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.

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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

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.

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