Halle, Universiẗat, Diss., 2001.

Thesis (M.Sc.)(Microbiology)--University of Pretoria, 2004. / Title from opening screen (viewed 12th March, 2004). Summaries in English and Afrikaans. Includes bibliographical references.

No description available.

M.Sc. (Biochemistry) / Please refer to full text to view abstract

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)

The black shank (Phytophthora parasitica var. nicotianae) fungus is a very destructive tobacco disease which is responsible for great losses to farmers worldwide. This disease is also a problem in South Africa, as the most popular South African air-cured tobacco cultivar, CDL28, is very susceptible to black shank. This diallel study focussed on finding the most suitable black shank resistance source to include in a resistance breeding programme with CDL28. Four cultivars were crossed in all possible combinations and planted as an F1 field and greenhouse trial. The F1's were selfed to obtain a segregating F2 population, planted in a greenhouse, to be compared with the mean values of the F1 trials. The field trial was exposed to natural infection while the greenhouse trials were root inoculated. The general combining ability effects of the four parent cultivars differed significantly from each other. The specific combining ability effects of the F1 trials were non significant. These experimental results suggest that additive genetic effects were involved in black shank resistance. The Beinhart 1000-1 source of resistance was significantly better than the Florida 301 source. Burley 37, which possesses the Florida 301 as well as another source of resistance performed better than Domkrag with only the Florida 301 source of resistance. In order to incorporate black shank resistance in CDL28, Beinhart 1000-1 and Burley 37 can be used in a backcross breeding programme with CDL28 which can solve the problems encountered in the cultivation of CDL28 in the presence of black shank. / Dissertation (MSc (Genetics))--University of Pretoria, 2006. / Genetics / unrestricted

The tobacco-adapted form of the green peach aphid, Myzus persicae (Sulzer), is one of the most important insect pests of tobacco in the United States and around the world. Insecticides play a major role in controlling the aphid on tobacco because natural enemies usually fail to maintain its populations below damaging levels. The aphid has a history of developing resistance to many insecticides. Therefore, baseline information on the aphid's susceptibility to imidacloprid and other insecticides is critical for developing future resistant management programs to minimize losses attributed to the aphid. Studies were conducted on colonies of the tobacco-adapted form of the green peach aphid collected from nine states in the eastern United States in 2004-2007. The susceptibility of 151 colonies to imidacloprid was determined in serial leaf-dip bioassays. When combined over the four years, 18, 14, and 4% of the colonies had 10- to 20-fold, 20- to 30-fold, and 30- to 90-fold resistance ratios, respectively, suggesting that high levels of resistance to imidacloprid are present in field populations of the aphid. A colony collected near Clayton, NC had the highest LC50 value (31 ppm) combined over six tests and three years, but the average resistance ratios for the first three runs was over 130-fold (48 ppm). Geographic location had little effect on susceptibility to imidacloprid. Aphid colonies (136) including red, green, and orange color morphs were screened for total esterase activity using microplate assay with 1-Naphthyl acetate as the substrate. The green morphs generally had lower esterase levels than the red and orange morphs. All orange morphs had among the highest esterase activities. Esterase activities of red and green morphs were positively correlated with LC50 values as determined by leaf-dip bioassays for acephate and methomyl. All 25 colonies tested for esterase gene amplification had either E4 or FE4 gene amplification. The amplification of both E4 and FE4 seen as an 865-bp band characteristic of the FE4 gene and an additional 381-bp band characteristic of a deleted upstream region of the E4 gene occurred in all (4) orange morphs and one (1 of 9) green morph. Target-site insensitivity of acetylcholinesterase (AChE), as modified AChE resistance (MACE) was assessed in 65 colonies of field-collected tobacco-adapted forms of M. persicae. Eight colonies over a range of AChE activity were selected to study inhibition of AChE in the presence of two carbamate insecticides, methomyl and pirimicarb. IC50 values for methomyl ranged from 0.35 to 2.4 μM while six of eight colonies had lower values with a range of 0.16 to 0.30 μM for pirimicarb. Two colonies that were inhibited by methomyl had very high IC50 values of 40.4 and 98.6 μM for pirimicarb. Such insensitivity may be due to mutations in the ace2 gene, but this needs to be confirmed by genetic and molecular analysis. Glutathione S- transferases (GSTs), isoenzymes that are involved in the metabolism and detoxification of many xenobiotic compounds were quantified for 100 colonies by CDNB conjugation. There was a wide range of GST activity for the red (8 to 343 pmol/min/mg protein) and green (15.3 to 330 pmol/min mg protein) morphs, but all six orange morphs collected in 2007 had a narrower range (160 to 211 pmol/min/mg protein). About 45% of the red morphs had GST activity from 200-300 pmol/min/mg of protein, while 53% of the green morphs had a range of 100-200 pmol/min/mg protein. The influence of temperature-mediated synergisms on the toxicity of insecticides in red and green color morphs of the tobacco-adapted from of M. persicae were evaluated using leaf-dip bioassay procedures in laboratory incubators. Post-exposure temperatures of 15, 20, and 25°C were evaluated for four classes of insecticides, acephate, imidacloprid, lambda-cyhalothrin, and methomyl. The temperature change from 15 to 20°C caused almost a 3-fold increase in toxicity to the red and green color morphs for methomyl, acephate, and imidacloprid. In contrast, the toxicity of lambda-cyhalothrin decreased as the temperature increased, showing a negative temperature coefficient. Bioassay experiments conducted with the red morph for indirect estimates of imidacloprid concentrations in flue-cured tobacco showed that leaf position, imidacloprid rate and time after application affected the concentration of the toxicant in the leaf. The differences in aphid mortality between the lower and upper leaf positions indicate that the concentration of imidacloprid and its metabolites were unevenly distributed with the lowest mortality for aphids feeding on the younger, upper leaves and the highest for those feeding on the older, lower leaves. In field experiments, higher aphid populations occurred on tobacco treated with imidacloprid less than the field recommended rate of 41.4 ml/1,000 plants. The development of aphid populations in the field was consistent with the laboratory bioassays. Field trials were conducted to evaluate the performance of various insecticides currently registered for aphid control on tobacco. Imidacloprid applied as a tray drench treatment and acephate as foliar sprays were the most effective treatments. Moderate declines in control with imidacloprid were observed at 75-87 d after transplanting in 2006 and 2007. Aldicarb gave good to excellent control in one of three years, but only fair to poor control in the other two years. Methomyl and lambda-cyhalothrin gave good control in all three years except the residual was shorter. The poor performance of aldicarb in the two years may have been related to the presence of E4 or FE4 resistance in the naturally occurring TGPA in the experimental plots. / Ph. D.

