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

Espécies do gênero Phytophthora são responsáveis por doenças de importância nas principais culturas no mundo. A citricultura brasileira possui grande importância no agronegócio nacional. Uma das doenças que afeta a cultura é a gomose, causada principalmente por Phytophthora nicotianae e Phytophthora citrophthora. O controle da doença é feito basicamente por meio de medidas preventivas e curativas, com o uso de fungicidas. Uma das opções de manejo da doença seria induzir os mecanismos de defesa existentes na plantas. Esses mecanismos podem ser potencializados mediante o uso de indutores de resistência. O objetivo deste trabalho foi identificar possíveis indutores de resistência para esse patossistema e avaliar aspectos fisiológicos e bioquímicos envolvidos. Inicialmente, foi realizado um teste para selecionar o isolado mais agressivo. Uma vez selecionado o isolado, foram testados métodos de inoculação: punção no caule, imersão de raízes em suspensão de zoósporos com e sem ferimento. O método de inoculação selecionado foi utilizado para se fazer testes de seleção de substâncias indutoras em plantas de tangerina Sunki, sendo avaliados o peso da matéria fresca e seca dos tecidos das plantas. Finalmente, com a escolha da substância indutora, foi avaliada a indução de resistência na interação citros-Phytophthora spp. em plantas de tangerina Sunki e citrumelo Swingle. As variáveis bioquímicas consideradas foram -1,3- glucanase, peroxidase, catalase, proteína, açúcares totais e açúcares redutores. Foram estudados seis isolados de P. citrophthora e oito de P. nicotianae, sendo que os isolados selecionados foram 02/02 de P. citrophthora e 07/06 de P. nicotianae. O método de inoculação selecionado foi a imersão por zoósporos, o qual apresentou diferenças estatísticas nas variáveis avaliadas. Nos testes in vitro os indutores Phytogard®, silicato de potássio e fosfitos apresentaram redução significativa no desenvolvimento de P. nicotianae e P. citrophthora. Três substâncias indutoras Bion®, Seacrop® e Phytogard® apresentaram diferenças estatísticas nas variáveis, peso da matéria seca da parte aérea e da raiz em relação ao controle. Por tanto, a substância selecionada foi o Phytogard®, com base nos resultados e registros existentes sobre fosfitos, como possíveis indutores de resistência. Na interação citros- Phytophthora spp, submetida ao tratamento para resistência induzida, encontrou-se aumento no teor de proteína nas plantas de tangerina Sunki inoculadas com P. nicotianae, 24 dias após a inoculação. Efeitos na concentração de carboidratos e um incremento significativo na concentração de açúcar redutor em plantas de citrumelo Swingle foram observados em plantas inoculadas com os dois patógenos. Incremento significativo na atividade da -1,3-glucanase foi observado 24 dias após a inoculação em plantas inoculadas com P. nicotianae, sem o indutor. A atividade da catalase não apresentou diferenças em nenhum tratamento. A atividade de guaiacol peroxidase apresentou alteração significativa em plantas de tangerina Sunki 24 horas após da inoculação com P. citrophthora. Por sua vez, a incidência da doença foi menor nas plantas tratadas com o Phytogard® nos dois patossistemas. Não foram encontradas alterações na atividade das enzimas avaliadas e o teor de proteínas, açúcares totais e redutores, que explicassem a indução nas plantas tratadas com fosfitos, e os mecanismos de defesa envolvidos nesse patossistema. / Species of the genus Phytophthora are responsible for major diseases in crops around the world. The Brazilian citrus industry plays an important role in the agribusiness. One of the diseases that affects the culture is the root rot, mainly caused by Phytophthora nicotianae and Phytophthora citrophthora. The disease control is carried out mainly through preventive and curative measures, with the use of fungicides. One of the options for management of the disease would be the induction of defense mechanisms existing in the plants. These mechanisms can be induced through the use of resistance inducers. The aim of this study was to identify potential resistance inducers for this pathosystem and evaluate physiological and biochemical aspects involved. Initially, a test was performed to select the most aggressive oomycete isolate. Afterwards, inoculation methods were tested: stem puncture, root immersion in a suspension of zoospores with and without injury. The inoculation method was used to in an experiment to select inducer substances in plants of Sunki tangerine, and it was evaluated the wet and dry weight of plant tissues. Finally, with the choice of the inducing substance, it was assessed the induction of resistance in the interaction citrus-Phytophthora spp. in Sunki tangerine and Swingle citromelo plants. The biochemical variables considered were -1,3- glucanase, peroxidase, catalase, protein, total sugars and reducing sugars. Six isolates of P. citrophthora and eight P. nicotianae were studied, and the selected isolates were 02/02 of P. citrophthora and 07/06 of P. nicotianae. The inoculation method selected was immersion in zoospore suspensions. The inducers Phytogard, potassium silicate and phosphite have shown siginificative reduction on the development of P. nicotianae and P. citrophthora on in vitro tests. Three inducing substances Bion®, Seacrop® and Phytogard® showed statistical differences in variables of dry weight of shoot and root. Therefore, the substance selected was Phytogard®, based on the results and existing records of phosphite as potential inducers of resistance. In the interaction citrus-Phytophthora spp. submitted to the treatment for induced resistance, it was found an increase in protein content in plants of Sunki tangerine inoculated with P. nicotianae 24 days after inoculation. Effects on carbohydrate concentration and significant increase in concentration of reducing sugar in plants of Swingle citromelo were observed in plants inoculated with both pathogens. Significant increase in the activity of -1,3-glucanase was observed 24 days after inoculation in plants inoculated with P. nicotianae, without the inducer. The catalase activity showed no differences in any treatment. The activity of guaiacol peroxidase showed significant changes in Sunki tangerine plants 24 hours after inoculation with P. citrophthora. Disease incidence was lower in plants treated with Phytogard® in both pathosystems. Thus there were no changes in enzyme activity assessed and in the protein content, total and reducing sugars, which could explain the induction in plants treated with phosphite, and the defense mechanisms involved in this pathosystem.

