Submitted by Mario BC (mario@bc.ufrpe.br) on 2017-10-03T14:42:11Z No. of bitstreams: 1 Kleyton Danilo da Silva Costa.pdf: 1289095 bytes, checksum: 9861fbf632f584701f964a72b5670215 (MD5) / Made available in DSpace on 2017-10-03T14:42:11Z (GMT). No. of bitstreams: 1 Kleyton Danilo da Silva Costa.pdf: 1289095 bytes, checksum: 9861fbf632f584701f964a72b5670215 (MD5) Previous issue date: 2017-07-14 / The bacterial wilt in the tomato crop is a disease that has local, national and world importance. This disease is difficult to control and can cause damage that can compromise the entire crop. Genetic resistance within integrated management is the primary measure of bacterial wilt control. In this sense, the knowledge of the genetic control of resistance in breeding programs tends to improve the efficiency in its planning and in the choice of the best method to be adopted. The objective of this thesis was to study the genetic control of resistance of 'Yoshimatsu' tomato to Ralstonia pseudosolanacearum and Ralstonia solanacearum. In the first stage two experiments were conducted with Yoshimatsu, IPA-7 and the F1, F2, RC11 and RC21 generations, using a randomized block design with four replicates. In the second stage two experiments were conducted with 43 progenies F2:3 and their parents with the same experimental design of the first stage. At each stage the two species of the R. solanacearum complex were inoculated in independent experiments. The incidence and severity of bacterial wilt were evaluated by means of a descriptive scale of notes at 10 and 20 days after inoculation. Genetic control of the resistance of tomato 'Yoshimatsu' to R. pseudosolanacearum involves two genes of greater effect with independent segregation of additive effects only, plus polygenes with additive and dominance effects, in which resistance is associated with recessive alleles. On the other hand, the genetic control of the resistance of the tomato 'Yoshimatsu' to R. solanacearum involves two genes of greater effect with independent segregation of additive effects and dominance, plus polygenes with additive and dominance effects, in which resistance is also associated To recessive alleles. In this study, the selection of plants resistant to R. pseudosolanacearum and R. solanacearum is indicated mainly at 20 days after inoculation. / A murcha bacteriana na cultura do tomateiro é uma doença que tem importância local, nacional e mundial. Esta doença é de difícil controle e pode provocar prejuízos que podem comprometer toda a lavoura. A resistência genética dentro do manejo integrado é a principal medida de controle da murcha bacteriana. Neste sentido, o conhecimento do controle genético da resistência em programas de melhoramento tende a aprimorar a eficiência em seu planejamento e na escolha do melhor método a ser adotado. O objetivo com esta tese foi estudar o controle genético da resistência do tomateiro ‘Yoshimatsu’ à Ralstonia pseudosolanacearum e Ralstonia solanacearum. Na primeira etapa dois experimentos foram conduzidos com os genitores Yoshimatsu, IPA-7 e as gerações F1, F2, RC11 e RC21, utilizando o delineamento em blocos casualisados com quatro repetições. Na segunda etapa dois experimentos foram conduzidos com 43 progênies F2:3 e seus genitores com o mesmo delineamento experimental da primeira etapa. Em cada etapa foram inoculadas as duas espécies do complexo R. solanacearum, em experimentos independentes. Foram avaliadas a incidência e severidade da murcha bacteriana por meio de escala descritiva de notas aos 10 e 20 dias após a inoculação. O controle genético da resistência do tomateiro ‘Yoshimatsu’ à R. pseudosolanacearum envolve dois genes de efeito maior com segregação independente de efeitos aditivos apenas, mais poligenes com efeitos aditivos e de dominância, em que a resistência está associada a alelos recessivos. Por outro lado, o controle genético da resistência do tomateiro ‘Yoshimatsu’ à R. solanacearum envolve dois genes de efeito maior com segregação independente de efeitos aditivos e de dominância, mais poligenes com efeitos aditivos e de dominância, em que a resistência também está associada a alelos recessivos. Neste estudo, a seleção de plantas resistentes a R. pseudosolanacearum e R. solanacearum é indicada principalmente aos 20 dias após a inoculação.