Blackman (1987) used life cycle and morphology to separate Myzus nicotianae Blackman, a tobacco-feeding species of aphid, from Myzus persicae (Sulzer). In the present study, the first objective was to investigate the influence of temperature and host plant on the morphology of M. nicotianae and M. persicae. The second objective was to assess Blackman's 1987 key to Myzus for separating tobacco and non-tobacco originating morphs under different environmental conditions. Four host plants were used: tobacco, turnip, pepper, and okra, and three temperatures, 15°C, 20°C, and 25°C. The intraclonal plasticity of two tobacco collected morphs and one turnip collected morph was investigated in relation to these combinations of host and temperature in a 4 x 3 x 3 factorial experimental design. Fifth generation mature apterous aphids were mounted on slides and 10 different morphological structures utilized in morphometric analysis were measured. Data support a morphologically distinct, host-adapted tobacco race but not a separate tobacco-feeding species of M. persicae. The key developed by Blackman (1987) did not discriminate between the tobacco and non-tobacco originating clones but the canonical variates generated from the analysis successfully separated the tobacco and non-tobacco groups. Other studies have used many different clones to investigate the possible distinctions between M. persicae and M. nicotianae; the objective here was to see how much morphological perturbation may be induced within a clone by rearing at different temperatures and on different host plants. Temperature and host plant had substantial influences on the morphology of these aphids. The physiological interactions of temperature-host plant-aphid morphology are very complex yet controlling only for temperature and host plant was sufficient to group specimens according to these independent variables with remarkable accuracy using the linear discriminant functions generated with these data. Percent of aphids in which rearing temperature was correctly identified using linear discriminant functions generated for temperature classes was 87%, 63%, and 64% for 15°C, 20°C, and 25°C, respectively. Random designations would be 33%. Correct identification of host plant was 65%, 45%, 47%, and 48% successful for tobacco, turnip, pepper, and okra, respectively. Random designations for host plant would be 25%. Canonical variates produced clusters by host, temperature, morph, and combinations of these independent variables with varying degrees of discreteness. CV1 by CV2 for host plants gave a very distinct cluster for tobacco and also separate groupings for aphids reared on turnip and pepper. Aphids from the host plant okra were scattered quite widely across the CV1 by CV2 graph. CV1 by CV2 for temperature conditions showed a tight cluster for aphids from 15°C and still distinct though less closely grouped clusters for both 20°C and 25°C rearing temperatures. CV1 by CV2 for the three morphs gave substantial overlap for the two tobacco originating morphs and a more separate cluster for the morph originally collected from turnip. / Master of Science

Kentucky is the nation’s leading producer of burley tobacco and the crop’s most economically important disease is black shank, caused by Phytophthora nicotianae (Pn). Current management is effective, however, problems with expense and pathogen persistence are issues. Two alternative methods for control of Pn were examined: biofumigation and soil application of an organic, yeast fermentation‐derived product (Soil‐Set). Field studies in 2009 and 2010 found no effect on populations of fungi, disease severity of Pn, and yield between mustard‐ and wheat‐amended plots. Experiments in the greenhouse suggested that survival of Pn was impacted by biomass rather than biofumigation. Biofumigation is not a viable option for controlling black shank in tobacco production. Soil‐Set was inhibitory against mycelial growth of Pn on corn meal agar rather than V8 juice. Results from a greenhouse study indicated that increasing the dose of Soil‐Set by four times what is recommended held the most potential for suppression of Pn in a burley variety with no resistance. A field study in 2012 found no differences among treatments in reducing severity of Pn in a variety with high resistance. More field and greenhouse studies need to be conducted to examine the potential of Soil‐Set in tobacco production.

