertilidade e a razão sexual da geração F1. Estes resultados foram usados em uma projeção matricial que gerou dados como as taxas líquidas reprodutivas, taxas de incremento populacional e tempo de geração para cada tratamento. Os resultados indicaram um maior feito do inseticida para o casal tratado, seguido por fêmea ou macho tratado e por último o controle. Ainda assim, o inseticida azadiractina se mostrou seguro para o predador B. pallescens e, portanto um candidato para ser usado no controle de T. absoluta permitindo a compatibilização entre uso deste inseticida sem comprometimento da ação predadora sobre esta espécie praga.

No description available.