Submitted by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2017-12-19T15:11:24Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertação - Brunno dos Santos Fernandes.pdf: 1141872 bytes, checksum: 677ea8ad97b157d84045b98ea4db976d (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2017-12-19T15:11:37Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertação - Brunno dos Santos Fernandes.pdf: 1141872 bytes, checksum: 677ea8ad97b157d84045b98ea4db976d (MD5) / Made available in DSpace on 2017-12-19T15:11:37Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertação - Brunno dos Santos Fernandes.pdf: 1141872 bytes, checksum: 677ea8ad97b157d84045b98ea4db976d (MD5) Previous issue date: 2016-01-29 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The bacterial wilt (Ralstonia solanacearum) is a major disease of tomato (Solanum lycopersicum). The control is difficult because there are no resistant cultivars not recommended chemicals. The objective of this study was to evaluate the use of cubiu (Solanum sessiliflorum), jurubeba (Solanum viarum) and cultivar of tomato Yoshimatsu (tolerant) as rootstocks to control bacterial wilt. The experiment was conducted in a greenhouse was set up in a completely randomized design with five replicates, consisting of the treatments „Santa Cruz Kada Gigante‟/cubiu, „Santa Cruz Kada Gigante‟/jurubeba, „Santa Cruz Kada Gigante‟/‟Yoshimatsu‟, „Santa Cruz Kada Gigante‟ autograft and „Santa Cruz Kada Gigante‟ ungrafted, inoculated with 10 mL of the bacterial suspension at a concentration of 108 ufc.mL-1 race 1 (biovar 1) of Ralstonia solanacerum, the substrate around plant lap, whose roots were slightly injured with a scalpel. The witness consisted of plants treated with all combinations sterile distilled water. The experiment conducted in the field was set up in soil infested with Ralstonia solanacearum in a randomized block design with five replications, consisting of the same treatments used in the greenhouse. For both experiments, the assessment was made on a daily basis according to the incidence of the disease and the development of symptoms. It was also rated the stem diameter (mm) at the time of the colon, stem diameter 2 cm above the grafting point and plant height of the neck to the pointer (cm), these every seven days using digital calipers and tape measure, respectively . Still it was quantified the number of flowers per plant, number of flowers per inflorescence, number of fruits per plant, number of fruits per inflorescence, early flowering, early fruiting and designated rig plants above the eighth inflorescence of weeks after grafting. Data were statistically analyzed by Assistat version 7.7 program, and the averages compared by Tukey test at 5% probability. Tomato plants grafted on cubiu not developed symptoms of bacterial wilt and may be suitable for the production of seedlings and planting in areas contaminated with R. solanacearum. Cultivar yoshimatso showed the greatest growth and productivity of plants, providing partial resistance in grafted plants with this rootstock and can be used within a management program of the disease in tomato cultivation. / A murcha bacteriana (Ralstonia solanacearum) é uma das principais doenças do tomateiro (Solanum lycopersicum). O controle é difícil, pois não existem cultivares resistentes nem produtos químicos recomendados. O objetivo deste trabalho foi avaliar o uso de cubiu (Solanum sessiliflorum), jurubeba (Solanum viarum) e da cultivar de tomateiro Yoshimatsu (tolerante) como porta-enxertos para o controle da murcha bacteriana. O experimento foi conduzido em casa de vegetação em delineamento inteiramente casualizado com cinco repetições, constituído dos tratamentos „Santa Cruz Kada Gigante‟/cubiu, „Santa Cruz Kada Gigante‟/jurubeba, „Santa Cruz Kada Gigante‟/‟Yoshimatsu‟, „Santa Cruz Kada Gigante‟ auto-enxerto e „Santa Cruz Kada Gigante‟ pé-franco. As plantas foram inoculadas com 10 mL da suspensão bacteriana na concentração de 108 ufc.mL-1 da raça 1 (biovar 1) de R. solanacerum, depositada no substrato ao redor do colo da planta, cujas raízes foram levemente feridas com um bisturi. A testemunha constou de plantas de todas as combinações tratadas com água destilada esterilizada. O