Espécies do gênero Phytophthora são responsáveis por doenças de importância nas principais culturas no mundo. A citricultura brasileira possui grande importância no agronegócio nacional. Uma das doenças que afeta a cultura é a gomose, causada principalmente por Phytophthora nicotianae e Phytophthora citrophthora. O controle da doença é feito basicamente por meio de medidas preventivas e curativas, com o uso de fungicidas. Uma das opções de manejo da doença seria induzir os mecanismos de defesa existentes na plantas. Esses mecanismos podem ser potencializados mediante o uso de indutores de resistência. O objetivo deste trabalho foi identificar possíveis indutores de resistência para esse patossistema e avaliar aspectos fisiológicos e bioquímicos envolvidos. Inicialmente, foi realizado um teste para selecionar o isolado mais agressivo. Uma vez selecionado o isolado, foram testados métodos de inoculação: punção no caule, imersão de raízes em suspensão de zoósporos com e sem ferimento. O método de inoculação selecionado foi utilizado para se fazer testes de seleção de substâncias indutoras em plantas de tangerina Sunki, sendo avaliados o peso da matéria fresca e seca dos tecidos das plantas. Finalmente, com a escolha da substância indutora, foi avaliada a indução de resistência na interação citros-Phytophthora spp. em plantas de tangerina Sunki e citrumelo Swingle. As variáveis bioquímicas consideradas foram -1,3- glucanase, peroxidase, catalase, proteína, açúcares totais e açúcares redutores. Foram estudados seis isolados de P. citrophthora e oito de P. nicotianae, sendo que os isolados selecionados foram 02/02 de P. citrophthora e 07/06 de P. nicotianae. O método de inoculação selecionado foi a imersão por zoósporos, o qual apresentou diferenças estatísticas nas variáveis avaliadas. Nos testes in vitro os indutores Phytogard®, silicato de potássio e fosfitos apresentaram redução significativa no desenvolvimento de P. nicotianae e P. citrophthora. Três substâncias indutoras Bion®, Seacrop® e Phytogard® apresentaram diferenças estatísticas nas variáveis, peso da matéria seca da parte aérea e da raiz em relação ao controle. Por tanto, a substância selecionada foi o Phytogard®, com base nos resultados e registros existentes sobre fosfitos, como possíveis indutores de resistência. Na interação citros- Phytophthora spp, submetida ao tratamento para resistência induzida, encontrou-se aumento no teor de proteína nas plantas de tangerina Sunki inoculadas com P. nicotianae, 24 dias após a inoculação. Efeitos na concentração de carboidratos e um incremento significativo na concentração de açúcar redutor em plantas de citrumelo Swingle foram observados em plantas inoculadas com os dois patógenos. Incremento significativo na atividade da -1,3-glucanase foi observado 24 dias após a inoculação em plantas inoculadas com P. nicotianae, sem o indutor. A atividade da catalase não apresentou diferenças em nenhum tratamento. A atividade de guaiacol peroxidase apresentou alteração significativa em plantas de tangerina Sunki 24 horas após da inoculação com P. citrophthora. Por sua vez, a incidência da doença foi menor nas plantas tratadas com o Phytogard® nos dois patossistemas. Não foram encontradas alterações na atividade das enzimas avaliadas e o teor de proteínas, açúcares totais e redutores, que explicassem a indução nas plantas tratadas com fosfitos, e os mecanismos de defesa envolvidos nesse patossistema. / Species of the genus Phytophthora are responsible for major diseases in crops around the world. The Brazilian citrus industry plays an important role in the agribusiness. One of the diseases that affects the culture is the root rot, mainly caused by Phytophthora nicotianae and Phytophthora citrophthora. The disease control is carried out mainly through preventive and curative measures, with the use of fungicides. One of the options for management of the disease would be the induction of defense mechanisms existing in the plants. These mechanisms can be induced through the use of resistance inducers. The aim of this study was to identify potential resistance inducers for this pathosystem and evaluate physiological and biochemical aspects involved. Initially, a test was performed to select the most aggressive oomycete isolate. Afterwards, inoculation methods were tested: stem puncture, root immersion in a suspension of zoospores with and without injury. The inoculation method was used to in an experiment to select inducer substances in plants of Sunki tangerine, and it was evaluated the wet and dry weight of plant tissues. Finally, with the choice of the inducing substance, it was assessed the induction of resistance in the interaction citrus-Phytophthora spp. in Sunki tangerine and Swingle citromelo plants. The biochemical variables considered were -1,3- glucanase, peroxidase, catalase, protein, total sugars and reducing sugars. Six isolates of P. citrophthora and eight P. nicotianae were studied, and the selected isolates were 02/02 of P. citrophthora and 07/06 of P. nicotianae. The inoculation