Made available in DSpace on 2016-06-02T18:57:50Z (GMT). No. of bitstreams: 1 5948.pdf: 2099671 bytes, checksum: a2a82b16af89dab4eb5f364794eebc2f (MD5) Previous issue date: 2014-05-29 / Financiadora de Estudos e Projetos / The species of fungi belonging to the genus Trichoderma can be found in many environments, especially in the rhizosphere, as colonizers of roots and plant growth promoters, for their ability in facilitating the absorption of nutrients and protecting them against attack by pathogens. Thus, this study aimed to test isolates of Trichoderma spp. for application as growth promoting agents and inducers of resistance to Phytophthora nicotianae in citrus rootstocks. First, eighteen isolates of Trichoderma were tested in vitro for their ability in inhibiting mycelial growth of P. nicotianae, in terms of: production of volatiles or termostable antifungical substances and cell free compounds. The growth promotion test was done with three rootstocks: Sunki tangerine, Swingle citrumelo and Rangpur lime, whose height and number of leaves were measured at 30, 60, 90, 120, 150 and 180 days after treatment with the antagonistic; and in the end of the test, the measuring of dry weight for shoots and roots of plants was measured. The seeds of the rootstocks were treated with the Trichoderma spp isolates and, 180 days later, was tested their capacity of resistance induction through pathogen inoculation on stem, assessed by lesion length. The Phytophthora control assay was carried out by seeds microbiolization of tangerine Sunki and Rangpur lime. By the results, it was found that all Trichoderma spp. isolates provided more than 50% inhibition of the pathogen colony, however, no one produced volatile metabolites. Five isolates produced cell free metabolites and, only the ACB-38 produced termostable antifungal substances, capable of inhibiting the pathogen growth. Trichoderma spp. has the potential for rootstocks growth promoting and resistence inducing to P. nicotianae, but the responses vary according to the plant genotype and the type of isolate antagonist applied. Most isolates of Trichoderma spp. tested were able to control P. nicotianae in Sunki tangerine and Rangpur lime plants. / As espécies de fungos pertencentes ao gênero Trichoderma podem ser encontradas em muitos ambientes, principalmente na rizosfera, como colonizadores de raízes de plantas e promotores de crescimento das mesmas, por facilitarem a absorção de nutrientes, além de protegê-las contra o ataque de fitopatógenos. Sendo assim, esse trabalho teve por objetivo selecionar isolados de Trichoderma spp. com potencial de aplicação como agentes promotores de crescimento e indutores de resistência à Phytophthora nicotianae em porta-enxertos de citros. Primeiramente, avaliou-se in vitro a produção de metabólitos voláteis, termoestáveis e livres de células de dezoito isolados de Trichoderma spp., assim como, a atividade antagônica sobre o crescimento micelial do patógeno. Os testes in vivo compreenderam estudos de promoção de crescimento de três porta-enxertos: tangerina Sunki, citrumelo Swingle e limão Cravo, avaliando-se a altura e o número de folhas aos 30, 60, 90, 120, 150 e 180 dias após o tratamento com o antagônico e, a produção de massa seca da parte aérea e raiz das plantas. Microbiolizou-se as sementes dos porta-enxertos e testou-se a indução de resistência pela inoculação no caule, 180 dias após o tratamento, avaliada pelo comprimento de lesão. O teste de controle de Phytophthora foi realizado pela microbiolização de sementes de tangeriana Sunki e limão Cravo. Pelos resultados dos testes in vitro, verificouse que, todos os isolados de Trichoderma spp. proporcionaram mais de 50 % de inibição na colônia do fitopatógeno, porém, nenhum produziu metabólitos voláteis. Cinco isolados produziram metabólitos livres de células e, somente o ACB-38 produziu substâncias antifúngicas termoestáveis, com capacidade para inibir o desenvolvimento do patógeno. Trichoderma spp. têm potencial para promover crescimento de porta-enxertos cítricos, bem como induzir a resistência à P. nicotianae, porém, as respostas variam de acordo com o genótipo da planta e com o isolado do antagonista utilizado. A maioria dos isolados de Trichoderma spp. testada foi capaz de controlar P. nicotianae em plantas de tangerina Sunki e limão Cravo.