experimento conduzido em campo foi montado em solo naturalmente infestado com R. solanacearum, com delineamento em blocos casualizados com cinco repetições, constituído dos mesmos tratamentos utilizados em casa de vegetação. Para ambos os experimentos, a avaliação foi feita diariamente em função da incidência da doença e do desenvolvimento dos sintomas. Foi avaliado também o diâmetro do caule (mm) na altura do colo, diâmetro do caule 2 cm acima do ponto de enxertia e a altura das plantas do colo ao ponteiro (cm), a cada sete dias utilizando paquímetro digital e trena, respectivamente. Foi quantificado o número de flores por planta, número de flores por inflorescência, número de frutos por planta, número de frutos por inflorescência, início da floração, início da frutificação e capação das plantas acima da oitava inflorescência, em semanas após a enxertia. Os dados foram analisados estatisticamente pelo programa Assistat versão 7.7, e as médias comparadas pelo teste de Tukey a 5% de probabilidade. Plantas de tomate enxertadas em cubiu não desenvolveram sintomas de murcha bacteriana, podendo ser indicada para produção de mudas e plantio em áreas contaminadas com R. solanacearum. A cultivar yoshimatso apresentou o maior crescimento e produtividade das plantas, proporcionando resistência parcial em plantas enxertadas com este porta-enxerto, podendo ser usada dentro de um programa de manejo da doença em cultivo de tomateiro.

M.Sc. / Field grown plants are constantly challenged with a variety of stressful factors, such as high temperatures, drought and pathogen infection that adversely affect crop production and quality. These stresses seldom occur as single entities in plants and in warm climates, heat stress is often a common dominator in combinatorial stress. The heat shock (HS) response in plants has priority over other stress responses, including the pathogen-induced stress response. Activation of the HS response prevents the normal plant defence strategy, leaving the plant vulnerable to pathogen attack. However, prior exposure to elevated temperatures confers protection from subsequent, otherwise lethal, temperatures (thermotolerance) and a variety of other stress conditions including heavy-metals, chilling injury and certain pathogens (cross tolerance). In general, litterature supports a central role for heat shock proteins (HSP), in particular the 70 kDa HSP (Hsp70), in thermotolerance. Incompatible host-pathogen interactions lead to the activation of an array of defence mechanisms, including the promotion of phenylpropanoid metabolism. Phenylalanine ammonia-lyase is a key regulator of this metabolic pathway, influencing the production of salicylic acid, lignin and phytoalexins among other essential defence products. In this study it was hypothesised that prior exposure to non-lethal HS confers protection from subsequent heat-related suppression of the phenylpropanoid pathway, induced as a defence mechanism during an incompatible plant-pathogen interaction. This hypothesis was verified by analysing the effect of thermotolerance on pathogen-related stimulation of PAL promoter activity, enzyme activity and lignin deposition. The tomato, Lycopersicon esculentum cultivar UC82B and Ralstonia solanacearum, the causative agent of bacterial wilt, were used as host-pathogen model. Specific objectives in the study were: (1) Development of PAL promoter-GUS reporter transformed Lycopersicon esculentum. (2) Establishment of a thermotolerance protocol that ensures optimal Hsp70 levels at subsequent HS. (3) Evaluation of the influence of prior heat treatment on phenylpropanoid metabolism after exposure to HS in combination with Ralstonia solanacearum. Results obtained support the hypothesis indicating that thermotolerance protects phenylpropanoid metabolism, in particular PAL promoter and enzyme activity, and to a certain extent lignin production, induced by avirulent Ralstonia solanacearum during a second severe HS. In contrast, HS without a prior heat treatment, suppressed phenylpropanoid metabolism. The protective potential of prior heat treatment during subsequent infection under hyperthermic conditions support the application of HSP in the development of novel plant protection strategies.