method selected was immersion in zoospore suspensions. The inducers Phytogard, potassium silicate and phosphite have shown siginificative reduction on the development of P. nicotianae and P. citrophthora on in vitro tests. Three inducing substances Bion®, Seacrop® and Phytogard® showed statistical differences in variables of dry weight of shoot and root. Therefore, the substance selected was Phytogard®, based on the results and existing records of phosphite as potential inducers of resistance. In the interaction citrus-Phytophthora spp. submitted to the treatment for induced resistance, it was found an increase in protein content in plants of Sunki tangerine inoculated with P. nicotianae 24 days after inoculation. Effects on carbohydrate concentration and significant increase in concentration of reducing sugar in plants of Swingle citromelo were observed in plants inoculated with both pathogens. Significant increase in the activity of -1,3-glucanase was observed 24 days after inoculation in plants inoculated with P. nicotianae, without the inducer. The catalase activity showed no differences in any treatment. The activity of guaiacol peroxidase showed significant changes in Sunki tangerine plants 24 hours after inoculation with P. citrophthora. Disease incidence was lower in plants treated with Phytogard® in both pathosystems. Thus there were no changes in enzyme activity assessed and in the protein content, total and reducing sugars, which could explain the induction in plants treated with phosphite, and the defense mechanisms involved in this pathosystem.

Flue-cured tobacco (Nicotiana tabacum L.) is a high value-per-acre crop that is intensively managed to optimize the yield of high quality cured leaf. Aerial remote sensing, specifically unmanned aerial vehicles (UAVs), present flue-cured tobacco producers and researchers with a potential tool for scouting and crop management. A two-year study, conducted in Southside Virginia at the Southern Piedmont Agricultural Research and Extension Center and on commercial farms, assessed the potential of aerial remote sensing in flue-cured tobacco. The effort encompassed two key objectives. First, examine the use of the enhanced normalized difference vegetation index (ENDVI) for separating flue-cured tobacco varieties and nitrogen rates. Secondly, develop hyperspectral indices and/or machine learning classification models capable of detecting Phytophthora nicotianae (black shank) incidence in flue-cured tobacco. In 2017, UAV-acquired ENDVI surveys demonstrated the ability to consistently separate between flue-cured tobacco varieties and nitrogen rates from topping to harvest. In 2018, ENDVI revealed significant differences among N-rates as early as 34 days after transplanting. Two hyperspectral indices were developed to detect black shank incidence based on differences in the spectral profiles of asymptomatic flue-cured tobacco plants compared to those with black shank symptoms. Testing of the indices showed significant differences between the index values of healthy and symptomatic plants (alpha = 0.05). In addition, the indices were able to detect black shank symptoms pre-symptomatically (alpha = 0.09). Subspace linear discriminant analysis, a machine learning classification, was also used for prediction of black shank incidence with up to 85.7% classification accuracy. / Master of Science / Unmanned Aerial Vehicle’s (UAVs) or drones, as they are commonly referred to, may have potential as a tool in flue-cured tobacco research and production. UAVs combined with sensors and cameras provide the opportunity to gather a large amount of data on a particular crop, which may be useful in crop management. Given the intensive management of flue-cured tobacco, producers may benefit from extra insight on how to better assess threats to yield such as under-fertilization and disease pressure. A two-year study was conducted in Southside Virginia at the Southern Piedmont Agricultural Research and Extension Center and on commercial farms. There were two objectives to this effort. First, assess the ability of UAV-acquired multispectral near-infrared imagery to separate flue-cured tobacco varieties and nitrogen rates. Secondly, develop hyperspectral indices and machine learning models that can accurately predict the incidence of black shank in flue-cured tobacco. Flue-cured tobacco nitrogen rates were significantly different in 2017 from 59 days after transplanting to harvest using UAV-acquired near-infrared imagery. In 2018, heavy rainfall may have led to nitrogen leaching from the soil resulting in nitrogen rates being significantly different as early as 34 days after transplanting. The imagery also showed a significant relationship with variety maturation type in the late stages of crop development during ripening. Two hyperspectral indices were developed and one machine learning model was trained. Each had the ability to detect black shank incidence in fluecured tobacco pre-symptomatically, as well as separated black shank infested plants from healthy plants.