M.Sc. / Plants are in constant conflict with pathogens and have evolved intricate mechanisms to protect themselves against pathogens. The gene-for-gene response is regarded as the first line of defence when plant and pathogen meet. This interaction leads to the induction of defence proteins such as PR proteins that protect the plant from invading pathogens. A seemingly unrelated topic to plants and pathogens is heat shock proteins (HSP). HSP are a highly conserved group of defence proteins induced in all organisms in response to a variety of environmental stresses to provide protection from, and adaptation to cellular stress. HSP are in general not considered to be part of the defence response classically induced by avirulent pathogens and whether they are induced and play a role in plant-pathogen interactions is controversial. The protective chaperoning capacity of HSP makes them ideal proteins to exploit to target as endogenous defence proteins in the search for new strategies in the management of infectious diseases. In humans, HSP induction during infection is a complex phenomenon depending on the pathogen, whether the infection is acute or chronic, the host cell type and its differentiative state as well as environmental factors. In this investigation the expression of the inducible and constitutive isoforms of the 70kDa HSP (Hsp70/Hsc70) was investigated in tomato, Lycopersicon esculentum in response to virulent and avirulent strains of Ralstonia solanacearum, the causative agent of bacterial wilt. Expression of Hsp70 was studied in conjunction with the accumulation of PR-la and host cell viability. A quick, non-toxic, tetrazolium-based assay was developed from the Alamar Blue assay, commonly used in mammalian cells, and applied for the evaluation of host cell viability. The results shown suggest Hsp70/Hsc70 is significantly induced in tomato cell suspensions during an incompatible interaction 24h to 48 h following co-cultivation with the avirulent R. solanacearum strain compared to normal levels at this interval in cells exposed to the virulent strain. In both compatible and incompatible interactions Hsp70/Hsc70 levels eventually (72 h) accumulated correlating significantly with decreased viability. PR-la accumulation was significantly induced from 6 h to 18 h by the virulent as well as the avirulent R. solanacearum strains. In general, comparable results were obtained using leaf discs as an in vivo model. Based upon the differential induction of Hsp70/Hsc70 by virulent and avirulent pathogens it is proposed that HSP may play an important role in determining the outcome of the interaction between tomato and R. solanacearum. Successful defence may not only involve a limited number of defence genes but may result from a concerted action of a large number of defence genes.

Heat shock proteins (HSP), although not considered classical defence proteins, have general cytoprotective properties, which promote survival of cells and organisms. Hsp70, in particular, provides resistance to the harmful consequences of various forms of otherwise damaging or even lethal stress including pathogen infection. Increased levels of Hsp70, due to stable transfection of cells with hsp70 genes, or elevated expression in response to stress, generally correlate with the hindrance of cell death processes triggered by a variety of noxious stimuli or toxic agents. The effect of lipopolysaccharides (LPS), the major constituent of the outer membrane of the cell wall (envelope) of almost all Gram-negative bacteria, on Hsp70/Hsc70 expression in plants is unknown. In various mammalian systems, LPS has been shown to induce Hsp70 accumulation, along with programmed (apoptotic) cell death. Contrary to the effects of LPS on animal hosts however, LPS does not elicit cell death in plants, but rather pre-treatment with LPS fraction can prevent or delay the so-called hypersensitive response (HR), thus sensitizing plant tissue to respond more rapidly, or to a greater extent, to subsequently inoculated phytopathogenic bacteria. Elevated levels of reactive oxygen species (ROS) reportedly contribute to stress sensing and hsp gene activation, and subsequent Hsp70 induction, during the stress response. Increased ROS production can also trigger cell death via either programmed cell death (PCD), an active (i.e., energy-dependent) physiological suicide mechanism that is genetically regulated, or uncontrolled necrosis, an accidental, lytic form of cell destruction passively triggered by severe trauma or injury. In plants specifically, ROS may be involved in PCD activation during the HR. As a pathogen-associated molecular pattern (PAMP) or general elicitor of defence or resistance-related responses, LPS may trigger some defence-related responses, including an oxidative burst (manifest as increased levels of reactive oxygen species or ROS) in certain plant cells as it does in animal systems. However, there is conflicting evidence that shows that LPS treatment does not elicit an oxidative burst in plants. The aim of this study was to determine the effect of LPS isolated from Ralstonia solanacearum, an extremely harmful soil-borne bacterium that causes Southern wilt in over 200 plant species by infecting the host’s roots and invading the xylem vessels, on Hsp70/Hsc70 accumulation, cell death and ROS production in suspension-cultured tobacco (Nicotiana tabacum) cells, in order to gain a better understanding of the inter-relationship between these three phenomena in plant cells responding to LPS(Ralstonia). Western (immuno)blot analysis was used to study the unknown effect of LPS(Ralstonia) on Hsp70/Hsc70 accumulation in tobacco cell suspensions. LPS(Ralstonia) (all concentrations and time periods studied) generally suppressed Hsp70/Hsc70 accumulation. However, only exposure to 100 μg LPS/ml for 3 h caused a significant reduction (P < 0.05). Therefore, there was an early suppression of Hsp70/Hsc70 accumulation in response to 100 μg LPS(Ralstonia)/ml. To determine whether the observed LPS-mediated attenuation in Hsp70/Hsc70 accumulation was due to an increase in cell death in these cells, we investigated the effect of LPS(Ralstonia) on i) the general viability of the cells, and ii) the integrity of nuclear or genomic DNA extracted from LPS-treated suspension-cultured tobacco cells. The AlamarBlue™ (AB) assay was used to investigate the general cell viability in response to LPS(Ralstonia) treatment. LPS(Ralstonia) (all concentrations and time intervals studied) did not significantly affect the overall viability of the cells. Because treatment of tobacco cell suspensions with LPS(Ralstonia) did not result in a significant decrease (P < 0.05) in AB reduction, it was presumed that LPS(Ralstonia) did not appreciably compromise metabolic activity and was therefore not particularly toxic to these cells. Genomic DNA from cells undergoing PCD-associated internucleosomal DNA fragmentation (IDF) typically runs as a ladder of internucleosomal-sized DNA fragments corresponding to multimers of ca. 180 bp in agarose gels. In contrast, random DNA cleavage, usually manifest as smearing of nuclear DNA following agarose gel electrophoresis, is a token of uncontrolled necrosis. Therefore, if so-called “DNA laddering” is observed following agarose gel electrophoresis of genomic DNA extracted from suspension-cultured tobacco cells exposed to LPS(Ralstonia) then it can be assumed that LPS(Ralstonia) induced PCD. Alternatively, if a long, continuous “necrotic smear” is evident after electrophoretic separation of nuclear DNA from LPS-treated cells then LPS(Ralstonia) clearly induced uncontrolled necrosis. Whether or not LPS(Ralstonia) induced PCD-associated IDF or necrotic smearing was determined by investigating genomic DNA fragmentation (or DNA integrity) in response to LPS(Ralstonia) iii treatment. Although no typical DNA ladders were detected following electrophoresis of DNA isolated from LPS-treated cells, PCD may still have transpired. However, this is highly unlikely. No necrotic smearing was evident in LPS-treated samples either, which verifies the hypothesis that LPS(Ralstonia) (25–100 μg/ml) did not induce uncontrolled necrosis in suspension-cultured tobacco cells. In fact, these concentrations of LPS(Ralstonia) did not seem to significantly compromise DNA integrity given that LPS(Ralstonia) (25–100 μg/ml) generally had no appreciable effect on genomic DNA fragmentation (compared to untreated control samples). Incidentally, 24-h exposure of tobacco cell suspensions to higher concentrations of LPS(Ralstonia) (500 and 1000 μg/ml) may have resulted in partial DNA cleavage and/or degradation. Exposure of tobacco cell suspensions to 400 μg LPS(Burkholderia)/ml for 7 days may also have evoked partial DNA cleavage and/or degradation. Whether this cleavage and/or degradation occurred deliberately by means of a fixed or predetermined mechanism or randomly by an uncontrolled mechanism remains uncertain. Finally, the H2DCF-DA (2′, 7′-dihydrodichlorofluorescein-diacetate) fluorescence assay was used to investigate the effect of LPS(Ralstonia) on ROS production, a common factor in the regulation of HSP expression and cell death activation. LPS(Ralstonia) treatment (25–100 μg/ml) generally increased ROS levels in suspension-cultured tobacco cells (compared to untreated control cells). Exposure to 75 μg LPS(Ralstonia)/ml resulted in a particularly prominent elevation in ROS levels almost instantaneously. Incidentally, higher concentrations of LPS(Ralstonia) (500 and 1000 μg/ml) resulted in decreased ROS levels at some point during the assay. Although LPS(Ralstonia) (100 μg/ml for 3 h) significantly decreased Hsp70/Hsc70 accumulation in tobacco cell suspensions, cell death did not appear to be induced. In fact, LPS(Ralstonia) had no effect on general cell viability and appeared to be ineffective at causing PCD-associated IDF (DNA laddering) or necrotic smearing regardless of concentration or time of exposure. Despite these findings, treatment of suspension-cultured tobacco cells with LPS(Ralstonia) (≤ 100 μg/ml) resulted in a mild increase in ROS production. Although the exact mechanism(s) by which LPS(Ralstonia) suppressed Hsp70/Hsc70 accumulation is elusive, our results suggest that the suppression is not related to excessive LPS-mediated injury caused by excessively high ROS levels or increased cell death. We speculate that the prevention of HR-related PCD often observed in plants that are pre-treated with LPS and subsequently inoculated with phytopathogenic bacteria may be dependent on the LPS-mediated suppression of cytosolic Hsp70 expression. / Dr. M.J. Cronje

Please read the abstract in the section 07back of this document / Dissertation (MSc (Plant Pathology))--University of Pretoria, 2006. / Microbiology and Plant Pathology / unrestricted

Nous présentons l’étude de l’interaction entre la bactérie pathogène racinaire Ralstonia solanacearum et la légumineuse modèle Medicago truncatula. Un pathosystème avec les lignées A17 et F83005.5, respectivement sensible et résistante à la souche GMI1000, a été mis en place avec une procédure d’inoculation sur racines intactes. Ce dispositif expérimental nous a permis de suivre le processus infectieux, de la pénétration de la bactérie par l’extrémité racinaire au développement des symptômes foliaires. L’analyse des étapes précoces de l’interaction a permis de décrire l’apparition de symptômes racinaires qui se mettent en place rapidement après l’infection, que les lignées soient résistantes ou sensibles à la bactérie. Un arrêt de croissance de la racine s'observe dès 24 heures post-inoculation, ainsi qu’une mortalité de l’épiderme de l’extrémité racinaire. Ces phénotypes sont notés suite à des inoculations avec de faibles concentrations bactériennes, et ce sur plusieurs espèces hôtes ou non-hôtes testées. La mise en place des symptômes racinaires est dépendante de l’appareil de sécrétion de type III. Un crible de mutants d’effecteurs de type III de la souche GMI1000, basé sur l’apparition des symptômes racinaires, a permis de montrer que des pools différents d’effecteurs interviennent chez A17 et F83005.5. Chez la lignée sensible A17, deux effecteurs sont principalement impliqués, Gala7 et AvrA. L’étude de la colonisation de cette lignée a montré que le mutant gala7 ne pénètre pas la plante et n’induit pas de symptômes de flétrissement. Le mutant avrA s’est révélé capable d’induire la maladie chez la lignée A17 mais de manière nettement réduite par rapport à la souche sauvage. L’analyse des extrémités racinaires des lignées sensible et résistante infectées par la souche GMI1000 a révélé qu’au niveau des parois de l’endoderme, la présence de lignine est induite de manière plus précoce chez la lignée résistante. Des phénomènes de division cellulaire ont été identifiés autour du cylindre central et semblent également liés à une restriction de la propagation bactérienne. Au niveau du contenu cellulaire, une autofluorescence et une production de ROS semblent liés à une phase nécrotrophe de la bactérie lors de sa propagation dans la zone corticale de l’extrémité racinaire. L’étude de la colonisation bactérienne en s’affranchissant de l’étape de pénétration a révélé que des mécanismes de résistances peuvent intervenir au niveau de collet chez la lignée F83005.5 et lors de la colonisation racinaire des vaisseaux conducteurs suite à une inoculation avec le mutant gala7 / Manquant

Ralstonia solanacearum, the causal agent of bacterial wilt disease, has been found to affect numerous economically important plants. Understanding the molecular basis of resistance, tolerance and susceptibility of plants to pathogens such as R. solanacearum is a major goal of molecular plant pathologists. Prior to this study it was thought that Arabidopsis accession Kil-0 shows gene-for-gene “resistance” to an African Eucalyptus isolate of R. solanacearum, BCCF402. However, a subsequent preliminary study indicated that Kil-0 may exhibit “tolerance” which is defined as the plant’s ability to support high pathogen numbers without displaying disease symptoms or a reduction in host fitness. The aim of this study was to determine if Kil-0 was tolerant to R. solanacearum BCCF402. The bacterial load of R. solanacearum was quantified in accessions Kil-0 and Be-0 using dilution plating and quantitative PCR methods. The cytC gene region was used to quantify R. solanacearum in Arabidopsis plants and the amount of bacterial DNA was normalized to “alien” DNA that was spiked into each sample. High bacterial concentrations of BCCF402 were found in Kil-0 but plants exhibited no wilting symptoms. Additionally, Kil-0 plants inoculated with BCCF402 showed no significant reduction in fitness compared to control Kil-0 plants. In contrast, high bacterial numbers and severe disease symptoms were observed in the susceptible Be-0 plants, whereas Nd1 plants contained a low number of bacteria and no disease symptoms indicative of a resistance response. These results illustrated that Kil-0 is tolerant to R. solanacearum isolate BCCF402. A tool for the visualization of R. solanacearum in Arabidopsis plants was designed. R. solanacearum isolate BCCF402 was tagged with two mCherry-containing plasmids under the constitutive expression of the tac promoter. The expression levels of mCherry were suitable for successful visualization in planta. BCCF402 cells transformed with the mCherry-containing plasmids were not affected in terms of virulence or disease progression compared to wildtype BCCF402 cells. A plasmid loss of 30-35% was observed in mCherry-tagged BCCF402 cells at later stages of Arabidopsis infection. mCherry-tagged BCCF402 was successfully visualized in Kil-0 leaves at early infection stages. / Dissertation (MSc)--University of Pretoria, 2014. / gm2014 / Plant Science / unrestricted

Unlike mammals, plants do not have specific immune cells. However, plants can still recognize pathogens and defend themselves. They do that by recognizing microbial-associated molecular patterns (MAMPs) and secreted pathogen proteins, called effectors. MAMP-triggered immunity (MTI) relies on recognition of MAMPs by leucine-rich repeats (LRRs) pattern-recognition receptors (PRRs). The best-studied LRR PRR is Flagellin-Sensitive 2 (Fls2), the receptor of a 22-amino acid long epitope of bacterial flagellin, called flg22. In this project, alleles of FLS2 of different tomato cultivars were sequenced and compared to each other to get insight into natural selection acting on FLS2 and to identify residues important for ligand binding. This information may be used in the future to engineer Fls2 for improved ability to recognize flagellin. MTI can be suppressed by effectors secreted by bacteria into plant cells through the type III secretion system. On the other hand, plants are equipped with repertoires of resistance proteins, which can recognize some pathogen effectors. If a pathogen carries an effector that is recognized, effector-triggered immunity (ETI) is activated and the plant is resistant. Here, eggplant breeding lines were screened for their ability to activate ETI upon recognition of effectors of the soil borne pathogen Ralstonia solanacearum, a causative agent of bacterial wilt. Four effectors were found to trigger plant defenses in some of the lines. This is the first step in cloning the genes coding for the responsible resistance proteins. These genes may be used in the future for engineering tomato and potato for resistance to bacterial wilt. / Master of Science in Life Sciences

Submitted by Gabriela Lopes (gmachadolopesufpel@gmail.com) on 2017-06-20T16:50:30Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertação Dediel.pdf: 766607 bytes, checksum: 1d7fec62b5b0587e0accd22056511b85 (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-06-21T18:58:18Z (GMT) No. of bitstreams: 2 Dissertação Dediel.pdf: 766607 bytes, checksum: 1d7fec62b5b0587e0accd22056511b85 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-06-21T18:58:18Z (GMT). No. of bitstreams: 2 Dissertação Dediel.pdf: 766607 bytes, checksum: 1d7fec62b5b0587e0accd22056511b85 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2012-03-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / A busca por alternativas no controle de doenças em substituição ao uso intensivo de agrotóxicos tem sido objeto da pesquisa agrícola. Rizobactérias são conhecidas pelo biocontrole de doenças e promoção de crescimento em diversos cultivos. Os objetivos deste trabalho foram avaliar a eficácia de seis rizobactérias, pré- selecionadas, no controle de Ralstonia solanacearum e Fusarium oxysporum f. sp. lycopersici (FOL), em casa de vegetação e relacionar este comportamento a produção de compostos “in vitro”, bem como avaliar a proteção de plantas de tomate contra doenças da parte aérea que ocorreram naturalmente em condições de campo. Foi avaliada a capacidade destas rizobactérias em produzir quitinases, amilases, lipases, compostos antibióticos e de solubilizar fosfato. A microbiolização das sementes com a rizobactéria DFs1420 (Bacillus sp.) reduziu a severidade da murcha bacteriana em 66% aos 14 dias após a inoculação, no primeiro ensaio e valores de AACPD em 70%, no segundo ensaio. Este controle pode ser associado à produção de compostos responsáveis pela antibiose observada “in vitro”. Streptomyces (DFs1296 e DFs1315) e Bacillus sp. (DFs1414) reduziram significativamente a murcha de fusário. O controle observado pode ser atribuído à atividade quitinolítica e/ou antibiótica por compostos voláteis. Foram instalados três ensaios de campo. A severidade das doenças foliares foi monitorada ao longo do tempo. As rizobactérias foram capazes de proteger as plantas contra Septoria lycopersici. DFs1414 e DFs1421 (Pseudomonas sp.) foram as mais estáveis, proporcionando proteção em dois ensaios consecutivos. As rizobactérias DFs1296 e DFs1420 foram capaz de controlar a requeima (Phytophthora infestans) em dois ensaios. A bactéria DFs1296 também apresentou capacidade de proteção contra estas e outras doenças quando pulverizada semanalmente na parte aérea. De modo geral, o controle alcançado por estas bactérias, microbiolizadas às sementes ou em aplicação foliar, não diferiu dos tratamentos utilizando produtos químicos recomendados para a cultura em aplicações semanais. / The search on alternative control of diseases to replace the use of pesticides has been the subject of agricultural research. Rhizobacteria are known by controlling diseases and promoting growth in several crops. The objectives of this work were to evaluate the efficacy of six rhizobacteria pre-selected for the control of Ralstonia solanacearum and Fusarium oxysporum f. sp. lycopersici (FOL) in greenhouse, and to relate this behavior with the production of compounds "in vitro", as well as evaluate the protection of tomato plants against foliar diseases under field conditions. It was evaluated the ability of these rhizobacteria to produce chitinases, amylases, lipases, antibiotic compounds and to solubilize phosphate. The seed microbiolization with rhizobacterium DFs1420 (Bacillus sp.) reduced the severity of the bacterial wilt 66% at 14 days after inoculation and AUDPC 70%, respectively, the first and second assays. This control may be associated with the production of compounds responsible for the antibiosis observed "in vitro". Streptomyces (DFs1296 and DFs1315) and Bacillus sp. (DFs1414) significantly reduced fusarium wilt. The control observed can be attributed to the chitinolytic activity and/or antibiosis in the presence of volatile compounds. Three field trials were carried out in field. The foliar diseases severity was monitored over time. The rhizobacteria were capable of protecting plants against Septoria lycopersici. DFs1414 and DFs1421 (Pseudomonas sp.) were the most stable, providing protection in two consecutive trials. The rhizobacteria DFs1296 and DFs1420 were able to control late blight (Phytophthora infestans) in two trials. DFs1296 also had the ability to protect against these and other diseases when sprayed weekly in the plant canopy.In general, the control achieved by these rhizobacteria, by seed microbiolization or by foliar application, did not differ from treatments using recommended chemical in weekly applications.